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Antarctic DNA moving forward: genomic plasticity and biotechnological potential

Cecilia Martínez-Rosales, Natalia Fullana, Héctor Musto, Susana Castro-Sowinski
DOI: http://dx.doi.org/10.1111/j.1574-6968.2012.02531.x 1-9 First published online: 1 June 2012

Abstract

Antarctica is the coldest, driest, and windiest continent, where only cold-adapted organisms survive. It has been frequently cited as a pristine place, but it has a highly diverse microbial community that is continually seeded by nonindigenous microorganisms. In addition to the intromission of ‘alien’ microorganisms, global warming strongly affects microbial Antarctic communities, changing the genes (qualitatively and quantitatively) potentially available for horizontal gene transfer. Several mobile genetic elements have been described in Antarctic bacteria (including plasmids, transposons, integrons, and genomic islands), and the data support that they are actively involved in bacterial evolution in the Antarctic environment. In addition, this environment is a genomic source for the identification of novel molecules, and many investigators have used culture-dependent and culture-independent approaches to identify cold-adapted proteins. Some of them are described in this review. We also describe studies for the design of new recombinant technologies for the production of ‘difficult’ proteins.

Keywords
  • Antarctic
  • cold-adapted
  • mobile genetic element

Introduction

Antarctica is the coldest, driest, and windiest continent, where the temperature can reach −30 °C, the annual precipitation is only 200 mm and the highest recorded wind velocity is 327 km h−1. It has the highest average elevation of all the continents, and about 98% of its 14.0 million km2 is covered by ice 1.6 km thick. In these extreme conditions, only cold-adapted organisms survive, including plants, animals, and microorganisms. The continent remained largely abandoned because of its hostile environment, lack of resources and isolation, but after the signing of the Antarctic Treaty (1959; entering into force in 1961 and eventually signed by 47 countries), human activities have increased with 1000–5000 nonpermanent human residents (now living at the research stations spread through the continent). Antarctica is a protected continent, where research is freely conducted and where military activity is forbidden. For information about the Antarctic legislative and bio-security framework, biodiversity and biogeography, transfer of species, and the biological implications of Antarctic glacial history, we recommend reading Hugues & Convey (2010) and Convey et al. (2008).

The Antarctic continent has been frequently cited as a pristine place, with a rather limited diversity of plants and animals, but with a highly diverse microbial community (Tindall, 2004). In particular, it was reported that aquatic environments (sea, sea ice, lakes from freshwater to highly saline) were more diverse compared with soils. However, recent applications of molecular methods have revealed a very wide diversity of microbial taxa in soil, many of which are uncultured and taxonomically unique (Cary et al., 2010; Margesin & Miteva, 2010). Thus, this continent could be considered to be of great importance for several reasons, among them because it might be regarded as a reservoir for novel genetic resources that could be of use in the development of new biotechnological products. In addition, Antarctica might be considered a natural laboratory to understand the genetic and structural basis of adaptation of eukaryotic and prokaryotic cells to extreme conditions.

This review presents recent information about the genomic elements that have been found to act in the evolution of the Antarctic prokaryotic genomes and their potential for biotechnological exploitation.

The misleading notion of pristinity of the Antarctic environment

Currently, it is accepted that the notion that the Antarctic continent is a pristine environment is misleading because of the input of airborne microorganisms and the anthropogenic transport and dissemination of microorganisms, as an inevitable consequence of human presence and activity. These ‘alien’ microorganisms do indeed influence the microbial diversity, giving insight into the complexity of the balance between evolution, extinction, and colonization of microorganisms in this extreme environment (Vincent, 2000; Pearce et al., 2009; Cowan et al., 2011). The continent is continuously seeded by nonindigenous microorganisms including mesophilic species that, although they will probably not establish viable populations, they contribute to the environmental pool of DNA available through one of the major forces in the evolution of the prokaryotic genome, horizontal gene transfer (HGT).

