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April 21, 2020

Methicillin-Resistant Staphylococcus aureus Blood Isolates Harboring a Novel Pseudo-staphylococcal Cassette Chromosome mec Element.

The aim of this work was to assess a novel pseudo-staphylococcal cassette chromosome mec (?SCCmec) element in methicillin-resistant Staphylococcus aureus (MRSA) blood isolates. Community-associated MRSA E16SA093 and healthcare-associated MRSA F17SA003 isolates were recovered from the blood specimens of patients with S. aureus bacteremia in 2016 and in 2017, respectively. Antimicrobial susceptibility was determined via the disk diffusion method, and SCCmec typing was conducted by multiplex polymerase chain reaction. Whole genome sequencing was carried out by single molecule real-time long-read sequencing. Both isolates belonged to sequence type 72 and agr-type I, and they were negative for Panton-Valentine leukocidin and toxic shock syndrome toxin. The spa-types of E16SA093 and F17SA003 were t324 and t2460, respectively. They had a SCCmec IV-like element devoid of the cassette chromosome recombinase (ccr) gene complex, designated as ?SCCmecE16SA093. The element was manufactured from SCCmec type IV and the deletion of the ccr gene complex and a 7.0- and 31.9-kb portion of each chromosome. The deficiency of the ccr gene complex in the SCCmec unit is likely resulting in mobility loss, which would be an adaptive evolutionary mechanism. The dissemination of this clone should be monitored closely.

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April 21, 2020

A Pathovar of Xanthomonas oryzae Infecting Wild Grasses Provides Insight Into the Evolution of Pathogenicity in Rice Agroecosystems

Xanthomonas oryzae (Xo) are critical rice pathogens. Virulent lineages from Africa and Asia and less virulent strains from the US have been well characterized. X. campestris pv. leersiae (Xcl), first described in 1957, causes bacterial streak on the perennial grass, Leersia hexandra, and is a close relative of Xo. L. hexandra, a member of the Poaceae, is highly similar to rice phylogenetically, is globally ubiquitous around rice paddies, and is a reservoir of pathogenic Xo. We used long read, single molecule, real time (SMRT) genome sequences of five strains of Xcl from Burkina Faso, China, Mali and Uganda to determine the genetic relatedness of this organism with Xo. Novel Transcription Activator-Like Effectors (TALEs) were discovered in all five strains of Xcl. Predicted TALE target sequences were identified in the L. perrieri genome and compared to rice susceptibility gene homologs. Pathogenicity screening on L. hexandra and diverse rice cultivars confirmed that Xcl are able to colonize rice and produce weak but not progressive symptoms. Overall, based on average nucleotide identity, type III effector repertoires and disease phenotype, we propose to rename Xcl to X. oryzae pv. leersiae (Xol) and use this parallel system to improve understanding of the evolution of bacterial pathogenicity in rice agroecosystems.

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April 21, 2020

Prediction of Host-Specific Genes by Pan-Genome Analyses of the Korean Ralstonia solanacearum Species Complex.

The soil-borne pathogenic Ralstonia solanacearum species complex (RSSC) is a group of plant pathogens that is economically destructive worldwide and has a broad host range, including various solanaceae plants, banana, ginger, sesame, and clove. Previously, Korean RSSC strains isolated from samples of potato bacterial wilt were grouped into four pathotypes based on virulence tests against potato, tomato, eggplant, and pepper. In this study, we sequenced the genomes of 25 Korean RSSC strains selected based on these pathotypes. The newly sequenced genomes were analyzed to determine the phylogenetic relationships between the strains with average nucleotide identity values, and structurally compared via multiple genome alignment using Mauve software. To identify candidate genes responsible for the host specificity of the pathotypes, functional genome comparisons were conducted by analyzing pan-genome orthologous group (POG) and type III secretion system effectors (T3es). POG analyses revealed that a total of 128 genes were shared only in tomato-non-pathogenic strains, 8 genes in tomato-pathogenic strains, 5 genes in eggplant-non-pathogenic strains, 7 genes in eggplant-pathogenic strains, 1 gene in pepper-non-pathogenic strains, and 34 genes in pepper-pathogenic strains. When we analyzed T3es, three host-specific effectors were predicted: RipS3 (SKWP3) and RipH3 (HLK3) were found only in tomato-pathogenic strains, and RipAC (PopC) were found only in eggplant-pathogenic strains. Overall, we identified host-specific genes and effectors that may be responsible for virulence functions in RSSC in silico. The expected characters of those genes suggest that the host range of RSSC is determined by the comprehensive actions of various virulence factors, including effectors, secretion systems, and metabolic enzymes.

