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

Abstract

SummaryThe 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 phosphor ylation, 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 phosphor ylation-dependent activity, but did not affect the high-level, constitutive, UhpBCindependent 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 phosphor ylation-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 highlevel 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 unphosphor ylated 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.

Phosphorylation-dependent conformational changes and domain rearrangements in Staphylococcus aureus VraR activation

“…Additional complexity is introduced by evidence of oligomerization of some NarL/ FixJ transcription factors on DNA, implying that multiple surfaces of a receiver domain might be involved in protomer-protomer contacts. Indeed, mutagenesis studies suggest that the α1 helix of Escherichia coli UhpA, a response regulator that mediates sugar phosphate uptake, is involved in oligomerization (20). However, there are some indications from the crystal packing arrangements of more closely related VraR homologs, that the mode of receiver domain dimerization seen in the active VraR structure is not simply an outlier, but could represent a conserved dimerization mode shared by a subset of the NarL/FixJ family.…”

The Linker Region Plays an Important Role in the Interdomain Communication of the Response Regulator OmpR

“…Both the amino and carboxyl termini of OmpR are necessary for its function, and neither domain is inhibitory (36). Similarly, although the FixJ family members UhpA and NarL share high sequence similarity, the carboxyl terminus of NarL is inhibited by its amino terminus, whereas that of UhpA is not (37,38). NarL has a short interdomain linker of 6 amino acid residues, and the linker of UhpA is 16 residues long (38).…”

“…Similarly, although the FixJ family members UhpA and NarL share high sequence similarity, the carboxyl terminus of NarL is inhibited by its amino terminus, whereas that of UhpA is not (37,38). NarL has a short interdomain linker of 6 amino acid residues, and the linker of UhpA is 16 residues long (38). Thus, even within the FixJ family of response regulators to which UhpA and NarL belong, there is a correlation between linker length and functional differences in the activation mechanism.…”

Cooperativity in Signal Transfer through the Uhp System ofEscherichia coli

“…The E. coli strains (K-12 derivatives) used in this study were RK5115 [⌽(uhpT-lacZ) ⌬uhpT], RK5163 [⌽(uhpT-lacZ)], and RK1307 [⌬uhp(B60-C437) ⌽(uhpT-lacZ[Km])], kindly provided by R. J. Kadner (27,34). Cells were grown aerobically at 37°C in Luria-Bertani (LB) medium (20) or in MOPS (morpholinepropanesulfonic acid) minimal medium (21) containing either 30 mM succinate, 10 mM glucose, or 40 mM pyruvate as the carbon source, 14 mM NH 4 Cl as the nitrogen source, and (unless indicated otherwise) 2 mM K 2 HPO 4 as the phosphate source and supplemented with thiamine (20 g ml Ϫ1 ).…”