CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16.

BACKGROUND: Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2. RESULTS: In this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB- 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml. CONCLUSION: The new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry.

IS6 family insertion sequences promote optrA dissemination between plasmids varying in transfer abilities.

Plasmids are the primary vectors for intercellular transfer of the oxazolidinone and phenicol cross-resistance gene optrA, while insertion sequences (ISs) are mobile genetic elements that can mobilize plasmid-borne optrA intracellularly. However, little is known about how the IS-mediated intracellular mobility facilitates the dissemination of the optrA gene between plasmid categories that vary in transfer abilities, including non-mobilizable, mobilizable, and conjugative plasmids. Here, we performed a holistic genomic study of 52 optrA-carrying plasmids obtained from searches guided by the Comprehensive Antibiotic Resistance Database. Among the 132 ISs identified within 10 kbp from the optrA gene in the plasmids, IS6 family genes were the most prevalent (86/132). Homologous gene arrays containing IS6 family genes were shared between different plasmids, especially between mobilizable and conjugative plasmids. All these indicated the central role of IS6 family genes in disseminating plasmid-borne optrA. Thirty-three of the 52 plasmids were harbored by Enterococcus faecalis found mainly in humans and animals. By Nanopore sequencing and inverse PCR, the potential of the enterococcal optrA to be transmitted from a mobilizable plasmid to a conjugative plasmid mediated by IS6 family genes was further confirmed in Enterococcus faecalis strains recovered from the effluents of anaerobic digestion systems for treating chicken manure. Our findings highlight the increased intercellular transfer abilities and dissemination risk of plasmid-borne optrA gene caused by IS-mediated intracellular mobility, and underscore the importance of routinely monitoring the dynamic genetic contexts of clinically important antibiotic resistance genes to effectively control this critical public health threat. KEY POINTS: • IS6 was prevalent in optrA-plasmids varying in intercellular transfer abilities. • Enterococcal optrA-plasmids were widespread among human, animal, and the environment. • IS6 elevated the dissemination risk of enterococcal optrA-plasmids.

Local Multiple-site Injections of a Plasmid Encoding Human MnSOD Mitigate Radiation-induced Skin Injury by Inhibiting Ferroptosis.

BACKGROUND: Most patients who undergo radiotherapy develop radiation skin injury, for which effective treatment is urgently needed. MnSOD defends against reactive oxygen species (ROS) damage and may be valuable for treating radiation-induced injury. Here, we (i) investigated the therapeutic and preventive effects of local multiple-site injections of a plasmid, encoding human MnSOD, on radiation-induced skin injury in rats and (ii) explored the mechanism underlying the protective effects of pMnSOD. METHODS: The recombinant plasmid (pMnSOD) was constructed with human cytomegalovirus (CMV) promoter and pUC-ori. The protective effects of pMnSOD against 20-Gy X-ray irradiation were evaluated in human keratinocytes (HaCaT cells) by determining cell viability, ROS levels, and ferroptosisrelated gene expression. In therapeutic treatment, rats received local multiple-site injections of pMnSOD on days 12, 19, and 21 after 40-Gy γ-ray irradiation. In preventive treatment, rats received pMnSOD injections on day -3 pre-irradiation and on day 4 post-irradiation. The skin injuries were evaluated based on the injury score and pathological examination, and ferroptosis-related gene expression was determined. RESULTS: In irradiated HaCaT cells, pMnSOD transfection resulted in an increased SOD2 expression, reduced intracellular ROS levels, and increased cell viability. Moreover, GPX4 and SLC7A11 expression was significantly upregulated, and erastin-induced ferroptosis was inhibited in HaCaT cells. In the therapeutic and prevention treatment experiments, pMnSOD administration produced local SOD protein expression and evidently promoted the healing of radiation-induced skin injury. In the therapeutic treatment experiments, the injury score in the high-dose pMnSOD group was significantly lower than in the PBS group on day 33 post-irradiation (1.50 vs. 2.80, P < 0.05). In the prevention treatment experiments, the skin injury scores were much lower in the pMnSOD administration groups than in the PBS group from day 21 to day 34. GPX4, SLC7A11, and Bcl-2 were upregulated in irradiated skin tissues after pMnSOD treatment, while ACSL4 was downregulated. CONCLUSION: The present study provides evidence that the protective effects of MnSOD in irradiated HaCaT cells may be related to the inhibition of ferroptosis. The multi-site injections of pMnSOD had clear therapeutic and preventive effects on radiation-induced skin injury in rats. pMnSOD may have therapeutic value for the treatment of radiation-induced skin injury.

