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

The incidence of foodborne disease caused by Salmonella enterica is increasing yearly, despite the growing body of information regarding these most common pathogens. We examined the growth of, and lipopolysaccharide (LPS) expression of three Salmonella species (S. typhimurium, S. enteritidis , and S. blockley) grown at different pH values and temperatures and to different stages of the growth cycle. Growth conditions were found to affect the compositions of the LPS molecules produced by the organisms. The Tricine-SDS-PAGE and immunoblot profiles of LPS from the three Salmonella species were in accordance with the chemical data. Altered LPS profiles at different growth conditions were noted. Such differences among the profiles include variations within core oligosaccharides and the presence of O-side chains. The results also show that S. enteritidis and S. blockley grown to their exponential and stationary phases of the growth cycle produced longer O antigens and greater amounts of O antigens than did S. typhimurium grown at different growth stages. Tricine-SDS-PAGE allowed excellent resolution of low molecular weight LPS and the core region of LPS.

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<I>Escherichia coli</I> mutants impaired in growth and survival at low external pH in minimal medium were selected and attempts made to identify the disrupted genes. This study suggested that <I>clpX</I>, encoding a heat-shock induced protease and molecular chaperone, was functional in survival of <I>E. coli </I>at pH 3.3. Promoter probe plasmid libraries of <I>Salmonella typhimurium </I>LT2 DNA were created in <I>E. coli </I>and screened for acid-inducible transcriptional elements, and transcriptionally active fragments of degradative amino-acid decarboxylase genes recovered. Chromosomal gene fusions to the reporter gene <I>lacZ </I>in <I>E. coli</I> generated by Mu DII 1734 insertion were screened in a similar way and suggested that the gene encoding adenylate cyclase (<I>cya</I>) could be induced by mild cytoplasmic acidification. The sequence of a gene known to be inducible by cytoplasmic acidification, <I>inaA, </I>became available during the course of this study. The 5' region of this gene was used to generate a set of plasmids carrying fragments of the acid-inducible promoter transcriptionally fused to a luciferase based reporter system. Elements of the sequence required for induction by cytoplasmic acidification were identified. One of these reporter constructs was used to screen an <I>E. coli </I>Tn<I>10</I> chromosomal insertional mutant library for genes involved in the regulation of <I>inaA. </I>One such mutant had a multiple antibiotic resistant (<I>mar</I>) phenotype. The disrupted loci in 2 other mutants were identified by inverse PCR, sequence analysis and database searches. Both were known only as open reading frames (ORFs) discovered during the sequencing of the entire <I>E. coli</I> genome, and were tentatively identified as <I>yddB </I>(closely linked to <I>gadB </I>and <I>gadC</I>; required for glutamate dependent acid resistance) and <I>f300</I> (closely linked to <I>pldA; </I>required for detergent resistance). The promoter of <I>f300</I> was shown to be sensitive to cytoplasmic acidification. The <I>inaA</I> promoter was also demonstrated to be induced at the onset of stationary phase, and to be independent of the stationary phase and weak-acid inducible σ factor RpoS and also of cAMP levels.

Salmonella species are among the major foodborne intestinal pathogens that are of public concern with respect to food safety. The ability of intestinal pathogens to resist gastric acidity corresponds to their oral infective dose (ID). The survival and lipopolysaccharide (LPS) profiles of Salmonella typhimurium grown at different pH values and to different phases of growth were examined in simulated gastric fluid (pH 1.5), ileal fluid (pH 7.0), colon fluid (pH 8.0). The survival and growth of S. typhimurium were also examined during sequential passage through all three fluids. Viable cells were rapidly reduced from 106 CFU.ml-1 to <10 CFU.ml-1 within 4 min in gastric fluid. Cells inoculated directly into ilea] and colon fluids survived and multiplied extensively. When low numbers of viable cells of Salmonella in contact with gastric fluid (0.5 min of contact) were transferred sequentially to ileal and colon fluids, only the early and late stationary phase cells were capable of recovery and growth to high numbers. The harsh environment of the gastric fluid did not change the LPS profiles of the inoculated Salmonella cells. Entrapment of S. typhimurium in calcium alginate beads and chocolate increased its survival in gastric fluid. This implies that Salmonella cells are protected from killing when ingested with food. These results may explain why Salmonella species have a very low ID when consumed as part of some contaminated food sources.

Over the past decades, there has been increase in the consumption of the fresh tomato in the United States; this has been attributed to the nutritional benefits of fresh tomato, its widespread use in cooking and its availability throughout the year. In a Food and Agricultural Organization report, the United States was ranked as one of the largest producers of the fresh tomato in the world. In spite of its large production capacity, large quantities of the tomatoes are still being imported to the United States annually from Mexico. Series of multistate outbreaks of Salmonella infection have been associated with consumption of the fresh tomatoes; traceback of the tomatoes implicated in salmonellosis has been traced to tomatoes grown domestically. However, a survey conducted by U.S. Department of Agriculture on both domestic and imported tomatoes determined that imported fresh tomato was Salmonella positive. The purpose of this study was to determine the microbiological quality of fresh tomatoes imported from Mexico to the United States. The study consisted of sampling surfaces of cleaned tomatoes in Mexico prior to packing and shipping to the United States, and sampling of the tomato wash water at the end of the work shift at a Mexican tomato packinghouse. Four tomatoes were randomly sampled prior to packing, and they were rinsed with Universal Preenrichment Broth (UPB), this was repeated 10 times per working shift, with 2 shifts per day. 102 l of tomato wash water were collected and sampled with the aid of Modified Moore’s Swab (MMS) and membrane filter. The tomato wash water was collected at the end of shift twice daily. Both fruit and wash water samplings were repeated 3 times during the tomato harvesting season. Both the tomato UPB rinsates and the membrane filter were assayed for the E. coli and enterococci populations. Additionally, the tomato UPB rinsates and MMS were assayed for the presence of Salmonella. The results of the microbiological analysis on the UPB rinsates showed that no Salmonella was present, E. coli was not detectable (< 1.0), and the mean populations of enterococci were log 3.8, 2.6, and 1.0 CFU/g in sampling trials 1, 2, and 3 respectively. In the tomato wash water, no Salmonella was present, and no E. coli and enterococci were detected. Therefore, it was concluded that the microbiological quality of the tomatoes that were sampled and tested were high, this was due to the fact that all the samples collected tested negative to Salmonella analysis, and no E. coli was detected in any of the samples.