Cold-loving Bacteria from Antarctic and Arctic: Occurrence, Survival and Usefulness

By: S. Shivaji
Psychrophilic or cold loving bacteria are the most predominant type of bacteria, confined to all cold habitats of the world viz. Antarctic and Arctic. These bacteria are unique due to their ability to survive at temperatures below the freezing point of water, as they possess various strategies which facilitate their survival at low temperature. The present review is an attempt to highlight the bacterial diversity of the Antarctic and the Arctic, the biotechnological potential of the cold-loving bacteria and to also understand their survival strategies with an emphasis on the research carried out in India, at the Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India.
Policy

Introduction

Psychrophilic (cold-loving) bacteria are extremophiles that define the lower limits of temperature for the survival of life forms. They survive and multiply at temperatures as low as -20oC and rarely survive above 30oC. Such bacteria are predominant in habitats that are permanently cold, such as Antarctic, Arctic, glaciers, deep sea etc. Psychrophiles play a key role in the cold habitats by facilitating nutrient recycling and producing bioactive molecules, biopharmaceuticals and extremozymes. Psychrophiles could thus serve as potential workhorses in biotechnology and this has led to extensive research on these psychrophilic microorganisms from Antarctic, Arctic and the Himalayan Glaciers so as to be able to establish their identity, to exploit their untapped natural resource and also to understand their molecular basis of survival and multiplication at low temperatures. As a part of the Indian Antarctic and Arctic research programmes research on psychrophiles at the CCMB, Hyderabad, has essentially centred around the following aspects:

  1. Biodiversity of bacteria from Antarctic and the Arctic.
  2. Biotechnological potential of bacteria from Antarctic and the Arctic.
  3. Molecular basis of the survival of cold loving bacteria.

 

 

Bacterial Biodiversity of Antarctic

Microbial diversity of Antarctic using the culturable approach

Erik Ekelöf (1908), the father of Antarctic microbiology was the first to demonstrate the presence of bacteria, yeasts and fungi in the soil and air at Snow Hill Island, Antarctic Peninsula. During the last hundred years Antarctic microbiology changed from enumeration of bacteria to identifying these unique microorganisms in varied habitats such as coastal waters, sea-ice, soil, glaciers and lakes. The abundance of cultivable psychrophilic bacteria in Antarctic varies depending on the specific habitat, season of the year and the water or the nutrient content of the habitat. The abundance may range from a minimum of <100 colony forming units in case of glacial ice to as many as 0.6 X 1012 Cfu g-1 in case of sea ice (Delille and Gleizon, 2003; Straka and Stokes, 1960). These viable bacteria could be identified by polyphasic taxonomy. As of now, the world over, only about 250 odd new species of Gram-negative and Gram-positive bacteria from various habitats in Antarctic have been identified up to the species level. Out of this, 30 new species affiliated to Psychrobacter, Pseudomonas, Pseudonocardia, Sphingobacterium, Halomonas, Marinomonas, Marinobacter, Planococcus, Kocuria, Arthrobacter, Leifsonia, Sporosarcina and Exiguobacterium (Table 1) were identified by our group at the CCMB, Hyderabad, India (Shivaji et al., 1988; 1989a; 1989b; Shivaji and Reddy, 2009). These novel psychrophiles could be differentiated from the closely related species based on their phenotypic and chemotaxonomic characteristics and also at the 16S rRNA gene sequence level (Figure 1). All the Antarctic isolates of bacteria were psychrophilic, oligotrophic, produced cold-active enzymes, varied in their antibiotic sensitivity when compared to the nearest phylogenetic neighbour and produced greater proportion of unsaturated fatty acids. The Indian studies focused on soil samples from Schirmacher oasis, Antarctic (Shivaji et al., 2004a) and viable bacteria from a sediment core of a lake in Schirmacher Oasis, Antarctic (Shivaji et al., 2011b).

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Microbial diversity of Antarctic using the culture independent approach

The advent of the culture-independent identification of microorganisms by directly cloning the 16S rRNA gene sequences from environmental samples led to the realisation that bacterial diversity of Antarctic is far greater than that detected by the cultivable approach. As of now there have been only three studies from India on the bacterial diversity of Antarctic using the rRNA approach using a soil sample from Schirmacher Oasis, Antarctic (Shivaji et al., 2004a), a water sample from the sea (Prabagaran et al., 2007) and a lake sediment core (Shivaji et al., 2011b).

