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Bioprospecting and Biotechnology in Antarctica

David S. Nichols, Kevin Sanderson, Alex Buia, Jodie van de Kamp, Paul Holloway, John P. Bowman, Matthew Smith, Carol Mancuso Nichols, Peter D. Nichols and Tom A. McMeekin
INTRODUCTION Bioprospecting may be defined as the search for new or better bioproducts or technological processes from biological sources. In particular, bioprospecting relies on provision of a bioresource, a supply of novel biodiversity. Molecules derived from natural products, particularly those produced by plants and microorganisms, have an excellent record of providing novel chemical structures for development as new pharmaceuticals. Many of the world's most successful and valuable pharmaceuticals have been derived directly, or indirectly, from natural product sources eg. acetylsalicilic acid (aspirin) from willow bark and penicillin from the fungus Penicillium. The screening of microbial natural products continues to represent an important route to the discovery of novel bioactive and therapeutic chemicals. Of increasing interest is the evaluation of the potential of lesser-known and/or new microbial taxa. There is, therefore, an opportunity within Australia to make important contributions in the field of biotechnology through the acquisition and screening of Antarctic and other novel microbiota. MICROBIAL BIOPROSPECTING Microorganisms represent the largest reservoir of undescribed biodiversity, and hence possess the greatest potential for the discovery of new natural products. It is estimated that the Earth currently supports between 3 and 30 million species of organisms. Of these, approximately 1.4 million have been described by science. This includes virtually all the species of birds and mammals (~ 13,500). In contrast only around 200,000 of the estimated 1.0 to 1.5 million species of fungi have been characterised (ie. 13-20%). For the bacteria this percentage is even lower with estimates ranging from only 1-10% of probable species being described in culture (Bull et al. 1992, 2000). Figure 1 represents the division level diversity of the bacterial domain based on 16S rDNA sequences. Several of the divisions (eg. Proteobacteria, Actinobacteria) are well represented by cultivated strains. However the majority are
Nichols, D.S. .et al. "Bioprospecting and Biotechnology in Antarctica" in Jabour-Green, J. & Haward, M. (Eds). The Antarctic: Past, Present and Future. Antarctic CRC Research Report #28. Hobart, 2002, pp.85-103.

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poorly represented by cultured organisms. In fact thirteen of the thirty-six divisions are defined by environmental sequences only (Hugenholtz et al. 1998). The proportion of cultivated versus uncultivated sequences obtained from environmental molecular analyses may be used as an indication of the level of described biodiversity for a particular division. For the Actinobacteria, the percentage of uncultivated environmental sequences has been estimated at around 20% of total known sequences. It is perhaps an indication of the efforts committed to the isolation of Actinobacteria that this percentage is the lowest of all the cosmopolitan bacterial divisions (Hugenholtz et al. 1998). Hence, the search for novel Actinobacteria biodiversity now centres on the bioprospecting of more unusual or extreme environments, such as Antarctica. Relatively few Antarctic microorganisms have been described (approximately 30 species of bacteria and 3 species of Archaea) with around 20 of these isolated in the recent past (Table 1). Most have derived from studies of marine environments (sea ice, seawater) or the marine-like saline lakes of the Vestfold Hills, Eastern Antarctica. Few studies have concentrated on the soil environments of ice-free areas for the isolation of Actinobacteria. BIODIVERSITY OF ANTARCTIC ACTINOBACTERIA Streptomyces sp. are the most cosmopolitan of the Actinobacteria. They are readily isolated from most aerobic soil and some aquatic environments. Due to their ease of isolation and the intensive screening programs carried out over several decades on them, there is growing interest in non-streptomycete Actinobacteria as sources of novel compounds. Much of the search for these rarer genera has focused on environments other than terrestrial soil, eg. inshore marine sediments (Takizawa et al. 1993) rather than extreme environments. We began our investigation of Actinobacterial biodiversity in Antarctica by looking at the ice free soils of the Vestfold Hills. This was later expanded to include plant and soil from Macquarie Island (a sub Antarctic island). During these investigations 769 Actinobacteria were isolated and purified. These were identified to the genus or group level by morphological examination of spore structures. Results demonstrated that a large number of non-streptomycete Actinobacteria were present in Antarctic environments, although as expected the majority of isolates were Streptomyces sp. (Table 2). To further assess the biodiversity of these isolates, 93 were characterised by 16S rDNA sequencing. Twenty two novel 16S rDNA sequences representing nine genera were identified in this group (Table 3). These novel sequences probably represent new species although further biochemical and genetic studies are required

