Electron transfer and binding of the c-type cytochrome TorC to the trimethylamine N-oxide reductase in Escherichia coli.

Reduction of trimethylamine N-oxide (E'(0(TMAO/TMA)) = +130 mV) in Escherichia coli is carried out by the Tor system, an electron transfer chain encoded by the torCAD operon and made up of the periplasmic terminal reductase TorA and the membrane-anchored pentahemic c-type cytochrome TorC. Although the role of TorA in the reduction of trimethylamine N-oxide (TMAO) has been clearly established, no direct evidence for TorC involvement has been presented. TorC belongs to the NirT/NapC c-type cytochrome family based on homologies of its N-terminal tetrahemic domain (TorC(N)) to the cytochromes of this family, but TorC contains a C-terminal extension (TorC(C)) with an additional heme-binding site. In this study, we show that both domains are required for the anaerobic bacterial growth with TMAO. The intact TorC protein and its two domains, TorC(N) and TorC(C), were produced independently and purified for a biochemical characterization. The reduced form of TorC exhibited visible absorption maxima at 552, 523, and 417 nm. Mediated redox potentiometry of the heme centers of the purified components identified two negative midpoint potentials (-177 and -98 mV) localized in the tetrahemic TorC(N) and one positive midpoint potential (+120 mV) in the monohemic TorC(C). In agreement with these values, the in vitro reconstitution of electron transfer between TorC, TorC(N), or TorC(C) and TorA showed that only TorC and TorC(C) were capable of electron transfer to TorA. Surprisingly, interaction studies revealed that only TorC and TorC(N) strongly bind TorA. Therefore, TorC(C) directly transfers electrons to TorA, whereas TorC(N), which probably receives electrons from the menaquinone pool, is involved in both the electron transfer to TorC(C) and the binding to TorA.

The torYZ (yecK bisZ) operon encodes a third respiratory trimethylamine N-oxide reductase in Escherichia coli.

The bisZ gene of Escherichia coli was previously described as encoding a minor biotin sulfoxide (BSO) reductase in addition to the main cytoplasmic BSO reductase, BisC. In this study, bisZ has been renamed torZ based on the findings that (i) the torZ gene product, TorZ, is able to reduce trimethylamine N-oxide (TMAO) more efficiently than BSO; (ii) although TorZ is more homologous to BisC than to the TMAO reductase TorA (63 and 42% identity, respectively), it is located mainly in the periplasm as is TorA; (iii) torZ belongs to the torYZ operon, and the first gene, torY (formerly yecK), encodes a pentahemic c-type cytochrome homologous to the TorC cytochrome of the TorCAD respiratory system. Furthermore, the torYZ operon encodes a third TMAO respiratory system, with catalytic properties that are clearly different from those of the TorCAD and the DmsABC systems. The torYZ and the torCAD operons may have diverged from a common ancestor, but, surprisingly, no torD homologue is found in the sequences around torYZ. Moreover, the torYZ operon is expressed at very low levels under the conditions tested, and, in contrast to torCAD, it is not induced by TMAO or dimethyl sulfoxide.

Molecular analysis of the trimethylamine N-oxide (TMAO) reductase respiratory system from a Shewanella species.

Trimethylamine N-oxide (TMAO) is an abundant compound of tissues of marine fish and invertebrates. During fish spoilage, certain marine bacteria can reduce TMAO to nauseous trimethylamine (TMA). One such bacterium has been isolated and identified as a new Shewanella species, and called Shewanella massilia. The anaerobic growth of S. massilia is greatly increased when TMAO is added, indicating that TMAO reduction involves a respiratory pathway. The TorA enzyme responsible for TMAO reduction is a molybdenum cofactor-containing protein of 90 kDa located in the periplasm. Whereas TorA is induced by both TMAO and dimethylsulfoxide (DMSO), this enzyme has a high substrate specificity and appears to only efficiently reduce TMAO as a natural compound. The structural torA gene encoding the TMAO reductase (TorA) and its flanking regions were amplified using PCR techniques. The torA gene is the third gene of a TMAO-inducible operon (torECAD) encoding the TMAO respiratory components. The torC gene, located upstream from torA encodes a pentahemic c-type cytochrome, likely to be involved in electron transfer to the TorA terminal reductase. TorC was shown to be anchored to the membrane and, like TorA, is induced by TMAO. Except for the TorE protein, which is encoded by the first gene of the torECAD operon, all the tor gene products are homologous to proteins found in the TMAO/DMSO reductase systems from Escherichia coli and Rhodobacter species. In addition, the genetic organization of these systems is similar. Although these bacteria are found in different ecological niches, their respiratory systems appear to be phylogenetically related, suggesting that they come from a common ancestor.

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 torCAD operon, is investigated. Construction of a mutant, in which the torD 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.

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.

High substrate specificity and induction characteristics of trimethylamine-N-oxide reductase of Escherichia coli.

Using a wide variety of N- and S-oxide compounds we have shown by kinetic analysis that only two N-oxides, trimethylamine-N-oxide and 4-methylmorpholine-N-oxide, can be considered good substrates for trimethylamine-N-oxide (TMAO) reductase on the basis of their kcat/Km ratio. This result demonstrates that TMAO reductase possesses a high substrate specificity. Induction of the torCAD operon using the same S- and N-oxide compounds was also analyzed. We demonstrate that there is no correlation between the ability for a compound to be reduced by TMAO reductase and to induce TMAO reductase synthesis.

