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

ABSTRACT: 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.

SUBMITTER: Surdova K

PROVIDER: S-EPMC3889613 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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

Surdova Katarina K Gamba Pamela P Claessen Dennis D Siersma Tjalling T Jonker Martijs J MJ Errington Jeff J Hamoen Leendert W LW

Journal of bacteriology 20131004 24

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 wil ...[more]

PMID: 24097947

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Project description: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 ftsZ, which is essential for sporulation. For Bacillus subtilis, it was shown that WhiA is essential when certain cell division genes are deleted. However, in B. subtilis, 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 B. subtilis WhiA remains elusive. We noticed that whiA 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 whiA 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 ?whiA mutant is highly sensitive for DNA-damaging agents. Further tests revealed that the deletion of parAB induces the SOS response, including the cell division inhibitor YneA. When yneA was inactivated, the viability of the synthetic lethal ?whiA ?parAB 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.IMPORTANCE 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 B. subtilis The function of this protein in B. subtilis remains unclear. We noticed that a whiA 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.

Project description:By chance, we discovered a window of extracellular magnesium (Mg2+) availability that modulates the division frequency of Bacillus subtilis without affecting its growth rate. In this window, cells grown with excess Mg2+ produce shorter cells than do those grown in unsupplemented medium. The Mg2+-responsive adjustment in cell length occurs in both rich and minimal media as well as in domesticated and undomesticated strains. Of other divalent cations tested, manganese (Mn2+) and zinc (Zn2+) also resulted in cell shortening, but this occurred 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 being observed 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 the 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 an enhanced expression of a large number of SigB-regulated genes and the activation of the Fur, MntR, and Zur regulons. Thus, by manipulating the availability of one nutrient, we were able to uncouple the growth rate from the division frequency and identify transcriptional changes that suggest that cell division is accompanied by the general stress response and an enhanced demand to sequester and/or increase the uptake of iron, Mn2+, and Zn2+. IMPORTANCE The signals that cells use to trigger cell division are unknown. Although division is often considered intrinsic to the cell cycle, microorganisms can continue to grow and repeat rounds of DNA replication without dividing, indicating that cycles of division can be skipped. Here, we show that by manipulating a single nutrient, namely, Mg2+, cell division can be uncoupled from the growth rate. This finding can be applied to investigate the nature of the cell division signal(s).

Project description:The conserved protein WhiA is present in most Gram-positive bacteria and plays a role in cell division. WhiA contains a DNA-binding motif and is a transcription regulator of the key cell division gene ftsZ in actinomycetes. In Bacillus subtilis, the absence of WhiA influences both cell division and chromosome segregation; however, the protein does not regulate any gene involved in these processes. In this study, we addressed three alternative mechanisms by which WhiA might exert its activity in B. subtilis and examined whether WhiA influences either (i) central carbon metabolism, (ii) fatty acid composition of the cell membrane, or (iii) chromosome organization. Mutations in glycolytic enzymes have been shown to influence both cell division and DNA replication. To measure the effect of WhiA on carbon metabolism, we tested different carbon sources and measured exometabolome fluxes. This revealed that the absence of WhiA does not affect glycolysis but does influence the pool of branched-chain fatty acid precursors. Due to the effect of WhiA on chromosome segregation, we examine chromosome organization in a ∆whiA mutant using chromosome conformation capture (Hi-C) analysis. This revealed a local reduction in short-range chromosome interactions. Together, these findings provide new avenues for future research into how this protein works in the non-actinomycete firmicutes.IMPORTANCEWhiA is a conserved DNA-binding protein that influences cell division in many Gram-positive bacteria and, in B. subtilis, also chromosome segregation. How WhiA works in Bacillus subtilis is unknown. Here, we tested three hypothetical mechanisms using metabolomics, fatty acid analysis, and chromosome confirmation capture experiments. This revealed that WhiA does not influence cell division and chromosome segregation by modulating either central carbon metabolism or fatty acid composition. However, the inactivation of WhiA reduces short-range chromosome interactions. These findings provide new avenues to study the molecular mechanism of WhiA in the future.

Dataset Information

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

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

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