1887

Abstract

is is an essential gene involved in cell division. This paper shows that and its homologue in are functionally equivalent. The genes are syntenous, and share significant homology in both their coding and non-coding DNA sequences. Transcription site mapping showed that the two genes possess near-identical promoter elements, and they displayed comparable promoter strengths in a reporter gene assay. The two proteins show near identity in their C-terminus, and polyclonal antiserum to WhmD specifically cross-reacts with a ∼15 kDa band in lysates. Following overexpression of sense and anti-sense constructs in their cognate mycobacterial hosts, and transformants displayed a small-colony phenotype, exhibited filamentation, and showed a reduction in viability. These observations reveal that the two proteins are functionally homologous and that their intracellular concentration is critical for septation in mycobacteria. Colonies of overexpressing were spherical and glossy, suggesting a change in composition of the cell envelope. Filaments of the conditionally complemented mutant were non-acid-fast, also indicating changes in characteristics of surface lipids. transformants carrying a fusion showed a diffuse pattern of fluorescence, consistent with the putative role of WhmD as a regulator. These observations strongly suggest that is an essential gene and its protein product in all likelihood regulates the expression of genes involved in the cell division cascade.

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2006-09-01
2019-09-21
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References

  1. Anilkumar, G., Srinivasan, R., Anand, S. P. & Ajitkumar, P. ( 2001; ). Bacterial cell division protein FtsZ is a specific substrate for the AAA family protease FtsH. Microbiology 147, 516–517.
    [Google Scholar]
  2. Anilkumar, G., Srinivasan, R. & Ajitkumar, P. ( 2004; ). Genomic organization and in vivo characterization of proteolytic activity of FtsH of Mycobacterium smegmatis SN2. Microbiology 150, 2629–2639.[CrossRef]
    [Google Scholar]
  3. Aravind, L., Anantharaman, V., Balaji, S., Babu, M. M. & Iyer, L. M. ( 2005; ). The many faces of the helix-turn-helix domain: transcription regulation and beyond. FEMS Microbiol Rev 29, 231–262.
    [Google Scholar]
  4. Bachrach, G., Colston, M. J., Bercovier, H., Bar-Nir, D., Anderson, C. & Papavinasasundaram, K. G. ( 2000; ). A new single-copy mycobacterial plasmid, pMF1, from Mycobacterium fortuitum which is compatible with the pAL5000 replicon. Microbiology 146, 297–303.
    [Google Scholar]
  5. Betts, J. C., Lukey, P. T., Robb, L. C., McAdam, R. A. & Duncan, K. ( 2002; ). Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol Microbiol 43, 717–731.[CrossRef]
    [Google Scholar]
  6. Bradford, M. M. ( 1976; ). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef]
    [Google Scholar]
  7. Chaba, R., Raje, M. & Chakraborti, P. K. ( 2002; ). Evidence that a eukaryotic-type serine/threonine protein kinase from Mycobacterium tuberculosis regulates morphological changes associated with cell division. Eur J Biochem 269, 1078–1085.[CrossRef]
    [Google Scholar]
  8. Colangeli, R., Helb, D., Sridharan, S. & 9 other authors ( 2005; ). The Mycobacterium tuberculosis iniA gene is essential for activity of an efflux pump that confers drug tolerance to both isoniazid and ethambutol. Mol Microbiol 55, 1829–1840.[CrossRef]
    [Google Scholar]
  9. 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]
  10. Datta, P., Dasgupta, A., Bhakta, S. & Basu, J. ( 2002; ). Interaction between FtsZ and FtsW of Mycobacterium tuberculosis. J Biol Chem 277, 24983–24987.[CrossRef]
    [Google Scholar]
  11. Davis, N. K. & Chater, K. F. ( 1992; ). The Streptomyces coelicolor whiB gene encodes a small transcription factor-like protein dispensable for growth but essential for sporulation. Mol Gen Genet 232, 351–358.
    [Google Scholar]
  12. Deol, P., Vohra, R., Saini, A. K., Singh, A., Chandra, H., Chopra, P., Das, T. K., Tyagi, A. K. & Singh, Y. ( 2005; ). Role of Mycobacterium tuberculosis Ser/Thr kinase PknF: implications in glucose transport and cell division. J Bacteriol 187, 3415–3420.[CrossRef]
    [Google Scholar]
  13. Dubnau, E., Chan, J., Mohan, V. P. & Smith, I. ( 2005; ). Responses of Mycobacterium tuberculosis to growth in the mouse lung. Infect Immun 73, 3754–3757.[CrossRef]
    [Google Scholar]
  14. Dziadek, J., Madiraju, M. V., Rutherford, S. A., Atkinson, M. A. & Rajagopalan, M. ( 2002; ). Physiological consequences associated with overproduction of Mycobacterium tuberculosis FtsZ in mycobacterial hosts. Microbiology 148, 961–971.
