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The genome sequence of Psychrobacter arcticus 273-4, a psychroactive Siberian permafrost 1

bacterium reveals mechanisms for adaptation to low temperature growth. 2

3

4

Running title: Psychrobacter arcticus genome sequence 5

6

7

Héctor L. Ayala-del-Río1,2*

, Patrick S. Chain3,5

, Joseph J. Grzymski4, Monica A. Ponder

1, 8

Natalia Ivanova5, Peter W. Bergholz

1, Genevive Di Bartolo

5, Loren Hauser

6, Miriam Land

6, 9

Corien Bakermans1, Debora Rodrigues

1, Joel Klappenbach

1, Dan Zarka

1, Frank Larimer

6, Paul 10

Richardson5, Alison Murray

4, Michael Thomashow

1 and James M. Tiedje

1 11

12

13

Center for Microbial Ecology, Michigan State University1, Department of Biology, University of 14

Puerto Rico at Humacao2, Lawrence Livermore National Laboratory

3, Desert Research Institute, 15

Reno Nevada4, Joint Genome Institute

5, Genome Analysis and Systems Modeling, Life Sciences 16

Division, Oak Ridge National Laboratory6, 17

Michigan State University, East Lansing, Michigan, 48824-1325. 18

19

20

21

22 *Corresponding author. Present address: University of Puerto Rico at Humacao, Department of 23

Biology, 100 – 908th

RD, CUH postal station, Humacao, PR 00791. Phone: (787)-850-9388. 24

Fax: 787-850-9439. E-mail: [email protected] 25

26

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.02101-09 AEM Accepts, published online ahead of print on 12 February 2010

on January 8, 2019 by guesthttp://aem

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Abstract 27

Psychrobacter arcticus strain 273-4, which grows at temperatures as low as -10º C, was 28

the first cold adapted bacterium from a terrestrial environment whose genome has been 29

sequenced. Analysis of the 2.65MB genome suggests that some of the strategies employed by P. 30

arcticus 273-4 for cold and stress survival include changes in membrane composition, synthesis 31

of cold shock proteins and accumulation of poly-phosphate as an energy source. Comparative 32

genome analysis indicates that a significant portion of the P. arcticus proteome shows a 33

reduction in the use of the acidic amino acids and proline and arginine, which is consistent with 34

increased protein flexibility at low temperatures. Differential amino acid usage was spread 35

across all gene categories, but it was favored in gene categories essential for cell growth and 36

reproduction suggesting that P. arcticus has evolved to grow at low temperatures. The 37

combination of amino acid adaptations and gene content likely evolved in response to the long-38

term freezing temperatures (-10 to -12º C) of the Kolyma (Siberia) permafrost soil from where 39

this strain was isolated. Intracellular water likely does not freeze at this in situ temperature, 40

providing the means for P. arcticus to live at subzero temperatures. 41

42

43

44

45


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Introduction 46

Temperature is one of the most important parameters that determines the distribution and 47

extent of life on earth by affecting cell structure and function. High temperatures break covalent 48

bonds and ionic interactions between molecules, inactivating proteins and disrupting cell 49

structures. Low temperatures reduce biochemical reaction rates, substrate transport, and induce 50

the formation of ice that damages cell structures. Not surprisingly, an organism’s compatibility 51

with habitat temperature is ultimately determined by its underlying genetic architecture. 52

The strong emphasis on mesophile biology, in the 20-37ºC range, gives us a 53

misimpression of the importance of cold on earth. However, 70% of the Earth’s surface is 54

covered by oceans with average temperatures between 1 to 5ºC (11), 20% of Earth’s terrestrial 55

surface is permafrost (47), and a still larger portion undergoes seasonal freezing, making our 56

planet a predominantly cold environment. Hence cold adaptation in the microbial world is to be 57

expected (55). 58

Permafrost is defined as soils or sediments continuously exposed to a temperature of 0º C 59

or below for at least 2 years (44). Permafrost temperatures range from -10º to -20º C in the 60

Arctic to -10º to -65º C in the Antarctic, have low water activity, often low amounts of carbon 61

(0.85-1%) and prolonged exposure to damaging gamma radiation from 40

K in soil minerals (49). 62

Liquid water exists as a very thin, salty layer surrounding the soil particles within the frozen 63

layer. Despite the challenges of the permafrost a variety of microorganisms successfully 64

colonize this environment, and many have been isolated (54, 70). Bacterial taxa most frequently 65

isolated from the Kolyma permafrost of northeast Siberia, include Arthrobacter, 66

Exiguobacterium, Flavobacterium, Sphingomonas, and Psychrobacter (71). Rhode and Price 67

(56) proposed that microorganisms can survive in frozen ice for very long periods due to the very 68