 

  1. Bacterial diversity of a soil sample from Schirmacher Oasis, Antarctic using the 16S rRNA approach: The bacterial diversity of a soil sample collected in the vicinity of Lake Zub, Schirmacher Oasis, Antarctic, determined by cloning the total 16S rDNA of the soil indicated that the bacteria belonged to the classes— Proteobacteria, Proteobacteria, Proteobacteria, Gemmatimonas, Bacteriodetes, Actinobacteria, Chloroflexi and Chlamydiae (Shivaji et al., 2004a). In addition, seven clones were categorised as unidentified and unculturable in the classes of Proteobacteria, Actinobacteria, Chloroflexi and Chlamydiae. These results identified for the first time the presence of bacteria belonging to the genera Brevundimonas, Microbacterium, Rhodococcus, Serratia, Enterobacter, Rhodopseudomonas, Sphingomonas, Acidovorax, Burkholderia, Nevskia, Gemmatimonas, Xanthomonas and Flexibacter in Antarctic. Further, comparison of the Antarctic soil bacterial diversity with other cold habitats of Antarctic like from sediments, ice and cyanobacterial mat samples indicated that the bacterial diversity in soil was similar to the diversity observed in the continental shelf sediment sample. The Antarctic soil clones also resembled the bacterial diversity of soils from other geographical regions, but were unique in that none of the clones from the soil belonged to the uncultured Y, O, G, A and B groups common to all soil samples.1 Theme C article_S Shivaji 2
  2. 2. Bacterial diversity of pristine seawater and seawater contaminated with crude oil off Ushuaia, subAntarctic, using the rRNA approach: Bacterial diversity in sub-Antarctic seawater, collected off Ushuaia, Argentina, was examined using a culture independent approach (Prabagaran et al., 2007). In both libraries, clones representing the Proteobacteria, Proteobacteria, the Cytophaga-Flavobacterium-Bacteroidetes group and unculturable bacteria were dominant. Clones associated with the genera Roseobacter, Sulfitobacter, Staleya, Glaciecola, Colwellia, Marinomonas and Cytophaga were common to both the libraries (Table 2). However, clones associated with Psychrobacter, Arcobacter, Formosa algae, Polaribacter, Ulvibacter and Tenacibaculum were found only in seawater contaminated with hydrocarbons. Increase in their numbers may imply that crude oil addition enriched these clones but whether they are involved in biodegradation needs to be demonstrated. Further, the percentage of clones of Roseobacter, Sulfitobacter and Glaceicola was high in seawater (43 per cent, 90 per cent and 12 per cent respectively) compared to seawater contaminated with hydrocarbons (35 per cent, 4 per cent and 9 per cent respectively) probably implying that crude oil is toxic to these bacteria (Prabagaran et al., 2007). It was also observed that clones related to Psychromonas, Vibrio, Cytophaga and Maribacter which were present in seawater were no longer detectable in seawater with crude oil probably implying toxic effects of the hydrocarbons present. However, a common feature was the predominance of unculturable bacteria in seawater independent of the presence of hydrocarbons. Some of these unculturable bacteria were phylogenetically related to Antarctobacter, Roseobacter, Staleya, Loktanella, Sulfitobacter and Agrobacterium. So far, none of these bacteria have been implicated in hydrocarbon biodegradation. Therefore, one of the challenges would be to cultivate these organisms so as to be able to understand better the role of these organisms in the ecosystem. One of the clones F2C63 showed 100 per cent similarity with Marinomonas ushuaiensis (Prabagaran et al., 2005) a bacterium identified by us from the same site.1 Theme C article_S Shivaji 3
  3. 3. Vertical distribution of bacteria in a lake sediment from Antarctic using the 16S rRNA approach: Bacterial diversity of the sub-surface (18-22 cm), middle (60-64 cm) and bottom (100-104 cm) of a 136 cm long sediment core sampled from a fresh water lake in Antarctic, was also determined by T-RFLP (Figure 2) and 16S rRNA gene clone libraries (Shivaji et al., 2011b). Stratification of bacteria along the depth of the sediment was observed both with the T-RFLP and the 16S rRNA gene clone library methods (Table 3). For instance the aerobic Janthinobacterium and Polaromonas are confined to the surface of the sediment where as the anaerobic Caldisericum was present only in the bottom portion of the core (Table 3). It may be concluded that the bacterial diversity of an Antarctic lake sediment core sample varies along the length of the core depending on the oxic-anoxic conditions of the sediment.

