Minimal and hybrid hydrogenases are active from archaea.

Microbial hydrogen (H2) cycling underpins the diversity and functionality of diverse anoxic ecosystems. Among the three evolutionarily distinct hydrogenase superfamilies responsible, [FeFe] hydrogenases were thought to be restricted to bacteria and eukaryotes. Here, we show that anaerobic archaea encode diverse, active, and ancient lineages of [FeFe] hydrogenases through combining analysis of existing and new genomes with extensive biochemical experiments. [FeFe] hydrogenases are encoded by genomes of nine archaeal phyla and expressed by H2-producing Asgard archaeon cultures. We report an ultraminimal hydrogenase in DPANN archaea that binds the catalytic H-cluster and produces H2. Moreover, we identify and characterize remarkable hybrid complexes formed through the fusion of [FeFe] and [NiFe] hydrogenases in ten other archaeal orders. Phylogenetic analysis and structural modeling suggest a deep evolutionary history of hybrid hydrogenases. These findings reveal new metabolic adaptations of archaea, streamlined H2 catalysts for biotechnological development, and a surprisingly intertwined evolutionary history between the two major H2-metabolizing enzymes.

Structural basis for bacterial energy extraction from atmospheric hydrogen.

Diverse aerobic bacteria use atmospheric H2 as an energy source for growth and survival1. This globally significant process regulates the composition of the atmosphere, enhances soil biodiversity and drives primary production in extreme environments2,3. Atmospheric H2 oxidation is attributed to uncharacterized members of the [NiFe] hydrogenase superfamily4,5. However, it remains unresolved how these enzymes overcome the extraordinary catalytic challenge of oxidizing picomolar levels of H2 amid ambient levels of the catalytic poison O2 and how the derived electrons are transferred to the respiratory chain1. Here we determined the cryo-electron microscopy structure of the Mycobacterium smegmatis hydrogenase Huc and investigated its mechanism. Huc is a highly efficient oxygen-insensitive enzyme that couples oxidation of atmospheric H2 to the hydrogenation of the respiratory electron carrier menaquinone. Huc uses narrow hydrophobic gas channels to selectively bind atmospheric H2 at the expense of O2, and 3 [3Fe-4S] clusters modulate the properties of the enzyme so that atmospheric H2 oxidation is energetically feasible. The Huc catalytic subunits form an octameric 833 kDa complex around a membrane-associated stalk, which transports and reduces menaquinone 94 Å from the membrane. These findings provide a mechanistic basis for the biogeochemically and ecologically important process of atmospheric H2 oxidation, uncover a mode of energy coupling dependent on long-range quinone transport, and pave the way for the development of catalysts that oxidize H2 in ambient air.

Multiple energy sources and metabolic strategies sustain microbial diversity in Antarctic desert soils.

Numerous diverse microorganisms reside in the cold desert soils of continental Antarctica, though we lack a holistic understanding of the metabolic processes that sustain them. Here, we profile the composition, capabilities, and activities of the microbial communities in 16 physicochemically diverse mountainous and glacial soils. We assembled 451 metagenome-assembled genomes from 18 microbial phyla and inferred through Bayesian divergence analysis that the dominant lineages present are likely native to Antarctica. In support of earlier findings, metagenomic analysis revealed that the most abundant and prevalent microorganisms are metabolically versatile aerobes that use atmospheric hydrogen to support aerobic respiration and sometimes carbon fixation. Surprisingly, however, hydrogen oxidation in this region was catalyzed primarily by a phylogenetically and structurally distinct enzyme, the group 1l [NiFe]-hydrogenase, encoded by nine bacterial phyla. Through gas chromatography, we provide evidence that both Antarctic soil communities and an axenic Bacteroidota isolate (Hymenobacter roseosalivarius) oxidize atmospheric hydrogen using this enzyme. Based on ex situ rates at environmentally representative temperatures, hydrogen oxidation is theoretically sufficient for soil communities to meet energy requirements and, through metabolic water production, sustain hydration. Diverse carbon monoxide oxidizers and abundant methanotrophs were also active in the soils. We also recovered genomes of microorganisms capable of oxidizing edaphic inorganic nitrogen, sulfur, and iron compounds and harvesting solar energy via microbial rhodopsins and conventional photosystems. Obligately symbiotic bacteria, including Patescibacteria, Chlamydiae, and predatory Bdellovibrionota, were also present. We conclude that microbial diversity in Antarctic soils reflects the coexistence of metabolically flexible mixotrophs with metabolically constrained specialists.

