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AIMS: The autoimmune-mediated destruction of pancreatic beta-cells in Type 1 diabetes mellitus renders patients deficient in two glucoregulatory peptide hormones, insulin and amylin. With insulin replacement alone, most patients do not achieve glycaemic goals. We aimed to determine the long-term efficacy and safety of adjunctive therapy with pramlintide, a synthetic human amylin analogue, in patients with Type 1 diabetes. METHODS: In a double-blind, placebo-controlled, parallel-group, multicentre study, 651 patients with Type 1 diabetes (age 41 +/- 13 years, HbA(1c) 8.9 +/- 1.0%, mean +/- sd) were randomized to mealtime injections of placebo or varying doses of pramlintide, in addition to their insulin therapy, for 52 weeks. RESULTS: Addition of pramlintide [60 microg three times daily (TID) or four times daily (QID)] to insulin led to significant reductions in HbA(1c) from baseline to Week 52 of 0.29% (P < 0.011) and 0.34% (P < 0.001), respectively, compared with a 0.04% reduction in placebo group. Three times the proportion of pramlintide- than placebo-treated patients achieved an HbA(1c) of < 7%. The greater reduction in HbA(1c) with pramlintide was achieved without an increase in concomitant insulin use and was accompanied by a significant reduction in body weight from baseline to Week 52 of 0.4 kg in the 60 microg TID (P < 0.027) or QID (P < 0.040) pramlintide treatment groups, compared with a 0.8-kg gain in body weight in the placebo group. The most common adverse event in pramlintide-treated patients was transient, mild-to-moderate nausea. CONCLUSIONS: These results show that mealtime amylin replacement with pramlintide, as an adjunct to insulin therapy, improves long-term glycaemic and weight control in patients with Type 1 diabetes. [ABSTRACT FROM AUTHOR]

Fluoride is an environmental toxin prevalent in water, soil, and air. A fluoride transporter called Fluoride EXporter (FEX) has been discovered across all domains of life, including bacteria, single cell eukaryotes, and all plants, that is required for fluoride tolerance. How FEX functions to protect multicellular plants is unknown. In order to distinguish between different models, the dynamic movement of fluoride in wildtype (WT) and fex mutant plants was monitored using [ 18 F]fluoride with positron emission tomography. Significant differences were observed in the washout behavior following initial fluoride uptake between plants with and without a functioning FEX. [ 18 F]Fluoride traveled quickly up the floral stem and into terminal tissues in WT plants. In contrast, the fluoride did not move out of the lower regions of the stem in mutant plants resulting in clearance rates near zero. The roots were not the primary locus of FEX action, nor did FEX direct fluoride to a specific tissue. Fluoride efflux by WT plants was saturated at high fluoride concentrations resulting in a pattern like the fex mutant. The kinetics of fluoride movement suggested that FEX mediates a fluoride transport mechanism throughout the plant where each individual cell benefits from FEX expression., (© 2024. The Author(s).)

Ligand specificity is an essential requirement for all riboswitches. Some variant riboswitches utilize a common structural motif, yet through subtle sequence differences, they are able to selectively respond to different small molecule ligands and regulate downstream gene expression. These variants discriminate between structurally and chemically similar ligands. Crystal structures provide insight into how specificity is achieved. However, ligand specificity cannot always be explained solely by nucleotides in direct contact with the ligand. The cyclic dinucleotide variant family contains two classes, cyclic-di-GMP and cyclic-AMP-GMP riboswitches, that were distinguished based on the identity of a single nucleotide in contact with the ligand. Here we report a variant riboswitch with a mutation at a second ligand-contacting position that is promiscuous for both cyclic-di-GMP and cyclic-AMP-GMP despite a predicted preference for cyclic-AMP-GMP. A high-throughput mutational analysis, SMARTT, was used to quantitatively assess thousands of sites in the first- and second-shells of ligand contact for impacts on ligand specificity and promiscuity. In addition to nucleotides in direct ligand contact, nucleotides more distal from the binding site, within the J1/2 linker and the terminator helix, were identified that impact ligand specificity. These findings provide an example of how nucleotides outside the ligand binding pocket influence the riboswitch specificity. Moreover, these distal nucleotides could be used to predict promiscuous sequences.

Riboswitches function as important translational regulators in bacteria. Comprehensive mutational analysis of transcriptional riboswitches has been used to probe the energetic intricacies of interplay between the aptamer and expression platform, but translational riboswitches have been inaccessible to massively parallel techniques. The guanidine-II (gdm-II) riboswitch is an exclusively translational class. We have integrated RelE cleavage with next-generation sequencing to quantify ligand-dependent changes in translation initiation for all single and double mutations of the Pseudomonas aeruginosa gdm-II riboswitch, a total of more than 23,000 variants. This extensive mutational analysis is consistent with the prominent features of the bioinformatic consensus. These data indicate, unexpectedly, that direct sequestration of the Shine-Dalgarno sequence is dispensable for riboswitch function. Additionally, this comprehensive data set reveals important positions not identified in previous computational and crystallographic studies. Mutations in the variable linker region stabilize alternate conformations. The double mutant data reveal the functional importance of the previously modeled P0b helix formed by the 5' and 3' tails that serves as the basis for translational control. Additional mutations to GU wobble base pairs in both P1 and P2 reveal how the apparent cooperativity of the system involves an intricate network of communication between the two binding sites. This comprehensive examination of a translational riboswitch's expression platform illuminates how the riboswitch is precisely tuned and tunable with regard to ligand sensitivity, the amplitude of expression between ON and OFF states, and the cooperativity of ligand binding., (© 2023 Focht et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Fluoride is highly abundant in the environment. Many organisms have adapted specific defense mechanisms against high concentrations of fluoride, including the expression of proteins capable of removing fluoride from cells. However, these fluoride transporters have not been identified in all organisms, and even organisms that express fluoride transporters vary in tolerance capabilities across species, individuals, and even tissue types. This suggests that alternative factors influence fluoride tolerance. We screened for adaptation against fluoride toxicity through an unbiased mutagenesis assay conducted on Saccharomyces cerevisiae lacking the fluoride exporter FEX, the primary mechanism of fluoride resistance. Over 80 independent fluoride-hardened strains were generated, with anywhere from 100- to 1200-fold increased fluoride tolerance compared to the original strain. The whole genome of each mutant strain was sequenced and compared to the wild type. The fluoride-hardened strains utilized a combination of phenotypes that individually conferred fluoride tolerance. These included intracellular acidification, cellular dormancy, nutrient storage, and a communal behavior reminiscent of flocculation. Of particular importance to fluoride resistance was intracellular acidification, which served to reverse the accumulation of fluoride and lead to its excretion from the cell as HF without the activity of a fluoride-specific protein transporter. This transport mechanism was also observed in wild-type yeast through a manual mutation to lower their cytoplasmic pH. The results demonstrate that the yeast developed a protein-free adaptation for removing an intracellular toxicant.

