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April 21, 2020  |  

Characterization of the genome of a Nocardia strain isolated from soils in the Qinghai-Tibetan Plateau that specifically degrades crude oil and of this biodegradation.

A strain of Nocardia isolated from crude oil-contaminated soils in the Qinghai-Tibetan Plateau degrades nearly all components of crude oil. This strain was identified as Nocardia soli Y48, and its growth conditions were determined. Complete genome sequencing showed that N. soli Y48 has a 7.3?Mb genome and many genes responsible for hydrocarbon degradation, biosurfactant synthesis, emulsification and other hydrocarbon degradation-related metabolisms. Analysis of the clusters of orthologous groups (COGs) and genomic islands (GIs) revealed that Y48 has undergone significant gene transfer events to adapt to changing environmental conditions (crude oil contamination). The structural features of the genome might provide a competitive edge for the survival of N. soli Y48 in oil-polluted environments and reflect the adaptation of coexisting bacteria to distinct nutritional niches.Copyright © 2018. Published by Elsevier Inc.

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April 21, 2020  |  

TranscriptClean: variant-aware correction of indels, mismatches and splice junctions in long-read transcripts.

Long-read, single-molecule sequencing platforms hold great potential for isoform discovery and characterization of multi-exon transcripts. However, their high error rates are an obstacle to distinguishing novel transcript isoforms from sequencing artifacts. Therefore, we developed the package TranscriptClean to correct mismatches, microindels and noncanonical splice junctions in mapped transcripts using the reference genome while preserving known variants.Our method corrects nearly all mismatches and indels present in a publically available human PacBio Iso-seq dataset, and rescues 39% of noncanonical splice junctions.All Python and R scripts used in this paper are available at https://github.com/dewyman/TranscriptClean.

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April 21, 2020  |  

TaF: a web platform for taxonomic profile-based fungal gene prediction.

The accurate prediction and annotation of gene structures from the genome sequence of an organism enable genome-wide functional analyses to obtain insight into the biological properties of an organism.We recently developed a highly accurate filamentous fungal gene prediction pipeline and web platform called TaF. TaF is a homology-based gene predictor employing large-scale taxonomic profiling to search for close relatives in genome queries.TaF pipeline consists of four processing steps; (1) taxonomic profiling to search for close relatives to query, (2) generation of hints for determining exon-intron boundaries from orthologous protein sequence data of the profiled species, (3) gene prediction by combination of ab inito and evidence-based prediction methods, and (4) homology search for gene models.TaF generates extrinsic evidence that suggests possible exon-intron boundaries based on orthologous protein sequence data, thus reducing false-positive predictions of gene structure based on distantly related orthologs data. In particular, the gene prediction method using taxonomic profiling shows very high accuracy, including high sensitivity and specificity for gene models, suggesting a new approach for homology-based gene prediction from newly sequenced or uncharacterized fungal genomes, with the potential to improve the quality of gene prediction.TaF will be a useful tool for fungal genome-wide analyses, including the identification of targeted genes associated with a trait, transcriptome profiling, comparative genomics, and evolutionary analysis.

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April 21, 2020  |  

Genome Sequence of Jaltomata Addresses Rapid Reproductive Trait Evolution and Enhances Comparative Genomics in the Hyper-Diverse Solanaceae.

Within the economically important plant family Solanaceae, Jaltomata is a rapidly evolving genus that has extensive diversity in flower size and shape, as well as fruit and nectar color, among its ~80 species. Here, we report the whole-genome sequencing, assembly, and annotation, of one representative species (Jaltomata sinuosa) from this genus. Combining PacBio long reads (25×) and Illumina short reads (148×) achieved an assembly of ~1.45?Gb, spanning ~96% of the estimated genome. Ninety-six percent of curated single-copy orthologs in plants were detected in the assembly, supporting a high level of completeness of the genome. Similar to other Solanaceous species, repetitive elements made up a large fraction (~80%) of the genome, with the most recently active element, Gypsy, expanding across the genome in the last 1-2 Myr. Computational gene prediction, in conjunction with a merged transcriptome data set from 11 tissues, identified 34,725 protein-coding genes. Comparative phylogenetic analyses with six other sequenced Solanaceae species determined that Jaltomata is most likely sister to Solanum, although a large fraction of gene trees supported a conflicting bipartition consistent with substantial introgression between Jaltomata and Capsicum after these species split. We also identified gene family dynamics specific to Jaltomata, including expansion of gene families potentially involved in novel reproductive trait development, and loss of gene families that accompanied the loss of self-incompatibility. This high-quality genome will facilitate studies of phenotypic diversification in this rapidly radiating group and provide a new point of comparison for broader analyses of genomic evolution across the Solanaceae.

