April 21, 2020  |  

Biochemical characterization of a novel cold-adapted agarotetraose-producing a-agarase, AgaWS5, from Catenovulum sediminis WS1-A.

Although many ß-agarases that hydrolyze the ß-1,4 linkages of agarose have been biochemically characterized, only three a-agarases that hydrolyze the a-1,3 linkages are reported to date. In this study, a new a-agarase, AgaWS5, from Catenovulum sediminis WS1-A, a new agar-degrading marine bacterium, was biochemically characterized. AgaWS5 belongs to the glycoside hydrolase (GH) 96 family. AgaWS5 consists of 1295 amino acids (140 kDa) and has the 65% identity to an a-agarase, AgaA33, obtained from an agar-degrading bacterium Thalassomonas agarivorans JAMB-A33. AgaWS5 showed the maximum activity at a pH and temperature of 8 and 40 °C, respectively. AgaWS5 showed a cold-tolerance, and it retained more than 40% of its maximum enzymatic activity at 10 °C. AgaWS5 is predicted to have several calcium-binding sites. Thus, its activity was slightly enhanced in the presence of Ca2+, and was strongly inhibited by EDTA. The Km and Vmax of AgaWS5 for agarose were 10.6 mg/mL and 714.3 U/mg, respectively. Agarose-liquefication, thin layer chromatography, and mass and NMR spectroscopic analyses demonstrated that AgaWS5 is an endo-type a-agarase that degrades agarose and mainly produces agarotetraose. Thus, in this study, a novel cold-adapted GH96 agarotetraose-producing a-agarase was identified.


April 21, 2020  |  

Chlorella vulgaris genome assembly and annotation reveals the molecular basis for metabolic acclimation to high light conditions.

Chlorella vulgaris is a fast-growing fresh-water microalga cultivated at the industrial scale for applications ranging from food to biofuel production. To advance our understanding of its biology and to establish genetics tools for biotechnological manipulation, we sequenced the nuclear and organelle genomes of Chlorella vulgaris 211/11P by combining next generation sequencing and optical mapping of isolated DNA molecules. This hybrid approach allowed to assemble the nuclear genome in 14 pseudo-molecules with an N50 of 2.8 Mb and 98.9% of scaffolded genome. The integration of RNA-seq data obtained at two different irradiances of growth (high light-HL versus low light -LL) enabled to identify 10,724 nuclear genes, coding for 11,082 transcripts. Moreover 121 and 48 genes were respectively found in the chloroplast and mitochondrial genome. Functional annotation and expression analysis of nuclear, chloroplast and mitochondrial genome sequences revealed peculiar features of Chlorella vulgaris. Evidence of horizontal gene transfers from chloroplast to mitochondrial genome was observed. Furthermore, comparative transcriptomic analyses of LL vs HL provide insights into the molecular basis for metabolic rearrangement in HL vs. LL conditions leading to enhanced de novo fatty acid biosynthesis and triacylglycerol accumulation. The occurrence of a cytosolic fatty acid biosynthetic pathway can be predicted and its upregulation upon HL exposure is observed, consistent with increased lipid amount under HL. These data provide a rich genetic resource for future genome editing studies, and potential targets for biotechnological manipulation of Chlorella vulgaris or other microalgae species to improve biomass and lipid productivity.This article is protected by copyright. All rights reserved.


April 21, 2020  |  

The Chinese chestnut genome: a reference for species restoration

Forest tree species are increasingly subject to severe mortalities from exotic pests, diseases, and invasive organisms, accelerated by climate change. Forest health issues are threatening multiple species and ecosystem sustainability globally. While sources of resistance may be available in related species, or among surviving trees, introgression of resistance genes into threatened tree species in reasonable time frames requires genome-wide breeding tools. Asian species of chestnut (Castanea spp.) are being employed as donors of disease resistance genes to restore native chestnut species in North America and Europe. To aid in the restoration of threatened chestnut species, we present the assembly of a reference genome with chromosome-scale sequences for Chinese chestnut (C. mollissima), the disease-resistance donor for American chestnut restoration. We also demonstrate the value of the genome as a platform for research and species restoration, including new insights into the evolution of blight resistance in Asian chestnut species, the locations in the genome of ecologically important signatures of selection differentiating American chestnut from Chinese chestnut, the identification of candidate genes for disease resistance, and preliminary comparisons of genome organization with related species.


