Novel transcriptional regulator OxtR1 regulates potential ferrodoxin in response to oxygen stress in Treponema denticola.

OBJECTIVE: Treponema denticola has been strongly implicated in the pathogenesis of chronic periodontitis. Previously, we reported that the potential transcriptional regulator TDE_0259 (oxtR1) is upregulated in the bacteriocin ABC transporter gene-deficient mutant. OxtR1 may regulate genes to adapt to environmental conditions during colonization; however, the exact role of the gene in T. denticola has not been reported. Therefore, we investigated its function using an oxtR1-deficient mutant. METHODS: The growth rates of the wild-type and oxtR1 mutant were monitored under anaerobic conditions; their antibacterial agent susceptibility and gene expression were assessed using a liquid dilution assay and DNA microarray, respectively. An electrophoretic mobility shift assay was performed to investigate the binding of OxtR1 to promoter regions. RESULTS: The growth rate of the bacterium was accelerated by the inactivation of oxtR1, and the mutant exhibited an increased minimum inhibitory concentration against ofloxacin. We observed a relative increase in the expression of genes associated with potential ferrodoxin (TDE_0260), flavodoxin, ABC transporters, heat-shock proteins, DNA helicase, iron compounds, and lipoproteins in the mutant. OxtR1 expression increased upon oxygen exposure, and oxtR1 complementation suppressed the expression of potential ferrodoxin. Our findings also suggested that OxtR1 binds to a potential promoter region of the TDE_0259-260 operon. Moreover, the mutant showed a marginal yet significantly faster growth rate than the wild-type strain under H2O2 exposure. CONCLUSION: The oxygen-sensing regulator OxtR1 plays a role in regulating the expression of a potential ferrodoxin, which may contribute to the response of T. denticola to oxygen-induced stress.

Localization and pathogenic role of the cysteine protease dentipain in Treponema denticola.

The Msp protein complex and the serine protease dentilisin are the best-characterized virulence factors in Treponema denticola, the major etiological agent of chronic periodontitis. In addition to these outer sheath factors, the cysteine protease dentipain contributes to pathogenicity, but its secretion, processing, cellular localization, and role in T. denticola virulence are not fully understood. In this study, we found that full-sized dentipain (74-kDa) and the 52-kDa truncated form of the enzyme are located, respectively, in the outer sheath derived from T. denticola dentilisin- and the Msp-deficient mutants. Furthermore, dentipain was barely detected in the wild-type strain. These results suggest that dentilisin and Msp, the major outer sheath proteins, are involved in the secretion and maturation of dentipain. Inactivation of the dentipain gene slowed the growth of T. denticola, and the effect was more profound in serum-free medium than in serum-containing medium. Several genes, including those encoding transporters and methyl-accepting chemotaxis proteins, were differentially expressed in the dentipain-deficient mutant. Furthermore, the mutant strain was more hydrophobic than the wild-type strain. Finally, the mutant showed less autoaggregation activity and adhesion to IgG in a serum-free medium than the wild-type strain. These findings suggest that dentipain contributes to the virulence of T. denticola by facilitating adhesion and acquisition of nutrients essential for colonization and proliferation in the gingival crevice under serum-rich conditions.

Effect of Treponema Denticola Infection on Epithelial Cells.

Chronic periodontitis is an infectious disease caused by periodontopathic bacteria in subgingival plaque. One major pathogen of this disease, Treponema denticola, has several virulence factors, including a major surface protein (Msp) and the surface protease dentilisin. The cytopathic effects of periodontopathic bacteria on epithelial cells disrupt the integrity of the barrier junction, resulting in the inflammation of periodontal tissue. The aim of this study was to investigate the effect of T. denticola virulence factors dentilisin and Msp on epithelial cells. The effects of T. denticola wild-type, Msp-mutant, and dentilisin-mutant strains on the contact junction in Madin-Darby canine kidney epithelial cells was evaluated based on ohmic values. Cultured oral carcinoma epithelial cells were scratched and exposed to the selected T. denticola strains and cell migration determined. Subsequent degradation of adherence proteins and proteins in the contact junctions was evaluated. Dissociation of cell contact junctions was detected in cells infected with wild-type T. denticola approximately 30 min after infection, but not in those exposed to the mutants. Inhibition of migration was observed in the wild-type and Msp-deficient mutants. The adherent proteins focal adhesion kinase, ZO-1, and paxillin were hydrolyzed by infection with the wild-type and Msp mutants. These results indicate that T. denticola disrupts the function of epithelial cells by hydrolyzing proteins at the intercellular junction and inhibiting healing of epithelial cells via hydrolyzed proteins associated with focal adhesion; Msp was also associated with these effects.

