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September 22, 2019  |  

Complete genome sequence and analysis of the industrial Saccharomyces cerevisiae strain N85 used in Chinese rice wine production.

Chinese rice wine is a popular traditional alcoholic beverage in China, while its brewing processes have rarely been explored. We herein report the first gapless, near-finished genome sequence of the yeast strain Saccharomyces cerevisiae N85 for Chinese rice wine production. Several assembly methods were used to integrate Pacific Bioscience (PacBio) and Illumina sequencing data to achieve high-quality genome sequencing of the strain. The genome encodes more than 6,000 predicted proteins, and 238 long non-coding RNAs, which are validated by RNA-sequencing data. Moreover, our annotation predicts 171 novel genes that are not present in the reference S288c genome. We also identified 65,902 single nucleotide polymorphisms and small indels, many of which are located within genic regions. Dozens of larger copy-number variations and translocations were detected, mainly enriched in the subtelomeres, suggesting these regions may be related to genomic evolution. This study will serve as a milestone in studying of Chinese rice wine and related beverages in China and in other countries. It will help to develop more scientific and modern fermentation processes of Chinese rice wine, and explore metabolism pathways of desired and harmful components in Chinese rice wine to improve its taste and nutritional value.© The Author(s) 2018. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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September 22, 2019  |  

Enhancing the adaptability of the deep-sea bacterium Shewanella piezotolerans WP3 to high pressure and low temperature by experimental evolution under H2O2 stress.

Oxidative stresses commonly exist in natural environments, and microbes have developed a variety of defensive systems to counteract such events. Although increasing evidence has shown that high hydrostatic pressure (HHP) and low temperature (LT) induce antioxidant defense responses in cells, there is no direct evidence to prove the connection between antioxidant defense mechanisms and the adaptation of bacteria to HHP and LT. In this study, using the wild-type (WT) strain of a deep-sea bacterium, Shewanella piezotolerans WP3, as an ancestor, we obtained a mutant, OE100, with an enhanced antioxidant defense capacity by experimental evolution under H2O2 stress. Notably, OE100 exhibited better tolerance not only to H2O2 stress but also to HHP and LT (20 MPa and 4°C, respectively). Whole-genome sequencing identified a deletion mutation in the oxyR gene, which encodes the transcription factor that controls the oxidative stress response. Comparative transcriptome analysis showed that the genes associated with oxidative stress defense, anaerobic respiration, DNA repair, and the synthesis of flagella and bacteriophage were differentially expressed in OE100 compared with the WT at 20 MPa and 4°C. Genetic analysis of oxyR and ccpA2 indicated that the OxyR-regulated cytochrome c peroxidase CcpA2 significantly contributed to the adaptation of WP3 to HHP and LT. Taken together, these results confirmed the inherent relationship between antioxidant defense mechanisms and the adaptation of a benthic microorganism to HHP and LT.IMPORTANCE Oxidative stress exists in various niches, including the deep-sea ecosystem, which is an extreme environment with conditions of HHP and predominantly LT. Although previous studies have shown that HHP and LT induce antioxidant defense responses in cells, direct evidence to prove the connection between antioxidant defense mechanisms and the adaptation of bacteria to HHP and LT is lacking. In this work, using the deep-sea bacterium Shewanella piezotolerans WP3 as a model, we proved that enhancement of the adaptability of WP3 to HHP and LT can benefit from its antioxidant defense mechanism, which provided useful insight into the ecological roles of antioxidant genes in a benthic microorganism and contributed to an improved understanding of microbial adaptation strategies in deep-sea environments.

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September 22, 2019  |  

Rhizospheric microbial communities are driven by Panax ginseng at different growth stages and biocontrol bacteria alleviates replanting mortality

The cultivation of Panax plants is hindered by replanting problems, which may be caused by plant-driven changes in the soil microbial community. Inoculation with microbial antagonists may efficiently alleviate replanting issues. Through high-throughput sequencing, this study revealed that bacterial diversity decreased, whereas fungal diversity increased, in the rhizosphere soils of adult ginseng plants at the root growth stage under different ages. Few microbial community, such as Luteolibacter, Cytophagaceae, Luteibacter, Sphingomonas, Sphingomonadaceae, and Zygomycota, were observed; the relative abundance of microorganisms, namely, Brevundimonas, Enterobacteriaceae, Pandoraea, Cantharellales, Dendryphion, Fusarium, and Chytridiomycota, increased in the soils of adult ginseng plants compared with those in the soils of 2-year-old seedlings. Bacillus subtilis 50-1, a microbial antagonist against the pathogenic Fusarium oxysporum, was isolated through a dual culture technique. These bacteria acted with a biocontrol efficacy of 67.8%. The ginseng death rate and Fusarium abundance decreased by 63.3% and 46.1%, respectively, after inoculation with B. subtilis 50-1. Data revealed that microecological degradation could result from ginseng-driven changes in rhizospheric microbial communities; these changes are associated with the different ages and developmental stages of ginseng plants. Biocontrol using microbial antagonists alleviated the replanting problem.

