Insights into the genomic evolution and the alkali tolerance mechanisms of Agaricus sinodeliciosus by comparative genomic and transcriptomic analyses.

Agaricus sinodeliciosus is a rare wild edible mushroom from northwest China, and grows naturally in mild saline-alkali soil, which is also unusual in mushrooms. A. sinodeliciosus represents a potential model organism for explaining saline-alkali tolerance mechanisms and revealing related physiological processes in mushrooms. Here, we provide a high-quality genome of A. sinodeliciosus. Comparative genomic analyses reveal A. sinodeliciosus has numerous changes to its genome organization after a solitary evolutionary history under saline-alkali environments, such as gene family contraction, retrotransposon expansion and rapid evolution of adaptative genes. Our saline and alkali tolerance tests show that mycelium growth and fruit body formation of this species are effected by mild alkalinity. Transcriptomic analyses reveal that genes involved in carbon and nitrogen utilization, cell stability and fruit body formation of A. sinodeliciosus could be activated under mildly alkaline conditions. In particular, the 'starch and sucrose metabolism', 'biosynthesis of amino acids' and 'phenylpropanoid biosynthesis' pathways are important for mildly alkaline tolerance of A. sinodeliciosus. Like plants and arbuscular mycorrhizal fungi, in the rot fungus A. sinodeliciosus, the biosynthesis of intracellular small molecules could be enhanced to counter osmotic and oxidative stresses caused by mild alkalinity, and the biosynthesis of monolignol could be suppressed to increase cell wall infiltrates under mildly alkaline conditions. This research provides an understanding of the genomic evolution and mechanisms of A. sinodeliciosus in tolerance to saline-alkali environments. The A. sinodeliciosus genome constitutes a valuable resource for evolutionary and ecological studies of Agaricus.

SARS-CoV-2 RNAs are processed into 22-nt vsRNAs in Vero cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global pandemic, resulting in great fatalities around the world. Although the antiviral roles of RNA interference (RNAi) have been well studied in plants, nematodes and insects, the antiviral roles of RNAi in mammalians are still debating as RNAi effect is suspected to be suppressed by interferon (IFN) signaling pathways in most cell types. To determine the role of RNAi in mammalian resistance to SARS-CoV-2, we studied the profiling of host small RNAs and SARS-CoV-2 virus-derived small RNAs (vsRNAs) in the early infection stages of Vero cells, an IFN-deficient cell line. We found that host microRNAs (miRNAs) were dysregulated upon SARS-CoV-2 infection, resulting in downregulation of microRNAs playing antiviral functions and upregulation of microRNAs facilitating viral proliferations. Moreover, vsRNA peaked at 22 nt at negative strand but not the positive strand of SARS-CoV-2 and formed successive Dicer-spliced pattern at both strands. Similar characteristics of vsRNAs were observed in IFN-deficient cell lines infected with Sindbis and Zika viruses. Together, these findings indicate that host cell may deploy RNAi pathway to combat SARS-CoV-2 infection in IFN-deficient cells, informing the alternative antiviral strategies to be developed for patients or tissues with IFN deficiency.

Identification of a Putative CodY Regulon in the Gram-Negative Phylum Synergistetes.

CodY is a dominant regulator in low G + C, Gram-positive Firmicutes that governs the regulation of various metabolic pathways and cellular processes. By using various bioinformatics analyses and DNA affinity precipitation assay (DAPA), this study confirmed the presence of CodY orthologues and corresponding regulons in Gram-negative Synergistetes. A novel palindromic sequence consisting of AT-rich arms separated by a spacer region of variable length and sequence was identified in the promoters of the putative codY-containing operons in Synergistetes. The consensus sequence from genera Synergistes and Cloacibacillus (5'-AATTTTCTTAAAATTTCSCTTGATATTTACAATTTT) contained three AT-rich regions, resulting in two palindromic sequences; one of which is identical to Firmicutes CodY box (5'-AATTTTCWGAAAATT). The function of the consensus sequence was tested by using a recombinant CodY protein (His-CodYDSM) of Cloacibacillus evryensis DSM19522 in DAPA. Mutations in the central AT-rich sequence reduced significantly the binding of His-CodYDSM, whereas mutations in the 5' or 3' end AT-rich sequence slightly reduced the binding, indicating that CodYDSM could recognize both palindromic sequences. The proposed binding sequences were found in the promoters of multiple genes involved in amino acids biosynthesis, metabolism, regulation, and stress responses in Synergistetes. Thus, a CodY-like protein from Synergistetes may function similarly to Firmicutes CodY.

