Project description:The conserved WhiA protein family is present in most Gram-positive bacteria and plays a role in cell division. WhiA contains a DNA-binding motive and has been identified as a transcription factor in the actinomycetes. In Bacillus subtilis, a deletion of whiA influences cell division and chromosome segregation. However, WhiA does not seem to function as a transcription factor in this organism, and it is still unclear how WhiA influences these processes. The B. subtilis whiA gene is part of an operon and flanked by two metabolic genes, including yvcK required for growth under gluconeogenic conditions, and the gene encoding the catabolite repressor Crh. Therefore, B. subtilis WhiA might play a role in carbon metabolism which could indirectly affect cell division and chromosome maintenance. To study this, we followed a metabolomics approach and measured exo-metabolome flux during growth on different carbon sources, using nuclear magnetic resonance spectroscopy. The carbon utilization of wild type and whiA mutant cells was similar, however, the whiA mutant exhibited depleted pools of branched-chain fatty acid precursors and higher levels of acetate and 2-oxoglutarate. Transcriptome data could not link these effects to gene regulatory differences, but the reduction in branched-chain fatty acid precursors influenced the fatty acid composition of the cell membrane. In conclusion, WhiA does not participate in carbon catabolite regulation, but affects fatty acid composition of the membrane by an as yet unknown mechanism. We speculate that the effect on the membrane might be related to the pleiotropic phenotype of the B. subtilis whiA mutant.
Project description:One of the first steps in bacterial cell division is the polymerization of the tubulin-like protein FtsZ at midcell. The dynamics of FtsZ polymerization is regulated by a set of proteins among which ZapA. 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 absent. Three transposon insertions were found in yvcL. 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 polymerization, 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. WhiA is required for septation of aerial hyphae during sporulation. Using GFP fusions, we show that YvcL localizes at the nucleoid. Surprisingly, transcriptome analyses in combination with a ChIP on chip assay did not provide clear evidence 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 M-bM-^HM-^FyvcL M-bM-^HM-^FzapA mutant. Transposon insertions in gtaB and pgcA restored normal cell division of the double mutant. The corresponding proteins have been implemented 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. Comparing wild tpe Bascillus subtilus (n=3) with Bascillus Subtilis KS400 (n=2) and Bascillus subtilis KS696 (n=2)
Project description:One of the first steps in bacterial cell division is the polymerization of the tubulin-like protein FtsZ at midcell. The dynamics of FtsZ polymerization is regulated by a set of proteins among which ZapA. 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 absent. Three transposon insertions were found in yvcL. 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 polymerization, 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. WhiA is required for septation of aerial hyphae during sporulation. Using GFP fusions, we show that YvcL localizes at the nucleoid. Surprisingly, transcriptome analyses in combination with a ChIP on chip assay did not provide clear evidence 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 mutant. Transposon insertions in gtaB and pgcA restored normal cell division of the double mutant. The corresponding proteins have been implemented 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.
Project description:Identification of the specific WalR (YycF) binding regions on the B. subtilis chromosome during exponential and phosphate starvation growth phases. The data serves to extend the WalRK regulon in Bacillus subtilis and its role in cell wall metabolism, as well as implying a role in several other cellular processes.
Project description:Identification of the specific WalR (YycF) binding regions on the B. subtilis chromosome during exponential and phosphate starvation growth phases. The data serves to extend the WalRK regulon in Bacillus subtilis and its role in cell wall metabolism, as well as implying a role in several other cellular processes. For each sample analyzed in this study three biological replicates were performed. Three different samples were taken from a strain expressing the WalR-SPA protein as well as from wild-type (168) without a tagged WalR. Samples were taken from exponentially growing cells in low phosphate medium (LPDM) as well as from phosphate-limited cells (T2). Each sample compares ChIP DNA vs. Total DNA from the same cells.
Project description:Comparison of the transcriptome of Bacillus subtilis under going membrane protein overproduction in the wild type, sigW and cssRS deletion strains. We demonstrate that the dynamics of the stress systems involved in membrane overproduction are far more complicated than was first hypothesised and that many more systems than SigW and CssRS are involved in membrane protein overexpression stress. Interestingly the cssRS genes are repressed in the sigW deletion strain.
Project description:Coordination of chromosome segregation and cytokinesis is crucial for efficient cell proliferation. In Bacillus subtilis the nucleoid occlusion protein Noc protects chromosomes by associating with the chromosome and preventing cell division in its vicinity. Using protein localization, ChAP-on-Chip and bioinformatics, we have identified a consensus Noc-binding DNA sequence (NBS), and show that Noc is targeted to about 70 discrete regions scattered around the chromosome, though absent from a large region around the replication terminus. Purified Noc bound specifically to an NBS in vitro. NBSs inserted near the replication terminus bound Noc-YFP and caused a delay in cell division. An autonomous plasmid carrying an NBS recruited Noc-YFP and conferred a severe Noc-dependent inhibition of cell division. This shows that Noc is a potent inhibitor of division but that its activity is strictly localized by interaction with NBS sites in vivo. We propose that Noc not only serves as a spatial regulator of cell division to protect the nucleoid, but also a timing device with an important role in the co-ordination of chromosome segregation and cell division.
Project description:To investigate the possible genes regulated by the DNA binding protein MraZ The bacterial division and cell wall (dcw) cluster is a highly conserved region of the genome which encodes several essential cell division factors including the central divisome protein FtsZ. Understanding the regulation of this region is key to our overall understanding of the division process. mraZ is found at the 5’ end of the dcw cluster and previous studies have described MraZ as a sequence-specific DNA binding protein. In this article, we investigate MraZ to elucidate its role in Bacillus subtilis. Through our investigation, we demonstrate that increased levels of MraZ result in lethal filamentation due to repression of its own operon (mraZ-mraW-ftsL-pbpB). We observe rescue of filamentation upon decoupling ftsL expression, but not other genes in the operon, from MraZ control. Our data suggests that regulation of the mra operon may be an alternative way for cells to quickly arrest cytokinesis potentially during entry into stationary phase and in the event of DNA replication arrest. Furthermore, through timelapse microscopy we were able to identify that overexpression of mraZ or depletion of FtsL results in de-condensation of the FtsZ ring (Z-ring). Using fluorescent D-amino acid labelling, we also observed that coordinated peptidoglycan insertion at division site is dysregulated in the absence of FtsL. Thus, we reveal the precise role of FtsL is in Z-ring maturation and focusing septal peptidoglycan synthesis.