Mycobacterium smegmatis RoxY is a repressor of oxyS and contributes to resistance to oxidative stress and bactericidal ubiquitin-derived peptides.

The mycobactericidal properties of macrophages include the generation of reactive oxygen intermediates and the delivery of bacteria to a hydrolytic lysosome enriched in bactericidal ubiquitin-derived peptides (Ub-peptides). To better understand the interactions of ubiquitin-derived peptides with mycobacteria and identify putative mycobacterial intrinsic resistance mechanisms, we screened for transposon mutants with increased susceptibility to the bactericidal Ub-peptide Ub2. We isolated 27 Mycobacterium smegmatis mutants that were hypersusceptible to Ub2. Two mutants were isolated that possessed mutations in the msmeg_0166 gene, which encodes a transcriptional regulator. The msmeg_0166 mutants were also hypersusceptible to other host antimicrobial peptides and oxidative stress. In characterizing msmeg_0166, we found that it encodes a repressor of oxyS, and therefore we have renamed the gene roxY. We demonstrate that RoxY and OxyS contribute to M. smegmatis resistance to oxidative stress. An ahpD transposon mutant was also isolated in our screen for Ub-peptide hypersusceptibility. Overexpression of oxyS in M. smegmatis reduced transcription of the ahpCD genes, which encode a peroxide detoxification system. Our data indicate that RoxY, OxyS, and AhpD play a role in the mycobacterial oxidative stress response and are important for resistance to host antimicrobial peptides.

Abundance of the delta subunit of RNA polymerase is linked to the virulence of Streptococcus agalactiae.

Group B streptococcus (GBS) remains a major cause of morbidity and mortality among newborn children. The bacterium is a commensal organism colonizing the rectum and the gastrointestinal and urogenital tracts of adults, but it can be transmitted to neonates by an ascending infection of the maternal genital tract or during parturition. We previously reported that a transposon insertion disrupting rpoE resulted in the decreased survival of the mutant in the neonatal rat sepsis model of GBS infection. rpoE encodes the delta protein, a subunit of RNA polymerase (RNAP) that has been characterized in Bacillus species. In this study, we confirm the association of the delta protein with purified GBS RNAP and show that it is expressed in strains representing all nine serotypes. Flow cytometric analysis of a reporter strain containing a transcriptional fusion of the rpoE promoter to gfp revealed that, in vitro, this gene is continuously expressed. Analysis of delta expression in the transposon mutant by quantitative Western blotting revealed a 10-fold reduction in relative abundance (which was linked to the attenuation in virulence that was observed for this mutant) compared to that for the wild-type strain. These data suggest that a minimum intracellular concentration of delta is necessary for this organism to cause disease.