Characterization of the Trehalose Utilization Operon in Streptococcus mutans Reveals that the TreR Transcriptional Regulator Is Involved in Stress Response Pathways and Toxin Production.

Streptococcus mutans, the organism most frequently associated with the development of dental caries, is able to utilize a diverse array of carbohydrates for energy metabolism. One such molecule is trehalose, a disaccharide common in human foods, which has been recently implicated in enhancing the virulence of epidemic strains of the pathogen Clostridium difficile In this study, mutants with deletions of all three genes in the putative S. mutans trehalose utilization operon were characterized, and the genes were shown to be required for wild-type levels of growth when trehalose was the only carbohydrate source provided. Interestingly, the TreR transcriptional regulator appeared to be critical for responding to oxidative stress and for mounting a protective stress tolerance response following growth at moderately acidic pH. mRNA sequencing (RNA-seq) of a treR deletion mutant suggested that in S. mutans, TreR acts as a trehalose-sensing activator of transcription of the tre operon, rather than as a repressor, as described in other species. In addition, deletion of treR caused the downregulation of a number of genes involved in genetic competence and bacteriocin production, supporting the results of a recent study linking trehalose and the S. mutans competence pathways. Finally, deletion of treR compromised the ability of S. mutans to inhibit the growth of the competing species Streptococcus gordonii and Lactococcus lactis Taking the results together, this study solidifies the role of the S. mutans tre operon in trehalose utilization and suggests novel functions for the TreR regulator, including roles in the stress response and competitive fitness.IMPORTANCES. mutans is the primary etiologic agent of dental caries, which globally is the most common chronic disease. S. mutans must be able to outcompete commensal organisms in its dental plaque niche in order to establish persistence and pathogenesis. To that end, S. mutans metabolizes a diverse array of carbohydrates to generate acid and impede its acid-sensitive neighbors. Additionally, S. mutans utilizes quorum signaling through genetic competence-associated pathways to induce production of toxins to kill its rivals. This study definitively shows that the S. mutans trehalose utilization operon is required for growth in trehalose. Furthermore, this study suggests that the S. mutans TreR transcriptional regulator has a novel role in virulence through regulation of genes involved in genetic competence and toxin production.

A Drug Repositioning Approach Reveals that Streptococcus mutans Is Susceptible to a Diverse Range of Established Antimicrobials and Nonantibiotics.

Streptococcus mutans is the primary causative agent of dental caries and contributes to the multispecies biofilm known as dental plaque. An adenylate kinase-based assay was optimized for S. mutans to detect cell lysis when exposed to the Selleck library (Selleck Chemical, Houston, TX) of 853 FDA-approved drugs in, to our knowledge, the first high-throughput drug screen in S. mutans We found 126 drugs with activity against S. mutans planktonic cultures, and they were classified into six categories: antibacterials (61), antineoplastics (23), ion channel effectors (9), other antimicrobials (7), antifungals (6), and other (20). These drugs were also tested for activity against S. mutans biofilm cultures, and 24 compounds were found to inhibit biofilm formation, 6 killed preexisting biofilms, 84 exhibited biofilm inhibition and killing activity, and 12 had no activity against biofilms. The activities of 9 selected compounds that exhibited antimicrobial activity were further characterized for their activity against S. mutans planktonic and biofilm cultures. Together, our results suggest that S. mutans exhibits a susceptibility profile to a diverse array of established and novel antibacterials.

Vitamin D Compounds Are Bactericidal against Streptococcus mutans and Target the Bacitracin-Associated Efflux System.

