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The arsenite oxidase (AioAB) from Pseudorhizobium banfieldiae sp. strain NT-26 catalyzes the oxidation of arsenite to arsenate and transfers electrons to its cognate electron acceptor cytochrome c 552 (cytc 552). This activity underpins the ability of this organism to respire using arsenite present in contaminated environments. The crystal structure of the AioAB/cytc 552 electron transfer complex reveals two A2B2/(cytc 552)2 assemblies per asymmetric unit. Three of the four cytc 552 molecules in the asymmetric unit dock to AioAB in a cleft at the interface between the AioA and AioB subunits, with an edge-to-edge distance of 7.5 Å between the heme of cytc 552 and the [2Fe–2S] Rieske cluster in the AioB subunit. The interface between the AioAB and cytc 552 proteins features electrostatic and nonpolar interactions and is stabilized by two salt bridges. A modest number of hydrogen bonds, salt bridges and relatively small, buried surface areas between protein partners are typical features of transient electron transfer complexes. Interestingly, the fourth cytc 552 molecule is positioned differently between two AioAB heterodimers, with distances between its heme and the AioAB redox active cofactors that are outside the acceptable range for fast electron transfer. This unique cytc 552 molecule appears to be positioned to facilitate crystal packing rather than reflecting a functional complex.

The anaerobic bacterium Chrysiogenes arsenatis respires using the oxyanion arsenate (AsO43-) as the terminal electron acceptor, where it is reduced to arsenite (AsO33-) while concomitantly oxidizing various organic (e.g., acetate) electron donors. This respiratory activity is catalyzed in the periplasm of the bacterium by the enzyme arsenate reductase (Arr), with expression of the enzyme controlled by a sensor histidine kinase (ArrS) and a periplasmic-binding protein (PBP), ArrX. Here, we report for the first time, the molecular structure of ArrX in the absence and presence of bound ligand arsenate. Comparison of the ligand-bound structure of ArrX with other PBPs shows a high level of conservation of critical residues for ligand binding by these proteins; however, this suite of PBPs shows different structural alterations upon ligand binding. For ArrX and its homologue AioX (from Rhizobium sp. str. NT-26), which specifically binds arsenite, the structures of the substrate-binding sites in the vicinity of a conserved and critical cysteine residue contribute to the discrimination of binding for these chemically similar ligands.

Bacteriophages (phages) are obligate parasites that use host bacterial translation machinery to produce viral proteins. However, some phages have alternative genetic codes with reassigned stop codons that are predicted to be incompatible with bacterial translation systems. We analysed 9,422 phage genomes and found that stop-codon recoding has evolved in diverse clades of phages that infect bacteria present in both human and animal gut microbiota. Recoded stop codons are particularly over-represented in phage structural and lysis genes. We propose that recoded stop codons might function to prevent premature production of late-stage proteins. Stop-codon recoding has evolved several times in closely related lineages, which suggests that adaptive recoding can occur over very short evolutionary timescales.

Bacteriophages (phages) are obligate parasites that use host bacterial translation machinery to produce viral proteins. However, some phages have alternative genetic codes with reassigned stop codons that are predicted to be incompatible with bacterial translation systems. We analysed 9,422 phage genomes and found that stop-codon recoding has evolved in diverse clades of phages that infect bacteria present in both human and animal gut microbiota. Recoded stop codons are particularly over-represented in phage structural and lysis genes. We propose that recoded stop codons might function to prevent premature production of late-stage proteins. Stop-codon recoding has evolved several times in closely related lineages, which suggests that adaptive recoding can occur over very short evolutionary timescales.

The genetic code is a highly conserved feature of life. However, some “alternative” genetic codes use reassigned stop codons to code for amino acids. Here, we survey stop codon recoding across bacteriophages (phages) in human and animal gut microbiomes. We find that stop codon recoding has evolved in diverse clades of phages predicted to infect hosts that use the standard code. We provide evidence for an evolutionary path towards recoding involving reduction in the frequency of TGA and TAG stop codons due to low GC content, followed by acquisition of suppressor tRNAs and the emergence of recoded stop codons in structural and lysis genes. In analyses of two distinct lineages of recoded virulent phages, we find that lysis-related genes are uniquely biased towards use of recoded stop codons. This convergence supports the inference that stop codon recoding is a strategy to regulate the expression of late stage genes and control lysis timing. Interestingly, we identified prophages with recoded stop codons integrated into genomes of bacteria that use standard code, and hypothesize that recoding may control the lytic-lysogenic switch. Alternative coding has evolved many times, often in closely related lineages, indicating that genetic code is plastic in bacteriophages and adaptive recoding can occur over very short evolutionary timescales.

Summary Lak phages with alternatively coded ∼540 kbp genomes were recently reported to replicate in Prevotella in microbiomes of humans that consume a non-Western diet, baboons, and pigs. Here, we explore Lak phage diversity and broader distribution using diagnostic polymerase chain reaction and genome-resolved metagenomics. Lak phages were detected in 13 animal types, including reptiles, and are particularly prevalent in pigs. Tracking Lak through the pig gastrointestinal tract revealed significant enrichment in the hindgut compared to the foregut. We reconstructed 34 new Lak genomes, including six curated complete genomes, all of which are alternatively coded. An anomalously large (∼660 kbp) complete genome reconstructed for the most deeply branched Lak from a horse microbiome is also alternatively coded. From the Lak genomes, we identified proteins associated with specific animal species; notably, most have no functional predictions. The presence of closely related Lak phages in diverse animals indicates facile distribution coupled to host-specific adaptation., Graphical abstract, Highlights • PCR and genome-resolved metagenomics revealed Lak phages in 13 animal species • Lak is abundant in the pig hindgut, with potential relevance to growth performance • Thirty-four new Lak phage genomes present expanded size range and phylogeny • Unique Lak protein families from specific microbiomes suggest animal host adaptation, Virology; Microbiome; Omics

