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

Giffard, Philip M.; Simpson, Christine L.; Milward, Carolyn P.; Jacques, Nicholas A.

doi:10.1099/00221287-137-11-2577

Cited by 64 publications

(70 citation statements)

References 24 publications

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“…Comparison of the amino acid sequences of GTFs from oral streptococci synthesizing primarily SG (S. mutans GS5 GTF-S and S. downei GTF-S [6]) or IG (S. mutans GTF-I, S. salivarius GTF-I [5], and S. downei GTF-I [3]) revealed several positions that appear to be conserved for each group of enzymes (Fig. 2).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 1994

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The glucosyltransferases (GTFs) of mutans streptococci are important virulence factors in the sucrosedependent 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 T1O, 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.

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Section: Methodsmentioning

confidence: 99%

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

et al. 1994

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“…Plasmids were maintained in Escherichia coli strain NM522 (Gough & Murray, 1983) and derivatives of bacteriophage 1 were grown on E. coli strain LE392 (Murray et al, 1977). All S. salivarius DNA was derived from ll-A33, which has been described previously (Pitty et al, 1989;Giffard et al, 1991). E. coli cells were routinely grown on Luria Bertani (LB) medium (Miller, 1972) supplemented with ampicillin (100 pg ml-'), isopropyl P-Dthiogalactoside (IPTG) (1 mM), or 5-bromo-4-chloro-3-indolyl p-Dgalactoside (X-Gal) (100 pg ml-') as appropriate.…”

Section: Methodsmentioning

confidence: 99%

Sequence of the gtfK gene of Streptococcus salivarius ATCC 25975 and evolution of the gtf genes of oral streptococci

Giffard¹,

Allen²,

Milward³

et al. 1993

Journal of General Microbiology

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Many strains of oral streptococci secrete glucosyltransferases (GTFs) that polymerize sucrose into glucans that form an integral part of the plaque matrix on the tooth surface. Recently, we reported the cloning of two closely linked GTF-encoding genes (gtfJ and gtfly) from Streptococcus salivarius ATCC 25975 as well as the sequence of gt'J, which encodes a primer-dependent GTF that synthesizes an insoluble product (a GTF-I). In this communication we report the sequence of gtflv, which encodes a primer-dependent GTF that synthesizes a soluble product (a GTF-S), as well as the sequence of a small downstream open reading frame of unknown function. The deduced sequence of GtfK was compared with those of seven other streptococcal Gtfs and an unrooted phylogenetic tree constructed. This analysis suggested that Gtfs with similar product specificities do not form phylogenetic clusters and was consistent with currently accepted phylogenetic schemes. The tree was tested by constructing a series of 'sub-trees' from different blocks of the alignment. Evidence was obtained for recombination events involving g t f l and gtfr: from S. mutans GS-5, gtfJ and gtflv from S. salivarius, as well as the gtfl genes from S. downei and S. sobrinus. The recombination events between g t f l and gt&, and between the two gtfl genes, were confirmed by examining divergences at silent sites.

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“…Genes coming from other related species were also tested by Southern hybridization under low-stringency conditions. Because S. salivarius is closely related to S. thermophilus (4,17), probes specific to the ftf and gtfJ genes (19,20) of S. salivamius were hybridized on total DNA of A054 strain, but no hybridization signal was detected. The gene involved in DNA repair, hexA, cloned from Streptococcus pneumoniae (45), was also hybridized with S. thermophilus DNA.…”

Section: Sf3mentioning

confidence: 99%

Physical and genetic map of Streptococcus thermophilus A054

Roussel¹,

Pébay²,

Guédon³

et al. 1994

J Bacteriol

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The three restriction endonucleases Sfil, BssHl, and SmaI were found to generate fragments with suitable size distributions for mapping the genome of Streptococcus thermophilus A054. A total of 5, 8, and 24 fragments were produced with SfiI, BssHI, and SmaI, respectively. An average genome size of 1,824 kb was determined by summing the total fragment sizes obtained by digestions with these three enzymes. Partial and multiple digestions of genomic DNA in conjunction with Southern hybridization were used to map SfI, BssHII, and SmaI fragments. All restriction fragments were arranged in a unique circular chromosome. Southern hybridization analysis with specific probes allowed 23 genetic markers to be located on the restriction map. Among them, six rrn loci were precisely located. The area of the chromosome containing the ribosomal operons was further detailed by mapping some of the ApaI and SgrAI sites. Comparison of macrorestriction patterns from three clones derived from strain A054 revealed two variable regions in the chromosome. One was associated with the tandem rinD and rnnE loci, and the other was mapped in the region of the lactose operon.Streptococcus thermophilus is an economically important lactic acid bacterium used in the manufacture of yoghurt and some hard cheeses. It is a gram-positive, facultative anaerobic microorganism that is exclusively isolated from milk and fermented dairy products. Despite its importance in the dairy industry, genetic studies of S. thermophilus have been limited until recently. To date, basic methods of gene transfer by transformation, transduction, or conjugative mobilization have been reported (34). Several genes have been cloned and analyzed, including genes involved in glucide metabolism (lacZ, lacS, galE, galM, and ldh [5,25,43,44,51]) and DNA recombination (recA [15]), ribosomal sequences (4, 23, 38), and the aspartate racemase gene (62). A marked restriction polymorphism has been detected between different strains of this species by analysis of hybridization patterns obtained with a species-specific probe (13) and with probes by genes coding for rRNA (38). Despite the availability of gene transfer systems and cloned genes, the structure and organization of the S. thermophilus genome are not well defined.Pulsed-field gel electrophoresis (PFGE) (9, 52) has proved to be an efficient method for genome size estimation and construction of chromosomal maps, as well as being a useful tool for the characterization of bacterial species. Preliminary results from PFGE have indicated a genome size of 1.75 Mb for strain STi of S. thermophilus (31) and allowed restriction polymorphism within S. thermophilus to be observed (48).In this study, we used PFGE to estimate the size of the chromosome of S. thermophilus A054 and to construct a physical map by using the restriction enzymes SMaI, Sfil, BssHII, ApaI, and SgrAI. Hybridization probes were used to locate 23 genetic markers on the chromosomal map. Genetic polymorphism within strain A054 was analyzed by comparing the macrorestric...

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

Cited by 64 publications

References 24 publications

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

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

Sequence of the gtfK gene of Streptococcus salivarius ATCC 25975 and evolution of the gtf genes of oral streptococci

Physical and genetic map of Streptococcus thermophilus A054

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