Gérard Giordano

A novel Sec‐independent periplasmic protein translocation pathway in Escherichia coli

The trimethylamine N‐oxide (TMAO) reductase of Escherichia coli is a soluble periplasmic molybdoenzyme. The precursor of this enzyme possesses a cleavable N‐terminal signal sequence which contains a twin‐arginine motif. By using various moa, mob and mod mutants defective in different steps of molybdocofactor biosynthesis, we demonstrate that acquisition of the molybdocofactor in the cytoplasm is a prerequisite for the translocation of the TMAO reductase. The activation and translocation of the TMAO reductase precursor are post‐translational processes, and activation is dissociable from translocation. The export of the TMAO reductase is driven mainly by the proton motive force, whereas sodium azide exhibits a limited effect on the export. The most intriguing observation is that translocation of the TMAO reductase across the cytoplasmic membrane is independent of the SecY, SecE, SecA and SecB proteins. Depletion of Ffh, a core component of the signal recognition particle of E.coli, appears to have a slight effect on the export of the TMAO reductase. These results strongly suggest that the translocation of the molybdoenzyme TMAO reductase into the periplasm uses a mechanism fundamentally different from general protein translocation.

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.

Tellurite reductase activity of nitrate reductase is responsible for the basal resistance of Escherichia coli to tellurite

Tellurite and selenate reductase activities were identified in extracts of Escherichia coli. These activities were detected on non-denaturing polyacrylamide gels using an in situ methyl viologen activity-staining technique. The activity bands produced from membrane-protein extracts had the same RF values as those of nitrate reductases (NRs) A and Z. Tellurite and selenate reductase activities were absent from membranes obtained from mutants deleted in NRs A and Z. Further evidence of the tellurite and selenate reductase activities of NR was demonstrated using rocket immunoelectrophoresis analysis, where the tellurite and selenate reductase activities corresponded to the precipitation arc of NR. Additionally, hypersensitivity to potassium tellurite was observed under aerobic growth conditions in nar mutants. The tac promoter expression of NR A resulted in elevated tellurite resistance. The data obtained also imply that a minimal threshold level of NR A is required to increase resistance. Under anaerobic growth conditions additional tellurite reductase activity was identified in the soluble fraction on non-denaturing gels. Nitrate reductase mutants were not hypersensitive under anaerobic conditions, possibly due to the presence of this additional reductase activity.

Involvement of the narJ and mob gene products in distinct steps in the biosynthesis of the molybdoenzyme nitrate reductase in Escherichia coli

The Escherichia coli mob locus is required for synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide. The mobB gene is not essential for molybdenum cofactor biosynthesis because a deletion of both mob genes can be fully complemented by just mobA. Inactive nitrate reductase, purified from a mob strain, can be activated in vitro by incubation with protein FA (the mobA gene product), GTP, MgCl2, and a further protein fraction, factor X. Factor X activity is present in strains that lack MobB, indicating that it is not an essential component of factor X, but over‐expression of MobB increases the level of factor X. MobB, therefore, can participate in nitrate reductase activation. The narJ protein is not a component of mature nitrate reductase but narJ mutants cannot express active nitrate reductase A. Extracts from narJ strains are unable to support the in vitro activation of purified mob nitrate reductase: they lack factor X activity. Although the mob gene products are necessary for the biosynthesis of all E. coli molybdoenzymes as a result of their requirement for molybdopterin guanine dinucleotide, NarJ action is specific for nitrate reductase A. The inactive nitrate reductase A derivative in a narJ strain can be activated in vitro following incubation with cell extracts containing the narJ protein. NarJ acts to activate nitrate reductase after molybdenum cofactor biosynthesis is complete.

NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli

The formation of active membrane‐bound nitrate reductase A in Escherichia coli requires the presence of three subunits, NarG, NarH and NarI, as well as a fourth protein, NarJ, that is not part of the active nitrate reductase. In narJ strains, both NarG and NarH subunits are associated in an unstable and inactive NarGH complex. A significant activation of this complex was observed in vitro after adding purified NarJ‐6His polypeptide to the cell supernatant of a narJ strain. Once the apo‐enzyme NarGHI of a narJ mutant has become anchored to the membrane via the NarI subunit, it cannot be reactivated by NarJ in vitro. NarJ protein specifically recognizes the catalytic NarG subunit. Fluorescence, electron paramagnetic resonance (EPR) spectroscopy and molybdenum quantification based on inductively coupled plasma emission spectroscopy (ICPES) clearly indicate that, in the absence of NarJ, no molybdenum cofactor is present in the NarGH complex. We propose that NarJ is a specific chaperone that binds to NarG and may thus keep it in an appropriate competent‐open conformation for the molybdenum cofactor insertion to occur, resulting in a catalytically active enzyme. Upon insertion of the molybdenum cofactor into the apo‐nitrate reductase, NarJ is then dissociated from the activated enzyme.

TorD, A Cytoplasmic Chaperone That Interacts with the Unfolded Trimethylamine N-Oxide Reductase Enzyme (TorA) in Escherichia coli

Reduction of trimethylamine N-oxide (TMAO) in Escherichia coli involves the terminal molybdoreductase TorA, located in the periplasm, and the membrane anchored c type cytochrome TorC. In this study, the role of the TorD protein, encoded by the third gene of torCADoperon, is investigated. Construction of a mutant, in which thetorD gene is interrupted, showed that the absence of TorD protein leads to a two times decrease of the final amount of TorA enzyme. However, specific activity and biochemical properties of TorA enzyme were similar to those of the enzyme produced in the wild type. Excess of TorD protein restores the normal level of TorA enzyme, and also, leads to the appearance of a new cytoplasmic form of TorA on SDS-polyacrylamide gel electrophoresis using gentle conditions. This probably indicates a new folding state of the cytoplasmic TorA protein when TorD is overexpressed. BIAcore techniques demonstrated direct specific interaction between the TorA and TorD proteins. This interaction was enhanced when TorA was previously unfolded by heating. Finally, as TorA is a molybdoenzyme, we demonstrated that TorD can interact with TorA before the molybdenum cofactor has been inserted. As TorD homologue encoding genes are found in various TMAO reductase loci, we propose that TorD is a chaperone protein specific for the TorA enzyme. It belongs to a family of TorD-like chaperones present in several bacteria, and, probably, involved in TMAO reductase folding.

Molecular genetic analysis of the moa operon of Escherichia coli K-12 required for molybdenum cofactor biosynthesis

A 3.2 kb chromosomal DNA fragment which complements the defects in a series of twelve moa::Mucts insertion mutants has been sequenced. Five open reading frames (ORFs) were identified and these are arranged in a manner consistent with their forming an operon. The encoded proteins (MoaA‐MoaE) have predicted molecular weights of 37346, 18665, 17234, 8843 and 16981 respectively. Examination of subclones of the whole locus in an expression system demonstrated the predicted products. N‐terminal amino acid sequences for the moa A, B, C and E products confirmed the translational starts. Genetic analysis distinguished four classes of moa mutants corresponding to genes moaA, C, D and E. Potential promoter sequences upstream of moaA and a possible transcription termination signal have been identified. Genetic analysis of the chlA1 and chlM mutants, which have been biochemically characterized as defective in molybdopterin biosynthesis, indicates that these carry lesions in moaA and moaD respectively. The moa locus is orientated clockwise at 17.7 minutes in the chromosome.

The coordination and function of the redox centres of the membrane-bound nitrate reductases

Abstract. Under anaerobic conditions and in the presence of nitrate, the facultative anaerobe Escherichia coli synthesises an electron-transport chain comprising a primary dehydrogenase and the terminal membrane-bound nitrate reductase A (NarGHI). This review focuses on recent advances obtained on the structure and function of the three protein subunits of membrane-bound nitrate reductases. We discuss a global architecture for the Mo-bisMGD-containing subunit (NarG) and a coordination model for the four [Fe–S] centres of the electron-transfer subunit (NarH) and for the two b-type haems of the anchor subunit NarI.

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.