Improved 93-11 Genome and Time-Course Transcriptome Expand Resources for Rice Genomics.

In 2002, the first crop genome was published using the rice cultivar 93-11, which is the progenitor of the first super-hybrid rice. The genome sequence has served as a reference genome for the indica cultivars, but the assembly has not been updated. In this study, we update the 93-11 genome assembly to a gap-less sequence using ultra-depth single molecule real-time (SMRT) reads, Hi-C sequencing, reference-guided, and gap-closing approach. The differences in the genome collinearity and gene content between the 93-11 and the Nipponbare reference genomes confirmed to map the indica cultivar sequencing data to the 93-11 genome, instead of the reference. Furthermore, time-course transcriptome data showed that the expression pattern was consistently correlated with the stages of seed development. Alternative splicing of starch synthesis-related genes and genomic variations of waxy make it a novel resource for targeted breeding. Collectively, the updated high quality 93-11 genome assembly can improve the understanding of the genome structures and functions of Oryza groups in molecular breeding programs.

Genome Assembly and Pathway Analysis of Edible Mushroom Agrocybe cylindracea.

Agrocybe cylindracea, an edible mushroom, is widely cultivated for its abundance of nutrients and flavor, and many of its metabolites are reported to have beneficial roles, such as medicinal effects on tumors and chronical illnesses. However, the lack of genomic information has hindered further molecular studies on this fungus. Here, we present a genome assembly of A. cylindracea together with comparative genomics and pathway analyses of Agaricales species. The draft, generated from both next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing platforms to overcome high genetic heterozygosity, is composed of a 56.5 Mb sequence and 15,384 predicted genes. This mushroom possesses a complex reproductive system, including tetrapolar heterothallic and secondary homothallic mechanisms, and harbors several hydrolases and peptidases for gradual and effective degradation of various carbon sources. Our pathway analysis reveals complex processes involved in the biosynthesis of polysaccharides and other active substances, including B vitamins, unsaturated fatty acids, and N-acetylglucosamine. RNA-seq data show that A. cylindracea stipes tend to synthesize carbohydrate for carbon sequestration and energy storage, whereas pilei are more active in carbon utilization and unsaturated fatty acid biosynthesis. These results reflect diverse functions of the two anatomical structures of the fruiting body. Our comprehensive genomic and transcriptomic data, as well as preliminary comparative analyses, provide insights into the molecular details of the medicinal effects in terms of active compounds and nutrient components.

Cardiac arrhythmias and cardiac arrest related to mushroom poisoning: A case report.

BACKGROUND: Mushroom exposure is a global health issue. The manifestations of mushroom poisoning (MP) may vary. Some species have been reported as rhabdomyolytic, hallucinogenic, or gastrointestinal poisons. Critical or even fatal MPs are mostly attributable to Amanita phalloides, with the development of severe liver or renal failure. Myocardial injury and even cases mimicking ST-segment elevation myocardial infarction (STEMI) have been previously reported, while cardiac arrhythmia or cardiac arrest is not commonly seen. CASE SUMMARY: We report a 68-year-old woman with MP who suffered from delirium, seizure, long QT syndrome on electrocardiogram (ECG), severe cardiac arrhythmias of multiple origins, and cardiac arrest. She was intubated and put on blood perfusion. Her kidney and liver functions were intact; creatine kinase-MB was mildly elevated, and then fell within normal range during her hospital stay. We sent the mushrooms she left for translation elongation factor subunit 1α, ribosomal RNA gene sequence, and internal transcribed spacer sequence analyses. There were four kinds of mushrooms identified, two of which were found to be toxic. CONCLUSION: This is the first time that we found cardiac toxicity caused by Panaeolus subbalteatus and Conocybe lactea, which were believed to be toxic to the liver, kidney, and brain. We suggest that intensive monitoring and ECG follow-up are essential to diagnose prolonged QT interval and different forms of tachycardia in MP patients, even without the development of severe liver or renal failure. The mechanisms need to be further investigated and clarified based on animal experiments and molecular signal pathways.

