PpiD is a player in the network of periplasmic chaperones in Escherichia coli
© Matern et al; licensee BioMed Central Ltd. 2010
Received: 11 June 2010
Accepted: 29 September 2010
Published: 29 September 2010
The inner membrane-anchored periplasmic folding factor PpiD is described as a parvulin-like peptidyl prolyl isomerase (PPIase) that assists in the maturation of the major beta-barrel outer membrane proteins (OMPs) of Escherichia coli. More recent work however, calls these findings into question. Here, we re-examined the role of PpiD in the E. coli periplasm by analyzing its functional interplay with other folding factors that influence OMP maturation as well as general protein folding in the periplasmic compartment of the cell, such as SurA, Skp, and DegP.
The analysis of the effects of both deletion and overexpression of ppiD on cell envelope phenotypes revealed that PpiD in contrast to prior observations plays only a minor role, if any, in the maturation of OMPs and cannot compensate for the lack of SurA in the periplasm. On the other hand, our results show that overproduction of PpiD rescues a surA skp double mutant from lethality. In the presence of increased PpiD levels surA skp cells show reduced activities of both the SigmaE-dependent and the Cpx envelope stress responses, and contain increased amounts of folded species of the major OMP OmpA. These effects require the anchoring of PpiD in the inner membrane but are independent of its parvulin-like PPIase domain. Moreover, a PpiD protein lacking the PPIase domain also complements the growth defects of an fkpA ppiD surA triple PPIase mutant and exhibits chaperone activity in vitro. In addition, PpiD appears to collaborate with DegP, as deletion of ppiD confers a temperature-dependent conditional synthetic phenotype in a degP mutant.
This study provides first direct evidence that PpiD functions as a chaperone and contributes to the network of periplasmic chaperone activities without being specifically involved in OMP maturation. Consistent with previous work, our data support a model in which the chaperone function of PpiD is used to aid in the early periplasmic folding of many newly translocated proteins.
It is well established that numerous chaperones, folding catalysts and proteases exist in the periplasm of E. coli and cooperate in protein folding and protein quality control in this cellular compartment of the cell. At least three of these factors, SurA, Skp and DegP, assist in the maturation of integral β-barrel outer membrane proteins (OMPs), a major class of proteins in the E. coli outer membrane, and are thought to be responsible for the transport of OMP folding intermediates through the periplasm to the OMP assembly site, a multi-protein complex in the outer membrane .
The chaperone and peptidyl-prolyl isomerase (PPIase) SurA is specialized for the biogenesis of OMPs. SurA preferentially interacts with newly-synthesized OMPs in vitro by specifically recognizing and binding to peptide sequences that are characteristic of OMPs [3, 4]. Only a subset of OMPs however, appears to directly depend on SurA for maturation . The two biochemical activities of SurA reside in distinct regions of the protein . The PPIase activity is carried in the second of two iterative parvulin-like domains (domain I and domain II) located in the C-terminal half of the protein [2, 6]. The chaperone activity, which is required and sufficient for the so far known biological role of SurA, is contained in a module formed by its N-terminal region and a short C-terminal sequence . Lack of SurA gives phenotypes that are indicative of disturbances in OMP biogenesis and of a defective cell envelope. Such phenotypes are reduced levels of the major OMPs OmpA, LamB, and OmpC in the outer membranes of the cells, increased sensitivity to hydrophobic agents, such as SDS/EDTA, bile salts, and the antibiotic novobiocin, and a constitutively induced σE-dependent envelope stress response [6–8]. The σE pathway together with the Cpx signal transduction pathway monitors and controls the protein folding state in the cell envelope .
The small periplasmic chaperone Skp and the protease-chaperone DegP affect general protein folding in the periplasm and assist in the biogenesis of OMPs. A skp mutant shows phenotypes that are similar to but less severe than those of a surA mutant . Moreover, deletion of skp confers synthetic lethality in a surA mutant, as does deletion of degP[2, 10]. degP skp double mutants on the other hand are viable. It has therefore been proposed that DegP and Skp act together in one pathway of OMP maturation whereas SurA acts in a separate parallel pathway [5, 10]. Skp has been shown to interact with early OMP folding intermediates at the periplasmic side of the inner membrane [11, 12] and to keep immature OMPs in a soluble state [13, 14]. DegP on the other hand, was found to bind to and stabilize folded OMP monomers  and thus appears to act downstream of Skp in the proposed Skp/DegP pathway for OMP maturation.
