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bioRxiv2018

Detection of low-frequency mutations and removal of heat-induced artifactual mutations using Duplex Sequencing

We present a genome-wide comparative and comprehensive analysis of three different sequencing methods (conventional next generation sequencing (NGS), tag-based single strand sequencing (eg. SSCS), and Duplex Sequencing for investigating mitochondrial mutations in human breast epithelial cells. Duplex Sequencing produces a single strand consensus sequence (SSCS) and a duplex consensus sequence (DCS) analysis, respectively. Our study validates that although high-frequency mutations are detectable by all the three sequencing methods with the similar accuracy and reproducibility, rare (low-frequency) mutations are not accurately detectable by NGS and SSCS. Even with conservative bioinformatical modification to overcome the high error rate of NGS, the NGS frequency of rare mutations is 7.0x10-4. The frequency is reduced to 1.3x10-4 with SSCS and is further reduced to 1.0x10-5 using DCS. Rare mutation context spectra obtained from NGS significantly vary across independent experiments, and it is not possible to identify a dominant mutation context. In contrast, rare mutation context spectra are consistently similar in all independent DCS experiments. We have systematically identified heat-induced artifactual mutations and corrected the artifacts using Duplex Sequencing. All of these artifacts are stochastically occurring rare mutations. C>A/G>T, a signature of oxidative damage, is the most increased (170-fold) heat-induced artifactual mutation type. Our results strongly support the claim that Duplex Sequencing accurately detects low-frequency mutations and identifies and corrects artifactual mutations introduced by heating during DNA preparation.

genomics↗

bioRxiv2018

Metagenomic Insights Unveil the Dominance of Undescribed Actinobacteria in Pond Ecosystem of an Indian Shrine

Metagenomic analysis holds immense potential for identifying rare and uncharacterized microorganisms from many ecological habitats. Actinobacteria have been proved to be an excellent source of novel antibiotics for several decades. The present study was designed to delineate and understand the bacterial diversity with special focus on Actinobacteria from pond sediment collected from Sanjeeviraya Hanuman Temple, Ayyangarkulam, Kanchipuram, Tamil Nadu, India. The sediment had an average temperature (25.32%), pH (7.13), salinity (0.960 mmhos/cm) and high organic content (10.7%) posing minimal stress on growth condition of the microbial community. Subsequent molecular manipulations, sequencing and bioinformatics analysis of V3 and V4 region of 16S rRNA metagenomics analysis confirmed the presence of 40 phyla, 100 classes, 223 orders, 319 families and 308 genera in the sediment sample dominated by Acidobacteria (18.14%), Proteobacteria (15.13%), Chloroflexi (12.34), Actinobacteria (10.84%), Cyanobacteria (5.58%), Verrucomicrobia (3.37%), Firmicutes (2.28%), and, Gemmatimonadetes (1.63%). Among the Actinobacteria phylum, Acidothermus (29.68%) was the predominant genus followed by Actinospica (17.65%), Streptomyces (14.64%), Nocardia (4.55%) and Sinomonas (2.9%). Culture-dependent isolation of Actinobacteria yielded all strains of similar morphology to that of Streptomyces genus which clearly indicating that the traditional based technique is incapable of isolating majority of the non-Streptomyces or the so called rare Actinobacteria. Although Actinobacteria were among the dominant phylum, a close look at the species level indicated that only 15.2% within the Actinobacterial phylum could be assigned to cultured species. This leaves a vast majority of the Actinobacterial species yet to be explored with possible novel metabolites have special pharmaceutical and industrial application. It also indicates that the microbial ecology of pond sediment is neglected fields which need attention.

microbiology↗

bioRxiv2018

Identification of single nucleotide variants using position-specific error estimation in deep sequencing data

