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April 21, 2020

Genome-wide systematic identification of methyltransferase recognition and modification patterns.

Genome-wide analysis of DNA methylation patterns using single molecule real-time DNA sequencing has boosted the number of publicly available methylomes. However, there is a lack of tools coupling methylation patterns and the corresponding methyltransferase genes. Here we demonstrate a high-throughput method for coupling methyltransferases with their respective motifs, using automated cloning and analysing the methyltransferases in vectors carrying a strain-specific cassette containing all potential target sites. To validate the method, we analyse the genomes of the thermophile Moorella thermoacetica and the mesophile Acetobacterium woodii, two acetogenic bacteria having substantially modified genomes with 12 methylation motifs and a total of 23 methyltransferase genes. Using our method, we characterize the 23 methyltransferases, assign motifs to the respective enzymes and verify activity for 11 of the 12 motifs.

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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

Strain-level metagenomic assignment and compositional estimation for long reads with MetaMaps.

Metagenomic sequence classification should be fast, accurate and information-rich. Emerging long-read sequencing technologies promise to improve the balance between these factors but most existing methods were designed for short reads. MetaMaps is a new method, specifically developed for long reads, capable of mapping a long-read metagenome to a comprehensive RefSeq database with >12,000 genomes in <16?GB or RAM on a laptop computer. Integrating approximate mapping with probabilistic scoring and EM-based estimation of sample composition, MetaMaps achieves >94% accuracy for species-level read assignment and r2?>?0.97 for the estimation of sample composition on both simulated and real data when the sample genomes or close relatives are present in the classification database. To address novel species and genera, which are comparatively harder to predict, MetaMaps outputs mapping locations and qualities for all classified reads, enabling functional studies (e.g. gene presence/absence) and detection of incongruities between sample and reference genomes.

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April 21, 2020

Extensive intraspecific gene order and gene structural variations in upland cotton cultivars.

Multiple cotton genomes (diploid and tetraploid) have been assembled. However, genomic variations between cultivars of allotetraploid upland cotton (Gossypium hirsutum L.), the most widely planted cotton species in the world, remain unexplored. Here, we use single-molecule long read and Hi-C sequencing technologies to assemble genomes of the two upland cotton cultivars TM-1 and zhongmiansuo24 (ZM24). Comparisons among TM-1 and ZM24 assemblies and the genomes of the diploid ancestors reveal a large amount of genetic variations. Among them, the top three longest structural variations are located on chromosome A08 of the tetraploid upland cotton, which account for ~30% total length of this chromosome. Haplotype analyses of the mapping population derived from these two cultivars and the germplasm panel show suppressed recombination rates in this region. This study provides additional genomic resources for the community, and the identified genetic variations, especially the reduced meiotic recombination on chromosome A08, will help future breeding.

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April 21, 2020

Urinary tract colonization is enhanced by a plasmid that regulates uropathogenic Acinetobacter baumannii chromosomal genes.

Multidrug resistant (MDR) Acinetobacter baumannii poses a growing threat to global health. Research on Acinetobacter pathogenesis has primarily focused on pneumonia and bloodstream infections, even though one in five A. baumannii strains are isolated from urinary sites. In this study, we highlight the role of A. baumannii as a uropathogen. We develop the first A. baumannii catheter-associated urinary tract infection (CAUTI) murine model using UPAB1, a recent MDR urinary isolate. UPAB1 carries the plasmid pAB5, a member of the family of large conjugative plasmids that represses the type VI secretion system (T6SS) in multiple Acinetobacter strains. pAB5 confers niche specificity, as its carriage improves UPAB1 survival in a CAUTI model and decreases virulence in a pneumonia model. Comparative proteomic and transcriptomic analyses show that pAB5 regulates the expression of multiple chromosomally-encoded virulence factors besides T6SS. Our results demonstrate that plasmids can impact bacterial infections by controlling the expression of chromosomal genes.

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April 21, 2020

Comprehensive identification of the full-length transcripts and alternative splicing related to the secondary metabolism pathways in the tea plant (Camellia sinensis).

Flavonoids, theanine and caffeine are the main secondary metabolites of the tea plant (Camellia sinensis), which account for the tea’s unique flavor quality and health benefits. The biosynthesis pathways of these metabolites have been extensively studied at the transcriptional level, but the regulatory mechanisms are still unclear. In this study, to explore the transcriptome diversity and complexity of tea plant, PacBio Iso-Seq and RNA-seq analysis were combined to obtain full-length transcripts and to profile the changes in gene expression during the leaf development. A total of 1,388,066 reads of insert (ROI) were generated with an average length of 1,762?bp, and more than 54% (755,716) of the ROIs were full-length non-chimeric (FLNC) reads. The Benchmarking Universal Single-Copy Orthologue (BUSCO) completeness was 92.7%. A total of 93,883 non-redundant transcripts were obtained, and 87,395 (93.1%) were new alternatively spliced isoforms. Meanwhile, 7,650 differential expression transcripts (DETs) were identified. A total of 28,980 alternative splicing (AS) events were predicted, including 1,297 differential AS (DAS) events. The transcript isoforms of the key genes involved in the flavonoid, theanine and caffeine biosynthesis pathways were characterized. Additionally, 5,777 fusion transcripts and 9,052 long non-coding RNAs (lncRNAs) were also predicted. Our results revealed that AS potentially plays a crucial role in the regulation of the secondary metabolism of the tea plant. These findings enhanced our understanding of the complexity of the secondary metabolic regulation of tea plants and provided a basis for the subsequent exploration of the regulatory mechanisms of flavonoid, theanine and caffeine biosynthesis in tea plants.

