Members of the betaproteobacterial ‘Aromatoleum’/Azoarcus/Thauera cluster contribute to the biodegradation of aromatic and other recalcitrant compounds in anoxic soils and sediments. The metabolically versatile ‘Aromatoleumaromaticum’ strain EbN1 represents a model organism for this cluster, having already been studied on the physiological, proteogenomic and biochemical level. Here we report the development of a genetic system for ‘A. aromaticum’ strain EbN1 enabling unmarked deletion mutagenesis by heterologous recombination and subsequent complementation. The antibiotic sensitivity of strain EbN1 was characterized and optimal conditions for efficient cultivation on solid medium were established. A procedure for introducing foreign DNA into strain EbN1 by conjugation was developed. The effectiveness of the genetic system was demonstrated by unmarked in-frame deletion of ebdC2, encoding the γ-subunit of a paralogous ethylbenzene dehydrogenase.

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