BackgroundTargeted deep sequencing is a highly effective technology to identify known and novel single nucleotide variants (SNVs) with many applications in translational medicine, disease monitoring and cancer profiling. However, identification of SNVs using deep sequencing data is a challenging computational problem as different sequencing artifacts limit the analytical sensitivity of SNV detection, especially at low variant allele frequencies (VAFs).\n\nMethodsTo address the problem of relatively high noise levels in amplicon-based deep sequencing data (e.g. with the Ion AmpliSeq technology) in the context of SNV calling, we have developed a new bioinformatics tool called AmpliSolve. AmpliSolve uses a set of normal samples to model position-specific, strand-specific and nucleotide-specific background artifacts (noise), and deploys a Poisson model-based statistical framework for SNV detection.\n\nResultsOur tests on both synthetic and real data indicate that AmpliSolve achieves a good trade-off between precision and sensitivity, even at VAF below 5% and as low as 1%. We further validate AmpliSolve by applying it to the detection of SNVs in 96 circulating tumor DNA samples at three clinically relevant genomic positions and compare the results to digital droplet PCR experiments.\n\nConclusionsAmpliSolve is a new tool for in-silico estimation of background noise and for detection of low frequency SNVs in targeted deep sequencing data. Although AmpliSolve has been specifically designed for and tested on amplicon-based libraries sequenced with the Ion Torrent platform it can, in principle, be applied to other sequencing platforms as well. AmpliSolve is freely available at https://github.com/dkleftogi/AmpliSolve.

genomics↗

bioRxiv2018

Mutational analysis of N-ethyl-N-nitrosourea (ENU) in the fission yeast Schizosaccharomyces pombe.

Forward genetics has boosted our knowledge on genic function in a multitude of biological models and it has significantly contributed to the understanding of genetic bases of development, ageing and human diseases. With the advent of the next generation sequencing and use of powerful bioinformatic tools, this traditional genetic strategy has acquired a new impulse. At present, whole genome sequencing assisted by in silico analysis allows the rapid and efficient identification of gene variants that are responsible for a particular phenotype. In this experimental pipeline, it is crucial to start by provoking a large number of random changes in the genome of the model organisms to be screened. A range of chemical mutagens are used to this end because most of them display particular reactivity properties and act differently over DNA. Here we use N-ethyl-N-nitrosourea (ENU) as a mutagen for the first time to our knowledge in the fission yeast Schizosaccharomyces pombe. By comparison to the extensively used Ethyl methanesulfonate (EMS) in a phenotype-based study, we conclude that ENU is a very potent and easy-to-use mutagen. Judging from DNA sequence analysis of the identified mutants, ENU induces base changes rather than indels and the mutational spectrum in the fission yeast seems similar to that found in mice but different to that described in other single-celled organisms such as budding yeast and E. coli. Using ENU in S. pombe, we have gathered a collection of 49 auxotrophic mutants including two deleterious alleles of ATIC human ortholog. Defective alleles of this gene are causative of AICA-Ribosiduria, a severe genetic disease. We have also identified 5 aminoglycoside-resistance inactivating mutations in APH genes. All these mutations reported here may be of interest in the metabolism and antibiotic resistance research fields.

genetics↗

bioRxiv2018

Genetics of adaptation of the ascomycetous fungus Podospora anserina to submerged cultivation

Podospora anserina is a model ascomycetous fungus which shows pronounced phenotypic senescence when grown on solid medium but possesses unlimited lifespan under submerged cultivation. In order to study the genetic aspects of adaptation of P. anserina to submerged cultivation, we initiated a long-term evolution experiment. In the course of the first four years of the experiment, 125 single-nucleotide substitutions and 23 short indels were fixed in eight independently evolving populations. Six proteins that affect fungal growth and development evolved in more than one population; in particular, the G-protein alpha subunit FadA evolved in seven out of eight experimental populations. Parallel evolution at the level of genes and pathways, an excess of nonsense and missense substitutions, and an elevated conservation of proteins and their sites where the changes occurred suggest that many of the observed allele replacements were adaptive and driven by positive selection.\n\nAuthor summaryLiving beings adapt to novel conditions that are far from their original environments in different ways. Studying mechanisms of adaptation is crucial for our understanding of evolution. The object of our interest is a multicellular fungus Podospora anserina. This fungus is known for its pronounced senescence and a definite lifespan, but it demonstrates an unlimited lifespan and no signs of senescence when grown under submerged conditions. Soon after transition to submerged cultivation, the rate of growth of P. anserina increases and its pigmentation changes. We wanted to find out whether there are any genetic changes that contribute to adaptation of P. anserina to these novel conditions and initiated a long-term evolutionary experiment on eight independent populations. Over the first four years of the experiment, 148 mutations were fixed in these populations. Many of these mutations lead to inactivation of the part of the developmental pathway in P. anserina, probably reallocating resources to vegetative proliferation in liquid medium. Our observations imply that strong positive selection drives changes in at least some of the affected protein-coding genes.\n\nData AvailabilityGenome sequence data have been deposited at DDBJ/ENA/GenBank under accessions QHKV00000000 (founder genotype A; version QHKV01000000) and QHKU00000000 (founder genotype B; version QHKU01000000), with the respective BioSample accessions SAMN09270751 and SAMN09270757, under BioProject PRJNA473312. Sequencing data have been deposited at the SRA with accession numbers SRR7233712-SRR7233727, under the same BioProject.\n\nFundingExperimental work and sequencing were supported by the Russian Foundation for Basic Research (grants no. 16-04-01845a and 18-04-01349a). Bioinformatic analysis was supported by the Russian Science Foundation (grant no. 16-14-10173). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

