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Streptococcus agalactiae is a major cause of invasive infections in human newborns. To satisfy its growth requirements, S. agalactiae takes up 9 of the 20 proteinogenic amino acids from the environment. Defined S. agalactiae mutants in one or several of four putative peptide permease systems were constructed and tested for peptide uptake, growth in various media, and expression of virulence traits. Oligopeptide uptake by S. agalactiae was shown to be mediated by the ABC transporter OppA1-F, which possesses two substrate-binding proteins (OppA1 and OppA2) with overlapping substrate specificities. Dipeptides were found to be taken up in parallel by the oligopeptide permease OppA1-F, by the dipeptide ABC transporter DppA-E, and by the dipeptide symporter DpsA. Reverse transcription-PCR analysis revealed a polycistronic organization of the genes oppA1-F and dppA-E and a monocistronic organization of dpsA in S. agalactiae. The results of quantitative real-time PCR revealed a medium-dependent expression of the operons dppA-E and oppA1-F in S. agalactiae. Growth of S. agalactiae in human amniotic fluid was shown to require an intact dpsA gene, indicating an important role of DpsA during the infection of the amniotic cavity by S. agalactiae. Deletion of the oppB gene reduced the adherence of S. agalactiae to epithelial cells by 26%, impaired its adherence to fibrinogen and fibronectin by 42 and 33%, respectively, and caused a 35% reduction in expression of the fbsA gene, which encodes a fibrinogen-binding protein in S. agalactiae. These data indicate that the oligopeptide permease is involved in modulating virulence traits and virulence gene expression in S. agalactiae.

Characterization of classic type II restriction-modification systems (RMS) (restriction endonucleases and modification methyltransferases) was carried out in isolates of Staphylococcus aureus and Streptococcus agalactiae obtained from clinical material. Among the 100 isolates of S. aureus two different RMS type II were detected. The first was expressed in isolates 32 and 33 (Sau32 I and Sau33 I); the targeting sequence was determined as 5'-GGN CC-3' (Sau96 I isoschizomer). The second was found in isolates no. 90, 93, 96*, and 98 (Sau90 I, Sau93 I, Sau96* I, Sau98 I) and enzymes recognized sequence 5'-CTY RAG-3' (SmlI isoschizomer). Analysis of 40 isolates of S. agalactiae revealed only one RMS; it was detected in two isolates (no. 16 and 23; Sag16 I and Sag23 I). Restriction endonuclease expressed by these isolates cleaved DNA in sequence 5'-CTG CA/G-3' (PstI isoschizomer). In RMS-positive S. aureus and S. agalactiae isolates plasmid DNA capable of replication in Escherichia coli and Bacillus subtilis was also detected and isolated.

Capsular polysaccharides (CPs) of several pathogenic bacteria are thought to be good materials for the development of new therapeutic reagents. These polysaccharides can be used as vaccines against infection of pathogenic bacteria and are also useful as inhibitors for disease caused by aberrant and abnormal cell-cell interaction, such as cancer metastasis and inflammation. Since bacterial CPs are diverse in structure and these bacteria have a variety of sugar transferases responsible for the synthesis of CPs, bacterial CP synthesis (cps) genes have attracted much interest as a source of glycosyltransferases useful for glycoengineering. In this review, we describe physiological effects of the bacterial CPs on mammalian cells, and the structure and function of the cps genes, by focusing on group B streptococci, Streptococcus agalactiae type Ia and Ib, that produce high-molecular weight polysaccharides consisting of the following pentasaccharide repeating units: -->4)-[alpha-D-NeupNAc-(2-->3)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-(1--> and -->4)-[alpha-D-NeupNAc-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->3)]-beta-D-Galp-(1-->4)beta-D-Glcp-(1-->, respectively.

A high-virulence clone (HVC) was proposed as causing much of the morbidity and mortality when a collection of group B Streptococcus (GBS) isolates was examined by multi-locus enzyme electrophoresis. HVC isolates could be further distinguished by their inability to grow at 40 degrees C, and a temperature-sensitive aldolase was identified as responsible for this characteristic. In the present study, the HVC was sought in a collection of 57 GBS isolates by hybridization with a probe containing a putative aldolase gene on genomic DNA restriction enzyme digests. Isolates were initially classified as HVC or non-HVC by their inability to grow at 40 degrees C. Three serotype III invasive isolates had the HVC control restriction/hybridization pattern. They were also unable to grow at 40 degrees C. The remaining 11 invasive and all carrier isolates showed a pattern identical to that of the non-HVC control. These results provide additional support for the existence of a highly virulent clonal group among serotype III isolates and suggest that hybridization with a probe containing the aldolase gene on DNA restriction enzyme digests can be an alternative method for identifying highly virulent isolates.

Group B streptococci (GBS) remain the most significant bacterial pathogen causing neonatal sepsis, pneumonia, and meningitis in the United States despite the chemoprophylaxis strategies for preventing infection recommended by the Centers for Disease Control and Prevention. Using signature-tagged transposon mutagenesis to screen for novel virulence factors, we identified the rpoE gene as essential for development of sepsis in a neonatal rat model of GBS infection. An rpoE allelic replacement mutant displayed attenuated virulence in the sepsis model of infection identical to that of the transposon mutant, confirming linkage of the phenotype to the mutation in rpoE. The rpoE mutants also displayed increased sensitivity to killing in whole-blood bactericidal assays, which may explain the attenuated virulence. The mutants were otherwise phenotypically identical to the wild-type strain, including growth rate in plasma, indicating that a growth defect is not responsible for the attenuated virulence. rpoE is found only in gram-positive bacterial species and encodes the delta peptide, a subunit of RNA polymerase. Previous in vitro studies in other bacteria suggest that the delta peptide plays a role in maintaining transcriptional fidelity by blocking RNA polymerase binding at all but the strongest promoters, thereby inhibiting initiation of transcription. Despite the availability of rpoE mutants for several gram-positive bacterial species, a role for the peptide in vivo has not been defined, though it has been postulated that the delta peptide may be important for long-term survival in vitro or during growth phase transitions. Our data represent the first report of a phenotype relevant to virulence for rpoE mutants.

During characterization of the surface antigens of serotype III group B streptococci (GBS), a protein with an apparent M(r) of approximately 173,500 migrating on a SDS--polyacrylamide gel was found to have an N-terminal amino acid sequence identical to that of the plasmin receptor (Plr) of group A streptococci, a surface-localized glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This work begins to characterize GBS GAPDH and to assess its functional activity on the cell surface. The 1.0-kb gapC gene of GBS was amplified by PCR. plr and gapC demonstrated 87% homology. An anti-Plr monoclonal antibody reacted with GBS whole cells, suggesting GBS GAPDH is surface localized. Multiple serotypes of GBS demonstrated functional GAPDH on their surfaces. The anti-Plr monoclonal antibody recognized GBS protein bands of approximately 41 and 173.5 kDa, by Western blot. Presumably, these represent monomeric and tetrameric forms of the GAPDH molecule. GBS GAPDH was demonstrated by Western blot analysis to interact with lys- and glu-plasminogens. Fluid-phase GBS GAPDH interacted, by means of ELISA, with immobilized lys-plasminogen, glu-plasminogen, actin, and fibrinogen. Enzymatically active GAPDH, capable of binding cytoskeletal and extracellular matrix proteins, is expressed on the surface of GBS.