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small films of water surrounding the cell that serves as a reserve of substrates. Permafrost is a 69

more favorable environment than ice as a result of heterogeneous soil particles and larger 70

reservoirs of nutrients. 71

The genus Psychrobacter comprises a group of Gram-negative, rod shaped, heterotrophic 72

bacteria with many species capable of growth at low temperatures. Members of this genus can 73

grow at temperatures between -10º to 42º C and they have been frequently isolated from various 74

cold environments including: Antarctic sea ice, ornithogenic soil and sediments, stomach content 75

of Antarctic krill (Euphausia), deep sea water and permafrost (9, 36, 57, 70, 71, 76) 76

(http://www.bacterio.cict.fr/p/psychrobacter.html). Psychrobacter arcticus 273-4 is a recently 77

described species (4) that was isolated from a 20,000-30,000 year continuously frozen 78

permafrost horizon from the Kolyma region in Siberia that was not exposed to temperatures 79

higher than 4ºC during isolation (70). This strain, the type strain of the species, grows from -10º 80

to 28º C, has a generation time of 3.5 days at -2.5ºC, possesses excellent long-term freeze 81

survival and exhibits temperature dependent physiological modifications in membrane 82

composition and carbon source utilization (50). The fact that Psychrobacter has been found to 83

be an indicator genus for permafrost and other polar environments (66) suggests that many of its 84

members are adapted to low temperature and increased osmotica, and have evolved molecular 85

level changes to aid its low temperature survival. 86

Early studies on cold adaptation in microorganisms revealed physiological strategies to 87

deal with low temperatures such as changes in membrane saturation, accumulation of compatible 88

solutes, cold shock proteins (CSPs), and many other proteins of general function (62). However, 89

many of the studies were conducted with mesophilic microorganisms, which limits the generality 90

of the conclusions. We have re-addressed the question of cold adaptation by studying 91


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microorganisms isolated from subzero environments using physiologic and genomic methods. 92

We chose P. arcticus as our model because of its subzero growth and widespread prevalence in 93

permafrost. This paper focuses on the more novel of the potential adaptations. 94

95

MATERIALS AND METHODS 96

Cell preparation and genome sequencing. The genome of Psychrobacter arcticus 273-4 97

(ATCC BAA1226) was sequenced by the Joint Genome Institute (JGI, Walnut Creek, California) 98

using their standard shotgun method and Sanger sequencing (13). Coding sequences (CDS) were 99

identified by combining the results from Critica (3) and Glimmer (17) gene modelers using the 100

Oakridge National Laboratory Genome Analysis Pipeline. CDS identification was confirmed 101

and polished by comparison of amino acid translations against GenBank’s non-redundant 102

database using the basic local alignment search tool for proteins (BLASTP) (2) and manual 103

identification of ribosomal binding sites using Artemis (60). Genes encoding tRNAs were 104

identified with tRNAScanSE tool (34), while 16S and 23S ribosomal RNAs were identified by 105

comparing genome fragments against a rRNA database using the BLASTN tool. Structural 106

RNAs (e.g. 5S rRNA, rnpB, tmRNA, SRP RNA) were identified using the Infernal search tool 107

(18). 108

Functional assignment of each CDS was made manually by the authors comparing each 109

CDS against KEGG (74), InterPro (77),

TIGRFams (24), PFams (65), and Clusters of 110

Orthologous Groups of Proteins (COGs) (67) databases; and using a hierarchy system that 111

considered database ranking, sequence identity, and alignment quality (available upon request). 112

Finally, the annotation was polished by the Integrated Microbial Genomes annotation group 113

(http://img.jgi.doe.gov/cgi-bin/pub/main.cgi). 114


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Amino acid usage analysis. Cold adaptation at the amino acid level was examined by 115

comparisons against the SwissProt database using several cold adaptation indicators (23). 116

Briefly, P. arcticus amino acid sequences were searched against the SwissProt database (7) using 117

BLAST, and the five most similar protein sequences with an expect value < 10-15

were extracted 118

from the database. SwissProt was used for this analysis because it is a manually curated 119

database with high quality annotations. Cold adaptation was measured in each P. arcticus amino 120

acid sequence and the corresponding SwissProt proteins using the following parameters known 121

to contribute to cold adaptation (55, 58): GRAVY (grand average of hydropathicity) and 122

aliphaticity (that were calculated using the ExPASy website http://us.expasy.org); proline 123

content; acidic residues content; and the Arg/Lys ratio were calculated using in-house PERL 124

scripts. A one-sample t test was used to evaluate if there were statistically significant differences 125

between the P. arcticus 273-4 amino acid sequence queried and the five most similar proteins of 126

the SwissProt database for any given parameter. Genes that showed significant differences were 127

classified as cold or hot adapted depending on the direction of the change. 128

To determine if there was enrichment of cold adapted genes in the P. arcticus genome a 129

ratio (total genes cold adapted / total gene hot adapted) was calculated for each cold adaptation 130

descriptor. A chi-squared analysis was used to determine if there was a significant difference 131

between the number of cold and hot adapted genes in the P. arcticus genome using R version 132

