The global regulator Hfq participates in the singlet oxygen stress response of Rhodobacter sphaeroides

The phototrophic bacterium Rhodobacter sphaeroides is an established model organism for studying the response to singlet oxygen (¹O₂), a highly reactive oxygen species, generated by illumination of the photosynthetic apparatus under oxic conditions. In a previous RNA-seq study, we have identified five sRNAs which were either induced or processed under ¹O₂ stress (1, 2). Their induction depends on the RpoE and RpoH_II sigma factors, which are known to be the major regulators of the ¹O₂ response. Accordingly, ¹O₂ dependent regulatory networks, comprised of sigma factors and sRNAs, exist in R. sphaeroides. The conserved RNA-chaperone Hfq is one of the key players in sRNA-mediated regulation in many bacteria. To get insights into the possible roles of Hfq in R. sphaeroides and the ¹O₂ response and to identify the direct sRNA and mRNA binding partners of Hfq in this bacterium, we used a co-immunoprecipitation strategy combined with deep sequencing as previously described for Salmonella (3) and confirmed the Hfq-dependency of several known and also newly identified sRNAs by Northern blot analysis. Strikingly, >70% of the Hfq-associated sRNAs were ¹O₂-affected. Among Hfq-associated mRNAs we found several mRNAs for cell division and ribosomal proteins. In addition, gel-based proteomics revealed an influence of Hfq on RpoH_II-dependent genes, amino acid transport/metabolism, and ATP synthase. Overall, this study suggests Hfq to be a global regulator like in other bacteria and largely explains the pleiotropic phenotype of strain 2.4.1Δhfq. The extensive work on sRNAs in R. sphaeroides will help to solve the question of how photosynthetic bacteria manage an effective ¹O₂ stress response.