Protein target highlights in CASP15: Analysis of models by structure providers.

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.

Developing high-affinity, oxygen-insensitive [NiFe]-hydrogenases as biocatalysts for energy conversion.

The splitting of hydrogen (H2) is an energy-yielding process, which is important for both biological systems and as a means of providing green energy. In biology, this reaction is mediated by enzymes called hydrogenases, which utilise complex nickel and iron cofactors to split H2 and transfer the resulting electrons to an electron-acceptor. These [NiFe]-hydrogenases have received considerable attention as catalysts in fuel cells, which utilise H2 to produce electrical current. [NiFe]-hydrogenases are a promising alternative to the platinum-based catalysts that currently predominate in fuel cells due to the abundance of nickel and iron, and the resistance of some family members to inhibition by gases, including carbon monoxide, which rapidly poison platinum-based catalysts. However, the majority of characterised [NiFe]-hydrogenases are inhibited by oxygen (O2), limiting their activity and stability. We recently reported the isolation and characterisation of the [NiFe]-hydrogenase Huc from Mycobacterium smegmatis, which is insensitive to inhibition by O2 and has an extremely high affinity, making it capable of oxidising H2 in air to below atmospheric concentrations. These properties make Huc a promising candidate for the development of enzyme-based fuel cells (EBFCs), which utilise H2 at low concentrations and in impure gas mixtures. In this review, we aim to provide context for the use of Huc for this purpose by discussing the advantages of [NiFe]-hydrogenases as catalysts and their deployment in fuel cells. We also address the challenges associated with using [NiFe]-hydrogenases for this purpose, and how these might be overcome to develop EBFCs that can be deployed at scale.

Protease-associated import systems are widespread in Gram-negative bacteria.

Bacteria have evolved sophisticated uptake machineries in order to obtain the nutrients required for growth. Gram-negative plant pathogens of the genus Pectobacterium obtain iron from the protein ferredoxin, which is produced by their plant hosts. This iron-piracy is mediated by the ferredoxin uptake system (Fus), a gene cluster encoding proteins that transport ferredoxin into the bacterial cell and process it proteolytically. In this work we show that gene clusters related to the Fus are widespread in bacterial species. Through structural and biochemical characterisation of the distantly related Fus homologues YddB and PqqL from Escherichia coli, we show that these proteins are analogous to components of the Fus from Pectobacterium. The membrane protein YddB shares common structural features with the outer membrane ferredoxin transporter FusA, including a large extracellular substrate binding site. PqqL is an active protease with an analogous periplasmic localisation and iron-dependent expression to the ferredoxin processing protease FusC. Structural analysis demonstrates that PqqL and FusC share specific features that distinguish them from other members of the M16 protease family. Taken together, these data provide evidence that protease associated import systems analogous to the Fus are widespread in Gram-negative bacteria.

Structural basis of substrate recognition and catalysis by fucosyltransferase 8.

Fucosylation of the innermost GlcNAc of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here we describe several structures of mouse and human FUT8 in the apo state and in complex with GDP, a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80-2.50 Å resolution. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process.

Target highlights in CASP14: Analysis of models by structure providers.

The biological and functional significance of selected Critical Assessment of Techniques for Protein Structure Prediction 14 (CASP14) targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modeled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins.

FusC, a member of the M16 protease family acquired by bacteria for iron piracy against plants.

