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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

Discovery of new genes involved in curli production by a uropathogenic Escherichia coli strain from the highly virulent O45:K1:H7 lineage.

Curli are bacterial surface-associated amyloid fibers that bind to the dye Congo red (CR) and facilitate uropathogenic Escherichia coli (UPEC) biofilm formation and protection against host innate defenses. Here we sequenced the genome of the curli-producing UPEC pyelonephritis strain MS7163 and showed it belongs to the highly virulent O45:K1:H7 neonatal meningitis-associated clone. MS7163 produced curli at human physiological temperature, and this correlated with biofilm growth, resistance of sessile cells to the human cationic peptide cathelicidin, and enhanced colonization of the mouse bladder. We devised a forward genetic screen using CR staining as a proxy for curli production and identified 41 genes that were required for optimal CR binding, of which 19 genes were essential for curli synthesis. Ten of these genes were novel or poorly characterized with respect to curli synthesis and included genes involved in purine de novo biosynthesis, a regulator that controls the Rcs phosphorelay system, and a novel repressor of curli production (referred to as rcpA). The involvement of these genes in curli production was confirmed by the construction of defined mutants and their complementation. The mutants did not express the curli major subunit CsgA and failed to produce curli based on CR binding. Mutation of purF (the first gene in the purine biosynthesis pathway) and rcpA also led to attenuated colonization of the mouse bladder. Overall, this work has provided new insight into the regulation of curli and the role of these amyloid fibers in UPEC biofilm formation and pathogenesis.IMPORTANCE Uropathogenic Escherichia coli (UPEC) strains are the most common cause of urinary tract infection, a disease increasingly associated with escalating antibiotic resistance. UPEC strains possess multiple surface-associated factors that enable their colonization of the urinary tract, including fimbriae, curli, and autotransporters. Curli are extracellular amyloid fibers that enhance UPEC virulence and promote biofilm formation. Here we examined the function and regulation of curli in a UPEC pyelonephritis strain belonging to the highly virulent O45:K1:H7 neonatal meningitis-associated clone. Curli expression at human physiological temperature led to increased biofilm formation, resistance of sessile cells to the human cationic peptide LL-37, and enhanced bladder colonization. Using a comprehensive genetic screen, we identified multiple genes involved in curli production, including several that were novel or poorly characterized with respect to curli synthesis. In total, this study demonstrates an important role for curli as a UPEC virulence factor that promotes biofilm formation, resistance, and pathogenesis. Copyright © 2018 Nhu et al.

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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

Population genomics of Culiseta melanura, the principal vector of Eastern equine encephalitis virus in the United States.

Eastern Equine Encephalitis (EEE) (Togaviridae, Alphavirus) is a highly pathogenic mosquito-borne arbovirus that circulates in an enzootic cycle involving Culiseta melanura mosquitoes and wild Passeriformes birds in freshwater swamp habitats. Recently, the northeastern United States has experienced an intensification of virus activity with increased human involvement and northward expansion into new regions. In addition to its principal role in enzootic transmission of EEE virus among avian hosts, recent studies on the blood-feeding behavior of Cs. melanura throughout its geographic range suggest that this mosquito may also be involved in epizootic / epidemic transmission to equines and humans in certain locales. Variations in blood feeding behavior may be a function of host availability, environmental factors, and/or underlying genetic differences among regional populations. Despite the importance of Cs. melanura in transmission and maintenance of EEE virus, the genetics of this species remains largely unexplored.To investigate the occurrence of genetic variation in Cs. melanura, the genome of this mosquito vector was sequenced resulting in a draft genome assembly of 1.28 gigabases with a contig N50 of 93.36 kilobases. Populations of Cs. melanura from 10 EEE virus foci in the eastern North America were genotyped with double-digest RAD-seq. Following alignment of reads to the reference genome, variant calling, and filtering, 40,384 SNPs were retained for downstream analyses. Subsequent analyses revealed genetic differentiation between northern and southern populations of this mosquito species. Moreover, limited fine-scale population structure was detected throughout northeastern North America, suggesting local differentiation of populations but also a history of ancestral polymorphism or contemporary gene flow. Additionally, a genetically distinct cluster was identified predominantly at two northern sites.This study elucidates the first evidence of fine-scale population structure in Cs. melanura throughout its eastern range and detects evidence of gene flow between populations in northeastern North America. This investigation provides the groundwork for examining the consequences of genetic variations in the populations of this mosquito species that could influence vector-host interactions and the risk of human and equine infection with EEE virus.

