Search

Corals’ tolerance to high temperature stress largely depends on their symbiotic microalgae (Symbiodiniaceae). However, the contributing microalgal traits and their relationships to one another are largely unclear. Here we compare the in vitro cellular profiles of seven Cladocopium C1acro microalgal strains (derived from the same ancestral strain) during a four-week exposure to 27°C or 31°C. One was an unselected wild-type strain (WT), three were selected at 31°C for nine years and shown to confer thermal tolerance on the coral host (SS+) and three others were similarly selected but did not confer tolerance (SS-). Flow cytometry was used to measure the staining intensities of cells treated with dyes representing the intracellular stress indicators reactive oxygen species (ROS), reduced glutathione (rGSH) and mitochondrial activity, as well as cell size/shape and photosynthetic pigments. Cell densities and photosynthetic efficiency (ϕPSII, Fv/Fm) were also measured. WT showed the highest levels of intracellular ROS and mitochondrial activity, lowest rGSH and largest cell sizes at both temperatures. SS+ strains had the lowest ROS and highest rGSH values and a unique pattern of correlations among parameters at 31°C. Our results support previous reports implicating the role of microalgal ROS, mitochondria and rGSH in holobiont thermal tolerance and suggest flow cytometry is a useful screening tool for identifying microalgal strains with enhanced thermal tolerance.

Understanding the cumulative risk of chemical mixtures at environmentally realistic concentrations is a key challenge in honey bee ecotoxicology. Ecotoxicogenomics, including transcriptomics, measures responses in individual organisms at the molecular level which can provide insights into the mechanisms underlying phenotypic responses induced by one or more stressors and link impacts on individuals to populations. Here, fifth instar honey bee larvae were sampled from a previously reported field experiment exploring the phenotypic impacts of environmentally realistic chronic exposures of the pesticide imidacloprid (5 μg.kg

Reef-building corals are cnidarian animals (Anthozoa, Scleractinia) that mostly live in colonies composed of hundreds of thousands of tiny coral polyps. The polyps house dinoflagellate algal photosymbionts of the family Symbiodiniaceae inside their gastrodermal cells (Fig. 1 A ), and this mutualistic association builds the three-dimensional structure of the reefs via deposition of calcium carbonate skeletons. Scleractinian corals thus support a tremendous diversity and biomass of coral reef organisms. However, much of the biology of corals is poorly understood, partly due to a lack of advanced genetic tools for both the corals and the Symbiodiniaceae. In PNAS, Cleves et al. (1) catapult the field forward by describing an efficient protocol for applying the CRISPR technology to the coral Acropora millepora . They show that a CRISPR knockout of the gene encoding Heat Shock Transcription Factor 1 ( HSF1 ) in larvae of this coral reduces their heat shock tolerance, thus confirming a role for HSF1 in protecting corals from heat stress. Fig. 1. The scleractinian coral, Acropora millepora . ( A ) Close-up of individual polyps showing the endosymbiotic Symbiodiniaceae algae as brown pigmentation and as individual cells in the Inset . Symbiodiniaceae cells are ∼5 to 10 µm in diameter. Image credit: Katarina Damjanovic (photographer). ( B ) Bleached and unbleached adult colonies during a natural bleaching event on the Great Barrier Reef. Image credit: Ray Berkelmans (photographer). ( C ) Aposymbiotic larva. Image credit: Kate Quigley (photographer). Gaining a deeper understanding of the coral heat stress response is urgent given that climate warming, and particularly the resulting increased frequency and severity of summer heat waves, are responsible for the alarmingly rapid loss of coral reefs worldwide. Exposure to elevated temperature can result in the loss of the symbiotic algae from the coral … [↵][1]1To whom correspondence may be addressed. Email: madeleine.van{at}unimelb.edu.au. [1]: #xref-corresp-1-1

Background Insights into the genetic capacities of species to adapt to future climate change can be gained by using comparative genomic and transcriptomic data to reconstruct the genetic changes associated with such adaptations in the past. Here we investigate the genetic changes associated with adaptation to arid environments, specifically climatic extremes and new cactus hosts, through such an analysis of five repleta group Drosophila species. Results We find disproportionately high rates of gene gains in internal branches in the species’ phylogeny where cactus use and subsequently cactus specialisation and high heat and desiccation tolerance evolved. The terminal branch leading to the most heat and desiccation resistant species, Drosophila aldrichi, also shows disproportionately high rates of both gene gains and positive selection. Several Gene Ontology terms related to metabolism were enriched in gene gain events in lineages where cactus use was evolving, while some regulatory and developmental genes were strongly selected in the Drosophila aldrichi branch. Transcriptomic analysis of flies subjected to sublethal heat shocks showed many more downregulation responses to the stress in a heat sensitive versus heat resistant species, confirming the existence of widespread regulatory as well as structural changes in the species’ differing adaptations. Gene Ontology terms related to metabolism were enriched in the differentially expressed genes in the resistant species while terms related to stress response were over-represented in the sensitive one. Conclusion Adaptations to new cactus hosts and hot desiccating environments were associated with periods of accelerated evolutionary change in diverse biochemistries. The hundreds of genes involved suggest adaptations of this sort would be difficult to achieve in the timeframes projected for anthropogenic climate change. Electronic supplementary material The online version of this article (10.1186/s12864-018-5413-3) contains supplementary material, which is available to authorized users.

Background Bactrocera tryoni and Bactrocera neohumeralis mate asynchronously; the former mates exclusively around dusk while the latter mates during the day. The two species also differ in the colour of the post-pronotal lobe (callus), which is predominantly yellow in B. tryoni and brown in B. neohumeralis. We have examined the genetic relationship between the two characters in hybrids, backcrosses and multigeneration hybrid progeny. Results Our analysis of the mating time of the parental species revealed that while B. tryoni mate exclusively at dusk, B. neohumeralis females pair with B. neohumeralis males during the day and with B. tryoni males at dusk. We found considerable variance in mating time and callus colour among hybrid backcross individuals of both sexes but there was a strong although not invariant trend for callus colour to co-segregate with mating time in both sexes. To genetically separate these two phenotypes we allowed the interspecific F1 hybrids to propagate for 25 generations (F25) without selection for mating time or callus colour, finding that the advanced hybrid population had moved towards B. tryoni phenotypes for both traits. Selection for day mating in replicate lines at F25 resulted in significant phenotypic shifts in both traits towards B. neohumeralis phenotypes in F26. However, we were unable to completely recover the mating time profile of B. neohumeralis and relaxation of selection for day mating led to a shift back towards dusk mating, but not yellow callus colour, by F35. Conclusion We conclude that the inheritance of the two major species-defining traits is separable but tightly linked and involves more than one gene in each case. It also appears that laboratory conditions select for the B. tryoni phenotypes for mating time. We discuss our findings in relation to speciation theory and the likely effects of domestication during the generation of mass release strains for sterile insect control programmes.

Many Drosophila species differ widely in their distributions and climate niches, making them excellent subjects for evolutionary genomic studies. Here, we have developed a database of high-quality assemblies for 46 Drosophila species and one closely related Zaprionus. Fifteen of the genomes were newly sequenced, and 20 were improved with additional sequencing. New or improved annotations were generated for all 47 species, assisted by new transcriptomes for 19. Phylogenomic analyses of these data resolved several previously ambiguous relationships, especially in the melanogaster species group. However, it also revealed significant phylogenetic incongruence among genes, mainly in the form of incomplete lineage sorting in the subgenus Sophophora but also including asymmetric introgression in the subgenus Drosophila. Using the phylogeny as a framework and taking into account these incongruences, we then screened the data for genome-wide signals of adaptation to different climatic niches. First, phylostratigraphy revealed relatively high rates of recent novel gene gain in three temperate pseudoobscura and five desert-adapted cactophilic mulleri subgroup species. Second, we found differing ratios of nonsynonymous to synonymous substitutions in several hundred orthologues between climate generalists and specialists, with trends for significantly higher ratios for those in tropical and lower ratios for those in temperate-continental specialists respectively than those in the climate generalists. Finally, resequencing natural populations of 13 species revealed tropics-restricted species generally had smaller population sizes, lower genome diversity and more deleterious mutations than the more widespread species. We conclude that adaptation to different climates in the genus Drosophila has been associated with large-scale and multifaceted genomic changes.

The cuticular layer of the insect exoskeleton contains diverse compounds that serve important biological functions, including the maintenance of homeostasis by protecting against water loss, protection from injury, pathogens and insecticides, and communication. Bactrocera tryoni (Froggatt) is the most destructive pest of fruit production in Australia, yet there are no published accounts of this species&rsquo, cuticular chemistry. We here provide a comprehensive description of B. tryoni cuticular chemistry. We used gas chromatography-mass spectrometry to identify and characterize compounds in hexane extracts of B. tryoni adults reared from larvae in naturally infested fruits. The compounds found included spiroacetals, aliphatic amides, saturated/unsaturated and methyl branched C12 to C20 chain esters and C29 to C33 normal and methyl-branched alkanes. The spiroacetals and esters were found to be specific to mature females, while the amides were found in both sexes. Normal and methyl-branched alkanes were qualitatively the same in all age and sex groups but some of the alkanes differed in amounts (as estimated from internal standard-normalized peak areas) between mature males and females, as well as between mature and immature flies. This study provides essential foundations for studies investigating the functions of cuticular chemistry in this economically important species.

The Queensland fruit fly, Bactrocera tryoni, is a major pest of Australian horticulture which has expanded its range in association with the spread of horticulture over the last ~ 150 years. Its distribution in northern Australia overlaps that of another fruit fly pest to which some authors accord full species status, Bactrocera aquilonis. We have used reduced representation genome-wide sequencing to genotype 359 individuals taken from 35 populations from across the current range of the two taxa, plus a further 73 individuals from six of those populations collected 15–22 years earlier. We find significant population differentiation along an east–west transect across northern Australia which likely reflects limited but bidirectional gene flow between the two taxa. The southward expansion of B. tryoni has led to relatively little genetic differentiation, and most of it is associated with a move into previously marginal inland habitats. Two disjunct populations elsewhere in Australia and three on Melanesian islands are each clearly differentiated from all others, with data strongly supporting establishment from relatively few founders and significant isolation subsequently. Resequencing of historical samples from one of the disjunct Australian populations shows that its genetic profile has changed little over a 15-year period, while the Melanesian data suggest a succession of ‘island hopping’ events with progressive reductions in genetic diversity. We discuss our results in relation to the control of B. tryoni and as a model for understanding the genetics of invasion and hybridisation processes.

Coral bleaching tolerance can be enhanced through directed evolution of their microalgal symbionts., Coral reefs worldwide are suffering mass mortalities from marine heat waves. With the aim of enhancing coral bleaching tolerance, we evolved 10 clonal strains of a common coral microalgal endosymbiont at elevated temperatures (31°C) for 4 years in the laboratory. All 10 heat-evolved strains had expanded their thermal tolerance in vitro following laboratory evolution. After reintroduction into coral host larvae, 3 of the 10 heat-evolved endosymbionts also increased the holobionts’ bleaching tolerance. Although lower levels of secreted reactive oxygen species (ROS) accompanied thermal tolerance of the heat-evolved algae, reduced ROS secretion alone did not predict thermal tolerance in symbiosis. The more tolerant symbiosis exhibited additional higher constitutive expression of algal carbon fixation genes and coral heat tolerance genes. These findings demonstrate that coral stock with enhanced climate resilience can be developed through ex hospite laboratory evolution of their microalgal endosymbionts.

