Plasmid-mediated antibiotic resistance gene transfer under environmental stresses: Insights from laboratory-based studies.

Although clinical settings play a major role in the current global dissemination of antibiotic resistance, once antibiotic resistance bacteria and genes are released into the environment, their fate will be subject to complex ecological processes. One of the processes prevalent in microbial communities - horizontal gene transfer - can largely facilitate the dissemination of antibiotic resistance genes (ARGs) across phylogenetic and ecological boundaries. Especially, plasmid transfer has aroused increasing concern as it has been proved a significant role in promoting ARG dissemination. As a multi-step process, plasmid transfer can be influenced by various factors, among which those stresses caused by environmental pollutants are important elements affecting the plasmid mediated ARG transfer in the environment. In fact, diverse traditional and emerging pollutants are continuously entering the environment nowadays, as evidenced by the global occurrence of pollutants like metals and pharmaceuticals in aquatic and terrestrial systems. It is therefore imperative to understand to what extent and in which way the plasmid mediated ARG dissemination can be influenced by these stresses. Over the past decades, numerous research endeavours have been made to understand the plasmid mediated ARG transfer under various environmental relevant pressures. In this review, progress and challenges of studies on environmental stress regulating plasmid mediated ARG dissemination will be discussed, with specific focus on emerging pollutants like antibiotics and non-antibiotic pharmaceuticals, metals and their nanoparticles, disinfectants and disinfection by-products, as well as the emerging particulate matter like microplastics. Despite the previous efforts, we are still lacking insights into the in situ plasmid transfer under environmental stresses, which can be addressed by future studies considering environmental relevant pollution status and multi-species microbial communities. We believe that future development of standardized high-throughput screening platforms will assist in rapidly identifying which pollutants enhance plasmid transfer and also which ones may block such gene transfer processes.

An omics-based framework for assessing the health risk of antimicrobial resistance genes.

Antibiotic resistance genes (ARGs) are widespread among bacteria. However, not all ARGs pose serious threats to public health, highlighting the importance of identifying those that are high-risk. Here, we developed an 'omics-based' framework to evaluate ARG risk considering human-associated-enrichment, gene mobility, and host pathogenicity. Our framework classifies human-associated, mobile ARGs (3.6% of all ARGs) as the highest risk, which we further differentiate as 'current threats' (Rank I; 3%) - already present among pathogens - and 'future threats' (Rank II; 0.6%) - novel resistance emerging from non-pathogens. Our framework identified 73 'current threat' ARG families. Of these, 35 were among the 37 high-risk ARGs proposed by the World Health Organization and other literature; the remaining 38 were significantly enriched in hospital plasmids. By evaluating all pathogen genomes released since framework construction, we confirmed that ARGs that recently transferred into pathogens were significantly enriched in Rank II ('future threats'). Lastly, we applied the framework to gut microbiome genomes from fecal microbiota transplantation donors. We found that although ARGs were widespread (73% of genomes), only 8.9% of genomes contained high-risk ARGs. Our framework provides an easy-to-implement approach to identify current and future antimicrobial resistance threats, with potential clinical applications including reducing risk of microbiome-based interventions.

Source tracking of antibiotic resistance genes in the environment - Challenges, progress, and prospects.

Antibiotic resistance has become a global public health concern, rendering common infections untreatable. Given the widespread occurrence, increasing attention is being turned toward environmental pathways that potentially contribute to antibiotic resistance gene (ARG) dissemination outside the clinical realm. Studies during the past decade have clearly proved the increased ARG pollution trend along with gradient of anthropogenic interference, mainly through marker-ARG detection by PCR-based approaches. However, accurate source-tracking has been always confounded by various factors in previous studies, such as autochthonous ARG level, spatiotemporal variability and environmental resistome complexity, as well as inherent method limitation. The rapidly developed metagenomics profiles ARG occurrence within the sample-wide genomic context, opening a new avenue for source tracking of environmental ARG pollution. Coupling with machine-learning classification, it has been demonstrated the potential of metagenomic ARG profiles in unambiguously assigning source contribution. Through identifying indicator ARG and recovering ARG-host genomes, metagenomics-based analysis will further increase the resolution and accuracy of source tracking. In this review, challenges and progresses in source-tracking studies on environmental ARG pollution will be discussed, with specific focus on recent metagenomics-guide approaches. We propose an integrative metagenomics-based framework, in which coordinated efforts on experimental design and metagenomic analysis will assist in realizing the ultimate goal of robust source-tracking in environmental ARG pollution.

