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Diversity and cold-active hydrolytic enzymes of culturable bacteria associated with Arctic sea ice, Spitzbergen

Abstract

The diversity of culturable bacteria associated with sea ice from four permanently cold fjords of Spitzbergen, Arctic Ocean, was investigated. A total of 116 psychrophilic and psychrotolerant strains were isolated under aerobic conditions at 4°C. The isolates were grouped using amplified rDNA restriction analysis fingerprinting and identified by partial sequencing of 16S rRNA gene. The bacterial isolates fell in five phylogenetic groups: subclasses α and γ of Proteobacteria, the BacillusClostridium group, the order Actinomycetales, and the Cytophaga–Flexibacter–Bacteroides (CFB) phylum. Over 70% of the isolates were affiliated with the Proteobacteria γ subclass. Based on phylogenetic analysis (<98% sequence similarity), over 40% of Arctic isolates represent potentially novel species or genera. Most of the isolates were psychrotolerant and grew optimally between 20 and 25°C. Only a few strains were psychrophilic, with an optimal growth at 10–15°C. The majority of the bacterial strains were able to secrete a broad range of cold-active hydrolytic enzymes into the medium at a cultivation temperature of 4°C. The isolates that are able to degrade proteins (skim milk, casein), lipids (olive oil), and polysaccharides (starch, pectin) account for, respectively, 56, 31, and 21% of sea-ice and seawater strains. The temperature dependences for enzyme production during growth and enzymatic activity were determined for two selected enzymes, α-amylase and β-galactosidase. Interestingly, high levels of enzyme productions were measured at growth temperatures between 4 and 10°C, and almost no production was detected at higher temperatures (20–30°C). Catalytic activity was detected even below the freezing point of water (at −5°C), demonstrating the unique properties of these enzymes.

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References

  1. Abyzov SS, Mitskevich IN, Poglasova MN (1998) Microflora of the deep glacier horisonts of central Africa. Microbiology 67:451–458

    CAS  Google Scholar 

  2. Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cell without cultivation. Microbiol Rev 59:143–169

    CAS  PubMed  Google Scholar 

  3. Bano N, Hollibaugh JT (2002) Phylogenetic composition of bacterioplankton assemblages from the Arctic Ocean. Appl Environ Microbiol 68:505–518

    Article  CAS  PubMed  Google Scholar 

  4. Barbaro SE, Trevors JT, Inniss WE (2001) Effects of low temperature, cold shock, and various carbon sources on esterase and lipase activities and exopolysaccharide production by a psychrotrophic Acinetobacter sp. Can J Microbiol 47:194–205

    Article  CAS  PubMed  Google Scholar 

  5. Bernfeld P (1955) Amylases α and β. Methods Enzymol 1:149–155

    Article  Google Scholar 

  6. Bianchi A, Giuliano L (1996) Enumeration of viable bacteria in marine pelagic environment. Appl Environ Microbiol 62:174–177

    CAS  Google Scholar 

  7. Bowman JP, McCammon SA, Brown JL, Nichols PD, McMeekin TA (1997a) Psychroserpens burtonensis gen. nov., sp. nov., and Gelidibacter algens gen. nov., sp. nov., psychrophilic bacteria isolated from Antarctic lacustrine and sea ice habitats. Int J Syst Bacteriol 47:670–677

    CAS  PubMed  Google Scholar 

  8. Bowman JP, McCammon SA, Brown MV, Nichols DS, McMeekin TA (1997b) Diversity and association of psychrophilic bacteria in Antarctic sea ice. Appl Environ Microbiol 63:3068–3078

    CAS  PubMed  Google Scholar 

  9. Bowman JP, Rea SM, McCammon SA, McMeekin TA (2000) Diversity and community structure within anoxic sediment from marine salinity meromictic lakes and a coastal meromictic marine basin, Vestfold Hilds, Eastern Antarctica. Environ Microbiol 2:227–237

    Article  CAS  PubMed  Google Scholar 

  10. Brambilla E, Hippe H, Hagelstein A, Tindall BJ, Stackebrandt E (2001) 16S rDNA diversity of cultured and uncultured prokaryotes of a mat sample from Lake Fryxell, McMurdo Dry Valleys, Antarctica. Extremophiles 5:23–33

    Article  CAS  PubMed  Google Scholar 

  11. Brown MV, Bowman JP (2001) A molecular phylogenetic survey of sea-ice microbial communities (SIMCO). FEMS Microbiol Ecol 35:267–275

    Article  CAS  PubMed  Google Scholar 

  12. Buchholz-Cleven BEE, Rattunde B, Straub KL (1997) Screening for genetic diversity of isolates of anaerobic Fe(II)-oxidizing bacteria using DGGE and whole-cell hybridization. Syst Appl Microbiol 20:301–309

    Google Scholar 

  13. Buchon L, Laurent P, Gounot AM, Guespin-Michel JF (2000) Temperature dependence of extracellular enzymes production by psychrotrophic and psychrophilic bacteria. Biotech Lett 22:1577–1581

