1887

Abstract

This paper is the first detailed description of the development and use of new genetic tools specifically for the safe manipulation of highly pathogenic subsp. . Most of these tools are also demonstrated to work with other subspecies. Kanamycin and hygromycin resistance determinants that function as genetic markers in subsp. strain Schu and sets of episomal shuttle vectors that are either unstable or stably maintained in the absence of selection were developed. In addition, the gene, expressed from the promoter, was successfully used as a marker for transposon mutagenesis. This work also includes the development of -based suicide plasmids expressing kanamycin resistance that can be used for electroporation-mediated allelic exchange of unmarked mutations in Schu and the live vaccine strain (LVS). Using these plasmids, the two predicted -lactamase genes, and , in Schu and LVS were deleted. Only the Δ mutants had increased susceptibility to ampicillin, and this phenotype was complemented by a plasmid expressing . The results suggest that the -lactam antibiotic resistance phenotype of Schu and LVS is likely due to only one of the two -lactamase genes present and that ampicillin resistance can be used as an additional selectable marker in -lactamase deletion mutants. The collection of tools presented in this report will be helpful for the genetic analyses of subsp. pathogenesis.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.29121-0
2006-11-01
2020-08-13
Loading full text...

Full text loading...

/deliver/fulltext/micro/152/11/3425.html?itemId=/content/journal/micro/10.1099/mic.0.29121-0&mimeType=html&fmt=ahah

References

  1. Ausubel F. M, Brent R, Kingston R. E, Moore D. D, Seidman J. G, Smith J. A, Struhl K. 1987; Current Protocols in Molecular Biology New York: Greene Publishing Associates and Wiley-Interscience;
    [Google Scholar]
  2. Baker C. N, Hollis D. G, Thornsberry C. 1985; Antimicrobial susceptibility testing of Francisella tularensis with a modified Mueller-Hinton broth. J Clin Microbiol22:212–215
    [Google Scholar]
  3. Cowley S. C, Gray C. J, Nano F. E. 2000; Isolation and characterization of Francisella novicida mutants defective in lipopolysaccharide biosynthesis. FEMS Microbiol Lett182:63–67[CrossRef]
    [Google Scholar]
  4. Dennis D. T, Inglesby T. V, Henderson D. A.15 other authors 2001; Tularemia as a biological weapon: medical and public health management. JAMA285:2763–2773[CrossRef]
    [Google Scholar]
  5. Derbyshire K. M, Takacs C, Huang J. 2000; Using the EZ : : TN transposome for transposon mutagenesis in Mycobacterium smegmatis . Epicentre Forum7:1–4
    [Google Scholar]
  6. Eigelsbach H. T, Downs C. M. 1961; Prophylactic effectiveness of live and killed tularemia vaccines. I. Production of vaccine and evaluation in the white mouse and guinea pig. J Immunol87:415–425
    [Google Scholar]
  7. Ericsson M, Golovliov I, Sandstrom G, Tarnvik A, Sjostedt A. 1997; Characterization of the nucleotide sequence of the groE operon encoding heat shock proteins chaperone-60 and -10 of Francisella tularensis and determination of the T-cell response to the proteins in individuals vaccinated with F. tularensis . Infect Immun65:1824–1829
    [Google Scholar]
  8. Flores A. R, Parsons L. M, Pavelka M. S. Jr. 2005a; Characterization of novel Mycobacterium tuberculosis and Mycobacterium smegmatis mutants hypersusceptible to beta-lactam antibiotics. J Bacteriol187:1892–1900[CrossRef]
    [Google Scholar]
  9. Flores A. R, Parsons L. M, Pavelka M. S. Jr. 2005b; Genetic analysis of the beta-lactamases of Mycobacterium tuberculosis and Mycobacterium smegmatis and susceptibility to beta-lactam antibiotics. Microbiology151:521–532[CrossRef]
    [Google Scholar]
  10. Garcia Del Blanco N, Dobson M. E, Vela A. I.7 other authors 2002; Genotyping of Francisella tularensis strains by pulsed-field gel electrophoresis, amplified fragment length polymorphism fingerprinting, and 16S rRNA gene sequencing. J Clin Microbiol40:2964–2972[CrossRef]
    [Google Scholar]
  11. Gerdes K, Christensen S. K, Lobner-Olesen A. 2005; Prokaryotic toxin-antitoxin stress response loci. Nat Rev Microbiol3:371–382[CrossRef]
    [Google Scholar]
  12. Golovliov I, Sjostedt A, Mokrievich A, Pavlov V. 2003; A method for allelic replacement in Francisella tularensis . FEMS Microbiol Lett222:273–280[CrossRef]
    [Google Scholar]
  13. Goryshin I. Y, Jendrisak J, Hoffman L. M, Meis R, Reznikoff W. S. 2000; Insertional transposon mutagenesis by electroporation of released Tn 5 transposition complexes. Nat Biotechnol18:97–100[CrossRef]
    [Google Scholar]
  14. Hollis D. G, Weaver R. E, Steigerwalt A. G, Wenger J. D, Moss C. W, Brenner D. J. 1989; Francisella philomiragia comb. nov. (formerly Yersiniaphilomiragia) and Francisella tularensis biogroup novicida (formerly Francisella novicida) associated with human disease. J Clin Microbiol27:1601–1608
    [Google Scholar]
  15. Johansson A, Goransson I, Larsson P, Sjostedt A. 2001; Extensive allelic variation among Francisella tularensis strains in a short-sequence tandem repeat region. J Clin Microbiol39:3140–3146[CrossRef]
    [Google Scholar]
  16. Johansson A, Farlow J, Larsson P.8 other authors 2004; Worldwide genetic relationships among Francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis. J Bacteriol186:5808–5818[CrossRef]
    [Google Scholar]
  17. Kawula T. H, Hall J. D, Fuller J. R, Craven R. R. 2004; Use of transposon-transposase complexes to create stable insertion mutant strains of Francisella tularensis LVS. Appl Environ Microbiol70:6901–6904[CrossRef]
    [Google Scholar]
  18. Lauriano C. M, Barker J. R, Nano F. E, Arulanandam B. P, Klose K. E. 2003; Allelic exchange in Francisella tularensis using PCR products. FEMS Microbiol Lett229:195–202[CrossRef]
    [Google Scholar]
  19. Leboul J, Davies J. 1982; Enzymatic modification of hygromycin B in Streptomyces hygroscopicus . J Antibiot35:527–528[CrossRef]
    [Google Scholar]
  20. Maier T. M, Havig A, Casey M, Nano F. E, Frank D. W, Zahrt T. C. 2004; Construction and characterization of a highly efficient Francisella shuttle plasmid. Appl Environ Microbiol70:7511–7519[CrossRef]
    [Google Scholar]
  21. Maier T. M, Pechous R, Casey M, Zahrt T. C, Frank D. W. 2006; In vivo Himar1-based transposon mutagenesis of Francisella tularensis . Appl Environ Microbiol72:1878–1885[CrossRef]
    [Google Scholar]
  22. Malpartida F, Zalacain M, Jimenez A, Davies J. 1983; Molecular cloning and expression in Streptomyces lividans of a hygromycin B phosphotransferase gene from Streptomyces hygroscopicus . Biochem Biophys Res Commun117:6–12[CrossRef]
    [Google Scholar]
  23. Nano F. E, Zhang N, Cowley S. C.8 other authors 2004; A Francisella tularensis pathogenicity island required for intramacrophage growth. J Bacteriol186:6430–6436[CrossRef]
    [Google Scholar]
  24. Norqvist A, Kuoppa K, Sandstrom G. 1996; Construction of a shuttle vector for use in Francisella tularensis . FEMS Immunol Med Microbiol13:257–260[CrossRef]
    [Google Scholar]
  25. Pavelka M. S. Jr, Jacobs W. R. Jr. 1999; Comparison of the construction of unmarked deletion mutations in Mycobacterium smegmatis , Mycobacterium bovis bacillus Calmette-Guerin, and Mycobacterium tuberculosis H37Rv by allelic exchange. J Bacteriol181:4780–4789
    [Google Scholar]
  26. Pavlov V. M, Mokrievich A. N, Volkovoy K. 1996; Cryptic plasmid pFNL10 from Francisella novicida -like F6168: the base of plasmid vectors for Francisella tularensis . FEMS Immunol Med Microbiol13:253–256[CrossRef]
    [Google Scholar]
  27. Pomerantsev A. P, Golovliov I. R, Ohara Y, Mokrievich A. N, Obuchi M, Norqvist A, Kuoppa K, Pavlov V. M. 2001a; Genetic organization of the Francisella plasmid pFNL10. Plasmid46:210–222[CrossRef]
    [Google Scholar]
  28. Pomerantsev A. P, Obuchi M, Ohara Y. 2001b; Nucleotide sequence, structural organization, and functional characterization of the small recombinant plasmid pOM1 that is specific for Francisella tularensis . Plasmid46:86–94[CrossRef]
    [Google Scholar]
  29. Shaw K. J, Rather P. N, Hare R. S, Miller G. H. 1993; Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol Rev57:138–163
    [Google Scholar]
  30. Sjostedt A, Tarnvik A, Sandstrom G. 1996; Francisella tularensis : host-parasite interaction. FEMS Immunol Med Microbiol13:181–184[CrossRef]
    [Google Scholar]
  31. Stover C. K, Fuerst T. R, de la Cruz V. F. 28 other authors 1991; New use of BCG for recombinant vaccines. Nature351:456–460[CrossRef]
    [Google Scholar]
  32. Titball R. W, Johansson A, Forsman M. 2003; Will the enigma of Francisella tularensis virulence soon be solved?. Trends Microbiol11:118–123[CrossRef]
    [Google Scholar]
  33. Twine S, Bystrom M, Chen W.9 other authors 2005; A mutant of Francisella tularensis strain SCHU S4 lacking the ability to express a 58-kilodalton protein is attenuated for virulence and is an effective live vaccine. Infect Immun73:8345–8352[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.29121-0
Loading
/content/journal/micro/10.1099/mic.0.29121-0
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

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