Isolation and characterization of novel F-plasmid sopB mutants defective in gene silencing

Abstract.

The product of the sopB gene on the Escherichia coli F-plasmid has been shown to silence genes in the vicinity of its binding region, sopC, when overexpressed. We searched for mutants defective in SopB-dependent silencing by screening for a plasmid incompatibility phenotype, in order to examine the relationship between gene silencing and the intracellular localization of SopB, as revealed by a green fluorescent protein (GFP)-SopB fusion. Nine new mutants were isolated. One of them, in which leucine 92 is replaced by proline, was completely compatible with a sopC-carrying plasmid and was defective in other silencing activities. When expressed as a GFP fusion protein, the L92P mutant was found to be uniformly distributed in the cell. This implies a link between silencing and SopB localization, supporting the view that a high local concentration of SopB drives non-specific DNA binding in segments of the plasmid adjacent to sopC. Despite the lack of apparent localization of GFP fluorescence, the mutant protein, like the wild-type SopB, was found mostly in the inner membrane fraction, indicating that the association with the inner membrane was retained.