1887

Abstract

We have established a reliable procedure for electroporation in the marine bacterium . Plasmids carrying the P15A replicon were found to be stably maintained in the cells, and chloramphenicol, kanamycin or tetracycline were used for selection. Since we found that the cells excrete DNase into the culture medium, cells were subjected to osmotic shock before extensive washing in order to remove the DNase from the periplasmic space. This manipulation resulted in about a 10-fold increase in the efficiency of transformation. In addition, cells were washed in the presence of 5-10 mM Mgin order to stabilize the outer membrane. The efficiency of transformation was found to be optimal when cells were harvested at early stationary phase, and when electroporation was carried out at an electric field strength between 5-0 and 7.5 kV cm. Under optimal conditions, about 10transformants per μg of input DNA were reproducibly obtained, which is tolerable for cloning.

Loading

Article metrics loading...

/content/journal/micro/10.1099/13500872-140-9-2355
1994-09-01
2024-04-23
Loading full text...

Full text loading...

/deliver/fulltext/micro/140/9/mic-140-9-2355.html?itemId=/content/journal/micro/10.1099/13500872-140-9-2355&mimeType=html&fmt=ahah

References

  1. Allen R.D., Baumann P. 1971; Structure and arrangement of flagella in species of the genus Beneckea and Photobacterium fischeri. J Bacteriol 107:295–302
    [Google Scholar]
  2. Allison C., Hughes C. 1991; Bacterial swarming: an example of prokaryotic differentiation and multicellular behaviour. Sci Progl S403–422
    [Google Scholar]
  3. Atsumi T., McCarter L., Imae Y. 1992; Polar and lateral flagellar motors of marine Vibrio are driven by different ion-motive forces. Nature 355:182–184
    [Google Scholar]
  4. Bartolome B., Jubete Y., Martinez E., Cruz F. D. 1991; Construction and properties of a family of pACYC184-derived cloning vectors compatible with pBR322 and its derivatives. Gene 102:75–78
    [Google Scholar]
  5. Belas R., Mileham A., Simon M., Silverman M. 1984; Transposon mutagenesis of marine Vibrio spp. J Bacteriol 158:890–896
    [Google Scholar]
  6. Belas R., Mileham A., Cohn D., Hilmen M., Simon M., Silverman M. 1986; Bacterial bioluminescence: isolation and expression of the luciferase genes from Vibrio harveyi. Science 218:791–793
    [Google Scholar]
  7. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. 1977; Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113
    [Google Scholar]
  8. Chang A.C.Y., Cohen S. N. 1978; Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156
    [Google Scholar]
  9. Dower W. J., Miller J. F., Ragsdale C. W. 1988; High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16:6127–6145
    [Google Scholar]
  10. Focareta T., Manning P. A. 1987; Extracellular proteins of Vibrio cholerae: molecular cloning, nucleotide sequence and characterization of the deoxyribonuclease (DNase) together with its periplasmic localization in Escherichia coli. Gene 53:31–40
    [Google Scholar]
  11. Grant S. G., Lessee J., Bloom F. R., Hanahan D. 1990; Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci USA 87:4645–4649
    [Google Scholar]
  12. Hamashima H., Nakao T., Tamura S., Arai T. 1990; Genetic transformation of Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio cholerae non-01 with plasmid DNA by electroporation.. Microbiol Immunol 34:703–708
    [Google Scholar]
  13. Henrichsen J. 1972; Bacterial surface translocation: a survey and a classification. Bacteriol Rep 36:478–503
    [Google Scholar]
  14. Imae Y., Atsumi T. 1989; Na+-driven bacteria flagellar motors.. J Bioenerg Biomembr 21:705–716
    [Google Scholar]
  15. Kaneko T., Colwell R. T. 1973; Ecology of 17brio parahaemolyticus in Chesapeake Bay. J Bacteriol 113:24–32
    [Google Scholar]
  16. Marcus H., Ketley J. M., Kaper J. B., Holmes R. H. 1990; Effects of DNase production, plasmid size, and restriction barriers on transformation of Vibrio cholerae by electroporation and osmotic shock.. FEMS Microbiol Eett 68:149–154
