title: Novel regulatory roles of cAMP receptor proteins in fast-growing environmental mycobacteria.
abstract: Mycobacterium smegmatis 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 crp1 mutant of M. smegmatis was readily obtained, but crp2 could not be deleted, suggesting it was essential for growth. A total of 239 genes were differentially regulated in response to crp1 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 M. smegmatis, the crp2 gene was overexpressed (gain of function) and transcriptional profiling studies revealed that 58 genes were differentially regulated. Identification of the CRP promoter consensus in M. smegmatis showed that both Crp1 and Crp2 recognized the same consensus sequence (TGTGN8CACA). 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, sdh1). Expression of the sdh1 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 sdh1 promoter was cAMP-independent whereas Crp2 binding was cAMP-dependent. These data suggest that Crp1 and Crp2 respond to distinct signalling pathways in M. smegmatis to coordinate gene expression in response to carbon and energy supply.
authors.affiliation: Department of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Auckland 1042, New Zealand.
authors.forename: Htin Lin
authors.initials: HL
authors.lastname: Aung
authors.affiliation: The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Laura L
authors.initials: LL
authors.lastname: Dixon
authors.affiliation: The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Smith
authors.affiliation: Division of Mycobacterial Research, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
authors.forename: Nathan P
authors.initials: NP
authors.lastname: Sweeney
authors.affiliation: Department of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Robson
authors.affiliation: Department of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Berney
authors.affiliation: Division of Mycobacterial Research, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
authors.forename: Roger S
authors.initials: RS
authors.lastname: Buxton
authors.affiliation: The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Green
authors.affiliation: Department of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Auckland 1042, New Zealand gregory.cook@otago.ac.nz.
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Cook
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D009165:Mycobacterium Infections, Nontuberculous / Q000382:microbiology; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D009876:Operon; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D002244:Carbon