The sequences of the 5' untranslated regions (5'-UTRs) of mRNA alter gene expression across domains of life. Transcriptional modulators can be easily assayed through transcription termination, but translational regulators often require indirect, laborious methods. We have leveraged RelE's ribosome-dependent endonuclease activity to develop a quantitative assay to monitor translation initiation of cis-regulatory mRNAs. RelE cleavage accurately reports ligand-dependent changes in ribosome association for two translational riboswitches and provides quantitative information about each switch's sensitivity and range of response. RelE accurately reads out sequence-driven changes in riboswitch specificity and function and is quantitatively dependent upon ligand concentration. RelE cleavage similarly captures differences in translation initiation between yeast 5'-UTR isoforms. RelE cleavage can thus reveal a plethora of information about translation initiation in different domains of life., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Fluoride is everywhere in the environment, yet it is toxic to living things. How biological organisms detoxify fluoride has been unknown until recently. Fluoride-specific ion transporters in both prokaryotes (Fluoride channel; Fluc) and fungi (Fluoride Exporter; FEX) efficiently export fluoride to the extracellular environment. FEX homologs have been identified throughout the plant kingdom. Understanding the function of FEX in a multicellular organism will reveal valuable knowledge about reducing toxic effects caused by fluoride. Here, we demonstrate the conserved role of plant FEX (FLUORIDE EXPORTER) in conferring fluoride tolerance. Plant FEX facilitates the efflux of toxic fluoride ions from yeast cells and is required for fluoride tolerance in plants. A CRISPR/Cas9-generated mutation in Arabidopsis thaliana FEX renders the plant vulnerable to low concentrations (100-µM) of fluoride at every stage of development. Pollen is particularly affected, failing to develop even at extremely low levels of fluoride in the growth medium. The action of the FEX membrane transport protein is the major fluoride defense mechanism in plants., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Alginates gel rapidly under ambient conditions and have widely documented potential to form protective matrices for sensitive bioactive cargo. Most commonly, alginate gelation occurs via calcium mediated electrostatic crosslinks between the linear polyuronic acid polymers. A recent breakthrough to form crosslinked alginate microcapsules (CLAMs) by in situ gelation during spray drying ("CLAMs process") has demonstrated applications in protection and controlled delivery of bioactives in food, cosmetics, and agriculture. The extent of crosslinking of alginates in CLAMs impacts the effectiveness of its barrier properties. For example, higher crosslinking extents can improve oxidative stability and limit diffusion of the encapsulated cargo. Crosslinking in CLAMs can be controlled by varying the calcium to alginate ratio; however, the choice of alginates used in the process also influences the ultimate extent of crosslinking. To understand how to select alginates to target crosslinking in CLAMs, we examined the roles of alginate molecular properties. A surprise finding was the formation of alginic acid gelling in the CLAMs that is a consequence of simultaneous and rapid pH reduction and moisture removal that occurs during spray drying. Thus, spray dried CLAMs gelation is due to calcium crosslinking and alginic acid formation, and unlike external gelation methods, is insensitive to the molecular composition of the alginates. The 'extent of gelation' of spray dried CLAMs is influenced by the molecular weights of the alginates at saturating calcium concentrations. Alginate viscosity correlates with molecular weight; thus, viscosity is a convenient criterion for selecting commercial alginates to target gelation extent in CLAMs., Competing Interests: TJ’s research group has received research support from BASF. Malvern Panalytical’s involvement in facilitating GPC/SEC data collection does not alter our adherence to PLOS ONE policies on sharing data and materials. KAW is an employee of Malvern Panalytical. TJ, SAS, and HBS are inventors on the ‘CLAMs process’ patent US9700519B2; TJ, BMA, and HBS are inventors on a pending patent applying CLAMs to encapsulate bacteria (US Patent Application 63,033,305); TJ, SAS, DW and HBS are inventors on a pending patent for a process improvement to the CLAMs process (US Patent Application 62/775,331); TJ, SAS and HBS are inventors on a pending patent applying the CLAMs process to encapsulate oxygen labile cargo (US Patent Application 62/837,399). The authors’ inventor status does not alter our adherence to PLOS ONE policies on sharing data and materials.

Disruption of the tumor suppressor PTEN, either at the protein or genomic level, plays an important role in human cancer development. The high frequency of PTEN deficiency reported across several cancer subtypes positions therapeutic approaches that exploit PTEN loss-of-function with the ability to significantly impact the treatment strategies of a large patient population. Here, we report that an endophytic fungus isolated from a medicinal plant produces an inhibitor of DNA double-strand-break repair. Furthermore, the novel alkaloid product, which we have named irrepairzepine ( 1 ), demonstrated synthetic lethal targeting in PTEN-deficient glioblastoma cells. Our results uncover a new therapeutic lead for PTEN-deficient cancers and an important molecular tool toward enhancing the efficacy of current cancer treatments.

Hydrofluoric acid elicits cell cycle arrest through a mechanism that has long been presumed to be linked with the high affinity of fluoride to metals. However, we have recently found that the acid stress from fluoride exposure is sufficient to elicit many of the hallmark phenotypes of fluoride toxicity. Here we report the systematic screening of genes involved in fluoride resistance and general acid resistance using a genome deletion library in Saccharomyces cerevisiae . We compare these to a variety of acids - 2,4-dinitrophenol, FCCP, hydrochloric acid, and sulfuric acid - none of which has a high metal affinity. Pathways involved in endocytosis, vesicle trafficking, pH maintenance, and vacuolar function are of particular importance to fluoride tolerance. The majority of genes conferring resistance to fluoride stress also enhanced resistance to general acid toxicity. Genes whose expression regulate Golgi-mediated vesicle transport were specific to fluoride resistance, and may be linked with fluoride-metal interactions. These results support the notion that acidity is an important and underappreciated principle underlying the mechanisms of fluoride toxicity., (Copyright © 2020 Johnston, Nallur, Gordon, Smith and Strobel.)

