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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  |  

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  |  

Comparative genomics reveal a flagellar system, a type VI secretion system and plant growth-promoting gene clusters unique to the endophytic bacterium Kosakonia radicincitans.

The recent worldwide discovery of plant growth-promoting (PGP) Kosakonia radicincitans in a large variety of crop plants suggests that this species confers significant influence on plants, both in terms of yield increase and product quality improvement. We provide a comparative genome analysis which helps to unravel the genetic basis for K. radicincitans’ motility, competitiveness and plant growth-promoting capacities. We discovered that K. radicincitans carries multiple copies of complex gene clusters, among them two flagellar systems and three type VI secretion systems (T6SSs). We speculate that host invasion may be facilitated by different flagella, and bacterial competitor suppression by effector proteins ejected via T6SSs. We found a large plasmid in K. radicincitans DSM 16656T, the species type strain, that confers the potential to exploit plant-derived carbon sources. We propose that multiple copies of complex gene clusters in K. radicincitans are metabolically expensive but provide competitive advantage over other bacterial strains in nutrient-rich environments. The comparison of the DSM 16656T genome to genomes of other genera of enteric plant growth-promoting bacteria (PGPB) exhibits traits unique to DSM 16656T and K. radicincitans, respectively, and traits shared between genera. We used the output of the in silico analysis for predicting the purpose of genomic features unique to K. radicincitans and performed microarray, PhyloChip, and microscopical analyses to gain deeper insight into the interaction of DSM 16656T, plants and associated microbiota. The comparative genome analysis will facilitate the future search for promising candidates of PGPB for sustainable crop production.

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September 22, 2019  |  

Comparison of highly and weakly virulent Dickeya solani strains, with a view on the pangenome and panregulon of this species.

Bacteria belonging to the genera Dickeya and Pectobacterium are responsible for significant economic losses in a wide variety of crops and ornamentals. During last years, increasing losses in potato production have been attributed to the appearance of Dickeya solani. The D. solani strains investigated so far share genetic homogeneity, although different virulence levels were observed among strains of various origins. The purpose of this study was to investigate the genetic traits possibly related to the diverse virulence levels by means of comparative genomics. First, we developed a new genome assembly pipeline which allowed us to complete the D. solani genomes. Four de novo sequenced and ten publicly available genomes were used to identify the structure of the D. solani pangenome, in which 74.8 and 25.2% of genes were grouped into the core and dispensable genome, respectively. For D. solani panregulon analysis, we performed a binding site prediction for four transcription factors, namely CRP, KdgR, PecS and Fur, to detect the regulons of these virulence regulators. Most of the D. solani potential virulence factors were predicted to belong to the accessory regulons of CRP, KdgR, and PecS. Thus, some differences in gene expression could exist between D. solani strains. The comparison between a highly and a low virulent strain, IFB0099 and IFB0223, respectively, disclosed only small differences between their genomes but significant differences in the production of virulence factors like pectinases, cellulases and proteases, and in their mobility. The D. solani strains also diverge in the number and size of prophages present in their genomes. Another relevant difference is the disruption of the adhesin gene fhaB2 in the highly virulent strain. Strain IFB0223, which has a complete adhesin gene, is less mobile and less aggressive than IFB0099. This suggests that in this case, mobility rather than adherence is needed in order to trigger disease symptoms. This study highlights the utility of comparative genomics in predicting D. solani traits involved in the aggressiveness of this emerging plant pathogen.

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September 22, 2019  |  

Genomic analysis for heavy metal resistance in S. maltophilia

Stenotrophomonas maltophilia is highly resistant to heavy metals, but the genetic knowledge of metal resistance in S. maltophilia is poorly understood. In this study, the genome of S. maltophilia Pho isolated from the contaminated soil near a metalwork factory was sequenced using PacBio RS II. Its genome is composed of a single chromosome with a GC content of 66.4% and 4434 protein-encoding genes. Comparative analysis revealed high syntney between S. maltophilia Pho and the model strain, S. maltophilia K279a. Then, the type and number of mechanisms of heavy metal uptake were analyzed firstly. Results showed that 7 unspecific ion transporter genes and 13 specific ion transporter genes, most of which were involved in iron transport. But the sulfate permeases belonging to the family of SulT/CysP that can uptake chromate and the high affinity ZnuABC/SitABCD were absent. Secondly, the putative genes controlling metal efflux were analyzed. Results showed that this bacterium encoded 5 CDFs, 1 copper exporting ATPase and 4 RND systems, including 2 CzcABC efflux pumps. Moreover, the putative metal transformation genes including arsenate and mercury detoxification genes were also identified. This study may provide useful information on the metal resistance mechanisms of S. maltophilia.