Conflicting results have been reported regarding the involvement of the periplasmic PpiD protein in the biogenesis of OMPs. PpiD is anchored to the inner membrane by an N-terminal transmembrane segment and consists of a single parvulin domain flanked by large N- and C-terminal protein regions. The N-terminal region shares sequence similarity with the N-terminal region of SurA, which comprises the major part of the SurA chaperone module ([16–19]; see additional file 1). Several previous findings suggested that PpiD and SurA have overlapping functions in OMP biogenesis . First, a ppiD mutant was documented to have phenotypes that are similar to those of a surA mutant and are suppressed by multicopy surA. Second, the simultaneous deletion of ppiD and surA was reported to cause lethality. More recently however, surA ppiD mutants were shown to display no visible growth defects . Finally and most importantly, ppiD was isolated as a multicopy suppressor in a surA mutant. Remarkably however, whereas the surA phenotypes result from loss of chaperone function , a high PPIase activity of PpiD was identified as the complementing biochemical activity . Most recently, this result was disputed by the finding that the isolated parvulin domain of PpiD is devoid of detectable PPIase activity . Here, we analyzed the functional interplay of PpiD with SurA, Skp, and DegP to define its role in the E. coli periplasm.
Re-examination of PpiD function in the biogenesis of OMPs
To resurvey the role of PpiD in OMP maturation we analyzed the physiological consequences of both inactivation and overexpression of ppiD in wild-type cells and in the surA and skp mutants, respectively, using phenotypes known to report on OMP biogenesis and outer membrane integrity, such as σE activity, resistance of the cells to SDS/EDTA and to the antibiotic novobiocin, as well as the levels of major OMPs in their outer membranes. In contrast to previous work  we found that expression of multicopy ppiD from the IPTG-inducible Ptrc promoter does not suppress the surA mutant phenotypes but rather interferes with cell growth (data not shown). We therefore used a plasmid (pPpiD) that carries ppiD under control of its natural promoter, which is positively regulated by the classical cytoplasmic σ32-dependent heat-shock response and by the Cpx two-component system [18, 21].
Plating efficiencies on SDS/EDTA
Efficiency of platinga on 0.5 mM EDTA
+ 0.5% SDS
+ 2% SDS
0.54 ± 0.146
0.93 ± 0.061
8.0, 0.028, and 0.011 [× 10-3]
1.0 ± 0.13
5.8, 0.011, and 0.032 [× 10-3]
0.66 ± 0.156
0.96 ± 0.087
0.42 ± 0.184
0.81 ± 0.067
2.6, 7.2, and 0.66 [× 10-3]
0.7 ± 0.02
4.1, 2.6, and 0.25 [× 10-3]
0.87 ± 0.02
0.57 ± 0.042
1.01 ± 0.06
0.17 ± 0.042
Effects of inactivation and overexpression of ppiD on the Cpx envelope stress response
The σE signal transduction pathway partially overlaps with the CpxA/R pathway in sensing and responding to folding stress in the cell envelope . Since ppiD is a member of the Cpx regulon  we asked whether the Cpx system would respond to inactivation or increased expression of ppiD. As shown in Figure 1B, inactivation of ppiD had no significant effect on Cpx activity in any of the tested strains, indicating that PpiD is not specifically involved in cell envelope functions that are monitored by the Cpx stress response pathway. In contrast, lack of SurA induced the Cpx response ~4-fold, as is consistent with the involvement of SurA in OMP and pilus biogenesis  and with misfolding pilus subunits being sensed by the Cpx signaling system . The presence of ppiD in multicopy led to an about 2-fold induction of the Cpx response in all strains but the surA single and the surA ppiD double mutants. In the surA ppiD double mutant increased expression of ppiD from pPpiD slightly reduced Cpx activity, whereas it showed no significant effect on Cpx activity in the surA single mutant.
