Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Feb;173(4):1561–1564. doi: 10.1128/jb.173.4.1561-1564.1991

A novel approach to insertional mutagenesis of Haemophilus influenzae.

C Sharetzsky 1, T D Edlind 1, J J LiPuma 1, T L Stull 1
PMCID: PMC207296  PMID: 1847369

Abstract

Insertional mutagenesis of the Haemophilus influenzae chromosome was accomplished by a novel method employing a 2.2-kbp element, TSTE. This element, consisting of the neo gene of Tn5 flanked by Haemophilus-specific uptake sequences, was ligated to circularized chromosomal fragments before transformation into the homologous strain. Eight mutants defective in the production of haemocin were detected. This strategy provides an efficient mechanism for the insertional mutagenesis of H. influenzae.

Full text

PDF
1561

Images in this article

1563Image
on p.1563

Selected References

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

  1. ALEXANDER H. E., LEIDY G. Induction of streptomycin resistance in sensitive Hemophilus influenzae by extracts containing desoxyribonucleic acid from resistant Hemophilus influenzae. J Exp Med. 1953 Jan;97(1):17–31. doi: 10.1084/jem.97.1.17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bachmann B. J. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev. 1972 Dec;36(4):525–557. doi: 10.1128/br.36.4.525-557.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Bukhari A. I. Bacteriophage mu as a transposition element. Annu Rev Genet. 1976;10:389–412. doi: 10.1146/annurev.ge.10.120176.002133. [DOI] [PubMed] [Google Scholar]
  5. Danner D. B., Deich R. A., Sisco K. L., Smith H. O. An eleven-base-pair sequence determines the specificity of DNA uptake in Haemophilus transformation. Gene. 1980 Nov;11(3-4):311–318. doi: 10.1016/0378-1119(80)90071-2. [DOI] [PubMed] [Google Scholar]
  6. Danner D. B., Smith H. O., Narang S. A. Construction of DNA recognition sites active in Haemophilus transformation. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2393–2397. doi: 10.1073/pnas.79.7.2393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gawron-Burke C., Clewell D. B. Regeneration of insertionally inactivated streptococcal DNA fragments after excision of transposon Tn916 in Escherichia coli: strategy for targeting and cloning of genes from gram-positive bacteria. J Bacteriol. 1984 Jul;159(1):214–221. doi: 10.1128/jb.159.1.214-221.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Genilloud O., Blázquez J., Mazodier P., Moreno F. A clinical isolate of transposon Tn5 expressing streptomycin resistance in Escherichia coli. J Bacteriol. 1988 Mar;170(3):1275–1278. doi: 10.1128/jb.170.3.1275-1278.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jorgensen R. A., Rothstein S. J., Reznikoff W. S. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet. 1979;177(1):65–72. doi: 10.1007/BF00267254. [DOI] [PubMed] [Google Scholar]
  10. Joshi V. P., Notani N. K. Cloning of novr gene in Haemophilus influenzae. Indian J Exp Biol. 1984 Dec;22(12):625–628. [PubMed] [Google Scholar]
  11. Kathariou S., Metz P., Hof H., Goebel W. Tn916-induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes. J Bacteriol. 1987 Mar;169(3):1291–1297. doi: 10.1128/jb.169.3.1291-1297.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kauc L., Goodgal S. H. Introduction of transposon Tn916 DNA into Haemophilus influenzae and Haemophilus parainfluenzae. J Bacteriol. 1989 Dec;171(12):6625–6628. doi: 10.1128/jb.171.12.6625-6628.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Klaer R., Kühn S., Fritz H. J., Tillmann E., Saint-Girons I., Habermann P., Pfeifer D., Starlinger P. Studies on transposition mechanisms and specificity of IS4. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):215–224. doi: 10.1101/sqb.1981.045.01.034. [DOI] [PubMed] [Google Scholar]
