Identification of amino acid residues in Streptococcus mutans glucosyltransferases influencing the structure of the glucan product.

The glucosyltransferases (GTFs) of mutans streptococci are important virulence factors in the sucrose-dependent colonization of tooth surfaces by these organisms. To investigate the structure-function relationship of the GTFs, an approach was initiated to identify amino acid residues of the GTFs which affect the incorporation of glucose residues into the glucan polymer. Conserved amino acid residues were identified in the GTF-S and GTF-I enzymes of the mutans streptococci and were selected for site-directed mutagenesis in the corresponding enzymes from Streptococcus mutans GS5. Conversion of six amino acid residues of the GTF-I enzyme to those present at the corresponding positions in GTF-S, either singly or in multiple combinations, resulted in enzymes synthesizing increased levels of soluble glucans. The enzyme containing six alterations synthesized 73% water-soluble glucan in the absence of acceptor dextran T10, while parental enzyme GTF-I synthesized no such glucan product. Conversely, when residue 589 of the GTF-S enzyme was converted from Thr to either Asp or Glu, the resulting enzyme synthesized primarily water-insoluble glucan in the absence of the acceptor. Therefore, this approach has identified several amino acid positions which influence the nature of the glucan product synthesized by GTFs.     

Mechanism of Streptococcus mutans glucosyltransferases: hybrid-enzyme analysis.

Streptococcus mutans GS5 expresses three glucosyltransferases (GTFs): GTF-I and GTF-SI, which synthesize water-insoluble glucans in a primer-independent manner, and GTF-S, which is responsible for the formation of primer-dependent soluble glucan. The amino acid sequences of the GTF-I and GTF-S enzymes exhibit approximately 50% sequence identity. Various hybrid genes were constructed from the structural genes for the enzymes, and their products were analyzed. Three different approaches were used to construct the hybrid enzymes: (i) ligation of DNA fragments containing compatible endonuclease restriction sites of the two genes at homologous positions; (ii) in vivo recombination between the homologous regions of each gene; and (iii) random fusion of DNA fragments from each gene generated following exonuclease III digestion of tandemly arranged fragments corresponding to the two functional domains of each enzyme. Hybrid GTFs composed of the sucrose-binding domain of one enzyme (GTF-I or GTF-S) with the glucan-binding domain of the other synthesized insoluble glucan exclusively in the absence of primer dextran. Insoluble glucan synthesis by some, but not all, of the GTF-S:GTF-I chimeric enzymes was stimulated by primer dextran T10 addition. In addition, glucan binding by the former but not latter group of hybrid GTFs was demonstrated. These results suggest that the glucan-binding domain alone does not solely determine primer dependence or independence or the structure of the resulting glucan product, although this carboxyl-terminal domain containing direct repeating units does appear to play a significant role in primer dependence.     

Utilization of a mini-mu transposon to construct defined mutants in Streptococcus mutans

The gtfB gene coding for glucosyltransferase-I (GTF-I) activity previously isolated from Streptococcus mutans GS-5 was insertionally inactivated with the newly constructed transposon MudE in an Escherichia coli background. Insertion of MudE into various regions of the gtfB gene led to inactivation of GTF-I activity. The altered gene was introduced back into S. mutans GS-5 by transformation and produced mutants defective in insoluble glucan synthesis as well as the ability to colonize smooth surfaces in the presence of sucrose. Therefore, the MudE transposon can be utilized to produce specific mutants in oral streptococci as well as in other transformable Gram-positive bacteria expressing an erythromycin-resistance marker.     

Molecular basis for the spontaneous generation of colonization-defective mutants of Streptococcus mutans

Spontaneous mutants of Streptococcus mutans GS-5 defective in sucrose-dependent colonization of smooth surfaces are generated at frequencies above the spontaneous mutation rate. Southern blot analysis of such mutants suggested rearrangement of the genes coding for glucosyltransferase (GTF) activity. Two strain GS-5 homologous tandem genes, gtfB and gtfC, coding for GTF-I and GTF-S activities respectively, were demonstrated to undergo recombination when introduced into recombination-proficient Escherichia coli transformants. However, the two genes were quite stable when transformed on a single DNA fragment into a recA mutant of E. coli. The DNA fragment coding for GTF activity from one S. mutans colonization-defective mutant, SP2, was isolated and shown also to have undergone recombination between the gtfB and gtfC genes, resulting in reduced GTF activity. These results are discussed relative to the in vivo generation of colonization-defective mutants in cultures of S. mutans.     

Peptide sequences for sucrose splitting and glucan binding within Streptococcus sobrinus glucosyltransferase (water-insoluble glucan synthetase).

