Abiotic and Biotic Drivers of Soil Fungal Communities in Response to Dairy Manure Amendment.

Modern agriculture often relies on large inputs of synthetic fertilizers to maximize crop yield potential, yet their intensive use has led to nutrient losses and impaired soil health. Alternatively, manure amendments provide plant available nutrients, build organic carbon, and enhance soil health. However, we lack a clear understanding of how consistently manure impacts fungal communities, the mechanisms via which manure impacts soil fungi, and the fate of manure-borne fungi in soils. We assembled soil microcosms using five soils to investigate how manure amendments impact fungal communities over a 60-day incubation. Further, we used autoclaving treatments of soils and manure to determine if observed changes in soil fungal communities were due to abiotic or biotic properties, and if indigenous soil communities constrained colonization of manure-borne fungi. We found that manure amended soil fungal communities diverged from nonamended communities over time, often in concert with a reduction in diversity. Fungal communities responded to live and autoclaved manure in a similar manner, suggesting that abiotic forces are primarily responsible for the observed dynamics. Finally, manure-borne fungi declined quickly in both live and autoclaved soil, indicating that the soil environment is unsuitable for their survival. IMPORTANCE Manure amendments in agricultural systems can impact soil microbial communities via supplying growth substrates for indigenous microbes or by introducing manure-borne taxa. This study explores the consistency of these impacts on soil fungal communities and the relative importance of abiotic and biotic drivers across distinct soils. Different fungal taxa responded to manure among distinct soils, and shifts in soil fungal communities were driven largely by abiotic factors, rather than introduced microbes. This work demonstrates that manure may have inconsistent impacts on indigenous soil fungi, and that abiotic properties of soils render them largely resistant to invasion by manure-borne fungi.

Small molecule AX-024 reduces T cell proliferation independently of CD3ϵ/Nck1 interaction, which is governed by a domain swap in the Nck1-SH3.1 domain.

Activation of the T cell receptor (TCR) results in binding of the adapter protein Nck (noncatalytic region of tyrosine kinase) to the CD3ϵ subunit of the TCR. The interaction was suggested to be important for the amplification of TCR signals and is governed by a proline-rich sequence (PRS) in CD3ϵ that binds to the first Src homology 3 (SH3) domain of Nck (Nck-SH3.1). Inhibition of this protein/protein interaction ameliorated inflammatory symptoms in mouse models of multiple sclerosis, psoriasis, and asthma. A small molecule, AX-024, was reported to inhibit the Nck/CD3ϵ interaction by physically binding to the Nck1-SH3.1 domain, suggesting a route to develop an inhibitor of the Nck1/CD3ϵ interaction for modulating TCR activity in autoimmune and inflammatory diseases. We show here that AX-024 reduces T cell proliferation upon weak TCR stimulation but does not significantly affect phosphorylation of Zap70 (ζ chain of T cell receptor-associated protein kinase 70). We also find that AX-024 is likely not involved in modulating the Nck/TCR interaction but probably has other targets in T cells. An array of biophysical techniques did not detect a direct interaction between AX-024 and Nck-SH3.1 in vitro Crystal structures of the Nck-SH3.1 domain revealed its binding mode to the PRS in CD3ϵ. The SH3 domain tends to generate homodimers through a domain swap. Domain swaps observed previously in other SH3 domains indicate a general propensity of this protein fold to exchange structural elements. The swapped form of Nck-SH3.1 is unable to bind CD3ϵ, possibly representing an inactive form of Nck in cells.

Inhibitory and nutrient use phenotypes among coexisting Fusarium and Streptomyces populations suggest local coevolutionary interactions in soil.