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September 22, 2019

First draft genome assembly of the Argane tree (Argania spinosa)

Background: The Argane tree (Argania spinosa L. Skeels) is an endemic tree of southwestern Morocco that plays an important socioeconomic and ecologic role for a dense human population in an arid zone. Several studies confirmed the importance of this species as a food and feed source and as a resource for both pharmaceutical and cosmetic compounds. Unfortunately, the argane tree ecosystem is facing significant threats from environmental changes (global warming, over-population) and over-exploitation. Limited research has been conducted, however, on argane tree genetics and genomics, which hinders its conservation and genetic improvement. Methods: Here, we present a draft genome assembly of A. spinosa. A reliable reference genome of A. spinosa was created using a hybrid de novo assembly approach combining short and long sequencing reads. Results: In total, 144 Gb Illumina HiSeq reads and 7.2 Gb PacBio reads were produced and assembled. The final draft genome comprises 75 327 scaffolds totaling 671 Mb with an N50 of 49 916 kb. The draft assembly is close to the genome size estimated by k-mers distribution and covers 89% of complete and 4.3 % of partial Arabidopsis orthologous groups in BUSCO. Conclusion: The A. spinosa genome will be useful for assessing biodiversity leading to efficient conservation of this endangered endemic tree. Furthermore, the genome may enable genome-assisted cultivar breeding, and provide a better understanding of important metabolic pathways and their underlying genes for both cosmetic and pharmacological purposes.

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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

The hpRNA/RNAi pathway is essential to resolve intragenomic conflict in the Drosophila male germline.

Intragenomic conflicts are fueled by rapidly evolving selfish genetic elements, which induce selective pressures to innovate opposing repressive mechanisms. This is patently manifest in sex-ratio (SR) meiotic drive systems, in which distorter and suppressor factors bias and restore equal transmission of X and Y sperm. Here, we reveal that multiple SR suppressors in Drosophila simulans (Nmy and Tmy) encode related hairpin RNAs (hpRNAs), which generate endo-siRNAs that repress the paralogous distorters Dox and MDox. All components in this drive network are recently evolved and largely testis restricted. To connect SR hpRNA function to the RNAi pathway, we generated D. simulans null mutants of Dcr-2 and AGO2. Strikingly, these core RNAi knockouts massively derepress Dox and MDox and are in fact completely male sterile and exhibit highly defective spermatogenesis. Altogether, our data reveal how the adaptive capacity of hpRNAs is critically deployed to restrict selfish gonadal genetic systems that can exterminate a species. Copyright © 2018 Elsevier Inc. All rights reserved.

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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

Groundnut entered post-genome sequencing era: Opportunities and challenges in translating genomic information from genome to field

Cultivated groundnut or peanut (Arachis hypogaea) is an allopolyploid crop with a large complex genome and genetic barrier for exchanging genetic diversity from its wild relatives due to ploidy differences. Optimum genetic and genomic resources are key for accelerating the process for trait mapping and gene discovery and deploying diagnostic markers in genomics-assisted breeding. The better utilization of different aspects of peanut biology such as genetics, genomics, transcriptomics, proteomics, epigenomics, metabolomics, and interactomics can be of great help to groundnut genetic improvement program across the globe. The availability of high-quality reference genome is core to all the “omics” approaches, and hence optimum genomic resources are a must for fully exploiting the potential of modern science into conventional breeding. In this context, groundnut is passing through a very critical and transformational phase by making available the required genetic and genomic resources such as reference genomes of progenitors, resequencing of diverse lines, transcriptome resources, germplasm diversity panel, and multi-parent genetic populations for conducting high-resolution trait mapping, identification of associated markers, and development of diagnostic markers for selected traits. Lastly, the available resources have been deployed in translating genomic information from genome to field by developing improved groundnut lines with enhanced resistance to root-knot nematode, rust, and late leaf spot and high oleic acid. In addition, the International Peanut Genome Initiative (IPGI) have made available the high-quality reference genome for cultivated tetraploid groundnut which will facilitate better utilization of genetic resources in groundnut improvement. In parallel, the development of high-density genotyping platforms, such as Axiom_Arachis array with 58 K SNPs, and constitution of training population will initiate the deployment of the modern breeding approach, genomic selection, for achieving higher genetic gains in less time with more precision.

