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This is to certify that the
dissertation entitled

CHARACTERIZATION OF PHYSIOLOGICAL AND
TRANSCRIPTOME CHANGES IN THE ANCIENT SIBERIAN
PERMAFROST BACTERIUM Psychrobacter arcticum 273-4 WITH
LOW TEMPERATURE AND INCREASED OSMOTICA

presented by

Monica A. Ponder

has been accepted towards fulfillment
of the requirements for the

Ph.D. degree in Microbiology and Molecular
A Genetics

 

 

    

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/ M' or Professor’s éijnature

MSU is an Affirmative Action/Equal Opportunity Institution

PLACE IN RETURN BOX to remove this checkout from your record.
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2/05 Jam.m.as

CHARACTERIZATION OF PHYSIOLOGICAL AND TRANSCRIPTOME CHANGES
IN THE ANCIENT SIBERIAN PERMAFROST BACTERIUM Psychrobacter arcticum
273-4 WITH LOW TEMPERATURE AND INCREASED OSMOTICA

By

Monica A. Ponder

A DISSERTATION

Submitted to
Michigan State University
In partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology and Molecular Genetics

2005

ABSTRACT

CHARACTERIZATION OF PHYSIOLOGICAL AND TRANSCRIPTOME CHANGES
IN THE ANCIENT SIBERIAN PERMAFROST BACTERIUM Psychrobacter arcticum
2734 WITH LOW TEMPERATURE AND INCREASED OSMOTICA
By

Monica A. Ponder

The Siberian permafi'ost is an extreme, yet stable environment due to its
continuously frozen state. Average temperatures of ~10 to -12°C concentrate solutes to
an aw = 0.90 (5 osm), and limit nutrient diffusion into cells. The isolation of viable
Psychrobacter arcticum 273-4 and Exiguobacterium sibericum 255-15 from ancient
permafrost suggests that these bacteria have maintained some level of metabolic activity
for thousands of years and are likely cryo-adapted. Past studies Of cold-acclimated
bacteria have focused primarily on organisms not capable of sub-zero growth. Siberian
permafrost isolates E. sibericum 255-15 and P. arcticum 273-4, which grow at subzero
temperatures, were used to study cold-acclimated physiology. Changes in membrane
composition and exopolysaccharides were defined as a function of growth at 24, 4 and —
2.5 °C in the presence and absence of 5% NaCl. As expected, there was a decrease in
fatty acid saturation and chain length at the colder temperatures and a further decrease in
the degree of saturation at higher osmolarity. A shift in carbon source utilization and
antibiotic resistance occurred at 4 versus 24 °C growth, perhaps due to changes in the
membrane transport. Both isolates had excellent survival after one year of being frozen

at -20°C and moderate ice nucleation activity.

To gain knowledge of the features for adaptation to low temperature and water
activity, the genome Of a Siberian permafrost isolate was sequenced. Psychrobacter
arcticum 273-4, a gamma-Proteobacterium grows at temperatures as low as -—10°C and
displays marked physiological changes under low temperature vs. mesophilic growth and
in the presence of increased osmotica (salt). Differential gene expression analyses at 4°C
and 22°C, using microarrays specific for the predicted genes of P. arcticum 273-4, reveal
that only a few genes known to be low-temperature and salt-responsive in mesophiles and

in some psychrotrophs are differentially expressed in P. arcticum. Total respiratory
activity increased in the presence of salt, due to increased number of Na+ dependent

dehydrogenase and decarboxylase transcripts suggesting that P. arcticum has adapted for
energy generation in the low temperature and low water activity of the permafrost
environment. Growth in salt-amended l/2 TSB leads to induction of capsule genes and a
capsule is visible around cells grown at both 22 and 4°C, which could allow P. arcticum
to adhere to soil particles within the permafrost. Capsule production, lipid modification
and compatible solute accumulation to balance internal osmolytes may allow P. arcticum
to remain metabolically active within the low water activity of the Siberian permafrost.
The majority of P. arcticum cells had functioning electron transport systems, intact

membranes, aerobically respire and synthesize new proteins at the salinity (2.79m NaCl,
aw= 0.91) of the permafrost. The gene expression profiles and physiological responses

of P. arcticum to low temperature and increased osmotica indicate that they have adapted
to the low water activity and subzero temperatures (—10 to —12 °C) of the Kolyma region

permafrost environment, where intracellular water is likely not frozen.

Copyright by
Monica A. Ponder

2005

ACKNOWLEDGMENTS

I would like to thank my advisor Dr. James M. Tiedje for the opportunity to
realize my dream of earning a Ph.D. in Microbiology and Molecular Genetics. His
support and trust has provided me with the confidence necessary to realize my scientific
goals. I would like to thank all the members Of the NASA astrobiology institute’s Center
for Genomic and Evolutionary Studies on Microbial Life at Low Temperatures. Their
guidance, critical questions and encouragement have been vital for my scientific
development.

I thank the members Of my guidance committee: Drs. John Breznak, Sophia
Kathariou, John McGrath and Michael Thomashow for their discussion, encouragement
and advice throughout this research. Special thanks to Dr. Frank Dazzo for sharing his
microscopy expertise and making available the CMEIAS software for statistical analysis.

I thank the members Of the Tiedje lab for all their helpful advice, encouragement
and especially friendship through the last five years. I would like to especially
acknowledge Drs. Hector Ayala-del-Rio and J oonhong Park for their critical discussion
and willingness to share their expertise as often as I as asked. I thank Chia- Kai Chang
and Gisel Rodrigues, who it was my privilege to mentor, and with their assistance much
Of this work was expedited. Thanks to the CME staff: Lisa Pline, Pat Engelhart, Nikki
Mulvaney and Joyce Wildenthal for their wonderful assistance and support throughout

the years.

I owe the utmost gratitude to Dr. Renate Snider, who has allowed me to develop

as a scientific writer. Special thanks to Steven Forbes Tuckey, who always provides a

laugh and valuable suggestions about my writing. Thank you to Dr. John Urbance, for
his enthusiasm and willingness to mentor my teaching experiences.

I would like to thank Dr. Marcia Lee, who gave me my first taste of microbiology
research as an undergraduate. Without her encouragement and assistance, I would have
missed the fascinating world of microbiology. I am grateful for her continuing interest
and encouragement of my career.

I can never thank my family enough for their encouragement and understanding
throughout the years. They have never stopped believing in me, which provided me the
self-confidence necessary to succeed in life. I am eternally gratefiil to my husband,
Kevin who has supported me emotionally and financially throughout the last five years. I
am very grateful to the sacrifices he has made so that I may pursue my dream. His level-
headed outlook and optimistic dreaming has enriched my life. Thanks to my friends
Katherine Goliver, Kelly Harness, Sandy Johnson and Nora Meijia—-without your

willingness to listen, laugh and attend any cultural event, life would have been very dull.

vi

PREFACE

The whole genome sequence of Psychrobacter arcticum 273-4 was generated by
the Joint Genome institute, pipe-line annotated by the computational division of the Oak
Ridge National Lab Life Science’s Division. The genome was finished by Patrick Chain
of Lawrence Livermore National Labs and manually annotated by Monica Ponder, Peter
Bergholz, Debora Rodrigues and Drs. Hector Ayala del-Rio, Loren Hauser, Joel
Klappenbach, Corien Bakermans and Dan Zarka. Oligonucleotides for whole genome
microarrays for Psychrobacter arcticum 2734 were designed and synthesized by Operon
technologies. Microarrays were spotted by Jeff Landgraf of the GTSF at Michigan State
University (MSU).

Ice nucleation activity measurements were performed in the laboratory of Dr.
Marcia Lee at Miami University. Fatty acid profiling was performed in the laboratory of
Dr. Rawle Hollingsworth at MSU. UV-C resistance measurements were performed by
Peter Bergholz in the laboratory of George Sundin at MSU.

Nuclear magnetic resonance (NMR) measurements were performed by Dr. Carol
Mindock at MSU. GC/MS was performed by Beverly Chamberlin at the Center for Mass
Spectrometry at MSU. Presences of compatible solutes were detected by Dr. Gary Smith
0f the University of California, Davis. Transmission electron microscopy pictures
Presented within this dissertation were taken by Alicia Pastor of the Center for Advanced
Microscopy at MSU.

CaPSule compositions presented within were determined by Dr. Parastoo Azadi of the

Complex Carbohydrate Research Group of the University of Georgia.

vii

TABLE OF CONTENTS

LIST of FIGURES ................................................................................ xi

LIST of TABLES ............................................................................... xiii

CHAPTER 1: INTRODUCTION .............................................................. 1
Thesis overview ............................................................................ 6
Experimental design ........................................................................ 7
Conclusions and future perspectives ..................................................... 9
References .................................................................................. 10

CHAPTER 2: MICROBIAL LIFE in PERMAFROST: EXTENDED TIMES in

EXTREME CONDITIONS ............................................................. . ....... 13
Introduction .................................................................................... 14
Permafrost physical characteristics ........................................................................... 15
Permafrost diversity ................................................................................................... 17

History of permafrost microbial isolations ........................................................ 19
Bacteria .................................................................................... 20
Cyanobacteria and algae ................................................................ 25
Archaea .................................................................................... 27
Eukaryotic Microorganisms ............................................................................... 27
Mechanisms of survival .............................................................................................. 29
Physical characteristics of permafrost ............................................................... 29
Physiological adaptation .................................................................................... 3O
Membrane adaptation ................................................................... 31
Production of cold-induced proteins... ... . . . .. . ..- .................................... 33
Compatible solute production ......................................................... 34
Alternate physiological adaptations to permafi'ost conditions ..................... 38
Metabolic activity ........................................................................................................ 40
Anabiosis theory ................................................................................................ 40
Low rate of in situ metabolic activity theory ..................................................... 41
Importance of the permafrost environment ................................................. 43
References ...................................................................................... 46

CHAPTER 3: CHARACTERIZATION of POTENTIAL STRESS RESPONSES in

ANCIENT SIBERIAN PERMAFROST PSYCHROACTIVE BACTERIA ......... 56
Introduction ................................................................................. 58
Materials and Methods ..................................................................... 6O

Isolation and phylogenetic characterization .................................... 60
Growth rates ........................................................................ 61
Cell morphology and size ....................................................... 62
Effect of temperature and salinity on lipid and polysaccharide composition
....................................................................................... 63

viii

Freezing tolerance ................................................................ 64

Temperature dependent nutrient utilization ................................... 65
Antibiotic susceptibility ......................................................... 65
UVC survival ..................................................................... 6
Ice nucleation activity ............................................................ 67
Results ....................................................................................... 67
Effect of temperature on the maximal growth rate and cellular morphology
....................................................................................... 68
Effect of increased osmolarity on growth of two Siberian permafrost
isolates .............................................................................. 68
Membrane compositional changes ............................................. 71
Polysaccharide composition ..................................................... 73
Freezing tolerance ............................................. . .................... 77
Temperature effect on antibiotic susceptibility ................................ 79
UVC resistance ................................................................... 81
Ice nucleation activity ............................................................ 85
Discussion ................................................................................... 86
References .................................................................................. 94

CHAPTER 4: CRYO-ADAPTATION of Psychrobacter arcticum 2734 AS
DETERMINED THROUGH TRANSCRIPTOME PROFLING AT LOW

TEMPERATURE and INCREASED OSMOTICA ........................................ 98
Introduction ................................................................................ 99
Materials and Methods .................................................................... 102

Bacterial strain and genome sequence ....................................... 102
Microarray construction ...................................................... 102
Cell Growth and Preservation ................................................. 103
RNA extraction protocol ....................................................... 103
Amplification protocol .......................................................... 104
Hybridization .................................................................... 104
Image acquisition and analysis ................................................ 106
Quantitative real-time reverse transcription PCR (Q RT-PCR) ........... 106
NADH dehydrogenase assay ................................................... 108
Transmission electron microscopy ........................................... 109
Results and Discussion .................................................................. 110
Activity of NADH dehydrogenase in different salinities and temperatures
.................................................................................... 113
Compatible solute transcripts and salt response of Psychrobacter arcticum
.................................................................................... 1 1 8
Fatty acid and phospholipid synthesis ....................................... 121
Polysaccharide composition associated transcripts ........................ 123

Verification of microarray fold change using quantitative real time PCR
...................................................................................... 125

ix

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Comparison of transcriptome and proteome profiles ....................... 130
Conclusions ............................................................................... 130
References ................................................................................. 143

CHAPTER 5: METABOLIC ACTIVITY of SIBERIAN PERMAFROST
ISOLATES, Psychrobacter arcticum 273-4 and Exiguobacterium sibericum 255-15,

AT LOW WATER ACTIVITIES ............................................................ 149
Introduction ............................................................................... 150
Materials and Methods ................................................................... 152

Isolation and phylogenetic characterizations ................................ 151
Culture conditions ............................................................... 153
Electron shuttling activity by CTC ........................................... 154
Membrane permeability by LIV E/DEAD staining ......................... 154
Aerobic respiration by resazurin reduction .................................. 155
Tritiated leucine incorporation assay ......................................... 155
Transmission electron microscopy ........................................... 156
Results ...................................................................................... 157
Growth in 1/2 TSB + 2.79m NaCl .............................................. 157
Electron transport ............................................................... 158
Membrane permeability ........................................................ 158
Aerobic respiration .............................................................. 161
Incorporation of tritiated leucine into protein ............................... 163
Transmission electron microscopy ........................................... 166
Discussion ................................................................................ 168
References ................................................................................ 174
CHAPTER 6: CONCLUSIONS and FUTURE DIRECTIONS ...................... 177
Conclusions ..................................................................... 177
Future Directions ............................................................... 180
References ........................................................................ l 83

APPENDIX A: CONTRIBUTIONS TO THE DESCRIPTION and GENOME

ANALYSIS of Psychrobacter arcticum ....................................................... 184
Contributions to the description of Psychrobacter arcticum ...................... 184
Contributions to the genome annotation of Psychrobacter arcticum ............ 186

Annotation of genes involved in carbohydrate metabolism ............... 188
Annotation of P. arcticum transcription related genes ..................... 189
References ................................................................................ 190

APPENDIX B: EXPRESSION PROFILES of P. arcticum 2734 in DIFFERENT
SALINITIES and TEMPERATURES ....................................................... 193

APPENDIX C: EXPERIMENTAL CONDITIONS FOR PROTEOMICS ......... 210

LIST OF FIGURES

FIGURE 2.1 Microbes apparently encased in biofilm in frozen state in permafrost soil.
environmental scannning electron microscope. (Courtesy of Dr. Vera Soina) ............. 30

FIGURE 2.2 This plot shows predicted microbial plasmolysis during freezing at various
cooling rates. ......................................................................................... 39

FIGURE 3.1 An Arrhenius plot of growth of Exiguobacterium (392-28 and 255-15) and
Psychrobacter (273—4 and 215-51) at a range of temperatures between 42°-
O.5°C .................................................................................................. 70

FIGURE 3.2 Comparison of average growth rates of two permafrost isolates at different
temperatures and osmolarities .................................................................... 72

FIGURE 4.1 Venn diagram of the number of ORFs whose expression changed more
than two-fold as a function of temperature or salt ............................................ 112

FIGURE 4.2 Differential expression of the Na+ dependent NADH dehydrogenase
operon in P. arcticum ........................................................................... 116

FIGURE 4.3 Transmission electron microscopy pictures of P. arcticum revealing
presence of capsules when grown in the presence of 5% NaCl at 4 (A) or 22°C (B) and no
capsules surrounding cells grown at 4 (C) or 22 °C (D) in l/2 TSB ........................ 126

FIGURE 4.4 Comparison of microarray fold change and difference in copy number by
real time PCR. .................................................................................... 129

FIGURE 5.1 Average percent of cells with intact membranes afier 10 days incubation
1n 1/2 TSB + 2.79 M NaCl at different temperature .......................................... 161

FIGURE 5.2A Resazurin reduction at 22°C by E. sibericum 255-15 and P. arcticum
273-4 1n ‘/2 TSB+ 2.79 M NaCl ................................................................ 162

xi

FIGURE 5.2B Resazurin reduction measured by absorbance decrease at 4°C by E.
sibericum 255-15 and P. arcticum 273-4 in l/2 TSB + 2.79m NaCl ..................... 163

FIGURE 5.3A Incorporation of 3H- leucine into TCA precipitable fraction of cells
grown at 22°C for permafrost isolates and 37°C for E. coli 1n 1/2 TSB + 2. 79m NaCl.
................................................................................................................. 165

FIGURE 5.33 Incorporation of 3H- Leucine into The TCA precipitable fraction of cells
over 30 days in ‘/2 TSB + 2.79m NaCl at 4°C in permafrost isolated P. arcticum and E.
sibericum. ........................................................................................ 166

FIGURE 5.4 Negative stained transmission electronmicrographs of P. arcticum 273-4.
. ..................................................................................................... 167

FIGURE A-l Scanning electron micrograph of Psychrobacter arcticum 273-4 grown at
22°C in ‘/2 TSB .................................................................................... 185

FIGURE A-2 Phylogenetic tree of the genus Psychrobacter. Psychrobacter 273-4 and
Psychrobacter cryopegella are highlighted as their genomes are currently being

sequenced by the Department of Energy Joint Genome institute. (Courtesy of H. Ayala
del-Rio) ............................................................................................ 186

FIGURE A-3 Distribution of Psychrobacter arcticum 273-4 predicted open reading
frames arranged by cluster of orthologus gene groups ...................................... 187

xii

ll

LIST OF TABLES

TABLE 2.1 Diversity of organisms obtained fiom Permafrost soils ..................... 20

TABLE 2.2 Phylogenetic group vs. age (in thousands of years) of Permafrost sediment

.......................................................................................................... 25
TABLE 2.3 Listing of common compatible solutes accumulating bacteria and
conditions under which their increased uptake or synthesis occurs ....................... 37
TABLE 3.1 Effect of salt and temperature on the cellular morphology of
Psychrobacter sp. 273-4 ........................................................................... 69

TABLE 3.2 Fatty acid methyl ester composition (% of average total peak area) of two
permafrost isolates at different temperatures and osmolarities .......................................... 74

TABLE 3.3 Soluble polysaccharide composition of two permafrost isolates grown at
different temperatures and osmolarities .......................................................... 76

TABLE 3.4 Effect of growth temperature on survival of permafrost isolates afier one
year at -20 C ........................................................................................ 78

TABLE 3.5 Carbon sources uniquely utilized at the indicated temperatures ........... 80

TABLE 3.6 The susceptibilities of selected permafrost strains to five classes of
antibiotics .......................................................................................... 82

TABLE 3.7 Average percent survival of permafrost isolates exposed to UVC ........ 84

TABLE 3.8 Ice nucleation activity (X10'9) per cell of selected permafrost isolates
after growth at 24 C followed by exposure to 4 and -10°C .................................. 85

xiii

TABLE 4.1 The primer sequences used for quantitative real-time PCR .......... 107

TABLE 4.2 Comparison of the number of transcriptome changes of exponential cells
of P. arcticum, Synechocystis sp. PCC 6803, Saccharomyces cerevisiae and Shewenalla
oenedensis when grown at low temperatures or increased salinity ....................... 113

TABLE 4.3 The activity of NADH dehydrogenase obtained from P. arcticum 2734 at
different temperatures and salinities ......................................................... 114

TABLE 4.4 Alditiol acetate profile of P. arcticum at different temperatures and
salinities ............................................................................................ 127

TABLE 4.5 List of P. arcticum proteins and genes that agree in their expression
pattern and are above 2-fold different in the transcriptome profile ........................ 131

TABLE 4.6 List of P. arcticum proteins and genes that disagree in their expression
pattern and are above 2—fold different in the transcriptome profile ........................ 132

TABLE 4.7 List of differentially expressed proteins that are not 2-fold differentially
expressed in transcriptome analysis ........................................................... 133

TABLE 4.8 The list of genes and predicted functions with differentially expressed
proteins .............................................................................................. 134

TABLE 4.9 F old-change of known low temperature and increased osmotica stress-
responsive genes in P. arcticum at different temperatures and salinities . . . . . . . . . . .........136

TABLE 5.1 Percent of the metabolically active cells measured by CTC reduction vs.
total cells DAPI stained for two permafrost isolates and E. coli at different temperatures
and osmotica afier 10 days at 22°C and 14 days at 4°C160

TABLE B-l: Average number of copies of mRNA for select genes at different salinities
and temperatures as determined by quantitative real- time PCR ............................ 193

xiv

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TABLE B-2 Average fold change of genes differentially expressed when comparing
22°C ‘/2 TSB + 5% NaCl versus 22°C 1/2 TSB growth in P. arcticum... ...........194

TABLE B-3 average fold change of genes with 2 fold difference for P. arcticum cells
grown at 4°C 1/2 TSB + 5% NaCl versus 4°C 1/2 TSB .................................... 196

TABLE B-4 Average fold change of ORFS with at least 2-fold difference between P.
arcticum cells grown at 22°C in ‘/z TSB + 5% NaCl versus 4°C in 1/2 TSB + 5%

NaCl .............................................................................................. 199

TABLE B-5 Average fold change of genes with two fold difference when grown in
22°C 1/2 TSB Versus 4°C 1/2 TSB. Average of six biological replicates and dye swapped p
value <0.05 ....................................................................................... 201

TABLE B-6 Average fold change of P. arcticum ORFS that were differentially
expressed above two fold when grown at 22 °C in V2 TSB ................................... 203

TABLE B-7 Average fold change of P. arcticum genes that were differentially

expressed two fold or more when grown in 22°C ‘/2 TSB +5% NaCl ...................... 204
TABLE B-8 Average fold change of ORFS differentially expressed 2-fold or more in P.
arcticum cells grown at 4°C in ‘/2 TSB + 5% NaCl ........................................... 204

TABLE B-9 Average fold change of ORF S differentially expressed two-fold or greater
when P. arcticum was grown at 4°C in l/2 TSB ................................................ 205

TABLE B-10 ORFS that are not expressed with different salinities and
temperatures ......................................................................................... 205

XV

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CHAPTER 1

INTRODUCTION

The quest to understand whether Earth alone in the universe, can sustain life
compels scientists to search not only the skies but also Earth-analogues that resemble
astral bodies in temperature, water availability, geography or mineral composition to
better understand their potential for life. The low temperatures and thin atmosphere of
Mars are predicted to result in layers of frozen water within the Martian sediments, 500-
3000 m below the surface, depending on geographic location (Ostroumov, 1995). This
layer, referred to as permafrost, can be composed of soil, bedrock, sands, and sediments
that remain frozen for a period of two years or more (Mueller, 1973). While only 20-
25% of the Earth’s land surface is permafrost (Pewe, 1995), these regions serve as an
Earth-analogue to Marian permafrost due to low temperatures (-10 to ~30 °C), low water
content (1-7%; water activity = 0.9), low carbon availability, and long term exposure to
radiation. Throughout this dissertation, the Siberian permafrost environment is used as a
natural Earth-analogue in the study of bacterial survival and adaptive mechanisms to low
temperature and increased osmotica.

The thickness of terrestrial permafrost is governed by the balance between its
formation and sublimation, due to constant heat flow from the Earth’s core and variable
surface conditions. Regional thickness varies from a few meters in sub-Arctic regions, to
hundreds of meters in the northern Arctic (Gilichinsky et al., 1992). At a depth of 1000m,

Martian permafrost is predicted to be at a temperature of -10°C, corresponding to the

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temperature and water activity of the permafrost within the Kolyma Lowland region of
Siberia (Ostroumov, 1995).

The composition of permafrost varies greatly in ice content, organic carbon
content and physical characteristics. It is possible for permafrost to contain no moisture,
thus no ice, in dry, arid regions (e. g., the Ross desert of Antarctica). In other regions, the
high salinity of permafrost may inhibit ice formation or growth well below freezing
temperatures. However, the majority of permafrost in the Arctic and Antarctic regions is
firmly cemented by ice. Ice content varies according to the physical and chemical content
of the soil. Sandy, well-drained soils ofien contain between 15—25% ice, while loamy
soils have between 30—60% ice (Gilichinsky et al., 1993).

Beneath the active layer, freezing and thawing each year, lies the permafrost
table. Permafrost is a physical barrier, restricting water percolation and solute penetration
due to the presence of ice-filled pores. The absence of continuous, water-filled horizons

reduces the possibility of penetration of modern microorganisms into the ancient frozen
layers. The remaining water persists in an unfrozen state, due to the concentration of
organic solutes and mineral particles. Generally, the amount of unfrozen water present in
permafrost decreases with temperature (Ostroumov, 1992). In Arctic permafrost soils,
which experience temperatures between -10 to -12°C, the amount of unfrozen water is
estimated to be between 3-5% (Gilichinsky et al., 1995). The ice-filled soil horizons
prevent microbial movement because the thickness of the unfrozen water film (5-75 A) is
insufficient for the passage of a 0.5-1.0 um microorganism (Gilichinsky, 1993).

The presence of polygonal ice wedges, a unique water ratio of 16O:'8O, and the

Presence of pollen grains in the soil horizons indicate that these soils have remained

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continually frozen (Gilichinsky et al., 1992; Vasilchuk et al., 1997) from the time they
were buried below the active (annually thawed) layer. Soil age is determined by
radiocarbon dating for the more recent soils, though the examination of the geologic
record and extracted pollen is used to date soils older than 7,000 years. Permafrost
provides a unique opportunity to examine the microorganisms that have resided in
ancient soils due to the stability provided by its continually frozen state.

It might be construed that such harsh conditions would minimize the diversity of
microbial life and cause the small number of remaining cells to exist only in a resting
state. However, this is not the case. Viable microorganisms have been isolated from
permafrost soils all over the world that would not be culturable unless DNA damage
caused by the inherent y-radiation emitted by the soil is repaired (Horowitz et aI. , 1972;
Gilichinsky et al., 1995). Bacteria dominate in all layers of the permafrost. Their
diversity and numbers decrease with soil age, but large numbers (108 CFU/ml) of viable
bacteria were obtained from some of the oldest samples (3 million years) (Vishnivetskaya
et al., 2000). Recent studies in our laboratory have revealed a preponderance of non-
spore-forming bacteria in ancient Siberian permafrost isolates from the Kolyma-Indigirka
lowland, located adjacent to the East Siberian Sea (V ishnivetskaya et al. , in preparation).
The long-term survival of these ancient bacteria under such harsh conditions suggests that
the surviving populations contain the necessary stress responses for maintaining cell
viability until conditions improve allowing cell grth and division. The existence of
some stress responses has long been known for non-psychroactive (unable to grow below

0° C) bacteria, but little is known about the responses of cold-acclimated bacteria in the

permafrost environment. Studies of these responses will prove important for the
astrobiological and microbiological communities.

The activation of stress-specific and universal stress responses allow microbes to
respond to low temperature and low water activity in the permafrost environment. Ice
formation increases solute concentration by decreasing the amount of free water available
for biological use. This produces an environment with low water activity, similar to
those seen in desiccated and salt-stressed habitats. As a result, cell physiology responds
to these stress conditions. Successful adaptations include: membrane fatty acid
composition changes to maintain membrane fluidity; increases in the surface-to-volume
ratio to increase nutrient uptake; and production of compatible solute molecules to
balance the cell’s osmolarity with its surrounding environment (Poindexter, ; Russell,
1990; deSantos et al., 1998). Studies of psychrotrophs and psychrophiles have revealed
the compatible solutes glycine betaine, glutamate, and proline (Russell, 1990). Studies of
select permafrost isolates indicate that glutamate and proline were produced in high
concentrations when the cells were grown at low temperatures (Galinski, 1999). Changes
in membrane surface area and composition must also maintain necessary interactions
with membrane proteins to allow normal functions, such as transport, to continue.

The ability of microbes to maintain membrane, cell structure, transport, and
enzymatic fimction at cold temperatures is necessary for continued cellular activity
within a permafrost environment. Protein chaperones, proteases, and DNA binding
proteins have been described as cold and osmotic stress responsive molecules. One of
the most well studied general stress responses, particularly in Escherichia coli and

Bacillus subtilis, is called the stringent response, which is activated when the cell

6133

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experiences limiting amounts of amino acids and carbon sources. Growth stops
temporarily while metabolic adjustments are made to halt the synthesis of cellular
constituents and allow rapid recovery once conditions again become favorable. The
stringent response offers cross-protection to heat, oxidative stress, and osmotic stress
(Spector et al., 1993). Several common proteins (e.g., GroEL, DnaK) act as chaperones
when expressed under low temperatures, increased osmotica and low nutrient conditions
in E. coli, Pseudomonas and permafrost isolate 23-9 (Snyder et al., 1997; Chong et al. ,
2000). In addition, the cell experiences identical physiological changes in stress
conditions. Little information is available about the cellular response to the interaction of
the different stress conditions.

To gain knowledge of microbial adaptation to low temperature and water activity,
the genome of a Siberian permafrost isolate was sequenced and gene expression studies
undertaken. Psychrobacter arcticum 2 73-4, a psychroactive gamma-Proteobacterium,
displays marked physiological change under low temperature vs. mesophilic growth and
in the presence of increased osmotica. Genome sequencing technology allowed the
identification of predicted open reading frames, including those homologous to
previously characterized cold- and salt-responsive genes. Microarray technology made it
possible to examine gene expression of an entire genome under multiple conditions
through comparison of the relative abundance of mRNA between differently treated
samples (Eisen et al., 1999). Microarrays were constructed which consist of 70-mer
Oligonucleotides for 1,993 of the 2,056 predicted genes of P. arcticum.

The genome and transcriptome profiles of an ancient Siberian permafrost

bacterium, Psychrobacter arcticum 273-4, were examined in order to better understand

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the mechanisms of cold and salt acclimation. This work serves to examine the
mechanisms of long-term survival (especially cold and salt acclimation) under
laboratory-simulated conditions that allow for P. arcticum, but not all other ttmdra
organisms to continue to live in permafrost.
Thesis overview

The main purpose of this research is to gain insight into the mechanisms used by
permafrost microorganisms to survive the stresses of low temperature and low water
activity. Psychrobacter arcticum 273-4 was isolated from 20,000 year old permafrost,
suggesting the likelihood that it is adapted to low temperature and increased osmotica.
To determine if these two stress conditions result in similar physiological responses, the
following questions were investigated:

1. How does the transcriptome of P. arcticum 273-4 respond to low temperature and
increased osmotica during exponential growth phase? What is the degree of
commonality between genes expressed at cold temperatures and those expressed
at increased osmotica?

2. What are the physiological responses to increased osmotica and low temperature
of P. arcticum 273-4 and how do the observed transcriptome and proteome
differences reflect the physiological responsiveness?

3. Do the two permafrost model isolates P. arcticum 273-4 and Exiguobacterium
sibericum 25 5-1 5 maintain metabolic activity at low water activity conditions that
mimic the Siberian permafrost?

In order to investigate these questions, the following experimental design was employed.

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Experimental desig

Factors:
Temperature (two levels: optimal growth temperature (22°C) and 4°C, aW = 0.99)
Salinity (two levels: no salt amendment (0.076m NaCl) and 0.86m NaCl (5% NaCl, 1.47

osm, aW = 0.97)

Past studies of cold-acclimated bacteria primarily have focused on organisms not
capable of sub-zero growth. Siberian permafrost isolates Exiguobacterium sibericum
255-15 and Psychrobacter arcticum 273-4, which grow at -5°C, were used to study cold
and salt acclimated physiology, and this is discussed within Chapter 3, originally
published in FEMS Microbiology Ecology, 2005. P. arcticum 273-4 and E. sibericum
255-15 were chosen for genomic sequencing based on the following criteria: rapid low-
temperature growth rates, ability to grow at -2.5°C, long-term freeze survival, grth in
high salt concentrations, age of the strain, and small genome size.

The Department of Energy Joint Genome Institute (J GI) performed the genomic
sequencing. Genomic coverage of 8 times resulted and closure was carried out by Patrick
Chain of Lawrence Livermore National Laboratory using inverse PCR, direct sequencing
0f the chromosomal DNA template and ligation-mediated PCR as used previously to
close genomes (Offiinga etal., 1995; Prod'hom et al., 1998; Frangeul et al., 1999).
Afterwards, annotation using three gene finding programs followed by open reading
frame determination by BLAST match was used to automatically assign possible gene
functions to the sequences by Oak Ridge Laboratory Department of Biosciences. I

Participated with a group of Michigan State University scientists in a manual annotation

IA-
final

of the Psychrobacter arcticum 273-4 in order to ensure that computational calls closely
matched those in existing databases. One limitation was the small number of assigned
protein sequences within the databases. To fill gaps in metabolic pathways Natalia
Ivanonva of Lawrence Berkeley National laboratory used computational methods to
identify possible open reading frame candidates. The genome information was used to
construct microarrays for the majority of predicted ORFS, including hypotheticals, in P.
arcticum. The microarrays were used to identify changes in the transcriptome profile of
P. arcticum when grown at its optimal temperature (22°C) compared to 4°C and when
grown in nutrient rich media (172 tryptic soy broth) versus the same media amended with
5% NaCl. These profile changes are discussed in Chapter 4. To determine if
transcriptome changes yielded functional changes, enzyme analysis and proteomic
analyses (undertaken by Suping Zheng and David Lubman of the University of
Michigan) were performed and compared to the transcriptome changes also discussed in
Chapter 4.

Psychrobacter arcticum 273-4 and Exiguobacterium sibericum 255-15 bacteria
must maintain metabolic activity within the low water activity conditions of the Siberian
permafrost to retain viability. In order to determine the response to permafrost
conditions, we simulated the water activity of permafrost using 1/2 TSB+ 2.79 m NaCl (5
osm) at 22°C and 4°C. Chapter 5, presents studies on the membrane potential, electron
transport shuttling and aerobic respiration, coupled with incorporation of radio-labeled
leucine into cell material when incubated in high osmolarity media. The evidence
rePorted in this chapter shows that some of the population is metabolically active under

simulated in situ conditions.

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Conclusions and future perspectives

This research broadens the understanding of microbial survival under extreme
conditions, particularly with respect to the combination of low temperature and low water
activity stresses found in most cold environments, including permafrost, ice and frozen or
refrigerated foods. Investigating long term survival and adaptation to low temperature
and low water activity may lead to advances in control of food borne pathogens, many of
which are resistant to cold and increased osmotica. Identification of cold-active proteins
can lead to the development of cold active laundry detergents, extend refrigeration times
of foods and extend the shelf life of blood products and organs by protection and control
of the ice crystallization process. The issue of long-term survival under extreme
conditions is of particular interest in the field of astrobiology. Organisms that survive in

such hostile environments can be used as models for understanding cellular responses on

astral bodies.

 

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References

Chong, B., J. Kim, D. Lubman, J. Tiedje and S. Kathariou (2000). "Use of Non-Porous
Reversed-Phase High-Performance Liquid Chromatography for Protein Profiling
and Isolation of Proteins Induced by Temperature Variations for Siberian
Permafrost Bacteria with Identification by Matrix-Assisted Laser
Desorption/Ionization Time-of-F light Mass Spectrometry and Capillary
Electrophoresis-Electrospray Ionization Mass Spectrometry." Journal of
Chromatography B 748: 167-77.

deSantos, H. and E. A. Galinski (1998). An Overview of the Role and Diversity of
Compatible Solutes in Bacteria and Archaea. Biotechnology of Extremophiles. G.
Antranikan. New York, Springer. 61: 117-49.

Eisen, M. B. and P. 0. Brown (1999). DNA Arrays for Analysis of Gene Expression.
Patterns of mRNA Expression. Washington DC, ASM Press: 179-205.

Frangeul, L., K. Nelson, C. Buchrieser, A. Danchin, P. Glaser and F. Kunst (1999).
"Cloning and Assembly Strategies in Microbial Genome Projects." Microbiology
145: 2625-34.

Galinski, E. A. (1999). "Unpublished Communication."

Gilichinsky, D. (1993). y_iable Microorganisms in Permafrost: The Spectrum of Possible
Applications to Investigatiorp in Science for Cold Regio_ns. Fourth International
symposium on Thermal engineering and science for cold regions, US. Army cold
regions research and engineering laboratory Hanover, NH, US Army corps of
engineers.

Gilichinsky, D., E. Vorobyova, L. G. Erokhina, D. G. Fyordorov-Dayvdov and N. R.
Chaikovskaya (1992). "Long Term Preservation of Microbial Ecosystems in
Permafrost." Advances in Space Reseaih 12(4): 255-63.

Gilichinsky, D., S. Wagener and T. Vishnivetskaya (1995). "Permafrost Microbiology."
Permafrost and Periglacial Processes 6: 281-91.

Gilichinsky, D. A., V. S. Soina and M. A. Petrova (1993). "Cryoprotective Proprties of
Water in the Earth Cryolithosphere and Its Role in Exobiology." M
Evol. Biosphere 23: 65-75.

10

Hem

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‘5"). r
i nib"!

0
h“\.

Prat

Poi:

(I)
J

Horowitz, N. H., R. E. Cameron and J. S. Hubbard (1972). "Microbiology of the Dry
Valleys of Antarctica." Science 176: 242-45.

 

Mueller, S. (1973). Permfiost of Permanently Frozen Ground and Related Engineering
Problems United States Army.

 

Offringa, R. and F. Van der Lee (1995). "Isolation and Characterization of Plant Genomic
DNA Sequences Via (Inverse) Pcr Amplification." Method_s of Molecular Biology
49: 181-95.

Ostroumov, V. E. (1992). Unfrozen Water Types in Frozen Soils. Joint Russian-
American seminar on Cryopedology and global change, Pushchino, Russia,
Russian Academy of Sciences.

Ostroumov, V. E. (1995). "A Physical and Chemical Characterization of Martian
Permafrost as a Possible Habitat for Viable Microorganisms." Advances in Space
Research 15(3): 229-36.

Pewe, T. (1995). Permafrost. Encyclopedia Britannica. 20: 752-59.

Poindexter, J. (1981). "Oligotrophy: Fast and Famine Existence." Advances in Microbfl
Ecology 5: 63-89.

Prod'hom, G., B. Lagier, V. Pelicic, A. J. Hance, B. Gicquel and C. Guilhot (1998). "A
Reliable Amplification Technique for the Characterization of Genomic DNA

Sequences Flanking Insertion Sequences." FEMS Microbiology Letters 158(75-
81).

Russell, N. J. (1990). "Cold Adaptation of Microorganisms." Phil. Traps, R. Soc. Lond. B
326: 595-611.

Snyder, L. and W. Champness (1997). Molecular Genetics of Bgacteria. Washington DC,
ASM Press.