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September 22, 2019  |  

Genomics of Corynebacterium striatum, an emerging multidrug-resistant pathogen of immunocompromised patients.

Corynebacterium striatum is an emerging multidrug-resistant (MDR) pathogen of immunocompromised and chronically ill patients. The objective of these studies was to provide a detailed genomic analysis of disease-causing C. striatum and determine the genomic drivers of resistance and resistance-gene transmission.A multi-institutional and prospective pathogen genomics programme flagged seven MDR C. striatum infections occurring close in time, and specifically in immunocompromised patients with underlying respiratory diseases. Whole genome sequencing was used to identify clonal relationships among strains, genetic causes of antimicrobial resistance, and their mobilization capacity. Matrix-assisted linear desorption/ionization-time-of-flight analyses of sequenced isolates provided curated content to improve rapid clinical identification in subsequent cases.Epidemiological and genomic analyses identified a related cluster of three out of seven C. striatum among lung transplant patients who had common procedures and exposures at an outlying institution. Genomic analyses further elucidated drivers of the MDR phenotypes, including resistance genes mobilized by IS3504 and ISCg9a-like insertion sequences. Seven mobilizable resistance genes were localized to a common chromosomal region bounded by unpaired insertion sequences, suggesting that a single recombination event could spread resistance to aminoglycosides, macrolides, lincosamides and tetracyclines to naive strains.In-depth genomic studies of MDR C. striatum reveal its capacity for clonal spread within and across healthcare institutions and identify novel vectors that can mobilize multiple forms of drug resistance, further complicating efforts to treat infections in immunocompromised populations. Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. All rights reserved.

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September 22, 2019  |  

Involvement of Burkholderiaceae and sulfurous volatiles in disease-suppressive soils.

Disease-suppressive soils are ecosystems in which plants suffer less from root infections due to the activities of specific microbial consortia. The characteristics of soils suppressive to specific fungal root pathogens are comparable to those of adaptive immunity in animals, as reported by Raaijmakers and Mazzola (Science 352:1392-3, 2016), but the mechanisms and microbial species involved in the soil suppressiveness are largely unknown. Previous taxonomic and metatranscriptome analyses of a soil suppressive to the fungal root pathogen Rhizoctonia solani revealed that members of the Burkholderiaceae family were more abundant and more active in suppressive than in non-suppressive soils. Here, isolation, phylogeny, and soil bioassays revealed a significant disease-suppressive activity for representative isolates of Burkholderia pyrrocinia, Paraburkholderia caledonica, P. graminis, P. hospita, and P. terricola. In vitro antifungal activity was only observed for P. graminis. Comparative genomics and metabolite profiling further showed that the antifungal activity of P. graminis PHS1 was associated with the production of sulfurous volatile compounds encoded by genes not found in the other four genera. Site-directed mutagenesis of two of these genes, encoding a dimethyl sulfoxide reductase and a cysteine desulfurase, resulted in a loss of antifungal activity both in vitro and in situ. These results indicate that specific members of the Burkholderiaceae family contribute to soil suppressiveness via the production of sulfurous volatile compounds.

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September 22, 2019  |  

Complete genome sequence of Cd(II)-resistant Arthrobacter sp. PGP41, a plant growth-promoting bacterium with potential in microbe-assisted phytoremediation.

Microbe-assisted phytoremediation has great potential for practical applications. Plant growth-promoting bacteria (PGPB) with heavy metal (HM) resistance are important for the implementation of PGPB-assisted phytoremediation of HM-contaminated environments. Arthrobacter sp. PGP41 is a Cd(II)-resistant bacterium isolated from the rhizosphere soils of a Cd(II) hyperaccumulator plant, Solanum nigrum. Strain PGP41 can significantly improve plant seedling and root growth under Cd(II) stress conditions. This bacterium exhibited the ability to produce high levels of indole-3-acetic acid (IAA), as well as the ability to fix nitrogen and solubilize phosphate, and it possessed 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Here, we present the complete genome sequence of strain PGP41. The genome consists of a single chromosome with a G+C content of 65.38% and no plasmids. The genome encodes 3898 genes and contains 49 tRNA and 12 rRNA genes. Multiple genes associated with plant growth promotion were identified in the genome. The whole genome sequence of PGP41 provides information useful for further clarifying the molecular mechanisms behind plant growth promotion by PGPB and facilitates its potential use as an inoculum in the bioremediation of HM-contaminated environments.

