Abstract
Members of the genus Mycobacterium respire using two terminal respiratory oxidases: a cytochrome bd-type menaquinol oxidase (encoded by cydABDC) or an aa3-type menaquinol-cytochrome c oxidase. During the transition to hypoxic (< 0.1% air saturation) conditions, cytochrome bd is induced in Mycobacterium smegmatis but the molecular mechanism governing this induction remains unknown. Analysis of the proposed cydABDC operon in M. smegmatis under hypoxic conditions revealed that cydAB and cydDC were not polycistronic. A transposon-mediated random mutagenesis was performed and revealed genes involved in redox homeostasis and defense against oxidase stress including KatG and PruC were required for cydAB operon expression. DNA-binding experiments were performed using a cydA promoter as target DNA and a DNA-binding histone-like protein (Hlp) was identified as protein bound to the cydAB promoter region. A cAMP receptor protein (CRP)-binding site (GTGAN6CCACA) was identified in the regulatory region of the cydAB operon and mutations in this sequence caused a significant reduction in cydA-lacZ expression.
The M. smegmatis mc2155 genome has two copies of CRP [Msmeg_0539 (Crp1) and Msmeg_6189 (Crp2)]. Phylogenetic analysis suggested that Crp1 is uniquely present in fast-growing mycobacteria, whilst Crp2 is closely related to CRP from other slow- growing mycobacteria. In addition, only Crp1 but not Crp2 could complement a Δcrp mutant of the Gram-negative bacterium, Pectobacterium atrosepticum. To dissect the physiological role of each Crp protein, transcriptional profiling studies using deletion mutants were conducted. Microarray analysis of the crp1 deletion mutant revealed 239 genes were differentially regulated including those involved in carbon catabolism and energy metabolism. However, a crp2-deletion mutant could not be obtained suggesting that this gene is essential for growth in M. smegmatis. Therefore, crp2 was conditionally expressed and transcriptional profiling studies were performed. Microarray analysis of the crp2 overexpression strain showed 58 genes were differentially regulated including genes from many biological processes. Identification of the CRP promoter consensus in M. smegmatis revealed that both Crp1 and Crp2 recognise the same consensus sequence yet the regulon of Crp1 is distinct from that of Crp2. Taken together, these data demonstrate that each Crp could be activated in response to unique signals. The mechanism of how each Crp is activated remains to be elucidated.