Impact of growth pH and glucose concentrations on the CodY regulatory network in Streptococcus salivarius.

BACKGROUND: Streptococcus salivarius is an abundant isolate of the human oral microbiota. Since both pH and glucose availability fluctuate frequently in the oral cavity, the goal of this study was to investigate regulation by CodY, a conserved pleiotropic regulator of Gram positive bacteria, in response to these two signals. The chemostat culture system was employed to precisely control the growth parameters, and the transcriptomes of wild-type S. salivarius 57.I and its CodY-null derivative (ΔcodY) grown at pH 7 and 5.5, with limited and excessive glucose supply were determined. RESULTS: The transcriptomic analysis revealed that CodY was most active at pH 7 under conditions of glucose limitation. Based on whether a CodY binding consensus could be located in the 5' flanking region of the identified target, the transcriptomic analysis also found that CodY shaped the transcriptome via both direct and indirect regulation. Inactivation of codY reduced the glycolytic capacity and the viability of S. salivarius at pH 5.5 or in the presence of H2O2. Studies using the Galleria mellonella larva model showed that CodY was essential for the toxicity generated from S. salivarius infection, suggesting that CodY regulation was critical for immune evasion and systemic infections. Furthermore, the CodY-null mutant strain exhibited a clumping phenotype and reduced attachment in biofilm assays, suggesting that CodY also modulates cell wall metabolism. Finally, the expression of genes belonging to the CovR regulon was affected by codY inactivation, but CodY and CovR regulated these genes in opposite directions. CONCLUSIONS: Metabolic adaptation in response to nutrient availability and growth pH is tightly linked to stress responses and virulence expression in S. salivarius. The regulation of metabolism by CodY allows for the maximal utilization of available nutrients and ATP production. The counteractive regulation of the CovR regulon could fine tune the transcriptomes in response to environmental changes.

The complete mitochondrial genome of Gossypium hirsutum and evolutionary analysis of higher plant mitochondrial genomes.

BACKGROUND: Mitochondria are the main manufacturers of cellular ATP in eukaryotes. The plant mitochondrial genome contains large number of foreign DNA and repeated sequences undergone frequently intramolecular recombination. Upland Cotton (Gossypium hirsutum L.) is one of the main natural fiber crops and also an important oil-producing plant in the world. Sequencing of the cotton mitochondrial (mt) genome could be helpful for the evolution research of plant mt genomes. METHODOLOGY/PRINCIPAL FINDINGS: We utilized 454 technology for sequencing and combined with Fosmid library of the Gossypium hirsutum mt genome screening and positive clones sequencing and conducted a series of evolutionary analysis on Cycas taitungensis and 24 angiosperms mt genomes. After data assembling and contigs joining, the complete mitochondrial genome sequence of G. hirsutum was obtained. The completed G.hirsutum mt genome is 621,884 bp in length, and contained 68 genes, including 35 protein genes, four rRNA genes and 29 tRNA genes. Five gene clusters are found conserved in all plant mt genomes; one and four clusters are specifically conserved in monocots and dicots, respectively. Homologous sequences are distributed along the plant mt genomes and species closely related share the most homologous sequences. For species that have both mt and chloroplast genome sequences available, we checked the location of cp-like migration and found several fragments closely linked with mitochondrial genes. CONCLUSION: The G. hirsutum mt genome possesses most of the common characters of higher plant mt genomes. The existence of syntenic gene clusters, as well as the conservation of some intergenic sequences and genic content among the plant mt genomes suggest that evolution of mt genomes is consistent with plant taxonomy but independent among different species.

Insight into the specific virulence related genes and toxin-antitoxin virulent pathogenicity islands in swine streptococcosis pathogen Streptococcus equi ssp. zooepidemicus strain ATCC35246.

