Streptococci and lactococci synthesize large amounts of HPr(Ser-P)(His~P)

HPr is a protein of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). In gram-positive bacteria, HPr can be phosphorylated on Ser-46 by the kinase/phosphorylase HprK/P and on His-15 by phospho-enzyme I (EI~P) of the PTS. In vitro studies with purified HPrs from Bacillus subtilis, Enterococcus faecalis, and Streptococcus salivarius have indicated that the phosphorylation of one residue impedes the phosphorylation of the other. However, a recent study showed that while the rate of Streptococcus salivarius HPr phosphorylation by EI~P is reduced at acidic pH, the phosphorylation of HPr(Ser-P) by EI~P, generating HPr(Ser-P)(His~P), is stimulated. This suggests that HPr(Ser-P)(His~P) synthesis may occur in acidogenic bacteria unable to maintain their intracellular pH near neutrality. Consistent with this hypothesis, significant amounts of HPr(Ser-P)(His~P) have been detected in some streptococci. The present study was aimed at determining whether the capacity to synthesize HPr(Ser-P)(His~P) is common to streptococcal species, as well as to lactococci, which are also unable to maintain their intracellular pH near neutrality in response to a decrease in extracellular pH. Our results indicated that unlike Staphylococcus aureus, B. subtilis, and E. faecalis, all the streptococcal and lactococcal species tested were able to synthesize large amounts of HPr(Ser-P)(His~P) during growth. We also showed that Streptococcus salivarius IIABLMan, a protein involved in sugar transport by the PTS, could be efficiently phosphorylated by HPr(Ser-P)(His~P).     

Phosphorylation-induced torsion-angle strain in the active center of HPr, detected by NMR and restrained molecular dynamics refinement.

The structure of the phosphorylated form of the histidine-containing phosphocarrier protein HPr from Escherichia coli has been solved by NMR and compared with that of unphosphorylated HPr. The structural changes that occur upon phosphorylation of His 15, monitored by changes in NOE patterns, 3JNHH alpha-coupling constants, and chemical shifts, are limited to the region around the phosphorylation site. The His15 backbone torsion angles become strained upon phosphorylation. The release of this strain during the phosphoryl-transfer to Enzyme II facilitates the transport of carbohydrates across the membrane. From an X-ray study of Streptococcus faecalis HPr (Jia Z, Vandonselaar M, Quail JW, Delbaere LTJ, 1993, Nature 361:94-97), it was proposed that the observed torsion-angle strain at residue 16 in unphosphorylated S. faecalis HPr has a role to play in the protein's phosphocarrier function. The model predicts that this strain is released upon phosphorylation. Our observations on E. coli HPr in solution, which shows strain only after phosphorylation, and the fact that all other HPrs studied thus far in their unphosphorylated forms show no strain either, led us to investigate the possibility that the crystal environment causes the strain in S. faecalis HPr. A 1-ns molecular dynamics simulation of S. faecalis HPr, under conditions that mimic the crystal environment, confirms the observations from the X-ray study, including the torsion-angle strain at residue 16. The strain disappeared, however, when S. faecalis HPr was simulated in a water environment, resulting in an active site configuration virtually the same as that observed in all other unphosphorylated HPrs. This indicates that the torsion-angle strain at Ala 16 in S. faecalis HPr is a result of crystal contacts or conditions and does not play a role in the phosphorylation-dephosphorylation cycle.     

Zymographic characterization of bacteriolytic enzymes produced by oral streptococci

Zymographic analysis was performed to know the bacteriolytic enzyme profiles of 4% SDS extracts of oral streptococci, Streptococcus mutans, S. sobrinus, S. sanguis, S. mitis and S. salivarius. We investigated the five strains in each species and found that the profile was very similar among strains of the same species except for S. salivarius(the profile was classified into two types). On the other hand, the profile was considerably different among species. Two major bacteriolytic enzymes of S. mutans showing molecular mass of 80 and 100 kDa were found using SDS-boiled S. mutans or S. sobrinus cells as substrate. These bacteriolytic activities were less apparent in the gel containing S. mitis or S. salivarius, and also not detectable in the gel containing S. sanguis. S. sobrinus extract showed only one bacteriolytic band (78 kDa) as strong activity using S. sobrinus cells as substrate. S. sanguis extract showed no bacteriolytic bands using any streptococcal cells. Extracts of either S. mitis or S. salivarius showed weak activity by using respective strains as substrate.     

