Aromatic Metabolism Research Articles

Functional genomic analysis of bacterial lignin degraders: diversity in mechanisms of lignin oxidation and metabolism.

Although several bacterial lignin-oxidising enzymes have been discovered in recent years, it is not yet clear whether different lignin-degrading bacteria use similar mechanisms for lignin oxidation and degradation of lignin fragments. Genome sequences of 13 bacterial lignin-oxidising bacteria, including new genome sequences for Microbacterium phyllosphaerae and Agrobacterium sp., were analysed for the presence of lignin-oxidising enzymes and aromatic degradation gene clusters that could be used to metabolise the products of lignin degradation. Ten bacterial genomes contain DyP-type peroxidases, and ten bacterial strains contain putative multi-copper oxidases (MCOs), both known to have activity for lignin oxidation. Only one strain lacks both MCOs and DyP-type peroxidase genes. Eleven bacterial genomes contain aromatic degradation gene clusters, of which ten contain the central β-ketoadipate pathway, with variable numbers and types of degradation clusters for other aromatic substrates. Hence, there appear to be diverse metabolic strategies used for lignin oxidation in bacteria, while the β-ketoadipate pathway appears to be the most common route for aromatic metabolism in lignin-degrading bacteria.

The effect of grazing on the microbiome of two commercially important agarophytes, Gracilaria firma and G. salicornia (Gracilariaceae, Rhodophyta)

Grazing, which leads to losses in biomass and drastic declines in total crop production, is one of the main concerns in seaweed aquaculture. This is also thought to affect the composition of the associated bacterial communities which are believed to play a crucial role in determining the host’s health and development. Apart from morphological impairment, studying changes in the prokaryotic microbiome composition and predicted functional responses to grazing will allow us to understand the underlying effects of grazing on the seaweed host. This study is the first report of the effect of grazing on the prokaryotic microbiome of two economically important agarophytes, Gracilaria firma and Gracilaria salicornia, by high-throughput sequencing targeting the V3-V4 variable region of the 16S rRNA gene. The results indicated that for G. firma, the microbiome composition of tissues grazed by marine herbivores had significantly more agarolytic bacteria Marinagarivorans sp. and Algisphaera sp. than in ungrazed tissues. The predictive functional metagenomics for this species revealed that grazing escalated the pathway activities related to nucleotide degradation, aromatic compound degradation and aerobic sugar metabolism, while pathways associated with cell wall synthesis, aerobic respiration, vitamin biosynthesis and amino acid biosynthesis were reduced. However, for G. salicornia, the bacterial communities were not significantly affected by grazing. Nevertheless, pathways relating to anaerobic respiration and amino acid, coenzyme and vitamin B-6 biosynthesis in this species were predicted to be more active in grazed tissues, whereas the microbiome of ungrazed tissues had higher activities in bacteriochlorophyll a, fatty acid, secondary metabolite and heme biosynthesis.

The complete genome sequence of the nitrile biocatalyst Rhodococcus rhodochrous ATCC BAA-870

BackgroundRhodococci are industrially important soil-dwelling Gram-positive bacteria that are well known for both nitrile hydrolysis and oxidative metabolism of aromatics. Rhodococcus rhodochrous ATCC BAA-870 is capable of metabolising a wide range of aliphatic and aromatic nitriles and amides. The genome of the organism was sequenced and analysed in order to better understand this whole cell biocatalyst.ResultsThe genome of R. rhodochrous ATCC BAA-870 is the first Rhodococcus genome fully sequenced using Nanopore sequencing. The circular genome contains 5.9 megabase pairs (Mbp) and includes a 0.53 Mbp linear plasmid, that together encode 7548 predicted protein sequences according to BASys annotation, and 5535 predicted protein sequences according to RAST annotation. The genome contains numerous oxidoreductases, 15 identified antibiotic and secondary metabolite gene clusters, several terpene and nonribosomal peptide synthetase clusters, as well as 6 putative clusters of unknown type. The 0.53 Mbp plasmid encodes 677 predicted genes and contains the nitrile converting gene cluster, including a nitrilase, a low molecular weight nitrile hydratase, and an enantioselective amidase.Although there are fewer biotechnologically relevant enzymes compared to those found in rhodococci with larger genomes, such as the well-known Rhodococcus jostii RHA1, the abundance of transporters in combination with the myriad of enzymes found in strain BAA-870 might make it more suitable for use in industrially relevant processes than other rhodococci.ConclusionsThe sequence and comprehensive description of the R. rhodochrous ATCC BAA-870 genome will facilitate the additional exploitation of rhodococci for biotechnological applications, as well as enable further characterisation of this model organism. The genome encodes a wide range of enzymes, many with unknown substrate specificities supporting potential applications in biotechnology, including nitrilases, nitrile hydratase, monooxygenases, cytochrome P450s, reductases, proteases, lipases, and transaminases.