evolutionary biology↗

bioRxiv2018

Molecular convergence and positive selection associated with the evolution of symbiont transmission mode in stony corals

Heritable symbioses are thought to be important for the maintenance of mutually beneficial relationships (1), and for facilitating major transitions in individuality, such as the evolution of the eukaryotic cell (2, 3). In stony corals, vertical transmission has evolved repeatedly (4), providing a unique opportunity to investigate the genomic basis of this complex trait. We conducted a comparative analysis of 25 coral transcriptomes to identify orthologous genes exhibiting both signatures of positive selection and convergent amino acid substitutions in vertically transmitting lineages. The frequency of convergence events tends to be higher among vertically transmitting lineages, consistent with the proposed role of natural selection in driving the evolution of convergent transmission mode phenotypes (5). Of the 10,774 total orthologous genes identified, 403 exhibited at least one molecular convergence event and evidence of positive selection in at least one vertically transmitting lineage. Functional enrichments among these top candidate genes include processes previously implicated in mediating the coral-Symbiodiniaceae symbiosis including endocytosis, immune response, cytoskeletal protein binding and cytoplasmic membrane-bounded vesicles (6). We also identified 100 genes showing evidence of positive selection at the particular convergence event. Among these, we identified several novel candidate genes, highlighting the value of our approach for generating new insight into the mechanistic basis of the coral symbiosis, in addition to uncovering host mechanisms associated with the evolution of heritable symbioses.\n\nDATA ARCHIVAL LOCATIONRaw sequencing data generated for this study have been uploaded to NCBIs SRA: PRJNA395352. All bioinformatic scripts and input files can be found at https://github.com/grovesdixon/convergent_evo_coral.

evolutionary biology↗

bioRxiv2018

Comparative Transcriptomes Analysis of Taenia pisiformis at Different Development Stages

To understand the characteristics of the transcriptional group of Taenia pisiformis at different developmental stages, and to lay the foundation for the screening of vaccine antigens and drug target genes, the transcriptomes of adult and larva of T. pisiformis were assembled and analyzed using bioinformatic tools. A total of 36,951 unigenes with a mean length of 950bp were formed, among which 12,665, 8,188, 7,577, and 6,293 unigenes have been annotated respectively by sequence similarity analysis with four databases (NR, Swiss-Prot, KOG, and KEGG). It should be noted there are 5,662 unigenes that share good similarity with the four databases and get a relatively perfect functional annotation. Besides, a total of 10,247 differentially expressed genes were screened. To be specific, 6,910 unigenes were up-regulated in the larva stage while 3,337 were down-regulated in the adult stage. To sum up, this study sequenced and analyzed the transcriptomes of the larval and adult stages of T. pisiformis. The results of differentially expressed genes in these two stages could provide basis for functional genomics, immunology and gene expression profiles of T. pisiformis.

molecular biology↗

bioRxiv2018

Real-time capture of horizontal gene transfers from gut microbiota by engineered CRISPR-Cas acquisition

