Purification and characterization of an ATP-dependent protein kinase from Streptococcus faecalis

Abstract An enzyme catalyzing the ATP-dependent phosphorylation of HPr of the bacterial phosphotransferase system has been purified from Streptococcus faecalis . Size exclusion chromatography and sodium dodecyl sulfate (SDS) polyacrylamide gels revealed an M _r of 65000. Beside HPr of S. faecalis the protein kinase also phosphorylates HPr of Streptococcus lactis, Streptococcus pyogenes, Bacillus subtilis and Streptococcus aureus , but not HPr of Escherichia coli . The kinase is largely inhibited by P_i and EDTA. Mg²⁺ and Mn²⁺ could overcome inhibition by EDTA. 2-Phosphoglycerate and glucose-6-phosphate, previously reported to stimulate kinase activity in crude extracts, had no effect on the purified enzyme. Fructose-1,6-diphosphate stimulated the protein kinase.     

Properties of ATP-dependent protein kinase from Streptococcus pyogenes that phosphorylates a seryl residue in HPr, a phosphocarrier protein of the phosphotransferase system.

Transport of sugars across the cytoplasmic membranes of gram-positive bacteria appears to be regulated by the action of a metabolite-activated, ATP-dependent protein kinase that phosphorylates a seryl residue in the phosphocarrier protein of the phosphotransferase system, HPr. We have developed a quantitative assay for measuring the activity of this enzyme from Streptococcus pyogenes. The product of the in vitro protein kinase-catalyzed reaction was shown to be phosphoseryl-HPr by several independent criteria (rates of hydrolysis in the presence of various agents, detection of serine-phosphate in acid hydrolysates, immunological assay, and electrophoretic migration rates). HPrs isolated from four different gram-positive bacteria (S. pyogenes, Streptococcus faecalis, Staphylococcus aureus, and Bacillus subtilis) were shown to be phosphorylated by the kinase from S. pyogenes. In contrast, Escherichia coli HPr was not a substrate of this enzyme. The soluble kinase released from the particulate fraction of the cells with high salt in the presence of a protease inhibitor was shown to have an approximate molecular weight of 60,000 as estimated by gel filtration. Its activity was dependent on divalent cations, with Mg2+ and Mn2+ being most active. EDTA, Pi, and high concentrations of salt were strongly inhibitory. The enzyme was optimally active at pH 7.0, exhibited high affinity for its substrates, and was dependent on the presence of one of several metabolites. Of these compounds, fructose 1-6-diphosphate was most active, with gluconate 6-phosphate, 2-phosphoglycerate, 2,3-diphosphoglycerate, phosphoenolpyruvate, and pyruvate exhibiting moderate to low stimulatory activities. Other compounds tested, including a variety of sugar phosphates, pyridine nucleotides, and other metabolites were without effect. The ATP-dependent phosphorylation of HPr on the seryl residue was strongly inhibited by phosphoenolpyruvate-dependent phosphorylation of the active histidyl residue of this protein. Treatment of the kinase with diethyl pyrocarbonate strongly inhibited the ATP-dependent phosphorylation activity, although the sulfhydryl reagents N-ethylmaleimide, p-chloromercuribenzoate, and iodoacetate were without effect. These results serve to characterize the HPr (serine) kinase, which apparently regulates the rates of carbohydrate transport in streptococcal cells via the phosphotransferase system. A primary role of this kinase in the control of cellular inducer levels and carbohydrate metabolic rates is proposed.     

The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase

The HPr kinase of Gram-positive bacteria is an ATP-dependent 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-Ser-HPr 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.     

Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr

The amino acid sequence of histidine-containing protein (HPr) from Streptococcus faecalis has been determined by direct Edman degradation of intact HPr and by amino acid sequence analysis of tryptic peptides, V8 proteolytic peptides, thermolytic peptides, and cyanogen bromide cleavage products. HPr from S. faecalis was found to contain 89 amino acid residues, corresponding to a molecular weight of 9438. The amino acid sequence of HPr from S. faecalis shows extended homology to the primary structure of HPr proteins from other bacteria. Besides the phosphoenolpyruvate-dependent phosphorylation of a histidyl residue in HPr, catalyzed by enzyme I of the bacterial phosphotransferase system, HPr was also found to be phosphorylated at a seryl residue in an ATP-dependent protein kinase catalyzed reaction [Deutscher, J., & Saier, M. H., Jr. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6790-6794]. The site of ATP-dependent phosphorylation in HPr of S. faecalis has now been determined. [32P]P-Ser-HPr was digested with three different proteases, and in each case, a single labeled peptide was isolated. Following digestion with subtilisin, we obtained a peptide with the sequence -(P)Ser-Ile-Met-. Using chymotrypsin, we isolated a peptide with the sequence -Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-Gly-Val-Met-. The longest labeled peptide was obtained with V8 staphylococcal protease. According to amino acid analysis, this peptide contained 36 out of the 89 amino acid residues of HPr. The following sequence of 12 amino acid residues of the V8 peptide was determined: -Tyr-Lys-Gly-Lys-Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of dihydroxyacetone and glycerol kinase activity in Streptococcus faecalis by phosphoenolpyruvate-dependent phosphorylation catalyzed by enzyme I and HPr of the phosphotransferase system.

Recently we reported the phosphoenolpyruvate (PEP)-dependent phosphorylation of a 55-kilodalton protein of Streptococcus faecalis catalyzed by enzyme I and histidine-containing protein (HPr) of the phosphotransferase system (J. Deutscher, FEMS Microbiol. Lett. 29:237-243, 1985). The purified 55-kilodalton protein was found to exhibit dihydroxyacetone kinase activity. Glycerol was six times more slowly phosphorylated than dihydroxyacetone. The Kms were found to be 0.7 mM for ATP, 0.45 mM for dihydroxyacetone, and 0.9 mM for glycerol. PEP-dependent phosphorylation of dihydroxyacetone kinase stimulated phosphorylation of both substrates about 10-fold. Fructose 1,6-diphosphate at concentrations higher than 2 mM inhibited the activity of phosphorylated and unphosphorylated dihydroxyacetone kinase in a noncompetitive manner. The rate of PEP-dependent phosphorylation of dihydroxyacetone kinase was about 200-fold slower than the phosphorylation rate of III proteins (also called enzyme III or factor III), which so far have been considered the only phosphoryl acceptors of histidyl-phosphorylated HPr. P-Dihydroxyacetone kinase was found to be able to transfer its phosphoryl group in a backward reaction to HPr. Following [32P]PEP-dependent phosphorylation and tryptic digestion of dihydroxyacetone kinase, we isolated a labeled peptide composed of 37 amino acids, as determined by amino acid analysis. The single histidyl residue of this peptide most likely carries the phosphoryl group in phosphorylated dihydroxyacetone kinase.     

HPr proteins of different microorganisms studied by hydrogen-1 high-resolution nuclear magnetic resonance: similarities of structures and mechanisms

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.     

Bacterial phosphoenolpyruvate-dependent phosphotransferase system: P-Ser-HPr and its possible regulatory function?

HPr of the bacterial phosphotransferase system is a histidine-containing phospho-carrier protein. It is phosphorylated at a single histidyl residue with phosphoenolpyruvate (PEP) and enzyme I and transfers the histidyl-bound phosphoryl group to a variety of factor III proteins. Recently, we described an HPr phosphorylated at a seryl residue (P-Ser-HPr), which is formed in an adenosine 5'-triphosphate dependent reaction catalyzed by a protein kinase [Deutscher, J., & Saier, M.-H., Jr. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6790-6794]. Now we demonstrate that this P-Ser-HPr is an altered substrate of phosphorylated enzyme I and factor III proteins compared to unphosphorylated HPr. Thus, P-Ser-HPr of Streptococcus lactis is phosphorylated about 5000 times slower by PEP and enzyme I than HPr. The slow phosphorylation by PEP and enzyme I can be overcome when factor III protein specific for gluconate (factor III(Gct)) of Streptococcus faecalis is added. Most likely, a complex of P-Ser-HPr and factor III(Gct) is formed which then becomes phosphorylated as fast as free HPr. Factor III protein specific for lactose (factor III(Lac)) of Staphylococcus aureus also enhances the phosphorylation of P-Ser-HPr by enzyme I and PEP, but its effect is lower. Thus, P-Ser-HPr is phosphorylated 70-100-fold slower in the presence of factor III(Lac) than in the presence of factor III(Gct). The described interaction of P-Ser-HPr with enzyme I in the presence of different factor III proteins could account for the regulation of sugar uptake within the phosphotransferase system. Some of the phosphoenolpyruvate-dependent phosphotransferase system sugars like glucose are known to be taken up in preference to others, for example, lactose.     