Online searching platform for the antibiotic resistome in bacterial tree of life and global habitats.

Metagenomic analysis reveals that antibiotic-resistance genes (ARGs) are widely distributed in both human-associated and non-human-associated habitats. However, it is difficult to equally compare ARGs between samples without a standard method. Here, we constructed a comprehensive profile of the distribution of potential ARGs in bacterial tree of life and global habitats by investigating ARGs in 55 000 bacterial genomes, 16 000 bacterial plasmid sequences, 3000 bacterial integron sequences and 850 metagenomes using a standard pipeline. We found that >80% of all known ARGs are not carried by any plasmid or integron sequences. Among potential mobile ARGs, tetracycline and beta-lactam resistance genes (such as tetA, tetM and class A beta-lactamase gene) distribute in multiple pathogens across bacterial phyla, indicating their clinical relevance and importance. We showed that class 1 integrases (intI1) display a poor linear relationship with total ARGs in both non-human-associated and human-associated environments. Furthermore, both total ARGs and intI1 genes show little correlation with the degree of anthropogenicity. These observations highlight the need to differentiate ARGs of high clinical relevance. This profile is published on an online platform (ARGs-OSP, http://args-osp.herokuapp.com/) as a valuable resource for the most challenging topics in this field, i.e. the risk, evolution and emergence of ARGs.

Fate of Labile Organic Carbon in Paddy Soil Is Regulated by Microbial Ferric Iron Reduction.

Global paddy soil is the primary source of methane, a potent greenhouse gas. It is therefore highly important to understand the carbon cycling in paddy soil. Microbial reduction of iron, which is widely found in paddy soil, is likely coupled with the oxidation of dissolved organic matter (DOM) and suppresses methanogenesis. However, little is known about the biotransformation of small molecular DOM accumulated under flooded conditions and the effect of iron reduction on the biotransformation pathway. Here, we carried out anaerobic incubation experiments using field-collected samples amended with ferrihydrite and different short-chain fatty acids. Our results showed that less than 20% of short-chain fatty acids were mineralized and released to the atmosphere. Using Fourier transform ion cyclotron resonance mass spectrometry, we further found that a large number of recalcitrant molecules were produced during microbial consumption of these short-chain fatty acids. Moreover, the biotransformation efficiency of short-chain fatty acids decreased with the increasing length of carbon chains. Ferrihydrite addition promoted microbial assimilation of short-chain fatty acids as well as enhanced the activation and biotransformation of indigenous stable carbon in the soil replenished with formate. This study demonstrates the significance of ferrihydrite in the biotransformation of labile DOM and promotes a more comprehensive understanding of the coupling of iron reduction and carbon cycling in paddy soils.

Effects of Genipin on the expression of uncoupling protein 1 in brown adipose and white adipose tissues in mice / 中国应用生理学杂志