  14. Labarre J., Chauvat F., Thuriaux P. Insertional mutagenesis by random cloning of antibiotic resistance genes into the genome of the cyanobacterium Synechocystis strain PCC 6803. J Bacteriol. 1989 Jun;171(6):3449–3457. doi: 10.1128/jb.171.6.3449-3457.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lereclus D., Mahillon J., Menou G., Lecadet M. M. Identification of Tn4430, a transposon of Bacillus thuringiensis functional in Escherichia coli. Mol Gen Genet. 1986 Jul;204(1):52–57. doi: 10.1007/BF00330186. [DOI] [PubMed] [Google Scholar]
  16. LiPuma J. J., Richman H., Stull T. L. Haemocin, the bacteriocin produced by Haemophilus influenzae: species distribution and role in colonization. Infect Immun. 1990 Jun;58(6):1600–1605. doi: 10.1128/iai.58.6.1600-1605.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lodge J. K., Weston-Hafer K., Berg D. E. Transposon Tn5 target specificity: preference for insertion at G/C pairs. Genetics. 1988 Nov;120(3):645–650. doi: 10.1093/genetics/120.3.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McCarthy D. Cloning of the rec-2 locus of Haemophilus influenzae. Gene. 1989 Jan 30;75(1):135–143. doi: 10.1016/0378-1119(89)90390-9. [DOI] [PubMed] [Google Scholar]
  19. Munson R., Jr, Hunt A. Isolation and characterization of a mutant of Haemophilus influenzae type b deficient in outer membrane protein P1. Infect Immun. 1989 Mar;57(3):1002–1004. doi: 10.1128/iai.57.3.1002-1004.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rowji P., Gromkova R., Koornhof H. Genetic transformation in encapsulated clinical isolates of Haemophilus influenzae type b. J Gen Microbiol. 1989 Oct;135(10):2775–2782. doi: 10.1099/00221287-135-10-2775. [DOI] [PubMed] [Google Scholar]
  21. Sasakawa C., Yoshikawa M. A series of Tn5 variants with various drug-resistance markers and suicide vector for transposon mutagenesis. Gene. 1987;56(2-3):283–288. doi: 10.1016/0378-1119(87)90145-4. [DOI] [PubMed] [Google Scholar]
  22. Senghas E., Jones J. M., Yamamoto M., Gawron-Burke C., Clewell D. B. Genetic organization of the bacterial conjugative transposon Tn916. J Bacteriol. 1988 Jan;170(1):245–249. doi: 10.1128/jb.170.1.245-249.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sengstag C., Arber W. A cloned DNA fragment from bacteriophage P1 enhances IS2 insertion. Mol Gen Genet. 1987 Feb;206(2):344–351. doi: 10.1007/BF00333593. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Spencer H. T., Herriott R. M. Development of competence of Haemophilus influenzae. J Bacteriol. 1965 Oct;90(4):911–920. doi: 10.1128/jb.90.4.911-920.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stull T. L., Mendelman P. M., Haas J. E., Schoenborn M. A., Mack K. D., Smith A. L. Characterization of Haemophilus influenzae type b fimbriae. Infect Immun. 1984 Dec;46(3):787–796. doi: 10.1128/iai.46.3.787-796.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  28. Tomb J. F., Barcak G. J., Chandler M. S., Redfield R. J., Smith H. O. Transposon mutagenesis, characterization, and cloning of transformation genes of Haemophilus influenzae Rd. J Bacteriol. 1989 Jul;171(7):3796–3802. doi: 10.1128/jb.171.7.3796-3802.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tu C. P., Cohen S. N. Translocation specificity of the Tn3 element: characterization of sites of multiple insertions. Cell. 1980 Jan;19(1):151–160. doi: 10.1016/0092-8674(80)90396-7. [DOI] [PubMed] [Google Scholar]
  30. Vandeyar M. A., Zahler S. A. Chromosomal insertions of Tn917 in Bacillus subtilis. J Bacteriol. 1986 Aug;167(2):530–534. doi: 10.1128/jb.167.2.530-534.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  32. Youngman P. J., Perkins J. B., Losick R. Genetic transposition and insertional mutagenesis in Bacillus subtilis with Streptococcus faecalis transposon Tn917. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2305–2309. doi: 10.1073/pnas.80.8.2305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zerbib D., Gamas P., Chandler M., Prentki P., Bass S., Galas D. Specificity of insertion of IS1. J Mol Biol. 1985 Oct 5;185(3):517–524. doi: 10.1016/0022-2836(85)90068-3. [DOI] [PubMed] [Google Scholar]

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

RESOURCES