Lak phages with alternatively coded ~540 kbp genomes were recently reported to replicate in Prevotella in the gut microbiomes of humans that consume a non-western diet, baboons and some pigs. Here, we investigate the diversity and broader distribution of Lak phages in human and animal microbiomes using diagnostic PCR and genome-resolved metagenomics. Lak phages were detected in 13 different animal types and are particularly prevalent in pigs, with significant enrichment in the hindgut compared to foregut. We reconstructed 34 new Lak genomes, including six curated complete genomes, all of which are alternatively coded. The most deeply branched Lak is from a horse faecal sample and is the largest phage genome from an animal microbiome (~660 kbp). From the Lak genomes, we identified families of hypothetical proteins associated with specific animal types. Overall, we substantially expanded Lak phage diversity and demonstrate their occurrence in a variety of human and animal microbiomes.

The family Rhizobiaceae includes many genera of soil bacteria, often isolated for their association with plants. Herein, we investigate the genomic diversity of a group of Rhizobium species and unclassified strains isolated from atypical environments, including seawater, rock matrix or polluted soil. Based on whole-genome similarity and core genome phylogeny, we show that this group corresponds to the genus Pseudorhizobium. We thus reclassify Rhizobium halotolerans, R. marinum, R. flavum and R. endolithicum as P. halotolerans sp. nov., P. marinum comb. nov., P. flavum comb. nov. and P. endolithicum comb. nov., respectively, and show that P. pelagicum is a synonym of P. marinum. We also delineate a new chemolithoautotroph species, P. banfieldiae sp. nov., whose type strain is NT-26 (=DSM 106348 =CFBP 8663 ). This genome-based classification was supported by a chemotaxonomic comparison, with increasing taxonomic resolution provided by fatty acid, protein and metabolic profiles. In addition, we used a phylogenetic approach to infer scenarios of duplication, horizontal transfer and loss for all genes in the Pseudorhizobium pangenome. We thus identify the key functions associated with the diversification of each species and higher clades, shedding light on the mechanisms of adaptation to their respective ecological niches. Respiratory proteins acquired at the origin of Pseudorhizobium were combined with clade-specific genes to enable different strategies for detoxification and nutrition in harsh, nutrient-poor environments. [Abstract copyright: Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.]

SARS-CoV-2 has a zoonotic origin and was transmitted to humans via an undetermined intermediate host, leading to infections in humans and other mammals. To enter host cells, the viral spike protein (S-protein) binds to its receptor, ACE2, and is then processed by TMPRSS2. Whilst receptor binding contributes to the viral host range, S-protein:ACE2 complexes from other animals have not been investigated widely. To predict infection risks, we modelled S-protein:ACE2 complexes from 215 vertebrate species, calculated changes in the energy of the complex caused by mutations in each species, relative to human ACE2, and correlated these changes with COVID-19 infection data. We also analysed structural interactions to better understand the key residues contributing to affinity. We predict that mutations are more detrimental in ACE2 than TMPRSS2. Finally, we demonstrate phylogenetically that human SARS-CoV-2 strains have been isolated in animals. Our results suggest that SARS-CoV-2 can infect a broad range of mammals, but few fish, birds or reptiles. Susceptible animals could serve as reservoirs of the virus, necessitating careful ongoing animal management and surveillance.

The evolution of aerobic respiration was likely linked to the origins of oxygenic Cyanobacteria. Close phylogenetic neighbors to Cyanobacteria, such as Margulisbacteria (RBX-1 and ZB3), Saganbacteria (WOR-1), Melainabacteria and Sericytochromatia, may constrain the metabolic platform in which aerobic respiration arose. Here, we analyze genomic sequences and predict that sediment-associated Margulisbacteria have a fermentation-based metabolism featuring a variety of hydrogenases, a streamlined nitrogenase, and electron bifurcating complexes involved in cycling of reducing equivalents. The genomes of ocean-associated Margulisbacteria encode an electron transport chain that may support aerobic growth. Some Saganbacteria genomes encode various hydrogenases, and others may be able to use O2 under certain conditions via a putative novel type of heme copper O2 reductase. Similarly, Melainabacteria have diverse energy metabolisms and are capable of fermentation and aerobic or anaerobic respiration. The ancestor of all these groups may have been an anaerobe in which fermentation and H2 metabolism were central metabolic features. The ability to use O2 as a terminal electron acceptor must have been subsequently acquired by these lineages., Most cyanobacteria are oxygenic photoautotrophs, and fermenters under dark anoxic conditions. Here, the authors analyse genomic sequences of related uncultivated bacteria, inferring their metabolic potential, and supporting that their common ancestor was an anaerobe capable of fermentation and H2 metabolism.

Arsenic contamination of drinking water affects more than 140 million people worldwide. While toxic to humans, inorganic forms of arsenic (arsenite and arsenate), can be used as energy sources for microbial respiration. AioX and its orthologues (ArxX and ArrX) represent the first members of a new sub-family of periplasmic-binding proteins that serve as the first component of a signal transduction system, that’s role is to positively regulate expression of arsenic metabolism enzymes. As determined by X-ray crystallography for AioX, arsenite binding only requires subtle conformational changes in protein structure, providing insights into protein-ligand interactions. The binding pocket of all orthologues is conserved but this alone is not sufficient for oxyanion selectivity, with proteins selectively binding either arsenite or arsenate. Phylogenetic evidence, clearly demonstrates that the regulatory proteins evolved together early in prokaryotic evolution and had a separate origin from the metabolic enzymes whose expression they regulate.

