Michèle Lepelletier

TMAO anaerobic respiration in Escherichia coli: involvement of the tor operon

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 polypeptide encoded by the third open reading frame does not share any similarity with proteins listed in data banks.

The TorR High-Affinity Binding Site Plays a Key Role in Both torR Autoregulation and torCAD Operon Expression in Escherichia coli

In the presence of trimethylamine N-oxide (TMAO), the TorS-TorR two-component regulatory system induces the torCAD operon, which encodes the TMAO respiratory system of Escherichia coli. The sensor protein TorS detects TMAO and transphosphorylates the response regulator TorR which, in turn, activates transcription of torCAD. The torR gene and the torCAD operon are divergently transcribed, and the short torR-torC intergenic region contains four direct repeats (the tor boxes) which proved to be TorR binding sites. The tor box 1-box 2 region covers the torR transcription start site and constitutes a TorR high-affinity binding site, whereas box 3 and box 4 correspond to low-affinity binding sites. By using torR-lacZ operon fusions in different genetic backgrounds, we showed that the torR gene is negatively autoregulated. Surprisingly, TorR autoregulation is TMAO independent and still occurs in a torS mutant. In addition, this negative regulation involves only the TorR high-affinity binding site. Together, these data suggest that phosphorylated as well as unphosphorylated TorR binds the box 1-box 2 region in vivo, thus preventing RNA polymerase from binding to the torR promoter whatever the growth conditions. By changing the spacing between box 2 and box 3, we demonstrated that the DNA motifs of the high- and low-affinity binding sites must be close to each other and located on the same side of the DNA helix to allow induction of the torCAD operon. Thus, prior TorR binding to the box 1-box 2 region seems to allow cooperative binding of phosphorylated TorR to box 3 and box 4.

Rapid Dephosphorylation of the TorR Response Regulator by the TorS Unorthodox Sensor in Escherichia coli

Induction of the torCAD operon, encoding the trimethylamine N-oxide (TMAO) respiratory system, is tightly controlled by the TorS-TorR phosphorelay system in response to TMAO availability. TorS is an unorthodox sensor that contains three phosphorylation sites and transphosphorylates TorR via a four-step phosphorelay, His443-->Asp723-->His850-->Asp(TorR). In this study, we provide genetic evidence that TorS can dephosphorylate phospho-TorR when TMAO is removed. Dephosphorylation probably occurs by a reverse phosphorelay, Asp(TorR)-->His850-->Asp723, since His850 and Asp723 are both essential in this process. By using reverse transcriptase PCR, we also show that TMAO removal results in shutoff of tor operon transcription in less than 2 min. Based on our results and on analogy to other phosphorelay signal transduction systems, we propose that reverse phosphotransfer could be a rapid and efficient mechanism to inactivate response regulators.

Nitrate reductase and cytochrome bnitrate reductase structural genes as parts of the nitrate reductase operon

SummaryThe existence of a nitrate-reductase operon in the tryptophane region was deduced from the effects of prophage insertion in each of chl I and chl C genes and from transposition of the Mu-mediated host DNA fragments on F-prime. This operon appears to be polarized from chlC to chlI and the gene order in the region is trp-chlI-chlC-purB.

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

The trimethylamine N‐oxide (TMAO) anaerobic respiratory system of Escherichia coli comprises a periplasmic terminal TMAO reductase (TorA) and a pentahaem c‐type cytochrome (TorC), which is involved in electron transfer to TorA. The structural proteins are encoded by the torCAD operon whose expression is induced in the presence of TMAO through the TorS/TorR two‐component system. By using a genomic library cloned into a multicopy plasmid, we identified 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 cytochrome. This led us to propose that, after translocation, TorC apocytochrome downregulates the tor 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 tor operon expression. Dithiothreitol (DTT), which reduces disulphide bonds and thus prevents the first step in c‐type cytochrome formation, also strongly decreases the tor promoter activity. The DTT effect is TorC dependent, as it is abolished when torC is disrupted. In contrast, overexpression of the c‐type cytochrome maturation (ccm ) genes relieved the tor operon of the negative control and allowed the bacteria to produce a higher amount of TorC holocytochrome. Therefore, the TorC negative autoregulation probably means that maturation of the c‐type cytochrome is a limiting step for Tor system biogenesis. Genetic experiments have provided evidence that TorC control is mediated by the TorS/TorR two‐component system and different from the tor anaerobic control. In our working model, TMAO and apoTorC bind to the periplasmic side of TorS, but TMAO activates TorS autophosphorylation, whereas apoTorC inhibits the TorS kinase activity.

Genetic analysis of mutants of Escherichia coli K12 and Salmonella typhimurium LT2 deficient in hydrogenase activity

SummaryA genetic, study of mutants deficient in hydrogenase activity was performed. In E. coli, the affected gene (hyd) is located at 51 min, between cys C and nal B; in S. typhimurium, it probably lies in the homologous region of the chromosome.

Tn10 insertions directed in the pyr D-ser C region and improved mapping of pep N in Escherichia coli K12

SummaryTn10 transposons were intergrated near ser C at 20 units on the chromosomal map of E. coli; pep N was mapped close to ser C between this gene and pyr D, one minute away from its previously assumed position.

Nitrite reduction in Escherichia coli: Genetic analysis of nir mutants

SummaryFive mutants of Escherichia coli impaired on nitrite reduction were studied. All have lost NADH-nitrite reductase activity but have retained the capacity to synthesize all or part of their cytochrome c552. Three genes, nir C, nir D, and nir E were mapped at 26, 72.5 and 49.5 min, respectively. Another gene, nir F was tentatively localized around 52 min.

Genetic analysis of mutants of Salmonella typhimurium deficient in formate dehydrogenase activity

SummaryA genetical study of mutants of Salmonella typhimurium deficient in formate dehydrogenase activity was performed. The affected gene was designated fdh A and mapped at 116 min, the order of genes in that region being xyl-fdh A-mtl-cys E.