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.
LaPointe
G
G
Agriculture Canada, Food Research and Development, St. Hyacinthe, Quebec.
Leblanc
D
D
Morin
A
A
D000581:Amidohydrolases / Q000096:biosynthesis* / Q000235:genetics*; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D001673:Biodegradation, Environmental; D015342:DNA Probes / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006827:Hydantoins / Q000378:metabolism; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D011548:Pseudomonadaceae / Q000201:enzymology / Q000235:genetics; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics*; D012231:Rhizobium / Q000201:enzymology / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
D015342:DNA Probes; D004269:DNA, Bacterial; D006827:Hydantoins; D000581:Amidohydrolases; C020047:dihydropyrimidinase