ppiD is a multicopy suppressor of the lethal surA skp phenotype
Suppression of surA skp lethality does not require the parvulin domain but the membrane-localization of PpiD
The lethal phenotype of surA skp cells has been suggested to result from loss of periplasmic chaperone activity . Consistent with this assumption, we found that the chaperone module of SurA (SurAN-Ct), which is devoid of any PPIase activity , is sufficient to fully complement the growth defect of the P Llac-O1 -surA Δskp strain in the absence of IPTG (Figure 2B). To also dissect the activities and regions of PpiD required for complementation of surA skp lethality, we substituted amino acids G347 and I350 in its parvulin domain with alanine, generating the proteins PpiDG347A and PpiDI350A, respectively. These mutations had originally been reported to effectively eliminate PPIase activity of PpiD and to result in the loss of its reported surA complementing function . In addition, we completely deleted the parvulin domain from the protein, resulting in PpiDΔParv (Figure 2A). Only most recently, while this manuscript was in preparation, PpiD and its isolated parvulin domain have been shown to be devoid of PPIase activity . However, because G347 and I350 are located at the peptide binding site of the parvulin domain, it was suggested that substrate binding to this domain is important for the in vivo function of PpiD.
Finally, we asked if PpiD must be anchored to the inner membrane to function in vivo. Neither production of soluble N-terminally His6-tagged PpiD (PpiDΔTM) at a level similar to that of PpiDΔParv nor its production from pASKssPpiD at different inducer concentrations restored growth of surA skp cells (Figure 2, and data not shown). pASKssPpiD has also been used to produce and purify soluble His6-PpiD from the periplasmic fraction of E. coli, thus confirming the periplasmic location of the protein. As soluble His6-PpiD is functional in vitro (see below and ), these results suggest that the function of PpiD in vivo requires the protein to be anchored in the inner membrane.
Overproduction of PpiD lowers folding stress in the cell envelope of surA skp cells
Previous studies suggested that the lethal phenotype of a surA skp mutant is caused by severe protein folding stress in the periplasmic compartment of the cells [10, 25]. To determine whether increased PpiD levels restore viability of surA skp cells by counteracting folding stress in the cell envelope, we monitored the activities of the σE and Cpx stress pathways over time once growth of P Llac-O1 -surA Δskp cells had leveled off in the absence of IPTG (time interval indicated in Figure 2C). At this time point, SurA was hardly detectable in the cells (Figure 3B), indicating that SurA had efficiently been depleted from the cells. During the course of the depletion of SurA in Δskp cells both the Cpx pathway and, as also reported previously , the σE-dependent pathway were strongly induced (Figure 3A). The σE and Cpx activities were 4- to 6-fold increased in SurA-depleted Δskp cells (surA skp pASK75) relative to those of SurA-depleted wild-type cells (surA pASK75). This is also reflected in further increased levels of DegP (Figure 3B, lane 4 versus lane 2), whose gene is positively controlled by the σE and Cpx stress responses [27, 28]. In Δskp cells that overproduced PpiD during the course of SurA depletion, σE and Cpx activities were significantly lower, being only 1.5- to 3-fold induced relative to the respective activities in surA cells. Consistent herewith, the level of DegP was lower in these cells than in surA skp cells that not overproduced PpiD but slightly higher than the DegP level in surA cells (Figure 3B, lane 5 versus lanes 4 and 2, respectively). Production of PpiDΔParv during the course of SurA depletion in Δskp cells reduced the σE and Cpx activities slightly less effectively and production of soluble His6-PpiD (PpiDΔTM), which does not rescue surA skp cells from lethality, further induced both stress responses (Figure 3A). Thus, only increased levels of membrane-anchored PpiD proteins dampen the strong response of the σE and the Cpx envelope stress signal transduction pathways to the simultaneous loss of SurA and Skp chaperone activity.
Taking advantage of the fact that overproduction of the outer membrane lipoprotein NlpE specifically induces the Cpx response  and that the Cpx pathway negatively controls expression of the σE regulatory components  we also asked whether down-regulation of σE activity alone would be sufficient to restore growth of surA skp cells. Indeed, the presence of multicopy nlpE during the course of SurA depletion in Δskp cells led to a further induction of the Cpx response and down-regulated σE activity to a similar extent as overproduction of PpiD (see additional files 3 and 4). Overexpression of nlpE even slightly improved cell growth in liquid media but it did not restore growth of surA skp cells on solid plates. Thus, Cpx-mediated repression of σE alone is not sufficient to restore surA skp cell viability.
