Displaying publications 21 - 40 of 102 in total

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  1. Short homologies efficiently generate detectable homologous recombination events
    Osahor AN, Tan CY, Sim EU, Lee CW, Narayanan K
    Anal Biochem, 2014 Oct 1;462:26-8.
    PMID: 24929088 DOI: 10.1016/j.ab.2014.05.030
    When recombineering bacterial artificial chromosomes (BACs), it is common practice to design the ends of the donor molecule with 50 bp of homology specifying its insertion site. We demonstrate that desired recombinants can be produced using intermolecular homologies as short as 15 bp. Although the use of shorter donor end regions decreases total recombinants by several fold, the frequency of recombinants with correctly inserted donor molecules was high enough for easy detection by simple polymerase chain reaction (PCR) screening. This observation may have important implications for the design of oligonucleotides for recombineering, including significant cost savings, especially for high-throughput projects that use large quantities of primers.
    Matched MeSH terms: Plasmids/genetics
  2. Construction of a novel inducible expression vector for Lactococcus lactis M4 and Lactobacillus plantarum Pa21
    Maidin MS, Song AA, Jalilsood T, Sieo CC, Yusoff K, Rahim RA
    Plasmid, 2014 Jul;74:32-8.
    PMID: 24879963 DOI: 10.1016/j.plasmid.2014.05.003
    A vector that drives the expression of the reporter gusA gene in both Lactobacillus plantarum and Lactococcus lactis was constructed in this study. This vector contained a newly characterized heat shock promoter (Phsp), amplified from an Enterococcus faecium plasmid, pAR6. Functionality and characterization of this promoter was initially performed by cloning Phsp into pNZ8008, a commercial lactococcal plasmid used for screening of putative promoters which utilizes gusA as a reporter. It was observed that Phsp was induced under heat, salinity and alkaline stresses or a combination of all three stresses. The newly characterized Phsp promoter was then used to construct a novel Lactobacillus vector, pAR1801 and its ability to express the gusA under stress-induced conditions was reproducible in both Lb. plantarum Pa21 and L. lactis M4 hosts.
    Matched MeSH terms: Plasmids/genetics*
  3. Combination of essential oils and antibiotics reduce antibiotic resistance in plasmid-conferred multidrug resistant bacteria
    Yap PS, Lim SH, Hu CP, Yiap BC
    Phytomedicine, 2013 Jun 15;20(8-9):710-3.
    PMID: 23537749 DOI: 10.1016/j.phymed.2013.02.013
    In this study we investigated the relationship between several selected commercially available essential oils and beta-lactam antibiotics on their antibacterial effect against multidrug resistant bacteria. The antibacterial activity of essential oils and antibiotics was assessed using broth microdilution. The combined effects between essential oils of cinnamon bark, lavender, marjoram, tea tree, peppermint and ampicillin, piperacillin, cefazolin, cefuroxime, carbenicillin, ceftazidime, meropenem, were evaluated by means of the checkerboard method against beta-lactamase-producing Escherichia coli. In the latter assays, fractional inhibitory concentration (FIC) values were calculated to characterize interaction between the combinations. Substantial susceptibility of the bacteria toward natural antibiotics and a considerable reduction in the minimum inhibitory concentrations (MIC) of the antibiotics were noted in some paired combinations of antibiotics and essential oils. Out of 35 antibiotic-essential oil pairs tested, four of them showed synergistic effect (FIC≤0.5) and 31 pairs showed no interaction (FIC>0.5-4.0). The preliminary results obtained highlighted the occurrence of a pronounced synergistic relationship between piperacillin/cinnamon bark oil, piperacillin/lavender oil, piperacillin/peppermint oil as well as meropenem/peppermint oil against two of the three bacteria under study with a FIC index in the range 0.26-0.5. The finding highlighted the potential of peppermint, cinnamon bark and lavender essential oils being as antibiotic resistance modifying agent. Reduced usage of antibiotics could be employed as a treatment strategy to decrease the adverse effects and possibly to reverse the beta-lactam antibiotic resistance.