SPeCtor, M. P. and J. W. Foster (1993). Starvation-Stress Response (ssr) of Salmonella
Typhimurium. Gene Expression and Survival Nutrient Starvation. Starvation in
Bacteria. S. Kjelleberg. NY, Plenum Press: 201-24.

11

Vail.”

 

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\D' '

uu'll

Vasilchuk, Y. K. and A. C. Vasilchuk (1997). "Radiocarbon Dating and Oxygen Isotope
Variations in Late Pleistocene Syngenetic Ice-Wedges, Northern Siberia."
Permafrost and Periglacial Processes 8(3): 335-45.

Vishnivetskaya, T., S. Kathariou, J. McGrath and J. M. Tiedje (2000). "Low Temperature
Recovery Strategies for the Isolation of Bacteria from Ancient Permafrost
Sediments." Extremophiles 4: 165-73.

Vishnivetskaya, T., M. A. Petrova, J. Urbance, C. Moyer, M. Ponder and J. Tiedje (in
preparation). "Phylogenetic Diversity of Bacteria inside the Arctic Permafrost."

12

CHAPTER 2

MICROBIAL LIFE IN PERMAFROST: EXTENDED TIMES IN EXTREME

CONDITIONS

This chapter has been published in its entirety and is available as detailed within the

citation below

Ponder, M., T. Vishnivetskaya, J. McGrath and J. Tiedje. (2004). Microbial life in
permafrost: extended times in extreme conditions. L_ifa in tpeyfrogr sta_te_. B. J.

Fuller, N. Lane and E. E. Benson. Boca Raton, CRC Press: 151-165.

13

MICROBIAL LIFE IN PERMAFROST: EXTENDED TIMES IN EXTREME

CONDITIONS

Introduction
The permafrost microbial community has been characterized as a “community of

survivors” (F riedmann, 1994), based on their continued viability after hundreds to
millions of years in the frozen state. A vast diversity of microorganisms have been
described from even the deepest layers of permafi'ost. These layers contain members of
the bacteria, archaea and some green algae, other eukaryotes have not been described in
permafrost layers older than 10,000 years. The response of permafrost entrapped
microbes to any of the extreme conditions of low temperature, low nutrients and low
water activity associated with this environment may prolong their survival. This
response often involves physiological changes to membrane composition, protein

production, cell size and production of internal osmolytes.

Dispute remains about whether microbes simply persist in the long-term or
whether they actively survive in non—growth promoting conditions. A particularly
controversial topic is whether sustained or intermittent metabolic activity occurs within
the permafrost. Evidence presented below supports both intermittent metabolic activity
and the existence of a sustained anabiotic state. The remarkable ability to survive in a
Continuously frozen matrix of permafrost for millions of years makes the permafrost
c0mmunity unique for several practical and scientific reasons. For example, the perma-

fi‘Ost microbes may harbor novel enzymes of biotechnological importance. They also beg

l4

deep questions about the nature of life itself: How long can cells survive in continually
frozen conditions? What is the lowest metabolic activity necessary to retain viability? Is
life likely to exist on the cooler outer planets?

The purpose of this chapter is to highlight the nature of the physical environment
while primarily focusing on the microbial community and the metabolic state of these

microbes in situ.

Permafrost physical characteristics

The permafrost environment is composed of soil, bedrock, sands, and sediments
that are exposed to temperatures of 0°C or below for a period of at least two years. Over
20% of the Earth’s land surfaces are subjected to these permanently cold conditions
including, 85% of Alaska, 55% of Russia and Canada, 20% of China, and the majority of

Antarctica (Pewe, 1995).

The active layer of permafrost is the surface layer that freezes in the winter and
thaws in the summer. The depth of the active layer is dependent on moisture content and
can vary from several feet in well drained soils to less than 1 foot in bog environments
(Gilichinsky, 1993; Pewe, 1995).

The thickness of the permafrost is governed by the balance between its formation
and its loss to the atmosphere, due to heat flow from the earth’s core through the
permafrost layers. Regional thickness varies from a few metres in sub-Arctic regions, to
hundreds of meters in the northern Arctic (Gilichinsky et al., 1992). Permafrost is often
much thicker in Antarctic regions due to longer periods of sub-zero temperatures.
Topography and vegetation also contribute to differing local variations in permafrost

thiekness. Vegetation and snow cover serve to insulate, by preventing heat from leaving

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the ground, and thereby reduce the permafrost thickness (Pewe, 1995; Spirina et aI. ,
1998)

Permafrost soil represents an extreme environment due to low temperatures
(-10°C to -20°C (Arctic) to -65°C (Antarctic), low water content (1—7%), low carbon
availability, and long term exposure to gamma radiation. The composition of permafrost
varies greatly in ice, organic carbon content and physical characteristics. It is possible for
permafrost to contain no moisture, thus no ice, in dry, arid regions, such as the Ross
desert of Antarctica. In other regions, the high salinity of permafrost may inhibit ice
formation or growth well below freezing temperatures. However, the majority of
permafrost in the Arctic and Antarctic regions are firmly cemented by ice. Ice content
varies according to the physical and ionic content of the soil. Sandy, well-drained soils
often contain between 15—25% ice, while loam soils vary between 30—60% ice
(Gilichinsky, 1993). Sands of the Antarctic Dry valley contain between 25—40% ice, most
likely due to the ability of the coarse grains to cement (Wynn-Williams, 1989).

The concentration of organic carbon varies greatly in localized areas of
permafrost. Many sites within Northeastern Siberia are oligotrophic, with total organic
compositions (TOC) between 0.85—1% (Matsumoto et al., 1995); others contain higher
TOC (Gilichinsky et al., 1992), due to the presence of detritus layers. Late Pliocene and
Pleistocene Siberian soils often contain peat, detritus layers, semi-decayed plants and
humic-rich areas, which can cause large variations in organic carbon concentrations
(Gilichinsky et al., 1992). High TOC contents and a low C/N ratio are common in the

soils of East Antarctica (Beyer et al., 2000).

16

Beneath the active layer lies the permafrost table. This is a physical barrier,
restricting water percolation and solute penetration due to the presence of ice-filled pores.
The absence of continuous water-filled horizons reduces the possibility of penetration of
modern microorganisms into the ancient frozen layers. The remaining water remains in
an unfi'ozen state, due to the concentration of organic solutes and mineral particles. The
amount of unfrozen water present in permafrost decreases with temperature (Ostroumov,
1992). In Arctic permafrost soils which experience temperatures between -10 to -12°C,
the amount of unfrozen water is estimated to be between 3—5% (Gilichinsky et al., 1995).
In Antarctic soils where temperatures are commonly below -25°C, unfrozen water is
ofien undetected (Ostroumov, 1992). The ice-filled soil horizons prevent microbial
movement because the thickness of the unfrozen water film (5—75 A) is insufficient for
the passage of 0.5—1 .0 micron microorganisms (Gilichinsky, 1993). The presence of
polygonal ice wedges, unique 160: 180 ratios, and the presence of pollen grains only from
tundra plants in the soil horizons indicate that these soils have remained continually
frozen (Gilichinsky et al., 1992; Vasilchuk et al., 1997). Soil age is determined by
radiocarbon dating of the extracted pollen and other organic matter. Permafrost provides
a unique opportunity to examine the microbial diversity present in ancient soils due to its
continually frozen state.

Permafrost diversigy

A vast diversity of microorganisms has been isolated from all layers of
permafrost—some as old as 3 to 5 million years (see Table 2.1). Microbial diversity and
numbers seem to be related to the age of the soil and are, therefore, dependent on the

length of time frozen.

l7

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It might be construed that such harsh conditions would limit life to a very low
diversity and cause the small number of remaining cells to exist only in a resting state.
However, this is not the case. Omelansky isolated the first Siberian permafrost
microorganisms in 1911, while excavating a mammoth carcass. This discovery was
followed by isolation of viable microorganisms fi'om permafrost soils all over the world.
Microbes were soon characterized from snow and ice in Antarctica, Canadian permafrost
subsoil, and Alaskan permafrost (Boyd and Boyd, 1964; James and Sutherland, 1942;
McLean, 1918). Four different genera of bacteria were isolated from 20,000 to 70,000-
year-old Alaskan permafrost cores (Becker and Volkmann, 1961). This was followed
shortly by the first quantitative study of Alaskan permafrost. The active layer showed the
presence of 55,000 viable bacteria per grams of soil, whereas the permafiost (8 to 15 ft in
depth) revealed the presence of 5 to 130 cells/gm soil when incubated at 22°C on nutrient

broth (Boyd and Boyd, 1964). These early studies also highlighted the differences in
numbers of viable fungi (103/g soil) and bacteria (104/g soil) isolated from the active

permafrost layer when incubated at 24°C (Kjoller and Odum, 1971). The first viable
microflora older than the late Pleistocene (1 to 2 million years) were isolated in
Antarctica by Cameron and Morrelli (1974).

These initial studies included few controls to ensure that contaminant
microorganisms were not introduced into the samples. Early attempts to control the
introduction of outside microorganisms relied on the surface sterilization of the core with
a flame and then drilling into the center of the core with flame-sterilized drill bits, though
Some speculation remains about possible contamination by the drilling fluid (Cameron

and Morrelli, 1974). At present, standardized methods adapted from temperate subsurface

l8

sampling processes minimize contamination risks (Beeman and Suflita, 1990). Samples
are obtained by slow, rotary drilling without the use of fluid to prevent melting and
contamination by surface organisms. In addition, the outside of the drill is coated with an
indicator microorganism, Serratia marcescens. Internal segments of the cores are
obtained by aseptically removing the outermost 1 cm under frozen conditions. These
internal segments are then tested for the presence of the indicator strain (Khlebnikova et

al., 1990).

History of Permafrost Microbial Isolations

It might be construed that such harsh conditions would limit life to a very small
diversity and cause the small number of remaining cells to exist only in a resting state.
However, this is not the case. Omelansky isolated the first Siberian permafrost
microorganisms in 1911, while excavating a mammoth carcass. This discovery was
followed by isolation of viable microorganisms fi'om permafrost soils all over the world.
Microbes were soon characterized from snow and ice in Antarctica, Canadian permafrost
subsoil and Alaskan permafrost (McLean, 1918; James et al., 1942) (Boyd et al., 1964).
Four different genera of bacteria were isolated from 20,000—70,000 year old Alaskan
permafrost cores (Becker et al., 1961).

This was followed shortly by the first quantitative study of Alaskan permafrost.
The active layer showed the presence of 55,000 viable bacteria/ gm soil, while the
permafrost (8—1 5 ft in depth) revealed the presence of 5—130 cells/ gm soil when
incubated at 22°C on nutrient broth (Boyd et al., 1964). These early studies also
highlighted the differences in numbers of viable fungi (103/g soil), and bacteria (104/g

soil) isolated from the active permafrost layer when incubated at 24°C (Kjoller et aI. ,

l9

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1971). The first viable microflora older than the late Pleistocene (1-2 million years) were
isolated in Antarctica by Cameron and Morrelli (Cameron et al., 1974).

These initial studies included few controls to ensure that contaminant
microorganisms were not introduced into the samples. Early attempts to control the
introduction of outside microorganisms relied on the surface sterilization of the core with
a flame and then drilling into the centre of the core with flame sterilized drill bits, though
some speculation remains about possible contamination by the drilling fluid (Cameron et
al., 1974). Currently, standardized methods adapted from temperate subsurface sampling
processes minimize contamination risks (Beeman et al., 1990). Samples are obtained by
slow, rotary drilling without the use of fluid to prevent melting and contamination by
surface organisms. In addition, the outside of the drill is coated with an indicator
microorganism, Serratia marcescens. Internal segments of the cores are obtained by
aseptically removing the outermost 1-centimeter under frozen conditions. These internal
segments are then tested for the presence of the indicator strain (Khlebnikova et al. ,
1990)

TABLE 2.1 Diversity of Organisms Obtained from Permafrost Soils

Number isolated Oldest Depth

 

sample
(cells/gm soil) (years) (meters) Reference
5 7 Rivkina et al., 1998; Tiedje et
ArChaea 10 —10 2 M 43 al., 1998
Eukaryotes 103—105
Algae 103—105 3 M Vishnivetskaya et al., 2001
Fungi and 103 .
yeasts 3 M 78 Dmrtrev et al., 1997
Protists 103 Modern 0.3 Vishniac, 1993
Bacteria 107—109 3 M 43 Vishnivetskaya et al., 2000

20

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Bacteria
Bacteria dominate in all layers of the permafi'ost. Their diversity and numbers

decrease with soil age, but large numbers (108 cells/g) of viable bacteria were obtained

from the oldest samples (3 million years) (V orobyova et al., 1997; Vishnivetskaya et al.,
2000). The majority of studies of the psychrotolerant permafrost community focus on
aerobic systems where up to 108 cells/g can be isolated (V orobyova et al., 1997)
(V ishnivetskaya et al. , 2000). A small number of anaerobic studies reveal that permafrost
samples also contain a diverse population of anaerobic bacteria, accounting for 102—106
cell/g (Rivkina et al., 1998). Psychrophilic bacteria are surprisingly absent from
permafrost communities, with the majority of isolates possessing psychrotolerant
properties (V ishiniac, 1993).

A discrepancy exists between the number of bacteria that can be cultured and

those present in the permafrost soils. Arctic permafrost sediment often contain between

107—109 cells/g dry weight (dw), as determined by acridine orange direct microscopy
counts, while Antarctic permafrost contains between 107—108 cells/g dw. However, only

a small fraction of these bacteria (102-108 cfu/ g dw) are viable by selected culturing
methods (Khlebnikova et al., 1990) (V orobyova et al., 1997). Studies utilizing low
nutrient media such as peptone yeast glucose vitamin media or 1/ 10 tryptic soy broth,
obtained greater numbers of isolates, but a smaller diversity of morphotypes, than studies
using rich media (Siebert et al. , 1988); (V ishnivetskaya et al. , 2000). However,
incubation at cold temperatures was shown to increase both the microbial diversity and

the numbers of bacteria isolated from Arctic permafrost (V ishnivetskaya et al., 2000).

21

Characterization of the viable and culturable permafrost community has revealed
the presence of equal numbers of Gram positive and Gram negative bacterial isolates in
some soils and a preponderance of non-spore-forming Gram positive bacteria in other
permafrost soil samples (V ishnivetskaya et al., 2000). Previous studies by Russian
scientists suggest that Gram-positive bacteria such as actinomycetes (85%), and cocci
(5%), including the genera Arthrobacter, Rhodococcus, Micrococcus, Deinococcus,
Brevibacterium and Streptomyces, were predominant in Siberian permafrost (Zvyagintsev
et al., 1990). Gram-negative rods represented about 12% of aerobic isolates with the
majority of isolates being members of the Pseudomonas and F lavobacterium genera
(Zvyagintsev et al., 1990); (V orobyova et al., 1997). The majority of aerobic permafrost-
bacterial isolates are non-spore forming bacteria, especially in the most ancient
permafrost soils and therefore this classical persistence structure does not appear to be
vital for microbial survival in permafrost.

In late Pliocene (1 .8—3 million years old) permafrost, the mesophilic
actinomycetes and related high-GC Gram-positive bacteria were still found to be the
predominant (SS—75%) culturable isolates. However, the total number of cells isolated
decreased with age of the soil (6 x104—l .4 x105 cell/g). Classification based on
culturable, morphological, and chemotaxonomical characteristics placed the isolates
within the genera Arthrobacter, Kocuria, Aureobacterium, Gordona, Nocardia,
Rhodococcus, Mycobacterium, Nocardioides and Streptomyces. In the aforementioned
study, a number of bacteria isolated differed from previously described genera in their

cell wall composition, suggesting that the bacteria may belong to a new species or genera

22

 

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(Karasev et al., 1998). Bacteria of the genus Bacillus were often isolated from old
permafrost samples (Kuz'min et al., 1996).

Only a few studies have examined the presence of anaerobes in permafrost despite
their presence in large numbers (107—108 total cells/g) (Vorobyova et al. , 1997).

Denitrifiers, sulfate reducers and F e(III) reducers were isolated by viable plate counts and
quantified by presence of end products. The largest numbers of anaerobes were detected
in the younger soils (Rivkina et al., 1998). The anaerobic bacteria were identified as
Propionibacterium species (sp.) (Karasev et al., 1998), Desulfotomaculum 31)., as well as
nitrifying and denitrifying bacteria of the genera Nitrosospira, Nitrosovibrio, and
Nitrobacter (Rivkina et al., 1998).

Phylogenetic studies of Siberian permafi'ost isolates reveal a vast diversity of
microorganisms belonging to the high G+C Gram positive, low G+C Gram positive, and
Proteobacteria phylogenetic groups. Arthrobacter and a member of the Micrococcus
subclass were the most common isolates independent of soil age within the high G+C
Gram positive group. Exiguobacterium represented the low G+C Gram positive group
throughout the soil column, while Planococcus was isolated only from the younger soils.
Members of the a-proteobacteria, Sphingomonas, were found in soils up to 3 million
years old, while members of the Flavobacterium—Bacteroides—Cytophaga group and y-
proteobacteria, F lavobacterium and Psychrobacter, were present only in the younger
soils (V ishnivetskaya et al., in preparation). This age relationship is currently
unexplained, but it may suggest that those genera present in ancient soils possess cold
acclimation properties not present in the strains of younger origin. Shi et al. (1997)

examined 29 viable permafrost isolates. Among them, 16 (55%) were Gram-negative and

23

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13 (45%) Gram-positive. Phylogenetic analysis revealed that the isolates fell into four
categories: high-GC Gram-positive bacteria, B-proteobacteria, y-proteobacteria, and low-
GC Gram-positive bacteria. Most high-GC Gram-positive bacteria and B-proteobacteria,
and all y-proteobacteria, came from samples with an estimated age of 1.8—3.0 million
years. Most low-GC Gram-positive bacteria came from samples with an estimated age of
5,000—8,000 years (Shi et al., 1997).

Recent studies have shown that the number of viable but non-culturable
microorganisms present in permafrost is considerable. Extraction of genomic DNA from
the active layer followed by observations of 16s rRNA restriction fragment length
polymorphism (RF LP) patterns indicate that the Gram negative bacteria are most often
amplified (60.5%) compared with the Gram positive bacteria that often dominate in
viable diversity studies. The Proteobacteria dominate the Gram negative isolates with
clones belonging to the a (20.9%), 6, (25.6%), B (9.3%) and y (4.7%). Overall, 70% of
clones were 5—15% different at the 16s rDNA level from the strains in current databases,
while 7% differed more than 20% (Zhou et al., 1997) in 16s rDNA content. This would
seem to indicate that the physiology and function of the dominant members of the
community are unknown. The reports about isolation of viable bacteria from Antarctic
permafrost are numerous and are thoroughly reviewed by Vishinac (1993) in Antarctic
Microbiology. Bacteria are able to survive in Antarctic permafrost at levels of nearly 105
cell/g, with the genera of Bacillus, Arthrobacter and Streptomyces predominating

(Vorobyova et al. , 1997).

24

[h

TABLE 2.2 Phylogenetic group vs. Age (in Thousands of Years) of Permafrost
Sediment

Flavobacteria/Cytophaga/ 6 1 4 l
Bacteroides group

Fibrobacter group 7 7

Proteobacteria 43 26 1 5 l 10
(31131138) (1 1) (9) (1) (1)
Chem) (10) (4) (1) (5)
(gammaS) (1 1) (2) (5) (4)
(deltas) (11) (11)

Bacillus/Clostridia group 20 2 8 5 1 1 3
(Bacillus) (1 l) ( l) (8) (2)
(Exiguobacterium) (4) (1) (l) (1) (l)
(Planococcus) (5) (4) (l)

Actinomycetes 32 1 l3 4 14
(Arthrobacter) (24) (12) (1) (1 1)
(Microbacteriaceae) (6) (l ) (3) (2)
(Rhodococcus) (2) (1) (1)

Note. Compiled from phylogenetic diversity literature of Shi et al. 1997, Vishnivetskaya,
in preparation, and Zhou et al. 1997.

Qanoficteria and alga;
Viable green algae and cyanobacteria were isolated from numerous permafrost

samples, which varied in lithology genesis and depth. Permafi'ost samples were obtained
from the Kolyma-lowland, Siberia and Antarctic dry valleys (Victoria Land). The
frequency of discovery of viable algae has been lower than bacteria (see Table 2.1), and
decreases with increasing permafrost age. On the other hand, the presence of viable algae
did not depend on the depth of the soil core, when samples of identical age were
examined. Green algae and cyanobacteria were isolated more often from fine lake-swamp
or lake—alluvium loams and sandy loams. No viable algae were isolated from coarse

marine and channel-filled sands. The biodiversity of green algae and cyanobacteria was

25

 

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highest in the Holocene sediments (10,000 years old). Their occurrence in the Arctic
early Pleistocene and late Pliocene, and in any Antarctic frozen sediments, can be
characterized as sporadic. Statistically, green algae species were found more frequently
and with greater species diversity than cyanobacteria. This may reflect the ability of the
green algae to survive in the ancient trmdra environments. Numerous studies to date have
shown that green algae are dominant in the modern tundra environments in quantity and
biodiversity (Zenova et al., 1990); (Getsen, 1990) (Getsen, 1994).

Unicellular green algae Pseudococcomyxa and Chlorella were often isolated from
different ages of Arctic permafiost samples. Non-motile globular cells of Chlorella were
more abundant among green algae isolates. Other Chlorella were dominant in the
permafrost samples. Viable green algae fiom the Arctic permafiost were represented by
Chlorella sp., Chlorella vulgaris, Chlorella sacchorophilla, Pseudococcomyxa sp.,
Mychonastes sp., Chlorococcum sp., Chodatia sp., Chodatia tetrallontoidea,
Stichococcus sp., Scotiellopsis sp. Unicellular green algae of Mychonastes sp. and
Pedinomonas sp. were the only algae discovered in the Antarctic permafrost. Green algae
isolates were unicellular coccoidal, elliptical and rod-like. All cyanobacteria were
filamentous in morphology and belonged to the Oscillatoriales and Nostocales orders.
Among them Nosctoc, Anabaena, Phormidium and two different species of Oscillatoria
were identified. Nastoc and Oscillatoria with straight, narrow trichomes were most often
isolated form Arctic permafrost. Viable cyanobacteria have not been isolated from
Antarctic permafrost samples to date (V ishnivetskaya et al., 2001).

Viable algal isolates possessed low growth rates, with doubling times of 10—14

days. Green algae strains were better able to grow at 27°C, 20°C and 4°C, but

26

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cyanobacteria possessed good growth only at 25°C. Cyanobacteria Nostoc and
Anabaena were capable of nitrogen fixation and chromatic adaptation (Erokhina et al. ,
1999); (V ishnivetskaya et al., 2001).

It is amazing that green algae and cyanobacteria survive in deep permafrost
sediments devoid of light and below the freezing point for thousands to millions of years.
These algae may exist in some readily reversible dormant state from which they may

easily regain photoautotrophic capabilities when light and water again become available.

Archaea
Archaea are present in great numbers in permafrost. Methanogens are present

between 105—107 total counts/gm (Rivkina et al., 1998). Phylogenetic diversity of
Archaea from Siberian permafrost aged from 100,000 to 2 million years has been studied
by RFLP distribution analysis. The archaea] diversity was demonstrated to be
significantly lower than that of archaea found in surface tundra soils from the same
region. The majority of archaea] amplicons belonged to a novel lineage that was deeply
rooted in the Crenarchaeota. Three amplicons were closely related to Methanosacina sp.,
contained in the Euryarchaeota (Tiedje et al., 1998). Since this study, 12 additional
strains of methanogenic archaea have been isolated from permanently cold environments,

including permafrost (Nozhevnikova er al. , 2001).

Eukaryotic Microorganisms
Fungi are found predominantly in the upper strata of permafrost, while yeast can

be isolated from all layers of permafrost. Fungi have yet to be isolated from samples

older than 10,000 years in age (Zvyagintsev et al., 1990; Gilichinsky et al., 1992). A

27

complete overview of early taxonomic studies of fungi and yeasts isolated from Antarctic

permafrost are described elsewhere (V ishiniac, 1993).

Electron microscopic investigations of deep permafrost samples show a decrease
in the stability of eukaryotes under cryopreserved conditions. Samples older than 10,000
years contain low numbers of eukaryotic cells, all showing damaged internal structures

but intact cell walls (Soina et al. , 1995).

Recently yeasts have been shown to comprise 20—25% of the aerobic heterotrophs
in some 2—3 million-year-old Siberian permafrost soils. The large number of yeast (9,700
cfii/g) and the absence of yeast from adjacent layers indicates that these yeast are not
contaminants or percolated through from upper layers (Dmitriev et al., 1997). Large
numbers of yeasts (17,000—70,000 cells/g) are more commonly encountered in soils that
contain large amounts of un-decomposed plant material (Spirina et al., 1998). Yeast
abundance increases in coastal Antarctica due to increased moisture contents and plant
cover and around human inhabited areas, such as McMurdo station (Atlas et al., 1978).
Some studies have revealed yeasts to be the predominant microbe in the soil, however
there seems to be no correlation between any of the physical characteristics of the soils
and the microbial diversity (Horowitz et al., 1972; Atlas et al., 1978). The yeast isolated
from permafrost are generally obligate psychrophiles, unlike the viable bacteria which

tend to be psychrotrophic (Atlas et al., 1978).

Despite their smaller numbers throughout the permafrost column, the presence of
eukaryotic microorganisms indicates their ability to survive extreme conditions for

extended periods of time.

28

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Mechanisms of survival
Throughout the scientific community there remains much dispute about what the

existence of microbes in the permafrost indicates about the potential for long-term
survival in non-growth promoting conditions. This section seeks to express some of the

current theories about cell preservation and the possibilities of in situ metabolic activity.

Physical characteristics of firmafrost
The soil composition itself can impact survival of bacteria at low temperatures.

Soil particles decrease the depth of penetration of thermal oscillations and therefore help
to buffer the microbes from rapid temperature changes. Soils with larger pore spaces,
such as sand, have been shown to contain less unfrozen water and yield fewer viable
bacteria compared to peat containing soils (Gilichinsky et al., 1995). Recent studies
reveal a correlation between bacterial numbers and the total organic carbon and clay
content of soil (Beyer et al., 2000). Increased content of organic carbon and clay will
provide nutrients and have greater water holding capacity, preventing desiccation of the
cells.

Unfrozen water surrounding the cells acts as a nutrient medium because as ice
forms it concentrates solutes. Additionally, this unfiozen film acts as a cryoprotectant by
preventing invasion fi'om extracellular ice crystals. The amount of unfiozen bound water
surrounding microbes was shown to decrease with temperature (Gilichinsky et al., 1995).

In addition to the ameliorating effect of unfrozen water and organic composition
the physical structure of soil particles may increase microbial survival. This is
particularly apparent when one compares the high number of cells isolated from frozen

soil to the low numbers from pure ice. Recent studies of Lake Vostok accretion ice

29

indicate that the number of viable microbes increases when dust particles are present in
the ice cores (Karl et al., 1999). This is further supported by evidence in cryopreserved
soils modelled to resemble permafrost environments, which shows that the ability of cells
to adhere to soil particles increases cell survival (Sidyakina er al., 1992). Scanning
environmental microscopy firrther reveals the tight association of bacteria with the

surrounding Siberian permafrost (Soina V.S. unpublished observation; see Figure 2.1).

 

FIGURE 2.1 Microbes apparently encased in biofilm in frozen state in permafrost soil.
Environmental scannning electron microscope. (Courtesy of Dr. Vera Soina).

30

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Physiological adaptation
Physiological responses to extreme conditions, including low temperature,

desiccation and low nutrients have been well described (see also chapters by Elster and
Benson, Tan and van Ingen, and Pearce, Life in the Frozen State). Many cellular responses
to a single stress are also protective against other types of stress. The low water activity of
the permafrost environment may induce physiological changes that allow cells to survive
radiation exposure as seen in Deinococcus radiodurans (V enkateswaran et al., 2000). The
response of permafrost entrapped microbes to one or more of these conditions may prolong

their survival.

Membrane adaptation

Biological membranes are composed of multiple types of phospholipids and proteins
that are transitioned between a highly ordered state (crystalline gel) and a fluid state (liquid
crystalline). A reduction in temperature often leads to an ordering of the membrane system
resulting in a solid-state (gel) membrane. In order to maintain membrane fluidity,
microorganisms induce changes in membrane composition at low temperatures (Russell,
1990); (Wagener et al.). These membrane changes allow normal functions, such as
transport, to continue by maintaining necessary interactions with membrane proteins. In
general, a decrease in temperature brings about a subsequent decrease in saturation of fatty
acids, acyl chain length, and proportion of cyclic fatty acids. Altogether, these changes

function to lower the gel—liquid crystalline transition temperature (Morris et al., 1987).

Membrane unsaturation is accomplished via changes in the synthesis of new
lipids, as seen in Escherichia coli and also by alteration of existing fatty acids, as seen in

Bacillus subtilis and Synechocystis. E. coli has a 30-second response time before

31

production of unsaturated fatty acids occurs after a temperature downshift (Ingraham et
al., 1996), performed by regulating the enzyme B-keto-acyl ACP synthase. The alteration
of existing membranes may be a more rapid, efficient response to cold temperature stress.
The desaturase genes responsible for introducing the double bonds between specific C
moietes allow for rapid cellular response to cold temperatures, and are constitutively
expressed to allow for immediate response to low temperature stress (Aguilar et al.,
1999). A two component signal transduction system, composed of a sensor kinase and
response regulator is responsible for the cold induction of des genes in B. subtilis and
Synechocystis (Aguilar et al., 1999); (Suzuki et al., 2000). The inactivation of two sensor
kinase domains decreases the amount of low temperature induction of genes encoding 86
and 63 desaturases (Suzuki et al., 2001). Shortly after, the response regulator was
identified in B. subtilis and shown to be involved in control of gene expression. It is also
inactivated by the presence of unsaturated fatty acids however, the exact mechanism is
unknown (Aguilar et al., 2001).

An important consequence of membrane adaptation is the increased efficiency of
nutrient uptake at cold temperatures in strains of permanently cold origin. Glucose
utilization/uptake increases at 0°C in a cold-adapted psychrotroph, while a psychrotroph
exposed to temperature fluctuations shows a decrease in uptake at 0°C (Ellis-Evans et al.,
1985). This suggests that fluctuations in temperature favour a different fatty acid
composition of the membrane than the cold-acclimated membrane.

A temperature minimum does exist where simple membrane changes no longer
maintain the spatial organization to allow transport molecule interactions, effectively

allowing the cell to starve even at high nutrient concentrations (Nedwell, 1999).

32

 

 

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Membrane adaptation may function to exclude ice crystals and allow continued nutrient
transport, while maintaining cellular integrity. However the enzymatic and structural
function of proteins not associated with the cell membrane must be maintained,

necessitating other molecular mechanisms of cold adaptation.

Production of cold-induced proteins
The ability to maintain structure, transport, storage, and enzymatic firnction at

cold temperatures is necessary for continued cellular activity. Low temperature and
nutrient conditions have been shown to induce high levels of expression of three types of
proteins: cold-shock, antifreeze and ice nucleation (Sun et al., 1995; Chong et al., 2000);
(N emecek-Marshall et al. , 1993).

Cold—shock proteins control many different types of functions from transcription
and translation to general metabolism and recombination. A complete review of cold
shock proteins was made by Phadtare et al. (Phadtare et al., 2000). It is believed that their
primary role is to halt protein synthesis until the cells have acclimated to their new
environment. The signals necessary to restart protein synthesis are unknown (Russell,
1990)

Ice nucleation proteins are proteins whose structure mimics an ice crystal,
providing a lattice for crystallization of water (Zachariassen et al., 1976). This allows for
crystallization of extracellular water at higher temperatures, releasing a small amount of
heat, which may aid in delaying internal damage until the temperature is increased. The
small extracellular crystals may be too small to penetrate the membrane and initiate
fi'eezing (Mazur, 1977). In addition to the surface-catalyzed theories of intracellular ice

formation, ice can form inside cells as osmotic pressure rises during extracellular

33

fi'eezing, which may lead to the rupturing of the membrane (Toner et al., 1990);
(Muldrew et al., 1994). Harmfirl intracellular ice formation is believed to be averted with
ice nucleation active proteins because the small size of the formed crystals prevents
membrane damage, the primary cause of cell death in freezing systems. These small,
thermodynamically unstable crystals have a tendency to reform into large, damaging
structures, indicating that INA proteins are unlikely to offer long-term survival benefits
(Mazur, 1984). Recently, the discovery of a protein that is capable of both ice nucleation
and antifreeze activity has been described, and may present a solution to this dilemma
(Xu et al., 1998).

Antifreeze proteins have long been known to prevent the freezing intracellular
water in fish by impeding the addition of water molecules to the existing crystal (DeVries
et al., 1969). Such thermal hysteresis activity can result in lowering of the freezing
temperatures of water by as much as 9—18°C. Conversely, the freezing of extracellular
water can also protect some terrestrial arthropods (Duman, 2001) and invertebrates (see
Storey and Storey, this volume), presumably by enabling the intracellular environment to
vitrify while maintaining ice in ‘safe’ extracellular spaces. The ice nucleation domain of
the dual-action protein is believed to cause the formation of extracellular ice, while the
antifreeze domain maintains these crystals at a non-damaging size (Xu et al. , 1998). The
ability of some plant- and animal-derived antifreeze proteins to inhibit microbial ice-
nucleation activity suggests that antifreeze proteins may lower the supercooling point of
water within the cells. However, no direct evidence supports this theory (Duman et al. ,

1991); (Griffith et al., 1995).

34

 

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Compatible solute production
Ice formation increases solute concentration by decreasing the amount of free

water available for biological use. This produces an environment with low water activity,
similar to those seen in desiccation and salt-stressed environments. Halophilic and
halotolerant microorganisms have developed mechanisms to allow their continued
growth in low water activities and high salinity. The mechanism excludes environmental
solutes from the cell, while accumulating other solutes (compatible solutes) that control
osmotic balance and are compatible with the organism’s metabolism (Brown, 1990).

Compatible solutes reduce the normal cell shrinkage associated with desiccation
(McGrath et al., 1994). Compatible solutes fall into five classes of compounds: sugars,
polyols, betaines, ectoines, and some amino acids (D'Souza-Ault et al., 1993);(deSantos
et al., 1998); (F rings et al., 1993); (Severin et al., 1992); (Larsen et al., 1987); (Csonka,
1989); (Glaasker et al., 1996); (Welsh, 2000); (Ruffert et al., 1997); (K0 et al., 1994);
(Smith et al., 1989); (Galinski et al., 1990). Studies of psychrotrophs and psychrophiles
have revealed the presence of compatible solutes such as glycine betaine, glutamate and
proline (Russell, 1990). Proline has been shown to depress the freezing point of water in
some plants (Aspinall et al., 1981). In addition, Gould and Measures have shown a
correlation between the concentration of proline and grth at low water activities, such
as those seen at freezing temperatures (Measures, 1975). It is believed that compatible
solutes such as proline intercalate between membrane phospholipids, helping to stabilize
membranes by maintaining hydrogen bonding (Rudolph et al., 1986).

Studies of selected permafrost isolates indicate the dominance of glutamate and
proline as compatible solutes, produced in high concentrations when exposed to low

temperatures (Galinski, unpublished). However this has not been the case for one

35

 

 

 

 

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Arthrobacter sp. isolated from 600 thousand year old permafrost core (Mindock et al.,
2001). Instead, in this case, the concentration of compatible solutes actually decreased
with lowered temperatures. This curious finding might be explained by the idea that the
remaining water in the interior of the cell does not behave like bulk water, but is instead
ordered along the surface of the membrane, producing a high enthalpic potential, which
prevents the reordering of molecules, and so ice formation. Solutes are excluded from the
ordered regions, which would result in a decrease in the concentration of compatible
solutes necessary to maintain homeostasis. The combination of ordered peripheral water
molecules and centralized solute accumulation inhibits freezing (Mindock et al., 2001). It
remains to be seen whether compatible solutes will be detected in other permafrost
bacterial isolates.

ABC-transporter proteins mediate uptake of most compatible solutes. The only
well-defined osmotic responsive ABC transporter is OpuA, responsible for glycine
betaine uptake in Lactococcus lactis. The uptake of glycine betaine by OpuA, is
dependent on the interactions of the lipid and transporter interactions (van der Heide et
al., 2000). As the outside osmolarity increases, the proteoliposomes react by decreasing
their surface to volume ratio, therefore reducing the electrostatic interactions between the
lipid headgroups and the protein, resulting in increased glycine betaine uptake (van der
Heide et al., 2001). Similar systems are believed to exist in the ion-linked transporters
ProP and BetP, involved in proline and betaine uptake respectively (van der Heide et al.,

2001).

36

TABLE 2.3 Listing of Common Compatible Salutes Accumulating Bacteria and Conditions
under which Their Increased Uptake or Synthesis Occurs

Compatible Solute and Bacteria Known to Accumulate Conditions References
N-acetylglutaminylglutamine amide
Pseudomonas aeruginosa Osmotic l8
Ectoine
Brevibacterium Osmotic 24
Halomonas Osmotic 72
Halovibrio Osmotic 72
Marinococcus Osmotic 72
Pseudomonas halophila Osmotic 72
Vibrio costicola Osmotic 72
Glutamate
Arthrobacter Cold 25
Escherichia coli Osmotic 48
Exiguobacterium Cold 25
Vibrio* Osmotic 14
Klebsiella>l< Osmotic l4
Lactobacillus Osmotic 33
Glycine betaine
Chromatium Osmotic 95
Corynebacterium Osmotic 70
Ectothiorhodospira Osmotic 72
Enterobacteriaceae family Osmotic 14
Listeria monocytogenes Osmotic, cold 45
Rhizobium meliloti Osmotic 76
Pseudomonas aeruginosa Osmotic 14
Streptomyces griselous Osmotic 72
Thiobacillus ferrooxidans Osmotic 72
Proline
Salmonella>l< Osmotic 72
Coreynebacteriurn Osmotic 70
E. coli Osmotic, cold 38
Exiguobacterium Cold 25
Klebsiella Osmotic 14
Lactobacillus Osmotic 14
Pseudomonas aeurginosa Osmotic, cold 14
Staphylococcus aureus Osmotic 14
Serratia marcescens Osmotic 14
Thiobacillus ferroxidans Osmotic 14
Trehalose
E. coli* Osmotic, cold 25, 38
Ectothiorhodospira halochris Osmotic 24
Chlorobium Osmotic 95

37

Alternate physiological adaptations to permafrost conditions
It seems unlikely that membrane adaptation, compatible solute production or cold

induced proteins are alone responsible for adaptation to permafrost conditions.
Compatible solutes may act to stabilize proteins and membranes by substituting for
hydrogen bonds until the membrane composition has changed. Selective dehydration may
also be used to expel intracellular water and therefore reduce the chance of frozen
intracellular water, as seen in spores to promote heat resistance (Gould et al., 1977). A
decrease in cell size and ability to adhere to the surrounding microenvironment could
greatly facilitate survival. A decrease in cell volume 14-fold is seen for one permafrost
isolate when grown at 4°C compared with 24°C (Mindock et al., 2001). The reduction in
metabolism required for cell survival at low temperatures is facilitated by cell
dehydration, caused by a combination of fieezing and nutrient limitation. Theoretically,
the small size should decrease the probability of intracellular ice formation, but
modelling studies suggest that permafrost cells are small enough to dehydrate rather than
form intracellular ice at the fi'eezing rates likely to be experienced in the environment
(McGrath et al., 1994). The diminished size of the cells would require a lower rate of
basal metabolism, which could allow the cell to survive given the diminishing levels of
energy caused by a decrease in active transport (Herbert, 1986). Decreased active
transport of solutes has been shown to occur with decreasing temperature through the use

of oxidation of radio-labelled substrates (Herbert, 1986).