OBJECTIVE@#To investigate the effects of genipin on promoting brown adipose tissue activation and white adipose tissue browning.@*METHODS@#The male C57BL/6J mice were divided into three groups: normal control group, genipin group and cold-stimulus group.Genipin group were treated consecutively with genipin at a dose of 15 mg/kg once a day for 9 days, normal control group were treated with the saline.The mice with cold-stimulus were exposed to 4℃ environment for 5 days.Daily food amount and body weight were measured.Morphological changes were observed in the subscapular region, inguinal region and epididymis around the adipose tissue.The expression of uncoupling protein 1 (UCP1) was determined by real-time PCR and Western blot respectively.@*RESULTS@#The wet weight of white fat in genipin-treated mice was decreased by 16% , and 28% in that of cold-stimulus mice, compared with the normal control group (P＜0.05).After treatments of genipin and cold-stimulus, the color of white adipose tissues was darker, and the size of lipid droplets in adipocytes was smaller, whereas the number was increased.Compared with the normal control group, UCP1 expression was increased obviously in fat tissues, including the subcutaneous and visceral white adipose tissues, and brown adipose tissue after treated with genipin and cold-stimulus (P＜0.05).@*CONCLUSION@#Genipin promoted activation of brown adipose tissue and browning of white adipose tissue by upregulating UCP1 expression, which could contribute to the loss of body weight against obesity.

Conserved phylogenetic distribution and limited antibiotic resistance of class 1 integrons revealed by assessing the bacterial genome and plasmid collection.

BACKGROUND: Integrons, especially the class 1 integrons, are major contributors to the acquisition and dissemination of antibiotic resistance genes (ARGs). However, comprehensive knowledge of the types, content, and distribution of integrons in bacterial taxa is lacking to evaluate their contribution. RESULTS: We have constructed a new integrase database and developed a pipeline that provides comprehensive recovery of class 1 integrons. Previous PCR-based techniques might only detect one fourth of the integron-integrases and integrons recovered in this study. By exploring the class 1 integrons in over 73,000 currently available complete and draft bacterial genomes, the contribution of class 1 integrons in spreading and acquiring ARGs was evaluated. Firstly, the host species of class 1 integrons are highly conserved within (96%) in class Gammaproteobacteria, dominated by four pathogenic species of "ESKAPE." Secondly, more than half of class 1 integrons are embedded in chromosomes with less potential for horizontal gene transfer. Finally, ARGs that have been acquired by these integrons only cover 11% of all the ARG genotypes detected in bacterial genomes. CONCLUSIONS: The above observations indicated that there are both biological and ecological limitations to class 1 integrons in acquiring and spreading ARGs across different classes of the domain Bacteria.

Tracking antibiotic resistance gene pollution from different sources using machine-learning classification.

BACKGROUND: Antimicrobial resistance (AMR) has been a worldwide public health concern. Current widespread AMR pollution has posed a big challenge in accurately disentangling source-sink relationship, which has been further confounded by point and non-point sources, as well as endogenous and exogenous cross-reactivity under complicated environmental conditions. Because of insufficient capability in identifying source-sink relationship within a quantitative framework, traditional antibiotic resistance gene (ARG) signatures-based source-tracking methods would hardly be a practical solution. RESULTS: By combining broad-spectrum ARG profiling with machine-learning classification SourceTracker, here we present a novel way to address the question in the era of high-throughput sequencing. Its potential in extensive application was firstly validated by 656 global-scale samples covering diverse environmental types (e.g., human/animal gut, wastewater, soil, ocean) and broad geographical regions (e.g., China, USA, Europe, Peru). Its potential and limitations in source prediction as well as effect of parameter adjustment were then rigorously evaluated by artificial configurations with representative source proportions. When applying SourceTracker in region-specific analysis, excellent performance was achieved by ARG profiles in two sample types with obvious different source compositions, i.e., influent and effluent of wastewater treatment plant. Two environmental metagenomic datasets of anthropogenic interference gradient further supported its potential in practical application. To complement general-profile-based source tracking in distinguishing continuous gradient pollution, a few generalist and specialist indicator ARGs across ecotypes were identified in this study. CONCLUSION: We demonstrated for the first time that the developed source-tracking platform when coupling with proper experiment design and efficient metagenomic analysis tools will have significant implications for assessing AMR pollution. Following predicted source contribution status, risk ranking of different sources in ARG dissemination will be possible, thereby paving the way for establishing priority in mitigating ARG spread and designing effective control strategies.

MinION Nanopore Sequencing Enables Correlation between Resistome Phenotype and Genotype of Coliform Bacteria in Municipal Sewage.