The bacterial toxin RelE cleaves mRNA in the ribosomal A site. Although it shares a global fold with other microbial RNases, the active site contains several positively charged residues instead of histidines and glutamates that are typical of ribonucleases. The pH dependences of wild-type and mutant RelE indicate it uses general acid-base catalysis, but either the general acid (proposed to be R81) or the general base must have a substantially downshifted p K a . However, which group is shifted cannot be determined using available structural and biochemical data. Here, we use a phosphorothiolate at the scissile phosphate to remove the need for a general acid. We show this modification rescues nearly all of the defect of the R81A mutation, supporting R81 as the general acid. We also find that the observed p K a of the general base is dependent on the charge of the side chain at position 81. This indicates that positive charge in the active site contributes to a general base p K a downshifted by more than 5 units. Although this modestly reduces the effectiveness of general acid-base catalysis, it is strongly supplemented by the role of the positive charge in stabilizing the transition state for cleavage. Furthermore, we show that the ribosome is required for cleavage but not binding of mRNA by RelE. Ribosome functional groups do not directly contact the scissile phosphate, indicating that positioning and charge interactions dominate RelE catalysis. The unusual RelE active site catalyzes phosphoryl transfer at a rate comparable to those of similar enzymes, but in a ribosome-dependent fashion.

Glycine riboswitches utilize both single- and tandem-aptamer architectures. In the tandem system, the relative contribution of each aptamer toward gene regulation is not well understood. To dissect these contributions, the effects of 684 single mutants of a tandem ON switch from Bacillus subtilis were characterized for the wild-type construct and binding site mutations that selectively restrict ligand binding to either the first or second aptamer. Despite the structural symmetry of tandem aptamers, the response to these mutations was frequently asymmetrical. Mutations in the first aptamer often significantly weakened the K 1/2 , while several mutations in the second aptamer improved the amplitude. These results demonstrate that this ON switch favors ligand binding to the first aptamer. This is in contrast to the tandem OFF switch variant from Vibrio cholerae , which was previously shown to have preferential binding to its second aptamer. A bioinformatic analysis of tandem glycine riboswitches revealed that the two binding pockets are differentially conserved between ON and OFF switches. Altogether, this indicates that tandem ON switch variants preferentially utilize binding to the first aptamer to promote helical switching, while OFF switch variants favor binding to the second aptamer. The data set also revealed a cooperative glycine response when both binding pockets were maximally stabilized with three GC base pairs. This indicates a cooperative response may sometimes be obfuscated by a difference in the affinities of the two aptamers. This conditional cooperativity provides an additional layer of tunability to tandem glycine riboswitches that adds to their versatility as genetic switches., (© 2020 Torgerson et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The modularity of protein domains is well-known, but the existence of independent domains that confer function in RNA is less established. Recently, a family of RNA aptamers termed ykkC was discovered; they bind at least four ligands of very different chemical composition, including guanidine, phosphoribosyl pyrophosphate (PRPP), and guanosine tetraphosphate (ppGpp) (graphical abstract). Structures of these aptamers revealed an architecture characterized by two coaxial helical stacks. The first helix appears to be a generic scaffold, while the second helix forms the most contacts to the ligands. To determine if these two regions within the aptamer are modular units for ligand recognition, we swapped the ligand-binding coaxial stacks of a guanidine aptamer and a PRPP aptamer. This operation, in combination with a single mutation in the scaffold domain, achieved full switching of ligand specificity. This finding suggests that the ligand-binding helix largely dictates the ligand specificity of ykkC RNAs and that the scaffold coaxial stack is generally compatible with various ykkC ligand-binding modules. This work presents an example of RNA domain modularity comparable to that of a ligand-binding protein, showcasing the versatility of RNA as an entity capable of molecular evolution through adaptation of existing motifs.

Fluoride is ubiquitously present throughout the world. It is released from minerals, magmatic gas, and industrial processing, and travels in the atmosphere and water. Exposure to low concentrations of fluoride increases overall oral health. Consequently, many countries add fluoride to their public water supply at 0.7-1.5 ppm. Exposure to high concentrations of fluoride, such as in a laboratory setting often exceeding 100 ppm, results in a wide array of toxicity phenotypes. This includes oxidative stress, organelle damage, and apoptosis in single cells, and skeletal and soft tissue damage in multicellular organisms. The mechanism of fluoride toxicity can be broadly attributed to four mechanisms: inhibition of proteins, organelle disruption, altered pH, and electrolyte imbalance. Recently, there has been renewed concern in the public sector as to whether fluoride is safe at the current exposure levels. In this review, we will focus on the impact of fluoride at the chemical, cellular, and multisystem level, as well as how organisms defend against fluoride. We also address public concerns about fluoride toxicity, including whether fluoride has a significant effect on neurodegeneration, diabetes, and the endocrine system.

Organisms are exposed to fluoride in the air, water, and soil. Yeast and other microbes utilize fluoride channels as a method to prevent intracellular fluoride accumulation and mediate fluoride toxicity. Consequently, deletion of fluoride exporter genes (FEX) in S. cerevisiae resulted in over 1000-fold increased fluoride sensitivity. We used this FEX knockout strain to identify genes, that when overexpressed, are able to partially relieve the toxicity of fluoride exposure. Overexpression of five genes, SSU1, YHB1, IPP1, PHO87, and PHO90, increase fluoride tolerance by 2- to 10-fold. Overexpression of these genes did not provide improved fluoride resistance in wild-type yeast, suggesting that the mechanism is specific to low fluoride toxicity in yeast. Ssu1p and Yhb1p both function in nitrosative stress response, which is induced upon fluoride exposure along with metal influx. Ipp1p, Pho87p, and Pho90p increase intracellular orthophosphate. Consistent with this observation, fluoride toxicity is also partially mitigated by the addition of high levels of phosphate to the growth media. Fluoride inhibits phosphate import upon stress induction and causes nutrient starvation and organelle disruption, as supported by gene induction monitored through RNA-Seq. The combination of observations suggests that transmembrane nutrient transporters are among the most sensitized proteins during fluoride-instigated stress.

Riboswitches contain structured aptamer domains that, upon ligand binding, facilitate helical switching in their downstream expression platforms to alter gene expression. To fully dissect how riboswitches function requires a better understanding of the energetic landscape for helical switching. Here, we report a sequencing-based high-throughput assay for monitoring in vitro transcription termination and use it to simultaneously characterize the functional effects of all 522 single point mutants of a glycine riboswitch type-1 singlet. Mutations throughout the riboswitch cause ligand-dependent defects, but only mutations within the terminator hairpin alter readthrough efficiencies in the absence of ligand. A comprehensive analysis of the expression platform reveals that ligand binding stabilizes the antiterminator by just 2-3 kcal/mol, indicating that the competing expression platform helices must be extremely close in energy to elicit a significant ligand-dependent response. These results demonstrate that gene regulation by this riboswitch is highly constrained by the energetics of ligand binding and conformational switching. These findings exemplify the energetic parameters of RNA conformational rearrangements driven by binding events., (© 2018 Torgerson et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Two classes of riboswitches related to the ykkC guanidine-I riboswitch bind phosphoribosyl pyrophosphate (PRPP) and guanosine tetraphosphate (ppGpp). Here we report the co-crystal structure of the PRPP aptamer and its ligand. We also report the structure of the G96A point mutant that prefers ppGpp over PRPP with a dramatic 40,000-fold switch in specificity. The ends of the aptamer form a helix that is not present in the guanidine aptamer and is involved in the expression platform. In the mutant, the base of ppGpp replaces G96 in three-dimensional space. This disrupts the S-turn, which is a primary structural feature of the ykkC RNA motif. These dramatic differences in ligand specificity are achieved with minimal mutations. ykkC aptamers are therefore a prime example of an RNA fold with a rugged fitness landscape. The ease with which the ykkC aptamer acquires new specificity represents a striking case of evolvability in RNA., Competing Interests: AK, CR, SS No competing interests declared, (© 2018, Knappenberger et al.)

