Siderophore receptor PupA as a marker to monitor wild-type Pseudomonas putida WCS358 in natural environments
Raaijmakers, J.M. | Bitter, W. | Punte, H.L.M. | Bakker, P.A.H.M. | Weisbeek, P.J. | Schippers, B.
For application of genetically engineered fluorescent Pseudomonas spp., specific markers are required for monitoring of wild-type Pseudomonas strains and their genetically modified derivatives in natural environments. In this study, the specific siderophore receptor PupA of plant growth-promoting Pseudomonas putida WCS358 was used as a marker to monitor wild-type strain WCS358. After introduction into natural soil and rhizosphere environments, strain WCS358 could be recovered efficiently on a medium amended with 300 micromolar pseudobactin 358. Although low population densities of indigenous pseudomonads (less than or equal 10(3) of soil or root) were recovered on the pseudobactin 358-amended medium, subsequent agglutination assays with a WCS358-specific polyclonal antiserum enabled accurate monitoring of populations of wild-type strain WCS358 over a range of approximately 10(3) to 10(7) CFU/g of soil or root. Genetic analysis of the background population by PCR and Southern hybridization revealed that natural occurrence of the pupA gene was limited to a very small number of indigenous Pseudomonas spp. which are very closely related to P. putida WCS358. The PupA marker system enabled the study of differences in rhizosphere colonization among wild-type strain WCS358, rifampin-resistant derivative WCS358rr, and Tn5 mutant WCS358:xylE. Chromosomally mediated rifampin resistance did not affect the colonizing ability of P. putida WCS358. However, Tn5 mutant WCS358:xylE colonizes the radish rhizosphere significantly less than did its parental strain.
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