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to confirm this. The very high rate of novel species obtained is due in part to the fact that only isolates identified by the preliminary morphological characterisation as being unusual were chosen for sequencing. Interestingly, these preliminary results indicate that novel species isolated from Antarctic soil are distinct from related, novel species isolated from Macquarie Island (Figure 2). This research has demonstrated that Antarctic terrestrial environments are a rich source of both novel species and rare genera of Actinobacteria. Many of the novel Antarctic species belong to genera with very strong track records for producing pharmaceutically active compounds (eg Streptomyces, Nocardia, Micromonospora). Over the coming years it will be interesting to observe if this pool of novel biodiversity translates into novel bioproducts. MICROBIAL ADAPTATIONS BIOTECHNOLOGY TO ANTARCTICA AND

Microbial adaptation to a permanently cold environment includes the optimisation of basic cell processes necessary for growth and survival. These can be summarised into three categories; enzyme function, nutrient transport and cell membrane function. The adaptations of cellular processes in each of these areas represent potential biotechnology products for exploitation. Two examples are the production of polyunsaturated fatty acids (PUFA) and the production of cold-active enzymes by bacteria from Antarctic sea ice. Investigations of sea ice microbial communities have demonstrated the dominance of bacterial populations highly adapted to life at constant low temperature. These bacteria are known as psychrophiles. The term "psychrophile" (psychro- cold, phile- loving) was first coined in 1902 to describe bacteria capable of growth and survival at 0C (Russell 1992). However, it soon became apparent that a more stringent definition was required to separate those organisms requiring cold temperatures for growth (true psychrophiles) from others that were capable of growth at or near 0C, but possessed quite high optimal growth temperatures (around 20 - 30C). The latter organisms became known as psychrotolerant. The term psychrophile is therefore an operational definition, to describe the temperature-growth relationship of microorganisms. While there is not necessarily a phylogenetic relationship between psychrophilic organisms, such relationships may exist through the evolution of adaptive strategies for low temperature growth. An example is the genus Shewanella which includes a number of psychrophilic species (S. pealeana, S. hanedai, S. benthica) including two isolated from Antarctic sea ice (S. gelidimarina, S. frigidimarina), in addition to species which are non-cold-adapted,

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or mesophilic (S. putrefaciens, S. alga, S. baltica, S. woodyi, S. oneidensis, S. amazonensis; Figure 3). BIOTECHNOLOGY: POLYUNSATURATED FATTY ACIDS (PUFA) PUFA have remained as a natural product due to the synthetic difficulty of reproducing the methylene interrupted double bond sequence (Figure 4) by industrial chemistry. Their significance to humans lies in their biological activity, as precursors for groups of nutritionally important compounds and as essential cellular components. PUFAs act as precursors to groups of compounds known as eicosanoid hormones encompassing the prostaglandins, leukotrienes and thromboxanes. These hormones are short-lived, but have a diversity of effects in mediating cardiovascular disease conditions (Torssell 1983). They are also essential in animals for the proper development of retinal and nervous tissue (Takahata et al. 1998). Of importance is the fact that higher animals lack the ability to synthesise 3 PUFA de novo, hence these components or their immediate C18 precursors, must be provided in the diet (Meyer et al. 1999). In bacteria PUFA are component fatty acids of certain phospholipids which occur in the cell membrane. Generically, long-chain PUFA consist of a long chain of carbon atoms (usually C20 or C22) containing a number (usually 4-6) methylene interrupted double bonds. Studies of psychrophilic bacteria from the Antarctic have shown a that high proportion of cold-adapted bacteria from sea ice possess the ability to produce PUFA, such as eicosapentaenoic acid (20:5 3; EPA) or docosahexaenoic acid (22:6 3; DHA) (see Nichols et al. 1999, Russell and Nichols 1999). It is considered that the low melting temperature of these highly unsaturated membrane components, combined with their unique molecular geometry in the membrane, yields a particular advantage at low temperature in balancing the competing homeoviscous and homeophasic forces in the cell membrane (Russell and Nichols 1999). It can be inferred that PUFA production represents a particular physiological strategy for cold-adaptation. This is supported by empirical observations such as in the genus Shewanella. Here, there is a good correlation between those species which are cold-adapted and produce PUFA (highlighted in bold, Figure 3), in contrast to those which do not produce PUFA and grow at higher temperatures (mesophiles) (highlighted in grey, Figure 3). The current commercial sources of EPA and DHA are restricted to fish and algal-derived oils. Problems exist with both of these sources. Commercial fish stocks are likely to decline in the future, with the demand for fish oil in the

