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THE PSYCHROPHILIC RESPONSE TO GROWTH AT COLD
TEMPERATURES: FUNCTIONAL GENOMICS OF THE
PERMAFROST BACTERIUM PSYCHROBACTER ARCTICUS
273-4

presented by

PETER WARREN BERGHOLZ

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6/07 p:/CIRC/DateDue.indd-p.1

THE PSYCHROPHILIC RESPONSE To GROWTH AT COLD
TEMPERATURES: FUNCTIONAL GENOMICS or THE PERMAFROST
BACTERIUM PSYCHROBA CTER ARCTICUS 2734
By

Peter Warren Bergholz

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

2007

ABSTRACT
THE PSYCHROPHILIC RESPONSE TO GROWTH AT COLD TEMPERATURES:
FUNCTIONAL GENOMICS OF THE PERMAFROST BACTERIUM
PSYCHROBACTER ARCTICUS 273-4
By
Peter Warren Bergholz
Permafrost is an environment that imposes three selective pressures limiting the
metabolism of resident microbes: low temperature, low water activity, and low nutrient
availability. Furthermore, resident microbes are exposed to low continuous doses of
background y-irradiation which should shear genomic DNA. In spite of these
characteristics, microbes have been isolated from Siberian permafrost 2-3 million yr in
age. The present work aims to elucidate the cold acclimation responses that permitted.
maintenance of viability over thousands of years in Psychrobacter arcticus 273-4, an
isolate from Siberian permafrost. P. arcticus could have survived long term storage in
the permafrost by inducing a dormant survival stage upon exposure to cold. I
demonstrate that this is not the case. Rather survival of lethal stresses decreases with
decreasing growth temperature. The transcriptome response to grth at 4°C, a cardinal
temperature used in cold acclimation studies was narrow in the scope of genes
differentially expressed. However, when P. arcticus was challenged with growth at
subzero temperatures in a high salt defined medium, global expression decreased for
genes involved in energy metabolism, anabolic metabolism, and the basal machinery of
transcription and translation. Temperature compensation via gene expression was
observed in expression for carbon and nitrogen incorporation, sulfur metabolism,

biosynthesis of key amino acids and cell envelope dynamics. Changes in amino acid

metabolism could be correlated to bias in amino acid content of genes highly expressed at
subzero temperatures, suggesting that the observed changes have ecological relevance for

P. arcticus in cryo-environments.

Copyright by
Peter
Bergholz
2007

T 0 Natalie and Warren Bergholz, Rebecca and Jamie Glasgow, Rick Bergholz, Charles
T isdale, the Cunningham family, and my grandparents, who have throughout my life
inspired confidence and optimism while encouraging my curiosity for the natural world.

iv

ACKNOWLEDGEMENTS

First and foremost, I would like to acknowledge the guidance and support of my advisor,
Dr. Jim Tiedje, whose efforts at promoting my work and providing me with opportunities
was essential to my success in pursuing this degree. Having worked with Jim has
provided me with an example that I am certain will educate me far into the future as I
pursue my career.

I appreciate the efforts of my research guidance committee, Dr. Robert Britton,
Dr. Terence Marsh, Dr. Thomas Schmidt and Dr. Michael Thomashow. My committee
meetings were a source of much inspiration to push forward the quality of my work
through the incorporation of further experiments and new analyses. Their guidance
provided me with the invaluable lesson to challenge my own assumptions in every
scientific endeavor. I’d also like to thank other MSU faculty who took an interest in my
progress without taking a formal role in my education. Dr. Tom Whittam was a frequent
source of advice and friendship during the course of my education. Dr. Tom Comer was
my mentor during my teaching assistantship, and shepherded me through learning to see
matters from the point of view of my students.

I would also like to acknowledge the support and friendship of my colleagues in
the Center for Genomic and Evolutionary Studies on Microbial Life at Low
Temperatures, who contributed in innumerable ways every day to the progress of
knowledge of life in the permafrost. None of this work could have been accomplished

without the support of the staff scientists in the Research Technology and Support

Facility (RTSF) at Michigan State. In particular, the functional genomics support staff
and the staff of the Macromolecular Structure, Sequencing and Synthesis Facility. The
genome sequence of Psychrobacter arcticus was completed and initially analyzed by the
Department of Energy Joint Genome Institute and I would like to acknowledge Patrick
Chain and Natalia Ivanova for their contributions to the closure of the genome sequence
and metabolic analysis of the genome based on gene content, respectively. Teresa
Bergholz, Lukas Wick and the members of the Tiedje lab Microarray ThinkTank (MaTT)
were instrumental in the design of my gene expression rnicroarray experiments. The
remaining members of the Tiedje lab contributed to an exhilarating and diverse
environment in which to do research, and the importance of that cannot be overstated.
Finally, I would like to acknowledge the NASA Astrobiology Institute for funding
the work presented in this dissertation. Early in the course of my degree, I have to
confess that I did not fully appreciate the value of the NAI research structure which
forced geochemists, microbiologists, astronomers, physicists and engineers to interact on
a monthly basis in extremely formalized meetings and discussions. However, I’m
confident that such an integrative approach to science will ultimately carry biology
forward into an age of new global theories of biological systems, and I feel that I’ve
gained the capacity to converse with scientists from extremely diverse fields as a result of

my participation on this project.

vi

PREFACE

The research on UV-survival was carried out in the laboratory of Dr. George Sundin at
Michigan State University. The slow freeze—thaw experiment was performed by Chia-
Kai Chang under my direction. Control Escherichia coli strains were provided by the

laboratory of Dr. Richard Lenski at Michigan State University.

vii

TABLE OF CONTENTS

LIST OF TABLES ..................................................................................................... x
LIST OF FIGURES ................................................................................................... xi
CHAPTER 1.

MICROBIAL LIFE IN CRYO-ENVIRONMENTS ................................................. 1
Cryo-environments and permafrost ............................................................... 2
Challenges in cryo-environments .................................................................. 4
Psychrophilic adaptations to low temperature ............................................... 6
Psychrobacter arcticus 273-4 ........................................................................ 12
Goals of the present research ......................................................................... 14
References ...................................................................................................... 1 5

CHAPTER 2.

GROWTH AT LOW TEMPERATURES DOES NOT IMPROVE
SURVIVAL OF LETHAL STRESSES BY

PSYCHROBACT ER ARCTICUS 273-4 ..................................................................... 23
Introduction .................................................................................................... 25
Materials and Methods ................................................................................... 27
Results ............................................................................................................ 29
Discussion ...................................................................................................... 34
References ...................................................................................................... 37

CHAPTER 3.

GROWTH AT 4°C INDUCES A LIMITED TRANSCRIPTIONAL
RESPONSE IN THE PSYCHROPHILE

PS YCHROBA CT ER ARC TIC US 273-4 ..................................................................... 41
Introduction .................................................................................................... 43
Materials and Methods ................................................................................... 44
Results ............................................................................................................ 49
Discussion ...................................................................................................... 56
References ...................................................................................................... 61

CHAPTER 4.

RESOURCE EFFICIENCY AND MOLECULAR DYNAMICS
DOMINATE THE TRANSCRIPTOME RESPONSE FOR

GROWTH BELOW 0°C IN PSYCHROBACTER ARCTICUS 273-4 ....................... 66
Introduction .................................................................................................... 68
Materials and Methods ................................................................................... 70
Results ............................................................................................................ 76
Discussion ...................................................................................................... 96

viii

References ...................................................................................................... 106

CHAPTER 5.

TEMPERATURE INDUCED DIFFERENTIAL EXPRESSION
IN GLYCINE, METHIONINE, AND LEUCINE METABOLISM
CORRESPOND TO BIAS IN AMINO ACID CONTENT OF
GENES HIGHLY EXPRESSED BELOW 0°C BY

PS YCHROBA CT ER ARC TIC US 273-4 ..................................................................... 112
Introduction .................................................................................................... 1 14
Materials and Methods ................................................................................... 116
Results ............................................................................................................ 120
Discussion ...................................................................................................... 129
References ...................................................................................................... 140

CHAPTER 6.

SUMMARY AND FUTURE PERSPECTIVE .......................................................... 143
The psychrophilic response to low temperature and its implications for
Psychrobacter ecology ................................................................................... 144
Future perspective for research on Psychrobacter arcticus ........................... 147
References ...................................................................................................... 150

ix

LIST OF TABLES

TABLE 3.1. Genes with decreased expression during grth at 4°C.
Genes are grouped by functional category ....................................................................... 51

TABLE 3.2. Genes increasing in expression during grth at 4°C.
Genes are grouped by functional category ....................................................................... 52

TABLE 3.3. Growth rates of Psychrobacter arcticus 273-4
on defined basal medium with various carbon sources. .................................................. 54

TABLE 3.4. Genes with > 1.5-fold change in mean expression between
22°C and 4°C. .................................................................................................................. 55

TABLE 4.1. Strains, primers and plasmids used in this study. ...................................... 75

TABLE 4.2. Amino acid biosynthesis genes that are differentially
expressed (P < 0.01) in fold change from 17°C. .............................................................. 88

TABLE 4.3. tRNA synthetase genes that are differentially expressed
(P < 0.01) in fold change from 17°C. .............................................................................. 89

TABLE 4.4. Expression profiles of P. arcticus genes involved
in protein folding and disaggregation. Differentially expressed genes
have a P-value < 0.01. ...................................................................................................... 90

TABLE 5.1. Strains, primers and plasmids used in this study ....................................... 117

TABLE 5.2. Ratios of average growth rates in cultures
supplemented with amino acids to unsupplemented cultures. ......................................... 124

TABLE 5.3. Compiled data from expression of amino acid biosynthesis
genes their proportion in -6°C vs. 17°C proteomes and the effect on
growth of supplementation with those amino acids ......................................................... 127

SUPPLEMENTARY TABLE 5.1. Genes included in the proteome

amino acid content analysis. Genes were differentially

up-regulated > 2-fold at the indicated temperature and highly expressed

based on transcriptome analysis ....................................................................................... 138

SUPPLEMENTARY TABLE 5.2. Genes included in the predicted
17°C (Topt) proteome. Genes were selected for analysis based on
differential up-regulation > 2-fold and were highly expressed transcripts. ..................... 139

LIST OF FIGURES

FIGURE 1.1. Mechanisms of cold acclimation in Gram negative bacteria .................... 9

FIGURE 2.1. Log survival difference over increasing UVC dose ................................. 30
FIGURE 2.2. Log survival ratios obtained during slow freeze-thaw experiment .......... 31
FIGURE 2.3. Log survival ratios obtained during fast freeze-thaw experiment ............ 33

FIGURE 3.1. M vs. A plot in P. arcticus grown at 4°C (positive M-values)
and 22°C (negative M-values). ........................................................................................ 49

FIGURE 3.2. M vs. A plot comparing the transcriptome at
4°C (positive M-values) to the transcriptome at 22°C (negative M-values)
in acetate medium. ........................................................................................................... 55

FIGURE 4.1. Experimental design of temperature comparisons using microarray
hybridizations ................................................................................................................... 73

FIGURE 4.2. Square—root transformed growth rates plotted
over temperature. ............................................................................................................. 77

FIGURE 4.3. Plots of normalized fluorescence values across
15 cDNA populations hybridized from each temperature. .............................................. 79

FIGURE 4.4. Counts of differentially expressed genes compared with 17°C
by COGS category. .......................................................................................................... 80

FIGURE 4.5. Heat map of differentially expressed genes. Images in this
dissertation are presented in color .................................................................................... 82

FIGURE 4.6. Heat map of selected differentially expressed genes. Images
in this dissertation are presented in color. ........................................................................ 84

FIGURE 4.7. Exchange of gene expression over temperature. ...................................... 86

FIGURE 4.8. Conceptual model of the temperature response
in the transcriptome of P. arcticus. .................................................................................. 93

FIGURE 4.9. Growth rate distributions for knockout mutants and
wild-type P. arcticus controls. ......................................................................................... 95

xi

FIGURE 5.1. Growth characteristics of P. arcticus 273-4 wild-type
strain and ArelA mutant strain PB0343 ............................................................................ 122

FIGURE 5.2. Overlaid histograms of amino acid proportions in the
-6°C and 17°C proteomes. ............................................................................................... 126

FIGURE 5.3. PCA comparing data trends in amino acid content,
growth rate effect, and gene expression ........................................................................... 129

SUPPLEMENTARY FIGURE 5.1. Growth of P. arcticus reIA knockout
compared with wild-type P. arcticus 273-4. .................................................................... 137

xii

CHAPTER 1

MICROBIAL LIFE IN CRYO-ENVIRONMENTS

Cryo-environments and permafrost

Cold environments, those with average temperatures less than or equal to 4°C,
represent nearly the entire ocean and approximately 25% of the terrestrial surface
area. In Spite of the clear impact of cold temperatures on the biosphere, studies of the
composition and function of microbial communities in these environments have been
limited until recent years (17, 24, 69). Cryo-environments are a subset of cold
environments defined by the predominance of frozen water. Polar cryo-environments
can be stable on the order of 103-107 yr. Rates of carbon and nutrient turnover in
these environments are slow. Microbes have been detected and isolated from all
known cryo-environments including sea ice, glaciers, subglacial lakes, snow, and
permafrost (8, 26, 34, 62, 68). The biomass and diversity of these environments are
dominated by microbial populations. While no one disputes that the rates of
microbially driven geochemical processes are lower in cryo-environments, diverse
microbial communities and activities are detected in cryoenvironments. For example,
incorporation of acetate was observed by Rivkina and colleagues in Antarctic
permafrosts at -20°C (52). Motility of a sea ice microbe Colwellia psychrerythrea
was observed at -10°C (30). Growth and carbon incorporation by Psychrobacter spp.
isolated from cryopegs in Siberian permafrosts have been reported (2, 27). Biogenic
methanecorresponding microbes have been detected in Siberian permafrosts (22, 64).
Sulfate reducing bacteria have been detected in Lake Fryxell, Antarctica (32).
Rhizobial and cyanobacterial nitrogen fixation have been also been described (19,

75).

Permafrost selects for organisms capable of persisting at subzero temperatures
with concomitant reduced rates of biological activity, reduced water activity, and
reduced nutrient availability. In spite of these three stressors, microbes have been
isolated from permafrost soil cores as old as 3 million years (68). In situ radiation
levels in permafrost, resulting from soil minerals, dictate that a dormant microbe
should only be able to survive on the order of 104 yr without the capacity to repair
damage to the genome (24, 69). Therefore, organisms that can be resuscitated afier
residing in permafrost for 104-106 yr must have been carrying out at least survival
level metabolism, defined as metabolism necessary to repair damaged cellular
constituents (49). Considering the natural limits on microbial life in cryo—
environments, it has been assumed that microbial communities in permafrost are
populated by specialized genera of microbes. Examination of permafrost microbial
communities with both molecular and culture based techniques have detected
members of common soil associated microbial phyla including alpha, beta and
gamma Proteobacteria, Cytophaga-Flavobacterium, and High and Low GC Gram
Positive Bacteria (58, 68). However, some genera are more easily detected and
isolated from the Siberian permafrost suggesting these genera such as Psychrobacter,
Exiguobacterium, and Arthrobacter spp may have some specialized traits allowing
the maintenance of viability under these conditions. The preceding genera contain
members adapted for growth at warmer temperatures with varying degrees loss of
psychrotolerance when compared to permafrost species. This introduces the
possibility of whole genome comparison as a tool for examination of evolutionary

adaptations to cryo-environments (15, 67).

Permafrost is also a major sink of fixed carbon. As much as one-third of the
Earth’s soil carbon is contained in Arctic permafrosts according to the National Snow
and Ice Data Center’s “State of the Cryosphere” report on permafrosts

(http://www.nsidc.org/sotc/permafrost.html). Such a high level of stored carbon

 

makes permafrost communities important from the perspective of climate change. A
small increase in temperature of only a few degrees Celsius could have major
implications for microbial carbon turnover in permafrost. The prolonged frozen state
and observed persistence of microbes over intervals of millions of years makes
permafrost communities particularly interesting as models for hypothetical microbial
communities in extraterrestrial habitats such as underneath the polar caps on Mars
(51). How do permafrost microbes maintain viability for thousands of yr at
conditions near the limits of known life? Are these organisms surviving in the
dormant state in the permafrost, or are resident microbes specially adapted to function
at rates that could support grth in this extreme environment? Understanding the
adaptive traits of microbial species from permafrost will yield insights into the
function of microbes in cold environments and also potentially the responses of

microbes and communities in temperate soils to transient freezing during winter.

Challenges in cryo—environments

Cold temperatures impact biology on the molecular level by imposing constraints on
the rates and magnitude of both chemical and physical processes. Processes as
diverse as diffusion, elasticity, turbulent flow, microbial growth rate, biogeochemical

processes, and chemical reaction rates all decrease with temperature (18, 49). Central

to our understanding of the behavior of microbes and biochemistry over temperature

is the Arrhenius equation (Eq. 1).

k = AOe-Ea/RT (Equation 1)

Here, k is the rate constant of a reaction, A0 is some basal rate constant specific to the

process, Ea is the activation energy of a reaction, R is the gas constant and T is the

temperature. Thus, rates of chemical processes decrease exponentially with
temperature.

There are special exceptions at the extremes of functional temperature for
microbial cultures which result in observations of inflections in growth rate near the
temperature limits of microbial growth. Ratkowsky proposed a thermodynamic
model that takes into account the probability of “denaturation” as a function of
distance from the temperature optimum of growth. This equation models the
behavior of microbial grth of temperature better than previous Bélehradek or
square-root models (50, 54). These thermodynamic models still fail to model growth
rate behavior accurately over temperature when growth temperatures are near the
limits for an organism. The weakness of the thermodynamic model for microbial
growth at the thermal limits is probably due to its inherent assumption that function of
a Single enzyme is limiting growth rate across the entire temperature range of growth.
Rather, microbial cells growing near the temperature limit of growth modify their
gene expression and physiology to cope with the low temperature condition.

The effects of reduced temperatures can be greatly exasperated below the

freezing point of water. During freezing, water molecules are incorporated into ice

crystals, rendering them biologically inaccessible. Solutes and microbes concentrate
into the remaining volume. Freezing of extracellular solutions reduces water activity
and extracellular osmotic pressure and increases the heterogeneity of solutions by
generating physical barriers to already slow diffusion of solutes (65). The effect of
sudden osmotic shock on microbial cells due to fieezing can be lethal due to loss of
water from the hydrated shell of proteins (33). Nucleation of intracellular ice crystals
can lead to cell death through physical disruption of the cell membrane (40, 63).
However, water in microbial cells is confined due to their small volume, so the
probability of intracellular ice nucleation is very small in microbial cells (42). The
combined effects of life in cryo-environments force microbes in permafrost to adapt
to the interacting stresses of low temperature, low water activity and low nutrient
availability. The nature of these adaptations on a genome-wide scale in a model

organism is the focus of this dissertation.

Psychrophilic adaptations to low temperature

 

Physiological acclimation or adaptation to different thermal ranges is a major factor
in defining the biogeography of species. Originally, cold growing microbes were
divided into broad thermal categories based on cutoffs in optimal temperatures as
psychrophiles (Top, ~ 15°C, Tmax ~ 20°C, Tmin S 0°C) and psychrotrophs (Tom > 20°C,
Tmin < 4°C). The psychrophile classification persists today, though increased
knowledge of the diversity of cold-growing microbes has led to new definitions based
on both the biogeography and the growth temperature limits. Microbes which are

rarely isolated from cold environments, but exhibit grth at 4°C are considered

broadly as psychrotolerant. Microbes which are commonly isolated from cold
environments are now referred to as psychrophiles, and can be subdivided into steno-
psychrophiles with a narrow physiological temperature range and eury-psychrophiles
with a broad physiological temperature range (20).

Organisms can adapt for function at a temperature through evolution of alleles
specific for function at physiological temperatures or evolution of mechanisms for
regulation of genes that permit acclimation to different temperature ranges.
Evolutionary pressure for allele specialization in the cold should be stronger in stem-
rather than eury-psychrophiles. In contrast, eury-psychrophiles should be under
pressure to maintain thermostability and function of those alleles at low temperature
(23). Bury-psychrophiles would also be under selective pressure to maintain
regulatory mechanisms that allow for acclimation for growth to both psychrophilic
and mesophilic temperature ranges. Our discussion here will examine both allelic
adaptations for low temperature function and gene expression responses to the cold.

Allelic adaptation for low temperature function. The critical challenge to
enzyme function at low temperatures is loss of flexibility in the peptide chain. Recent
years have witnessed an explosion in research on thermal adaptation of psychrophilic
enzymes. Increased flexibility results in decreased enthalpy, AH', of the enzyme
activation energy thus increasing the reaction rates at low temperatures (23). One of
the first methods of understanding adaptations for increased flexibility was to
examine amino acid contents of psychrophilic enzymes (55). Genome-wide amino
acid bias analyses in psychrophilic bacteria and archaea have demonstrated that

psychrophiles have increased proportions of polar amino acids in both surface

exposed and buried amino acid positions when compared to mesophilic relatives.
Surface exposed residues in these genomes exhibit decreased charged amino acid
content. Buried residues are biased away fi'om hydrophobic amino acid content
coupled with slight increases in charged amino acid content (41, 56). Other
adaptations for increased flexibility of enzymes at low temperature can include
glycine clusters particularly near the boundaries of secondary structure elements, and
loss of proline residues in coils (35, 39). However, global trends in amino acid
content are far less important than the position of amino acid substitutions. Glycine
to proline substitutions were found to be most important in the context of the local
structure in a psychrophilic chitinase where one such substitution reduced kw at low
temperature and another improved it (38). Similarly, a recent modeling of molecular
dynamics of cold adapted enzymes showed higher flexibility localized to coil regions,
and that insertions in psychrophilic enzymes were commonly highly disordered (59).
Sensing low temperatures. Virtually every cellular system is impacted by
low temperatures, so sensing of low temperatures need not rely on membrane bound
histidine kinases (Fig. 1.1) (57). Nevertheless, such histidine kinases have been
identified in Synechocystis and Bacillus subtilis where it is thought that they respond
to cytoplasmic membrane fluidity (29, 61). RNA thermometers also play a significant
role in cold shock and acclimation. Cold temperatures can induce the formation of
secondary structures. Secondary structures impact the stability of mRNAs and their
translational efficiency. This strategy 5 involved in the regulation of cold inducible
proteins such as cspA, pnp, rnr, and the bkd operon (10, 37, 45, 70, 73). One open

question in cold sensing involves the evolutionary modification of thresholds for

temperature responses. Experiments in psychrophilic microbes suggest that the heat
shock response is induced in psychrophiles at lower temperatures than in mesophiles

(16,71).

 

 

 

 

 

 

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Figure 1.1. Mechanisms of cold acclimation in Gram negative bacteria. Cold temperatures reduce
the inherent disorder of molecular systems, decreasing enzymatic reaction rates and membrane
fluidity and stabilizing hydrogen bonds in nucleic acids. Depicted genes are up-regulated in
diverse mesophilic and psychrophilic microbes including E. coli, B. subtilis, Synechococcus sp.
PCC7942, M. burtonii, and Psychrobacter cryohalolentis. See text for references.

Translation and protein folding responses in the cold. A high proportion
of genes up-regulated during cold acclimation or shock have to do with translation
functions (Fig. 1.1). RNA chaperones, e. g. the major cold shock proteins, are among
the most highly conserved examples. RNA chaperones are up-regulated
constitutively in some psychrophiles during growth at low temperatures (1, 4).

DEAD-box RNA helicases play a complementary role in unwinding secondary

structures in RNA. Together these genes ensure that RNAs are free from secondary
structure, so that translation can proceed. Changes in ribosomal protein complement
have previously been detected in organisms growing in the cold, including structural
ribosomal proteins, elongation factors, initiation factors and ribosome-binding factor
( 1, 9, 28, 74). Cold shock induced changes in Listeria monocytogenes ribosomes led
to decreased thermostability and presrunably improved ribosome function in the cold
(3).

Mesophiles commonly up-regulate heat shock chaperones during cold shock
and express transcripts and proteins thereof at elevated levels during grth at sub-
optimal temperatures (9, 36, 46). The chaperone ClpB is strongly induced during low
temperature growth presumably due to its role in protein disaggregation and repair
(46, 48). While psychrophiles do not increase expression of heat shock chaperones in
the cold, increased expression of peptidyl-prolyl cis-trans isomerases has been
observed in both psychrophiles and mesophiles during grth at suboptimal
temperatures (9, 28, 60). Thus, psychrophiles may not experience protein
denaturation at low temperatures, but do exhibit increased response for the proline
dependent folding of peptides.

Maintenance of membrane fluidity. The fluidity of biological membranes is
considered a key factor in dictating metabolic activity levels (Fig. 1.1). Transport
proteins and the key proteins of respiratory metabolism are associated with the cell
membrane. The activity of these proteins is thus controlled strongly by the viscosity
of the cell membrane (44). Maintenance of membrane fluidity is one of the dominant

factors in the response to cold shock and for growth in the cold (14). Expression of

10

fatty acid desaturases and reduction in the activity of long-chain acyl-CoA ligase at
low temperatures are common mechanisms of maintaining membrane fluidity in
mesophiles (29). Cyclopropane fatty acid synthases are also up-regulated during low
temperature grth in mesophiles and can stabilize double-bonds in membrane lipids
against oxidative damage. Increased synthesis of branched chain fatty acids has been
supposed to result from increased production of isobutyryl-CoA in B. subtilis, though
this induction appears to occur only during cold Shock rather than cold acclimated
grth (9, 45). In theory, changes in phospholipids head-group chemistry could also
introduce steric hindrance to lipid packing though this has been only rarely reported
(14).

