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September 22, 2019

Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis.

Astragalus membranaceus, also known as Huangqi in China, is one of the most widely used medicinal herbs in Traditional Chinese Medicine. Traditional Chinese Medicine formulations from Astragalus membranaceus have been used to treat a wide range of illnesses, such as cardiovascular disease, type 2 diabetes, nephritis and cancers. Pharmacological studies have shown that immunomodulating, anti-hyperglycemic, anti-inflammatory, antioxidant and antiviral activities exist in the extract of Astragalus membranaceus. Therefore, characterising the biosynthesis of bioactive compounds in Astragalus membranaceus, such as Astragalosides, Calycosin and Calycosin-7-O-ß-d-glucoside, is of particular importance for further genetic studies of Astragalus membranaceus. In this study, we reconstructed the Astragalus membranaceus full-length transcriptomes from leaf and root tissues using PacBio Iso-Seq long reads. We identified 27 975 and 22 343 full-length unique transcript models in each tissue respectively. Compared with previous studies that used short read sequencing, our reconstructed transcripts are longer, and are more likely to be full-length and include numerous transcript variants. Moreover, we also re-characterised and identified potential transcript variants of genes involved in Astragalosides, Calycosin and Calycosin-7-O-ß-d-glucoside biosynthesis. In conclusion, our study provides a practical pipeline to characterise the full-length transcriptome for species without a reference genome and a useful genomic resource for exploring the biosynthesis of active compounds in Astragalus membranaceus.

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September 22, 2019

Isoform evolution in primates through independent combination of alternative RNA processing events.

Recent RNA-seq technology revealed thousands of splicing events that are under rapid evolution in primates, whereas the reliability of these events, as well as their combination on the isoform level, have not been adequately addressed due to its limited sequencing length. Here, we performed comparative transcriptome analyses in human and rhesus macaque cerebellum using single molecule long-read sequencing (Iso-seq) and matched RNA-seq. Besides 359 million RNA-seq reads, 4,165,527 Iso-seq reads were generated with a mean length of 14,875?bp, covering 11,466 human genes, and 10,159 macaque genes. With Iso-seq data, we substantially expanded the repertoire of alternative RNA processing events in primates, and found that intron retention and alternative polyadenylation are surprisingly more prevalent in primates than previously estimated. We then investigated the combinatorial mode of these alternative events at the whole-transcript level, and found that the combination of these events is largely independent along the transcript, leading to thousands of novel isoforms missed by current annotations. Notably, these novel isoforms are selectively constrained in general, and 1,119 isoforms have even higher expression than the previously annotated major isoforms in human, indicating that the complexity of the human transcriptome is still significantly underestimated. Comparative transcriptome analysis further revealed 502 genes encoding selectively constrained, lineage-specific isoforms in human but not in rhesus macaque, linking them to some lineage-specific functions. Overall, we propose that the independent combination of alternative RNA processing events has contributed to complex isoform evolution in primates, which provides a new foundation for the study of phenotypic difference among primates.© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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September 22, 2019

Assessment of an organ-specific de novo transcriptome of the nematode trap-crop, Solanum sisymbriifolium

Solanum sisymbriifolium, also known as “Litchi Tomato” or “Sticky Nightshade,” is an undomesticated and poorly researched plant related to potato and tomato. Unlike the latter species, S. sisymbriifolium induces eggs of the cyst nematode, Globodera pallida, to hatch and migrate into its roots, but then arrests further nematode maturation. In order to provide researchers with a partial blueprint of its genetic make-up so that the mechanism of this response might be identified, we used single molecule real time (SMRT) sequencing to compile a high quality de novo transcriptome of 41,189 unigenes drawn from individually sequenced bud, root, stem, and leaf RNA populations. Functional annotation and BUSCO analysis showed that this transcriptome was surprisingly complete, even though it represented genes expressed at a single time point. By sequencing the 4 organ libraries separately, we found we could get a reliable snapshot of transcript distributions in each organ. A divergent site analysis of the merged transcriptome indicated that this species might have undergone a recent genome duplication and re-diploidization. Further analysis indicated that the plant then retained a disproportionate number of genes associated with photosynthesis and amino acid metabolism in comparison to genes with characteristics of R-proteins or involved in secondary metabolism. The former processes may have given S. sisymbriifolium a bigger competitive advantage than the latter did. Copyright © 2018 Wixom et al.

