Complementation Analysis of the Dichelobacter nodosus fimN , fimO , and fimP Genes in Pseudomonas aeruginosa and Transcriptional Analysis of the fimNOP Gene Region

Abstract

ABSTRACT The causative agent of ovine footrot, the gram-negative anaerobe Dichelobacter nodosus , produces polar type IV fimbriae, which are the major protective antigens. The D. nodosus genes fimN , fimO , and fimP are homologs of the Pseudomonas aeruginosa fimbrial assembly genes, pilB , pilC , and pilD , respectively. Both the pilD and fimP genes encode prepilin peptidases that are responsible for cleavage of the leader sequence from the immature fimbrial subunit. To investigate the functional similarity of the fimbrial biogenesis systems from these organisms, the D. nodosus genes were introduced into P. aeruginosa strains carrying mutations in the homologous genes. Analysis of the resultant derivatives showed that the fimP gene complemented a pilD mutant of P. aeruginosa for both fimbrial assembly and protein secretion. However, the fimN and fimO genes did not complement pilB or pilC mutants, respectively. These results suggest that although the PilD prepilin peptidase can be functionally replaced by the heterologous FimP protein, the function of the PilB and PilC proteins may require binding or catalytic domains specific for the P. aeruginosa fimbrial assembly system. The transcriptional organization and regulation of the fimNOP gene region were also examined. The results of reverse transcriptase PCR and primer extension analysis suggested that these genes form an operon transcribed from two ς 70 -type promoters located upstream of ORFM, an open reading frame proximal to fimN . Transcription of the D. nodosus fimbrial subunit was found to increase in cells grown on solid media, and it was postulated that this regulatory effect may be of significance in the infected footrot lesion.