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COMPARATIVE ANALYSIS OF BACTERIAL COMMUNITY
COMPOSITION IN SIBERIAN PERMAFROST AND
ANTARCTIC POND SEDIMENTS

presented by

BRAD CHlA-KAI CHANG

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2/05 p:/ClRC/Date0ue.indd-p.1

COMPARATIVE ANALYSIS OF BACTERIAL COMMUNITY COMPOSITION

IN SIBERIAN PERMAFROST AND ANTARCTIC POND SEDIMENTS

By

Brad Chia-Kai Chang

A THESIS

Submitted to
Michigan State University
In partial ﬁilﬁllment of the requirements
For the degree of

MASTER OF SCIENCE

Department of Crop and Soil Sciences

2006

ABSTRACT

COMPARATIVE ANALYSIS OF BACTERIAL COMMUNITY COMPOSITION IN
SIBERIAN PERMAFROST AND ANTARCTIC POND SEDIMENTS

By

Brad Chia-Kai Chang

Understanding the microbial diversity under very cold and usually frozen
environments such as Siberia and Antarctica may lead to the discovery of traits important
for life in extreme environments. We compared the bacterial community structure of
Siberian permafrost and Arctic pond sediment by building 16S rRNA gene clone
libraries, a culture-independent approach. Our goal was to explore the composition and to
compare the diversity of these communities by utilizing an RDP high throughput pipeline
tool and the following programs: DOTUR, Estimates, Libshuff, MEGA and Classiﬁer.

Four Siberia samples were selected according to the age which ranged from
twelve thousand to three million years old, and from freshwater alluvial versus marine
horizon. There were two samples from the Antarctic McMurdo Ice Shelf. They are pond
surface sediments collected from Fresh pond and Brack pond on Bratina Island. These
two ponds are next to each other but represent very different salinities in pond water.
From the alpha diversity analysis, the Siberia samples showed lower diversities than
Antarctica samples. Both Arctic and Antarctica sediments with the higher salinities
showed lower diversity in bacterial community. The results from RDP Classiﬁer showed

that the Proteobacteria were the most dominant phylum in both regions.

C0pyright by
BRAD CHIA-KAI CHANG

2006

This work is dedicated to my parents,
who have continuously

loved and supported me.

iv

ACKNOWLEDGEMENTS

I would like to thank Dr. James Tiedje, my major advisor, for his intellectual
guidance and continuous help. His patience has been most appreciated while I struggled
throughout my study, and I could never thank him enough for offering me such great
learning experience.

I would also like to thank my committee members, Dr. Michael Thomashow and
Dr. Terence L. Marsh, for their valuable suggestions and comments.

I am grateful to Dr. Monica A. Ponder, Peter Bergholz and Debora Rodrigues for
introducing me into the ﬁeld of Astrobiology. Their advices, encouragements and
friendship have enriched my life here at MSU. I also want to recognize Erick Cardenas
for spending so much time arguing and discussing all my questions with me. Without
them, this work would not have been possible.

Most of all, I wish to express my deepest gratitude to my parents and both my
sisters’ families for giving me so much support, ﬁnancially and spiritually. I could not

make it without their love.

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. vii
LIST OF FIGURES ................................................................................ viii
CHAPTER I.

COMPARATIVE ANALYSIS OF BACTERIAL COMMUNITY COMPOSITION IN
SIBERIAN PERMAFROST AND ANTARCTIC POND SEDIMENTS BY THE
CULTURE-INDEPENDENT METHOD

Introduction ................................................................................... 1

Materials and Methods ............................................................................................ 4

Results ............................................................................................................... .10

Conclusion and Discussion ................................................................ 27
APPENDIX A.

The Effects of Sodium Ion Concentration on the Growth of Psychrobacter
arcticus 2 73 -4

Introduction .................................................................................. 32
Materials and Methods ..................................................................... 35
Results and Discussion .................................................................... 37
LITERATURE CITED .............................................................................. 43

vi

LIST OF TABLES

CHAPTER I
Table 1. Characteristics of the sites sampled for DNA extraction .............................. 6
Table 2. DNA concentration ﬁ'om studied sites and clone library information ............. 11
Table 3. Alpha diversity indices ................................................................... 13
Table 4. The result (P-value) for ILIBSHUFFZO

Table 5. Overview of the clone result for Antarctica and Siberia bacteria diversity from
sediments at class level ................................................................... 21
Table 6. Comparison of microbial community composition on three similar studies of

Arctic-area permafrost plus the Antarctica pond sediments ......................... 30

APPENDIX A

Table l. The maximum OD600 reading for various concentrations acetate in the media

with 30 mM NaCl ........................................................................ 39

vii

LIST OF FIGURES

CHAPTER I

Figure I. Rarefaction curve for Antarctic ponds and Siberian permafrost communities..12

Figure 2. Shannon index result .................................................................... 14
Figure 3. Simpson index result .................................................................... 15
Figure 4. Chaol index result ....................................................................... 16
Figure 5. Rank-abundance curve for Antarctica and Siberia communities .................. 18
Figure 6. Clustering of Antarctica and Siberia samples ....................................... 19

Figure 7. The phylum distribution of Siberia samples by clone sequence number. The
Proteobacteria and Actinobacteria are signiﬁcantly highly dominant. . . . . . ..22

Figure 8. Phylogenetic tree shows the relationship of the Psychrobacter Type strains and

Psychrobacter clones from this study ................................................ 26
Figure 9. The relationship of OTU numbers to the age and salinity of samples. . . . . . . . ....28
APPENDIX A

Figure 1. The gene expression ratio for sodium dependent syrnporter (ORF1600). The
culture medium base is 1/2 TSB (from Ponder, 2005) ............................... 33

