Intragenic suppressors of an OmpF assembly mutant and assessment of the roles of various OmpF residues in assembly through informational suppressors

We employed two separate genetic approaches to examine the roles of various OmpF residues in assembly. In one approach, intragenic suppressors of a te mperature-sensitive OmpF assembly mutant carrying a W214E substitution were sought at 42 degreesC, or at 37 degreesC in a genetic background lacking t he periplasmic folding factor SurA. In the majority of cases (58 out of 61 revertants), the suppressors mapped either at the original site (position 2 14) or two residues downstream from it. In the remaining three revertants t hat were obtained in a surA background, an alteration of N230Y was located 16 residues away from the original site. The N230Y suppressor also correcte d OmpF315 assembly at 42 degreesC in a surA(+) background, indicating that the two different physiological environments imposed similar assembly const raints. The specificity of N230Y was tested against five different residues , at position 214 of mature OmpF. Clear specificity was displayed, with max imum suppression observed for the original substitution at position 214 (E2 14) against which the N230Y suppressor was isolated, and no negative effect on OmpF assembly was noted when the wild-type W214 residue was present. Th e mechanism of suppression may involve compensation for a specific conforma tional defect. The second approach involved the application of informationa l suppressors (Su-tRNA) in combination with ompF amber mutations to generat e variant OmpF proteins. In this approach we targeted the Y40, Q66, W214, a nd Y231 residues of mature OmpF and replaced them with S, Q, L, and Y throu gh the action of Su-tRNAs. Thus, a total of 16 variant OmpF proteins were g enerated, of which three were identical to the parental protein, and two va riants carrying W214Q and Y231Q substitutions were similar to assembly-defe ctive proteins isolated previously (R Misra, J. Bacteriol. 175:5049-5056, 1 993). The results obtained from these analyses provided useful information regarding the compatibility of various alterations in OmpF assembly.