Streptococcus sobrinus has four gtf genes, gtfI, gtfS, gtfT, and gtfU, on the chromosome. These genes correspond respectively to the enzymes GTF-I, GTF-S1, GTF-S2, and GTF-S3. An Escherichia coli MD66 clone that contained the S. sobrinus gtfU gene was characterized. Immunological properties showed that the protein produced by the E. coli MD66 clone was similar to S. sobrinus GTF-S1. Biological properties and a linkage analysis of the glucans by 13C NMR spectrometry revealed that the protein produced by the E. coli MD66 clone was GTF-S1.

The enzyme UTP-glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, UDPG:PP) is synthesized by practically all organisms, although prokaryotic UDPG:PPs are evolutionarily unrelated to the eukaryotic counterparts. The primary structure of prokaryotic UDPG:PPs is well conserved, although little information exists on the polymorphism of the genes coding for these enzymes. It has been reported that the galU gene encoding the Streptococcus pneumoniae UDPG:PP is absolutely required for the synthesis of the capsular polysaccharide, a sine qua non prerequisite for virulence. A 594 bp fragment covering 66% of the galU gene from 37 pneumococcal isolates and the type strains of Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii, Streptococcus sanguinis, Streptococcus salivarius, and Streptococcus sobrinus has been amplified by PCR and sequenced. Up to 21 different alleles were found in S. pneumoniae. They possess a mosaic-like structure and belong to, at least, two evolutionarily distinct families that show a sequence divergence of 15-20%. In spite of its marked polymorphism, phylogenetic relationships among pneumococcal strains deduced from the galU gene matched those previously established by using alternative approaches. Comparison of the pneumococcal galU alleles with those from other streptococci indicated the existence of a complex network of genetic interchange. The galU gene represents an informative marker to be used alone or in conjunction with other molecular typing methods.

The dnaJ and gyrB nucleotide sequences were determined for members of the genus Streptococcus. The average similarity between the species tested was 76.4% (69.7-100%) for dnaJ and 75.9 (70.1-98.7%) for gyrB. These data indicated that the dnaJ and gyrB genes are more divergent and more discriminatory than the 16S rDNA gene. Furthermore, the variation in the dnaJ nucleotide sequences among the mitis group was greater than that of the gyrB nucleotide sequences, especially between Streptococcus pneumoniae and Streptococcus mitis. Subsequently, the high discrimination power of dnaJ within the mitis group was confirmed. Thus, we conclude that the dnaJ and gyrB genes are efficient alternative targets for the classification of the genus Streptococcus, and that dnaJ is suitable for phylogenetic analysis of closely related Streptococcus strains.

The near full-length sequences of the groESL genes were determined and analysed among eight reference strains (serotypes a to h) representing five species of mutans group streptococci. The groES sequences from these reference strains revealed that there are two lengths (285 and 288 bp) in the five species. The intergenic spacer between groES and groEL appears to be a unique marker for species, with a variable size (ranging from 111 to 310 bp) and sequence. Phylogenetic analysis of groES and groEL separated the eight serotypes into two major clusters. Strains of serotypes b, c, e and f were highly related and had groES gene sequences of the same length, 288 bp, while strains of serotypes a, d, g and h were also closely related and their groES gene sequence lengths were 285 bp. The groESL sequences in clinical isolates of three serotypes of S. mutans were analysed for intraspecies polymorphism. The results showed that the groESL sequences could provide information for differentiation among species, but were unable to distinguish serotypes of the same species. Based on the determined sequences, a PCR assay was developed that could differentiate members of the mutans streptococci by amplicon size and provide an alternative way for distinguishing mutans streptococci from other viridans streptococci.

A 761-bp portion of the tuf gene (encoding the elongation factor Tu) from 28 clinically relevant streptococcal species was obtained by sequencing amplicons generated using broad-range PCR primers. These tuf sequences were used to select Streptococcus-specific PCR primers and to perform phylogenetic analysis. The specificity of the PCR assay was verified using 102 different bacterial species, including the 28 streptococcal species. Genomic DNA purified from all streptococcal species was efficiently detected, whereas there was no amplification with DNA from 72 of the 74 nonstreptococcal bacterial species tested. There was cross-amplification with DNAs from Enterococcus durans and Lactococcus lactis. However, the 15 to 31% nucleotide sequence divergence in the 761-bp tuf portion of these two species compared to any streptococcal tuf sequence provides ample sequence divergence to allow the development of internal probes specific to streptococci. The Streptococcus-specific assay was highly sensitive for all 28 streptococcal species tested (i.e., detection limit of 1 to 10 genome copies per PCR). The tuf sequence data was also used to perform extensive phylogenetic analysis, which was generally in agreement with phylogeny determined on the basis of 16S rRNA gene data. However, the tuf gene provided a better discrimination at the streptococcal species level that should be particularly useful for the identification of very closely related species. In conclusion, tuf appears more suitable than the 16S ribosomal RNA gene for the development of diagnostic assays for the detection and identification of streptococcal species because of its higher level of species-specific genetic divergence.