Pseudomonas syringae coffee blight is associated with the horizontal transfer of plasmid-encoded type III effectors.

The emergence of new pathogens is an ongoing threat to human health and agriculture. While zoonotic spillovers received considerable attention, the emergence of crop diseases is less well studied. Here, we identify genomic factors associated with the emergence of Pseudomonas syringae bacterial blight of coffee. Fifty-three P. syringae strains from diseased Brazilian coffee plants were sequenced. Comparative and evolutionary analyses were used to identify loci associated with coffee blight. Growth and symptomology assays were performed to validate the findings. Coffee isolates clustered in three lineages, including primary phylogroups PG3 and PG4, and secondary phylogroup PG11. Genome-wide association study of the primary PG strains identified 37 loci, including five effectors, most of which were encoded on a plasmid unique to the PG3 and PG4 coffee strains. Evolutionary analyses support the emergence of coffee blight in PG4 when the coffee-associated plasmid and associated effectors derived from a divergent plasmid carried by strains associated with other hosts. This plasmid was only recently transferred into PG3. Natural diversity and CRISPR-Cas9 plasmid curing were used to show that strains with the coffee-associated plasmid grow to higher densities and cause more severe disease symptoms in coffee. This work identifies possible evolutionary mechanisms underlying the emergence of a new lineage of coffee pathogens.

Plasmids, a molecular cornerstone of antimicrobial resistance in the One Health era.

Antimicrobial resistance (AMR) poses a substantial threat to human health. The widespread prevalence of AMR is, in part, due to the horizontal transfer of antibiotic resistance genes (ARGs), typically mediated by plasmids. Many of the plasmid-mediated resistance genes in pathogens originate from environmental, animal or human habitats. Despite evidence that plasmids mobilize ARGs between these habitats, we have a limited understanding of the ecological and evolutionary trajectories that facilitate the emergence of multidrug resistance (MDR) plasmids in clinical pathogens. One Health, a holistic framework, enables exploration of these knowledge gaps. In this Review, we provide an overview of how plasmids drive local and global AMR spread and link different habitats. We explore some of the emerging studies integrating an eco-evolutionary perspective, opening up a discussion about the factors that affect the ecology and evolution of plasmids in complex microbial communities. Specifically, we discuss how the emergence and persistence of MDR plasmids can be affected by varying selective conditions, spatial structure, environmental heterogeneity, temporal variation and coexistence with other members of the microbiome. These factors, along with others yet to be investigated, collectively determine the emergence and transfer of plasmid-mediated AMR within and between habitats at the local and global scale.

Apoptin and apoptotic protease-activating factor 1 plasmid-assisted multi-functional nanoparticles in hepatocellular carcinoma therapy.

Cancer drugs usually have side effects in chemotherapy. Apoptin, a protein recognized by its good therapeutical effect on tumors and innocuous to body, is employed to treat hepatocellular carcinoma (HCC). As our previous data shown, the efficiency of apoptin protein might be limited by the protein of apaf-1. Therefore, we designed the multi-functional nanoparticles (MFNPs) encapsulating apoptin and apaf-1 plasmids by layer-by layer assembly. The NPs could release drugs into tumor site specifically and had good compatibility to normal cells and tissues. The groups of biotin, ε-polylysine, and nuclear localization signal in MFNPs conferred NPs the capabilities to enter cancer cells specifically, escape lysosome and enter the nucleus, respectively. In vitro inhibition experiment and in vivo anti-tumor therapy confirmed MFNPs as an excellent carrier to treat HCC. In addition, the dual-drug system was superior to any of the single-drug system. The mechanism analysis proved that supplement of the protein of apaf-1 might enhance apoptosome formation, causing the increase of therapeutical efficacy.

