Bacterial dichloropropene degradation in soil; screening of soils and involvement of plasmids carrying the dhlA gene

Abstract

The nematocide cis-1,3-dichloropropene is widely used in intensive agricultural practice against root knot nematodes. As a result of the regular application of dichloropropene to six out of eight different soils, the compound was rapidly degraded to CO 2, H 2O and chloride ions in subsequent applications (adapted soils). Such degradation occurred at a lower rate in the same soil types without a history of dichloropropene use (unadapted soils). In two soils, the effect of repeated dichloropropene treatment was not found, since dichloropropene was already rapidly decomposed in the untreated controls. Since the dehalogenating enzyme haloalkane dehalogenase, encoded by the dhlA gene, is possibly involved in the degradation of dichloropropene, a specific PCR detection system combined with a dhlA probe was developed to detect dhlA in soil DNA extracts. Five of the eight adapted soils as well as three unadapted ones showed a response to this system, indicating the presence of DNA with sequence similarity to dhlA. Fifteen bacterial strains with dichloropropene-degradative capacity were isolated from enrichment cultures grown in the presence of this compound and inoculated with adapted soil (five soil types). Six selected isolates were identified as, respectively, Alcaligenes paradoxus, Pseudomonas cichorii I, Pseudomonas corrugata (2 ×), Pseudomonas putida and Pseudomonas sp. Each of the six strains harboured a plasmid of 50–60 kb in size and all but one carried resistance to HgCl 2. Filter matings were performed to investigate the possible cotransfer of biodegradative genes and Hg resistance to suitably-marked P. fluorescens R2f recipient strains. Three plasmid donor strains, P. cichorii I, P. corrugata and Pseudomonas sp., transferred the Hg resistance and the plasmid to the recipient used, suggesting that the Hg resistance marker was located on the plasmid. In addition, transconjugants produced with P. cichorii I and P. corrugata donor strains had also acquired 1,3-dichloropropene-degrading capability. PCR amplification with dhlA specific primers, of the plasmids obtained both from the original P. cichorii I and P. corrugata strains and from the respective transconjugant P. fluorescens R2f strains generated 400–500 bp amplification products. After cloning of the product of the P. cichorii I plasmid and sequencing of 180 bases, extensive homology with the dhlA gene was detected. These results suggest that a plasmid-located dhlA-like gene may be involved in dichloropropene degradation in soil by soil bacteria, but the extent to which the gene is involved in the process in situ is not clear.