Properties of a Streptococcus salivarius spontaneous mutant in which the methionine at position 48 in the protein HPr has been replaced by a valine.

HPr is a protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) that participates in the concomitant transport and phosphorylation of sugars in bacteria. In gram-positive bacteria, HPr is also reversibly phosphorylated at a seryl residue at position 46 (Ser-46) by a metabolite-activated ATP-dependent kinase and a Pi-dependent HPr(Ser-P) phosphatase. We report in this article the isolation of a spontaneous mutant (mutant A66) from a streptococcus (Streptococcus salivarius) in which the methionine at position 48 (Met-48) in the protein HPr has been replaced by a valine (Val). The mutation inhibited the phosphorylation of HPr on Ser-46 by the ATP-dependent kinase but did not prevent phosphorylation of HPr by enzyme I or the phosphorylation of enzyme II complexes by HPr(His-P). The results, however, suggested that replacement of Met-48 by Val decreased the affinity of enzyme I for HPr or the affinity of enzyme II proteins for HPr(His-P) or both. Characterization of mutant A66 demonstrated that it has pleiotropic properties, including the lack of IIILman, a specific protein of the mannose PTS; decreased levels of HPr; derepression of some cytoplasmic proteins; reduced growth on PTS as well as on non-PTS sugars; and aberrant growth in medium containing a mixture of sugars.     

The phosphotransferase system (PTS) of Streptomyces coelicolor identification and biochemical analysis of a histidine phosphocarrier protein HPr encoded by the gene ptsH.

HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS) controls sugar uptake and carbon utilization in low-GC Gram-positive bacteria and in Gram-negative bacteria. We have purified HPr from Streptomyces coelicolor cell extracts. The N-terminal sequence matched the product of an S. coelicolor orf, designated ptsH, sequenced as part of the S. coelicolor genome sequencing project. The ptsH gene appears to form a monocistronic operon. Determination of the evolutionary relationship revealed that S. coelicolor HPr is equally distant to all known HPr and HPr-like proteins. The presumptive phosphorylation site around histidine 15 is perfectly conserved while a second possible phosphorylation site at serine 47 is not well-conserved. HPr was overproduced in Escherichia coli in its native form and as a histidine-tagged fusion protein. Histidine-tagged HPr was purified to homogeneity. HPr was phosphorylated by its own enzyme I (EI) and heterologously phosphorylated by EI of Bacillus subtilis and Staphylococcus aureus, respectively. This phosphoenolpyruvate-dependent phosphorylation was absent in an HPr mutant in which histidine 15 was replaced by alanine. Reconstitution of the fructose-specific PTS demonstrated that HPr could efficiently phosphorylate enzyme IIFructose. HPr-P could also phosphorylate enzyme IIGlucose of B. subtilis, enzyme IILactose of S. aureus, and IIAMannitol of E. coli. ATP-dependent phosphorylation was detected with HPr kinase/phosphatase of B. subtilis. These results present the first identification of a gene of the PTS complement of S. coelicolor, providing the basis to elucidate the role(s) of HPr and the PTS in this class of bacteria.     

Loss of protein kinase-catalyzed phosphorylation of HPr, a phosphocarrier protein of the phosphotransferase system, by mutation of the ptsH gene confers catabolite repression resistance to several catabolic genes of Bacillus subtilis.

