Trace gas oxidation sustains energy needs of a thermophilic archaeon at suboptimal temperatures.

Diverse aerobic bacteria use atmospheric hydrogen (H2) and carbon monoxide (CO) as energy sources to support growth and survival. Such trace gas oxidation is recognised as a globally significant process that serves as the main sink in the biogeochemical H2 cycle and sustains microbial biodiversity in oligotrophic ecosystems. However, it is unclear whether archaea can also use atmospheric H2. Here we show that a thermoacidophilic archaeon, Acidianus brierleyi (Thermoproteota), constitutively consumes H2 and CO to sub-atmospheric levels. Oxidation occurs across a wide range of temperatures (10 to 70 °C) and enhances ATP production during starvation-induced persistence under temperate conditions. The genome of A. brierleyi encodes a canonical CO dehydrogenase and four distinct [NiFe]-hydrogenases, which are differentially produced in response to electron donor and acceptor availability. Another archaeon, Metallosphaera sedula, can also oxidize atmospheric H2. Our results suggest that trace gas oxidation is a common trait of Sulfolobales archaea and may play a role in their survival and niche expansion, including during dispersal through temperate environments.

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.

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.

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.

Microbial oxidation of atmospheric trace gases.

The atmosphere has recently been recognized as a major source of energy sustaining life. Diverse aerobic bacteria oxidize the three most abundant reduced trace gases in the atmosphere, namely hydrogen (H2), carbon monoxide (CO) and methane (CH4). This Review describes the taxonomic distribution, physiological role and biochemical basis of microbial oxidation of these atmospheric trace gases, as well as the ecological, environmental, medical and astrobiological importance of this process. Most soil bacteria and some archaea can survive by using atmospheric H2 and CO as alternative energy sources, as illustrated through genetic studies on Mycobacterium cells and Streptomyces spores. Certain specialist bacteria can also grow on air alone, as confirmed by the landmark characterization of Methylocapsa gorgona, which grows by simultaneously consuming atmospheric CH4, H2 and CO. Bacteria use high-affinity lineages of metalloenzymes, namely hydrogenases, CO dehydrogenases and methane monooxygenases, to utilize atmospheric trace gases for aerobic respiration and carbon fixation. More broadly, trace gas oxidizers enhance the biodiversity and resilience of soil and marine ecosystems, drive primary productivity in extreme environments such as Antarctic desert soils and perform critical regulatory services by mitigating anthropogenic emissions of greenhouse gases and toxic pollutants.

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.

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.

BonA from Acinetobacter baumannii Forms a Divisome-Localized Decamer That Supports Outer Envelope Function.

Acinetobacter baumannii is a high-risk pathogen due to the rapid global spread of multidrug-resistant lineages. Its phylogenetic divergence from other ESKAPE pathogens means that determinants of its antimicrobial resistance can be difficult to extrapolate from other widely studied bacteria. A recent study showed that A. baumannii upregulates production of an outer membrane lipoprotein, which we designate BonA, in response to challenge with polymyxins. Here, we show that BonA has limited sequence similarity and distinct structural features compared to lipoproteins from other bacterial species. Analyses through X-ray crystallography, small-angle X-ray scattering, electron microscopy, and multiangle light scattering demonstrate that BonA has a dual BON (Bacterial OsmY and Nodulation) domain architecture and forms a decamer via an unusual oligomerization mechanism. This analysis also indicates this decamer is transient, suggesting dynamic oligomerization plays a role in BonA function. Antisera recognizing BonA shows it is an outer membrane protein localized to the divisome. Loss of BonA modulates the density of the outer membrane, consistent with a change in its structure or link to the peptidoglycan, and prevents motility in a clinical strain (ATCC 17978). Consistent with these findings, the dimensions of the BonA decamer are sufficient to permeate the peptidoglycan layer, with the potential to form a membrane-spanning complex during cell division. IMPORTANCE The pathogen Acinetobacter baumannii is considered an urgent threat to human health. A. baumannii is highly resistant to treatment with antibiotics, in part due to its protective cell envelope. This bacterium is only distantly related to other bacterial pathogens, so its cell envelope has distinct properties and contains components distinct from those of other bacteria that support its function. Here, we report the discovery of BonA, a protein that supports A. baumannii outer envelope function and is required for cell motility. We determine the atomic structure of BonA and show that it forms part of the cell division machinery and functions by forming a complex, features that mirror those of distantly related homologs from other bacteria. By improving our understanding of the A. baumannii cell envelope this work will assist in treating this pathogen.

Mycobacteria Tolerate Carbon Monoxide by Remodeling Their Respiratory Chain.

