Analysis of the pilU gene for the prepilin peptidase involved in the biogenesis of type IV pili encoded by plasmid R64

ABSTRACT The type IV pili of plasmid R64 belonging to the type IVB group are required only for liquid mating. They consist of the major and minor components PilS pilin and PilV adhesin, respectively. PilS pilin is first synthesized as a 22-kDa prepilin from the pilS gene and is then processed to a 19-kDa mature pilin by PilU prepilin peptidase. In a previous genetic analysis, we identified four classes of the pilS mutants (T. Horiuchi and T. Komano, J. Bacteriol. 180:4613-4620, 1998). The products of the class I pilS mutants were not processed by prepilin peptidase; the products of the class II mutants were not secreted; in the class III mutants type IV pili with reduced activities in liquid mating were produced; and in the class IV mutants type IV pili with normal activities were produced. Here, we describe a novel class, class V, of pilS mutants. Mutations in the pilS gene at Gly-56 or Tyr-57 produced type IV pili lacking PilV adhesin, which were inactive in liquid mating. Residues 56 and 57 of PilS pilin are suggested to function as an interface of PilS-PilV interactions.

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FppA, a Novel Pseudomonas aeruginosa Prepilin Peptidase Involved in Assembly of Type IVb Pili

Journal of Bacteriology ◽

10.1128/jb.00345-06 ◽

2006 ◽

Vol 188 (13) ◽

pp. 4851-4860 ◽

Cited By ~ 47

Author(s):

Sophie de Bentzmann ◽

Marianne Aurouze ◽

Geneviève Ball ◽

Alain Filloux

Keyword(s):

Pseudomonas Aeruginosa ◽

Cell Surface ◽

Molecular Mechanisms ◽

Type Iv Pili ◽

Reading Frame ◽

Type Iv ◽

Bacterial Aggregation ◽

Epithelial Cell Surface ◽

Prepilin Peptidase ◽

Pilus Assembly

ABSTRACT Several subclasses of type IV pili have been described according to the characteristics of the structural prepilin subunit. Whereas molecular mechanisms of type IVa pilus assembly have been well documented for Pseudomonas aeruginosa and involve the PilD prepilin peptidase, no type IVb pili have been described in this microorganism. One subclass of type IVb prepilins has been identified as the Flp prepilin subfamily. Long and bundled Flp pili involved in tight adherence have been identified in Actinobacillus actinomycetemcomitans, for which assembly was due to a dedicated machinery encoded by the tad-rcp locus. A similar flp-tad-rcp locus containing flp, tad, and rcp gene homologues was identified in the P. aeruginosa genome. The function of these genes has been investigated, which revealed their involvement in the formation of extracellular Flp appendages. We also identified a gene (designated by open reading frame PA4295) outside the flp-tad-rcp locus, that we named fppA, encoding a novel prepilin peptidase. This is the second enzyme of this kind found in P. aeruginosa; however, it appears to be truncated and is similar to the C-terminal domain of the previously characterized PilD peptidase. In this study, we show that FppA is responsible for the maturation of the Flp prepilin and belongs to the aspartic acid protease family. We also demonstrate that FppA is required for the assembly of cell surface appendages that we called Flp pili. Finally, we observed an Flp-dependent bacterial aggregation process on the epithelial cell surface and an increased biofilm phenotype linked to Flp pilus assembly.

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The Pseudomonas aeruginosa Pathogenicity Island PAPI-1 Is Transferred via a Novel Type IV Pilus

Journal of Bacteriology ◽

10.1128/jb.00041-10 ◽

2010 ◽

Vol 192 (13) ◽

pp. 3249-3258 ◽

Cited By ~ 63

Author(s):

Michelle Qiu Carter ◽

Jianshun Chen ◽

Stephen Lory

Keyword(s):

Pseudomonas Aeruginosa ◽

Pathogenicity Island ◽

Genomic Islands ◽

Virulence Determinants ◽

Type Iv Pilus ◽

Conjugation System ◽

Type Iv ◽

Infection Models ◽

Prepilin Peptidase ◽

Plasmid R64

ABSTRACT Pseudomonas aeruginosa is a major cause of nosocomial infections, particularly in immunocompromised patients or in individuals with cystic fibrosis. The notable ability of P. aeruginosa to inhabit a broad range of environments, including humans, is in part due to its large and diverse genomic repertoire. The genomes of most strains contain a significant number of large and small genomic islands, including those carrying virulence determinants (pathogenicity islands). The pathogenicity island PAPI-1 of strain PA14 is a cluster of 115 genes, and some have been shown to be responsible for virulence phenotypes in a number of infection models. We have previously demonstrated that PAPI-1 can be transferred to other P. aeruginosa strains following excision from the chromosome of the donor. Here we show that PAPI-1 is transferred into recipient P. aeruginosa by a conjugative mechanism, via a type IV pilus, encoded in PAPI-1 by a 10-gene cluster which is closely related to the genes in the enterobacterial plasmid R64. We also demonstrate that the precursor of the major pilus subunit, PilS2, is processed by the chromosomally encoded prepillin peptidase PilD but not its paralog FppA. Our results suggest that the pathogenicity island PAPI-1 may have evolved by acquisition of a conjugation system but that because of its dependence on an essential chromosomal determinant, its transfer is restricted to P. aeruginosa or other species capable of providing a functional prepilin peptidase.