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September 22, 2019

Single molecule RNA sequencing uncovers trans-splicing and improves annotations in Anopheles stephensi.

Single molecule real-time (SMRT) sequencing has recently been used to obtain full-length cDNA sequences that improve genome annotation and reveal RNA isoforms. Here, we used one such method called isoform sequencing from Pacific Biosciences (PacBio) to sequence a cDNA library from the Asian malaria mosquito Anopheles stephensi. More than 600 000 full-length cDNAs, referred to as reads of insert, were identified. Owing to the inherently high error rate of PacBio sequencing, we tested different approaches for error correction. We found that error correction using Illumina RNA sequencing (RNA-seq) generated more data than using the default SMRT pipeline. The full-length error-corrected PacBio reads greatly improved the gene annotation of Anopheles stephensi: 4867 gene models were updated and 1785 alternatively spliced isoforms were added to the annotation. In addition, six trans-splicing events, where exons from different primary transcripts were joined together, were identified in An. stephensi. All six trans-splicing events appear to be conserved in Culicidae, as they are also found in Anopheles gambiae and Aedes aegypti. The proteins encoded by trans-splicing events are also highly conserved and the orthologues of these proteins are cis-spliced in outgroup species, indicating that trans-splicing may arise as a mechanism to rescue genes that broke up during evolution.© 2017 The Royal Entomological Society.

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September 22, 2019

Contemporary evolution of a Lepidopteran species, Heliothis virescens, in response to modern agricultural practices.

Adaptation to human-induced environmental change has the potential to profoundly influence the genomic architecture of affected species. This is particularly true in agricultural ecosystems, where anthropogenic selection pressure is strong. Heliothis virescens primarily feeds on cotton in its larval stages, and US populations have been declining since the widespread planting of transgenic cotton, which endogenously expresses proteins derived from Bacillus thuringiensis (Bt). No physiological adaptation to Bt toxin has been found in the field, so adaptation in this altered environment could involve (i) shifts in host plant selection mechanisms to avoid cotton, (ii) changes in detoxification mechanisms required for cotton-feeding vs. feeding on other hosts or (iii) loss of resistance to previously used management practices including insecticides. Here, we begin to address whether such changes occurred in H. virescens populations between 1997 and 2012, as Bt-cotton cultivation spread through the agricultural landscape. For our study, we produced an H. virescens genome assembly and used this in concert with a ddRAD-seq-enabled genome scan to identify loci with significant allele frequency changes over the 15-year period. Genetic changes at a previously described H. virescens insecticide target of selection were detectable in our genome scan and increased our confidence in this methodology. Additional loci were also detected as being under selection, and we quantified the selection strength required to elicit observed allele frequency changes at each locus. Potential contributions of genes near loci under selection to adaptive phenotypes in the H. virescens cotton system are discussed.© 2017 John Wiley & Sons Ltd.

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September 22, 2019

Plasmodium knowlesi: a superb in vivo nonhuman primate model of antigenic variation in malaria.

Antigenic variation in malaria was discovered in Plasmodium knowlesi studies involving longitudinal infections of rhesus macaques (M. mulatta). The variant proteins, known as the P. knowlesi Schizont Infected Cell Agglutination (SICA) antigens and the P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) antigens, expressed by the SICAvar and var multigene families, respectively, have been studied for over 30 years. Expression of the SICA antigens in P. knowlesi requires a splenic component, and specific antibodies are necessary for variant antigen switch events in vivo. Outstanding questions revolve around the role of the spleen and the mechanisms by which the expression of these variant antigen families are regulated. Importantly, the longitudinal dynamics and molecular mechanisms that govern variant antigen expression can be studied with P. knowlesi infection of its mammalian and vector hosts. Synchronous infections can be initiated with established clones and studied at multi-omic levels, with the benefit of computational tools from systems biology that permit the integration of datasets and the design of explanatory, predictive mathematical models. Here we provide an historical account of this topic, while highlighting the potential for maximizing the use of P. knowlesi – macaque model systems and summarizing exciting new progress in this area of research.

