Pattern of distribution of Prevotella species/phylotypes associated with healthy gingiva and periodontal disease.

The purpose of the present study was to obtain diverse profiles of Prevotella species associated with gingival sites in an isolated Aboriginal and an urban community by phylogenetic analysis and to establish patterns of association of identified Prevotella species in gingival sites. Species/phylotypes identified from the phylogenetic analysis of near full-length Bacteroidetes 16S rRNA gene sequences cloned from subgingival plaque samples obtained from an Aboriginal community were compared with those from an ethnically diverse urban metropolitan population suffering from periodontal disease. Specific primer sets were designed and validated for 22 distinct Prevotella species from the 24 species/phylotypes identified from both populations. Within the isolated Aboriginal community, gingival sites in adults were colonised by a mean of 15 different Prevotella species. Prevotella sp. oral clone P4PB24, Prevotella intermedia, Prevotella oralis, Prevotella denticola and Prevotella sp. strain P4P62 had the highest association with increasing probing depth in diseased sites (p < 0.05). P. intermedia and Prevotella sp. oral clone P4PB24, the Prevotella species significantly associated with increasing probing depth in diseased gingival sites and also strongly associated with P. gingivalis load (p < 0.05) in diseased gingival sites, showed significant correlation for co-colonisation (r = 0.6). Prevotella sp. oral clone B31FD, showing strong association with P. gingivalis load (p < 0.05) in diseased gingival sites, showed no significant correlation for co-colonisation with any other Prevotella species. This study provides a comprehensive analysis of Prevotella species associated with gingival sites for the informative evaluation of the epidemiology of infection by this genus.

Deletion of competence-induced genes over-expressed in biofilms caused transformation deficiencies in Streptococcus mutans.

Previous studies identified nine genes with increased expression in Streptococcus mutans biofilms of which six possessed putative ComX promoter sequences and were homologous to competence-induced genes in Streptococcus pneumoniae, Streptococcus gordonii and Bacillus subtilis. As competence increases in biofilms, a study was undertaken into the roles that these biofilm-induced genes might play in transformation. Only five of the nine gene deletions had a significant effect on transformation efficiency. Deletion of the genes for recombinase A, recA, DNA processing protein, dprA and single-stranded DNA-binding protein, ssbA, produced results comparable with those from other bacteria, supporting the contention that these proteins have similar functions in S. mutans competence. The uncharacterized genes SMU.769 and SMU.836 produced results in variance to deletion mutants of putative homologues in S. pneumoniae. Deletion of SMU.769 reduced chromosomal transformation 2.3-fold. SMU.769 belongs to a family of conserved genes induced by the competence-stimulating peptide and which have no established function. In contrast, deletion of SMU.836 reduced transformation of both plasmid and chromosomal DNA to <3%. Homology searches suggested that Smu.836 belongs to a family of competence-induced peptidoglycan hydrolases with a conserved enzyme domain and a species-variable cell-binding domain for which the best characterized member is the choline-binding protein D, CbpD, of S. pneumoniae.

Up-regulation of competence- but not stress-responsive proteins accompanies an altered metabolic phenotype in Streptococcus mutans biofilms.

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.

Proteome analysis of oral pathogens.

The oral environment contains diverse communities of micro-organisms including bacteria, fungi, protozoa, and viruses. Studies of oral ecology have led to an appreciation of the complexity of the interactions that oral micro-organisms have with the host in both health and disease. Despite this, diseases such as dental caries and periodontal diseases are still worldwide human ailments, resulting in a high level of morbidity and an economic burden to society. Proteomics offers a new approach to the understanding of holistic changes occurring as oral micro-organisms adapt to environmental change within their habitats in the mouth.

Mutation of aspartic acid residues in the fructosyltransferase of Streptococcus salivarius ATCC 25975.

