The chaperone-assisted membrane release and folding pathway is sensed by two signal transduction systems
1997; Springer Nature; Volume: 16; Issue: 21 Linguagem: Inglês
10.1093/emboj/16.21.6394
ISSN1460-2075
Autores Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle1 November 1997free access The chaperone-assisted membrane release and folding pathway is sensed by two signal transduction systems C. Hal Jones C. Hal Jones Department of Molecular Microbiology, Washington University Medical School, Box 8230, 660 South Euclid Avenue, St Louis, MO, 63110 USA Search for more papers by this author Paul N. Danese Paul N. Danese Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Jerome S. Pinkner Jerome S. Pinkner Department of Molecular Microbiology, Washington University Medical School, Box 8230, 660 South Euclid Avenue, St Louis, MO, 63110 USA Search for more papers by this author Thomas J. Silhavy Thomas J. Silhavy Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Scott J. Hultgren Corresponding Author Scott J. Hultgren Department of Molecular Microbiology, Washington University Medical School, Box 8230, 660 South Euclid Avenue, St Louis, MO, 63110 USA Search for more papers by this author C. Hal Jones C. Hal Jones Department of Molecular Microbiology, Washington University Medical School, Box 8230, 660 South Euclid Avenue, St Louis, MO, 63110 USA Search for more papers by this author Paul N. Danese Paul N. Danese Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Jerome S. Pinkner Jerome S. Pinkner Department of Molecular Microbiology, Washington University Medical School, Box 8230, 660 South Euclid Avenue, St Louis, MO, 63110 USA Search for more papers by this author Thomas J. Silhavy Thomas J. Silhavy Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ, 08544 USA Search for more papers by this author Scott J. Hultgren Corresponding Author Scott J. Hultgren Department of Molecular Microbiology, Washington University Medical School, Box 8230, 660 South Euclid Avenue, St Louis, MO, 63110 USA Search for more papers by this author Author Information C. Hal Jones1, Paul N. Danese2, Jerome S. Pinkner1, Thomas J. Silhavy2 and Scott J. Hultgren 1 1Department of Molecular Microbiology, Washington University Medical School, Box 8230, 660 South Euclid Avenue, St Louis, MO, 63110 USA 2Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ, 08544 USA The EMBO Journal (1997)16:6394-6406https://doi.org/10.1093/emboj/16.21.6394 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The assembly of interactive protein subunits into extracellular structures, such as pilus fibers in the Enterobacteriaceae, is dependent on the activity of PapD-like periplasmic chaperones. The ability of PapD to undergo a β zippering interaction with the hydrophobic C-terminus of pilus subunits facilitates their folding and release from the cytoplasmic membrane into the periplasm. In the absence of the chaperone, subunits remained tethered to the membrane and were driven off-pathway via non-productive interactions. These off-pathway reactions were detrimental to cell growth; wild-type growth was restored by co-expression of PapD. Subunit misfolding in the absence of PapD was sensed by two parallel pathways: the Cpx two-component signaling system and the σE modulatory pathway. Introduction Periplasmic chaperones are an essential component of the machinery required for biogenesis of surface-localized structures such as bacterial pili (Hultgren et al., 1991, 1993, 1996, and references therein). These structures are typically associated with virulence, since they contain adhesins which mediate binding to complementary surfaces of host receptors (Hultgren et al., 1993, 1996; Roberts et al., 1994). P pili are produced specifically by uropathogenic strains of Escherichia coli and have been studied extensively as a model system to understand macromolecular assembly (Hultgren et al., 1991, 1993, 1996). Subunits destined for assembly into pili pass through a periplasmic intermediate state (Hultgren et al., 1991, 1996). Stability of pilus subunits in the periplasm is dependent on the activity of the PapD chaperone, which binds to each of the P pilus subunits to form bimolecular complexes (Hultgren et al., 1991, 1996). Periplasmic complexes between PapD and PapA, PapE, PapF, PapG and PapK have been identified and characterized (Hultgren et al., 1991, 1993, 1996; unpublished data). Once formed, chaperone-subunit complexes are targeted to outer membrane assembly sites, composed of proteins known as ushers, where pilus biogenesis takes place (Dodson et al., 1993). In the absence of the chaperone the subunits collapse into off-pathway aggregates that are proteolytically degraded (Bullitt et al., 1996; Hultgren et al., 1996). The studies described in this report provide new insights into the mechanism of how the chaperone guides translocation of subunits across membranes and facilitates their folding and assembly into pili. PapD is the prototype member of a family of 26 periplasmic chaperones present in most members of the Enterobacteriaceae as well as in other bacteria (Hultgren et al., 1996; Hung et al., 1996). The 3-dimensional structure of PapD revealed that it possesses two globular immunoglobulin-like domains oriented such that a deep cleft is formed between the domains (Kuehn et al., 1993). The structural basis for part of the chaperone-subunit interaction was solved by co-crystallizing PapD with a peptide corresponding to the C-terminus of PapG (Kuehn et al., 1993). Utilizing an in vitro assay we demonstrate that PapD facilitates the targeting of pilus subunits into the periplasm and drives the subunits down a productive pathway such that the subunits are kinetically partitioned away from an aggregative fate. We present data suggesting that PapD facilitates the final tertiary fold of the PapG adhesin. We also discovered that misfolded subunits activate one or both signal transduction pathways (Mecsas et al., 1993; Danese et al., 1995) that induce synthesis of multiple factors, such as the DegP protease, for recruitment to the periplasm. One pathway operates via a classic two component regulatory system, Cpx (Danese et al., 1995; Danese and Silhavy, 1997; Pogliano et al., 1997), while the second pathway results in direct modulation of a σ factor, σE (Mecsas et al., 1993; Raina et al., 1995; Rouviere et al., 1995; De Las Penas et al., 1997; Missiakis and Raina, 1997; Missiakis et al., 1997). Results An assay to study the role of PapD in pilus biogenesis Synthesis of P pilin subunits in the absence of PapD results in their proteolytic degradation (Hultgren et al., 1989; Slonim et al., 1992). We employed an isogenic pair of strains KS272 (MC1000) and KS474 (KS272 degP::kan) to test the role of the periplasmic protease DegP (Strauch et al., 1989; Lipinska et al., 1990) in degradation of subunits. It was anticipated that in this genetic background new insights into the mechanism of action of PapD could be gained, since subunits would accumulate in the absence of the chaperone and be more amenable to study. In KS272 high level synthesis of PapG and PapE in the absence of PapD caused a profound growth defect, whereas PapA and PapK were tolerated when produced at similar or higher levels than PapE or PapG (Figure 1A–C and unpublished data). In KS474 PapA synthesis was found to be highly toxic, while PapK was tolerated when synthesized at similar or higher levels (Figure 1A and C). As expected from the results in KS272, PapG and PapE were highly toxic in KS474 (Figure 1B and D). Subunit synthesis (PapG, PapE and PapA) resulted in growth arrest; affected cultures showed no change in viable counts (∼1×107 c.f.u./ml throughout the 6 h assay) when plated on LB agar lacking IPTG (unpublished data). We hypothesize that interactive surfaces on subunits promote non-productive interactions in the absence of the chaperone, leading to growth arrest. The inability of PapK synthesis to inhibit growth may be related to its inability to self-associate (Bullitt et al., 1996), which makes it less aggregative. The reason for PapG toxicity is not readily apparent. However, this protein is larger than the other pilins and previous studies have revealed a potential for it to aggregate in vitro (Kuehn et al., 1991; see also below). Figure 1.Subunit synthesis is toxic, resulting in a severe growth defect. (A) PapA synthesis is toxic in KS474 (pHJ2, filled