In gram-positive bacteria, HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), is phosphorylated by an ATP-dependent, metabolite-activated protein kinase on seryl residue 46. In a Bacillus subtilis mutant strain in which Ser-46 of HPr was replaced with a nonphosphorylatable alanyl residue (ptsH1 mutation), synthesis of gluconate kinase, glucitol dehydrogenase, mannitol-1-P dehydrogenase and the mannitol-specific PTS permease was completely relieved from repression by glucose, fructose, or mannitol, whereas synthesis of inositol dehydrogenase was partially relieved from catabolite repression and synthesis of alpha-glucosidase and glycerol kinase was still subject to catabolite repression. When the S46A mutation in HPr was reverted to give S46 wild-type HPr, expression of gluconate kinase and glucitol dehydrogenase regained full sensitivity to repression by PTS sugars. These results suggest that phosphorylation of HPr at Ser-46 is directly or indirectly involved in catabolite repression. A strain deleted for the ptsGHI genes was transformed with plasmids expressing either the wild-type ptsH gene or various S46 mutant ptsH genes (S46A or S46D). Expression of the gene encoding S46D HPr, having a structure similar to that of P-ser-HPr according to nuclear magnetic resonance data, caused significant reduction of gluconate kinase activity, whereas expression of the genes encoding wild-type or S46A HPr had no effect on this enzyme activity. When the promoterless lacZ gene was put under the control of the gnt promoter and was subsequently incorporated into the amyE gene on the B. subtilis chromosome, expression of beta-galactosidase was inducible by gluconate and repressed by glucose. However, we observed no repression of beta-galactosidase activity in a strain carrying the ptsH1 mutation. Additionally, we investigated a ccpA mutant strain and observed that all of the enzymes which we found to be relieved from carbon catabolite repression in the ptsH1 mutant strain were also insensitive to catabolite repression in the ccpA mutant. Enzymes that were repressed in the ptsH1 mutant were also repressed in the ccpA mutant.     

The ptsH gene from Bacillus thuringiensis israelensis. Characterization of a new phosphorylation site on the protein HPr.

The ptsH gene from Bacillus thuringiensis israelensis (Bti), coding for the phosphocarrier protein HPr of the phosphotransferase system has been cloned and overexpressed in Escherichia coli. Comparison of its primary sequence with other HPr sequences revealed that the conserved His15 and Ser46 residues were shifted by one amino acid and located at positions 14 and 45, respectively. The biological activity of the protein was not affected by this change. When expressed in a Bacillus subtilis ptsH deletion strain, Bti HPr was able to complement the functions of HPr in sugar uptake and glucose catabolite repression of the gnt and iol operons. A modified form of HPr was detected in Bti cells, and also when Bti ptsH was expressed in E. coli or B. subtilis. This modification was identified as phosphorylation, because alkaline phosphatase treatment converted the modified form to unmodified HPr. The phosphoryl bond in the new form of in vivo phosphorylated HPr was resistant to alkali treatment but sensitive to acid treatment, suggesting phosphorylation at a histidine residue. Replacement of His14 with alanine in Bti HPr prevented formation of the new form of phosphorylated HPr. The phosphorylated HPr was stable at 60 degrees C, in contrast with HPr phosphorylated at the N delta 1 position of His14 with phosphoenolpyruvate and enzyme I. (31)P-NMR spectroscopy was used to show that the new form of P-HPr carried the phosphoryl group bound to the N epsilon 2 position of His14 of Bti HPr. Phosphorylation of HPr at the novel site did not occur when Bti HPr was expressed in an enzyme I-deficient B. subtilis strain. In addition, P-(N epsilon 2)His-HPr did not transfer its phosphoryl group to the purified glucose-specific enzyme IIA domain of B. subtilis.     