2.3.1 (25). Cluster analysis using Euclidian distance and average linkage was performed on the 133

significance data using the Cluster 3.0 (16) and were visualized using Java Treeview (61). 134

Repeat analysis. RepeatFinder was used to identify and classify repeated sequences of length > 135

25 bp (33, 72). MultiFASTA output from RepeatFinder.pl was BLASTed against the P. arcticus 136

273-4 genome sequence using blastall 2.2.5 with word size 7 and low complexity sequence 137


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filters turned off. Repeated sequences were identified in the genome by having alignments with 138

more than 94% nucleotide sequence identity over more than 85% of the length of the query 139

sequence. Hits by multiple repeat sequences to the same base pair coordinates in a genome were 140

resolved by deleting the shorter repeat. If a pair of repeat loci overlapped by more than 25% of 141

the length of either member of that pair, then the repeats were merged into a single sequence. 142

The most abundant repeat class, class 1, was subclassified by multiple alignment using ClustalW 143

1.81 (68). Secondary structure of the class 1 repeats was predicted using MFOLD 3.1.2 (79). 144

Predictions were carried out assuming a temperature of 22°C and all other parameters were the 145

default. Code is available upon request from P.B. 146

Correspondence Analysis and Model-Based Clustering: The FactoMineR R package was 147

used to perform Correspondence Analysis (CA) of amino acid frequencies to identify what 148

factors determine amino acid usage (53). After selecting protein sequences with more than 100 149

residues, the first 10 and last 5 residues were removed and normalized amino acid frequencies 150

were generated using a custom PERL script. Model based clustering, as in Riley et al. (53), was 151

performed on the scores from the first six dimensions from the CA using the Mclust R package. 152

Development of a defined medium. Using the genome sequence information a basal defined 153

medium was developed for growth of P. arcticus that would also be suitable for transcriptome 154

experiments. The medium contained: 20 mM D,L-lactic acid or 100 mM sodium pyruvate, 5 155

mM glutamate or 5 mM NH4Cl, 1 mM K2HPO4, 1X Wolin’s vitamins (75), MOPS, and 1X trace 156

minerals (31). Buffers tested to support growth include: HEPES, PIPES, MOPSO, MOPS and 1 157

and 2 mM K2HPO4 buffer, all at pH 7.0. P. arcticus was cultured serially in marine broth 158

containing 5% sea salts and then in the basal defined medium containing 20 mM lactate. A 1% 159

inoculum was used to inoculate the basal defined medium containing the carbon source to be 160


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tested, and growth was measured by OD600. Growth rates were estimated from the data in the 161

OD600 range from 0.03-0.3. Cultures were considered negative for growth on a substrate if no 162

growth was observed after 5 days at 22° C or 10 days at 4°C. 163

Nucleotide sequence accession number. The complete genome sequence of P. arcticus 273-4 164

is available under GenBank accession number CP000082. 165

166

RESULTS AND DISCUSSION 167

Genome summary. P. arcticus 273-4 contains a single replication unit of 2,650,701 bp. which 168

encodes 2,147 putative proteins (Supplementary materials Table 1). Genes were evenly 169

distributed between forward (53.5%) and reverse (46.5%) strands, with an average CDS length 170

of 994 bp. Eighty-two percent of the coding bases account for putative proteins. Ribosomal 171

RNA operons were found in the positive [1] and negative [3] strands. The G+C content was 172

stable across the genome with an average ratio of 42.8% (Fig. 1). An unusually high G+C 173

content was detected in a 20,147 bp region between 1,959,325 and 1,979,472 bp. with an average 174

ratio of 56.9%. This region encodes a putative protein of 6,715 amino acids with possible 175

membrane function. The genome is divided in two symmetric replichores (Fig. 1) as indicated 176

by GC skew analysis. The GC skew was weakly correlated with the G+C spikes present. 177

Mobile elements (e.g. phage, transposons and insertion sequences) could be responsible for the 178

changes in G+C content and GC skew since some of them lie in the vicinity where spikes 179

occurred. 180

The genome has 48 transposon/IS elements and 25 phage related genes. The majority of 181

the IS elements classify into one of four distinct major families: IS3 [27], IS4 [10], IS30 [4], and 182

IS mutator [3]. Although many of them seem to be truncated, based on small ORF sizes (35), 183


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they could still be active (45). IS elements seem to be randomly distributed in the genome (Fig. 184