Protein phosphorylation is essential for the regulation of cell growth, division, and differentiation in both prokaryotes and eukaryotes. Signal transduction in prokaryotes was previously thought to occur primarily by histidine kinases, involved in two-component signaling pathways. Lately, bacterial homologues of eukaryotic-type serine/threonine kinases and phosphatases have been found to be necessary for cellular functions such as growth, differentiation, pathogenicity, and secondary metabolism. The Gram-positive bacteria Streptococcus agalactiae (group B streptococci, GBS) is an important human pathogen. We have identified and characterized a eukaryotic-type serine/threonine protein kinase (Stk1) and its cognate phosphatase (Stp1) in GBS. Biochemical assays revealed that Stk1 has kinase activity and localizes to the membrane and that Stp1 is a soluble protein with manganese-dependent phosphatase activity on Stk1. Mutations in these genes exhibited pleiotropic effects on growth, virulence, and cell segregation of GBS. Complementation of these mutations restored the wild type phenotype linking these genes to the regulation of various cellular processes in GBS. In vitro phosphorylation of cell extracts from wild type and mutant strains revealed that Stk1 is essential for phosphorylation of six GBS proteins. We have identified the predominant endogenous substrate of both Stk1 and Stp1 as a manganese-dependent inorganic pyrophosphatase (PpaC) by liquid chromatography/tandem mass spectrometry. These results suggest that these eukaryotic-type enzymes regulate pyrophosphatase activity and other cellular functions of S. agalactiae.

Although bovine testicular hyaluronidase (BTH) has been used in several medical fields for many years, these drugs are poorly characterized. We compared pharmaceutical BTH preparations (Neopermease, Hylase "Dessau") and a hyaluronate lyase from Streptococcus agalactiae. The BTH preparations were complex mixtures of proteins (SDS-PAGE, gel filtration) with enzymatic activity in different fractions. In the case of Neopermease the highest specific activity was found in the 58 kDa fraction (optimum at pH 3.6), whereas the 77 and 33 kDa fractions showed markedly lower specific activities at an optimal pH of 6.2. Maximum specific activity of the bacterial enzyme (approx. 1000 micromol min(-1) mg(-1)) was found at pH 5.0, being 410- and 5100-times higher compared to Neopermease and Hylase "Dessau", respectively. The hyaluronate lyase preparation was separated into two main proteins [100 kDa (pI=8.9) and 85 kDa (pI=9.2)] which were enzymatically active in SDS substrate-PAGE. Zymography after limited proteolysis of the bacterial enzyme with trypsin revealed active fragments (75-50 kDa). Our results suggest that hyaluronate lyase is an alternative for BTH, of which there has been a shortage, since companies have stopped the production of BTH preparations due to the risk of BSE.

Streptococcus agalactiae remains an important pathogen of dairy herds in Québec, but data about antigenic characteristics of this microorganism are sparse. This study was conducted to determine the variety of S. agalactiae serotypes in dairy herds in Québec. Two hundred and ninety-five isolates cultured from the milk of individual cows from 7 regions of Québec were serotyped. Sixty-two percent of the isolates were untypable. Among the 38% of typeable isolates, serotype III was found most frequently. In conclusion, the heterogeneity found among antigenic determinants of isolates from bovine milk suggests that an immunological method for the detection of S. agalactiae performed directly on bovine milk would not be a practical approach.

The main causes of microbial death after heat exposure are not well understood. Here, it is shown that the heat-shock protein ClpP plays a major role in heat-induced growth arrest in Streptococcus agalactiae. A mutant lacking the ClpP protease was more sensitive to the inhibitory effects of heat, salt and oxidative stress than the isogenic wild-type strain. During growth arrest, this mutant displayed important modifications of its total protein content, including a decreased level of essential metabolic enzymes such as the alcohol dehydrogenase. Analysis of protein carbonylation demonstrated that the ClpP protease plays a role in preventing accelerated protein oxidation. Higher levels of oxidized DnaK, a key modulator of the heat-shock regulon, were observed in the ClpP mutant and these were increased following heat shock. Accumulation of oxidized/inactivated DnaK might explain why the ClpP mutant was unable to properly synthesize DNA and proteins, and why it exhibited an aberrant cell morphology. Even though ClpP plays a minor role in the virulence of S. agalactiae in a murine infection model, the data presented here point to the importance of ClpP in oxidative stress defence in preventing heat-induced cell alterations.

N-acetylneuraminate synthase (NeuAc-synthase; E.C. 4.1.3.19) is one of the two enzymes responsible for sialic acid (N-acetylneuraminic acid) synthesis in bacteria. Potential genes encoding NeuAc synthase in Streptococcus agalactiae and Bacillus subtilis were identified from a BLAST search of the EMBL/GenBank/DDBJ database using the E. coli neuB gene sequence as a probe and the genes cloned and expressed at high level in Escherichia coli. The neuB gene of S. agalactiae was shown to encode an active NeuAc synthase, whereas the spsE gene product from B. subtilis did not have this activity. Expression of the native S. agalactiae neuB gene product enzyme in E. coli resulted in a product that was prone to proteolysis during purification so the protein was tagged with a hexa-histidine tag at its N-terminus and the enzyme was rapidly purified to homogeneity by ammonium sulphate fractionation and Ni-chelating affinity chromatography in two steps. Measurement of the subunit molecular mass by electrospray ionisation mass spectrometry (M(r) = 38, 987 +/- 3) and of the native molecular mass by gel filtration chromatography (M(r) = 78,000) clearly demonstrated that the enzyme is dimeric. The effects of EDTA, temperature, and pH on the activity of the S. agalactiae NeuAc synthase were examined. Enzyme activity was maximal at pH 7 and was dependent on the presence of metal ions such as Mg(2+), Mn(2+) or Co(2+). The purified enzyme was inhibited by the reagent phenylglyoxal and the substrates N-acetyl mannosamine or phosphoenol pyruvate afforded protection against this inhibition, suggesting that one or more arginine residues are involved in substrate recognition and binding. The ease of expression and the properties of the enzyme should now permit a thorough study of the specificity of the enzyme and provide the prerequisites for attempts to alter this specificity by directed evolution for the production of novel sialic acid analogues., (Copyright 2002 Elsevier Science (USA))

A new method for the identification of oligosaccharides obtained by enzymatic digestion of hyaluronic acid (HA) with bacterial hyaluronidase (HA lyase, E.C. 4.2.2.1, from Streptococcus agalactiae) using online capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS) is presented. A fused-silica capillary coated with polyacrylamide was used with a 40 mM ammonium acetate buffer at pH 9.0 and a separation voltage of +30 kV applied to the inlet. Separation was achieved for oligosaccharides containing 4-16 monomers. The migration behavior follows the chain length of the oligomers, regardless of charge state. However, no linear relationship was found for the relation between mobility and chain length. Using an ion trap mass analyzer, complementary structural information was obtained by MS/MS and MS(n) experiments., (Copyright 2003 John Wiley & Sons, Ltd.)

