Regulation of the Escherichia coli antiterminator protein BglG by phosphorylation at multiple sites and evidence for transfer of phosphoryl groups between monomers

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.     

Catabolite control of Escherichia coli regulatory protein BglG activity by antagonistically acting phosphorylations.

In bacteria various sugars are taken up and concomitantly phosphorylated by sugar-specific enzymes II (EII) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The phosphoryl groups are donated by the phosphocarrier protein HPr. BglG, the positively acting regulatory protein of the Escherichia coli bgl (beta-glucoside utilization) operon, is known to be negatively regulated by reversible phosphorylation catalyzed by the membrane spanning beta-glucoside-specific EIIBgl. Here we present evidence that in addition BglG must be phosphorylated by HPr at a distinct site to gain activity. Our data suggest that this second, shortcut route of phosphorylation is used to monitor the state of the various PTS sugar availabilities in order to hierarchically tune expression of the bgl operon in a physiologically meaningful way. Thus, the PTS may represent a highly integrated signal transduction network in carbon catabolite control.     

Directed evolution of cellobiose utilization in Escherichia coli K12

The cellobiose catabolic system of Escherichia coli K12 is being used to study the role of cryptic genes in evolution of new functions. Escherichia coli does not use beta-glucoside sugars; however, mutations in several loci can activate the cryptic bgl operon and permit growth on the beta-glucoside sugars arbutin and salicin. Such Bgl+ mutants do not use cellobiose, which is the most common beta-glucoside in nature. We have isolated a Cel+ (cellobiose-utilizing) mutant from a Bgl+ mutant of E. coli K12. The Cel+ mutant grows well on cellobiose, arbutin, and salicin. Genes for utilization of these beta-glucosides are located at 37.8 min on the E. coli map. The genes of the bgl operon are not involved in cellobiose utilization. Introduction of a deletion covering bgl does not affect the ability to utilize cellobiose, arbutin, or salicin, indicating that the new Cel+ genes provide all three functions. Spontaneous cellobiose negative mutants also become arbutin and salicin negative. Analysis of beta-glucoside positive revertants of these mutants indicates that there are separate loci for utilization of each of the beta-glucoside sugars. The genes are closely linked and may be activated from a single locus. A fourth gene at an unknown location increases the growth rate on cellobiose. The cel genes constitute a second cryptic system for beta-glucoside utilization in E. coli K12.      

Inducible System for the Utilization of β-Glucosides in Escherichia coli I. Active Transport and Utilization of β-Glucosides

Wild-type Escherichia coli strains (beta-gl(-)) do not split beta-glucosides, but inducible mutants (beta-gl(+)) can be isolated which do so. This inducible system consists of a beta-glucoside permease and an aryl beta-glucoside splitting enzyme. Both can be induced by aryl and alkyl beta-glucosides. In beta-gl(-) and noninduced beta-gl(+) cells, C(14)-labeled thioethyl beta-glucoside (TEG) is taken up by a constitutive permease, apparently identical with a glucose permease (GP). This permease has a high affinity for alpha-methyl glucoside and a low affinity for aryl beta-glucosides. No accumulation of TEG occurs in a beta-gl(-) strain lacking glucose permease (GP(-)). In induced beta-gl(+) strains, there appears a second beta-glucoside permease with low affinity for alpha-methyl glucoside and high affinity for aryl beta-glucosides. Autoradiography shows that TEG is accumulated by the beta-glucoside permease and glucose permease in two different forms (one being identical with TEG, the other probably phosphorylated TEG). In GP(+) beta-gl(+) strains with high GP activity, alkyl beta-glucosides induce the enzyme and the beta-glucoside permease after a prolonged induction lag, and they competitively inhibit the induction by aryl beta-glucosides. The induction lag and competition do not exist in GP(-) beta-gl(+) strains. It is assumed that phosphorylated alkyl and thioalkyl beta-glucosides inhibit the induction, and that this inhibition is responsible for the induction lag.