USE OF MOLECULAR TECHNIQUES TO EVALUATE THE SURVIVAL OF A MICROORGANISM INJECTED INTO AN AQUIFER

A PCR primer set and an internal probe that are specific for Pseudomon as sp. strain B13, a 3-chlorobenzoate-metabolizing strain, were develo ped. Using this primer set and probe, we were able to detect Pseudomon as sp. strain B13 DNA sequences in DNA extracted from aquifer samples 14.5 months after Pseudomonas sp. strain B13 had been injected into a sand and gravel aquifer. This primer set and probe were also used to a nalyze isolates from 3-chlorobenzoate enrichments of the aquifer sampl es by Southern blot analysis. Hybridization of Southern blots with the Pseudomonas sp. strain B13-specific probe and a catabolic probe in co njunction with restriction fragment length polymorphism (RFLP) analysi s of ribosome genes was used to determine that viable Pseudomonas sp. strain B13 persisted in this environment. We isolated a new 3-chlorobe nzoate-degrading strain from one of these enrichment cultures. The B13 -specific probe does not hybridize to DNA from this isolate. The new s train could be the result of gene exchange between Pseudomonas sp. str ain B13 and an indigenous bacterium. This speculation is based on an R FLP pattern of ribosome genes that differs from that of Pseudomonas sp . strain B13, the fact that identically sized restriction fragments hy bridized to the catabolic gene probe, and the absence of any enrichabl e 3-chlorobenzoate-degrading strains in the aquifer prior to inoculati on.