Cold-Adapted Microorganisms [1 ed.] 9781908230904, 9781908230263

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Cold-adapted Microorganisms

Edited by Isao Yumoto National Institute of Advanced Industrial Science and Technology (AIST) Sapporo Japan

Caister Academic Press

Copyright © 2013 Caister Academic Press Norfolk, UK www.caister.com British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-1-908230-26-3 (Hardback) ISBN: 978-1-908230-90-4 (ebook) Description or mention of instrumentation, software, or other products in this book does not imply endorsement by the author or publisher. The author and publisher do not assume responsibility for the validity of any products or procedures mentioned or described in this book or for the consequences of their use. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission of the publisher. No claim to original U.S. Government works. Cover image courtesy of Yoshinobu Nodasaka. Printed and bound in Great Britain

Contents Contributorsv Prefaceix Part I Biodiversity in Cold Ecosystems 1

Diversity of Bacteria in Permafrost

2

Ecology and Taxonomy of Psychrotolerant Bacteria in Artificial Cold Environments

13

3

Psychrophilic Microorganisms In Marine Environments

33

4

Fungi in Cryosphere: Their Adaptations to Environments

51

Shannon Hinsa-Leasure and Corien Bakermans

Isao Yumoto and Koji Yamazaki Yuichi Nogi

Tamotsu Hoshino, Nan Xiao, Yuka Yajima and Oleg B. Tkachenko

1

Part II Physiology and Molecular Adaptation Mechanisms 5

Energy Metabolism at Low-temperature and Frozen Conditions in Cold-adapted Microorganisms

71

6

Proteins Involved in Cold Adaptation

97

7

Heat Shock Response in Psychrophilic Microorganisms

111

8

Catalysis and Protein Folding in Psychrophiles

137

Pierre Amato

Kazuaki Yoshimune, Jun Kawamoto and Tatsuo Kurihara Seiji Yamauchi, Shinsuke Fukata and Hidenori Hayashi Charles Gerday

iv | Contents

Part III Biomolecules Related to Cold 9

Psychrotolerant H2O2-resistant Bacteria and Environmental Adaptation of their Catalases

161

Microorganisms in a Permafrost Ice Wedge and their Resuscitation-promoting Factors

177

Lipids in Cold-adapted Microorganisms

189

Isao Yumoto and Isao Hara

10

Taiki Katayama and Michiko Tanaka

11

Ahmad Iskandar Bin Haji Mohd Taha, Rifat Zubair Ahmed, Taro Motoigi, Kentaro Watanabe, Norio Kurosawa and Hidetoshi Okuyama

Index215

Contributors

Rifat Zubair Ahmed Centre for Molecular Genetics University of Karachi Karachi Pakistan

Shinsuke Fukata Graduate School of Science and Technology Ehime University Matsuyama Japan

[email protected]

[email protected]

Pierre Amato Clermont University Blaise Pascal University Institute of Chemistry of Clermont-Ferrand (ICCF) Clermont-Ferrand; CNRS UMR 6296 ICCF Aubière France

Charles Gerday Laboratory of Biochemistry University of Liege Liege Belgium

[email protected] Corien Bakermans Altoona College Pennsylvania State University Altoona, PA USA [email protected] Ahmad Iskandar Bin Haji Mohd Taha Division of Biosphere Science Graduate School of Environmental Science Hokkaido University Sapporo Japan [email protected]

[email protected] Isao Hara Technology Research Laboratory Shimadzu Corporation Seika-cho Soraku-gun Kyoto Japan [email protected] Hidenori Hayashi Cell-Free Science and Technology Research Center Ehime University Matsuyama Japan [email protected]

vi | Contributors

Shannon Hinsa-Leasure Grinnell College Grinnell, IA USA [email protected] Tamotsu Hoshino National Institute of Advanced Industrial Science and Technology (AIST) Toyohira-ku and Graduate School of Life Sciences Hokkaido University Sapporo Hokkaido Japan [email protected] Taiki Katayama Institute for Geo-Resources and Environment National Institute of Advanced Industrial Science and Technology Tsukuba Ibaraki Japan [email protected] Jun Kawamoto Institute for Chemical Research Kyoto University Uji Kyoto Japan [email protected] Tatsuo Kurihara Institute for Chemical Research Kyoto University Uji Kyoto Japan [email protected]

