Transcriptome analysis of Streptococcus gordonii Challis DL1 indicates a role for the biofilm-associated fruRBA operon in response to Candida albicans.

Multiple levels of interkingdom signaling have been implicated in maintaining the ecological balance between Candida albicans and commensal streptococci to assure a state of oral health. To better understand the molecular mechanisms involved in the initial streptococcal response to the presence of C. albicans that can initiate oral surface colonization and biofilm formation, hypha-forming cells were incubated with Streptococcus gordonii cells for 30 min to assess the streptococcal transcriptome response. A genome-wide microarray analysis and quantitative polymerase chain reaction validation of S. gordonii transcripts identified a number of genes, the majority of which were involved in metabolic functions that were differentially expressed in the presence of hyphae. The fruR, fruB, and fruA genes encoding the transcriptional regulator, fructose-1-phosphate kinase, and fructose-specific permease, respectively, of the phosphoenolpyruvate-dependent fructose phosphotransferase system, were consistently upregulated. An S. gordonii mutant in which these genes were deleted by allelic replacement formed an architecturally distinct, less robust biofilm with C. albicans than did parental strain cells. Complementing the mutant with plasmid borne fruR, fruB, and fruA genes caused phenotype reversion, indicating that the genes in this operon played a role in dual-species biofilm formation. This genome-wide analysis of the S. gordonii transcriptional response to C. albicans has identified several genes that have potential roles in interkingdom signaling and responses.

Streptococcus mutans out-competes Streptococcus gordonii in vivo.

Streptococcus gordonii and Streptococcus mutans avidly colonize teeth. S. gordonii glucosyltransferase (GtfG) and amylase-binding proteins (AbpA/AbpB), and S. mutans glucosyltransferase (GtfB), affect their respective oral colonization abilities. We investigated their interrelationships and caries association in a rat model of human caries, examining the sequence of colonization and non- vs. high-sucrose diets, the latter being associated with aggressive decay in humans and rats. Virulence-characterized wild-types of both species and well-defined mutants of S. gordonii with interrupted abpA and gtfG genes were studied. While both S. gordonii and S. mutans were abundant colonizers of rat's teeth in the presence of either diet, if inoculated singly, S. mutans always out-competed S. gordonii on the teeth, independent of diet, strain of S. mutans, simultaneous or sequential inoculation, or presence/absence of mutations of S. gordonii's abpA and gtfG genes known to negatively or positively affect its colonization and to interact in vitro with S. mutans GtfB. S. mutans out-competed S. gordonii in in vivo plaque biofilm. Caries induction reflected S. mutans or S. gordonii colonization abundance: the former highly cariogenic, the latter not. S. gordonii does not appear to be a good candidate for replacement therapy. These results are consistent with human data.

Molecular analysis of 16S rRNA genes identifies potentially periodontal pathogenic bacteria and archaea in the plaque of partially erupted third molars.

PURPOSE: Small subunit rRNA sequencing and phylogenetic analysis were used to identify cultivable and uncultivable microorganisms present in the dental plaque of symptomatic and asymptomatic partially erupted third molars to determine the prevalence of putative periodontal pathogens in pericoronal sites. MATERIALS AND METHODS: Template DNA prepared from subgingival plaque collected from partially erupted symptomatic and asymptomatic mandibular third molars and healthy incisors was used in polymerase chain reaction with broad-range oligonucleotide primers to amplify 16S rRNA bacterial and archaeal genes. Amplicons were cloned, sequenced, and compared with known nucleotide sequences in online databases to identify the microorganisms present. RESULTS: Two thousand three hundred two clones from the plaque of 12 patients carried bacterial sequences from 63 genera belonging to 11 phyla, including members of the uncultivable TM7, SR1, and Chloroflexi, and difficult-to-cultivate Synergistetes and Spirochaetes. Dialister invisus, Filifactor alocis, Fusobacterium nucleatum, Porphyromonas endodontalis, Prevotella denticola, Tannerella forsythia, and Treponema denticola, which have been associated with periodontal disease, were found in significantly greater abundance in pericoronal compared with incisor sites. Dialister invisus and F nucleatum were found in greater abundance in sites exhibiting clinical symptoms. The archaeal species, Methanobrevibacter oralis, which has been associated with severe periodontitis, was found in 3 symptomatic patients. CONCLUSIONS: These findings have provided new insights into the complex microbiota of pericoronitis. Several bacterial and archaeal species implicated in periodontal disease were recovered in greater incidence and abundance from the plaque of partially erupted third molars compared with incisors, supporting the hypothesis that the pericoronal region may provide a favored niche for periodontal pathogens in otherwise healthy mouths.