Iron is essential for life. Accessing iron from the environment can be a limiting factor that determines success in a given environmental niche. For bacteria, access of chelated iron from the environment is often mediated by TonB-dependent transporters (TBDTs), which are ß-barrel proteins that form sophisticated channels in the outer membrane. Reports of iron-bearing proteins being used as a source of iron indicate specific protein import reactions across the bacterial outer membrane. The molecular mechanism by which a folded protein can be imported in this way had remained mysterious, as did the evolutionary process that could lead to such a protein import pathway. How does the bacterium evolve the specificity factors that would be required to select and import a protein encoded on another organism's genome? We describe here a model whereby the plant iron-bearing protein ferredoxin can be imported across the outer membrane of the plant pathogen Pectobacterium by means of a Brownian ratchet mechanism, thereby liberating iron into the bacterium to enable its growth in plant tissues. This import pathway is facilitated by FusC, a member of the same protein family as the mitochondrial processing peptidase (MPP). The Brownian ratchet depends on binding sites discovered in crystal structures of FusC that engage a linear segment of the plant protein ferredoxin. Sequence relationships suggest that the bacterial gene encoding FusC has previously unappreciated homologues in plants and that the protein import mechanism employed by the bacterium is an evolutionary echo of the protein import pathway in plant mitochondria and plastids.

The structure of the bacterial iron-catecholate transporter Fiu suggests that it imports substrates via a two-step mechanism.

The ferric iron uptake (Fiu) transporter from Escherichia coli functions in the transport of iron-catecholate complexes across the bacterial outer membrane, providing the bacterium with iron, which is essential for growth. Recently it has become clear that Fiu also represents a liability for E. coli because its activity allows import of antimicrobial compounds that mimic catecholate. This inadvertent import suggests the potential utility of antimicrobial catechol siderophore mimetics in managing bacterial infections. However, to fully exploit these compounds, a detailed understanding of the mechanism of transport through Fiu and related transporters is required. To address this question, we determined the crystal structure of Fiu at 2.1-2.9 Å and analyzed its function in E. coli Through analysis of the Fiuo crystal structure, in combination with in silico docking and mutagenesis, we provide insight into how Fiu and related transporters bind catecholate in a surface-exposed cavity. Moreover, through determination of the structure of Fiu in multiple crystal states, we revealed the presence of a large, selectively gated cavity in the interior of this transporter. This chamber is large enough to accommodate the Fiu substrate and may allow import of substrates via a two-step mechanism. This would avoid channel formation through the transporter and inadvertent import of toxic molecules. As Fiu and its homologs are the targets of substrate-mimicking antibiotics, these results may assist in the development of these compounds.

Two uptake hydrogenases differentially interact with the aerobic respiratory chain during mycobacterial growth and persistence.

To persist when nutrient sources are limited, aerobic soil bacteria metabolize atmospheric hydrogen (H2). This process is the primary sink in the global H2 cycle and supports the productivity of microbes in oligotrophic environments. H2-metabolizing bacteria possess [NiFe] hydrogenases that oxidize H2 to subatmospheric concentrations. The soil saprophyte Mycobacterium smegmatis has two such [NiFe] hydrogenases, designated Huc and Hhy, that belong to different phylogenetic subgroups. Both Huc and Hhy are oxygen-tolerant, oxidize H2 to subatmospheric concentrations, and enhance bacterial survival during hypoxia and carbon limitation. Why does M. smegmatis require two hydrogenases with a seemingly similar function? In this work, we resolved this question by showing that Huc and Hhy are differentially expressed, localized, and integrated into the respiratory chain. Huc is active in late exponential and early stationary phases, supporting energy conservation during mixotrophic growth and transition into dormancy. In contrast, Hhy is most active during long-term persistence, providing energy for maintenance processes following carbon exhaustion. We also show that Huc and Hhy are obligately linked to the aerobic respiratory chain via the menaquinone pool and are differentially affected by respiratory uncouplers. Consistently, these two enzymes interacted differentially with the respiratory terminal oxidases. Huc exclusively donated electrons to, and possibly physically associated with, the proton-pumping cytochrome bcc-aa3 supercomplex. In contrast the more promiscuous Hhy also provided electrons to the cytochrome bd oxidase complex. These results indicate that, despite their similar characteristics, Huc and Hhy perform distinct functions during mycobacterial growth and survival.

The flagellotropic bacteriophage YSD1 targets Salmonella Typhi with a Chi-like protein tail fibre.

