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September 22, 2019

Vegetative compatibility groups partition variation in the virulence of Verticillium dahliae on strawberry.

Verticillium dahliae infection of strawberry (Fragaria x ananassa) is a major cause of disease-induced wilting in soil-grown strawberries across the world. To understand what components of the pathogen are affecting disease expression, the presence of the known effector VdAve1 was screened in a sample of Verticillium dahliae isolates. Isolates from strawberry were found to contain VdAve1 and were divided into two major clades, based upon their vegetative compatibility groups (VCG); no UK strawberry isolates contained VdAve1. VC clade was strongly related to their virulence levels. VdAve1-containing isolates pathogenic on strawberry were found in both clades, in contrast to some recently published findings. On strawberry, VdAve1-containing isolates had significantly higher virulence during early infection, which diminished in significance as the infection progressed. Transformation of a virulent non-VdAve1 containing isolate, with VdAve1 was found neither to increase nor decrease virulence when inoculated on a susceptible strawberry cultivar. There are therefore virulence factors that are epistatic to VdAve1 and potentially multiple independent routes to high virulence on strawberry in V. dahliae lineages. Genome sequencing a subset of isolates across the two VCGs revealed that isolates were differentiated at the whole genome level and contained multiple changes in putative effector content, indicating that different clonal VCGs may have evolved different strategies for infecting strawberry, leading to different virulence levels in pathogenicity tests. It is therefore important to consider both clonal lineage and effector complement as the adaptive potential of each lineage will differ, even if they contain the same race determining effector.

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September 22, 2019

Multidrug-resistant Escherichia albertii: Co-occurrence of ß-lactamase and MCR-1 encoding genes.

Escherichia albertii is an emerging member of the Enterobacteriaceae causing human and animal enteric infections. Antimicrobial resistance among enteropathogens has been reported to be increasing in the past years. The purpose of this study was to investigate antibiotic resistance and resistance genes in E. albertii isolated from Zigong city, Sichuan province, China. The susceptibility to 21 antimicrobial agents was determined by Kirby-Bauer disk diffusion method. The highest prevalence was tetracycline resistance with a rate of 62.7%, followed by resistance to nalidixic acid and streptomycin with a rate of 56.9 and 51.0%, respectively. All isolates were sensitive or intermediate susceptible to imipenem, meropenem, amoxicillin-clavulanic acid, and levofloxacin. Among 51 E. albertii isolates, 15 were extended-spectrum ß-lactamase-producing as confirmed by the double disk test. The main ß-lactamase gene groups, i.e., blaTEM, blaSHV, and blaCTX-M, were detected in17, 20, and 22 isolates, respectively. Furthermore, four colistin-resistant isolates with minimum inhibitory concentrations of 8 mg/L were identified. The colistin-resistant isolates all harbored mcr-1 and blaCTX-M-55. Genome sequencing showed that E. albertii strain SP140150 carried mcr-1 and blaCTX-M-55 in two different plasmids. This study provided significant information regarding antibiotic resistance profiles and identified the co-occurrence of ß-lactamase and MCR-1 encoding genes in E. albertii isolates.

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September 22, 2019

Analyzing AbrB-knockout effects through genome and transcriptome sequencing of Bacillus licheniformis DW2.

As an industrial bacterium, Bacillus licheniformis DW2 produces bacitracin which is an important antibiotic for many pathogenic microorganisms. Our previous study showed AbrB-knockout could significantly increase the production of bacitracin. Accordingly, it was meaningful to understand its genome features, expression differences between wild and AbrB-knockout (?AbrB) strains, and the regulation of bacitracin biosynthesis. Here, we sequenced, de novo assembled and annotated its genome, and also sequenced the transcriptomes in three growth phases. The genome of DW2 contained a DNA molecule of 4,468,952 bp with 45.93% GC content and 4,717 protein coding genes. The transcriptome reads were mapped to the assembled genome, and obtained 4,102~4,536 expressed genes from different samples. We investigated transcription changes in B. licheniformis DW2 and showed that ?AbrB caused hundreds of genes up-regulation and down-regulation in different growth phases. We identified a complete bacitracin synthetase gene cluster, including the location and length of bacABC, bcrABC, and bacT, as well as their arrangement. The gene cluster bcrABC were significantly up-regulated in ?AbrB strain, which supported the hypothesis in previous study of bcrABC transporting bacitracin out of the cell to avoid self-intoxication, and was consistent with the previous experimental result that ?AbrB could yield more bacitracin. This study provided a high quality reference genome for B. licheniformis DW2, and the transcriptome data depicted global alterations across two strains and three phases offered an understanding of AbrB regulation and bacitracin biosynthesis through gene expression.

