ABSTRACT
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.
This review discusses the diversity among the aquatic ubiquitous γ-proteobacterial Shewanella genus, which acts on several cycles of Matter and forms biological interactions with both prokaryotes and eukaryotes, in addition to being implicated in ecosystem resilience and biotechnological applications.
Clostridium autoethanogenum, the bacterial model for biological conversion of waste gases into biofuels, grows under extreme carbon-monoxide (CO) concentrations. The strictly anaerobic bacterium ...derives its entire cellular energy and carbon from this poisonous gas, therefore requiring efficient molecular machineries for CO-conversion. Here, we structurally and biochemically characterized the key enzyme of the CO-converting metabolism: the CO-dehydrogenase/Acetyl-CoA synthase (CODH/ACS). We obtained crystal structures of natively isolated complexes from fructose-grown and CO-grown C. autoethanogenum cultures. Both contain the same isoforms and if the overall structure adopts the classic α2β2 architecture, comparable to the model enzyme from Moorella thermoacetica, the ACS binds a different position on the CODH core. The structural characterization of a proteolyzed complex and the conservation of the binding interface in close homologs rejected the possibility of a crystallization artefact. Therefore, the internal CO-channeling system, critical to transfer CO generated at the C-cluster to the ACS active site, drastically differs in the complex from C. autoethanogenum. The 1.9-Å structure of the CODH alone provides an accurate picture of the new CO-routes, leading to the ACS core and reaching the surface. Increased gas accessibility would allow the simultaneous CO-oxidation and acetyl-CoA production. Biochemical experiments showed higher flexibility of the ACS subunit from C. autoethanogenum compared to M. thermoacetica, albeit monitoring similar CO-oxidation and formation rates. These results show a reshuffling of internal CO-tunnels during evolution of these Firmicutes, putatively leading to a bidirectional complex that ensure a high flux of CO-conversion toward energy conservation, acting as the main cellular powerplant.
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•Structure of the CODH/ACS complex solved from a waste gas-converting bacterium.•The CODH-ACS interface drastically differs from the model of Moorella thermoacetica.•The CODH dimer structure reveals the organization of C-clusters and CO-channels.•Internal CO tunneling system is reshuffled to fit the novel binding mode.•A porous gas channeling would allow bi-directionality during CO-autotrophic growth.
Microbial anaerobic oxidation of alkanes intrigues the scientific community by way of its impact on the global carbon cycle, and its biotechnological applications. Archaea are proposed to degrade ...short- and long-chain alkanes to CO2 by reversing methanogenesis, a theoretically reversible process. The pathway would start with alkane activation, an endergonic step catalyzed by methyl-coenzyme M reductase (MCR) homologues that would generate alkyl-thiols carried by coenzyme M. While the methane-generating MCR found in methanogens has been well characterized, the enzymatic activity of the putative alkane-fixing counterparts has not been validated so far. Such an absence of biochemical investigations contrasts with the current explosion of metagenomics data, which draws new potential alkane-oxidizing pathways in various archaeal phyla. Therefore, validating the physiological function of these putative alkane-fixing machines and investigating how their structures, catalytic mechanisms, and cofactors vary depending on the targeted alkane have become urgent needs. The first structural insights into the methane- and ethane-capturing MCRs highlighted unsuspected differences and proposed some explanations for their substrate specificity. This Perspective reviews the current physiological, biochemical, and structural knowledge of alkyl-CoM reductases and offers fresh ideas about the expected mechanistic and chemical differences among members of this broad family. We conclude with the challenges of the investigation of these particular enzymes, which might one day generate biofuels for our modern society.
