Use of a polymerase-chain-reaction-amplified DNA probe from Pseudomonas putida to detect D-hydantoinase-producing microorganisms by direct colony hybridization.

Pseudomonas putida strain DSM 84 produces N-carbamyl-D-amino acids from the corresponding D-5-monosubstituted hydantoins. The sequence of the D-hydantoinase gene from this strain (GenBank accession number L24157) was used to develop a DNA probe of 122 base pairs (bp) that could detect D-hydantoinase genes in other bacterial genera by DNA and by colony hybridization. Under conditions tolerating 32% mismatch, the probe was specific for all strains that expressed D-hydantoinase activity. These include Pseudomonadaceae of all rRNA groups, and bacteria belonging to the genera Agrobacterium, Serratia, Corynebacterium, and Arthrobacter. Environmental sampling was simulated by screening a mixture of unknown microorganisms from commercial inocula for the biodegradation of industrial, municipal and domestic wastes. The 122-bp probe was specific for microorganisms that subsequently demonstrated D-hydantoinase activity. Bacterial species from four different genera were detected, which were Pseudomonas, Klebsiella, Enterobacter, and Enterococcus.