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

Abstract

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.

Regulation of the Escherichia coli Antiterminator Protein BglG by Phosphorylation at Multiple Sites and Evidence for Transfer of Phosphoryl Groups between Monomers

“…This was important, because the intracellular concentration of BglG is crucial for its undisturbed regulation by the PTS (18). As expected, expression of the bglG-bglF cassette in the ptspositive strain led to only low enzyme activities (Fig.…”

“…1A, bar 1) close to the background level of this system (ϭ 37 units, the activity detected with plasmid pFDY226 alone; Ref. 18). In contrast, activities up to ϳ1600 units at maximum expression levels (1 mM IPTG) were obtained in the absence of bglF or when the bglF-C24S allele was present (Fig.…”

“…1A, bars 2 and 3). These results reflect negative control of BglG by wild-type EII Bgl as well as its escape from this control in the presence of mutant EII Bgl -C24S (13,18). Expression of the bglG as well as the bglG-bglF-C24S cassette in the pts-negative strain led to drastically reduced activities when compared with the ptspositive strain (bars 2 and 3) due to the second level of activity control of BglG, which requires phosphorylation by HPr to be active (18).…”

“…Antitermination activity was tested using reporter plasmid pFDY226 (Fig. 1), which allows quantitative monitoring of BglG activity (18). The ␤-galactosidase enzyme activities were determined in the presence of different concentrations of IPTG (0, 0.05, 0.1, and 1 mM) to compare the antitermination activities at different expression levels (Fig.…”

“…In Vivo Protein Phosphorylation-Cells were labeled with H 3 [ 32 P]O 4 as described (18). For induction of tacOP-controlled gene expression, 0.1 mM IPTG was added.…”