Domestication of CO
2
-fixation became a worldwide priority enhanced by the will to convert this greenhouse gas into fuels and valuable chemicals. Because of its high stability, CO
2
...-activation/fixation represents a true challenge for chemists. Autotrophic microbial communities, however, perform these reactions under standard temperature and pressure. Recent discoveries shine light on autotrophic acetogenic bacteria and hydrogenotrophic methanogens, as these anaerobes use a particularly efficient CO
2
-capture system to fulfill their carbon and energy needs. While other autotrophs assimilate CO
2
via carboxylation followed by a reduction, acetogens and methanogens do the opposite. They first generate formate and CO by CO
2
-reduction, which are subsequently fixed to funnel the carbon toward their central metabolism. Yet their CO
2
-reduction pathways, with acetate or methane as end-products, constrain them to thrive at the “thermodynamic limits of Life”. Despite this energy restriction acetogens and methanogens are growing at unexpected fast rates. To overcome the thermodynamic barrier of CO
2
-reduction they apply different ingenious chemical tricks such as the use of flavin-based electron-bifurcation or coupled reactions. This mini-review summarizes the current knowledge gathered on the CO
2
-fixation strategies among acetogens. While extensive biochemical characterization of the acetogenic formate-generating machineries has been done, there is no structural data available. Based on their shared mechanistic similarities, we apply the structural information obtained from hydrogenotrophic methanogens to highlight common features, as well as the specific differences of their CO
2
-fixation systems. We discuss the consequences of their CO
2
-reduction strategies on the evolution of Life, their wide distribution and their impact in biotechnological applications.
Whilst widespread in the microbial world, the hybrid cluster protein (HCP) has been paradoxically a long-time riddle for microbiologists. During three decades, numerous studies on a few model ...organisms unravelled its structure and dissected its metal-containing catalyst, but the physiological function of the enzyme remained elusive. Recent studies on bacteria point towards a nitric oxide reductase activity involved in resistance during nitrate and nitrite reduction as well as host infection. In this study, we isolated and characterised a naturally highly produced HCP class I from a marine methanogenic archaeon grown on ammonia. The crystal structures of the enzyme in a reduced and partially oxidised state, obtained at a resolution of 1.45 and 1.36-Å, respectively, offered a precise picture of the archaeal enzyme intimacy. There are striking similarities with the well-studied enzymes from
species regarding sequence, kinetic parameters, structure, catalyst conformations, and internal channelling systems. The close phylogenetic relationship between the enzymes from
and many
corroborates this similarity. Indeed,
HCPs are closer to these bacterial homologues than to any other archaeal enzymes. The relatively high constitutive production of HCP in
, in the absence of a notable nitric oxide source, questions the physiological function of the enzyme in these ancient anaerobes.
How to feed an enzyme ethane
When released from ocean floor seeps, small hydrocarbons are rapidly consumed by micro-organisms. Methane is highly abundant and is both produced and consumed by microbes ...through well understood biochemical pathways. Less well understood is how ethane, also a major natural component of gaseous hydrocarbons, is metabolized. To understand how microbes take advantage of this energy and carbon source, Hahn
et al.
solved the x-ray crystal structures of an enzyme they call ethyl coenzyme-M reductase, which converts ethane into the thioether ethyl-coenzyme M as the entry point for catabolism. They found an expanded active site and, using a xenon gas derivatization experiment, a distinctive tunnel through the protein that is proposed to permit access of the gaseous substrate.
Science
, abg1765, this issue p.
118
Crystal structures reveal key features in a metalloenzyme from ocean floor archaea that activates ethane.
Ethane, the second most abundant hydrocarbon gas in the seafloor, is efficiently oxidized by anaerobic archaea in syntrophy with sulfate-reducing bacteria. Here, we report the 0.99-angstrom-resolution structure of the proposed ethane-activating enzyme and describe the specific traits that distinguish it from methane-generating and -consuming methyl-coenzyme M reductases. The widened catalytic chamber, harboring a dimethylated nickel-containing F
430
cofactor, would adapt the chemistry of methyl-coenzyme M reductases for a two-carbon substrate. A sulfur from methionine replaces the oxygen from a canonical glutamine as the nickel lower-axial ligand, a feature conserved in thermophilic ethanotrophs. Specific loop extensions, a four-helix bundle dilatation, and posttranslational methylations result in the formation of a 33-angstrom-long hydrophobic tunnel, which guides the ethane to the buried active site as confirmed with xenon pressurization experiments.
