Characterization of single-stranded DNA-binding proteins from the psychrophilic bacteria Desulfotalea psychrophila, Flavobacterium psychrophilum, Psychrobacter arcticus, Psychrobacter cryohalolentis, Psychromonas ingrahamii, Psychroflexus torquis, and Photobacterium profundum

BMC Microbiology

Józef Kur¹  Marta Śpibida¹  Marcin Olszewski¹  Marta Nowak¹ 

[1] Department of Microbiology, Faculty of Chemistry, Gdańsk University of Technology, ul. Narutowicza 11/12, 80-233 Gdańsk, Poland

关键词: Psychrophiles;    Thermostability;    SSB;    Psychrophilic microorganism;    Expression;    DNA replication;   

Others  :  1141442
DOI  :  10.1186/1471-2180-14-91

 received in 2014-02-26, accepted in 2014-03-31,  发布年份 2014

【 摘 要 】

Background

Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination and repair in Bacteria, Archaea and Eukarya. In recent years, there has been an increasing interest in SSBs, since they find numerous applications in diverse molecular biology and analytical methods.

Results

We report the characterization of single-stranded DNA-binding proteins from the psychrophilic bacteria Desulfotalea psychrophila (DpsSSB), Flavobacterium psychrophilum (FpsSSB), Psychrobacter arcticus (ParSSB), Psychrobacter cryohalolentis (PcrSSB), Psychromonas ingrahamii (PinSSB), Photobacterium profundum (PprSSB), and Psychroflexus torquis (PtoSSB). The proteins show a high differential within the molecular mass of their monomers and the length of their amino acid sequences. The high level of identity and similarity in respect to the EcoSSB is related to the OB-fold and some of the last amino acid residues. They are functional as homotetramers, with each monomer encoding one single stranded DNA binding domain (OB-fold). The fluorescence titrations indicated that the length of the ssDNA-binding site size is approximately 30 ± 2 nucleotides for the PinSSB, 31 ± 2 nucleotides for the DpsSSB, and 32 ± 2 nucleotides for the ParSSB, PcrSSB, PprSSB and PtoSSB. They also demonstrated that it is salt independent. However, when the ionic strength was changed from low salt to high, binding-mode transition was observed for the FpsSSB, at 31 ± 2 nucleotides and 45 ± 2 nucleotides, respectively. As expected, the SSB proteins under study cause duplex DNA destabilization. The greatest decrease in duplex DNA melting temperature was observed in the presence of the PtoSSB 17°C. The SSBs in question possess relatively high thermostability for proteins derived from cold-adapted bacteria.

Conclusion

The results showed that SSB proteins from psychrophilic microorganisms are typical bacterial SSBs and possess relatively high thermostability, offering an attractive alternative to other thermostable SSBs in molecular biology applications.