1) with the exception of the IS3 family elements that were found in pairs from 50bp to 12Kb 185

apart. The close proximity of the IS3 elements could be indicative of arrival via a transposition 186

event or that they are still active contributing to genome rearrangements. 187

There are two almost complete phage genomes in P. arcticus genome. The first is a 33.3 188

Kb lambda-like phage located between 551,715 and 585,095 bp, and the second is most similar 189

to a 45.7 Kb MU phage and is located between 1,177,957 and 1,223,710 bp. Both phage regions 190

contain many, but not all, phage proteins needed for replication. A total of 57 hypothetical 191

proteins were present in the phage regions accounting for 17% of all hypothetical proteins. 192

Within the phage MU region a possible neutral zinc metallopeptidase (GI:71065546), that is 193

most similar to a hypothetical protein from Haemophilus influenzae, was identified, indicating 194

that phage have likely helped shape the current genome structure by the addition of genes so far 195

unique to P. arcticus. Although there were indications that gene transfer occurred, due to IS 196

elements and phage genes, the GC skew data indicates that no major genome rearrangements or 197

horizontal movement of genes have occurred (22). This finding is surprising since the 198

Psychrobacter genus is characterized as naturally transformable (28). The lack of horizontal 199

transfer and gene rearrangements could be indicative of the strong selective pressure exerted by 200

the cold niche early in the evolution of this genome resulting in the current structure. 201

Correspondence analysis (CA) revealed differences in the amino acid frequencies in the 202

P. arcticus genome. P. arcticus proteins group in two clusters along the first dimension 203

(Supplementary material Fig. 1). An inertia analysis of the amino acids, i.e. their relative 204

contribution in the CA, indicates that hydrophobic (F, L and W) and charged (E, D and K) 205

residues are important to generate cluster A and B, respectively (Supplementary material Table 206


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2). Pascal and coworkers observed the same amino acid distributions also causing two similar 207

clusters when model genomes were analyzed using CA (46). They concluded that 208

hydrophobicity is a discriminant factor for protein location, since all proteins in the cluster 209

equivalent to cluster A were integral inner membrane proteins. The separation is likely due to 210

the higher abundance of hydrophobic residues. Interestingly, in Psychromonas ingrahamii 37 211

the proteins derived form the genome sequence did not form two clusters possibly because of a 212

lower hydrophobic character (53). No well-defined clusters were formed along the second and 213

third dimensions of the P. arcticus CA. The inertia of those two axes indicates that aromatic 214

residues and glutamine, respectively, influence the protein distribution along those two 215

dimensions. Different from Psychromonas ingrahamii, aspargine did not occur as frequently in 216

the third dimension suggesting that differences in the habitat of the two microorganisms, 217

terrestrial vs marine, could affect amino acid preferences. 218

Riley and coworkers (53) analyzed the CA results of Psychromonas ingrahamii using 219

Model-Based Clustering (MBC), and found six classes of proteins. MBC of P. arcticus CA 220

results classified proteins in four clusters, similar to many microorganisms (Supplementary 221

material Fig. 2). Different from Psychromonas ingrahamii, in which the bulk of the proteins 222

formed three clusters, in P. arcticus 60 percent of the proteins were in cluster 2, followed by 223

cluster 4 with 19.6% (Supplementary materials Table 3). Representatives of all COG categories 224

were present in both cluster 2 and 4, but were highly abundant in Cluster 2 except for transport 225

proteins that where more abundant in cluster 3. Cluster A proteins in CA correspond to cluster 3 226

proteins in MBC, supporting the higher number of transport proteins present in cluster 3. The 227

good separation of proteins in cluster 3, different from Psychromonas ingrahamii, is indicative 228

of distinct frequencies of hydrophobic residues in that cluster compared with entire proteome in 229


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P. arcticus. Finally, cluster 1 groups most transposons and a moderate number of hypothetical 230

and conserved hypothetical proteins. The number of clusters, and their composition indicate 231

differences in amino acid frequencies between P. arcticus and Psychromonas ingrahamii 232

although both of them are cold-adapted microorganisms. 233

234

Known adaptations to cold. Based on past work on cold adaptation we expect membrane 235

modifications, compatible solute accumulation and cold shock proteins to be features present in 236

the P. arcticus genome. Lipid analyses of P.arcticus grown at warm and cold temperatures 237

revealed that P. arcticus decreases the acyl chain length and saturation of the membrane fatty 238

acids at low temperatures (50). P. arcticus genome sequence revealed genes for the synthesis of 239

saturated and unsaturated fatty acids; and genes for the control of the acyl chain length. 240

Unsaturated fatty acids can be synthesized either de novo or via a fatty acid desaturase, 241

suggesting that fatty acid unsaturation is an important characteristic for P. arcticus to grow at 242

low temperatures. Colwellia psychrerythraea 34H, a psychrophile from the marine environment, 243

possesses a similar strategy by employing multiple polyunsaturated fatty acid synthases (40). 244

