Construction and characterisation of lux-marked bacteria as biosensors
Many sites world-wide are contaminated with a range of pollutants of environmental concern. Bioremediation has the potential to be a cost-effective and efficient alternative clean-up technology. The success of bioremediation is influenced by both biological and environmental factors. A site, therefore, needs extensive characterisation to determine the extent of contamination and to evaluate the potential for bioremediation. Chemical analysis has traditionally been used to determine pollutant concentrations, but it provides no information about the bioavailability of the pollutants. Bioassays are able to complement chemical analyses by showing the bioavailability and ecotoxico logical effects of pollutants. Bioluminescent bacteria have been adapted as biosensors where the response to environmental stresses is monitored by a reduction in light output. Only metabolically active cells produce light and any substance or environmental condition which impairs cell metabolism and, thus, compromises cellular activity and viability will lead to a reduction in light output. Naturally bioluminescent marine bacteria (e.g. Vibrio fischeri) have been used for ecotoxicity testing, but soil and freshwater bacteria that have been marked with lux genes have several advantages. These genetically modified biosensors do not require high salinity and a neutral pH, and they have environmental relevance. A suite of biosensors has been developed at the University of Aberdeen that responds to a wide range of pollutants. These biosensors have been successfully used for acute ecotoxicity measurements. The research carried out in this study was part of a larger ICI project for the assessment and management of bioremediation of a BTEX contaminated site. The aim of this study was to develop a lux-marked biosensor based on a BTEX-degrader. None of the existing biosensors are known to be degraders. Environmental isolates from the ICI site that were able to degrade BTEX were characterised and assessed for their suitability for lux-marking. An appropriate isolate was selected for lux-marking, but the marking was not successful. It was concluded that there are several problems associated with obtaining suitable isolates in pure culture from a site. The enrichment, isolation, identification and characterisation of isolates is laborious and time-consuming, and the lack of characterisation of the isolates can complicate the Iwc-marking attempts. Selecting a well-characterised bacterium for lux-marking avoids these problems. Therefore, Pseudomonas putida FI was selected as the bacterium for lux-marking as a biosensor in this study. It was selected because it is a toluene-degrader and the degradative genes are located on the chromosome. P. putida FI also has environmental relevance for the ICI site. P. putida FI and P. putida FI06 (an isogenic mutant of P. putida FI) were lux-marked with the plasmid pUCD607, and P. putida FI and FI06 pUCD607 were characterised. Characterisation of P. putida FI and FI06 pUCD607 suggested that pUCD607 was not stable even under selective conditions due to segregational instability. This study, therefore, concluded that the plasmid pUCD607 is not appropriate for lux-marking bacteria as biosensors. P. putida FI was lux-marked with the mini-Tn5 luxCDABE transposon and P. putida FI Tn5 luxCDABE was characterised. The integration of the mini-Tn5 luxCDABE cassette did not affect growth of P. putida FI Tn5 luxCDABE and luminescence levels were higher than in P. putida FI pUCD607. P. putida FI Tn5 luxCDABE was also stable in the absence of selective pressure over time. This study, therefore, concluded that the mini-TnJ luxCDABE transposon is appropriate for lux-marking bacteria as biosensors.