On the evolution and physiology of cable bacteria.

Cable bacteria of the family Desulfobulbaceae form centimeter-long filaments comprising thousands of cells. They occur worldwide in the surface of aquatic sediments, where they connect sulfide oxidation with oxygen or nitrate reduction via long-distance electron transport. In the absence of pure cultures, we used single-filament genomics and metagenomics to retrieve draft genomes of 3 marine Candidatus Electrothrix and 1 freshwater Ca. Electronema species. These genomes contain >50% unknown genes but still share their core genomic makeup with sulfate-reducing and sulfur-disproportionating Desulfobulbaceae, with few core genes lost and 212 unique genes (from 197 gene families) conserved among cable bacteria. Last common ancestor analysis indicates gene divergence and lateral gene transfer as equally important origins of these unique genes. With support from metaproteomics of a Ca. Electronema enrichment, the genomes suggest that cable bacteria oxidize sulfide by reversing the canonical sulfate reduction pathway and fix CO2 using the Wood-Ljungdahl pathway. Cable bacteria show limited organotrophic potential, may assimilate smaller organic acids and alcohols, fix N2, and synthesize polyphosphates and polyglucose as storage compounds; several of these traits were confirmed by cell-level experimental analyses. We propose a model for electron flow from sulfide to oxygen that involves periplasmic cytochromes, yet-unidentified conductive periplasmic fibers, and periplasmic oxygen reduction. This model proposes that an active cable bacterium gains energy in the anodic, sulfide-oxidizing cells, whereas cells in the oxic zone flare off electrons through intense cathodic oxygen respiration without energy conservation; this peculiar form of multicellularity seems unparalleled in the microbial world.

The Tubular Sheaths Encasing Methanosaeta thermophila Filaments Are Functional Amyloids.

Archaea are renowned for their ability to thrive in extreme environments, although they can be found in virtually all habitats. Their adaptive success is linked to their unique cell envelopes that are extremely resistant to chemical and thermal denaturation and that resist proteolysis by common proteases. Here we employ amyloid-specific conformation antibodies and biophysical techniques to show that the extracellular cell wall sheaths encasing the methanogenic archaea Methanosaeta thermophila PT are functional amyloids. Depolymerization of sheaths and subsequent MS/MS analyses revealed that the sheaths are composed of a single major sheath protein (MspA). The amyloidogenic nature of MspA was confirmed by in vitro amyloid formation of recombinant MspA under a wide range of environmental conditions. This is the first report of a functional amyloid from the archaeal domain of life. The amyloid nature explains the extreme resistance of the sheath, the elastic properties that allow diffusible substrates to penetrate through expandable hoop boundaries, and how the sheaths are able to split and elongate outside the cell. The archaeal sheath amyloids do not share homology with any of the currently known functional amyloids and clearly represent a new function of the amyloid protein fold.

Against the odds? De novo structure determination of a pilin with two cysteine residues by sulfur SAD.

Exploiting the anomalous signal of the intrinsic S atoms to phase a protein structure is advantageous, as ideally only a single well diffracting native crystal is required. However, sulfur is a weak anomalous scatterer at the typical wavelengths used for X-ray diffraction experiments, and therefore sulfur SAD data sets need to be recorded with a high multiplicity. In this study, the structure of a small pilin protein was determined by sulfur SAD despite several obstacles such as a low anomalous signal (a theoretical Bijvoet ratio of 0.9% at a wavelength of 1.8 Å), radiation damage-induced reduction of the cysteines and a multiplicity of only 5.5. The anomalous signal was improved by merging three data sets from different volumes of a single crystal, yielding a multiplicity of 17.5, and a sodium ion was added to the substructure of anomalous scatterers. In general, all data sets were balanced around the threshold values for a successful phasing strategy. In addition, a collection of statistics on structures from the PDB that were solved by sulfur SAD are presented and compared with the data. Looking at the quality indicator R(anom)/R(p.i.m.), an inconsistency in the documentation of the anomalous R factor is noted and reported.

Mutational analysis of the yeast RNA helicase Sub2p reveals conserved domains required for growth, mRNA export, and genomic stability.

