is a fast-growing, saprophytic, mycobacterial species that contains two cAMP-receptor protein (CRP) homologues designated herein as Crp1 and Crp2. Phylogenetic analysis suggests that Crp1 (Msmeg_0539) is uniquely present in fast-growing environmental mycobacteria, whereas Crp2 (Msmeg_6189) occurs in both fast- and slow-growing species. A mutant of was readily obtained, but could not be deleted, suggesting it was essential for growth. A total of 239 genes were differentially regulated in response to deletion (loss of function), including genes coding for mycobacterial energy generation, solute transport and catabolism of carbon sources. To assess the role of Crp2 in , the gene was overexpressed (gain of function) and transcriptional profiling studies revealed that 58 genes were differentially regulated. Identification of the CRP promoter consensus in showed that both Crp1 and Crp2 recognized the same consensus sequence (TGTGNCACA). Comparison of the Crp1- and Crp2-regulated genes revealed distinct but overlapping regulons with 11 genes in common, including those of the succinate dehydrogenase operon (MSMEG_0417-0420, ). Expression of the operon was negatively regulated by Crp1 and positively regulated by Crp2. Electrophoretic mobility shift assays with purified Crp1 and Crp2 demonstrated that Crp1 binding to the promoter was cAMP-independent whereas Crp2 binding was cAMP-dependent. These data suggest that Crp1 and Crp2 respond to distinct signalling pathways in to coordinate gene expression in response to carbon and energy supply.


Article metrics loading...

Loading full text...

Full text loading...



