Characterization of Glucosyltransferase Expressed from a Streptococcus Sobrinus Gene Cloned in Escherichia Coli

Abstract

The gene encoding a glucosyltransferase which synthesized water-insoluble glucan, g t f l , previously cloned from Streptococcus sobrinus strain MFe28 (mutans serotype h) into a bacteriophage 2 vector, was subcloned into the plasmid pBR322. The recombinant plasmid was stable in Escherichia coli and gtjlr 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-Cr-Dglycosyl residues (-90%). An internal fragment of the g t f l 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. I N T R O D U C T I O NThe glucosyltransferases (GTFs) of the mutans group of oral streptococci are extracellular enzymes responsible for the synthesis of complex mixtures of water-soluble and insoluble glucans. These glucans are believed to be important in the formation and metabolism of dental plaque (Hamada & Slade, 1980). It was first shown by Guggenheim & Newbrun (1969) that a single strain could produce a number of electrophoretically distinct GTFs. Ciardi et al. (1976Ciardi et al. ( , 1977 showed that some of these enzymes synthesized soluble, and others insoluble, polymers but progress towards understanding the reason for the multiplicity of enzymes has been considerably hampered by technical problems, including the fact that GFTs exist in highmolecular-mass aggregates and have a strong tendency to bind to separation matrices commonly used in column chromatography (Germaine et al., 1977;Russell, 1979; Figures & Edwards, 1981 ; Ono et al., 1984). The latter fact has, however, proved useful in purification procedures involving affinity chromatography (McCabe & Smith, 1977;Russell, 1979;McCabe, 1985). At least some of the heterogeneity of GTFs is a consequence of proteolytic degradation (Asem et al., 1986;Mukasa, 1986;Russell et al., 1986), but many questions remain concerning the number of distinct GTFs and their individual functions.Most progress has been made with strains that originally constituted Streptococcus mutans serotypes d and g, which are now classified as Streptococcus sobrinus (Coykendall, 1983; Coykendall & Gustafson, 1986). The purification from strains of these serotypes of GTFs capable of synthesizing soluble glucans (GTF-S) or insoluble glucans (GTF-I) has been described by a number of workers (Ciardi et al., 1977;Germaine et al., 1977;Hare et al., 1978; Figures& Edwards, 1981 ; Fukushimaetal., 1981 ; Fukui etal., 1982; Kogaetal., 1983;Robyt & Martin, 1983;Furuta et al., 1985;McCabe, 1985; Namiki et al., 1985;Mukasa, 1986). The glucan synthesized by GTF-S (dextran) consists mainly of 1,6-a-linked glucose units while that

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

“…Even in the C-terminal region there is a highly homologous region (94%) between residues 1181 and 1320, which is shown to be essential for glucan binding in ISG synthesis (see above). These two proteins are indeed indistinguishable in antigenicity (27). By contrast, either of these proteins shows a rather low homology with proteins of gtJB and gtfC from S. mutans GS-5 (-60%).…”

“…Glucan-binding fragments were isolated by tryptic digestion of GTF proteins from S. sobrinus, but none of them showed glucan-synthesizing activity (18,22). Of several molecular cloning studies of GTF genes from mutans streptococci (1,7,10,13,14,25,27,29,31), Ferretti et al (7) * Corresponding author.were the first to determine the complete nucleotide sequence of the GTF-I gene from a strain of S. sobrinus (MFe28); they have located a glucan-binding site in a carboxy-terminal region of the GTF-I protein, which is essential for the enzyme activity. However, it is not yet known what roles the regions near the N terminus play or how they are correlated with the roles of C-terminal regions in GTF-I activity.…”

“…Glucan-binding fragments were isolated by tryptic digestion of GTF proteins from S. sobrinus, but none of them showed glucan-synthesizing activity (18,22). Of several molecular cloning studies of GTF genes from mutans streptococci (1,7,10,13,14,25,27,29,31), Ferretti et al (7) * Corresponding author.…”

Streptococcus downei sp. nov. for Strains Previously Described as Streptococcus mutans Serotype h

“…Recently, it has been reported that a 3.3-kilobase fragment of chromosomal DNA of MFe2ST reveals differences between MFe28T and S. sobrinus strains in Southern blotting hybridization experiments (21). Despite the relatively high degree of DNA homology observed between the monkey plaque strains and strains of S. sobrinus, compared with representatives of the other species within the mutans streptococci, the results show that the monkey strains and S. sobrinus are sufficiently dissimilar to warrant separate species status, for which we propose the name Streptococcus downei.…”

DentalCariesVaccines:Prospects andConcerns

“…Genes responsible for glucan synthesis in S. mutans are gtfB (Shiroza et al, 1987), which synthesizes an ␣-1,3-linked insoluble glucan, gtfC (Pucci et al, 1987), which synthesizes glucan with both ␣-1,3 and ␣-1,6 linkages, and gtfD (Honda et al, 1990), which synthesizes a soluble ␣-1,6-linked glucan. Similarly, the products of gtfI (Russell et al, 1987) and gtfS (Gilmore et al, 1990) genes of S. sobrinus synthesize insoluble and soluble glucan products, respectively. Mutational inactivation techniques have shown that each of these gene products is important to the cariogenicity of the respective mutans streptococcal strain.…”