Vitamin D analogs were identified as compounds that induced lysis of planktonic cultures of Streptococcus mutans in a high-throughput screen of FDA-approved drugs. Previous studies have demonstrated that certain derivatives of vitamin D possess lytic activity against other bacteria, though the mechanism has not yet been established. Through the use of a combinatorial approach, the vitamin D derivative doxercalciferol was shown to act synergistically with bacitracin, a polypeptide-type drug that is known to interfere with cell wall synthesis, suggesting that doxercalciferol may act in a bacitracin-related pathway. Innate resistance to bacitracin is attributed to efflux by a conserved ABC-type transporter, which in S. mutans is encoded by the mbrABCD operon. S. mutans possesses two characterized mechanisms of resistance to bacitracin, the ABC transporter, S. mutans bacitracin resistance (Mbr) cassette, consisting of MbrABCD, and the rhamnose-glucose polysaccharide (Rgp) system, RgpABCDEFGHI. Loss of function of the transporter in ΔmbrA and ΔmbrD mutants exacerbated the effect of the combination of doxercalciferol and bacitracin. Despite conservation of a transporter homologous to mbrABCD, the combination of doxercalciferol and bacitracin appeared to be synergistic only in streptococcal species. We conclude that vitamin D derivatives possess lytic activity against S. mutans and act through a mechanism dependent on the bacitracin resistance mechanism of MbrABCD.

RgpF Is Required for Maintenance of Stress Tolerance and Virulence in Streptococcus mutans.

Bacterial cell wall dynamics have been implicated as important determinants of cellular physiology, stress tolerance, and virulence. In Streptococcus mutans, the cell wall is composed primarily of a rhamnose-glucose polysaccharide (RGP) linked to the peptidoglycan. Despite extensive studies describing its formation and composition, the potential roles for RGP in S. mutans biology have not been well investigated. The present study characterizes the impact of RGP disruption as a result of the deletion of rgpF, the gene encoding a rhamnosyltransferase involved in the construction of the core polyrhamnose backbone of RGP. The ΔrgpF mutant strain displayed an overall reduced fitness compared to the wild type, with heightened sensitivities to various stress-inducing culture conditions and an inability to tolerate acid challenge. The loss of rgpF caused a perturbation of membrane-associated functions known to be critical for aciduricity, a hallmark of S. mutans acid tolerance. The proton gradient across the membrane was disrupted, and the ΔrgpF mutant strain was unable to induce activity of the F1Fo ATPase in cultures grown under low-pH conditions. Further, the virulence potential of S. mutans was also drastically reduced following the deletion of rgpF The ΔrgpF mutant strain produced significantly less robust biofilms, indicating an impairment in its ability to adhere to hydroxyapatite surfaces. Additionally, the ΔrgpF mutant lost competitive fitness against oral peroxigenic streptococci, and it displayed significantly attenuated virulence in an in vivoGalleria mellonella infection model. Collectively, these results highlight a critical function of the RGP in the maintenance of overall stress tolerance and virulence traits in S. mutansIMPORTANCE The cell wall of Streptococcus mutans, the bacterium most commonly associated with tooth decay, is abundant in rhamnose-glucose polysaccharides (RGP). While these structures are antigenically distinct to S. mutans, the process by which they are formed and the enzymes leading to their construction are well conserved among streptococci. The present study describes the consequences of the loss of RgpF, a rhamnosyltransferase involved in RGP construction. The deletion of rgpF resulted in severe ablation of the organism's overall fitness, culminating in significantly attenuated virulence. Our data demonstrate an important link between the RGP and cell wall physiology of S. mutans, affecting critical features used by the organism to cause disease and providing a potential novel target for inhibiting the pathogenesis of S. mutans.

Loss of NADH Oxidase Activity in Streptococcus mutans Leads to Rex-Mediated Overcompensation in NAD+ Regeneration by Lactate Dehydrogenase.