Microencapsulation of plant-beneficial bacteria, such as pink pigmented facultative methylotrophs (PPFM), may greatly extend the shelf life of these Gram-negative microorganisms and facilitate their application to crops for sustainable agriculture. A species of PPFM designated Methylobacterium radiotolerans was microencapsulated in cross-linked alginate microcapsules (CLAMs) prepared by an innovative and industrially scalable process that achieves polymer cross-linking during spray-drying. PPFM survived the spray-drying microencapsulation process with no significant loss in viable population, and the initial population of PPFM in CLAMs exceeded 10 10 CFU/g powder. The PPFM population in CLAMs gradually declined by 4 to 5 log CFU/g over one year of storage. The extent of alginate cross-linking, modulated by adjusting the calcium phosphate content in the spray-dryer feed, did not influence cell viability after spray-drying, viability over storage, or dry particle size. However, particle size measurements and light microscopy of aqueous CLAMs suggest that enhanced crosslinking may limit the release of encapsulated bacteria. This work demonstrates an industrially scalable method for producing alginate-based inoculants that may be suitable for on-seed or foliar spray applications., Competing Interests: No competing financial interests exist.

Cyclic dinucleotides are second messenger molecules produced by both prokaryotes and eukaryotes in response to external stimuli. In bacteria, these molecules bind to RNA riboswitches and several protein receptors ultimately leading to phenotypic changes such as biofilm formation, ion transport and secretion of virulence factors. Some cyclic dinucleotide analogs bind differentially to biological receptors and can therefore be used to better understand cyclic dinucleotide mechanisms in vitro and in vivo. However, production of some of these analogs involves lengthy, multistep syntheses. Here, we describe a new, simple method for enzymatic synthesis of several 3', 5' linked cyclic dinucleotide analogs of c-di-GMP, c-di-AMP and c-AMP-GMP using the cyclic-AMP-GMP synthetase, DncV. The enzymatic reaction efficiently produced most cyclic dinucleotide analogs, such as 2'-amino sugar substitutions and phosphorothioate backbone modifications, for all three types of cyclic dinucleotides without the use of protecting groups or organic solvents. We used these novel analogs to explore differences in phosphate backbone and 2'-hydroxyl recognition between GEMM-I and GEMM-Ib riboswitches.

The guanidine-II riboswitch, also known as mini-ykkC , is a conserved mRNA element with more than 800 examples in bacteria. It consists of two stem-loops capped by identical, conserved tetraloops that are separated by a linker region of variable length and sequence. Like the guanidine-I riboswitch, it controls the expression of guanidine carboxylases and SugE-like genes. The guanidine-II riboswitch specifically binds free guanidinium cations and functions as a translationally controlled on-switch. Here we report the structure of a P2 stem-loop from the Pseudomonas aeruginosa guanidine-II riboswitch aptamer bound to guanidine at 1.57 Å resolution. The hairpins dimerize via the conserved tetraloop, which also contains the binding pocket. Two guanidinium molecules bind near the dimerization interface, one in each tetraloop. The guanidinium cation is engaged in extensive hydrogen bonding to the RNA. Contacts include the Hoogsteen face of a guanine base and three nonbridging phosphate oxygens. Cation-π interactions and ionic interactions also stabilize ligand binding. The guanidine-II riboswitch utilizes the same recognition strategies as the guanidine-I riboswitch while adopting an entirely different and much smaller RNA fold., (© 2017 Reiss and Strobel; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Antagonistic microorganisms produce antimicrobials to inhibit the growth of competitors. Although water-soluble antimicrobials are limited to proximal interactions via aqueous diffusion, volatile antimicrobials are able to act at a distance and diffuse through heterogeneous environments. Here, we identify the mechanism of action of Muscodor albus , an endophytic fungus known for its volatile antimicrobial activity toward a wide range of human and plant pathogens and its potential use in mycofumigation. Proposed uses of the Muscodor species include protecting crops, produce, and building materials from undesired fungal or bacterial growth. By analyzing a collection of Muscodor isolates with varying toxicity, we demonstrate that the volatile mycotoxin, N -methyl- N -nitrosoisobutyramide, is the dominant factor in Muscodor toxicity and acts primarily through DNA methylation. Additionally, Muscodor isolates exhibit higher resistance to DNA methylation compared with other fungi. This work contributes to the evaluation of Muscodor isolates as potential mycofumigants, provides insight into chemical strategies that organisms use to manipulate their environment, and provokes questions regarding the mechanisms of resistance used to tolerate constitutive, long-term exposure to DNA methylation., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The fluoride export protein (FEX) in yeast and other fungi provides tolerance to fluoride (F-), an environmentally ubiquitous anion. FEX efficiently eliminates intracellular fluoride that otherwise would accumulate at toxic concentrations. The FEX homolog in bacteria, Fluc, is a 'double-barreled' channel formed by dimerization of two identical or similar subunits. FEX in yeast and other eukaryotes is a monomer resulting from covalent fusion of the two subunits. As a result, both potential fluoride pores are created from different parts of the same protein. Here we identify FEX proteins from two multicellular eukaryotes, a plant Arabidopsis thaliana and an animal Amphimedon queenslandica, by demonstrating significant fluoride tolerance when these proteins are heterologously expressed in the yeast Saccharomyces cerevisiae. Residues important for eukaryotic FEX function were determined by phylogenetic sequence alignment and functional analysis using a yeast growth assay. Key residues of the fluoride channel are conserved in only one of the two potential fluoride-transporting pores. FEX activity is abolished upon mutation of residues in this conserved pore, suggesting that only one of the pores is functional. The same topology is conserved for the newly identified FEX proteins from plant and animal. These data suggest that FEX family of fluoride channels in eukaryotes are 'single-barreled' transporters containing one functional pore and a second non-functional vestigial remnant of a homologous gene fusion event.

