Gene Cloning of Oral Streptococcal Glucanases

Cariogenic bacterium Streptococcus mutans synthesizes extracellularly adhesive water-insolubleglucans (mutan,ad-WIG from sucrose by the combined action of two or three types of glucosyltransferase （GTase）resulting in the adherence of cells to smooth surfaces and cariogenic plaque formation. There aremany experimental evidences with respect to the inhibition of ad-WIG and plaque formation byglucanases . thus,in order to realize the mass production of glucanases and the gene expression in oral cavity，the cloning of glucanase genes from oral streptococci was carried out in this investigation.Ad-WIGwas enzymatically synthesized using GTases purified from S mutans B13-Nand <alpha> -1,3 glucan was prepared by chemical treatments of ad-WIG. Mutanase-producing bacterium（turmed MU-strain） was isolated from human oral-fluids，by the use of BHI agar plates supplemented with ad-WIG. This isolate was identified as Streptococcusmitior,from its morphological and biochemical characteristics. The MU-strain produced extracellulerlydextranase,in addition to intracelluler mutanase. Both glucanases partially purified and used as antigensfor preparation of the antiserums. From the mutanolysin-treated cells，high-moleculer-weight (> 20kb) chromosomal DNA was prepared, digested with sau3AI，ligated with BamHI fragments of <lambda> L47.1 DNA，体外packaging was done with a GIGA pack system. Consequently，the usefull clone bank was obtained. the clone bank was screened for glucanase clones by animmunobloting method using a mixture of anti-dextranase and anti-mutanase serums. Seven phage clonesstained by peroxidase were isolated. But，none of the antigen-positive clones expressed any glucanase activitywhen the phage lyzates were incubated at 37c for 48h with <alpha> -1,3 glucan or dextran T-2000。

Cariogenic bacterium Streptococcus mutans synthesizes extracellularly adhesive water-insoluble glucans (mutan, ad-WIG) from sucrose by the combined action of two or three types of glucosyltransferase (GTase), resulting in the adherence of cells to smooth surfaces and cariogenic plaque formation. There are many experimental evidences with respect to the inhibition of ad-WIG and plaque formation by glucanases. Thus, in order to realize the mass production of glucanases and the gene expression in oral cavity, the cloning of glucanase genes from oral streptococci was carried out in this investigation.Ad-WIG was enzymatically synthesized using GTases purified from S mutans B13-N, and <alpha> -1,3 glucan was prepared by chemical treatments of ad-WIG. Mutanase-producing bacterium (turmed MU-strain) was isolated from human oral-fluids, by the use of BHI agar plates supplemented with ad-WIG. This isolate was identified as Streptococcus mitior, from its morphological and biochemical characteristics. The MU-strain produced extracellulerly dextranase, in addition to intracelluler mutanase. Both glucanases were partially purified and used as antigens for preparation of the antiserums. From the mutanolysin-treated cells, high-moleculer-weight (>20 Kb) chromosomal DNA was prepared, digested with sau3AI, ligated with BamHI fragments of <lambda> L47.1 DNA, and in vitro packaging was done with a GIGA pack system. Consequently, the usefull clone bank was obtained. The clone bank was screened for glucanase clones by an immunobloting method using a mixture of anti-dextranase and anti-mutanase serums. Seven phage clones stained by peroxidase were isolated. But, none of the antigen-positive clones expressed any glucanase activity, when the phage lyzates were incubated at 37゜C for 48 h with <alpha> -1,3 glucan or dextran T-2000.