Occurrence and diversity of the oxidative hydroxyhydroquinone pathway for the anaerobic degradation of aromatic compounds in nitrate-reducing bacteria

The nitrate-reducing betaproteobacteria Azoarcus anaerobius and Thauera aromatica AR-1 use an oxidative mechanism to anaerobically degrade resorcinol and 3,5-dihydroxybenzoate (3,5-DHB), respectively, rendering hydroxyhydroquinone as intermediate. The first pathway step is performed by a dimethylsulphoxide-reductase family hydroxylase. The gene cluster coding for the pathway is homologous in these strains. Only these two Rhodocyclales are known to follow this anaerobic pathway, and nothing is known about its distribution in prokaryotes. To determine the relevance and diversity of this strategy in nature, we enriched for bacteria able to oxidize resorcinol or 3,5-DHB under denitrifying conditions. Nitrate-reducing bacteria able to degrade these compounds were present in soil, aquifer and marine sediments. We were able to isolate a number of strains with this capacity from soil and aquifer samples. Amplicon libraries of rehL, the gene encoding the first step of this pathway, showed an overall low diversity, most sequences clustering with either pathway enzyme. Isolates belonging to the Beta- and Gammaproteobacteria able to grow on these substrates revealed rehL homologues only in strains belonging to Thauera and Azoarcus. Analysis of sequenced genomes in the databases detected the presence of highly similar clusters in two additional betaproteobacteria and in the gammaproteobacterium Sedimenticola selenatireducens, although anaerobic growth on a dihydroxyaromatic could only be confirmed in Thauera chlorobenzoica 3CB-1. The presence of mobile elements in the flanking sequences of some of the clusters suggested events of horizontal gene transfer, probably contributing to expand the pathway to a broader host range within the Proteobacteria.