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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

Laboratorio de Microbiología, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, Casilla 114-D, Santiago, Chile¹
Division of Microbiology, National Research Centre for Biotechnology – GBF, Braunschweig, Germany²

Author for correspondence: Bernardo González. Tel: +56 2 6862845. Fax: +56 2 2225515. e-mail: bgonzale{at}genes.bio.puc.cl

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 degradation of these compounds. During growth of strain JMP134 in liquid medium containing 3-CB, a derivative strain harbouring a 95 kb plasmid was isolated. This derivative, designated JMP134(pJP4-F3), had an improved ability to grow on 3-CB, but had lost the ability to grow on 2,4-D. Sequence analysis of pJP4-F3 indicated that the plasmid had undergone a deletion of 16 kb, which included the tfdA–tfdS intergenic region, spanning the tfdA gene to a previously unreported IS1071 element. The loss of the tfdA gene explains the failure of the derivative to grow on 2,4-D. A 23 kb duplication of the region spanning tfdR-tfdD_IIC_IIE_IIF_II-tfdB_II-tfdK-ISJP4-tfdT-tfdC_ID_IE_IF_I-tfdB_I, giving rise to a 51-kb-long inverted repeat, was also observed. The increase in gene copy number for the tfdCD(DC)EF gene cluster may provide an explanation for the derivative strain’s improved growth on 3-CB. These observations are additional examples of the metabolic plasticity of R. eutropha JMP134, one of the more versatile pollutant-degrading bacteria.

Keywords: chloroaromatics, catabolic plasmid, IS1071 insertion sequence, chlorocatechol pathway

Abbreviations: 3-CB, 3-chlorobenzoate; 2,4-D, 2,4-dichlorophenoxyacetate

The GenBank accession numbers for the 3115 nt BamHI-F and 2833 nt EcoRI-F fragments of pJP4 and the 4037 nt EcoRI-E' fragment of pJP4-F3 are AF225972, AF225973 and AF225974, respectively.

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