Figure 2. The gene expression ratio for Na+ pumps NADH dehydrogenases (ORF1687-

viii

ORF 1691). The culture medium base is 1/2 TSB (from Ponder, 2005) ........... 34
Figure 3. OD600 for Psychrobacter arcticus 273-4 grown in 20 mM acetate media with
different sodium concentration ........................................................ 37
Figure 4. The growth rates (generation/h) for four different sodium concentrations of 20
mM acetate media ....................................................................... 38
Figure 5. The total protein yields for different sodium concentrations in 11 mM acetate
medium. The bars represent total protein concentration (pg/ml) and the blue
dots represent the ﬁnal (optimal) optical density for each different
sample .................................................................................... 40
Figure 6. The protein yield presented by relative turbidity (OD600). The values are shown
on the top of the bars. The media used are 11 mM acetate media with the

indicated NaCl concentrations .......................................................... 41

ix

CHAPTER I

COMPARATIVE ANALYSIS OF BACTERIAL COMMUNITY COMPOSITION
IN SIBERIAN PERMAFROST AND ANTARCTIC POND SEDIMENTS BY THE

CULTURE-INDEPENDENT METHOD

INTRODUCTION

The permafrost environment is composed of soil, bedrock, sands, and sediments
that remain frozen for a period of two years or more (Mueller, 1973). Over 20% of the
earth’s land surface is subjected to these permanently cold conditions including, 85% of
Alaska, 55% of Russia and Canada, 20% of China, and the majority of Antarctica (Pewe,
1995). Members of the genera Psychrobacter are commonly isolated from environments
that are low in temperature or high in salinity (Juni, 2002), such as permafrost.
Psychrobacter arcticus 273-4 was isolated (Vishnivetskaya et al., 2000) from polar
region permafrost soil of the Kolyma-Indigirka lowland (152-162°E, 68-72°N), adjacent
to the Siberian Sea. The sediment from the sampled permafrost layer has been ﬁ'ozen for
at least 20-30,000 years.

Siberian permafrost soil represents an extreme environment that is characterized by
low temperatures (-10 to -12°C), low water activity (aW = 0.9), and low nutrient
availability (Gilichinsky, 2001). The low water activity is due to the increased
concentration of solutes in the remaining unfrozen water. The water activity of 0.9
corresponds to a salt concentration of 2.79 m NaCl (5 osm). The permafrost environment

in contrast to many surface environments is very stable, with a constant set of stresses

that may have acted as selective factors on the survivors, including adaptation to a long-
term frozen environment and the associated desiccation (Gilichinsky, 2001; Willerslev, et
al., 2004). The Kolyma permafrost temperatures of -1 0 to -12°C would not be expected
to freeze the cytosol of bacterial cells (McGrath, 2004) and hence continued biochemical
catalysis could be expected, albeit the ﬂuid would be viscous and reaction rates very slow
(Ponder, et al., 2004).

The Antarctic ecosystem is also considered an extreme environment. The
McMurdo Ice Shelf comprises an area of about 1500 km2 with a thickness from 10 to 50
m (Howard-Williams, et al., 1990). During the summer, the area is covered with a
network of meltwater ponds that vary in size, shape and physicochemical conditions,
even though some of them are only several meters apart (De Mora, et al., 1991)

The goal was to analyze the bacterial community diversity from these two
geologically separated but low temperature and usually frozen environments. Our
approach was to use a culture-independent, 16S rRNA gene clone library comparison
method. The 16S rRNA genes in bacteria are highly conserved. This character makes
them a good target for establishing phylogenetic relationships of microbes in ecological
studies (Lane, et al., 1985). The analysis of the total-community genomic DNA via 16S
rRN A gene sequence has been used to characterize Archaea within the permafrost
sediments (Tiedj e, et al., 1998). A recent study of Vishnivetskaya et a1. (V ishnivetskaya,
et al., 2006) used both culture-dependent and culture-independent methods of
characterizing a Siberian permafrost bacterial community. The results of two methods
from above study yield very different perspective about the bacterial community

structure. Thus, additional community comparison of Antarctic and Siberian communities

is important for a greater understanding of cold adapted communities and to see if the

composition of bacterial communities is similar for both habitats.

MATERIALS AND METHODS

Characterization of the Antarctica and Siberian sediments

The samples from Antarctic were collected by Peter Bergholz during January
2006. The sampling sites were on the south portion of Bratina Island (165°35'E, 78°00'S)
on the McMurdo Ice Shelf. The samples are sediments from two ponds lying within 50
meters of each other. These two ponds are discrete and were designated with the
unofﬁcial names, Brack Pond and Fresh Pond. They have electrical conductivities of
about 20,000 and 1,200 micro-Siemens per centimeter respectively (20,000 microS/cm =
~219 mmol/L of NaCl). The average temperature of Brack pond was 59°C, and ranged
from 3°C to 95°C for January 2006. The Fresh pond had a lower average temperature,
37°C, and ranged from 1.5°C to 68°C (Table 1).

The samples from the Siberian permafrost were collected by David Gilichinsky
and his team ﬁom the Cryobiology Laboratory, Russian Academy of Sciences,
Pushchino. Four permafrost samples were collected from borehole 17/99, 04/01, 01/01
and 02/92. The second pair of digits identiﬁes the year the core was collected. Borehole
17/99 is located in Lake Yakutskoe by East Siberian Sea coast (159°29'E, 69°51 'N). This
is a timdra zone in the Kolyma lowland. The texture of this sediment sample is described
as sandy and sandy-loams. The core was from a depth of 16.8-16.9 meter and is a middle
Pleistocene marine horizon that contains cryopegs, and dated as 100 — 120 thousand years
old. Borehole 04/01 is located in a tundra zone in the Cape Svyatoi Nos by Laptev Sea
coast (140°10'E, 72°55'N). This alluvial sample is sandy-loam with icy complex. The

depth where the sample was collected is 9.0 meter and dated as 300 — 400 thousand years

old from middle Pleistocene epoch. Borehole 01/01 is located in a tundra zone on Cape
Bykovskii by the Laptev Sea coast (129°30'E, 71°40'N). This sample was collected from
13.0 meter deep and is late Pleistocene alluvial sandy-loath with icy complex. It is dated
as 12-40 thousand years old. Borehole 02/92 is also located in a tundra zone. It is from

the right bank of the mouth of the Malaya Kon’kovaya River, Kolyma lowland (158°28’E,
69°23 ’N). This sediment sample was collected from 18.7 meter deep. It is lake-swamp
loam from the late Pliocene epoch and dated as 2-3 million years old. All of these
sediment samples were stored in a -20°C freezer and were not exposed to room

temperature for more than 5 min while weighing the soil needed for DNA extraction.

