Isolation and characterization of novel F-plasmid sopB mutants defective in gene silencing.
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.
Kubo
Y
Y
Department of Chemistry, and Frontier Project Life's Adaptation Strategies to Environmental Changes, Rikkyo (St. Paul's) University, 3-34-1 Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, Japan.
Kim
S-K
SK
Hanai
R
R
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D020868:Gene Silencing; D005798:Genes, Bacterial; D049452:Green Fluorescent Proteins; D007763:Lac Operon; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C060972:SopB protein, Bacteria; C489223:SopB protein, E coli; D049452:Green Fluorescent Proteins