The present study was performed to investigate streptococci of serological group B obtained from various sources and group B streptococcal reference strains for serotype, hyaluronate lyase enzyme activity, the occurrence of the hylB gene and the insertion sequence IS1548. All group B streptococci were identified by cultural, biochemical, and serological properties and by polymerase chain reaction amplification of species-specific parts of the 16S-23S rDNA intergenic spacer region, the 16S rRNA gene and the CAMP-factor (cfb) gene. Of the 73 group B streptococci investigated, 59 strains displayed hyaluronate lyase enzyme activity. All hyaluronate-lyase-positive strains and three phenotypically hyaluronate-lyase-negative strains had a hylB gene with an amplicon size of 3.3kb. Eleven of the 14 phenotypically hyaluronate-lyase-negative strains generated a hylB gene PCR product with a size of 4.6kb, and 10 of these strains displayed a IS1548 amplicon with a size of 0.98kb. The hyaluronate-lyase-negative isolates were mainly observed among group B streptococci of serotype III/Rib. All strains harbouring IS1548 had an additional copy of IS1548 located downstream of the C5a peptidase (scpB) gene.

Streptococcus agalactiae hyaluronate lyase degrades primarily hyaluronan, the main polysaccharide component of the host connective tissues, into unsaturated disaccharide units as the end product. Such function of the enzyme destroys the normal connective tissue structure of the host and exposes the tissue cells to various bacterial toxins. The crystal structure of hexasaccharide hyaluronan complex with the S. agalactiae hyaluronate lyase was determined at 2.2 A resolution; the mechanism of the catalytic process, including the identification of specific residues involved in the degradation of hyaluronan, was clearly identified. The enzyme is composed structurally and functionally from two distinct domains, an alpha-helical alpha-domain and a beta-sheet beta-domain. The flexibility of the protein was investigated by comparing the crystal structures of the S. agalactiae and the Streptococcus pneumoniae enzymes, and by using essential dynamics analyses of CONCOORD computer simulations. These revealed important modes of flexibility, which could be related to the protein function. First, a rotation/twist of the alpha-domain relative to the beta-domain is potentially related to the mechanism of processivity of the enzyme; this twist motion likely facilitates shifting of the ligand along the catalytic site cleft in order to reposition it to be ready for further cleavage. Second, a movement of the alpha- and beta-domains with respect to each other was found to contribute to a change in electrostatic characteristics of the enzyme and appears to facilitate binding of the negatively charged hyaluronan ligand. Third, an opening/closing of the substrate binding cleft brings a catalytic histidine closer to the cleavable substrate beta1,4-glycosidic bond. This opening/closing mode also reflects the main conformational difference between the crystal structures of the S. agalactiae and the S. pneumoniae hyaluronate lyases.

Streptococcus agalactiae (GBS) is a major cause of serious newborn bacterial infections. Crucial to GBS evasion of host immunity is the production of a capsular polysaccharide (CPS) decorated with sialic acid, which inactivates the alternative complement pathway. The CPS operons of serotypes Ia and III GBS have been described, but the CPS sialyltransferase gene was not identified. We identified cpsK, an open reading frame in the CPS operon of most serotypes, which was homologous to the lipooligosaccharide (LOS) sialyltransferase gene, lst, of Haemophilus ducreyi. To determine if cpsK might encode a sialyltransferase, we complemented a H. ducreyi lst mutant with cpsK. CpsK was expressed in H. ducreyi and LOS was isolated and analysed for sialic acid content by SDS-PAGE and high-performance liquid chromatography (HPLC). Sialo-LOS was seen in the wild-type, cpsK- or lst-complemented mutant strains, but not in the mutant without cpsK. Addition of Neu5Ac to the LOS was confirmed by mass spectroscopy. Lectin binding studies detected terminal Neu5Ac(alpha 2-->3)Gal(beta 1- on LOS produced by the wild-type, cpsK or lst-complemented mutant strain LOS, compared with the mutant alone. Our data characterize the first sialyltransferase gene from a Gram- positive bacterium and provide compelling evidence that its product catalyses the alpha2,3 addition of Neu5Ac to H. ducreyi LOS and therefore the terminal side-chain of GBS CPS. Phylogenetic studies further indicated that lst and cpsK are related but distinct from sialyltransferases of most other bacteria and, along with their similar codon usage bias and G + C content, suggests acquisition by lateral transfer from an ancestral low G + C organism.

Streptococcus agalactiae hyaluronate lyase is a virulence factor that helps this pathogen to break through the biophysical barrier of the host tissues by the enzymatic degradation of hyaluronan and certain chondroitin sulfates at beta-1,4 glycosidic linkages. Crystal structures of the native enzyme and the enzyme-product complex were determined at 2.1- and 2.2-A resolutions, respectively. An elongated cleft transversing the middle of the molecule has been identified as the substrate-binding place. Two product molecules of hyaluronan degradation were observed bound to the cleft. The enzyme catalytic site was identified to comprise three residues: His(479), Tyr(488), and Asn(429). The highly positively charged cleft facilitates the binding of the negatively charged polymeric substrate chain. The matching between the aromatic patch of the enzyme and the hydrophobic patch of the substrate chain anchors the substrate chain into degradation position. A pair of proton exchanges between the enzyme and the substrate results in the cleavage of the beta-1,4 glycosidic linkage of the substrate chain and the unsaturation of the product. Phe(423) likely determines the size of the product at the product release side of the catalytic region. Hyaluronan chain is processively degraded from the reducing end toward the nonreducing end. The unsulfated or 6-sulfated regions of chondroitin sulfate can also be degraded in the same manner as hyaluronan.

Superoxide dismutases convert superoxide anions to molecular oxygen and hydrogen peroxide, which, in turn, is metabolized by catalases and/or peroxidases. These enzymes constitute one of the major defense mechanisms of cells against oxidative stress and hence play a role in the pathogenesis of certain bacteria. We previously demonstrated that group B streptococci (GBS) possess a single Mn-cofactored superoxide dismutase (SodA). To analyze the role of this enzyme in the pathogenicity of GBS, we constructed a sodA-disrupted mutant of Streptococcus agalactiae NEM316 by allelic exchange. This mutant was subsequently cis complemented by integration into the chromosome of pAT113/Sp harboring the wild-type sodA gene. The SOD specific activity detected by gel analysis in cell extracts confirmed that active SODs were present in the parental and complemented strains but absent in the sodA mutant. The growth rates of these strains in standing cultures were comparable, but the sodA mutant was extremely susceptible to the oxidative stress generated by addition of paraquat or hydrogen peroxide to the culture medium and exhibited a higher mutation frequency in the presence of rifampin. In mouse bone marrow-derived macrophages, the sodA mutant showed an increased susceptibility to bacterial killing by macrophages. In a mouse infection model, after intravenous injection the survival of the sodA mutant in the blood and the brain was markedly reduced in comparison to that of the parental and complemented strains whereas only minor effects on survival in the liver and the spleen were observed. These results suggest that SodA plays a role in GBS pathogenesis.

