The Role of the Nucleotides in the Insertion of the bis-Molybdopterin Guanine Dinucleotide Cofactor into apo-Molybdoenzymes.

The role of the GMP nucleotides of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor of the DMSO reductase family has long been a subject of discussion. The recent characterization of the bis-molybdopterin (bis-Mo-MPT) cofactor present in the E. coli YdhV protein, which differs from bis-MGD solely by the absence of the nucleotides, now enables studying the role of the nucleotides of bis-MGD and bis-MPT cofactors in Moco insertion and the activity of molybdoenzymes in direct comparison. Using the well-known E. coli TMAO reductase TorA as a model enzyme for cofactor insertion, we were able to show that the GMP nucleotides of bis-MGD are crucial for the insertion of the bis-MGD cofactor into apo-TorA.

Shewanella decolorationis LDS1 Chromate Resistance.

The genus Shewanella is well known for its genetic diversity, its outstanding respiratory capacity, and its high potential for bioremediation. Here, a novel strain isolated from sediments of the Indian Ocean was characterized. A 16S rRNA analysis indicated that it belongs to the species Shewanella decolorationis It was named Shewanella decolorationis LDS1. This strain presented an unusual ability to grow efficiently at temperatures from 24°C to 40°C without apparent modifications of its metabolism, as shown by testing respiratory activities or carbon assimilation, and in a wide range of salt concentrations. Moreover, S. decolorationis LDS1 tolerates high chromate concentrations. Indeed, it was able to grow in the presence of 4 mM chromate at 28°C and 3 mM chromate at 40°C. Interestingly, whatever the temperature, when the culture reached the stationary phase, the strain reduced the chromate present in the growth medium. In addition, S. decolorationis LDS1 degrades different toxic dyes, including anthraquinone, triarylmethane, and azo dyes. Thus, compared to Shewanella oneidensis, this strain presented better capacity to cope with various abiotic stresses, particularly at high temperatures. The analysis of genome sequence preliminary data indicated that, in contrast to S. oneidensis and S. decolorationis S12, S. decolorationis LDS1 possesses the phosphorothioate modification machinery that has been described as participating in survival against various abiotic stresses by protecting DNA. We demonstrate that its heterologous production in S. oneidensis allows it to resist higher concentrations of chromate.IMPORTANCEShewanella species have long been described as interesting microorganisms in regard to their ability to reduce many organic and inorganic compounds, including metals. However, members of the Shewanella genus are often depicted as cold-water microorganisms, although their optimal growth temperature usually ranges from 25 to 28°C under laboratory growth conditions. Shewanella decolorationis LDS1 is highly attractive, since its metabolism allows it to develop efficiently at temperatures from 24 to 40°C, conserving its ability to respire alternative substrates and to reduce toxic compounds such as chromate or toxic dyes. Our results clearly indicate that this novel strain has the potential to be a powerful tool for bioremediation and unveil one of the mechanisms involved in its chromate resistance.

Modulating the Molybdenum Coordination Sphere of Escherichia coli Trimethylamine N-Oxide Reductase.

The well-studied enterobacterium Escherichia coli present in the human gut can reduce trimethylamine N-oxide (TMAO) to trimethylamine during anaerobic respiration. The TMAO reductase TorA is a monomeric, bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor-containing enzyme that belongs to the dimethyl sulfoxide reductase family of molybdoenzymes. We report on a system for the in vitro reconstitution of TorA with molybdenum cofactors (Moco) from different sources. Higher TMAO reductase activities for TorA were obtained when using Moco sources containing a sulfido ligand at the molybdenum atom. For the first time, we were able to isolate functional bis-MGD from Rhodobacter capsulatus formate dehydrogenase (FDH), which remained intact in its isolated state and after insertion into apo-TorA yielded a highly active enzyme. Combined characterizations of the reconstituted TorA enzymes by electron paramagnetic resonance spectroscopy and direct electrochemistry emphasize that TorA activity can be modified by changes in the Mo coordination sphere. The combination of these results together with studies of amino acid exchanges at the active site led us to propose a novel model for binding of the substrate to the molybdenum atom of TorA.