A genetic determinant in Streptococcus gordonii Challis encodes a peptide with activity similar to that of enterococcal sex pheromone cAM373, which facilitates intergeneric DNA transfer.

Enterococcus faecalis strains secrete multiple peptides representing different sex pheromones that induce mating responses by bacteria carrying specific conjugative plasmids. The pheromone cAM373, which induces a response by the enterococcal plasmid pAM373, has been of interest because a similar activity is also secreted by Streptococcus gordonii and Staphylococcus aureus. The potential to facilitate intergeneric DNA transfer from E. faecalis is of concern because of extensive multiple antibiotic resistance, including vancomycin resistance, that has emerged among enterococci in recent years. Here, we characterize the related pheromone determinant in S. gordonii and show that the peptide it encodes, gordonii-cAM373, does indeed induce transfer of plasmid DNA from E. faecalis into S. gordonii. The streptococcal determinant camG encodes a lipoprotein with a leader sequence, the last 7 residues of which represent the gordonii-cAM373 heptapeptide SVFILAA. Synthetic forms of the peptide had activity similar to that of the enterococcal cAM373 AIFILAS. The lipoprotein moiety bore no resemblance to the lipoprotein encoded by E. faecalis. We also identified determinants in S. gordonii encoding a signal peptidase and an Eep-like zinc metalloprotease (lspA and eep, respectively) similar to those involved in processing certain pheromone precursors in E. faecalis. Mutations generated in camG, lspA, and eep each resulted in the ablation of gordonii-cAM373 activity in culture supernatants. This is the first genetic analysis of a potential sex pheromone system in a commensal oral streptococcal species, which may have implications for intergeneric gene acquisition in oral biofilms.

Regulation of gene expression in a mixed-genus community: stabilized arginine biosynthesis in Streptococcus gordonii by coaggregation with Actinomyces naeslundii.

Interactions involving genetically distinct bacteria, for example, between oral streptococci and actinomyces, are central to dental plaque development. A DNA microarray identified Streptococcus gordonii genes regulated in response to coaggregation with Actinomyces naeslundii. The expression of 23 genes changed >3-fold in coaggregates, including that of 9 genes involved in arginine biosynthesis and transport. The capacity of S. gordonii to synthesize arginine was assessed using a chemically defined growth medium. In monoculture, streptococcal arginine biosynthesis was inefficient and streptococci could not grow aerobically at low arginine concentrations. In dual-species cultures containing coaggregates, however, S. gordonii grew to high cell density at low arginine concentrations. Equivalent cocultures without coaggregates showed no growth until coaggregation was evident (9 h). An argH mutant was unable to grow at low arginine concentrations with or without A. naeslundii, indicating that arginine biosynthesis was essential for coaggregation-induced streptococcal growth. Using quantitative reverse transcriptase PCR, the expression of argC, argG, and pyrA(b) was strongly (10- to 100-fold) up-regulated in S. gordonii monocultures after 3 h of growth when exogenous arginine was depleted. Cocultures without induced coaggregation showed similar regulation. However, within 1 h after coaggregation with A. naeslundii, the expression of argC, argG, and pyrA(b) in S. gordonii was partially up-regulated although arginine was plentiful, and mRNA levels did not increase further when arginine was diminished. Thus, A. naeslundii stabilizes S. gordonii expression of arginine biosynthesis genes in coaggregates but not cocultures and enables aerobic growth when exogenous arginine is limited.

Streptococcus gordonii's sequenced strain CH1 glucosyltransferase determines persistent but not initial colonization of teeth of rats.

OBJECTIVE: Extracellular glucan synthesis from sucrose by Streptococcus gordonii, a major dental plaque biofilm bacterium, is assumed important for colonization of teeth; but this hypothesis is un-tested in vivo. METHODS: To do so, we studied an isogenic glucosyltransferase (Gtf)-negative mutant (strain AMS12, gtfG(-)) of S. gordonii sequenced wild type (WT, strain Challis CH1, gtfG(+)), comparing their in vitro abilities to grow in the presence of glucose and sucrose and, in vivo, to colonize and persist on teeth and induce caries in rats. Weanling rats of two breeding colonies, TAN:SPFOM(OM)BR and TAN:SPFOM(OMASF)BR, eating high sucrose diet, were inoculated with either the WT (gtfG(+)), its isogenic gtfG(-) mutant, or reference strains of Streptococcus mutans. Control animals were not inoculated. RESULTS: In vitro, the gtfG(-) strain grew at least as rapidly in the presence of sucrose as its WT gtfG(+) progenitor, but formed soft colonies on sucrose agar, consistent with its lack of insoluble glucan synthesis. It also had a higher growth yield due apparently to its inability to channel carbon flow into extracellular glucan. In vivo, the gtfG(-) mutant initially colonized as did the WT but, unlike the WT, failed to persist on the teeth as shown over time. By comparison to three S. mutans strains, S. gordonii WT, despite its comparable ecological success on the teeth, was associated with only modest caries induction. Failure of the gtfG(-) mutant to persistently colonize was associated with slight diminution of caries scores by comparison with its gtfG(+) WT. CONCLUSIONS: Initial S. gordonii colonization does not depend on Gtf-G synthesis; rather, Gtf-G production determines S. gordonii's ability to persist on the teeth of sucrose-fed rats. S. gordonii appears weakly cariogenic by comparison with S. mutans reference strains.

Genome-wide transcriptional changes in Streptococcus gordonii in response to competence signaling peptide.

Streptococcus gordonii is a primary colonizer of the multispecies biofilm on tooth surfaces forming dental plaque and a potential agent of endocarditis. The recent completion of the genome sequence of the naturally competent strain Challis allowed the design of a spotted oligonucleotide microarray to examine a genome-wide response of this organism to environmental stimuli such as signal peptides. Based on temporal responses to synthetic competence signaling peptide (CSP) as indicated by transformation frequencies, the S. gordonii transcriptome was analyzed at various time points after CSP exposure. Microarray analysis identified 35 candidate early genes and 127 candidate late genes that were up-regulated at 5 and 15 min, respectively; these genes were often grouped in clusters. Results supported published findings on S. gordonii competence, showing up-regulation of 12 of 16 genes that have been reported to affect transformation frequencies in this species. Comparison of CSP-induced S. gordonii transcriptomes to results published for Streptococcus pneumoniae strains identified both conserved and species-specific genes. Putative intergenic regulatory sites, such as the conserved combox sequence thought to be a binding site for competence sigma factor, were found preceding S. gordonii late responsive genes. In contrast, S. gordonii early CSP-responsive genes were not preceded by the direct repeats found in S. pneumoniae. These studies provide the first insights into a genome-wide transcriptional response of an oral commensal organism. They offer an extensive analysis of transcriptional changes that accompany competence in S. gordonii and form a basis for future intra- and interspecies comparative analyses of this ecologically important phenotype.

Phylogenetic analysis of bacterial and archaeal species in symptomatic and asymptomatic endodontic infections.