The glycine riboswitch often occurs in a tandem architecture, with two ligand-binding domains (aptamers) followed by a single expression platform. Based on previous observations, we hypothesized that "singlet" versions of the glycine riboswitch, which contain only one aptamer domain, are able to bind glycine if appropriate structural contacts are maintained. An initial alignment of 17 putative singlet riboswitches indicated that the single consensus aptamer domain is flanked by a conserved peripheral stem-loop structure. These singlets were sorted into two subtypes based on whether the active aptamer domain precedes or follows the peripheral stem-loop, and an example of each subtype of singlet riboswitch was characterized biochemically. The singlets possess glycine-binding affinities comparable to those of previously published tandem examples, and the conserved peripheral domains form A-minor interactions with the single aptamer domain that are necessary for ligand-binding activity. Analysis of sequenced genomes identified a significant number of singlet glycine riboswitches. Based on these observations, we propose an expanded model for glycine riboswitch gene control that includes singlet and tandem architectures., (© 2016 Ruff et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Fungi have highly active secondary metabolic pathways which enable them to produce a wealth of sesquiterpenoids that are bioactive. One example is Δ6-protoilludene, the precursor to the cytotoxic illudins, which are pharmaceutically relevant as anticancer therapeutics. To date, this valuable sesquiterpene has only been identified in members of the fungal division Basidiomycota. To explore the untapped potential of fungi belonging to the division Ascomycota in producing Δ6-protoilludene, we isolated a fungal endophyte Diaporthe sp. BR109 and show that it produces a diversity of terpenoids including Δ6-protoilludene. Using a genome sequencing and mining approach 17 putative novel sesquiterpene synthases were identified in Diaporthe sp. BR109. A phylogenetic approach was used to predict which gene encodes Δ6-protoilludene synthase, which was then confirmed experimentally. These analyses reveal that the sesquiterpene synthase and its putative sesquiterpene scaffold modifying cytochrome P450(s) may have been acquired by inter-phylum horizontal gene transfer from Basidiomycota to Ascomycota. Bioinformatic analyses indicate that inter-phylum transfer of these minimal sequiterpenoid secondary metabolic pathways may have occurred in other fungi. This work provides insights into the evolution of fungal sesquiterpenoid secondary metabolic pathways in the production of pharmaceutically relevant bioactive natural products., (Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

The ability of bacteria to sense environmental cues and adapt is essential for their survival. The use of second-messenger signaling molecules to translate these cues into a physiological response is a common mechanism employed by bacteria. The second messenger 3'-5'-cyclic diadenosine monophosphate (c-di-AMP) has been linked to a diverse set of biological processes involved in maintaining cell viability and homeostasis, as well as pathogenicity. A complex network of both protein and RNA receptors inside the cell activates specific pathways and mediates phenotypic outputs in response to c-di-AMP. Structural analysis of these RNA and protein receptors has revealed the different recognition elements employed by these effectors to bind the same small molecule. Herein, using a series of c-di-AMP analogues, we probed the interactions made with a riboswitch and a phosphodiesterase protein to identify the features important for c-di-AMP binding and recognition. We found that the ydaO riboswitch binds c-di-AMP in two discrete sites with near identical affinity and a Hill coefficient of 1.6. The ydaO riboswitch distinguishes between c-di-AMP and structurally related second messengers by discriminating against an amine at the C2 position more than a carbonyl at the C6 position. We also identified phosphate-modified analogues that bind both the ydaO RNA and GdpP protein with high affinity, whereas symmetrically modified ribose analogues exhibited a substantial decrease in ydaO affinity but retained high affinity for GdpP. These ligand modifications resulted in increased resistance to enzyme-catalyzed hydrolysis by the GdpP enzyme. Together, these data suggest that these c-di-AMP analogues could be useful as chemical tools to specifically target subsections of second-messenger signaling pathways.

Endophytic fungi are plant tissue-associated fungi that represent a rich resource of unexplored biological and chemical diversity. As part of an ongoing effort to characterize Amazon rainforest-derived endophytes, numerous fungi were isolated and cultured from plants collected in the Yasuní National Park in Ecuador. Of these samples, phylogenetic and morphological data revealed a previously undescribed fungus in the order Pleosporales that was cultured from the tropical tree Duroia hirsuta. Extracts from this fungal isolate displayed activity against Staphylococcus aureus and were thus subjected to detailed chemical studies. Two compounds with modest antibacterial activity were isolated, and their structures were elucidated using a combination of NMR spectroscopic analysis, LC-MS studies, and chemical degradation. These efforts led to the identification of stelliosphaerols A (1) and B (2), new sesquiterpene-polyol conjugates that are responsible, at least in part, for the S. aureus inhibitory activity of the fungal extract.

Fungal endophytes colonize every major lineage of land plants without causing apparent harm to their hosts. Despite their production of interesting and potentially novel compounds, endophytes-particularly those inhabiting stem tissues-are still a vastly underexplored component of microbial diversity. In this study, we explored the diversity of over 1500 fungal endophyte isolates collected from three Ecuadorian ecosystems: lowland tropical forest, cloud forest, and coastal dry forest. We sought to determine whether Ecuador's fungal endophytes are hyperdiverse, and whether that biological diversity is reflected in the endophytes' chemical diversity. To assess this chemical diversity, we analyzed a subset of isolates for their production of volatile organic compounds (VOCs), a representative class of natural products. This study yielded a total of 1526 fungal ITS sequences comprising some 315 operational taxonomic units (OTUs), resulting in a non-asymptotic OTU accumulation curve and characterized by a Fisher's α of 120 and a Shannon Diversity score of 7.56. These figures suggest that the Ecuadorian endophytes are hyperdiverse. Furthermore, the 113 isolates screened for VOCs produced more than 140 unique compounds. These results present a mere snapshot of the remarkable biological and chemical diversity of stem-inhabiting endophytic fungi from a single neotropical country.

The bacterial toxin RelE is a ribosome-dependent endoribonuclease. It is part of a type II toxin-antitoxin system that contributes to antibiotic resistance and biofilm formation. During amino acid starvation, RelE cleaves mRNA in the ribosomal A-site, globally inhibiting protein translation. RelE is structurally similar to microbial RNases that employ general acid-base catalysis to facilitate RNA cleavage. The RelE active site is atypical for acid-base catalysis, in that it is enriched with positively charged residues and lacks the prototypical histidine-glutamate catalytic pair, making the mechanism of mRNA cleavage unclear. In this study, we use a single-turnover kinetic analysis to measure the effect of pH and phosphorothioate substitution on the rate constant for cleavage of mRNA by wild-type RelE and seven active-site mutants. Mutation and thio effects indicate a major role for stabilization of increased negative change in the transition state by arginine 61. The wild-type RelE cleavage rate constant is pH-independent, but the reaction catalyzed by many of the mutants is strongly dependent on pH, suggestive of general acid-base catalysis. pH-rate curves indicate that wild-type RelE operates with the pK(a) of at least one catalytic residue significantly downshifted by the local environment. Mutation of any single active-site residue is sufficient to disrupt this microenvironment and revert the shifted pK(a) back above neutrality. pH-rate curves are consistent with K54 functioning as a general base and R81 as a general acid. The capacity of RelE to effect a large pK(a) shift and facilitate a common catalytic mechanism by uncommon means furthers our understanding of other atypical enzymatic active sites.

