1887

Abstract

Mycoloyltransferases (Myts) play an essential role in the biogenesis of the cell envelope of members of the , a group of bacteria that includes the mycobacteria and corynebacteria. While the existence of several functional genes has been demonstrated in both mycobacteria and corynebacteria (), the disruption of any of these genes has at best generated cell-wall-defective but always viable strains. To investigate the importance of Myts on the physiology of members of the , a double mutant of was constructed by deleting and , and the consequences of the deletion on the viability of the mutant, the transfer of corynomycoloyl residues onto its cell-wall arabinogalactan and trehalose derivatives, and on its cell envelope ultrastructure were determined. The double mutant strain failed to grow at 34 °C and exhibited a growth defect and formed segmentation-defective cells at 30 °C. Biochemical analyses showed that the double mutant elaborated 60 % less cell-wall-bound corynomycolates and produced less crystalline surface layer proteins associated with the cell surface than the parent and -inactivated mutant strains. Freeze-fracture electron microscopy showed that the Δ Δ double mutant, unlike the wild-type and -inactivated single mutant strains, frequently exhibited an additional fracture plane that propagated within the plasma membrane and rarely exposed the S-layer protein. Ultra-thin sectioning of the double mutant cells showed that they were totally devoid of the outermost layer. Complementation of the double mutant with the wild-type or gene restored completely or partially this phenotype. The data indicate that Myts are important for the physiology of and reinforce the concept that these enzymes would represent good targets for the discovery of new drugs against the pathogenic members of the .

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2004-01-01
2024-03-29
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References

  1. Aggerbeck L. P., Gulik-Krzywicki T. 1986; Studies of lipoproteins by freeze-fracture and etching electron microscopy. Methods Enzymol 128:457–472
    [Google Scholar]
  2. Armitige L. Y., Jagannath C., Wanger A. R., Norris S. J. 2000; Disruption of the genes encoding antigen 85A and antigen 85B of Mycobacterium tuberculosis H37Rv: effect on growth in culture and in macrophages. Infect Immun 68:767–778 [CrossRef]
    [Google Scholar]
  3. 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: Wiley;
  4. Belisle J. T., Vissa V. D., Sievert T., Takayama K., Brennan P. J., Besra G. S. 1997; Role of the major antigen of Mycobacterium tuberculosis in the cell wall biogenesis. Science 276:1420–1422 [CrossRef]
    [Google Scholar]
  5. Bligh E., Dyer W. J. 1959; Extraction des lipides. Can J Biochem Physiol 37:88–97
    [Google Scholar]
  6. Bonamy C., Guyonvarch A., Reyes O., David F., Leblon G. 1990; Interspecies electro-transformation in corynebacteria. FEMS Microbiol Lett 66:263–270 [CrossRef]
    [Google Scholar]
  7. Brand S., Niehaus K., Pühler A., Kalinowski J. 2003; Identification and functional analysis of six mycolyltransferase genes of Corynebacterium glutamicum ATCC 13032: the genescop1, cmt1, and cmt2 can replace each other in the synthesis of trehalose dicorynomycolate, a component of the mycolic acid layer of the cell envelope. Arch Microbiol 180:33–44 [CrossRef]
    [Google Scholar]
  8. Brennan P. J., Nikaido H. 1995; The envelope of mycobacteria. Annu Rev Biochem 64:29–63 [CrossRef]
    [Google Scholar]
  9. Chami M., Bayan N., Peyret J. L., Gulik-Krzywicki T., Leblon G., Shechter E. 1997; The S-layer protein of Corynebacterium glutamicum is anchored to the cell wall by its C-terminal hydrophobic domain. Mol Microbiol 23:483–492 [CrossRef]
    [Google Scholar]
  10. 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]
  11. Collins M. D., Goodfellow M., Minnikin D. E. 1982; A survey of the structures of mycolic acids in Corynebacterium and related taxa. J Gen Microbiol 128:129–149
    [Google Scholar]
  12. Daffé M., Draper P. 1998; The envelope layers of mycobacteria with reference to their pathogenicity. Adv Microb Physiol 39:131–203
    [Google Scholar]