Wastewater treatment plants (WWTPs) functioned as the intersection between the human society and nature environment, are receiving increasingly more attention on risk assessment of the acquisition of environmental antibiotic resistance genes (ARGs) by pathogenetic populations during treatment. However, because of the general lack of robust resistome profiling methods, genotype, and resistance phenotype is still poorly correlated in human pathogens of sewage samples. Here we applied MinION sequencing to quantify the resistance genes of multiple antibiotic resistant (MAR) coliform bacteria, a common indicator for human enteric pathogens in sewage samples. Our pipeline could deliver the results within 30 h from sample collection and the resistome quantification was consistent to that based on the Illumina platform. Additionally, the long nanopore reads not only enabled a simultaneous identification of the carrier populations of ARGs detected, but also facilitated the genome reconstruction of a representative MAR strain, from which we identified an instance of chromosomal integration of environmental resistance gene obtained by plasmid exchange with a porcine pathogen. This study demonstrated the utilization of MinION sequencing in quick monitoring and simultaneous phylogenetic tracking of environmental ARGs to address potential health risk associated with them.

Antibiotic Resistance Genes and Correlations with Microbial Community and Metal Resistance Genes in Full-Scale Biogas Reactors As Revealed by Metagenomic Analysis.

Digested residues from biogas plants are often used as biofertilizers for agricultural crops cultivation. The antibiotic resistance genes (ARGs) in digested residues pose a high risk to public health due to their potential spread to the disease-causing microorganisms and thus reduce the susceptibility of disease-causing microorganisms to antibiotics in medical treatment. A high-throughput sequencing (HTS)-based metagenomic approach was used in the present study to investigate the variations of ARGs in full-scale biogas reactors and the correlations of ARGs with microbial communities and metal resistance genes (MRGs). The total abundance of ARGs in all the samples varied from 7 × 10-3 to 1.08 × 10-1 copy of ARG/copy of 16S-rRNA gene, and the samples obtained from thermophilic biogas reactors had a lower total abundance of ARGs, indicating the superiority of thermophilic anaerobic digestion for ARGs removal. ARGs in all the samples were composed of 175 ARG subtypes; however, only 7 ARG subtypes were shared by all the samples. Principal component analysis and canonical correspondence analysis clustered the samples into three groups (samples from manure-based mesophilic reactors, manure-based thermophilic reactors, and sludge-based mesophilic reactors), and substrate, temperature, and hydraulic retention time (HRT) as well as volatile fatty acids (VFAs) were identified as crucial environmental variables affecting the ARGs compositions. Procrustes analysis revealed microbial community composition was the determinant of ARGs composition in biogas reactors, and there was also a significant correlation between ARGs composition and MRGs composition. Network analysis further revealed the co-occurrence of ARGs with specific microorganisms and MRGs.

Co-occurrence of antibiotic and metal resistance genes revealed in complete genome collection.

The high frequency of antibiotic resistance is a global public health concern. More seriously, widespread metal pressure in the environment may facilitate the proliferation of antibiotic resistance via coselection of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs). Given the lack of comprehensive understanding of the ARG and MRG coselection, in this study both abundance relationship and genetic linkage between ARGs and MRGs were rigorously investigated by performing a genomic analysis of a large complete genome collection. Many more ARGs were enriched in human-associated bacteria compared with those subjected to less anthropogenic interference. The signatures of ARG and MRG co-occurrence were much more frequent and the distance linkages between ARGs and MRGs were much more intimate in human pathogens than those less human-associated bacteria. Moreover, the co-occurrence structures in the habitat divisions were significantly different, which could be attributed to their distinct gene transfer potentials. More exogenous ARGs and MRGs on the genomes of human pathogens indicated the importance of recent resistance acquisition in resistome development of human commensal flora. Overall, the study emphasizes the potential risk associated with ARG and MRG coselection of both environmental and medical relevance.

Metagenomic analysis revealed highly diverse microbial arsenic metabolism genes in paddy soils with low-arsenic contents.

