The barA and sirA genes of Salmonella enterica serovar Typhimurium encode a two-component sensor kinase and a response regulator, respectively. This system increases the expression of virulence genes and decreases the expression of motility genes. In this study, we examined the pathways by which SirA affects these genes. We found that the master regulator of flagellar genes, flhDC, had a positive regulatory effect on the primary regulator of intestinal virulence determinants, hilA, but that hilA had no effect on flhDC. SirA was able to repress flhDC in a hilA mutant and activate hilA in an flhDC mutant. Therefore, although the flhDC and hilA regulatory cascades interact, sirA affects each of them independently. A form of BarA lacking the two Nterminal membrane-spanning domains, BarA198, autophosphorylates in the presence of ATP and transfers the phosphate to purified SirA. Phosphorylated SirA was found to directly bind the hilA and hilC promoters in gel mobility shift assays but not the flhD, fliA, hilD, and invF promoters. Given that the CsrA/csrB system is known to directly affect flagellar gene expression, we tested the hypothesis that SirA affects flagellar gene expression indirectly by regulating csrA or csrB. The sirA gene did not regulate csrA but did activate csrB expression. Consistent with these results, phosphorylated SirA was found to directly bind the csrB promoter but not the csrA promoter. We propose a model in which SirA directly activates virulence expression via hilA and hilC while repressing the flagellar regulon indirectly via csrB.
The sirA gene of Salmonella enterica serovar Typhimurium encodes a two-component response regulator of the FixJ family that has a positive regulatory influence on the expression of type III secretion genes involved with epithelial cell invasion and the elicitation of bovine gastroenteritis. SirA orthologs in Pseudomonas, Vibrio, and Erwinia control the expression of distinct virulence genes in these genera, but an evolutionarily conserved target of SirA regulation has never been identified. In this study we tested the hypothesis that sirA may be an ancient member of the flagellar regulon. We examined the effect of a sirA mutation on transcriptional fusions to flagellar promoters (flhD, fliE, fliF, flgA, flgB, fliC, fliD, motA, and fliA) while using fusions to the virulence gene sopB as a positive control. SirA had only small regulatory effects on all fusions in liquid medium (less than fivefold). However, in various types of motility agar plates, sirA was able to activate a sopB fusion by up to 63-fold while repressing flagellar fusions by values exceeding 100-fold. Mutations in the sirA orthologs of Escherichia coli, Vibrio cholerae, Pseudomonas fluorescens, and Pseudomonas aeruginosa result in defects in either motility or motility gene regulation, suggesting that control of flagellar regulons may be an evolutionarily conserved function of sirA orthologs. The implications for our understanding of virulence gene regulation in the gamma Proteobacteria are discussed.
The design of real-time polymerase chain reaction (PCR) assays for the detection of meat in processed products has focused on using small amplicons, often to the detriment of specificity. However, the relationship between amplification rates and the amplicon size for processed meat products has yet to be determined. To investigate this relationship, real-time PCR assays were designed to give a series of amplicons of increasing size. These assays were then used to assess amplification rates, in relation to amplicon size, in processed meat matrices. Although the most sensitive assays were those that used the smallest amplicons, amplification was still observed using amplicons of 351 base pairs for highly processed samples. It was found, therefore, that although in general, amplicons should be as small as possible, larger amplicons give efficient amplification and that small amplicons should not be chosen if they compromise assay specificity.
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