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From Daniela Giordano, Daniela Coppola, Roberta Russo, Mariana Tinajero-Trejo,
Guido di Prisco, Federico Lauro, Paolo Ascenzi, Cinzia Verde, The Globins of Cold-
Adapted Pseudoalteromonas haloplanktis TAC125: From the Structure to the
Physiological Functions. In Robert K. Poole, editor: Advances in Microbial
Physiology, Vol. 63,
Amsterdam, The Netherlands: Academic Press, 2013, pp. 1-47.
ISBN: 978-0-12-407693-8
© Copyright 2013 Elsevier Ltd
Elsevier
CHAPTER EIGHT
The Globins of Cold-Adapted
Pseudoalteromonas haloplanktis
TAC125: From the Structure to the
Physiological Functions
Daniela Giordano*, Daniela Coppola*, Roberta Russo*,
Mariana Tinajero-Trejo
†
, Guido di Prisco*, Federico Lauro
{
,
Paolo Ascenzi*
,}
, Cinzia Verde*
,},1
*Institute of Protein Biochemistry, CNR, Naples, Italy
†
Department of Molecular Biology & Biotechnology, The University of Sheffield, Sheffield, United Kingdom
{
School of Biotechnology & Biomolecular Sciences, The University of New South Wales, Sydney, New South
Wales, Australia
}
Interdepartmental Laboratory for Electron Microscopy, University Roma 3, Rome, Italy
}
Department of Biology, University Roma 3, Rome, Italy
1
Corresponding author: e-mail address: c.verde@ibp.cnr.it
Contents
1. The Polar Environments 331
2. Phylogeny and Biogeography of Cold-Adapted Marine Microorganisms 334
3. The Role of Temperature in Evolutionary Adaptations 337
4. Bacterial Globins 340
4.1 Flavohaemoglobins 341
4.2 Single-domain globins 344
4.3 Truncated haemoglobins 345
5. The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125:
A Case Study 348
5.1 General aspects 348
5.2 Genomic and post-genomic insights 350
5.3 Excess of O
2
and metabolic constraints 355
5.4 Biotechnological applications 357
6. P. haloplanktis TAC125 Globins 358
6.1 General aspects 358
6.2 Structure–function relationships of Ph-2/2HbO 360
7. Conclusion and Perspectives 372
Acknowledgements 374
References 374
Advances in Microbial Physiology, Volume 63 #2013 Elsevier Ltd
ISSN 0065-2911 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-407693-8.00008-X
329
Author's personal copy
Abstract
Evolution allowed Antarctic microorganisms to grow successfully under extreme
conditions (low temperature and high O
2
content), through a variety of structural
and physiological adjustments in their genomes and development of programmed
responses to strong oxidative and nitrosative stress. The availability of genomic
sequences from an increasing number of cold-adapted species is providing insights
to understand the molecular mechanisms underlying crucial physiological processes
in polar organisms. The genome of Pseudoalteromonas haloplanktis TAC125 contains
multiple genes encoding three distinct truncated globins exhibiting the 2/2 a-helical
fold. One of these globins has been extensively characterised by spectroscopic
analysis, kinetic measurements and computer simulation. The results indicate unique
adaptive structural properties that enhance the overall flexibility of the protein, so that
the structure appears to be resistant to pressure-induced stress. Recent results on a
genomic mutant strain highlight the involvement of the cold-adapted globin in
the protection against the stress induced by high O
2
concentration. Moreover, the
protein was shown to catalyse peroxynitrite isomerisation in vitro. In this review,
we first summarise how cold temperatures affect the physiology of microorganisms
and focus on the molecular mechanisms of cold adaptation revealed by recent
biochemical and genetic studies. Next, since only in a very few cases the physiological
role of truncated globins has been demonstrated, we also discuss the structural
and functional features of the cold-adapted globin in an attempt to put into perspective
what has been learnt about these proteins and their potential role in the biology of
cold-adapted microorganisms.
ABBREVIATIONS
CAP cold-adapted protein
CSP cold-shock protein
Cygb cytoglobin
FHb flavohaemoglobin
Hb haemoglobin
Hmp FHb from E. coli
HSP heat-shock protein
Mb myoglobin
Ngb neuroglobin
PhTAC125 Pseudoalteromonas haloplanktis TAC125
PTS phosphoenolpyruvate-dependent phosphotransferase system
RNS reactive nitrogen species
ROS reactive oxygen species
SDgb single-domain globin
TBDTs TonB-dependent transport systems
TF trigger factor
330 Daniela Giordano et al.
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1. THE POLAR ENVIRONMENTS
Although many cold-ocean species have been studied, we still have
limited knowledge about adaptation to low temperatures in sea water. In
the light of the ongoing climate change, there is growing interest in polar
marine organisms and how they have evolved at constantly cold tempera-
tures. The rate of the impact of current climate change in relation to the
capacity of species to acclimate or adapt is a crucial study area for managing
polar ecosystems in the future.
While the early biological works focussed on specific aspects of molec-
ular adaptation of single genes and proteins to cold, progress in genomics and
postgenomics, as well as the availability of genomic sequences of several
model species, allowed highlighting the adaptation mechanisms permitting
species evolution in polar regions (Peck, 2011; Somero, 2010).
The planet is currently losing sea ice, most notably in the Arctic region,
because of warming trends over the last century (Moritz, Bitz, & Steig,
2002). The dramatic sea-ice decrease is progressing from the Barents and
Bering Seas to the central Arctic Ocean.
Isolation of the two polar oceans has occurred to a different extent. The
land masses surrounding the Arctic Ocean have partially limited water
exchanges with other oceans for the last 60 million years or so. The Southern
Ocean was isolated much more thoroughly by the Antarctic Circumpolar
Current, the strongest current system in the world, since 20–40 million years
ago (Eastman, 2005).
Geography, oceanography and biology of species inhabiting Arctic and
Antarctic polar regions have often been intercompared (see Dayton,
Mordida, & Bacon, 1994) to detect and outline differences between the
two ecosystems. The northern polar region is characterised by extensive,
shallow shelf sea areas of the land masses that surround a partially land-locked
ocean; in contrast, the Antarctic region comprises a dynamic open ocean
that surrounds the continent, and a continental shelf (Smetacek & Nicol,
2005) that is very deep because of the enormous weight exerted on the con-
tinent by the covering ice sheet, which has a thickness of 2–4000 m.
Although the climate drivers acting on the biota are relatively similar, the
two polar environments are quite different from each other. One of the main
differences is the freshwater supply. Arctic surface waters are modified by the
331The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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input of large rivers that influence the nutrient regimes and their differences.
In the Southern Ocean, the influence of freshwater, mostly from glaciers, is
much smaller.
The Arctic is currently experiencing some of the most rapid and
severe climate changes on Earth. Some effects of increasing temperature
on marine ecosystems are already evident (Rosenzweig et al., 2008). Over
the next 100 years, warming is expected to accelerate, contributing to
major physical, ecological, social and economic changes. In fact, all models
forecast reduction of cold areas and expansion of the warmer ones, with
consequent threat for cold-adapted organisms. However, there is still little
understanding regarding how the loss of a species or groups of species will
affect ecosystem services.
The most important Arctic characters include seasonality in light, cold
temperatures with winter extremes, and extensive shelf seas around a deep
central ocean basin. The Arctic comprises a vast ocean surrounded by the
northern coasts of three continents, open to influx of warm water from
the Atlantic and, to a lesser extent, from the Pacific. The “permanent
cap” of ice, composed of multi- and first-year ice that forms annually and
extends and retreats seasonally, is probably the most important feature of
Arctic marine systems (Polyak et al., 2010). The major decline in sea ice that
began to take place in the Arctic since 2000 is the most important climate-
change signal (Comiso, Parkinson, Gersten, & Stock, 2008). Expectations
are that summer sea ice will continue to decline in the future. Climate
models have indicated that the Arctic Ocean might essentially be ice-free
during summer by the later half of the twenty-first century (Overpeck
et al., 2006; Wang & Overland, 2009), with dramatic and potentially dev-
astating effects on a number of species associated with the sea ice (Moline
et al., 2008) and significant biological consequences (Clarke et al., 2007).
In terms of constantly low temperatures, the southern polar environment
is considered the most extreme on our planet. Antarctica has the capacity to
influence the Earth’s climate and ocean-ecosystem function, and from this
standpoint, it is the world’s most important continent. As such, its palaeo-
and current geological and climatic history, physical and biological
oceanography, as well as marine and terrestrial ecosystems have been—
and are—the target of a wealth of studies. As a frozen deep mass of ice, most
of Antarctica reflects the sun’s radiation, buffering global warming trends.
The current lack of warming is also due to the shielding effect produced
by the stratospheric winds driven by the human-induced Ozone Hole.
These winds generate a Polar Vortex extending to the surface that acts as
332 Daniela Giordano et al.
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a strong barrier, keeping warm and moist air away (Turner et al., 2009).
When and if the Ozone Hole closes, such a protection will no longer be
efficient, and warming caused by greenhouse gases, another anthropogenic
contribution, will take the lead. However, shielding by the Polar Vortex is
not taking place in the Antarctic Peninsula, especially on the western side.
Similar to the Arctic, the Peninsula is thus experiencing one of the fastest
rates of warming on the planet (Convey et al., 2009; Turner et al., 2009).
The consequences are already being seen on land (Convey, 2006, 2010),
where ice loss leads to new land becoming available for rapidly occurring
colonisation. Reduction of sea ice causes displacement of key invertebrate
and fish species, whose reproductive processes, closely associated with sea
ice, are upset (Moline et al., 2008). Such migrations have consequences
on the whole food chain.
The Southern Ocean is the planet’s fourth largest ocean. Over geological
time, environmental conditions and habitats in the Antarctic have changed
dramatically. Two key events allowed the establishment of the powerful
Antarctic CircumpolarCurrent: (i) the opening of the Tasman Seaway, which
occurred approximately 32–35 million years ago, according to tectonics and
marine geology (Kennett, 1977; Lawver & Gahagan, 2003); and (ii) the
opening of the Drake Passage, between South America and the Antarctic
Peninsula, dated between 40 and 17 million years ago (Scher & Martin,
2006; Thomson, 2004). The Antarctic Polar Front, a roughly circular oceanic
feature running between 50S and 60S, is the northern boundary of the Cir-
cumpolar Current. Along the Front, the surface layers of the north-moving
Antarctic waters sink beneath the less cold and less dense sub-Antarctic waters,
generating almost permanent turbulence. Just north of the Front, the surface
water temperature is ca. 2–3 C warmer. Separating warm northern waters
from cold southern waters, the Front acts as a barrier for migration of marine
organisms between Antarctica and the lower latitudes.
Thus, Antarctica is a closed system, shielded by the Antarctic
Circumpolar Current from the influence of waters from latitudes lower
than 60(Eastman, 2005). These conditions promoted adaptive evolution
to low temperature and extreme seasonality to develop in isolation, and
led to the current composition of the Antarctic marine biota
(Eastman, 2005). The earliest cold-climate marine fauna is thought to date
from the late Eocene–Oligocene (about 35 million years ago). The
water column south of the Front is close to O
2
saturation at all depths.
O
2
solubility increases with temperature decrease, thus the cold seas are
an O
2
-rich habitat.
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The dominant feature of the modern continent is its ice sheet, covering
most of the land area. In the sea, during wintertime, ice coverage currently
extends from the Antarctic coastline northward to approximately 60S.
Antarctic marine environments are thus considered the most extreme on
Earth in combining the globally lowest and most stable temperatures with
the highest O
2
content, and at the same time great variability in light inten-
sity, ice cover and phytoplankton productivity (Peck, Convey, & Barnes,
2006). Antarctic marine habitats include sea water and sediments at near
1C and the sea ice, where internal fluids remain liquid to 35 C
during winter.
2. PHYLOGENY AND BIOGEOGRAPHY OF COLD-
ADAPTED MARINE MICROORGANISMS
Global warming is expected to increase the microbial activity and
decrease the availability of energy and food for organisms that are at higher
levels in the food chain (Kirchman, Mora
´n, & Ducklow, 2009). The role of
microorganisms in polar waters is essential. Microbial processes in polar eco-
systems are highly sensitive to small environmental changes and influence
ecosystem functioning. Cold marine environments are colonised by a wide
diversity of microorganisms including bacteria, archaea, yeasts, fungi and
algae (Margesin & Miteva, 2011; Murray & Grzymski, 2007). Sea-ice
microbial communities at the two poles display closely related organisms
(Brinkmeyer et al., 2003) and are continually seeded by alien microorgan-
isms, including mesophilic species that contribute to the potential environ-
mental pool of DNA (Cowan et al., 2011).
In the past, understanding bacterial, archaeal and viral diversity in polar
marine environments has been somewhat impaired by the difficulty of
accessing sampling locations year-round. In comparison, rapid advances
in microbial ecological theory have been achieved through results from tem-
perate oceans, particularly, with respect to long-term microbial observato-
ries such as BATS (Bermuda Atlantic Time Series) (Steinberg et al., 2001)
and Station HOT (Hawaii Ocean Time Series) (DeLong et al., 2006).
Some of the drawbacks in data acquisition have been overcome with the
advent of culture-independent molecular tools and large-scale community
sequencing. Studies based on denaturing gradient gel electrophoresis
(Abell & Bowman, 2005; Giebel, Brinkhoff, Zwisler, Selje, & Simon,
2009) have revolutionised our views of diversity in Antarctic waters, making
the Southern Ocean one of the focal regions of microbial ecology.