In addition to the intromission of ‘alien’ microorganisms, climate changes like global warming strongly affect microbial Antarctic communities. Yergeau et al. (2012) showed an increase in the abundance of fungi and bacteria and in the ratio of Alphaproteobacteria to Acidobacteria in response to experimental field warming, which might result in an increase in soil respiration. On the other hand, Jung et al. (2011) reported diminished fungal and archaeal communities in response to warming temperatures. But, whether there is an increase or decline in a group of microorganisms, the shift in Antarctic microbial communities is not in doubt. As mentioned before, this change as well as the intromission of foreign DNA in the Antarctic environment is changing qualitatively and quantitatively the genes potentially available for HGT.

DNA promiscuity, a powerful command in evolution

HGT is an important force modulating bacterial evolution and depends on the number of transferred genes and their maintenance in the host cells by means of positive selection. In this way, genes coding for new proteins with novel properties are preserved while nonbeneficial genes tend to be removed. Also, it depends on the extent of the phenomenon, in both evolutionary time and phylogenetic distance between the organisms involved (Boto, 2010). Although HGT is a widespread phenomenon among bacteria, there are few reports on gene transfer in extreme cold environments probably due to our lack of knowledge and understanding of polar microbial diversity. There are a few reports concerning gene transfer from bacteria to arthropods (Song et al., 2010), crustacea (Kiko, 2010), or prokaryotes. Table 1 summarizes examples of HGT in Antarctic prokaryotes. The transfer of genes associated with antibacterial metabolites such as the biosynthesis of violacein (Hakvåg et al., 2009), hydrocarbon biodegradation (Ma et al., 2006; Pini et al., 2007), signal transduction (López-García et al., 2004; Allen et al., 2009), vitamin metabolism (López-García et al., 2004; Moreira et al., 2006), central metabolism (López-García et al., 2004; Allen et al., 2009), and hydrolytic enzyme production (Xiao et al., 2005) illustrates the crucial role of HGT in the evolution and the adaptation of bacterial communities in a changing environment. In the oligotrophic Antarctic environment, the production of the hydrolytic enzyme chitinase, which breaks down glycosidic bonds, might confer a fitness improvement to a microbe that can now use the chitin found in the outer skeleton of invertebrates as a C- and N-source. Another recalcitrant substrate available for microorganisms in Antarctica is fossil fuels. It is used for human activities and has led to hydrocarbon contamination, a serious environmental problem because of their persistence and high toxicity in biological systems. Studies carried out by Flocco et al. (2009) showed a relative abundance of ndo genes in polluted soils from anthropogenic sources compared to noncontaminated sites. In those sites, the transfer of genes related to hydrocarbon degradation clearly has an impact on the bacterial fitness. It is very likely that the acquisition of genes related to antibiotics, biodegradation of carbon and nitrogen supplies, or contaminants, plays a key role in such environmental conditions.

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Table 1

Examples of prokaryotic horizontal gene transfer in Antarctica

HostGenes and functionsHGT supported byReference
Collimonas sp.Antibacterial compound active against Micrococcus luteusvioA and vioBPhylogenetic analysis. Identity to vioA and vioB of Janthinobacterium lividum and Duganella sp.Hakvåg et al. (2009)
Methanococcoides burtoniiSignal transduction genes; envelope biogenesis genes; central metabolism and a new aconitase geneComplete genome sequencing and identification of predicted HGT eventsAllen et al. (2009)
Pseudomonas sp.Poly-hydroxyburyrate (PHB) production — phaRBACPhylogenetic analysis. Burkholderiales species and Azotobacter vinelandiiAyub et al. (2007)
Rhodococcus and Alcaligenes speciesAlkane mono-oxygenase — alkB — involved in n-alkane biodegradationPhylogenetic analysis. alkB sequences do not follow the bacterial 16S rRNA-based phylogenyPini et al. (2007)
Rahnella sp.Naphthalene dioxygenase — polycyclic aromatic hydrocarbon degradation — ndoPhylogenetic analysis. Pseudomonas sp.Ma et al. (2006)
Pseudomonas spp.Naphthalene dioxygenase — polycyclic aromatic hydrocarbon degradation — ndoNo apparent phylogenetic correlation between ndo and 16S phylogenetic treesMa et al. (2006)
Bacterial 16S-rDNA-containing clone belonging to the Deltaprotobacteria, DeepAnt-32C6Adenosylmethionine-8-amino-7-oxononanoate aminotransferase — transferase that participate in biotin metabolismPhylogenetic positions and discrepancies of G+C contentMoreira et al. (2006)
Bacterial 16S-rDNA-containing clone belonging to the Deltaprotobacteria, DeepAnt-1F12Conserved transposase domain of Gammaprotobacteria; Cys-rich protein related to eukaryotic and Myxococcus xanthus homologues; acetyltransferasePhylogenetic positions and discrepancies of G+C contentMoreira et al. (2006)
Janthinobacterium, Stenotrophomonas, Cytophaga, Streptomyces and Norcardiopsis speciesChitinase — chiA — digest chitin as a nutrient source for growthPhylogenetic analysis. chiA sequences do not follow the bacterial 16S rRNA-based phylogenyXiao et al. (2005)
16S plus 23S-rDNA-containing 33.3-Kb archaeota fragmentSugar kinase, ribokinase, menaquinone (vitamine K) biosynthesis protein, adenylate cyclase, among othersPhylogenetic analysis of protein genesLópez-García et al. (2004)