Expulsion of plasmid-mediated antibiotic resistance genes in E. coli by ethidium bromide and acridine orange treatment.

Plasmid borne antibiotics resistance is the global threat to healthcare facilities. Such antibiotics resistance is inherited stably within the same bacterial generations and transmitted horizontally to other species of bacteria. The elimination of such resistance plasmid is of great importance to contain dispersal of antibiotics resistance. E. coli strains were identified, screened for the presence of antibiotics resistance by disc diffusion method, and cured by sub-lethal concentrations of Ethidium bromide and Acridine orange. After curing, again antibiotic resistance was determined. Before and after curing, plasmids were extracted by column spin Kit and subjected to 1% agarose gel electrophoresis and antibiotic resistance genes were identified by PCR. The Ethidium bromide was more effective than Acridine orange in eliminating antibiotics resistance and resistance genes bearing plasmids (4, 5, 6, 8, 9, 10 and <10kb). The most frequently eliminated antibiotic resistance was against Imipenem and Meropenem followed by Cefoperazone-sulbactam, Amikacin and cephalosporins in sequence. The loss of antibiotic resistance was associated with the elimination of plasmid-borne antibiotic resistance genes; bla-TEM, bla-SHV, bla-CTX-M, qnrA, qnrB, qnrC and qnrD. Some E. coli strains did not show the removal of antibiotics resistance and plasmids, suggesting the presence of resistance genes on main chromosome and or non-curable plasmids.

Impact of veterinary antibiotics on plasmid-encoded antibiotic resistance transfer.

OBJECTIVES: Resistance genes can be genetically transmitted and exchanged between commensal and pathogenic bacterial species, and in different compartments including the environment, or human and animal guts (One Health concept). The aim of our study was to evaluate whether subdosages of antibiotics administered in veterinary medicine could enhance plasmid transfer and, consequently, resistance gene exchange in gut microbiota. METHODS: Conjugation frequencies were determined with Escherichia coli strains carrying IncL- (blaOXA-48) or IncI1-type (blaCTX-M-1) plasmids subjected to a series of subinhibitory concentrations of antibiotics used in veterinary medicine, namely amoxicillin, ceftiofur, apramycin, neomycin, enrofloxacin, colistin, erythromycin, florfenicol, lincomycin, oxytetracycline, sulfamethazine, tiamulin and the ionophore narasin. Treatments with subinhibitory dosages were performed with and without supplementation with the antioxidant edaravone, known as a mitigator of the inducibility effect of several antibiotics on plasmid conjugation frequency (PCF). Expression of SOS-response associated genes and fluorescence-based reactive oxygen species (ROS) detection assays were performed to evaluate the stress oxidative response. RESULTS: Increased PCFs were observed for both strains when treating with florfenicol and oxytetracycline. Increased expression of the SOS-associated recA gene also occurred concomitantly, as well as increased ROS production. Addition of edaravone to the treatments reduced their PCF and also showed a decreasing effect on SOS and ROS responses for both plasmid scaffolds. CONCLUSIONS: We showed here that some antibiotics used in veterinary medicine may induce transfer of plasmid-encoded resistance and therefore may contribute to the worldwide spread of antibiotic resistance genes.

Analysis of the Plasmid-Based ts-Mutant ΔfabA/pTS-fabA Reveals Its Lethality under Aerobic Growth Conditions That Is Suppressed by Mild Overexpression of desA at a Restrictive Temperature in Pseudomonas aeruginosa.