The site-directed mutated fructosyltransferases (Ftfs) of Streptococcus salivarius ATCC 25975, D312E, D312S, D312N and D312K were all active at 37 degrees C, indicating that Asp-312 present in the 'sucrose box' was not the nucleophilic Asp residue responsible for the formation of a covalent fructosyl-enzyme intermediate required for enzyme activity. Analysis of the kinetic constants of the purified mutated forms of the enzyme showed that Asp-312 was most likely an essential amino acid involved in determining acceptor recognition and/or stabilizing a beta-turn in the protein. In contrast, when the Asp-397 of the Ftf present in the conserved triplet RDP motif of all 60 bacterial and plant family-32 glycosylhydrolases was mutated to a Ser residue, both sucrose hydrolysis and polymerization ceased. Tryptophan emission spectra confirmed that this mutation did not alter protein structure. Comparison of published data from other site-directed mutated enzymes implicated the Asp residue in the RDP motif as the one that may form a transient covalent fructosyl intermediate during the catalysis of sucrose by the Ftf of S. salivarius.

Purification and enzymic properties of the fructosyltransferase of Streptococcus salivarius ATCC 25975.

The recombinant fructosyltransferase (Ftf) of Streptococcus salivarius was expressed in Escherichia coli and purified to electrophoretic homogeneity after a combination of adsorption, ion-exchange and gel-filtration chromatography. The N-terminal signal sequence of the Ftf was removed by E. coli at the same site as in its natural host. The purified Ftf exhibited maximum activity at pH 6.0 and 37 degrees C, was activated by Ca2+, but inhibited by the metal ions Cu2+, Zn2+, Hg2+ and Fe3+. The enzyme catalysed the transfer of the fructosyl moiety of sucrose to a number of acceptors, including water, glucose and sucrose via a Ping Pong mechanism involving a fructosyl-enzyme intermediate. While this mechanism of catalysis is utilized by the levansucrases of Bacillus subtilis and Acetobacter diazotrophicus and the values of the kinetic constants for the three enzymes are similar, sucrose was a far more efficient fructosyl-acceptor for the Ftf of S. salivarius than for the two other enzymes.

Role of C-terminal domains in surface attachment of the fructosyltransferase of Streptococcus salivarius ATCC 25975.

The cell-associated beta-D-fructosyltransferase of Streptococcus salivarius, which is devoid of the cell wall anchoring motif, LPXTG, is released on exposure to its substrate, sucrose. Deletions within the C terminus of the enzyme implicated both the hydrophobic and the proline-glycine-serine-threonine-rich wall-associated domain in stabilizing the enzyme on the cell surface.

Development of a technique for multiple site-directed mutagenesis of the ftf gene of Streptococcus salivarius containing palindromic sequences.

Attempts at site-directed mutagenesis of the fructosyltransferase (ftf) gene of Streptococcus salivarius ATCC 25975 using standard protocols were unsuccessful and resulted in a series of deletions. These deletions appeared to commence at points within the ftf gene where there were palindromic sequences which were capable of forming closed loop structures that acted as terminators under the conditions of mutagenesis. To overcome this problem, two modified mutagenic techniques were developed. They made use of T4 DNA polymerase in conjunction with either T7 DNA polymerase at 37 degrees C or Vent DNA polymerase from Thermococcus litoralis at an elevated temperature. These methods eliminated the need for a single-stranded DNA template and allowed polymerisation through palindromic sequences to rapidly produce multiple site-directed mutations.

Streptococcus salivarius ATCC 25975 possesses at least two genes coding for primer-independent glucosyltransferases.