Natural products remain an important source of new therapeutics for emerging drug-resistant pathogens like Candida albicans, which particularly affects immunocompromised patients. A bioactive 3-decalinoyltetramic acid, pyrrolocin A, was isolated from extracts of a novel Amazonian fungal endophyte, E6927E, of the Diaporthales family. The structure of the natural product was solved using NMR and CD spectroscopy and it is structurally related to the fungal setins, equisetin and phomasetin, which are well-characterized tetramic acid antibiotics specific for Gram-positive organisms. We show that the compound inhibits growth of Staphylococcus aureus and Enterococcus faecalis. It shows selective and potent bioactivity against fungal strains, with an MIC of 4 μg/mL for C. albicans, 100 μg/mL for Aspergillus sp. and greater than 100 μg/mL for Saccharomyces cerevisiae. Further, the compound is less toxic to mammalian cells (IC50 = 150 μg/mL), with an inhibitory concentration greater than forty times that for C. albicans. Pyrrolocin A retained potent activity against eight out of seventeen strains of clinical Candida sp. isolates tested.

Fluoride is a ubiquitous environmental toxin with which all biological species must cope. A recently discovered family of fluoride export (FEX) proteins protects organisms from fluoride toxicity by removing it from the cell. We show here that FEX proteins in Saccharomyces cerevisiae function as ion channels that are selective for fluoride over chloride and that these proteins are constitutively expressed at the yeast plasma membrane. Continuous expression is in contrast to many other toxin exporters in yeast, and this, along with the fact that two nearly duplicate proteins are encoded in the yeast genome, suggests that the threat posed by fluoride ions is frequent and detrimental. Structurally, eukaryotic FEX proteins consist of two homologous four-transmembrane helix domains folded into an antiparallel dimer, where the orientation of the two domains is fixed by a single transmembrane linker helix. Using phylogenetic sequence conservation as a guide, we have identified several functionally important residues. There is substantial functional asymmetry in the effect of mutation at corresponding sites in the two domains. Specifically, mutations to residues in the C-terminal domain proved significantly more detrimental to function than did similar mutations in the N-terminal domain. Our data suggest particular residues that may be important to anion specificity, most notably the necessity of a positive charge near the end of TMH1 in the C-terminal domain. It is possible that a cationic charge at this location may create an electrostatic well for fluoride ions entering the channel from the cytoplasm., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Over a thousand extracts were tested for phenotypic effects in developing zebrafish embryos to identify bioactive molecules produced by endophytic fungi. One extract isolated from Fusarium sp., a widely distributed fungal genus found in soil and often associated with plants, induced an undulated notochord in developing zebrafish embryos. The active compound was isolated and identified as fusaric acid. Previous literature has shown this phenotype to be associated with copper chelation from the active site of lysyl oxidase, but the ability of fusaric acid to bind copper ions has not been well described. Isothermal titration calorimetry revealed that fusaric acid is a modest copper chelator with a binding constant of 4.4 × 10(5) M(-1). These results shed light on the toxicity of fusaric acid and the potential teratogenic effects of consuming plants infected with Fusarium sp.

Recent advances in the biological production of fuels have relied on the optimization of pathways involving genes from diverse organisms. Several recent articles have highlighted the potential to expand the pool of useful genes by looking to filamentous fungi. This review highlights the enzymes and organisms used for the production of a variety of fuel types and commodity chemicals with a focus on the usefulness and promise of those from filamentous fungi.

An endophytic fungus was isolated that produces a series of volatile natural products, including terpenes and odd chain polyenes. Phylogenetic analysis of the isolate using five loci suggests that it is closely related to Nigrograna mackinnonii CBS 674.75. The main component of the polyene series was purified and identified as (3E,5E,7E)-nona-1,3,5,7-tetraene (NTE), a novel natural product. Non-oxygenated hydrocarbons of this chain length are uncommon and desirable as gasoline-surrogate biofuels. The biosynthetic pathway for NTE production was explored using metabolic labeling and gas chromatography time of flight mass spectometer (GCMS). Two-carbon incorporation (13)C acetate suggests that it is derived from a polyketide synthase (PKS) followed by decarboxylation. There are several known mechanisms for such decarboxylation, though none have been discovered in fungi. Towards identifying the PKS responsible for the production of NTE, the genome of N. mackinnonii E5202H (ATCC SD-6839) was sequenced and assembled. Of the 32 PKSs present in the genome, 17 are predicted to contain sufficient domains for the production of NTE. These results exemplify the capacity of endophytic fungi to produce novel natural products that may have many uses, such as biologically derived fuels and commodity chemicals.

