Genetic and genomic approaches have been successfully used to assign genes to distinct regulatory networks. However, the present challenge of distinguishing differentially regulated genes within a network is particularly hard because members of a given network tend to have similar regulatory features. We have addressed this challenge by developing a method, termed Gene Promoter Scan, that discriminates coregulated promoters by simultaneously considering both multiple cis promoter features and gene expression. Here, we apply this method to probe the regulatory networks governed by the PhoP͞PhoQ two-component system in the enteric bacteria Escherichia coli and Salmonella enterica. Our analysis uncovered members of the PhoP regulon and interactions with other regulatory systems that were not discovered in previous approaches. The predictions made by Gene Promoter Scan were experimentally validated to establish that the PhoP protein uses multiple mechanisms to control gene transcription, regulates acid resistance determinants, and is a central element in a highly connected network.promoter ͉ machine learning ͉ gene transcription ͉ acid pH

A fundamental question in signal transduction is how an organism integrates multiple signals into a cellular response. Here we report the mechanism by which the Salmonella PmrA/PmrB two-component system responds to the signal controlling the PhoP/PhoQ two-component system. We establish that the PhoP-activated PmrD protein binds to the phosphorylated form of the response regulator PmrA, preventing both its intrinsic dephosphorylation and that promoted by its cognate sensor kinase PmrB. This results in PmrA-mediated transcription because phosphorylated PmrA exhibits higher affinity for its target promoters than unphosphorylated PmrA. A PmrD-independent form of the PmrA protein was resistant to PmrB-catalyzed dephosphorylation and promoted transcription of PmrA-activated genes in the absence of inducing signals. This is the first example of a protein that enables a two-component system to respond to the signal governing a different two-component system by protecting the phosphorylated form of a response regulator.[Keywords: PhoP; PhoQ; PmrA; PmrB; PmrD; protein phosphorylation; signal transduction] Supplemental material is available at http://www.genesdev.org.

Transcription profile microarray analysis in Escherichia coli was performed to identify the member genes of the Mg 2؉ stimulon that respond to the availability of external Mg 2؉ in a PhoP/PhoQ two-component systemdependent manner. The mRNA levels of W3110 in the presence of 30 mM MgCl 2 , WP3022 (phoP defective), and WQ3007 (phoQ defective) were compared with those of W3110 in the absence of MgCl 2 . The expression ratios of a total of 232 genes were <0.75 in all three strains (the supplemental data are shown at http://www.nara-.kindai.ac.jp/nogei/seiken/array.html), suggesting that the PhoP/PhoQ system is involved directly or indirectly in the transcription of these genes. Of those, 26 contained the PhoP box-like sequences with the direct repeats of (T/G)GTTTA within 500 bp upstream of the initiation codon. Furthermore, S1 nuclease assays of 26 promoters were performed to verify six new Mg 2؉ stimulon genes, hemL, nagA, rstAB, slyB, vboR, and yrbL, in addition to the phoPQ, mgrB, and mgtA genes reported previously. In gel shift and DNase I footprinting assays, all of these genes were found to be regulated directly by PhoP. Thus, we concluded that the phoPQ, mgrB, mgtA, hemL, nagA, rstAB, slyB, vboR, and yrbL genes make up the Mg 2؉ stimulon in E. coli.

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SUMMARY Gram-negative bacteria often modify their lipopolysaccharide (LPS) thereby increasing resistance to antimicrobial agents and avoidance of the host immune system. However, it is unclear how bacteria adjust the levels and activities of LPS-modifying enzymes in response to the modification status of their LPS. We now address this question by investigating the major regulator of LPS modifications in Salmonella enterica. We report that the PmrA/PmrB system controls expression of a membrane peptide that inhibits the activity of LpxT, an enzyme responsible for increasing the LPS negative charge. LpxT’s inhibition and the PmrA-dependent incorporation of positively charged L-4-aminoarabinose into the LPS decrease Fe3+ binding to the bacterial cell. Because Fe3+ is an activating ligand for the sensor PmrB, transcription of PmrA-dependent LPS-modifying genes is reduced. This mechanism enables bacteria to sense their cell surface by its effect on the availability of an inducing signal for the system regulating cell surface modifications.