RNA regulatory networks diversified through curvature of the PUF protein scaffold
Abstract not provided
Wilinski, Daniel↗
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Structural basis of glycan specificity in neonate-specific bovine-human reassortant rotavirus
We report that strain-dependent variation of glycan recognition during initial cell attachment of viruses is a critical determinant of host specificity, tissue-tropism and zoonosis. Rotaviruses (RVs), which cause life-threatening gastroenteritis in infants and children, display significant genotype-dependent variations in glycan recognition resulting from sequence alterations in the VP8* domain of the spike protein VP4. The structural basis of this genotype-dependent glycan specificity, particularly in human RVs, remains poorly understood. Here, from crystallographic studies, we show how genotypic variations configure a novel binding site in the VP8* of a neonate-specific bovine-human reassortant to uniquely recognize either type I or type II precursor glycans, and to restrict type II glycan binding in the bovine counterpart. In conclusion, such a distinct glycan-binding site that allows differential recognition of the precursor glycans, which are developmentally regulated in the neonate gut and abundant in bovine and human milk provides a basis for age-restricted tropism and zoonotic transmission of G10P[11] rotaviruses.
59 BASIC BIOLOGICAL SCIENCES↗
Metagenomics uncovers gaps in amplicon-based detection of microbial diversity
Our view of microbial diversity has expanded greatly over the past 40 years, primarily through the wide application of PCR-based surveys of the small-subunit ribosomal RNA (SSU rRNA) gene. Yet significant gaps in knowledge remain due to well-recognized limitations of this method. Here in this paper, we systematically survey primer fidelity in SSU rRNA gene sequences recovered from over 6,000 assembled metagenomes sampled globally. Our findings show that approximately 10% of environmental microbial sequences might be missed from classical PCR-based SSU rRNA gene surveys, mostly members of the Candidate Phyla Radiation (CPR) and as yet uncharacterized Archaea. In conclusion, these results underscore the extent of uncharacterized microbial diversity and provide fruitful avenues for describing additional phylogenetic lineages.
59 BASIC BIOLOGICAL SCIENCES↗
Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales
Hydraulic fracturing is the industry standard for extracting hydrocarbons from shale formations. Attention has been paid to the economic benefits and environmental impacts of this process, yet the biogeochemical changes induced in the deep subsurface are poorly understood. Recent single-gene investigations revealed that halotolerant microbial communities were enriched after hydraulic fracturing. Here the reconstruction of 31 unique genomes coupled to metabolite data from the Marcellus and Utica shales revealed that methylamine cycling supports methanogenesis in the deep biosphere. Fermentation of injected chemical additives also sustains long-term microbial persistence, while sulfide generation from thiosulfate represents a poorly recognized corrosion mechanism in shales. Extensive links between viruses and microbial hosts demonstrate active viral predation, which may contribute to the release of labile cellular constituents into the extracellular environment. Our analyses show that hydraulic fracturing provides the organismal and chemical inputs for colonization and persistence in the deep terrestrial subsurface.