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September 22, 2019

Assembly and analysis of a qingke reference genome demonstrate its close genetic relation to modern cultivated barley.

Qingke, the local name of hulless barley in the Tibetan Plateau, is a staple food for Tibetans. The availability of its reference genome sequences could be useful for studies on breeding and molecular evolution. Taking advantage of the third-generation sequencer (PacBio), we de novo assembled a 4.84-Gb genome sequence of qingke, cv. Zangqing320 and anchored a 4.59-Gb sequence to seven chromosomes. Of the 46,787 annotated ‘high-confidence’ genes, 31 564 were validated by RNA-sequencing data of 39 wild and cultivated barley genotypes with wide genetic diversity, and the results were also confirmed by nonredundant protein database from NCBI. As some gaps in the reference genome of Morex were covered in the reference genome of Zangqing320 by PacBio reads, we believe that the Zangqing320 genome provides the useful supplements for the Morex genome. Using the qingke genome as a reference, we conducted a genome comparison, revealing a close genetic relationship between a hulled barley (cv. Morex) and a hulless barley (cv. Zangqing320), which is strongly supported by the low-diversity regions in the two genomes. Considering the origin of Morex from its breeding pedigree, we then demonstrated a close genomic relationship between modern cultivated barley and qingke. Given this genomic relationship and the large genetic diversity between qingke and modern cultivated barley, we propose that qingke could provide elite genes for barley improvement.© 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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September 22, 2019

Functional genomics of lipid metabolism in the oleaginous yeast Rhodosporidium toruloides.

The basidiomycete yeast Rhodosporidium toruloides (also known as Rhodotorula toruloides) accumulates high concentrations of lipids and carotenoids from diverse carbon sources. It has great potential as a model for the cellular biology of lipid droplets and for sustainable chemical production. We developed a method for high-throughput genetics (RB-TDNAseq), using sequence-barcoded Agrobacterium tumefaciens T-DNA insertions. We identified 1,337 putative essential genes with low T-DNA insertion rates. We functionally profiled genes required for fatty acid catabolism and lipid accumulation, validating results with 35 targeted deletion strains. We identified a high-confidence set of 150 genes affecting lipid accumulation, including genes with predicted function in signaling cascades, gene expression, protein modification and vesicular trafficking, autophagy, amino acid synthesis and tRNA modification, and genes of unknown function. These results greatly advance our understanding of lipid metabolism in this oleaginous species and demonstrate a general approach for barcoded mutagenesis that should enable functional genomics in diverse fungi.

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September 22, 2019

Biodegradation of di-n-butyl phthalate (DBP) by a novel endophytic Bacillus megaterium strain YJB3.