Usually, among prokaryotes, HGT is facilitated by a number of genetic elements, including plasmids, transposons, and integrons, and most attention has been focused on the first two. However, considering that nonindigenous microorganisms are not likely to be metabolically active, natural transformation might be the predominant form of HGT in Antarctic soils (Cowan et al., 2011). Recent evidence shows the presence of genetic elements related to recruitment and mobilization of genes, such as integrons, reinforcing the relevance of the capture-cassette-gene recombinase systems in the dissemination of genes in the Antarctic environment (Berlemont et al., 2011). Integrons are DNA platforms that capture exogenous gene cassettes containing open reading frames (ORFs) and assemble them under the control of a promoter that ensures gene functionality. They are composed of three elements: a gene (intI) encoding an integrase belonging to the tyrosine-recombinase family; a primary recombination site (attI); and an outward-orientated promoter (Pc) that directs transcription of the captured genes (Mazel, 2006). These assembling platforms have a major role in the spread of genes and have been described in Antarctic environments. Several ORFs, homologous to putative or hypothetical transposases, transcription elongation factors, alkylmercury lyase, transcription regulators, penicillin-binding protein, integrases, recombinase/topoisomerase and many unknown proteins, have been described (Stokes et al., 2001; Berlemont et al., 2011). Because integrons are widespread in bacterial populations, it is clear that the pool of ORFs represents a genomic resource for bacterial adaptation because they are ready for mobilization, reshuffling, and expression of genes.

Genomic islands (GIs) are genetic elements, usually acquired by HGT, that also play a major role in microbial evolution and have been found in cold-adapted bacteria. A new bacteriocin biosynthetic cluster was located in a GI of Carnobacterium sp. AT7 (Voget et al., 2011). Interestingly, Ayub et al. (2007) found a GI containing polybetahydroxyalkanoate (PHA) biosynthetic genes, numerous mobile elements, an integrase, insertion sequences, a bacterial group II intron, a complete Type I protein secretion system, and IncP plasmid-related proteins in a mosaic distribution structure, in the Antarctic Pseudomonas sp. 14-3. PHA has a role in stress alleviation, mainly environmental stress. PHA is a carbon and energy storage compound that is accumulated during suboptimal growth conditions, and their degraded elements can be used rapidly for numerous metabolic needs, enhancing fitness during stressful environmental conditions (Kadouri et al., 2005). Taken together, these results support the idea that horizontal transfer of pha genes is a mechanism of adaptability in the Antarctic environment.

The huge potential of Antarctic bacterial DNA

On the basis of its microbial diversity and extreme environmental conditions, the Antarctic continent has been described as a genomic resource for the identification of novel molecules, in particular cold-active enzymes, for biotechnological uses.