Horizontal gene transfer (HGT) is central to the adaptation and evolution of bacteria. However, our knowledge about the flow of genetic material within complex microbiomes is lacking; most studies of HGT rely on bioinformatic analyses of genetic elements maintained on evolutionary timescales or experimental measurements of phenotypically trackable markers (e.g. antibiotic resistance). Consequently, our knowledge of the capacity and dynamics of HGT in complex communities is limited. Here, we utilize the CRISPR-Cas spacer acquisition process to detect HGT events from complex microbiota in real-time and at nucleotide resolution. In this system, a recording strain is exposed to a microbial sample, spacers are acquired from foreign transferred elements and permanently stored in genomic CRISPR arrays. Subsequently, sequencing and analysis of these spacers enables identification of the transferred elements. This approach allowed us to quantify transfer frequencies of individual mobile elements without the need for phenotypic markers or post-transfer replication. We show that HGT in human clinical fecal samples can be extensive and rapid, often involving multiple different plasmid types, with the IncX type being the most actively transferred. Importantly, the vast majority of transferred elements did not carry readily selectable phenotypic markers, highlighting the utility of our approach to reveal previously hidden real-time dynamics of mobile gene pools within complex microbiomes.

microbiology↗

bioRxiv2018

METAGENOMIC SEQUENCING FOR COMBINED DETECTION OF RNA AND DNA VIRUSES IN RESPIRATORY SAMPLES FROM PAEDIATRIC PATIENTS

IntroductionViruses are the main cause of respiratory tract infections. Metagenomic next-generation sequencing (mNGS) enables the unbiased detection of all potential pathogens in a clinical sample, including variants and even unknown pathogens. To apply mNGS in viral diagnostics, there is a need for sensitive and simultaneous detection of RNA and DNA viruses. In this study, the performance of an in-house mNGS protocol for routine diagnostics of viral respiratory infections, with single tube DNA and RNA sample-pre-treatment and potential for automated pan-pathogen detection was studied.\n\nMaterials and MethodsThe sequencing protocol and bioinformatics analysis was designed and optimized including the optimal concentration of the spike-in internal controls equine arteritis virus (EAV) and phocine-herpes virus-1 (PhHV-1).The whole genome of PhHV-1 was sequenced and added to the NCBI database. Subsequently, the protocol was retrospectively validated using a selection of 25 respiratory samples with in total 29 positive and 346 negative PCR results, previously sent to the lab for routine diagnostics.\n\nResultsThe results demonstrated that our protocol using Illumina Nextseq 500 sequencing with 10 million reads showed high repeatability. The NCBI RefSeq database as opposed to the NCBI nucleotide database led to enhanced specificity of virus classification. A correlation was established between read counts and PCR cycle threshold value, demonstrating the semi-quantitative nature of viral detection by mNGS. The results as obtained by mNGS appeared condordant with PCR based diagnostics in 25 out of the 29 (86%) respiratory viruses positive by PCR and in 315 of 346 (91%) PCR-negative results. Viral pathogens only detected by mNGS, not present in the routine diagnostic workflow were influenza C, KI polyomavirus, and cytomegalovirus.\n\nConclusionsSensitivity and analytical specificity of this mNGS protocol was comparable with PCR and higher when considering off-PCR target viral pathogens. All potential viral pathogens were detected in one single test, while it simultaneously obtained detailed information on detected viruses.

microbiology↗

bioRxiv2018

Uncovering the unexplored diversity of thioamidated ribosomal peptides in Actinobacteria using the RiPPER genome mining tool

The rational discovery of new specialized metabolites by genome mining represents a very promising strategy in the quest for new bioactive molecules. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural product that derive from genetically encoded precursor peptides. However, RiPP gene clusters are particularly refractory to reliable bioinformatic predictions due to the absence of a common biosynthetic feature across all pathways. Here, we describe RiPPER, a new tool for the family-independent identification of RiPP precursor peptides and apply this methodology to search for novel thioamidated RiPPs in Actinobacteria. Until now, thioamidation was believed to be a rare post-translational modification, which is catalyzed by a pair of proteins (YcaO and TfuA) in Archaea. In Actinobacteria, the thioviridamide-like molecules are a family of cytotoxic RiPPs that feature multiple thioamides, and it has been proposed that a YcaO-TfuA pair of proteins also catalyzes their formation. Potential biosynthetic gene clusters encoding YcaO and TfuA protein pairs are common in Actinobacteria but the chemical diversity generated by these pathways is almost completely unexplored. A RiPPER analysis reveals a highly diverse landscape of precursor peptides encoded in previously undescribed gene clusters that are predicted to make thioamidated RiPPs. To illustrate this strategy, we describe the first rational discovery of a new family of thioamidated natural products, the thiovarsolins from Streptomyces varsoviensis.