    Article  CAS  Google Scholar 

  14. Chrost RJ (1991) Environmental control of the synthesis and activity of aquatic microbial ectoenzymes, In: Chrost RJ (ed) Microbial enzymes in aquatic environments. Springer, Berlin Heidelberg New York, pp 60–83

    Google Scholar 

  15. Cleveland TE, Cotty PJ (1991) Invasiveness of Aspergillus flavus isolates in wounded cotton bolls is associated with production of a specific fungal polygalacturonase. Phytopathology 81:155–158

    CAS  Google Scholar 

  16. DeLong EF, Wu KY, Prezelin BB, Jovine RV (1994) High abundance of Archaea in Antarctic marine picoplankton. Nature 371:695–697

    Article  CAS  PubMed  Google Scholar 

  17. Eilers H, Pernthaler J, Amann R (2000) Succession of pelagic marine bacteria during enrichment: a close look at cultivation-induced shifts. Appl Environ Microbiol 66:4634–4640

    Article  CAS  PubMed  Google Scholar 

  18. Feller G, Gerday C (1997) Psychrophilic enzymes: molecular basis of cold adaptation. Cell Mol Life Sci 53:830–841

    Article  CAS  PubMed  Google Scholar 

  19. Feller G, Lonhienne T, Deroanne C, Libioulle C, Van Beeumen J, Gerday C (1992) Purification, characterization, and nucleotide sequence of the thermolabile alpha-amylase from the Antarctic psychrotroph Alteromonas haloplanctis A23. J Biol Chem 267:5217–5221

    CAS  PubMed  Google Scholar 

  20. Felsenstein J (1993) PHYLIP (PHYlogenetic Inference Package) version 3.57c. Department of Genetics, University of Washington, Seattle

  21. Fridmann EI (1980) Endolithic microbial life in hot and cold deserts. Orig Life 10:223–235

    PubMed  Google Scholar 

  22. Gerday C, Aittaleb M, Bentahir M, Chessa JP, Claverie P, Collins T, D’Amico S, Dumont J, Garsoux G, Georlette D, Hoyoux A, Lonhienne T, Meuwis MA, Feller G (2000) Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol 18:103–107

    Article  CAS  PubMed  Google Scholar 

  23. Giuliano L, De Domenico M, De Domenico E, Hofle MG, Yakimov MM (1999) Identification of culturable oligotrophic bacteria within naturally occurring bacterioplankton communities of the Ligurian sea by 16S rRNA sequencing and probing. Microb Ecol 37:77–85

    Article  CAS  PubMed  Google Scholar 

  24. Gugi B, Orange N, Hellio F, Burini JF, Guillou C, Leriche F, Guespin-Michel JF (1991) Effect of growth temperature on several exported enzyme activities in the psychrotrophic bacterium Pseudomonas fluorescens. J Bacteriol 173:3814–3820

    CAS  PubMed  Google Scholar 

  25. Helmke E, Weyland H (1995) Bacteria in sea ice and underlying water of the eastern Weddell Sea in midwinter. Mar Ecol Prog Ser 117:269–288

    Google Scholar 

  26. Hiraishi A, Ueda Y (1994) Intragenetic structure of the genus Rhodobacter: transfer of Rhodobacter sulfidophilus and related marine species to the genus Rhodovulum gen. nov. Int J Syst Bacteriol 44:15–23

    Google Scholar 

  27. Hoppe HG (1991) Microbial extracellular enzyme activity: a new key parameter in aquatic ecology. In: Chrost RJ (ed) Microbial enzymes in aquatic environments. Springer, Berlin Heidelberg New York, pp 60–83

    Google Scholar 

  28. Huston AL, Krieger-Brockett BB, Deming JW (2000) Remarkably low temperature optima for extracellular enzyme activity from Arctic bacteria and sea ice. Environ Microbiol 2:383–388

    Article  CAS  PubMed  Google Scholar 

  29. Jorgensen S, Vorgias CE, Antranikian G (1997) Cloning, sequencing, characterization, and expression of an extracellular alpha-amylase from the hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli and Bacillus subtilis. J Biol Chem 272:16335–16342

    Article  CAS  PubMed  Google Scholar 

  30. Junge K, Imhoff F, Staley T, Deming JW (2002) Phylogenetic diversity of numerically important Arctic sea-ice bacteria cultured at subzero temperature. Microb Ecol 43:315–328

    Article  CAS  PubMed  Google Scholar 

  31. Knoblauch C, Jorgensen BB (1999) Effect of temperature on sulphate reduction, growth rate and growth yield in five psychrophilic sulphate-reducing bacteria from Arctic sediments. Environ Microbiol 1:457–467

    Article  CAS  PubMed  Google Scholar 

  32. Knoblauch C, Jorgensen BB, Harder J (1999) Community size and metabolic rates of psychrophilic sulfate-reducing bacteria in Arctic marine sediments. Appl Environ Microbiol 65:4230–4233

    CAS  PubMed  Google Scholar 

  33. Kottmeier ST, Sullivan CW (1990) Bacterial biomass and production in pack ice of Antarctic marginal ice age zones. Deep-Sea Res 37:1311–1330

    Google Scholar 

  34. Kouker G, Jaeger KE (1987) Specific and sensitive plate assay for bacterial lipases. Appl Environ Microbiol 53:211–213