Sub2p/UAP56 is a highly conserved DEAD-box RNA helicase involved in the packaging and nuclear export of mRNA/protein particles (mRNPs). In Saccharomyces cerevisiae, Sub2p is recruited to active chromatin by the pentameric THO complex and incorporated into the larger transcription-export (TREX) complex. Sub2p also plays a role in the maintenance of genome integrity as its inactivation causes severe transcription-dependent recombination of DNA. Despite the central role of Sub2p in early mRNP biology, little is known about its function. Here, we report the presence of an N-terminal motif (NTM) conserved specifically in the Sub2p branch of RNA helicases. Mutation of the NTM causes nuclear accumulation of poly(A)(+) RNA and impaired growth without affecting core helicase functions. Thus, the NTM functions as an autonomous unit. Moreover, two sub2 mutants, that are deficient in ATP binding, act in a trans-dominant negative fashion for growth and induce high recombination rates in vivo. Although wild-type Sub2p is prevented access to transcribed loci in such a background, this does not mechanistically explain the phenotype.

High-resolution structure of a type IV pilin from the metal-reducing bacterium Shewanella oneidensis.

BACKGROUND: Type IV pili are widely expressed among Gram-negative bacteria, where they are involved in biofilm formation, serve in the transfer of DNA, motility and in the bacterial attachment to various surfaces. Type IV pili in Shewanella oneidensis are also supposed to play an important role in extracellular electron transfer by the attachment to sediments containing electron acceptors and potentially forming conductive nanowires. RESULTS: The potential nanowire type IV pilin PilBac1 from S. oneidensis was characterized by a combination of complementary structural methods and the atomic structure was determined at a resolution of 1.67 Å by X-ray crystallography. PilBac1 consists of one long N-terminal α-helix packed against four antiparallel ß-strands, thus revealing the core fold of type IV pilins. In the crystal, PilBac1 forms a parallel dimer with a sodium ion bound to one of the monomers. Interestingly, our PilBac1 crystal structure reveals two unusual features compared to other type IVa pilins: an unusual position of the disulfide bridge and a straight α-helical section, which usually exhibits a pronounced kink. This straight helix leads to a distinct packing in a filament model of PilBac1 based on an EM model of a Neisseria pilus. CONCLUSIONS: In this study we have described the first structure of a pilin from Shewanella oneidensis. The structure possesses features of the common type IV pilin core, but also exhibits significant variations in the α-helical part and the D-region.

Structural basis of Δ9-THC analog activity at the Cannabinoid 1 receptor.

Δ9-tetrahydrocannabinol (THC) is the principal psychoactive compound derived from the cannabis plant Cannabis sativa and approved for emetic conditions, appetite stimulation and sleep apnea relief. THC's psychoactive actions are mediated primarily by the cannabinoid receptor CB1. Here, we determine the cryo-EM structure of HU210, a THC analog and widely used tool compound, bound to CB1 and its primary transducer, Gi1. We leverage this structure for docking and 1,000 ns molecular dynamics simulations of THC and 10 structural analogs delineating their spatiotemporal interactions at the molecular level. Furthermore, we pharmacologically profile their recruitment of Gi and ß-arrestins and reversibility of binding from an active complex. By combining detailed CB1 structural information with molecular models and signaling data we uncover the differential spatiotemporal interactions these ligands make to receptors governing potency, efficacy, bias and kinetics. This may help explain the actions of abused substances, advance fundamental receptor activation studies and design better medicines.

Protein expression, crystallization and preliminary X-ray crystallographic analysis of the isolated Shigella flexneri VapC toxin.

Upon release from the stable complex formed with its antitoxin VapB, the toxin VapC (MvpT) of the Gram-negative pathogen Shigella flexneri is capable of globally down-regulating translation by specifically cleaving initiator tRNA(fMet) in the anticodon region. Recombinant Shigella flexneri VapC(D7A) harbouring an active-site mutation was overexpressed in Escherichia coli, purified to homogeneity and crystallized by the vapour-diffusion technique. A preliminary X-ray crystallographic analysis shows that the crystals diffracted to at least 1.9 Å resolution at a synchrotron X-ray source and belonged to the trigonal space group in the hexagonal setting, H3, with unit-cell parameters a = b = 120.1, c = 52.5 Å, α = ß = 90, γ = 120°. The Matthews coefficient is 2.46 Å(3) Da(-1), suggesting two molecules per asymmetric unit and corresponding to a solvent content of 50.0%.

Structure, folding and flexibility of co-transcriptional RNA origami.

