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Psychrophiles
Article in Annual Review of Earth and Planetary Sciences · May 2012
DOI: 10.1146/annurev-earth-040610-133514
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EA41CH06-Cavicchioli ARI 7 February 2013 14:43
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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
1
School of Biotechnology and Biomolecular Sciences and 2
Evolution and Ecology Research
Center, 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 is
online 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, microbial
diversity, Antarctica
Abstract
Psychrophilic (cold-adapted) microorganisms make a major contribution
to Earth’s biomass and perform critical roles in global biogeochemical cy-
cles. The vast extent and environmental diversity of Earth’s cold biosphere
has selected for equally diverse microbial assemblages that can include ar-
chaea, bacteria, eucarya, and viruses. Underpinning the important ecological
roles of psychrophiles 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 protein structure that enables biocatalysis
in nonaqueous solvents. In this review, we examine the ecology of Antarctic
psychrophiles, physiological adaptation of psychrophiles, and properties of
cold-adapted proteins, and we provide a view of how these characteristics
inform studies of astrobiology.
6.1
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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 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 deep sea, geographically dis-
persed alpine regions, geologically specific subterranean caverns, climatically challenged regions
of permafrost, and biogeochemically diverse polar reaches (Figure 1). Proliferating throughout
these 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 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 ecosystem 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 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 –160ºC
Subsurface ocean: ?ºC
Alpine
< 10°C Deep sea
1 to 4°C
Figure 1
Terrestrial and extraterrestrial cold environments. Representative temperatures are shown.
6.2 Siddiqui et al.

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 climate change. The review concludes with a section
reflecting 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 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 organisms 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
Quéré et al. 2007), and reduced nutrient supply particularly at higher latitudes caused by increased
stratification (Sarmiento & Le Quéré 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 microbial mat communities (Wilkins et al. 2012b). These lakes
include subglacial, epiglacial, and surface systems that range in salinity 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 Blood Falls dating from 1.5 Mya (Mikucki et al. 2009); and waters in the depths of
subglacial 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 properties
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
www.annualreviews.org • Psychrophiles 6.3

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6.4 Siddiqui et al.

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 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 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 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 (López-Bueno et al. 2009; Ng
et al. 2010; Lauro et al. 2011b; Yau et al. 2011; Brown et al. 2012; Gryzmski 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 methanogens and aerobic haloarchaea (Bowman et al. 2000a,b; Purdy et al. 2003;
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
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 Fjord
by 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 Ace
Lake formed after a blizzard behind quad bikes (used for transport between the lake and Davis Research Base located 15 km away) and
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
temperatures 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 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 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 of
Stephenson Glacier 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.

Glatz et al. 2006; Karr et al. 2006; Kurasawa 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, 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 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; Säwström et al. 2007; López-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, Deinococcus-Thermus, Firmicutes, fungi, green
algae, Planctomycetes, Proteobacteria, and Verrucomicrobia (Brambilla et al. 2001; Van Trappen et al.
2002; Taton et al. 2003, 2006; Jungblut et al. 2005; Fernández-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 Crenarchaeota (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).
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, 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, and
nutrient gradients (Eicken 2003, Mock & Thomas 2005).
6.6 Siddiqui et al.

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/thermophiles, 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, oxygen, 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, are the only
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 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., 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 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 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
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 genomes (Table 1). Csps contain
a nucleic-acid-binding domain, 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 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’Amico
et al. 2006). Overexpression of cspA of Psychromonas arctica was shown to increase cold resistance
of Escherichia coli 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).

Table 1 Characteristics of selected bacterial and archaeal psychrophiles
Species and strain Origin of strain Type Phylogeny
csp or
ctr
genesa
Total
genes
Genome
size (Mb)
Cenarchaeum
symbiosum A
Marine sponge
symbiont, off
California coast
Eurypsychrophilic
archaeon
Crenarchaeota (or
Thaumarchaeota),
Marine Group I,
Cenarchaeales
1 csp 2,066 2.05
Colwellia psychrerythraea
34H
Arctic marine
sediments, off
Greenland
Stenopsychrophilic
bacterium
Proteobacteria,
Gammaproteobacteria,
Alteromonadales
4 csp 5,066 5.37
Desulfotalea psychrophila
LSv54
Arctic marine
sediments, off
Svalbard
Eurypsychrophilic
bacterium
Proteobacteria,
Deltaproteobacteria,
Desulfobacterales
7 csp 3,332 3.66
Exiguobacterium
sibiricum 255–15
Permafrost,
Siberia, Russia
Eurypsychrophilic
bacterium
Firmicutes, Bacilli,
Bacillales
6 csp 3,151 3.04
Flavobacterium
psychrophilum
JIP02/86
Fish pathogen Eurypsychrophilic
bacterium
Bacteroidetes,
Flavobacteria,
Flavobacteriales
1 csp 2,505 2.86
Halorubrum
lacusprofundi ATCC
49239
Deep Lake
sediments,
Antarctica
Eurypsychrophilic
archaeon
Euryarchaeota,
Halobacteria,
Halobacteriales
3 csp 3,725 3.69
Idiomarina loihiensis
L2TR
Hydrothermal
vent, Loihi
Seamount, off
Hawai’i
Eurypsychrophilic
bacterium
Proteobacteria,
Alteromonadales
2 csp 2,706 2.84
Listeria monocytogenes
LO28
Foodborne
pathogen
Eurypsychrophilic
bacterium
Firmicutes, Bacilli,
Bacillales
2 csp 2,455 2.91
Mariprofundus
ferrooxydans PV-1
Hydrothermal
vent, Loihi
Seamount, off
Hawai’i
Eurypsychrophilic
bacterium
Proteobacteria,
Zetaproteobacteria,
Mariprofundales
2 csp 2,920 2.87
Methanococcoides
burtonii DSM 6242
Ace Lake
sediments,
Antarctica
Eurypsychrophilic
archaeon
Euryarchaeota,
Methanomicrobia,
Methanosarcinales
3 ctr 2,506 2.58
Octadecabacter
antarcticus 307
Sea ice off
Antarctica
Stenopsychrophilic
bacterium
Proteobacteria,
Alphaproteobacteria,
Rhodobacterales
3 csp 5,544 4.91
Photobacterium
profundum SS9
Sulu Trough
deep-sea
sediments
Stenopsychrophilic
bacterium
Proteobacteria,
Vibrionales
8 csp 5,754 6.40
Polaribacter irgensii
23-P
Subsurface
seawater, off
Antarctica
Stenopsychrophilic
bacterium
Bacteroidetes,
Flavobacteria,
Flavobacteriales
3 csp 2,602 2.75
(Continued)
6.8 Siddiqui et al.

