Complementation analysis of the Dichelobacter nodosus fimN, fimO, and fimP genes in Pseudomonas aeruginosa and transcriptional analysis of the fimNOP gene region

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 sigma70-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.

FIG. 1

Genetic organization of the fimNOP gene region. Relevant plasmids are shown to scale. Products of RT-PCR experiments and their sizes are indicated above the map. The position of the Ω insertion in pJIR1263 is shown. The two promoters, P1 and P2, are indicated. Abbreviations: B, BamHI; C, ClaI; EV, EcoRV; H, HpaI; Hf, HinfI; H2, HindII; K, KpnI; N, NarI; Nd, NdeI; Nr, NruI; P, PstI; S, SalI; Sp, SphI.

FIG. 2

Complementation analysis of a P. aeruginosa pilD mutant. Electron micrographs (Magnification, ×27,900) of P. aeruginosa PAK (A), the P. aeruginosa pilD mutant PAKDΩ (29) (B), P. aeruginosa PAKDΩ(pJIR1042) (carries fimP) (C), and P. aeruginosa PAKDΩ(pJIR1263) (carries fimPΩ) (D).

FIG. 3

RT-PCR analysis of fimN and fimO transcripts from P. aeruginosa. RT-PCR was performed on RNA extracted from PAKBΩ(pJIR1332) (lanes 1 and 2) and PAKCΩ(pJIR1068) (lanes 4 and 5) cells grown overnight on 2YT agar containing 1 mM IPTG. The resultant samples were analyzed by agarose gel electrophoresis. Lanes 1 and 4 contain the products of the RT-PCRs. Lanes 2 and 5 contain control reactions performed in an identical manner except that no RT was added. Lanes 3 and 6 contain positive controls, the products of PCRs performed with the respective plasmid templates. The standards (S) consist of HindIII-digested λcI857 DNA (measured in kilobases).

FIG. 4

Nucleotide sequence of the upstream region of ORFM. Putative RBSs are marked by a line above the sequence. Nucleotides corresponding to the transcription start sites mapped by primer extension are indicated by asterisks. The two promoters, P1 and P2, are boxed. An incomplete inverted repeat sequence is indicated by arrows below the sequence. Numbers to the right of the sequence refer to the number of nucleotides or amino acid residues, respectively.

FIG. 5

RNA dot blot hybridization analysis of fimN and fimA transcripts from D. nodosus. Cells were grown on Eugon agar (a) or in Eugonbroth with 10% horse serum (b). RNA concentrations (in micrograms per milliliter) are indicated. A 16S rRNA probe was included as a control.