Number of transformants recovered when complementation vector in <i>M</i>. <i>tuberculosis</i> Δ<i>Rv1460 attB</i>::pMV1460 was swopped with vector indicated.

<p>Number of transformants recovered when complementation vector in <i>M</i>. <i>tuberculosis</i> Δ<i>Rv1460 attB</i>::pMV1460 was swopped with vector indicated.</p

Rv1460 is a repressor of the <i>suf</i> operon.

<p>(A) Location of RT and gene specific primers used for RT-qPCR (Table B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200145#pone.0200145.s001" target="_blank">S1 File</a>). (B) Relative gene expression in H37Rv (wild-type), Δ<i>Rv1460</i>stop_5.20 mutant and complemented strain. (C) Relative gene expression using the Rv1460RT and Rv1461RT primers and gene specific <i>Rv1460</i> and <i>Rv1461</i> primers in H37Rv. The results shown are the mean and standard deviation of three experiments and stars indicate significant differences determined by unpaired t-test: * p≤0.05, ** p ≤0.01, ***p≤0.001**** p ≤0.0001.</p

Rv1460 is a SufR homologue.

<p>Maestro Multiple Sequence Viewer multiple alignment of Rv1460 [ACJ83238] with homologues in selected mycobacteria and SufR homologues present in other organisms. The re-annotated start site of Rv1460 is indicated by the red box. Conserved cysteine residues are indicated by the orange boxes. <i>Mycobacterium smegmatis</i>, transcriptional regulator [WP_011728830]; <i>Mycobacterium leprae</i>, transcriptional regulator [WP_010907825]; <i>Mycobacterium marinum</i> E11, transcriptional regulatory protein [CDM76334]; <i>Mycobacterium tuberculosis</i> CDC1551, conserved hypothetical protein [AAK45771]; <i>Mycobacterium avium</i> subsp. paratuberculosis K-10, hypothetical protein MAP_1186 [AAS03503]; <i>Mycobacterium avium</i> 104, DNA-binding protein [ABK68668]; <i>Rhodococcus fascians</i>, transcriptional regulator [WP_037190040]; <i>Nocardia veteran</i>, transcriptional regulator [WP_051031599] and <i>Synechocystis</i> sp. PCC 6803, SufR [WP 020862050].</p

Rv1460, a SufR homologue, is a repressor of the <i>suf </i>operon in <i>Mycobacterium tuberculosis</i>

<div><p>Iron–sulphur (Fe-S) clusters are ubiquitous co-factors which require multi-protein systems for their synthesis. In <i>Mycobacterium tuberculosis</i>, the <i>Rv1460-Rv1461-Rv1462-Rv1463-csd-Rv1465-Rv1466</i> operon (<i>suf</i> operon) encodes the primary Fe-S cluster biogenesis system. The first gene in this operon, <i>Rv1460</i>, shares homology with the cyanobacterial SufR, which functions as a transcriptional repressor of the <i>sufBCDS</i> operon. Rv1460’s function in <i>M</i>. <i>tuberculosis</i> has however not been determined. In this study, we demonstrate that <i>M</i>. <i>tuberculosis</i> mutants lacking a functional Rv1460 protein are impaired for growth under standard culture conditions. Elevated expression of <i>Rv1460</i> and <i>Rv1461</i> was observed in the mutant, implicating Rv1460 in the regulation of the <i>suf</i> operon. Binding of an Fe-S cluster to purified recombinant Rv1460 was confirmed by UV-visible spectroscopy and circular dichroism. Furthermore, three conserved cysteine residues, C203, C216 and C244, proposed to provide ligands for the coordination of an Fe-S cluster, were shown to be required for the function of Rv1460 in <i>M</i>. <i>tuberculosis</i>. Rv1460 therefore seems to be functionally analogous to cyanobacterial SufR.</p></div

Succinate dehydrogenase and aconitase activity is not impaired in Rv1460 truncation mutants.

<p>(A) Succinate dehydrogenase activity and (B) aconitase activity in the H37Rv (wild-type), Δ<i>Rv1460</i>stop_5.20 and complemented strains cultured in 7H9 OADC. Activity was standardised relative to total protein. The results shown are the mean and standard deviation of five and three experiments respectively.</p

Rv1460 truncation mutants are not impaired for growth under iron-limiting conditions.

<p>Growth of H37Rv (wild-type), three truncation mutants (Δ<i>Rv1460</i>stop) and complemented strains in (A–C) MM + Fe⁺³ and (D–F) MM for three growth cycles (subcultured on day 14 to an OD_600nm of 0.05). The results shown are the mean and standard deviation of three experiments.</p