Properties of a phosphocarrier protein (HPr) extracted from intact cells of Streptococcus sanguis

Cells of Streptococcus sanguis strain Challis were incubated with sodium lauroylsarcosinate to extract surface proteins. A polypeptide of apparent molecular mass 16 kDa comprising about 12% of the extract was purified using anion-exchange chromatography. The polypeptide was shown to be a phosphocarrier protein (HPr) that could also be found in the soluble (cytoplasmic) fraction from cells broken by homogenization with glass beads. In vivo labelling of S. sanguis cells with 32Pi showed that the polypeptide carried a heat- and acid-stable phosphorylation and that during sucrose starvation the HPr became dephosphorylated. Antiserum raised to the S. sanguis HPr reacted on Western blots with HPrs from all oral streptococci tested, together with strains of S. pyogenes and S. salivarius, but not with HPrs from S. faecalis or S. bovis, nor with proteins from Staphylococcus aureus, Bacillus subtilis, Actinomyces viscosus and various lactobacilli. The S. sanguis HPr had a high content of alanine (17.2%) and was similar in overall amino acid composition to the HPrs from S. mutans an S. salivarius. The N-terminal residues (to 37) of the S. sanguis HPr showed strong sequence identity (82%) with the N-terminal sequence of S. faecalis HPr. It is suggested that HPr in S. sanguis is associated closely with the cytoplasmic membrane. Non-disruptive methods of removing cell-surface proteins from streptococci effect release of HPr and possibly other cytoplasmic components.     

Two-dimensional 1H NMR studies on HPr protein from Staphylococcus aureus: complete sequential assignments and secondary structure

Complete sequence-specific assignments of the 1H NMR spectrum of HPr protein from Staphylococcus aureus were obtained by two-dimensional NMR methods. Important secondary structure elements that can be derived from the observed nuclear Overhauser effects are a large antiparallel beta-pleated sheet consisting of four strands, A, B, C, D, a segment SAB consisting of an extended region around the active-center histidine (His-15) and an alpha-helix, a half-turn between strands B and C, a segment SCD which shows no typical secondary structure, and the alpha-helical, C-terminal segment S(term). These general structural features are similar to those found earlier in HPr proteins from different microorganisms such as Escherichia coli, Bacillus subtilis, and Streptococcus faecalis.     

Regulatory protein phosphorylation in Mycoplasma pneumoniae. A PP2C-type phosphatase serves to dephosphorylate HPr(Ser-P)

Among the few regulatory events in the minimal bacterium Mycoplasma pneumoniae is the phosphorylation of the HPr phosphocarrier protein of the phosphotransferase system. In the presence of glycerol, HPr is phosphorylated in an ATP-dependent manner by the HPr kinase/phosphorylase. The role of the latter enzyme was studied by constructing a M. pneumoniae hprK mutant defective in HPr kinase/phosphorylase. This mutant strain no longer exhibited HPr kinase activity but, surprisingly, still had phosphatase activity toward serine-phosphorylated HPr (HPr(Ser-P)). An inspection of the genome sequence revealed the presence of a gene (prpC) encoding a presumptive protein serine/threonine phosphatase of the PP2C family. The phosphatase PrpC was purified and its biochemical activity in HPr(Ser-P) dephosphorylation demonstrated. Moreover, a prpC mutant strain was isolated and found to be impaired in HPr(Ser-P) dephosphorylation. Homologues of PrpC are present in many bacteria possessing HPr(Ser-P), suggesting that PrpC may play an important role in adjusting the cellular HPr phosphorylation state and thus controlling the diverse regulatory functions exerted by the different forms of HPr.     

Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme IIImtl of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme IImtl of Escherichia coli

Enzyme IIImtl is part of the mannitol phosphotransferase system of Staphylococcus aureus and Staphylococcus carnosus and is phosphorylated by phosphoenolpyruvate in a reaction sequence requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase) and the histidine-containing protein HPr. In this paper, we report the isolation of IIImtl from both S. aureus and S. carnosus and the characterization of the active center. After phosphorylation of IIImtl with [32P]PEP, enzyme I, and HPr, the phosphorylated protein was cleaved with endoproteinase Glu(C). The amino acid sequence of the S. aureus peptide carrying the phosphoryl group was found to be Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp- Asp. The corresponding peptide from S. carnosus shows an equal sequence except that the first residue is Ala instead of Gln. These peptides both contain a single histidyl residue which we assume to carry the phosphoryl group. All proteins of the PTS so far investigated indeed carry the phosphoryl group attached to a histidyl residue. According to sodium dodecyl sulfate gels, the molecular weight of the IIImtl proteins was found to be 15,000. We have also determined the N-terminal sequence of both proteins. Comparison of the IIImtl peptide sequences and the C-terminal part of the enzyme IImtl of Escherichia coli reveals considerable sequence homology, which supports the suggestion that IImtl of E. coli is a fusion protein of a soluble III protein with a membrane-bound enzyme II. In particular, the homology of the active-center peptide of IIImtl of S. aureus and S. carnosus with the enzyme IImtl of E. coli allows one to predict the N-3 histidine phosphorylation site within the E. coli enzyme.     

Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in Gram-positive bacteria

CcpA, the repressor/activator mediating carbon catabolite repression and glucose activation in many Gram-positive bacteria, has been purified from Bacillus megaterium after fusing it to a His tag. CcpA-his immobilized on a Ni-NTA resin specifically interacted with HPr phosphorylated at seryl residue 46. HPr, a phosphocarrier protein of the phosphoenolpyruvate: glycose phosphotransferase system (PTS), can be phosphorylated at two different sites: (i) at His-15 in a PEP-dependent reaction catalysed by enzyme I of the PTS; and (ii) at Ser-46 in an ATP-dependent reaction catalysed by a metabolite-activated protein kinase. Neither unphosphorylated HPr nor HPr phosphorylated at His-15 nor the doubly phosphorylated HPr bound to CcpA. The interaction with seryl-phosphorylated HPr required the presence of fructose 1,6-bisphosphate. These findings suggest that carbon catabolite repression in Gram-positive bacteria is a protein kinase-triggered mechanism. Glycolytic intermediates, stimulating the corresponding protein kinase and the P-ser-HPr/CcpA complex formation, provide a link between glycolytic activity and carbon catabolite repression. The sensitivity of this complex formation to phosphorylation of HPr at His-15 also suggests a link between carbon catabolite repression and PTS transport activity.     

Purification of proteins similar to HPr and enzyme I from the oral bacterium Streptococcus salivarius. Biochemical and immunochemical properties

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is made of several proteins. Two of them are designated general proteins because they are required for the transport and phosphorylation of all sugars of the PTS. These two proteins are found in the soluble fraction of cellular extracts and are termed HPr and enzyme I (EI). We reported in this work the purification and the characterization of these two proteins from Streptococcus salivarius ATCC 25975. HPr was purified by DEAE-cellulose chromatography, molecular sieving on Ultrogel AcA44, and carboxymethylcellulose chromatography. Sodium dodecyl sulfate electrophoresis in the presence of urea revealed a single band with a molecular weight of 6700. The protein contained no tryptophan and had a pI of 4.8. The purification scheme of EI was as follows: DEAE-cellulose chromatography, hydroxylapatite chromatography, DEAE-Sephadex A-50 chromatography, preparative electrophoresis, and molecular sieving on Ultrogel AcA34. The five-step purification for EI produced a 199-fold purified preparation with a specific activity of 530 mumol of HPr phosphorylated per minute per milligram of protein at 37 degrees C. The fraction obtained after filtration on Ultrogel AcA34 gave one band (68 000) on sodium dodecyl sulfate - polyacrylamide gel electrophoresis. The molecular weight of the native enzyme determined by gel filtration at 4 degrees C was 135 000, suggesting that it was a dimer. Enzyme I had a pI of 4.2, a pH optimum of 6.7, a Km for HPr of about 27 microM, a Km for phosphoenolpyruvate of 0.48 mM, and kinetics that were consistent with a Ping-Pong mechanism. Evidence had been obtained which indicated that S. salivarius enzyme I was antigenically very similar to enzyme I from various strains of Streptococcus mutans, but not to the enzyme from Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis, and Escherichia coli.     