April 21, 2020  |  

Complete genome sequence of Bacillus velezensis JT3-1, a microbial germicide isolated from yak feces

Bacillus velezensis JT3-1 is a probiotic strain isolated from feces of the domestic yak (Bos grunniens) in the Gansu province of China. It has strong antagonistic activity against Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella Typhimurium, Mannheimia haemolytica, Staphylococcus hominis, Clostridium perfringens, and Mycoplasma bovis. These properties have made the JT3-1 strain the focus of commercial interest. In this study, we describe the complete genome sequence of JT3-1, with a genome size of 3,929,799 bp, 3761 encoded genes and an average GC content of 46.50%. Whole genome sequencing of Bacillus velezensis JT3-1 will lay a good foundation for elucidation of the mechanisms of its antimicrobial activity, and for its future application.


April 21, 2020  |  

Evolution and global transmission of a multidrug-resistant, community-associated MRSA lineage from the Indian subcontinent

The evolution and global transmission of antimicrobial resistance has been well documented in Gram-negative bacteria and healthcare-associated epidemic pathogens, often emerging from regions with heavy antimicrobial use. However, the degree to which similar processes occur with Gram-positive bacteria in the community setting is less well understood. Here, we trace the recent origins and global spread of a multidrug resistant, community-associated Staphylococcus aureus lineage from the Indian subcontinent, the Bengal Bay clone (ST772). We generated whole genome sequence data of 340 isolates from 14 countries, including the first isolates from Bangladesh and India, to reconstruct the evolutionary history and genomic epidemiology of the lineage. Our data shows that the clone emerged on the Indian subcontinent in the early 1970s and disseminated rapidly in the 1990s. Short-term outbreaks in community and healthcare settings occurred following intercontinental transmission, typically associated with travel and family contacts on the subcontinent, but ongoing endemic transmission was uncommon. Acquisition of a multidrug resistance integrated plasmid was instrumental in the divergence of a single dominant and globally disseminated clade in the early 1990s. Phenotypic data on biofilm, growth and toxicity point to antimicrobial resistance as the driving force in the evolution of ST772. The Bengal Bay clone therefore combines the multidrug resistance of traditional healthcare-associated clones with the epidemiological transmission of community-associated MRSA. Our study demonstrates the importance of whole genome sequencing for tracking the evolution of emerging and resistant pathogens. It provides a critical framework for ongoing surveillance of the clone on the Indian subcontinent and elsewhere.Importance The Bengal Bay clone (ST772) is a community-acquired and multidrug-resistant Staphylococcus aureus lineage first isolated from Bangladesh and India in 2004. In this study, we show that the Bengal Bay clone emerged from a virulent progenitor circulating on the Indian subcontinent. Its subsequent global transmission was associated with travel or family contact in the region. ST772 progressively acquired specific resistance elements at limited cost to its fitness and continues to be exported globally resulting in small-scale community and healthcare outbreaks. The Bengal Bay clone therefore combines the virulence potential and epidemiology of community-associated clones with the multidrug-resistance of healthcare-associated S. aureus lineages. This study demonstrates the importance of whole genome sequencing for the surveillance of highly antibiotic resistant pathogens, which may emerge in the community setting of regions with poor antibiotic stewardship and rapidly spread into hospitals and communities across the world.


April 21, 2020  |  

Plantibacter flavus, Curtobacterium herbarum, Paenibacillus taichungensis, and Rhizobium selenitireducens Endophytes Provide Host-Specific Growth Promotion of Arabidopsis thaliana, Basil, Lettuce, and Bok Choy Plants.

