IDENTIFICATION AND TEMPERATURE REGULATION OF LEGIONELLA-PNEUMOPHILA GENES INVOLVED IN TYPE-IV PILUS BIOGENESIS AND TYPE-II PROTEIN SECRETION

Previously, we had isolated by transposon mutagenesis a Legionella pne umophila mutant that appeared defective for intracellular iron acquisi tion. While sequencing in the proximity of the mini-Tn10 insertion, we found a locus that had a predicted protein product with strong simila rity to PilB from Pseudomonas aeruginosa. PIE is a component of the ty pe II secretory pathway, which is required for the assembly of type IV pill. Consequently, the locus was cloned and sequenced. Within this 4 -kb region were three genes that appeared to be organized in an operon and encoded homologs of P. aeruginosa PilB, PilC, and PilD, proteins essential for pilus production and type II protein secretion. Northern blot analysis identified a transcript large enough to include all thr ee genes and showed a substantial increase in expression of this opero n when L. pneumophila was grown at 30 degrees C as opposed to 37 degre es C. The latter observation was then correlated with an increase in p iliation when bacteria were grown at the lower temperature. Southern h ybridization analysis indicated that the pilB locus was conserved with in L. pneumophila serogroups and other Legionella species. These data represent the first isolation of type II secretory genes from an intra cellular parasite and indicate that the legionellae express temperatur e-regulated type IV pili.