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Purification, characterization, and molecular analysis of the gene encoding glucosyltransferase from Streptococcus oralis.

ABSTRACT: Streptococcus oralis is a member of the oral streptococcal family and an early-colonizing microorganism in the oral cavity of humans. S. oralis is known to produce glucosyltransferase (GTase), which synthesizes glucans from sucrose. The enzyme was purified chromatographically from a culture supernatant of S. oralis ATCC 10557. The purified enzyme, GTase-R, had a molecular mass of 173 kDa and a pI of 6.3. This enzyme mainly synthesized water-soluble glucans with no primer dependency. The addition of GTase markedly enhanced the sucrose-dependent resting cell adhesion of Streptococcus mutans at a level similar to that found in growing cells of S. mutans. The antibody against GTase-R inhibited the glucan-synthesizing activities of Streptococcus gordonii and Streptococcus sanguis, as well as S. oralis. The N-terminal amino acid sequence of GTase-R exhibited no similarities to known GTase sequences of oral streptococci. Using degenerate PCR primers, an 8.1-kb DNA fragment, carrying the gene (gtfR) coding for GTase-R and its regulator gene (rgg), was cloned and sequenced. Comparison of the deduced amino acid sequence revealed that the rgg genes of S. oralis and S. gordonii exhibited a close similarity. The gtfR gene was found to possess a species-specific nucleotide sequence corresponding to the N-terminal 130 amino acid residues. Insertion of erm or aphA into the rgg or gtfR gene resulted in decreased GTase activity by the organism and changed the colony morphology of these transformants. These results indicate that S. oralis GTase may play an important role in the subsequent colonizing of mutans streptoccoci.

SUBMITTER: Fujiwara T

PROVIDER: S-EPMC97449 | biostudies-literature | 2000 May

REPOSITORIES: biostudies-literature

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Purification, characterization, and molecular analysis of the gene encoding glucosyltransferase from Streptococcus oralis.

Fujiwara T T Hoshino T T Ooshima T T Sobue S S Hamada S S

Infection and immunity 20000501 5

Streptococcus oralis is a member of the oral streptococcal family and an early-colonizing microorganism in the oral cavity of humans. S. oralis is known to produce glucosyltransferase (GTase), which synthesizes glucans from sucrose. The enzyme was purified chromatographically from a culture supernatant of S. oralis ATCC 10557. The purified enzyme, GTase-R, had a molecular mass of 173 kDa and a pI of 6.3. This enzyme mainly synthesized water-soluble glucans with no primer dependency. The addition ...[more]

PMID: 10768934

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Project description:The oral streptococcal group (mitis phylogenetic group) currently consists of nine recognized species, although the group has been traditionally difficult to classify, with frequent changes in nomenclature over the years. The pneumococcus (Streptococcus pneumoniae), an important human pathogen, is traditionally distinguished from the most closely related oral streptococcal species Streptococcus mitis and Streptococcus oralis on the basis of three differentiating characteristics: optochin susceptibility, bile solubility, and agglutination with antipneumococcal polysaccharide capsule antibodies. However, there are many reports in the literature of pneumococci lacking one or more of these defining characteristics. Sometimes called "atypical" pneumococci, these isolates can be the source of considerable confusion in the clinical laboratory. Little is known to date about the genetic relationships of such organisms with classical S. pneumoniae isolates. Here we describe these relationships based on sequence analysis of housekeeping genes in comparison with previously characterized isolates of S. pneumoniae, S. mitis, and S. oralis. While most pneumococci were found to represent a closely related group these studies identified a subgroup of atypical pneumococcal isolates (bile insoluble and/or "acapsular") distinct from, though most closely related to, the "typical" pneumococcal isolates. However, a large proportion of isolates, found to be atypical on the basis of capsule reaction alone, did group with typical pneumococci, suggesting that they have either lost capsule production or represent as-yet-unrecognized capsular types. In contrast to typical S. pneumoniae, isolates phenotypically identified as S. mitis and S. oralis, which included isolates previously characterized in taxonomic studies, were genetically diverse. While most of the S. oralis isolates did fall into a well-separated group, S. mitis isolates did not cluster into a well-separated group. During the course of these studies we also identified a number of potentially important pathogenic isolates, which were frequently associated with respiratory disease, that phenotypically and genetically are most closely related to S. mitis but which harbor genes encoding the virulence determinants pneumolysin and autolysin classically associated with S. pneumoniae.

Project description:BackgroundFlavonol glycosides (FGs) are major components of soybean leaves and there are substantial differences in FG composition among genotypes. The first objective of this study was to identify genes responsible for FG biosynthesis and to locate them in the soybean genome. The second objective was to clone the candidate genes and to verify their function. Recombinant inbred lines (RILs) were developed from a cross between cultivars Nezumisaya and Harosoy.ResultsHPLC comparison with authentic samples suggested that FGs having glucose at the 2″-position of glucose or galactose that is bound to the 3-position of kaempferol were present in Nezumisaya, whereas FGs of Harosoy were devoid of 2″-glucose. Conversely, FGs having glucose at the 6″-position of glucose or galactose that is bound to the 3-position of kaempferol were present in Harosoy, whereas these FGs were absent in Nezumisaya. Genetic analysis suggested that two genes control the pattern of attachment of these sugar moieties in FGs. One of the genes may be responsible for attachment of glucose to the 2″-position, probably encoding for a flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase. Nezumisaya may have a dominant whereas Harosoy may have a recessive allele of the gene. Based on SSR analysis, linkage mapping and genome database survey, we cloned a candidate gene designated as GmF3G2″Gt in the molecular linkage group C2 (chromosome 6). The open reading frame of GmF3G2″Gt is 1380 bp long encoding 459 amino acids with four amino acid substitutions among the cultivars. The GmF3G2″Gt recombinant protein converted kaempferol 3-O-glucoside to kaempferol 3-O-sophoroside. GmF3G2″Gt of Nezumisaya showed a broad activity for kaempferol/quercetin 3-O-glucoside/galactoside derivatives but it did not glucosylate kaempferol 3-O-rhamnosyl-(1 → 4)-[rhamnosyl-(1 → 6)-glucoside] and 3-O-rhamnosyl-(1 → 4)-[glucosyl-(1 → 6)-glucoside].ConclusionGmF3G2″Gt encodes a flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase and corresponds to the Fg3 gene. GmF3G2″Gt was designated as UGT79B30 by the UGT Nomenclature Committee. Based on substrate specificity of GmF3G2″Gt, 2″-glucosylation of flavonol 3-O-glycoside may be irreconcilable with 4″-glycosylation in soybean leaves.

Dataset Information

Purification, characterization, and molecular analysis of the gene encoding glucosyltransferase from Streptococcus oralis.

Publications

Purification, characterization, and molecular analysis of the gene encoding glucosyltransferase from Streptococcus oralis.

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