Marie-Claire Pascal

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.

A mutation leading to the total lack of nitrite reductase activity in Escherichia coli K 12

SummaryMutants of E. coli, completely devoid of nitrite reductase activity with glucose or formate as donor were studied. Biochemical analysis indicates that they are simultaneously affected in nitrate reductase, nitrite reductase, fumarate reductase and hydrogenase activities as well as in cytochrome c552 biosynthesis. The use of an antiserum specific for nitrate reductase shows that the nitrate reductase protein is probably missing. A single mutation is responsible for this phenotype: the gene affected, nir R, is located close to tyr R i.e. at 29 min on the chromosomal map.

The inducible trimethylamine N-oxide reductase of Escherichia coli K12: its localization and inducers

We used an anti-trimethylamine-N-oxide reductase (EC 1.6.6.9) serum and different immunological techniques (Ouchterlony, rocket immunoelectrophoresis, immunoblotting) to show that dimethylsulphoxide (DMSO), tetrahydrothiophene 1-oxide (THTO) and pyridine N-oxide (PNO) were effective inducers of the inducible form of trimethylamine N-oxide reductase. We confirmed this genetically and biochemically using a strain in which phage MudII 1734 carrying lacZ was inserted into torA, the structural gene for inducible trimethylamine-N-oxide reductase. By subcellular fractionation and quantitation with rocket immunoelectrophoresis, we showed that the enzyme was principally localized in the periplasmic fraction. Constitutive trimethylamine-N-oxide reductase was localized in the membrane fraction and, like the inducible enzyme showed a broad specificity with respect to various compounds such as DMSO, THTO and PNO. Apart from their immunological properties, the two enzymes could be clearly differentiated by their temperature stability.

Binding of the TorR regulator to cis‐acting direct repeats activates tor operon expression

The expression of the Escherichia coli torCAD operon, which encodes the anaerobically expressed trimethylamine N‐oxide (TMAO) reductase respiratory system, requires the presence of TMAO in the medium. The response regulator, TorR, has recently been identified as the regulatory protein that controls the expression of the torCAD operon in response to TMAO. The torC regulatory region contains four direct repeats of a decameric consensus motif designated the tor boxes. Alteration by base substitutions of any of the four tor boxes in a plasmid containing a torC′‐lacZ fusion dramatically reduces TorR‐dependent torC expression. In addition, deletion of the distal tor box (box1) abolishes torC induction whereas the presence of a DNA fragment starting three bases upstream from box1 suffices for normal torC expression. Footprinting and gel‐retardation experiments unambiguously demonstrated that TorR binds to the torC regulatory region. Three distinct regions are protected by TorR binding. One of approximately 24 nucleotides covers the first two tor boxes (box1 and box2); the second is located upstream from the −35 promoter sequence and includes the third tor box (box3); the last is found downstream from the −35 sequence and corresponds to the fourth tor box (box4). Binding to the upstream tor boxes (box1 and box2) appears to be stronger than binding to the downstream tor boxes (box3 and box4) since only the upstream region is protected at the lower concentration of TorR used in the footprinting experiments.

Operon fusions in the nitrate reductase operon and study of the control gene nir R in Escherichia coli

SummaryStrains carrying operon fusions between the promotor of the chl I gene and the lac structural genes were constructed. From these strains in which the expression of the lac genes is under the control of both nitrate and oxygen, spontaneous regulatory mutants were selected: (i) mutants which synthesize β-galactosidase constitutively in anaerobiosis; (ii) mutants in which β-galactosidase synthesis is no longer repressed by oxygen.Introduction of the nir R mutated allele into strains carrying these fusions resulted in the total loss of β-galactosidase synthesis, confirming that nir R is a regulatory gene controlling the expression of the biosynthesis of the nitrate reductase.

Regulation of the nitrate reductase operon: Effect of mutations in chl A, B, D and E genes

SummaryIntroduction of chlA, B or E mutant alleles into strains carrying fusions between the lac structural genes and the promoter of the nitrate reductase operon led to the partial or total constitutive expression of the fusion. Presence of chlD mutated alleles in the same strains did not result in constitutive expression of the fusion and allowed full induction by nitrate only in the presence of molybdenum. It is proposed that the molybdenum cofactor, Mo-X, of the nitrate reductase is also corepressor of the operon. The chlA, B and E genes would be involved in the biosynthesis of the X-moity. Mutations in these genes would give an altered X-moity which still binds to molybdenum but leads to a less effcient repressor complex; chlD gene would code for an enzyme inserting molybdenum in the X-moity of the cofactor. Mutations in chlD give an empty cofactor leading to a complex which permanently represses the operon unless molybdenum is added.

Mutants of Escherichia coli K12 with defects in anaerobic pyruvate metabolism.

A strain of Escherichia coli with a mutation in the ana gene was shown to lack acetaldehyde dehydrogenase and alcohol dehydrogenase. The requirement of this strain for an external oxidant to grow anaerobically on glucose shows that the reduction of acetyl-CoA is the principal means of reoxidation of NADH produced during glycolysis in E. coli. Further mutants derived from the ana strain were shown to be affected in the enzymes involved in the fermentation of pyruvate (pyruvate formate-lyase, phosphotransacetylase, acetate kinase). A gene controlling acetate kinase (ackB) activity has been located at 39 min on the chromosomal map. Evidence is presented that anaerobic nitrite reduction with pyruvate involves at least the dehydrogenase subunit of the pyruvate dehydrogenase complex.

Regulation of the trimethylamine N-oxide (TMAO) reductase in Escherichia coli: analysis of tor::Mud1 operon fusion

SummaryMud1 insertion mutants of Escherichia coli were obtained in which the lac structural genes were fused to the promoter of torA, a gene encoding the trimethylamine N-oxide (TMAO) reductase. Expression of the fusion is induced by TMAO and repressed by oxygen. However, in contrast to the nar operon which codes for the nitrate reductase structural genes, the tor::Mud1 fusion was found to be independent of the positive control exerted by the nirR gene product and not repressed by the molybdenum cofactor. The torA gene which is strongly linked to pyrF (28.3 U) is different from any tor gene already described in E. coli or in Salmonella typhimurium.

A Mutant of Escherichia coli Deficient in Pyruvate Formate Lyase

SummaryA genetic study was performed on a mutation which has caused loss of pyruvate formate lyase. The gene affected is designated pfl and is located close to ser C, i.e. at 20 mn on the chromosomal map of E. coli.

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.