Renée M. Tsolis

Identification of a new iron regulated locus of Salmonella typhi

In order to identify genes belonging to the Fur regulon of Salmonella typhi which are absent from Escherichia coli K-12, a plasmid gene bank consisting of 4000 independent clones was screened for Fur regulated promoters using the Fur titration assay (FURTA). DNA probes generated from FURTA positive plasmids were then used for hybridization with chromosomal DNA from S. typhi, Salmonella typhimurium and E. coli. Using these techniques we identified an iron regulated locus present in S. typhi and S. typhimurium but not in E. coli. Further cloning and nucleotide sequence analysis identified two open reading frames, termed iroBC, organized in a typical operon structure. The genes iroBC were located at 4 and 57 centisomes on the physical maps of Salmonella typhi and S. typhimurium, respectively. This region of the S. typhimurium chromosome contains a large DNA loop which is absent from the corresponding area of the E. coli chromosome. Finally, we developed a new method for generation of single copy transcriptional fusions. A suicide vector was constructed, which allows for the generation of chromosomal fusions to the promoterless E. coli lacZYA genes. By integration of this construct at the iro locus we could establish iron responsive expression of iroBC.

Fimbrial Adhesins of Salmonella Typhimurium

S. typhimurium initiates infection of its mammalian host by attachment to mucosal surfaces in the intestine and subsequent invasion of epithelial cells. To date, three S. typhimurium fimbrial operons, fim, lpf and pef, have been characterized. This analysis suggests that fimbrial adhesins fulfill multiple functions during the initial phase of an infection. In addition to their role in colonization of the small intestine, adhesins contribute to the tissue tropism for Peyers patches, which is characteristic for Salmonella infections. Furthermore, by mediating the initial contact to epithelial cells, fimbrial adhesins appear to be necessary for invasion and possibly for elicitation of an inflammatory response. Thus, fimbriae are important virulence factors of S. typhimurium and their future analysis promises to yield fascinating new insights into host-parasite interactions of this pathogen.

Mutagenesis and variant selection in Salmonella.

Publisher Summary This chapter describes methods for mutagenesis of the Salmonella typhimurium genome and potential methods for screening a bank of mutants and genetic analysis of interesting variants. The chapter focuses on frequently used systems for transposon mutagenesis and variant selection. Variant selection describes in vitro cell culture assays designed to enrich a pool of mutants for those with defects in genes related to pathogenesis. Although the chapter focuses on methods developed in S. typhimurium , many of these are also applicable to other Salmonella serovars. Variants that have alterations in a defined region of the genome have been created in two independent ways: plasmid curing and construction of hybrids between different Salmonella strains. The breakthrough in creating large numbers of defined mutants that are easy to analyze came with the introduction of transposon mutagenesis. These mobile genetic elements insert more or less randomly in the genome, thus disrupting the function of the gene into which they have inserted. Transposons that are used for mutagenesis contain selectable markers. This important feature allows transfer of the mutation into a clean background, ensuring that an observed phenotype can be attributed to a single mutation.