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 temperature-sensitive OmpF assembly mutant carrying a W214E substitution were sought at 42 degrees C, or at 37 degrees C in a genetic background lacking the periplasmic folding factor SurA. In the majority of cases (58 out of 61 revertants), the suppressors mapped either at the original site (position 214) or two residues downstream from it. In the remaining three revertants that were obtained in a surA background, an alteration of N230Y was located 16 residues away from the original site. The N230Y suppressor also corrected OmpF315 assembly at 42 degrees C in a surA(+) background, indicating that the two different physiological environments imposed similar assembly constraints. The specificity of N230Y was tested against five different residues at position 214 of mature OmpF. Clear specificity was displayed, with maximum suppression observed for the original substitution at position 214 (E214) against which the N230Y suppressor was isolated, and no negative effect on OmpF assembly was noted when the wild-type W214 residue was present. The mechanism of suppression may involve compensation for a specific conformational defect. The second approach involved the application of informational suppressors (Su-tRNA) in combination with ompF amber mutations to generate variant OmpF proteins. In this approach we targeted the Y40, Q66, W214, and Y231 residues of mature OmpF and replaced them with S, Q, L, and Y through the action of Su-tRNAs. Thus, a total of 16 variant OmpF proteins were generated, of which three were identical to the parental protein, and two variants carrying W214Q and Y231Q substitutions were similar to assembly-defective proteins isolated previously (R. Misra, J. Bacteriol. 175:5049-5056, 1993). The results obtained from these analyses provided useful information regarding the compatibility of various alterations in OmpF assembly.

asmB, a suppressor locus for assembly-defective OmpF mutants of Escherichia coli, is allelic to envA (lpxC).

A novel genetic scheme allowed us to isolate extragenic suppressor mutations that restored mutant OmpF assembly. One group of these mutations, termed asmB for assembly suppressor mutation B, permitted mutant OmpF assembly in a non-allele-specific manner. Genetic mapping analyses placed the asmB mutations at the 2-min region of the Escherichia coli K-12 chromosome. Further analyses revealed that the asmB mutations map within the envA (lpxC) gene, which encodes an enzyme needed for the synthesis of the lipid A moiety of lipopolysaccharide (LPS). Nucleotide sequence analysis showed that the asmB mutations caused a change from F-50 to S (F50S substitution) (asmB2 and asmB3) or a G210S substitution (asmB1) in EnvA. Cells bearing the asmB alleles displayed increased sensitivity to various hydrophobic compounds and detergents, suggesting an alteration within the outer membrane. Direct examination (of the LPS showed that its amounts were reduced by the asmB mutations, with asmB1 exerting a greater effect than asmB2 or asmB3. Thus, it appears that the asmB mutations achieve mutant OmpF assembly suppression by reducing LPS levels, which in turn may alter membrane fluidity.

Assembly of LamB and OmpF in deep rough lipopolysaccharide mutants of Escherichia coli K-12.

Assembly of the OmpF and LamB proteins was kinetically retarded in deep rough lipopolysaccharide mutants of Escherichia coli K-12. OmpF assembly was affected at the step of conversion of metastable trimers to stable trimers, whereas LamB assembly was influenced both at the monomer-to-metastable trimer and metastable-to-stable trimer steps. These assembly defects were reversed in the presence of the sfaA1 and sfaB3 suppressor alleles, which were isolated by using ompF assembly mutants.

Role of the rfaG and rfaP genes in determining the lipopolysaccharide core structure and cell surface properties of Escherichia coli K-12.

Deletions which removed rfa genes involved in lipopolysaccharide (LPS) core synthesis were constructed in vitro and inserted into the chromosome by linear transformation. The deletion delta rfa1, which removed rfaGPBI, resulted in a truncated LPS core containing two heptose residues but no hexose and a deep rought phenotype including decreased expression of major outer membrane proteins, hypersensitivity to novobiocin, and resistance to phage U3. In addition, delta rfa1 resulted in the loss of flagella and pili and a mucoid colony morphology. Measurement of the synthesis of beta-galactosidase from a cps-lacZ fusion showed that the mucoid phenotype was due to rcsC-dependent induction of colanic acid capsular polysaccharide synthesis. Complementation of delta rfa1 with rfaG+ DNA fragments resulted in a larger core and restored the synthesis of flagella and pili but did not reverse the deep rough phenotype or the induction of cps-lacZ, while complementation with a fragment carrying only rfaP+ reversed the deep rough phenotype but not the loss of flagella and pili. A longer deletion which removed rfaQGPBIJ was also constructed, and complementation studies with this deletion showed that the product of rfaQ was not required for the functions of rfaG and rfaP. Thus, the function of rfaQ remains unknown. Tandem mass spectrometric analysis of LPS core oligosaccharides from complemented delta rfa1 strains indicated that rfaP+ was necessary for the addition of either phosphoryl (P) or pyrophosphorylethanolamine (PPEA) substituents to the heptose I residue, as well as for the partial branch substitution of heptose II by heptose III. The substitution of heptose II is independent of the type of P substituent present on heptose I, and this results in four different core structures. A model is presented which relates the deep rough phenotype to the loss of heptose-linked P and PPEA.