squares) but tolerated in the parent strain KS272 (pHJ2, filled circles). (B) PapG synthesis is toxic in both KS272 and KS474 (pHJ8, filled symbols). (C) PapK synthesis is not toxic in either KS272 or KS474. (D) PapE synthesis is toxic (pHJ13, open circles) and toxicity is suppressed by co-synthesis of PapD (pHJ13+pHJ9203, open squares). (E) Non-aggregative PapA derivatives PapA-G150T (pHJ33, open circles), PapA-Y162L (pHJ34, filled triangles) and PapA-C22S (pHJ35, dashed line) are non-toxic in KS474. (F) Removal of the signal sequence (pRS4A, filled squares) renders PapA non-toxic in KS474. Download figure Download PowerPoint Toxicity is a result of exposure of the subunit β zipper motif and other interactive surfaces The conserved C-terminus of subunits has been implicated in forming an interactive surface that drives subunit–subunit interactions (Bullitt et al., 1996). A Leu substitution at Tyr162 (Y162L) and a Thr substitution at Gly150 (G150T) in the C-terminal β zipper motif of PapA have been shown to abolish the subunit–subunit interactions necessary for formation of pilus rods (Bullitt et al., 1996). However, both the mutant and wild-type proteins accumulated in the periplasmic space of both strains, KS272 and KS474, to the same extent when co-synthesized with the PapD chaperone (Bullitt et al., 1996). A Ser substitution for Cys22 (C22S) in PapA, which forms a conserved disulfide with Cys61, also abolished the subunit–subunit interactions necessary for pilus formation (R.Striker and S.J.Hultgren, personal communication), but was stable when expressed in strain KS474. Since these mutations disrupt interactive surfaces on the subunit, we tested whether these mutations would also abolish the toxic effect associated with expression of the subunit in the absence of the chaperone. Figure 1E shows that the non-aggregative PapA mutants (Y162L, G150T and C22S) are non-toxic when expressed in KS474, whereas a conserved substitution, Y162F, which assembles into pili, although to a lesser degree, remains toxic. These data suggest that exposure of the C-terminal β zipper in the periplasm causes the observed toxic phenotype in strain KS474. Toxicity arises from the periplasm The growth defect resulting from subunit production in the absence of PapD suggests that the toxic effect originates from the periplasm. Further support for this hypothesis was garnered by demonstrating that signal sequence processing is required for the observed toxicity. A PapA mutant lacking a signal sequence is not toxic when expressed in KS474 (Figure 1F). These results suggest that engagement of the Sec machinery and transport across the inner membrane is required for toxicity. Toxicity suppression and correlation with complex formation If the role of the periplasmic chaperone is to protect interactive subunit surfaces and block their aggregation, then production of the chaperone along with the toxic subunit should be sufficient to suppress toxicity. In both strains, KS272 and KS474, co-expression of the periplasmic chaperone PapD suppressed the toxicity of pilin subunit synthesis (Figures 1D, E, 2A and B and unpublished data). Figure 2.Subunit toxicity is relieved by co-synthesis of the PapD periplasmic chaperone. (A) PapG toxicity (pHJ31, open circles) is suppressed by co-synthesis of PapD (pL5101, filled circles). (B) PapA toxicity (pHJ2, open circles) is suppressed by co-synthesis of PapD (pHJ9203, filled circles) but not by co-synthesis of R8G PapD mutant protein (pHJ9204, open squares). Download figure Download PowerPoint The invariant residues Arg8 and Lys112 in the crevice of the PapD cleft form a critical part of the subunit binding site (Slonim et al., 1992; Kuehn et al., 1993). Substitutions at Arg8 and Lys112 had no effect on PapD accumulation in the periplasm (Slonim et al., 1992; Kuehn et al., 1993). We tested the effect of mutating the invariant Arg8 residue to Gly (R8G) on the ability of PapD to suppress the toxic effects of PapA. The R8G mutation in PapD blocked its ability to suppress PapA toxicity (Figure 2B). These data confirm that the interactions, via the cleft residues, known to be essential for chaperone–subunit complex formation are also required for