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September 22, 2019  |  

Loss of stomach, loss of appetite? Sequencing of the ballan wrasse (Labrus bergylta) genome and intestinal transcriptomic profiling illuminate the evolution of loss of stomach function in fish.

The ballan wrasse (Labrus bergylta) belongs to a large teleost family containing more than 600 species showing several unique evolutionary traits such as lack of stomach and hermaphroditism. Agastric fish are found throughout the teleost phylogeny, in quite diverse and unrelated lineages, indicating stomach loss has occurred independently multiple times in the course of evolution. By assembling the ballan wrasse genome and transcriptome we aimed to determine the genetic basis for its digestive system function and appetite regulation. Among other, this knowledge will aid the formulation of aquaculture diets that meet the nutritional needs of agastric species.Long and short read sequencing technologies were combined to generate a ballan wrasse genome of 805 Mbp. Analysis of the genome and transcriptome assemblies confirmed the absence of genes that code for proteins involved in gastric function. The gene coding for the appetite stimulating protein ghrelin was also absent in wrasse. Gene synteny mapping identified several appetite-controlling genes and their paralogs previously undescribed in fish. Transcriptome profiling along the length of the intestine found a declining expression gradient from the anterior to the posterior, and a distinct expression profile in the hind gut.We showed gene loss has occurred for all known genes related to stomach function in the ballan wrasse, while the remaining functions of the digestive tract appear intact. The results also show appetite control in ballan wrasse has undergone substantial changes. The loss of ghrelin suggests that other genes, such as motilin, may play a ghrelin like role. The wrasse genome offers novel insight in to the evolutionary traits of this large family. As the stomach plays a major role in protein digestion, the lack of genes related to stomach digestion in wrasse suggests it requires formulated diets with higher levels of readily digestible protein than those for gastric species.

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September 22, 2019  |  

Predominant gut Lactobacillus murinus strain mediates anti-inflammaging effects in calorie-restricted mice.

Calorie restriction (CR), which has a potent anti-inflammaging effect, has been demonstrated to induce dramatic changes in the gut microbiota. Whether the modulated gut microbiota contributes to the attenuation of inflammation during CR is unknown, as are the members of the microbial community that may be key mediators of this process.Here, we report that a unique Lactobacillus-predominated microbial community was rapidly attained in mice within 2 weeks of CR, which decreased the levels of circulating microbial antigens and systemic inflammatory markers such as tumour necrosis factor alpha (TNF-a). Lactobacillus murinus CR147, an isolate in the most abundant operational taxonomic unit (OTU) enriched by CR, downregulated interleukin-8 production in TNF-a-stimulated Caco-2 cells and significantly increased the lifespan and the brood size of the nematode Caenorhabditis elegans. In gnotobiotic mice colonized with the gut microbiota from old mice, this strain decreased their intestinal permeability and serum endotoxin load, consequently attenuating the inflammation induced by the old microbiota.Our study demonstrated that a strain of Lactobacillus murinus was promoted in CR mice and causatively contributed to the attenuation of ageing-associated inflammation.

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September 22, 2019  |  

Microsatellite polymorphism in the endangered snail kite reveals a panmictic, low diversity population

Genetic structure and genetic diversity are key population characteristics that can inform conservation decisions, such as delineating management units or assessing potential risks for inbreeding depression. Evidence of genetic structuring or low genetic diversity in the critically endangered snail kite (Rostrhamus sociabilis plumbeus) would have implications for monitoring and planning decisions. Recent work on understanding connectivity across the snail kite range indicated that there is less dispersal between northern and southern parts of the current range, and that dispersal is shaped by individual habitat preference. We examine whether there is neutral genetic structure and the amount of genetic variation in the population by non-lethally sampling 235 nestlings from unique nests across the entire breeding range between 2013 and 2014. Data on 15 microsatellite revealed low diversity (e.g., Na?=?2.54, He?=?0.37) and range-wide panmixia based on AMOVA, Bayesian clustering, spatial autocorrelation, isolation by distance, and spatially explicit ordination analyses. Our results emphasize that long-term recovery goals and management strategies should be based on viewing snail kites as a single genetic population, despite evidence for non-random dispersal between wetlands over ecological time scales. These results also highlight the need to understand potential effects of low genetic diversity on population dynamics and viability of snail kites. More broadly, these results add to the growing evidence for potential discrepancies between dispersal and genetic patterns, emphasizing that care should be taken if using one to interpret the other, particularly for widely-ranging species.

