It has recently been suggested that bactericidal antibiotics, including aminoglycoside antibiotics (AGAs), and toxic small molecules, such as hydroxyurea (HU), kill bacteria the same way, namely, by generating reactive oxygen species (ROS) via a process requiring activation of the Cpx stress response. We suggest an opposite, protective role for Cpx. We have confirmed the initial finding that cpxA null mutations confer resistance to HU. However, the two-component sensor CpxA is both a kinase and a phosphatase, and previous work from our lab has shown that removing CpxA can activate the stress response owing to buildup of the phosphorylated response regulator (CpxRϳP) that occurs in the absence of the phosphatase activity. We show that a dominant cpxA* mutation that constitutively activates the Cpx stress response confers a high level of resistance to both HU and AGAs in a CpxR-dependent manner. In contrast, inactivating the CpxR response regulator by mutating the phosphorylation site (D51A) or the putative DNA-binding motif (M199A) does not increase resistance to HU or AGAs. Taken together, these results demonstrate that activation of the Cpx stress response can protect cells from HU and AGAs. However, the Cpx response does not increase resistance to all classes of bactericidal antibiotics, as the cpxA* mutants are not significantly more resistant to fluoroquinolones or ␤-lactams than wild-type cells. Thus, it seems unlikely that all bactericidal antibiotics kill by the same mechanism.

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.

The biogenesis of the outer membrane (OM) of Escherichia coli is a conserved and vital process. The assembly of integral ␤-barrel outer membrane proteins (OMPs), which represent a major component of the OM, depends on periplasmic chaperones and the heteropentameric ␤-barrel assembly machine (Bam complex) in the OM. However, not all OMPs are affected by null mutations in the same chaperones or nonessential Bam complex members, suggesting there are categories of substrates with divergent requirements for efficient assembly. We have previously demonstrated two classes of substrates, one comprising large, lowabundance, and difficult-to-assemble substrates that are heavily dependent on SurA and also Skp and FkpA, and the other comprising relatively simple and abundant substrates that are not as dependent on SurA but are strongly dependent on BamB for assembly. Here, we describe novel mutations in bamD that lower levels of BamD 10-fold and >25-fold without altering the sequence of the mature protein. We utilized these mutations, as well as a previously characterized mutation that lowers wildtype BamA levels, to reveal a third class of substrates. These mutations preferentially cause a marked decrease in the levels of multimeric proteins. This susceptibility of multimers to lowered quantities of Bam machines in the cell may indicate that multiple Bam complexes are needed to efficiently assemble multimeric proteins into the OM. IMPORTANCEThe outer membrane (OM) of Gram-negative bacteria, such as Escherichia coli, serves as a selective permeability barrier that prevents the uptake of toxic molecules and antibiotics. Integral ␤-barrel proteins (OMPs) are assembled by the ␤-barrel assembly machine (Bam), components of which are conserved in mitochondria, chloroplasts, and all Gram-negative bacteria, including many clinically relevant pathogenic species. Bam is essential for OM biogenesis and accommodates a diverse array of client proteins; however, a mechanistic model that accounts for the selectivity and broad substrate range of Bam is lacking. Here, we show that the assembly of multimeric OMPs is more strongly affected than that of monomeric OMPs when essential Bam complex components are limiting, suggesting that multiple Bam complexes are needed to assemble multimeric proteins. The assembly of integral ␤-barrel outer membrane proteins (OMPs) into the outer membrane (OM) of Gram-negative bacteria is essential for cell growth and viability. OMP targeting and OM integration depend on an assembly pathway comprising semiredundant periplasmic chaperones and the heteropentameric OM-associated ␤-barrel assembly machine (Bam). The primary constituents of the periplasmic chaperone network in Escherichia coli are the proline isomerases SurA and FkpA, the bifunctional protease/chaperone DegP, and the prefoldin-like chaperone Skp. The Bam complex is composed of BamA (itself a ␤-barrel) and four associated lipoproteins, BamB, BamC, BamD, and BamE (1-3).Although OMPs require the Bam complex for folding into the OM, the individ...