The anaerobic monoterpene metabolism in Castellaniella defragrans 65Phen

The betaproteobacterium Castellaniella defragrans 65Phen belongs to a handful of bacterial strains able to mineralize monoterpenes under anaerobic denitrifying conditions. It utilizes acyclic, monocyclic and bicyclic monoterpenes as sole carbon and energy sources. Whether C. defragrans uses parallel pathways for the degradation of the different monoterpene families or whether the metabolism of all its substrates is centralized in a single pathway was elucidated in this study. Monocyclic monoterpenes were formed from the bicyclic monoterpenes A A /--pinene, 3-carene and sabinene in cell cultures and in in vitro assays using cell lysates. This enzyme activity was stimulated by ATP and the first evidence suggested a membrane association. Transposon mutants and the proteome of bicyclic monoterpene-degrading cells supported that the cyclic monoterpene degradation pathway is used for the degradation of all monoterpenes in C. defragrans. Additional evidence was provided by the in vitro formation of monocyclic monoterpene intermediates from monoterpene alcohols. Here, the in vitro dehydration of A A /--terpineol and terpinen-4-ol was observed for the first time. This dehydratase activity together with a second alcohol dehydratase acting on linalool were partially purified and characterized. An anaerobic limonene dehydrogenase (CtmAB) was purified and characterized from limonene-grown C. defragrans. CtmAB is a heterodimeric enzyme and carries two tightly-bound flavins per dimer. CtmAB catalyzes the reversible anaerobic hydroxylation of limonene to perillyl alcohol. CtmAB reduced ferrocenium ions using several other monocyclic monoterpenes including those formed during acyclic and bicyclic monoterpene degradation. Thus, C. defragrans channels all monocyclic monoterpene dienes with a sp2-hybridized C1 atom via the limonene dehydrogenase into the monocyclic monoterpene degradation pathway for their mineralization. A novel efflux pump of the Resistance-Nodulation Division (RND) superfamily was analyzed to prove its function as a monoterpene detoxification mechanism in C defragrans. The RND transporter encoded by genes ameABCD is likely forming a proton gradient-dependent tetrapartite efflux complex. The deletion of genes ameABCD significantly reduced C. defragransa tolerance towards monoterpenes and increased intracellular accumulation of the fluorescent dye Nile Red. Thus, we suggest that AmeABCD is actively exporting monoterpenes and other non-polar substrates.