Phthalic acid esters (PAEs) are a group of recalcitrant and hazardous organic compounds that pose a great threat to both ecosystem and human beings. A novel endophytic strain YJB3 that could utilize a wide range of PAEs as the sole carbon and energy sources for cell growth was isolated from Canna indica root tissue. It was identified as Bacillus megaterium based on morphological characteristics and 16S rDNA sequence homology analysis. The degradation capability of the strain YJB3 was investigated by incubation in mineral salt medium containing di-n-butyl-phthalate (DBP), one of important PAEs under different environmental conditions, showing 82.5% of the DBP removal in 5days of incubation under the optimum conditions (acetate 1.2g·L-1, inocula 1.8%, and temperature 34.2°C) achieved by two-step sequential optimization technologies. The DBP metabolites including mono-butyl phthalate (MBP), phthalic acid (PA), protocatechuic acid (PCA), etc. were determined by GC-MS. The PCA catabolic genes responsible for the aromatic ring cleavage of PCA in the strain YJB3 were excavated by whole-genome sequencing. Thus, a degradation pathway of DBP by the strain YJB3 was proposed that MBP was formed, followed by PA, and then the intermediates were further utilized till complete degradation. To our knowledge, this is the first study to show the biodegradation of PAEs using endophyte. The results in the present study suggest that the strain YJB3 is greatly promising to act as a competent inoculum in removal of PAEs in both soils and crops. Copyright © 2017 Elsevier B.V. All rights reserved.

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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

Identification of the streptothricin and tunicamycin biosynthetic gene clusters by genome mining in Streptomyces sp. strain fd1-xmd.

The genus Streptomyces have been highly regarded for their important source of natural products. Combined with the technology of genome sequencing and mining, we could identify the active ingredients from fermentation broth quickly. Here, we report on Streptomyces sp. strain fd1-xmd, which was isolated from a soil sample collected in Shanghai. Interestingly, the fermentation broth derived from this strain demonstrated broad-spectrum antimicrobial activity against gram-positive bacteria, gram-negative bacteria, and eukaryotes. To identify the antimicrobial substances and their biosynthetic gene clusters, we sequenced the fd1-xmd strain and obtained a genome 7,929,999 bp in length. The average GC content of the chromosome was 72.5 mol%. Knockout experiments demonstrated that out of eight biosynthetic gene clusters we could identify, two are responsible for the biosynthesis of the antibiotics streptothricin (ST) and tunicamycin (TM). The ST biosynthetic gene cluster from fd1-xmd was verified via successful heterologous expression in Streptomyces coelicolor M1146. ST production had a yield of up to 0.5 g/L after the optimization of culture conditions. This study describes a novel producer of ST and TM and outlines the complete process undertaken for Streptomyces sp. strain fd1-xmd genome mining.

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September 22, 2019

The hardy rubber tree genome provides insights into the evolution of polyisoprene biosynthesis.

Eucommia ulmoides, also called hardy rubber tree, is an economically important tree; however, the lack of its genome sequence restricts the fundamental biological research and applied studies of this plant species. Here, we present a high-quality assembly of its ~1.2-Gb genome (scaffold N50 = 1.88 Mb) with at least 26 723 predicted genes for E. ulmoides, the first sequenced genome of the order Garryales, which was obtained using an integrated strategy combining Illumina sequencing, PacBio sequencing, and BioNano mapping. As a sister taxon to lamiids and campanulids, E. ulmoides underwent an ancient genome triplication shared by core eudicots but no further whole-genome duplication in the last ~125 million years. E. ulmoides exhibits high expression levels and/or gene number expansion for multiple genes involved in stress responses and the biosynthesis of secondary metabolites, which may account for its considerable environmental adaptability. In contrast to the rubber tree (Hevea brasiliensis), which produces cis-polyisoprene, E. ulmoides has evolved to synthesize long-chain trans-polyisoprene via farnesyl diphosphate synthases (FPSs). Moreover, FPS and rubber elongation factor/small rubber particle protein gene families were expanded independently from the H. brasiliensis lineage. These results provide new insights into the biology of E. ulmoides and the origin of polyisoprene biosynthesis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Genotype assembly, biological activity and adaptation of spatially separated isolates of Spodoptera litura nucleopolyhedrovirus.