Bacteria and fungi are key components of virtually all natural habitats, yet the significance of fungal-bacterial inhibitory interactions for the ecological and evolutionary dynamics of specific bacterial and fungal populations in natural habitats have been overlooked. More specifically, despite the broad consensus that antibiotics play a key role in providing a fitness advantage to competing microbes, the significance of antibiotic production in mediating cross-kingdom coevolutionary interactions has received relatively little attention. Here, we characterize reciprocal inhibition among Streptomyces and Fusarium populations from prairie soil, and explore antibiotic inhibition in relation to niche overlap among sympatric and allopatric populations. We found evidence for local adaptation between Fusarium and Streptomyces populations as indicated by significantly greater inhibition among sympatric than allopatric populations. Additionally, for both taxa, there was a significant positive correlation between the strength of inhibition against the other taxon and the intensity of resource competition from that taxon among sympatric but not allopatric populations. These data suggest that coevolutionary antagonistic interactions between Fusarium and Streptomyces are driven by resource competition, and support the hypothesis that antibiotics act as weapons in mediating bacterial-fungal interactions in soil.

Core Rhizosphere Microbiomes of Dryland Wheat Are Influenced by Location and Land Use History.

The Inland Pacific Northwest is one of the most productive dryland wheat production areas in the United States. We explored the bacterial and fungal communities associated with wheat in a controlled greenhouse experiment using soils from multiple locations to identify core taxa consistently associated with wheat roots and how land use history influences wheat-associated communities. Further, we examined microbial co-occurrence networks from wheat rhizospheres to identify candidate hub taxa. Location of origin and land use history (long-term no-till versus noncropped Conservation Reserve Program [CRP]) of soils were the strongest drivers of bacterial and fungal communities. Wheat rhizospheres were especially enriched in many bacterial families, while only a few fungal taxa were enriched in the rhizosphere. There was a core set of bacteria and fungi that was found in >95% of rhizosphere or bulk soil samples, including members of Bradyrhizobium, Sphingomonadaceae, Massilia, Variovorax, Oxalobacteraceae, and Caulobacteraceae Core fungal taxa in the rhizosphere included Nectriaceae, Ulocladium, Alternaria, Mortierella, and Microdochium Overall, there were fewer core fungal taxa, and the rhizosphere effect was not as pronounced as with bacteria. Cross-domain co-occurrence networks were used to identify hub taxa in the wheat rhizosphere, which included bacterial and fungal taxa (e.g., Sphingomonas, Massilia, Knufia, and Microdochium). Our results suggest that there is a relatively small group of core rhizosphere bacteria that were highly abundant on wheat roots regardless of soil origin and land use history. These core communities may play important roles in nutrient uptake, suppressing fungal pathogens, and other plant health functions.IMPORTANCE Plant-associated microbiomes are critical for plant health and other important agroecosystem processes. We assessed the bacterial and fungal microbiomes of wheat grown in soils from across a dryland wheat cropping systems in eastern Washington to identify the core microbiome on wheat roots that is consistent across soils from different locations and land use histories. Moreover, cross-domain co-occurrence network analysis identified core and hub taxa that may play important roles in microbial community assembly. Candidate core and hub taxa provide a starting point for targeting microbiome components likely to be critical to plant health and for constructing synthetic microbial communities for further experimentation. This work is one of the first examples of identifying a core microbiome on a major field crop grown across hundreds of square kilometers over a wide range of biogeographical zones.

Biosolids and Tillage Practices Influence Soil Bacterial Communities in Dryland Wheat.