Norio Kurosawa Department of Environmental Engineering for Symbiosis Soka University Hachioji Tokyo Japan [email protected] Taro Motoigi Division of Biosphere Science Graduate School of Environmental Science Hokkaido University Sapporo Japan [email protected] Yuichi Nogi Extremobiosphere Research Program Institute of Biogeosciences Japan Agency for Marine-Earth Science and Technology Yokosuka Japan [email protected] Hidetoshi Okuyama Division of Biosphere Science Graduate School of Environmental Science and Laboratory of Environmental Molecular Biology Faculty of Environmental Earth Science Hokkaido University Sapporo Japan [email protected] Michiko Tanaka Laboratory of Novel Microbial Function Research Graduate School of Agriculture Hokkaido University Sapporo Hokkaido Japan [email protected]

Contributors | vii

Oleg B. Tkachenko Main Botanical Garden named after N.V. Tsitsin, Russian Academy of Sciences Moscow Russia [email protected]

Seiji Yamauchi Cell-Free Science and Technology Research Center Ehime University Matsuyama Japan [email protected]

Kentaro Watanabe Bioscience Group National Institute of Polar Research Tachikawa Tokyo Japan

Koji Yamazaki Reaearch Faculty of Fisheries Hokkaido University Hakodate Japan

[email protected]

[email protected]

Nan Xiao Kochi Core Center Japan Agency for Marine-Earth Science and Technology ( JAMSTEC) Nakoku Kochi Japan

Kazuaki Yoshimune Department of Applied Molecular Chemistry Nihon University Narashino Chiba Japan

[email protected] Yuka Yajima National Institute of Advanced Industrial Science and Technology (AIST) Toyohira-ku Sapporo Hokkaido Japan [email protected]

[email protected] Isao Yumoto Bioproduction Research Institute National Institute of Advanced Industrial Science and Technology (AIST) Toyohira-ku Sapporo Japan [email protected]

Current books of interest Myxobacteria: Genomics, Cellular and Molecular Biology2014 Next Generation Sequencing: Current Technologies and Applications2014 Omics in Soil Science2014 Mollicutes: Molecular Biology and Pathogenesis2014 Genome Analysis: Current Procedures and Applications2014 Bacterial Membranes: Structural and Molecular Biology2014 Bacterial Toxins: Genetics, Cellular Biology and Practical Applications 2013 Fusarium: Genomics, Molecular and Cellular Biology 2013 Prions: Current Progress in Advanced Research 2013 RNA Editing: Current Research and Future Trends 2013 Real-Time PCR: Advanced Technologies and Applications 2013 Microbial Efflux Pumps: Current Research 2013 Cytomegaloviruses: From Molecular Pathogenesis to Intervention 2013 Oral Microbial Ecology: Current Research and New Perspectives 2013 Bionanotechnology: Biological Self-assembly and its Applications 2013 Real-Time PCR in Food Science: Current Technology and Applications2012 Bacterial Gene Regulation and Transcriptional Networks2012 Bioremediation of Mercury: Current Research and Industrial Applications2012 Neurospora: Genomics and Molecular Biology2012 Rhabdoviruses2012 Horizontal Gene Transfer in Microorganisms2012 Microbial Ecological Theory: Current Perspectives2012 Two-Component Systems in Bacteria2012 Foodborne and Waterborne Bacterial Pathogens2012 Yersinia: Systems Biology and Control2012 Stress Response in Microbiology2012 Bacterial Regulatory Networks2012 Systems Microbiology: Current Topics and Applications2012 Quantitative Real-time PCR in Applied Microbiology2012 Bacterial Spores: Current Research and Applications2012 Small DNA Tumour Viruses2012 Extremophiles: Microbiology and Biotechnology2012 Full details at www.caister.com

