Two new loci affecting cell division identified as suppressors of an ftsQ-null mutation in Streptomyces coelicolor A3(2)

We have cloned and sequenced the gene encoding the surface protein antigen PAa (antigen I/II family) from Streptococcus cricetus E49 (serotype a) using degenerate PCR. The deduced amino acid sequence of PAa reveals two repeating regions (A region; alanine-rich region, P region; proline-rich region). Two additional tandem repeats were found in the A region and part of the P region was deleted compared to antigen I/II. Homology and phylogenetic analyses reveal that PAa is homologous to Streptococcus sobrinus PAg rather than Streptococcus mutans PAc. Using degenerate PCR a gene homologous to PAc was identified in Streptococcus intermedius, but not found in Streptococcus rattus or Streptococcus anginosus.     

Immunogenicity and protective effect against oral colonization by Streptococcus mutans of synthetic peptides of a streptococcal surface protein antigen

Streptococcus mutans is known to be a major causative organism of human dental caries. A surface protein Ag with a molecular mass of 190 kDa of S. mutans (PAc) is receiving attention as an anticaries vaccine. We have recently determined the complete nucleotide sequence of the gene for PAc. In this study, four peptides were synthesized on the basis of amino acid sequence of PAc. Among these peptides, PAc(301-319) corresponding to the alanine-rich repeating amino acid region was the most strongly bound by polyclonal murine anti-rPAc antibodies. The peptide partially inhibited the binding of polyclonal anti-rPAc antibodies to rPAc. The peptide induced the proliferation of T cells from BALB/c mice immunized with rPAc. Subcutaneous immunization with PAc(301-319) or rPAc emulsified in CFA/IFA induced high serum IgG responses to rPAc and PAc(301-319). In addition, serum IgG responses to a surface protein Ag with a molecular mass of 210 kDa of Streptococcus sobrinus were elicited in mice immunized by s.c. injection with PAc(301-319) or rPAc. Intranasal immunization with PAc(301-319) coupled to cholera toxin B subunit (CTB) or with rPAc and free CTB induced high serum IgG responses to rPAc. The immunization with PAc(301-319) coupled to CTB or rPAc and free CTB suppressed the colonization of murine teeth by S. mutans. These results suggest that intranasal immunization with the peptide or rPAc may be effective for the prevention of dental caries.     

Complete nucleotide sequence of the sr gene from Streptococcus mutans OMZ 175

The nucleotide sequence encoding the SR protein of Streptococcus mutans OMZ 175 (serotype f) has been determined. The sr gene consists of 4667 bp and codes for a 171177 Da protein. Comparison of the inferred amino acid sequence with the one of PAc antigen from S. mutans MT 8148 (serotype c) indicates a 88% conservation of amino acid residues which reflects the close relatedness of both proteins. Major differences in amino acid composition are located at the C-terminal part of the sequence where only 298 amino acids of the terminal 420 are conserved.     

Sequence analysis of the gtfB gene from Streptococcus mutans.

The nucleotide sequence of the gtfB gene from Streptococcus mutans GS-5, coding for glucosyltransferase I activity, was determined. The gene codes for a strongly hydrophilic protein with a molecular size of 165,800 daltons. The deduced amino acid sequence revealed a typical gram-positive bacterial signal sequence at the NH2 terminus of the protein and 3.5 direct repeating units (each containing 65 amino acids) at the COOH terminus. Nucleotide sequencing of the region immediately downstream from the gtfB gene revealed the presence of a putative gene coding for an extracellular protein. This open reading frame is partially homologous to the gtfB gene.     

Contribution of cell surface protein antigen PAc of Streptococcus mutans to bacteremia