microbiology↗

bioRxiv2018

Uncultured marine cyanophages encode for active NblA, phycobilisome proteolysis adaptor protein

Phycobilisomes (PBS) are large water-soluble membrane-associated complexes in cyanobacteria and some chloroplasts that serve as a light-harvesting antennas for the photosynthetic apparatus. When short of nitrogen or sulfur, cyanobacteria readily degrade their phycobilisomes allowing the cell to replenish the vanishing nutrients. The key regulator in the degradation process is NblA, a small protein (~6 kDa) which recruits proteases to the PBS. It was discovered previously that not only do cyanobacteria possess nblA genes but also that they are encoded by genomes of some freshwater cyanophages. A recent study, using assemblies from oceanic metagenomes, revealed genomes of a novel uncultured marine cyanophage lineage which contain genes coding for the PBS degradation protein. Here, we examine the functionality of nblA-like genes from these marine cyanophages by testing them in a freshwater model cyanobacterial nblA knockout. One of the viral NblA variants could complement the non-bleaching phenotype and restore PBS degradation. Our findings reveal a functional NblA from a novel marine cyanophage lineage. Furthermore, we shed new light on the distribution of nblA genes in cyanobacteria and cyanophages.\n\nOriginality-Significance StatementThis is the first study to examine the distribution and function of nblA genes of marine cyanophage origin. We describe as well the distribution of nblA-like genes in marine cyanobacteria using bioinformatic methods.

microbiology↗

bioRxiv2018

Identifying the Biosynthetic Gene Cluster for Triacsins with an N-hydroxytriazene Moiety

Triacsins are a family of natural products containing an N-hydroxytriazene moiety not found in any other known secondary metabolites. Though many studies have examined the biological activity of triacsins in lipid metabolism, the biosynthesis of triacsins has remained unknown. Here, we report the identification of the triacsin biosynthetic gene cluster in Streptomyces aureofaciens ATCC 31442. Bioinformatic analysis of the gene cluster led to the discovery of the tacrolimus producer Streptomyces tsukubaensis NRRL 18488 as a new triacsin producer. In addition to targeted gene disruption to identify necessary genes for triacsin production, stable isotope feeding was performed in vivo to advance the understanding of N-hydroxytriazene biosynthesis.

biochemistry↗

bioRxiv2018

Phase transition and amyloid formation by a viral protein as an additional molecular mechanism of virus-induced cell toxicity

Henipaviruses are severe human pathogens responsible for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We have previously reported a biophysical characterization of the Henipavirus V proteins and shown that they interact with DDB1, a cellular protein that is a component of the ubiquitin ligase E3 complex. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquidhydrogel phase transition. By combining experimental and bioinformatics approaches, we have identified the V region responsible for this phenomenon. This region (referred to as PNT3), which falls within the long intrinsically disordered region of V, was further investigated using a combination of biophysical and structural approaches. ThioflavinT and Congo red binding assays, together with negative-staining electron microscopy studies, show that this region forms amyloid-like, {beta}-enriched structures. Such structures are also formed in mammal cells transfected to express PNT3. Those cells also exhibit a reduced viability in the presence of a stress agent. Interestingly, mammal cells expressing a rationally designed, non-amyloidogenic PNT3 variant (PNT33A), appear to be much less sensitive to the stress agent, thus enabling the establishment of a link between fibril formation and cell toxicity. The present findings therefore pinpoint a so far never reported possible mechanism of virus-induced cell toxicity.

biophysics↗

bioRxiv2018

Id proteins promote a cancer stem cell phenotype in triple negative breast cancer via Robo1-dependent c-Myc activation

