Cloning and expression in Escherichia coli K-12 of the structural gene for outer membrane PhoE protein from Enterobacter cloacae

Abstract

In Escherichia coli K-12, the phoE gene encodes an outer membrane pore protein, which is induced by phosphate starvation. The corresponding gene of Enterobacter cloacae was transferred to E. coli K-12 by using RP4::mini Mu plasmid pULB113 and selecting for R-prime plasmids that carry the genes proA and proB, which are closely linked to phoE in E. coli K-12. The phoE gene was subcloned into the multicopy vector pACYC184, and the location of the gene was determined by analysis of in vitro constructed deletion plasmids and mutant plasmids generated by gamma delta insertions. The E. cloacae phoE gene is normally expressed in E. coli K-12, and the regulation of the expression is similar to that of the E. coli phoE gene. Functionally, the products of the phoE genes of E. coli K-12 and E. cloacae behave very similarly since they form pores in the outer membrane with a recognition site for negatively charged compounds and they serve as (part of) the receptor for phage TC45.