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Journal of Bacteriology, May 2001, p. 3149-3159, Vol. 183, No. 10
Department of Microbiology, School of
Medicine, University of Virginia, Charlottesville, Virginia
22908-0734
Received 1 November 2000/Accepted 27 February 2001
Bacterial two-component regulatory systems control the expression
of target genes through regulated changes in protein phosphorylation. Signal reception alters the ability of a membrane-bound histidine kinase (HK) protein to transfer phosphate from ATP to a highly conserved histidine residue. The transfer of phosphate from the histidine to an aspartate residue on the cognate response regulator (RR) changes the ability of the latter protein to bind to target DNA
sequences and to alter gene transcription. UhpB is the HK protein which
controls production of the sugar phosphate transporter UhpT. Elevated
expression of full-length UhpB or of a soluble hybrid protein, GST-Bc,
which is glutathione S-transferase (GST) fused to the
cytoplasmic C-terminal portion of UhpB, results in complete blockage of
uhpT expression in a uhp+ strain.
This dominant-negative interference could result from the ability of
GST-Bc to bind and sequester the RR UhpA and to accelerate its
dephosphorylation. The portion of GST-Bc responsible for the
interference phenotype was localized using truncation, linker
insertion, and point mutations to the region between residues 293 and
366 flanking His-313, the putative site of autophosphorylation. Point
mutations which allow GST-Bc to activate uhpT expression or
which relieve the interference phenotype were obtained at numerous sites throughout this region. This region of UhpB is related to the
phosphoryl transfer domain of EnvZ, which forms half of an interdimer
four-helix bundle and is responsible for dimerization of its
cytoplasmic domain. The expression of GST fusion proteins carrying the
corresponding portions of EnvZ strongly interfered with the activation
of porin gene expression by OmpR. The GST-Bc protein accelerated
dephosphorylation of P-UhpA. Reverse transfer of phosphate from P-UhpA
to GST-Bc was observed in the presence of the metal chelator EDTA and
depended on the presence of His-313. Phosphate transfer from P-UhpA to
the liberated phosphoryl transfer domain also occurred. Taken together,
these results indicate that the phosphoryl transfer-dimerization domain
of UhpB participates in the specific binding of UhpA, in the control of
autokinase activity, and in the dephosphorylation of P-UhpA.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.10.3149-3159.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The Phosphoryl Transfer Domain of UhpB Interacts
with the Response Regulator UhpA
*
Corresponding author. Mailing address: Department of
Microbiology, University of Virginia School of Medicine, P.O. Box
800734, Charlottesville, VA 22908-0734. Phone: (804) 924 2532. Fax:
(804) 982 1071. E-mail: rjk{at}virginia.edu.
Journal of Bacteriology, May 2001, p. 3149-3159, Vol. 183, No. 10
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.10.3149-3159.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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