Biomedical subjects

Robyn L Woodbury

Publications and source records attributed to Robyn L Woodbury.

4 recordsLinked to original sources

PubMed2006

Sigma X induces competence gene expression in Streptococcus pyogenes.

Although not thought to become competent for DNA uptake, the bacterium Streptococcus pyogenes appears to encode within its genome the DNA uptake and recombination machinery required for competence. The promoters of these genes contain a conserved sequence, CIN-box, which is recognized by the S. pyogenes alternative RNA polymerase sigma factor X. Using microarray technology, we found that sigma X induced the expression of competence genes in S. pyogenes. Real-time RT-PCR was performed to confirm that the expression of these transcripts was induced 2-265-fold by sigma X. Of the eight CIN-box loci induced, femB, the ortholog a virulence factor in Staphylococcus aureus, was shown to be transcribed in vitro by RNA polymerase containing sigma X, indicating that sigma X directly activates expression of this CIN-box gene. Sigma X-dependent induction of genes encoding competence machinery in S. pyogenes raises the possibility that some strains of this human pathogen can develop competence for genetic transformation.

Bacillus subtilis↗

PubMed2005

Coexpression patterns of sigma(B) regulators in Bacillus subtilis affect sigma(B) inducibility.

RsbT is an essential component of the pathway that activates the Bacillus subtilis sigma(B) transcription factor in response to physical stress. rsbT is located within an operon that includes the genes for its principal negative regulator (RsbS) and the stress pathway component that it activates (RsbU), as immediate upstream and downstream neighbors. In the current work we demonstrate that RsbT's ability to function is strongly influenced by coexpression with these adjoining genes. When rsbT is expressed at a site displaced from rsbS and rsbU, RsbT accumulates but it is unable to activate sigma(B) following stress. RsbT activity is restored if rsbT is cotranscribed at the alternative site with the genes that normally abut it. Additionally, an rsbS allele whose product allows constitutively high RsbT-dependent sigma(B) activity displays this activity in rsbS merodiploid strains only when cotranscribed with rsbT and is recessive to a wild-type rsbS allele only if the wild-type rsbS gene is not cotranscribed with an rsbT gene of its own. The data suggest that RsbS and RsbT are synthesized in equivalent amounts and interact coincidently with their synthesis to form stable regulatory complexes that maintain RsbT in a state from which it can be stress activated.

Bacillus subtilis↗

PubMed2004

Associations between Bacillus subtilis sigmaB regulators in cell extracts.

The general stress regulon of Bacillus subtilis is induced by the activation of the sigma(B) transcription factor. Activation of sigma(B) occurs as a consequence of the dephosphorylation of its positive regulator RsbV by one of two phosphatases that respond to either physical or nutritional stress. The physical stress phosphatase (RsbU) requires a second protein (RsbT) for activity. Stress is thought to initiate a process that triggers the release of RsbT from a large inhibitory complex composed of multiple copies of two protein species, RsbR (and/or its paralogues) and RsbS. The stress-derived signal driving RsbT release is unknown, but it fails to develop in B. subtilis lacking either ribosome protein L11 or the ribosome-associated protein Obg. RsbR, RsbS, RsbT, Obg and ribosomes elute in common high-molecular-mass fractions during gel-filtration chromatography of crude B. subtilis extracts. This paper reports the investigation of the basis of this coelution by the examining of associations between these proteins in extracts prepared from wild-type and mutant B. subtilis, and Escherichia coli engineered to express RsbR, RsbS and RsbT. Large RsbR/RsbS complexes, distinct from ribosomes, were detected in extracts of both B. subtilis and E. coli. In E. coli, high-molecular-mass forms of RsbS were less abundant when RsbR was absent, but in B. subtilis, only when both RsbR and its principal paralogues were missing from the extract was this form less abundant. This finding is consistent with the notion that the RsbR paralogues, present in B. subtilis but not E. coli, can substitute for RsbR in such complexes. RsbT was not bound to RsbR/RsbS in any extract that was examined, including one prepared from a B. subtilis strain with an RsbS variant (RsbS59SA) that is believed to continuously associate with RsbT. The high-molecular-mass forms of RsbT were found to be Triton-sensitive and independent of any other B. subtilis protein for their formation. These probably represent RsbT aggregates. The data suggest that the contribution of ribosomes/Obg to sigma(B) activation does not involve formation of a stable association between these proteins and the Rsb complex. In addition, the binding of RsbT to RsbS/RsbR appears to be more labile than the binding between the previously analysed Rsb proteins which form inhibitory complexes. This, and the apparent proclivity of RsbT to aggregate, suggests an inherent instability in RsbT which may play a role in its regulation.

Bacillus subtilis↗

PubMed2004

Mutational analysis of RsbT, an activator of the Bacillus subtilis stress response transcription factor, sigmaB.

SigmaB, the stress-activated sigma factor of Bacillus subtilis, requires the RsbT protein as an essential positive regulator of its physical stress pathway. Stress triggers RsbT to both inactivate the principal negative regulator of the physical stress pathway (RsbS) by phosphorylation and activate a phosphatase (RsbU) required for sigmaB induction. Neither the regions of RsbT that are involved in responding to stress signaling nor those required for downstream events have been established. We used alanine scanning mutagenesis to examine the contributions of RsbT's charged amino acids to the protein's stability and activities. Eleven of eighteen rsbT mutations blocked sigmaB induction by stress. The carboxy terminus of RsbT proved to be particularly important for accumulation in Bacillus subtilis. Four of the five most carboxy-terminal mutations yielded rsbT alleles whose products were undetectable in B. subtilis extracts. Charged amino acids in the central region of RsbT were less critical, with four of the five substitutions in this region having no measurable effect on RsbT accumulation or activity. Only when the substitutions extended into a region of kinase homology was sigmaB induction affected. Six other RsbT variants, although present at levels adequate for activity, failed to activate sigmaB and displayed significant changes in their ability to interact with RsbT's normal binding partners in a yeast dihybrid assay. These changes either dramatically altered the proteins' tertiary structure without affecting their stability or defined regions of RsbT that are involved in multiple interactions.

Alleles↗