These cold-active enzymes have high activities at low temperatures, and this enables their application in certain industrial processes that can be performed at room or tap water temperature, thus allowing energy savings. In addition, their relatively high thermo sensitivity provides the possibility of a rapid inactivation, preserving in this way the quality of some products (Pulicherla et al., 2011). For information on the commercial value and application of cold-active enzymes, we suggest reading Marx et al. (2007).

One of the major adaptations of cold-proteins includes modifications of structural features that increase flexibility, and specific amino acids have emerged as key elements (Marx et al., 2007). Glycine has been reported as an important residue to improve the flexibility of protein structure, providing more amplitude to the relative movements between elements of the secondary structure. In pioneering work, Saunders et al. (2003) compared the global proteomes of two cold-adapted Archaea (Methanogenium frigidum and Methanococcoides burtonii) with mesophilic proteomes. They found that these cold-adapted prokaryotes displayed higher frequencies of charged polar residues (mainly Gln and Thr) and a lower frequency of hydrophobic amino acids, mainly Leu. Using a different approach, Gianese et al. (2001) showed that, among psychrophilic enzymes, Ala and Asn were increased and Arg decreased at exposed sites, and some other differences were found within α-helices and β-strands. More recently, Grzymski et al. (2006) showed that the most significant changes found in Antarctic bacterial protein sequences were a reduction of Pro, stabilizing hydrophobic clusters, and in salt-bridge-forming residues (Arg, Glu, and Asp). The availability of more genome sequences from psychrophilic microorganisms will be crucial for understanding the adaptation of proteins to a cold environment, which in turn will have an obvious biotechnological application.

Relevant biotechnological cold-active bacterial enzymes have been identified using culture-dependent studies (Margesin & Schinner, 1994; Vazquez et al., 2004; Martínez-Rosales & Castro-Sowinski, 2011; among many others). Currently, however, the most promising approach is based upon metagenomics, a culture-independent genomic analysis. Functional metagenomics relies on the extraction of environmental DNA and subsequent cloning to eventually identify the entire genetic set of a habitat. This allows the analysis of a wide diversity of genes and their products as well as the study of their potential for biotechnological use (Schmeisser et al., 2007). Through metagenomics, several cold-active enzymes with many potential biotechnological applications have been identified, cloned in heterologous hosts and characterized. Examples include lipases and esterases (Cieslinski et al., 2009; Heath et al., 2009; Yuhong et al., 2009; Berlemont et al., 2011; Yu et al., 2011; Hu et al., 2012), proteases (Berlemont et al., 2011; Zhang et al., 2011), cellulases (Berlemont et al., 2011), and glycosyl hydrolases (Berlemont et al., 2009, 2011).

Expressing cold-active enzymes in heterologous hosts

A variety of bacterial hosts and promoter systems have been used for heterologous protein production (Table 2), with Escherichia coli being the most widely used because its genetic background is by far the best known (Chen, 2011). However, it is common that heterologous proteins fail to fold correctly at optimal E. coli growth temperatures, resulting in formation of insoluble aggregates known as inclusion bodies. A possible solution is recombinant protein expression at reduced growth temperatures, increasing the solubility of aggregation-prone recombinant proteins, but this is accompanied by a reduction in metabolic rate. The use of cold-shock expression systems, such as pCold, allowed high-level expression of soluble proteins in E. coli.

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Table 2

Examples of enzymes from Antarctic microorganisms expressed in heterologous hosts

EnzymeAntarctic microorganismHeterologous hostReference
ProteasePseudoalteromonas haloplanktis TAC125Escherichia colide Pascale et al. (2010)
ProteasePseudoalteromonas sp. QI-1E. coliXu et al. (2011)
β-galactosidaseArthrobacter sp. 32c-E. coli and Pichia pastorisHildebrandt et al. (2009)
β-galactosidasePseudoalteromonas sp.22bE. coliCieslinski et al. (2005)
LipasePsychrobacter sp. TA144E. coliDe Santi et al. (2010)
LipasePseudomonas sp. 7323E. coliZhang & Zeng (2008)
LipasePsychrobacter sp. GE. coliCui et al. (2011)
α-amylaseAlteromonas haloplanktisE. coliFeller et al. (1998)
α-amylasePseudomonas stutzeri 7193E. coliZhang & Zeng (2011)
Alkaline phosphatasePsychrophilic bacterium strain TAB 5E. coliRina et al. (2000)
EndochitinaseSanguibacter antarcticus KCTC 13143P. pastorisLee et al. (2010)