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

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 metabolism 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
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
nutrients 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
6.10 Siddiqui et al.

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, 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, and Shewanella) 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 originating from diverse cold habitats that include
Antarctic lakes, symbionts of sea sponges, marine sediment, permafrost, marshes, fish pathogens,
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 functional
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◦
C to 28◦
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 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 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
oxidative stress manifests in the cell. The study also revealed that protein profiles at temperatures
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, hydrogenases, isomerases, nucleic acid-modifying enzymes), have evolved to function

e–
e–e–
TT
GalT
Glycosylation
Ig-like
protein
Mxal-like
protein
YVTN/NHL
(β propeller)
protein
METHANOGENESIS
AMINO ACID
METABOLISM
Flavoproteins
Biomass
Energy
Biomass
Energy
MdrA Isf
Hcp
Sm-like
ClpB
DnaJ
DnaK
Catalase
CatalaseDUF1608
UspA
RadA
FMN
reductase
Proteasome
Exosome
Ctr (TRAM)
proteins
RNA
helicase
PPlase
S-layer proteins
Cohesin and
dockerin proteins
Ribosome
Chaperonin
complex
Misfolded
proteins
Denatured
protein
S-layerS-layerS-layer
CYTOPLASM
CYTOPLASMIC MEMBRANE
QUASIPERIPLASMIC SPACE
Superoxide reductase
Superoxide
reductase
ROS
mRNA
RNase
DNA
NH3
Redox
imbalance
–2°C
COLD STRESS
1–16°C
COLD ADAPTATION
23–28°C
HEAT STRESS
SPFH
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
uridylyltransferase; Hcp, hybrid-cluster protein; Isf, iron-sulfur flavoprotein; MdrA, protein disulfide reductase; mRNA, messenger
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. Reproduced with permission
from 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, 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

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., López-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 facilitatesubsequent targeted analyses to assess specificfeatures 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 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 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 exam-
ple, α-amylases from the psychrophilic bacterium Pseudoalteromonas haloplanktis and from the
thermophilic 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 is
alanine 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 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 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 the
mechanisms 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 reaction
occurring in a mesophile at 37◦
C, a drop in temperature from 37◦
C to 0◦
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

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

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 hydrogen
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),
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 subzero
temperatures (D’Amico et al. 2003, Xu et al. 2003), and some oligomeric or cofactor that requires
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 decomposition of soil
organic matter (SOM) (German et al. 2012). Predictions are that the carbon sequestered in SOM

is at least four times higher than the carbon content in the atmosphere and living plants. 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◦
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 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
microbialdiversityhasbeenidentifiedinthedeepsea.Themicrobiotaincludebacterialmembersof
Alpha-, Beta-, Delta-, Epsilon-, and Gammaproteobacteria as well as Actinobacteria, Bacteroidetes, Chlo-
roflexi, Planctomycetes, and Verrucomicrobia. 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 (Enceladus) and Jupiter’s (Europa, Ganymede) icy moons; possibly Mars

0
500
1,000
1,500
2,000
2,500
3,000
3,500
4,000
–4 0 4 8
Polar
Temperate
Tropical
12 16 20 24 28
Depth(m)
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 Titan, 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
conversion rate (kcat/kuncat = 2.3 × 1015
) (Thaler et al. 2006).
Mandelate racemase from the mesophilic bacterium Pseudomonas putida has been reported to be
active at low temperatures (−30◦
C) in the presence of cryosolvents such as saturated ammonium
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

Table 3 Characteristics of some planets and moons from Earth’s solar system with the potential to
harbor psychrophilic lifea
Planet/moon
Atmospheric, surface, and subsurface
composition Surface temperature
Earthb 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 surface
ice 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 its
surface
−193 to −108◦C
Titan (Saturn) N2, H2, CH4
CH4 and C2H6 exist in all three states as
gas, liquid, and solid
−179◦C
Enceladus (Saturn) H2O, N2, CO2, CH4
Water ice
−240 to −128◦C
a
Data taken from Chown (2011).
b
The 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
presence 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 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

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
tardigrades (“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
FOTON-M3 mission to examine their resistance to the effects of outer space (LIFE-TARSE
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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