PMID:20545868

Citation	Rybniker, J, Nowag, A, van Gumpel, E, Nissen, N, Robinson, N, Plum, G and Hartmann, P (2010) Insights into the function of the WhiB-like protein of mycobacteriophage TM4--a transcriptional inhibitor of WhiB2. Mol. Microbiol. 77:642-57
Abstract	WhiB-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 apo-WhiB2 of Mycobacterium tuberculosis and apo-WhiBTM4 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.
Links	PubMed Online version:10.1111/j.1365-2958.2010.07235.x
Keywords	Amino Acid Motifs; Amino Acid Sequence; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Down-Regulation; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Mycobacteriophages/chemistry; Mycobacteriophages/genetics; Mycobacteriophages/metabolism; Mycobacterium smegmatis/genetics; Mycobacterium smegmatis/metabolism; Mycobacterium smegmatis/virology; Mycobacterium tuberculosis/chemistry; Mycobacterium tuberculosis/genetics; Mycobacterium tuberculosis/metabolism; Mycobacterium tuberculosis/virology; Protein Binding; Sequence Alignment; Transcription Factors/chemistry; Transcription Factors/genetics; Transcription Factors/metabolism; Transcription, Genetic; Viral Proteins/chemistry; Viral Proteins/genetics; Viral Proteins/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BPMT4:WHIB		GO:0006355: regulation of transcription, DNA-templated	ECO:0000314:		P		The protein WhiBTM4 from mycobacteriophage TM4 serves as a transcription regulator in bacteria such as M. smegmatis, in which it inhibits WhiB2. When WhiBTM4 was expressed in M. smegmatis, it caused the cells to lose acid-fast properties and created long, branched cells. WhiBTM4 caused these changes, in which the results were similar to a WhiB2 knockout protein (Fig. 2). When M. smegmatis contained the protein WhiBTM4, WhiB2 mRNA levels (measured using quantitative RT-PCR) greatly decreased compared to the wildtype, which only contained pSD24 (Fig. 3B). A decrease in mRNA levels suggests that the mycobacteriophage TM4 protein WhiBTM4 inhibits transcription of WhiB2 in M. smegmatis. WhiBTM4 binds only to a DNA WhiB2 promoter, which shows that transcription is regulated through DNA (Fig 6E). This is shown by the band created by the protein/DNA complex in figure 6 B-E.	complete CACAO 13120

References

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