    Matched MeSH terms: Plasmids/genetics
  4. Fulltext Mechanisms of resistance in nontyphoidal Salmonella enterica strains exhibiting a nonclassical quinolone resistance phenotype
    Gunell M, Webber MA, Kotilainen P, Lilly AJ, Caddick JM, Jalava J, et al.
    Antimicrob Agents Chemother, 2009 Sep;53(9):3832-6.
    PMID: 19596880 DOI: 10.1128/AAC.00121-09
    Nontyphoidal Salmonella enterica strains with a nonclassical quinolone resistance phenotype were isolated from patients returning from Thailand or Malaysia to Finland. A total of 10 isolates of seven serovars were studied in detail, all of which had reduced susceptibility (MIC > or = 0.125 microg/ml) to ciprofloxacin but were either susceptible or showed only low-level resistance (MIC < or = 32 microg/ml) to nalidixic acid. Phenotypic characterization included susceptibility testing by the agar dilution method and investigation of efflux activity. Genotypic characterization included the screening of mutations in the quinolone resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE by PCR and denaturing high-pressure liquid chromatography and the amplification of plasmid-mediated quinolone resistance (PMQR) genes qnrA, qnrB, qnrS, qnrD, aac(6')-Ib-cr, and qepA by PCR. PMQR was confirmed by plasmid analysis, Southern hybridization, and plasmid transfer. No mutations in the QRDRs of gyrA, gyrB, parC, or parE were detected with the exception of a Thr57-Ser substitution within ParC seen in all but the S. enterica serovar Typhimurium strains. The qnrA and qnrS genes were the only PMQR determinants detected. Plasmids carrying qnr alleles were transferable in vitro, and the resistance phenotype was reproducible in Escherichia coli DH5alpha transformants. These data demonstrate the emergence of a highly mobile qnr genotype that, in the absence of mutation within topoisomerase genes, confers the nontypical quinolone resistance phenotype in S. enterica isolates. The qnr resistance mechanism enables bacteria to survive elevated quinolone concentrations, and therefore, strains carrying qnr alleles may be able to expand during fluoroquinolone treatment. This is of concern since nonclassical quinolone resistance is plasmid mediated and therefore mobilizable.
    Matched MeSH terms: Plasmids/genetics
  5. An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production
    Low KO, Mahadi NM, Rahim RA, Rabu A, Abu Bakar FD, Murad AM, et al.
    J Ind Microbiol Biotechnol, 2011 Sep;38(9):1587-97.
    PMID: 21336875 DOI: 10.1007/s10295-011-0949-0
    Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 μM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage.
    Matched MeSH terms: Plasmids/genetics
  6. Constructions of expression vectors of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and transient expression of transgenes in immature oil palm embryos
    Ariffin N, Abdullah R, Rashdan Muad M, Lourdes J, Emran NA, Ismail MR, et al.
    Plasmid, 2011 Sep;66(3):136-43.
    PMID: 21827784 DOI: 10.1016/j.plasmid.2011.07.002
    Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a polyhydroxyalkanoate (PHA) bioplastic group with thermoplastic properties is thus high in quality and can be degradable. PHBV can be produced by bacteria, but the process is not economically competitive with polymers produced from petrochemicals. To overcome this problem, research on transgenic plants has been carried out as one of the solutions to produce PHBV in economically sound alternative manner. Four different genes encoded with the enzymes necessary to catalyze PHBV are bktB, phaB, phaC and tdcB. All the genes came with modified CaMV 35S promoters (except for the tdcB gene, which was promoted by the native CaMV 35S promoter), nos terminator sequences and plastid sequences in order to target the genes into the plastids. Subcloning resulted in the generation of two different orientations of the tdcB, pLMIN (left) and pRMIN (right), both 17.557 and 19.967 kb in sizes. Both plasmids were transformed in immature embryos (IE) of oil palm via Agrobacterium tumefaciens. Assays of GUS were performed on one-week-old calli and 90% of the calli turned completely blue. This preliminary test showed positive results of integration. Six-months-old calli were harvested and RNA of the calli were isolated. RT-PCR was used to confirm the transient expression of PHBV transgenes in the calli. The bands were 258, 260, 315 and 200 bp in size for bktB, phaB, phaC and tdcB transgenes respectively. The data obtained showed that the bktB, phaB, phaC and tdcB genes were successfully integrated and expressed in the oil palm genome.
