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September 22, 2019

Genomic structural variations affecting virulence during clonal expansion of Pseudomonas syringae pv. actinidiae biovar 3 in Europe.

Pseudomonas syringae pv. actinidiae (Psa) biovar 3 caused pandemic bacterial canker of Actinidia chinensis and Actinidia deliciosa since 2008. In Europe, the disease spread rapidly in the kiwifruit cultivation areas from a single introduction. In this study, we investigated the genomic diversity of Psa biovar 3 strains during the primary clonal expansion in Europe using single molecule real-time (SMRT), Illumina and Sanger sequencing technologies. We recorded evidences of frequent mobilization and loss of transposon Tn6212, large chromosome inversions, and ectopic integration of IS sequences (remarkably ISPsy31, ISPsy36, and ISPsy37). While no phenotype change associated with Tn6212 mobilization could be detected, strains CRAFRU 12.29 and CRAFRU 12.50 did not elicit the hypersensitivity response (HR) on tobacco and eggplant leaves and were limited in their growth in kiwifruit leaves due to insertion of ISPsy31 and ISPsy36 in the hrpS and hrpR genes, respectively, interrupting the hrp cluster. Both strains had been isolated from symptomatic plants, suggesting coexistence of variant strains with reduced virulence together with virulent strains in mixed populations. The structural differences caused by rearrangements of self-genetic elements within European and New Zealand strains were comparable in number and type to those occurring among the European strains, in contrast with the significant difference in terms of nucleotide polymorphisms. We hypothesize a relaxation, during clonal expansion, of the selection limiting the accumulation of deleterious mutations associated with genome structural variation due to transposition of mobile elements. This consideration may be relevant when evaluating strategies to be adopted for epidemics management.

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September 22, 2019

Comparative genomics of cocci-shaped Sporosarcina strains with diverse spatial isolation.

Cocci-shaped Sporosarcina strains are currently one of the few known cocci-shaped spore-forming bacteria, yet we know very little about the genomics. The goal of this study is to utilize comparative genomics to investigate the diversity of cocci-shaped Sporosarcina strains that differ in their geographical isolation and show different nutritional requirements.For this study, we sequenced 28 genomes of cocci-shaped Sporosarcina strains isolated from 13 different locations around the world. We generated the first six complete genomes and methylomes utilizing PacBio sequencing, and an additional 22 draft genomes using Illumina sequencing. Genomic analysis revealed that cocci-shaped Sporosarcina strains contained an average genome of 3.3 Mb comprised of 3222 CDS, 54 tRNAs and 6 rRNAs, while only two strains contained plasmids. The cocci-shaped Sporosarcina genome on average contained 2.3 prophages and 15.6 IS elements, while methylome analysis supported the diversity of these strains as only one of 31 methylation motifs were shared under identical growth conditions. Analysis with a 90% identity cut-off revealed 221 core genes or ~?7% of the genome, while a 30% identity cut-off generated a pan-genome of 8610 genes. The phylogenetic relationship of the cocci-shaped Sporosarcina strains based on either core genes, accessory genes or spore-related genes consistently resulted in the 29 strains being divided into eight clades.This study begins to unravel the phylogenetic relationship of cocci-shaped Sporosarcina strains, and the comparative genomics of these strains supports identification of several new species.

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September 22, 2019

Genetic relationships among multidrug-resistant Salmonella enterica serovar Typhimurium strains from humans and animals.

We identified 20 to 22 resistance genes, carried in four incompatibility groups of plasmids, in each of five genetically closely related Salmonella enterica serovar Typhimurium strains recovered from humans, pigs, and chickens. The genes conferred resistance to aminoglycosides, chloramphenicol, sulfonamides, trimethoprim, tetracycline, fluoroquinolones, extended-spectrum cephalosporins and cefoxitin, and azithromycin. This study demonstrates the transmission of multidrug-resistant Salmonella strains among humans and food animals and may be the first identification of mphA in azithromycin-resistant Salmonella strains in Taiwan. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Double insertion of transposable elements provides a substrate for the evolution of satellite DNA.