It is uncertain whether PA1610|fabA is essential or dispensable for growth on LB-agar plates under aerobic conditions in Pseudomonas aeruginosa PAO1. To examine its essentiality, we disrupted fabA in the presence of a native promoter-controlled complementary copy on ts-plasmid. In this analysis, we showed that the plasmid-based ts-mutant ΔfabA/pTS-fabA failed to grow at a restrictive temperature, consistent with the observation by Hoang and Schweizer (T. T. Hoang, H. P. Schweizer, J Bacteriol 179:5326-5332, 1997, https://doi.org/10.1128/jb.179.17.5326-5332.1997), and expanded on this by showing that ΔfabA exhibited curved cell morphology. On the other hand, strong induction of fabA-OE or PA3645|fabZ-OE impeded the growth of cells displaying oval morphology. Suppressor analysis revealed a mutant sup gene that suppressed a growth defect but not cell morphology of ΔfabA. Genome resequencing and transcriptomic profiling of sup identified PA0286|desA, whose promoter carried a single-nucleotide polymorphism (SNP), and transcription was significantly upregulated (level increase of >2-fold, P < 0.05). By integration of the SNP-bearing promoter-controlled desA gene into the chromosome of ΔfabA/pTS-fabA, we showed that the SNP is sufficient for ΔfabA to phenocopy the sup mutant. Furthermore, mild induction of the araC-PBAD-controlled desA gene but not desB rescued ΔfabA. These results validated that mild overexpression of desA fully suppressed the lethality but not the curved cell morphology of ΔfabA. Similarly, Zhu et al. (Zhu K, Choi K-H, Schweizer HP, Rock CO, Zhang Y-M, Mol Microbiol 60:260-273, 2006, https://doi.org/10.1111/j.1365-2958.2006.05088.x) showed that multicopy desA partially alleviated the slow growth phenotype of ΔfabA, the difference in which was that ΔfabA was viable. Taken together, our results demonstrate that fabA is essential for aerobic growth. We propose that the plasmid-based ts-allele is useful for exploring the genetic suppression interaction of essential genes of interest in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen whose multidrug resistance demands new drug development. Fatty acids are essential for viability, and essential genes are ideal drug targets. However, the growth defect of essential gene mutants can be suppressed. Suppressors tend to be accumulated during the construction of essential gene deletion mutants, hampering the genetic analysis. To circumvent this issue, we constructed a deletion allele of fabA in the presence of a native promoter-controlled complementary copy in the ts-plasmid. In this analysis, we showed that ΔfabA/pTS-fabA failed to grow at a restrictive temperature, supporting its essentiality. Suppressor analysis revealed desA, whose promoter carried a SNP and whose transcription was upregulated. We validated that both the SNP-bearing promoter-controlled and regulable PBAD promoter-controlled desA suppressed the lethality of ΔfabA. Together, our results demonstrate that fabA is essential for aerobic growth. We propose that plasmid-based ts-alleles are suitable for genetic analysis of essential genes of interest.

Per/polyfluoroalkyl substances modulate plasmid transfer of antibiotic resistance genes: A balance between oxidative stress and energy support.

Emerging contaminants can accelerate the transmission of antibiotic resistance genes (ARGs) from environmental bacteria to human pathogens via plasmid conjugation, posing a great challenge to the public health. Although the toxic effects of per/polyfluoroalkyl substances (PFAS) as persistent organic pollutants have been understood, it is still unclear whether and how PFAS modulate the transmission of ARGs. In this study, we for the first time reported that perfluorooctanoic acid (PFOA), perfluorododecanoic acid (PFDoA) and ammonium perfluoro (2-methyl-3-oxahexanoate) (GenX) at relatively low concentrations (0.01, 0.1 mg/L) promoted the conjugative transfer of plasmid RP4 within Escherichia coli, while the plasmid conjugation was inhibited by PFOA, PFDoA and GenX at relatively high concentrations (1, 10 mg/L). The non-unidirectional conjugation result was ascribed to the co-regulation of ROS overproduction, enhanced cell membrane permeability, shortage of energy support as well as l-arginine pool depletion. Taking the well-known PFOA as an example, it significantly enhanced the conjugation frequency by 1.4 and 3.4 times at relatively low concentrations (0.01, 0.1 mg/L), respectively. Exposure to PFOA resulted in enhanced cell membrane permeability and ROS overproduction in donor cells. At high concentrations of PFOA (1, 10 mg/L), although enhanced oxidative stress and cell membrane permeability still occurred, the ATP contents in E. coli decreased, which contributed to the inhibited conjugation. Transcriptome analysis further showed that the expression levels of genes related to arginine biosynthesis (argA, argC, argF, argG, argI) and transport (artJ, artM, artQ) pathways were significantly increased. Intracellular l-arginine concentration deficiency were observed at high concentrations of PFOA. With the supplementary exogenous arginine, it was demonstrated that arginine upregulated conjugation transfer- related genes (trfAp, trbBp) and restores the cell number of transconjugants in PFOA-treated group. Therefore, the inhibited conjugation at high concentrations PFOA were attributed to the shortage of ATP and the depletion of L-arginine pool. These findings provide important insights into the effect environmental concentrations of PFAS on the conjugative transfer of ARGs, and update the regulation mechanism of plasmid conjugation, which is critical for the management of antibiotic resistance in aquatic environments.

