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

PhoP is a response regulator of the PhoQ-PhoP two-component system controlling a set of the Mg(II)-response genes in Escherichia coli . Here we demonstrate the mode of transcription regulation by phosphorylated PhoP of divergently transcribed mgtA and treR genes, each encoding a putative Mg(II) transporter and a repressor for the trehalose utilization operon respectively. Under Mg(II)-limiting conditions in vivo , two promoters, the upstream constitutive P2 and the downstream inducible P1, were detected for the mgtA gene. Gel-shift analysis in vitro using purified PhoP indicates its binding to a single DNA target, centred between -43 and -24 of the mgtA P1 promoter. This region includes the PhoP box, which consists of a direct repeat of the heptanucleotide sequence (T)G(T)TT(AA). Site-directed mutagenesis studies indicate the critical roles for T (position 3), T (position 4) and A (position 6) for PhoP-dependent transcription from mgtA P1. DNase I footprinting assays reveal weak binding of PhoP to this PhoP box, but the binding becomes stronger in the simultaneous presence of RNA polymerase. Likewise the RNA polymerase binding to the P1 promoter becomes stronger in the presence of PhoP. For the PhoP-assisted formation of open complex at the mgtA P1 promoter, however, the carboxy-terminal domain of alpha subunit (alphaCTD) is not needed. For transcription in vivo of the treR gene, four promoters were identified. The most upstream promoter treR P4 divergently overlaps with the mgtA P1 promoter, sharing the same sequence as the respective -10 signal in the opposite direction. In vitro transcription using mutant promoters support this prediction. In the presence of PhoP, transcription from the promoter treR P3 was repressed with concomitant activation of mgtA P1 transcription. The PhoP box is located between - 46 and -30 with respect to treR P3, and the alphaCTD is needed for this repression.