Project description:The Rok protein of Bacillus subtilis was identified as a negative regulator of competence development. Here we show that Rok binds to extended areas of the B. subtilis genome that are characterized by a high A+T content and are known or believed to have been acquired by horizontal gene transfer, e.g. mobile elements. A deletion of rok results in higher excision of one such element, ICEBs1. The preferential association of Rok with DNA with a high A+T content is also observed in a Gram-negative host, E. coli, and depends on a conserved C-terminal region of the protein. Based on our findings, we propose that Rok is a nucleoid-associated protein that fulfills a function analogous to H-NS, a protein absent from most Gram-positive bacteria.
Project description:RNase Y is a key endoribonuclease affecting global mRNA stability in Bacillus subtilis. Its characterization provided the first evidence that endonucleolytic cleavage plays a major role in the mRNA metabolism of this organism. RNase Y shares important functional features with the RNA decay initiating RNase E from Escherichia coli, notably a similar cleavage specificity and a preference for 5' monophosphorylated substrates. We used high-resolution tiling arrays to analyze the effect of RNase Y depletion on RNA abundance covering the entire genome. The data confirm that this endoribonuclease plays a key role in initiating the decay of a large number of mRNAs as well as non coding RNAs. The downstream cleavage products are likely to be degraded by the 5' exonucleolytic activity of RNases J1/J2 as we show for a specific case. Comparison of the data with that of two other recent studies revealed very significant differences. About two thirds of the mRNAs upregulated following RNase Y depletion were different when compared to either one of these studies and only about 10% were in common in all three studies. This highlights that experimental conditions and data analysis play an important role in identifying RNase Y substrates by global transcriptional profiling. Our data confirmed already known RNase Y substrates and due to the precision and reproducibility of the profiles allow an exceptionally detailed view of the turnover of hundreds of new RNA substrates.
Project description:The Rok protein of Bacillus subtilis was identified as a negative regulator of competence development. Here we show that Rok binds to extended areas of the B. subtilis genome that are characterized by a high A+T content and are known or believed to have been acquired by horizontal gene transfer, e.g. mobile elements. A deletion of rok results in higher excision of one such element, ICEBs1. The preferential association of Rok with DNA with a high A+T content is also observed in a Gram-negative host, E. coli, and depends on a conserved C-terminal region of the protein. Based on our findings, we propose that Rok is a nucleoid-associated protein that fulfills a function analogous to H-NS, a protein absent from most Gram-positive bacteria. The genome-wide binding profile of the transcription factor Rok and the nucleoid-binding protein HBsu were determined. Three biological replicates were analyzed per strain (one per array). Binding profiles were determined in exponentially growing cells. Enrichment in immunoprecipitated samples versus total genomic DNA were determined.
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:RNase Y is an essential endoribonuclease affecting global mRNA stability in Bacillus subtilis. We used high-resolution tiling arrays to analyze the effect of RNase Y depletion on RNA abundance covering the entire genome. The data confirm that this endoribonuclease plays a key role in initiating the decay of a large number of mRNAs as well as non coding RNAs. Comparison of the data with that of two other recent studies revealed very significant differences. About two thirds of the mRNAs upregulated following RNase Y depletion were different when compared to either one of these studies and only about 10% of were in common in all three studies.Our data confirmed already known RNase Y substrates and due to the precision and reproducibility of the profiles allows an exceptionally detailed view of the turnover of hundreds of new RNA substrates.
Project description:The availability of the complete sequence of the Bacillus subtilis chromosome (F. Kunst et al., Nature 390:249-256, 1997) makes possible the construction of genome-wide DNA arrays and the study of this organism on a global scale. Because we have a long-standing interest in the effects of scoC on late-stage developmental phenomena as they relate to aprE expression, we studied the genome-wide effects of a scoC null mutant with the goal of furthering the understanding of the role of scoC in growth and developmental processes. In the present work we compared the expression patterns of isogenic B. subtilis strains, one of which carries a null mutation in the scoC locus (scoC4). The results obtained indicate that scoC regulates, either directly or indirectly, the expression of at least 560 genes in the B. subtilis genome. ScoC appeared to repress as well as activate gene expression. Changes in expression were observed in genes encoding transport and binding proteins, those involved in amino acid, carbohydrate, and nucleotide and/or nucleoside metabolism, and those associated with motility, sporulation, and adaptation to atypical conditions. Changes in gene expression were also observed for transcriptional regulators, along with sigma factors, regulatory phosphatases and kinases, and members of sensor regulator systems. In this report, we discuss some of the phenotypes associated with the scoC mutant in light of the transcriptome changes observed.