    Matched MeSH terms: Plasmids/genetics*
  7. Fulltext The mazEF toxin-antitoxin system as an attractive target in clinical isolates of Enterococcus faecium and Enterococcus faecalis
    Soheili S, Ghafourian S, Sekawi Z, Neela VK, Sadeghifard N, Taherikalani M, et al.
    Drug Des Devel Ther, 2015;9:2553-61.
    PMID: 26005332 DOI: 10.2147/DDDT.S77263
    The toxin-antitoxin (TA) system is a regulatory system where two sets of genes encode the toxin and its corresponding antitoxin. In this study, the prevalence of TA systems in independently isolated clinical isolates of Enterococcus faecium and Enterococcus faecalis was determined, the dominant TA system was identified, different virulence genes in E. faecium and E. faecalis were surveyed, the level of expression of the virulence and TA genes in normal and stress conditions was determined, and finally their associations with the TA genes were defined. Remarkably, the analysis demonstrated higBA and mazEF in all clinical isolates, and their locations were on chromosomes and plasmids, respectively. On the other hand, a quantitative analysis of TA and virulence genes revealed that the expression level in both genes is different under normal and stress conditions. The results obtained by anti-mazF peptide nucleic acids demonstrated that the expression level of virulence genes had decreased. These findings demonstrate an association between TA systems and virulence factors. The mazEF on the plasmids and the higBA TA genes on the chromosomes of all E. faecium and E. faecalis strains were dominant. Additionally, there was a decrease in the expression of virulence genes in the presence of anti-mazF peptide nucleic acids. Therefore, it is suggested that mazEF TA systems are potent and sensitive targets in all E. faecium and E. faecalis strains.
    Matched MeSH terms: Plasmids/genetics
  8. Improved Fab presentation on phage surface with the use of molecular chaperone coplasmid system
    Loh Q, Leong SW, Tye GJ, Choong YS, Lim TS
    Anal Biochem, 2015 May 15;477:56-61.
    PMID: 25769419 DOI: 10.1016/j.ab.2015.02.026
    The low presentation efficiency of Fab (fragment antigen binding) fragments during phage display is largely due to the complexity of disulphide bond formation. This can result in the presentation of Fab fragments devoid of a light chain during phage display. Here we propose the use of a coplasmid system encoding several molecular chaperones (DsbA, DsbC, FkpA, and SurA) to improve Fab packaging. A comparison was done using the Fab fragment from IgG and IgD. We found that the use of the coplasmid during phage packaging was able to improve the presentation efficiency of the Fab fragment on phage surfaces. A modified version of panning using the coplasmid system was evaluated and was successful at enriching Fab binders. Therefore, the coplasmid system would be an attractive alternative for improved Fab presentation for phage display.
    Matched MeSH terms: Plasmids/genetics*
  9. The influence of antibiotic resistance gene carriage on biofilm formation by two Escherichia coli strains associated with urinary tract infections
    Teh AH, Wang Y, Dykes GA
    Can J Microbiol, 2014 Feb;60(2):105-11.