Fractionation of the culture medium showed that Streptococcus salivarius ATCC 25975 secreted a glucosyltransferase (Gtf) that was primer independent. On the basis of this observation, a gene library of S. salivarius chromosomal DNA cloned into lambda L47.1 was screened for a gene(s) coding for such an activity. As a result of this screening process, two new gtf genes, gtfL and gtfM, both of which coded for primer-independent Gtf activities, were isolated. GtfL produced an insoluble glucan that was refractory to digestion by the endo-(1-->6)-alpha-D-glucanase. of Chaetonium gracile, while GtfM produced a soluble glucan that was readily degraded by the glucanase. Comparison of the deduced amino acid sequences of gtfL and gtfM with 10 other available Gtf sequences allowed the relatedness of the conserved catalytic regions to be assessed. This analysis showed that the 12 enzymes did not form clusters based on their primer dependencies or on their product solubilities. Further analysis of the YG repeats in the C-terminal glucan-binding domains of GtfJ, GtfK, GtfL, and GtfM from S. salivarius showed that there was strong homology between a block of contiguous triplet YG repeats present in the four alleles. These blocks of YG repeats were coded for by a region of each gene that appeared to have arisen as a result of a recent duplication event(s).

Definition of a fundamental repeating unit in streptococcal glucosyltransferase glucan-binding regions and related sequences.

The C-termini of the glucosyltransferases (Gtfs) of oral streptococci are responsible for glucan binding. These glucan-binding domains (GBDs) are composed of a series of repeated sequences that have been classified into four different classes (A-D) by virtue of sequence similarity and which, by inference, have been suggested to be of functional importance. In contrast, we propose that repeat sequences evolve in response to selection for an increase in the number of copies of a particular domain through multiple duplication events occurring at different times. According to this hypothesis, repeats should possess various degrees of similarity, especially if only key residues are of functional importance. Analysis of the GBDs of the Gtfs indicated that a common fundamental repeat, designated the "YG" repeat, could be discerned within the "A", "B", "C", and "D" repeats. Similar elements were also conserved in the ligand-binding repeats of the Clostridium difficile toxins and the lysins and the PspA protein of Streptococcus pneumoniae, suggesting that similar selective pressures had also been imposed on these sequences. Analysis of the "YG" repeats present in the GtfJ and GtfK of Streptococcus salivarius indicated that some of the "YG" repeats in the GBDs of these proteins had arisen as a result of duplication events involving a series of three sequential "YG" repeats.

The glucosyltransferases of Streptococcus salivarius.

This review covers some of the more recent developments in the understanding of the different glucosyltransferases (GTFs) secreted by oral streptococci, particularly those produced by Streptococcus salivarius--a species that has been intensively studied at the Institute of Dental Research in Sydney.

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

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 gtfK) from Streptococcus salivarius ATCC 25975 as well as the sequence of gtfJ, which encodes a primer-dependent GTF that synthesizes an insoluble product (a GTF-I). In this communication we report the sequence of gtfK, 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 gtfB and gtfC from S. mutans GS-5, gtfJ and gtfK from S. salivarius, as well as the gtfI genes from S. downei and S. sobrinus. The recombination events between gtfB and gtfC, and between the two gtfI genes, were confirmed by examining divergences at silent sites.

The cell-bound fructosyltransferase of Streptococcus salivarius: the carboxyl terminus specifies attachment in a Streptococcus gordonii model system.

The ftf gene, coding for the cell-bound beta-D-fructosyltransferase (FTF) of Streptococcus salivarius ATCC 25975, has been analyzed, and its deduced amino acid sequence has been compared with that of the secreted FTF of Streptococcus mutans and the levansucrases (SacBs) of Bacillus species. A unique proline-rich region detected at the C terminus of the FTF of S. salivarius preceded a hydrophobic terminal domain. This proline-rich region was shown to possess strong homology to the product of the prgC gene from pCF10 in Enterococcus faecalis, which encodes a pheromone-responsive protein of unknown function, as well as homology to the human proline-rich salivary protein PRP-4. A series of 3'-OH deletions of the S. salivarius ftf gene expressed in Streptococcus gordonii Challis LGR2 showed that the C terminus was required for cell surface attachment in this heterologous organism, as only the complete gene product was cell bound. This cell-bound activity was released in the presence of sucrose, suggesting that the mode of attachment and release of the S. salivarius FTF in S. gordonii was similar to that in its native host.