Arsenite oxidase is thought to be an ancient enzyme, originating before the divergence of the Archaea and the Bacteria. We have investigated the nature of the molybdenum active site of the arsenite oxidase from the Alphaproteobacterium Rhizobium sp. str. NT-26 using a combination of X-ray absorption spectroscopy and computational chemistry. Our analysis indicates an oxidized Mo(VI) active site with a structure that is far from equilibrium. We propose that this is an entatic state imposed by the protein on the active site through relative orientation of the two molybdopterin cofactors, in a variant of the Rây-Dutt twist of classical coordination chemistry, which we call the pterin twist hypothesis. We discuss the implications of this hypothesis for other putatively ancient molybdopterin-based enzymes.

The family Rhizobiaceae includes many genera of soil bacteria, often isolated for their association with plants. Herein, we investigate the genomic diversity of a group of Rhizobium species and unclassified strains isolated from atypical environments, including seawater, rock matrix or polluted soil. Based on whole-genome similarity and core genome phylogeny, we show that this group corresponds to the genus Pseudorhizobium. We thus reclassify Rhizobium halotolerans, R. marinum, R. flavum and R. endolithicum as P. halotolerans comb. nov., P. marinum comb. nov., P. flavum comb. nov. and R. endolithicum comb. nov., respectively, and show that P. pelagicum is a synonym of P. marinum. We also delineate a new chemolithoautotroph species, P. banfieldiae sp. nov., whose type strain is NT-26T (= DSM 106348T = CFBP 8663T). This genome-based classification was supported by a chemotaxonomic comparison, with gradual taxonomic resolution provided by fatty acid, protein and metabolic profiles. In addition, we used a phylogenetic approach to infer scenarios of duplication, horizontal transfer and loss for all genes in the Pseudorhizobium pangenome. We thus identify the key functions associated with the diversification of each species and higher clades, shedding light on the mechanisms of adaptation to their respective ecological niches. Respiratory proteins acquired at the origin of Pseudorhizobium are combined with clade-specific genes to encode different strategies for detoxification and nutrition in harsh, nutrient-poor environments. Finally, we predict diagnostic phenotypes for the distinction of P. banfieldiae from other Pseudorhizobium species, including autotrophy and sensitivity to the azo dye Congo Red, which we experimentally validated.

Bacteriophages (phages) dramatically shape microbial community composition, redistribute nutrients via host lysis and drive evolution through horizontal gene transfer. Despite their importance, much remains to be learned about phages in the human microbiome. We investigated the gut microbiomes of humans from Bangladesh and Tanzania, two African baboon social groups and Danish pigs; many of these microbiomes contain phages belonging to a clade with genomes >540 kilobases in length, the largest yet reported in the human microbiome and close to the maximum size ever reported for phages. We refer to these as Lak phages. CRISPR spacer targeting indicates that Lak phages infect bacteria of the genus Prevotella. We manually curated to completion 15 distinct Lak phage genomes recovered from metagenomes. The genomes display several interesting features, including use of an alternative genetic code, large intergenic regions that are highly expressed and up to 35 putative transfer RNAs, some of which contain enigmatic introns. Different individuals have distinct phage genotypes, and shifts in variant frequencies over consecutive sampling days reflect changes in the relative abundance of phage subpopulations. Recent homologous recombination has resulted in extensive genome admixture of nine baboon Lak phage populations. We infer that Lak phages are widespread in gut communities that contain the Prevotella species, and conclude that megaphages, with fascinating and underexplored biology, may be common but largely overlooked components of human and animal gut microbiomes.

Phage typically have small genomes and depend on their bacterial hosts for replication. DNA sequenced from many diverse ecosystems revealed hundreds of huge phage genomes, between 200 kbp and 716 kbp in length. Thirty-four genomes were manually curated to completion, including the largest phage genomes yet reported. Expanded genetic repertoires include diverse and new CRISPR-Cas systems, tRNAs, tRNA synthetases, tRNA modification enzymes, translation initiation and elongation factors, and ribosomal proteins. Phage CRISPR-Cas systems have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phage may repurpose bacterial CRISPR-Cas systems to eliminate competing phage. We phylogenetically define major clades of huge phage from human and other animal microbiomes, oceans, lakes, sediments, soils and the built environment. We conclude that their large gene inventories reflect a conserved biological strategy, observed over a broad bacterial host range and across Earth’s ecosystems.

Arsenate respiration by bacteria was discovered over two decades ago and is catalyzed by diverse organisms using the well-conserved Arr enzyme complex. Until now, the mechanisms underpinning this metabolism have been relatively opaque. Here, we report the structure of an Arr complex (solved by X-ray crystallography to 1.6-Å resolution), which was enabled by an improved Arr expression method in the genetically tractable arsenate respirer Shewanella sp. ANA-3. We also obtained structures bound with the substrate arsenate (1.8 Å), the product arsenite (1.8 Å), and the natural inhibitor phosphate (1.7 Å). The structures reveal a conserved active-site motif that distinguishes Arr [(R/K)GRY] from the closely related arsenite respiratory oxidase (Arx) complex (XGRGWG). Arr activity assays using methyl viologen as the electron donor and arsenate as the electron acceptor display two-site ping-pong kinetics. A Mo(V) species was detected with EPR spectroscopy, which is typical for proteins with a pyranopterin guanine dinucleotide cofactor. Arr is an extraordinarily fast enzyme that approaches the diffusion limit (K(m) = 44.6 ± 1.6 μM, k(cat) = 9,810 ± 220 seconds(−1)), and phosphate is a competitive inhibitor of arsenate reduction (K(i) = 325 ± 12 μM). These observations, combined with knowledge of typical sedimentary arsenate and phosphate concentrations and known rates of arsenate desorption from minerals in the presence of phosphate, suggest that (i) arsenate desorption limits microbiologically induced arsenate reductive mobilization and (ii) phosphate enhances arsenic mobility by stimulating arsenate desorption rather than by inhibiting it at the enzymatic level.