Phylogenetic analysis of bacterial and archaeal 16S rRNA was used to examine polymicrobial communities within infected root canals of 20 symptomatic and 14 asymptomatic patients. Nucleotide sequences from approximately 750 clones amplified from each patient group with universal bacterial primers were matched to the Ribosomal Database Project II database. Phylotypes from 37 genera representing Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria and Proteobacteria were identified. Results were compared to those obtained with species-specific primers designed to detect Prevotella intermedia, Porphyromonas gingivalis, Porphyromonas endodontalis, Peptostreptococcus micros, Enterococcus sp., Streptococcus sp., Fusobacterium nucleatum, Tannerella forsythensis and Treponema denticola. Since members of the domain Archaea have been implicated in the severity of periodontal disease, and a recent report confirms that archaea are present in endodontic infections, 16S archaeal primers were also used to detect which patients carried these prokaryotes, to determine if their presence correlated with severity of the clinical symptoms. A Methanobrevibacter oralis-like species was detected in one asymptomatic and one symptomatic patient. DNA from root canals of these two patients was further analysed using species-specific primers to determine bacterial cohabitants. Trep. denticola was detected in the asymptomatic but not the symptomatic patient. Conversely, Porph. endodontalis was found in the symptomatic but not the asymptomatic patient. All other species except enterococci were detected with the species-specific primers in both patients. These results confirm the presence of archaea in root canals and provide additional insights into the polymicrobial communities in endodontic infections associated with clinical symptoms.

Glucan-binding proteins of the oral streptococci.

The synthesis of extracellular glucan is an integral component of the sucrose-dependent colonization of tooth surfaces by species of the mutans streptococci. In investigators' attempts to understand the mechanisms of plaque biofilm development, several glucan-binding proteins (GBPs) have been discovered. Some of these, the glucosyltransferases, catalyze the synthesis of glucan, whereas others, designated only as glucan-binding proteins, have affinities for different forms of glucan and contribute to aspects of the biology of their host organisms. The functions of these latter glucan-binding proteins include dextran-dependent aggregation, dextranase inhibition, plaque cohesion, and perhaps cell wall synthesis. In some instances, their glucan-binding domains share common features, whereas in others the mechanism for glucan binding remains unknown. Recent studies indicate that at least some of the glucan-binding proteins modulate virulence and some can act as protective immunogens within animal models. Overall, the multiplicity of GBPs and their aforementioned properties are testimonies to their importance. Future studies will greatly advance the understanding of the distribution, function, and regulation of the GBPs and place into perspective the facets of their contributions to the biology of the oral streptococci.

An amino acid change near the carboxyl terminus of the Streptococcus gordonii regulatory protein Rgg affects its abilities to bind DNA and influence expression of the glucosyltransferase gene gtfG.

The Streptococcus gordonii glucosyltransferase structural gene, gtfG, is located immediately downstream from its positive transcriptional regulatory determinant, rgg. Recent genetic studies have indicated that the 3' end of rgg is involved either directly as a binding site or indirectly, e.g. by playing a role in secondary structure, in the interaction of Rgg with the gtfG promoter. A previously identified spontaneous mutant with a point mutation near the 3' end of rgg had only approximately 25% of the parental level of glucosyltransferase activity. To determine if this decreased activity was due to a change in the DNA binding site of trans-acting Rgg, or due to a change in the Rgg protein itself, complementation analyses and DNA-binding studies were performed. In Rgg-deficient strains, the chromosomal rgg point mutation did not influence the ability of plasmid-borne rgg to increase glucosyltransferase expression. However, plasmids carrying parental rgg were able to increase glucosyltransferase activity and expression of a gtfG promoter fusion to a greater extent than plasmids carrying the mutant allele, indicating that the mutant Rgg protein had decreased activity. The ability of NH(2)-terminal (hexahistidine) tagged proteins to bind to a 107 bp dsDNA fragment corresponding to the region immediately upstream of gtfG was demonstrated by surface plasmon resonance. Despite their differences in activity, both mutant and parental recombinant Rgg proteins bound to this dsDNA, albeit with different strengths. These studies provide insights into functional domains of S. gordonii Rgg which influence glucosyltransferase expression, and may have implications for Rgg-like regulatory proteins in related bacteria.

Genetic analysis of the rgg-gtfG junctional region and its role in Streptococcus gordonii glucosyltransferase activity.