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April 21, 2020

Closing the Yield Gap for Cannabis: A Meta-Analysis of Factors Determining Cannabis Yield.

Until recently, the commercial production of Cannabis sativa was restricted to varieties that yielded high-quality fiber while producing low levels of the psychoactive cannabinoid tetrahydrocannabinol (THC). In the last few years, a number of jurisdictions have legalized the production of medical and/or recreational cannabis with higher levels of THC, and other jurisdictions seem poised to follow suit. Consequently, demand for industrial-scale production of high yield cannabis with consistent cannabinoid profiles is expected to increase. In this paper we highlight that currently, projected annual production of cannabis is based largely on facility size, not yield per square meter. This meta-analysis of cannabis yields reported in scientific literature aimed to identify the main factors contributing to cannabis yield per plant, per square meter, and per W of lighting electricity. In line with previous research we found that variety, plant density, light intensity and fertilization influence cannabis yield and cannabinoid content; we also identified pot size, light type and duration of the flowering period as predictors of yield and THC accumulation. We provide insight into the critical role of light intensity, quality, and photoperiod in determining cannabis yields, with particular focus on the potential for light-emitting diodes (LEDs) to improve growth and reduce energy requirements. We propose that the vast amount of genomics data currently available for cannabis can be used to better understand the effect of genotype on yield. Finally, we describe diversification that is likely to emerge in cannabis growing systems and examine the potential role of plant-growth promoting rhizobacteria (PGPR) for growth promotion, regulation of cannabinoid biosynthesis, and biocontrol.

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April 21, 2020

Whole Genome Analysis of Lactobacillus plantarum Strains Isolated From Kimchi and Determination of Probiotic Properties to Treat Mucosal Infections by Candida albicans and Gardnerella vaginalis.

Three Lactobacillus plantarum strains ATG-K2, ATG-K6, and ATG-K8 were isolated from Kimchi, a Korean traditional fermented food, and their probiotic potentials were examined. All three strains were free of antibiotic resistance, hemolysis, and biogenic amine production and therefore assumed to be safe, as supported by whole genome analyses. These strains demonstrated several basic probiotic functions including a wide range of antibacterial activity, bile salt hydrolase activity, hydrogen peroxide production, and heat resistance at 70°C for 60 s. Further studies of antimicrobial activities against Candida albicans and Gardnerella vaginalis revealed growth inhibitory effects from culture supernatants, coaggregation effects, and killing effects of the three probiotic strains, with better efficacy toward C. albicans. In vitro treatment of bacterial lysates of the probiotic strains to the RAW264.7 murine macrophage cell line resulted in innate immunity enhancement via IL-6 and TNF-a production without lipopolysaccharide (LPS) treatment and anti-inflammatory effects via significantly increased production of IL-10 when co-treated with LPS. However, the degree of probiotic effect was different for each strain as the highest TNF-a and the lowest IL-10 production by the RAW264.7 cell were observed in the K8 lysate treated group compared to the K2 and K6 lysate treated groups, which may be related to genomic differences such as chromosome size (K2: 3,034,884 bp, K6: 3,205,672 bp, K8: 3,221,272 bp), plasmid numbers (K2: 3, K6 and K8: 1), or total gene numbers (K2: 3,114, K6: 3,178, K8: 3,186). Although more correlative inspections to connect genomic information and biological functions are needed, genomic analyses of the three strains revealed distinct genomic compositions of each strain. Also, this finding suggests genome level analysis may be required to accurately identify microorganisms. Nevertheless, L. plantarum ATG-K2, ATG-K6, and ATG-K8 demonstrated their potential as probiotics for mucosal health improvement in both microbial and immunological contexts.

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April 21, 2020

Long-Read Sequencing Emerging in Medical Genetics

The wide implementation of next-generation sequencing (NGS) technologies has revolutionized the field of medical genetics. However, the short read lengths of currently used sequencing approaches pose a limitation for identification of structural variants, sequencing repetitive regions, phasing alleles and distinguishing highly homologous genomic regions. These limitations may significantly contribute to the diagnostic gap in patients with genetic disorders who have undergone standard NGS, like whole exome or even genome sequencing. Now, the emerging long-read sequencing (LRS) technologies may offer improvements in the characterization of genetic variation and regions that are difficult to assess with the currently prevailing NGS approaches. LRS has so far mainly been used to investigate genetic disorders with previously known or strongly suspected disease loci. While these targeted approaches already show the potential of LRS, it remains to be seen whether LRS technologies can soon enable true whole genome sequencing routinely. Ultimately, this could allow the de novo assembly of individual whole genomes used as a generic test for genetic disorders. In this article, we summarize the current LRS-based research on human genetic disorders and discuss the potential of these technologies to facilitate the next major advancements in medical genetics.