The presence of two forms of the phosphocarrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system in streptococci.

The protein, HPr, a necessary component of the phosphoenolpyruvate phosphotransferase system (PTS) in bacteria, was purified from Streptococcus salivarius by column chromatography. The purified preparation gave only one band when analyzed by sodium dodecylsulfate gel electrophoresis or by isoelectric focusing in polyacrylamide gel (pI = 4.85). However, electrophoresis in Tris-containing buffers under non-denaturing conditions revealed 2 bands that could be phosphorylated by PEP in the presence of enzyme I of the PTS or by ATP with the HPr kinase. Homogeneous preparations of these 2 forms could be obtained by preparative electrophoresis. Each preparation exhibited only 1 band when analyzed by electrophoresis under non-denaturing conditions, indicating that the doublet observed before preparative electrophoresis was not an electrophoretic artefact. The electrophoretic mobility of each protein was not modified following heat-treatment at 100 degrees C for 20 min or storage at -40 degrees C for several months. Both HPr proteins catalyzed in vitro the PEP-dependent phosphorylation of glucose, but at a rate slightly lower than that observed with a preparation of HPr containing both forms of the protein. Both forms were also able to transfer the phosphate group from PEP to the other specific PTS proteins known in S salivarius. Rabbit polyclonal antibodies directed against each form reacted with both proteins. The presence of the 2 forms of HPr was detected in fresh cellular extracts of S salivarius; however, their intracellular ratio varied according to growth conditions. A doublet was also found in many other streptococcal species tested (S mutans, S sobrinus, S sanguis, S thermophilus, S bovis, S rattus) and also in L lactis.(ABSTRACT TRUNCATED AT 250 WORDS)     

The doubly phosphorylated form of HPr, HPr(Ser~P)(His-P), is abundant in exponentially growing cells of Streptococcus thermophilus and phosphorylates the lactose transporter LacS as efficiently as HPr(His~P)

In Streptococcus thermophilus, lactose is taken up by LacS, a transporter that comprises a membrane translocator domain and a hydrophilic regulatory domain homologous to the IIA proteins and protein domains of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The IIA domain of LacS (IIALacS) possesses a histidine residue that can be phosphorylated by HPr(His~P), a protein component of the PTS. However, determination of the cellular levels of the different forms of HPr, namely, HPr, HPr(His~P), HPr(Ser-P), and HPr(Ser-P)(His~P), in exponentially lactose-growing cells revealed that the doubly phosphorylated form of HPr represented 75% and 25% of the total HPr in S. thermophilus ATCC 19258 and S. thermophilus SMQ-301, respectively. Experiments conducted with [32P]PEP and purified recombinant S. thermophilus ATCC 19258 proteins (EI, HPr, and IIALacS) showed that IIALacS was reversibly phosphorylated by HPr(Ser-P)(His~P) at a rate similar to that measured with HPr(His~P). Sequence analysis of the IIALacS protein domains from several S. thermophilus strains indicated that they can be divided into two groups on the basis of their amino acid sequences. The amino acid sequence of IIALacS from group I, to which strain 19258 belongs, differed from that of group II at 11 to 12 positions. To ascertain whether IIALacS from group II could also be phosphorylated by HPr(His~P) and HPr(Ser-P)(His~P), in vitro phosphorylation experiments were conducted with purified proteins from Streptococcus salivarius ATCC 25975, which possesses a IIALacS very similar to group II S. thermophilus IIALacS. The results indicated that S. salivarius IIALacS was phosphorylated by HPr(Ser-P)(His~P) at a higher rate than that observed with HPr(His~P). Our results suggest that the reversible phosphorylation of IIALacS in S. thermophilus is accomplished by HPr(Ser-P)(His~P) as well as by HPr(His~P).     

