1887

Abstract

The gene encoding -acetyl-1---inosityl-2-amino-2-deoxy---glucopyranoside deacetylase (MshB) is a key enzyme in mycothiol biosynthesis. Disruption of in resulted in decreased production of mycothiol (5–10 % of the parent strain mc155) but did not abolish mycothiol synthesis completely. Complementation of the MshB mutants with the gene resulted in increased mycothiol production towards the exponential and stationary phases of the bacterial growth cycle. These results suggest that another enzyme is capable of mycothiol biosynthesis by providing -acetylglucosaminylinositol deacetylation activity in the absence of MshB. One of the candidate enzymes capable of carrying out such reactions is the MshB orthologue mycothiol amide hydrolase, MCA. However, epichromosomal expression of in the MshB mutants did not restore mycothiol levels to the level of the parent strain. Unlike other mutants, which have little or no detectable levels of mycothiol, the MshB mutant did not exhibit increased resistance to isoniazid. However, the MshB mutant was resistant to ethionamide. Phenotypic analysis of other mutants lacking mycothiol revealed that MshA mutants also exhibit ethionamide resistance but that a MshCmutant was sensitive to ethionamide, suggesting that mycothiol or its early intermediates influence ethionamide activation.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26084-0
2003-05-01
2019-10-17
Loading full text...

Full text loading...

/deliver/fulltext/micro/149/5/mic1491341.html?itemId=/content/journal/micro/10.1099/mic.0.26084-0&mimeType=html&fmt=ahah