    PMID: 24498987 DOI: 10.1139/cjm-2013-0633
    Urinary tract infections (UTI) caused by uropathogenic Escherichia coli are one of the most common forms of human disease. In this study, the effect of the presence of newly acquired antibiotic resistance genes on biofilm formation of UTI-associated E. coli strains was examined. Two clinical UTI-associated E. coli strains (SMC18 and SMC20) carrying different combinations of virulence genes were transformed with pGEM-T, pGEM-T::KmΔAmp, or pGEM-T::Km to construct ampicillin-resistant (Km(S)Amp(R)), kanamycin-resistant (Km(R)Amp(S)), or ampicillin- and kanamycin-resistant (Km(R)Amp(R)) strains. Transformed and wild-type strains were characterized for biofilm formation, bacterial surface hydrophobicity, auto-aggregation, morphology, and attachment to abiotic surfaces. Transformation with a plasmid carrying an ampicillin resistance gene alone decreased (p < 0.05) biofilm formation by SMC18 (8 virulence marker genes) but increased (p < 0.05) biofilm formation by SMC20 (5 virulence marker genes). On the other hand, transformation with a plasmid carrying a kanamycin resistance gene alone or both ampicillin and kanamycin resistance genes resulted in a decrease (p < 0.05) in biofilm formation by SMC18 but did not affect (p > 0.05) the biofilm formation by SMC20. Our results suggest that transformation of UTI-associated E. coli with plasmids carrying different antibiotic resistance gene(s) had a significant impact on biofilm formation and that these effects were both strain dependent and varied between different antibiotics.
    Matched MeSH terms: Plasmids/genetics
  10. Fulltext Surface display of glycosylated Tyrosinase related protein-2 (TRP-2) tumour antigen on Lactococcus lactis
    Kalyanasundram J, Chia SL, Song AA, Raha AR, Young HA, Yusoff K
    BMC Biotechnol, 2015;15:113.
    PMID: 26715153 DOI: 10.1186/s12896-015-0231-z
    The exploitation of the surface display system of food and commensal lactic acid bacteria (LAB) for bacterial, viral, or protozoan antigen delivery has received strong interest recently. The Generally Regarded as Safe (GRAS) status of the Lactococcus lactis coupled with a non-recombinant strategy of in-trans surface display, provide a safe platform for therapeutic drug and vaccine development. However, production of therapeutic proteins fused with cell-wall anchoring motifs is predominantly limited to prokaryotic expression systems. This presents a major disadvantage in the surface display system particularly when glycosylation has been recently identified to significantly enhance epitope presentation. In this study, the glycosylated murine Tyrosinase related protein-2 (TRP-2) with the ability to anchor onto the L. lactis cell wall was produced in suspension adapted Chinese Hamster Ovary (CHO-S) cells by expressing TRP-2 fused with cell wall anchoring LysM motif (cA) at the C-terminus.
    Matched MeSH terms: Plasmids/genetics
  11. Fulltext Genetic profiling of multidrug-resistant Acinetobacter baumannii from a tertiary care center in Malaysia
    Shahari AS, Palanisamy NK, Mohd Nor F
    Microbiol Spectr, 2025 Feb 04;13(2):e0087224.
    PMID: 39704504 DOI: 10.1128/spectrum.00872-24
    Genetic characterization of multidrug-resistant (MDR) Acinetobacter baumannii remains scarce in Malaysia. This study aimed to characterize antibiotic resistance, genomic location, and genetic relatedness among the A. baumannii isolates obtained from a tertiary hospital in Malaysia. A total of 128 MDR A. baumannii isolates were collected from patients admitted to various wards (intensive care unit [ICU], neonatal intensive care unit, coronary care unit, high dependency ward [HDW], and general wards). The isolates were identified by Vitek 2 and PCR amplification of the 16S rRNA gene followed by sequencing. The isolates were tested against imipenem, ceftazidime, amikacin, gentamicin, ampicillin, and ciprofloxacin using disk diffusion, Epsilometer test, and broth microdilution. The antibiotic