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Bacterial Biodiversity of Arctic

Viable bacteria from Arctic: Research in Arctic Microbiology was initiated by S Shivaji as a Member of the First Indian Expedition to Arctic in August, 2007. The primary aim was to study the microbial biodiversity of the Arctic glaciers and the Fjord and also use the microorganisms for prospecting, for biomolecules of importance to the biotechnology industry. In this connection, the work thus far completed has demonstrated the presence of a wide variety of bacteria and yeasts in the vicinity of Midre Lovenbreen glacier and a Fjord (Kongsfjorden), in the Arctic.

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  1. 1. Bacterial Diversity of Culturable Bacteria of Kongsfjorden and Ny-Ålesund, Svalbard, Arctic: In the marine sediments of Kongsfjorden and the two samples collected from the shore of Ny-Ålesund a total of 103 bacterial isolates were obtained and these isolates could be grouped in to 47 phylotypes based on the 16S rRNA gene sequence belonging to 4 phyla namely Actinobacteria, Bacilli, Bacteroidetes and Proteobacteria (Table 4). Representatives of the 47 phylotypes varied in their growth temperature range (4-37oC), in their tolerance to NaCl (0.3-2 M NaCl) and growth pH range (2-11) (Srinivas et al., 2009). In addition representatives of 26 phylotypes exhibited amylase and lipase activity either at 5 or 20oC or at both the temperatures. Most of the phylotypes were pigmented. Fatty acid profile studies indicated that short chain fatty acids, unsaturated fatty acids, branched fatty acids, the cyclic and the cis fatty acids are predominant in the psychrophilic bacteria (Srinivas et al., 2009). The study also led to the discovery of two new species of bacteria from arctic namely Cyclobacterium qasimii and Oceanisphaera arcticum (Table 1).
  2. Bacterial Diversity of Culturable Bacteria of Midtre Lovenbreen glacier, an Arctic glacier, Ny-Ålesund, Svalbard, Arctic: Culturable bacterial diversity of Midtre Lovenbreen glacier, an Arctic glacier, was studied using 12 sediment samples collected from different points, along a transect, from the snout of Midtre Lovenbreen glacier up to the convergence point of the melt water stream with the sea (Reddy et al., 2009b). A total of 117 bacterial strains were isolated from the sediment samples, and categorised into 32 groups, (Table 5). Representatives of the phylotypes varied in their growth temperature range (4-37oC), in their tolerance to NaCl (0.1-1 M NaCl) and in the growth pH range (2-13) and 14 of 32 representative strains exhibited amylase, lipase and (or) protease activity and only one isolate (AsdM4-6) showed all three enzyme activities at 5 and 20oC respectively. More than half of the isolates were pigmented. Fatty acid profile studies indicated that short-chain fatty acids, unsaturated fatty acids, branched fatty acids, cyclic and cis fatty acids are predominant in the psychrophilic bacteria (Reddy et al., 2009b).

 

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Biotechnological Potential of Microbes
from Antarctic and Arctic

Psychrophilic microorganisms produce unique lipids, fatty acids, pigments and enzymes which find a prominent application in the biotechnology industry (Herbert 1992). Recently it was also demonstrated that psychrophilic bacteria from Antarctic are capable of synthesising silver nanoparticles (Shivaji et al., 2011d). Prasad et al. (2011) have also studied the ability of bacteria to produce anti-microbial compounds and demonstrated that it is a phenomenon dependent on the growth temperature of microbes. The enzymes produced by psychrophiles are unique in that they are heat-labile and cold-active with optimum activity between 10 to 30oC. Such enzymes would be best suited for biotechnological processes at low temperatures and lead to savings in energy requirements and would be very appropriate for the food processing industry, perfume industry and cold wash detergent industry. Psychrophilic enzymes have also been used in cheese manufacture and in milk processing (Cavicchioli et al., 2002).

Biotechnological potential of Antarctic microbes is also dependent on the ability of psychrophiles to synthesise polyunsaturated fatty acids (PUF) such as eicosopentaenoic acid (EPA), linolenic acid (GLA) and archidonic acid (AA) which are essential fatty acids for human beings and are used especially to supplement the diets of patients suffering from eczema, cardiovascular disease and diabetic neuropathy since these individuals are unable to convert lineoleic acid to GLA. PUFAs also play an important role in modulating membrane fluidity, a physiological strategy for cold adaptation. Further, PUFA producing bacteria could also serve as model organisms to identify and understand the genes and enzymes involved in PUFA production.

