Oxidative Catabolism of (+)-Pinoresinol Is Initiated by an Unusual Flavocytochrome Encoded by Translationally Coupled Genes within a Cluster of (+)-Pinoresinol-Coinduced Genes in Pseudomonas sp. Strain SG-MS2.

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 Km 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 ß-ß' 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.

Genomic changes associated with adaptation to arid environments in cactophilic Drosophila species.

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.

Detoxification Genes Differ Between Cactus-, Fruit-, and Flower-Feeding Drosophila.

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.

Are feeding preferences and insecticide resistance associated with the size of detoxifying enzyme families in insect herbivores?

The size of gene families associated with xenobiotic detoxification in insects may be associated with the complexity of their diets and their propensities to develop insecticide resistance. We test these hypotheses by collating the annotations of cytochrome P450, carboxyl/cholinesterase and glutathione S-transferase genes in 65 insect species with data on their host use and history of insecticide resistance. We find 2-4 fold variation across the species in the numbers of these genes and, in some orders, especially the Hymenoptera, there is a clear relationship between the numbers of genes and feeding preferences. However in other orders, in particular the Lepidoptera, no such relationship is apparent. The size of these three gene families also tend to correlate with insecticide resistance propensity but this may not be an independent effect because species with broader host ranges are more likely to be pests that are heavily sprayed with insecticides.

Phylogenetic and kinetic characterization of a suite of dehydrogenases from a newly isolated bacterium, strain SG61-1L, that catalyze the turnover of guaiacylglycerol-ß-guaiacyl ether stereoisomers.

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-ß-guaiacyl ether (GGE), which contains a key ß-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.

Insights into Ongoing Evolution of the Hexachlorocyclohexane Catabolic Pathway from Comparative Genomics of Ten Sphingomonadaceae Strains.

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.

Whole-genome expression analysis in the third instar larval midgut of Drosophila melanogaster.

Survival of insects on a substrate containing toxic substances such as plant secondary metabolites or insecticides is dependent on the metabolism or excretion of those xenobiotics. The primary sites of xenobiotic metabolism are the midgut, Malpighian tubules, and fat body. In general, gene expression in these organs is reported for the entire tissue by online databases, but several studies have shown that gene expression within the midgut is compartmentalized. Here, RNA sequencing is used to investigate whole-genome expression in subsections of third instar larval midguts of Drosophila melanogaster. The data support functional diversification in subsections of the midgut. Analysis of the expression of gene families that are implicated in the metabolism of xenobiotics suggests that metabolism may not be uniform along the midgut. These data provide a starting point for investigating gene expression and xenobiotic metabolism and other functions of the larval midgut.

Identification of candidate odorant degrading gene/enzyme systems in the antennal transcriptome of Drosophila melanogaster.

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.

Draft Genome Sequence of Ralstonia sp. Strain GA3-3, Isolated from Australian Suburban Soil.

Ralstonia sp. strain GA3-3 is a hexachlorocyclohexane (HCH)-degrading bacterial strain isolated from suburban soil in Canberra, Australia. The genome of strain GA3-3 was sequenced to investigate its ability to degrade α-HCH. Here, we report the annotated genome sequence of this strain.

Draft Genome Sequence of Pandoraea sp. Strain SD6-2, Isolated from Lindane-Contaminated Australian Soil.

Pandoraea sp. strain SD6-2 is a δ-hexachlorocyclohexane-degrading bacterial strain isolated from lindane-contaminated soil in Queensland, Australia. The genome of SD6-2 was sequenced to investigate its ability to degrade δ-hexachlorocyclohexane. Here we report the annotated genome sequence of this strain.

Genome sequence of the newly isolated chemolithoautotrophic Bradyrhizobiaceae strain SG-6C.

Strain SG-6C (DSM 23264, CCM 7827) is a chemolithoautotrophic bacterium of the family Bradyrhizobiaceae. It can also grow heterotrophically under appropriate environmental conditions. Here we report the annotated genome sequence of this strain in a single 4.3-Mb circular scaffold.