The genus
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
It was named
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,
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,
LDS1 degrades different toxic dyes, including anthraquinone, triarylmethane, and azo dyes. Thus, compared to
, 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
and
S12,
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
allows it to resist higher concentrations of chromate.
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
genus are often depicted as cold-water microorganisms, although their optimal growth temperature usually ranges from 25 to 28°C under laboratory growth conditions.
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.
Massive efforts are invested in developing innovative CO2‐sequestration strategies to counter climate change and transform CO2 into higher‐value products. CO2‐capture by reduction is a chemical ...challenge, and attention is turned toward biological systems that selectively and efficiently catalyse this reaction under mild conditions and in aqueous solvents. While a few reports have evaluated the effectiveness of isolated bacterial formate dehydrogenases as catalysts for the reversible electrochemical reduction of CO2, it is imperative to explore other enzymes among the natural reservoir of potential models that might exhibit higher turnover rates or preferential directionality for the reductive reaction. Here, we present electroenzymatic catalysis of formylmethanofuran dehydrogenase, a CO2‐reducing‐and‐fixing biomachinery isolated from a thermophilic methanogen, which was deposited on a graphite rod electrode to enable direct electron transfer for electroenzymatic CO2 reduction. The gas is reduced with a high Faradaic efficiency (109±1 %), where a low affinity for formate prevents its electrochemical reoxidation and favours formate accumulation. These properties make the enzyme an excellent tool for electroenzymatic CO2‐fixation and inspiration for protein engineering that would be beneficial for biotechnological purposes to convert the greenhouse gas into stable formate that can subsequently be safely stored, transported, and used for power generation without energy loss.
Crystallization is the limiting step in X-ray structure determination of biological macromolecules. As crystallization experiments can be largely automatized, the diversity of precipitant solutions ...is often the determinant factor to obtain crystals of high quality. Here, we introduce a 96-well screening kit of crystallization conditions, centered on three ethoxylate-based organic polymers as precipitants and various additional compounds to promote crystal formation. This crystallization screen was tested on various non-standard proteins from bacteria and archaea. Structure determination succeeded for seven out of thirteen targets based on crystals that frequently diffracted to a higher resolution than those obtained with commercially available screening kits. Crystallization hits were rarely similar among the three ethoxylate-based organic polymers and, in comparison, with already available crystallization screens. Hence, the presented crystallization screen is an efficient tool to complement other screens and increase the likelihood of growing crystals suitable for X-ray structure determination.
Summary
To infect plants, viruses rely heavily on their host's machinery. Plant genetic resistances based on host factor modifications can be found among existing natural variability and are widely ...used for some but not all crops. While biotechnology can supply for the lack of natural resistance alleles, new strategies need to be developed to increase resistance spectra and durability without impairing plant development. Here, we assess how the targeted allele modification of the Arabidopsis thaliana translation initiation factor eIF4E1 can lead to broad and efficient resistance to the major group of potyviruses. A synthetic Arabidopsis thaliana eIF4E1 allele was designed by introducing multiple amino acid changes associated with resistance to potyvirus in naturally occurring Pisum sativum alleles. This new allele encodes a functional protein while maintaining plant resistance to a potyvirus isolate that usually hijacks eIF4E1. Due to its biological functionality, this synthetic allele allows, at no developmental cost, the pyramiding of resistances to potyviruses that selectively use the two major translation initiation factors, eIF4E1 or its isoform eIFiso4E. Moreover, this combination extends the resistance spectrum to potyvirus isolates for which no efficient resistance has so far been found, including resistance‐breaking isolates and an unrelated virus belonging to the Luteoviridae family. This study is a proof‐of‐concept for the efficiency of gene engineering combined with knowledge of natural variation to generate trans‐species virus resistance at no developmental cost to the plant. This has implications for breeding of crops with broad‐spectrum and high durability resistance using recent genome editing techniques.
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