Terpenes are an important and diverse class of secondary metabolites widely produced by fungi. Volatile compound screening of a fungal endophyte collection revealed a number of isolates in the family Xylariaceae, producing a series of terpene molecules, including 1,8-cineole. This compound is a commercially important component of eucalyptus oil used in pharmaceutical applications and has been explored as a potential biofuel additive. The genes that produce terpene molecules, such as 1,8-cineole, have been little explored in fungi, providing an opportunity to explore the biosynthetic origin of these compounds. Through genome sequencing of cineole-producing isolate E7406B, we were able to identify 11 new terpene synthase genes. Expressing a subset of these genes in Escherichia coli allowed identification of the hyp3 gene, responsible for 1,8-cineole biosynthesis, the first monoterpene synthase discovered in fungi. In a striking example of convergent evolution, mutational analysis of this terpene synthase revealed an active site asparagine critical for water capture and specificity during cineole synthesis, the same mechanism used in an unrelated plant homologue. These studies have provided insight into the evolutionary relationship of fungal terpene synthases to those in plants and bacteria and further established fungi as a relatively untapped source of this important and diverse class of compounds., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Muscodor albus belongs to a genus of endophytic fungi that inhibit and kill other fungi, bacteria, and insects through production of a complex mixture of volatile organic compounds (VOCs). This process of mycofumigation has found commercial application for control of human and plant pathogens, but the mechanism of the VOC toxicity is unknown. Here, the mode of action of these volatiles was investigated through a series of genetic screens and biochemical assays. A single-gene knockout screen revealed high sensitivity for Escherichia coli lacking enzymes in the pathways of DNA repair, DNA metabolic process, and response to stress when exposed to the VOCs of M. albus. Furthermore, the sensitivity of knockouts involved in the repair of specific DNA alkyl adducts suggests that the VOCs may induce alkylation. Evidence of DNA damage suggests that these adducts lead to breaks during DNA replication or transcription if not properly repaired. Additional cytotoxicity profiling indicated that during VOC exposure, E. coli became filamentous and demonstrated an increase in cellular membrane fluidity. The volatile nature of the toxic compounds produced by M. albus and their broad range of inhibition make this fungus an attractive biological agent. Understanding the antimicrobial effects and the VOC mode of action will inform the utility and safety of potential mycofumigation applications for M. albus., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The glycine riboswitch predominantly exists as a tandem structure, with two adjacent, homologous ligand-binding domains (aptamers), followed by a single expression platform. The recent identification of a leader helix, the inclusion of which eliminates cooperativity between the aptamers, has reopened the debate over the purpose of the tandem structure of the glycine riboswitch. An equilibrium dialysis-based assay was combined with binding-site mutations to monitor glycine binding in each ligand-binding site independently to understand the role of each aptamer in glycine binding and riboswitch tertiary interactions. A series of mutations disrupting the dimer interface was used to probe how dimerization impacts ligand binding by the tandem glycine riboswitch. While the wild-type tandem riboswitch binds two glycine equivalents, one for each aptamer, both individual aptamers are capable of binding glycine when the other aptamer is unoccupied. Intriguingly, glycine binding by aptamer-1 is more sensitive to dimerization than glycine binding by aptamer-2 in the context of the tandem riboswitch. However, monomeric aptamer-2 shows dramatically weakened glycine-binding affinity. In addition, dimerization of the two aptamers in trans is dependent on glycine binding in at least one aptamer. We propose a revised model for tandem riboswitch function that is consistent with these results, wherein ligand binding in aptamer-1 is linked to aptamer dimerization and stabilizes the P1 stem of aptamer-2, which controls the expression platform., (© 2014 Ruff and Strobel; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The toxin RelE is a ribosome-dependent endoribonuclease implicated in diverse cellular processes, including persistence. During amino acid starvation, RelE inhibits translation by cleaving ribosomal A-site mRNA. Although RelE is structurally similar to other microbial endoribonucleases, the active-site amino acid composition differs substantially and lacks obvious candidates for general acid-base functionality. Highly conserved RelE residues (Lys52, Lys54, Arg61, Arg81, and Tyr87) surround the mRNA scissile phosphate, and specific 16S rRNA contacts further contribute to substrate positioning. We used a single-turnover kinetic assay to evaluate the catalytic importance of individual residues in the RelE active site. Within the context of the ribosome, RelE rapidly cleaves A-site mRNA at a rate similar to those of traditional ribonucleases. Single-turnover rate constants decreased between 10(2)- and 10(6)-fold for the RelE active-site mutants of Lys52, Lys54, Arg61, and Arg81. RelE may principally promote catalysis via transition-state charge stabilization and leaving-group protonation, in addition to achieving in-line substrate positioning in cooperation with the ribosome. This kinetic analysis complements structural information to provide a foundation for understanding the molecular mechanism of this atypical endoribonuclease.

Fluorine is an abundant element and is toxic to organisms from bacteria to humans, but the mechanisms by which eukaryotes resist fluoride toxicity are unknown. The Escherichia coli gene crcB was recently shown to be regulated by a fluoride-responsive riboswitch, implicating it in fluoride response. There are >8,000 crcB homologs across all domains of life, indicating that it has an important role in biology. Here we demonstrate that eukaryotic homologs [renamed FEX (fluoride exporter)] function in fluoride export. FEX KOs in three eukaryotic model organisms, Neurospora crassa, Saccharomyces cerevisiae, and Candida albicans, are highly sensitized to fluoride (>200-fold) but not to other halides. Some of these KO strains are unable to grow in fluoride concentrations found in tap water. Using the radioactive isotope of fluoride, (18)F, we developed an assay to measure the intracellular fluoride concentration and show that the FEX deletion strains accumulate fluoride in excess of the external concentration, providing direct evidence of FEX function in fluoride efflux. In addition, they are more sensitive to lower pH in the presence of fluoride. These results demonstrate that eukaryotic FEX genes encode a previously unrecognized class of fluoride exporter necessary for survival in standard environmental conditions.

Endophytes isolated from tropical plants represent a largely untapped reservoir of bioactive secondary metabolites. We screened a library of fungal endophyte extracts for inhibition of the plant pathogen, Pythium ultimum , and purified an active compound using bioassay-guided fractionation. A new nonenolide, ( 4S,7S,8S,9R )-4- O -succinyl-7,8-dihydroxy-9-heptyl-nonen-9-olide, was isolated and named xyolide. The structure was elucidated by a combination of 1D and 2D NMR methods and the absolute configuration was determined by exciton-coupled circular dichroism. The MIC of xyolide against P. ultimum was 425 µM.

The bacterial second messenger signaling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) controls important biological processes such as biofilm formation, virulence response, and motility. This second messenger is sensed by macromolecular targets inside the cell, both protein and RNA, which induce specific phenotypic responses critical for bacterial survival. One class of enzymes responsible for regulating the intracellular concentration of c-di-GMP, and therefore the physiological behavior of the cell, consists of the EAL domain phosphodiesterases, which degrade the second messenger to its linear form, pGpG. Here, we investigate how base and backbone modifications of c-di-GMP affect the rate of cyclic dinucleotide degradation by an EAL domain protein (CC3396 from Caulobacter crescentus). The doubly substituted thiophosphate analogue is highly resistant to hydrolysis by this metabolizing enzyme but can still bind c-di-GMP riboswitch targets. We used these findings to develop a novel ribosyl phosphate-modified derivative of c-di-GMP containing 2'-deoxy and methylphosphonate substitutions that is charge neutral and demonstrate that this analogue is also resistant to EAL domain-catalyzed degradation. This suggests a general strategy for designing c-di-GMP derivatives with increased enzymatic stability that also possess desirable properties for development as chemical probes of c-di-GMP signaling.

