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September 22, 2019  |  

Full-length transcriptome sequences of ephemeral plant Arabidopsis pumila provides insight into gene expression dynamics during continuous salt stress.

Arabidopsis pumila is native to the desert region of northwest China and it is extraordinarily well adapted to the local semi-desert saline soil, thus providing a candidate plant system for environmental adaptation and salt-tolerance gene mining. However, understanding of the salt-adaptation mechanism of this species is limited because of genomic sequences scarcity. In the present study, the transcriptome profiles of A. pumila leaf tissues treated with 250 mM NaCl for 0, 0.5, 3, 6, 12, 24 and 48 h were analyzed using a combination of second-generation sequencing (SGS) and third-generation single-molecule real-time (SMRT) sequencing.Correction of SMRT long reads by SGS short reads resulted in 59,328 transcripts. We found 8075 differentially expressed genes (DEGs) between salt-stressed tissues and controls, of which 483 were transcription factors and 1157 were transport proteins. Most DEGs were activated within 6 h of salt stress and their expression stabilized after 48 h; the number of DEGs was greatest within 12 h of salt stress. Gene annotation and functional analyses revealed that expression of genes associated with the osmotic and ionic phases rapidly and coordinately changed during the continuous salt stress in this species, and salt stress-related categories were highly enriched among these DEGs, including oxidation-reduction, transmembrane transport, transcription factor activity and ion channel activity. Orphan, MYB, HB, bHLH, C3H, PHD, bZIP, ARF and NAC TFs were most enriched in DEGs; ABCB1, CLC-A, CPK30, KEA2, KUP9, NHX1, SOS1, VHA-A and VP1 TPs were extensively up-regulated in salt-stressed samples, suggesting that they play important roles in slat tolerance. Importantly, further experimental studies identified a mitogen-activated protein kinase (MAPK) gene MAPKKK18 as continuously up-regulated throughout salt stress, suggesting its crucial role in salt tolerance. The expression patterns of the salt-responsive 24 genes resulted from quantitative real-time PCR were basically consistent with their transcript abundance changes identified by RNA-Seq.The full-length transcripts generated in this study provide a more accurate depiction of gene transcription of A. pumila. We identified potential genes involved in salt tolerance of A. pumila. These data present a genetic resource and facilitate better understanding of salt-adaptation mechanism for ephemeral plants.


September 22, 2019  |  

Analysis of transcripts and splice isoforms in red clover (Trifolium pratense L.) by single-molecule long-read sequencing.

Red clover (Trifolium pratense L.) is an important cool-season legume plant, which is the most widely planted forage legume after alfalfa. Although a draft genome sequence was published already, the sequences and completed structure of mRNA transcripts remain unclear, which limit further explore on red clover.In this study, the red clover transcriptome was sequenced using single-molecule long-read sequencing to identify full-length splice isoforms, and 29,730 novel isoforms from known genes and 2194 novel isoforms from novel genes were identified. A total of 5492 alternative splicing events was identified and the majority of alter spliced events in red clover was corrected as intron retention. In addition, of the 15,229 genes detected by SMRT, 8719 including 186,517 transcripts have at least one poly(A) site. Furthermore, we identified 4333 long non-coding RNAs and 3762 fusion transcripts.We analyzed full-length transcriptome of red clover with PacBio SMRT. Those new findings provided important information for improving red clover draft genome annotation and fully characterization of red clover transcriptome.


September 22, 2019  |  

Transcription-associated mutation promotes RNA complexity in highly expressed genes – A major new source of selectable variation.

Alternatively spliced transcript isoforms are thought to play a critical role for functional diversity. However, the mechanism generating the enormous diversity of spliced transcript isoforms remains unknown, and its biological significance remains unclear. We analyzed transcriptomes in saker falcons, chickens, and mice to show that alternative splicing occurs more frequently, yielding more isoforms, in highly expressed genes. We focused on hemoglobin in the falcon, the most abundantly expressed genes in blood, finding that alternative splicing produces 10-fold more isoforms than expected from the number of splice junctions in the genome. These isoforms were produced mainly by alternative use of de novo splice sites generated by transcription-associated mutation (TAM), not by the RNA editing mechanism normally invoked. We found that high expression of globin genes increases mutation frequencies during transcription, especially on nontranscribed DNA strands. After DNA replication, transcribed strands inherit these somatic mutations, creating de novo splice sites, and generating multiple distinct isoforms in the cell clone. Bisulfate sequencing revealed that DNA methylation may counteract this process by suppressing TAM, suggesting DNA methylation can spatially regulate RNA complexity. RNA profiling showed that falcons living on the high Qinghai-Tibetan Plateau possess greater global gene expression levels and higher diversity of mean to high abundance isoforms (reads per kilobases per million mapped reads?=18) than their low-altitude counterparts, and we speculate that this may enhance their oxygen transport capacity under low-oxygen environments. Thus, TAM-induced RNA diversity may be physiologically significant, providing an alternative strategy in lifestyle evolution.


