1887

Abstract

is able to use a variety of carbon sources and current knowledge suggests that cholesterol is used as a carbon source during infection. The catabolized cholesterol is used both as an energy source (ATP generation) and as a source of precursor molecules for the synthesis of complex methyl-branched fatty acids. In previous studies, we described a TetR-type transcriptional repressor, , that controls the expression of a number of genes involved in cholesterol catabolism. In this study, we describe a second TetR-type repressor, which we call We knocked this gene out in and used microarrays and quantitative RT-PCR to examine the effects on gene expression. We identified a palindromic regulatory motif for KstR2, showed that this motif is present in three promoter regions in mycobacteria and rhodococcus, and demonstrated binding of purified KstR2 to the motif. Using a combination of motif location analysis, gene expression analysis and the examination of gene conservation, we suggest that controls the expression of a 15 gene regulon. Like , and the regulon are highly conserved among the actinomycetes and studies in rhodococcus suggest a role for these genes in cholesterol catabolism. The functional significance of the regulon and implications for the control of cholesterol utilization are discussed.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.034538-0
2010-05-01
2020-09-26
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/5/1362.html?itemId=/content/journal/micro/10.1099/mic.0.034538-0&mimeType=html&fmt=ahah

References

  1. Bailey T. L., Elkan C.. 1994; Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol2:28–36
    [Google Scholar]
  2. Bailey T. L., Gribskov M.. 1998; Combining evidence using p-values: application to sequence homology searches. Bioinformatics14:48–54
    [Google Scholar]
  3. Benjamini Y.. 1995; Controlling false discovery rate: a practical and powerful approach to multiple testing. J Roy Stat Soc B57:289–300
    [Google Scholar]
  4. Brzostek A., Dziadek B., Rumijowska-Galewicz A., Pawelczyk J., Dziadek J.. 2007; Cholesterol oxidase is required for virulence of Mycobacterium tuberculosis. FEMS Microbiol Lett275:106–112
    [Google Scholar]
  5. Camus J. C., Pryor M. J., Medigue C., Cole S. T.. 2002; Re-annotation of the genome sequence of Mycobacterium tuberculosis H37Rv. Microbiology148:2967–2973
    [Google Scholar]
  6. Capyk J. K., D'Angelo I., Strynadka N. C., Eltis L. D.. 2009; Characterization of 3-ketosteroid 9 α-hydroxylase, a Rieske oxygenase in the cholesterol degradation pathway of Mycobacterium tuberculosis. J Biol Chem284:9937–9946
    [Google Scholar]
  7. Carver T. J., Rutherford K. M., Berriman M., Rajandream M. A., Barrell B. G., Parkhill J.. 2005; ACT: the Artemis Comparison Tool. Bioinformatics21:3422–3423
    [Google Scholar]
  8. Chang J. C., Harik N. S., Liao R. P., Sherman D. R.. 2007; Identification of mycobacterial genes that alter growth and pathology in macrophages and in mice. J Infect Dis196:788–795
    [Google Scholar]
  9. Chang J. C., Miner M. D., Pandey A. K., Gill W. P., Harik N. S., Sassetti C. M., Sherman D. R.. 2009; igr genes and Mycobacterium tuberculosis cholesterol metabolism. J Bacteriol191:5232–5239
    [Google Scholar]
  10. Cole S. T.. 1999; Learning from the genome sequence of Mycobacterium tuberculosis H37Rv. FEBS Lett452:7–10
    [Google Scholar]
  11. Crooks G. E., Hon G., Chandonia J. M., Brenner S. E.. 2004; WebLogo: a sequence logo generator. Genome Res14:1188–1190
    [Google Scholar]
  12. Dubnau E., Fontan P., Manganelli R., Soares-Appel S., Smith I.. 2002; Mycobacterium tuberculosis genes induced during infection of human macrophages. Infect Immun70:2787–2795
    [Google Scholar]
  13. Dubnau E., Chan J., Mohan V. P., Smith I.. 2005; Responses of Mycobacterium tuberculosis to growth in the mouse lung. Infect Immun73:3754–3757
    [Google Scholar]
  14. Honer zu Bentrup K., Russell D. G.. 2001; Mycobacterial persistence: adaptation to a changing environment. Trends Microbiol9:597–605
    [Google Scholar]
  15. Hu Y., van der Geize R., Besra G. S., Gurcha S. S., Liu A., Rohde M., Singh M., Coates A.. 2010; 3-Ketosteroid 9 α-hydroxylase is an essential factor in the pathogenesis of Mycobacterium tuberculosis. Mol Microbiol75:107–121
    [Google Scholar]
  16. Kendall S. L., Withers M., Soffair C. N., Moreland N. J., Gurcha S., Sidders B., Frita R., Ten Bokum A., Besra G. S.. other authors 2007; A highly conserved transcriptional repressor controls a large regulon involved in lipid degradation in Mycobacterium smegmatis and Mycobacterium tuberculosis. Mol Microbiol65:684–699
    [Google Scholar]
  17. Knol J., Bodewits K., Hessels G. I., Dijkhuizen L., van der Geize R.. 2008; 3-Keto-5 α-steroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1 and its orthologue in Mycobacterium tuberculosis H37Rv are highly specific enzymes that function in cholesterol catabolism. Biochem J410:339–346
    [Google Scholar]
  18. Lack N. A., Kawamura A., Fullam E., Laurieri N., Beard S., Russell A. J., Evangelopoulos D., Westwood I., Sim E.. 2009; Temperature stability of proteins essential for the intracellular survival of Mycobacterium tuberculosis. Biochem J418:369–378
