GREEN FLUORESCENT PROTEIN AS A MARKER FOR PSEUDOMONAS SPP

The development of sensitive methods for observing individual bacteria l cells in a population in experimental models and natural environment s, such as in biofilms or on plant roots, is of great importance for s tudying these systems, We report the construction of plasmids which co nstitutively express a bright mutant of the green fluorescent protein of the jellyfish Aequorea victoria and are stably maintained in Pseudo monas spp, We demonstrate the utility of these plasmids to detect indi vidual cells in two experimental laboratory systems: (i) the examinati on of a mixed bacterial population of Pseudomonas aeruginosa and Burkh olderia cepacia attached to an abiotic surface and (ii) the associatio n of Pseudomonas fluorescens WCS365 with tomato seedling roots, We als o show that two plasmids, pSMC2 and pGB5, are particularly useful, bec ause they are stable in the absence of antibiotic selection, they plac e an undetectable metabolic burden on cells that carry the plasmids, a nd cells carrying these constructs continue to fluoresce even after 7 days in culture without the addition of fresh nutrients, The construct ion of improved Escherichia coli-Pseudomonas shuttle vectors which car ry multiple drug resistance markers also is described.