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aquaculture industry alone estimated to exceed supply by 2010 (Lewis et al. 1999). Algal-derived oils require a relatively high investment of technology and expense compared to the prospect of bacterial fermentation, although bacteria contain a lower proportion of lipid (Nichols et al. 1996). A key advantage of bacterial PUFA production is that only a single PUFA is produced, rather than the complex mixture yielded from fish or algal oils (Russell and Nichols 1999). Thus bacterial sources of PUFA remove the expense of preparative purification in the production of highpurity PUFA oils. In addition to their potential use as cell factories bacteria in particular offer the biotechnological opportunity to investigate the genes and enzymes responsible for PUFA production. Interest in the production of PUFA from alternative sources for use in aquaculture feeds and human nutraceuticals (health supplements) has fuelled recent research into the molecular biology of PUFA production in prokaryotes. Insight has also been gained from related research into the mechanisms of gene expression in deep-sea microorganisms (Nakasone et al. 1998) many of which belong to PUFAproducing genera (Kato et al. 1998). The transfer of a gene cluster from Shewanella putrefaciens SCRC-2738 into Escherichia coli and a Synechococcus sp. resulted in the successful expression of EPA in these organisms (Yazawa 1996; Takeyama et al. 1997). However, the level of expression achieved was low. The gene cluster used in both cases consisted of a 38 kb fragment containing eight open reading frames with three of these possessing homology with genes that encode for enzymes involved in fatty acid biosynthesis (Figure 5). These studies were extended to other PUFA-producing strains (Moritella (Vibrio) marinus, Photobacterium (sic) and Shewanella sp.). Further characterisation using these organisms has identified five genes responsible for PUFA biosynthesis, four of which may have been grouped as a possible operon within the DNA fragment. Strong homologies of both DNA composition and organization were noted between the strains, with PUFA genes from either EPA or DHA producing bacteria able to replace and/or complement each other in E. coli. Furthermore, a single gene (orf 8) controlled the synthesis of either EPA or DHA in the strains examined (Figure 5). In future years it will be interesting to observe whether the transgenic potential of bacterial PUFA genes is realised. The strong homology of PUFA genes marries with the correlation of the current 16S rDNA phylogenetic tree for the genus Shewanella (Bowman 2001) and with the known ability of different species to produce PUFA (Russell and Nichols 1999). This correlation implies that the progenitor of the genus possessed this ability. It also appears that while the ability to synthesise PUFA has been retained by all the psychrophilic and several of the psychrotrophic species, the ability (or the expression of PUFA synthesis) has disappeared on two distinct occasions during

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the divergence of psychrotrophic and/or mesophilic species (Figure 4). The apparent conservation of PUFA genes in this genus may point to the potential utilisation of these functional genes as phylogenetic markers in cultureindependent ecological studies. BIOTECHNOLOGY: COLD-ACTIVE ENZYMES Cold-active or cold adapted enzymes may be produced by organisms existing in permanently cold habitats located in polar zones, at high altitudes or in the deep sea. These enzymes provide opportunities to study the adaptation of life to low temperature and the potential for biotechnological exploitation (Aguilar 1996, Morita et al. 1997). Applications exist in a range of industries for cold-active enzyme applications, eg. cleaning agents, leather processing, degradation of xenobiotic compounds in cold climes, food processing (fermentation, cheese manufacture, bakery, confectionery, meat tenderisation) and molecular biology (heterologous gene expression) (Margesin and Schinner 1999). Cold-active enzymes typically have maximal catalytic activity at temperatures below 40C and usually display some degree of thermolability. Recent research has focused on determining the structural characteristics, which confer cold adaptation in enzymes. The tertiary and quaternary structures of cold-active enzymes have more open and flexible arrangements, thus providing better access of substrates to the active site at lower temperatures. Rigid secondary structures and disulfide bridges are practically absent, thus accounting for increased thermolability (Feller et al. 1997, Feller and Gerday 1997). Individual enzyme types possess different structural strategies to gain overall increased flexibility. Certain structural features thought to be indicative of cold adaptation have also been found in similar non-cold adapted enzymes (Schroder et al. 1998). In addition the same enzyme from different organisms, containing an identical amino acid sequence, have been found to possess different thermal properties (Love et al. 1998). This suggests that protein folding has a critical role in conferring activity at low temperature. With more knowledge of the mechanisms of cold adaptation our fundamental understanding of protein function and structure will be advanced. Preliminary data has been obtained for a variety of psychrophilic and psychrotolerant (cold-tolerant) enzymes from Antarctic bacterial isolates (Buia 1997). The isolates were obtained from sea ice and lake habitats in the Vestfold Hills. Whole cell and cell-free assays indicated the presence of protease, galactosidase, phosphatase, and amylase enzyme(s) exhibiting strong cold adaptation in several strains (Table 4). The Cytophaga-like strain IC166 showed particular promise as it elaborates several cold adapted enzymes. From this