The mob locus of Escherichia coli K12 required for molybdenum cofactor biosynthesis is expressed at very low levels.

The mob locus of Escherichia coli encodes functions which catalyse the synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide, from molybdopterin and GTP. Reporter translational lac fusion mutations in the mobA gene have been constructed using lambda placMu9 mutagenesis. The mob locus is expressed at very low levels under both aerobic and anaerobic growth conditions. Neither additions to the growth media (nitrate, tungstate or molybdate) nor secondary mutations at the moa, mob, mod, moe or mog loci affected the level of expression. Two transcription initiation sites and their associated promoter regions have been identified upstream of mobA. Both of the promoter regions show a poor match to the -35 and -10 consensus sequences for sigma 70 promoters. A 2.2 kb chromosomal DNA fragment which complemented all available mob mutants has been sequenced. Two ORFs were identified, arranged as a single transcription unit. The encoded polypeptides have predicted molecular masses of 21642 Da and 19362 Da, respectively. The DNA has been subcloned into a T7 overexpression system and the predicted products identified. The mobA gene encodes protein FA, which has been purified to homogeneity and brings about the activation of inactive molybdoenzymes in cell extracts of mob mutants. The mobB gene encodes a polypeptide with a putative nucleotide binding site. All available mob mutations which have been selected for by their ability to grow anaerobically in the presence of chlorate are located in the mobA gene.

A reassessment of the range of c-type cytochromes synthesized by Escherichia coli K-12.

Five different c-type cytochromes have been detected during anaerobic growth of various Escherichia coli strains in different media. None of these cytochromes was detectable in aerobically-grown cultures. Only a single, 43 kDa cytochrome was synthesized in response to the presence of trimethylamine-N-oxide: synthesis of this cytochrome was unaffected by the presence of nitrate or nitrite, was repressed by oxygen, but was dependent upon a functional tor operon located at minute 22 (coordinate 1070 kb) on the E. coli chromosome. The other four cytochromes, masses 16, 18, 24 and 50 kDa, were induced by nitrite coordinately with formate-dependent nitrite reductase activity, but repressed by oxygen and nitrate. As only the 18 kDa and 50 kDa cytochromes are encoded by the nrf operon located at minute 92 (coordinate 4366 kb), there must be other loci, possibly essential for formate-dependent nitrite reduction, encoding the 16 kDa and 24 kDa cytochromes. No other c-type cytochrome was detected under any growth condition tested.

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

Molybdoenzyme biosynthesis in Escherichia coli: in vitro activation of purified nitrate reductase from a chlB mutant.

All molybdoenzyme activities are absent in chlB mutants because of their inability to synthesize molybdopterin guanine dinucleotide, which together with molybdate constitutes the molybdenum cofactor in Escherichia coli. The chlB mutants are able to synthesize molybdopterin. We have previously shown that the inactive nitrate reductase present in a chlB mutant can be activated in a process requiring protein FA and a heat-stable low-molecular-weight substance. We show here that purified nitrate reductase from the soluble fraction of a chlB mutant can be partially activated in a process that requires protein FA, GTP, and an additional protein termed factor X. It appears that the molybdopterin present in the nitrate reductase of a chlB mutant is converted to molybdopterin guanine dinucleotide during activation. The activation is absolutely dependent upon both protein FA and factor X. Factor X activity is present in chlA, chlB, chlE, and chlG mutants.

Purification and further characterization of the second nitrate reductase of Escherichia coli K12.

Two nitrate reductases, nitrate reductase A and nitrate reductase Z, exist in Escherichia coli. The nitrate reductase Z enzyme has been purified from the membrane fraction of a strain which is deleted for the operon encoding the nitrate reductase A enzyme and which harbours a multicopy plasmid carrying the nitrate reductase Z structural genes; it was purified 219 times with a yield of about 11%. It is an Mr-230,000 complex containing 13 atoms iron and 12 atoms labile sulfur/molecule. The presence of a molybdopterin cofactor in the nitrate reductase Z complex was demonstrated by reconstitution experiments of the molybdenum-cofactor-deficient NADPH-dependent nitrate reductase activity from a Neurospora crassa nit-1 mutant and by fluorescence emission and excitation spectra of stable derivatives of molybdoterin extracted from the purified enzyme. Both nitrate reductases share common properties such as relative molecular mass, subunit composition and electron donors and acceptors. Nevertheless, they diverge by two properties: their electrophoretic migrations are very different (RF of 0.38 for nitrate reductase Z versus 0.23 for nitrate reductase A), as are their susceptibilities to trypsin. An immunological study performed with a serum raised against nitrate reductase Z confirmed the existence of common epitopes in both complexes but unambiguously demonstrated the presence of specific determinants in nitrate reductase Z. Furthermore, it revealed a peculiar aspect of the regulation of both nitrate reductases: the nitrate reductase A enzyme is repressed by oxygen, strongly inducible by nitrate and positively controlled by the fnr gene product; on the contrary, the nitrate reductase Z enzyme is produced aerobically, barely induced by nitrate and repressed by the fnr gene product in anaerobiosis.