BACKGROUND: Streptococcus equi ssp. zooepidemicus (S. zooepidemicus) is an important pathogen causing swine streptococcosis in China. Pathogenicity islands (PAIs) of S. zooepidemicus have been transferred among bacteria through horizontal gene transfer (HGT) and play important roles in the adaptation and increased virulence of S. zooepidemicus. The present study used comparative genomics to examine the different pathogenicities of S. zooepidemicus. RESULTS: Genome of S. zooepidemicus ATCC35246 (Sz35246) comprises 2,167,264-bp of a single circular chromosome, with a GC content of 41.65%. Comparative genome analysis of Sz35246, S. zooepidemicus MGCS10565 (Sz10565), Streptococcus equi. ssp. equi. 4047 (Se4047) and S. zooepidemicus H70 (Sz70) identified 320 Sz35246-specific genes, clustered into three toxin-antitoxin (TA) systems PAIs and one restriction modification system (RM system) PAI. These four acquired PAIs encode proteins that may contribute to the overall pathogenic capacity and fitness of this bacterium to adapt to different hosts. Analysis of the in vivo and in vitro transcriptomes of this bacterium revealed differentially expressed PAI genes and non-PAI genes, suggesting that Sz35246 possess mechanisms for infecting animals and adapting to a wide range of host environments. Analysis of the genome identified potential Sz35246 virulence genes. Genes of the Fim III operon were presumed to be involved in breaking the host-restriction of Sz35246. CONCLUSION: Genome wide comparisons of Sz35246 with three other strains and transcriptome analysis revealed novel genes related to bacterial virulence and breaking the host-restriction. Four specific PAIs, which were judged to have been transferred into Sz35246 genome through HGT, were identified for the first time. Further analysis of the TA and RM systems in the PAIs will improve our understanding of the pathogenicity of this bacterium and could lead to the development of diagnostics and vaccines.

Marine sediment bacteria harbor antibiotic resistance genes highly similar to those found in human pathogens.

The ocean is a natural habitat for antibiotic-producing bacteria, and marine aquaculture introduces antibiotics into the ocean to treat infections and improve aquaculture production. Studies have shown that the ocean is an important reservoir of antibiotic resistance genes. However, there is a lack of understanding and knowledge about the clinical importance of the ocean resistome. We investigated the relationship between the ocean bacterial resistome and pathogenic resistome. We applied high-throughput sequencing and metagenomic analyses to explore the resistance genes in bacterial plasmids from marine sediments. Numerous putative resistance determinants were detected among the resistance genes in the sediment bacteria. We also found that several contigs shared high identity with transposons or plasmids from human pathogens, indicating that the sediment bacteria recently contributed or acquired resistance genes from pathogens. Marine sediment bacteria could play an important role in the global exchange of antibiotic resistance.

The association between H3K4me3 and antisense transcription.

Histone H3 lysine 4 trimethylation (H3K4me3) is well known to occur in the promoter region of genes for transcription activation. However, when investigating the H3K4me3 profiles in the mouse cerebrum and testis, we discovered that H3K4me3 also has a significant enrichment at the 3' end of actively transcribed (sense) genes, named as 3'-H3K4me3. 3'-H3K4me3 is associated with ~15% of protein-coding genes in both tissues. In addition, we examined the transcriptional initiation signals including RNA polymerase II (RNAPII) binding sites and 5'-CAGE-tag that marks transcriptional start sites. Interestingly, we found that 3'-H3K4me3 is associated with the initiation of antisense transcription. Furthermore, 3'-H3K4me3 modification levels correlate positively with the antisense expression levels of the associated sense genes, implying that 3'-H3K4me3 is involved in the activation of antisense transcription. Taken together, our findings suggest that H3K4me3 may be involved in the regulation of antisense transcription that initiates from the 3' end of sense genes. In addition, a positive correlation was also observed between the expression of antisense and the associated sense genes with 3'-H3K4me3 modification. More importantly, we observed the 3'-H3K4me3 enrichment among genes in human, fruitfly and Arabidopsis, and found that the sequences of 3'-H3K4me3-marked regions are highly conserved and essentially indistinguishable from known promoters in vertebrate. Therefore, we speculate that these 3'-H3K4me3-marked regions may serve as potential promoters for antisense transcription and 3'-H3K4me3 appear to be a universal epigenetic feature in eukaryotes. Our results provide a novel insight into the epigenetic roles of H3K4me3 and the regulatory mechanism of antisense transcription.

