The periplasm of Escherichia coli contains many proteins proposed to have redundant functions in protein folding. Using depletion analysis, we directly demonstrated that null mutations in skp and surA, as well as in degP and surA, result in synthetic phenotypes, suggesting that Skp, SurA, and DegP are functionally redundant. The ⌬skp surA::kan combination has a bacteriostatic effect and leads to filamentation, while the degP::Tn10 surA::kan combination is bactericidal. The steady-state levels of several envelope proteins are greatly reduced upon depletion of a wild-type copy of surA in both instances. We suggest that the functional redundancy of Skp, SurA, and DegP lies in the periplasmic chaperone activity. Taken together, our data support a model in which the periplasm of E. coli contains parallel pathways for chaperone activity. In particular, we propose that Skp and DegP are components of the same pathway and that SurA is a component of a separate pathway. The loss of either pathway has minimal effects on the cell, while the loss of both pathways results in the synthetic phenotypes observed.In Escherichia coli proteins are targeted to four distinct cellular locations: two very different aqueous environments, the cytoplasm and the periplasm; and two distinct membranes, the inner membrane and the outer membrane. The outer membrane serves as a barrier that protects the cell from its external environment. Generally speaking, this membrane contains three types of proteins: lipoproteins; surface organelles, such as pili; and the ␤-barrel proteins.The folding of the ␤-barrel proteins is not well understood. These proteins are initially synthesized in the cytoplasm with an N-terminal signal sequence that directs them to the secretion machinery at the inner membrane (30). After translocation from the cytoplasm and signal sequence cleavage, the path that the ␤-barrel proteins follow en route to the outer membrane is unclear. However, there is a good deal of evidence which supports a periplasmic intermediate model in which the ␤-barrel proteins pass through the periplasm in soluble form before localizing to the outer membrane (for a review see reference 5), where many of them, such as LamB and OmpF, serve as pores through which solutes can diffuse into the cell.To date, several groups of periplasmic factors have been implicated in the folding and targeting of various extracytoplasmic proteins. These include factors involved in the formation and isomerization of disulfide bonds, peptidyl-prolyl cistrans isomerases (PPIases), and chaperones. The formation of appropriate disulfide bonds in the oxidizing environment of the periplasm is critical for proper folding of many noncytoplasmic proteins (for a review see reference 24). However, disulfide bond formation is not required for proper folding of the porins LamB and OmpF.

In Escherichia coli, FkpA, PpiA, PpiD, and SurA are the four known periplasmic cis-trans prolyl isomerases. These isomerases facilitate proper protein folding by increasing the rate of transition of proline residues between the cis and trans states. Genetic inactivation of all four periplasmic isomerases resulted in a viable strain that exhibited a decreased growth rate and increased susceptibility to certain antibiotics. Levels of the outer membrane proteins LamB and OmpA in the quadruple mutant were indistinguishable from those in the surA single mutant. In addition, expression of P and type 1 pili (adhesive organelles produced by uropathogenic strains of E. coli and assembled by the chaperone/usher pathway) were severely diminished in the absence of the four periplasmic isomerases. Maturation of the usher was significantly impaired in the outer membranes of strains devoid of all four periplasmic isomerases, resulting in a defect in pilus assembly. Moreover, this defect in pilus assembly and usher stability could be attributed to the absence of SurA. The data presented here suggest that the four periplasmic isomerases are not essential for growth under laboratory conditions but may have significant roles in survival in environmental and pathogenic niches, as indicated by the effect on pilus production.

DegP is a periplasmic protease that is a member of both the E and Cpx extracytoplasmic stress regulons of Escherichia coli and is essential for viability at temperatures above 42°C. [U-14 C]acetate labeling experiments demonstrated that phospholipids were degraded in degP mutants at elevated temperatures. In addition, chloramphenicol acetyltransferase, ␤-lactamase, and ␤-galactosidase assays as well as sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis indicated that large amounts of cellular proteins are released from degP cells at the nonpermissive temperature. A mutation in pldA, which encodes outer membrane phospholipase A (OMPLA), was found to rescue degP cells from the temperature-sensitive phenotype. pldA degP mutants had a normal plating efficiency at 42°C, displayed increased viability at 44°C, showed no degradation of phospholipids, and released far lower amounts of cellular protein to culture supernatants. degP and pldA degP mutants containing chromosomal lacZ fusions to Cpx and E regulon promoters indicated that both regulons were activated in the pldA mutants. The overexpression of the envelope lipoprotein, NlpE, which induces the Cpx regulon, was also found to suppress the temperature-sensitive phenotype of degP mutants but did not prevent the degradation of phospholipids. These results suggest that the absence of OMPLA corrects the degP temperature-sensitive phenotype by inducing the Cpx and E regulons rather than by inactivating the phospholipase per se.Extracytoplasmic stress, such as that caused by heat shock or the overproduction of outer membrane proteins in Escherichia coli, is believed to be caused by the accumulation and aggregation of denatured and misfolded proteins in the membranes and periplasm. Under these conditions the Cpx two-component signal transduction pathway and the alternative sigma factor E direct the synthesis of several proteins that are involved in the degradation and refolding of these denatured and misfolded periplasmic proteins, leading to alleviation of the stress (for a review, see reference 50).The rpoE gene encoding E is essential for the viability of cells at all temperatures (22).E is known to direct the transcription of degP (htrA), fkpA, rpoE, rpoH, and many others (12,14,15,26,34). DegP is a protease/chaperone that digests abnormal proteins in the periplasm and has been demonstrated to be necessary for cell viability at temperatures of 42°C and above (33,35,55,57,58,59), and FkpA is a peptidyl prolyl cis/trans isomerase (28, 41). RseA, RseB, and RseC are involved in regulating the transcription of genes in the E regulon (21, 42). Under heat shock conditions or upon overexpression of outer membrane proteins, denatured and misfolded proteins in the periplasm are sensed by RseA and/or RseB (38). E is then released by the cytoplasmic domain, allowing it to direct transcription of the genes in the E regulon.

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