Incorporating plasmid biology and metagenomics into a holistic model of the human gut microbiome.

The human gut microbiome is often described as the collection of bacteria, archaea, fungi, protists, and viruses associated with an individual, with no acknowledgement of the plasmid constituents. However, like viruses, plasmids are autonomous intracellular replicating entities that can influence the genotype and phenotype of their host and mediate trans-kingdom interactions. Plasmids are frequently noted as vehicles for horizontal gene transfer and for spreading antibiotic resistance, yet their multifaceted contribution to mutualistic and antagonistic interactions within the human microbiome and impact on human health is overlooked. In this review, we highlight the importance of plasmids and their biological properties as overlooked components of microbiomes. Subsequent human microbiome studies should include dedicated analyses of plasmids, particularly as a holistic understanding of human-microbial interactions is required before effective and safe interventions can be implemented to improve human well-being.

Assessment of plasmids for relating the 2020 Salmonella enterica serovar Newport onion outbreak to farms implicated by the outbreak investigation.

BACKGROUND: The Salmonella enterica serovar Newport red onion outbreak of 2020 was the largest foodborne outbreak of Salmonella in over a decade. The epidemiological investigation suggested two farms as the likely source of contamination. However, single nucleotide polymorphism (SNP) analysis of the whole genome sequencing data showed that none of the Salmonella isolates collected from the farm regions were linked to the clinical isolates-preventing the use of phylogenetics in source identification. Here, we explored an alternative method for analyzing the whole genome sequencing data driven by the hypothesis that if the outbreak strain had come from the farm regions, then the clinical isolates would disproportionately contain plasmids found in isolates from the farm regions due to horizontal transfer. RESULTS: SNP analysis confirmed that the clinical isolates formed a single, nearly-clonal clade with evidence for ancestry in California going back a decade. The clinical clade had a large core genome (4,399 genes) and a large and sparsely distributed accessory genome (2,577 genes, at least 64% on plasmids). At least 20 plasmid types occurred in the clinical clade, more than were found in the literature for Salmonella Newport. A small number of plasmids, 14 from 13 clinical isolates and 17 from 8 farm isolates, were found to be highly similar (> 95% identical)-indicating they might be related by horizontal transfer. Phylogenetic analysis was unable to determine the geographic origin, isolation source, or time of transfer of the plasmids, likely due to their promiscuous and transient nature. However, our resampling analysis suggested that observing a similar number and combination of highly similar plasmids in random samples of environmental Salmonella enterica within the NCBI Pathogen Detection database was unlikely, supporting a connection between the outbreak strain and the farms implicated by the epidemiological investigation. CONCLUSION: Horizontally transferred plasmids provided evidence for a connection between clinical isolates and the farms implicated as the source of the outbreak. Our case study suggests that such analyses might add a new dimension to source tracking investigations, but highlights the need for detailed and accurate metadata, more extensive environmental sampling, and a better understanding of plasmid molecular evolution.

High prevalence of Escherichia coli co-harboring conjugative plasmids with colistin- and carbapenem resistance genes in a wastewater treatment plant in China.

