BglF, the sensor of the E.coli bgl system, uses the same site to phosphorylate both a sugar and a regulatory protein

Abstract

The Escherichia coli BglF protein is a sugar permease that is a member of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). It catalyses transport and phosphorylation of beta-glucosides. In addition to its ability to phosphorylate its sugar substrate, BglF has the unusual ability to phosphorylate and dephosphorylate the transcriptional regulator BglG according to beta-glucoside availability. By controlling the phosphorylation state of BglG, BglF controls the dimeric state of BglG and thus its ability to bind RNA and antiterminate transcription of the bgl operon. BglF has two phosphorylation sites. The first site accepts a phosphoryl group from the PTS protein HPr; the phosphoryl group is then transferred to the second phosphorylation site, which can deliver it to the sugar. We provide both in vitro and in vivo evidence that the same phosphorylation site on BglF, the second one, is in charge not only of sugar phosphorylation but also of BglG phosphorylation. Possible mechanisms that ensure correct phosphoryl delivery to the right entity, sugar or protein, depending on environmental conditions, are discussed.

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

“…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).…”

“…For comparison, plasmids were analyzed that carried wild-type bglG alone or together with bglF or its mutant with a C24S exchange. Cys-24 represents the second phosphorylation site of EII Bgl and is essential for negative regulation of BglG (13). To decide about the role of the PRDs in dimerization, a plasmid, which encoded only the CAT domain was additionally employed.…”

“…These data correlate well with the results of the genetic analysis of the mutants shown above (Table III). Those proteins that were not (mutant H101L; lane 21) or only weakly (mutants C76R, D100(G,N,V), E153V; lanes 7,15,17,19,25) phosphorylated by EII Bgl , were also completely independent of negative control, whereas those proteins that were more efficiently phosphorylated (mutants I80T, Q91R, S97P, F104V, and H160R; lanes 9,11,13,23,27), were still subject to residual repression by EII Bgl (Table III). This correlation suggests that the level of escape from repression seen for the individual mutant proteins may indeed be caused by a corresponding less efficient EII Bgl -catalyzed phosphorylation.…”

“…The antiterminator becomes dephosphorylated by its EII and thereby reactivated when substrate becomes available, as has been demonstrated first for the BglG/EII Bgl pair in Escherichia coli (11,12). In this case, a direct transfer of phosphoryl groups from the phosphorylation site cysteine 24 in IIB Bgl to BglG has been proposed (13). BglG regulates the bgl operon, which codes for BglG itself, EII Bgl , and other functions for uptake and utilization of aryl-␤-glucosides (14,15).…”

Modulation of transcription antitermination in the bgl operon of Escherichia coli by the PTS

“…We attempted to reproduce transcription antitermination in this minimal sys-tem by adding purified and soluble BglG fused to maltosebinding protein (MBP). MBP-BglG was shown to be active in bgl antitermination in vivo and to be regulated by BglF in vitro and in vivo (20,22). As a template, we used a 532-bp PCR fragment containing the bgl promoter activated by a point mutation in the CAP-binding site (23), the leader region, which contains a transcription terminator, and the very beginning of the bglG gene (encoding the first 19 aa of BglG).…”