Effect of PpiD overproduction in surA skp cells on OMP biogenesis
PpiD has in vitro chaperone activity
PpiDΔParv complements the growth defect of an fkpA ppiD surA triple mutant
Lack of PpiD confers increased temperature-sensitivity in a degP mutant
PpiD is a SurA-like multidomain chaperone
To date, four representatives of the three major families of PPIases are known to exist in the periplasm of E. coli: the cyclophilin PpiA , the FKBP-like protein FkpA , and the parvulin-like proteins PpiD  and SurA [6–8]. In addition to PPIase activity, SurA and FkpA also exhibit prolyl isomerase-independent chaperone activity [2, 36] and the major function of SurA in the maturation of the integral β-barrel OMPs actually is that of a chaperone . While PpiD has also been implicated in OMP biogenesis, the biochemical activity required for this function was reported to be a PPIase activity carried in its parvulin domain . A chaperone activity has so far not been demonstrated for either PpiD or PpiA.
In this study we for the first time directly demonstrate, both in vitro and in vivo, that PpiD exhibits a PPIase-independent chaperone activity that resides in the N- and/or C-terminal regions of the protein. The parvulin domain of PpiD is neither required for function in vivo nor for chaperone activity in vitro, as a PpiD protein lacking this domain fully complements the growth defect of an fkpA ppiD surA triple mutant and protects citrate synthase from thermal aggregation even more effectively than wild-type PpiD. In addition, these results show that a catalytic prolyl isomerase activity plays no major role for the function of PpiD in vivo. This conflicts with previous results  but is consistent with most recent data showing that the parvulin domain of PpiD is devoid of detectable PPIase activity in vitro.
The chaperone function of PpiD is most likely carried in its N-terminal region, which shares sequence similarity with the N-terminal region of SurA (see additional file 1A; [16–18]) and thus with a substantial part of the SurA chaperone module . Model structures of this region of PpiD generated by alignment based as well as by automated three-dimensional homology modeling (see additional file 1, C and D) show some deviation from the crystal structure of the SurA chaperone module mainly in the helix 1-helix 2 and the helix 3-helix 4 interconnecting loop regions. Interestingly, whereas the chaperone module of SurA lacks sequence similarity with the structurally homologous chaperone domain of the E. coli cytosolic Trigger factor (TF) , the predicted helix 1-loop-helix 2 region of PpiD shows similarity on the amino acid level with the corresponding region of TF (24.1% identity between regions 43-121 and 295-371 of PpiD and TF, respectively; see additional file 1, B and E).
The similarities in sequence and predicted structure between PpiD, SurA and TF suggest that PpiD contains a conserved SurA-like chaperone module. However, for a complete chaperone active module the region of PpiD that would correspond to the C-terminal helix of SurA still needs to be identified. As an integral element of the conserved module structure this helix is indispensable for the stability and activity of SurA [2, 42] and presumably also of other members of this family of chaperones. The C-terminal helix of SurA was originally identified as the stabilizing region of the protein as it is very basic (predicted isoelectric points of 10.5) as compared to the rather acidic N-terminal region (predicted isoelectric point 5.3) . Similarly, the corresponding helix in the chaperone domain of TF is rather basic as opposed to the rest of the module (predicted isoelectric points of 8.4 and 4.7, respectively). Finally, the N-terminal region of PpiD is acidic too (predicted isoelectric point of 4.7) and therefore the single basic region of the protein which is located in the C-terminal domain (amino acids 511-560, predicted isoelectric point of 10) and is predicted to be rich in α-helical secondary structure, would be a primary candidate for the stabilizing region. Taken together, all indications are that PpiD is a membrane-anchored SurA-like multidomain chaperone, which like SurA combines a conserved chaperone module with an inactive parvulin domain. Different from SurA however, PpiD lacks a second active parvulin domain and instead contains a C-terminal domain, whose function remains to be determined.
