BglG, the response regulator of the Escherichia coli bgl operon, is phosphorylated on a histidine residue.

We have shown previously that the activity of BglG, the response regulator of the bgl system, as a transcriptional antiterminator is modulated by the sensor BglF, which reversibly phosphorylates BglG. We show here that the phosphoryl group on BglG is present as a phosphoramidate, based on the sensitivity of phosphorylated BglG to heat, hydroxylamine, and acidic but not basic conditions. By analyzing the products of base-hydrolyzed phosphorylated BglG by thin-layer chromatography, we show that the phosphorylation occurs on a histidine residue. This result supports the notion that the bgl system is a member of a new family of bacterial sensory systems

Efficient and accurate in vitro processing of simian virus 40-associated small RNA

Nuclei were isolated from simian virus 40 (SV40)-infected cells with a hypotonic, detergent-free buffer and incubated in vitro in a high-ionic-strength buffer containing [alpha-32P]UTP. The labeled viral RNAs produced were analyzed by gel electrophoresis together with 3-h-labeled viral RNAs extracted from SV40-infected cells. The in vitro-synthesized RNA contained a major RNA species of 62 to 64 nucleotides that appeared on the gel at the same position as in vivo-synthesized SV40-associated small RNA (SAS-RNA). Analyses of the in vitro-synthesized 62- to 64-nucleotide RNA by hybridization to restriction fragments and by the use of an SAS-RNA deletion mutant clearly identified it as SAS-RNA. The intensity of the band of the in vitro-synthesized SAS-RNA increased with an increase in the labeling time or when a short pulse was followed by a chase. Moreover, the SAS-RNA band disappeared when ITP replaced GTP in the transcription reaction mixture. These results indicate that SAS-RNA is processed from a precursor molecule and that an RNA secondary structure could be an element recognized by the processing enzyme.</jats:p

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

BglG, which regulates expression of the β-glucoside utilization (bgl) operon in Escherichia coli, represents a family of RNA-binding transcriptional antiterminators that positively regulate transcription of sugar utilization genes in Gram-negative and Gram-positive organisms. BglG is negatively regulated by the β-glucoside phosphotransferase, BglF, by means of phosphorylation and physical association, and it is positively regulated by the general phosphoenolpyruvate phosphotransferase system (PTS) proteins, enzyme I (EI) and HPr. We studied the positive regulation of BglG both in vitro and in vivo. Here, we show that although EI and HPr are essential for BglG activity, this mode of activation does not require phosphorylation of BglG by HPr, as opposed to the phosphorylation-mediated activation of many BglG-like antiterminators in Gram-positive organisms. The effect of EI and HPr on BglG is not mediated by BglF. Nevertheless, the release of BglG from BglF, which is stimulated by the extracellular sugar in a sugar uptake-independent manner, is a prerequisite for BglG activation. Taken together, the results indicate that activation of BglG is a 2-stage process: a sugar-stimulated release from the membrane-bound sugar sensor followed by a phosphorylation-independent stimulatory effect exerted by the general PTS proteins

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

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

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

The Escherichia coli BglF protein is a sugar-sensor that controls the activity of the transcriptional antiterminator BglG by reversibly phosphorylating it, depending on β-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 β-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

Supplementary Material for: A Search for Ribonucleic Antiterminator Sites in Bacterial Genomes: Not Only Antitermination

BglG/LicT-like proteins are transcriptional antiterminators that prevent termination of transcription at intrinsic terminators by binding to ribonucleic antiterminator (RAT) sites and stabilizing an RNA conformation which is mutually exclusive with the terminator structure. The known RAT sites, which are located in intergenic regions of sugar utilization operons, show low sequence conservation but significant structural analogy. To assess the prevalence of RATs in bacterial genomes, we employed bioinformatic tools that describe RNA motifs based on both sequence and structural constraints. Using descriptors with different stringency, we searched the genomes of Escherichiacoli K12, uropathogenic E. coli and Bacillus subtilis for putative RATs. Our search identified all known RATs and additional putative RAT elements. Surprisingly, most putative RATs do not overlap an intrinsic terminator and many reside within open reading frames (ORFs). The ability of one of the putative RATs, which is located within an antiterminator-encoding ORF and does not overlap a terminator, to bind to its cognate antiterminator protein in vitro and in vivo was confirmed experimentally. Our results suggest that the capacity of RAT elements has been exploited during evolution to mediate activities other than antitermination, for example control of transcription elongation or of RNA stability