Cold-active and heat-labile enzymes

In India, studies on cold active and heat labile enzymes were initiated at least a decade ago and the enzymes were either partially (alkaline phosphatase) or totally purified (protease, RNAse RNA polymerase and trmE GTPase) and studied with respect to their thermal properties (Ray et al., 1992; Reddy et al., 1994a; Chattopadhyay et al., 1995; Uma et al., 1999; Singh and Shivaji, 2010). The five enzymes studied were active at low temperature (2 to 5oC) and were characteristically heat-labile compared to the mesophilic enzyme. Several reviews have provided a molecular understanding of the cold-activity enzymes from psychrophiles (Lonhienne et al. 2000; Russell et al. 2000; Smal et al. 2000; Gerday et al. 2000))

 

Molecular Basis of the Survival of
Antarctic Microbes

a. Changes in composition of fatty acids in psychrophilic Nostoc: Studies using Antarctic bacteria have revealed that their adaptation to low temperature is dependent on a number of survival strategies such as their ability to modulate membrane fluidity, ability to carry out biochemical reactions at low temperatures, ability to regulate gene expression at low temperatures and ability to sense temperature (Ray et al. 1994b; 1994c; Chintalapati, et al., 2004; Shivaji and Prakash, 2010; Shivaji et al. 2007; Prakash et al., 2010).

Fatty acid desaturation is the most common mechanism used by mesophilic cyanobacteria, to modulate membrane fluidity in response to low temperature stress. It has been demonstrated unequivocally that polyunsaturated fatty acids, the enzymes that convert saturated to unsaturated fatty acids and the corresponding genes are essential for growth at low temperatures. But our studies using a psychrophilic Nostoc and Oscillatoria from Antarctic also indicate increase in C18:3 at the expense of C18:1 and C18:2 in cells grown at 10oC compared to cells grown at 25oC implying that polyunsaturated fatty acids are crucial for growth of microorganisms at low temperature (Chintalapati et al., 2007). Detailed studies on the psychrophilic Nostoc have indicated that the upregulation in the synthesis of C18:3 was due to increase in the activity of DesA and DesB enzymes. At the transcription level interestingly it was noticed that all the three desaturase genes in psychrophilic Nostoc namely desA, desB and desC are constitituvely expressed at all temperatures, unlike the mesophilic cyanobacteria in which desA and desB are upregulated in expression (Chintalapati et al., 2007). The study also led to the discovery of a new desaturase gene desC2 which introduces a double bond in C16:0 (Chintalapati et al., 2006). Studies have also indicated that psychrophilic Pseudomonas syringae require trans monounsaturated fatty acid for growth at higher temperature and Cis-trans isomerase gene is constitutively expressed during growth and under conditions of temperature and solvent stress (Kiran et al. 2004; 2005).

b. Changes in composition of carotenoids: A large number of Antarctic bacteria have been found to contain carotenoid type of pigments in their membrane (Chauhan and Shivaji, 1994; Jagannadham et al., 1991; 1996a; 1996b; 2000; Chattopadhyay et al., 1997; Strand et al., 1997). Furthermore, it has also been demonstrated that polar carotenoids stabilise the membrane and the non-polar carotenoids increase the fluidity of the membrane. Therefore, it is indeed very interesting when it was observed by a series of experiments that in two psychrophilic bacteria from Antarctic (Sphingobacterium antarcticum and Micrococcus roseus) the synthesis of carotenoids was dependent on the temperature of growth, with an increase in the synthesis of polar carotenoids at low temperature of growth so as to stabilise the membrane and a concomitant decrease in the synthesis of non-polar carotenoids (Jagannadham et al., 1991; 1996a; 1996b; 2000; Chattopadhyay et al., 1997; Strand et al., 1997).

