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J. Bacteriol. doi:10.1128/JB.01729-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Heterologous expression and identification of the genes involved in anaerobic degradation of 1,3-dihydroxybenzene (resorcinol) in Azoarcus anaerobius

Universität Konstanz, Fachbereich Biologie, D-78457 Konstanz, Germany; Estación Experimental del Zaidín, C/. Profesor Albareda 1, E-18008 Granada, Spain

^* To whom correspondence should be addressed. Email: bodo.philipp{at}uni-konstanz.de.

	Abstract

Azoarcus anaerobius, a strictly anaerobic gram-negative bacterium, utilizes resorcinol as sole carbon and energy source with nitrate as 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 formation of hydroxyhydroquinone (HHQ, 1,2,4-benzenetriol, BT) and 2-hydroxy-1,4-benzoquinone (HBQ). This study presents the evidence for the further degradation of HBQ in cell free extracts to form acetic and malic acid. To identify A. anaerobius genes required for anaerobic resorcinol catabolism, a cosmid library with genomic DNA was constructed and transformed into the phylogenetically related Thauera aromatica that 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 twenty-two putative genes. In cell-free extracts of T. aromatica transconjugants, resorcinol was degraded to HHQ, HBQ, and acetate, suggesting that cosmid R⁺ carried all genes necessary for resorcinol degradation. Based on physiological characterization of T. aromatica transconjugants carrying transposon insertions in different genes of cosmid R⁺, eight ORFs were found to be essential for resorcinol mineralization. Resorcinol hydroxylase coding genes were assigned based on sequence analysis and enzyme assays with two mutants. Putative genes for hydroxyhydroquinone dehydrogenase and enzymes involved in ring fission have been also proposed. This work provides the first example of identifiying genes involved in anaerobic degradation of aromatic compounds by heterologous expression of a cosmid library in a phylogenetically related organism.