Adaptation of Cornamonas testosteroni TAM1 to utilize phenol: organization and regulation of the genes involved in phenol degradatio

Abstract

Comamonas testosteroni TAU1 was not able to grow on phenol as a sole carbon and energy source, but it gained the ability to utilize phenol after a 2-3-week incubation in a medium containing phenol. Phenol hydroxylase (PH) and catechol2,3-dioxygenase (C230) were highly induced by phenol in the adapted strain designated as strain P1, suggesting that phenol was degraded via the meta-pathway. Gene clusters for phenol degradation were isolated from both strains TAU1 and P1. The structural genes encoding multicomponent PH and C230 (aphKLMNOPQB), and a regulatory gene of the NtrC family (aphR), were located in a divergent transcriptional organization. The cloned aphKLMNOPQl3 genes from either strain TAU1 or strain P1 produced active PH and C230 enzymes in strain TA441. No difference was found between the strains in the sequences of aphR and the intergenic promoter region of aphK and aphR. However, the transcriptional activities of the aphK and aphR promoters were higher in strain P1 than in strain TA441. The aphK-promoter activity was not observed in aphR mutant strains and these strains could not grow on phenol. The aphR mutant of strain P1 was able to grow on phenol after transformation with a recombinant aphR gene but strain TAM1 was not, suggesting that the expression of the aph genes is silenced by an unidentified repressor in strain TAU1 and that this repressor is modified in strain P1.

Meta-cleavage enzyme gene tesB is necessary for testosterone degradation in Comamonas testosteroni TA441

“…This yellow colour suggests that TA441 degrades testosterone via a meta-cleavage reaction. Our preliminary experiments showed that TA441 had a metacleavage enzyme which is produced when TA441 is grown on testosterone, in addition to the meta-cleavage enzymes already characterized in this strain (aphB and mhpB) (Arai et al, 1998(Arai et al, , 1999, and that the aphBmhpB -double knock-out mutant of TA441 still retained the ability to utilize testosterone as the sole carbon source. From these results, we considered that a metacleavage enzyme different from AphB and MhpB was involved in testosterone degradation in TA441, and we proceeded to partially purify the protein and determine the N-terminal sequence of the enzyme, as described in Methods.…”

“…The tesB gene in pCP311 was disrupted by insertion of a SmaI fragment from pSKKm r , containing the Km-resistance gene, into the EcoRV site in tesB. The resultant plasmid, pTesB-Km r , which encodes the Km-resistance gene in the same transcriptional direction as tesB, was used for inactivation of the tesB gene in TA441 by homologous recombination according to the method described previously (Arai et al, 1998). A Km-resistant and carbenicillin-sensitive TA441 mutant was selected and designated strain TesB − .…”

“…Comamonas testosteroni strains were grown at 30 mC in LB medium or C medium (Arai et al, 1998) with suitable carbon sources. Carbon sources (testosterone, 1,4-androstadiene-3,17-dione or p-hydroxybenzoate) were added as a filtered DMSO solution at a final concentration of 0n1% (w\v).…”

“…TA441 can also utilize testosterone and some other steroids as the sole carbon and energy source. The aph genes and mhp genes, which code for enzymes involved in the degradation of phenols and 3-(3-hydroxyphenyl)-propionic acid in TA441, have previously been identified and characterized (Arai et al, 1998(Arai et al, , 1999, whereas the genes encoding the enzymes involved in testosterone degradation have not yet been characterized. Cultures of TA441 show a yellow colour during growth on testosterone.…”

Complete nucleotide sequence of the catechol metabolic region of plasmid pTDN1.

“…Aph, Arai et al, 1998Arai et al, , 2000Atd, Takeo et al, 1998;Cdo, Parales et al, 1997 and Data base accession no. AF 190463;Dmp, Singler et al, 1992;Nah, Bosch et al, 2000;Xyl, Harayama and Rekik, 1990;CbzX, Mars et al, 1999;AAG04733, probable P. aeruginosa SCD, Stover et al, 2000. FED, plant-type ferredoxin; SCD, short-chain dehydrogenase.…”