Evaluation of Cellular Responses of Heterotrophic Escherichia coli Cultured with Autotrophic Chlamydomonas reinhardtii as a Nutrient Source by Analyses Based on Microbiology and Transcriptome.

Heterotrophic microorganism Escherichia coli LS5218 was cultured with flesh green alga Chlamydomonas reinhardtii C-9: NIES-2235 as a nutrient supplier. In order to evaluate the cell response of Escherichia coli with Chlamydomonas reinhardtii, Escherichia coli was evaluated with microbial methods and comprehensive gene transcriptional analyses. Escherichia coli with Chlamydomonas reinhardtii showed a specific growth rate (µmax) of 1.04 ± 0.27, which was similar to that for cells growing in Luria-Bertani medium (µmax = 1.20 ± 0.40 h-1). Furthermore, comparing the cellular responses of Escherichia coli in a green-algae-containing medium with those in the Luria-Bertani medium, transcriptomic analysis showed that Escherichia coli upregulated gene transcription levels related to glycolysis, 5-phospho-d-ribosyl-1-diphosphate, and lipid synthesis; on the other hand, it decreased the levels related to lipid degradation. In particular, the transcription levels were increased by 103.7 times on pgm (p * < 0.05 (p = 0.015)) in glycolysis, and decreased by 0.247 times on fadE (p * < 0.05 (p = 0.041)) in lipolysis. These genes are unique and could regulate the direction of metabolism; these responses possibly indicate carbon source assimilation as a cellular response in Escherichia coli. This paper is the first report to clarify that Escherichia coli, a substance-producing strain, directly uses Chlamydomonas reinhardtii as a nutrient supplier by evaluation of the cellular responses analyzed with microbial methods and transcriptome analysis.

Inhibitory effect of the combination of xylitol and funoran on Streptococcus mutans biofilm formation on the uncoated surface.

We investigated the effect of xylitol or/and funoran on biofilm formation by Streptococcus mutans, one of cariogenic bacteria, on the surfaces coated and non-coated with saliva. Effects of xylitol and/or funoran were observed on biofilm formation of S. mutans in non-coated and salivary components-coated polystyrene microtiter 96-well plates (s-plate) and flow cell system. Xylitol did not strongly affect biofilm formation of S. mutans UA159 on non-coated and s-plates and, however, changed the quality of the biofilm on the cells in a flow cell system. Funoran had effects on biofilm formation, and the combination of xylitol and funoran strongly inhibited S. mutans biofilm formation on non-coated plates. In particular, funoran had inactivation effects on membrane vesicles (MVs) and inhibited MV-dependent biofilm formation of S. mutans on non-coated plate surfaces but not on the s-plate. These findings suggest that the combination of xylitol and funoran might be useful to remove the oral biofilm formation in elderly individuals with decreased saliva production. This result suggests that the synergistic effect of funoran and xylitol might be useful for the prevention of biofilm-associated diseases such as dental caries in saliva-decreased patients such as elderly patients.

Effects of pH on the Properties of Membrane Vesicles Including Glucosyltransferase in Streptococcus mutans.

Streptococcus mutans releases membrane vesicles (MVs) and induces MV-dependent biofilm formation. Glucosyltransferases (Gtfs) are bound to MVs and contribute to the adhesion and glucans-dependent biofilm formation of early adherent bacteria on the tooth surface. The biofilm formation of S. mutans may be controlled depending on whether the initial pH tends to be acidic or alkaline. In this study, the characteristics and effects of MVs extracted from various conditions {(initial pH 6.0 and 8.0 media prepared with lactic acid (LA) and acetic acid (AA), and with NaOH (NO), respectively)} on the biofilm formation of S. mutans and early adherent bacteria were investigated. The quantitative changes in glucans between primary pH 6.0 and 8.0 conditions were observed, associated with different activities affecting MV-dependent biofilm formation. The decreased amount of Gtfs on MVs under the initial pH 6.0 conditions strongly guided low levels of MV-dependent biofilm formation. However, in the initial pH 6.0 and 8.0 solutions prepared with AA and NO, the MVs in the biofilm appeared to be formed by the expression of glucans and/or extracellular DNA. These results suggest that the environmental pH conditions established by acid and alkaline factors determine the differences in the local pathogenic activities of biofilm development in the oral cavity.

Potential Risk of Spreading Resistance Genes within Extracellular-DNA-Dependent Biofilms of Streptococcus mutans in Response to Cell Envelope Stress Induced by Sub-MICs of Bacitracin.