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However, the applicability of these investigations to the peculiar character-
istics of the polar microbiota is largely unknown. Deeper understanding is
necessary in light of the important role that polar waters play in global carbon
cycling. The microbial component represents up to 90% of cellular DNA
(Paul, Jeffrey, & DeFlaun, 1985) and is estimated to be responsible for up
to 80% of the primary carbon production (Douglas, 1984; Ducklow,
1999; Li et al., 1983) and for most of the carbon flux between the sea water
and the atmosphere (Azam, 1998; Azam & Malfatti, 2007).
Environmental genomics revealed that heterotrophic bacteria play a key
role in controlling carbon fluxes within oceans. These bacteria dominate
biogeochemical cycles and are part of the microbial loop which, at least
in part, causes the response of oceanic ecosystems to climate change
(Kirchman et al., 2009). Recent diversity studies that employed sequencing
of ribosomal RNA genes (Galand, Casamayor, Kirchman, & Lovejoy, 2009;
Ghiglione & Murray, 2012; Kirchman, Cottrell, & Lovejoy, 2010), and
metagenomics and metaproteomics (Grzymski et al., 2012; Wilkins et al.,
2013; Williams et al., 2012, 2013) have clarified some of the aspects of
the interactions between microorganisms and the polar environment, which
is unique in terms of environmental parameters such as temperature, day
length and trophic interactions.
Antarctic marine waters harbour taxa of heterotrophic microbes similar
to those found in temperate and tropical waters. Among these, the most
dominant are a-Proteobacteria and, in particular, specific phylotypes of
SAR11 (Brown et al., 2012), g-Proteobacteria, Flavobacteria and
ammonia-oxidising Marine Group I Crenarchaeota (Grzymski et al.,
2012; Wilkins et al., 2013; Williams et al., 2012). However, the emerging
view is that, while the taxa present might be distributed worldwide, there are
clear signatures of allopatric speciation, which are only evident at a finer
phylogenetic scale (Brown et al., 2012).
The geographic separation necessary for such evolutionary events is pro-
vided by sharp transitions in chemicophysical parameters that mark and iso-
late water masses (Agogue
´, Lamy, Neal, Sogin, & Herndl, 2011). The
Antarctic Polar Front provides one of the most dramatic examples of such
transitions. Here, the water drops 3C in temperature over a space of less
than 30 miles which results in abrupt shifts in the microbial community
composition and functional gene distribution (Wilkins et al., 2013).
Moreover, certain taxa become transiently dominant in response to par-
ticular seasonal changes in environmental parameters such as the Marine
Group I Crenarchaeota which show a dramatic increase in relative
335The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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abundance and activity during the winter in the Antarctic Peninsula
(Grzymski et al., 2012; Williams et al., 2012). Similarly, bacterial clades
within the Rhodobacteraceae, uncultivated g-Proteobacteria and
Bacteriodetes show large seasonal variations between samples from summer
and winter from both the Antarctic Peninsula and the sub-Antarctic Kergue-
len Islands (Ghiglione & Murray, 2012). On top of these oscillations, over
shorter time and spatial scales, Flavobacteria can become dominant in
response to algal blooms (Ghiglione & Murray, 2012; Grzymski et al.,
2012; Williams et al., 2013).
The composition of the sea-ice microbiota is also unique as a result of its
seasonal nature and physicochemical environment (Bowman et al., 2012;
Brown & Bowman, 2001). It has an important role in providing the “seed
populations” for the productive springtime microbial communities. It is still
unclear whether the selective pressure within the winter sea ice generates sig-
nificant genetic bottlenecks on different microbial species (Connelly,
Tilburg,& Yager, 2006; Junge, Imhoff, Staley, & Deming, 2002). What is clear
is that, when compared to surrounding seawater, the species richness in sea ice
is lower than in surrounding waters (Bowman et al., 2012), which in turn has
been shown to decrease when moving from lower to higher latitudes (Sul,
Oliver, Ducklow, Amaral-Zettler, & Sogin, 2013). This lower richness might
not provide enough resilience in case of future climatic changes.
Compared to the heterotrophic community, the latitudinal distribution
and temporal variation of primary producers are even more extreme. Cyano-
bacteria, for example, Synechococcus sp. and Prochlorococcus sp., are fundamental
in carbon fixation and responsible for more than half of primary production in
oligotrophic ocean waters (Liu et al., 1998; Liu, Nolla, & Campbell, 1997).
A consistent trend is the progressive disappearance of Prochlorococcus populations
south of the Polar Front and the appearance of specific clades of Synechococcus
which dominate at higherlatitudes (Scanlan et al., 2009). This trend holds true
at both poles. In fact, bipolar distribution of organisms is the rule rather than the
exception amongst microbial taxa (Suletal.,2013). In microeukaryotes, the
observation of pheromone cross signallingamongst Arctic and Antarctic strains
of the polar protozoan ciliate Euplotes nobilii suggests mechanisms for recent
genetic exchange (Di Giuseppe et al., 2011). If associated with the strong bipo-
lar biogeographical patterns, this could be true for all classes of organisms living
at low Reynolds numbers, with the caveat that deep-sea currents, in particular
those associated with thermohaline circulation, are allowing an ongoing
genetic exchange between the poles (Lauro, Chastain, Blankenship,
Yayanos, & Bartlett, 2007).
336 Daniela Giordano et al.
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Complementing these culture-independent studies, the last few years
have seen an increase in genomic sequences of cultured isolates. The study
of individual genomes facilitates the characterisation of physiological adap-
tations to the specific polar conditions. Nevertheless, in contrast with the
large diversity observed with molecular techniques, the phylogenetic
breadth of the taxa with at least one representative genome sequence is lim-
ited to a few genera (Fig. 8.1).
In view of the high degree of temporal and spatial variability observed in
polar environments, which positively correlates with changes in microbial
community structure and function, there is a pressing need for increasing
culturing efforts and single-cell genomic analysis targeted at under-
represented phyla. These should be integrated within the larger framework
of global organismal biogeography and ocean models.
3. THE ROLE OF TEMPERATURE IN EVOLUTIONARY
ADAPTATIONS
The bulk of the Earth’s biosphere is cold (e.g. 90% of the ocean is
below 5 C), sustaining a broad diversity of microbial life. Evolution under
extreme conditions has been marked by a suite of adaptations (evolutionary
gains) including the development of proteins that function optimally in the
cold. A commonly accepted view for protein cold adaptation is the
activity/stability/flexibility relationships. Although active sites are generally
highly conserved among homologous proteins, adaptive changes may
occur at recognition site(s). These alterations in the strength of subunit
interactions may affect thermal stability and energy changes associated with
conformational transitions due to ligand binding (D’Amico, Collins, Marx,
Feller, & Gerday, 2006).
Comparative genome analysis indicates that the cold-adapted lifestyle is
generally conferred by a collection of changes in the overall genome content
and composition. The flexible structures of enzymes from cold-adapted bac-
teria compensate for the environment’s low kinetic energy.
In cold environments, challenges to cellular function and structural
integrity include low rates of transcription, translation and cell division,
inappropriate protein folding and cold denaturation, as well as intracellular
ice formation (D’Amico et al., 2006). The ability of an organism to survive
and grow in the cold is dependent on a number of adaptive strategies
(Table 8.1) to maintain vital cellular functions at cold temperatures
(Rodrigues & Tiedje, 2008). These strategies include the synthesis of
337The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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Figure 8.1 Bacterial and Archaeal phylogenetic tree adapted from the Silva compre-
hensive ribosomal RNA database (http://www.arb-silva.de/). Blue: phyla containing at
least one sequenced polar genome. Green: phyla containing polar isolates or numeri-
cally relevant in culture-independent surveys of polar regions.
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Table 8.1 Molecular adaptations in cold-adapted bacteria
Molecular adaptations Explanation or consequence Reference
Protection against reactive oxygen species (ROS):
lower frequency of oxidisable amino acids;
oxidoreductases, superoxide dismutases, catalases,
peroxidases
Due to increased solubility
of O
2
at low temperatures
forming increased ROS
Rabus et al. (2004),Me
´digue et al. (2005),Methe
´
et al. (2005),Bakermans et al. (2007),Duchaud
et al. (2007),Ayub, Tribelli, and Lopez (2009)
and Piette et al. (2010)
Enzymes Maintain catalytic efficiency
at low temperatures
Georlette et al. (2004)
Membranes: increased unsaturation and decreased
chain length of fatty acids, carotenoids,
desaturases
Increase the fluidity of
membranes
Jagannadham, Rao, and Shivaji (1991),Chauhan
and Shivaji (1994) and Ray et al. (1998)
Synthesis of specific elements: cold-shock
proteins, molecular chaperones, compatible
solutes
Maintain vital cellular
functions at cold
temperatures
Motohashi, Watanabe, Yohda, and Yoshida
(1999),Cavicchioli, Thomas, and Curmi (2000),
Watanabe and Yoshida (2004) and Pegg (2007)
Molecular mechanisms involved in protein
flexibility Consequence Reference
Decreased number of H bonds and salt bridges Increased flexibility Feller and Gerday (1997)
Reduced proline and arginine content Increased molecular entropy Ray et al. (1998),Russell (2000),D’Amico et al.
(2002),Cavicchioli, Siddiqui, Andrews, and
Sowers (2002) and Rodrigues and Tiedje (2008)
Reduced frequency of surface, inter-domain and
inter-subunit ionic linkages and ion-network
Increased conformational
flexibility and reduced
enthalphic contribution to
stability
D’Amico et al. (2006)
Adapted from Casanueva, Tuffin, Cary, and Cowan (2010).
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cold-shock proteins (CSPs) (Cavicchioli et al., 2000) and molecular
chaperones (Motohashi et al., 1999), solutes (Pegg, 2007) and structural
modifications for maintaining membrane fluidity (Chintalapati, Kiran, &
Shivaji, 2004; Russell, 1998). Moreover, cold-adapted organisms must
develop an effective and intricate network of defence mechanisms against
oxidative stress: an increasing number of oxidoreductases, superoxide dis-
mutases, catalases and peroxidases can be seen in this perspective (Ayub
et al., 2009; Bakermans et al., 2007; Duchaud et al., 2007; Me
´digue
et al., 2005; Methe
´et al., 2005; Piette et al., 2010; Rabus et al., 2004).
In addition to adaptations at the cellular level, a key adaptive strategy is
the maintenance of adequate reaction rates at thermal extremes; therefore,
adequate features of catalytic processes become crucial. Enzyme catalytic
rates at low temperatures depend on increased protein flexibility and con-
comitant increase in thermolability (Georlette et al., 2004).
In this respect, a suite of factors contribute to maintaining enzyme molecular
flexibility in all cold-adapted organisms. In bacterial enzymes, Ser, Asp, Thr and
Ala are over-represented in the coil regions of secondary structures. On the
other hand, in the helical regions, aliphatic, basic, aromatic and hydrophilic
residues are generally under-represented (Cavicchioli et al., 2002; D’Amico
et al., 2002; Ray et al., 1998; Rodrigues & Tiedje, 2008; Russell, 2000). More-
over, a reduction of surface, inter-domain, inter-subunit ionic linkages and a
decreased number of hydrogen bonds and salt bridges are key mechanisms to
induce an increasing of conformational flexibility of psychrophilic enzymes
(D’Amico et al., 2006; Feller & Gerday, 1997).
Cold adaptation is also strongly linked to the capacity of the organism to
sense temperature changes, perhaps by virtue of mechanisms linked with the
lipid composition of the cell membrane and alterations in the DNA and
RNA topology. The latter may enhance (or halt) the replication, transcrip-
tion and translation processes (Eriksson, Hurme, & Rhen, 2002). Although
increased unsaturation and decreased chain length of fatty acids are the major
modifications of cell membranes, other membrane-associated components
may well play important roles in adaptation to low temperatures
(Jagannadham et al., 1991; Ray et al., 1998). Studies of Antarctic psy-
chrotrophic bacteria in vitro have shown that carotenoids may have a func-
tion in buffering membrane fluidity (Jagannadham et al., 1991).
4. BACTERIAL GLOBINS
The traditional view of the exclusive role of haemoglobin (Hb) as O
2
carrier in vertebrates is obsolete. The discovery of globin genes in prokaryotic
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and eukaryotic microorganisms, including bacteria, yeasts, algae, protozoa
and fungi, suggests that the globin superfamily is exceptionally flexible in
terms of biological roles and possible functions. The number of “globin-
like” proteins is currently increasing as different genomes from microorgan-
isms are sequenced and annotated (Vinogradov et al., 2005; Vinogradov
& Moens, 2008; Vinogradov, Tinajero-Trejo, Poole, & Hoogewijs, 2013).
Non-vertebrate globins display high variability in primary and tertiary
structures, which probably indicates their adaptations to additional functions
with respect to their vertebrate homologues (Vinogradov et al., 2005;
Vinogradov & Moens, 2008; Vinogradov et al., 2013).
The most recent bioinformatic survey of globin-like sequences in prokary-
otic genomes revealed that over half of the more than 2200 bacterial genomes
sequenced so far contain putative globins (Vinogradov et al., 2013). A new
global nomenclature including prokaryotic and eukaryotic globins has been
proposed, and globins have been classified within three families:
(i) myoglobin (Mb)-like family (M) (displaying the classical three-on-three
(3/3) a-helical sandwich motif ) containing flavohaemoglobins (FHbs) and
single-domain globins (SDgb); (ii) sensor globins family (S); and
(iii) truncated haemoglobins family (T), showing the two-on-two (2/2)
a-helical sandwich motif (Vinogradov et al., 2013).
