Identification of a Novel Gene, Pilz, Essential for Type 4 Fimbrial

JOURNAL OF BACTERIOLOGY, Jan. 1996, p. 46–53 Vol. 178, No. 1 0021-9193/96/$04.0010 Copyright q 1996, American Society for Microbiology Identification of a Novel Gene, pilZ, Essential for Type 4 Fimbrial Biogenesis in Pseudomonas aeruginosa RICHARD A. ALM, AMANDA J. BODERO, PATRICIA D. FREE, AND JOHN S. MATTICK* Centre for Molecular and Cellular Biology, University of Queensland, Brisbane, Queensland 4072, Australia Downloaded from Received 5 June 1995/Accepted 24 October 1995 The opportunistic pathogen Pseudomonas aeruginosa produces type 4 fimbriae which promote adhesion to epithelial cells and are associated with a form of surface translocation called twitching motility. We have used transposon mutagenesis to identify loci required for fimbrial assembly or function by screening for mutants that lack the spreading colony morphology characteristic of twitching motility. A subset of these mutants is resistant to fimbria-specific phage. One of these mutants (R270) was found to contain a transposon insertion in a new gene, termed pilZ, which is located on chromosomal SpeI fragment I at about 40 min on the P. http://jb.asm.org/ aeruginosa map, a position remote from other loci involved in fimbrial biogenesis. pilZ appears to be linked to and possibly forms an operon with a gene, holB*, which is homologous to the gene encoding the d* subunit of Escherichia coli DNA polymerase III. The product of the pilZ gene is a protein of 118 amino acids (predicted molecular weight, 12,895) which probably has a cytoplasmic location. PilZ appears to be a new class of protein which has not hitherto been represented in the sequence databases, and its function is unknown. Comple- mentation studies indicate that pilZ is able to restore the expression of fimbriae on the surface of P. aeruginosa, as well as twitching motility and sensitivity to fimbria-specific phage when provided in trans to the R270 mutant. on September 19, 2016 by University of Queensland Library Type 4 fimbriae (or common pili) are flexible, filamentous motility and apparent hyperfimbriation (47, 48). About 10 kb surface appendages which are found in a number of important from pilT and pilU is a cluster of genes (pilGHIJK) which pathogenic bacteria, including Neisseria gonorrhoeae, Neisseria encode a set of proteins with close resemblance to members of meningitidis, Moraxella bovis, Dichelobacter nodosus, and Pseu- the enteric chemotactic network, which suggests a related sig- domonas aeruginosa, and which mediate adherence to host nal transduction system that regulates fimbrial function, pos- epithelial tissue during colonization (33, 40, 42). Type 4 fim- sibly the directional control of twitching motility (8–10). The briae were originally defined by their polar arrangement on the third fimbria-associated region is found on SpeI fragment B at cell and their ability to promote a form of motion called twitch- ;2 min on the physical map and contains an operon of five ing motility (14, 32). Type 4 fimbriae are also characterized by genes, pilMNOPQ, all of which are required for fimbrial as- conserved features of the structural subunit proteins which sembly (25, 26). make up the fimbrial strand. These include a short, positively The infrastructure and machinery required to produce and charged leader sequence of six to eight amino acids which is assemble type 4 fimbriae appear to be well conserved among removed during fimbrial assembly, a modified amino acid, N- the different bacterial species that possess these structures. methylphenylalanine, as the first residue in the mature protein, This is indicated by the observation that P. aeruginosa is capa- and a highly conserved hydrophobic amino-terminal domain ble of producing fimbriae composed of heterologous subunits (7, 42). expressed from cloned subunit genes from D. nodosus, M. The biogenesis of type 4 fimbriae is a complex process in- bovis, and N. gonorrhoeae (3, 19, 27). A number of homologs to volving a large network of genes that have thus far been local- the fimbria-associated genes of P. aeruginosa have been iden- ized to three separate regions of the Pseudomonas chromo- tified in other type 4 fimbriate bacteria (20, 24). However, it is some. The largest locus is located on SpeI fragment E at about also clear that a number of genes involved in the biogenesis 70 min on the physical map and contains the fimbrial subunit and function of type 4 fimbriae remain unidentified. In this gene (pilA), two ancillary genes (pilB and pilC), the specific paper, analysis of a bacteriophage-resistant fimbrial mutant leader peptidase gene (pilD), and the sensor/regulator genes has enabled identification and characterization of a novel pro- (pilS and pilR) responsible for the transcriptional activation of tein that is essential for correct fimbrial export in P. aeruginosa. the fimbrial subunit gene (16, 21, 41). Recently, two further genes (pilE and pilV) have been localized to this region of the MATERIALS AND METHODS genome, both of which encode proteins possessing prepilin- like leader sequences that appear to be cleaved by the