Dynamic Localization of a Transcription Factor in Bacillus subtilis: the LicT Antiterminator Relocalizes in Response to Inducer Availability

Abstract

Bacillus subtilis transports ␤-glucosides such as salicin by a dedicated phosphotransferase system (PTS). The expression of the ␤-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 RNAbinding transcription factor.

The General Phosphotransferase System Proteins Localize to Sites of Strong Negative Curvature in Bacterial Cells

“…Still, one difference between the two organisms is that in E. coli cells, the EI proteins from both species localize to the cell pole independent of growth conditions, whereas in B. subtilis cells, EI localization seems to depend on growth conditions. Thus, when grown in LB, the native B. subtilis EI showed very little if any distinct localization (data not shown), similar to the distribution documented previously (17). The reason for the dependence of the B. subtilis EI localization pattern on the medium, which is in contrast to the lack of such dependence for E. coli EI (15), is currently not known.…”

“…The PTS proteins known to localize to the poles are all soluble. They include EI, which remains at the poles independent of environmental conditions, HPr, which discharges from the poles in response to the presence of PTS sugars in the growth medium, and transcription factors that regulate PTS operons, i.e., BglG in E. coli and LicT in B. subtilis , which are recruited to the poles depending on the availability of sugars that are transported and metabolized by the products of the operons that they regulate (15, 17). The membrane-bound components of the PTS, the sugar permeases, do not localize specifically to the poles; rather, they are distributed around the cell circumference (15, 16).…”

“…Thus, BglG, a transcription factor that positively regulates transcription of the β-glucoside utilization operon ( bgl ) in E. coli , is sequestered near the membrane in an inactive form via interaction with the β-glucoside transporter, BglF (16); in the presence of β-glucosides, BglG is dephosphorylated by BglF and migrates to the cell poles, where it is activated by EI and HPr; consequently, the dimeric form of BglG is released to the cytoplasm, where it binds to the emerging bgl transcript and antiterminates transcription of the bgl operon (15). Similarly, LicT, a BglG homologue from B. subtilis , has recently been shown to be localized near the poles upon addition of β-glucosides to the medium (17). Spatiotemporal control of other transcription regulators by sugar permeases was also documented, i.e., repression of Mlc and MalT activity as positive regulators of glucose and maltose utilization genes in E. coli via interaction and membrane sequestration with the PTS glucose permease (18) and the maltose ABC transporter MalFGK 2 (19), respectively, and activation of MtlR as a positive regulator of mannitol operon expression in B. subtilis via interaction with the mannitol permease (20).…”

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

“…In the absence of a substrate, LicT is phosphorylated in its PRD1 by the PϳEIIB Bgl domain, and the antiterminator is equally distributed in the cell. In the presence of a substrate, LicT is no longer phosphorylated by the PϳEIIB Bgl domain and is located in subpolar regions (173). Although there is no evidence that the presence or absence of EIIB Bgl or PϳEIIB Bgl affects the activity of LicT by direct interaction, phosphorylation of the PRD1 of LicT by PϳEIIB Bgl seems to play an important role in the cellular localization of the antiterminator.…”

Selection-Driven Accumulation of Suppressor Mutants in Bacillus subtilis: The Apparent High Mutation Frequency of the Cryptic gudB Gene and the Rapid Clonal Expansion of gudB+ Suppressors Are Due to Growth under Selection

“…Plasmids pCFPbglS and pYFPbglS served as templates for PCR to amplify the fluorophore-encoding cfp and yfp genes, respectively [54]. The plasmid pGP1870 was used for the construction of a gudB-gfp fusion [55] (Table S2). …”