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September 22, 2019

Genomic analysis of the insect-killing fungus Beauveria bassiana JEF-007 as a biopesticide.

Insect-killing fungi have high potential in pest management. A deeper insight into the fungal genes at the whole genome level is necessary to understand the inter-species or intra-species genetic diversity of fungal genes, and to select excellent isolates. In this work, we conducted a whole genome sequencing of Beauveria bassiana (Bb) JEF-007 and characterized pathogenesis-related features and compared with other isolates including Bb ARSEF2860. A large number of Bb JEF-007 genes showed high identity with Bb ARSEF2860, but some genes showed moderate or low identity. The two Bb isolates showed a significant difference in vegetative growth, antibiotic-susceptibility, and virulence against Tenebrio molitor larvae. When highly identical genes between the two Bb isolates were subjected to real-time PCR, their transcription levels were different, particularly in heat shock protein 30 (hsp30) gene which is related to conidial thermotolerance. In several B. bassiana isolates, chitinases and trypsin-like protease genes involved in pathogenesis were highly conserved, but other genes showed noticeable sequence variation within the same species. Given the transcriptional and genetic diversity in B. bassiana, a selection of virulent isolates with industrial advantages is a pre-requisite, and this genetic approach could support the development of excellent biopesticides with intellectual property protection.

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September 22, 2019

A miR172 target-deficient AP2-like gene correlates with the double flower phenotype in roses.

One of the well-known floral abnormalities in flowering plants is the double-flower phenotype, which corresponds to flowers that develop extra petals, sometimes even containing entire flowers within flowers. Because of their highly priced ornamental value, spontaneous double-flower variants have been found and selected for in a wide range of ornamental species. Previously, double flower formation in roses was associated with a restriction of AGAMOUS expression domain toward the centre of the meristem, leading to extra petals. Here, we characterized the genomic region containing the mutation associated with the switch from simple to double flowers in the rose. An APETALA2-like gene (RcAP2L), a member of the Target Of EAT-type (TOE-type) subfamily, lies within this interval. In the double flower rose, two alleles of RcAP2L are present, one of which harbours a transposable element inserted into intron 8. This insertion leads to the creation of a miR172 resistant RcAP2L variant. Analyses of the presence of this variant in a set of simple and double flower roses demonstrate a correlation between the presence of this allele and the double flower phenotype. These data suggest a role of this miR172 resistant RcAP2L variant in regulating RcAGAMOUS expression and double flower formation in Rosa sp.

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September 22, 2019

Genomic analysis of Sparus aurata reveals the evolutionary dynamics of sex-biased genes in a sequential hermaphrodite fish

Sexual dimorphism is a fascinating subject in evolutionary biology and mostly results from sex-biased expression of genes, which have been shown to evolve faster in gonochoristic species. We report here genome and sex-specific transcriptome sequencing of Sparus aurata, a sequential hermaphrodite fish. Evolutionary comparative analysis reveals that sex-biased genes in S. aurata are similar in number and function, but evolved following strikingly divergent patterns compared with gonochoristic species, showing overall slower rates because of stronger functional constraints. Fast evolution is observed only for highly ovary-biased genes due to female-specific patterns of selection that are related to the peculiar reproduction mode of S. aurata, first maturing as male, then as female. To our knowledge, these findings represent the first genome-wide analysis on sex-biased loci in a hermaphrodite vertebrate species, demonstrating how having two sexes in the same individual profoundly affects the fate of a large set of evolutionarily relevant genes.

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September 22, 2019

Phenotypic and genomic properties of Brachybacterium vulturis sp. nov. and Brachybacterium avium sp. nov.

