Survival of a lacZY-marked strain of Pseudomonas corrugata following a field release

Abstract

Pseudomonas corrugata, strain 2140, a biological control agent of take-all disease of wheat, was originally isolated from an acidic red-brown earth soil in New South Wales, Australia. A spontaneous rifampicin-resistant mutant of this bacterium was marked with the disarmed transposon, Tn 7:: lacZY. This marked strain (2140R lacZY) was introduced into a calcareous sandy loam soil (pH 8) in South Australia. Up to 4 years after its release, P. corrugata 2140R lacZY cells were re-isolated, single colony purified and stored at −80°C. Re-isolated bacteria, including re-isolates obtained 3 (22 re-isolates) and 4 (3 re-isolates) years after release, were examined for stability of the lacZY insert site and for gross chromosomal changes. Hybridization of a cloned lacZY fragment to DNA extracted from the soil re-isolates did not reveal any major changes to the lacZY insert site. Gross chromosomal changes were further examined by restriction endonuclease fingerprinting and PCR based on repetitive sequences (repetitive extragenic palindromic-, enterobacterial repetitive intergeneric consensus- and BOX-PCR). MspI digests distinguished the lacZY-marked strain from the parental strain. None of the genetic techniques used revealed any polymorphisms between the original 2140R lacZY-marked strain and the soil re-isolates. The results demonstrated that the chromosomal landscape within and around the insertion site of the lacZY construct had not altered in the re-isolated bacteria during the 4 years the organism had been in the field.