Assembly of CS1 pili: the role of specific residues of the major pilin, CooA

CS1 pili are important virulence factors of enterotoxigenic Escherichia coli strains associated with human diarrheal disease. They are the prototype for a family of pili that share extensive sequence similarity among their structural and assembly proteins. Only four linked genes, cooB, cooA, cooC, and cooD, are required to produce CS1 pili in E. coli K-12. To identify amino acids important for the function of the major pilin CooA, we used alanine substitution mutagenesis targeting conserved residues in the N and C termini of the protein. To test function, we examined cooA mutants for the ability to agglutinate bovine erythrocytes. Each hemagglutination-negative (HA(-)) cooA mutant was examined to identify its assembly pathway defect. CooA has been shown to be degraded in the absence of CooB (K. Voegele, H. Sakellaris, and J. R. Scott, Proc. Natl. Acad. Sci. USA 94:13257-13261, 1997). We found several HA(-) cooA mutants that produced no detectable CooA, suggesting that recognition by CooB is mediated by residues in both the N and C termini of CooA. In addition, we found that alanine substitution for some of the conserved residues in the C-terminal motif "AGxYxG(x(6))T," which is found in all subunits of this pilus family, had no effect on pilus formation. However, alanine substitution for some of the alternating hydrophobic residues within this motif prevented CooA from interacting with CooD, which serves as both the tip adhesin and nucleation protein for pilus formation. Thus, it appears that some, but not all, of the residues in both the N and C termini of CooA play a critical role in the intermolecular interactions of the major pilin with the other structural and assembly proteins. We anticipate that the results obtained here for CS1 pili in enterotoxigenic E. coli will help develop an understanding of the pilus assembly pathway used by CS1 family members in several important human pathogens.     

New tools in an old trade: CS1 pilus morphogenesis

CS1 pili serve as the prototype for a large class of serologically distinct pili associated with enterotoxigenic Escherichia coli that cause diarrhoea in humans. The four genes essential for CS1 pilus morphogenesis, cooB, A, C and D, are arranged in an operon and encode structural and assembly proteins unlike those of other pilus systems commonly associated with Gram-negative bacteria. CS1 pili are composed primarily of the major pilin subunit, CooA, which determines the serological type of the pilus. The major pilin subunit is assembled into pili by the proteins CooB, CooC and CooD. CooD is both a minor component found at the pilus tip and an essential assembly protein, whereas CooC is an outer membrane protein thought to be involved in pilin transport. CooB is a novel periplasmic chaperone-like protein that forms intermolecular complexes with and stabilizes the major and minor pilins. Unlike other pilin chaperones, CooB also stabilizes the outer membrane component of the assembly system, CooC. The proteins of CS1 pili have no significant homology to those of the well-characterized Pap (pyelonephritis-associated) pili and related systems, although most of the features of pilus morphogenesis are similar. Therefore, these appear to be among the rare cases of convergent evolution. Thus, for CS1 pili, enterotoxigenic E. coli use new protein 'tools' in the old 'trade' of forming functional pili.     

Products of three accessory genes, pilB, pilC, and pilD, are required for biogenesis of Pseudomonas aeruginosa pili.

The polar pili of Pseudomonas aeruginosa are composed of monomers of the pilin structural subunits. The biogenesis of pili involves the synthesis of pilin precursor, cleavage of a six-amino-acid leader peptide, membrane translocation, and assembly of monomers into a filamentous structure extending from the bacterial surface. This report describes three novel genes necessary for the formation of pili. DNA sequences adjacent to pilA, the pilin structural gene, were cloned and mutagenized with transposon Tn5. Each of the insertions were introduced into the chromosome of P. aeruginosa PAK by gene replacement. The effect of the Tn5 insertions in the bacterial chromosome on pilus assembly was assessed by electron microscopy and sensitivity of mutants to a pilus-specific bacteriophage. The resultant mutants were also tested for synthesis and membrane localization of the pilin antigen in order to define the genes required for maturation, export, and assembly of pilin. A 4.0-kilobase-pair region of DNA adjacent to the pilin structural gene was found to be essential for formation of pili. This region was sequenced and found to contain three open reading frames coding for 62-, 38- to 45-, and 28- to 32-kilodalton proteins (pilB, pilC, and pilD, respectively). Three proteins of similar molecular weight were expressed in Escherichia coli from the 4.0-kilobase-pair fragment flanking pilA with use of a T7 promoter-polymerase expression system. The results of the analyses of the three genes and the implications for pilin assembly and maturation are discussed.     