    [Google Scholar]
  19. Ladunga I.. 2002; Finding homologs to nucleotide sequences using network blast searches. Curr Protoc Bioinformatics26:3.3.1–3.3.26
    [Google Scholar]
  20. McKinney J. D., Honer zu Bentrup K., Munoz-Elias E. J., Miczak A., Chen B., Chan W. T., Swenson D., Sacchettini J. C., Jacobs W. R. Jr, Russell D. G.. 2000; Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature406:735–738
    [Google Scholar]
  21. Munoz-Elias E. J., McKinney J. D.. 2005; Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat Med11:638–644
    [Google Scholar]
  22. Munoz-Elias E. J., Upton A. M., Cherian J., McKinney J. D.. 2006; Role of the methylcitrate cycle in Mycobacterium tuberculosis metabolism, intracellular growth, and virulence. Mol Microbiol60:1109–1122
    [Google Scholar]
  23. Nesbitt N. M., Yang X., Fontan P., Kolesnikova I., Smith I., Sampson N. S., Dubnau E.. 2010; A thiolase of Mycobacterium tuberculosis is required for virulence and production of androstenedione and androstadienedione from cholesterol. Infect Immun78:275–282
    [Google Scholar]
  24. Nevozhay D., Adams R. M., Murphy K. F., Josic K., Balazsi G.. 2009; Negative autoregulation linearizes the dose-response and suppresses the heterogeneity of gene expression. Proc Natl Acad Sci U S A106:5123–5128
    [Google Scholar]
  25. Pandey A. K., Sassetti C. M.. 2008; Mycobacterial persistence requires the utilization of host cholesterol. Proc Natl Acad Sci U S A105:4376–4380
    [Google Scholar]
  26. Parish T., Stoker N. G.. 2000; Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology146:1969–1975
    [Google Scholar]
  27. Parish T., Gordhan B. G., McAdam R. A., Duncan K., Mizrahi V., Stoker N. G.. 1999; Production of mutants in amino acid biosynthesis genes of Mycobacterium tuberculosis by homologous recombination. Microbiology145:3497–3503
    [Google Scholar]
  28. Rengarajan J., Bloom B. R., Rubin E. J.. 2005; Genome-wide requirements for Mycobacterium tuberculosis adaptation and survival in macrophages. Proc Natl Acad Sci U S A102:8327–8332
    [Google Scholar]
  29. Sacchettini J. C., Rubin E. J., Freundlich J. S.. 2008; Drugs versus bugs: in pursuit of the persistent predator Mycobacterium tuberculosis. Nat Rev Microbiol6:41–52
    [Google Scholar]
  30. Sambrook J., Russell D.. 2001; Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor: Cold Spring Harbor Laboratory Press;
  31. Sassetti C. M., Rubin E. J.. 2003; Genetic requirements for mycobacterial survival during infection. Proc Natl Acad Sci U S A100:12989–12994
    [Google Scholar]
  32. Schnappinger D., Ehrt S., Voskuil M. I., Liu Y., Mangan J. A., Monahan I. M., Dolganov G., Efron B., Butcher P. D.. other authors 2003; Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: insights into the phagosomal environment. J Exp Med198:693–704
    [Google Scholar]
  33. Shen-Orr S. S., Milo R., Mangan S., Alon U.. 2002; Network motifs in the transcriptional regulation network of Escherichia coli. Nat Genet31:64–68
    [Google Scholar]
  34. Smyth G. K., Michaud J., Scott H. S.. 2005; Use of within-array replicate spots for assessing differential expression in microarray experiments. Bioinformatics21:2067–2075
    [Google Scholar]
  35. 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 Microbiol4:1911–1919
    [Google Scholar]
  36. Tailleux L., Waddell S. J., Pelizzola M., Mortellaro A., Withers M., Tanne A., Castagnoli P. R., Gicquel B., Stoker N. G.. other authors 2008; Probing host pathogen cross-talk by transcriptional profiling of both Mycobacterium tuberculosis and infected human dendritic cells and macrophages. PLoS One3:e1403
    [Google Scholar]
  37. Talaat A. M., Lyons R., Howard S. T., Johnston S. A.. 2004; The temporal expression profile of Mycobacterium tuberculosis infection in mice. Proc Natl Acad Sci U S A101:4602–4607
    [Google Scholar]
  38. Thompson J. D., Higgins D. G., Gibson T. J.. 1994; clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res22:4673–4680
    [Google Scholar]
  39. Van der Geize R., Yam K., Heuser T., Wilbrink M. H., Hara H., Anderton M. C., Sim E., Dijkhuizen L., Davies J. E.. other authors 2007; A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages. Proc Natl Acad Sci U S A104:1947–1952
    [Google Scholar]
  40. Wernisch L., Kendall S. L., Soneji S., Wietzorrek A., Parish T., Hinds J., Butcher P. D., Stoker N. G.. 2003; Analysis of whole-genome microarray replicates using mixed models. Bioinformatics19:53–61
    [Google Scholar]
  41. Yam K. C., D'Angelo I., Kalscheuer R., Zhu H., Wang J. X., Snieckus V., Ly L. H., Converse P. J., Jacobs W. R. Jr. other authors 2009; Studies of a ring-cleaving dioxygenase illuminate the role of cholesterol metabolism in the pathogenesis of Mycobacterium tuberculosis. PLoS Pathog5:e1000344
    [Google Scholar]
  42. Yang X., Nesbitt N. M., Dubnau E., Smith I., Sampson N. S.. 2009; Cholesterol metabolism increases the metabolic pool of propionate in Mycobacterium tuberculosis. Biochemistry48:3819–3821
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.034538-0
Loading
/content/journal/micro/10.1099/mic.0.034538-0
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