Growing up cold and hungry? In cryo-environments, solutes are
concentrated into a limited volume of remaining water; in Siberian pennafiost, 3-8%
of water is unfrozen (25). While substrates are concentrated in briny veins and
pockets of water, the rates at which they can be taken up and metabolized is limited at
low temperatures (44, 57). Indeed, enzymes in frozen and otherwise heterogeneous
solutions exhibit oscillations in activity due to local depletion of substrate due to
decreased diffusion rates (65). From the microorganismal perspective, induction of a
starvation response, with its consequent reduction in overall metabolic activity is an
important strategy to ensure that resources are not exhausted. Such a response can
take the form of the stringent response, which controls gene expression during a
variety of substrate limited growth conditions (12, 13). Alternatively, permafrost
microbes could activate a general stress response such as that controlled by RpoS or

SigB. Such responses lead to growth arrest, but also activate a variety of damage

11

resistance and repair mechanisms, essentially hardening the cell against
environmental sources of damage (9, 43, 66). However, proteome studies in a
psychrophilic archaeon and a psychrophilic bacterium have detected increased
expression of genes involved in carbon catabolism and energy metabolism during
growth at sub-optimal temperatures (1, 28). These results suggest that substrate
uptake is not limiting for psychrophiles, rather energy conversion must be
compensated to maximize growth rate. Directing energy toward damage resistance or
growth metabolism could both enable the survival of organisms in permafrost, but the
extent to which psychrophilic organisms have adopted either strategy is poorly

understood.

Psychrobacter arcticus 273-4: A model organismfor sybzero metabolism
Psychrobacter arcticus 273-4 was isolated from a depth of 12.5 m of a bore hole in
the Kolyma lowland (68). This depth places P. arcticus firmly in the Edoma suite, a
syngenetically frozen layer resulting from alluvial flooding 20,000 to 40,000 years
ago in this location. It is a gamma Proteobacterium with an optimal growth
temperature of 17°C to 22°C dependent on medium conditions and can be cultured at
temperatures ranging from 28°C to -10°C. P. arcticus is also capable of metabolism
in high salt concentrations up to 2.8 osmolal (47). It has also been found to increase
the proportion of unsaturated fatty acids in its cytoplasmic membrane during growth
at 4°C and on increased salt and exhibits slight ice nucleation activity (47).

Members of the genus Psychrobacter are Gram negative, heterotrophic,

aerobic gamma Proteobacteria that are defined by their ability to grow at 4°C and the

12

ability of a competent strain to take up exogenous DNA from candidate
Psychrobacter strains (31). Psychrobacter species have been detected in clone
libraries from Antarctic sea ice (6). In addition to Siberian permafrost, isolates of
Psychrobacter species have been obtained from a variety of polar environments (7,
11, 21), sea water and marine worms (53), and salted meats (5, 72). The combined
tolerance in this genus for low temperatures and reduced water activity make them
excellent candidates for model cryo-adapted bacteria.

The genome sequence of P. arcticus 273-4 was obtained by the DOE Joint
Genome Institute (JGI) in 2002 (http://genomeoml.gov/microbial/psvc). This
completed sequence was manually annotated by a team of scientists from Michigan
State University and J GI. Interesting metabolic features of the genome include the
lack of a glycolysis pathway and the presence of a rare glyoxylate metabolism
pathway. The P. arcticus genome is also very repetitive with a single 139 bp
dispersed repeat that accounts for 1.92% of the genome sequence and is highly
dominant among repeated sequences in the genome (evenness of all repeated
sequences = 0.11). Among the potential cryo-adaptive features of the genome are
three cold shock proteins, two DEAD box RNA helicases, glycine-betaine
biosynthesis pathways, fatty acid desaturase, anti-freeze domain proteins, and a hill

complement of proteases and chaperones.

13

Goals of the present research
The central question of this dissertation is:
How does P. arcticus acclimate to life near the lower temperature limit?

I seek to answer this question through a combination of survival studies, rnicroarray
gene expression experiments, genome analysis, and gene knockout experiments. In
Chapter 2, I test the effect of low temperature incubation on survival of lethal
stresses. In Chapters 3 and 4, I present analysis of transcriptomes during low
temperature incubation in both rich and mineral media developed based on the P.
arcticus genome. In Chapter 5, I examine changes in amino acid metabolism as a
result of low temperature incubation and present evidence for the ecological

relevance of the observed changes.

14

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22

CHAPTER 2

GROWTH AT LOW TEMPERATURES DOES NOT IMPROVE SURVIVAL OF

LETHAL STRESSES BY PS YCHROBAC T ER ARC TIC US 273-4

23

Abstract

Permafrost microbes experience continuous low temperatures, dessication and low levels
of radiation in situ. In spite of these conditions, Psychrobacter arcticus 273-4 was
isolated following 10,000-40,000 yr inhabitation of the Kolyma lowland permafrosts.
We examined low temperature effects on survival of UVC and freeze-thaw stress. As
growth temperature decreased, P. arcticus exhibited decreased survival after both UVC.
P. arcticus survived freeze-thaw at higher rates than E. coli regardless of growth
temperature, but E. coli survived UVC at higher rates than P. arcticus under all
conditions. In contrast to repeated observations of increased freeze-thaw survival
following cold acclimation in mesophiles and Gram-positive permafrost bacteria, no
increase in stress resistance was observed in P. arcticus during low temperature growth.
Therefore, P. arcticus may not respond to low temperature by increasing resistance to
lethal stress.

The UVC survival analysis portion of this work was carried out by P. W. Bergholz and
published in:

Ponder, MA. et al (2004). Characterization of potential stress responses in ancient

Siberian permafrost psychroactive bacteria. FEMS Microbiol. Ecol. 53(1): 103-15.

24

Introduction

The permafrost soils of the Kolyma lowland, Siberia, Russia have been frozen for periods
between 10,000 and 3.5 million yr, depending on the geologic stratum sampled (28).
Owing to their frozen state, these soils represent a set of stable environmental stresses to
the resident microbes including constant low temperatures (-10°C to -12°C), continuously
low water activity and low doses of background radiation from soil minerals at a rate of
1-6 kGy yr'1 (7, 8). Recent advances in our understanding of the biogeochemical flux in
these environments have shed light on the potential for microbial survival, maintenance
and even growth in permafrosts and other cryoenvironments, i.e. environments which
undergo long-term freezing on the order of years (4, 21).

While background levels of y-radiation are low in the permafrost of the Kolyma
lowland, exposure to even low levels of background radiation over 104-106 yr would
significantly damage genomic DNA (7, 31). Exposure to ultraviolet radiation can lead to
direct DNA damage in the form of pyrimidine dimers and DNA strand breaks, and can
also lead to generalized damage to the cell through the formation of reactive oxygen
species (13). If radiation-induced damage is an important selective factor in permafrost,
one would expect to isolate organisms that are adapted either for resistance to damage
from radiation, or improved capacity to repair damage due to radiation. Price and
Sowers proposed an alternative hypothesis that psychrophiles might be resilient against
DNA damage by adopting a survival level metabolism, defined as the metabolic rate
necessary to repair damage to the genome, in nutrient-limited and cold environments

(20). Both such strategies could yield organisms with long survival times in permafrost.

25

Microbes must also undergo freezing upon entry into the permafrost. While
survival in a frozen soil matrix over 103-104 yr cannot be directly assayed, freeze-thaw
cycling can be used to assess the cryotolerance of microorganisms. Freeze-thaw stress is
thought to impact cells through two principle damage mechanisms: the formation of
intracellular ice and the formation of extracellular ice with concomitant increase in solute
concentrations due to loss of liquid water (15). Formation of intracellular ice is unlikely
in the case of bacteria, because water in the bacterial cell is confined in a very small
volume and interacts with proteins and the cytoplasmic membrane (17). However,
osmotic stress can be particularly damaging as it leads to the denaturation of cytoplasmic
proteins and the damage of cell membranes (12, 25). Diverse permafrost strains survived
a single freeze-thaw cycle for 1 yr with less viable cell loss following growth at 4°C
versus growth at 25°C (19). Exiguobacterium species isolated from the Kolyma lowland
were recently demonstrated to increase survival after repeatedifreeze-thaw cycles when
cultured at low temperatures (29). The prevalence of increased resistance to freeze-thaw
stress in permafrost isolates after growth at low temperatures remains unknown.

We examined the Gram-negative permafrost isolate, Psychrobacter arcticus 273-
4 to determine if incubation at low temperatures improved survival of freeze-thaw
cycling or UVC irradiation. P. arcticus was isolated from 10,000-40,000 yr Kolyma
lowland permafrost. It is capable of grth at temperatures ranging from -10°C to 28°C
and salinities ranging from 10 mM to 1.3 M NaCl (2, 19). These combined capacities
suggest that P. arcticus is adapted to inhabit cryoenviromnents. If P. arcticus exhibits
increased resistance to lethal stresses following growth at low temperatures, then this

resistance response may explain its capacity for survival over 103-104 yr in this

26

environment. Alternatively, P. arcticus may not respond to low temperatures by
increasing resistance to radiation or freeze-thaw. The lack of such a response would
indicate that active metabolism at some low rate was required for survival in the

permafrost.

Materials and Methods

UVC Survival Assay. Cultures of Psychrobacter arcticus 273-4 and Escherichia coli
B606 were grown to late-log phase (OD600 = 0.6 to 0.9 under all conditions) in 50 ml of
V2 Tryptic Soy Broth (TSB, Difco). P. arcticus was cultured at either 25°C or 4°C. E.
coli was cultured at 37°C. E. coli was grown to late log phase (OD600 = 0.9) in ‘/2 TSB at
25°C. Fifteen ml of each culture was mixed with an equal volume 0.85% NaCl and
centrifuged at 6000 x g for 30 min at their cultured temperature. The culture supernatant
was discarded, and cell pellets were resuspended in 15 ml 0.85% NaCl. Cells were stored
on ice until experiments were completed (never more than 1.5 hours).

Cells were pipetted into a sterile glass Petri plate, and exposed to a UVC
fluorescent lamp (equivalent dose 1.5 J m"2 s") with constant mixing. Cell suspensions
were cumulatively exposed to 0, 25, 50, and 100 J m'z. At each exposure level, a 100 pl
cell suspension was collected. After all exposures were completed, cell suspensions from
each exposure level were decimally diluted to 10'8 of their original concentration in
0.85% NaCl. Triplicate 50 pl aliquots of cell suspension at each dilution level were spot
plated onto a 1/2 TSA plate. Plates were incubated in the dark at culture temperature. All
steps from UVC exposure through incubation were carried out in the dark. All solutions

were kept at the same temperature at which cells were cultured. Colony forming units

27

were counted on the spot plates after 48 hours of incubation at 25°C or two weeks
incubation at 4°C respectively.

Slow freeze thaw survival. P. arcticus 273-4 and E. coli B606 were grown from
-80°C freezer stocks on 1/2 TSA at 25°C and 37°C respectively. Starter cultures were
inoculated by transfer of single colonies from agar medium to 5 ml ‘/2 Tryptic Soy Broth
(1/2 TSB, Difco). Two starter cultures were grown to stationary phase (OD600 E 0.8 for
P. arcticus and ODboo E 2 for E. coli) at 25°C or 4°C for P. arcticus and 37°C for E. coli.
Two 25 ml ‘/2 TSB sample cultures were inoculated with 250 pl of starter culture and
these cultures were allowed to grow at the same temperature as the starter cultures until
exponential phase (OD600 5 0.2). Each sample culture was split into 17 x 1 m1 aliquots in
1 ml Corning cryovials (Corning, Corning, NY). These aliquots were transferred to an -
15°C incubator and allowed to freeze at that temperature. Samples were incubated at -
15°C for 24 hours each freeze-thaw cycle. Each day, aliquots were thawed at room
temperature for approximately 10 minutes. After each freeze-thaw cycle, one aliquot of
each sample was decimally diluted and plate counts were performed by spreading 100 pl
each dilution from 10'1 to 10'6 on '/2 TSB. The remaining aliquots were returned to the -
15°C incubator for refreezing. Plated cells were allowed to grow at the original culture
temperature of the sample. After 4 days at 25°C or 15 days at 4°C for P. arcticus plates
or 1 day at 37°C for E. coli, colony forming units (CFU) were enumerated on each plate.
Fast freeze thaw survival. P. arcticus 273-4 was inoculated from -80°C freezer stock
onto a marine agar (MA) plates and grown at 4°C. Marine agar consisted of 5 g l’1
tryptone (Difco), 1 g 1'1 yeast extract (Difco) and 30 g 1'1 sea salts (Sigma-Aldrich).

Starter cultures were inoculated by transferring a single colony for each of six replicate

28

samples to 5 ml marine broth (MB, Difco) at 25°C, 4°C, and -6°C. Starter cultures were
grown to stationary phase (OD600 5 0.8). Freeze-thaw samples were grown at each
temperature by inoculating 5 m1 MB in a 18 mm culture tube with 50 ul starter culture.
Freeze-thaw sample cultures were allowed to reach exponential phase (OD600 a 0.2).
Samples were split into 8 x 0.5 ml aliquots in 1.5 ml Eppendorf tubes. Freeze-thaw
cycles were carried out by immersing the vials in a dry ice and ethanol bath for 1 minute.
Aliquots were then thawed at 25°C in a water bath for 3 minutes. This fieeze-thaw
process was repeated for 15 cycles. At 0, 1, 3, 5, 10, and 15 freeze-thaw cycles, one
aliquot was retained, decimally diluted, and spread plated at 10'3 to 1045 dilutions on MA.
MA plates were incubated at 4°C. CFU were enumerated after 15 days incubation at
4°C.

Log Survival Calculations and Analysis. For all survival experiments, CFU ml"1 of
culture were estimated to be plated volume'l - CFU per plate - dilution factor. Log
survival was calculated as Log10(CFU ml'lfina.) - Log10(CFU ml'lim-u-al). T-test
comparisons were carried out in the UVC and fast freeze-thaw survival studies
comparing log survival of E. coli and P. arcticus grown at 25°C. P. arcticus cultures
grown at 25°C versus cultures at lower temperatures were also tested. A P-value < 0.05

was considered statistically significant.

Results

UVC survival. Cumulative damage due to long term exposure to low levels of
background radiation could be an important selective pressure on microbial permafrost

communities. We tested P. arcticus to determine if it was more resistant to UVC

29

radiation than a related mesophilic bacterium, E. coli B606. We also tested whether there
was an increase in survival of UVC irradiation as growth temperature decreased.

Since P. arcticus cultured in 1/2 TSA exhibits a growth rate inflection at 6°C
indicative of cold acclimated growth, P. arcticus at two grth temperatures (25°C and
4°C) and E. coli B606 at 37°C were exposed to cumulative UVC doses equivalent to 0,
25, 50, and 100 J m'z. Average log survival at each of the UVC dosage levels was used
to compare P. arcticus grown at 25°C to both of the other two samples using a one-tailed
two sample t-test assuming unequal variances. The log survival of the 25°C sample of P.
arcticus was significantly greater than that of the 4°C at 50 and 100 J In2 dosage levels (P
< 0.05) (Fig. 2.1.). The E. coli B606 sample was found to exhibit greater survival (P <

0.05) than P. arcticus grown at 25°C at the 100 J m‘2 dose.

 

 

 

 

V3
l
o g“. o
g .
N I
>
.E Ca
.2 “- -
O
:7; V2- I "
.3 -- l T
O
:3- l
.n T
N" o
' .L
I I I l l l
0 20 40 60 80 100
UVC Dose (J m-Z)

Figure 2.1. Log survival difference over increasing UVC dose. Counts of
E. coli B606 (0) and P. arcticus grown at 25°C (0) and 4°C (0) after
UVC up to 100 J m’z. CFU ml"1 was estimated from cultures grown on '/2
TSA in the dark. Error bars represent one standard deviation for each

sample (n = 4).

30

Slow Freeze-thaw survival. P. arcticus cultures were exposed to slow freeze-thaw
stress, by freezing 1 ml aliquots of culture at -15°C and thawing at room temperature.
Freezing of cell aliquots was verified after 2 h incubation at -15°C. Cooling rates were
measured in to be 01°C min'1 in proxy aliquots when averaged over the freezing period.
The freezing point of '/2 TSB under our experimental conditions was -7.5°C. Thawing
rates at room temperature were also not strictly controlled in the slow fieeze-thaw
regime. Thawing required approximately 10 minutes. Therefore, warming rates were

approximately 2°C min'l averaged over the thawing period.

 

 

 

 

vvvvvv XVAX8§A
-e§ IAA‘.A"O Vv
O .. AAVVV V
.2 9
267‘
at. e
*3 '°
EC?-
m
2
our?-
3 e
‘T-
e
'IP-i .
I r r I
O 5 10 15

F reeze/T haw Cycle

Figure 2.2. Log survival ratios obtained during slow freeze-
thaw experiment. Counts of two replicates each of P. arcticus
grown at 25°C (open downward triangle, V) and 4°C (open
upward triangle A) in V2 TSB medium. Survival of the
psychrophile P. arcticus is compared with the mesophile E.
coli B606 (closed circle 0). All assays were carried out with
plating on '/2 TSA agar.

E. coli B606 cultures were grown at 37°C. P. arcticus cultures were grown at

25°C and 4°C to evaluate the effect of low temperature growth below the critical

31

temperature (Tm, ~ 6 degC) on cryotolerance. CFU ml'1 was estimated for each sample
for two replicates after each freeze-thaw cycle by decimal dilution and plating on 1/2 TSA.
P. arcticus cultures were found to be cryotolerant, with no detectable decline in viable
cells over multiple freeze-thaw cycles in this slow freeze regime (Fig. 2.2). Similar
survival and Slight growth in P. arcticus populations was observed regardless of the
growth temperature. In contrast, E. coli B606 was much more sensitive to freeze-thaw
stress. E. coli CFU were undetectable after 5 freeze-thaw cycles. The detection limit of
our assay was approximately 102 CFU ml". The average log survival rate of E. coli in
this experiment was -0.9 CFU freeze-thaw cycle".

Fast freeze-thaw survival. Use of ‘/2 TSA as our plating medium for fast fi'eeze-thaw
experiments failed to yield stable CFU ml'1 estimates after several attempts. Therefore,
MB was used as the growth and plate count medium for this fast freeze-thaw survival
experiment. Sample cultures with separate colonies as source inocula were grown to
mid-exponential phase under three temperature conditions, 25°C, 4°C and in the subzero
temperature range at -6°C. Six replicate cultures inoculated from separate colonies were
assayed at each temperature. Under the conditions of this experiment, cooling and
heating rates were strictly controlled by the use of dry ice/ethanol baths and water baths
heated to 25°C. The cooling rate was estimated to be greater than -30°C min'1 in the
interval of our fieezing regime from 25°C to -7.5°C. This is a conservative estimate as

freezing was evaluated visually in these samples.

32

 

 

 

 

V?

0.1

q
ofi‘i

O O T

'5 .L 3

22:?- l I l l

S I -

°-c>.

2")

EDT“ e T
o. l T ‘
a." - 1
m l
(\j—l
' I I j I

o 5 10 15

Freeze/T haw Cycle

Figure 2.3. Log survival ratio obtained during fast freeze-thaw
experiment. CFU ml‘l estimates of P. arcticus cultures grown at
25°C (closed squares I), 4°C (closed triangles A) and -6°C (closed
circles O). The error bars indicate standard deviations for each
sample at each temperature (n = 6). CFU ml'l was estimated using
MA as the growth medium.

P. arcticus survived at significantly higher rates at 25°C and 4°C than at -6°C
(Fig. 2.3). Cultures grown at 25°C and 4°C were statistically indistinguishable in log
survival until the freeze-thaw cycle 15 where 4°C grown cells exhibited statistically
lower log survival with P < 0.05. Log survival behaved as a log-linear decay in CFU ml’l
over the interval of 0-5 freeze-thaw cycles for -6°C samples and 0-10 freeze-thaw cycles
for 25°C and 4°C samples. The loss of the expected decay behavior in -6°C samples at
10 freeze-thaw cycles is probably due to CFU ml'l estimates which approach the limit of
detection at these time points (detection limit 2 104 CFU ml"). Log survival rates were -
0.07 Log“) CFU freeze-thaw cycle", 011 Logo CFU freeze-thaw cycle", and -0.31

Loglo CFU freeze—thaw cycle'1 at 22°C, 4°C and -6°C respectively.

33

Discussion

We tested the effect of growth temperature on stress resistance by measuring survival of
P. arcticus following UVC and fi'eeze-thaw exposure. UVC was chosen because it
causes both pyrimidine dimers and occasional single strand DNA breakage, and DNA
damage is a relevant stress in the permafrost (7, 13). While estimation of temperature
effects on cryotolerance over 103-104 yr is not possible in a laboratory setting, we used
resistance to repeated freeze-thaw as a proxy for cryotolerance. Furthermore, exposure to
cold is known to improve cryotolerance in plants and mesophilic bacteria (3, 5, 6, 22, 24).

Freeze-thaw conditions combine the challenges of desiccation and low
temperatures. Gene expression responses to low temperatures and desiccation have been
linked on a functional genomic level in Arabidopsis thaliana (22). Accumulation of
compatible solutes is a prevalent response to dessication stress. Compatible solutes
protect proteins fiom denaturation, but may destabilize cell membranes as was observed
in model membranes during freeze-thaw assays (10). Nevertheless, some bacteria
accumulate compatible solutes to high levels after cold shock. Exposure to 10°C1ed to
expression of glycine-betaine and camitine uptake transporters in Listeria monocytogenes
(1, 16). E. coli survival at low temperatures is dependent on the ability to synthesize
trehalose (11).

The characteristics of the permafrost soil matrix may also impact survival; thin
layers of liquid water will be retained around soil particles far below the freezing point
(9). While Gram-positive bacteria appeared to benefit from the presence of a soil matrix
in freeze-thaw assays, Gram-negative organisms survived better in liquid environments

or high moisture content soils (18, 23). Therefore, we did not assess the effect of

34

presence vs. absence of a soil matrix on freeze-thaw survival. However, attachment to
permafrost soil particles could afford P. arcticus protection in ordered liquid water bound
to particle surfaces (9).

Our results demonstrate primarily that low temperature growth of P. arcticus cells
does not yield a benefit to freeze-thaw or UVC survival. Survival at 25°C was greatest in
all experiments and survival at low temperatures, 4°C or -6°C, was never greater than at
25°C. While E. coli exhibited higher survival in the UVC experiment, P. arcticus
performed better than E. coli in all freeze-thaw conditions. P. arcticus may be
evolutionarily adapted to survive freeze-thaw stress more successfully than E. coli, but
this difference in survival is not improved by acclimation of P. arcticus for growth at
lower temperatures.

The P. arcticus genome contains factors for nucleotide excision repair and
recombination repair of DNA including but not limited to recA, uvrBCD, mats and mutL.
The oxidative stress response gene complement in the P. arcticus genome includes katE,
soxA, ahpC, and msrA. This genome also contains factors for freeze-thaw damage and
osmotic shock resistance including betAB, betaine/choline/camitine uptake transporters,
small and large conductance mechanosensitive ion channels, and two proteins containing
antifreeze domains. P. arcticus was also reported to possess significant ice nucleation
activity that improved with incubation at 4°C versus 25°C (19). The preceding factors
may explain P. arcticus inherently higher survival of freeze-thaw challenge versus E.
coli.

Cold shock improves freeze-thaw survival of lactic acid bacteria and E. coli

0157:H7 (3, 6). Mesophiles such as E. coli commonly harden against lethal stresses

35

when exposed to grth inhibitory conditions. The stationary phase sigma factor RpoS
controls expression of approximately 10% of E. coli genes in response to starvation, low
pH, cold, heat, osmotic and oxidative stress responses (27, 30). For example, a Vibrio
parahaemolyticus ArpoS mutant exhibited substantially decreased survival vs. the wild-
type during low temperature storage (26). Similarly, an E. coli 0157:H7 ArpoS mutant
did not exhibit increased freeze-thaw survival following cold shock (14).

No rpoS homologue has been detected in the P. arcticus genome, and this may
explain the lack of a improved general stress resistance at low temperature. Growth
temperature had no influence on freeze-thaw survival in the slow freeze-thaw experiment.
In contrast, the fast freeze-thaw experiment resulted in decreased survival as growth
temperature decreased. Growth at low temperatures reduces P. arcticus ability to cope
with exposure to fast freeze-thaw and UVC. Reduced growth temperature may be
negatively affecting survival by decreasing the capacity to resist or repair damage.

Exiguobacterium Species isolated from the Kolyma lowland permafrosts were
recently tested for freeze-thaw tolerance (29). In contrast to our results, low temperature
growth improved freeze tolerance of Exguobacterium spp. Vishnivetskaya and
colleagues observed the formation of highly refractive, spore-like cells at subzero
temperatures, and these differentiated cells may have contributed to improved freeze-
thaw survival at low temperatures. However, the P. arcticus data demonstrate that
increased stress resistance after low temperature incubation is not a universal strategy
selected for in permafrost microbes.

Exposure to UVC following growth at 25°C and 4°C was used to evaluate the

ability of P. arcticus 273-4 to survive DNA damage. A decrease in UVC survival at 4°C

36

relative to 25°C was observed. This indicates that unusual capacity to withstand DNA
damage was not necessary for P. arcticus to survive and be resuscitated in the lab after
1. 04 to 106 yr in permafiost. The theoretical time to break a 50% G+C genome into ~ 100
bp fragments is 81,000 yr at -10°C (31). Therefore, one must conclude that some level of
repair must have been occurring in situ. Price and Sowers compiled data indicating that
in situ survival metabolism by microbial communities is Slightly higher than theoretical
rates of DNA depurination over time (20). This suggests that exceptional ability of
microbes to resist DNA damage would only be required by organisms that are incapable
of survival level metabolism, defined as a state in which cells “can repair macromolecular
damage but are probably largely dormant” in permafrost conditions. They reported that
survival metabolism would only need to be proportionally 10'6 of the rates required for
growth metabolism.