RNA origami is a method for designing RNA nanostructures that can self-assemble through co-transcriptional folding with applications in nanomedicine and synthetic biology. However, to advance the method further, an improved understanding of RNA structural properties and folding principles is required. Here we use cryogenic electron microscopy to study RNA origami sheets and bundles at sub-nanometre resolution revealing structural parameters of kissing-loop and crossover motifs, which are used to improve designs. In RNA bundle designs, we discover a kinetic folding trap that forms during folding and is only released after 10 h. Exploration of the conformational landscape of several RNA designs reveal the flexibility of helices and structural motifs. Finally, sheets and bundles are combined to construct a multidomain satellite shape, which is characterized by individual-particle cryo-electron tomography to reveal the domain flexibility. Together, the study provides a structural basis for future improvements to the design cycle of genetically encoded RNA nanodevices.

How to grow your cable bacteria: Establishment of a stable single-strain culture in sediment and proposal of Candidatus Electronema aureum GS.

Cable bacteria are multicellular filamentous bacteria within the Desulfobulbaceae that couple the oxidation of sulfide to the reduction of oxygen over centimeter distances via long distance electron transport (LDET). So far, none of the freshwater or marine cable bacteria species have been isolated into pure culture. Here we describe a method for establishing a stable single-strain cable bacterium culture in partially sterilized sediment. By repeated transfers of a single cable bacterium filament from freshwater pond sediment into autoclaved sediment, we obtained strain GS, identified by its 16S rRNA gene as a member of Ca. Electronema. This strain was further propagated by transferring sediment clumps, and has now been stable within its semi-natural microbial community for several years. Its metagenome-assembled genome was 93% complete, had a size of 2.76 Mbp, and a DNA G + C content of 52%. Average Nucleotide Identity (ANI) and Average Amino Acid Identity (AAI) suggest the affiliation of strain GS to Ca. Electronema as a novel species. Cell size, number of outer ridges, and detection of LDET in the GS culture are likewise consistent with Ca. Electronema. Based on these combined features, we therefore describe strain GS as a new cable bacterium species of the candidate genus Electronema, for which we propose the name Candidatus Electronema aureum sp.nov. Although not a pure culture, this stable single-strain culture will be useful for physiological and omics-based studies; similar approaches with single-cell or single-filament transfers into natural medium may also aid the characterization of other difficult-to-culture microbes.

Predicted Loop Regions Promote Aggregation: A Study of Amyloidogenic Domains in the Functional Amyloid FapC.

Protein fibrillation is traditionally associated with misfolding, loss of functional phenotype, and gain of toxicity in neurodegenerative diseases. However, many organisms exploit fibrils in the form of functional amyloids (FA), as seen in bacteria, such as E. coli, Salmonella, Bacillus, and Pseudomonas. Here, we provide structural information and mechanistic data for fibrillation of the smallest amyloidogenic truncation unit along with the full-length version (FL) of the major amyloid protein FapC from Pseudomonas, predicted to consist of three ß-hairpin-forming imperfect repeats separated by disordered regions. Using a series of truncation mutants, we establish that the putative loops (linkers) increase the rate of aggregation. The minimal aggregation unit consisting of a single repeat with flanking disordered regions (R3C) aggregates in a pathway dominated by secondary nucleation, in contrast to the primary nucleation favored by full-length (FL) FapC. SAXS on FapC FL, R3C, and remaining truncation constructs resolves two major coexisting species in the fibrillation process, namely pre-fibrillar loosely aggregated monomers, and cylindrical, elliptical cross-section fibrils. Solid-state NMR spectra identified rigid parts of the FapC fibril. We assigned Cα-Cß chemical shifts, indicative of a predominant ß-sheet topology with some α-helix or loop chemical shifts. Our work emphasizes the complex nature of FapC fibrillation. In addition, we are able to deduce the importance of non-repeat regions (i.e., predicted loops), which enhance the amyloid protein aggregation and their influence on the polymorphism of the fibril architecture.

An Hfq-like protein in archaea: crystal structure and functional characterization of the Sm protein from Methanococcus jannaschii.

