Molecular characterization of an Enterobacter cloacae gene (romA) which pleiotropically inhibits the expression of Escherichia coli outer membrane proteins

Abstract

The introduction of a newly cloned Enterobacter cloacae chromosomal gene romA, into Escherichia coli and E. cloacae resulted in enhancement of resistance to quinolones, beta-lactams, chloramphenicol, and tetracycline. The primary effect of romA on a multicopy vector in E. coli was almost complete inhibition of OmpF expression in the outer membrane. From the experiments with ompR and envZ mutants or with ompF-lacZ and ompC-lacZ fusion plasmids, it was concluded that this inhibition is posttranscriptional. The introduction of romA on a multicopy vector into strains with micF deletion elicited only a moderate decrease in OmpF protein expression. This indicates that reduction of OmpF expression by romA is partly mediated posttranscriptionally by the activation of micF. Moreover, the overexpression of RomA protein from an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter resulted in nearly complete inhibition of expression of OmpC and OmpA, as well as OmpF. Taken together with an observation in a recent study that overexpressed OmpC inhibited the synthesis of OmpA and LamB, a possible inhibitory mechanism at the translational stage of the synthesis of outer membrane proteins should also be considered. By Southern hybridization, romA was generally detected in the chromosomes of all E. cloacae strains tested but not in the E. coli K-12 chromosome. Sequence data show that there is an open reading frame specifying 368 amino acids residues including a putative signal peptide. RomA appears to belong to the outer membrane protein family since it was extractable from an outer membrane preparation, but no sequence homology to other outer membrane proteins was detected.