Bacteria possess a signal transduction system, referred to as a two-component system, for adaptation to external stimuli. Each two-component system consists of a sensor protein-histidine kinase (HK) and a response regulator (RR), together forming a signal transduction pathway via histidyl-aspartyl phospho-relay. A total of 30 sensor HKs, including as yet uncharacterized putative HKs (BaeS, BasS, CreC, CusS, HydH, RstB, YedV, and YfhK), and a total of 34 RRs, including putative RRs (BaeR, BasR, CreB, CusR, HydG, RstA, YedW, YfhA, YgeK, and YhjB), have been suggested to exist in Escherichia coli. We have purified the carboxyl-terminal catalytic domain of 27 sensor HKs and the full-length protein of all 34 RRs to apparent homogeneity. Selfphosphorylation in vitro was detected for 25 HKs. The rate of self-phosphorylation differed among HKs, whereas the level of phosphorylation was generally co-related with the phosphorylation rate. However, the phosphorylation level was low for ArcB, HydH, NarQ, and NtrB even though the reaction rate was fast, whereas the level was high for the slow phosphorylation species BasS, CheA, and CreC. By using the phosphorylated HKs, we examined trans-phosphorylation in vitro of RRs for all possible combinations. Trans-phosphorylation of presumed cognate RRs by HKs was detected, for the first time, for eight pairs, BaeS-BaeR, BasS-BasR, CreC-CreB, CusS-CusR, HydH-HydG, RstB-RstA, YedV-YedW, and YfhK-YfhA. All trans-phosphorylation took place within less than 1/2 min, but the stability of phosphorylated RRs differed, indicating the involvement of de-phosphorylation control. In addition to the trans-phosphorylation between the cognate pairs, we detected trans-phosphorylation between about 3% of non-cognate HK-RR pairs, raising the possibility that the cross-talk in signal transduction takes place between two-component systems.

No abstract

No abstract

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.

The Tar chemoreceptor of Escherichia coli is a membrane-bound sensory protein that facilitates bacterial chemotaxis in response to aspartate. The EnvZ molecule has a membrane topology similar to Tar and is a putative osmosensor that is required for osmoregulation of the genes for the major outer membrane porin proteins, OmpF and OmpC. The cytoplasmic signaling domain of Tar was replaced with the carboxyl portion of EnvZ, and the resulting chimeric receptor activated transcription of the ompC gene in response to aspartate. The activation of ompC by the chimeric receptor was absolutely dependent on OmpR, a transcriptional activator for ompF and ompC.

Novel mode of transcription regulation of divergently overlapping promoters by PhoP, the regulator of two-component system sensing external magnesium availability opposite direction. In vitro transcription using mutant promoters support this prediction. In the presence of PhoP, transcription from the promoter treRP3 was repressed with concomitant activation of mgtAP1 transcription. The PhoP box is located between -46 and -30 with respect to treRP3, and the aCTD is needed for this repression. IntroductionThe PhoP-PhoQ system, a bacterial two-component system, senses the extracellular Mg(II) level and regulates transcription of a number of genes in response to the increase or decrease in external Mg(II) level (for a review, see Groisman, 2001). In Salmonella typhimurium, the PhoP-PhoQ system governs the expression of at least 40 genes (Miller and Mekalanos, 1990), including the genes for Mg(II) transporters, factors mediating resistance to antimicrobial peptides, and virulence factors. PhoP and PhoQ of Escherichia coli are 93% and 86% identical in amino acid sequence to the corresponding proteins from S. typhimurium (Kasahara et al., 1992). So far, five E. coli genes, phoPQ, mgtA, mgrB and mgrC have been identified to be under the control of the PhoP-PhoQ system (Groisman et al., 1992;Kato et al., 1999). Extracellular Mg(II) binds to the periplasmic domain of PhoQ and, as a result, the PhoQ protein becomes fixed in a conformation inactive in signalling (Waldburger and Sauer, 1996;Vescovi et al., 1997). As in the case of other two-component systems, the C-terminal domain of the E. coli PhoQ protein has autophosphorylation activity, and phosphorylated PhoQ transfers the phosphate moiety to the PhoP protein (Castelli et al., 2000;Yamamoto et al., 2000a; K. Yamamoto and A. Ishihama, unpublished data). Promoter regions of the genes under the control of PhoP contain a direct repeat with the consensus sequence (T/G)GTTTA, called the 'PhoP box' (Kato et al., 1999; also reviewed in Groisman, 2001), but until now, neither direct binding of PhoP to the PhoP box nor transcription activation by PhoP have been experimentally demonstrated (Groisman, 2001).In this report, we describe for the first time the mechanism of transcription activation of the putative Mg(II) transporter gene, mgtA, by the E. coli PhoP protein. S1 mapping of total RNA from exponential-phase E. coli cells indicates the presence of two promoters for the mgtA gene, of which the downstream mgtAP1 promoter is activated under Mg(II)-limiting conditions. Results of in vitro transcription assays, together with gel-shift and DNase-I footprinting analyses, using purified PhoP, indicate that the phosphorylated form of PhoP activates transcription from mgtAP1 at the step of open complex formation. Using reconstituted mutant RNA polymerases, we also demonstrate that the C-terminal domain of the s subunit (sCTD), but not the C-terminal domain of the a subunit (aCTD), is required for the recruitment of RNA polymerase, even though the aCTD is needed to achieve the maxim...

Two-component signal-transduction systems (TCSs) of bacteria are considered to form an intricate signal network to cope with various environmental stresses. One example of such a network in Escherichia coli is the signal transduction cascade from the EvgS/EvgA system to the PhoQ/PhoP system, where activation of the EvgS/ EvgA system promotes expression of PhoP-activated genes. As a factor connecting this signal transduction cascade, we have identified a small inner membrane protein (65 aa), B1500. Expression of the b1500 gene is directly regulated by the EvgS/EvgA system, and b1500 expression from a heterologous promoter simultaneously activated the expression of mgtA and other PhoP regulon genes. This activation was PhoQ/PhoP-dependent and EvgS/EvgA-independent. Furthermore, deletion of b1500 from an EvgS-activated strain suppressed mgtA expression. B1500 is localized in the inner membrane, and bacterial two-hybrid data showed that B1500 formed a complex with the sensor PhoQ. These results indicate that the small membrane protein, B1500, connected the signal transduction between EvgS/EvgA and PhoQ/PhoP systems by directly interacting with PhoQ, thus activating the PhoQ/PhoP system. two-component signal transduction

We identified Mg2+-responsive promoters of thephoPQ, mgtA, and mgrB genes ofEscherichia coli K-12 by S1 nuclease analysis. Expression of these genes was induced by magnesium limitation and depended on PhoP and PhoQ. The transcription start sites were also determined, which allowed us to find a (T/G)GTTTA direct repeat in their corresponding promoter regions.