Structure of the Bacterial Sex F Pilus Reveals an Assembly of a Stoichiometric Protein-Phospholipid Complex.

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.

Clades of huge phages from across Earth's ecosystems.

Bacteriophages typically have small genomes1 and depend on their bacterial hosts for replication2. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is-to our knowledge-the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR-Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR-Cas systems of phages 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 phages may repurpose bacterial CRISPR-Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth's ecosystems.

Arsenite oxidase in complex with antimonite and arsenite oxyanions: Insights into the catalytic mechanism.

Arsenic contamination of groundwater is among one of the biggest health threats affecting millions of people in the world. There is an urgent need for efficient arsenic biosensors where the use of arsenic metabolizing enzymes can be explored. In this work, we have solved four crystal structures of arsenite oxidase (Aio) in complex with arsenic and antimony oxyanions and the structures determined correspond to intermediate states of the enzymatic mechanism. These structural data were complemented with density-functional theory calculations providing a unique view of the molybdenum active site at different time points that, together with mutagenesis data, enabled to clarify the enzymatic mechanism and the molecular determinants for the oxidation of As(III) to the less toxic As(V) species.

Decoding huge phage diversity: a taxonomic classification of Lak megaphages.

High-throughput sequencing for uncultivated viruses has accelerated the understanding of global viral diversity and uncovered viral genomes substantially larger than any that have so far been cultured. Notably, the Lak phages are an enigmatic group of viruses that present some of the largest known phage genomes identified in human and animal microbiomes, and are dissimilar to any cultivated viruses. Despite the wealth of viral diversity that exists within sequencing datasets, uncultivated viruses have rarely been used for taxonomic classification. We investigated the evolutionary relationships of 23 Lak phages and propose a taxonomy for their classification. Predicted protein analysis revealed the Lak phages formed a deeply branching monophyletic clade within the class Caudoviricetes which contained no other phage genomes. One of the interesting features of this clade is that all current members are characterised by an alternative genetic code. We propose the Lak phages belong to a new order, the 'Grandevirales'. Protein and nucleotide-based analyses support the creation of two families, three sub-families, and four genera within the order 'Grandevirales'. We anticipate that the proposed taxonomy of Lak megaphages will simplify the future classification of related viral genomes as they are uncovered. Continued efforts to classify divergent viruses are crucial to aid common analyses of viral genomes and metagenomes.

Structural and Functional Investigation of the Periplasmic Arsenate-Binding Protein ArrX from Chrysiogenes arsenatis.

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.

Structural and mechanistic analysis of the arsenate respiratory reductase provides insight into environmental arsenic transformations.

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 (Km = 44.6 ± 1.6 µM, kcat = 9,810 ± 220 seconds-1), and phosphate is a competitive inhibitor of arsenate reduction (Ki = 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.

Reservoirs of faecal indicator bacteria in well-head hand pumps in Bangladesh.

The majority of the population of Bangladesh (90%) rely on untreated groundwater for drinking and domestic use. At the point of collection, 40% of these supplies are contaminated with faecal indicator bacteria (FIB). Recent studies have disproved the theory that latrines discharging to shallow aquifers are the major contributor to this contamination. In this study, we tested the hypothesis that hand pumps are a reservoir of FIB. We sampled the handle, spout, piston and seal from 19 wells in Araihazar Upazila, Bangladesh and identified that the spout and seal were reservoirs of FIB. These findings led to our recommendation that well spouts be regularly cleaned, including the removal of precipitated deposits, and that the seals be regularly changed. It is envisaged that one or both of these interventions will reduce the numbers of FIB in drinking water, thereby reducing the burden of diarrhoeal disease in Bangladesh.

Electron transfer through arsenite oxidase: Insights into Rieske interaction with cytochrome c.

