MUTATIONS IN THE 5TH-POSITION GLUTAMATE IN PSEUDOMONAS-AERUGINOSA PILIN AFFECT THE TRANSMETHYLATION OF THE N-TERMINAL PHENYLALANINE

The pili of Pseudomonas aeruginosa are composed of 15-kDa pilin monome rs that are synthesized in the cytoplasm and assembled in the membrane . Processing occurs between the synthesis and assembly steps. The prop ilin is cleaved by a unique leader peptidase encoded by pilD, which is adjacent to the pilin structural gene pilA. This generates an N-termi nal phenylalanine that is subsequently methylated by an as yet unchara cterized transmethylase. The pili of P. aeruginosa belong to the type IV class of pilins, which share a highly conserved N-terminal region 3 5 amino acids in length, containing a short leader of 6 or 7 amino aci ds. Two site-specific mutants in the N-terminal region of the mature p ilin were constructed. Reestablishing the fifth-position glutamate in a four amino acid deletion mutant (amino acids 4-7) restored the leade r peptidase cleavage but not the methylation. A mutation of the fifth- position glutamate to alanine decreased the degree of methylation of t he N-terminal phenylalanine. Pili were not assembled by these mutants as assessed by electron microscopy and sensitivity to pilus-specific b acteriophage. Methylation may be required for recognition of the pilin by the assembly machinery and is not residue specific. The fifth-posi tion glutamate appears to play an important role in transmethylase rec ognition of the pilin subunit.