Streptococcus gordonii Challis binds salivary amylase, an interaction which may play a role in plaque formation. S. gordonii Challis exhibits natural competence providing a convenient gene transfer system in which to study the genetic basis of amylase binding. S. gordonii Challis was transformed with the conjugative transposon Tn916. A transformation rate of approximately 10-4 was observed, and 6000 colonies were initially screened for the non-amylase-binding phenotype. Of these, 30 colonies appeared to be amylase-binding deficient. A secondary screening resulted in the isolation of a single mutant with the desired phenotype. The binding of amylase to this mutant was found to be nearly identical to that of S. sanguis 10556, which does not bind amylase, and significantly less than that of the wild type. The morphology of the mutant and wild type colonies were indistinguishable when grown on blood or Mitis-Salivarius agars. In addition, no differences were noted between the mutant and wild type by the API Rapid Strep kit. Comparison of trypsin digests as well as culture supernatants of the wild type and mutant bacteria analyzed by SDS-PAGE/immunoblotting after incubation with antibodies generated against the 20-kDa amylase-binding component described previously clearly demonstrated the absence of the immunoreactive components on the mutant cells. An amylase overlay assay in which Western blots were probed first with whole saliva followed by an antibody against amylase confirmed that the mutants failed to express a functional amylase receptor. Genetic linkage of the Tn916 insertion and the loss of amylase binding was established by isolating genomic DNA from the mutant and transforming the wild type S. gordonii Challis with the mutant genomic DNA. Random screening of 25 transformants found none of them capable of binding amylase. Southern blot analysis indicated a single copy of Tn916 to be inserted into the genome of each cell. In conclusion in this work conducted in the laboratory of Dr. Frank Scannapieco an amylase-binding deficient mutant of S. gordonii Challis generated by a single insertion of Tn916 has been isolated.
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