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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

Genome analysis of the rice coral Montipora capitata.

Corals comprise a biomineralizing cnidarian, dinoflagellate algal symbionts, and associated microbiome of prokaryotes and viruses. Ongoing efforts to conserve coral reefs by identifying the major stress response pathways and thereby laying the foundation to select resistant genotypes rely on a robust genomic foundation. Here we generated and analyzed a high quality long-read based ~886 Mbp nuclear genome assembly and transcriptome data from the dominant rice coral, Montipora capitata from Hawai’i. Our work provides insights into the architecture of coral genomes and shows how they differ in size and gene inventory, putatively due to population size variation. We describe a recent example of foreign gene acquisition via a bacterial gene transfer agent and illustrate the major pathways of stress response that can be used to predict regulatory components of the transcriptional networks in M. capitata. These genomic resources provide insights into the adaptive potential of these sessile, long-lived species in both natural and human influenced environments and facilitate functional and population genomic studies aimed at Hawaiian reef restoration and conservation.

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April 21, 2020

Within-host evolution of Helicobacter pylori shaped by niche-specific adaptation, intragastric migrations and selective sweeps.

The human pathogen Helicobacter pylori displays extensive genetic diversity. While H. pylori is known to evolve during infection, population dynamics inside the gastric environment have not been extensively investigated. Here we obtained gastric biopsies from multiple stomach regions of 16 H. pylori-infected adults, and analyze the genomes of 10 H. pylori isolates from each biopsy. Phylogenetic analyses suggest location-specific evolution and bacterial migration between gastric regions. Migration is significantly more frequent between the corpus and the fundus than with the antrum, suggesting that physiological differences between antral and oxyntic mucosa contribute to spatial partitioning of H. pylori populations. Associations between H. pylori gene polymorphisms and stomach niches suggest that chemotaxis, regulatory functions and outer membrane proteins contribute to specific adaptation to the antral and oxyntic mucosa. Moreover, we show that antibiotics can induce severe population bottlenecks and likely play a role in shaping the population structure of H. pylori.

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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

Arcobacter cryaerophilus Isolated From New Zealand Mussels Harbor a Putative Virulence Plasmid.

A wide range of Arcobacter species have been described from shellfish in various countries but their presence has not been investigated in Australasia, in which shellfish are a popular delicacy. Since several arcobacters are considered to be emerging pathogens, we undertook a small study to evaluate their presence in several different shellfish, including greenshell mussels, oysters, and abalone (paua) in New Zealand. Arcobacter cryaerophilus, a species associated with human gastroenteritis, was the only species isolated, from greenshell mussels. Whole-genome sequencing revealed a range of genomic traits in these strains that were known or associated virulence factors. Furthermore, we describe the first putative virulence plasmid in Arcobacter, containing lytic, immunoavoidance, adhesion, antibiotic resistance, and gene transfer traits, among others. Complete genome sequence determination using a combination of long- and short-read genome sequencing strategies, was needed to identify the plasmid, clearly identifying its benefits. The potential for plasmids to disseminate virulence traits among Arcobacter and other species warrants further consideration by researchers interested in the risks to public health from these organisms.

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April 21, 2020

Multi-platform discovery of haplotype-resolved structural variation in human genomes.

The incomplete identification of structural variants (SVs) from whole-genome sequencing data limits studies of human genetic diversity and disease association. Here, we apply a suite of long-read, short-read, strand-specific sequencing technologies, optical mapping, and variant discovery algorithms to comprehensively analyze three trios to define the full spectrum of human genetic variation in a haplotype-resolved manner. We identify 818,054 indel variants (<50?bp) and 27,622 SVs (=50?bp) per genome. We also discover 156 inversions per genome and 58 of the inversions intersect with the critical regions of recurrent microdeletion and microduplication syndromes. Taken together, our SV callsets represent a three to sevenfold increase in SV detection compared to most standard high-throughput sequencing studies, including those from the 1000 Genomes Project. The methods and the dataset presented serve as a gold standard for the scientific community allowing us to make recommendations for maximizing structural variation sensitivity for future genome sequencing studies.

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April 21, 2020

Occurrence and Characterization of mcr-1-Positive Escherichia coli Isolated From Food-Producing Animals in Poland, 2011-2016.