Although there are still some uncertainties about the evolutionary
relationship between these three classes of microbial globins, it has been
proposed that prokaryotic and eukaryotic globins, including vertebrate a/
bglobins, Mb, neuroglobin (Ngb), cytoglobin (Cygb), and invertebrate
and plant Hbs, emerged from a common ancestor (Vinogradov et al., 2005).
4.1. Flavohaemoglobins
Chimeric globins seem to have kept their original enzymatic functions in
prokaryotes, plants and some unicellular eukaryotes. Therefore, the FHb
sub-family has been the only one able to adapt to different functions more
extensively than the other two globin families. Moreover, the presence of
Hbs in unicellular organisms suggests that O
2
transport in metazoans is a rel-
atively recent evolutionary acquisition and that the early Hb functions have
been enzymatic and O
2
sensing (Vinogradov & Moens, 2008).
FHbs are widely present in bacteria, yeasts and fungi and belong to the
ferredoxin reductase-like protein family. They consist of an N-terminal
haem-globin domain fused with C-terminal reductase domain binding
NAD(P)H and FAD (Bolognesi, Bordo, Rizzi, Tarricone, & Ascenzi,
1997; Bonamore & Boffi, 2008).
341The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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Sequence alignments in Gram-negative and Gram-positive bacteria and
unicellular eukaryotes indicate that the FHbs family is a very homogeneous
group of proteins that share highly conserved active sites in both domains.
Amino acid residues building up the haem domain and the flavin-binding
region are widely conserved, and the architecture of FHb domains is closely
similar to those of globins and flavodoxin-reductase proteins. This finding sug-
gests that the haem domain displays globin-like functional properties and that
the flavin moiety acts as an electron-transfer module from NADH to the haem.
However, sequence alignments on separate domains strongly diverge towards
the homologous proteins, suggesting that the rise of FHbs comes from fusion of
a protoglobin ancestor and a flavin-binding domain (Bonamore & Boffi, 2008).
Details of the structure, function and reaction mechanism of purified
native or recombinant FHbs from bacteria and yeast are available (Lewis,
Corker, Gollan, & Poole, 2008). FHb from Escherichia coli (Hmp) is the best
characterised member of the family. Hmp is distributed into both the cyto-
plasmic and periplasmic space, although the biochemically active protein is
exclusively found in the cytoplasmic fraction (Vasudevan, Tang, Dixon, &
Poole, 1995). It is subject to complex control (reviewed by Spiro, 2007;
Poole, 2008), being dramatically up-regulated in response to NO and
nitrosating agents (Membrillo-Herna
´ndez et al., 1999; Membrillo-
Herna
´ndez, Coopamah, Channa, Hughes, & Poole, 1998; Poole et al.,
1996). In particular, the hmp gene is predominantly regulated at the tran-
scriptional level by NO-sensitive transcription factors, especially NsrR
and Fnr (Spiro, 2007). Remarkably, the fine-tuning of Hmp synthesis
appears critical for E. coli survival. In fact, the constitutive expression of
Hmp in the absence of NO generates oxidative stress because of partial
O
2
oxidation by the haem to superoxide and peroxide anion; accumulation
of O
2
radicals has been stressed both in kinetic studies on the purified protein
(Orii, Ioannidis, & Poole, 1992; Poole, Rogers, D’mello, Hughes, & Orii,
1997; Wu, Corker, Orii, & Poole, 2004) and by detecting the
superoxide-generating activity of Hmp in vivo (Membrillo-Herna
´ndez,
Ioannidis, & Poole, 1996). Similar results have also been obtained in
Salmonella enterica where the constitutive expression of Hmp makes cells
hypersensitive to paraquat and H
2
O
2
(Gilberthorpe, Lee, Stevanin,
Read, & Poole, 2007), as well as to peroxynitrite (ONOO
)(McLean,
Bowman, & Poole, 2010).
FHbs display a pivotal role in NO detoxification (Poole & Hughes,
2000). NO, involved in many beneficial and/or dangerous physiological
and pathological processes, is a signalling and defence molecule of great
342 Daniela Giordano et al.
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importance in biological systems, and nowadays has an important role in
contemporary medicine, physiology, biochemistry and microbiology.
The behaviour of NO is made particularly complex by its ability to be
oxidised to the nitrosonium cation (NO
þ
) or reduced to the nitroxyl anion
(NO
) and to react with O
2
to form nitrite (NO2). Moreover, the reac-
tions of NO led to production of reactive nitrogen species (RNS) (reviewed
by Bowman, McLean, Poole, & Fukuto, 2011; Poole & Hughes, 2000),
such as ONOO
, formed by the reaction of NO with superoxide anion.
Extensive literature deals with NO and related species, especially
considering that NO plays vital anti-microbial roles in innate immunity
(Granger, Perfect, & Durack, 1986; Green, Meltzer, Hibbs, & Nacy, 1990;
Iyengar, Stuehr, & Marletta, 1987; Liew, Millott, Parkinson, Palmer, &
Moncada, 1990; Marletta, Yoon, Iyengar, Leaf, & Wishnok, 1988; Stuehr,
Gross, Sakuma, Levi, & Nathan, 1989) and that microorganisms have evolved
a large number of NO-sensitive targets and defence mechanisms against its
toxic effects.
FHbs catalyse reaction of NO with O
2
to yield the relatively innocuous
NO3(Gardner, 2005; Mowat, Gazur, Campbell, & Chapman, 2010;
Poole & Hughes, 2000) by a dioxygenase (Gardner et al., 2006, 2000;
Gardner, Gardner, Martin, & Salzman, 1998) or denitrosylase (Hausladen,
Gow, & Stamler, 1998, 2001). Anaerobically, Hmp shows low
NO-reductase activity, converting NO to N
2
O(Kim, Orii, Lloyd,
Hughes, & Poole, 1999; Liu, Zeng, Hausladen, Heitman, & Stamler,
2000; Poole & Hughes, 2000; Vinogradov & Moens, 2008).
Deletion of the hmp gene alone abolishes the NO-consuming activity
(Liu et al., 2000) and is sufficient to render bacteria hypersensitive to NO
and related compounds, not only in vitro (Membrillo-Herna
´ndez et al.,
1999) but also in vivo (Stevanin, Poole, Demoncheaux, & Read, 2002).
Similar to E. coli (Stevanin, Read, & Poole, 2007), the S. enterica serovar
Typhimurium mutant defective in FHb shows enhanced sensitivity to
mouse and human-macrophage microbicidal activity (Gilberthorpe et al.,
2007; Stevanin et al., 2002), suggesting that the globin contributes to pro-
tection from NO-mediated toxicity in macrophages.
FHbs from several other bacteria show protective functions against
RNS, such as those from Ralstonia eutropha,Bacillus subtilis,Pseudomonas
aeruginosa,Deinococcus radiodurans,S. enterica and Klebsiella pneumoniae
(reviewed by Frey, Farres, Bollinger, & Kallio, 2002).
Although FHbs protect pathogenic microorganisms from the host
immune systems, they also defend non-pathogenic organisms from
343The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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endogenous NO generated by nitrate and nitrite reduction, under anaerobic
conditions (Rogstam, Larsson, Kjelgaard, & von Wachenfeldt, 2007).
Vinogradov and Moens (2008) also propose that FHb may be involved in
the coordination of intracellular NO concentration with extracellular O
2
levels, as suggested by the mitochondrial generation of NO in Saccharomyces
exposed to hypoxia, with concomitant localisation of FHb in the pro-
mitochondrial matrix (Castello, David, McClure, Crook, & Poyton, 2006).
Although FHbs are involved in NO detoxification, other physiological
functions have been identified; for instance, Hmp has alkyl hydroperoxide
reductase activity under anaerobic conditions, suggesting that, together with
the unique lipid-binding properties, this globin is capable of catalysing the
reduction of lipid-membrane hydroperoxides into the corresponding alco-
hols using NADH as electron donor and may thus be involved in the repair
of the lipid-membrane oxidative damage generated during oxidative/
nitrosative stress (Bonamore & Boffi, 2008; D’Angelo et al., 2004).
4.2. Single-domain globins
The first identified and sequenced SDgb was the Hb of Vitreoscilla (Vgb),
whose expression is significantly increased under microaerobic conditions.
It comprises a single domain, unmistakably globin-like, and the protohaem;
it lacks the reductase domain seen in FHbs. Despite evidence that its expres-
sion in heterologous hosts can provide some protection from nitrosative
stress, the generally accepted view is that Vgb facilitates O
2
utilisation,
perhaps by directly interacting with a terminal oxidase (Wu, Wainwright,
Membrillo-Herna
´ndez, & Poole, 2004, Wu, Wainwright, & Poole, 2003).
Another example of SDgb is offered by the microaerophilic, foodborne,
pathogenic bacterium Campylobacter jejuni, exposed to NO and other
nitrosating species during host infection. This globin (Cgb) is dramatically
up-regulated in response to nitrosative stress (Elvers et al., 2005,
Elvers, Wu, Gilberthorpe, Poole, & Park, 2004; Monk, Pearson,
Mulholland, Smith, & Poole, 2008; Smith, Shepherd, Monk, Green, &
Poole, 2011) and provides a specific and inducible defence against NO
and nitrosating agents (Poole, 2005); it detoxifies NO and presents a
peroxidase-like haem-binding cleft. In contrast to Vgb, there is no evidence
that Cgb functions in O
2
delivery.
Since a detailed overview of SDgbs is beyond the goal of this contribu-
tion, the reader is directed elsewhere (Bowman et al., 2011; Frey et al., 2002;
344 Daniela Giordano et al.
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Frey & Kallio, 2003, Frey, Shepherd, Jokipii-Lukkari, Haggman, & Kallio,
2011; Vinogradov et al., 2005, 2013).
4.3. Truncated haemoglobins
Members of the T family are found in eubacteria, cyanobacteria, protozoa
and plants but not in animals (Milani et al., 2005; Wittenberg, Bolognesi,
Wittenberg, & Guertin, 2002). The N-termini of these globins are
20–40-residue shorter; these globins display the 2/2 a-helical sandwich fold
(composed of helices B, E, G and H), which has been recognised as a subset
of the classical 3/3 a-helical sandwich (Milani et al., 2005; Wittenberg et al.,
2002). The 2/2 a-helical sandwich fold results in four a-helices (B/E and
G/H) arranged in antiparallel pairs connected by an extended polypeptide
loop that replaces the a-helix F (Fig. 8.2).
It is noteworthy that the haem-proximal helix F is replaced by a poly-
peptide segment (Milani et al., 2001; Pesce et al., 2000). The residues com-
prising the F loop affect the orientation of the proximal HisF8 modulating
the O
2
-binding properties (Milani et al., 2005; Pathania, Navani, Gardner,
Gardner, & Dikshit, 2002). Helix E is very close to the haem distal site;
therefore, residues at positions B10, CD1, E7, E11, E15 and/or G8
(Table 8.2) modulate ligand binding (Milani et al., 2005).
On the basis of phylogenetic analysis, the T family can be further divided
into three distinct sub-families: TrHbI (or N), TrHbII (or O) and TrHbIII
(or P); specific structural features that depend on residues of the distal haem
pocket distinguish each group (Table 8.2). In group I, the hydrogen bond
network stabilising the haem-bound ligand involves the B10, E7 and E11
residues. Strongly conserved Tyr B10 plays a key role in ligand stabilisation
through OH pointing directly to the heam-bound ligand. Normally, com-
plete stabilisation by the H-bond network is provided by Glu located at E7
or E11, or at both positions. In group II, TrpG8 is fully conserved, contrib-
uting to ligand stabilisation by the H bond linking the indole nitrogen atom
and the ligand.
Further, Tyr at CD1 in some TrHbIIs drastically modifies the interaction
network (Pesce et al., 2000). In group III, for example, C. jejuni TrHbIII
(Ctb), the hydrogen bond network stabilising the haem-bound ligand
involves TyrB10 and TrpG8 (Nardini et al., 2006). Interestingly, the affinity
of cyanide for the ferrous derivative of C. jejuni Ctb is higher than that
reported for any known (in)vertebrate ferrous globin and is reminiscent
345The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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of that of ferrous horseradish peroxidase, suggesting that this globin may par-
ticipate in cyanide detoxification (Bolli et al., 2008).
Strikingly, TrHbs belonging to group III (or N) host a protein matrix
tunnel system offering a potential path for ligand diffusion to and from
the haem distal site. The apolar tunnel/cavity system, extending for approx-
imately 28 A
˚through the protein matrix, is conserved in TrHbs belonging
to group N, although with modulation of its size and/or structure (Milani
et al., 2001; Pesce, Milani, Nardini, & Bolognesi, 2008). It has been pro-
posed that in Mycobacterium tuberculosis HbN, the haem-Fe/O
2
stereochem-
istry and the protein matrix tunnel may promote O
2
/NO chemistry in vivo,
as a M. tuberculosis defence mechanism against macrophage nitrosative stress
(Milani et al., 2001).
Unlike HbN, M. tuberculosis HbO does not host the protein matrix tun-
nel but two topologically equivalent matrix cavities. Moreover, the small
apolar Ala E7 residue leaves room for ligand access to the haem distal site
through the conventional E7 path (Pesce et al., 2008), as proposed for Mb.