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September 22, 2019  |  

Genome annotation and comparative genomic analysis of Bacillus subtilis MJ01, a new bio-degradation strain isolated from oil-contaminated soil.

One of the main challenges in elimination of oil contamination from polluted environments is improvement of biodegradation by highly efficient microorganisms. Bacillus subtilis MJ01 has been evaluated as a new resource for producing biosurfactant compounds. This bacterium, which produces surfactin, is able to enhance bio-accessibility to oil hydrocarbons in contaminated soils. The genome of B. subtilis MJ01 was sequenced and assembled by PacBio RS sequencing technology. One big contig with a length of 4,108,293 bp without any gap was assembled. Genome annotation and prediction of gene showed that MJ01 genome is very similar to B. subtilis spizizenii TU-B-10 (95% similarity). The comparison and analysis of orthologous genes carried out between B. subtilis MJ01, reference strain B. subtilis subsp. subtilis str. 168, and close relative spizizenii TU-B-10 by microscope platform and various bioinformatics tools. More than 88% of 4269 predicted coding sequences in MJ01 had at least one similar sequence in genome of reference strain and spizizenii TU-B-10. Despite this high similarity, some differences were detected among encoding sequences of non-ribosome protein and bacteriocins in MJ01 and spizizenii TU-B-10. MJ01 has unique nucleotide sequences and a novel predicted lasso-peptide bacteriocin; it also has not any similar nucleotide sequence in non-redundant nucleotide data base.

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September 22, 2019  |  

Genomic analysis of multidrug-resistant Escherichia coli ST58 causing urosepsis.

Sequence type 58 (ST58) phylogroup B1 Escherichia coli have been isolated from a wide variety of mammalian and avian hosts but are not noted for their ability to cause serious disease in humans or animals. Here we determined the genome sequences of two multidrug-resistant E. coli ST58 strains from urine and blood of one patient using a combination of Illumina and Single Molecule, Real-Time (SMRT) sequencing. Both ST58 strains were clonal and were characterised as serotype O8:H25, phylogroup B1 and carried a complex resistance locus/loci (CRL) that featured an atypical class 1 integron with a dfrA5 (trimethoprim resistance) gene cassette followed by only 24 bp of the 3′-CS. CRL that carry this particular integron have been described previously in E. coli from cattle, pigs and humans in Australia. The integron abuts a copy of Tn6029, an IS26-flanked composite transposon encoding blaTEM, sul2 and strAB genes that confer resistance to ampicillin, sulfathiazole and streptomycin, respectively. The CRL resides within a novel Tn2610-like hybrid Tn1721/Tn21 transposon on an IncF, ColV plasmid (pSDJ2009-52F) of 138 553 bp that encodes virulence associated genes implicated in life-threatening extraintestinal pathogenic E. coli (ExPEC) infections. Notably, pSDJ2009-52F shares high sequence identity with pSF-088-1, a plasmid reported in an E. coli ST95 strain from a patient with blood sepsis from a hospital in San Francisco. These data suggest that extraintestinal infections caused by E. coli carrying ColV-like plasmids, irrespective of their phylogroup or ST, may pose a potential threat to human health, particularly to the elderly and immunocompromised. Copyright © 2018. Published by Elsevier B.V.

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September 22, 2019  |  

Isolation, development, and genomic analysis of Bacillus megaterium SR7 for growth and metabolite production under supercritical carbon dioxide

Supercritical carbon dioxide (scCO2) is an attractive substitute for conventional organic solvents due to its unique transport and thermodynamic properties, its renewability and labile nature, and its high solubility for compounds such as alcohols, ketones, and aldehydes. However, biological systems that use scCO2 are mainly limited to in vitro processes due to its strong inhibition of cell viability and growth. To solve this problem, we used a bioprospecting approach to isolate a microbial strain with the natural ability to grow while exposed to scCO2. Enrichment culture and serial passaging of deep subsurface fluids from the McElmo Dome scCO2 reservoir in aqueous media under scCO2 headspace enabled the isolation of spore-forming strain Bacillus megaterium SR7. Sequencing and analysis of the complete 5.51 Mbp genome and physiological characterization revealed the capacity for facultative anaerobic metabolism, including fermentative growth on a diverse range of organic substrates. Supplementation of growth medium with L-alanine for chemical induction of spore germination significantly improved growth frequencies and biomass accumulation under scCO2 headspace. Detection of endogenous fermentative compounds in cultures grown under scCO2 represents the first observation of bioproduct generation and accumulation under this condition. Culturing development and metabolic characterization of B. megaterium SR7 represent initial advancements in the effort toward enabling exploitation of scCO2 as a sustainable solvent for in vivo bioprocessing.

