Abstract
Activity of antiterminator protein BglG regulating the beta-glucoside operon in Escherichia coli is controlled by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in a dual manner. It requires HPr phosphorylation to be active, whereas phosphorylation by the beta-glucoside-specific transport protein EIIBgl inhibits its activity. BglG and its relatives carry two PTS regulation domains (PRD1 and PRD2), each containing two conserved histidines. For BglG, histidine 208 in PRD2 was reported to be the negative phosphorylation site. In contrast, other antiterminators of this family are negatively regulated by phosphorylation of the first histidine in PRD1, and presumably activated by phosphorylation of the histidines in PRD2. In this work, a screen for mutant BglG proteins that escape repression by EIIBgl yielded exchanges of nine residues within PRD1, including conserved histidines His-101 and His-160, and C-terminally truncated proteins. Genetic and phosphorylation analyses indicate that His-101 in PRD1 is phosphorylated by EIIBgl and that His-160 contributes to negative regulation. His-208 in PRD2 is essential for BglG activity, suggesting that it is phosphorylated by HPr. Surprisingly, phosphorylation by HPr is not fully abolished by exchanges of His-208. However, phosphorylation by HPr is inhibited by exchanges in PRD1 and the phosphorylation of these mutants is restored in the presence of wild-type BglG. These results suggest that the activating phosphoryl group is transiently donated from HPr to PRD1 and subsequently transferred to His-208 of a second BglG monomer. The active His-208-phosphorylated BglG dimer can subsequently be inhibited in its activity by EIIBgl-catalyzed phosphorylation at His-101.
Highlights
□S The on-line version of this article contains Supplemental Data and Tables V and VI
These results suggest that the activating phosphoryl group is transiently donated from HPr to PRD1 and subsequently transferred to His-208 of a second BglG monomer
To investigate whether the fusion protein is still subject to negative regulation, its gene was placed on a plasmid downstream of the IPTG-inducible tacOP either alone or together with the bglF gene in tandem
Summary
For phenotypic screening of mutant BglG proteins, strain R2243 was constructed as follows: First, the PL-bglt2-lacZ cassette of plasmid pFDX3128 (see below) was integrated into the attachment site (attB) of strain R1279 as recently described [27]. From the resulting strain (R1735) a streptomycin-resistant mutant derivative was isolated, into which the FЈ lacIq lacZ⌬M15 lacYϩ proAϩ proBϩ episome was introduced by conjugation with strain BMH71-18. Strain constructions followed the procedures given by Miller [28]. Mutant plasmids isolated in the screen were sequenced using primers GK2 and GK3. These plasmids and their derivatives are listed, and oligonucleotides used in this study are given in Table VI under
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