  13. Daffé M., Lanéelle M.-A., Asselineau C., Lévy-Frébault V., David H. L. 1983; Intérêt taxonomique des acides gras des Mycobactéries: proposition d'une méthode d'analyse. Ann Microbiol (Paris 134B:241–256
    [Google Scholar]
  14. De Sousa-D'Auria C., Kacem R., Puech V., Tropis M., Leblon G., Houssin C., Daffe M. 2003; New insights into the biogenesis of the cell envelope of corynebacteria: identification and functional characterization of five new mycoloyltransferase genes in Corynebacterium glutamicum. FEMS Microbiol Lett 224:35–44 [CrossRef]
    [Google Scholar]
  15. Dittmer J. C. F., Lester R. L. 1964; A simple specific spray for the detection of phospholipids on thin-layer chromatograms. J Lipid Res 15:126–127
    [Google Scholar]
  16. Draper P. 1998; The outer parts of the mycobacterial envelope as permeability barriers. Front Biosci 3:D1253–D1261
    [Google Scholar]
  17. Dubnau E., Chan J., Raynaud C., Mohan V. P., Lanéelle M.-A., Yu K., Quémard A., Smith I., Daffe M. 2000; Oxygenated mycolic acids are necessary for virulence of Mycobacterium tuberculosis in mice. Mol Microbiol 36:630–637
    [Google Scholar]
  18. Glickman M. S., Cox J. S., Jacobs W. R. Jr 2000; A novel mycolic acid cyclopropane synthetase is required for cording, persistence, and virulence of Mycobacterium tuberculosis. Mol Cell 5:717–727 [CrossRef]
    [Google Scholar]
  19. Harth G., Horwitz M. A., Tabatadze D., Zamecnik P. C. 2002; Targeting the Mycobacterium tuberculosis 30/32-kDa mycolyl transferase complex as a therapeutic strategy against tuberculosis: proof of principle by using antisense technology. Proc Natl Acad Sci U S A 99:15614–15619 [CrossRef]
    [Google Scholar]
  20. Jackson M., Raynaud C., Lanéelle M.-A., Guilhot C., Laurent-Winter C., Ensergueix D., Gicquel B., Daffe M. 1999; Inactivation of the antigen 85C gene profoundly affects the mycolate content and alters the permeability of the Mycobacterium tuberculosis cell envelope. Mol Microbiol 31:1573–1587 [CrossRef]
    [Google Scholar]
  21. Joliff G., Mathieu L., Hahn V., Bayan N., Duchiron F., Renaud M., Shechter E., Leblon G. 1992; Cloning and nucleotide sequence of the csp1 gene encoding PS1, one of the two major secreted proteins of Corynebacterium glutamicum: the deduced N-terminal region of PS1 is similar to the Mycobacterium antigen 85 complex. Mol Microbiol 6:2349–2362 [CrossRef]
    [Google Scholar]
  22. Kartmann B., Stenger S., Niederweis M., Stengler S. 1999; Porins in the cell wall of Mycobacterium tuberculosis. J Bacteriol 181:6543–6546
    [Google Scholar]
  23. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
    [Google Scholar]
  24. Lemassu A., Daffé M. 1994; Structural features of the exocellular polysaccharides of Mycobacterium tuberculosis. Biochem J 297:351–357
    [Google Scholar]
  25. Lichtinger T., Burkovski A., Niederweis M., Krämer R., Benz R. 1998; Biochemical and biophysical characterization of the cell wall porin of Corynebacterium glutamicum: the channel is formed by a low molecular mass polypeptide. Biochemistry 37:15024–15032 [CrossRef]
    [Google Scholar]
  26. Lichtinger T., Heym B., Maier E., Eichner H., Cole S. T., Benz R. 1999; Evidence for a small anion-selective channel in the cell wall of Mycobacterium bovis BCG besides a wide cation-selective pore. FEBS Lett 454:349–355 [CrossRef]
    [Google Scholar]
  27. Lichtinger T., Rieß F. G., Burkovski A., Engelbrecht F., Hess D., Kratzin H. D., Krämer R., Benz R. 2001; The low-molecular-mass subunit of the cell wall channel of the Gram-positive Corynebacterium glutamicum. Immunological localization, cloning and sequencing of its gene porA. Eur J Biochem 268:462–469
    [Google Scholar]
  28. Liu J., Rosenberg E. Y., Nikaido H. 1995; Fluidity of the lipid domain of cell wall from Mycobacterium chelonae. Proc Natl Acad Sci U S A 92:11254–11258 [CrossRef]
    [Google Scholar]
  29. Minnikin D. E. 1982; Lipids: complex lipids, their chemistry, biosynthesis and roles. In The Biology of the Mycobacteria vol 1 pp. 95–184Edited by Ratledge C., Stanford J. L. London: Academic Press;
    [Google Scholar]
  30. Mukhopadhyay S., Basu D., Chakrabarti P. 1997; Characterization of a porin from Mycobacterium smegmatis. J Bacteriol 179:6205–6207