    [Google Scholar]
  15. Dziadek, J., Rutherford, S. A., Madiraju, M. V., Atkinson, M. A. & Rajagopalan, M. ( 2003; ). Conditional expression of Mycobacterium smegmatis ftsZ, an essential cell division gene. Microbiology 149, 1593–1603.[CrossRef]
    [Google Scholar]
  16. Frohman, M. A. ( 1993; ). Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE. Methods Enzymol 218, 340–356.
    [Google Scholar]
  17. Goehring, N. W. & Beckwith, J. ( 2005; ). Diverse paths to midcell: assembly of the bacterial cell division machinery. Curr Biol 15, 514–526.[CrossRef]
    [Google Scholar]
  18. Gomez, J. E. & Bishai, W. R. ( 2000; ). whmD is an essential mycobacterial gene required for proper septation and cell division. Proc Natl Acad Sci U S A 97, 8554–8559.[CrossRef]
    [Google Scholar]
  19. Greendyke, R., Rajagopalan, M., Parish, T. & Madiraju, M. V. ( 2002; ). Conditional expression of Mycobacterium smegmatis dnaA, an essential DNA replication gene. Microbiology 148, 3887–3900.
    [Google Scholar]
  20. Harada, K. ( 1976; ). The nature of mycobacterial acid-fastness. Stain Technol 51, 255–260.
    [Google Scholar]
  21. Jacobs, C. & Shapiro, L. ( 1999; ). Bacterial cell division: a moveable feast. Proc Natl Acad Sci U S A 96, 5891–5893.[CrossRef]
    [Google Scholar]
  22. Jain, S., Kaushal, D., DasGupta, S. K. & Tyagi, A. K. ( 1997; ). Construction of shuttle vectors for genetic manipulation and molecular analysis of mycobacteria. Gene 190, 37–44.[CrossRef]
    [Google Scholar]
  23. Jennings, L. D., Foreman, K. W., Rush, T. S., 3rd & 12 other authors ( 2004; ). Design and synthesis of indolo[2,3-a]quinolizin-7-one inhibitors of the ZipA-FtsZ interaction. Bioorg Med Chem Lett 14, 1427–1431.[CrossRef]
    [Google Scholar]
  24. Kang, C. M., Abbott, D. W., Park, S. T., Dascher, C. C., Cantley, L. C. & Husson, R. N. ( 2005; ). The Mycobacterium tuberculosis serine/threonine kinases PknA and PknB: substrate identification and regulation of cell shape. Genes Dev 19, 1692–1704.[CrossRef]
    [Google Scholar]
  25. Kim, T. H., Park, J. S., Kim, H. J., Kim, Y., Kim, P. & Lee, H. S. ( 2005; ). The whcE gene of Corynebacterium glutamicum is important for survival following heat and oxidative stress. Biochem Biophys Res Commun 337, 757–764.[CrossRef]
    [Google Scholar]
  26. Manabe, Y. C., Chen, J. M., Ko, C. G., Chen, P. & Bishai, W. R. ( 1999; ). Conditional sigma factor expression, using the inducible acetamidase promoter, reveals that the Mycobacterium tuberculosis sigF gene modulates expression of the 16-kilodalton alpha-crystallin homologue. J Bacteriol 181, 7629–7633.
