Biochemical characterization and crystal structure of Synechocystis arogenate dehydrogenase provide insights into catalytic reaction

The extreme diversity in substrate specificity, and in the regulation mechanism of arogenate/prephenate dehydrogenase enzymes in nature, makes a comparative structural study of these enzymes of great interest. We report here on the biochemical and structural characterization of arogenate dehydrogenase from Synechocystis sp. (TyrAsy). This work paves the way for the understanding of the structural determinants leading to diversity in substrate specificity, and of the regulation mechanisms of arogenate/prephenate dehydrogenases. The overall structure of TyrAsy in complex with NADP was refined to 1.6 A. The asymmetric unit contains two TyrAsy homodimers, with each monomer consisting of a nucleotide binding N-terminal domain and a particularly unique alpha-helical C-terminal dimerization domain. The substrate arogenate was modeled into the active site. The model of the ternary complex enzyme-NADP-arogenate nicely reveals at the atomic level the concerted mechanism of the arogenate/prephenate dehydrogenase reaction.     

Spawning energetics and otolith microchemistry provide insights into the stock structure of bonga shad Ethmalosa fimbriata

The gross energy content of spawning batches and the microchemistry of sagittal otoliths in individual female bonga shad Ethmalosa fimbriata were compared between contrasting sampling sites at the Senegalese southern coast and inside the hypersaline Sine Saloum Estuary. Results show that females spawning in the estuary's middle reaches invested almost three times more energy into reproduction (115 ± 65 J g⁻¹ body mass) than their neritic counterparts (39 ± 34 J g⁻¹ body mass). Also, female otolith levels of Ba:Ca, Sr:Ca and Zn:Ca either differed significantly between study sites or could be linked to heterogeneous environmental variables. A quadratic discriminant function analysis provided evidence of segregated spawning populations of E. fimbriata in southern Senegalese waters.     

Fish species introductions provide novel insights into the patterns and drivers of phylogenetic structure in freshwaters

Despite long-standing interest of terrestrial ecologists, freshwater ecosystems are a fertile, yet unappreciated, testing ground for applying community phylogenetics to uncover mechanisms of species assembly. We quantify phylogenetic clustering and overdispersion of native and non-native fishes of a large river basin in the American Southwest to test for the mechanisms (environmental filtering versus competitive exclusion) and spatial scales influencing community structure. Contrary to expectations, non-native species were phylogenetically clustered and related to natural environmental conditions, whereas native species were not phylogenetically structured, likely reflecting human-related changes to the basin. The species that are most invasive (in terms of ecological impacts) tended to be the most phylogenetically divergent from natives across watersheds, but not within watersheds, supporting the hypothesis that Darwin''s naturalization conundrum is driven by the spatial scale. Phylogenetic distinctiveness may facilitate non-native establishment at regional scales, but environmental filtering restricts local membership to closely related species with physiological tolerances for current environments. By contrast, native species may have been phylogenetically clustered in historical times, but species loss from contemporary populations by anthropogenic activities has likely shaped the phylogenetic signal. Our study implies that fundamental mechanisms of community assembly have changed, with fundamental consequences for the biogeography of both native and non-native species.     

Gall-inducing insects provide insights into plant systematic relationships

Field surveys of cynipid gall-inducer occurrences on Quercus species were conducted in Florida, North Carolina, and Pennsylvania, USA. All cynipids demonstrated strong host species and organ fidelity. One result of this specialization is effective niche partitioning among cynipids. The host-association patterns of these specialist herbivores should reflect similarities among oaks, thus we clustered oak species according to their cynipid distributions. Cynipids distinguished small differences among their hosts. A dendrogram of oak species based on cynipid distributions was largely congruent with botanical arrangernents. Cynipid occurrences offer information helpful to resolving some aspects of oak systematics. Collaborative efforts between taxonomic botanists and entomologists will be useful in resolving a variety of plant and insect systematic problems.     

