Cloning and sequence analysis of two catechol-degrading gene clusters from the aniline-assimilating bacterium...

The aniline-assimilating bacterium Frateuria species ANA-18 produced two catechol 1,2-dioxygenases, CD I and CD II, and two muconate cycloisomerases, MC I and MC II. The catA genes catA1 and catA2 encoding CD I and CD II, respectively, were cloned from a gene library of this bacterium. The catA1 gene was clustered with catB1 encoding MC I, catC1 encoding muconolactone isomerase (MI), catD encoding beta-ketoadipate enol-lactone hydrolase (ELH), and ORFR1 encoding a putative LysR-type regulator. The organization of these genes was ORFR1catB1C1D. The catA2 gene also constructed a gene cluster involving catB2 encoding MC II, catC2 encoding MI, and ORFR2 encoding a putative LysR-type regulator with the alignment of ORFR2catB2A2C2. The intergenic regions of ORFR1-catB1 and ORFR2-catB2 contained homologous sequences with the catR-catB intergenic region containing a repression binding site and activation binding site of CatR in Pseudomonas putida. These findings suggest that the two cat clusters were regulated independently in their expression. When a product of cloned catD was added to a reaction mixture containing beta-ketoadipate enol-lactone, beta-ketoadipate was produced. This observation showed that the cloned catD encoded ELH and was expressed in Escherichia coli. We found that Frateuria sp. ANA-18 had a large plasmid with a molecular size more than 100kb. Polymerase chain reaction amplifying partial catA genes and Southern hybridization analyses with probes containing catA genes were conducted, to examine the localization of the two catA genes. We concluded that the catA1 and catA2 genes were located on the chromosomal and large plasmid DNAs, respectively, in Frateuria sp. ANA-18.