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 Arfal, 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, Arfal resulted in the loss of flagella and pili and a mucoid colony morphology. Measurement of the synthesis of 13-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 lrfial 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 ofcps-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 ofrfaQ was not required for the functions of fiaG and rifaP. Thus, the function of rfaQ remains unknown. Tandem mass spectrometric analysis of LPS core oligosaccharides from complemented rfial 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.

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 sfaAl and sfaB3 suppressor alleles, which were isolated by using ompF assembly mutants.The cell envelope surrounding an Escherichia coli K-12 cell is a complex structure composed of inner and outer membranes with an aqueous periplasmic space sandwiched between these membranes. The outer membrane is composed of proteins, lipids, and lipopolysaccharide (LPS). LPS is found almost entirely within the external leaflet of the outer membrane (17), where it serves as an important component of the permeability barrier on the exterior of gram-negative bacteria (19). LPS is closely associated with the outer membrane proteins OmpF and LamB (1, 7). These trimeric proteins, also known as porins, form water-filled channels to allow diffusion of substrates into the cell (3). It has been shown that deep rough mutants, which lack part of the LPS inner core, possess greatly reduced amounts of porin proteins (1, 12). In' addition, LPS mutants lacking several rfa genes that are responsible for the synthesis of LPS outer core and for phosphorylating the heptose residues of the inner core produced reduced levels of porins (20). Biochemical studies from Nikaido's laboratory showed the in vitro role of LPS during the trimerization of OmpF (27, 28). It has also been shown that in vitro, these proteins can be trimerized in the absence of LPS (8).OmpF is initially synthesized as a precursor containing a 22-amino-acid signal sequence at the amino-terminal end. Cleavage of the signal sequence at the periplasmic face of the inner membrane results in the release of mature monomers (2). Prior to assembling into heat-stable trimers, these monomers form heat-labile dimers and metastable trimers (9, 23). The site of OmpF trimerization remains unclear. The heatstable form of trimer functions as a nonspecific channel through the outer membrane (18) and serves as the receptor for bacteriophage K20 (30 Bacterial strains and genetic techniques. The strains used in this study were constructed in an MC4100 background (5). The two rfa deletions, Aifa-2057 and Arfa-2058, were moved by P1 transductions using chloramphenicol-resistant (Cmr) markers linked to the deletions. These deletions were moved from CAS2057 and CAS2058, respectively (20). Strains containing rfa deletions were checked for resistance to the bacteriophage U3, which utilizes wild-type LPS as the receptor. These strains were transduced with cps::TnlO or rcsB::Kanr because the deletion of the rfa locus resulted in the overproduction of capsule. In addition, all strains used here were transduced with an ompC::lacZ fusion. The removal of a functional ompC gene was necessary to avoid cross-reactivity ...

SummaryThe assembly defect of a mutant outer membrane protein, OmpF315, can be corrected by suppressor mutations that lower lipopolysaccharide (LPS) levels and indirectly elevate phospholipid levels. One such assembly suppressor mutation, asmB1, is an allele of lpxC (envA) whose product catalyses the first ratelimiting step in the lipid A (LPS) biosynthesis pathway. Besides reducing LPS levels, asmB1 confers sensitivity to MacConkey medium. A mutation, sabA1, that reverses the MacConkey sensitivity phenotype of asmB1 maps within fabZ (whose product is needed for phospholipid synthesis from a precursor) is also required for lipid A synthesis. In addition to reversing MacConkey sensitivity, the sabA1 mutation reverses the OmpF315 assembly suppression phenotype of asmB1. These results show that OmpF315 assembly suppression by asmB1, which is achieved by lowering LPS levels, can be averted by a subsequent aberration in phospholipid synthesis at a point where the biosynthetic pathways for these two lipid molecules split. OmpF315 assembly suppression can also be achieved in an asmB þ background where FabZ expression is increased. The data obtained in this study provide genetic evidence that elevated phospholipid levels and/or phospholipid to LPS ratios are necessary for assembly suppression.

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.