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Psychrophiles

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EA41CH06-Cavicchioli ARI 7 February 2013 14:43

RE V I E W

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PsychrophilesKhawar S. Siddiqui,1 Timothy J. Williams,1

David Wilkins,1 Sheree Yau,1 Michelle A. Allen,1

Mark V. Brown,1,2 Federico M. Lauro,1

and Ricardo Cavicchioli11School of Biotechnology and Biomolecular Sciences and 2Evolution and Ecology ResearchCenter, The University of New South Wales, Sydney, New South Wales 2052, Australia;email: r.cavicchioli@unsw.edu.au

Annu. Rev. Earth Planet. Sci. 2013. 41:6.1–6.29

The Annual Review of Earth and Planetary Sciences isonline at earth.annualreviews.org

This article’s doi:10.1146/annurev-earth-040610-133514

Copyright c© 2013 by Annual Reviews.All rights reserved

Keywords

microbial cold adaptation, cold-active enzymes, metagenomics, microbialdiversity, Antarctica

Abstract

Psychrophilic (cold-adapted) microorganisms make a major contributionto Earth’s biomass and perform critical roles in global biogeochemical cy-cles. The vast extent and environmental diversity of Earth’s cold biospherehas selected for equally diverse microbial assemblages that can include ar-chaea, bacteria, eucarya, and viruses. Underpinning the important ecologicalroles of psychrophiles are exquisite mechanisms of physiological adaptation.Evolution has also selected for cold-active traits at the level of molecularadaptation, and enzymes from psychrophiles are characterized by specificstructural, functional, and stability properties. These characteristics of en-zymes from psychrophiles not only manifest in efficient low-temperatureactivity, but also result in a flexible protein structure that enables biocatalysisin nonaqueous solvents. In this review, we examine the ecology of Antarcticpsychrophiles, physiological adaptation of psychrophiles, and properties ofcold-adapted proteins, and we provide a view of how these characteristicsinform studies of astrobiology.

6.1

Review in Advance first posted online on February 14, 2013. (Changes may still occur before final publication online and in print.)

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INTRODUCTION

Much of life on Earth has evolved to colonize low-temperature environments. In fact, at tem-peratures permanently below 5◦C, the cold biosphere represents by far the largest fraction ofthe global biosphere (Feller & Gerday 2003, Cavicchioli 2006, Siddiqui & Cavicchioli 2006,Casanueva et al. 2010, Margesin & Miteva 2011). Consistent with representative size, the coldbiosphere consists of diverse types of environments—vast tracts of the deep sea, geographically dis-persed alpine regions, geologically specific subterranean caverns, climatically challenged regionsof permafrost, and biogeochemically diverse polar reaches (Figure 1). Proliferating throughoutthese cold realms is a plethora of psychrophilic (cold-adapted) microorganisms—archaea, bacte-ria, eucarya, and viruses. A small proportion of the isolated microorganisms from naturally coldenvironments have a restricted growth temperature range with an upper growth temperature limitless than ∼20◦C (stenopsychrophile), whereas the majority of isolates have a broader temperaturerange, tolerating warmer temperatures (eurypsychrophile).

Particularly through the application of molecular genetics approaches, most notably small sub-unit ribosomal RNA (SSU rRNA) sequencing, fluorescent in situ hybridization (FISH), and DNAsequencing of whole environmental samples (metagenomics), the cold biosphere has been discov-ered to harbor a diverse range of microbial groups. In recent years, the application of metagenomicsand associated meta-functional approaches (metaproteomics and metatranscriptomics) has shedlight on whole microbial community composition dynamics and microbial processes that are be-ing driven by the resident psychrophiles. Genomic, physiological, and biochemical analyses ofpsychrophilic isolates and their cellular components have also gleaned valuable information aboutthe diverse molecular mechanisms of cold adaptation. As a result, whether driven by global ques-tions concerning the impact of ecosystem change on microbial communities in cold environments,fundamental studies of molecular structure and function, or biotechnologically driven pursuits ofnovel cold-active biocatalysts, the field of psychrophiles has made great advances.

This review aims to cover topics relevant to studies of earth and planetary sciences by providingknowledge about physiological and protein adaptation—characteristics that speak to fundamentalprinciples of biological adaptation to the cold and provide insight into survivability. A perspectiveon microbial ecology of Antarctic systems opens the review, particularly focusing on lake, sea-ice,and deep-sea environments—systems that include a broad range of physicochemical conditions

Polar

e.g. Deep Lake,Antarctica

–20°C

Extraterrestrial

e.g. Europa

Surface: –200 to –160ºCSubsurface ocean: ?ºC

Alpine

< 10°C Deep sea

1 to 4°C

Figure 1Terrestrial and extraterrestrial cold environments. Representative temperatures are shown.

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that provide knowledge about the diversity of microbial life that is sustained under a rangeof cold and abiotically varied environmental extremes. Also provided is a brief perspective onpsychrophiles and global warming, providing a glimpse into the use of cold-active enzymes andits impact on psychrophiles in relation to climate change. The review concludes with a sectionreflecting on microbial extremes and cold-active enzymes and their relevance to astrobiology.

ANTARCTIC PSYCHROPHILES

Antarctic Aquatic Ecosystems

Both southern and northern polar regions are delicately balanced ecosystems that are easily affectedby ecosystem changes (Moline et al. 2004, Murray & Grzymski 2007, Wilkins et al. 2012b), andglobal warming is expected to cause changes that will flow through to organisms right up thefood chain (Kirchman et al. 2009). In the Antarctic, global warming has particularly impactedthe Antarctic Peninsula and West Antarctica (Meredith & King 2005, Murray & Grzymski 2007,Cavalieri & Parkinson 2008, Whitehouse et al. 2008, Reid et al. 2009, Steig et al. 2009, Hogget al. 2011), and Antarctic sea-ice extent has decreased by at least ∼20% since the early 1950s andis projected to continue to decrease (Curran et al. 2003, Liu & Curry 2010). Ocean acidification(Kintisch & Stoksta 2008, McNeil & Matear 2008, Falkowski 2012), reduced CO2 absorption (LeQuere et al. 2007), and reduced nutrient supply particularly at higher latitudes caused by increasedstratification (Sarmiento & Le Quere 1996, Wignall & Twitchett 1996, Matear & Hirst 1999)are all effects linked to global warming. As the ocean microorganisms are critical for sequesteringanthropogenic CO2 (Sabine et al. 2004, Mikaloff Fletcher et al. 2006) and transporting it to thebenthic zones (Thomalla et al. 2011), the changes taking place in polar waters are of great concernfor the health of the global ecosystem.

Even though only 50,850 km2 (0.4%) of Antarctica is seasonally ice free (Poland et al. 2003,Cary et al. 2010), a broad range of lake systems are distributed around Antarctica that maintainice, water column, sediment, and microbial mat communities (Wilkins et al. 2012b). These lakesinclude subglacial, epiglacial, and surface systems that range in salinity from fresh to saturatedand from mixed to permanently stratified. The evolutionary history of these lakes is as varied asthe lakes themselves, which include the hundreds of marine-derived systems in the Vestfold Hills,which were isolated ∼3,000–7,000 years ago from the ocean (Gibson 1999) (Figure 2); subglacialoutflow from Blood Falls dating from 1.5 Mya (Mikucki et al. 2009); and waters in the depths ofsubglacial Lake Vostok, which are probably even older (Siegert et al. 2001).