Role of PpiD in the periplasm
PpiD was previously reported to be redundant in function with SurA in the maturation of OMPs . Our results however, establish that PpiD plays no major role in the biogenesis of OMPs and that it cannot compensate for lack of SurA in the periplasm. In addition, PpiD differs from SurA in that it requires to be anchored in the inner membrane to function in vivo whereas SurA is functional both in a soluble and in a membrane-anchored state (S. Behrens-Kneip, unpublished results). Then again, ppiD in multicopy suppresses the surA skp caused deficiencies. The strong induction of the σE and Cpx stress pathways during the course of depletion of SurA from Δskp cells is significantly reduced by simultaneous overproduction of PpiD. This suggests that increased levels of PpiD rescue surA skp cells from lethality by counteracting the severe folding stress in the cell envelope which results from the loss of periplasmic chaperone activity. We cannot formally eliminate the possibility that the observed effects result from a response of (an)other known or still to be identified regulatory pathway(s) to increased levels of PpiD in the inner membrane. Such regulatory mechanisms may, for instance, induce periplasmic protease activity that reduces folding stress by protein degradation. However, they would not readily explain our observation that PpiD overproducing surA skp cells contain higher levels of folded forms of OmpA even though they lack two of three chaperones critical for OMP folding. The third OMP chaperone, DegP, appears to interact preferentially with OMPs that already contain substantial levels of folded structure  and would thus be expected to predominantly assist in late steps of OMP folding. Moreover, since DegP levels in surA skp cells are reduced by overproduction of PpiD it seems implausible that DegP is responsible for the observed effect on OmpA folding. This, together with our finding that PpiD has chaperone activity in vitro leads us to suggest that PpiD, when present at sufficient levels, is able to partially compensate for the simultaneous loss of SurA and Skp chaperone function.
But why would PpiD promote the folding of OmpA in a surA skp double mutant but have no discernable impact on OMP folding in the respective surA and skp single mutants? We believe that this effect is due to overlapping substrate specificities but yet distinct roles of these chaperones in the periplasm, as has also been suggested for the SurA and Skp chaperones [5, 26]. Both SurA and Skp interact with unfolded major OMPs [2, 43] and facilitate their biogenesis, yet they cannot functionally substitute one another in the cell (Figure 1 and our unpublished data) and are thought to act in parallel pathways of OMP folding [5, 26]. The peptide binding specificity of PpiD has been shown to overlap with that of SurA but to be less specific , suggesting that PpiD is capable of interacting with a broader range of substrates. Thus, while unfolded major OMPs obviously are no preferred substrates of PpiD, they may still effectively interact with PpiD for folding in the absence of the competing chaperones SurA and Skp. In this context it is important to mention, that overproduction of PpiD does not restore viability of a surA degP double mutant (S. Behrens-Kneip, unpublished results). This suggests that, when overproduced in surA skp cells, PpiD compensates for the lack of Skp upstream of DegP in the proposed Skp/DegP branch of protein folding rather than for the lack of SurA. The magnitude of suppression of the surA skp phenotypes elicited by multicopy ppiD and the additive phenotypes of the ppiD degP and skp ppiD double mutants described in this work are in support of this notion. Moreover, direct evidence for partially overlapping functions of the membrane-anchored PpiD and the soluble Skp comes from the recent finding that both proteins interact with OMP polypeptides very early during translocation and promote their release from the SecYEG translocation channel [12, 14, 24].
It has recently been proposed that PpiD is a periplasmic gatekeeper of the Sec translocon responsible for newly translocated OMPs . Our work agrees with and refines this assumption, as it shows that PpiD exhibits the requisite chaperone activity for such a function, that this function is not preferentially directed at folding of OMPs, and that PpiD cooperates with SurA, Skp, FkpA and DegP in mediating protein folding in the periplasmic compartment of the cell. We suggest that the role of PpiD is to assist in the initial periplasmic folding events of many newly secreted envelope proteins.