Breast cancers display phenotypic and functional heterogeneity and several lines of evidence support the existence of cancer stem cells (CSCs) in certain breast cancers, a minor population of cells capable of tumor initiation and metastatic dissemination. Identifying factors that regulate the CSC phenotype is therefore important for developing strategies to treat metastatic disease. The Inhibitor of Differentiation Protein 1 (Id1) and its closely related family member Inhibitor of Differentiation 3 (Id3) (collectively termed Id) are expressed by a diversity of stem cells and are required for metastatic dissemination in experimental models of breast cancer. In this study, we show that ID1 is expressed in rare neoplastic cells within ER-negative breast cancers. To address the function of Id1 expressing cells within tumors, we developed two independent murine models of Triple Negative Breast Cancer (TNBC) in which a genetic reporter permitted the prospective isolation of Id1+ cells. Id1+ cells are enriched for self-renewal in tumorsphere assays in vitro and for tumor initiation in vivo. Conversely, depletion of Id1 and Id3 in the 4T1 murine model of TNBC demonstrates that Id1/3 are required for cell proliferation and self-renewal in vitro, as well as primary tumor growth and metastatic colonization of the lung in vivo. Using combined bioinformatic analysis, we have defined a novel mechanism of Id protein function via negative regulation of the Roundabout Axon Guidance Receptor Homolog 1 (Robo1) leading to activation of a Myc transcriptional programme.

cancer biology↗

bioRxiv2018

Detection of a novel mutation G511T in the 530 loop in 16S rRNA in multi drugs resistant Mycobacterium tuberculosis isolated from Sudanese patients

BackgroundTuberculosis (TB) is a bacterial disease considered as a global public health emergency by the World Health Organization (WHO) since 1993. In Sudan, MDR-TB represents a growing threat and one of the most important challenges that faced national tuberculosis program to establish a comprehensive multidrug-resistant tuberculosis management system.\n\nObjectiveTo characterize the diversity and frequency of mutations in Sudanese MDR-TB strains isolated from Wad Madani, Al-Gadarif and Khartoum using 16S rRNA and phylogeny approach.\n\nMaterial and MethodsA total of 60 MDR-TB isolates from Wad-Madani, Al-Gadarif and Khartoum were tested with molecular LPA (Genotype MTBDR plus) and GeneXpert MTB/RIF assay and Spoligotyping to confirm their resistance to RIF and INH. Sequencing and phylogenetic analysis was carried out using in silico tools.\n\nResultThis study revealed the circulation of different Sudanese MDR-TB strains isolated from Wad Madani and Al-Gadarif belonging to two distinct common ancestors. Two isolates from Wad Madani (isolate3 and isolate11) found in one main group which characterized by a novel mutation G511T in the 530 loop.\n\nConclusionThe recurrence of C217A mutation in Wad Madani (isolate11) indicates the spread of this mutation in Sudanese MDR-TB strains and the diversity of this inheritance leading to generate new G511T novel mutation. So, understanding the molecular characterization of resistance mechanisms in MD-TB can facilitate the early detection of resistance, the choice of appropriate treatment and ultimately the management of MD-TB transmission. Bioinformatics approaches provide helpful tools for analyzing molecular mechanisms of resistance in pathogens.

microbiology↗

bioRxiv2018

Accessory genome contributes to the virulence and resistance of the ocular isolate of Pseudomonas aeruginosa: A complete genome analysis

Bacteria can acquire an accessory genome through the horizontal transfer of genetic elements from non-parental lineages. This leads to rapid genetic evolution allowing traits such as antibiotic resistance and virulence to spread through bacterial communities. The study of complete genomes of bacterial strains helps to understand the genomic traits associated with virulence and antibiotic resistance. We aimed to investigate the complete accessory genome of an ocular isolate of P. aeruginosa. We obtained the complete genome of the ocular isolate strain PA34 of P. aeruginosa utilising genome sequence reads from Illumina and Oxford Nanopore Technology followed by PCR to close any identified gaps. In-depth genomic analysis was performed using various bioinformatics tools. The phenotypic properties of susceptibility to heavy metals and cytotoxicity were determined to confirm expression of certain traits. The complete genome of PA34 includes a chromosome of 6.8 Mbp and two plasmids of 95.4 Kbp (pMKPA34-1) and 26.8 Kbp (pMKPA34-2). PA34 had a large accessory genome of 1,213 genes and had 543 unique genes not present in other strains. These exclusive genes encoded features related to metal and antibiotic resistance, phage integrase and transposons. At least 24 GIs were predicated in the complete chromosome, of which two were integrated into novel sites. Eleven GIs carried virulence factors or replaced pathogenic genes. A bacteriophage carried the aminoglycoside resistance gene (aac(3)-IId). The two plasmids carried other six antibiotic resistance genes. The large accessory genome of this ocular isolate plays a large role in shaping its virulence and antibiotic resistance.