resistance genes, blaOXA-23, blaOXA-24, blaADC, blaVIM, and blaIMP, were detected in chromosomal and plasmid DNA using PCR. Insertion sequence ISAba1/blaOXA-23 gene was detected on chromosomal DNA only. Isolates with different antibiotic susceptibility patterns and PCR profiles were subjected to multi-locus sequence typing. MDR A. baumannii was predominantly found in HDW (39.84%), general wards (29.69%), and ICU (28.13%). All isolates conferred resistance to carbapenem and more than 90% resistance to the remaining antibiotics. The antibiotic resistance genes blaOXA-23, blaVIM, and blaADC were detected in both chromosomal and plasmid DNA. The ISAba1/blaOXA-23 gene was detected in 99.22% of the isolates. Four sequence types (STs) were distinguished: ST2 (76.67%), ST164 (10%), ST642 (10%), and ST643 (3.33%). ST164 and ST642 were unique and represent a significant finding in Malaysia's surveillance data. These STs are associated with acquired blaOXA-23, indicating an evolutionary adaptation of A. baumannii within the hospital setting.IMPORTANCEAcinetobacter baumannii is a ubiquitous Gram-negative coccobacillus bacterium that is primarily associated with nosocomial infections that can colonize biotic and abiotic surfaces to enhance cell-to-cell adhesion, ensuring the establishment of infections. To date, the spread of multidrug-resistant A. baumannii (MDRAB) has become rampant and a great concern in the hospital setting, as the available antibiotics are insufficient to treat infections. The antibiotic resistance island resides in a mobile element and rapidly evolved. The antibiotic susceptibility data with its resistance mechanisms would contribute to and facilitate the management and infection control caused by MDRAB.
    Matched MeSH terms: Plasmids/genetics
  12. Fulltext Charting the Voyages of Acinetobacter baumannii in Southeast Asia: Mapping the Regional Genomic Landscape
    Lean SS, Palittapongarnpim P, Clarke SC, Yeo CC
    Microbiologyopen, 2026 Dec;15(6).
    PMID: 42961112 DOI: 10.1002/mbo3.70446
    Acinetobacter baumannii is a critical-priority nosocomial pathogen, yet its genomic epidemiology in Southeast Asia remains fragmented. We analysed publicly available genomes to define the regional A. baumannii population structure, resistome, mobile genetic elements, and phage defence repertoire. Following quality filtering, 853 genomes from Southeast Asia which were retrieved from PubMLST and NCBI, were subjected to multilocus sequence typing, pangenome-based phylogenetic analysis, antimicrobial resistance gene screening, plasmid and resistance island characterisation, and defence-system profiling. The dataset spanned 1996-2024 and included genomes from seven Southeast Asian countries, with Thailand contributing the largest number. The Global Clone 2 (GC2) lineage dominated the regional A. baumannii population and formed major country-associated clades, although non-dominant GCs and non-GC isolates persisted throughout the sampling period. blaOXA-23, blaADC-73, and tetB were the most prevalent acquired resistance determinants, while the gyrA-S81L and parC-S84L mutations, which are associated with fluoroquinolone resistance, were widespread across the Southeast Asian isolates. AbaR-type resistance islands were confined to GC1, whereas AbGRI-type islands predominated in GC2, especially AbGRI1-5. R3-type plasmids were the most widely distributed plasmid family overall, although RP-type plasmids predominated in Indonesia. Phage defence systems were lineage-associated, with GC2 enriched for SspBCDE and Gao-type systems and other lineages showing distinct defence combinations. Southeast Asian A. baumannii is therefore characterised by dominant GC2 circulation, broad dissemination of key resistance determinants, diverse mobile genetic elements, and heterogeneous phage defence repertoires. These findings provide a regional genomic baseline for surveillance and highlight the need for broader, geographically representative sampling across Southeast Asia.