We have demonstrated that tolerance of P. arcticus to two kinds of lethal stresses
does not improve with decreased growth temperature. P. arcticus was more resistant to
freeze-thaw induced damage than E. coli, but this was not the case when UVC damage
tolerance was tested. Low temperature does not induce damage resistance phenotypes in
P. arcticus. P. arcticus does grow under subzero temperatures and, so may not sense low
temperature growth as stress resistance inducing condition. P. arcticus may not possess
an rpoS gene, and this may also explain its lack of a general stress resistance response.
Rates of damage due to the cold, desiccation, and irradiation stresses present in the
permafrost must be slow, and the lack of a cold-induced survival response suggests that
P. arcticus is sufficiently adapted under low temperature conditions to these conditions to

at least survive damage in situ for periods of ~104 yr.

37

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40

CHAPTER 3

GROWTH AT 4°C INDUCES A LIMITED TRANSCRIPT IONAL RESPONSE IN

THE PSYCHROPHILE PSYCHROBA C TER ARC TIC US 273-4

41

Abstract

Eurypsychrophiles are capable of growth at a wide-range of temperatures ranging from <
0°C to optimum growth temperatures near 30°C. Growth at cold temperatures, T S 4°C,
impacts functions of all molecules in the cell by reducing rates and amplitude of
molecular motion. The transcriptome response to grth at 4°C and 22°C was analyzed
in the eurypsychrophilic permafrost bacterium Psychrobacter arcticus 273-4. Gene
expression for increased production of unsaturated fatty acids, increased metabolism of
oxidized substrates, and ribosomal adaptation to the cold were detected during growth at
4°C in rich V2 TSB medium. A defined mineral medium was developed to allow analysis
of the biosynthetic demands placed on the cell during growth at low temperatures.
During growth in the mineral medium, no statistical differences in gene expression were
detected in the comparison between 22°C and 4°C. The results of this study indicate that
P. arcticus does not differentiate transcriptomes strongly between growth at Top, and at
4°C, a temperature difference of 18°C. This lack of response is probably the result of

specialization in P. arcticus for life in low temperature environments.

42

Introduction

Low temperature is a global stress on organisms that leads to reduced enzymatic reaction
rates, increased viscosity, liquid-crystalline phase transitions of biological membranes,
denaturation of proteins due to weakening of hydrophobic interactions, increased
formation of secondary structures in DNA and RNA due to stabilization of hydrogen
bonds, increased steady state concentrations of reactive oxygen species, and reduced
diffusion rates of solutes (6, 36). Microorganisms subjected to selection at low
temperatures have evolved proteins and membrane lipid fatty acid compositions for
function in either constantly high or low temperature environments (28, 29). However,
microbes adapted to a broader thermal range must respond to these stressors by
expressing cold acclimation proteins. Known cold acclimation proteins have roles in
unwinding secondary structures, ribosome function at low temperature, protein folding,
expression of alternate enzymes that function at low temperature, and membrane lipid
modification (3, 4, 14, 21, 24). Transcription of these genes is typically induced at least 2
to 8-fold during cold shock and continues after cold shock at elevated levels typically in
the range of 1.5 to 3-fold during cold acclimated growth (15, 22). However, gene
expression in a low temperature versus optimal growth state may depend on the
evolutionary history of an organism. Psychrophiles likely do not experience cold stress
in the same temperature thresholds as mesophiles and are probably differentially adapted
to cope with growth at low and moderate temperatures.

Eurypsychrophiles have a great potential for expanding our knowledge of the
functional genomics of thermal adaptation because they are capable of metabolism at

temperatures ranging from below 0°C up into the mesophilic range (25°C to 40°C).

43

Psychrobacter arcticus is such an organism with a growth temperature range of -10°C to
28°C (1). P. arcticus adjusts its membrane lipid fatty acid composition, capsular
carbohydrate composition, and substrate utilization profile during growth at 4°C versus
growth at 25°C (23). However, the transcriptional changes contributing to this modified
state at 4°C are unclear. Here we present a transcriptome analysis of P. arcticus growing
under rich medium conditions at 22°C compared to 4°C. A mineral medium was also
developed to permit analysis of the biosynthetic challenges of life at low temperature
during grth on a single carbon source. This medium enabled us to analyze
transcriptome changes for the same temperature comparison in the context of

biosynthetic pathways that may not be required in a complex grth medium.

Materials and Methods

Culture conditions for V2 TSB temperature comparison. Four biological replicate
cultures of Psychrobacter arcticus 273-4 were inoculated from V2 tryptic soy agar plate
(V2 TSA, BD Biosciences, San Jose, CA) into a 5 m1 starter culture of V2 tryptic soy broth
(V2 TSB, BD Biosciences). Experimental cultures consisted of 50 m1 V2 TSB and were
inoculated with 50 ul stationary phase starter culture. Cultures were grown at either 22°C
(Topt) or 4°C with shaking at 150 rpm to mid-exponential phase (OD600 E 0.1) in a 300 m1
Nephelo flask (Bellco Biotech., Vineland, NJ) for cell harvest and RNA extraction
(below).

Medium development. A basal mineral medium was developed that allowed for growth
of Psychrobacter in a medium containing 3% sea salts, 20 mM D,L-lactic acid or sodium

pyruvate, 5 mM NH4C1 or 5 mM glutamate, 1 mM KZHPO4, 1X Wolfe’s Vitamins and

44

1X Trace Minerals. Wolfe’s Vitamins (1000X) and trace minerals (1000X) were
produced as described by Kostka and Nealson (18). HEPES, PIPES, MOPSO, MOPS
and Phosphate buffer all at pH 7.0 were tested for ability to support growth.
Concentrations of 1 mM and 2 mM KZHPO4 were tested, but higher concentrations could
not be used due to precipitation of phosphate salts particularly in high sea salts medium.
For grth rate determinations, P. arcticus was cultured initially in marine broth
(MB) containing 5% sea salts, 5 g l'1 tryptone, and 1 g l'1 yeast extract until it reached
stationary phase about 40 h after inoculation. A 1% inoculum was added to the mineral
medium containing 5% sea salts and 20 mM lactate, and cells were grown to stationary
phase. Cells were then cultured to stationary phase in mineral medium containing the
carbon source to be tested. After two successive passages to condition P. arcticus to the
selected carbon source, growth was measured by OD600 in a Spec20D visible light
spectrophotometer. Growth rates were estimated from the data in the OD600 range from
0.03-0.3. Cultures were considered negative for grth on a substrate if no growth was
observed after 5 days at 22°C or 10 days at 4°C.
Culture conditions for acetate medium temperature comparison. Three biological
replicate cultures were inoculated from marine agar (MA) plates into a 5 ml starter
culture in marine broth (MB) at 22°C. One liter MB contained 5 g tryptone, 1 g yeast
extract and 30 g sea salts. P. arcticus was acclimated to mineral medium containing 20
mM acetate and 30 g 1'1 sea salts by growth to stationary phase (OD600 ~ 0.8) through
three passages in 50 ml acetate medium with shaking at 150 rpm. Cultures for analysis

by microarrays were grown in 50 ml acetate medium with Shaking at 150 rpm in a 300 ml

45

Nephelo flask at either 22°C or 4°C until the cultures reached rrrid-exponential phase
growth (OD600 ~ 0.1).
Microarray Characteristics. The Psychrobacter arcticus 273-4 microarray consists of
2144 70-mer oligonucleotide probes (Operon, Huntsville, AL). Arrays were printed in
duplicate on CMT-UltraGAPS slides (Corning, Corning, NY). Each array consists of
1998 probes for P. arcticus genes, 12 random oligonucleotides with no matching
sequence to any gene in the probe database, 10 SpotArray (Stratagene, La Jolla, CA)
oligonucleotide probes to assay spike-in positive controls, and 10 cloned human
sequences with no homology to P. arcticus genes. The remaining spots contained only
0.3X SSC printing buffer.
Cell Harvest, RNA Extraction and cDNA synthesis. When cultures reached mid
exponential phase, 25 ml of culture was preserved with 2 volumes isothermal
RNAprotect Bacteria Reagent (Qiagen, Valencia, CA). Cells in RNAprotect were
incubated at room temperature for 5 min then pelleted by centrifugation in a Sorvall RC-
5B centrifuge (Dupont) at 4°C for 25 min at 8000 x g in an SLA-1500 rotor. Cells were
resuspended in 1 ml room temperature RNAprotect and pelleted again in an Eppendorf
5417R centrifuge at 4°C at 8000 x g for 10 min. Supernatant was decanted and the cell
pellet was frozen at -80°C.

RNA extractions were performed according to the RNEasy mini kit (Qiagen) with
the following modifications: Specificially, lysozyrne digestion was carried out for 30
min. Following cell lysis using buffer RLT, 1 ul each of two-fold diluted SpotReport

mRNAs (Stratagene) ranging from 1 ng to 1 pg of RNA were added to the lysate as

46

controls for RNA degradation during sample preparation. Purified RNAs were analyzed
by gel electrophoresis in a 1X FA gel containing 0.67% formaldehyde and 1.2% agarose.

Three x 5 pg aliquots of total RNA were denatured with 6 pg random hexarners
(Invitrogen, Carlsbad, CA) in a 17.5 pl volume for 10 min at 70°C and snap-cooled 5 min
on ice. Denatured total RNA mixtures were reverse transcribed to amino-ally] labeled
cDNAs by combining the total RNA mixture with 6 pl 5X First Strand Synthesis Buffer,
3 pl 0.1 M dithiothreotol, 1 pl RNaseOUT (Invitrogen), 1.2 pl 25X dNTPs (3 aa-dUTP:2
dTTP), and 2 p1 Superscript II reverse transcriptase (Invitrogen). Amino-allyl dUTP was
obtained from Arnbion (Austin, TX). Reactions were incubated at 42°C overnight and
stopped by addition of 10 pl 0.5 M EDTA. RNA was hydrolyzed by incubation with 10
pl 1 M NaOH at 65°C for 15 min. RNA hydrolysis was neutralized with 10 pl 1 M HCl.
Amino-ally] labeled cDNAs were pruified according the amino-ally] labeling protocol
posted by The Institute for Genomic Research (12).
Microarray Hybridization. Amino-allyl labeled cDNAs were resuspended in 4.5 pl 0.1
M NazCO3, pH 9.0 for 10 min at room temperature and combined with 4.5 pl Cy3 or Cy5
NHS-ester in DMSO (GE Biosciences, Piscataway, NJ). Dye incorporation reactions
were incubated at 25°C for 1.5 h. Equipicomolar nucleotide amounts of dye-labeled
cDNA samples to be hybridized such that neither Cy3 nor Cy5 exceeded 550 pmoles.
cDNAs were dried in a SpeedVac for 1.5 h. Hybridization mix was prepared by
resuspending cDNA pellets in 50 pl 5X SSC, 25% forrnamide, 0.1% SDS, 0.1 mg ml'1
Salmon Testes DNA.

Glass slide arrays were prehybridized by incubation in 5X SSC, 0.1% SDS, 0.1

mg ml'1 BSA for 60 min at 49°C. Slides were then washed twice in room temperature

47

0.1X SSC for 5 min. Slides were washed twice for 30 sec in sterile water. Prehybridized
slides were then immediately dried by centrifugation for 3 min at 1600 rpm in a Sorvall
RT 6000D centrifuge with an H1000B hanging basket rotor. Lifierslips (22 x 60 mm,
Erie Scientific, Portsmouth, NH) were washed in sterile water followed by 100% ethanol
and dried under filtered forced air. Hybridization mix was preheated to 95°C for 10 min,
then pipetted onto the slide. Hybridization cassettes were sealed and immediately placed
in a 49°C water bath shaking at 30 rpm for 20 h.

Hybridized slides were first washed in 2X SSC, 0.1% SDS for 5 min at 49°C. A
second wash was performed twice in 0.1X SSC, 0.1% SDS at room temperature for 5
min. A third wash was performed four times in 0.1X SSC for 1 min each. The final
wash was in 0.01X SSC for 10 sec. Washed slides were dried by centrifugation as
described above. Slides were scanned using a Genepix 40003 scanner (Molecular
Devices, Sunnyvale, CA). Images were analyzed and quality control analysis was
performed using Genepix 6.0 software. Only slides passing the quality control statistical
analysis were included in the biological data analysis.

Microarray data analysis. Fluorescence data from rnicroarray hybridizations were
analyzed in the LIMMA 2.7.10 package in R (33). Minimum background correction was
applied and data were normalized by print-tip lowess normalization (37). Differential
expression was estimated using a simple linear model to estimate the effect of 22°C or
4°C incubation on gene expression. Statistical analysis was performed by t-test with
false discovery rate control correction for multiple hypothesis tests (25). Genes were

considered differentially expressed with P < 0.05.

48

Accession Numbers. The P. arcticus genome was deposited under GenBank accession

number CP000082 by H.L. Ayala-del-Rio et al.

Results

Analysis of differentially expressed genes at 22°C and 4°C in rich medium.
Transcriptomes of P. arcticus grown at 22°C (Top,) and 4°C in V2 TSB medium were
compared using oligonucleotide microarrays. Gene expression changes were analyzed
using the LIMMA 2.7.10 with false-discovery rate control for multiple hypothesis tests.
We deemed a post correction P-value cutoff of 0.05 to be significant in these experiments
across four biological replicates. Only 44 genes were differentially expressed with a P <

0.05 (Fig. 3.1).

 

 

 

 

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Figure 3.1. M vs. A plot in P. arcticus grown at 4°C
(positive M-values) and 22°C (negative M-values). M is
defined as log2(4°C/22°C). A is log2(4°C - 22°C)”2). Only
spots with mean intensity (A) greater than 7 were considered
relevant for our analysis due to our use of background
correction. The dashed line represents the intensity cut-off.
Black points are genes that were statistically differentially
expressed (P < 0.05).

49

Eighteen genes were down-regulated during grth at 4°C with fold change ranging
from 1.3 to 2-fold. These included genes for the isobutyryl-CoA degradation, signal
transduction, and fatty acid biosynthesis (Table 3.1). Isobutyryl-CoA may be required in
higher concentrations by the cell at 4°C, possibly as a substrate for branched chain fatty
acid biosynthesis. Other fatty acid biosynthesis genes which were down-regulated
included a long-chain fatty acid acyl-CoA ligase and an enoyl-ACP reductase which
might indicate decreased need for long-chain and saturated fatty acids at low temperature.
Signal transduction functions in nitrogen metabolism and twitching motility were down-
regulated. Dicarboxylate uptake (Psyc_1965) and malic enzyme (Psyc_1 36 7) were
down-regulated indicating that dicarboxylic acids may be the substrate of choice for
grth at 22°C but not at 4°C.

In contrast, up-regulated genes at 4°C had roles in transport, translation,
transcription and energy metabolism. Up—regulated genes at 4°C exhibited fold changes
ranging from 1.2 to 4.8-fold. Psyc_1653, a glycolate and lactate uptake symporter was
up-regulated, as was the periplasmic binding subunit of a basic amino acid ABC
transporter (Table 3.2). The septum ring formation antagonist minE was up-regulated at
4°C. Several genes involved in energy metabolism and the metabolism of glycolate or
glyoxylate were also up—regulated at 4°C including genes for the B-hydroxyaspartate
pathway of glyoxylate metabolism, an alternative to malate synthase driven glyoxylate
metabolism. Two out of three acetone carboxylase subunits were strongly up-regulated
during grth at 4°C. A peptidyl-prolyl-cis-trans isomerase was up-regulated at 4°C

possibly to speed peptide folding.

50

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52

Development of a mineral medium. We concluded that the observation of only a small
number of differentially expressed genes in the rich medium might be due to the
nutritional complexity of ‘/2 TSB. Hence, a mineral medium was developed to analyze
biosynthetic challenges resulting from growth at 4°C. This medium formulation was
based on hypotheses from a metabolic pathway reconstruction which predicted that P.
arcticus possesses the necessary gene complement for growth on proline, serine,
propionate, malonate, glycolate, acetate, fatty acids, lactate, and dicarboxylic TCA cycle
intermediates (N .N. Ivanova, personal communication). Lactate was chosen as the
carbon source for development of the medium because P. arcticus contains a lactate
dehydrogenase cytochrome which was not differentially expressed in the 22°C vs 4°C
comparison of growth in 1/2 TSB. Furthermore, transport of lactate was up-regulated at
4°C, indicating that this substrate might be more important to P. arcticus in the cold
(Table 3.2). After several trials with various buffers and salt mixtures, a basal medium
was developed for growth on 20 mM lactate. The medium contained 5% sea salts (w/v),
50 mM MOPS, pH 7.0, 5 mM NH4C1, 1 mM K2HP04, 1X Wolfe’s Vitamins, and 1X
trace minerals mix. Psychrobacter was tested for growth on a variety of carbon sources
(Table 3.3). No growth was observed on glucose, isovalerate, isobutyrate, glycerol,
malate, succinate, citrate, glycolate, glyoxylate, serine, glycine or propionate at 22°C or

4°C after repeated attempts using inocula pre-cultured on acetate, lactate and marine

broth.

53

Table 3.3. Growth rates of Psychrobacter arcticus
273-4 on defined basal medium with various sole

a
carbon sources

 

-1
Growth Rate (AlongDxm hr J

Substrate 22°C 4°52
Acetate 0.069 d: 0.003 0.016 t 0.001
Acetate + 5 mM 0.073 i 0.002 d
ND
Glutamate
d
Malonate 0.067 t 0.003 ND
Pyruvate 0.003 t 0.0008 0.005 d: 0.0006
Lactate 0.027 t 0.002 0.010 :1: 0.002
Lactate + 5 mM 0.028 t 0.004 d
ND
Glutamate
Butanoic Acid 0.041 :E 0.001 0.011 :t 0.0008
Decanoic Acidc 0.014 3: 0.004 0.006 a: 0.002
b
Palmitic Acidc 0.007 NG
d
Glutamate 0.040 a 0.004 ND
Marine Broth, 5% 0.106 i 0.006 0.026 :1: 0.0008

Sea Salts

1/2 TSB + 5% NaCl 0.019 :t 0.0004 0.005 a; 0.0007
a Unless otherwise noted, all carbon sources were at 20
mM concentration. No growth was observed when
glyoxylate, glycolate, glycine, proline, serine, glycerol,
isovaleric acid, isobutyric acid, propionic acid, citrate,
succinate, malate or glucose at 22°C or 4°C with

b
NG = No growth after 10 days at 4°C

 

c
No significant growth observed on 0.5% Ethanol

introduced to cultures as solvent for decanoic and

d ND = No data collected under this condition.

We hypothesized from the microarray data that lactate would support the fastest
growth rates. However, cultures in lactate medium grew at only 39% of the rates
achieved on acetate. P. arcticus had the fastest rate of grth on acetate and malonate
media, the two basic units of fatty acid metabolism, and growth rate decreased as fatty
acid chain length increased. Acetate enabled grth at 65% of the rate observed in the

complex marine broth with 5% sea salts. Supplementation of the medium with 5 mM

54

glutamate yielded only a slight increase in grth rate over medium containing acetate or
lactate alone.

Analysis of differentially expressed genes at 22°C and 4°C in a defined medium.
Microarray hybridizations were performed on cultures grown to mid-exponential phase at
22°C and 4°C in the 3% sea salts medium with acetate as the sole source of heterotrophic
carbon. We expected that by culturing P. arcticus in the acetate medium, temperature
specific biosynthetic challenges could be elucidated through transcriptome analysis.
However, no differential gene expression was observed between the two temperatures

with an FDR corrected P < 0.05 (Fig. 3.2).

 

 

 

 

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A

Figure 3.2. M vs. A plot comparing the transcriptome a 4°C
(positive M values) to the transcriptome at 22°C (negative M
values) in acetate medium. No statistically significant
differential expression was observed between the two
temperatures this experiment.

55

Table 3.4. Genes with >1 .S-fold change in mean expression between 22°C and 4°C

M .Gflg mm mam“ m
Psyc_0397 Psyc_03 97 hypothetical protein 2.1 1 1.000
Psyc_0286 Psyc_0286 probable RNA pseudouridine synthase -2.93 1.000
Psyc_1872 Psyc_] 872 conserved hypothetical protein -2.65 1.000
Psyc_0243 Psyc_0243 conserved hypothetical protein -1 .70 1.000
Psyc_0876 Psyc_08 76 conserved hypothetical protein -2. l 3 1.000
Psyc_1126 Psyc_1126 probable exonuclease V, alpha subunit -l.76 1.000
Psyc_13 77 Psyc_ 1 3 7 7 conserved hypothetical protein 1 .73 1.000
Psyc 0721 Psyc 0721 hypothetical protein 2.13 1.000

 

a Fold change is expressed relative to 4°C.

In an attempt to learn as much as possible about the temperature response in the
nutritionally simple medium, we relaxed our differential expression criteria to include
any gene with an average fold change > 1.5 in this experiment. Only eight genes were
detected as differentially expressed based on fold change alone (Table 3.4). Psyc_0286
and Psyc_1126 had functions in rRNA maturation and recombination respectively. The

remaining six genes were hypothetical proteins with no known function.

Discussion

In spite of genomic capacity to acquire and metabolize a variety of amino acids and
organic acids, P. arcticus demonstrated a clear preference for growth supported by a
limited range of oxidized mono-carboxylic acids and fatty acids. When rich and mineral
media were used as a growth medium for transcriptome comparisons at 22°C and 4°C,
only small magnitude changes in < 50 genes were observed. This suggests that 4°C does
not significantly alter growth physiology compared with growth at 22°C (Topt). It seems
likely from our transcriptome results that adjustments to metabolism at 4°C might be

better explained on the level of posttranscriptional regulation.

56

P. arcticus may be optimized to conserve energy and carbon by utilizing acetate,
a ubiquitous substrate, as the basis for its biosynthesis and energy metabolism. Acetate
could originate in permafrost from numerous biological and chemical processes including
the degradation and metabolism of reduced molecules. Acetate metabolism has several
features that may be desirable at low temperatures. Uptake of acetate can occur directly
via the cytoplasmic membrane. P. arcticus does not contain a homologue of ActP, the
acetate uptake transporter (11). Utilization of the glyoxylate shunt can allow P. arcticus
to conserve much of the carbon from this substrate. Metabolic activation of acetate
requires only one to two enzymes to generate acetyl-CoA from acetate and coenzyme A,
so the net energy investment in acetate metabolism is low though this may be offset by
increased net cost to synthesize other key cell constituents such as carbohydrates or
amino acids.

The reasons for lack of growth on dicarboxylates and uncharged amino acids are
unclear. Transporters are present for the dicarboxylates (TRAP-T dicarboxylate uptake
transporter Psyc_1963-1965), glycolate (glycolate:H+ symporter Psyc_1653), and
glycine (BCCT uptake transporter Psyc_1301). P. arcticus has only a 2.7 Mbp genome,
and small genomes possess smaller proportions of regulatory genes than large genomes
(16). So, it is possible that culture conditions used here were unable to induce utilization
of these substrates for growth, or that unpredicted aspects of the metabolic network of P.
arcticus prevented growth on these substrates despite the presence of the complete gene
complement. Growth on palmitic acid was only observed at 22°C. The reasons for this

seeming temperature preference are unclear, but could be due to a combination of

57

reduced water solubility of the compound at low temperatures and reduced uptake of the
compound due to decreased membrane fluidity at 4°C.

A slight inflection of growth rate decline over temperature was observed at 6°C in
P. arcticus growing in ‘/2 TSB (23). In the same study, P. arcticus was reported to
decrease fatty acid chain length, increase the proportion of unsaturated fatty acids in
membrane lipids and to increase cell length and cell volume during growth at 4°C. The
transcriptome in ‘/2 TSB generally supports the hypotheses from the results of the
physiological analyses that showed fatty acid metabolism changes. Genes for long chain
fatty acid synthesis and saturation of fatty acids during biosynthesis were
transcriptionally down-regulated at 4°C. In Bacillus subtilis, degradation of valine to
isobutyryl-CoA was up-regulated during cold shock (15). An increase in the metabolic
pool of isobutyryl-CoA could lead to increased formation of iso-branched chain fatty
acids. Down-regulation of genes for isobutyryl-CoA degradation may indicate increased
need for this branched-chain fatty acid substrate. However, only a small but statistically
insignificant increase in branched chain fatty acids was observed in P. arcticus at 4°C
(M. Ponder, personal communication).

MinE antagonizes the formation of the cell division septum. Location of the
division septum formation by FtsZ polymerization is controlled by diffusion of MinCD,
and MinE from pole to pole in rod shaped cells (26, 30). Diffiision rates decrease over
decreasing temperature but an increase in concentration at the high end of a concentration
gradient can increase diffusion rates. Therefore, the increase in cell length observed by

Ponder et a] may be due to overcompensation by increased expression of minE.

58

P. arcticus has a rare mode of glyoxylate metabolism in which glyoxylate,
resulting from isocitrate lyase activity, is transformed to glycine then condensed with
another glyoxylate to form B-hydroxyaspartate (17). B-hydroxyaspartate aldolase
produces oxaloacetate from beta-hydroxyaspartate. Activities associated with this
pathway were up-regulated at 4°C. This increase can be interpreted as a demand to
process the products of acetate or glyoxylate metabolism faster or to regenerate
oxaloacetate as a precursor for gluconeogenesis. Acetone carboxylase was also up-
regulated at low temperature which carboxylates acetone or isopropanol to organic acids
acids (32). However, the source of acetone in the cell is unclear.

The ribosome responds to cold shock in a variety of organisms. For example,
Listeria monocytogenes ribosomes become more thermally sensitive after cold shock (2).
Translation was also clearly affected in P. arcticus by growth at 4°C. Four ribosomal
proteins were detected as up-regulated at 4°C. Loss of ribosomal protein L30 is reported
to produce an E. coli mutant deficient in ribosome assembly at 20°C (9). Ribosomal
proteins 813, 811 and L13 have not been reported to be involved in cold acclimated
ribosome biogenesis. RNA binding genes associated with translation in the cold, e.g.
cspA, csdA, were not induced during growth at 4°C, but P. arcticus did induce one PPIase
at low temperature which may indicate that P. arcticus is increasing expression for
peptide folding.