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April 21, 2020  |  

The conservation of polyol transporter proteins and their involvement in lichenized Ascomycota.

In lichen symbiosis, polyol transfer from green algae is important for acquiring the fungal carbon source. However, the existence of polyol transporter genes and their correlation with lichenization remain unclear. Here, we report candidate polyol transporter genes selected from the genome of the lichen-forming fungus (LFF) Ramalina conduplicans. A phylogenetic analysis using characterized polyol and monosaccharide transporter proteins and hypothetical polyol transporter proteins of R. conduplicans and various ascomycetous fungi suggested that the characterized yeast’ polyol transporters form multiple clades with the polyol transporter-like proteins selected from the diverse ascomycetous taxa. Thus, polyol transporter genes are widely conserved among Ascomycota, regardless of lichen-forming status. In addition, the phylogenetic clusters suggested that LFFs belonging to Lecanoromycetes have duplicated proteins in each cluster. Consequently, the number of sequences similar to characterized yeast’ polyol transporters were evaluated using the genomes of 472 species or strains of Ascomycota. Among these, LFFs belonging to Lecanoromycetes had greater numbers of deduced polyol transporter proteins. Thus, various polyol transporters are conserved in Ascomycota and polyol transporter genes appear to have expanded during the evolution of Lecanoromycetes. Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

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April 21, 2020  |  

Snf2 controls pulcherriminic acid biosynthesis and antifungal activity of the biocontrol yeast Metschnikowia pulcherrima.

Metschnikowia pulcherrima synthesises the pigment pulcherrimin, from cyclodileucine (cyclo(Leu-Leu)) as a precursor, and exhibits strong antifungal activity against notorious plant pathogenic fungi. This yeast therefore has great potential for biocontrol applications against fungal diseases; particularly in the phyllosphere where this species is frequently found. To elucidate the molecular basis of the antifungal activity of M. pulcherrima, we compared a wild-type strain with a spontaneously occurring, pigmentless, weakly antagonistic mutant derivative. Whole genome sequencing of the wild-type and mutant strains identified a point mutation that creates a premature stop codon in the transcriptional regulator gene SNF2 in the mutant. Complementation of the mutant strain with the wild-type SNF2 gene restored pigmentation and recovered the strong antifungal activity. Mass spectrometry (UPLC HR HESI-MS) proved the presence of the pulcherrimin precursors cyclo(Leu-Leu) and pulcherriminic acid and identified new precursor and degradation products of pulcherriminic acid and/or pulcherrimin. All of these compounds were identified in the wild-type and complemented strain, but were undetectable in the pigmentless snf2 mutant strain. These results thus identify Snf2 as a regulator of antifungal activity and pulcherriminic acid biosynthesis in M. pulcherrima and provide a starting point for deciphering the molecular functions underlying the antagonistic activity of this yeast. © 2019 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.

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April 21, 2020  |  

Function and Distribution of a Lantipeptide in Strawberry Fusarium Wilt Disease-Suppressive Soils.

Streptomyces griseus S4-7 is representative of strains responsible for the specific soil suppressiveness of Fusarium wilt of strawberry caused by Fusarium oxysporum f. sp. fragariae. Members of the genus Streptomyces secrete diverse secondary metabolites including lantipeptides, heat-stable lanthionine-containing compounds that can exhibit antibiotic activity. In this study, a class II lantipeptide provisionally named grisin, of previously unknown biological function, was shown to inhibit F. oxysporum. The inhibitory activity of grisin distinguishes it from other class II lantipeptides from Streptomyces spp. Results of quantitative reverse transcription-polymerase chain reaction with lanM-specific primers showed that the density of grisin-producing Streptomyces spp. in the rhizosphere of strawberry was positively correlated with the number of years of monoculture and a minimum of seven years was required for development of specific soil suppressiveness to Fusarium wilt disease. We suggest that lanM can be used as a diagnostic marker of whether a soil is conducive or suppressive to the disease.

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April 21, 2020  |  

Penicillium purpurogenum Produces a Set of Endoxylanases: Identification, Heterologous Expression, and Characterization of a Fourth Xylanase, XynD, a Novel Enzyme Belonging to Glycoside Hydrolase Family 10.