Eukaryotic genomes are replete with repeated sequences in the form of transposable elements (TEs) dispersed across the genome or as satellite arrays, large stretches of tandemly repeated sequences. Many satellites clearly originated as TEs, but it is unclear how mobile genetic parasites can transform into megabase-sized tandem arrays. Comprehensive population genomic sampling is needed to determine the frequency and generative mechanisms of tandem TEs, at all stages from their initial formation to their subsequent expansion and maintenance as satellites. The best available population resources, short-read DNA sequences, are often considered to be of limited utility for analyzing repetitive DNA due to the challenge of mapping individual repeats to unique genomic locations. Here we develop a new pipeline called ConTExt that demonstrates that paired-end Illumina data can be successfully leveraged to identify a wide range of structural variation within repetitive sequence, including tandem elements. By analyzing 85 genomes from five populations of Drosophila melanogaster, we discover that TEs commonly form tandem dimers. Our results further suggest that insertion site preference is the major mechanism by which dimers arise and that, consequently, dimers form rapidly during periods of active transposition. This abundance of TE dimers has the potential to provide source material for future expansion into satellite arrays, and we discover one such copy number expansion of the DNA transposon hobo to approximately 16 tandem copies in a single line. The very process that defines TEs-transposition-thus regularly generates sequences from which new satellites can arise.© 2018 McGurk and Barbash; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019

Multiple convergent supergene evolution events in mating-type chromosomes.

Convergent adaptation provides unique insights into the predictability of evolution and ultimately into processes of biological diversification. Supergenes (beneficial gene linkage) are striking examples of adaptation, but little is known about their prevalence or evolution. A recent study on anther-smut fungi documented supergene formation by rearrangements linking two key mating-type loci, controlling pre- and post-mating compatibility. Here further high-quality genome assemblies reveal four additional independent cases of chromosomal rearrangements leading to regions of suppressed recombination linking these mating-type loci in closely related species. Such convergent transitions in genomic architecture of mating-type determination indicate strong selection favoring linkage of mating-type loci into cosegregating supergenes. We find independent evolutionary strata (stepwise recombination suppression) in several species, with extensive rearrangements, gene losses, and transposable element accumulation. We thus show remarkable convergence in mating-type chromosome evolution, recurrent supergene formation, and repeated evolution of similar phenotypes through different genomic changes.

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September 22, 2019

In vitro DNA SCRaMbLE.

The power of synthetic biology has enabled the expression of heterologous pathways in cells, as well as genome-scale synthesis projects. The complexity of biological networks makes rational de novo design a grand challenge. Introducing features that confer genetic flexibility is a powerful strategy for downstream engineering. Here we develop an in vitro method of DNA library construction based on structural variation to accomplish this goal. The “in vitro SCRaMbLE system” uses Cre recombinase mixed in a test tube with purified DNA encoding multiple loxPsym sites. Using a ß-carotene pathway designed for expression in yeast as an example, we demonstrate top-down and bottom-up in vitro SCRaMbLE, enabling optimization of biosynthetic pathway flux via the rearrangement of relevant transcription units. We show that our system provides a straightforward way to correlate phenotype and genotype and is potentially amenable to biochemical optimization in ways that the in vivo system cannot achieve.

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September 22, 2019

Precise control of SCRaMbLE in synthetic haploid and diploid yeast.

Compatibility between host cells and heterologous pathways is a challenge for constructing organisms with high productivity or gain of function. Designer yeast cells incorporating the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system provide a platform for generating genotype diversity. Here we construct a genetic AND gate to enable precise control of the SCRaMbLE method to generate synthetic haploid and diploid yeast with desired phenotypes. The yield of carotenoids is increased to 1.5-fold by SCRaMbLEing haploid strains and we determine that the deletion of YEL013W is responsible for the increase. Based on the SCRaMbLEing in diploid strains, we develop a strategy called Multiplex SCRaMbLE Iterative Cycling (MuSIC) to increase the production of carotenoids up to 38.8-fold through 5 iterative cycles of SCRaMbLE. This strategy is potentially a powerful tool for increasing the production of bio-based chemicals and for mining deep knowledge.

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September 22, 2019

Genome-wide analysis of Mycoplasma bovirhinis GS01 reveals potential virulence factors and phylogenetic relationships.

Mycoplasma bovirhinis is a significant etiology in bovine pneumonia and mastitis, but our knowledge about the genetic and pathogenic mechanisms of M. bovirhinis is very limited. In this study, we sequenced the complete genome of M. bovirhinis strain GS01 isolated from the nasal swab of pneumonic calves in Gansu, China, and we found that its genome forms a 847,985 bp single circular chromosome with a GC content of 27.57% and with 707 protein-coding genes. The putative virulence determinants of M. bovirhinis were then analyzed. Results showed that three genomic islands and 16 putative virulence genes, including one adhesion gene enolase, seven surface lipoproteins, proteins involved in glycerol metabolism, and cation transporters, might be potential virulence factors. Glycerol and pyruvate metabolic pathways were defective. Comparative analysis revealed remarkable genome variations between GS01 and a recently reported HAZ141_2 strain, and extremely low homology with others mycoplasma species. Phylogenetic analysis demonstrated that M. bovirhinis was most genetically close to M. canis, distant from other bovine Mycoplasma species. Genomic dissection may provide useful information on the pathogenic mechanisms and genetics of M. bovirhinis. Copyright © 2018 Chen et al.