    [Google Scholar]
  27. Margalit, D. N., Romberg, L., Mets, R. B., Hebert, A. M., Mitchison, T. J., Kirschner, M. W. & RayChaudhuri, D. ( 2004; ). Targeting cell division: small-molecule inhibitors of FtsZ GTPase perturb cytokinetic ring assembly and induce bacterial lethality. Proc Natl Acad Sci U S A 101, 11821–11826.[CrossRef]
    [Google Scholar]
  28. Miller, J. H. ( 1972; ). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  29. Mizuguchi, Y., Ogawa, M. & Udou, T. ( 1985; ). Morphological changes induced by beta-lactam antibiotics in Mycobacterium avium-intracellulare complex. Antimicrob Agents Chemother 27, 541–547.[CrossRef]
    [Google Scholar]
  30. Morris, R. P., Nguyen, L., Gatfield, J. & 9 other authors ( 2005; ). Ancestral antibiotic resistance in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 102, 12200–12205.[CrossRef]
    [Google Scholar]
  31. Nguyen, L., Chinnapapagari, S. & Thompson, C. J. ( 2005; ). FbpA-dependent biosynthesis of trehalose dimycolate is required for the intrinsic multidrug resistance, cell wall structure, and colonial morphology of Mycobacterium smegmatis. J Bacteriol 187, 6603–6611.[CrossRef]
    [Google Scholar]
  32. North, R. J. & Izzo, A. A. ( 1993; ). Mycobacterial virulence. Virulent strains of Mycobacterium tuberculosis have faster in vivo doubling times and are better equipped to resist growth-inhibiting functions of macrophages in the presence and absence of specific immunity. J Exp Med 177, 1723–1733.[CrossRef]
    [Google Scholar]
  33. 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]
  34. Patterson, J. H., Waller, R. F., Jeevarajah, D., Billman-Jacobe, H. & McConville, M. J. ( 2003; ). Mannose metabolism is required for mycobacterial growth. Biochem J 372, 77–86.[CrossRef]
    [Google Scholar]
  35. Raghunand, T. R. & Bishai, W. R. ( 2006; ). Mapping essential domains of Mycobacterium smegmatis WhmD: insights into WhiB structure and function. J Bacteriol (in press).
    [Google Scholar]
  36. Rajagopalan, M., Maloney, E., Dziadek, J., Poplawska, M., Lofton, H., Chauhan, A. & Madiraju, M. V. ( 2005; ). Genetic evidence that mycobacterial FtsZ and FtsW proteins interact, and colocalize to the division site in Mycobacterium smegmatis. FEMS Microbiol Lett 250, 9–17.[CrossRef]
    [Google Scholar]
  37. Ramakrishnan, L., Federspiel, N. A. & Falkow, S. ( 2000; ). Granuloma-specific expression of Mycobacterium virulence proteins from the glycine-rich PE-PGRS family. Science 288, 1436–1439.[CrossRef]
    [Google Scholar]
  38. Roy, S. & Ajitkumar, P. ( 2005; ). Transcriptional analysis of the principal cell division gene, ftsZ, of Mycobacterium tuberculosis. J Bacteriol 187, 2540–2550.[CrossRef]
    [Google Scholar]
  39. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  40. Saviola, B., Woolwine, S. C. & Bishai, W. R. ( 2003; ). Isolation of acid-inducible genes of Mycobacterium tuberculosis with the use of recombinase-based in vivo expression technology. Infect Immun 71, 1379–1388.[CrossRef]
    [Google Scholar]
  41. Soliveri, J. A., Gomez, J., Bishai, W. R. & Chater, K. F. ( 2000; ). Multiple paralogous genes related to the Streptomyces coelicolor developmental regulatory gene whiB are present in Streptomyces and other actinomycetes. Microbiology 146, 333–343.
    [Google Scholar]
  42. Steyn, A. J., Collins, D. M., Hondalus, M. K., Jacobs, W. R., Jr, Kawakami, R. P. & Bloom, B. R. ( 2002; ). Mycobacterium tuberculosis WhiB3 interacts with RpoV to affect host survival but is dispensable for in vivo growth. Proc Natl Acad Sci U S A 99, 3147–3152.[CrossRef]
    [Google Scholar]
  43. Stover, C. K., de la Cruz, V. F., Fuerst, T. R. & other authors ( 1991; ). New use of BCG for recombinant vaccines. Nature 351, 456–460.[CrossRef]
    [Google Scholar]
  44. Wheeler, P. R. & Ratledge, C. ( 1994; ). Metabolism of Mycobacterium tuberculosis. In Tuberculosis: Pathogenesis, Protection and Control, pp. 353–385. Edited by B. B. Bloom. Washington, DC: American Society for Microbiology.
  45. White, E. L., Suling, W. J., Ross, L. J., Seitz, L. E. & Reynolds, R. C. ( 2002; ). 2-Alkoxycarbonylaminopyridines: inhibitors of Mycobacterium tuberculosis FtsZ. J Antimicrob Chemother 50, 111–114.[CrossRef]
    [Google Scholar]
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