Carbon catabolite repression (CCR) is the prototype of a signal transduction mechanism. In enteric bacteria, cAMP was considered to be the second messenger in CCR by playing a role reminiscent of its actions in eukaryotic cells. However, recent results suggest that CCR in Escherichia coli is mediated mainly by an inducer exclusion mechanism. In many Gram-positive bacteria, CCR is triggered by fructose-1,6-bisphosphate, which activates HPr kinase, presumed to be one of the most ancient serine protein kinases. We here report cloning of the Bacillus subtilis hprK and hprP genes and characterization of the encoded HPr kinase and P-Ser-HPr phosphatase. P-Ser-HPr phosphatase forms a new family of phosphatases together with bacterial phosphoglycolate phosphatase, yeast glycerol-3-phosphatase, and 2-deoxyglucose-6-phosphate phosphatase whereas HPr kinase represents a new family of protein kinases on its own. It does not contain the domain structure typical for eukaryotic protein kinases. Although up to now the HPr modifying͞demodifying enzymes were thought to exist only in Gram-positive bacteria, a sequence comparison revealed that they also are present in several Gram-negative pathogenic bacteria.Carbon catabolite repression (CCR) is the paradigm of signal transduction. It allows bacteria to alter catabolic gene expression in response to the availability of rapidly metabolizable carbon sources. Discovered in the early 1940s in Bacillus subtilis and termed the ''diauxic phenomenon'' (1), one type of molecular mechanism was deciphered in the 1960s in Escherichia coli; in enteric bacteria, changes in the level of cAMP were thought to provide the signal for CCR (2). However, recent results on lacZ expression in E. coli suggest that an increase in the cAMP level reduces only the lag phase of diauxic growth but that the major CCR mechanism is based on inducer exclusion mediated by EIIA Glc of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) (3). It was only in the last decade that the molecular mechanisms underlying CCR in bacilli and other Gram-positive bacteria were partly elucidated (refs. 4-7; for a review, see ref. 8). In these organisms, the complex regulatory cascade is triggered by the ATP-dependent, fructose-1,6-bisphosphate (FBP)-stimulated phosphorylation of Ser-46 in histidine-containing protein (HPr) (9-11), a phosphocarrier protein implicated in carbohydrate transport effected via PTS (12). Signal transduction in CCR continues with a phosphorylation-controlled proteinprotein interaction between HPr and the transcriptional repressor͞activator catabolite control protein A (CcpA) (13,14). ATP-dependent phosphorylation at Ser-46 is a prerequisite for the interaction of HPr with CcpA whereas phosphoenolpyruvate-dependent phosphorylation of HPr at His-15 prevents the complex formation, thus linking PTS-mediated sugar transport to CCR (13). The protein complex formed between CcpA and P-Ser-HPr interacts specifically with an operator site called catabolite responsive element (cre) (15,16). A recent...

SummaryThe HPr kinase of Gram-positive bacteria is an ATPdependent serine protein kinase, which phosphorylates the HPr protein of the bacterial phosphotransferase system (PTS) and is involved in the regulation of carbohydrate metabolism. The hprK gene from Enterococcus faecalis was cloned via polymerase chain reaction (PCR) and sequenced. The deduced amino acid sequence was confirmed by microscale Edman degradation and mass spectrometry combined with collision-induced dissociation of tryptic peptides derived from the HPr kinase of E. faecalis. The gene was overexpressed in Escherichia coli, which does not contain any ATP-dependent HPr kinase or phosphatase activity. The homogeneous recombinant protein exhibits the expected HPr kinase activity as well as a P-SerHPr phosphatase activity, which was assumed to be a separate enzyme activity. The bifunctional HPr kinase/ phosphatase acts preferentially as a kinase at high ATP levels of 2 mM occurring in glucose-metabolizing Streptococci. At low ATP levels, the enzyme hydrolyses P-Ser-HPr. In addition, high concentrations of phosphate present under starvation conditions inhibit the HPr kinase activity. Thus, a putative function of the enzyme may be to adjust the ratio of HPr and P-Ser-HPr according to the metabolic state of the cell; P-Ser-HPr is involved in carbon catabolite repression and regulates sugar uptake via the phosphotransferase system (PTS). Reinvestigation of the previously described Bacillus subtilis HPr kinase revealed that it also possesses P-Ser-HPr phosphatase activity. However, contrary to the E. faecalis enzyme, ATP alone was not sufficient to switch the phosphatase activity of the B. subtilis enzyme to the kinase activity. A change in activity of the B. subtilis HPr kinase was only observed when fructose-1,6-bisphosphate was also present.

The HPr proteins of Streptococcus lactis, Streptococcus faecalis, Bacillus subtilis, and Escherichia coli were studied by 1H NMR at 360 MHz. The "active-center" histidines of all HPr proteins are characterized by a low pK value between 5.6 and 6.1 and similar spectral parameters. Phosphorylation of the histidyl residues leads to an increase of the pK value of 2-3 units and spectral changes characteristic for N-1 phosphorylation of the histidyl ring. The spectra of the HPr proteins of S. lactis, S. Faecalis, B. subtilis, and Staphylococcus aureus reveal many similarities, whereas the spectrum of the E. coli protein is different with exception of the active-center histidine. The HPr protein of S. lactis is formylated at its terminal amino group.