The ability of bacteria to adapt to a changing environment is essential for their survival. One mechanism used to facilitate behavioral adaptations is the second messenger signaling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). c-di-GMP is widespread throughout the bacterial domain and plays a vital role in regulating the transition between the motile planktonic lifestyle and the sessile biofilm forming state. This second messenger also controls the virulence response of pathogenic organisms and is thought to be connected to quorum sensing, the process by which bacteria communicate with each other. The intracellular concentration of c-di-GMP is tightly regulated by the opposing enzymatic activities of diguanlyate cyclases and phosphodiesterases, which synthesize and degrade the second messenger, respectively. The change in the intracellular concentration of c-di-GMP is directly sensed by downstream targets of the second messenger, both protein and RNA, which induce the appropriate phenotypic response. This review will summarize our current state of knowledge of c-di-GMP signaling in bacteria with a focus on protein and RNA binding partners of the second messenger. Efforts towards the synthesis of c-di-GMP and its analogs are discussed as well as studies aimed at targeting these macromolecular effectors with chemically synthesized cyclic dinucleotide analogs.

We used computational linguistic and content analyses to explore the concept of project ownership for undergraduate research. We used linguistic analysis of student interview data to develop a quantitative methodology for assessing project ownership and applied this method to measure degrees of project ownership expressed by students in relation to different types of educational research experiences. The results of the study suggest that the design of a research experience significantly influences the degree of project ownership expressed by students when they describe those experiences. The analysis identified both positive and negative aspects of project ownership and provided a working definition for how a student experiences his or her research opportunity. These elements suggest several features that could be incorporated into an undergraduate research experience to foster a student's sense of project ownership.

Preferential binding of an enzyme to the transition state relative to the ground state is a key strategy for enzyme catalysis. When there is a difference between the ground and transition state charge distributions, enzymes maximize electrostatic interactions to achieve this enhanced transition state binding. Although the transition state is difficult to observe directly by structural methods, the chemical details of this transient species can be characterized by studies of substituent effects (Brønsted, Hammett, Swain-Scott, etc.) and isotope effects. Brønsted analysis can provide an estimate of transition state charges for the nucleophile and leaving group of a reaction. This Account will discuss the theoretical basis of Brønsted analysis and describe its practical application to the study of transacylase enzyme systems including the peptidyl transferase reaction of the ribosome. The Brønsted coefficient is derived from the linear free energy relationship (LFER) that correlates the acidity (pK(a)) of a reactive atom to the log of its rate constant. The Brønsted coefficient establishes the change in atomic charge as the reaction proceeds from the ground state to the transition state. Bonding events alter the electrostatics of atoms and the extent of bonding can be extrapolated from transition state charges. Therefore, well-defined nucleophile and leaving group transition state charges limit the number of mechanisms that are consistent with a particular transition state. Brønsted results are most informative when interpreted in the context of other mechanistic data, especially for enzymatic studies where an active site may promote a transition state that differs significantly from a prediction based on uncatalyzed solution reactions. Here we review Brønsted analyses performed on transacylases to illustrate how these data enhanced the enzymatic mechanistic studies. Through a systematic comparison of five enzymes, we reveal a wide spectrum of Brønsted values that are possible for what otherwise appear to be similar chemical reactions. The variations in the Brønsted coefficients predict different transition states for the various enzymes. This Account explores an overriding theme in the enzymatic mechanisms that catalysis enhances commensurate bond formation and proton abstraction events. The extent of the two bonding events in relationship to each other can be inferred from the Brønsted coefficient. When viewed in the context of recent ribosomal studies, this interpretation provides mechanistic insights into peptide bond formation.

The cyclic dinucleotide c-di-GMP regulates lifestyle transitions in many bacteria, such as the change from a free motile state to a biofilm-forming community. Riboswitches that bind this second messenger are important downstream targets in this bacterial signaling pathway. The breakdown of c-di-GMP in the cell is accomplished enzymatically and results in the linear dinucleotide pGpG. The c-di-GMP-binding riboswitches must be able to discriminate between their cognate cyclic ligand and linear dinucleotides in order to be selective biological switches. It has been reported that the c-di-GMP-I riboswitch binds c-di-GMP 5 orders of magnitude better than the linear pGpG, but the cause of this large energetic difference in binding is unknown. Here we report binding data and crystal structures of several linear c-di-GMP analogues in complex with the c-di-GMP-I riboswitch. These data reveal the parameters for phosphate recognition and the structural basis of linear dinucleotide binding to the riboswitch. Additionally, the pH dependence of binding shows that exclusion of pGpG is not due to the additional negative charge on the ligand. These data reveal principles that, along with published work, will contribute to the design of c-di-GMP analogues with properties desirable for use as chemical tools and potential therapeutics.

The microbial conversion of solid cellulosic biomass to liquid biofuels may provide a renewable energy source for transportation fuels. Endophytes represent a promising group of organisms, as they are a mostly untapped reservoir of metabolic diversity. They are often able to degrade cellulose, and they can produce an extraordinary diversity of metabolites. The filamentous fungal endophyte Ascocoryne sarcoides was shown to produce potential-biofuel metabolites when grown on a cellulose-based medium; however, the genetic pathways needed for this production are unknown and the lack of genetic tools makes traditional reverse genetics difficult. We present the genomic characterization of A. sarcoides and use transcriptomic and metabolomic data to describe the genes involved in cellulose degradation and to provide hypotheses for the biofuel production pathways. In total, almost 80 biosynthetic clusters were identified, including several previously found only in plants. Additionally, many transcriptionally active regions outside of genes showed condition-specific expression, offering more evidence for the role of long non-coding RNA in gene regulation. This is one of the highest quality fungal genomes and, to our knowledge, the only thoroughly annotated and transcriptionally profiled fungal endophyte genome currently available. The analyses and datasets contribute to the study of cellulose degradation and biofuel production and provide the genomic foundation for the study of a model endophyte system., Competing Interests: CK currently works at 454 Life Sciences. All of the work reported in this manuscript was completed when he was in residence at the Broad Institute. ME currently works at Pall Corporation. All of the work reported in this manuscript was completed when he was at 454 Life Sciences..

Peptide bond formation during ribosomal protein synthesis involves an aminolysis reaction between the aminoacyl α-amino group and the carbonyl ester of the growing peptide via a transition state with a developing negative charge, the oxyanion. Structural and molecular dynamic studies have suggested that the ribosome may stabilize the oxyanion in the transition state of peptide bond formation via a highly ordered water molecule. To biochemically investigate this mechanistic hypothesis, we estimated the energetic contribution to catalytic charge stabilization of the oxyanion using a series of transition state mimics that contain different charge distributions and hydrogen bond potential on the functional group mimicking the oxyanion. Inhibitors containing an oxyanion mimic that carried a neutral charge and a mimic that preserved the negative charge but could not form hydrogen bonds had less than a 3-fold effect on inhibitor binding affinity. These observations argue that the ribosome provides minimal transition state charge stabilization to the oxyanion during peptide bond formation via the water molecule. This is in contrast to the substantial level of oxyanion stabilization provided by serine proteases. This suggests that the oxyanion may be neutralized via a proton shuttle, resulting in an uncharged transition state.