September 22, 2019  |  

Basic characterization of natural transformation in a highly transformable Haemophilus parasuis strain SC1401.

Haemophilus parasuis causes Glässer’s disease and pneumonia, incurring serious economic losses in the porcine industry. In this study, natural competence was investigated in H. parasuis. We found competence genes in H. parasuis homologous to ones in Haemophilus influenzae and a high consensus battery of Sxy-dependent cyclic AMP (cAMP) receptor protein (CRP-S) regulons using bioinformatics. High rates of natural competence were found from the onset of stationary-phase growth condition to mid-stationary phase (OD600 from 0.29 to 1.735); this rapidly dropped off as cells reached mid-stationary phase (OD600 from 1.735 to 1.625). As a whole, bacteria cultured in liquid media were observed to have lower competence levels than those grown on solid media plates. We also revealed that natural transformation in this species is stable after 200 passages and is largely dependent on DNA concentration. Transformation competition experiments showed that heterogeneous DNA cannot outcompete intraspecific natural transformation, suggesting an endogenous uptake sequence or other molecular markers may be important in differentiating heterogeneous DNA. We performed qRT-PCR targeting multiple putative competence genes in an effort to compare bacteria pre-cultured in TSB++ vs. TSA++ and SC1401 vs. SH0165 to determine expression profiles of the homologs of competence-genes in H. influenzae. Taken together, this study is the first to investigate natural transformation in H. parasuis based on a highly naturally transformable strain SC1401.


September 22, 2019  |  

Multidrug-resistant Escherichia albertii: Co-occurrence of ß-lactamase and MCR-1 encoding genes.

Escherichia albertii is an emerging member of the Enterobacteriaceae causing human and animal enteric infections. Antimicrobial resistance among enteropathogens has been reported to be increasing in the past years. The purpose of this study was to investigate antibiotic resistance and resistance genes in E. albertii isolated from Zigong city, Sichuan province, China. The susceptibility to 21 antimicrobial agents was determined by Kirby-Bauer disk diffusion method. The highest prevalence was tetracycline resistance with a rate of 62.7%, followed by resistance to nalidixic acid and streptomycin with a rate of 56.9 and 51.0%, respectively. All isolates were sensitive or intermediate susceptible to imipenem, meropenem, amoxicillin-clavulanic acid, and levofloxacin. Among 51 E. albertii isolates, 15 were extended-spectrum ß-lactamase-producing as confirmed by the double disk test. The main ß-lactamase gene groups, i.e., blaTEM, blaSHV, and blaCTX-M, were detected in17, 20, and 22 isolates, respectively. Furthermore, four colistin-resistant isolates with minimum inhibitory concentrations of 8 mg/L were identified. The colistin-resistant isolates all harbored mcr-1 and blaCTX-M-55. Genome sequencing showed that E. albertii strain SP140150 carried mcr-1 and blaCTX-M-55 in two different plasmids. This study provided significant information regarding antibiotic resistance profiles and identified the co-occurrence of ß-lactamase and MCR-1 encoding genes in E. albertii isolates.


September 22, 2019  |  

Analyzing AbrB-knockout effects through genome and transcriptome sequencing of Bacillus licheniformis DW2.