Table 1. Characteristics of the sites sampled for DNA extraction.

 

 

 

 

 

 

 

 

Sample Sampling Site Depth Temp. Age Other
(m.) (°C) (year old)
AB Antarctic Brack Pond Surface 3~9.5* < 100 High salt
AF Antarctic Fresh Pond Surface 1.5~6.8* < 100
S17M Siberian borehole 17/99 16.85 -10~-12 100~120 Marine
thousand horizon
SO4F Siberian borehole 04/01 9 -10~-12 300~400
thousand
SOlY Siberian borehole 01/01 13 -10~-12 12~40 Young
thousand
S020 Siberian borehole 02/92 18.7 -10~-12 2~3 million Old

 

 

 

 

 

 

* Average temperature during austral summer 2006

 

Total DNA extraction from sediment

The sediment samples (0.8 g) were placed in sterile ceramic mortars. After several
repeated rounds of freezing and grinding steps, they were ground to powder condition.
The total DNA was extracted using the F astDNA SPIN KIT for Soil (B10 101 system
from Q-BIO Gene Company) according to the manufacturer’s protocol. The
concentration of DNA was estimated at 260 nm by NanoDrop spectrophotometer. In case
of extraction failure, two replicates were performed for each sediment sample and the

replicate with better DNA quality was used for 16S rRNA gene ampliﬁcation.

16S rRNA gene ampliﬁcation from genomic DNA

The 16S rRNA genes were ampliﬁed by the using universal primer set 27F and
1392R in six replicates. Ampliﬁcations were performed in a reaction mixture containing
5 pl of 5X GoTaq Flexi Buffer (Promega), 1.0 mM MgC12, 0.4 mM each
deoxynucleotide triphosphate(1nvitrogen), 5 pg of bovine serum albumin (BSA) (New
England Biolab), 0.4 pM each of forward and reverse primers, 25 ng of total community
genomic DNA, 1.25 U of GoTaq DNA polymerase (Promega), and molecular level
DNase-free H20 (Sigma) to a total volume of 25 p1. The temperature proﬁle for the
polymerase chain reaction (PCR) was set to an initial denaturation at 95°C for 9 min
followed by 25 cycles of 95°C for 1 min, 59°C (optimum temperature of the primer set)

for 1 min, and 72°C for 1 min 40 3, plus a ﬁnal extension at 72°C for 10 min.

Cloning by using T 0P0 TA cloning kit

The ampliﬁed PCR products were veriﬁed in a 1% agarose gel electrophoresis
and the three reactions with better quality were cut and puriﬁed by a Qiaquick Gel
Puriﬁcation kit (Qiagen). The concentration of puriﬁed PCR product was determined by
a NanoDrop spectrophotometer at 260 nm. The puriﬁcation steps resulted in loss of
deoxyadenosine (A) at the 3’ end of the PCR product that is needed for successful
ligation; therefore, a reaction for adding the 3 ’ end A was preformed. This reaction
mixture contained 0.1 pl of Taq DNA polymerase (Invitrogen), 2.5 pl of 10X PCR
Buffer, 1.5 pl of 50 mM MgC12, 1 mM of deoxyadenosine triphosphate (dATP), and
molecular level DNase-free H20 (Sigma) to a total volume of 25 pl. The reactions were
incubated in a water bath at 72°C for 10 min and the products were used directly for
cloning. The PCR products were ligated into pCRTM 4.0 vector using TOPO TA cloning
kit (Invitrogen) and transformed into chemically competent E. coli according to the
manufacturer’s instructions. Luria-Bertani (LB) agar containing 100 pg/ml of kanamycin
was used for clone selection. Aﬁer overnight incubation at 37°C, selected colonies were
picked with toothpicks and placed in a 96-well growth block which contained 1.2 ml LB
selective (100 p g/ml of kanamycin) freezing buffer (4.4% glycerol). The growth block
was covered by airpore tape and the cells allowed to grow overnight on a shaker (200
rpm) at 37°C. Transfer of 150 pl of the grown cell culture was made into a Grainer 96-
well plate. An ethanol-ﬂamed colony copier was used to stamp colonies from the Grainer
plate onto an OmniTray LB agar plate containing 100 pg/ml of kanamycin. The

OmniTray plates were incubated at 37°C overnight before sending to Macrogen Inc.

(Seoul, Korea; www.macrogen.com) for sequencing. The Grainer plates were sealed by

aluminum tape and stored in -20°C freezer to retain stock cultures.

Clone Library Analyses

Raw sequence data were processed by using pipeline quality ﬁlter tools on the
Ribosomal Database Project II (RDP-II) website (Cole et al., 2005;
http://rdp.cme.msu.edu). The RDP pipeline tool automatically processes entire libraries
of single sequences from raw sequencer output to analysis. The base-calling work is done
by PHRED. The quality trimming and vector removal are done by LUCY program. The
Q20 cutoff was set to 0.8 and the minimum sequence length of 400 was used. Sequence
alignment, library distance matrix ﬁle and input ﬁle for Estimates (Colwell, 2005;
http://viceroy.eeb.uconn.edu/EstimateS) software were downloaded via RDP pipeline
tool. The distance matrix ﬁle was used with Distance-Based Operational Taxonomic Unit
and Richness (DOTUR) determination program (Schloss and Handelsman, 2005;
http://www.plantpath.wisc.edu/fac/joh/dotur.html) for obtaining operational taxonomic
units (OTUs) and alpha diversities, the biodiversity within a particular community. The
Estimates software was used for obtaining beta diversity, which is a measure of
biodiversity between ecosystems. The MEGA (Kumar, Tamura and Nei, 2004) program
was used for clustering libraries following the Estimates analysis. The distance matrix
was used for comparing similarity between clone libraries via a statistical test provided
by the LIBSHUFF program (Singleton et al., 2001).