Evolutionary adaptation of Aspergillus niger for increased ferulic acid tolerance.

AimsTo create an Aspergillus niger mutant with increased tolerance against ferulic acid using evolutionary adaptation.Methods and ResultsEvolutionary adaptation of A. niger N402 was performed by consecutive growth on increasing concentrations of ferulic acid in the presence of 25 mmol l−1 d‐fructose, starting from 0·5 mmol l−1 and ending with 5 mmol l−1 ferulic acid. The A. niger mutant obtained after six months, named Fa6, showed increased ferulic acid tolerance compared to the parent. In addition, Fa6 has increased ferulic acid consumption and a higher conversion rate, suggesting that the mutation affects aromatic metabolism of this species. Transcriptome analysis of the evolutionary mutant on ferulic acid revealed a distinct gene expression profile compared to the wild type. Further analysis of this mutant and the parent strain provided the first experimental confirmation that A. niger converts coniferyl alcohol to ferulic acid.ConclusionsThe evolutionary adaptive A. niger mutant Fa6 has beneficial mutations that increase the tolerance, conversion rate and uptake of ferulic acid.Significance and Impact of the StudyThis study demonstrates that evolutionary adaptation is a powerful tool to modify micro‐organisms towards increased tolerance to harsh conditions, which is beneficial for various industrial applications.

The White-Rot Basidiomycete Dichomitus squalens Shows Highly Specific Transcriptional Response to Lignocellulose-Related Aromatic Compounds.

Lignocellulosic plant biomass is an important feedstock for bio-based economy. In particular, it is an abundant renewable source of aromatic compounds, which are present as part of lignin, as side-groups of xylan and pectin, and in other forms, such as tannins. As filamentous fungi are the main organisms that modify and degrade lignocellulose, they have developed a versatile metabolism to convert the aromatic compounds that are toxic at relatively low concentrations to less toxic ones. During this process, fungi form metabolites some of which represent high-value platform chemicals or important chemical building blocks, such as benzoic, vanillic, and protocatechuic acid. Especially basidiomycete white-rot fungi with unique ability to degrade the recalcitrant lignin polymer are expected to perform highly efficient enzymatic conversions of aromatic compounds, thus having huge potential for biotechnological exploitation. However, the aromatic metabolism of basidiomycete fungi is poorly studied and knowledge on them is based on the combined results of studies in variety of species, leaving the overall picture in each organism unclear. Dichomitus squalens is an efficiently wood-degrading white-rot basidiomycete that produces a diverse set of extracellular enzymes targeted for lignocellulose degradation, including oxidative enzymes that act on lignin. Our recent study showed that several intra- and extracellular aromatic compounds were produced when D. squalens was cultivated on spruce wood, indicating also versatile aromatic metabolic abilities for this species. In order to provide the first molecular level systematic insight into the conversion of plant biomass derived aromatic compounds by basidiomycete fungi, we analyzed the transcriptomes of D. squalens when grown with 10 different lignocellulose-related aromatic monomers. Significant differences for example with respect to the expression of lignocellulose degradation related genes, but also putative genes encoding transporters and catabolic pathway genes were observed between the cultivations supplemented with the different aromatic compounds. The results demonstrate that the transcriptional response of D. squalens is highly dependent on the specific aromatic compounds present suggesting that instead of a common regulatory system, fine-tuned regulation is needed for aromatic metabolism.