Antarctic Microorganisms Colonize Diverse Cold Niches

Microbial populations vary in accordance with the wide range of physical and chemical propertiesof Antarctic lakes. In some marine-derived lakes, such as Ace Lake, the marine origin and, possibly,subsequent seeding from marine waters can be seen in the community composition of some partsof the water column (Lauro et al. 2011b) (Figure 2). However, this stratified system harbors vastlydifferent communities in other parts of the lake where very different physicochemical conditionsexist (Lauro et al. 2011b), including a highly purified population of green sulfur bacteria at the lake’soxycline interface (Ng et al. 2010). The microbial communities in Lake Bonney have evolved inresponse to physical distinctions occurring in two different lobes of the lake (Glatz et al. 2006). Bothof these examples illustrate how seed populations have diverged in response to ecosystem changes.

The transition from a marine to a hypersaline environment at Deep Lake provides an extremeexample of ecosystem change (Figure 2). Situated in the Vestfold Hills, Deep Lake is ∼55 m belowsea level, 36-m deep, hypersaline (3.6–4.8 M), ice free, and perennially cold (e.g., −20◦C) (Ferris &Burton 1988, Franzmann et al. 1988). The system appears on the border of sustaining life; scientific

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records indicate it has been extremely unproductive (<10 g C m−2 year−1) (Campbell 1978).The microbial diversity in the lake is extremely low, dominated by members of the haloarchaea(Bowman et al. 2000a). Ongoing studies of this system have identified a range of genomic traitsand ecology of the system that are unique compared with hypersaline or cold aquatic systemselsewhere in the world (R. Cavicchioli, unpublished results).

Subsurface lake systems include subglacial lakes, such as Lake Vostok (Siegert et al. 2001), andepiglacial lakes that result from glacier melt and form where mountains (e.g., Framnes Moun-tains) penetrate the polar ice surface and may harbor microorganisms that are ancient or recent(postglacial) inhabitants (Gibson 2006, Cavicchioli 2007). Avoiding contamination in the pursuitof studying such pristine systems is a significant logistical challenge, and lessons learned aboutdrilling into Lake Vostok and other subglacial lakes (Inman 2005, Wingham et al. 2006, Alekhinaet al. 2007, Lukin & Bulat 2011, Gramling 2012, Jones 2012) should provide wisdom for guidingcontemplation of future endeavors, including extraterrestrial studies.

Antarctic Aquatic Microorganisms

Our understanding of community composition in Antarctic aquatic systems has been greatlyfacilitated by molecular-based studies (Wilkins et al. 2012b). These have included analyses usingdenaturing gradient gel electrophoresis (Pearce 2003, 2005; Pearce et al. 2003, 2005; Karr et al.2005; Unrein et al. 2005; Glatz et al. 2006; Mikucki & Priscu 2007; Mosier et al. 2007; Schiaffinoet al. 2009; Villaescusa et al. 2010), rRNA genes (Bowman et al. 2000a,b, 2003; Gordon et al.2000; Christner et al. 2001; Purdy et al. 2003; Karr et al. 2003, 2005, 2006; Matsuzaki et al. 2006;Kurosawa et al. 2010; Bielewicz et al. 2011), functional genes (Olsen et al. 1998, Voytek et al.1999, Mikucki et al. 2009), and metagenomics and metaproteomics (Lopez-Bueno et al. 2009; Nget al. 2010; Lauro et al. 2011b; Yau et al. 2011; Brown et al. 2012; Gryzmski et al. 2012; Varinet al. 2012; Wilkins et al. 2012a; Williams et al. 2012a,b).

Molecular signatures of archaea have been detected in a range of Antarctic lakes, includingstrictly anaerobic methanogens and aerobic haloarchaea (Bowman et al. 2000a,b; Purdy et al. 2003;

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 2Antarctic lake systems. (a–k) Lakes in the Vestfold Hills (68◦33′0′ ′ S, 78◦15′0′ ′ E) and (l–n) Heard Island (53◦6′0′ ′ S, 73◦31′0′ ′E).(a) Ace Lake, a marine-derived meromictic system (Gibson 1999, Cavicchioli 2006) that is separated from marine waters of Long Fjordby only several hundred meters ( foreground ). Sea ice and icebergs are present in the early-mid austral summer 2008 (background ).Among other species, green sulfur bacteria play a particularly important role in this lake’s ecosystem (Ng et al. 2010, Lauro et al.2011b). (b) Ace Lake at the end of summer 2006 after the lake ice and sea ice have melted and begun to refreeze. (c) Snow drifts on AceLake formed after a blizzard behind quad bikes (used for transport between the lake and Davis Research Base located 15 km away) andmobile work shelters (MWSs) that were used for sample collection and protection from the weather. (d ) Organic Lake, a hypersalinemeromictic system where the waters are −13◦C below the surface ice; photo taken in 2008 (Gibson 1999). The novel and importantrole of virophages was discovered in this lake (Yau et al. 2011). (e) Drilling through surface ice on Organic Lake prior to the positioningof MWSs for sample collection. ( f ) Foam generated by the wind blowing across the organically rich waters of Organic Lake in 2006.Shown are microbial biofilms (orange) in the water and on rocks as well as penguin feathers (white) near the edge of the lake. ( g) DeepLake panorama in September 2008 after a cold winter (−40◦C) (photo credit: Mark Milnes). (h) Deep Lake is hypersaline, and watertemperatures reach −20◦C and do not freeze. (i ) Deep Lake is ∼55 m below sea level, marked by the flat hill line in the background.( j ) The Vestfold Hills region contains hundreds of lakes and ponds positioned between the coastline and the edge of the Antarcticcontinental ice mass (background ). (k) MWSs, dinghy, and research equipment at Deep Lake. Water pumped into drums on board thedinghy at the center of the lake (∼800 m from shore) was transported back to the MWSs for processing. (l ) Brown Lagoon at the baseof Brown Glacier, Heard Island, in 2008 contains glacier meltwater and is separated from ocean waters by a narrow strip of beach.(m) Winston Lagoon at the base of Winston Glacier is open to the ocean, allowing water exchange. (n) Water formed at the base ofStephenson Glacier contains slabs of recently melted glacier. The melted sections and large lake of water that were not present inprevious seasons are overt signs of ecosystem change as a result of global warming.

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Glatz et al. 2006; Karr et al. 2006; Kurasawa et al. 2010; Lauro et al. 2011b), of which several havebeen brought into axenic culture (Franzmann et al. 1988, 1992, 1997). A large number of studieshave focused on Antarctic bacteria, and diverse taxa have been identified, including members ofthe groups Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Chlorobi, Chloroflexi, Cyanobacteria,Deltaproteobacteria, Firmicutes, Gammaproteobacteria, Bacteroidetes [Cytophaga-Flavobacterium-Bacteroides (CFB) group], Planctomycetes, Spirochaetes, and Verrucomicrobia (Bowman et al. 2000a,b;Glatz et al. 2006; Mosier et al. 2007; Kurosawa et al. 2010; Pearce 2005; Pearce et al. 2003,2005; Schiaffino et al. 2009; Lauro et al. 2011b). Eucarya, particularly algal phototrophs, are alsoimportant in Antarctic lakes, although fungi and silicoflagellates have also been identified (Unreinet al. 2005, Mosier et al. 2007, Bielewicz et al. 2011, Lauro et al. 2011b, Yau et al. 2011). Virusesof Antarctic eucarya, bacteria, and archaea have also been identified (Lauro et al. 2011b, Yau et al.2011). The absence of higher trophic level organisms in Antarctic lake systems indicates virusesmay play an important role in the microbial loop (Kepner et al. 1998; Anesio & Bellas 2011;Laybourn-Parry et al. 2001, 2007; Madan et al. 2005; Sawstrom et al. 2007; Lopez-Bueno et al.2009). Specific impacts on bacterial hosts have been linked to mechanisms of cellular resistance;uncharacteristically low levels of viruses (Lauro et al. 2011b); and roles for virophage predationof algal viruses, which is predicted to increase overall primary production and net carbon flow inthe lake system (Yau et al. 2011) (Figure 2).

