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Asset Tag: NGS

April 21, 2020

Thermosynechococcus as a thermophilic photosynthetic microbial cell factory for CO2 utilisation.

Thermophilic unicellular cyanobacterium Thermosynechococcus elongatus PKUAC-SCTE542, has been developed as a thermophilic photosynthetic microbial cell factory for CO2 utilisation. The strain exhibits its highest growth rate around 55?°C, can withstand up to 15% CO2, and up to 0.5?M concentration of sodium bicarbonate. The strain is also capable of resisting a 200?ppm concentration of NO and SO2 in simulated flue gasses, and these compounds have a positive effect on its growth. Whole genome sequencing of the strain revealed the presence of numerous forms of active transport of nutrients and additional chaperones acting as the predominant mechanism of strain adaptation to high temperatures. Based on the sequenced genome, two neutral gene insertion sites have been identified and engineered using modular vectors. Site-specific knock-ins and knock-outs have been performed using the spectinomycin resistance gene and proved functional, enabling future application of the strain to produce biofuels and biochemicals from waste CO2. Copyright © 2019 Elsevier Ltd. All rights reserved.

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April 21, 2020

Heteroresistance to the model antimicrobial peptide polymyxin B in the emerging Neisseria meningitidis lineage 11.2 urethritis clade: mutations in the pilMNOPQ operon.

Clusters of Neisseria meningitidis (Nm) urethritis among primarily heterosexual males in multiple US cities have been attributed to a unique non-encapsulated meningococcal clade (the US Nm urethritis clade, US_NmUC) within the hypervirulent clonal complex 11. Resistance to antimicrobial peptides (AMPs) is a key feature of urogenital pathogenesis of the closely related species, Neisseria gonorrhoeae. The US_NmUC isolates were found to be highly resistant to the model AMP, polymyxin B (PmB, MICs 64-256 µg ml-1 ). The isolates also demonstrated stable subpopulations of heteroresistant colonies that showed near total resistant to PmB (MICs 384-1024 µg ml-1 ) and colistin (MIC 256 µg ml-1 ) as well as enhanced LL-37 resistance. This is the first observation of heteroresistance in N. meningitidis. Consistent with previous findings, overall PmB resistance in US_NmUC isolates was due to active Mtr efflux and LptA-mediated lipid A modification. However, whole genome sequencing, variant analyses and directed mutagenesis revealed that the heteroresistance phenotypes and very high-level AMP resistance were the result of point mutations and IS1655 element movement in the pilMNOPQ operon, encoding the type IV pilin biogenesis apparatus. Cross-resistance to other classes of antibiotics was also observed in the heteroresistant colonies. High-level resistance to AMPs may contribute to the pathogenesis of US_NmUC. © 2018 John Wiley & Sons Ltd.

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April 21, 2020

Engineering and modification of microbial chassis for systems and synthetic biology.

Engineering and modifying synthetic microbial chassis is one of the best ways not only to unravel the fundamental principles of life but also to enhance applications in the health, medicine, agricultural, veterinary, and food industries. The two primary strategies for constructing a microbial chassis are the top-down approach (genome reduction) and the bottom-up approach (genome synthesis). Research programs on this topic have been funded in several countries. The ‘Minimum genome factory’ (MGF) project was launched in 2001 in Japan with the goal of constructing microorganisms with smaller genomes for industrial use. One of the best examples of the results of this project is E. coli MGF-01, which has a reduced-genome size and exhibits better growth and higher threonine production characteristics than the parental strain [1]. The ‘cell factory’ project was carried out from 1998 to 2002 in the Fifth Framework Program of the EU (European Union), which tried to comprehensively understand microorganisms used in the application field. One of the outstanding results of this project was the elucidation of proteins secreted by Bacillus subtilis, which was summarized as the ‘secretome’ [2]. The GTL (Genomes to Life) program began in 2002 in the United States. In this program, researchers aimed to create artificial cells both in silico and in vitro, such as the successful design and synthesis of a minimal bacterial genome by John Craig Venter’s group [3]. This review provides an update on recent advances in engineering, modification and application of synthetic microbial chassis, with particular emphasis on the value of learning about chassis as a way to better understand life and improve applications.

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April 21, 2020

BREX system of Escherichia coli distinguishes self from non-self by methylation of a specific DNA site.

Prokaryotes evolved numerous systems that defend against predation by bacteriophages. In addition to well-known restriction-modification and CRISPR-Cas immunity systems, many poorly characterized systems exist. One class of such systems, named BREX, consists of a putative phosphatase, a methyltransferase and four other proteins. A Bacillus cereus BREX system provides resistance to several unrelated phages and leads to modification of specific motif in host DNA. Here, we study the action of BREX system from a natural Escherichia coli isolate. We show that while it makes cells resistant to phage ? infection, induction of ? prophage from cells carrying BREX leads to production of viruses that overcome the defense. The induced phage DNA contains a methylated adenine residue in a specific motif. The same modification is found in the genome of BREX-carrying cells. The results establish, for the first time, that immunity to BREX system defense is provided by an epigenetic modification.