Transcriptome changes in the phenylpropanoid pathway in senescing leaves of Toona sinensis.

Toona sinensis is a deciduous tree native to eastern and southeastern Asia that has important culinary and cultural values. To expand current knowledge of the transcriptome and functional genomics in this species, a de novo transcriptome sequence analysis of young and mature leaf tissues of T. sinensis was performed using the Illumina platform. Over 8.1 Gb of data were generated, assembled into 64,541 unigenes, and annotated with known biological functions. Proteins involved in primary metabolite biosynthesis were identified based on similarities to known proteins, including some related to biosynthesis of carbohydrates, amino acids, lipids, and energy. Analysis of unigenes differentially expressed between young and mature leaves (transcriptomic libraries ‘YL’ and ‘ML’, respectively) showed that the KEGG pathways of phenylpropanoid, naringenin, lignin, cutin, suberin, and wax biosynthesis were significantly enriched in mature leaves. These results not only expand knowledge of transcriptome characteristics for this valuable species, but also provide a useful transcriptomic dataset to accelerate the researches on its metabolic mechanisms and functional genomics. This study can also further the understanding of unique aromatic metabolism and Chinese medicinal properties of T. sinensis.Electronic supplementary materialThe online version of this article (10.1007/s11738-019-2915-9) contains supplementary material, which is available to authorized users.

An Integrated Analysis of the Rice Transcriptome and Metabolome Reveals Differential Regulation of Carbon and Nitrogen Metabolism in Response to Nitrogen Availability.

Nitrogen (N) is an extremely important macronutrient for plant growth and development. It is the main limiting factor in most agricultural production. However, it is well known that the nitrogen use efficiency (NUE) of rice gradually decreases with the increase of the nitrogen application rate. In order to clarify the underlying metabolic and molecular mechanisms of this phenomenon, we performed an integrated analysis of the rice transcriptome and metabolome. Both differentially expressed genes (DEGs) and metabolite Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that carbon and nitrogen metabolism is significantly affected by nitrogen availability. Further analysis of carbon and nitrogen metabolism changes in rice under different nitrogen availability showed that high N inhibits nitrogen assimilation and aromatic metabolism pathways by regulating carbon metabolism pathways such as the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway (PPP). Under low nitrogen, the TCA cycle is promoted to produce more energy and α-ketoglutarate, thereby enhancing nitrogen transport and assimilation. PPP is also inhibited by low N, which may be consistent with the lower NADPH demand under low nitrogen. Additionally, we performed a co-expression network analysis of genes and metabolites related to carbon and nitrogen metabolism. In total, 15 genes were identified as hub genes. In summary, this study reveals the influence of nitrogen levels on the regulation mechanisms for carbon and nitrogen metabolism in rice and provides new insights into coordinating carbon and nitrogen metabolism and improving nitrogen use efficiency in rice.

Complete Genome Sequence of Serratia quinivorans Strain 124R, a Facultative Anaerobe Isolated on Organosolv Lignin as a Sole Carbon Source.

The complete genome sequence of the gammaproteobacterial isolate Serratia quinivorans 124R consists of 5 Mb over 2 scaffolds and a G+C content of 52.85%. Genes relating to aromatic metabolism reflect its isolation on organosolv lignin as a sole carbon source under anoxic conditions as well as the potential for lignin biorefinery applications.

Comparative Genomic Analysis of the Regulation of Aromatic Metabolism in Betaproteobacteria.

