Characterization of the product of the gtfS gene of Streptococcus downei a primer-independent enzyme synthesizing oligo-isomaltosaccharides

Russell, R. P. B.; Gilpin, M. L.; Hanada, Nobuhiro; Yamashita, Yoshihisa; Shibata, Yukie; Takehara, Tadamichi

doi:10.1099/00221287-136-8-1631

Cited by 12 publications

(14 citation statements)

References 25 publications

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“…The S. downei MFe28 rGTFS expressed in E. coli synthesizes oligoisomaltosaccharides from sucrose (15). However, the rGTF synthesizes high-molecular-weight, water-soluble Western blot analysis of the gtf gene product (rGTF).…”

Section: Discussionmentioning

confidence: 99%

“…Although four GTF peaks of strain OMZ176 appeared from the chromatofocusing column (11), antisera against four kinds of GTFs are not yet available from strain OMZ176. In the previous study (15), antisera derived from S. sobrinus AHT were tested. The anti-GTF P2 serum, which reacted with rGTFS extracted from E. coli (pMLG60) in the previous study, did not react with the rGTF extracted from E. coli(pGT72).…”

Section: Discussionmentioning

confidence: 99%

“…In this experiment, 26% of the GTF activity was found in the periplasmic fraction and most (67%) of the GTF activity was in the cytoplasmic fraction. The fractionation of crude extracts from E. coli HB101 carrying pMLG60 (15) revealed that most (73%) of the GTF activity was found in the membrane fraction, compared with only 23% in the cytoplasmic fraction and only 5% in the periplasmic fraction. Since both the gtf and gtfS genes code for extracellular GTFs, we are interested in their differences in secretion.…”

Section: Discussionmentioning

confidence: 99%

“…Western blot analysis was carried out as described by Towbin et al (20). Because the four types of antisera raised against the S. sobrinus OMZ176 GTFs were not yet available, we used previously described S. sobrinus AHT antisera (15). Before use, the antisera were absorbed with heat-killed cells of E. coli HB101 carrying pBR322.…”

Section: Methodsmentioning

confidence: 99%

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Cloning of a Streptococcus sobrinus gtf gene that encodes a glucosyltransferase which produces a high-molecular-weight water-soluble glucan

et al. 1991

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The gtf gene coding for glucosyltransferase (GTF), which produces a water-soluble glucan, was cloned from Streptococcus sobrinus OMZ176 (serotype d) into plasmid vector pBR322. This gene was expressed in Escherichia coli, and the product was purified to near homogeneity. The antigenicity of recombinant GTF (rGTF) was examined with the antisera raised against purified GTF P1, P2, P3, and P4 obtained from S. sobrinus AHT (serotype g). The rGTF reacted only with anti-GTF P1 serum in a Western blot (immunoblot) analysis. The rGTF closely resembled GTF P1 in its molecular mass, Km value for sucrose, optimal pH, primer dependency, and immunological properties. The high-molecular-weight, water-soluble glucan produced by the rGTF also resembled that of GTF P1, which is the most efficient primer donor for primer-dependent, water-insoluble glucan synthesis. Properties of the rGTF were also compared with those of rGTFS, which was purified from E. coli carrying the gtJS gene isolated from Streptococcus downei (previously S. sobrinus serotype h) MFe28. Both rGTF and rGTFS synthesized water-soluble glucan from sucrose without primer dextran, but their characteristics in Km values for sucrose, optimal pHs, and polymer sizes of the glucan were different. Furthermore, the gtfgene did not hybridize with the gtfS gene in a Southern blot analysis. These results showed that rGTF is similar to S. sobrinus AHT GTF P1 but distinct from rGTFS that has been previously purified from E. coli carrying the gtJS gene. Mutans streptococci are essential to the etiology of dental caries (12). This group of bacteria consists of seven species (23). These organisms produce extracellular glucosyltransferases (GTFs; EC 2.4.1.5) which catalyze glucan synthesis from dietary sucrose. These glucans are believed to be important in the pathogenicity of these bacteria (12). Three kinds of gtf genes (gtfB, gtfC, and gtJD) which code for GTFs have been isolated from Streptococcus mutans GS5 chromosomal DNA (1, 7, 8). Several immunological studies have showed that the GTF enzymes of S. mutans are distinct from those of Streptococcus sobrinus and Streptococcus cricetus (21, 22, 25). Many workers have reported the purification of GTF enzymes from S. sobrinus and S. cricetus (3, 14). Recently, four GTFs (GTF P1, P2, P3, and P4) were purified from the culture supernatant of S. sobrinus AHT (24, 25). These four GTFs are different in their enzymatic characteristics and immunoreactivities. These results suggest that four gtfgenes must exist in S. sobrinus AHT. Up to now, two gtf genes coding for GTFS and GTFI which were similar to those of S. sobrinus and S. cricetus (15, 16) have been isolated from S. downei (previously S. sobrinus serotype h) MFe28 (6, 15, 16, 23) and their nucleotide sequences have been reported (4, 5). In this report, we describe the properties of the recombinant GTF (rGTF) purified from Escherichia coli carrying the gtf gene isolated from S. sobrinus OMZ176 compared with GTF P1 from S. sobrinus AHT.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Cloning of a Streptococcus sobrinus gtf gene that encodes a glucosyltransferase which produces a high-molecular-weight water-soluble glucan

