Role of the Eikenella corrodens pilA locus in pilus function and phase variation

The human pathogen Eikenella corrodens expresses type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying pilus structure and function in the clinical isolate E. corrodens VA1. Earlier work defined the pilA locus which includes pilA1, pilA2, pilB, and hagA. Both pilA1 and pilA2 predict a type IV pilin, whereas pilB predicts a putative pilus assembly protein. The role of hagA has not been clearly established. That work also confirmed that pilA1 encodes the major pilus protein in this strain and showed that the phase variation involves a posttranslational event in pilus formation. In this study, the function of the individual genes comprising the pilA locus was examined using a recently developed protocol for targeted interposon mutagenesis of S-phase variant VA1-S1. Different pilA mutants were compared to S-phase and L-phase variants for several distinct aspects of phase variation and type IV pilus biosynthesis and function. S-phase cells were characterized by surface pili, competence for natural transformation, and twitching motility, whereas L-phase cells lacked these features. Inactivation of pilA1 yielded a mutant that was phenotypically indistinguishable from L-phase variants, showing that native biosynthesis of the type IV pilus in strain VA1 is dependent on expression of pilA1 and proper export and assembly of PilA1. Inactivation of pilA2 yielded a mutant that was phenotypically indistinguishable from S-phase variants, indicating that pilA2 is not essential for biosynthesis of functionally normal pili. A mutant inactivated for pilB was deficient for twitching motility, suggesting a role for PilB in this pilus-related phenomenon. Inactivation of hagA, which may encode a tellurite resistance protein, had no effect on pilus structure or function.     

Genetic complementation analysis of Escherichia coli type 1 somatic pilus mutants.

A genetic complementation analysis of 75 stable nonpiliated mutants of a type 1 piliated strain of Escherichia coli K-12, AW405, was performed. Strains containing pairs of pil mutations were constructed by the infectious transfer of an F101 plasmid containing one pil mutation into E. coli K-12 AW 405 containing another pil mutation. The presence or absence of type 1 pili on the merodiploid strains was determined by agglutination with type 1 pilus antiserum. All 75 mutants fell into one of four complementation groups. The pattern of complementation defined three cistrons involved in pilus formation, pilA, pilB, and pilC. The fourth complementation group was composed of a large number of mutants defective in both pilA and pilB functions.     

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.     

Characterization of type IV pilus genes in plant growth-promoting Pseudomonas putida WCS358.

In a search for factors that could contribute to the ability of the plant growth-stimulating Pseudomonas putida WCS358 to colonize plant roots, the organism was analyzed for the presence of genes required for pilus biosynthesis. The pilD gene of Pseudomonas aeruginosa, which has also been designated xcpA, is involved in protein secretion and in the biogenesis of type IV pili. It encodes a peptidase that processes the precursors of the pilin subunits and of several components of the secretion apparatus. Prepilin processing activity could be demonstrated in P. putida WCS358, suggesting that this nonpathogenic strain may contain type IV pili as well. A DNA fragment containing the pilD (xcpA) gene of P. putida was cloned and found to complement a pilD (xcpA) mutation in P. aeruginosa. Nucleotide sequencing revealed, next to the pilD (xcpA) gene, the presence of two additional genes, pilA and pilC, that are highly homologous to genes involved in the biogenesis of type IV pili. The pilA gene encodes the pilin subunit, and pilC is an accessory gene, required for the assembly of the subunits into pili. In comparison with the pil gene cluster in P. aeruginosa, a gene homologous to pilB is lacking in the P. putida gene cluster. Pili were not detected on the cell surface of P. putida itself, not even when pilA was expressed from the tac promoter on a plasmid, indicating that not all the genes required for pilus biogenesis were expressed under the conditions tested. Expression of pilA of P. putida in P. aeruginosa resulted in the production of pili containing P. putida PilA subunits.     

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.     

Regulation of expression of the pilA gene in Myxococcus xanthus.