c. Role of trmE in cold adaptation: Transposon mutagenesis of Pseudomonas syringae (Lz4W) a psychrophilic bacterium capable of growing between 2 to 30oC, yielded thirty cold sensitive mutants. CSM1, one of the cold sensitive mutants, was characterised. Growth of CSM1 is retarded when cultured at 4°C but not when cultured at 22°C and 28°C compared to the wild type cells thus indicating that CSM1 is a cold sensitive mutant of Pseudomonas syringae (Lz4W) (Figure 3). The mutated gene in CSM1 was identified as trmE (coding for transfer RNA modification GTPase) and evidence was provided to indicate that this gene is induced at low temperature (Figure 3). Further, the cold inducible nature of the trmE promoter has been demonstrated. In addition, the transcription start site and the various regulatory elements of the trmE promoter such as the -10 region, -35 region, UP element, cold-box and DEAD-box have been identified and the importance of these regulatory elements in promoter activity have been confirmed. This is the first report demonstrating that trmE is required for growth at low temperature and this is further confirmed by gain of the cold resistant phenotype in CSM1 complemented with the trmE gene (Figure 3) (Singh et al. 2009).

d. Aspartate aminotransferase is involved in cold adaptation in psychrophilic Pseudomonas syringae: CSM2, a cold-sensitive mutant of psychrophilic Pseudomonas syringae, grows like wild type cells when cultured at 22°C and 28°C; but at 4°C the growth is retarded (Figure 4). In CSM2, AAT (coding for aspartate aminotransferase) is identified as the mutated gene (Sundareswaran et al., 2010). The expression of AAT and activity of AAT in Pseudomonas syringae was transiently enhanced when cells were shifted from 22°C to 4°C indicating that AAT expression and AAT activity are cold-inducible (Figure 4). Complementation of the mutated AAT transformed CSM2 from a cold-sensitive phenotype to a cold-resistant phenotype like the wild type cells thus providing evidence for the first time that AAT is required for low temperature growth (Figure 4) (Sundareswaran et al., 2010).

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Modern Approach to Biodiversity and
Biotechnology of Psychrophiles

The ‘Great Plate Count Anamoly’ wherein only a miniscule fraction of microorganisms (< 5 per cent) are detected using the culture dependent method as compared to in situ detection methods has now encouraged methods based on culture independent methods, such as the ‘rRNA approach’. This latter approach has indeed revealed a greater degree of biodiversity but suffers from the fact that other characteristics of living organisms cannot be ascertained. Therefore, there is a need to have a method, which not only reflects the phylogeny of the bacterium, but also its biotechnological potential and this indeed is possible using the ‘metagenome’ approach (Rondon et al., 2000; Holmes et al., 2003). Metagenome represents the genomes of the total microbiota found in nature and involves cloning of total DNA isolated from an environment sample and expressing the genes in a suitable host. Thus the metagenome approach apart from providing phylogenetic data based on 16S rRNA gene sequence analysis and enumerating the extent of microbial diversity, also provides access to genetic information of uncultured microorganisms. In fact, metagenome libraries could form the basis for identification of novel genes from uncultured microorganisms. Recognising the potential of this approach, molecular biologists and chemists have undertaken a project to establish the metagenome of ‘whole earth’ (soil) to catalog genes for useful natural products such as streptomycin, actinomycin D, immunosuppressants and anticancer agents. Thus it may not be too long before this approach is also customised to hunt for genes of interest in metagenomes from various habitats, such as in Antarctic.

 

 

Conclusions

Antarctic microbiology dates back to the early twentieth century. But what we know today about these microbes is very limited and were confined to establishing their distribution, abundance, seasonality, ecology, identity etc. We know comparatively very little about those surviving in the depths of Antarctic as in the frozen lakes below the ice shelf, those associated with ice cores, permafrost, cryoconites etc. Even less is known about the anaerobes present in the sediments or deep below. The deep biosphere of the Antarctic, Arctic and Himalayan glaciers are areas that need to be studied and such microbes could define the limits of life with respect to low temperature, water activity and nutrients. Psychrophiles are a bioresource and establishing their identity would go beyond academics to identifying them as workhorses of biotechnology. Compared to heat-stable enzymes, the cold-active enzymes are limited in their application but it is their uniqueness of catalysing reactions at temperatures close to the freezing point which make them the most wanted enzymes for processes which operate at low temperature. A point in case would be their ability to remediate oil spills or any chemical contaminant at temperatures below 8oC, a temperature at which mesophilic bacteria are totally inactive. Basic research focused to unravel the molecular basis of their survival under freezing conditions would enhance our understanding of the lower temperature limits of life, would unravel mechanisms operating at the level of gene expression and would reveal the unique temperature driven regulation of gene expression in life forms. This information could also become the basis for future activities related to crop survival and yield under low temperature conditions. Thus the three simple questions that need to be addressed on a continuing basis are :

  1. What are the microbes surviving in the cold habitats including their identity, seasonality, distribution, ecology etc?
  2. What processes could be utilised in biotechnology and may be in low temperature agriculture?
  3. What is the molecular basis for their survival and multiplication?