In addition to the water column, rich microbial communities are found in Antarctic mats andcan make important contributions to biomass and productivity (Vincent 2000, Moorhead et al.2005, Laybourn-Parry & Pearce 2007). Microorganisms identified in Antarctic mats includemembers of Actinobacteria, CFB, Cyanobacteria, Deinococcus-Thermus, Firmicutes, fungi, greenalgae, Planctomycetes, Proteobacteria, and Verrucomicrobia (Brambilla et al. 2001; Van Trappen et al.2002; Taton et al. 2003, 2006; Jungblut et al. 2005; Fernandez-Valiente et al. 2007; Sutherland2009; Borghini et al. 2010; Verleyen et al. 2010; Anderson et al. 2011; Callejas et al. 2011;Fernandez-Carazo et al. 2011; Hawes et al. 2011; Peeters et al. 2011, 2012; Antibus et al. 2012a,b;Varin et al. 2012). Mats are interesting features of lakes because they provide mineral andbiological records of the ecosystem, thereby also providing insight into the evolution of past andextant species (Bomblies et al. 2001, Sutherland & Hawes 2009, Anderson et al. 2011, Hawes et al.2011).

The taxa in Antarctic marine waters are, on the whole, similar to those in temperate or tropicalocean waters and include a high proportion of Alphaproteobacteria (e.g., SAR11 clade), Flavobacteria,Gammaproteobacteria, and ammonia oxidizing Marine Group I Crenarchaeota (Wilkins et al. 2012b).However, although many common taxa are found, the indigenous Antarctic populations havegenetic and physiological traits that enable them to compete effectively at low temperatures andunder the specific physicochemical regimes that prevail (e.g., Brown et al. 2012).

Molecular analyses offer insight into microbial communities because they can canvass largecross sections of the community (e.g., pyrotag sequencing of SSU rRNA genes) and particularlybecause they report on the whole community irrespective of whether the microorganisms areamenable to cultivation—the majority of which are not (Amann et al. 1995). However, althoughmolecular analyses have proven useful for studying sea-ice microorganisms, a high proportionof these communities are culturable and, hence, amenable to laboratory study. Antarctic isolatesinclude members of the genera Arthrobacter, Colwellia, Gelidibacter, Glaciecola, Halobacillus,Halomonas, Hyphomonas, Marinobacter, Planococcus, Pseudoalteromonas, Pseudomonas, Psychrobacter,Psychroflexus, Psychroserpens, Shewanella, and Sphingomonas (Bowman et al. 1997a–c, 1998a,b).Sea-ice communities have adapted to a range of location-specific physicochemical conditions,including temperature (0 to −35◦C), salinity (up to seven times seawater salinity), pH, light, andnutrient gradients (Eicken 2003, Mock & Thomas 2005).

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PHYSIOLOGICAL ADAPTATIONS IN PSYCHROPHILES

Overview

Physiological adaptations to growth temperature can be identified by comparing the propertiesof microorganisms that grow naturally at different temperatures. However, compared with pro-tein adaptation (see below) where insight can be gained by comparing the properties of proteinsbetween psychrophiles and hyper/thermophiles, physiological adaptation is more complicatedowing to the greater number of factors that can impact the complex variety of components in acell and ultimately cause an adaptive response. The cell’s physiology is dictated by its genomiccomplement of genes and the regulation of gene expression in response to environmental stimuli.Depending on the environment, a large number of biotic (e.g., predation by grazers and viruses,antibiotics, cell-cell interactions), abiotic (e.g., pH, salinity, oxygen, nutrient flux), and broaderecological factors (e.g., sea ice versus seawater, particle attached versus free living) can greatlyinfluence the selection and growth properties of individual microorganisms. In addition, the di-versity of microorganisms colonizing Earth’s biosphere, the majority of which is cold, is enormous.As a result, a variety of physiotypes have evolved to colonize cold environments successfully. Inaddition, very few classes of microorganisms that can successfully colonize both low- and high-temperature extremes have evolved. Methanogens, which are members of Archaea, are the onlygroup known to have individual species that span the growth temperature range from subzero to122◦C (Saunders et al. 2003, Cavicchioli 2006, Reid et al. 2006, Takai et al. 2008). Thus, thereare limited opportunities to compare the adaptive traits of psychrophiles and hyper/thermophilesthat belong to the same genus or family.

As a result, most of our knowledge about physiological adaptations has been gained by ex-amining the response of individual microorganisms to different growth temperatures (e.g., highversus low temperature). In this respect, global expression studies (e.g., proteomics, transcrip-tomics) linked to knowledge of direct physiological measurements (e.g., temperature and nutrientperturbation of morphology, growth rate, rates of macromolecular synthesis, solute composition,membrane lipid composition, modification of nucleic acids) have proven particularly valuablefor determining the mechanisms of psychrophile adaptation (see, for example, Cavicchioli 2006).Examples of knowledge gained are described below.

Cellular Mechanisms of Cold Adaptation

Low temperature can impede transcription and translation owing to the increased stability ofadventitious secondary structures of transcripts. Preventing or resolving inhibitory secondarystructures of RNA can be achieved by RNA chaperones. Cold shock proteins (Csps) are smallproteins that bind to RNA to preserve its single-stranded conformation ( Jones & Inouye 1994).DEAD box RNA helicases are capable of unwinding secondary structures in an ATP-dependentmanner and are upregulated during cold growth in some psychrophiles (Lim et al. 2000). Psy-chrophiles vary widely in the number of csp genes present in their genomes (Table 1). Csps containa nucleic-acid-binding domain, known as the cold shock domain (CSD), and have additional rolesbesides serving as RNA chaperones. Individual CSD-containing proteins can regulate the coldshock response or play a major role in subsequent growth at low temperatures in mesophiles(Hebraud & Potier 1999). Thus, many of the Csps act as cold-adaptive proteins in psychrophiles,because they are constitutively rather than transiently expressed at low temperatures (D’Amicoet al. 2006). Overexpression of cspA of Psychromonas arctica was shown to increase cold resistanceof Escherichia coli at low temperatures ( Jung et al. 2010). Additionally, one of three Csps appearsto be important in the low-temperature growth of Shewanella oneidensis (Gao et al. 2006).

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Table 1 Characteristics of selected bacterial and archaeal psychrophiles

Species and strain Origin of strain Type Phylogeny

csp orctr

genesaTotalgenes

Genomesize (Mb)