Class B biosolids are used in dryland wheat (Triticum aestivum L.) production in eastern Washington as a source of nutrients and to increase soil organic matter, but little is known about their effects on bacterial communities and potential for harboring human pathogens. Moreover, conservation tillage is promoted to reduce erosion and soil degradation. We explored the impacts of biosolids or synthetic fertilizer in combination with traditional (conventional) or conservation tillage on soil bacterial communities. Bacterial communities were characterized from fresh biosolids, biosolid aggregates embedded in soil, and soil after a second application of biosolids using high-throughput amplicon sequencing. Biosolid application significantly affected bacterial communities, even 4 years after their application. Bacteria in the families Clostridiaceae, Norcardiaceae, Anaerolinaceae, Dietziaceae, and Planococcaceae were more abundant in fresh biosolids, biosolid aggregates, and soils treated with biosolids than in synthetically fertilized soils. Taxa identified as Turcibacter, Dietzia, Clostridiaceae, and Anaerolineaceae were highly abundant in biosolid aggregates in the soil and likely originated from the biosolids. In contrast, Oxalobacteriaceae, Streptomyceteaceae, Janthinobacterium, Pseudomonas, Kribbella, and Bacillus were rare in the fresh biosolids, but relatively abundant in biosolid aggregates in the soil, and probably originated from the soil to colonize the substrate. However, tillage had relatively minor effects on bacterial communities, with only a small number of taxa differing in relative abundance between traditional and conventional tillage. Although biosolid-associated bacteria persisted in soil, potentially pathogenic taxa were extremely rare and no toxin genes for key groups (Salmonella, Clostridium) were detectable, suggesting that although fecal contamination was apparent via indicator taxa, pathogen populations had declined to low levels. Thus, biosolid amendments had profound effects on soil bacterial communities both by introducing gut- or digester-derived bacteria and by enriching potentially beneficial indigenous soil populations.

Location, Root Proximity, and Glyphosate-Use History Modulate the Effects of Glyphosate on Fungal Community Networks of Wheat.

Glyphosate is the most-used herbicide worldwide and an essential tool for weed control in no-till cropping systems. However, concerns have been raised regarding the long-term effects of glyphosate on soil microbial communities. We examined the impact of repeated glyphosate application on bulk and rhizosphere soil fungal communities of wheat grown in four soils representative of the dryland wheat production region of Eastern Washington, USA. Further, using soils from paired fields, we contrasted the response of fungal communities that had a long history of glyphosate exposure and those that had no known exposure. Soil fungal communities were characterized after three cycles of wheat growth in the greenhouse followed by termination with glyphosate or manual clipping of plants. We found that cropping system, location, year, and root proximity were the primary drivers of fungal community compositions, and that glyphosate had only small impacts on fungal community composition or diversity. However, the taxa that responded to glyphosate applications differed between rhizosphere and bulk soil and between cropping systems. Further, a greater number of fungal OTUs responded to glyphosate in soils with a long history of glyphosate use. Finally, fungal co-occurrence patterns, but not co-occurrence network characteristics, differed substantially between glyphosate-treated and non-treated communities. Results suggest that most fungi influenced by glyphosate are saprophytes that likely feed on dying roots.

Succession of Fungal and Oomycete Communities in Glyphosate-Killed Wheat Roots.

The successional dynamics of root-colonizing microbes are hypothesized to be critical to displacing fungal pathogens that can proliferate after the use of some herbicides. Applications of glyphosate in particular, which compromises the plant defense system by interfering with the production of aromatic amino acids, are thought to promote a buildup of root pathogens and can result in a "greenbridge" between weeds or volunteers and crop hosts. By planting 2 to 3 weeks after spraying, growers can avoid most negative impacts of the greenbridge by allowing pathogen populations to decline, but with the added cost of delayed planting dates. However, the specific changes in microbial communities during this period of root death and the microbial taxa likely to be involved in displacing pathogens are poorly characterized. Using high-throughput sequencing, we characterized fungal and oomycete communities in roots after applications of herbicides with different modes of action (glyphosate or clethodim) and tracked their dynamics over 3 weeks in both naturally infested soil and soil inoculated with Rhizoctonia solani AG-8. We found that many unexpected taxa were present at high relative abundance (e.g., Pythium volutum and Myrmecridium species) in live and dying wheat roots and may play an under-recognized role in greenbridge dynamics. Moreover, communities were highly dynamic over time and had herbicide-specific successional patterns, but became relatively stable by 2 weeks after herbicide application. Network analysis of communities over time revealed patterns of interactions among taxa that were both common and unique to each herbicide treatment and identified two primary groups of taxa with many positive associations within-groups but negative associations between-groups, suggesting that these groups are antagonistic to one another in dying roots and may play a role in displacing pathogen populations during greenbridge dynamics.

