Alteration of a single amino acid residue reverses fosfomycin resistance of recombinant MurA from

The EMBL accession number for the sequence in this paper is X96711.

Free

Abstract

has innate resistance to a range of broad-spectrum antimicrobial agents. This may in part reflect the relative impermeability of the mycobacterial cell wall, but additional specific mechanisms may also be important. In the case of fosfomycin, it has been suggested that a key difference in the active site of the MurA enzyme might confer resistance. In , fosfomycin covalently binds to a cysteine normally involved in the enzymic activity, while protein alignments predict an aspartate at this position in the MurA. In the present study, it is demonstrated that the wild-type MurA is indeed resistant to fosfomycin, and that it becomes sensitive following replacement of the aspartate residue in position 117 by a cysteine. In addition, the study illustrates the use of an inducible expression system in mycobacteria to allow functional characterization of an enzyme that is unstable during constitutive expression.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-145-11-3177
1999-11-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/145/11/1453177a.html?itemId=/content/journal/micro/10.1099/00221287-145-11-3177&mimeType=html&fmt=ahah

References

  1. Ainsa J. A., Perez E., Pelicic V., Berthet F. X., Gicquel B., Martin C. 1997; Aminoglycoside 2′-N-acetyltransferase genes are universally present in mycobacteria: characterization of the aac(2′)-Ic gene from Mycobacterium tuberculosis and the aac(2′)-Id gene from Mycobacterium smegmatis. Mol Microbiol 24:431–441 [CrossRef]
    [Google Scholar]
  2. Andersson G. E., Sharp P. M. 1996; Codon usage in the Mycobacterium tuberculosis complex. Microbiology 142:915–925 [CrossRef]
    [Google Scholar]
  3. Arca P., Reguera G., Hardisson C. 1997; Plasmid-encoded fosfomycin resistance in bacteria isolated from the urinary tract in a multicentre survey. J Antimicrob Chemother 40:393–399 [CrossRef]
    [Google Scholar]
  4. Ashbridge K. R., Booth R. J., Watson J. D., Lathigra R. B. 1989; Nucleotide sequence of the 19 kDa antigen gene from Mycobacterium tuberculosis. Nucleic Acids Res 17:1249 [CrossRef]
    [Google Scholar]
  5. Bardarov S., Kriakov J., Carriere C., Yu S., Vaamonde C., McAdam R. A., Bloom B. R., Hatfull G. F., Jacobs W. R. Jr 1997; Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis. Proc Natl Acad Sci USA 94:10961–10966 [CrossRef]
    [Google Scholar]
  6. Boshoff H. I., Mizrahi V. 1998; Purification, gene cloning, targeted knockout, overexpression, and biochemical characterization of the major pyrazinamidase from Mycobacterium smegmatis. J Bacteriol 180:5809–5814
    [Google Scholar]
  7. Christensen B. G., Leanza W. J., Beattie T. R., Patchett A. A., Arison B. H., Ormond R. E., Kuehl F. A. Jr, Albers Schonberg G., Jardetzky O. 1969; Phosphonomycin: structure and synthesis. Science 166:123–125 [CrossRef]
    [Google Scholar]
  8. Cole S. T., Brosch R., Parkhill J.39 other authors 1998; Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544 [CrossRef]
    [Google Scholar]
  9. Garbe T. R., Barathi J., Barnini S., Zhang Y., Abou Zeid C., Tang D., Mukherjee R., Young D. B. 1994; Transformation of mycobacterial species using hygromycin resistance as selectable marker. Microbiology 140:133–138 [CrossRef]
    [Google Scholar]
  10. Hendlin D., Stapley E. O., Jackson M.11 other authors 1969; Phosphonomycin, a new antibiotic produced by strains of streptomyces. Science 166:122–123 [CrossRef]
    [Google Scholar]
  11. Itaya K., Ui M. 1966; A new micromethod for the colorimetric determination of inorganic phosphate. Clin Chim Acta 14:361–366 [CrossRef]
    [Google Scholar]
  12. Jacobs W. R. Jr, Tuckman M., Bloom B. R. 1987; Introduction of foreign DNA into mycobacteria using a shuttle phasmid. Nature 327:532–535 [CrossRef]
    [Google Scholar]
  13. Kahan F. M., Kahan J. S., Cassidy P. J., Kropp H. 1974; The mechanism of action of fosfomycin (phosphonomycin). Ann N Y Acad Sci 235:364–386 [CrossRef]
