Green fluorescent protein as a marker to monitor survival of phenanthrene-mineralizing Pseudomonas sp. UG14Gr in creosote-contaminated soil

Abstract

The green fluorescent protein (encoded by gfp gene) was used as a marker to estimate survival of a phenanthrene-degrading Pseudomonas sp. UG14Gr in a creosote-contaminated soil from Alberta, Canada. The gfp was integrated via Tn5 transposition into the chromosome of UG14r and resultant gfp-marked colonies were identified by green fluorescence emission under UV light. The gfp was stably maintained in UG14Gr and the gfp insertion had no apparent adverse effect on the metabolic capability of the marked isolate, which showed unhindered ability to mineralize 14C-labelled phenanthrene. Colony forming units were used to monitor parent strain UG14r and gfp-marked UG14Gr in creosote-contaminated soil at 22°C. Green fluorescence in the gfp-marked colonies allowed detection of UG14Gr, on antibiotic-amended and non-amended Trypticase soy agar (TSA), up to 13 months after inoculation into soil while the parent UG14r strain could be detected, on TSA amended with rifampicin, for only up to 25 days after inoculation into soil. Our studies indicated that the gfp marker can be used for survival studies of marked bacterial cells in contaminated soil. When the gfp-marked UG14Gr strain was co-inoculated with a lux-lac-marked Pseudomonas aeruginosa UG2Lr strain, both strains were unambiguously detected by their respective markers. Comparison of the effect of water holding capacity (WHC) of soil on [14C]phenanthrene mineralization showed that mineralization proceeded faster in treatments at 60% WHC than at 85% WHC. Nitrogen amendment in soils had no effect on [14C]phenanthrene mineralization at 60% WHC while treatments at 85% WHC showed higher mineralization in unamended soils than in N-amended soils.