The gene encoding glucosyltransferase responsible for water-insoluble glucan synthesis (GTF-I) of Streptococcus sobrinus (formerly Streptococcus mutans 6715) was cloned, expressed, and sequenced. A gene bank from S. sobrinus 6715 DNA was constructed in vector pUC18 and screened with anti-GTF-I antibody to detect clones producing GTF-I peptide. Five immunopositive clones were isolated, all of which produced peptides that bound alpha-1,6 glucan. GTF-I activity was found in only two large peptides: one stretching over the full length of the GTF-I peptide and composed of about 1,600 amino acid residues (AB1 clone) and the other lacking about 80 N-terminal residues and about 260 C-terminal residues (AB2 clone). A deletion study of the AB2 clone indicated that specific glucan binding, which is essential for water-insoluble glucan synthesis, was lost prior to sucrase activity with an increase in deletion from the 3' end of the GTF-I gene. These results suggest that the GTF-I peptide consists of three segments: that for sucrose splitting (approximately 1,100 residues), that for glucan binding (approximately 240 residues), and that of unknown function (approximately 260 residues), in order from the N terminus. The primary structure of the GTF-I peptide, deduced by DNA sequencing of the AB1 clone, was found to be very similar to that of the homologous protein from another strain of S. sobrinus.     

Immunological relationships between glucosyltransferases synthesizing insoluble glucan from Streptococcus cricetus, Streptococcus sobrinus and Streptococcus downei.

The Mr values and isoelectric points of glucosyltransferases synthesizing insoluble glucan (GTF-Is) were determined, and the immunological relationships between them studied. The GTF-I enzymes were from Streptococcus cricetus (mutans group serotype a), Streptococcus sobrinus (mutans group serotypes d and g) and Streptococcus downei (mutans group serotype h). By double immunodiffusion tests, the GTF-I enzymes from the three species possessed a common antigenic determinant; in addition, the GTF-I enzymes of serotypes d, g and h shared a further determinant. The S. sobrinus serotypes d and g GTF-I enzymes were immunologically identical. The GTF-I enzymes of S. sobrinus serotypes d and g, and of S. downei, had an Mr of 161,000 and isoelectric points of 4.8-4.9, while S. cricetus GTF-I had a lower Mr (150,000) and a higher isoelectric point (5.2). This suggests that the S. cricetus GTF-I enzyme may lack a sequence of amino acids which include the determinant shared by S. sobrinus and S. downei GTF-I enzymes. Antibodies specific to the determinant shared by all four serotypes inhibited the homologous and heterologous enzymes by 94-100%.     

Molecular characterization of a cluster of at least two glucosyltransferase genes in Streptococcus salivarius ATCC 25975.

The oral micro-organism Streptococcus salivarius ATCC 25975 synthesizes extracellular glucosyltransferases (GTFs) which polymerize the glucose moiety of sucrose into glucan polymers. Two separate genes encoding the activities of a GTF-I (a GTF that synthesizes an insoluble product) and a GTF-S (a GTF that synthesizes soluble product) were cloned into bacteriophage lambda L47.1. The inserts in the lambda-clones were characterized by restriction mapping and Southern hybridization and were found to overlap, implying that the two genes lay very close to one another on the S. salivarius chromosome. Both genes were subcloned into phagemid vector pIBI30 where they were expressed at a high level. The GTF-I-encoding gene was named gtfJ and the GTF-S-encoding gene, gtfK. Nucleotide sequencing showed that gtfJ and most probably gtfK were closely related to the gtf genes of the mutans streptococci. Sequence alignment also indicated that gtfK lay very close to and downstream from gtfJ, and that both were transcribed in the same direction.     

Cloning and nucleotide sequence analysis of the Streptococcus sobrinus gtfU gene that produces a highly branched water-soluble glucan

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.     

Identification of essential amino acids in the Streptococcus mutans glucosyltransferases.

A comparison of the amino acid sequences of the glucosyltransferases (GTFs) of mutans streptococci with those from the alpha-amylase family of enzymes revealed a number of conserved amino acid positions which have been implicated as essential in catalysis. Utilizing a site-directed mutagenesis approach with the GTF-I enzyme of Streptococcus mutans GS-5, we identified three of these conserved amino acid positions, Asp413, Trp491, and His561, as being important in enzymatic activity. Mutagenesis of Asp413 to Thr resulted in a GTF which expressed only about 12% of the wild-type activity. In contrast, mutagenesis of Asp411 did not inhibit enzyme activity. In addition, the D413T mutant was less stable than was the parental enzyme when expressed in Escherichia coli. Moreover, conversion of Trp491 or His561 to either Gly or Ala resulted in enzymes devoid of GTF activity, indicating the essential nature of these two amino acids for activity. Furthermore, mutagenesis of the four Tyr residues present at positions 169 to 172 which are part of a subdomain with homology to the direct repeating sequences present in the glucan-binding domain of the GTFs had little overall effect on enzymatic activity, although the glucan products appeared to be less adhesive. These results are discussed relative to the mechanisms of catalysis proposed for the GTFs and related enzymes.     