Cenarchaeumsymbiosum A

Marine spongesymbiont, offCalifornia coast

Eurypsychrophilicarchaeon

Crenarchaeota (orThaumarchaeota),Marine Group I,Cenarchaeales

1 csp 2,066 2.05

Colwellia psychrerythraea34H

Arctic marinesediments, offGreenland

Stenopsychrophilicbacterium

Proteobacteria,Gammaproteobacteria,Alteromonadales

4 csp 5,066 5.37

Desulfotalea psychrophilaLSv54

Arctic marinesediments, offSvalbard

Eurypsychrophilicbacterium

Proteobacteria,Deltaproteobacteria,Desulfobacterales

7 csp 3,332 3.66

Exiguobacteriumsibiricum 255–15

Permafrost,Siberia, Russia

Eurypsychrophilicbacterium

Firmicutes, Bacilli,Bacillales

6 csp 3,151 3.04

FlavobacteriumpsychrophilumJIP02/86

Fish pathogen Eurypsychrophilicbacterium

Bacteroidetes,Flavobacteria,Flavobacteriales

1 csp 2,505 2.86

Halorubrumlacusprofundi ATCC49239

Deep Lakesediments,Antarctica

Eurypsychrophilicarchaeon

Euryarchaeota,Halobacteria,Halobacteriales

3 csp 3,725 3.69

Idiomarina loihiensisL2TR

Hydrothermalvent, LoihiSeamount, offHawai’i

Eurypsychrophilicbacterium

Proteobacteria,Gammaproteobacteria,Alteromonadales

2 csp 2,706 2.84

Listeria monocytogenesLO28

Foodbornepathogen

Eurypsychrophilicbacterium

Firmicutes, Bacilli,Bacillales

2 csp 2,455 2.91

Mariprofundusferrooxydans PV-1

Hydrothermalvent, LoihiSeamount, offHawai’i

Eurypsychrophilicbacterium

Proteobacteria,Zetaproteobacteria,Mariprofundales

2 csp 2,920 2.87

Methanococcoidesburtonii DSM 6242

Ace Lakesediments,Antarctica

Eurypsychrophilicarchaeon

Euryarchaeota,Methanomicrobia,Methanosarcinales

3 ctr 2,506 2.58

Octadecabacterantarcticus 307

Sea ice offAntarctica

Stenopsychrophilicbacterium

Proteobacteria,Alphaproteobacteria,Rhodobacterales

3 csp 5,544 4.91

Photobacteriumprofundum SS9

Sulu Troughdeep-seasediments

Stenopsychrophilicbacterium

Proteobacteria,Gammaproteobacteria,Vibrionales

8 csp 5,754 6.40

Polaribacter irgensii23-P

Subsurfaceseawater, offAntarctica

Stenopsychrophilicbacterium

Bacteroidetes,Flavobacteria,Flavobacteriales

3 csp 2,602 2.75

(Continued)

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Table 1 (Continued)

Species and strain Origin of strain Type Phylogeny

csp orctr

genesaTotalgenes

Genomesize (Mb)

Polaromonasnaphthalenivorans CJ2

Coal-tar-contaminatedsurfacesediments fromSouth GlensFalls, New York

Eurypsychrophilicbacterium

Proteobacteria,Betaproteobacteria,Burkholderiales

1 csp 5,000 5.37

Pseudoalteromonashaloplanktis TAC125

Subsurfaceseawater, offAntarctica

Eurypsychrophilicbacterium

Proteobacteria,Gammaproteobacteria,Alteromonadales

9 csp 3,634 3.85

Psychrobacter arcticus273–4

Permafrost,Siberia, Russia

Eurypsychrophilicbacterium

Proteobacteria,Gammaproteobacteria,Pseudomonadales

3 csp 2,215 2.65

Psychrobacter cryohalentisKS

Permafrost,Siberia, Russia

Eurypsychrophilicbacterium

Proteobacteria,Gammaproteobacteria,Pseudomonadales

4 csp 2,582 3.10

Psychroflexus torquisATCC 700755

Sea ice algalassemblage, offAntarctica

Stenopsychrophilicbacterium

Bacteroidetes,Flavobacteria,Flavobacteriales

2 csp 6,835 6.01

Psychromonasingrahamii 37

Sea ice, offnorthern Alaska

Stenopsychrophilicbacterium

Proteobacteria,Gammaproteobacteria,Alteromonadales

12 csp 3,877 4.56

Rhodoferax ferrireducensT118

Aquifersediments,Virginia

Eurypsychrophilicbacterium

Proteobacteria,Betaproteobacteria,Burkholderiales

0 4,561 4.97

Shewanella oneidensisMR-1

Lake Oneidasediments,New York

Eurypsychrophilicbacterium

Proteobacteria,Gammaproteobacteria,Alteromonadales

4 csp 4,657 5.13

Shewanella violaceaDSS12

Ryukyu Trench,deep-seasediments

Stenopsychrophilicbacterium

Proteobacteria,Gammaproteobacteria,Alteromonadales

6 csp 4,515 4.96

aAbbreviations: csp, cold shock protein; ctr, cold-responsive TRAM protein.

Not all bacteria and archaea capable of growing at low temperatures have known homologs ofCsps (Table 1). For example, Rhodoferax (Albidoferax) ferrireducens lacks identifiable csp genes, eventhough csp genes are present in other members of the Burkholderiales (Betaproteobacteria), includingPolaromonas strains. csp genes are present in the archaea Methanogenium frigidum (stenopsy-chrophile) and Halorubrum lacusprofundi (eurypsychrophile) but absent from Methanococcoidesburtonii, a eurypsychrophilic archaeon isolated from the same Antarctic lake as M. frigidum(Giaquinto et al. 2007). For M. burtonii, small proteins composed of a single RNA-bindingTRAM domain were upregulated at low temperatures and proposed to serve as RNA chaperonesin an analogous manner to Csps (Williams et al. 2010a, 2011). These putative RNA chaperoneshave been termed Ctr (cold-responsive TRAM domain) proteins and are unique to a subset ofarchaea (Table 1). The abundance of Ctr proteins in M. burtonii is particularly high at very lowgrowth temperature (−2◦C), and a role in facilitating cell function during cold stress has been

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proposed (Williams et al. 2011). The upregulation of Ctr proteins in M. burtonii in response togrowth in the presence of the solvent methanol further suggests a wider role in the cell as stressresponse proteins (Williams et al. 2010a).

Small RNA-binding proteins (Rbps) can facilitate cold adaptation, but similar to Csps, theycan also have other functional roles in the cell (Maruyama et al. 1999, Christiansen et al. 2004).These Rbps accumulate following cold stress and play important roles in regulating transcriptiontermination (Mori et al. 2003), Rbps are small proteins that contain a single glycine-rich RNA-binding motif. They are prevalent in cyanobacteria but rare in other bacteria (Maruyama et al.1999, Ehira et al. 2003). The mesophilic cyanobacterium Anabaena variabilis has eight rbp genes,all but one of which are cold regulated (Maruyama et al. 1999). Osmotic stress also enhances rbpgene expression in Anabaena sp. PCC 7120: Responses to cold and osmotic stresses overlap becausethey both decrease the availability of free water (Mori et al. 2003). Rbp proteins may also play arole in thermal adaptation in psychrophilic cyanobacteria, as expression of rbp genes increases atlow temperatures in the Antarctic strain Oscillatoria sp. SU1 (Ehira et al. 2003).

Nucleoside modifications can affect the stability of tRNA. As a result, the extent of modificationtends to be high in hyperthermophilic archaea and bacteria (Dalluge et al. 1997, Noon et al. 2003).However, dihydrouridine can enhance tRNA flexibility and is elevated in some psychrophilicbacteria and archaea (Dalluge et al. 1997, Noon et al. 2003).

Enzymes involved in the degradation of RNA and proteins are upregulated during low-temperature growth in some psychrophilic bacteria and archaea, including RNases and pro-teases from the permafrost bacterium Psychrobacter arcticus (Bergholz et al. 2009) and M. burtonii(Williams et al. 2010b). This has been interpreted as a strategy to conserve biosynthetic precursors(Bergholz et al. 2009) or as enhanced quality control of irreparably damaged RNA and proteins(Williams et al. 2010b), although the two are not mutually exclusive.

Energy conservation and biosynthetic pathways can be regulated in response to low-temperature growth. Psychrobacter cryohalolentis, a eurypsychrophilic bacterium isolated fromSiberian permafrost, increases the cytoplasmic pool of ATP and ADP to offset reduced ATP-dependent reaction rates (Amato & Christner 2009). Specific carbon substrate utilization pathways(e.g., methanol versus trimethylamine) are differentially regulated with growth temperature in M.burtonii (Williams et al. 2010a,b). In P. arcticus, a large number of energy metabolism genes aredownregulated at low temperatures (Bergholz et al. 2009), whereas P. cryohalolentis shows upreg-ulation of glyoxylate cycle enzymes (Bakermans et al. 2007). These examples highlight the varietyand complexity of metabolic responses of individual psychrophiles.