Type IV pili are required for social gliding motility in Myxococcus xanthus. In this work, the expression of pilin (the pilA gene product) during vegetative growth and fruiting-body development was examined. A polyclonal antibody against the pilA gene product (prepilin) was prepared, along with a pilA-lacZ fusion, and was used to assay expression of pilA in M. xanthus in different mutant backgrounds. pilA expression required the response regulator pilR but was negatively regulated by the putative sensor kinase pilS. pilA expression did not require pilB, pilC, or pilT. pilA was also autoregulated; a mutation which altered an invariant glutamate five residues from the presumed prepilin processing site eliminated this autoregulation, as did a deletion of the pilA gene. Primer extension and S1 nuclease analysis identified a sigma54 promoter upstream of pilA, consistent with the homology of pilR to the NtrC family of response regulators. Expression of pilA was found to be developmentally regulated; however, the timing of this expression pattern was not entirely dependent on pilS or pilR. Finally, pilA expression was induced by high nutrient concentrations, an effect that was also not dependent on pilS or pilR.     

The frequency and rate of pilin antigenic variation in Neisseria gonorrhoeae

The pilin antigenic variation (Av) system of Neisseria gonorrhoeae (Gc) mediates unidirectional DNA recombination from silent gene copies into the pilin expression locus. A DNA sequencing assay was developed to accurately measure pilin Av in a population of Gc strain FA1090 arising from a defined pilin progenitor under non-selective culture conditions. This assay employs a piliated parental Gc variant with a recA allele whose promoter is replaced by lac-regulatory elements, allowing for controlled induction of pilin Av. From this assay, the frequency of pilin Av was measured as 0.13 recombination events per cell, with a corresponding rate of pilin Av of 4x10(-3) events per cell per generation. Most pilin variants retained the parental piliation phenotype, providing the first comprehensive analysis of piliated variants arising from a piliated progenitor. Sequence analysis of pilin variants revealed that a subset of possible recombination events predominated, which differed between piliated and non-piliated progeny. Pilin Av exhibits the highest reported frequency of any pathogenic gene conversion system and can account for the extensive pilin variation detected during human infection.     

Gonococci exit apically and basally from polarized epithelial cells and exhibit dynamic changes in type IV pili

Type IV pili are a major virulence factor of the obligate human pathogen Neisseria gonorrhoeae (the gonococcus; Gc). Pili facilitate bacterial adherence to epithelial cells, but their participation in later steps of epithelial infection, particularly intracellular replication and exit, is poorly understood. Using polarized T84 cells as a model for mature mucosal epithelia, pilus dynamics in piliated, Opa-expressing Gc were examined over time. T84 infection was characterized by a several-hour delay in the growth of cell-associated bacteria and by non-directional exit of Gc, the first time these phenomena have been reported. During infection, non-piliated progeny arose stochastically from piliated progenitors. Piliated and non-piliated Gc replicated and exited from T84 cell monolayers equally well, demonstrating that piliation did not influence Gc survival during epithelial infection. The frequency with which pilin variants arose from a defined piliated progenitor during T84 cell infection was found to be sufficiently high to account for the extensive pilin variation reported during human infection. However, the repertoire of variants appearing in association with T84 cells was similar to what was seen in the absence of cells, demonstrating that polarized epithelial cells can support Gc replication without selecting for a subset of pilin variants or piliation states.     

Phase variation of gonococcal pili by frameshift mutation in pilC, a novel gene for pilus assembly.

Pili prepared from Neisseria gonorrhoeae contain minor amounts of a 110 kd outer membrane protein denoted PilC. The corresponding gene exists in two copies, pilC1 and pilC2, in most strains of N.gonorrhoeae. In the piliated strain MS11(P+), only one of the genes, pilC2, was expressed. Inactivation of pilC2 by a mTnCm insertion resulted in a nonpiliated phenotype, while a mTnCm insertion in pilC1 had no effect on piliation. Expression of pilC was found to be controlled at the translational level by frameshift mutations in a run of G residues positioned in the region encoding the signal peptide. Nonpilated (P-), pilin expressing colony variants that did not express detectable levels of PilC were selected; all P+ backswitchers from these P-, PilC- clones were found to be PilC+. The structural gene for pilin, pilE, was sequenced and found to be identical in one P-, PilC- and P+, PilC+ pair. Most PilC- cells were completely bald whereas the PilC+ backswitcher had 10-40 pili per cell. Thus, a turn ON and turn OFF in the expression of PilC results in gonococcal pili phase variation. These results suggest that PilC is required for pilus assembly and/or translocation across the gonococcal outer membrane.     