As an industrial bacterium, Bacillus licheniformis DW2 produces bacitracin which is an important antibiotic for many pathogenic microorganisms. Our previous study showed AbrB-knockout could significantly increase the production of bacitracin. Accordingly, it was meaningful to understand its genome features, expression differences between wild and AbrB-knockout (?AbrB) strains, and the regulation of bacitracin biosynthesis. Here, we sequenced, de novo assembled and annotated its genome, and also sequenced the transcriptomes in three growth phases. The genome of DW2 contained a DNA molecule of 4,468,952 bp with 45.93% GC content and 4,717 protein coding genes. The transcriptome reads were mapped to the assembled genome, and obtained 4,102~4,536 expressed genes from different samples. We investigated transcription changes in B. licheniformis DW2 and showed that ?AbrB caused hundreds of genes up-regulation and down-regulation in different growth phases. We identified a complete bacitracin synthetase gene cluster, including the location and length of bacABC, bcrABC, and bacT, as well as their arrangement. The gene cluster bcrABC were significantly up-regulated in ?AbrB strain, which supported the hypothesis in previous study of bcrABC transporting bacitracin out of the cell to avoid self-intoxication, and was consistent with the previous experimental result that ?AbrB could yield more bacitracin. This study provided a high quality reference genome for B. licheniformis DW2, and the transcriptome data depicted global alterations across two strains and three phases offered an understanding of AbrB regulation and bacitracin biosynthesis through gene expression.


September 22, 2019  |  

Comparative genomics of the wheat fungal pathogen Pyrenophora tritici-repentis reveals chromosomal variations and genome plasticity.

Pyrenophora tritici-repentis (Ptr) is a necrotrophic fungal pathogen that causes the major wheat disease, tan spot. We set out to provide essential genomics-based resources in order to better understand the pathogenicity mechanisms of this important pathogen.Here, we present eight new Ptr isolate genomes, assembled and annotated; representing races 1, 2 and 5, and a new race. We report a high quality Ptr reference genome, sequenced by PacBio technology with Illumina paired-end data support and optical mapping. An estimated 98% of the genome coverage was mapped to 10 chromosomal groups, using a two-enzyme hybrid approach. The final reference genome was 40.9 Mb and contained a total of 13,797 annotated genes, supported by transcriptomic and proteogenomics data sets.Whole genome comparative analysis revealed major chromosomal segmental rearrangements and fusions, highlighting intraspecific genome plasticity in this species. Furthermore, the Ptr race classification was not supported at the whole genome level, as phylogenetic analysis did not cluster the ToxA producing isolates. This expansion of available Ptr genomics resources will directly facilitate research aimed at controlling tan spot disease.


September 22, 2019  |  

Heterologous expression guides identification of the biosynthetic gene cluster of chuangxinmycin, an indole alkaloid antibiotic.

The indole alkaloid antibiotic chuangxinmycin, from Actinobacteria Actinoplanes tsinanensis, containing a unique thiopyrano[4,3,2- cd]indole scaffold, is a potent and selective inhibitor of bacterial tryptophanyl-tRNA synthetase. The chuangxinmycin biosynthetic gene cluster was identified by in silico analysis of the genome sequence, then verified by heterologous expression. Systemic gene inactivation and intermediate identification determined the minimum set of genes for unique thiopyrano[4,3,2- cd]indole formation and the concerted action of a radical S-adenosylmethionine protein plus an unknown protein for addition of the 3-methyl group. These findings set a solid foundation for comprehensively investigating the biosynthesis, optimizing yield, and generating new analogues of chuangxinmycin.


September 22, 2019  |  

Phenotypic diversification by enhanced genome restructuring after induction of multiple DNA double-strand breaks.

DNA double-strand break (DSB)-mediated genome rearrangements are assumed to provide diverse raw genetic materials enabling accelerated adaptive evolution; however, it remains unclear about the consequences of massive simultaneous DSB formation in cells and their resulting phenotypic impact. Here, we establish an artificial genome-restructuring technology by conditionally introducing multiple genomic DSBs in vivo using a temperature-dependent endonuclease TaqI. Application in yeast and Arabidopsis thaliana generates strains with phenotypes, including improved ethanol production from xylose at higher temperature and increased plant biomass, that are stably inherited to offspring after multiple passages. High-throughput genome resequencing revealed that these strains harbor diverse rearrangements, including copy number variations, translocations in retrotransposons, and direct end-joinings at TaqI-cleavage sites. Furthermore, large-scale rearrangements occur frequently in diploid yeasts (28.1%) and tetraploid plants (46.3%), whereas haploid yeasts and diploid plants undergo minimal rearrangement. This genome-restructuring system (TAQing system) will enable rapid genome breeding and aid genome-evolution studies.


September 22, 2019  |  

Precise control of SCRaMbLE in synthetic haploid and diploid yeast.

