Regulation of the expression of whiB1 in Mycobacterium tuberculosis: role of cAMP receptor protein

Abstract

The wbl (whiB-like) genes encode putative transcription factors unique to actinomycetes. This study characterized the promoter element of one of the seven wbl genes of Mycobacterium tuberculosis, whiB1 (Rv3219c). The results reveal that whiB1 is transcribed by a class I-type cAMP receptor protein (CRP)-dependent promoter, harbouring a CRP-binding site positioned at "58?5 with respect to its transcription start point. In vivo promoter activity analysis and electrophoretic mobility shift assays suggest that the expression of whiB1 is indeed regulated by cAMP-dependent binding of CRP M (encoded by the M. tuberculosis gene Rv3676) to the whiB1 59 untranslated region (59UTR). b-Galactosidase gene fusion analysis revealed induction of the whiB1 promoter in M. tuberculosis on addition of exogenous dibutyric cAMP (a diffusible cAMP analogue) only when an intact CRP-binding site was present. These results indicate that M. tuberculosis whiB1 transcription is regulated in part by cAMP levels via direct binding of cAMP-activated CRP M to a consensus CRP-binding site in the whiB1 59UTR. INTRODUCTIONMycobacterium tuberculosis, the aetiological agent of tuberculosis, accounts for nearly 2 million human deaths every year. Approximately one-third of the world's population is latently infected with M. tuberculosis, and 7 to 8 million new TB cases occur annually (Dye et al., 1999). M. tuberculosis can survive in diverse surroundings ranging from the human host to droplet nuclei in the atmosphere. Adaptation to such diverse conditions requires controlled regulation of the expression of key genes that allow the bacillus to alter its physiology in response to changes in environmental stimuli. Sequencing of the M. tuberculosis genome has revealed the presence of more than 100 regulatory proteins, 13 sigma factors and 11 two-component systems (Cole et al., 1998), which provide the bacterium with a high degree of adaptability.The Wbl (WhiB-like) family of proteins is present throughout the actinomycetes but absent from all other organisms evaluated so far (Molle et al., 2000;Soliveri et al., 2000). Due to the presence of a conserved helix-turn-helix motif, these proteins are believed to function as DNA-binding transcription regulators. The first of these proteins, WhiB, was identified in Streptomyces coelicolor, a Gram-positive sporulating bacterium closely related to M. tuberculosis. S. coelicolor whiB mutants produce abnormally long, tightly coiled aerial hyphae that are completely blocked in their ability to form sporulation septa (Chater, 1972;Davis & Chater, 1992;Flardh et al., 1999). Studies of whiB orthologues in mycobacteria have shown that the M. smegmatis whiB2 gene (also called whmD) is essential ; M. tuberculosis whiB3 plays a role in virulence and its gene product may interact with a sigma factor of RNA polymerase (Steyn et al., 2002); whiB7 of M. tuberculosis is involved in multi-drug resistance (Morris et al., 2005). Each of the Wbl family of proteins contains four invariant cysteine residues, which are believed to be inv...

Mycobacterium tuberculosis WhiB3 responds to O 2 and nitric oxide via its [4Fe-4S] cluster and is essential for nutrient starvation survival

“…The latter is supported by elegant studies showing that in response to carbon source starvation, E. coli FNR and ArcA (24,25) regulate the synthesis of key enzymes involved in glycolysis and tricarboxylic acid cycle. Finally, the severe growth impairment of Mtb⌬whiB3 on various carbon sources as well as the induction of WhiB2 (15), WhiB1 (26), and WhiB3 (27) upon nutrient limitation, by exogenous cAMP, and during infection, respectively, strongly suggests that some Mtb WhiB family members play an important role in sensing fluctuations in the intracellular redox state.…”

Genomic mapping of cAMP receptor protein (CRPMt) inMycobacterium tuberculosis: relation to transcriptional start sites and the role of CRPMtas a transcription factor

“…The corresponding M. tuberculosis CRP Mt , encoded by Rv3676, recognizes a similar binding motif and has been shown to regulate transcription of several promoters—for example, the serC promoter (13), the rpfA promoter (14), the whiB1 promoter (15,16) and the frdA promoter (17). Computational approaches and in vitro DNA-binding studies suggest that M. tuberculosis CRP Mt has multiple targets and is likely to share the global profile of the E. coli homologue (18–20).…”

Mycobacterium Tuberculosis Signaling via c-AMP

“…Expression of M. tuberculosis whiB1, a member of the Wbl (WhiB-like) family, is controlled positively and negatively by CRP Mt Agarwal et al, 2006). A CRP Mt binding site (CRP Mt 1) was detected in whiB1's upstream regulatory region (Smith et al, 2010) (Rickman et al, 2005).…”

“…A CRP Mt binding site (CRP Mt 1) was detected in whiB1's upstream regulatory region (Smith et al, 2010) (Rickman et al, 2005). Reporter assays with native and mutated promoter sequences indicated that transcription from the native, but not the mutant, promoter was affected by cAMP levels via the direct binding with CRP Mt (Agarwal et al, 2006 CtpE, methyl-accepting chemotaxis protein Table 2. Mycobaterium genes belonging to CRP regulon and their associated function regulatory effect inhibiting whiB1 expression .…”