UNLABELLED: Previous studies of the oral pathogen Streptococcus mutans have determined that this Gram-positive facultative anaerobe mounts robust responses to both acid and oxidative stresses. The water-forming NADH oxidase (Nox; encoded by nox) is thought to be critical for the regeneration of NAD(+), for use in glycolysis, and for the reduction of oxygen, thereby preventing the formation of damaging reactive oxygen species. In this study, the free NAD(+)/NADH ratio in a nox deletion strain (Δnox) was discovered to be remarkably higher than that in the parent strain, UA159, when the strains were grown in continuous culture. This unanticipated result was explained by significantly elevated lactate dehydrogenase (Ldh; encoded by ldh) activity and ldh transcription in the Δnox strain, which was mediated in part by the redox-sensing regulator Rex. cDNA microarray analysis of S. mutans cultures exposed to simultaneous acid stress (growth at a low pH) and oxidative stress (generated through the deletion of nox or the addition of exogenous oxygen) revealed a stress response synergistically heightened over that with either stress alone. In the Δnox strain, this elevated stress response included increased glucose phosphoenolpyruvate phosphotransferase system (PTS) activity, which appeared to be due to elevated manL transcription, mediated in part, like elevated ldh transcription, by Rex. While the Δnox strain does possess a membrane composition different from that of the parent strain, it did not appear to have defects in either membrane permeability or ATPase activity. However, the altered transcriptome and metabolome of the Δnox strain were sufficient to impair its ability to compete with commensal peroxigenic oral streptococci during growth under aerobic conditions. IMPORTANCE: Streptococcus mutans is an oral pathogen whose ability to outcompete commensal oral streptococci is strongly linked to the formation of dental caries. Previous work has demonstrated that the S. mutans water-forming NADH oxidase is critical for both carbon metabolism and the prevention of oxidative stress. The results of this study show that upregulation of lactate dehydrogenase, mediated through the redox sensor Rex, overcompensates for the loss of nox. Additionally, nox deletion led to the upregulation of mannose and glucose transport, also mediated through Rex. Importantly, the loss of nox rendered S. mutans defective in its ability to compete directly with two species of commensal streptococci, suggesting a role for nox in the pathogenic potential of this organism.

Streptococcus mutans NADH oxidase lies at the intersection of overlapping regulons controlled by oxygen and NAD+ levels.

NADH oxidase (Nox, encoded by nox) is a flavin-containing enzyme used by the oral pathogen Streptococcus mutans to reduce diatomic oxygen to water while oxidizing NADH to NAD(+). The critical nature of Nox is 2-fold: it serves to regenerate NAD(+), a carbon cycle metabolite, and to reduce intracellular oxygen, preventing formation of destructive reactive oxygen species (ROS). As oxygen and NAD(+) have been shown to modulate the activity of the global transcription factors Spx and Rex, respectively, Nox is potentially poised at a critical junction of two stress regulons. In this study, microarray data showed that either addition of oxygen or loss of nox resulted in altered expression of genes involved in energy metabolism and transport and the upregulation of genes encoding ROS-metabolizing enzymes. Loss of nox also resulted in upregulation of several genes encoding transcription factors and signaling molecules, including the redox-sensing regulator gene rex. Characterization of the nox promoter revealed that nox was regulated by oxygen, through SpxA, and by Rex. These data suggest a regulatory loop in which the roles of nox in reduction of oxygen and regeneration of NAD(+) affect the activity levels of Spx and Rex, respectively, and their regulons, which control several genes, including nox, crucial to growth of S. mutans under conditions of oxidative stress.

Repression of the TreR transcriptional regulator in Streptococcus mutans by the global regulator, CcpA.

Streptococcus mutans, the etiologic agent of dental caries in humans, is considered a dominating force in the oral microbiome due to its highly-evolved propensity for survival. The oral pathogen encodes an elaborate array of regulatory elements, including the carbon catabolite-responsive regulator, CcpA, a global regulator key in the control of sugar metabolism and in stress tolerance response mechanisms. The recently characterized trehalose utilization operon, integral for the catabolism of the disaccharide trehalose, is controlled by a local regulator, TreR, which has been implicated in a number of cellular functions outside of trehalose catabolism. Electrophoretic mobility shift assays demonstrated that CcpA bound a putative cre site in the treR promoter. Loss of ccpA resulted in elevated expression of treR in cultures of the organism grown in glucose or trehalose, indicating that CcpA not only acts as a repressor of trehalose catabolism genes, but also the local regulator. The loss of both CcpA and TreR in S. mutans resulted in an impaired growth rate and fitness response, supporting the hypothesis that these regulators are involved in carbon catabolism control and in induction of components of the organism's stress response.

