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 the phytoparasitic ring nematode, Mesocriconema xenoplax. Tn5 transposon mutagenesis was utilized to generate five BG33R mutants 246, 1122, 1233, 1397, and 1917, which lacked ovicidal activity and also exhibited altered fluorescent siderophore and protease production. Southern blot analysis revealed that each of the five mutants contained a single unique Tn5 insertion. Mobilization of selected BG33R genomic cosmid clones into mutants 246 and 1917 restored egg-kill activity and restored normal protease and siderophore production. Furthermore, a genomic cosmid clone hybridizing to the Tn5 insertion 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 phenotype. Sequence analysis was performed on the genetic regions flanking the Tn5 insertion site in all five egg-kill-negative mutants. Open reading frame analysis and amino acid comparative database searches of the Tn5 insertion sites in the five mutants revealed a high degree of homology to the vgrG gene, the two-component sensor kinase protein, rtpA (gacS analog), a UDP-glucose 4-epimerase gene, a siderophore receptor protein, and the 50S ribosomal protein, L21. The involvement of these genes in the production of the egg-kill factor is discussed.