Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1984 Oct;160(1):239–244. doi: 10.1128/jb.160.1.239-244.1984

Molecular cloning of the hemolysin determinant from Vibrio cholerae El Tor.

S L Goldberg, J R Murphy
PMCID: PMC214707  PMID: 6090415

Abstract

Vibrio cholerae El Tor RV79 is phenotypically nonhemolytic; however, strongly hemolytic convertants are occasionally observed on blood agar plates. We have cloned DNA sequences corresponding to the hemolysin determinant from RV79 (Hly+) in the lambda L47.1 and pBR322 vectors. A 2.3-kilobase fragment of V. cholerae DNA was found to be necessary for hemolytic activity. This cloned DNA sequence was used as a probe in Southern blot hybridization analysis of chromosomal restriction digests of a variety of El Tor and classical biotype V. cholerae strains. In all cases, DNA fragments with the same electrophoretic mobilities hybridized to the Hly probe. The results presented demonstrate that the cloned hemolysin determinant is the hly locus. By using mutator vibriophage VcA-3 insertion to promote high-frequency transfer, the hly locus was mapped between arg and ilv on the V. cholerae RV79 chromosome.

Full text

PDF
239

Images in this article

241Image
on p.241
242Image
on p.242

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barrett T. J., Blake P. A. Epidemiological usefulness of changes in hemolytic activity of Vibrio cholerae biotype El Tor during the seventh pandemic. J Clin Microbiol. 1981 Jan;13(1):126–129. doi: 10.1128/jcm.13.1.126-129.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  3. Brenner D. J., Fanning G. R., Johnson K. E., Citarella R. V., Falkow S. Polynucleotide sequence relationships among members of Enterobacteriaceae. J Bacteriol. 1969 May;98(2):637–650. doi: 10.1128/jb.98.2.637-650.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clarke L., Carbon J. A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome. Cell. 1976 Sep;9(1):91–99. doi: 10.1016/0092-8674(76)90055-6. [DOI] [PubMed] [Google Scholar]
  5. Coleman K., Dougan G., Arbuthnott J. P. Cloning, and expression in Escherichia coli K-12, of the chromosomal hemolysin (phospholipase C) determinant of Pseudomonas aeruginosa. J Bacteriol. 1983 Feb;153(2):909–915. doi: 10.1128/jb.153.2.909-915.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Collins J., Brüning H. J. Plasmids useable as gene-cloning vectors in an in vitro packaging by coliphage lambda: "cosmids". Gene. 1978 Oct;4(2):85–107. doi: 10.1016/0378-1119(78)90023-9. [DOI] [PubMed] [Google Scholar]
  7. DE MOOR C. E. A NON-HAEMOLYTIC EL TOR VIBRIO AS THE CAUSE OF AN OUTBREAK OF PARACHOLERA IN WEST NEW GUINEA. THE EL TOR PROBLEM AND PANDEMIC PARACHOLERA IN THE WEST PACIFIC. Trop Geogr Med. 1963 Jun;15:97–107. [PubMed] [Google Scholar]
  8. Gallut J. La septième pandémie cholérique 1961-1966, 1970. Bull Soc Pathol Exot Filiales. 1971 Sep-Oct;64(5):551–560. [PubMed] [Google Scholar]
  9. Goldberg S., Murphy J. R. Molecular epidemiological studies of United States Gulf Coast Vibrio cholerae strains: integration site of mutator vibriophage VcA-3. Infect Immun. 1983 Oct;42(1):224–230. doi: 10.1128/iai.42.1.224-230.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hohn B., Murray K. Packaging recombinant DNA molecules into bacteriophage particles in vitro. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3259–3263. doi: 10.1073/pnas.74.8.3259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Honda T., Finkelstein R. A. Purification and characterization of a hemolysin produced by Vibrio cholerae biotype El Tor: another toxic substance produced by cholera vibrios. Infect Immun. 1979 Dec;26(3):1020–1027. doi: 10.1128/iai.26.3.1020-1027.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Iwanaga M., Mori K., Kaviti J. N. Vibrio cholerae O1 isolated in Kenya. J Clin Microbiol. 1982 Oct;16(4):742–743. doi: 10.1128/jcm.16.4.742-743.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Johnson S. R., Romig W. R. Transposon-facilitated recombination in Vibrio cholerae. Mol Gen Genet. 1979 Feb 16;170(1):93–101. doi: 10.1007/BF00268584. [DOI] [PubMed] [Google Scholar]
  15. Kaper J. B., Bradford H. B., Roberts N. C., Falkow S. Molecular epidemiology of Vibrio cholerae in the U.S. Gulf Coast. J Clin Microbiol. 1982 Jul;16(1):129–134. doi: 10.1128/jcm.16.1.129-134.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Karn J., Brenner S., Barnett L., Cesareni G. Novel bacteriophage lambda cloning vector. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5172–5176. doi: 10.1073/pnas.77.9.5172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kehoe M., Duncan J., Foster T., Fairweather N., Dougan G. Cloning, expression, and mapping of the Staphylococcus aureus alpha-hemolysin determinant in Escherichia coli K-12. Infect Immun. 1983 Sep;41(3):1105–1111. doi: 10.1128/iai.41.3.1105-1111.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kreft J., Berger H., Härtlein M., Müller B., Weidinger G., Goebel W. Cloning and expression in Escherichia coli and Bacillus subtilis of the hemolysin (cereolysin) determinant from Bacillus cereus. J Bacteriol. 1983 Aug;155(2):681–689. doi: 10.1128/jb.155.2.681-689.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Loenen W. A., Brammar W. J. A bacteriophage lambda vector for cloning large DNA fragments made with several restriction enzymes. Gene. 1980 Aug;10(3):249–259. doi: 10.1016/0378-1119(80)90054-2. [DOI] [PubMed] [Google Scholar]
  20. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  21. Moseley S. L., Falkow S. Nucleotide sequence homology between the heat-labile enterotoxin gene of Escherichia coli and Vibrio cholerae deoxyribonucleic acid. J Bacteriol. 1980 Oct;144(1):444–446. doi: 10.1128/jb.144.1.444-446.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Parker C., Richardson S. H., Romig W. R. Production of Bacteriophage-Associated Materials by Vibrio cholerae: Possible Correlation with Pathogenicity. Infect Immun. 1970 Apr;1(4):417–420. doi: 10.1128/iai.1.4.417-420.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. ROY C., MUKERJEE S. Variability in the haemolytic property of El Tor vibrios. Ann Biochem Exp Med. 1962 Nov;22:295–296. [PubMed] [Google Scholar]
  24. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  25. Rüther U. Construction and properties of a new cloning vehicle, allowing direct screening for recombinant plasmids. Mol Gen Genet. 1980;178(2):475–477. doi: 10.1007/BF00270503. [DOI] [PubMed] [Google Scholar]
  26. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  27. Sporecke I., Castro D., Mekalanos J. J. Genetic mapping of Vibrio cholerae enterotoxin structural genes. J Bacteriol. 1984 Jan;157(1):253–261. doi: 10.1128/jb.157.1.253-261.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wagner W., Vogel M., Goebel W. Transport of hemolysin across the outer membrane of Escherichia coli requires two functions. J Bacteriol. 1983 Apr;154(1):200–210. doi: 10.1128/jb.154.1.200-210.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES