1887

Abstract

is a fastidious Gram-negative bacterium responsible for the zoonotic disease tularemia. Investigation of the biology and molecular pathogenesis of has been limited by the difficulties in manipulating such a highly pathogenic organism and by a lack of genetic tools. However, recent advances have substantially improved the ability of researchers to genetically manipulate this organism. To expand the molecular toolbox we have developed two systems to stably integrate genetic elements in single-copy into the genome. The first system is based upon the ability of transposon Tn to insert in both a site- and orientation-specific manner at high frequency into the Tn site located downstream of the highly conserved gene. The second system consists of a -based suicide plasmid used for allelic exchange of unmarked elements with the gene, encoding a -lactamase, resulting in the replacement of with the element and the loss of ampicillin resistance. To test these new tools we used them to complement a novel -glutamate auxotroph of LVS, created using an improved -based allelic exchange plasmid. These new systems will be helpful for the genetic manipulation of in studies of tularemia biology, especially where the use of multi-copy plasmids or antibiotic markers may not be suitable.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.022491-0
2009-04-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/155/4/1152.html?itemId=/content/journal/micro/10.1099/mic.0.022491-0&mimeType=html&fmt=ahah

References

  1. Ashiuchi M., Nishikawa Y., Matsunaga K., Yamamoto M., Shimanouchi K., Misono H. 2007; Genetic design of conditional d-glutamate auxotrophy for Bacillus subtilis : use of a vector-borne poly- γ -glutamate synthetic system. Biochem Biophys Res Commun 362:646–650
    [Google Scholar]
  2. 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: Green Publishing & Wiley Interscience;
  3. Bardarov S., Bardarov S. Jr, Pavelka M. S. Jr, Sambandamurthy V., Larsen M., Tufariello J., Chan J., Hatfull G., Jacobs W. R. Jr 2002; Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis , M.bovis BCG and M. smegmatis . Microbiology 148:3007–3017
    [Google Scholar]
  4. Bina X. R., Wang C., Miller M. A., Bina J. E. 2006; The Bla2 β -lactamase from the live-vaccine strain of Francisella tularensis encodes a functional protein that is only active against penicillin-class β -lactam antibiotics. Arch Microbiol 186:219–228
    [Google Scholar]
  5. Boyer H. W., Roulland-Dussoix D. 1969; A complementation analysis of the restriction and modification of DNA in Escherichia coli . J Mol Biol 41:459–472
    [Google Scholar]
  6. Buchan B. W., McLendon M. K., Jones B. D. 2008; Identification of differentially regulated Francisella tularensis genes using a newly developed Tn 5 -based transposon delivery system. Appl Environ Microbiol 74:2637–2645
    [Google Scholar]
  7. Choi K. H., Gaynor J. B., White K. G., Lopez C., Bosio C. M., Karkhoff-Schweizer R. R., Schweizer H. P. 2005; A Tn 7 -based broad-range bacterial cloning and expression system. Nat Methods 2:443–448
    [Google Scholar]
  8. Choi K. H., DeShazer D., Schweizer H. P. 2006; Mini-Tn 7 insertion in bacteria with multiple glmS -linked att Tn 7 sites: example Burkholderia mallei ATCC 23344. Nat Protoc 1:162–169
    [Google Scholar]
  9. Choi K. H., Mima T., Casart Y., Rholl D., Kumar A., Beacham I. R., Schweizer H. P. 2008; Genetic tools for select-agent-compliant manipulation of Burkholderia pseudomallei . Appl Environ Microbiol 74:1064–1075
    [Google Scholar]
  10. Cormack B. P., Valdivia R. H., Falkow S. 1996; FACS-optimized mutants of the green fluorescent protein (GFP. Gene 173:33–38
    [Google Scholar]
  11. DeBoy R. T., Craig N. L. 1996; Tn 7 transposition as a probe of cis interactions between widely separated (190 kilobases apart) DNA sites in the Escherichia coli chromosome. J Bacteriol 178:6184–6191
    [Google Scholar]
  12. Dennis D. T., Inglesby T. V., Henderson D. A., Bartlett J. G., Ascher M. S., Eitzen E., Fine A. D., Friedlander A. M., Hauer J. other authors 2001; Tularemia as a biological weapon: medical and public health management. JAMA (J Am Med Assoc) 285:2763–2773
    [Google Scholar]
  13. Dodd D., Reese J. G., Louer C. R., Ballard J. D., Spies M. A., Blanke S. R. 2007; Functional comparison of the two Bacillus anthracis glutamate racemases. J Bacteriol 189:5265–5275
    [Google Scholar]
  14. Doublet P., van Heijenoort J., Bohin J. P., Mengin-Lecreulx D. 1993; The murI gene of Escherichia coli is an essential gene that encodes a glutamate racemase activity. J Bacteriol 175:2970–2979
    [Google Scholar]
  15. Ellis J., Oyston P. C., Green M., Titball R. W. 2002; Tularemia. Clin Microbiol Rev 15:631–646
    [Google Scholar]
  16. Gallagher L. A., Ramage E., Jacobs M. A., Kaul R., Brittnacher M., Manoil C. 2007; A comprehensive transposon mutant library of Francisella novicida , a bioweapon surrogate. Proc Natl Acad Sci U S A 104:1009–1014
    [Google Scholar]