Large-scale snake genome analyses provide insights into vertebrate development

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Kinetic analyses of the magnesium chelatase provide insights into the mechanism, structure, and formation of the complex

The metabolic pathway known as (bacterio)chlorophyll biosynthesis is initiated by magnesium chelatase (BchI, BchD, BchH). This first step involves insertion of magnesium into protoporphyrin IX (proto), a process requiring ATP hydrolysis. Structural information shows that the BchI and BchD subunits form a double hexameric enzyme complex, whereas BchH binds proto and can be purified as BchH-proto. We utilized the Rhodobacter capsulatus magnesium chelatase subunits using continuous magnesium chelatase assays and treated the BchD subunit as the enzyme with both BchI and BchH-proto as substrates. Michaelis-Menten kinetics was observed with the BchI subunit, whereas the BchH subunit exhibited sigmoidal kinetics (Hill coefficient of 1.85). The BchI.BchD complex had intrinsic ATPase activity, and addition of BchH greatly increased ATPase activity. This was concentration-dependent and gave sigmoidal kinetics, indicating there is more than one binding site for the BchH subunit on the BchI.BchD complex. ATPase activity was approximately 40-fold higher than magnesium chelatase activity and continued despite cessation of magnesium chelation, implying one or more secondary roles for ATP hydrolysis and possibly an as yet unknown switch required to terminate ATPase activity. One of the secondary roles for BchH-stimulated ATP hydrolysis by a BchI.BchD complex is priming of BchH to facilitate correct binding of proto to BchH in a form capable of participating in magnesium chelation. This porphyrin binding is the rate-limiting step in catalysis. These data suggest that ATP hydrolysis by the BchI.BchD complex causes a series of conformational changes in BchH to effect substrate binding, magnesium chelation, and product release.     

Crystal structure of AmyA lacks acidic surface and provide insights into protein stability at poly-extreme condition

Here we report the first crystal structure of a protein, AmyA, a secretory alpha-amylase isolated from Halothermothrix orenii, which is both halophilic and thermophilic. The crystal structure was determined at 1.6 A resolution. AmyA lacks the conserved acidic surface, which is considered essential for protein stability at high salinity. Sedimentation velocity and CD experiments on AmyA reveal the formation of unique reversible poly-dispersed oligomers that show unusually high thermal stability. These studies provide valuable insight into the structural elements that contribute to the stability of AmyA at both physical and chemical extremes and their functional implications.     

Feature embeddings from the BirdNET algorithm provide insights into avian ecology

Bioacoustics has become widely used in the study of acoustically active animals, and machine learning algorithms have emerged as efficient and effective strategies to identify species vocalizations. Current applications of machine learning in bioacoustics often identify acoustic events to the species-level but fail to capture the complex acoustic repertoires animals use to communicate, which can inform habitat associations, demography, behavior, and the life history of cryptic species. The penultimate layer of most machine learning algorithms results in a vector of numbers describing the input, called feature embeddings. Here, we demonstrate that the feature embeddings generated by the BirdNET algorithm can enable within-species classifications of acoustic events. First, we successfully differentiated adult and juvenile Great Gray Owls; second, we identified three unique sounds associated with Great Spotted Woodpeckers (series call, alarm call, and drumming). These applications of BirdNET feature embeddings suggest that researchers can classify vocalizations into groups when group membership is unknown, and that within-species grouping is possible even when target signals are extremely rare. These applications of a relatively “black-box” aspect of machine learning algorithms can be used to derive ecologically informative acoustic classifications, which can inform the conservation of cryptic and otherwise difficult to study species.     

Omics technologies provide new insights into the molecular physiopathology of equine osteochondrosis

Background

Osteochondrosis (OC(D)) is a juvenile osteo-articular disorder affecting several mammalian species. In horses, OC(D) is considered as a multifactorial disease and has been described as a focal disruption of endochondral ossification leading to the development of osteoarticular lesions. Nevertheless, OC(D) physiopathology is poorly understood. Affected horses may present joint swelling, stiffness and lameness. Thus, OC(D) is a major concern for the equine industry. Our study was designed as an integrative approach using omics technologies for the identification of constitutive defects in epiphyseal cartilage and/or subchondral bone associated with the development of primary lesions to further understand OC(D) pathology. This study compared samples from non-affected joints (hence lesion-free) from OC(D)-affected foals (n = 5, considered predisposed samples) with samples from OC-free foals (n = 5) considered as control samples. Consequently, results are not confounded by changes associated with the evolution of the lesion, but focus on altered constitutive molecular mechanisms. Comparative proteomics and micro computed tomography analyses were performed on predisposed and OC-free bone and cartilage samples. Metabolomics was also performed on synovial fluid from OC-free, OC(D)-affected and predisposed joints.

Results

Two lesion subtypes were identified: OCD (lesion with fragment) and OC (osteochondral defects). Modulated proteins were identified using omics technologies (2-DE proteomics) in cartilage and bone from affected foals compare to OC-free foals. These were associated with cellular processes including cell cycle, energy production, cell signaling and adhesion as well as tissue-specific processes such as chondrocyte maturation, extracellular matrix and mineral metabolism. Of these, five had already been identified in synovial fluid of OC-affected foals: ACTG1 (actin, gamma 1), albumin, haptoglobin, FBG (fibrinogen beta chain) and C4BPA (complement component 4 binding protein, alpha).

Conclusion

This study suggests that OCD lesions may result from a cartilage defect whereas OC lesions may be triggered by both bone and cartilage defects, suggesting that different molecular mechanisms responsible for the equine osteochondrosis lesion subtypes and predisposition could be due to a defect in both bone and cartilage. This study will contribute to refining the definition of OC(D) lesions and may improve diagnosis and development of therapies for horses and other species, including humans.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-947) contains supplementary material, which is available to authorized users.     

Deadwood-rich managed forests provide insights into the old-forest association of wood-inhabiting fungi

A major question in fungal conservation is why many species are confined to old forests, and how they could be supported by contemporary landscape matrix. Specifically, forestry that retains large biological legacies across landscape could reduce old-forest dependencies to species that require unusual substrate conditions. We sampled polypores in 112 2 ha plots (both old and harvested stands) in a semi-natural forestry context in Estonia and modelled the habitat factors of species confined to old growth. The results confirmed that old-growth assemblages emerged mostly due to diverse and abundant substrate supply (notably downed CWD). Only 10 species (five spruce-dwellers) were confined to old growth; of these, only Fomitopsis rosea and Oxyporus corticola were additionally affected by forest connectivity. The forestry system studied appeared particularly favourable for the species inhabiting deciduous wood. To better address habitat degradation in conservation, expert lists of ‘old-forest (indicator) fungi’ should be replaced with evidence-based focal taxa.     

Phylogeography of Asparagopsis taxiformis revisited: Combined mtDNA data provide novel insights into population structure in Japan

Six intraspecific lineages (Lineages 1–6) of Asparagopsis taxiformis have been previously established based on mitochondrial cox2‐cox3 intergenic spacer and a partial cox1 sequences. ‘Lineage 2’ (L2) was suggested to be a recent introduction to the Mediterranean Sea, but its source population has not yet been identified. In order to clarify the nature of northwestern Pacific populations, we performed extensive sampling in Japan (60 individuals from 16 locations) and molecular phylogenetic analyses based on mitochondrial sequences. Sixteen additional individuals, collected from eight locations in the Indo‐Pacific, Caribbean, and Mediterranean regions, were also analyzed. Combined sequence analyses revealed that the Japanese populations only consisted of L2. Out of 19 combined haplotypes identified within L2, two are shared between Japan and the Mediterranean Sea and the Hawaiian Islands, and 12 were identified as endemic to Japan. Genetic analyses of population differentiation suggested that Japanese populations are genetically isolated from the Mediterranean and the Hawaiian populations. A genetic disjunction appears to separate two subpopulations within Japan: one between Toi and Kagoshima and the other between Ojikajima Island and Kagoshima in the Kyushu area.     

