Membrane proteins that are integrated into the outer membrane of Gram-negative bacteria typically contain a unique “β barrel” structure that serves as a membrane spanning segment. A conserved “β signal” motif is located at the C terminus of the β barrel of many outer membrane proteins (OMPs), but the function of this sequence is unclear. We found that mutations in the β signal slightly delayed the assembly of three model Escherichia coli OMPs by reducing their affinity for the barrel assembly machinery (Bam) complex, a heterooligomer that catalyzes β barrel insertion, and led to the degradation of a fraction of the protein in the periplasm. Interestingly, the absence of the periplasmic chaperone SurA amplified the effect of the mutations and caused the complete degradation of the mutant proteins. In contrast, the absence of another periplasmic chaperone (Skp) suppressed the effect of the mutations and considerably enhanced the efficiency of assembly. Our results reveal the existence of two parallel OMP targeting mechanisms that rely on a cis-acting peptide (the β signal) and a trans-acting factor (SurA), respectively. Our results also challenge the long-standing view that periplasmic chaperones are redundant and provide evidence that they have specialized functions. IMPORTANCE Proteins that are embedded in the outer membrane of Gram-negative bacteria (OMPs) play an important role in protecting the cell from harmful chemicals. OMPs share a common architecture and often contain a conserved sequence motif (β motif) of unknown function. Although OMPs are escorted to the outer membrane by proteins called chaperones, the exact function of the chaperones is also unclear. Here, we show that the β motif and the chaperone SurA both target OMPs to the β barrel insertion machinery in the outer membrane. In contrast, the chaperone Skp delivers unintegrated OMPs to protein degradation complexes. Our results challenge the long-standing view that chaperones are functionally redundant and strongly suggest that they have specialized roles in OMP targeting and quality control.

Substitutions in the LiaFSR membrane stress pathway are frequently associated with the emergence of antimicrobial peptide resistance in both Enterococcus faecalis and Enterococcus faecium. Cyclic di-AMP (c-di-AMP) is an important signal molecule that affects many aspects of bacterial physiology, including stress responses. We have previously identified a mutation in a gene (designated yybT) in E. faecalis that was associated with the development of daptomycin resistance, resulting in a change at position 440 (yybT I440S ) in the predicted protein. Here, we show that intracellular c-di-AMP signaling is present in enterococci, and on the basis of in vitro physicochemical characterization, we show that E. faecalis yybT encodes a cyclic dinucleotide phosphodiesterase of the GdpP family that exhibits specific activity toward c-di-AMP by hydrolyzing it to 5=pApA. The E. faecalis GdpP I440S substitution reduces c-di-AMP phosphodiesterase activity more than 11-fold, leading to further increases in c-di-AMP levels. Additionally, deletions of liaR (encoding the response regulator of the LiaFSR system) that lead to daptomycin hypersusceptibility in both E. faecalis and E. faecium also resulted in increased c-di-AMP levels, suggesting that changes in the LiaFSR stress response pathway are linked to broader physiological changes. Taken together, our data show that modulation of c-di-AMP pools is strongly associated with antibiotic-induced cell membrane stress responses via changes in GdpP activity or signaling through the LiaFSR system.

OsmY is a widely conserved but poorly understood 20 kDa periplasmic protein. Using a folding biosensor, we previously obtained evidence that OsmY has molecular chaperone activity. To discover natural OsmY substrates, we screened for proteins that are destabilized and thus present at lower steady-state levels in an osmY-null strain. The abundance of an outer membrane protein called antigen 43 was substantially decreased and its β-barrel domain was undetectable in the outer membrane of an osmY-null strain. Antigen 43 is a member of the diffuse adherence family of autotransporters. Like strains that are defective in antigen 43 production, osmY-null mutants failed to undergo cellular autoaggregation. In vitro, OsmY assisted in the refolding of the antigen 43 β-barrel domain and protected it from added protease. Finally, an osmY-null strain that expressed two members of the diffuse adherence family of autotransporters that are distantly related to antigen 43, EhaA and TibA, contained reduced levels of the proteins and failed to undergo cellular autoaggregation. Taken together, our results indicate that OsmY is involved in the biogenesis of a major subset of autotransporters, a group of proteins that play key roles in bacterial pathogenesis.