et al. 1991

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“…Pure recombinant GTF‐I, on its own, produces insoluble glucan, which contains >90% 1,3‐α‐linked glucose residues ( 33) so this raises the question of the involvement of the other glucosyltransferases. GTF‐S catalyses the formation of 1,6‐α‐linked linear chains consisting of approximately 25 glucose units ( 14, 32). GTF‐T and GTF‐U have not been characterized in S. downei but their properties can be deduced from studies of S. sobrinus .…”

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confidence: 99%

Effect of inactivation of gtf genes on adherence of Streptococcus downei

Colby

McLaughlin

Ferretti

et al. 1999

Oral Microbiology and Immunology

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The activity of glucosyltransferases (GTF), a group of enzymes that synthesize water-soluble and -insoluble glucans from sucrose, significantly contributes to the cariogenicity of mutans streptococci. Streptococcus downei produces four glucosyltransferases, GTFI, which produces insoluble glucan, and GTFS, GTFT, and GTFU, which synthesize soluble glucans. We have previously reported that inactivation of gtfS results in altered adherence and have now examined its interaction with other enzymes by constructing mutants which were gtfS, gtfS/gtfT, gtfS/gtfI and gtfI. The mutants were tested for their ability to accumulate on wires and on plastic microtiter trays in the presence of sucrose. The gtfS mutant displayed a reduced ability to adhere compared to the wild type but there was no further reduction of adherence in a gtfS/gtfT mutant. In contrast, the gtfS/gtfI double mutant showed a drastic reduction in adherence and when gtfI alone was inactivated, bacteria were unable to adhere to a hard surface. The results confirmed that insoluble glucan is required for strong adherence to a smooth surface but that the amount and structure of this glucan is dependent upon the availability of soluble glucans to act as primer molecules.

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Molecular genetics of glucan metabolism in oral streptococci

Russell

1990

Archives of Oral Biology

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Characterization of the product of the gtfS gene of Streptococcus downei a primer-independent enzyme synthesizing oligo-isomaltosaccharides

Cited by 12 publications

References 25 publications

Cloning of a Streptococcus sobrinus gtf gene that encodes a glucosyltransferase which produces a high-molecular-weight water-soluble glucan

Cloning of a Streptococcus sobrinus gtf gene that encodes a glucosyltransferase which produces a high-molecular-weight water-soluble glucan

Effect of inactivation of gtf genes on adherence of Streptococcus downei

Molecular genetics of glucan metabolism in oral streptococci

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