Protection of the general stress response σS factor by the CrsR regulator allows a rapid and efficient adaptation of Shewanella oneidensis.

To cope with environmental stresses, bacteria have evolved various strategies, including the general stress response (GSR). GSR is governed by an alternative transcriptional σ factor named σS (RpoS) that associates with RNA polymerase and controls the expression of numerous genes. Previously, we have reported that posttranslational regulation of σS in the aquatic bacterium Shewanella oneidensis involves the CrsR-CrsA partner-switching regulatory system, but the exact mechanism by which CrsR and CrsA control σS activity is not completely unveiled. Here, using a translational gene fusion, we show that CrsR sequesters and protects σS during the exponential growth phase and thus enables rapid gene activation by σS as soon as the cells enter early stationary phase. We further demonstrate by an in vitro approach that this protection is mediated by the anti-σ domain of CrsR. Structure-based alignments of CsrR orthologs and other anti-σ factors identified a CsrR-specific region characteristic of a new family of anti-σ factors. We found that CrsR is conserved in many aquatic proteobacteria, and most of the time it is associated with CrsA. In conclusion, our results suggest that CsrR-mediated protection of σS during exponential growth enables rapid adaptation of S. oneidensis to changing and stressful growth conditions, and this ability is probably widespread among aquatic proteobacteria.

The General Stress Response σS Is Regulated by a Partner Switch in the Gram-negative Bacterium Shewanella oneidensis.

Here, we show that a partner-switching system of the aquatic Proteobacterium Shewanella oneidensis regulates post-translationally σS (also called RpoS), the general stress response sigma factor. Genes SO2118 and SO2119 encode CrsA and CrsR, respectively. CrsR is a three-domain protein comprising a receiver, a phosphatase, and a kinase/anti-sigma domains, and CrsA is an anti-sigma antagonist. In vitro, CrsR sequesters σS and possesses kinase and phosphatase activities toward CrsA. In turn, dephosphorylated CrsA binds the anti-sigma domain of CrsR to allow the release of σS This study reveals a novel pathway that post-translationally regulates the general stress response sigma factor differently than what was described for other proteobacteria like Escherichia coli We argue that this pathway allows probably a rapid bacterial adaptation.

The sulfur carrier protein TusA has a pleiotropic role in Escherichia coli that also affects molybdenum cofactor biosynthesis.

The Escherichia coli L-cysteine desulfurase IscS mobilizes sulfur from L-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a ΔtusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the ΔtusA mutant. Characterization of the ΔtusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes.

Molybdenum enzymes, their maturation and molybdenum cofactor biosynthesis in Escherichia coli.

Molybdenum cofactor (Moco) biosynthesis is an ancient, ubiquitous, and highly conserved pathway leading to the biochemical activation of molybdenum. Moco is the essential component of a group of redox enzymes, which are diverse in terms of their phylogenetic distribution and their architectures, both at the overall level and in their catalytic geometry. A wide variety of transformations are catalyzed by these enzymes at carbon, sulfur and nitrogen atoms, which include the transfer of an oxo group or two electrons to or from the substrate. More than 50 molybdoenzymes were identified in bacteria to date. In molybdoenzymes Mo is coordinated to a dithiolene group on the 6-alkyl side chain of a pterin called molybdopterin (MPT). The biosynthesis of Moco can be divided into four general steps in bacteria: 1) formation of the cyclic pyranopterin monophosphate, 2) formation of MPT, 3) insertion of molybdenum into molybdopterin to form Moco, and 4) additional modification of Moco with the attachment of GMP or CMP to the phosphate group of MPT, forming the dinucleotide variant of Moco. This review will focus on molybdoenzymes, the biosynthesis of Moco, and its incorporation into specific target proteins focusing on Escherichia coli. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.

