Search for: All records

ABSTRACT Almost all integral membrane proteins that reside in the outer membrane (OM) of gram-negative bacteria contain a closed amphipathic β sheet (“β barrel”) that serves as a membrane anchor. The membrane integration of β barrel structures is catalyzed by a highly conserved heterooligomer called thebarrelassemblymachine (BAM). Although charged residues that are exposed to the lipid bilayer are infrequently found in outer membrane protein β barrels, the β barrels of OmpC/OmpF-type trimeric porins produced by Enterobacterales contain multiple conserved lipid-facing basic residues located near the extracellular side of the OM. Here, we show that these residues are required for the efficient insertion of theEscherichia coliOmpC protein into the OMin vivo. We found that the mutation of multiple basic residues to glutamine or alanine slowed insertion and reduced insertion efficiency. Furthermore, molecular dynamics simulations provided evidence that the basic residues promote the formation of hydrogen bonds and salt bridges with lipopolysaccharide, a unique glycolipid located exclusively in the outer leaflet of the OM. Taken together, our results support a model in which hydrophilic interactions between OmpC and LPS help to anchor the protein in the OM when the local environment is perturbed by BAM during membrane insertion and suggest a surprising role for membrane lipids in the insertion reaction.IMPORTANCEThe assembly (folding and membrane insertion) of bacterial outer membrane proteins (OMPs) is an essential cellular process that is a potential target for novel antibiotics. A heterooligomer called thebarrelassemblymachine (BAM) plays a major role in catalyzing OMP assembly. Here, we show that a group of highly conserved lipid-facing basic residues inEscherichia coliOmpC, a member of a major family of abundant OMPs known as trimeric porins, is required for the efficient integration of the protein into the outer membrane (OM). Based on our work and previous studies, we propose that the basic residues form interactions with a unique OM lipid (lipopolysaccharide) that promotes the insertion reaction. Our results provide strong evidence that interactions between specific membrane lipids and at least a subset of OMPs are required to supplement the activity of BAM and facilitate the integration of the proteins into the membrane. 

ABSTRACT Bacteria live in spatially organized aggregates during chronic infections, where they adapt to the host environment, evade immune responses, and resist therapeutic interventions. Although it is known that environmental factors such as polymers influence bacterial aggregation, it is not clear how bacterial adaptation during chronic infection impacts the formation and spatial organization of aggregates in the presence of polymers. Here, we show that in an in vitro model of cystic fibrosis (CF) containing the polymers extracellular DNA (eDNA) and mucin, O-specific antigen is a major factor determining the formation of two distinct aggregate assembly types of Pseudomonas aeruginosa due to alterations in cell surface hydrophobicity. Our findings suggest that during chronic infection, the interplay between cell surface properties and polymers in the environment may influence the formation and structure of bacterial aggregates, which would shed new light on the fitness costs and benefits of O-antigen production in environments such as CF lungs. IMPORTANCE During chronic infection, several factors contribute to the biogeography of microbial communities. Heterogeneous populations of Pseudomonas aeruginosa form aggregates in cystic fibrosis airways; however, the impact of this population heterogeneity on spatial organization and aggregate assembly is not well understood. In this study, we found that changes in O-specific antigen determine the spatial organization of P. aeruginosa cells by altering the relative cell surface hydrophobicity. This finding suggests a role for O-antigen in regulating P. aeruginosa aggregate size and shape in cystic fibrosis airways.