Oral Microbiota Composition Predicts Early Childhood Caries Onset.

As the most common chronic disease in preschool children in the United States, early childhood caries (ECC) has a profound impact on a child's quality of life, represents a tremendous human and economic burden to society, and disproportionately affects those living in poverty. Caries risk assessment (CRA) is a critical component of ECC management, yet the accuracy, consistency, reproducibility, and longitudinal validation of the available risk assessment techniques are lacking. Molecular and microbial biomarkers represent a potential source for accurate and reliable dental caries risk and onset. Next-generation nucleotide-sequencing technology has made it feasible to profile the composition of the oral microbiota. In the present study, 16S ribosomal RNA (rRNA) gene sequencing was applied to saliva samples that were collected at 6-mo intervals for 24 mo from a subset of 56 initially caries-free children from an ongoing cohort of 189 children, aged 1 to 3 y, over the 2-y study period; 36 children developed ECC and 20 remained caries free. Analyses from machine learning models of microbiota composition, across the study period, distinguished between affected and nonaffected groups at the time of their initial study visits with an area under the receiver operating characteristic curve (AUC) of 0.71 and discriminated ECC-converted from healthy controls at the visit immediately preceding ECC diagnosis with an AUC of 0.89, as assessed by nested cross-validation. Rothia mucilaginosa, Streptococcus sp., and Veillonella parvula were selected as important discriminatory features in all models and represent biomarkers of risk for ECC onset. These findings indicate that oral microbiota as profiled by high-throughput 16S rRNA gene sequencing is predictive of ECC onset.

Association between Oral Candida and Bacteriome in Children with Severe ECC.

Candida albicans is an opportunistic fungal organism frequently detected in the oral cavity of children with severe early childhood caries (S-ECC). Previous studies suggested the cariogenic potential of C. albicans, in vitro and in vivo, and further demonstrated its synergistic interactions with Streptococcus mutans. In combination, the 2 organisms are associated with higher caries severity in a rodent model. However, it remains unknown whether C. albicans influences the composition and diversity of the entire oral bacterial community to promote S-ECC onset. With 16s rRNA amplicon sequencing, this study analyzed the microbiota of saliva and supragingival plaque from 39 children (21 S-ECC and 18 caries-free [CF]) and 33 mothers (17 S-ECC and 16 CF). The results revealed that the presence of oral C. albicans is associated with a highly acidogenic and acid-tolerant bacterial community in S-ECC, with an increased abundance of plaque Streptococcus (particularly S. mutans) and certain Lactobacillus/Scardovia species and salivary/plaque Veillonella and Prevotella, as well as decreased levels of salivary/plaque Actinomyces. Concurrent with this microbial community assembly, the activity of glucosyltransferases (cariogenic virulence factors secreted by S. mutans) in plaque was significantly elevated when C. albicans was present. Moreover, the oral microbial community composition and diversity differed significantly by disease group (CF vs. S-ECC) and sample source (saliva vs. plaque). Children and mothers within the CF and S-ECC groups shared microbiota composition and diversity, suggesting a strong maternal influence on children's oral microbiota. Altogether, this study underscores the importance of C. albicans in association with the oral bacteriome in the context of S-ECC etiopathogenesis. Further longitudinal studies are warranted to examine how fungal-bacterial interactions modulate the onset and severity of S-ECC, potentially leading to novel anticaries treatments that address fungal contributions.

Oral Sciences PhD Program Enrollment, Graduates, and Placement: 1994 to 2016.

