doi:

DOI: 10.3724/SP.J.1085.2013.00223

Advances in Polar Science 2013/24:4 PP.223-230

Transposon mutagenesis of Psychrobacter cryohalolentis PAMC 21807 by tri-parental conjugation


Abstract:
Random mutagenesis is commonly used to study gene function.The screening of mutants exhibiting specific phenotypes assists in the identification of phenotype-related genes.In the current study,we isolated Antarctic bacteria,and developed a transposon Tn5 mutagenesis system.A total of 26 strains were isolated from seawater and freshwater near Antarctic King Sejong Research Station,King George Island.Six Psychrobacter strains were identified as psychrophilic,with optimal growth temperatures of 10℃or 15℃Psychrobacter cryohalolentis PAMC 21807 with a high growth rate at 4℃was selected for transposon mutagenesis.Tri-parental conjugation with a plasmid containing Tn5 produced 13 putative recombinants containing the selectable marker.Genomic Southern hybridization confirmed Tn5 existed as episomes for seven recombinants,and for a single recombinant,Tn5 was integrated into the genome of Psychrobacter cryohalolentis PAMC 21807.The result indicates that the mutagenesis method,although successful,has a relatively low rate.The psychrophilic bacteria isolated in this study may be a useful resource for studying cold adaptation mechanisms,and the mutagenesis method can be applied to genetic analysis.

Key words:cold adaptation,Psychrobacter,psychrophilic bacteria,tri-parental conjugation,transposon mutagenesis

ReleaseDate:2015-04-16 13:27:16



1 Siddiqui K S, Poljak A, Cavicchioli R. Improved activity and stability of alkaline phosphatases from psychrophilic and mesophilic organisms by chemically modifying aliphatic or amino groups using tetracar-boxy-benzophenone derivatives. Cell Mol Biol, 2004, 50(5): 657-667.

2 Wagner D, Lipski A, Embacher A, et al. Methane fluxes in permafrost habitats of the lena delta: Effects of microbial community structure and organic matter quality. Environ Microbiol, 2005, 7: 1582-1592.

3 Chattopadhyay M K. Mechanism of bacterial adaptation to low temperature. JBiosci, 2006, 31(1): 157-165.

4 Fulco A J. The biosynthesis of unsaturated fatty acids by Bacilli. III. Uptake andutilization of exogenous palmitate. J Biol Chem, 1972, 247: 3503-3510.

5 Fulco A J. Metabolic alterations of fatty acids. Annu Rev Biochem, 1974, 43: 215-248.

6 Schindelin H, Marahiel M A, Heinemann U. Universal nucleic acid-binding domain revealed by crystal structure of the B. subtilis major cold-shock protein. Nature, 1993, 364(6433): 164-168.

7 Thieringer H A, Jones P G, Inouye M. Cold shock and adaptation. BioEssays, 1998, 20(1): 49-57.

8 Gilbert J A, Hill P J, Dodd C E R, et al. Demonstration of antifreeze protein activity in Antarctic lake bacteria. Microbiology, 2004, 150(1): 171-180.

9 Bergholz P W, Bakermans C, Tiedje J M. Psychrobacter arcticus 273-4 uses resource efficiency and molecular motion adaptations for subzero temperature growth. J Bacteriol, 2009, 191(7): 2340-2352.

10 Nichols D S, Nichols P D, McMeekin T A. Ecology and physiology of psychrophilic bacteria from Antarctic saline lakes and sea ice. Sci Prog, 1995, 78: 311-347.

11 Bowman J P, Cavanagh J, Austin J J, et al. Novel Psychrobacter species from Antarctic ornithogenic soils. Int J Syst Bacteriol, 1996, 46(4): 841-848.

12 Denner E B, Mark B, Busse H J, et al. Psychrobacter proteolyticus sp. nov., a psychrotrophic, halotolerant bacterium isolated from the Antarctic krill Euphausia superba Dana, excreting a cold画adapted metalloprotease. Syst Appl Microbiol, 2001, 24(1): 44-53.

