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April 21, 2020  |  

Real time monitoring of Aeromonas salmonicida evolution in response to successive antibiotic therapies in a commercial fish farm.

Our ability to predict evolutionary trajectories of pathogens in response to antibiotic pressure is one of the promising leverage to fight against the present antibiotic resistance worldwide crisis. Yet, few studies tackled this question in situ at the outbreak level, due to the difficulty to link a given pathogenic clone evolution with its precise antibiotic exposure over time. In this study, we monitored the real-time evolution of an Aeromonas salmonicida clone in response to successive antibiotic and vaccine therapies in a commercial fish farm. The clone was responsible for a four-year outbreak of furunculosis within a Recirculating Aquaculture System Salmo salar farm in China, and we reconstructed the precise tempo of mobile genetic elements (MGEs) acquisition events during this period. The resistance profile provided by the acquired MGEs closely mirrored the antibiotics used to treat the outbreak, and we evidenced that two subclonal groups developed similar resistances although unrelated MGE acquisitions. Finally, we also demonstrated the efficiency of vaccination in outbreak management and its positive effect on antibiotic resistance prevalence. Our study provides unprecedented knowledge critical to understand evolutionary trajectories of resistant pathogens outside the laboratory. © 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020  |  

Genome assembly of a tropical maize inbred line provides insights into structural variation and crop improvement.

Maize is one of the most important crops globally, and it shows remarkable genetic diversity. Knowledge of this diversity could help in crop improvement; however, gold-standard genomes have been elucidated only for modern temperate varieties. Here, we present a high-quality reference genome (contig N50 of 15.78?megabases) of the maize small-kernel inbred line, which is derived from a tropical landrace. Using haplotype maps derived from B73, Mo17 and SK, we identified 80,614 polymorphic structural variants across 521 diverse lines. Approximately 22% of these variants could not be detected by traditional single-nucleotide-polymorphism-based approaches, and some of them could affect gene expression and trait performance. To illustrate the utility of the diverse SK line, we used it to perform map-based cloning of a major effect quantitative trait locus controlling kernel weight-a key trait selected during maize improvement. The underlying candidate gene ZmBARELY ANY MERISTEM1d provides a target for increasing crop yields.

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April 21, 2020  |  

Chromosome-level genome assembly of Triplophysa tibetana, a fish adapted to the harsh high-altitude environment of the Tibetan Plateau.

Triplophysa is an endemic fish genus of the Tibetan Plateau in China. Triplophysa tibetana, which lives at a recorded altitude of ~4,000 m and plays an important role in the highland aquatic ecosystem, serves as an excellent model for investigating high-altitude environmental adaptation. However, evolutionary and conservation studies of T. tibetana have been limited by scarce genomic resources for the genus Triplophysa. In the present study, we applied PacBio sequencing and the Hi-C technique to assemble the T. tibetana genome. A 652-Mb genome with 1,325 contigs with an N50 length of 3.1 Mb was obtained. The 1,137 contigs were further assembled into 25 chromosomes, representing 98.7% and 80.47% of all contigs at the base and sequence number level, respectively. Approximately 260 Mb of sequence, accounting for ~39.8% of the genome, was identified as repetitive elements. DNA transposons (16.3%), long interspersed nuclear elements (12.4%) and long terminal repeats (11.0%) were the most repetitive types. In total, 24,372 protein-coding genes were predicted in the genome, and ~95% of the genes were functionally annotated via a search in public databases. Using whole genome sequence information, we found that T. tibetana diverged from its common ancestor with Danio rerio ~121.4 million years ago. The high-quality genome assembled in this work not only provides a valuable genomic resource for future population and conservation studies of T. tibetana, but it also lays a solid foundation for further investigation into the mechanisms of environmental adaptation of endemic fishes in the Tibetan Plateau. © 2019 John Wiley & Sons Ltd.

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April 21, 2020  |  

Analysis of the Complete Genome Sequence of a Novel, Pseudorabies Virus Strain Isolated in Southeast Europe.

Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease giving rise to significant economic losses worldwide. Many countries have implemented national programs for the eradication of this virus. In this study, long-read sequencing was used to determine the nucleotide sequence of the genome of a novel PRV strain (PRV-MdBio) isolated in Serbia.In this study, a novel PRV strain was isolated and characterized. PRV-MdBio was found to exhibit similar growth properties to those of another wild-type PRV, the strain Kaplan. Single-molecule real-time (SMRT) sequencing has revealed that the new strain differs significantly in base composition even from strain Kaplan, to which it otherwise exhibits the highest similarity. We compared the genetic composition of PRV-MdBio to strain Kaplan and the China reference strain Ea and obtained that radical base replacements were the most common point mutations preceding conservative and silent mutations. We also found that the adaptation of PRV to cell culture does not lead to any tendentious genetic alteration in the viral genome.PRV-MdBio is a wild-type virus, which differs in base composition from other PRV strains to a relatively large extent.

