A DNA PROBE SUITABLE FOR MONITORING GENETICALLY ENGINEERED MICROORGANISMS IN THE ENVIRONMENT

[摘要] A bacterial haloacetate dehalogenase (H-1 type enzyme, EC 3.8.1.3) was proved to be one of the useful probes for detecting a genetically engineered microorganism (GEM) from environmental samples. A mono-fluoroacetate assimilating bacterium (Pseudomonas sp.), characteristically encoding H-1 gene on its plasmid pUO12, was applied to two sorts of intact paddy soil and was detected by colony hybridization probing 32P-labeled H-1 DNA fragment (1.6kb). Target bacteria could be quantitatively counted by autoradiography with the samples of no less than 10-3-fold soil dilution while using nutrient agar plates for colonization. Thus, the detection limit of the target bacteria was regarded as 400 CFU (colony-forming unit) per gram of dry weight of soil introducing the principle of most-probable-number method. When the same experiment was carried out using minimal agar containing monofluoroacetate as a sole source of carbon, target colonies were clearly detected even in the 10-1 soil dilution and thus, the detection limit was improved to as low as 4 CFU per gram of dry weight of soil. These redults suggested that H-1 gene probe was very valuable for tracing a GEM in the environment because of its unique nucleotide sequence and high sensitivity.

[学科分类] 微生物学和免疫学