Sites of positive and negative regulation in the Bacillus subtilis antiterminators LicT and SacY

Abstract

The Bacillus subtilis homologous transcriptional antiterminators LicT and SacY control the inducible expression of genes involved in aryl β‐glucoside and sucrose utilization respectively. Their RNA‐binding activity is carried by the N‐terminal domain (CAT), and is regulated by two similar C‐terminal domains (PRD1 and PRD2), which are the targets of phosphorylation reactions catalysed by the phosphoenolpyruvate: sugar phosphotransferase system (PTS). In the absence of the corresponding inducer, LicT is inactivated by BglP, the PTS permease (EII) specific for aryl β‐glucosides, and SacY by SacX, a negative regulator homologous to the EII specific for sucrose. LicT, but not SacY, is also subject to a positive control by the general PTS components EI and HPr, which are thought to phosphorylate LicT in the absence of carbon catabolite repression. Construction of SacY/LicT hybrids and mutational analysis enabled the location of the sites of this positive regulation at the two phosphorylatable His207 and His269 within LicT‐PRD2, and suggested that the presence of negative charges at these sites is sufficient for LicT activation in vivo. The BglP‐mediated inhibition process was found to essentially involve His100 of LicT‐PRD1, with His159 of the same domain playing a minor role in this regulation. In vitro experiments indicated that His100 could be phosphorylated directly by the general PTS proteins, this phosphorylation being stimulated by P∼BglP. We confirmed that, similarly, the corresponding conserved His99 residue in SacY is the major site of the negative control exerted by SacX on SacY activity. Thus, for both antiterminators, the EII‐mediated inhibition process seems to rely primarily on the presence of a negative charge at the first conserved histidine of the PRD1.

Regulation of the Escherichia coli Antiterminator Protein BglG by Phosphorylation at Multiple Sites and Evidence for Transfer of Phosphoryl Groups between Monomers

“…In this case it was concluded that the conserved His-208 residue in PRD2 is the target of negatively acting phosphorylation by EII Bgl (26). Thus, no general rules appear to exist concerning the molecular mechanisms governing the regulation of this protein family (8,20).…”

“…Where investigated, the first conserved histidine in PRD1 was found to be crucial for negative control. Exchange of this histidine leads to constitutively high antitermination activity in vivo even in the absence of the specific substrate (20,24,25). The second histidine in PRD1 participates in negative control of LicT and LacT (20,25).…”

“…Exchange of this histidine leads to constitutively high antitermination activity in vivo even in the absence of the specific substrate (20,24,25). The second histidine in PRD1 participates in negative control of LicT and LacT (20,25). In contrast, the histidine(s) in PRD2 were shown to be necessary for activity of the dually controlled proteins SacT, LicT, and LacT (6,20,25), and preferential phosphorylation of these residues by HPr has been demonstrated for LicT (19).…”

“…The second histidine in PRD1 participates in negative control of LicT and LacT (20,25). In contrast, the histidine(s) in PRD2 were shown to be necessary for activity of the dually controlled proteins SacT, LicT, and LacT (6,20,25), and preferential phosphorylation of these residues by HPr has been demonstrated for LicT (19). Based on these observations, it has been proposed that these proteins are stimulated by HPr-dependent phosphorylation of the histidines in PRD2 and inhibited by EII-dependent phosphorylation of the first histidine in PRD1.…”

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

“…Thus, for BglG and LevR, the enzyme II-mediated inhibition process seems to rely on the presence of a negative charge on a conserved histidine in PRD2. In contrast, the site suggested for the phosphorylation of LicT and SacY by their respective enzymes II, BglP and SacX, is a conserved histidine in PRD1 (29,31). Hence, the site on which the antiterminators are phosphorylated by their cognate enzymes II seems to be a conserved histidine in either PRD1 or PRD2.…”