Bacteriophages (phages) dramatically shape microbial community composition, redistribute nutrients via host lysis and drive evolution through horizontal gene transfer. Despite their importance, much remains to be learned about phages in the human microbiome. We investigated the gut microbiomes of humans from Bangladesh and Tanzania, two African baboon social groups and Danish pigs; many of these microbiomes contain phages belonging to a clade with genomes 540 kilobases in length, the largest yet reported in the human microbiome and close to the maximum size ever reported for phages. We refer to these as Lak phages. CRISPR spacer targeting indicates that Lak phages infect bacteria of the genus Prevotella. We manually curated to completion 15 distinct Lak phage genomes recovered from metagenomes. The genomes display several interesting features, including use of an alternative genetic code, large intergenic regions that are highly expressed and up to 35 putative transfer RNAs, some of which contain enigmatic introns. Different individuals have distinct phage genotypes, and shifts in variant frequencies over consecutive sampling days reflect changes in the relative abundance of phage subpopulations. Recent homologous recombination has resulted in extensive genome admixture of nine baboon Lak phage populations. We infer that Lak phages are widespread in gut communities that contain the Prevotella species, and conclude that megaphages, with fascinating and underexplored biology, may be common but largely overlooked components of human and animal gut microbiomes.

Bacteriophage (phage) dramatically shape microbial community composition, redistribute nutrients via host lysis, and drive evolution through horizontal gene transfer. Despite their importance, much remains to be learned about phage in the human microbiome. We investigated gut microbiomes of humans from Bangladesh and Tanzania, two African baboon social groups, and Danish pigs, and report that many contain phage belonging to a clade with genomes >540 kb in length, the largest yet reported in the human microbiome and close to the maximum size ever reported for phage. We refer to these as Lak phage. CRISPR spacer targeting indicates that the Lak phage infect bacteria of the genus Prevotella. We manually curated to completion 15 distinct Lak phage genomes recovered from metagenomes. The genomes display several interesting features, including use of an alternative genetic code, large intergenic regions that are highly expressed, and up to 35 putative tRNAs, some of which contain enigmatic introns. Different individuals have distinct phage genotypes, and shifts in variant frequencies over consecutive sampling days reflect changes in relative abundance of phage sub-populations. Recent homologous recombination has resulted in extensive genome admixture of nine baboon Lak phage populations. We infer that Lak phage are widespread in gut communities that contain Prevotella species, especially in individuals in the developing world, and conclude that megaphage, with fascinating and underexplored biology, may be common but largely overlooked components of human and animal gut microbiomes.

The metabolic machinery from which microbial aerobic respiration evolved is tightly linked to the origins of oxygenic Cyanobacteria (Oxyphotobacteria). Even though the majority of Oxyphotobacteria are photoautotrophs and can use carbohydrates with oxygen (O2) as the electron acceptor, all are fermenters under dark anoxic conditions. Studies suggest that the ancestor of Oxyphotobacteria may have used hydrogen (H2) as an electron donor and that two types of NiFe hydrogenases are essential for its oxidation. Melainabacteria and Sericytochromatia, close phylogenetic neighbors to Oxyphotobacteria comprise fermentative and aerobic representatives, or organisms capable of both. Margulisbacteria (candidate divisions RBX-1 and ZB3) and Saganbacteria (candidate division WOR-1), a novel cluster of bacteria phylogenetically related to Melainabacteria, Sericytochromatia and Oxyphotobacteria may further constrain the metabolic platform in which oxygenic photosynthesis and aerobic respiration arose. Here, we predict the metabolisms of Margulisbacteria and Saganbacteria from new and published metagenome-assembled genomes (MAGs) and single amplified genomes (SAGs), and compare them to their phylogenetic neighbors. Sediment-associated Margulisbacteria are predicted to have a fermentation-based metabolism featuring a variety of hydrogenases, a nitrogenase for nitrogen (N2) fixation, and electron bifurcating complexes involved in cycling of ferredoxin and NAD(P)H. Overall, the genomic features suggest the capacity for metabolic fine-tuning under strictly anoxic conditions. In contrast, the genomes of Margulisbacteria from the ocean ecosystem encode an electron transport chain that supports aerobic growth. Similarly, some Saganbacteria genomes encode various hydrogenases, and others may have the ability to use O2 under certain conditions via a putative novel type of heme copper O2 reductase. Like Melainabacteria and Sericytochromatia, Margulisbacteria and Saganbacteria have diverse energy metabolisms capable of fermentation, and aerobic or anaerobic respiration. In summary, our findings support the hypothesis that the ancestor of these groups was an anaerobe in which fermentation and H2 metabolism were central metabolic features. Our genomic data also suggests that contemporary lineages sibling to the Oxyphotobacteria may have acquired the ability to use O2 as a terminal electron acceptor under certain environmental conditions.

The toxic element arsenic (As) occurs widely in solid and liquid mine wastes. Aqueous forms of arsenic are taken up in As-bearing sulfides, arsenides, sulfosalts, oxides, oxyhydroxides, Fe-oxides, -hydroxides, -oxyhydroxides and -sulfates, and Fe-, Ca-Fe- and other arsenates. Although a considerable body of research has demonstrated that microbes play a significant role in the precipitation and dissolution of these As-bearing minerals, and in the alteration of the redox state of As, in natural and simulated mining environments, the molecular-scale mechanisms of these interactions are still not well understood. Further research is required using traditional and novel mineralogical, spectroscopic and microbiological techniques to further advance this field, and to help design remediation schemes.