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April 21, 2020  |  

A global survey of full-length transcriptome of Ginkgo biloba reveals transcript variants involved in flavonoid biosynthesis

Ginkgo biloba, which contains flavonoids as bioactive components, is widely used in traditional Chinese medicine. Increasing the flavonoid production of medicinal plants through genetic engineering generally focuses on the key genes involved in flavonoid biosynthesis. However, the molecular mechanisms underlying such biosynthesis are not yet well understood. To understand these mechanisms, a combination of second-generation sequencing (SGS) and single-molecule real-time (SMRT) sequencing was applied to G. biloba. Eight tissues were sampled for SMRT sequencing to generate a high-quality, full-length transcriptome database. From 23.36 Gb clean reads, 12,954 alternative polyadenylation events, 12,290 alternative splicing events, 929 fusion transcripts, 2,286 novel transcripts, and 1,270 lncRNAs were predicted by removing redundant reads. Further studies reveal that 7 AS, 5 lncRNA, and 6 fusion gene events were identified in flavonoid biosynthesis. A total of 12 gene modules were revealed to be involved in flavonoid metabolism structural genes and transcription factors by constructing co-expression networks. Weighted gene coexpression network analysis (WGCNA) analysis reveals that some hub genes operate during the biosynthesis by identifying transcription factors (TFs) and structure genes. Seven key hub genes were also identified by analyzing the correlation between gene expression level and flavonoids content. The results highlight the importance of SMRT sequencing of the full-length transcriptome in improving genome annotation and elucidating the gene regulation of flavonoid biosynthesis in G. biloba by providing a comprehensive set of reference transcripts.

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April 21, 2020  |  

SMRT sequencing reveals differential patterns of methylation in two O111:H- STEC isolates from a hemolytic uremic syndrome outbreak in Australia.

In 1995 a severe haemolytic-uremic syndrome (HUS) outbreak in Adelaide occurred. A recent genomic analysis of Shiga toxigenic Escherichia coli (STEC) O111:H- strains 95JB1 and 95NR1 from this outbreak found that the more virulent isolate, 95NR1, harboured two additional copies of the Shiga toxin 2 (Stx2) genes encoded within prophage regions. The structure of the Stx2-converting prophages could not be fully resolved using short-read sequence data alone and it was not clear if there were other genomic differences between 95JB1 and 95NR1. In this study we have used Pacific Biosciences (PacBio) single molecule real-time (SMRT) sequencing to characterise the genome and methylome of 95JB1 and 95NR1. We completely resolved the structure of all prophages including two, tandemly inserted, Stx2-converting prophages in 95NR1 that were absent from 95JB1. Furthermore we defined all insertion sequences and found an additional IS1203 element in the chromosome of 95JB1. Our analysis of the methylome of 95NR1 and 95JB1 identified hemi-methylation of a novel motif (5′-CTGCm6AG-3′) in more than 4000 sites in the 95NR1 genome. These sites were entirely unmethylated in the 95JB1 genome, and included at least 177 potential promoter regions that could contribute to regulatory differences between the strains. IS1203 mediated deactivation of a novel type IIG methyltransferase in 95JB1 is the likely cause of the observed differential patterns of methylation between 95NR1 and 95JB1. This study demonstrates the capability of PacBio SMRT sequencing to resolve complex prophage regions and reveal the genetic and epigenetic heterogeneity within a clonal population of bacteria.

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April 21, 2020  |  

Extended insight into the Mycobacterium chelonae-abscessus complex through whole genome sequencing of Mycobacterium salmoniphilum outbreak and Mycobacterium salmoniphilum-like strains.