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September 22, 2019

Multiple large inversions and breakpoint rewiring of gene expression in the evolution of the fire ant social supergene.

Supergenes consist of co-adapted loci that segregate together and are associated with adaptive traits. In the fire ant Solenopsis invicta, two ‘social’ supergene variants regulate differences in colony queen number and other traits. Suppressed recombination in this system is maintained, in part, by a greater than 9 Mb inversion, but the supergene is larger. Has the supergene in S. invicta undergone multiple large inversions? The initial gene content of the inverted allele of a supergene would be the same as that of the wild-type allele. So, how did the inversion increase in frequency? To address these questions, we cloned one extreme breakpoint in the fire ant supergene. In doing so, we found a second large (greater than 800 Kb) rearrangement. Furthermore, we determined the temporal order of the two big inversions based on the translocation pattern of a third small fragment. Because the S. invicta supergene lacks evolutionary strata, our finding of multiple inversions may support an introgression model of the supergene. Finally, we showed that one of the inversions swapped the promoter of a breakpoint-adjacent gene, which might have conferred a selective advantage relative to the non-inverted allele. Our findings provide a rare example of gene alterations arising directly from an inversion event.© 2018 The Author(s).

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September 22, 2019

Signatures of host specialization and a recent transposable element burst in the dynamic one-speed genome of the fungal barley powdery mildew pathogen.

Powdery mildews are biotrophic pathogenic fungi infecting a number of economically important plants. The grass powdery mildew, Blumeria graminis, has become a model organism to study host specialization of obligate biotrophic fungal pathogens. We resolved the large-scale genomic architecture of B. graminis forma specialis hordei (Bgh) to explore the potential influence of its genome organization on the co-evolutionary process with its host plant, barley (Hordeum vulgare).The near-chromosome level assemblies of the Bgh reference isolate DH14 and one of the most diversified isolates, RACE1, enabled a comparative analysis of these haploid genomes, which are highly enriched with transposable elements (TEs). We found largely retained genome synteny and gene repertoires, yet detected copy number variation (CNV) of secretion signal peptide-containing protein-coding genes (SPs) and locally disrupted synteny blocks. Genes coding for sequence-related SPs are often locally clustered, but neither the SPs nor the TEs reside preferentially in genomic regions with unique features. Extended comparative analysis with different host-specific B. graminis formae speciales revealed the existence of a core suite of SPs, but also isolate-specific SP sets as well as congruence of SP CNV and phylogenetic relationship. We further detected evidence for a recent, lineage-specific expansion of TEs in the Bgh genome.The characteristics of the Bgh genome (largely retained synteny, CNV of SP genes, recently proliferated TEs and a lack of significant compartmentalization) are consistent with a “one-speed” genome that differs in its architecture and (co-)evolutionary pattern from the “two-speed” genomes reported for several other filamentous phytopathogens.

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September 22, 2019

Gene duplication and evolution dynamics in the homeologous regions harboring multiple prolamin and resistance gene families in hexaploid wheat.

Improving end-use quality and disease resistance are important goals in wheat breeding. The genetic loci controlling these traits are highly complex, consisting of large families of prolamin and resistance genes with members present in all three homeologous A, B, and D genomes in hexaploid bread wheat. Here, orthologous regions harboring both prolamin and resistance gene loci were reconstructed and compared to understand gene duplication and evolution in different wheat genomes. Comparison of the two orthologous D regions from the hexaploid wheat Chinese Spring and the diploid progenitor Aegilops tauschii revealed their considerable difference due to the presence of five large structural variations with sizes ranging from 100 kb to 2 Mb. As a result, 44% of the Ae. tauschii and 71% of the Chinese Spring sequences in the analyzed regions, including 79 genes, are not shared. Gene rearrangement events, including differential gene duplication and deletion in the A, B, and D regions, have resulted in considerable erosion of gene collinearity in the analyzed regions, suggesting rapid evolution of prolamin and resistance gene families after the separation of the three wheat genomes. We hypothesize that this fast evolution is attributed to the co-evolution of the two gene families dispersed within a high recombination region. The identification of a full set of prolamin genes facilitated transcriptome profiling and revealed that the A genome contributes the least to prolamin expression because of its smaller number of expressed intact genes and their low expression levels, while the B and D genomes contribute similarly.

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September 22, 2019

Extensive exchange of transposable elements in the Drosophila pseudoobscura group.

