~~~The oral micro-organism Streptococcus safivarius ATCC 25975 synthesizes extracellular glucosyltransferases (GTFs) which polymerize the glucose moiety of sucrose into glucan polymers. Two separate genes encoding the activities of a GTF-I (a GTF that synthesizes an insoluble product) and a GTF-S (a GTF that synthesizes soluble product) were cloned into bacteriophage IIA7.1. The inserts in the I-clones were characterized by restriction mapping and Southern hybridization and were found to overlap, implying that the two genes lay very close to one another on the S. safiuarius chromosome. Both genes were subcloned into phagemid vector pIBI30 where they were expressed at a high level. The GTF-I-encoding gene was named gtfJ and the GTF-S-encoding gene, gtfK. Nucleotide sequencing showed that gtfJ and most probably gtfK were closely related to thegtf genes of the mutans streptococci. Sequence alignment also indicated that gtfK lay very close to and downstream from gtfJ, and that both were transcribed in the same direction.

Mature biofilm and planktonic cells of Streptococcus mutans cultured in a neutral pH environment were subjected to comparative proteome analysis. Of the 242 protein spots identified, 48 were significantly altered in their level of expression (P<0?050) or were unique to planktonic or biofilm-grown cells. Among these were four hypothetical proteins as well as proteins known to be associated with the maintenance of competence or found to possess a cin-box-like element upstream of their coding gene. Most notable among the non-responsive genes were those encoding the molecular chaperones DnaK, GroEL and GroES, which are considered to be up-regulated by sessile growth. Analysis of the rest of the proteome indicated that a number of cellular functions associated with carbon uptake and cell division were down-regulated. The data obtained were consistent with the hypothesis that a reduction in the general growth rate of mature biofilms of S. mutans in a neutral pH environment is associated with the maintenance of transformation without the concomitant stress response observed during the transient state of competence in bacterial batch cultures.

The four recombinant glucosyltransferases (GTFs), GtfJ, GtfK, GtfL and GtfM, that had previously been cloned from Streptococcus salivarius ATCC 25975, were individually expressed in Escherichia coli and their glucan products and kinetic properties were analysed. GtfJ was a primer-dependent GTF which synthesized an insoluble glucan composed mainly of a-(1 + 3)-linked glucosyl residues in the presence of dextran 1-10. GtfK was primer-stimulated, and produced a linear soluble dextran without any detectable branch points both in the absence and in the presence of dextran 1-10. GtfL was primerindependent and produced a mixed-linkage insoluble glucan composed of approximately equal proportions of a-(l-+ 3)-and a-(1 + 6)-linked glucosyl residues. GtfL was inhibited by dextran T-10. G t f M was primer-independent and produced a soluble dextran with approximately 5% a-(1 + 3)-linked glucosyl residues. G t f M was essentially unaffected by the presence of dextran T-10. The results confirmed that each enzyme represented one of the four possible combinations of primer-dependency and product solubility and that each possessed unique biosynthetic properties. The soluble dextrans formed by GtfK and GtfM, as well as the mixed-linkage insoluble glucan formed by GtfL, were also capable of acting as primers for the primer-dependent GtfJ and the primer-stimulated GtfK. Unexpectedly, the linear dextran produced by GtfK was by far the least effective either at priming itself or at activating and priming the pr i mer-de pendent GtfJ .

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.

Compared with traditional two-dimensional (2D) proteome analysis of Streptococcus mutans grown as a biofilm from a planktonic culture at steady state (Rathsam et al., Microbiol. 2005, 151, 1823-1837), the use of 2D fluorescence difference gel electrophoresis (DIGE) led to a 3-fold increase in the number of identified protein spots that were significantly altered in their level of expression (P < 0.050). Of the 73 identified proteins, only nine were up-regulated in biofilm grown cells. The results supported the previously surmised hypothesis that general metabolic functions were down-regulated in response to a reduction in growth rate in mature S. mutans biofilms. Up-regulation of competence proteins without any concomitant increase in stress-responsive proteins was confirmed, while the levels of glucosyltransferase C (GtfC), involved in glucan formation, O-acetylserine sulfhyrylase (cysteine synthetase A; CsyK), implicated in the formation of [Fe-S] clusters, and a hypothetical protein encoded by the open reading frame, SMu0188, were also up-regulated.