Bead-linked transposomes enable a normalization-free workflow for NGS library preparation.

BACKGROUND: Transposome-based technologies have enabled the streamlined production of sequencer-ready DNA libraries; however, current methods are highly sensitive to the amount and quality of input nucleic acid. RESULTS: We describe a new library preparation technology (Nextera DNA Flex) that utilizes a known concentration of transposomes conjugated directly to beads to bind a fixed amount of DNA, and enables direct input of blood and saliva using an integrated extraction protocol. We further report results from libraries generated outside the standard parameters of the workflow, highlighting novel applications for Nextera DNA Flex, including human genome builds and variant calling from below 1 ng DNA input, customization of insert size, and preparation of libraries from short fragments and severely degraded FFPE samples. Using this bead-linked library preparation method, library yield saturation was observed at an input amount of 100 ng. Preparation of libraries from a range of species with varying GC levels demonstrated uniform coverage of small genomes. For large and complex genomes, coverage across the genome, including difficult regions, was improved compared with other library preparation methods. Libraries were successfully generated from amplicons of varying sizes (from 50 bp to 11 kb), however, a decrease in efficiency was observed for amplicons smaller than 250 bp. This library preparation method was also compatible with poor-quality DNA samples, with sequenceable libraries prepared from formalin-fixed paraffin-embedded samples with varying levels of degradation. CONCLUSIONS: In contrast to solution-based library preparation, this bead-based technology produces a normalized, sequencing-ready library for a wide range of DNA input types and amounts, largely obviating the need for DNA quantitation. The robustness of this bead-based library preparation kit and flexibility of input DNA facilitates application across a wide range of fields.

The explosive-degrading cytochrome P450 XplA: biochemistry, structural features and prospects for bioremediation.

XplA is a cytochrome P450 that mediates the microbial metabolism of the military explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). It has an unusual structural organisation comprising a heme domain that is fused to its flavodoxin redox partner. XplA along with its partnering reductase XplB are plasmid encoded and the gene xplA has now been found in divergent genera across the globe with near sequence identity. Importantly, it has only been detected at explosives contaminated sites suggesting rapid dissemination of this novel catabolic activity, possibly within the 50-year period since the introduction of RDX into the environment. The X-ray structure of XplA-heme has been solved, providing fundamental information on the heme binding site. Interestingly, oxygen is not required for the degradation of RDX, but its presence determines the final degradation products, demonstrating that the degradation chemistry is flexible with both anaerobic and aerobic pathways resulting in the release of nitrite from the substrate. Transgenic plants expressing xplA are able to remove saturating levels of RDX from soil leachate and may provide a low cost sustainable remediation strategy for contaminated military sites.

An explosive-degrading cytochrome P450 activity and its targeted application for the phytoremediation of RDX.

The widespread presence in the environment of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), one of the most widely used military explosives, has raised concern owing to its toxicity and recalcitrance to degradation. To investigate the potential of plants to remove RDX from contaminated soil and water, we engineered Arabidopsis thaliana to express a bacterial gene xplA encoding an RDX-degrading cytochrome P450 (ref. 1). We demonstrate that the P450 domain of XplA is fused to a flavodoxin redox partner and catalyzes the degradation of RDX in the absence of oxygen. Transgenic A. thaliana expressing xplA removed and detoxified RDX from liquid media. As a model system for RDX phytoremediation, A. thaliana expressing xplA was grown in RDX-contaminated soil and found to be resistant to RDX phytotoxicity, producing shoot and root biomasses greater than those of wild-type plants. Our work suggests that expression of xplA in landscape plants may provide a suitable remediation strategy for sites contaminated by this class of explosives.

Identification and characterisation of Arabidopsis glycosyltransferases capable of glucosylating coniferyl aldehyde and sinapyl aldehyde.

This study describes the substrate recognition profile of UGT72E1, an UDP-glucose:glycosyltransferase of Arabidopsis thaliana that is the third member of a branch of glycosyltransferases, capable of conjugating lignin monomers and related metabolites. The data show that UGT72E1, in contrast to the two closely related UGTs 72E2 and 72E3, is specific for sinapyl and coniferyl aldehydes. The biochemical properties of UGT72E1 are characterised, and are compared with that of UGT72E2, which is capable of glycosylating the aldehydes as well as coniferyl and sinapyl alcohols.

The use of abscisic acid analogues to analyse the substrate selectivity of UGT71B6, a UDP-glycosyltransferase of Arabidopsis thaliana.

This study analyses the activity of an Arabidopsis thaliana UDP-glycosyltransferase, UGT71B6 (71B6), towards abscisic acid (ABA) and its structural analogues. The enzyme preferentially glucosylated ABA and not its catabolites. The requirement for a specific chiral configuration of (+)-ABA was demonstrated through the use of analogues with the chiral centre changed or removed. The enzyme was able to accommodate extra bulk around the double bond of the ABA ring but not alterations to the 8'- and 9'-methyl groups. Interestingly, the ketone of ABA was not required for glucosylation. Bioactive analogues, resistant to 8'-hydroxylation, were also poor substrates for conjugation by UGT71B6. This suggests the compounds may be resistant to both pathways of ABA inactivation and may, therefore, prove to be useful agrochemicals for field applications.

The role of oxophytodienoate reductases in the detoxification of the explosive 2,4,6-trinitrotoluene by Arabidopsis.

The explosive 2,4,6-trinitrotoluene (TNT) is a significant environmental pollutant that is both toxic and recalcitrant to degradation. Phytoremediation is being increasingly proposed as a viable alternative to conventional remediation technologies to clean up explosives-contaminated sites. Despite the potential of this technology, relatively little is known about the innate enzymology of TNT detoxification in plants. To further elucidate this, we used microarray analysis to identify Arabidopsis (Arabidopsis thaliana) genes up-regulated by exposure to TNT and found that the expression of oxophytodienoate reductases (OPRs) increased in response to TNT. The OPRs share similarity with the Old Yellow Enzyme family, bacterial members of which have been shown to transform explosives. The three predominantly expressed forms, OPR1, OPR2, and OPR3, were recombinantly expressed and affinity purified. Subsequent biochemical characterization revealed that all three OPRs are able to transform TNT to yield nitro-reduced TNT derivatives, with OPR1 additionally producing the aromatic ring-reduced products hydride and dihydride Meisenheimer complexes. Arabidopsis plants overexpressing OPR1 removed TNT more quickly from liquid culture, produced increased levels of transformation products, and maintained higher fresh weight biomasses than wild-type plants. In contrast, OPR1,2 RNA interference lines removed less TNT, produced fewer transformation products, and had lower biomasses. When grown on solid medium, two of the three OPR1 lines and all of the OPR2-overexpressing lines exhibited significantly enhanced tolerance to TNT. These data suggest that, in concert with other detoxification mechanisms, OPRs play a physiological role in xenobiotic detoxification.

Exploring the biochemical properties and remediation applications of the unusual explosive-degrading P450 system XplA/B.

Widespread contamination of land and groundwater has resulted from the use, manufacture, and storage of the military explosive hexa-hydro-1,3,5-trinitro-1,3,5-triazine (RDX). This contamination has led to a requirement for a sustainable, low-cost method to remediate this problem. Here, we present the characterization of an unusual microbial P450 system able to degrade RDX, consisting of flavodoxin reductase XplB and fused flavodoxin-cytochrome P450 XplA. The affinity of XplA for the xenobiotic compound RDX is high (K(d) = 58 muM) and comparable with the K(m) of other P450s toward their natural substrates (ranging from 1 to 500 muM). The maximum turnover (k(cat)) is 4.44 per s, only 10-fold less than the fastest self-sufficient P450 reported, BM3. Interestingly, the presence of oxygen determines the final products of RDX degradation, demonstrating that the degradation chemistry is flexible, but both pathways result in ring cleavage and release of nitrite. Carbon monoxide inhibition is weak and yet the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) is a potent inhibitor. To test the efficacy of this system for the remediation of groundwater, transgenic Arabidopsis plants expressing both xplA and xplB were generated. They are able to remove saturating levels of RDX from liquid culture and soil leachate at rates significantly faster than those of untransformed plants and xplA-only transgenic lines, demonstrating the applicability of this system for the phytoremediation of RDX-contaminated sites.

The glucosyltransferase UGT72E2 is responsible for monolignol 4-O-glucoside production in Arabidopsis thaliana.

The phenylpropanoid pathway in plants leads to the synthesis of a wide range of soluble secondary metabolites, many of which accumulate as glycosides. In Arabidopsis, a small cluster of three closely related genes, UGT72E1-E3, encode glycosyltransferases shown to glucosylate several phenylpropanoids in vitro, including monolignols, hydroxycinnamic acids and hydroxycinnamic aldehydes. The role of these genes in planta has now been investigated through genetically downregulating the expression of individual genes or silencing the entire cluster. Analysis of these transgenic Arabidopsis plants showed that the levels of coniferyl and sinapyl alcohol 4-O-glucosides that accumulate in light-grown roots were significantly reduced. A 50% reduction in both glucosides was observed in plants in which UGT72E2 was downregulated, whereas silencing the three genes led to a 90% reduction, suggesting some redundancy of function within the cluster. The gene encoding UGT72E2 was constitutively overexpressed in transgenic Arabidopsis to determine whether increased glucosylation of monolignols could influence flux through the soluble phenylpropanoid pathway. Elevated expression of UGT72E2 led to increased accumulation of monolignol glucosides in root tissues and also the appearance of these glucosides in leaves. In particular, coniferyl alcohol 4-O-glucoside accumulated to massive amounts (10 micromol g(-1) FW) in root tissues of these plants. Increased glucosylation of other phenylpropanoids also occurred in plants overexpressing this glycosyltransferase. Significantly changing the pattern of glycosides in the leaves also led to a pronounced change in accumulation of the hydroxycinnamic ester sinapoyl malate. The data demonstrate the plasticity of phenylpropanoid metabolism and the important role that glucosylation of secondary metabolites can play in cellular homeostasis.

Arabidopsis glycosyltransferases as biocatalysts in fermentation for regioselective synthesis of diverse quercetin glucosides.

Regioselectivity of glycosyltransferases offers an important means to overcome the limitations of chemical synthesis of small molecule glycosides. In this study we explore a large multigene family of UDP-glucose:glycosyltransferases of Arabidopsis for their potential as novel biocatalysts for in vitro synthesis and whole-cell catalysis. We used quercetin as a substrate for this study because the flavonol and its glycosides have important medicinal properties and the metabolite provides a complex structure for regioselective glucosylation. We analyzed the activity of 91 recombinant enzymes for in vitro activity toward quercetin and discovered 29 that are capable of glucosylating the substrate. We demonstrate the first enzymic synthesis of a range of glucosides in vitro, including the 3-O-, 7-O-, 3'-O-, and 4'-O-monoglucosides, 3,7-di-O-glucoside, and 7,3'-di-O-glucoside. We also show that the regioselectivity of glucosylation can be maintained when the enzymes are used as whole-cell biocatalysts in Escherichia coli.

Over-expression of an Arabidopsis gene encoding a glucosyltransferase of indole-3-acetic acid: phenotypic characterisation of transgenic lines.

An analysis of the multigene family of Group 1 glucosyltransferases (UGTs) of Arabidopsis thaliana revealed a gene, UGT84B1, whose recombinant product glucosylated indole-3-acetic acid (IAA) in vitro. Transgenic Arabidopsis plants constitutively over-expressing UGT84B1 under the control of the CaMV 35S promoter have been constructed and their phenotype analysed. The transgenic lines displayed a number of changes that resembled those described previously in lines in which auxin levels were depleted. A root elongation assay was used as a measure of auxin sensitivity. A reduced sensitivity of the transgenic lines compared to wild-type was observed when IAA was applied. In contrast, application of 2,4-dichlorophenoxyacetic acid (2,4-D), previously demonstrated not to be a substrate for UGT84B1, led to a wild-type response. These data suggested that the catalytic specificity of the recombinant enzyme in vitro was maintained in planta. This was further confirmed when levels of IAA metabolites and conjugates were measured in extracts of the transgenic plants and 1-O-IAGlc was found to be elevated to approximately 50 pg mg-1 FW, compared to the trace levels characteristic of wild-type plants. Surprisingly, in the same extracts, levels of free IAA were also found to have accumulated to some 70 pg mg-1 FW compared to approximately 15 pg mg-1 FW in extracts of wild-type plants. Analysis of leaves at different developmental stages revealed the auxin gradient, typical of wild-type plants, was not observed in the transgenic lines, with free IAA levels in the apex and youngest leaves at a lower level compared to wild-type. In total, the data reveal that significant changes in auxin homeostasis can be caused by overproduction of an IAA-conjugating enzyme.

Evolution of substrate recognition across a multigene family of glycosyltransferases in Arabidopsis.

The complete sequence of the Arabidopsis genome enables definitive characterization of multigene families and analysis of their phylogenetic relationships. Using a consensus sequence previously defined for glycosyltransferases that use small-molecular-weight acceptors, 107 gene sequences were identified in the Arabidopsis genome and used to construct a phylogenetic tree. Screening recombinant proteins for their catalytic activities in vitro has revealed enzymes active toward physiologically important substrates, including hormones and secondary metabolites. The aim of this study has been to use the phylogenetic relationships across the entire family to explore the evolution of substrate recognition and regioselectivity of glucosylation. Hydroxycoumarins have been used as the model substrates for the analysis in which 90 sequences have been assayed and 48 sequences shown to recognize these compounds. The study has revealed activity in 6 of the 14 phylogenetic groups of the multigene family, suggesting that basic features of substrate recognition are retained across substantial evolutionary periods.