In the cytosol, the folding of newly synthesized proteins is initiated by the ribosome-associated chaperone TF [45, 46]. Of note, PpiD and TF show some interesting analogies. First, similar to PpiD TF is composed of three domains: an N-terminal ribosome-binding domain, a central FKBP-like PPIase domain, and a C-terminal chaperone module which is structurally homologous to the chaperone module of SurA [41, 47] and, as outlined above, shows sequence similarity with the N-terminal putative chaperone region of PpiD. Second, TF associates with the ribosome to sequester and protect polypeptides just as they emerge from the peptide exit tunnel  and this association is crucial for its in vivo function . PpiD on the other hand, is anchored in the inner membrane and interacts with newly translocated polypeptides that emerge from the periplasmic exit site of the Sec translocon  and according to our data, the anchoring of PpiD in the membrane is required for its function in vivo. Third, TF is dispensable for cell viability and a deletion of the tig gene confers a discernable phenotype only in combination with a mutation of the dnaK gene for the cytosolic chaperone DnaK . Likewise, lack of PpiD gives a discernable phenotype only in cells with already compromised periplasmic chaperone activity, such as in the fkpA ppiD surA triple mutant and in the degP ppiD and ppiD skp double mutants. Finally, the amino acid sequence pattern of known PpiD binding peptides  resembles that of the peptide binding motifs identified for the cytosolic chaperones TF and DnaK, consisting of a central patch of hydrophobic amino acids flanked by positively charged amino acids . Altogether, we speculate that PpiD may represent the periplasmic counterpart of TF. Its fixed localization in the inner membrane not necessarily conflicts with such a function, as it may provide a local enrichment of the binding partners but still allows PpiD to dynamically interact with and cycle on and off its interaction partners by lateral diffusion in the membrane, just as it is the hallmark of TF function on translating ribosomes . Further detailed studies on the interaction of PpiD with both the Sec translocation machinery and translocating polypeptides as well as the analysis of the structure-function relationship in PpiD are needed to ascertain its real function in the periplasm.
This study for the first time directly demonstrates that PpiD functions as a chaperone and that its previous classification as a folding factor for OMPs must be revised. PpiD appears to belong to the SurA-like family of chaperones but different from SurA it plays no major role in the maturation of OMPs. A biochemical capability of PpiD to also assist the folding of OMPs becomes relevant only in the absence of both chaperones for unfolded OMPs, SurA and Skp. In addition, the role of PpiD in the periplasm appears to be restricted to folding events that take place in close proximity to the inner membrane, as only membrane-anchored PpiD functions in vivo. Taken together, our data are in line with the recently proposed role of PpiD as a periplasmic gatekeeper of the Sec translocon , as they suggest that it acts as a chaperone for initial folding events of many newly exported proteins. We speculate that PpiD may have a role at the periplasmic exit site of the Sec translocon similar to that of TF at the exit site of the translating ribosome.
Media and growth conditions
Luria-Bertani (LB) media were prepared as described . Ampicillin (Ap), chloramphenicol (Cm), kanamycin (Kan), spectinomycin (Spec), and tetracycline (Tc) were used at final concentrations of 100, 20, 30, 50 and 10 μg ml-1, respectively. For assaying β-galactosidase activity in cpxP-lacZ reporter strains the medium was buffered with 100 mM sodium phosphate to a pH of 7.0, at which cpxP transcription, which is affected by extracellular alkaline pH, is induced to a medium level . Strains were grown at 37°C with aeration unless noted otherwise.