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research it is apparent Antarctic bacteria, especially those isolated from ice, are good sources of cold-active enzymes (Buia 1997). Identification of industrial requirements for cold-active enzymes is essential to establish a committed effort in isolating cold-adapted enzymes with commercial potential. When a target enzyme type (ie. one with commercial potential) has been established, extensive screening of bacterial isolates can be initiated. Examples may include lipases and proteases as they have the broadest applicability. Screening would be simple and rapid to obtain the best possible strains. The ideal strain would demonstrate a high endogenous enzyme activity and the enzyme itself would ideally show strong activity at low temperatures, but not be overly thermolabile as this would hamper storage. With appropriate candidates, purified enzymes would be tested for their effectiveness and scale-up fermentations designed and implemented to test the product. We anticipate this will be a focus in future research and development. In summary, Antarctic bacteria offer a unique biological resource. To date a high proportion of isolates represent novel biodiversity. Biotechnological opportunities have been identified in the areas of novel Actinobacteria, the production of PUFA and cold-active enzymes. ACKNOWLEDGEMENTS Aspects of this research have been supported by the Antarctic Scientific Advisory Council, the Australian Research Council, the British Council, the Australian Society for Microbiology Research Trust and Cerylid Biosciences Ltd. REFERENCES Aguilar A: Extremophile research in the European Union: from fundamental aspects to industrial expectations. FEMS Microbiol Rev 1996, 18:89-92. Bowman JP: Genus Shewanella. In Bergey's Manual of Systematic Bacteriology. Edited by Krieg NR, Holt JG. Baltimore: Williams & Wilkins; 2001 (in press): Bowman JP, McCammon SA, Nichols DS, Skerratt JS, Rea SM, Nichols PD, McMeekin TA: Shewanella gelidimarina sp. nov. and Shewanella frigidimarina sp. nov. - novel species with the ability to produce eicosapentaenoic acid (20:5w3) and grow anaerobically by dissimilatory Fe(III) reduction. Int J Syst Bacteriol 1997A, 47:1040-1047. Bowman JP, McCammon SA, Brown JL, Nichols PD, McMeekin TA: Psychroserpens burtonensis gen. nov., sp. nov., and Gelidibacter algens gen.