When taken together, the transcription response to growth at 4°C in V2 TSB
suggests only a fine-tuned compensation for translation and carbon catabolism, coupled
with a membrane fluidity response. Irnportantly, a proteome comparison of P. arcticus

growth at 22°C and 4°C shows similarities in the functional categories of up—regulated

59

genes at 4°C including increased expression of PPIases, PhoH, and acetone carboxylase
(39).

When P. arcticus was placed in a medium with a single source of carbon, no
change gene expression due to grth temperature was observed. In the permafrost,
responses to low temperatures, low water activity and decreased nutrient availability may
be linked (10). Exposure to low temperatures is known to effect survival during osmotic
shock and starvation (8, 19, 20). Therefore, carbon source limitation or salt content of
the mineral medium may have moderated the effect of temperature on the transcriptional
response of P. arcticus to growth at 4°C. Furthermore, the Q10, or change in growth rate
over 10°C, in ‘/2 TSB is ~2.25 versus ~2.15 in the acetate medium and may not demand
adjustments in the cell metabolism to support different growth rates. Growth at
temperatures exhibiting a Q10 > 4 would be expected to yield major physiological
changes over temperature and likely exhibit substantial changes to the transcriptome (6).

Effects of temperature on physiology and the differentiation of species is a long
researched area in biological science (27, 34). Adaptations of polar species are of
particular interest because their physical environment is different in many ways from
those in temperate or tropical environments (35). Polar environments are commonly
energy and nutrient limited systems with reduced biomass and slower rates of microbial
activity (5). Numerous adaptations to these environments have been elucidated including
observations of a reduced threshold to induce heat shock responses, reduced hemoglobin
content in Antarctic fishes, production of antifreeze and ice nucleation proteins, and
adaptation of proteins for high activity rates at low temperatures with concomitant

increased thermolability of those proteins (7, 13, 31, 38). Adaptation to the permafrost

60

environment has likely impacted the P. arcticus response to temperature. P. arcticus has
undergone selection in the cold and may not sense a temperature or growth stress at 4°C
relative to growth at 22°C. P. arcticus may have regulatory mechanisms that modify the
activity of specific enzymes in response to temperatures. Indeed, evidence for post-
translational modifications was observed in a P. arcticus proteome comparison at 22°C
and 4°C (39). Furthermore, reductions in enzyme activity can be attained due to simple
temperature effects on enzymes without changes to transcription. Therefore, changes in
physiology observed by Ponder et al may be regulated more directly on a post-

transcriptional level.

61

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65

CHAPTER 4

RESOURCE EFFICIENCY AND MOLECULAR DYNAMICS DOMINATE THE

TRAN SCRIPT OME RESPONSE FOR GROWTH BELOW 0°C IN

PS Y CHROBA C T ER ARC TIC US 273-4

66

Abstract

We analyzed the trancriptome of Psychrobacter arcticus 273-4 grown at two low
temperatures: 0°C and -6°C, and two high temperature: 17°C and 22°C using a mixed
effects ANOVA model. Genes involved in transcription, translation, energy production
and the majority of biosynthetic pathways were down-regulated at low temperatures.
However, specific genes in the transcriptome were up-regulated at 0°C and -6°C
presumably to compensate for reduced function of their products. Evidence of enzyme
exchange was detected during growth at low vs high temperature, particularly for DD-
peptidase, DEAD-box RNA helicase and energy efficient substrate incorporation
pathways for ammonium and acetate. Other genes exhibited compensatory up-regulation
at low temperature including specific genes for uradine biosynthesis and the biosynthesis
of proline, tryptophan, methionine, and histidine. RNases and peptidases were generally
up-regulated at low temperatures. In contrast to results observed in other psychrophiles
and mesophiles, only clpB and hsp33 were up-regulated at low temperature with no up-
regulation of other chaperones and peptidyl-prolyl isomerases. Knockout mutants of
katE, msrA, csdA, and dac2 were all deficient in low temperature growth, but a mutant in
dad was deficient in growth at 22°C. Our data suggest that the majority of the basal
biological machinery including translation, transcription and energy metabolism are well
adapted to function across the -6°C to 22°C growth range of P. arcticus and temperature
compensation by gene expression was employed to address specific challenges to low

temperature life.

67

Introduction

Growth at low temperatures presents several challenges for life including reduced
chemical reaction rates, rates of physical processes and water activity (53). Metabolism
at subzero temperatures has been proven for psychrotrophs and psychrophiles isolated
from cryoenvironments, i.e. environments which exhibit transient or long-term freezing
conditions, including sea ice, cryopegs, and permafrost soils (9, 15, 37). Proteomic
studies of psychrophile responses to growth at suboptimal temperatures have identified
differentially expressed genes including energy metabolism, translation factors and
peptidyl-prolyl isomerases (29, 51). It remains unclear how a cryoadapted microbe must
globally modify metabolism for growth at subzero temperatures. Gerday and others have
reasoned that eurypsychrophiles could be most energy efficient if their key metabolic
proteins were able to function under the full growth temperature range (14, 21, 48).
Enzyme adaptations for localized flexibility of the peptide chain surrounding the active
site and counteradaptations for thermostability of the peptide chain can allow proteins to
function over a wide range of temperatures (21, 50). However, an organism approaching
the lower temperature limit of its growth would benefit from differential gene expression
as a means of metabolic flexibility versus the alternative of growth inhibition. Organisms
can increase expression of genes in order to compensate for reduced biochemical reaction
rates or can differentially express isozymes to adapt to temperatures as has been observed
in fish and some bacteria (43, 55). Gene expression studies on organisms growing at
suboptimal growth temperatures have identified increased expression of genes important

to several responses necessary for life in the cold, including DNA and RNA binding

68

proteins to destabilize secondary structures, maintenance of membrane fluidity,
chaperones, uptake of compatible solutes, and energy production (3, 6, 46, 57).

Psychrobacter arcticus 273-4 was isolated from Kolyma lowland permafrost
sediments continuously frozen for 10,000 to 40,000 yr (4, 60). Permafrost poses four
continuous stressors on resident microbes including continuous cold, dessication, reduced
nutrient availability and low continuous levels of radiation resulting from soil minerals
(22). Ponder et al (2005) showed that P. arcticus was capable of growth below 0°C, and
that it exhibited modified membrane fatty acid profiles and substrate utilization profiles
during growth in the cold (47). P. arcticus grows in an experimentally confirmed
temperature range of -10°C to 28°C and in a salinity range from 10 mM to 1.3 M NaCl
(4). The capacity to endure temperatures and salinities in these ranges suggests that P.
arcticus is adapted for life in cryoenvironments, which are characterized by low water
activity and low temperature. Therefore, we consider P. arcticus 273-4 to be a good
model for a cryo-adapted eurypsychrophile, i.e. a psychrophile capable of growth at a
wide-range of temperatures (12).

Here we assayed the genome-wide modifications in transcription that are
necessary for P. arcticus to grow in ice-like conditions at -6°C and used these data to
learn about the physiology of P. arcticus under these conditions. We cultured P. arcticus
to mid-exponential phase at two warm temperatures: 22°C and 17°C, and two cold
temperatures: 0°C, and -6°C in a mineral medium with acetate as the sole source of
heterotrophic carbon. We used our transcriptome data to develop a conceptual model of
growth at subzero temperatures, in which the first analysis of the transcriptome of a

bacterium growing at temperatures below 0°C.

69

Materials and Methods

Microarray Characteristics and Printing. The Psychrobacter arcticus 273-4
rnicroarray consists of 2144 70-mer oligonucleotide probes (Operon, Huntsville, AL).
Arrays were printed in duplicate on CMT-UltraGAPS slides (Corning, Corning, NY).
Each array consists of 1998 probes for P. arcticus genes, 12 random oligonucleotides
with no matching sequence to any gene in the probe database, 10 SpotArray (Stratagene,
La Jolla, CA) oligonucleotide probes to assay spike-in positive controls, and 10 cloned
human sequences with no homology to P. arcticus genes. Additional spots contained
only 0.3X SSC printing buffer.
Strains, Media and Culture Conditions For all experiments, single colonies of
Psychrobacter arcticus 273-4 were first grown to OD600 2 0.8 at 4°C in marine broth
medium consisting of 3% (w/v) sea salts (Sigma-Aldrich, St. Louis, MO), 5 g/l tryptone
(BD, Sparks, MD) and 1 g/l yeast extract (Difco). A 1% (v/v) inoculum of this starter
culture was transferred to 7 ml of acetate medium. One liter acetate medium contained
75 g 1'1 sea salts (Sigma-Aldrich), 50 mM MOPS buffer (Sigma-Aldrich), 20 mM sodium
acetate (Baker), 5 mM NH4Cl (Baker), pH 7.0 with NaOH, 1 mM KzHPO4 (Baker), 1X
Wolfe’s Vitamins, and 1X trace metal mix. Wolfe’s Vitamins (1000X) and trace
minerals solutions were made according to M1 defined medium from Kostka and
Nealson (3 8).

P. arcticus 273-4 was acclimatized to the acetate medium through two passages to
OD600 _>. 0.8 at 4°C shaken at 150 rpm before inoculation into experimental samples. A 5
ml inoculum from the final starter culture of P. arcticus in acetate medium was

introduced to 500 ml acetate medium in 300 cm2 untreated and vent-capped Falcon tissue

70

culture flask. Cultures were allowed to grow without shaking at -6°C, 0°C, 17°C or 22°C
until they reached an OD600 between 0.09 and 0.25 (mid-exponential phase at all
temperatures).

Cell Harvest, RNA Extraction and cDNA synthesis. When cultures reached mid-
exponential phase, 25 ml of culture was preserved with 2 volumes of isothermal
RNAprotect Bacteria Reagent (Qiagen, Valencia, CA). Cells in RNAprotect were
incubated at room temperature for 5 min then pelleted by centrifugation in a Sorvall RC-
5B centrifuge at 4°C. Cells were resuspended in 1 m1 room temperature RNAprotect and
pelleted again in an Eppendorf 5417R centrifuge at 4°C. Supernatant was decanted and
the cell pellet was frozen at -80°C.

RNA extractions were performed according to the RNEasy mini kit (Qiagen) with
some modifications. Specificially, lysozyme digestion was carried out for 30 min.
Following cell lysis using buffer RLT (Qiagen), 1 ul each of two-fold diluted SpotReport
mRNAs (Stratagene) ranging from 1 ng to 1 pg of RNA were added to the lysate as
controls for RNA degradation during sample preparation. Purified RNAs were analysed
by gel electrophoresis in a 1X FA gel containing 0.67% formaldehyde and 1.2% agarose.

Three 5 ug aliquots of total RNA were denatured with 6 ug random hexamers
(Invitrogen, Carlsbad, CA) in a 17.5 It] volume for 10 min at 70°C and snap-cooled for 5
min on ice. Denatured total RNA mixtures were reverse transcribed to amino-allyl
labeled cDNAs by combining the total RNA mixture with 6 ul 5X First Strand Synthesis
Buffer, 3 ul 0.1 M dithiothreitol, 1 ul RNaseOUT (Invitrogen), 1.2 ul 25X dNTPs (3 aa-
dUTP:2 dTTP), and 2 ul Superscript II reverse transcriptase (Invitrogen). Amino-allyl

dUTP was obtained from Ambion (Austin, TX). Reactions were incubated at 42°C

71

overnight and stopped by addition of 10 III 0.5 M EDTA. RNA was hydrolyzed by
incubation with 10 ul 1 M NaOH at 65°C for 15 min. RNA hydrolysis was neutralized
with 10 ul 1 M HCl. Amino-ally] labeled cDNAs were purified according to the amino-
allyl labeling protocol posted by The Institute for Genomic Research (TIGR) (33).
Microarray Hybridization. Amino-ally] labeled cDNAs were resuspended in 4.5 III 0.1
M NazCO3, pH 9.0 for 10 min at room temperature and combined with 4.5 ul Cy3 or Cy5
NHS-ester in DMSO (GE Biosciences, Piscataway, NJ). Dye incorporation reactions
were incubated at 25°C for 1.5 h. Equipicomolar nucleotide amounts of dye-labeled
cDNA samples were hybridized such that neither Cy3 nor Cy5 exceeded 550 pmoles.
cDNAs were dried in a SpeedVac for 1.5 h. Hybridization mix was prepared by
resuspending cDNA pellets in 50 ul 5X SSC, 25% forrnarnide, 0.1% SDS and 0.1 mg ml'
1 Salmon Testes DNA (Sigma-Aldrich).

Glass slide arrays were prehybridized by incubation in 5X SSC, 0.1% SDS, 0.1
mg ml'1 BSA for 60 min at 49°C. Slides were then washed twice in room temperature
0.1X SSC for 5 min. Slides were washed twice for 30 sec in sterile water. Prehybridized
slides were then immediately dried by centrifugation for 3 min at 1600 rpm in a Sorvall
RT 6000D centrifuge with an HIOOOB hanging basket rotor. Lifterslips (22 x 60 mm,
Erie Scientific, Portsmouth, NH) were washed in sterile water followed by 100% ethanol
and dried under filtered forced air. Hybridization mix was preheated to 95 °C for 10 min,
then pipeted onto the slide. Hybridization cassettes were sealed and immediately placed

in a 49°C water bath shaking at 30 rpm for 20 h.

72

2.

/‘\

-6°C‘ +17°C

\./

0°C

Figure 4.1. Experimental design of temperature
comparisons using rnicroarray hybridizations. The
experiment consisted of five biological replicates at
each temperature. Three cDNAs were synthesized
from each RNA extract resulting from a biological
replicate and hybridizations were carried out for each
biological replicate in direct comparison to samples
from all temperatures.

 

 

 

 

Hybridizations were performed for all growth temperature comparisons with five
biological replicates at each temperature (Fig. 4.1). Hybridized slides were first washed
in 2X SSC, 0.1% SDS for 5 min at 49°C. A second wash was performed twice in 0.1X
SSC, 0.1% SDS at room temperature for 5 min. A third wash was performed four times
in 0.1X SSC for 1 min each. The final wash was in 0le SSC for 10 sec. Washed
slides were dried by centrifugation as described above. Slides were scanned using a
Genepix 40008 scanner (Molecular Devices, Sunnyvale, CA). Images were analyzed
using Genepix 6.0 software and results were analyzed for quality using the marray and
arrayQuality packages in R 2.3.0 (http:llwww.r—proicct.orgi’). Only slides passing the
quality control statistical analysis were included in the biological data analysis.

Fluorescence data were analyzed using MAANOVA 1.0 to determine statistical
significance and LIMMA 2.7.10 to calculate pairwise comparisons of gene expression.

In both cases data were normalized using regional (print-tip) lowess normalization (16,

73

62). Normalized data were fit to a mixed-effects ANOVA model with gene, dye and
temperature as fixed terms. Array, i.e. slide, and sample, i.e. biological replicate, were
random (17). F tests were carried out using the Fs statistic in R/MAANOVA to
determine the statistical significance of differential expression (18). Genes were
considered differentially expressed if P < 0.01 after False Discovery Rate Control (FDR)
adjustment for multiple hypothesis testing (17). All pairwise temperature comparison
coefficients were estimated using LIMMA (54).
Genetics. Knockout mutants katE (Psyc_5 70), dacCI (Psyc_068 7), dacCZ
(Psyc_O 704), msrA (Psyc_1043), and csdA (Psyc_1082) were generated using essentially
the strategy described in Denef et a1 (2006). Primers, plasmids and strains for this study
are described in Table 4.1. Flanking regions of each gene were PCR amplified with
primers containing restriction sites for insertion into pJ K100. Upstream flanking regions
were amplified with primers containing Bng and N011 restriction sites on the forward
and reverse primers respectively. Downstream flanking regions were amplified with
SacII and Sacl site containing primers on the forward and reverse primers respectively.

P. arcticus 273-4 cultures for conjugation were grown in Luria Bertani (LB) broth
at 22°C for 36 h. E. coli WM3064 containing the suicide vector for knockout was grown
at 37°C in LB supplemented with 25 pg ml" kanamycin (Kan), 20 ug ml'1 tetracyclin

(Tet) and 100 ug ml'1 diaminopimellic acid (DAP) overnight.

74

Strain. Primer or Plasmid

Table 4.1: Strains, primers and plasmids used in this study.

. . a
Descnptron

Reference or Source

Strains
Psychrobacter arcticus
273-4 Wild type Bakerrnans et al (2006a)
PBOS 70 AkatE :zkan This study
PBO687 AdacCl :2kan This study
PBO704 AdacCZ ::kan This study
PBlO43 AmsrA ::kan This study

Escherichia coli WM3064

Primers

de1570UF AGCTAGATCTgcgtatttcaaaaccgatcc This study
de1570UR AGCTGCGGCCGCccattgccacgttccatatt This study
de1570DF AGCTGAATTCaaggttcgtgaccttgccta This study
de1570DR AGCTCATATGacatcacatcgacaccctga This study
del687UF AGCTAGATCTgccctttattgatacgctgttt This study
del687UR AGCTGCGGCCGCcacaatcgttggcagactgt This study
del687DF AGCTGAA'ITCgctgggcacaaaacttatt This study
del687DR AGCTCATATGaagtgggatggtctgactgc This study
del704UF AGCTAGATCTgcgagcatacggctaatacc This study
del704UR AGCTGCGGCCGCcactgcccaaactcaatgaa This study
del704DF AGCTGAA'ITngtgccaatagctcagcaac This study
del704DR AGCTCATATGtagatgcacgccaataccaa This study
dell 043UF AGCTAGATCTgaaaggtatgccggctcata This study
dell 043UR AGCTGCGGCCGCgtggcaaatccgtaaacgtc This study
de11043DF AGCTGAATTCatggaagcagcaggttatgg This study
de11043UR AGCTCATATthgngcgcacattggttac This study
de11082UF AGCTAGATCTctgcgattttgggtctagga This study
de11082UR AGCTGCGGCCGCgtttggggtgtcagtgcttt This study
de11082DF AGCTGAA'ITthcaagtggtagccaaggtc This study
de11082 DR AGCTCATATGccataaatctgcgagccaat This study
kanF GAAGATCTctcaaaatctctgatgttacattgc This study
kanR GAAGATCTtggttgatgagagctttgttgtaggt This study
Plasmids

pJKlOO Allelic Exchange Vector Denef et al (2006)
p57OD pJKlOO with Psyc_05 70 downstream flanking sequence This study
p570UD pJ K100 with both Psyc_05 70 flanking sequences This study
p687D pJKl 00 with Psyc_068 7 downstream flanking sequence This study
p687UD pJKlOO with both Psyc_068 7 flanking sequences This study
p704D pJKlOO with Psyc_0704 downstream flanking sequence This study
p704UD pJ K100 with both Psyc_0704 flanking sequences This study
p1043D pJKlOO with Psyc_1043 downstream flanking sequence This study
plO43UD pJKl 00 with both Psyc_/043 flanking sequences This study
p1082D pl K100 with Psyc_/082 downstream flanking sequence This study
p1082UD pJKlOO with both Psyc_]082 flanking sequences This study

lhrBl 004 pro thi rpsL hst lacZ A1415 RP4-l360
A(araBAD)567 AdapA 1341::[erm pir]

Denef et al (2006)

 

Capital letters Include a 4 nucleotide extensron on the 5' end of pnmers and a restnctron srte for Insertion Into pJ K100.

Cells were combined in a ratio of 200 III E. coli donor and either 200 pl or 800 1.11 P.
arcticus 273-4 recipient. Conjugants were allowed to grow for 72 h at 22°C. Putative

knockouts were isolated on LB plates with 25 pg ml'l Kan and screened for a Kanr Tets

75

phenotype. PCR screening of knockouts revealed amplicons of the expected product size
and no wild-type amplicons were observed in mutant strains used for growth
experiments.
Mutant Growth Experiments. Representative strains of the knockout mutants were
grown in MB supplemented with 25 pg ml'1 Kan for 48 h at 22°C. Two subsequent
rounds of culturing were carried out in acetate medium with 25 pg ml'1 Kan for 72 h.
Mutant growth rates were assayed using 96 well plates. Eight replicates of each
mutant were inoculated into 200 pl acetate medium in each well. Sixteen replicates of
wild-type P. arcticus 273-4 were present on each plate along with 12 blank wells. Plates
were incubated at 22°C, 17°C, 4°C, 0°C, and -2.5°C. Blank subtracted OD600 data were
collected using a SpectraMax M2 spectrophotometer (Molecular Devices). Data were
compiled using a Perl script to parse data by growth temperature, mutant strain and time
point. T-tests and plotting of data were carried out using R 2.4.0. The script calculated
average growth rates for each replicate of each mutant for optical density increases
between OD600 values 0.03 and 0.4. Two-tailed t-tests were performed to determine if
growth rates were statistically different between wild-type P. arcticus and PB0343 within
a temperature. Strains were considered statistically different if P < 0.05 after Bonferoni
multiple-testing correction over the five hypothesis tests within a temperature.
Results
Growth physiology changes with temperature. Growth rate measurements were
performed at -6°C, 0°C, 4°C, 17°C and 22°C in acetate medium (Fig. 4.2). Comparison
of grth rates revealed that the Q10 of growth rate, defined as the fold—decline in

growth rate over 10°C, exhibited the expected relationship of Q10 ~ 2 between growth

76

rates at 17°C and 4°C but not at 22°C or at temperatures below 4°C. The inflection in
growth rate behavior between 17°C and 22°C suggested that growth at 22°C was being
impacted by heat-stress on the culture. The Q10 in the temperature range 17°C to 0°C and
0°C to -6°C were 6.2 and 8.4, respectively. Change in growth rates of cultures grown at
0°C and -6°C were not continuous with changes in growth rates of cultures grown at
higher temperatures. The observed inflection in growth rate decline at T < 4°C indicates
that some changes in P. arcticus physiology have occurred resulting from incubation at
0°C or -6°C.

Temperature(°C)
-2 2 J 12 17 22

r r
i .
O
I

 

1

 

I/'

I l T I I I
270 275 280 285 290 295
Temperature(K)
Figure 4.2. Square-root transformed of growth rates plotted
over temperature. P. arcticus was grown at 22°C, 17°C, 4°C,

0°C, and -6°C. Lines are the result of fitting the square-root
growth rates to a Belehradek-type model.

JAIOgm(OD(,oo) Girl)
005 010 015 020 025

L

 

 

 

Microarray Results. Microarray analysis of growth in acetate medium at -6°C, 0°C,
17°C, and 22°C was performed to elucidate differences in gene expression between
growth at the optimal growth temperature of 17°C and 22°C, cold acclimated grth at

0°C, and near the limits of P. arcticus grth at -6°C (Fig. 4.1). One-thousand-eighty-

77

five coding sequences (CDS) were differentially expressed during growth at one or more
temperatures with P < 0.01. LIMMA analysis identified 92.9% of those genes identified
by R/MAANOVA, but R/MAANOVA identified only 73.1% of the genes identified by
LIMMA as differentially expressed. This highlights the more conservative nature of the
ANOVA analysis, which models sources of variance in expression values (1).
Temperature comparison plots of the microarray results demonstrate that transcriptomes
at 0°C and -6°C are more similar to each other than to the trancriptomes at 22°C and
17°C (Fig. 4.3). The largest observed number of differentially expressed genes, 572
(27% of the P. arcticus genome), were in the comparison of -6°C vs. 17°C. Only 88
genes were differentially expressed between cells growing at 22°C vs. 17°C, and 24
genes were differentially expressed between cells grown at 0°C vs. -6°C. The trends in
number of differentially expressed genes with temperature show that the P. arcticus
transcriptome can be classified into the high temperature transcriptome at 17°C and 22°C
and the low temperature transcriptome at 0°C and -6°C. When grouped by functional
category, hypothetical and conserved hypothetical genes accounted for 28% of the
differentially expressed genes (Fig. 4.4). For ease of results communication, all fold-

changes are expressed relative to growth at 17°C unless otherwise noted.

78

0°C vs 17°C Normalized Data 0°C vs 22°C Normalized Data

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

 

 

 

 

 

 

If} If]
v' - v‘ -
O. 0.
v - <1- _
It} In
. "’ ' .U '3 ‘
O. ° 0.
m - m _
V2 V2
N " N _
c. O.
N ‘ N -
I I T r I l
2.0 2.5 3.0 3.5 4.0 4.5
17°C
22'C vs 17°C Normalized Data
o
v' - S; _
It} In
U m - U «i —
a O. ‘P o
M ‘ y; -4
V} W
N " N .1
O. C.
N " N
W V}
v' - :5 ~
6. c.
v - v -
V3 ll)
C.) vi - U N; _
:9 o :9 o
m' - r'i -
V: n.
N " N _
O. C:
N " N .1
I I I I l I I I I I I I
2.0 2.5 3.0 3.5 4.0 4.5 2.0 2.5 3.0 3.5 4.0 4.5
17°C 22°C

Figure 4.3. Plots of normalized average fluorescence values across 15 cDNA populations hybridized
from each temperature. Fluorescence data are logm transformed.

79

 

 

 

a 22‘C
I 0°C
0 -6°C

 

 

 

ofiwes+tesosexoeoowoee

Figure 4.4. Counts of differentially expressed genes compared with
17°C by COGS category. Up—regulated genes are plotted above the
zero line, down-regulated genes are plotted below the zero line. C
energy conversion, CS previously reported cold shock genes, V
defense mechanisms, N cell motility, J translation, ribosomal structure
and biogenesis, K transcription, F nucleotide transport and
metabolism, H coenzyme transport and metabolism, P inorganic ion
transport and metabolism, Q secondary metabolite biosynthesis,
transport and metabolism, T signal transduction, E amino acid
transport and metabolism, I lipid transport and metabolism, 0
posttranslational modification, protein turnover and chaperones, M
cell wall, membrane and envelope biogenesis, G carbohydrate
transport and metabolism, D cell cycle control, cell division, and
chromosome partitioning, L DNA replication, recombination and
repair, U intracellular trafficking, secretion, and vesicular transport, R
conserved hypothetical proteins, S hypothetical protein.

