Studies on the heterologous expression of BstVI restriction endonuclease in Escherichia coli.

Abstract

Bacterial restriction and modification systems must be regulated to avoid self-restriction. It is generally accepted that cognate DNA methyltransferases normally protects both, the host's chromosome and extrachromosomal elements from the activity of their endonuclease counterparts. When the bstVIRM genes from Bacillus stearothermophilusV were subcloned into Escherichia coli, several clones exhibiting a r+m- phenotype were originated. The present work was undertaken to analyze the possibility that mechanisms other than DNA methylation could account for the viability of these cells. No evidence was found for an inhibitory agent or endonuclease compartmentation. In vivo experiments showed that phage multiplication was poorly restricted by the heterologous enzyme. The restricting activity against the incoming phage increased however when phage adsortion was performed at higher temperatures. Analogous experiments in which a DNA-repair deficient strain was used as a host for the thermophilic R-M system suggested, to some extent, the participation of the repair machinery in the viability of r+m- clones.