Defining the regulatory mechanisms of sigma factor RpoS degradation in Azotobacter vinelandii and Pseudomonas aeruginosa

In several Gram‐negative bacteria, the general stress response is mediated by the alternative sigma factor RpoS, a subunit of RNA polymerase that confers promoter specificity. In Escherichia coli, regulation of protein levels of RpoS involves the adaptor protein RssB, which binds RpoS for presenting it to the ClpXP protease for its degradation. However, in species from the Pseudomonadaceae family, RpoS is also degraded by ClpXP, but an adaptor has not been experimentally demonstrated. Here, we investigated the role of an E. coli RssB‐like protein in two representative Pseudomonadaceae species such as Azotobacter vinelandii and Pseudomonas aeruginosa. In these bacteria, inactivation of the rssB gene increased the levels and stability of RpoS during exponential growth. Downstream of rssB lies a gene that encodes a protein annotated as an anti‐sigma factor antagonist (rssC). However, inactivation of rssC in both A. vinelandii and P. aeruginosa also increased the RpoS protein levels, suggesting that RssB and RssC work together to control RpoS degradation. Furthermore, we identified an in vivo interaction between RssB and RpoS only in the presence of RssC using a bacterial three‐hybrid system. We propose that both RssB and RssC are necessary for the ClpXP‐dependent RpoS degradation during exponential growth in two species of the Pseudomonadaceae family. In bacteria belonging to the Pseudomonadaceae family, such as Azotobacter vinelandii and Pseudomonas aeruginosa, the regulation of proteolysis of the alternative sigma factor RpoS remains poorly understood. Here, we demonstrate that during the exponential phase of growth, two proteins (RssB and RssC) are necessary for the degradation of RpoS by the proteolytic complex ClpXP.