Impact Investigation of a Mercury Reducing GEM in Stream Microcosms & Construction of Mercury Reducing Reporter Strains Based on the Safety Strain Ps. putida KT2440

The impact of the Hg reducing GEM Pseudomonas putida KT2442::mer73 on the native bacterial community in Elbe River water and sediment was investigated in terms of survival and persistence, effect on community composition as depicted by 16S rDNA-TGGE patterns and gene transfer. For this purpose a stream microcosm was designed and characterised regarding its flow. Both, the effect of longterm inoculation of low GEM numbers and a cell shock with high GEM numbers were investigated. Gene transfer was studied with and without phenyl mercuric acetate (PMA) as selective pressure. Survival was not detected in water, however, a tendency to higher colony forming units (cfu) was observed in the sediment. Neither in water nor in sediment did the GEM show an effect on the indigenous bacterial population or did it transfer its recombinant genes. Although Ps. putida KT2442::mer73 was shown to be highly resistant to Hg, it failed to establish stable biofilms in laboratory scale bioreactors (von Canstein et al. 2002). New GEMs were thus constructed that were selected for high Hg resistance and were in addition provided with green fluorescent protein (GFP). A promoterless cassette consisting of the mer operon and gfp was inserted into the genome of the fully sequenced safety strain Ps. putida KT2440, so that both functions were under the transcriptional control of the same host promoter. This way, the GFP tag permits easy detection of the new strains and monitoring of Hg resistance expression through the fluorescence. The site of integration was identified to yield genetic information that could be used to develop a highly specific, PCR-based detection method. Furthermore, knowledge of the construct genotype allowed implications regarding the phenotype.