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April 21, 2020

Toxin and genome evolution in a Drosophila defensive symbiosis.

Defenses conferred by microbial symbionts play a vital role in the health and fitness of their animal hosts. An important outstanding question in the study of defensive symbiosis is what determines long term stability and effectiveness against diverse natural enemies. In this study, we combine genome and transcriptome sequencing, symbiont transfection and parasite protection experiments, and toxin activity assays to examine the evolution of the defensive symbiosis between Drosophila flies and their vertically transmitted Spiroplasma bacterial symbionts, focusing in particular on ribosome-inactivating proteins (RIPs), symbiont-encoded toxins that have been implicated in protection against both parasitic wasps and nematodes. Although many strains of Spiroplasma, including the male-killing symbiont (sMel) of Drosophila melanogaster, protect against parasitic wasps, only the strain (sNeo) that infects the mycophagous fly Drosophila neotestacea appears to protect against parasitic nematodes. We find that RIP repertoire is a major differentiating factor between strains that do and do not offer nematode protection, and that sMel RIPs do not show activity against nematode ribosomes in vivo. We also discovered a strain of Spiroplasma infecting a mycophagous phorid fly, Megaselia nigra. Although both the host and its Spiroplasma are distantly related to D. neotestacea and its symbiont, genome sequencing revealed that the M. nigra symbiont encodes abundant and diverse RIPs, including plasmid-encoded toxins that are closely related to the RIPs in sNeo. Our results suggest that distantly related Spiroplasma RIP toxins may perform specialized functions with regard to parasite specificity and suggest an important role for horizontal gene transfer in the emergence of novel defensive phenotypes.

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April 21, 2020

Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the 16S-ITS-23S rRNA operon.

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286-37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (~1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full-length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost-effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput. © 2019 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020

The complete mitochondrial genome sequence of Schisandra chinensis (Austrobaileyales: Schisandraceae)

Chinese magnolia vine (Schisandra chinensis) is an economically important oriental medicinal plant that belongs to the Schisandraceae family. The complete mitochondrial genome sequence of S. chinensis was 946,141bp in length. A total of 45 genes was annotated, including 30 protein-coding genes, 12 tRNA genes, and 3 rRNA genes. A phylogenetic tree based on the mitochondrial genome demonstrated that S. chinensis was most closely related to Schisandra sphenanthera of the Schisandraceae family.

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April 21, 2020

Long-read sequence capture of the haemoglobin gene clusters across codfish species.

Combining high-throughput sequencing with targeted sequence capture has become an attractive tool to study specific genomic regions of interest. Most studies have so far focused on the exome using short-read technology. These approaches are not designed to capture intergenic regions needed to reconstruct genomic organization, including regulatory regions and gene synteny. Here, we demonstrate the power of combining targeted sequence capture with long-read sequencing technology for comparative genomic analyses of the haemoglobin (Hb) gene clusters across eight species separated by up to 70 million years. Guided by the reference genome assembly of the Atlantic cod (Gadus morhua) together with genome information from draft assemblies of selected codfishes, we designed probes covering the two Hb gene clusters. Use of custom-made barcodes combined with PacBio RSII sequencing led to highly continuous assemblies of the LA (~100 kb) and MN (~200 kb) clusters, which include syntenic regions of coding and intergenic sequences. Our results revealed an overall conserved genomic organization of the Hb genes within this lineage, yet with several, lineage-specific gene duplications. Moreover, for some of the species examined, we identified amino acid substitutions at two sites in the Hbb1 gene as well as length polymorphisms in its regulatory region, which has previously been linked to temperature adaptation in Atlantic cod populations. This study highlights the use of targeted long-read capture as a versatile approach for comparative genomic studies by generation of a cross-species genomic resource elucidating the evolutionary history of the Hb gene family across the highly divergent group of codfishes. © 2018 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

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April 21, 2020

The complete chloroplast genome sequence of watercress (Nasturtium officinale R. Br.): Genome organization, adaptive evolution and phylogenetic relationships in Cardamineae.

