Luminescence-based detection of genetically modified Erwinia carotovora subspecies carotovora
Methods currently under evaluation for detecting and enumerating genetically modified microorganisms in the environment include culturing methods, direct microscopic detection and nucleic acid hybridisation techniques. The aim of this project was to develop a luminescence-based detection and enumeration system for genetically modified Erwinia carotovora subspecies carotovora in soil. Plasmid vectors encoding the genes for luciferase from Vibrio fischeri were introduced into E.carotovora subsp. carotovora using recombinant DNA techniques. Light output, determined by luminometry, from luminescent strains of E.carotovora subsp. carotovora, constitutively expressing the luciferase genes, was used for specific estimation of bacterial biomass during growth, both in liquid culture and in soil. In liquid culture, the lower detection limit was approximately 102 cfu ml-1. For exponentially growing cells inoculated into soil, the sensitivity was reduced. When light output was measured directly from 1g of soil, the lower detection limit was aproximately 106 cfu g soil-1, while recording light from a soil suspension increased the sensitivity to 104 cfu g soil-1. However, luminometry was only of value for enumerating growing cultures of E.carotovora subsp. carotovora as light output per cell declined when growth ceased. Light output from luminescent strains of E.carotovora subsp. carotovora, in both liquid culture and following inoculation into soil was correlated with microbial activity, measured by the dehydrogenase assay. Compared to conventional activity assays which measure the net activity of the total soil population, the bioluminescence reporter system enabled the activity of the marked population to be determined. In addition, the assay was non-invasive, non-destructive and rapid, with microbial activity determined within 5 min of sampling. Luminescent strains of E.carotovora subsp. carotovora were enumerated within a background soil population, in the absence of selection, by detecting luminescent colonies after plating onto solid medium. Detection was possible when luminescence-marked organisms were outnumbered by the indigenous soil population, although the efficiency of quantitative detection decreased significantly with decreasing population size. Luminescence-based marker techniques possess all the advantages of other molecular-based marker systems. Detection of marked cells is possible by hybridisation with nucleic acid probes and dilution plate counting is also possible through visible luminescence of colonies. In addition, luminometry provides a sensitive, rapid, non-extractive technique for the measurement of the microbial activity of the marked strain.