Members of the Mycobacterium chelonae-abscessus complex (MCAC) are close to the mycobacterial ancestor and includes both human, animal and fish pathogens. We present the genomes of 14 members of this complex: the complete genomes of Mycobacterium salmoniphilum and Mycobacterium chelonae type strains, seven M. salmoniphilum isolates, and five M. salmoniphilum-like strains including strains isolated during an outbreak in an animal facility at Uppsala University. Average nucleotide identity (ANI) analysis and core gene phylogeny revealed that the M. salmoniphilum-like strains are variants of the human pathogen Mycobacterium franklinii and phylogenetically close to Mycobacterium abscessus. Our data further suggested that M. salmoniphilum separates into three branches named group I, II and III with the M. salmoniphilum type strain belonging to group II. Among predicted virulence factors, the presence of phospholipase C (plcC), which is a major virulence factor that makes M. abscessus highly cytotoxic to mouse macrophages, and that M. franklinii originally was isolated from infected humans make it plausible that the outbreak in the animal facility was caused by a M. salmoniphilum-like strain. Interestingly, M. salmoniphilum-like was isolated from tap water suggesting that it can be present in the environment. Moreover, we predicted the presence of mutational hotspots in the M. salmoniphilum isolates and 26% of these hotspots overlap with genes categorized as having roles in virulence, disease and defense. We also provide data about key genes involved in transcription and translation such as sigma factor, ribosomal protein and tRNA genes.

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April 21, 2020  |  

Genome-wide mutational biases fuel transcriptional diversity in the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex (MTBC) members display different host-specificities and virulence phenotypes. Here, we have performed a comprehensive RNAseq and methylome analysis of the main clades of the MTBC and discovered unique transcriptional profiles. The majority of genes differentially expressed between the clades encode proteins involved in host interaction and metabolic functions. A significant fraction of changes in gene expression can be explained by positive selection on single mutations that either create or disrupt transcriptional start sites (TSS). Furthermore, we show that clinical strains have different methyltransferases inactivated and thus different methylation patterns. Under the tested conditions, differential methylation has a minor direct role on transcriptomic differences between strains. However, disruption of a methyltransferase in one clinical strain revealed important expression differences suggesting indirect mechanisms of expression regulation. Our study demonstrates that variation in transcriptional profiles are mainly due to TSS mutations and have likely evolved due to differences in host characteristics.

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April 21, 2020  |  

Meiotic sex in Chagas disease parasite Trypanosoma cruzi.

Genetic exchange enables parasites to rapidly transform disease phenotypes and exploit new host populations. Trypanosoma cruzi, the parasitic agent of Chagas disease and a public health concern throughout Latin America, has for decades been presumed to exchange genetic material rarely and without classic meiotic sex. We present compelling evidence from 45 genomes sequenced from southern Ecuador that T. cruzi in fact maintains truly sexual, panmictic groups that can occur alongside others that remain highly clonal after past hybridization events. These groups with divergent reproductive strategies appear genetically isolated despite possible co-occurrence in vectors and hosts. We propose biological explanations for the fine-scale disconnectivity we observe and discuss the epidemiological consequences of flexible reproductive modes. Our study reinvigorates the hunt for the site of genetic exchange in the T. cruzi life cycle, provides tools to define the genetic determinants of parasite virulence, and reforms longstanding theory on clonality in trypanosomatid parasites.

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April 21, 2020  |  

Modulation of metabolome and bacterial community in whole crop corn silage by inoculating homofermentative Lactobacillus plantarum and heterofermentative Lactobacillus buchneri.

The present study investigated the species level based microbial community and metabolome in corn silage inoculated with or without homofermentative Lactobacillus plantarum and heterofermentative Lactobacillus buchneri using the PacBio SMRT Sequencing and time-of-flight mass spectrometry (GC-TOF/MS). Chopped whole crop corn was treated with (1) deionized water (control), (2) Lactobacillus plantarum, or (3) Lactobacillus buchneri. The chopped whole crop corn was ensiled in vacuum-sealed polyethylene bags containing 300 g of fresh forge for 90 days, with three replicates for each treatment. The results showed that a total of 979 substances were detected, and 316 different metabolites were identified. Some metabolites with antimicrobial activity were detected in whole crop corn silage, such as catechol, 3-phenyllactic acid, 4-hydroxybenzoic acid, azelaic acid, 3,4-dihydroxybenzoic acid and 4-hydroxycinnamic acid. Catechol, pyrogallol and ferulic acid with antioxidant property, 4-hydroxybutyrate with nervine activity, and linoleic acid with cholesterol lowering effects, were detected in present study. In addition, a flavoring agent of myristic acid and a depression mitigation substance of phenylethylamine were also found in this study. Samples treated with inoculants presented more biofunctional metabolites of organic acids, amino acids and phenolic acids than untreated samples. The Lactobacillus species covered over 98% after ensiling, and were mainly comprised by the L. acetotolerans, L. silagei, L. parafarraginis, L. buchneri and L. odoratitofui. As compared to the control silage, inoculation of L. plantarum increased the relative abundances of L. acetotolerans, L. buchneri and L. parafarraginis, and a considerable decline in the proportion of L. silagei was observed; whereas an obvious decrease in L. acetotolerans and increases in L. odoratitofui and L. farciminis were observed in the L. buchneri inoculated silage. Therefore, inoculation of L. plantarum and L. buchneri regulated the microbial composition and metabolome of the corn silage with different behaviors. The present results indicated that profiling of silage microbiome and metabolome might improve our current understanding of the biological process underlying silage formation.

