Premium
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
Author(s) -
Russell Mary A.,
Darztns Aldis
Publication year - 1994
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1994.tb00489.x
Subject(s) - pilin , pilus , biology , cosmid , peptide sequence , plasmid , genetics , nucleic acid sequence , gene , mutant , integron , open reading frame , microbiology and biotechnology , virulence
Summary A new locus required for type 4 pilus biogenesis by Pseudomonas aeruginosa has been identified. A pilE mutant, designated MJ‐6, was broadly resistant to pili‐specific phages and unable to translocate across solid surfaces by the pilus‐dependent mechanism of twitching motility (Twt − ). Immunoblot analysis demonstrated that MJ‐6 was devoid of pili (Pil − ) but was unaffected in the production of unassembled pilin pools. Genetic studies aimed at localizing the pilE mutation on the P. aeruginosa PAO chromosome demonstrated a strong co‐linkage between MJ‐6 phage resistance and the proB marker located at 71 min. Cloning of the pilE gene was facilitated by the isolation and identification of a proB + ‐containing plasmid from a PAO1 cosmid library. Upon introduction of the PA01 proB + cosmid clone into MJ‐6, sensitivity to pili‐specific phage, twitching motility and pilus production were restored. The nucleotide sequence of a 1 kb Eco RV‐ Clal fragment containing the pilE region revealed a single complete open reading frame with characteristic P. aeruginosa codon bias. PilE, a protein with a molecular weight of 15278, showed significant sequence identity to the pilin precursors of P. aeruginosa and to other type 4 prepilin proteins. The region of highest 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 pep‐tidase and a largely hydrophobic region which contained tyrosine residues (Tyr‐24 and Tyr‐27) previously implicated in maintaining pilin subunit‐subunit interactions. The requirement of PilE in pilus biogenesis was confirmed by demonstrating that chromosomal pilE insertion mutants were pilus‐ and twitching‐motility deficient.