Environmental Molecular Sciences Laboratory↗
Standardizng Data
To enable the rational design of nanomaterials for improved efficacy and safety, it is critical to understand and exploit the physicochemical properties that drive biological behavior (Morris, 2010). Data mining and computer simulation are essential for deriving information about nanomaterial behavior; however, the datasets needed to support such studies are sparse and stored across a variety of repositories and resources. Schrurs and Lison (2012) have expressed the need for more coherence and structure in the conduct of nanotechnology research. Additionally, the lack of common reporting standards and non-uniformity of information reported have proven to be significant barriers to such data sharing and re-use. The Nanotechnology Working Group (Nano WG), of the US National Institutes of Health National Cancer Informatics Program, has been focused on addressing these barriers. The Nano WG - which includes representatives from over 20 organizations including government agencies, academia, industry, standards organizations, and alliances -has developed ISA-TAB-Nano (Thomas et al, 2013), a general framework for representing and integrating diverse types of data related to the description and characterization of nanomaterials. Recognizing that nanoparticle characterization studies have many of the same challenges as ‘omics-based assays, the Nano WG joined the ISA Commons (Sansone et al., 2012) to leverage lessons learned in ‘omics data sharing. The ISA Commons community brings together 50 collaborators at over 30 scientific organizations around the globe, including regulatory and industrial participants in an increasingly diverse set of life science domains. At the core of the ISA Commons is the ISA metadata tracking framework which forms the basis for the ISA-TAB-Nano extension. The extension of the ISA framework to nanotechnology domain illustrates the power of a synergistic approach that seeks the interoperability of data across multiple research disciplines. To increase adoption, especially in the commercial arena from vendors and manufactures, the ISA-Tab-Nano data-sharing specification has also been submitted for consideration as a standard to the American Society for Testing and Materials (ASTM). Delivering a community-driven specification is only the first phase of the process. To be useful and used, ISA-TAB-Nano must be implemented by tools and databases to assist researchers in reporting their data accordingly, shielding them from unnecessary complexity. Our next step is to extend the open source ISA Software Suite to provide user-oriented tools for the collection, curation, and storage of data compliant with the ISA-TAB-Nano specification. Future work will also focus on the application of the ISA-TAB-Nano format to support emerging standards on minimal information about nanomaterials in biological research (Ostraat et al, 2012; MinChar). ISA-TAB-Nano development is a community-driven effort and we welcome new contributions, collaborations and domain expertise. We invite researchers, software developers, vendors, and other stakeholders to work with us to implement the ISA-Tab-Nano format in their existing systems and research. Likewise, we welcome engagement of regulators, funding agencies, editors, and other policy makers to discuss how this standard can be used to facilitate the sharing and reuse of nanotechnology data across a wide range of disciplines. More information about the ISA-TAB-Nano project can be found online at https://wiki.nci.nih.gov/display/ICR/ISA-TAB-Nano.
informatics↗
Resin-assisted enrichment of thiols as a general strategy for proteomic profiling of cysteine-based reversible modifications
Reversible modifications on cysteine thiols play a significant role in redox signaling and regulation. A number of reversible redox modifications, including disulfide formation, S-nitrosylation, and S-glutathionylation, have been recognized for their significance in various physiological and pathological processes. Here we describe in detail a resin-assisted thiol-affinity enrichment protocol for both biochemical and proteomics applications. This protocol serves as a general approach for specific isolation of thiol-containing proteins or peptides derived from reversible redox-modified proteins. This approach utilizes thiol-affinity resins to directly capture thiol-containing proteins or peptides through a disulfide exchange reaction followed by on-resin protein digestion and on-resin multiplexed isobaric labeling to facilitate LC-MS/MS based quantitative site-specific analysis of redox modifications. The overall approach requires a much simpler workflow with increased specificity compared to the commonly used biotin switch technique. By coupling different selective reduction strategies, the resin-assisted approach provides the researcher with a useful tool capable of enriching different types of reversible modifications on protein thiols. Procedures for selective enrichment and analyses of S-nitrosylation and total reversible cysteine oxidation are presented to demonstrate the utility of this general strategy.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Mechanism of mismatch recognition revealed by human MutS[beta] bound to unpaired DNA loops
DNA mismatch repair corrects replication errors, thus reducing mutation rates and microsatellite instability. Genetic defects in this pathway cause Lynch syndrome and various cancers in humans. Binding of a mispaired or unpaired base by bacterial MutS and eukaryotic MutS{alpha} is well characterized. We report here crystal structures of human MutS{beta} in complex with DNA containing insertion-deletion loops (IDL) of two, three, four or six unpaired nucleotides. In contrast to eukaryotic MutS{alpha} and bacterial MutS, which bind the base of a mismatched nucleotide, MutS{beta} binds three phosphates in an IDL. DNA is severely bent at the IDL; unpaired bases are flipped out into the major groove and partially exposed to solvent. A normal downstream base pair can become unpaired; a single unpaired base can thereby be converted to an IDL of two nucleotides and recognized by MutS{beta}. The C-terminal dimerization domains form an integral part of the MutS structure and coordinate asymmetrical ATP hydrolysis by Msh2 and Msh3 with mismatch binding to signal for repair.