OxyR inactivation reduces the growth rate and oxidative stress defense in Capnocytophaga ochracea.

OBJECTIVE: The human oral cavity harbors several bacteria. Among them, Capnocytophaga ochracea, a facultative anaerobe, is responsible for the early phase of dental plaque formation. In this phase, the tooth surface or tissue is exposed to various oxidative stresses. For colonization in the dental plaque phase, a response by hydrogen peroxide (H2O2)-sensing transcriptional regulators, such as OxyR, may be necessary. However, to date, no study has elucidated the role of OxyR protein in C. ochracea. METHODS: Insertional mutagenesis was used to create an oxyR mutant, and gene expression was evaluated by reverse transcription-polymerase chain reaction and quantitative real-time reverse transcription-polymerase chain reaction. Bacterial growth curves were generated by turbidity measurement, and the sensitivity of the oxyR mutant to H2O2 was assessed using the disc diffusion assay. Finally, a two-compartment system was used to assess biofilm formation. RESULTS: The oxyR mutant grew slower than the wild-type under anaerobic conditions. The agar diffusion assay revealed that the oxyR mutant had increased sensitivity to H2O2. The transcript levels of oxidative stress defense genes, sod, ahpC, and trx, were lower in the oxyR mutant than in the wild-type strain. The turbidity of C. ochracea, simultaneously co-cultured with Streptococcus gordonii, was lower than that observed under conditions of homotypic growth. Moreover, the percentage decrease in growth of the oxyR mutant was significantly higher than that of the wild-type. CONCLUSIONS: These results show that OxyR in C. ochracea regulates adequate in vitro growth and escapes oxidative stress.

Crawling motility of Treponema denticola modulated by outer sheath protein.

Treponema denticola, a helically shaped motile microorganism, is a major pathogen of chronic periodontitis. Major surface protein (Msp) and dentilisin are virulence factors of T. denticola that are located on the outer sheath. The motility of T. denticola is deeply involved in colonization on and invasion into the host tissue. The outer sheath is located at the interface between the environment and T. denticola, and its components may also contribute to its motility via interaction with the materials outside the cells. The study aimed to clarify whether Msp or dentilisin contributes to the motility of T. denticola on solid surfaces, termed crawling, by investigating their effects using Msp-deficient and dentilisin-deficient T. denticola strains. Motility was analyzed by measuring the colony size in agar plates and velocity was analyzed using dark-field microscopy. The colony area of the mutant strains was smaller than that of the wild-type strain. The crawling velocity of the mutant strains was lower than that of the wild-type strain, with the lowest velocity observed in the dentilisin-deficient strain. Additionally, the ratio of the crawling distance by one revolution to the protoplasmic cylinder pitch (an indicator of the crawling efficiency) in the dentilisin mutant was significantly lower than that in the wild type strain and the Msp mutant. Together, these results indicate that dentilisin facilitates the crawling-dependent surface spreading of T. denticola.

Taxonomic and Gene Category Analyses of Subgingival Plaques from a Group of Japanese Individuals with and without Periodontitis.

