In an initial application of the method, we have added the sequences encoding the FLAG and 3xFLAG and influenza virus hemagglutinin epitopes to various genes of Salmonella enterica serovar Typhimurium, including putative and established pathogenic determinants present in prophage genomes. Epitope fusion proteins were detected in bacteria growing in vitro, tissue culture cells, and infected mouse tissues. This work identified a prophage locus specifically expressed in bacteria growing intracellularly. The procedure described here should be applicable to a wide variety of Gram-negative bacteria and is particularly suited for the study of intracellular pathogens.

A relevant, yet little recognized feature of antisense regulation is the possibility of switching roles between regulatory and regulated RNAs. Here we show that induction of a Salmonella gene relies on the conversion of a small RNA from effector to regulatory target. The chiP gene (formerly ybfM), identified and characterized in the present study, encodes a conserved enterobacterial chitoporin required for uptake of chitin-derived oligosaccharides. In the absence of inducer, chiP is kept silent by the action of a constitutively made small RNA, ChiX (formerly SroB, RybC), which pairs with a sequence at the 59 end of chiP mRNA. Silencing is relieved in the presence of chitooligosaccharides due to the accumulation of an RNA that pairs with ChiX and promotes its degradation. Anti-ChiX RNA originates from an intercistronic region of the chb operon, which comprises genes for chitooligosaccharide metabolism and whose transcription is activated in the presence of these sugars. We present evidence suggesting that the chb RNA destabilizes ChiX sRNA by invading the stem of its transcription terminator hairpin. Overall, our findings blur the distinction between effector and target in sRNA regulation, raising the possibility that some of the currently defined targets could actually be inhibitors of sRNA function.[Keywords: Antisense regulation; chitoporin; chitobiose; Hfq; small RNA] Supplemental material is available at http://www.genesdev.org.

Gene transfer between separate lineages of a bacterial pathogen can promote recombinational divergence and the emergence of new pathogenic variants. Temperate bacteriophages, by virtue of their ability to carry foreign DNA, are potential key players in this process. Our previous work has shown that representative strains of Salmonella typhimurium (LT2, ATCC14028 and SL1344) are lysogenic for two temperate bacteriophages: Gifsy‐1 and Gifsy‐2. Several lines of evidence suggested that both elements carry genes that contribute to Salmonella virulence. One such gene, on the Gifsy‐2 prophage, codes for the [Cu, Zn] superoxide dismutase SodCI. Other putative pathogenicity determinants were uncovered more recently. These include genes for known or presumptive type III‐translocated proteins and a locus, duplicated on both prophages, showing sequence similarity to a gene involved in Salmonella enteropathogenesis (pipA). In addition to Gifsy‐1 and Gifsy‐2, each of the above strains was found to harbour a specific set of prophages also carrying putative pathogenicity determinants. A phage released from strain LT2 and identified as phage Fels‐1 carries the nanH gene and a novel sodC gene, which was named sodCIII. Strain ATCC14028 releases a lambdoid phage, named Gifsy‐3, which contains the phoP/phoQ‐activated pagJ gene and the gene for the secreted leucine‐rich repeat protein SspH1. Finally, a phage specifically released from strain SL1344 was identified as SopEΦ. Most phage‐associated loci transferred efficiently between Salmonella strains of the same or different serovars. Overall, these results suggest that lysogenic conversion is a major mechanism driving the evolution of Salmonella bacteria.