Construction and characterization of a deletion mutant lacking micF, a proposed regulatory gene for OmpF synthesis in Escherichia coli

Abstract

A method is presented for the construction of a deletion mutant lacking the micF gene, which has been proposed to negatively regulate expression of the ompF gene. The method includes (i) construction of a temperature-sensitive plasmid containing a chromosomal fragment that carries both flanking regions of the micF gene but does not carry micF itself and (ii) replacement of the corresponding chromosomal domain with the fragment. The method is applicable to construction of a deletion mutant for any Escherichia coli chromosomal gene provided that it is dispensable. The micF deletion was confirmed by genetic and biochemical tests, including nucleotide sequence analysis. ompF expression in the micF deletion mutant thus constructed was normally regulated and was not enhanced. When micF was cloned into a high-copy-number plasmid it repressed ompF gene expression, whereas when cloned into a low-copy-number plasmid it did not. From these results, it is concluded that a single copy of the micF gene on the E. coli chromosome does not play a critical role in ompF gene expression.