Periodontitis is an inflammation of tooth-supporting tissues, which is caused by bacteria in the subgingival plaque (biofilm) and the host immune response. Traditionally, subgingival pathogens have been investigated using methods such as culturing, DNA probes, or PCR. The development of next-generation sequencing made it possible to investigate the whole microbiome in the subgingival plaque. Previous studies have implicated dysbiosis of the subgingival microbiome in the etiology of periodontitis. However, details are still lacking. In this study, we conducted a metagenomic analysis of subgingival plaque samples from a group of Japanese individuals with and without periodontitis. In the taxonomic composition analysis, genus Bacteroides and Mycobacterium demonstrated significantly different compositions between healthy sites and sites with periodontal pockets. The results from the relative abundance of functional gene categories, carbohydrate metabolism, glycan biosynthesis and metabolism, amino acid metabolism, replication and repair showed significant differences between healthy sites and sites with periodontal pockets. These results provide important insights into the shift in the taxonomic and functional gene category abundance caused by dysbiosis, which occurs during the progression of periodontal disease.

Role of Hyalin-like Protein in Gliding and Biofilm Formation by Capnocytophaga Ochracea.

Capnocytophaga ochracea possesses a type-IX secretion system that exports proteins which have a gliding motility-associated C-terminal (CTD) domain. This system is found in several species of the Bacteroidetes phylum. Hyalin, a large protein encoded by Coch_0033 in C. ochracea ATCC 27872, has a CTD domain and is posited to be involved in quorum sensing according to the database of the Kyoto Encyclopedia of Genes and Genomes. This suggests that it plays a role in biofilm formation via interbacterial communication. The aim of this study was to investigate the potential role of the hyalin-like protein coded by the Coch_0033 gene in gliding and biofilm formation of C. ochracea. A hyalin-like protein-deficient mutant strain of C. ochracea, designated mutant WR-1, was constructed through insertion of the ermF-ermAM cassette into the target gene. The spreading feature at the edge of the colony was lost in the mutant strain. Crystal violet and confocal laser scanning microscopy revealed no difference between the quantity of biofilm organized by the mutant and that organized by the wild-type strain. These data suggest that the hyalin-like protein encoded by the Coch_0033 gene is indeed involved in C. ochracea gliding activity.

Inhibitory Effect of Resveratrol on Candida albicans Biofilm Formation.

Candida albicans is the primary candidiasis-causing fungal pathogen in humans, and one of its most important virulence factors is the ability to form biofilms. Moreover, these biofilms are often resistant to antifungal agents, so there is a need to develop alternative elimination strategies and therapeutic agents for such infections. The antifungal activity of resveratrol, a phytoalexin polyphenolic compound, impairs the morphological transition of C. albicans under various hypha-inducing conditions and inhibits growth of the yeast-form and mycelia. The purpose of this study was to investigate the effect of resveratrol against C. albicans biofilm formation. The developmental, sustained, and mature stages of biofilm formation were affected or inhibited by resveratrol. Exposure to resveratrol at the developmental stage inhibited growth of C. albicans in a dose-dependent manner. A >30% reduction was observed in sustained biofilm growth in the presence of 200 µg/ml resveratrol in comparison with in its absence. In terms of disruption of matured biofilm, 6.25-100 µg/ml resveratrol significantly reduced cell viability of C. albicans compared with in a control sample (p<0.05). The present results indicate that resveratrol has the potential to serve as an anti-Candida treatment and preventive tool which functions by inhibiting existing or under-forming C. albicans biofilms.

Investigation of the potential regulator proteins associated with the expression of major surface protein and dentilisin in Treponema denticola.

Objective Treponema denticola is involved in 'chronic' periodontitis pathogenesis. The mechanism underlying the regulation of the expression of its virulence factors, such as major surface protein (Msp) and prolyl-phenylalanine specific protease (dentilisin) is yet to be clarified. We determined the gene expression profiles of Msp- and dentilisin-deficient mutants of T. denticola to identify the regulation network of gene expression concomitant with the inactivation of these virulence genes. Methods Gene expression profiles of T. denticola ATCC 35405 (wild type), dentilisin-deficient mutant K1, and msp-deficient mutant DMSP3 were determined using DNA microarray analysis and quantitative real-time reverse transcription PCR (qRT-PCR). Msp and dentilisin protein levels were determined by immunoblotting and proteolytic activity assays. Results In addition to several differentially expressed genes, dentilisin expression was reduced in DMSP3; msp expression was significantly reduced in K1 (p < 0.05), both at the gene and protein levels. To identify the regulatory system involved, the expression levels of the potential regulators whose expression showed changes in the mutants were evaluated using qRT-PCR. Transcriptional regulators TDE_0127 and TDE_0814 were upregulated in K1, and the potential repressor, TDE_0344, was elevated in DMSP3. Conclusions Dentilisin and Msp expression were interrelated, and gene expression regulators, such as TDE_0127, may be involved in their regulation.