Redundancy of pathways ensures that an essential phenotypic trait gets expressed when needed, 245

as expected in low-nutrient and/or harsh environments like soil (12). Low temperatures also 246

increase the accumulation of compatible solutes such as proline, glutamate and glycine betaine in 247

P. arcticus (48). Transcriptomic analysis of the P. arcticus genome revealed that genes related 248

to transport and synthesis of compatible solutes were up-regulated at low temperatures in the 249

presence of salt (48). Since P. arcticus comes from a low temperature and low water activity 250

soil environment compatible solute accumulation likely represents an important adaptation to life 251

in the permafrost niche. Finally, cold shock genes cspE, cspA, capB and unnamed cold shock 252


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binding domain protein were identified in P. arcticus genome. Transcriptomic analysis of cspA 253

revealed that it is constitutively expressed regardless of the temperature (48). Hence, cspA 254

appears to be a required element for protein synthesis in P. arcticus. 255

256

Distinctive features. We performed metabolic reconstruction of the central carbon metabolism 257

to better understand the organism’s potential metabolism. Although P. arcticus grows on 258

complex media, Psychrobacter sp. in general don’t grow on carbohydrates (8). Metabolic 259

reconstruction revealed that P. arcticus lacks the genes for any of the known versions of 260

glycolysis (see Supplementary Discussion), and PTS-type sugar transporters, suggesting that P. 261

arcticus is unable to utilize sugars. However, both “committed” gluconeogenic enzymes, 262

fructose-1,6-bisphosphatase and phosphoenolpyruvate synthase, are present in P. arcticus 263

(Supplementary material Fig. 3), which indicates that oxidized substrates such as acetate, malate, 264

oxaloacetate and other mono- and dicarboxylic acids could be the preferred carbon sources. 265

A defined medium was developed to test hypotheses that P. arcticus should prefer 266

oxidized substrates instead of sugars. Acetate supported more than two-fold faster growth than 267

lactate (Table 1). No growth was observed on glucose, isovalerate, isobutyrate, glycerol, malate, 268

succinate, citrate, glycolate, glyoxylate, serine, glycine or propionate at 22°C or 4°C after 269

repeated attempts using inocula grown on acetate, lactate and marine broth (Table 1). 270

Transporters are present for the dicarboxylates (TRAP-T dicarboxylate uptake transporter GIs: 271

71066208-71066208), glycolate (glycolate:H+ symporter GI: 71066208), and glycine (BCCT 272

uptake transporter GI:71065858). However, it is possible that culture conditions were unable to 273

induce use of these substrates for growth, or that unpredicted aspects of the metabolism 274

prevented growth on these substrates despite the presence of the complete gene compliment. 275


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Growth on palmitic acid was only observed at 22°C for unknown reasons, though could be due 276

to a combination of reduced water solubility of the compound at low temperatures and reduced 277

uptake of the compound due to decreased membrane fluidity at 4°C. 278

P. arcticus may be optimized to conserve energy by utilizing acetate, a substrate expected 279

during the waterlogged conditions of the tundra summer, as the basis for its biosynthesis and 280

energy metabolism. Acetate yielded the highest growth rate and yield of all carbon sources 281

tested, which was 65% of the rate observed in the complex, rich marine broth with 5% sea salts. 282

Supplementation of the medium with 5 mM glutamate yielded only a slight increase in growth 283

rate over medium containing acetate or lactate alone. Acetate metabolism has several features 284

that may be desirable at low temperatures. Uptake of acetate occurs through the cytoplasmic 285

membrane with no transporter required, although transporters may assist (5). Utilization of the 286

glyoxylate shunt can allow P. arcticus to conserve all of the carbon from this substrate. 287

Metabolism of acetate requires only one to two enzymes to generate acetyl-CoA from acetate 288

and HS-CoA. 289

A less known but likely important factor in cold adaptation is the synthesis of wax esters. 290

Wax esters are commonly found in plants and animals (30). Acinetobacter sp. accumulates large 291

amounts of wax esters to be used later as a carbon source for growth (26, 29, 52). P. arcticus 292

genome has a wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (GI: 71064803). In 293

Psychrobacter urativorans (formerly known as Micrococcus cryophilus) wax esters represent 294

14% of the total lipid content and were found associated with the cell membrane (59). 295

Furthermore, a decrease in growth temperature from 20 to 1ºC resulted in a significant increase 296

in unsaturation, and a decrease in average chain length of the wax associated fatty acids 297

suggesting that they are important for low temperature growth (59). The wax ester synthase of 298


