Outer membrane protein (OMP) biogenesis in is a robust process essential to the life of the organism. It is catalyzed by the β-barrel assembly machine (Bam) complex, and a number of quality control factors, including periplasmic chaperones and proteases, maintain the integrity of this trafficking pathway. Little is known, however, about how periplasmic proteases recognize and degrade OMP substrates when assembly is compromised or whether different proteases recognize the same substrate at distinct points in the assembly pathway. In this work, we use well-defined assembly-defective mutants of LptD, the essential lipopolysaccharide assembly translocon, to show that the periplasmic protease DegP degrades substrates with assembly defects that prevent or impair initial contact with Bam, causing the mutant protein to accumulate in the periplasm. In contrast, another periplasmic protease, BepA, degrades a LptD mutant substrate that has engaged the Bam complex and formed a nearly complete barrel. Furthermore, we describe the role of the outer membrane lipoprotein YcaL, a protease of heretofore unknown function, in the degradation of a LptD substrate that has engaged the Bam complex but is stalled at an earlier step in the assembly process that is not accessible to BepA. Our results demonstrate that multiple periplasmic proteases monitor OMPs at distinct points in the assembly process. OMP assembly is catalyzed by the essential Bam complex and occurs in a cellular environment devoid of energy sources. Assembly intermediates that misfold can compromise this essential molecular machine. Here we demonstrate distinctive roles for three different periplasmic proteases that can clear OMP substrates with folding defects that compromise assembly at three different stages. These quality control factors help ensure the integrity of the permeability barrier that contributes to the intrinsic resistance of Gram-negative organisms to many antibiotics.

The periplasmic chaperone SurA is critical for the biogenesis of outer membrane proteins (OMPs) and, thus, the maintenance of membrane integrity in Escherichia coli. The activity of this modular chaperone has been attributed to a core chaperone module, with only minor importance assigned to the two SurA peptidyl-prolyl isomerase (PPIase) domains. In this work, we used synthetic phenotypes and covalent tethering to demonstrate that the activity of SurA is regulated by its PPIase domains and, furthermore, that its activity is correlated with the conformational state of the chaperone. When combined with mutations in the ␤-barrel assembly machine (BAM), SurA mutations resulting in deletion of the second parvulin domain (P2) inhibit OMP assembly, suggesting that P2 is involved in the regulation of SurA. The first parvulin domain (P1) potentiates this autoinhibition, as mutations that covalently tether the P1 domain to the core chaperone module severely impair OMP assembly. Furthermore, these inhibitory mutations negate the suppression of and biochemically stabilize the protein specified by a well-characterized gain-of-function mutation in P1, demonstrating that SurA cycles between distinct conformational and functional states during the OMP assembly process. IMPORTANCEThis work reveals the reversible autoinhibition of the SurA chaperone imposed by a heretofore underappreciated parvulin domain. Many ␤-barrel-associated outer membrane (OM) virulence factors, including the P-pilus and type I fimbriae, rely on SurA for proper assembly; thus, a mechanistic understanding of SurA function and inhibition may facilitate antibiotic intervention against Gram-negative pathogens, such as uropathogenic Escherichia coli, E. coli O157:H7, Shigella, and Salmonella. In addition, SurA is important for the assembly of critical OM biogenesis factors, such as the lipopolysaccharide (LPS) transport machine, suggesting that specific targeting of SurA may provide a useful means to subvert the OM barrier.T he Gram-negative bacterial outer membrane (OM) is an essential, selectively permeable barrier between the cell and its external environment. This asymmetric bilayer affords Gramnegative bacteria resistance to a wide range of antimicrobial compounds and membrane perturbants due to strong lateral interactions between lipopolysaccharides (LPS) in the outer leaflet (1, 2). In order to maintain selective permeability, the OM is replete with integral ␤-barrel outer membrane proteins (OMPs), some of which serve as pores for nutrient diffusion. The transport and assembly of these ␤-barrel proteins comprise a complicated multistep process, from their secretion through the inner membrane translocase (Sec) machinery to transport across the aqueous periplasm and assembly into the OM by the ␤-barrel assembly machine (BAM) complex (3).Prior to their assembly in the OM by BAM, these aggregationprone, insoluble ␤-barrel substrates must be maintained in a folding-competent state as they traverse the aqueous, oxidizing periplasm. This maintenance is ...

The periplasmic chaperone Skp has long been implicated in the assembly of outer membrane proteins (OMPs) in Escherichia coli. It has been shown to interact with unfolded OMPs, and the simultaneous loss of Skp and the main periplasmic chaperone in E. coli, SurA, results in synthetic lethality. However, a ⌬skp mutant displays only minor OMP assembly defects, and no OMPs have been shown to require Skp for their assembly. Here, we report a role for Skp in the assembly of the essential OMP LptD. This role may be compensated for by other OMP assembly proteins; in the absence of both Skp and FkpA or Skp and BamB, LptD assembly is impaired. Overexpression of SurA does not restore LptD levels in a ⌬skp ⌬fkpA double mutant, nor does the overexpression of Skp or FkpA restore LptD levels in the ⌬surA mutant, suggesting that Skp acts in concert with SurA to efficiently assemble LptD in E. coli. Other OMPs, including LamB, are less affected in the ⌬skp ⌬fkpA and ⌬skp bamB::kan double mutants, suggesting that Skp is specifically necessary for the assembly of certain OMPs. Analysis of an OMP with a domain structure similar to that of LptD, FhuA, suggests that common structural features may determine which OMPs require Skp for their assembly.

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