59 BASIC BIOLOGICAL SCIENCES↗
Structural basis of evasion of cellular adaptive immunity by HIV-1 Nef
The HIV-1 protein Nef inhibits antigen presentation by class I major histocompatibility complex (MHC-I). We determined the mechanism of this activity by solving the crystal structure of a protein complex comprising Nef, the MHC-I cytoplasmic domain (MHC-I CD) and the {mu}1 subunit of the clathrin adaptor protein complex 1. A ternary, cooperative interaction clamps the MHC-I CD into a narrow binding groove at the Nef-{mu}1 interface, which encompasses the cargo-recognition site of {mu}1 and the proline-rich strand of Nef. The Nef C terminus induces a previously unobserved conformational change in {mu}1, whereas the N terminus binds the Nef core to position it optimally for complex formation. Positively charged patches on {mu}1 recognize acidic clusters in Nef and MHC-I. The structure shows how Nef functions as a clathrin-associated sorting protein to alter the specificity of host membrane trafficking and enable viral evasion of adaptive immunity.
59 BASIC BIOLOGICAL SCIENCES↗
Structural basis for diverse N-glycan recognition by HIV-1-neutralizing V1-V2-directed antibody PG16
HIV-1 uses a diverse N-linked-glycan shield to evade recognition by antibody. Select human antibodies, such as the clonally related PG9 and PG16, recognize glycopeptide epitopes in the HIV-1 V1–V2 region and penetrate this shield, but their ability to accommodate diverse glycans is unclear. Here we report the structure of antibody PG16 bound to a scaffolded V1–V2, showing an epitope comprising both high mannose–type and complex-type N-linked glycans. We combined structure, NMR and mutagenesis analyses to characterize glycan recognition by PG9 and PG16. Three PG16-specific residues, arginine, serine and histidine (RSH), were critical for binding sialic acid on complex-type glycans, and introduction of these residues into PG9 produced a chimeric antibody with enhanced HIV-1 neutralization. Although HIV-1–glycan diversity facilitates evasion, antibody somatic diversity can overcome this and can provide clues to guide the design of modified antibodies with enhanced neutralization.
Pancera, Marie↗
Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env
As the sole viral antigen on the HIV-1–virion surface, trimeric Env is a focus of vaccine efforts. In this paper, we present the structure of the ligand-free HIV-1–Env trimer, fix its conformation and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C 433C (DS) variant specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer bound by a single CD4 without the typical antigenic hallmarks of CD4 induction. Finally, antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like-particle and soluble formats providing a new generation of vaccine antigens.
59 BASIC BIOLOGICAL SCIENCES↗
Recognition of the bacterial alarmone ZMP through long-distance association of two RNA subdomains
The bacterial alarmone 5-aminoimidazole-4-carboxamide riboside 5'-triphosphate (AICAR triphosphate or ZTP), derived from the monophosphorylated purine precursor ZMP, accumulates during folate starvation. ZTP regulates genes involved in purine and folate metabolism through a cognate riboswitch. The linker connecting this riboswitch's two subdomains varies in length by over 100 nucleotides. In this paper, we report the cocrystal structure of the Fusobacterium ulcerans riboswitch bound to ZMP, which spans the two subdomains whose interface also comprises a pseudoknot and ribose zipper. The riboswitch recognizes the carboxamide oxygen of ZMP through an unprecedented inner-sphere coordination with a Mg 2+ ion. We show that the affinity of the riboswitch for ZMP is modulated by the linker length. Notably, ZMP can simultaneously bind to the two subdomains even when they are synthesized as separate RNAs. Finally, the ZTP riboswitch demonstrates how specific small-molecule binding can drive association of distant noncoding-RNA domains to regulate gene expression.
