Efficient turnover of chlorocatechols is essential for growth of Ralstonia eutropha JMP134(pJP4) in 3-chlorobenzoic acid.
Ralstonia eutropha JMP134(pJP4) degrades 3-chlorobenzoate (3-CB) by using two not completely isofunctional, pJP4-encoded chlorocatechol degradation gene clusters, tfdC(I)D(I)E(I)F(I) and tfdD(II)C(II)E(II)F(II). Introduction of several copies of each gene cluster into R. eutropha JMP222, which lacks pJP4 and thus accumulates chlorocatechols from 3-CB, allows the derivatives to grow in this substrate. However, JMP222 derivatives containing one chromosomal copy of each cluster did not grow in 3-CB. The failure to grow in 3-CB was the result of accumulation of chlorocatechols due to the limiting activity of chlorocatechol 1,2-dioxygenase (TfdC), the first enzyme in the chlorocatechol degradation pathway. Micromolar concentrations of 3- and 4-chlorocatechol inhibited the growth of strains JMP134 and JMP222 in benzoate, and cells of strain JMP222 exposed to 3 mM 3-CB exhibited a 2-order-of-magnitude decrease in viability. This toxicity effect was not observed with strain JMP222 harboring multiple copies of the tfdC(I) gene, and the derivative of strain JMP222 containing tfdC(I)D(I)E(I)F(I) plus multiple copies of the tfdC(I) gene could efficiently grow in 3-CB. In addition, tfdC(I) and tfdC(II) gene mutants of strain JMP134 exhibited no growth and impaired growth in 3-CB, respectively. The introduction into strain JMP134 of the xylS-xylXYZL genes, encoding a broad-substrate-range benzoate 1,2-dioxygenase system and thus increasing the transformation of 3-CB into chlorocatechols, resulted in derivatives that exhibited a sharp decrease in the ability to grow in 3-CB. These observations indicate that the dosage of chlorocatechol-transforming genes is critical for growth in 3-CB. This effect depends on a delicate balance between chlorocatechol-producing and chlorocatechol-consuming reactions.
Pérez-Pantoja
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D
Laboratorio de Microbiología, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile.
Ledger
T
T
Pieper
D H
DH
González
B
B
D001426:Bacterial Proteins; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism; D002396:Catechols / Q000378:metabolism*; D002455:Cell Division / Q000235:genetics; D002723:Chlorobenzoates / Q000378:metabolism*; D020118:Cupriavidus necator / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D004268:DNA-Binding Proteins; D049308:Dioxygenases; D043364:Endo-1,4-beta Xylanases; D018628:Gene Dosage; D008969:Molecular Sequence Data; D005810:Multigene Family; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014995:Xylosidases / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D002396:Catechols; D002723:Chlorobenzoates; D004268:DNA-Binding Proteins; D015534:Trans-Activators; C078720:XylS protein, Pseudomonas putida; C036427:3-chlorobenzoic acid; C031137:3-chlorocatechol; D010088:Oxidoreductases; D010105:Oxygenases; D049308:Dioxygenases; C044173:chlorocatechol 1,2-dioxygenase; D044925:Oxidoreductases Acting on CH-CH Group Donors; C021530:maleylacetate reductase; D002265:Carboxylic Ester Hydrolases; C046130:carboxymethylenebutenolidase; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases; C114374:XYLY xylanase; C038861:4-chlorocatechol