Streptococcus sobrinus, one agent of dental caries, secretes a protein that induces lymphocyte polyclonal activation of the host as a mechanism of immune evasion. We have isolated from culture supernatants of this bacterium a protein with murine B-cell-stimulatory properties and subsequently cloned the relevant gene. It contains an open reading frame of 825 bp encoding a polypeptide with 275 amino acid residues and a molecular mass of 30 kDa. The protein displays high sequence homology with NAD(+) synthetases from several organisms, including a conserved fingerprint sequence (SGGXD) characteristic of ATP pyrophosphatases. The polypeptide was expressed in Escherichia coli as a hexahistidine-tagged protein and purified in an enzymatically active form. The recombinant NAD(+) synthetase stimulates murine B cells after in vitro treatment of spleen cell cultures, as demonstrated by its ability to induce up-regulation of the expression of CD69, an early marker of lymphocyte activation. Stimulation with the recombinant NAD(+) synthetase was also observed with other B-cell markers, such as CD19(+), B220(+), and CD21(+). Cell proliferation follows the activation induced by the recombinant NAD(+) synthetase.

Cystic fibrosis (CF) is a multiorgan disease, with the majority of mortalities resulting from pulmonary failure due to repeated pulmonary exacerbations. Recently, members of the Streptococcus anginosus group (S. anginosus, S. constellatus, and S. intermedius), herein referred to as the "Streptococcus milleri group" (SMG) have been implicated as important etiological pathogens contributing to pulmonary exacerbations in CF patients. This is partly due to better microbiological detection of the SMG species through the development of a novel specific medium termed "McKay agar." McKay agar demonstrated that SMG has been an underreported respiratory pathogen contributing to lung exacerbations. Our aim was to develop a real-time PCR assay to expedite the detection of SMG within diagnostic samples. The cpn60 gene was chosen as a target, with all three members amplified using a single hybridization probe set. SMG strain analysis showed that speciation based on melting curve analysis allowed for the majority of the S. constellatus (96%), S. intermedius (94%), and S. anginosus (60%) strains to be correctly identified. To increase specificity for S. anginosus, two 16S rRNA real-time PCR assays were developed targeting the 16S rRNA gene. The 16s_SA assay is specific for S. anginosus (100%), while the 16s_SCI assay is specific for S. constellatus and S. intermedius (100%). These assays can detect <10 genome equivalents in pure culture and >10(4) genome equivalents in sputum samples, making this a great tool for assessment of the presence of SMG in complex polymicrobial samples. Novel molecular methods were developed providing detection ability for SMG, an emerging opportunistic pathogen.

Phylogenetic analysis and species identification of members of the genus Streptococcus were carried out using partial sequence comparison of the 16S rRNA gene (1468-1478 bp), rpoB, encoding the beta subunit of RNA polymerase (659-680 bp), sodA, encoding the manganese-dependent superoxide dismutase (435-462 bp), groEL, encoding the 60 kDa heat-shock protein (757 bp), and gyrB, encoding the Beta subunit of DNA gyrase (458-461 bp). For the first time, most species within the genus Streptococcus were represented in the study (65 strains, representing 58 species and nine subspecies). Phylogenies inferred from rpoB, sodA, gyrB and groEL sequence comparisons were more discriminative than those inferred from 16S rRNA gene sequence comparison, and showed common clusters. The minimal interspecies divergence was 0.3, 2.7, 0, 2.5 and 3.4 % for the 16S rRNA gene, rpoB, sodA, gyrB and groEL, respectively. In general, groEL partial gene sequence comparison represented the best tool for identifying species and subspecies and for phylogenetic analysis.