Preface

If we define environments lower than 10°C as cold environments, most of the earth would consist of cold environments. These include the conventional cold environments such as the subarctic regions in winter, most of the sea environments, and specific environments such as the Arctic regions, soils and ice wedges in permafrost, deep sea, sea ice and glaciers, and soils in high alpine regions. Some of these specific cold environments are far from human activities and exhibit totally natural ecosystems. Analyses of microbiota in such cold environments reveal that certain microbiota adaptable to certain environments are composed of various cold-adapted microorganisms, although most of the mesophilic microorganisms living in the moderate temperature environments would find it difficult to propagate in such cold environments. Studies of cold ecosystems need to be carried out to further our understanding of the interaction between microorganisms and surrounding organisms such as plants or other microorganisms, or between microorganisms and environmental factors such as oxidative stress, hydrostatic pressure or desiccation in cold environments. There are specific relationships between microorganisms and other organisms in cold environments. In addition, microorganisms adapted to such cold ecosystems are suitable for the investigation of the various strategies they use to adapt to cold environments only or to cold environments concomitant with other environmental factors such as those mentioned above. Although microorganisms are able to thrive at a temperature of approximately 5°C, all the activities of microorganisms stop under freezing conditions (e.g. −20°C). The analyses of microbiota in such environments may provide a clue to the microorganisms that existed more than 10,000 years ago. On the other hand, if microorganisms have evolved by the accumulation of subtle mutational changes during generational changes, in cold environments where microorganisms can barely metabolize these microorganisms will exhibit different evolutional strategies because of their longer generation times compared with microorganisms living in mesophilic or thermophilic environments. Thus, studies of microorganisms in cold environments may provide the most comprehensive information on the life of microorganisms during the course of their evolution over 10,000 years. Furthermore, bacterial adaptation mechanisms in such extreme environments will provide new findings regarding bacterial environmental adaptation and evolution. Certain cold-adapted microorganisms are adapted to temperature fluctuating environments. Such microorganisms are sensitive to ambient temperature changes and demonstrate exquisite gene regulation mechanisms by which proteins corresponding to the changes in ambient temperature are expressed. The microorganisms are able to grow more effectively than the surrounding organisms when the temperature rises in spring after a long period

x | Preface

of low temperatures in winter. On the other hand, a stable appropriately low temperature (e.g. 5–10°C) is suitable for promoting certain biological activities because exhaustion of nutrients at low temperatures is slower than that at ordinary ambient temperature. Some psychrophiles adapt well only to cold environments and their functions degenerate in respond to fluctuations of the ambient temperature. In cold sea environments, the temperature is stable, the decomposition of ambient nutrients is slow, and the concentration of dissolved oxygen is high. Indeed, microbial activities flourish in cold sea water and cold sea ice. On the other hand, very low temperatures such as subzero temperatures provide dormant conditions for microorganisms. Under such conditions, although the biological activities are low, the microorganisms have a chance of having an extended lifetime. Thus, as described above, cold environments do not only lower metabolic activities and are barren but they also provide microorganisms with beneficial niches from several aspects. Although cold environments are quite common on earth, biological activities and biological mass are not always abundant in such environments especially at temperatures lower than −2°C. This is because of the fact that biological activities are based on chemical reactions, and such reactions need a certain activation energy depending on temperature. Therefore, cold-adapted microorganisms are not able to exhibit energy production rates equivalent to those in low to moderate temperature adapted microorganisms. Certain cold adaptation strategies at the molecular level are necessary for organisms to live in cold environments. Indeed, ecosystems in moderate cold (e.g. around 5°C) embrace various microorganisms. They possess exquisite molecular mechanisms for adaptation to cold environments such as the production of cold active enzymes via the metabolic pathways or enzymes for hydrolysing substrates and cold shock proteins to accelerate the transcription and translation of genes at low temperatures. In addition, the maintenance of appropriate membrane fluidity at low temperatures is indispensable for survival because membrane-binding proteins have important roles in energy production in most aerobic microorganisms. These enzymes are able to react with each other only in the presence of the appropriate membrane fluidity. As described above, cold environments do not simply provide negative factors for microorganisms that delay their metabolism, they also give niches for microorganisms owing to stable environments, less competition with organisms living in their surroundings and less heat induced exhaustion due a decreased metabolic turnover rate. In this book, prominent authors present basic and new knowledge and concepts concerning cold-adapted microorganisms from the aspects of each area of expertise. The editor would like sincerely to thank all the authors for their excellent contributions. I believe that each chapter will reveal to the readers quite fascinating research horizons of cold-adapted microorganisms. I also thank Horizon Press, particularly Dr Hugh Griffin, for giving me the opportunity to edit this book and his continuous support and encouragement to hasten the publication of this book. Isao Yumoto Sapporo, Japan