The emergence of plasmid-mediated colistin resistance (mcr genes) threatens the effectiveness of polymyxins, which are last-resort drugs to treat infections by multidrug- and carbapenem-resistant Gram-negative bacteria. Based on the occurrence of colistin resistance the aims of the study were to determine possible resistance mechanisms and then characterize the mcr-positive Escherichia coli. The research used material from the Polish national and EU harmonized antimicrobial resistance (AMR) monitoring programs. A total of 5,878 commensal E. coli from fecal samples of turkeys, chickens, pigs, and cattle collected in 2011-2016 were screened by minimum inhibitory concentration (MIC) determination for the presence of resistance to colistin (R) defined as R > 2 mg/L. Strains with MIC = 2 mg/L isolated in 2014-2016 were also included. A total of 128 isolates were obtained, and most (66.3%) had colistin MIC of 2 mg/L. PCR revealed mcr-1 in 80 (62.5%) isolates recovered from 61 turkeys, 11 broilers, 2 laying hens, 1 pig, and 1 bovine. No other mcr-type genes (including mcr-2 to -5) were detected. Whole-genome sequencing (WGS) of the mcr-1-positive isolates showed high diversity in the multi-locus sequence types (MLST) of E. coli, plasmid replicons, and AMR and virulence genes. Generally mcr-1.1 was detected on the same contig as the IncX4 (76.3%) and IncHI2 (6.3%) replicons. One isolate harbored mcr-1.1 on the chromosome. Various extended-spectrum beta-lactamase (blaSHV-12, blaCTX-M-1, blaCTX-M-15, blaTEM-30, blaTEM-52, and blaTEM-135) and quinolone resistance genes (qnrS1, qnrB19, and chromosomal gyrA, parC, and parE mutations) were present in the mcr-1.1-positive E. coli. A total of 49 sequence types (ST) were identified, ST354, ST359, ST48, and ST617 predominating. One isolate, identified as ST189, belonged to atypical enteropathogenic E. coli. Our findings show that mcr-1.1 has spread widely among production animals in Poland, particularly in turkeys and appears to be transferable mainly by IncX4 and IncHI2 plasmids spread across diverse E. coli lineages. Interestingly, most of these mcr-1-positive E. coli would remain undetected using phenotypic methods with the current epidemiological cut-off value (ECOFF). The appearance and spread of mcr-1 among various animals, but notably in turkeys, might be considered a food chain, and public health hazard.

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April 21, 2020

Platanus-allee is a de novo haplotype assembler enabling a comprehensive access to divergent heterozygous regions.

The ultimate goal for diploid genome determination is to completely decode homologous chromosomes independently, and several phasing programs from consensus sequences have been developed. These methods work well for lowly heterozygous genomes, but the manifold species have high heterozygosity. Additionally, there are highly divergent regions (HDRs), where the haplotype sequences differ considerably. Because HDRs are likely to direct various interesting biological phenomena, many genomic analysis targets fall within these regions. However, they cannot be accessed by existing phasing methods, and we have to adopt costly traditional methods. Here, we develop a de novo haplotype assembler, Platanus-allee ( http://platanus.bio.titech.ac.jp/platanus2 ), which initially constructs each haplotype sequence and then untangles the assembly graphs utilizing sequence links and synteny information. A comprehensive benchmark analysis reveals that Platanus-allee exhibits high recall and precision, particularly for HDRs. Using this approach, previously unknown HDRs are detected in the human genome, which may uncover novel aspects of genome variability.

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April 21, 2020

Complete Assembly of the Genome of an Acidovorax citrulli Strain Reveals a Naturally Occurring Plasmid in This Species.

Acidovorax citrulli is the causal agent of bacterial fruit blotch (BFB), a serious threat to cucurbit crop production worldwide. Based on genetic and phenotypic properties, A. citrulli strains are divided into two major groups: group I strains have been generally isolated from melon and other non-watermelon cucurbits, while group II strains are closely associated with watermelon. In a previous study, we reported the genome of the group I model strain, M6. At that time, the M6 genome was sequenced by MiSeq Illumina technology, with reads assembled into 139 contigs. Here, we report the assembly of the M6 genome following sequencing with PacBio technology. This approach not only allowed full assembly of the M6 genome, but it also revealed the occurrence of a ~53 kb plasmid. The M6 plasmid, named pACM6, was further confirmed by plasmid extraction, Southern-blot analysis of restricted fragments and obtention of M6-derivative cured strains. pACM6 occurs at low copy numbers (average of ~4.1 ± 1.3 chromosome equivalents) in A. citrulli M6 and contains 63 open reading frames (ORFs), most of which (55.6%) encoding hypothetical proteins. The plasmid contains several genes encoding type IV secretion components, and typical plasmid-borne genes involved in plasmid maintenance, replication and transfer. The plasmid also carries an operon encoding homologs of a Fic-VbhA toxin-antitoxin (TA) module. Transcriptome data from A. citrulli M6 revealed that, under the tested conditions, the genes encoding the components of this TA system are among the highest expressed genes in pACM6. Whether this TA module plays a role in pACM6 maintenance is still to be determined. Leaf infiltration and seed transmission assays revealed that, under tested conditions, the loss of pACM6 did not affect the virulence of A. citrulli M6. We also show that pACM6 or similar plasmids are present in several group I strains, but absent in all tested group II strains of A. citrulli.

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