 

 

Acknowledgements

This work was supported by the Indo-French Centre for the Promotion of Advanced Research, New Delhi, India; the Department of Biotechnology, Government of India, New Delhi, India; National Centre for Antarctic and Ocean Research, Goa, India and the India Japan Cooperative Science Programme of Department of Science and Technology, India and the Japanese Society for Promotion of Science, Japan. The following have also contributed to the research activity in my lab either as a collaborator, a colleague, a research staff or a student : T.N.R. Srinivas, Zareena Begum, Suman Pradhan, M.S. Pratibha, K. Hara Kishore, Ashish K. Singh, G.S.N. Reddy, V.R. Sundareswaran, M.K. Chattopadhyay, M.V. Jagannadham, J.S.S. Prakash, R. Manorama, M.D. Kiran, S. Chintalapati, P. Chaturvedi, P. Gupta, K. Suresh, K. Prabahar, S. Uma, G. Seshu Kumar, P.K. Pindi, M.K. Ray, S. Madhu, S. Mayilraj, P. Manasa, Sathish Prasad, P.V.V. Reddy, S.S.S.N. Rao, S.R. Prabagaran, S. Buddhi, Kiran Kumari, P.S. Rao, S. Dube, A.K.K. Pathan, B. Bhadra, R. Sreenivas Rao, S.M. Singh, B. Kavya, P. Anil Kumar, B. Sunil Reddy, S.I. Alam, G.S. Prasad, P.U.M. Raghavan, N.B. Sarita, S. Annapoorni, K. Kannan, T. Sitaramamma, K. Janiyani, Sarita Chauhan, Saisree, N. Shyamala Rao, R.P. Aduri, R. Kutty and P.M.Bhargava.

 

 