Two strains, VM2412T and VR2415T, were isolated from the feces of an Andean condor (Vultur gryphus) living in Seoul Grand Park, Gyeonggi-do, South Korea. Cells of both strains were observed to be Gram-stain positive, non-motile, aerobic, catalase positive and oxidase negative. Growth was found to occur at 10-30°C, showing optimum growth at 30°C. The strains could tolerate up to 15% (w/v) NaCl concentration and grow at pH 6-9. The strains shared 99.3% 16S rRNA gene sequence similarity to each other but were identified as two distinct species based on 89.0-89.2% ANIb, 90.3% ANIm, 89.7% OrthoANI and 38.0% dDDH values calculated using whole genome sequences. Among species with validly published names, Brachybacterium ginsengisoli DCY80T shared high 16S rRNA gene sequence similarities with strains VM2412T (98.7%) and VR2415T (98.4%) and close genetic relatedness with strains VM2412T (83.3-83.5% ANIb, 87.0% ANIm, 84.3% OrthoANI and 27.8% dDDH) and VR2415T (82.8-83.2% ANIb, 86.7% ANIm, 83.9% OrthoANI and 27.2% dDDH). The major fatty acid of the two strains was identified as anteiso-C15:0 and the polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, presumptively phosphatidylethanolamine and three unidentified glycolipids. Strain VR2415T also produced an unidentified phospholipid. The cell walls of the two strains contained meso-diaminopimelic acid as diagnostic diamino acid and the whole cell sugars were ribose, glucose, and galactose. The strains contained MK-7 as their predominant menaquinone. The genomes of strains VM2412T, VR2415T, and B. ginsengisoli DCY80T were sequenced in this study. The genomic G+C contents of strains VM2412T and VR2415T were determined to be 70.8 and 70.4 mol%, respectively. A genome-based phylogenetic tree constructed using an up-to-date bacterial core gene set (UBCG) showed that the strains formed a clade with members of the genus Brachybacterium, supporting their taxonomic classification into the genus Brachybacterium. Based on phenotypic and genotypic analyses in this study, strains VM2412T and VR2415T are considered to represent two novel species of the genus Brachybacterium and the names Brachybacterium vulturis sp. nov. and Brachybacterium avium sp. nov. are proposed for strains VM2412T (=KCTC 39996T = JCM 32142T) and VR2415T (=KCTC 39997T = JCM 32143T), respectively.

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September 22, 2019

Large plasmidome of dairy Lactococcus lactis subsp. lactis biovar diacetylactis FM03P encodes technological functions and appears highly unstable.

Important industrial traits have been linked to plasmids in Lactococcus lactis.The dairy isolate L. lactis subsp. lactis biovar diacetylactis FM03P was sequenced revealing the biggest plasmidome of all completely sequenced and published L. lactis strains up till now. The 12 plasmids that were identified are: pLd1 (8277 bp), pLd2 (15,218 bp), pLd3 (4242 bp), pLd4 (12,005 bp), pLd5 (7521 bp), pLd6 (3363 bp), pLd7 (30,274 bp), pLd8 (47,015 bp), pLd9 (15,313 bp), pLd10 (39,563 bp), pLd11 (9833 bp) and pLd12 (3321 bp). Structural analysis of the repB promoters and the RepB proteins showed that eleven of the plasmids replicate via the theta-type mechanism, while only plasmid pLd3 replicates via a rolling-circle replication mechanism. Plasmids pLd2, pLd7 and pLd10 contain a highly similar operon involved in mobilisation of the plasmids. Examination of the twelve plasmids of L. lactis FM03P showed that 10 of the plasmids carry putative genes known to be important for growth and survival in the dairy environment. These genes encode technological functions such as lactose utilisation (lacR-lacABCDFEGX), citrate uptake (citQRP), peptide degradation (pepO and pepE) and oligopeptide uptake (oppDFBCA), uptake of magnesium and manganese (2 mntH, corA), exopolysaccharides production (eps operon), bacteriophage resistance (1 hsdM, 1 hsdR and 7 different hsdS genes of a type I restriction-modification system, an operon of three genes encoding a putative type II restriction-modification system and an abortive infection gene) and stress resistance (2 uspA, cspC and cadCA). Acquisition of these plasmids most likely facilitated the adaptation of the recipient strain to the dairy environment. Some plasmids were already lost during a single propagation step signifying their instability in the absence of a selective pressure.Lactococcus lactis FM03P carries 12 plasmids important for its adaptation to the dairy environment. Some of the plasmids were easily lost demonstrating that propagation outside the dairy environment should be minimised when studying dairy isolates of L. lactis.

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September 22, 2019

Complete genome of streamlined marine actinobacterium Pontimonas salivibrio strain CL-TW6T adapted to coastal planktonic lifestyle.

Pontimonas salivibrio strain CL-TW6T (=KCCM 90105?=?JCM18206) was characterized as the type strain of a new genus within the Actinobacterial family Microbacteriaceae. It was isolated from a coastal marine environment in which members of Microbactericeae have not been previously characterized.The genome of P. salivibrio CL-TW6T was a single chromosome of 1,760,810 bp. Genomes of this small size are typically found in bacteria growing slowly in oligotrophic zones and said to be streamlined. Phylogenetic analysis showed it to represent a lineage originating in the Microbacteriaceae radiation occurring before the snowball Earth glaciations, and to have a closer relationship with some streamlined bacteria known through metagenomic data. Several genomic characteristics typical of streamlined bacteria are found: %G?+?C is lower than non-streamlined members of the phylum; there are a minimal number of rRNA and tRNA genes, fewer paralogs in most gene families, and only two sigma factors; there is a noticeable absence of some nonessential metabolic pathways, including polyketide synthesis and catabolism of some amino acids. There was no indication of any phage genes or plasmids, however, a system of active insertion elements was present. P. salivibrio appears to be unusual in having polyrhamnose-based cell wall oligosaccharides instead of mycolic acid or teichoic acid-based oligosaccharides. Oddly, it conducts sulfate assimilation apparently for sulfating cell wall components, but not for synthesizing amino acids. One gene family it has more of, rather than fewer of, are toxin/antitoxin systems, which are thought to down-regulate growth during nutrient deprivation or other stressful conditions.Because of the relatively small number of paralogs and its relationship to the heavily characterized Mycobacterium tuberculosis, we were able to heavily annotate the genome of P. salivibrio CL-TW6T. Its streamlined status and relationship to streamlined metagenomic constructs makes it an important reference genome for study of the streamlining concept. The final evolutionary trajectory of CL-TW6 T was to adapt to growth in a non-oligotrophic coastal zone. To understand that adaptive process, we give a thorough accounting of gene content, contrasting with both oligotrophic streamlined bacteria and large genome bacteria, and distinguishing between genes derived by vertical and horizontal descent.

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September 22, 2019

The complete mitochondrial genome of the early flowering plant Nymphaea colorata is highly repetitive with low recombination.

Mitochondrial genomes of flowering plants (angiosperms) are highly dynamic in genome structure. The mitogenome of the earliest angiosperm Amborella is remarkable in carrying rampant foreign DNAs, in contrast to Liriodendron, the other only known early angiosperm mitogenome that is described as ‘fossilized’. The distinctive features observed in the two early flowering plant mitogenomes add to the current confusions of what early flowering plants look like. Expanded sampling would provide more details in understanding the mitogenomic evolution of early angiosperms. Here we report the complete mitochondrial genome of water lily Nymphaea colorata from Nymphaeales, one of the three orders of the earliest angiosperms.Assembly of data from Pac-Bio long-read sequencing yielded a circular mitochondria chromosome of 617,195 bp with an average depth of 601×. The genome encoded 41 protein coding genes, 20 tRNA and three rRNA genes with 25 group II introns disrupting 10 protein coding genes. Nearly half of the genome is composed of repeated sequences, which contributed substantially to the intron size expansion, making the gross intron length of the Nymphaea mitochondrial genome one of the longest among angiosperms, including an 11.4-Kb intron in cox2, which is the longest organellar intron reported to date in plants. Nevertheless, repeat mediated homologous recombination is unexpectedly low in Nymphaea evidenced by 74 recombined reads detected from ten recombinationally active repeat pairs among 886,982 repeat pairs examined. Extensive gene order changes were detected in the three early angiosperm mitogenomes, i.e. 38 or 44 events of inversions and translocations are needed to reconcile the mitogenome of Nymphaea with Amborella or Liriodendron, respectively. In contrast to Amborella with six genome equivalents of foreign mitochondrial DNA, not a single horizontal gene transfer event was observed in the Nymphaea mitogenome.The Nymphaea mitogenome resembles the other available early angiosperm mitogenomes by a similarly rich 64-coding gene set, and many conserved gene clusters, whereas stands out by its highly repetitive nature and resultant remarkable intron expansions. The low recombination level in Nymphaea provides evidence for the predominant master conformation in vivo with a highly substoichiometric set of rearranged molecules.