Watercress (Nasturtium officinale R. Br.), an aquatic leafy vegetable of the Brassicaceae family, is known as a nutritional powerhouse. Here, we de novo sequenced and assembled the complete chloroplast (cp) genome of watercress based on combined PacBio and Illumina data. The cp genome is 155,106?bp in length, exhibiting a typical quadripartite structure including a pair of inverted repeats (IRA and IRB) of 26,505?bp separated by a large single copy (LSC) region of 84,265?bp and a small single copy (SSC) region of 17,831?bp. The genome contained 113 unique genes, including 79 protein-coding genes, 30 tRNAs and 4 rRNAs, with 20 duplicate in the IRs. Compared with the prior cp genome of watercress deposited in GenBank, 21 single nucleotide polymorphisms (SNPs) and 27 indels were identified, mainly located in noncoding sequences. A total of 49 repeat structures and 71 simple sequence repeats (SSRs) were detected. Codon usage showed a bias for A/T-ending codons in the cp genome of watercress. Moreover, 45 RNA editing sites were predicted in 16 genes, all for C-to-U transitions. A comparative plastome study with Cardamineae species revealed a conserved gene order and high similarity of protein-coding sequences. Analysis of the Ka/Ks ratios of Cardamineae suggested positive selection exerted on the ycf2 gene in watercress, which might reflect specific adaptations of watercress to its particular living environment. Phylogenetic analyses based on complete cp genomes and common protein-coding genes from 56 species showed that the genus Nasturtium was a sister to Cardamine in the Cardamineae tribe. Our study provides valuable resources for future evolution, population genetics and molecular biology studies of watercress. Copyright © 2019 Elsevier B.V. All rights reserved.

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April 21, 2020

Comparative genomic analysis of Lactobacillus mucosae LM1 identifies potential niche-specific genes and pathways for gastrointestinal adaptation.

Lactobacillus mucosae is currently of interest as putative probiotics due to their metabolic capabilities and ability to colonize host mucosal niches. L. mucosae LM1 has been studied in its functions in cell adhesion and pathogen inhibition, etc. It demonstrated unique abilities to use energy from carbohydrate and non-carbohydrate sources. Due to these functions, we report the first complete genome sequence of an L. mucosae strain, L. mucosae LM1. Analysis of the pan-genome in comparison with closely-related Lactobacillus species identified a complete glycogen metabolism pathway, as well as folate biosynthesis, complementing previous proteomic data on the LM1 strain. It also revealed common and unique niche-adaptation genes among the various L. mucosae strains. The aim of this study was to derive genomic information that would reveal the probable mechanisms underlying the probiotic effect of L. mucosae LM1, and provide a better understanding of the nature of L. mucosae sp. Copyright © 2017 Elsevier Inc. All rights reserved.

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April 21, 2020

Vaccine-induced protection from homologous tier 2 SHIV challenge in nonhuman primates depends on serum-neutralizing antibody titers.

Passive administration of HIV neutralizing antibodies (nAbs) can protect macaques from hard-to-neutralize (tier 2) chimeric simian-human immunodeficiency virus (SHIV) challenge. However, conditions for nAb-mediated protection after vaccination have not been established. Here, we selected groups of 6 rhesus macaques with either high or low serum nAb titers from a total of 78 animals immunized with recombinant native-like (SOSIP) Env trimers. Repeat intrarectal challenge with homologous tier 2 SHIVBG505 led to rapid infection in unimmunized and low-titer animals. High-titer animals, however, demonstrated protection that was gradually lost as nAb titers waned over time. An autologous serum ID50 nAb titer of ~1:500 afforded more than 90% protection from medium-dose SHIV infection. In contrast, antibody-dependent cellular cytotoxicity and T cell activity did not correlate with protection. Therefore, Env protein-based vaccination strategies can protect against hard-to-neutralize SHIV challenge in rhesus macaques by inducing tier 2 nAbs, provided appropriate neutralizing titers can be reached and maintained. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

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April 21, 2020

Population Genome Sequencing of the Scab Fungal Species Venturia inaequalis, Venturia pirina, Venturia aucupariae and Venturia asperata.

The Venturia genus comprises fungal species that are pathogens on Rosaceae host plants, including V. inaequalis and V. asperata on apple, V. aucupariae on sorbus and V. pirina on pear. Although the genetic structure of V. inaequalis populations has been investigated in detail, genomic features underlying these subdivisions remain poorly understood. Here, we report whole genome sequencing of 87 Venturia strains that represent each species and each population within V. inaequalis We present a PacBio genome assembly for the V. inaequalis EU-B04 reference isolate. The size of selected genomes was determined by flow cytometry, and varied from 45 to 93 Mb. Genome assemblies of V. inaequalis and V. aucupariae contain a high content of transposable elements (TEs), most of which belong to the Gypsy or Copia LTR superfamilies and have been inactivated by Repeat-Induced Point mutations. The reference assembly of V. inaequalis presents a mosaic structure of GC-equilibrated regions that mainly contain predicted genes and AT-rich regions, mainly composed of TEs. Six pairs of strains were identified as clones. Single-Nucleotide Polymorphism (SNP) analysis between these clones revealed a high number of SNPs that are mostly located in AT-rich regions due to misalignments and allowed determining a false discovery rate. The availability of these genome sequences is expected to stimulate genetics and population genomics research of Venturia pathogens. Especially, it will help understanding the evolutionary history of Venturia species that are pathogenic on different hosts, a history that has probably been substantially influenced by TEs.Copyright © 2019 Le Cam et al.