Cold-shock expression vectors (named pColdI, II, III, and IV) are plasmids in which protein expression is under the control of the cspA (cold-shock protein A) promoter in a pUC118 background, with the cspA 5′-UTR and the cpsA 3′end transcription terminator site. All pCold vectors contain the lac operator sequence immediately upstream of the cspA transcription initiation site, allowing the cold-shock induction of gene expression by simultaneous addition of IPTG and temperature downshift in E. coli (Qing et al., 2004). These vectors have been used for expressing successfully cold-adapted proteins in E. coli, for example the protease from Pseudoalteromonas sp. QI-1 (Xu et al., 2011), β-galactosidase from Arthrobacter spychrolactaphilus (Nakagawa et al., 2007), and lipase from Psychrobacter sp. G (Lin et al., 2010), among others. However, enzyme aggregation and accumulation in inclusion bodies cannot be entirely solved by this approach. Cui et al. (2011) successfully improved the yield of soluble cold-active lipase in the E. coli cytoplasm by co-expression with molecular chaperones. The biotechnological implication of this finding is clear.

Designing cold-adapted expression vectors from Antarctic bacterial DNA

The production of recombinant proteins in cold-adapted bacteria such as Pseudoalteromonas circumvents the slowdown in metabolic rate imposed by the temperature downshift in mesophilic bacteria such as E. coli, thus increasing productivity, and probably solubility and stability. In this regard, authors have developed new vectors to produce heterologous proteins at low temperature using Antarctic genetic resources as described below.

The occurrence of bacterial plasmids in Antarctic bacterial isolates was early studied by Kobori et al. (1984). They found that 48 of 155 isolates (31%) carried at least one plasmid and concluded that bacterial plasmids are ubiquitous in this environment. These endogenous plasmids could be used for the development of cloning systems, mainly by genetic engineering and for the overproduction of heat-labile proteins. Tutino et al. (2000) reported for the first time the isolation and characterization of a cold-adapted plasmid, named pTAUp, from the Antarctic gram-negative Psychrobacter sp. strain TA144. This plasmid duplicates in vivo by a rolling-circle mechanism, and several functional and structural features of the Rep initiator protein suggest the existence of a novel subfamily of RC replicons (Tutino et al., 2000). Later, Tutino et al. (2001) and Zhao et al. (2011) designed cold-adapted expression vectors by cloning into mesophilic conjugative plasmids cold-adapted replication sequences from cryptic plasmids isolated from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 and the Arctic Pseudoalteromonas sp. BSi20429. Some of the recent studies of cold-adapted expression vectors that are able to direct the expression of thermo-labile and psychrophilic proteins in psychrophilic bacteria are summarized in Table 3. Papa et al. (2007) constructed a cold-inducible expression system by cloning into the vector pUCLT/Rtem (Tutino et al., 2002) a regulatory region from P. haloplanktis TAC125 that regulates a functional two-component system involved in the expression of a C4-dicarboxylate transporter, which is induced by l-malate (Papa et al., 2009). The inducible expression vector (pUCRP) contains a σ54-dependent promoter that is activated by the transcription factor, MalR, in response to the presence of l-malate. It has provided a valuable system for the production of ‘difficult’ proteins and biopharmaceuticals such as antibodies (Papa et al., 2007; Giuliani et al., 2011). These developments illustrate the great value of Antarctic plasmids as cold-adapted expression vectors and the huge potential of Antarctic bacteria, such as Pseudoaltermonas strains, in the development of stable expression systems for high-level production of recombinant proteins. We recommend Rippa et al. (2012) and Parrilli et al. (2008) for a full description of effective inducible expression systems in cold-adapted bacteria and evaluation of optimal production of homologous or heterologous proteins.