The mreA gene from Streptococcus agalactiae COH31 gamma/delta, resistant to macrolides and clindamycin by active efflux, has recently been cloned in Escherichia coli, where it was reported to confer macrolide resistance (J. Clancy, F. Dib-Hajj, J. W. Petitpas, and W. Yuan, Antimicrob. Agents Chemother. 41:2719--2723, 1997). Cumulative data suggested that the mreA gene was located on the chromosome of S. agalactiae COH31 gamma/delta. Analysis of the deduced amino acid sequence of mreA revealed significant homology with several bifunctional flavokinases/(flavin adenine dinucleotide (FAD) synthetases, which convert riboflavin to flavin mononucleotide (FMN) and FMN to FAD, respectively. High-performance liquid chromatography experiments showed that the mreA gene product had a monofunctional flavokinase activity, similar to that of RibR from Bacillus subtilis. Sequences identical to those of the mreA gene and of a 121-bp upstream region containing a putative promoter were detected in strains of S. agalactiae UCN4, UCN5, and UCN6 susceptible to macrolides. mreA and its allele from S. agalactiae UCN4 were cloned on the shuttle vector pAT28. Both constructs were introduced into E. coli, where they conferred a similar two- to fourfold increase in the MICs of erythromycin, spiramycin, and clindamycin. The MICs of a variety of other molecules, including crystal violet, acriflavin, sodium dodecyl sulfate, and antibiotics, such as certain cephalosporins, chloramphenicol, doxycycline, nalidixic acid, novobiocin, and rifampin, were also increased. In contrast, resistance to these compounds was not detected when the constructs were introduced into E. faecalis JH2-2. In conclusion, the mreA gene was probably resident in S. agalactiae and may encode a metabolic function. We could not provide any evidence that it was responsible for macrolide resistance in S. agalactiae COH31 gamma/delta; broad-spectrum resistance conferred by the gene in E. coli could involve multidrug efflux pumps by a mechanism that remains to be elucidated.

Streptococcus agalactiae is a primary cause of neonatal morbidity and mortality. Essential to the virulence of this pathogen is the production of a type-specific capsular polysaccharide (CPS) that enables the bacteria to evade host immune defenses. The identification, cloning, sequencing, and functional characterization of seven genes involved in type III capsule production have been previously reported. Here, we describe the cloning and sequencing of nine additional adjacent genes, cps(III)FGHIJKL, neu(III)B, and neu(III)C. Sequence comparisons suggested that these genes are involved in sialic acid synthesis, pentasaccharide repeating unit formation, and oligosaccharide transport and polymerization. The type III CPS (cpsIII) locus was comprised of 16 genes within 15.5 kb of contiguous chromosomal DNA. Primer extension analysis and investigation of mRNA from mutants with polar insertions in their cpsIII loci supported the hypothesis that the operon is transcribed as a single polycistronic message. The translated cpsIII sequences were compared to those of the S. agalactiae cpsIa locus, and the primary difference between the operons was found to reside in cps(III)H, the putative CPS polymerase gene. Expression of cps(III)H in a type Ia strain resulted in suppression of CPS Ia synthesis and in production of a CPS which reacted with type III-specific polyclonal antibody. Likewise, expression of the putative type Ia polymerase gene in a type III strain reduced synthesis of type III CPS with production of a type Ia immunoreactive capsule. Based on the similar structures of the oligosaccharide repeating units of the type Ia and III capsules, our observations demonstrated that cps(Ia)H and cps(III)H encoded the type Ia and III CPS polymerases, respectively. Additionally, these findings suggested that a single gene can confer serotype specificity in organisms that produce complex polysaccharides.

The hyaluronan lyase of group B streptococci rapidly cleaves hyaluronan by an elimination mechanism to yield the unsaturated disaccharide 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose. Additionally, it has been shown that the enzyme has limited specificity for achondroitin sulphate and cleaves the chain at unsulphated sites [Baker,Yu, Morrison, Averett and Pritchard (1997) Biochem. J. 327,65-71]. In the present extension of that study it was found that 6-sulphated regions of chondroitin sulphate are also susceptible to cleavage by this hyaluronan lyase. Of the four 6- and/or 4-sulphated tetrasaccharides which can be isolated from testicular hyaluronidase digests of chondroitin sulphate, only those two tetrasaccharides with a6-sulphated disaccharide at the reducing end were cleaved. From thisand other data, a model is proposed for the cleavage specificity of hyaluronan lyase on a chondroitin sulphate. Evidence is presented in support of an action pattern for hyaluronan lyase which involves aninitial random endolytic cleavage followed by rapid exolytic and processive release of unsaturated disaccharide. Since the on lyoligosaccharides which tend to accumulate in near-complete digests of hyaluronan are unsaturated, it is argued that the processive cleavage occurs from the non-reducing to the reducing end of a hyaluronan chain. This detailed knowledge of substrate specificity contributes to our understanding of the enzyme's role in Group B streptococcal pathogenesis. In addition, the hyaluronan lyase may find application in sequence studies of chondroitin sulphates.

The bacteria Streptococcus agalactiae, part of normal human flora, produce an enzyme, hyaluronate lyase, which appears to contribute to the invasive capacity of this pathogen by degrading hyaluronan and chondroitin sulfates of the extracellular matrix of host tissues. The native enzyme, the product of the hylB(3502) allele, has a molecular mass of 111 kDa but undergoes an autocatalytic conversion to a smaller enzymatically active 92 kDa form. To initiate the determination of the catalytic mechanism of action of these enzymes, the 111 and 92 kDa enzymes were crystallized by a vapor-diffusion method using polyethylene glycol monomethyl ether 5000 and potassium thiocyanate as precipitating agents. The 111 kDa enzyme crystals are of poor quality and diffract X-rays to a very low resolution. However, the crystals of the truncated 92 kDa enzyme diffract X-rays to 2.1 A resolution. The crystal symmetry is C222(1) and the unit-cell parameters are a = 51.69, b = 157.03, c = 239.20 A (alpha = beta = gamma = 90 degrees ). The V(m) of 2.64 A(3) Da(-1) is consistent with the presence of one molecule of hyaluronate lyase in the asymmetric unit and a crystal solvent content of 53%. An isomorphous ethylmercuricthiosalicylic acid heavy-atom derivative diffraction data set has been obtained in order to solve the structure.

A hyaluronic acid splitting enzyme of Streptococcus agalactiae was characterized by splitting mechanism, Michaelis-constant and inhibition type for sulfated hyaluronic acid: The enzyme splits hyaluronic acid as a hyaluronate lyase [EC 4.2.2.1]. The Km = 8 x 10(-4) mg ml-1 was determined with the influence of substrate inhibition constant Kiu = 2 x 10(-6) mg ml-1. Sulfated hyaluronic acid inhibits the enzyme in a partially non-competitive way. The inhibition constant is Ki = 5.47 x 10(-4) mg ml-1. The GBS-hyaluronate lyase cleaves hyaluronic acid as an endoglycosidase. The work is related with the intention to establish a hyaluronate lyase of microbial origin as a therapeutical enzyme replacing bovine hyaluronidase.