Microbe-mediated arsenic (As) metabolism plays a critical role in global As cycle, and As metabolism involves different types of genes encoding proteins facilitating its biotransformation and transportation processes. Here, we used metagenomic analysis based on high-throughput sequencing and constructed As metabolism protein databases to analyze As metabolism genes in five paddy soils with low-As contents. The results showed that highly diverse As metabolism genes were present in these paddy soils, with varied abundances and distribution for different types and subtypes of these genes. Arsenate reduction genes (ars) dominated in all soil samples, and significant correlation existed between the abundance of arr (arsenate respiration), aio (arsenite oxidation), and arsM (arsenite methylation) genes, indicating the co-existence and close-relation of different As resistance systems of microbes in wetland environments similar to these paddy soils after long-term evolution. Among all soil parameters, pH was an important factor controlling the distribution of As metabolism gene in five paddy soils (p = 0.018). To the best of our knowledge, this is the first study using high-throughput sequencing and metagenomics approach in characterizing As metabolism genes in the five paddy soil, showing their great potential in As biotransformation, and therefore in mitigating arsenic risk to humans.

Metagenomic Assembly Reveals Hosts of Antibiotic Resistance Genes and the Shared Resistome in Pig, Chicken, and Human Feces.

The risk associated with antibiotic resistance disseminating from animal and human feces is an urgent public issue. In the present study, we sought to establish a pipeline for annotating antibiotic resistance genes (ARGs) based on metagenomic assembly to investigate ARGs and their co-occurrence with associated genetic elements. Genetic elements found on the assembled genomic fragments include mobile genetic elements (MGEs) and metal resistance genes (MRGs). We then explored the hosts of these resistance genes and the shared resistome of pig, chicken and human fecal samples. High levels of tetracycline, multidrug, erythromycin, and aminoglycoside resistance genes were discovered in these fecal samples. In particular, significantly high level of ARGs (7762 ×/Gb) was detected in adult chicken feces, indicating higher ARG contamination level than other fecal samples. Many ARGs arrangements (e.g., macA-macB and tetA-tetR) were discovered shared by chicken, pig and human feces. In addition, MGEs such as the aadA5-dfrA17-carrying class 1 integron were identified on an assembled scaffold of chicken feces, and are carried by human pathogens. Differential coverage binning analysis revealed significant ARG enrichment in adult chicken feces. A draft genome, annotated as multidrug resistant Escherichia coli, was retrieved from chicken feces metagenomes and was determined to carry diverse ARGs (multidrug, acriflavine, and macrolide). The present study demonstrates the determination of ARG hosts and the shared resistome from metagenomic data sets and successfully establishes the relationship between ARGs, hosts, and environments. This ARG annotation pipeline based on metagenomic assembly will help to bridge the knowledge gaps regarding ARG-associated genes and ARG hosts with metagenomic data sets. Moreover, this pipeline will facilitate the evaluation of environmental risks in the genetic context of ARGs.

Cloning and expression analysis of CtbZIP20 transcription factor gene from Carthamus tinctorius and construction of plant expression vector / 中草药

Objective: To clone bZIP20 (basic region/leucine zipper motif) gene from Carthamus tinctorius, analyze the expression level in different plant tissues, and construct the plant expression vector. Methods: The bZIP20 gene was cloned by RT-PCR techniques, and the protein characteristics were analyzed by bioinformatics, and phylogenetic tree was constructed. The expression of bZIP20 gene in different tissues and the roots after inoculated by Fusarium oxysporum were analyzed using real time-PCR, and the plant expression vector pBASTA-bZIP20 was constructed. Results: The ORF sequence of bZIP20 gene was 981 bp, encoded a protein of 326 amino acids (GenBank: KT692605). Sequence alignment and phylogenetic tree analyses showed that bZIP20 had 85.41% and 83.99% of consistency with bZIP of Sesamum indicum and Camellia assamica. Real-time PCR results showed significant differences, the highest expression level of bZIP20 gene was detected in flower, and was highest in the bud period, bZIP20 gene was significantly increased in root tissue inoculated with F. oxysporum. The plant expression vector pBASTA-bZIP20 was obtained. Conclusion: The bZIP20 gene of safflower is successfully cloned, and the expression is analyzed. The plant expression vector pBASTA-bZIP20 is constructed.