Burkholderia sp. strain SG-MS1 and Pseudomonas sp. strain SG-MS2 have previously been found to mineralize (+)-pinoresinol through a common catabolic pathway. Here, we used comparative genomics, proteomics, protein semipurification, and heterologous expression to identify a flavoprotein from the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family in SG-MS2 that carries out the initial hydroxylation of (+)-pinoresinol at the benzylic carbon. The cognate gene is translationally coupled with a downstream cytochrome gene, and the cytochrome is required for activity. The flavoprotein has a unique combination of cofactor binding and cytochrome requirements for the VAO/PCMH family. The heterologously expressed enzyme has a K(m) of 1.17 μM for (+)-pinoresinol. The enzyme is overexpressed in strain SG-MS2 upon exposure to (+)-pinoresinol, along with 45 other proteins, 22 of which were found to be encoded by genes in an approximately 35.1-kb cluster also containing the flavoprotein and cytochrome genes. Homologs of 18 of these 22 genes, plus the flavoprotein and cytochrome genes, were also found in a 38.7-kb cluster in SG-MS1. The amino acid identities of four of the other proteins within the SG-MS2 cluster suggest they catalyze conversion of hydroxylated pinoresinol to protocatechuate and 2-methoxyhydroquinone. Nine other proteins upregulated in SG-MS2 on exposure to (+)-pinoresinol appear to be homologs of proteins known to comprise the protocatechuate and 2-methoxyhydroquinone catabolic pathways, but only three of the cognate genes lie within the cluster containing the flavoprotein and cytochrome genes. IMPORTANCE (+)-Pinoresinol is an important plant defense compound, a major food lignan for humans and some other animals, and the model compound used to study degradation of the β-β′ linkages in lignin. We report a gene cluster, in one strain each of Pseudomonas and Burkholderia, that is involved in the oxidative catabolism of (+)-pinoresinol. The flavoprotein component of the α-hydroxylase which heads the pathway belongs to the 4-phenol oxidizing (4PO) subgroup of the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family but constitutes a novel combination of cofactor and electron acceptor properties for the family. It is translationally coupled with a cytochrome gene whose product is also required for activity. The work casts new light on the biology of (+)-pinoresinol and its transformation to other bioactive molecules. Potential applications of the findings include new options for deconstructing lignin into useful chemicals and the generation of new phytoestrogenic enterolactones from lignans.

Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F420, and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F420 is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F420 in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F420, determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F420., F420 is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F420 in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F420 biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-phospholactate stimulates F420 biosynthesis. Analysis of F420 biosynthesis intermediates present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F420-0. To determine the structural basis of dehydro-F420-0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo-, substrate-, and product-bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic precomplex of this enzyme and demonstrate that dehydro-F420-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F420 in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance. IMPORTANCE Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F420, and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F420 is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F420 in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F420, determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F420.

Significance Helicoverpa armigera is a major agricultural and horticultural pest that recently spread from its historical distribution throughout much of the Old World to the Americas, where it is already causing hundreds of millions of dollars in damage every year. The species is notoriously quick to generate and disseminate pesticide resistance throughout its range and has a wider host range than the native Helicoverpa zea. Hybridization between the two species increases the opportunity for novel, agriculturally problematic ecotypes to emerge and spread through the Americas., Within the mega-pest lineage of heliothine moths are a number of polyphagous, highly mobile species for which the exchange of adaptive traits through hybridization would affect their properties as pests. The recent invasion of South America by one of the most significant agricultural pests, Helicoverpa armigera, raises concerns for the formation of novel combinations of adaptive genes following hybridization with the closely related Helicoverpa zea. To investigate the propensity for hybridization within the genus Helicoverpa, we carried out whole-genome resequencing of samples from six species, focusing in particular upon H. armigera population structure and its relationship with H. zea. We show that both H. armigera subspecies have greater genetic diversity and effective population sizes than do the other species. We find no signals for gene flow among the six species, other than between H. armigera and H. zea, with nine Brazilian individuals proving to be hybrids of those two species. Eight had largely H. armigera genomes with some introgressed DNA from H. zea scattered throughout. The ninth resembled an F1 hybrid but with stretches of homozygosity for each parental species that reflect previous hybridization. Regions homozygous for H. armigera-derived DNA in this individual included one containing a gustatory receptor and esterase genes previously associated with host range, while another encoded a cytochrome P450 that confers insecticide resistance. Our data point toward the emergence of novel hybrid ecotypes and highlight the importance of monitoring H. armigera genotypes as they spread through the Americas.

Summary Vegetable oils extracted from oilseeds are an important component of foods, but are also used in a range of high value oleochemical applications. Despite being biodegradable, nontoxic and renewable current plant oils suffer from the presence of residual polyunsaturated fatty acids that are prone to free radical formation that limit their oxidative stability, and consequently shelf life and functionality. Many decades of plant breeding have been successful in raising the oleic content to ~90%, but have come at the expense of overall field performance, including poor yields. Here, we engineer superhigh oleic (SHO) safflower producing a seed oil with 93% oleic generated from seed produced in multisite field trials spanning five generations. SHO safflower oil is the result of seed‐specific hairpin‐based RNA interference of two safflower lipid biosynthetic genes, FAD2.2 and FATB, producing seed oil containing less than 1.5% polyunsaturates and only 4% saturates but with no impact on lipid profiles of leaves and roots. Transgenic SHO events were compared to non‐GM safflower in multisite trial plots with a wide range of growing season conditions, which showed no evidence of impact on seed yield. The oxidative stability of the field‐grown SHO oil produced from various sites was 50 h at 110°C compared to 13 h for conventional ~80% oleic safflower oils. SHO safflower produces a uniquely stable vegetable oil across different field conditions that can provide the scale of production that is required for meeting the global demands for high stability oils in food and the oleochemical industry.