In contrast to TrHbs I and II (Milani et al., 2004, 2001; Nardini et al.,
2006; Pesce et al., 2008), Ctb does not display protein matrix tunnel/cavity
systems at all (Nardini et al., 2006). Although the gating role of HisE7 in
the modulation of ligand access into and out of the heam pocket is debated
Figure 8.2 A stereo view of sperm whale Mb (3/3) (pdb:1JP6, Urayama, Gruner, &
Phillips, 2002) and B. subtilis TrHbII (HbO) (pdb:1UX8, Ilari, Giangiacomo, Boffi, &
Chiancone, 2005) tertiary structures, including the haem group and helices. Modifica-
tions of the conventional 3/3 Mb fold occur particularly at helix A and in the CD-D
regions, which are virtually absent, and in the EF-F regions of TrHbs. A very short seg-
ment linking helices C and E forces the haem-distal helix E very close to the haem distal
side.
346 Daniela Giordano et al.
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(Lu, Egawa, Wainwright, Poole, & Yeh, 2007; Nardini et al., 2006), this
mechanism appears to be operative in the C. jejuni TrHbIII (Nardini
et al., 2006).
The sequence identity between TrHbs belonging to the three phyloge-
netic groups is very low (<20%) (Nardini, Pesce, Milani, & Bolognesi, 2007;
Vuletich & Lecomte, 2006; Wittenberg et al., 2002), but may be higher than
80% within a given group. Analysis of the distribution of TrHbs suggests a
scenario for the evolution of the different groups where the group II gene is
ancestral and group-I and group-III genes are the results of duplications and
transfer events (Vuletich & Lecomte, 2006).
Table 8.2 Amino acid residues building up the proximal (F8) and distal (B10, CD1, E7,
E11, E15 and G8) haem pocket of sperm whale Mb and TrHbs belonging to groups I, II
and III
Protein Group B10 CD1 E7 E11 E15 F8 G8
Sperm whale Mb L F H V L H I
Nostoc commune HF QL L HI
Paramecium caudatum YF QTL HV
Chlamydomonas eugametos IYFQQLHV
Synechocystis YF QQLHV
Mycobacterium tuberculosis NYFLQFHV
Mycobacterium avium NYFLQFHV
Mycobacterium avium OYYALLHW
Mycobacterium tuberculosis OYYALLHW
Mycobacterium smegmatis OYYALLHW
Mycobacterium leprae II Y Y A L L H W
Thermobifida fusca YY AL L HW
Bacillus subtilis YF TQLHW
Arabidopsis thaliana YF AQFHW
Thiobacillus ferrooxidans YF HLWHW
Mycobacterium avium PYFHMWHW
Campylobacter jejuni III Y F H I W H W
Bordetella pertussis 1YFHLWHW
Adapted from Milani et al. (2005).
347The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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TrHbs belonging to the three groups may coexist in some bacteria,
suggesting distinct functions. These globins have been hypothesised to store
ligands and/or substrates, to facilitate NO detoxification, to sense O
2
/NO,
to display (pseudo)enzymatic activities and to deliver O
2
under hypoxic
conditions (Vinogradov & Moens, 2008; Wittenberg et al., 2002). The high
O
2
affinity suggests that some TrHbs may function as O
2
scavengers rather
than O
2
transporters (Ouellet et al., 2003; Wittenberg et al., 2002).
5. THE ANTARCTIC MARINE BACTERIUM
PSEUDOALTEROMONAS HALOPLANKTIS TAC125:
A CASE STUDY
5.1. General aspects
Despite the fact that the Antarctic marine environment is characterised by
permanent low temperatures, the surface water and the sea-ice zones host
a surprisingly high level of microbial activity.
A typical representative of g-Proteobacteria found in the Antarctic is the
marine cold-adapted psychrophile P. haloplanktis TAC125 (PhTAC125), a
Gram-negative bacterium, isolated in Antarctic coastal sea water in
the vicinity of the French station Dumont d’Urville, Terre Ade
´lie
(66400S; 140010E). As in many marine g-Proteobacteria, its genome is
made up of two chromosomes (Me
´digue et al., 2005). This strain thrives
between 2C and 4 C, but is also able to survive long-term frozen con-
ditions when entrapped in the winter sea ice. PhTAC125 can grow in a wide
temperature range (4–25 C) (Fig. 8.3A) and achieve very high cell density
even under uncontrolled laboratory conditions (Fig. 8.3B). In a marine
broth, PhTAC125 displays a doubling time of about 4 h at 4 C and 5 h
15 min at 0 C. At higher temperatures, the bacterium divides actively
and the generation time decreases moderately (e.g. 1 h 40 min at 18 C),
with increase in the biomass produced at the stationary phase (Piette
et al., 2011). In contrast, higher temperatures cause a drastic reduction in
cell density at the stationary phase, suggesting that the heat-induced stress
affects the growth (Piette et al., 2011).
The doubling time of PhTAC125 at 16 C is approximately 2 h, almost
three times faster than that of E. coli under similar growth conditions (Piette
et al., 2010). Consistent with the high growth rate, at room temperature,
PhTAC125 shows a very efficient chemotactic response, 10 times faster than
that of E. coli, allowing it to exploit nutrient patches in the marine
348 Daniela Giordano et al.
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environment before they dissipate (Stocker, Seymour, Samadani, Hunt, &
Polz, 2008). PhTAC125 grows with extremely high growth rates in defined
sea water medium, with peptone as the only carbon and nitrogen source,
suggesting that these growth conditions resemble the favoured natural envi-
ronment of the marine bacterium, which can be easily isolated from dam-
aged tissues of fishes or molluscs, where such substrates are available (Wilmes
et al., 2011). PhTAC125 lacks a cyclic AMP (cAMP)–catabolite activator
protein complex, that regulates carbon availability in related organisms,
and a phosphoenolpyruvate-dependent phosphotransferase system (PTS)
Figure 8.3 (A) Temperature dependence of the doubling time of Antarctic bacterium
PhTAC125, grown in marine broth (solid line and circles), compared to a typical growth
curve of E.coli RR1 strain, obtained in LB broth (dashed line). (B) Growth curves of
PhTAC125, performed at 4 C (open circles), 18 C (filled circles) and 26 C
(filled squares). Adapted from Piette et al. (2011).
349The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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for the transport and first metabolic step of so-called PTS sugars (Me
´digue
et al., 2005; Wilmes et al., 2011), making the bacterium unable to grow
on glucose.
Approximately 13% of the identified proteins in the periplasmic protein
fraction of PhTAC125 are transport-related proteins, mostly belonging to
TonB-dependent transport systems (TBDTs) (Wilmes et al., 2011). The
high amount of TBDTs in the genome (Me
´digue et al., 2005) and in the
periplasmic proteome (Wilmes et al., 2011) support the idea that these trans-
porters permit efficient use and scavenge the large variety of substrates found
in the marine environment, probably representing an important prerequisite
for fast growth under nutrient-rich conditions. Besides TBDTs, three ABC
transporters, four porins and the transporter TolB are the other detected
putative substrate-transport-related proteins (Me
´digue et al., 2005;
Wilmes et al., 2011).
PhTAC125 is also able to grow in anaerobiosis, although with lower
yields (Me
´digue et al., 2005). It is worth noting that lower duplication rate
and poor growth of PhTAC125 in micro-aerobiosis have been observed
(Parrilli, Giuliani, Giordano, et al., 2010). Due to lower O
2
solubility at
15 C than at 4 C, OD
600, max
is approximately 7.2 at 15 C, and 4.3 at
4C, in extreme aerobiosis; moreover, OD
600, max
is approximately 1.25
at 4 C and 0.38 at 15 C, in micro-aerobiosis.
5.2. Genomic and post-genomic insights
Over the last decade, several genomes from psychrophilic bacteria and
Archaea have been sequenced (Casanueva et al., 2010). Some of these
(Allen et al., 2009; Ayala-del-Rio et al., 2010; Duchaud et al., 2007;
Me
´digue et al., 2005; Methe
´et al., 2005; Rabus et al., 2004; Riley et al.,
2008; Rodrigues et al., 2008; Saunders et al., 2003) have been analysed with
respect to cold adaptation through proteomic and transcriptomic approaches
(Bakermans et al., 2007, Bergholz, Bakermans, & Tiedje, 2009; Campanaro
et al., 2011; Goodchild, Raftery, Saunders, Guilhaus, & Cavicchioli, 2005;
Goodchild et al., 2004; Kawamoto, Kurihara, Kitagawa, Kato, & Esaki,
2007; Piette et al., 2011, 2010; Qiu, Kathariou, & Lubman, 2006; Ting
et al., 2010; Williams et al., 2010; Wilmes et al., 2011; Zheng et al., 2007).
Through the MaGe annotation platform (http://www.genoscope.cns.fr/
agc/mage/wwwpkgdb/Login/log.php?pid¼7#ancreLogin), and by in silico
and in vivo analyses, several exceptional genomic and metabolic features have
been identified in PhTAC125 (Me
´digue et al., 2005).
350 Daniela Giordano et al.
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PhTAC125 is an interesting model for investigating strategies adopted to
survive at low temperature (Me
´digue et al., 2005). The available genome
sequence, combined with remarkable versatility and fast growth (Duilio,
Tutino, & Marino, 2004), makes PhTAC125 an attractive model to study
protein-secretion mechanisms in marine environments, in addition to its
use as a non-conventional host for recombinant production of thermal-
labile and aggregation-prone proteins at low temperature (Cusano et al.,
2006; Gasser et al., 2008; Parrilli, Duilio, & Tutino, 2008; Parrilli,
Giuliani, Pezzella, et al., 2010; Vigentini, Merico, Tutino, Compagno, &
Marino, 2006). Actually, low temperatures improve the quality of the prod-
ucts, removing the negative effects of high temperatures on protein folding,
due to the strong temperature dependence of hydrophobic interactions that
mainly drive the aggregation (Kiefhaber, Rudolph, Kohler, & Buchner,
1991). The growth of E. coli at lower temperatures to minimise aggregation
has not been successful, probably because sub-optimal temperatures act neg-
atively on cell performance (Gasser et al., 2008).
The efficiency of the cold-adapted PhTAC125 expression system was
demonstrated by the production of biologically active soluble products,
for example, a yeast a-glucosidase, the mature human nerve growth factor
and a cold-adapted lipase (de Pascale et al., 2008; Parrilli et al., 2008).
At the genome level, a relatively large number of rRNA genes (nine
rRNA gene clusters) and tRNA genes (106 genes, organised in long runs
of repeated sequences) have been observed in PhTAC125 (Me
´digue
et al., 2005), similar to Colwellia psychrerythraea (Methe
´et al., 2005) and
Psychromonas ingrahamii (Riley et al., 2008). This finding may be explained
as a response to the limited speed of transcription/translation at low temper-
ature, allowing fast growth in the cold. However, it has recently been spec-
ulated that a high number of rRNA genes may reflect an ecological bacterial
strategy to improve the response to perturbations in nutrient resources
(Klappenbach, Dunbar, & Schmidt, 2000). Moreover, PhTAC125 contains
19 genes presumably encoding known RNA-binding proteins or RNA
chaperones. An unexpected feature is the prominent absence of hns,an
RNA/nucleoid-associated cold-shock gene found in all g-Proteobacteria.
In contrast, the presence of many RNA helicases (three copies of rhlE,
and probably a fourth one, PSHAa0641, and two copies of srmB) instead
of the single one in E. coli, suggests that the control of RNA folding and
degradation is important at low temperature (Me
´digue et al., 2005). In fact,
RNA helicases have been found to be over-expressed at low temperature in
many other psychrophilic microorganisms, such as Methanococcoides burtonii
351The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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(Lim, Thomas, & Cavicchioli, 2000), Exiguobacterium sibiricum (Rodrigues
et al., 2008), Sphingopyxis alaskensis (Ting et al., 2010) and Psychrobacter
arcticus (Bergholz et al., 2009; Zheng et al., 2007). This feature may reflect
the need for help to unwind the RNA secondary structures for highly effi-
cient translation in the cold (Cartier, Lorieux, Allemand, Dreyfus, &
Bizebard, 2010).
Sequence analyses using genomic and metagenomic data clearly show
that different mechanisms of adaptation to cold, including a bias towards
specific residues, occur. The analysis of the amino acid composition of
g-Proteobacteria from different biotopes reveals a similar trend in the var-
ious genomes, Leu being most abundant, and Trp, Cys, His and Met
most unusual. The proteome of thermophilic microorganisms shows several
differences when compared to those of mesophilic and psychrophilic spe-
cies, for example, Gln is poorly represented in thermophilic species, whereas
mesophilic and psychrophilic species prefer Ala (except in Oceanobacillus
iheyensis). A few remarkable differences have been identified between
mesophilic and psychrophilic species, in particular, that some Asn and
Gln are pivotal for bacteria growth in cold environments.
Specifically, proteins from PhTAC125 are rich in Asn (Me
´digue et al.,
2005) and contain few hydrophilic uncharged residues bearing an often ther-
molabile amide group (Stratton et al., 2001; Weintraub & Manson, 2004;
Zhou, Cocco, Russ, Brunger, & Engelman, 2000), which undergo
deamidating cyclisation, a process extremely sensitive to temperature
(Daniel, Dines, & Petach, 1996). The richness in Asn in the PhTAC125
proteome makes this Antarctic bacterium an organism of choice for foreign
protein production when deamidation ought to be at a minimum
(Weintraub & Manson, 2004).