Strains used in this study
Source, reference, donor strain
MC1061 ϕλ[rpoH P3::lacZ]
CAG16037 surA:: Tn10d Cm
C.A. Gross laboratory
CAG16037 Δskp zae-502:: Tn10
C.A. Gross laboratory
MC4100 surA:: Tn10d Cm slyD1 zhg:: Tn10; CmR, TcR
argE3 his-4 leuB6 thr-1 ara-14 galK2 lacY1 mtl-1 xyl-5 thi-1 rpsL31 tsx-33 supE44 Δ(recC ptr recB recD):: P lac -bet exo kan
CAG24029 ppiD:: Tn10
This study; donor MC4100 ppiD:: Tn10 (T. Silhavy)
CAG33398 Ω::spec-P Llac-O1 -surA pPLT13
CAG33398 ppiD:: Tn10
This study; donor MC4100 ppiD:: Tn10 (T. Silhavy)
CAG33398 surA:: Tn10d Cm
This study; donor CAG24029
This study; donor JW0431 
SB11069 ppiD:: Tn10
This study; donor MC4100 ppiD:: Tn10 (T. Silhavy)
This study; donor JW3309 
This study; donor JW3309 
This study; donor JW0431 
CAG33398 Δskp zae-502:: Tn10
This study; donor CAG37057
CAG37057 Ω::spec-P Llac-O1 -surA
CAG37057 Ω::spec-P Llac-O1 -surA pPLT13
CAG16037 Ω::spec-P Llac-O1 -surA pPLT13
CAG16037 ppiD:: Tn10
This study; donor MC4100 ppiD:: Tn10 (T. Silhavy)
This study; donor JW0431 
This study; donor JW0431 
SB44451 ppiD::kan pACLacI
This study; donor JW0431 
This study; donor JW0157 
This study; donor JW0157 
CAG44080 Ω::spec-P Llac-O1 -surA pPLT13
Plasmids used in this study
pACYC184 derivative with lacI q ; CmR
vector, P/O tet , tetR, ColEI ori; ApR
surA gene in pASK75; ApR
surAN-Ct fusion from pSurAN-Ct  in pASK75; ApR
surA signal sequence-his6-ppiD (codons 34-623) fusion in pASK75; ApR
Ω::spec-P Llac-O1 surA in pUC18; ApR; SpecR
skp gene region of E. coli MC1061 (corresponding to nucleotides 199495-201937 of the E. coli MG1655 genomec) in pSU18; CmR
mini-F carrying lacI q ; KanR
ppiD gene and promoter of E. coli MC1061 (corresponding to nucleotides 460852-463020 of the E. coli MG1655 genomec) in pTrc99A (lacI q and P trc sequences deleted); ApR
pPpiD carrying mutation I350 to A; ApR
pPpiD carrying mutation G347 to A; ApR
surA signal sequence-his6-ppiD (codons 34-623) fusion in pPpiD; ApR
pPpiD with a frameshift mutation in ppiD that generates a stop at codon 173; ApR
C-His6 fusion vector, P T5 /O lac , ColEI ori; ApR
skp gene and skp promoter of E. coli MC1061 (corresponding to nucleotides 200073-201801 of the E. coli MG1655 genomec) in pQE60 (PT5/Olac deleted); ApR
Expression vector, P trc , ColEI ori; ApR
Assay of susceptibility to SDS/EDTA
The sensitivity of the strains to SDS/EDTA was determined in plating assays as previously described . The efficiency of plating was calculated from the colony count after incubation at 37°C for 24-48 h. A minimum of three experiments were performed for each strain and condition.
Spot dilution assays
SurA-depletion strains were freshly transformed with the required plasmids and were grown overnight at 37°C in selective LB containing 1 mM IPTG. Overnight cultures were adjusted to an optical density at 600 nm (OD600) of 4.0 and 10-fold serially diluted with IPTG-free LB. Ten microlitres of the 10-1, 10-3, 10-5, and 10-7 dilutions were spotted on LB ± 1 mM IPTG plates supplemented with the appropriate antibiotics and incubated at 37°C for 16-24 h. To test for temperature sensitivity, strains were grown overnight at 30°C in LB and were diluted and spotted on LB plates as described above.
SurA depletion in vivo
SB44452 or SB44997 were freshly transformed with the appropriate plasmids and grown overnight at 37°C in LB/Ap/Kan/Spec (buffered at a pH of 7.0, if required) supplemented with 1 mM IPTG and 0.2% (w/v) maltose to induce expression of the maltoporin LamB. Two milliliters of each overnight culture were pelleted in a microcentrifuge and were washed three times in 2 ml of LB to remove IPTG from the cells. The washed cultures were then diluted to an OD600 of 0.01 into 50 ml of LB/Ap/Kan ± 1 mM IPTG. These pre-cultures were grown for 4-5 cell generations with shaking in a gyratory water bath at 37°C and diluted into fresh LB/Ap/Kan ± 1 mM IPTG to an OD600 of 0.005. Aliquots were sampled for β-galactosidase assays, for western blot analysis, and for the preparation of OmpA folding intermediates at the indicated time points after the second sub-culturing and processed as described below.
