Biological characterization of an Enterobacter cloacae outer membrane protein (OmpX)

Abstract

We have described a gene coding for an Enterobacter cloacae protein, provisionally called OmpX (J. Stoorvogel, M. J. A. W. M. van Bussel, J. Tommassen, and J. A. M. van de Klundert, J. Bacteriol. 173:156-160, 1991). In the work reported here, OmpX was localized in the cell envelope by means of sucrose gradient fractionation of membrane vesicles. Overproduction of OmpX in Escherichia coli from a multicopy plasmid resulted in a reduction in the amount of OmpF. No accumulation of OmpF, of its uncleft precursor, or of its degradation products could be detected in various cell fractions by Western immunoblot analysis using monoclonal antibodies produced in response to OmpF. A decrease in the rate of synthesis of ompF mRNA was indicated by a beta-galactosidase assay in an ompF-lacZ fusion strain containing the cloned ompX gene and by Northern (RNA) blot analysis. These results indicate that the inhibition is at the level of transcription. Colony hybridization, using an internal ompX fragment as a probe, showed a widespread distribution of the ompX gene among clinical isolates of members of the family Enterobacteriaceae. To study the function of the OmpX protein and its role in the regulation of porin protein synthesis, the ompX gene was deleted from the Enterobacter cloacae chromosome and replaced by the aphA gene. The absence of the ompX gene had no apparent effect on cell growth or on the regulation of the porin proteins.

Detection and Prevalence of Active Drug Efflux Mechanism in Various Multidrug-Resistant Klebsiella pneumoniae Strains from Turkey

“…K. pneumoniae ATCC 11296 was used as the reference strain in determinations of MICs for the various clinical isolates. K. pneumoniae Kp63, a porin-deficient clinical strain (2); K. pneumoniae ATCC 11296; E. coli BW5104 (26), which expresses AcrA at a basal level; and Enterobacter aerogenes ATCC 13048(pJS04) (ompX mutant with its promoter [29]), a strain that overexpresses OmpX, were used as controls for protein analysis. Bacteria were grown either in MuellerHinton (MH) broth and agar or in Luria-Bertani (LB) broth or nutrient broth (Difco Laboratories, Detroit, Mich.) at 37°C.…”

“…In contrast, the expression of OmpK36 porin was elevated in all isolates, and high-osmolality conditions did not modify its biosynthesis level. Expression of OmpX has been previously reported to negatively regulate porin synthesis (29). Therefore, we determined its synthesis by immunodetection in our isolates.…”

Porin alteration and active efflux: two in vivo drug resistance strategies used by Enterobacter aerogenes

“…Expression of this outer-membrane protein has been reported to regulate porin synthesis negatively at a posttranscriptional level in E. cloacae (Stoorvogel et al, 1991). We identified OmpX expression by using immunological tools.…”

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

“…It is therefore likely that the major outer membrane protein whose level was reduced by the introduction of romA corresponds to E. coli OmpF. Stoorvogel et al (30) reported the cloning of a DNA fragment of the E. cloacae chromosome that caused an increase in the level of P-lactam resistance of host bacteria (30). This resistance was associated with a decrease in the concentration of outer membrane proteins OmpF and OmpC in E. coli and of a 37-kDa outer membrane protein in E. cloacae.…”