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September 22, 2019

Temperature responses of mutation rate and mutational spectrum in an Escherichia coli strain and the correlation with metabolic rate.

Temperature is a major determinant of spontaneous mutation, but the precise mode, and the underlying mechanisms, of the temperature influences remain less clear. Here we used a mutation accumulation approach combined with whole-genome sequencing to investigate the temperature dependence of spontaneous mutation in an Escherichia coli strain. Experiments were performed under aerobic conditions at 25, 28 and 37 °C, three temperatures that were non-stressful for the bacterium but caused significantly different bacterial growth rates.Mutation rate did not differ between 25 and 28 °C, but was higher at 37 °C. Detailed analyses of the molecular spectrum of mutations were performed; and a particularly interesting finding is that higher temperature led to a bias of mutation to coding, relative to noncoding, DNA. Furthermore, the temperature response of mutation rate was extremely similar to that of metabolic rate, consistent with an idea that metabolic rate predicts mutation rate.Temperature affects mutation rate and the types of mutation supply, both being crucial for the opportunity of natural selection. Our results help understand how temperature drives evolutionary speed of organisms and thus the global patterns of biodiversity. This study also lend support to the metabolic theory of ecology for linking metabolic rate and molecular evolution rate.

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September 22, 2019

Reprogramming of the antimycin NRPS-PKS assembly lines inspired by gene evolution.

Reprogramming of the NRPS/PKS assembly line is an attractive method for the production of new bioactive molecules. However, it is usually hampered by the loss of intimate domain/module interactions required for the precise control of chain transfer and elongation reactions. In this study, we first establish heterologous expression systems of the unique antimycin-type cyclic depsipeptides: JBIR-06 (tri-lactone) and neoantimycin (tetra-lactone), and engineer their biosyntheses by taking advantage of bioinformatic analyses and evolutionary insights. As a result, we successfully accomplish three manipulations: (i) ring contraction of neoantimycin (from tetra-lactone to tri-lactone), (ii) ring expansion of JBIR-06 (from tri-lactone to tetra-lactone), and (iii) alkyl chain diversification of JBIR-06 by the incorporation of various alkylmalonyl-CoA extender units, to generate a set of unnatural derivatives in practical yields. This study presents a useful strategy for engineering NRPS-PKS module enzymes, based on nature’s diversification of the domain and module organizations.

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September 22, 2019

A gene-rich fraction analysis of the Passiflora edulis genome reveals highly conserved microsyntenic regions with two related Malpighiales species.

Passiflora edulis is the most widely cultivated species of passionflowers, cropped mainly for industrialized juice production and fresh fruit consumption. Despite its commercial importance, little is known about the genome structure of P. edulis. To fill in this gap in our knowledge, a genomic library was built, and now completely sequenced over 100 large-inserts. Sequencing data were assembled from long sequence reads, and structural sequence annotation resulted in the prediction of about 1,900 genes, providing data for subsequent functional analysis. The richness of repetitive elements was also evaluated. Microsyntenic regions of P. edulis common to Populus trichocarpa and Manihot esculenta, two related Malpighiales species with available fully sequenced genomes were examined. Overall, gene order was well conserved, with some disruptions of collinearity identified as rearrangements, such as inversion and translocation events. The microsynteny level observed between the P. edulis sequences and the compared genomes is surprising, given the long divergence time that separates them from the common ancestor. P. edulis gene-rich segments are more compact than those of the other two species, even though its genome is much larger. This study provides a first accurate gene set for P. edulis, opening the way for new studies on the evolutionary issues in Malpighiales genomes.

