CHARACTERIZATION OF A 5-GENE CLUSTER REQUIRED FOR THE BIOGENESIS OF TYPE-4 FIMBRIAE IN PSEUDOMONAS-AERUGINOSA

The opportunistic pathogen Pseudomonas aeruginosa produces type 4 fimb riae which promote adhesion to epithelial cells and are associated wit h a form of surface translocation called twitching motility. Transposo n mutagenesis was used to identify loci required for fimbrial assembly or function by screening for mutants that lack the spreading colony m orphology characteristic of twitching motility, Six mutants were isola ted that contain transposon insertions upstream of the previously char acterized gene pilQ. This region contains four genes: pilM-P, which en code proteins with predicted sizes of 37.9, 22.2, 22.8 and 19.0kDa, re spectively, pilM-P appear to form an operon and to be expressed from a promoter in the intergenic region between pilM and the divergently tr anscribed upstream gene ponA. PilM-P were found to be required for fim brial biogenesis by complementation studies using twitching motility a nd sensitivity to fimbrial-specific phage as indicators of the presenc e of functional fimbriae, This was confirmed by electron microscopy, P llO and PllP did not have homologues in the sequence databases, but th e predicted PilN amino acid sequence displayed similarity to XpsL from Xanthamonas campestris, a protein required for protein secretion. Pll P contained a hydrophobic leader sequence characteristic of lipoprotei ns, while PilN and PilO have long internal hydrophobic domains which m ay serve to localize them to the cytoplasmic membrane, PllM has shared sequence motifs with the cell division protein FtsA from Bacillus sub tilis and Escherichia coil, as well as the rod-shape-determining prote in MreB from E. coli. These motifs are also conserved in eukaryotic ac tin, in which they; are involved in forming an ATPase domain. Deletion mutants of pilM and pilQ displayed a dominant negative phenotype when transformed into wild-type cells, suggesting that these genes encode proteins involved in multimeric structures.