Identification and characterization of a gene product that regulates type 1 piliation in Escherichia coli.

The recombinant plasmid pSH2 confers type 1 piliation (Pil+) on a nonpiliated (Pil-) strain of Escherichia coli K-12. At least four plasmid-encoded gene products are involved in pilus biosynthesis and expression. We present evidence which indicates that one gene encodes an inhibitor of piliation. Hyperpiliated (Hyp) mutants were isolated after Tn5 insertion mutagenesis of pSH2 and introduction of the plasmid DNA into a Pil- strain of E. coli as unique small, compact colonies. Also, Hyp mutants clumped during growth in static broth and were piliated under several cultural conditions that normally suppressed piliation. Electron microscopic examination of Hyp mutants associated an observed 40-fold increase in pilin antigen with an increase in the number and length of pili per cell. All Hyp mutants examined failed to produce a 23-kilodalton protein that was encoded by a gene adjacent to the structural (pilin) gene for type 1 pili, and all Tn5 insertion mutations that produced the Hyp phenotype mapped in this region (hyp). Piliation in Hyp mutants could be reduced to near parental levels by introducing a second plasmid containing a parental hyp gene. Thus the 23-kilodalton (hyp) protein appears to act in trans to regulate the level of piliation.     

Type IV pilus genes pilA and pilC of Pseudomonas stutzeri are required for natural genetic transformation, and pilA can be replaced by corresponding genes from nontransformable species

Pseudomonas stutzeri lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation-deficient mutants were isolated from a P. stutzeri JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of Pseudomonas aeruginosa, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The pilC mutant had no pili and was defective in twitching motility. Further sequencing revealed that pilC is clustered in an operon with genes homologous to pilB and pilD of P. aeruginosa, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a pilA gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of pilA abolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of (3)H-labeled P. stutzeri DNA that were bound to competent parental cells and taken up were strongly reduced in the pilC and pilA mutants. Remarkably, the cloned pilA genes from nontransformable organisms like Dichelobacter nodosus and the PAK and PAO strains of P. aeruginosa fully restored pilus formation and transformability of the P. stutzeri pilA mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium P. stutzeri function in DNA uptake for transformation and that their role in this process is not confined to the species-specific pilin.     

Maturation of IncP pilin precursors resembles the catalytic Dyad-like mechanism of leader peptidases

The pilus subunit, the pilin, of conjugative IncP pili is encoded by the trbC gene. IncP pilin is composed of 78 amino acids forming a ring structure (R. Eisenbrandt, M. Kalkum, E.-M. Lai, C. I. Kado, and E. Lanka, J. Biol. Chem. 274:22548-22555, 1999). Three enzymes are involved in maturation of the pilin: LepB of Escherichia coli for signal peptide removal and a yet-unidentified protease for removal of 27 C-terminal residues. Both enzymes are chromosome encoded. Finally, the inner membrane-associated IncP TraF replaces a four-amino-acid C-terminal peptide with the truncated N terminus, yielding the cyclic polypeptide. We refer to the latter process as "prepilin cyclization." We have used site-directed mutagenesis of trbC and traF to unravel the pilin maturation process. Each of the mutants was analyzed for its phenotypes of prepilin cyclization, pilus formation, donor-specific phage adsorption, and conjugative DNA transfer abilities. Effective prepilin cyclization was determined by matrix-assisted laser desorption-ionization-mass spectrometry using an optimized sample preparation technique of whole cells and trans-3-indolyl acrylic acid as a matrix. We found that several amino acid exchanges in the TrbC core sequence allow prepilin cyclization but disable the succeeding pilus assembly. We propose a mechanism explaining how the signal peptidase homologue TraF attacks a C-terminal section of the TrbC core sequence via an activated serine residue. Rather than cleaving and releasing hydrolyzed peptides, TraF presumably reacts as a peptidyl transferase, involving the N terminus of TrbC in the aminolysis of a postulated TraF-acetyl-TrbC intermediate. Under formal loss of a C-terminal tetrapeptide, a new peptide bond is formed in a concerted action, connecting serine 37 with glycine 114 of TrbC.     