Aromatic compounds are a common carbon and energy source for many microorganisms, some of which can even degrade toxic chloroaromatic xenobiotics. This comparative study of aromatic metabolism in 32 Betaproteobacteria species describes the links between several transcription factors (TFs) that control benzoate (BenR, BenM, BoxR, BzdR), catechol (CatR, CatM, BenM), chlorocatechol (ClcR), methylcatechol (MmlR), 2,4-dichlorophenoxyacetate (TfdR, TfdS), phenol (AphS, AphR, AphT), biphenyl (BphS), and toluene (TbuT) metabolism. We characterize the complexity and variability in the organization of aromatic metabolism operons and the structure of regulatory networks that may differ even between closely related species. Generally, the upper parts of pathways, rare pathway variants, and degradative pathways of exotic and complex, in particular, xenobiotic compounds are often controlled by a single TF, while the regulation of more common and/or central parts of the aromatic metabolism may vary widely and often involves several TFs with shared and/or dual, or cascade regulation. The most frequent and at the same time variable connections exist between AphS, AphR, AphT, and BenR. We have identified a novel LysR-family TF that regulates the metabolism of catechol (or some catechol derivative) and either substitutes CatR(M)/BenM, or shares functions with it. We have also predicted several new members of aromatic metabolism regulons, in particular, some COGs regulated by several different TFs.

The nature of the progression of drought stress drives differential metabolomic responses in Populus deltoides.

The use of woody crops for Quad-level (approx. 1 × 1018 J) energy production will require marginal agricultural lands that experience recurrent periods of water stress. Populus species have the capacity to increase dehydration tolerance by lowering osmotic potential via osmotic adjustment. The aim of this study was to investigate how the inherent genetic potential of a Populus clone to respond to drought interacts with the nature of the drought to determine the degree of biochemical response. A greenhouse drought stress study was conducted on Populus deltoides 'WV94' and the resulting metabolite profiles of leaves were determined by gas chromatography-mass spectrometry following trimethylsilylation for plants subjected to cyclic mild (-0.5 MPa pre-dawn leaf water potential) drought vs. cyclic severe (-1.26 MPa) drought in contrast to well-watered controls (-0.1 MPa) after two or four drought cycles, and in contrast to plants subjected to acute drought, where plants were desiccated for up to 8 d. The nature of drought (cyclic vs. acute), frequency of drought (number of cycles) and the severity of drought (mild vs. severe) all dictated the degree of osmotic adjustment and the nature of the organic solutes that accumulated. Whereas cyclic drought induced the largest responses in primary metabolism (soluble sugars, organic acids and amino acids), acute onset of prolonged drought induced the greatest osmotic adjustment and largest responses in secondary metabolism, especially populosides (hydroxycinnamic acid conjugates of salicin). The differential adaptive metabolite responses in cyclic vs. acute drought suggest that stress acclimation occurs via primary metabolism in response to cyclic drought, whereas expanded metabolic plasticity occurs via secondary metabolism following severe, acute drought. The shift in carbon partitioning to aromatic metabolism with the production of a diverse suite of higher order salicylates lowers osmotic potential and increases the probability of post-stress recovery.

Lignolytic-consortium omics analyses reveal novel genomes and pathways involved in lignin modification and valorization

BackgroundLignin is a heterogeneous polymer representing a renewable source of aromatic and phenolic bio-derived products for the chemical industry. However, the inherent structural complexity and recalcitrance of lignin makes its conversion into valuable chemicals a challenge. Natural microbial communities produce biocatalysts derived from a large number of microorganisms, including those considered unculturable, which operate synergistically to perform a variety of bioconversion processes. Thus, metagenomic approaches are a powerful tool to reveal novel optimized metabolic pathways for lignin conversion and valorization.ResultsThe lignin-degrading consortium (LigMet) was obtained from a sugarcane plantation soil sample. The LigMet taxonomical analyses (based on 16S rRNA) indicated prevalence of Proteobacteria, Actinobacteria and Firmicutes members, including the Alcaligenaceae and Micrococcaceae families, which were enriched in the LigMet compared to sugarcane soil. Analysis of global DNA sequencing revealed around 240,000 gene models, and 65 draft bacterial genomes were predicted. Along with depicting several peroxidases, dye-decolorizing peroxidases, laccases, carbohydrate esterases, and lignocellulosic auxiliary (redox) activities, the major pathways related to aromatic degradation were identified, including benzoate (or methylbenzoate) degradation to catechol (or methylcatechol), catechol ortho-cleavage, catechol meta-cleavage, and phthalate degradation. A novel Paenarthrobacter strain harboring eight gene clusters related to aromatic degradation was isolated from LigMet and was able to grow on lignin as major carbon source. Furthermore, a recombinant pathway for vanillin production was designed based on novel gene sequences coding for a feruloyl-CoA synthetase and an enoyl-CoA hydratase/aldolase retrieved from the metagenomic data set.ConclusionThe enrichment protocol described in the present study was successful for a microbial consortium establishment towards the lignin and aromatic metabolism, providing pathways and enzyme sets for synthetic biology engineering approaches. This work represents a pioneering study on lignin conversion and valorization strategies based on metagenomics, revealing several novel lignin conversion enzymes, aromatic-degrading bacterial genomes, and a novel bacterial strain of potential biotechnological interest. The validation of a biosynthetic route for vanillin synthesis confirmed the applicability of the targeted metagenome discovery approach for lignin valorization strategies.