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September 22, 2019

Complete genome sequence of Sphingobium baderi DE-13, an alkyl-substituted aniline-mineralizing bacterium.

Alkyl-substituted aniline is an important aniline derivative that may be associated with serious environmental risks. Previously, Sphingobium baderi DE-13, a bacterium that can mineralize alkyl substituted anilines such as 2,6-dimethylaniline, 2,6-diethylaniline, 2-methyl-6-ethylaniline, 2-methylaniline, and 2-ethylaniline, was isolated from active sludge. Here, we report the complete genome sequence of strain DE-13. It contains one circular chromosome and eight circular plasmids with total 4,583,422 bp and GC content of 62.41%. The reported and predicted genes involved in the catabolism of alkyl-substituted anilines are indicated. This study will provide insights into the bacterial catabolism of alkyl substituted anilines.

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September 22, 2019

Genome analysis of Taraxacum kok-saghyz Rodin provides new insights into rubber biosynthesis

The Russian dandelion Taraxacum kok-saghyz Rodin (TKS), a member of the Composite family and a potential alternative source of natural rubber (NR) and inulin, is an ideal model system for studying rubber biosynthesis. Here we present the draft genome of TKS, the first assembled NR-producing weed plant. The draft TKS genome assembly has a length of 1.29 Gb, containing 46,731 predicted protein-coding genes and 68.56% repeats, in which the LTR-RT elements predominantly contribute to the genome enlargement. We analyzed the heterozygous regions/genes, suggesting its possible involvement in inbreeding depression. Through comparative studies between rubber-producing and non-rubber-producing plants, we found that enzymes of the mevalonate (MVA) pathway and rubber elongation might be critical for rubber biosynthesis, and several key isoforms have been isolated showing predominantly expressed in the latex, indicating their crucial functions in rubber biosynthesis. Moreover, for two important families in rubber elongation, the CPT/CPTL and REF/SRPP families, diverse evolutionary tracks have been revealed. These results provide valuable resources and new insights into the mechanism of NR biosynthesis, and facilitate the development of alternative NR producing crops.

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September 22, 2019

Prey range and genome evolution of Halobacteriovorax marinus predatory bacteria from an estuary

Halobacteriovorax strains are saltwater-adapted predatory bacteria that attack Gram-negative bacteria and may play an important role in shaping microbial communities. To understand how Halobacteriovorax strains impact ecosystems and develop them as biocontrol agents, it is important to characterize variation in predation phenotypes and investigate Halobacteriovorax genome evolution. We isolated Halobacteriovorax marinus BE01 from an estuary in Rhode Island using Vibrio from the same site as prey. Small, fast-moving, attack-phase BE01 cells attach to and invade prey cells, consistent with the intraperiplasmic predation strategy of the H. marinus type strain, SJ. BE01 is a prey generalist, forming plaques on Vibrio strains from the estuary, Pseudomonas from soil, and Escherichia coli. Genome analysis revealed extremely high conservation of gene order and amino acid sequences between BE01 and SJ, suggesting strong selective pressure to maintain the genome in this H. marinus lineage. Despite this, we identified two regions of gene content difference that likely resulted from horizontal gene transfer. Analysis of modal codon usage frequencies supports the hypothesis that these regions were acquired from bacteria with different codon usage biases than H. marinus. In one of these regions, BE01 and SJ carry different genes associated with mobile genetic elements. Acquired functions in BE01 include the dnd operon, which encodes a pathway for DNA modification, and a suite of genes involved in membrane synthesis and regulation of gene expression that was likely acquired from another Halobacteriovorax lineage. This analysis provides further evidence that horizontal gene transfer plays an important role in genome evolution in predatory bacteria. IMPORTANCE Predatory bacteria attack and digest other bacteria and therefore may play a role in shaping microbial communities. To investigate phenotypic and genotypic variation in saltwater-adapted predatory bacteria, we isolated Halobacteriovorax marinus BE01 from an estuary in Rhode Island, assayed whether it could attack different prey bacteria, and sequenced and analyzed its genome. We found that BE01 is a prey generalist, attacking bacteria from different phylogenetic groups and environments. Gene order and amino acid sequences are highly conserved between BE01 and the H. marinus type strain, SJ. By comparative genomics, we detected two regions of gene content difference that likely occurred via horizontal gene transfer events. Acquired genes encode functions such as modification of DNA, membrane synthesis and regulation of gene expression. Understanding genome evolution and variation in predation phenotypes among predatory bacteria will inform their development as biocontrol agents and clarify how they impact microbial communities.

