Horizontal transfer of dehalogenase genes on IncP1β plasmids during bacterial adaptation to degrade α-halocarboxylic acids

Abstract

The diversity of bacterial α-halocarboxylic acid (αHA) dehalogenases from a polluted soil was investigated. Polymerase chain reaction (PCR) primers designed to amplify group I and group II dehalogenase ( deh) gene sequences were used to screen bacterial isolates, nine β-Proteobacteria and one γ-Proteobacterium, from soil enrichments. Primers successfully amplified deh sequences from all 10 αHA-utilising isolates. Bacteria isolated at 15 or 30°C on chloroacetic acid or 2-chloropropionic acid from the same polluted soil were shown to contain up to four plasmids, some of these common between isolates. Analysis of deletion mutants and Southern hybridisation showed that each isolate contained an apparently identical IncP1β plasmid c. 80 kb in size, carrying group I deh genes in addition to an associated insertion sequence element. Moreover, an identical conjugative catabolic plasmid was isolated exogenously in several transconjugants independently selected from biparental matings between Ralstonia eutropha JMP222 and enrichment samples. PCR cloning and sequencing of deh genes directly from enrichment cultures inoculated with the same soil revealed that an identical deh gene was present in both primary, secondary and tertiary enrichment cultures, although this deh could not be amplified directly from soil. Two αHA-utilising bacteria isolated at lower temperature were found also to contain group II deh genes. Transfer of the deh catabolic phenotype to R. eutropha strain JMP222 occurred at high frequencies for four strains tested, a result that was consistent with assignment of the plasmids to the IncP1 incompatibility group. The promiscuous nature and broad host range of IncP plasmids make them likely to be involved in horizontal gene transfer during adaptation of bacteria to degrade organohalogens.