2 μg/ml ATc before β-galactosidase activity was measured (arbitrary units) as described . The data correspond to the means of three independent experiments performed in duplicate, and the error bars represent standard deviations. Discussion We identified CacA, encoded on a plasmid clone, as a novel connector-like factor that activated the CpxR/CpxA system from screening a library of high-copy-number Selumetinib in vitro plasmids containing Selleck Adriamycin various Salmonella chromosomal DNA fragments. CacA appears to exclusively act on the CpxR/CpxA system because a similar induction was not observed in other TCS reporter strains with the same clone. This observation was not just
an artifact of CacA overexpression or from its expression driven by a heterologous Selonsertib promoter because deleting this gene revealed a moderate decrease in transcription of the cpxP and spy genes, which are directly regulated by the CpxR/CpxA system. Moreover, the activation
of the cacA gene promoter is, at least in part, dependent on RpoS, the stability of which is subject to RssB/ClpXP-mediated processability and the -10 region sequence. Taken together, we hypothesize that CacA may integrate information about the regulatory status of RssB/RpoS into the CpxR/CpxA system (Figure 5). However, future investigations are necessary to fully elucidate the mechanism of CacA-mediated CpxR/CpxA activation. Figure 5 A model for the regulatory interactions between RssB/RpoS and the CpxR/CpxA system. RpoS accumulates during stationary phase and log phase, when the small anti-adopter protein IraP inhibits the RssB/ClpXP-mediated degradation of RpoS in low Mg2+ conditions . RpoS induces expression of CacA, which stimulates the CpxR/CpxA system thus activating cpxP transcription. TrxA functionally associates with CacA-mediated Cpx induction. Several assessments of how the CacA selleck chemicals protein activates CpxR-regulated genes were attempted. However, we did not detect a physical association between CacA and the CpxR/CpxA system. For example, no significant interaction was observed between the CacA
protein and the CpxR/CpxA system in our bacterial two-hybrid system analyses (data not shown), although we cannot completely dismiss that these proteins do not interact directly. Instead, thioredoxin 1 amino acid sequences were recovered by our pull-down assay. trxA inactivation impacted the activation of the CpxR/CpxA system by CacA, which possesses the conserved cysteine residues. This is in contrast to a report that demonstrated that a dsbD mutation activated the CpxR/CpxA system in Vibrio cholerae, where the DsbC-DsbD pathway promotes proper folding of substrate proteins with disulfide bond(s) at the periplasm using the cytoplasmic reducing ability of thioredoxin . Moreover, the cysteine residues of NlpE are critical for activating the CpxR/CpxA system in E. coli, and a periplasmic LolA derivative with an artificial disulfide bond activates the CpxR/CpxA system .