Arsenic is a widely distributed environmental toxin whose presence in drinking water poses a threat to >140 million people worldwide. The respiratory enzyme arsenite oxidase from various bacteria catalyses the oxidation of arsenite to arsenate and is being developed as a biosensor for arsenite. The arsenite oxidase from Rhizobium sp. str. NT-26 (a member of the Alphaproteobacteria) is a heterotetramer consisting of a large catalytic subunit (AioA), which contains a molybdenum centre and a 3Fe-4S cluster, and a small subunit (AioB) containing a Rieske 2Fe-2S cluster. Stopped-flow spectroscopy and isothermal titration calorimetry (ITC) have been used to better understand electron transfer through the redox-active centres of the enzyme, which is essential for biosensor development. Results show that oxidation of arsenite at the active site is extremely fast with a rate of >4000s-1 and reduction of the electron acceptor is rate-limiting. An AioB-F108A mutation results in increased activity with the artificial electron acceptor DCPIP and decreased activity with cytochrome c, which in the latter as demonstrated by ITC is not due to an effect on the protein-protein interaction but instead to an effect on electron transfer. These results provide further support that the AioB F108 is important in electron transfer between the Rieske subunit and cytochrome c and its absence in the arsenite oxidases from the Betaproteobacteria may explain the inability of these enzymes to use this electron acceptor.

The H-bond network surrounding the pyranopterins modulates redox cooperativity in the molybdenum-bisPGD cofactor in arsenite oxidase.

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 Mo(V) 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 Mo(V) 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.

Electrochemically driven catalysis of Rhizobium sp. NT-26 arsenite oxidase with its native electron acceptor cytochrome c552.

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.

Arsenite oxidase also functions as an antimonite oxidase.

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 Vmax 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.

Isolation of an arsenate-respiring bacterium from a redox front in an arsenic-polluted aquifer in West Bengal, Bengal Basin.

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 reach >1000 µg/L.

Heterologously expressed arsenite oxidase: a system to study biogenesis and structure/function relationships of the enzyme family.

Studies of native arsenite oxidases from Ralstonia sp. S22 and Rhizobium sp. NT-26 raised two major questions. The first one concerns the mode of the enzyme's membrane-association. It has been suggested that a hypothetical not conserved protein could account for this variable association. Expression of the wild type arsenite oxidase in Escherichia coli allowed us to study the cellular localization of this enzyme in the absence of such a hypothetical partner. The results with the Ralstonia sp. S22 enzyme suggest that no additional protein is required for membrane association. The second question addresses the influence of the disulfide bridge in the small Rieske subunit, conspicuously absent in the Rhizobium sp. NT-26 enzyme, on the properties of the [2Fe-2S] center. The disulfide bridge is considered to be formed only after translocation of the enzyme to the periplasm. To address this question we thus first expressed the enzyme in the absence of its Twin-arginine translocation signal sequence. The spectral and redox properties of the cytoplasmic enzyme are unchanged compared to the periplasmic one. We finally studied a disulfide bridge mutant, Cys106Ala, devoid of the first Cys involved in the disulfide bridge formation. This mutation, proposed to have a strong effect on redox and catalytic properties of the Rieske protein in Rieske/cytb complexes, had no significant effect on properties of the Rieske protein from arsenite oxidase. Our present results demonstrate that the effects attributed to the disulfide bridge in the Rieske/cytb complexes are likely to be secondary effects due to conformational changes.

Mechanisms of bioleaching: iron and sulfur oxidation by acidophilic microorganisms.

Bioleaching offers a low-input method of extracting valuable metals from sulfide minerals, which works by exploiting the sulfur and iron metabolisms of microorganisms to break down the ore. Bioleaching microbes generate energy by oxidising iron and/or sulfur, consequently generating oxidants that attack sulfide mineral surfaces, releasing target metals. As sulfuric acid is generated during the process, bioleaching organisms are typically acidophiles, and indeed the technique is based on natural processes that occur at acid mine drainage sites. While the overall concept of bioleaching appears straightforward, a series of enzymes is required to mediate the complex sulfur oxidation process. This review explores the mechanisms underlying bioleaching, summarising current knowledge on the enzymes driving microbial sulfur and iron oxidation in acidophiles. Up-to-date models are provided of the two mineral-defined pathways of sulfide mineral bioleaching: the thiosulfate and the polysulfide pathway.

The structure of the complex between the arsenite oxidase from Pseudorhizobium banfieldiae sp. strain NT-26 and its native electron acceptor cytochrome c552.

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 c552 (cytc552). This activity underpins the ability of this organism to respire using arsenite present in contaminated environments. The crystal structure of the AioAB/cytc552 electron transfer complex reveals two A2B2/(cytc552)2 assemblies per asymmetric unit. Three of the four cytc552 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 Šbetween the heme of cytc552 and the [2Fe-2S] Rieske cluster in the AioB subunit. The interface between the AioAB and cytc552 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 cytc552 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 cytc552 molecule appears to be positioned to facilitate crystal packing rather than reflecting a functional complex.