38

0.8 -

0.6 P

Norrnaiized Volume

0.2 -

 

255 260 265 Temp
K

FIGURE 2.2 This plot shows predicted microbial plasmolysis during freezing at various
cooling rates. The vertical axis represents the volume of the cytoplasm normalized with
respect to the cytoplasm volume before freezing. The results show that the cytoplasm
shrinks in response to external fi'eezing. The right-most curve is for an extremely slow
freezing rate (0.01°C/min), indistinguishable from equilibrium freezing at infinitely slow
rates. The faster the cooling rate, the more displaced the curves are to the left in this
figure. The cooling rates here are 5°, 10°, 25°, and 50°C/min, which would be high rates
of cooling for most cases in the environment. The fact that the cell retains more of its
initial volume for freezing at faster rates corresponds to the fact that more water remains
“trapped” within the cell, making it more likely to freeze internally. Overall, these
predictions indicate that permafrost microbes are not likely to form intracellular ice.
Instead, they will respond to freezing in their environment by exosmosis. This will create
a dehydrated, but unfrozen, intracellular state. It is assumed that the intracellular
concentration of compatible solutes is 2.0 osmolal. The initial cell diameter is taken to be
1.0 pm, and it is assumed that 50% of the intracellular volume is incapable of
participating in osmosis. Membrane water permeability and its temperature dependence
are taken as representative values for yeast published in the literature.

The production of capsular polysaccharides is common in permafrost bacteria
(Soina et al., 1995); (V orobyova et al. , 1996). The capsules decrease in thickness at cold
temperatures. The benefits of a decrease in capsule thickness are two-fold: it conserves
the energy-rich components, while decreasing the diffusion barrier encountered by
nutrients and other small molecules. Capsules also play a role in adherence. The survival
rates of non-motile bacteria have been shown to increase after a rapid freeze, compared

With liquid environments where no soil particles are present for adherence (Sidyakina et

39

al., 1992). Regardless of thickness, the presence of capsular layers seen in some
permafrost isolates could provide an extra barrier similar to the membrane to hinder
penetration of extracellular ice (Mazur, 1977); (Zvyagintsev et al., 1985).

The production of pigment proteins is a known consequence of general stress
responses in microorganisms. Therefore, it is of no surprise that the majority of
permafrost isolates are highly pigmented (Siebert et al., 1988; Vishnivetskaya et al. ,
2000). Antarctic red pigmented isolates are more tolerant of alkaline conditions and
intense UV light exposure than their non-pigmented relatives (Siebert et al. , 1988).
Recent studies have shown that ancient Siberian permafrost isolates decrease in pigment
intensity while maintaining rapid growth rates with decreased temperature and nutrient
level (V ishnivetskaya et al. , in preparation). The cause of this response is unknown. It
seems likely that the cells are limiting metabolism to allow expression of only a few

necessary proteins as the energy available to the cell decreases.

Metabolic activigy

The fact that many of these microorganisms are viable and culturable leads to the
question of what adaptations they possess to allow their survival in such harsh conditions.
Bacteria might survive either by a very slow rate of metabolism, or by existing in a state
of anabiosis. Both theories have supporting and contradictory evidence, and presumably

both are true of some microorganisms.

Anabiosis theog
The theory of anabiosis, or dormancy, is popular at least partly because little

evidence exists for metabolic activity within the permafrost. The presence of intact
membranes and autoregulatory factors that keep the cell in a resting state reinforce the

anabiosis theory (Soina et al., 1995). Phenol lipids of acylresorcine, which have been

40

shown to accompany dormancy of both spore and non-spore formers in response to
starvation, have been detected in permafrost isolates (V orobyova et al., 1996).

Many studies have shown the presence of some enzyme driven reactions, but
were unable to determine if the cells producing the enzymes were still viable. Early
studies failed to detect the presence of radio-labelled carbon dioxide in Antarctic soil,
after the soil had been amended with a substrate, allowing the authors to conclude that
metabolic activity was absent (Horowitz et al., 1972). However, in surface soils some
radio-labelled glucose was assimilated even though a very small number of bacteria were
cultivated (Horowitz et al., 1972). This activity was attributed to a combination of factors
including the presence of non-culturable microorganisms, the presence of inorganic
catalysts, and the presence of dead, albeit still enzymatically active cells (Horowitz et al.,
1972).

Activity at cold temperatures has been characterized for several enzymes. The
best studied are isocitrate dehydrogenase and catalase (V orobyova et al., 1996). Isocitrate
dehydrogenase is cold labile and is used as a measure of the cryoprotective properties of
other proteins (Koda et al., 2000). Within the permafrost ionizing radiation produces
hydroxyl radicals upon contact with superoxides and peroxide the continued activity of
catalase could be beneficial for organisms within the permafrost since the role of catalase

is to protect cells fiom harrnfirl hydrogen peroxide by reducing it to water and oxygen.

Low ram: of in situ metabolic activity theory
For a number of years, only circumstantial evidence of metabolic activity was

available. The presence of large numbers of viable microorganisms isolated fiom ancient

permafrost soils indicated that DNA- and membrane-repair mechanisms must exist to

41

protect the cells from long-term exposure to free radicals produced by the y—radiation
emitted fi'om the potassium-40 in surrounding rock (F riedmann, 1994). In addition, the
presence of sufficient unfrozen water for a small rate of biological activity has also been
detected (Rivkina et al., 2000; Zvyagintsev et al., 1990). By combining studies of bound
water concentrations, the thin surface layer of water that surrounds the microbes, and
measures of membrane lipid incorporation at decreasing temperatures, Rivinka et al.
(2000) illustrated that the availability of free water diminishes with decreased
temperature (well below Siberian permafi'ost in situ temperatures). This creates a
diffusion barrier, effectively starving the cells (Rivkina etal., 2000).

A number of recent studies have shown the presence of metabolic activity at cold
temperatures in snow, ice, and soil. Slow growth of microorganisms has been detected in
Siberian permafrost at —8° to —10°C over an 18-month period (Gilichinsky et al., 1993).
In addition, doubling rates of 1 d at 5°C, 20 d at —10°C, and 160 d at —20°C were
measured by membrane lipid incorporation (Rivkina et al., 2000).

Low rates of protein and DNA synthesis were indicated by biological incorporation of
[3H] leucine (proteins) and methyl [3H] thymidine (DNA) between —12° and —17°C in
arctic snow (Carpenter et al., 2000). Furthermore, radioisotopic studies of Lake Vostok
accretion ice indicate the presence of metabolic activity, with only a small portion
resulting fi'om macromolecular synthesis (Karl et al., 1999). These findings indicate that
the microbes maintain only enough basal metabolism to allow repairs to membranes and
DNA molecules but do not actively divide under the oligotrophic, freezing environment.

Amino acid racemization studies of aspartic acid extracted from 35,000-year-old

permafiost indicate ongoing repair processes. Racemization studies examine the

42

frequency of D-aspartic acid, which is formed after cell death when the hydrogen of the
chiral a-C is detached and rejoined in the opposite orientation. In the presence of
metabolically active organisms this D form is repaired to the biologically active L form
of aspartic acid. The estimated ratio of D-aspartic acid, resulting from dead, inactive
cells, to L-aspartic acid was smaller than predicted by mathematical models, allowing the
authors to conclude that some metabolic activity was occurring to produce the diminished

levels of D-aspartic acid (Brinton et al., 2002).

Metabolic activity of anaerobes and nitrifying microorganisms, has been shown in
permafrost frozen for up to 2 million years by measurement of metabolic end products.
The youngest soils were shown to contain the highest amounts of anaerobic metabolic
end products (ferrous iron, sulfide, and methane), and these decreased with the age of the
soil (Rivkina et al., 1998). As the age of the soil increases, the concentration of the
substrate, ammonium, increases, whereas the products of nitrification are found in smaller
amounts (Janssen and Bock, 1994). However, it is uncertain whether these end products
were formed before freezing and then simply trapped in the soil layers after fi'eezing
because of the low gas diffusion seen in pennafi'ost.

Importance of the permafrost environment

Low temperatures provide a stable environment for sustaining life in the
permafrost. The permafrost is therefore likely to be a depository for ancient biomarkers
such as biogenic gases, polyaromatic hydrocarbons, biominerals, biological pigments,
lipids, enzymes, proteins, nucleotides, RNA and DNA fragments, and molecules,

microfossils and viable cells (V orobyova et al., 1997). The presence of potentially deadly

43

human viruses also exists in permafrost (Reid et al., 1999). This makes the permafiost
important for future advances in cryobiology, palaeontology and exobiology.

The continually frozen nature of permafrost aids the study of microbial evolution
because closely related microorganisms have been isolated from soils of different periods
of geologic time. Discovery of similar species using phylogenetic methods may allow for
better representations of the evolutionary clock in cold environments, as determined by
the mutation rates of particular organisms in that environment (Gilichinsky et al., 1992).

Permafrost is believed to extend over many of the planets of our Solar system,
including the majority of the planet Mars. The low temperature, low water-containing,
highly reducing and continuous exposures to radiation of Arctic and Antarctic permafrost
are very similar to conditions on Mars (Malin et al., 2000). Therefore, studies of the long-
term preservation of microbiota in Earth’s permafrost can provide a benchmark, from
which searches for extraterrestrial life can be launched (Soina et al., 1995). The age of
the Earth’s permafrost is much younger than the 3 billion-year-old estimate for Martian
surface, but it may still be used as a model to examine the long-term preservation of cells
that cannot be modelled accurately.

Permafrost might also provide a better understanding of the types of microbes
likely to survive on meteors or spacecraft, which may be able to regain their activity
when conditions are again agreeable. This is the basis of the theory of panspermia, in
which life is argued to survive as spores in space, and underpins the nascent science of
astrobiology. The low temperatures, low nutrients and radiation exposure found in the

permafrost is similar to that of space, at least within meteors or other debris (Mastrapa et

al., 2001).

44

The remarkable ability to survive in a continuously frozen matrix of permafrost for
millions of years makes the permafrost community unique, not only for its implications
for the discovery of novel enzymes of biotechnological importance, but also to begin to
answer basic science questions. How long can a cell survive in continually fi'ozen
conditions? What is the lowest metabolic activity necessary to retain viability? And does

life exist outside the Earth?

45

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CHAPTER 3:

CHARACTERIZATION OF POTENTIAL STRESS RESPONSES IN ANCIENT
SIBERIAN PERMAFROST PSYCHROACTIVE BACTERIA

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56

, —
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“I i ~' a.» ~‘ ’ -
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. t. J- a," ti
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4’ ' a
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' MICROBIOLOGY
“ " Ecology

FEMS Microbiology Ecology 53 (2005) 103—1 15

 

Characterization of potential stress responses in ancient
Siberian permafrost psychroactive bacteria

aed ab

b 9
Monica A. Ponder a, , Sarah J. Gilmour , Peter W. Bergholz , Carol A.
abs.

C C
Mindock , Rawle Hollingsworth , Michael F. Thomashow ,

ahad.
James M. Tiedje

a Center for Genomic and Evolutionary Studies on Microbial Life at Low Temperatures,
Michigan State University, East Lansing, MI 48823, USA

b Department of Microbiology and Molecular Genetics, Michigan State University, East
Lansing, MI 48823, USA

c Department of Crops and Soil Sciences, Michigan State University, East Lansing, MI
48823, USA

d MSU-DOE Plant Research Lab, Michigan State University, East Lansing, MI 48823,
USA

c Department of Chemistry, Michigan State University, East Lansing, MI 48823, USA

Received 30 June 2004; received in revised form 4 December 2004; accepted 6
December 2004

First published online 27 December 2004

Abstract

Past studies of cold-acclimated bacteria have focused primarily on organisms not
capable of sub-zero growth. Siberian permafrost isolates Exiguobacterium sp. 25 5-1 5 and
Psychrobacter sp. 273-4, which grow at subzero temperatures, were used to study cold-

acclimated physiology. Changes in membrane composition and exopolysaccharides were

57

defined as a fimetion of growth at 24, 4 and 2.5 °C in the presence and absence of 5%
NaCl. As expected, there was a decrease in fatty acid saturation and chain length at the
colder temperatures and a further decrease in the degree of saturation at higher
osmolarity. A shift in carbon source utilization and antibiotic resistance occurred at 4
versus 24 °C growth, perhaps due to changes in the membrane transport. Some carbon
substrates were used uniquely at 4 °C and, in general, increased antibiotic sensitivity was
observed at 4 °C. All the permafrost strains tested were resistant to long-term freezing (1
year) and were not particularly unique in their UVC tolerance. Most of the tested isolates
had moderate ice nucleation activity, and particularly interesting was the fact that the
Gram-positive Exiguobacterium showed some soluble ice nucleation activity. In general
the features measured suggest that the Siberian organisms have adapted to the conditions
of long-term freezing at least for the temperatures of the Kolyma region which are 10 to

12 °C where intracellular water is likely not frozen.

2004 Federation of European Microbiological Societies. Published by Elsevier B.V.

All rights reserved.

Keywords: Permafrost; Cryotolerance; Low temperature; Cold response; Psychroaetivity;

Salt response; Psychrobacter; Exiguobacterium

Introduction

The majority of the Earths surface is permanently cold, with approximately 70% of the
surface covered by oceans with an average temperature of 4 °C and over 20% of the

terrestrial area occupied by permafrost including, 85% of Alaska, 55% of Russia and

58

Canada, 20% of China, and the majority of Antarctica. Soils, sediments and rock exposed

to temperatures of 0 °C or below for a period of at least 2 years are defined as permafrost

[1]-

A variety of microorganisms have been isolated from buried permafrost of the Kolyma

region of northeast Siberia indicating that these organisms can survive subzero

temperatures (10 to 12 °C), low water activity (aW = 0.9), low nutrient availability, and

the cumulative effect from background 1 radiation from soil minerals that ranges fiom 1

to 6 kGy [2]. Depending on geologic strata these microbes have been in a continuously
fiozen environment for 20,000 to 3—5 million years [3]. In contrast to many oceanic
isolates, permafrost isolates are not psychrophiles, but psychrotolerant in that they can
grow at 4 °C and above 20 °C [4,5]. Most previous studies of permafrost microbes have

been done with strains that have been isolated and grown at room temperature and in

nutrient rich media. We previously isolated 238 bacterial strains from different age layers

of Siberian permafrost without exposure to temperatures above 4 °C and using several
isolation strategies, including low nutrient media and cryo-protectants [3]. Previous char-
acterization has indicated that many of these isolates are psychroactive in that they grow

at 2.5 °c [6].

Physiological responses to low temperatures of some mesophilic bacteria,
psychrotrophic food-borne pathogens, and environmental isolates have been
characterized [7]. The majority of studies have focused on membrane composition
changes and cold shock protein production in response to lower temperatures. While

most studies have defined responses to temperatures below the organism's temperature

59

optimum, only a few of these mesophilic bacteria are capable of growth at temperatures
below 4 °C. Those that can are Listeria monocytogenes [8], several Pseudomonas [9] and
Arthrobacter strains [10], and some un-identified Arctic isolates [11] from non-
permafi'ost environments.

The permafrost environment in contrast to many surface environments, is very stable,
with a constant set of stresses that may have acted as selective factors on the survivors,

including adaptation to a very long-term frozen environment and the associated
desiccation, and to cumulative effects of 7 radiation which would be expected to damage

cell DNA [2,12]. The Kolyma permafrost temperatures of 10 to 12 °C would not be
expected to freeze the cytosol of bacterial cells [13] and hence continued biochemical
catalysis could be expected, albeit the fluid would be viscous and reaction rates very
slow. The objective of this study is to characterize features of a selected set of these
isolates that may play a role in or reflect their adaptation to permafrost conditions.
Particular emphasis is placed on a Gram negative Psychrobacter strain isolated from a
20,000— 30,000 year old permafrost layer and a Gram positive Exiguobacterium strain

isolated from a 2—3 million year old layer.

2. Materials and methods

2.1. Isolation and phylogenetic characterization

Permafrost samples were obtained from the polar region of the Kolyma-Indigirka
lowland (152-162E, 68—72N), located adjacent to the East Siberian Sea by David
Gilichinsky and team (Cryobiology Laboratory, Russian Academy of Sciences,

Pushchino). The isolation conditions and characteristics of sampling sites and cores

60

chosen for bacterial isolation, were detailed previously by Vishnivetskaya et al. [3]. The
first 500 bp of the 16S rRNA genes of the isolates were sequenced to determine
phylogeny [6]. The isolates included members of the order Actinomycetales (genus
Arthrobacter and Family Microbacteriaeea) division Firmicutes (the genera
Exiguobacterium and Planomicrobium), genus Flavobacterium, and division
Proteobacteria (Psychrobacter and Sphingomonas). Analysis of the complete 16S rRN A
gene of Psychrobacter sp. 273-4 and Exiguobacterium sp. 255-15 isolates confirmed their
identity and established their similarity to other previously studied members of their
respective clades (H. Ayala del-Rio and D. Rodrigues, respectively, unpublished

observation). BOX PCR profiles revealed that Psychrobacter sp. 273-4 and sp. 215-51
were different strains, likely different species. Isolates were chosen fi'om the larger set for
further studies based on ease of culturability at 4 °C, growth at 2.5 °C, age of permafrost

strata and for being representative of different taxa found.

2.2. Growth rates

Growth rates, as firnctions of temperature were measured for Exiguobacterium strain
255-15 and Psychrobacter strains 273-4 and 215-51 by optical density in shaken flasks
(200 rpm) of tryptic soy broth (TSB) (Difco, Detroit, MI), at temperatures from 45 to 0.5
°C using three biological replicates per temperature. Arrhenius plots of Exiguobacterium
sp. 255-15 and Psychrobacter sp. 273-4 and sp. 215-51 were prepared by plotting the
natural logarithm of the growth rate against the reciprocal of absolute temperature [14]
using Statview with Lowess fitted lines (SAS Institute, Cary, NC). A Be'lehra‘dek

growth model was also constructed by plotting the square root of the growth rate against

61

the absolute temperature and the minimmn temperature determined by the method of
Ratkowsky et al. [15]. Growth rates at 2.5 °C were obtained by viable plate count in

triplicate non-shaken flasks. This data was not included in the Arrhenius and

Be'lehra‘dek models because of the different growth condition. Growth rates were

calculated from the slope of four or more time points.

Growth rates as a fimction of low water activity were measured by optical density in
1/10 TSB supplemented with NaCl. Colony formation was scored after one week on 1/10
TSA media supplemented with 0.125, 0.250, 0.5, 1.0, 1.5, 2.0, or 2.50 M NaCl. These
concentrations correspond to internal osmotic pressures of 0.217, 0.433, 0.873, 1.78,
2.72, 3.70 and 4.70 osm, respectively, as extrapolated from Rand et al. [16]. Several
isolates were chosen based on grth at high concentrations of NaCl to further examine

growth using sucrose as an alternate osmolyte.

2.3. Cell morphology and size

Psychrobacter sp. 273-4 was grown to an OD600 of 0.2 in 1/2 TSB and 1/2 TSB + 5%
NaCl (1.61 osm) at 4 and 22 °C. Twenty microliters of the culture was spotted onto 10
agar coated microscope slides and viewed by light microscopy. Digital images were
analyzed for cell size and morphology using the Center for Microbial Ecology Image
Analysis software (CMEIAS) [17]. Approximately 250 cells were analyzed for each

treatment.

62

2.4. Effect of temperature and salinity on lipid and polysaccharide composition

Psychrobacter sp. 273-4 and Exiguobacterium sp. 25515 were incubated at 24, 4 or 2.5
°C in 1/2 TSB or 1/2 TSB + 5% NaCl with shaking until an OD600 of 0.5 was obtained.
All cells were harvested by centrifugation and washed four times in sterile 1X PBS. Total
lipids were extracted by the method of Mindock et al. [1 8]. The aqueous layers were kept
for polysaccharide analysis. The organic layers containing polar lipids were analyzed by
NMR spectroscopy (using d4-methanol as the solvent) and by thin layer chromatography
(TLC) on silica plates using 10:4:2:2:1 chloroform: acetone: methanol: acetic acid: water.
The spots were sprayed either with orcinol or 10% phosphomolybdic acid in ethanol and
heated at 120 °C to visualize the organic components. NMR spectroscopy provided
confirmation of identities obtained by TLC mobility compared to standards. NMR spectra
were recorded on a Varian VXR-500 spectrometer (500 MHz). Chemical shifts for
samples in d4-methanol are quoted relative to the proton resonances at 4.78 and 3.30
ppm, and for samples in D20 relative to the proton resonance at 4.65 ppm. For the
double quantum filtered-correlated spectroscopy (DQF-COSY) experiments, a total of
256 data sets with 24 transients at 2048 points each were acquired. The total correlated
spectroscopy (TOCSY) experiments were also performed by using a total of 256 data sets
with 24 transients at 2048 data points each and with a mixing time of 90 ms.

Fatty acid methyl ester analysis was done using a portion of the fatty acid containing
organic layer of the total lipid extract. The extract was incubated in acidified methanol at
75 °C for 36 h, dried and re-suspended in 2:1:0.7 hexane: chloroform: H20. After
vigorous shaking, the mixture was centrifuged, the organic layer removed and

concentrated to dryness. The resulting fatty acid methyl esters were analyzed by gas

63

chromatography (GC) and GC—MS using a 30-m DB1 column (0.32 mm inner diameter,
0.25 1m film). The temperature program increased from 50 to 150 °C at5/min, 150—185 at
2/min, 185-250 at 5/min. After an initial analysis 2 ng of 2-OH dodecanoate (Sigma-
Aldrich, St. Louis, MO), not present in any of the samples, was added to whole cells as
an internal standard.

Polysaccharides were isolated from the aqueous layers of the total lipid extracts by
precipitation with ethanol. The precipitated polysaccharide was recovered with a glass
rod and dissolved in 10 ml of water, 10 mg MgC12, and 10 units each of RNase A and
DNase (Sigma—Aldrich) and incubated overnight at 22 °C. The polysaccharides were
then dialyzed against water for 4—5 h, changing the water twice during that time,
lyophilized and the resulting solids were weighed. These polymers were analyzed by gas
chromatography—mass spectrometry (GC-MS) after converting them to alditol acetate
derivatives [19]. The remaining aqueous alcoholic solution was centrifuged to remove
any solids. The supematants which contained free amino acids and carbohydrates were

removed and also analyzed by GC—MS.
2.5. Freezing tolerance

To determine if temperature acclimation influences freezing survival, the strains were

grown at 24 and at 4 °C to a cell density of 108 CFU/ml in 1/2 TSB and subsequently
fiozen slowly (0.2 °C/min) to 20 °C. Viable plate counts were performed initially before
freezing and compared to those obtained after slow thawing (0.3 °C/min) of samples held
at 20 °C for 1 year. Plates were incubated at the same temperature at which the strains

were originally grown. An unpaired t test was used to determine statistical significance

64

(Statview version 5.0, SAS institute).
2.6. Temperature dependent nutrient utilization

Biolog plates (Biolog, Inc, Hayward, CA) were used to assess whether temperature
affected the utilization of 95 different carbon sources. Psychrobacter sp. 273-4 and

Exiguobacterium sp. 255-15 were grown at 24 or 4 °C in 150 ml flasks, with 40 ml of
1/10 TSB with shaking until an OD600 of 0.5 was obtained. All cells were harvested by
centrifugation and washed four times in sterile 1X PBS. Exiguobacterium sp. 255-15 was
ali-quoted into GP inoculating broth (Biolog Inc.) to match the percent transmittance
(33%) of the GP standard prepared by Biolog. The same procedure was followed with
Psychrobacter sp. 2734 with the exception that GN (non-enteric) broth was used for the
inoculation standard. After inoculating the GP (Exiguobacterium) and GN
(Psychrobacter) Biolog plates, they were then incubated at the temperature of inoculum
growth. Six biological replicates (with two technical replicates each) were used per strain
and temperature.

The OD595 of the wells was determined with a micro-plate reader every 8 h until the
absorbance did not increase (4 days at 24 °C and 3 weeks at 4 °C). Average of the 95
values for each carbon source were determined for each set of replicates per time point.
Replicates which showed a standard deviation >15% were excluded and the averages
recalculated. Carbon sources which showed at least an average absorbance change of 0.2
or more from the blank plate were considered utilized.

2.7. Antibiotic susceptibility

The effect of temperature on antibiotic resistance was assessed using the impregnated

65

disk method at both 24 and 4 °C of triplicate experiments [20]. Antibiotic disks (BBL
Microbiology) were placed on lawns of bacteria on Mueller Hinton agar, incubated for 2
days at 24 °C or 2 weeks at 4 °C. After this time the size of clearings around the disks

was measured. Resistant cells grew significantly closer to the antibiotic disk than
susceptible cells while clearing size cutoffs for each antibiotic were used to determine

resistance categories described by Barry and Thomsberry [20].

2.8. UVC survival

The effect of temperature on UVC survival was assessed using cultures of Psychrobacter
sp. 273-4 and Exiguobacterium sp. 255-15 grown to late-log phase 600 = 0.6—0.9) in 1/2
TSB at 25 and 4 °C. UVC was chosen because it causes similar damage to low doses of
y—radiation [21,22], and the ease of application of small doses. Escherichia coli B606 was
used as a comparison strain, because it is a mesophilic El-Proteobacterium related to
Psychrobacter sp. 273-4. Bacillus subtilis PY79 was selected as a related mesophilic
Firmicute for comparison to Exiguobacterium sp. 255-15. Both mesophilic strains were
grown to late log phase (OD600 = 0.9) in 1/2 TSB at 24 °C.

A 15 ml culture at late log phase was collected by centrifugation at their cultured
temperature, re-suspended in 15 ml 0.85% NaCl and stored on ice until use. Cells were

then pipetted into a sterile glass Petri plate and exposed to a UVC fluorescent lamp
2 1
(equivalent dose 1.5 J m s ) with constant mixing. Psychrobacter sp. 273—4 and E. coli
2
B606 were cumulatively exposed to 0, 25, 50, and 100 J m . Exiguobacterium sp. 255-15

2
and B. subtilis PY79 were cumulatively exposed to 0, 100, 250, and 500 J m . At each

exposure level, plate counts were performed on 1/2 TSA plates and incubated in the dark

66

at the respective culturing temperatures. All steps from UVC exposure through
incubation were carried out in the dark and all solutions were kept at the same
temperature at which cells were cultured. After 48 h at 24 °C or 2 weeks incubation at 4
°C, CFU/ml were estimated. Average percent survival of four replicates at each of the
UVC dosage levels was used to compare Psychrobactersp. 273-4 grown at 24 °C to both
of the other two samples using a one-tailed two sample t test assuming unequal variances.

2.9. Ice nucleation activity

Ice nucleation studies were undertaken to determine the effect of temperature on ice

nucleation activity in seven permafrost strains. The strains were grown at 24 °C on 1/10
TSA and 1/ 10 TSA supplemented with 5% glycerol, which has been shown to optimize
ice nucleation activity (INA) in many bacteria [23,24]. INA was measured by the

freezing drop method [25]. Suspensions of bacterial cells were prepared in sterile buffer

and 60 10 11 drops of the suspension were placed on an aluminum boat in a circulating
ethanol cold bath set to 10 °C. The fieezing of droplets at or above 10 °C indicated INA.
After initial determination of INA in cultures grown at 24 °C, the suspensions were
subjected to 3.5 h at 4 °C and l h at 10 °C and then reassessed for INA by the fieezing
drop method. Pseudomonas syringae ATTC 35421 and E. coli ATTC 39524 were used as
positive and negative controls, respectively, for INA. The ice nucleation activity per cell

was determined with the method of Pooley and Brown [26].

Lesults

67

3.1. Effect of temperature on the maximal growth rate and cellular morphology

The Exiguobacterium and two Psychrobacter strains tested both grew at 2.5 °C with
generations times of 5.5 and 3.5 days, respectively, but the Exiguobacterium strain had a
much higher grth maximum (42 °C) than Psychrobacter strains (26 °C) (Fig. 3.1).
Arrhenius plots of the Exiguobacterium growth profile reveal a change in slope occurring
at 24 °C, while the Psychrobacter showed changes in slope at 22 and 6 °C. Minimum

growth temperatures of 15, 12, and 7 °C were estimated using the Be'lehra‘dek growth
model for Psychrobacter sp. 273-4 and sp. 215-51 and Exiguobacterium, respectively,
and agreed with extrapolations from Arrhenius plots.
Psychrobacter sp. 273-4 exhibited a change in cell size and shape when subjected to
either 5% salt or low temperature (Table 3.1). In general, the cells were slightly larger
when grown at 4 °C or in the presence of salt. At the optimum grth temperature of 22
°C, the cells are rod shaped and 1.8 1m in length. The introduction of 5% salt resulted in a
significant increase in width and decrease surface/volume ratio of the cells. The average
bio-volume of Psychrobacter increased at low temperature but most significantly in the

presence of salt (Table 3.1). Cells were somewhat more pleomorphic when grown in the
presence of salt. No differences in cell size or morphology were seen in initial
observations of Exiguobacterium sp. 255-15 in response to 5% NaCl or 4 °C so further
ZMEIAS analysis was not performed.

.2. Effect of increased osmolarity on growth of two Siberian permafrost isolates

Given the low water activity of permafrost, salt tolerance was assessed in

rychrobacter sp. 273-4 and Exiguobacterium sp. 255-15 using NaCl as an osmolyte.

68

Addition of salt dramatically reduced the growth rates of both strains at 24 °C and

somewhat for Exiguobacterium at 4 °C (Fig. 3.2). In contrast, the growth rate was

relatively constant as a function of salt concentration for Psychrobacter sp. 273-4 at 4 °C.

Growth rate and lag time did not differ significantly between cells grown in sucrose or

NaCl as an osmolyte ensuring that ion toxicity did not influence growth capabilities

(results not shown). As expected, lag phase increased for both strains with exposure to

increasing salt concentration and low temperature. For Exiguobacterium, the lag phase

increased from 0.67 h at 24 °C to 6.7 h at 4 °C (1/2 TSB), while the lag phase in high salt

(1.78 m) increased more dramatically at 24 °C to 21 h when compared to 4 °C where an

increase to 82 h occurred. Psychrobacter lag times also increased at 4 °C from 2.5 h to 22

h in 1/2 TSB, and incubation in 1.78 m salt increased lag times to 21 and 144 h at 24 and

4 °C, respectively.

Table 3.1: Effect Of Salt And Temperature On

Psychrobacter Sp. 27 3-4.

The Cellular Morphology Of

 

 

 

Median Median Median Median
Median Median
Medium length biovolume biosurface surface/
width (pm) length/width
(um) (um’) (um’) volume
3 1’ 1/2 TSB 1.80 :1: 0.12 0.77 :t 0.03 2.35 a: 0.15 1.00 :t 0.12 5.23 d: 0.48 5.00 t 0.3
+5%NaCI 1.74 d: 0.09 0.84 at 0.031' 1.99 :t 0.09‘1 1.15 :t 0.13 5.59 :l: 0.50 4.77 :t 0.1‘I

 

 

 

 

 

 

 

 

69

 

 

 

 

Median Median Median Median
Median Median
Medium length biovolume biosurface surface!
width (um) length/width
(um) (um’) (um’) volume
22°C 1/2 TSB 1.62 d: 0.13 0.73 d: 0.03 2.25 :1: 0.15 0.85 i 0.12‘I 4.44 :1: 0.48 5.26 a: 0.3
+5%NaCl 1.86 :1: 0.09 0.82 :i: 0.03‘ 2.20 :1: 0.091‘ 1.17 :1: 0.13 5.74 :t 0.59 4.94 i 0.1'

 

 

 

 

 

 

 

 

All results reflect the average of 250 cells analyzed by CMEIAS.

a p-Value below 0.05 for 5%NaCI.

In generation] h

 

.254,

a
I
0 a
1
4

-1 .251

i

4.5%

 

-1.75-'

 

Cl
0

 

h
i
p
l
l

3115' 52 V3215 , 3'3 V3315 - 3'4 V345 - 3'5 V3515 ' 36 see 317
Temperature (Mr X 10e4)
Figure 3. 1: An Arrhenius Plot Of Grth Of Exiguobacterium (392-28 And 255-15)

And Psychrobacter (273-4 And 215-51) At A Range Of Temperatures Between 42°-

O.5°C. No growth occurred in Psychrobacter isolates at temperatures above 28°C.

70

 

0.5—1

 

 

 

 

 

 

 

 

0.45 ~
0.4 —
if 0.35 —
5
it 0.3 -
i.
g 025 i -O-Exiguobacterium 255-1540
-0- Exiguobacterium 255-15 24C
.C 0 2 “ -Ci-Psychrobacter 273—4 40
E -I- Psychrobacter 273-4 240
c) 0.15 —
0.1 —
0.05 — K: _ \
0 T . . ' .
0 0 5 1 1 5 2
Osmolarity

Figure 3.2: Comparison of Average Growth Rates of Two Permafrost Isolates at
Different Temperatures and Osmolarities. The umax at 24°C of Exiguobacterium 255-15
was 0.9.

3.3. Membrane composition under stress

Requirements for membrane fluidity at permafrost temperature and water activity should
be reflected in membrane composition for organisms adapted to these conditions. Fatty
acid profiles for Psychrobacter sp. 273-4 included both straight chain and unsaturated
fatty acids but branch chained fatty acids were not detected. The overall composition of

unsaturated fatty acids increases at lower temperatures and when grown in presence of

71

5% salt (Table 3.2a). The dominant fatty acid was a C18, saturated at 24 °C and
unsaturated in the presence of 5% NaCl and 4 °C (Table 3.2a). A C18:2 fatty acid was
present between 1 and 3% under all the growth condition (not shown). An unsaturated
C16 was dominant at subzero temperatures alone. An increase in the amount of C17
correlated with increasing salinity, at the expense of C18 at 4 °C. The second most
abundant fatty acid species were the C16 methyl esters. Saturated 18:0 was predominant
at 24 °C, while in the presence of NaCl or 4 °C growth unsaturation was favored.
However, the combination of NaCl and 4 °C resulted in an increase in the amount of
C17:l at 4 °C rather than 16:1 as seen in the single stress conditions (Table 3.2).

Phosphatidylglycerol and phosphatidylethanolamine were detected at 4 and 24 °C.

Diacylglycerol and additional spots not corresponding to standards were seen at 4 °C

under both conditions (data not shown). In addition, spots were present at the origins that

were not soluble in the TLC solution.

In Exiguobacterium sp. 255-15, the presence of 5% NaCl or low temperature
conditions alone shifted the fatty acids from saturated to unsaturated as expected at 4 °C,
however unsaturated fatty acids were not de-acid in Exiguobacterium sp. 25 5-1 5 at
mesophilic tected from three biological replicates grown at and subzero temperatures but
at 4 °C a shift occurred 2.5 °C (Table 3.2b). C16:0 was the predominant fatty to
isoCl7:0. The interaction between 4 °C and salt results in a further increase in
unsaturation with the predominant fatty acid becoming Cl8:1. Fatty acids below 5% of
the total were not included in Table 3.2 and included: isoC13:0, anteisoC15z0, isoC15:0,
isoCl6:O, C12:0, C13:0,C14:0, C17:0 and C22:0. The phospholipid profile contained

phosphatidylglycerol, diacylglycerol and phosphatidylethanolamine at 24 and 4 °C, with

72

additional unidentified spots. Unidentified carbohydrate containing spots were present
when cells were grown at 24 °C. Only one spot, identified as phosphatidylglycerol, was

shared in 4 and 24 °C grown cells (data not shown).

3.4. Polysaccharide composition

The soluble polysaccharide composition of Psychrobacter sp. 273-4 and

Exiguobacterium sp. 255-15 differed with temperature and presence of 5% NaCl (I able

3.3). Psychrobacter sp. 273-4 polysaccharides consisted of rhamnose, glucose, mannose,
galactose, xylose, fucose, ribose and arabinose when grown at 24 °C. When the cells
were grown in 5% NaCl arabinose increased and fucose decreased. Lower temperatures
resulted in an increase in predominance of glucose, mannose and a decrease in rhamnose
(Table 3.3).

The predominant sugar in Exiguobacterium sp. 25515 at all three temperatures was
glucose. Rhamnose, galactose, mannose, ribose, fircose, arabinose, xylose and an
unknown amine sugar were also present (in decreasing percentages). Ribose decreased in
dominance with low temperature, while arabinose and mannose increased. The presence
of 5% NaCl resulted in a shift with mannose and arabinose (2.5 °C) increasing in percent

composition at the expense of galactose and arabinose (Table 3.3).