    [Google Scholar]
  14. Kempsell K. E., Ji Y. E., Estrada I. C., Colston M. J., Cox R. A. 1992; The nucleotide sequence of the promoter, 16S rRNA and spacer region of the ribosomal RNA operon of Mycobacterium tuberculosis and comparison with Mycobacterium leprae precursor rRNA. J Gen Microbiol 138:1717–1727 [CrossRef]
    [Google Scholar]
  15. Kim D. H., Lees W. J., Kempsell K. E., Lane W. S., Duncan K., Walsh C. T. 1996; Characterization of a Cys115 to Asp substitution in the cell wall biosynthetic enzyme UDP-GlcNAc enolpyruvyl transferase (MurA) that confers resistance to inactivation by the antibiotic fosfomycin. Biochemistry 35:4923–4928 [CrossRef]
    [Google Scholar]
  16. Kumar D., Srivastava B. S., Srivastava R. 1998; Genetic rearrangements leading to disruption of heterologous gene expression in mycobacteria: an observation with Escherichia coli beta-galactosidase in Mycobacterium smegmatis and its implication in vaccine development. Vaccine 16:1212–1215 [CrossRef]
    [Google Scholar]
  17. Mahenthiralingam E., Draper P., Davis E. O., Colston M. J. 1993; Cloning and sequencing of the gene which encodes the highly inducible acetamidase of Mycobacterium smegmatis. J Gen Microbiol 139:575–583 [CrossRef]
    [Google Scholar]
  18. O’Gaora P., Barnini S., Hayward C., Filley E., Rook G., Young D., Thole J. 1997; Mycobacteria as immunogens: development of expression vectors for use in multiple mycobacterial species. Med Princ Pract 6:91–96
    [Google Scholar]
  19. Pablos-Mendez A., Raviglione M. C., Laszlo A.8 other authors 1998; Global surveillance for antituberculosis-drug resistance, 1994–1997. World Health Organization-International Union against Tuberculosis and Lung Disease Working Group on Anti-Tuberculosis Drug Resistance Surveillance. N Engl J Med 338:1641–1649 [CrossRef]
    [Google Scholar]
  20. Parish T., Mahenthiralingam E., Draper P., Davis E. O., Colston M. J. 1997; Regulation of the inducible acetamidase gene of Mycobacterium smegmatis. Microbiology 143:2267–2276 [CrossRef]
    [Google Scholar]
  21. Pelicic V., Jackson M., Reyrat J. M., Jacobs W. R. Jr, Gicquel B., Guilhot C. 1997; Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc Natl Acad Sci USA 94:10955–10960 [CrossRef]
    [Google Scholar]
  22. Prammananan T., Sander P., Springer B., Bottger E. C. 1999; RecA-mediated gene conversion and aminoglycoside resistance in strains heterozygous for rRNA. Antimicrob Agents Chemother 43:447–453 [CrossRef]
    [Google Scholar]
  23. Skarzynski T., Mistry A., Wonacott A., Hutchinson S. E., Kelly V. A., Duncan K. 1996; Structure of UDP-N-acetylglucosamine enolpyruvyl transferase, an enzyme essential for the synthesis of bacterial peptidoglycan, complexed with substrate UDP-N-acetylglucosamine and the drug fosfomycin. Structure 4:1465–1474 [CrossRef]
    [Google Scholar]
  24. Stover C. K., de la Cruz V. F., Fuerst T. R.11 other authors 1991; New use of BCG for recombinant vaccines. Nature 351:456–460 [CrossRef]
    [Google Scholar]
  25. Takiff H. E., Cimino M., Musso M. C., Weisbrod T., Martinez R., Delgado M. B., Salazar L., Bloom B. R., Jacobs W. R. Jr 1996; Efflux pump of the proton antiporter family confers low-level fluoroquinolone resistance in Mycobacterium smegmatis. Proc Natl Acad Sci USA 93:362–366 [CrossRef]
    [Google Scholar]
  26. Triccas J. A., Parish T., Britton W. J., Gicquel B. 1998; An inducible expression system permitting the efficient purification of a recombinant antigen from Mycobacterium smegmatis. FEMS Microbiol Lett 167:151–156 [CrossRef]
    [Google Scholar]
  27. Zhang Y., Lathigra R., Garbe T., Catty D., Young D. 1991; Genetic analysis of superoxide dismutase, the 23 kilodalton antigen of Mycobacterium tuberculosis. Mol Microbiol 5:381–391 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-145-11-3177
Loading
/content/journal/micro/10.1099/00221287-145-11-3177
Loading

Data & Media loading...

Most cited Most Cited RSS feed