    [Google Scholar]
  17. McCarter L.L., Silverman M. 1987; Phosphate regulation of gene expression in Vibrio parahaemolyticus. J Bacteriol 169:3441–3449
    [Google Scholar]
  18. McCarter L.L., Silverman M. 1990; Surface-induced swarmer cell differentiation of Vibrio parahaemolyticus. Mol Microbiol 4:1057–1062
    [Google Scholar]
  19. McCarter L., Hilmen M., Silverman M. 1988; Flagellar dynamometer controls swarmer cell differentiation of V. parahaemolyticus. Cell 54:345–351
    [Google Scholar]
  20. Muramatsu K., Matsumoto H. 1991; Two generalized transducing phages in V. parahaemolyticus and V. alginolyticus.. Microbiol Immunol 35:1073–1084
    [Google Scholar]
  21. Neu H.C., Heppel L. A. 1965; The release of enzyme from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem 240:3685–3692
    [Google Scholar]
  22. Newland J. W., Green B. A., Foulds J., Holmes R. K. 1985; Cloning of extracellular DNase and construction of a DNase negative strain of Vibrio cholerae.. Infect Immun 47:691–696
    [Google Scholar]
  23. Sambrook J., Fritsch E. F., Maniatis T. 1987 Molecular Cloning: a Laboratory Manual, 2 nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  24. Shinoda S., Okamoto K. 1977; Formation and function of Vibrio parahaemolyticus lateral flagella. J Bacteriol 129:1266–1271
    [Google Scholar]
  25. Silhavy T. J., Berman M. L., Enquist L. W. 1984 Experiments with Gene Fusions. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  26. Takeshita S., Sato M., Masahashi W., Hashimoto-Gotoh T. 1987; High -copy-number and low-copy-number plasmid vector for lac Zα-complementation and chloramphenicol or kanamvcin resistance selection. Gene 61:63–74
    [Google Scholar]
  27. Tokuda H. 1986; Sodium translocation by NADH oxidase of Vibrio alginolyticus: isolation and characterization of the sodium pump-defective mutants. Methods Enyymol 125:520–530
    [Google Scholar]
  28. Tokuda H. 1989; Respiratory Na+ pump and Na+-dependent energetics in Vibrio alginolyticus.. J Bioenerg Biomembr 21:693–704
    [Google Scholar]
  29. Tokudo H., Unemoto T. 1982; Characterization of the respiration-dependent Na+ pump in the marine bacterium Vibrio alginolyticus. J Biol Chem 257:10007–10014
    [Google Scholar]
  30. Tokuda H., Nakamura T., Unemoto T. 1981; Potassium ion is required for the generation of pH-dependent membrane potential and ApH by the marine bacterium Vibrio alginolyticus.. Biochemistry 20:4198–4203
    [Google Scholar]
  31. Tokuda H., Asano M., Shimamura Y., Unemoto T., Sugiyama S., Imae Y. 1988; Roles of the respiratory Na+ pump in bioenergetics of Vibrio alginolyticus.. J Biochem 103:650–655
    [Google Scholar]
  32. Tsuchiya T., Shinoda S. 1985; Respiration-driven Na+ pump and Na+ circulation in Vibrio parahaemolyticus. J Bacteriol 162:794–798
    [Google Scholar]
  33. Unemoto T., Hayashi M. 1989; Sodium-transport NADH-quinone reductase of a marine l^ibrio alginolyticus.. J Bioenerg Biomembr 21:649–662
    [Google Scholar]
  34. Unemoto T., MacLeod R.A. 1975; Capacity of the outer membrane of a gram-negative marine bacterium in the presence of cations to prevent lysis by Triton X-100. J Bacteriol 121:800–806
    [Google Scholar]
  35. Unemoto T., Tsuruoka T., Hayashi M. 1973; Role of Na+ and K+ in preventing lysis of a slightly halophilic 17brio alginolyticus.. Can J Microbiol 19:563–571
    [Google Scholar]
  36. Vieira J., Messing J. 1982; The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
    [Google Scholar]
  37. Vieira J., Vieira J., Messing J. 1991; New pUC-derived cloning vectors with different selectable markers and DNA replicon origins. Gene 100:189–194
    [Google Scholar]
  38. Yu C., Lee A. M., Bassler B. L., Roseman S. 1991; Chitin utilization by marine bacteria. J Biol Chem 266:24260–24267
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-140-9-2355
Loading
/content/journal/micro/10.1099/13500872-140-9-2355
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error