Emergence and dissemination of resistance to last-resort antibiotics such as carbapenem and colistin is a growing, global health concern. Wastewater treatment plants (WWTPs) link human activities and the environment, can act as reservoirs and sources for emerging antibiotic resistance, and likely play a large role in antibiotic resistance transmission. The aim of this study was to investigate occurrence and characteristics of colistin- and carbapenem-resistant Escherichia coli (CCREC) in wastewater and sludge samples collected over a one-year period from different functional areas of an urban WWTP in Jinan city, Shandong, China. A total of 8 CCREC were isolated from 168 samples with selective agar and PCR, corresponding to high prevalence of 4.8%, co-harboring carbapenem resistance genes (blaNDM) and colistin resistance gene (mcr-1) and subsequently whole-genome sequenced. Additionally, all isolates were multidrug-resistant by antimicrobial susceptibility testing and carried a variety of antibiotic resistance genes. Two isolates carrying virulence genes associated with avian pathogenic E. coli were identified, one belonging to the high-risk clone O101:H9-ST167. Southern blotting was used to characterize CCREC isolates and plasmids carrying blaNDM-genes or mcr-1 could be transferred to a recipient strain E. coli J53 by in vitro conjugation assays. Resistance to other antibiotic classes were sporadically co-transferred to the transconjugant. Transposition of and mcr-1-carrying element from a transferable IncHI2-plasmid was observed among two CCREC clones isolated within 4 days of each other. The occurrence of multidrug-resistant CCREC capable of transferring their antibiotic resistance genotypes via conjugative plasmids is alarming. WWTPs bring bacteria from different sources together, providing opportunities for horizontal exchange of DNA among compatible hosts. Further dissemination of the colistin-, carbapenem-, or both colistin- and carbapenem resistant E. coli could lead to a serious threat to public health.

Construction and heterologous expression of the di-AFN A1 biosynthetic gene cluster in Streptomyces model strains.

Natural cyclohexapeptide AFN A1 fromStreptomyces alboflavus 313 has moderate antibacterial and antitumor activities. An artificial designed AFN A1 homodimer, di-AFN A1, is an antibiotic exhibiting 10 to 150 fold higher biological activities, compared with the monomer. Unfortunately, the yield of di-AFN A1 is very low (0.09 ± 0.03 mg·L-1) in the engineered strain Streptomyces alboflavus 313_hmtS (S. albo/313_hmtS), which is not friendly to be genetically engineered for titer improvement of di-AFN A1 production. In this study, we constructed a biosynthetic gene cluster for di-AFN A1 and increased its production through heterologous expression. During the collection of di-AFN A1 biosynthetic genes, the afn genes were located at three sites of S. alboflavus 313 genome. The di-AFN A1 biosynthetic gene cluster (BGC) was first assembled on one plasmid and introduced into the model strain Streptomyces lividans TK24, which produced di-AFN A1 at a titer of 0.43 ± 0.01 mg·L-1. To further increase the yield of di-AFN A1, the di-AFN A1 BGC was multiplied and split to mimic the natural afn biosynthetic genes, and the production of di-AFN A1 increased to 0.62 ± 0.11 mg·L-1 in S. lividans TK24 by the later strategy. Finally, different Streptomyces hosts were tested and the titer of di-AFN A1 increased to 0.81 ± 0.17 mg·L-1, about 8.0-fold higher than that in S. albo/313_hmtS. Successful heterologous expression of di-AFN A1 with a remarkable increased titer will greatly facilitate the following synthetic biological study and drug development of this dimeric cyclohexapeptide.

Extract of Acanthopanax senticosus and Its Components Interacting with Sulfide, Cysteine and Glutathione Increase Their Antioxidant Potencies and Inhibit Polysulfide-Induced Cleavage of Plasmid DNA.

Aqueous root extract from Acanthopanax senticosus (ASRE) has a wide range of medicinal effects. The present work was aimed at studying the influence of sulfide, cysteine and glutathione on the antioxidant properties of ASRE and some of its selected phytochemical components. Reduction of the 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazol-1-yloxy-3-oxide (●cPTIO) stable radical and plasmid DNA (pDNA) cleavage in vitro assays were used to evaluate antioxidant and DNA-damaging properties of ASRE and its individual components. We found that the interaction of ASRE and its two components, caffeic acid and chlorogenic acid (but not protocatechuic acid and eleutheroside B or E), with H2S/HS-, cysteine or glutathione significantly increased the reduction of the ●cPTIO radical. In contrast, the potency of ASRE and its selected components was not affected by Na2S4, oxidized glutathione, cystine or methionine, indicating that the thiol group is a prerequisite for the promotion of the antioxidant effects. ASRE interacting with H2S/HS- or cysteine displayed a bell-shaped effect in the pDNA cleavage assay. However, ASRE and its components inhibited pDNA cleavage induced by polysulfides. In conclusion, we suggest that cysteine, glutathione and H2S/HS- increase antioxidant properties of ASRE and that changes of their concentrations and the thiol/disulfide ratio can influence the resulting biological effects of ASRE.