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  29. _______Smita Dube, Reddy, G.S.N. and Shivaji, S. (2004), ‘Pseudonocardia Antarctic sp. nov., an Actinomycetes from McMurdo Dry Valleys, Antarctic’, Systematic and Applied Microbiology, 27, 66-71.
  30. Prakash, J.S.S., Kanesaaki, Y., Suzuki, I., Shivaji, S. and Murata, N. (2010), ‘An RNA helicase, CrhR, regulates the low-temperature-inducible expression of three genes for molecular chaperones in Synechocystis sp. PCC 6803’, Microbiology, 156, 442-451.
  31. Prasad, S., Manasa, P., Buddhi, S., Singh, S.M. and Shivaji, S. (2011), ‘Antagonistic interaction networks among bacteria from a cold soil environment’, FEMS Microbiology Ecology, 78, 376-385.
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  33. ________ Seshu Kumar, G. and Shivaji, S. (1994a), ‘Phosphorylation of membrane proteins in response to temperature in an Antarctic Pseudomonas syringae’, Microbiology, 140, 3217-3223.
  34. _______ (1994b), ‘Tyrosine phosphorylation of a cytosolic protein from the antarctic psychrotrophic bacterium Pseudomonas syringae, FEMS Microbiology Letters, 122, 49-54.
  35. _______ (1994c), ‘Phosphorylation of lipopolysaccharides in the Antarctic psychrotroph Pseudomonas syringae: A possible role in temperature adaptation’, Journal of Bacteriology, 176, 4243-4249.
  36. Reddy, G.S.N., Rajagopalan, G. and Shivaji, S. (1994), ‘Thermolabile ribonucleases from antarctic psychrotrophic bacteria: Detection of the enzyme in various bacteria and purification from Pseudomonas fluorescens’, FEMS Microbiology Letters, 122, 122-126
  37. _______ Aggarwal, R.K., Matsumoto, G.I. and Shivaji, S. (2000), ‘Arthrobacter flavus sp. nov., a psychrotropic bacterium isolated from a pond in Mc Murdo Dry Valley, Antarctic’, International Journal of Systematic and Evolutionary Microbiology, 50, 1553-1561.
  38. ______ Prakash, J.S.S., Matsumoto, G.I., Stackebrandt, E. and Shivaji, S. (2002a),
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  39. ______ Prakash, J.S.S., Vairamani, M., Prabhakar, S., Matsumoto, G.I. and Shivaji, S. (2002b), ‘Planococcus antarcticus and Planococcus psychrophilus spp. nov. isolated from cyanobacterial mat samples collected from ponds in Antarctic’, Extremophiles, 6, 253-261.
  40. _______ Prakash, J.S.S., Prabahar, V., Matsumoto, G.I., Stackebrandt, E. and Shivaji, S. (2003a), ‘Kocuria polaris sp. nov., an orange pigmented psychrophilic bacterium isolated from an Antarctic cyanobacterial mat sample’, International Journal of Systematic and Evolutionary Microbiology, 53, 183-187.
  41. ______ Prakash, J.S.S., Srinivas, R., Matsumoto, G.I. and Shivaji, S. (2003b), ‘Leifsonia rubra sp. nov. and Leifsonia aurea sp. nov. psychrophilic species isolated from a pond in Antarctic’, International Journal of Systematic and Evolutionary Microbiology, 53, 977-984.
  42. _______ Matsumoto, G.I. and Shivaji, S. (2003c), ‘Sporosarcina macmurdoensis sp. nov., from a cyanobacterial mat sample from a pond in Mcmurdo Dry Valleys, Antarctic’, International Journal of Systematic and Evolutionary Microbiology, 53, 1363-1367.
  43. _______ Raghavan, P.U.M., Sarita, N.B., Prakash, J.S.S., Nagesh, N., Delille, D. and Shivaji, S. (2003d), ‘Halomonas glaciei sp. nov. isolated from fast ice of Adelie Land, Antarctic’, Extremophiles, 7, 55-61.
  44. ______ Matsumoto, G.I., Schumann, P., Stackebrandt, E. and Shivaji, S. (2003e), ‘Psychrophiic Pseudomonads from Antarctic: Pseudomonas Antarctic sp. nov., Pseudomonas meridiana sp. nov. and Pseudomonas proteolytica sp. nov.’, International Journal of Systematic and Evolutionary Microbiology, 54, 713-719.
  45. Reddy, P.V.V., Rao, S.S.S., Pratibha, M.S., Sailaja, B., Kavya, B., Manorama, R., Singh, S.M., Srinivas, T.N.R. and Shivaji, S. (2009b), ‘Bacterial diversity and bioprospecting for cold-active enzymes from culturable bacteria associated with sediment of melt water stream of Midtre Lov´enbreen glacier, an Arctic glacier’, Research in Microbiology, 160, 538-546.
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  47. Russell, N.J. (2000), ‘Towards a molecular understanding of cold activity of enzymes from psychrophiles’, Extremophiles, 4, 83-90.
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  49. ______ (1989a), ‘Isolates of Arthrobacter from the soils of Schirmacher Oasis, Antarctic’, Polar Biology, 10, 225-229.
  50. _________(1989b), ‘Isolation and identification of Pseudomonas species from Schirmacher Oasis, Antarctic’, Applied and Environmental Microbiology, 55, 767-771.
  51. Shivaji, S., Ray, M.K., Seshu Kumar, G., Reddy, G.S.N., Saisree, L. and Wynn Williams, D.D. (1991), ‘Identification of Janthinobacterium lividum from the soils of the islands of Scotia Ridge and from Antarctic peninsula’, Polar Biology, 11, 267-272.
