Insights into the function of the WhiB‐like protein of mycobacteriophage TM4 – a transcriptional inhibitor of WhiB2

Abstract

SummaryWhiB-like proteins of actinomycetes are known to co-ordinate iron-sulfur (Fe-S) clusters and are believed to have regulatory functions in many essential bacterial processes. The systematic determination of the genome sequences of mycobacteriophages has revealed the presence of several whiB-like genes in these viruses. Here we focussed on the WhiB-like protein of mycobacteriophage TM4, WhiBTM4. We provide evidence that this viral protein is capable of co-ordinating a Fe-S cluster. The UV-visible absorption spectra obtained from freshly purified and reconstituted WhiBTM4 were consistent with the presence of an oxygen sensitive [2Fe-2S] cluster. Expression of WhiBTM4 in the mycobacterial host led to hindered septation resembling a WhiB2 knockout phenotype whereas basal expression of WhiBTM4 led to superinfection exclusion. The quantification of mRNA-levels during phage infection showed that whiBTM4 is a highly transcribed early phage gene and a dominant negative regulator of WhiB2. Strikingly, both apoWhiB2 of Mycobacterium tuberculosis and apoWhiBTM4 were capable of binding to the conserved promoter region upstream of the whiB2 gene indicating that WhiB2 regulates its own synthesis which is inhibited in the presence of WhiBTM4. Thus, we provide substantial evidence supporting the hypothesis of viral and bacterial WhiB proteins being important Fe-S containing transcriptional regulators with DNA-binding capability.

ϕ 2 GFP10, a High-Intensity Fluorophage, Enables Detection and Rapid Drug Susceptibility Testing of Mycobacterium tuberculosis Directly from Sputum Samples

“…For example, mycobacteriophage D29 yields 120 new phages from a single infected cell of M. tuberculosis in 3 h, whereas the doubling time of M. tuberculosis itself is approximately 24 h (15). The new reporter phage vector, phAE159, is also deleted for the TM4-gp49 gene, which encodes a product that may inhibit bacteriophage superinfection (37). Deletion of TM4-gp49 might be responsible for the enhanced per-cell signal at higher MOI (Fig.…”

WhiB5, a Transcriptional Regulator That Contributes to Mycobacterium tuberculosis Virulence and Reactivation

“…A similar strategy was used previously to determine the regulon of WhiB7 (23). The fact that whiB5 induction resulted in the increased expression of 58 genes and in no downregulated genes (see Table S2 in the supplemental material) underscores its role as a positive regulator of transcription, as already suggested for other members of this family of proteins (32,35), even if our experimental design did not allow us to discriminate between genes regulated directly or indirectly by WhiB5. Analysis of the transcriptional start sites of three genes belonging to its regulon revealed a conserved consensus promoter sequence that we hypothesize might include the region bound by WhiB5 (Fig.…”

“…WhiB1 and WhiB2 are known to be autoregulated (32,35). To determine whether WhiB5 can also directly or indirectly regulate transcription of its own structural gene, we constructed a replicative plasmid in which the whiB5 upstream region containing its promoter was transcriptionally fused to a promoterless green fluorescent protein (GFP)-encoding gene.…”

“…The interaction of WhiB5 with several transposases suggests that its function might have been hijacked from mobile genetic elements, which have an evolutionary advantage in inducing their mobility under conditions of stress endangering the bacterial host. It was recently demonstrated that the TM4 mycobacteriophage genome encodes a WhiB2-like protein whose role is to inhibit transcription of whiB2 during the early phase of infection, causing bacteriostasis, demonstrating a relationship between WhiB-like proteins and mobile genetic elements (32). Further studies are needed to clarify the physiological role of the interaction of WhiB5 with these proteins.…”

“…A conditional mutant of this gene in M. smegmatis was shown to produce filamentous cells under nonpermissive conditions (27,28). Recently, both WhiB2 and a WhiB2 homolog encoded by a mycobacteriophage were shown to bind the whiB2 promoter to inhibit its transcription (32).…”