Mycobacterium tuberculosis is a major pathogen that has the ability to establish, and emerge from, a persistent state. Wbl family proteins are associated with developmental processes in actinomycetes, and M. tuberculosis has seven such proteins. In the present study it is shown that the M. tuberculosis H37Rv whiB1 gene is essential. The WhiB1 protein possesses a [4Fe-4S]2+ cluster that is stable in air but reacts rapidly with eight equivalents of nitric oxide to yield two dinuclear dinitrosyl-iron thiol complexes. The [4Fe-4S] form of WhiB1 did not bind whiB1 promoter DNA, but the reduced and oxidized apo-WhiB1, and nitric oxide-treated holo-WhiB1 did bind to DNA. Mycobacterium smegmatis RNA polymerase induced transcription of whiB1 in vitro; however, in the presence of apo-WhiB1, transcription was severely inhibited, irrespective of the presence or absence of the CRP (cAMP receptor protein) Rv3676, which is known to activate whiB1 expression. Footprinting suggested that autorepression of whiB1 is achieved by apo-WhiB1 binding at a region that overlaps the core promoter elements. A model incorporating regulation of whiB1 expression in response to nitric oxide and cAMP is discussed with implications for sensing two important signals in establishing M. tuberculosis infections.

SummaryIn many species of bacteria most inducible DNA repair genes are regulated by LexA homologues and are dependent on RecA for induction. We have shown previously by analysing the induction of recA that two mechanisms for the induction of gene expression following DNA damage exist in Mycobacterium tuberculosis . Whereas one of these depends on RecA and LexA in the classical way, the other mechanism is independent of both of these proteins and induction occurs in the absence of RecA. Here we investigate the generality of each of these mechanisms by analysing the global response to DNA damage in both wild-type M. tuberculosis and a recA deletion strain of M. tuberculosis using microarrays. This revealed that the majority of the genes that were induced remained inducible in the recA mutant stain. Of particular note most of the inducible genes with known or predicted functions in DNA repair did not depend on recA for induction. Amongst these are genes involved in nucleotide excision repair, base excision repair, damage reversal and recombination. Thus, it appears that this novel mechanism of gene regulation is important for DNA repair in M. tuberculosis .

SummaryDeletion of gene Rv3676 in Mycobacterium tuberculosis coding for a transcription factor belonging to the cAMP receptor protein (CRP) family caused growth defects in laboratory medium, in bone marrowderived macrophages and in a mouse model of tuberculosis. Transcript profiling of M. tuberculosis grown in vitro identified 16 genes with significantly altered expression in the mutant compared with the wild type. Analysis of the DNA sequences upstream of the corresponding open reading frames revealed that 12 possessed sequences related to a consensus CRP binding site that could represent the sites of action of Rv3676. These included rpfA , lprQ , whiB1 and ahpC among genes with enhanced expression in the wild type, and Rv3616c-Rv3613c, Rv0188 and lipQ among genes exhibiting enhanced expression in the mutant. The activity of an rpfA :: lacZ promoter fusion was lowered in the Rv3676 mutant and by mutation of the predicted Rv3676 binding site. Moreover, the product of Rv3676 (isolated as a TrxA fusion protein) interacted specifically with the rpfA promoter, and binding was inhibited by mutation of the Rv3676 site. Although Rv3676 retains four of the six amino acid residues that bind cAMP in Escherichia coli CRP addition of cAMP did not enhance Rv3676 binding at the rpfA promoter in vitro . In summary, it has been shown that Rv3676 is a direct regulator of rpfA expression, and because rpfA codes for a resuscitation promoting factor this may implicate Rv3676 in reactivation of dormant M. tuberculosis infections.

The reactivity of protein bound iron−sulfur clusters with nitric oxide (NO) is well documented, but little is known about the actual mechanism of cluster nitrosylation. Here, we report studies of members of the Wbl family of [4Fe−4S] containing proteins, which play key roles in regulating developmental processes in actinomycetes, including Streptomyces and Mycobacteria, and have been shown to be NO responsive. Streptomyces coelicolor WhiD and Mycobacterium tuberculosis WhiB1 react extremely rapidly with NO in a multiphasic reaction involving, remarkably, 8 NO molecules per [4Fe−4S] cluster. The reaction is 104-fold faster than that observed with O2 and is by far the most rapid iron−sulfur cluster nitrosylation reaction reported to date. An overall stoichiometry of [Fe4S4(Cys)4]2− + 8NO → 2[FeI2(NO)4(Cys)2]0 + S2− + 3S0 has been established by determination of the sulfur products and their oxidation states. Kinetic analysis leads to a four-step mechanism that accounts for the observed NO dependence. DFT calculations suggest the possibility that the nitrosylation product is a novel cluster [FeI4(NO)8(Cys)4]0 derived by dimerization of a pair of Roussin’s red ester (RRE) complexes.

No abstract