CHARACTERIZATION OF TYPE-IV PILUS GENES IN PLANT GROWTH-PROMOTING PSEUDOMONAS-PUTIDA WCS358

In a search for factors that could contribute to the ability of the pl ant growth-stimulating Pseudomonas putida WCS358 to colonize plant roo ts, the organism was analyzed for the presence of genes required for p ilus biosynthesis. The pilD gene of Pseudomonas aeruginosa, which has also been designated xcpA, is involved in protein secretion and in the biogenesis of type IV pill. It encodes a peptidase that processes the precursors of the pilin subunits and of several components of the sec retion apparatus. Prepilin processing activity could be demonstrated i n P. putida WCS358, suggesting that this nonpathogenic strain may cont ain type IV pill as well. A DNA fragment containing the pilD (xcpA) ge ne of P. putida was cloned and found to complement a pilD (xcpA) mutat ion in P. aeruginosa. Nucleotide sequencing revealed, next to the pilD (xcpA) gene, the presence of two additional genes, pilA and pilC, tha t are highly homologous to genes involved in the biogenesis of type TV pili. The pilA gene encodes the pilin subunit, and pilC is an accesso ry gene, required for the assembly of the subunits into pill. In compa rison with the pil gene cluster in P. aeruginosa, a gene homologous to pilB is lacking in the P. putida gene cluster. Pill were not detected on the cell surface of P. putida itself, not even when pilA was expre ssed from the tac promoter on a plasmid, indicating that not all the g enes required for pilus biogenesis were expressed under the conditions tested. Expression of pilA of P. putida in P. aeruginosa resulted in the production of pill containing P. putida PilA subunits.