Antibiotics are used to treat or prevent some types of bacterial infection. The inappropriate use of antibiotics unnecessarily promotes antibiotic resistance and increases resistant bacteria, and controlling these bacteria is difficult. While the emergence of drug-resistant bacteria is a serious problem, the behavior of drug-resistant bacteria is not fully understood. In this study, we investigated the behavior of Streptococcus mutans, a major etiological agent of dental caries that is resistant to bacitracin, which is a cell wall-targeting antibiotic, and focused on biofilm formation in the presence of bacitracin. S. mutans UA159 most strongly induced extracellular DNA (eDNA)-dependent biofilm formation in the presence of bacitracin at 1/8× MIC. The ΔmbrC and ΔmbrD mutant strains, which lack bacitracin resistance, also formed biofilms in the presence of bacitracin at 1/2× MIC. This difference between the wild type and the mutants was caused by the induction of atlA expression in the mid-log phase. We also revealed that certain rgp genes involved in the synthesis of rhamnose-glucose polysaccharide related to cell wall synthesis were downregulated by bacitracin. In addition, glucosyltransferase-I was also involved in eDNA-dependent biofilm formation. The biofilm led to increased transformation efficiencies and promoted horizontal gene transfer. Biofilms were also induced by ampicillin and vancomycin, antibiotics targeting cell wall synthesis, suggesting that cell envelope stress triggers biofilm formation. Therefore, the expression of the atlA and rgp genes is regulated by S. mutans, which forms eDNA-dependent biofilms, promoting horizontal gene transfer in response to cell envelope stress induced by sub-MICs of antibiotics.IMPORTANCE Antibiotics have been reported to induce biofilm formation in many bacteria at subinhibitory concentrations. Accordingly, it is conceivable that the MIC against drug-sensitive bacteria may promote biofilm formation of resistant bacteria. Since drug-resistant bacteria have spread, it is important to understand the behavior of resistant bacteria. Streptococcus mutans is bacitracin resistant, and the 1/8× MIC of bacitracin, which is a cell wall-targeted antibiotic, induced eDNA-dependent biofilm formation. The ΔmbrC and ΔmbrD strains, which are not resistant to bacitracin, also formed biofilms in the presence of bacitracin at 1/2× MIC, and biofilms of both the wild type and mutants promoted horizontal gene transfer. Another cell wall-targeted antibiotic, vancomycin, showed effects on biofilms and gene transfer similar to those of bacitracin. Thus, treatment with cell wall-targeted antibiotics may promote the spread of drug-resistant genes in biofilms. Therefore, the behavior of resistant bacteria in the presence of antibiotics at sub-MICs should be investigated when using antibiotics.

Roles of membrane vesicles from Streptococcus mutans for the induction of antibodies to glucosyltransferase in mucosal immunity.

Glucosyltransferase (Gtf) B and GtfC from Streptococcus mutans are key enzymes for the development of biofilm-associated diseases such as dental caries. Gtfs are involved in membrane vesicles (MVs) and function in the formation of biofilms by initial colonizers such as Streptococcus mitis and Streptococcus oralis on the tooth surface. Therefore, MVs may be important virulence factors and targets for the prevention of biofilm-associated disease. To clarify how GtfB encoded by gtfB and GtfC encoded by gtfC associate with MVs and whether MVs are effective as a mucosal immunogen to induce the production of antibodies against Gtfs, MVs from S. mutans UA159 wild-type (WT), gtfB-, gtfC- and gtfB-C- were extracted from culture supernatants by ultracentrifugation and observed by scanning electron microscopy. Compared with GtfB, GtfC was mainly contained in MVs and regulated the size and aggregation of MVs, and the biofilm formation of S. mutans. The intranasal immunization of BALB/c mice with MVs plus a TLR3 agonist, poly(I-C), was performed 2 or 3 times for 5 weeks, with an interval of 2 or 3 weeks. MVs from all strains caused anti-MV IgA and IgG antibody production. In quality analysis of these antibodies, the IgA and IgG antibodies produced by immunization with MVs from WT and gtfB- strains reacted with Gtfs in the saliva, nasal wash and serum but those produced by immunization with MVs from gtfC- and gtfB-C- strains did not. S. mutans MVs mainly formed by GtfC are an intriguing immunogen for the production of anti-Gtf antibodies in mucosal immunogenicity.

Effects of Complex DNA and MVs with GTF Extracted from Streptococcus mutans on the Oral Biofilm.

Streptococcus mutans is one of the principal pathogens for the development of dental caries. Oral biofilms formed by S. mutans are constructed of insoluble glucan formation induced by the principal enzymes, GTF-I and GTF-SI, in sucrose-containing conditions. However, as another means of biofilm formation, extracellular DNA (eDNA) and membrane vesicles (MVs) are also contributors. To explore the roles of eDNA and MVs for biofilm formation, short and whole size pure DNAs, two types of sub-purified DNAs and MVs were extracted from S. mutans by beads destruction, treatment of proteinase K, and ultracentrifugation of culture supernatant, and applied into the biofilm formation assay using the S. mutans UA159 gtfBC mutant, which lost GTF-I and GTF-SI, on a human saliva-coated 96 well microtiter plate in sucrose-containing conditions. Sub-purified DNAs after cell lysis by beads destruction for total 90 and 180 s showed a complex form of short-size DNA with various proteins and MVs associated with GTF-I and GTF-SI, and induced significantly higher biofilm formation of the S. mutans UA159.gtfBC mutant than no sample (p < 0.05). Short-size pure DNA without proteins induced biofilm formation but whole-size pure DNA did not. Moreover, the complex form of MV associated with GTFs and short-size DNA showed significantly higher biofilm formation of initial colonizers on the human tooth surface such as Streptococcus mitis than no sample (p < 0.05). The short-size DNAs associated with MVs and GTFs are important contributors to the biofilm formation and may be one of additional targets for the prevention of oral biofilm-associated diseases.