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Table 3

Cold-adapted expression vectors designed using Antarctic bacterial DNA and recombinant proteins expressed in psychrophilic bacteria

Cold-adapted expression vectorsDescription of vectorRecombinant protein producedReference
pA6Mesophilic vector pαH12 derivative, containing the T/R box. Replicative in psychrophiles and E. coliCold-adapted α-amylaseTutino et al. (2001)
pFFamypUCL derivative containing the T/R box and promoter and termination sequences from the P. haloplanktis TAC125 aspC geneCold-adapted α-amylaseTutino et al. (2002)
pFFamyΔCtPsychrophilic recombinant secretion system, based on pFFamy vector devoid of α-amylase C-terminal propeptide and with two restrictions sites to allow in frame cloning downstream of amylase linker encoding sequenceHyper-thermophilic indole-3-glycerol-phosphate synthase
mesophilic β-lactamase
psychrophilic disulfide oxidoreductase
Cusano et al. (2006)
pUCRPpUCLT/Rterm-derived vector, containing a cold-active promoter (PSHAb0363) from P. haloplanktis TAC125, strongly induced by l-malatePsychrophilic β-galactosidase
mesophilic yeast α-glucosidase
Papa et al. (2007)
Fab 3H6Giuliani et al. (2011)
pWD2pUC19 containing the regions essential for plasmid replication and stability of pSM429 isolated from psychrophilic Pseudoalteromonas sp. BSi20429Cold-adapted cellulaseZhao et al. (2011)
  • pαH12: pUC12 derivative containing the P. haloplanktis α-amylase gene.

  • T/R Box: DNA fragment containing a cold-adapted origin of replication derived from pMtBL cryptic plasmid and the origin for conjugative transfer from plasmid pJB3.

  • pUCLT/Rterm: pUC18 vector, containing the T/R box and transcription termination signal from PhTAC125 aspC gene.

The mesophile to psychrophile metamorphosis

It has been shown that the expression of only a few genes from cold-adapted microorganisms in mesophilic hosts allows them to grow at much lower temperatures, and they even become heat-sensitive. For example, the heterologous expression of chaperonin-encoding cpn60 and cpn10 genes from the psychrophilic bacterium Oleispira antarctica enables E. coli to grow at 5 °C (Ferrer et al., 2003). Substitution of psychrophilic gene orthologs of ligA (NAD-dependent DNA ligase) into the mammalian pathogenic strains Francisella tularensis, Salmonella enterica and Mycobacterium smegmatis, resulted in temperature-sensitive phenotypes (Duplantis et al., 2010). On the basis of these reports, Lorenzo (2010) argues that cold adaptation is just a survival trait that can be acquired by HGT of only a few genes among various bacterial species and thus changes their niche specificity leaving the rest of the genetic and physiological chassis untouched.

Concluding remarks

Antarctica possesses a flourishing bacterial population actively modulated by many evolutionary forces. Genetic elements including plasmids, transposons, integrons, and GIs have been shown to be present in Antarctic bacterial communities. In addition, the intromission of ‘alien’ microorganisms and global warming are strongly affecting microbial Antarctic populations, giving us an insight into new genetic evolutionary forces. This changing environment, rich in cold-adapted bacteria, is a genomic source for the identification of novel molecules and provides DNA elements suitable for the design of new recombinant technologies. Extensive research has shown the potential of the Antarctic bacterial DNA in the development of genetic engineering vectors to produce heterologous proteins at low temperature. The isolation by either culture-dependent or culture-independent approaches of genes responsible for producing cold-active enzymes with many potential biotechnological applications had also been successful. Antarctic bacterial DNA is a valuable resource that is a substantial biotechnological resource that must be preserved.

Author's contribution

C.M.-R. and N.F. contributed equally to this work.

Acknowledgements

Authors thank Programa De Desarrollo de las Ciencias Básicas (PEDECIBA), Uruguay, and Instituto Antártico Uruguayo (IAU). C.M.-R. was supported by Agencia Nacional de Investigación e Innovación (ANII).

References

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