  52. ______ Ray, M.K., Shyamala Rao, N., Saisree, L., Jagannadham, M.V., Seshu Kumar, G., Reddy, G.S.N. and Bhargava, P.M. (1992), ‘Sphingobacterium antarcticus sp. nov. a psychrotrophic bacterium from the soils of Schirmacher Oasis, Antarctic’, International Journal of Systematic Bacteriology, 42, 102-116.
  53. _______ Reddy, G.S.N., Aduri, R.P., Kutty, R. and Ravenschlag, K. (2004a), ‘Bacterial diversity of a soil sample from Schiracher Oasis, Antarctic’, Cell and Molecular Biology, 50, 525-536.
  54. _______ Reddy, G.S.N., Raghavan, P.U.M., Sarita, N.B. and Delille, D. (2004b), ‘Psychrobacter salsus sp. nov. and Psychrobacter adeliensis sp. nov. isolated from fast ice from Adelie Land, Antarctic’, Systematic and Applied Microbiology, 27, 628-635.
  55. _______ Reddy, G.S.N., Suresh, K., Gupta, P., Chintalapati, S., Schumann, P., Stackebrandt, E. and Matsumoto, G. (2005a), ‘Psychrobacter vallis sp. nov. and Psychrobacter aquaticus sp. nov. from Antarctic’, International Journal of Systematic and Evolutionary Microbiology, 55, 757-762.
  56. ______ Gupta, P., Chaturvedi, P., Suresh, K., Delille, D. (2005c), ‘Marinobacter maritimus sp. nov., a psychrotolerant strain isolated from sea water off the subantarctic Kerguelen Island’, International Journal of Systematic and Evolutionary Microbiology, 55, 1453-1456.
  57. ______ Kiran, M.D. and Chintalapati, S. (2007), ‘Perception and transduction of low temperature in bacteria’; Physiology and Biochemistry of Extremophiles, Ed. C. Gerday & N. Glansdorff, ASM Press: Washington D.C, 194-207.
  58. _____ and Reddy, G.S.N. (2009), ‘Bacterial biodiversity of Antarctic : Conventional, Polyphasic and rRNA approaches’, Polar Microbiology: The Ecology, Biodiversity and Bioremediation Potential of Microorganisms in Extremely Cold Environments: CRC Press, 61-94.
  59. ______ and Prakash, J.S.S. (2010), ‘How do bacteria sense and respond to low temperature?’, Archives of Microbiology, 192, 85-95
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  61. _______ Kiran Kumari, Hara Kishore, K., Pindi, P.K., Rao, P.S., Srinivas, T.N.R., Asthana, R. and Ravindra, R. (2011b), ‘Vertical distribution of bacteria in a lake sediment from Antarctic by culture-independent and culture-dependent approaches’, Research in Microbiology, 162, 191-203.
  62. _______ Madhu, S. and Singh, S. (2011d), ‘Extracellular synthesis of silver nanoparticles using psychrophilic bacteria’, Process Biochemistry, 46, 1800-1807.
  63. Singh, A.K. and Shivaji, S. (2010), ‘A cold-active and a heat-labile t-RNA modification GTPase from a psychrophilic bacterium Pseudomonas syringae (Lz4W)’, Research in Microbiology, 161, 46-50.
  64. ______ Pindi, P.K., Dube, S., Sundareswaran, V.R. and Shivaji, S. (2009), ‘In the psychrophilic Pseudomonas syringae, trmE is important for low temperature growth’, Applied and Environmental Microbiology, 75, 4419-4426.
  65. Smal, A.O., Leiros, H.K.S., Os, V. and Willassen, N.P. (2000), ‘Cold adapted enzymes’, Biotechnology Annual Review, 6, 1-57.
  66. Srinivas, T.N.R., Nageswara Rao, S.S.S., Vishnu Vardhan Reddy, P., Pratibha, M.S., Sailaja, B., Kavya, B., Hara Kishore, K., Begum, Z., Singh, S.M. and Shivaji, S. (2009), ‘Bacterial diversity and bioprospecting for cold-active lipases, amylases and proteases, from culturable bacteria of Kongsfjorden and Ny-Ålesund, Svalbard, Arctic’, Current Microbiology, 59, 537-547.
  67. ______ Prasad, S., Manasa, P., Sailaja, B., Begum, Z. and Shivaji, S. (2012), ‘Lacinutrix himadriensis sp. nov., a psychrophilic bacterium isolated from a marine sediment of Kongsfjorden, Svalbard, Arctic’, International Journal of Systematic and Evolutionary Microbiology: In press.
  68. _______ Reddy, P.V.V., Begum, Z., Manasa, P. and Shivaji, S. (2011a), ‘Oceanisphaera arctica sp. nov., isolated from a marine sediment of Kongsfjorden, Svalbard, Arctic’, International Journal of Systematic and Evolutionary Microbiology: In press.
  69. Straka, R.P. and Stokes, L.L. (1960), ‘Psychrophilic bacteria from Antarctic’, Journal of Bacteriology, 80, 622-625.
  70. Strand, A., Shivaji, S. and Liaaen-Jensen, S. (1997), ‘Bacterial carotenoids 55. C50-Carotenoids 25: Revised structures of carotenoids associated with membranes in psychrotrophic Micrococcus roseus’, Biochemical Systematics and Ecology, 25, 547-552.
  71. Sundareswaran, V.R., Singh, A.K., Dube, S. and Shivaji, S. (2010), ‘Aspartate aminotransferase is involved in cold adaptation in psychrophilic Pseudomonas syringae’, Archives of Microbiology, 192, 663-672.
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