A collection of bacterial endophytes isolated from stem tissues of plants growing in soils highly contaminated with petroleum hydrocarbons were screened for plant growth-promoting capabilities. Twenty-seven endophytic isolates significantly improved the growth of Arabidopsis thaliana plants in comparison to that of uninoculated control plants. The five most beneficial isolates, one strain each of Curtobacterium herbarum, Paenibacillus taichungensis, and Rhizobium selenitireducens and two strains of Plantibacter flavus were further examined for growth promotion in Arabidopsis, lettuce, basil, and bok choy plants. Host-specific plant growth promotion was observed when plants were inoculated with the five bacterial strains. P. flavus strain M251 increased the total biomass and total root length of Arabidopsis plants by 4.7 and 5.8 times, respectively, over that of control plants and improved lettuce and basil root growth, while P. flavus strain M259 promoted Arabidopsis shoot and root growth, lettuce and basil root growth, and bok choy shoot growth. A genome comparison between P. flavus strains M251 and M259 showed that both genomes contain up to 70 actinobacterial putative plant-associated genes and genes involved in known plant-beneficial pathways, such as those for auxin and cytokinin biosynthesis and 1-aminocyclopropane-1-carboxylate deaminase production. This study provides evidence of direct plant growth promotion by Plantibacter flavusIMPORTANCE The discovery of new plant growth-promoting bacteria is necessary for the continued development of biofertilizers, which are environmentally friendly and cost-efficient alternatives to conventional chemical fertilizers. Biofertilizer effects on plant growth can be inconsistent due to the complexity of plant-microbe interactions, as the same bacteria can be beneficial to the growth of some plant species and neutral or detrimental to others. We examined a set of bacterial endophytes isolated from plants growing in a unique petroleum-contaminated environment to discover plant growth-promoting bacteria. We show that strains of Plantibacter flavus exhibit strain-specific plant growth-promoting effects on four different plant species.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Whole Genome Sequencing and Analysis of Chlorimuron-Ethyl Degrading Bacteria Klebsiella pneumoniae 2N3.

Klebsiella pneumoniae 2N3 is a strain of gram-negative bacteria that can degrade chlorimuron-ethyl and grow with chlorimuron-ethyl as the sole nitrogen source. The complete genome of Klebsiella pneumoniae 2N3 was sequenced using third generation high-throughput DNA sequencing technology. The genomic size of strain 2N3 was 5.32 Mb with a GC content of 57.33% and a total of 5156 coding genes and 112 non-coding RNAs predicted. Two hydrolases expressed by open reading frames (ORFs) 0934 and 0492 were predicted and experimentally confirmed by gene knockout to be involved in the degradation of chlorimuron-ethyl. Strains of ?ORF 0934, ?ORF 0492, and wild type (WT) reached their highest growth rates after 8-10 hours in incubation. The degradation rates of chlorimuron-ethyl by both ?ORF 0934 and ?ORF 0492 decreased in comparison to the WT during the first 8 hours in culture by 25.60% and 24.74%, respectively, while strains ?ORF 0934, ?ORF 0492, and the WT reached the highest degradation rates of chlorimuron-ethyl in 36 hours of 74.56%, 90.53%, and 95.06%, respectively. This study provides scientific evidence to support the application of Klebsiella pneumoniae 2N3 in bioremediation to control environmental pollution.


April 21, 2020  |  

Genome assembly and annotation of the Trichoplusia ni Tni-FNL insect cell line enabled by long-read technologies.

Trichoplusiani derived cell lines are commonly used to enable recombinant protein expression via baculovirus infection to generate materials approved for clinical use and in clinical trials. In order to develop systems biology and genome engineering tools to improve protein expression in this host, we performed de novo genome assembly of the Trichoplusiani-derived cell line Tni-FNL.By integration of PacBio single-molecule sequencing, Bionano optical mapping, and 10X Genomics linked-reads data, we have produced a draft genome assembly of Tni-FNL.Our assembly contains 280 scaffolds, with a N50 scaffold size of 2.3 Mb and a total length of 359 Mb. Annotation of the Tni-FNL genome resulted in 14,101 predicted genes and 93.2% of the predicted proteome contained recognizable protein domains. Ortholog searches within the superorder Holometabola provided further evidence of high accuracy and completeness of the Tni-FNL genome assembly.This first draft Tni-FNL genome assembly was enabled by complementary long-read technologies and represents a high-quality, well-annotated genome that provides novel insight into the complexity of this insect cell line and can serve as a reference for future large-scale genome engineering work in this and other similar recombinant protein production hosts.


April 21, 2020  |  

Parallels between natural selection in the cold-adapted crop-wild relative Tripsacum dactyloides and artificial selection in temperate adapted maize.

