Cloning of Acinetobacter calcoaceticus chromosomal region involved in catechin degradation

Abstract

Acinetobacter calcoaceticus utilizes catechin as sole carbon source. The chromosomal region involved in catechin catabolism was cloned in Escherichia coli DH5α from the genomic DNA of A. calcoaceticus. A recombinant E. coli containing 9.2 kb DNA fragment of A. calcoaceticus inserted in pUC19 showed a halo zone around the colony in plate assays, indicating the catechin utilizing ability of the clone. Enzyme assays revealed the expression of the cloned DNA fragment of A. calcoaceticus. High performance thin layer chromatography confirmed protocatechuic acid and phloroglucinol carboxylic acid as cleavage products of catechin in A. calcoaceticus and the catechin degrading ability of the clones. A. calcoaceticus followed the β-ketoadipate pathway for catechin degradation. The sub-clone (pASC1) of this insert was sequenced and analyzed. The sequence showed three major ORFs but only ORF 2 showed similarities to other aromatic oxygenases and the sequence of ORF 2 was submitted to GenBank (AF369935).