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Mutational Analysis of Plasmid R64 Thin Pilus Prepilin: the Entire Prepilin Sequence Is Required for Processing by Type IV Prepilin Peptidase

Journal of Bacteriology ◽

10.1128/jb.180.17.4613-4620.1998 ◽

1998 ◽

Vol 180 (17) ◽

pp. 4613-4620 ◽

Cited By ~ 26

Author(s):

Takayuki Horiuchi ◽

Teruya Komano

Keyword(s):

Mutational Analysis ◽

Minor Component ◽

Site Directed Mutagenesis ◽

Class I ◽

Missense Mutations ◽

Class Iii ◽

Mating Activity ◽

Type Iv ◽

Prepilin Peptidase ◽

Plasmid R64

ABSTRACT The thin pili of IncI1 plasmid R64, which is required for conjugation in liquid media, belong to the type IV pilus family. They consist of a major subunit, the pilS product, and a minor component, one of the seven pilV products. ThepilS product is first synthesized as a 22-kDa prepilin, processed to a 19-kDa mature pilin by the function of thepilU product, and then secreted outside the cell. The mature pilin is assembled to form a thin pilus with thepilV product. To reveal the relationship between the structure and function of the pilS product, 27 missense mutations, three N-terminal deletions, and two C-terminal deletions were constructed by PCR and site-directed mutagenesis. The characteristics of 32 mutant pilS products were analyzed. Four pilS mutant phenotype classes were identified. The products of 10 class I mutants were not processed by prepilin peptidase; the extracellular secretion of the products of two class II mutants was inhibited; from 11 class III mutants, thin pili with reduced activities in liquid mating were formed; from 9 class IV mutants, thin pili with mating activity similar to that of the wild-type pilS gene were formed. The point mutations of the class I mutants were distributed throughout the prepilin sequence, suggesting that processing of the pilS product requires the entire prepilin sequence.

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Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacteriumStreptococcus sanguinis

10.1101/459388 ◽

2018 ◽

Author(s):

Jamie-Lee Berry ◽

Ishwori Gurung ◽

Jan Haug Anonsen ◽

Ingrid Spielman ◽

Elliot Harper ◽

...

Keyword(s):

Structural Analysis ◽

3D Structure ◽

Type Iv Pili ◽

Gram Positive ◽

Post Translational Modifications ◽

Type Iv ◽

C Terminus ◽

Evolutionary Relatedness ◽

Bona Fide ◽

Prepilin Peptidase

AbstractType IV pili (Tfp) are functionally versatile filaments, widespread in prokaryotes, that belong to a large class of filamentous nanomachines known as type IV filaments (Tff). Although Tfp have been extensively studied in several Gram-negative pathogens where they function as key virulence factors, many aspects of their biology remain poorly understood. Here, we performed a global biochemical and structural analysis of Tfp in a recently emerged Gram-positive model,Streptococcus sanguinis. In particular, we focused on the five pilins and pilin-like proteins involved in Tfp biology inS. sanguinis. We found that the two major pilins, PilE1 and PilE2, (i) follow widely conserved principles for processing by the prepilin peptidase PilD and for assembly into filaments; (ii) display only one of the post-translational modifications frequently found in pilins,i.e. a methylated N-terminus; (iii) are found in the same hetero-polymeric filaments; and (iv) are not functionally equivalent. The 3D structure of PilE1, solved by NMR, revealed a classical pilin fold with a highly unusual flexible C-terminus. Intriguingly, PilE1 more closely resembles pseudopilins forming shorter Tff thanbona fideTfp-forming major pilins, underlining the evolutionary relatedness among different Tff. Finally, we show thatS. sanguinisTfp contain a low abundance of three additional proteins processed by PilD, the minor pilins PilA, PilB, and PilC. These findings provide the first global biochemical and structural picture of a Gram-positive Tfp and have fundamental implications for our understanding of a widespread class of filamentous nanomachines.

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Demonstration of Type IV Pilus Expression and a Twitching Phenotype by Haemophilus influenzae

Infection and Immunity ◽

10.1128/iai.73.3.1635-1643.2005 ◽

2005 ◽

Vol 73 (3) ◽

pp. 1635-1643 ◽

Cited By ~ 82

Author(s):

Lauren O. Bakaletz ◽

Beth D. Baker ◽

Joseph A. Jurcisek ◽

Alistair Harrison ◽

Laura A. Novotny ◽

...

Keyword(s):

Haemophilus Influenzae ◽

Gene Cluster ◽

Defined Medium ◽

Type Iv Pili ◽

Homologous Gene ◽

Type Ii Secretion ◽

Twitching Motility ◽

Type Iv Pilus ◽

Type Iv ◽

Prepilin Peptidase

ABSTRACT Haemophilus influenzae is considered a nonmotile organism that expresses neither flagella nor type IV pili, although H. influenzae strain Rd possesses a cryptic pilus locus. We demonstrate here that the homologous gene cluster pilABCD in an otitis media isolate of nontypeable H. influenzae strain 86-028NP encodes a surface appendage that is highly similar, structurally and functionally, to the well-characterized subgroup of bacterial pili known as type IV pili. This gene cluster includes a gene (pilA) that likely encodes the major subunit of the heretofore uncharacterized H. influenzae-expressed type IV pilus, a gene with homology to a type IV prepilin peptidase (pilD) as well as two additional uncharacterized genes (pilB and pilC). A second gene cluster (comABCDEF) was also identified by homology to other pil or type II secretion system genes. When grown in chemically defined medium at an alkaline pH, strain 86-028NP produces approximately 7-nm-diameter structures that are near polar in location. Importantly, these organisms exhibit twitching motility. A mutation in the pilA gene abolishes both expression of the pilus structure and the twitching phenotype, whereas a mutant lacking ComE, a Pseudomonas PilQ homologue, produced large appendages that appeared to be membrane bound and terminated in a slightly bulbous tip. These latter structures often showed a regular pattern of areas of constriction and expansion. The recognition that H. influenzae possesses a mechanism for twitching motility will likely profoundly influence our understanding of H. influenzae-induced diseases of the respiratory tract and their sequelae.

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