Novel mode of transcription regulation of divergently overlapping promoters by PhoP, the regulator of two‐component system sensing external magnesium availability

Abstract

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the phosphate moiety to the PhoP protein (Castelli et al., 2000;Yamamoto et al., 2000a; K. Yamamoto and A. Ishihama, unpublished data).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...

Transcription factor function and promoter architecture govern the evolution of bacterial regulons

“…The ␣CTD was dispensable for PhoP-dependent mgtA transcription (Fig. S7), as expected for a class II promoter and consistent with previous findings reported for the Escherichia coli mgtA promoter and the E. coli PhoP protein (22). By contrast, the ␣CTD was necessary for PhoP-dependent transcription of the class I ugtL promoter (Fig.…”

“…We began by investigating Yersinia's ability to promote transcription of the S. enterica mgtA and ugtL genes, which have no orthologs in Y. pestis and harbor distinct promoter architectures (Fig. 1A) (21)(22)(23). Y. pestis produced fluorescence levels similar to those of S. enterica when transcription of a promoterless gfp gene was driven by the promoter of the mgtA gene (Fig.…”

“…(H-M) Single-round in vitro transcription assays with linear templates corresponding to the mgtA (H-J) and ugtL (K-M) promoters, E. coli RNA polymerase, and increasing amounts of phosphorylated Salmonella or Yersinia PhoP proteins. The upper band in H-I corresponds to treR, a PhoP-repressed transcript going in the reverse orientation (22). The upper band in K and L corresponds to a spurious transcript observed in vitro but not in vivo (21,45).…”

A connector of two-component regulatory systems promotes signal amplification and persistence of expression

“…17; J. C. Perez and E.A.G., unpublished data). The induction ratio ranged from 38 to 128 for the genes regulated via the connector-mediated pathway, which is much higher than the 4 to 20 induction ratio for the genes regulated via the direct pathway ( Fig.…”

The transcription regulator AllR senses both allantoin and glyoxylate and controls a set of genes for degradation and reutilization of purines

“…For preparation of total RNA for primer extension analysis, overnight cultures were diluted 100-fold in 100 ml LB medium and cells were grown to OD 600 0.6-0.8 (exponential phase). RNA purification was carried out as described previously (Yamamoto et al, 2002). Primer extension analysis was performed using fluorescently labelled probes according to a published procedure (Yamada et al, 1998).…”