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September 22, 2019

Genome characterization of oleaginous Aspergillus oryzae BCC7051: A potential fungal-based platform for lipid production.

The selected robust fungus, Aspergillus oryzae strain BCC7051 is of interest for biotechnological production of lipid-derived products due to its capability to accumulate high amount of intracellular lipids using various sugars and agro-industrial substrates. Here, we report the genome sequence of the oleaginous A. oryzae BCC7051. The obtained reads were de novo assembled into 25 scaffolds spanning of 38,550,958 bps with predicted 11,456 protein-coding genes. By synteny mapping, a large rearrangement was found in two scaffolds of A. oryzae BCC7051 as compared to the reference RIB40 strain. The genetic relationship between BCC7051 and other strains of A. oryzae in terms of aflatoxin production was investigated, indicating that the A. oryzae BCC7051 was categorized into group 2 nonaflatoxin-producing strain. Moreover, a comparative analysis of the structural genes focusing on the involvement in lipid metabolism among oleaginous yeast and fungi revealed the presence of multiple isoforms of metabolic enzymes responsible for fatty acid synthesis in BCC7051. The alternative routes of acetyl-CoA generation as oleaginous features and malate/citrate/pyruvate shuttle were also identified in this A. oryzae strain. The genome sequence generated in this work is a dedicated resource for expanding genome-wide study of microbial lipids at systems level, and developing the fungal-based platform for production of diversified lipids with commercial relevance.

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September 22, 2019

Stalking a lethal superbug by whole-genome sequencing and phylogenetics: Influence on unraveling a major hospital outbreak of carbapenem-resistant Klebsiella pneumoniae.

From July 2010-April 2013, Leipzig University Hospital experienced the largest outbreak of a Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing Klebsiella pneumoniae (KPC-2-Kp) strain observed in Germany to date. After termination of the outbreak, we aimed to reconstruct transmission pathways by phylogenetics based on whole-genome sequencing (WGS).One hundred seventeen KPC-2-Kp isolates from 89 outbreak patients, 5 environmental KPC-2-Kp isolates, and 24 K pneumoniae strains not linked to the outbreak underwent WGS. Phylogenetic analysis was performed blinded to clinical data and based on the genomic reads.A patient from Greece was confirmed as the source of the outbreak. Transmission pathways for 11 out of 89 patients (12.4%) were plausibly explained by descriptive epidemiology, applying strict definitions. Five of these and an additional 15 (ie, 20 out of 89 patients [22.5%]) were confirmed by phylogenetics. The rate of phylogenetically confirmed transmissions increased significantly from 8 out of 66 (12.1% for the time period before) to 12 out of 23 patients (52.2% for the time period after; P?<.001) after implementation of systematic screening for KPC-2-Kp (33,623 screening investigations within 11 months). Using descriptive epidemiology, systematic screening showed no significant effect (7 out of 66 [10.6%] vs 4 out of 23 [17.4%] patients; P?=?.465). The phylogenetic analysis supported the assumption that a contaminated positioning pillow served as a reservoir for the persistence of KPC-2-Kp.Effective phylogenetic identification of transmissions requires systematic microbiologic screening. Extensive screening and phylogenetic analysis based on WGS should be started as soon as possible in a bacterial outbreak situation. Copyright © 2018 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Complete genome sequence of Geobacillus thermodenitrificans T12, a potential host for biotechnological applications.

