Rudolf Allmansberger

Catabolite repression of the operon for xylose utilization from Bacillus subtilis W23 is mediated at the level of transcription and depends on a cis site in the xylA reading frame

SummaryThe Bacillus subtilis xyl operon encoding enzymes for xylose utilization is repressed in the absence of xylose and in the presence of glucose. Transcriptional fusions of spoVG-lacZ to this operon show regulation of β-galactosidase expression by glucose, indicating that glucose repression operates at the level of transcription. A similar result is obtained when glucose is replaced by glycerol, thus defining a general catabolite repression mechanism. A deletion of xylR, which encodes the xylose-sensitive repressor of the operon, does not affect glucose repression. The cis element mediating glucose repression was identified by Bal31 deletion analysis. It is confined to a 34 by segment located at position + 125 downstream of the xyl promoter in the coding sequence for xylose isomerase. Cloning of this segment in the opposite orientation leads to reduced catabolite repression. The homology of this element to various proposed consensus sequences for catabolite repression in B. subtilis is discussed.

Regulation of the Bacillus subtilis W23 xylose utilization operon : interaction of the Xyl repressor with the xyl operator and the inducer xylose

SummaryA crude protein extract of Bacillus subtilis W23 contains a sequence-specific DNA binding activity for the xyl operator as detected by the gel mobility shift assay. A xylR determinant encoded on a multicopy plasmid leads to increased expression of this binding activity. In situ footprinting analysis of the protein-DNA complex in a polyacrylamide gel shows that the xyl operator is sequence-specifically bound and protected from cleavage by copper-phenanthroline at 26 phosphodiester bonds on each strand. Quantitative competition assays for repressor binding reveal that a 25 by synthetic xyl operator cloned into a polylinker is bound with the same affinity as the operator in the wild-type xyl regulatory region. This confirms that no additional sites in the wild-type sequence contribute to repressor binding. The xyl operator consists of ten palindromic base pairs flanking five central non-palindromic base pairs. A mutational analysis shows that the sequence of the central base pairs contributes to recognition by the repressor protein and that the spacing of the palindromic elements is crucial for repressor binding. An operator half site is not bound by the repressor. In vivo and in vitro induction studies suggest that, of several structurally similar sugars, xylose is the only molecular inducer of the Xyl repressor.

Transcription of the nfrA-ywcH Operon from Bacillus subtilis Is Specifically Induced in Response to Heat

The NfrA protein, an oxidoreductase from the soil bacterium Bacillus subtilis, is synthesized during the stationary phase and in response to heat. Analysis of promoter mutants revealed that the nfrA gene belongs to the class III heat shock genes in B. subtilis. An approximate 10-fold induction at both the transcriptional and the translational levels was found after thermal upshock. This induction resulted from enhanced synthesis of mRNA. Genetic and Northern blot analyses revealed that nfrA and the gene downstream of nfrA are transcribed as a bicistronic transcriptional unit. The unstable full-length transcript is processed into two short transcripts encoding nfrA and ywcH. The nfrA-ywcH operon is not induced by salt stress or by ethanol. According to previously published data, the transcription of class III genes in general is activated in response to the addition of these stressors. However, this conclusion is based on experiments which lacked a valid control. Therefore, it seems possible that the transcription of all class III genes is specifically induced by heat shock.

The sigmaD-dependent transcription of the ywcG gene from Bacillus subtilis is dependent on an excess of glucose and glutamate.

We investigated the function and transcriptional regulation of ywcG. The protein is essential for Bacillus subtilis. Biochemical characterization of the protein revealed that it is an FMN‐containing NADPH oxidase. ywcG is transcribed throughout the whole life cycle of B. subtilis. The start point of transcription is preceded by potential promoter sequences for σA, σB and σD. A boost in transcription occurs at the beginning of stationary phase in complex media containing glutamate and glucose. The induction of transcription at the beginning of stationary phase needs the activity of a different alternative σ‐factor σD. ywcG is, therefore, the first gene with a putative role in energy metabolism from B. subtilis that is transcribed in a σD‐dependent fashion, but its regulation is unique and the reverse of that described for all other σD‐dependent genes.

DEGRADATION OF THE BACILLUS SUBTILIS XYNA TRANSCRIPT IS ACCELERATED IN RESPONSE TO STRESS

A popular method for the investigation of transcriptional regulation of gene expression is direct measurement of mRNA levels. As an internal control the level of a transcript from a constitutively expressed gene is often determined. To measure the induction rate of stress-responsive genes fromBacillus subtilis the transcript of the constitutively expressedxynA gene was used as a control. But the results presented in this communication prove that the degradation rate of thexynA transcript rises considerably in response to different kinds of stress. This response to stress is not dependent on protein biosynthesis.