THE PILE GENE-PRODUCT OF PSEUDOMONAS-AERUGINOSA, REQUIRED FOR PILUS BIOGENESIS, SHARES AMINO-ACID-SEQUENCE IDENTITY WITH THE N-TERMINI OF TYPE-4 PREPILIN PROTEINS

A new locus required for type 4 pilus biogenesis by Pseudomonas aerugi nosa has been identified. A pilE mutant, designated MJ-6, was broadly resistant to pili-specific phages and unable to translocate across sol id surfaces by the pilus-dependent mechanism of twitching motility (Tw t(-)). Immunoblot analysis demonstrated that MJ-6 was devoid of pill ( Pil(-)) but was unaffected in the production of unassembled pilin pool s. Genetic studies aimed at localizing the pilE mutation on the P. aer uginosa PAO chromosome demonstrated a strong co-linkage between MJ-6 p hage resistance and the proB marker located at 71 min. Cloning of the pilE gene was facilitated by the isolation and identification of a pro B(+)-containing plasmid from a PAO1 cosmid library. Upon introduction of the PAO1 proB(+) cosmid clone into MJ-6, sensitivity to pili-specif ic phage, twitching motility and pilus production were restored. The n ucleotide sequence of a 1 kb EcoRV-ClaI fragment containing the pilE r egion revealed a single complete open reading frame with characteristi c P. aeruginosa codon bias. PilE, a protein with a molecular weight of 15278, showed significant sequence identity to the pilin precursors o f P. aeroginosa and to other type 4 prepilin proteins. The region of h ighest homology was localized to the N-terminal 40 amino acid residues . The putative PilE N-terminus contained a seven-residue basic leader sequence followed by a consensus cleavage site for prepilin peptidase and a largely hydrophobic region which contained tyrosine residues (Ty r-24 and Tyr-27) previously implicated in maintaining pilin subunit-su bunit interactions. The requirement of PilE in pilus biogenesis was co nfirmed by demonstrating that chromosomal pilE insertion mutants were pilus- and twitching-motility deficient.