Glucans synthesized by glucosyltransferase enzymes of oral streptococci facilitate bacterial accumulation on surfaces. The Streptococcus gordonii glucosyltransferase gene, gtfG, is positively regulated by rgg, which encodes a putative cytoplasmic protein. The gtfG promoter and ribosomal binding sequences are located within a DNA inverted repeat immediately downstream of rgg. Polycistronic rgg-gtfG as well as rgg- and gtfG-specific transcripts are associated with this chromosomal region. Previous studies have shown that the rgg product acts in trans near the gtfG promoter to increase the level of gtfG transcript, but it does not affect the level of rgg-gtfG transcript. To further analyze regulation by rgg, a series of strain Challis derivatives was constructed and glucosyltransferase activities were determined. Strains in which rgg was separated from gtfG by integrated vector sequences had decreased levels of glucosyltransferase activity; plasmid-borne rgg could not increase activity to parental levels. As expected, strains with chromosomal deletions involving the rgg structural gene and either the rgg or gtfG promoter also showed decreased glucosyltransferase activity. Plasmid-borne rgg could increase glucosyltransferase activity only in strains which had a 36-bp chromosomal region beginning 72 nucleotides upstream of the gtfG transcriptional start site. Results suggest that these nucleotides, located within the 3' end of rgg, are necessary, either by direct involvement in binding or by indirectly affecting secondary structure, for Rgg to increase glucosyltransferase activity. Surprisingly, the presence of the rgg promoter upstream of this 36-bp region significantly increased the effects of plasmid-borne rgg. Implications for glucosyltransferase regulation and applicability to other rgg-like determinants are considered.

Initial characterization of the Streptococcus gordonii htpX gene.

Examination of the Streptococcus gordonii chromosomal region, which lies immediately upstream of the glucosyltransferase positive regulatory determinant rgg, revealed two open reading frames. Based on nucleotide sequences, these genes were similar to the Listeria monocytogenes lemA gene, which is involved in antigen presentation, and the Escherichia coli htpX heat shock gene, which has an unknown function. Northern hybridization analysis indicated that S. gordonii lemA and htpX genes were associated with a ca. 1.7-kb polycistronic transcript. Although levels of the lemA/htpX transcript did not increase in response to heat to levels seen with dnaK controls, insertional inactivation of htpX resulted in changes in adhesiveness, cellular morphology and detergent-extractable surface antigens in cells grown at 41 degrees C, implying that htpX may be involved in surface protein expression. Insertional inactivation of lemA and htpX indicated that, despite their proximity to rgg and the structural gene, gtfG, these upstream genes do not affect S. gordonii glucosyltransferase activity.

Characterization of the Streptococcus gordonii chromosomal region immediately downstream of the glucosyltransferase gene.

The Streptococcus gordonii glucosyltransferase gene, gtfG, is positively regulated by the upstream determinant rgg. In the present study, two ORFs, transcribed on the opposite DNA strand, were identified immediately downstream of gtfG. The first, designated dsg, shares a convergent putative transcriptional terminator with gtfG, and encodes a predicted 46 kDa transmembrane protein similar to the Yersinia enterocolitica TrsA involved in polysaccharide biosynthesis. Insertional inactivation of dsg resulted in only approximately approximately 60% of the parental level of glucosyltransferase activity. The 870 bp gene 5' to dsg is similar to the gtfG regulatory determinant. Designated rggD, this rgg-like determinant downstream of gtfG encodes a putative 33.6 kDa cytoplasmic protein. Despite their sequence similarity, the functions of rgg and rggD appear specific. Strains in which rggD was insertionally inactivated and strains containing plasmid-borne rggD had parental levels of glucosyltransferase activity. Northern blot hybridization analyses showed approximately 1.3 kb dsg-specific and approximately 1.0 kb rggD-specific mRNA transcripts associated with this region; no polycistronic transcript was observed. Although rgg-like gene products have been demonstrated to function as positive transcriptional regulators of adjacent genes in several streptococcal species, Northern blot analysis suggested that rggD did not influence the transcription of dsg or the divergent downstream ylbN-like determinant under the conditions in the present study. Comparison of this S. gordonii chromosome region to other streptococcal genomes, which do not contain the rgg/rggD-flanked region involved in glucan synthesis, raised intriguing possibilities about the origins of this chromosomal region, and also suggested that rggD might regulate a distally located gene.