At temperatures low enough for ice to form, cells are subjected to additional stressors such asice damage, oxidative insult, and osmotic imbalance (Tanghe et al. 2003; Williams et al. 2010b,2011). Extracellular polymeric substances (EPS) can offer protection against mechanical disruptionto the cell membrane caused by ice. Sea-ice bacteria such as Colwellia psychrerythraea producepolysaccharide-rich EPS (Thomas & Dieckmann 2002, Junge et al. 2004). The resulting biofilmsmay afford protection against invasive ice crystal damage as well as facilitate the acquisition ofnutrients within the channels that form within the sea ice (Thomas & Dieckmann 2002; Jungeet al. 2004; Mancuso Nichols et al. 2005a,b). At low temperatures, psychrophilic archaea such as H.lacusprofundi and M. burtonii also form multicellular aggregates embedded in EPS (Reid et al. 2006).

Low temperatures decrease membrane fluidity and permeability. In response, the elastic liq-uid crystalline nature of the cell membrane is replaced by a gel-phase state that can impair thebiological functions of the membrane, including transport (Phadtare 2004). This can be offset byincreasing the proportion of unsaturated fatty acids in the lipid bilayer, resulting in a more looselypacked array (Russell 2008). Increasing the proportion of unsaturated fatty acids can be achievedby decreasing the saturation of pre-existing fatty acids or by synthesizing fewer saturated fatty acids

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de novo. The eurypsychrophilic bacterium Exiguobacterium sibiricum has higher fatty acid desat-urase gene expression at low temperatures (Ponder et al. 2005, Rodrigues et al. 2008). M. burtonii,which lacks a fatty acid desaturase, alters expression of several lipid biosynthesis genes, resultingin fewer saturated isoprenoid lipid precursors (Nichols et al. 2004). Unsaturated isoprenoid lipidshave also been detected in H. lacusprofundi (Gibson et al. 2005). Many psychrophilic membersof Gammaproteobacteria (e.g., species of Colwellia, Moritella, Photobacterium, Psychromonas, Mari-nomonas, and Shewanella) are characterized by a high proportion of unsaturated fatty acids in theircell membranes (Margesin & Miteva 2011). In a metagenomic analysis, a microbial assemblage inglacier ice was found to be relatively enriched for genes involved in the maintenance of membranefluidity (Simon et al. 2009). Membrane lipid changes appear to be a generally conserved featurefor cellular adaptation to the cold.

Adaptation of Psychrophiles Viewed Through Genomes and Global GeneExpression Profiles

Many of the advances in understanding adaptive mechanisms have come from studies involvingthe genome sequences of psychrophiles. Approximately 30 bacterial and 4 archaeal genomesequences are available for psychrophiles originating from diverse cold habitats that includeAntarctic lakes, symbionts of sea sponges, marine sediment, permafrost, marshes, fish pathogens,and Kimchi (Lauro et al. 2011a). In addition to providing genomic blueprints that describethe capacity of psychrophiles, genomes provide the basis for targeted and global functionalstudies (e.g., proteomics and transcriptomics). The capacity to overview global responses isgreatly accelerating the ways in which knowledge is being gained about adaptive mechanisms, inparticular, as researchers define general characteristics of psychrophilic microorganisms versusspecific traits of individual psychrophiles.

Good illustrations of what can be defined by these approaches include recent analyses of expres-sion profiles across multiple growth temperatures. An analysis of P. arcticus (growth temperaturerange from −10◦C to 28◦C) used transcriptomics to identify differences in mRNA abundancebetween four growth temperatures (−6, 0, 17, and 22◦C) (Bergholz et al. 2009), and a multiplexproteomics study of M. burtonii quantitated changes occurring across seven growth temperaturesthat span the organism’s complete growth temperature range (−2◦C to 28◦C) (Williams et al.2011) (Figure 3). In the latter study, by including growth temperature extremes as well as tem-peratures in between, researchers were able to infer stressful versus nonstressful physiologicalstates. Interestingly, the upregulation of oxidative stress proteins at both upper and lower tem-perature extremes demonstrated the important, yet distinct, ways in which temperature-inducedoxidative stress manifests in the cell. The study also revealed that protein profiles at temperaturesin which M. burtonii grew fastest (Topt) were similar to those at maximum growth temperature(Tmax). These findings highlighted the extent to which this psychrophile was heat stressed at thesetemperatures, which is consistent with a number of other studies that suggest that psychrophilesgrowing at Topt are likely to be heat stressed (Feller & Gerday 2003; Bakermans & Nealson 2004;Goodchild et al. 2004; Cavicchioli 2006; Williams et al. 2010b, 2011).

PROTEIN ADAPTATION TO THE COLD

Overview

Many types of proteins, including diverse classes of enzymes (e.g., glucanases, hydrolases, oxidore-ductases, hydrogenases, isomerases, nucleic acid-modifying enzymes), have evolved to function

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e–

e–e–

TT

GalT

Glycosylation

Ig-likeprotein

Mxal-likeprotein

YVTN/NHL(β propeller)

protein

METHANOGENESIS

AMINO ACIDMETABOLISM

Flavoproteins

Biomass

Energy

Biomass

Energy

MdrA Isf

Hcp

Sm-like

ClpB

DnaJ

DnaK

Catalase

CatalaseDUF1608

UspA

RadA

FMN reductase

Proteasome

Exosome

Ctr (TRAM) proteins

RNAhelicase

PPlase

S-layer proteins

Cohesin anddockerin proteins

Ribosome

Chaperonincomplex

Misfolded proteins

Denatured protein

S-layerS-layerS-layer

CYTOPLASM

CYTOPLASMIC MEMBRANE

QUASIPERIPLASMIC SPACE

Superoxide reductase

Superoxidereductase

ROS

mRNA

RNaseDNA

NH3

Redoximbalance

–2°CCOLD STRESS

1–16°CCOLD ADAPTATION

23–28°CHEAT STRESS

SPFH

Figure 3Temperature-dependent physiological states in the Antarctic archaeon, Methanococcoides burtonii. Shown are the cellular processes mostinfluenced during cold stress (−2◦C), cold adaptation (1, 4, 10, and 16◦C), and heat stress (23 and 28◦C) states of the cell.Abbreviations: ClpB, chaperone; Ctr, cold-responsive TRAM protein; DnaK/DnaJ, chaperones; DUF1608, S-layer protein containingdomain of unknown function; e−, electron (or reducing equivalent); FMN, flavin mononucleotide; GalT, galactose-1-phosphateuridylyltransferase; Hcp, hybrid-cluster protein; Isf, iron-sulfur flavoprotein; MdrA, protein disulfide reductase; mRNA, messengerRNA; MxaI-like, methanol dehydrogenase small subunit homolog; PPIase, peptidyl-prolyl cis/trans isomerase; RadA, DNA repairprotein; RNase, ribonuclease; ROS, reactive oxygen species; Sm-like, RNA-binding protein homolog; SPFH, degradation-relatedprotein; UspA, universal stress protein A; YVTN/NHL, S-layer protein containing cell adhesion domain. Reproduced with permissionfrom Williams et al. (2011) (Society for Applied Microbiology and Blackwell Publishing Ltd).

effectively at temperatures ranging from subzero to well above 100◦C (Adams & Kelly 1994,Demirjian et al. 2001, Siddiqui & Cavicchioli 2006). By comparing the structure, activity, andstability properties of the same type of proteins (preferably orthologs with high sequence identity)from different thermal classes, investigators have gained useful insight into how proteins evolvedand what features appear to be important for conferring specific thermal properties.