73

I‘able 3.2. Fatty Acid Methyl Ester Composition (% Of Average Total Peak Area) Of

Two Permafiost Isolates At Different Temperatures And Osmolarities

 

FAME

TSB

TSB + 5% NaCI

 

24°C

 

4°C

 

-2.5°C

 

24°C

 

4°C

 

-2.5°C

 

 

(a) Average percent fatty acid methyl ester profiles of Psychrobacter sp. 27 3-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4

crezo 28.39 1.3g 40.29 1.25a 283 2.9g
C18:0 44.29 12.412 20.012 1.2a 1.13 23.412
C16:1 1.0g 20.012 13.21! 32.41! 1.30n 29.911
mm 1.09 13.012 1.0a 2.631! 17.312 1.0‘—‘
C18:1 1.75 42.0‘2 7.09 52.2! 72.012 43.49
Straight chain fatty acids

%oftotal 72.5 13.7 60.2 2.5 3.9 26.3
Unsaturated fatty acids

%oftotal 3.7 75.0 21.2 87.2 90.6 74.3

 

 

 

 

 

 

 

(b) Average Exiguobacterium sp. 255-15 fatty acid methyl ester profiles

 

C1620

26.3a

15.012

31.3‘E

23.09

9.712

31.8g

 

Cl8:0

 

6.0g

 

5.0!

36.19

 

 

13.612

 

14.012

 

27.9b

 

74

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FAME TSB TSB + 5% NaCI

24°C 4°C -2.5°C 24°C 4°C -2.5°C
C15:OIso 7.0! 4.5! 10.5! 6.8! 1.3! 11.3!-
Clm 4.0! 7.0! 1.6! 4.2! 4.7! 0.9!
Anteiso
C17:OIso 16.5! 26.0! 6.6! 22.0! 18.2! 7.5!
C16:l 1.0! 13.0! * 2.5! 4.0! t
C18:1 5.4! 19.0! =1 13.0! 29.0! t
Straight chain saturated fatty acids
%oftotal 32.3 20.0 67.4 36.6 23.7 597
Branched chain fatty acids
%oftotal 27.5 37.5 18.7 33.0 24.7 197
Unsaturated fatty acids
%oftotal 6.4 32 * 15.5 33 *

 

Average of three biological replicates.

° Standard deviation < 0.5.

" 0.5 > Standard deviation < 2.0.
c 2.0 > Standard deviation < 5.0.

* Indicates trace amounts below 5% of total percentage.

75

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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77

3.5. Freezing tolerance

Twelve different permafrost isolates (including Psychrobacter and Exiguobacterium)

were examined for their ability to survive freezing at 20 C after a period of 1 year (Table

5 s
4). Most striking is that all strains showed excellent survival rates with 10 —10 CFU/ml

8
found from 10 CFU/ml after 1 year. Prior cold acclimation (growth at 4 °C) increased

freeze survival in 9 of 12 strains, although only 3 strains (Planococcus sp. 21568, 45-18

and Rathayibacter sp. 190-4) were statistically significant. Both results suggest that these

strains are already adapted to subzero environments.

Table 3.4. Effect Of Growth Temperature On Survival Of Permafrost Isolates After One ‘

 

 

 

 

 

 

 

 

 

 

 

Year At -20 C
J Average cell loss after 365 days at -20 °C Significance of growth
Strain
[ (log CFU/ml) temperature (p-value)
{ Growth temperature (°C)
I l 4 24
Arthrobacter sp. 255-12 2.6 1.1 0.08
ll ' Arthrobacter sp. LTER 2.3 l 1.0 0.04
{ Arthrobacter sp. 33-1 1.0 1.8 0.35
{ Exiguobacterium sp. 392-28 1.2 1.7 0.04
Exiguobacterium sp. 190-11 0.9 2.1 0.15
Exiguobacterium sp. 255-15 1.2 2.4 0.42

 

 

 

 

 

*7

78

 

 

 

 

 

 

 

 

 

 

 

 

Average cell loss after 365 days at -20 °C Significance of growth
Strain
(log CFU/ml) temperature (p-value)
Growth temperature (°C)
4 N 24
F lavobacterium sp. 309-37 1.3 1.7 0.58
Planococcus sp. 109-1 3.3 1.9 f 0.09
Planococcus sp. 215—68 0.6 2.5 0.03
Planococcus sp. 45-18 0.3 2.1 0.003
Psychrobacter sp. 215-51 0.7 1.4 0.35
Rathayibacter sp. 1904 0.5 2.0 0.006

 

 

 

 

 

 

 

3.6. Temperature dependent carbon utilization

Psychrobacter sp. 273-4 and Exiguobacterium sp. 25515 were tested for their ability to

use 95 different carbon sources at either 4 or 24 °C (Table 3.5). Psychrobacter sp. 273-4

utilized 32 different carbon sources at 24 and 4 °C. Six of these carbon sources were used

only at 4 °C, while 12 were used only at 24 °C. Exiguobacterium sp. 255-15 was able to

 

utilize 42 different carbon sources at 24 °C, while at 4 °C only 36 were used. Seven of

these carbon sources were used only at 4 °C and 13 carbon sources were used exclusively

at 24 °c (Table 3.5).

79

Table 3. 5. Carbon Sources Uniquely Utilized At The Indicated Temperatures

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Psychrobacter sp. 273-4 Exiguobacterham sp. 255-15
24 C 4 C 24 C 4 C
D-Arabitol D,L-lactic acid L-Fucose Inulin
D—Galactose Propionic acid a-Methyl D-glucoside Tween 40
Gentibiose a-Ketoglutaric acid Palatinose Tween 80
m-Inositiol u-Ketovaleric acid B-Hydroxybutyric acid Amygladin
a-D-Lactose B-Hydroxybutyric acid D-Malic acid Raffinose
D-Glucuronic
y-Aminobutyric acid L-Malic acid D-Xylosc
acid
D-Saccharic
Pyruvic acid Acetic acid
acid
Glucoronamide D-Alanine
L-Aspartic acid L-Alanine
L-Arabinose L-Asparganine
D-Gluconic
L-Glutamic acid
acid
Cellobiose Cellobiose
Glycerol

 

 

 

 

 

80

 

3.7. Temperature effect on antibiotic susceptibility

Six different permafrost isolates were tested for naturally occurring resistance at 4 and

24 °C to five antibiotics (Table 3.6). Psychrobacter sp. 273-4 isolates showed no
resistance to any antibiotics tested. Arthrobacter sp. 45-3 resistance was maintained at 4
°C with the five antibiotics tested, while Arthrobacter sp. 255-12 showed decreased
resistance to ampicillin, chloramphenicol and tetracycaline at 4 °C. Only

Exiguobacterium sp. 7-3 and Planococcus sp. 215-68 showed decreased resistance to

streptomycin at 4 °C. No strains showed differential resistance to erythromycin with

temperature. Resistance was maintained at 4 °C only in those strains possessing a high

level of resistance at 24 °C (Table 3.6).

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3.8. UVC resistance

Because ionizing and UV radiation share some similarities in DNA damage, we tested
the capacity of two permafrost isolates and reference mesophiles to remain culturable
after exposure to UVC. The percent survival of Psychrobacter sp. 273-4 grown at 24 °C
was significantly greater than those grown at 4 °C at all dosages (Table 3.7).
Psychrobacter and E. coli B606 had similar sensitivities to UVC at mesophilic
temperatures, except at the highest UV dose where E. coli was more resistant to UVC (p
< 0.05). Exiguobacterium sp. 255-15, as well as B. subtilis PY79, exhibited more UVC
tolerance than Psychrobacter sp. 273-4, in cells grown at 24 and 4 °C. Exiguobacterium

sp. 255-15 grown at 4 °C was more sensitive to UVC than when grown at 24 °C at both
2
100 (p < 0.005) and 250 J m (p < 0.02). Exiguobacterium sp. 255-15 at 24 °C exhibited
‘ 5
significantly greater survival than B. subtilis PY79 at both the 100 (p < 7.9 - 10 ) and at

2 4
the250Jm(p<8-10).

83

 

 

Table 3.7. Average percent survival of permafrost isolates exposed to UVC

 

 

 

 

 

 

 

 

Strain and temperature UVC dose (J/mz)

0 25 50 100 250
lam-mega” sp. 2734 40c 100 26.9 e 13.9g 13.3 :1: 6.6! 0.29 a: 0.2IQ ND
7’sychrobacter sp. 2734 24°C 100 74.7 a: 22.6”- 37.1 e 9.6a 1.46 e 0.8“ ND
r5. coIiB606 24°C 100 68.6 r. 8.4 34.2 a: 0.4 5.30 e 1.09 ND
Exiguobacterium sp. 255-15 4°C 100 ND ND 2.20 :1: .329 0.01 :1: 0.019
Exiguobacterium sp. 255-15 24°C 100 ND ND 91.1 i 13.1"“4 2.72 a: 1.97‘5’g
T9. subtilis pm 24°C 100 ND ND 11.7 e 1.64 0.08 e 0.01‘1

 

 

 

ND, not determined for this UVC dose.

’ p—Value (24 °C > 4 °C) < 0.1.

b p-Value (E. coli > 2734) < 0.05.
c p-Value (24 °C > 4 °C) < 0.01.

d

p-Value (24 °C > B. subtilis) < 0.02.

84

 

 

 

 

 

 

3.9. Ice nucleation activity

Nine different permafrost isolates were tested for their ice nucleation activity, which

may provide protection from harmful intracellular ice accumulation. Five isolates
(Flavobacterium sp. 309-37 and 23-9, Psychrobacter sp. 215-51 and 273-4,
Sphingomonas sp. 190-14 and 3361-2) possessed weak ice nucleation activity when
grown at 24 °C. Exposure to 4 °C firrther increased ice nucleation activity in all strains
except Psychrobacter sp. 215-51 and Sphingomonas sp. 3361-2 (Table 8). This cold
shock treatment also induced INA in Exiguobacterium sp. 7-3, which exhibited no
measurable ice nucleation ability at 24 °C. Cold shock at 10 °C resulted in a further

increase in ice nucleation of in all six of the above strains (Table 3.8).

Table 3.8. Ice Nucleation Activity (X10'9) Per Cell Of Selected Permafiost Isolates After

Growth At 24 C Followed By Exposure To 4 And -10°C

 

 

 

 

 

 

 

 

 

 

 

Strain Exposure temperature
24 °C 4 °C -10 °C
Exiguobacterium
7-3 0 10.5 :t 0.1 18.2 :1: 0.2

1».

X F lavobacterium sp. 23-9 3.4 :1: 0.1 10.4 :1: 0.05 61.5 i: 0.4
Flavobacterium sp. 309-37 7.4 i 0.4 17.5 :1: 0.1 2.8 d: 0.04
Psychrobactersp. 273-4 252 i 4.3 360 i 18 170 i 12
Psychrobacter-sp. 215-51 16.2 i 3.4 0.8 :1: 0.1 3.4 i 0.05
Sphingomonas sp. 190-14 10.4 i 1.2 314 i 20 17.0 :h 0.8

 

 

 

 

 

 

85

 

 

 

Strain Exposure temperature

 

24 °C 4 °C -10 °C

 

Sphingomonas sp. 3361-2 54.8 i 5.6 19.4 i 0.21 5.2 d: 0.1

 

 

 

Escherichia coli Migula 0 0 0
Pseudomonas

Sijderius 411 411 411
syringae

 

 

 

 

 

 

 

Mean value of two replicates. If no standard deviation given there was no difference in

number of drops frozen between replicates.

Discussion

Permafrost provides an opportunity to obtain microbes that have experienced long term
exposure to cold temperatures, decreased water activities, 7 radiation and low carbon
availability. The Psychrobacter and Exiguobacterium strains, as diverse representatives
of the permafrost community, should carry traits that have allowed them to adapt to these
conditions. The genomes of these two organisms are currently being sequenced and the
characterization of physiological traits potentially important to cryo-adaptation is
important for beginning understand these adaptations at the genome and proteome levels.
Psychroaetivity is common for bacteria isolated from cold environments such as
sea ice. One such isolate, Psychromonas ingrahamii, grows at a temperature of 12 °C
with a generation time of 240 h, the lowest growth of any organism authenticated by a

growth curve [27]. Recently, growth of another Psychrobacter from Siberian permafrost

86

 

 

was reported at 10 °C (0.016 day-1 [28]), the temperature of the Kolyma permafrost.
Tolerance to low water activity coupled with sub-zero growth, including the minimum
growth temperature prediction of 7and 15 °C suggests that Exiguobacterium sp. 255-15
and Psychrobacter sp. 2734 could be active in their native habitat. Our Psychrobacter
and Exiguobacterium isolates grew over a moderate to broad temperature range, (15 °C
calculated Tmin) 5t026 °C and (7 °C calculated min) 2.5 to 42 °C, respectively, the later
being especially interesting because of its temperature span of at least 45 °C. Permafrost
strains typically must live for thousands of years in the surface (active) soil layer with
annual freeze and thaw cycles of +10 °C to 20 °C, which may have been the selective
force for the broad grth range typical of permafrost isolates.

These two isolates exhibit different growth patterns as determined by the linear portion
of the Arrhenius plot. Switch points in the slope of grth rates, indicative of a

physiological shift in metabolism, occurred at 22 °C in Psychrobacter sp. 273-4, 24 °C in

Exiguobacterium sp. 255-15 and also at 6 °C in Psychrobacter. These two isolates exhibit
different growth characteristic as has been reported in Pseudomonas fluorescens MFO
where the “thermometer temperature” of 17 °C triggers a change in physiological
characteristics such as increased membrane permeability to B-lactamine and an increase

in enzymatic activity of extracellular protease, lipase and periplasmic phosphatases [29].

While cell sizes, antibiotic susceptibility and membrane composition were not assessed at

the switch points in our study, marked differences in these traits were seen between cells

grown at 4 and 24 °C.

Minor but significant cell morphology and size changes in Psychrobacter sp. 273-4

occurred with exposure to low temperature and increased salt. The decrease in surface to

87

 

volume ratio observed in Psychrobacter sp. 273-4 with low temperature and salt is
similar to that seen in other bacteria isolated from low water activity environments [30].
An increase in surface to volume ratio is seldom encountered in cells isolated from low
water activity environments since the presence of compatible solutes, which balance the
external and internal solute concentrations, tend to swell cells. In contrast, another
permafrost isolate, Arthrobacter sp. 45-3, showed a large decrease in cell size (l4-fold)
after incubation at 4 °C [18]. Members of the genus Arthrobacter are known to change
cellular morphologies with stress and development stages, suggesting that this may be a
common adaptation in this genus [31].

Low temperature and high osmolarity are known to induce changes in membrane
composition that maintain membrane fluidity [32,13]. A decrease in temperature resulted
in a decrease in the saturation of fatty acids and acyl chain lengths in Psychrobacter sp.
273-4 and Exiguobacterium sp. 255-15, as occurs in other bacteria shifted to lower
temperatures. Together these changes function to lower the gel-liquid crystalline
transition temperature, i.e., homeoviscous adaptation [33]. Unsaturated membrane fatty
acids are less abundant in these psychroactive Siberian permafrost isolates grown at

mesophilic temperatures compared to the psychrotolerant, Oleispira antarctica. This

difference in unsaturation may account for the Psychrobacters narrower range of growth

temperatures [34]. These Siberian permafrost isolates show a more dramatic shifi to
unsaturated fatty acids than previously described for a permafrost isolate, Arthrobacter
sp. 33-1 [18]. Exiguobacterium sp. 255-15 exhibits the same shift to shorter branched
chain fatty acids, with an increase in anteiso-C15:0 at the expense of anteiso-c1720,

which was also seen in Arthrobacter sp. 33-1 and L. monocytogenes [35].

88

 

Exposure to increased osmolarity resulted in increased fatty acid unsaturation and
acyl chain length in these Siberian permafrost isolates. Moderate halophiles have been
described as showing increased membrane fatty acid saturation [36] rather than the
increased unsaturation seen in this experiment. The present study concurs with the trend
shown in Vibrio costicola indicating that the combination of high salinity and low tem-
peratures results in increased unsaturation of fatty acids and an increased ratio of
phosphotidylethanolamine: phosphotidylglycerol; both of these metabolic changes are
commonly seen only under low temperature conditions [37]. The changes in sugar
composition with temperature and salinity reflect additional adaptations, likely in the
membrane-associated exopolysaccharide that may improve survival. Recent reports of
exopolysaccharide accumulation in sea ice supports the stabilizing role for the cold active

Cowellia ColAP, thought to aid environmental survival [3 8].

An important consequence of membrane changes at cold temperatures is the effect on

membrane transport which could explain the differences in carbon source utilization and

antibiotic sensitivity seen at low temperatures. Glucose utilization increased at 0 °C in an
unidentified cold-adapted psychrotroph [39]. Uptake rates of NH4 in psychrophilic Vibrio
increased when the organism was grown at temperatures between 0 and 15 °C compared
to 24 °C, resulting in an increased max for the NH+ transport system [40]. Alternatively,
cold shock increased the activity of malate dehydrogenase and glucose-6-phosphate

dehydrogenase in Lactococcus lactis and Rhizobium [41,42]. In addition to alterations in

enzyme efficiency and in Vmax of uptake, a more universal change in membrane

permeability may explain the differential use of certain carbon sources in the permafrost

89

 

bacteria tested. Studies on glycerol uptake, which is transported both by facilitated and
passive diffusion, indicate an increase in both types of uptake in E. coli when membrane
fluidity is increased [43]. Molecules, such as cellobiose, may not enter the cell at 4 °C
due to changes in transport-associated proteins which are influenced by membrane
composition. Additional substrates may also be bound when membrane and protein
flexibility increases as demonstrated recently for the arabinose binding protein [44]. An
increase in flexibility commonly occurs in proteins at low temperatures raising the

possibility that additional substrates could be bound by other binding proteins. If a single
transporter handles a range of substrates then decreased efficiency of such an enzyme
would decrease uptake at low temperatures. The presence of temperature dependent
nutrient uptake efficiencies suggests that microbes have developed adaptations to low
temperatures which will counteract the unfavorable effects of decreased diffusion,
allowing adapted microbes to be more competitive for nutrients under unfavorable

conditions.

Continual exposure to sub-zero temperatures within the permafrost for organisms with
long-term freezing survival, a trait noted in all permafrost isolates tested. Cryotolerance
has been reported in another Arctic-isolated Pseudomonas when pre-conditioned at 4 °C

prior to freezing at 20 °C for 24 h [9]. All permafrost strains possessed excellent survival
5 8
rates with bacterial numbers of 10 —10 CFU/ml observed afier 1 year at 20 °C from an

8
original population of 10 CFU/ml. This is substantially higher than E. coli [45], although
it is similar to the survival reported for the food-associated, L. lactis [46]. Pre-

conditioning to cold temperatures is known to increase freezing survival in many food-

90

 

 

associated microbes, including E. coli OlS7:H7 [45] and L. lactis [46] though after only
28 days a one log decrease in survival occurs. This effect may be due to expression of

cold-responsive genes and cryoprotectant molecules that function to enhance the survival
of the microbe through the stress of freezing and thawing conditions [47—49]. However
pre-conditioning to cold temperatures, does not increase freeze survival in all bacteria, as
seen in this experiment and for lactic acid bacteria [47]. Permafrost organisms could
already be selected for life in this continuously frozen environment since they don't need
to respond to temperature cycles.

Ice nucleation activity, enhanced by exposure to lower temperatures, may be another
mechanism of survival in permafrost. Ice nucleation activity results from outer membrane
proteins whose structure mimics an ice crystal, providing a lattice for crystallization of
water [50] at higher temperatures, and preventing harmful intracellular ice formation
because the crystals are too large to penetrate the membrane and initiate intracellular

fieezing [51]. The formation of these small, thermodynamically unstable molecules

makes ice nucleation proteins unlikely to offer long-term survival due to the crystals

tendency to reform into large, damaging structures [52]. The discovery of a protein
capable of both ice nucleation and antifreeze activity in Pseudomonas putida may present
a solution to this dilemma. The ice nucleation domain is believed to cause the formation
of extracellular ice, while the antifreeze domain maintains the crystals at a non-damaging
size [53]. The ability of some plant and animal antifreeze proteins to inhibit microbial ice
nucleation activity suggests that antifreeze proteins may lower the supercooling point of
water within the cells. However, no direct evidence supports this theory [54,55]. The

presence of small amounts of thermal hysteresis activity in Psychrobacter sp. 273-4

91

 

 

suggests it may possess antifreeze proteins or a combination anti-freeze/ice nucleation
protein, as seen in y—Protcobacterium relative, P. putida (J. Duman, personal
communication). The detection of ice nucleation activity in the Gram positive
Exiguobacterium sp. 255-15 is surprising since the ice nucleation protein is located
within the outer membrane in all bacteria known to date. Ice nucleation activity was
maintained after filtering (results not shown) suggesting a soluble protein may be
responsible in Exiguobacterium sp. 255-15.
Exposure to UVC under two growth temperatures was used to evaluate the ability of
Psychrobacter sp. 273-4 and Exiguobacterium sp. 255-15 to survive DNA damage
resulting from reactive oxygen species and occasional single stranded DNA breaks such
as would occur from ionizing radiation. A decrease in UVC survival at 4 °C relative to 24
°C was observed in both strains, and UVC survival during grth at low temperature was
not greater than that of either mesophilic comparison strain. This result appears to

indicate that unusual capacity to withstand DNA damage was not necessary for either

organism to survive and be resuscitated in the lab after 104—106 years in permafi'ost. One
must conclude that some level of repair must have been occurring in situ. Recently, Price
and Sowers compiled data indicating that in situ survival metabolism, defined as a
metabolic state in which cells “can repair macromolecular damage but are probably
largely dormant’ ’, is higher than theoretical rates of DNA depurination over decreasing

temperature [56] their calculated rates of survival metabolism were found to be

6
approximately 10 times lower than that required for measured metabolic rates required
for growth at similar temperatures. This very low metabolic requirement could be met in

:itu by Psychrobacter sp. 273-4 or Exiguobacterium sp. 255-15 and hence allow them to

92

 

repair DNA damage.

The ability of permafrost isolated bacteria to respond to laboratory-simulated
permafrost conditions suggests that these organisms possess adaptations to low

temperature, increased osmotica and have efficient repair mechanisms that allow for

these and not other ttmdra organisms to continue to live in permafrost.

Acknowledgement

This research was funded by the National Astrobiology Institute of NASA. We would
also acknowledge the assistance of Chia-Kai Chang, Gisel Rodriguez, Debora Rodrigues
and Alexa Turke. We also thank Marcia Lee for assistance with the ice nucleation
activity studies, Frank Dazzo for CMEIAS expertise, George Sundin for assistance with
UVC exposure experiments and Rich Lenski and Lee Kroos for gifts of strains E. coli
606 and B. subtilis PY79 respectively. All GC/MS analysis was performed by Beverly

Chamberlin at the Center for Mass Spectrometry of Michigan State University.

93

 

 

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Cryobiology 37, 86—91.

[47] Kim, W. and Dunn, NW. (1997) Identification of a cold shock gene in lactic acid
bacteria and the effect of cold shock on cryotolerance. Current Microbiol. 35, 59—63.

[48] Hong, SH. and Marshall, RT (2001) Natural exopolysaccharides enhance survival
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bulgaricus CIP 101027T. Cryobiology 40, 264—269.

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an antifreeze protein with ice nucleation activity from the plant growth promoting
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[54] Duman, J.G., Xu, L., Neven, T.G., Tursman, D. and Wu, D.W. (1991) Hemolymph
proteins involved in insect sub-zero temperature tolerance: ice nucleators and
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pp. 94— 127. Chapman and Hall, New York.

[55] Griffith, M. and Ewart, K.V. (1995) Antifreeze proteins and their potential uses in
frozen foods. Biotechnol. Adv. 13, 375—402.

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4636.

97

 

CHAPTER 4

CRYO-ADAPTATION OF Psychrobacter arcticum 273—4 AS DETERMINED
THROUGH TRANSCRIPTOME PROFILING AT LOW TEMPERATURE AND
INCREASED OSMOTICA

Abstract

Siberian permafrost bacteria must maintain activity at low temperatures (-10°C) and the
increased solutes in the remaining unfrozen water films (aw=0.85-0.9). To gain
knowledge of microbial adaptation to low temperature and water activity, the genome of
a Siberian permafrost isolate was sequenced and microarrays consisting of 70-mer
Oligonucleotides were constructed to the majority of the predicted genes for
Psychrobacter arcticum 273-4. P. arcticum, a gamma-Proteobacterium is psychroactive
and displayed marked physiological changes under low temperature versus mesophilic
growth and in the presence of increased osmotic pressure. Only a few genes known to be
low temperature and salt responsive in mesophiles and some psychrotrophs were
differentially expressed in P. arcticum. Experiments reveal 2-fold or greater number of
transcripts at 4°C for transport-associated and metabolic genes. Genes for increased
synthesis and uptake of compatible solute were upregulated in the presence of salt to help
balance the cell’s internal osmolarity. Several genes with unknown functions were also
upregulated under both stresses, which may indicate their importance in stress survival.
Growth in salt amended 1/2 TSB leads to induction of capsule synthesis genes. A capsule

is visible around cells grown at both 22 and 4°C that could allow P. arcticum to adhere to

98

 

soil particles within the permafrost. In addition, total respiratory activity increased in the
presence of salt, due to increased expression of genes encoding Na+ dependent

dehydrogenase and a decarboxylase suggesting that P. arcticum has adapted for energy
generation in the low temperature and low water activity of the permafrost environment.
Introduction

The continually frozen nature of the Siberian permafrost provides a stable
environment selecting for microorganisms able to withstand low temperature and low
water activity. Psychrobacter arcticum 273-4 was isolated from a 20-40 thousand-year-
old Siberian permafrost core (V ishnivetskaya et al., 2000) obtained from the Kolyma-
lndigirka lowland, Siberia by David Gilichinsky (Institute of Soil Cryosciences,
Pushchino, Russia). P. arcticum grows over a range of temperatures from a maximum of
28°C to as low as -10°C (Ponder et al., 2005). It possesses tolerance to high salt
concentrations, similar to other reported Psychobacter isolates (Bowman, 2005),
requiring at least 10mM but no more than 1.3 M NaCl when grown in ‘/2 tryptic soy
broth. In the presence of high salt concentrations the cells become more pleomorphic and
increase their size (1.9 um x 0.8 pm).

Psychrobacter arcticum 273-4 was selected for genome sequencing based on its
excellent survival after exposure to one year frozen at -20°C, rapid grth at sub-zero
temperatures and age of the permafrost sediment from which it was cultured. The G + C
content of P. arcticum 2734 DNA is 42.8 mol% with a genome size of 2.7 Mb, with
2,104 predicted genes. The genome contains a large number of homologs to known
stress responsive genes. Thirty two percent of the predicted open reading frames in the

genome were hypothetical in nature, and functionally uncharacterized. A number of

99

these hypothetical proteins may have roles in stress response. The response to low
temperature of mesophilic bacteria has been well characterized; however the mechanisms
of adaptation by cryo-adapted bacteria remain poorly understood.

The activation of stress-specific and universal stress responses allow microbes to
respond to low temperature and low water activity in the permafrost environment. Ice
formation increases solute concentration by decreasing the amount of free water available
for biological use. This produces an environment with low water activity, similar to
those seen in desiccated and salt-stressed environments. As a result, cell physiology
responds to these stress conditions with the following successful adaptations:
composition changes in membrane fatty acids maintain membrane fluidity; increases in
the surface-to-volume ratio improve nutrient uptake; and production of compatible solute
molecules balances the cell’s osmolarity with its surrounding environment (Poindexter, ;
Russell, 1990; deSantos et al., 1998). Studies of psychrotrophs and psychrophiles have
revealed the compatible solutes glycine, betaine, glutamate, and proline (Russell, 1990).
Studies of select permafrost isolates indicate that glutamate and proline were produced in
high concentrations when the cells were grown at low temperatures (Galinski, 1999).
Changes in membrane surface area and composition must also maintain necessary
interactions with membrane proteins to allow normal fimctions, such as transport, to
continue.

Microbial ability to maintain membrane, cell structure, transport, and enzymatic
function at cold temperatures is necessary for continued cellular activity within a
permafrost environment. Protein chaperones, proteases, and DNA binding proteins have

been described as cold and osmotic stress response mechanisms. One of the most well

100

studied general stress responses, particularly in Escherichia coli and Bacillus subtilis, is
called stringent control, which is activated when the cell experiences limiting amounts of
amino acids and carbon sources. Growth stops temporarily while metabolic adjustments
are made to halt the synthesis of cellular constituents and allow rapid recovery once
conditions again become favorable. The stringent response offers cross-protective
properties to heat, oxidative stress, and osmotic stress (Spector et al., 1993). Several
common proteins (e.g., GroEL, DnaK) act as chaperones when expressed under low
temperatures, increased osmotica and low nutrient conditions in E. coli, Pseudomonas and
permafrost isolate 23-9 (Snyder et al., 1997; Chong et al., 2000). In addition, the cell
experiences identical physiological changes in all three stress conditions. Little
information is available about the cellular response to the interaction of the different
stress conditions.

In order to investigate microbial adaptation to low temperature and water activity,
the genome of a Siberian permafrost isolate was sequenced. Genome sequencing
technology allows the identification of predicted open reading fi‘ames, including those
homologous to previously characterized cold- and salt-responsive genes in the genome.
Microarray technology makes it possible to examine gene expression of an entire genome
under multiple conditions through comparison of the relative abundance of mRNA
between differently treated samples (Eisen et al., 1999). Using these two techniques,
microarrays were constructed which consist of 70-mer Oligonucleotides for 1,993 of the
2,056 predicted genes of P. arcticum.

The genome and transcriptome profiles of an ancient Siberian permafrost

bacterium, Psychrobacter arcticum 273—4, were examined in order to better understand

101

the mechanisms of cold and salt acclimation. This work examines the mechanisms of
long-term survival (i.e., cold and salt acclimation) that allow these tundra organisms to
continue to live in laboratory simulated permafrost conditions.
Materials and Methods
Bacterial strain and genome sequence
Psychrobacter arcticum 273-4 was isolated from 20,000- 40,000 year old Kolyma
Lowland permafrost from the mouth of the Malaya Kon’kovaya River (69°5’N, 158°6’E)
(V ishnivetskaya et al., 2000) and stored at -80°C to provide a uniform culture population.
An 8x coverage genome sequence draft was produced by the Department of Energy's
Joint Genome Institute and automatically annotated by Oak Ridge National Laboratory's
Computational Genomics Group. The annotation used in this work is available at
(JGI/ORNL annotation February 2003). The complete genome sequence and analysis is
available at http://genome.ornl.gov/microbial/psyc/.
Microarray con_struction

Oligonucleotides of 70mers were designed to non-redundant regions of the 2,056
predicted open reading frames (ORFS) in the December, 2002 annotation of
Psychrobacter arcticum 2734 (available at
http://cme.msu.edu/tiedjelab/nasagroup.shtml). Designed oligos were compared against
the genome using BLAST to insure that non-specific hybridization did not occur.
Oligonucleotides were synthesized by Operon (Huntsville, Alabama) for 1993 of the
predicted ORFs (93.9% of the genome). The 70 mer oligos were spotted in triplicate
onto UltraGaps slides (Corning, Corning NY) along with the controls provided in the

SpotArray kit (Stratagene, LaJolla,CA) and UV cross-linked at 120 m].

102

Cell Growth and Preservation

Psychrobacter arcticum was acclimated to low water activity and low temperature
by culturing at least four times in '/2 TSB or 1/2 TSB +5% NaCl at 4 or 22°C,. The
permafrost isolate was grown to an absorbance (OD500) of 0.3 in 1/2 TSB +5% NaCl or '/2
TSB at 4 and 22°C in 325 ml of media in four 500ml Erylenmeyer flasks shaken at 250
rpm. A 25ml culture fi'om a given treatment was removed from each flask and combined.
The remaining 1200mL of culture was spun down for proteomics.

Cells were harvested by decanting into sterile centrifuge bottles containing a one
third volume of RNAlater (chilled for low temperature experiments) (Ambion, Austin,
TX) and centrifuged at 10,000 x g for 10 minutes under the same temperature conditions
as grown. A 13.3ml volume of total culture cells was then re-suspended in 1ml of a 3:1
mix of sterile DEPC treated leBS/RNAlater. The cells were stored at -20°C until used
in the microarray and real-time PCR experiment.

RNA extraction protocol:

Cells were lysed in 400 ug/ml of lysozyme in Tris-EDTA buffer, incubated at
37°C for 30 minutes to degrade cell walls. The RNA was extracted using the RNeasy kit
(Qiagen, Valencia, CA) according to manufacturer directions. The integrity of the RNA
and the absence of DNA were verified by 1.2% agarose gel electrophoresis.

Amplification protocol:

Amino-ally] labeling was performed as adapted from a protocol of The Institute
for Genomic Research (http://www.tiggpgg/tdb/microarrjy/protocolsTIGR.shtml).
Briefly, 2 ug of total RNA was converted to cDNA overnight at 45°C using 6 ug (mol

Concentration) of random primers (Invitrogen, Carlsbad, CA), of 5-(3 -amino-allyl)-dUTP

103

and dTTP (3:2 ratio) (Sigma-Aldrich, St. Louis, MO), and Superscript II reverse
transcriptase (Invitrogen), and subsequently labeled by coupling reactive Cy5 or Cy3
fluorophores (Amersham, Piscataway, NJ) to the amino-allyl groups. Purification after
enzymatic incorporation and chemical coupling was performed by using QiaQuick PCR
purification columns (Qiagen) as described in The Institute for Genomic Research
protocol. The quantity of labeled cDNA and the fluororophore incorporation efficiency
were determined by using UV-visible spectrophotometry. A portion of the single
stranded cDNA was then denatured and analyzed by gel electrophoresis to assure quality
of cDNA. Equal amounts (400pmol each) of Cy 5 and Cy 3 labeled cDNA were
combined and dried in the speed vacuum.

Hvbridizgtion

Prembridization step:

Slides were submerged in a pre-heated solution of 5 x SSC, 0.1% SDS, 0.1% BSA
at 50°C for 45 minutes. The slides were rinsed in Millipore filtered water (Millipore, ),
dipped in isopropanol and dried by low speed centrifugation. Lifterslip (Erie Scientific,
Portsmouth, NH) cover glasses were soaked in isopropanol, washed in 0.1% SDS, rinsed
in filtered water and dried under a stream of nitrogen gas. The slides were then pre-
heated at 48°C within Corning hybridization chambers containing 10 [IL of 3x SSC to
maintain humidity. Pre-hybridization steps occurred no more than 15 minutes before
hybridization.

Hybridization step:

The labeled cDNA was re-suspended in 7111 of nuclease free water and combined with

211L of herring sperm DNA (10 mg/ml) and incubated at 96°C for 3 minutes to denature.

104

Pre-heated Glass Hyb buffer (Clonetech, Palo Alto, CA) was added to the denatured
cDNA and returned to 48°C for 5 minutes. The solution was then applied at the edge of
the Lifterslip nearest the printed portion of array and capillary action evenly distributed
the hybridization solution. The hybridization chambers were then incubated in a shaking
water bath at 48°C for 16 hours. For each treatment, twelve hybridizations fiom six
biological replicates and two technical replicates (dye-swap) were performed.

Post-hybridization step:

After hybridization the slide was dipped into pre-heated low stringency wash buffer (1x
SSC, 0.1% SDS) to remove the Liflerslip. Washing steps were performed for 5 minutes
on a shaking platform, with one wash in low stringency wash buffer (1x SSC, 0.1%
SDS), followed by two washes in medium stringency wash buffer (0.1x SSC, 0.1%SDS),
and finally washed in high stringency wash buffer (0.05x SSC) in 48°C pre-heated
buffers. Nuclease free water was then used to remove excess SSC and the slides were
dried by low speed centrifugation.

Image acquisition and analysis

Slides were scanned with an Axon 4000B scanner and GenePix 5.0 used for spot
finding. Only spots with more than 80% of pixels greater than background plus ZSD in
either cy5 or cy3 channel were used for analysis. Analysis was performed with the

Limma (Linear models for microarray data) library (Smyth, 2004) of R version 1.8.1

(Team, 2003). Data was normalized both within and between arrays using the Lowess

method, and a linear model was fit to each gene. Empirical Bayes statistics were

calculated to determine differential expression and p-values adjusted for false

105

discovery. Those genes that showed a change in gene expression (p<0.05) were
considered significant.
Quititaftive real-time reverse transcription PCR (0 RT-PCR)

Q RT-PCR was performed for ten selected genes (Table 4.1) to verify microarray
results. Primers of 22m in length, specific for each gene were designed using Primer 3
software th_ttp://frodo.wi.mit.edu/cgi-bin/primer3/primer3 www.cgi, (Rozen et a1. ,
2000)) to an amplicon of 100-120 bp. The template RNA was extracted fi'om cells
grown for microarray analysis. The total RNA was converted to cDNA using the same
methods as microarrays, with the exception that only dTI'P was used instead of aa-
dUTP/dTTP mixture. The remaining RNA was then hydrolyzed and the total cDNA
purified using the Qiagen PCR purification kit and quantified by UV-
spectrophotometry. The primer and template concentrations were optimized for each
gene in a 1x SYBR Master mixture (Applied Biosystems) using an ABI 7900 Sequence
Detection System (Applied Biosystems). The reaction specificity was determined for
each gene by constructing a dissociation curve afler each PCR run. A standard curve of
each gene was constructed using purified PCR product. Duplicate runs were performed

for each treatment.

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NADH dehydrogenase assay
The NADH dehydrogenase activity was assayed from fresh cells grown under the
same conditions used for microarrays. Membrane vesicles were prepared as described

previously (Singer, 1974). Briefly, 500 ml of cells were harvested (0.3 OD 600) by

centrifugation at 4°C, washed once with sucrose/TES/Mg2+ (0.25M, 0.1M, 0.02M), re-

suspended in 1 g dry weight per 5ml buffer and disrupted by sonication. The protein was
collected by centrifugation at 25,000 x g for 1 hour, re-suspended in 20mM Tris-HCl pH
7.5, 10mM NaCl, 10% glycerol and stored at -80°C until assayed. The protein was then
quantified using the Bio-Rad protein assay, per manufacturer’s instructions (Bio-Rad,
Hercules, CA). The standard cuvette NADH assay was adapted to microplate volumes as
follows. Standard concentration scale reagents were prepared using varying
concentrations, 0.5, 0.2, 0.125, 0.08, 0.06, 0.05 ml, of ferricyanide (0.01M K3Fe(CN)6,
(Fisher Scientific, Fairlawn, NJ)) 1 ml triethanolarnine buffer (0.12M, pH 7.8 (Sigma—
Aldrich)), and water to give a 3m] final volume. Two hundred microliters of the solution
was then aliquoted into three columns of the 96 well plate, per sample, per standard
concentration. The plate was incubated at 30°C and a baseline determined using a
SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA) at 30°C. Fresh
NADH (3 11M [3- NADH in 0.1% (w/v) KHC03 (Sigrna)) was added to the columns,

ensuring that only six columns are used per reading, and the blank rate calculated from

OD420nm readings every 103 for 1 m. The membrane protein was next added (1.7 pg),
and kinetic measurements recorded every 20 sec for 40 min at OD420nm. An additional

negative control that consisted of membrane protein, ferricyanide and KHCO3, ensured

108

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that reduction of fenicyanide was NADH dependent. The enzymatic rate was determined
by subtracting the blank rate fi'om he initial slope and plotting reciprocal absorbance
change per minute against the reciprocal volume of ferricyanide and extrapolating to
Vmax (ferricyanide). Units per mg total protein were determined using Beer’s Law using
the Vmax. Two biological replicates were performed with three technical replicates per
ferricyanide concentration.