Other aspects of cold-adapted proteins (CAPs) are (i) the significantly
high level of non-charged polar Gln and Thr, and the low content of hydro-
phobic residues (particularly Leu) in the archaeal psychrophiles
Methanogenium frigidum and M. burtonii (Saunders et al., 2003); (ii) the low
content of polar residues such as Ser, the replacement of Asp with Glu,
and the general decrease in charged residues in the proteins of
C. psychrerythraea (Methe
´et al., 2005); (iii) the reduction of Pro and Arg
codons in the P. arcticus genome, in particular, in the cell-growth and repro-
duction genes (Ayala-del-Rio et al., 2010); and (iv) decrease in Ala, Pro and
Arg in Shewanella halifaxensis and S. sediminis (Zhao, Deng, Manno, &
Hawari, 2010). These findings support the hypothesis of the increased flex-
ibility and reduced thermostability of CAPs (Methe
´et al., 2005). However,
352 Daniela Giordano et al.
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no substitution promoting cold adaptation has been found in the Desulfotalea
psychrophila genome (Rabus et al., 2004).
The proteomes expressed by PhTAC125 at 4 and 18 C were com-
pared (Piette et al., 2010, 2011) to identify the cold-acclimation proteins,
that is, those continuously over-expressed at high level at low tempera-
tures, and to highlight the numerous down-regulated cellular functions
(Piette et al., 2011). Interestingly, three proteins (Pnp, TypA and Tig,
involved in distinct functions such as degradosome, membrane integrity
and protein folding, respectively) have been identified as CAPs in
PhTAC125 (Piette et al., 2010) and as CSPs in mesophiles. Moreover, sev-
eral CSP homologues have been reported in other cold-adapted bacteria
(Bakermans et al., 2007; Bergholz et al., 2009; Kawamoto et al., 2007),
suggesting striking similarities between CSPs in mesophiles and CAPs in
psychrophiles. In agreement with these results, it has been proposed that
“from an evolutionary point of view, one of the adaptive mechanisms
for growth in the cold is to regulate the cold-shock response, shifting from
a transient expression of CSPs to a continuous synthesis of at least some of
them” (Piette et al., 2010, 2011).
The proteomic analyses of PhTAC125 revealed that 30% of the identi-
fied CAPs are directly related to protein synthesis, covering all essential steps,
from transcription (including RNA polymerase RpoB) to translation (i.e.
methionyl-tRNA synthetase MetG that can be connected to the need of
an increased pool of initiation tRNA to promote protein synthesis) and
folding (i.e. the trigger factor—TF—Tig that acts on proteins synthesised
by the ribosome; and PpiD, involved in the folding of outer membrane pro-
teins). The genes pnp and rpsA also encode components of the degradosome
that regulate transcript lifetimes and two putative proteases (PSHAa2492
and PSHAa2260), identified as CAPs, likely to be involved in the proteolysis
of misfolded proteins (Piette et al., 2010). Other identified CAPs are both
components and regulators of the outer membrane architecture (Piette
et al., 2010). In particular, the TonB-dependent receptor is indicative of
sensing and exchanges with the external medium, and TypA (involved in
lipopolysaccharides core synthesis) and Pal (a peptidoglycan-associated pro-
tein) are involved in the outer membrane stability and integrity (Abergel,
Walburger, Chenivesse, & Lazdunski, 2001).
These results strongly suggest that low temperatures impair protein syn-
thesis and folding, resulting in up-regulation of the associated functions and
indicating that both cellular processes are limiting factors for bacterial devel-
opment in cold environments (Piette et al., 2010).
353The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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Similar amounts of ribosomal and translation-specific proteins have also
been revealed in mesophilic fast-growing bacteria, such as B. subtilis or
B. licheniformis, under optimal growth conditions (Buttner et al., 2001;
Voigt et al., 2004; Wilmes et al., 2011), suggesting that also the expression
of these proteins, directly related to protein synthesis, is likely growth-rate
dependent (Klumpp, Zhang, & Hwa, 2009).
Based on the cytoplasmic proteome and the available genome sequence, the
analysis of the amino acid degradation pathways showed that all the common
degradation routes are present in PhTAC125, with the exception of those
involved in Trp and Lys catabolism (Wilmes et al., 2011). Since the Antarctic
genome contains coding sequences of biosynthetic enzymes for all
20 proteinogenic amino acids, it is likely that the degradation of Trp and Lys
occurs by reversal of the biosynthetic routes. For instance, an alternative way
to use Lys may be decarboxylation to cadaverine via PSHAa1094 (annotated
as a putative basic amino acid decarboxylase) (Wilmes et al., 2011). Further, a
relatively high abundance of the tricarboxylic acid cycle enzymes in the cyto-
plasmic proteome analysed at 16 C, needed for efficient catabolism of the
peptone-based amino acids, is in line with the extremely high growth rate
(maximal rate being 0.35 h
1
) of the Antarctic bacterium (Wilmes et al., 2011).
The major CAP, 37-fold over-expressed at 4 C(Piette et al., 2010), is the
TF Tig, a CSP in E. coli (Kandror & Goldberg, 1997). TF is the first molecular
chaperone interacting with virtually all newly synthesised polypeptides on
the ribosome; it assists folding by delaying premature chain compaction
and maintaining the elongating polypeptide in a non-aggregated state until
adequate structural information for correct folding is available, and later
promotes protein folding (Hartl & Hayer-Hartl, 2009; Martinez-Hackert &
Hendrickson, 2009; Merz et al., 2008). TF also possesses a peptidyl-prolyl
cis–trans isomerase activity (Kramer et al., 2004), the rate-limiting step in
the folding of a wide range of proteins (Baldwin, 2008). In PhTAC125,
the peptidyl-prolyl cis–trans isomerase involved in protein folding is
up-regulated at low temperature (Piette et al., 2010).
The major heat-shock proteins (HSPs), such as the chaperone DnaK, the
chaperonin GroEL/ES and the chaperone Hsp90, are cold-repressed in the
proteome of PhTAC125. However, their expression is up-regulated when
the bacterium is grown at higher temperature, indicating heat-induced cel-
lular stress (Goodchild et al., 2005; Rosen & Ron, 2002). Synthesis of HSPs
is also repressed during growth at low temperatures (Kandror & Goldberg,
1997)inE. coli. Accordingly, the observed cold repression of HSPs would be
beneficial not only to PhTAC125 but also to E. coli.
354 Daniela Giordano et al.
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TF is transiently expressed in mesophilic bacteria but continuously over-
expressed in psychrophiles to achieve cold adaptation, rescuing the chaper-
one function at low temperatures (Piette et al., 2010, 2011).
Either PPiases or TF act as potential CAPs in the proteome of most cold-
adapted microorganisms analysed so far (Goodchild et al., 2005, 2004;
Kawamoto et al., 2007; Qiu et al., 2006; Suzuki, Haruki, Takano,
Morikawa, & Kanaya, 2004; Ting et al., 2010), suggesting that the constraint
imposed by protein folding at low temperature and the cellular responses are
common traits in most psychrophiles (Piette et al., 2010). In contrast, an
almost inverse regulation was found in P. arcticus where GroEL/ES
chaperonins and repression of TF are up-regulated under cold conditions
(Bergholz et al., 2009; Zheng et al., 2007). Increased synthesis of
chaperonins has also been reported in S. alaskensis (Ting et al., 2010)
possessing two sets of dnaK–dnaJ–grpE gene clusters; proteomic analysis sug-
gests that one of these sets functions as a low-temperature chaperone system
whereas the other functions at higher temperatures (Ting et al., 2010).
At low temperature, in accordance with reduced biomass, almost half of
the down-regulated proteins are involved in general bacterial metabolism.
Most of these proteins are involved in oxidative metabolism, including gly-
colysis, the pentose phosphate pathway, the Kreb’s cycle and the electron
chain transporters (Piette et al., 2011; Wilmes et al., 2011).
The PhTAC125 genome contains genes putatively involved in NO
metabolism, such as NO reductase, PSHAa2417,andNO
2reductase,
PSHAa1477 (Me
´digue et al., 2005). In this context, the presence of multiple
genes in distinct positions on chromosome I encoding three TrHbs (annotated
as PSHAa0030,PSHAa0458,PSHAa2217)andaFHb(PSHAa2880)
(Giordano et al., 2007; Me
´digue et al., 2005) may be pivotal for cell protection
(see Section 6).
5.3. Excess of O
2
and metabolic constraints
Gases (e.g. O
2
) and radicals (e.g. NO) are highly soluble and stable at
low temperature with visible consequences in genome annotations in
cold-adapted bacteria, having developed responses to strong oxidative stress
(see Casanueva et al., 2010).
The apparent benefits of easier O
2
supply are contrasted by the adverse
effects of low temperature on (macro)molecular functions and on the
increased production of RNS and reactive oxygen species (ROS)
(Casanueva et al., 2010; D’Amico et al., 2006). In fact, although RNS and
355The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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ROS could act as signalling molecules during cell differentiationand cell cycle
progression, and in response to extracellular stimuli (Sauer, Wartenberg, &
Hescheler, 2001), they are potentially toxic for cells (Finkel, 2003), being
involved in a large number of pathological mechanisms.
Several mechanisms to cope with RNS and ROS have been proposed for
cold-adapted bacteria. They include slightly lower frequency of oxidisable res-
idues in protein sequences, occurrence of specific reductases, presence of
dioxygenases and deletion of RNS- and ROS-producing metabolic pathways
(see Casanueva et al., 2010). Interestingly, acyl desaturases (that introduce a dou-
ble bond into fatty-acyl chains, using O
2
as substrate) combine the elimination
of toxic O
2
withtheimprovementofmembranefluidity(Zhang & Rock,
2008). Therefore, the augmented capacity in antioxidant defence is likely an
important component of evolutionary adaptation to a cold and O
2
-rich envi-
ronment (Ayub et al., 2009; Bakermans et al., 2007; Duchaud et al., 2007;
Me
´digue et al., 2005; Methe
´et al., 2005; Piette et al., 2010; Rabus et al., 2004).
The cold environment raises the question of how PhTAC125 can cope
with RNS and ROS. We have evidence proving that, in order to prevent
significant damage to cellular structures, PhTAC125 improves the redox
buffering capacity of the cytoplasm, and glutathione synthetase is strongly
up-regulated at low temperature (Piette et al., 2010). These adjustments
in antioxidant defences are needed to maintain the steady-state concentra-
tion of ROS and may be important components in evolutionary adaptations
in cold and O
2
-rich environments (Chen et al., 2008).
The main adaptive strategy used by PhTAC125, exposed to permanent
oxidative stress, is expected to be increased production of enzymes active
against hydrogen peroxide and superoxide. Surprisingly, in the genome
of PhTAC125, only two genes, encoding an iron superoxide dismutase
(sodB;PSHAa1215) and a catalase (katB, with the possible homologue
PSHAa1737), have been identified. This catalase has very high similarity
to catalases from other a-, b- and g-Proteobacteria, for example, Psy-
chrobacter,Mannheimia,Haemophilus and Neisseria (Me
´digue et al., 2005).
Moreover, while the O
2
-responding OxyR control has been found in
PhTAC125, SoxR regulation is absent (Me
´digue et al., 2005).
Proteomic analyses of PhTAC125 reveal that oxidative stress-related
proteins, such as catalase, glutathione reductase and peroxiredoxin (Piette
et al., 2011), are repressed at 4 C. However, because PhTAC125 metabo-
lism is stimulated at 18 C, it should be mentioned that, although these pro-
teins would be repressed at 4 C, they would most likely be induced at 18 C
(Piette et al., 2011).
356 Daniela Giordano et al.
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In contrast, the Arctic bacterium C. psychrerythraea has developed an
enhanced antioxidant capacity owing to the presence of three copies of cat-
alase genes as well as two superoxide-dismutase genes, one of which codes
for a nickel-containing superoxide-dismutase, never reported before in
proteobacteria (Methe
´et al., 2005).
PhTAC125 copes with increased O
2
solubility by deleting entire meta-
bolic pathways that generate ROS as side products. It is worth noting
that, despite the availability of molybdate in sea water (Hille, 2002),
PhTAC125 not only lacks the molybdate biosynthetic and transport genes
but also genes encoding enzymes using molybdate as cofactor, for example,
trimethylamine N-oxide reductase, xanthine oxidase, biotin sulphoxide
reductase and oxido-reductase YedY (Loschi et al., 2004).
The PhTAC125 genome also contains all genes required for the pentose
phosphate pathway (Me
´digue et al., 2005); moreover, glucose-6-phosphate
dehydrogenase (Zwf; PSHAa1140), transketolase (TktA; PSHAa0671) and
transaldolase (TalB; PSHAa2559) have been found in the cytoplasmic
proteome (Wilmes et al., 2011). This feature increases the concentration
of NADPH, which in turn provides high levels of reduced thioredoxin that
can help to protect against the toxic effects of O
2
. Therefore, to develop
better oxidative stress adaptation in the cold, PhTAC125 can use the pentose
phosphate pathway for carbohydrate inter-conversion.
In order to cope with the improved stability of ROS at low tempera-
tures, iron-related proteins are down-regulated at 4 C(Piette et al.,
2011), presumably to avoid oxidative cell damage induced by the deleterious
Fenton reaction (Valko, Morris, & Cronin, 2005). Cell protection may be
achieved by dioxygenases that are coded in large number in both chromo-
somes (Me
´digue et al., 2005). Moreover, O
2
-consuming lipid desaturases
protect against toxic O
2
by increasing the membrane fluidity at low temper-
ature (Me
´digue et al., 2005).