The Classiﬁer tool provided by RDP II is used to assign 16S rRNA sequences to

the taxonomical hierarchy. The default rank conﬁdence threshold of 80% was used.

RESULTS

Total community genomic DNA yield

The total community genomic DNA extractions for Siberia permafrost samples
had yields of 1.95, 1.89, 3.23 and 3.26 pg DNA per gram of sediment for borehole 17/99,
04/01, 01/01 and 02/92, respectively. The genomic DNA yields from Antarctic Brack

pond and Fresh pond samples were 9.84 and 1.69 pg/ g of sediment (Table 2).

168 rRNA gene sequences analyses for positive sequences and GT Us

Six plates, one plate (96 clones) for each sample, were sent to Macrogen Inc. for
sequencing. The raw sequencer output is processed by RDP pipeline tools. The numbers
of clones that passed the quality control of RDP pipeline ranged from 92 to 96 clones per
plate. This provided a total of 561 16S rRNA gene sequences cloned from total-
community genomic DNA extracted from the six sediment samples. DOTUR program
generated the OTU numbers and calculated alpha diversity indices. The OTU difference
threshold for all DOTUR calculations was set at 97% similarity. The OTU numbers were
6, 13, 43 and 6 OTUs for Siberia 17/99, 04/01, 01/01 and 02/92 samples, respectively.
There are 41 and 58 OTUs for sediment samples from Antarctica Brack pond and Fresh
pond (Table 2). Higher OTU numbers appeared in Siberia 01/01 (young sample) and both

Antarctica samples.

10

Table 2. DNA concentration from studied sites and clone library information.

 

 

 

Sample" DNA concentration Sequences 0T Us
(ﬁg/g-soil)

Sl7M 1.95 93 6

SO4F 1.89 92 13

SOlY 3.23 92 44

S020 3.26 95 6
AB 9.84 93 41
AF 1.69 96 ‘ 58

Total P 561 168

 

* See Table 1 for identiﬁcation of sample code

11

The rarefaction curves showed that samples S17M, S04F and S020 were close to
full coverage of the community (Fig. 1). On the other hand, samples AB, AF and SOlY

need more clones for adequate coverage. Images in this thesis are presented in color.

 

Rarefaction curve

70

60

50

40

Number of OTUs

30

20

  

16111621263136414651566166717681869196
Number of sequences

 

 

 

Figure 1. Rarefaction curve for Antarctic ponds and Siberian permafrost communities.

Alpha diversity indices and rank-abundance curves

Alpha-diversity indices include Shannon index, Simpson index and Chao 1 index.

Alpha diversity indices are calculated for comparing diversity within a community and

the values for indices were shown in Table 3.

Table 3. Alpha diversity indices.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Number Number of
SAMPLE Shannon (11') Simpson (D) Simpson (l-D) Chaol
of OTUs sequences
$17M 6 93 0.728 0.648 0.352 9
S04F 13 92 1.81 0.212 0.788 23.5
SOlY 44 92 3.42 0.0394 0.961 83
8020 6 95 1.34 0.315 0.685 6
AB 41 93 3.49 0.0262 0.974 58.1
AF 58 96 3.9 0.0136 0.986 103

 

13

 

The Shannon index is most commonly used for presenting biodiversity. This
index takes into account the number of species and the evenness of the species. The index
(diversity) is increased either by having more unique species, or by having greater species
evenness.

The results for Antarctica samples were 3.49 for Brack pond and 3.9 for Fresh
pond. They were signiﬁcantly higher than the value for three of the Siberia samples, 0.7
for sample $17M, 1.8 for SO4F and 1.3 for S020 (Figure 2). However, the result for
sample S01Y was 3.4, which was higher than other Siberian samples and was comparable

to Antarctica samples.

 

Shannonindex

 

 

 

 

 

 

 

 

Figure 2. Shannon index result.

For the Simpson index, value (D) represents the probability that two randomly
selected individuals in the community belong to the same species. Therefore, (l-D)
represents the probability of two different species being picked. The higher the (1 -D)
value, the higher the diversity of the community. For example, sample S17M was low in
biodiversity (l-D), the chance for picking up two individuals as same species (D) is as
high as 64.8% (Figure 3).

 

Simpson index

 

AB AF _ - sow

    

 

 

 

 

Figure 3. Simpson index result.

The Chaol index is a nonparametric estimator suitable for microbial diversity
analysis. It estimates the richness of a community.

Sample S17M and S020 had lower OTU numbers than others and thus had lower
values for richness (Figure 4). Within the Antarctica communities, sample AB showed

lower richness than sample AF.

 

 

 

 

 

 

 

Chao1
200
AF

150 T SQ1Y
100 — AB

SO4F
50 g S17M

* 8020

0 _

 

 

 

Figure 4. Chaol index result.

Rank-abundance curves were generated for easier visualization of species
abundance in a community (Figure 5). The ﬁgures clearly show that Siberian samples had
higher abundance of species than Antarctica samples. There were 74 clone sequences
from only 1 OTU in the Siberia marine sample; 31 clone sequences from only 1 OTU in
the Siberia fresh sample; and 43 clone sequences from only 1 OTU in the Siberia 02 (old)

sample.

