strain CTM degrades 2-methylaniline mainly via the -cleavage pathway. Conversion of the metabolite 3-methylcatechol was catalysed by an 156 000 catechol 2,3-dioxygenase (C23OI) comprising four identical subunits of 39000. The corresponding gene was detected by using an oligonucleotide as a gene probe. This oligonucleotide was synthesized on the basis of a partial amino acid sequence obtained from the purified enzyme from . The structural gene of C23OI was located on a 3.5 kb /II restriction fragment of plasmid pTCl. On the same restriction fragment the gene for a second catechol 2,3-dioxygenase, designated C23OII. was found. This gene coded for the synthesis of the 40000 polypeptide of the 158000 tetrameric C23OII. More precise mapping of the structural genes showed that the C23OI gene was located on a 1.2 kb /II I fragment and the C23OII gene on the adjacent 1.15 kb I fragment. Comprehensive substrate range analysis showed that C23OII accepted all the substrates that C23OI did, but additionally cleaved 2,3-dihydroxybiphenyl and catechols derived from phenylcarboxylic acids. C23OI exhibited highest activity towards methylcatechols, whereas C23OII cleaved unsubstituted catechol preferentially.


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