The RdoA-dependent Phosphoproteome Profile of Salmonella enterica
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RdoA, a serine/threonine kinase, is a member of the Cpx regulon, a stress response pathway, in Salmonella enterica serovar Typhimurium. Phenotypic characterization of rdoA null mutants suggested that RdoA kinase activity affects a wide range of cell functions, which could be the result of both direct and indirect phosphorylation of targets. In a search for RdoA’s target(s), the phosphoproteome profile of wild-type and rdoA null S. enterica was examined through phosphoprotein enrichment followed by 2-dimensional gel electrophoresis coupled with phospho-specific fluorescent stains and western blots using phospho-specific antibodies. Three different phosphoprotein enrichment protocols, all based on metal-ion affinity chromatography, were compared for yield and phosphoprotein specificity to determine which would be the most suitable for S. enterica. This study showed that the Phostag Enrich Phosphoprotein kit (PerkinElmer) gave the highest yield, the majority of which were phosphoproteins. These studies also showed that western blots using phospho-specific antibodies were more sensitive than phosphoprotein-specific fluorescent stain ProQ Diamond in detecting phosphoproteins. The phosphoproteome profile of S. typhimurium cells grown under Cpx activating conditions included phosphoproteins involved in the heat shock response, cellular metabolism and protein synthesis. This work also identified changes in the phosphoproteome that were dependent upon the presence or absence of RdoA. Phosphoproteins that showed a significant change in phosphorylation were identified by mass spectroscopy using peptide mass fingerprinting. Proteins identified included protein foldases (DnaK and GroEL), proteins involved in metabolism (glycerol kinase, enolase and E1 subunit of the pyruvate dehydrogenase complex), and in protein synthesis (elongation factor-Tu). These proteins may be phosphorylated in an RdoA-dependent manner to allow normal cell functioning under envelope stress. Several proteins unlikely to be phosphoproteins were also RdoA-dependent. SrgA, encoded on the virulence plasmid, is a disulfide oxidoreductase specific for the PEF fimbriae that was shown to be repressed by RdoA. This work also showed that integration host factor, previously suggested to be an RdoA target, was not affected in terms of expression or phosphorylation by RdoA. The several RdoA-dependent changes in protein expression levels and phosphorylation that were identified contribute to the elucidation of RdoA’s role in the envelope stress response and provided further insight in determining RdoA target(s).