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nov., sp. nov, psychrophilic bacteria isolated from antarctic lacustrine and sea ice habitats. Int J Syst Bacteriol 1997B, 47:670-677. Bowman JP, Nichols DS, McMeekin TA: Psychrobacter glacincola sp. nov., a halotolerant, psychrophilic bacterium isolated from antarctic sea ice. Syst Appl Microbiol 1997C, 20:209-215. Bowman JP, McCammon SA, Skerratt JH: Methylosphaera hansonii gen. nov., sp. nov., a psychrophilic, group I methanotroph from Antarctic marine-salinity, meromictic lakes. Microbiology 1997D, 143:1451-1459. Bowman JP, Gosink JJ, McCammon SA, Lewis TE, Nichols DS, Nichols PD, Skerratt JH, Staley JT, McMeekin TA: Colwellia demingae sp. nov., Colwellia hornerae sp. nov., Colwellia rossensis sp. nov. and Colwellia psychrotropica sp. nov.: psychrophilic Antarctic species with the ability to synthesize docosahexaenoic acid (22:6 n-3). Int J Syst Bacteriol 1998B, 48:11711180. Bowman JP, McCammon SA, Brown JL, McMeekin TA: Glaciecola punicea gen. nov., sp. nov. and Glaciecola pallidula gen. nov., sp. nov.: psychrophilic bacteria from Antarctic sea-ice habitats. Int J Syst Bacteriol 1998A, 48:12051212. Bowman JP: Pseudoalteromonas prydzensis sp. nov., a psychrotrophic, halotolerant bacterium from Antarctic sea ice. Int J Syst Bacteriol 1998D, 48:1037-1041. Bowman JP, McCammon SA, Lewis TE, Skerratt JH, Brown JL, Nichols DS, McMeekin TA: Psychroflexus torquis gen. nov., sp. nov., a psychrophilic bacterium from Antarctic Sea ice with the ability to form polyunsaturated fatty acids and the reclassification of Flavobacterium gondwanense Dobson, Franzmann 1993 as Psychroflexus gondwanense gen. nov., comb. nov. Microbiology 1998C, 144:1601-1609. Bozal N, Tudela E, Rossell-Mora R, Lalucat J, Guinea J: Pseudoalteromonas antarctica sp. nov., isolated from an Antarctic coastal environment. Int J Syst Bacteriol 1997, 47:345-351. Buia A, Psychrophilic enzymes of Antarctic sea-ice bacteria, BSc (Hons) Thesis, University of Tasmania, 1997. Bull AT, Goodfellow M and Slater JH. Biodiversity as a Source of Innovation in Biotechnology. Annu Rev Microbiol 1992;46:219-252 Bull A. T., Ward A. C. and Goodfellow M. Search and Discovery Strategies for Biotechnology: the Paradigm Shift Microbiology and Molecular Biology Reviews. 2000 46(3) 573-606

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Feller G, Arpigny JL, Narinx E, Gerday C: Molecular adaptations of enzymes from psychrophilic organisms. Comp Biochem Physiol 1997, 118A:495-499. Feller G, Gerday C: Psychrophilic enzymes - molecular basis of cold adaptation. Cell Mol Life Sci 1997, 53:830-841. Franzmann PD. Liu YT. Balkwill DL. Aldrich HC. Demacario EC. Boone DR. (1997) Methanogenium frigidum sp. nov., a psychrophilic, H-2-using methanogen from ace lake, Antarctica. International Journal of Systematic Bacteriology. 47:1068-1072. Gosink JJ, Herwig RP, Staley JT: Octadecobacter arcticus gen. nov., sp. nov., and O. antarcticus, sp. nov., nonpigmented, psychrophilic gas vacuolate bacteria from polar sea ice and water. Syst Appl Microbiol 1997, 20:356-365. Gosink JJ, Woese CR, Staley JT: Polaribacter gen. nov., with three new species, P. irgensii sp. nov., P. franzmannii sp. nov., and P. filamentus sp. nov., gas vacuolate polar marine bacteria of the Cytophaga-Flavobacterium-Bacteroides group and reclassification of Flectobacillus glomeratus as Polaribacter glomeratus comb. nov. Int J Syst Bacteriol 1998, 48:223-235. Hugenholtz, P., Goebel, B.M. and Pace, N.R. (1998) Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. Journal of Bacteriology, 180, 4765-4774. Kato C, Li L, Nogi Y, Nakamura Y, Tamaoka J, Horikoshi K: Extremely barophilic bacteria isolated from the Mariana Trench, Challenger Deep, at a depth of 11,000 meters. Appl Environ Microbiol 1998, 64:1510-1513. Labrenz M, Collins MD, Lawson PA, Tindall BJ, Braker G, Hirsch P: Antarctobacter heliothermus gen. nov., sp. nov., a budding bacterium from hypersaline and heliothermal Ekho Lake. Int J Syst Bacteriol 1998, 48:13631372. Labrenz M. Collins MD. Lawson PA. Tindall BJ. Schumann P. Hirsch P. (1999) Roseovarius tolerans gen. nov., sp. nov., a budding bacterium with variable bacteriochlorophyll a production from hypersaline Ekho Lake. International Journal of Systematic Bacteriology. 49:137-147. Labrenz M. Tindall BJ. Lawson PA. Collins MD. Schumann P. Hirsch P. (2000) Staleya guttiformis gen. nov., sp nov and Sulfitobacter brevis sp nov., alpha-3Proteobacteria from hypersaline, heliothermal and meromictic Antarctic Ekho Lake. International Journal of Systematic & Evolutionary Microbiology. 50:303-313. Lewis, T.E., Nichols, P.D. and McMeekin, T.A. (1999) The biotechnological