Study on expression of metallothionein 2 gene in safflower / 中草药

Objective: To obtain the transgenic safflower plants which expressed Arabidopsis thaliana metallothionein 2 (MT2) gene, and lay a foundation for development of MT products. Methods: The oleosin-MT gene was obtained from pEASY-oleosin-MT by Nco I/Hind III, then was inserted into plant expression vector pOP. The recombinant plasmid named pOP-oleosin-MT was transferred into Agrobacterium tumefaciens EHA105.The oleosin-MT gene was introduced into safflowers via Agrobacterium-mediated method and positive transgenic plants were determined by PCR analysis. Results: The recombinant plasmid pOP-oleosin-MT was successfully constructed. PCR and Southern blotting analysis confirmed that MT gene was integrated into the genome of safflower plant and three transgenic plants were obtained. Conclusion: The safflower regeneration system is constructed successfully and MT gene is successfully transformed into safflower plant.

Inspiratory muscle training followed by non-invasive positive pressure ventilation in patients with severe chronic obstructive pulmonary disease: a randomized controlled trial / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of inspiratory muscle training followed by non-invasive positive pressure ventilation in patients with severe chronic obstructive pulmonary disease (COPD).</p><p><b>METHODS</b>This investigator-initiated randomized, controlled trial recruited 88 patients with stable GOLD stage IV COPD, who were randomized into 4 equal groups to continue oxygen therapy (control group) or to receive inspiratory muscle training followed by non-invasive positive pressure ventilation (IMT-NPPV group), inspiratory muscle training only (IMT group), or noninvasive positive pressure ventilation only (NPPV group) for at least 8 weeks. The outcomes of the patients were assessed including the quality of life (SRI scores), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), dyspnea (MRC scores), 6-min walking distance (6MWD) and lung function.</p><p><b>RESULTS</b>s Compared to baseline values, SRI scores, 6MWT and MRC scores increased significantly after 8 weeks in IMT-NPPV, IMT and NPPV groups, and the improvements were significantly greater in IMT-NPPV group than in IMT and NPPV groups (P<0.05 for all). In IMT-NPPV and IMT groups, MIP and MEP increased significantly after the training (P<0.05), and the improvement was more prominent in IMT-NPPV group (P<0.05). No significant changes were found in pulmonary functions in the groups after 8 weeks of treatment (P>0.05).</p><p><b>CONCLUSION</b>Inspiratory muscle training followed by non-invasive positive pressure ventilation, compared with inspiratory muscle training or non-invasive positive pressure ventilation alone, can better enhance the quality of life, strengthen the respiratory muscles, improve exercise tolerance and relieve the dyspnea in patients with COPD.</p>

Cloning and expression analysis of tocopherol cyclase gene in Carthamus tinctorius / 中国中药杂志

The tocopherol cyclase was one of the key enzymes in plant vitamin E biosynthesis pathway. According to the study of Carthamus tinctorius transcriptome data,the Tocopherol cyclase gene was obtained using RT-PCR techniques and named CtTC . Bioinformatics analysis showed theopen reading frame （ORF）of CtTC was 1 524 bp. The putative protein contained 507 amino acids with a predicted molecular mass of 62.9 kDa and theoretically isoelectric point was 5.01.Signal peptide analysis showed that it was a non secretory protein, and there was no signal peptide. The subcellular localization showed that the CtTC protein was located in the chloroplast. The expression of CtTC gene in safflower seeds at different development stages was determined by quantitative real-time PCR, it was found that the highest expression level of CtTC gene was detected in 50 DAF.Quantitative RT-PCR analysis suggested that expression of CtTC is induced and strengthened by drought stresses. This research provided a candidate gene for metabolic engineering of vitamin E and resisting stress.