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April 21, 2020  |  

Programmable mutually exclusive alternative splicing for generating RNA and protein diversity.

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.

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April 21, 2020  |  

Interspecies conservation of organisation and function between nonhomologous regional centromeres.

Despite the conserved essential function of centromeres, centromeric DNA itself is not conserved. The histone-H3 variant, CENP-A, is the epigenetic mark that specifies centromere identity. Paradoxically, CENP-A normally assembles on particular sequences at specific genomic locations. To gain insight into the specification of complex centromeres, here we take an evolutionary approach, fully assembling genomes and centromeres of related fission yeasts. Centromere domain organization, but not sequence, is conserved between Schizosaccharomyces pombe, S. octosporus and S. cryophilus with a central CENP-ACnp1 domain flanked by heterochromatic outer-repeat regions. Conserved syntenic clusters of tRNA genes and 5S rRNA genes occur across the centromeres of S. octosporus and S. cryophilus, suggesting conserved function. Interestingly, nonhomologous centromere central-core sequences from S. octosporus and S. cryophilus are recognized in S. pombe, resulting in cross-species establishment of CENP-ACnp1 chromatin and functional kinetochores. Therefore, despite the lack of sequence conservation, Schizosaccharomyces centromere DNA possesses intrinsic conserved properties that promote assembly of CENP-A chromatin.

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April 21, 2020  |  

Complete Genome Sequence of Sequevar 14M Ralstonia solanacearum Strain HA4-1 Reveals Novel Type III Effectors Acquired Through Horizontal Gene Transfer.

Ralstonia solanacearum, which causes bacterial wilt in a broad range of plants, is considered a “species complex” due to its significant genetic diversity. Recently, we have isolated a new R. solanacearum strain HA4-1 from Hong’an county in Hubei province of China and identified it being phylotype I, sequevar 14M (phylotype I-14M). Interestingly, we found that it can cause various disease symptoms among different potato genotypes and display different pathogenic behavior compared to a phylogenetically related strain, GMI1000. To dissect the pathogenic mechanisms of HA4-1, we sequenced its whole genome by combined sequencing technologies including Illumina HiSeq2000, PacBio RS II, and BAC-end sequencing. Genome assembly results revealed the presence of a conventional chromosome, a megaplasmid as well as a 143 kb plasmid in HA4-1. Comparative genome analysis between HA4-1 and GMI1000 shows high conservation of the general virulence factors such as secretion systems, motility, exopolysaccharides (EPS), and key regulatory factors, but significant variation in the repertoire and structure of type III effectors, which could be the determinants of their differential pathogenesis in certain potato species or genotypes. We have identified two novel type III effectors that were probably acquired through horizontal gene transfer (HGT). These novel R. solanacearum effectors display homology to several YopJ and XopAC family members. We named them as RipBR and RipBS. Notably, the copy of RipBR on the plasmid is a pseudogene, while the other on the megaplasmid is normal. For RipBS, there are three copies located in the megaplasmid and plasmid, respectively. Our results have not only enriched the genome information on R. solanacearum species complex by sequencing the first sequevar 14M strain and the largest plasmid reported in R. solanacearum to date but also revealed the variation in the repertoire of type III effectors. This will greatly contribute to the future studies on the pathogenic evolution, host adaptation, and interaction between R. solanacearum and potato.

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April 21, 2020  |  

A study of the extraordinarily strong and tough silk produced by bagworms.

Global ecological damage has heightened the demand for silk as ‘a structural material made from sustainable resources’. Scientists have earnestly searched for stronger and tougher silks. Bagworm silk might be a promising candidate considering its superior capacity to dangle a heavy weight, summed up by the weights of the larva and its house. However, detailed mechanical and structural studies on bagworm silks have been lacking. Herein, we show the superior potential of the silk produced by Japan’s largest bagworm, Eumeta variegata. This bagworm silk is extraordinarily strong and tough, and its tensile deformation behaviour is quite elastic. The outstanding mechanical property is the result of a highly ordered hierarchical structure, which remains unchanged until fracture. Our findings demonstrate how the hierarchical structure of silk proteins plays an important role in the mechanical property of silk fibres.

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