The Sm and Sm-like proteins are conserved in all three domains of life and have emerged as important players in many different RNA-processing reactions. Their proposed role is to mediate RNA-RNA and/or RNA-protein interactions. In marked contrast to eukaryotes, bacteria appear to contain only one distinct Sm-like protein belonging to the Hfq family of proteins. Similarly, there are generally only one or two subtypes of Sm-related proteins in archaea, but at least one archaeon, Methanococcus jannaschii, encodes a protein that is related to Hfq. This archaeon does not contain any gene encoding a conventional archaeal Sm-type protein, suggesting that Hfq proteins and archaeal Sm-homologs can complement each other functionally. Here, we report the functional characterization of M. jannaschii Hfq and its crystal structure at 2.5 A resolution. The protein forms a hexameric ring. The monomer fold, as well as the overall structure of the complex is similar to that found for the bacterial Hfq proteins. However, clear differences are seen in the charge distribution on the distal face of the ring, which is unusually negative in M. jannaschii Hfq. Moreover, owing to a very short N-terminal alpha-helix, the overall diameter of the archaeal Hfq hexamer is significantly smaller than its bacterial counterparts. Functional analysis reveals that Escherichia coli and M. jannaschii Hfqs display very similar biochemical and biological properties. It thus appears that the archaeal and bacterial Hfq proteins are largely functionally interchangeable.

The Cell Envelope Structure of Cable Bacteria.

Cable bacteria are long, multicellular micro-organisms that are capable of transporting electrons from cell to cell along the longitudinal axis of their centimeter-long filaments. The conductive structures that mediate this long-distance electron transport are thought to be located in the cell envelope. Therefore, this study examines in detail the architecture of the cell envelope of cable bacterium filaments by combining different sample preparation methods (chemical fixation, resin-embedding, and cryo-fixation) with a portfolio of imaging techniques (scanning electron microscopy, transmission electron microscopy and tomography, focused ion beam scanning electron microscopy, and atomic force microscopy). We systematically imaged intact filaments with varying diameters. In addition, we investigated the periplasmic fiber sheath that remains after the cytoplasm and membranes were removed by chemical extraction. Based on these investigations, we present a quantitative structural model of a cable bacterium. Cable bacteria build their cell envelope by a parallel concatenation of ridge compartments that have a standard size. Larger diameter filaments simply incorporate more parallel ridge compartments. Each ridge compartment contains a ~50 nm diameter fiber in the periplasmic space. These fibers are continuous across cell-to-cell junctions, which display a conspicuous cartwheel structure that is likely made by invaginations of the outer cell membrane around the periplasmic fibers. The continuity of the periplasmic fibers across cells makes them a prime candidate for the sought-after electron conducting structure in cable bacteria.

Saposin-Lipoprotein Scaffolds for Structure Determination of Membrane Transporters.

Membrane proteins depend on their natural lipid environment for function, which makes them more difficult to study in isolation. A number of approaches that mimic the lipid bilayer of biological membranes have been described (nanodiscs, SMALPs), enabling novel ways to assay activity and elucidate structures of this important class of proteins. More recently, the use of saposin A, a protein that is involved in lipid transport, to form Salipro (saposin-lipid-protein) complexes was demonstrated for a range of membrane protein targets (Frauenfeld et al., 2016). The method is fast and requires few resources. The saposin-lipid-scaffold adapts to various sizes of transmembrane regions during self-assembly, forming a minimal lipid nanoparticle. This results in the formation of a well-defined membrane protein-lipid complex, which is desirable for structural characterization. Here, we describe a protocol to reconstitute the sarco-endoplasmic reticulum calcium ATPase (SERCA) into Salipro nanoparticles. The complex formation is analyzed using negative stain electron microscopy (EM), allowing to quickly determine an initial structure of the membrane protein and to evaluate sample conditions for structural studies using single-particle cryo-EM in a detergent-free environment.

A taxonomic framework for cable bacteria and proposal of the candidate genera Electrothrix and Electronema.