AtlA is a major cell-lytic enzyme called autolysin in Streptococcus mutans. In this study, we identified the atlg gene-encoding autolysin (Atlg), consisting of 863 residues from Streptococcus sobrinus 6715DP, and confirmed lytic activity of recombinant Atlg by zymography of S. sobrinus cells. An atlA-inactivated mutant was constructed in S. mutans Xc, and the atlg gene product was characterized by plasmid complementation. Microscopic analysis, saliva-induced aggregation assay and autolysis assay of static cultures in air revealed that the atlg gene product partially complemented the role of AtlA. Furthermore, the capability of biofilm formation of the atlA-deficient mutant cultivated in air was restored by plasmid comprising the atlg gene. These findings suggest that Atlg may be involved in cell separation and biofilm formation in S. sobrinus.

The complete nucleotide sequence of the gene for a cell surface protein antigen (SpaA) of Streptococcus sobrinus MT3791 (serotype g) was determined. The spaA gene consisted of 4,698 bp and coded for a protein of 170,202 Da. A putative signal peptide was found in the amino-terminal end of the protein. A potential promoter sequence and a putative Shine-Dalgarno sequence preceded the open reading frame. Two internal repeating amino acid sequences were present in SpaA. One repeating region, located in the amino-terminal region, was rich in alanine, and the other, located in the central region, was rich in proline. The molecular structure of SpaA was very similar to that of the surface protein antigen of Streptococcus mutans.

An active-site peptide containing an aspartic acid implicated in catalysis has been isolated and sequenced from two Streptococcus sobrinus extracellular glucosyltransferases: sucrose:1,3-alpha-D-glucan 3-alpha-D-glucosyltransferase (GTase-I) and sucrose:1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase (GTase-S). The sequenced peptides, tagged with radiolabeled glucose, were isolated from a pepsin digest of a stabilized glucosylenzyme complex prepared by rapidly denaturing a reaction of enzyme and radiolabeled sucrose. The glucosyl linkage had previously been characterized as a beta-anomer bound to an active-site carboxyl group. Purified GTase-I and GTase-S glucosyl-peptides had the following similar but not identical sequences: GTase-I, Asp-Ser-Ile-Arg-Val-Asp-Ala-Val-Asp; and GTase-S, Asp-Gly-Val-Arg-Val-Asp-Ala-Val-Asp. Each has 3 aspartic acids as potential sites of glucose conjugation, but the relevant residue was not identified in sequence analysis because the highly base-labile glucosyl bond was cleaved in the first sequence cycle. As an alternative, the GTase-I glucosyl-peptide was partially digested at the N terminus with cathepsin C and at the C terminus with carboxypeptidase P. Analysis of the truncated products by fast atom bombardment mass spectrometry localized the glucosyl group to Asp-6 i the GTase-I peptide. In the native enzyme, this sequence is found near the N terminus, well-removed from the glucan-binding site located on a 60-kDa domain at the C terminus. The catalysis-dependent method of incorporating a glucosyl label implicates the aspartic acid as the residue involved in stabilizing an oxocarbonium ion transition state. The peptide segment is highly conserved and homologous to a peptide from sucrase-isomaltase labeled by site-directed irreversible inhibition and peptide segments common to a broad array of alpha-glucosidases and related transferases.

Streptococcus sobrinus exhibits marked dextran-dependent aggregation mediated by glucan-binding proteins (GBPs). In contrast to Streptococcus mutans, in which the gbpC gene responsible for dextran-dependent aggregation of this organism has been characterized, genes encoding the S. sobrinus GBPs have not yet been identified. Recently, we identified the gbpC gene homologue from Streptococcus macacae using polymerase chain reaction primers based on the conserved regions of the gbpC sequence exhibiting intraspecies variations. This method was applied to amplify a S. sobrinus homologue. Unexpectedly, two gbpC gene homologues were identified in S. sobrinus strain 100-4. One homologue, named gbpC, was more similar to the S. mutans gbpC gene than the other and was approximately half the molecular size of its homologue with similar regions interrupted by several non-similar stretches. However, the dextran-binding activity of the protein expressed from gbpC in Escherichia coli was not detected in contrast to the other homologue, a protein designated as Dbl, expressing this activity. The gbpC gene was shown to be intact on the chromosome of strain OMZ176, which does not exhibit dextran-dependent aggregation, while the dbl gene of this strain contained a single adenine nucleotide insertion at approximately one-third the distance from the 5'-end. The insertion mutation in the dbl gene resulted in translation of a premature protein missing its LPXTG sequence signature sequence of the wall-anchored proteins. These results suggest that the dbl gene is very likely responsible for S. sobrinus dextran-dependent aggregation.