Part I Biodiversity in Cold Ecosystems

Diversity of Bacteria in Permafrost Shannon Hinsa-Leasure and Corien Bakermans

1

Abstract In the cold, challenging environment of the permafrost, bacteria have found a way to survive and grow for thousands to millions of years. In this chapter, we explore bacterial diversity in permafrost from around the world, identified through culture-dependent and -independent techniques. Members of the phylum Actinobacteria, Firmicutes and Proteobacteria have been found in every environment studied thus far, indicating that these bacteria are well suited for life at low temperatures with low water activity. Also, unique species specific to individual environments have been discovered at each site. Researchers are faced with the challenge of determining which bacteria are active in the permafrost and which are in a dormant state. The ability of bacteria to reside in a dormant state further complicates culture-independent experimental results, as DNA from both dormant and active cells has been analysed. We are only beginning to understand the metabolic capabilities of permafrost bacteria, many discoveries are still to come. Introduction We live on a planet that is dominated by low-temperature environments. These low-temperature environments, including 90% of oceans and 26% of terrestrial soil ecosystems, once thought too cold for life have been shown to support diverse microbial communities (Gilichinsky et al., 1995; Zhang et al., 2008b). In this chapter we focus on bacterial diversity in permafrost, soil that has been at or below 0°C for at least two years. Permafrost is an extreme type of environment due to stable low temperatures, low water activity, and radiation exposure (Gilichinsky, 2002a,b). Omelyansky first described viable permafrost microorganisms in 1911 (Omelyansky, 1911). In 1942, James and Sutherland isolated aerobic and anaerobic bacteria from Canadian permafrost ( James and Sutherland, 1942). These studies were followed by investigations in the permafrost of Alaska and Antarctica (Becker and Volkmann, 1961; Boyd and Boyd, 1964; Cameron and Morelli, 1974) and laid the groundwork for the now thriving study of microbial diversity in permafrost environments. Researchers have found the permafrost to be full of bacteria, with counts ranging from 105 to 108 cells per gram dry weight (gdw) permafrost (Bai et al., 2006; Gilichinsky, 2002b; Hansen et al., 2007; Kochkina et al., 2001; Steven et al., 2008b; Wilhelm et al., 2011). Questions arose about these bacterial cells- were they metabolically active or dormant in a spore form or cyst-like state? Soina and colleagues characterized cell structure in Arctic and Antarctic permafrost by microscopy and found no spores or sporulating cells, instead they observed cells with

2 | Hinsa-Leasure and Bakermans

thickened cell walls, compact nucleoids, and altered cytoplasm indicative of dormant cells (Soina et al., 2004). In contrast, metabolic activity has been measured in the Siberian permafrost from −10°C to −20°C (Panikov and Sizova, 2007; Rivkina et al., 2000, 2004). Work now focuses on identifying which bacteria are metabolically active in the permafrost. Both culture-dependent and -independent techniques have been employed to characterize bacterial diversity in permafrost environments around the world. Typically bacterial viability decreases with increasing age of the permafrost (Gilichinsky et al., 2007; Shi et al., 1997; Zhang et al., 2007b). Few if any isolates recovered from permafrost are psychrophiles; instead isolates are psychrotolerant, often with optimal growth at temperatures much higher than in situ temperatures (Bakermans et al., 2003; Hinsa-Leasure et al., 2010; Shi et al., 1997; Steven et al., 2007; Wilhelm et al., 2011). Cultivation on diluted media at low temperatures has been shown to result in the highest number of CFU per gram of permafrost, although bacteria grown on rich media and/or at higher temperatures presented more morphological diversity (Hansen et al., 2007; Hinsa-Leasure et al., 2010; Steven et al., 2007; Vishnivetskaya et al., 2000). Several types of media have been employed in bacterial isolations from the permafrost, with R2A, PYGV (peptone, yeast extract, glucose, and vitamin) and tryptone soy agar being the most common (Bai et al., 2006; Bakermans et al., 2003; Gilichinsky et al., 2007; Hansen et al., 2007; Hinsa-Leasure et al., 2010; Reasoner and Geldreich, 1985; Shi et al., 1997; Steven et al., 2007, 2008b; Vishnivetskaya et al., 2000, 2006; Wilhelm et al., 2011; Zhang et al., 2007b). Culture-independent techniques are increasingly applied to the study of bacterial diversity in permafrost (Gilichinsky et al., 2007; Hansen et al., 2007; Hinsa-Leasure et al., 2010; Steven et al., 2007, 2008a; Vishnivetskaya et al., 2006; Wilhelm et al., 2011; Yergeau et al., 2010) but face significant challenges. Because low temperatures preserve cells and DNA (Lindahl, 1993; Poinar et al., 1996; Soina et al., 1995; Willerslev et al., 2004) extracted DNA does not necessarily represent active populations of cells. In the following sections we will describe bacterial diversity found within permafrost from around the world (Fig. 1.1 and Table 1.1). Both culture-dependent and -independent studies are described; as is permafrost from Siberia, the high Arctic, Antarctica and Central Asia (Fig. 1.1). The active layer (the uppermost layer which does not remain frozen year-round), while an integral part of permafrost, is beyond the scope of this review. Siberian permafrost The Siberian permafrost is one of the best-studied permafrost areas on earth. Early work by Russian scientists determined that up to 108 bacterial cells could be found in each gram of permafrost (Gilichinsky et al., 1995). At depth, Siberian permafrost maintains a constant temperature from −10°C to −12°C and has a high total organic content (1–1.3%). Diverse bacteria have been isolated from the Siberian permafrost including: Actinobacteria, Firmicutes, sulfate-reducing bacteria, aerobic and anaerobic bacteria, and Proteobacteria, including nitrite-oxidizing Betaproteobacteria (Table 1.1) (Alawi et al., 2007; Bakermans et al., 2006; Hinsa-Leasure et al., 2010; Liebner et al., 2009; Liebner and Wagner, 2007; Shi et al., 1997; Vatsurina et al., 2008; Vishnivetskaya et al., 2000, 2006; Wagner et al., 2009). Many isolates have been shown to tolerate high salt concentrations, a state bacterial cells are likely to encounter in the unfrozen water of the Siberian permafrost (Bakermans et al., 2003; Hinsa-Leasure et al., 2010; Vishnivetskaya et al., 2000). Studies conducted on samples from the active and permafrost table layers have found a rich diversity of bacteria including type