Summary Conjugative pili are widespread bacterial appendages that play important roles in horizontal gene transfer, in spread of antibiotic resistance genes, and as sites of phage attachment. Among conjugative pili, the F “sex” pilus encoded by the F plasmid is the best functionally characterized, and it is also historically the most important, as the discovery of F-plasmid-mediated conjugation ushered in the era of molecular biology and genetics. Yet, its structure is unknown. Here, we present atomic models of two F family pili, the F and pED208 pili, generated from cryoelectron microscopy reconstructions at 5.0 and 3.6 Å resolution, respectively. These structures reveal that conjugative pili are assemblies of stoichiometric protein-phospholipid units. We further demonstrate that each pilus type binds preferentially to particular phospholipids. These structures provide the molecular basis for F pilus assembly and also shed light on the remarkable properties of conjugative pili in bacterial secretion and phage infection., Graphical Abstract Image 1, Highlights • The atomic structures of the F pilus and an F-like pilus are solved by cryo-EM • The structure reveals an assembly of a protein-phospholipid complex unit • Mutations at subunit-lipid interfaces affect phage infection and conjugation • The structure elucidates the molecular basis for DNA transfer and phage attachment, Conjugative pili, the appendages that enable DNA transfer between bacteria, are not made only of protein but, as near-atomic resolution cryoEM reveals, are assemblies of stoichiometric protein-phospholipid units, with the phospholipid component suited to electrochemically facilitating DNA transport.

NT-26 is a chemolithoautotrophic arsenite oxidizer. Understanding the mechanisms of arsenite signalling, tolerance and oxidation by NT-26 will have significant implications for its use in bioremediation and arsenite sensing. We have identified the histidine kinase (AroS) and the cognate response regulator (AroR) involved in the arsenite-dependent transcriptional regulation of the arsenite oxidase aroBA operon. AroS contains a single periplasmic sensory domain that is linked through transmembrane helices to the HAMP domain that transmits the signal to the kinase core of the protein. AroR belongs to a family of AAA+ transcription regulators that interact with DNA through a helix-turn-helix domain. The presence of the AAA+ domain as well as the RNA polymerase σ54-interaction sequence motif suggests that this protein regulates transcription through interaction with RNA polymerase in a σ54-dependent fashion. The kinase core of AroS and the receiver domain of AroR were heterologously expressed and purified and their autophosphorylation and transphosphorylation activities were confirmed. Using site-directed mutagenesis, we have identified the phosphorylation sites on both proteins. Mutational analysis in NT-26 confirmed that both proteins are essential for arsenite oxidation and the AroS mutant affected growth with arsenite, also implicating it in the regulation of arsenite tolerance. Lastly, arsenite sensing does not appear to involve thiol chemistry.

Here, we describe the characterization of the [2Fe-2S] clusters of arsenite oxidases from Rhizobium sp. NT-26 and Ralstonia sp. 22. Both reduced Rieske proteins feature EPR signals similar to their homologs from Rieske-cyt b complexes, with g values at 2.027, 1.88, and 1.77. Redox titrations in a range of pH values showed that both [2Fe-2S] centers have constant Em values up to pH 8 at ∼+210 mV. Above this pH value, the Em values of both centers are pH-dependent, similar to what is observed for the Rieske-cyt b complexes. The redox properties of these two proteins, together with the low Em value (+160 mV) of the Alcaligenes faecalis arsenite oxidase Rieske (confirmed herein), are in line with the structural determinants observed in the primary sequences, which have previously been deduced from the study of Rieske-cyt b complexes. Since the published Em value of the Chloroflexus aurantiacus Rieske (+100 mV) is in conflict with this sequence analysis, we re-analyzed membrane samples of this organism and obtain a new value (+200 mV). Arsenite oxidase activity was affected by quinols and quinol analogs, which is similar to what is found with the Rieske-cyt b complexes. Together, these results show that the Rieske protein of arsenite oxidase shares numerous properties with its counterpart in the Rieske-cyt b complex. However, two cysteine residues, strictly conserved in the Rieske-cyt b-Rieske and considered to be crucial for its function, are not conserved in the arsenite oxidase counterpart. We discuss the role of these residues.

A biosensor for arsenite has been developed using molybdenum-containing arsenite oxidase, prepared from the chemolithoautotroph NT-26 that oxidizes arsenite to arsenate. The enzyme was galvanostatically deposited for 10 min at 10 microA onto the active surface of a multiwalled carbon nanotube modified glassy carbon electrode. The resulting biosensor enabled direct electron transfer, i.e., effecting reduction and then reoxidization of the enzyme without an artificial electron-transfer mediator. Arsenite was detected within 10 s at an applied potential of 0.3 V with linearity up to 500 ppb and a detection limit of 1 ppb. The biosensor exhibited excellent reproducibility, 2% at 95% confidence interval for 12 repeated analyses of 25 ppb arsenite. Copper, a severe interfering species commonly found in groundwater, did not interfere, and the biosensor was applicable for repeated analysis of spiked arsenite in tap water, river water, and a commercial mineral water.

