A chromosomal gene of Enterobacter cloacae affecting the synthesis of major outer membrane proteins in E. cloacae and Escherichia coli was cloned by using selection for resistance to cefoxitin in E. coli. The presence of the gene, when plasmid-borne, led to a decrease in the amount of porin F in E. cloacae and the amount of OmpF in E. coli and caused 2- to 32-fold increases in the MICs of chloramphenicol, tetracycline, quinolones, and beta-lactam antibiotics. The gene encoded a 33-kDa protein, similar (83% identity) to the protein Rob involved in the initiation of DNA replication in E. coli, which was called RobA(EC1) by analogy. RobA from E. cloacae was found to inhibit ompF expression at the posttranscriptional level via activation of micF, a gene also apparently present in E. cloacae, as detected by PCR. As with its homolog from E. coli, RobA(EC1) is related to the XylS-AraC class of positive transcriptional regulators, along with MarA and SoxS, which also cause a micF-mediated decrease in the level of ampF expression.