Carbon monoxide (CO) gas is infamous for its acute toxicity. This toxicity predominantly stems from its tendency to form carbonyl complexes with transition metals, thus inhibiting the heme-prosthetic groups of proteins, including respiratory terminal oxidases. While CO has been proposed as an antibacterial agent, the evidence supporting its toxicity toward bacteria is equivocal, and its cellular targets remain poorly defined. In this work, we investigate the physiological response of mycobacteria to CO. We show that Mycobacterium smegmatis is highly resistant to the toxic effects of CO, exhibiting only minor inhibition of growth when cultured in its presence. We profiled the proteome of M. smegmatis during growth in CO, identifying strong induction of cytochrome bd oxidase and members of the dos regulon, but relatively few other changes. We show that the activity of cytochrome bd oxidase is resistant to CO, whereas cytochrome bcc-aa 3 oxidase is strongly inhibited by this gas. Consistent with these findings, growth analysis shows that M. smegmatis lacking cytochrome bd oxidase displays a significant growth defect in the presence of CO, while induction of the dos regulon appears to be unimportant for adaptation to CO. Altogether, our findings indicate that M. smegmatis has considerable resistance to CO and benefits from respiratory flexibility to withstand its inhibitory effects.IMPORTANCE Carbon monoxide has an infamous reputation as a toxic gas, and it has been suggested that it has potential as an antibacterial agent. Despite this, how bacteria resist its toxic effects is not well understood. In this study, we investigated how CO influences growth, proteome, and aerobic respiration of wild-type and mutant strains of Mycobacterium smegmatis We show that this bacterium produces the CO-resistant cytochrome bd oxidase to tolerate poisoning of its CO-sensitive complex IV homolog. Further, we show that aside from this remodeling of its respiratory chain, M. smegmatis makes few other functional changes to its proteome, suggesting it has a high level of inherent resistance to CO.

Cofactor F420: an expanded view of its distribution, biosynthesis and roles in bacteria and archaea.

Many bacteria and archaea produce the redox cofactor F420. F420 is structurally similar to the cofactors FAD and FMN but is catalytically more similar to NAD and NADP. These properties allow F420 to catalyze challenging redox reactions, including key steps in methanogenesis, antibiotic biosynthesis and xenobiotic biodegradation. In the last 5 years, there has been much progress in understanding its distribution, biosynthesis, role and applications. Whereas F420 was previously thought to be confined to Actinobacteria and Euryarchaeota, new evidence indicates it is synthesized across the bacterial and archaeal domains, as a result of extensive horizontal and vertical biosynthetic gene transfer. F420 was thought to be synthesized through one biosynthetic pathway; however, recent advances have revealed variants of this pathway and have resolved their key biosynthetic steps. In parallel, new F420-dependent biosynthetic and metabolic processes have been discovered. These advances have enabled the heterologous production of F420 and identified enantioselective F420H2-dependent reductases for biocatalysis. New research has also helped resolve how microorganisms use F420 to influence human and environmental health, providing opportunities for tuberculosis treatment and methane mitigation. A total of 50 years since its discovery, multiple paradigms associated with F420 have shifted, and new F420-dependent organisms and processes continue to be discovered.

Trace gas oxidizers are widespread and active members of soil microbial communities.

Soil microorganisms globally are thought to be sustained primarily by organic carbon sources. Certain bacteria also consume inorganic energy sources such as trace gases, but they are presumed to be rare community members, except within some oligotrophic soils. Here we combined metagenomic, biogeochemical and modelling approaches to determine how soil microbial communities meet energy and carbon needs. Analysis of 40 metagenomes and 757 derived genomes indicated that over 70% of soil bacterial taxa encode enzymes to consume inorganic energy sources. Bacteria from 19 phyla encoded enzymes to use the trace gases hydrogen and carbon monoxide as supplemental electron donors for aerobic respiration. In addition, we identified a fourth phylum (Gemmatimonadota) potentially capable of aerobic methanotrophy. Consistent with the metagenomic profiling, communities within soil profiles from diverse habitats rapidly oxidized hydrogen, carbon monoxide and to a lesser extent methane below atmospheric concentrations. Thermodynamic modelling indicated that the power generated by oxidation of these three gases is sufficient to meet the maintenance needs of the bacterial cells capable of consuming them. Diverse bacteria also encode enzymes to use trace gases as electron donors to support carbon fixation. Altogether, these findings indicate that trace gas oxidation confers a major selective advantage in soil ecosystems, where availability of preferred organic substrates limits microbial growth. The observation that inorganic energy sources may sustain most soil bacteria also has broad implications for understanding atmospheric chemistry and microbial biodiversity in a changing world.

