A genomewide mutagenesis screen identifies multiple genes contributing to Vi capsular expression in Salmonella enterica serovar Typhi

Abstract

A transposon-based, genomewide mutagenesis screen exploiting the killing activity of a lytic ViII bacteriophage was used to identify Salmonella enterica serovar Typhi genes that contribute to Vi polysaccharide capsule expression. Genes enriched in the screen included those within the viaB locus (tviABCDE and vexABCDE) as well as oxyR, barA/sirA, and yrfF, which have not previously been associated with Vi expression. The role of these genes in Vi expression was confirmed by constructing defined null mutant derivatives of S. Typhi, and these were negative for Vi expression as determined by agglutination assays with Vi-specific sera or susceptibility to Vi-targeting bacteriophages. Transcriptome analysis confirmed a reduction in expression from the viaB locus in these S. Typhi mutant derivatives and defined regulatory networks associated with Vi expression.

Fig 1

Mapping of transposon insertion reads within the mutants studied in detail. (A) Insertions in the viaB locus; (B) insertions in ompR and envZ; (C) insertions in rcsB; (D) insertions in barA; (E) insertions in sirA; (F) insertions in yrfF (igaA); (G) insertions in oxyR; (H) insertions in greA. The scale on the right indicates the number of reads for any insertion point shown. For some insertion points in the viaB locus, this exceeded 20,000 reads, while for ompR the maximum was approximately 500 reads at one insertion point for the transposon. Mapping of the insertion points to the sequence was done using the ARTEMIS program.

Fig 2

Gene expression levels within the viaB locus in S. Typhi barA, greA, oxyR, rcsB, sirA, and yrfF mutant strains, based upon RNA transcription microarrays. Note the significant difference between the barA, greA, and sirA mutants in one grouping and the rcsB, yrfF, and oxyR mutants in another. wt, wild type.