Kiyoshi Nishikawa

Objective: Simulated microgravity (SMG) alters fluid dynamics and may influence microbial aggregation. However, its effects on oral bacteria remain unclear. This study investigated the effects of SMG on the growth and aggregation of representative oral bacteria. Methods: Four facultative oral bacteria representing distinct ecological and adhesive properties—Staphylococcus aureus(opportunistic pathogen), Streptococcus mutans (glucan-producing cariogenic bacterium), Streptococcus salivarius (commensal fructan-producing bacterium), and Streptococcus sanguinis (early colonizer)—together with the periodontal pathogen Porphyromonas gingivalis were cultured under SMG (10⁻³G) or normal gravity (NG). Streptococci were grown with or without sucrose to evaluate the role of extracellular polysaccharides. Growth was assessed by optical density, and aggregation was quantified by image analysis. Results: SMG induced aggregation in a species-dependent manner. S. mutans and S. salivarius formed spherical aggregates under SMG, whereas little or no aggregate formation was observed in S. aureus, S. sanguinis, or P. gingivalis. Aggregation of S. mutans was markedly reduced without sucrose, indicating dependence on glucan-mediated adhesion. S. salivarius also showed reduced aggregation under sucrose-free conditions, although limited aggregation persisted. Growth kinetics were virtually unaffected by SMG. Conclusions: SMG promotes aggregation of selected oral bacteria through species-specific mechanisms. These findings suggest that microgravity may alter the architecture of oral microbial communities by enhancing particular intercellular adhesion pathways.

Porphyromonas gingivalis (Pg) utilizes FimA fimbriae to colonize the gingival sulcus and evade the host immune system. The biogenesis of all FimA-related components is positively regulated by the FimS–FimR two-component system, making the FimS sensory protein an attractive target for preventing Pg infection. However, the specific environmental signal received by FimS remains unknown. We constructed random Pg mutant libraries to identify critical amino acid residues for signal sensing by FimS. Optimized error-prone polymerase chain reaction (PCR) was used to introduce a limited number of random mutations in the periplasmic-domain-coding sequence of fimS, and expression vectors carrying various mutants were generated by inverse PCR. More than 500 transformants were obtained from the fimS-knockout Pg strain using the Escherichia coli–Pg conjugal transfer system, whereas only ~100 transformants were obtained using electroporation. Four and six transformant strains showed increased and decreased fimA expression, respectively. Six strains had single amino acid substitutions in the periplasmic domain, indicating critical residues for signal sensing by FimS. This newly developed strategy should be generally applicable and contribute to molecular genetics studies of Pg, including the elucidation of structure–function relationships of proteins of interest.

Tannerella forsythia is a Gram-negative oral anaerobe closely associated with both periodontal and periapical diseases. The ORF TF0022 of strain ATCC 43037 encodes a hybrid two-component system consisting of an N-terminal histidine kinase and a C-terminal response regulator. Disruption of the TF0022 locus enhanced autoaggregation of the broth-cultured cells. Comparative proteome analyses revealed that two S-layer proteins in the TF0022 mutant exhibited decreased apparent masses by denaturing gel electrophoresis, suggesting a deficiency in post-translational modification. Furthermore, the mutant decreased the production of a glycosyltransferase encoded by TF1061 that is located in a putative glycosylation-related gene cluster. Quantitative real-time PCR revealed reduced transcription of TF1061 and the associated genes in the TF0022 mutant. These results indicate that TF0022 upregulates the expression of the glycosylation-related genes and suggest modulation of the autoaggregation of T. forsythia cells by a possible post-translational modification of cell-surface components.