A Plasmid-Based Fluorescence Reporter System for Monitoring Oxidative Damage in E. coli.

Quantitating intracellular oxidative damage caused by reactive oxygen species (ROS) is of interest in many fields of biological research. The current systems primarily rely on supplemented oxygen-sensitive substrates that penetrate the target cells, and react with ROS to produce signals that can be monitored with spectroscopic or imaging techniques. The objective here was to design a new non-invasive analytical strategy for measuring ROS-induced damage inside living cells by taking advantage of the native redox sensor system of E. coli. The developed plasmid-based sensor relies on an oxygen-sensitive transcriptional repressor IscR that controls the expression of a fluorescent marker in vivo. The system was shown to quantitatively respond to oxidative stress induced by supplemented H2O2 and lowered cultivation temperatures. Comparative analysis with fluorescence microscopy further demonstrated that the specificity of the reporter system was equivalent to the commercial chemical probe (CellROX). The strategy introduced here is not dependent on chemical probes, but instead uses a fluorescent expression system to detect enzyme-level oxidative damage in microbial cells. This provides a cheap and simple means for analysing enzyme-level oxidative damage in a biological context in E. coli.

Genomic characterization of antimicrobial resistance in mcr-carrying ESBL-producing Escherichia coli from pigs and humans.

Whole-genome sequencing (WGS) was conducted to characterize mcr-carrying extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (n=7). These E. coli isolates originated from two pigs (TH2 and TH3) and two humans (TH8 and TH9) from Thailand, and three pigs from Lao PDR (LA1, LA2 and LA3). Four E. coli sequence types/serotypes - ST6833/H20 (TH2 and TH3), ST48/O160:H40 (TH8 and TH9), ST5708/H45 (LA1) and ST10562/O148:H30 (LA2 and LA3) - were identified. The plasmid replicon type IncF was identified in all isolates. The point mutations Ser31Thr in PmrA and His2Arg in PmrB were found concurrently in all isolates (colistin MIC=4-8 µg ml-1). LA1 contained up to five point mutations in PmrB, and the colistin MIC was not significantly different from that for the other isolates. All mcr-1.1 was located in the ISApl1-mcr-1-pap2 element, while all mcr-3.1 was located in the TnAs2-mcr-3.1-dgkA-ISKpn40 element. The mcr-3.1 and blaCTX-M-55 genes were co-localized on the same plasmid, which concurrently contained cml, qnrS1 and tmrB. The blaCTX-M-55 and mcr-3.1 genes were located on conjugative plasmids and could be transferred horizontally under selective pressure from ampicillin or colistin. In conclusion, comprehensive insights into the genomic information of ESBL-producing E. coli harbouring mcr were obtained. As mcr-carrying ESBL-producing E. coli were detected in pigs and humans, a holistic and multisectoral One Health approach is required to contain antimicrobial resistance (AMR).

Role of Plasmid in Pesticide Degradation and Metal Tolerance in Two Plant Growth-Promoting Rhizobacteria Bacillus cereus (NCIM 5557) and Bacillus safensis (NCIM 5558).

Disha A (Bacillus cereus) and Disha B (Bacillus safensis) were isolated from pesticide-infested agricultural field and showed tolerance against pesticides, heavy metals, and antibiotics. The isolates exhibited PGPR activities in vitro as well as in field conditions in lentil (Lens culinaris) and cow pea (Vigna unguiculata). Both the Bacillus species could not be grown in mineral salt medium but efficiently grown in the media supplemented with pesticide (imidacloprid/carbendazim) demonstrating the utilization of pesticide as carbon/nitrogen source. The HPLC studies exhibited the pesticide (imidacloprid/carbendazim) degradation by both the bacteria. B. safensis showed better degradation of carbendazim (88.93%) and imidacloprid (82.48%) than that of B. cereus 78.07% and 49.12%, respectively. The bacterial isolates showed high concentration of heavy metal tolerance viz. lead, molybdenum, cadmium, copper, cobalt, and zinc, except mercury. Both the bacteria possessed single plasmid. The plasmid-cured isolates of B. cereus did not tolerate any pesticide, whereas that of B. safensis tolerated all the pesticides, like wild strains. The plasmid curing experiments did not affect the heavy metal tolerance ability of both the bacteria indicating the genomic nature of heavy metal tolerance genes, whereas pesticide resistance genes are plasmid-dependent in B. cereus but genomic in B. safensis.