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September 22, 2019  |  

Genomic analysis of oral Campylobacter concisus strains identified a potential bacterial molecular marker associated with active Crohn’s disease.

Campylobacter concisus is an oral bacterium that is associated with inflammatory bowel disease (IBD) including Crohn’s disease (CD) and ulcerative colitis (UC). C. concisus consists of two genomospecies (GS) and diverse strains. This study aimed to identify molecular markers to differentiate commensal and IBD-associated C. concisus strains. The genomes of 63 oral C. concisus strains isolated from patients with IBD and healthy controls were examined, of which 38 genomes were sequenced in this study. We identified a novel secreted enterotoxin B homologue, Csep1. The csep1 gene was found in 56% of GS2 C. concisus strains, presented in the plasmid pICON or the chromosome. A six-nucleotide insertion at the position 654-659?bp in csep1 (csep1-6bpi) was found. The presence of csep1-6bpi in oral C. concisus strains isolated from patients with active CD (47%, 7/15) was significantly higher than that in strains from healthy controls (0/29, P?=?0.0002), and the prevalence of csep1-6bpi positive C. concisus strains was significantly higher in patients with active CD (67%, 4/6) as compared to healthy controls (0/23, P?=?0.0006). Proteomics analysis detected the Csep1 protein. A csep1 gene hot spot in the chromosome of different C. concisus strains was found. The pICON plasmid was only found in GS2 strains isolated from the two relapsed CD patients with small bowel complications. This study reports a C. concisus molecular marker (csep1-6bpi) that is associated with active CD.

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September 22, 2019  |  

Developing collaborative works for faster progress on fungal respiratory infections in cystic fibrosis.

Cystic fibrosis (CF) is the major genetic inherited disease in Caucasian populations. The respiratory tract of CF patients displays a sticky viscous mucus, which allows for the entrapment of airborne bacteria and fungal spores and provides a suitable environment for growth of microorganisms, including numerous yeast and filamentous fungal species. As a consequence, respiratory infections are the major cause of morbidity and mortality in this clinical context. Although bacteria remain the most common agents of these infections, fungal respiratory infections have emerged as an important cause of disease. Therefore, the International Society for Human and Animal Mycology (ISHAM) has launched a working group on Fungal respiratory infections in Cystic Fibrosis (Fri-CF) in October 2006, which was subsequently approved by the European Confederation of Medical Mycology (ECMM). Meetings of this working group, comprising both clinicians and mycologists involved in the follow-up of CF patients, as well as basic scientists interested in the fungal species involved, provided the opportunity to initiate collaborative works aimed to improve our knowledge on these infections to assist clinicians in patient management. The current review highlights the outcomes of some of these collaborative works in clinical surveillance, pathogenesis and treatment, giving special emphasis to standardization of culture procedures, improvement of species identification methods including the development of nonculture-based diagnostic methods, microbiome studies and identification of new biological markers, and the description of genotyping studies aiming to differentiate transient carriage and chronic colonization of the airways. The review also reports on the breakthrough in sequencing the genomes of the main Scedosporium species as basis for a better understanding of the pathogenic mechanisms of these fungi, and discusses treatment options of infections caused by multidrug resistant microorganisms, such as Scedosporium and Lomentospora species and members of the Rasamsonia argillacea species complex.

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September 22, 2019  |  

Multi-omics approach identifies novel pathogen-derived prognostic biomarkers in patients with Pseudomonas aeruginosa bloodstream infection

Pseudomonas aeruginosa is a human pathogen that causes health-care associated blood stream infections (BSI). Although P. aeruginosa BSI are associated with high mortality rates, the clinical relevance of pathogen-derived prognostic biomarker to identify patients at risk for unfavorable outcome remains largely unexplored. We found novel pathogen-derived prognostic biomarker candidates by applying a multi-omics approach on a multicenter sepsis patient cohort. Multi-level Cox regression was used to investigate the relation between patient characteristics and pathogen features (2298 accessory genes, 1078 core protein levels, 107 parsimony-informative variations in reported virulence factors) with 30-day mortality. Our analysis revealed that presence of the helP gene encoding a putative DEAD-box helicase was independently associated with a fatal outcome (hazard ratio 2.01, p = 0.05). helP is located within a region related to the pathogenicity island PAPI-1 in close proximity to a pil gene cluster, which has been associated with horizontal gene transfer. Besides helP, elevated protein levels of the bacterial flagellum protein FliL (hazard ratio 3.44, p < 0.001) and of a bacterioferritin-like protein (hazard ratio 1.74, p = 0.003) increased the risk of death, while high protein levels of a putative aminotransferase were associated with an improved outcome (hazard ratio 0.12, p < 0.001). The prognostic potential of biomarker candidates and clinical factors was confirmed with different machine learning approaches using training and hold-out datasets. The helP genotype appeared the most attractive biomarker for clinical risk stratification due to its relevant predictive power and ease of detection.