Permafrost Bacteria | 3

Figure 1.1 Location of permafrost bacterial diversity studies summarized in Table 1.1. Positions are marked at the approximate site where samples were collected. A, Kolyma lowland, Siberia; B, Lena Delta, Siberia; C, Ellesmere Island, Canada; D, Axel Heiberg Island, Canada; E, Spitsbergen, Norway; F, Dry Valleys, Antarctica; G, Tian Shan Mountains, China; H, Qinghai–Tibet Plateau, China.

I and type II methane-oxidizing bacteria, community members that will be of vital importance as temperatures rise and methane production increases (Liebner et al., 2009; Liebner and Wagner, 2007). Culture-dependent studies have commonly isolated Arthrobacter, Planococcus, Exiguobacterium and Psychrobacter species from several sites, while Sporosarcina, Bacillus, Paenibacillus, Flavobacterium, Sphingobacterium, Pseudomonas, Serratia, Escherichia, Alcaligenes and Sphingomonas have been identified at lower frequencies (Hinsa-Leasure et al., 2010; Shi et al., 1997; Vishnivetskaya et al., 2006). However, when culture-independent techniques were employed, Planomicrobium and Carnobacterium rose to prominence in the study by Hinsa-Leasure and colleagues, while Xanthomonadaceae sequences were identified at high frequency from sites studied by Vishnivetskaya and colleagues (Hinsa-Leasure et al., 2010; Vishnivetskaya et al., 2006). The differences found between culture-dependent and -independent studies could reflect any combination of the following issues: the challenges faced in culturing permafrost bacteria in the laboratory, bias in DNA extraction and amplification, and the presence of non-viable cells. Further examination of species from two genera commonly isolated from permafrost demonstrates their ubiquity in and adaptation to the permafrost environment. Exiguobacterium and Psychrobacter species have been detected in a majority of Siberian permafrost samples from cores ranging from 5000 to 3 million years old (Rodrigues et al., 2009;

I(14)

I(21)

I(34)

C, I(47)

I(31)

C, I(10)

I(4)

C, I(29)

I(10)

Vishnivetskaya (2006)

Shi (1997)

Bakermans (2003)

I(24)

I(37)

I(39)

C(6)

C(1), I(4)

C(1)

C(72), I(45)

C(19), I(51)

C(1)

C(4)

C(8), I(4)

C(17)

C(2)

C(12)

C(1)

C(3)