Phosphorylation of Streptococcus salivarius lactose permease (LacS) by HPr(His ~ P) and HPr(Ser-P)(His ~ P) and effects on growth

The oral bacterium Streptococcus salivarius takes up lactose via a transporter called LacS that shares 95% identity with the LacS from Streptococcus thermophilus, a phylogenetically closely related organism. S. thermophilus releases galactose into the medium during growth on lactose. Expulsion of galactose is mediated via LacS and stimulated by phosphorylation of the transporter by HPr(His approximately P), a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). Unlike S. thermophilus, S. salivarius grew on lactose without expelling galactose and took up galactose and lactose concomitantly when it is grown in a medium containing both sugars. Analysis of the C-terminal end of S. salivarius LacS revealed a IIA-like domain (IIA(LacS)) almost identical to the IIA domain of S. thermophilus LacS. Experiments performed with purified proteins showed that S. salivarius IIA(LacS) was reversibly phosphorylated on a histidine residue at position 552 not only by HPr(His approximately P) but also by HPr(Ser-P)(His approximately P), a doubly phosphorylated form of HPr present in large amounts in rapidly growing S. salivarius cells. Two other major S. salivarius PTS proteins, IIAB(L)(Man) and IIAB(H)(Man), were unable to phosphorylate IIA(LacS). The effect of LacS phosphorylation on growth was studied with strain G71, an S. salivarius enzyme I-negative mutant that cannot synthesize HPr(His approximately P) or HPr(Ser-P)(His approximately P). These results indicated that (i) the wild-type and mutant strains had identical generation times on lactose, (ii) neither strain expelled galactose during growth on lactose, (iii) both strains metabolized lactose and galactose concomitantly when grown in a medium containing both sugars, and (iv) the growth of the mutant was slightly reduced on galactose.     

The phylogenetic position of Streptococcus and Enterococcus

Streptococcus pyogenes, S. equinus, S. bovis, S. salivarius, S. sanguis, S. mutans, S. rattus, S. cricetus, S. lactis, S. raffinolactis and Enterococcus faecalis have been characterized by oligonucleotide cataloguing of their 16S ribosomal RNA. All the organisms form a loose but coherent group that is phylogenetically equivalent to those of lactobacilli, bacilli, the Brochothrix and Listeria group, and related taxa that constitute one of several sublines within the 'Clostridium' branch of Gram-positive eubacteria. Within the Steptococcus-Enterococcus group, organisms fall into three moderately related clusters defined by Enterococcus, the lactic acid streptococci and streptococci of the pyogenic and oral groups, respectively.     

Functional interactions between proteins of the phosphoenolpyruvate:sugar phosphotransferase systems of Bacillus subtilis and Escherichia coli.

Proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Bacillus subtilis were overexpressed, purified to near homogeneity, and characterized. The proteins isolated include Enzyme I, HPr, the glucose-specific IIA domain of the glucose-specific Enzyme II (IIAglc), and the mannitol-specific IIA protein, IIAmtl. Site specific mutant proteins of IIAglc and HPr were also overexpressed and purified, and their properties were compared with those of the wild type proteins. These proteins and their phosphorylated derivatives were characterized with respect to their immunological cross-reactivities employing the Western blot technique and in terms of their migratory behavior during sodium dodecyl sulfate-gel electrophoresis, nondenaturing gel electrophoresis, and isoelectric focusing. The interactions between homologous and heterologous Enzymes I and HPrs, between homologous and heterologous HPrs and the IIAglc proteins, and between homologous and heterologous IIAglc proteins and IIBCscr of B. subtilis as well as IICBglc of Escherichia coli were defined and compared kinetically. The mutant HPrs and IIAglc proteins were also characterized kinetically as PTS phosphocarrier proteins and/or as inhibitors of the phosphotransferase reactions of the PTS. These studies revealed that complexation of IIAglc with the mutant form of HPr in which serine 46 was replaced by aspartate (S46D) did not increase the rate of phosphoryl transfer from phospho Enzyme I to S46D HPr more than when IIAmtl was complexed to S46D HPr. These findings do not support a role for HPr(Ser-P) in the preferential utilization of one PTS carbohydrate relative to another. Functional analyses in E. coli established that IIAglc of B. subtilis can replace IIAglc of E. coli with respect both to sugar transport and to regulation of non-PTS permeases, catabolic enzymes, and adenylate cyclase. Site-specific mutations in histidyl residues 68 and 83 (H68A and H83A) inactivated IIAglc of B. subtilis with respect to phosphoryl transfer and its various regulatory roles.     