Impacts of Repeated Glyphosate Use on Wheat-Associated Bacteria Are Small and Depend on Glyphosate Use History.

Glyphosate is the most widely used herbicide worldwide and a critical tool for weed control in no-till cropping systems. However, there are concerns about the nontarget impacts of long-term glyphosate use on soil microbial communities. We investigated the impacts of repeated glyphosate treatments on bacterial communities in the soil and rhizosphere of wheat in soils with and without long-term history of glyphosate use. We cycled wheat in the greenhouse using soils from 4 paired fields under no-till (20+-year history of glyphosate) or no history of use. At each cycle, we terminated plants with glyphosate (2× the field rate) or by removing the crowns, and soil and rhizosphere bacterial communities were characterized. Location, cropping history, year, and proximity to the roots had much stronger effects on bacterial communities than did glyphosate, which only explained 2 to 5% of the variation. Less than 1% of all taxa were impacted by glyphosate, more in soils with a long history of use, and more increased than decreased in relative abundance. Glyphosate had minimal impacts on soil and rhizosphere bacteria of wheat, although dying roots after glyphosate application may provide a "greenbridge" favoring some copiotrophic taxa.IMPORTANCE Glyphosate (Roundup) is the most widely used herbicide in the world and the foundation of Roundup Ready soybeans, corn, and the no-till cropping system. However, there have been recent concerns about nontarget impacts of glyphosate on soil microbes. Using next-generation sequencing methods and glyphosate treatments of wheat plants, we described the bacterial communities in the soil and rhizosphere of wheat grown in Pacific Northwest soils across multiple years, different locations, and soils with different histories of glyphosate use. The effects of glyphosate were subtle and much less than those of drivers such as location and cropping systems. Only a small percentage of the bacterial groups were influenced by glyphosate, and most of those were stimulated, probably because of the dying roots. This study provides important information for the future of this important tool for no-till systems and the environmental benefits of reducing soil erosion and fossil fuel inputs.

Disease Suppressive Soils: New Insights from the Soil Microbiome.

Soils suppressive to soilborne pathogens have been identified worldwide for almost 60 years and attributed mainly to suppressive or antagonistic microorganisms. Rather than identifying, testing and applying potential biocontrol agents in an inundative fashion, research into suppressive soils has attempted to understand how indigenous microbiomes can reduce disease, even in the presence of the pathogen, susceptible host, and favorable environment. Recent advances in next-generation sequencing of microbiomes have provided new tools to reexamine and further characterize the nature of these soils. Two general types of suppression have been described: specific and general suppression, and theories have been developed around these two models. In this review, we will present three examples of currently-studied model systems with features representative of specific and general suppressiveness: suppression to take-all (Gaeumannomyces graminis var. tritici), Rhizoctonia bare patch of wheat (Rhizoctonia solani AG-8), and Streptomyces. To compare and contrast the two models of general versus specific suppression, we propose a number of hypotheses about the nature and ecology of microbial populations and communities of suppressive soils. We outline the potential and limitations of new molecular techniques that can provide novel ways of testing these hypotheses. Finally, we consider how this greater understanding of the phytobiome can facilitate sustainable disease management in agriculture by harnessing the potential of indigenous soil microbes.

How High Can You Go?: Retrograde Nailing of Proximal Femur Fractures.

There are no data-supported recommendations on how proximal is too proximal for retrograde nailing (RGN). At six level 1 trauma centers, patients with femur fractures within the proximal one-third of the femur treated with RGN were included. This article describes a proximal segment capture ratio (PSCR) and nail segment capture ratio to evaluate RGN of proximal fractures. The study included 107 patients. The average follow-up was 44 weeks. There were two nonunions and three malunions. There was no significant difference between PSCR of 0.3 or less and need for secondary procedures or time to full weight bearing (p>.05). In this study, a smaller (< 0.3) PSCR was not associated with an increased number of complications. A higher Orthopaedic Trauma Association classification was predictive of malunion and increased time to union. These data demonstrate that retrograde nailing is safe and effective for the treatment of supraisthmal femur fractures.