References

  1. Anderberg, S., Newton, G. L. & Fahey, R. C. ( 1998; ). Mycothiol biosynthesis and metabolism: cellular levels of potential intermediates in the biosynthesis and degradation of mycothiol. J Biol Chem 273, 30391–30397.[CrossRef]
    [Google Scholar]
  2. Baulard, A. R., Betts, J. C., Engohang-Ndong, J., Quan, S., McAdam, R. A., Brennan, P. J., Locht, C. & Besra, G. S. ( 2000; ). Activation of the pro-drug ethionamide is regulated in mycobacteria. J Biol Chem 275, 28326–28331.
    [Google Scholar]
  3. Billman-Jacobe, H., McConville, M. J., Haites, R. E., Kovacevic, S. & Coppel, R. L. ( 1999; ). Identification of a peptide synthetase involved in the biosynthesis of glycopeptidolipids of Mycobacterium smegmatis. Mol Microbiol 33, 1244–1253.
    [Google Scholar]
  4. Bornemann, C., Jardine, M. A., Spies, H. S. C. & Steenkamp, D. J. ( 1997; ). Biosynthesis of mycothiol: elucidation of the sequence of steps in Mycobacterium smegmatis. Biochem J 325, 623–629.
    [Google Scholar]
  5. 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]
  6. De Barber, A. E., Mdluli, K., Bosman, M., Bekker, L. & Barry, C. E., III ( 2000; ). Ethionamide activation and sensitivity in multidrug-resistant Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 97, 9677–9682.[CrossRef]
    [Google Scholar]
  7. De Smet, K. A., Kempsell, K. E., Gallagher, A., Duncan, K. & Young, D. B. ( 1999; ). Alteration of a single amino acid residue reverses fosfomycin resistance of recombinant MurA from Mycobacterium tuberculosis. Microbiology 145, 3177–3184.
    [Google Scholar]
  8. Koledin, T., Newton, G. L. & Fahey, R. C. ( 2002; ). Identification of the mycothiol synthase gene (mshD) encoding the acetyltransferase producing mycothiol in actinomycetes. Arch Microbiol 178, 331–337.[CrossRef]
    [Google Scholar]
  9. Larsen, M. H. ( 2000; ). Some common methods in mycobacterial genetics. In Molecular Genetics of Mycobacteria. Edited by G. F. Hatfull & W. R. Jacobs, Jr. Washington, DC: American Society for Microbiology.
  10. Newton, G. L. & Fahey, R. C. ( 2002; ). Mycothiol biochemistry. Arch Microbiol 178, 388–394.[CrossRef]
    [Google Scholar]
  11. Newton, G. L., Arnold, K., Price, M. S. & 7 other authors ( 1996; ). Distribution of thiols in microorganisms: mycothiol is a major thiol in most actinomycetes. J Bacteriol 178, 1990–1995.
    [Google Scholar]
  12. Newton, G. L., Unson, M., Anderberg, S., Aguilera, J. A., Oh, N. N., del Cardayré, S., Av-Gay, Y. & Fahey, R. C. ( 1999; ). Characterization of Mycobacterium smegmatis mutants defective in 1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside and mycothiol biosynthesis. Biochem Biophys Res Commun 255, 239–244.[CrossRef]
    [Google Scholar]
  13. Newton, G. L., Av-Gay, Y. & Fahey, R. C. ( 2000a; ). A novel mycothiol-dependent detoxification pathway in mycobacteria involving mycothiol S-conjugate amidase. Biochemistry 39, 10739–10746.[CrossRef]
    [Google Scholar]
  14. Newton, G. L., Av-Gay, Y. & Fahey, R. C. ( 2000b; ). N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside deacetylase (MshB) is a key enzyme in mycothiol biosynthesis. J Bacteriol 24, 6958–6963.
    [Google Scholar]
  15. Newton, G. L., Koledin, T., Gorovitz, B., Rawat, M., Fahey, R. C. & Av-Gay, Y. ( 2003; ). The glycosyltransferase gene encoding the enzyme catalyzing the first step of mycothiol biosynthesis (MshA). J Bacteriol (in press).
    [Google Scholar]
  16. O'Gaora, P., Barnini, S., Hayward, C., Filley, E., Rook, G., Young, D. & Thole, J. ( 1997; ). Mycobacteria as immunogens: development of expression vectors in multiple mycobacterial species. Med Princ Pract 6, 91–96.
    [Google Scholar]
  17. Prentki, P. & Krisch, H. M. ( 1984; ). In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29, 303–313.[CrossRef]
    [Google Scholar]
  18. Rawat, M., Newton, G. L., Ko, M., Martinez, G. J., Fahey, R. C. & Av-Gay, Y. ( 2002; ). Mycothiol-deficient Mycobacterium smegmatis mutants are hypersensitive to alkylating agents, free radicals, and antibiotics. Antimicrob Agents Chemother 46, 3348–3355.[CrossRef]
    [Google Scholar]
  19. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  20. Sareen, D., Steffek, M., Newton, G. L. & Fahey, R. C. ( 2002; ). ATP-dependent L-cysteine : 1d-myo-inosityl 2-amino-2-deoxy-α-d-glucopyranoside ligase, mycothiol biosynthesis enzyme, MshC, is related to Class I cysteinyl-tRNA synthetases. Biochemistry 41, 6885–6890.[CrossRef]
    [Google Scholar]
  21. Snapper, S. B., Lugosi, L., Jekkel, A., Melton, R. E., Kieser, T., Bloom, B. R. & Jacobs, W. R., Jr ( 1988; ). Lysogeny and transformation in mycobacteria: stable expression of foreign genes. Proc Natl Acad Sci U S A 85, 6987–6991.[CrossRef]
    [Google Scholar]
  22. Vanelli, T., Dykman, A. & de Montellano, P. ( 2002; ). The antituberculosis drug ethionamide is activated by a flavoprotein monooxygenase. J Biol Chem 277, 12824–12829.[CrossRef]
    [Google Scholar]
  23. Vetting, M. W., Hegde, S. S., Javid-Majd, F., Blanchard, J. S. & Roderick, S. L. ( 2002; ). Aminoglycoside 2′-N-acetyltransferase from Mycobacterium tuberculosis in complex with coenzyme A and aminoglycoside substrates. Nat Struct Biol 9, 653–658.[CrossRef]
    [Google Scholar]
  24. Young, D. B. & Duncan, K. ( 1995; ). Prospects for new interventions in the treatment and prevention of mycobacterial disease. Annu Rev Microbiol 49, 641–673.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.26084-0
Loading
/content/journal/micro/10.1099/mic.0.26084-0
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