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September 22, 2019

Comparative genomics reveals diverse capsular polysaccharide synthesis gene clusters in emerging Raoultella planticola.

Raoultella planticola is an emerging zoonotic pathogen that is associated with rare but life-threatening cases of bacteremia, biliary tract infections, and urinary tract infections. Moreover, increasing antimicrobial resistance in the organism poses a potential threat to public health. In spite of its importance as a human pathogen, the genome of R. planticola remains largely unexplored and little is known about its virulence factors. Although lipopolysaccharides has been detected in R. planticola and implicated in the virulence in earlier studies, the genetic background is unknown. Here, we report the complete genome and comparative analysis of the multidrug-resistant clinical isolate R. planticola GODA. The complete genome sequence of R. planticola GODA was sequenced using single-molecule real-time DNA sequencing. Comparative genomic analysis reveals distinct capsular polysaccharide synthesis gene clusters in R. planticola GODA. In addition, we found bla TEM-57 and multiple transporters related to multidrug resistance. The availability of genomic data in open databases of this emerging zoonotic pathogen, in tandem with our comparative study, provides better understanding of R. planticola and the basis for future work.

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September 22, 2019

Genome-wide SNP and InDel mutations in Mycobacterium tuberculosis associated with rifampicin and isoniazid resistance

Objective: Multiple resistances to isoniazid and rifampicin lead to the majority of death associated with M. tuberculosis infection. This study aimed to characterize the single nucleotide polymorphisms (SNPs) and insertion and deletion (InDel) mutations associated with isoniazid and rifampicin resistance. Methods: The M. tuberculosis strain H37Rv was cultured and treated with isoniazid or rifampicin for generations. Total DNA samples from different generations were extracted for construction of DNA library, and the SNP and InDel mutation in different samples were detected by whole genome sequencing. Bioinformatics analysis such as phylogenetic tree and heap map were also performed. Results: Totally 58 nonsynonymous SNP mutations, 64 synonymous SNP mutations, and 99 SNP mutations in intergenic regions were detected in M. tuberculosis strains treated with rifampicin or isoniazid. Seven InDel mutations were found in the intergenic regions, and also six frameshift InDel mutation and three non- frameshift InDel mutations were also characterized. The phylogenetic tree showed clustering of all samples into three main subgroups. A great number of known and newly identified genes associated with drug resistance were detected in M. tuberculosis, showing distinct mutation patterns. Conclusion: By whole genome sequencing, many genetic mutations in both known and new genes associated with isoniazid and rifampicin resistance were charac- terized in M. tuberculosis.

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September 22, 2019

Characterization of Haemophilus parasuis serovar 2 CL120103, a moderately virulent strain in China

Haemophilus parasuis is an important bacterium affecting pigs, causing Glässer’s disease. To further characterize this species, we determined the complete genomic sequence of H. parasuis CL120103, which was isolated from diseased pigs. The strain H. parasuis CL120103 was identified as serovar 2. The size of the largest scaffold is 2,326,318 bp and contains 145 large contigs, with the N50 contig being 20,573 bp in length. The complete genome of H. parasuis CL120103 is 2,305,354 bp in length with 39.97% GC content and contains 2227 protein-coding genes, 19 ribosomal rRNA operons and 60 tRNA genes. Sequence similarity of the genome of H. parasuis CL120103 to the previously sequenced genome of H. parasuis was up to 96% and query cover to 86%. Annotation of the genome of H. parasuis CL120103 identified a number of genes encoding potential virulence factors. These virulence factors are involved in metabolism, adhesion, secretion and LPS biosynthesis. These related genes pave the way to better understand mechanisms underlying metabolic capabilities. The comprehensive genetic and phylogenetic analysis shows that H. parasuis is closely related to Actinobacillus pleuropneumoniae and provides a foundation for future experimental confirmation of the virulence and pathogen-host interactions in H. parasuis.