The process of screening bacterial transformants for recombinant plasmids is made more rapid and simple by the use of vectors with visually detectable reporter genes. In such systems, an alteration in colony phenotype occurs when a vector-borne indicator gene is interrupted with exogenous DNA. Although the lacZ system has been used extensively for this purpose in E. coli, analogous systems for use in Gram-positive bacteria remain uncommon. We have developed a Gram-positive cloning vector that utilizes the interruption of an alkaline phosphatase gene, phoZ, to identify recombinant plasmids. To facilitate introduction of foreign DNA, a multiple cloning site (MCS) was inserted distal to the region coding for the putative signal peptide of phoZ. Alkaline phosphatase expressed from the derivative phoZ gene (phoZMCS) retained activity similar to that of the native protein. The phoZMCS was transferred to pJS3, a well-characterized, high-copy number, and broad-host-range plasmid, to produce pDC123. In pDC123, phoZMCS was transcriptionally linked to the chloramphenicol acetyl transferase (cat) gene under the control of the constitutively expressed tetM and cat promoters that drive cat expression in pJS3. S. agalactiae (Group B streptococci, GBS), E. faecalis, S. pyogenes, S. gordonii, and E. coli containing pDC123 displayed a blue colonial phenotype on agar containing 5-bromo-4-chloro-3-indolyl phosphate (X-p), which was readily distinguished from that of colonies containing the parent plasmid pJS3. Introduction of foreign DNA into the MCS of phoZMCS produced a white colonial phenotype in E. coli and GBS on agar containing X-p and allowed discrimination between transformants containing recombinant plasmids versus those maintaining self-annealed or uncut vector. We have used pDC123 to subclone the cpsE gene from the plasmid pCER111, which carries a 9.0-kb fragment of the GBS capsular polysaccharide synthesis locus. The plasmid pDC123 containing cpsE was isolated by direct electroporation into GBS strain A909 with selection of transformants containing recombinant plasmids achieved by 'blue/white' screening, without the use of an intermediate host. This new cloning vector should improve the efficiency of performing recombinant DNA experiments in Gram-positive bacteria.

We report the cloning, sequencing and expression analysis of the Streptococcus agalactiae strain 4755 hylB4755 allele, the first chromosomally-encoded streptococcal hyaluronate lyase gene to be cloned and sequenced completely. This gene lies in a region homologous to that found in S. mutans, between the mutX and rmlB genes, a region involved in the synthesis of the serotype c-specific polysaccharide antigen of this organism. Sequencing of hylB4755 revealed a 3216-bp open reading frame that encodes a 121.2-kDa polypeptide possessing a 30-amino acid signal sequence which was theoretically predicted and experimentally confirmed. A recombinant plasmid, pHYB100, containing hylB4755 together with its promoter and terminator was constructed and used to analyze the expression of the gene in Escherichia coli. In Northern hybridization experiments, hylB4755 was found to be transcribed as 3.3-kb monocistronic mRNA from its own promoter which exhibits an extended, sigma70-like 10 consensus sequence. Transcript mapping by primer extension analysis placed the major transcription initiation site leading to the longest transcript 38 bp upstream of the translational initiation codon, ATG. E. coli TG1(pHYB100) efficiently synthesized hyaluronan-cleaving enzyme activity at approximately 7000 working units/109 cells, with lyase activity detectable in all principle cellular locations. Zymography and Western analysis identified functional activity in TG1(pHYB100) to be associated with approximately 118, 110 and 94-kDa polypeptides, with the two low molecular weight species constituting the major components of the enzyme purified from the culture supernatant fluid of S. agalactiae 4755. The 118-kDa form was shown to represent the undegraded mature enzyme, whereas the smaller species are likely to arise from proteolytic cleavage in the N-terminal part of the mature protein. The HylB4755 protein showed extensive sequence identity to the homologous enzymes from S. agalactiae 3502 and S. pneumoniae characterized by others but sequence comparisons clearly show that incomplete genes truncated at their 5' ends had been isolated from these two organisms., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

We have cloned and sequenced a 3103-bp DNA fragment carrying the gene encoding the Mn-SOD from Streptococcus agalactiae NEM318 serotype III. This DNA fragment contained four orfs that have the same polarity of transcription. Orf1 was truncated by molecular cloning and the corresponding 228-aa-long polypeptide did not exhibit any significant homology with other cognate proteins. Orf2 encodes a protein of 345 aa that displays some similarity (29% identity) with the YqeN peptide of Bacillus subtilis, the function of which is unknown. Orf3 encodes the 202-aa-long Mn-SOD which was functionally expressed in Escherichia coli. Orf4 was also truncated by molecular cloning and encodes 99 aa of the N-terminal moiety of a protein that displays significant homology (40% f identity) with the antiterminator LicT of B. subtilis. Transcriptional analysis revealed that the sodA gene of S. agalactiae NEM318 was transcribed monocistronically from a promoter, the activity of which is neither regulated by pH, O2, nor CO2 concentrations of the culture medium. Analysis by high resolution agarose gel electrophoresis of the AluI DNA polymorphism of the sodA locus in wild-type strains of S. agalactiae belonging to serogroups I, II, or III revealed no detectable difference.

The purification and properties of a hyaluronate lyase secreted by Streptococcus agalactiae, which is believed to facilitate the invasion of host tissues by the organism, have been described previously [Pritchard, Lin, Willingham and Baker (1994) Arch. Biochem. Biophys. 315, 431-436]. The specificity of the limited cleavage of chondroitin sulphate by the enzyme is the subject of this report. To simplify the task, a chondroitin sulphate from the Swarm rat chondrosarcoma, which contains only 4-sulphated and unsulphated disaccharide repeats, was used in this study. Tetrasaccharides from an ovine testicular hyaluronidase digest of the chondroitin sulphate were isolated, identified and tested as substrates of the streptococcal hyaluronate lyase. Only tetrasaccharides with an unsulphated disaccharide at the reducing end were cleaved (by elimination at the N-acetylgalactosaminidic bond). Thus chondroitin sulphate chains are cleaved by the action of this lyase at every unsulphated disaccharide repeat, but release of unsaturated unsulphated disaccharides only occurs from sites where two or more sequential unsulphated disaccharide repeats are present. Analysis of the chondrosarcoma chondroitin sulphate showed that of approximately five unsulphated disaccharide repeats per chain, two are clustered. The ability of group-B streptococcal hyaluronate lyase to cleave chondroitin sulphate may allow the organisms to invade tissues more efficiently. The demonstrated specific and highly limited cleavage of chondroitin sulphate by this bacterial lyase promises to be a useful tool in the determination of chondroitin sulphate structure and variability.

A collection of 54 unrelated Streptococcus agalactiae strains isolated from cerebrospinal fluid samples from neonates and 60 unrelated strains isolated from carriers that had been previously studied by multilocus enzyme electrophoresis (R. Quentin, H. Huet, F.-S. Wang, P. Geslin, A. Goudeau, and R. K. Selander, J. Clin. Microbiol. 33:2576-2581, 1995) were characterized by randomly amplified polymorphic DNA (RAPD) assay. Four primers, 5'AGGGGGTTCC3', 5'AACGCGCAAC3', 5'GCATCAATCT3', and 5'AGTCGGGTGG3', named OPS16, AP42, A4, and OPS11, respectively, were selected from 29 primers tested. This investigation identified 71 RAPD types. The three families of strains defined by multilocus enzyme electrophoresis analysis, which contain most of the cerebrospinal fluid isolates, were also identified by clustering analysis of RAPD data. Each of these three groups exhibits specific RAPD patterns or fragments. The discriminatory power of the RAPD typing method was also evaluated. The simplest typing scheme was obtained by the combination of RAPD typing done with primers AP42 and OPS11 and serotyping (index of discrimination, 0.97).