Substrate uptake and activation. Genes for central metabolism of acetate (TCA cycle
with glyoxylate shunt) were down-regulated at low temperatures at least 2-fold.
However, concurrent 2-fold up-regulation of acetyl-CoA synthetase and down-regulation
of acetate kinase and phosphotransacetylase were observed at low temperatures (Fig.
4.7A). Likewise, there was concurrent up-regulation of glutamate dehydrogenase and

down-regulation of glutamate synthase subunits at low temperatures (Table 4.2). These

80

data indicate that the exchange of expression of pathways for acetate activation and
glutamate biosynthesis differentiates low temperature and high temperature growth.
Psyc_2044, for the uptake of ammonium and inorganic phosphate uptake transporters,
Psyc_1926-1930, were not differentially expressed at low temperatures. Genes for the
uptake of sulfate, Psyc_1070 the periplasmic subunit for an ABC sulfate transporter and
Psyc_2041 the sulfate uptake transporter associated with the isc operon in P. arcticus,
were up-regulated 2.1-fold and 2.3-fold respectively at -6°C. Phosphate and ammonium
starvation were probably not significant in the cold, but sulfate uptake was being
compensated.

mRNA and Protein Turnover. Twelve peptidases and five ribonucleases were up-
regulated at low temperatures at least 2-fold (Fig. 4.5A). Genes for targeting RNAs for
degradation were up-regulated at low temperatures including pan at 2-fold, and ssrA at
1.6-fold. Only two RNases were down-regulated at 0°C and -6°C: Psyc_0292, a cold
shock RNA binding domain containing ribonuclease (2.1-fold at -6°C), and
polynucleotide phosphorylase (PNPase) (1 .8-fold at -6°C).

Oxidative Stress Response. The oxidative stress response was up-regulated at low
temperatures (Fig. 4.5B). In P. arcticus this included an ABC transporter for zinc uptake
Psyc_2033-2035, ahpC, hsp33, the isc operon Psyc_14 77-1482, and peptide methionine
sulfoxide reductases Psyc_1043 and Psyc_1950. Genes for Fe2+ uptake transporters,
Psyc_1546-154 7, were down-regulated, consistent with the oxidative stress responses.
Likewise, aconitase A was upregulated 2.2-fold at -6°C whereas aconitase B was down-

regulated 5.6-fold at -6°C (Fig. 4.7B).

81

-3.0 O +3.0

22°C 0°C -6°C
Psyc_2129 possible OmpA family protein
Psyc_2128 conserved hypothetical protein
. Psyc_2127 band 7 protein
Psyc_l92l metallopeptidase
Psyc_1921 metallopeptidase
Psyc_1842 peptidase M16 family
Psyc_ 165 9 ATP- -dependent protease La
Psyc_ 1270 probable serine protease
= Psyc_ 0986 neutral zinc metallopeptidase
Psyc_ 0282 carboxyltenninal protease
Psyc_0043 cytosol aminopeptidase
‘ Psyc_2101 exoribonuclease RNase R
Psyc_1865 ribonuclease PH 3 exoribonuclease
Psyc_1633 ribonuclease
Psyc_0912 ribonuclease H11
Psyc_048 7 ribonuclease EG
Psyc_0325 small stable RNA binding protein
Psyc_0074 PNPase
Psyc_0292 cold-shock DNA binding ribonuclease
' : . Psyc_0114 Poly(A) polymerase

A)

 

 

 

B) i“ ' N '7 Psyc_ 2035 ABC zinc transporter ZnuB
A“ “ ‘» Psyc_ 2034 ABC zinc transporter ZnuC

Psyc _2033 transcriptional regulator, FUR family
Psyc_ 2006 conserved h othetical protein
Psyc_l950 peptide methionine sulfoxide reductase
Psyc_1938 superoxide dismutase
Psyc_1797 alkyl hydroperoxide reductase, subunit C
Psyc_154 7 ABC iron transporter
Psyc_1546 ABC iron transporter
Psyc_l482 ferredoxin, 2Fe2S type
Psyc_1481 heat shock protein Hsp70
Psyc_1480 co-chaperone Hsc20
Psyc_ 14 79 HesB/YadR/thF family protein
Psyc_ I 4 78 nitrogen-fixing NifU- like
Psyc_ I 4 77 putative cysteine desulfurase
Psyc_ I 4 76 transcriptional re lator
Psyc_ 1109 metal Ion (Mn2+ e2+) transporter
_ Psyc_1097 metal Ion (Mn2+/Fe2+) transporter
Psyc_1043 peptide methionine sulfoxide reductase
Psyc_0 769 peroxiredoxin
Psyc_0741 cyclopropane fatty acyl phospholipid synthase
Psyc_0674 magnesium and cobalt transport protein CorA
., Psyc_03 79 organic h droperoxide resistance protein

Psyc_0302 heat shock protein, Hsp33

Psyc_0265 FUR family regulator

Figure 4.5. Heat map of selected differentially expressed genes. A) Peptidases and RNases. B)
Oxidative stress response. Temperature comparisons with 17°C are mapped in columns , genes are
mapped in rows. Values are expressed as log; ratio of expression. All genes were differentially
expressed in at least one contrast with P < 0.01. Images in this dissertation are presented in color.

 

 

82

Energy Conversion and Metabolism. All NADH dehydrogenase, ATP synthase
and Na+-translocating NADH-ubiquinone oxidoreductase subunits were down-regulated
at low temperatures (Fig. 4.6A). Psyc_1515 cytochrome bd ubiquinol oxidase was up-
regulated at low temperature with a 1.8-fold induction. C-type cytrochromes were either
down-regulated or not differentially expressed in the cold except Psyc_0260 which was
upregulated 2.3-fold in the cold. All TCA cycle and glyoxylate shunt genes were down-
regulated in the cold (Fig. 4.6A). Succinate and lactate dehydrogenase cytochromes,
Psyc_0098-0101 and Psyc_1654, respectively, were down-regulated approximately 2-
fold and 1.4-fold at -6°C.

Membrane Lipid and Cell Wall Response. Fatty acid biosynthesis pathways were
down-regulated at low temperatures (Fig. 4.6B). Only acyl-carrier protein, Psyc_0522,
was up-regulated 3.8-fold at -6°C, indicating that this central protein in fatty acid chain
growth is being compensated. Fatty acid desaturase, Psyc_1365, was up-regulated 2.7-
fold at low temperatures and cyclopropane fatty acid synthase was up-regulated 2.8-fold
at -6°C. Phosphatidylserine decarboxylase, Psyc_1925, which encodes
phosphatidylethanolamine biosynthesis, was up-regulated 3.5-fold at -6°C. Up-
regulation of cardiolipin synthase at 0°C and -6°C was also observed at 1.6-fold. This
change in phospholipids head-group biosynthesis may be indicative of modifications to

increase membrane interactions with cytoplasmic water.

83

-3.0

0 +3.0

_ fl

22°C
A)

    
  
  
 
 

24:125.!

P3? '8
— Psyc_ 1309 short-chain dehydrogenase

° 0
0 C -6 C

_ Psyc_ 2104- 2109 NADH-ubiquinone oxidoreductase
Psyc_ 0584- 0596 NADH dehydrogenase] subunits
Psyc_ 0555-0556 NAD(P) transhydrogenasc

-P Psyc_ 2028- 2031 ATP synthase F0 subunits
Psyc_ 2024- 2027 ATP synthase F 1 subunits

. J.,. _. Pry c0_152lc flavin oxidoredqas“

Psyc_ 1516 cytochrome bd ubiquinol oxidase, subunit II

Psyc_ 1515 cytochrome bd ubiquinol oxidase, submit 1

[=2_ Psyc_1482 ferredoxiln, 2Fe2S type
Ps scy _l422 ubiqu Inolcytochrome cl reductase
Psyc_1421 cytochrome 1), b6 subunit
Psyc_1420 ubiquinolcytochrome e reductase

F— Psyc_ 0751 aconitate hydratase B

., Psyc_! 763 malate tegenasedehydm

c1657 ante h dratase class II

Psyc_1643 malate sanhase
Psyc_! 627 isocitrate lyase

r _. .3; Psyc_ 0105-0106 suecinyl-CoA synthetase subunits
Psyc_ 0103 ' ‘ '

 

Psyc_ 0098-0101 succinate dehydrogenase subunits
E ] Psyc_ 0097 citrate synthase

transglycosylase

transpeptidase
D

ligase

A

dccarboxylase

li ase

Psyc_1299 far?! acyl-CoA reductase prote
a onate decarboxylase, alphai subunit

Pry 1146 m
Psyc_ 144 maol nate decarbox x,ylase beta subunit
Psyc_ 101 Acetyl- -CoA carboxylase, biotin carboxylase

 

Psyc_r 052 FabB family proteIn

Psyc_r 045 eno l-ACP reductase

Psyc_ 965 acy- -CoA dehydrogenase

Psyc_ 964 acyl-CoA dehydrog enase

Psyc_t 903 eno l- CoA h dratasefisomerase protein
Psyc_ 901ac -CoAde dro en ase

Psyc_ 08223 ydro xyacy -Co dehydrogenase

Psyc_ 0743a acyl-CoA throester hydrolase

Psyc _ 521 3-oxoac acyl ACP reductase

520 malonyl-CoA Pasetransacyl

Psyc_ 356 1-acyl-glycerol-3-phosphate acyltransferase
313 acyl- -CoA dehydro genase

Psyc_ 310 eno l- CoA hydratase/isomerase

» Psyc_ 309ac ac-y —CoA carboxylase (beta subunit) proteIn
, 1. Psyc acetyl-CoA acyltransferases

Psyc_ 137 acyl- -CoA dehydrogenase

 

Figure 4.6. Heat map of selected differentially expressed genes. A) Energy conversion. B) Fatty acid
and peptidoglycan metabolism. C) Transcription. Temperature comparisons with 17°C are mapped in
columns , genes are mapped in rows. Values are expressed as log; ratio of expression. All genes were
differentially expressed in at least one comparison with P < 0.01. Figure continued on next page.
Images in this dissertation

are presented in color

84

6&3 0 33.0
22°C 0°C -6°C
C) Psyc_20 70 NusB antitermination factor
.... .5 Psyc_2022 two component response regulator
Psyc_1923 two component response regulator
Psyc_1887 RNA polymerase. beta subunit
Psyc_/886 RNA polymerase, alpha subunit
Psyc_1844 transcriptional regulator, LysR family
:- Ps_vc_l820 two component response regulator
Psyc_1791 transcriptional regulator. ArsR family
I, — Psyc_] 7 75 stress transcriptional regulator, PspC
Psyc_1724 RNA polymerase, omega subunit
Psyc_1626 transcription regulator, LysR family
Psyc_/484 transcription repair coupling factor
— Psyc_] 4 76 transcriptional regulator
Psyc_1446 transcriptional regulator, luxR family
7 Psyc_/381 possible transcription factor
, Psyc_1351 transcriptional regulator, TetR famil
Psyc_]056 transcriptional regulator, LysR fami y
Psyc_0952 transcriptional regulator, ArsR family
Psyc_0905 transcriptional regulator, lclR family
Psyc_0514 RNA polymerase, alpha chain
Psyc_0386 transcriptional regulator, MerR family
Psyc_0343 RelA/SpoT family protein
Psyc_0216 transcriptional regulator, LysR family
Psyc_0207 Sigma 32 (RpoH)

Figure 4.6 Continued. Images in this dissertation are presented in color.

Peptidoglycan biosynthesis for murein disaccharides, transglycosylation reactions,
and transpeptidation of peptidoglycan strands were down-regulated (Fig. 4.6B). In
contrast, genes for the elastic growth of the cell wall including soluble and membrane-
bound lytic transglycosylases, Psyc_12 72 and Psyc_12 73, were up-regulated 3.5-fold and

1.6-fold respectively.

85

 

A) Acetate Activation B) Aconitase
1.75- 1‘75 t--.‘
I
1.25
075 ’63.: '3
1"
I"."'
..I'~0.2.5 I.’ l'."’
a. 'l
15 a? 7" 7 LT " 7 I0 25
g 10 ‘5 -0.250 ‘ ’5 ‘~ IO I5 20 23 -
..»A'
11.1 K 0.751 N‘ """ .
Q) ....... Psyc_0821 ,’ —0— Psyc_1112
g 7 --o -- Psyc_0197 K -~o-- Psyc_égfrl
a -l...5' __.___ Psyc_0196 -125 *- Psyc_ )1
a
E C) D""“~""D""‘“i"e C"b°“""’“d“° D) ATP-dependent DEAD box RNAlIelicase
a) 1.75: ,
I"‘ 1.75
N
g) 1.25 1,5)
\
hi .. \0‘ ’ln\\‘fl .lo '\.\
.TSK.‘ \‘ ,” ("0.75- .\.\ ’Iu
\- I
025 ’7’. -‘ "~ [In
,I N...“ 0.25 )1
’ ‘ I" ‘ "" ‘ . " a“ . In--- .\. ,___. _
"0 '5-0.25}’ 5 ‘0 15 2° 25 10 -5 0250 ,9’ 10 151.20 25
I ' ' ' ‘.
I . fl
/-’0 75- ' ‘9’
' - .75
d ‘ U’ 0
--._- Psyc_0687 --0--- Psyc_1082
-1.25 _'.H- Psyc_0704 475 —-o—- Psyc_0943

Temperature (°C)

Figure 4.7. Exchange of gene expression over temperature. Low temperature upregulated genes are
depicted with closed symbols and high temperature upregulated genes are depicted with open symbols.
A) Acetate activation pathways. B) Aconitase, C) D-alanyl-D-alanine carboxypeptidase. D) ATP-
dependent DEAD-box RNA helicase. In total, eleven pairs of homologous genes were found to switch
expression with temperature. Presented genes have high confidence annotations in the Psychrobacter
arcticus genome database.

Also related to growth of the cell wall, two putative D-alanyl-D-alanine
carboxypeptidase isozymes were differentially expressed with temperature. Psyc_0704
(dac2) was up-regulated 1.9-fold at -6°C and Psyc_0687(dac1) was down-regulated 3.2-
fold at -6°C (Fig. 4.7C). dac2 and dad are 27% identical and 50% similar in their amino
acid sequence from amino acid positions 42-273 of due] by use of BLAST homology
searches against the genome (2).

Primary structure disorder was computed using

DisEMBL 1.5 with the default parameters (41). To compare the results of DisEMBL

86

analysis from the two genes, the disorder predictions for each peptide were manually
aligned by adjusting for gaps in BLAST alignments of the two amino acid sequences.
dac2 features a predicted coil of 64 amino acid residues near the N-terminal end of the
DD-peptidase domain that is not present in dac]. Two additional coils are predicted in
the DD-peptidase domain of dac2 which are not present in due] from positions 210-225
and positions 354-361 of the aligned disorder data. dac2 contained 155 disordered amino
acid residues in the aligned sequence region whereas dacI contains only 128. Only 32
amino acids positions were predicted to reside in hot loops, i.e. highly disordered amino
acid positions, in the aligned region dacI sequence, but DisEMBL predicted 125 amino
acids to reside in hot loops in the dac2. P. arcticus may exchange the expression of these
two isozymes with temperature because the more disordered dac2 probably has higher
activity rates in the cold but is likely more therrnolabile.
Nucleotide metabolism and transcription. Purine, pyrimidine and nucleotide
biosynthesis were downregulated in the cold or not differentially expressed. Psyc_201],
thymidylate synthase and Psyc_1263, ribonucleotide reductase, were down-regulated
most strongly at 1.9-fold and 1.8-fold at -6°C.

The alpha and beta subunits of RNA polymerase were down-regulated in the cold.
Sigma 70 was not differentially expressed, but a sigma 32 homolog was up-regulated 2.6-
fold at low temperatures, and 1.5-fold at 22°C. The omega subunit of RNA polymerase,
rpoZ, was 5-fold up-regulated in the cold. Nine transcription factors out of 17 in the
genome were up-regulated at low temperature, though the role of many of these factors is

unclear from their annotation and genome context (Fig. 4.6C).

87

Table 4.2. Amino acid biosynthesis genes that are differentially expressed ( P < 0.01) in fold change from 17°C.

 

 

 

Fold Changea
9222
Amino Acid 9&1: Nam; Annotation 22°C 0°C ;_6:’£
Arg Psyc_0815 car/1 putative carbamoyl-phosphate synthase, small -1 . 13 -2.46 -3.27
subunn
Psyc_0814 carB carbamoyl-phosphate synthase, large subunit -1.25 -3. 12 -3.29
Psyc_0957 argF probable omithine carbamoyltransferase -0.98 -1.84 -l .79
Glu Psyc_1062 gdh probable bacterial NAD specific-glutamate -l .06 2.14 2.03
dehydrogenase
Psyc_l 873 putative glutamate synthase (NADPH-dependent), -1.20 -1.96 -1.92
small subunit
Psyc_1874 putative glutamate synthase (NADPH-dependent) -1.37 -1.84 -1.91
large chain
Gly Psyc_0798 gch glycine cleavage 11 protein -1.24 -3.05 -3.23
Psyc_0796 gcrH glycine dehydrogenase -1. 15 -2.46 -3.07
Psyc_0799 gch putative glycine cleavage T protein (aminomethyl 1.09 -l .34 -1.42
transferase)
Ala Psyc_0177 ang possible serine-pyruvate aminotransferase -1. 18 -2.20 -2.36
lle, Len, Val Psyc_0951 iII'A probable threonine dehydratase 1 -1.29 -2. 16 —2.62
Psyc_0527 ilvG putative acetolactate synthase, large subunit -1 . 16 -2.73 -2.87
Psyc_0528 ilvM putative acetolactate synthase, small subunit -1.25 -2.57 -2.77
Psyc_0529 iva Ketol-acid reductoisomerase -1.21 -2.16 -2.30
Leu Psyc_1410 leuB 3-isopmpy1malate dehydrogenase -1.21 -2.36 -2.28
Psyc_l413 leuC 3-isopropy1malate dehydratase large subunit -1.35 -2.20 -2.01
Psyc_1412 leuD 3-isopropy1malate dehydratase small subunit -1 .27 -2.1 1 -2.08
Lys Psyc_0028 dapB probable dihydrodipicolinate reductase -1.27 -l .83 -2.38
Psyc_1523 asd putative aspartate semialdehyde dehydrogenase -l . 16 -2. 17 -2.66
Psyc_0200 dapF probable diaminopimelate epimerase 1.06 1.42 1.45
Pro Psyc_l 391 ocd possible ornithine cyclodeaminase -1.30 1.44 1.27
Psyc_0293 proA putative gamma-glutamyl phosphate reductase 1.01 2.19 2.41
Tip Psyc_0973 trpG probable anthranilate phosphoribosyl transferase 1.28 1.33 1.48
Psyc_0974 trpD putative anthranilate synthase component 11 1.36 1.33 1.52
Met Psyc_0792 metZ probable O-succinylhomoserine sulfnydrylase -l .04 1.87 1.41
His Psyc_1902 hisD putative histidinol dehydrogenase 1.06 1.36 1.29
Ser Psyc_0369 serA putative D-3 phosphoglycerate dehydrogenase -1.38 -l .41 -2.53

 

a Data are expressed in fold change compared to 17°C.

Amino Acid Biosynthesis and the Stringent Response. Among regulators, a relA
homolog was detected as differentially expressed with 2-fold up-regulation at -6°C vs
17°C (Fig. 4.6C). Such a result suggests that P. arcticus may be responding to deficits in
amino-acylated tRNAs (27). Genes for the biosynthesis of proline, methionine, histidine
and tryptophan were up-regulated during low temperature growth (Table 4.2). Genes for

the biosynthesis of branched-chain amino acids, arginine, and lysine were down-

88

regulated in the cold. The glycine cleavage system genes gch and gch were down-
regulated 3-fold at low temperature, suggesting glycine limitation. Differential
expression of amino acyl tRNA synthetases was also observed suggesting that
cytoplasmic pools of amino acids are different between low temperature and high

temperature growth (Table 4.3).

Table 4.3. tRNA synthetase genes that are differentially expressed (P < 0.01) in fold change from 17°C.

 

 

 

Fold Changed
Amino Acid _O_Rj tRNA Smthetase 22°C mt; ;_6:(_'I_
Gln Psyc_0694 glutaminyl-tRNA synthetase -l .06 1.68 1.58
Gly Psyc_1774 glycyl-tRN A synthetase, alpha subunit 1.32 1.46 1.67
Gly Psyc_] 7 73 glycyl-tRNA synthetase, beta subunit 1.40 1.39 1.89
Tyr Psyc_1970 tyrosyl-tRNA synthetase -l.53 -3.89 -5.46
Phe Psyc_] 995 phenylalanyl-IRN A synthetase, beta subunit -1 .36 -2.97 -3.94
Phe Psyc_1996 phenylalanyl-tRNA synthetase, alpha subunit -1.31 -2.36 -2.99
Cys Psyc_] 3 61 cysteinyl-IRNA synthetase, class la -1.21 -1.60 -2.35
Trp Psyc_1568 tryptophanyl-tRNA synthetase, class 1b -1 .27 -1.78 -2. l 7
Ala Psyc_/460 alanyl-tRNA synthetase, class 11c -1 .37 -2.38 -2.46
Pro Psyc_0429 prolyl-tRNA synthetase, class 11a -1.20 -1.93 -2.20
Ile Psyc_0364 isoleucyl-tRNA synthetase, class la -1 .01 -1.71 -l .84
Ser Psyc_1618 seryl-tRNA synthetase, class 11a -1.15 -l .68 -1.43
His Psyc_0683 histidyl-tRNA synthetase, class 11a -1 .04 -1.59 -l .33
Arg Psyc_l946 arginyl tRNA synthetase 1.15 -1.42 -1.17
Met Psyc_1432 methionyl-tRNA synthetase -1.09 -1.04 1.06

 

a Data are expressed in fold change compared to 17°C.

Compatible Solute Uptake and Biosynthesis.

Proline, glutamate and glycine are

potential compatible solutes for which data were presented in the previous section. P.
arcticus also contains genes for the biosynthesis and uptake of camitine, betaine and
choline. Psyc_0728, choline dehydrogenase was up-regulated 2.1-fold at 22°C, and
Psyc_0729, betaine aldeyde dehydrogenase was upregulated 2.3-fold at 22°C and down-
regulated 1.4-fold at -6°C. The P. arcticus genome contains three annotated
betaine/camitine/choline type transporters: Psyc_O 727 was not differentially expressed

with temperature, Pysc_0826 was down-regulated 1.5-fold at -6°C, and Psyc_1301 was

upregulated 1.8-fold at -6°C.

89

Table 4.4. Expression profiles of P. arcticus genes involved in protein folding and disaggregation. Difiemntially expressed genes

have a P-value < 0.010 .

b
Fold Chan e

 

QLF Gene Name Annotation 22°C Q_°_§ fl
Psyc_0034 dsbB possible disulfide bond formation protein 1.27 1.41" 1.67“
Psyc_0254 dst possible thiolzdisulfide interchange protein 1.27 1.26 1.56"
Psyc_1560 dst probable periplasmic protein thiolzdisulfide oxidoreductase -1 . 12 -2.27* -2. 14*

Dst
Psyc_0552 groES putative Chaperonin HSPlO -1 . 18 -1.09 -1.41
Psyc_0553 groEL putative Chaperonin HSP60 family 1.04 1.04 -1.28
Psyc_0027 probable chaperone protein dnaJ -1.21 -2.38* -2.33*
Psyc_0039 dnaJ probable heat shock protein DnaJ -1.01 1.15 1.06
Psyc_2132 dnaK chaperone protein dnaK 1.19 1.18 1.04
Psyc_2133 grpE heat shock protein GrpE, hsp70 family 1.09 -1 . 16 -1.35*
Psyc_0823 clpB putative Chaperonin clpAlB 1.82” 1.40 1.93“
Psyc_! 941 prP putative ATP—dependent ClpP protease, proteolytic subunit ~1.06 -1.06 -1.61"‘
Psyc_] 942 cle putative ATP-dependent Cle protease -l .1 l -1.27 -1.66*
Psyc_] 719 hth probable chaperone protein hth (Hsp90) 1.07 -1.91* —2.41 *
Psyc__1940 tig trigger factor -1 .36 -1.67* -2.22"‘
Psyc_085 7 peptidyl-prolyl isomerase -1 .22 -l .13 -l .09
Psyc_06 93 ppiB peptidyl-prolyl cis—trans isomerase, cyclophilin type -1 .29 1.12 -1.58*
Psyc_0283 slyD putative peptidyl-prolyl cis-trans isomerase, FKBP-type -l .04 1.08 1.05
Psyc_03 58 probable peptidyl-prolyl cis-trans isomerase protein, FKBP- -l .01 1.10 -1.08

type
Psyc_14 73 ppiC possible PpiC-type peptidyl-prolyl cis-trans isomerase -1 . 17 -1.79* -l .67“

 

a . . . . .
P-values are estrmated from an across srx contrasts In the expenment. Three contrasts are presented In the table.

Data are expressed in fold change compared to 17°C.
*A significant fold change (P < 0.01).

Protein Folding Response: Peptidyl-prolyl cis-trans isomerases (PPIases), trigger factor
and the major heat shock associated chaperones were down-regulated or not differentially
expressed at 22°C, 0°C and -6°C (Table 4.4). Only chaperones associated with iron-
sulfur cluster biosynthesis, oxidative protein damage, and a clpB chaperone homolog
were up-regulated at low temperatures (Fig. 4.5B and Table 4.4). Genes with functions
in disulfide bridge formation were differentially expressed with temperature. dsbB and
dst were up-regulated approximately 1.6-fold at low temperature, but the periplasmic

enzyme gene (1st was down-regulated (Table 4.4).