In attempt to obtain a thermophilic host for the conversion of lignocellulose derived substrates into lactic acid, Geobacillus thermodenitrificans T12 was isolated from a compost heap. It was selected from over 500 isolates as a genetically tractable hemicellulolytic lactic acid producer, requiring little nutrients. The strain is able to ferment glucose and xylose simultaneously and can produce lactic acid from xylan, making it a potential host for biotechnological applications. The genome of strain T12 consists of a 3.64 Mb chromosome and two plasmids of 59 and 56 kb. It has a total of 3.676 genes with an average genomic GC content of 48.7%. The T12 genome encodes a denitrification pathway, allowing for anaerobic respiration. The identity and localization of the responsible genes are similar to those of the denitrification pathways found in strain NG80-2. The hemicellulose utilization (HUS) locus was identified based on sequence homology against G. stearothermophilus T-6. It appeared that T12 has all the genes that are present in strain T-6 except for the arabinan degradation cluster. Instead, the HUS locus of strain T12 contains genes for both an inositol and a pectate degradation pathway. Strain T12 has complete pathways for the synthesis of purine and pyrimidine, all 20 amino acids and several vitamins except D-biotin. The host-defense systems present comprise a Type II and a Type III restriction-modification system, as well as a CRISPR-Cas Type II system. It is concluded that G. thermodenitrificans T12 is a potentially interesting candidate for industrial applications.

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September 22, 2019

Identification of the biosynthetic pathway for the antibiotic bicyclomycin.

Diketopiperazines (DKPs) make up a large group of natural products with diverse structures and biological activities. Bicyclomycin is a broad-spectrum DKP antibiotic with unique structure and function: it contains a highly oxidized bicyclic [4.2.2] ring and is the only known selective inhibitor of the bacterial transcription termination factor, Rho. Here, we identify the biosynthetic gene cluster for bicyclomycin containing six iron-dependent oxidases. We demonstrate that the DKP core is made by a tRNA-dependent cyclodipeptide synthase, and hydroxylations on two unactivated sp(3) carbons are performed by two mononuclear iron, a-ketoglutarate-dependent hydroxylases. Using bioinformatics, we also identify a homologous gene cluster prevalent in a human pathogen Pseudomonas aeruginosa. We detect bicyclomycin by overexpressing this gene cluster and establish P. aeruginosa as a new producer of bicyclomycin. Our work uncovers the biosynthetic pathway for bicyclomycin and sheds light on the intriguing oxidation chemistry that converts a simple DKP into a powerful antibiotic.

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September 22, 2019

The genome sequence of the soft-rot fungus Penicillium purpurogenum reveals a high gene dosage for lignocellulolytic enzymes.

The high lignocellulolytic activity displayed by the soft-rot fungus Penicillium purpurogenum has made it a target for the study of novel lignocellulolytic enzymes. We have obtained a reference genome of 36.2 Mb of non-redundant sequence (11,057 protein-coding genes). The 49 largest scaffolds cover 90% of the assembly, and Core Eukaryotic Genes Mapping Approach (CEGMA) analysis reveals that our assembly captures almost all protein-coding genes. RNA-seq was performed and 93.1% of the reads aligned to the assembled genome. These data, plus the independent sequencing of a set of genes of lignocellulose-degrading enzymes, validate the quality of the genome sequence. P. purpurogenum shows a higher number of proteins with CAZy motifs, transcription factors and transporters as compared to other sequenced Penicillia. These results demonstrate the great potential for lignocellulolytic activity of this fungus and the possible use of its enzymes in related industrial applications.

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September 22, 2019

Complete genome sequence of Enterobacter cloacae R11 reveals multiple genes potentially associated with high-level polymyxin E resistance.

Enterobacter cloacae strain R11 is a multidrug-resistant bacterium isolated from sewage water near a swine feedlot in China. Strain R11 can survive in medium containing up to 192 µg/mL polymyxin E, indicating a tolerance for this antibiotic that is significantly higher than that reported for other gram-negative bacteria. In this study, conjugation experiments showed that partial polymyxin E resistance could be transferred from strain R11 to Escherichia coli strain 25922, revealing that some genes related to polymyxin E resistance are plasmid-based. The complete genome sequence of this strain was determined, yielding a total of 4?993?008 bp (G+C content, 53.15%) and 4908 genes for the circular chromosome and 4 circular plasmids. Genome analysis revealed a total of 73 putative antibiotic resistance genes, including several polymyxin E resistance genes and genes potentially involved in multidrug resistance. These data provide insights into the genetic basis of the polymyxin E resistance and multidrug resistance of E. cloacae.

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