17

 

Antarctica Brack pond w Siberia marine

o—ONQ#UIO>N®

 

0 ,
1357911131517192123252729313335373941 1 2 3 4 5 6

 

Antarctica Fresh pond

501°)

CD

1

    

# ofsequences

 

 

 

 

    

0 I . .
1471013161922252831343140434649525558| 1 2 3 4 5 6 7 a 9 10111213

16 Siberia 01 (young) Siberia 02 (old)

14

12

10

    

 

 

 

 

147101316192225283134374043 1 2 3 4 5 6

 

 

#ofOTUs

Figure 5. Rank-abundance curve for Antarctica and Siberia communities.

18

Beta-diversity and clustering
Beta-diversity is a method for comparing diversity between different communities.
The Antarctic samples clustered separately from the Siberian samples (Figure 6) by
MEGA program. Also, the Siberian 17M and 4F samples are clustered within the
Siberian cluster. However, even though the clusters can be recognized, the distance
between any two communities is larger than 0.86 (817M and 84F), meaning that the

highest similarity is about 14%.

 

S17M
84F
820
SW
AB
AF

 

 

 

 

 

 

 

 

 

 

1 l l
l l I I

0.4 0.3 0.2 0.1 0.0

#—

 

 

 

Figure 6. Clustering of Antarctica and Siberia samples.

19

LIBSHUFF

LIBSHUF F is a computer program designed to compare two libraries of 16S
rRNA gene sequences and determine if they are signiﬁcantly different. I—LIBSHUFF is
another version of LIBSHUFF that can compare several libraries at once. The P value
represents the rank of two communities being the same. When the P value is smaller than
0.05, samples are considered signiﬁcantly different. The results from I-LIBSHUFF
analysis showed that all six libraries from Siberian and Antarctic sites are signiﬁcantly

different from each other (Table 4).

Table 4. The result (P-value) for I—LIBSHUFF.

 

 

P - value AB AF 817M SO4F $01Y 8020
AB - 0.000 0.000 0.000 0.000 0.000
AF 0.000 - 0.000 0.000 0.000 0.000

$17M 0.000 0.000 - 0.000 0.000 0.000
SO4F 0.000 0.000 0.000 - 0.000 0.000
SOlY 0.000 0.000 0.000 0.000 - 0.000
8020 0.000 0.000 0.000 0.000 0.000 -

 

 

 

 

20

RDP classiﬁer

From the total of 561 sequences cloned from the Siberian and Antarctic sediment
samples, there were total of 167 OTUs differentiated by the 97% sequence identity cut-
off. These OTUs were distributed among 10 phyla and at least 17 classes (Table 5). There
were 7.3% of unclassiﬁed bacteria sequences and they only appeared in the Siberian-04

(young) sediment and in both Antarctic libraries.

Table 5. Overview of the clone result for Antarctica and Siberia bacteria diversity from

sediments at class level.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Phylum Class AB AF S17M SO4F SO1Y $020

Proteobacteria Alpha 16 2 1 2 8
Beta 7 11
Gamma 1 5 90 61 1 86
Delta 4 5

Bacteroidetes Sphingobacteria 3 1 1 1
Flavobacteria 3 16
Unclas. Bacteroidetes 15 6

Firmicutes Clostridia 5 14 1
Mollicutes 1
Bacilli 5 1 1 1 3

Actinobacteria Actinobacteria 7 22 2 28 54

Gem matimonadetes Gemmatimonadetes 1 2

Cyanobacteria Cyanobacteria 7

Chloroﬂexi Anaerolineae 12

Deinococcus-Therm us Deinococci 4

Genera_incertae‘TM7 TM7 1

Spirocheta S pirochaetes 1

Thermom icrobia Therm om icrobia 1

Unclas. Bacteria 8 5 28

Total 93 96 93 92 92 95

 

21

For Siberia samples, there were 69 OTUs in 372 cloned sequences. These OTUs
were distributed among six known phyla and at least eight classes (Figure 7). The
Proteobacteria and Actinobacteria were most dominant and contributed 66.9% and 22.6%
of clone sequences, respectively. The Antarctica samples were more evenly distributed.
They had 99 OTUs in 189 clone sequences among ten known phyla and at least sixteen

classes.

 

Phylum distribution of Siberia samples

 

I Proteobacmria

I Acti nobacteria

El unclass Bacteria

I Firmicutes

I Bacteroides

I Gemmtimonadetes
I Thermom'crobia

 

 

 

 

 

 

Figure 7. The phylum distribution of Siberia samples by clone sequence number. The

Proteobacteria and Actinobacteria are signiﬁcantly highly dominant.

22

Proteobacteria

The Proteobacteria were the most dominant phylum in both Siberian and
Antarctic libraries, representing 67% and 27%, respectively. Garnma-Proteobacteria were
the only class that was found in every library. A total of 96.8% of the clones for sample
817M, 66.3% of the clones for SO4F and 90.5% of the clones for 8020 were Gamma
Proteobacteria. For communities $17M and SO4F, all the Gamma-Proteobacteria were
Pseudomonadales. For community 8020, 31.6% were Xanthomonadales, 45.3% were
Pseudomonadales and 13.7% were Enterobacteriales. Pseudomonadales was not found in
Antarctic libraries. At the family level, sample S17M had 4.3% of Pseudomonadaceae
and 92.5% of Moraxellaceae, sample SO4F had 12% of Pseudomonadaceae and 53.3% of
Moraxellaceae, sample S020 had 45.3% of Pseudomonadaceae. At the genus level, the
result showed that all of these Moraxellaceae were Psychrobacter. The Psychrobacter
was dominant in S17M and SO4F clone libraries. However, Psychrobacter was only
found in these two libraries. Alpha-Proteobacteria was commonly found in these
communities except the 817M library. At the family level, Hyphomicrobiaceae and
Rhizobiaceae were found in Siberian libraries, while Beijerinckiaceae, Rhodobacteraceae
and Caulobacteraceae appeared in Antarctic libraries. While Alpha- and Gamma
Proteobacteria were common in both habitats, Beta- and Delta-Proteobacteria appeared in
Antarctic libraries only. For Beta Proteobacteria, only Burkholderiales were present. For
Deltaproteobacteria, Desulfobulbaceae was the only one represented in library AB. In
sample AF, Geobacteraceae, Desulﬁrromonaceae, one unclassiﬁed Myxococcales and

one unclassiﬁed Deltaproteobacteria were present.

