April 21, 2020  |  

Tandem repeats lead to sequence assembly errors and impose multi-level challenges for genome and protein databases.

The widespread occurrence of repetitive stretches of DNA in genomes of organisms across the tree of life imposes fundamental challenges for sequencing, genome assembly, and automated annotation of genes and proteins. This multi-level problem can lead to errors in genome and protein databases that are often not recognized or acknowledged. As a consequence, end users working with sequences with repetitive regions are faced with ‘ready-to-use’ deposited data whose trustworthiness is difficult to determine, let alone to quantify. Here, we provide a review of the problems associated with tandem repeat sequences that originate from different stages during the sequencing-assembly-annotation-deposition workflow, and that may proliferate in public database repositories affecting all downstream analyses. As a case study, we provide examples of the Atlantic cod genome, whose sequencing and assembly were hindered by a particularly high prevalence of tandem repeats. We complement this case study with examples from other species, where mis-annotations and sequencing errors have propagated into protein databases. With this review, we aim to raise the awareness level within the community of database users, and alert scientists working in the underlying workflow of database creation that the data they omit or improperly assemble may well contain important biological information valuable to others. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.


April 21, 2020  |  

Genome-Wide Screening for Enteric Colonization Factors in Carbapenem-Resistant ST258 Klebsiella pneumoniae.

A diverse, antibiotic-naive microbiota prevents highly antibiotic-resistant microbes, including carbapenem-resistant Klebsiella pneumoniae (CR-Kp), from achieving dense colonization of the intestinal lumen. Antibiotic-mediated destruction of the microbiota leads to expansion of CR-Kp in the gut, markedly increasing the risk of bacteremia in vulnerable patients. While preventing dense colonization represents a rational approach to reduce intra- and interpatient dissemination of CR-Kp, little is known about pathogen-associated factors that enable dense growth and persistence in the intestinal lumen. To identify genetic factors essential for dense colonization of the gut by CR-Kp, we constructed a highly saturated transposon mutant library with >150,000 unique mutations in an ST258 strain of CR-Kp and screened for in vitro growth and in vivo intestinal colonization in antibiotic-treated mice. Stochastic and partially reversible fluctuations in the representation of different mutations during dense colonization revealed the dynamic nature of intestinal microbial populations. We identified genes that are crucial for early and late stages of dense gut colonization and confirmed their role by testing isogenic mutants in in vivo competition assays with wild-type CR-Kp Screening of the transposon library also identified mutations that enhanced in vivo CR-Kp growth. These newly identified colonization factors may provide novel therapeutic opportunities to reduce intestinal colonization by CR-KpIMPORTANCEKlebsiella pneumoniae is a common cause of bloodstream infections in immunocompromised and hospitalized patients, and over the last 2 decades, some strains have acquired resistance to nearly all available antibiotics, including broad-spectrum carbapenems. The U.S. Centers for Disease Control and Prevention has listed carbapenem-resistant K. pneumoniae (CR-Kp) as an urgent public health threat. Dense colonization of the intestine by CR-Kp and other antibiotic-resistant bacteria is associated with an increased risk of bacteremia. Reducing the density of gut colonization by CR-Kp is likely to reduce their transmission from patient to patient in health care facilities as well as systemic infections. How CR-Kp expands and persists in the gut lumen, however, is poorly understood. Herein, we generated a highly saturated mutant library in a multidrug-resistant K. pneumoniae strain and identified genetic factors that are associated with dense gut colonization by K. pneumoniae This study sheds light on host colonization by K. pneumoniae and identifies potential colonization factors that contribute to high-density persistence of K. pneumoniae in the intestine. Copyright © 2019 Jung et al.


April 21, 2020  |  

Complete Genome Sequence of Saccharospirillum mangrovi HK-33T Sheds Light on the Ecological Role of a Bacterium in Mangrove Sediment Environment.

We present the genome sequence of Saccharospirillum mangrovi HK-33T, isolated from a mangrove sediment sample in Haikou, China. The complete genome of S. mangrovi HK-33T consisted of a single-circular chromosome with the size of 3,686,911 bp as well as an average G?+?C content of 57.37%, and contained 3,383 protein-coding genes, 4 operons of 16S-23S-5S rRNA genes, and 52 tRNA genes. Genomic annotation indicated that the genome of S. mangrovi HK-33T had many genes related to oligosaccharide and polysaccharide degradation and utilization of polyhydroxyalkanoate. For nitrogen cycle, genes encoding nitrate and nitrite reductase, glutamate dehydrogenase, glutamate synthase, and glutamine synthetase could be found. For phosphorus cycle, genes related to polyphosphate kinases (ppk1 and ppk2), the high-affinity phosphate-specific transport (Pst) system, and the low-affinity inorganic phosphate transporter (pitA) were predicted. For sulfur cycle, cysteine synthase and type III acyl coenzyme A transferase (dddD) coding genes were searched out. This study provides evidence about carbon, nitrogen, phosphorus, and sulfur metabolic patterns of S. mangrovi HK-33T and broadens our understandings about ecological roles of this bacterium in the mangrove sediment environment.


