Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Jul 15;373(Pt 2):465–474. doi: 10.1042/BJ20030248

Transposon-5 mutagenesis transforms Corynebacterium matruchotii to synthesize novel hybrid fatty acids that functionally replace corynomycolic acid.

Kuni Takayama 1, Barry Hayes 1, Matha M Vestling 1, Randall J Massey 1
PMCID: PMC1223520  PMID: 12879902

Abstract

Enzymes within the biosynthetic pathway of mycolic acid (C(60)-C(90) a-alkyl,b-hydroxyl fatty acid) in Mycobacterium tuberculosis are attractive targets for developing new anti-tuberculosis drugs. We have turned to the simple model system of Corynebacterium matruchotii to study the terminal steps in the anabolic pathway of a C32 mycolic acid called corynomycolic acid. By transposon-5 mutagenesis, we transformed C. matruchotii into a mutant that is unable to synthesize corynomycolic acid. Instead, it synthesized two related series of novel fatty acids that were released by saponification from the cell wall fraction and from two chloroform/methanol-extractable glycolipids presumed to be analogues of trehalose mono- and di-corynomycolate. By chemical analyses and MS, we determined the general structure of the two series to be 2,4,6,8,10-penta-alkyl decanoic acid for the larger series (C(70)-C(77)) and 2,4,6,8-tetra-alkyl octanoic acid for the smaller series (C(52)-C(64)), both containing multiple keto groups, hydroxy groups and double bonds. The mutant was temperature-sensitive, aggregated extensively, grew very slowly relative to the wild type, and was resistant to the presence of lysozyme. We suggest that a regulatory protein that normally prevents the transfer of the condensation product back to b-ketoacyl synthase in the corynomycolate synthase system of the wild type was inactivated in the mutant. This will result in multiple Claisen-type condensation and the formation of two similar series of these complex hybrid fatty acids. A similar protein in M. tuberculosis would be an attractive target for new drug discovery.

Full Text

The Full Text of this article is available as a PDF (293.5 KB).