Failure to Identify Somatic Mutations in Monozygotic Twins Discordant for Schizophrenia by Whole Exome Sequencing / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Schizophrenia (SCZ) is a severe, debilitating, and complex psychiatric disorder with multiple causative factors. An increasing number of studies have determined that rare variations play an important role in its etiology. A somatic mutation is a rare form of genetic variation that occurs at an early stage of embryonic development and is thought to contribute substantially to the development of SCZ. The aim of the study was to explore the novel pathogenic somatic single nucleotide variations (SNVs) and somatic insertions and deletions (indels) of SCZ.</p><p><b>METHODS</b>One Chinese family with a monozygotic (MZ) twin pair discordant for SCZ was included. Whole exome sequencing was performed in the co-twin and their parents. Rigorous filtering processes were conducted to prioritize pathogenic somatic variations, and all identified SNVs and indels were further confirmed by Sanger sequencing.</p><p><b>RESULTS</b>One somatic SNV and two somatic indels were identified after rigorous selection processes. However, none was validated by Sanger sequencing.</p><p><b>CONCLUSIONS</b>This study is not alone in the failure to identify pathogenic somatic variations in MZ twins, suggesting that exonic somatic variations are extremely rare. Further efforts are warranted to explore the potential genetic mechanism of SCZ.</p>

Exploring antibiotic resistance genes and metal resistance genes in plasmid metagenomes from wastewater treatment plants.

Plasmids operate as independent genetic elements in microorganism communities. Through horizontal gene transfer (HGT), they can provide their host microorganisms with important functions such as antibiotic resistance and heavy metal resistance. In this study, six metagenomic libraries were constructed with plasmid DNA extracted from influent, activated sludge (AS) and digested sludge (DS) of two wastewater treatment plants (WWTPs). Compared with the metagenomes of the total DNA extracted from the same sectors of the wastewater treatment plant, the plasmid metagenomes had significantly higher annotation rates, indicating that the functional genes on plasmids are commonly shared by those studied microorganisms. Meanwhile, the plasmid metagenomes also encoded many more genes related to defense mechanisms, including ARGs. Searching against an antibiotic resistance genes (ARGs) database and a metal resistance genes (MRGs) database revealed a broad-spectrum of antibiotic (323 out of a total 618 subtypes) and MRGs (23 out of a total 23 types) on these plasmid metagenomes. The influent plasmid metagenomes contained many more resistance genes (both ARGs and MRGs) than the AS and the DS metagenomes. Sixteen novel plasmids with a complete circular structure that carried these resistance genes were assembled from the plasmid metagenomes. The results of this study demonstrated that the plasmids in WWTPs could be important reservoirs for resistance genes, and may play a significant role in the horizontal transfer of these genes.

Cloning and prokaryotic expression of transcription factor CtMYB1 gene in Carthamus tinctorius / 中草药

Objective: To obtain a transcription factor gene MYB, clone a CtMYB1 gene from the safflower petals of Carthamus tinctorius, and performe its sequence analysis and prokaryotic expression vector construction. Methods: According to high expression Unigene123993 sequence in safflower transcriptome, MYB gene was cloned from safflower by RT-PCR and RACE methods. The full-length cDNA sequences CtMYB1 gene as templates, the open reading frame (ORF) of cDNA sequences was obtained by PCR. Prokaryotic expression vector pEASY-E1-CtMYB1 was constructed and the expression in E. coli BL21 (DE3) was transformed. Results: A MYB gene was successfully cloned from the safflower petals of C. tinctorius, named CtMYB1 (GenBank accession No. KJ524853). A full length cDNA of CtMYB1 was 893 bp and ORF was 750 bp, encoding a protein of 249 amino acid. The prokaryotic expression vector was obtained. SDS-PAGE results showed that the molecular weight was 30000, same with the relative molecular weight of predicted protein. Conclusion: CtMYB1 is cloned from safflower and the prokaryotic expression vector is constructed, which preliminarily proves that the gene is successfully expressed in E. coli.