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September 22, 2019

Comparative genomics of Salmonella enterica serovar Montevideo reveals lineage-specific gene differences that may influence ecological niche association.

Salmonella enterica serovar Montevideo has been linked to recent foodborne illness outbreaks resulting from contamination of products such as fruits, vegetables, seeds and spices. Studies have shown that Montevideo also is frequently associated with healthy cattle and can be isolated from ground beef, yet human salmonellosis outbreaks of Montevideo associated with ground beef contamination are rare. This disparity fuelled our interest in characterizing the genomic differences between Montevideo strains isolated from healthy cattle and beef products, and those isolated from human patients and outbreak sources. To that end, we sequenced 13 Montevideo strains to completion, producing high-quality genome assemblies of isolates from human patients (n=8) or from healthy cattle at slaughter (n=5). Comparative analysis of sequence data from this study and publicly available sequences (n=72) shows that Montevideo falls into four previously established clades, differentially occupied by cattle and human strains. The results of these analyses reveal differences in metabolic islands, environmental adhesion determinants and virulence factors within each clade, and suggest explanations for the infrequent association between bovine isolates and human illnesses.

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September 22, 2019

Distinct genomic features characterize two clades of Corynebacterium diphtheriae: Proposal of Corynebacterium diphtheriae subsp. diphtheriae subsp. nov. and Corynebacterium diphtheriae subsp. lausannense subsp. nov.

Corynebacterium diphtheriae is the etiological agent of diphtheria, a disease caused by the presence of the diphtheria toxin. However, an increasing number of records report non-toxigenic C. diphtheriae infections. Here, a C. diphtheriae strain was recovered from a patient with a past history of bronchiectasis who developed a severe tracheo-bronchitis with multiple whitish lesions of the distal trachea and the mainstem bronchi. Whole-genome sequencing (WGS), performed in parallel with PCR targeting the toxin gene and the Elek test, provided clinically relevant results in a short turnaround time, showing that the isolate was non-toxigenic. A comparative genomic analysis of the new strain (CHUV2995) with 56 other publicly available genomes of C. diphtheriae revealed that the strains CHUV2995, CCUG 5865 and CMCNS703 share a lower average nucleotide identity (ANI) (95.24 to 95.39%) with the C. diphtheriae NCTC 11397T reference genome than all other C. diphtheriae genomes (>98.15%). Core genome phylogeny confirmed the presence of two monophyletic clades. Based on these findings, we propose here two new C. diphtheriae subspecies to replace the lineage denomination used in previous multilocus sequence typing studies: C. diphtheriae subsp. lausannense subsp. nov. (instead of lineage-2), regrouping strains CHUV2995, CCUG 5865, and CMCNS703, and C. diphtheriae subsp. diphtheriae subsp. nov, regrouping all other C. diphtheriae in the dataset (instead of lineage-1). Interestingly, members of subspecies lausannense displayed a larger genome size than subspecies diphtheriae and were enriched in COG categories related to transport and metabolism of lipids (I) and inorganic ion (P). Conversely, they lacked all genes involved in the synthesis of pili (SpaA-type, SpaD-type and SpaH-type), molybdenum cofactor and of the nitrate reductase. Finally, the CHUV2995 genome is particularly enriched in mobility genes and harbors several prophages. The genome encodes a type II-C CRISPR-Cas locus with 2 spacers that lacks csn2 or cas4, which could hamper the acquisition of new spacers and render strain CHUV2995 more susceptible to bacteriophage infections and gene acquisition through various mechanisms of horizontal gene transfer.

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