【 授权许可】

   
2014 Nowak et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Greipel J, Urbanke C, Maass G: The single-stranded DNA binding protein of Escherichia coli. Physicochemical properties and biological functions. In Protein-Nucleic Acid Interaction. Edited by Saenger W, Heinemann U. London: Macmillan; 1989:61-86.
• [2]Alani E, Tresher R, Griffith JD, Kolodner RD: Characterization of DNA-binding and strand-exchange stimulation properties of y-RPA, a yeast single-strand-DNA-binding protein. J Mol Biol 1992, 227:54-71.
• [3]Lohman TM, Overman LB: Two binding modes in Escherichia coli single strand binding protein-single stranded DNA complexes. Modulation by NaCl concentration. J Biol Chem 1985, 260:3594-3603.
• [4]Meyer RR, Laine PS: The single-stranded DNA-binding protein of Escherichia coli. Microbiol Rev 1990, 54:342-380.
• [5]Shereda RD, Kozlov AG, Lohman TM, Cox MM, Keck JL: SSB as an organizer/mobilizer of genome maintenance complexes. Crit Rev Biochem Mol 2009, 43:289-318.
• [6]Murzin AG: OB (oligonucleotide/oligosaccharide binding)-fold: common structural and functional solution for non-homologous sequences. EMBO J 1993, 2:861-867.
• [7]Olszewski M, Nowak M, Cyranka-Czaja A, Kur J: Identification and characterization of single-stranded DNA-binding protein from the facultative psychrophilic bacteria Pseudoalteromonas haloplanktis. Microbiol Res 2014, 169:139-147.
• [8]Nogi Y, Masui N, Kato C: Photobacterium profundum sp. nov., a new, moderately barophilic bacterial species isolated from a deep-sea sediment. Extremophiles 1998, 2:1-7.
• [9]Bartlett D, Wright M, Yayanos AA, Silverman M: Isolation of a gene regulated by hydrostatic pressure in a deep-sea bacterium. Nature 1989, 342:572-574.
• [10]Knoblauch C, Sahm K, Jorgensen BB: Psychrophilic sulfate-reducing bacteria isolated from permanently cold Arctic marine sediments description of Desulfofrigus oceanense gen. nov., sp. nov., Desulfofrigus fragile sp. nov., Desulfofaba gelida gen. nov., sp. nov., Desulfotalea psychrophila gen. nov., sp. nov. and Desulfotalea arctica sp. nov. Int J Syst Bacteriol 1999, 49:1631-1643.
• [11]Alvarez B, Secades P, McBride M, Guijarro J: Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum. Appl Envir Microb 2004, 70:581-587.
• [12]Bakermans C, Ayala-del-Rio HL, Ponder MA, Vishnivetskaya T, Gilichinsky D, Thomashow MF, Tiedje JM: Psychrobacter cryohalolentis sp. nov. and Psychrobacter arcticus sp. nov., isolated from Siberian permafrost. Int J Syst Evol Microbiol 2006, 56:1285-1291.
• [13]Bergholz PW, Bakermans C, Tiedje JM: Psychrobacter arcticus 273–4 Uses resource efficiency and molecular motion adaptations for subzero temperature growth. J Bacteriol 2009, 191:2340-2352.
• [14]Auman AJ, Breezee JL, Gosink JJ, Kämpfer P, Staley JT: Psychromonas ingrahamii sp. nov., a novel gas vacuolate, psychrophilic bacterium isolated from Arctic polar sea ice. Int J Syst Evol Microbiol 2006, 56:1001-1007.
• [15]Bowman JP, McCammon SA, Lewis T, Skerratt JH, Brown JL, Nichols DS, McMeekin TA: Psychroflexus torquis gen. nov., sp. nov., a psychrophilic species from Antarctic sea ice, and reclassification of Flavobacterium gondwanense (Dobson et al. 1993) as Psychroflexus gondwanense gen. nov., comb. nov. Microbiology 1998, 144:1601-1609.
• [16]Rabus R, Ruepp A, Frickey T, Rattei T, Fartmann B, Stark M, Bauer M, Zibat A, Lombardot T, Becker I, Amann J, Gellner K, Teeling H, Leuschner WD, Glockner F-O, Lupas AN, Amann R, Klenk H-P: The genome of Desulfotalea psychrophila, a sulfate-reducing bacterium from permanently cold Arctic sediments. Environ Microbiol 2004, 6:887-902.
