Heterologous Expression and Identification of the Genes Involved in Anaerobic Degradation of 1,3-Dihydroxybenzene (Resorcinol) in Azoarcus anaerobius

摘要

Azoarcus anaerobius, a strictly anaerobic, gram-negative bacterium, utilizes resorcinol as a sole carbon and\udenergy source with nitrate as an electron acceptor. Previously, we showed that resorcinol degradation by this\udbacterium is initiated by two oxidative steps, both catalyzed by membrane-associated enzymes that lead to the\udformation of hydroxyhydroquinone (HHQ; 1,2,4-benzenetriol) and 2-hydroxy-1,4-benzoquinone (HBQ). This\udstudy presents evidence for the further degradation of HBQ in cell extracts to form acetic and malic acids. To\udidentify the A. anaerobius genes required for anaerobic resorcinol catabolism, a cosmid library with genomic\udDNA was constructed and transformed into the phylogenetically related species Thauera aromatica, which\udcannot grow with resorcinol. By heterologous complementation, a transconjugant was identified that gained the\udability to metabolize resorcinol. Its cosmid, designated R , carries a 29.88-kb chromosomal DNA fragment\udcontaining 22 putative genes. In cell extracts of T. aromatica transconjugants, resorcinol was degraded to HHQ,\udHBQ, and acetate, suggesting that cosmid R carried all of the genes necessary for resorcinol degradation. On\udthe basis of the physiological characterization of T. aromatica transconjugants carrying transposon insertions\udin different genes of cosmid R , eight open reading frames were found to be essential for resorcinol mineralization.\udResorcinol hydroxylase-encoding genes were assigned on the basis of sequence analysis and enzyme\udassays with two mutants. Putative genes for hydroxyhydroquinone dehydrogenase and enzymes involved in ring\udfission have also been proposed. This work provides the first example of the identification of genes involved in\udthe anaerobic degradation of aromatic compounds by heterologous expression of a cosmid library in a\udphylogenetically related organism.