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). / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-11-06 10:51:01.253

RdoA is a eukaryotic-like serine/threonine protein kinase found in Salmonella typhimurium. It is a downstream effector of the Cpx stress response pathway and has been phenotypically characterized to have a functional role in flagellin phase variation and long-term bacterial survivability. Structurally, RdoA is homologous to, choline kinase and aminoglycoside (3’) phosphotransferase IIIa (APH[3’]IIIa). These kinases all belong to a protein kinase superfamily and share highly conserved residues/motifs in their catalytic domain. In RdoA seven of these conserved amino acids were proposed to have functional roles in the phosphotransfer mechanism. Mutation of these proposed catalytic domain residues resulted in a loss of in vitro kinase activity and in vivo RdoA function for a majority of the mutants. Four of the mutants also exhibited decreased levels of stable RdoA compared to wildtype. Many protein kinases regulate activity through phosphorylation of an activation loop. Although RdoA does not contain a canonical activation loop, its carboxyl terminus is proposed to play a similar regulatory function. Mutations of a putative autophosphorylation target in the carboxyl terminus resulted in loss of in vitro kinase activity. Truncations of this region also resulted in loss of kinase activity, as well as decreasing RdoA stability. The length of the carboxyl terminus in the kinase was shown to be an important determinant in the overall structural stability of RdoA. Mutational analyses of conserved amino acid residues surrounding the putative substrate-binding cleft of RdoA revealed site specific mutants with diminished in vitro phosphorylation activity and/or RdoA levels. A subset of these mutants for which no in vitro kinase activity was detected were still able to complement RdoA function in vivo. Taken together these results indicate that this region of the protein is important for RdoA function. In summary, this work has generated a panel of RdoA mutants with several unique phenotypes that will facilitate characterization of RdoA function and of regions of the protein / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2010-09-29 21:35:42.815