Variations in the expression of pili: the effect on adherence of Neisseria meningitidis to human epithelial and endothelial cells

The effect of variations in Neisseria meningitidis pili on bacterial interactions with three epithelial cell lines as well as human umbilical vein endothelial cells was studied using a panel of seven strains expressing Class I or Class II pili. Comparison of adherence of piliated and pilus-deficient variants of each strain to epithelial cells suggested that Class I pili may mediate bacterial adherence with all three epithelial cell lines. In contrast, Class II pili of the strains used did not increase bacterial adherence to Hep-2 larynx carcinoma cells, although an increase in adherence to Chang conjunctival and A549 lung carcinoma epithelial cells was observed in the Class II pili-expressing strains. In addition to these interclass functional variations, differences in adherence to epithelial cells were also observed among Class I and Class II strains. Functionally different pilin variants of one Class I strain, MC58, were obtained by single colony isolation. One piliated variant was identified which had concurrently lost the ability to adhere to both Chang and Hep-2 cells ('non-adherent' phenotype; adherence of less than 2 bacteria per cell). In addition, several adherent pilin variants were isolated from non-adherent Pil- and Pil+ bacteria by selection on Chang cells (adherence of 10-25 bacteria per cell). In contrast to epithelial cells, all variant pili, whether of Class I or Class II, adhered to endothelial cells in substantially larger numbers (greater than 50 bacteria per cell) and therefore implied the existence of distinct mechanisms in pilus-facilitated interactions of N. meningitidis with endothelial and epithelial cells.     

Regulating pilin expression reveals a threshold for S motility in Myxococcus xanthus

An isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter was constructed in Myxococcus xanthus. The single-copy pilA gene encodes pilin, the monomer unit of M. xanthus type IV pili. To vary the level of pilA expression, we cloned its promoter in front of the lac operator, and a plasmid containing the construct was inserted into the chromosome of a DeltapilA strain. Induction of pilin expression increased smoothly as the dose of IPTG added to the culture was increased. IPTG-induced pilin rescued S motility of the DeltapilA strain to wild-type levels. The rate of S-motile swarming was found to be proportional to the number of pili (shear-sensitive pilin) produced rather than to the level of total pilin. In fact, S motility was not rescued until the total level of pilin was more than 50% of the wild-type level. This observation implies that a threshold concentration of pilin must be exceeded before the shear-sensitive material (pili) is polymerized in M. xanthus.     

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.     

High adhesiveness of encapsulated Neisseria meningitidis to epithelial cells is associated with the formation of bundles of pili

Pili are indispensable in adhesion of encapsulated Neisseria meningitidis (MC) to eukaryotic cells. Intrastrain variability with respect to the degree of adhesion is the result of pilin antigenic variation. We have localized the region responsible for this variability to the 20-amino-acid hypervariable domain of pilin. The replacement of an aspartic acid, located in the hypervariable region of a low-adhesive variant by a lysine restored high adhesiveness. To assess whether hyperadhesiveness confered by some pilin variants was related to the generation of a new pilus-associated ligand, high- and low-adhesive variants were purified. In a first step, low- and high-adhesive pilins were fused to maltose binding protein (MBP). These hybrid proteins bound epithelial cells with the same affinity. Truncated MBP pilin fusions identified a cell-binding domain within the 77 residues of the N-terminal end of mature pilin. This region of the protein is common to low- and high-adhesive derivatives used in this work, thus eliminating the possibility that high adhesiveness confered by some pilin variants was because of the generation of a new pilus-associated ligand. Electron-microscopic examination showed that low-adhesive derivatives expressed long and distinct pili and adhered as single cells. In contrast, pili of derivatives expressing high-adhesive pilins, either wild type or mutagenized from the low-adhesive variant, formed large bundles which bound bacteria and caused them to grow as colonies on infected mono-layers. These data demonstrate that aggregative pili promote high adhesiveness of encapsulated MC.     