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potential of Thraustochytrids. Marine Biotechnology, 1, 580-587. Love CA, Marshall CJ: Cloning, expression, purification and crystallization of lactate dehydrogenase from Antarctic fish. VII SCAR International Biology Symposium, Christchurch, New Zealand 1998, 23:108.61 Margesin, R. and Schinner, F. (1999) Biotechnological Applications of ColdAdapted Organisms. pp. 338, Springer-Verlag, Berlin. Meyer, B.J., Tsivis, E., Howe, P.R.C., Tapsell, L. and Calvert, G.D. (1999) Polyunsaturated fatty acid content of foods: differentiating between long and short chain onega-3 fatty acids. Food Australia, 51, 82-95. Morita Y, Nakamura T, Hasan Q, Murakami Y, Yokoyama K, Tamiya E: Coldactive enzymes from cold-adapted bacteria. J Am Oil Chem Soc 1997, 74:441444. Mountfort DO, Rainey FA, Burghardt J, Kaspar HF, Stackebrandt E: Psychromonas antarcticus gen. nov., sp. nov., a new aerotolerant anaerobic, halophilic psychrophile isolated from pond sediment of the McMurdo ice shelf, Antarctica. Archives of Microbiology 1998, 169:231-238. Nakasone K, Ikegami A, Kato C, Usami R, Horikoshi K: Mechanisms of gene expression controlled by pressure in deep-sea microorganisms. Extremophiles 1998, 2:149-154. Nichols, D.S., Hart, P., Nichols, P.D. and McMeekin, T.A. (1996) Enrichment of the rotifer Brachionus plicatilis fed an Antarctic bacterium containing polyunsaturated fatty acids. Aquaculture, 147, 115-125. Nichols, D.S., Sanderson, K., Bowman, J., Lewis, T., Mancuso, C.A., McMeekin, T.A. and Nichols, P.D. (1999) Developments with Antarctic microorganisms: PUFA, culture collections, bioactivity screening and cold adapted enzymes. Current Opinions in Biotechnology, 10, 240-246. Nichols, D.S. and McMeekin, T.A. (2001) Biomarker techniques to screen for bacteria that produce polyunsaturated fatty acids. Journal of Microbiological Methods, 1533, (in press). Russell, N.J. (1992) Physiology and molecular biology of psychrophilic microorganisms. In: Herbert, R.A. & Sharpe, R.J. (eds). Molecular Biology and Biotechnology of Extremeophiles, pp. 203-224, Blackie, Glasgow. Russell, N.J. and Nichols, D.S. (1999) Polyunsaturated fatty acids in marine Bacteria - a dogma rewritten. Microbiology, 145, 767-779. Schroder HK, Willassen NP, Smalas AO: Structure of a non-psychrophilic trypsin

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from a cold-adapted fish species. Acta Crystallogr D 1998, 54:780-798. Takahata, K., Monobe, K., Tada, M. and Weber, P.C. (1998) The benefits and risks of n-3 polyunsaturated fatty acids. Bioscience, Biotechnology and Biochemistry, 62, 2079-2085. Takeyama H, Takeda D, Yazawa K, Yamada A, Matsunaga T: Expression of the eicosapentaenoic acid synthesis gene cluster from Shewanella sp. in a transgenic marine cyanobacterium, Synechococcus sp. Microbiology 1997, 143:2725-2731. Takizawa, M., Colwell, R.R. and Hill, R.T. 1993. Isolation and diversity of Actinomycetes in the Chesapeake Bay. Applied and Environmental Microbiology 59:997-1002. Tanaka, M., Ueno, A., Kawasaki, K., Yumoto, I., Ohgiya, S., Hoshino, T., Ishizaki, K. and Morita, N. (1999) Isolation of clustered genes that are notably homologous to the eicosapentaenoic acid biosynthesis gene cluster from the docosahexaenoic acid-producing bacterium Vibrio marinus strain MP-1. Biotechnology Letters, 21, 939-945. Torssell, K.B.G. (1983) Natural Product Chemistry, pp. 401. John Wiley and Sons, New York. Yazawa K: Production of eicosapentaenoic acid from marine bacteria. Lipids 1996, 31:S297-S300.