Detection and enumeration of Lak megaphages in microbiome samples by endpoint and quantitative PCR.

Lak megaphages are prevalent across diverse gut microbiomes and may potentially impact animal and human health through lysis of Prevotella. Given their large genome size (up to 660 kbp), Lak megaphages are difficult to culture, and their identification relies on molecular techniques. Here, we present optimized protocols for identifying Lak phages in various microbiome samples, including procedures for DNA extraction, followed by detection and quantification of genes encoding Lak structural proteins using diagnostic endpoint and SYBR green-based quantitative PCR, respectively. For complete details on the use and execution of this protocol, please refer to Crisci et al., (2021).

Global genomic analysis of microbial biotransformation of arsenic highlights the importance of arsenic methylation in environmental and human microbiomes.

Arsenic is a ubiquitous toxic element, the global cycle of which is highly affected by microbial redox reactions and assimilation into organoarsenic compounds through sequential methylation reactions. While microbial biotransformation of arsenic has been studied for decades, the past years have seen the discovery of multiple new genes related to arsenic metabolism. Still, most studies focus on a small set of key genes or a small set of cultured microorganisms. Here, we leveraged the recently greatly expanded availability of microbial genomes of diverse organisms from lineages lacking cultivated representatives, including those reconstructed from metagenomes, to investigate genetic repertoires of taxonomic and environmental controls on arsenic metabolic capacities. Based on the collection of arsenic-related genes, we identified thirteen distinct metabolic guilds, four of which combine the aio and ars operons. We found that the best studied phyla have very different combinations of capacities than less well-studied phyla, including phyla lacking isolated representatives. We identified a distinct arsenic gene signature in the microbiomes of humans exposed or likely exposed to drinking water contaminated by arsenic and that arsenic methylation is important in soil and in human microbiomes. Thus, the microbiomes of humans exposed to arsenic have the potential to exacerbate arsenic toxicity. Finally, we show that machine learning can predict bacterial arsenic metabolism capacities based on their taxonomy and the environment from which they were sampled.

Widespread stop-codon recoding in bacteriophages may regulate translation of lytic genes.

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 small subunit AroB of arsenite oxidase: lessons on the [2Fe-2S] Rieske protein superfamily.

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 E(m) values up to pH 8 at approximately +210 mV. Above this pH value, the E(m) 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 E(m) 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 E(m) 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.

Quinol-cytochrome c oxidoreductase and cytochrome c4 mediate electron transfer during selenate respiration in Thauera selenatis.

Selenate reductase (SER) from Thauera selenatis is a periplasmic enzyme that has been classified as a type II molybdoenzyme. The enzyme comprises three subunits SerABC, where SerC is an unusual b-heme cytochrome. In the present work the spectropotentiometric characterization of the SerC component and the identification of redox partners to SER are reported. The mid-point redox potential of the b-heme was determined by optical titration (E(m) + 234 +/- 10 mV). A profile of periplasmic c-type cytochromes expressed in T. selenatis under selenate respiring conditions was undertaken. Two c-type cytochromes were purified ( approximately 24 and approximately 6 kDa), and the 24-kDa protein (cytc-Ts4) was shown to donate electrons to SerABC in vitro. Protein sequence of cytc-Ts4 was obtained by N-terminal sequencing and liquid chromatography-tandem mass spectrometry analysis, and based upon sequence similarities, was assigned as a member of cytochrome c(4) family. Redox potentiometry, combined with UV-visible spectroscopy, showed that cytc-Ts4 is a diheme cytochrome with a redox potential of +282 +/- 10 mV, and both hemes are predicted to have His-Met ligation. To identify the membrane-bound electron donors to cytc-Ts4, growth of T. selenatis in the presence of respiratory inhibitors was monitored. The specific quinol-cytochrome c oxidoreductase (QCR) inhibitors myxothiazol and antimycin A partially inhibited selenate respiration, demonstrating that some electron flux is via the QCR. Electron transfer via a QCR and a diheme cytochrome c(4) is a novel route for a member of the DMSO reductase family of molybdoenzymes.