The fungus Penicillium purpurogenum grows on a variety of natural carbon sources and secretes a large number of enzymes which degrade the polysaccharides present in lignocellulose. In this work, the gene coding for a novel endoxylanase has been identified in the genome of the fungus. This gene (xynd) possesses four introns. The cDNA has been expressed in Pichia pastoris and characterized. The enzyme, XynD, belongs to family 10 of the glycoside hydrolases. Mature XynD has a calculated molecular weight of 40,997. It consists of 387 amino acid residues with an N-terminal catalytic module, a linker rich in ser and thr residues, and a C-terminal family 1 carbohydrate-binding module. XynD shows the highest identity (97%) to a putative endoxylanase from Penicillium subrubescens but its highest identity to a biochemically characterized xylanase (XYND from Penicillium funiculosum) is only 68%. The enzyme has a temperature optimum of 60 °C, and it is highly stable in its pH optimum range of 6.5-8.5. XynD is the fourth biochemically characterized endoxylanase from P. purpurogenum, confirming the rich potential of this fungus for lignocellulose biodegradation. XynD, due to its wide pH optimum and stability, may be a useful enzyme in biotechnological procedures related to this biodegradation process.

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April 21, 2020  |  

Natural product drug discovery in the genomic era: realities, conjectures, misconceptions, and opportunities.

Natural product discovery from microorganisms provided important sources for antibiotics, anti-cancer agents, immune-modulators, anthelminthic agents, and insecticides during a span of 50 years starting in the 1940s, then became less productive because of rediscovery issues, low throughput, and lack of relevant new technologies to unveil less abundant or not easily detected drug-like natural products. In the early 2000s, it was observed from genome sequencing that Streptomyces species encode about ten times as many secondary metabolites as predicted from known secondary metabolomes. This gave rise to a new discovery approach-microbial genome mining. As the cost of genome sequencing dropped, the numbers of sequenced bacteria, fungi and archaea expanded dramatically, and bioinformatic methods were developed to rapidly scan whole genomes for the numbers, types, and novelty of secondary metabolite biosynthetic gene clusters. This methodology enabled the identification of microbial taxa gifted for the biosynthesis of drug-like secondary metabolites. As genome sequencing technology progressed, the realities relevant to drug discovery have emerged, the conjectures and misconceptions have been clarified, and opportunities to reinvigorate microbial drug discovery have crystallized. This perspective addresses these critical issues for drug discovery.

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April 21, 2020  |  

Toxin and genome evolution in a Drosophila defensive symbiosis.

Defenses conferred by microbial symbionts play a vital role in the health and fitness of their animal hosts. An important outstanding question in the study of defensive symbiosis is what determines long term stability and effectiveness against diverse natural enemies. In this study, we combine genome and transcriptome sequencing, symbiont transfection and parasite protection experiments, and toxin activity assays to examine the evolution of the defensive symbiosis between Drosophila flies and their vertically transmitted Spiroplasma bacterial symbionts, focusing in particular on ribosome-inactivating proteins (RIPs), symbiont-encoded toxins that have been implicated in protection against both parasitic wasps and nematodes. Although many strains of Spiroplasma, including the male-killing symbiont (sMel) of Drosophila melanogaster, protect against parasitic wasps, only the strain (sNeo) that infects the mycophagous fly Drosophila neotestacea appears to protect against parasitic nematodes. We find that RIP repertoire is a major differentiating factor between strains that do and do not offer nematode protection, and that sMel RIPs do not show activity against nematode ribosomes in vivo. We also discovered a strain of Spiroplasma infecting a mycophagous phorid fly, Megaselia nigra. Although both the host and its Spiroplasma are distantly related to D. neotestacea and its symbiont, genome sequencing revealed that the M. nigra symbiont encodes abundant and diverse RIPs, including plasmid-encoded toxins that are closely related to the RIPs in sNeo. Our results suggest that distantly related Spiroplasma RIP toxins may perform specialized functions with regard to parasite specificity and suggest an important role for horizontal gene transfer in the emergence of novel defensive phenotypes.

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April 21, 2020  |  

Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the 16S-ITS-23S rRNA operon.

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286-37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (~1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full-length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost-effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput. © 2019 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020  |  

Population Genome Sequencing of the Scab Fungal Species Venturia inaequalis, Venturia pirina, Venturia aucupariae and Venturia asperata.

The Venturia genus comprises fungal species that are pathogens on Rosaceae host plants, including V. inaequalis and V. asperata on apple, V. aucupariae on sorbus and V. pirina on pear. Although the genetic structure of V. inaequalis populations has been investigated in detail, genomic features underlying these subdivisions remain poorly understood. Here, we report whole genome sequencing of 87 Venturia strains that represent each species and each population within V. inaequalis We present a PacBio genome assembly for the V. inaequalis EU-B04 reference isolate. The size of selected genomes was determined by flow cytometry, and varied from 45 to 93 Mb. Genome assemblies of V. inaequalis and V. aucupariae contain a high content of transposable elements (TEs), most of which belong to the Gypsy or Copia LTR superfamilies and have been inactivated by Repeat-Induced Point mutations. The reference assembly of V. inaequalis presents a mosaic structure of GC-equilibrated regions that mainly contain predicted genes and AT-rich regions, mainly composed of TEs. Six pairs of strains were identified as clones. Single-Nucleotide Polymorphism (SNP) analysis between these clones revealed a high number of SNPs that are mostly located in AT-rich regions due to misalignments and allowed determining a false discovery rate. The availability of these genome sequences is expected to stimulate genetics and population genomics research of Venturia pathogens. Especially, it will help understanding the evolutionary history of Venturia species that are pathogenic on different hosts, a history that has probably been substantially influenced by TEs.Copyright © 2019 Le Cam et al.

