A novel compartment, the 'subqpical stem' of the aerial hyphae, is the location of a sigN-dependent, developmentally distinct transcription in Streptomyces coelicolor.

Streptomyces coelicolor has nine SigB-like RNA polymerase sigma factors, several of them implicated in morphological differentiation and/or responses to different stresses. One of the nine, SigN, is the focus of this article. A constructed sigN null mutant was delayed in development and exhibited a bald phenotype when grown on minimal medium containing glucose as carbon source. One of two distinct sigN promoters, sigNP1, was active only during growth on solid medium, when its activation coincided with aerial hyphae formation. Transcription from sigNP1 was readily detected in several whi mutants (interrupted in morphogenesis of aerial mycelium into spores), but was absent from all bld mutants tested, suggesting that sigNP1 activity was restricted to the aerial hyphae. It also depended on sigN, thus sigN was autoregulated. Mutational and transcription studies revealed no functional significance to the location of sigN next to sigF, encoding another SigB-like sigma factor. We identified another potential SigN target, nepA, encoding a putative small secreted protein. Transcription of nepA originated from a single, aerial hyphae-specific and sigN-dependent promoter. While in vitro run-off transcription using purified SigN on the Bacillus subtilis ctc promoter confirmed that SigN is an RNA polymerase sigma factor, SigN failed to initiate transcription from sigNP1 and from the nepA promoter in vitro. Additional in vivo data indicated that further nepA upstream sequences, which are likely to bind a potential activator, are required for successful transcription. Using a nepA–egfp transcriptional fusion we located nepA transcription to a novel compartment, the ‘subapical stem’ of the aerial hyphae. We suggest that this newly recognized compartment defines an interface between the aerial and vegetative parts of the Streptomyces colony and might also be involved in communication between these two compartments

A mutant of Salmonella enterica serovar Typhimurium RNA polymerase extracytoplasmic stress response sigma factor σE with altered promoter specificity

The alternative sigma factor σE is critical for envelope stress response and plays a role in pathogenicity of a variety of different bacteria. We previously identified several critical nucleotides in the Salmonella enterica serovar Typhimurium (S. Typhimurium) σE-dependent rpoEp3 promoter that corresponded to the most conserved nucleotides in the σE consensus sequence of the −10 and −35 promoter elements. In the present study, we exploited a previously established Escherichia coli (E. coli) two-plasmid system with an error-prone PCR mutagenesis to identify mutants in the rpoE gene that suppress the mutation of the most conserved residue A-30G of the rpoEp3 promoter. This analysis identified amino-acid changes in the conserved arginine residue (R171G, R171C) located in the conserved region 4.2 of σE that enabled efficient recognition of the mutated rpoEp3 promoter. However, the change of this conserved arginine to alanine (R171A) resulted in an almost complete loss of σE activity. The activity of the mutant σE factors in directing transcription of the wild-type (WT) and the A-30G mutated rpoEp3 promoters was investigated by S1-nuclease mapping using RNA isolated from the E. coli two-plasmid system. In addition to suppression of the A-30G mutated rpoEp3 promoter, both mutant sigma factors (R171G, R171C) also efficiently directed transcription from the WT rpoEp3 promoter and from the rpoEp3 promoter with other mutations in the −35 element, indicating relaxed recognition of the σE-dependent promoters by both mutants. The activity of both mutant σE factors was confirmed in vivo in S. Typhimurium. In conclusion, replacement of the conserved R171 residue in σE by different amino-acid residues exhibited intriguingly different phenotypes; R171A almost completely abolished sigma factor activity, whereas R171G and R171C impart a relaxed recognition phenotype to σE

Characterization of the micA gene encoding a small regulatory σE-dependent RNA in Salmonella enterica serovar Typhimurium

The role of MicA (repressing small regulatory non-coding RNAs of two Salmonella porins) was determined in virulence of Salmonella enterica serovar Typhimurium. Transcriptional analysis revealed that the expression of the micA gene is driven by a single sigma(E)-dependent promoter, micAp. Its activity increased towards stationary phase; in exponential phase, the activity was induced by several stresses by a DegS-dependent mechanism. Although phenotypic analysis revealed no significant differences between wild-type and the micA mutant strains, in vivo studies showed that this mutant is more virulent in the mouse mode

Phenotypic analysis of salmonella enterica serovar typhimurium rpoE mutants encoding RNA polymerase extracytoplasmic stress response sigma factors σE with altered promoter specificity

We previously identified mutants in the rpoE gene of Salmonella enterica serovar Typhimurium (S. Typhimurium) encoding RNA polymerase extracytoplasmic stress response sigma factors σE with altered promoter specificity. The replacement of the conserved R171 residue in the conserved region 4.2 of σE by different amino acid residues exhibited different phenotypes. While R171A almost completely abolished sigma factor activity, R171G and R171C mutant changes imparted a relaxed recognition phenotype to the sigma factor. In the present study, we introduced these mutations into the S. Typhimurium chromosome to investigate their phenotype during ethanol stress and in promoter recognition. Both relaxed sigma factors were found to initiate transcription from a high number of artificial promoters in the S. Typhimurium genome. Both mutants had substantially decreased activity under stress conditions. However, this decreased activity and also the recognition of atypical promoters had no significant effect upon growth, even in stressful conditions