Streptococcus mutans, a major cariogenic bacterium, is occasionally isolated from the blood of patients with bacteremia and infective endocarditis. Mutant strains of S. mutans MT8148, defective in the major surface proteins glucosyltransferase (GTF) B-, C-, and D-, and protein antigen c (PAc), were constructed by insertional inactivation of each respective gene with an antibiotic resistant cassette. Susceptibility to phagocytosis was determined by analyses of interactions of the bacteria with human polymorphonuclear leukocytes, and the PAc-defective mutant strain (PD) showed the lowest rate of phagocytosis. Further, when PD and MT8148 were separately injected into the jugular veins of Sprague-Dawley rats, PD was recovered in significantly larger numbers and for a longer duration, and caused more severe systemic inflammation than MT8148, indicating that S. mutans PAc is associated with its systemic virulence in blood. Next, 100 S. mutans clinical isolates from 100 Japanese children and adolescents were analyzed by Western blotting using antisera raised against recombinant PAc, generated based on the pac sequence of MT8148. Four of the 100 strains showed no positive band and each exhibited a significantly lower phagocytosis rate than that of 25 randomly selected clinical strains (P < 0.01). In addition, three of the 100 strains possessed a lower molecular weight PAc and a significantly lower rate of phagocytosis than the 25 reference strains (P < 0.05). These results suggest that S. mutans PAc may be associated with phagocytosis susceptibility to human polymorphonuclear leukocytes, with approximately 7% of S. mutans clinical isolates possible high-risk strains for the development of bacteremia.     

Homology between surface protein antigen genes of Streptococcus sobrinus and Streptococcus mutans

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.     

Application of in vitro mutagenesis to identify the gene responsible for cold agglutination phenotype of Streptococcus mutans

A previously unidentified protein with an apparent molecular mass of 120 kDa was detected in some Streptococcus mutans strains including the natural isolate strain Z1. This protein was likely involved in the cold-agglutination of the strain, since a correlation between this phenotype and expression of the 120 kDa protein was found. We have applied random mutagenesis by in vitro transposition with the Himar1 minitransposon and isolated three cold-agglutination-negative mutants of this strain from approximately 2,000 mutants screened. A 2.5 kb chromosomal fragment flanking the minitransposon in one of the three mutants was amplified by PCR-based chromosome walking and the minitransposon insertion in the other two mutants occurred also within the same region. Nucleotide sequencing of the region revealed a 1617 nt open reading frame specifying a putative protein of 538 amino acid residues with a calculated molecular weight of 57,192. The deduced eight amino acid sequence following a putative signal sequence completely coincided with the N-terminal octapeptide sequence of the 120 kDa protein determined by the Edman degradation. Therefore, the 1617 nt gene unexpectedly encoded the 120 kDa protein from S. mutans. Interestingly, this gene encoded a collagen adhesin homologue. In vitro mutagenesis using the Himar1 minitransposon was successfully applied to S. mutans.     

A family of clostridial and streptococcal ligand-binding proteins with conserved C-terminal repeat sequences

Analysis of the derived amino acid sequences of toxins A and B from Clostridium difficile has identified an extraordinarily large number of repeat amino acid units in the C-terminal regions of the proteins. Nearly one third of each of the proteins consist of repeating units which appear, at least in the case of toxin A, to be responsible for carbohydrate binding. Similar repeat units are also found in the C-terminal region of four glucosyltransferases from Streptococcus mutans and Streptococcus downei, and in four lytic enzymes from Streptococcus pneumoniae and its bacteriophages (HB-3, Cp-1 and Cp-9). In each case the repeats constitute the ligand-binding portion of the respective enzymes. A glucan-binding protein from S. mutans, which lacks enzymatic activity, has similar repeats spanning almost the entire molecule. This family of ligand-binding proteins appears to be of modular design, with one module consisting of a repetitive ligand-binding domain located in the C-terminal region and the other module(s) providing enzymatic functions.     

Sequence analysis of the gtfC gene from Streptococcus mutans GS-5

The nucleotide sequence of the gtfC gene, which codes for glucosyltransferase synthesizing both water-soluble and water-insoluble glucans, and its flanking regions from Streptococcus mutans GS-5, was determined. Although the gtfC gene (4218 bp) is preceded by a Shine-Dalgarno (SD) sequence, a promoter-like sequence for this gene could not be identified. The gtfC gene product composed of 1375 amino acid residues (approx. 153 kDa) is generally hydrophilic with three small hydrophobic domains. Two direct repeating units were found near the C terminus of the peptide. The gtfC gene has extensive homology with the previously sequenced gtfB gene. The homologous regions correspond to the signal sequence, an internal region, and the direct repeating units of the peptide. An open reading frame preceded by an SD sequence and followed by an inverted repeat sequence was found immediately downstream from the gtfC gene. The combined sequences of the gtfB and gtfC genes as well as flanking regions suggest that the two gtf genes and the small downstream coding region could be coordinately expressed within an operon. The possible evolution of the gtfC gene in S. mutans GS-5 is also discussed.     