Studies have involved characterization of enzymes purified from representative organisms aswell as genomic surveys of the protein complement. In recent years, genomics has been applied

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to microbial communities from whole environmental samples (metagenomics), thereby providingDNA sequence information for proteins from uncultivated microorganisms. Metagenomics ofsamples from cold environments has included the generation of large data sets obtained by shotgunsequencing (e.g., Lopez-Bueno et al. 2009, Lauro et al. 2011b, Yau et al. 2011, Brown et al. 2012,Varin et al. 2012, Wilkins et al. 2012a, Williams et al. 2012b) and functional screening of clonesfor cold-active enzymes (e.g., Elenda et al. 2007, Kim et al. 2009). Genomic and metagenomicanalyses facilitate subsequent targeted analyses to assess specific features of individual proteins (e.g.,site-directed mutagenesis). Broad-spectrum modification (e.g., mutagenesis by directed evolution,chemical modification of particular amino acid side groups) and assessment of changes in thermalproperties of individual enzymes have also been used to identify structural properties that play rolesin conferring thermal activity/stability (Cavicchioli et al. 2006, Siddiqui et al. 2006). Collectively,these types of studies have revealed a great deal about the adaptation of proteins to temperature.

To achieve sufficient structural flexibility to afford enzyme activity at low temperatures, en-zymes have evolved specific compositional biases (i.e., amino acid composition) and secondary,tertiary, and/or quaternary structural properties (Feller & Gerday 2003, D’Amico et al. 2006,Siddiqui & Cavicchioli 2006, Feller 2008). In contrast, proteins from hyper/thermophiles requiresufficient structural rigidity to resist unfolding, which is also manifested through specific com-positional and structural properties (Daniel et al. 2008). In general terms, the features associatedwith adaptation (e.g., proportion of specific amino acids, hydrophobicity of exposed surfaces) tendto have opposite trends between proteins from psychrophiles and those from hyper/thermophiles(Siddiqui & Cavicchioli 2006, Feller 2008).

Proteins from psychrophiles have higher activity and thermolability compared with mesophilicand thermophilic homologues (Demirjian et al. 2001, Siddiqui & Cavicchioli 2006). For exam-ple, α-amylases from the psychrophilic bacterium Pseudoalteromonas haloplanktis and from thethermophilic bacterium Bacillus amyloliquefaciens have an optimal temperature of activity (Topt)of 28◦C and 84◦C, respectively (D’Amico et al. 2003). A striking example of cold adaptation isalanine racemase from Bacillus psychrosaccharolyticus, which has a Topt of 0◦C (Okubo et al. 1999). Be-cause low-temperature environments present significant problems for enzyme and, more broadly,protein function, the unique properties of cold-active enzymes has attracted both academic andcommercial interest (Cavicchioli et al. 2002, Feller & Gerday 2003, Cavicchioli & Siddiqui 2006,Siddiqui & Cavicchioli 2006, Feller 2008, Cavicchioli et al. 2011). This has led to rapid growthin the description of enzymes from a broad range of psychrophiles, with a concomitant devel-opment of biochemical and biophysical approaches attuned to their characterization (Feller &Gerday 2003, Cavicchioli et al. 2006, Siddiqui & Cavicchioli 2006). Below we discuss some of themechanisms by which thermal adaptation at low temperatures is attained.

Mechanisms of Enzyme Adaptation to the Cold

In low-temperature environments, there is insufficient kinetic energy to overcome enzyme acti-vation barriers, thus resulting in very slow rates of chemical reactions. For a biochemical reactionoccurring in a mesophile at 37◦C, a drop in temperature from 37◦C to 0◦C results in a 20–80-foldreduction in enzyme activity. This is the main factor preventing growth at low temperatures.However, organisms adapted to low temperatures have evolved several ways to overcome thisconstraint, including the energetically costly strategy of enhanced enzyme production (Crawford& Powers 1992) and seasonal expression of isoenzymes (Somero 1995). However, the most com-mon adaptive feature of cold-active enzymes is a reaction rate (kcat) that is largely independentof temperature. The majority of psychrophilic enzymes achieve temperature-insensitive kcat bydecreasing the activation energy barrier between the ground state (substrate) and activated state

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Table 2 Activity-stability relationship of some thermally adapted enzymesa

Enzyme kcat (min−1) Km (mM)Topt

(◦C) Tm (◦C) t1/2 (min) Referenceα-AmylasePsychrophileMesophileThermophile

(10◦C)17,6405,820840

(10◦C)0.230.06–

285384

445286

0.23 (43◦C)0.23 (60◦C)0.23 (80◦C)

D’Amico et al. 2003

CellulasePsychrophileMesophile

(4◦C)110.6

(4◦C)6.01.5

3756

––

(45◦C)40Unaffected

Garsoux et al. 2004

AminopeptidasePsychrophileMesophile

(10◦C)950114

––

3949

4758

(46◦C)1100,000

Huston et al. 2008

ImidasePsychrophileMesophile

(25◦C)25,7001,500

(25◦C)1.61.0

55>65

––

(40◦C)1502,880

Huang & Yang 2003

Lactate dehydrogenasePsychrophileThermophile

13,800 (0◦C)105,000 (44◦C)40,500 (90◦C)

0.16 (0◦C)0.41 (44◦C)0.16 (90◦C)

5090

5090

–––

Coquelle et al. 2007

Alkaline phosphatasePsychrophileMesophile

(37◦C)48,7406,954

(37◦C)0.130.11

4056

––

(50◦C)1038

Siddiqui et al. 2004b

akcat , turnover number of substrate molecules per minute per active site. Km, affinity for substrate; lower values imply higher binding affinity. Topt

(optimum temperature), temperature at which maximum enzyme activity is observed. Tm (melting temperature), temperature at which 50% of theprotein structure is in an unfolded state. t1/2 (half-life of inactivation), time needed to lose 50% of the enzyme activity at a specified temperature. Dashesindicate data not available.

(TS#). For example, reducing the activation energy from 70 kJ mol−1 for a thermophilic α-amylaseto 35 kJ mol−1 for a psychrophilic α-amylase enhanced kcat by 21-fold at 10◦C (D’Amico et al.2003). To aid substrate binding at a low energy cost, the active sites of cold-active enzymes tendto be larger and more accessible to substrates. As a result, the binding affinity of substrates forcold-active enzymes is generally lower (higher Km) than that of their thermophilic counterparts(Siddiqui & Cavicchioli 2006).

High rates of catalysis at low temperatures are generally achieved by the flexible structure andconcomitant low stability of cold-active enzymes, which is referred to as an activity-stability trade-off (Siddiqui & Cavicchioli 2006) (Table 2). Many cold-active enzymes have a more labile andflexible catalytic region than does the remainder of the protein structure, i.e., localized flexibility(Siddiqui et al. 2005, Feller 2008). Accordingly, in an environment characterized by low kineticenergy and retarded molecular motion, cold-active enzymes rely on greater disorder as a means ofmaintaining molecular dynamics and, hence, function (Feller 2007). For example, a psychrophilicalanine racemase that is very active at low temperatures and has very low thermal stability wasfound to have a hydrophilic region located at a surface loop surrounding the active site (Okubo et al.1999). The surface hydrophilic and polar regions are likely to promote solvent interactions, therebyreducing compactness and destabilizing the enzyme (Okubo et al. 1999, Siddiqui & Cavicchioli2006).

The α-amylase from P. haloplanktis, AHA, has become a model to study the structure, function,and stability relationship in cold-adapted enzymes (D’Amico et al. 2001, 2003; Feller & Gerday2003; Siddiqui & Cavicchioli 2006; Siddiqui et al. 2005, 2006; Feller 2008). Collectively, the studies

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indicate that the structure of AHA has evolved to have relatively few electrostatic interactions inorder to provide sufficient conformational flexibility to afford activity at low temperatures, whileretaining a sufficient level of overall protein structural integrity.

