Regulation of carbon metabolism in gram-positive bacteria by protein phosphorylation.

The main function of the bacterial phosphotransferase system is to transport and to phosphorylate mono- and disaccharides as well as sugar alcohols. However, the phosphotransferase system is also involved in regulation of carbon metabolism. In Gram-positive bacteria, it is implicated in carbon catabolite repression and regulation of expression of catabolic genes by controlling either catabolic enzyme activities, transcriptional activators or antiterminators. All these different regulations follow a protein phosphorylation mechanism.

Phosphotransfer functions mutated Bacillus subtilis HPr-like protein Crh carrying a histidine in the active site.

The Bacillus subtilis protein Crh exhibits strong similarity to HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). HPr phosphorylated at His-15 can transfer its phosphoryl group to several EIIAs of the PTS for sugar transport and phosphorylation. In addition, it phosphorylates and activates transcriptional regulators containing PTS regulation domains (PRDs). In Gram-positive bacteria, it also controls the enzyme glycerol kinase. Since in Crh the active site His-15 of HPr is replaced with a glutamine, Crh was not able to carry out the catalytic and regulatory functions mediated by P approximately His-HPr. However, when Gln-15 of Crh was replaced with a histidine, Crh gained most of the catalytic and regulatory functions exerted by HPr. To allow CrhQ15H to efficiently phosphorylate and activate the PRD-containing antiterminator LicT, which controls the expression of the bgIS gene and the bgIPH operon, it was sufficient to express the crhQ15H allele under control of the spac promoter in monocopy. By contrast, to phosphorylate and activate glycerol kinase and to allow a ptsH deletion strain (devoid of HPr) to slowly grow on the non-PTS substrate glycerol and to efficiently utilize the PTS sugars glucose and mannitol, the crhQ15H allele had to be expressed from a multicopy plasmid.

Cloning and sequencing of two enterococcal glpK genes and regulation of the encoded glycerol kinases by phosphoenolpyruvate-dependent, phosphotransferase system-catalyzed phosphorylation of a single histidyl residue.

The glpK genes of Enterococcus casseliflavus and Enterococcus faecalis, encoding glycerol kinase, the key enzyme of glycerol uptake and metabolism in bacteria, have been cloned and sequenced. The translated amino acid sequences exhibit strong homology to the amino acid sequences of other bacterial glycerol kinases. After expression of the enterococcal glpK genes in Escherichia coli, both glycerol kinases were purified and were found to be phosphorylated by enzyme I and the histidine-containing protein of the phosphoenolpyruvate:glycose phosphotransferase system. Phosphoenolpyruvate-dependent phosphorylation caused a 9-fold increase in enzyme activity. The site of phosphorylation in glycerol kinase of E. casseliflavus was determined as His-232. Site-specific mutagenesis was used to replace His-232 in glycerol kinase of E. casseliflavus with an alanyl, glutamate, or arginyl residue. The mutant proteins could no longer be phosphorylated confirming that His-232 of E. casseliflavus glycerol kinase represents the site of phosphorylation. The His232 --> Arg glycerol kinase exhibited an about 3-fold elevated activity compared with wild-type glycerol kinase. Fructose 1,6-bisphosphate was found to inhibit E. casseliflavus glycerol kinase activity. However, neither EIIAGlc from E. coli nor the EIIAGlc domain of Bacillus subtilis had an inhibitory effect on glycerol kinase of E. casseliflavus.