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September 22, 2019  |  

Redefinition and unification of the SXT/R391 family of integrative and conjugative elements.

Integrative and conjugative elements (ICEs) of the SXT/R391 family are key drivers of the spread of antibiotic resistance in Vibrio cholerae, the infectious agent of cholera, and other pathogenic bacteria. The SXT/R391 family of ICEs was defined based on the conservation of a core set of 52 genes and site-specific integration into the 5′ end of the chromosomal gene prfC Hence, the integrase gene int has been intensively used as a marker to detect SXT/R391 ICEs in clinical isolates. ICEs sharing most core genes but differing by their integration site and integrase gene have been recently reported and excluded from the SXT/R391 family. Here we explored the prevalence and diversity of atypical ICEs in GenBank databases and their relationship with typical SXT/R391 ICEs. We found atypical ICEs in V. cholerae isolates that predate the emergence and expansion of typical SXT/R391 ICEs in the mid-1980s in seventh-pandemic toxigenic V. cholerae strains O1 and O139. Our analyses revealed that while atypical ICEs are not associated with antibiotic resistance genes, they often carry cation efflux pumps, suggesting heavy metal resistance. Atypical ICEs constitute a polyphyletic group likely because of occasional recombination events with typical ICEs. Furthermore, we show that the alternative integration and excision genes of atypical ICEs remain under the control of SetCD, the main activator of the conjugative functions of SXT/R391 ICEs. Together, these observations indicate that substitution of the integration/excision module and change of specificity of integration do not preclude atypical ICEs from inclusion into the SXT/R391 family.IMPORTANCEVibrio cholerae is the causative agent of cholera, an acute intestinal infection that remains to this day a world public health threat. Integrative and conjugative elements (ICEs) of the SXT/R391 family have played a major role in spreading antimicrobial resistance in seventh-pandemic V. cholerae but also in several species of Enterobacteriaceae Most epidemiological surveys use the integrase gene as a marker to screen for SXT/R391 ICEs in clinical or environmental strains. With the recent reports of closely related elements that carry an alternative integrase gene, it became urgent to investigate whether ICEs that have been left out of the family are a liability for the accuracy of such screenings. In this study, based on comparative genomics, we broaden the SXT/R391 family of ICEs to include atypical ICEs that are often associated with heavy metal resistance. Copyright © 2018 American Society for Microbiology.

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September 22, 2019  |  

Genome analysis of the ancient tracheophyte Selaginella tamariscina reveals evolutionary features relevant to the acquisition of desiccation tolerance.

Resurrection plants, which are the “gifts” of natural evolution, are ideal models for studying the genetic basis of plant desiccation tolerance. Here, we report a high-quality genome assembly of 301 Mb for the diploid spike moss Selaginella tamariscina, a primitive vascular resurrection plant. We predicated 27 761 protein-coding genes from the assembled S. tamariscina genome, 11.38% (2363) of which showed significant expression changes in response to desiccation. Approximately 60.58% of the S. tamariscina genome was annotated as repetitive DNA, which is an almost 2-fold increase of that in the genome of desiccation-sensitive Selaginella moellendorffii. Genomic and transcriptomic analyses highlight the unique evolution and complex regulations of the desiccation response in S. tamariscina, including species-specific expansion of the oleosin and pentatricopeptide repeat gene families, unique genes and pathways for reactive oxygen species generation and scavenging, and enhanced abscisic acid (ABA) biosynthesis and potentially distinct regulation of ABA signaling and response. Comparative analysis of chloroplast genomes of several Selaginella species revealed a unique structural rearrangement and the complete loss of chloroplast NAD(P)H dehydrogenase (NDH) genes in S. tamariscina, suggesting a link between the absence of the NDH complex and desiccation tolerance. Taken together, our comparative genomic and transcriptomic analyses reveal common and species-specific desiccation tolerance strategies in S. tamariscina, providing significant insights into the desiccation tolerance mechanism and the evolution of resurrection plants. Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.

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September 22, 2019  |  

Horizontal transfer of BovB and L1 retrotransposons in eukaryotes.

Transposable elements (TEs) are mobile DNA sequences, colloquially known as jumping genes because of their ability to replicate to new genomic locations. TEs can jump between organisms or species when given a vector of transfer, such as a tick or virus, in a process known as horizontal transfer. Here, we propose that LINE-1 (L1) and Bovine-B (BovB), the two most abundant TE families in mammals, were initially introduced as foreign DNA via ancient horizontal transfer events.Using analyses of 759 plant, fungal and animal genomes, we identify multiple possible L1 horizontal transfer events in eukaryotic species, primarily involving Tx-like L1s in marine eukaryotes. We also extend the BovB paradigm by increasing the number of estimated transfer events compared to previous studies, finding new parasite vectors of transfer such as bed bug, leech and locust, and BovB occurrences in new lineages such as bat and frog. Given that these transposable elements have colonised more than half of the genome sequence in today’s mammals, our results support a role for horizontal transfer in causing long-term genomic change in new host organisms.We describe extensive horizontal transfer of BovB retrotransposons and provide the first evidence that L1 elements can also undergo horizontal transfer. With the advancement of genome sequencing technologies and bioinformatics tools, we anticipate our study to be a valuable resource for inferring horizontal transfer from large-scale genomic data.