Compatibility between host cells and heterologous pathways is a challenge for constructing organisms with high productivity or gain of function. Designer yeast cells incorporating the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system provide a platform for generating genotype diversity. Here we construct a genetic AND gate to enable precise control of the SCRaMbLE method to generate synthetic haploid and diploid yeast with desired phenotypes. The yield of carotenoids is increased to 1.5-fold by SCRaMbLEing haploid strains and we determine that the deletion of YEL013W is responsible for the increase. Based on the SCRaMbLEing in diploid strains, we develop a strategy called Multiplex SCRaMbLE Iterative Cycling (MuSIC) to increase the production of carotenoids up to 38.8-fold through 5 iterative cycles of SCRaMbLE. This strategy is potentially a powerful tool for increasing the production of bio-based chemicals and for mining deep knowledge.


September 22, 2019  |  

Genome-wide analysis of Mycoplasma bovirhinis GS01 reveals potential virulence factors and phylogenetic relationships.

Mycoplasma bovirhinis is a significant etiology in bovine pneumonia and mastitis, but our knowledge about the genetic and pathogenic mechanisms of M. bovirhinis is very limited. In this study, we sequenced the complete genome of M. bovirhinis strain GS01 isolated from the nasal swab of pneumonic calves in Gansu, China, and we found that its genome forms a 847,985 bp single circular chromosome with a GC content of 27.57% and with 707 protein-coding genes. The putative virulence determinants of M. bovirhinis were then analyzed. Results showed that three genomic islands and 16 putative virulence genes, including one adhesion gene enolase, seven surface lipoproteins, proteins involved in glycerol metabolism, and cation transporters, might be potential virulence factors. Glycerol and pyruvate metabolic pathways were defective. Comparative analysis revealed remarkable genome variations between GS01 and a recently reported HAZ141_2 strain, and extremely low homology with others mycoplasma species. Phylogenetic analysis demonstrated that M. bovirhinis was most genetically close to M. canis, distant from other bovine Mycoplasma species. Genomic dissection may provide useful information on the pathogenic mechanisms and genetics of M. bovirhinis. Copyright © 2018 Chen et al.


September 22, 2019  |  

Insect symbionts as valuable grist for the biotechnological mill: an alkaliphilic silkworm gut bacterium for efficient lactic acid production.

Insects constitute the most abundant and diverse animal class and act as hosts to an extraordinary variety of symbiotic microorganisms. These microbes living inside the insects play critical roles in host biology and are also valuable bioresources. Enterococcus mundtii EMB156, isolated from the larval gut (gut pH >10) of the model organism Bombyx mori (Lepidoptera: Bombycidae), efficiently produces lactic acid, an important metabolite for industrial production of bioplastic materials. E. mundtii EMB156 grows well under alkaline conditions and stably converts various carbon sources into lactic acid, offering advantages in downstream fermentative processes. High-yield lactic acid production can be achieved by the strain EMB156 from renewable biomass substrates under alkaline pretreatments. Single-molecule real-time (SMRT) sequencing technology revealed its 3.01 Mbp whole genome sequence. A total of 2956 protein-coding sequences, 65 tRNA genes, and 6 rRNA operons were predicted in the EMB156 chromosome. Remarkable genomic features responsible for lactic acid fermentation included key enzymes involved in the pentose phosphate (PP)/glycolytic pathway, and an alpha amylase and xylose isomerase were characterized in EMB156. This genomic information coincides with the phenotype of E. mundtii EMB156, reflecting its metabolic flexibility in efficient lactate fermentation, and established a foundation for future biotechnological application. Interestingly, enzyme activities of amylase were quite stable in high-pH broths, indicating a possible mechanism for strong EMB156 growth in an alkaline environment, thereby facilitating lactic acid production. Together, these findings implied that valuable lactic acid-producing bacteria can be discovered efficiently by screening under the extremely alkaline conditions, as exemplified by gut microbial symbionts of Lepidoptera insects.


September 22, 2019  |  

Clinical Staphylococcus argenteus develops to small colony variants to promote persistent infection.