A widely distributed hydrogenase oxidises atmospheric H2 during bacterial growth.

Diverse aerobic bacteria persist by consuming atmospheric hydrogen (H2) using group 1h [NiFe]-hydrogenases. However, other hydrogenase classes are also distributed in aerobes, including the group 2a [NiFe]-hydrogenase. Based on studies focused on Cyanobacteria, the reported physiological role of the group 2a [NiFe]-hydrogenase is to recycle H2 produced by nitrogenase. However, given this hydrogenase is also present in various heterotrophs and lithoautotrophs lacking nitrogenases, it may play a wider role in bacterial metabolism. Here we investigated the role of this enzyme in three species from different phylogenetic lineages and ecological niches: Acidithiobacillus ferrooxidans (phylum Proteobacteria), Chloroflexus aggregans (phylum Chloroflexota), and Gemmatimonas aurantiaca (phylum Gemmatimonadota). qRT-PCR analysis revealed that the group 2a [NiFe]-hydrogenase of all three species is significantly upregulated during exponential growth compared to stationary phase, in contrast to the profile of the persistence-linked group 1h [NiFe]-hydrogenase. Whole-cell biochemical assays confirmed that all three strains aerobically respire H2 to sub-atmospheric levels, and oxidation rates were much higher during growth. Moreover, the oxidation of H2 supported mixotrophic growth of the carbon-fixing strains C. aggregans and A. ferrooxidans. Finally, we used phylogenomic analyses to show that this hydrogenase is widely distributed and is encoded by 13 bacterial phyla. These findings challenge the current persistence-centric model of the physiological role of atmospheric H2 oxidation and extend this process to two more phyla, Proteobacteria and Gemmatimonadota. In turn, these findings have broader relevance for understanding how bacteria conserve energy in different environments and control the biogeochemical cycling of atmospheric trace gases.

The architecture and stabilisation of flagellotropic tailed bacteriophages.

Flagellotropic bacteriophages engage flagella to reach the bacterial surface as an effective means to increase the capture radius for predation. Structural details of these viruses are of great interest given the substantial drag forces and torques they face when moving down the spinning flagellum. We show that the main capsid and auxiliary proteins form two nested chainmails that ensure the integrity of the bacteriophage head. Core stabilising structures are conserved in herpesviruses suggesting their ancestral origin. The structure of the tail also reveals a robust yet pliable assembly. Hexameric rings of the tail-tube protein are braced by the N-terminus and a ß-hairpin loop, and interconnected along the tail by the splayed ß-hairpins. By contrast, we show that the ß-hairpin has an inhibitory role in the tail-tube precursor, preventing uncontrolled self-assembly. Dyads of acidic residues inside the tail-tube present regularly-spaced motifs well suited to DNA translocation into bacteria through the tail.

Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F420-0 in Mycobacteria.

F420 is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F420 in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F420 biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-phospholactate stimulates F420 biosynthesis. Analysis of F420 biosynthesis intermediates present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F420-0. To determine the structural basis of dehydro-F420-0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo-, substrate-, and product-bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic precomplex of this enzyme and demonstrate that dehydro-F420-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F420 in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance.IMPORTANCE Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F420, and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F420 is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F420 in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F420, determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F420.

The crystal structure of the TonB-dependent transporter YncD reveals a positively charged substrate-binding site.

The outer membrane of Gram-negative bacteria is highly impermeable to hydrophilic molecules of larger than 600 Da, protecting these bacteria from toxins present in the environment. In order to transport nutrients across this impermeable membrane, Gram-negative bacteria utilize a diverse family of outer-membrane proteins called TonB-dependent transporters. The majority of the members of this family transport iron-containing substrates. However, it is becoming increasingly clear that TonB-dependent transporters target chemically diverse substrates. In this work, the structure and phylogenetic distribution of the TonB-dependent transporter YncD are investigated. It is shown that while YncD is present in some enteropathogens, including Escherichia coli and Salmonella spp., it is also widespread in Gammaproteobacteria and Betaproteobacteria of environmental origin. The structure of YncD was determined, showing that despite a distant evolutionary relationship, it shares structural features with the ferric citrate transporter FecA, including a compact positively charged substrate-binding site. Despite these shared features, it is shown that YncD does not contribute to the growth of E. coli in pure culture under iron-limiting conditions or with ferric citrate as an iron source. Previous studies of transcriptional regulation in E. coli show that YncD is not induced under iron-limiting conditions and is unresponsive to the ferric uptake regulator (Fur). These observations, combined with the data presented here, suggest that YncD is not responsible for the transport of an iron-containing substrate.

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.

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.

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.

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.

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.