Browsing by Author "Rak, Bodo"

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.

Amino sugars are essential precursor molecules for the biosynthesis of bacterial cell walls. Their synthesis pathway is initiated by glucosamine-6-phosphate (GlcN-6-P) synthase (GlmS) which catalyses the rate limiting reaction. We report here that expression of the Escherichia coli glmS gene is negatively feedback regulated by its product GlcN-6-P at the post-transcriptional level. Initially, we observed that mutants defective for yhbJ, a gene of the rpoN operon, overproduce GlmS. Concomitantly, a glmS mRNA accumulates that is derived from processing of the primary glmUS transcript at the glmU stop codon by RNase E. A transposon mutagenesis screen in the yhbJ mutant identified the small RNA GlmZ (formerly RyiA or SraJ) to be required for glmS mRNA accumulation. GlmZ, which is normally processed, accumulates in its full-length form in the yhbJ mutant. In the wild type, a decrease of the intracellular GlcN-6-P concentration induces accumulation of the glmS transcript in a GlmZ-dependent manner. Concomitantly, GlmZ accumulates in its unprocessed form. Hence, we conclude that the biological function of GlmZ is to positively control the glmS mRNA in response to GlcN-6-P concentrations and that YhbJ negatively regulates GlmZ. As in yhbJ mutants GlcN-6-P has no effect, YhbJ is essential for sensing this metabolite.

The histidine protein (HPr) is the energy-coupling protein of the phosphoenolpyruvate (PEP)-dependent carbohydrate: phosphotransferase system (PTS), which catalyzes sugar transport in many bacteria. In its functions, HPr interacts with a number of evolutionarily unrelated proteins. Mainly, it delivers phosphoryl groups from enzyme I (EI) to the sugar-specific transporters (Ells). HPr proteins of different bacteria exhibit almost identical structures, and, where known, they use similar surfaces to interact with their target proteins. Here we studied the in vivo effects of the replacement of HPr and El of Escherichia coli with the homologous proteins from Bacillus subtilis, a gram-positive bacterium. This replacement resulted in severe growth defects on PTS sugars, suggesting that HPr of B. subtilis cannot efficiently phosphorylate the Ells of E. coli. In contrast, activation of the E. coli BgIG regulatory protein by HPr-catalyzed phosphorylation works well with the B. subtilis HPr protein. Random mutations were introduced into B. subtilis HPr, and a screen for improved growth on PTS sugars yielded amino acid changes in positions 12, 169 17, 20, 24, 279 47, and 51, located in the interaction surface of HPr. Most of the changes restore intermolecular hydrophobic interactions and salt bridges normally formed by the corresponding residues in E. coli HPr. The residues present at the targeted positions differ between HPrs of gram-positive and -negative bacteria, but within each group they are highly conserved. Therefore, they may constitute a signature motif that determines the specificity of HPr for either gram-negative or -positive Ells.

The bacterial sugar: phosphotransferase system (PTS) delivers phosphoryl groups via proteins EI and HPr to the EII sugar transporters. The antitermination protein LicT controls beta-glucoside utilization in Bacillus subtilis and belongs to a family of bacterial transcriptional regulators that are antagonistically controlled by PTS-catalyzed phosphorylations at two homologous PTS regulation domains (PRDs). LicT is inhibited by phosphorylation of PRD1, which is mediated by the beta-glucoside transporter EIIBgl. Phosphorylation of PRD2 is catalyzed by HPr and stimulates LicT activity. Here, we report that LicT, when artificially expressed in the nonrelated bacterium Escherichia coli, is likewise phosphorylated at both PRDs, but the phosphoryl group donors differ. Surprisingly, E. coli HPr phosphorylates PRD1 rather than PRD2, while the stimulatory phosphorylation of PRD2 is carried out by the HPr homolog NPr. This demonstrates that subtle differences in the interaction surface of HPr can switch its affinities toward the PRDs. NPr transfers phosphoryl groups from EINtr to EIIA(Ntr). Together these proteins form the paralogous PTSNtr, which controls the activity of K+ transporters in response to unknown signals. This is achieved by binding of dephosphorylated EIIA(Ntr) to other proteins. We generated LicT mutants that were controlled either negatively by HPr or positively by NPr and were suitable bio-bricks, in order to monitor or to couple gene expression to the phosphorylation states of these two proteins. With the aid of these tools, we identified the stringent starvation protein SspA as a regulator of EIIA(Ntr) phosphorylation, indicating that PTSNtr represents a stress-related system in E. coli.