  1. Agarwal N., Lamichhane G., Gupta R., Nolan S., Bishai W. R. (2009). Cyclic AMP intoxication of macrophages by a Mycobacterium tuberculosis adenylate cyclase. Nature 460, 98102. [View Article][PubMed] [Google Scholar]
  2. Aung H. L., Berney M., Cook G. M. (2014). Hypoxia-activated cytochrome bd expression in Mycobacterium smegmatis is cyclic AMP receptor protein dependent. J Bacteriol 196, 30913097. [View Article][PubMed] [Google Scholar]
  3. Bai G., McCue L. A., McDonough K. A. (2005). Characterization of Mycobacterium tuberculosis Rv3676 (CRPMt), a cyclic AMP receptor protein-like DNA binding protein. J Bacteriol 187, 77957804. [View Article][PubMed] [Google Scholar]
  4. Bai G., Schaak D. D., McDonough K. A. (2009). cAMP levels within Mycobacterium tuberculosis and Mycobacterium bovis BCG increase upon infection of macrophages. FEMS Immunol Med Microbiol 55, 6873. [View Article][PubMed] [Google Scholar]
  5. Bai G., Schaak D. D., Smith E. A., McDonough K. A. (2011). Dysregulation of serine biosynthesis contributes to the growth defect of a Mycobacterium tuberculosis crp mutant. Mol Microbiol 82, 180198. [View Article][PubMed] [Google Scholar]
  6. Bai N. J., Pai M. R., Murthy P. S., Venkitasubramanian T. A. (1976). Pathways of glucose catabolism in Mycobacterium smegmatis . Can J Microbiol 22, 13741380. [View Article][PubMed] [Google Scholar]
  7. Berg O. G., von Hippel P. H. (1988). Selection of DNA binding sites by regulatory proteins. II. The binding specificity of cyclic AMP receptor protein to recognition sites. J Mol Biol 200, 709723. [View Article][PubMed] [Google Scholar]
  8. Bergmeyer H. U., Bergmeyer J. r., Grassl M. (1983).Methods of Enzymatic Analysis, 3rd edn. Weinheim: Verlag Chemie. [Google Scholar]
  9. Berney M., Cook G. M. (2010). Unique flexibility in energy metabolism allows mycobacteria to combat starvation and hypoxia. PLoS ONE 5, e8614. [View Article][PubMed] [Google Scholar]
  10. Berney M., Weimar M. R., Heikal A., Cook G. M. (2012). Regulation of proline metabolism in mycobacteria and its role in carbon metabolism under hypoxia. Mol Microbiol 84, 664681. [View Article][PubMed] [Google Scholar]
  11. Berney M., Greening C., Conrad R., Jacobs W. R. Jr, Cook G. M. (2014). An obligately aerobic soil bacterium activates fermentative hydrogen production to survive reductive stress during hypoxia. Proc Natl Acad Sci U S A 111, 1147911484. [View Article][PubMed] [Google Scholar]
  12. Blokpoel M. C., Murphy H. N., O’Toole R., Wiles S., Runn E. S., Stewart G. R., Young D. B., Robertson B. D. (2005). Tetracycline-inducible gene regulation in mycobacteria. Nucleic Acids Res 33, e22. [View Article][PubMed] [Google Scholar]
  13. 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, 537544. [View Article][PubMed] [Google Scholar]
  14. Dass B. K., Sharma R., Shenoy A. R., Mattoo R., Visweswariah S. S. (2008). Cyclic AMP in mycobacteria: characterization and functional role of the Rv1647 ortholog in Mycobacterium smegmatis . J Bacteriol 190, 38243834. [View Article][PubMed] [Google Scholar]
  15. de Carvalho L. P., Zhao H., Dickinson C. E., Arango N. M., Lima C. D., Fischer S. M., Ouerfelli O., Nathan C., Rhee K. Y. (2010). Activity-based metabolomic profiling of enzymatic function: identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase. Chem Biol 17, 323332. [View Article][PubMed] [Google Scholar]
  16. Dehal P. S., Joachimiak M. P., Price M. N., Bates J. T., Baumohl J. K., Chivian D., Friedland G. D., Huang K. H., Keller K. & other authors (2010). MicrobesOnline: an integrated portal for comparative and functional genomics. Nucleic Acids Res 38 (Database issue), D396D400. [View Article][PubMed] [Google Scholar]
  17. Downing K. J., Betts J. C., Young D. I., McAdam R. A., Kelly F., Young M., Mizrahi V. (2004). Global expression profiling of strains harbouring null mutations reveals that the five rpf-like genes of Mycobacterium tuberculosis show functional redundancy. Tuberculosis (Edinb) 84, 167179. [View Article][PubMed] [Google Scholar]
  18. Downing K. J., Mischenko V. V., Shleeva M. O., Young D. I., Young M., Kaprelyants A. S., Apt A. S., Mizrahi V. (2005). Mutants of Mycobacterium tuberculosis lacking three of the five rpf-like genes are defective for growth in vivo and for resuscitation in vitro . Infect Immun 73, 30383043. [View Article][PubMed] [Google Scholar]
  19. Flamholz A., Noor E., Bar-Even A., Liebermeister W., Milo R. (2013). Glycolytic strategy as a tradeoff between energy yield and protein cost. Proc Natl Acad Sci U S A 110, 1003910044. [View Article][PubMed] [Google Scholar]
  20. Gazdik M. A., McDonough K. A. (2005). Identification of cyclic AMP-regulated genes in Mycobacterium tuberculosis complex bacteria under low-oxygen conditions. J Bacteriol 187, 26812692. [View Article][PubMed] [Google Scholar]