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April 21, 2020  |  

Complete Genome Sequence of Saccharospirillum mangrovi HK-33T Sheds Light on the Ecological Role of a Bacterium in Mangrove Sediment Environment.

We present the genome sequence of Saccharospirillum mangrovi HK-33T, isolated from a mangrove sediment sample in Haikou, China. The complete genome of S. mangrovi HK-33T consisted of a single-circular chromosome with the size of 3,686,911 bp as well as an average G?+?C content of 57.37%, and contained 3,383 protein-coding genes, 4 operons of 16S-23S-5S rRNA genes, and 52 tRNA genes. Genomic annotation indicated that the genome of S. mangrovi HK-33T had many genes related to oligosaccharide and polysaccharide degradation and utilization of polyhydroxyalkanoate. For nitrogen cycle, genes encoding nitrate and nitrite reductase, glutamate dehydrogenase, glutamate synthase, and glutamine synthetase could be found. For phosphorus cycle, genes related to polyphosphate kinases (ppk1 and ppk2), the high-affinity phosphate-specific transport (Pst) system, and the low-affinity inorganic phosphate transporter (pitA) were predicted. For sulfur cycle, cysteine synthase and type III acyl coenzyme A transferase (dddD) coding genes were searched out. This study provides evidence about carbon, nitrogen, phosphorus, and sulfur metabolic patterns of S. mangrovi HK-33T and broadens our understandings about ecological roles of this bacterium in the mangrove sediment environment.

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April 21, 2020  |  

A chromosome-scale genome assembly reveals a highly dynamic effector repertoire of wheat powdery mildew.

Blumeria graminis f. sp. tritici (B.g. tritici) is the causal agent of the wheat powdery mildew disease. The highly fragmented B.g. tritici genome available so far has prevented a systematic analysis of effector genes that are known to be involved in host adaptation. To study the diversity and evolution of effector genes we produced a chromosome-scale assembly of the B.g. tritici genome. The genome assembly and annotation was achieved by combining long-read sequencing with high-density genetic mapping, bacterial artificial chromosome fingerprinting and transcriptomics. We found that the 166.6 Mb B.g. tritici genome encodes 844 candidate effector genes, over 40% more than previously reported. Candidate effector genes have characteristic local genomic organization such as gene clustering and enrichment for recombination-active regions and certain transposable element families. A large group of 412 candidate effector genes shows high plasticity in terms of copy number variation in a global set of 36 isolates and of transcription levels. Our data suggest that copy number variation and transcriptional flexibility are the main drivers for adaptation in B.g. tritici. The high repeat content may play a role in providing a genomic environment that allows rapid evolution of effector genes with selection as the driving force. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

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April 21, 2020  |  

The developmental dynamics of the Populus stem transcriptome.

The Populus shoot undergoes primary growth (longitudinal growth) followed by secondary growth (radial growth), which produces biomass that is an important source of energy worldwide. We adopted joint PacBio Iso-Seq and RNA-seq analysis to identify differentially expressed transcripts along a developmental gradient from the shoot apex to the fifth internode of Populus Nanlin895. We obtained 87 150 full-length transcripts, including 2081 new isoforms and 62 058 new alternatively spliced isoforms, most of which were produced by intron retention, that were used to update the Populus annotation. Among these novel isoforms, there are 1187 long non-coding RNAs and 356 fusion genes. Using this annotation, we found 15 838 differentially expressed transcripts along the shoot developmental gradient, of which 1216 were transcription factors (TFs). Only a few of these genes were reported previously. The differential expression of these TFs suggests that they may play important roles in primary and secondary growth. AP2, ARF, YABBY and GRF TFs are highly expressed in the apex, whereas NAC, bZIP, PLATZ and HSF TFs are likely to be important for secondary growth. Overall, our findings provide evidence that long-read sequencing can complement short-read sequencing for cataloguing and quantifying eukaryotic transcripts and increase our understanding of the vital and dynamic process of shoot development. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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