Artificial selection has produced varieties of domesticated maize that thrive in temperate climates around the world. However, the direct progenitor of maize, teosinte, is indigenous only to a relatively small range of tropical and subtropical latitudes and grows poorly or not at all outside of this region. Tripsacum, a sister genus to maize and teosinte, is naturally endemic to the majority of areas in the western hemisphere where maize is cultivated. A full-length reference transcriptome for Tripsacum dactyloides generated using long-read Iso-Seq data was used to characterize independent adaptation to temperate climates in this clade. Genes related to phospholipid biosynthesis, a critical component of cold acclimation in other cold-adapted plant lineages, were enriched among those genes experiencing more rapid rates of protein sequence evolution in T. dactyloides. In contrast with previous studies of parallel selection, we find that there is a significant overlap between the genes that were targets of artificial selection during the adaptation of maize to temperate climates and those that were targets of natural selection in temperate-adapted T. dactyloides. Genes related to growth, development, response to stimulus, signaling, and organelles were enriched in the set of genes identified as both targets of natural and artificial selection. © 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.


April 21, 2020  |  

Genomic and Functional Characterization of the Endophytic Bacillus subtilis 7PJ-16 Strain, a Potential Biocontrol Agent of Mulberry Fruit Sclerotiniose.

Bacillus sp. 7PJ-16, an endophytic bacterium isolated from a healthy mulberry stem and previously identified as Bacillus tequilensis 7PJ-16, exhibits strong antifungal activity and has the capacity to promote plant growth. This strain was studied for its effectiveness as a biocontrol agent to reduce mulberry fruit sclerotiniose in the field and as a growth-promoting agent for mulberry in the greenhouse. In field studies, the cell suspension and supernatant of strain 7PJ-16 exhibited biocontrol efficacy and the lowest disease incidence was reduced down to only 0.80%. In greenhouse experiments, the cell suspension (1.0?×?106 and 1.0?×?105 CFU/mL) and the cell-free supernatant (100-fold and 1000-fold dilution) stimulated mulberry seed germination and promoted mulberry seedling growth. In addition, to accurately identify the 7PJ-16 strain and further explore the mechanisms of its antifungal and growth-promoting properties, the complete genome of this strain was sequenced and annotated. The 7PJ-16 genome is comprised of two circular plasmids and a 4,209,045-bp circular chromosome, containing 4492 protein-coding genes and 116 RNA genes. This strain was ultimately designed as Bacillus subtilis based on core genome sequence analyses using a phylogenomic approach. In this genome, we identified a series of gene clusters that function in the synthesis of non-ribosomal peptides (surfactin, fengycin, bacillibactin, and bacilysin) as well as the ribosome-dependent synthesis of tasA and bacteriocins (subtilin, subtilosin A), which are responsible for the biosynthesis of numerous antimicrobial metabolites. Additionally, several genes with function that promote plant growth, such as indole-3-acetic acid biosynthesis, the production of volatile substances, and siderophores synthesis, were also identified. The information described in this study has established a good foundation for understanding the beneficial interactions between endophytes and host plants, and facilitates the further application of B. subtilis 7PJ-16 as an agricultural biofertilizer and biocontrol agent.


April 21, 2020  |  

Genome sequence and genetic transformation of a widely distributed and cultivated poplar.

Populus alba is widely distributed and cultivated in Europe and Asia. This species has been used for diverse studies. In this study, we assembled a de novo genome sequence of P. alba var. pyramidalis (= P. bolleana) and confirmed its high transformation efficiency and short transformation time by experiments. Through a process of hybrid genome assembly, a total of 464 M of the genome was assembled. Annotation analyses predicted 37 901 protein-coding genes. This genome is highly collinear to that of P. trichocarpa, with most genes having orthologs in the two species. We found a marked expansion of gene families related to histone and the hormone auxin but loss of disease resistance genes in P. alba if compared with the closely related P. trichocarpa. The genome sequence presented here represents a valuable resource for further molecular functional analyses of this species as a new tree model, poplar breeding practices and comparative genomic analyses across different poplars. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.


April 21, 2020  |  

Oenococcus sicerae sp. nov., isolated from French cider.

Two Gram-stain-positive, small ellipsoidal cocci, non-motile, oxidase- and catalase-negative, and facultative anaerobic strains (UCMA15228T and UCMA17102) were isolated in France, from fermented apple juices (ciders). The 16S rRNA gene sequence was identical between the two isolates and showed 97 % similarity with respect to the closest related species Oenococcus oeni and O. kitaharae. Therefore, the two isolates were classified within the genus Oenococcus. The phylogeny based on the pheS gene sequences also confirmed the position of the new taxon. DNA-DNA hybridizations based on in silico genome-to-genome comparisons (GGDC) and Average Nucleotide Identity (ANI) values, as well as species-specific PCR, validated the novelty of the taxon. Various phenotypic characteristics such as the optimum temperature and pH for growth, the ability to metabolise sugars, the aptitude to perform the malolactic fermentation, and the resistance to ethanol and NaCl, revealed that the two strains are distinguishable from the other members of the Oenococcus genus. The combined genotypic and phenotypic data support the classification of strains UCMA15228T and UCMA17102 into a novel species of Oenococcus, for which the name O. sicerae sp. nov. is proposed. The type strain is UCMA15228T (=DSM107163T=CIRM-BIA2288T).Copyright © 2018 Elsevier GmbH. All rights reserved.


