Analysis of genes involved in exopolysaccharide production of Rothia mucilaginosa clinically isolated from a persistent apical periodontitis lesion

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Rothia mucilaginosa (R. mucilaginosa) strain DY-18 isolated from a persistent apical periodontitis lesion has dense meshwork-like structures around cells, which is typical for exopolysaccharide (EPS)-producing bacteria. In this study, to identify the genes responsible for EPS production in R. mucilaginosa, we isolated three meshwork-like structures defective mutants (strains FA1, FA7 and FA8) by random transposon mutagenesis using the Epicentre EZ-TN5 transposome system. EPS-producing ability was evaluated by measuring the viscosity of spent culture media. The viscosity of the spent culture medium obtained from strains FA1, FA7 and FA8 cultures was significantly lower than the strain DY-18 culture. The transposon insertion region of strain FA1 was determined by PCR direct sequencing analysis. The sequence of the insertion region was then compared with the published complete genome sequence of strain DY-18 on a database. Strain FA1 had a disruption in the RMDY 18_10020 gene encoding P-loop-containing kinase. This gene and downstream genes (RMDY 18_10030 and RMDY 18_10040) showed high homology to the genes yvcJ, yvcK and yvcL respectively. These genes are involved in competence and gene regulation in the stationary phase in Bacillus subtilis. These results suggest that the RMDY 18_10020 gene homologous to yvcJ is involved in EPS biosynthesis and thus plays an important role in biofilmformation of R. mucilaginosa.