A selective medium for quantitative determination of Streptococcus agalactiae in bulk milk samples was tested. Among the medium bases, Edwards medium (EW) supplemented with 6 to 9% (v/v) of carefully washed sheep erythrocytes was proved to be best (Tab. V). The production of sphingomyelinasis C (BHE, Tab. II) or D (COREX, Tab. I) as a supplement supporting the formation of specific hemolytic zones in the medium, was tested in four strains of Staphylococcus aureus and seven strains of Corynebacterium pseudotuberculosis. Submersion aerated cultivation was used for enzyme production; optimum cultivation time is about 100 hours (Figs. 1, 2). To determine the activity of the enzymes produced a screening method was developed applying hemolytic interactions with CAMP factor of Str. agalactiae. Method sensitivity and reproducibility are sufficiently high for the purposes of observation (Tab. III). The produced enzymes did not show any changes in their activity over the whole period of storage under all conditions of storage (32 days/7 degrees C, 48 weeks/-18 degrees C, lyophilizate 48 weeks/20 degrees C). Sphingomyelinasis C is more resistant to heat denaturation, not losing its activity even after being warmed to 85 degrees C for 5 min; sphingomyelinasis D is sensitive to temperatures higher than 50 degrees C (Fig. 3). COREX was selected (sphingomyelinasis D), produced by the strain CNTCC 18/62 of C. pseudotuberculosis. The enzyme was applied either in raw filtrate of the medium (sterilization by repeated membrane filtration) or in prepurified form obtained by cold acetone precipitation, providing the same results. The medium is highly specific and likely enough selective for Str. agalactiae, unlike BHE it does not provide any positive results with UBERIS-factor+strains of Str. uberis (Tab. V). Optimum cultivation time is 18 hours; prolongation of cultivation for more than 24 hours brings about the risk of falsely positive readings (Tab. VI) in the strains of accompanying microflora (Morganella morganii ssp. morganii, delta-hemolytic staphylococci).

Hyaluronate lyase produced by group B streptococci (GBS) degrades hyaluronan completely to unsaturated disaccharide units and also cleaves unsulfated regions of chondroitin sulfate. The enzyme is rapidly inactivated by diethyl pyrocarbonate and enzymatic activity is restored by treatment with hydroxylamine, suggesting that a histidine residue is present in the active site. Amino acid sequence comparisons of GBS hyaluronate lyase and four other related enzymes revealed that one of the 16 histidine residues of the enzyme (His-479) is present in a highly conserved region. Conversion of His-479 to a glycine by site-directed mutagenesis resulted in a complete loss of enzymatic activity of the modified protein. We propose that His-479 is in the active site of GBS hyaluronate lyase and participates in the initial abstraction of hydrogen ions from the glucuronic acid residues of hyaluronan.

The glnA gene from the human pathogen Streptococcus agalactiae was cloned from a genomic library prepared with the lambda phage vector lambdaDASHII. A 4.6-kb DNA fragment of one of the recombinant phages was subcloned in pUC18. This Escherichia coli clone expressed a 52-kDa protein encoded by a 1,341-bp open reading frame. The nucleotide sequence of the open reading frame and the deduced amino acid sequence shared a significant degree of homology with the sequences of other glutamine synthetases (GS). The highest homology was between our deduced protein and GS of gram-positive bacteria such as Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus. Plasmids with the cloned streptococcal glnA were able to complement E. coli glnA mutants grown on minimal media. Rabbit antisera to streptococcal GS recombinant protein recognized not only the recombinant protein but also a similar-sized band in mutanolysin extracts of all group B streptococcal strains tested, regardless of polysaccharide type or surface protein profile. The amino acid sequence of the deduced protein had similarities to other streptococcal cell-surface-bound proteins. The possible functional role of the immunological features of streptococcal GS is discussed.

The serum and urine proteins responsible for enhanced pigment production in Streptococcus agalactiae in culture media were purified by chromatography and were identified as amylases by comparison of their amino acid composition with that calculated for proteins with known sequences. Similar pigment-enhancing activity was displayed by other amylases of nonanimal origin and by maltooligosaccharides.

Group B streptococci were recently reported to possess a cell-associated collagenase. Although the enzyme hydrolyzed the synthetic collagen-like substrate N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala, we found that neither the highly purified enzyme nor crude group B streptococcal cell lysate solubilized a film of reconstituted rat tail collagen, an activity regarded as obligatory for a true collagenase. We cloned and sequenced the gene for the enzyme (pepB). The deduced amino acid sequence showed 66.4% identity to the PepF oligopeptidase from Lactococcus lactis, a member of the M3 or thimet family of zinc metallopeptidases. The group B streptococcal enzyme also showed oligopeptidase activity and degraded a variety of small bioactive peptides, including bradykinin, neurotensin, and peptide fragments of substance P and adrenocorticotropin.

The chromosome of group B streptococci (GBS) contains a gene which is related to the C5a peptidase gene (scpA) of group A streptococci (GAS). scpA encodes a surface-associated peptidase (group A streptococcal C5a peptidase [SCPA]) which specifically cleaves C5a, a major chemoattractant generated in serum by activation of complement. The entire scpA-like gene (scpB) was cloned from a GBS strain and sequenced. The gene encodes an open reading frame of 3,450 bp, which corresponds to a deduced protein (SCPB) of 1,150 amino acids with a molecular weight of 126,237 Da. Nucleotide and deduced amino acid sequences of SCPB were found to be highly homologous to those of SCPAs from GAS. Unexpectedly, scpA12 is more similar to scpB than to another GAS gene, scpA49. The sequence 5' of the open reading frame, including transcription start and a termination site in the signal sequence, is also similar to that of scpA, although less conserved than the coding sequences. The near identity of GBS and GAS peptidases is consistent with horizontal transmission of the scp gene between these species. Recombinant SCPB was expressed in Escherichia coli by using the expression vector plasmid pGEX-4T-1 and was shown to be identical in size to the enzyme extracted from the parental GBS strain 78-471.

Group B Streptococcus (GBS) is the foremost cause of neonatal sepsis and meningitis in the United States. A major virulence factor for GBS is its capsular polysaccharide, a high molecular weight polymer of branched oligosaccharide subunits. N-acetylneuraminic acid (Neu5Ac or sialic acid), at the end of the polysaccharide side chains, is critical to the virulence function of the capsular polysaccharide. Neu5Ac must be activated by CMP-Neu5Ac synthetase before it is incorporated into the polymer. We showed previously that a transposon mutant of a serotype III GBS strain which had no detectable capsular Neu5Ac was deficient in CMP-Neu5Ac-synthetase activity (Wessels et al., 1992). In this paper, we report the identification and characterization of cpsF, a gene interrupted by transposon insertion in the previously described Neu5Ac-deficient mutant. The predicted amino acid sequence of the cpsF gene product shares 57% similarity and 37% identity with CMP-Neu5Ac synthase encoded by the Escherichia coli K1 gene, neuA. The enzymatic function of the protein encoded by cpsF was established by cloning the gene E. coli under the control of the T7 polymerase/promoter. Lysates of E. coli in which the cpsF gene product was expressed, catalysed the condensation of CTP with Neu5Ac to form CMP-Neu5Ac. In addition, when CMP-Neu5Ac synthetase-deficient mutant of E. coli K1 was transformed with cpsF, K1 antigen expression was restored. We concluded that cpsF encodes CMP-Neu5Ac synthetase in type III GBS, and that the GBS enzyme can function in the capsule-synthesis of a heterologous bacterial species.