Iron limitation indirectly reduces the Escherichia coli torCAD operon expression by a reduction of molybdenum cofactor availability.

The expression of most molybdoenzymes in Escherichia coli has so far been revealed to be regulated by anaerobiosis and requires the presence of iron, based on the necessity of the transcription factor FNR to bind one [4Fe-4S] cluster. One exception is trimethylamine-N-oxide reductase encoded by the torCAD operon, which has been described to be expressed independently from FNR. In contrast to other alternative anaerobic respiratory systems, the expression of the torCAD operon was shown not to be completely repressed by the presence of dioxygen. To date, the basis for the O2-dependent expression of the torCAD operon has been related to the abundance of the transcriptional regulator IscR, which represses the transcription of torS and torT, and is more abundant under aerobic conditions than under anaerobic conditions. In this study, we reinvestigated the regulation of the torCAD operon and its dependence on the presence of iron and identified a novel regulation that depends on the presence of the bis-molybdopterin guanine dinucleotide (bis-MGD) molybdenum cofactor . We confirmed that the torCAD operon is directly regulated by the heme-containing protein TorC and is indirectly regulated by ArcA and by the availability of iron via active FNR and Fur, both regulatory proteins that influence the synthesis of the molybdenum cofactor. Furthermore, we identified a novel regulation mode of torCAD expression that is dependent on cellular levels of bis-MGD and is not used by other bis-MGD-containing enzymes like nitrate reductase.IMPORTANCEIn bacteria, molybdoenzymes are crucial for anaerobic respiration using alternative electron acceptors. FNR is a very important transcription factor that represents the master switch for the expression of target genes in response to anaerobiosis. Only Escherichia coli trimethylamine-N-oxide (TMAO) reductase escapes this regulation by FNR. We identified that the expression of TMAO reductase is regulated by the amount of bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor synthesized by the cell itself, representing a novel regulation pathway for the expression of an operon coding for a molybdoenzyme. Furthermore, TMAO reductase gene expression is indirectly regulated by the presence of iron, which is required for the production of the bis-MGD cofactor in the cell.

Molybdopterin dinucleotide biosynthesis in Escherichia coli: identification of amino acid residues of molybdopterin dinucleotide transferases that determine specificity for binding of guanine or cytosine nucleotides.

The molybdenum cofactor is modified by the addition of GMP or CMP to the C4' phosphate of molybdopterin forming the molybdopterin guanine dinucleotide or molybdopterin cytosine dinucleotide cofactor, respectively. The two reactions are catalyzed by specific enzymes as follows: the GTP:molybdopterin guanylyltransferase MobA and the CTP:molybdopterin cytidylyltransferase MocA. Both enzymes show 22% amino acid sequence identity and are specific for their respective nucleotides. Crystal structure analysis of MobA revealed two conserved motifs in the N-terminal domain of the protein involved in binding of the guanine base. Based on these motifs, we performed site-directed mutagenesis studies to exchange the amino acids to the sequence found in the paralogue MocA. Using a fully defined in vitro system, we showed that the exchange of five amino acids was enough to obtain activity with both GTP and CTP in either MocA or MobA. Exchange of the complete N-terminal domain of each protein resulted in the total inversion of nucleotide specificity activity, showing that the N-terminal domain determines nucleotide recognition and binding. Analysis of protein-protein interactions showed that the C-terminal domain of either MocA or MobA determines the specific binding to the respective acceptor protein.

The Tol-Pal system of Escherichia coli plays an unexpected role in the import of the oxyanions chromate and phosphate.