Characterization of a novel potential peptide import system in Treponema denticola.

Treponema denticola is a major etiologic agent of chronic periodontitis. On the outer sheath of T. denticola, several proteins, such as the major outer sheath protein and dentilisin were detected, and among them, a 95 kDa protein which has not yet been characterized. The aim of this study was to characterize the function of this 95 kDa protein containing gene cluster. A gene encoding this 95 kDa protein (TDE_1072) of T. denticola was inactivated by homologous recombination. We compared growth curves between the TDE_1072 mutant and wild-type strains as well as differences in gene expression by DNA microarray analysis. Differential expression of genes identified by microarray analysis was confirmed by quantitative reverse transcription-polymerase chain reaction. The proteins encoded by TDE_1072, TDE_1073, TDE_1074, TDE_1075, and TDE_1076 shared respective similarities to the substrate-binding domain (DppA) of an ABC-type dipeptide/oligopeptide/nickel transport system, and to the permease components (DppB and DppC) and ATPase components (DppD and DppF) of an ABC-type dipeptide/oligopeptide/nickel transport system. Inactivation of dppA attenuated the growth of T. denticola and dppA-dppF were co-transcribed. In contrast, expression of oppB-oppF was up-regulated in the mutant. Our findings indicate that TDE_1072 may be a potential periplasmic solute binding protein encoded by dppA that is involved in the organization of a peptide uptake system with dppB-dppF.

[Possible Involvement of Surface Antigen Protein 2 in the Morphological Transition and Biofilm Formation of Candida albicans].

Surface antigen protein 2 (Csa2) is a member of the Candida albicans Common in Fungal Extracellular Membranes (CFEM) protein superfamily. We previously established its role in iron acquisition in C. albicans. However, the other roles of Csa2 remain unknown. Here, we compared growth, morphological transition, and biofilm formation among wild-type, Csa2-mutant, and complemented strains of C. albicans. Deletion of the Csa2 gene resulted in smaller and reduced colony growth, significant attenuation of the dimorphic transition under serum-inducing conditions, and reduced biofilm formation; complementation restored these levels to those of the wild-type. Our findings demonstrated that Csa2 participated in yeast-to-hyphae morphological switching under serum-inducing conditions and contributed to the biofilm formation of C. albicans. This work, therefore, provides novel insights into the potential roles of Csa2 in virulence of C. albicans.

Antimicrobial susceptibility profiles of oral Treponema species.

Treponemes occur in the microflora of the dental plaque. Certain Treponema species that are frequently isolated from chronic periodontitis lesions are involved in its initiation and progression. In addition to mechanical instrumentation, antimicrobial agents are used as an adjunctive treatment modality for periodontitis. Despite its importance for successful antimicrobial treatment, information about susceptibility is limited for Treponema species. The aim of this study was to assess the susceptibility of Treponema denticola strains, Treponema socranskii, and Treponema vincentii to eleven antimicrobial agents. The minimum inhibitory and minimum bactericidal concentrations of these antimicrobial agents revealed strain-specific variation. Doxycycline, minocycline, azithromycin, and erythromycin were effective against all Treponema species tested in this study, whereas fluoroquinolones only exhibited an equivalent effectiveness on T. socranskii. The susceptibility of one T. denticola strain, T. socranskii, and T. vincentii to kanamycin was influenced by prior exposure to aerobic conditions. The susceptibility to quinolone drugs varied among strains of T. denticola, although they share an amino acid sequence identity of greater than 99% for DNA gyrase (type II topoisomerase) subunit A. In addition, an ATP-binding cassette (ABC) transporter inhibitor assay for T. denticola indicated that the transport of quinolone drugs is partially related to this transporter, although there may be parallel transport mechanisms. Our results provide important insights into antimicrobial agent-Treponema dynamics and establish a basis for developing an appropriate adjunctive therapy for periodontal disease.