Characterization of an HPr kinase mutant of Staphylococcus xylosus

The Staphylococcus xylosus gene hprK, encoding HPr kinase (HPrK), has been isolated from a genomic library. The HPrK enzyme, purified as a His(6) fusion protein, phosphorylated HPr, the phosphocarrier protein of the bacterial phosphotransferase system, at a serine residue in an ATP-dependent manner, and it also catalyzed the reverse reaction. Therefore, the enzyme constitutes a bifunctional HPr kinase/phosphatase. Insertional inactivation of the gene in the genome of S. xylosus resulted in the concomitant loss of both HPr kinase and His serine-phosphorylated-HPr phosphatase activities in cell extracts, strongly indicating that the HPrK enzyme is also responsible for both reactions in vivo. HPrK deficiency had a profound pleiotropic effect on the physiology of S. xylosus. The hprK mutant strain showed a severe growth defect in complex medium upon addition of glucose. Glucose uptake in glucose-grown cells was strongly enhanced compared with the wild type. Carbon catabolite repression of three tested enzyme activities by glucose, sucrose, and fructose was abolished. These results clearly demonstrate the prominent role of HPr kinase in global control to adjust catabolic capacities of S. xylosus according to the availability of preferred carbon sources.     

ATP-dependent protein kinase activities in the oral pathogen Streptococcus mutans

ATP-dependent protein kinase activities were detected in both membrane and cytoplasmic fractions from the oral pathogen Streptococcus mutans. Different polypeptides were phosphorylated by endogenous kinase(s) in the two fractions. In membranes, five phosphoproteins were detected with apparent masses of 82, 37, 22, 12, and 10 kilodaltons (KD). In cytoplasm, two major acid-stable phosphoproteins were found. One was identified as HPr of the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS), while the other had an apparent mass of 61 KD. Both of these proteins were phosphorylated on a seryl residue. Fructose 1,6-bisphosphate stimulated phosphorylation of HPr by the kinase and inhibited phosphorylation of the 61-KD protein. In contrast, fructose 1-phosphate, 2-phosphoglycerate, 3-phosphoglycerate, and dihydroxyacetone phosphate inhibited phosphorylation of HPr and stimulated phosphorylation of the 61-KD protein. Several other glycolytic intermediates as well as inorganic phosphate inhibited phosphorylation of either or both proteins. Preincubation of cytoplasm with PEP prior to incubation with ATP reduced the amount of phospho-(seryl)-HPr formed, but not that of the 61-KD phosphoprotein. The latter protein has not yet been identified but has properties that suggest that it may be the protein kinase itself. These results provide evidence for one or more soluble ATP-dependent protein kinases in S mutans that are regulated by glycolytic intermediates and that may play a role in the modulation of carbohydrate uptake and metabolism in this organism. A model for feedback regulation of sugar transport in S mutans, mediated by an allosterically regulated kinase, is presented.     

The HPr(Ser) Kinase of Streptococcus salivarius: Purification, Properties, and Cloning of the hprK Gene

In gram-positive bacteria, HPr, a protein of the phosphoenolpyruvate:sugar phosphotransferase system, is phosphorylated on a serine residue at position 46 by an ATP-dependent protein kinase. The HPr(Ser) kinase of Streptococcus salivarius ATCC 25975 was purified, and the encoding gene (hprK) was cloned by using a nucleotide probe designed from the N-terminal amino acid sequence. The predicted amino acid sequence of the S. salivarius enzyme showed 45% identity with the Bacillus subtilis enzyme, the conserved residues being located mainly in the C-terminal half of the protein. The predicted hprK gene product has a molecular mass of 34,440 Da and a pI of 5.6. These values agree well with those found experimentally by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, molecular sieve chromatography in the presence of guanidine hydrochloride, and chromatofocusing using the purified protein. The native protein migrates on a Superdex 200 HR column as a 330,000-Da protein, suggesting that the HPr(Ser) kinase is a decamer. The enzyme requires Mg²⁺ for activity and functions optimally at pH 7.5. Unlike the enzyme from other gram-positive bacteria, the HPr(Ser) kinase from S. salivarius is not stimulated by FDP or other glycolytic intermediates. The enzyme is inhibited by inorganic phosphate, and its K_ms for HPr and ATP are 31 μM and 1 mM, respectively.