Complete genome and transcriptomes of Streptococcus parasanguinis FW213: phylogenic relations and potential virulence mechanisms.

Streptococcus parasanguinis, a primary colonizer of the tooth surface, is also an opportunistic pathogen for subacute endocarditis. The complete genome of strain FW213 was determined using the traditional shotgun sequencing approach and further refined by the transcriptomes of cells in early exponential and early stationary growth phases in this study. The transcriptomes also discovered 10 transcripts encoding known hypothetical proteins, one pseudogene, five transcripts matched to the Rfam and additional 87 putative small RNAs within the intergenic regions defined by the GLIMMER analysis. The genome contains five acquired genomic islands (GIs) encoding proteins which potentially contribute to the overall pathogenic capacity and fitness of this microbe. The differential expression of the GIs and various open reading frames outside the GIs at the two growth phases suggested that FW213 possess a range of mechanisms to avoid host immune clearance, to colonize host tissues, to survive within oral biofilms and to overcome various environmental insults. Furthermore, the comparative genome analysis of five S. parasanguinis strains indicates that albeit S. parasanguinis strains are highly conserved, variations in the genome content exist. These variations may reflect differences in pathogenic potential between the strains.

Genome sequence of Agrobacterium tumefaciens strain F2, a bioflocculant-producing bacterium.

Agrobacterium tumefaciens F2 is an efficient bioflocculant-producing bacterium. But the genes related to the metabolic pathway of bioflocculant biosynthesis in strain F2 are unknown. We present the draft genome of A. tumefaciens F2. It could provide further insight into the biosynthetic mechanism of polysaccharide-like bioflocculant in strain F2.

Complete genome sequence of Streptococcus equi subsp. zooepidemicus strain ATCC 35246.

Streptococcus equi subsp. zooepidemicus is an opportunistic pathogen. It has caused a very large economic loss in the swine industry of China and has become a threat to human health. We announce the complete genome sequence of S. equi subsp. zooepidemicus strain ATCC 35246, which provides opportunities to understand its pathogenesis mechanism and genetic basis.

Complete genome sequence of the ureolytic Streptococcus salivarius strain 57.I.

Streptococcus salivarius 57.I is one of the most abundant and highly ureolytic bacteria in the human mouth. It can utilize urea as the sole nitrogen source via the activity of urease. Complete genome sequencing of S. salivarius 57.I revealed a chromosome and a phage which are absent in strain SK126.

Effectiveness of 10 polymorphic microsatellite markers for parentage and pedigree analysis in plateau pika (Ochotona curzoniae).

BACKGROUND: The plateau pika (Ochotona curzoniae) is an underground-dwelling mammal, native to the Tibetan plateau of China. A set of 10 polymorphic microsatellite loci has been developed earlier. Its reliability for parentage assignment has been tested in a plateau pika population. Two family groups with a known pedigree were used to validate the power of this set of markers. RESULTS: The error in parentage assignment using a combination of these 10 loci was very low as indicated by their power of discrimination (0.803 - 0.932), power of exclusion (0.351 - 0.887), and an effectiveness of the combined probability of exclusion in parentage assignment of 99.999%. CONCLUSION: All the offspring of a family could be assigned to their biological mother; and their father or relatives could also be identified. This set of markers therefore provides a powerful and efficient tool for parentage assignment and other population analyses in the plateau pika.

A comparison between ribo-minus RNA-sequencing and polyA-selected RNA-sequencing.