Molecular cloning and structural analysis of key enzymes in Tetrastigma hemsleyanum for resveratrol biosynthesis.

Resveratrol (RES), a plant antitoxin, has antioxidant, anti-inflammatory, anti-cancer and cardiovascular protection effects. It has been reported that RES can be stably detected in a Chinese herbal medicinal plant Tetrastigma hemsleyanum. At present, the research of T. hemsleyanum mainly focused on the discovery of new compounds and pharmacology. However, there were few studies on the molecular mechanism of the synthesis of secondary metabolites in T. hemsleyanum. In this experiment, four key enzymes (ThPAL/ThC4H/Th4CL/ThRS) involved in the RES biosynthesis pathway were cloned and obtained. They contained an open reading frame (ORF) of 2139 bp, 1518 bp, 1716 bp and 1035 bp, encoding 712, 505, 571 and 344 amino acids, separately. Various bioinformatics tools were used to analyze these deduced protein domains, secondary structures, three-dimensional (3D) structures and phylogenetic trees. Subsequently, quantitative primers were designed to conduct the tissue-specific expression. Quantitative results displayed that the four genes were expressed in all tested tissues, and their expression in root tubers was more stable. Moreover, the subcellular localization of the four genes was studied by constructed recombinant green fluorescent expression vectors. Herein, by digging out the key enzyme genes in the biosynthesis of RES in T. hemsleyanum, this experiment tried to reveal the expression patterns of these key enzyme genes. It also provided the basis for the research on the molecular level, which will help people further illuminate and clarify the biosynthesis and regulation mechanism of secondary metabolites in T. hemsleyanum.

GHRH expression plasmid improves osteoporosis and skin damage in aged mice.

The hormone secretion of GHRH-GH-IGF-1 axis in animals was decreased as aging. These hormones play an important role in maintaining bone mass and bone structure, and also affect the normal structure and function of the skin. We used plasmid-based technology to deliver growth hormone releasing hormone (GHRH) to elderly mice. In the current study, 80 and 120 µg/kg pVAX-GHRH plasmid expression plasmid were injected into old mice, the serum GHRH and insulin-like growth factor-1(IGF-1) content were increased within three weeks (P < 0.05). In the groups of 80 and 120 µg/kg plasmid, the content of procollagen type I N-terminal pro-peptide (PINP) in the serum was increased(P < 0.05), and the content of C-terminal telopeptides of type I collagen (CTX-1) in the serum was reduced significantly (P < 0.05). Furthermore, the expression of osteoprotegerin (OPG) and osteocalcin (OCN) in the femur also was increased(P < 0.05). The bone mineral density(BMD)、trabecular bone volume (BV/TV) and trabecular number(Tb.N) of mouse femur were increased significantly (P < 0.05) and trabecular separation(Tb.Sp) was decreased(P < 0.05). There were more trabecular bones in the bone marrow cavity and the trabecular bones are thicker in the groups of 80 and 120 µg/kg plasmid relative to control. The superoxide dismutase (SOD) content in the skin was increased(P < 0.05), and the malondialdehyde (MDA) content was reduced significantly (P < 0.05). Meanwhile, the skin moisture content also increased significantly(P < 0.05). Moreover, the expression of matrix metalloproteinase 3(MMP3) and matrix metalloproteinase 9(MMP9) was decreased in the skin(P < 0.05). The thickness of the dermis and epidermis of the skin had increased significantly(P < 0.05). Skin structure is more dense and complete in the two groups. These results indicate that 80 and 120 µg/kg plasmid-mediated GHRH supplementation can improve osteoporosis and skin aging in aged mice.