Genomic analyses of psychrophilic archaea have revealed proteins characterized by a highercontent of noncharged polar amino acids (especially Gln and Thr), a lower content of hydrophobicamino acids (particularly Leu), increased exposure of hydrophobic residues, and a decreased chargethat is associated with destabilizing the surface of psychrophilic proteins (Saunders et al. 2003).Evolutionary selection of amino acid usage enabled such adaptation (Allen et al. 2009). Somewhatdifferent trends have been noted via genome surveys of marine Gammaproteobacteria where cold-adapted strains were reported to have lower contents of Ala, Arg, and Pro as well as higher contentsof Ile, Lys, and Asn (Zhao et al. 2010). Among these, Pro and Arg are associated with an abilityto confer increased stability by restricting backbone rotations and by forming multiple hydrogenbonds and salt bridges, respectively (Feller & Gerday 2003).

Psychrophilic proteins are characterized by decreased core hydrophobicity, increased surfacehydrophobicity, increased surface hydrophilicity, a lower arginine/lysine ratio, weaker interdo-main and intersubunit interactions, more and longer loops, decreased secondary structure con-tent, more glycine residues, fewer prolines in loops, more prolines in α-helices, fewer and weakermetal-binding sites, fewer disulfide bridges, fewer electrostatic interactions (H-bonds, salt bridges,cation-pi interactions, aromatic-aromatic interactions), reduced oligomerization, and an increasein the conformational entropy of the unfolded state (Siddiqui & Cavicchioli 2006). Some cold-adapted proteins also tend to have flexible 5-turn and strand secondary structures, and they possesslarge cavities lined predominantly by acidic residues to accommodate water molecules (Paredeset al. 2011). However, although the abovementioned structural features can be associated withpsychrophilic proteins, any one protein will have a limited number of, and specific context for,these structural features (Siddiqui & Cavicchioli 2006).

Other Factors Influencing Enzyme Adaptation

A cell’s cytoplasm contains high concentrations of both low- and high-molecular-weight com-pounds that lead to molecular crowding (Chebotareva et al. 2004), and under natural environ-mental conditions, microorganisms are often exposed to more than one abiotic constraint (seealso Physiological Adaptations in Psychrophiles, above). Consistent with this, the stability andactivity of enzymes are affected by the presence of organic solutes (amino acids and sugars) andpolymers (proteins and polysaccharides) (Thomas et al. 2001, Siddiqui et al. 2002, Somero 2003,Faria et al. 2008), protein-protein interactions (Thomas et al. 2001), viscosity of the intracellularand extracellular environment (Demchenko et al. 1989, Siddiqui et al. 2004a, Karan et al. 2012),and the combined effects of temperature and pressure (Saito & Nakayama 2004, Kato et al. 2008)or temperature and salt (Srimathi et al. 2007, Yan et al. 2009).

A limited number of heat-labile enzymes can also be cold-labile enzymes near or below subzerotemperatures (D’Amico et al. 2003, Xu et al. 2003), and some oligomeric or cofactor that requiresenzymes (e.g., tryptophanase) can be reversibly inactivated at lower temperatures as a result ofsubunit and cofactor dissociation (Kogan et al. 2009). Therefore, if a key cellular enzyme is coldinactivated or cold denaturated, it could define the lower temperature limit for growth rather thanthe freezing point of the aqueous environment in which the organism grows.

COLD-ADAPTED ENZYMES AND CLIMATE CHANGE

A major source of CO2 input into the atmosphere is caused by the microbial decomposition of soilorganic matter (SOM) (German et al. 2012). Predictions are that the carbon sequestered in SOM

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is at least four times higher than the carbon content in the atmosphere and living plants. Globalwarming has a particularly strong effect on polar and alpine environments, wherein ∼30% of theglobal soil carbon pool resides. The degradation of cellulose, hemicellulose, and humic substancesin SOM by extracellular enzymes (e.g., glucanases, ligninases) into dissolved organic compoundsrepresents the rate-limiting step in carbon release (Weedon et al. 2011, German et al. 2012).The kinetic and thermodynamic properties of extracellular enzymes, including their responsesto environmental factors (e.g., nutrient supply, nitrogen and oxygen availability, phenolics andsubstrate concentration, soil moisture, permafrost melting, and temperature), are now beginningto be incorporated into predictive models describing the effects of global warming on carboncycling (Davidson & Janssens 2006, Weedon et al. 2011, German et al. 2012).

In view of such issues associated with global warming, it is important to recognize that cold-adapted enzymes work efficiently at low temperatures and therefore help to reduce CO2 emissionsby reducing electricity consumption associated with heating (Cavicchioli et al. 2002, 2011). Forexample, washing machines utilize a high proportion of a household’s electricity budget, and∼80% of the electricity is used to heat water (Nielsen 2005). Using cold-active enzymes, washingtemperatures can be reduced from 40◦C to 30◦C, resulting in a 30% decrease in electricity usage.Importantly, washing temperatures set 10◦C lower reduces the CO2 emissions associated with theburning of fossil fuels for energy generation by 100 g per wash (Nielsen 2005). The application ofcold-adapted enzymes in a range of other industries such as textile, food, waste-water treatment,and paper and pulp also helps to reduce toxic by-products, electricity usage, and CO2 emissions(EuropaBio Rep. 2009, Cavicchioli et al. 2011).

MICROBIAL EXTREMES, COLD-ACTIVE ENZYMES,AND ASTROBIOLOGY

The deep sea offers a unique perspective on cold environments (Figure 4), but more manned expe-ditions to outer space have been performed than trips to the deepest reaches of the ocean. There-fore, the experience gained in overcoming issues with deep-sea exploration may translate to thedevelopment of tractable systems for biological exploration of extraterrestrial environments. Sam-pling cold deep-sea environments is logistically challenging, particularly at depths below 6,000–8,000 m, where the length of wire cable that can be carried on an oceanographic vessel is exceeded(Lauro & Bartlett 2008). As a result, in addition to the use of cable-tethered Niskin bottles for sam-ple collection (Martin-Cuadrado et al. 2007), autonomous underwater vehicles (e.g., Takami et al.1997) and free vehicles (e.g., Eloe et al. 2011b) have been developed. Arising from a limited numberof molecular studies that have been performed using such sampling designs (DeLong et al. 2006;Lauro & Bartlett 2008; Brown et al. 2009; Agogue et al. 2011; Eloe et al. 2010, 2011a), a high level ofmicrobial diversity has been identified in the deep sea. The microbiota include bacterial members ofAlpha-, Beta-, Delta-, Epsilon-, and Gammaproteobacteria as well as Actinobacteria, Bacteroidetes, Chlo-roflexi, Planctomycetes, and Verrucomicrobia. Also included are archaeal members of EuryarchaeotaMarine Groups II and III, Crenarchaeota Marine Group I, Methanopyri, and novel alveolate GroupsI and II of eucarya that include endoparasitic dinoflagellates. The capacity of microorganisms tothrive under a range of combined extremes, such as in the deep sea where adaptation to cold, highhydrostatic pressure, and nutrient limitation is required, broadens the horizons for the scope oflocations that may be considered in the search for extraterrestrial life (Cavicchioli 2002).

Cold-active enzymes may be useful for specific applications in studies aimed at searching forsigns of life in extraterrestrial environments where liquid water is known or inferred to exist,such as on Saturn’s (Enceladus) and Jupiter’s (Europa, Ganymede) icy moons; possibly Mars

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0

500

1,000

1,500

2,000

2,500

3,000

3,500

4,000– 4 0 4 8

PolarTemperateTropical

12 16 20 24 28

De

pth

(m

)

Temperature (°C)

Figure 4Annual mean temperature at ocean depths in the Southern Hemisphere. Plots are for temperature datacollected at 2.5◦S (tropical), 37.5◦S (temperate), and 67.5◦S (polar). Similar trends occur in the NorthernHemisphere. The plots were generated from data in Levitus (1982).

