Generation of duplication or deletion mutants in Streptococcus mutans following homologous recombination-mediated random mutagenesis by integrational vector pVA891.

Abstract

Integration vectors have been used as genetic tools for mutagenesis in a number of organisms. We introduced random mutation into Streptococcus mutans with one of the vectors pVA891, and screened the mutants for the glucan-dependent aggregation-negative phenotype to isolate the genes involved in this property. Insertion of pVA891 containing a Sau3AI-digested host DNA fragment into the chromosome occurs following homologous recombination via a Campbell-like mechanism. This type of integration should generate a direct repeat of the homologous region at both ends of inserted pVA891. However, most mutants that we obtained do not have such a duplication and appear to have resulted from chromosomal rearrangements. For instance, the gbpC gene encoding the cell-bound glucan-binding protein was identified in the region deleted from the chromosome in one mutant. Several dozen other mutants exhibiting the nonaggregation phenotype harbored the intact gbpC gene and were shown to be diploid for large chromosomal regions. Based on the analysis of the chromosome from the duplication mutants, we propose that these deletions and duplications were generated by an unequal cross-over between two pVA891 sequences following multiple integrations of the vectors by a Campbell-like mechanism.