April 21, 2020  |  

Liriodendron genome sheds light on angiosperm phylogeny and species-pair differentiation.

The genus Liriodendron belongs to the family Magnoliaceae, which resides within the magnoliids, an early diverging lineage of the Mesangiospermae. However, the phylogenetic relationship of magnoliids with eudicots and monocots has not been conclusively resolved and thus remains to be determined1-6. Liriodendron is a relict lineage from the Tertiary with two distinct species-one East Asian (L. chinense (Hemsley) Sargent) and one eastern North American (L. tulipifera Linn)-identified as a vicariad species pair. However, the genetic divergence and evolutionary trajectories of these species remain to be elucidated at the whole-genome level7. Here, we report the first de novo genome assembly of a plant in the Magnoliaceae, L. chinense. Phylogenetic analyses suggest that magnoliids are sister to the clade consisting of eudicots and monocots, with rapid diversification occurring in the common ancestor of these three lineages. Analyses of population genetic structure indicate that L. chinense has diverged into two lineages-the eastern and western groups-in China. While L. tulipifera in North America is genetically positioned between the two L. chinense groups, it is closer to the eastern group. This result is consistent with phenotypic observations that suggest that the eastern and western groups of China may have diverged long ago, possibly before the intercontinental differentiation between L. chinense and L. tulipifera. Genetic diversity analyses show that L. chinense has tenfold higher genetic diversity than L. tulipifera, suggesting that the complicated regions comprising east-west-orientated mountains and the Yangtze river basin (especially near 30°?N latitude) in East Asia offered more successful refugia than the south-north-orientated mountain valleys in eastern North America during the Quaternary glacial period.


April 21, 2020  |  

The complete genome sequence of Ethanoligenens harbinense reveals the metabolic pathway of acetate-ethanol fermentation: A novel understanding of the principles of anaerobic biotechnology.

Ethanol-type fermentation is one of three main fermentation types in the acidogenesis of anaerobic treatment systems. Non-spore-forming Ethanoligenens is as a typical genus capable of ethanol-type fermentation in mixed culture (i.e. acetate-ethanol fermentation). This genus can produce ethanol, acetate, CO2, and H2 using carbohydrates, and has application potential in anaerobic bioprocesses. Here, the complete genome sequences and methylome of Ethanoligenens harbinense strains with different autoaggregative and coaggregative abilities were obtained using the PacBio single-molecule real-time sequencing platform. The genome size of E. harbinense strains was about 2.97-3.10?Mb with 55.5% G+C content. 3020-3153 genes were annotated, most of which were methylated at specific sites or motifs. The methylation types included 6mA, 4mC, and unknown types. Comparative genomic analysis demonstrated low levels of genetic similarity between E. harbinense and other well-known hydrogen-producing bacteria (i.e., Clostridium and Thermoanaerobacter) in phylogenesis. Hydrogen production of E. harbinense was catalyzed by genes that encode [FeFe]-hydrogenases and that were synthesized by three maturases of [FeFe]-H2ase. The metabolic mechanism of H2-ethanol co-production fermentation, catalyzed by pyruvate ferredoxin oxidoreductase was proposed. This study provides genetic and evolutionary information of a model genus for the further investigation of the metabolic pathway and regulatory network of ethanol-type fermentation and anaerobic bioprocesses for waste or wastewater treatment.Copyright © 2019. Published by Elsevier Ltd.


April 21, 2020  |  

Genetic basis of functional variability in adhesion G protein-coupled receptors.

The enormous sizes of adhesion G protein-coupled receptors (aGPCRs) go along with complex genomic exon-intron architectures giving rise to multiple mRNA variants. There is a need for a comprehensive catalog of aGPCR variants for proper evaluation of the complex functions of aGPCRs found in structural, in vitro and animal model studies. We used an established bioinformatics pipeline to extract, quantify and visualize mRNA variants of aGPCRs from deeply sequenced transcriptomes. Data analysis showed that aGPCRs have multiple transcription start sites even within introns and that tissue-specific splicing is frequent. On average, 19 significantly expressed transcript variants are derived from a given aGPCR gene. The domain architecture of the N terminus encoded by transcript variants often differs and N termini without or with an incomplete seven-helix transmembrane anchor as well as separate seven-helix transmembrane domains are frequently derived from aGPCR genes. Experimental analyses of selected aGPCR transcript variants revealed marked functional differences. Our analysis has an impact on a rational design of aGPCR constructs for structural analyses and gene-deficient mouse lines and provides new support for independent functions of both, the large N terminus and the transmembrane domain of aGPCRs.


April 21, 2020  |  

A chromosome-level genome assembly of Cydia pomonella provides insights into chemical ecology and insecticide resistance.