• [17]Duchaud E, Boussaha M, Loux V, Bernardet JF, Michel C, Kerouault B, Mondot S, Bossy R, Caron C, Bessieres P, Gibrat JF, Dumetz F, Le Henaff M, Benmansour A: Complete genome sequence of the fish pathogen Flavobacterium psychrophilum. Nat Biotech 2007, 25:763-769.
• [18]Ayala-del-Rio HL, Chain PS, Grzymski JJ, Ponder MA, Ivanova N, Bergholz PW, Di Bartolo G, Hauser L, Land M, Bakermans C, Rodrigues D, Klappenbach J, Zarka D, Larimer F, Richardson P, Murray A, Thomashow M, Tiedje JM: The genome sequence of Psychrobacter arcticus 273–4, a psychroactive Siberian permafrost bacterium reveals mechanisms for adaptation to low temperature growth. Appl Environ Microbiol 2010, 76:2304-2312.
• [19]Riley M, Staley JT, Danchin A, Wang TZ, Brettin TS, Hauser LJ, Land ML, Thompson LS: Genomics of an extreme psychrophile, Psychromonas ingrahamii. BMC Genomics 2008, 9:210. BioMed Central Full Text
• [20]Vezzi A, Campanaro S, D'Angelo M, Simonato F, Vitulo N, Lauro FM, Cestaro A, Malacrida G, Simionati B, Cannata N, Romualdi C, Bartlett DH, Valle G: Life at depth: Photobacterium profundum genome sequence and expression analysis. Science 2005, 307:1459-1461.
• [21]Lindner C, Nijland R, van Hartskamp M, Bron S, Hamoen LW, Kuipers OP: Differential expression of two paralogous genes of Bacillus subtilis encoding single-stranded DNA binding protein. J Bacteriol 2004, 186:1097-1105.
• [22]Makemson JC, Fulayfil NR, Landry W, Van Ert LM, Wimpee CF, Widder EA, Case JF: Shewanella woodyi sp. nov., an exclusively respiratory luminous bacterium isolated from the Alboran Sea. Int J Syst Bacteriol 1997, 47:1034-1039.
• [23]Riley M, Abe T, Arnaud MB, Berlyn MK, Blattner FR, Chaudhuri RR, Glasner JD, Horiuchi T, Keseler IM, Kosuge T, Mori H, Perna NT, Plunkett G 3rd, Rudd KE, Serres MH, Thomas GH, Thomson NR, Wishart D, Wanner BL: Escherichia coli K-12: a cooperatively developed annotation snapshot--2005. Nucleic Acids Res 2006, 34:1-9.
• [24]Barbe V, Cruveiller S, Kunst F, Lenoble P, Meurice G, Sekowska A, Vallenet D, Wang T, Moszer I, Médigue C, Danchin A: From a consortium sequence to a unified sequence: the Bacillus subtilis 168 reference genome a decade later. Microbiology 2009, 155:1758-1775.
• [25]Bao Q, Tian Y, Li W, Xu Z, Xuan Z, Hu S, Dong W, Yang J, Chen Y, Xue Y, Xu Y, Lai X, Huang L, Dong X, Ma Y, Ling L, Tan H, Chen R, Wang J, Yu J, Yang H: A complete sequence of the T. tengcongensis genome. Genome Res 2002, 12:689-700.
• [26]Nelson KE, Clayton RA, Gill SR, Gwinn ML, Dodson RJ, Haft DH, Hickey EK, Peterson JD, Nelson WC, Ketchum KA, McDonald L, Utterback TR, Malek JA, Linher KD, Garrett MM, Stewart AM, Cotton MD, Pratt MS, Phillips CA, Richardson D, Heidelberg J, Sutton GG, Fleischmann RD, Eisen JA, White O, Salzberg SL, Smith HO, Venter JC, Fraser CM: Evidence for lateral gene transfer between Archaea and bacteria from genome sequence of Thermotoga maritima. Nature 1999, 399:323-329.
• [27]Chilukuri LN, Bartlett DH: Isolation and characterization of the gene encoding single-stranded-DNA-binding protein (SSB) from four marine Shewanella strains that differ in their temperature and pressure optima for growth. Microbiology 1997, 143:1163-1174.
• [28]Olszewski M, Grot A, Wojciechowski M, Nowak M, Mickiewicz M, Kur J: Characterization of exceptionally thermostable single-stranded DNA-binding proteins from Thermotoga maritima and Thermotoga neapolitana. BMC Microbiology 2010, 10:260. BioMed Central Full Text