59 BASIC BIOLOGICAL SCIENCES↗
Recognition of distinct RNA motifs by the clustered CCCH zinc fingers of neuronal protein Unkempt
Unkempt is an evolutionarily conserved RNA-binding protein that regulates translation of its target genes and is required for the establishment of the early bipolar neuronal morphology. Here we determined the X-ray crystal structure of mouse Unkempt and show that its six CCCH zinc fingers (ZnFs) form two compact clusters, ZnF1–3 and ZnF4–6, that recognize distinct trinucleotide RNA substrates. Both ZnF clusters adopt a similar overall topology and use distinct recognition principles to target specific RNA sequences. Structure-guided point mutations reduce the RNA binding affinity of Unkempt both in vitro and in vivo, ablate Unkempt's translational control and impair the ability of Unkempt to induce a bipolar cellular morphology. Furthermore, our study unravels a new mode of RNA sequence recognition by clusters of CCCH ZnFs that is critical for post-transcriptional control of neuronal morphology.
59 BASIC BIOLOGICAL SCIENCES↗
Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme
CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gating of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.
59 BASIC BIOLOGICAL SCIENCES↗
Strong succession in arbuscular mycorrhizal fungal communities
Abstract The ecology of fungi lags behind that of plants and animals because most fungi are microscopic and hidden in their substrates. Here, we address the basic ecological process of fungal succession in nature using the microscopic, arbuscular mycorrhizal fungi (AMF) that form essential mutualisms with 70–90% of plants. We find a signal for temporal change in AMF community similarity that is 40-fold stronger than seen in the most recent studies, likely due to weekly samplings of roots, rhizosphere and soil throughout the 17 weeks from seedling to fruit maturity and the use of the fungal DNA barcode to recognize species in a simple, agricultural environment. We demonstrate the patterns of nestedness and turnover and the microbial equivalents of the processes of immigration and extinction, that is, appearance and disappearance. We also provide the first evidence that AMF species co-exist rather than simply co-occur by demonstrating negative, density-dependent population growth for multiple species. Our study shows the advantages of using fungi to test basic ecological hypotheses (e.g., nestedness v. turnover, immigration v. extinction, and coexistence theory) over periods as short as one season.
Gao, Cheng (ORCID:0000000325227909)↗
Impacts of microbial assemblage and environmental conditions on the distribution of anatoxin-a producing cyanobacteria within a river network
Blooms of planktonic cyanobacteria have long been of concern in lakes, but more recently, harmful impacts of riverine benthic cyanobacterial mats been recognized. As yet, we know little about how various benthic cyanobacteria are distributed in river networks, or how environmental conditions or other associated microbes in their consortia affect their biosynthetic capacities. We performed metagenomic sequencing for 22 Oscillatoriales-dominated (Cyanobacteria) microbial mats collected across the Eel River network in Northern California and investigated factors associated with anatoxin-a producing cyanobacteria. All microbial communities were dominated by one or two cyanobacterial species, so the key mat metabolisms involve oxygenic photosynthesis and carbon oxidation. Only a few metabolisms fueled the growth of the mat communities, with little evidence for anaerobic metabolic pathways. We genomically defined four cyanobacterial species, all which shared <96% average nucleotide identity with reference Oscillatoriales genomes and are potentially novel species in the genus Microcoleus . One of the Microcoleus species contained the anatoxin-a biosynthesis genes, and we describe the first anatoxin-a gene cluster from the Microcoleus clade within Oscillatoriales. Occurrence of these four Microcoleus species in the watershed was correlated with total dissolved nitrogen and phosphorus concentrations, and the species that contains the anatoxin-a gene cluster was found in sites with higher nitrogen concentrations. Microbial assemblages in mat samples with the anatoxin-a gene cluster consistently had a lower abundance of Burkholderiales (Betaproteobacteria) species than did mats without the anatoxin-producing genes. The associations of water nutrient concentrations and certain co-occurring microbes with anatoxin-a producing Microcoleus motivate further exploration for their roles as potential controls on the distributions of toxigenic benthic cyanobacteria in river networks.