microbiology↗

bioRxiv2018

Transcriptomic responses to thermal stress and varied phosphorus conditions in Symbiodinium kawagutii

Symbiodinium species are essential symbionts of tropical reef-building corals and the disruption of their symbiosis with corals as a consequence of seawater warming and other stress conditions leads to the globally widespread coral bleaching. As coral reefs live in the oligotrophic environment, Symbiodinium photosynthesis can also face nutrient stress. How metabolic pathways in Symbiodinium respond to thermal stress and phosphate depletion is poorly understood and underexplored for many species. Here we conducted RNA-seq analysis to investigate transcriptomic responses to thermal stress, phosphate deprivation and glycerol-3-phosphate (Gro3P) replacement in S. kawagutii. RNA-seq and bioinformatic analysis were conducted for the above-mentioned three treatments and a control. We identified 221 (2.04%) genes showing no significant differential expression among all conditions, and defined them as \"core\" genes of S. kawagutii, which mostly were in the Gene Ontology terms of catalytic activity and binding. Using algorithms edgeR and NOIseq in combination, we identified a set of differentially expressed genes (DEGs) for each treatment relative to the control. Under heat stress 357 (4.42%) DEGs were found, with predicted roles in active molecular (protein-protein/RNA/DNA) interaction, cell wall modulation and transport (including nutrients, iron, and oxygen). About as many DEGs (396, 4.73%) were identified under P deprivation while nearly double of that (671, 8.05%) were detected under Gro3P utilization; in both cases most of the DEGs were up-regulated and predicted to function in photosystem and defensome. Further KEGG pathway comparison revealed different molecular responses between phosphate deprivation and Gro3P utilization. Catalytic activity and binding seem to be two important core functions in S. kawagutii. The most significant transcriptional response in S. kawagutii to heat stress was regulation of molecular interaction, cell wall modulation, and transport of iron, oxygen, and major nutrients, suggesting that this species uses a unique mechanism to cope with heat stress, possibly conferring thermal tolerance. The greatest transcriptomic impact of phosphate deprivation and Gro3P replacement were the up-regulation of photosystem and defense. This study provides new clues about molecular mechanisms underpinning responses in Symbiodinium to temperature and nutrient stresses, which will generate new hypotheses and set a new framework for future investigations.

microbiology↗

bioRxiv2018

Functional evaluation of transposable elements as transcriptional enhancers in mouse embryonic and trophoblast stem cells

The recurrent invasion and expansion of transposable elements (TEs) throughout evolution brought with it a vast array of coding and non-coding sequences that can serve as substrates for natural selection. Namely, TEs are thought to have contributed to the establishment of gene regulatory networks via their cis-acting elements. Both the embryonic and extraembryonic lineages of the early mouse embryo are thought to have benefited from the co-option of TEs as distal enhancer elements. However, there is little to no evidence that these particular TEs play significant roles in the regulation of gene expression. Here we tested for roles of TEs as enhancers in mouse embryonic and trophoblast stem cells by combining bioinformatic analyses with genetic and epigenetic editing experiments. Epigenomic and transcriptomic data from wildtype cells suggested that a large number of TEs played a role in the establishment of highly tissue-specific gene expression programmes. Through genetic editing of individual TEs we confirmed a subset of these regulatory relationships. However, a wider survey via CRISPR interference of RLTR13D6 elements in embryonic stem cells revealed that only a minority play significant roles in gene regulation. Our results suggest that a small proportion of TEs contribute to the mouse pluripotency regulatory network, and highlight the importance of functional experiments when evaluating the role of TEs in gene regulation.

genetics↗