DETECTION AND ENUMERATION OF A TAGGED PSEUDOMONAS-FLUORESCENS STRAIN BY USING SOIL WITH MARKERS ASSOCIATED WITH AN ENGINEERED CATABOLIC PATHWAY

Previously we described a novel gene tagging method, using the moc (ma nnityl opine catabolism) region from the Agrobacterium tumefaciens Ti plasmid pTi15955, to identify microorganisms destined for release into the environment. Here, we used the engineered strain Pseudomonas fluo rescens PF5MT12 carrying the moc region integrated into the bacterial chromosome to demonstrate the usefulness of the markers for detection and direct selection of marked organisms present in soil samples, Usin g this system, we routinely detected population levels as low as 10(2) CFU per g of soil sampled, In addition to direct selection, we develo ped an immunologically based assay using MOP cyclase, a unique enzyme associated with moc, as the epitope for detecting the tagged organism. The colony immunoblot assay proved to be highly specific and without any false-positive signals when used to identify organisms cultured fr om soil on nonselective medium. The numbers of colonies that were immu noreactive with the anti-MOP cyclase antibody were essentially equal t o those that grew out on selection plates. This indicates that MOP cyc lase can be used as a marker and that we can use nonselective medium t o retrieve the marked genetically engineered microorganisms and then i dentify them by using colony immunoblot assays, These direct selection and colony immunoblot methods provide a sensitive and accurate strate gy for identifying and enumerating marked organisms recovered from soi l samples. We also developed a rapid assay for MOP cyclase that does n ot require cell permeabilization with toluene, This assay can be used to verify tagged organisms isolated by other methods or to screen larg e numbers of colonies for the tag following nonselective isolation.