F420 is a low-potential redox cofactor that mediates the transformations of a wide range of complex organic compounds. Considered one of the rarest cofactors in biology, F420 is best known for its role in methanogenesis and has only been chemically identified in two phyla to date, the Euryarchaeota and Actinobacteria. In this work, we show that this cofactor is more widely distributed than previously reported. We detected the genes encoding all five known F420 biosynthesis enzymes (cofC, cofD, cofE, cofG and cofH) in at least 653 bacterial and 173 archaeal species, including members of the dominant soil phyla Proteobacteria, Chloroflexi and Firmicutes. Metagenome datamining validated that these genes were disproportionately abundant in aerated soils compared with other ecosystems. We confirmed through high-performance liquid chromatography analysis that aerobically grown stationary-phase cultures of three bacterial species, Paracoccus denitrificans, Oligotropha carboxidovorans and Thermomicrobium roseum, synthesized F420, with oligoglutamate sidechains of different lengths. To understand the evolution of F420 biosynthesis, we also analyzed the distribution, phylogeny and genetic organization of the cof genes. Our data suggest that although the Fo precursor to F420 originated in methanogens, F420 itself was first synthesized in an ancestral actinobacterium. F420 biosynthesis genes were then disseminated horizontally to archaea and other bacteria. Together, our findings suggest that the cofactor is more significant in aerobic bacterial metabolism and soil ecosystem composition than previously thought. The cofactor may confer several competitive advantages for aerobic soil bacteria by mediating their central metabolic processes and broadening the range of organic compounds they can synthesize, detoxify and mineralize.

Oligomerization has been suggested to be an important mechanism for increasing or maintaining the thermostability of proteins. Although it is evident that protein-protein contacts can result in substantial stabilization in many extant proteins, evidence for evolutionary selection for oligomerization is largely indirect and little is understood of the early steps in the evolution of oligomers. A laboratory-directed evolution experiment that selected for increased thermostability in the αE7 carboxylesterase from the Australian sheep blowfly, Lucilia cuprina, resulted in a thermostable variant, LcαE7-4a, that displayed increased levels of dimeric and tetrameric quaternary structure. A trade-off between activity and thermostability was made during the evolution of thermostability, with the higher-order oligomeric species displaying the greatest thermostability and lowest catalytic activity. Analysis of monomeric and dimeric LcαE7-4a crystal structures revealed that only one of the oligomerization-inducing mutations was located at a potential protein-protein interface. This work demonstrates that by imposing a selective pressure demanding greater thermostability, mutations can lead to increased oligomerization and stabilization, providing support for the hypothesis that oligomerization is a viable evolutionary strategy for protein stabilization.

We use annotated genomes of 14 Drosophila species covering diverse host use phenotypes to test whether 4 gene families that often have detoxification functions are associated with host shifts among species. Bark, slime flux, flower, and generalist necrotic fruit-feeding species all have similar numbers of carboxyl/cholinesterase, glutathione S-transferase, cytochrome P450, and UDP-glucuronosyltransferase genes. However, species feeding on toxic Morinda citrifolia fruit and the fresh fruit-feeding Drosophila suzukii have about 30 and 60 more, respectively. ABC transporters show a different pattern, with the flower-feeding D. elegans and the generalist necrotic fruit and cactus feeder D. hydei having about 20 and >100 more than the other species, respectively. Surprisingly, despite the complex secondary chemistry we find that 3 cactophilic specialists in the mojavensis species cluster have variably fewer genes than any of the other species across all 4 families. We also find 82 positive selection events across the 4 families, with the terminal D. suzukii and M. citrifolia-feeding D. sechellia branches again having the highest number of such events in proportion to their respective branch lengths. Many of the genes involved in these host-use-specific gene number differences or positive selection events lie in specific clades of the gene families that have been recurrently associated with detoxification. Several genes are also found to be involved in multiple duplication and/or positive selection events across the species studied regardless of their host use phenotypes; the most frequently involved are the ABC transporter CG1718, which is not in a specific clade associated with detoxification, and the α-esterase gene cluster, which is.

Carbamate kinases catalyze the conversion of carbamate to carbamoyl phosphate, which is readily transformed into other compounds. Carbamate forms spontaneously from ammonia and carbon dioxide in aqueous solutions, so the kinases have potential for sequestrative utilization of the latter compounds. Here, we compare seven carbamate kinases from mesophilic, thermophilic, and hyperthermophilic sources. In addition to the known enzymes from Enterococcus faecalis and Pyrococcus furiosus , the previously unreported enzymes from the hyperthermophiles Thermococcus sibiricus and Thermococcus barophilus , the thermophiles Fervidobacterium nodosum and Thermosipho melanesiensis , and the mesophile Clostridium tetani were all expressed recombinantly, each in high yield. Only the clostridial enzyme did not show catalysis. In direct assays of carbamate kinase activity, the three hyperthermophilic enzymes display higher specific activities at elevated temperatures, greater stability, and remarkable substrate turnover at alkaline pH (9.9 to 11.4). Thermococcus barophilus and Thermococcus sibiricus carbamate kinases were found to be the most active when the enzymes were tested at 80°C, and maintained activity over broad temperature and pH ranges. These robust thermococcal enzymes therefore represent ideal candidates for biotechnological applications involving aqueous ammonia solutions, since nonbuffered 0.0001 to 1.0 M solutions have pH values of approximately 9.8 to 11.8. As proof of concept, here we also show that carbamoyl phosphate produced by the Thermococcus barophilus kinase is efficiently converted in situ to carbamoyl aspartate by aspartate transcarbamoylase from the same source organism. Using acetyl phosphate to simultaneously recycle the kinase cofactor ATP, at pH 9.9 carbamoyl aspartate is produced in high yield and directly from solutions of ammonia, carbon dioxide, and aspartate. IMPORTANCE Much of the nitrogen in animal wastes and used in fertilizers is commonly lost as ammonia in water runoff, from which it must be removed to prevent downstream pollution and evolution of nitrogenous greenhouse gases. Since carbamate kinases transform ammonia and carbon dioxide to carbamoyl phosphate via carbamate, and carbamoyl phosphate may be converted into other valuable compounds, the kinases provide a route for useful sequestration of ammonia, as well as of carbon dioxide, another greenhouse gas. At the same time, recycling the ammonia in chemical synthesis reduces the need for its energy-intensive production. However, robust catalysts are required for such biotransformations. Here we show that carbamate kinases from hyperthermophilic archaea display remarkable stability and high catalytic activity across broad ranges of pH and temperature, making them promising candidates for biotechnological applications. We also show that carbamoyl phosphate produced by the kinases may be efficiently used to produce carbamoyl aspartate.

Lignin is a complex aromatic polymer found in plant cell walls that makes up 15 to 40% of plant biomass. The degradation of lignin substructures by bacteria is of emerging interest because it could provide renewable alternative feedstocks and intermediates for chemical manufacturing industries. We have isolated a bacterium, strain SG61-1L, that rapidly degrades all of the stereoisomers of one lignin substructure, guaiacylglycerol-β-guaiacyl ether (GGE), which contains a key β- O -4 linkage found in most intermonomer linkages in lignin. In an effort to understand the rapid degradation of GGE by this bacterium, we heterologously expressed and kinetically characterized a suite of dehydrogenase candidates for the first known step of GGE degradation. We identified a clade of active GGE dehydrogenases and also several other dehydrogenases outside this clade that were all able to oxidize GGE. Several candidates exhibited stereoselectivity toward the GGE stereoisomers, while others had higher levels of catalytic performance than previously described GGE dehydrogenases for all four stereoisomers, indicating a variety of potential applications for these enzymes in the manufacture of lignin-derived commodities.

Hexachlorocyclohexane (HCH), a synthetic organochloride, was first used as a broad-acre insecticide in the 1940s, and many HCH-degrading bacterial strains have been isolated from around the globe during the last 20 years. To date, the same degradation pathway (the lin pathway) has been implicated in all strains characterized, although the pathway has only been characterized intensively in two strains and for only a single HCH isomer. To further elucidate the evolution of the lin pathway, we have biochemically and genetically characterized three HCH-degrading strains from the Czech Republic and compared the genomes of these and seven other HCH-degrading bacterial strains. The three new strains each yielded a distinct set of metabolites during their degradation of HCH isomers. Variable assembly of the pathway is a common feature across the 10 genomes, eight of which (including all three Czech strains) were either missing key lin genes or containing duplicate copies of upstream lin genes (linA-F). The analysis also confirmed the important role of horizontal transfer mediated by insertion sequence IS6100 in the acquisition of the pathway, with a stronger association of IS6100 to the lin genes in the new strains. In one strain, a linA variant was identified that likely caused a novel degradation phenotype involving a shift in isomer preference. This study identifies a number of strains that are in the early stages of lin pathway acquisition and shows that the state of the pathway can explain the degradation patterns observed.

Three Australian tephritid fruit flies (Bactrocera tryoni - Q-fly, Bactrocera neohumeralis - NEO, and Bactrocera jarvisi - JAR) are promising models for genetic studies of pest status and invasiveness. The long history of ecological and physi- ological studies of the three species has been augmented by the development of a range of genetic and genomic tools, including the capacity for forced multigeneration crosses between the three species followed by selection experiments, a draft genome for Q-fly, and tissue- and stage-specific transcriptomes. The Q-fly and NEO species pair is of particular interest. The distribution of NEO is contained entirely within the wider distribution of Q-fly and the two species are ecologically extremely similar, with no known differences in pheromones, temperature tolerance, or host-fruit utilisation. However there are three clear differences be- tween them: humeral callus colour, complete pre-mating isolation based on mating time-of-day, and invasiveness. NEO is much less invasive, whereas in historical times Q-fly has invaded southeastern Australia and areas of Western Australia and the North- ern Territory. In southeastern fruit-growing regions, microsatellites suggest that some of these outbreaks might derive from ge- netically differentiated populations overwintering in or near the invaded area. Q-fly and NEO show very limited genome diffe- rentiation, so comparative genomic analyses and QTL mapping should be able to identify the regions of the genome controlling mating time and invasiveness, to assess the genetic bases for the invasive strains of Q-fly, and to facilitate a variety of improve- ments to current sterile insect control strategies for that species (Current Zoology 61 (3): 477-487, 2015). Keywords Bactrocera, Genomics, STERILE-insect-technique, Interspecific-hybridisation

The amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible.

Pinoresinol is a dimer of two β-β′-linked coniferyl alcohol molecules. It is both a plant defense molecule synthesized through the shikimic acid pathway and a representative of several β-β-linked dimers produced during the microbial degradation of lignin in dead plant material. Until now, little has been known about the bacterial catabolism of such dimers. Here we report the isolation of the efficient (+)-pinoresinol-mineralizing Pseudomonas sp. strain SG-MS2 and its catabolic pathway. Degradation of pinoresinol in this strain is inducible and proceeds via a novel oxidative route, which is in contrast to the previously reported reductive transformation by other bacteria. Based on enzyme assays and bacterial growth, cell suspension, and resting cell studies, we provide conclusive evidence that pinoresinol degradation in strain SG-MS2 is initiated by benzylic hydroxylation, generating a hemiketal via a quinone methide intermediate, which is then hydrated at the benzylic carbon by water. The hemiketal, which stays in equilibrium with the corresponding keto alcohol, undergoes an aryl-alkyl cleavage to generate a lactone and 2-methoxyhydroquinone. While the fate of 2-methoxyhydroquinone is not investigated further, it is assumed to be assimilated by ring cleavage. The lactone is further metabolized via two routes, namely, lactone ring cleavage and benzylic hydroxylation via a quinone methide intermediate, as described above. The resulting hemiketal again exists in equilibrium with a keto alcohol. Our evidence suggests that both routes of lactone metabolism lead to vanillin and vanillic acid, which we show can then be mineralized by strain SG-MS2. IMPORTANCE The oxidative catabolism of (+)-pinoresinol degradation elucidated here is fundamentally different from the reductive cometabolism reported for two previously characterized bacteria. Our findings open up new opportunities to use lignin for the biosynthesis of vanillin, a key flavoring agent in foods, beverages, and pharmaceuticals, as well as various new lactones. Our work also has implications for the study of new pinoresinol metabolites in human health. The enterodiol and enterolactone produced through reductive transformation of pinoresinol by gut microbes have already been associated with decreased risks of cancer and cardiovascular diseases. The metabolites from oxidative metabolism we find here also deserve attention in this respect.

