Molecular characterization of a deletion/duplication rearrangement in tfd genes from Ralstonia eutropha JMP134(pJP4) that improves growth on 3-chlorobenzoic acid but abolishes growth on 2,4-dichlorophenoxyacetic acid

Ralstonia eutropha JMP134(pJP4) is able to grow on minimal media containing the pollutants 3-chlorobenzoate (3-CB) or 2,4-dichlorophenoxyacetate (2,4- D). tfd genes from the 88 kb plasmid pJP4 encode enzymes involved in the de gradation of these compounds. During growth of strain JMP134 in liquid medi um containing 3-CB, a derivative strain harbouring a similar to 95 kb plasm id was isolated. This derivative, designated JMP134(pJP4-F3), had an improv ed ability to grow on 3-CB, but had lost the ability to grow on 2,4-D. Sequ ence analysis of pJP4-F3 indicated that the plasmid had undergone a deletio n of similar to 16 kb, which included the tfdA-tfdS intergenic region, span ning the tfdA gene to a previously unreported IS1071 element. The loss of t he tfdA gene explains the failure of the derivative to grow on 2,4-D. A sim ilar to 23 kb duplication of the region spanning tfdR-tfdD(II)C(II)E(II)F(I I)-tfdB(II)-tfdK-ISJP4-tfdT-tfdC(I)D(I)E(I)F(I)-tfdB(I), giving rise to a 5 1-kb-long inverted repeat, was also observed. The increase in gene copy num ber for the tfdCD(DC)EF gene cluster may provide an explanation for the der ivative strain's improved growth on 3-CB. These observations are additional examples of the metabolic plasticity of R. eutropha JMP134, one of the mor e versatile pollutant-degrading bacteria.