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The conserved DNA-binding protein WhiA is involved in cell division in Bacillus subtilis

ABSTRACT: 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.

ORGANISM(S): Bacillus subtilis

PROVIDER: GSE45824 | GEO | 2013/11/28

SECONDARY ACCESSION(S): PRJNA196524

REPOSITORIES: GEO

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The conserved DNA-binding protein WhiA is involved in cell division in Bacillus subtilis

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)

2013-11-28 | E-GEOD-45824 | biostudies-arrayexpress

Free SepF blocks recruitment of late cell division proteins

Project description:The cell division protein SepF aligns polymers formed by the key cell division protein FtsZ during synthesis of the (Fts)Z-ring at midcell, the first stage in cytokinesis. In addition, SepF acts as a membrane anchor for the Z-ring. SepF is conserved in Gram-positive and cyanobacteria. Recently, it was shown that SepF overproduction in Mycobacterium smegmatis blocks cell division. Here we investigated this in more detail using the Gram-positive model system Bacillus subtilis. Surprisingly, overproduction of SepF does not interfere with assembly of the Z-ring, but blocks assembly of the late cell division proteins responsible for septum synthesis. Transposon mutagenesis suggested that SepF overproduction inactivates the WalKR two-component system involved in cell division. Indeed, SepF overproduction impairs WalK localization, possibly because septal WalK localization requires late cell division proteins. Unexpectedly, transcriptome analysis showed that WalKR activity was not affected. Another surprise was that the cell division phenotype occurs when SepF does not bind to FtsZ. Further analyses provided an explanation for the contradictory transposon and transcriptome results, and suggested that SepF competes with other cell division proteins for binding to FtsZ. Our data show that an imbalance in early cell division proteins can interfere with recruitment of late cell division proteins.

2018-02-02 | GSE94788 | GEO

Expression profiling of B.subtils ftsZ depletion cells with ftsZ induced or uninduced for 1 or 2hrs.

Project description:The past twenty years have seen tremendous advances in our understanding of the mechanisms underlying bacterial cytokinesis, particularly the composition of the division machinery and the factors controlling its assembly. At the same time, however, we understand very little about the relationship between cell division and other cell cycle events in bacteria. Here we report that inhibiting division in Bacillus subtilis and Staphylococcus aureus quickly leads to an arrest in the initiation of new rounds of DNA replication followed by a complete arrest in cell growth. Arrested cells are metabolically active but unable to initiate new rounds of either DNA replication or division when shifted to permissive conditions. Inhibiting DNA replication results in entry into a similar quiescent state, in which cells are unable to resume growth or division when returned to permissive conditions. Our findings suggest the presence of two cell cycle control points: one linking division to the initiation of DNA replication and another linking the initiation of DNA replication to division. Significantly, this evidence contradicts the prevailing view of the bacterial cell cycle as a series of coordinated but uncoupled events. Importantly, the terminal nature of the cell cycle arrest validates the bacterial cell cycle machinery as an effective target for antimicrobial development. Four-condition experiment: ftsZ induced for 1hr, ftsZ depleted for 1hr, ftsZ induced for 2hrs, ftsZ depleted for 2hrs. Biological replicates: 3-4 for each sample. Reference: a mixture of wt RNA from different growth phases and wt backgrounds.

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Project description:In summary, we have identified and characterized FtsR as a transcriptional activator of the essential cell division protein FtsZ in C. glutamicum, providing a novel regulatory player in the process of cell division.

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MraZ is a transcriptional inhibitor of cell division in Bacillus subtilis

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.

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Project description:Comparative transposon sequencing of a bacterial cell division mutant

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Genes required for aerial growth, cell division, and chromosome segregation are targets of WhiA before sporulation in Streptomyces venezuelae.

Project description:WhiA is a highly unusual transcriptional regulator related to a family of eukaryotic homing endonucleases. WhiA is required for sporulation in the filamentous bacterium Streptomyces, but WhiA homologues of unknown function are also found throughout the Gram-positive bacteria. To better understand the role of WhiA in Streptomyces development and its function as a transcription factor, we identified the WhiA regulon through a combination of chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray transcriptional profiling, exploiting a new model organism for the genus, Streptomyces venezuelae, which sporulates in liquid culture. The regulon encompasses ~240 transcription units, and WhiA appears to function almost equally as an activator and as a repressor. Bioinformatic analysis of the upstream regions of the complete regulon, combined with DNase I footprinting, identified a short but highly conserved asymmetric sequence, GACAC, associated with the majority of WhiA targets. Construction of a null mutant showed that whiA is required for the initiation of sporulation septation and chromosome segregation in S. venezuelae, and several genes encoding key proteins of the Streptomyces cell division machinery, such as ftsZ, ftsW, and ftsK, were found to be directly activated by WhiA during development. Several other genes encoding proteins with important roles in development were also identified as WhiA targets, including the sporulation-specific sigma factor (WhiG) and the diguanylate cyclase CdgB. Cell division is tightly coordinated with the orderly arrest of apical growth in the sporogenic cell, and filP, encoding a key component of the polarisome that directs apical growth, is a direct target for WhiA-mediated repression during sporulation.

2016-05-20 | E-MTAB-4673 | biostudies-arrayexpress

Magnesium modulates Bacillus subtilis cell division frequency

Project description:By chance, we discovered a window of extracellular magnesium (Mg2+) availability that modulates Bacillus subtilis division frequency without affecting growth rate. In this window, cells grown with excess Mg2+ produce shorter cells than those grown in unsupplemented medium. The Mg2+-responsive adjustment in cell length occurs in both rich and minimal media and in domesticated and undomesticated strains. Of other divalent cations tested, manganese (Mn2+) and zinc (Zn2+) also resulted in cell shortening, but only at concentrations that affected growth. Cell length decreased proportionally with increasing Mg2+ from 0.2 mM to 4.0 mM, with little or no detectable change in labile, intracellular Mg2+ based on a riboswitch reporter. Cells grown in excess Mg2+ had fewer nucleoids and possessed more FtsZ-rings per unit cell length, consistent with increased division frequency. Remarkably, when shifting cells from unsupplemented to supplemented medium, more than half of the cell length decrease occurred in the first 10 min, consistent with rapid division onset. Relative to unsupplemented cells, cells growing at steady-state with excess Mg2+ showed enhanced expression of a large number of SigB-regulated genes and activation of the Fur, MntR, and Zur regulons. Thus, by manipulating the availability of one nutrient, we were able to uncouple growth rate from division frequency and identify transcriptional changes suggesting cell division is accompanied by the general stress response and an enhanced demand to sequester and/or increase uptake of iron, Mn2+, and Zn2+.

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