  21. Gazdik M. A., Bai G., Wu Y., McDonough K. A. (2009). Rv1675c (cmr) regulates intramacrophage and cyclic AMP-induced gene expression in Mycobacterium tuberculosis-complex mycobacteria. Mol Microbiol 71, 434448. [View Article][PubMed] [Google Scholar]
  22. Gebhard S., Tran S. L., Cook G. M. (2006). The Phn system of Mycobacterium smegmatis: a second high-affinity ABC-transporter for phosphate. Microbiology 152, 34533465. [View Article][PubMed] [Google Scholar]
  23. Green J., Scott C., Guest J. R. (2001). Functional versatility in the CRP-FNR superfamily of transcription factors: FNR and FLP. Adv Microb Physiol 44, 134. [View Article][PubMed] [Google Scholar]
  24. Green J., Stapleton M. R., Smith L. J., Artymiuk P. J., Kahramanoglou C., Hunt D. M., Buxton R. S. (2014). Cyclic-AMP and bacterial cyclic-AMP receptor proteins revisited: adaptation for different ecological niches. Curr Opin Microbiol 18, 17. [View Article][PubMed] [Google Scholar]
  25. Griffin J. E., Gawronski J. D., Dejesus M. A., Ioerger T. R., Akerley B. J., Sassetti C. M. (2011). High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism. PLoS Pathog 7, e1002251. [View Article][PubMed] [Google Scholar]
  26. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. (1989). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 5159. [View Article][PubMed] [Google Scholar]
  27. Hümpel A., Gebhard S., Cook G. M., Berney M. (2010). The SigF regulon in Mycobacterium smegmatis reveals roles in adaptation to stationary phase, heat, and oxidative stress. J Bacteriol 192, 24912502. [View Article][PubMed] [Google Scholar]
  28. Hunt D. M., Saldanha J. W., Brennan J. F., Benjamin P., Strom M., Cole J. A., Spreadbury C. L., Buxton R. S. (2008). Single nucleotide polymorphisms that cause structural changes in the cyclic AMP receptor protein transcriptional regulator of the tuberculosis vaccine strain Mycobacterium bovis BCG alter global gene expression without attenuating growth. Infect Immun 76, 22272234. [View Article][PubMed] [Google Scholar]
  29. Kahramanoglou C., Cortes T., Matange N., Hunt D. M., Visweswariah S. S., Young D. B., Buxton R. S. (2014). Genomic mapping of cAMP receptor protein (CRPMt) in Mycobacterium tuberculosis: relation to transcriptional start sites and the role of CRPMt as a transcription factor. Nucleic Acids Res 42, 83208329.[CrossRef] [Google Scholar]
  30. Kana B. D., Gordhan B. G., Downing K. J., Sung N., Vostroktunova G., Machowski E. E., Tsenova L., Young M., Kaprelyants A. & other authors (2008). The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro . Mol Microbiol 67, 672684. [View Article][PubMed] [Google Scholar]
  31. Kolb A., Busby S., Buc H., Garges S., Adhya S. (1993). Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem 62, 749797. [View Article][PubMed] [Google Scholar]
  32. Körner H., Sofia H. J., Zumft W. G. (2003). Phylogeny of the bacterial superfamily of Crp-Fnr transcription regulators: exploiting the metabolic spectrum by controlling alternative gene programs. FEMS Microbiol Rev 27, 559592. [View Article][PubMed] [Google Scholar]
  33. Kovárová-Kovar K., Egli T. (1998). Growth kinetics of suspended microbial cells: from single-substrate-controlled growth to mixed-substrate kinetics. Microbiol Mol Biol Rev 62, 646666.[PubMed] [Google Scholar]
  34. Krawczyk J., Kohl T. A., Goesmann A., Kalinowski J., Baumbach J. (2009). From Corynebacterium glutamicum to Mycobacterium tuberculosis – towards transfers of gene regulatory networks and integrated data analyses with MycoRegNet. Nucleic Acids Res 37, e97. [View Article][PubMed] [Google Scholar]
  35. Kumar P., Joshi D. C., Akif M., Akhter Y., Hasnain S. E., Mande S. C. (2010). Mapping conformational transitions in cyclic AMP receptor protein: crystal structure and normal-mode analysis of Mycobacterium tuberculosis apo-cAMP receptor protein. Biophys J 98, 305314. [View Article][PubMed] [Google Scholar]
  36. Larsson C., Luna B., Ammerman N. C., Maiga M., Agarwal N., Bishai W. R. (2012). Gene expression of Mycobacterium tuberculosis putative transcription factors whiB1–7 in redox environments. PLoS ONE 7, e37516. [View Article][PubMed] [Google Scholar]
  37. Lowrie D. B., Jackett P. S., Ratcliffe N. A. (1975). Mycobacterium microti may protect itself from intracellular destruction by releasing cyclic AMP into phagosomes. Nature 254, 600602. [View Article][PubMed] [Google Scholar]
  38. McCue L. A., McDonough K. A., Lawrence C. E. (2000). Functional classification of cNMP-binding proteins and nucleotide cyclases with implications for novel regulatory pathways in Mycobacterium tuberculosis . Genome Res 10, 204219. [View Article][PubMed] [Google Scholar]
  39. Padh H., Venkitasubramanian T. A. (1976). Cyclic adenosine 3′, 5′-monophosphate in mycobacteria. Indian J Biochem Biophys 13, 413414.[PubMed] [Google Scholar]
  40. Padh H., Venkitasubramanian T. A. (1980). Lack of adenosine-3′,5′-monophosphate receptor protein and apparent lack of expression of adenosine-3′,5′-monophosphate functions in Mycobacterium smegmatis CDC 46. Microbios 27, 6978.[PubMed] [Google Scholar]