Identification of methionine-processed HPr in the equine pathogen Streptococcus equi

Using preparative electrophoresis, a low molecular weight protein has been partially purified from a cell extract of the equine pathogen Streptococcus equi susp. equi. N-terminal sequence analysis and Western blotting revealed the protein to be HPr, a central component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Interestingly, the only form of the HPr protein detected in S. equi was one with the amino-terminal methionine removed, a modification that has previously been associated with surface localization of streptococcal HPr proteins.     

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)     

Role of Bacteriocin During Plaque Formation by Streptococcus salivarius and Streptococcus sanguis on a Tooth in an Artificial Mouth

Bacteriocinogenic strains of Streptococcus salivarius antagonized Strep. sanguis on blood agar and in Todd-Hewitt broth with, but not without, sucrose. Each organism produced plaque in vitro but, after a mixed inoculum with both organisms, the numbers of Strep. sanguis rapidly fell to <0.01% plaque organisms. A non-bacterio-cinogenic mutant of Strep. salivarius was itself inhibited by Strep. sanguis in the plaque-producing system; derivatives of Strep. sanguis partially resistant to bacteriocin in the plate test nevertheless failed to co-habit plaque with bacteriocinogenic Strep. salivarius. The latter could suppress Strep. sanguis in established monoculture plaque but only if sucrose were continuously supplied. It was concluded that the effect of bacteriocin in plaque formation by these streptococci is linked to other as yet unknown properties which may account for the absence of Strep. salivarius from plaque in vivo .     

Inhibition of Streptococcus mutans biofilm formation by Streptococcus salivarius FruA

The oral microbial flora consists of many beneficial species of bacteria that are associated with a healthy condition and control the progression of oral disease. Cooperative interactions between oral streptococci and the pathogens play important roles in the development of dental biofilms in the oral cavity. To determine the roles of oral streptococci in multispecies biofilm development and the effects of the streptococci in biofilm formation, the active substances inhibiting Streptococcus mutans biofilm formation were purified from Streptococcus salivarius ATCC 9759 and HT9R culture supernatants using ion exchange and gel filtration chromatography. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis was performed, and the results were compared to databases. The S. salivarius HT9R genome sequence was determined and used to indentify candidate proteins for inhibition. The candidates inhibiting biofilms were identified as S. salivarius fructosyltransferase (FTF) and exo-beta-d-fructosidase (FruA). The activity of the inhibitors was elevated in the presence of sucrose, and the inhibitory effects were dependent on the sucrose concentration in the biofilm formation assay medium. Purified and commercial FruA from Aspergillus niger (31.6% identity and 59.6% similarity to the amino acid sequence of FruA from S. salivarius HT9R) completely inhibited S. mutans GS-5 biofilm formation on saliva-coated polystyrene and hydroxyapatite surfaces. Inhibition was induced by decreasing polysaccharide production, which is dependent on sucrose digestion rather than fructan digestion. The data indicate that S. salivarius produces large quantities of FruA and that FruA alone may play an important role in multispecies microbial interactions for sucrose-dependent biofilm formation in the oral cavity.     

Characterization of the 1-phosphohistidinyl residue in the phosphocarrier protein HPr of the phosphoenolpyruvate: sugar phosphotransferase system of Streptococcus faecalis

The phosphocarrier protein HPr of the bacterial phosphoenolpyruvate:sugar phosphotransferase system contains 1-phosphohistidine at residue 15. This residue and the active site residue Arg-17 are conserved in HPrs isolated from both Gram-positive and -negative bacteria. The pH- and temperature-dependent hydrolysis of the 1-phosphohistidinyl residue in P-HPr from Streptococcus faecalis has been investigated. The results show that the hydrolysis properties are very similar to those previously reported for P-HPr from Escherichia coli. It was postulated that the unusual hydrolysis properties were due to the presence of a carboxyl group at the active site, and it is now known that in HPr from Escherichia coli the C-terminal residue Glu-85 is present. The results in this paper suggest that a similar carboxyl group is present at the active site in HPr from Streptococcus faecalis.     