Nucleotide sequence and analysis of the Vibrio alginolyticus sucrose uptake-encoding region

The nucleotide sequence of the Vibrio alginolyticus sucrose uptake-encoding region was determined, and contained two genes, scrA and scrK. The scrA gene encodes an enzyme IISucrose (EIIScr) protein of the phosphoenolpyruvate dependent phosphotransferase system and the scrK gene encodes a fructokinase. The deduced amino acid (aa) sequence for the V. alginolyticus EIIScr protein was homologous with the EIIScr proteins from Streptococcus mutans, Salmonella typhimurium (pUR400 system) and Bacillus subtilis. The deduced aa sequence for the V. alginolyticus fructokinase was homologous with the Escherichia coli enzymes, 6-phosphofructokinase (isoenzyme 2) and ribokinase. Transposon phoA mutagenesis experiments indicated that the EIIScr protein was a membrane-bound protein with a region that extended into the periplasm.     

Epitope mapping of Streptococcus mutans SR protein and human IgG cross-reactive determinants, by using recombinant proteins and synthetic peptides.

alpha-Hemolytic oral streptococci are known to possess a family of cell surface cross-reactive proteins termed Ag I/II, having a molecular mass of approximately 180 to 210 kDa. These proteins are implicated in bacterial adherence to various oral tissues, and we showed recently that the SR protein, an I/II Ag-related protein, from Streptococcus mutans OMZ 175 serogroup f possesses Ag mimicry with human IgG. In this study, regions of the SR protein encoding the cross-reactive epitope(s) were analyzed by expressing selected restriction fragments from the cloned sr gene. The three SR-derived polypeptides reacted in ELISA with anti-SR rabbit IgG, whereas only the two polypeptides located along the carboxyl-terminal two thirds of the SR protein reacted with anti-human IgG rabbit IgG. In order to locate more precisely the human IgG-cross-reactive region, we synthesized six peptides, on the basis of the recently determined complete nucleotide sequence of the sr gene. Among these peptides, peptide 2, corresponding to the alanine-rich repeating amino-terminal region, peptide 3, located in the three tandem proline-rich regions, and peptide 6, located near the cell wall-spanning region, were the most interesting in term of antigenicity and immunogenicity. Anti-peptide 2, 3, and 6 rabbit IgG reacted with free SR and with cell wall-associated SR. Peptide 1, located near the amino terminus, was poorly immunogenic. Peptides 4 and 5, located in the putative human IgG-cross-reactive region, were immunogenic; however, anti-peptide 4 rabbit IgG reacted only weakly with SR or human IgG, whereas anti-peptide 5 rabbit IgG reacted strongly with SR and human IgG, and peptide 5 was recognized by anti-SR and anti-human IgG rabbit IgG. These results confirm the cell surface accessibility of this epitope and its potential participation in eliciting, in rabbits, anti-SR IgG cross-reactive with human IgG.     

Molecular characterization of dextranase from Streptococcus rattus

The complete nucleotide sequence of the dextranase gene of Streptococcus rattus ATCC19645 was determined. An open reading frame of the dextranase gene was 2,760 bp long and encoded a dextranase protein consisting of 920 amino acids with a molecular weight of 100,163 Da and an isoelectric point of 4.67. The S. rattus dextranase purified from recombinant Escherichia coli cells showed dextran-hydrolyzing activity with optimal pH (5.0) and temperature (40 C) similar to those of dextranases from Streptococcus mutans and Streptococcus sobrinus. The deduced amino acid sequence of the S. rattus dextranase revealed that the dextranase molecule consists of two variable regions and a conserved region. The variable regions contained an N-terminal signal peptide and a C-terminal cell wall sorting signal; the conserved region contained two functional domains, catalytic and dextran-binding sites. This structural feature of the S. rattus dextranase is quite similar to that of other cariogenic species such as S. mutans, S. sobrinus, and Streptococcus downei.     