suppression of toxicity. Periplasmic localization is dependent on PapD In the light of precedents established with other systems, several explanations can be offered for the toxicity caused by overproduction of the Pap subunits. For example, it is possible that high level production of these subunits causes a deleterious 'jamming' of the general secretion machinery. This would inhibit envelope biogenesis and cause accumulation of the precursor forms of secreted proteins in the cytoplasm (Snyder and Silhavy, 1992). We ruled out the 'jamming' hypothesis by showing that the kinetics of signal sequence processing for periplasmic maltose binding protein (MBP) were unaffected by induction of either PapA or PapG (unpublished data). We also ruled out the related hypothesis that overproduced Pap subunits might interfere with discharge of proteins from the secretion machinery by showing that MBP release into the periplasm was also unaffected (unpublished data). These results demonstrate that overproduction of Pap subunits does not interfere with the function of the general protein secretion machinery. The toxicity of overproduced Pap subunits could also result from improper targeting of these proteins to the periplasm (Matsuyama et al., 1995). Accordingly, we tested the periplasmic localization of pilin subunits when expressed in KS474 in the absence or presence of PapD (Figure 3A). Production of PapA, PapE, PapG and PapK in the absence of PapD resulted in poor accumulation of the subunits in the periplasm. These results suggest that efficient release of pilin subunits into the periplasm is dependent on PapD–subunit interactions. Figure 3.Targeting of pilin subunits to the periplasmic space is dependent on PapD. (A) Periplasmic fractions prepared from KS474 strains producing pilin subunits alone (odd numbered lanes) or producing subunits and PapD (even numbered lanes). Coomassie blue staining of SDS-PAGE clearly demonstrates that accumulation of stable pilin subunits PapA, PapE, PapG and PapK is dependent on the chaperone. (B) Western blot with anti-PapD–PapG antisera demonstrating PapD-mediated targeting of PapG to the periplasmic space. Fractionation of KS474/pHJ8/pHJ9203 reveals that PapG resides in the inner membrane fraction when synthesized in the absence (lane 4) of PapD, but is efficiently targeted to the periplasmic space by PapD (lane 2). In lanes 3 and 5 the samples were not exposed to β-mercaptoethanol prior to SDS-PAGE, demonstrating that cysteine disulfides are formed in PapG prior to interaction with PapD. Download figure Download PowerPoint The above results suggest that subunits synthesized alone are either not produced efficiently or are associated with another compartment of the cell, such as the inner membrane. The fate of subunits expressed in the absence of the chaperone was investigated by fractionating KS474/pHJ8 (papG)/pHJ9203 (papD) into periplasmic and inner membrane fractions (Figure 3B). The fractions were immunoblotted with polyclonal antisera directed against the PapD–PapG complex. As shown in Figure 3B, PapG is only detectable in the periplasm following co-induction of PapD synthesis; PapG is associated with the inner membrane fraction when produced in the absence of PapD. Similar results were obtained with PapA and PapE (unpublished data). We conclude that the chaperone facilitates release of the subunit from an inner membrane location to the soluble periplasmic compartment. Spheroplast assay for testing the targeting function of PapD To further define the role of the chaperone in mediating subunit compartmentalization we established a semi-in vitro assay utilizing spheroplasts. A related spheroplast system was employed by Matsuyama et al. (1995) to define the activity of the periplasmic lipoprotein carrier protein P20. In our system spheroplasts were prepared from KS474/pHJ8; the spheroplasts were induced and pulse labeled in the presence or absence of purified PapD added to the supernatant. As shown in Figure 4A, addition of purified PapD to the spheroplast suspension increased the amount of immunoprecipitable PapG in the supernatant by >30-fold (Figure 4A, compare lanes 1 and 2). Figure 4.Spheroplast release assay. PapG