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September 22, 2019  |  

Complete genome sequencing and comparative genomic analysis of Helicobacter apodemus isolated from the wild Korean striped field mouse (Apodemus agrarius) for potential pathogenicity

The Helicobacter bacterial genus comprises of spiral-shaped gram-negative bacteria with flagella that colonize the gastro-intestinal (GI) tract of humans and various mammals (Solnick and Schauer, 2001). In particular, Helicobacter pylori was classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC) in 1994, and has been shown to occur with a high prevalence in humans, although this varies between geographical regions, ethnic groups, and various populations (Kusters et al., 2006; Goh et al., 2011). To date, more than 37 Helicobacter species have been identified in addition to H. pylori (Péré-Védrenne et al., 2017). Furthermore, non-H. pylori Helicobacters (NHPH) have been shown to infect both humans and animals, and NHPH infections are associated with intestinal carcinoma, and mucinous adenocarcinoma (Swennes et al., 2016). Despite the demonstrated association between NHPH and disease, most studies to date have investigated H. pylori in humans; thus, it is necessary to characterize NHPH and elucidate its role in the GI tract of wild rodents which are potential Helicobacter carriers (Taylor et al., 2007; Mladenova-Hristova et al., 2017).

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September 22, 2019  |  

An outbreak of a rare Shiga-toxin-producing Escherichia coli serotype (O117:H7) among men who have sex with men.

Sexually transmissible enteric infections (STEIs) are commonly associated with transmission among men who have sex with men (MSM). In the past decade, the UK has experienced multiple parallel STEI emergences in MSM caused by a range of bacterial species of the genus Shigella, and an outbreak of an uncommon serotype (O117?:?H7) of Shiga-toxin-producing Escherichia coli (STEC). Here, we used microbial genomics on 6 outbreak and 30 sporadic STEC O117?:?H7 isolates to explore the origins and pathogenic drivers of the STEC O117?:?H7 emergence in MSM. Using genomic epidemiology, we found that the STEC O117?:?H7 outbreak lineage was potentially imported from Latin America and likely continues to circulate both in the UK MSM population and in Latin America. We found genomic relationships consistent with existing symptomatic evidence for chronic infection with this STEC serotype. Comparative genomic analysis indicated the existence of a novel Shiga toxin 1-encoding prophage in the outbreak isolates, and evidence of horizontal gene exchange among the STEC O117?:?H7 outbreak lineage and other enteric pathogens. There was no evidence of increased virulence in the outbreak strains relative to contextual isolates, but the outbreak lineage was associated with azithromycin resistance. Comparing these findings with similar genomic investigations of emerging MSM-associated Shigella in the UK highlighted many parallels, the most striking of which was the importance of the azithromycin phenotype for STEI emergence in this patient group.

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September 22, 2019  |  

Potential survival and pathogenesis of a novel strain, Vibrio parahaemolyticus FORC_022, isolated from a soy sauce marinated crab by genome and transcriptome analyses.

Vibrio parahaemolyticus can cause gastrointestinal illness through consumption of seafood. Despite frequent food-borne outbreaks of V. parahaemolyticus, only 19 strains have subjected to complete whole-genome analysis. In this study, a novel strain of V. parahaemolyticus, designated FORC_022 (Food-borne pathogen Omics Research Center_022), was isolated from soy sauce marinated crabs, and its genome and transcriptome were analyzed to elucidate the pathogenic mechanisms. FORC_022 did not include major virulence factors of thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). However, FORC_022 showed high cytotoxicity and had several V. parahaemolyticus islands (VPaIs) and other virulence factors, such as various secretion systems (types I, II, III, IV, and VI), in comparative genome analysis with CDC_K4557 (the most similar strain) and RIMD2210633 (genome island marker strain). FORC_022 harbored additional virulence genes, including accessory cholera enterotoxin, zona occludens toxin, and tight adhesion (tad) locus, compared with CDC_K4557. In addition, O3 serotype specific gene and the marker gene of pandemic O3:K6 serotype (toxRS) were detected in FORC_022. The expressions levels of genes involved in adherence and carbohydrate transporter were high, whereas those of genes involved in motility, arginine biosynthesis, and proline metabolism were low after exposure to crabs. Moreover, the virulence factors of the type III secretion system, tad locus, and thermolabile hemolysin were overexpressed. Therefore, the risk of foodborne-illness may be high following consumption of FORC_022 contaminated crab. These results provided molecular information regarding the survival and pathogenesis of V. parahaemolyticus FORC_022 strain in contaminated crab and may have applications in food safety.

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September 22, 2019  |  

The integrative conjugative element clc (ICEclc) of Pseudomonas aeruginosa JB2.

