Development of a Heterodimer Plasmid System for the Introduction of Heterologous Genes into Streptococci

Abstract

We have previously constructed a model secretion system for oral streptococci using the secretory domain of theStreptococcus mutansGS-5gtfB gene. As an initial step in developing systems for secreting protein fusions containing a glucan-binding polypeptide from streptococci, a DNA fragment corresponding to the glucan-binding domain (GBD) of the glucosyltransferase-S enzyme from strain GS-5 was fused to thegtfB secretory domain. However, it was not possible to clone the hybrid gene intoEscherichia coli–streptococcal shuttle plasmids usingE. colias host cells. Integration of the hybrid GBD gene into theStreptococcus gordoniichromosome was directly accomplished utilizing a strategy involving a novel heterodimeric plasmid system. The heterodimer was constructed by ligating together two plasmids each containing DNA fragments homologous with a corresponding region of theS. gordoniichromosome flanking the hybrid GBD gene following a double crossover recombination event. However, this single-copy integrant secreted limited quantities of the GBD protein. In order to achieve high-level expression of the protein, the hybrid GBD gene was integrated into resident plasmids inS. gordoniifollowing construction of intermediate heterodimeric plasmids. These plasmids contained the GBD gene flanked by sequences homologous to regions of the resident plasmids. The presence of the GBD protein in culture fluids from transformants harboring the multicopy plasmids demonstrated the secretion of the functional GBD protein. The strategy successfully developed for secreting the GBD in the streptococci should be adaptable for other organisms for which gene transfer systems are available. In addition, these systems will allow the direct introduction of genetic constructs when the constructs cannot be stably maintained in shuttle vectors within intermediate hosts such asE. coli.