For decades, dental schools in the United States have endured a significant faculty shortage. Studies have determined that the top 2 sources of dental faculty are advanced education programs and private practice. Those who have completed both DDS and PhD training are considered prime candidates for dental faculty positions. However, there is no national database to track those trainees and no evidence to indicate that they entered academia upon graduation. The objective of this study was to assess outcomes of dental school-affiliated oral sciences PhD program enrollment, graduates, and placement between 1994 and 2016. Using the American Dental Association annual survey of advanced dental education programs not accredited by the Commission on Dental Accreditation and data obtained from 22 oral sciences PhD programs, we assessed student demographics, enrollment, graduation, and placement. Based on the data provided by program directors, the average new enrollment was 33, and graduation was 26 per year. A total of 605 graduated; 39 did not complete; and 168 were still in training. Among those 605 graduates, 211 were faculty in U.S. academic institutions, and 77 were faculty in foreign institutions. Given that vacant budgeted full-time faculty positions averaged 257 per year during this period, graduates from those oral sciences PhD programs who entered academia in the United States would have filled 9 (3.6%) vacant faculty positions per year. Therefore, PhD programs have consistently generated only a small pipeline of dental school faculty. Better mentoring to retain talent in academia is necessary. Stronger support and creative funding plans are essential to sustain the PhD program. Furthermore, the oral sciences PhD program database should be established and maintained by dental professional organizations to allow assessments of training models, trends of enrollment, graduation, and placement outcomes.

Adaptation of oral streptococci to low pH.

The strategies employed by oral streptococci to resist the inimical influences of acidification reflect the diverse and dynamic niches of the human mouth. All of the oral streptococci are capable of rapid degradation of sugar to acidic end-products. As a result, the pH value of their immediate environment can plummet to levels where glycolysis and growth cease. At this point, the approaches for survival in acid separate the organisms. Streptococcus mutans, for example, relies on its F-ATPase, to protect itself from acidification by pumping protons out of the cells. S. salivarius responds by degrading urea to ammonia and S. sanguis produces ammonia by arginolysis. The mechanisms by which these organisms regulate their particular escape route are now being explored experimentally. The picture that emerges is that the acid-adaptive regulatory mechanisms of the oral streptococci differ markedly from those employed by Gram-negative bacteria. What remains to be elucidated are the breadth of the acid-response systems in these organisms and how they permit the microbes to sustain themselves in the face of low pH and the bacterial competition present in their respective niches. In this article, we summarize reports concerning the means by which oral streptococci either utilize acidification to subdue their competitors or protect themselves until pH values return to a more favorable level.

Regulation of fatty acid biosynthesis by the global regulator CcpA and the local regulator FabT in Streptococcus mutans.

SMU.1745c, encoding a putative transcriptional regulator of the MarR family, maps to a location proximal to the fab gene cluster in Streptococcus mutans. Deletion of the SMU.1745c (fabTS m ) coding region resulted in a membrane fatty acid composition comprised of longer-chained, unsaturated fatty acids (UFA), compared with the parent strain. Previous reports have indicated a role for FabT in regulation of genes in the fab gene cluster in other organisms, through binding to a palindromic DNA sequence. Consensus FabT motif sequences were identified in S. mutans in the intergenic regions preceding fabM, fabTSm and fabK in the fab gene cluster. Chloramphenicol acetyltransferase (cat) reporter fusions, using the fabM promoter, revealed elevated transcription in a ∆fabTS m background. Transcription of fabTS m was dramatically elevated in cells grown at pH values of 5 and 7 in the ∆ fabTS m background. Transcription of fabTS m was also elevated in a strain carrying a deletion for the carbon catabolite repressor CcpA. Purified FabTS m and CcpA bound to the promoter regions of fabTS m and fabM. Hence, the data indicate that FabTS m acts as a repressor of fabM and fabTS m itself and the global regulator CcpA acts as a repressor for fabTS m .