April 21, 2020  |  

Penicillium purpurogenum Produces a Set of Endoxylanases: Identification, Heterologous Expression, and Characterization of a Fourth Xylanase, XynD, a Novel Enzyme Belonging to Glycoside Hydrolase Family 10.

The fungus Penicillium purpurogenum grows on a variety of natural carbon sources and secretes a large number of enzymes which degrade the polysaccharides present in lignocellulose. In this work, the gene coding for a novel endoxylanase has been identified in the genome of the fungus. This gene (xynd) possesses four introns. The cDNA has been expressed in Pichia pastoris and characterized. The enzyme, XynD, belongs to family 10 of the glycoside hydrolases. Mature XynD has a calculated molecular weight of 40,997. It consists of 387 amino acid residues with an N-terminal catalytic module, a linker rich in ser and thr residues, and a C-terminal family 1 carbohydrate-binding module. XynD shows the highest identity (97%) to a putative endoxylanase from Penicillium subrubescens but its highest identity to a biochemically characterized xylanase (XYND from Penicillium funiculosum) is only 68%. The enzyme has a temperature optimum of 60 °C, and it is highly stable in its pH optimum range of 6.5-8.5. XynD is the fourth biochemically characterized endoxylanase from P. purpurogenum, confirming the rich potential of this fungus for lignocellulose biodegradation. XynD, due to its wide pH optimum and stability, may be a useful enzyme in biotechnological procedures related to this biodegradation process.


April 21, 2020  |  

Microbial community and fermentation characteristic of Italian ryegrass silage prepared with corn stover and lactic acid bacteria.

The bacterial community determined via PacBio single molecule, real-time sequencing technology (SMRT) and the fermentation characteristics of Italian ryegrass (IR, 82% moisture) silage prepared with corn stover (CS) were investigated. A selected strain of Lactobacillus plantarum (L694) and a commercial inoculant stain of Lactobacillus plantarum (LP) were used as additives. Lactic acid bacteria (LAB) effectively improved silage quality. After fermentation, Lactobacillus plantarum was the dominant species in IR?+?LP and IR?+?L694 treatments, which led to higher (P?


April 21, 2020  |  

The complete genome sequence of Ethanoligenens harbinense reveals the metabolic pathway of acetate-ethanol fermentation: A novel understanding of the principles of anaerobic biotechnology.

Ethanol-type fermentation is one of three main fermentation types in the acidogenesis of anaerobic treatment systems. Non-spore-forming Ethanoligenens is as a typical genus capable of ethanol-type fermentation in mixed culture (i.e. acetate-ethanol fermentation). This genus can produce ethanol, acetate, CO2, and H2 using carbohydrates, and has application potential in anaerobic bioprocesses. Here, the complete genome sequences and methylome of Ethanoligenens harbinense strains with different autoaggregative and coaggregative abilities were obtained using the PacBio single-molecule real-time sequencing platform. The genome size of E. harbinense strains was about 2.97-3.10?Mb with 55.5% G+C content. 3020-3153 genes were annotated, most of which were methylated at specific sites or motifs. The methylation types included 6mA, 4mC, and unknown types. Comparative genomic analysis demonstrated low levels of genetic similarity between E. harbinense and other well-known hydrogen-producing bacteria (i.e., Clostridium and Thermoanaerobacter) in phylogenesis. Hydrogen production of E. harbinense was catalyzed by genes that encode [FeFe]-hydrogenases and that were synthesized by three maturases of [FeFe]-H2ase. The metabolic mechanism of H2-ethanol co-production fermentation, catalyzed by pyruvate ferredoxin oxidoreductase was proposed. This study provides genetic and evolutionary information of a model genus for the further investigation of the metabolic pathway and regulatory network of ethanol-type fermentation and anaerobic bioprocesses for waste or wastewater treatment.Copyright © 2019. Published by Elsevier Ltd.


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