Type III group B streptococci (GBS) can be subdivided into three subtypes, RDP III-1, III-2, and III-3, on the basis of numerical analysis of HindIII restriction endonuclease digestion patterns (HindIII RDP) with their chromosomal DNAs. In the present study, the effect of C5a on opsonophagocytic killing of a representative strain from each RDP type was investigated by using a novel optical method for determining opsonophagocytic killing, and the effect of C5a-ase treatment of C5a on opsonophagocytic killing was also investigated. Pre-stimulation of polymorphonuclear leukocytes (PMNs) with C5a significantly increased opsonophagocytic killing of all three strains. The increase in killing was abolished by pretreating the C5a with GBS that express C5a-ase, a treatment that also destroyed the chemoattractant activity of the C5a. The kinetics of killing of the RDP III-2 strain differed from those of the other two strains. The survival of the RDP III-2 bacteria continued to decline over the entire 60-min incubation of the opsonophagocytic assay when PMNs were prestimulated with C5a or with C5a that had been inactivated with GBS C5a-ase (dC5a). In contrast, killing of the RDP III-1 and III-3 strains almost ceased after 20 or 60 min when PMNs were prestimulated with dC5a or C5a, respectively. A difference in bacterial killing between the III-2 strain and the III-1 and III-3 strains therefore became increasingly apparent with prolonged incubation time. The percentage of bacteria surviving in the extracellular fluid was approximately the same as the percentages of bacteria surviving in both intracellular and extracellular locations when PMNs were prestimulated with either C5a or dC5a. These data imply that the majority of bacterial killing occurred following phagocytosis and suggest that the enhanced killing of GBS following prestimulation of PMNs with C5a resulted from increased ingestion of the bacteria.

The potency of selected antibacterials on mastitis-causing Escherichia coli, Streptococcus agalactine and Streptococcus uberis in milk, whey and Iso-sensitest broth (ISB) was compared, based on the suppression of bacterial beta-glucuronidase production. The beta-glucuronidase activity in the samples was analysed by substrate-defined fluorometry where the turbidity of milk does not disturb the assay. In ISB, all four E. coli strains were susceptible to enrofloxacin and gentamicin, sulfadoxin-trimethoprim and tetracycline. S. agalactiae and S. uberis strains were susceptible in ISB to most of the antibacterials tested. The antibacterial potency of sulfadoxin-trimethoprim, tetracycline, novobiocin, gentamicin and enrofloxacin on E. coli and S. agalactiae were considerably decreased in milk as compared with that in ISB. However, S. uberis seemed to be more susceptible to antibacterials in milk or whey than in ISB. Regression analysis of the sigmoidal dose-response curves of sulfadoxin-trimethoprim showed that slopes of the linearized lines seemed to become less steep in milk than in the artificial broth medium, indicating a shift of the bactericidic effect in ISB towards a bacteriostatic effect in milk.

The chromosomal genotypes of 277 isolates of 16 serotypes of Streptococcus agalactiae were characterized by analysis of electrophoretically demonstrable allele profiles at 12 metabolic enzyme loci. The collection comprised the type strain and 276 strains recovered from French symptomatic and asymptomatic subjects. Sixty-one distinctive electrophoretic types (ETs), representing multilocus clonal genotypes, were identified. Cluster analysis of the ETs revealed two primary phylogenetic divisions separated by a genetic distance of 0.62, Division I contained 67 isolates which could be assigned to 13 ETs. Twenty-seven of these isolates were from samples of cerebrospinal fluid (CSF) from neonatal meningitis patients. Two ETs, separated by a genetic distance of 0.217, contained 26 of these 27 isolates. Division II contained 210 isolates, of which 27 were isolated from CSF. This division was more polymorphic and included 48 ETs. Spanning a genetic distance of 0.3, three clusters and one ET were identified within this group. Twenty-four of 27 strains isolated from CSF belonged to one cluster, and 19 of them belonged to two adjacent ETs with a genetic distance of 0.083. Fifty-five of the 68 serotype Ia strains and 24 of the 26 serotype Ib strains were each confined to one of the evolutionary lineages, and 85 of the 86 strains which carried protein antigen c belonged to phylogenetic division II. Most of the type III organisms were assigned to two clone families. The characteristics of this French population argue for the existence of particular groups of strains responsible for neonatal meningitis and demonstrate that serotyping can supply information about the genetic distribution of strains.

A new HaeIII isoschizomer from Streptococcus agalactiae was isolated by a single-step purification method. The highly active restriction endonuclease, SagI, was free of nonspecific nuclease activity and was suitable for use in molecular biology procedures. The rapid isolation procedure may be applicable for the recovery of other restriction endonucleases from bacteria.

Group B streptococci (GBS) are a major cause of serious human perinatal infections. Most clinical isolates of GBS secrete hyaluronate lyase, and production of high levels of the enzyme has been associated with strain virulence. Degenerate oligonucleotide primers, designed on the basis of the amino acid sequences of tryptic peptides prepared from the purified enzyme, permitted the polymerase chain reaction amplification from GBS chromosomal DNA of a 363-base pair internal DNA fragment of the GBS hyaluronate lyase gene (hylB). This DNA fragment was used as a probe to screen a lambda phage library of GBS chromosomal DNA fragments. Sequence analysis of positive clones identified an open reading frame capable of coding for a 111-kDa protein. Since no single clone was found to contain the entire gene it was necessary to reconstruct the gene from two plasmids containing inserts with suitable overlapping sequences. When this reconstructed gene was transformed into Escherichia coli, high level expression of hyaluronate lyase activity was obtained.

The capsular polysaccharide is a critical virulence factor for group B streptococci associated with human infections, yet little is known about capsule biosynthesis. We detected CMP-Neu5Ac synthetase, the enzyme which activates N-acetylneuraminic acid (Neu5Ac, or sialic acid) for transfer to the nascent capsular polysaccharide, in multiple group B streptococcus serotypes, all of which elaborate capsules containing Neu5Ac. CMP-Neu5Ac synthetase isolated from a high-producing type Ib strain was purified 87-fold. The enzyme had apparent Km values of 7.6 for Neu5Ac and 1.4 for CTP and a pH optimum of 8.3 to 9.4, required magnesium, and was stimulated by dithiothreitol. This is the first characterization of an enzyme involved in group B streptococcus capsular polysaccharide biosynthesis.

