Role for Skp in LptD Assembly in Escherichia coli

Schwalm, Jaclyn; Mahoney, Tara F.; Soltes, Garner R.; Silhavy, Thomas J.

doi:10.1128/jb.00431-13

Cited by 46 publications

(50 citation statements)

References 54 publications

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“…Our conclusion of the different degrees of importance of SurA, Skp, and FkpA for the biogenesis of OMPs at the normal temperature (i.e., SurA Ͼ Skp Ͼ FkpA) also finds support from a recent study by Schwalm et al, who observed that the single deletion of surA rather than skp or fkpA significantly decreased the level of folded LptD and that the double deletion of bamB and skp led to a more severe defect in LptD folding than the double deletion of bamB and fkpA (45). Notably, they also reported that the double deletion of skp and fkpA led to a significant reduction in the levels of LptD and FhuA and that the overexpression of FkpA was unable to restore the LptD assembly defect of a ⌬surA mutant under normal growth temperatures (45).…”

Section: Discussionsupporting

confidence: 63%

“…Notably, they also reported that the double deletion of skp and fkpA led to a significant reduction in the levels of LptD and FhuA and that the overexpression of FkpA was unable to restore the LptD assembly defect of a ⌬surA mutant under normal growth temperatures (45). We noticed that LptD and FhuA are large (87 kDa and 79 kDa, respectively), both containing a transmembrane ␤-barrel domain and a soluble periplasmic domain.…”

Section: Discussionmentioning

confidence: 86%

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Identification of FkpA as a Key Quality Control Factor for the Biogenesis of Outer Membrane Proteins under Heat Shock Conditions

Lyu

Liu

et al. 2014

J Bacteriol

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The outer membrane proteins (OMPs) of Gram-negative bacterial cells, as well as the mitochondrion and chloroplast organelles, possess unique and highly stable ␤-barrel structures. Biogenesis of OMPs in Escherichia coli involves such periplasmic chaperones as SurA and Skp. In this study, we found that the ⌬surA ⌬skp double-deletion strain of E. coli, although lethal and defective in the biogenesis of OMPs at the normal growth temperature, is viable and effective at the heat shock temperature. We identified FkpA as the multicopy suppressor for the lethal phenotype of the ⌬surA ⌬skp strain. We also demonstrated that the deletion of fkpA from the ⌬surA cells resulted in only a mild decrease in the levels of folded OMPs at the normal temperature but a severe decrease as well as lethality at the heat shock temperature, whereas the deletion of fkpA from the ⌬skp cells had no detectable effect on OMP biogenesis at either temperature. These results strongly suggest a functional redundancy between FkpA and SurA for OMP biogenesis under heat shock stress conditions. Mechanistically, we found that FkpA becomes a more efficient chaperone for OMPs under the heat shock condition, with increases in both binding rate and affinity. In light of these observations and earlier reports, we propose a temperature-responsive OMP biogenesis mechanism in which the degrees of functional importance of the three chaperones are such that SurA > Skp > FkpA at the normal temperature but FkpA > SurA > Skp at the heat shock temperature.

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Section: Discussionsupporting

confidence: 63%

Section: Discussionmentioning

confidence: 86%

Identification of FkpA as a Key Quality Control Factor for the Biogenesis of Outer Membrane Proteins under Heat Shock Conditions

Lyu

Liu

et al. 2014

J Bacteriol

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“…It had been previously demonstrated that many of the OMP assembly factors, including chaperones and BAM complex members, function in overlapping and redundant pathways (4,14,27). Indeed, a role for the SurA P1 domain in vivo was revealed only when surA10 was isolated in a background defective for BAM complex function (27).…”

Section: Deletion Of the Second Parvulin Domain Results In Inhibition Ofmentioning

confidence: 99%

“…SurA is also thought to be the terminal chaperone in the assembly process, as it is the only chaperone known to interact with the periplasmic polypeptide-transport-associated (POTRA) domains of the BAM insertase BamA and, as the deletion of surA affects OMP maturation in the same manner as the loss of BamB, one of the four BamAassociated lipoprotein members of the BAM complex (4,11,12). Despite its central role in OMP assembly, SurA is not essential, because a redundant network of other chaperones, notably Skp, FkpA, and the chaperone/protease DegP, is sufficient to maintain viability in the absence of SurA (13,14).The functions of several periplasmic chaperones were better elucidated when it was discovered that their three-dimensional structures resemble those of well-known cytoplasmic chaperones and folding factors. The trimeric jellyfish structure of the functional Skp chaperone, for example, resembles that of cytosolic prefoldin, and Skp sequesters substrates in a manner mechanisti- …”

mentioning

confidence: 99%

The Activity of Escherichia coli Chaperone SurA Is Regulated by Conformational Changes Involving a Parvulin Domain

et al. 2016

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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 ...

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“…It functions as a chaperone in vivo, promoting assembly of OMPs. FkpA function overlaps Skp function at least in part, and both seem to be involved in assembly of the essential OMP LptD (37), which is in turn responsible for inserting lipopolysaccharide (LPS) into the OM of Gram-negative bacteria like E. coli (38,39). In the present study, we found that disruption of fkpA leads to a 2-to 3-fold reduction in whole-cell ␤-galactosidase activity in cells expressing the malF-lacZ102 fusion in the absence of DsbA.…”

Section: Discussionmentioning

confidence: 99%

Folding LacZ in the Periplasm of Escherichia coli

et al. 2014

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b Targeted, translational LacZ fusions provided the initial support for the signal sequence hypothesis in prokaryotes and allowed for selection of the mutations that identified the Sec translocon. Many of these selections relied on the fact that expression of targeted, translational lacZ fusions like malE-lacZ and lamB-lacZ42-1 causes lethal toxicity as folded LacZ jams the translocation pore. However, there is another class of targeted LacZ fusions that do not jam the translocon. These targeted, nonjamming fusions also show toxic phenotypes that may be useful for selecting mutations in genes involved in posttranslocational protein folding and targeting; however, they have not been investigated to the same extent as their jamming counterparts. In fact, it is still unclear whether LacZ can be fully translocated in these fusions. It may be that they simply partition into the inner membrane where they can no longer participate in folding or assembly. In the present study, we systematically characterize the nonjamming fusions and determine their ultimate localization. We report that LacZ can be fully translocated into the periplasm, where it is toxic. We show that this toxicity is likely due to LacZ misfolding and that, in the absence of the periplasmic disulfide bond catalyst DsbA, LacZ folds in the periplasm. Using the novel phenotype of periplasmic ␤-galactosidase activity, we show that the periplasmic chaperone FkpA contributes to LacZ folding in this nonnative compartment. We propose that targeted, nonjamming LacZ fusions may be used to further study folding and targeting in the periplasm of Escherichia coli.

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Role for Skp in LptD Assembly in Escherichia coli

Cited by 46 publications

References 54 publications

Identification of FkpA as a Key Quality Control Factor for the Biogenesis of Outer Membrane Proteins under Heat Shock Conditions

Identification of FkpA as a Key Quality Control Factor for the Biogenesis of Outer Membrane Proteins under Heat Shock Conditions

The Activity of Escherichia coli Chaperone SurA Is Regulated by Conformational Changes Involving a Parvulin Domain

Folding LacZ in the Periplasm of Escherichia coli

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