synthesis was induced in spheroplasts produced from KS474/pHJ8 by addition of 1 mM IPTG. (A) Prior to pulse labeling with 35S Trans label (cysteine + methionine), 7 μg purified PapD (lane 2), the R8A PapD derivative (lane 3) or purified histidine-tagged PapD (lane 6) were added and allowed to incubate for 10 min. Following a 10 min chase with unlabeled cysteine + methionine the spheroplast suspension was subjected to centrifugation to remove the spheroplasts and the supernatant immunoprecipitated with antibody containing reactivity to PapG. In lanes 4 and 5 purified PapD was added 10 (lane 4) or 30 (lane 5) min following the cold chase. (B) The F314S substitution in PapG reduces the efficiency of targeting relative to wild-type PapG (compare lanes 3 and 5). Both the double mutant F314S+G302V and deletion of the C-terminal 100 amino acids relieve PapG dependence on PapD for efficient partitioning to the supernatant fraction. Download figure Download PowerPoint In a separate experiment PapD was added 10 or 30 min following the chase to test if targeting required that PapD interact with PapG during or shortly after translocation. In this experiment PapD-facilitated targeting would require that PapD act on post-translocated PapG that had accumulated in the inner membrane. PapD was fully capable of releasing PapG into the soluble fraction 10 and 30 min post-chase (Figure 4A, lanes 4 and 5). Furthermore, trypsin treatment of spheroplasts producing PapG released an ∼25 kDa fragment of PapG; demonstrating that a portion of PapG is exposed on the surface of the spheroplast (unpublished data). These findings suggest that membrane-associated PapG is stable and a substrate for PapD and we conclude that membrane-associated PapG is an intermediate in the biogenesis pathway. FimC, HifB and SfaE, the periplasmic chaperones required for assembly of type 1, Haemophilus and S pili respectively, and the newly discovered EcpD (E.coli PapD) (Raina et al., 1993) were purified and tested in the spheroplast assay. None of these chaperones were able to stimulate release of PapG into the supernatant fraction (unpublished data). The structural basis of the targeting reaction was investigated by testing the effect of site-directed mutations in both PapD and PapG of residues that are known to be critical for PapD–PapG complex formation based on the co-crystal structure of PapD with the PapG C-terminal peptide (Kuehn et al., 1993). Arg8 forms a critical part of the invariant subunit binding site in the chaperone cleft. The targeting activity of PapD was severely compromised by an R8A substitution, arguing that the invariant cleft is critical for this function (Figure 4A, lane 3). Phe314 and Gly302 are two conserved C-terminal PapG residues that are part of the conserved β zipper motif that is recognized by PapD. The Phe was changed to Ser (F314S PapG) as a single mutation and it was also combined with a Gly mutation to Val at position 302 (F314S+G302V PapG) as part of a double mutation. The C-terminal 100 residues of PapG were also deleted, yielding the PapGΔ100 truncate. The F314S mutation in PapG reduced the efficiency with which PapD released this derivative into the supernatant (Figure 4B, lane 5). F314S+G302V PapG was efficiently released to the supernatant in the absence of PapD, in contrast to wild-type PapG (Figure 4B, lanes 6 and 7). Moreover, the presence of PapD had no effect on release of this double mutant protein. Similarly, deletion of the C-terminal 100 amino acids resulted in a stable 24 kDa PapG derivative that was released into the supernatant independent of PapD (Figure 4B, lanes 8 and 9). These results argue that the β zipper region of a subunit facilitates its retention in the inner membrane in the absence of PapD and that binding of PapD to this surface is required for release from the membrane. Overproduction of PapG activates a two component signal transduction system Since the Cpx and σE modulatory systems both respond to overproduction of extracytoplasmic proteins (Mecsas et al., 1993; Danese et al., 1995; Danese and Silhavy, 1997; De Las Penas et al., 1997; Missiakis and Raina, 1997; Missiakis et al., 1997), we wished to determine if these systems were also affected by overproduction of the PapE and PapG subunits. Accordingly, we transformed strains PND2000 (MC4100, degP–lacZ), SP558 (PND2000, cpxA::cam) and SP559 (PND2000, cpxR::Ω) with pHJ8 (overproducing PapG) and pMMB66 (vector control for pHJ8) and then determined the amount of degP–lacZ transcription generated from these transformants. Lanes 1 and 2 of Figure 5A show that overproduction of PapG stimulates degP–lacZ transcription 6.7-fold. Interestingly, overproduction of PapG only stimulates degP–lacZ transcription 2.4-fold in the absence of CpxA (compare lanes 3 and 4 of Figure 5A). Finally, in a cpxR−, cpxA− background (SP559) the stimulatory effect of PapG overproduction on degP–lacZ transcription is only 2.2-fold (compare lanes 5 and 6 of Figure 5A). Thus overproduction of PapG stimulates degP transcription, in part, by activation of the Cpx signal transduction system. Figure 5.Pilus subunit synthesis in the absence of PapD results in promoter activation via the Cpx and σE signal transduction pathways. (A) degP–lacZ promoter activation as a result of PapG production utilizes both the Cpx and σE modulation pathways. degP–lacZ activity was monitored in strains PND2000, SP558 and SP559 following induction of synthesis of PapG from pHJ8. (B) Co-synthesis of PapD abrogates degP–lacZ promoter activation. degP–lacZ activity was monitored in PND2000 following induction of PapG synthesis (pHJ9208) or PapG and PapD synthesis (pHJ9208 + pHJ6). (C) Synthesis of PapG activates the rpoHP3–lacZ promoter, indicating that PapG misfolding is sensed by the σE modulatory system. rpoHP3–lacZ activity was monitored in SP616 following induction of PapG synthesis (pHJ8). (D) PapG activation of the cpxP–LacZ promoter is Cpx dependent. cpxP–lacZ activity was monitored in SP594 and SP620 following induction of PapG synthesis (pHJ8). (E) Synthesis of PapE activates the degP–lacZ promoter in a Cpx-dependent manner. degP–lacZ activity was monitored in PND2000, SP558 and SP559 following induction of PapE synthesis (pHJ13). (F) The cpxP–lacZ promoter is activated by PapE synthesis and is Cpx dependent. cpxP–lacZ activity was monitored in SP594, SP619 and SP620 following induction of PapE synthesis (pHJ13). (G) Pilus biogenesis is monitored by the Cpx and σE signal transduction pathways. degP–lacZ activity was monitored in PND2000, SP558 and SP559 following induction of pilus synthesis from pFJ29 (pap) and pFJ29-71 (papD−). degP–lacZ promoter activation is seen, especially following induction of the pilus operon lacking a functional papD gene (pFJ29-71, papD−). (H) degP–lacZ promoter activation resulting from initiation of pilus biogenesis is not entirely Cpx dependent, as seen in triggering of the σE modulatory pathway. rpoHP3–lacZ activity was monitored following induction of the pilus operon from pFJ29 and pFJ29-71 in SP616. Download figure Download PowerPoint The results presented in Figures 1 and 2 indicated that co-synthesis of the PapD chaperone blocks the growth defect associated with overproduction of P pilin subunits. Thus we hypothesized that co-synthesis of PapG and PapD would suppress PapG-mediated induction of degP transcription. As shown in Figure 5B, production of PapG with simultaneous synthesis of PapD significantly reduced the amount of degP transcription generated from PND2000. This result implies that E.coli monitors the assembly status of PapG and uses this information to modulate degP transcription. PapG synthesis stimulates σE activity Figure 5A shows that synthesis of PapG causes a residual induction of degP transcription even in the absence of the Cpx pathway (compare lanes 1 and 2 with lanes 5 and 6). Therefore, a second signal transduction system must also sense the unfolded state of PapG and stimulate degP transcription. The σE modulatory system has been shown to activate both the degP and the rpoHP3 promoters (Mecsas et al., 1993). Because the rpoHP3 promoter is solely controlled by σE and not by the Cpx system (Danese et al., 1995), we used an rpoHP3–lacZ operon fusion to determine if PapG synthesis also stimulated σE activity. Figure 5C shows that PapG synthesis (from pHJ8) stimulates rpoHP3–lacZ [SP616 (MC4100, rpoHP3–lacZ)] transcription 2.4-fold, which closely agrees with the ∼2.2-fold stimulation of degP transcription in the absence of the Cpx proteins (Figure 5A, lanes 5 and 6). Production of PapG stimulates transcription from a second Cpx-regulated locus In an effort to corroborate the conclusion that synthesis of unchaperoned PapG stimulates the Cpx pathway, we assayed a second Cpx-regulated locus, cpxP, whose transcription is wholly dependent on CpxR and is independent of σE (P.N.Danese, doctoral thesis, Princeton University). To this end, we determined the β-galactosidase activities of strains SP594 (MC4100, cpxP–lacZ) and SP620 (SP594, cpxR::Ω) following induction of PapG synthesis. As shown in Figure 5D, PapG synthesis in the parental strain (SP594) stimulates cpxP transcription 10.3-fold. In a cpxR−, cpxA− background (SP620) cpxP transcription is not induced following PapG synthesis. PapE synthesis stimulates Cpx but not σE activity We also performed the assays described above with the P pilus fibrillar component PapE (pHJ13). Surprisingly, unlike PapG, PapE production stimulates only the Cpx signal transduction pathway. As shown in Figure 5E, induction of PapE synthesis in PND2000 caused a 4-fold stimulation of degP transcription (compare lanes 1 and 2), whereas in SP558 and SP559 the amount of degP–lacZ transcription is not elevated above the background level (compare lanes 3 and 4 and lanes 5 and 6). High level synthesis of PapE also stimulated cpxP–lacZ transcription in a Cpx-dependent fashion (Figure 5F) and failed to stimulate transcription from the rpoHP3–lacZ fusion (unpublished data). Thus while the Cpx pathway can monitor the level of both PapE and PapG, the σE modulatory system can only monitor PapG levels. The Cpx pathway as a monitor of pilus assembly We also observed a significant activation of degP–lacZ transcription when the entire P pilus operon was expressed in the absence of the PapD chaperone (Figure 5G, compare lanes 1, 2 and 3). This P pilus-mediated induction of degP transcription is significantly reduced in the absence of the Cpx pathway (compare lanes 2 and 3 with lanes 8 and 9); however, a residual induction in degP transcription was still detected (compare lanes 7–9). This residual induction (∼2.1-fold) appears to be due to an increase in σE activity because high level expression of the P pilus operon also stimulated rpoHP3–lacZ transcription ∼2.1-fold (Figure 5H). The implication from these results is that the Cpx and σE modulatory systems monitor the assembly status of the P pilus subunits and use this information to modulate synthesis of the periplasmic protease DegP in response to this input. Chaperone folding assay The presence of misfolded or partially denatured proteins in the periplasm is thought to be a signal that leads to activation of degP transcription. PapD is known to be required to stabilize pilus subunits, so we decided to investigate further the folded state of pilin subunits synthesized in the absence of PapD. PapG activity can be monitored in vitro by binding to the Galα(1-4)Gal digalactoside (galabiose) receptor (Hultgren et al., 1991, 1996). Lectin binding activity has been shown to be a function of tertiary structure (Weis et al., 1988). We tested the ability of periplasmically localized full-length PapG to bind to galabiose when produced alone or together with wild-type PapD. Nearly 100% of the immunoprecipitable PapG in the periplasm bound to digalactoside when PapD was present. However, when PapD was absent <10% of the immunoprecipitable periplasmic PapG bound to receptor (Figure 6). These results indicate that in the absence of PapD, PapG is unable to achieve or be maintained in a native-like receptor binding conformation. Figure 6.PapD-mediated folding of PapG as assayed by galabiose chromatography. (A) Labeled periplasms containing PapG only (lanes 1 and 7) or PapG along with PapD (lanes 2 and 8), PapD R8A (lanes 3 and 9), PapD K112A (lanes 4 and 10), FimC (lanes 5 and 11) or the PapD–FimC chimera (lanes 6 and 12) were subjected to immunoprecipitation (lanes 1–6) or galabiose–Sepharose chromatography (lanes 7–12). (B) Phosphorimager quantificati
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