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April 21, 2020

Comparative transcriptome analyses of genes involved in sulforaphane metabolism at different treatment in Chinese kale using full-length transcriptome sequencing.

Sulforaphane is a natural isothiocyanate available from cruciferous vegetables with multiple characteristics including antioxidant, antitumor and anti-inflammatory effect. Single-molecule real-time (SMRT) sequencing has been used for long-read de novo assembly of plant genome. Here, we investigated the molecular mechanism related to glucosinolates biosynthesis in Chinese kale using combined NGS and SMRT sequencing.SMRT sequencing produced 185,134 unigenes, higher than 129,325 in next-generation sequencing (NGS). NaCl (75?mM), methyl jasmonate (MeJA, 40?µM), selenate (Se, sodium selenite 100?µM), and brassinolide (BR, 1.5?µM) treatment induced 6893, 13,287, 13,659 and 11,041 differentially expressed genes (DEGs) in Chinese kale seedlings comparing with control. These genes were associated with pathways of glucosinolates biosynthesis, including phenylalanine, tyrosine and tryptophan biosynthesis, cysteine and methionine metabolism, and glucosinolate biosynthesis. We found NaCl decreased sulforaphane and glucosinolates (indolic and aliphatic) contents and downregulated expression of cytochrome P45083b1 (CYP83b1), S-alkyl-thiohydroximatelyase or carbon-sulfur lyase (SUR1) and UDP-glycosyltransferase 74B1 (UGT74b1). MeJA increased sulforaphane and glucosinolates contents and upregulated the expression of CYP83b1, SUR1 and UGT74b1; Se increased sulforaphane; BR increased expression of CYP83b1, SUR1 and UGT74b1, and increased glucosinolates contents. The desulfoglucosinolate sulfotransferases ST5a_b_c were decreased by all treatments.We confirmed that NaCl inhibited the biosynthesis of both indolic and aliphatic glucosinolates, while MeJA and BR increased them. MeJA and BR treatments, conferred the biosynthesis of glucosinolates, and Se and MeJA contributed to sulforaphane in Chinese kale via regulating the expression of CYP83b1, SUR1 and UGT74b1.

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April 21, 2020

Genomics-driven discovery of a biosynthetic gene cluster required for the synthesis of BII-Rafflesfungin from the fungus Phoma sp. F3723.

Phomafungin is a recently reported broad spectrum antifungal compound but its biosynthetic pathway is unknown. We combed publicly available Phoma genomes but failed to find any putative biosynthetic gene cluster that could account for its biosynthesis.Therefore, we sequenced the genome of one of our Phoma strains (F3723) previously identified as having antifungal activity in a high-throughput screen. We found a biosynthetic gene cluster that was predicted to synthesize a cyclic lipodepsipeptide that differs in the amino acid composition compared to Phomafungin. Antifungal activity guided isolation yielded a new compound, BII-Rafflesfungin, the structure of which was determined.We describe the NRPS-t1PKS cluster ‘BIIRfg’ compatible with the synthesis of the cyclic lipodepsipeptide BII-Rafflesfungin [HMHDA-L-Ala-L-Glu-L-Asn-L-Ser-L-Ser-D-Ser-D-allo-Thr-Gly]. We report new Stachelhaus codes for Ala, Glu, Asn, Ser, Thr, and Gly. We propose a mechanism for BII-Rafflesfungin biosynthesis, which involves the formation of the lipid part by BIIRfg_PKS followed by activation and transfer of the lipid chain by a predicted AMP-ligase on to the first PCP domain of the BIIRfg_NRPS gene.

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April 21, 2020

Reconstruction of the full-length transcriptome atlas using PacBio Iso-Seq provides insight into the alternative splicing in Gossypium australe.

Gossypium australe F. Mueller (2n?=?2x?=?26, G2 genome) possesses valuable characteristics. For example, the delayed gland morphogenesis trait causes cottonseed protein and oil to be edible while retaining resistance to biotic stress. However, the lack of gene sequences and their alternative splicing (AS) in G. australe remain unclear, hindering to explore species-specific biological morphogenesis.Here, we report the first sequencing of the full-length transcriptome of the Australian wild cotton species, G. australe, using Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq) from the pooled cDNA of ten tissues to identify transcript loci and splice isoforms. We reconstructed the G. australe full-length transcriptome and identified 25,246 genes, 86 pre-miRNAs and 1468 lncRNAs. Most genes (12,832, 50.83%) exhibited two or more isoforms, suggesting a high degree of transcriptome complexity in G. australe. A total of 31,448 AS events in five major types were found among the 9944 gene loci. Among these five major types, intron retention was the most frequent, accounting for 68.85% of AS events. 29,718 polyadenylation sites were detected from 14,536 genes, 7900 of which have alternative polyadenylation sites (APA). In addition, based on our AS events annotations, RNA-Seq short reads from germinating seeds showed that differential expression of these events occurred during seed germination. Ten AS events that were randomly selected were further confirmed by RT-PCR amplification in leaf and germinating seeds.The reconstructed gene sequences and their AS in G. australe would provide information for exploring beneficial characteristics in G. australe.