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P. arcticus shares 52% amino acid identity with the wax synthase of Acinetobacter ADP1 and 299

our transcriptomic studies show that the gene is expressed constitutively regardless of growth 300

temperature (48). It may play a role in membrane fluidity in P. arcticus. 301

The production of extracellular polymeric substances (EPS) is seen in many prokaryotic 302

species. Typically EPS is found either closely associated with the cell wall, forming a capsule, 303

or loosely attached, forming slime (73). Although there are many possible functions for EPS one 304

general trend is that aids cell survival by forming biofilms, retaining water (especially important 305

in a frozen soil) and serve as a cryoprotectant (27). Similar to psychrohiles Colwellia 306

psychroerythraea 34H (40), and Psychromonas ingrahamii 37 (53), P. arcticus posses genes for 307

the production of capsular type EPS (GI: 71065213, 71065215, 71065219, and 71065223). P. 308

arcticus formed a capsule in the presence of salt (48), suggesting that this could be an adaptation 309

to grow in the permafrost environment. 310

P. arcticus has a highly repetitive genome containing four superfamilies of dispersed 311

repeat loci of length > 25 bp: the long tandem repeat constituent sequences (total length ~16.7 312

kb), transposon and insertion sequence (IS) repetitive elements (1.86 % of repeated sequences), 313

repeats internal to non-mobile genes (3 genes), and AT-rich intergenic repeated loci. The 314

Shannon-Weaver index and evenness estimator, that were calculated to test for dominance of 315

particular repeat sequences, were 0.79 and 0.11 respectively, indicating that the repeat sequence 316

assemblage was dominated by one or a few sequences. 317

A single AT-rich intergenic repeat sequence of 69-149 bp, the class 1 repeats, accounted 318

for 305 of 336 AT-rich repeat loci or 1.92% of the genome sequence. Class 1 sequences were 319

characterized by inverted terminal repeats that were at least 34 bp long and ending in TA 320

dinucleotide repeats at both ends. Class 1 repeats were predicted to form secondary structure at 321


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22ºC (Supplementary material Fig. 4) by MFOLD 3.1.2 (37, 79). The 6-bp terminal end 322

sequence, TATAGT, and the predicted hairpin loop structures of the class 1 repeat are similar to 323

nemis elements of pathogenic Neiserria spp. (10, 14, 15, 38). Nemis elements of Neiserria are 324

thought to participate in recombination and are known to bind to the integration host factor (IHF) 325

and to be cleaved by RNase III where transcribed (10, 15). Predicted secondary structure of the 326

nemis and the class 1 repeat elements are similar, suggesting that class 1 P. arcticus repeats may 327

exhibit the above activities. It has long been known that salt stress, growth temperature and 328

temperature shock strongly effect DNA topology (21, 32, 42, 43). Hence, the dominant repeats 329

in P. arcticus 273-4 may associate with DNA binding proteins to alter the temperature and salt 330

effect on the topology of the chromosome in yet unknown ways. 331

332

Traits with evidence of cold adaptation. Comparisons of P. arcticus 273-4 proteome 333

substitutions against the Swiss-Prot database revealed strategies used to cope with the limitations 334

of low temperature. Between 476 (38%) and 1074 (84%) of P. arcticus amino acid sequences 335

showed a statistically significant difference when compared against the SwissProt database for 336

all cold adaptation indicators, i.e. were less hydrophobic; or had less proline residues; or were 337

less aliphatic; or had less acidic residues; or had low Arg and increased Lys (Fig. 2). Ratios 338

(total cold adapted genes/total hot adapted genes) calculated for hydrophobicity, proline, acidic 339

residues, and Arg/Lys were 1.35, 1.69, 3.0 and 1.42 respectively, indicating a higher amount of 340

cold adapted genes in the P. arcticus genome. Aliphacity was the only indicator with a ratio 341

below one (0.78). A total of 1,212 genes (56% of the genome) had at least one adaptation and 342

the average was three cold adaptive qualities per gene. Different from other studies (1, 39) that 343

used proportional changes, we evaluated cold adaptation by measuring what substitutions are 344


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more likely in cold adapted microbes compared to mesophilic microorganisms. Measurement of 345

amino acid substitutions proved to be a very sensitive strategy, compared to the examination of 346

proportional changes in amino acid composition, since four out of the five indicators used 347

suggest that the P. arcticus proteome is cold adapted. Metpally and Reddy (2009) (41) 348

compared amino acid substitution patterns across psychrophilic, and mesophilic genomes, with 349

the former including Psychrobacter cryohalolentis K5, Colwellia psychroerythraea 34H, and 350

Psychromonas ingrahamii 37. They concluded that psychrophile proteins contained less 351

hydrophilic, acidic, and proline residues, consistent with our findings (41, 69). 352