Localizing the subunit pool for the temporally regulated polar pili of Caulobacter crescentus

The pili of the stalked bacterium Caulobacter crescentus are assembled at a specific time in the life cycle at one pole of the cell and are composed of the monomer protein, pilin. A previous study demonstrated that the onset of pilin synthesis occurs well before pili appear on the surface, suggesting that pilin accumulates within the cell. In the present study, an electron microscope immunocytochemistry assay was used to determine the subcellular location of this unassembled pilin and its fate during pilus assembly and cell division. Populations of synchronously growing cells were embedded in epoxy resin at selected times during the cell cycle. Ultrathin sections were treated with pilin-specific antibody, followed by protein A coupled to colloidal gold. It was determined that the cellular location for unassembled pilin was the cell cytoplasm. All cell membranes and regions of nuclear material were poorly labeled. Quantitation demonstrated that label density increased during the period of pilin synthesis and declined during the period of pilus assembly and maintenance. The pilin pool was not unequally segregated at division; e.g., to the daughter cell that is elaborating pili. Mutants which have simultaneously lost the ability to produce flagella, pili, and other polar organelles, possibly due to alterations in the specialized region of polar organelle assembly, were also examined by the immunocytochemistry technique. There was no significant difference in the pilin pool size relative to the wild type, indicating that pilin synthesis continues in the absence of a functioning assembly site. This pattern of synthesis and assembly for the pilus is significantly different from that of the polar flagellum which is produced at the same time and location on the cell surface. These findings are discussed in relation to the hypothesized organization center at the cell pole which may have a major role in directing the assembly of all the polar structures.     

Isopeptide bonds of the major pilin protein BcpA influence pilus structure and bundle formation on the surface of Bacillus cereus

Bacillus cereus strains elaborate pili on their surface using a mechanism of sortase-mediated cross-linking of major and minor pilus components. Here we used a combination of electron microscopy and atomic force microscopy to visualize these structures. Pili occur as single, double or higher order assemblies of filaments formed from monomers of the major pilin, BcpA, capped by the minor pilin, BcpB. Previous studies demonstrated that within assembled pili, four domains of BcpA - CNA(1), CNA(2), XNA and CNA(3) - each acquire intramolecular lysine-asparagine isopeptide bonds formed via catalytic glutamic acid or aspartic acid residues. Here we showed that mutants unable to form the intramolecular isopeptide bonds in the CNA(2) or CNA(3) domains retain the ability to form pilus bundles. A mutant lacking the CNA(1) isopeptide bond assembled deformed pilin subunits that failed to associate as bundles. X-ray crystallography revealed that the BcpA variant Asp(312) Ala, lacking an aspartyl catalyst, did not generate the isopeptide bond within the jelly-roll structure of XNA. The Asp(312) Ala mutant was also unable to form bundles and promoted the assembly of deformed pili. Thus, structural integrity of the CNA(1) and XNA domains are determinants for the association of pili into higher order bundle structures and determine native pilus structure.     

Crystal Structure of the Minor Pilin CofB,the Initiator of CFA/III Pilus Assembly in Enterotoxigenic Escherichia coli

Type IV pili are extracellular polymers of the major pilin subunit. These subunits are held together in the pilus filament by hydrophobic interactions among their N-terminal α-helices, which also anchor the pilin subunits in the inner membrane prior to pilus assembly. Type IV pilus assembly involves a conserved group of proteins that span the envelope of Gram-negative bacteria. Among these is a set of minor pilins, so named because they share their hydrophobic N-terminal polymerization/membrane anchor segment with the major pilins but are much less abundant. Minor pilins influence pilus assembly and retraction, but their precise functions are not well defined. The Type IV pilus systems of enterotoxigenic Escherichia coli and Vibrio cholerae are among the simplest of Type IV pilus systems and possess only a single minor pilin. Here we show that the enterotoxigenic E. coli minor pilins CofB and LngB are required for assembly of their respective Type IV pili, CFA/III and Longus. Low levels of the minor pilins are optimal for pilus assembly, and CofB can be detected in the pilus fraction. We solved the 2.0 Å crystal structure of N-terminally truncated CofB, revealing a pilin-like protein with an extended C-terminal region composed of two discrete domains connected by flexible linkers. The C-terminal region is required for CofB to initiate pilus assembly. We propose a model for CofB-initiated pilus assembly with implications for understanding filament growth in more complex Type IV pilus systems as well as the related Type II secretion system.     