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September 22, 2019  |  

The genome of Naegleria lovaniensis, the basis for a comparative approach to unravel pathogenicity factors of the human pathogenic amoeba N. fowleri.

Members of the genus Naegleria are free-living eukaryotes with the capability to transform from the amoeboid form into resting cysts or moving flagellates in response to environmental conditions. More than 40 species have been characterized, but only Naegleria fowleri (N. fowleri) is known as a human pathogen causing primary amoebic meningoencephalitis (PAM), a fast progressing and mostly fatal disease of the central nervous system. Several studies report an involvement of phospholipases and other molecular factors, but the mechanisms involved in pathogenesis are still poorly understood. To gain a better understanding of the relationships within the genus of Naegleria and to investigate pathogenicity factors of N. fowleri, we characterized the genome of its closest non-pathogenic relative N. lovaniensis.To gain insights into the taxonomy of Naegleria, we sequenced the genome of N. lovaniensis using long read sequencing technology. The assembly of the data resulted in a 30 Mb genome including the circular mitochondrial sequence. Unravelling the phylogenetic relationship using OrthoMCL protein clustering and maximum likelihood methods confirms the close relationship of N. lovaniensis and N. fowleri. To achieve an overview of the diversity of Naegleria proteins and to assess characteristics of the human pathogen N. fowleri, OrthoMCL protein clustering including data of N. fowleri, N. lovaniensis and N. gruberi was performed. GO enrichment analysis shows an association of N. fowleri specific proteins to the GO terms “Membrane” and “Protein Secretion.”In this study, we characterize the hitherto unknown genome of N. lovaniensis. With the description of the 30 Mb genome, a further piece is added to reveal the complex taxonomic relationship of Naegleria. Further, the whole genome sequencing data confirms the hypothesis of the close relationship between N. fowleri and N. lovaniensis. Therefore, the genome of N. lovaniensis provides the basis for further comparative approaches on the molecular and genomic level to unravel pathogenicity factors of its closest human pathogenic relative N. fowleri and possible treatment options for the rare but mostly fatal primary meningoencephalitis.

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September 22, 2019  |  

The linear mitochondrial genome of the quarantine chytrid Synchytrium endobioticum; insights into the evolution and recent history of an obligate biotrophic plant pathogen.

Chytridiomycota species (chytrids) belong to a basal lineage in the fungal kingdom. Inhabiting terrestrial and aquatic environments, most are free-living saprophytes but several species cause important diseases: e.g. Batrachochytrium dendrobatidis, responsible for worldwide amphibian decline; and Synchytrium endobioticum, causing potato wart disease. S. endobioticum has an obligate biotrophic lifestyle and isolates can be further characterized as pathotypes based on their virulence on a differential set of potato cultivars. Quarantine measures have been implemented globally to control the disease and prevent its spread. We used a comparative approach using chytrid mitogenomes to determine taxonomical relationships and to gain insights into the evolution and recent history of introductions of this plant pathogen.We assembled and annotated the complete mitochondrial genome of 30 S. endobioticum isolates and generated mitochondrial genomes for five additional chytrid species. The mitochondrial genome of S. endobioticum is linear with terminal inverted repeats which was validated by tailing and PCR amplifying the telomeric ends. Surprisingly, no conservation in organisation and orientation of mitochondrial genes was observed among the Chytridiomycota except for S. endobioticum and its sister species Synchytrium microbalum. However, the mitochondrial genome of S. microbalum is circular and comprises only a third of the 72.9 Kbp found for S. endobioticum suggesting recent linearization and expansion. Four mitochondrial lineages were identified in the S. endobioticum mitochondrial genomes. Several pathotypes occur in different lineages, suggesting that these have emerged independently. In addition, variations for polymorphic sites in the mitochondrial genome of individual isolates were observed demonstrating that S. endobioticum isolates represent a community of different genotypes. Such communities were shown to be complex and stable over time, but we also demonstrate that the use of semi-resistant potato cultivars triggers a rapid shift in the mitochondrial haplotype associated with increased virulence.Mitochondrial genomic variation shows that S. endobioticum has been introduced into Europe multiple times, that several pathotypes emerged multiple times, and that isolates represent communities of different genotypes. Our study represents the most comprehensive dataset of chytrid mitogenomes, which provides new insights into the extraordinary dynamics and evolution of mitochondrial genomes involving linearization, expansion and reshuffling.

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