  41. Pecsi I., Hards K., Ekanayaka N., Berney M., Hartman T., Jacobs W. R. Jr, Cook G. M. (2014). Essentiality of succinate dehydrogenase in Mycobacterium smegmatis and its role in the generation of the membrane potential under hypoxia. MBio 5, e01093-14. [View Article][PubMed] [Google Scholar]
  42. Pelicic V., Jackson M., Reyrat J. M., Jacobs W. R. Jr, Gicquel B., Guilhot C. (1997). Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis . Proc Natl Acad Sci U S A 94, 1095510960. [View Article][PubMed] [Google Scholar]
  43. Perrenoud A., Sauer U. (2005). Impact of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on glucose catabolism in Escherichia coli . J Bacteriol 187, 31713179. [View Article][PubMed] [Google Scholar]
  44. Peterson J. D., Umayam L. A., Dickinson T., Hickey E. K., White O. (2001). The Comprehensive Microbial Resource. Nucleic Acids Res 29, 123125. [View Article][PubMed] [Google Scholar]
  45. Pinter J. K., Hayashi J. A., Watson J. A. (1967). Enzymic assay of glycerol, dihydroxyacetone, and glyceraldehyde. Arch Biochem Biophys 121, 404414. [View Article][PubMed] [Google Scholar]
  46. Rickman L., Scott C., Hunt D. M., Hutchinson T., Menéndez M. C., Whalan R., Hinds J., Colston M. J., Green J., Buxton R. S. (2005). A member of the cAMP receptor protein family of transcription regulators in Mycobacterium tuberculosis is required for virulence in mice and controls transcription of the rpfA gene coding for a resuscitation promoting factor. Mol Microbiol 56, 12741286. [View Article][PubMed] [Google Scholar]
  47. Saier M. H. Jr (1989). Protein phosphorylation and allosteric control of inducer exclusion and catabolite repression by the bacterial phosphoenolpyruvate: sugar phosphotransferase system. Microbiol Rev 53, 109120.[PubMed] [Google Scholar]
  48. Saier M. H. Jr, Reizer J. (1994). The bacterial phosphotransferase system: new frontiers 30 years later. Mol Microbiol 13, 755764. [View Article][PubMed] [Google Scholar]
  49. Saitou N., Nei M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406425.[PubMed] [Google Scholar]
  50. Sambrook J., Russell D. W. (2001).Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. [Google Scholar]
  51. Schultz S. C., Shields G. C., Steitz T. A. (1991). Crystal structure of a CAP–DNA complex: the DNA is bent by 90 degrees. Science 253, 10011007. [View Article][PubMed] [Google Scholar]
  52. Shenoy A. R., Visweswariah S. S. (2006). Mycobacterial adenylyl cyclases: biochemical diversity and structural plasticity. FEBS Lett 580, 33443352. [View Article][PubMed] [Google Scholar]
  53. Shenoy A. R., Sivakumar K., Krupa A., Srinivasan N., Visweswariah S. S. (2004). A survey of nucleotide cyclases in actinobacteria: unique domain organization and expansion of the class III cyclase family in Mycobacterium tuberculosis . Comp Funct Genomics 5, 1738. [View Article][PubMed] [Google Scholar]
  54. Shimada T., Fujita N., Yamamoto K., Ishihama A. (2011). Novel roles of cAMP receptor protein (CRP) in regulation of transport and metabolism of carbon sources. PLoS ONE 6, e20081. [View Article][PubMed] [Google Scholar]
  55. 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, 19111919. [View Article][PubMed] [Google Scholar]
  56. Stapleton M., Haq I., Hunt D. M., Arnvig K. B., Artymiuk P. J., Buxton R. S., Green J. (2010). Mycobacterium tuberculosis cAMP receptor protein (Rv3676) differs from the Escherichia coli paradigm in its cAMP binding and DNA binding properties and transcription activation properties. J Biol Chem 285, 70167027. [View Article][PubMed] [Google Scholar]
  57. 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, 456460. [View Article][PubMed] [Google Scholar]
  58. Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S. (2011). mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 27312739. [View Article][PubMed] [Google Scholar]
  59. Titgemeyer F., Amon J., Parche S., Mahfoud M., Bail J., Schlicht M., Rehm N., Hillmann D., Stephan J. & other authors (2007). A genomic view of sugar transport in Mycobacterium smegmatis and Mycobacterium tuberculosis . J Bacteriol 189, 59035915. [View Article][PubMed] [Google Scholar]
  60. Tran S. L., Cook G. M. (2005). The F1Fo-ATP synthase of Mycobacterium smegmatis is essential for growth. J Bacteriol 187, 50235028. [View Article][PubMed] [Google Scholar]
  61. Weber I. T., Steitz T. A. (1987). Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2.5 Å resolution. J Mol Biol 198, 311326. [View Article][PubMed] [Google Scholar]
  62. You C., Okano H., Hui S., Zhang Z., Kim M., Gunderson C. W., Wang Y. P., Lenz P., Yan D., Hwa T. (2013). Coordination of bacterial proteome with metabolism by cyclic AMP signalling. Nature 500, 301306. [View Article][PubMed] [Google Scholar]

Data & Media loading...


Supplementary Data

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