Hyaluronate lyase is one of several proteins secreted by group B streptococci which are believed to contribute to strain virulence. Characterization of the purified enzyme revealed that it degrades hyaluronan by a mechanism different from that of other previously studied hyaluronidases. Instead of randomly cleaving hyaluronan chains leading to a continuous decrease in average chain size, the group B streptococcal enzyme initially yields primarily unsaturated disaccharides. The observation that most of the free reducing ends generated during group B streptococcal hyaluronate lyase digestion are present in the unsaturated disaccharide units supports the conclusion that they are released primarily from the ends of the hyaluronan chains. Furthermore, the experimental evidence is consistent with a mode of action by which the enzyme initially makes a random cut in a hyaluronan chain and then processively moves along the chain releasing disaccharide units. Group B streptococcal hyaluronate lyase also slowly degrades chondroitin sulfate, and its desulfation greatly increases the reaction rate. A preferential cleavage of unsulfated residues is consistent with the observed extensive release of free chondroitin sulfate chains following very limited digestion of aggrecan from bovine nasal cartilage.

Hyaluronidase from two different strains of Streptococcus agalactiae was purified and characterized. The purification was performed successively by chromatography and rechromatography on phenylsepharose, gel filtration with FPLC on Superdex G 200 and isoelectric focusing. The purified hyaluronidase had an isoelectric point of 8.75 and a molecular weight of approximately 116,000 D. It showed maximal enzyme activity at pH 6.30 and 40 degrees C. The Michaelis constant was estimated to be 8.17 x 10(-2) mg/ml. Hyaluronidase was stimulated only by Mg++ and inhibited by Zn++, Al , Cu++ and Fe++ at a final concentration of 10 mmol/l, respectively. The enzyme splitted hyaluronic acid and in low amounts dermatan sulphate and chondroitin sulphate A. Additionally, synthetic polyanions (like polymers of gentisic acid with formaldehyde and hydroxy sulphonic acid with formaldehyde) turned out to be also potent inhibitors of the enzyme.

An assay based on the utilization of degenerate primers that enable enzymatic amplification of an internal fragment of cat genes known to be present in gram-positive cocci was developed to identify the genes encoding chloramphenicol resistance in streptococci and enterococci. The functionality of this system was illustrated by the detection of cat genes belonging to four different hydridization classes represented by the staphylococcal genes catpC221, catpC194, catpSCS7, and the clostridial gene catP, and by the characterization of a new streptococcal cat gene designated catS. A sequence related to the clostridial catQ gene, which was present in one streptococcal strain, was not detected by this assay. These results reveal that these six cat genes account for chromosomal-borne chloramphenicol resistance in 12 group A, B, and G streptococci tested. By contrast, only three of these six cat genes (catpC221, catpC194, and catpSCS7) were detected on the 10 enterococcal plasmids studied here that encode resistance to chloramphenicol.

The extracellular group B streptococcal enzyme described in numerous reports as a neuraminidase is really a hyaluronidase. Over the past 25 years, the enzyme was routinely assayed with bovine submaxillary mucin as the substrate and by the thiobarbituric acid procedure to measure released sialic acid. Characterization of the actual compound released by the enzyme revealed it to be an alpha,beta-unsaturated derivative of hyalobiuronic acid that was derived from hyaluronic acid contaminating the mucin preparation. Previous reports describing an association of elevated levels of extracellular neuraminidase with virulent strains of group B streptococci must be reevaluated with the recognition that the enzyme is really a hyaluronidase.

Most strains of group B streptococci (GBS) elaborate a cell surface-associated enzyme that rapidly inactivates the human complement-derived chemoattractants C5a and C5adesarg by cleaving the His-Lys bond at positions 67 and 68 in the C5a molecule. We have suggested that rapid inactivation of C5a and C5adesarg by this enzyme, called C5a-ase, can hinder the inflammatory response at sites of GBS infection. We tested the ability of GBS C5a-ase to inactivate C5a preparations from various animal species to determine the proper species for studying the role of GBS C5a-ase in the pathogenesis of GBS infections. Exposure of C5a preparations from humans, monkeys, and cows to GBS caused inhibition of C5a functional activity as measured by the ability of C5a to stimulate human polymorphonuclear leukocyte (PMN) adherence and human PMN chemotaxis. Bovine PMN chemotaxis to bovine C5a was also abolished after exposure of bovine C5a to GBS. In contrast, mouse, rat, guinea pig, rabbit, pig, and sheep C5a preparations retained full functional activity after exposure to GBS as measured by chemotaxis of human PMNs, PMNs from the same animal species, or both. These data suggest that there are structural differences between C5a proteins from different species which alter their susceptibility to GBS C5a-ase and indicate that most commonly used animal models of human GBS infection are inadequate for detection of a contribution of GBS C5a-ase to GBS virulence.

Group B streptococci (GBS) and group A streptococci (GAS) are known to have surface-associated peptidase activity which specifically cleaves C5a (C5a-ase), the primary chemotaxin for polymorphonuclear leukocytes. Amplification products were obtained from GBS genomic DNA template by using the polymerase chain reaction and primers corresponding to the C5a peptidase gene of GAS (scpA12). A restriction map of the GBS full-length amplified gene (scpB) was developed. The scpB restriction map was found to be highly similar to that of the analogous gene in GAS. A 50-bp deletion was located near the 5' end of scpB in a region where repeat sequences are found in scpA12. Hybridization experiments confirmed that other serotypes of GBS also carry an scpB-like gene. These results show that GBS contain a gene, scpB, which is very similar to that harbored by M12 GAS. The probability that scpB encodes the C5a-ase protein is discussed.

Most strains of group B streptococci (GBS) possess an enzyme that inactivates the human anaphylatoxin C5a by cleaving a heptapeptide from the carboxyl terminus of C5a. This enzyme, called GBS C5a-ase, has been purified to homogeneity and cleaves and inactivates C5a in physiologic buffer. The enzymatic activity of soluble C5a-ase is completely inhibited, however, in the presence of plasma or serum from normal human adults. The neutralization of soluble C5a-ase by plasma and serum results largely from naturally occurring IgG antibodies directed against C5a-ase. IgG does not neutralize C5a-ase present on intact encapsulated type III GBS but does neutralize the C5a-ase activity associated with a transposon-induced mutant strain of type III GBS that lacks capsule. The location of GBS C5a-ase on the surface of encapsulated type III GBS permits the C5a-ase to inactivate C5a while evading neutralization by IgG antibodies.

We previously reported that group B streptococci (GBS) possess a cell-associated activity that inactivates the chemotactic activity generated in zymosan-activated serum by cleaving a specific site within the carboxy termini of C5a and C5adesarg. This inactivates the major chemoattractants for neutrophils that are generated when serum complement is activated. We now report the isolation of the enzyme responsible for the proteolytic cleavage of C5a. Treatment of GBS with mutanolysin, an endo-N-acetyl muramidase, released activity from GBS which destroyed the functional activity of C5a. The soluble activity was purified to homogeneity by hydroxyapatite, ion-exchange and gel-filtration chromatography. Analysis by SDS-PAGE showed that the enzyme (GBS C5a-ase) has an Mr of approx. 120,000. The GBS C5a-ase appears to be a serine esterase on the basis of its sensitivity to di-isopropyl fluorophosphate. This enzyme is distinct from the C5a-cleaving enzyme produced by group A streptococci, since the two bacterial products migrate differently on SDS-PAGE, and lack antigenic cross reactivity. This enzyme may play a role in the pathogenesis of group B streptococcal disease through its ability to rapidly inactivate the potent neutrophil agonist, C5a, at sites of infection.