Desulfobacterium autotrophicum HRM2 is a metabolically versatile sulfate-reducing bacterium, capable of heterotrophic (e.g. with organic acids and alcohols) and chemolithoautotrophic growth (with H(2)/CO(2)). It employs the Wood-Ljungdahl pathway for complete oxidation of acetyl-CoA to CO(2) and for CO(2) fixation. Here, we investigated substrate-dependent regulation at different levels of anaerobic carbon catabolism in this bacterium. (a) Whole-cell adaptation studies indicated an inducibleutilization of short-chained alcohols, agreeing with a substrate-specific abundance increase (up to 40-fold) of alcohol dehydrogenase Adh4. Simultaneous utilization of lactate and 1-propanol was paralleled by adh4 expression and Adh4 formation, respectively. (b) Degradation of propionate generally involves methylmalonyl-CoA mutase (Sbm). Expression of sbm was upregulated during growth with 1-propanol, but not with a mixture of lactate and 1-propanol. Correspondingly, propionate was excreted during growth with this substrate mixture. (c) CO dehydrogenase, the key enzyme of the Wood-Ljungdahl pathway, is encoded by several genes (cdhC, cdh1 and cdh2) located at different genomic positions. Expression of all of these genes during heterotrophic and autotrophic growth points to a reversible operation of the Wood-Ljungdahl pathway. In summary, the different regulatory patterns displayed by Db. autotrophicum HRM2 at the tested metabolic levels point to a multi-layered regulatory network.