The trimethylamine N-oxide (TMAO) respiratory system is subject to a strict positive control by the substrate. This property was exploited in the performance of miniMu replicon-mediated in vivo cloning of the promoter region of gene(s) positively regulated by TMAO. This region, located at 22 min on the chromosome, was shown to control the expression of a transcription unit composed of three open reading frames, designated torC, torA and torD, respectively. The presence of five putative c-type haem-binding sites within the TorC sequence, as well as the specific biochemical characterization, indicated that torC encodes a 43,300 Da c-type cytochrome. The second open reading frame, torA, was identified as the structural gene for TMAO reductase. A comparison of the predicted amino-terminal sequence of the torA gene product to that of the purified TMAO reductase indicated cleavage of a 39 amino acid signal peptide, which is in agreement with the periplasmic location of the enzyme. The predicted TorA protein contains the five molybdenum cofactor-binding motifs found in other molybdoproteins and displays extensive sequence homology with BisC and DmsA proteins. As expected, insertions in torA led to the loss of TMAO reductase. The 22,500 Da polypeptides encoded by the third open reading frame does not share any similarity with proteins listed in data banks.