Deletions in the carboxyl-terminal region of Streptococcus gordonii glucosyltransferase affect cell-associated enzyme activity and sucrose-associated accumulation of growing cells.

The single glucosyltransferase (GTF) of Streptococcus gordonii Challis CH1 makes alpha 1,3- and alpha 1,6-linked glucans from sucrose. The GTF carboxyl-terminal region has six direct repeats thought to be involved in glucan binding. Strains with defined mutations in this region have been described recently (M. M. Vickerman, M. C. Sulavik, P. E. Minick, and D. B. Clewell, Infect. Immun. 64:5117-5128, 1996). Strain CH107 GTF has three internal direct repeats deleted; the 59 carboxyl-terminal amino acids are identical to those of the parental strain. This deletion resulted in decreased enzyme activity but did not affect the amount of cell-associated GTF protein. The GTFs of strains CH2RPE and CH4RPE have six and eight direct repeats, respectively, but are both missing the 14 carboxyl-terminal amino acids. Strain CH2RPE had significantly decreased levels of cell-associated GTF; this decrease was not obviated by the increased number of direct repeats in strain CH4RPE. Thus, the carboxyl-terminal amino acids appeared to influence the amount of cell-associated GTF more than the direct repeats. The qualitative and quantitative differences in the GTFs did not affect the abilities of these strains to accumulate on hydroxyapatite beads in the absence of sucrose. However, when sucrose was added as a substrate for GTF, the mutant strains were unable to accumulate on these surfaces to the same extent as the parent. These differences in sucrose-associated accumulation may be due to changes in the nature of the glucans produced by the different enzymes and/or cohesive interactions between these glucans and the GTF on the surfaces of the growing streptococci.

Molecular analysis of representative Streptococcus gordonii Spp phase variants reveals no differences in the glucosyltransferase structural gene, gtfG.

Streptococcus gordonii glucosyltransferase polymerizes sucrose to form glucans, which confer a hard, sucrose-promoted phenotype (Spp+) to colonies on sucrose agar plates. The glucosyltransferase structural gene, gtfG, is positively regulated by the upstream determinant, rgg. Strain Challis undergoes a spontaneous, reversible phase variation between high (Spp+) and low (Spp-) levels of glucosyltransferase activity. Representative strains were examined to gain insights into the basis of glucosyltransferase phase variation. Western blots indicated that the level of glucosyltransferase activity was related to the amount of extracellular glucosyltransferase protein produced by Spp- and Spp+ strains. The nucleotide sequence of rgg and gtfG of the Spp- strain CH97 was found to be identical to that of the Spp+ parent, indicating that DNA differences in these regions are not the basis for glucosyltransferase phase variation. Indeed, 13C-NMR spectroscopy suggested that glucans synthesized by strain CH97 glucosyltransferase were similar to those synthesized by glucosyltransferase of the Spp+ parental strain, indicating a quantitative rather than qualitative change. However, one Spp- strain, CH1C1, had a point mutation in rgg; replacement of the parent rgg with the CH1C1 allele resulted in decreased levels of glucosyltransferase protein and activity. The results indicate that glucosyltransferase phase variation can occur in more than one way, and suggest that glucosyltransferase regulation may involve distally located regulatory gene(s) that affect rgg and/or gtfG expression.

Nucleotide sequence analysis of the Streptococcus gordonii glucosyltransferase gene, gtfG.

Streptococcus gordonii has an extracellular glucosyltransferase (GTF) that polymerizes the glucose moiety of sucrose to form both water-soluble and water-insoluble glucans. Whereas multiple gtf genes have been identified in strains of mutans streptococci and Streptococcus salivarius, a single gene, designated gtfG, encodes the GTF of S. gordonii Challis. gtfG is also unique among the characterized gtfs in that it has a described regulatory determinant, rgg. Furthermore, the GTF activity in S. gordonii undergoes reversible phase variation between high and low levels. In order to gain insight into this novel GTF system, the nucleotide sequence of gtfG was determined and found to consist of a 4,734 base pair open reading frame encoding a protein with a deduced molecular weight of ca. 174,000. gtfG was similar to other sequenced gtfs with a conserved signal sequence followed by a ca. 600-bp region distinctive for gtfG, a conserved region encoding a putative catalytic active site and a series of six direct repeats in the carboxyl terminal region implicated in glucan binding. Although comparison of gtfG to other gtfs did not show a basis for the primer-independence of the encoded enzyme or the nature of the glucan products, the gtfG sequence data provide an important basis for further studies of these enzymes.

