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Salmonella are major food-borne pathogens responsible for causing a broad spectrum of human disease, ranging from mild gastroenteritis to life-threatening systemic disease. We are interested in analysing the function of the putative mrd operon in Salmonella, whose gene products include PBP2 and RodA; two proteins which are involved in longitudinal cell wall synthesis and essential for rod shapemaintenance in Salmonella. Although PBP2 is fairly well described, surprisingly little is known about the function of RodA or the other mrd operon proteins. This study aimed to characterise the mrd operon, elucidating its contribution to the biology and pathogenicity of Salmonella. Possible links between the structural integrity of the cell wall and the functionality of wall-spanning virulence organelles were also examined. Precise knockout mutations of the mrd operon genes were constructed in Salmonella enterica serovars Typhi and Typhimurium. A range of subsequent phenotypic analyses have revealed that the round-cell ΔpbpA (PBP2) and ΔrodA mutants, though viable and able to stably propagate, are defective in the expression of flagella and the Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS), although the SPI-2 T3SS remains active. These virulence defects were successfully complemented by expressing the major SPI-1 and flagella regulators in trans from inducible promoters. A transposon mutagenesis screen was subsequently developed to search for global regulators of the SPI-1 and motility phenotypes in round-cell mutants. This led to the identification of several candidate regulators, which ongoing work seeks to further analyse. This study has demonstrated that round-cell mrd mutants almost completely down-regulate the expression of key invasion-associated virulence genes, although the cell wall in these mutants remains sufficiently robust to enable the assembly and functioning of wall-spanning multi-protein complexes. Collectively these data provide important insights into both the physiological state of mrd operon mutants, and the function of mrd operon genes.

A collection of three newly isolated Salmonella bacteriophages, vB_SenS-Ent1, vB_SenS-Ent2 and vB_SenS-Ent3 was established. These bacteriophages were characterised by electron microscopy, host range, sensitivity to restriction enzymes and profiles of structural proteins on SDS-PAGE gels. The complete genome sequences of each bacteriophage were established to greater than 30x coverage and bioinformatics analysis identified the functions of a number of coding sequences. Since the last update of virus taxonomy by the ICTV a number of additional genome sequences for bacteriophages infecting the genus Salmonella have been reported in the literature. To date, all but one of the Siphoviridae comprising the Salmonella bacteriophages with fully sequenced genomes remain unclassified by the ICTV. Comparative genomic analysis reveals that a number of these phages form a coherent group within the Siphoviridae and supports the establishment of a new genus, the “Setp3likeviruses”. The proposed genus includes 5 bacteriophages infecting Salmonella; SETP3, vB_SenS-Ent1, SE2, wksl3 and SS3e, and 5 infecting Escherichia; K1G, K1H, K1ind1, K1ind2 and K1ind3. This group share identical virion morphology, have terminally redundant, circularly permuted genomes ranging between 42-45 kb in size and are characterised by high nucleotide sequence similarity, shared homologous proteins and conservation of gene order. Bioluminescent bacterial reporters, transformed to express the luxCDABE operon of Photorhabdus luminescens, were used to establish the activity of the vB_SenS-Ent bacteriophages in microtitre broth lysis assays, efficacy as biological control agents for the removal of Salmonella in contaminated foods and for spatial measurements of plaque expansion in agar overlays.

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No description available.