1887

Abstract

Mycolic acids are vital components of the cell wall and are essential for survival. While most components of the fatty acid synthase-II (FAS-II) enzymic machinery that synthesizes these long chain -alkyl, -hydroxy fatty acids have been identified, the gene encoding the -hydroxyacyl-acyl carrier protein (ACP) dehydratase activity has remained elusive. Recent bioinformatics-based studies and drug inhibition experiments have identified the gene as a promising candidate for this role. Using a recently described, specialized transduction-based genetic tool we now demonstrate that , the homologue of , is an essential gene; null mutants of the gene could only be generated in a merodiploid strain which contained a second integrated acetamide-inducible copy of . Growth of the conditional mutant in the absence of acetamide resulted in loss of mycolic acid biosynthesis and eventually loss of viability due to cell lysis. Null mutants could also be generated in a strain containing an integrated copy of , indicating that was the functional counterpart of in . Our results demonstrate that is an essential gene involved in mycolic acid biosynthesis and encodes the FAS-II -hydroxyacyl-ACP dehydratase.

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2007-12-01
2024-03-28
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References

  1. Banerjee A., Dubnau E., Quemard A., Balasubramanian V., Um K. S., Wilson T., Collins D., de Lisle G., Jacobs W. R. Jr 1994; inhA , a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis . Science 263:227–230
    [Google Scholar]
  2. Banerjee A., Sugantino M., Sacchettini J. C., Jacobs W. R. Jr 1998; The mabA gene from the inhA operon of Mycobacterium tuberculosis encodes a 3-ketoacyl reductase that fails to confer isoniazid resistance. Microbiology 144:2697–2704
    [Google Scholar]
  3. Bardarov S., Bardarov S., Pavelka M. S., Sambandamurthy V., Larsen M., Tufariello J., Chan J., Hatfull G., Jacobs W. R. 2002; Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis , M.bovis BCG and M. smegmatis . Microbiology 148:3007–3017
    [Google Scholar]
  4. Bhatt A., Kremer L., Dai A. Z., Sacchettini J. C., Jacobs W. R. Jr 2005; Conditional depletion of KasA, a key enzyme of mycolic acid biosynthesis, leads to mycobacterial cell lysis. J Bacteriol 187:7596–7606
    [Google Scholar]
  5. Brennan P. J., Nikaido H. 1995; The envelope of mycobacteria. Annu Rev Biochem 64:29–63
    [Google Scholar]
  6. Brown A. K., Sridharan S., Kremer L., Lindenberg S., Dover L. G., Sacchettini J. C., Besra G. S. 2005; Probing the mechanism of the Mycobacterium tuberculosis β -ketoacyl-acyl carrier protein synthase III mt FabH: factors influencing catalysis and substrate specificity. J Biol Chem 280:32539–32547
    [Google Scholar]
  7. Brown A. K., Papaemmanouil A., Bhowruth V., Bhatt A., Dover L. G., Besra G. S. 2007; Flavonoid inhibition as novel anti-mycobacterial agents targeting Rv0636, a putative dehydratase enzyme involved in Mycobacterium tuberculosis fatty acid synthase-II. Microbiology 153:3314–3322
    [Google Scholar]
  8. Castell A., Johansson P., Unge T., Jones T. A., Backbro K. 2005; Rv0216, a conserved hypothetical protein from Mycobacterium tuberculosis that is essential for bacterial survival during infection, has a double hotdog fold. Protein Sci 14:1850–1862
    [Google Scholar]
  9. Cole S. T., Brosch R., Parkhill J., Garnier T., Churcher C., Harris D., Gordon S. V., Eiglmeier K., Gas S. other authors 1998; Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544
    [Google Scholar]
  10. Daugelat S., Kowall J., Mattow J., Bumann D., Winter R., Hurwitz R., Kaufmann S. H. 2003; The RD1 proteins of Mycobacterium tuberculosis : expression in Mycobacterium smegmatis and biochemical characterization. Microbes Infect 5:1082–1095
    [Google Scholar]
  11. Dover L. G., Cerdeno-Tarraga A. M., Pallen M. J., Parkhill J., Besra G. S. 2004; Comparative cell wall core biosynthesis in the mycolated pathogens, Mycobacterium tuberculosis and Corynebacterium diphtheriae . FEMS Microbiol Rev 28:225–250
    [Google Scholar]
  12. Dye C. 2006; Global epidemiology of tuberculosis. Lancet 367:938–940
    [Google Scholar]
  13. Gande R., Gibson K. J., Brown A. K., Krumbach K., Dover L. G., Sahm H., Shioyama S., Oikawa T., Besra G. S., Eggeling L. 2004; Acyl-CoA carboxylases ( accD2 and accD3 ), together with a unique polyketide synthase ( Cg - pks ), are key to mycolic acid biosynthesis in Corynebacterianeae such as Corynebacterium glutamicum and Mycobacterium tuberculosis . J Biol Chem 279:44847–44857
    [Google Scholar]
  14. Kaye K., Frieden T. R. 1996; Tuberculosis control: the relevance of classic principles in an era of acquired immunodeficiency syndrome and multidrug resistance. Epidemiol Rev 18:52–63
    [Google Scholar]
  15. Kikuchi S., Kusaka T. 1984; Purification of NADPH-dependent enoyl-CoA reductase involved in the malonyl-CoA dependent fatty acid elongation system of Mycobacterium smegmatis . J Biochem (Tokyo 96:841–848
    [Google Scholar]