A further tool, possibly related to the peculiar features of the Antarctic
habitat, may be the synthesis of globins facilitating several biological
functions, including protection from nitrosative and oxidative stress (see
Section 6.2.4).
5.4. Biotechnological applications
Microorganisms are an interesting source of cold-active enzymes endowed
with biotechnological potential. Enzymes from psychrophiles have recently
received increasing attention, because they offer novel opportunities in
357The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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several industrial processes where high enzymatic activity or peculiar stereo-
specificity at low temperature is required. The high specific activity of cold-
adapted enzymes is due to the lack of a number of non-covalent stabilising
interactions, providing improved flexibility of the conformation (Feller,
2010; Feller & Gerday, 2003; Siddiqui & Cavicchioli, 2006); this is a key
adaptation to compensate for the exponential decrease in chemical reaction
rates at lower temperatures. For instance, cold-active esterases and lipases
found in the genome of PhTAC125 (Aurilia, Parracino, Saviano,
Rossi, & D’Auria, 2007; de Pascale et al., 2008; Me
´digue et al., 2005)
can be added to detergents for use at low temperatures and to biocatalysts
for biotransformation of heat-labile compounds (Margesin & Schinner,
1994). Cold-active Lip1 lipase (de Pascale et al., 2008), encoded by the
PSHAa0051 gene, was functionally over-expressed in PhTAC125 at 4 C
(Duilio et al., 2004). In contrast, in mesophilic E. coli, the recombinant pro-
duction was always found associated with the inclusion bodies and refolding
was unsuccessful (de Pascale et al., 2008).
Engineered PhTAC125 expressing a toluene-o-xylene mono-oxygenase
from mesophilic Pseudomonas sp. OX1 (Bertoni, Bolognese, Galli, &
Barbieri, 1996), combined with the endogenous laccase-like protein
induced by copper, can convert several aromatic compounds into non-toxic
metabolites (Papa, Parrilli, & Sannia, 2009; Parrilli, Papa, Tutino, & Sannia,
2010; Siani, Papa, Di Donato, & Sannia, 2006). This strategy endows
PhTAC125 with degrading capabilities and wide potentiality in bioremedi-
ation applications, for example, removal of organic pollutants from chem-
ically contaminated marine environments and cold effluents of industrial
processes (Parrilli, Papa, et al., 2010).
6. P. haloplanktis TAC125 GLOBINS
6.1. General aspects
Three TrHbs were identified in PhTAC125: one TrHbI (encoded by the
PSHAa0458 gene) and two TrHbsII (encoded by PSHAa0030 and
PSHAa2217)(Giordano et al., 2007). The sequence identity between
TrHbs from different groups is generally low but may be higher within a
given group. The identity between the two TrHbs belonging to group II
is 24%, suggesting that these proteins may have different function(s) in bac-
terial cellular metabolism. Moreover, a FHb, annotated as PSHAa2880, has
been found in PhTAC125 (Giordano et al., 2007).
The distribution of globins in polar bacteria is very different (Table 8.3).
358 Daniela Giordano et al.
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Interestingly, the C. psychrerythraea and P. cryohalolentis K5 genomes do
not possess genes encoding TrHbs, but contain a gene for a single FHb;
E. sibiricum 255-15 (Rodrigues et al., 2008), P. ingrahamii 37 (Riley et al.,
2008) and Marine actinobacterium PHSC20C1 genomes contain one gene
encoding a TrHb and one encoding an FHb; S. frigidimarina NCMB400
contains two genes encoding TrHbs and one encoding a FHb; P. arcticus
273-4, Oleispira antarctica RB-8, D. psychrophila LSv54, Psychroflexus torquis
ATCC 700755, Polaribacter filamentous 215 and Polaribacter irgensii 23-P do
not possess globin genes (Table 8.3).
The presence of multiple genes encoding globins may be considered a
mechanism of defence against oxidative and nitrosative stress also in
mesophilic organisms.
Table 8.3 Distribution of FHb and TrHbs in polar bacteria
Polar bacteria Strain origin FHb TrHbs
Colwellia psychrerythraea 34H Arctic marine sediments 1 –
Shewanella frigidimarina
NCMB400
Sea ice, sea water, Antarctica 1 2
Psychrobacter arcticus 273-4 Siberian permafrost – –
Psychrobacter cryohalolentis K5 Siberian permafrost 1 –
Oleispira antarctica RB-8 Rod Bay, Ross Sea Antarctica – –
Pseudoalteromonas haloplanktis
TAC125
Coastal Antarctic sea water, Terre
Ade
´lie
13
Desulfotalea psychrophila LSv54 Arctic marine sediments, Svalbard – –
Exiguobacterium sibiricum 255-
15
Siberian permafrost 1 1
Psychroflexus torquis ATCC
700755
Sea-ice algal assemblage Prydz Bay,
Antarctica
––
Polaribacter filamentous 215 Surface sea water, north of Deadhorse,
Alaska
––
Polaribacter irgensii 23-P Nearshore marine waters off Antarctic
Peninsula
––
Psychromonas ingrahamii 37 Sea ice, off Point Barrow, Northern
Alaska
11
Marine actinobacterium
PHSC20C1
Nearshore marine waters of Antarctic
Peninsula
11
359The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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M. tuberculosis carries both a TrHbI (HbN) and a TrHbII (HbO) (Milani
et al., 2005). It is worth noting that HbN efficiently protects M. tuberculosis
from nitrosative damage, contributing to its survival in the host macrophage
(Bidon-Chanal et al., 2006), whereas HbO may have an oxidation/reduc-
tion function because it has peroxidase activity with formation of ferryl
intermediates (Ouellet et al., 2007). On the other hand, Mycobacterium leprae
only displays HbO, which is capable of protecting against NO as well as
oxidative stress (Ascenzi, De Marinis, Coletta, & Visca, 2008). B. subtilis
(Ouellet et al., 2007) and Thermobifida fusca (Bonamore et al., 2005) encode
both a TrHbII and an FHb. To our knowledge, PhTAC125 is the first
example of coexistence of genes encoding a FHb and three TrHbs (see
Section 6.2.2).
A transcriptional analysis of the PSHAa0030,PSHAa0458 and
PSHAa2217 genes encoding the TrHbs and the FHb-encoding gene was
carried out on PhTAC125 wild type and on a mutant strain in which
PSHAa0030 was inactivated. In PhTAC125 wild-type cells, PHSAa0030
is expressed at 4 C and 15 C. PSHAa0458 and PSHAa2217 encoding
the other TrHbs are expressed in both strains under all conditions, whereas
transcription of the FHb-encoding gene is detectable only in mutant cells
grown at 4 C in micro-aerobiosis (Parrilli, Giuliani, Giordano, et al.,
2010) (see Section 6.2.4).
To date, only group II of the TrHbs encoded by PSHAa0030 (hereafter
named Ph-2/2HbO) has been thoroughly investigated from the structural
and functional viewpoints (Coppola et al., 2013; Giordano et al., 2007,
2011; Howes et al., 2011; Parrilli, Giuliani, Giordano, et al., 2010; Russo
et al., 2013). The gene was selected as the first of the three because its posi-
tion on chromosome I is very close to the origin of replication of the bac-
terium, indicating an important physiological role (Giordano et al., 2007).
Since transcription of FHb-encoding genes is usually directly or indi-
rectly induced by NO (Hausladen et al., 1998; Spiro, 2007), the observed
FHb-gene expression only in a PhTAC125 mutant strain is suggestive of
occurrence of an NO-induced stress related to the absence of the TrHb
encoded by PSHAa0030 (Parrilli, Giuliani, Giordano, et al., 2010; see
Section 6.2.4).
6.2. Structure–function relationships of Ph-2/2HbO
6.2.1 Structure
Group II of TrHbs is by far the most populated of the three and is
characterised by specific residues building up the haem cavity (Vuletich &
360 Daniela Giordano et al.
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Lecomte, 2006; Wittenberg et al., 2002). The crystal structures of TrHbsII
from B. subtilis (Giangiacomo, Ilari, Boffi, Morea, & Chiancone, 2005),
T. fusca (Bonamore et al., 2005), Geobacillus stearothermophilus (Ilari et al.,
2007) and M. tuberculosis (Milani et al., 2005, 2003) show a network of inter-
actions between polar residues and the haem-Fe atom that may explain the
high O
2
affinity of these globins (Bonamore et al., 2005; Giangiacomo et al.,
2005; Ilari et al., 2007; Milani et al., 2005; Mukai, Savard, Ouellet,
Guertin, & Yeh, 2002; Ouellet et al., 2003).
Ph-2/2HbO displays structural features typical of TrHbII (Giordano
et al., 2007). In particular, Ph-2/2HbO has Trp at G8, and Tyr at both
CD1 and B10 (Fig. 8.4;Howes et al., 2011). These three positions are pivotal
for the stabilisation of the haem-bound O
2
in TrHbsII (Milani et al., 2005). It
is worth noting that CD1 Phe, that wedges the haem into its pocket, is con-
sidered a conserved residue among globins, unlike members of TrHbsII from
M. tuberculosis,Mycobacterium avium,M. leprae,Mycobacterium smegmatis,Strep-
tomyces coelicolor,Corynebacterium diphtheriae and T. fusca, which host Tyr
instead (Table 8.2;Bonamore et al., 2005; Milani et al., 2005).
Figure 8.4 Sequence alignment of some representative TrHbs of group II. Identical
functionally important residues of the distal haem pocket (B9, B10, CD1, E7, E11 and
G8) and the proximal His F8 are highlighted in grey. The Gly-Gly motifs typical of TrHbs
are highlighted in black. Adapted from Howes et al. (2011).
361The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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Ph-2/2HbO shows structural differenceswith respect to other TrHbsII. In
particular, the insertion of three residues in the CD loop (Howes et al., 2011;
Fig. 8.4) confers higher flexibility that may facilitate its action at low temper-
ature, providing greater freedom for the correct positioning of ligand(s)
(Feller & Gerday, 2003; Siddiqui & Cavicchioli, 2006). In contrast to TrHbsI,
the E7 and E11 positions are occupied by non-polar residues, Ile and Phe,
respectively, precluding haem-bound ligand stabilisation. On the proximal
side, HisF8, conserved in all members of the globin superfamily (Howes
et al., 2011), is coordinated to the haem-Fe atom (Table 8.2 and Fig. 8.4).
Ph-2/2HbO shows an unusual extension of 15 residues at the
N-terminus (pre-helix A), similar to M. tuberculosis HbN and to many
slow-growing species of Mycobacterium, such as M. bovis,M. avium,
M. microti,M. marinum (Lama et al., 2009) and Shewanella oneidensis
(Vuletich & Lecomte, 2006). The pre-A motif of M. tuberculosis HbN does
not significantly contribute to the structural integrity of the protein, pro-
truding out of the compact globin fold (Milani et al., 2001). However,
the deletion of this motif reduces the ability of M. tuberculosis to scavenge
NO (Lama et al., 2009). Unlike in M. tuberculosis HbN, the deletion of
the N-terminal extension of Ph-2/2HbO does not seem to reduce the
NO scavenging activity (Coppola et al., 2013) (see Section 6.2.4).
6.2.2 Hexacoordination
Ph-2/2HbO displays hexacoordination of the ferric and ferrous haem-Fe atom
(Giordano et al., 2011; Howes et al., 2011). Hydrostatic pressure enhances
hexacoordination in both oxidation states of the haem-Fe atom, as previously
shown in other haem proteins (Hamdane et al., 2005), indicating that a flexible
protein allows structural changes (Russo et al., 2013).
Binding of O
2
to Mb and Hb occurs on the distal side of the
pentacoordinated haem-Fe atom, where O
2
establishes a sixth coordination
bond to the Fe atom, whereas the fifth coordination position is occupied by
invariant HisF8 (Fig. 8.5). The haem-Fe-bound O
2
is generally stabilised by
interaction(s) with distal residues. The main O
2
stabilising interaction is
usually provided by an H bond donated by HisE7 (Fig. 8.5).
In hexacoordinated globins, where, in the absence of external ligands,
the sixth position is taken by an internal residue, exogenous ligands (e.g.
O
2
, CO and NO) compete with the internal ligand to bind Fe, this behav-
iour being the basis of the control of Fe reactivity (Smagghe, Trent, &
Hargrove, 2008; Trent, Hvitved, & Hargrove, 2001).
Haem-Fe hexacoordination is widespread in globins, having been found
in unicellular eukaryotes (Wittenberg et al., 2002), plants (Watts et al.,
362 Daniela Giordano et al.
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2001), invertebrates (Dewilde et al., 2006), but only a few cases have been
reported in bacterial TrHbs (Falzone, Christie Vu, Scott, & Lecomte, 2002;
Razzera et al., 2008; Scott et al., 2002; Vinogradov & Moens, 2008; Visca
et al., 2002). Hexacoordination has also been found in higher vertebrates, for
example, in ferric b-chains of tetrameric Antarctic fish Hbs (Riccio,
Vitagliano, di Prisco, Zagari, & Mazzarella, 2002; Vergara et al., 2007;
Vergara, Vitagliano, Verde, di Prisco, & Mazzarella, 2008; Vitagliano
et al., 2004, 2008) and in the ferric and ferrous states of mammalian
(Pesce et al., 2003; Vallone, Nienhaus, Brunori, & Nienhaus, 2004) and
Antarctic fish (Giordano et al., 2012) Ngbs and Cygbs (de Sanctis et al.,
2004; Alessia Riccio et al., unpublished results). The occurrence of ferrous
(haemochrome) and ferric (haemichrome) oxidation states in members of
the Hb superfamily is not uniform, suggesting that the functional roles of
these states are multiple, possibly being a tool for modulating ligand-binding
or redox properties (Vergara et al., 2008; Vitagliano et al., 2008). Exchange
between haemichrome and pentacoordinated forms may play a physiolog-
ical role in Antarctic fish due to higher peroxidase activity (Vergara et al.,
2008; Vitagliano et al., 2008).