(Trent 2000, Cavicchioli 2002); and Saturn’s moon Titan, which reportedly contains nonpolarliquid (Ogino 2008) (Table 3). Nonenzymatic chemical reactions tend to be racemic, producingequal amounts of right- and left-handed enantiomers of a chiral molecule. However, enzymaticreactions tend to produce or incorporate homochiral forms (either right- or left-handed formsof a molecule), such as D-sugars and L-amino acids. Owing to these distinctions, homochiralitymay be useful as a biomarker. Polarimeters measure changes in optical rotation (change in left- orright-handedness of a chiral molecule) and may be useful for assessing changes taking place overtime in an extraterrestrial sample. Investigations into this type of application have been assessedthrough studies of mandelate (C8H7O3

−, R-2-hydroxy-2-phenylacetate), which is a simple chiralmolecule that is racemized by mandelate racemase in a reaction that has a very high enzymeconversion rate (kcat/kuncat = 2.3 × 1015) (Thaler et al. 2006).

Mandelate racemase from the mesophilic bacterium Pseudomonas putida has been reported to beactive at low temperatures (−30◦C) in the presence of cryosolvents such as saturated ammoniumsalts and water-in-oil microemulsions (Thaler et al. 2006). However, in water-miscible organiccosolvents, the enzyme is inactive owing to instability and very high Km (Cartwright & Waley 1987,Thaler et al. 2006). Psychrophilic enzymes not only are more efficient at low temperatures, but alsotend to be comparatively stable in mixed aqueous-organic or nonaqueous solvents. This derivesfrom their inherent flexibility, which counteracts the destabilizing effects of low water activity inorganic solvents (Owusu-Apenten 1999, Sellek & Chaudhuri 1999, Gerday et al. 2000). In fact,cold temperatures affect the properties of bulk water as well as the hydration shell surroundingthe protein surface. As temperature decreases, water molecules around a protein become moreordered and are less available to interact with the protein surface, thereby destabilizing the proteintoward the unfolded state. The loss of critical water molecules is one of the main reasons for theloss of activity in organic solvents. Cold-adapted enzymes tend to interact strongly with available

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Table 3 Characteristics of some planets and moons from Earth’s solar system with the potential toharbor psychrophilic lifea

Planet/moonAtmospheric, surface, and subsurface

composition Surface temperatureEarthb O2, N2, CO2

Water exists in all three states (gas, liquid,solid)

−89 to 58◦C

Mars CO2, N2

Polar water and CO2 ice caps−140 to 20◦C

Europa ( Jupiter) O2

Liquid water ocean may exist under surfaceice sheet

−223 to −148◦C

Ganymede ( Jupiter) O2

Water ice−203 to −121◦C

Callisto ( Jupiter) CO2 (99%), O2 (1%)Liquid water ocean may exist beneath itssurface

−193 to −108◦C

Titan (Saturn) N2, H2, CH4

CH4 and C2H6 exist in all three states asgas, liquid, and solid

−179◦C

Enceladus (Saturn) H2O, N2, CO2, CH4

Water ice−240 to −128◦C

aData taken from Chown (2011).bThe lowest temperature recorded on Earth was at the Russian Research Station, Vostok, Antarctica, on July 21, 1983.

water. As a result, the enzymes retain their activity in nonaqueous systems (Karan et al. 2012).Organic solvents also decrease the polarity of the medium Thus, the conditions of the mediumbecome more favorable for the buried hydrophobic core to interact with the surrounding medium,thereby causing unfolding of the protein. Enhanced stability in water-miscible organic solventscan be achieved by making the surface of the enzyme more hydrophobic (Siddiqui et al. 1999,Ogino 2008). Because cold-adapted enzymes contain a relatively high proportion of hydrophobicresidues on their surface (Siddiqui & Cavicchioli 2006), they tend to resist unfolding in organicsolvents. As a result, a mandelate racemase from a psychrophile is likely to be a good replacementfor the P. putida enzyme, finding application in the development of assays for use of polarimetersand possibly for use in the processing of extraterrestrial samples as a biosensor to detect thepresence of homochiral mandelate.

As discussed in the previous section (see Protein Adaptation to Low Temperature, above) cold-active enzymes achieve higher activities (kcat) by reducing the activation energy barrier betweenthe ground and the transition state. However, although enzymes from psychrophiles are active attheir environmental temperatures, selection pressures operate at the whole-cell level, and naturalenvironments do not tend to select for the maximum achievable low-temperature activity forevery cellular component. As a result, higher activities can be achieved for individual psychrophilicenzymes at low temperatures by artificially manipulating the enzymes. This capacity is relevant tothe application of enzymes for use in space or on extraterrestrial bodies where temperatures canbe much lower than those encountered on Earth.

Improvements in low-temperature activity can be achieved by bypassing enthalpy-entropycompensation. Enthalpy-entropy compensation implies that a decrease in �H# is accompanied

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by a decrease in �S# so that an overall small increase in kcat is achieved (Siddiqui & Cavicchioli2006). However, the gain in kcat would be massive if the decrease in �H# was not accompanied bya corresponding decrease in �S# or even more so if an increase in �S# occurred. Theoretically,by maintaining a constant �S# and decreasing �H# by only 20 kJ mol−1, a 50,000-fold increasein kcat would occur at 15◦C (Lonhienne et al. 2000). Experimental work has shown that theenthalpy-entropy compensation relationship does not always hold true in cold-adapted lipasesfrom Candida antarctica, particularly in supercritical CO2 and an organic solvent (3-hexanol) wherehigher activity was associated with both negative �H# and positive �S# (Ottosson et al. 2001,2002a,b). Supercritical fluids may function as useful, nonaqueous solvents for enzyme catalysis,and they occur naturally on some planets (Mesiano et al. 1999, Comm. Origins Evol. Life Natl.Res. Counc. 2007).

kcat of a cold-adapted enzyme could be further enhanced by simultaneously decreasing �H#

and increasing �S#; this condition could be achieved on an extraterrestrial body where a water-likepolar solvent is present by indirectly increasing the entropy of the system via solvent displacement(Wolfenden & Snider 2001, Snider et al. 2002). If more solvent molecules are released uponbinding to the transition state of the enzyme than upon binding to the ground-state substrate,then there will be considerable entropic benefit for the formation of an enzyme-transition-statecomplex that has a concomitant increase in activity (Wolfenden & Snider 2001).

To design highly active enzymes from antibodies (catalytic antibodies), reaction rates canbe enhanced by promoting the release of water from the binding pocket during formation ofthe transition state and thereby producing an increase in �S# (Houk et al. 2003). Similarly,an enhanced rate of reaction for ribosome-mediated peptide bond formation can be achievedby effective substrate positioning and/or by water exclusion from within the active site, whichcreates an increase in �S# (Wolfenden 2011). Therefore, in theory, enzyme reactions, biologicalprocesses, and metabolically active life may be achievable under very cold planetary conditions,provided that a decrease in �H# is accompanied by either no change or an increase in �S# duringenzyme catalysis (i.e., surmounting enthalpy-entropy compensation).

Although this review focuses on unicellular microorganisms, as a parting note we highlightthe remarkable properties of the small (∼0.1–1 mm in length) metazoans (panarthropods) calledtardigrades (“waterbears”). Tardigrades are adapted to multiple extremes, and in both their hy-drated (active) and dehydrated (tun) forms, they are resistant to very cold temperatures. Antarctictardigrades have survived exposure to −22◦C for 600 and 3,040 days in active and tun states,respectively, with some in their tun state surviving up to 14 days at −180◦C (Somme & Meier1995). Given their tolerance to cold and other extremes, tardigrades are recognized as valuablemetazoan models for astrobiological research (Horikawa et al. 2008): They were used aboard theFOTON-M3 mission to examine their resistance to the effects of outer space (LIFE-TARSEproject) (Rebecchi et al. 2009).

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

This work was supported by the Australian Research Council and the Australian Antarctic ScienceProgram. We thank Mark Milnes for the panoramic image of Deep Lake in Figure 2.

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