To examine what specific groups of genes show signs of cold adaptation, all the genes 353

from the previous analysis were separated according to COG categories. A chi-squared analysis 354

of the number of cold and hot adapted genes (Supplementary material table 4), and cluster 355

analysis of the results revealed that acidic residues (14 categories) and proline (10 categories) are 356

the two most frequent cold adaptation indicators across all COG categories (Fig. 3) 357

(Supplementary material table 5). The COG categories with a higher number of adaptations 358

include: No COG designation; replication, recombination and repair; amino acid transport and 359

metabolism; lipid transport and metabolism; transcription; translation, ribosomal structure and 360

biogenesis; and signal transduction mechanisms (Fig. 3). 361

One of the biggest challenges for proteins at low temperatures is to have sufficient 362

flexibility to increase their interactions with substrate reducing the required activation energy. 363

Abundance of proline residues has been related to increased protein stability due to the rigid 364

nature of the N-Cα bond (19, 51). Hence, the decrease in the amount of proline suggests cold 365

adaptation, supporting a trend seen in smaller scale studies (69). Arginine is also considered a 366

structurally stabilizing residue by forming salt-bridges and hydrogen bonds with side chains (1). 367


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Substituting arginine by lysine has been proposed as a more flexible substitution. The negatively 368

charged (acidic) residues, glutamic acid and aspartic acid, favor salt-bridge formation on protein 369

surfaces, thus favoring a stable protein structure (23). Removal of acidic residues will increase 370

protein flexibility (20). Finally, an increase in hydrophobicity of core amino acids increases 371

protein stability at higher temperatures (64), while an overall reduction has been observed in 372

cold-active enzymes (63). Our results suggest that P. arcticus adaptation to low temperatures 373

involves multiple amino acid substitutions that lower protein stability presumably yielding 374

enzymes more active at low temperatures. The only parameter not consistent with this 375

conclusion was aliphacity since it showed an opposite response to cold. This inconsistent result, 376

which has been previously observed could be due to the analysis strategy that did not separate 377

exposed versus buried residues (23, 41, 69). In support of this, Metpally and Reddy (2009) (41) 378

recently showed a high frequency of aliphatic residues in coil/loop regions of psychrophilic 379

genomes. 380

To further validate our cold adaptation indicators we analyzed genes that were 381

upregulated at 4ºC as measured by 2D liquid (proteome) mapping (78). A total of 11 out of 14 382

proteins with at least two-fold greater expression when cells were grown at 4ºC versus 22ºC 383

show at least two adaptations and an average of 3.6 cold adaptations per gene. COG categories 384

related to essential processes such as transcription, translation, repair, amino acid metabolism, 385

and lipid metabolism had a higher number of adaptations consistent with the importance of these 386

processes for life in the cold. Considering that microbial metabolism in permafrost is controlled 387

by substrate diffusion from a thin layer of water around the cell (56) it seems logical that genes 388

essential for survival would show several cold adaptation strategies. Overall, the results suggest 389


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that increased protein flexibility, by using amino acids that promote structural instability, is a 390

major adaptation employed by P. arcticus to maintain activity at low temperatures. 391

392

Conclusions. Psychrobacter arcticus 273-4 is a Siberian permafrost psychroactive bacterium 393

capable of growth at -10º C. Members of this genus have been frequently isolated from cold 394

environments and recent work on the genus by quantitative PCR indicates that its distribution 395

and density is strongly skewed to cold environments, with its densities highest in Antarctic 396

sediments (55). To survive the continuous gamma radiation emitted by soil particles it is 397

necessary to invest ATP in repair. P. arcticus prefers acetate, a simple ubiquitous substrate, as 398

the basis for its biosynthesis and energy metabolism. Acetate easily diffuses into the cell without 399

the need for costly transport systems. Low temperatures in the permafrost make mRNAs more 400

stable and less efficient for translation. P. arcticus possess three CSPs, RNA chaperones that 401

enhance translation processes by avoiding the formation of secondary structures in the mRNA. 402

Multiple pathways to increase the unsaturation of membrane lipids, and increase acyl chain 403

length maintain its membrane fluidity. P. arcticus compensates for the effects of low 404

temperatures on enzyme activity by structural modifications that increase flexibility of at least 405

50% of its proteome, thus reducing the energetic requirements for activity. Transcriptome 406

analysis following growth at subzero temperatures detected increased expression for fatty acid 407

unsaturation, growth rate control mechanisms and isozyme exchange, with more a structurally 408

flexible DEAD-box RNA helicase and D-alanyl-D-alanine carboxypeptidase upregulated at cold 409

temperatures (6). The low nutrients, low water activity and low temperatures characteristic of 410

the permafrost environment should favor microorganisms with multiple, low cost coping 411

strategies. The combination of those above and probably more, especially the many cold-412