C(8)

C(41)

C(16)

C(2)

I(100)

C(18)

C(11)

C(4), I(70)

C(13)

C(2)

Q(3.5)

Q(40)

Q(6.5)

Q(1)

Q(11

Q(38)

Steven Yergeauc (2008) (2010)

C(38),I(26)

Hinsa-Leasure Steven (2010) (2007)

Norway

C(15)

C(2)

C(1)

I(6.5)

I(6.5)

C(15)

C(2)

C(1)

C(15)

I(33)

C(2)

C(28), I(53)

C(20

Wilhelm (2011)

C, I(4)

C

I(1)

C, I(6)

C

C

I(4)

C

C,I(3)

C,I(81)

Hansend (2007)

Axel Heiberg Spitsbergen Island (D) (E)

Antarctica

Central Asia

C

C, I

I

C C

C,I

C

C

C

C

C, I

C

Gilichinsky (2007)

I(14)

I(10) I(6)

I(6)

I(16)

I(49)

Bai (2007)

Dry Valleys Tian Shan (F) Mountains (G)

I(15)

I(18) I(12)

I(6)

II(36) I(12)

Zhang (2007)

Qinghai–Tibet Plateau (H)

diversity as identified in a clone library (C), isolates (I), or through Q-PCR (Q). The percentage of sequences belonging to each phylum is given in parenthesis, if available from the study. letters behind the location name identify the location in Fig. 1.1. cDiversity shown from the sample that did not receive multiple displacement amplification. dDiversity represents that found in original sample, enrichments not included. eCFB, Cytophaga–Flexibacter–Bacteroides.

bThe

aBacterial

Unclassified

Verrucomicrobia

TM7

Gamma

Delta

Beta

Alpha

Proteobacteria

Planctomyces

OP10

OD1

Nitrospira

Gemmatinonadetes

Firmicutes

Fibrobacteres

Chloroflexi

Clostridia

CFBe

Bacteroides

Actinobacteria

Acidobacteria

Phylum

Ellesmere Island (C)

Kolyma lowland (A)

Lena Delta (B)

Canadian high Arctic

Siberia

Locationb

Table 1.1 Bacterial diversity in permafrosta

Permafrost Bacteria | 5

Rodrigues and Tiedje, 2007; Vishnivetskaya et al., 2009). In the laboratory, two permafrost isolates, Psychrobacter arcticus and Exiguobacterium sibiricum, have been shown to be metabolically active when under low water activity conditions, conditions which simulate environmental conditions (Ponder et al., 2008). E. sibericum and P. arcticus have also been shown to alter membrane composition by decreasing fatty acid saturation and chain length, in accordance with decreasing temperatures, which affect carbon utilization and antibiotic resistance at low temperatures (Ponder et al., 2005). Further characterization of these two species has revealed additional adaptations to low temperatures which likely allow them to actively survive within permafrost (Ayala-del-Rio et al., 2010; Bergholz et al., 2009; Rodrigues et al., 2008). Owing to the age of Siberian permafrost and the ability of its low temperatures to preserve material, study of Siberian permafrost isolates can provide historical information that would not be available otherwise. For example, Petrova and colleagues isolated a novel integron, a mobile DNA element which confers antibiotic and chromate resistance, from a Pseudomonas isolate recovered from 15,000- to 40,000-year-old permafrost (Petrova et al., 2011). This work demonstrated that the presence of antibiotic resistance genes predates human manufacturing and use of antibiotics. Additionally, Mindlin and colleagues identified mercury resistance transposons in Pseudomonas isolates from permafrost aged 15,000–40,000 years (Mindlin et al., 2005). The mercury resistance transposons from permafrost are very similar to those found today, suggesting that mercury resistance gene content and distribution have not changed significantly over the last 40,000 years. A distinct environment within the Siberian permafrost is the cryopeg, a pocket of highly saline (170–300 g/l) liquid water sandwiched between layers of permafrost. Cryopegs contain about 107 cells/ml (direct counts) and a diversity of prokaryotes (from heterotrophs to sulfate-reducers to methanogens) have been cultivated from them (Bakermans et al., 2003; Gilichinsky et al., 2003). Bakermans and colleagues characterized isolates from several cryopegs and determined that Psychrobacter and Arthrobacter species dominated the non-spore-forming isolates, while Bacillus and Paenibacillus species made up the sporeforming bacterial isolates (Bakermans et al., 2003). Another study (Gilichinsky et al., 2005) also found that isolates were dominated by spore-forming rod-shaped cells when incubated anaerobically in heterotrophic media. Two isolates from this study, Psychrobacter murincola 2pS and Clostridia sp. 14D, both reproduced at −5°C (Gilichinsky et al., 2005). More spore-forming bacteria were isolated from cryopegs than have typically been isolated in the terrestrial permafrost. Further, two genera found in a majority of permafrost environments, Planococcus and Exiguobacterium were not isolated (Bakermans et al., 2003; Gilichinsky et al., 2005). These studies suggest that the high salinity and larger liquid volume found in cryopegs has selected for a different bacterial community than that found in the surrounding permafrost. Canadian high Arctic permafrost Low temperatures and high carbon content characterize high Arctic permafrost regions in Nunavut, Canada. Several studies conducted on unique environments in this region have found remarkably similar bacterial communities (Table 1.1) (Steven et al., 2007, 2008a; Wilhelm et al., 2011; Yergeau et al., 2010). New species have been characterized including a psychrotolerant anaerobic bacterium, Clostridium tagluense, which was isolated from