• [29]Feller G, Arpigny JL, Narinx E, Gerday C: Molecular adaptations of enzymes from psychrophilic organisms. Comp Biochem Phys A 1997, 118:495-499.
• [30]Feller G, Payan F, Theys F, Qian M, Haser R, Gerday C: Stability and structural analysis of alpha-amylase from the antarctic psychrophile Alteromonas haloplanctis A23. Eur J Biochem 1994, 222:441-447.
• [31]Feller G, Thiry M, Gerday C: Nucleotide sequence of the lipase gene lip2 from the antarctic psychrotroph Moraxella TA144 and site-specific mutagenesis of the conserved serine and histidine residues. DNA Cell Biol 1991, 10:381-388.
• [32]Feller G, Gerday C: Psychrophilic enzymes: molecular basis of cold adaptation. Cell Mol Life Sci 1997, 53:830-841.
• [33]Van Petegem F, Collins T, Meuwis MA, Gerday C, Feller G, Van Beeumen J: The structure of a cold-adapted family 8 xylanase at 1.3 A resolution: structural adaptations to cold and investigation of the active site. J Biol Chem 2003, 278:7531-7539.
• [34]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: Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol 2000, 18:103-107.
• [35]Russell NJ: Toward a molecular understanding of cold activity of enzymes from psychrophiles. Extremophiles 2000, 4:83-90.
• [36]Matthews BW, Nicholson H, Becktel WJ: Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc Natl Acad Sci USA 1987, 84:6663-6667.
• [37]Korolev S, Nayal M, Barnes WM, Di Cera E, Waksman G: Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5-A resolution: structural basis for thermostability. Proc Natl Acad Sci USA 1995, 92:9264-9268.
• [38]Zuber H: Temperature adaptation of lactate dehydrogenase. Structural, functional and genetic aspects. Biophys Chem 1988, 29:171-179.
• [39]Metpally RPR, Reddy BVB: Comparative proteome analysis of psychrophilic versus mesophilic bacterial species: Insights into the molecular basis of cold adaptation of proteins. BMC Genomics 2009, 10:11. BioMed Central Full Text
• [40]Williams KR, Murphy JB, Chase JW: Characterization of the structural and functional defect in the Escherichia coli single-stranded DNA binding protein encoded by the ssb-1 mutant gene. Expression of the ssb-1 gene under lambda pL regulation. J Biol Chem 1984, 259:11804-11811.
• [41]Genschel J, Litz L, Thole H, Roemling U, Urbanke C: Isolation, sequencing and overproduction of the single-stranded DNA binding protein from Pseudomonas aeruginosa PAO. Gene 1996, 182:137-143.
• [42]Dabrowski S, Olszewski M, Piatek R, Brillowska-Dabrowska A, Konopa G, Kur J: Identification and characterization of single-stranded-DNA-binding proteins from Thermus thermophilus and Thermus aquaticus - new arrangement of binding domains. Microbiology 2002, 148:3307-3315.
• [43]Dabrowski S, Kur J: Cloning, overexpression, and purification of the recombinant His-tagged SSB protein of Escherichia coli and use in polymerase chain reaction amplification. Protein Expr Purif 1999, 16:96-102.
• [44]Curth U, Greipel J, Urbanke C, Maass G: Multiple binding modes of the single-stranded DNA binding protein from Escherichia coli as detected by tryptophan fluorescence and site-directed mutagenesis. Biochemistry 1993, 32:2585-2591.
• [45]Schwarz G, Watanabe F: Thermodynamics and kinetics of co-operative protein-nucleic acid binding. I. General aspects of analysis of data. J Mol Biol 1983, 163:467-484.
• [46]Augustyns K, Van Aerschot A, Van Schepdael A, Urbanke C, Herdewijn P: Influence of the incorporation of (S)-9-(3,4-dihydroxybutyl)adenine on the enzymatic stability and base-pairing properties of oligodeoxynucleotides. Nucleic Acids Res 1991, 19:2587-2593.