Functional profiling in Streptococcus mutans: construction and examination of a genomic collection of gene deletion mutants.

A collection of tagged deletion mutant strains was created in Streptococcus mutans UA159 to facilitate investigation of the aciduric capability of this oral pathogen. Gene-specific barcoded deletions were attempted in 1432 open reading frames (representing 73% of the genome), and resulted in the isolation of 1112 strains (56% coverage) carrying deletions in distinct non-essential genes. As S. mutans virulence is predicated upon the ability of the organism to survive an acidic pH environment, form biofilms on tooth surfaces, and out-compete other oral microflora, we assayed individual mutant strains for the relative fitness of the deletion strain, compared with the parent strain, under acidic and oxidative stress conditions, as well as for their ability to form biofilms in glucose- or sucrose-containing medium. Our studies revealed a total of 51 deletion strains with defects in both aciduricity and biofilm formation. We have also identified 49 strains whose gene deletion confers sensitivity to oxidative damage and deficiencies in biofilm formation. We demonstrate the ability to examine competitive fitness of mutant organisms using the barcode tags incorporated into each deletion strain to examine the representation of a particular strain in a population. Co-cultures of deletion strains were grown either in vitro in a chemostat to steady-state values of pH 7 and pH 5 or in vivo in an animal model for oral infection. Taken together, these data represent a mechanism for assessing the virulence capacity of this pathogenic microorganism and a resource for identifying future targets for drug intervention to promote healthy oral microflora.

Cloning and expression in Escherichia coli of the form II ribulose 1,5-bisphosphate carboxylase/oxygenase gene from Rhodopseudomonas sphaeroides.

The gene encoding the form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) from Rhodopseudomonas (R.) sphaeroides has been identified on a 3-kb EcoRI fragment and cloned into a broad-host-range, high-copy-number plasmid, using the gene from Rhodospirillum (Rs.) rubrum as a hybridization probe. Subclones of the gene from R. sphaeroides in pBR322 and pUC8 show substantial levels of expression and enzymatic activity in whole cells and crude cell extracts of Escherichia coli. This enzymatic activity has been shown to be similar in many respects to that of the protein purified from R. sphaeroides.

In vivo inactivation of the Streptococcus mutans recA gene mediated by PCR amplification and cloning of a recA DNA fragment.

The inactivation of the RecA protein in pathogenic oral streptococci would facilitate genetic analysis of potential virulence factors in these strains. Comparison of recA nucleotide (nt) sequences from a number of bacteria has suggested that two regions of highly conserved RecA amino acid (aa) sequence could be used as a basis for synthesizing degenerate oligodeoxyribonucleotide primers with which to amplify recA homologues from the streptococci. Accordingly, primer mixtures were used to amplify a 693-bp fragment of the Streptococcus mutans chromosome by PCR. The amplified fragment was cloned and its identity confirmed via hybridization to an Escherichia coli recA gene probe and by nt sequence determination. The recA homologue fragment from S. mutans GS-5 was 63% and 75% homologous to the deduced aa sequences of the E. coli and Bacillus subtilis RecA enzymes, respectively. The S. mutans recA fragment was mutagenized in vitro via insertional inactivation and returned to the chromosome using allelic exchange. The resulting strains of S. mutans were shown to be substantially more sensitive to UV irradiation than the wild-type strain. Further, the ability to incorporate linear markers into the chromosome was abolished in putative S. mutans recA strains, thus indicating the functional inactivation of RecA in these microorganisms.

Cloning and nucleotide sequence analysis of the Streptococcus mutans membrane-bound, proton-translocating ATPase operon.