Metabolism of aromatics by Trichosporon oleaginosus while remaining oleaginous

BackgroundThe oleaginous yeast, Trichosporon oleaginosus, has been extensively studied for its ability to metabolize non-conventional feedstocks. These include phenol-containing waste streams, such as distillery wastewater, or streams consisting of non-conventional sugars, such as hydrolyzed biomass and various bagasse. An initial BLAST search suggests this yeast has putative aromatic metabolizing genes. Given the desirability to valorize underutilized feedstocks such as lignin, we investigated the ability of T. oleaginosus to tolerate and metabolize lignin-derived aromatic compounds.ResultsTrichosporon oleaginosus can tolerate and metabolize model lignin monoaromatics and associated intermediates within funneling pathways. Growth rates and biomass yield were similar to glucose when grown in 4-hydroxybenzoic acid (pHBA) and resorcinol, but had an increased lag phase when grown in phenol. Oleaginous behavior was observed using resorcinol as a sole carbon source. Fed-batch feeding resulted in lipid accumulation of 69.5% on a dry weight basis.ConclusionsThough the exact pathway of aromatic metabolism remains to be determined for T. oleaginosus, the results presented in this work motivate use of this organism for lignin valorization and phenolic wastewater bioremediation. Trichosporon oleaginosus is the first yeast shown to be oleaginous while growing on aromatic substrates, and shows great promise as a model industrial microbe for biochemical and biofuel production from depolymerized lignin.

Conversion of lignin model compounds by Pseudomonas putida KT2440 and isolates from compost

Starting from mature vegetable compost, four bacterial strains were selected using a lignin-rich medium. 16S ribosomal RNA identification of the isolates showed high score similarity with Pseudomonas spp. for three out of four isolates. Further characterization of growth on mixtures of six selected lignin model compounds (vanillin, vanillate, 4-hydroxybenzoate, p-coumarate, benzoate, and ferulate) was carried out with three of the Pseudomonas isolates and in addition with the strain Pseudomonas putida KT2440 from a culture collection. The specific growth rates on benzoate, p-coumarate, and 4-hydroxybenzoate were considerably higher (0.26–0.27 h−1) than those on ferulate and vanillate (0.21 and 0.22 h−1), as were the uptake rates. There was no direct growth of P. putida KT2440 on vanillin, but instead, vanillin was rapidly converted into vanillate at a rate of 4.87 mmol (gCDW h)−1 after which the accumulated vanillate was taken up. The growth curve reflected a diauxic growth when mixtures of the model compounds were used as carbon source. Vanillin, 4-hydroxybenzoate, and benzoate were preferentially consumed first, whereas ferulate was always the last substrate to be taken in. These results contribute to a better understanding of the aromatic metabolism of P. putida in terms of growth and uptake rates, which will be helpful for the utilization of these bacteria as cell factories for upgrading lignin-derived mixtures of aromatic molecules.

Draft Genome Sequence of Pseudonocardia autotrophica Strain DSM 43083, an Efficient Producer of Peroxidases for Lignin Modification

ABSTRACTPseudonocardia autotrophica strain DSM 43083 is a filamentous actinobacterium and was described to degrade or modify lignin. Here, we present its draft genome sequence, with a size of 5.8 Mb, to unravel the gene set coding for promising monooxygenases, dioxygenases, and DyP-type peroxidases associated with aromatic metabolism and lignin modification.