Integrative conjugative elements (ICE) are a diverse group of chromosomally integrated, self-transmissible mobile genetic elements (MGE) that are active in shaping the functions of bacteria and bacterial communities. Each type of ICE carries a characteristic set of core genes encoding functions essential for maintenance and self-transmission, and cargo genes that endow on hosts phenotypes beneficial for niche adaptation. An important area to which ICE can contribute beneficial functions is the biodegradation of xenobiotic compounds. In the biodegradation realm, the best-characterized ICE is ICEclc, which carries cargo genes encoding for ortho-cleavage of chlorocatechols (clc genes) and aminophenol metabolism (amn genes). The element was originally identified in the 3-chlorobenzoate-degrader Pseudomonas knackmussii B13, and the closest relative is a nearly identical element in Burkholderia xenovorans LB400 (designated ICEclc-B13 and ICEclc-LB400, respectively). In the present report, genome sequencing of the o-chlorobenzoate degrader Pseudomonas aeruginosa JB2 was used to identify a new member of the ICEclc family, ICEclc-JB2. The cargo of ICEclc-JB2 differs from that of ICEclc-B13 and ICEclc-LB400 in consisting of a unique combination of genes that encode for the utilization of o-halobenzoates and o-hydroxybenzoate as growth substrates (ohb genes and hyb genes, respectively) and which are duplicated in a tandem repeat. Also, ICEclc-JB2 lacks an operon of regulatory genes (tciR-marR-mfsR) that is present in the other two ICEclc, and which controls excision from the host. Thus, the mechanisms regulating intracellular behavior of ICEclc-JB2 may differ from that of its close relatives. The entire tandem repeat in ICEclc-JB2 can excise independently from the element in a process apparently involving transposases/insertion sequence associated with the repeats. Excision of the repeats removes important niche adaptation genes from ICEclc-JB2, rendering it less beneficial to the host. However, the reduced version of ICEclc-JB2 could now acquire new genes that might be beneficial to a future host and, consequently, to the survival of ICEclc-JB2. Collectively, the present identification and characterization of ICEclc-JB2 provides insights into roles of MGE in bacterial niche adaptation and the evolution of catabolic pathways for biodegradation of xenobiotic compounds.

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September 22, 2019  |  

Identification of the DNA methyltransferases establishing the methylome of the cyanobacterium Synechocystis sp. PCC 6803.

DNA methylation in bacteria is important for defense against foreign DNA, but is also involved in DNA repair, replication, chromosome partitioning, and regulatory processes. Thus, characterization of the underlying DNA methyltransferases in genetically tractable bacteria is of paramount importance. Here, we characterized the methylome and orphan methyltransferases in the model cyanobacterium Synechocystis sp. PCC 6803. Single molecule real-time (SMRT) sequencing revealed four DNA methylation recognition sequences in addition to the previously known motif m5CGATCG, which is recognized by M.Ssp6803I. For three of the new recognition sequences, we identified the responsible methyltransferases. M.Ssp6803II, encoded by the sll0729 gene, modifies GGm4CC, M.Ssp6803III, encoded by slr1803, represents the cyanobacterial dam-like methyltransferase modifying Gm6ATC, and M.Ssp6803V, encoded by slr6095 on plasmid pSYSX, transfers methyl groups to the bipartite motif GGm6AN7TTGG/CCAm6AN7TCC. The remaining methylation recognition sequence GAm6AGGC is probably recognized by methyltransferase M.Ssp6803IV encoded by slr6050. M.Ssp6803III and M.Ssp6803IV were essential for the viability of Synechocystis, while the strains lacking M.Ssp6803I and M.Ssp6803V showed growth similar to the wild type. In contrast, growth was strongly diminished of the ?sll0729 mutant lacking M.Ssp6803II. These data provide the basis for systematic studies on the molecular mechanisms impacted by these methyltransferases.

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September 22, 2019  |  

Chromosomally encoded mcr-5 in colistin non-susceptible Pseudomonas aeruginosa.

Whole genome sequencing (WGS) of historical Pseudomonas aeruginosa clinical isolates identified a chromosomal copy of mcr-5 within a Tn3-like transposon in P. aeruginosa MRSN 12280. The isolate was non-susceptible to colistin by broth microdilution and genome analysis revealed no mutations known to confer colistin resistance. To the best of our knowledge, this is the first report of mcr in colistin non-susceptible P. aeruginosa.

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September 22, 2019  |  

A synthetic-diploid benchmark for accurate variant-calling evaluation.

Existing benchmark datasets for use in evaluating variant-calling accuracy are constructed from a consensus of known short-variant callers, and they are thus biased toward easy regions that are accessible by these algorithms. We derived a new benchmark dataset from the de novo PacBio assemblies of two fully homozygous human cell lines, which provides a relatively more accurate and less biased estimate of small-variant-calling error rates in a realistic context.

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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  |  

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  |  

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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