PilD Bacterial strains, plasmids, and media. The P. aeruginosa mutant R270 is a peptidase (2, 35). A second group of fimbria-associated genes derivative of wild-type strain PAK (David Bradley, Memorial University of is found on SpeI fragment H at ;20 min on the physical map. Newfoundland, St. Johns, Newfoundland, Canada) and constitutes part of the transposon mutant collection held in our laboratory. The Escherichia coli strain These include the pilT and pilU genes which encode two po- XL-1 Blue (supE44 hsdR17 recA1 endA1 gyrA46 thi relA1 lac [F9, proA1B1 lacIq tential nucleotide-binding proteins involved in fimbrial func- lacZDM15 Tn10(tetr)]) was used as a host for all manipulations including gen- tion, since mutations in either gene cause loss of twitching eration of single-stranded DNA templates prior to DNA sequencing. The DNA inserts of recombinant plasmids used throughout this study are shown in Fig. 1, and were contained in either pBluescript (Stratagene) or in the shuttle vectors pUCP18/19 (kindly provided by H. Schweizer) which replicate stably in both * Corresponding author. Phone: (61-7) 336-54446. Fax: (61-7) 336- Pseudomonas and E. coli hosts (38). Bacteriophage PO4 is a PAK fimbria-specific 54388. Electronic mail address: [email protected]. bacteriophage described elsewhere (5), and sensitivity to infection by this bac- 46 VOL. 178, 1996 TYPE 4 FIMBRIAL BIOGENESIS IN P. AERUGINOSA 47 transferred to nylon membrane (Hybond N; Amersham) and probed with the 1.8-kb insert from pAB6 by standard procedures (36). Preparation of RNA and Northern (RNA) blotting. Total RNA was extracted from P. aeruginosa strains by the method described previously (16). RNA was electrophoresed under denaturing conditions and transferred to nylon membrane (Hybond N; Amersham) as described elsewhere (16). The probe used was the purified insert contained within plasmid pRIC369 labelled with [32P]dCTP, and hybridizations and washes were performed according to the protocol of the manufacturer. RESULTS Downloaded from Identification of the pil-specific mutant and cloning of the affected locus. To genetically define all of the components involved in the biogenesis of type 4 fimbriae in P. aeruginosa, the wild-type strain PAK was subjected to transposon mutagenesis with Tn5-B21 (16). The initial screening method was to identify mutants that had become deficient in twitching motility, a phenotype that requires the presence of functional fimbriae. Such mutants exhibit an altered colony morphology which appears smooth and domed, in contrast to the rough- http://jb.asm.org/ FIG. 1. Schematic representation of pAB1 and the R270 fimbrial gene locus. The relevant restriction sites are shown, and the site of transposon insertion is edged spreading colony morphology of wild-type Pseudomonas indicated by an arrowhead. The relevant plasmid inserts are shown diagrammat- cells. These mutants were then classified according to their ically, and their orientations with respect to the external lac or T7 promoters are sensitivity or resistance to infection by the fimbria-dependent indicated if relevant. bacteriophage PO4. Hybridization analyses of the 28 bacteriophage resistant mutants with the transposon grouped these mutants into six categories based on the size of the BglII teriophage was determined quantitatively as described elsewhere (2). The PAO restriction fragment into which the transposon had inserted cosmid library was generously provided by B. Holloway (Monash University, (16). One of these groups, R6, contained eight mutants which on September 19, 2016 by University of Queensland Library Clayton, Victoria, Australia). All bacterial strains were maintained on LB me- contained large hybridizing BglII fragments and which were dium (36) containing 100 mg of ampicillin (E. coli) or 500 mg of carbenicillin (P. aeruginosa) per ml if maintenance of plasmids was required. likely to be heterogeneous. This has proved to be the case, and Recombinant DNA techniques and transformation. Preparation of plasmid two of these mutants have been recently localized to the pilV DNA, digestion with restriction endonucleases (New England Biolabs, Beverly, gene (2). Mass.), ligations, and transformation in E. coli hosts were carried out under The flanking chromosomal DNA from mutant R270 (a standard conditions as described by Sambrook et al. (36). Preparation of P. aeruginosa competent cells and transformation were achieved as described pre- member of the R6 group) was cloned by marker rescue, i.e., 1 viously (2). digestion of the genomic DNA, ligation into pBluescriptSK , Subsurface twitching assay. The quantitative assay for twitching motility was and subsequent selection for the tetracycline resistance gene an adaptation of previous methods described by McMichael (28), with modifi- encoded on Tn5-B21. This plasmid, pR270, was used to probe cations described elsewhere (2), except that the agar was not removed after staining. a wild-type PAO1 cosmid library to obtain an intact copy of the Protein expression. Plasmid-encoded proteins were visualized by subcloning affected locus.

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