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.

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.

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.

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.

Inhibition of the expression of cell-associated fructosyltransferase in Streptococcus salivarius by octyl beta-D-glucopyranoside.

Octyl beta-D-glucopyranoside prevented the expression of cell-associated fructosyltransferase activity in Streptococcus salivarius ATCC 25975 grown in batch culture or incubated in nonproliferating cell suspension medium. This effect was not due to the direct inhibition of enzyme activity nor due to the loss of active enzyme into the external medium. The prevention of enzyme expression did not appear to be due to the inhibition of a general translocation mechanism for protein secretion, since fructosyltransferase activity was not detected within the cytoplasm of lysed cells grown in the presence of octyl beta-D-glucopyranoside; nor was there any observed inhibition of the secretion of the extracellular enzyme glucosyltransferase. These and other observations supported the view that fructosyltransferase was not secreted across the cytoplasmic membrane in an active form before becoming associated with the cell surface.

Membrane perturbation by cerulenin modulates glucosyltransferase secretion and acetate uptake by Streptococcus salivarius.

Cerulenin and dodecanoic acid prevented the synthesis and secretion of glucosyltransferase in non-proliferating cell suspensions of Streptococcus salivarius ATCC 25975 under conditions that also inhibited the incorporation of radioactively labelled acetate into the cell. In the presence of Tween 80, acetate incorporation was not markedly affected by cerulenin despite the fact that glucosyltransferase secretion was still inhibited. Cerulenin and dodecanoic acid were found to prevent the incorporation of radioactively labelled acetate by affecting the uptake of acetate by the cell. In the case of cerulenin, the inhibition of uptake of acetate by the cell was partially relieved by the addition of Tween 80. These and other observations strongly suggested that cerulenin inhibited glucosyltransferase secretion and acetate incorporation by perturbing the membrane, rather than by directly inhibiting lipid synthesis.

The influence of incorporation of octadecenoic acid on the cell-associated fructosyltransferase and the extracellular glucosyltransferase activities of Streptococcus salivarius.

The rate of expression of the cell-associated fructosyltransferase (FTFm) activity of Streptococcus salivarius ATCC 25975 grown in continuous culture was linearly related to the rate of octadecenoic acid (C18:1) incorporation into the membrane lipids irrespective of the presence or absence of Tween 80 in the growth medium. This observation was confirmed with data obtained from cells grown in the presence of a series of n-alkanols. The results suggested that cosynthesis of lipids containing C18:1 residues was necessary for FTFm expression and accounted for the slight stimulation of enzyme expression by Tween 80 at all growth rates. In contrast, addition of Tween 80 to the growth medium resulted in several-fold increases in extracellular glucosyltransferase (GTFe) production irrespective of the growth rate. Following the addition of the surfactant to the growth medium, an exponential relation between the increased rate of GTFe production and the concomitant net increase in the rate of C18:1 incorporation was noted. The results obtained in continuous culture emphasized the underlying effect growth rate had on GTFe production, especially when Tween 80 was added to the growth medium. In the presence of n-alkanols, the rate of GTFe production plotted as a single 'U'-shaped curve with respect to the rate of C18:1 incorporation irrespective of the chain length of the n-alkanol studied. Rapid analyses of the extracellular proteins by SDS-PAGE suggested that hexan-1-ol and Tween 80 specifically stimulated the synthesis and secretion of GTFe and no other extracellular protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Does an increase in membrane unsaturated fatty acids account for Tween 80 stimulation of glucosyltransferase secretion by Streptococcus salivarius?

When Streptococcus salivarius was grown in batch culture in the presence of various Tween detergents, the fatty acid moiety of the detergent was incorporated into the lipids of its membrane. Tween 80 (containing primarily oleic acid) markedly stimulated the production of extracellular glucosyltransferase and also increased the degree of unsaturation of the membrane lipid fatty acids. The possibility that an increase in membrane unsaturated fatty acids promoted extracellular glucosyltransferase production was examined by growing cells at different temperatures in the presence or absence of Tween 80. The membrane lipids of cells grown at 30 degrees C, 37 degrees C and 40 degrees C without Tween 80 exhibited unsaturated/saturated fatty acid ratios of 2.06, 1.01 and 0.87 respectively. A significant increase in the production of extracellular glucosyltransferase was observed at 30 degrees C compared to cells grown at 40 degrees C. However, cells produced much more exoenzyme at all temperatures when grown with Tween 80. The results indicated that an increase in the unsaturated fatty acid content of the membrane lipids was not by itself sufficient to account for the stimulation of extracellular glucosyltransferase production by Tween 80, but that the surfactant also had to be present.

Effect of Tween 80 on the morphology and physiology of Lactobacillus salivarius strain IV CL-37 grown in a chemostat under glucose limitation.

The effect of Tween 80 on Lactobacillus salivarius strain IV CL-37 growing in a chemostat under various conditions was investigated. The organisms could grow under glucose limitation in the absence of Tween 80 at pH 6.0 or lower anaerobically but not aerobically. Aerobic growth under glucose limitation and in the presence of Tween 80 occurred in complete MRS medium but not in the dialysable fraction of MRS medium. The morphology of cells differed from coccal to filamentous and branched structures according to the growth condition. The possible effect of Tween 80 on membrane components was examined by estimating the cellular and extracellular lipoteichoic acid contents. In both batch and continuous culture the amounts of cellular lipoteichoic acid were inversely related to the amount of Tween 80 whereas the amounts of extracellular lipoteichoic acid were influenced by other factors in addition to Tween 80.

Characterization of two strains of cariogenic lactobacilli.

Two strains of lactobacilli that initiate dental caries in conventional animals were exained for their physiological and serological characteristics. The strain designated V CL-25 was identified as Lactobacillus fermentum and belonged to serological group F. The strain designated IV CL-37 was a Lactobacillus salivarius, but it could not be further identified as either of the known subspecies, nor did it belong to serological group G.

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.