The arsenic (As) drinking water crisis in south and south-east Asia has stimulated intense study of the microbial processes controlling the redox cycling of As in soil-water systems. Microbial oxidation of arsenite is a critical link in the global As cycle, and phylogenetically diverse arsenite-oxidizing microorganisms have been isolated from various aquatic and soil environments. However, despite progress characterizing the metabolism of As in various pure cultures, no functional gene approaches have been developed to determine the importance and distribution of arsenite-oxidizing genes in soil-water-sediment systems. Here we report for the first time the successful amplification of arsenite oxidase-like genes (aroA/asoA/aoxB) from a variety of soil-sediment and geothermal environments where arsenite is known to be oxidized. Prior to the current work, only 16 aroA/asoA/aoxB-like gene sequences were available in GenBank, most of these being putative assignments from homology searches of whole genomes. Although aroA/asoA/aoxB gene sequences are not highly conserved across disparate phyla, degenerate primers were used successfully to characterize over 160 diverse aroA-like sequences from 10 geographically isolated, arsenic-contaminated sites and from 13 arsenite-oxidizing organisms. The primer sets were also useful for confirming the expression of aroA-like genes in an arsenite-oxidizing organism and in geothermal environments where arsenite is oxidized to arsenate. The phylogenetic and ecological diversity of aroA-like sequences obtained from this study suggests that genes for aerobic arsenite oxidation are widely distributed in the bacterial domain, are widespread in soil-water systems containing As, and play a critical role in the biogeochemical cycling of As.

A facultative chemoautotrophic bacterium, strain MLHE-1T, was isolated from Mono Lake, an alkaline hypersaline soda lake in California, USA. Cells of strain MLHE-1T were Gram-negative, short motile rods that grew with inorganic electron donors (arsenite, hydrogen, sulfide or thiosulfate) coupled with the reduction of nitrate to nitrite. No aerobic growth was attained with arsenite or sulfide, but hydrogen sustained both aerobic and anaerobic growth. No growth occurred when nitrite or nitrous oxide was substituted for nitrate. Heterotrophic growth was observed under aerobic and anaerobic (nitrate) conditions. Cells of strain MLHE-1T could oxidize but not grow on CO, while CH4 neither supported growth nor was it oxidized. When grown chemoautotrophically, strain MLHE-1T assimilated inorganic carbon via the Calvin–Benson–Bassham reductive pentose phosphate pathway, with the activity of ribulose 1,5-bisphosphate carboxylase (RuBisCO) functioning optimally at 0.1 M NaCl and at pH 7.3. Strain MLHE-1T grew over broad ranges of pH (7.3–10.0; optimum, 9.3), salinity (15–190 g l−1; optimum 30 g l−1) and temperature (13–40 °C; optimum, 30 °C). Phylogenetic analysis of 16S rRNA gene sequences placed strain MLHE-1T in the class Gammaproteobacteria (family Ectothiorhodospiraceae) and most closely related to Alkalispirillum mobile (98.5 %) and Alkalilimnicola halodurans (98.6 %), although none of these three haloalkaliphilic micro-organisms were capable of photoautotrophic growth and only strain MLHE-1T was able to oxidize As(III). On the basis of physiological characteristics and DNA–DNA hybridization data, it is suggested that strain MLHE-1T represents a novel species within the genus Alkalilimnicola for which the name Alkalilimnicola ehrlichii is proposed. The type strain is MLHE-1T (=DSM 17681T=ATCC BAA-1101T). Aspects of the annotated full genome of Alkalilimnicola ehrlichii are discussed in the light of its physiology.

International audience; While the molybdenum cofactor in the majority of bisPGD enzymes goes through two consecutive 1-electron redox transitions, previous protein-film voltammetric results indicated the possibility of cooperative (n = 2) redox behavior in the bioenergetic enzyme arsenite oxidase (Aio). Combining equilibrium redox titrations, optical and EPR spectroscopies on concentrated samples obtained via heterologous expression, we unambiguously confirm this claim and quantify Aio's redox cooperativity. The stability constant, Ks, of the MoV semi-reduced intermediate is found to be lower than 10− 3. Site-directed mutagenesis of residues in the vicinity of the Mo-cofactor demonstrates that the degree of redox cooperativity is sensitive to H-bonding interactions between the pyranopterin moieties and amino acid residues. Remarkably, in particular replacing the Gln-726 residue by Gly results in stabilization of (low-temperature) EPR-observable MoV with KS = 4. As evidenced by comparison of room temperature optical and low temperature EPR titrations, the degree of stabilization is temperature-dependent. This highlights the importance of room-temperature redox characterizations for correctly interpreting catalytic properties in this group of enzymes.Geochemical and phylogenetic data strongly indicate that molybdenum played an essential biocatalytic roles in early life. Molybdenum's redox versatility and in particular the ability to show cooperative (n = 2) redox behavior provide a rationale for its paramount catalytic importance throughout the evolutionary history of life. Implications of the H-bonding network modulating Molybdenum's redox properties on details of a putative inorganic metabolism at life's origin are discussed.

Acidithiobacillus ferrooxidans is an acidophile that thrives in metal-contaminated environments and tolerates high levels of uranium. To gain a better understanding of the processes involved in U(VI) resistance, comparative proteomics was used. The proteome of A. ferrooxidans was grown in the presence and absence of 0.5 mM U(VI); expression of 17 proteins was upregulated and one was downregulated. Most proteins with increased expression are part of the general stress response or are involved in reactive oxygen species detoxification. Four novel proteins showed increased expression in the presence of U(VI) and may contribute to U(VI) resistance via thiol homoeostasis and U(VI) binding.

