The BglF sensor recruits the BglG transcription regulator to the membrane and releases it on stimulation

Abstract

The Escherichia coli BglF protein is a sugar-sensor that controls the activity of the transcriptional antiterminator BglG by reversibly phosphorylating it, depending on ␤-glucoside availability. BglF is a membrane-bound protein, whereas BglG is a soluble protein, and they are both present in the cell in minute amounts. How do BglF and BglG find each other to initiate signal transduction efficiently? Using bacterial two-hybrid systems and the Far-Western technique, we demonstrated unequivocally that BglG binds to BglF and to its active site-containing domain in vivo and in vitro. Measurements by surface plasmon resonance corroborated that the affinity between these proteins is high enough to enable their stable binding. To visualize the subcellular localization of BglG, we used fluorescence microscopy. In cells lacking BglF, the BglG-GFP fusion protein was evenly distributed throughout the cytoplasm. In contrast, in cells producing BglF, BglG-GFP was localized to the membrane. On addition of ␤-glucoside, BglG-GFP was released from the membrane, becoming evenly distributed throughout the cell. Using mutant proteins and genetic backgrounds that impede phosphorylation of the Bgl proteins, we demonstrated that BglG-BglF binding and recruitment of BglG to the membrane sensor requires phosphorylation but does not depend on the individual phosphorylation sites of the Bgl proteins. We suggest a mechanism for rapid response to environmental changes by preassembly of signaling complexes, which contain transcription regulators recruited by their cognate sensors-kinases, under nonstimulating conditions, and release of the regulators to the cytoplasm on stimulation. This mechanism might be applicable to signaling cascades in prokaryotes and eukaryotes.

The compartmentalized vessel

“…25 Notably, BglG forms a precomplex with BglF at the membrane in the absence of the stimulating sugar. 17 It is very tempting to suggest that targeting of operon transcripts, which usually code for protein products that operate in the same pathway, is a universal mechanism that facilitates assembly of these products into complexes. Notably, when bglG is transcribed without bglF, its transcripts localize to the poles, 25 where the BglG protein forms a complex with the general PTS proteins after it is released from the membrane pre-complex upon sugar stimulation.…”

“…We showed that localization of the BglG transcription factor, which enables expression of a PTS sugar utilization operon, bgl, is determined by its consecutive interaction with different PTS components, i.e., with the PTS sugar permease BglF, which localizes around the cell circumference, and with the general PTS proteins at the poles. 12,17 Hence, upon sugar stimulation, BglG migrates from the cell periphery to the cytoplasm through the poles (Fig. 1).…”

Dynamic localization of membrane proteins in Bacillus subtilis

“…Penicillinbinding proteins involved in cell wall synthesis have also been shown to localize in several patterns, including to division septa and discrete foci along the long axis of the cell, reflecting the role of these proteins in specific stages of cell wall synthesis (Scheffers et al, 2004). However, analysis of the localization of the E. coli Sec protein secretion machinery, BglF sugar sensor and B. subtilis phage w29 DNA replication protein p16.7 indicates that these proteins are homogeneously distributed around the cytoplasmic membrane, with no reported preference for cell poles or other observable domains (Brandon et al, 2003;Lopian et al, 2003;Meijer et al, 2001). Certainly, the images presented in these papers indicate that the localization pattern of these proteins creates a clear outline of the cytoplasmic membrane.…”

Interactions between the PTS Regulation domains of the BglG Transcriptional Antiterminator from Escherichia coli

“…Hence, in the absence of ␤-glucoside, BglG exists in the cell as a phosphorylated inactive monomer, whereas in the presence of the sugar in the growth medium, BglG exists as an active non-phosphorylated dimer. We have recently shown that in the absence of the stimulating sugar, BglF recruits BglG to the membrane, releasing it to the cytoplasm upon the addition of ␤-glucoside (7).…”