Chromate is a toxic metal that enters bacteria by using oxyanion importers. Here, we show that each mutant of the Tol-Pal system of Escherichia coli exhibited increased chromate resistance. This system, which spans the cell envelope, plays a major role in envelope integrity and septation. The ΔtolQR mutant accumulated three-fold less chromate than the wild-type. Addition of phosphate but not sulfate to rich medium drastically reduced chromate toxicity and import in the wild-type strain. Furthermore, the intracellular concentration of free inorganic phosphate was significantly reduced for the ΔtolR mutant in comparison to the wild-type strain. Moreover, extracellular labeled phosphate was significantly less incorporated into the ΔtolR mutant. Finally, two distinct TolQR mutant complexes, specifically affected in Tol-Pal energization without affecting the TolQRA complex structure, did not complement the ΔtolQR mutant for inorganic phosphate accumulation. We thus propose that, while the Pst system is well known to import inorganic phosphate, the Tol-Pal system participates to phosphate uptake in particular at medium to high extracellular phosphate concentrations. Since mutations disabling the Tol-Pal system lead to pleiotropic effects, chromate resistance and reduced inorganic phosphate import could occur from an indirect effect of mutations in components of the Tol-Pal system.

Defining Two Chemosensory Arrays in Shewanella oneidensis.

Shewanella oneidensis has 2 functional chemosensory systems named Che1 and Che3, and 27 chemoreceptors. Che3 is dedicated to chemotaxis while Che1 could be involved in RpoS post-translational regulation. In this study, we have shown that two chemoreceptors Aer2so and McpAso, genetically related to the Che1 system, form distinct core-signaling units and signal to Che1 and Che3, respectively. Moreover, we observed that Aer2so is a cytoplasmic dynamic chemoreceptor that, when in complex with CheA1 and CheW1, localizes at the two poles and the centre of the cells. Altogether, the results obtained indicate that Che1 and Che3 systems are interconnected by these two chemoreceptors allowing a global response for bacterial survival.

YcdY protein of Escherichia coli, an atypical member of the TorD chaperone family.

The TorD family of specific chaperones is divided into four subfamilies dedicated to molybdoenzyme biogenesis and a fifth one, exemplified by YcdY of Escherichia coli, for which no defined partner has been identified so far. We propose that YcdY is the chaperone of YcdX, a zinc protein involved in the swarming motility process of E. coli, since YcdY interacts with YcdX and increases its activity in vitro.

Electrochemical Trimethylamine N-Oxide Biosensor with Enzyme-Based Oxygen-Scavenging Membrane for Long-Term Operation under Ambient Air.

An amperometric trimethylamine N-oxide (TMAO) biosensor is reported, where TMAO reductase (TorA) and glucose oxidase (GOD) and catalase (Cat) were immobilized on the electrode surface, enabling measurements of mediated enzymatic TMAO reduction at low potential under ambient air conditions. The oxygen anti-interference membrane composed of GOD, Cat and polyvinyl alcohol (PVA) hydrogel, together with glucose concentration, was optimized until the O2 reduction current of a Clark-type electrode was completely suppressed for at least 3 h. For the preparation of the TMAO biosensor, Escherichia coli TorA was purified under anaerobic conditions and immobilized on the surface of a carbon electrode and covered by the optimized O2 scavenging membrane. The TMAO sensor operates at a potential of -0.8 V vs. Ag/AgCl (1 M KCl), where the reduction of methylviologen (MV) is recorded. The sensor signal depends linearly on TMAO concentrations between 2 µM and 15 mM, with a sensitivity of 2.75 ± 1.7 µA/mM. The developed biosensor is characterized by a response time of about 33 s and an operational stability over 3 weeks. Furthermore, measurements of TMAO concentration were performed in 10% human serum, where the lowest detectable concentration is of 10 µM TMAO.

Unexpected chemoreceptors mediate energy taxis towards electron acceptors in Shewanella oneidensis.

