Identification of genetic loci in a rhizosphere-inhabiting, species of Pseudomonas involved in expression of a phytoparasitic nematode ovicidal factor

Pseudomonas sp. BG33R was isolated from peach orchard soil suppressive to t he phytoparasitic ring nematode, Mesocriconema xenoplax. Tn5 transposon mut agenesis was utilized to generate five BG33R mutants 246, 1122, 1233, 1397, and 1917, which lacked ovicidal activity and also exhibited altered fluore scent siderophore and protease production, Southern blot analysis revealed that each of the five mutants contained a single unique Tn5 insertion. Mobi lization of selected BG33R genomic cosmid clones into mutants 246 and 1917 restored egg-kill activity and restored normal protcase and siderophore pro duction. Furthermore, a genomic cosmid clone hybridizing to the Tn5 inserti on site in 1122 also complemented mutant 1917. Site-directed mutagenesis of BG33R using the cloned Tn5 insertion site from mutant 1397 resulted in the loss of ovicidal activity and restoration of the hyperfluorescence phenoty pe. Sequence analysis was performed on the genetic regions flanking the Tn5 insertion site in all five egg-kill-negative mutants. Open reading frame a nalysis and amino acid comparative database searches of the Tn5 insertion s ites in the five mutants revealed a high degree of homology to the vgrG gen e, the two-component sensor kinase protein, rtpA (gacS analog), a UDP-gluco se 4-epimerase gene, a siderophore receptor protein, and the 50S ribosomal protein, L21. The involvement of these genes in the production of the egg-k ill factor is discussed. (C) 2001 Elsevier Science Ltd. All rights reserved .