23

Bacteroidetes

Clones of the Bacteroidetes phylum were common in Antarctic communities.
They were among class Sphingobacteria, Flavobacteria and some unclassiﬁed
Bacteroidetes. Total of 23.3% of Antarctic clones were Bacteroidetes. Only two clones
ﬁ'om Siberian libraries were Bacteroidetes; they were Sphingobacteriales and appeared in

SO4F and S01Y libraries.

Firmicutes

Antarctic libraries contained 13.8% clones from the phylum of F irmicutes while
the other four Siberian libraries only had 1.6% of clones in this phylum. Class of Bacillii
was detected in all libraries, except library S020. The other classes were Clostridia found
in both Antarctic libraries and Siberian 8020, and class of Molicutes was found only in

Antarctic Brack pond (AB) library.

Actinobacteria

Actinobacteria was another phylum widely distributed. This phylum was seen in
ﬁve of the six libraries of this study. The Antarctic and Siberian libraries contain 15.3%
and 22.6% of Actinobacteria. All of these clones fall into the class of Actinobacteria as
well. At the order level, Actinomycetales was found in all ﬁve libraries. Acidirnicrobiales
was found in library AF and S01Y, Rubrobacterales was found in S01Y only. Two and

ﬁve unclassiﬁed Actinobacteria clones were found in library AF and S01Y, respectively.

24

Overall, Arthrobacter was the most dominant genus within Actinobacteria by clone
sequence number but was only found in sample S17M and SO4F.
Other phyla

Clones from phylum Gemmatimonadetes appeared in both libraries AF and SOlY
with one and two clones, respectively. There were seven clones from library AF that
appeared in phylum Cyanobacteria. There were twelve clones in library AB appearing in

phylum Chloroﬂexi and they were all genus Anaerolinea.

Phylogenetic tree for Psychrobacter

A bootstrap consensus phylogenetic tree was constructed that combines all
published Psychrobacter type strains plus the Psychrobacter clones from this study by
MEGA program (Fig. 8). The Psychrobacter type strains 16S rRNA gene sequences were
collected and edited by Dr. Hector Ayala-del-Rio. The construction of a tree is for better
understanding on the relationship of the clones to other Psychrobacter. Sib17-Al 1-12
represents one OTU from library 817M that contains 12 clones. This OTU is closely
related to another OTU ﬁ'om library SO4F (Sib4-A1-42). A nucleotide BLAST (Basic
Local Alignment Search Tool) search indicated that these two OTUs were closely related
to Psychrobacter maritimus and P. aquaticus. There were 74 clone sequences represented
by Sibl 7-A1-74. The BLAST result on nucleotides showed that this OTU was closely
related to Psychrobacter psychrophilus and the Sib4-C10-5 had very high possibility to
be P. glacincola. Psychrobacter arcticus 273-4, which was isolated from another

borehole in the Kolyma region, is also present in this phylogenetic tree. There was

25

another OTU that is less related to the majority and was more closely related to

    
     
     
   
    
   
  

Pseudomonas.
50 Sib4-A1-42
[:1 Sim Sib17-A11-12
61
1:] $04F Sib17—A1_74
- h'I
| : Isolates from 51 psychrop ”15
CME 54 P. maritimus

 

I: P. halophilus
9 P. submarinus

. glacialis

44

P. uratiwrans

  

P. glacincola

64 P. paciﬁcensis

P. phenylpyruvicum

 

 

 

P. arenosus

ISIb4-F1-1 I

Escherichia coli K12

 

 

 

 

0.'10 0.08 0.06 0.04 0.02 0.00

Figure 8. Phylogenetic tree shows the relationship of the Psychrobacter type strains and

Psychrobacter clones from this study.

26

CONCLUSION AND DISCUSSION

When the age of sediment was plotted versus the number of OTUs, a trend of
OTU number to the age of sediment sample is seen (Figure 9). The younger sediment
samples had a higher yield of OTUs. Another noticeable trend ﬁom this ﬁgure and
Shannon index (Fig. 2) or Chaol index (Fig. 4) is the salinity effect. While comparing
Antarctic communities, the AB community showed lower number in OTUs, lower
diversity by Shannon index and lower community richness from Chaol index. Same
trend was also apparent in Siberian communities; the S17M sample presents the lowest
OTUs, diversity and richness among other three Siberia samples.

However, the total community DNA yield does not show the same trend when
compared to the data in Table 2. This could be due to the DNA degradation over time.
After the 16S rRNA gene is degraded into fragments, the pieces can still be extracted
from soil and contribute to the DNA yield. However, these fragments would not be

ampliﬁed during PCR or would be cleaned out during the gel puriﬁcation step I used.

27

 

Trend of OTUs

I Higher salinity

 

58 SO 1Y S17M SO4F $020

,_44 A W _

 

OTU number

 

 

0.1 0.1 26 110 350 2500
Age (thousand yrs)

 

 

 

Figure 9. The relationship of OTU numbers to the age and salinity of samples.

28

One recently published paper by Tatiana A. Vishnivetskaya et a1. (2006) used
both culture-dependent and culture-independent methods for characterizing Siberian
permafrost bacterial communities. Another study done by Aviaja A. Hansen et a1.
(Hansen, Ph.D. dissertation, University of Aarhus, 2006) used media enrichment to
characterize the composition and diversity of the bacterial community in Spitsbergen soil,
a high Arctic permafrost soil. A comparison of my study to these two studies shows a
similar trend in composition of Siberia permafrost samples (Table 6).