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induced genes of unknown function, appears to allow P. arcticus to live long lives at subzero 413

temperatures. 414

415

Acknowledgements 416

This work was supported by the NASA Astrobiology Institute cooperative agreement NCC2-417

1274 and the DOE’s Joint Genome Institute. We thank Iván Dávila Marcano for statistical 418

advice; and Ting Zhang Wang and Antoine Danching for help with the Correspondence Analysis 419

and Model-Based Clustering. 420


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647


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Figure captions 1

Fig 1: Circular representation of the Psychrobacter arcticus 273-4 genome. The outer two rings 2

(1 and 2) represent the genes in forward and reverse strands, respectively, colored by function 3

categories. The next two rings (3 and 4) represent the Class 1 repeat element in the forward and 4

reverse strand respectively. The next two circles (5 and 6) represent transposons/IS elements 5

(green) and prophage (red) in the forward and reverse strands respectively. Ring 7, G+C content, 6

ring 8, GC skew. 7

8

Fig 2: Cold adaptation. Upper graph, cold adaptation ratios were calculated using all the genes 9

with a statistically significant difference against the Swiss-Prot database in favor (cold adapted) 10

or against (hot adapted). A ratio of one indicates an equal proportion of genes in the two 11

categories. Lower graph, total number of genes used to generate the ratio. 12

13

Fig 3: Distribution of cold adaptation qualities across COG categories. Statistical differences 14

between the amount of genes with cold and hot qualities in each COG category were determined 15

using the chi-squared test. Results were ranked in a scale from 0-3, where a p-value < 0.001 =3, 16

a p-value <0.05 =2, a p-value <0.10=1, and a p-value >0.10 =0. Enrichment of cold adapted 17

qualities received a positive sign and enrichment of hot adapted qualities received a negative 18

sign. Rankings were used for cluster analysis of the COG categories. 19

20

Table 1; P. articus 273-4 growth response to diverse 21

growth substrates 22

23


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0100,000

200,000

300,000

400,000

500,000

600,000

700,000

800,000

900,000

1,000,000

1,100,000

1,200,000

1,300,0001,400,000

1,500,000

1,600,000

1,700,000

1,800,000

1,900,000

2,000,000

2,100,000

2,200,000

2,300,000

2,400,000

2,500,000

2,600,000

Fig. 1


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Cold adaptation ratio

Cold adapted genes

Hot adapted genes

Total P. arcticus

Genes tested

200

400

600

800

1000

1200

Hydrophobicity

Proline

Aliphacity

Acidic Residues

Arg/Lys

0

0.5 1

1.5 2

2.5 3

3.5

Fig

. 2

( )


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s

No Cog designation

[L] Replication, recombination and repair

[E] Amino acid transport and metabolism

[I] Lipid transport and metabolism

[K] Transcription

[J] Translation, ribosomal structure and biogenesis

[M] Cell wall/membrane/envelope biogenesis

[S] Function unknown

[G] Carbohydrate transport and metabolism

[H] Coenzyme transport and metabolism

[F] Nucleotide transport and metabolism

[C] Energy production and conversion

[D] Cell cycle control, cell division, chromosome partitioning

[P] Inorganic ion transport and metabolism

[O] Posttranslational modification, protein turnover

[Q] Secondary metabolites biosynthesis and transport.

[U] Intracellular trafficking and secretion

[T] Signal transduction mechanisms

[R] General function prediction only

[V] Defense mechanisms

<0

.00

1

<0

.00

1

<0

.05

<0

.05

<.0

.10

<.0

.10

0.0

Cold

adapted

Hot

adapted

p-value

Fig. 3


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Table 1. P. articus 273-4 growth response to diverse growth substrates

Growth Rate (µ hr-1)

Substrate 22°C 4°C

Acetate 0.159 ± 0.007 0.037 ± 0.002

Acetate + 5 mM

Glutamate 0.168 ± 0.002 NDd

Malonate 0.154 ± 0.007 NDd

Pyruvate 0.007 ± 0.002 0.005 ± 0.0006

Lactate 0.062 ± 0.005 0.023 ± 0.005

Lactate + 5 mM


Butanoic Acid 0.094 ± 0.002 0.025 ± 0.002

Decanoic Acid 0.032 ± 0.009 0.014 ± 0.005

Palmitic Acid 0.014 NGb


Marine Broth, 5% Sea

Salts 0.244 ± 0.014 0.060 ± 0.002

1/2 TSB + 5% NaCl 0.044 ± 0.0009 0.012 ± 0.002 aUnless otherwise noted, all carbon sources were at 20 mM

concentration. bNG = No growth after 10 days at 4°C

cNo significant growth observed on 0.5% Ethanol introduced to

cultures as solvent for decanoic and palmitic acid.

dND = No data collected under this

condition.


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Documents

1/24 The genome sequence of Psychrobacter arcticus 273-4, a