California Condor Microbiomes

We present a metagenomic analysis of California condor (Gymnogyps californianus) microbiomes with a goal of developing indicators of condor and ecosystem health. 16S ribosomal DNA (rDNA) and shotgun metagenomic DNA sequences were acquired from fecal and cloacal samples of captive birds. Detailed classification of shotgun DNA sequence data with the Sequedex analysis package provided both phylogenetic and functional profiles of all samples. We observed numerous bacterial species previously associated with birds and gut microbiomes, including both virulent and opportunistic pathogens such as Clostridium perfringens, Propionibacterium acnes, Shigella flexneri, and Fusobacterium mortiferum, probiotics such as Lactobacillus johnsonii, Lactobacillus ruminus, and Bacteroides vulgatus, and mucosal microbes such as Delftia acidovorans, Stenotrophomonas maltophilia, and Corynebacterium falsnii. Classification using shotgun metagenomic reads from phylogenetic marker genes was consistent with, but more specific than, analysis based on 16S data. Classification of samples based on functional profiles of genomic fragments differentiated three distinct types of samples: fecal, mature cloacal, and immature cloacal. Statistical analysis of the determinants of sample classification indicated that fecal samples were enriched in metabolism-related genes involved in glycolysis, methionine degradation, maltose utilization, and butyrate production, while cloacal samples were enriched in aromatic metabolism, virulence genes, and membrane transporters. Our analysis was further supported by the recovery of protein sequences for phylogenetic analysis, such as the RNA polymerase of Delftia, a Shigella toxin, a nitrate reductase of Delftia, and beta lactamases from several species. Our work identifies several strategies by which metagenomic sequencing of condor microbiomes may inform us about condor diet, infections, intoxication, and other types of stress.

Influence of a lignification and mineralization of leaf tissues on resistance to a brown rust in common wheat plants

Aromatic substances in plant tissues can have protective effect against fungal diseases. One of the key enzymes in aromatic metabolism of plants, CAD (cinnamyl-alcohol dehydrogenase, dehydrogenase of cinnamyl alcohol; EC 1.1.1.195), at a number of species of plants exerts impact on the content of aromatic substances and on protective properties of tissues from fungal infections. When studying a collection of cultivars of bread wheat Triticum aestivum L., polymorphic on CAD, distinctions on extent of defeat are found by brown rust (with Puccinia recondita f. sp. Tritici as a causative agent). The purpose of the work was studying of features of structure and chemical composition of the tissues of a leaf promoting increased resistance. On a phytopathologic plot, against artificial infection with spores of a brown rust, two samples of spring bread wheat 3-13-15-4 and 3-4-14-3 were affected 1–5 % and 30 %, respectively. An analysis of various substances content in the leaf tissue at the contrast samples was conducted. Large plaques and spot consisting of mineral compounds were observed on the leaf surface of the more resistant plant. In ashes of leaves and ashes of a lignin differences in the maintenance of a number of mineral elements were also found. Lignin content on the dry mass of a leaf differed slightly (14.2 % vs 12.3 %), however there are differences in chemical composition. It is possible that the observed differences lead to afflict the plants with leaf rust to such different degrees. In that case these characteristics can be used for diagnostics of potential resistance of cultivars to fungal infection.

Draft Genome Sequence of Pseudomonas mosselii Gil3, Isolated from Catfish and Antagonistic against Hypervirulent Aeromonas hydrophila.

Pseudomonas mosselii Gil3 was isolated from a catfish that survived from lethal challenge with hypervirulent Aeromonas hydrophila (vAh). When assayed in vitro, the bacterium showed antagonism against vAh. Sequence analysis revealed that the genome of P. mosselii Gil3 encodes numerous aromatic metabolism pathways and proteins for biosynthesis of antimicrobial compounds.