To compare the two RNA-sequencing protocols, ribo-minus RNA-sequencing (rmRNA-seq) and polyA-selected RNA-sequencing (mRNA-seq), we acquired transcriptomic data-52 and 32 million alignable reads of 35 bases in length-from the mouse cerebrum, respectively. We found that a higher proportion, 44% and 25%, of the uniquely alignable rmRNA-seq reads, is in intergenic and intronic regions, respectively, as compared to 23% and 15% from the mRNA-seq dataset. Further analysis made an additional discovery of transcripts of protein-coding genes (such as Histone, Heg1, and Dux), ncRNAs, snoRNAs, snRNAs, and novel ncRNAs as well as repeat elements in rmRNA-seq dataset. This result suggests that rmRNA-seq method should detect more polyA- or bimorphic transcripts. Finally, through comparative analyses of gene expression profiles among multiple datasets, we demonstrated that different RNA sample preparations may result in significant variations in gene expression profiles.

A modified enrichment method to construct microsatellite library from plateau pika genome (Ochotona curzoniae).

A microsatellite-enriched library of plateau pika (Ochotona curzoniae) was constructed according to the strong affinity between biotin and streptavidin. Firstly, genomic DNA was fragmented by ultrasonication, which is a major improvement over traditional methods. Linker-ligated DNA fragments were hybridized with biotinylated microsatellite probes, and then were subjected to streptavidin-coated magnetic beads. PCR amplification was performed to obtain double-stranded DNA fragments containing microsatellites. Ligation and transformation were carried out by using the pGEM-T Vector System I and Escherichia coli DH10B competent cells. Sequencing results showed that 80.2% of clones contained microsatellite repeat motif. Several modifications make this protocol time-efficient and technically easier than the traditional ones; particularly, composition and relative abundance of microsatellite repeats in plateau pika genome were truly represented through the optimized PCR conditions. This method has also been successfully applied to construct microsatellite-enriched genomic libraries of Chinese hamster (Cricetulus griseus) and small abalone [Haliotis diversicolor (Reeve)] with high rates of positive clones, demonstrating its feasibility and stability.

The Genomes of Oryza sativa: a history of duplications.

We report improved whole-genome shotgun sequences for the genomes of indica and japonica rice, both with multimegabase contiguity, or almost 1,000-fold improvement over the drafts of 2002. Tested against a nonredundant collection of 19,079 full-length cDNAs, 97.7% of the genes are aligned, without fragmentation, to the mapped super-scaffolds of one or the other genome. We introduce a gene identification procedure for plants that does not rely on similarity to known genes to remove erroneous predictions resulting from transposable elements. Using the available EST data to adjust for residual errors in the predictions, the estimated gene count is at least 38,000-40,000. Only 2%-3% of the genes are unique to any one subspecies, comparable to the amount of sequence that might still be missing. Despite this lack of variation in gene content, there is enormous variation in the intergenic regions. At least a quarter of the two sequences could not be aligned, and where they could be aligned, single nucleotide polymorphism (SNP) rates varied from as little as 3.0 SNP/kb in the coding regions to 27.6 SNP/kb in the transposable elements. A more inclusive new approach for analyzing duplication history is introduced here. It reveals an ancient whole-genome duplication, a recent segmental duplication on Chromosomes 11 and 12, and massive ongoing individual gene duplications. We find 18 distinct pairs of duplicated segments that cover 65.7% of the genome; 17 of these pairs date back to a common time before the divergence of the grasses. More important, ongoing individual gene duplications provide a never-ending source of raw material for gene genesis and are major contributors to the differences between members of the grass family.

Gene expression profiling in porcine fetal thymus.

To obtain an initial overview of gene diversity and expression pattern in porcine thymus, 11,712 ESTs (Expressed Sequence Tags) from 100-day-old porcine thymus (FTY) were sequenced and 7,071 cleaned ESTs were used for gene expression analysis. Clustered by the PHRAP program, 959 contigs and 3,074 singlets were obtained. Blast search showed that 806 contigs and 1,669 singlets (totally 5,442 ESTs) had homologues in GenBank and 1,629 ESTs were novel. According to the Gene Ontology classification, 36.99% ESTs were cataloged into the gene expression group, indicating that although the functional gene (18.78% in defense group) of thymus is expressed in a certain degree, the 100-day-old porcine thymus still exists in a developmental stage. Comparative analysis showed that the gene expression pattern of the 100-day-old porcine thymus is similar to that of the human infant thymus.