    Matched MeSH terms: Plasmids/genetics
  13. Cell Wall-Treated Lactococcus lactis Increases the Plasmid Transfer Efficiency of Internal Ribosome Entry Site-Incorporated Lactococcal Bicistronic Vector into DF1 Cells
    Mat Isa N, Abdul Mutalib NE, Alitheen NB, Song AA, Rahim RA
    J. Mol. Microbiol. Biotechnol., 2017;27(4):246-251.
    PMID: 29055951 DOI: 10.1159/000481257
    This study demonstrates that cell wall treatment of Lactococcus lactis harbouring the internal ribosome entry site-incorporated lactococcal bicistronic vector pNZ:VIG mediated the delivery of genes into an eukaryotic cell line, DF1 cells, through bactofection. Bactofection analysis showed that the pNZ:VIG plasmid in L. lactis can be transferred into DF1 cells and that both the VP2 and gfp genes cloned in the plasmid can be transcribed and translated. The protein band relative to the Mr of VP2 protein (49 kDa) was successfully detected via Western blot analysis, while green fluorescence was successfully detected using a fluorescence microscope. The intensity of the bands detected increased for samples treated with both 1.5% (w/v) glycine and 10 μg/mL of lysozyme when compared to L. lactis treated with glycine alone and without treatment. Cell wall treatment of L. lactis with a combination of both glycine and lysozyme was not only shown to mediate plasmid transfer to DF1 cells, but also to increase the plasmid transfer efficiency.
    Matched MeSH terms: Plasmids/genetics*
  14. Fulltext Amenable epigenetic traits of dental pulp stem cells underlie high capability of xeno-free episomal reprogramming
    Thekkeparambil Chandrabose S, Sriram S, Subramanian S, Cheng S, Ong WK, Rozen S, et al.
    Stem Cell Res Ther, 2018 03 20;9(1):68.
    PMID: 29559008 DOI: 10.1186/s13287-018-0796-2
    BACKGROUND: While a shift towards non-viral and animal component-free methods of generating induced pluripotent stem (iPS) cells is preferred for safer clinical applications, there is still a shortage of reliable cell sources and protocols for efficient reprogramming.

    METHODS: Here, we show a robust episomal and xeno-free reprogramming strategy for human iPS generation from dental pulp stem cells (DPSCs) which renders good efficiency (0.19%) over a short time frame (13-18 days).

    RESULTS: The robustness of DPSCs as starting cells for iPS induction is found due to their exceptional inherent stemness properties, developmental origin from neural crest cells, specification for tissue commitment, and differentiation capability. To investigate the epigenetic basis for the high reprogramming efficiency of DPSCs, we performed genome-wide DNA methylation analysis and found that the epigenetic signature of DPSCs associated with pluripotent, developmental, and ecto-mesenchymal genes is relatively close to that of iPS and embryonic stem (ES) cells. Among these genes, it is found that overexpression of PAX9 and knockdown of HERV-FRD improved the efficiencies of iPS generation.

    CONCLUSION: In conclusion, our study provides underlying epigenetic mechanisms that establish a robust platform for efficient generation of iPS cells from DPSCs, facilitating industrial and clinical use of iPS technology for therapeutic needs.

    Matched MeSH terms: Plasmids/genetics*
  15. Heterologous expression of the tetracycline resistance gene tetX to enhance degradability and safety in doxycycline degradation
    Wen X, Huang J, Cao J, Xu J, Mi J, Wang Y, et al.
    Ecotoxicol Environ Saf, 2020 Mar 15;191:110214.
    PMID: 31968275 DOI: 10.1016/j.ecoenv.2020.110214
    Microbial remediation has the potential to inexpensively yet effectively decontaminate and restore contaminated environments, but the virulence of pathogens and risk of resistance gene transmission by microorganisms during antibiotic removal often limit its implementation. Here, a cloned tetX gene with clear evolutionary history was expressed to explore doxycycline (DOX) degradation and resistance variation during the degradation process. Phylogenetic analysis of tetX genes showed high similarity with those of pathogenic bacteria, such as Riemerella sp. and Acinetobacter sp. Successful tetX expression was performed in Escherichia coli and confirmed by SDS-PAGE and Western blot. Our results showed that 95.0 ± 1.0% of the DOX (50 mg/L) was degraded by the recombinant strain (ETD-1 with tetX) within 48 h, which was significantly higher than that for the control (38.9 ± 8.7%) and the empty plasmid bacteria (8.8 ± 5.1%) (P  0.05). The efficient and safe DOX-degrading capacity of the recombinant strain ETD-1 makes it valuable and promising for antibiotic removal in the environment.