Over the years, haemichromes in tetramers have been considered
precursors of Hb denaturation (Rifkind, Abugo, Levy, & Heim, 1994);
however, haemichromes can be obtained under non-denaturing as well as
physiological conditions (Vergara et al., 2008). It has also been suggested that
haemichromes can be involved in Hb protection from peroxide attack (Feng
et al., 2005), given that the haemichrome species of human a-subunits com-
plexed with the a-helix-stabilising protein do not exhibit peroxidase activity
(Feng et al., 2005).
Figure 8.5 Schematical representation of the iron coordination in hexacoordinated,
pentacoordinated and oxygenated forms. The protein is in hexacoordinated conforma-
tion when, in the absence of external ligands, there is an amino acid residue as internal
ligand. The sixth ligand is usually provided by His. Upon addition of gaseous ligands, for
example, O
2
, there is competition between the external ligand and the sixth ligand, with
replacement of the internal ligand with O
2
.
363The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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Hexacoordination of the haem-Fe atom may suggest a common physi-
ological mechanism for protecting cells against oxidative chemistry in
response to high O
2
concentration. Several roles have been hypothesised
for hexacoordinated Ngb and Cygb, for example, as O
2
scavengers under
hypoxic conditions (Burmester, Ebner, Weich, & Hankeln, 2002;
Burmester, Weich, Reinhardt, & Hankeln, 2000), as terminal oxidases
(Sowa, Guy, Sowa, & Hill, 1999), as O
2
-sensor proteins (Kriegl et al.,
2002), and in NO metabolism (Smagghe et al., 2008). It was recently
reported that Ngb over-expression and intracellular localisation confer pro-
tection to neurons, both in vitro and in vivo, against oxidative stress and
enhance cell survival under anoxia and ischaemic conditions (Fiocchetti,
De Marinis, Ascenzi, & Marino, 2013). However, their physiological role
is still a matter of debate.
Hexacoordinated globins are characterised by specific electronic
absorption bands in the UV–visible spectra, clearly indicating the electronic
structure of the Fe atom and its axial ligands (Dewilde et al., 2001).
The electronic-absorption spectrum of ferric Ph-2/2HbO is
characterised by hexacoordinated high-spin (bands at 503 nm and charge-
transfer transition at 635 nm) and low-spin forms (bands at 533 and
570 nm) (Fig. 8.6A), the latter being characteristic of a Tyr coordinated
400 450
Absorption
500 550 600
X5 X5
Wavelength (nm) Wavelen
g
th
(
nm
)
AB
650 400 450 500 550 600 650
Figure 8.6 Overlay of absorption spectra of (A) ferric hexacoordinated Ph-2/2HbO
(black line) with ferric Mb (red line) and (B) deoxy ferrous hexacoordinated Ph-2/
2HbO (black line) with ferrous Mb (red line). All measurements were at pH 7.6 and
25 C. The ferrous samples were prepared by adding 2 ml of sodium dithionite
(10 mg ml
1
) to 600 ml of deoxygenated buffered solution of ferric globins, obtained
flushing the ferric forms with nitrogen. The protein concentration was 10 mM on a haem
basis.
364 Daniela Giordano et al.
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to the Fe atom (Howes et al., 2011). The ferrous state shows a mixture of a
predominant hexacoordinated low-spin state (Soret band at 421 nm and
Q bands at 528 and 559 nm) and a pentacoordinated high-spin state (shoul-
der at 440 nm) (Fig. 8.6B; Giordano et al., 2011). These spectra are in mar-
ked contrast to those of monomeric Mb, in which the Fe atom is
pentacoordinated. In fact, the deoxygenated ferrous form has a broad peak
at 556 nm (Fig. 8.6B), whereas the ferric form exhibits two peaks at 504 and
632 nm (Fig. 8.6A; Antonini & Brunori, 1971).
Based on the spectroscopic data and molecular-dynamics simulation
(Howes et al., 2011), it has been shown that either TyrCD1 or TyrB10
can coordinate the ferrous atom. Although His is the most common residue
that coordinates the Fe atom, Tyr coordinates Fe of ferrous Herbaspirillum
seropedicae Hb (Razzera et al., 2008) and of ferrous and ferric Chlamydomonas
Hb (Couture et al., 1999; Das et al., 1999; Milani et al., 2005).
6.2.3 Reactivity
Reversible hexa- to pentacoordination of the haem-Fe atom modulates the
reactivity of Ph-2/2HbO; in fact, the cleavage of the haem distal
Fe-TyrCD1 or Fe-TyrB10 bonds is the rate-limiting step for the association
of exogenous ligands (e.g. O
2
, CO and NO) and (pseudo)enzymatic activ-
ities (Russo et al., 2013).
CO binding to Ph-2/2HbO displays a rapid spectroscopic phase inde-
pendent of CO concentration, followed by standard bimolecular recombi-
nation. CO-rebinding kinetics show an unusually slow geminate phase,
which becomes dominant at low temperature. While geminate recombina-
tion usually occurs on the ns timescale, Ph-2/2HbO displays a component of
about 1 ms that accounts for half of the geminate phase at 8 C, indicative of a
relatively slow internal ligand binding (Russo et al., 2013).
After ligand escape, bimolecular recombination takes place. Second-
order rebinding indicates two major conformations at 25 C, characterised
by CO-association rates that differ by a factor of 20, with pH-dependent
relative fractions. A dynamic equilibrium was found between a predominant
hexacoordinated low-spin state and a pentacoordinated high-spin state.
A shift in the equilibrium between the two conformations may also provide
a large change in the ligand affinity. The second-order rate constant of the
fast phase (Russo et al., 2013) is of the order of 10
7
M
1
s
1
and closely sim-
ilar to that of human Ngb (Uzan et al., 2004), whereas the second-order rate
constant of the slow process (Russo et al., 2013) is compatible with that of
Mb (Table 8.4), being in the range of 10
5
M
1
s
1
(Springer, Sligar,
365The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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Olson, & Phillips, 1994). The relatively fast CO-dependent kinetic
process is unusual for a TrHbs of group II; the second-order rate constant
of the fast phase for carbonylation of M. tuberculosis HbO is in the range
of 10
5
M
1
s
1
, whereas the second-order rate constant of the slow phase
is in the range of 10
4
M
1
s
1
(Ouellet et al., 2003). Thus the proteins dis-
plays two conformations that greatly differ in the ligand association rate,
suggesting that they may switch between two distinct functional levels.
At 8 C, 85% of the CO bimolecular recombination occurs on the ms
timescale at a rate similar to that of a 3/3 Mb (3.5 10
5
M
1
s
1
), whereas
the remaining kinetic component is faster (10
7
M
1
s
1
)(Russo et al.,
2013), as observed at 25 C(Giordano et al., 2011;Table 8.4).
Hexacoordination of the haem-Fe atom of Ph-2/2HbO via distal Tyr is
only partial (the Tyr equilibrium affinity is close to 1), indicating a weak inter-
action between Tyr and the Fe atom under atmospheric pressure (Russo et al.,
2013). The fast binding and dissociation of Tyr from the Fe atom can be a
molecular event that triggers the shift of the globin between two
Table 8.4 Values of kinetic parameters for O
2
and CO binding to penta and
hexacoordinated Hbs.
haem protein
k
on
O
2
(mM
1
s
1
)
k
off
O
2
(s
1
)
k
on
CO
(mM
1
s
1
)
k
off
CO
(s
1
)
P
50
O
2
(Torr) Reference
Hexacoordinated
Ph-2/2HbO
(8 C)
0.9 1.0 0.35 – 1 Russo et al.
(2013)
Ph-2/2HbO
(25 C)
4.2 0.69
(slow)
12.0 (fast)
–– Giordano
et al. (2011)
Ngb 170 0.7 40 – 6.8
(0.9 S–S)
Uzan et al.
(2004)
Pentacoordinated
Sperm whale
Mb
14 12 0.51 0.019 0.51 Springer et al.
(1994)
M. tuberculosis
HbO*
0.11 0.0014 0.014 0.004 – Ouellet et al.
(2003)
(80%) (78%) (79%) (60%)
0.85 0.0058 0.18 0.0015 –
(20%) (22%) (21%) (40%)
*The relative percentage of the two rate constants are reported in parentheses.
366 Daniela Giordano et al.
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conformations (penta- vs. hexacoordinated haem) with different redox poten-
tials. This behaviour may be considered as an ancestral mechanism for mod-
ulating a conformational switch between two functional species (Russo
et al., 2013).
Ph-2/2HbO is quickly oxidised in the presence of O
2
, probably due to
the superoxide character of the haem-Fe-O
2
adduct, affected by the pres-
ence of the surrounding hydrogen-bond donor residues (Milani et al.,
2001, 2003).
The O
2
affinity, poorly affected by competition with Tyr, is about 1 Torr
at 8 C, pH 7.0 (Table 8.4). The O
2
affinity of Ph-2/2HbO is compatible
with the in vivo conditions (Fig. 8.7A), considering that the PhTAC125 bac-
terial metabolism must cope with high O
2
concentration and high-salinity
conditions at low temperature. However, Mb-like functions do not seem
to be possible for Ph-2/2HbO, requiring a still unknown efficient reducing
system, and a local high globin concentration (Russo et al., 2013).
Figure 8.7 Some postulated functions of Ph-2/2HbO. (A) O
2
carrier even though this
function requires the presence of a still unknown coupled reductase system for high turn-
over reaction; (B) Ph-2/2HbO may convert NO to nitrate and (C) nitrite to NO; (D) it
may detoxify RNS and (E) ROS; (F) it may function as electron transfer coupled to an accep-
tor molecule. The model of Ph-2/2HbO was kindly provided by L. Boechi.
367The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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Thus Ph-2/2HbO is likely to be involved in a redox reaction(s) associ-
ating diatomic ligands and their derived oxidative species. Such a reaction is
not unusual, since globins generally display NO dioxygenase activity leading
to nitrate synthesis (Flo
¨gel, Merx, Go
¨decke, Decking, & Schrader, 2001;
Gardner et al., 2006;Fig. 8.7B). It is noteworthy that Ph-2/2HbO provides
protection against NO and related reactive species (Fig. 8.7D), under aero-
bic conditions (Coppola et al., 2013; see Section 6.2.4). Moreover, Ph-2/
2HbO exhibits a twofold higher nitrite-reductase activity than horse Mb
at pH 7.0, 25 C. This evidence suggests (Fig. 8.7C) that, during an anaer-
obic phase, Ph-2/2HbO may supply NO via nitrite reduction (Russo
et al., 2013).
Other reactions (Fig. 8.7E) may involve complex ROS chemistry
(Flo
¨gel, Go
¨decke, Klotz, & Schrader, 2004). O
2
is necessary for bacterial
metabolism, but can become poisonous if it is responsible for oxidative-stress
burst. A number of reactions take place between ROS and the haem-Fe
atom since O
2
is reduced by four e
before yielding a water molecule with
three ROS intermediates, whereas the haem-Fe atom is susceptible to oxi-
dation from þ2toþ4. In general, pentacoordinated globins are more prone
to ROS oxidation than hexacoordinated forms (Herold, Kalinga, Matsui, &
Watanabe, 2004; Lardinois, Tomer, Mason, & Deterding, 2008). However,
in Ph-2/2HbO, due to the low affinity of the haem for distal Tyr (weak pro-
tection) in ferrous and ferric states, the protection against deleterious oxida-
tion at high ROS concentration (autocatalytic oxidations leading to
irreversible haem oxidation and globin degradation) is not expected
(Russo et al., 2013).
The redox state of Ph-2/2HbO in vivo will depend on the presence of
specific reductases and on the O
2
levels. The redox state could be involved
in an electron-transfer reaction or in a regulatory mechanism with the pro-
tein acting as a redox sensor. In fact, at high O
2
concentration, in the pres-
ence of a reducing system that can compensate for autoxidation, the globin
will be mainly ferrous, but under intermediate conditions it could be in the
ferric form. This behaviour has been observed in pentacoordinated sperm
whale and pig Mb (II), probably upon nucleophile attack such as that medi-
ated by water (Brantley, Smerdon, Wilkinson, Singleton, & Olson, 1993),
and in hexacoordinated bis-His form of the globin GLB-26 of the nematode
worm Caenorhabditis elegans and of human Ngb, which promotes O
2
reduc-
tion (Kiger et al., 2011). By analogy, the hexacoordinated His-Fe-Tyr
adduct of Ph-2/2HbO (Fig. 8.7F) may be involved in electron transfer
(Russo et al., 2013).
368 Daniela Giordano et al.
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6.2.4 NO detoxification
The remarkably high number of TrHbs in the PhTAC125 genome strongly
suggests that these globins fulfil important functional roles associated with
the extreme features of the Antarctic habitat. The involvement of cold-
adapted Ph-2/2HbO in detoxification of RNS and ROS may be a mech-
anism associated with high production of toxic species upon cold stress.