Selected References

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

  1. Bates J. H. Tuberculosis chemotherapy. The need for new antituberculosis drugs is urgent. Am J Respir Crit Care Med. 1995 Apr;151(4):942–943. doi: 10.1164/ajrccm/151.4.942. [DOI] [PubMed] [Google Scholar]
  2. Bloom B. R., Murray C. J. Tuberculosis: commentary on a reemergent killer. Science. 1992 Aug 21;257(5073):1055–1064. doi: 10.1126/science.257.5073.1055. [DOI] [PubMed] [Google Scholar]
  3. Castro K. G. Tuberculosis as an opportunistic disease in persons infected with human immunodeficiency virus. Clin Infect Dis. 1995 Aug;21 (Suppl 1):S66–S71. doi: 10.1093/clinids/21.supplement_1.s66. [DOI] [PubMed] [Google Scholar]
  4. Datta A. K., Takayama K. Biosynthesis of a novel 3-oxo-2-tetradecyloctadecanoate-containing phospholipid by a cell-free extract of Corynebacterium diphtheriae. Biochim Biophys Acta. 1993 Aug 11;1169(2):135–145. doi: 10.1016/0005-2760(93)90198-i. [DOI] [PubMed] [Google Scholar]
  5. Datta A. K., Takayama K., Nashed M. A., Anderson L. An improved synthesis of trehalose 6-mono- and 6,6'-di-corynomycolates and related esters. Carbohydr Res. 1991 Sep 30;218:95–109. doi: 10.1016/0008-6215(91)84089-w. [DOI] [PubMed] [Google Scholar]
  6. Dubnau E., Chan J., Raynaud C., Mohan V. P., Lanéelle M. A., Yu K., Quémard A., Smith I., Daffé M. Oxygenated mycolic acids are necessary for virulence of Mycobacterium tuberculosis in mice. Mol Microbiol. 2000 May;36(3):630–637. doi: 10.1046/j.1365-2958.2000.01882.x. [DOI] [PubMed] [Google Scholar]
  7. George K. M., Yuan Y., Sherman D. R., Barry C. E., 3rd The biosynthesis of cyclopropanated mycolic acids in Mycobacterium tuberculosis. Identification and functional analysis of CMAS-2. J Biol Chem. 1995 Nov 10;270(45):27292–27298. doi: 10.1074/jbc.270.45.27292. [DOI] [PubMed] [Google Scholar]
  8. Gleissberg V. The threat of multidrug resistance: is tuberculosis ever untreatable or uncontrollable? Lancet. 1999 Mar 20;353(9157):998–999. doi: 10.1016/S0140-6736(99)01439-7. [DOI] [PubMed] [Google Scholar]
  9. Glickman M. S., Cox J. S., Jacobs W. R., Jr A novel mycolic acid cyclopropane synthetase is required for cording, persistence, and virulence of Mycobacterium tuberculosis. Mol Cell. 2000 Apr;5(4):717–727. doi: 10.1016/s1097-2765(00)80250-6. [DOI] [PubMed] [Google Scholar]
  10. Goryshin I. Y., Jendrisak J., Hoffman L. M., Meis R., Reznikoff W. S. Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes. Nat Biotechnol. 2000 Jan;18(1):97–100. doi: 10.1038/72017. [DOI] [PubMed] [Google Scholar]
  11. Iseman M. D. Evolution of drug-resistant tuberculosis: a tale of two species. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2428–2429. doi: 10.1073/pnas.91.7.2428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jackson M., Raynaud C., Lanéelle M. A., Guilhot C., Laurent-Winter C., Ensergueix D., Gicquel B., Daffé M. Inactivation of the antigen 85C gene profoundly affects the mycolate content and alters the permeability of the Mycobacterium tuberculosis cell envelope. Mol Microbiol. 1999 Mar;31(5):1573–1587. doi: 10.1046/j.1365-2958.1999.01310.x. [DOI] [PubMed] [Google Scholar]
  13. Kremer L., Douglas J. D., Baulard A. R., Morehouse C., Guy M. R., Alland D., Dover L. G., Lakey J. H., Jacobs W. R., Jr, Brennan P. J. Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in Mycobacterium tuberculosis. J Biol Chem. 2000 Jun 2;275(22):16857–16864. doi: 10.1074/jbc.M000569200. [DOI] [PubMed] [Google Scholar]
  14. Liu J., Barry C. E., 3rd, Besra G. S., Nikaido H. Mycolic acid structure determines the fluidity of the mycobacterial cell wall. J Biol Chem. 1996 Nov 22;271(47):29545–29551. doi: 10.1074/jbc.271.47.29545. [DOI] [PubMed] [Google Scholar]
  15. Liu J., Rosenberg E. Y., Nikaido H. Fluidity of the lipid domain of cell wall from Mycobacterium chelonae. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):11254–11258. doi: 10.1073/pnas.92.24.11254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Qureshi N., Takayama K., Jordi H. C., Schnoes H. K. Characterization of the purified components of a new homologous series of alpha-mycolic acids from Mycobacterium tuberculosis H37Ra. J Biol Chem. 1978 Aug 10;253(15):5411–5417. [PubMed] [Google Scholar]
  17. Shimakata T., Iwaki M., Kusaka T. In vitro synthesis of mycolic acids by the fluffy layer fraction of Bacterionema matruchotii. Arch Biochem Biophys. 1984 Feb 15;229(1):329–339. doi: 10.1016/0003-9861(84)90159-0. [DOI] [PubMed] [Google Scholar]
  18. Shimakata T., Minatogawa Y. Essential role of trehalose in the synthesis and subsequent metabolism of corynomycolic acid in Corynebacterium matruchotii. Arch Biochem Biophys. 2000 Aug 15;380(2):331–338. doi: 10.1006/abbi.2000.1924. [DOI] [PubMed] [Google Scholar]
  19. Shimakata T., Tsubokura K., Kusaka T., Shizukuishi K. Mass-spectrometric identification of trehalose 6-monomycolate synthesized by the cell-free system of Bacterionema matruchotii. Arch Biochem Biophys. 1985 May 1;238(2):497–508. doi: 10.1016/0003-9861(85)90193-6. [DOI] [PubMed] [Google Scholar]
  20. Staunton J., Weissman K. J. Polyketide biosynthesis: a millennium review. Nat Prod Rep. 2001 Aug;18(4):380–416. doi: 10.1039/a909079g. [DOI] [PubMed] [Google Scholar]
  21. Walker R. W., Prome J. C., Lacave C. S. Biosynthesis of mycolic acids. Formation of a C32 beta-keto ester from palmitic acid in a cell-free system of Corynebacterium diphtheriae. Biochim Biophys Acta. 1973 Oct 17;326(1):52–62. doi: 10.1016/0005-2760(73)90027-1. [DOI] [PubMed] [Google Scholar]
  22. Wang L., Slayden R. A., Barry C. E., 3rd, Liu J. Cell wall structure of a mutant of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids. J Biol Chem. 2000 Mar 10;275(10):7224–7229. doi: 10.1074/jbc.275.10.7224. [DOI] [PubMed] [Google Scholar]
  23. Winder F. G., Collins P. B. Inhibition by isoniazid of synthesis of mycolic acids in Mycobacterium tuberculosis. J Gen Microbiol. 1970 Sep;63(1):41–48. doi: 10.1099/00221287-63-1-41. [DOI] [PubMed] [Google Scholar]
  24. Winder F. G., Collins P. B., Whelan D. Effects of ethionamide and isoxyl on mycolic acid synthesis in Mycobacterium tuberculosis BCG. J Gen Microbiol. 1971 Jun;66(3):379–380. doi: 10.1099/00221287-66-3-379. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES