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seminars in C E L L & D E V E L OP M E N T A L B I OL OG Y, Vol 11, 2000: pp. 27᎐34 doi:10.1006rscdb.1999.0348, available online at http:rrwww.idealibrary.com on PapD-like chaperones and pilus biogenesis Frederic G. Sauer a, Stefan D. Knight c, Gabriel Waksman b and Scott J. Hultgrena,U tion, and assembly.1 The PapD-like superfamily of periplasmic chaperones directs the assembly of an array of adhesive surface organelles that mediate attachment to host tissues, a critical early step in the development of disease,1,2 ŽTable 1.. The elucidation of the function of PapD-like chaperones has revealed molecular details of a mechanism that has contributed to our understanding both of general principles of organelle development and of the pathogenesis of a variety of bacterial diseases. The more than 30 known PapD-like chaperones facilitate the assembly of both pilus and non-pilus organelles2 ŽTable 1.. Much of our knowledge of the function of these molecules comes from the study of PapD and FimC, which assemble P and type 1 pili, respectively, in E. coli 3 ᎐ 5 ŽFigure 1.. Genes important in P Ž papA᎐papK . and type 1 Ž fimA᎐fimH . pilus biogenesis are organized in the pap and fim operons, respectively.1 P pili consist of six structural proteins that interact to form a composite fiber with two subassemblies: a 7-nm-thick pilus rod comprised of PapA subunits arranged in a right-handed helical cylinder, and a thin fibrillum comprised primarily of PapE subunits arranged in an open helical configuration.6,7 The PapK adaptor protein links the two subassemblies.8 The PapG adhesin, a virulence factor in uropathogenic E. coli, is joined to the distal end of the tip fibrillum by the PapF adaptor protein.8 ᎐ 10 Type 1 pili are also composite fibers consisting of a short tip fibrillum containing the FimH adhesin, joined to the distal end of a pilus rod.11 During pilus biogenesis, the chaperone interacts with each nascently translocated pilus subunit in the periplasm ŽFigure 1. via a mechanism termed donor strand complementation Žsee below.. This interaction facilitates the release of the subunit from the cytoplasmic membrane in a process that my be driven by its folding directly on the chaperone template. The chaperone remains bound to the folded subunit, stabilizing it and capping its interactive surfaces and The assembly of adhesive pili from individual subunits by periplasmic PapD-like chaperones in Gram-negative bacteria offers insight into the complex process of organelle biogenesis. PapD-like chaperones bind, stabilize, and cap interactive surfaces of subunits until they are assembled into the pilus. Subunits lack the seventh -strand necessary to complete their immunoglobulin-like folds; the chaperone supplies this missing strand. Indeed, the chaperone may act as a template, providing steric information to facilitate subunit folding. In the mature pilus, each subunit is thought to supply the missing strand to complete the fold of its neighbor. Thus, one general function of chaperones in organelle biogenesis may be to cap highly interactive surfaces of subunits until they reach the proper assembly site. Key words: chaperone r pilus r organelle biogenesis r protein folding 䊚2000 Academic Press Introduction A FUNDAMENTAL QUESTION in molecular biology is how proteins fold into domains that can serve as assembly modules for building up larger macromolecular structures. The biogenesis of large fibrous organelles, called pili, on the surface of Gram-negative bacteria requires the orchestration of a complex process that includes protein synthesis, folding, secre- From the aDepartments of Molecular Microbiology, bBiochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA, and cDepartment of Molecular Biology, Uppsala Biomedical Center, Swedish University U of Agricultural Sciences, Uppsala, Sweden. Corresponding author. 䊚2000 Academic Press 1084-9521r 00 r 010027q 08 $35.00r 0 27 F. G. Sauer et al. Table 1. Bacterial surface organelles assembled by PapD-like chaperones Organelle Žreference. Fibers P pilus Prs pilus Type 1 pilus Organism Chaperone PapC PrsC FimD Pyelonephritis, cystitis Cystitis? Cystitis FocC SfaE HifB HafB FocD SfaF HifC HafC Cystits? UTI, NBM Otitis media, meningitis Brazilian purpuric fever FimB ŽFhaD. PefD LpfB MrpD PmfD AftB AfrC FanE FimC ŽFhaA. PefC LpfC MrpC PmfC AftC AfrB FanD E. coli E. coli ETEC K. pneumoniae FaeE FasB F17D MrkB FaeD FasD F17papC MrkC Whopping cough Gastroenteritis, samonellosis Gastroenteritis?, samonellosis? Nosocomial UTI Nosocomial UTI UTI Diarrhea in rabbits Neonatal diarrhea in calves, lambs, piglets Neonatal diarrhea in piglets Diarrhea in piglets Diarrhea in piglets Pneumonia E. coli E. coli E. coli E. coli NfaE AfaB DraE BmaB NfaC AfaC DraD BmaC UTI, NBM Pyelonephritis UTI, diarrhea Pyelonephritis ETEC ETEC ETEC ETEC S. enteritidis Yersinia pestis Y. pestis, Y. pseudotuberculosis Y. enteritidis Cs3-1 ClpE CssC AggD SefB Caf1M PsaB Cs3-2 ClpD CssD? AggC SefC Caf1A PsaC Traveler’s diarrhea Diarrhea Diarrhea Diarrhea Gastroenteritis, salmonellosis Plague Plague MyfB MyfC Entercolitis RalE RalD Diarrhea in rabbits Type 2 and 3 pili Pef pilus Lpf pilus MRrP pilus PMF pilus Aft pilus AFrR1 pilus38 K99 pilus K88 pilus 987P pilus F17 pilus MRrK ŽType 3. pilus Non-fimbrial Adhesins NFA1-6 family Afa-1 DrrAfa-111 M Atypical structures CS3 CS31A pilus CS6 pilus AAFr1 Sef F1 antigen PH6 antigen Myf Unknown structures ? Disease PapD PrsD FimC E. coli E. coli E. coli, Salmonella ssp., Klebsiella pneumoniae E. coli E. coli Hemophilus influenzae H. influenzae biogroup aegyptius Bordetella pertussis S. typhimurium S. typhimurium Proteus mirabilis P. mirabilis P. mirabilis E. coli E. coli F1C pilus S pilus Hif pilus Haf pilus Usher REPEC 17 Notes. Adapted from Table 1 in Thanassi et al. See refs 2 and 17 for references not listed. UTI, urinary tract infection; NBM, newborn meningitis; ETEC, enterotoxigenic E. coli; REPEC, rabbit enteropathogenic E. coli. thus preventing premature aggregation in the periplasm.12 ᎐ 16 Each PapD-like chaperone functions in concert with a corresponding outer membrane usher protein.17 Chaperone᎐subunit complexes are specifically targeted to the usher, which forms a 2᎐3nm diameter donut-shaped channel, large enough to allow the passage of pilus subunits.18,19 In the fim and pap systems, it has been shown that the chaperone᎐ adhesin complex binds most rapidly and tightly to the usher in a process that is thought to initiate pilus assembly. Formation of a chaperone᎐adhesin᎐usher ternary complex induces a conformational change in the usher to an assembly-competent form that is maintained throughout pilus assembly.20 The usher facilitates chaperone uncapping to expose the interactive surfaces on the subunits that drive their assembly into the pilus. The pilus is thought to grow through the usher as a linear fiber that packages into its final quaternary structure upon reaching the bacterial surface.19 28 Chaperones and pilus biogenesis Figure 1. Schematic of P pilus biogenesis. P pili are members of a large family of surface organelles assembled by PapD-like chaperones and their corresponding ushers. Electron micrographs of a P pilus and a type 1 pilus from E. coli are shown Ž1.. Pilus subunits enter the periplasm through the Sec transport system. In the absence of the PapD periplasmic chaperone, subunits aggregate and are degraded Ž2., inducing signal transduction in the bacterium. In the presence of PapD, subunits form stable chaperone᎐subunit complexes Ž3. through a mechanism termed donor strand complementation Žthe PapD᎐PapK chaperone᎐subunit complex is shown. Ž4., in which the chaperone donates its G1 -strand to complete the Ig fold of the subunit ŽFigure 2.. This interaction permits subunit folding and prevents both subunit aggregation and premature subunit polymerization. The chaperone᎐subunit complexes are then targeted to an outer membrane protein pore termed the usher ŽPapC., where the subunits assemble into a pilus. The chaperone᎐adhesin complex ŽPapD᎐PapG. binds most rapidly and tightly to the usher, initiating pilus assembly Ž5.. Assembly of subsequent subunits Ž6. occurs through a mechanism termed donor strand exchange Ž7., in which the disordered N-terminus of an incoming subunit displaces the G1 strand of the chaperone in the most recently assembled chaperone᎐subunit complex ŽFigure 3.. Thus, in the mature pilus, the Ig fold of a subunit is completed by the N-terminal strand of the adjacent subunit. The rod subunits ŽPapA. wind into their final helical form once outside the cell Ž8., a process that is thought to facilitate the outward growth of the pilus. An electron micrograph of an unwound pilus rod is shown Ž9.. known as the Cpx pathway.12,21 The Cpx pathway responds to periplasmic stress by inducing the activation of a variety of genes encoding periplasmic protein folding factors ŽDsbA and prolyl isomerases. and periplasmic proteases ŽDegP..21 ᎐ 23 DegP degrades misfolded pilins, preventing their toxic buildup in The Cpx pathway In the absence of the chaperone, subunits are unstable and misfold, aggregate, and are proteolytically degraded. OFF-pathway subunits have been shown to activate a two-component signal transduction system 29 F. G. Sauer et al. the periplasm. DsbA catalyzes disulfide bond formation in proteins required for the assembly of P pili.24 Thus, Cpx is part of a pilus biogenesis sensorrcontrol circuit ŽFigure 1. that ensures that toxic OFF-pathway products do not accumulate.12 Chaperone–subunit complexes: donor strand complementation The crystal structure of PapD and the solution structure of FimC, and more recently, the crystal structures of the FimC᎐FimH chaperone᎐adhesin complex and the PapD᎐PapK chaperone᎐pilin complex have all been solved.25 ᎐ 28 The chaperone consists of two immunoglobulin-like ŽIg. domains oriented in an L-shape to form a cleft between them. The apo and complexed forms of the chaperone are virtually superimposable, with the exception of the F1 ᎐G1 loop, as discussed below. The FimH adhesin consists of two domains: a pilin domain and a receptor-binding domain. The PapK pilin and the pilin domain of FimH have Ig folds; however, they lack the seventh and C-terminal -strand Žstrand G. present in canonical Ig folds ŽFigure 2.. The absence of this strand produces a deep groove along the surface of the pilin domain and exposes its hydrophobic coreᎏhence the instability of pilins when expressed without the chaperone. In the chaperone᎐subunit complex, the G1 strand of the chaperone and a portion of the F1 ᎐G1 loop, which extends to lengthen the G1 strand, complete the Ig fold of the subunit by occupying the groove. This donor strand complementation interaction stabilizes the subunit by shielding its hydrophobic core. Indeed, alternating hydrophobic residues in the G1 strand become an integral part of the hydrophobic core of the subunit. The G1 strand provides the missing strand of the subunit Ig domain by interacting with strands A2 Žalso called A⬙ . and F located on either side of the groove, while the Cterminal carboxyl group of the subunit, at the end of the F strand, is anchored between the conserved Arg and Lys residues29 in the cleft of the chaperone ŽFigure 2.. The Ig fold that is produced is atypical since the G1 strand of the chaperone runs parallel, rather than anti-parallel, to the F strand, as it does in a canonical Ig fold.27,28,30 While stabilizing the subunit and completing its fold, the chaperone simultaneously caps one of its interactive surfaces. Mutational and biochemical studies indicate that the groove of the pilus subunit that participates in donor strand complementation is the same surface that interacts Figure 2. Donor strand complementation interactions in the FimC᎐FimH chaperone᎐adhesin complex. FimC is shown in black and FimH is in gray. The G1 -strand completes the Ig fold of the FimH pilin domain between the A⬙ Ži.e. A2. and F strands. The G1 -strand runs parallel to the F strand. The alternating hydrophobic residues of the G1 -strand ŽLeu103, Leu105, Ile107 in FimC. form part of the hydrophobic core of the pilin domain and may facilitate the collapse of the hydrophobic core during subunit folding. The C-terminal carboxyl group ŽCOOH. is anchored in the cleft between the two domains of FimC by the conserved Arg8 and Lys112 residues. with other subunits in the pilus.14,16 Thus, the G1 strand of the chaperone, by occupying the groove, prevents premature pilus formation in the periplasm. Donor strand exchange and pilus biogenesis Pilus subunits have an N-terminal extension Žresidues 1᎐13 in PapK, for example. with a highly conserved alternating hydrophobic motif that has been shown to participate in subunit᎐subunit interactions.16 This motif is similar to the alternating hydrophobic motif present in the G1 -strand of the chaperone that participates in donor strand complementation. The N-terminal extension does not contribute to the Ig fold of the subunit, but rather projects away from the rest of the pilin domain where it would be free to interact with another subunit.28 Based on these observations, we propose that during pilus biogenesis, the N-terminal extension of one subunit displaces the chaperone G1 strand from its neighboring subunit in a mechanism termed donor strand exchange27,28 ŽFigure 3.. The pilin domain of the adhesin lacks this N-terminal extension, consistent 30 Chaperones and pilus biogenesis one prevents non-productive interactions, thereby keeping it ON pathway. However, the PapD-like chaperones may play a more active role by providing a structural context for subunit folding. Donor strand complementation might occur either early or late in the folding pathway, but once the subunit is folded, the chaperone remains bound, capping the critical subunit groove. In other words, the chaperone may couple the folding of the subunit with the capping of the groove. Then, during donor strand exchange, the N-terminal extension of the neighboring subunit would replace the G1 strand of the chaperone in the groove, thus preventing the exposure of the interactive groove at any time during the entire folding and assembly pathway. -sheet formation is thought to be determined in a large part by tertiary context, even at solvent-accessible sites, and not by intrinsic secondary structural preferences.31 Donation of the G1 strand of the chaperone may provide the necessary tertiary context for the formation of the G1 , F, C1 -sheet,16 which forms half of the immunoglobulin fold of the pilin domain.27,28 Indeed, PapD is known to induce the formation of -strands in peptides corresponding to the C-terminal F strands of subunits.15,16,32 During chaperone᎐subunit complex formation, the interaction between the C-terminal carboxyl group at the end of the subunit F strand and the conserved Arg and Lys residues in the chaperone cleft would position the F strand correctly in relation to the chaperone G1 strand during subunit folding ŽFigure 2.. At the same time, this interaction would position the hydrophobic side chains of strand F to allow them to make the appropriate contacts with the alternating hydrophobic residues in the chaperone G1 strand. Thus, by providing the correct context for beta sheet formation, the G1 strand of the chaperone may facilitate the proper collapse of the hydrophobic core of the subunit ŽFigure 2.. Figure 3. Proposed model of subunit᎐subunit interactions in a pilus. The N-terminal extension of one subunit replaces the chaperone G1 strand and completes the Ig fold of the preceding subunit, running anti-parallel to the F strand. with its location at the tip of the pilus.27 The subsequent order of subunits in the pilus may at least in part be determined by the stereochemical complementarity between the N-terminal extension of the subunit and the groove of its neighbor.27,28 The known geometry of pilus rods indicates that the Nterminal strand would insert anti-parallel to the F strand of the neighboring subunit, unlike the chaperone G1 strand, which inserts in a parallel fashion.27,28 The mature pilus would thus consist of an array of perfectly canonical Ig domains, each of which contributes a strand to the fold of the preceding subunit to produce the organelle. Protein folding with a novel twist Donor strand complementation simultaneously stabilizes the pilus subunit and caps the interactive groove. In addition, donor strand complementation permits subunit folding. In the absence of the chaperone, subunits aggregate in the periplasm and are degraded by the DegP protease.12 These non-productive interactions are thought to interfere with the proper folding pathway. For example, PapG expressed in the absence of the chaperone in a degPy strain does not fold into a native receptor binding conformation.12 Therefore, by providing the seventh G strand to complete the subunit Ig fold, the chaper- Cytoplasmic and periplasmic chaperones The PapD-like chaperones differ in several respects from the DnaJrDnaKrGrpE and GroELrGroES cytoplasmic chaperone systems reviewed in this issue Žsee preceding paper by Agashe and Hartl. as well as in ref 33. DnaJ and DnaK bind to semiunfolded polypeptides and maintain them in folding-competent conformations by preventing them from engaging in non-productive interactions. DnaJ 31 F. G. Sauer et al. A model for organelle biogenesis? is thought to be released from the ternary complex, leaving DnaK tightly bound to the polypeptide. GrpE is then thought to facilitate the release of the polypeptide from DnaK, allowing it to fold in solution. In the GroELrGroES system, semi-unfolded or misfolded polypeptides interact with the hydrophobic walls of the GroEL chamber. This is thought to prevent non-productive interactions andror to facilitate the folding of misfolded proteins.33,34 Release of the folding-competent polypeptide from the hydrophobic walls allows folding to occur in solution. In contrast, in the PapD-like chaperone system, pilus subunits may fold directly on the chaperone template and remain bound to the chaperone in their fully folded states until they are assembled into a pilus at the usher. Also, the DnaJrDnaKrGrpE and GroELrGroES systems require ATP hydrolysis, while the PapD-like chaperones function in the absence of known cellular energy sources.35 Finally, the cytoplasmic and periplasmic chaperones differ in how they facilitate folding. Both the DnaJrDnaKrGrpE and GroELrGroES systems appear to prevent or overcome the misfolding of proteins, rather than contribute steric information during substrate folding.33 PapD-like chaperones, on the other hand, may contribute steric information during the folding of their substrates by providing an integral part of their Ig fold. In this model, the complete information required to produce a correctly folded Ig domain would reside not in a single polypeptide chain but rather in two distinct polypeptides.27 The various surface organelles assembled by Gramnegative bacteria, including pili, flagella, and other macromolecular structures, have to withstand the considerable forces that they experience in carrying out their functions. A flagellum that breaks apart daring the first stroke is of little use to the cell. A pilus that breaks upon attachment loses its adhesiveness. The proposed pilus structure explains how such a sturdy organelle can be built. The contribution of a portion of the fold from one molecule to another, as in the pilus model, produces a very tight interaction, one that can be repeated over many molecules to produce large structures. Protein polymerization by domain swapping could also lead to the formation of robust macromolecular assemblies.36 Indeed, any relatively strong interaction between two molecules that can be repeated over a network of molecules in one or more dimensions could be used to build an organelle. Many variations of such interactions could be imagined to produce different structures. Such interactions might play a role in the formation of detrimental structures as well, for example the amyloid fibers that characterize a variety of human diseases. The coordinated assembly of such organelles, however, requires that the cell allow the subunits to interact only at the site of assembly, since interactions elsewhere might be non-productive. In pilus biogenesis, the PapD-like chaperone assures that subunits interact with each other only at the usher. The FlgN Table 2. Partial list of chaperones involved in organelle biogenesis Organelle Representative chaperone Pilus CS1 pilus family PapD CooB Curli Flagellum Flagellum Flagellum Type II secretion system Type III secretion system Type III secretion system Type III secretion system Organelle subunit Reference 2,17, Table 1 herein Reviewed in ref 39 CsgG? FlgN FliT? FliJ PulS Pilus subunits CooA, CooD Žpilus subunits. CooC Žouter membrane protein. CsgA,CsgB FlgK, FlgL Žhook associated proteins. FliD Žfilament cap protein. FlgD, FlgE Žhook proteins., FliE? PulD Žouter membrane protein. SycD YopB, YopD 43,44 YscB YopN 45 VirG? YscC Žouter membrane channel protein. 46 40 37 37 41 42 Notes. Only representative chaperones from homologous families are listed. Chaperones may or may not be part of the final assembled organelle. 32 Chaperones and pilus biogenesis 12. Jones CH, Danese PN, Pinkner JS, Silhavey TJ, Hultgren SJ Ž1997. The chaperone-assisted membrane release and folding pathway is sensed by two signal transduction systems. EMBO J 16:6394᎐6406 13. Kuehn MJ, Normark S, Hultgren SJ Ž1991. Immunoglobulinlike PapD chaperone caps and uncaps interactive surfaces of nascently translocated pilus subunits. Proc Natl Acad Sci USA 88:10586᎐10590 14. Bullitt E, Jones CH, Striker R, Soto G, Jacob-Dubuisson F, Pinkner J, Wick MJ, Makowski L, Hultgren SJ Ž1996. Development of pilus organelle subassemblies in vitro depends on chaperone uncapping of a beta zipper. Proc Natl Acad Sci USA 93:12890᎐12895 15. Kuehn MJ, Ogg DJ, Kihlberg J, Slonim LN, Flemmer K, Bergfors, T, Hultgren SJ Ž1993. Structural basis of pilus subunit recognition by the PapD chaperone. Science 262:1234᎐1241 16. Soto GE, Dodson KW, Ogg D, Liu C, Heuser J, Knight S, Kihlberg J, Jones CH, Hultgren SJ Ž1998. Periplasmic chaperone recognition motif of subunits mediates quaternary interactions in the pilus. EMBO J 17:6155᎐6167 17. Thanassi DG, Saulino ET, Hultgren SJ Ž1998. The chaperonerusher pathway: a major terminal branch of the general secretory pathway. Curr Opin Microbiol 1:223᎐231 18. Dodson KW, Jacob-Dubuisson F, Striker RT, Hultgren SJ Ž1993. Outer membrane PapC molecular usher discriminately recognizes periplamic chaperone᎐pilus subunit complexes. Proc Natl Acad Sci USA 90:3670᎐3674 19. Thanassi DG, Saulino ET, Lombardo M-J, Roth R, Heuser J, Hultgren SJ Ž1998. The PapC usher forms an oligomeric channel: implications for pilus biogenesis across the outer membrane. Proc Natl Acad Sci USA 95:3146᎐3151 20. Saulino ET, Thanassi DG, Pinkner JS, Hultgren SJ Ž1998. Ramifications of kinetic partitioning on usher-mediated pilus biogenesis. EMBO J 17:2177᎐2185 21. Raivio TL, Silhavy TJ Ž1999. The sigmaE and Cpx regulatory pathways: overlapping but distinct envelope stress responses. Curr Opin Microbiol 2:159᎐165 22. Danese PN, Silhavy TJ Ž1997. The sigmaŽE. and Cpx signal transduction systems control the synthesis of periplasmic protein-folding enzymes in Escherichia coli. Genes Dev 11:1183᎐1193 23. Danese PN, Snyder WB, Cosma CL, Davis LJ, Silhavy TJ Ž1995. The Cpx two-component signal transduction pathway of Escherichia coli regulates transcription of the gene specifying the stress inducible periplasmic protease DegP. Genes Dev 9:387᎐398 24. Jacob-Dubuisson F, Pinkner J, Xu Z, Striker R, Padmanhaban A, Hultgren SJ Ž1994. PapD chaperone function in pilus biogenesis depends on oxidant and chaperone-like activities of DsbA. Proc Natl Acad Sci USA 91:11552᎐11556 25. Holmgren A, Branden C-I Ž1989. Crystal structure of chaperone protein PapD reveals an immunoglobulin fold. Nature 342:248᎐251 26. Pellecchia M, Guntert P, Glockshuber R, Wuthrich K Ž1998. NMR solution structure of the periplasmic chaperone FimC. Nat Struct Biol 5:885᎐890 27. Choudhury D, Thompson A, Stojanoff V, Langermann S, Pinkner J, Hultgren SL, Knight SD Ž1999. X-ray structure of the FimC᎐FimH chaperone᎐adhesin complex from uropathogenic Escherichia coli. Science 285:1061᎐1066 28. Sauer FG, Futterer K, Pinkner JS, Dodson K, Hultgren SJ, Waksman G Ž1999. Structural basis of chaperone function and pilus biogenesis. Science 285:1058᎐1061 29. Slonim LN, Pinkner JS, Branden C-I, Hultgren SJ Ž1992. Interactive surface in the PapD chaperone cleft is conserved in pilus chaperone superfamily and essential in subunit recognition and assembly. EMBO J 11:4747᎐4756 and FliT chaperones have been proposed to function analogously in flagellar biogenesis.37 Thus, one common function of the growing number of chaperones known to be involved in organelle biogenesis ŽTable 2. may be to regulate subunit assembly. By capping the interactive surfaces of subunits until they reach the appropriate assembly site, chaperones would guarantee that organelle assembly proceeds in an orderly fashion. Acknowledgements SJH is supported by NIH grants RO1DK51406 and RO1AI29549, GW by NIH grant RO1GM54033, and SDK by grants from the Swedish Research Council NFR and the Swedish Foundation for Strategic Research ŽStructural Biology Network.. FGS was supported by an NSF Predoctoral Fellowship. References 1. Hultgren SJ, Jones CH, Normark S Ž1996. Bacterial adhesins and their assembly, in Escherichia coli and Salmonella Cellular and Molecular Biology ŽNeidhardt FC ed. 2nd ed, pp 2730᎐2756. ASM Press, Washington, DC 2. Hung DL, Knight SD, Woods RM, Pinkner JS, Hultgren SJ Ž 1996 . M olecu lar basis of tw o subfam ilies of immunoglobulin-like chaperones. EMBO J 15:3792᎐3805 3. Lindberg F, Tennent JM, Hultgren SJ, Lund B, Normark S Ž1989. PapD, a periplasmic transport protein in P-pilus biogenesis. 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