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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.