The DtxR protein from Corynebacterium diphtheriae is an iron-dependent repressor that regulates transcription from the tox, IRP1, and IRP2 promoters. A gene from virulent Mycobacterium tuberculosis H37Rv was recently shown to encode a protein, here designated iron-dependent regulator (IdeR), that is almost 60% homologous to DtxR from C. diphtheriae. A 750-bp PCR-derived DNA fragment carrying the M. tuberculosis ideR allele was subcloned to both high-and low-copy-number vectors. In Escherichia coli, transcription from the C. diphtheriae tox, IRP1, and IRP2 promoters was strongly repressed by ideR under high-iron conditions, and ideR restored normal iron-dependent expression of the corynebacterial siderophore in the C. diphtheriae dtxR mutant C7()hm723. The M. tuberculosis IdeR protein was overexpressed in E. coli and purified to near homogeneity by nickel affinity chromatography. Gel mobility shift experiments revealed that IdeR bound to a DNA fragment that carried the C. diphtheriae tox promoter/operator sequence. DNase I footprint analysis demonstrated that IdeR, in the presence of Cd 2؉ , Co 2؉ , Fe 2؉ , Mn 2؉ , Ni 2؉ , or Zn 2؉ , protected an approximately 30-bp region on DNA fragments carrying the tox, IRP1, or IRP2 promoter/operator sequences. IdeR reacted very weakly in Western blots (immunoblots) with antiserum against the C. diphtheriae DtxR protein, suggesting that the immunodominant epitopes of DtxR may be located in its poorly conserved carboxylterminal domain.
SummarysigA encodes a factor of the 70 family, A , that is found in all mycobacterial species. As A shows high similarity to the primary factor in Streptomyces coelicolor, it was postulated that A has the same role in mycobacteria. However, a point mutation in sigA, resulting in the replacement of arginine 522 by histidine, was found responsible for the attenuated virulence of the Mycobacterium bovis strain ATCC 35721. This raised the possibility that A was an alternative factor specifically required for virulence gene expression. In this work, we show that sigA can not be disrupted in Mycobacterium smegmatis unless an extra copy of the gene is provided at another chromosomal site, which demonstrates that sigA is essential. To characterize the pattern of sigA expression during exponential and stationary phase in M. smegmatis, we measured the -galactosidase activity in a strain carrying a sigA-lacZ transcriptional fusion and monitored A levels using Western blotting. Our results indicate that sigA is expressed throughout the growth of the culture. The essential character of sigA and its pattern of expression corroborate the hypothesis that sigA codes for the primary factor in M. smegmatis and, most likely, in all mycobacteria.
SinR is a pleiotropic DNA binding protein that is essential for the late-growth processes of competence and motility in Bacillus subtilis and is also a repressor of others, e.g., sporulation and subtilisin synthesis. In this report, we show that SinR, in addition to being an inhibitor of sporulation stage II gene expression, is a repressor of the key early sporulation gene spo0A. The sporulation-specific rise in spo0A expression at time zero is absent in a SinR-overproducing strain and is much higher than normal in strains with a disrupted sinR gene. This effect is direct, since SinR binds specifically to spo0A in vitro, in a region overlapping the ؊10 region of the sporulation-specific P s promoter that is recognized by E-H polymerase. Methyl interference and sitedirected mutagenesis studies have identified guanine residues that are important for SinR recognition of this DNA sequence. Finally, we present evidence that SinR controls sporulation through several independent genes, i.e., spo0A, spoIIA, and possibly spoIIG and spoIIE.
A search for Mycobacterium smegmatis genes showing similarity to the conserved family encoding major sigma factors in diverse prokaryotes has identified two such determinants. Both genes are expressed in exponentially growing cells, as judged by Western immunoassays. A series of chromatographic steps was used to purify M. smegmatis RNA polymerase holoenzyme and it was shown that its ability to initiate in vitro transcription with a heterologous Bacillus subtilis promoter is dependent on the presence of these sigma factor(s). Reconstitution of specific in vitro transcription activity was obtained upon mixing of M. smegmatis core RNA polymerase with the major sigma factor of Bacillus subtilis. We also demonstrated in vitro transcription of the M. smegmatis rrnB promoter by the M. smegmatis RNA polymerase. Significantly, highly active B. subtilis RNA polymerase holoenzyme was unable to transcribe this gene.
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