90

Cold acclimation and cold shock gene expression. P. arcticus possesses two major
cold shock protein homologs. One cspA homolog, Psyc_531, was expressed at high
levels at all temperatures. The other cold shock protein, Psyc_942, and the adjacent
DEAD box helicase gene, Psyc_0943, are down-regulated 3.1—fold and 2.4—fold
respectively at -6°C. A second DEAD-box helicase, Psyc_1082, was up-regulated 2-fold
at cold temperatures (Fig. 4.7D).

Psyc_1082 (csdA) and Psyc_0943 are annotated as homologous ATP-dependent
DEAD-box RNA helicases with high scoring alignments to the TIGRfam DEAD-box
helicase equivalog. These genes are predicted to encode proteins of 396 and 567 amino
acids respectively. The csdA amino acid translation has a C-terminal extension rich in
glycine, serine and arginine, supporting its function at low temperature similar to
mesophilic and psychrophilic cold associated DEAD box helicases (36). The amino acid
sequences of the two genes were 40% identical over a 375 amino acid homologous region
which included the DEAD-box RNA helicase domain of each gene by BLAST
alignment. While these DEAD box helicases may act on different RNA substrates, we
hypothesized that the amino acid sequence of the low temperature up-regulated csdA
would be more disordered owing to putative activity in the cold. DisEMBL 1.5 was used
to predict disordered regions in the two genes in the conserved DEAD box helicase
domains. csdA contained two predicted coils in a sequence region from positions 272-
336 which were not in Psyc_0943 and another predicted coil in positions 180-200 of the
aligned sequences. In contrast, Psyc_0943 contained only one predicted coil from
positions 109-127 which was not present in csdA. Over the aligned length of the

sequences, 0561/! contained 213 amino acid positions in predicted coils versus 195 in the -

91

6°C down-regulated Psyc_0943. Hot loop predictions by DisEMBL revealed a larger
difference in protein disorder over the aligned sequences, with csdA containing a
predicted 120 hot loop amino acid positions versus only 70 in Psyc_0943. These results
are consistent with cold adaptation of csdA, the DEAD-box helicase up-regulated at —6°C.

Transcription and translation factors associated with cold shock were observed to
be up-regulated at -6°C including rbfA ribosomal binding factor at 1.8-fold, nusB and
nusA at 1.7-fold and 2.6-fold respectively, and translation initiation factor IF-Z at 1.8-fold
(Fig. 4.6C). However, no previously reported cold shock associated genes involved in
recombination or carbon catabolism were up-regulated in the cold. The anti-aggregation
chaperone ClpB was the only previously identified cold expressed chaperone to be up-
regulated, at 1.9-fold, during -6°C growth in P. arcticus.

Global model and knockout mutants. Considering the overall differential
expression trends in the data, a global conceptual model differentiating high temperature
growth from low temperature growth was developed (Fig. 4.8). Fast growth rate was the
simplest explanation for all of the up-regulated genes at 22°C and 17°C. Heat stress is a
possible explanation for up-regulation of chaperone and PPIase genes at high
temperature. At low temperature, explanations are more diverse as cells must respond to

the multiple stresses associated with low temperature growth at 0°C and -6°C.

92

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Based on this conceptual model and to test the role of exchanged putative
isozymes in the different temperature regimes, knockout mutant strains were generated
using the suicide vector pJ K1 00. Catalase (Psyc_05 70, katE) and methionine sulfoxide
reductase (Psyc_1043, msrA) knockout mutants were generated to examine the effect of
loss of function of these genes on growth under increased oxidative stress at low
temperature. To examine the effects of gene expression exchange with temperature,
mutants were constructed in the two DD-peptidases, Psyc_0687(dac1) and Psyc_O 704
(dac2), which were respectively up-regulated at high and low temperature. The cold up-
regulated DEAD box helicase (Psyc_1082, csdA) was also deleted with the hypothesis
that loss of this gene would result in reduced growth rates at low temperature.

No mutant growth rates were statistically different from wild-type under optimal
growth conditions at 17°C or 4°C (Fig. 4.98 and C). At 0°C and ~2.5°C, AkatE, AdacZ,
and AcsdA grew slower than wild-type as predicted (Fig. 4.9D and E, P < 0.05). AmsrA
also grew significantly slower than wild-type at -2.5°C (Fig. 4.913, P < 0.05). At 17°C,
growth was negatively effected in Adac] and AmsrA (Fig. 4.9A, P < 0.05). Conversely,
AdacZ exhibited improved growth over wild-type at 22°C (Fig. 4.9A, P < 0.05). Whereas
Adac2 grew more slowly than the wild-type P. arcticus at low temperature, Adac]
exhibited no statistical difference in grth rate versus wild-type at low temperature.
This switch in growth rate phenotype between Adam? and Adac] with temperature is
consistent with the both expression data and bioinformatics data suggesting the dac2
activity is favored at low temperature and dac] activity is favored at high temperature.
The observation of improved grth over wild-type at high temperature in Adac2

coupled with wild-type growth of AdacI at low temperature suggests that these two DD-

94

peptidases may interact competitively. This competition may be relieved by deletion of

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 4.9. Growth rate distributions for knockout mutants and wild-type P. arcticus controls. Panels
contain data fi'om different growth temperatures: A) 22°C, B) 17°C, C) 4°C, D) 0°C, and E) -2.5°C.
Bars represent the population medians (n = 8). Boxes are plotted from the 25th to 7 5th percentiles, and
whiskers are plotted from to the 10th to the 90th percentiles. Outliers are plotted as dots.

9S

Discussion

This study assessed the temperature effect on P. arcticus of gene expression during
growth under nutrient poor, briny conditions as expected in cryo-environments including
permafrost (22, 23). Our data demonstrate several important aspects of P. arcticus
adaptation for function in the cold. The threshold temperature at which P. arcticus
demonstrates acclimation to the cold is lower than 4°C under the conditions used for this
experiment. Exchange of isozyme expression during growth in high versus low
temperatures was not widespread in the genome, but it did occur as did up-regulation of
expression of energy efficient substrate incorporation pathways at low temperature.
Multiple adjustments to gene expression were employed to respond to growth in the cold.
Based on our array data, oxidative stress, amino acid biosynthesis, and cell wall and
membrane dynamics appeared to be the growth rate limiting factors for which
compensation was necessary instead of energy conversion, transport, translation or
peptide chaperoning. Clearly these modes of acclimation to low temperature life warrant
further investigation.

A grth rate inflection point was reported at 6°C for P. arcticus during growth
in nutritionally complex and less saline 1/2 TSB broth with or without 5% NaCl added
(47). Our medium elicited no such response, indicating that the elevated salt
concentration in our media may have shifted the cold acclimation threshold substantially
downward. Our growth experiments show P. arcticus, a eurypsychrophile, grows under a
similar physiology at 4°C as at 17°C. Cold shock and cold acclimation studies of
psychrotolerant microbes have typically been carried out in the comparisons of various

warm temperatures to 4°C (7, 8, 29). The threshold for low temperature response in P.

96

arcticus, in this experiment, is lower than the threshold for other organisms such as
Shewanella onez’densz's MR-l at 8°C, Acinetobacter Hl-Il at 4°C, Methanococcoides
burtonii at 4°C, and Bacillus psychrotrophicus at 4°C (7, 20, 29, 61). The reduced
threshold of the acclimation response calls into question the usefulness of the benchmark
4°C temperature in studies of low temperature physiology of psychrophiles. The shift in
growth temperature response in P. arcticus seems dependent on medium composition,
and so one should consider the scientific question at hand before deciding on how to treat
cells for low temperature growth.

Numerous proteome studies have been performed on cold shocked organisms. It
is known that posttranscriptional control is an important factor in both E. coli and B.
subtilis during cold shock and cold acclimated growth (10, 24, 32). Only a few proteome
studies have been performed on cold acclimated growth of psychrophiles (6, 30). A
transcriptome approach offers the strength of analyzing data for 93% of the genes in P.
arcticus during cold acclimated growth across statistically relevant numbers of replicates
(1). The thoroughness of this approach has allowed us to identify the complex metabolic
responses to prolonged low temperature exposure. While transcriptome analyses can
provide insight into coarse metabolic changes, they do not take into account translational
and post-translational regulation. Thus, we have directed our discussion toward
outcomes resulting from complex gene expression responses such as resource and energy
efficiency, biosynthetic pathways, and regulation of molecular dynamic motion at low
temperature. Our transcriptome data show that transcription control plays a role in
differentiating cold-acclimated growth state at 0°C and -6°C from that at higher

temperatures.

97

Proteome studies in Psychrobacteria

Two proteome studies have been carried out with Psychrobacter species which
examined temperature effects. Zheng et al (2006) reported a small number of proteins,
14, were detected as differentially expressed between 22°C and 4°C in V2 TSB + 5%
NaCl (64). This culture condition was most similar to the conditions of our experiment.
Alkyl-hydroperoxide reductase, ahpC, was strongly upregulated in the comparison, and
we observed upregulation of its transcript at low temperature. Downregulated genes
included the chaperone HSP60 and peptidyl-prolyl cis-trans isomerases whose transcripts
were also observed to be downregulated in our experiment.

Bakermans et al (2006) carried out proteome analysis of Psychrobacter
cryohalolentis, a related permafrost isolate from cryopegs in the Kolyma permafi'ost (6).
In addition to up-regulation of energy production enzymes, proteins for enhancing
translation including CspA, EF-Ts and EF-Tu were upregulated during growth at -4°C in
P. cryohalolentz's. Our trascriptome data suggest that P. arcticus has unique adaptive
features for low temperature growth among permafrost Psychrobacteria with basal
energy and translation machinery that function well at low temperature due to a
combination of cold adaptation of the enzymes and constitutive expression of the genes.
Alternatively, it is possible that a degree of posttranscriptional control is allowing genes
that are transcriptionally downregulated to be translated more efficiently at low
temperature. Other instances of posttranscriptional control have favored translational
down-regulation of transcriptionally highly expressed genes at high temperature, e. g.

cspA and pnp (25, 45).

98

Resource efficiency responses

Glutamate dehydrogenase is known to be a slow, energy efficient pathway to
produce glutamate from 2-oxoglutarate and ammonium. Glutamate synthase, in contrast,
is dependent on glutamine synthetase which requires ATP and glutamine (34, 35).
Acetyl-CoA synthetase is also potentially an energy saving pathway for the activation of
acetate. Acetyl-CoA synthethase performs the same function as both acetate kinase and
phosphotransacetylase which were up-regulated at warm temperatures. The differential
expression of these two gene systems has been observed in E. coli which utilizes ackA
and pta during fast growth to excrete acetate and acetyl-CoA synthetase to metabolize
acetate when other carbon sources have been depleted (39). Our data suggest that the
regulation of these two pathways has been coupled to temperature changes in P. arcticus
under acetate-utilizing conditions. So, P. arcticus may save energy by inducing acetate
metabolism via acetyl-CoA synthetase and nitrogen incorporation via glutamate
dehydrogenase in the cold. Under cold conditions, where generation times are long,
energy efficiency is likely more important than growth at slightly faster rates than
competitors.

Increased expression for energy production and ATP synthesis were observed in
two psychrophiles growing at low temperature, Psychrobacter cryohalolentis and
Methanococcoides burtonii (6, 30). In the P. arcticus transcriptome, genes for energy
conversion and metabolism were most often down-regulated at low temperature,
suggesting that NADH and ATP production are not the limiting metabolic pathways at
low temperature. An exception to energy conversion expression in P. arcticus was

cytochrome bd ubiquinol oxidese (Psyc_1515-1516). Cytochrome bd is a high affinity

99

oxidase, and E. coli knockout mutants of cytochrome bd oxidase are sensitive to
hydrogen peroxide (26). Therefore, the upregulation of cytochrome bd in P. arcticus was
interpreted to be responding to higher steady state concentrations of reactive oxygen
species. The P. arcticus response for energy metabolism suggests that this psychrophile
is differently adapted for growth at low temperature in comparison to other psychrophiles
examined to date.

TCA cycle genes for the catabolism of acetate and regeneration of biosynthetic
intermediates were down-regulated in the cold. The down-regulation of carbon catabolic
reactions is different from previous findings during cold acclimation in E. coli, S.
oneidensis MR-l, P. cryohalolentis, and M. burtoniz’ in which increased expression of
genes especially in glycolysis and some TCA cycle genes was detected (6, 20, 30, 46,
56). Down-regulation of catabolic pathways was observed in B. subtilis, where it was
concluded that this is probably a result of reduced growth rates (10).

Down-regulation of thymine biosynthesis, and ribonucleotide reductase genes
suggests that P. arcticus is modifying nucleotide metabolism to support more RNA
synthesis than DNA replication. The result is consistent with the observation of
increased synthesis of RNA and protein incorporation rates normalized per cell division
with no change in DNA synthesis rates during growth at subzero temperatures (5). P.
arcticus also exhibited high levels of RNase and peptidase expression in general during
growth at low temperature. RNase R and PNPase of E. coli are induced during cold
shock (11, 46). PNPase is also essential for the growth of Yersinia entercolitica at low
temperature (31). RNase PH, PNPase and RNase R were reported as induced during

grth at 15°C in B. subtilis (10). In constrast, PNPase was one of two RNases down-

100

regulated at low temperature in P. arcticus during cold acclimated growth. Increased
expression of RNases and peptidases during cold acclimated growth would result in
increased turnover RNA and proteins to address the need for transcriptome and proteome
flexibility during growth with generation times on the order of days or weeks at
temperatures where inherent half—lives of transcripts and peptides will be longer.
Differential expression of hz'sJ and trpG was detected in L. monocytogenes
growing at 10°C (42). Genes for arginine and aromatic amino acid biosynthesis are up-
regulated during cold shock and cold acclimated grth in both E. coli and B. subtilis
(10, 46). Growth arrest in E. coli induced the arginine and histidine biosynthetic
pathways, but not those of other amino acids in a starvation experiment (13). The
observed down-regulation and energy metabolism, translation and transcription
machinery with concurrent up-regulation of rpoZ and relA is consistent with the stringent
response for growth arrest (13). Up—regulation was also observed in Vibrio cholerae in a
Q-RT-PCR comparison between the VBNC state induced at 4°C and a starvation stressed
growth state at 15°C (28). Simultaneous examination of amino acid biosynthetic genes
and tRNA synthetase genes showed that in P. arcticus genes for arginine, the branched
chain amino acids and lysine were down-regulated at all temperatures relative to 17°C.
However, genes for proline, histidine, methionine and tryptophan were up-regulated.
This suggests that P. arcticus attempts to compensate for hypothesized reductions in
amino acid biosynthesis activity by up-regulating specific genes in a pathway. Down-
regulation of the glycine cleavage system is consistent with reduced cytoplasmic

concentration of glycine. Up-regulation of the stringent response may be involved in

101

slowing the growth of P. arcticus in order to allow the organism to maintain an adequate
supply of amino acids and nucleotides.
Comparisons to cold acclimated growth in mesophiles

In addition to the major cold shock proteins and RNA helicases, the cold shock
response in E. coli leads to up-regulation of pyruvate dehydrogenase complex, recA,
chaperones, rbfA, translation initiation factors, and trehalose biosynthesis (32, 46). Cold
inducible genes like rbfA, IF-2, and nusA were up-regulated during growth in the cold in
P. arcticus. P. arcticus expresses one cold shock protein homolog constitutively with
temperature. Differential expression of membrane fatty acid desaturase and the oxidative
stress response are also commonly observed during cold acclimated expression studies
(46, 64). The genes shared with mesophilic cold acclimation responses are those
involved in the initiation and stable function of transcription and translation but not those
in the basal machinery such as RNA polymerase subunits and ribosomal proteins.

DEAD box RNA helicases function to unwind duplex RNA and possibly to
displace RNA-protein complexes. These enzymes exhibit functions as diverse as
ribosome biogenesis, RNA degradation and translation initiation (49). CsdA from E. coli
has roles in all of the former processes whereas other DEAD box RNA helicases have
specific RNA targets (36). Comparison of inversely expressed DEAD box RNA helicase
genes csdA (Psyc_1082) and Psyc_0943 showed that the low temperature up-regulated
csdA is predicted to be more disordered, i.e. more flexible, than Psyc_0943. The reduced
growth rate of our P. arcticus csdA mutant further solidifies the role of this DEAD box
helicase in cold acclimated growth. While the two alleles of DEAD box RNA helicase in

P. arcticus may not target the same RNA populations, it was clear from our analyses that

102

csdA (Psyc_1082) encodes a gene important to the low temperature grth of P. arcticus.
Another interesting feature of this P. arcticus csdA is that it possesses a highly disordered
C-terminal extension rich in glycine, serine and arginine. A similar C-terminal extension
was observed in the cold regulated DEAD box helicase of the psychrophilic methanogen
Methanococcoides burtonii (40). The structural similarity of these two cold adapted
RNA helicases from disparate microbes isolated from opposite hemispheres of the planet
highlights that some specific alleles can contribute significantly to psychrophily. Perhaps
such a protein can be transformed into more cold sensitive organisms to improve cold
tolerance as was observed in E. coli expressing a cold adapted Cpn60/10 chaperone (56).
Protein misfolding and aggregation are thought to lead to the induction of
chaperones at low temperature and PPIase at low temperature (6, 10, 30, 46, 56, 58).
However, PPIases and heat-shock associated chaperones were down-regulated or not
differentially expressed during growth in the cold in P. arcticus. This result suggests that
in P. arcticus, protein folding stress is only minimal at low temperature. Only clpB
chaperone and hsp33 were up-regulated in our experiment. The hsp33 chaperone is
activated by oxidation and responds transcriptionally to heat shock and oxidation stress
(14). It is most likely playing a role in the oxidative stress response in P. arcticus. The
clpB chaperone is thought to play a role in protein disaggregation and it is expressed
highly in B. subtilis and E. coli and L. monocytogenes during cold acclimated growth at
15°C or 10°C (10, 42, 46). ClpB induction occurs during cold acclimation of
Synecchococcus sp. PCC 7942, and deletion of clpB reduced survival and photosynthesis

during cold acclimated growth at 25°C in this cold sensitive bacterium (48).

103

Conservation of clpB up-regulation in P. arcticus demonstrates the universal nature of

protein aggregation stress at low temperature.

Oxidative Response

Differential expression in response to reactive oxygen species was one of the
strongest magnitude changes in the transcriptome with temperature. Genes involved
were consistent with those observed in the oxidative stress stimulon in E. coli (19).
Aconitase isozymes A and B were also exchanged with temperature in P. arcticus.
Aconitase A and B in E. coli were exchanged in response to superoxide (59). Knockout
of two oxidative stress genes decreased growth rates of P. arcticus at subzero
temperatures.
Cell Wall Response

DD-peptidases control the degree of cross-linking between peptidoglycan strand
in the cell wall by catalyzing the removal of one D-alanine residue from peptidoglycan
peptides before cross-linking can occur via transpeptidase activity (44, 52). Lytic
transglycosylases are involved in the dynamic insertion of new peptidoglycan strands into
the cell wall. The peptidoglycan cell wall is a elastic structure whose elasticity is a
function cross-bridging between peptidoglycan strands (63). Low temperature decreases
elasticity, so differential expression of genes with functions in cross-bridging control
could adjust the elasticity of the cell wall at low temperature. Consistent with reduced
grth rates, we observed down-regulation of peptidoglycan strand biosynthesis genes.
In contrast, lytic transglycosylases were upregulated in the cold. Interestingly, isozyme

exchange of DD-peptidases was also observed. If these isozymes yield different levels

104

of cross-linking in the cell wall, they may contribute to control of elasticity in the cell
wall with temperature. Our observation of isozyme exchange and differential effects of
knockout of these isozymes reveals a new role for control of cell wall elasticity as a
mechanism of cold adaptation.
Conclusion

We summarize our conceptual model of the low temperature response in P.
arcticus in Fig. 4.8. We attribute up-regulation of genes at warm temperatures solely to
growth rate effects or possibly heat in the case of chaperone up-regulation at high
temperature. The transcriptome at 0°C and -6°C was attributed to a conglomerate of
effects of life in the cold including increased dissolved oxygen concentrations in our
cultures, acclimation of metabolic pathways for resource and energy efficiency,
biosynthetic demand or reduced supply of specific amino acids, and expression to
increase the dynamic behaviors of the cell wall and cytoplasmic membrane. Altogether,
these data suggest the concept that P. arcticus is evolutionarily adapted for firnction in
both temperature ranges in genes for central cellular processes and components as diverse
as membrane and peptidoglycan biosynthesis, energy metabolism, carbon catabolism,
nucleotide biosynthesis, amino acid biosynthesis, RNA polymerase, and the ribosome.
Compensation was reserved for specific processes such as cell wall and membrane
dynamics, differential expression of a small number of isozyme pairs, and specific

biosynthetic steps in amino acid and macromolecular turnover at low temperature.

105

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11]

CHAPTER 5

TEMPERATURE-INDUCED DIFFERENTIAL EXPRESSION IN GLYCINE,
METHIONINE AND LEUCINE METABOLISM CORRESPOND TO BIAS IN
AMINO ACID CONTENT OF GENES HIGHLY EXPRESSED BELOW 0°C BY

PS Y CHROBA C T ER ARC T I C US 273-4

112

Abstract

Growth at S 0 °C induces numerous changes in the transcriptome of the
eurypsychrophilic permafrost microbe Psychrobacter arcticus 273-4. Among the
changes previously observed was induction of the stringent response concomitant with
gene expression patterns consistent with metabolism to increase abundance of six amino
acids. A AreIA knockout mutant exhibited decreased grth rates during mid-late
exponential phase relative to the wild-type P. arcticus strain. We examined the
relationship between amino acid biosynthesis expression, the effect of supplementation
with amino acids on growth rate and the biases in amino acid content predicted from
genes highly expressed in the transcriptome at subzero temperatures. Results for gene
expression and amino acid supplementation experiments are consistent with increased
demand for glycine and methionine in cells growing at subzero temperatures. The
subzero proteome had the strongest biases for reduced glycine and methionine content
and increased leucine content. Based on these data, we hypothesize that glycine and
methionine biosynthesis are negatively impacted by subzero temperature growth whereas
leucine biosynthesis may have been favored over valine and isoleucine in the same
condition. Metabolic flux analysis may yield further insights into magnitude and causes

of these hypothesized changes in amino acid pool.

113

Introduction

Eurypsychrophilic (i.e. “broad cold-loving”) microbes can commonly grow in a 30—40°C
temperature range by adjusting their physiology to cope with growth at both at sub- and
supra-optimal temperatures (12). Growth rates of microbial cultures exhibit exponential
decline as temperatures depart fiom the optimal growth temperature (Topt) except near the
low temperature limit of growth, where an inflection is observed in the growth rate trend
(21). The temperature at which this growth rate inflection occurs is known as the critical
temperature (Tom); the temperature at which optimal growth physiology is exchanged for
low temperature acclimated physiology. Below Tom, transcriptomic, proteomic, and
enzymatic regulation contribute to adaptations for low temperature growth such as
increased membrane fluidity, differential expression of biosynthesis pathways, and
changes to energy metabolism (6, 15, 18). While energy consumption and rates of
macromolecular synthesis decrease below Tcm, these processes represent a larger
investment of energy per cell division than at temperature above Tc,“ (3, 5).
Transcriptome analysis of the eurypsychrophilic permafrost bacterium
Psychrobacter arcticus growing at temperatures on either side of Teri, revealed the
expected expression increases in genes for homeoviscous adaptation of membranes and
oxidative stress response genes in addition to downregulation of transcription, translation
and nucleotide biosynthesis (Chapter 4). Surprising results included up-regulation of
genes for dynamics of the cell wall, increased expression of ribonucleases and peptidases,
and down-regulation of chaperones. A mineral medium with no amino acids was utilized

in the transcriptome experiment to enable discovery of challenges to biosynthesis below

114

0°C. P. arcticus exhibited increased expression of six species of amino acids: proline,
glycine, tryptophan, histidine and sulfirr-containing amino acids.

The stringent response defines a regulon devoted to energy and carbon
conservation under nutrient limited conditions. Differential expression of genes under
stringent response control was also observed in P. arcticus during growth at 0°C and -
6°C, corroborating the potential importance of the amino acid biosynthesis response at
low temperatures. When the stringent response is induced by the presence of uncharged-
tRNAs, RelA, a ribosome associated protein, catalyzes the synthesis of the stringent
response “alarmone” (p)ppGpp (7). In Escherichia coli and Bacillus subtilis, (p)ppGpp
represses expression of tRNAs, rRNAs, ribosomal proteins, RNA polymerase subunits,
DNA replication and ATP synthase (8, 10). These modifications of gene expression are
not observed in relA deletion strains (10). Induction of the stringent response transiently
arrests grth and is thought to permit carbon and energy conservation under short term
nutrient-limited conditions while avoiding entry into stationary phase (or sporulation in
Bacillus), a comprehensive dormancy state that includes the expression of several
damage resistance responses.

Up-regulation of amino acid biosynthesis genes has been observed in several
bacteria growing at low temperatures, but the relevance of these expression responses to
low temperature physiology has not been examined. Amino acid biosynthesis is a major
fraction of energy cost to the cell dming growth under energy and nutrient limiting
conditions. Indeed, metabolic costs of amino acid biosynthesis have been shown to

influence the amino acid content of microbial proteomes (1). Therefore, to understand

115

life in cold, energy and nutrient limited environments like permafrost, both the effect of
biosynthetic hurdles and strategies for coping with such stress must be understood.