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September 22, 2019

Orphan legumes growing in dry environments: Marama bean as a case study.

Plants have developed morphological, physiological, biochemical, cellular, and molecular mechanisms to survive in drought-stricken environments with little or no water caused by below-average precipitation. In this mini-review, we highlight the characteristics that allows marama bean [Tylosema esculentum (Burchell) Schreiber], an example of an orphan legume native to arid regions of southwestern Southern Africa, to flourish under an inhospitable climate and dry soil conditions where no other agricultural crop competes in this agro-ecological zone. Orphan legumes are often better suited to withstand such harsh growth environments due to development of survival strategies using a combination of different traits and responses. Recent findings on questions on marama bean speciation, hybridization, population dynamics, and the evolutionary history of the bean and mechanisms by which the bean is able to extract and conserve water and nutrients from its environment as well as aspects of morphological and physiological adaptation will be reviewed. The importance of the soil microbiome and the genetic diversity in this species, and their interplay, as a reservoir for improvement will also be considered. In particular, the application of the newly established marama bean genome sequence will facilitate both the identification of important genes involved in the interaction with the soil microbiome and the identification of the diversity within the wild germplasm for genes involved drought tolerance. Since predicted future changes in climatic conditions, with less water availability for plant growth, will severely affect agricultural productivity, an understanding of the mechanisms of unique adaptations in marama bean to such conditions may also provide insights as to how to improve the performance of the major crops.

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September 22, 2019

Comprehensive evaluation of the host responses to infection with differentially virulent classical swine fever virus strains in pigs.

Classical swine fever virus (CSFV) infection causes most variable clinical syndromes from chronic or latent infection to acute death, and it is generally acknowledged that the course of disease is affected by both virus and host factors. To compare host immune responses to differentially virulent CSFV strains in pigs, fifteen 8-week-old specific-pathogen-free pigs were randomly divided into four groups and inoculated with the CSFV Shimen strain (a highly virulent strain), the HLJZZ2014 strain (a moderately virulent strains), C-strain (an avirulent strain), and DMEM (mock control), respectively. Infection with the Shimen or HLJZZ2014 strain resulted in fever, clinical signs and histopathological lesions, which were not observed in the C-strain-inoculated pigs, though low viral genome copies were detected in the peripheral blood and tissue samples. The data showed that the virulence of the strains affected the outcome of duration and intensity of the disease rather than the tissue tropism of the virus. Furthermore, leukopenia, lymphocytopenia, differentiation of T-cells, and the secretion of cytokines associated with inflammation or apoptosis such as interferon alpha (IFN-a), tumor necrosis factor alpha (TNF-a), interleukin 2 (IL-2), IL-4, IL-6, and IL-10 were induced by the virulent CSFV infection, the differences reflected in onset and extent of the regulation. Taken together, our results revealed that the major differences among the three strains resided in the kinetics of host response to the infection: severe and immediate with the highly virulent strain, while progressive and delayed with the moderately virulent one. This comparative study will help to dissect the pathogenesis of CSFV. Copyright © 2018 Elsevier B.V. All rights reserved.

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September 22, 2019

Integration of genomic data with NMR analysis enables assignment of the full stereostructure of neaumycin B, a potent inhibitor of glioblastoma from a marine-derived Micromonospora.

The microbial metabolites known as the macrolides are some of the most successful natural products used to treat infectious and immune diseases. Describing the structures of these complex metabolites, however, is often extremely difficult due to the presence of multiple stereogenic centers inherent in this class of polyketide-derived metabolites. With the availability of genome sequence data and a better understanding of the molecular genetics of natural product biosynthesis, it is now possible to use bioinformatic approaches in tandem with spectroscopic tools to assign the full stereostructures of these complex metabolites. In our quest to discover and develop new agents for the treatment of cancer, we observed the production of a highly cytotoxic macrolide, neaumycin B, by a marine-derived actinomycete bacterium of the genus Micromonospora. Neaumycin B is a complex polycyclic macrolide possessing 19 asymmetric centers, usually requiring selective degradation, crystallization, derivatization, X-ray diffraction analysis, synthesis, or other time-consuming approaches to assign the complete stereostructure. As an alternative approach, we sequenced the genome of the producing strain and identified the neaumycin gene cluster ( neu). By integrating the known stereospecificities of biosynthetic enzymes with comprehensive NMR analysis, the full stereostructure of neaumycin B was confidently assigned. This approach exemplifies how mining gene cluster information while integrating NMR-based structure data can achieve rapid, efficient, and accurate stereostructural assignments for complex macrolides.