EσE and Cpx pathway activities were assayed by monitoring the β-galactosidase activity resulting from the expression of the chromosomal σE- and Cpx-dependent reporter fusions rpoH P3::lacZ and cpxP-lacZ, respectively, in cells growing in LB medium (buffered at pH 7.0 for Cpx assays) at 37°C. Overnight cultures were diluted to an OD600 of 0.005 into fresh media and grown with shaking in a gyratory water bath at 37°C. Duplicate samples (0.5 ml) were taken throughout the early exponential phase of the growth curve (OD600 = 0.08-0.4) and β-galactosidase activity was measured by the standard assay . EσE and Cpx activities shown in Figure 1 were determined from the slope on the line of a differential plot of β-galactosidase activity in 0.5 ml of culture versus OD600 and normalized to the wild-type case. In Figure 3, the average β-galactosidase activity/OD600 (Miller Units) was calculated and normalized to that of wild-type. Statistical analysis was performed using a Student's t-test.
Western blot analysis
Whole cell extracts were prepared by resuspending cells in urea protein sample buffer (8 M urea, 200 mM Tris-Base, 200 mM DTT, 2% SDS, 0.02% bromphenol blue) followed by short sonication and heating of the sample to 95°C for 10 min. Extracts from equal numbers of cells were run on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with dilutions of rabbit polyclonal antisera raised against SurA (1:10 000), PpiD (1:10 000), DegP (1:20 000), Hsc66 (1:20 000), LamB (1:3000), and with mouse monoclonal antibodies raised against OmpA (1:500), respectively. Alkaline phosphatase conjugated goat anti-rabbit and anti-mouse IgGs (Sigma, 1.10 000 dilutions), respectively, served as secondary antibodies. They were visualized by incubating the blots in reaction buffer (100 mM Tris-HCl, pH 8.8, 100 mM NaCl, 5 mM MgCl2, 37.5 μg/ml nitro blue tetrazolium, 150 μg/ml 5-bromo-4-chloro-3-indolyl phosphate). Signal intensities were quantified using ImageJ software http://rsb.info.nih.gov/ij/. Hsc66 and MalE were used as the internal standard for each lane. Experiments were repeated a minimum of two times for each strain and condition, and data for one representative experiment are shown.
Preparation of OmpA folding intermediates
During the course of SurA depletion, samples corresponding to an equal number of cells were harvested by centrifugation and immediately frozen in a dry ice/ethanol bath. Folded and unfolded OmpA folding intermediates were isolated by gentle lysis as previously described . Samples were mixed with protein sample buffer (3% SDS, 10% glycerol, 5% β-mercaptoethanol in 70 mM Tris, HCl, pH 6.8), heated to 37°C for 10 min and loaded onto 12.5% SDS-polyacrylamide gels. Electrophoresis was performed at 50 V and OmpA intermediates were detected by Western blot analysis as described above.
N-terminally His6-tagged PpiD proteins and C-terminally His6-tagged SurA were produced in E. coli CAG44102 from pASKssPpiD, pASKssPpiDΔParv and pASKSurA, respectively, and purified from the periplasmic fraction by affinity chromatography on Ni2+-chelating sepharose as previously described . Protein concentrations were determined by UV absorbance at 280 nm using absorption coefficients of 36 270 M-1 cm-1, 26 740 M-1 cm-1 and 29 450 M-1 cm-1 for His6-PpiD, His6-PpiDΔParv and SurA-His6, respectively, as calculated according to .
Analysis of chaperone function in vitro
Effects of PpiD proteins on the thermal aggregation of citrate synthase were determined according to . Aggregation was monitored on a Hitachi F-4500 spectrofluorometer with both excitation and emission wavelengths set to 500 nm at a spectral bandwidth of 2.5 nm. Data points were recorded every 0.5 s.
We thank C.A. Gross for providing strains and plasmids, D. Kahne and T. Silhavy for sharing strains, M. Ehrmann for the gift of plasmids and antibodies, and A. Charbit, E. Deuerling, B. Bukau, and K. Williams for providing antibodies. We also thank W. Kramer for helpful discussions. The work was supported by grants of the DFG to S.B.
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