Marc Chippaux

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.

Nitrate reductase of Escherichia coli: Completion of the nucleotide sequence of the nar operon and reassessment of the role of the α and β subunits in iron binding and electron transfer

SummaryThe nucleotide sequence of the narGHJI operon that encodes the nitrate reductase of Escherichia coli was completed. It encodes four polypeptides NarG, NarH, NarJ and NarI of molecular weight 138.7, 57.7, 26.5 and 25.5 kDa, respectively. The analysis of deduced amino acid sequence failed to reveal any structure capable of binding iron within the NarG polypeptide. In contrast, cysteine arrangements typical of iron-sulfur centers were found in the NarH polypeptide. This suggested that the latter is an electron transfer unit of the nitrate reductase complex. Such a view is opposite to the current description of the nitrate reductase. The findings allowed us to propose a model for the electron transfer steps that occur during nitrate reduction. The NarG polypeptide was found to display a high degree of homology with numerous E. coli molybdoproteins. Moreover, the same genetic and functional organizations as well as the presence of highly conserved stretches of amino acids were noted between both NarG/NarH and DmsA/DmsB (encoding the dimethyl sulfoxide reductase) pairs.

Nitrate reductases of Escherichia coli: sequence of the second nitrate reductase and comparison with that encoded by the narGHJI operon.

SummaryThe structural genes for NRZ, the second nitrate reductase of Escherichia coli, have been sequenced. They are organized in a transcription unit, narZYWV, encoding four subunits, NarZ, NarY, NarW and NarV. The transcription unit is homologous (73% identity) to the narGHJI operon which encodes the genes for NRA, the better characterized nitrate reductase of this organism. The level of homology between the corresponding polypeptides ranges from 69% for the NarW/NarJ pair to 86% for the NarV/ Narl pair. The NarZ polypeptide contains the five conserved regions present in all other known molybdoproteins of E. coli and their relative order is the same. The NarY polypeptide, which contains the same four cysteine clusters in the same order as NarH, is probably an electron transfer unit of the complex. Upstream of narZ, an open reading frame, ORFA, is present which could encode a product which has homology (73% identity) with the COON-terminal end of NarK. The ORFA-narZ intergenic region, however, is about 80 nucleotides long and does not contain the cis-acting elements, NarL and Fnr boxes, nor the terC4 terminator sequence present in the 500 nucleotide narK-narG intergenic region. This might explain why the nar-ZYWV and the narGHJI operons are regulated differently. Our results tend to support the hypothesis that a DNA fragment larger than that encompassing the narGHJI genes has been duplicated.

Periplasmic disulphide bond formation is essential for cellulase secretion by the plant pathogen Erwinia chrysanthemi

Secretion to the cell exterior of cellulase EGZ and of at least six pectinases enables the Gram‐negative Erwinia chrysanthemi to cause severe plant disease. The C‐terminal cellulose‐binding domain (CBD) of EGZ was found to contain a disulphide bond which forms, in the periplasm, between residues Cys‐325 and Cys‐382. Dithiothreitol (DTT)‐treatment of native EGZ showed that the disulphide bond was dispensable, both for catalysis and cellulose binding. Adding DTT to E. chrysanthemi cultures led to immediate arrest of secretion of EGZ which accumulated in the periplasm where the CBD was eventually proteolysed. Site‐directed mutagenesis that affected Cys residues involved in disulphide bond formation resulted in molecules that were catalytically active and able to bind to cellulose but were no longer secreted, Instead they accumulated in the periplasm. Interestingly, the region around EGZ Cys‐325 is conserved in two pectinases secreted by the same pathway as EGZ. We conclude that the conserved Cys, and possibly adjacent residues, bear essential information for EGZ to be secreted and that periplasmic disulphide bond formation is an obligatory step which provides a pre‐folded functional form of EGZ with secretion competence.

Involvement of the narJ or narW gene product in the formation of active nitrate reductase in Escherichia coli.

