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

Abstract

Azoarcus anaerobius, a strictly anaerobic, gram-negative bacterium, utilizes resorcinol as a sole carbon and energy source with nitrate as an electron acceptor. Previously, we showed that resorcinol degradation by this bacterium is initiated by two oxidative steps, both catalyzed by membrane-associated enzymes that lead to the formation of hydroxyhydroquinone (HHQ; 1,2,4-benzenetriol) and 2-hydroxy-1,4-benzoquinone (HBQ). This study presents evidence for the further degradation of HBQ in cell extracts to form acetic and malic acids. To identify the A. anaerobius genes required for anaerobic resorcinol catabolism, a cosmid library with genomic DNA was constructed and transformed into the phylogenetically related species Thauera aromatica, which cannot grow with resorcinol. By heterologous complementation, a transconjugant was identified that gained the ability to metabolize resorcinol. Its cosmid, designated R ؉ , carries a 29.88-kb chromosomal DNA fragment containing 22 putative genes. In cell extracts of T. aromatica transconjugants, resorcinol was degraded to HHQ, HBQ, and acetate, suggesting that cosmid R ؉ carried all of the genes necessary for resorcinol degradation. On the basis of the physiological characterization of T. aromatica transconjugants carrying transposon insertions in different genes of cosmid R؉ , eight open reading frames were found to be essential for resorcinol mineralization. Resorcinol hydroxylase-encoding genes were assigned on the basis of sequence analysis and enzyme assays with two mutants. Putative genes for hydroxyhydroquinone dehydrogenase and enzymes involved in ring fission have also been proposed. This work provides the first example of the identification of genes involved in the anaerobic degradation of aromatic compounds by heterologous expression of a cosmid library in a phylogenetically related organism.

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

“…Consistent with this, the bqdhLSM gene products show similarity to the E1, E2, and E3 components of multicomponent enzymes, such as the pyruvate dehydrogenase, and they have been proposed to be involved in the ring cleavage reaction of HBQ in A. anaerobius (Fig. 5) (77). Although a tentative pathway for anaerobic resorcinol degradation in A. anaerobius has been postulated based on the putative function of the key gene products (Fig.…”

“…5). Whereas the btdhL gene product is similar to putative ␤-hydroxy acid dehydrogenases, the btdhS gene product seems to be a membrane protein, which would explain why the HHQ dehydrogenase activity was measured in the membrane fraction (77). The further conversion of HBQ to acetate and malate requires the splitting of two C-C bonds and releases two electrons.…”

“…As in the case of the transhydroxylase, the RhL and RhS subunits contain putative binding sites for two molybdopterin guanine dinucleotide cofactors and two iron-sulfur clusters, respectively (77). Although resorcinol hydroxylase is predicted to be soluble from the amino acid sequence, it was measured almost exclusively in the membranes of A. anaerobius cells and T. aromatica transconjugants, suggesting the existence of an asyet-unknown membrane anchor in its molecule (77).…”

“…The best match for the rhL and rhS gene products are the large and small subunits of the heterodimeric pyrogallolphloroglucinol transhydroxylase from Pelobacter acidigallici, a member of the dimethyl sulfoxide reductase family (244). As in the case of the transhydroxylase, the RhL and RhS subunits contain putative binding sites for two molybdopterin guanine dinucleotide cofactors and two iron-sulfur clusters, respectively (77). Although resorcinol hydroxylase is predicted to be soluble from the amino acid sequence, it was measured almost exclusively in the membranes of A. anaerobius cells and T. aromatica transconjugants, suggesting the existence of an asyet-unknown membrane anchor in its molecule (77).…”

“…This 30-kb gene cluster is organized into three operon-like units (Fig. 5) The mapping of transposon insertions in cosmid R ϩ derivatives of those mutants impaired in resorcinol utilization revealed that eight genes, rhLS, bqdhLS, btdhLS, orf13, and orf14, encode enzymes specific for resorcinol degradation in A. anaerobius (77). The rhL and rhS genes encode the large and small subunits of the resorcinol hydroxylase, respectively (Fig.…”