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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 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)

ORGANISM(S): Bacillus subtilis

SUBMITTER: Paul Wackers

PROVIDER: E-GEOD-45824 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 ∆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.

2013-11-28 | GSE45824 | GEO

The conserved WhiA protein influences the fatty acid composition of the Bacillus subtilis cell membrane

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.

2019-10-18 | GSE121479 | GEO

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

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.

2015-01-01 | E-GEOD-56753 | 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

Targeted transcriptome analysis of the SigB regulon in Bacillus subtilis

Project description:Bacillus subtilis is exposed to a wide range of transitory stress and starvation conditions. Here we investigate the expression changes observed in the B. subtilis wild type strain 168 and its isogenic sigB mutant(BSM29) with respect to each stress condition tested. Gene expression was queried for the stress conditions: ethanol-, butanol-, osmotic- and oxidative stress, heat shock, low temperature growth, glucose as well as oxygen limitation. For butanol-, ethanol-, osmotic-, and oxidative stress as well as heat shock : time points (0min, 5min, 10min, 15min and 20min) ; for glucose limitation and oxygen limitation : time points (0min, 15min, 30min, 45min, 60min or 90min) and for low temperature growth, samples for recording of expression values were taken during mid-exponential growth at OD540 0.9 and 1.0.

2012-03-14 | E-GEOD-32895 | biostudies-arrayexpress

Expression analysis of an amoxicillin resistant Escherichia coli strain

Project description:Comparison of the whole genome gene expression level of an amoxicillin resistant E. coli strain with the wildtype it was derived from. The process of amoxicillin adaptation of E. coli MG1655 wildtype cells is further descibed in van der Horst, M, J.M. Schuurmans, M. C. Smid, B. B. Koenders, and B. H. ter Kuile (2011) in Microb. Drug Resist. 17:141-147. Resistance to amoxicillin was induced in E. coli by growth in the presence of stepwise increasing antibiotic concentrations. To investigate consequences of the aquisition of amoxicillin resistance the transcriptomic profile of sensitive and resistant cells was compared in the absence and presence of sub-inhibitory (0.25xMIC) amoxicillin concentrations was compared. Total RNA of 3 biological replicates of E. coli MG1655 wildtype cells and amoxicillin resistant cells cultured with (0.25xMIC) or without amoxicillin was hybridized on a 12x135k custom designed microarraychip against one common reference.

2013-05-27 | E-GEOD-47221 | biostudies-arrayexpress

Expression analysis of an enrofloxacin and a tetracline resistant Escherichia coli strain

Project description:Comparison of the whole genome gene expression level of an enrofloxacin and tetracycline resistant E. coli strain with the wildtype it was derived from. The process of drug adaptation of E. coli MG1655 wildtype cells is further descibed in van der Horst, M, J.M. Schuurmans, M. C. Smid, B. B. Koenders, and B. H. ter Kuile (2011) in Microb. Drug Resist. 17:141-147. Resistance to amoxicillin was induced in E. coli by growth in the presence of stepwise increasing antibiotic concentrations. To investigate consequences of the aquisition of amoxicillin resistance the transcriptomic profile of sensitive and resistant cells was compared in the absence and presence of sub-inhibitory (0.25xMIC) amoxicillin concentrations was compared. Total RNA of 3 biological replicates of E. coli MG1655 wildtype cells and drug resistant cells cultured with (0.25xMIC) or without the drug was hybridized on a 12x135k custom designed microarraychip against one common reference.

2014-04-26 | E-GEOD-57084 | biostudies-arrayexpress

Effect of multiple longitudinal biopsy sampling in individual mice

Project description:Small scale study to asses the effects of taking multiple skinbiopts from individual mice in time. Results showed no influence on expression levels in time from older samplings on newer samplings. skin biopts from 4 individual mice in taken at 6 points in time

2014-09-01 | E-GEOD-51347 | biostudies-arrayexpress

UV-B dose and time range finding study on mouse biopsy skin samples

Project description:In molecular biology, the design of mechanistic experiments has to be optimized by considering statistical and biological principles. In contrast to statistical principles, biological principles of experimental design are not universally formulated. In an attempt to pinpoint generally acceptable rules, we investigated the importance of determining the optimal ranges of scale of i.e. dose and time in gene expression experiments. We propose a protocol for executing small scale, genome wide, range finding studies, covering a wide range of the potentially relevant part of the design space to find the optimal ranges of experimentation. This protocol is executed and a proof-of-concept is presented, where this approach is tested for both an in-vitro and an in-vivo study that aim to unravel DNA repair mechanisms provoked after UV radiation. We identified four challenges of range finding studies in omics experimentation; (1) the modularity of biological processes, (2) their dynamics, (3) the extent to which end-points indicate biological processes, and (4) the costs associated with the assays, which are all addressed by our approach. 57 skin biopt samples taken from 12 individual mice on 8 timepionts and for 6 different UV-B doses. Per mouse 5 skin biopts were samples in time

2014-09-01 | E-GEOD-51348 | biostudies-arrayexpress

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