Shewanella oneidensis uses a wide range of terminal electron acceptors for respiration. In this study, we show that the chemotactic response of S. oneidensis to anaerobic electron acceptors requires functional electron transport systems. Deletion of the genes encoding dimethyl sulphoxide and trimethylamine N-oxide reductases, or inactivation of these molybdoenzymes as well as nitrate reductase by addition of tungstate, abolished electron acceptor taxis. Moreover, addition of nigericin prevented taxis towards trimethylamine N-oxide, dimethyl sulphoxide, nitrite, nitrate and fumarate, showing that this process depends on the DeltapH component of the proton motive force. These data, together with those concerning response to metals (Bencharit and Ward, 2005), support the idea that, in S. oneidensis, taxis towards electron acceptors is governed by an energy taxis mechanism. Surprisingly, energy taxis in S. oneidensis is not mediated by the PAS-containing chemoreceptors but rather by a chemoreceptor (SO2240) containing a Cache domain. Four other chemoreceptors also play a minor role in this process. These results indicate that energy taxis can be mediated by new types of chemoreceptors.

The Shewanella genus: ubiquitous organisms sustaining and preserving aquatic ecosystems.

The Gram-negative Shewanella bacterial genus currently includes about 70 species of mostly aquatic γ--proteobacteria, which were isolated around the globe in a multitude of environments such as surface freshwater and the deepest marine trenches. Their survival in such a wide range of ecological niches is due to their impressive physiological and respiratory versatility. Some strains are among the organisms with the highest number of respiratory systems, depending on a complex and rich metabolic network. Implicated in the recycling of organic and inorganic matter, they are important components of organism-rich oxic/anoxic interfaces, but they also belong to the microflora of a broad group of eukaryotes from metazoans to green algae. Examples of long-term biological interactions like mutualism or pathogeny have been described, although molecular determinants of such symbioses are still poorly understood. Some of these bacteria are key organisms for various biotechnological applications, especially the bioremediation of hydrocarbons and metallic pollutants. The natural ability of these prokaryotes to thrive and detoxify deleterious compounds explains their use in wastewater treatment, their use in energy generation by microbial fuel cells and their importance for resilience of aquatic ecosystems.

Reconstitution of Molybdoenzymes with Bis-Molybdopterin Guanine Dinucleotide Cofactors.

Molybdoenzymes are ubiquitous and play important roles in all kingdoms of life. The cofactors of these enzymes comprise the metal, molybdenum (Mo), which is bound to a special organic ligand system called molybdopterin (MPT). Additional small ligands are present at the Mo atom, including water, hydroxide, oxo-, sulfido-, or selenido-functionalities, and in some enzymes, amino acid ligand, such as serine, aspartate, cysteine, or selenocysteine that coordinate the cofactor to the peptide chain of the enzyme. The so-called molybdenum cofactor (Moco) is deeply buried within the protein at the end of a narrow funnel, giving access only to the substrate. In 1974, an assay was developed by Nason and coworkers using the pleiotropic Neurospora crassa mutant, nit-1, for the reconstitution of molybdoenzyme activities from crude extracts. These studies have led to the understanding that Moco is the common element in all molybdoenzymes from different organisms. The assay has been further developed since then by using specific molybdenum enzymes as the source of Moco for the reconstitution of diverse purified apo-molybdoenzymes. Alternatively, the molybdenum cofactor can be synthesized in vitro from stable intermediates and subsequently inserted into apo-molybdoenzymes with the assistance of specific Moco-binding chaperones. A general working protocol is described here for the insertion of the bis-molybdopterin guanine dinucleotide cofactor (bis-MGD) into its target molybdoenzyme using the example of Escherichia coli trimethylamine N-oxide (TMAO) reductase.

The regulation of Moco biosynthesis and molybdoenzyme gene expression by molybdenum and iron in bacteria.

