Genetic and functional characterization of the gene cluster specifying expression of Pseudomonas aeruginosa pili

Abstract

The genetic organization of the gene cluster containing pilA, the structural gene for type IV pilin of Pseudomonas aeruginosa, as well as the accessory genes pilB, pilC, and pilD, has been studied. DNA sequences capable of initiating transcription when fused to a promoterless lacZ gene have been identified in the pilA-pilB and pilB-pilC intergenic regions. Unlike pilA, which requires rpoN (encoding the sigma 54 subunit of RNA polymerase) and products of two regulatory genes, pilS and pilR, expression of pilB, pilC, or pilD did not depend on any of these transcriptional regulators. Moreover, transcription of pilA from the tac promoter in an rpoN mutant background resulted in piliated bacteria, suggesting that the RpoN-based regulatory network is specific for pilA and does not control expression of any other genes necessary for formation of pili. Insertion of the omega fragment containing strong transcriptional terminators into pilB, pilC, and pilD failed to have a polar effect on expression of downstream genes, as determined by the ability of each cloned gene to complement, in trans, the corresponding insertionally inactivated chromosomal copy. Insertions into pilC, however, resulted in decreased synthesis of PilD as determined by quantitation of PilD enzymatic activity in processing prepilin in vitro and by immunoassay. This finding suggests that PilD may require PilC for its optimal stability or correct membrane localization.