The discovery of a Salmonella-targeting phage from the waterways of the United Kingdom provided an opportunity to address the mechanism by which Chi-like bacteriophage (phage) engages with bacterial flagellae. The long tail fibre seen on Chi-like phages has been proposed to assist the phage particle in docking to a host cell flagellum, but the identity of the protein that generates this fibre was unknown. We present the results from genome sequencing of this phage, YSD1, confirming its close relationship to the original Chi phage and suggesting candidate proteins to form the tail structure. Immunogold labelling in electron micrographs revealed that YSD1_22 forms the main shaft of the tail tube, while YSD1_25 forms the distal part contributing to the tail spike complex. The long curling tail fibre is formed by the protein YSD1_29, and treatment of phage with the antibodies that bind YSD1_29 inhibits phage infection of Salmonella. The host range for YSD1 across Salmonella serovars is broad, but not comprehensive, being limited by antigenic features of the flagellin subunits that make up the Salmonella flagellum, with which YSD1_29 engages to initiate infection.

Engineering bacteriocin-mediated resistance against the plant pathogen Pseudomonas syringae.

The plant pathogen, Pseudomonas syringae (Ps), together with related Ps species, infects and attacks a wide range of agronomically important crops, including tomato, kiwifruit, pepper, olive and soybean, causing economic losses. Currently, chemicals and introduced resistance genes are used to protect plants against these pathogens but have limited success and may have adverse environmental impacts. Consequently, there is a pressing need to develop alternative strategies to combat bacterial disease in crops. One such strategy involves using narrow-spectrum protein antibiotics (so-called bacteriocins), which diverse bacteria use to compete against closely related species. Here, we demonstrate that one bacteriocin, putidacin L1 (PL1), can be expressed in an active form at high levels in Arabidopsis and in Nicotiana benthamiana in planta to provide effective resistance against diverse pathovars of Ps. Furthermore, we find that Ps strains that mutate to acquire tolerance to PL1 lose their O-antigen, exhibit reduced motility and still cannot induce disease symptoms in PL1-transgenic Arabidopsis. Our results provide proof-of-principle that the transgene-mediated expression of a bacteriocin in planta can provide effective disease resistance to bacterial pathogens. Thus, the expression of bacteriocins in crops might offer an effective strategy for managing bacterial disease, in the same way that the genetic modification of crops to express insecticidal proteins has proven to be an extremely successful strategy for pest management. Crucially, nearly all genera of bacteria, including many plant pathogenic species, produce bacteriocins, providing an extensive source of these antimicrobial agents.

Plumage redness signals mitochondrial function in the house finch.

Carotenoid coloration is widely recognized as a signal of individual condition in various animals, but despite decades of study, the mechanisms that link carotenoid coloration to condition remain unresolved. Most birds with red feathers convert yellow dietary carotenoids to red carotenoids in an oxidation process requiring the gene encoding the putative cytochrome P450 enzyme CYP2J19. Here, we tested the hypothesis that the process of carotenoid oxidation and feather pigmentation is functionally linked to mitochondrial performance. Consistent with this hypothesis, we observed high levels of red ketolated carotenoids associated with the hepatic mitochondria of moulting wild house finches (Haemorhous mexicanus), and upon fractionation, we found the highest concentration of ketolated carotenoids in the inner mitochondrial membrane. We further found that the redness of growing feathers was positively related to the performance of liver mitochondria. Structural modelling of CYP2J19 supports a direct role of this protein in carotenoid ketolation that may be functionally linked to cellular respiration. These observations suggest that feather coloration serves as a signal of core functionality through inexorable links to cellular respiration in the mitochondria.

A Highly Conserved Bacterial D-Serine Uptake System Links Host Metabolism and Virulence.

The ability of any organism to sense and respond to challenges presented in the environment is critically important for promoting or restricting colonization of specific sites. Recent work has demonstrated that the host metabolite D-serine has the ability to markedly influence the outcome of infection by repressing the type III secretion system of enterohaemorrhagic Escherichia coli (EHEC) in a concentration-dependent manner. However, exactly how EHEC monitors environmental D-serine is not understood. In this work, we have identified two highly conserved members of the E. coli core genome, encoding an inner membrane transporter and a transcriptional regulator, which collectively help to "sense" levels of D-serine by regulating its uptake from the environment and in turn influencing global gene expression. Both proteins are required for full expression of the type III secretion system and diversely regulated prophage-encoded effector proteins demonstrating an important infection-relevant adaptation of the core genome. We propose that this system acts as a key safety net, sampling the environment for this metabolite, thereby promoting colonization of EHEC to favorable sites within the host.