F420 is a microbial cofactor that mediates a wide range of physiologically important and industrially relevant redox reactions, including in methanogenesis and tetracycline biosynthesis. This deazaflavin comprises a redox-active isoalloxazine headgroup conjugated to a lactyloligoglutamyl tail. Here we studied the catalytic significance of the oligoglutamate chain, which differs in length between bacteria and archaea. We purified short-chain F420 (two glutamates) from a methanogen isolate and long-chain F420 (five to eight glutamates) from a recombinant mycobacterium, confirming their different chain lengths by HPLC and LC/MS analysis. F420 purified from both sources was catalytically compatible with purified enzymes from the three major bacterial families of F420-dependent oxidoreductases. However, long-chain F420 bound to these enzymes with a six- to ten-fold higher affinity than short-chain F420. The cofactor side chain also significantly modulated the kinetics of the enzymes, with long-chain F420 increasing the substrate affinity (lower Km) but reducing the turnover rate (lower kcat) of the enzymes. Molecular dynamics simulations and comparative structural analysis suggest that the oligoglutamate chain of F420 makes dynamic electrostatic interactions with conserved surface residues of the oxidoreductases while the headgroup binds the catalytic site. In conjunction with the kinetic data, this suggests that electrostatic interactions made by the oligoglutamate tail result in higher-affinity, lower-turnover catalysis. Physiologically, we propose that bacteria have selected for long-chain F420 to better control cellular redox reactions despite tradeoffs in catalytic rate. Conversely, this suggests that industrial use of shorter-length F420 will greatly increase the rates of bioremediation and biocatalysis processes relying on purified F420-dependent oxidoreductases.

An unusual aspect of actinobacterial metabolism is the use of the redox cofactor F420. Studies have shown that actinobacterial F420H2-dependent reductases promiscuously hydrogenate diverse organic compounds in biodegradative and biosynthetic processes. These enzymes therefore represent promising candidates for next-generation industrial biocatalysts. In this work, we undertook the first broad survey of these enzymes as potential industrial biocatalysts by exploring the extent, as well as mechanistic and structural bases, of their substrate promiscuity. We expressed and purified 11 enzymes from seven subgroups of the flavin/deazaflavin oxidoreductase (FDOR) superfamily (A1, A2, A3, B1, B2, B3, B4) from the model soil actinobacterium Mycobacterium smegmatis. These enzymes reduced compounds from six chemical classes, including fundamental monocycles such as a cyclohexenone, a dihydropyran, and pyrones, as well as more complex quinone, coumarin, and arylmethane compounds. Substrate range and reduction rates varied between the enzymes, with the A1, A3, and B1 groups exhibiting greatest promiscuity. Molecular docking studies suggested that structurally diverse compounds are accommodated in the large substrate-binding pocket of the most promiscuous FDOR through hydrophobic interactions with conserved aromatic residues and the isoalloxazine headgroup of F420H2. Liquid chromatography-mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS) analysis of derivatized reaction products showed reduction occurred through a common mechanism involving hydride transfer from F420H- to the electron-deficient alkene groups of substrates. Reduction occurs when the hydride donor (C5 of F420H-) is proximal to the acceptor (electrophilic alkene of the substrate). These findings suggest that engineered actinobacterial F420H2-dependent reductases are promising novel biocatalysts for the facile transformation of a wide range of α,β-unsaturated compounds.

An unusual aspect of actinobacterial metabolism is the use of the redox cofactor F420. Studies have shown that actinobacterial F420H2-dependent reductases promiscuously hydrogenate diverse organic compounds in biodegradative and biosynthetic processes. These enzymes therefore represent promising candidates for next-generation industrial biocatalysts. In this work, we undertook the first broad survey of these enzymes as potential industrial biocatalysts by exploring the extent, as well as mechanistic and structural bases, of their substrate promiscuity. We expressed and purified 11 enzymes from seven subgroups of the flavin/deazaflavin oxidoreductase (FDOR) superfamily (A1, A2, A3, B1, B2, B3, B4) from the model soil actinobacterium Mycobacterium smegmatis. These enzymes reduced compounds from six chemical classes, including fundamental monocycles such as a cyclohexenone, a dihydropyran, and pyrones, as well as more complex quinone, coumarin, and arylmethane compounds. Substrate range and reduction rates varied between the enzymes, with the A1, A3, and B1 groups exhibiting greatest promiscuity. Molecular docking studies suggested that structurally diverse compounds are accommodated in the large substrate-binding pocket of the most promiscuous FDOR through hydrophobic interactions with conserved aromatic residues and the isoalloxazine headgroup of F420H2. Liquid chromatography-mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS) analysis of derivatized reaction products showed reduction occurred through a common mechanism involving hydride transfer from F420H- to the electron-deficient alkene groups of substrates. Reduction occurs when the hydride donor (C5 of F420H-) is proximal to the acceptor (electrophilic alkene of the substrate). These findings suggest that engineered actinobacterial F420H2-dependent reductases are promising novel biocatalysts for the facile transformation of a wide range of α,β-unsaturated compounds.