Changes in the carboxyl-terminal repeat region affect extracellular activity and glucan products of Streptococcus gordonii glucosyltransferase.

Glucans produced by the glucosyltransferase (GTF) of Streptococcus gordonii confer a hard, cohesive phenotype (Spp+) on colonies grown on sucrose agar plates. S. gordonii strains with specific mutations in the region of gtfG that encodes the GTF carboxyl terminus were characterized. In the parental strain Challis CH1, this region included a series of six direct repeats thought to function in glucan binding. The spontaneous mutant strain CH107 had a 585-bp deletion resulting in the loss of three internal direct repeats. Insertional mutagenesis was used to construct strain CH2RPE, which had the parental repeat region but was missing 14 carboxyl-terminal amino acids. The similarly constructed strain CH4RPE had an in-frame addition of 390 nucleotides encoding two additional direct repeats. Although strains CH1, CH2RPE, and CH4RPE all had similar levels of extracellular GTF activity, strain CH107 had less than 15% of the parental activity; however, Western blots (immunoblots) indicated that the amounts of extracellular GTF protein in all four strains were similar. 13C NMR analyses indicated that partially purified GTFs from the Spp+ strains CH1, CH2RPE, and CH4RPE all produced glucans with similar ratios of alpha1,6 and alpha1,3 glucosidic linkages, whereas the Spp- strain CH107 GTF produced primarily alpha1,6-linked glucans. Transformation of strain CH107 with pAMS57, which carries the gtfG positive regulatory determinant, rgg, increased the amount of GTF activity and GTF antibody-reactive protein ca. fivefold but did not confer a hard colony phenotype on sucrose agar plates, suggesting that the type of glucan product affects the sucrose-promoted colony phenotype.

Multiple phase variation in haemolytic, adhesive and antigenic properties of Streptococcus gordonii.

Streptococcus gordonii gave rise to beta-haemolytic variants (Bhp+ for beta-haemolysin production) at frequencies of 10(-4)-10(-3) on agar medium containing washed horse erythrocytes. Bhp+ variants reverted to the wild-type alpha-haemolytic phenotype (Bhp-) at the same frequencies. There was a significant probability (> or = 0.1) that phase variation in Bhp and phase variation in the previously described Spp (sucrose promoted phenotype) would occur concomitantly, but there was no correlation between these phenotypes. There was evidence also of independent phase variation in adhesion to saliva-coated hydroxyapatite (Asp for adhesion to salivary pellicles), in lactose-sensitive coaggregation (Cls for coaggregation, lactose-sensitive) and in the concentrations of particular cell surface antigens (Cap for cell antigen profile) in strains that had undergone phase changes in Spp and/or Bhp. Phase variation in all these phenotypes were transitions between high and low levels of activity and each appeared to occur as an independent event. Significant associations (P << 0.0001 by contingency table analysis) between particular phenotypes such as Bhp and Asp and between Asp, Cls and Cap phenotypes, however, were apparent. The results suggest that S. gordonii cells become predisposed to phase variation and that the resulting independent phenotypic changes may give rise to phenotypically diverse streptococcal populations able to accommodate rapid and transient environmental changes in the mouth.

Oral streptococci with genetic determinants similar to the glucosyltransferase regulatory gene, rgg.

The Streptococcus gordonii Challis glucosyltransferase structural gene, gtfG, is positively regulated by the upstream gene, rgg, the only described gtf regulatory determinant in oral streptococci. Southern hybridization analyses indicated that rgg-like and gtfG-like determinants were present on the same HindIII fragment in strains of S. gordonii, Streptococcus sanguis, and Streptococcus oralis, whereas no rgg-like determinants were detected in mutans streptococci, Streptococcus mitis, and Streptococcus salivarius.