The modular nature of the ß-barrel assembly machinery, illustrated in Borrelia burgdorferi.

Diderm bacteria have an outer membrane that provides defense against environmental factors including antibiotics. Understanding the process of outer membrane biogenesis is, therefore, of critical importance in order to envisage new treatments of these bacterial pathogens. Borrelia burgdorferi is the pathogen responsible for Lyme disease. Its outer membrane contains integral, ß-barrel proteins as well as swathes of externally exposed lipoproteins. Previous work has demonstrated that the ß-barrel assembly machine (BAM complex) in B. burgdorferi and other Spirochetes shares several similarities with the BAM complex in other bacterial lineages, such as the Proteobacteria that includes Escherichia coli. However, Iqbal et al. () have identified the inner membrane protein TamB as a subunit of the BAM complex in Spirochetes. This latest study highlights the modular nature of the BAM complex, and suggests that in some bacterial lineages the BAM complex and translocation and assembly module (the TAM) function as a single unit.

Discovery, characterization and in vivo activity of pyocin SD2, a protein antibiotic from Pseudomonas aeruginosa.

Increasing rates of antibiotic resistance among Gram-negative pathogens such as Pseudomonas aeruginosa means alternative approaches to antibiotic development are urgently required. Pyocins, produced by P. aeruginosa for intraspecies competition, are highly potent protein antibiotics known to actively translocate across the outer membrane of P. aeruginosa. Understanding and exploiting the mechanisms by which pyocins target, penetrate and kill P. aeruginosa is a promising approach to antibiotic development. In this work we show the therapeutic potential of a newly identified tRNase pyocin, pyocin SD2, by demonstrating its activity in vivo in a murine model of P. aeruginosa lung infection. In addition, we propose a mechanism of cell targeting and translocation for pyocin SD2 across the P. aeruginosa outer membrane. Pyocin SD2 is concentrated at the cell surface, via binding to the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide (LPS), from where it can efficiently locate its outer membrane receptor FpvAI. This strategy of utilizing both the CPA and a protein receptor for cell targeting is common among pyocins as we show that pyocins S2, S5 and SD3 also bind to the CPA. Additional data indicate a key role for an unstructured N-terminal region of pyocin SD2 in the subsequent translocation of the pyocin into the cell. These results greatly improve our understanding of how pyocins target and translocate across the outer membrane of P. aeruginosa. This knowledge could be useful for the development of novel anti-pseudomonal therapeutics and will also support the development of pyocin SD2 as a therapeutic in its own right.

Lectin-like bacteriocins from Pseudomonas spp. utilise D-rhamnose containing lipopolysaccharide as a cellular receptor.

Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of D-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing D-rhamnose and not D-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins.

Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment.

Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

Structure of the atypical bacteriocin pectocin M2 implies a novel mechanism of protein uptake.

The colicin-like bacteriocins are potent protein antibiotics that have evolved to efficiently cross the outer membrane of Gram-negative bacteria by parasitizing nutrient uptake systems. We have structurally characterized the colicin M-like bacteriocin, pectocin M2, which is active against strains of Pectobacterium spp. This unusual bacteriocin lacks the intrinsically unstructured translocation domain that usually mediates translocation of these bacteriocins across the outer membrane, containing only a single globular ferredoxin domain connected to its cytotoxic domain by a flexible α-helix, which allows it to adopt two distinct conformations in solution. The ferredoxin domain of pectocin M2 is homologous to plant ferredoxins and allows pectocin M2 to parasitize a system utilized by Pectobacterium to obtain iron during infection of plants. Furthermore, we identify a novel ferredoxin-containing bacteriocin pectocin P, which possesses a cytotoxic domain homologous to lysozyme, illustrating that the ferredoxin domain acts as a generic delivery module for cytotoxic domains in Pectobacterium.