A similar function has been reported in HbN of M. tuberculosis (Pathania
et al., 2002) and M. bovis (Ouellett et al., 2002) and M. leprae HbO
(Fabozzi, Ascenzi, Renzi, & Visca, 2006), which protect pathogenic bacteria
from the toxic activity of macrophage-generated RNS and ROS.
The physiological role of Ph-2/2HbO has been investigated using a geno-
mic approach, by the construction of a PhTAC125 mutant strain in which the
PSHAa0030 gene was inactivated by insertional mutagenesis. The mutant
strain was grown under controlled conditions and its growth behaviour was
compared to that of wild-type cells, changing O
2
pressure in solution and
growth temperature (4 and 15 C). Regardless of temperature, growth of
the mutant strain in extreme aerobiosis is lower than that of the wild type, also
in terms of biomass. The presence of Ph-2/2HbO in wild-type cells is thus an
advantage when cells are grown at high O
2
concentration. In micro-aerobiosis,
both strains slow down their replication kinetics. At 4 C, the wild-type cells
appear better suited to the challenging conditions, reaching higher biomass
than the mutant cells (Parrilli, Giuliani, Giordano, et al., 2010).
The inactivation of the Ph-2/2HbO gene makes the mutant strain sen-
sitive to high O
2
levels, hydrogen peroxide and nitrosating agents (Parrilli,
Giuliani, Giordano, et al., 2010), suggesting involvement of the protein in
protection from oxidative and nitrosative stress. Moreover, the transcription
of the FHb-encoding gene occurs only in the mutant in which PSHAa0030
is inactivated, when grown in micro-aerobiosis at 4 C, suggesting that the
occurrence of the NO-induced stress is probably related to the absence of
Ph-2/2HbO (Parrilli, Giuliani, Giordano, et al., 2010). In micro-aerobiosis,
PhTAC125 may endogenously produce NO, due to a gene encoding a
nitrite reductase (PSHAa1477), as reported in other Gram-negative bacteria
(Corker & Poole, 2003; Ji & Hollocher, 1988). Further, NO may accumu-
late when its spontaneous oxidation is limited by low O
2
availability. In
micro-aerobiosis, O
2
is further reduced when the biomass is increased, that
is, in the late exponential phase, and NO accumulation may become a seri-
ous threat for cell viability. Induction of the FHb gene may be viewed as a
suitable strategy aimed at counteracting NO-induced stress due to the
absence of Ph-2/2HbO (Parrilli, Giuliani, Giordano, et al., 2010).
369The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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An up-to-date approach set up by Coppola et al. (2013) to establish the
participation of Ph-2/2HbO in RNS detoxification, tested the influence of
heterologous expression of the PSHAa0030 gene in vivo on protection from
NO toxicity in a NO-sensitive E. coli strain (E. coli hmp, defective in the
FHb) (see Section 4.1).
The growth properties and O
2
uptake of E. coli hmp having the
PSHAa0030 gene was analysed in an attempt to demonstrate that Ph-2/
2HbO offers resistance to nitrosative stress. Wild-type E. coli and a hmp mutant,
carrying the PSHAa0030 gene or not, were grown at 25 C under aerobic
conditions and treated with either the NO-releaser DETA-NONOate or
the nitrosating agent GSNO. As expected, exposure to these sources of NO
has no effect on the growth of wild-type E. coli or the expression of Hmp from
the complemented plasmid, and a comparable level of resistance is evident in
cells expressing Ph-2/2HbO. In contrast, in the absence of Ph-2/2HbO,
expression results in severe growth inhibition (Coppola et al., 2013).
Moreover, Coppola et al. (2013) demonstrated that upon addition of
NO, E. coli hmp not expressing Ph-2/2HbO shows prolonged inhibition
of O
2
uptake (Fig. 8.8A) until the NO level falls steadily. In contrast, in
the mutant strain carrying hmp
þ
, the addition of NO does not inhibit O
2
uptake (Fig. 8.8B), confirming that Hmp is able to detoxify NO, as reported
previously (Membrillo-Herna
´ndez et al., 1999; Mills, Sedelnikova, Søballe,
Hughes, & Poole, 2001; Stevanin et al., 2000). Upon addition of NO to the
E. coli mutant carrying Ph-2/2HbO, only very short periods of inhibition of
respiration are observed and, again, the disappearance of NO is very fast
(Fig. 8.8C). When NO reaches negligible levels, the O
2
uptake is brought
back to a rate similar to that occurring before NO addition, unlike in the
cells bearing the empty vector (Fig. 8.8A). Following exhaustion of O
2
, fur-
ther addition of NO results in a larger signal and a slower rate of consump-
tion (Coppola et al., 2013).
Under aerobic conditions, over-expression of Ph-2/2HbO provides sig-
nificant resistance to NO and nitrosating agents and distinct NO consump-
tion ability to the NO-sensitive E. coli hmp mutant. In contrast, growth
curves and cellular respiration are strongly inhibited in E. coli hmp not
expressing the Antarctic globin gene. These results are clear evidence of a
very important physiological role of Ph-2/2HbO in PhTAC125.
In vitro kinetic studies of peroxynitrite isomerisation by the ferric protein
support the NO and O2detoxification activity as a possible functional
role of the cold-adapted globin, thus confirming the involvement of Ph-2/
2HbO in the protection from nitrosative stress. The high reactivity of
370 Daniela Giordano et al.
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Figure 8.8 NO uptake and respiration of E. coli hmp cells (A) carrying the empty vector
pBAD/HisA, or (B) expressing Hmp, or (C) Ph-2/2HbO. Respiration was followed in a
Clark-type O
2
electrode (solid lines) upon addition of 1 mM Proli-NONOate (arrows).
NO uptake was measured simultaneously with NO electrode (dashed lines). Adapted
from Coppola et al. (2013).
371The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
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ferric Ph-2/2HbO towards peroxynitrite at low temperature
(k
on
¼3.510
4
M
1
s
1
at 5 C and 2.910
4
M
1
s
1
at 20 C) suggests
that the protection of the psychrophile PhTAC125 against RNS and ROS
may happen also in the cold Antarctic environment (Coppola et al., 2013).
The k
on
values for the ferric Ph-2/2HbO-mediated peroxynitrite
isomerisation are similar to those reported for catalysis of the ferric equine-
heart Mb (2.9 10
4
M
1
s
1
,Herold & Kalinga, 2003), sperm-whale Mb
(1.610
4
M
1
s
1
,Herold et al., 2004) and human Hb (1.210
4
M
1
s
1
,
Herold & Kalinga, 2003), whereas they are lower than those of horse-heart
native and carboxymethylated cytochrome cin the presence of saturating
cardiolipin (3.210
5
and 5.3 10
5
M
1
s
1
, respectively), horse-heart car-
boxymethylated cytochrome c(6.8 10
4
M
1
s
1
), and human serum
haem-albumin (4.110
5
M
1
s
1
)(Ascenzi, Bolli, et al., 2011; Ascenzi,
Bolli, Gullotta, Fanali, & Fasano, 2010; Ascenzi, Ciaccio, Sinibaldi,
Santucci, & Coletta, 2011a, 2011b; Ascenzi et al., 2009). As reported for
several haemoproteins, the acceleration of the peroxynitrite-isomerisation rate
by ferric Ph-2/2HbO seems to be caused by reaction of peroxynitrite with the
ferric penta-coordinated derivative only (Ascenzi, Bolli, et al., 2011, 2010;
Ascenzi et al., 2011a, 2011b, 2009; Goldstein, Lind, & Mere
´nyi, 2005;
Herold & Kalinga, 2003; Herold et al., 2004).
Taken together, the in vivo and in vitro evidence suggests that, under aer-
obic conditions, Ph-2/2HbO supplies cell protection against RNS and
ROS, compensating for the defect in NO detoxification of E. coli hmp,
which lacks the major NO-scavenging protein.
Finally, attempting to ascertain whether the N-terminal motif of Ph-2/
2HbO is a requirement for efficient NO scavenging, similar to M. tuberculosis
HbN (Lama et al., 2009; see Section 6.2.1), Coppola et al. (2013) investi-
gated the effect of deletion of the first 20 residues of the N-terminus of
the protein on the ability of E. coli hmp strain to deal with nitrosative stress;
while full-length Ph-2/2HbO restores the ability to survive and grow under
nitrosative-stress conditions, the protein without the N-terminal extension
does not significantly contribute to NO detoxification, since its deletion
does reduce the globin NO-scavenging ability in the heterologous host,
unlike in M. tuberculosis HbN.
7. CONCLUSION AND PERSPECTIVES
The Antarctic exhibits stable living conditions due to substantial
isolation, also by virtue of the Polar Front. The evolutionary processes of
372 Daniela Giordano et al.
Author's personal copy
Antarctic organisms and the time scales in the context of geological and cli-
matic changes have been extensively analysed and discussed (Peck, 2011).
The structure and function of proteins are the basis for understanding the
evolutionary forces operating at sub-zero temperature, and—in this
context—the knowledge gained at the molecular level is also crucial for
predictions of the evolutionary consequences of global warming. In fact,
at all analysed levels, the functional adaptation to permanently low temper-
ature appears to require maintenance of flexibility of molecules in order to
adequately support the cellular functioning. Proteins are one main factor of
the ensuing mechanisms of adaptation.
Temperature is the prime driver that shaped the current structure and
function of polar communities. Amongst abiotic factors influenced by tem-
perature, O
2
and CO
2
, and their concentrations, play an important role in
life-sustaining processes. Due to low temperature, their concentrations are
several-fold higher than in temperate and tropical marine habitats. The
temperature-dependent balance between O
2
demand/supply and the
associated functional capacity for specific functions of macromolecules shape
the performance window in polar species (Po
¨rtner et al., 2007). In polar
environments, the benefits of O
2
levels (high by default) are indeed largely
apparent, because they are counterbalanced by the kinetics of biological pro-
cesses operating at low temperature (D’Amico et al., 2006) which decreases
the rates, and by increased production of ROS. O
2
is obviously necessary for
aerobic bacterial metabolism, but it can become poisonous in triggering
oxidative-stress bursts.
In view of these considerations, in all Antarctic organisms, biological
processes envisaging O
2
(respiration, transport/release, scavenging, reactive
species, etc.) and other gases are bound to attract the interest of biologists.
In the realm of microbial life, the cold-adapted bacterial protein Ph-2/
2HbO displays hexacoordination of the ferric and ferrous haem-Fe atom
(Giordano et al., 2011; Howes et al., 2011). Investigating the features of this
globin, in an attempt to shed light on possible multiplicity of functions, has
been an important task. For instance, Ph-2/2HbO appears to exhibit a
pseudo-enzymatic function in which O
2
is involved (Russo et al., 2013)
and is available for reactions with NO to produce nitrate anions. In a single
molecule, multiple conformations (penta- vs. hexacoordinated states) may
account for multiple functions: under aerobic conditions, on one hand,
Ph-2/2HbO provides cells with protection against NO and related RNS;
on the other, during the anaerobic phase, Ph-2/2HbO may provide
NO via nitrite reduction. The evidence summarised here indicates that
373The Antarctic Marine Bacterium Pseudoalteromonas haloplanktis TAC125: A Case Study
Author's personal copy
Ph-2/2HbO displays unique adaptive structural properties ensuring higher
flexibility, thus facilitating its function at low temperatures, for example, by
enhancing the capacity for correct positioning of ligand(s), which would be
made more difficult by a rigid structure. In summary, this globin is a notable
case study of relationship between molecular structure, cold adaptation and a
wide range of equally important biological functions.
Hexacoordinated globins in Antarctic microorganisms call for efforts in
shedding light on their place and role in the context of evolution. Knowl-
edge of the range of their functions and physiological role in vivo is still
incomplete and a matter of lively debate. However, modern concepts of
biological sciences appear more and more to support the idea that the phys-
iological role of a given molecule is not restricted to a single aspect, although
one aspect may well be predominant. Based on this assumption, the current
knowledge summarised in this review seems to be a useful starting point to
achieve progress by further investigations aimed at increasing our albeit
incomplete understanding of the biological function of a fundamentally
important class of macromolecules such as globins, not only Ph-2/
2HbO—a valuable case study—but also other globins, for example, Ngb
and Cygb, whose biomedical significance is steadily growing.
ACKNOWLEDGEMENTS
The authors wish to thank the Centre de Ressources Biologiques de l’Institut Pasteur, Paris,
France (http://www.crbip.pasteur.fr) for supplying the P. haloplanktis CIP 108707 strain.
This study was carried out in the framework of the SCAR programme “Antarctic
Thresholds–Ecosystem Resilience and Adaptation” (AnT-ERA), and of the “Coordination
Action for Research Activities on Life in Extreme Environments” (CAREX), European
Commission FP7 call ENV.2007.2.2.1.6. It was financially supported by the Italian
National Programme for Antarctic Research (PNRA).
F. M. is supported by a fellowship from the Australian Research Council (DE120102610).
M. T.-T. is supported by Consejo Nacional de Ciencia y Tecnologia (CONACyT)
(Mexico) through grant number 99171 and Consejo Estatal de Ciencia, Tecnologı
´ae
Innovacio
´n de Michoaca
´n (CECTI) through grant number 007. Thanks are due to Prof.
Robert K. Poole for kindly inviting us to submit this review. We apologise in case we
have neglected references relevant to the subject.
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