Mutation analysis of the different tfd genes for degradation of chloroaromatic compounds in Ralstonia eutropha JMP134

Ralstonia eutropha JMP134 possesses two sets of similar genes for degradation of chloroaromatic compounds, tfdCDEFB (in short: tfd(I) cluster) and tfdD(II)C(II)E(II)F(II)B(II) (tfd(II) cluster). The significance of two sets of tfd genes for the organism has long been elusive. Here, each of the tfd genes in the two clusters on the original plasmid pJP4 was replaced by double recombination with a gene fragment in which a kanamycin resistance gene was inserted into the respective tfd gene's reading frame. The insertion mutants were all tested for growth on 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), and 3-chlorobenzoate (3-CBA). None of the tfdD(II)C(II)E(II)F(II)B(II) genes appeared to be essential for growth on 2,4-D or on 3-CBA. Mutations in tfdC, tfdD and tfdF also did not abolish but only retarded growth on 2,4-D, indicating that they were redundant to some extent as well. Of all tfd genes tested, only tfdE and tfdB were absolutely essential, and interruption of those two reading frames abolished growth on 2,4-D, 3-CBA (tfdE only), and MCPA completely. Interestingly, strains with insertion mutations in the tfd(I) cluster and those in tfdD(II), tfdC(II), tfdE(II) and tfdB(II) were severely effected in their growth on MCPA, compared to the wild-type. This indicated that not only the tfd(I) cluster but also the tfd(II) cluster has an essential function for R. eutropha during growth on MCPA. In contrast, insertion mutation of tfdD(II) resulted in better growth of R. eutropha JMP134 on 3-CBA, which is most likely due to the prevention of toxic metabolite production in the absence of TfdD(II) activity.