Effects of the resistance plasmid R124 on the level of the OmpF outer membrane protein and on the response of Escherichia coli to environmental agents

Abstract

The introduction of the F-like resistance plasmid R124 into an ompC mutant of Escherichia coli K12 conferred altered sensitivity to a wide range of inhibitory agents. Sensitivity to ampicillin, chloramphenicol, ethionine, copper ions, deoxycholate, two fatty acids and colicins L and M was decreased by the plasmid. In contrast the plasmid-bearing ompC derivatives were more sensitive than the plasmid-free ompC mutant to erythromycin, cetyltrimethylammonium bromide and phenol. Introduction of R124 into the ompC strain also decreased the level of the OmpF protein and some (but not all) of the changed sensitivities listed above clearly resulted from this outer membrane protein deficiency. The presence in the ompC mutant of R124 (rather than the more efficient introduction of the plasmid into variants of the ompC strain) led to at least most of the changes described above because those tested were accentuated by the presence of a copy mutant of R124 and reversed by plasmid curing.

Suppression by the ColV, I-K94 Plasmid of a Growth Lesion in ompA Mutants of Escherichia coli

“…The plasmid ColV-M40(5) is a mutant form of ColV, I-K94 which forms virtually no VmpA protein in P678-54 ompA. The origin and characteristics of strains P678-54, PCO 479 and 1131 ompA were described by Rossouw & Rowbury (1984). From P678-54, ompA mutants were prepared by plating organisms with excess phage K3 and isolating resistant colonies.…”

“…Derivatives of strain 1131 and strain P678-54 ompA carrying F-like plasmids were made either by plasmid transfer in nutrient broth at 37 "C with gentle shaking (Rossouw & Rowbury, 1984) using derivatives of E. coli 1829 (Rowbury et al, 1985) as donors, or by filter conjugation. For this purpose, unless otherwise stated, donor strains were derivatives of strain 1829; 1 ml samples of donor and recipient strains grown to exponential phase (about 2 x lo8 organisms ml-I ) in nutrient broth at 37 "C were filtered onto 0.22 pm pore size Millipore filters (sterilized by UV irradiation) and filters incubated on nutrient agar plates for 2-3 h at 37 "C. After incubation, filters were transferred to 2 ml supplemented glucose MM and, after dilution, cells were plated on appropriate selective media.…”

“…For analysis of membranes for presence or absence of the OmpA protein, the organisms were grown in broth and membranes prepared and analysed by SDS-PAGE as described by Rossouw & Rowbury (1984). For separation of membrane fractions, organisms were lysed and membranes prepared as described by Ito et al (1977) and Burnell et al (1980).…”

“…For separation of membrane fractions, organisms were lysed and membranes prepared as described by Ito et al (1977) and Burnell et al (1980). Fractions were analysed as described by Rossouw & Rowbury (1984). For preparation and use of activated dextran, the procedure of Kamio & Nikaido (1977) was used, membrane fractions being prepared and analysed as above.…”

“…Outer membrane peptidoglycan complexes were prepared as described by Burnell et al (1980), washed in 50 mM-Tris/HCl pH 8.0 and then incubated in the extraction buffer of Rosenbusch (1974) for 30 min at 60 "C. The mixture was then diluted with an equal volume of the same buffer and centrifuged at 1 lOOOOg for 45 rnin at 20 "C. The proteins in the soluble fraction were precipitated with 95% (v/v) ethanol at -70 "C and collected by centrifugation (35000g for 30 min at 4 "C). The pellet was suspended in SDS sample buffer (Rossouw & Rowbury, 1984) heated at about 100 "C for 5 min and samples were run on a polyacrylamide gel. After electrophoresis, the required bands were sliced out, crushed and the crushed fragments wetted with a small volume of 0.1 M-Tris/HCl buffer pH 7.0 containing 0.1 % (w/v) SDS.…”