Epistasis Test in Meta-Analysis: A Multi-Parameter Markov Chain Monte Carlo Model for Consistency of Evidence.

Conventional genome-wide association studies (GWAS) have been proven to be a successful strategy for identifying genetic variants associated with complex human traits. However, there is still a large heritability gap between GWAS and transitional family studies. The “missing heritability” has been suggested to be due to lack of studies focused on epistasis, also called gene–gene interactions, because individual trials have often had insufficient sample size. Meta-analysis is a common method for increasing statistical power. However, sufficient detailed information is difficult to obtain. A previous study employed a meta-regression-based method to detect epistasis, but it faced the challenge of inconsistent estimates. Here, we describe a Markov chain Monte Carlo-based method, called “Epistasis Test in Meta-Analysis” (ETMA), which uses genotype summary data to obtain consistent estimates of epistasis effects in meta-analysis. We defined a series of conditions to generate simulation data and tested the power and type I error rates in ETMA, individual data analysis and conventional meta-regression-based method. ETMA not only successfully facilitated consistency of evidence but also yielded acceptable type I error and higher power than conventional meta-regression. We applied ETMA to three real meta-analysis data sets. We found significant gene–gene interactions in the renin–angiotensin system and the polycyclic aromatic hydrocarbon metabolism pathway, with strong supporting evidence. In addition, glutathione S-transferase (GST) mu 1 and theta 1 were confirmed to exert independent effects on cancer. We concluded that the application of ETMA to real meta-analysis data was successful. Finally, we developed an R package, etma, for the detection of epistasis in meta-analysis [etma is available via the Comprehensive R Archive Network (CRAN) at https://cran.r-project.org/web/packages/etma/index.html].

Use of Substrate-Induced Gene Expression in Metagenomic Analysis of an Aromatic Hydrocarbon-Contaminated Soil.

Metagenomics allows the study of genes related to xenobiotic degradation in a culture-independent manner, but many of these studies are limited by the lack of genomic context for metagenomic sequences. This study combined a phenotypic screen known as substrate-induced gene expression (SIGEX) with whole-metagenome shotgun sequencing. SIGEX is a high-throughput promoter-trap method that relies on transcriptional activation of a green fluorescent protein (GFP) reporter gene in response to an inducing compound and subsequent fluorescence-activated cell sorting to isolate individual inducible clones from a metagenomic DNA library. We describe a SIGEX procedure with improved library construction from fragmented metagenomic DNA and improved flow cytometry sorting procedures. We used SIGEX to interrogate an aromatic hydrocarbon (AH)-contaminated soil metagenome. The recovered clones contained sequences with various degrees of similarity to genes (or partial genes) involved in aromatic metabolism, for example, nahG (salicylate oxygenase) family genes and their respective upstream nahR regulators. To obtain a broader context for the recovered fragments, clones were mapped to contigs derived from de novo assembly of shotgun-sequenced metagenomic DNA which, in most cases, contained complete operons involved in aromatic metabolism, providing greater insight into the origin of the metagenomic fragments. A comparable set of contigs was generated using a significantly less computationally intensive procedure in which assembly of shotgun-sequenced metagenomic DNA was directed by the SIGEX-recovered sequences. This methodology may have broad applicability in identifying biologically relevant subsets of metagenomes (including both novel and known sequences) that can be targeted computationally by in silico assembly and prediction tools.

Genome wide transcriptional profiling of Herbaspirillum seropedicae SmR1 grown in the presence of naringenin.

Herbaspirillum seropedicae is a diazotrophic bacterium which associates endophytically with economically important gramineae. Flavonoids such as naringenin have been shown to have an effect on the interaction between H. seropedicae and its host plants. We used a high-throughput sequencing based method (RNA-Seq) to access the influence of naringenin on the whole transcriptome profile of H. seropedicae. Three hundred and four genes were downregulated and seventy seven were upregulated by naringenin. Data analysis revealed that genes related to bacterial flagella biosynthesis, chemotaxis and biosynthesis of peptidoglycan were repressed by naringenin. Moreover, genes involved in aromatic metabolism and multidrug transport efllux were actived.