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April 21, 2020

Divergent evolutionary trajectories following speciation in two ectoparasitic honey bee mites.

Multispecies host-parasite evolution is common, but how parasites evolve after speciating remains poorly understood. Shared evolutionary history and physiology may propel species along similar evolutionary trajectories whereas pursuing different strategies can reduce competition. We test these scenarios in the economically important association between honey bees and ectoparasitic mites by sequencing the genomes of the sister mite species Varroa destructor and Varroa jacobsoni. These genomes were closely related, with 99.7% sequence identity. Among the 9,628 orthologous genes, 4.8% showed signs of positive selection in at least one species. Divergent selective trajectories were discovered in conserved chemosensory gene families (IGR, SNMP), and Halloween genes (CYP) involved in moulting and reproduction. However, there was little overlap in these gene sets and associated GO terms, indicating different selective regimes operating on each of the parasites. Based on our findings, we suggest that species-specific strategies may be needed to combat evolving parasite communities. © The Author(s) 2019.

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April 21, 2020

Characterization of mcr-1-Harboring Plasmids from Pan Drug-Resistant Escherichia coli Strains Isolated from Retail Raw Chicken in South Korea

A number of studies from different countries have characterized mcr-1-harboring plasmids isolated from food; however, nothing has been reported about it in South Korea. In this study, we report the characterization of mcr-1 plasmids from pan drug-resistant (PDR) Escherichia coli strains isolated from retail food in the country. Colistin-resistant E. coli strains were isolated from retail raw chicken, and PCR was carried out to detect the mcr-1 gene. Whole genome sequencing of the mcr-1-positive strains was performed for further characterization. The results of whole genome sequencing revealed that all mcr-1 plasmids belonged to the IncI2 type. In addition to the mcr-1 plasmids, all of the isolates also carried additional plasmids possessing multiple antibiotic resistance genes, and the PDR was mediated by resistant plasmids except for fluoroquinolone resistance resulting from mutations in gyrA and parC. Interestingly, the mcr-1 plasmids were transferred by conjugation to other pathogenic strains including enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), Salmonella, and Klebsiella at the frequencies of 10−3−10−6, 10−2−10−5, 10−4−10−5, 10−4−10−6, and 10−5−10−6, respectively. The results showed that mcr-1 plasmids can be easily transmitted to pathogenic bacteria by conjugation.

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April 21, 2020

Improved annotation of the domestic pig genome through integration of Iso-Seq and RNA-seq data.

Our understanding of the pig transcriptome is limited. RNA transcript diversity among nine tissues was assessed using poly(A) selected single-molecule long-read isoform sequencing (Iso-seq) and Illumina RNA sequencing (RNA-seq) from a single White cross-bred pig. Across tissues, a total of 67,746 unique transcripts were observed, including 60.5% predicted protein-coding, 36.2% long non-coding RNA and 3.3% nonsense-mediated decay transcripts. On average, 90% of the splice junctions were supported by RNA-seq within tissue. A large proportion (80%) represented novel transcripts, mostly produced by known protein-coding genes (70%), while 17% corresponded to novel genes. On average, four transcripts per known gene (tpg) were identified; an increase over current EBI (1.9 tpg) and NCBI (2.9 tpg) annotations and closer to the number reported in human genome (4.2 tpg). Our new pig genome annotation extended more than 6000 known gene borders (5′ end extension, 3′ end extension, or both) compared to EBI or NCBI annotations. We validated a large proportion of these extensions by independent pig poly(A) selected 3′-RNA-seq data, or human FANTOM5 Cap Analysis of Gene Expression data. Further, we detected 10,465 novel genes (81% non-coding) not reported in current pig genome annotations. More than 80% of these novel genes had transcripts detected in >?1 tissue. In addition, more than 80% of novel intergenic genes with at least one transcript detected in liver tissue had H3K4me3 or H3K36me3 peaks mapping to their promoter and gene body, respectively, in independent liver chromatin immunoprecipitation data. These validated results show significant improvement over current pig genome annotations.

