Expression of the water-soluble Glucan Synthase Genes From S. sobrinus in S. anginosus

Objectives: Streptoccus sobrinus has known to as one of the cariogenic bacteria in human. This bacterium can secrete at least four glucosyltransferases (GTFs) which catalyze the formation of water-insoluble and soluble glucans (WIG and WIG, respectively). Previously, we have constructed a Streptococcus anginosus transformant, designated SS-03E, expressing the WIG synthase gene, gtfI, from S. sobrinus utilizing the integration technique. The aim of this study was to construct S. anginosus integrant capable of secreting the WSG synthase genes, gtfT and gtfU, from S. sobrinus using the same strategy and to assess the correlation of WIG and WSG synthesized GTFs to the cariogenic events.
Methods: Several kinds of plasmids were constructed by modifying the E. coli plasmid pACYC184, and used to transform S. anginosus. The transformation of S. anginosus could be accomplished by integration of linear DNA fragments possessing two homologous domains present in the host chromosome. To detect the enzymatic and immunological activities of the gene products, zymograph and western-blot analysis using anti GTFs antibodies were carried out following SDS-PAGE.
Results: The intact gtfT and gtfU gene from S. sobrinus was successfully integrated on the S. anginosus chromosome following sequential integration. The chromosomal structures of integrant including intermediates were confirmed by PCR. The final S. anginosus integrants, designated SS-05E and SS-07E, secreted active GTF proteins, however secretion levels of the GTFs from both integrants were lower to compare to those of the parental S. sobrinus. In sucrose dependent adherence test, both transformant were not exhibited the firm-adherent ability to glass surface, but when three integrants, SS-03E, -05E, and -07E were applied at the same time to the test, ratio of adherence was slightly increased.
Conclusions: It is likely that all four GTFs enzyme activities might be necessary to fully express sucrose-dependent adherence at the similar level as that of parental S. sobrinus strain. This study was supported in part by Grants-in-Aid for Scientific Research from JSPS.