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September 22, 2019  |  

The genome of the marine medaka Oryzias melastigma.

Marine medaka (Oryzias melastigma) is considered to be a useful fish model for marine and estuarine ecotoxicology studies and has good potential for field-based population genomics because of its geographical distribution in Asian estuarine and coastal areas. In this study, we present the first whole-genome draft of O. melastigma. The genome assembly consists of 8,602 scaffolds (N50 = 23.737 Mb) and a total genome length of 779.4 Mb. A total of 23,528 genes were predicted, and 12,670 gene families shared with three teleost species (Japanese medaka, mangrove killifish and zebrafish) were identified. Genome analyses revealed that the O. melastigma genome is highly heterozygous and contains a large number of repeat sequences. This assembly represents a useful genomic resource for fish scientists.© 2018 John Wiley & Sons Ltd.

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September 22, 2019  |  

Landscape of the genome and host cell response of Mycobacterium shigaense reveals pathogenic features.

A systems approach was used to explore the genome and transcriptome of Mycobacterium shigaense, a new opportunistic pathogen isolated from a patient with a skin infection, and the host response transcriptome was assessed using a macrophage infection model. The M. shigaense genome comprises 5,207,883?bp, with 67.2% G+C content and 5098 predicted coding genes. Evolutionarily, the bacterium belongs to a cluster in the phylogenetic tree along with three target opportunistic pathogenic strains, namely, M. avium, M. triplex and M. simiae. Potential virulence genes are indeed expressed by M. shigaense under culture conditions. Phenotypically, M. shigaense had similar infection and replication capacities in a macrophage model as the opportunistic species compared to M. tuberculosis. M. shigaense activated NF-?B, TNF, cytokines and chemokines in the host innate immune-related signaling pathways and elicited an early response shared with pathogenic bacilli except M. tuberculosis. M. shigaense upregulated specific host response genes such as TLR7, CCL4 and CXCL5. We performed an integrated and comparative analysis of M. shigaense. Multigroup comparison indicated certain differences with typical pathogenic bacilli in terms of gene features and the macrophage response.

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September 22, 2019  |  

A comprehensive understanding of the biocontrol potential of Bacillus velezensis LM2303 against Fusarium head blight.

Fusarium head blight (FHB) mainly caused by F. graminearum, always brings serious damage to wheat production worldwide. In this study, we found that strain LM2303 had strong antagonist activity against F. graminearum and significantly reduced disease severity of FHB with the control efficiency of 72.3% under field conditions. To gain a comprehensive understanding of the biocontrol potential of strain LM2303 against FHB, an integrated approach of genome mining and chemical analysis was employed. The whole genome of strain LM2303 was obtained and analyzed, showing the largest number of genes/gene clusters associated with biocontrol functions as compared with the known biocontrol strains (FZB42, M75, CAU B946). And strain LM2303 was accurately determined as a member of the B. velezensis clade using the phylogenomic analysis of single-copy core genes. Through genome mining, 13 biosynthetic gene clusters(BGCs) encoding secondary metabolites with biocontrol functions were identified, which were further confirmed through chemical analyses such as UHPLC-ESI-MS, including three antifungal metabolites (fengycin B, iturin A, and surfactin A), eight antibacterial metabolites (surfactin A, butirosin, plantazolicin and hydrolyzed plantazolicin, kijanimicin, bacilysin, difficidin, bacillaene A and bacillaene B, 7-o-malonyl macrolactin A and 7-o-succinyl macrolactin A), the siderophore bacillibactin, molybdenum cofactor and teichuronic acid. In addition, genes/gene clusters involved in plant colonization, plant growth promotion and induced systemic resistance were also found and analyzed, along with the corresponding metabolites. Finally, four different mechanisms of strain LM2303 involved in the biocontrol of FHB were putatively obtained. This work provides better insights into a mechanistic understanding of strain LM2303 in control of FHB, reinforcing the higher potential of this strain as a powerful biocontrol strain agent (BCA) for FHB control. The results also provide scientific reference and comparison for other biocontrol strains.