The cotton leafworm Spodoptera litura is a polyphagous insect. It has recently made a comeback as a primary insect pest of cotton in Pakistan due to reductions in pesticide use on the advent of genetically modified cotton, resistant to Helicoverpa armigera. Spodoptera litura nucleopolyhedrovirus (SpltNPV) infects S. litura and is recognized as a potential candidate to control this insect. Twenty-two NPV isolates were collected from S. litura from different agro-ecological zones (with collection sites up to 600?km apart) and cropping systems in Pakistan to see whether there is spatial dispersal and adaptation of the virus and/or adaptation to crops. Therefore, the genetic make-up and biological activity of these isolates was measured. Among the SpltNPV isolates tested for speed of kill in 3rd instar larvae of S. litura, TAX1, SFD1, SFD2 and GRW1 were significantly faster killing isolates than other Pakistani isolates. Restriction fragment length analysis of the DNA showed that the Pakistan SpltNPV isolates are all variants of a single SpltNPV biotype. The isolates could be grouped into three genogroups (A-C). The speed of kill of genogroup A viruses was higher than in group C according to a Cox’ proportional hazards analysis. Sequence analysis showed that the Pakistan SpltNPV isolates are more closely related to each other than to the SpltNPV type species G2 (Pang et al., 2001). This suggests a single introduction of SpltNPV into Pakistan. The SpltNPV-PAK isolates are distinct from Spodoptera littoralis nucleopolyhedrovirus. There was a strong correlation between geographic spread and the genetic variation of SpltNPV, and a marginally significant correlation between the latter and the cropping system. The faster killing isolates may be good candidates for biological control of S. litura in Pakistan. Copyright © 2018 Elsevier Inc. All rights reserved.

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September 22, 2019

Analysis of the Aedes albopictus C6/36 genome provides insight into cell line utility for viral propagation.

The 50-year-old Aedes albopictus C6/36 cell line is a resource for the detection, amplification, and analysis of mosquito-borne viruses including Zika, dengue, and chikungunya. The cell line is derived from an unknown number of larvae from an unspecified strain of Aedes albopictus mosquitoes. Toward improved utility of the cell line for research in virus transmission, we present an annotated assembly of the C6/36 genome.The C6/36 genome assembly has the largest contig N50 (3.3 Mbp) of any mosquito assembly, presents the sequences of both haplotypes for most of the diploid genome, reveals independent null mutations in both alleles of the Dicer locus, and indicates a male-specific genome. Gene annotation was computed with publicly available mosquito transcript sequences. Gene expression data from cell line RNA sequence identified enrichment of growth-related pathways and conspicuous deficiency in aquaporins and inward rectifier K+ channels. As a test of utility, RNA sequence data from Zika-infected cells were mapped to the C6/36 genome and transcriptome assemblies. Host subtraction reduced the data set by 89%, enabling faster characterization of nonhost reads.The C6/36 genome sequence and annotation should enable additional uses of the cell line to study arbovirus vector interactions and interventions aimed at restricting the spread of human disease.

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September 22, 2019

Genomic insights into the Acidobacteria reveal strategies for their success in terrestrial environments.

Members of the phylum Acidobacteria are abundant and ubiquitous across soils. We performed a large-scale comparative genome analysis spanning subdivisions 1, 3, 4, 6, 8 and 23 (n?=?24) with the goal to identify features to help explain their prevalence in soils and understand their ecophysiology. Our analysis revealed that bacteriophage integration events along with transposable and mobile elements influenced the structure and plasticity of these genomes. Low- and high-affinity respiratory oxygen reductases were detected in multiple genomes, suggesting the capacity for growing across different oxygen gradients. Among many genomes, the capacity to use a diverse collection of carbohydrates, as well as inorganic and organic nitrogen sources (such as via extracellular peptidases), was detected – both advantageous traits in environments with fluctuating nutrient environments. We also identified multiple soil acidobacteria with the potential to scavenge atmospheric concentrations of H2 , now encompassing mesophilic soil strains within the subdivision 1 and 3, in addition to a previously identified thermophilic strain in subdivision 4. This large-scale acidobacteria genome analysis reveal traits that provide genomic, physiological and metabolic versatility, presumably allowing flexibility and versatility in the challenging and fluctuating soil environment.© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019

Deciphering lignocellulose deconstruction by the white rot fungus Irpex lacteus based on genomic and transcriptomic analyses.

