Involvement of the amino-terminal phosphorylation module of UhpA in activation of uhpT transcription in Escherichia coli.

The UhpA protein is required for expression of the sugar phosphate transporter UhpT in Escherichia coli and is regulated by phosphate transfer from the transmembrane UhpBC sensor kinase complex. UhpA action requires the sensor kinase complex and the site of phosphorylation, Asp-54, under normal conditions, but not when UhpA is overexpressed. Directed mutagenesis of the uhpA gene allowed examination of the role of several residues of UhpA in response to phosphorylation and in transcription activation. Residues Asp-9, Asp-54, and Lys-101 are highly conserved and required for function in other response regulators. Changes at any of these residues in UhpA resulted in complete loss of phosphorylation-dependent activity, but did not affect the high-level, constitutive, UhpBC-independent expression when the UhpA variants were overexpressed. Thus, these residues are important for the response to the phosphorylation pathway but not for transcription activation. Eight independent uhpA mutants selected for activity in the absence of UhpBC function carried the F17-->V or H170-->Y substitutions. Other substitutions for Phe-17 conferred various phenotypes, ranging from inducible to high-level constitutive behaviour. Residues in helix-1 flanking Phe-17 were converted to Ala or other residues. Alanine substitutions at Val-13, Arg-14, and Leu-20 resulted in complete loss of phosphorylation-dependent activation. Change of Gly-16 to Ala had no effect, but changes to other residues resulted in loss of function. Alanine substitutions at Phe-17 and at Gln-19 resulted in high-level constitutive expression, and changes at Ala-18 and Leu-21 had only modest effects. Most interesting was the L20-->A substitution, which conferred low uhpT expression when overexpressed and interfered with action of the wild-type chromosomal allele. The combination of the L20-->A change with changes at Phe-17, Asp-54 and His-170 indicated that the trans-dominant action of L20-->A occurred at several steps. The observations that UhpA can activate uhpT transcription in its unphosphorylated state are consistent with its occupancy of low-affinity binding sites necessary for promoter function. We propose that the effect of phosphorylation of UhpA is to enhance its oligomerization on the DNA surface to extend to the low-affinity sites, and that helix-1 participates in the process of oligomer formation.