Use of a xylE marker gene to monitor survival of recombinant Pseudomonas putida populations in lake water by culture on nonselective media.

IncQ marker plasmids were previously constructed to enable the analysis of the survival of populations of Pseudomonas putida released into lake water (C. Winstanley, J. A. W. Morgan, R. W. Pickup, J. G. Jones, and J. R. Saunders, Appl. Environ. Microbiol. 55:771-777, 1989). We constructed equivalent IncP plasmids, pLV1016 and pLV1017, to provide conjugative alternative systems. Detection of the xylE gene carried by marker plasmids was found to be a valid indicator to use for studying the survival of released populations by culturing on nonselective media. These plasmids were used to study the survival of populations of Pseudomonas putida in both sterile and untreated lake water. The effects of inoculum size, the metabolic burden imposed on the cell by the unregulated expression of xylE, and an auxotrophic mutation carried by the host strain were studied. We also assessed the reproducibility and hence the predictability of the survival of released populations. Model systems with a single lake water sample and model systems with three different lake water samples, taken from the same site in consecutive months, were used to analyze variability between replicates and to assess differences caused by host strain or water sample. A large variability was found depending on which water sample was used. These findings imply that it will be difficult to predict accurately the survival of released populations in the natural environment.