As species diverge, so does their transposable element (TE) content. Within a genome, TE families may eventually become dormant due to host-silencing mechanisms, natural selection and the accumulation of inactive copies. The transmission of active copies from a TE families, both vertically and horizontally between species, can allow TEs to escape inactivation if it occurs often enough, as it may allow TEs to temporarily escape silencing in a new host. Thus, the contribution of horizontal exchange to TE persistence has been of increasing interest.Here, we annotated TEs in five species with sequenced genomes from the D. pseudoobscura species group, and curated a set of TE families found in these species. We found that, compared to host genes, many TE families showed lower neutral divergence between species, consistent with recent transmission of TEs between species. Despite these transfers, there are differences in the TE content between species in the group.The TE content is highly dynamic in the D. pseudoobscura species group, frequently transferring between species, keeping TEs active. This result highlights how frequently transposable elements are transmitted between sympatric species and, despite these transfers, how rapidly species TE content can diverge.

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September 22, 2019

Diversity and evolution of the emerging Pandoraviridae family.

With DNA genomes reaching 2.5?Mb packed in particles of bacterium-like shape and dimension, the first two Acanthamoeba-infecting pandoraviruses remained up to now the most complex viruses since their discovery in 2013. Our isolation of three new strains from distant locations and environments is now used to perform the first comparative genomics analysis of the emerging worldwide-distributed Pandoraviridae family. Thorough annotation of the genomes combining transcriptomic, proteomic, and bioinformatic analyses reveals many non-coding transcripts and significantly reduces the former set of predicted protein-coding genes. Here we show that the pandoraviruses exhibit an open pan-genome, the enormous size of which is not adequately explained by gene duplications or horizontal transfers. As most of the strain-specific genes have no extant homolog and exhibit statistical features comparable to intergenic regions, we suggest that de novo gene creation could contribute to the evolution of the giant pandoravirus genomes.

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September 22, 2019

A reference genome of the European beech (Fagus sylvatica L.).

The European beech is arguably the most important climax broad-leaved tree species in Central Europe, widely planted for its valuable wood. Here, we report the 542 Mb draft genome sequence of an up to 300-year-old individual (Bhaga) from an undisturbed stand in the Kellerwald-Edersee National Park in central Germany.Using a hybrid assembly approach, Illumina reads with short- and long-insert libraries, coupled with long Pacific Biosciences reads, we obtained an assembled genome size of 542 Mb, in line with flow cytometric genome size estimation. The largest scaffold was of 1.15 Mb, the N50 length was 145 kb, and the L50 count was 983. The assembly contained 0.12% of Ns. A Benchmarking with Universal Single-Copy Orthologs (BUSCO) analysis retrieved 94% complete BUSCO genes, well in the range of other high-quality draft genomes of trees. A total of 62,012 protein-coding genes were predicted, assisted by transcriptome sequencing. In addition, we are reporting an efficient method for extracting high-molecular-weight DNA from dormant buds, by which contamination by environmental bacteria and fungi was kept at a minimum.The assembled genome will be a valuable resource and reference for future population genomics studies on the evolution and past climate change adaptation of beech and will be helpful for identifying genes, e.g., involved in drought tolerance, in order to select and breed individuals to adapt forestry to climate change in Europe. A continuously updated genome browser and download page can be accessed from beechgenome.net, which will include future genome versions of the reference individual Bhaga, as new sequencing approaches develop.

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September 22, 2019

Raising the stakes: Loss of efflux pump regulation decreases meropenem susceptibility in Burkholderia pseudomallei

Burkholderia pseudomallei, the causative agent of the high-mortality disease melioidosis, is a gram-negative bacterium that is naturally resistant to many antibiotics. There is no vaccine for melioidosis, and effective eradication is reliant on biphasic and prolonged antibiotic administration. The carbapenem drug meropenem is the current gold standard option for treating severe melioidosis. Intrinsic B. pseudomallei resistance toward meropenem has not yet been documented; however, resistance could conceivably develop over the course of infection, leading to prolonged sepsis and treatment failure.We examined our 30-year clinical collection of melioidosis cases to identify B. pseudomallei isolates with reduced meropenem susceptibility. Isolates were subjected to minimum inhibitory concentration (MIC) testing toward meropenem. Paired isolates from patients who had evolved decreased susceptibility were subjected to whole-genome sequencing. Select agent-compliant genetic manipulation was carried out to confirm the molecular mechanisms conferring resistance.We identified 11 melioidosis cases where B. pseudomallei isolates developed decreased susceptibility toward meropenem during treatment, including 2 cases not treated with this antibiotic. Meropenem MICs increased from 0.5-0.75 µg/mL to 3-8 µg/mL. Comparative genomics identified multiple mutations affecting multidrug resistance-nodulation-division (RND) efflux pump regulators, with concomitant overexpression of their corresponding pumps. All cases were refractory to treatment despite aggressive, targeted therapy, and 2 were associated with a fatal outcome.This study confirms the role of RND efflux pumps in decreased meropenem susceptibility in B. pseudomallei. These findings have important ramifications for the diagnosis, treatment, and management of life-threatening melioidosis cases.

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