Assembly proteins of CS1 pili of enterotoxigenic Escherichia coli

Some strains of enterotoxigenic Escherichia coli associated with human diarrhoeal disease produce a class of pili represented by those called CS1. For the assembly of the major-pilin subunit, CooA, into pili, each of four linked genes, cooB,A,C, and D, is required. In this study, we have determined the subcellular localization of CooB, C and D, and investigated the molecular interactions of these proteins using specific antisera. CooD appears to be an integral pilus protein because it co-purifies with, and is strongly associated with, CS1 pili. In keeping with its role as an assembly protein, the CooD minor pilin (when overexpressed in CS1-piliated strains) was detected in periplasmic inter-molecular complexes with the major-pilin subunit CooA. CooB is an assembly protein found exclusively in the periplasm of CS1-piliated strains. CooB also forms periplasmic intermolecular complexes with CooA, but does not constitute part of the final pilus structure. Immunoblot analysis of cell fractions showed that CooC is an outer membrane protein of CS1-piliated E. coli. Based on this information, we have proposed a model for CS1 -pilus assembly which is very similar to the model for polymerization of the PapA pilin of uropathogenic E. coli. As the assembly proteins of Pap and CS1 pili are structurally unrelated, this may represent a case of convergent evolution.     

Biogenesis of E. coli Pap pili: papH, a minor pilin subunit involved in cell anchoring and length modulation

The biogenesis of Escherichia coli Pap pili, encoded by the pap gene cluster, was studied. A novel gene, papH, was identified and found to encode a weakly expressed pilin-like protein. PapH was dispensable for digalactoside-specific binding and for formation of Pap pili. However, in papH deletion mutants 50%-70% of total pilus antigen was found free of the cells. We present evidence showing coregulation of papH and the adjacent gene, papA, which encodes the major pilin subunit. A decrease in the PapA to PapH ratio resulted in a large fraction of cells producing shortened pili, whereas overproduction of PapA relative to PapH resulted in cells with lengthened pili. The data show that PapH has roles in anchoring the pilus to the cell and in modulating pilus length.     

Assembly of pili on the surface of Corynebacterium diphtheriae

Pili of Gram-negative pathogens are formed from pilin precursor molecules by non-covalent association within the outer membrane envelope. Gram-positive microbes employ the cell wall peptidoglycan as a surface organelle for the covalent attachment of proteins, however, an assembly pathway for pili has not yet been revealed. We show here that pili of Corynebacterium diphtheriae are composed of three pilin subunits, SpaA, SpaB and SpaC. SpaA, the major pilin protein, is distributed uniformly along the pilus shaft, whereas SpaB is observed at regular intervals and SpaC seems positioned at the pilus tip. Assembled pili are released from the bacterial surface by treatment with murein hydrolase, suggesting that the pilus fibres may be anchored to the cell wall envelope. All three pilin subunit proteins are synthesized as precursors carrying N-terminal signal peptides and C-terminal sorting signals. Some, but not all, of the six sortase genes encoded in the genome of C. diphtheriae are required for precursor processing, pilus assembly or cell wall envelope attachment. Pilus assembly is proposed to occur by a mechanism of ordered cross-linking, whereby pilin-specific sortase enzymes cleave precursor proteins at sorting signals and involve the side chain amino groups of pilin motif sequences to generate links between pilin subunits. This covalent tethering of adjacent pilin subunits appears to have evolved in many Gram-positive pathogens that encode sortase and pilin subunit genes with sorting signals and pilin motifs.