Effect of extracytoplasmic function sigma factors on autoaggregation, hemagglutination, and cell surface properties of Porphyromonas gingivalis.

Porphyromonas gingivalis is a bacterium frequently isolated from chronic periodontal lesions and is involved in the development of chronic periodontitis. To colonize the gingival crevice, P. gingivalis has to adapt to environmental stresses. Microbial gene expression is regulated by transcription factors such as those in two-component systems and extracytoplasmic function (ECF) sigma factors. ECF sigma factors are involved in the regulation of environmental stress response genes; however, the roles of individual ECF sigma factors are largely unknown. The purpose of this study was to investigate the functions, including autoaggregation, hemagglutination, gingipain activity, susceptibility to antimicrobial agents, and surface structure formation, of P. gingivalis ECF sigma factors encoded by SigP (PGN_0274), SigCH (PGN_0319), PGN_0450, PGN_0970, and SigH (PGN_1740). Various physiological aspects of the sigP mutant were affected; autoaggregation was significantly decreased at 60 min (p < 0.001), hemagglutination activity was markedly reduced, and enzymatic activities of Kgp and Rgps were significantly decreased (p < 0.001). The other mutants also showed approximately 50% reduction in Rgps activity. Kgp activity was significantly reduced in the sigH mutant (p < 0.001). No significant differences in susceptibilities to tetracycline and ofloxacin were observed in the mutants compared to those of the wild-type strain. However, the sigP mutant displayed an increased susceptibility to ampicillin, whereas the PGN_0450 and sigH mutants showed reduced susceptibility. Transmission electron microscopy images revealed increased levels of outer membrane vesicles formed at the cell surfaces of the sigP mutant. These results indicate that SigP is important for bacterial surface-associated activities, including gingipain activity, autoaggregation, hemagglutination, vesicle formation, and antimicrobial susceptibility.

Involvement of luxS in Biofilm Formation by Capnocytophaga ochracea.

Capnocytophaga ochracea is present in the dental plaque biofilm of patients with periodontitis. Biofilm cells change their phenotype through quorum sensing in response to fluctuations in cell-population density. Quorum sensing is mediated by auto-inducers (AIs). AI-2 is involved in intercellular signaling, and production of its distant precursor is catalyzed by LuxS, an enzyme involved in the activated methyl cycle. Our aim was to clarify the role of LuxS in biofilm formation by C. ochracea. Two luxS-deficient mutants, TmAI2 and LKT7, were constructed from C. ochracea ATCC 27872 by homologous recombination. The mutants produced significantly less AI-2 than the wild type. The growth rates of these mutants were similar to that of the wild-type in both undiluted Tryptic soy broth and 0.5 × Tryptic soy broth. However, according to crystal violet staining, they produced significantly less biofilm than the wild type. Confocal laser scanning microscopy and scanning electron microscopy showed that the biofilm of the TmAI2 strain had a rougher structure than that of the wild type. Complementation of TmAI-2 with extrinsic AI-2 from the culture supernatant of wild-type strain did not restore biofilm formation by the TmAI2 strain, but complementation of LKT7 strain with luxS partially restored biofilm formation. These results indicate that LuxS is involved in biofilm formation by C. ochracea, and that the attenuation of biofilm formation by the mutants is likely caused by a defect in the activated methyl cycle rather than by a loss of AI-2.

Additional Glucose-PTS Induction in Streptococcus mutans Mutant Deficient in Mannose- and Cellobiose-PTS.