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.     

Diversity of Streptococcus salivarius ptsH mutants that can be isolated in the presence of 2-deoxyglucose and galactose and characterization of two mutants synthesizing reduced levels of HPr, a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase system

In streptococci, HPr, a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS), undergoes multiple posttranslational chemical modifications resulting in the formation of HPr(His approximately P), HPr(Ser-P), and HPr(Ser-P)(His approximately P), whose cellular concentrations vary with growth conditions. Distinct physiological functions are associated with specific forms of HPr. We do not know, however, the cellular thresholds below which these forms become unable to fulfill their functions and to what extent modifications in the cellular concentrations of the different forms of HPr modify cellular physiology. In this study, we present a glimpse of the diversity of Streptococcus salivarius ptsH mutants that can be isolated by positive selection on a solid medium containing 2-deoxyglucose and galactose and identify 13 amino acids that are essential for HPr to properly accomplish its physiological functions. We also report the characterization of two S. salivarius mutants that produced approximately two- and threefoldless HPr and enzyme I (EI) respectively. The data indicated that (i) a reduction in the synthesis of HPr due to a mutation in the Shine-Dalgarno sequence of ptsH reduced ptsI expression; (ii) a threefold reduction in EI and HPr cellular levels did not affect PTS transport capacity; (iii) a twofold reduction in HPr synthesis was sufficient to reduce the rate at which cells metabolized PTS sugars, increase generation times on PTS sugars and to a lesser extent on non-PTS sugars, and impede the exclusion of non-PTS sugars by PTS sugars; (iv) a threefold reduction in HPr synthesis caused a strong derepression of the genes coding for alpha-galactosidase, beta-galactosidase, and galactokinase when the cells were grown at the expense of a PTS sugar but did not affect the synthesis of alpha-galactosidase when cells were grown at the expense of lactose, a noninducing non-PTS sugar; and (v) no correlation was found between the magnitude of enzyme derepression and the cellular levels of HPr(Ser-P).     

Synthesis of HPr(Ser-P)(His-P) by enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system of Streptococcus salivarius

HPr is a protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). In Gram-positive bacteria, HPr can be phosphorylated on Ser(46) by HPr(Ser) kinase/phosphorylase (HPrK/P) and on His(15) by enzyme I (EI) of the PTS. In vitro studies have shown that phosphorylation on one residue greatly inhibits the second phosphorylation. However, streptococci contain significant amounts of HPr(Ser-P)(His approximately P) during exponential growth, and recent studies suggest that phosphorylation of HPr(Ser-P) by EI is involved in the recycling of HPr(Ser-P)(His approximately P). We report in this paper a study on the phosphorylation of Streptococcus salivarius HPr, HPr(Ser-P), and HPr(S46D) by EI. Our results indicate that (i) the specificity constant (k(cat)/K(m)) of EI for HPr(Ser-P) at pH 7.9 was approximately 5000-fold smaller than that observed for HPr, (ii) no metabolic intermediates were able to stimulate HPr(Ser-P) phosphorylation, (iii) the rate of HPr phosphorylation decreased at pHs below 6.5, while that of HPr(Ser-P) increased and was almost 10-fold higher at pH 6.1 than at pH 7.9, (iv) HPr(S46D), a mutated HPr alleged to mimic HPr(Ser-P), was also phosphorylated more efficiently under acidic conditions, and, lastly, (v) phosphorylation of Bacillus subtilis HPr(Ser-P) by B. subtilis EI was also stimulated at acidic pH. Our results suggest that the high levels of HPr(Ser-P)(His approximately P) in streptococci result from the combination of two factors, a high physiological concentration of HPr(Ser-P) and stimulation of HPr(Ser-P) phosphorylation by EI at acidic pH, an intracellular condition that occurs in response to the acidification of the external medium during growth of the culture.     