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April 21, 2020

High Quality Draft Genome of Arogyapacha (Trichopus zeylanicus), an Important Medicinal Plant Endemic to Western Ghats of India.

Arogyapacha, the local name of Trichopus zeylanicus, is a rare, indigenous medicinal plant of India. This plant is famous for its traditional use as an instant energy stimulant. So far, no genomic resource is available for this important plant and hence its metabolic pathways are poorly understood. Here, we report on a high-quality draft assembly of approximately 713.4 Mb genome of T. zeylanicus, first draft genome from the genus Trichopus The assembly was generated in a hybrid approach using Illumina short-reads and Pacbio longer-reads. The total assembly comprised of 22601 scaffolds with an N50 value of 433.3 Kb. We predicted 34452 protein coding genes in T. zeylanicus genome and found that a significant portion of these predicted genes were associated with various secondary metabolite biosynthetic pathways. Comparative genome analysis revealed extensive gene collinearity between T. zeylanicus and its closely related plant species. The present genome and annotation data provide an essential resource to speed-up the research on secondary metabolism, breeding and molecular evolution of T. zeylanicus. Copyright © 2019 Chellappan et al.

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April 21, 2020

De Novo Genome Sequence Assembly of Dwarf Coconut (Cocos nucifera L. ‘Catigan Green Dwarf’) Provides Insights into Genomic Variation Between Coconut Types and Related Palm Species.

We report the first whole genome sequence (WGS) assembly and annotation of a dwarf coconut variety, ‘Catigan Green Dwarf’ (CATD). The genome sequence was generated using the PacBio SMRT sequencing platform at 15X coverage of the expected genome size of 2.15 Gbp, which was corrected with assembled 50X Illumina paired-end MiSeq reads of the same genome. The draft genome was improved through Chicago sequencing to generate a scaffold assembly that results in a total genome size of 2.1 Gbp consisting of 7,998 scaffolds with N50 of 570,487 bp. The final assembly covers around 97.6% of the estimated genome size of coconut ‘CATD’ based on homozygous k-mer peak analysis. A total of 34,958 high-confidence gene models were predicted and functionally associated to various economically important traits, such as pest/disease resistance, drought tolerance, coconut oil biosynthesis, and putative transcription factors. The assembled genome was used to infer the evolutionary relationship within the palm family based on genomic variations and synteny of coding gene sequences. Data show that at least three (3) rounds of whole genome duplication occurred and are commonly shared by these members of the Arecaceae family. A total of 7,139 unique SSR markers were designed to be used as a resource in marker-based breeding. In addition, we discovered 58,503 variants in coconut by aligning the Hainan Tall (HAT) WGS reads to the non-repetitive regions of the assembled CATD genome. The gene markers and genome-wide SSR markers established here will facilitate the development of varieties with resilience to climate change, resistance to pests and diseases, and improved oil yield and quality.Copyright © 2019 Lantican et al.

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April 21, 2020

Analysis of two pheromone-responsive conjugative multiresistance plasmids carrying the novel mobile optrA locus from Enterococcus faecalis

Background: The acquired optrA gene, which encodes a ribosomal protection protein of the ABC-F family, can confer cross-resistance to linezolid and florfenicol, posing a serious therapeutic challenge to both human and veterinary medicine. Purpose: The objective of this study was to investigate the two Enterococcus faecalis (E. faecalis) plasmids for their fine structure, their transferability and the presence of mobile antimicrobial resistance loci. Methods: To elucidate their fine structure, the two plasmids were completely sequenced and the sequences analysed. Besides conjugation experiments, inverse PCR assays were conducted to see whether minicircles are produced from the mobile antimicrobial resistance loci. Results: Two pheromone-responsive conjugative optrA-carrying plasmids from E. faecalis, pE211 and pE508 were identified, which can transfer with frequencies of 2.6 ×10-2 and 3.7 ×10-2 (transconjugant per donor), respectively. In both plasmids, optrA was located on the novel mobile optrA locus with different sizes (12,834 bp in pE211 and 7,561 bp in pE508, respectively), flanked by two copies of IS1216 genes in the same orientation. Inverse PCR revealed that circular forms can be generated, consisting of optrA and one copy of IS1216, indicating they are all active. The 77,562 bp plasmid pE211 also carried Tn558 and a mobile bcrABDR locus, and the 84,468 bp plasmid pE508 also harbored the genes fexA, tet(L), tet(O/W/32/O) and a mobile aac(A)-aph(D) locus. Conclusion: The presence of mobile genetic elements in these plasmids renders them flexible and these elements will aid to the persistence and dissemination of these plasmids among enterococci and potentially also other gram-positive bacteria.

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