The function of the membrane-bound ATPase in S. mutans is to regulate cytoplasmic pH values for the purpose of maintaining delta pH. Previous studies have shown that as part of its acid-adaptive ability, S. mutans is able to increase H(+)-ATPase levels in response to acidification. As part of the study of ATPase regulation in S. mutans, we have cloned the ATPase operon and determined its genetic organization. The structural genes from S. mutans were found to be in the order: c, a, b, delta, alpha, gamma, beta, and epsilon; where c and a were reversed from the more typical bacterial organization. The operon contained no I gene homologue but was preceded by a 239-bp intergenic space. Deduced aa sequences from open reading frames indicated that genes encoding homologues of glycogen phosphorylase and nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase flank the H(+)-ATPase operon, 5' and 3' respectively. Sequence analysis indicated the presence of three inverted-repeat nt sequences in the glgP-uncE intergenic space. Primer extension analysis of mRNAs prepared from batch-grown or steady-state cultures demonstrated that the transcriptional start site did not change as a function of culture pH value. The data suggest that potential stem-and-loop structures in the promoter region of the operon do not function to alter the starting position of ATPase-specific mRNA transcription.

Polymerase chain reaction amplification, cloning, sequence determination and homologies of streptococcal ATPase-encoding DNAs.

The highly conserved portion of the catalytic subunit (beta-subunit) of the membrane-bound, proton-translocating ATPase from three strains of oral streptococci has been amplified via the polymerase chain reaction. Hybridization studies demonstrated the existence of homology between Escherichia coli and Bacillus megaterium beta-subunit probes at the streptococcal DNA level. Highly degenerate primers, based on the E. coli and B. megaterium amino acid (aa) sequences, were used to amplify the homologues in Streptococcus mutans, S. sanguis and S. sobrinus. The 600 bp fragment from S. sobrinus has been cloned and its nucleotide (nt) sequence determined. Comparison of its nt and deduced aa sequence to that of E. coli and B. megaterium reveals a high degree of homology at the aa level.

Physiologic homeostasis and stress responses in oral biofilms.

Studies performed since the early, 1970s have yielded tremendous amounts of information about the physiology, genetics, and interactions of oral bacteria. This pioneering work has provided a solid foundation to begin to apply the knowledge and technologies developed using suspended populations for studying oral bacteria under conditions that more closely mimic conditions in the oral cavity, in biofilms. Our current understanding of phenotypic capabilities of individual and complex mixtures of adherent oral bacteria is in its infancy. There is ample evidence that oral streptococci have different patterns of gene expression than planktonic cells, but we have little understanding of the basis for these observations. Even in biofilmforming bacteria with very well-developed genetic systems it is only very recently that genetic loci involved in biofilm formation and responses to surface growth have been identified. A comprehensive study of the physiology and gene expression characteristics of adherent oral bacteria not only will enhance our abilities to control oral diseases, but it will provide critical information that can be applied to a variety of other pathogenic microorganisms.

Raffinose-induced mutanase production from Trichoderma harzianum.

The enzyme alpha(1-->3),3-glucanohydrolase (referred to as mutanase) from the filamentous fungus Trichoderma harzianum OMZ 779 is capable of degrading the water-insoluble glucan in dental plaque. Previously, it was necessary to produce the glucan (referred to as mutan) in vitro for use as the sole carbon source and inducer of mutanase synthesis in fungal cultures. We report here that raffinose also induces the production of mutanase. The metabolism of raffinose differed from that of other sugars in metabolic end products and secreted protein profile. In addition to mutanase, we observed an approximately 15,000 M(r) protein that was also regulated by carbon source and by illumination conditions.

Shifts in membrane fatty acid profiles associated with acid adaptation of Streptococcus mutans.

Cells of Streptococcus mutans UA159 physiologically adapted to acidification during growth at pH 5 in glucose-limited chemostat cultures were enriched in mono-unsaturated and longer chain fatty acids compared with unadapted cells grown under the same conditions but at pH 7. Ratios of unsaturated to saturated fatty acids in the cells were, respectively, 1.2 and 0.3. Cyclopropane fatty acids were not detected. Streptococcus sobrinus 6715, which is known to have minimal acid-adaptive capacity, showed only minimal change in membrane fatty acids.