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EA41CH05-Cavicchioli ARI 30 April 2013 11:9

Psychrophiles

Khawar 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 Cavicchioli1

1School of Biotechnology and Biomolecular Sciences and 2Evolution and Research Center, The University of New South Wales, Sydney, New South Wales 2052, Australia; email: [email protected]

Annu. Rev. Planet. Sci. 2013. 41:87–115 Keywords First published online as a Review in Advance on microbial cold adaptation, cold-active , , microbial February 14, 2013 diversity, The Annual Review of Earth and Planetary Sciences is online at earth.annualreviews.org Abstract This article’s doi: Psychrophilic (cold-adapted) make a major contribution 10.1146/annurev-earth-040610-133514 to Earth’s biomass and perform critical roles in global biogeochemical cy- Copyright c 2013 by Annual Reviews. cles. The vast extent and environmental diversity of Earth’s cold biosphere All rights reserved has selected for equally diverse microbial assemblages that can include ar- Access provided by University of Nevada - Reno on 05/25/15. For personal use only.

Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org chaea, , eucarya, and viruses. Underpinning the important ecological roles of are exquisite mechanisms of physiological adaptation. Evolution has also selected for cold-active traits at the level of molecular adaptation, and enzymes from psychrophiles are characterized by specific structural, functional, and stability properties. These characteristics of en- zymes from psychrophiles not only manifest in efficient low-temperature activity, but also result in a flexible structure that enables biocatalysis in nonaqueous solvents. In this review, we examine the ecology of psychrophiles, physiological adaptation of psychrophiles, and properties of cold-adapted , and we provide a view of how these characteristics inform studies of .

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INTRODUCTION Much of 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 of the global biosphere (Feller & Gerday 2003, Cavicchioli 2006, Siddiqui & Cavicchioli 2006, Casanueva et al. 2010, Margesin & Miteva 2011). Consistent with representative size, the cold biosphere consists of diverse types of environments—vast tracts of the , geographically dis- persed alpine regions, geologically specific subterranean caverns, climatically challenged regions of , and biogeochemically diverse polar reaches (Figure 1). Proliferating throughout these cold realms is a plethora of psychrophilic (cold-adapted) microorganisms—, bacte- ria, eucarya, and viruses. A small proportion of the isolated microorganisms from naturally cold environments have a restricted growth temperature range with an upper growth temperature limit less than ∼20◦C (stenopsychrophile), whereas the majority of isolates have a broader temperature range, 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 DNA sequencing 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 metagenomics and associated meta-functional approaches (metaproteomics and metatranscriptomics) has shed light on whole microbial community composition dynamics and microbial processes that are be- ing driven by the resident psychrophiles. Genomic, physiological, and biochemical analyses of psychrophilic isolates and their cellular components have also gleaned valuable information about the diverse molecular mechanisms of cold adaptation. As a result, whether driven by global ques- tions concerning the impact of change on microbial communities in cold environments, fundamental studies of molecular structure and function, or biotechnologically driven pursuits of novel 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 providing knowledge about physiological and protein adaptation—characteristics that speak to fundamental principles of biological adaptation to the cold and provide insight into survivability. A perspective on of Antarctic systems opens the review, particularly focusing on , sea-ice, and deep-sea environments—systems that include a broad range of physicochemical conditions

Polar Alpine Extraterrestrial < 10°C e.g. Deep Lake, Deep sea e.g. Europa Access provided by University of Nevada - Reno on 05/25/15. For personal use only.

Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org Antarctica 1 to 4°C Surface: –200 to –160ºC –20°C Subsurface : ?ºC

Figure 1 Terrestrial 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 range of cold and abiotically varied environmental extremes. Also provided is a brief perspective on psychrophiles and global warming, providing a glimpse into the use of cold-active enzymes and its impact on psychrophiles in relation to change. The review concludes with a section reflecting on microbial extremes and cold-active enzymes and their relevance to astrobiology.

ANTARCTIC PSYCHROPHILES

Antarctic Aquatic Both southern and northern polar regions are delicately balanced ecosystems that are easily affected by ecosystem changes (Moline et al. 2004, Murray & Grzymski 2007, Wilkins et al. 2012b), and global warming is expected to cause changes that will flow through to right up the food chain (Kirchman et al. 2009). In the Antarctic, global warming has particularly impacted the 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, Hogg et al. 2011), and Antarctic sea-ice extent has decreased by at least ∼20% since the early 1950s and is 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 (Le Quer´ e´ et al. 2007), and reduced supply particularly at higher latitudes caused by increased stratification (Sarmiento & Le Quer´ e´ 1996, Wignall & Twitchett 1996, Matear & Hirst 1999) are all effects linked to global warming. As the ocean microorganisms are critical for sequestering

anthropogenic CO2 (Sabine et al. 2004, Mikaloff Fletcher et al. 2006) and transporting it to the benthic zones (Thomalla et al. 2011), the changes taking place in polar waters are of great concern for 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 maintain ice, water column, sediment, and communities (Wilkins et al. 2012b). These include subglacial, epiglacial, and surface systems that range in from fresh to saturated and from mixed to permanently stratified. The evolutionary history of these lakes is as varied as the 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); subglacial outflow from dating from 1.5 Mya (Mikucki et al. 2009); and waters in the depths of subglacial , 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 properties Access provided by University of Nevada - Reno on 05/25/15. For personal use only.

Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org of 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 parts of the water column (Lauro et al. 2011b) (Figure 2). However, this stratified system harbors vastly different communities in other parts of the lake where very different physicochemical conditions exist (Lauro et al. 2011b), including a highly purified population of green sulfur bacteria at the lake’s oxycline interface (Ng et al. 2010). The microbial communities in Lake Bonney have evolved in response to physical distinctions occurring in two different lobes of the lake (Glatz et al. 2006). Both of 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 extreme example of ecosystem change (Figure 2). Situated in the Vestfold Hills, Deep Lake is ∼55 m below sea 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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k Access provided by University of Nevada - Reno on 05/25/15. For personal use only. Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org

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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 (Bowman et al. 2000a). Ongoing studies of this system have identified a range of genomic traits and ecology of the system that are unique compared with hypersaline or cold aquatic systems elsewhere in the world (R. Cavicchioli, unpublished results). Subsurface lake systems include subglacial lakes, such as Lake Vostok (Siegert et al. 2001), and epiglacial lakes that result from 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 pursuit of studying such pristine systems is a significant logistical challenge, and lessons learned about drilling into Lake Vostok and other subglacial lakes (Inman 2005, Wingham et al. 2006, Alekhina et al. 2007, Lukin & Bulat 2011, Gramling 2012, Jones 2012) should provide wisdom for guiding contemplation of future endeavors, including extraterrestrial studies.

Antarctic Aquatic Microorganisms Our understanding of community composition in Antarctic aquatic systems has been greatly facilitated by molecular-based studies (Wilkins et al. 2012b). These have included analyses using denaturing 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; Schiaffino et 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; Ng et al. 2010; Lauro et al. 2011b; Yau et al. 2011; Brown et al. 2012; Grzymski et al. 2012; Varin et al. 2012; Wilkins et al. 2012a; Williams et al. 2012a,b). Molecular signatures of archaea have been detected in a range of Antarctic lakes, including strictly anaerobic and aerobic haloarchaea (Bowman et al. 2000a,b; Purdy et al. 2003;

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Figure 2 ◦   ◦   ◦   ◦   Antarctic 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 by only several hundred meters ( foreground ). Sea ice and icebergs are present in the early-mid austral summer 2008 (background ). Among other , 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) drifts on Ace

Access provided by University of Nevada - Reno on 05/25/15. For personal use only. Lake formed after a blizzard behind quad bikes (used for transport between the lake and Davis Research Base located 15 km away) and

Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org mobile work shelters (MWSs) that were used for sample collection and protection from the weather. (d ) Organic Lake, a hypersaline ◦ meromictic system where the waters are −13 C below the surface ice; photo taken in 2008 (Gibson 1999). The novel and important role of virophages was discovered in this lake (Yau et al. 2011). (e) Drilling through surface ice on Organic Lake prior to the positioning of 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) Deep Lake panorama in September 2008 after a cold winter (−40◦C) (photo credit: Mark Milnes). (h) Deep Lake is hypersaline, and water does not freeze despite reaching −20◦C. (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 Antarctic continental ice mass (background ). (k) MWSs, dinghy, and research equipment at Deep Lake. Water pumped into drums on board the dinghy at the center of the lake (∼800 m from shore) was transported back to the MWSs for processing. (l ) Brown Lagoon at the base of , 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 of contains slabs of recently melted glacier. The melted sections and large lake of water that were not present in previous 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; Kurosawa et al. 2010; Lauro et al. 2011b), of which several have been brought into axenic culture (Franzmann et al. 1988, 1992, 1997). A large number of studies have focused on Antarctic bacteria, and diverse taxa have been identified, including members of the groups Actinobacteria, , , Chlorobi, Chloroflexi, , Deltaproteobacteria, Firmicutes, , [Cytophaga-Flavobacterium- Bacteroides (CFB) group], Planctomycetes, Spirochaetes,andVerrucomicrobia (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 also important in Antarctic lakes, although fungi and silicoflagellates have also been identified (Unrein et al. 2005, Mosier et al. 2007, Bielewicz et al. 2011, Lauro et al. 2011b, Yau et al. 2011). Viruses of 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 viruses may 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; Sawstr¨ om¨ 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 predation of algal viruses, which is predicted to increase overall primary production and net carbon flow in the lake system (Yau et al. 2011) (Figure 2). In addition to the water column, rich microbial communities are found in Antarctic mats and can make important contributions to biomass and productivity (Vincent 2000, Moorhead et al. 2005, Laybourn-Parry & Pearce 2007). Microorganisms identified in Antarctic mats include members of Actinobacteria, CFB, Cyanobacteria, -, Firmicutes, fungi, green , Planctomycetes, ,andVerrucomicrobia (Brambilla et al. 2001; Van Trappen et al. 2002; Taton et al. 2003, 2006; Jungblut et al. 2005; Fernandez-Valiente´ et al. 2007; Sutherland 2009; 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 and biological records of the ecosystem, thereby also providing insight into the evolution of past and extant 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 tropical ocean waters and include a high proportion of Alphaproteobacteria (e.g., SAR11 clade), Flavobacteria, Gammaproteobacteria, and ammonia oxidizing Marine Group I (Wilkins et al. 2012b). However, although many common taxa are found, the indigenous Antarctic populations have genetic and physiological traits that enable them to compete effectively at low temperatures and under the specific physicochemical regimes that prevail (e.g., Brown et al. 2012). Access provided by University of Nevada - Reno on 05/25/15. For personal use only.

Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org Molecular analyses offer insight into microbial communities because they can canvass large cross sections of the community (e.g., pyrotag sequencing of SSU rRNA genes) and particularly because they report on the whole community irrespective of whether the microorganisms are amenable to cultivation—the majority of which are not (Amann et al. 1995). However, although molecular analyses have proven useful for studying sea-ice microorganisms, a high proportion of these communities are culturable and, hence, amenable to laboratory study. Antarctic isolates include members of the genera Arthrobacter, Colwellia, Gelidibacter, , Halobacillus, Halomonas, Hyphomonas, Marinobacter, Planococcus, Pseudoalteromonas, , , Psychroflexus, Psychroserpens, Shewanella,andSphingomonas (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 salinity), pH, light, and nutrient 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 properties of 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 proteins between psychrophiles and hyper/, physiological adaptation is more complicated owing to the greater number of factors that can impact the complex variety of components in a cell and ultimately cause an adaptive response. The cell’s physiology is dictated by its genomic complement 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, , nutrient flux), and broader ecological factors (e.g., sea ice versus seawater, particle attached versus free living) can greatly influence 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. In addition, very few classes of microorganisms that can successfully colonize both low- and high- temperature extremes have evolved. Methanogens, which are members of Archaea,aretheonly group known to have individual species that span the growth temperature range from subzero to 122◦C (Saunders et al. 2003, Cavicchioli 2006, Reid et al. 2006, Takai et al. 2008). Thus, there are limited opportunities to compare the adaptive traits of psychrophiles and hyper/thermophiles that belong to the same 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., high versus low temperature). In this respect, global expression studies (e.g., proteomics, transcrip- tomics) linked to knowledge of direct physiological measurements (e.g., temperature and nutrient perturbation of morphology, growth rate, rates of macromolecular synthesis, solute composition, membrane lipid composition, modification of nucleic acids) have proven particularly valuable for determining the mechanisms of 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 of adventitious secondary structures of transcripts. Preventing or resolving inhibitory secondary structures of RNA can be achieved by RNA chaperones. Cold shock proteins (Csps) are small Access provided by University of Nevada - Reno on 05/25/15. For personal use only.

Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org proteins 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-dependent manner 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 (Table 1). Csps contain a nucleic-acid-binding , known as the cold shock domain (CSD), and have additional roles besides serving as RNA chaperones. Individual CSD-containing proteins can regulate the cold shock response or play a major role in subsequent growth at low temperatures in (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’Amico et al. 2006). Overexpression of cspA of Psychromonas arctica was shown to increase cold resistance of at low temperatures ( Jung et al. 2010). Additionally, one of three Csps appears to 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 csp or ctr Total Species and strain Origin of strain Type Phylogeny genesa genes size (Mb) Cenarchaeum Marine sponge Eurypsychrophilic Crenarchaeota Marine 1 csp 2,066 2.05 symbiosum A symbiont, off archaeon Group I (or coast ), Cenarchaeales Colwellia psychrerythraea Arctic marine Stenopsychrophilic Proteobacteria, 4 csp 5,066 5.37 34H sediments, off bacterium Gammaproteobacteria, Desulfotalea psychrophila Arctic marine Eurypsychrophilic Proteobacteria, 7 csp 3,332 3.66 LSv54 sediments, off bacterium Deltaproteobacteria, Svalbard Desulfobacterales Exiguobacterium Permafrost, Eurypsychrophilic Firmicutes, Bacilli, 6 csp 3,151 3.04 sibiricum 255–15 Siberia, Russia bacterium Bacillales Flavobacterium Fish Eurypsychrophilic Bacteroidetes, 1 csp 2,505 2.86 psychrophilum bacterium Flavobacteria, JIP02/86 Halorubrum Deep Lake Eurypsychrophilic , 3 csp 3,725 3.69 lacusprofundi ATCC sediments, archaeon Halobacteria, 49239 Antarctica Halobacteriales loihiensis Hydrothermal Eurypsychrophilic Proteobacteria, 2 csp 2,706 2.84 L2TR vent, Loihi bacterium Gammaproteobacteria, Seamount, off Alteromonadales Hawai’i Foodborne Eurypsychrophilic Firmicutes, Bacilli, 2 csp 2,455 2.91 LO28 pathogen bacterium Bacillales Mariprofundus Hydrothermal Eurypsychrophilic Proteobacteria, 2 csp 2,920 2.87 ferrooxydans PV-1 vent, Loihi bacterium , Seamount, off Mariprofundales Hawai’i Methanococcoides Ace Lake Eurypsychrophilic Euryarchaeota, 3 ctr 2,506 2.58 burtonii DSM 6242 sediments, archaeon , Antarctica Octadecabacter Sea ice off Stenopsychrophilic Proteobacteria, 3 csp 5,544 4.91 antarcticus 307 Antarctica bacterium Alphaproteobacteria, Access provided by University of Nevada - Reno on 05/25/15. For personal use only.

Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org Rhodobacterales Photobacterium Sulu Trough Stenopsychrophilic Proteobacteria, 8 csp 5,754 6.40 profundum SS9 deep-sea bacterium Gammaproteobacteria, sediments Vibrionales irgensii Subsurface Stenopsychrophilic Bacteroidetes, 3 csp 2,602 2.75 23-P seawater, off bacterium Flavobacteria, Antarctica Flavobacteriales (Continued)

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Table 1 (Continued) csp or ctr Total Genome Species and strain Origin of strain Type Phylogeny genesa genes size (Mb) Polaromonas Coal-tar- Eurypsychrophilic Proteobacteria, 1 csp 5,000 5.37 naphthalenivorans CJ2 contaminated bacterium Betaproteobacteria, surface sediments from South Glens Falls, New York Pseudoalteromonas Subsurface Eurypsychrophilic Proteobacteria, 9 csp 3,634 3.85 haloplanktis TAC125 seawater, off bacterium Gammaproteobacteria, Antarctica Alteromonadales Psychrobacter arcticus Permafrost, Eurypsychrophilic Proteobacteria, 3 csp 2,215 2.65 273–4 Siberia, Russia bacterium Gammaproteobacteria, Psychrobacter cryohalentis Permafrost, Eurypsychrophilic Proteobacteria, 4 csp 2,582 3.10 KS Siberia, Russia bacterium Gammaproteobacteria, Pseudomonadales Psychroflexus torquis Sea ice algal Stenopsychrophilic Bacteroidetes, 2 csp 6,835 6.01 ATCC 700755 assemblage, off bacterium Flavobacteria, Antarctica Flavobacteriales Psychromonas Sea ice, off Stenopsychrophilic Proteobacteria, 12 csp 3,877 4.56 ingrahamii 37 northern Alaska bacterium Gammaproteobacteria, Alteromonadales ferrireducens Aquifer Eurypsychrophilic Proteobacteria, 0 4,561 4.97 T118 sediments, bacterium Betaproteobacteria, Virginia Burkholderiales Shewanella oneidensis Lake Oneida Eurypsychrophilic Proteobacteria, 4 csp 4,657 5.13 MR-1 sediments, bacterium Gammaproteobacteria, New York Alteromonadales Shewanella violacea Ryukyu Trench, Stenopsychrophilic Proteobacteria, 6 csp 4,515 4.96 DSS12 deep-sea bacterium Gammaproteobacteria, sediments Alteromonadales

aAbbreviations: csp, cold shock protein; ctr, cold-responsive TRAM protein. Access provided by University of Nevada - Reno on 05/25/15. For personal use only.

Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org Not all bacteria and archaea capable of growing at low temperatures have known homologs of Csps (Table 1). For example, Rhodoferax (Albidoferax) ferrireducens lacks identifiable csp genes, even though csp genes are present in other members of the Burkholderiales (Betaproteobacteria), including Polaromonas strains. csp genes are present in the archaea frigidum (stenopsy- chrophile) and Halorubrum lacusprofundi (eurypsychrophile) but absent from Methanococcoides burtonii, 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-binding TRAM domain were upregulated at low temperatures and proposed to serve as RNA chaperones in an analogous manner to Csps (Williams et al. 2010a, 2011). These putative RNA chaperones have been termed Ctr (cold-responsive TRAM domain) proteins and are unique to a subset of archaea (Table 1). The abundance of Ctr proteins in M. burtonii is particularly high at very low growth 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 to growth in the presence of the solvent methanol further suggests a wider role in the cell as stress response proteins (Williams et al. 2010a). Small RNA-binding proteins (Rbps) can facilitate cold adaptation, but similar to Csps, they can 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 transcription termination (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 rbp gene expression in Anabaena sp. PCC 7120. Responses to cold and osmotic stresses overlap because they both decrease the availability of free water (Mori et al. 2003). Rbp proteins may also play a role in thermal adaptation in psychrophilic cyanobacteria, as expression of rbp genes increases at low 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 modification tends 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 psychrophilic bacteria 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 from Siberian 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 genes are downregulated 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 variety and complexity of metabolic responses of individual psychrophiles. At temperatures low enough for ice to form, cells are subjected to additional stressors such as ice damage, oxidative insult, and osmotic imbalance (Tanghe et al. 2003; Williams et al. 2010b, 2011). Extracellular polymeric substances (EPS) can offer protection against mechanical disruption Access provided by University of Nevada - Reno on 05/25/15. For personal use only.

Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org to the cell membrane caused by ice. Sea-ice bacteria such as Colwellia psychrerythraea produce polysaccharide-rich EPS (Thomas & Dieckmann 2002, Junge et al. 2004). The resulting biofilms may afford protection against invasive ice crystal damage as well as facilitate the acquisition of within the channels that form within the sea ice (Thomas & Dieckmann 2002; Junge et 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 the biological functions of the membrane, including transport (Phadtare 2004). This can be offset by increasing the proportion of unsaturated fatty acids in the lipid bilayer, resulting in a more loosely packed array (Russell 2008). Increasing the proportion of unsaturated fatty acids can be achieved by decreasing the saturation of pre-existing fatty acids or by synthesizing fewer saturated fatty acids

96 Siddiqui et al. EA41CH05-Cavicchioli ARI 30 April 2013 11:9

de novo. The eurypsychrophilic bacterium Exiguobacterium sibiricum has higher 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, resulting in fewer saturated isoprenoid lipid precursors (Nichols et al. 2004). Unsaturated isoprenoid lipids have also been detected in H. lacusprofundi (Gibson et al. 2005). Many psychrophilic members of Gammaproteobacteria (e.g., species of Colwellia, Moritella, Photobacterium, Psychromonas, Mari- nomonas,andShewanella) are characterized by a high proportion of unsaturated fatty acids in their cell membranes (Margesin & Miteva 2011). In a metagenomic analysis, a microbial assemblage in glacier ice was found to be relatively enriched for genes involved in the maintenance of membrane fluidity (Simon et al. 2009). Membrane lipid changes appear to be a generally conserved feature for cellular adaptation to the cold.

