Genetic dissection of the divergent activities of the multifunctional membrane sensor BglF.
BglF catalyzes beta-glucoside phosphotransfer across the cytoplasmic membrane in Escherichia coli. In addition, BglF acts as a sugar sensor that controls expression of beta-glucoside utilization genes by reversibly phosphorylating the transcriptional antiterminator BglG. Thus, BglF can exist in two opposed states: a nonstimulated state that inactivates BglG by phosphorylation and a sugar-stimulated state that activates BglG by dephosphorylation and phosphorylates the incoming sugar. Sugar phosphorylation and BglG (de)phosphorylation are both catalyzed by the same residue, Cys24. To investigate the coordination and the structural requirements of the opposing activities of BglF, we conducted a genetic screen that led to the isolation of mutations that shift the balance toward BglG phosphorylation. We show that some of the mutants that are impaired in dephosphorylation of BglG retained the ability to catalyze the concurrent activity of sugar phosphotransfer. These mutations map to two regions in the BglF membrane domain that, based on their predicted topology, were suggested to be implicated in activity. Using in vivo cross-linking, we show that a glycine in the membrane domain, whose substitution impaired the ability of BglF to dephosphorylate BglG, is spatially close to the active-site cysteine located in a hydrophilic domain. This residue is part of a newly identified motif conserved among beta-glucoside permeases associated with RNA-binding transcriptional antiterminators. The phenotype of the BglF mutants could be suppressed by BglG mutants that were isolated by a second genetic screen. In summary, we identified distinct sites in BglF that are involved in regulating phosphate flow via the common active-site residue in response to environmental cues.
Monderer-Rothkoff
Galya
G
Department of Molecular Biology, The Hebrew University Medical School, P.O. Box 12272, Jerusalem 91120, Israel.
Amster-Choder
Orna
O
D019943:Amino Acid Substitution; D050260:Carbohydrate Metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary
C079638:BglF protein, E coli; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011494:Protein Kinases