6 | Hinsa-Leasure and Bakermans

permafrost in the Mackenzie Delta, Nunavut, mainland Canada (Suetin et al., 2009). This newly discovered species is similar to other cluster I psychrotolerant and psychrophilic Clostridium species. The bacterial diversity in the Canadian permafrost has only begun to be explored and it holds promise for the discovery of many new bacterial species. The permafrost on Eureka, Ellesmere Island, Canada, is a unique permafrost environment because it is more similar in temperature to the Antarctic permafrost, yet it has a high carbon content (total carbon of 2.19% dry weight of permafrost), similar to the Siberian permafrost (Gilichinsky et al., 1995, 2007; Steven et al., 2007). Two studies conducted by Steven and colleagues on separate permafrost samples from Eureka isolated a bacterial community dominated by spore-forming Bacillus, Paenibacillus and Sporosarcina. The dominance of spore-forming bacteria is in contrast to Siberian permafrost studies that have found far fewer spore-forming bacteria and significantly more Proteobacteria (Table 1.1) (Hansen et al., 2007; Hinsa-Leasure et al., 2010; Shi et al., 1997; Steven et al., 2007, 2008b; Vishnivetskaya et al., 2006). Three Gram-positive isolates from the Eureka permafrost sample were characterized as new species; Virgibacillus arcticus, Tumebacillus permanentifrigoris and Planococcus halocryophilus (Mykytczuk et al., 2011; Niederberger et al., 2009; Steven et al., 2008a). Unlike the isolate diversity, the diversity found in the 16S rRNA clone libraries was highly similar to the diversity found in the 16S rRNA clone libraries generated from Siberian permafrost (Steven et al., 2007, 2008b; Yergeau et al., 2010). The drastic differences between the isolated and the 16S rRNA clone library communities from this permafrost could be due to DNA surviving outside of cells in the permafrost or the presence of dead or unculturable cells in the permafrost. Another possible explanation could be differences in PCR amplification of environmental DNA. Yergeau and colleagues determined that the whole-community genome amplification techniques, often employed to increase DNA concentrations, led to a misrepresentation of bacterial diversity by increasing Bacteriodetes while decreasing Actinobacteria (Yergeau et al., 2010). To examine the metabolic diversity found in Ellesmere Island permafrost and the surrounding active layer, Yergeau and colleagues employed culture-independent techniques (Yergeau et al., 2010). Only type I methanotrophs were detected in metagenomic libraries from Eureka active-layer soil and permafrost; however, during qPCR experiments type II methanotrophs were found in these samples but at lower numbers than type I methanotrophs. This finding is in contrast to results from Siberian permafrost that found type I methanotrophs in the active-layer samples and type II methanotrophs distributed throughout the active layer and permafrost table (Liebner et al., 2009; Liebner and Wagner, 2007; Martineau et al., 2010). Genes known to be involved in ammonia oxidation and nitrogen fixation were also found in active-layer and permafrost (Yergeau et al., 2010). The bacterial diversity within an acidic wetland from Axel Heiberg Island, Nunavut, Canada, has been recently analysed (Wilhelm et al., 2011). This area has an annual temperature of −15°C and a pH