Dynamic localization of a transcription factor in Bacillus subtilis: the LicT antiterminator relocalizes in response to inducer availability.

Bacillus subtilis transports ß-glucosides such as salicin by a dedicated phosphotransferase system (PTS). The expression of the ß-glucoside permease BglP is induced in the presence of the substrate salicin, and this induction requires the binding of the antiterminator protein LicT to a specific RNA target in the 5' region of the bglP mRNA to prevent the formation of a transcription terminator. LicT is composed of an N-terminal RNA-binding domain and two consecutive PTS regulation domains, PRD1 and PRD2. In the absence of salicin, LicT is phosphorylated on PRD1 by BglP and thereby inactivated. In the presence of the inducer, the phosphate group from PRD1 is transferred back to BglP and consequently to the incoming substrate, resulting in the activation of LicT. In this study, we have investigated the intracellular localization of LicT. While the protein was evenly distributed in the cell in the absence of the inducer, we observed a subpolar localization of LicT if salicin was present in the medium. Upon addition or removal of the inducer, LicT rapidly relocalized in the cells. This dynamic relocalization did not depend on the binding of LicT to its RNA target sites, since the localization pattern was not affected by deletion of all LicT binding sites. In contrast, experiments with mutants affected in the PTS components as well as mutations of the LicT phosphorylation sites revealed that phosphorylation of LicT by the PTS components plays a major role in the control of the subcellular localization of this RNA-binding transcription factor.

Activation of Escherichia coli antiterminator BglG requires its phosphorylation.

Transcriptional antiterminator proteins of the BglG family control the expression of enzyme II (EII) carbohydrate transporters of the bacterial phosphotransferase system (PTS). In the PTS, phosphoryl groups are transferred from phosphoenolpyruvate (PEP) via the phosphotransferases enzyme I (EI) and HPr to the EIIs, which phosphorylate their substrates during transport. Activity of the antiterminators is negatively controlled by reversible phosphorylation catalyzed by the cognate EIIs in response to substrate availability and positively controlled by the PTS. For the Escherichia coli BglG antiterminator, two different mechanisms for activation by the PTS were proposed. According to the first model, BglG is activated by HPr-catalyzed phosphorylation at a site distinct from the EII-dependent phosphorylation site. According to the second model, BglG is not activated by phosphorylation, but solely through interaction with EI and HPr, which are localized at the cell pole. Subsequently BglG is released from the cell pole to the cytoplasm as an active dimer. Here we addressed this discrepancy and found that activation of BglG requires phosphorylatable HPr or the HPr homolog FruB in vivo. Further, we uniquely demonstrate that purified BglG protein becomes phosphorylated by FruB as well as by HPr in vitro. Histidine residue 208 in BglG is essential for this phosphorylation. These data suggest that BglG is in fact activated by phosphorylation and that there is no principal difference between the PTS-exerted mechanisms controlling the activities of BglG family proteins in Gram-positive and Gram-negative bacteria.

RNase Y in Bacillus subtilis: a Natively disordered protein that is the functional equivalent of RNase E from Escherichia coli.

The control of mRNA stability is an important component of regulation in bacteria. Processing and degradation of mRNAs are initiated by an endonucleolytic attack, and the cleavage products are processively degraded by exoribonucleases. In many bacteria, these RNases, as well as RNA helicases and other proteins, are organized in a protein complex called the RNA degradosome. In Escherichia coli, the RNA degradosome is assembled around the essential endoribonuclease E. In Bacillus subtilis, the recently discovered essential endoribonuclease RNase Y is involved in the initiation of RNA degradation. Moreover, RNase Y interacts with other RNases, the RNA helicase CshA, and the glycolytic enzymes enolase and phosphofructokinase in a degradosome-like complex. In this work, we have studied the domain organization of RNase Y and the contribution of the domains to protein-protein interactions. We provide evidence for the physical interaction between RNase Y and the degradosome partners in vivo. We present experimental and bioinformatic data which indicate that the RNase Y contains significant regions of intrinsic disorder and discuss the possible functional implications of this finding. The localization of RNase Y in the membrane is essential both for the viability of B. subtilis and for all interactions that involve RNase Y. The results presented in this study provide novel evidence for the idea that RNase Y is the functional equivalent of RNase E, even though the two enzymes do not share any sequence similarity.

Novel activities of glycolytic enzymes in Bacillus subtilis: interactions with essential proteins involved in mRNA processing.

Glycolysis is one of the most important metabolic pathways in heterotrophic organisms. Several genes encoding glycolytic enzymes are essential in many bacteria even under conditions when neither glycolytic nor gluconeogenic activities are required. In this study, a screening for in vivo interaction partners of glycolytic enzymes of the soil bacterium Bacillus subtilis was used to provide a rationale for essentiality of glycolytic enzymes. Glycolytic enzymes proved to be in close contact with several other proteins, among them a high proportion of essential proteins. Among these essential interaction partners, other glycolytic enzymes were most prominent. Two-hybrid studies confirmed interactions of phosphofructokinase with phosphoglyceromutase and enolase. Such a complex of glycolytic enzymes might allow direct substrate channeling of glycolytic intermediates. Moreover we found associations of glycolytic enzymes with several proteins known or suspected to be involved in RNA processing and degradation. One of these proteins, Rny (YmdA), which has so far not been functionally characterized, is required for the processing of the mRNA of the glycolytic gapA operon. Two-hybrid analyses confirmed the interactions between the glycolytic enzymes phosphofructokinase and enolase and the enzymes involved in RNA processing, RNase J1, Rny, and polynucleotide phosphorylase. Moreover RNase J1 interacts with its homologue RNase J2. We suggest that this complex of mRNA processing and glycolytic enzymes is the B. subtilis equivalent of the RNA degradosome. Our findings suggest that the functional interaction of glycolytic enzymes with essential proteins may be the reason why they are indispensable.