In addition, one of the discernable patterns from the two microarrays was that the three genes flanking the preAB operon: ygiW, STM3175, mdaB, were upregulated 37-, 21-, and ~7-fold, respectively (Table 2, column 2). Furthermore, in the preAB mutant background, we also observed upregulation of additional genes belonging to the PhoP/PhoQ and PmrA/PmrB regulons: pmrAB, udg, cptA (STM4118) and pagP. This further supports the connection between preAB and the

two major regulons controlling genes involved in LPS modifications and antimicrobial peptide resistance in Salmonella and provides confidence to the quality of our microarray experiments. qRT-PCR analysis and transcriptional organization of preAB and flanking genes To confirm the results of the microarray

and to examine the regulation of preAB and the genes surrounding it, we performed qRT-PCR. The preA gene find more was shown to be induced 344-fold in a ΔpreB strain vs. a wild type strain, furthering the previous finding of PreB acting primarily as a phosphatase when grown in LB and providing evidence of PreA-mediated positive autoregulation of preAB. The induction of preB in the microarray of the preA mutant background overexpressing preA also provided evidence of positive autoregulation of preAB (supplement Table 1). ygiW was strongly activated by PreA (355-fold) when comparing expression in a ΔpreAB/pBAD18-preA +strain vs. ΔpreAB/pBAD18. Using these same strains, ygiN was more weakly activated LGK-974 nmr by PreA (2.94-fold). Several other PreA-regulated genes including STM3175 (605.3-fold) and mdaB (32.5-fold) were also analyzed by qRT-PCR, all confirming the regulation observed in the microarrays (though not always matching the observed fold-change) (Table 2). The transcriptional organization of

the preAB operon and of the genes flanking it, which were strongly upregulated by PreA, Rebamipide were analyzed by RT-PCR. As shown in Fig. 1, PCR fragments spanning preA and preB, ygiW and STM3175, and mdaB and ygiN were observed, suggesting that these sets of genes are co-transcribed. While primers spanning preB and mdaB (separated by a 106 bp intergenic region) yielded PCR product using a DNA template, no such product was observed with cDNA, even with the use of multiple primer sets, suggesting that these genes are not co-transcribed. These data, coupled with the microarray results, suggest that PreA is necessary for the activation of the ygiW-STM3175, preA-preB, and mdaB-ygiN operons. Figure 1 Co-transcription analysis of the genes in the local chromosomal region surrounding preA. (A-D) The sets of genes examined are described above the ethidium bromide stained gels. The lane assignments in each set: (1) chromosomal DNA as a template; (2) cDNA as a template; (3) cDNA as a template, no reverse transcriptase. (E) A graphic representation of the preA-linked genes and the primers used for RT-PCR.