We hypothesized that up-regulation of amino acid biosynthesis is indicative of an
increased metabolic cost on P. arcticus during growth at low temperatures. Induction of
the stringent response at low temperatures could help moderate that cost by slowing
growth and resource consumption by P. arcticus. We examined the effect of deletion of
the stringent response signal gene relA on growth at low temperature and found that
grth rate was decreased at temperatures < 4°C. Wild-type P. arcticus cultures were
supplemented with extracellular pools of amino acids to test for increased growth rates,
some of these results indicated relief from the cost of biosynthesis of key amino acids.
Highly expressed genes from our transcriptome comparison of subzero growth and that at
Tom were compiled into predicted proteomes specific for those temperatures. The amino
acid content of predicted proteomes at Tom and subzero temperatures were analyzed to
detect amino acid content biases corroborating increased cost of synthesis of the six low

temperature favored amino acids.

Materials and Methods

Construction of a AreIA mutant P. arcticus strain. A knockout mutant of relA
(Psyc_0343) was generated essentially as described in Denef et al (9). Primers, plasmids
and strains for this study are described in Table 5.1. Flanking regions of each gene were
PCR amplified with primers containing restriction sites for insertion into pJKlOO.
Upstream flanking regions were amplified with primers containing BglII and NotI

restriction sites on the forward and reverse primers respectively. Downstream flanking

116

regions were amplified with SacII and SacI site containing primers on the forward and
reverse primers respectively.

Table 5.1. Strains, primers and plasmids used in this study.

Strains Descriptiona Source
Psychrobacter arcticus

273-4 Wild type

PBO343 ArelA ::kan This study
Escherichia coli IhrBl 004 pro (hi rpsl. )1st Incl AMIS RP4-l360 9
WM3064 A(araBAD)567 AdapA 1341::[erm pir]
Primers
del343UF AGCTAGATCTgcttatcgatccaccacgtt This study
del343UR AGCTGCGGCCGCttgcgtggtttgtccatcta This study
del343DF AGCTGAATTCctggcgcatttatccaagtt This study
del343DR AGCTCATATGctcgtttgatgggttgcttt This study
kanF GAAGATCTctcaaaatctctgatgttacattgc This study
kanR GAAGATCTtggttgatgagagctttgttgtaggt This study
Plasmids
pJKlOO Allelic Exchange Vector 9
p343U pJKlOO with Psyc_0343 upstream This study
p343UD pJKlOO wth Psyc 0343 up- and downstream This study

 

a
Capital letters include a 4 nucleotide extension on the 5' end of primers and a restriction site for insertion
into pJ K100.

P. arcticus 273-4 cultures for conjugation were grown in LB broth at 22°C for 36
hours. E. coli WM3064 containing the suicide vector for knockout was grown at 37°C in
Luria Bertani broth (LB) supplemented with 25 pg ml'l kanamycin (Kan), 20 pg ml'l
tetracyclin (Tet) and 100 pg ml'1 diaminopimellic acid (DAP) overnight. Cells were
combined in a ratio of 200 pl E. coli donor and either 200 p1 or 800 p1 P. arcticus 273-4
recipient. The mixtures were pelleted by centrifugation for 2 min at 4,000 x g in an
Eppendorf 5415D tabletop centrifuge. Pellets were resuspended in 100 pl LB and the full
volume was spot plated onto LB agar supplemented with 100 pg ml‘1 DAP. Plates were
incubated at 22°C for 24 hours. Cells were then harvested and resuspended in 500 pl LB.
This cell suspension was decimally diluted 102-fold and 100 pl of each dilution was

plated on LB agar supplemented only with 25 pg ml"1 Kan. Conjugants were allowed to

117

grow for 72 hours at 22°C. Putative knockout mutant strains were isolated on LB with 25
pg ml'1 Kan and screened for a Kanr Tets phenotype. PCR screening of knockouts
revealed amplicons of the expected gene deletion product size and these strains were used
for the temperature growth experiment.

Mutant Growth Experiment. A P. arcticus ArelA::kan strain, PBO343, was grown
in marine broth supplemented with 25 pg ml'1 Kan for 48 hr at 17°C. Two successive
cultures were carried out to acclimate the mutant to acetate mineral medium containing
7.5% sea salts with 25 pg ml'l Kan for 72 hr at 17°C. One liter acetate mineral medium
contained 75 g 1'1 sea salts, 50 mM MOPS, pH 7.0, 20 mM sodium acetate, 5 mM NH4C1,
1 mM KZHPO4, 1X Wolfe’s vitamins and 1X trace minerals (Chapter 3).

Growth rates were assayed using 96 well plates. Eight replicates of each mutant
were inoculated into 200 pl 7.5% sea salts acetate medium in each well. Sixteen
replicates of wild-type P. arcticus 273-4 were present on each plate and 12 blank wells.
Plates were incubated at 22°C, 17°C, 4°C, 0°C, and —2.5°C. Blank subtracted OD600 data
were collected using a SpectraMax M2 spectrophotometer (Molecular Devices,
Sunnyvale, CA). Data were compiled using a Perl script to parse data by growth
temperature, mutant strain and time point. T-tests and plotting of data were carried out
using R 2.4.0. The script calculated average growth rates for each replicate of each
mutant for optical density increases between OD600 values 0.03 and 0.4. Two-tailed t-
tests were performed to determine if growth rates were statistically different between
wild-type P. arcticus and PBO343 within a temperature. Growth rates were considered

statistically different if P < 0.05.

118

 

Amino acid supplementation experiment. In a 96 well plate, 200 pl cultures were
prepared in mineral medium with 20 mM acetate (see Chapter 4). Treatments consisted
of cultures supplemented with 1 mM concentrations of each of the following amino
acids: phenylalanine, tryptophan, proline, cysteine, alanine, glycine, leucine, histidine,
and methionine. Additional control cultures included 1% casamino acids and a defined
mixture composed of 1 mM concentrations of phenylalanine, tryptophan, proline,
cysteine, alanine, glycine, and methionine. A 1% inoculum from stationary phase culture
of wild-type P. arcticus 273-4 grown through three passages in acetate medium was
introduced to each medium. Four replicates of each nutrient supplementation condition
were examined during growth at 22°C, 17°C, 4°C, 0°C, and -2.5°C. Growth data were
parsed using a custom Perl script. Growth rates were calculated in the range 0.04 <
OD600 < 0.4. Two-tailed t-tests were performed comparing growth rates of amino acid
supplemented cultures versus the unsupplemented cultures at each temperature. Growth
rates were significantly different if P < 0.05.

Computational analysis of predicted proteomes from Top. and subzero
transcriptomes. Low temperature and optimal temperature proteome gene contents
were computed from the transcriptome at -6°C and 17°C or Top, (see Chapter 4). Genes
exhibiting high levels of transcript expression and a high magnitude of differential
expression were grouped into the predicted subzero and Top. proteomes (Supp. Tables 5.1

and 5.2). Cutoffs for inclusion in these groups were selected as A > 10 and -1 _>. M 2 1
where A = log2\/(-6°C - 17°C) and M = log2(-6°C /17°C) because these genes were on the

extremes of the P. arcticus transcriptome data and represent a large hypothetical amino

acid cost to the cell (Supp Table 5.1). The 17°C predicted proteome consisted of 105

119

genes and the -6°C predicted proteome consisted of 88 genes. Percent arrrino acid
content was calculated for each amino acid sequence to normalize for length of sequences
using a custom Perl script. Distributions of percent amino acid data were plotted for each
proteome in R 2.4.0. Amino acid bias in the proteomes was determined by log; ratio of
medians. If a median amino acid proportion differed from the total median of all amino
acids by more than 1 standard deviation, then we considered the difference between the

proteomes to be significant.

Results

Knockout mutation of relA. The P. arcticus relA (Psyc_0343) homologue increased
expression during growth at 0°C and -6°C compared to warmer temperatures in
transcriptome analysis. Energy, translation and transcription gene expression responses
of P. arcticus at low temperature were indicative of stringent response induction.
Analysis of gene expression indicated that genes for methionine, tryptophan, proline,
glycine, alanine and histidine accumulation were up-regulated in the cold (Chapter 4).
Branched chain amino acids, lysine and arginine biosynthesis were down-regulated at
low temperatures. The up-regulation of biosynthesis genes for six amino acids at 0°C
and -6°C led us to hypothesize that a depleted cytoplasmic pool of at least one amino acid
contributed to stringent response induction.

To examine the role of the stringent response during growth at low temperatures,
we generated a knockout mutant of relA (Psyc_0343) and compared the growth of this
mutant to the wild-type P. arcticus 273-4 (Fig 5.1). We hypothesized that the relA

knockout, designated PBO343, would exhibit either reduced growth rate or reduced

120

growth yield at low temperature. PBO343 exhibited a shorter lag phase of growth at all
temperatures similar to the relaxed control of grth in a B. subtilis ArelA strain (Fig
5.1A) (10). Our mutants were pre-cultured at 17°C and no effect on growth rate was
observed at 17°C (Fig 5.1A), so reduced lag time at low temperatures is possibly the
result of residual amino acid pools carried over in the inoculum. Growth of PBO343 at
the two temperature extremes, 22°C and -2.5°C, was characterized by a decrease in
growth rate before stationary phase between OD600 0.3 and 0.4 followed by an extended
period of growth rate reduction leading to growth cessation as compared to wild-type
where growth arrest occurred over a shorter time interval (Supp. Fig. 5.1 and Fig 5.1A);
stationary phase was observed at OD600 ~ 0.6 at 17°C and 4°C. Growth rates did not
differ at 22°C and 17°C between wild-type and PBO343 with median rates pwnd = 0.021
and pp30343 = 0.025 at 22°C and pwnd = pp30343 = 0.0143 at 17°C. Growth of PBO343
(HPBQ343 = 0.0061) at 4°C was slower on average than wild-type (pwnd = 0.0065), and the
variability in PBO343 growth rate was large (S.D.p30343 = 0.003) at this temperature.
Mutant growth was significantly slower than wild-type at 0°C and -2.5°C with P < 0.01
(Fig 5.1B). At 0C, pwnd = 0.0029 and pp30343 = 0.0022, 3 25% decrease in growth rate.
At -2.5C, pwnd = 0.0012 and pp30343 = 0.0010, a 20% decrease in growth rate. No
differences between wild-type and PBO343 were observed in growth yield at any
temperature, demonstrating that PBO343 was able to achieve biomass similar to wild-type
even though the starvation response was impacted by loss of the relA. Thus, the
temperature specific effect of relA deletion was observed at low temperatures as a growth

rate reduction.

121

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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1_b
l—l
_‘— _r_
O 0
Wild Ara/r
Mutant
4°C

 

 

 

 

 

 

 

 

 

 

 

 

 

 

l
l
° I
_L_
Wild ArelA
Mutant
-2.5°C
0
—I'_
I
E _._
+ i—i
—l_
I
Wild ArelA
Mutant

Growth characteristics of P. arcticus 273-4 wild-type strain and AreIA knockout strain
PBO343. A) Growth curves of the two strains, error bars represent one standard deviation from the mean
(n = 8). B) Box plots of growth rates calculated for the two strains. Bars indicate sample medians, boxes
are plotted from the 25‘h to the 75‘h percentiles, and whiskers are plotted to the 10m and 90th percentiles.

Dots represent outliers which resulted primarily from pipettor error during inoculation.

122

 

 

 

Amino acid effects on growth rate. We hypothesized that reduced growth rates at 0°C
and -2.5°C in the ArelA mutant were the result of loss of stringent response control of
growth in PBO343. In the wild-type strain, presence of relA yielded longer lag phase and
constant growth rates at 22°C and -2.5°C (Supp. Fig. 5.1A and Fig. 5.1A). Those results
corroborated our hypothesis that increased expression of amino acid biosynthesis genes
resulted from challenges to production of those amino acids specifically resulting from
incubation at subzero temperatures. The challenges of biosynthesis of the six amino
acids might be alleviated by supplying those amino acids in the growth medium.
Observation of temperature specific effects of an amino acid on growth rates would
clearly support the hypothesis that P. arcticus is starved for those amino acids at low
temperature.

P. arcticus was grown in the presence of individual amino acids and mixtures
thereof (Table 5.2). Glycine, proline, casamino acids and a defined mixture of amino
acids, designated “Complex”, increased grth rates at both optimal and sub-optimal
temperatures. Fewer significant changes were observed at 4°C than at the other
temperatures. This may be due to variability in the response to incubation at these
temperatures, which reside on either side of Tam. Interestingly, casamino acids did not
improve growth at -2.5°C compared to unsupplemented cultures. Phenylalanine and
methionine increased growth rates exclusively at -2.5°C. Leucine and tryptophan
decreased growth rates at -2.5°C. Histidine and alanine did not significantly increase

growth rates at any temperatures.

123

Table 5.2. Ratios of average growth rates in cultures

. . . a,b
supplemented wrth armno acrds to unsupplemented cultures .
Te_mggm_tu_rs

 

Amino acid 17°C fl mt; -2.5°C
Phe 1.17 1.10 1.04 2.18*
Trp NGC -1.19 -2.04* NGC

Pro 3.38* 1.46 1.68* 1.30*
Cys 1.68 1.57 1.89 -l.03
Ala 1.67 -1.01 1.13 1.24

Gly 248* 1.40 1.50 1.72“
Leu -1.14 1.00 1.05 -1.22*
Met 2.31 1.19 -1.05 1.58"
Casamino 3.85“ 2.11* 1.51* 1.16

Acids

His 1.24 1.13 1.25 1.29

Complex 264* 1.00 1.71* 1.71*

 

a . . . . .

Posrtrve ratios are ratio of growth rate in supplemented over unsupplemented
culture. For conditions in which unsupplemented cultures grew faster, ratios
are displayed as the negative inverse of supplemented to unsupplemented.

b
Statistically significant ratios (P < 0.05) are denoted by "'.

C N0 = No Growth
Analysis of amino acid bias in the predicted low temperature and high temperature
proteomes. If biosynthesis of key amino acids was limiting for translation, then we
expected that highly expressed proteins at low temperatures would contain lower
proportions of those amino acids to improve efficiency of grth in the cold. Therefore,
a negative correlation between ratio of amino acid content and ratio of gene expression
was expected between the predicted proteomes. Analysis of the predicted proteome was
performed to detect biases in protein amino acid content for genes highly expressed at
low temperature as compared to genes highly expressed at Topt. Genes were divided into
low and optimal temperature expressed proteomes based on their average intensity, A,
and their log-ratio of expression, M. Genes in the -6°C up-regulated proteome were

selected with A-value > 10 and M 2 1 (Supp. Tables 5.1). Genes with M S -1 and A > 10

124

were placed in the 17°C up-regulated transcriptome (Supp Table 5.2). Genes annotated
as membrane bound proteins were removed from the analysis, because the interactions of
membrane bound proteins with lipid rather than aqueous solutions could introduce biases
in amino acid composition due purely to evolution for function in different fluid matrices.
Such biases would appear temperature associated in our analysis because a much greater
proportion of 17°C up-regulated genes were membrane bound than those at low
temperature. Percent amino acid content of each predicted sequence was determined and
population distributions for each amino acid were compared (Fig. 5.2). Ratios of median
amino acid content in the -6°C to the 17°C proteome were calculated. Amino acid
contents were considered significantly different between the two populations if the log;
ratio of medians was greater than one standard deviation away from the median for all
log; ratios: 0.01 i 0.18. Glycine, methionine, arginine and valine were reduced in
proportion in the -6°C proteome while tryptophan, cysteine, and leucine increased in

proportion.

125

 

 

 

in '- NO
_. .n _ 2
:3 2 < a: O < m _ I < ._t on
._J < > F' < >- - < < '-
0 in °\° "1 in o\° U " N °\° > v
o o — o o
09 0 09 0 OS 0 OS 0
Kauanbard Kauanbarg Aauanbaid Kouanbmd

 

r11 IITII
0 8 12
%AA
HIS

 

THR
0 2 4 6 8
%AA
PHE
11111111
0 4 812
%AA
HP.
0 4 812

09

O
o
in
O
O
to
O
o
in
O

Kouonbarq Kauanbmg Aouanbard Kouonbarq

 

O
__ In N
—- N
u— w W
o —0
" -- < < <
33 - - <2 % °° *2 5 . s B . 2
O _ .n .\ < v .\ l— .\ —l m
m“ ° rmm ° rrrn'n ° nrrrn °
09 0 0s 0 0s 0 09 o
Kouonbarq Kauanbarq Aauanbar :1 Kouanbar :1

W
—' 2 st oo
o < < M < o
1% " < to?) S < E < 2
(I) . ,\° < o\" b- " .\° 0. "f
V‘ on
. -- N
o o o o
09 0 OS 0 09 0 09 0
Kouonbard Kauanbar :1 Kauanbmq Kouanbmq

AL
34444
5 10 15 20
°/oAA
GLN
,,
° al‘fifliq'fifi
0 4 8 12
°/oAA
ARG
,,
3115444
0 5 1015 20
%AA
MET
9;
. ilk—w
0 5 10 15

09 0
Aouanbarq Kauanbar :1 Aouonbar :1 Aouanbauq

[j -6°C
E 17°C

Figure 5.2. Overlaid histograms of amino acid proportions in the -6°C (El) and 17°C (I)
proteomes. Bin boundaries are defined by percent amino acid content (% AA) values.
Histograms have been overlaid with -6°C amino acid content in the foreground. Vertical lines
represent median amino acid proportions for -6°C (solid) and 17°C (dashed) proteomes.
Frequencies are representative of the number of proteins in each predicted proteome whose
percent content of an amino acid fell within a bin.

126

%AA

%AA

0/0 AA

% AA

%AA

Table 5.3. Compiled data from expression of amino acid biosynthesis genes, their
proportion in -6°C vs. 17°C proteomes and the effect on growth of supplementation
with those amino acids.

 

 

 

 

Amino Fold-QEHRC Expression ( _2. 5°C Growth Rate Ratio Ratio of Amino Acid

_Ac_id 6°C/17°C)a'b (AA added/no AA) Medians (-6°C/17°C)c
Gly 3.23d 1.72 -1.23
Pro 1.27 1.30 -1.06
Met 1.41 1.58 -1.21
Phe 1.00 2.18 -l.07
Cys 1.41 -l.03 1.20
Trp 1.48 NGe 1.34
Ala -2.36 1.24 1.00
His 1.29 1.29 1.05
Leu -2.28 -1.22 1.13

 

a
If more than one gene was differentially expressed in an amino acid pathway, then the gene closest to the
pathway product was used to represent fold-change behavior in the pathway.

b
Results from Chapter 4, Table 4.2
c Untransformed ratios of medians

Inverse of fold-change for glycine cleavage protein, gch

e
NC = No grth was observed in this trial.

Ratios of median percent amino acid content comparing the two proteomes were
calculated and compiled with data from the amino acid supplementation experiment
(Table 5.3). Principal components analysis (PCA) was performed on these data to group
amino acids based on data trends in these three experiments (Fig. 5.3). PCA delineated
the amino acid data into groups with similar trends. Those amino acids whose
biosynthesis genes increased greater than 1.4-fold also exhibited substantial changes in
proportion between 1.2 and 1.4-fold in the proteome comparison. Both methionine and
glycine biosynthesis are increased, and these amino acids increased growth rate when
supplemented into acetate medium. Both amino acids decrease their proportion 1.2-fold
in the low temperature proteome which supported our hypothesis that increases in

expression and growth rate would be correlated with bias against these amino acids in the

127

low temperature proteome. Thus, compensation by increasing transcription of glycine
and methionine biosynthesis appears insufficient to support translation of highly
expressed proteins at low temperature. In contrast, the biosynthesis of the least abundant
amino acids, cysteine and tryptophan, was up-regulated at low temperature, but the
proportions of these amino acids increased 1.2 to 1.3-fold in the low temperature
proteome. It may be that compensation by increased expression of biosynthesis genes is
sufficient to support translation, because these residues are rare in peptide sequences.
Alanine and histidine did not significantly effect grth at any temperature and their
proportion did not change in the low temperature proteome. Leucine increases in
proportion in the low temperature proteome while expression of leucine biosynthesis
genes decreases. This indicates that the biosynthesis of leucine is sufficient at low
temperatures. It is interesting to note that leucine proportion increased while valine and
isoleucine proportions decreased in the low temperature predicted proteome. The balance

of these inter-related biosynthesis pathways may have shifted at subzero temperatures.

128

 

 

 

d = 1
Ala
N Leu
*0
g Phe
t:
8
e In: -
8 ‘ His
'8
.9
o
.G
E

 

 

 

 

Principal component 1

Figure 5.3. PCA comparing data trends in amino acid content,
growth rate effect and gene expression. Amino acid loading on
principal component 1 is plotted on the horizontal axis and explains
the most variation across the three experiments. Principal component
2 explains the second most variation and is plotted on the vertical
axrs.

Discussion

Growth at low temperatures has been reported to induce genes for various
combinations of arginine, tryptophan, glutamate, glutamine, asparagines, cysteine and
leucine in diverse meSOphiles and psychrophiles (4, 6, 15, 18, 20). The biosynthetic cost
of amino acids as a function of both molecular weight and ATP have been shown to act
as a selective force on proteome evolution across taxa (1, 23). Here we expand this
hypothesis and search for evidence of effects of amino acid cost on the predicted

proteome specific to a physiologic condition, in this case temperature specific

129

metabolism. If an amino acid was limiting for grth at low temperatures, we expected
that i) low temperature expression for accumulation of that amino acid would increase, ii)
growth rates in cultures supplemented with that amino acid would increase as is observed
in auxotrophs which are also limited for an amino acid (13, 22), and iii) if this change in
metabolism is ecologically significant to P. arcticus, then selection against this amino
acid would be observed in the genes highly expressed at low temperature. This
relationship was observed for glycine and methionine. And the inverse was observed for
the branched chain amino acid leucine. These observations suggest that the metabolism
of glycine and methionine is limiting at low temperature and of leucine at optimal growth
temperature. Consequently, the production of these amino acids probably effects P.
arcticus fitness in the permafrost. The presented work is the first synthesis of global gene
expression data, growth experiments, and proteome prediction and analysis to elucidate
the relationships between gene expression at low temperatures and adaptations for
metabolism across the critical temperature.

The stringent response induces gene expression for growth rate reduction under
starvation conditions. The stringent response signal, (p)ppGpp, decreases the expression
of stable RNAs and consequently decreases grth rates and down-regulates central and
energy metabolism (7). Up-regulation of amino acid biosynthesis, in particular histidine
and arginine biosynthesis, has been linked to the stringent response, but recent global
transcriptome analysis confirmed that these responses are complicated and are probably
under metabolic control by feedback fiom cytoplasmic pools of these amino acids rather
than direct stringent response control (8). Reduced thermotolerance to supra-optimal

temperatures during growth on minimal media has been reported in E. coli ArelA mutants

130

I;

and these mutants also exhibited a long period of growth rate reduction before growth
arrest (26). A B. subtilis ArelA strain exhibited relaxed grth rate reduction and
decreased lag phase length compared to a wild-type strain (10), effects which were also
observed in P. arcticus AreIA growing at low temperature. Nutrient limitation is a
constant condition in permafrost and other cryo-environments (14), so association of the
stringent response with low temperature grth as hypothesized from our microarray
data is consistent with conditions to which P. arcticus is evolutionarily adapted. Our
observation that wild-type P. arcticus had a growth advantage compared with the ArelA
mutant PBO343 suggests that the stringent response plays a major role in regulating
growth rate at 0°C and -2.5°C. This advantage was negated at warmer temperatures
suggesting either that the stringent response is not as important in the optimal
temperature range from 22°C to 4°C, or that the growth rate effect of loss of relA can be
compensated by spoT activity under optimal growth conditions. SpoT is the primarily
involved in (p)ppGpp degradation, but can also act in its synthesis (7). However, the
former explanation seems most likely in the context of our rnicroarray observation that
the stringent response is induced in the cold.

Not surprisingly, casamino acid supplementation and that with a defined mixture
of amino acids improved growth at all temperatures. Clearly, provision of a mixed
population of amino acids alleviated some of the energetic cost of growth in the acetate
medium. Leucine exhibited a small negative effect on growth at 17°C and -2.5°C.
Excess leucine can induce starvation for other amino acids including tryptophan (13), and
this may explain decreased growth rates in the presence of excess leucine. Biosynthesis

of leucine was down-regulated in the subzero transcriptome indicating that biosynthesis

131

of leucine is sufficient to support growth at subzero temperatures. Furthermore, the
proportion of leucine in the subzero predicted proteome was higher than in the optimal
temperature highly expressed proteome corroborating the conclusion that leucine
biosynthesis is efficient at low temperature relative to optimal temperatures where
competition between translating ribosomes for leucine may lead to decreased proportion
of leucine per protein. Position of the methyl-group in branched chain amino acids may
have effects on flexibility of low temperature adapted enzymes, thus shifting the balance
between valine, isoleucine and leucine pools whose biosynthesis is interrelated.

Proline can act as a compatible solute and provision of up to 1 mM extracellular
proline can allow E. coli to grow in salt concentrations up to 1 M NaCl (16). Compatible
solute uptake and biosynthesis genes are up-regulated in bacteria following cold shock
and improve survival of psychrotolerant organisms at low temperatures where these
compounds can benefit both growth rate and protein stability (2, l9). Compatible solutes
provide the cell with a mechanism to maintain both protein interactions with intracellular
water and to maintain the turgor pressure which drives cell growth (16, 25). The medium
used in our grth experiments was 7.5% sea salts, a moderately high salinity.
Supplementation of P. arcticus cultures with 1 mM proline enhanced growth rates at all
temperatures and no significant change in proline proportion was observed in the
proteomes in spite of increased expression of two proline biosynthesis genes in our
transcriptome analysis. Proline is most likely serving a role as a compatible solute for
growth at low temperatures.