Two membrane‐bound nitrate reductases, NRA and NRZ, exist in Escherichia coil. Both isoenzymes are composed of three structural subunits, α, β and γ encoded by narG/narZ, narH/narY and narl/narV, respectively. The genes are in transcription units which also contain a fourth gene encoding a polypeptide, δ, which is not part of the final enzyme. A strain which is devoid of, or does not express, the nar genes, was used to investigate the role of the δ and γ polypeptides in the formation and/or processing of the nitrate reductase. When only the α and γ polypeptides are produced, an (αβ) complex exists which is inactive and soluble. When the α, β and δ and polypeptides are produced, the (αβ) complex is active with artificial donors such as benzyl viologen but is soluble. When the α, β, and δ polypeptides are produced, the (αβ) complex is inactive but partially binds the membrane. It was concluded that the γ polypeptide is involved in the binding of the (αβ) complex to the membrane while the δ polypeptide is indispensable for the (αβ) nitrate reductase activity. The activation by the δ polypeptide does not seem to involve the insertion of the redox centres of the enzyme since the purified inactive (αβ) complex was shown to contain the four iron–sulphur centres and the molybdenum cofactor, which are normally present in the native purified enzyme. The extreme sensitivity of this inactive complex to thermal denaturation or tryptic treatment favours the idea that the δ polypeptide promotes the correct assembly of the α and β subunits. Although this corresponds to the definition of a chaperone protein this possibility has been rejected. In this study we have also demonstrated that the δ or γ polypeptide encoded by one nar operon can be substituted succesfully for by its respective counterpart from the other nar operon to give an active membrane bound heterologous nitrate reductase enzyme.

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.

An unorthodox sensor protein (TorS) mediates the induction of the tor structural genes in response to trimethylamine N‐oxide in Escherichia coli

We isolated and characterized three spontaneous mutations leading to trimethylamine N‐oxide (TMAO)‐independent expression of the tor operon encoding the TMAO‐reductase anaerobic respiratory system in Escherichia coli. The mutations lie in a new tor regulatory gene, the torS gene, which probably encodes a sensor protein of a two‐component regulatory system. One mutation, which leads to full TMAO‐constitutive expression, is a 3‐amino‐acid deletion within the potential N‐terminal periplasmic region, suggesting that this region contains the TMAO‐detector site. For the other two mutations, a further induction of the tor operon is observed when TMAO is added. Both are single substitutions and affect the linker region located between the detector and the conserved transmitter domains. Thus, as proposed for other sensors, the TorS linker region might play an essential role in propagating conformational changes between the detector and the cytoplasmic signalling regions. The TorR histidine kinase is an unorthodox sensor that contains a receiver and a C‐terminal alternative transmitter domain in addition to the domains found in most sensors. Previously, we showed that TMAO induction of the tor operon requires the TorR response regulator and the TorT periplasmic protein. Additional genetic data confirm that torS encodes the sensor partner of TorR and TorT. First, insertion within torS abolishes tor operon expression whatever the growth conditions. Second, overexpressed TorR bypasses the requirement for torS, whereas the torT gene product is dispensable for tor operon expression in a torS constitutive mutant. This supports a signal‐transduction cascade from TorT to TorR via TorS.

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.

Resistance to Bacitracin in Bacillus subtilis: Unexpected Requirement of the BceAB ABC Transporter in the Control of Expression of Its Own Structural Genes

The Bacillus subtilis BceAB ABC transporter involved in a defense mechanism against bacitracin is composed of a membrane-spanning domain and a nucleotide-binding domain. Induction of the structural bceAB genes requires the BceR response regulator and the BceS histidine kinase of a signal transduction system. However, despite the presence of such a transduction system and of bacitracin, no transcription from an unaltered bceA promoter is observed in cells lacking the BceAB transporter. Expression in trans of the BceAB transporter in these bceAB cells restores the transcription from the bceA promoter. Cells possessing a mutated nucleotide-binding domain of the transporter are also no longer able to trigger transcription from the bceA promoter in the presence of bacitracin, although the mutated ABC transporter is still bound to the membrane. In these cells, expression of the bceA promoter can no longer be detected, indicating that the ABC transporter not only must be present in the cell membrane, but also must be expressed in a native form for the induction of the bceAB genes. Several hypotheses are discussed to explain the simultaneous need for bacitracin, a native signal transduction system, and an active BceAB ABC transporter to trigger transcription from the bceA promoter.