13 Bakermans C, Ayala-del-Rlo H L, Ponder M A, et al. Psychrobacter cryo-halolentis sp. nov. and Psychrobacter arcticus sp. nov., isolated from Siberian permafrost. Int J Syst Evol Microbiol, 2006, 56(6): 1285-1291.

14 Vishnivetskaya T, Kathariou S, McGrath J, et al. Low-temperature recovery strategies for the isolation of bacteria from ancient permafrost sediments. Extremophiles, 2000, 4(3): 165-173.

15 Shivaji S, Reddy G S N, Suresh K, et al. Psychrobacter vallis sp. nov. and Psychrobacter aquaticus sp. nov., from Antarctica. Int J Syst Evol Microbiol, 2005, 55(2): 757-762.

16 Bowman J P, Nichols D S, McMeekin T A. Psychrobacter glacinciola sp. nov., a halotolerant, psychrophilic bacterium isolated from Antarctic sea ice. Syst Appl Microbiol, 1997, 20(2): 209-215.

17 Bowman J P, McCammon S A, Brown M V, et al. Diversity and association of psychrophilic bacteria in Antarctic sea ice. Appl Environ Microbiol, 1997, 63(3): 3068-3078.

18 Bakermans C, Tollaksen S L, Giometti C S, et al. Proteomic analysis of Psychrobacter cryohalolentis K5 during growth at subzero temperatures. Extremophiles, 2007, 11(2): 343-354.

19 Ayala-del画Rio H L, Chain P S, Grzymski J J, et al. 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(7): 2304-2312.

20 Kim S J, Shin S C, Hong S G, et al. Genome sequence of a novel member of the Genus Psychrobacter isolated from Antarctic Soil. J Bacteriol, 2012, 194(9): 2403.

21 Stretton S, Techkarnjanaruk S, Mclennan A M, et al. Use of green fluorescent protein to tag and investigate gene expression in Marine Bacteria. Appl Environ Microbiol, 1998, 64(7): 2554-2559.

22 Bakermans C, Sloup R E, Zarka D G, et al. Development and use of genetic system to identify genes required for efficient low-temperature growth of Psychrobacter arcticus 273-4. Extremophiles, 2009, 13(1): 21-30.

23 Chun J, Lee J H, Jung Y, et al. EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol, 2007, 57(10): 2259-2261.

24 Felsenstein J. Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol, 1981, 17(6): 368-376.

25 Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol, 1987, 4(4): 406-425.

26 de Lorenzo V, Herrero M, Jakubzik U, et al. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol, 1990, 172(11): 6568-6572.

27 Herrero M, de Lorenzo V, Timmis K N. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes. Gene, 1990, 172(11): 6557-6567.

28 Ditta G, Stanfield S, Corbin D, et al. Broad-host range DNA cloning system for-gram negative bacteria: construction of gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA, 1980, 77(12): 7347-7451.

29 Whitta S, Sinclair M I, Holloway B W. Transposon mutagenesis in Me-thylobacterium AM1 (Pseudomonas AM1). J Gen Microbiol, 1985, 131(6): 1547-1549.

30 Pappas K M, Galani I, Typas M A. Transposon mutagenesis and strain construction in Zymomonas mobilis. Appl Microbiol, 1997, 82(3): 379-388.

31 Widanarni, Suwanto A, Sukenda, et al. Construction of recombinant Vibrio harveyi to study its adherence in Shrimp larvae//Walker P, Lester R, Bondad-Reantaso M G. Diseases in Asian aquaculture, 2005, 5: 465-474.

32 Guiney D G, Lanka E. Conjugative transfer of IncP plasmids//Thomas C M. Promiscuous plasmids of gram-negative bacteria. London: Academic Press, Inc. (London), Ltd., 27-56.

33 Ingraham J L. Growth of psychrophilic bacteria. J Bacteriol, 1958, 76(1): 75-80.