Natural pollution of groundwater by arsenic adversely affects the health of tens of millions of people worldwide, with the deltaic aquifers of SE Asia being particularly polluted. The pollution is caused primarily by, or as a side reaction of, the microbial reduction of sedimentary Fe(III)-oxyhydroxides, but the organism(s) responsible for As release have not been isolated. Here we report the first isolation of a dissimilatory arsenate reducer from sediments of the Bengal Basin in West Bengal. The bacterium, here designated WB3, respires soluble arsenate and couples its reduction to the oxidation of acetate; WB3 is therefore implicated in the process of arsenic pollution of groundwater, which is largely by arsenite. The bacterium WB3 is also capable of reducing dissolved Fe(III) citrate, solid Fe(III)-oxyhydroxide, and elemental sulfur, using acetate as the electron donor. It is a member of the Desulfuromonas genus and possesses a dissimilatory arsenate reductase that was identified using degenerate polymerase chain reaction primers. The sediment from which WB3 was isolated was brown, Pleistocene sand at a depth of 35.2 m below ground level (mbgl). This level was some 3 cm below the boundary between the brown sands and overlying reduced, gray, Holocene aquifer sands. The color boundary is interpreted to be a reduction front that releases As for resorption downflow, yielding a high load of labile As sorbed to the sediment at a depth of 35.8 mbgl and concentrations of As in groundwater that reach1000 μg/L.

Arsenic and antimony are toxic metalloids and are considered priority environmental pollutants by the U.S. Environmental Protection Agency. Significant advances have been made in understanding microbe-arsenic interactions and how they influence arsenic redox speciation in the environment. However, even the most basic features of how and why a microorganism detects and reacts to antimony remain poorly understood. Previous work with Agrobacterium tumefaciens strain 5A concluded that oxidation of antimonite [Sb(III)] and arsenite [As(III)] required different biochemical pathways. Here, we show with in vivo experiments that a mutation in aioA [encoding the large subunit of As(III) oxidase] reduces the ability to oxidize Sb(III) by approximately one-third relative to the ability of the wild type. Further, in vitro studies with the purified As(III) oxidase from Rhizobium sp. strain NT-26 (AioA shares 94% amino acid sequence identity with AioA of A. tumefaciens ) provide direct evidence of Sb(III) oxidation but also show a significantly decreased V max compared to that of As(III) oxidation. The aioBA genes encoding As(III) oxidase are induced by As(III) but not by Sb(III), whereas arsR gene expression is induced by both As(III) and Sb(III), suggesting that detection and transcriptional responses for As(III) and Sb(III) differ. While Sb(III) and As(III) are similar with respect to cellular extrusion (ArsB or Acr3) and interaction with ArsR, they differ in the regulatory mechanisms that control the expression of genes encoding the different Ars or Aio activities. In summary, this study documents an enzymatic basis for microbial Sb(III) oxidation, although additional Sb(III) oxidation activity also is apparent in this bacterium.

Heterotrophic arsenite oxidation by Hydrogenophaga sp. str. NT-14 is coupled to the reduction of oxygen and appears to yield energy for growth. Purification and partial characterization of the arsenite oxidase revealed that it (1). contains two heterologous subunits, AroA (86 kDa) and AroB (16 kDa), (2). has a native molecular mass of 306 kDa suggesting an alpha(3)beta(3) configuration, and (3). contains molybdenum and iron as cofactors. Although the Hydrogenophaga sp. str. NT-14 arsenite oxidase shares similarities to the arsenite oxidases purified from NT-26 and Alcaligenes faecalis, it differs with respect to activity and overall conformation. A c-551-type cytochrome was purified from Hydrogenophaga sp. str. NT-14 and appears to be the physiological electron acceptor for the arsenite oxidase. The cytochrome can also accept electrons from the purified NT-26 arsenite oxidase. A hypothetical electron transport chain for heterotrophic arsenite oxidation is proposed.

Arsenite [As(III)]-enriched anoxic bottom water from Mono Lake, California, produced arsenate [As(V)] during incubation with either nitrate or nitrite. No such oxidation occurred in killed controls or in live samples incubated without added nitrate or nitrite. A small amount of biological As(III) oxidation was observed in samples amended with Fe(III) chelated with nitrolotriacetic acid, although some chemical oxidation was also evident in killed controls. A pure culture, strain MLHE-1, that was capable of growth with As(III) as its electron donor and nitrate as its electron acceptor was isolated in a defined mineral salts medium. Cells were also able to grow in nitrate-mineral salts medium by using H 2 or sulfide as their electron donor in lieu of As(III). Arsenite-grown cells demonstrated dark 14 CO 2 fixation, and PCR was used to indicate the presence of a gene encoding ribulose-1,5-biphosphate carboxylase/oxygenase. Strain MLHE-1 is a facultative chemoautotroph, able to grow with these inorganic electron donors and nitrate as its electron acceptor, but heterotrophic growth on acetate was also observed under both aerobic and anaerobic (nitrate) conditions. Phylogenetic analysis of its 16S ribosomal DNA sequence placed strain MLHE-1 within the haloalkaliphilic Ectothiorhodospira of the γ- Proteobacteria . Arsenite oxidation has never been reported for any members of this subgroup of the Proteobacteria .

A new strictly anaerobic arsenate-respiring bacterium has been isolated from arseniccontaminated mud obtained from a gold mine in Bendigo, Australia. This organism, designated JMM-4, was found to be a Gram-positive, spore-forming rod, 0.6 2 2.5-3 w m, motile by means of flagella that are subpolar or along one side of the cell. JMM-4 grows using arsenate as the terminal electron acceptor and lactate as the electron donor. Arsenate is reduced to arsenite and the lactate is oxidized to CO2 via the intermediate, acetate. The doubling time for exponential growth with arsenate (5 mM) and lactate (5 mM) was 4.3 - 0.2 h. Alternative electron donors used by JMM-4 when grown with arsenate as the terminal electron acceptor are acetate, pyruvate, succinate, malate, glutamate, and hydrogen (with acetate as carbon source). Apart from arsenate, nitrate can serve as an alternative electron acceptor.Optimal growth occurs at pH 7.8 with a sodium chloride concentration of 1.2 g · l -1 . Based upon 16S rRNA gene sequence ana...