The structural gene (pag gene) for a 210 kDa protein antigen of Streptococcus sobrinus serotype g was cloned and compared with that (pac gene) of a 190 kDa protein antigen of Streptococcus mutans serotype c. Immunodiffusion analysis revealed that the product of the pag gene immunologically cross-reacted with that of the pac gene. Southern blot and nucleotide sequence analyses revealed that a significant homology existed between the middle regions of the two structural genes.

Mutans streptococci have been shown to give rise to variants in terms of expression of surface protein antigens by repeated subculturing of the organisms, which in turn induces changes in colonial morphologies. A 2,850-bp upstream region of the gene (pag) for a surface protein antigen, PAg, of Streptococcus sobrinus MT3791 was determined. Analysis of the nucleotide sequence revealed the existence of three open reading frames (ORFs) located upstream of the pag gene. ORF1 extended from an undetermined further upstream sequence to the termination codon TAG lying 1,943 bp upstream of the pag gene. ORF2, consisting of 609 bp lying 1,689 bp upstream of the pag gene, encoded a protein of 23,347 Da and a protein of 22,792 Da. The synthesis of these proteins (protein antigen regulators) was demonstrated by using the in vitro T7 RNA polymerase/promoter system. ORF3, extending from 314 bp upstream of the pag gene to 712 bp upstream of the pag gene, encoded a protein of 14,802 Da. Disruption of chromosomal ORF2 of parent strain MT3791 by allelic exchange resulted in isogenic mutants, termed PAREm-1 and PAREm-2, that synthesized larger amounts of cell-free and cell-associated PAg than did the parent strain. RNA dot blot analysis demonstrated that expression of PAg-specific mRNA transcripts by mutants PAREm-1 and PAREm-2 was about 32-fold higher than that by strain 3791. Mutants PAREm-1 and PAREm-2 were found to be more hydrophobic than strain MT3791. Resting cells of these mutants attached in larger numbers to saliva-coated hydroxyapatite than did those of the parent strain. These results suggest that protein antigen regulator regulates the expression of PAg gene in a negative fashion, affecting the colonization of tooth surfaces by the organism. Thus, ORF2 is concluded to be a negative regulator gene of PAg synthesis and was designated par.

Immunoblotting sera from cases of Streptococcus mutans or S. sobrinus endocarditis against an extract from S. sobrinus strain MUCOB 263 had identified three immunodominant antigenic bands at 190, 200 and 220 kDa. A lambda ZAPII DNA library was produced from the sheared genomic DNA of S. sobrinus MUCOB 263 and six identical positive clones were identified when this library was screened with serum from a patient with endocarditis caused by a bacterium from the mutans group of streptococci. On subcloning and sequencing, a protein containing 1548 amino acids was identified with a 99.2% homology to the SpaA antigen of S. sobrinus and 68.4% homology to the PAc antigen of S. mutans.

Some dextranase-deficient (Dex-) mutants of Streptococcus sobrinus UAB66 (serotype g) synthesize a substance which inhibits dextranase activity (S.-Y. Wanda, A. Camilli, H. M. Murchison, and R. Curtiss III, J. Bacteriol. 176:7206-7212, 1994). This substance produced by the Dex- mutant UAB108 was designated dextranase inhibitor (Dei) and identified as a protein. The Dei gene (dei) from UAB108 has been cloned into pACYC184 to yield pYA2651, which was then used to generate several subclones (pYA2653 to pYA2657). The DNA sequence of dei was determined by using Tn5seq1 transposon mutagenesis of pYA2653. The open reading frame of dei is 990 bp long. It encodes a signal peptide of 38 amino acids and a mature Dei protein of 292 amino acids with a molecular weight of 31,372. The deduced amino acid sequence of Dei shows various degrees of similarity with glucosyltransferases and glucan-binding protein and contains A and C repeating units probably involved in glucan binding. Southern hybridization results showed that the dei probe from UAB108 hybridized to the same-size fragment in S. sobrinus (serotype d and g) DNA, to a different-size fragment in S. downei (serotype h) and S. cricetus (serotype a), and not at all to DNAs from other mutans group of streptococci.

We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodA(int)) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (S. acidominimus, S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. constellatus, S. canis, S. cricetus, S. downei, S. dysgalactiae, S. equi subsp. equi, S. equi subsp. zooepidemicus, S. equinus, S. gordonii, S. iniae, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis, S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis, S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). Phylogenetic analysis of these sodA(int) fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of their sodA(int) fragments on the phylogenetic tree of the sodA(int) fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.