The codling moth Cydia pomonella, a major invasive pest of pome fruit, has spread around the globe in the last half century. We generated a chromosome-level scaffold assembly including the Z chromosome and a portion of the W chromosome. This assembly reveals the duplication of an olfactory receptor gene (OR3), which we demonstrate enhances the ability of C. pomonella to exploit kairomones and pheromones in locating both host plants and mates. Genome-wide association studies contrasting insecticide-resistant and susceptible strains identify hundreds of single nucleotide polymorphisms (SNPs) potentially associated with insecticide resistance, including three SNPs found in the promoter of CYP6B2. RNAi knockdown of CYP6B2 increases C. pomonella sensitivity to two insecticides, deltamethrin and azinphos methyl. The high-quality genome assembly of C. pomonella informs the genetic basis of its invasiveness, suggesting the codling moth has distinctive capabilities and adaptive potential that may explain its worldwide expansion.


April 21, 2020  |  

Mobilome of Brevibacterium aurantiacum Sheds Light on Its Genetic Diversity and Its Adaptation to Smear-Ripened Cheeses.

Brevibacterium aurantiacum is an actinobacterium that confers key organoleptic properties to washed-rind cheeses during the ripening process. Although this industrially relevant species has been gaining an increasing attention in the past years, its genome plasticity is still understudied due to the unavailability of complete genomic sequences. To add insights on the mobilome of this group, we sequenced the complete genomes of five dairy Brevibacterium strains and one non-dairy strain using PacBio RSII. We performed phylogenetic and pan-genome analyses, including comparisons with other publicly available Brevibacterium genomic sequences. Our phylogenetic analysis revealed that these five dairy strains, previously identified as Brevibacterium linens, belong instead to the B. aurantiacum species. A high number of transposases and integrases were observed in the Brevibacterium spp. strains. In addition, we identified 14 and 12 new insertion sequences (IS) in B. aurantiacum and B. linens genomes, respectively. Several stretches of homologous DNA sequences were also found between B. aurantiacum and other cheese rind actinobacteria, suggesting horizontal gene transfer (HGT). A HGT region from an iRon Uptake/Siderophore Transport Island (RUSTI) and an iron uptake composite transposon were found in five B. aurantiacum genomes. These findings suggest that low iron availability in milk is a driving force in the adaptation of this bacterial species to this niche. Moreover, the exchange of iron uptake systems suggests cooperative evolution between cheese rind actinobacteria. We also demonstrated that the integrative and conjugative element BreLI (Brevibacterium Lanthipeptide Island) can excise from B. aurantiacum SMQ-1417 chromosome. Our comparative genomic analysis suggests that mobile genetic elements played an important role into the adaptation of B. aurantiacum to cheese ecosystems.


April 21, 2020  |  

Genomics-driven discovery of a biosynthetic gene cluster required for the synthesis of BII-Rafflesfungin from the fungus Phoma sp. F3723.

Phomafungin is a recently reported broad spectrum antifungal compound but its biosynthetic pathway is unknown. We combed publicly available Phoma genomes but failed to find any putative biosynthetic gene cluster that could account for its biosynthesis.Therefore, we sequenced the genome of one of our Phoma strains (F3723) previously identified as having antifungal activity in a high-throughput screen. We found a biosynthetic gene cluster that was predicted to synthesize a cyclic lipodepsipeptide that differs in the amino acid composition compared to Phomafungin. Antifungal activity guided isolation yielded a new compound, BII-Rafflesfungin, the structure of which was determined.We describe the NRPS-t1PKS cluster ‘BIIRfg’ compatible with the synthesis of the cyclic lipodepsipeptide BII-Rafflesfungin [HMHDA-L-Ala-L-Glu-L-Asn-L-Ser-L-Ser-D-Ser-D-allo-Thr-Gly]. We report new Stachelhaus codes for Ala, Glu, Asn, Ser, Thr, and Gly. We propose a mechanism for BII-Rafflesfungin biosynthesis, which involves the formation of the lipid part by BIIRfg_PKS followed by activation and transfer of the lipid chain by a predicted AMP-ligase on to the first PCP domain of the BIIRfg_NRPS gene.


April 21, 2020  |  

The genome of the giant Nomura’s jellyfish sheds light on the early evolution of active predation.

Unique among cnidarians, jellyfish have remarkable morphological and biochemical innovations that allow them to actively hunt in the water column and were some of the first animals to become free-swimming. The class Scyphozoa, or true jellyfish, are characterized by a predominant medusa life-stage consisting of a bell and venomous tentacles used for hunting and defense, as well as using pulsed jet propulsion for mobility. Here, we present the genome of the giant Nomura’s jellyfish (Nemopilema nomurai) to understand the genetic basis of these key innovations.We sequenced the genome and transcriptomes of the bell and tentacles of the giant Nomura’s jellyfish as well as transcriptomes across tissues and developmental stages of the Sanderia malayensis jellyfish. Analyses of the Nemopilema and other cnidarian genomes revealed adaptations associated with swimming, marked by codon bias in muscle contraction and expansion of neurotransmitter genes, along with expanded Myosin type II family and venom domains, possibly contributing to jellyfish mobility and active predation. We also identified gene family expansions of Wnt and posterior Hox genes and discovered the important role of retinoic acid signaling in this ancient lineage of metazoans, which together may be related to the unique jellyfish body plan (medusa formation).Taken together, the Nemopilema jellyfish genome and transcriptomes genetically confirm their unique morphological and physiological traits, which may have contributed to the success of jellyfish as early multi-cellular predators.


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