Leveraging ecological theory to guide natural product discovery.

Technological improvements have accelerated natural product (NP) discovery and engineering to the point that systematic genome mining for new molecules is on the horizon. NP biosynthetic potential is not equally distributed across organisms, environments, or microbial life histories, but instead is enriched in a number of prolific clades. Also, NPs are not equally abundant in nature; some are quite common and others markedly rare. Armed with this knowledge, random 'fishing expeditions' for new NPs are increasingly harder to justify. Understanding the ecological and evolutionary pressures that drive the non-uniform distribution of NP biosynthesis provides a rational framework for the targeted isolation of strains enriched in new NP potential. Additionally, ecological theory leads to testable hypotheses regarding the roles of NPs in shaping ecosystems. Here we review several recent strain prioritization practices and discuss the ecological and evolutionary underpinnings for each. Finally, we offer perspectives on leveraging microbial ecology and evolutionary biology for future NP discovery.

Do tradeoffs structure antibiotic inhibition, resistance, and resource use among soil-borne Streptomyces?

BACKGROUND: Tradeoffs among competing traits are believed to be crucial to the maintenance of diversity in complex communities. The production of antibiotics to inhibit competitors and resistance to antibiotic inhibition are two traits hypothesized to be critical to microbial fitness in natural habitats, yet data on costs or tradeoffs associated with these traits are limited. In this work we characterized tradeoffs between antibiotic inhibition or resistance capacities and growth efficiencies or niche widths for a broad collection of Streptomyces from soil. RESULTS: Streptomyces isolates tended to have either very little or very high inhibitory capacity. In contrast, Streptomyces isolates were most commonly resistant to antibiotic inhibition by an intermediate number of other isolates. Streptomyces with either very high antibiotic inhibitory or resistance capacities had less efficient growth and utilized a smaller number of resources for growth (smaller niche width) than those with low inhibition or resistance capacities, suggesting tradeoffs between antibiotic inhibitory or resistance and resource use phenotypes. CONCLUSIONS: This work suggests that life-history tradeoffs may be crucial to the maintenance of the vast diversity of antibiotic inhibitory and resistance phenotypes found among Streptomyces in natural communities.

Plant community richness and microbial interactions structure bacterial communities in soil.

Plant species, plant community diversity and microbial interactions can significantly impact soil microbial communities, yet there are few data on the interactive effects of plant species and plant community diversity on soil bacterial communities. We hypothesized that plant species and plant community diversity affect soil bacterial communities by setting the context in which bacterial interactions occur. Specifically, we examined soil bacterial community composition and diversity in relation to plant "host" species, plant community richness, bacterial antagonists, and soil edaphic characteristics. Soil bacterial communities associated with four different prairie plant species (Andropogon gerardii, Schizachyrium scoparium, Lespedeza capitata, and' Lupinus perennis) grown in plant communities of increasing species richness (1, 4, 8, and 16 species) were sequenced. Additionally, soils were evaluated for populations of antagonistic bacteria and edaphic characteristics. Plant species effects on soil bacterial community composition were small and depended on plant community richness. In contrast, increasing plant community richness significantly altered soil bacterial community composition and was negatively correlated with bacterial diversity. Concentrations of soil carbon, organic matter, nitrogen, phosphorus, and potassium were similarly negatively correlated with bacterial diversity, whereas the proportion of antagonistic bacteria was positively correlated with soil bacterial diversity. Results suggest that plant species influences on soil bacterial communities depend on plant community diversity and are mediated through the effects of plant-derived resources on antagonistic soil microbes.

Mapping the conformational space accessible to catechol-O-methyltransferase.