Nine novel arsenite-oxidizing bacteria have been isolated from two different gold mine environments in Australia. Four of these organisms grow chemolithoautotrophically with oxygen as the terminal electron acceptor, arsenite as the electron donor, and carbon dioxide-bicarbonate as the sole carbon source. Five heterotrophic arsenite-oxidizing bacteria were also isolated, one of which was found to be both phylogenetically and physiologically identical to the previously described heterotrophic arsenite oxidizer misidentified as Alcaligenes faecalis . The results showed that this strain belongs to the genus Achromobacter . Phylogenetically, the arsenite-oxidizing bacteria fall within two separate subdivisions of the Proteobacteria . Interestingly, the chemolithoautotrophic arsenite oxidizers belong to the f - Proteobacteria , whereas the heterotrophic arsenite oxidizers belong to the g - Proteobacteria .

One of the issues facing the nuclear power industry is how to store spent nuclear fuel which is contaminated with radionuclides produced during nuclear fission, including caesium ((134)Cs(+), (135)Cs(+) and (137)Cs(+)) and cobalt ((60)Co(2+)). In this study, we have isolated Co(2+)- and Cs(+)-resistant bacteria from water collected from a nuclear fuel storage pond. The most resistant Cs(+) and Co(2+) isolates grew in the presence of 500 mM CsCl and 3 mM CoCl2. Strain Cs67-2 is resistant to fourfold more Cs(+) than Cupriavidus metallidurans str. CH34 making it the most Cs(+)-resistant strain identified to date. The Cs(+)-resistant isolates were closely related to bacteria in the Serratia and Yersinia genera, while the Co(2+)-resistant isolates were closely related to the Curvibacter and Tardiphaga genera. These new isolates could be used for bioremediation.

Two sulfate-reducing bacteria, which also reduce arsenate, were isolated; both organisms oxidized lactate incompletely to acetate. When using lactate as the electron donor, one of these organisms, Desulfomicrobium strain Ben-RB, rapidly reduced (doubling time = 8 h) 5.1 mM arsenate at the same time it reduced sulfate (9.6 mM). Sulfate reduction was not inhibited by the presence of arsenate. Arsenate could act as the terminal electron acceptor in minimal medium (doubling time = 9 h) in the absence of sulfate. Arsenate was reduced by a membrane-bound enzyme that is either a c-type cytochrome or is associated with such a cytochrome; benzyl-viologen-dependent arsenate reductase activity was greater in cells grown with arsenate/sulfate than in cells grown with sulfate only. The second organism, Desulfovibrio strain Ben-RA, also grew (doubling time = 8 h) while reducing arsenate (3.1 mM) and sulfate (8.3 mM) concomitantly. No evidence was found, however, that this organism is able to grow using arsenate as the terminal electron acceptor. Instead, it appears that arsenate reduction by the Desulfovibrio strain Ben-RA is catalyzed by an arsenate reductase that is encoded by a chromosomally-borne gene shown to be homologous to the arsC gene of the Escherichia coli plasmid, R773 ars system.

The arsenite oxidase (Aio) from the facultative autotrophic Alphaproteobacterium Rhizobium sp. NT-26 is a bioenergetic enzyme involved in the oxidation of arsenite to arsenate. The enzyme from the distantly related heterotroph, Alcaligenes faecalis, which is thought to oxidise arsenite for detoxification, consists of a large α subunit (AioA) with bis-molybdopterin guanine dinucleotide at its active site and a 3Fe-4S cluster, and a small β subunit (AioB) which contains a Rieske 2Fe-2S cluster. The successful heterologous expression of the NT-26 Aio in Escherichia coli has resulted in the solution of its crystal structure. The NT-26 Aio, a heterotetramer, shares high overall similarity to the heterodimeric arsenite oxidase from A. faecalis but there are striking differences in the structure surrounding the Rieske 2Fe-2S cluster which we demonstrate explains the difference in the observed redox potentials (+225 mV vs. +130/160 mV, respectively). A combination of site-directed mutagenesis and electron paramagnetic resonance was used to explore the differences observed in the structure and redox properties of the Rieske cluster. In the NT-26 AioB the substitution of a serine (S126 in NT-26) for a threonine as in the A. faecalis AioB explains a −20 mV decrease in redox potential. The disulphide bridge in the A. faecalis AioB which is conserved in other betaproteobacterial AioB subunits and the Rieske subunit of the cytochrome bc 1 complex is absent in the NT-26 AioB subunit. The introduction of a disulphide bridge had no effect on Aio activity or protein stability but resulted in a decrease in the redox potential of the cluster. These results are in conflict with previous data on the betaproteobacterial AioB subunit and the Rieske of the bc 1 complex where removal of the disulphide bridge had no effect on the redox potential of the former but a decrease in cluster stability was observed in the latter.

We describe the catalytic voltammograms of the periplasmic arsenite oxidase (Aio) from the chemolithoautotrophic bacterium Rhizobium sp. str. NT-26 that oxidizes arsenite to arsenate. Electrochemistry of the enzyme was accomplished using its native electron transfer partner, cytochrome c552 (cyt c552), as a mediator. The protein cyt c552 adsorbed on a mercaptoundecanoic acid (MUA) modified Au electrode exhibited a stable, reversible one-electron voltammetric response at +275mV vs NHE (pH6). In the presence of arsenite and Aio the voltammetry of cyt c552 is transformed from a transient response to an amplified sigmoidal (steady state) wave consistent with an electro-catalytic system. Digital simulation was performed using a single set of parameters for all catalytic voltammetries obtained at different sweep rates and various substrate concentrations. The obtained kinetic constants from digital simulation provide new insight into the kinetics of the NT-26 Aio catalytic mechanism.