P. arcticus may be limited for production of methionine and glycine at low

temperatures. Glycine is produced either via glycine hydroxyrnethyltransferase (glyA) or

132

serine—glyoxylate transaminase (sgaA) in this organism. During growth on acetate, P.
arcticus should produce substantial amounts of glyoxylate as part of the glyoxylate
bypass to regenerate amino acid precursors. Based on the genome sequence, the
glyoxylate bypass in P. arcticus could be completed via either malate synthase or via the
B-hydroxyaspartate pathway which produces glycine as an intermediate substrate (17).
As was observed in P. arcticus at subzero temperatures, expression of the glycine
cleavage system is repressed in response to glycine limitation in E. coli (24). Neither
egA nor sgaA were differentially expressed P. arcticus transcriptome. It seems likely
that glycine generation is significantly dictated by the flux of acetyl-CoA through the
TCA cycle, glyoxylate bypass, and B-hydroxyaspartate pathway. A consequence of life
over long generation times under limiting conditions is that the ratio between energy
production and structural precursor biosynthesis is likely shifted towards ATP. For
example, flux of acetyl-CoA through the complete TCA cycle may be increased at
subzero temperatures to produce energy, because more energy is required per generation
at low temperatures (5). Flux analysis with labeled acetate as the growth substrate will
shed light on this model of growth at low temperature.

Alternatively, glycine can also play a role in the cell as a compatible solute by
conversion to glycine-betaine (16). Utilization of glycine as a compatible solute is
consistent with growth rate ratios in glycine supplemented cultures and down-regulation
of glycine cleavage particularly if Cl-metabolism activities are decreased at low
temperatures. The proteome data for glycine could be explained under this compatible
solute alternative if the ribosome is at some competitive disadvantage for glycine versus

the betaine biosynthesis genes betAB. However, betAB expression was down-regulated at

133

low temperatures in our rnicroarray experiment whereas glycyl-tRNA synthetases
(glySQ) were up-regulated at low temperatures. Furthermore, the overall abundance of
egSQ transcript was 4-fold higher than betAB in that transcriptome analysis. For these
data to be resolved in the compatible solute model, betaine biosynthesis genes would
have to be much more efficient at subzero temperatures than the more central function of
the glycyl tRNA synthetases. Therefore, reduced glycine biosynthesis at low temperature
due to changes in acetate flux seems to be the most parsimonious explanation for the
results of gene expression, growth rate and subzero proteome glycine content.

The limitation of growth based on methionine is most likely related to sulfur
metabolism at low temperatures. Cysteine and methionine increased grth rates at all
temperatures, but effects were only statistically significant under methionine
supplementation at -2.5°C (Table 5.2). Low rates of biosynthesis would then lead to
decreased rates of reduced sulfur incorporation into coenzyme A, S-adenosylmethionine,
cysteine, glutathione, iron-sulfur clusters, and others thereby seriously impacting the
function of the cell. Sulfur incorporation rates at low temperature are particularly critical
in light of results indicating increased methionine biosynthesis. There was also increased
expression of genes for oxidative stress response (including iron sulfirr cluster
biosynthesis), sulfur incorporation for coenzyme A production and the periplasmic
binding protein for an ABC sulfate uptake transporter in our microarray experiment.
Addition of methionine to cultures of P. arcticus grown at -2.5°C could reduce the
energy investment required for sulfirr incorporation at subzero temperatures and enable

increased growth rates. The observed decrease in methionine proportion in the subzero

134

predicted proteome may be the result of competition between sulfur metabolism enzymes
and the ribosome for methionine.

The rarest amino acids in both the high and low temperature proteomes are
cysteine and tryptophan. Both amino acids increased significantly in proportion in the
low temperature up-regulated proteome. Cysteine biosynthesis is linked with methionine
metabolism. The increased proportion of oxidative stress proteins in the subzero
predicted proteome may explain the higher proportion of cysteine present in these
proteins, because cysteine plays a reactive role in many oxidation repair and resistance
functions (11). In contrast, tryptophan is the most energetically expensive amino acid to
synthesize and has the bulkiest side chain, so it would be expected represent a smaller
proportion of amino acid positions. Tryptophan supplementation would be expected to
increase growth rates. Our results for this study seem perplexing in light of this model.
It is possible that increased expression of anthranilate synthase compensates for
decreased rates of tryptophan synthesis. Tryptophan comprises only 0.9% of the amino
acid positions in proteins even in the subzero temperature proteome, so small increases in
gene expression might balance the cellular tryptophan budget. Another aromatic amino
acid, phenylalanine, did increase growth rates at low temperature and likely alleviated the
high cost of aromatic arrrino acid biosynthesis, but this result cannot be linked to potential
bias in the proteome.

Our results demonstrate that glycine, methionine and leucine metabolism gene
expression at temperatures is likely related to limitation for those amino acids due to
changes in cellular physiology below the critical temperature. Combined genome,

transcriptome and grth analysis have enabled exploration of the link between the

135

challenges to biosynthesis in the cold and the ecology of a permafrost microbe adapted to
exploit a niche near its lower thermal limit of growth and under nutrient limitation. P.
arcticus appears unable to compensate for putative reduced glycine and methionine
production at low temperature, but can most likely compensate for changes in tryptophan
and cysteine production. Analysis of the predicted highly expressed subzero temperature
proteome indicates that limitation for glycine and methionine has likely been a selective
factor in the evolution of the P. arcticus genome leading to modification of peptide
sequences to compensate for reduced cellular pools of these amino acids at subzero
temperatures. Metabolic flux analysis could confirm that the cellular pools of glycine
and methionine change at subzero temperatures and reveal the changes in metabolism
that account for depletion of these key amino acids in central and sulfirr metabolism

respectively.

136

22°C
[+Wild +relA]

 

1.2

 

1.0
I
-——-l

OD600

 

 

 

°9
o
l I/
so
o'd ./1
g _ z/A/i\‘[ o
/- ° ° 1
5! */_L 1
O "‘ O
o /°::/‘
o' _ A-6"”°
I I I I I I I I
0 20 40 60 80 100 120 140
Time (h)

Supplementary Figure 5.1. Growth of P. arcticus relA
knockout compared with wild-type P. arcticus 273-4.
Growth rates are not statistically different. The relA
knockout exhibits decreased lag time and relaxed growth
arrest.

137

Supplementary Table 5.1. Genes included in the proteome amino acid content analysis. Genes were
differentially up-regulated > 2-fold at the indicated temperature and highly expressed based on transcriptome

Psyc_002 I
Psyc_003 6
Ps yc_004 2
Psyc_004 3
Psyc_004 4
Psyc_OO 79
Psyc_008 I
Ps yc_01 I 2
Psyc_Ol l 4
Psyc_O l 24
Psyc_OI 28
Psyc_O I 3 0
Psyc_O I 3 2
Psyc_O I 3 5
Psyc_O l 5 1
Ps yc_01 5 4
Psyc_O I 5 6
Psyc_02 0 7
Psyc_ 02 5 0
Ps yc_02 5 I
Psyc_02 60
Psyc_03 02
Psyc_03 43
Psyc_ 03 6 0
Psyc_03 79
Psyc_03 96
Psyc_04 05
P3 yc_ 04 8 7
Psyc_ 05 2 2
Psyc_ 0523
Psyc_05 5 l
Psyc_ 0664
Psyc_06 98
Psyc_0 702
Psyc_O 71 5
Psyc_0 766
Psyc_O 7 70
Psyc_080]
Psyc_084 1
Psyc_09l 8
Psyc_092 I
Psyc_0962
Psyc_0985
Psyc_0986

analysis.

-6°C Predicted Proteome

conserved hypothetical protein
conserved hypothetical protein
possible DNA polymerase 11], Chi subunit
probable cytosol aminopeptidase
probable isochorismatase hydrolase
conserved hypothetical protein
conserved hypothetical protein
conserved hypothetical protein
Pan, poly(A) polymrase

probable RpoS regulator, TraR/DksA family
hypothetical protein

conserved hypothetical protein
conserved hypothetical protein
conserved hypothetical protein
hypothetical protein

hypothetical protein

conserved hypothetical protein
putative Sigma 32 (RpoH)
conserved hypothetical protein
conserved hypothetical protein
probable cytochrome c, class 1
possible heat shock protein, Hsp33
putative RelA/SpoT family protein
conserved hypothetcial protein
probable organic hydroperoxide resistance
methionine aminopeptidase, type 1
conserved hypothetical protein
putative ribonuclease EG

probable Acyl carrier protein (ACP)
conserved hypothetical protein
conserved hypothetical protein
probable Glycosyl transferase, group 1
conserved hypothetical protein
conserved hypothetical protein
conserved hypothetical protein
possible RNA methylase
hypothetical protein

hypothetical protein

monoamine oxidase

conserved hypothetical protein

qu family protein
bis(5'-nucleosyl)-tetraphosphatase
DNA binding protein , HTH

neutral zinc metallopeptidases

138

Psyc_1062
Psyc_! 080
Psyc_] l 18
Psyc_1228
Psyc_] 240
Psyc_ l 241
Psyc_1256
Psyc_125 7
Psyc_] 264
Psyc_] 2 70
Psyc_ I 2 74
Psyc_! 303
Psyc_] 3 l 7
Psyc_] 3 25
Psyc_] 33 I
Psyc_! 342
Psyc_! 349
Psyc_] 3 53
Psyc_! 354
Psyc_] 3 65
Psyc_] 3 66
Psyc_] 423
Psyc_] 43 3
Psyc_! 4 76
Psyc_ l 4 7 7
Psyc_] 4 78
Psyc_] 4 79
Psyc_] 4 80
Psyc_148l
Psyc_! 482
Psyc_] 506
Psyc_] 581
Psyc_] 658
Psyc_l65 9
Psyc_] 697
Psyc_] 712
Psyc_] 724
Psyc_] 762
Psyc_] 7 75
Psyc_] 7 7 7
Psyc_] 03 3
Psyc_ I 043
Psyc_] 05 6

probable glutamate dehydrogenase
glutathione S-transferase family

probable C-factor protein
hypothetical protein

putative glucose inhibited division A
hypothetical protein

conserved hypothetical protein

23S RNA pseudouridylate synthase
conserved hypothetical protein

probable serine protease

hypothetical protein

hypothetical protein

possible biopolymer transport protein

probable potassium channel, VIC family
conserved hypothetical protein

probable glycerate kinase
hypothetical protein

ThiJ/prl family protein

hypothetical protein

probable fatty acid desaturase

conserved hypothetical protein
starvation protein A

hypothetical protein

possible transcriptional regulator
putative cysteine desulfurase
nitrogen-fixing NiiU-like
HesB/YadR/thF

probable co—chaperone Hsc20

heat shock protein Hsp70
ferredoxin, 2Fe-2S type
hypothetical protein

hypothetical protein
5-forrnyltetrahydrofolate cyclo-ligase

ATP-dependent protease La

metal-dependent phosphoesterases

hypothetical protein

RNA polymerase, omega subunit

conserved hypothetical protein

putative PspC

conserved hypothetical protein
hypothetical protein

peptide methionine sulfoxide reductase
transcriptional regulator, LysR family

Supplementary Table 5.2. Genes included in the predicted 17°C (Topt) proteorrre. Genes were selected
for analysis based on differential up-regulation > 2-fold and were highly expressed transcripts.

 

 

17°C Predicted Proteome _
Psyc_002 7 chaperone protein dnaJ Psyc_] l 75 putative acetyl-CoA hydrolase/transferase
Psyc_0097 citrate synthase Psyc_1309 probable short-chain dehydrogenase
Psyc_0099 succinate dehydrogenase anchor Psyc_l326 probable outer membrane protein
Psyc_Ol 01 succinate dehydrogenase subunit Psyc_! 337 ribosomal protein S6
Psyc_0104 dihydrolipoamide dehydrogenase Psyc_/338 ribosomal protein 818
Psyc_0105 succinyl-CoA synthetase beta subunit Psyc_1339 ribosomal protein L9
Psyc_0106 succinyl-CoA synthetase alpha subunit Psyc_/358 putative pyridoxamine 5'-phosphate oxidase
Psyc_O I 2 7 conserved hypothetical protein Psyc_ l 3 5 9 phosphoglucomutase/phosphomannosemutase
Psyc_0/3 7 probable acyl-CoA dehydrogenase Psyc_l360 inosine 5'-monophosphate dehydrogenase
Psyc_017l ribosomal protein L25 Psyc_/36] cysteinyl-tRNA synthetase, class la
Psyc_Ol 72 ribose-phosphate pyrophosphokinase Psyc_l363 hypothetical protein
Psyc_Ol 77 serine-pyruvate aminotransferase Psyc_l410 3-isopropylmalate dehydrogenase
Psyc_OI 96 phosphate acetyltransferase Psyc_/412 3-isopropylmalate dehydratase small subunit
Psyc_025 9 conserved hypothetical protein Psyc_14l3 3-isopropylmalate dehydratase large subunit
Psyc_0292 ribonuclease, cold-shock domain Psyc_1460 alanyl-tRNA synthetase, class He
Psyc_03l 9 cell division protein Psyc_/468 penicillin-binding protein transpeptidase
Psyc_0369 D-3 phosphoglycerate dehydrogenase Psyc_] 48 7 ribosomal protein 820
Psyc_03 70 rhodanese-related sulfurtransferase Psyc_/492 hypothetical protein
Psyc_0429 prolyl-tRNA synthetase, class lla Psyc_l493 histone-like bacterial DNA-binding protein
Psyc_0453 hypothetical protein Psyc_/521 NADH-dependent flavin oxidoreductase
Psyc_0505 ribosomal protein L18 Psyc_/5 23 aspartate semialdehyde dehydrogenase
Psyc_05 06 ribosomal protein 85 Psyc_1534 ribosome recycling/releasing factor (RRF)
Psyc_0507 ribosomal protein L30 Psyc_]535 Uridylate Kinase
Psyc_0519 ribosomal protein L32 Psyc_/560 periplasmicthiolzdisulfide oxidoreductase Dst
Psyc_0527 acetolactate synthase, large subunit, Psyc_/561 cytochrome c-type biogenesis protein
Psyc_0528 acetolactate synthase, small subunit Psyc_]562 hypothetical protein
Psyc_0529 Ketol-acid reductoisomerase Psyc_/566 ribosomal protein S9
Psyc_055 6 NAD(P) transhydrogenase, alpha subunit Psyc_l572 ribosomal protein L27
Psyc_05 5 7 NAD(P) transhydrogenase, beta subunit Psyc_! 5 73 ribosomal protein [.21
Psyc_0649 tyrosine kinase Psyc_! 5 7 7 outer membrane efflux protein, OprM
Psyc_06 72 hypothetical protein Psyc_/588 Beta-lactamase-like superfamily protein
Psyc_06 73 tetrahydrofolate cyclohydrolase Psyc_/592 conserved hypothetical protein
Psyc_06 78 nucleoside diphosphate kinase Psyc_/594 hypothetical protein
Psyc_0687 D-alanyl-D-alanine carboxypeptidase Psyc_1598 hlyD family secretion protein
Psyc_0708 fructose-bisphosphate aldolase class 11 Psyc_1599 ABC drug efflux transporter
Psyc_0 709 Holliday junction DNA helicase RuvA Psyc_l600 outer membrane efflux protein
Psyc_O 71 7 aminotransferase Psyc_] 604 Pyridoxal phosphate-dependent deaminase
Psyc_075 I aconitate hydratase B Psyc_] 624 phosphoribosylforrnylglycinamidine cycloligase
Psyc_O 7 97 nitroreductase family protein Psyc_] 62 7 isocitrate lyase
Psyc_0798 glycine cleavage H-protein Psyc_/642 porin, gram-negative type
Psyc_0828 s-adenosylmethionine synthetase Psyc_/643 malate synthase
Psyc_0883 hypothetical protein Psyc_165 7 fumarate hydratase class II
Psyc_0884 hypothetical protein Psyc_] 6 70 NADP-specific glutamate dehydrogenase
Psyc_0928 possible rare lipoprotein A family Psyc_] 700 hypothetical protein
Psyc_0942 Cold-shock protein Psyc_] 719 chaperone protein hth (Hsp90)
Psyc_0943 ATP-dependent RNA helicase Psyc_1742 conserved hypothetical protein
Psyc_0956 ribose 5-phosphate isomerase Psyc_] 763 malate dehydrogenase
Psyc_0968 S-adenosyl-L-homocysteine hydrolase Psyc_18l4 MCP methyltransferase, CheR-type
Psyc_1066 3'-phosphoadenosine sulfotransferase Psyc_/815 chemotaxis sensory transducer
Psyc_1067 conserved hypothetical protein Psyc_l8 I 6 type IV pili signal transducer, CheW
Psyc_/102 acetyl-CoA carboxylase Psyc_] 8] 7 two-component response regulator
Psyc_/103 conserved hypothetical protein Psyc_/818 two component remnse regulator

 

139

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142

CHAPTER 6

SUMMARY AND FUTURE PERSPECTIVE

143

Psychrophilic response to low temperature and its implications for Psychrobacter
ecology

This work has addressed the phenomenon of grth in ice-like conditions in a
heterotrophic psychrophile isolated from Siberian permafrost. The strength of our model
organism is that it is capable of growth both at moderate and subzero temperatures in salt
concentrations roughly equivalent to twice that in seawater. I have demonstrated that
Psychrobacter arcticus exhibits a low temperature response directed towards slowing
metabolic rates with continuing growth at low temperatures as opposed to the alternative
of growth cessation with a generalized stress response to resist damage. Unlike all
mesophiles and some psychrophiles, P. arcticus does not exhibit an increased resistance
to freeze/thaw or DNA damage after low temperature incubation. P. arcticus was also
shown to exhibit only a narrow (44 genes) transcriptome response to incubation at 4°C
compared to 22°C, indicating that P. arcticus does not primarily regulate its response to
4°C by transcription control. Rather, the response to 4°C may be a fine-tuning response
involving the regulation of enzyme activity. Subzero transcriptome analysis indicates
that true to theories of functional enzyme adaptation to multiple temperature ranges (2),
energy metabolism, most anabolic pathways and the basal machinery of transcription and
translation decreased in expression suggesting that their function is adequate in the cold..
Some compensation was observed including expression for exchange of metabolic
pathways for carbon and nitrogen incorporation, increased expression of membrane
desaturase, increased expression for oxidative stress resistance, and a newly discovered
response to adjust cell wall dynamics. These observed changes in metabolism may be of

ecological relevance to P. arcticus. In the case of amino acid metabolism, differential

144

expression for glycine, methionine and leucine biosynthesis can be correlated to
differences in amino acid content of highly expressed genes at low temperature compared
to those highly expressed at Tom.

The strategy of adaptation for resource efficiency in P. arcticus is likely central to
its ecological strategy in cryo-environments. Indeed, P. arcticus is able to tolerate a 79-
fold decrease in average growth rate between Top, and -6°C without entering a dormant
state. Why would this strategy of slow growth near the physical limits of life be favored
in P. arcticus evolution? Presumably this allows P. arcticus to exploit a niche
unavailable to other heterotrophs. Entry into dormancy, with all of its advantages for
damage resistance, requires major changes in the expression of proteins and such
adjustments must be undone when conditions again become favorable for growth.
Perhaps, P. arcticus has specialized to live at the energy limits by continuing growth with
very low energy inputs. This might permit success over other organisms that will not
grow in an energy limited situation such as that expected in cryo-environments.
However, it is important to note that tolerance to energy limited growth and dormancy
strategies are both viable adaptations for survival in permafrost as indicated by the
isolation of Bacillus and Clostridium species, known spore-forming genera, from
permafrosts of various ages.

If such adaptation is critical to P. arcticus survival in cryo-environments, then
why has this species retained the ability to grow at warmer temperatures? We have an
extremely limited sense of the history of P. arcticus before it entered the permafrost. It is
unlikely that P. arcticus could have evolved under stably subzero temperatures previous

to incorporation into the permafrost community, because such stably cold environments

145

tend to be ephemeral on geological time scales longer than 105 yr. Furthermore,
Psychrobacter species have never been isolated from permafi'ost older than 110,000 yr.
While it is clear that aspects of P. arcticus adaptation have allowed substantially long
term survival in permafrost, continuously frozen soils may not represent the niche to
which Psychrobacter species are primarily adapted (1).

Perhaps P. arcticus is primarily adapted for success in environments that undergo
annual freezing cycles such as the active layer of permafrost soils or aquatic surface
waters which exhibit yearly freezing cycles and remain frozen for 9 months of the year in
many polar habitats. In the active layer of permafiost, fieezing occurs slowly over a
period of several months. Such a freezing process can create stable temperature
conditions at about 0°C until the freezing process is complete. Consumption of carbon
derived from soil microbiota increases in freezing tundra presumably due to death of
microbial populations during freezing (4). P. arcticus adaptations for grth over a
range of temperatures in variable water activities, and the displayed preference of
oxidized organic acids is consistent with an ecology supported by freeze-thaw cycles.
Thus, P. arcticus may represent only a small proportion of microbial populations
particularly in high activity soils during the summer, but be adapted to make substantial
gains in resources during the winter months while other microbial populations are merely
surviving or even dying. Such an ecological strategy is consistent with the lack of
anaerobic respiration pathways in the genome versus adaptation to the highly reducing

conditions of Siberian permafrosts which may contain no free oxygen (6).

146

 

Future perspectives for research on Psychrobacter arcticus
My experiments have not addressed the direct measurement of changes in metabolic
reactions with temperature. P. arcticus decreases growth rate 79-fold at -6°C versus
17°C, but transcriptome analysis showed decreased in energy metabolism genes of only 2
to 8-fold in the same comparison. The degree of down-regulation of transcription at low
temperature was, on average, higher than the degree of up-regulation (Fig. 4.3).
Undoubtedly, some of the discrepancy between growth rate decrease and transcriptome
changes can be explained by underestimation of gene expression changes with
temperature, a common problem in high throughput expression analyses which require
global normalization and estimation of the effects of multiple sources of variance over
thousands of data points. Another contributor to this discrepancy is certainly that the cell
is functioning more slowly at low temperature, so growth at low temperatures with
concomitant decreases in metabolic rates might lead to this skewing in the distribution of
gene expression changes. The relationship between transcription and magnitude of
compensation remains unclear and only physiological characterization of psychrophilic
grth at low temperature can elucidate this relationship. Nevertheless, my growth
experiments in both knockout mutants and wild-type cells, and the results of my genomic
analyses demonstrate that the hypotheses based on differential expression in the
transcriptome are testable.

The observed relationship between arrrino acid metabolism expression, grth
rate response to amino acid supplementation and proteome content suggests changes in
metabolism with temperature in our laboratory experiments may be of ecological

significance to P. arcticus. Thus, experiments constructed to understand the changes in

147

flux of nutrients through metabolic pathways and how changes in gene expression
contribute to that flux will yield important insights into the metabolism of P. arcticus in
cryo-environments. Specific experiments include analysis of how acetate is shunted
between energy production and biosynthesis with incubation at different temperatures
and how sulfur metabolism is regulated at low temperatures. The analysis of sulfur
metabolism at low temperatures is particularly interesting, because MgSO4 is the second
most abundant salt in our acetate medium and there was no reason to expect any sulfur
starvation to occur.

While gene expression and mutant data support a model of isozyme exchange to
maintain elasticity of the cell wall, many questions remain unanswered. First, is the
elasticity of cell walls from low temperature cultured cells greater than that from warm
temperature cultured cells, and how does this correlate to the degree of cross-linking?
How does cross-linking of peptidoglycan strands change in the isozyme mutants that we
generated? Based on mutant growth results, I propose that the DD-peptidase isozyme
deletion leads to increased cross-linking of the murien sacculus at the respective
functional temperatures of the isozymes. Biochemical characterization of the regulatory
process underlying transcriptional regulation of the DD-peptidases would enable us to
differentiate temperature regulation versus regulation by some property of the cell wall
controls their expression. Outer membrane lipoproteins are covalently anchored in the
cell wall and might contribute to sensing cell wall characteristics.

The model of P. arcticus as an organism adapted for annually fi'ozen
environments leads to testable hypotheses. P. arcticus in frozen microcosms with other

organisms should be able to incorporate carbon including that derived from microbial

148

biomass. P. arcticus should be able to outcompete other microbes in soils for carbon
under frozen conditions. Such hypotheses can be tested with stable isotope probing
combined with specific probes for P. arcticus cells and any competitor we might choose.
Additionally, because P. arcticus does not enter a dormant state, it may have an
advantage over competitors in acquiring resources immediately following the spring
thaw.

The ability to grow at very slow rates makes P. arcticus a candidate model
organism for metabolic analysis. Does metabolism at slow rates occur continuously or in
a stepwise fashion? There is a high probability that the metabolism of P. arcticus at low
temperatures exhibits interesting spectral qualities associated with discontinuous flux
through metabolic pathways. All biological systems from whole cells to enzymes exhibit
periodic behaviors. For instance, an enzyme in ice exhibits periodic fluctuations in
activity as substrate is locally depleted more quickly than it is replenished through
diffusion (5). The advantage of a model organism with very slow growth rates is that
such periodic shifts in the activity of the metabolic network could be analyzed at high
temporal resolution relative to the biological development of a cell. Spectral analyses
also permit the analysis of noise in biological behavior (3). Understanding the
contribution of noise to biology is one of the next great frontiers in biological science and
has implications for phenomena ranging from population diversity to behavioral control

in the single cell.

149

 

References

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cryohalolentis sp. nov. and Psychrobacter arcticus sp. nov., isolated from
Siberian permafrost. Int J Syst Evol Microbiol 56:1285-91.

2. Georlette, D., V. Blaise, T. Collins, S. D'Amico, E. Gratia, A. Hoyoux, J. C.
Marx, G. Sonan, G. Feller, and C. Gerday. 2004. Some like it cold: biocatalysis
at low temperatures. FEMS Microbiol Rev 28:25-42.

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temperature. Cell Mol Life Sci 56:398-414.

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and cultures from fossil ice and permafrost. Trends in Ecology & Evolution
19:141-147.

150

   

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