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Table 1 Recent novel bacterial taxa isolated from Antarctic marine environments. Psychrophilic species are denoted by *
Taxa Antarctobacter heliothermus Colwellia demingiae* Colwellia hornerae * Colwellia rossensis * Colwellia psychrotropica* Gelidibacter algens* Glaciecola punicea * Glaciecola pallidula* Methanogenium frigidum* Methylosphaera hansonii* Octadecobacter antarcticus* Polaribacter franzmannii* Polaribacter irgensii* Polaribacter glomeratus* Polaribacter filamentus* Pseudoalteromonas antarctica Pseudoalteromonas prydzensis Psychrobacter glacincola Psychroflexus torquis* Psychromonas antarcticus* Roseovarius tolerans Psychroserpens burtonensis* Shewanella frigidimarina* Shewanella gelidimarina* Staleya guttiformis Sulfitobacter brevis Isolation saline lake sea ice sea ice sea ice saline lake sea ice sea ice sea ice saline lake saline lake sea ice, seawater sea ice, seawater sea ice, seawater saline lake sea ice, seawater seawater sea ice sea ice, seawater, deep anchor ice sea ice pond mud saline lake saline lake sea ice sea ice saline lake saline lake Reference Labrenz et al. (1998) Bowman et al. (1998b) Bowman et al. (1998b) Bowman et al. (1998b) Bowman et al. (1998b) Bowman et al. (1997b) Bowman et al. (1998a) Bowman et al. (1998a) Framzmann et al. (1997) Bowman et al. (1997d) Gosink et al. (1997) Gosink et al. (1998) Gosink et al. (1998) Gosink et al. (1998) Gosink et al. (1998) Bozal et al. (1997) Bowman et al. (1998d) Bowman et al. (1997c) Bowman et al. (1998c) Mountfort et al. (1998) Labrenz et al. (1999) Bowman et al. (1997b) Bowman et al. (1997a) Bowman et al. (1997a) Labrenz et al. (2000) Labrenz et al. (2000)

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Table 2 The phylogenetic distribution of Actinobacteria (based on preliminary morphological identification) isolated from Antarctic material
Genus or group Number of Isolates Percentage

Streptomyces Unidentified Actinobacteria Nocardioforms Unidentified (non-spore-forming) Micromonospora Actinoplanetes Micropolyspora Maduromycetes Actinomadura Microbispora Nocardia Streptosporangium Actinopolyspora Amorphosporangium Dactylosporangium Elytrosporangium Geodermatophilus Kibdelosporangium Microtetraspora Excellospora Kineosporia Micrococcus Nocardiopsis Pseudonocardia

499 43 40 40 31 30 20 17 9 8 7 5 3 2 2 2 2 2 2 1 1 1 1 1

64.9 5.6 5.2 5.2 4 3.9 2.6 2.2 1.2 1 0.9 0.7 0.4 0.3 0.3 0.3 0.3 0.3 0.3 0.1 0.1 0.1 0.1 0.1

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Table 3 The numbers of novel species (assessed by 16S rDNA sequence) identified from ninety-three Actinobacterial isolates investigated
Genus Number of Novel Species Isolated

Kitasatospora Streptomyces Couchioplanes Nocardia Micromonospora Methylobacterium Terrabacter/Terracoccus Kineococcus Microtetraspora

sp. sp. sp. sp. sp. sp. sp. sp. sp.

7 6 2 2 1 1 1 1 1

Table 4 Optimal temperature and relative activity of cold-active enzymes from a variety of Antarctic sea ice bacteria. Adapted from Buia (1997) and Nichols et al. (1999)
Optimal % activity Temperature (C) 10oC 40oC ______________________________________________________________________________ Colwellia demingae protease (azocasein) 28 75 25 protease (azoalbumin) 30 39 30 trypsin 14 90 29 phosphatase 23 90 85 Colwellia-like strain trypsin 12 100 53 phosphatase 17 85 85 -galactosidase 26 75 70 Cytophaga-like strain protease (azocasein) 20 68 65 protease (azoalbumin) 27 70 55 trypsin 30 72 60 -galactosidase 15 100 46 -amylase 25 65 60 Cytophaga-like strain phosphatase 19 85 85 Pseudoalteromonas sp. protease (azocasein) 29 55 37 trypsin 22 90 23 Shewanella gelidimarina -galactosidase 24 65 < 20 Bacterial strains Enzyme

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Figure 1 Evolutionary distance tree of the bacterial domain (adapted from Hugenholtz et al. 1998). Divisions with cultured representatives are in black while white divisions contain environmental sequences only. The major PUFA producing genera are expanded from the Proteobacteria division (Shewanella, Colwellia and Moritella) together with those from the Cytophagales division (Flexibacter and Psychroflexus). For each genus biomarker fatty acids and the types of PUFA are noted. Abbreviations: PUFA, polyunsaturated fatty acid; AA, arachidonic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid. Adapted from Nichols and McMeekin (2001).