Characterization of glucosyltransferase expressed from a Streptococcus sobrinus gene cloned in Escherichia coli

The gene encoding a glucosyltransferase which synthesized water-insoluble glucan, gtfI, previously cloned from Streptococcus sobrinus strain MFe28 (mutans serotype h) into a bacteriophage lambda vector, was subcloned into the plasmid pBR322. The recombinant plasmid was stable in Escherichia coli and gtfI was efficiently expressed. The GTF-I expressed in E. coli was compared to the corresponding enzymes in S. sobrinus strains MFe28 (serotype h), B13 (serotype d) and 6715 (serotype g) and shown to resemble them closely in molecular mass and isoelectric point. The insoluble glucan produced by GTF-I from recombinant E. coli consisted of 1,3-alpha-D-glycosyl residues (approximately 90%). An internal fragment of the gtfI gene was used as a probe in hybridization experiments to demonstrate the presence of homologous sequences in chromosomal DNA of other streptococci of the mutans group.     

Sequence and phylogenetic analyses of novel glucosyltransferase genes of mutans streptococci isolated from pig oral cavity

Nucleotide sequences of water-insoluble glucan-producing glucosyltransferase (gtf) genes of new mutans streptococci isolated from pig oral cavity, Streptococcus orisuis JCM14035, and of Streptococcus criceti HS-6 were determined. The gtf gene of S. orisuis JCM14035 consisted of a 4,401 bp ORF encoding for a 1,466 amino acids, and was revealed to belong to the gtfI group. The percent homology of amino acid sequence of the GTF-I from S. orisuis and S. criceti are 95.0%, however, this score ranges from 77.0% to 78.0% when compared to Streptococcus sobrinus 6715. The deduced N-terminal amino acid sequence was considered responsible for the secretion of GTF-I in S. orisuis JCM14035 and S. criceti HS-6 with high similarity to known GTF proteins from other streptococci. In addition, two other conserved regions, i.e., N-terminal putative catalytic-site and C-terminal glucan binding domain, were also found in GTF-Is of S. orisuis JCM14035 and S. criceti HS-6. Phylogenetic analysis suggested that S. orisuis JCM14035 and S. criceti HS-6, closely related to each other, resemble S. sobrinus and S. downei based on the amino acid sequences of the GTFs.     

Inhibition of Streptococcus mutans 6715 glucosyltransferases by sucrose analogs modified at positions 6 and 6'

Sucrose derivatives modified at position 6 (6-deoxysucrose, 6-thiosucrose, 6,6'-dithiodisucrose, and 6,6'-dideoxy-6,6'-difluorosucrose) were tested as inhibitors of the two Streptococcus mutans 6715 glucosyltransferases. 6-Deoxysucrose was the best inhibitor studied, competitively inhibiting the soluble-D-glucan forming enzyme (GTF-S) and the insoluble-D-glucan forming enzyme (GTF-I) with Ki values one order of magnitude lower than the sucrose Km values. 6-Thiosucrose was also a competitive inhibitor for both enzymes. 6,6'-Dithiodisucrose and 6,6'-dideoxy-6,6'-difluorosucrose only inhibited GTF-I; 6,6'-dithiodisucrose gave mixed inhibition and 6,6'-dideoxy-6,6'-difluorosucrose gave uncompetitive inhibition. 6-Thiosucrose was a substrate for both enzymes to produce acceptor products when acceptors were present. GTF-I synthesized de novo a water-insoluble, (1----3)-6-thio-alpha-D-glucan from 6-thiosucrose.     

Synthesis of 4,6-dideoxysucrose, and inhibition studies of Leuconostoc and Streptococcus D-glucansucrases with deoxy and chloro derivatives of sucrose modified at carbon atoms 3, 4, and 6