Background Distinguishing orthologous and paralogous relationships between genes across multiple species is essential for comparative genomic analyses. Various computational approaches have been developed to resolve these evolutionary relationships, but strong trade-offs between precision and recall of orthologue prediction remains an ongoing challenge. Results Here we present Orthonome, an orthologue prediction pipeline, designed to reduce the trade-off between orthologue capture rates (recall) and accuracy of multi-species orthologue prediction. The pipeline compares sequence domains and then forms sequence-similar clusters before using phylogenetic comparisons to identify inparalogues. It then corrects sequence similarity metrics for fragment and gene length bias using a novel scoring metric capturing relationships between full length as well as fragmented genes. The remaining genes are then brought together for the identification of orthologues within a phylogenetic framework. The orthologue predictions are further calibrated along with inparalogues and gene births, using synteny, to identify novel orthologous relationships. We use 12 high quality Drosophila genomes to show that, compared to other orthologue prediction pipelines, Orthonome provides orthogroups with minimal error but high recall. Furthermore, Orthonome is resilient to suboptimal assembly/annotation quality, with the inclusion of draft genomes from eight additional Drosophila species still providing >6500 1:1 orthologues across all twenty species while retaining a better combination of accuracy and recall than other pipelines. Orthonome is implemented as a searchable database and query tool along with multiple-sequence alignment browsers for all sets of orthologues. The underlying documentation and database are accessible at http://www.orthonome.com. Conclusion We demonstrate that Orthonome provides a superior combination of orthologue capture rates and accuracy on complete and draft drosophilid genomes when tested alongside previously published pipelines. The study also highlights a greater degree of evolutionary conservation across drosophilid species than earlier thought. Electronic supplementary material The online version of this article (10.1186/s12864-017-4079-6) contains supplementary material, which is available to authorized users.

International audience; Background: Helicoverpa armigera and Helicoverpa zea are major caterpillar pests of Old and New World agriculture, respectively. Both, particularly H. armigera, are extremely polyphagous, and H. armigera has developed resistance to many insecticides. Here we use comparative genomics, transcriptomics and resequencing to elucidate the genetic basis for their properties as pests. Results: We find that, prior to their divergence about 1.5 Mya, the H. armigera/H. zea lineage had accumulated up to more than 100 more members of specific detoxification and digestion gene families and more than 100 extra gustatory receptor genes, compared to other lepidopterans with narrower host ranges. The two genomes remain very similar in gene content and order, but H. armigera is more polymorphic overall, and H. zea has lost several detoxification genes, as well as about 50 gustatory receptor genes. It also lacks certain genes and alleles conferring insecticide resistance found in H. armigera. Non-synonymous sites in the expanded gene families above are rapidly diverging, both between paralogues and between orthologues in the two species. Whole genome transcriptomic analyses of H. armigera larvae show widely divergent responses to different host plants, including responses among many of the duplicated detoxification and digestion genes. Conclusions: The extreme polyphagy of the two heliothines is associated with extensive amplification and neofunctionalisation of genes involved in host finding and use, coupled with versatile transcriptional responses on different hosts. H. armigera's invasion of the Americas in recent years means that hybridisation could generate populations that are both locally adapted and insecticide resistant.

Previous electrophysiological and behavioural studies implicate esterase 6 in the processing of the pheromone cis-vaccenyl acetate and various food odorants that affect aggregation and reproductive behaviours. Here we show esterase 6 has relatively high activity against many of the short-mid chain food esters, but negligible activity against cis-vaccenyl acetate. The crystal structure of esterase 6 confirms its substrate-binding site can accommodate many short-mid chain food esters but not cis-vaccenyl acetate. Immunohistochemical assays show esterase 6 is expressed in non-neuronal cells in the third antennal segment that could be accessory or epidermal cells surrounding numerous olfactory sensilla, including basiconics involved in food odorant detection. Esterase 6 is also produced in trichoid sensilla, but not in the same cell types as the cis-vaccenyl acetate binding protein LUSH. Our data support a model in which esterase 6 acts as a direct odorant degrading enzyme for many bioactive food esters, but not cis-vaccenyl acetate.

The metabolism of volatile signal molecules by odorant degrading enzymes (ODEs) is crucial to the ongoing sensitivity and specificity of chemoreception in various insects, and a few specific esterases, cytochrome P450s, glutathione S-transferases (GSTs) and UDP-glycosyltransferases (UGTs) have previously been implicated in this process. Significant progress has been made in characterizing ODEs in Lepidoptera but very little is known about them in Diptera, including in Drosophila melanogaster, a major insect model. We have therefore carried out a transcriptomic analysis of the antennae of D. melanogaster in order to identify candidate ODEs. Virgin male and female and mated female antennal transcriptomes were determined by RNAseq. As with the Lepidoptera, we found that many esterases, cytochrome P450 enzymes, GSTs and UGTs are expressed in D. melanogaster antennae. As olfactory genes generally show selective expression in the antennae, a comparison to previously published transcriptomes for other tissues has been performed, showing preferential expression in the antennae for one esterase, JHEdup, one cytochrome P450, CYP308a1, and one GST, GSTE4. These largely uncharacterized enzymes are now prime candidates for ODE functions. JHEdup was expressed heterologously and found to have high catalytic activity against a chemically diverse group of known ester odorants for this species. This is a finding consistent with an ODE although it might suggest a general role in clearing several odorants rather than a specific role in clearing a particular odorant. Our findings do not preclude the possibility of odorant degrading functions for other antennally expressed esterases, P450s, GSTs and UGTs but, if so, they suggest that these enzymes also have additional functions in other tissues.