Cable bacteria are long, multicellular filaments that can conduct electric currents over centimeter-scale distances. All cable bacteria identified to date belong to the deltaproteobacterial family Desulfobulbaceae and have not been isolated in pure culture yet. Their taxonomic delineation and exact phylogeny is uncertain, as most studies so far have reported only short partial 16S rRNA sequences or have relied on identification by a combination of filament morphology and 16S rRNA-targeted fluorescence in situ hybridization with a Desulfobulbaceae-specific probe. In this study, nearly full-length 16S rRNA gene sequences of 16 individual cable bacteria filaments from freshwater, salt marsh, and marine sites of four geographic locations are presented. These sequences formed a distinct, monophyletic sister clade to the genus Desulfobulbus and could be divided into six coherent, species-level clusters, arranged as two genus-level groups. The same grouping was retrieved by phylogenetic analysis of full or partial dsrAB genes encoding the dissimilatory sulfite reductase. Based on these results, it is proposed to accommodate cable bacteria within two novel candidate genera: the mostly marine "Candidatus Electrothrix", with four candidate species, and the mostly freshwater "Candidatus Electronema", with two candidate species. This taxonomic framework can be used to assign environmental sequences confidently to the cable bacteria clade, even without morphological information. Database searches revealed 185 16S rRNA gene sequences that affiliated within the clade formed by the proposed cable bacteria genera, of which 120 sequences could be assigned to one of the six candidate species, while the remaining 65 sequences indicated the existence of up to five additional species.

The crystal structure of the intact E. coli RelBE toxin-antitoxin complex provides the structural basis for conditional cooperativity.

The bacterial relBE locus encodes a toxin-antitoxin complex in which the toxin, RelE, is capable of cleaving mRNA in the ribosomal A site cotranslationally. The antitoxin, RelB, both binds and inhibits RelE, and regulates transcription through operator binding and conditional cooperativity controlled by RelE. Here, we present the crystal structure of the intact Escherichia coli RelB2E2 complex at 2.8 Å resolution, comprising both the RelB-inhibited RelE and the RelB dimerization domain that binds DNA. RelE and RelB associate into a V-shaped heterotetrameric complex with the ribbon-helix-helix (RHH) dimerization domain at the apex. Our structure supports a model in which relO is optimally bound by two adjacent RelB2E heterotrimeric units, and is not compatible with concomitant binding of two RelB2E2 heterotetramers. The results thus provide a firm basis for understanding the model of conditional cooperativity at the molecular level.

Crystal structure of the VapBC toxin-antitoxin complex from Shigella flexneri reveals a hetero-octameric DNA-binding assembly.

Toxin-antitoxin (TA) loci are common in archaea and prokaryotes and allow cells to rapidly adapt to changing environmental conditions through release of active regulators of metabolism. Many toxins are endonucleases that target cellular mRNA and tRNAs, while the antitoxins tightly wrap around the toxins to inhibit them under normal circumstances. The antitoxins also bind to operators in the promoter regions of the cognate TA operon and thereby regulate transcription. For enteric vapBC TA loci, the VapC toxins specifically cleave tRNA(fMet) and thus down-regulate protein synthesis. Here, we describe the crystal structure of the intact Shigella flexneri VapBC TA complex, determined to 2.7 Å resolution. Both in solution and in the crystal structure, four molecules of each protein combine to form a large and globular hetero-octameric assembly with SpoVT/AbrB-type DNA-binding domains at each end and a total molecular mass of about 100 kDa. The structure gives new insights into the inhibition of VapC toxins by VapB and provides the molecular basis for understanding transcriptional regulation through VapB dimerization.

Cyanobacteria contain a structural homologue of the Hfq protein with altered RNA-binding properties.

Hfq proteins are common in many species of enterobacteria, where they participate in RNA folding and translational regulation through pairing of small RNAs and messenger RNAs. Hfq proteins share the distinctive Sm fold, and form ring-shaped structures similar to those of the Sm/Lsm proteins regulating mRNA turnover in eukaryotes. However, bacterial Hfq proteins are homohexameric, whereas eukaryotic Sm/Lsm proteins are heteroheptameric. Recently, Hfq proteins with poor sequence conservation were identified in archaea and cyanobacteria. In this article, we describe crystal structures of the Hfq proteins from the cyanobacteria Synechocystis sp. PCC 6803 and Anabaena PCC 7120 at 1.3 and 2.3 A resolution, respectively, and show that they retain the classic Sm fold despite low sequence conservation. In addition, the intersubunit contacts and RNA-binding site are divergent, and we show biochemically that the proteins bind very weakly to known Escherichia coli Hfq target RNAs in vitro. Moreover, when expressed in E. coli, the proteins cannot mediate Hfq-dependent RNA regulation. It therefore appears that the cyanobacterial proteins constitute a specialized subfamily of Hfq proteins that bind relatively weakly to A/U-rich tracks of regulatory RNAs. The results have implications for our understanding of the evolution of the Sm fold and the Hfq proteins in the bacterial kingdom in general.