Nucleotide sequence of the Streptococcus mutans gtfD gene encoding the glucosyltransferase-S enzyme

The nucleotide sequence of the Streptococcus mutans GS-5 gtfD gene coding for the glucosyltransferase which synthesizes water-soluble glucan (GTF-S) has been determined. The complete gene contains 4293 base pairs and the unprocessed protein is composed of 1430 amino acids with a molecular mass of 159814 Da. The amino terminus of the unprocessed protein resembles the signal sequences of other extracellular proteins secreted by S. mutans and that of the GTF-I secreted by Streptococcus downei. In addition, the GTF-S protein exhibits high amino acid similarity with the strain GS-5 enzymes responsible for insoluble glucan synthesis (GTF-I, GTF-SI) previously isolated and sequenced in this laboratory. These results indicate that all three gtf genes evolved from a common ancestral gene.     

Sequence analysis of the wall-associated protein precursor of Streptococcus mutans antigen A

The nucleotide sequence has been determined for the Streptococcus mutans wall-associated protein A (wapA) gene from serotype c strains Ingbritt and GS5. The nucleotide sequence for each wapA gene was virtually identical, although the gene from strain GS5 contained a 24 base pair deletion. A 29 amino acid signal peptide was specified by each wapA gene with a mature protein of 424 amino acids (Mr, 45,276) for strain Ingbritt and 416 amino acids (Mr, 44,846) for strain GS5. In the C-terminal region of the wall-associated protein A, considerable sequence similarity was found with the membrane anchor region of proteins from other Gram-positive organisms such as the group A streptococcal M protein and the group G streptococcal IgG binding protein. Adjacent to the proposed membrane anchor is a highly hydrophilic region which may span the cell wall; both sequence data and experimental evidence indicate the existence of a region immediately outside the wall at which proteolytic cleavage occurs to release antigen A of Mr 29,000 into the culture supernatant. Thus, the wall-associated protein A is a precursor of the 29,000 Mr antigen A.     

Sequence, expression, and function of the gene for the nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase of Streptococcus mutans.

We report the sequencing of a 2,019-bp region of the Streptococcus mutans NG5 genome which contains a 1,428-bp open reading frame (ORF) whose putative translation product had 50% identity to the amino acid sequences of the nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenases (GAPN) from maize and pea. This ORF is located approximately 200 bp downstream of the ptsI gene coding for enzyme I of the phosphoenolpyruvate:sugar phosphotransferase transport system. Mutant BCH150, in which the putative gapN gene had been inactivated, lacked GAPN activity that was present in the wild-type strain, thus positively identifying the ORF as the S. mutans gapN gene. Another strain of S. mutans, DC10, which contains an insertionally inactivated ptsI gene, still possessed GAPN activity, as did S. salivarius ATCC 25975, which contains an insertion element between the ptsI and gapN genes. Since the wild-type S. mutans NG5 lacks both glucose-6-phosphate dehydrogenase and NADH:NADP oxidoreductase activities, the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase is important as a means of generating NADPH for biosynthetic reactions.     

Sequence Analysis of the Streptococcus mutans Ingbritt dexA Gene Encoding Extracellular Dextranase

The complete nucleotide sequence (3,747 bp) of the dextranase gene (dexA) and flanking regions of the chromosome of Streptococcus mutans Ingbritt (serotype c) were determined. The open reading frame for dexA was 2,550 bp, ending with a stop codon TGA. A putative ribosome-binding site, promoter preceding the start codon, and potential stem-loop structure were identified. The presumed dextranase protein (DexA) consisting of 850 amino acids was estimated to have a molecular size of 94,536 Da and a pI of 4.79. The nucleotide sequence and the deduced amino acid sequences of S. mutans dexA exhibited homologies of 57.8% and 47.0%, respectively, to those of Streptococcus sobrinus dex. The homologous region of dex of S. sobrinus was in the N-terminal half. The C terminus of DexA consisted of a hexapeptide LPQTGD, followed by 7 charged amino acids, 21 amino acids with a strongly hydrophobic character, and a charged hexapeptide tail, which have been reported as a common structure of C termini of not only the surface-associated proteins of Gram-positive cocci but also the extracellular enzymes such as β-fructosidase of S. mutans and dextranase of S. sobrinus. The DexA protein had no significant homology with the glucosyltransferases, the glucan-binding protein, or the dextranase inhibitor of mutans streptococci.