Methylation catalysed by catechol-O-methyltransferase (COMT) is the main pathway of catechol neurotransmitter deactivation in the prefrontal cortex. Low levels of this class of neurotransmitters are held to be causative of diseases such as schizophrenia, depression and Parkinson's disease. Inhibition of COMT may increase neurotransmitter levels, thus offering a route for treatment. Structure-based drug design hitherto seems to be based on the closed enzyme conformation. Here, a set of apo, semi-holo, holo and Michaelis form crystal structures are described that define the conformational space available to COMT and that include likely intermediates along the catalytic pathway. Domain swaps and sizeable loop movements around the active site testify to the flexibility of this enzyme, rendering COMT a difficult drug target. The low affinity of the co-substrate S-adenosylmethionine and the large conformational changes involved during catalysis highlight significant energetic investment to achieve the closed conformation. Since each conformation of COMT is a bona fide target for inhibitors, other states than the closed conformation may be promising to address. Crystallographic data for an alternative avenue of COMT inhibition, i.e. locking of the apo state by an inhibitor, are presented. The set of COMT structures may prove to be useful for the development of novel classes of inhibitors.

Diffuse symbioses: roles of plant-plant, plant-microbe and microbe-microbe interactions in structuring the soil microbiome.

A conceptual model emphasizing direct host-microbe interactions has dominated work on host-associated microbiomes. To understand plant-microbiome associations, however, broader influences on microbiome composition and functioning must be incorporated, such as those arising from plant-plant and microbe-microbe interactions. We sampled soil microbiomes associated with target plant species (Andropogon gerardii, Schizachyrium scoparium, Lespedeza capitata, Lupinus perennis) grown in communities varying in plant richness (1-, 4-, 8- or 16-species). We assessed Streptomyces antagonistic activity and analysed bacterial and Streptomyces populations via 454 pyrosequencing. Host plant species and plant richness treatments altered networks of coassociation among bacterial taxa, suggesting the potential for host plant effects on the soil microbiome to include changes in microbial interaction dynamics and, consequently, co-evolution. Taxa that were coassociated in the rhizosphere of a given host plant species often showed consistent correlations between operational taxonomic unit (OTU) relative abundance and Streptomyces antagonistic activity, in the rhizosphere of that host. However, in the rhizosphere of a different host plant species, the same OTUs showed no consistency, or a different pattern of responsiveness to such biotic habitat characteristics. The diversity and richness of bacterial and Streptomyces communities exhibited distinct relationships with biotic and abiotic soil characteristics. The rhizosphere soil microbiome is influenced by a complex and nested array of factors at varying spatial scales, including plant community, plant host, soil edaphics and microbial taxon and community characteristics.

Interactive transcriptome analyses of Northern Wild Rice (Zizania palustris L.) and Bipolaris oryzae show convoluted communications during the early stages of fungal brown spot development.

Fungal diseases, caused mainly by Bipolaris spp., are past and current threats to Northern Wild Rice (NWR) grain production and germplasm preservation in both natural and cultivated settings. Genetic resistance against the pathogen is scarce. Toward expanding our understanding of the global gene communications of NWR and Bipolaris oryzae interaction, we designed an RNA sequencing study encompassing the first 12 h and 48 h of their encounter. NWR activated numerous plant recognition receptors after pathogen infection, followed by active transcriptional reprogramming of signaling mechanisms driven by Ca2+ and its sensors, mitogen-activated protein kinase cascades, activation of an oxidative burst, and phytohormone signaling-bound mechanisms. Several transcription factors associated with plant defense were found to be expressed. Importantly, evidence of diterpenoid phytoalexins, especially phytocassane biosynthesis, among expression of other defense genes was found. In B. oryzae, predicted genes associated with pathogenicity including secreted effectors that could target plant defense mechanisms were expressed. This study uncovered the early molecular communication between the NWR-B. oryzae pathosystem, which could guide selection for allele-specific genes to boost NWR defenses, and overall aid in the development of more efficient selection methods in NWR breeding through the use of the most virulent fungal isolates.