Comparative adhesion of seven species of streptococci isolated from the blood of patients with sub-acute bacterial endocarditis to fibrin-platelet clots in vitro

The adhesion to fibrin-platelet clots in vitro of 21 strains of streptococci isolated from the blood of patients with sub-acute bacterial endocarditis (SABE) was measured. The species, in order of greatest adhesion, were Streptococcus faecalis, Streptococcus mutans, Streptococcus milleri, Streptococcus sanguis , dextran-positive Streptococcus mitior , dextran-negative Streptococcus mitior and Streptococcus salivarius. Individual strains within species, however, cannot be assumed to be representative of their species and may exhibit unusually high or low adhesion. Adhesion depended upon both bacterial concentration and period of contact. There was no simple relationship between ability to adhere and liability to cause endocarditis. Formation of dextran did not increase adhesion. The streptococci were more adhesive than strains of Escherichia coli and Neisseria sicca and less adhesive than strains of Staphylococcus aureus and Streptococcus pyogenes.     

Comparative adhesion of seven species of streptococci isolated from the blood of patients with sub-acute bacterial endocarditis to fibrin-platelet clots in vitro

The adhesion to fibrin-platelet clots in vitro of 21 strains of streptococci isolated from the blood of patients with sub-acute bacterial endocarditis (SABE) was measured. The species, in order of greatest adhesion, were Streptococcus faecalis, Streptococcus mutans, Streptococcus milleri, Streptococcus sanguis, dextran-positive Streptococcus mitior, dextran-negative Streptococcus mitior and Streptococcus salivarius. Individual strains within species, however, cannot be assumed to be representative of their species and may exhibit unusually high or low adhesion. Adhesion depended upon both bacterial concentration and period of contact. There was no simple relationship between ability to adhere and liability to cause endocarditis. Formation of dextran did not increase adhesion. The streptococci were more adhesive than strains of Escherichia coli and Neisseria sicca and less adhesive than strains of Staphylococcus aureus and Streptococcus pyogenes.     

Purification and characterization of the Streptococcus salivarius methionine aminopeptidase (MetAP)

Streptococcus salivarius methionine aminopeptidase (MetAP) was purified from a recombinant Escherichia coli strain containing the S. salivarius map gene, which codes for MetAP. S. salivarius map coded for a protein of 286 amino acids with a calculated molecular mass of 31,723 Da and a pI of 4.6. The native enzyme eluted from a Superdex column as a protein with a molecular mass of 30.6 kDa and cleaved N-terminal Met of peptide only when the penultimate amino acid was Gly, Ala, Ser, Val, Pro, or Thr. The enzyme was more active against tetrapeptides than tripeptides and did not recognize dipeptides. It required the presence of a metal cation for activity, with a preference for Co(2+) over Mn(2+). S. salivarius MetAP has a pH optimum of 8.0 and an optimal temperature at 50 degrees C. The S. salivarius protein had an extra sequence of 24 amino acids between two conserved aspartate residues involved in the coordination of the metal ion. A similar extra sequence is present in MetAP from other streptococci and from Lactococcus lactis, but not from other bacteria or eukaryotes.     

Streptococcal-host interactions. Structural and functional analysis of a Streptococcus sanguis receptor for a human salivary glycoprotein

Colonization of oral tissues by Streptococcus sanguis may be influenced by a mucin-like salivary glycoprotein (SAG) through a calcium-dependent interaction with a specific bacterial receptor. We report the nucleotide and deduced amino acid sequence of the S. sanguis receptor (SSP-5) and show that this protein may bind sialic acid residues of SAG. The SSP-5 protein contains three unique structural domains, two of which consist of repetitive amino acid sequences. The N-terminal domain is comprised of four tandem copies of an 82-residue repeat which exhibits homology to M protein of Streptococcus pyogenes. This region is highly charged and predicted to be alpha-helical. A second hydrophilic repetitive domain consists of three copies of a 39-amino acid sequence containing 30% proline flanked by nonrepetitive proline-rich sequence. The third domain consists of 48% proline and resides near the C terminus of the protein. Secondary structure analysis of the SSP-5 sequence also identified four potential helix-turn-helix motifs that resembled E-F hand calcium binding domains. The SSP-5 protein is highly homologous to a surface antigen expressed by the mutans streptococci and the domain structure of SSP-5 is conserved within this family of proteins. The interactions of SSP-5 and of intact S. sanguis with SAG were inhibited by neuraminidase digestion of the salivary glycoprotein and by simple sugars containing sialic acid, suggesting that sialic acid is the primary ligand involved in the binding reaction.