Characterization of the transcriptional regulator Rv3124 of Mycobacterium tuberculosis identifies it as a positive regulator of molybdopterin biosynthesis and defines the functional consequences of a non-synonymous SNP in the Mycobacterium bovis BCG orthologue

Abstract

A number of single-nucleotide polymorphisms (SNPs) have been identified in the genome of Mycobacterium bovis BCG Pasteur compared with the sequenced strain M. bovis 2122/97. The functional consequences of many of these mutations remain to be described; however, mutations in genes encoding regulators may be particularly relevant to global phenotypic changes such as loss of virulence, since alteration of a regulator's function will affect the expression of a wide range of genes. One such SNP falls in bcg3145, encoding a member of the AfsR/DnrI/SARP class of global transcriptional regulators, that replaces a highly conserved glutamic acid residue at position 159 (E159G) with glycine in a tetratricopeptide repeat (TPR) located in the bacterial transcriptional activation (BTA) domain of BCG3145. TPR domains are associated with protein–protein interactions, and a conserved core (helices T1–T7) of the BTA domain seems to be required for proper function of SARP-family proteins. Structural modelling predicted that the E159G mutation perturbs the third α-helix of the BTA domain and could therefore have functional consequences. The E159G SNP was found to be present in all BCG strains, but absent from virulent M. bovis and Mycobacterium tuberculosis strains. By overexpressing BCG3145 and Rv3124 in BCG and H37Rv and monitoring transcriptome changes using microarrays, we determined that BCG3145/Rv3124 acts as a positive transcriptional regulator of the molybdopterin biosynthesis moa1 locus, and we suggest that rv3124 be renamed moaR1. The SNP in bcg3145 was found to have a subtle effect on the activity of MoaR1, suggesting that this mutation is not a key event in the attenuation of BCG.

Horizontal acquisition of a hypoxia-responsive molybdenum cofactor biosynthesis pathway contributed to Mycobacterium tuberculosis pathoadaptation

“…The genomic region in green was found atypical in genomic signature analysis [ 9 , 24 ]. The moaA1-D1 operon is indicated by the orange strip; the transcriptional activator-encoding gene rv3124 / moaR1 [ 35 ] is indicated in cyan. Importantly, the divergence of the rv3108-13 cluster to pBVIE01 locally falls to values that can be described as “intragenomic variability” (red-colored strip for Mtb ).…”

“…The MoaR1 (Rv3124) transcriptional regulator, which is encoded by a gene in the Moco-1 genomic island, was shown to activate moaA1-D1 gene expression in recombinant Rv3124-overexpressing Mtb and Mycobacterium bovis bacillus Calmette Guérin (BCG) strains [ 35 ]. Here, we found that moaR1 expression was also induced by hypoxia ( Fig 4C ).…”

“…In this construct, GFP is expressed under the control of the rv3109 promoter, as previously defined by Mendoza Lopez et al . [ 35 ]. When transformed in Mtb , the construct is stably integrated in single copy at the att site of the L5 mycobacteriophage.…”

Functional Analysis of Molybdopterin Biosynthesis in Mycobacteria Identifies a Fused Molybdopterin Synthase in Mycobacterium tuberculosis

“…A putative binding site for this regulator was identified upstream of moaX (1); however, expression of this gene was not affected by loss of Rv3676 (35). Instead, a recent study revealed that Rv3124 acts as a positive transcriptional regulator of the moaA1-moaD1 operon in M. tuberculosis (27). The (32,53) or in NRP-1 and NRP-2 (29), and moeB1 is the only gene in the MoCo biosynthetic pathway that shows modest upregulation at a late stage of the enduring hypoxic response (39).…”

BCG Vaccines

“…The first examined a SNP found in BCG3145, an ortholog of Rv3124 in M. tuberculosis. BCG3145/Rv3124 are members of the AfsR/DnrI/SARP (Streptomyces antibiotic regulatory protein) class of transcriptional regulators and, by microarray and qRT-PCR analysis, were found to positively regulate the mao1 locus required for molybdopterin biosynthesis (39). Interestingly, the E159G (glutamic acid-to-glycine substitution) mutation in BCG3145 caused a reduction in activity but did not abolish induction of the moa1 locus.…”