  16. 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 other authors 2000; Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in Mycobacterium tuberculosis . J Biol Chem 275:16857–16864
    [Google Scholar]
  17. Kremer L., Nampoothiri K. M., Lesjean S., Dover L. G., Graham S., Betts J., Brennan P. J., Minnikin D. E., Locht C., Besra G. S. 2001; Biochemical characterization of acyl carrier protein (AcpM) and malonyl-CoA : AcpM transacylase (mtFabD), two major components of Mycobacterium tuberculosis fatty acid synthase II. J Biol Chem 276:27967–27974
    [Google Scholar]
  18. Lea-Smith D. J., Pyke J. S., Tull D., McConville M. J., Coppel R. L., Crellin P. K. 2007; The reductase that catalyzes mycolic motif synthesis is required for efficient attachment of mycolic acids to arabinogalactan. J Biol Chem 282:11000–11008
    [Google Scholar]
  19. 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
    [Google Scholar]
  20. Mdluli K., Slayden R. A., Zhu Y., Ramaswamy S., Pan X., Mead D., Crane D. D., Musser J. M., Barry C. E. III 1998; Inhibition of a Mycobacterium tuberculosis β -ketoacyl ACP synthase by isoniazid. Science 280:1607–1610
    [Google Scholar]
  21. Minnikin D. E. 1982; Lipids: complex lipids, their chemistry, biosynthesis and roles. In The Biology of the Mycobacteria: Physiology, Identification and Classification Edited by Ratledge C., Stanford J. London, UK: Academic Press;
    [Google Scholar]
  22. Paolo W. F. Jr, Nosanchuk J. D. 2004; Tuberculosis in New York city: recent lessons and a look ahead. Lancet Infect Dis 4:287–293
    [Google Scholar]
  23. Parish T., Roberts G., Laval F., Schaeffer M., Daffe M., Duncan K. 2007; Functional complementation of the essential gene fabG1 of Mycobacterium tuberculosis by Mycobacterium smegmatis fabG but not Escherichia coli fabG . J Bacteriol 189:3721–3728
    [Google Scholar]
  24. Portevin D., De Sousa-D'Auria C., Houssin C., Grimaldi C., Chami M., Daffe M., Guilhot C. 2004; A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms. Proc Natl Acad Sci U S A 101:314–319
    [Google Scholar]
  25. Portevin D., de Sousa-D'Auria C., Montrozier H., Houssin C., Stella A., Laneelle M. A., Bardou F., Guilhot C., Daffe M. 2005; The acyl-AMP ligase FadD32 and AccD4-containing acyl-CoA carboxylase are required for the synthesis of mycolic acids and essential for mycobacterial growth: identification of the carboxylation product and determination of the acyl-CoA carboxylase components. J Biol Chem 280:8862–8874
    [Google Scholar]
  26. Qin Y. M., Haapalainen A. M., Kilpelainen S. H., Marttila M. S., Koski M. K., Glumoff T., Novikov D. K., Hiltunen J. K. 2000; Human peroxisomal multifunctional enzyme type 2. Site-directed mutagenesis studies show the importance of two protic residues for 2-enoyl-CoA hydratase 2 activity. J Biol Chem 275:4965–4972
    [Google Scholar]
  27. Sambrook J., Russell D. W. 2001 Molecular Cloning: a Laboratory Manual , 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  28. Sassetti C. M., Boyd D. H., Rubin E. J. 2003; Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48:77–84
    [Google Scholar]
  29. Schaeffer M. L., Agnihotri G., Volker C., Kallender H., Brennan P. J., Lonsdale J. T. 2001; Purification and biochemical characterization of the Mycobacterium tuberculosis β -ketoacyl-acyl carrier protein synthases KasA and KasB. J Biol Chem 276:47029–47037
    [Google Scholar]
  30. Schroeder E. K., de Souza N., Santos D. S., Blanchard J. S., Basso L. A. 2002; Drugs that inhibit mycolic acid biosynthesis in Mycobacterium tuberculosis . Curr Pharm Biotechnol 3:197–225
    [Google Scholar]
  31. Smith S., Witkowski A., Joshi A. K. 2003; Structural and functional organization of the animal fatty acid synthase. Prog Lipid Res 42:289–317
    [Google Scholar]
  32. Snapper S. B., Melton R. E., Mustafa S., Kieser T., Jacobs W. R. Jr 1990; Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis . Mol Microbiol 4:1911–1919
    [Google Scholar]
  33. Stover C. K., de la Cruz V. F., Fuerst T. R., Burlein J. E., Benson L. A., Bennett L. T., Bansal G. P., Young J. F., Lee M. H. other authors 1991; New use of BCG for recombinant vaccines. Nature 351:456–460
    [Google Scholar]
  34. Takayama K., Wang C., Besra G. S. 2005; Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis . Clin Microbiol Rev 18:81–101
    [Google Scholar]
  35. Vilcheze C., Morbidoni H. R., Weisbrod T. R., Iwamoto H., Kuo M., Sacchettini J. C., Jacobs W. R. Jr 2000; Inactivation of the inhA -encoded fatty acid synthase II (FASII) enoyl-acyl carrier protein reductase induces accumulation of the FASI end products and cell lysis of Mycobacterium smegmatis . J Bacteriol 182:4059–4067
    [Google Scholar]
  36. Wright A., Bai G., Barrera L., Boulahbal F., Martín-Casabona N., Gilpin C., Drobniewski F., Havelková M., Lepe R. other authors 2006; Emergence of Mycobacterium tuberculosis with extensive resistance to second-line drugs–worldwide, 2000–2004. MMWR Morb Mortal Wkly Rep 55:301–305
    [Google Scholar]
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