The (+) marks indicate the presence of microbes at the taxonomic Class level. The
(l) marks represent the negative result in Tatiana’s clone study result but are positive in
the isolate result and can be assumed as false negative. Alpha-, Gamma-Proteobacteria,
Sphingobacteria, Bacilli and Actinobacteria are present in all studies. The Siberian clones
from this study covered Tatiana’s clone and isolate result at the class level, except
Flavobacteria, which was found in Antarctica samples. My clone study ageed with
Tatiana’s study and added Thermomicrobia into the list, which was also found in
Aviaja’s media-enriched clone study.

Delta-Proteobacteria and Cyanobacteria were only seen in the Antarctic Fresh
pond sediment sample. The reason for Cyanobacteria was seen in the Antarctica Fresh
pond sample could be because the Antarctica ponds have algal growth each austral
summer, which is not the case for the Siberian samples.

Over one-third of all sequences of permafrost strains from Tatiana’s study belong to a
single genus, Arthrobacter. This genus was dominant in phylum Actinobacteria from my

study (Fig. 7).

29

Overall, Antarctica samples showed higher diversity. Despite that these samples
are younger; this Antarctica habitat is a more dynamic environment due to the above zero

temperatures each austral summer.

Table 6. Comparison of microbial community composition on three similar studies of
Arctic-area permafrost plus the Antarctica pond sediments.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Arctic-area permafrost Antarctica
Phylum Class Tatiana Tatiana Aviaja Brad Brad
_ Isolate Clone Clone Clone Clone
Proteobacteria Alpha + l + + +
Beta + +
Gamma + + + + +
_ Delta +
Bacteroidetes Sphingobacteria + ! + + +
Flavobacteria + l + +
_ Unclas. Bacteroidetes + +
Firmicutes Clostridia + + + +
Mollicutes + +
Bacilli + + + + +
Actinobacteria Actinobacteria + + + + +
Gemmatimonadetes Gemmatimonadetes + +
Cyanobacteria Cyanobacteria +
Planctomycetes Planctomycetacia +
Chloroﬂexi Anaerolineae +
ﬁnucomicrobia Yerrucomicrobiae +
Deinococcus-Therrnus Deinococci +
Genera_inoertae_TM7 TM7 + +
Acidobacteria Acidobacteria +
_S_pirocheta Spirochaetes + +
Thermomicrobia Thermomicrobia + +
Unclass. Bacteria + + +

 

 

 

 

 

Label of (1) assumes existence of microbe in column: Tatiana Clone according to the

Tatiana Isolation evidence.

30

Appendix A

31

Appendix A

The Effects of Sodium Ion Concentration on the Growth of
Psychrobacter arcticus 2 73-4

INTRODUCTION

Members of the genus Psychrobacter are commonly isolated from environments
that are low in temperature or high in salinity (J uni, 2002), such as permafrost.
Psychrobacter arcticus 273-4 was isolated (Vishnivetskaya, et al., 2000) from polar
region permafrost soil of the Kolyma-Indigirka lowland (152-162°E, 68-72°N), adjacent
to the Siberian Sea collected by David Gilichinsky and team (Cryobiology Laboratory,
Russian Academy of Sciences, Pushchino). The sediment from the sampled permafrost
layer has been ﬁozen for at least 20-3 0,000 years. This environment has very low water
activity due to the concentration of solutes in water ﬁlms as a result of freezing.

Psychrobacter arcticus 273-4 is a Gram negative, non-motile, Gamma-
Proteobacteria (Bakermans et al., 2006) which grows at low temperature; 22°C ~ -10°C,
and is tolerant to long-term freeze. In order to grow, P. arcticus also requires a minimum
of 5 mM NaCl in the culture media. Understanding how P. arcticus has adapted to life in
the Siberian permafrost may lead to the discovery of traits important for life in extreme
environments.

Monica Ponder compared the expression of a series of functional genes from P.
arcticus under different conditions by microarray technology (Ponder, 2005). These

conditions include two factors: temperatures (22 °C and 4 °C) and NaCl added to half

32

strength tryptic soy broth (TSB) (Difco, Detroit, MI). The microarray analysis showed an
increased expression of the Na+ dependent symporter (ORF1600) and Na+ pumps NADH
dehydrogenases (ORF1687-ORF1691) under low temperature and high salinity condition

(Fig. 1 and Fig. 2). Images in this thesis are presented in color.

Sodium dependent symporter (ORF1600)
2.5

 

I ORF1600
expression

 

 

 

 

 

Gene expression ratio in fold

 

 

 

 

 

22°C vs. 4°C 22'C, NaCI added 4°C, NaCI addes 22°C. NaCl added
VS. VS. VS.
22°C 4°C 4°C, NaCI added
Figure l. The gene expression ratio for sodium dependent symporter (ORF1600). The

culture medium base is 1/2 TSB (from Ponder, 2005).

33

Na+ pumps NADH dehydrogenases

 

ORF1687 ORF1689 ORF1691

 

 

 

 

 

 

 

 

Gene expression ratio in fold

.4 _ I W,
I I22°0vs.4°6 7
'5 ‘ ' [122°C. NaCI added vs. 22°C
-6 a I EM'C. NaCI addes vs. 4°C ‘

 

LI 22°C. NaCI added vs. 4°C. NaCI added l

 

 

 

 

Figure 2. The gene expression ratio for Na+ pumps NADH dehydrogenases (ORF1687-
ORF1691). The culture medium base is 1/2 TSB (from Ponder, 2005).

These data suggest that P. arcticus may utilize the sodium motive force for energy
production. Hence I propose that P. arcticus will show enhanced growth ﬁom the sodium
motive force when grown under high salinity. This study tests the hypothesis from Dr.
Monica Ponder’s work that Na+ provides an energy beneﬁt to Psychrobacter arcticus

273-4.

34

MATERIALS AND METHODS
Growth and growth rate

In order to test the effects of sodium ion concentration on the growth of
Psychrobacter arcticus 273-4, a deﬁned medium developed by Peter Bergholz was
chosen to maintain stable control of the carbon source and other ingredient. The medium
contains 20 mM acetic acid (CH3COOH), 5 mM NH4C1, 1 mM KHzPO4, 1:1000 volumes
of Wolfe's Vitamins, 1:1000 volumes of trace metals, 25 mM trizma base (C4H11NO3) in
Milli-Q water.