Pilus-facilitated adherence of Neisseria meningitidis to human epithelial and endothelial cells: modulation of adherence phenotype occurs concurrently with changes in primary amino acid sequence and the glycosylation status of pilin

Adherence of capsulate Neisseria meningitidis to endothelial and epithelial cells is facilitated in variants that express pili. Whereas piliated variants of N. meningitidis strain C311 adhered to endothelial cells in large numbers (<150 bacteria/cell), derivatives containing specific mutations that disrupt pilE encoding the pilin subunit were both non-piliated and failed to adhere to endothelial cells (<1 bacterium/ cell). In addition, meningococcal pili recognized human endothelial and epithelial cells but not cells originating from other animals. Variants of strain C311 were obtained that expressed pilins of reduced apparent M_r and exhibited a marked increase in adherence to epithelial cells. Structural analysis of pilins from two hyper-adherent variants and the parent strain were carried out by DNA sequencing of their pilE genes. Deduced molecular weights of pilins were considerably tower compared with their apparent M_r values on SDS-PAGE. Hyper-adherent pilins shared unique changes in sequence including substitution of Asn-113 for Asp-113 and changes from Asn-Asp-Thr-Asp to Thr-Asp-Ala-Lys at residues 127-130 in mature pilin. Asn residues 113 and 127 of‘parental’pilin both form part of the typical eukaryotic N-glycosylation motif Asn-X-Ser/Thr and could potentially be glycosylated post-translationally. The presence of carbohydrate on pilin was demonstrated and when pilins were deglycosylated, their migration on SDS-PAGE increased, supporting the notion that variable glycosylation accounts for discrepancies in apparent and deduced molecular weights. Functionally distinct pilins produced by two fully piliated variants of a second strain (MC58) differed only in that the putative glycosylation motif Asn-60-Asn-61-Thr-62 in an adherent variant was replaced with Asp-60-Asn-61-Ser-62 in a non-adherent variant. Fully adherent backswitchers obtained from the non-adherent variant always regained Asn-60 but retained Ser-62. We propose, therefore, that functional variations in N. meningitidis pili may be modulated in large part by primary amino acid sequence changes that ablate or create N-linked glycosylation sites on the pilin subunit.     

Role of type IV pili in virulence of Pseudomonas syringae pv. tabaci 6605: correlation of motility, multidrug resistance, and HR-inducing activity on a nonhost plant

To investigate the role of type IV pili in the virulence of phytopathogenic bacteria, four mutant strains for pilus biogenesis-related genes were generated in Pseudomonas syringae pv. tabaci 6605. PilA encodes the pilin protein as a major subunit of type IV pili, and the pilO product is reported to be required for pilus assembly. The fimU and fimT genes are predicted to produce minor pilins. Western blot analysis revealed that pilA, pilO, and fimU mutants but not the fimT mutant failed to construct type IV pili. Although the swimming motility of all mutant strains was not impaired in liquid medium, they showed remarkably reduced motilities on semisolid agar medium, suggesting that type IV pili are required for surface motilities. Virulence toward host tobacco plants and hypersensitive response-inducing ability in nonhost Arabidopsis leaves of pilA, pilO, and fimU mutant strains were reduced. These results might be a consequence of reduced expression of type III secretion system-related genes in the mutant strains. Further, all mutant strains showed enhanced expression of resistance-nodulation-division family members mexA, mexB, and oprM, and higher tolerance to antimicrobial compounds. These results indicate that type IV pili are an important virulence factor of this pathogen.     

Metastable regulation of type 1 piliation in Escherichia coli and isolation and characterization of a phenotypically stable mutant.

Type 1 piliation in Escherichia coli exhibits phase variation due to the inversion of a small, ca. 300-base-pair, element that regulates pilA (fimA), the gene that encodes the structural subunit of pili (Abraham et al., Proc. Natl. Acad. Sci. USA 82:5724-5727, 1985). We have used the inversion as an assay to characterize a stably piliated mutant. The mutant strain did not exhibit the pilA ON and pilA OFF colonial variants characteristic of the wild type; rather, every clone produced a level of pilA expression intermediate between ON and OFF wild-type populations. The mutant phenotype was conferred by a lesion at a previously undescribed locus between hemA and trpA, which we have termed pilG. Examination of the pilA promoter region in four pilG mutant populations indicated that the phenotypic stability conferred by the pilG mutation was not due to an inability to carry out the inversion. Rather, all pilG mutant populations consisted of approximately equal mixtures of ON and OFF individuals. We suggest that pilG mutants may undergo such rapid switching of the pilA promoter that populations exhibit an intermediate level of pilA expression and phenotypic stability.