Adaptation of Psychrophiles Viewed Through Genomes and Global Gene Expression Profiles Many of the advances in understanding adaptive mechanisms have come from studies involving the genome sequences of psychrophiles. Approximately 30 bacterial and 4 archaeal genome sequences are available for psychrophiles that were obtained from diverse cold samples, including Antarctic lakes, sea sponges (i.e., symbionts), marine sediment, permafrost, marshes, fish (i.e., ), and kimchi (Lauro et al. 2011a). In addition to providing genomic blueprints that describe the capacity of psychrophiles, genomes provide the basis for targeted and global func- tional studies (e.g., proteomics and transcriptomics). The capacity to overview global responses is greatly accelerating the ways in which knowledge is being gained about adaptive mechanisms, in particular, as researchers define general characteristics of psychrophilic microorganisms versus specific 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 temperature range from −10◦Cto28◦C) used transcriptomics to identify differences in mRNA abundance between four growth temperatures (−6, 0, 17, and 22◦C) (Bergholz et al. 2009), and a multiplex proteomics study of M. burtonii quantitated changes occurring across seven growth temperatures that span the ’s complete growth temperature range (−2◦Cto28◦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 physiological states. Interestingly, the upregulation of oxidative stress proteins at both upper and lower tem- perature extremes demonstrated the important, yet distinct, ways in which temperature-induced

Access provided by University of Nevada - Reno on 05/25/15. For personal use only. oxidative stress manifests in the cell. The study also revealed that protein profiles at temperatures

Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org in 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 these temperatures, which is consistent with a number of other studies that suggest that psychrophiles

growing 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, , isomerases, nucleic acid-modifying enzymes), have evolved to function

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Glycosylation Cohesin and dockerin proteins S-layer proteins S-layerS-layer DUF1608 Catalase Superoxide reductase

–2°C 1–16°C 23–28°C COLD STRESS GalT COLD ADAPTATION HEAT STRESS

Redox Superoxide imbalance reductase e– ROS Biomass Biomass Energy Energy e– e– Catalase Flavoproteins

Hcp RNA MdrA RadA Isf Sm-like helicase DNA RNase FMN UspA reductase mRNA Ctr (TRAM) T T Chaperonin proteins DnaK complex Exosome DnaJ Ribosome SPFH METABOLISM NH3 Denatured protein

Misfolded proteins PPlase Proteasome CYTOPLASM ClpB

CYTOPLASMIC MEMBRANE QUASIPERIPLASMIC SPACE

YVTN/NHL (β propeller) protein Ig-like Mxal-like protein protein

Figure 3 Temperature-dependent physiological states in the Antarctic archaeon, Methanococcoides burtonii. Shown are the cellular processes most influenced 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 containing domain of unknown function; e−, electron (or reducing equivalent); FMN, flavin mononucleotide; GalT, galactose-1-phosphate

Access provided by University of Nevada - Reno on 05/25/15. For personal use only. uridylyltransferase; Hcp, hybrid-cluster protein; Isf, -sulfur flavoprotein; MdrA, protein disulfide reductase; mRNA, messenger Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org RNA; MxaI-like, methanol dehydrogenase small subunit homolog; PPIase, peptidyl-prolyl cis/trans isomerase; RadA, DNA repair protein; RNase, ribonuclease; ROS, reactive oxygen species; Sm-like, RNA-binding protein homolog; SPFH, degradation-related protein; UspA, universal stress protein A; YVTN/NHL, S-layer protein containing cell adhesion domain. Adapted with permission from Williams et al. (2011) (Society for Applied 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, and stability 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 evolved and what features appear to be important for conferring specific thermal properties. Studies have involved characterization of enzymes purified from representative organisms as well 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 providing DNA sequence information for proteins from uncultivated microorganisms. Metagenomics of samples from cold environments has included the generation of large data sets obtained by shotgun sequencing (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 clones for cold-active enzymes (e.g., Elenda et al. 2007, Kim et al. 2009). Genomic and metagenomic analyses 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 thermal properties of individual enzymes have also been used to identify structural properties that play roles in 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 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 require sufficient 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 associated with adaptation (e.g., proportion of specific amino acids, hydrophobicity of exposed surfaces) tend to 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 mesophilic and thermophilic homologues (Demirjian et al. 2001, Siddiqui & Cavicchioli 2006). For example, α-amylases from the psychrophilic bacterium Pseudoalteromonas haloplanktis and from the ther- ◦ mophilic bacterium amyloliquefaciens have an optimal temperature of activity (Topt)of28 C and 84◦C, respectively (D’Amico et al. 2003). A striking example of cold adaptation is alanine ◦ racemase from Bacillus psychrosaccharolyticus, which has a Topt of 0 C (Okubo et al. 1999). Because low-temperature environments present significant problems for enzyme and, more broadly, protein function, the unique properties of cold-active enzymes have attracted both academic and commercial 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 growth in the description of enzymes from a broad range of psychrophiles, with a concomitant development of biochemical and biophysical approaches attuned to their characterization (Feller & Gerday 2003, Cavicchioli et al. 2006, Siddiqui & Cavicchioli 2006). Below we discuss some of the mechanisms by which thermal adaptation at low temperatures is attained. Access provided by University of Nevada - Reno on 05/25/15. For personal use only. Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org 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 reaction occurring in a at 37◦C, a drop in temperature from 37◦Cto0◦C results in a 20–80-fold reduction 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 this constraint, 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 independent

of temperature. The majority of psychrophilic enzymes achieve temperature-insensitive kcat by decreasing 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

