Characterization of catechol catabolic genes from Rhodococcus erythropolis 1CP

Abstract

The biochemical characterization of the muconate and the chloromuconate cycloisomerases of the chlorophenol-utilizing Rhodococcus erythropolis strain 1CP previously indicated that efficient chloromuconate conversion among the gram-positive bacteria might have evolved independently of that among gram-negative bacteria. Based on sequences of the N terminus and of tryptic peptides of the muconate cycloisomerase, a fragment of the corresponding gene has now been amplified and used as a probe for the cloning of catechol catabolic genes from R. erythropolis. The clone thus obtained expressed catechol 1,2-dioxygenase, muconate cycloisomerase, and muconolactone isomerase activities. Sequencing of the insert on the recombinant plasmid pRER1 revealed that the genes are transcribed in the order catA catB catC. Open reading frames downstream of catC may have a function in carbohydrate metabolism. The predicted protein sequence of the catechol 1,2-dioxygenase was identical to the one from Arthrobacter sp. strain mA3 in 59% of the positions. The chlorocatechol 1,2-dioxygenases and the chloromuconate cycloisomerases of gram-negative bacteria appear to be more closely related to the catechol 1,2-dioxygenases and muconate cycloisomerases of the gram-positive strains than to the corresponding enzymes of gram-negative bacteria.

Characterization of the genes encoding the 3-carboxy-cis,cis-muconate-lactonizing enzymes from the 4-sulfocatechol degradative pathways of Hydrogenophaga intermedia S1 and Agrobacterium radiobacter S2

“…The plasmid vectors pJOE3075 and pAC28 were used for high levels of gene expression (Kholod & Mustelin, 2001;Stumpp et al, 2000). The characteristics of all plasmids used are given in Table 1. The genomic DNA of H. intermedia S1 was prepared after SDS lysis and phenol extraction as described by Eulberg et al (1997). The genomic DNA from A. radiobacter S2 was extracted using a DNeasy Tissue Kit (Qiagen) and the genomic DNA of P. putida using an E.Z.N.A Bacterial DNA Kit (Peqlab Biotechnologie).…”

Characterization of a gene cluster encoding the maleylacetate reductase from Ralstonia eutropha 335T, an enzyme recruited for growth with 4-fluorobenzoate

“…Genomic DNA from R. eutropha 335 T was available from a previous purification (Hinner, 1998). General methods of DNA manipulation as well as the overall cloning strategy and hybridization conditions used have been described previously (Eulberg et al, 1997;Seibert et al, 1998).…”

“…Genomic DNA from R. eutropha 335 T was available from a previous purification (Hinner, 1998). General methods of DNA manipulation as well as the overall cloning strategy and hybridization conditions used have been described previously (Eulberg et al, 1997;Seibert et al, 1998).An approximately 980 bp fragment of the maleylacetate reductase gene of R. eutropha 335 T was amplified from genomic DNA as template by using the same primers as for the cloning of macA of Rhodococcus opacus 1CP (Seibert et al …”

A linear megaplasmid, p1CP, carrying the genes for chlorocatechol catabolism of Rhodococcus opacus 1CP

“…Prolonged cultivation in the presence of 2-chlorophenol or 3-chlorobenzoate led to the isolation of a mutant also able to utilize these compounds as sole carbon source (Moiseeva et al, 1999). Gene clusters for the metabolism of catechol (catRABC), protocatechuate (pcaHGBLR), 4-chloro-/3,5-dichlorocatechol (clcBRAD), 3-chlorocatechol (clcA2D2B2F) and benzoate (benABCDK) were identified and their specific function has already been shown (Bauer, 2002;Eulberg et al, 1997Eulberg et al, , 1998aMoiseeva et al, 2001Moiseeva et al, , 2002. Available biochemical and sequence evidence suggests that chlorocatechol catabolism of strain 1CP evolved independently of that in proteobacteria (Eulberg et al, 1998a;Moiseeva et al, 2002;Solyanikova et al, 1995).…”

“…Even 2-chlorophenol and 3-chlorobenzoate are mineralized by a mutant of strain 1CP, which was selected after prolonged adaptation (Moiseeva et al, 1999). Whereas the non-chlorinated aromatic compounds are degraded via the catechol or the protocatechuate branch of the 3-oxoadipate pathway, encoded by cat and pca genes, respectively (Eulberg et al, , 1998b, chlorophenols and 3-chlorobenzoate are catabolized via the 4-chloro-/3,5-dichlorocatechol branch or the 3-chlorocatechol branch, which are encoded by the clc and the clc2 gene clusters, respectively (Eulberg et al, 1998a;Moiseeva et al, 2002). While the clcBRAD cluster includes genes for a chlorocatechol 1,2-dioxygenase, a chloromuconate cycloisomerase, a dienelactone hydrolase, and a presumed regulator, the clc2 cluster is unusual in comprising a 5-chloromuconolactone dehalogenase gene in addition to those for dioxygenase, cycloisomerase and dienelactone hydrolase.…”