Mapping the conformational space accessible to BACE2 using surface mutants and cocrystals with Fab fragments, Fynomers and Xaperones.

The aspartic protease BACE2 is responsible for the shedding of the transmembrane protein Tmem27 from the surface of pancreatic ß-cells, which leads to inactivation of the ß-cell proliferating activity of Tmem27. This role of BACE2 in the control of ß-cell maintenance suggests BACE2 as a drug target for diabetes. Inhibition of BACE2 has recently been shown to lead to improved control of glucose homeostasis and to increased insulin levels in insulin-resistant mice. BACE2 has 52% sequence identity to the well studied Alzheimer's disease target enzyme ß-secretase (BACE1). High-resolution BACE2 structures would contribute significantly to the investigation of this enzyme as either a drug target or anti-target. Surface mutagenesis, BACE2-binding antibody Fab fragments, single-domain camelid antibody VHH fragments (Xaperones) and Fyn-kinase-derived SH3 domains (Fynomers) were used as crystallization helpers to obtain the first high-resolution structures of BACE2. Eight crystal structures in six different packing environments define an ensemble of low-energy conformations available to the enzyme. Here, the different strategies used for raising and selecting BACE2 binders for cocrystallization are described and the crystallization success, crystal quality and the time and resources needed to obtain suitable crystals are compared.

Resource use of soilborne Streptomyces varies with location, phylogeny, and nitrogen amendment.

In this study, we explore variation in resource use among Streptomyces in prairie soils. Resource use patterns were highly variable among Streptomyces isolates and were significantly related to location, phylogeny, and nitrogen (N) amendment history. Streptomyces populations from soils less than 1 m apart differed significantly in their ability to use resources, indicating that drivers of resource use phenotypes in soil are highly localized. Variation in resource use within Streptomyces genetic groups was significantly associated with the location from which Streptomyces were isolated, suggesting that resource use is adapted to local environments. Streptomyces from soils under long-term N amendment used fewer resources and grew less efficiently than those from non-amended soils, demonstrating that N amendment selects for Streptomyces with more limited catabolic capacities. Finally, resource use among Streptomyces populations was correlated with soil carbon content and Streptomyces population densities. We hypothesize that variation in resource use among Streptomyces reflects adaptation to local resource availability and competitive species interactions in soil and that N amendments alter selection for resource use phenotypes.

Molecular recognition at the active site of catechol-O-methyltransferase (COMT): adenine replacements in bisubstrate inhibitors.

L-Dopa, the standard therapeutic for Parkinson's disease, is inactivated by the enzyme catechol-O-methyltransferase (COMT). COMT catalyzes the transfer of an activated methyl group from S-adenosylmethionine (SAM) to its catechol substrates, such as L-dopa, in the presence of magnesium ions. The molecular recognition properties of the SAM-binding site of COMT have been investigated only sparsely. Here, we explore this site by structural alterations of the adenine moiety of bisubstrate inhibitors. The molecular recognition of adenine is of special interest due to the great abundance and importance of this nucleobase in biological systems. Novel bisubstrate inhibitors with adenine replacements were developed by structure-based design and synthesized using a nucleosidation protocol introduced by Vorbrüggen and co-workers. Key interactions of the adenine moiety with COMT were measured with a radiochemical assay. Several bisubstrate inhibitors, most notably the adenine replacements thiopyridine, purine, N-methyladenine, and 6-methylpurine, displayed nanomolar IC(50) values (median inhibitory concentration) for COMT down to 6 nM. A series of six cocrystal structures of the bisubstrate inhibitors in ternary complexes with COMT and Mg(2+) confirm our predicted binding mode of the adenine replacements. The cocrystal structure of an inhibitor bearing no nucleobase can be regarded as an intermediate along the reaction coordinate of bisubstrate inhibitor binding to COMT. Our studies show that solvation varies with the type of adenine replacement, whereas among the adenine derivatives, the nitrogen atom at position 1 is essential for high affinity, while the exocyclic amino group is most efficiently substituted by a methyl group.