Emission electron microscopy

Negative staining, without any other pretreatment, was used to determine the
intact morphology of cell surface structures. A drop of cell suspension (10ml of culture
concentrated to one ml) was deposited onto formvar and carbon coated grids for 30
seconds. After that, the excess sample was removed with a filter paper. Samples were
then stained with a drop of 1% phosphotungstic acid (pH 7.0) for 15 sec. The excess
stain was wicked away with filter paper.

For ultrastructural studies, cells were suspended and fixed overnight with 2.5 %
glutaraldehyde in 0.1M cacodylate buffer at 4C. After fixation, cells were pelleted and re-
suspended in 2% agarose. Agarose blocks containing the cells were fixed 2 hours at room
temperature. The plugs were washed three times for 15 min in 0.1M cacodylate buffer,
post-fixed in 1% osmium tetroxide in 0.1M cacodylate bufier and washed an additional
three times for 15 min. in 0.1 M cacodylate buffer. The plugs were then dehydrated in a
series of 10 minutes acetone washes (30, 50, 70, 95, 100, 100, 100%). Infiltration was
then performed using 3:1 acetone: Poly/Bed 812, 1:1 acetone: Poly/Bed 812 and 1:3
acetone: Poly/Bed 812 followed by infiltration overnight in 100% Poly/Bed 812. Samples

were embedded in silicone molds and polymerized at 60C for 24 hours. Blocks were

109

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sectioned with a Power Tome XL ultramicrotome (RMC, US). 70 nm sections were
stained with 2% uranyl acetate and lead citrate (Reynolds formulation) and observed
using a JOEL 100 CX transmission electron microsc0pe (Japan) at an accelerating
voltage of 100 kV. Images were taken digitally with a Mega View 11 system. All sample
preparation and TEM was performed by the Center for Advanced Microscopy at
Michigan State University.
Results and Discussion

Transcriptome profiles of Psychrobacter arcticum 273-4 were compared for cells
in mid-exponential phase grown at 22°C, the optimal growth temperature, and 4°C, a
temperature chosen due to the diphasic shift in growth rate seen in Figure 3.1 and the
physiological differences reported in chapter 2. Comparison of cells grown at 4°C and
22°C (both in 1/2 TSB media) showed differential gene expression with changes in
temperature (Figure 4.1). Despite the marked physiological differences in growth
characteristics, fatty acid profiles and carbon source utilization, only 3.3% of the 1993
oligos (hereafter referred to as the transcriptome) were differentially expressed (2-fold or
greater) (Table B-5). of these differentially expressed open reading frames (orfs), 29 orfs
had more fluorescence (transcripts) when cells were grown at 4°C compared to those
cells grown at 22°C. Growth at 22°C resulted in more transcripts for 38 orfs. Several of
these differentially expressed orfs (8 at 22°C and 10 at 4°C) have no known function in
P. arcticum, or in other bacteria in which they have been identified. The remaining
differentially expressed orfs were chiefly involved in metabolism, transcription and
translation. A smaller portion of the transcriptome of exponential phase cells of P.

arcticum are differentially expressed at low temperatures compared to other

110

microorganisms growing at low temperatures that were not isolated from low temperature
environments (Table 4.2).

The comparison of P. arcticum growth at 4°C and 22°C (both in 1/2 TSB + 5%
NaCl) resulted in fewer differentially expressed orfs (2.9 % of the transcriptome) than the
temperature comparison in V2 TSB. The presence of salt in the medium resulted in 20
orfs having more transcripts when grown at 4°C and 39 orfs having more transcripts
when grown at 22°C (Table B-4). Several of the orfs have no known function but the
others are involved in energy generation, signal transduction, transport and cell envelope
biosynthesis.

Comparison of cells grown in V2 TSB and 1/2 TSB + 5% NaCl (both at 22°C),
revealed 47 orfs had more transcripts when grown in salt-amended media and 20 orfs had
more transcripts when grown in 1/2 TSB (Table B-2). Emotions of 21 of the salt
responsive orfs are unknown, while only one hypothetical protein is differentially
expressed at 22°C in ‘/2 TSB. This indicates that P. arcticum genes expressed at optimal
growth characteristics are well-defined, while those involved in salt response may be
unique to P. arcticum and other halo-adapted bacteria. There were 67 orfs with
differentially expressed when cells are grown at 22°C in 1/2 TSB +5% NaCl when
compared to grth in 1/2 TSB or at 4°C. Nine of these genes are increased with growth
at 22°C in salt amended media suggesting their importance in growth in 5% NaCl at
22°C. Cells that were grown at 4°C with NaCl amendment showed a similarly high
number of transcripts in comparison to those in ‘/2 TSB: 30 orfs had more transcripts
when grown in salt-amended medium, while 28 orfs produced more transcripts when

grown in l/2 TSB (Table B-3). Growth in salt-amended media at 4°C resulted in increased

111

 

 

 

Figure
LCast '

Cones]
111mb,-
gr 0111]
101d c]

expression for 9 proteins of unknown fimction. Three of these hypothetical proteins

(or2464, or2867, or825) have different expression patterns with temperature (Tables B-7,

B-8) suggesting they are important to salt response only at a given temperature.

 

 
  
 
     

 

 

     

         

47 more 22°C NaCl
20 fewer
5 more Table B-2 9
f more
2.23226 22 NaCl vs. 3 fewer
22 TSB Table B-7

 

 

  

 

 

 

22 NaCl vs. 4
NaCl

 

 

  
 

 

  
   
     
     
  
 

22 TSB vs. 4 TSB

 

 

   
 
  
    
  
 

 

 

 

39 more 4 NaCl
20 fewer
Table B-4

 

 

38 more 4TSB
29 fewer
Table B-5

 
  
  

 
  
      
 
  

9 more
10 fewer
Table B-8

4 NaCl vs.
4 TSB

  
 
 

4 more
0 fewer
Table B-9

 

 

 

   

30 more 4 NaCl
28 fewer
Table B-2

 
   
 

Figure 4.1. Venn Diagram Depicting The Number of Orfs Whose Expression Changed at
Least Two-F old As A Function of Temperature Or Salt. Overlapping areas of the circles
correspond to orfs which are differentially expressed in the shared growth condition. The
number of differentially expressed orfs is in reference to the number of transcripts when
grown under the first condition listed. Annotation of the differentially expressed orfs and

fold changes are listed in appendix tables as stated within the figure.

112

Table 4.2. Comparison of the number of transcriptome changes of exponential cells of P.
arcticum, synechocystis sp. PCC 6803, Saccharomyces cerevisiae and Shewenalla
oenedensis when grown at low temperatures or increased salinity.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Organism Condition ercent of Number of [Reference
enome ORFS
ifferentially
xpressed
ow temperature growth
P. arcticum 22°C vs. 4°C 3.3 1993
Synechocystis sp. 37°C vs. 22°C 2.2“ 3079 (Suzuki et al., 2001)
PCC 6803
Saccharomyces 30°C vs. 10°C 25 5829 (Sahara et al., 2002)
cerevisiae
25°C vs. 4°C 21 5829 (Homma et al., 2003)
ncreased osmotica growth
P. arcticum 0.9M NaCl .9 1993
Shewenella 0.5M NaCl 24 4200 (Liu et al., 2005)
oneidensis MR-I
Synechocystis sp. 0.5 M sorbitol 7 3079 (Mikami et al., 2002)
PCC 6803
4% NaCl 1.3 3079 (Marin et al. , 2004)

 

 

 

 

 

 

 

*- This study reported only those genes under control of the cold sensor Hik33, this may
not reflect the total number of low temperature responsive genes

Activity of NADH dehydrogenase in different salinitiega_n_d temperatures

Respiratory activities of the Psychrobacter arcticum 273-4 total protein
extractions were affected by the presence of salt and low temperature. The rate of
respiration in the cells grown at 22°C in V2 TSB + 5% NaCl was 3.1 and 2.2 times higher
than cells grown at 22°C in V2 TSB and at 4°C in l/2 TSB + 5% NaCl, respectively. The
Specific activity level of NADH dehydrogenase was significantly higher in the presence

of salt amendment and lower at 4°C (Table 4.3).

113

no

Table 4.3: The Activity of NADH Dehydrogenase Activity Obtained From
Psychrobacter 2734 At Different Temperatures And Salinities.

 

Medium Temperature Respiratory rate Specific Vmax
(°C) (Units) activity (M/min)

(units/mg
protein)

1/2 TSB + 5% NaCl 22 53 212 0.038

1/2 TSB + 5% NaCl 4 6 24 0.004

1/2 TSB 22 17 68 0.012

'/2 TSB 4 1 4 0.001

P. arctium encodes both Na+ and l-I+ pumps (or2161-or2167) and multiple NaVI-I+
antiporters who’s fimction would likely ensure the maintenance of both Na+ and H+
gradients on their cytoplasmic membrane. The presence of genes encoding primary Na+
pumps (or1686-or1691) indicates that these bacteria rely on the Na+ cycle for at least part
of their metabolic energy. The sodium motive force supplements the proton motive force

as an additional source of energy in these bacteria. The P. arcticum genome encodes
multiple NaJ’II-I+ antiporters (or1522, 2172), Na‘L/I-I+ symporters (or1600), which

theoretically allows the generation of an I-I+ gradient at the expense of an Na+ gradient,
and vice versa. This interchangeability of proton motive force and sodium motive force
is an important feature of the bioenergetics of nearly all bacteria whose genomes have
been sequenced. The availability of complete microbial genome sequences allowed the
identification of homologous genes encoding the sodium dependent pumps (N QR) in a
wide variety of bacteria by Hase et al., from E. coli to Chlamydia trachomatis, and even
in the genome of the aphid symbiont Buchnera sp. strain APS, the second smallest of all

known bacterial genomes (Shigenobu et al., 2000) (Hase et al., 2001).

The NQR complex located in the inner membrane is composed of six subunits

quA, quB, quC,quD, quE, and quF, which involve the co-factor FAD. The genes

114

CllC

n11

encoding NADH dehydrogenase activity (nqr- A, B, C, D, E, and F) in P. arcticum were
within an operon (or1686-or1691 are in the same gene order as was previously
described). Only three genes, nqu, D and F, were differentially expressed with a p-
value of 0.05 in response to salt (Figure 4.2). The NQR complex reduces ubiquinone-l to
ubiquinol by two successive reactions, which are coupled with the transport of Na+ ions
from the cytoplasm to the periplasm. The first step is catalyzed by nqu (or1691), which
accepts electrons from NADH and reduces ubiquinone-l to ubisemiquinone by a one
electron transfer pathway (Turk et al., 2004). More nqu transcripts were present in cells
grown in 1/2 TSB + 5% NaCl at 22°C compared to those grown in V2 TSB, which concurs
with increased dehydrogenase activity in the presence of 5% NaCl (Figure 4.2).

Subunits quB and quC each contain one FMN that is covalently linked to a threonine
residue (Hayashi et al., 2001) and may act as an electron acceptor for the Fe-S center.
The NQR subunit B and C showed different expression profiles, with only B showing
differential expression (p< 0.05) with more transcripts at 4°C compared to 22°C when
grown in V2 TSB + 5% NaCl. The gene nqu was also expressed 3-fold more at 4°C in
‘/2 TSB + 5% NaCl than in 22°C, and 2-fold more at 4°C and grown in 1/2 TSB. The
function of nqu has not been ascertained but it is known to be part of the complex. The

increased expression of NOR complex genes and the increased reduction of ferricyandide
in the presence of NADH and Na+ suggests a correlation between expression and

respiration. An increase in sodium motive force, generated through the successive
reduction of electrons across the membrane by the NADH dehydrogenase could be used

to accumulate energy and ultimately lead to increase in ATP.

115

Transcriptome analysis of Na’ NADH ubiquinone oxidoreductase operon

4

 

 

1689
I 1691

 

 

 

 

 

 

 

 

 

 

 

1690

 

 

 

 

 

 

[2122 m4 5% NaCI vs. 4TSB
4 ..._ 5%NaCI
vs. .22TSB

922 5% I 22TSB vs. 4 rse

NaCl vs. 4
1-

 

----- lllmj-l III-Ill
---- ----. III-II...-
----- III-II‘IIIIIIIII

 

 

 

 

 

 

 

 

 

 

Figure 4.2: Differential Expression of The Na+ Dependent NADH Dehydrogenase
Operon (or1686-or1691) In P. arcticum as determined through microarray analysis. Bars
depict the fold change when two conditions were compared. Positive fold changes reflect
more transcripts when grown at the first condition listed. Negative fold changes indicate
more transcripts when cells were grown in the second condition listed.

In addition to the primary Na+ pump, NQR, other Na+ pumps such as,
dicarboxylate decarboxylases are also present in the P. arcticum genome. The energy
generated by the decarboxylation reaction (AG°’= ca. -30 kJ/mol) could fuel the synthesis
of ATP molecules if an electrochemical ion gradient of [,tH+ or [J.Na+ is available to
accumulate energy from three decarboxylations (Buckel, 2001). In the presence of NaCl
at 4°C a 4.4-fold increase in transcripts of or1520, a glutamate decarboxylase occurred
while at 22°C in NaCl a 3-fold increase in malonyl-CoA decarboxylation (or339)

transcripts occurred. This increase in decarboxylation activity may act to conserve

energy and aid in the increase in cellular respiration seen in Table 4.3. There have been

116

reports that the cytochrome d terminal oxidase of E. coli is not an H+ pump but a Na+
pump (Bogachev et al., 1995) and may also function similarly in P. arcticum. It is
possible that the cytochrome d-type oxidases do not pump either Na+ or H+, and charge
the membrane solely by consuming H+ ions from the cytoplasm to produce H20. The
presence of these pumps suggests that, under conditions of low proton motive force and
high sodium motive force, a normally H+-transporting FoFl-type ATPase may fimction as
a Na+-transporting ATP synthetase, since the cation specificity of this enzyme was not
absolute. Na+-dependent ATP synthesis, requires a relatively large Na+ gradient and most
likely occurs only in a few bacteria, including V. alginolyticus and E. coli (Avetisyan et
al., 1993). Hence, the ability to generate anNa+ gradient through any primary Na+ pump
would be an important trait in helping the organism to synthesize ATP and ultimately

survive under certain unfavorable conditions.

Nutrient transport via sodium motive energetics is likely an adaptation to marine
and some permafrost environments; as such environments have Na+ concentrations that

are larger than H+ concentrations by up to a factor 106. The ability to generate sodium
motive force may be an adaptation to the low temperature of the permafrost since a
decrease in respiration is seen with growth at low temperature. While this decrease is
respiration is not alleviated with addition of salt total respiration does increase that may

be used to generate ATP. Within the permafrost of the Kolyma lowland, which has a

neutral pH approximately 10'7 M H30+ would be expected ( 10 '4 cmol kg '1) to be
present. In contrast Na+ is more abundant in Kolyma lowland permafi'ost (0.2 — 0.8 cmol

kg '1) (Alekseev et al., 2003). Since sodium is more abundant the level of sodium motive

ll7

force is unlikely to change as rapidly as proton motive force levels (due to more rapid
exhaustion), making sodium motive force a much more reliable source of energy. P.
arcticum encodes several Na+-dependent symporters for alanine (or 744, 745 and 1153),
proline (or 2760), and dicarboxcylic intermediates (or 988). The accumulation of proline
and glutamate occur in several bacteria when incubated in NaCl, with the function of

stabilizing macromolecular structures.

Compatible solute tra_n_scripts and salt respon_se of Psychrobacter arcticum

Microorganisms adapt to low osmotica via mechanisms of active extrusion
of solutes and the accumulation of compatible solutes, molecules that control osmotic
balance and are compatible with the organism’s metabolism (Roebler et al., 2001).
Compatible solutes act to stabilize macromolecular structures such as proteins by
preferential exclusion from the hydration shell (Rudolph et al. , 1986) and can assist in the -
refolding of polypeptides by chaperone proteins (Diamant et al., 2001). It is believed that
compatible solutes such as proline intercalate between membrane phospholipids, helping
to stabilize membranes by maintaining hydrogen bonding (Rudolph et al., 1986).

Studies of psychrotrophs and psychrophiles have revealed the presence of
compatible solutes such as glycine betaine, glutamate and proline (Russell, 1990). Proline
has been shown to depress the freezing point of water in some plants when present in
high concentrations (Aspinall et al., 1981). In addition, Gould and Measures (1975) have
shown a correlation between the concentration of proline and growth at low water
activities, such as those seen at freezing temperatures (Measures, 1975). In cyanobacteria
the accumulation of glycine betaine and glutamate betaine correlate with tolerance to salt

Concentrations as high as 2.7M NaCl (Reed et al., 1986). Sucrose and ectoine act as

118

indirect osmoprotectants in Sinorhizobium due not to bioaccumulation of these molecules
but their catabolic end product, glutamate which has high cystolic levels in salt stress
(Gouffi er al. , 1999). Osmoprotective disaccharide carbon sources, that when catabolized
yield glucose molecules were preferentially up-taken in salt stress compared to non-
osmoprotective carbohydrates. Accumulation of glycine betaine to a high internal
concentration by Escherichia coli cells in high osmolarity media restores growth rates by
suppressing the stringent response that decreases the initiation frequency of DNA
replication. The cell lengthening, likely due to disruption of cell turgor pressure is
reversed in glycine betaine (Meury, 1988).

In general, halotolerant bacteria prefer to uptake compatible solute molecules to
de novo biosynthesis for energetic reasons. ABC-transporter proteins mediate uptake of
most compatible solutes. The only well—defined osmotic responsive ABC transporter is
OpuA, responsible for glycine betaine uptake in Lactococcus lactis. The uptake of
glycine betaine by OpuA, is dependent on the interactions of the lipid and transporter
interactions (van der Heide et al., 2000). As the outside osmolarity increases, the
proteoliposomes react by decreasing their surface to volume ratio, therefore reducing the
electrostatic interactions between the lipid headgroups and the protein, resulting in
increased glycine betaine uptake (van der Heide et al., 2001). Similar systems are
believed to exist in the ion-linked transporters ProP and BetP, involved in proline and
betaine uptake respectively (van der Heide et al., 2001).

Psychrobacter arcticum genome analysis reveals homologs to genes associated
With compatible solute production in known compatible solute accumulating bacteria.

The pathways for choline oxidation to betaine and glycine production are both present in

119

Psychrobacter. Psychrobacter, like the majority of bacteria, also synthesizes proline
from the citrate cycle to produce omithine which becomes proline through the action of
omthine carbamoyl transferase, though the molar concentrations of proline for
compatible solute use is unlikely to be de novo synthesized due to the energetic
requirements. Psychrobacter arcticum does not have homologs for the production of
ectoine, trehalose or dimethylsulfonioprOpionate, which are known to accumulate with
salt stress in halophiles and with low temperature in some Gram negative bacteria.
Transcriptome analysis of Psychrobacter arcticum under conditions of V2 TSB or
5% NaCl growth at 4 and 22°C indicate that compatible solutes most likely accumulate
under low temperature and 5% NaCl growth conditions. Transport associated genes are
upregulated for proline (or608) and the glycine betaine precursor choline, increasing the
possibility of accumulation intracellulary. The transcripts for a choline transporter
(or2296), choline dehydrogenase (or2295) and betaine aldehyde dehydrogenase (or2294)
increase at least 2-fold as shown in Table 4.9. An increase in proline synthesis is unlikely
as, the transcripts for proline synthesis related genes were not differentially expressed.
The increase in a proline transport is likely to be responsible for proline detected by
NMR analysis (Smith, unpublished observation). NMR analysis indicated that proline
and glutamate accumulate significantly while glycine betaine is present in smaller
amounts when grown in 1A: TSB + 5% NaCl at 4°C. Growth at 4°C without salt
amendment or at 22°C in salt showed only minor accumulation of proline (G. Smith,
unpublished observation). The different expression patterns of transport associated genes
supports physiological evidence that differential carbon sources were utilized at the two

temperatures as catabolism genes for those carbon sources were not differentially

120

expressed. Differences in substrate binding capability have been detected that would
allow increased binding of L-arabinose and D-galactose with conversion of a proline to a
glycine (V errnersch et al., 1990).
Fittypcid and phospholipid synthesis
Low temperature and high osmolarity are known to induce changes in membrane
composition that maintain membrane fluidity (Russell, 1998),(McGrath et al., 1994). A
decrease in temperature resulted in a decrease in the saturation of fatty acids and acyl
chain lengths in Psychrobacter arcticum sp. 273-4 (Ponder et al., 2005). Together these
changes function to lower the gel-liquid crystalline transition temperature, i.e.
homeoviscous adaptation (Morris etal., 1987). The decrease in chain length at 4°C in P.
arcticum is likely due to the decreased transcripts of long chain fatty acid acyl carrier
protein ligase (or28l6), which have the role of increasing chain length. Unsaturated
membrane fatty acids were present in these psychroactive Siberian permafrost isolates
grown at mesophilic temperatures and expression of beta-ketoacyl-ACP synthase I FabB
(or528), FabH (or2507) and delta-9 fatty acid desaturase (or804) were constitutive and
did not increase with 4°C incubation or in the presence of salt. However the proteins
may be more stable at 4°C than 22°C so fewer transcripts would be required for increased
synthesis. This difference in unsaturation may account for the Psychrobacter’s narrower
range of growth temperatures (Y akimov et al., 2003). Control of the membrane
unsaturation may also be accomplished through controlling fatty acid degradation.
Transcripts for acyl-coA dehydrogenase (fadE, or 1519) which introduces a double bond,
and fad A (or1666) and fadB (or 1667), which reduce chain length were present in larger

numbers when grown at 4°C compared to 22°C in V2 TSB + 5% NaCl. P. arcticum has

121

two different acyl-CoA thiolases (fadA), the other or2682 produces fewer copies at 4°C
in V2 TSB + 5% NaCl compared to V2 TSB, which may prevent loss of two carbons and
account for the larger number of C18:1 fatty acids (72% in 5% NaCl, 42% in V2 TSB)
compared to Cl6:1 (1.3% in 5% NaCl and 20% in V2 TSB) when grown in V2 TSB + 5%
NaCl (Ponder et al., 2005).

Phospholipid biosynthesis genes were differentially expressed (more transcripts)
when cells were grown in V2 TSB + 5% NaCl compared to V2 TSB. This includes
increased conversion by @sA from of dihydroxyacetone phosphate to sn-glycerol-3-
phosphate. Two fatty acid moieties were next added by sn-glycerol-3- phosphate
acyltransferase (plsB, or1996), leading to formation of phosphatidic acid which is then
converted to cytidine diphosphoglyceride phosphatidate cytidyltrasnsferase @lsK, or
931). Cytidine diphosphoglyceride can then be converted to phosphatidylserine and
ultimately phosphatidylethanolamine or phosphatidylglycerol and ultimately cardiolipin.
The transcripts for conversion to cardiolipin from phosphatidylglycerol decrease in the
presence of salt at both 22 and 4°C, suggesting cardiolipin amounts were lower.
Transcripts encoding the conversion of phosphatidlyethanolamine increase in 5% NaCl at
22°C compared to V2 TSB, suggesting there may be less phosphatidylserine. This
concurs with physiological evidence that phosphatidylglycerol and
phosphatidylethanolamine but not phosphatidlyserine or cardiolipin were detected at 4°C

and 24°C (Ponder et al., 2005).

Wk composition associated transcripts

122

Psychrobacter arcticum 273-4 has adapted to the low temperature and high
osmotica environment of the permafrost by accumulating carbohydrates that may
maintain a fluid water layer in the presence of high salt and low temperature.
Carbohydrates with more hydroxyl groups will accumulate more unfrozen water per mole
of carbohydrate. Furthermore, hydration is governed by the compatibility of the pre-
existing three-dimensional hydrogen-bond network of water, to the relative distance of
next-nearest-neighbor oxygens of OH groups within a given carbohydrate molecule.
More water remains unfrozen in the case of the poorer fitness of carbohydrates into the
three-dimensional water structure (Galema et al., 1990). Arabinose, ribose, and xylose
that have lower measured values of unfrozen water (F uruki, 2000) were minor
components of P. arcticum polysaccharides. While the major components were glucose,
mannose and galactose that were measured to increase the amount of unfi'ozen water
associated with a carbohydrate. Glucose composition increases while rhamnose
decreases with increased salt and lower temperature. Mannose accumulation was more
significant in low temperature stress than in the presence of salt. When high osmotica
and low temperature were combined the percent mannose increased to 43% of the cells
alditol acetates. Galactose, measured to accumulate the most unfrozen water per mole of
carbohydrate by Furuki at -70°C, decreased in composition with low temperature and
salinity (F uruki, 2002).

Transcriptome analysis of Psychrobacter arcticum 273-4 reveals that the shift in
alditol acetate composition is due to the increased expression of dTDP-4-
dehydrorhamnose epimerase (or2613) in NaCl conditions (5x) and less strongly at 4°C

(2x). This increase in enzyme, which catalyzes the interconversion of dTDP-4-dehydro-

123

6-deoxy-D-glucose to dTDP-4-dehydro-6-deoxy-L-mannose,_would lead to the large
increase in mannose at 4°C in 5% NaCl (Table 4.4). The decrease in rhamnose is likely
due to the increased activity of dTDP-L-rhamnose synthetase (or261 1) which
interconverts NADPH + dTDP-4-dehydro-6-deoxy-L-mannose <=> NADP+ + dTDP-a-
L-rhamnose.

In the presence of increased osmotica, P. arcticum.273-4 is surrounded by a
capsule. The capsule also consists of sugars that will tightly bind unfrozen water with a
predominance of 4-linked glucopyranosyl residues when incubated at Sosm at 22 or 4°C.
In 5% NaCl the majority of residues were terminal glucopyranosyl residues,follwed by
terminal mannopyranosyl residue, 4-linked glucopyranosyl residues and 4 or 6 linked
mannospyranosyl residues. The general capsule synthesis genes, or495 and or708 were
also expressed in the presence of 5% NaCl but not in cells grown in V2 TSB.
Transmission electron microscopy pictures reveal an electron dense capsule surrounding
the cells grown at either 4 or 22°C in V2 TSB +5% NaCl (Figure 4.3 a and b). Linkage
group analysis of the capsule layers revealed a majority of 4- linked glucopyranosyl;
residues and less than 5% each of 6- linked mannosylpyranosyl and 3-linked
glucopyransosyl residues.

Verification of microarray fold change with the quantitative real time PCR

 

RNA extracted for microarray analysis was used to verify the magnitude of gene
Changes using quantitative real- time PCR and results were shown in Figure 4.4. Nine
genes were selected based on their transcriptome profile, including the housekeeping
gene spoU as a control for no differential expression. In general, microarray analysis

under estimated fold change (Figure 4.4: lines are qt-rtPCR results while bars are

124

microarray results) Growth at 22°C compared to 4°C lead to an increase in number of
transcripts of or 992, a gamma-glutamate-cysteine ligase involved in the biosynthesis of
glutathione (GSH) that protects against oxidative damage and participates in biosynthetic
or detoxification reactions. The increased growth rate and respiration of P. arcticum at

22°C would generate more hydroxyl radicals, that could generate oxidative damage.

are
i" ‘ ~‘L. h...
— .2

. J.,.
4“

' -

 

Figure 4.3. Transmission Electron Microscopy Pictures of P. arcticum Revealing
Presence of Thin Capsules When Grown In The Presence of 5% NaCl At 4°C (A) 0r

22°C (B) And No Capsules Surrounding Cells Grown At 4 (C) Or 22 °C (D) In V2 TSB.

125

 

o
1:
0 O .0 U .D
5 8 8 .o "2 «*2 b. b. —.
:- m g co —- so as In at
1: .— m m — N N
0
U)
o
a a .D .D a
g "1 ". .o a} ‘0. "'1 ". "1
E m N N — <1- N v m
v N N F" '— N '— v—4
0
U)
8 o o o
3 l‘. "l o O} .o N. O} .o
-' xo b o h m 05 b co
0 In 1~ m N N m N N
0
(h
o
H
O
.52 an .o .o as 0 V
a E D O. «a O: b. 9: .4
0 2 co co so so so ——
0
m
8 at to as .o to .o a
= «a D h. 09 <=>. to. —. °°.
l-I-r N Z c m in o m N
O
U)
E
' fl
"3 a ta at so ed at
E g D —. 0. or "a c: —:
Z v N N N — —
o. S) g)
E A U S) W U S) V‘. U S)
0 g) o N N O V N 0 V
i- v N I <- N I <1- N
2%
5 2.
° 6\
V) W
g + __ +
m U on CD
go (I: «I m m
m 1— Z 1— 1—

 

 

 

Table 4.4: Alditiol Acetate Differences of P. arcticum polysaccharides When Grown At

Different Temperatures And Salinities. " Standard deviation < 2%.b 2% > Standard
deviation < 5%.6 5% > Standard deviation < 10%.

126

 

lent

abur

inc;

inn

in:

In general both the hypothetical protein or 2389 and cation transporter or 24 had
fewer transcripts in 4°C except in salt amended media where the transcripts were more

abundant than those grown at 22°C due to the decrease when salt is added. The decrease
in cation transport at 22°C may serve to limit Na+ concentrations within the cell. The
increase in transcripts at 4°C in V2 TSB + 5% NaCl may serve to counteract the decrease
in respiration by helping to firel the Na+ motive force.

The cumulative effect of growth at 4°C and in the presence of 5% NaCl leads to a
4-fold increase in or 2380 which is not significantly expressed at any single condition.
Enoyl-CoA hydratase (or 23 80) catalyzes the hydration of 2-trans-enoyl-CoA into 3-
hydroxyacyl-CoA and shifts the 3- double bond of the intermediates of unsaturated fatty
acid oxidation to the 2-trans position. The conversion of naturally occurring cis-
unsaturated fatty acids to trans-unsaturated fatty acids results in better chain packing of
the phospholipid acyl chains, which raises the phase transition temperature and decreases
the permeability of the lipid bi-layer which would be important to limit salt
permeability(Cronan, 2002). Pseudomonad and Vibrio membranes shifted to high
temperatures, may become too fluid for optimal growth and the conversion of cUFA to
tUFA provides a means to rapidly decrease fluidity and permit better grth (Okuyama
et al., 1991, Heipieper, 1996 #682; Heipieper et al., 1996). The expression at 22°C of
this gene in P. arcticum may be important for decreasing fluidity of the unsaturated fatty
acids present even at optimal growth temperature. Growth in salt leads to increase in
transcripts of the permease component of an ABC-type molybdate transport system, (or

1013) especially at 4°C. Sodium motive force has been shown to fire] ABC transporters

127

in)

real

in"

W21

sug

 

in Vibrio (Hase et al. , 2001), which may explain the increase in transport in the presence
of NaCl.

Isocitrate lyase (or 2879) and cspA (or 2119) were included in the quantitative
real time PCR analysis because each have been shown to be differentially expressed in
low temperatures. Analysis of the P. arcticum genome revealed no significant expression
of the isocitrate lyase at 4°C or in the presence of 5% NaCl. The cold shock protein cspA
was induced with presence of 5% NaCl at 4°C, though not in1/2 TSB alone. This
suggests RNA-chaperone activity of cspA is not necessary for 4°C growth in acclimated

P. arcticum cells.

 

 

   

 
   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4C '6'3’3’3’39393‘; —t 220
1,2 m— 1,2
TSB TSB U or 992
—r
I 012389
40 112: : 220 ,
TSB], "2.012380
5%
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Fold change (log odds econ)

Figure 4.4: Comparison of microarray fold change and difference in copy number by
quantitative real time PCR. The bars correspond to microarray data, while the lines show
the range of quantitative copy number change.

128

 

9991

time
lulu.

Spec

difl

Comparison of transcriptome and proteome profiles

The cells for the transcriptome and proteome analysis were prepared at the same
time and frozen until proteins were extracted and analyzed by Suping Zheng and David
Lubman of University of Michigan. Proteome analysis revealed 44 proteins with distinct
spectra that were differentially expressed. Twenty of these proteins were not
differentially expressed above 2-fold in the transcriptome under any of the test conditions
(Table 4.7.). Ten proteins and transcripts had the same pattern of expression (Table 4.5),
while fourteen genes had different expression patterns in at least one condition examined
(Table 4.6). Ten hypothetical or conserved hypothetical proteins were detected,
indicating that these predicted genes were in fact expressed and likely important for cell
survival. Seven of the differentially expressed proteins were ribosomal proteins, however
only two or 1201 and 2098 had significantly different transcriptome profiles (Table 4.4).
Incubation at 22°C in salt led to an increase in or 1201 (L10) transcripts while or 2098
(L15) decreased. Both of the proteins were within the SOS ribosomal subunit, which is
assembled at low temperatures in E. coli through the association with csdA (Charollais et

al., 2004).

Conclusions

Psychrobacter arcticum. 273-4 displays a multi-faceted response to low
temperature and increased osmotica. The expression of additional energy generation,
compatible solute production and capsule synthesis genes only in the presence of
increased salinity suggest that these are adaptations to the low water activity of the
Siberian permafrost. P. arcticum 273-4 grows at low, even sub-zero temperatures- this

indicates that 4°C is not a severe stress for P. arcticum and therefore so called cold shock

129

proteins are not likely to be differentially expressed. Many other characterized
temperature stress proteins, such as heat shock proteins are expressed in the acclimation
phase after a shift in temperature (Inouye et al., 2004) and not expected to be seen in cells
which are in exponential growth phase. P. arcticum has not only adapted to increased
salt concentration but also to sub-zero temperatures, indicating that it will be a useful

organism to study adaptation strategies to astral conditions.

Table 4.5. List of P. arcticum Proteins And Genes That Agree In Their Expression
Pattern And Were Above 2-Fold Different In The Transcriptome Profile.

on Transcriptome Quantitation
number Protein Quantitation Comparison Comparison (fold change)

Jun-03 4/22 4SI228 228/22 4SI4 4SI22 4/22 4Sl22S 228/22 4814

148 3.1 1.1 2.2 0.8 2.4 -3.2 2.2 -0.2 -1.6
381 1.8 0.8 2.6 1.2 2.0 2.3 -1.2 -0.8 2.6
393 313.0 0.7 116.7 0.3 82.8 0.7 0.2 5.1 -3.2
518 2.3 0.6 4.4 1.2 2.7 2.1 0.3 -0.5 -0.7
1053 1.2 0.7 3.0 1.8 2.1 2.1 0.2 -1.9 2.2
1201 1.8 0.5 3.9 1.0 1.9 -1.5 1.9 2.1 -1.0
1407 0.6 1.0 1.8 3.2 1.9 —3.5 0.3 3.7 -0.3
1576 1.5 1.1 8.9 6.6 9.7 0.0 1.3 -1.9 3.2
1766 0.5 0.6 0.3 0.4 0.2 -2.5 1.0 -O.4 -0.6
1906 1.4 0.4 1.5 0.4 0.6 0.5 1.6 0.6. -2.1
2098 0.4 1.3 0.2 0.7 0.3 -1.7 0.7 -3.8 -0.4

 

 

130

so called cold shock proteins are not likely to be differentially expressed. Many other

characterized temperature stress proteins, such as heat shock proteins are expressed in

the acclimation phase after a shift in temperature (Inouye et al. , 2004) and not

expected to be seen in cells which are in exponential growth phase. P. arcticum has

not only adapted to increased salt concentration but also to sub-zero temperatures,

indicating that it will be a useful organism to study adaptation strategies to astral

conditions.

Table 4.5. List of P. arcticum proteins and genes that agree in their expression

pattern and were above 2-fold different in the transcriptome profile.
Transcriptome Quantitation

Comparison (fold change)
4I22 481228 228I22 48M

orf
number

Protein Quantitation Comparison
Jun-03 4I22 48l228 228/22 48I4 48122

 

148
381
393
518
1053
1201
1407
1576
1766
1906
2098

3.1
1.8
313.0
2.3
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1.8
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0.4

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6.6
0.4
0.4
0.7

131

2.4
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3.2
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-0.4

Table 4.6: List of P. arcticum proteins and genes that disagree in their expression
pattern and were above 2-fold different in the transcriptome profile.