  17. Golovliov I., Sjostedt A., Mokrievich A., Pavlov V. 2003; A method for allelic replacement in Francisella tularensis . FEMS Microbiol Lett 222:273–280
    [Google Scholar]
  18. Hoang T. T., Kutchma A. J., Becher A., Schweizer H. P. 2000; Integration-proficient plasmids for Pseudomonas aeruginosa : site-specific integration and use for engineering of reporter and expression strains. Plasmid 43:59–72
    [Google Scholar]
  19. Hoffmann B., Messer W., Schwarz U. 1972; Regulation of polar cap formation in the life cycle of Escherichia coli . J Supramol Struct 1:29–37
    [Google Scholar]
  20. 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 Microbiol 70:6901–6904
    [Google Scholar]
  21. Larsson P., Oyston P. C., Chain P., Chu M. C., Duffield M., Fuxelius H. H., Garcia E., Hälltorp G., Johansson D. other authors 2005; The complete genome sequence of Francisella tularensis , the causative agent of tularemia. Nat Genet 37:153–159
    [Google Scholar]
  22. Liu L., Yoshimura T., Endo K., Kishimoto K., Fuchikami Y., Manning J. M., Esaki N., Soda K. 1998; Compensation for d-glutamate auxotrophy of Escherichia coli WM335 by d-amino acid aminotransferase gene and regulation of murI expression. Biosci Biotechnol Biochem 62:193–195
    [Google Scholar]
  23. LoVullo E. D., Sherrill L. A., Perez L. L., Pavelka M. S. Jr 2006; Genetic tools for highly pathogenic Francisella tularensis subsp. tularensis . Microbiology 152:3425–3435
    [Google Scholar]
  24. LoVullo E. D., Sherrill L. A., Pavelka M. S. Jr 2008; Improved shuttle vectors for Francisella tularensis genetics. FEMS Microbiol Lett 291:95–102
    [Google Scholar]
  25. Ludu J. S., Nix E. B., Duplantis B. N., de Bruin O. M., Gallagher L. A., Hawley L. M., Nano F. E. 2008; Genetic elements for selection, deletion mutagenesis and complementation in Francisella spp. FEMS Microbiol Lett 278:86–93
    [Google Scholar]
  26. 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 Microbiol 70:7511–7519
    [Google Scholar]
  27. 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 Microbiol 72:1878–1885
    [Google Scholar]
  28. Maier T. M., Casey M. S., Becker R. H., Dorsey C. W., Glass E. M., Maltsev N., Zahrt T. C., Frank D. W. 2007; Identification of Francisella tularensis Himar1 -based transposon mutants defective for replication in macrophages. Infect Immun 75:5376–5389
    [Google Scholar]
  29. McKenzie G. J., Craig N. L. 2006; Fast, easy and efficient: site-specific insertion of transgenes into enterobacterial chromosomes using Tn 7 without need for selection of the insertion event. BMC Microbiol 6:39
    [Google Scholar]
  30. Miller J. H. 1972 Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  31. Mock M., Fouet A. 2001; Anthrax. Annu Rev Microbiol 55:647–671
    [Google Scholar]
  32. Peters J. E., Craig N. L. 2001; Tn 7 : smarter than we thought. Nat Rev Mol Cell Biol 2:806–814
    [Google Scholar]
  33. Piuri M., Hatfull G. F. 2006; A peptidoglycan hydrolase motif within the mycobacteriophage TM4 tape measure protein promotes efficient infection of stationary phase cells. Mol Microbiol 62:1569–1585
    [Google Scholar]
  34. Qin A., Mann B. J. 2006; Identification of transposon insertion mutants of Francisella tularensis tularensis strain Schu S4 deficient in intracellular replication in the hepatic cell line HepG2. BMC Microbiol 6:69
    [Google Scholar]
  35. Rasko D. A., Esteban C. D., Sperandio V. 2007; Development of novel plasmid vectors and a promoter trap system in Francisella tularensis compatible with the pFNL10 based plasmids. Plasmid 58:159–166
    [Google Scholar]
  36. Rodriguez S. A., Yu J. J., Davis G., Arulanandam B. P., Klose K. E. 2008; Targeted inactivation of Francisella tularensis genes by group II introns. Appl Environ Microbiol 74:2619–2626
    [Google Scholar]
  37. Stover C. K., de la Cruz V. F., Fuerst T. R., Burlein J. E., Benson L. A., Bennett L. T., Bansal G. P., Young J. F., Lee M. H. other authors 1991; New use of BCG for recombinant vaccines. Nature 351:456–460
    [Google Scholar]
  38. Su J., Yang J., Zhao D., Kawula T. H., Banas J. A., Zhang J. R. 2007; Genome-wide identification of Francisella tularensis virulence determinants. Infect Immun 75:3089–3101
    [Google Scholar]
  39. Titball R. W., Sjostedt A., Pavelka M. S. Jr, Nano F. E. 2007; Biosafety and selectable markers. Ann N Y Acad Sci 1105405–417
    [Google Scholar]
  40. Twine S., Byström M., Chen W., Forsman M., Golovliov I., Johansson A., Kelly J., Lindgren H., Svensson K. 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 Immun 73:8345–8352
    [Google Scholar]
  41. Weiss D. S., Brotcke A., Henry T., Margolis J. J., Chan K., Monack D. M. 2007; In vivo negative selection screen identifies genes required for Francisella virulence. Proc Natl Acad Sci U S A 104:6037–6042
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.022491-0
Loading
/content/journal/micro/10.1099/mic.0.022491-0
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