59 BASIC BIOLOGICAL SCIENCES↗
Mercury methylation by metabolically versatile and cosmopolitan marine bacteria
Microbes transform aqueous mercury (Hg) into methylmercury (MeHg), a potent neurotoxin that accumulates in terrestrial and marine food webs, with potential impacts on human health. This process requires the gene pair hgcAB, which encodes for proteins that actuate Hg methylation, and has been well described for anoxic environments. However, recent studies report potential MeHg formation in suboxic seawater, although the microorganisms involved remain poorly understood. In this study, we conducted large-scale multi-omic analyses to search for putative microbial Hg methylators along defined redox gradients in Saanich Inlet, British Columbia, a model natural ecosystem with previously measured Hg and MeHg concentration profiles. Analysis of gene expression profiles along the redoxcline identified several putative Hg methylating microbial groups, including Calditrichaeota, SAR324 and Marinimicrobia, with the last the most active based on hgc transcription levels. Marinimicrobia hgc genes were identified from multiple publicly available marine metagenomes, consistent with a potential key role in marine Hg methylation. Computational homology modelling predicts that Marinimicrobia HgcAB proteins contain the highly conserved amino acid sites and folding structures required for functional Hg methylation. Furthermore, a number of terminal oxidases from aerobic respiratory chains were associated with several putative novel Hg methylators. Our findings thus reveal potential novel marine Hg-methylating microorganisms with a greater oxygen tolerance and broader habitat range than previously recognized.
59 BASIC BIOLOGICAL SCIENCES↗
Reductive dissolution of pyrite by methanogenic archaea
Abstract The formation and fate of pyrite (FeS2) modulates global iron, sulfur, carbon, and oxygen biogeochemical cycles and has done so since early in Earth’s geological history. A longstanding paradigm is that FeS2 is stable at low temperature and is unavailable to microorganisms in the absence of oxygen and oxidative weathering. Here, we show that methanogens can catalyze the reductive dissolution of FeS2 at low temperature (≤38 °C) and utilize dissolution products to meet cellular iron and sulfur demands associated with the biosynthesis of simple and complex co-factors. Direct access to FeS2 is required to catalyze its reduction and/or to assimilate iron monosulfide that likely forms through coupled reductive dissolution and precipitation, consistent with close associations observed between cells and FeS2. These findings demonstrate that FeS2 is bioavailable to anaerobic methanogens and can be mobilized in low temperature anoxic environments. Given that methanogens evolved at least 3.46 Gya, these data indicate that the microbial contribution to the iron and sulfur cycles in ancient and contemporary anoxic environments may be more complex and robust than previously recognized, with impacts on the sources and sinks of iron and sulfur and other bio-essential and thiophilic elements such as nickel and cobalt.
59 BASIC BIOLOGICAL SCIENCES↗
Rationale for reconsidering current regulations restricting use of hybrids in orange juice
Huanglongbing (HLB) is a disease that has devastated the Florida citrus industry, threatens the entire U.S. citrus industry, and globally is rapidly spreading. Florida’s citrus production is 90% sweet orange, which is quite sensitive to HLB. The heavy reliance on sweet orange for Florida citrus production makes the industry especially vulnerable to diseases that are damaging to this type of citrus. Furthermore, 90% of Florida oranges are used in producing orange juice that is defined by a federal regulation known as the “orange juice standard”, specifying that at least 90% of “orange juice” must be derived from Citrus sinensis. Genomic analyses definitively reveal that sweet orange is not a true species, but just one of many introgression hybrids of C. reticulata and C. maxima, with phenotypic diversity resulting from accumulated mutations in this single hybrid, the “sweet orange”. No other fruit industry is limited by law to such a narrow genetic base. Fortunately, there are new citrus hybrids displaying reduced sensitivity to HLB, and in some cases they produce juice, alone or in blends, that consumers would recognize as “orange juice”. Reconsidering current regulations on orange juice standards may permit use of such hybrids in “orange juice”, providing greater latitude for commercialization of these hybrids, leading to higher-quality orange juice and a more sustainable Florida orange juice industry.
54 ENVIRONMENTAL SCIENCES↗
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