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September 22, 2019

Opposite polarity monospore genome de novo sequencing and comparative analysis reveal the possible heterothallic life cycle of Morchella importuna.

Morchella is a popular edible fungus worldwide due to its rich nutrition and unique flavor. Many research efforts were made on the domestication and cultivation of Morchella all over the world. In recent years, the cultivation of Morchella was successfully commercialized in China. However, the biology is not well understood, which restricts the further development of the morel fungus cultivation industry. In this paper, we performed de novo sequencing and assembly of the genomes of two monospores with a different mating type (M04M24 and M04M26) isolated from the commercially cultivated strain M04. Gene annotation and comparative genome analysis were performed to study differences in CAZyme (Carbohydrate-active enzyme) enzyme content, transcription factors, duplicated sequences, structure of mating type sites, and differences at the gene and functional levels between the two monospore strains of M. importuna. Results showed that the de novo assembled haploid M04M24 and M04M26 genomes were 48.98 and 51.07 Mb, respectively. A complete fine physical map of M. importuna was obtained from genome coverage and gene completeness evaluation. A total of 10,852 and 10,902 common genes and 667 and 868 endemic genes were identified from the two monospore strains, respectively. The Gene Ontology (GO) and KAAS (KEGG Automatic Annotation Serve) enrichment analyses showed that the endemic genes performed different functions. The two monospore strains had 99.22% collinearity with each other, accompanied with certain position and rearrangement events. Analysis of complete mating-type loci revealed that the two monospore M. importuna strains contained an independent mating-type structure and remained conserved in sequence and location. The phylogenetic and divergence time of M. importuna was analyzed at the whole-genome level for the first time. The bifurcation time of morel and tuber was estimated to be 201.14 million years ago (Mya); the two monospore strains with a different mating type represented the evolution of different nuclei, and the single copy homologous genes between them were also different due to a genetic differentiation distance about 0.65 Mya. Compared with truffles, M. importuna had an extension of 28 clusters of orthologous genes (COGs) and a contraction of two COGs. The two different polar nuclei with different degrees of contraction and expansion suggested that they might have undergone different evolutionary processes. The different mating-type structures, together with the functional clustering and enrichment analysis results of the endemic genes of the two different polar nuclei, imply that M. importuna might be a heterothallic fungus and the interaction between the endemic genes may be necessary for its complete life history. Studies on the genome of M. importuna facilitate a better understanding of morel biology and evolution.

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September 22, 2019

Molecular basis for the final oxidative rearrangement steps in chartreusin biosynthesis.

Oxidative rearrangements play key roles in introducing structural complexity and biological activities of natural products biosynthesized by type II polyketide synthases (PKSs). Chartreusin (1) is a potent antitumor polyketide that contains a unique rearranged pentacyclic aromatic bilactone aglycone derived from a type II PKS. Herein, we report an unprecedented dioxygenase, ChaP, that catalyzes the final a-pyrone ring formation in 1 biosynthesis using flavin-activated oxygen as an oxidant. The X-ray crystal structures of ChaP and two homologues, docking studies, and site-directed mutagenesis provided insights into the molecular basis of the oxidative rearrangement that involves two successive C-C bond cleavage steps followed by lactonization. ChaP is the first example of a dioxygenase that requires a flavin-activated oxygen as a substrate despite lacking flavin binding sites, and represents a new class in the vicinal oxygen chelate enzyme superfamily.

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