−1 ◦ ◦ Enzyme kcat (min ) Km (mM) Topt ( C) Tm ( C) t1/2 (min) Reference α-Amylase (10◦C) (10◦C) D’Amico et al. 2003 ◦ Psychrophile 17,640 0.23 28 44 0.23 (43 C) ◦ Mesophile 5,820 0.06 53 52 0.23 (60 C) ◦ 840 – 84 86 0.23 (80 C) Cellulase (4◦C) (4◦C) (45◦C) Garsoux et al. 2004 Psychrophile 11 6.0 37 – 40 Mesophile 0.6 1.5 56 – Unaffected ◦ ◦ Aminopeptidase (10 C) (46 C) Huston et al. 2008 Psychrophile 950 – 39 47 1 Mesophile 114 – 49 58 100,000 ◦ ◦ ◦ Imidase (25 C) (25 C) (40 C) Huang & Yang 2003 Psychrophile 25,700 1.6 55 – 150 > Mesophile 1,500 1.0 65 – 2,880 Lactate dehydrogenase Coquelle et al. 2007 ◦ ◦ Psychrophile 13,800 (0 C) 0.16 (0 C) 50 50 – ◦ ◦ 105,000 (44 C) 0.41 (44 C) – ◦ ◦ Thermophile 40,500 (90 C) 0.16 (90 C) 90 90 – Alkaline phosphatase (37◦C) (37◦C) (50◦C) Siddiqui et al. 2004b Psychrophile 48,740 0.13 40 – 10 Mesophile 6,954 0.11 56 – 38

a kcat, 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 the protein

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. Dashes indicate data not available.

(TS#). For example, reducing the activation energy from 70 kJ mol−1 for a thermophilic α-amylase −1 ◦ to 35 kJ mol 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 tend to be larger and more accessible to substrates. As a result, the binding affinity of substrates for

cold-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 and concomitant 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 and Access provided by University of Nevada - Reno on 05/25/15. For personal use only.

Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org flexible 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 kinetic energy and retarded molecular motion, cold-active enzymes rely on greater disorder as a means of maintaining molecular dynamics and, hence, function (Feller 2007). For example, a psychrophilic alanine racemase that is very active at low temperatures and has very low thermal stability was found 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, thereby reducing compactness and destabilizing the enzyme (Okubo et al. 1999, Siddiqui & Cavicchioli 2006). 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 & Gerday 2003; Siddiqui & Cavicchioli 2006; Siddiqui et al. 2005, 2006; Feller 2008). Collectively, the studies

100 Siddiqui et al. EA41CH05-Cavicchioli ARI 30 April 2013 11:9

indicate that the structure of AHA has evolved to have relatively few electrostatic interactions in order to provide sufficient conformational flexibility to afford activity at low temperatures, while retaining a sufficient level of overall protein structural integrity. Genomic analyses of psychrophilic archaea have revealed proteins characterized by a higher content of noncharged polar amino acids (especially Gln and Thr), a lower content of hydrophobic amino acids (particularly Leu), increased exposure of hydrophobic residues, and a decreased charge that 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). Somewhat different 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 contents of Ile, Lys, and Asn (Zhao et al. 2010). Among these, Pro and Arg are associated with an ability to confer increased stability by restricting backbone rotations and by forming multiple bonds and salt bridges, respectively (Feller & Gerday 2003). Psychrophilic proteins are characterized by decreased core hydrophobicity, increased surface hydrophobicity, 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 weaker metal-binding sites, fewer disulfide bridges, fewer electrostatic interactions (H-bonds, salt bridges, cation-pi interactions, aromatic-aromatic interactions), reduced oligomerization, and an increase in 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 possess large cavities lined predominantly by acidic residues to accommodate water molecules (Paredes et al. 2011). However, although the abovementioned structural features can be associated with psychrophilic 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 (see also Physiological Adaptations in Psychrophiles, above). Consistent with this, the stability and activity of enzymes are affected by the presence of organic solutes (amino acids and sugars) and polymers (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 intracellular and extracellular environment (Demchenko et al. 1989, Siddiqui et al. 2004a, Karan et al. 2012),

Access provided by University of Nevada - Reno on 05/25/15. For personal use only. and the combined effects of temperature and pressure (Saito & Nakayama 2004, Kato et al. 2008) Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org 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 subzero temperatures (D’Amico et al. 2003, Xu et al. 2003), and some oligomer or requiring enzymes (e.g., tryptophanase) can be reversibly inactivated at lower temperatures as a result of subunit and cofactor dissociation (Kogan et al. 2009). Therefore, if a key cellular enzyme is cold inactivated or cold denaturated, it could define the lower temperature limit for growth rather than the 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 of organic 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 . Global warming has a particularly strong effect on polar and alpine environments, wherein ∼30% of the global soil carbon pool resides. The degradation of cellulose, hemicellulose, and humic substances in SOM by extracellular enzymes (e.g., glucanases, ligninases) into dissolved organic compounds represents 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 responses to environmental factors (e.g., nutrient supply, nitrogen and oxygen availability, phenolics and substrate concentration, soil moisture, permafrost melting, and temperature), are now beginning to be incorporated into predictive models describing the effects of global warming on carbon cycling (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 emissions by reducing electricity consumption associated with heating (Cavicchioli et al. 2002, 2011). For example, 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, washing temperatures can be reduced from 40◦Cto30◦C, resulting in a 30% decrease in electricity usage. ◦ Importantly, washing temperatures set 10 C lower reduce the CO2 emissions associated with the burning of fossil fuels for energy generation by 100 g per wash (Nielsen 2005). The application of cold-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 the development 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 number of 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 of Access provided by University of Nevada - Reno on 05/25/15. For personal use only.

Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org microbial diversity has been identified in the deep sea. The microbiota include bacterial members of Alpha-, Beta-, Delta-, Epsilon-, and Gammaproteobacteria as well as Actinobacteria, Bacteroidetes, Chlo- roflexi, Planctomycetes,andVerrucomicrobia. Also included are archaeal members of Euryarchaeota Marine Groups II and III, Crenarchaeota Marine Group I, Methanopyri, and novel alveolate Groups I and II of eucarya that include endoparasitic dinoflagellates. The capacity of microorganisms to thrive under a range of combined extremes, such as in the deep sea where adaptation to cold, high hydrostatic pressure, and nutrient limitation is required, broadens the horizons for the scope of locations 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 for signs of life in extraterrestrial environments where liquid water is known or inferred to exist, such as on Saturn’s () and Jupiter’s (, Ganymede) icy moons; possibly

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0

500

1,000

1,500

2,000 Depth (m) 2,500

3,000 Tropical Temperate Polar 3,500

4,000 –4 0 4 8 12 16 20 24 28 Temperature (°C)

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

(Trent 2000, Cavicchioli 2002); and Saturn’s moon , which reportedly contains nonpolar liquid (Ogino 2008) (Table 3). Nonenzymatic chemical reactions tend to be racemic, producing equal amounts of right- and left-handed enantiomers of a chiral molecule. However, enzymatic reactions tend to produce or incorporate homochiral forms (either right- or left-handed forms of a molecule), such as D-sugars and L-amino acids. Owing to these distinctions, homochirality may be useful as a biomarker. Polarimeters measure changes in optical rotation (change in left- or right-handedness of a chiral molecule) and may be useful for assessing changes taking place over time in an extraterrestrial sample. Investigations into this type of application have been assessed − through studies of mandelate (C8H7O3 , R-2-hydroxy-2-phenylacetate), which is a simple chiral molecule that is racemized by mandelate racemase in a reaction that has a very high enzyme 15 conversion rate (kcat/kuncat = 2.3 × 10 ) (Thaler et al. 2006). Mandelate racemase from the mesophilic bacterium Pseudomonas putida hasbeenreportedtobe active at low temperatures (−30◦C) in the presence of cryosolvents such as saturated ammonium Access provided by University of Nevada - Reno on 05/25/15. For personal use only.

Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org salts and water-in-oil microemulsions (Thaler et al. 2006). However, in water-miscible organic

cosolvents, 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 also tend to be comparatively stable in mixed aqueous-organic or nonaqueous solvents. This derives from their inherent flexibility, which counteracts the destabilizing effects of low water activity in organic 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 surrounding the protein surface. As temperature decreases, water molecules around a protein become more ordered and are less available to interact with the protein surface, thereby destabilizing the protein toward the unfolded state. The loss of critical water molecules is one of the main reasons for the loss 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 to harbor psychrophilic lifea Atmospheric, surface, and subsurface Planet/moon composition Surface temperature b ◦ Earth O2,N2,CO2 −89 to 58 C Water exists in all three states (gas, liquid, solid) ◦ Mars CO2,N2 −140 to 20 C Polar water and CO2 ice caps ◦ Europa ( Jupiter) O2 −223 to −148 C Liquid water ocean may exist under surface ◦ Ganymede ( Jupiter) O2 −203 to −121 C Water ice ◦ Callisto ( Jupiter) CO2 (99%), O2 (1%) −193 to −108 C Liquid water ocean may exist beneath its surface ◦ Titan (Saturn) N2,H2,CH4 −179 C CH4 and C2H6 exist in all three states as gas, liquid, and solid ◦ Enceladus (Saturn) H2O, N2,CO2,CH4 −240 to −128 C Water ice

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 medium become 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 solvents can 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 hydrophobic residues on their surface (Siddiqui & Cavicchioli 2006), they tend to resist unfolding in organic solvents. As a result, a mandelate racemase from a psychrophile is likely to be a good replacement for the P. putida enzyme, finding application in the development of assays for use of polarimeters and possibly for use in the processing of extraterrestrial samples as a biosensor to detect the Access provided by University of Nevada - Reno on 05/25/15. For personal use only.

Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org presence of homochiral mandelate. As discussed in a previous section (see Protein Adaptation to the Cold, above) cold-active

enzymes achieve higher activities (kcat) by reducing the activation energy barrier between the ground and the transition state. However, although enzymes from psychrophiles are active at their environmental temperatures, selection pressures operate at the whole-cell level, and natural environments do not tend to select for the maximum achievable low-temperature activity for every cellular component. As a result, higher activities can be achieved for individual psychrophilic enzymes at low temperatures by artificially manipulating the enzymes. This capacity is relevant to the application of enzymes for use in space or on extraterrestrial bodies where temperatures can be much lower than those encountered on Earth. Improvements in low-temperature activity can be achieved by bypassing enthalpy-entropy compensation. Enthalpy-entropy compensation implies that a decrease in H# is accompanied