Streptococcus mutans utilizes maltooligosaccharides, including maltose derived from human dietary starch. We recently reported that the glucose-phosphotransferase system (Glc-PTS) was also involved in the metabolism of glucose derived from intracellular maltooligosaccharides in S. mutans. The activity of the Glc-PTS was mediated by the mannose-(manLMN) and cellobiose-PTSs (celABRCD) in this organism. The purpose of this study was to identify which kind of glucose transporter was involved in this process. A celD, manLM, and glk triple mutant, cm6vU1, was constructed and its growth in maltose or glucose broth measured. When cm6vU1 cells were inoculated into a fresh glucose broth following prolonged incubation with glucose, their growth rate was greater than that in the initial inoculum. This suggested that an additional Glc-PTS was induced in these cells. To investigate this possibility, permeabilized S. mutans cells were constructed and Glc-PTS activity examined by photometrical assay method. Activity in the cells was higher in the secondary inocula than in the initial inocula. These results suggest that S. mutans possesses an additional as yet uncharacterized PTS transporter for glucose in addition to the mannose- and cellobiose-PTSs.

Glucose-PTS Involvement in Maltose Metabolism by Streptococcus mutans.

Streptococcus mutans grows with starch-derived maltose in the presence of saliva. Maltose transported into the cells is mediated by the MalQ protein (4-alpha-glucanotransferase) to produce glucose and maltooligosaccharides. Glucose can be phosphorylated to glucose 6-phosphate, which can enter the glycolysis pathway. The MalQ enzyme is essential in the catabolism of maltose when it is the sole carbon source, suggesting the presence of a downstream glucokinase of the MalQ enzyme reaction. However, a glucokinase gene-inactivated mutant (glk mutant) grew with maltose as the sole carbon source, with no residual glucokinase activity. This left a phosphoenolpyruvate-dependent phosphotransferase system (PTS) as the only candidate pathway for the phosphorylation of glucose in its transport as a substrate. Our hypothesis was that intracellular glucose derived from maltose mediated by the MalQ protein was released into the extracellular environment, and that such glucose was transported back into the cells by a PTS. The mannose PTS encoded by the manL, manM, and manN genes transports glucose into cells as a high affinity system with concomitant phosphorylation. The purpose of this study was to investigate extracellular glucose by using an enzyme-linked photometrical method, monitoring absorbance changes at 340 nm in supernatant of S. mutans cells. A significant amount of glucose was detected in the extracellular fluid of a glk, manLM double mutant. These results suggest that the glk and manLMN genes participate in maltose catabolism in this organism. The significance of multiple metabolic pathways for important energy sources, including maltose, in the oral environment is discussed.

Dentilisin involvement in coaggregation between Treponema denticola and Tannerella forsythia.

Periodontitis arises from a biofilm consisting of gram-negative anaerobic rods and spirochetes. Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, termed the Red complex, have been co-isolated with high frequency from chronic periodontitis lesions, and these microorganisms are thought to be major pathogens of the disease. Coaggregation is an important strategy in the colonization of dental plaque biofilm by these bacteria. In the present study, we investigated the coaggregation of T. denticola strains with T. forsythia ATCC 43037 by use of visual grading or spectrophotometry. T. denticola ATCC 35405 coaggregated with T. forsythia, reaching a plateau at approximately 60 min. This coaggregation was inhibited by heat treatment of T. denticola ATCC 35405, but not of T. forsythia. Disaccharides such as sucrose, maltose, and lactose inhibited coaggregation by approximately 50%. The coaggregation reaction varied among T. denticola strains. There was somewhat less coaggregation between T. denticola ATCC 33520 and T. forsythia than between T. denticola ATCC 35405 and T. forsythia, although this difference was not statistically significant; T. denticola ATCC 33521 showed a trace level of coaggregation with T. forsythia. The magnitude of coaggregation among the three T. denticola strains was proportional to their dentilisin activities. Inactivation of dentilisin abolished coaggregation activity, but inactivation of the major outer sheath protein did not. In addition, phenylmethylsulfonyl fluoride did not affect coaggregation. These results indicate that dentilisin is involved indirectly in the coaggregation between T. denticola and T. forsythia, because its proteolytic activity is not required, possibly via ligand maturation.

Csa2, a member of the Rbt5 protein family, is involved in the utilization of iron from human hemoglobin during Candida albicans hyphal growth.

