A NOVEL GENE TAG FOR IDENTIFYING MICROORGANISMS RELEASED INTO THE ENVIRONMENT

A novel method using a moc (mannityl opine catabolism) region from the Agrobacterium tumefaciens Ti plasmid pTi15955 was developed as a tag to identify genetically modified microorganisms released into the envi ronment. Pseudomonas fluorescens 1855.344, a plant-gro,vth-promoting r hizosphere bacterium, was chosen as the organism in which to develop a nd test the system. moc genes carried by pYDH208, a cosmid clone conta ining a 20-kb segment of the octopine-mannityl opine-type Ti plasmid, conferred on P. fluorescens strains the capacity to utilize mannopine and agropine (AGR) as a sole source of carbon and energy. Modified P. fluorescens strains containing moc or mocunptII inserted into a chromo somal site were constructed by marker exchange. One such modified stra in, PF5MT12, utilized AGR as a sole carbon source and contained detect able levels of mannopine cyclase, an easily assayable enzyme encoded b y the moc region. Catabolism of AGR could be used to recover selective ly the marked strain from mixed populations containing a large excess of closely related bacteria. Nucleic acid-based detection strategies w ere developed on the basis of the unique fusion region between Agrobac terium DNA and Pseudomonas DNA in strain PF5MT12. The specificity and sensitivity of detection of PF5MT12 were enhanced by amplifying the fu sed DNA region by using PCR. The target fragment could be detected at levels of sensitivity comparable to those of other described PCR-based gene tags, even in the presence of high levels of Agrobacterium, Pseu domonas, or Escherichia coli DNA. This gene tag strategy gives a metho d for direct selection and enumeration of the marked strain from mixtu res containing a large excess of closely related bacteria and a sensit ive and highly specific system for detection by PCR amplification of t he target fragment even in the presence of large amounts of DNA from r elated or unrelated organisms.