104 Siddiqui et al. EA41CH05-Cavicchioli ARI 30 April 2013 11:9

# by a decrease in S so that an overall small increase in kcat is achieved (Siddiqui & Cavicchioli # 2006). However, the gain in kcat would be massive if the decrease in H was not accompanied by a 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 increase ◦ in kcat would occur at 15 C (Lonhienne et al. 2000). Experimental work has shown that the enthalpy-entropy compensation relationship does not always hold true in cold-adapted lipases

from Candida antarctica, particularly in supercritical CO2 and an organic solvent (3-hexanol) where higher 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-like polar 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 upon binding 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-state complex that has a concomitant increase in activity (Wolfenden & Snider 2001). To design highly active enzymes from antibodies (catalytic antibodies), reaction rates can be enhanced by promoting the release of water from the binding pocket during formation of the 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 achieved by effective substrate positioning and/or by water exclusion from within the active site, which creates an increase in S# (Wolfenden 2011). Therefore, in theory, enzyme reactions, biological processes, 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# during enzyme catalysis (i.e., surmounting enthalpy-entropy compensation). Although this review focuses on unicellular microorganisms, as a parting note we highlight the remarkable properties of the small (∼0.1–1 mm in length) metazoans (panarthropods) called (“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. Antarctic tardigrades 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 & Meier 1995). Given their tolerance to cold and other extremes, tardigrades are recognized as valuable metazoan models for astrobiological research (Horikawa et al. 2008): They were used aboard the

Access provided by University of Nevada - Reno on 05/25/15. For personal use only. FOTON-M3 mission to examine their resistance to the effects of outer space (LIFE-TARSE

Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org project) (Rebecchi et al. 2009).

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

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

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Annual Review of Earth and Planetary Sciences Volume 41, 2013 Contents

On Escalation Geerat J. Vermeij pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp1 The Meaning of Stromatolites Tanja Bosak, Andrew H. Knoll, and Alexander P. Petroff ppppppppppppppppppppppppppppppppp21 The Anthropocene William F. Ruddiman pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp45 Global Cooling by Grassland Soils of the Geological Past and Near Future Gregory J. Retallack pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp69 Psychrophiles Khawar S. Siddiqui, Timothy J. Williams, David Wilkins, Sheree Yau, Michelle A. Allen, Mark V. Brown, Federico M. Lauro, and Ricardo Cavicchioli pppppp87 Initiation and Evolution of Plate Tectonics on Earth: Theories and Observations Jun Korenaga ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp117 Experimental Dynamos and the Dynamics of Planetary Cores Peter Olson pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp153 Extracting Earth’s Elastic Wave Response from Noise Measurements Roel Snieder and Eric Larose ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp183

Access provided by University of Nevada - Reno on 05/25/15. For personal use only. Miller-Urey and Beyond: What Have We Learned About Prebiotic Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org Organic Synthesis Reactions in the Past 60 Years? Thomas M. McCollom pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp207 The Science of Geoengineering Ken Caldeira, Govindasamy Bala, and Long Cao ppppppppppppppppppppppppppppppppppppppppp231 Shock Events in the Solar System: The Message from Minerals in Terrestrial Planets and Asteroids Philippe Gillet and Ahmed El Goresy pppppppppppppppppppppppppppppppppppppppppppppppppppppp257 The Fossil Record of -Insect Dynamics Conrad C. Labandeira and Ellen D. Currano pppppppppppppppppppppppppppppppppppppppppppp287

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The Betic-Rif Arc and Its Orogenic Hinterland: A Review John P. Platt, Whitney M. Behr, Katherine Johanesen, and Jason R. Williams ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp313 Assessing the Use of Archaeal Lipids as Marine Environmental Proxies Ann Pearson and Anitra E. Ingalls pppppppppppppppppppppppppppppppppppppppppppppppppppppppp359 Heat Flow, Heat Generation, and the Thermal State of the Lithosphere Kevin P. Furlong and David S. Chapman pppppppppppppppppppppppppppppppppppppppppppppppp385 The Isotopic Anatomies of Molecules and Minerals John M. Eiler ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp411 The Behavior of the Lithosphere on Seismic to Geologic Timescales A.B. Watts, S.J. Zhong, and J. Hunter ppppppppppppppppppppppppppppppppppppppppppppppppppp443 The Formation and Dynamics of Super-Earth Planets Nader Haghighipour ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp469 Kimberlite Volcanism R.S.J. Sparks pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp497 Differentiated Planetesimals and the Parent Bodies of Chondrites Benjamin P. Weiss and Linda T. Elkins-Tanton ppppppppppppppppppppppppppppppppppppppppp529 Splendid and Seldom Isolated: The Paleobiogeography of Patagonia Peter Wilf, N. Rub´en C´uneo, Ignacio H. Escapa, Diego Pol, and Michael O. Woodburne pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp561 Electrical Conductivity of Mantle Minerals: Role of Water in Conductivity Anomalies Takashi Yoshino and Tomoo Katsura pppppppppppppppppppppppppppppppppppppppppppppppppppppp605 The Late Paleozoic Ice Age: An Evolving Paradigm Isabel P. Monta˜nez and Christopher J. Poulsen ppppppppppppppppppppppppppppppppppppppppppp629 Composition and State of the Core Kei Hirose, St´ephane Labrosse, and John Hernlund pppppppppppppppppppppppppppppppppppppp657 Access provided by University of Nevada - Reno on 05/25/15. For personal use only. Annu. Rev. Earth Planet. Sci. 2013.41:87-115. Downloaded from www.annualreviews.org Enceladus: An Active Ice World in the Saturn System John R. Spencer and Francis Nimmo pppppppppppppppppppppppppppppppppppppppppppppppppppppp693 Earth’s Background Free Oscillations Kiwamu Nishida pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp719 Global Warming and Neotropical Rainforests: A Historical Perspective Carlos Jaramillo and Andr´es C´ardenas pppppppppppppppppppppppppppppppppppppppppppppppppppp741 The Scotia Arc: Genesis, Evolution, Global Significance Ian W.D. Dalziel, Lawrence A. Lawver, Ian O. Norton, and Lisa M. Gahagan pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp767

Contents ix