The ftf gene encoding the cell-bound fructosyltransferase of Streptococcus salivarius ATCC 25975 is preceded by an insertion sequence and followed by FUR1 and clpP homologues.

Analysis of the region downstream of the ftf gene of Streptococcus salivarius led to the detection of two open reading frames (ORFs). The deduced amino acid sequences of these ORFs were homologous to proteins encoded by genes not previously described and/or sequenced in Gram-positive bacteria. The deduced amino acid sequence of the first of these (orf2) showed strong homology to the product of the FUR1 gene of Saccharomyces cerevisiae, which codes for a uracil phosphoribosyltransferase. The over-expression of the product of this gene appeared to be the source of the detrimental effect observed with phagemids carrying orf2 in Escherichia coli hosts. The deduced amino acid sequence of the second ORF (orf3) was homologous to the ClpP family of proteases. Examination of the upstream region of the ftf gene led to the discovery of a new insertion sequence-like element which has been designated IS1161.

A method for the isolation of RNA from Streptococcus salivarius and its application to the transcriptional analysis of the gtfJK locus.

The glucosyltransferases from oral streptococci cleave sucrose and polymerize the glucose moieties. In Streptococcus salivarius ATCC 25975, two glucosyltransferase-encoding genes, gtfJ and gtfK, are closely linked and transcribed in the same direction. A procedure for the isolation of intact RNA from this organism was devised. The procedure incorporated a high-temperature mutanolysin treatment and selective precipitation by LiCl. The RNA was subject to Northern hybridization and RNase protection assays and it was concluded that the two genes are transcribed separately. A potential factor-independent transcription terminator was located in the intergenic region.

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

The oral micro-organism Streptococcus salivarius 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 lambda L47.1. The inserts in the lambda-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. salivarius 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 the gtf 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.

Enumeration of oral streptococci on media containing different concentrations of sodium and potassium ions.

Laboratory "type" strains of oral streptococci were screened for their ability to grow on mitis-salivarius agar (MSA) in the presence of increasing concentrations of either Na+ or K+ up to 500 mmol/L. Strains were generally better able to withstand increasing concentrations of Na+ than K+, although low numbers of colony-forming units (cfus) were seen with the highest concentration of either cation. Two strains of Streptococcus mutans, Ingbritt 162 and Ingbritt 175, behaved differently when the concentration of cation was increased from 50 to 200 mmol/L; the latter showed a marked increase in the number of cfus when the Na+ concentration was increased from 50 to 200 mmol/L, whereas there was a decrease with strain Ingbritt 162. Strains of oral streptococci from the saliva of adults and children were isolated on modified MSA containing known concentrations of Na+ and K+ and further examined if they showed "mutans-like" colony morphology. The number of cfus generally dropped as the concentration of Na+ or K+ was increased from 200 to 350 or 500 mmol/L. Greater numbers of streptococci were tolerant to Na+ than to K+. Half of the isolates were members of the Streptococcus sanguis group (SSG), either Streptococcus mitis or S. sanguis II, and these were more tolerant to high concentrations of Na+ or K+ than other isolates that were identified as Streptococcus morbillorum, Streptococcus acidominimus, and Streptococcus milleri.

Secretion of fructosyltransferase by Streptococcus salivarius involves the sucrose-dependent release of the cell-bound form.

Three strains of Streptococcus salivarius including a recent clinical isolate were found to possess Ca2(+)-dependent fructosyltransferase (FTF) activity. The extracellular FTF activity of cells grown on sucrose increased as much as 9-fold compared with cells grown on either glucose, fructose or galactose. This increase in activity was due not to induction of FTF by sucrose, but to the release of the cell-bound form of the enzyme. Studies with washed cells of S. salivarius ATCC 25975 showed that the extent of release of the cell-bound FTF activity was dependent upon the sucrose concentration up to 4 mM, at which concentration maximum release (95%) of cell-bound FTF occurred. Several lines of evidence suggested that either substrate binding or de novo synthesis of fructan is required for the release of the cell-bound FTF activity.