 

orf Transcriptome Quantitation
number Protein Quantitation Comparison Comparison (log odd score)
Jun-03 4I22 48I228 228I22 48I4 48I22 4/22 4SI228 228I22 48I4
166 2.4 1.3 1.5 0.8 1.9 1.4 0.8 -O.8 3.4
237 3.1 1.1 2.2 0.8 2.4 -3.2 2.2 -0.2 -1.6
393 313.0 0.7 116.7 0.3 82.8 0.7 0.2 5.1 -3.2
414 59.6 1.5 49.1 1.2 72.6 -3.9 1.8 -0.9 -1.9
645 3.0 0.3 6.7 0.8 2.3 1.5 0.9 1.9 3.7
705 1.7 0.4 4.0 1.0 1.7 -8.2 3.5 1.6 -2.1
850 0.1 0.7 0.1 0.8 0.1 0.8 2.0 1.0 1.3
1053 1.2 0.7 3.0 1.8 2.1 2.1 0.2 -1.9 2.2
1164 1.5 0.7 1.0 0.5 0.7 -1.4 1.5 -2.0 -1.2
1407 0.6 1.0 1.8 3.2 1.9 -3.5 0.3 3.7 -0.3
1762 3.4 0.7 0.6 0.1 0.4 -2.8 -3.0 2.2 -1.5
1793 0.7 0.6 0.8 0.7 0.5 -1.5 0.2 -1.7 2.4
1964 0.9 0.7 0.5 0.4 0.4 0.1 0.2 2.0 -1.3
2268 1.9 1.2 0.8 0.5 0.9 -1.7 -0.4 -1.6 2.3
2392 2.5 1.0 2.0 0.8 2.0 -2.5 -1.6 1.5 -1.5

132

 

Table 4.7: List of differentially expressed proteins that were not 2-fold differentially
expressed in transcriptome analysis

 

orf Transcriptome Quantitation
number Protein Quantitation Comparison Comparison (log odd score)
Jun-03 4122 481228 228122 4814 48122 4122 481228 228122 4814
3 1.0 0.7 5.1 3.7 3.7 1.0 1.3 -1.5 1.6
536 1.7 0.6 3.1 1.0 1.8 0.4 0.2 0.7 0.6
539 2.5 0.5 5.5 1.2 2.9 1.7 0.4 0.5 1.1
767 4.5 1.5 6.3 2.2 9.7 0.0 .03 1.2 0.8
831 0.9 0.5 4.9 2.4 2.2 -1.6 -1.7 1.2 1.1
676 2.1 0.6 7.5 2.1 4.3 No oligo
902 3.0 0.7 6.2 1.9 5.6 0.6 0.2 0.3 0.1
935 4.9 1.4 4.9 1.4 6.7 -1.8 0.8 0.6 -1.7
1450 0.0 0.9 0.0 0.8 0.01 -O.8 1.5 -1.3 0.0
1640 0.7 0.7 0.5 0.5 0.4 -0.2 0.3 -1.3 1.1
1759 0.2 3.2 0.1 1.4 0.3 0.2 -0.9 0.4 0.7
2062 0.4 0.9 0.4 1.0 0.3 No oligo
2137 2.6 0.4 6.2 1.0 2.6 -O.6 0.7 -O.4 0.7
2395 1.1 1.1 7.5 7.7 8.3 1.8 -0.9 0.4 0.0
2443 0.2 2.3 0.5 6.2 1.1 0.9 0.5 0.0 -O.6
2509 3.2 1.2 3.8 1.4 4.4 0.4 0.0 -0.8 -0.6
2524 0.4 0.6 0.6 1.0 0.4 No oligo
2633 0.3 1.2 0.3 1.1 0.3 No oligo
2698 0.5 1.3 1.8 4.2 2.2 -O.4 -0.6 0.6 0.4
2701 1.8 1.2 3.9 2.6 4.6 0.3 -1.4 1.2 -1.6
2871 2.6 1.0 3.4 1.3 3.5 -1.7 -0.8 1.4 -O.5

133

 

Table 4.8: The list of differentially expressed proteins and predicted fimctions n P.

arcticum

134

Protein Name

gene3 Amino Acid Metabolism-Arginlnosuccinate synthase

gen01511 Transcription-putative two component response regulator

gene1576 Transport-putative ABC-type phosphate transport system. periplasmic
:omponent

gene1053 Amino Acid Metabolism-Putative phosphorlbosylfonnimlno-s-

3 carboxamide rlbotlde isomerase

gen61640 Signal Transduction-putative DnaK suppressor protein

gene1201 Translation-ribosomal protein L10

gene2524 Unasslgned-Ferritin:Bacterioferritln

gene1407 Carbohydrate Metabolism-Enolase

gen62392 Conserved Hypothetical-conserved hypothetical protein

gene1793 Metabolism of Cofactors-Probable ketopantoate
ransferase

gene876 Translation-Ribosomal protein 821

gene767 Conserved Hypothetical-conserved hypothetical protein

gene935 Conserved Hypothetical-conserved hypothetical protein
gen92633 Nucleotide Metabolism-Putative Adenylosucclnate lyase
gene1964 Amino Acid Metabolism-Acyl-CoA dehydrogenase

gene381 Metabolism of Cofactors-putative 3-Phosphoadenosine
gen0518 Nucleotide Metabolism-Nucleoside dlphosphate kinase

gene536 Unassigned & Other-Alkyl hydroperoxide reductasel Thiol specific

genez701 Hypothetical-hypothetical
gene1762 Conserved Hypothetical-conserved hypothetical protein

gene1766 Carbohydrate Metabolism-Probable fructose-bisphosphate aldolase
gen62871 Energy Metabolism-Aldehyde dehydrogenase family protein
gene2698 Translation-Ribosomal protein 86

gene831 Translation-putative peptidylprolyl isomerase. FKBP-type

gene645 Signal Transduction-Putative PhoH-llke protein, predicted ATPase

gene148 Unassigned 8 Other-Possible Acetone carboxylase gamma subunit
gene166 Unassigned-Putative outer membrane protein

991102137 Cellular processes-Putative Chaperonin l-lSPBO family

gene2509 Translation-Peptide chain release factor 1

gene2268 Unassigned 8 Other-Bacterial stress protein, probable tellurium
)

,gene539 Unassigned 8 Other-Probable Fst cytoplasmic membrane protein

gene1164 Cellular processes-putative superoxide dismutase (Mn/Fe binding)
gene1759 Translation-Ribosomal protein 815

gene705 Replication and Repair-putative Phage Tennlnase. large subunit
gene1450 Translation-Ribosomal protein L28

gen91906 Conserved Hypothetical-conserved hypothetical protein
gen62443 Hypothetical-hypothetical protein

gene855 Conserved Hypothetlcal-conserved hypothetical protein
gene2098 Translation-Ribosomal protein L15

gene2082 Translation-Ribosomal protein L2

gen6902 Hypothetical-Hypothetical

gene393 Conserved Hypothetical-Protein of unknown function DUF6
gene414 Conserved Hypothetical-Protein of unknown function DUF541

135

 

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CHAPTER 5

METABOLIC ACTIVITY OF SIBERIAN PERMAFROST ISOLATES, Psychrobacter
arcticum 273-4 AND Exiguobacterium sibericum 255-15, AT LOW WATER

ACTIVITIES

Abstract

The Siberian permafrost is an extreme, yet stable environment due to its
continuously frozen state. Average temperatures of -10 to -12°C concentrate solutes to
an aw = 0.90 (5 osm), and limit nutrient diffusion into cells. Viable microbes must
maintain membrane potential and respiratory activity in these stressful conditions. The
isolation of Psychrobacter arcticum 273-4 and Exiguobacterium sibericum 255-15 from
ancient permafrost suggests that these bacteria have maintained a maintenance level of
metabolic activity for thousands of years. In order to determine their response to
permafrost conditions, we simulated the water activity of permafrost using ‘/2 TSB+ 2.79
m NaCl (5 osm). Reduction of cyano-tetrazolium chloride (CTC) showed that many cells
maintained functioning electron transport systems after 10 and 14 days at 22°C and 4°C,
respectively. The lack of electron transport in some cells did not result from increased
membrane permeability, as more cells were determined to be intact by LIVE/DEAD
staining than were reducing CTC. Aerobic respiration rates in 5 osm media, as
determined by resazurin reduction for P. arcticum 273 -4, were 0.021and 0.015 (In OD600/
day) at 22°C and 4°C, respectively, and 0.016 and 0.008 (In OD6oo/ day) for E. sibericum

255-I5. at 22°C and 4°C, respectively. Incorporation of tritiated leucine into new

149

proteins indicated that metabolic activity was restricted to basal levels in 5 osm media at
4 and 22°C. The continued membrane potential, electron transport, aerobic respiration,
and incorporation of radio-labeled leucine into cell material when incubated in a medium
with the osmolarity of permafrost provides evidence that some of the population is
metabolically active under simulated in situ conditions.
Introduction

The majority of the Earth’s surface is cold, with approximately 70% covered by
oceans with an average temperature of 4°C and over 20% of land surfaces subjected to
consistently sub-zero conditions (Pewe, 1995). These soils, sediments and rock, which
were exposed to temperatures of 0°C or below for a period of at least 2 years, are referred
to as permafrost. Depending on the geologic stratum of permafrost sampled, microbes
from the Kolyma region of northeast Siberia have been subjected to continuously frozen
conditions (-10 to -12°C) for 20,000 to 3 million years (V ishnivetskaya et al., 2000).

The majority of microorganisms isolated from buried permafrost are bacteria,
although fungi, algae and Archaea have also been isolated (Gilichinsky et al., 1995). In
contrast to many sea ice isolates, most permafrost isolates are not psychrophilic, but
psychro-tolerant as they can grow at low, even subzero temperatures and also above 20°C
(Gilichinsky et al., 1995), (V ishiniac, 1993). Bacteria are more numerous in permafrost
than in sea ice with 108 colony forming units per gram of soil isolated from permafrost
soils up to 3 million years old (V ishnivetskaya et al., 2000). Studies of permafrost
microbes have generally focused on strains that were isolated and grown at room
temperature in nutrient-rich media. We previously isolated 238 bacterial strains from

different strata of 20-30,000 year old Kolyma region permafrost using several isolation

150

strategies, including low nutrient media and cryo-protectants with care to prevent
exposure to temperatures above 4°C (V ishnivetskaya et al., 2000). Previous
characterization has indicated that these isolates are psychroactive meaning that they
grow at -2.5°C, and are predicted, by Belehradek model, to grow at temperatures as low
as -12°C, the ambient temperature of the Kolyma region of permafrost (Ponder et al.,
2005)

Bacteria are theorized to survive in the permafrost either by a very slow metabolic
rate, or by existing in a state of anabiosis (V orobyova et al., 1997). Both theories have
supporting and contradictory evidence, and presumably both are true for different
microorganisms. The presence of such large numbers of viable microorganisms suggests
DNA and membrane repair mechanisms must exist to protect the cells from damage by
free radicals produced during long term exposure to y- radiation emitted from the

surrounding minerals (F riedmann, 1994).

Several recent studies indicate that, in addition to repair and basal metabolism,
microbial growth occurs at permafrost temperatures. Colony formation has been shown
to occur for Siberian permafrost isolates incubated at -8°C to -10°C over an l8-month
period (Gilichinsky, 1993). In vitro doubling rates for a mixed Siberian permafrost
community of 1 day at 5°C, 20 d at -10°C, and 160 d at -20°C were measured by
membrane lipid incorporation (Rivkina et al., 2000). The Siberian permafrost isolate
Psychrobacter cryohalolentis (formerly P. cryopegella) has a growth rate, determined by
plate count, of 0.16 day1 at -10°C (Bakermans et al., 2003). P. cryohanIenti, is closely

related to the Psychrobacter arcticum 273-4 the strain used in our study, (H. Ayala-del-

151

Rio, personal communication) suggesting that the latter may be active within the
permafrost.

Low temperature is not the only limiting stress within the Siberian permafrost.

Ice formation increases solute concentration by decreasing the amount of free water
available. This produces an environment with low water activity, similar to desiccated
and salt-stressed environments. The low water activity of 0.90 encountered in permafrost
corresponds to a solute concentration of 2.79 m NaCl (5 osm), extrapolated by Rand et al
(Rand, 2004). Low water activity may be a greater limiting stress than low temperature
because of the increased amount of turgor pressure necessary to maintain cellular
respiration and thus provide energy necessary for cellular processes.

We investigated the ability of Siberian permafrost-isolates Psychrobacter
arcticum 273-4 and Exiguobacterium sibericum 255-15 to maintain metabolic activity in
high osmotica medium by evaluating membrane permeability and electron transport
shuttling. These are necessary to maintain membrane potential. Metabolic activity
measurements were compared to the mesophilic, low-salt tolerating E. coli and the
obligate halophile, Dehalobacterium DSMZ 7990. The permafrost cells’ aerobic
respiration and incorporation of radio-labeled leucine suggest that they produce enough
energy to maintain basal metabolism, repaire cell damage, but perhaps not enough to
divide.

Materials and Methods

Isolation and phylogenetic characterizations

Kolyma-Indigirka lowland permafrost samples were obtained by David

Gilichinsky and colleagues (Cryobiology Laboratory, Russian Academy of Sciences,

152

Pushchino). Two isolates, 255-15 and 273-4 were chosen for further studies based on
ability to grow at -2.5°C, growth in increased salinities (to IM NaCl), ease of
culturability at 4°C, and age of permafrost strata (Ponder et al., 2004). Isolate 273-4 was
obtained from direct plating of permafrost soil from the mouth of the Malaya
Kon’kovaya River (69°5’N, 158°6’E) on tryptic soy broth (TSB) at 24°C from a 20,000-
30,000 YBP sample, which featured an ice content of 22% (V ishnivetskaya et al., 2000).
Isolate 255-15 was obtained from an icy (21%), 2-3 million year old sample from the
Bol’shaya Chucochya river (69°10’N, 158°4’E) after 12 weeks incubated at 4°C and
plating on TSB at 24°C (V ishnivetskaya et al., 2000). Analysis of the complete 16S
rDNA genes of isolates 273-4 and 255-15 indicated that the strains were members of y-
Proteobacteria (Psychrobacter) and F irmicutes (Exiguobacterium), respectively, and
established their similarity to previously studied members of their respective clades (H.
Ayala del-Rio and D. Rodrigues respectively, unpublished observation). Psychrobacter
cryohalolentis was isolated from a Kolyma lowland cryopeg water sample (12-14%
salinity) at 4°C on R2A media + 3% NaCl (Bakermans et al., 2003).
Culture conditions

Psychrobacter arcticum 273-4 and Exiguobacterium sibericum 25 5-1 5 were
acclimated to low water activity either at 4 or 22°C through four serial cultures in V2 TSB
+ 0.89 m NaCl (1.61 osm). The permafrost isolates were grown shaken at 250 rpm to an
absorbance (600 nm) of 0.3. Cells were pelleted and re-suspended in the same volume
of V2 TSB + 2.79 m NaCl or V2 TSB + 3.86 m sucrose. These concentrations correspond
to internal osmotic pressures of 5 osm, as extrapolated from Rand et al (Rand). E. coli B

was grown in 1/10 TSB to an OD600 = 0.3 and centrifuged to collect cells which were

153

then re-suspended in the same volume of V2 TSB + 2.79 m NaCl. Dehalobacterium DSM
7990 was grown to an OD600= 0.3 in Marinococcus albus medium (DSMZ medium 434)
and then transferred to an equal volume of V2 TSB + 2.79 m NaCl.
Electron shuttling activity by CTC

Acclirnated bacteria (108 CPU ml'l) were used to inoculate V2 TSB, V2 TSB + 2.79
m NaCl and V2 TSB + 3.86 m sucrose and incubated in the dark at 22°C for 10 days or
4°C for 14 days to be certain that cells have acclimated to the new conditions. Cyano-
tetrazolium chloride (CTC) (Polysciences, Warrington, PA) was added at inoculation to
achieve a total concentration of 5 mM. To determine whether CTC reduction was an
artifact of residual electron transport activity occurring immediately after osmotic shock,
two parallel batches of cells were inoculated for 4 and 24 h prior to addition of CTC.
Cells were fixed with formalin (3 7%) overnight at the end of the incubation, concentrated
onto black MilliPore GTBP filters, counterstained with DAPI (Sigma-Aldrich, St. Louis,
MO), and visualized with a Leitz fluorescence microscope. Three slides for each of three
biological replicates per sample were counted for the number of red (active) and blue
(inactive) cells (minimum of 100 cells per slide) in 10 fields per slide. The percent active
cells was then calculated and a student’s t-test was used to determine statistical
significance (Statview version 5.0, SAS institute).
Membrane permeability by LIVE/DEAD staining

Membrane permeability was assessed using the LIVE/DEAD BacLight Bacterial
viability kit (Molecular Probes, Eugene, OR). Samples were incubated at the original
growth temperature (4 or 22°C). Ten milliliter of cells were assayed, as described by the

manufacturer, immediately before transfer, and every 16 h and 48 h after transfer to 5

154

osm medium at 4 and 22°C, respectively. Three biological replicates per treatment were
used and three slides were viewed for each replicate. The numbers of red (dead) and
green (live) cells (minimum of 200 cells per slide) for ten fields per slide were counted
using a Leitz fluorescence microscope.
Aerobic respiration by resazurin reduction

Cells were grown overnight and diluted 1:100 into fresh media (V2 TSB + 0.91 m
NaCl and V2 TSB + 2.79 m NaCl) and then resazurin (Kodak, Rochester, NY) was added

(final concentration 0.001%). Resazurin (purple) reduction was measured by recording
the colorimetric shift at OD600 to its product, resorufin (pink) (Bakermans et al., 2003).

Measurements were made every 20 min at 22°C and every 4 h at 4°C. Controls consisted
of samples killed by autoclaving and of reagents only.
Tritiated leucine incorporation assay

P. arcticum, P. cryohalolentis and E. sibericum were grown to an OD600 of 0.3 in
V2 TSB + 0.91 m NaCl and transferred to the same volume of V2 TSB + 2.79 m NaCl.
Ten microCuries of 3H- leucine (40-60 mCi , Sigma-Aldrich) was added to each sample,

and incubated at 4°C or 22°C. Radiolabel incorporation was measured every 6 h at 22°C
for the first 72 h, then every 24 h for 7 days, while the 4°C cultures were sampled daily
for 30 days. Samples were pelleted by centrifugation (incorporated) and the supernatant
(unincorporated) filtered onto nitrocellulose filters (Millipore, Billerica, MA). Two
extractions with ice-cold 5% trichloroacetic acid were performed on both the pellet and
filter separately. The filter and pellet were then washed separately with ice-cold 80%
ethanol. The filters were dissolved in an alkaline solution (0.05 N NaOH, 25 mM EDTA,

0.1% SDS) for 60 min. at 90°C (Buesing et al., 2003). After cooling and centrifugation

155

at 14,000 x g for 10 min., 100 uL samples of supernatant were aliquoted into 6 ml of
Safety Solve scintillation fluid (Research Products International Corp, Mount Prospect,
IL). Radioactivity of the filtrate and dissolved filters (100 uL) was measured with a
Hewlett-Packard liquid scintillation counter. Protein concentrations were determined for
each sampling time point using the BioRad Quick protein determination assay kit
(BioRad, Hercules, CA).

Transmission electron microscopy

Negative staining, without any other pretreatment, was used to determine the
intact morphology of cell surface structures. A drop of cell suspension (10 ml of culture
concentrated to 1 ml) was deposited onto a formvar and carbon coated grids for 30 5.
After that, the excess sample was removed with filter paper. Samples were then stained
with a drop of 1% phosphotungstic acid (pH 7.0) for 15-30 s. The excess stain was
wicked away with filter paper.

For ultra-structure studies, cells were suspended and fixed overnight with 2.5 %
glutaraldehyde in 0.1 M cacodylate buffer at 4°C. After fixation, cells were pelleted and
re-suspended in 2% agarose. Agarose blocks containing the cells were fixed 2 h at room
temperature. The plugs were then washed three times for 15 min in 0.1 M cacodylate
buffer, post-fixed in 1% osmium tetroxide in 0.1M cacodylate buffer and washed an
additional three times for 15 min in 0.1 M cacodylate buffer. The plugs were then
dehydrated for 10 min in series of acetone (30, 50, 70, 95, 100, 100, 100%). Infiltration
was then performed using 3:1 acetone: Poly/Bed 812, 1:1 acetone: Poly/Bed 812 and 1:3
acetone: Poly/Bed 812 followed by infiltration overnight in 100% Poly/Bed 812. Samples

were embedded in silicone molds and polymerized at 60C for 24 h. Blocks were

156

sectioned with a Power Tome XL ultramicrotome (RMC, US). 70 nm sections were
stained with 2% uranyl acetate and lead citrate (Reynolds formulation) and then observed
using a JOEL 100 CX transmission electron microscope (Japan) at an accelerating
voltage of 100 kV. Images were taken digitally with a Mega View II system. All sample
preparation and TEM was performed by the Center for Advanced Microscopy at
Michigan State University.
3282M
Growth in V2 TSB + 2.79m NaCl

Despite recent reports of growth of Psychrobacter cryohalolentis at permafrost
temperatures (-10°C) (Bakermans et al., 2003), repeated attempts to grow the closely
related P. arcticum 273-4, P. cryohalolentis, as well as E. sibericum 255-15 at the
corresponding water activity in NaCl medium were unsuccessful. An increase in
turbidity was not detected in V2 TSB + 2.79 m NaCl for all three strains. during a three
month incubation period at 22 or 4°C (results not shown)
Electron transport

Electron shuttling activity, indicated by the red fluorescence of CTC, was
detected in 5 osm media (2.79 m NaCl or 3.86 m sucrose) for P. arcticum 273-4 and E.
sibericum 255-15. A substantially higher percentage of active cells was detected in the
permafrost isolates when compared to E. coli under all conditions (Table 5.1). In the
absence of salt stress the percent of active cells in both permafrost isolates did not differ
between 22°C and 4°C, while E. coli was significantly inhibited by the lower temperature
(p <0.05). Incubation in 5 osm medium resulted in a significant reduction of active cells

in all strains, with P. arcticum 273-4 and E. coli exhibiting a difference with both

157

temperature and increased osmotica. The largest decrease in percent of active cells in
high osmotica occurred for P. arcticum 273-4 at 4°C (50% loss), and E. sibericum 255-
15. at 22°C (34% loss). The use of both sucrose and salt as osmolytes gave comparable
decreases in activity (p <0.05) for all organisms suggesting that the decreased metabolic
activity was not due to ion toxicity as the salt was not more severe (Table 5.1). More
electron transport activity at 2.79 m NaCl was indicated by the red fluorescence of CTC
than those recently reported for particle- attached cells isolated from sea ice, which are
surrounded by only 10% brine (equivalent to 1.7 m NaCl) (Junge et al., 2004) suggesting
permafrost microbes may be better adapted to maintain electron transport at low water
activities.
Membrane permeability

Permafrost isolates show an overall greater membrane longevity in high osmotica
compared to E. coli, but more membrane permeability than the obligate halophile,
Dehalobacterium. It is likely that only the obligate halophile did not experience osmotic
shock. The majority of the permafrost isolates and the obligate halophile,
Dehalobacterium membranes were intact when incubated in the 5 osm media for 6 days
at 22 or 4 °C (Fig. 5.1). It is possible that the membrane changes with salinity may alter
the membrane permeability of cells leading to increased uptake of propidium iodide,
which quenches SYTO9 fluorscence. This would lead to an artificially high number of
cells appearing dead. Incubation at lower temperatures resulted in a statistically
significant decrease in live cells in 5 osm media for both P. arcticum 273-4 and E. coli (p
<0.004); however the incubation at 4°C increased survival of E. sibericum 255-15 cells

after 6 days (p <0.009) in 5 osm media (when compared to 22°C). There was no

158

significant difference (p= 0.89) in number of intact membranes between the halotolerant
permafrost isolates and E. coli until day 7 when significantly more E. coli membranes lost
integrity. Permafrost cell membranes were maintained longer within 5 osm media
compared to E. coli with 20-35% more permafrost cells intact at day 10. All the cells of
the obligate halophile, Dehalobacterium DSMZ 7990, retained intact membranes until
day 7, when death and lysis occurred due to exhaustion of nutrients. The slight
differences in membrane permeability with differing temperatures follow the same trend
as the electron transport results. The 4°C incubation showed increased metabolic activity
for E. sibericum sp. 255-15 and slightly decreased activity for P. arcticum 273-4.
However, more cell membranes remained intact than reduced CTC indicating that the
lack of electron transport in non-CTC stained cells was not exclusively due to loss of
membrane integrity. It is possible that the electron shuttling machinery of the non-
halophilic bacteria may have limited functionality in increased salinity. Halophilic
bacteria have different menaquinone complexes than those detected in the genome

sequence of P. arcticum (Marquez et al. , 1970).

Table 5.1: Percent of the metabolically active cells measured by CTC reduction vs. total
DAPI stained cells for two permafrost isolates and E. coli at different temperatures and
osmotica after 10 days at 22°C and 14 days at 4°C.

 

Medium Temp(°C) Organism Percent of
cells that
reduced
CTC

V2 TSB 4 E.coli 5.9

E. sibericum 64.2 b
P. arcticum 77.6”
22 E. coli 28 a
E. sibericum 62.3 b
P. arcticum 87.0 b

159

V2 TSB + 4 E. coli 1.98

2.79 m NaCl E. sibericum 35.1 b
(5 osm) P. arcticum 27.2 b
22 E. coli 1.8 a
E. sibericum 28.2 b
P. arcticum 57.0 1’
V2 TSB + 4 E. coli 1.6
3.86 m E. sibericum 47.9 b
sucrose P. arcticum 11.8 b
(5 osm) 22 E. coli 2.6 a
E. sibericum 21.5 b
P. arcticum 45.6 b

The median difference between percent active cells based on temperature was significant
(p < 0.05) for E. coli in V2 TSB media and for P. arcticum at 5 osm.

" Median difference in percent active cells between V2 and 5 osm media, also different
from other organisms: p < 0.05

b Median difference in percent active cells between V2 TSB and 5 osm media, no
difference between organisms: p < 0.05

160

 

 

 

35 - + P. arcticum at 220

Percent live stained cells

2 + E. siben'cum at 220
25 4 + 5.00” B at 30C

 

 

 

 

 

20 - -x- Dehalobacten'um at 300 .
15 _ -e— P. arcticum at 4C
10 1 +5. siben'cum at 4C
5 q -0— Eco" at 4C
0 ' I I T I r f r r
0 1 2 3 4 5 6 7 8 9 10

Time (days) in 2.79m NaCl

Figure 5.1. Average percent of cells with intact membranes after 10 days incubation in V2
TSB + 2.79 m NaCl at the indicated temperatures. Error bars represent standard
deviation from average of three biological replicates, three slides per replicate. Error bars
not visible are smaller than the symbol.
Aerobic respiration

The aerobic respiration rates, as measured by resazurin reduction in 5 osm
medium were higher for P. arcticum 273-4 than E. sibericum 255-15. (Figs 5.2a and
5.2b). Lower rates of respiration, determined by the slope of the reduced resazurin line,
occured afier incubation at 4°C compared to 22°C for both Psychrobacter arcticum 273-4
and Exiguobacterium sibericum 255-15 in Sosm medium. At 4°C, 5 osm respiration rates
reduced two fold in E. sibericum 255-15, while respiration rates for Psychrobacter

arcticum 273-4 were comparable to Exiguobacterium rates at 22°C (Figs. 5.2a and 5.2b).

Autoclaved controls showed no change in absorbance (data not shown). In V2 TSB,

161

without added salt, respiration and growth rates were significantly correlated (r2= 0.97

and 0.98 for E. sibericum 255-15 and P. arcticum 273-4, respectively) (data not shown).

 

 

 

 

 

 

 

 

0.1
0 2 - ‘
a
-0.1
O
02 ‘ a
§ -0.3
8 Q
g .04 Slgpe- 0.016
_ R =0.8864 ‘
-05 ‘
-0. 6 A E. siben’cum 0
e . '
P arcticum Slope= 0.021
.07 R2 = 0.9858
0
-0.8 . ' ' ' I I
0 1 2 3 4 5 6 7
Time (days)

Figure 5.2a: Resazurin reduction at 22°C by E. sibericum 255-15 and P. arcticum 273-4
in V2 TSB+ 2.79 m NaCl. The slope of the reduced resazurin line is 0.021, corresponding
to a mean reduction rate in 48 h for P. arcticum sp. 273-4. The rate of reduction for E.
sibericum 255-15 is 0.016, corresponding to a mean rate of 63 h. Error bars are smaller
than symbol except where shown.

162

 

0.5

    
  
 

 

 

 

0.4 J Slope= 0.008
0.3 -
o O 2 4
O
E
0.1 r A E. sibericum
0P. 3 ti
rc cum Slope=0.015
0 a
”0.1 r r r r r I r T 1 r r T T r
O 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time (days)

Figure 5.2b: Resazurin reduction measured by absorbance decrease at 4°C by E.
sibericum 255-15 and P. arcticum 273-4 in V2 TSB + 2.79 m NaCl. Error bars are
smaller than symbol except where shown.
Incorporation of tritiated leucine into protein

Incorporation of tritiated leucine as measured by disintegrations per minute
(DPM) increased in all the organisms tested at both 22 and 4°C in V2 TSB + 2.79 m NaCl
(Figs. 5.3a and 5.3b). There was however, no significant increase in protein number at
22°C for P. arcticum, E. sibericum, P. cryohalolentis or E. coli after 2 h in high osmotica
media, suggesting these may be shock proteins that either retained stability or were
necessary to continually re-synthesize. Protein number decreased with time at both 22
and 4°C for P. arcticum, P. cryohalolentis and E. sibericum. This net protein decrease

accompanied by an increase in dpm with time suggests that only proteins necessary for

survival in osmotic shock are synthesized. P. arcticum showed a larger net dpm

163

incorporation (6388 dpm) at 22 °C after 7 days which based on extrapolation, would
require 198 days at 4°C. Leucine incorporation for P. cryohalolentis, E. sibericum and
E. coli were not significantly different from each other at 22°C. The approximate
numbers of proteins synthesized were calculated based on a specific activity of 40-60
mCi and the average protein size of 36 kDa for E. coli. After 7 days at 22°C in 5 osm
medium ~300, 52, 40 and 30 protein molecules were synthesized for P. arcticum, P.
cryohalolentis , E. coli and E. sibericum, respectively. The number of protein molecules
synthesized at 4°C afler 30 days was ~275, 320 and 200 for P. arcticum, P.
cryohalolentis and E. sibericum (Fig. 5.3b). Incorporated 3H-leucine values were
significantly different with temperature, with 4°C improving incorporation for only P.
cryohalolentis and E. sibericum. The maximal incorporation of leucine for

Dehalobacterium was larger compared to the permafi'ost isolates (data not shown).

164

 

 

 

 

 

 

 

6000
5500 [Ii
5000 \
‘1 + mm
' 7' Re
4500 \ +P.cryohelotentts 1' 140°
\ ‘\ +5%», A
4000 \ ‘4 v +5.00! '1' 1200 E
\ \ -0- Auctteum E
g 3500 \\ ‘\\\ —a- Rambo” 1_ 1000 3
3000 \§\‘\ \ l --A- E. m .5
\\\ ' “ -9- 5.”. g
i

 

 

 

 

 

 

Time (days)

Figure 5.3a. Incorporation of 3H- leucine into TCA precipitable fraction of cells grown at
22°C for permafrost isolates and 37°C for E.coli in V2 TSB + 2.79m NaCl. The amount
of protein (open symbols) extracted is shown in the right axis with solid symbols. Data
are means and standard deviation is of triplicate samples .

165

 

 

 

 

 

 

2000 i -- 100
e P. arcticum
+ P. cryohalolentis b 80
+5. sibericum
150° ‘ +P. arcticum 60
5 -D- P. cryohalolentis :8-
g .l —a— E. Sibencum B
o 403
.E . .E
‘3’ 1000 - 8
2 2
3': . 20°-
... o
500 -
2: '20
0 2 T 40
O 5 10 15 20 25 30
Time (days)

Figure 5.3b. Incorporation of 3H- leucine into the tca precipitable fraction of cells over
30 days in V2 TSB + 2.79m NaCl grown at 4°C in permafrost isolated P. arcticums and E.
sibericum. The amount of protein (open symbols) extracted is shown in the right axis
with solid symbols. Data are means and standard deviation is of triplicate samples .
Transmission electron microscopy

TEM images of the cells stained with phosphotrmgstic acid revealed the presence
of capsules surrounding P. arcticum cells grown in the presence of high salt at both 22
and 4°C (Fig. 5.4a and b). P. arcticum grown at 22°C in V2 TSB showed no capsule (Fig.
5.4e), cells grown at 4°C in V2 TSB showed a polysaccharide coating that allowed cells to
adhere to each other but no capsule layer (Fig. 5.4d). Cross sections of cells grown at
4°C in the presence and absence of high salt reveal the cells have dispersed cytoplasm

(Figs. 5.4e and f). Some cells grown in V2 TSB + 2.79 m NaCl showed shrunken

cytoplasm and all showed thickened cell membranes.

166

 

!
-.r~
r
3.

‘ é _ r ' -|
500nm ‘ ‘ '- 500 nm

Figure 5.4. Negative stained transmission electronmicrographs of P. arcticum 27 3-4. a:
Capsule surrounding cells grown at 22°C in V2 TSB + 2.79 m NaCl b. Capsule
surrounding cells grown at 4°C in V2 TSB + 2.79 m NaCl. C. No capsule surrounds cells
grown at 22°C in V2 TSB. d. No capsule surrounds cells grown at 4°C in V2 TSB,
however a polysaccharide coating is visible that allows cells to adhere to each other. E.
Cross section of cell grown at 4°C in V2 TSB. F. Cross sections of cells grown at 4°C in
2.79 mNaCl reveal some cells with compacted cytoplasm, with all cells showing an
increased membrane thickness.

 

Discussion

167

Siberian permafrost isolates, Psychrobacter arcticum 273-4 and Exiguobacterium
sibericum 255-15 maintain membrane potential and electron transport, respire, and
synthesize new proteins when incubated in high osmolarity media. These results provide
‘ evidence that a portion of the cell population is metabolically active under simulated in
situ conditions, despite the lack of cell growth. The viability of microorganisms from
ancient Siberian permafrost supports the hypothesis that some low level of metabolic
activity must have continued during burial in this frozen, low water activity environment.
On-going repair processes have been recently demonstrated in 35,000-year-old Siberian
permafrost by amino acid racemization studies. The estimated ratio of D-aspartic acid
(resulting from dead or inactive cells) to L-aspartic acid was smaller than predicted by
mathematical models, allowing the authors to conclude that some metabolic activity was
occurring within the permafrost environment to convert the D form to the L form
(Brinton et al., 2002). Furthermore, membranes of native Siberian permafrost
communities are continually synthesized at permafrost temperatures (5°C to -20°C).
There is a direct correlation between the decreasing incorporation of 14C-acetate into
lipids and the thickness of the unfrozen water layer surrounding the loamy soil (Rivkina
et al. , 2000). The presence of unfrozen water would allow for mass transfer of nutrients
and elimination of waste products, until the diffusion gradients slow overall movement
(Ostroumov et al., 1996).

The high NaCl concentration may be toxic for growth, despite no major
differences between electron transport activity with sucrose as an alternative osmolyte. ).
While the turbidity did not change the number of viable cells determined by plate counts

decreased one log which appeared due to the initial shock occurring from transfer of the

168

cells to the high osmolarity medium. It is likely that the media used to simulate the
permafrost water activity does not in fact simulate permafrost conditions. Within the
permafrost adherence to soil minerals would provide access to thin films of water that
may have different osmolarities depending on the cation concentrations. These
permafrost isolates were capable of growth on solid or liquid media containing up to 1.3
m NaCl at 4°C (Ponder et al., 2005) but no growth was seen during a three month

incubation period at 22 or 4°C in medium with 2.79 m NaCl.

To maintain electron transport and cellular viability, bacteria must maintain turgor
pressure. Gram positive bacteria, such as E. sibericum 255-15, have thicker cell walls,
allowing them to withstand greater turgor pressures than Gram negative cells, in which
volume changes provide turgor pressure control (Brown, 1990). Despite the difference in
membrane composition between the Gram positive E. sibericum 255-15. cells and its
Gram negative counterpart, P. arcticum 273-4, there were no large differences in
membrane permeability in 5 osm medium. The dead cells were likely plasmolysed. Loss
of membrane integrity was not the exclusive cause of the decreased electron transport in
permafrost cells in 5 osm medium, as more cells remained intact than reduced CTC in the
case of the permafrost isolates and E. coli. Stability and function of permafrost bacteria
electron transport systems may be compromised by increased osmotica. Incubation in 5
osm media may decrease the turgor potential maintained in the permafrost isolates to the
point that cellular division is inhibited but proton motive force continues. Maintenance
of internal turgor pressure by extrusion of Na+ via H+/Na+ antiporters occurs in several

bacteria and Archaea (Albers et al. , 2001) and would be vital in the permafrost

169

environment. Recent genome sequencing of P. arcticum 273-4 and E. sibericum 255-15.
by the Joint Genome Institute (http://genome.0rnl.gov/microbial/) reveals the presence of
these H+/Na+ antiporters, perhaps allowing the maintenance of membrane potential
detected in the Live/Dead stained cells.

Transmission electron microscopy of P. arcticum 273-4 confirmed that the
majority of cells appeared to be intact afier 5 days. Furthermore, a capsule layer was
visible around all cells in 5 osm medium, while no cells showed capsules when incubated
in V2 TSB at 4 or 22°C (Figs 4a and b). The presence of capsular layers surrounding non-
spore-forrning bacteria occurs frequently in permafrost soils, and are believed to aid in
formation of cyst-like forms (Soina et al., 2004). TEM images of P. arcticum 273-4 do
not show the altered cytoplasm and compact nucleoids typical of resting cells.
Pleomorphy and encapsulation of cells in extracellular polymers is common and has been
recently been reported for unknown cells isolated from salty sabkhas incubated at low
water activities (Krumbein et al., 2004). Extracellular polysaccharides serve to bind
water, allowing access to nutrients for continuing exchange of metabolic end products.
For this reason, microbes isolated from hypersaline environments are commonly found in
biofilrns (Litchfield, 1998). The presence of capsules surrounding P. arcticum 273-4
may be an adaptation to the decreased water activity of the permafrost and may allow for
the adherence to_soi1 particles and, therefore, the access to unfrozen water layers for
metabolic activity.

Respiration within soils decreases with water activity, the resulting energy must
maintain a favorable osmotic balance within the cells and the remaining energy is

available for growth and division. In Vitro nucleic acid synthesis has been reported at low

170

temperatures, with water activity equivalent to the levels tested within this study, for both
mixed microbial communities and pure culture. Low rates of nucleic acid synthesis
indicated by biological incorporation of 3H leucine (protein) and methyl 3H thymidine
(nucleic acid) have been detected in Arctic snow at temperatures between -12°C to -l 7°C
(Carpenter et al., 2000) and in pure cultures inoculated into sea ice incubated at -15°C
(Christner, 2002). Furthermore, radioisotopic studies of Lake Vostok (Antarctica)
accretion ice indicate the presence of metabolic activity at -20°C with only a small
portion resulting from macromolecular synthesis, the remaining energy used for cellular
repair (Karl et al. , 1999). In this study the continuing incorporation of radio-labeled
leucine into new proteins indicates that a low rate of metabolic activity occurs in 5 osm
medium. The low number of proteins synthesized by the Psychrobacter isolates in the 5
osm media with time correspond with those protein numbers determined for an Antarctic
sea ice isolated Psychrobacter incubated at -15°C, which would have an even lower
associated water activity than this experiment (Christner, 2002). The energy necessary
for this continual protein production likely stems from aerobic respiration which is
reduced in 5 osm media and would result in less energy available for growth and division.
The increase in protein number at 4°C with time may not indicate actual cell growth, as
previous studies have shown that P. arcticum 273-4 increases in cell biovolume with
increased salinity (Ponder et al., 2005). The finding of the low numbers of proteins
synthesized in 5 osm media suggest that the microbes maintain only enough basal
metabolism to allow repairs to membranes and DNA molecules but do not actively divide

in this oligotrophic, freezing environment, as described by Price et al. (2004). This

171

continued metabolic activity under low water activity stress may be a survival mechanism

for these ancient permafrost isolates.
Reports of grth at -lO°C of P. cryohaloentsis are not supported by this study

investigating the water activity that permafrost bacteria would be expected to experience
at -lO°C. Several conditions of the permafrost were not modeled in this study.
Differences in ion concentration of the permafrost were not considered- only the
dominant cation was considered with NaCl used to increase osmotica. This study also

did not allow for adherence due to the shaking conditions. Within the permafrost, these

bacteria may adhere to soil minerals or form biofilms. The formation of biofilrns offers
protective properties from many stress conditions and are commonly found in high
salinity environments such as sabkhas and hypersaline mats (Krumbein et al., 2004).
Recent studies reveal a correlation between bacterial numbers and the total organic
carbon and clay content of soil (Beyer et al. , 2000). Increased content of organic carbon

and clay will provide nutrients and have greater water holding capacity, preventing

desiccation of the cells.
Unfrozen water surrounding the cells acts as a nutrient medium because as ice

forms it concentrates solutes. Additionally, this unfrozen film acts as a cryoprotectant by

preventing invasion from extracellular ice crystals. The amount of unfrozen bound water
surrounding microbes was shown to decrease with temperature (Gilichinsky et al., 1995)
This is further supported by evidence in cryopreserved soils modeled to resemble
permafrost environments, which shows that the ability of cells to adhere to soil particles

increases cell survival (Sidyakina et al., 1992). Scanning environmental microscopy

172

further reveals a tight association of bacteria with the surrounding Siberian permafrost
(Soina et al., 2004)

Measuring metabolic activity at low water activity, equivalent to -10°C, supports
the concept that the liquid inclusions present in permafrost (Gilichinsky et al., 2003) may
provide an adequate habitat for active microbial populations on Earth and possibly
elsewhere. The Siberian permafrost bacteria have lived in low temperature soil with a
high concentration of NaCl salts for thousands of years (V ishnivetskaya et al., 2000).
Despite the constant low water activity, obligate halophiles were not exclusively selected,
supporting continued metabolic activity in a resting state. Investigation of mechanisms
of microbial persistence within permanently cold environments is of great importance to
the fields of astrobiology and polar research. The remarkable ability of microbial
populations to survive in a continuously frozen matrix of permafrost for millions of years
makes the permafrost community unique. Our results raise questions on the survival time
of a cell in continually frozen conditions; the level of metabolic activity necessary to
retain viability; and the possibility of life existing on other cryogenic astral bodies.
Acknowledgements
This research was funded by the National Astrobiology Institute of NASA. We thank
Tatiana Vishnivetskaya her preliminary physiological data which allowed us to focus
further studies on a narrower number of interesting permafrost strains. We acknowledge
the assistance of Chia-Kai Chang, Gisel Rodriguez, and Matt Campbell. We thank

Richard Lenski, and Corien Bakermans for strains E. coli 606, and P. cryohalolentis,

respectively.

173

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CHAPTER 6

CONCLUSIONS AND FUTURE DIRECTIONS

CONCLUSIONS

This research broadens the understanding of microbial survival under extreme
conditions, particularly with respect to the combination of low temperature and low water
activity stresses found in most frozen environments, including permafrost, ice and frozen
foods. Investigating long term survival and adaptation to low temperature and low water
activity may lead to advances in control of food borne pathogens, many of which are
resistant to cold and increased osmotica. Identification of cold-active proteins can lead to
the development of cold active laundry detergents, extend refiigeration times of foods
and extend the shelf-life of blood products and organs by control of the ice crystallization
process. The issue of long-term survival under extreme conditions is of particular interest
in the field of astrobiology. Organisms that survive in such hostile environments can be

used as models for understanding cellular responses on astral bodies.

The major findings of this research can be summarized as follows:

1. In general the physiological properties of Siberian organisms indicate they have
adapted to the conditions of low temperature, at least for the 4°C temperature used in
this study. This adaptation may allow for survival of certain microbes in the

permafrost of the Kolyma region, which is -10 to -12 °C, where intracellular water is

likely not frozen.

177

 

2. A decrease in fatty acid saturation and chain length at the colder temperatures and a
further decrease in the degree of saturation at higher osmolarity may function to
maintain membrane fluidity. The decrease in chain length at 4°C in Psychrobacter
arcticum 273-4 is likely due to the decreased transcripts of long chain fatty acid acyl
carrier protein ligase. Unsaturated membrane fatty acids are present in these
psychroactive Siberian permafi'ost isolates grown at mesophilic temperatures and
expression of beta-ketoacyl-ACP synthase I,fabH, delta-9 fatty acid desaturase were
constitutive and did not increase with 4°C incubation or in the presence of salt.
However, the proteins may be more stable at 4°C than 22°C so fewer transcripts
would be required for increased synthesis. This difference in unsaturation may
account for Psychrobacter’s low upper growth temperature.

3. P. arcticum accumulates compatible solutes to balance external osmolytes under
growth conditions of V2 TSB + 5% NaCl at 4 and 22°C. Transport associated genes
are upregulated for proline, choline (the precursor to glycine betaine) and catabolism
genes that synthesize glycine betaine.

4. Proteins extracted from P. arcticum cells grown in V2 TSB + 5% NaCl had increased
respiratory activity. This increase in activity may be an adaptation to counteract the
low temperature related decrease in respiratory activity. The increase in expression
of NADH dehydrogenases and Na+ decarboxylases may establish an Na+ motive
force, producing energy necessary for the salt related physiological changes.

5. In the presence of increased osmotica, P. arcticum is surrounded by a capsule. This

accumulation of carbohydrates may maintain a fluid water layer in the presence of

178

high salt and low temperature — an adaptation to the high osmotica environment of the
permafrost.

Despite the marked physiological differences in growth characteristics, fatty acid
profiles and carbon source utilization, the majority of the genome was not
differentially expressed (at a cutoff level of 2- fold or greater difference in
transcripts). Only 3.1% of the P. arcticum transcriptome was differentially
expressed at 2-fold or greater level when cells were grown at 4°C and 22°C (both in
V2 TSB media). Comparing cells grown in V2 TSB and V2 TSB +5% NaCl (both at
22°C), 3.3% of the genome was differentially expressed. Halo-adaptation of P.
arcticum is indicated by a decreased number (2.9%) of differentially expressed genes
when cells were grown at 4°C and 22°C (both in V2 TSB media + 5% NaCl). P.
arcticum is both cryo- and halo-adapted, based on a smaller number (2.8%) of
differentially expressed genes, when cells were grown in V2 TSB media + 5% NaCl
and V2 TSB (both at 4°C).

Proteome analysis revealed that of the 44 proteins with distinct spectra that were
differentially expressed, 20 were not differentially expressed above 2-fold in the
transcriptome under any of the test conditions. Yet, 10 proteins and transcripts had
the same pattern of expression and 14 had different expression patterns in at least one
condition examined. These differences in proteome and transcriptome analysis
indicate that transcriptome changes are not the sole mechanism of acclimation; other
mechanisms such as increased stability and post-translational modification may also

be important.

179

8. P. arcticum is capable of metabolic activity under simulated in situ conditions as
indicated, by maintenance of membrane potential, electron transport, aerobic

respiration and the incorporation of radio-labeled leucine into newly synthesized
proteins when incubated in V2 TSB + 2.79m NaCl.

Future research

Although the aforementioned research provided new insights about cryo- and
halo-adaptation in P. arcticum 273-4, new questions emerged that are intriguing for
future investigations into mechanisms of survival in the permafrost environment.

A capsule layer was visible around all cells in salt-amended media, while no cells
showed capsules when incubated in V2 TSB at 4 or 22°C. Extracellular polysaccharides
bind water, allowing access to nutrients for continued exchange of metabolic end
products. For this reason, microbes isolated from hypersaline environments are
commonly found in biofilms (Litchfield, 1998). Capsular layers surrounding non-spore-
forrning bacteria occur frequently in permafrost soils, and are believed to aid in the
formation of cyst-like forms (Soina et al., 2004). Capsules surrounding P. arcticum may
be an adaptation to the decreased water activity of the permafrost, which may allow for
adherence to soil particles, and, therefore, the access to unfrozen water layers. Initial
observations of cells incubated in V2 TSB + 2.79m NaCl show cells adhering to the
culture flask. It is likely that these same polysaccharides may allow for the adherence to
soil particles. Future studies should examine the morphology and association of P.
arcticum in permafrost soil microcosms using scanning environmental microscopy to
determine if P. arcticum adheres. Measurement of aggregate stability would then

establish the importance of P. arcticum in permafrost soil stability.

180

 

 

While the capsule layer provides access to unfrozen water, P. arcticum
accumulates proline, glycine betaine and glutamate to maintain membrane and protein
stability in an increased osmotica environment. Compatible solute-associated genes —
proline transporter (or608), choline transporter (or2296), choline dehydrogenase (or2295)
and betaine aldehyde dehydrogenase (or2294) were revealed to be differentially
expressed by microarray analyses. Further examination of these genes is warranted using
quantitative real- time PCR to measure transcript quantities at different temperatures and
salinities, including the permafrost-simulated salinity. NMR analyses of cells from tracer
studies using incorporation of radio-labeled choline, proline and glutamate should also be
performed to further examine the correlation between increasing solute accumulation,
transcript amounts and environmental conditions.

Physiological differences of P. arcticum at low temperatures make the generation
of additional energy necessary for continued growth or activity (Bakermans et al., 2004).
The decrease in total respiratory activity at 4°C is alleviated by the addition of NaCl.
Increased expression of the Na+ pump, NADH dehydrogenase, and Na+ decarboxylases
may generate a sodium motive force that is harnessed for ATP production. ATP
production should be measured in addition to transcript quantities of associated genes
differentially expressed by microarray analyses (or1686- or1691, or1520, or339) at
different temperatures and salinities, including permafrost-simulated salinity.

The availability of the genome sequence and P. arcticum oligo-arrays make
possible investigation of stress responsive gene regulation by identifying clusters of
similarly responsive genes and the application of algorithms predicting promoter

sequence binding (Cases et al., 2003). Gene regulation has not been studied in cryo-

181

adapted microorganisms. This research provides several interesting gene targets for
mutational analysis to determine the important genes for survival in permafrost
conditions. The proteins and RNAs of P. arcticum have likely adapted to maintain
stability and function at the low temperature and increased osmotica of the permafrost
environment. Further study of this fascinating topic may lead to biotechnological

advances and the understanding of survival mechanisms in harsh environments.

182

References

Bakermans, C. and K. H. Nealson (2004). "Relationship of Critical Temperature to
Macromolecular Synthesis and Growth Yield in Psychrobacter cryopegella."
Journal of Bacteriology 186(8): 2340-45.

Cases, 1., D. W. Ussery and V. de Lorenzo (2003). "The 54 Regulon (Sigmulon) of
Pseudomonas putida." Environmental Microbiology 5(12): 1281-93.

Litchfield, C. (1998). "Survival Strategies for Microorganisms in Hypersaline
Environments and Their Relevance to Life on Early Mars." Meteoritics Planetary
Science 33: 813-19.

Soina, V. S., A. Mulyukin, E. V. Demkina, E. A. Vorobyova and G. el-Registan (2004).
"The Structure of Resting Bacterial Populations in Soil and Subsoil Permafrost."
Astrobiology 4(3): 345-58.

183

APPENDIX A:

BACKGROUND AND CONTRIBUTIONS TO THE DESCRIPTION AND GENOME
ANALYSIS OF Psychrobacter arcticum]

Contributions to the description of Psychrobacter arcticum
Psychrobacter is a member of the gamma Proteobacteria family. This genus is
commonly isolated from cold and marine environments, including soil, water brine lenses
(cryopegs) (Gilichinsky et al., 2003), sea-water (Bowman et al., 1997), glacial and ice
sheet cores (Christner, 2002) and the skin and gills of fish (Scholes et al., 1964). It has
also been associated with food spoilage and is often resistant to irradiation used for food
preservation (F irstenberg-Eden et al., 1980; Rodriguez-Calleja et al., 2005).
Psychrobacters have also been identified from human (Moss et al., 1988) and animal
(Vela et a1. , 2003) sources, and are occasionally pathogenic.

Psychrobacter sp. 273-4 is a small (1.6 pm x 0.7m), non-motile, coccoid rod
often found in pairs (Figure A-l). It is a Gram negative aerobe, producing smooth, non-
pigrnented colonies of ~2 mm. Psychrobacter sp. 273-4 grows over a range of
temperatures from a maximum of 28°C to as low as -10°C (Chapter 3). It possesses
tolerance to high salt concentrations, similar to other reported Psychrobacter isolates
(Bowman, 2005), requiring at least 10 mM but no more than 1.3 M NaCl when grown in
V2 tryptic soy broth. In the presence of high salt concentrations the cells become more

pleomorphic and increase their size (1.9 pm x 0.8 um).

 

‘ Derived from the portions I contributed to the papers which are team efforts of the MSU
NAI group.

184

Psychrobacter 273-4 was isolated from a 20-40 thousand-year-old Siberian
permafrost core (V ishnivetskaya et al., 2000). The permafrost samples were obtained
from the Kolyma-Indigirka lowland, Siberia, by David Gilichinsky (Institute of Soil
Cryosciences Laboratory, Russian Academy of Sciences, Pushchino, Russia). It was
selected for sequencing based on its excellent survival after exposure to a long-term
freeze, rapid growth at low temperatures (e. g. -2.5°C) and age of the permafrost sediment
from which it was cultured. Both l6S rRNA (Figure A-2) and Gyrase B gene sequence
analysis placed the isolate within the genus Psychrobacter; however, neither analysis
could unambiguously determine the branching order of the species in phylogenetic trees
(Ayala del-Rio and Bakermans, respectively, unpublished communications). DNA-DNA
hybridization data established that this isolate was distinct at the species level to any
previously described Psychrobacter species (Bakermans, unpublished communication).
These results support Psychrobacter sp. 273-4 is a novel species of Psychrobacter for
which we propose the name Psychrobacter arcticum sp. nov. that will be submitted to the

Internal Journal of Systematic and Evolutionary Microbiology for approval.

 

185

Figure A-l. Scanning Electron Micrograph of Psychrobacter arcticum 273-4 grown at

22°C in V2 TSB.

 

Moraxel/a aI/antae

Acineiobacter calcoaceticus

seudomonas aerugin

one/Iia psychroerythrea
Escherichia coli

  

 

 

 

     

Psychrobacter arenosus
Psychrobacter pacificensi s
Psychrobacter meningitidis
Psychrobacter phenylpyru vicum
Psychrobacter ni vimaris
Psychrobacter proteolyticus
Psychrobacter alimentan'us
Psychrobacter val/is
Psychrobacter aquatica
Psychrobacter maritimus
Psychrobacter psychrophilus
Psychrobacterjeot a/i
Psychrobacter 563
PS sychrobacter halophilus
Psychrobacter marincol a
Psychrobacter submarinus
Psychrob acter uraIivorans
Psychrobacter cib us
Psychrobacter sp. ikaite c11
Psychrobacter faecalisis
Psychrobacter pulmo
Psychrobacter glacinco/a sDSMZ T
Psychrobacter frigidicola
Psychrobacter immobilis
Psychrobacter IuII'
Psychrobacter okhots1kensis

 
 
  
 

   
   
   
 
 
  

 

 
 

Psychrobacte bzi
Ps chrobacter olacialis
Psych/obacter CIyopege/la

010

Figure A-2. 16S rDNA gene phylogenetic tree of the genus Psychrobacter.
Psychrobacter 273-4 and Psychrobacter cryopegella are highlighted as their genomes
have been sequenced by the Department of Energy Joint Genome Institute. (Courtesy of
H. Ayala del-Rio).

Contributions to the genome annotation of Psychrobacter arcticum
The G + C content of DNA of Psychrobacter arcticum 273-4 is 42.8 mol% with a

genome size of 2.7 Mb, with 2,155 predicted genes. The genome contains a large
number of homologs to known stress responsive genes (for a list see Table 4.8). Thirty

two percent of the predicted open reading frames in the genome are hypothetical in

186

nature, and functionally uncharacterized (Figure A-3). It is possible that some of these

hypothetical proteins may have roles in stress response.

 

Figure A-3: Distribution of Psychrobacter arcticum 273-4 predicted open reading

frames arranged by cluster of orthologus gene groups.

Amino Acid
Metabolism

Unassigned & Other
1°/0

 
   
 
 
  
 
  
  
  
   
   
   
    
 

Carbohydrate
Metabolism
2°/o

Unassigned
1 1%

Tra ns posons
2% Cellular processes
10%

Transport
2°/o
Translation
7°/o
Conserved

Hypothetical

8°/o

Transcription
3%
Signal Transduction
1%

RNAS
1%
Replication and
epair

4% Energy Metabolism
7%

Pseudo
1%

Nucleotide

Metabolism

2%

Metabolism of
Cofactors

4°/o

Upld Metabolism
3%

Hy potheticai
14°/o

 

 

 

I87

Annotation of genes involved in caabohydrge nLctabolism

Two percent of the predicted ORFs encoded by P. arcticum 27 3-4 (assuming
2,155 genes in genome) are involved in carbohydrate metabolism. P. arcticum does not
have the EMP pathway of glycolysis or the oxidative pathway of the pentose phosphate
shunt as it lacks the key enzymes 6-phosphofi'uctokinase and pyruvate kinase. P.
arcticum possesses the complete set of gluconeogenic enzymes suggesting it may use
acetate, pyruvate and oxidized sugars such as mono and di-carboxyclic acids and
intermediates of the TCA cycle as growth substrates, rather than glucose or other sugars.
The TCA cycle likely generates the reducing power necessary for the cell as the oxidative
pentose phosphate shunt is not present in P. arcticum. Biolog plate analysis revealed the
utilization of lactose by P. arcticum at 24°C but not 4°C. The presence of B- glucosidase
family enzymes suggest that lactose is broken down into galactose and glucose though
the enzymes necessary for the further breakdown of galactose are not present in the
genome. It is possible that the numerous unnamed epimerases and dehydrogenases may
be responsible for the catabolism of myo-inositol, arabitol, cellobiose and trehalose
which are positive for catabolism by Biolog reactions . .
Annotation of genes involved in arthacid metabolis’m

P. arcticum 273-4 is prototrophic for every amino acid. No complete pathways
have yet been identified for metabolism of the aromatic amino acids, though some
enzymes appearing as participants in these pathways are actually used to degrade short
chain mono and dicarboxyclic acids- indicating that these may be good carbon sources
for growth. P. arcticum 273-4 has complete pathways for the synthesis of valine, leucine

and isoleucine from pyruvate. However, it appears that there are several missing steps

188

yet to be identified in the pathways for the degradation of these amino acids. Leucine
and isoleucine degradation require either the enzyme 3- methyl-2-oxobutanoate
dehydrogenase or 2-oxoisovalerate dehydrogenase. The apparent lack of these enzymes
is interesting as these are required to carry any of the three amino acids to branched chain
fatty acid precursors- which have not been identified in P. arcticum grown in V2 TSB.
Serine can be synthesized from hydroxypyruvate or pyruvate. It can then be used to
synthesize the compatible solute glycine. Glycine may also be synthesized from
glyoxylate. Threonine, also known to bioaccumulate in Archaea, may be synthesized
from aspartate. Interestingly, no genes were identified for the metabolism of choline
except for betA and betB, which yield betaine— particularly important since only betaine
and proline are the only compatible solutes produced by Psychrobacter arcticum. It
appears that glycine, serine, glutamate, glutamine, aspartate, arginine, and proline are
likely to be good carbon and nitrogen sources for the growth of P. arcticum as all of these
amino acids have complete pathways linking them to the TCA cycle.
Annotation of P. arcticum transcription related genes

Three percent of the genome of P. arcticum 273-4 is involved in transcription.
Forty nine tRNA molecules were identified. The P. arcticum genome contains numerous
transcription regulatory factors which bind DNA through a helix-tum-helix motif in the
LysR, ArsR, GntR, TetR, Lrp and LuxR families. Sigma 32, 54 and 70 are the
transcriptional factors for P. arcticum 273-4. A negative regulatory element of sigma 54
was located adjacent to the transcription factor gene while another anti-sigma factor, with
weak similarity to chrR, was identified. Four cold shock genes were identified: cspE,

cspA, capA and an unnamed cold shock binding domain protein. Cold shock proteins are

189

RNA binding proteins having chaperone activity (cspA) and as transcription anti-
terminators (cspE) in E. coli (Phadtare et al., 2000; Phadtare et al., 2002). Twelve
histidine kinase/sensor domain pairs were also identified. Three anti-termination factor
homologs were detected with the NusB homolog adjacent to a GreA transcription

elongation factor.

190

References
Bowman, J. P. (2005). The Genus Psychrobacter. The Promotes.

Bowman, J. P., S. A. McCammon, M. V. Brown, D. S. Nichols and T. A. McMeekin
(1997). "Diversity and Association of Psychrophilic Bacteria in Antarctic Sea
Ice." Applied and Environmental Microbiology 63(8): 3068-78.

Christner, B. C. (2002). "Incorporation of DNA and Protein Precursors into
Macromolecules by Bacteria at -15°C." Applied and Environmental Microbiology
68(12): 6435-38.

Firstenberg-Eden, R., D. B. Rowley and G. E. Shattuck (1980). "Factors Affecting
Inactivation of Moraxella- Acientobacter Cells in an Irradiation Process." Applied
and EnvironmentaJfiMicrobiolgy 40: 480-85.

Gilichinsky, D., E. Rivkina, V. Shcherbakova, K. Laurinavichus and J. Tiedje (2003).
"Supercooled Water Brines within Permafrost- an Unknown Ecological Niche for
Microorganisms: A Model for Astrobiology." Astrobiology 3(4): 331-41.

Moss, C. W., P. L. Wallace, D. G. Hollis and R. E. Weaver (1988). "Cultural and
Chemical Characterization of Cdc Groups Eo-2, M-5 and M-6, Moraxella
(Moraxella) Species, Oligella urethralis, Acientobacter Sp. And Psychrobacter
immobilis." Journal of Clinical Microbiolgy 26: 484-92.

Phadtare, S., M. Inouye and K. Severinov (2002). "The Nucleic Acid Melting Activity of
Escherichia coli cspE Is Critical for Transcription Antiterrnination and Cold
Acclimation of Cells." Journal of Biological Chemist_ry 277(9): 7239-45.

Phadtare, S., K. Yamanaka and M. Inouye (2000). The Cold Shock Response. Bacterial
Stress Responses. G. Storaz and R. Hengge- Aronis. Washington DC, ASM
press: 33-47.

Rodriguez-Calleja, J. M., M. F. Patterson, I. Garcia-Lopez, J. A. Santos, A. Otero and M.
L. Garcia-Lopez (2005). "Incidence, Radioresistance, and Behavior of
Psychrobacter Spp. In Rabbit Meat." Journal of Food Protection 68(3): 538-43.

Scholes, R. B. and J. M. Shewan (1964). "The Present Status of Some Aspects of Marine
Microbiology." Advances in Marine Biology 2: 133-69.

191

Vela, A. I., M. D. Collins, M. V. Latre, A. Mateos, M. A. Moreno, R. Hutson, L.
Dominguez and J. F. Fernandez-Garayzabal (2003). "Psychrobacter pulmonis Sp.
Nov., Isolated from the Lungs of Lambs." International Journal of Systematic and

Evolutionary Microbiology 53(2): 415-19.

Vishnivetskaya, T., S. Kathariou, J. McGrath and J. M. Tiedje (2000). "Low Temperature
Recovery Strategies for the Isolation of Bacteria from Ancient Permafrost
Sediments." Extremophiles 4: 165-73.

192

APPENDIX B

Expression profiles of P. arcticum 273-4 in different salinities and temperatures.

Table B-1: Average number of copies of mRNA for select genes at different salinities
and temperatures as determined by quantitative real- time PCR

 

 

(D N O N O O O O O
V ‘- O (‘0 O C O O
m a a a a o
Q ‘- F' f- 8
v- '-
U
°n
Rise
V" N O O) O O O O
N N O CD 0 O O O
‘2 (‘0 co (‘0 O) O ‘- O
o on N no N N
m ‘- 2
90
NM
N2
00 N O O) O O N O O
CD (0 O (‘0 O O 3 O O
0') O) O G O N O
[x ‘- N N 00 o
Q ‘- N
‘- (O
9.0
#3
O 00 O O O) O O O O
O) 0‘) O 0 00 O O O O
1- 0') O) O (0 O O 00 O
‘2 in N as on a: o
0 ‘- N v N
In— N
00
o (0
V2
0) N M O O)
IN 3 w P w m N
g 00 to v o to co 0:
cl 8 ‘C u t c: a <2
8 O m 0 O O O O O

193

Table B-2. Average log odd score for genes differentially expressed when comparing
22°C V2 TSB + 5% NaCl versus 22°C V2 TSB growth in P. arcticum

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

22 NaCl 4 NaCl 22 NaCI 22TSB
Gene Predicted function vs. 22TSB vs. vs.
number 4TSB 4NaCl 4TSB
June
2003
1077 Probable UDP-N- -14.4 -5.2 -5.3 -2.5
acetylmuramoylalanyl-D-
glutamate-2,60iaminopimelate
masgpeptidoglycan synthetases)
1777 Probable succinate -7.8 -2.3 -2.5 -4.3
dehydrogenase
1897 Putative fimbriai protein pilin -4.6 -0.3 -2.0 0.0
1462 Reca bacterial DNA recombination 4.0 3.1 -1.9 07
protein
2611 Putative dtdp-4-dehydrorhamnose -3.9 -0.4 -1.7 2.9
reductase
938 Response regulator receiver -3.8 0.3 -1.5 -1.3
1899 Putative type IV prepilin -3.4 -2.2 -5.5 7.9
222 ATP/GTP-binding site motif A (P— -2.7 1.9 -2.2 0.0
100p):parb-Iike nucleasezparb-like
partition protein
843 Putative Bacterial type Ii secretion -2.6 -0.7 -1.0 -2.6
system protein E/Pilus retraction
protein pilt
38 Possible Copper-resistance protein -2.6 -2.6 1.7 2.3
copb
2548 Putative methylisocitrate lyase -2.4 4.6 0.5 -2.9
1071 Aconitate hydratase -2.3 7.0 1.5 -1.0
2389 Putative epimerase -2.3 -0.3 -5.9 5.3
1260 Putative two-component response -2.2 0.4 -4.0 -1.7
regulator (motility)
2238 Glycine cleavage H-protein -2.2 4.9 -1.4 -2.3
2464 Hypothetical protein -2.2 3.5 -3.6 -0.7
76 Probable Glutathione S- -2.2 5.6 -0.4 -4.1
transferase
1465 Predicted RNA-binding protein -2.1 1.0 32.3 -0.1
containigq KH domain
2146 Phoshatidyl diacylglycerol -2.1 1.2 -0.6 -1.5
acytransferase
210 Carbohydrate kinase, probable -2.0 -3.2 3.2 3.3
FGGYzGcherol kinase
597 Putative ribonuclease d -2.0 3.2 -1.4 -2.4
2867 Hypothetical protein 2.0 -3.2 2.5 1.0
813 Possible transcriptional regulator 2.0 0.7 0.2 -7.6
(homologof ng accessory factor)
1974 Putative 4Fe-48 ferredoxin 2.0 -2.4 2.9 0.0
331 Conserved hypothetical protein 2.1 -2.4 1.6 2.7
825 Hypothetical 2.3 -2.8 2.0 2.6

 

194

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table B—2 22 NaCl 4 NaCl 22 NaCi 22TSB
Gene Predicted function vs. 22T vs. vs. vs.
number 4TSB 4NaCl 4TSB
June
2003
1293 Conserved hypothetical (DUF81, 5 2.4 -2.3 1.7 3.3
transmembrane helices)
2763 Putative Ubiquinol-cytochrome c 2.7 -2.5 -2.7 -3.7
reductase, iron-sulfur subunit
2510 LysyI-trna synthetase, 3.0 0.6 -5.9 1.2
1649 Hypothetical protein 3.0 -2.8 1.8 3.3
1612 Conserved hypothetical (DUF81, 5 3.1 -2.6 1.7 -0.4
transmembrane helices)
1247 Conserved hypothetical (DUF81, 5 3.1 -3.5 1.8 1.6
transmembrane helices)
2702 Hypothetical protein 3.3 -3.6 1.8 0.1
2534 Possible DNA-binding protein 3.4 -2.7 2.0 -0.3
1494 Possible transcriptional regulator, 3.4 0.3 -1.5 -2.4
Iysr-family
2050 Putative tryptophan synthase, 3.5 5.2 -0.8 -3.1
alpha chain
2134 Putative flavodoxin 3.5 -5.0 1.8 3.1
1975 Adenosylmethionine—B-amino-7- 3.6 -4.7 1.7 -0.1
oxononanoate aminotransferase
2584 AcyI-coa dehydrogenase 3.6 -3.4 -0.6 0.6
2730 Probable serine-glyoxylate 3.7 0.1 -4.9 1.1
aminotransferase
210 Carbohydrate kinase, probable 3.7 -3.2 3.2 -0.8
FGGYzGcherol kinase
879 Sigma-70 factor family 3.7 -3.2 3.2 -0.8
1058 Putative topoisomerase IV subunit 3.8 -0.7 2.3 0.9
B
1024 Conserved hypothetical 3.8 -0.5 -1.9 -3.2
1598 Putative electron transfer 4.1 1.3 -1.3 -3.8
flavoprotein-ubiquinone
oxidoreductase
587 Hypothetical protein 4.1 -1.7 -5.7 0.0
25 Probable trka: K+ transport 4.2 0.1 1.7 5.3
systems with NAB-binding site
1654 Hypothetical (6 transmembrane 4.8 -3.7 -1.2 0.8
helices predicted)
2666 Hypothetical protein 4.8 -3.1 0.3 0.9
1630 Probable prolipoprotein 4.8 -1.2 0.4 -1.5
diacylglyceryl transferase
(lipoprotein biogenesis)
983 Conserved hypothetical 4.9 0.7 -1.4 -2.7
2263 Probable Mechanosensitive (MS) 4.9 -1.5 -1.8 -1.5
ion channel
779 Conserved hypothetical 5.0 -5.4 -1.7 11.6
1305 Conserved hypothetical protein 5.0 -2.1 0.8 3.1
393 Protein of unknown function DUF6 5.1 -3.2 -0.2 -O.7
1303 Putative phosphinothricin 5.2 -1.7 -1.7 -0.1
acetyltransferase

 

 

 

 

 

 

195

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table B-2 22 NaCI 4 NaCI 22 NaCI 22TSB
Gene Predicted function vs. 22T vs. vs. vs.
number 4TSB 4NaCl 4TSB
June
2003
2551 Conserved hypothetical, DUF453 5.2 -3.5 -0.9 1.1
family
1030 Probable formyl transferase 5.2 -9.2 0.2 0.3
1592 Conserved hypothetical protein 5.2 1.0 -2.2 -2.7
2705 Probable giycerate kinase 5.3 -0.6 0.4 2.7
1488 Transcriptional regulator, arsr 5.3 -5.4 -1.1 4.3
family
2582 Conserved hypothetical protein 5.5 -2.5 0.4 -0.8
2461 Putative phage Peptidase U7 5.5 -2.8 -0.4 7.1
24 Probable Cation transporter 5.5 -5.8 -4.1 0.8
1697 Possible ABC transporter 5.5 -5.2 -2.7 2.8
380 Conserved hypothetical protein 5.6 -1.6 -2.5 0.6
1244 Probable peptidase M16 familiy 6.0 -8.6 1.3 0.4
(inactive domain)
2715 Probable cobalmin 6.1 -1.2 2.9 5.9
adenosyltransferase
1013 Hypothetical protein 6.6 19.2 6.0 -1.0
2373 Major facilitator superfamily sugar 7.0 -5.6 0.6 1.1
transporter
1589 Putative ATP-dependent Clp 7.2 -4.7 1.5 2.1
protease, ATP-binding subunit
1169 Conserved hypothetical (metal 7.9 -1.6 -1.5 6.7
dependent phosphohydroiase)
1123 Probable neutral zinc 9.2 0.6 -1.2 5.4
metallopeptidase
2613 Putative dtdp—4-dehydrorhamnose 16.3 0.8 3.4 0.0

 

 

Table B-3. Average log odd score of genes with 2 fold difference for P. arcticum cells
grown at 4°C V2 TSB + 5% NaCl versus 4°C V2 TSB

 

 

 

 

 

 

 

 

 

 

 

 

June Predicted function 22 Nacl 4 NaCI 22 NaCI 22TSB
2003
Number
vs. 22T vs. vs. vs.
4TSB 4NaC| 4TSB
1030 Probable formyl transferase 5.2 -9.2 0.2 0.3
1244 Probable peptidase M16 familiy 6.0 -8.6 1.3 0.4
(inactive domain)
1436 Conserved hypothetical protein, -0.3 -8.4 5.4 -4.5
putative membrane protein
1950 Hypothetical protein 1.4 -6.8 2.2 2.4
24 Probable Cation transporter 5.5 -5.8 -4.1 0.8
2373 Major facilitator superfamily sugar 7.0 -5.6 0.6 1.1
transporter
779 Conserved hypothetical 5.0 -5.4 -1.7 11.6
1488 Transcriptional regulator, arsr 5.3 -5.4 -1.1 4.3
family
1077 Probable UDP-N- -14.4 -5.2 -5.3 -2.5

 

acetylmuramoylalanyi-D-

 

 

 

 

 

196

 

 

 

glutamate-2,6-diaminopimelate

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table B-3 Predicted function 22 Nael 4 NaCI 22 NaCI 22TSB
vs. 22T vs. vs. vs.
4TSB 4NaCI 4TSB
1697 Possible ABC transporter 5.5 -5.2 -2.7 2.8
2134 Putative flavodoxin 3.5 -5.0 1.8 3.1
1975 Adenosylmethionineu8-amino-7- 3.6 -4.7 1.7 -0.1
oxononanoate aminotransferase
1589 Putative ATP-dependent Clp 7.2 -4.7 1.5 2.1
protease, ATP-bindflg
1237 Hypothetical protein (2 1.8 -4.1 1.7 -1.7
transmembrane helices)
1654 Hypothetical (6 transmembrane 4.8 -3.7 -1.2 0.8
helices predicted)
2702 Hypothetical protein 3.3 -3.6 1 .8 0.1
1247 Conserved hypothetical (DUF81, 3.1 -3.5 1.8 1.6
5 transmembrane helices)
2551 Conserved hypothetical, DUF453 5.2 -3.5 -0.9 1.1
» family
2584 Acyl-coa dehydrogenase 3.6 -3.4 -0.6 0.6
342 Malonyl-coa ACP transacylase -1.9 -3.3 0.1 4.3
2867 Hypothetical protein 2.0 -3.2 2.5 1.0
210 Carbohydrate kinase, probable -2.0 -3.2 3.2 3.3
FGGY:Glycerol kinase
879 Sigma-70 factor family 3.7 -3.2 3.2 -0.8
393 Protein of unknown function 5.1 -3.2 -0.2 -0.7
DUF6
1155 Possible aspartate/glutamate 0.3 -3.2 2.5 3.9
racemase
2666 Hypothetical protein 4.8 -3.1 0.3 0.9
1649 Hypothetical protein 3.0 -2.8 1.8 3.3
825 Hypothetical 2.3 -2.8 2.0 2.6
2461 Putative phage Peptidase U7 5.5 -2.8 -0.4 7.1
2534 Possible DNA-binding protein 3.4 -2.7 2.0 -0.3
187 Probable patatin-related protein -1.6 -2.7 -2.1 1.9
38 Possible Copper-resistance -2.6 -2.6 1.7 2.3
protein copb
1612 Conserved hypothetical (DUF81, 3.1 -2.6 1.7 -O.4
5 transmembrane helices)
2763 Putative Ubiquinol-cytochrome c 2.7 -2.5 -2.7 -3.7
reductase, iron-sulfur subunit
1819 Possible serine protease 0.8 -2.5 2.2 2.2
2582 Conserved hypothetical protein 5.5 -2.5 0.4 -0.8
331 Conserved hypothetical protein 2.1 -2.4 1.6 2.7
1974 Putative 4Fe-4S ferredoxin 2.0 -2.4 2.9 0.0
1293 Conserved hypothetical (DUF81, 2.4 -2.3 1.7 3.3
5 transmembrane helices)
1777 Probable succinate -7.8 -2.3 -2.5 -4.3
dehydLoqenase
1899 Putative type IV prepilin -3.4 -2.2 -5.5 7.9
1305 Conserved hypothetical protein 5.0 -2.1 0.8 3.1
2629 Probable glucose dehydrogenase -0.6 -2.1 0.2 -0.1

 

(PQQ dependent)

 

 

 

 

 