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September 22, 2019  |  

T-independent IFN? and B cells cooperate to prevent mortality associated with disseminated Chlamydia muridarum genital tract infection.

CD4 T cells and antibody are required for optimal acquired immunity to C. muridarum genital tract infection, and T cell-mediated IFN? production is necessary to clear infection in the absence of humoral immunity. However, the role of T cell-independent immune responses during primary infection remains unclear. We investigated this question by inoculating wild-type and immune-deficient mice with C. muridarum CM001, a clonal isolate capable of enhanced extragenital replication. Genital inoculation of wild-type mice resulted in transient dissemination to the lungs and spleen that then was rapidly cleared from these organs. However, CM001 genital infection proved lethal for STAT1-/- and IFNG-/- mice, where IFN? signaling is absent, and for Rag1-/- mice that lack T and B cells, but retain innate IFN? signaling. In contrast, B cell-deficient muMT mice that can generate a Th1 response, and T cell-deficient mice with intact B cell and innate IFN? signaling survived. These data collectively indicate that IFN? prevents lethal CM001 dissemination in the absence of T cells and suggests a B cell co-requirement. Adoptive transfer of convalescent immune sera, but not naïve IgM, to Rag1-/- mice infected with CM001 significantly increased survival time, while transfer of naïve B cells completely rescued Rag1-/- mice from CM001 lethality. Protection was associated with a significant reduction in the lung chlamydial burden of genitally infected mice. These data reveal an important cooperation between T-independent B cell responses and innate IFN? in chlamydial host defense, and suggest interactions between T-independent antibody and IFN? are essential for limiting extragenital dissemination. Copyright © 2018 American Society for Microbiology.

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September 22, 2019  |  

Tumor-specific mitochondrial DNA variants are rarely detected in cell-free DNA.

The use of blood-circulating cell-free DNA (cfDNA) as a “liquid biopsy” in oncology is being explored for its potential as a cancer biomarker. Mitochondria contain their own circular genomic entity (mitochondrial DNA, mtDNA), up to even thousands of copies per cell. The mutation rate of mtDNA is several orders of magnitude higher than that of the nuclear DNA. Tumor-specific variants have been identified in tumors along the entire mtDNA, and their number varies among and within tumors. The high mtDNA copy number per cell and the high mtDNA mutation rate make it worthwhile to explore the potential of tumor-specific cf-mtDNA variants as cancer marker in the blood of cancer patients. We used single-molecule real-time (SMRT) sequencing to profile the entire mtDNA of 19 tissue specimens (primary tumor and/or metastatic sites, and tumor-adjacent normal tissue) and 9 cfDNA samples, originating from 8 cancer patients (5 breast, 3 colon). For each patient, tumor-specific mtDNA variants were detected and traced in cfDNA by SMRT sequencing and/or digital PCR to explore their feasibility as cancer biomarker. As a reference, we measured other blood-circulating biomarkers for these patients, including driver mutations in nuclear-encoded cfDNA and cancer-antigen levels or circulating tumor cells. Four of the 24 (17%) tumor-specific mtDNA variants were detected in cfDNA, however at much lower allele frequencies compared to mutations in nuclear-encoded driver genes in the same samples. Also, extensive heterogeneity was observed among the heteroplasmic mtDNA variants present in an individual. We conclude that there is limited value in tracing tumor-specific mtDNA variants in blood-circulating cfDNA with the current methods available. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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September 22, 2019  |  

N6-methyladenine DNA modification in the human genome.

DNA N6-methyladenine (6mA) modification is the most prevalent DNA modification in prokaryotes, but whether it exists in human cells and whether it plays a role in human diseases remain enigmatic. Here, we showed that 6mA is extensively present in the human genome, and we cataloged 881,240 6mA sites accounting for ~0.051% of the total adenines. [G/C]AGG[C/T] was the most significantly associated motif with 6mA modification. 6mA sites were enriched in the coding regions and mark actively transcribed genes in human cells. DNA 6mA and N6-demethyladenine modification in the human genome were mediated by methyltransferase N6AMT1 and demethylase ALKBH1, respectively. The abundance of 6mA was significantly lower in cancers, accompanied by decreased N6AMT1 and increased ALKBH1 levels, and downregulation of 6mA modification levels promoted tumorigenesis. Collectively, our results demonstrate that DNA 6mA modification is extensively present in human cells and the decrease of genomic DNA 6mA promotes human tumorigenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

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