TorC apocytochrome negatively autoregulates the trimethylamine N-oxide (TMAO) reductase operon in Escherichia coli

The trimethylamine N-oxide (TMAO) anaerobic respiratory system of Escherich ia coil comprises a periplasmic terminal TMAO reductase (TorA) and a pentah aem c-type cytochrome (TorC), which is involved in electron transfer to Tor A, The structural proteins are encoded by the torCAD operon whose expressio n is induced in the presence of TMAO through the TorS/TorR two-component sy stem. By using a genomic library cloned into a multicopy plasmid, we identi fied TorC as a possible negative regulator of the tor operon, Interestingly , in trans overexpression of torC not only decreased the activity of a torA '-'lacZ fusion, but also dramatically reduced the amount of mature TorC cyt ochrome, This led us to propose that, after translocation, TorC apocytochro me downregulates the for operon unless it is properly matured, In agreement with this hypothesis, we have shown that mini-Tn10 insertions within genes involved in the c-type cytochrome maturation pathway or haem biosynthesis decreased for operon expression, Dithiothreitol (DTT), which reduces disulp hide bonds and thus prevents the first step in c-type cytochrome formation, also strongly decreases the for promoter activity, The DTT effect is TorC dependent, as it is abolished when torC is disrupted, In contrast, overexpr ession of the c-type cytochrome maturation (ccm) genes relieved the for ope ron of the negative control and allowed the bacteria to produce a higher am ount of TorC holocytochrome, Therefore, the TorC negative autoregulation pr obably means that maturation of the c-type cytochrome is a limiting step fo r Tor system biogenesis. Genetic experiments have provided evidence that To rC control is mediated by the TorS/TorR two-component system and different from the for anaerobic control. In our working model, TMAO and apoTorC bind to the periplasmic side of TorS, but TMAO activates TorS autophosphorylati on, whereas apoTorC inhibits the TorS kinase activity.