Arabidopsis thaliana nucleosidase mutants provide new insights into nucleoside degradation

A central step in nucleoside and nucleobase salvage pathways is the hydrolysis of nucleosides to their respective nucleobases. In plants this is solely accomplished by nucleosidases (EC 3.2.2.x). To elucidate the importance of nucleosidases for nucleoside degradation, general metabolism, and plant growth, thorough phenotypic and biochemical analyses were performed using Arabidopsis thaliana T-DNA insertion mutants lacking expression of the previously identified genes annotated as uridine ribohydrolases (URH1 and URH2). Comprehensive functional analyses of single and double mutants demonstrated that both isoforms are unimportant for seedling establishment and plant growth, while one participates in uridine degradation. Rather unexpectedly, nucleoside and nucleotide profiling and nucleosidase activity screening of soluble crude extracts revealed a deficiency of xanthosine and inosine hydrolysis in the single mutants, with substantial accumulation of xanthosine in one of them. Mixing of the two mutant extracts, and by in vitro activity reconstitution using a mixture of recombinant URH1 and URH2 proteins, both restored activity, thus providing biochemical evidence that at least these two isoforms are needed for inosine and xanthosine hydrolysis. This mutant study demonstrates the utility of in vivo systems for the examination of metabolic activities, with the discovery of the new substrate xanthosine and elucidation of a mechanism for expanding the nucleosidase substrate spectrum.     

Hprt-targeted transgenes provide new insights into smooth muscle-restricted promoter activity

Mouse telokin and SM22 promoters have previously been shown to direct smooth muscle cell-specific expression of transgenes in vivo in adult mice. However, the activity of these promoters is highly dependent on the integration site of the transgene. In the current study, we found that the ectopic expression of telokin promoter transgenes could be abolished by flanking the transgene with insulator elements from the H19 gene. However, the insulator elements did not increase the proportion of mouse lines that exhibited consistent, detectable levels of transgene expression. In contrast, when transgenes were targeted to the hprt locus, both telokin and SM22 promoters resulted in reproducible patterns and levels of transgene expression in all lines of mice examined. Telokin promoter transgene expression was restricted to smooth muscle tissues in adult and embryonic mice. As reported previously, SM22 transgenes were expressed at high levels specifically in arterial smooth muscle cells; however, in contrast to randomly integrated transgenes, the hprt-targeted SM22 transgenes were also expressed at high levels in smooth muscle cells in veins, bladder, and gallbladder. Using hprt-targeted transgenes, we further analyzed elements within the telokin promoter required for tissue specific activity in vivo. Analysis of these transgenes revealed that the CArG element in the telokin promoter is required for promoter activity in all tissues and that the CArG element and adjacent AT-rich region are sufficient to drive transgene expression in bladder but not intestinal smooth muscle cells. visceral smooth muscle; development; myosin light chain kinase; embryos; CArG element     

Genomic analyses provide insights into the polyploidization-driven herbicide adaptation in Leptochloa weeds

Polyploidy confers a selective advantage under stress conditions; however, whether polyploidization mediates enhanced herbicide adaptation remains largely unknown. Tetraploid Leptochloa chinensis is a notorious weed in the rice ecosystem, causing severe yield loss in rice. In China, L. chinensis has only one sister species, the diploid L. panicea, whose damage is rarely reported. To gain insights into the effects of polyploidization on herbicide adaptation, we first assembled a high-quality genome of L. panicea and identified genome structure variations with L. chinensis. Moreover, we identified herbicide-resistance genes specifically expanded in L. chinensis, which may confer a greater herbicide adaptability in L. chinensis. Analysis of gene retention and loss showed that five herbicide target-site genes and several herbicide nontarget-site resistance gene families were retained during polyploidization. Notably, we identified three pairs of polyploidization-retained genes including LcABCC8, LcCYP76C1 and LcCYP76C4 that may enhance herbicide resistance. More importantly, we found that both copies of LcCYP76C4 were under herbicide selection during the spread of L. chinensis in China. Furthermore, we identified another gene potentially involved in herbicide resistance, LcCYP709B2, which is also retained during polyploidization and under selection. This study provides insights into the genomic basis of the enhanced herbicide adaptability of Leptochloa weeds during polyploidization and provides guidance for the precise and efficient control of polyploidy weeds.