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April 21, 2020

A hybrid de novo genome assembly of the honeybee, Apis mellifera, with chromosome-length scaffolds.

The ability to generate long sequencing reads and access long-range linkage information is revolutionizing the quality and completeness of genome assemblies. Here we use a hybrid approach that combines data from four genome sequencing and mapping technologies to generate a new genome assembly of the honeybee Apis mellifera. We first generated contigs based on PacBio sequencing libraries, which were then merged with linked-read 10x Chromium data followed by scaffolding using a BioNano optical genome map and a Hi-C chromatin interaction map, complemented by a genetic linkage map.Each of the assembly steps reduced the number of gaps and incorporated a substantial amount of additional sequence into scaffolds. The new assembly (Amel_HAv3) is significantly more contiguous and complete than the previous one (Amel_4.5), based mainly on Sanger sequencing reads. N50 of contigs is 120-fold higher (5.381 Mbp compared to 0.053 Mbp) and we anchor >?98% of the sequence to chromosomes. All of the 16 chromosomes are represented as single scaffolds with an average of three sequence gaps per chromosome. The improvements are largely due to the inclusion of repetitive sequence that was unplaced in previous assemblies. In particular, our assembly is highly contiguous across centromeres and telomeres and includes hundreds of AvaI and AluI repeats associated with these features.The improved assembly will be of utility for refining gene models, studying genome function, mapping functional genetic variation, identification of structural variants, and comparative genomics.

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April 21, 2020

Hybrid-Transcriptome Sequencing and Associated Metabolite Analysis Reveal Putative Genes Involved in Flower Color Difference in Rose Mutants.

Gene mutation is a common phenomenon in nature that often leads to phenotype differences, such as the variations in flower color that frequently occur in roses. With the aim of revealing the genomic information and inner mechanisms, the differences in the levels of both transcription and secondary metabolism between a pair of natural rose mutants were investigated by using hybrid RNA-sequencing and metabolite analysis. Metabolite analysis showed that glycosylated derivatives of pelargonidin, e.g., pelargonidin 3,5 diglucoside and pelargonidin 3-glucoside, which were not detected in white flowers (Rosa ‘Whilte Mrago Koster’), constituted the major pigments in pink flowers. Conversely, the flavonol contents of petal, such as kaempferol-3-glucoside, quercetin 3-glucoside, and rutin, were higher in white flowers. Hybrid RNA-sequencing obtained a total of 107,280 full-length transcripts in rose petal which were annotated in major databases. Differentially expressed gene (DEG) analysis showed that the expression of genes involved in the flavonoid biosynthesis pathway was significantly different, e.g., CHS, FLS, DFR, LDOX, which was verified by qRT-PCR during flowering. Additionally, two MYB transcription factors were found and named RmMYBAN2 and RmMYBPA1, and their expression patterns during flowering were also analyzed. These findings indicate that these genes may be involved in the flower color difference in the rose mutants, and competition between anthocyanin and flavonol biosynthesis is a primary cause of flower color variation, with its regulation reflected by transcriptional and secondary metabolite levels.

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April 21, 2020

Large Plasmid Complement Resolved: Complete Genome Sequencing of Lactobacillus plantarum MF1298, a Candidate Probiotic Strain Associated with Unfavorable Effect.

Considerable attention has been given to the species Lactobacillus plantarum regarding its probiotic potential. L. plantarum strains have shown health benefits in several studies, and even nonstrain-specific claims are allowed in certain markets. L. plantarum strain MF1298 was considered a candidate probiotic, demonstrating in vitro probiotic properties and the ability to survive passage through the human intestinal tract. However, the strain showed an unfavorable effect on symptoms in subjects with irritable bowel syndrome in a clinical trial. The properties and the genome of this strain are thus of general interest. Obtaining the complete genome of strain MF1298 proved difficult due to its large plasmid complement. Here, we exploit a combination of sequencing approaches to obtain the complete chromosome and plasmid assemblies of MF1298. The Oxford Nanopore Technologies MinION long-read sequencer was particularly useful in resolving the unusually large number of plasmids in the strain, 14 in total. The complete genome sequence of 3,576,440 basepairs contains 3272 protein-encoding genes, of which 315 are located on plasmids. Few unique regions were found in comparison with other L. plantarum genomes. Notably, however, one of the plasmids contains genes related to vitamin B12 (cobalamin) turnover and genes encoding bacterial reverse transcriptases, features not previously reported for L. plantarum. The extensive plasmid information will be important for future studies with this strain.

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