IADR Abstract Archives
Characterization of A Novel DNA Binding Protein of Treponema denticola
Objectives: Treponema denticola is a major pathogen of chronic periodontitis. T. denticola has several virulence factors such as surface protease (Dentilisin) and major outer sheath protein (Msp). T. denticola has to adapt a number of stressors such as O2 and antibodies to colonize and proliferate in gingival crevice. Gene regulation mechanisms such as quorum sensing and two components system are important to adapt these stressors. DNA binding proteins are involved in regulation of gene expression. However, little has been known about them in T. denticola. In this study, we constructed a gene deficient mutant of DNA binding protein of which expression was changed in T. denticola dentilisin-deficient mutant to clarify its role in the gene regulation of T. denticola.
Methods: T. denticola ATCC 35405 (wild type strain) was maintained in TYGVS medium. A DNA binding protein deficient mutant was constructed by homologous recombination. Flanking regions of the DNA binding protein was amplified by PCR from genomic DNA of T. denticola and an ermFermAM gene was inserted between fragments. Obtained fragments were transformed into T. denticola by the calcium chloride method. Potential mutant colonies were isolated from TYGVS plate containing 40 µg/ml erythromycin. The growth curve of the bacteria was evaluated by measuring OD660. The proteolytic activity of the cells was measured using synthetic substrates.
Results: We obtained 9 clones from 400 colonies deficient in DNA binding protein from TYGVS plate, and the inactivation of the gene was confirmed by sequencing. The growth rate of the mutant slightly increased compared to wildtype strain. Although the expression of the arginine-specific oligopeptidase was not attenuated in the mutant, the expression of the dentilisin showed a decrease compared with wildtype strain.
Conclusions: It was suggested that the DNA binding protein plays several roles in regulation of gene expressions including dentilisin in T. denticola.
Methods: T. denticola ATCC 35405 (wild type strain) was maintained in TYGVS medium. A DNA binding protein deficient mutant was constructed by homologous recombination. Flanking regions of the DNA binding protein was amplified by PCR from genomic DNA of T. denticola and an ermFermAM gene was inserted between fragments. Obtained fragments were transformed into T. denticola by the calcium chloride method. Potential mutant colonies were isolated from TYGVS plate containing 40 µg/ml erythromycin. The growth curve of the bacteria was evaluated by measuring OD660. The proteolytic activity of the cells was measured using synthetic substrates.
Results: We obtained 9 clones from 400 colonies deficient in DNA binding protein from TYGVS plate, and the inactivation of the gene was confirmed by sequencing. The growth rate of the mutant slightly increased compared to wildtype strain. Although the expression of the arginine-specific oligopeptidase was not attenuated in the mutant, the expression of the dentilisin showed a decrease compared with wildtype strain.
Conclusions: It was suggested that the DNA binding protein plays several roles in regulation of gene expressions including dentilisin in T. denticola.