    [Google Scholar]
  31. Niederweis M., Ehrt S., Heinz C., Klocker U., Karosi S., Swiderek K. M., Riley L. W., Benz R. 1999; Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis. Mol Microbiol 33:933–945 [CrossRef]
    [Google Scholar]
  32. Nilsson B., Uhlen M., Josephson S., Gatenbeck S., Philipson L. 1983; An improved positive selection plasmid vector constructed by oligonucleotide mediated mutagenesis. Nucleic Acids Res 11:8019–8030 [CrossRef]
    [Google Scholar]
  33. Peyret J.-L., Bayan N., Joliff G., Gulik-Krzywicki T., Mathieu L., Shechter E., Leblon G. 1993; Characterization of the cspB gene encoding PS2, an ordered surface-layer protein in Corynebacterium glutamicum. Mol Microbiol 9:97–109 [CrossRef]
    [Google Scholar]
  34. Puech V., Bayan N., Salim K., Leblon G., Daffe M. 2000; Characterization of the in vivo acceptors of the mycoloyl residues transferred by the corynebacterial PS1 and the related mycobacterial antigens 85. Mol Microbiol 35:1026–1041 [CrossRef]
    [Google Scholar]
  35. Puech V., Chami M., Lemassu A., Lanéelle M.-A., Schiffler B., Gounon P., Bayan N., Benz R., Daffe M. 2001; Structure of the cell envelope of corynebacteria: importance of the non-covalently bound lipids in the formation of the cell wall permeability barrier and fracture plane. Microbiology 147:1365–1382
    [Google Scholar]
  36. Puech V., Guilhot C., Perez E., Tropis M., Armitige L. Y., Gicquel B., Daffe M. 2002; Evidence for a partial redundancy of the fibronectin-binding proteins for the transfer of mycoloyl residues onto the cell wall arabinogalactan termini of Mycobacterium tuberculosis. Mol Microbiol 44:1109–1122 [CrossRef]
    [Google Scholar]
  37. Rastogi N. 1991; Recent observations concerning structure and function relationships in the mycobacterial cell envelope: elaboration of a model in terms of mycobacterial pathogenicity, virulence and drug-resistance. Res Microbiol 142:464–476 [CrossRef]
    [Google Scholar]
  38. Reyes O., Guyonvarch A., Bonamy C., Salti V., David F., Leblon G. 1991; ‘Integron’-bearing vectors: a method suitable for stable chromosomal integration in highly restrictive corynebacteria. Gene 107:61–68 [CrossRef]
    [Google Scholar]
  39. Rieß F. G., Lichtinger T., Cseh R., Yassin A. F., Schaal K. P., Benz R. 1998; The cell wall porin of Nocardia farcinica: biochemical identification of the channel-forming protein and biophysical characterization of the channel properties. Mol Microbiol 29:139–150 [CrossRef]
    [Google Scholar]
  40. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory;
  41. Senaratne R. H., Mobasheri H., Papavinasasundaram K. G., Jenner P., Lea E. J. A., Draper P. 1998; Expression of gene for a porin-like protein of the OmpA family from Mycobacterium tuberculosis H37Rv. . J Bacteriol 180:3541–3547
    [Google Scholar]
  42. Shimakata T., Minatogawa Y. 2000; Essential role of trehalose in the synthesis and subsequent metabolism of corynomycolic acid in Corynebacterium matruchotii. Arch Biochem Biophys 380:331–338 [CrossRef]
    [Google Scholar]
  43. Sweeley C. C., Bentley R., Makita M., Wells W. W. 1963; Gas-liquid chromatography of trimethylsilyl derivatives of sugars and related substances. J Am Chem Soc 85:2497–2507 [CrossRef]
    [Google Scholar]
  44. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354 [CrossRef]
    [Google Scholar]
  45. Trias J., Benz R. 1994; Permeability of the cell wall of Mycobacterium smegmatis. Mol Microbiol 14:283–290 [CrossRef]
    [Google Scholar]
  46. Trias J., Jarlier V., Benz R. 1992; Porins in the cell wall of mycobacteria. Science 258:1479–1481 [CrossRef]
    [Google Scholar]
  47. Tzvetkov M., Klopprogge C., Zelder O., Liebl W. 2003; Genetic dissection of trehalose biosynthesis in Corynebacterium glutamicum: inactivation of trehalose production leads to impaired growth and altered cell wall lipid composition. Microbiology 149:1659–1673 [CrossRef]
    [Google Scholar]
  48. Wolf A., Krämer R., Morbach S. 2003; Three pathways for trehalose metabolism in Corynebacterium glutamicum ATCC13032 and their significance in response to osmotic stress. Mol Microbiol 49:1119–1134 [CrossRef]
    [Google Scholar]
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