Csa2 is a member of both the Candida albicans Rbt5 protein family and the Common in Fungal Extracellular Membranes (CFEM) protein superfamily. CFEM proteins are characterized by an internal domain containing eight equally spaced cysteine residues. Csa2 is involved in iron uptake from hemoglobin and heme proteins; however, its precise role is unclear. Here, we provide quantitative evidence of the involvement of Csa2 in the utilization of iron from human hemoglobin during C. albicans hyphal growth. The ability of the hyphal form of the wild-type (wt), a homozygote csa2Δ mutant, and a complemented strain of C. albicans to utilize hemoglobin as an iron source under iron-restricted conditions was examined through growth studies and a crystal violet-staining assay. Hemoglobin-binding activity was assessed indirectly using a hemoglobin-sensitized tube method. Although hyphal growth of the wt and csa2Δ/Δ::CSA2 strains was completely recovered when a high concentration of human hemoglobin was added to the iron-restricted culture medium, the recovery of the csa2Δ/Δ mutant was significantly diminished. Furthermore, hemoglobin binding was impaired in the csa2Δ/Δ mutant compared with the wt and csa2Δ/Δ::CSA2 strains, revealing that Csa2 is involved in the utilization of hemoglobin as an iron source by the hyphal form of C. albicans.

The malQ gene is essential for starch metabolism in Streptococcus mutans.

BACKGROUND: The malQ and glgP genes, respectively, annotated as putative 4-α-glucanotransferase and putative glycogen phosphorylase are located with a 29 nucleotide overlap on the Streptococcus mutans genome. We found that the glgP gene of this organism was induced with maltose, and the gene likely constituted an operon with the upstream gene malQ. This putative operon was negatively regulated with the malR gene located upstream from the malQ gene and a MalR-binding consensus sequence was found upstream of the malQ gene. S. mutans is not able to catabolize starch. However, this organism utilizes maltose degraded from starch in the presence of saliva amylase. Therefore, we hypothesized that the MalQ/GlgP system may participate in the metabolism of starch-degradation products. METHODS: A DNA fragment amplified from the malQ or glgP gene overexpressed His-tagged proteins with the plasmid pBAD/HisA. S. mutans malQ and/or glgP mutants were also constructed. Purified proteins were assayed for glucose-releasing and phosphorylase activities with appropriate buffers containing maltose, maltotriose, maltodextrin, or amylodextrin as a substrate, and were photometrically assayed with a glucose-6-phosphate dehydrogenase-NADP system. RESULTS: Purified MalQ protein released glucose from maltose and maltotriose but did not from either maltodextrin or amylodextrin. The purified GlgP protein did not exhibit a phosphorylase reaction with maltose or maltotriose but generated glucose-1-phosphate from maltodextrin and amylodextrin. However, the GlgP protein released glucose-1-phosphate from maltose and maltotriose in the presence of the MalQ protein. In addition, the MalQ enzyme activity with maltose released not only glucose but also produced maltooligosaccharides as substrates for the GlgP protein. CONCLUSION: These results suggest that the malQ gene encodes 4-α-glucanotransferase but not α-1,4-glucosidase activity. The malQ mutant could not grow in the presence of maltose as a carbon source, which suggests that the malQ gene is essential for the utilization of starch-degradation products.

gbpC gene repertoire variation among mutans Streptococci.

The human dental caries pathogen Streptococcus mutans harbors one glucan-binding wall-anchored protein gene, gbpC, and another human pathogen, Streptococcus sobrinus, has 4 gbpC homologues. As no 4 gbpC homologues have so far been detected in other mutans streptococci, however, we cannot say whether those in S. sobrinus are paralogous or orthologous. Therefore, the purpose of this study was to identify and sequence the gbpC/dbl genes in Streptococcus downei and Streptococcus criceti. The findings revealed that the former harbored 5 gbpC/dbl homologues and the latter 4. In addition, another dbl homologue, the dblC gene, was found in some strains of S. sobrinus. We performed a phylogenetic analysis with the gbpC/dbl genes in these mutans streptococci. These results indicate that the gbpC and dbl genes have an orthologous relationship.