Biological characterization of an Enterobacter cloacae outer membrane protein (OmpX).
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.
Stoorvogel
J
J
Department of Medical Microbiology, University Hospital, Leiden, The Netherlands.
van Bussel
M J
MJ
van de Klundert
J A
JA
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry; D002501:Centrifugation, Zonal; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004754:Enterobacter / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005838:Genotype; D006867:Hydrolases; D008970:Molecular Weight; D010957:Plasmids; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; C482905:OmpX protein, E coli; D006867:Hydrolases