    CAS  PubMed  Google Scholar 

  35. Massana RT, Murray AE, Wu KY, Jeffrey WH, DeLong E (1998) Vertical distribution and temporal variation of marine planktonic archaea in the Gerlache Strain, Antarctica, during early spring. Limnol Oceanogr 43:607–617

    CAS  Google Scholar 

  36. Morita RY (1975) Psychrophilic bacteria. Bacteriol Rev 39:144–167

    CAS  PubMed  Google Scholar 

  37. Mountfort DO, Rainey FA, Burghardt J, Kaspar HF, Stackebrandt E (1998) Psychromonas antarcticus gen. nov., sp. nov., A new aerotolerant anaerobic, halophilic psychrophile isolated from pond sediment of the McMurdo Ice Shelf, Antarctica. Arch Microbiol 169:231–238

    Article  PubMed  Google Scholar 

  38. Palmisano AC, Garrison DL (1993) Microorganisms in Antarctic sea ice. In: Friedmann EI (ed) Antarctic microbiology. Wiley, New York, pp 167–219

    Google Scholar 

  39. Petri R, Imhoff JF (2001) Genetic analysis of sea ice bacterial communities of western Baltic Sea using an improved double gradient method. Polar Biol 24:252–257

    Article  Google Scholar 

  40. Priscu JC, Fritsen CH, Adams EE, Giovannoni SJ, Paerl HW, McKay CP, Doran PT, Gordon DA, Lanoil BD, Pinckney JL (1998) Perennial Antarctic lake ice: an oasis for life in a polar desert. Science 280:2095–2098

    Article  CAS  PubMed  Google Scholar 

  41. Ravenschlag K, Sahm K, Pernthaler J, Amann R (1999) High bacterial diversity in permanently cold marine sediments. Appl Environ Microbiol 65:3982–3989

    CAS  PubMed  Google Scholar 

  42. Reichardt W (1988) Impact of the Antarctic Benthic fauna on the enrichment of biopolymer degrading psychrophilic bacteria. Microb Ecol 15:311–321

    Google Scholar 

  43. Riemann L, Steward GF, Azam F (2000) Dynamics of bacterial community composition and activity during a mesocosm diatom bloom. Appl Environ Microbiol 66:578–587

    Article  CAS  PubMed  Google Scholar 

  44. Rivkin RB, Putt M, Alexander SP, Meritt D, Gaudet L (1989) Biomass and production in polar planktonic and sea ice microbial communities: a comparative study. Mar Biol 101:273–283

    CAS  Google Scholar 

  45. Russell NJ (2000) Toward a molecular understanding of cold activity of enzymes from psychrophiles. Extremophiles 4:83–90

    Article  CAS  PubMed  Google Scholar 

  46. Sahm K, Knoblauch C, Amann R (1999) Phylogenetic affiliation and quantification of psychrophilic sulfate-reducing isolates in marine Arctic sediments. Appl Environ Microbiol 65:3976–3981

    CAS  PubMed  Google Scholar 

  47. Schafer H, Servais P, Muyzer G (2000) Successional changes in the genetic diversity of a marine bacterial assemblage during confinement. Arch Microbiol 173:138–145

    Article  CAS  PubMed  Google Scholar 

  48. Schinner F, Margesin R, Pümpel T (1992) Extracellular protease-producing psychrotrophic bacteria from high Alpine habitats. Arctic Alpine Res 24:88–92

    Google Scholar 

  49. Smilbert RM (1994) Phenotypic characterization. In: Gerhardt P, Murray REG, Wood WA, Krieg NR (eds) Methods for general and molecular microbiology. American Society for Microbiology, Washington, pp 611–654

    Google Scholar 

  50. Smith MC, Bowman JP, Scott FJ, Line MA (2000) Sublithic bacteria associated with Antarctic quartz stones. Antarctic Sci 12:177–184

    Google Scholar 

  51. Staley JT, Gosink JJ (1999) Poles apart: biodiversity and biogeography of sea ice bacteria. Annu Rev Microbiol 53:189–215

    Article  CAS  PubMed  Google Scholar 

  52. Yumoto I, Kawasaki K, Iwata H, Matsuyama H, Okuyama H (1998) Assignment of Vibrio sp. strain ABE-1 to Colwellia maris sp. nov., a new psychrophilic bacterium. Int J Syst Bacteriol 48:1357–1362

    CAS  PubMed  Google Scholar 

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Acknowledgements

The authors are grateful to the Deutsche Bundesstiftung Umwelt and “Fonds der Chemischen Industrie” for financial support.

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Correspondence to Garabed Antranikian.

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Communicated by K. Horikoshi

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Groudieva, T., Kambourova, M., Yusef, H. et al. Diversity and cold-active hydrolytic enzymes of culturable bacteria associated with Arctic sea ice, Spitzbergen. Extremophiles 8, 475–488 (2004). https://doi.org/10.1007/s00792-004-0409-0

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Keywords

  • Arctic
  • Cold-active hydrolytic enzymes
  • Psychrotolerant bacteria
  • Sea ice