Starting from sucrose, 2,3,1',3',4',6'-hexa-O-benzoyl-6-deoxy-6-iodosucrose (1) was synthesized. Reaction of 1 with sulfuryl chloride in pyridine gave 2,3,1',3',4',6'-hexa-O-benzoyl-4-chloro-4,6-dideoxy-6-iodogalactosucr ose (2). Compound 2 was treated with tributyltin hydride in toluene in the presence of a radical initiator, alpha, alpha-azobis(isobutanonitrile) (AIBN), to remove iodine and chlorine groups and give hexa-O-benzoyl-4,6-dideoxysucrose. Benzoyl groups were removed by sodium methoxide in methanol to give 4,6-dideoxysucrose. Sucrose was modified at carbon atom 3, carbon atom 4, or carbon atoms 4 and 6, and these analogs were tested as inhibitors of the D-glucansucrases (D-glucosyltransferases) of Streptococcus mutans 6715 and Leuconostoc mesenteroides B-512F. Sucrose analogs used in this study are 4-deoxysucrose and 4-chloro-4-deoxygalactosucrose with S. mutans 6715 D-glucansucrases (GTF-S and GTF-I), and 3-deoxysucrose, 4-deoxysucrose, 4-chloro-4-deoxygalactosucrose, 6-deoxysucrose, and 4,6-dideoxysucrose with L. mesenteroides B-512F D-glucansucrase. The data indicate that 3-deoxysucrose, 4-deoxysucrose, and 4-chloro-4-deoxygalactosucrose are weak noncompetitive inhibitors for B-512F dextransucrase, with Ki values of 530, 201, and 202mM respectively. For the same enzyme, 6-deoxysucrose was a strong competitive inhibitor, with Ki of 1.60mM, and 4,6-dideoxysucrose was a good competitive inhibitor, with Ki of 20.3mM. 4-Deoxysucrose was a weak noncompetitive inhibitor for both GTF-I and GTF-S, with Ki values of 672 and 608mM, respectively. 4-Chloro-4-deoxygalactosucrose was also a weak noncompetitive inhibitor for GTF-I and GTF-S with Ki values of 391 and 308mM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Inhibition by maltose, isomaltose, and nigerose of the synthesis of high-molecular-weight D-glucans by the D-glucosyltransferases of Streptococcus sobrinus

Two D-glucosyltransferases are produced by Streptococcus sobrinus C211. One (GTF-S) catalyzes the conversion of sucrose into soluble alpha-(1----6)-linked alpha-(1----3)-branched D-glucans, and the other (GTF-I), of sucrose into alpha-(1----3)-linked alpha-(1----6)-branched D-glucans. These enzymes were studied by using maltose, isomaltose, and nigerose as inhibitors. Maltose and isomaltose were found to be competitive inhibitors of GTF-S, whereas nigerose has no effect on GTF-S activity. The Ki values for maltose and isomaltose were determined to be 11 and 15mM, respectively. Maltose, isomaltose, and nigerose competitively inhibit GTF-I. The Ki values for these inhibitors were found to be approximately 0.8, 2.5, and 15mM, respectively. The inhibitory properties of each disaccharide are interpreted in terms of conformational comparisons with sucrose.     

Production of glucooligosaccharides by an acceptor reaction using two types of glucansucrase from Streptococcus sobrinus

Glucooligosaccharides (GOS) were produced by using an acceptor reaction with two types of glucansucrase (GTF-S and GTF-I) from Streptococcus sobrinus. Acceptor reactions of GTF-S with maltose acceptor, gave a great number of GOS ranging from DP(degree of polymerization) 2 to DP15. At the both acceptor reactions with GTF-S or GTF-I, as the sucrose/maltose ratio was decreased, the amount of dextran and DP of oligosaccharides was decreased. A maximum GOS yield of 69% was achieved at the acceptor reaction with GTF-I and when the molar ratio of sucrose/maltose is 2:1, in which GOS of DP6~DP9 were major oligosaccharides and 17% of dextran. The polymeric size of GOS could be controlled by varying the ratio of sucrose to the acceptor (maltose in this work).     

Role of C-terminal direct repeating units of the Streptococcus mutans glucosyltransferase-S in glucan binding.

The C-terminal glucan-binding domain of the glucosyltransferase-S of Streptococcus mutans GS-5 contains five 65-amino-acid direct repeating units. A series of deletion derivatives of both the glucosyltransferase-S and its glucan-binding domain were constructed and analyzed. The results demonstrated that the four C-terminal direct repeating units constituted part of the minimum domain required for glucan binding.     

Molecular cloning and nucleotide sequencing of the Arthrobacter dextranase gene and its expression in Escherichia coli and Streptococcus sanguis

A bacterial strain, which assimilated dextran and water-insoluble glucan produced by Streptococcus mutans, was isolated from soil. The bacterium produced and secreted potent dextranase activity, which was identified as Arthrobacter sp. and named CB-8. The dextranase was purified and some enzymatic properties were characterized. The enzyme efficiently decomposed the water-insoluble glucan as well as dextran. A gene library from the bacteria was constructed with Escherichia coli, using plasmid pUC19, and clones producing dextranase activity were selected. Based on the result of nucleotide sequencing analysis, it was deduced that the dextranase was synthesized in CB-8 cells as a polypeptide precursor consisting of 640 amino acid residues, including 49 N-terminal amino acid residues which could be regarded as a signal peptide. In the E. coli transformant, the dextranase activity was detected mostly in the periplasmic space. The gene for the dextranase was introduced into Streptococcus sanguis, using an E. coli-S. sanguis shuttle vector that contained the promoter sequence of a gene for glucosyltransferase derived from a strain of S. mutans. The active dextranase was also expressed and accumulated in S. sanguis cells.     

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.