Irpex lacteus is one of the most potent white rot fungi for biological pretreatment of lignocellulose for second biofuel production. To elucidate the underlying molecular mechanism involved in lignocellulose deconstruction, genomic and transcriptomic analyses were carried out for I. lacteus CD2 grown in submerged fermentation using ball-milled corn stover as the carbon source.Irpex lacteus CD2 efficiently decomposed 74.9% lignin, 86.3% cellulose, and 83.5% hemicellulose in corn stover within 9 days. Manganese peroxidases were rapidly induced, followed by accumulation of cellulase and hemicellulase. Genomic analysis revealed that I. lacteus CD2 possessed a complete set of lignocellulose-degrading enzyme system composed mainly of class II peroxidases, dye-decolorizing peroxidases, auxiliary enzymes, and 182 glycoside hydrolases. Comparative transcriptomic analysis substantiated the notion of a selection mode of degradation. These analyses also suggested that free radicals, derived either from MnP-organic acid interplay or from Fenton reaction involving Fe2+ and H2O2, could play an important role in lignocellulose degradation.The selective strategy employed by I. lacteus CD2, in combination with low extracellular glycosidases cleaving plant cell wall polysaccharides into fermentable sugars, may account for high pretreatment efficiency of I. lacteus. Our study also hints the importance of free radicals for future designing of novel, robust lignocellulose-degrading enzyme cocktails.

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September 22, 2019

Tn2008-driven carbapenem resistance in Acinetobacter baumannii isolates from a period of increased incidence of infections in a Southwest Virginia hospital (USA).

The objectives of this study were (i) to determine the genetic basis for carbapenem resistance in multidrug-resistant (MDR) Acinetobacter baumannii strains isolated from patients affected by a sudden increase in the incidence of infections by such organisms in a tertiary care hospital in Virginia, USA, in 2009-2010 and (ii) to examine whether such strains are commonly encountered in the hospital setting.The whole genomes of one outbreak strain as well as one carbapenem-resistant and one carbapenem-sensitive strain from sporadic infections in 2010-2012 were sequenced and analysed. Then, 5 outbreak isolates and 57 sporadic isolates (of which 39 were carbapenem-resistant) were screened by PCR for relevant DNA elements identified in the genomics investigation.All three strains for which whole-genome sequences were obtained carried resistance genes linked to MDR phenotypes and a ca. 111-kbp plasmid (pCMCVTAb1) without drug resistance genes. Of these, the two carbapenem-resistant strains possessed a ca. 74-kbp plasmid (pCMCVTAb2) carrying a Tn2008 transposon that provides high-level carbapenem resistance. PCR analysis showed that all of the outbreak isolates carried both plasmids and Tn2008, and of the sporadic isolates 88% carried pCMCVTAb1, 25% contained pCMCVTAb2 and 50% of the latter group carried Tn2008.Carbapenem resistance in outbreak strains and 12% of sporadic isolates was due to the pCMCVTAb2-borne Tn2008. This is the first report of a Tn2008-driven outbreak of carbapenem-resistant A. baumannii infections in the Commonwealth of Virginia, which followed similar cases in Pennsylvania and Ohio. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. All rights reserved.

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September 22, 2019

The global distribution and spread of the mobilized colistin resistance gene mcr-1.

Colistin represents one of the few available drugs for treating infections caused by carbapenem-resistant Enterobacteriaceae. As such, the recent plasmid-mediated spread of the colistin resistance gene mcr-1 poses a significant public health threat, requiring global monitoring and surveillance. Here, we characterize the global distribution of mcr-1 using a data set of 457 mcr-1-positive sequenced isolates. We find mcr-1 in various plasmid types but identify an immediate background common to all mcr-1 sequences. Our analyses establish that all mcr-1 elements in circulation descend from the same initial mobilization of mcr-1 by an ISApl1 transposon in the mid 2000s (2002-2008; 95% highest posterior density), followed by a marked demographic expansion, which led to its current global distribution. Our results provide the first systematic phylogenetic analysis of the origin and spread of mcr-1, and emphasize the importance of understanding the movement of antibiotic resistance genes across multiple levels of genomic organization.

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