NaCl was added to achieve four different concentrations: 10 mM, 30 mM, 100
mM and 300 mM. Under low concentration of sodium chloride, polystyrene vacuum
ﬁlter systems were used for sterilization and polystyrene tissue culture ﬂasks with vented
cap were used for incubation in order to prevent addition of sodium ion from glassware.
The cultures were incubated in a shaker at 22 °C. The optical density was recorded at 600
nm by a Cary 50 UV-Vis Spectrophotometer (VARIAN, CA, USA) for triplicate

cultures.

Total protein yield

Another experiment was conducted to determine total protein yield of
Psychrobacter arcticus 273-4 at higher sodium concentrations. The same acetate medium
with the different sodium concentrations stated above plus 1 M was used. These
concentrations did not show growth inhibition by salt, which was shown to occur at or
above 1.3 M Na+ in preliminary experiments. Triplicate bacterial cultures were harvested

for each Na+ concentration when the maximum optical density for each sodium

35

concentration was achieved. Cells were centrifuged at 6,500x g for 10 min. The
supernatant was decanted and cell pellets were washed by re—suspended in sterilized
Milli-Q water and centrifuged again. The wash step was repeated to ensure removal of
residual medium. The cell pellets were stored at -80 °C until all samples were collected.
Aliquots of the frozen cells were mixed with an equal volume of solution containing 9 M
urea, 2% 2-mercaptoethanol, 2% ampholytes (pH 8-10, BioRad), and 4% Nonidet P40 (a
nonionic detergent). Soluble, denatured proteins were recovered in the supematants after
centrifugation of the samples at 43 5,000x g for 10 min using a Beckrnan TLlOO tabletop
ultracentrifuge. A modiﬁed Bradford protein assay was used to determine the

concentration of total protein (Ramagli and Rodriguez, 1985).

36

RESULTS AND DISCUSSION

Growth and growth rate

The growth of Psychrobacter arcticus str. 273-4 under aforementioned four
different salinities was measured by optical density (Fig. 3). Detectable grth under
higher salinity occurs slightly later than for the lower salinity cultures. However, no

signiﬁcant difference in growth yield was shown in the higher sodium media.

 

Growth of P. arcticus under different salin'rty

 

 

a, +10mM
E —I— 30mM
U

(u 100mM
4’ l
“ _._ 300mM‘
O ______l
o

to

D

O

0 20 40 60 80

 

 

Time incubated (hrs)

Figure 3. ODsoo for Psychrobacter arcticus 273-4 grown in 20 mM acetate media with

different sodium concentration

37

The growth rate for each different sodium concentration was calculated from the
exponential phase of growth (Fig. 4). The results showed no consistent trend although the
growth rate increased at the sodium concentration. However, the differences among these
growth rates were not signiﬁcant. Therefore, this experiment did not show a signiﬁcant

evidence to support that sodium can beneﬁt the growth of Psychrobacter arcticus 273—4.

 

Growth rate

Generation / h

 

10mM 30mM 100mM 300mM

NaCI concentration

 

 

 

Figure 4. The growth rates (generation/h) for four different sodium concentrations of 20

mM acetate media.

38

Total protein yield

Preliminary experiments were undertaken to determine the acetate concentration
that would provide a maximum OD600 of 0.6 to 0.8, to more sensitively measure the

effect of sodium to the total protein yield (Table 1).

Table 1. The maximum OD600 reading for various concentrations acetate in the media

with 30 mM NaCl.

 

Acetate media concentration (with 30 mM NaCl)

 

8mM 10mM 11mM 12mM 14mM

 

 

 

ODeoo 0.54 0.61 0.66 0.75 0.87

 

 

39

The 11 mM concentration of acetate was selected on the best ﬁt for the range of
cell yields expected for the total protein experiment. The results for protein yield are

presented together with ﬁnal bacterial culture maximum densities (Fig. 5).

 

Total protein assay

60.0 0.90 A
9 ii“ 0.80 °
E . , O
550-0 .. 70.70 g
340.0 0.60 3
-§ 300 1 0.50 2
§ ' 0.40 :-
£3200 ‘ 0.30 §
.. 0.20 «I
,2 10.0 0.10 E

0.0 0.00

 

1 0 30 1 00 300 1 000

NaCl concentration (mM)

 

 

 

Figure 5. The total protein yields for different sodium concentrations in 11 mM acetate
medium. The bars represent total protein concentration (pg/ml) and the blue dots

represent the ﬁnal (optimal) optical density for each different sample.

40

The protein yield can be present as protein concentration per cell optical density.

The values show a clear trend in that higher sodium concentration yields higher total

protein

(Figure 6).

 

 

Protein yield

 

Protein concentration I ODm

1 0 3O 1 00 300 1 000

NaCI concentration (mM)

 

 

Figure

6. The protein yield presented by relative turbidity (ODaoo). The values are shown
on the top of the bars. The media used are 11 mM acetate media with the

indicated NaCl concentrations.

41

In conclusion, the growth rate or the ﬁnal optical density results were not
consistent enough to answer the question whether sodium directly beneﬁts
Psychrobacter ’5 energy for growth. This could be due to the fact that Psychrobacter
arcticus 273-4 has been selected in high salinity environment and thus sodium ion
concentration does not show a strong effect on P. arcticus’ growth. The total-protein
yield result is signiﬁcant. However, the ﬁnal optical density did not support this
conclusion. A reasonable interpretation is that Psychrobacter arcticus 273-4 may produce
more protein but not cell turbidities, to help it survive under high salinity environments.
Nevertheless, more other evidences are needed to better characterize the effect of sodium

ion concentration to the growth of Psychrobacter arcticus 273-4.

42

10.

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44

   

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