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Pilimelia anulata Pilimelia terevasa Actinoplanes minutisporangius Catellatospora koreensis Catellatospora tsunoense Catellatospora citrea subsp methionotrophica Catellatospora citrea subsp citrea Catellatospora citrea Verrucosispora gifhornensis Actinoplanes ferrugineus Actinoplanes durhamensis Actinoplanes palleronii Actinoplanes utahensis Actinoplanes philippinensis Actinoplanes capillacius Actinoplanes auranticolor Actinoplanes missouriensis Actinoplanes derwentensis Actinoplanes italicus Actinoplanes cyaneus Actinoplanes digitatus Actinoplanes regularis Actinoplanes globisporus Actinoplanes consettensis Actinoplanes humidus Ma10000843 A10000347 A10000349 A10000348 A10000346 A10000316 Couchioplanes caeruleus subsp. azureus Couchioplanes caeruleus (type strain) Couchioplanes caeruleus subsp. caeruleus Actinoplanes deccanensis Actinoplanes brasiliensis Catenuloplanes japonicus Catenuloplanes crispus Spirilliplanes vamanashiensis Micromonospora olivasterospora Micromonospora carbonacea Ma10000913 Ma10000785 Ma10000915 Micromonospora matsumotoense Micromonospora halophytica Micromonospora echinospora Micromonospora echinospora Micromonospora inositola Micromonospora rosaria Micromonospora chersinia Micromonospora echinobrunnea Micromonospora purpureochromogenes Micromonospora halophytica Micromonospora aurantiaca Micromonospora chalcea Micromonospora coerulea Catellatospora ferruginea Dactylosporangium thailandense Dactylosporangium roseum Dactylosporangium fulvum Dactylosporangium salmoneum Dactylosporangium vinaceum Dactylosporangium matsuzakiense Dactylosporangium aurantiacum 0.05

Figure 2 Phylogenetic relationships between novel Antarctic Actinobacteria (bold type: prefix A, Antarctic mainland; Ma, Macquarie Island) and other members of the family Micromonosporineae based on comparison of 16S rDNA sequences. Distances were calculated using maximum likelihood and the tree was constructed using the Fitch Margolish method. The degree of difference between 16S rDNA sequences is shown by branch lengths, the value of which is indicated by the scale.

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S. putrefaciens Owens DNA Group I S. putrefaciens Owens DNA Group II S. oneidensis S. baltica
PUFA [-]

PUFA [+]

S. frigidimarina

S. alga S. amazonensis

S. benthica S. gelidimarina S. hanedai S. woodyi S. pealeana

Antarctic strains 519 & 520

Figure 3 Schematic of the phylogenetic relationship of the genus Shewanella based on 16S rDNA sequence. Species known to produce polyunsaturated fatty acids (PUFA) and their lines of descent are highlighted in bold. Species and lines of descent known not to produce PUFA are highlighted in grey. Arrows indicate sites of divergence where the expression of PUFA synthesis has been lost. Adapted from Russell and Nichols (1999).

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(a)
COOH CH3 (18:33; 18:3n3; 189,12,15) (-linolenic acid, ALA) COOH COOH CH3 (20:53; 20:5n3; 205,8,11,14,17) (eicosapentaenoic acid, EPA) CH3 (22:63; 22:6n3; 224,7,10,13,16,19) (docosahexaenoic acid, DHA) COOH CH3 (18:43; 18:4n3; 186,9,12,15) (stearidonic acid, SA)

(b)
COOH CH3 (18:26; 18:2n6; 189,12) (linoleic acid, LA) (18:36; 18:3n6; 186,9,12) (-linolenic acid, GLA) COOH CH3 (20:46; 20:4n6; 205,8,11,14) (arachidonic acid, AA) COOH CH3

Figure 4 Chemical structures of the common polyunsaturated fatty acids (PUFA) from (a) the 3 family and (b) the 6 family.

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Figure 5
Analysis of open reading frames encoding enzymes involved in polyunsaturated fatty acid biosynthesis in the genera Shewanella and Moritella. Based on the sequences provided by Yazawa (1996) and Tanaka et al. (1999).

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