The widely accepted paradigm for the development of insecticide resistance in field populations of insects is of selection for one or a very few genes of major effect. Limited genetic mapping data for organophosphate and pyrethroid resistance in heliothine and spodopteran pests generally agrees with this paradigm. However, other biochemical and transcriptomic data suggest a more complex set of changes in multiple P450 and esterase gene/enzyme systems in resistant strains of these species. We discuss possible explanations for this paradox, including the likely embedding of these genes in regulatory cascades and emerging evidence for their arrangement in large clusters of closely related genes. We conclude that there could indeed be an unusually large number of genetic options for evolving resistance in these species. © 2013 Society of Chemical Industry

Insect carboxylesterases from the α Esterase gene cluster, such as αE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina ( Lc αE7), play an important physiological role in lipid metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the importance of OPs to agriculture and the spread of insect-borne diseases, the molecular basis for the ability of α-carboxylesterases to confer OP resistance to insects is poorly understood. In this work, we used laboratory evolution to increase the thermal stability of Lc αE7, allowing its overexpression in Escherichia coli and structure determination. The crystal structure reveals a canonical α/β-hydrolase fold that is very similar to the primary target of OPs (acetylcholinesterase) and a unique N-terminal α-helix that serves as a membrane anchor. Soaking of Lc αE7 crystals in OPs led to the capture of a crystallographic snapshot of Lc αE7 in its phosphorylated state, which allowed comparison with acetylcholinesterase and rationalization of its ability to protect insects against the effects of OPs. Finally, inspection of the active site of Lc αE7 reveals an asymmetric and hydrophobic substrate binding cavity that is well-suited to fatty acid methyl esters, which are hydrolyzed by the enzyme with specificity constants (∼10 6 M −1 s −1 ) indicative of a natural substrate.

SUMMARY5-Deazaflavin cofactors enhance the metabolic flexibility of microorganisms by catalyzing a wide range of challenging enzymatic redox reactions. While structurally similar to riboflavin, 5-deazaflavins have distinctive and biologically useful electrochemical and photochemical properties as a result of the substitution of N-5 of the isoalloxazine ring for a carbon. 8-Hydroxy-5-deazaflavin (Fo) appears to be used for a single function: as a light-harvesting chromophore for DNA photolyases across the three domains of life. In contrast, its oligoglutamyl derivative F420is a taxonomically restricted but functionally versatile cofactor that facilitates many low-potential two-electron redox reactions. It serves as an essential catabolic cofactor in methanogenic, sulfate-reducing, and likely methanotrophic archaea. It also transforms a wide range of exogenous substrates and endogenous metabolites in aerobic actinobacteria, for example mycobacteria and streptomycetes. In this review, we discuss the physiological roles of F420in microorganisms and the biochemistry of the various oxidoreductases that mediate these roles. Particular focus is placed on the central roles of F420in methanogenic archaea in processes such as substrate oxidation, C1pathways, respiration, and oxygen detoxification. We also describe how two F420-dependent oxidoreductase superfamilies mediate many environmentally and medically important reactions in bacteria, including biosynthesis of tetracycline and pyrrolobenzodiazepine antibiotics by streptomycetes, activation of the prodrugs pretomanid and delamanid byMycobacterium tuberculosis, and degradation of environmental contaminants such as picrate, aflatoxin, and malachite green. The biosynthesis pathways of Foand F420are also detailed. We conclude by considering opportunities to exploit deazaflavin-dependent processes in tuberculosis treatment, methane mitigation, bioremediation, and industrial biocatalysis.

There is growing demand for high-throughput measurement of biochemical reactions in drug discovery and directed evolution programs. To meet this need, a powerful platform based on droplet-based bioreactors manipulated by microfluidic systems is being developed, which can overcome the limitations of scale and power encountered in conventional screening methods. This paper reports our progress in the synthesis of enzymes and assay of their activity within a microfluidic droplet system. The model system we use involves the organophosphorus hydrolase enzyme OpdA from Agrobacterium radiobacter and a robust microchip made from polymethyl methacrylate (PMMA). Synthesis of OpdA from cognate DNA within water-in-oil droplets was tested using both in-house and commercial in vitro transcription and translation (IVTT) kits. OpdA activity was measured using coumaphos as substrate and by monitoring the fluorescence released by its product, chlorferone. OpdA was demonstrated to be synthesized and assayed within the droplets using the commercial in vitro transcription and translation kit, although the activity measured within the droplets diminished over time, apparently due to leakage of chlorferone out of the droplets.

An efficient 3,4-dichloroaniline (3,4-DCA)-mineralizing bacterium has been isolated from enrichment cultures originating from a soil sample with a history of repeated exposure to diuron, a major metabolite of which is 3,4-DCA. This bacterium, Bacillus megaterium IMT21, also mineralized 2,3-, 2,4-, 2,5- and 3,5-DCA as sole sources of carbon and energy. These five DCA isomers were degraded via two different routes. 2,3-, 2,4- and 2,5-DCA were degraded via previously unknown dichloroaminophenol metabolites, whereas 3,4- and 3,5-DCA were degraded via dichloroacetanilide.

Evolutionary principles are now routinely incorporated into medicine and agriculture. Examples include the design of treatments that slow the evolution of resistance by weeds, pests, and pathogens, and the design of breeding programs that maximize crop yield or quality. Evolutionary principles are also increasingly incorporated into conservation biology, natural resource management, and environmental science. Examples include the protection of small and isolated populations from inbreeding depression, the identification of key traits involved in adaptation to climate change, the design of harvesting regimes that minimize unwanted life-history evolution, and the setting of conservation priorities based on populations, species, or communities that harbor the greatest evolutionary diversity and potential. The adoption of evolutionary principles has proceeded somewhat independently in these different fields, even though the underlying fundamental concepts are the same. We explore these fundamental concepts under four main themes: variation, selection, connectivity, and eco-evolutionary dynamics. Within each theme, we present several key evolutionary principles and illustrate their use in addressing applied problems. We hope that the resulting primer of evolutionary concepts and their practical utility helps to advance a unified multidisciplinary field of applied evolutionary biology.