Bacterial molybdoenzymes are key enzymes involved in the global sulphur, nitrogen and carbon cycles. These enzymes require the insertion of the molybdenum cofactor (Moco) into their active sites and are able to catalyse a large range of redox-reactions. Escherichia coli harbours nineteen different molybdoenzymes that require a tight regulation of their synthesis according to substrate availability, oxygen availability and the cellular concentration of molybdenum and iron. The synthesis and assembly of active molybdoenzymes are regulated at the level of transcription of the structural genes and of translation in addition to the genes involved in Moco biosynthesis. The action of global transcriptional regulators like FNR, NarXL/QP, Fur and ArcA and their roles on the expression of these genes is described in detail. In this review we focus on what is known about the molybdenum- and iron-dependent regulation of molybdoenzyme and Moco biosynthesis genes in the model organism E. coli. The gene regulation in E. coli is compared to two other well studied model organisms Rhodobacter capsulatus and Shewanella oneidensis.

Connected partner-switches control the life style of Pseudomonas aeruginosa through RpoS regulation.

Biofilm formation is a complex process resulting from the action of imbricated pathways in response to environmental cues. In this study, we showed that biofilm biogenesis in the opportunistic pathogen Pseudomonas aeruginosa depends on the availability of RpoS, the sigma factor regulating the general stress response in bacteria. Moreover, it was demonstrated that RpoS is post-translationally regulated by the HsbR-HsbA partner switching system as has been demonstrated for its CrsR-CrsA homolog in Shewanella oneidensis. Finally, it was established that HsbA, the anti-sigma factor antagonist, has a pivotal role depending on its phosphorylation state since it binds HsbR, the response regulator, when phosphorylated and FlgM, the anti-sigma factor of FliA, when non-phosphorylated. The phosphorylation state of HsbA thus drives the switch between the sessile and planktonic way of life of P. aeruginosa by driving the release or the sequestration of one or the other of these two sigma factors.

Same but different: Comparison of two system-specific molecular chaperones for the maturation of formate dehydrogenases.

The maturation of bacterial molybdoenzymes is a complex process leading to the insertion of the bulky bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor into the apo-enzyme. Most molybdoenzymes were shown to contain a specific chaperone for the insertion of the bis-MGD cofactor. Formate dehydrogenases (FDH) together with their molecular chaperone partner seem to display an exception to this specificity rule, since the chaperone FdhD has been proven to be involved in the maturation of all three FDH enzymes present in Escherichia coli. Multiple roles have been suggested for FdhD-like chaperones in the past, including the involvement in a sulfur transfer reaction from the l-cysteine desulfurase IscS to bis-MGD by the action of two cysteine residues present in a conserved CXXC motif of the chaperones. However, in this study we show by phylogenetic analyses that the CXXC motif is not conserved among FdhD-like chaperones. We compared in detail the FdhD-like homologues from Rhodobacter capsulatus and E. coli and show that their roles in the maturation of FDH enzymes from different subgroups can be exchanged. We reveal that bis-MGD-binding is a common characteristic of FdhD-like proteins and that the cofactor is bound with a sulfido-ligand at the molybdenum atom to the chaperone. Generally, we reveal that the cysteine residues in the motif CXXC of the chaperone are not essential for the production of active FDH enzymes.

Regulation of σ factors by conserved partner switches controlled by divergent signalling systems.

Partner-Switching Systems (PSS) are widespread regulatory systems, each comprising a kinase-anti-σ, a phosphorylatable anti-σ antagonist and a phosphatase module. The anti-σ domain quickly sequesters or delivers the target σ factor according to the phosphorylation state of the anti-σ antagonist induced by environmental signals. The PSS components are proteins alone or merged to other domains probably to adapt to the input signals. PSS are involved in major cellular processes including stress response, sporulation, biofilm formation and pathogenesis. Surprisingly, the target σ factors are often unknown and the sensing modules acting upstream from the PSS diverge according to the bacterial species. Indeed, they belong to either two-component systems or complex pathways as the stressosome or Chemosensory Systems (CS). Based on a phylogenetic analysis, we propose that the sensing module in Gram-negative bacteria is often a CS.