e Bacterial cell division is a highly coordinated process that begins with the polymerization of the tubulin-like protein FtsZ at midcell. FtsZ polymerization is regulated by a set of conserved cell division proteins, including ZapA. However, a zapA mutation does not result in a clear phenotype in Bacillus subtilis. In this study, we used a synthetic-lethal screen to find genes that become essential when ZapA is mutated. Three transposon insertions were found in yvcL. The deletion of yvcL in a wild-type background had only a mild effect on growth, but a yvcL zapA double mutant is very filamentous and sick. This filamentation is caused by a strong reduction in FtsZ-ring assembly, suggesting that YvcL is involved in an early stage of cell division. YvcL is 25% identical and 50% similar to the Streptomyces coelicolor transcription factor WhiA, which induces ftsZ and is required for septation of aerial hyphae during sporulation. Using green fluorescent protein fusions, we show that YvcL localizes at the nucleoid. Surprisingly, transcriptome analyses in combination with a ChIP-on-chip assay gave no indication that YvcL functions as a transcription factor. To gain more insight into the function of YvcL, we searched for suppressors of the filamentous phenotype of a yvcL zapA double mutant. Transposon insertions in gtaB and pgcA restored normal cell division of the double mutant. The corresponding proteins have been implicated in the metabolic sensing of cell division. We conclude that YvcL (WhiA) is involved in cell division in B. subtilis through an as-yet-unknown mechanism.

A previously optimized Escherichia coli two-plasmid system was used to identify Mycobacterium tuberculosis promoters recognized by RNA polymerase containing the M. tuberculosis stress response sigma factor sigma(F). The method allowed the identification of five new sigma(F)-dependent promoters. Transcriptional start points of the promoters were determined by high-resolution S1-nuclease mapping using RNA prepared from E. coli containing the two-plasmid system. The promoters were confirmed by an in vitro transcription assay. The Mycobacterium smegmatis and Mycobacterium tuberculosis core RNA polymerases, after complementation with sigma(F), were able to recognize all the five promoters. All the promoters contained sequences highly similar to the sequence of the previously identified M. tuberculosis sigma(F)-dependent promoter, usfXp1. Comparison of the promoters revealed a sigma(F) consensus sequence GtTtga-N(14-18)-GGGTAT. The sigma(F)-dependent promoters may govern expression of genes encoding a transcription regulator homologous to the response regulators of bacterial two-component signal transduction systems and proteins with unknown function.

The DNA binding protein WhiA is conserved in Gram-positive bacteria and is present in the genetically simple cell wall-lacking mycoplasmas. The protein shows homology to eukaryotic homing endonucleases but lacks nuclease activity. WhiA was first characterized in streptomycetes, where it regulates the expression of key differentiation genes, including the cell division gene , which is essential for sporulation. For, it was shown that WhiA is essential when certain cell division genes are deleted. However, in , WhiA is not required for sporulation, and it does not seem to function as a transcription factor, despite its DNA binding activity. The exact function of WhiA remains elusive. We noticed that mutants show an increased space between their nucleoids, and here, we describe the results of fluorescence microscopy, genetic, and transcriptional experiments to further investigate this phenomenon. It appeared that the deletion of is synthetic lethal when either the DNA replication and segregation regulator ParB or the DNA replication inhibitor YabA is absent. However, WhiA does not seem to affect replication initiation. We found that a Δ mutant is highly sensitive for DNA-damaging agents. Further tests revealed that the deletion of induces the SOS response, including the cell division inhibitor YneA. When was inactivated, the viability of the synthetic lethal Δ Δ mutant was restored. However, the nucleoid segregation phenotype remained. These findings underline the importance of WhiA for cell division and indicate that the protein also plays a role in DNA segregation. The conserved WhiA protein family can be found in most Gram-positive bacteria, including the genetically simple cell wall-lacking mycoplasmas, and these proteins play a role in cell division. WhiA has some homology with eukaryotic homing endonucleases but lacks nuclease activity. Because of its DNA binding activity, it is assumed that the protein functions as a transcription factor, but this is not the case in the model system The function of this protein in remains unclear. We noticed that a mutant has a mild chromosome segregation defect. Further studies of this phenomenon provided new support for a functional role of WhiA in cell division and indicated that the protein is required for normal chromosome segregation.

The previously established two-plasmid system in Escherichia coli for the identification of Mycobacterium tuberculosis promoters that are recognized by RNA polymerase containing the stress response sigma factor sigmaF was optimized. Expression of the M. tuberculosis sigmaF encoded by sigF gene was under the control of the isopropyl beta-D-thiogalactopyranoside (IPTG)-dependent Ptrc promoter. A low level of IPTG induced a nontoxic but sufficient level of sigmaF to interact with the core enzyme of RNA polymerase. Such an RNA polymerase holoenzyme recognized the known sigmaF-dependent promoter, usfXp1, which was cloned in the compatible promoter probe plasmid, upstream of a promoterless lacZalpha reporter gene. Primer extension analysis of the usfXp1 promoter in the E. coli two-plasmid system after IPTG-induced expression of M. tuberculosis sigF revealed a transcription start point that was identical as in M. tuberculosis. This new system has been shown to be useful for identification of M. tuberculosis sigmaF-dependent promoters.