Staphylococcus argenteus is a novel staphylococcal species (also considered as a part of Staphylococcus aureus complex) that is infrequently reported on, and clinical S. argenteus infections are largely unstudied. Here, we report a persistent and recurrent hip joint infection case in which a S. argenteus strain and its small colony variants (SCVs) strain were successively isolated. We present features of the two S. argenteus strains and case details of their pathogenicity, explore factors that induce S. argenteus SCVs formation in the course of anti-infection therapy, and reveal potential genetic mechanisms for S. argenteus SCVs formation. S. argenteus strains were identified using phenotypic and genotypic methods. The S. argenteus strain XNO62 and SCV strain XNO106 were characterized using different models. S. argenteus SCVs were induced by the administration of amikacin and by chronic infection course based on the clinical case details. The genomes of both strains were sequenced and aligned in a pair-wise fashion using Mauve. The case details gave us important insights on the characteristics and therapeutic strategies for infections caused by S. argenteus and its SCVs. We found that strain XNO62 and SCV strain XNO106 are genetically-related sequential clones, the SCV strain exhibits reduced virulence but enhanced intracellular persistence compared to strain XNO62, thus promoting persistent infection. The induction experiments for S. argenteus SCVs demonstrated that high concentrations of amikacin greatly induce S. argenteus XNO62 to form SCVs, while a chronic infection of S. argenteus XNO62 slightly induces SCVs formation. Potential genetic mechanisms for S. argenteus SCVs formation were revealed and discussed based on genomic alignments. In conclusion, we report the first case of infection caused by S. argenteus and its SCVs strain. More attention should be paid to infections caused by S. argenteus and its SCVs, as they constitute a challenge to current therapeutic strategies. The problem of S. argenteus SCVs should be noticed, in particular when amikacin is used or in the case of a chronic S. argenteus infection.


September 22, 2019  |  

Identification of natural product compounds as quorum sensing inhibitors in Pseudomonas fluorescens P07 through virtual screening.

Pseudomonas fluorescens, a Gram-negative psychrotrophic bacteria, is the main microorganism causing spoilage of chilled raw milk and aquatic products. Quorum sensing (QS) widely exists in bacteria to monitor their population densities and regulate numerous physiological activities, such as the secretion of siderophores, swarming motility and biofilm formation. Thus, searching for quorum sensing inhibitors (QSIs) may be another promising way to control the deterioration of food caused by P. fluorescens. Here, we screened a traditional Chinese medicine (TCM) database to discover potential QSIs with lesser toxicity. The gene sequences of LuxI- and LuxR-type proteins of P. fluorescens P07 were obtained through whole-genome sequencing. In addition, the protein structures built by homology modelling were used as targets to screen for QSIs. Twenty-one compounds with a dock score greater than 6 were purchased and tested by biosensor strains (Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136). The results showed that 10 of the compounds were determined as hits (hit rate: 66.67%). Benzyl alcohol, rhodinyl formate and houttuynine were effective QSIs. The impact of the most active compound (benzyl alcohol) on the phenotypes of P. fluorescens P07, including swimming and swarming motility, production of extracellular enzymes and siderophores, N-acylhomoserine lactone (AHLs) content and biofilm formation were determined. The inhibitory mechanism of benzyl alcohol on the QS system of P. fluorescens P07 is further discussed. This study reveals the feasibility of searching for novel QSIs through virtual screening. Copyright © 2018 Elsevier Ltd. All rights reserved.


September 22, 2019  |  

Temperature responses of mutation rate and mutational spectrum in an Escherichia coli strain and the correlation with metabolic rate.

Temperature is a major determinant of spontaneous mutation, but the precise mode, and the underlying mechanisms, of the temperature influences remain less clear. Here we used a mutation accumulation approach combined with whole-genome sequencing to investigate the temperature dependence of spontaneous mutation in an Escherichia coli strain. Experiments were performed under aerobic conditions at 25, 28 and 37 °C, three temperatures that were non-stressful for the bacterium but caused significantly different bacterial growth rates.Mutation rate did not differ between 25 and 28 °C, but was higher at 37 °C. Detailed analyses of the molecular spectrum of mutations were performed; and a particularly interesting finding is that higher temperature led to a bias of mutation to coding, relative to noncoding, DNA. Furthermore, the temperature response of mutation rate was extremely similar to that of metabolic rate, consistent with an idea that metabolic rate predicts mutation rate.Temperature affects mutation rate and the types of mutation supply, both being crucial for the opportunity of natural selection. Our results help understand how temperature drives evolutionary speed of organisms and thus the global patterns of biodiversity. This study also lend support to the metabolic theory of ecology for linking metabolic rate and molecular evolution rate.


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