I97

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table B-3 Predicted function 22 NaCI 4 NaCI 22 NaCI 22TSB
vs. 22T vs. vs. vs.
4TSB 4NaCI 4TSB
739 Possible chalcone synthase -0.4 2.0 0.5 -2.2
903 Possible 4-diphosphocytidyI-ZC- -0.2 2.0 -0.9 -2.7
methyl-D-erythritol kinase
1235 Conserved hypothetical (DUF74) 1.9 2.0 -2.6 -3.5
28 Conserved hypothetical protein -0.6 2.2 2.6 0.2
1689 Na+-translocating -1.4 2.2 -6.0 -3.1
NADquniquinone oxidoreductase
subunit qu4
1772 Conserved hypothetical protein 0.2 2.4 -0.9 -3.2
996 Conserved hypothetical -0.7 2.8 -1.4 -4.8
1391 Fumarate hydratase, class II -0.9 2.9 -2.2 -1.1
(fumarase)
1462 Reca bacterial DNA -4.0 3.1 -1.9 -0.7
recombination protein
597 Putative ribonuclease d -2.0 3.2 -1.4 -2.4
1657 Probable two-component sensor -0.2 3.3 -2.7 -1.1
histidine kinase
2464 Hypothetical protein -2.2 3.5 -3.6 -0.7
2382 Hypothetical protein with ATP 0.4 3.5 -4.4 -0.9
binding domain
868 Putative wax ester synthase/acyl- -1.7 3.6 1.3 -1.4
coazdiacylglycerol
acyltransferase; fatty acyl-coa
acyltransferase
132 Transcription-repair coupling 0.4 3.7 -2.3 -4.1
factor
2296 Putative 0.9 3.9 -0.5 -3.8
Choline/CamitinelBetaine
transporter (BCCT)
309 Probable integral membrane 1.7 4.0 -1.5 -3.4
protein, rard-like
1720 Conserved hypothetical -1.4 4.3 -2.2 -1.8
85 Cytidyltransferase -0.1 4.4 0.0 -1.9
2548 Putative methylisocitrate lyase -2.4 4.6 0.5 -2.9
1768 Porphobilinogen deaminase 1.8 4.8 -1.9 -3.5
2238 Glycine cleavage H-protein -2.2 4.9 -1.4 -2.3
2050 Putative tryptophan synthase, 3.5 5.2 -0.8 -3.1
alpha chain
299 Putative xanthine/uracillvitamin C -1.1 5.6 -3.8 -0.8
transport family protein
299 Putative xanthine/uraciI/vitamin C -1.1 5.6 -3.8 -0.8
transport family protein
76 Probable Glutathione S- -2.2 5.6 -0.4 -4.1
transferase
2385 Putative lipida disaccharide -1.8 5.7 -2.1 -2.5
gynthetase
2385 Putative lipida disaccharide -1.8 5.7 -2.1 -2.5
synthetase
310 Dihydroneopterin aldolase -1.4 6.6 -0.2 -3.9
familyzoihydroneopterin aldolase
1071 Aconitate hydratase -2.3 7.0 1.5 -1.0

 

 

 

 

 

 

198

 

 

1013

Hypothetical protein

6.6

19.2

6.0

-1.0

 

 

1735

 

Possible heat shock protein dnaj

 

-0.4

 

22.6

 

3.1

 

-9.0

 

Table B-4. Average log odds score for orfs with at least two-fold difference between P.
arcticum cells grown at 22°C in V2 TSB + 5% NaCl versus 4°C in V2 TSB + 5%

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

NaCI
3-Jun Predicted function 22 Nacl 4 NaCl 22 NaCI 22TSB
vs. 22T vs. vs. vs.
4TSB 4NaCI 4TSB
608 Amino acid permease-associated 1.5 1.3 -7.2 3.2
refln
1689 Na+-transiocating -1.4 2.2 -6.0 -3.1
NADquniquinone oxidoreductase
subunit qu4
2510 Lysyl-trna synthetase, 3.0 0.6 -5.9 1.2
2389 Putative epimerase -2.3 -0.3 -5.9 5.3
2397 CBS domain:Sugar isomerase 1.4 -1.5 -5.8 7.7
(SlS):kriflgutq family protein
587 Hypothetical protein 4.1 -1.7 -5.7 0.0
1037 Grpe protein -0.9 0.7 -5.6 -1.6
1899 Putative type IV prepilin -3.4 -2.2 -5.5 7.9
1077 Probable UDP-N- -14.4 -5.2 -5.3 -2.5
acetylmuramoylalanyi-D-
glutamate-2,6-diaminopimelate
ligase (peptidoglycan
synthetases)
1416 L-Iactatelglycoiate permease 1.8 -0.2 -5.1 -1.8
(H+:Iactate sym porter)
2730 Probable serine-glyoxylate 3.7 0.1 -4.9 1.1
aminotransferase
1962 Putative acyl-coa carboxyiase -0.1 0.9 -4.4 -2.3
alpha chain protein
2382 Hypothetical protein with ATP 0.4 3.5 -4.4 -0.9
binding domain
1520 Probable sodiumzproton 1.5 1.0 -4.2 -2.2
dicarboxylate (glutamate)
symporter
2380 EnoyI-coa hydratase/isomerase -1.1 0.4 -4.2 -1.6
family protein
2811 Ribosomal protein 820 1.3 0.7 -4.2 -0.7
24 Probable Cation transporter 5.5 -5.8 -4.1 0.8
1260 Putative two-com ponent response -2.2 0.4 -4.0 -1.7
regulator (motility)
299 Putative xanthine/uracillvitamin C -1.1 5.6 -3.8 -0.8
transport family protein
1599 Conserved hypothetical (HTH 1.5 0.8 -3.7 -2.7
motif, 2 transmembrane helices)
2464 Hypothetical protein -2.2 3.5 -3.6 -0.7
314 Straight chain dicarboxcylic acid -0.1 0.0 -3.2 -1.8
Putative acetyl-coa
hydrolaseltransferase family
protein
1697 Possible ABC transporter 5.5 -5.2 -2.7 2.8

 

199

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table B-4 Predicted function 22 Nacl 4 NaCI 22 NaCI 22TSB
vs. 22T vs. vs. vs.
4TSB 4NaCI 4TSB
1657 Probable two-component sensor -0.2 3.3 -2.7 -1.1
histidine kinase
2763 Putative Ubiquinol-cytochrome c 2.7 -2.5 -2.7 -3.7
reductase, iron-sulfur subunit
1235 Conserved hypothetical (DUF74) 1.9 2.0 -2.6 -3.5
29 Outer membrane efflux protein 0.5 1.2 -2.6 -1.5
380 Conserved hypothetical protein 5.6 -1.6 -2.5 0.6
551 Putative phosphate transporter 1.2 0.7 -2.5 -0.5
1777 Probable succinate -7.8 -2.3 -2.5 -4.3
dehydrogenase
132 Transcription-repair coupling 0.4 3.7 -2.3 -4.1
factor
1391 Fumarate hydratase, class II -0.9 2.9 -2.2 -1.1
(fumarase)
222 ATP/GTP-binding site motif A (P- -2.7 1.9 -2.2 0.0
loop):parb-like nucleasezparb-Iike
partition protein
1592 Conserved hypothetical protein 5.2 1.0 ~22 -2.7
1720 Conserved hypothetical -1.4 4.3 -2.2 -1.8
187 Probable patatin-related protein -1.6 -2.7 -2.1 1.9
2385 Putative lipida disaccharide -1.8 5.7 -2.1 -2.5
synthetase
825 Hypothetical 2.3 -2.8 2.0 2.6
2534 Possible DNA-binding protein 3.4 -2.7 2.0 -0.3
1819 Possible serine protease 0.8 -2.5 2.2 2.2
1950 Hypothetical protein 1.4 -6.8 2.2 2.4
1058 Putative topoisomerase IV 3.8 -0.7 2.3 0.9
subunit B
2867 Hypothetical protein 2.0 -3.2 2.5 1.0
1155 Possible aspartate/giutamate 0.3 -3.2 2.5 3.9
racemase
28 Conserved hypothetical protein -O.6 2.2 2.6 0.2
2388 Spermidine- putative 1.6 -1.5 2.7 3.2
saccharopine dehydrogenase
family protein
2715 Probable cobalmin 6.1 -1.2 2.9 5.9
adenosyltransferase
1974 Putative 4Fe-48 ferredoxin 2.0 -2.4 2.9 0.0
1735 Possible heat shock protein dnaj -0.4 22.6 3.1 -9.0
210 Carbohydrate kinase, probable 3.7 -3.2 3.2 -0.8
FGGYzGiycerol kinase
879 Sigma-70 factor family 3.7 -3.2 3.2 -0.8
2613 Putative dtdp-4- 16.3 0.8 3.4 0.0
dehydrorhamnose
1435 Conserved hypothetical protein -0.3 -8.4 5.4 -4.5
1436 Conserved hypothetical protein, -0.3 -8.4 5.4 -4.5
putative membrane protein
1013 Hypothetical protein 6.6 19.2 6.0 -1.0
1465 Predicted RNA-binding protein -2.1 1.0 32.3 -0.1

 

containing KH domain

 

 

 

 

 

200

 

Table B-5. Average fold change of genes with two fold difference when grown in 22°C
V2 TSB versus 4°C V2 TSB. Average of six biological replicates and dye swapped

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

p value <0.05
June Predicted function 22 NaCl 4 NaCl 22 NaCl 22st
2003
number
Vs. 22st vs. Vs. vs. 4st
4st 4NaCI
1735 Possible heat shock protein dnaj -0.4 22.6 3.1 -9.0
813 Possible transcriptional regulator 2.0 0.7 0.2 -7.6
3-jun Predicted function 22 nacl 4 nacl 22 nacl 22tsb
Vs. 22f vs. Vs. 4nacl vs. 4tsb
4tsb
200 Probable transmembrane protein 1.9 1.9 -1.0 -5.0
996 Conserved hypothetical -0.7 2.8 -1.4 -4.8
1435 Conserved hypothetical protein -0.3 -8.4 5.4 -4.5
1777 Probable succinate -7.8 -2.3 -2.5 -4.3
dehydrogenase
298 Hypothetical protein -1.3 0.7 -1.6 -4.2
76 Probable glutathione s- -2.2 5.6 -0.4 -4.1
transferase
132 Transcription-repair coupling 0.4 3.7 -2.3 -4.1
factor
310 Dihydroneopterin aldolase -1.4 6.6 -0.2 -3.9
familyzdihydroneopterin aldolase
2296 Putative choline/carnitine/betaine 0.9 3.9 -0.5 -3.8
transporter (bcct)
1598 Putative electron transfer 4.1 1.3 -1.3 -3.8
flavoprotein-ubiquinone
oxidoreductase
2763 Putative ubiquinol-cytochrome c 2.7 -2.5 -2.7 -3.7
reductase, iron-sulfur subunit
1235 Conserved hypothetical (duf74) 1.9 2.0 -2.6 -3.5
1768 Probable porphobilinogen 1.8 4.8 -1.9 -3.5
deaminase
309 Probable integral membrane 1.7 4.0 -1.5 -3.4
protein, rard-like
1772 Conserved hypothetical protein 0.2 2.4 09 -3.2
1024 Conserved hypothetical 3.8 -O.5 -1.9 -3.2
1689 Na+-translocating -1.4 2.2 -6.0 -3.1
nadhzuniquinone oxidoreductase
subunit n$4
2050 Putative tryptophan synthase, 3.5 5.2 -0.8 -3.1
alpha chain
2548 Putative methylisocitrate lyase -2.4 4.6 0.5 -2.9
1592 Conserved hypothetical protein 5.2 1.0 -2.2 -2.7
1599 Conserved hypothetical (hth 1.5 0.8 -3.7 -2.7
motif, 2 transmembrane helices)
983 Conserved hypothetical 4.9 0.7 -1.4 -2.7
903 Possible 4-diphosphocytidyl-2c- -0.2 2.0 -0.9 -2.7
methyl-d-erythritol kinase
2606 Probable glycosyl transferase, 0.8 0.5 -0.9 -2.6

 

201

 

 

family 2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table B-5 Predicted function 22 nacl 4 nacl 22 nacl 22tsb
Vs. 22t vs. Vs. 4nacl vs. 4tsb
4tsb
843 Putative bacterial type ii secretion -2.6 -0.7 -1.0 -2.6
system protein elpilus retraction
protein pilt
1077 Probable udp-n- -14.4 -5.2 -5.3 -2.5
acetylmuramoylalanyI-d-
glutamate-2,6-diaminopimelate
ligase (peptidoglycan
synthetases)
2385 Putative lipida disaccharide -1.8 5.7 -2.1 -2.5
synthetase
597 Putative ribonuclease d -2.0 3.2 -1.4 -2.4
1494 Possible transcriptional regulator, 3.4 0.3 -1.5 -2.4
iysr-family
549 Phosphate metabolism -1.0 0.4 -0.1 -2.3
1962 Putative acyl-coa carboxyiase -0.1 0.9 -4.4 -2.3
alpha chain protein
2238 Glycine cleavage h-protein -2.2 4.9 -1.4 -2.3
1520 Probable sodiumzproton 1.5 1.0 -4.2 -2.2
dicarboxylate (glutamate)
symporter
739 Possible chalcone synthase -0.4 2.0 0.5 -2.2
2864 Heavy metal response regulator: 0.3 1.7 1.1 -2.1
response regulator receiver ,
transcriptional regulatory protein
1589 Putative atp-dependent clp 7.2 —4.7 1.5 2.1
protease, atp-binding subunit
1819 Possible serine protease 0.8 -2.5 2.2 2.2
38 Possible copper-resistance -2.6 -2.6 1.7 2.3
protein copb
1950 Hypothetical protein 1.4 -6.8 2.2 2.4
825 Hypothetical 2.3 -2.8 2.0 2.6
331 Conserved hypothetical protein 2.1 -2.4 1.6 2.7
2705 Probable giycerate kinase 5.3 -O.6 0.4 2.7
1697 Possible abc transporter 5.5 -5.2 -2.7 2.8
2611 Putative dtdp-4- -3.9 -0.4 -1.7 2.9
dehydrorhamnose reductase
1305 Conserved hypothetical protein 5.0 -2.1 0.8 3.1
2134 Putative flavodoxin 3.5 -5.0 1.8 3.1
608 Amino acid permease-associated 1.5 1.3 -7.2 3.2
region
2388 Spermidine- putative 1.6 -1.5 2.7 3.2
saccharopine dehydrogenase
family protein
1649 Hypothetical protein 3.0 -2.8 1.8 3.3
1293 Conserved hypothetical (duf81, 5 2.4 -2.3 1.7 3.3
transmembrane helices)
210 Carbohydrate kinase, probable -2.0 -3.2 3.2 3.3
fggy_:glycerol kinase
931 Phospholipid/glycerol -0.8 1.6 -1.7 3.6

 

202

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

acyltransferase
Table B-5 Predicted function 22 NaCl 4 NaCl 22 NaCl 22st
Vs. 22st vs. Vs. vs. 4st
4st 4NaCI
1155 Possible aspartate/glutamate 0.3 -3.2 2.5 3.9
racemase
342 Malonyl-coa acp transacylase -1.9 -3.3 0.1 4.3
1488 Transcriptional regulator, arsr 5.3 -5.4 -1.1 4.3
family
2389 Putative epimerase -2.3 -0.3 -5.9 5.3
25 Probable trka : k+ transport 4.2 0.1 1.7 5.3
systems with had-binding site
1123 Probable neutral zinc 9.2 0.6 -1.2 5.4
metallopeptidase
2715 Probable cobalmin 6.1 -1.2 2.9 5.9
adenosyltransferase
1169 Conserved hypothetical (metal 7.9 -1.6 -1.5 6.7
dependent phosphohydroiase)
2461 Putative phage peptidase u7 5.5 -2.8 -0.4 7.1
2397 C05 domainzsugar isomerase 1.4 -1.5 -5.8 7.7
1899 Putative type iv prepilin -3.4 -2.2 -5.5 7.9
779 Conserved hypothetical 5.0 -5.4 -1.7 11.6

 

Table B-6. Average fold change for P. arcticum orfs that were differentially expressed
above two fold when grown at 22 °C in V2 TSB

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

June Predicted function 22 NaCI 4 NaCl 22 NaCl 22st
2003 vs. Vs.
Number Vs. 22st 4st 4NaCI vs. 4st
Fewer transcripts when grown at 220 in 1/2 tsb
813 Possible transcriptional regulator 2.0 0.7 0.2 -7.6
(homfig of bvg accessory factor)
200 Probable transmembrane protein 1.9 1.9 -1.0 -5.0
1598 Putative electron transfer flavoprotein- 4.1 1.3 -1.3 -3.8
ubiggiinone oxidoreductase
2763 Putative ubiquinol-cytochrome c 2.7 -2.5 -2.7 -3.7
reductase, iron-sulfur subunit
1024 Conserved hypothetical 3.8 -0.5 -1.9 -3.2
2050 Putative tryptophan synthase, alpha 3.5 5.2 0.8 -3.1
chain
1592 Conserved hypothetical protein 5.2 1.0 -2.2 -2.7
983 Conserved hypothetical 4.9 0.7 -1.4 -2.7
1494 Possible transcriptional regulator, 3.4 0.3 -1.5 -2.4
lysr-family
More transcripts grown at 220 in 1/2 tsb
38 Possible copper-resistance protein -2.6 -2.6 1.7 2.3
copb
2611 Putative dtdp-4-dehydrorhamnose -3.9 -0.4 -1.7 2.9
reductase
210 Carbohydrate kinase, probable -2.0 -3.2 3.2 3.3
fggy_:glycerol kinase
2389 Putative epimerase -2.3 -0.3 -5.9 5.3
1899 Putative type iv prepilin -3.4 -2.2 -5.5 7.9

 

 

 

 

 

 

203

 

 

Table B-7. Average fold change of P. arcticum genes that were differentially expressed
two fold or more when grown in 22°C V2 TSB +5% NaCI

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3-Jun Predicted function 22 Nacl 4 NaCl 22 NaCI 22TSB
vs. 22T vs. vs. vs.
4TSB 4NaCI 4TSB
Fewer transcripts when grown at 220 in 1/2 TSB + 5% nacl
1077 Probable UDP-N- -14.4 -5.2 -5.3 -2.5
acetylmuramoylalanyi-D-
glutamate-2.6-diaminopimelate
Ii ase (peptidoglycan)
1777 Probable succinate -7.8 -2.3 -2.5 -4.3
dehydrogenase
1897 Putative fimbriai protein pilin -4.6 -0.3 -2.0 0.0
1899 Putative type iV prepilin -3.4 -2.2 -5.5 7.9
222 ATP/GTP-binding site motif A (P- -2.7 1.9 -2.2 0.0
loop):parb-Iike nucleasezparb-like
partition protein
2389 Putative epimerase -2.3 -O.3 -5.9 5.3
1260 Putative two-component response -2.2 0.4 -4.0 -1.7
reglator (motility)
2464 Hypothetical protein -2.2 3.5 -3.6 -0.7
More transcripts grown at 22C in 1/2 TSB + 5% nacl
2867 Hypothetical protein 2.0 -3.2 2.5 1.0
1974 Putative 4F e-4S ferredoxin 2.0 -2.4 2.9 0.0
825 Hypothetical 2.3 -2.8 2.0 2.6
2534 Possible DNA-binding protein 3.4 -2.7 2.0 -0.3
879 Sigma-70 factor family 3.7 -3.2 3.2 -0.8
210 Carbohydrate kinase, probable 3.7 -3.2 3.2 -0.8
FGGYzGcherol kinase
1058 Putative topoisomerase lV subunit 3.8 -0.7 2.3 0.9
B
1013 Hypothetical protein 6.6 19.2 6.0 -1.0
2613 Putative dtdp-4-dehydrorham nose 16.3 0.8 3.4 0.0

 

Table B-8. Average fold change of orfs differentially expressed 2-fold or more in P.
arcticum cells grown at 4°C in V2 TSB + 5% NaCl

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3-Jun Predicted function 22 Nacl 4 NaCI 22 NaCI 22TSB
vs. 22T vs. vs. vs.
4TSB 4NaCI 4TSB
More transcripts when grown 40 in 1/2 TSB + 5% NaCI

2385 Putative lipida disaccharide -1.8 5.7 -2.1 -2.5
synthetase

1720 Conserved hypothetical -1.4 4.3 -2.2 -1.8

1391 F umarate hydratase, class II -0.9 2.9 -2.2 -1.1
(fumarase)

132 Transcription-repair coupling factor 0.4 3.7 -2.3 -4.1

299 Putative xanthine/uracil/vitamin C -1.1 5.6 -3.8 -0.8

 

204

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

transport family protein
2382 Hypothetical protein with ATP 0.4 3.5 -4.4 -0.9
binding domain
1689 Na+-translocating NADquniquinone -1.4 2.2 -6.0 -3.1
oxidoreductase subunit qu4
1657 Probable two-component sensor -0.2 3.3 -2.7 -1.1
histidine kinase
2464 Hypothetical protein -2.2 3.5 -3.6 -0.7
Fewer transcripts grown at 40 in 1/2 TSB + 5% nacl
1436 Conserved hypothetical protein, -0.3 -8.4 5.4 -4.5
putative membrane protein
210 Carbohydrate kinase, probable -2.0 -3.2 3.2 3.3
FGGYzGcheroi kinase
879 Sigma-70 factor family 3.7 -3.2 3.2 -0.8
1974 Putative 4Fe—48 ferredoxin 2.0 -2.4 2.9 0.0
1155 Possible aspartate/glutamate 0.3 -3.2 2.5 3.9
racemase
2867 Hypothetical protein 2.0 -3.2 2.5 1.0
1950 Hypothetical protein 1.4 -6.8 2.2 2.4
1819 Possible serine protease 0.8 -2.5 2.2 2.2
2534 Possible DNA-binding protein 3.4 -2.7 2.0 -0.3
825 Hypothetical 2.3 -2.8 2.0 2.6

 

Table B-9. Average fold change of orfs differentially expressed two-fold or greater when
P. arcticum was grown at 4°C in V2 TSB.

 

 

 

 

 

 

 

 

 

 

 

 

 

3-Jun Predicted function 22 Nacl 4 NaCl 22 NaCI 22TSB
vs. 22T vs. vs. vs.
4TSB 4NaCI 4TSB
More transcripts when grown 4C in 1/2 TSB +

1077 Probable UDP-N- -14.4 -5.2 -5.3 -2.5
acetylmuramoylalanyl-D-glutamate-
2,6-diaminopimelate ligase
(peptidoglycan synthetases)

2763 Putative Ubiquinol-cytochrome c 2.7 -2.5 -2.7 -3.7
reductase, iron-sulfur subunit

1777 Probable succinate dehydrogenase -7.8 -2.3 -2.5 -4.3

1436 Conserved hypothetical protein, -0.3 -8.4 5.4 -4.5
putative membrane protein

 

Table B-10. ORFS that are not expressed with different salinities and temperatures

Genes expressed with salt and 4°C but not in 22 V2 TSB

lune

annotation
2003

1301

Description
Malate dehydrogenase

 

 

1 706

Probable 8-amino-7-oxononanoate synthase

 

2357

Putative Type I restriction-modification system, M subunit

 

2582

Conserved hypothetical protein

 

 

2674

 

Probable lipb-like Biotin/Iipoate AIB protein iiggse domain

 

205

 

 

Table B-1 0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Genes ex ressed only with presence of 5% Nacl
35 Putative Cytochrome c heme-binding site
440 Protein of unknown function DUF218 domain
782 Putative oxidoreductase, aldo/keto reductase
808 Hypothetical protein
1108 Probable excinuclease ABC subunit C (UV damage repair)
2010 Conserved hypothetical protein
2050 Putative tryptqrhan smthase, alpha chain
2098 Ribosomal protein L15
2166 NADH dehydrogenase, F subunit
2600 Possible thioesterase
2739 Conserved Hypothetical protein
genes not expressed in 5% NaCl media but in V2 TSB
15 Transketolase, N terminalzTransketolase, central refin:
83 Putative Ribosome recycling factor
144 Chorismate synthase
180 Possible transcription factor
186 Probable transposase, lSSod6
189 hypothetical protein
195 Putative inosine 5'-monophosphate dehydrogenase
226 probable tetmacyldisaccharide-1-P 4'-kinase
putative type IV frmbriae assembly protein KO: K02676 type IV pilus assembly
229 protein PiIZ
247 Putative ATPase: ATP/GTP Binding Domain
636 Hypothetical protein
719 possible prophage minor tail protein
720 hypothetical
724 probable phage minor tail protein
739 possible chalcone synthase
960 hypothetical protein
979 Conserved hypothetical
995 Probable cytoplasmic peptidoglycan synthetases
1031 Conserved hypothetical
1032 Threonine synthase
1141 putative tyrosyl-tRNA synthetase
1157 probable S-adenosylmethionine-dependent methyltransferase function
1349 signal recognition particle protein
conserved hypothetical (carboxymuconolactone
1565 decarboxyiase:alkylhydrgperoxidase AhpD core domain)
1587 probable peptidylprolyl isomerase, FKBP-type (trigger factor)
1616 probable D-isomer specific 2-hydroxyacid dehydrogenase
1637 probable ABC-type Mn/Zn transport protein, ATPase component
1639 possible cyclic AMP phosphodiesterase (regulator of lacZ)
1647 probable phospholipase D/transphosphatidylase (cardiolipin synthases)
1689 Na+-translocating NADquniquinone oxidoreductase subunit qu4
1720 Conserved hypothetical
1734 Possible ABC transporter

 

 

 

206

 

Table B-10
1749 T

isomerase

1776 Probable succinate subunit

1782
1821

beta subunit

1876 Carbonic

1878
1913
1919
1931

1977 GTP
2127 Probable IS3/l891 1
2139 Putative Alanine

2140 Probable subunit
2150 Conserved

2158 Conserved

2416 ATP- RNA helicase RhIB

2422 Ribose isomerase

2426

5'-n

2623 Putative biotin carrier

2678
2714

m em

2719 Conserved

2720 Putative NADP malic
2760 Na+ fam

2761 ABC .AAA ATPase
2764 Probable c1

Genes expressed only at 22°C in V2 TSB

16

 

 

Conserved Hypothetical protein

 

27

Probable Bacterial outer membrane proteinzompa/motb domain

 

71

Putative ABC transporter

 

82

Putative Aspartate/glutamateluridylate kinase

 

105

Conserved Hypothetical protein

 

107

Possible Lipocalin-related protein and Bolean/Equ allergen

 

127

Putative Orotidine 5'-phosphate decarboxylase

 

149

Probable Acetone carboxyiase beta subunit

 

170

3-isopropyimalate dehydrogenase

 

208

ATP/GTP-binding site motif A (P-Ioop):Protein of unknown function DUF299

 

210

Carbohydrate kinase, probable FGGYzGcherol kinase

 

275

Glucose inhibited division protein A

 

279

Probable molybdopterin biosynthesis enzyme

 

286

Probable oxygen-independent coproporphyrinogen III oxidase

 

460

Probable Bacterial regulatory proteins, iclr family

 

462

Hypothetical protein

 

465

Putative Deoxyguanosinetriphosphate triphosphohydrolase

 

471

Transposase, mutator family

 

 

 

502

Neub family protein with antifreeze-like domains

 

207

 

 

Table B-10

 

509 Putative Glycosyl transferase, mup 1

 

516 Possible ATP phosphoribosyltransferase regulatory subunit KO: K02502

 

520 Conserved hypothetical protein 48:

 

542 Putative D-alanyl-D-alanine carboxypeLtidase

 

554 Probable two component sensor, histidine kinase

 

568 Putative uvrb excinuclease

 

601 Hypothetical protein

 

602 Atpase family protein

 

615 Probable Acyi-coenzyme A synthetases/AM P-(fatty) acid Iigases

 

621 Hypothetical protein

 

635 Probable glutathione S-transferase

 

638 Conserved hypothetical protein

 

683 Putative transposase

 

693 Putative glutamyI-trna(Gln) amidotransferase, C subunit

 

694 GIutamyI-trna(Gln) amidotransferase A subunit

 

701 Hypothetical

 

706 Probable phage portal protein

 

712 Hypothetical

 

713 Hypothetical

 

714 Hypothetical

 

727 Putative phage tail fiber protein

 

730 N-acetylmuramoyI-L-alanine amidase, putative

 

735 Proin-trna synthetase

 

772 Putative oxidoreductase, FAD binding

 

956 Possible ABC transporter

 

981 Possible phage integrase

 

990 Conserved hypothetical

 

1014 Probable oxidoreductase/dehydrogenase

 

1018 Possible SAM-dependent methyltransferase

 

1019 Conserved hypothetical

 

1023 Conserved hypothetical

 

1049 Hypothetical

 

1154 Conserved hypothetical protein

 

1188 Putative histidinol dehydrogenase (histidine metabolism)

 

1338 Chaperone protein htpg(Hsp90)

 

1359 Predicted metal-dependent phosphoesterases (PHP family)

 

1366 Hypothetical protein

 

1370 Mg-dependent deogribonuclease, tatd family

 

1381 NADP-specific glutamate dehydrogenase

 

1422 ATP-dependent protease La

 

1423 Putative Trp operon transcriptional repressor

 

1473 Putative virulence factor, mvin; MOP fiippase superfamily

 

1486 Transposase, orfa, lS3/18911family

 

1502 Hypothetical protein (3 transmembrane helices)

 

1512 Putative two-component sensor histidine kinase

 

1515 Putative outer membrane Iip0protein (antigenic properties)

 

 

 

1532 Conserved hypothetical (10 transmembrane helices)

 

208

 

 

Table B-10

 

1543 Hypothetical

 

1544 Putative biotin synthase

 

1558 Hypothetical protein

 

1569 Hypothetical protein (Iipolytic enzyme, G-D-S-L family, motig

 

1583 Conserved hypothetical (DUF72)

 

1584 Hypothetical

 

1595 Probable transglycolase (peptidoglycan binding domain 1)

 

1605 Conserved hypothetical protein

 

1646 Conserved hypothetical protein (transmembrane domains)

 

1648 Putative glutamyI-trna synthetase

 

1651 Hypothetical protein

 

1669 Hypothetical protein

 

1671 Hypothetical protein

 

1672 Conserved hypothetical DUF125—like

 

1702 Probable oxygen-independent coproporphyrinogen Ill oxidase

 

1707 Conserved hypothetical

 

1729 Probable chaperone protein dnaj

 

1730 Dihydrodipicolinate reductase

 

1737 Conserved hypothetical

 

1755 Ribosome-binding factor A

 

1756 Possible pseudouridylate synthase

 

1771 Conserved hypothetical

 

1774 Possible succinate dehydmenase, cytochrome 0 subunit

 

1785 Possible glucosephosphate uridylyltransferase

 

1806 Putative ABC transporter

 

1845 Putative N-ethylmaleimide reductase

 

1851 Probable phosphoribosylformylglycinamidine synthase

 

1852 Conserved hypothetical

 

1860 Possible site specific recombinase

 

1890 Possible competence protein, comf

 

1949 Possible phosphatase with TAT signal sequence

 

1972 Putative DNA ligase

 

1988 Putative large-conductance mechanosensitive channel

 

1990 Putative sensor histidine kinase/response regulator receiver

 

2139 Putative Alanine dehydrogenase

 

2193 Probable molybdenum ABC transporter, periplasmic bindigq protein

 

2196 Conserved hypothetical protein, probable bacterial transmembrane pair

 

2275 Conserved hypothetical protein, possible hydrolase of the HAD superfamily

 

2352 Unknown Protein with ompa/motb domain

 

2361 Putative Type I site specific deoxyribonuclease hsdr

 

2434 Glutamine synthetase class-I, adenylation site:

 

2626 Probable transcriptional regulator, asnc family

 

2630 Probable oxidoreductase

 

2636 Probable Biopglymer trangrort protein(toir)

 

2665 Probable branched chain amino acid permease

 

2682 Putative acyl-coa Thiolase

 

 

 

2686 Conserved hypothetical protein

 

209

 

 

Table B-10

 

2701 Hypothetical

 

2766 Putative Stringent starvation protein B

 

2779 Probable Formate dehydrgqenase, subunit fdhd

 

2805 Aminotransferase, class V

 

2807 Possible hesb/yadr/yfhf family protein

 

 

 

2840 Conserved Hypothetical protein

 

210

 

APPENDIX-C

EXPERIMENTAL CONDITIONS FOR PROTEOMICS

Performed by Suping Zheng of the University of Michigan

Chemicals

N-octyglucoside(OG), urea, thiourea, iminodiacetic acid, bis-tris propane, Trizma base
[Tris, tris(hydroxymethyl)aminomethane], phenylmethylsulphonylfluoride(PMSF),
ammonia bicarbonate, acetonitrile, trifluoroacetic acid(TFA), formic acid, sodium iodide,
sodium chloride, hydrogen chloride, iminodiacetic acid, alpha-cyano-4-hydroxycinnamic
acid (u-CHCA), isopropanol, and acetonitrile were purchased from Sigma (St. Louis,
MO), Tri(2-carboxyethyl)phosphine hydrochloride (TCEP) were obtained from
Pierce(Rockford, IL). Sequencing grade TPCK modified porcine trypsin was purchased
from Promega (Madison, WI). Standard buffer solution pH=4, pH=7 and pH=10was
obtained from Fisher Scientific(Burr Ridge, IL). Protein assay and bovine serum albumin
were purchased for Bio-Rad Laboratories(Richmond, CA). Start and elute buffer were
gifts fi'om Beckman coulter(Fullerton, CA), and pH were adjusted if necessary. The
water was purified by Milli-Q water filtration system (Millipore, Inc., Bedford, MA). The

reagents were used directly without further purification.

Column

PD-10 G-25 column were purchased from Amersham Pharmacia Biotech(Piscataway,

NJ), PF-2D column were gifts from Beckman Coulter.

211

LEL_C

A Beckman Coulter PF 2D system was employed for protein separation, with a Gilson
FC204 fraction collector collecting fractions after second dimension reverse phase
separation. A Beckman Coulter Gold model 126 binary pump and 166 UV detector were
used to separate proteins and introduce them into ESI mass spectrometry and UV,
fiactions fi'om UV detector were collected manually, the software which was used to

acquire the data from UV detector was written in-house.

Sample Lysis

The cells were harvested by centrifugation at the temperature under which they were
cultured. Then the cell pellet was resuspended in 4mL of 50mM tris-HCl
solution(pH=7.6) by vigorously vortex, then the solution was sonicated on ice for 10
seconds with 305 interval, repeat the procedure until the solution clear. The solution was
mixed with urea, thiourea, n-octyglucoside and TCEP, the final lysate contains 6M urea,
2M thiourea, 50mM tris-HCl, 2% n-octyglucoside, 5mM TCEP and 2mM PMSF. Then
the lysate was ultracentrifuged at 125,000g for 60 minutes, The supernatant was collected

and stored at —80°C refiigerator for further use.

Protein Quantification

The lysates were quantified using Bio-Rad protein assay based on Bradford method
with different dilutions of BSA as the standard as the procedure described in the manual.

The protein concentration of 4 cell lines varied from 2mg/ml to 12mg/ml.

212

Chromatofocuaflgand reverse phase(RP) separation

Chromatofocusing and reverse phase(RP) separation were carried out on PF2D system,
The pH of both start and elute buffer was adjusted by iminodiacetic acid or ammonia
hydroxide to 8.6 and 4.0, respectly. A PD-lO column was employed to exchange the
lysis buffer to start buffer before the sample was loaded on the column. The PF2D ID
column was equilibrated with start buffer at 0.2ml/min, then the exchanged sample(5mg
proteins) was loaded on the column, elution buffer was applied at flow rate 0.2ml/min 30
min later, 1M sodium chloride was used to elute the proteins bind on the column by
static electric interaction at 105min for 45 minutes, then water was applied to remove the
sodium chloride for two hours. The isopropanol was employed to clean the column if the
column was dirty. All the fiactions from CF were automatically collected by time with
2.5min interval, then the fractions collected by time were combined or not combined
based on the chromatogram. 250u1 of each fraction were loaded onto PF 2D 2D column
for second dimension separation by autosampler, solvent A was water with 0.1%TFA and
solvent B was acetonitrile with 0.1%TFA. The gradient was designed as the following:
5%B to 25%B in l min, 25% to 31%B in 1min, 31% to 37% B in 8min, 37% to 41%B in
8min, 41% to 67%B in 2min, to 100%B in 2 min, then hold 100%B for 1 min, 100%
goes back to 5%B in l min. The gradient was changed in linear mode with 0.75ml/min
flow rate. The column was held at 45°C. The fractions were collected by 0.93min interval
from 5min to 35min. The differential expressed proteins were found by comparing the
chromatograms from RP between fractions with the same pI or same retention time but

from different samples. The fi'actions of differential expressed proteins would be

213

divided into two parts for further analysis, one part was for direct infusion ESI-TOF MS,

the other was for MALDI-TOF MS.

ESI-TOFMS

Direct infusion of fractions fiom RP-HPLC was carried out on LCT ( Micromass,
Manchester, UK) using a syringe pump. The parameters were set as the following: ESI
positive mode, capillary voltage 3200V, sample cone 45V, RF lens 750.0V, extraction
cone 2.0V, desolvation temperature 150°C, source temperature 100°C, desolvation gas
flow 450L/hr. The external calibration was performed using direct infusion of NaI-Csl 2-
propanol solution by a syringe pump, spectra between m/z 500—4,000 were acquired, then

were deconvoluted into real mass spectrum using MaxEnt 1 provided by Masslynx.

On-line HPLC ESI-TOF MS was conducted on the fractions left from 1St dimension of
PF2D with the same retention time. A HPLC (Beckman Coulter Gold system) was
coupled with LCT ( Micromass, Manchester, UK), and the same PF2D 2D column was
used to separate proteins, the flow rate was 0.5ml/min, after column the flow was split
as 3:2 to be introduced into UV detector and ESI-TOF MS respectly, and the fi'actions

were collected by peaks manually.

Tgptic Digestion of the NPS RP-HPLC fractions

Each fraction collected from RP was concentrated to 80ul by a SpeedVac. lOul 1M

ammonia bicarbonate, Sul 0.1M DTT and 0.25ug TPCK modified trypsin were added

214

into the fractions, vortex, then the solution was incubated at 37 C for 12 hours. 50ul fresh

0.1% TFA was added to stop the digestion.

MALDI sample preparation and data acquisition

The tryptic digested peptides were cleaned by 2pm C18 Ziptips (Millipore), 50 frnol of
angiotensin I, ACTH 1-17 and ACTH(18-39) were added to the 1:4 dilution of saturated
CHCA as internal standard, 0.51.11 of the matix solution and 0.5ul sample were spotted on
the MALDI plate using two-layer method. MALDI -TOFMS was performed on MALDI-
TOF SPEC 2E(Micromass) in the delay-extraction reflectron positive ion mode. The
coarse laser energy was 20%-50%, and fine laser energy was 30%-90%, laser frequency
was 5. The source voltage was 20kV and extraction voltage was 19980V. The pulse
voltage was set 2300V, Reflectron voltage was 24500V, 15-20 spectra were collected

from 0 to 4000 m/z range for each spot.

MALDI Data analysis and database search

The spectrum was calibrated using internal standards, monoisotopic peptide mass list was
generated by Masslynx, and submitted to 273-4 unpublished database through MS-Fit
search engine for protein identification by the software written in-house, the search was
carried out allowing one missed cleavage and oxidation as modification. No limitation

was set for the molecular weight and pl.

215

 

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