Purification and Characterization of Extracellular Glucosyltransferase from Streptococcus mutans Serotype b (Subspecies rattus)

Abstract

An extracellular glucosyltransferase (GT-S) synthesizing water-soluble glucan was purified from the culture supernatant of Streptococcus mutans BHT (serotype b, subsp. rattus) by DEAE-Sepharose chromatography and preparative isoelectric focusing. The Mr of the enzyme was 155,000 and the pI was 4.5. The GT-S had a specific activity of 10.2 i.u. (mg protein)-1, an optimum pH of 6.0 and a Km value of 0.8 mM for sucrose, and was activated twofold by dextran T10. The GT-S was immunologically partially identical with the corresponding enzymes in crude preparations from serotypes c, e and f. The glucan synthesized de novo from sucrose by the GT-S was water-soluble and consisted of 29 mol% of non-reducing terminal, 49 mol% of 1,6-alpha-linked, 11 mol% of 1,3-alpha-linked and 11 mol% of 1,3,6-alpha-branched glucose residues.

Transcriptional repressor Rex is involved in regulation of oxidative stress response and biofilm formation by Streptococcus mutans

“…Complementation with a wild-type copy of rex , including its promoter region, on shuttle vector pDL278 (LeBanc & Lee, 1991) partially restored the phenotype of the wild-type (Figure 3). A phenol-sulfuric acid assay was also used to measure total glucans in the biofilms (Mukasa, et al , 1985, Kumada, et al , 1987, Ausubel, et al , 1992, Werning, et al , 2008). As expected, TW239 biofilms contained more than 2-fold glucose polymers than the parent strain, with an average of 30.62 (±5.7) µg/ml for UA159 and 72.45 (±15.85) µg/ml for TW239 ( P <0.001), respectively.…”

“…To measure the extracellular glucose polymers in biofilms, a phenol-sulfuric acid assay was used with known concentrations of glucose as the standards (Mukasa, et al , 1985, Kumada, et al , 1987, Ausubel, et al , 1992, Werning, et al , 2008). Briefly, 3-day biofilms were grown in BMGS on glass slides in 50 ml tubes as described elsewhere (Phan, et al , 2000, Wen, et al , 2010, Wen, et al , 2010).…”

Purification and Characterization of Cell-associated Glucosyltransferase Synthesizing Insoluble Glucan from Streptococcus mutans Serotype c

“…2), was free of the PI 7.4 and 8.4 GTF-S of similar M , (Fig. l), (as were the washed cells) and did not react with anti-GTF-S sera from serotype c and b streptococci (Mukasa et al, 1985;Kumada et al, 1987) (data not shown). These observations indicate that the preparation was not detectably contaminated with such proteins as GTF-S which might coprecipitate with GTF-I upon the IEF (seconddimension) run of the 2-DE.…”

“…After two washes with 75% ethanol containing 0.15 M-NaCl, the polysaccharides were analysed by gas-liquid chromatography (Mukasa et al, 19826) with dextran T10 (Pharmacia) and inulin (Tokyo Kasei) as standards. Total polysaccharides were measured by the phenol/sulphuric acid method (Dubois et al, 1956) with glucose as a standard, and fructan was measured according to the method of Van Handel (1967) as described by Kumada et al (1987) using fructose as a standard. The amount of glucan was estimated by subtracting the amount of fructan from the amount of total polysaccharides.…”

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

“…Tsumori et al, 1983b;Koga et al, 1983;McCabe, 1985;Mukasa, 1986). In Streptococcus rattus (mutans group serotype 6) and Streptococcus mutans (mutans group serotypes c, e andf), adherent insoluble polysaccharide is synthesized cooperatively by two GTFs; GTF-I, which synthesizes an insoluble glucan, and GTF-S, which synthesizes a soluble glucan ; and probably fructosyltransferases, which synthesize an inulin-type fructan (Scales et al, 1975;Mukasa et al, 1982bMukasa et al, , 1985Mukasa et al, , 1989Kenny & Cole, 1983;Mukasa, 1986;Shimamura et al, 1987;Kumada et al, 1987;Tsumori et al, 1989).…”