SIDEROPHORE RECEPTOR PUPA AS A MARKER TO MONITOR WILD-TYPE PSEUDOMONAS-PUTIDA WCS358 IN NATURAL ENVIRONMENTS

For application of genetically engineered fluorescent Pseudomonas spp. , specific markers are required for monitoring of wild-type Pseudomona s strains and their genetically modified derivatives in natural enviro nments. In this study, the specific siderophore receptor PupA of plant growth-promoting Pseudomonas putida WCS358 was used as a marker to mo nitor wild-type strain WCS358. After introduction into natural soil an d rhizosphere environments, strain WCS358 could be recovered efficient ly on a medium amended with 300 muM pseudobactin 358. Although low pop ulation densities of indigenous pseudomonads (less-than-or-equal-to 10 (3)/g of soil or root) were recovered on the pseudobactin 358-amended medium, subsequent agglutination assays with a WCS358-specific polyclo nal antiserum enabled accurate monitoring of populations of wild-type strain WCS358 over a range of approximately 10(3) to 10(7) CFU/g of so il or root. Genetic analysis of the background population by PCR and S outhern hybridization revealed that natural occurrence of the pupA gen e was limited to a very small number of indigenous Pseudomonas spp. wh ich are very closely related to P. putida WCS358. The PupA marker syst em enabled the study of differences in rhizosphere colonization among wild-type strain WCS358, rifampin-resistant derivative WCS358rr, and T n5 mutant WCS358=xylE. Chromosomally mediated rifampin resistance did not affect the colonizing ability of P. putida WCS358. However, Tn5 mu tant WCS358=xylE colonized the radish rhizosphere significantly less t han did its parental strain.