    Matched MeSH terms: Plasmids/genetics
  16. A Self-Replicating Linear DNA
    Liew PS, Tan TH, Wong YC, Sim EUH, Lee CW, Narayanan K
    ACS Synth Biol, 2020 04 17;9(4):804-813.
    PMID: 32196315 DOI: 10.1021/acssynbio.9b00478
    TelN and tos are a unique DNA linearization unit isolated from bacteriophage N15. While being transferable, the TelN cleaving-rejoining activities remained stable to function on tos in both bacterial and mammalian environments. However, TelN contribution in linear plasmid replication in mammalian cells remains unknown. Herein, we investigated the association of TelN in linear tos-containing DNA (tos-DNA) replication in mammalian cells. Additionally, the mammalian origin of replication (ori) that is well-known to initiate the replication event of plasmid vectors was also studied. In doing so, we identified that both TelN and mammalian initiation sites were essential for the replication of linear tos-DNA, determined by using methylation sensitive DpnI/MboI digestion and polymerase chain reaction (PCR) amplification approaches. Furthermore, we engineered the linear tos-DNA to be able to retain in mammalian cells using S/MAR technology. The resulting S/MAR containing tos-DNA was robust for at least 15 days, with (1) continuous tos-DNA replication, (2) correct splicing of gene transcripts, and (3) stable exogenous gene expression that was statistically comparable to the endogenous gene expression level. Understanding the activities of TelN and tos in mammalian cells can potentially provide insights for adapting this simple DNA linearization unit in developing novel genetic engineering tools, especially to the eukaryotic telomere/telomerase study.
    Matched MeSH terms: Plasmids/genetics
  17. Characterization of Burkholderia pseudomallei isolated in Thailand and Malaysia
    Radua S, Ling OW, Srimontree S, Lulitanond A, Hin WF, Yuherman, et al.
    Diagn Microbiol Infect Dis, 2000 Nov;38(3):141-5.
    PMID: 11109011
    A total of 35 Burkholderia pseudomallei isolates from Thailand (16 clinical and eight soil isolates) and Malaysia (seven animal, two isolate each from clinical and soil) were investigated by their antimicrobial resistance, plasmid profiles and were typed by randomly amplified polymorphic DNA analysis. All isolates were found to be resistant to six or more of the 12 antimicrobial agents tested. Only two small plasmids of 1.8 and 2.4 megadalton were detected in two clinical isolates from Thailand. RAPD analysis with primer GEN2-60-09 resulted in the identification of 35 RAPD-types among the 35 isolates. The constructed dendrogram differentiated the 35 isolates into two main clusters and a single isolate. The wide genetic biodiversity among the 35 isolates indicate that RAPD-PCR can be a useful method to differentiate unrelated B. pseudomallei in epidemiological investigation.
    Matched MeSH terms: Plasmids/genetics
  18. Genetic diversity of human isolates of Salmonella enterica serovar Enteritidis in Malaysia
    Bakeri SA, Yasin RM, Koh YT, Puthucheary SD, Thong KL
    J Appl Microbiol, 2003;95(4):773-80.
    PMID: 12969291
    The study was undertaken to determine clonal relationship and genetic diversity of the human strains of Salmonella enterica serovar Enteritidis isolated from 1995 to 2002 from different parts of Malaysia.
    Matched MeSH terms: Plasmids/genetics
  19. Characterization of Vibrio vulnificus isolated from cockles (Anadara granosa): antimicrobial resistance, plasmid profiles and random amplification of polymorphic DNA analysis
    Radu S, Elhadi N, Hassan Z, Rusul G, Lihan S, Fifadara N, et al.
    FEMS Microbiol Lett, 1998 Aug 01;165(1):139-43.
    PMID: 9711850
    Antibiotic susceptibility, plasmid profiles and random amplification of polymorphic DNA (RAPD) were used to study strains of Vibrio vulnificus isolated from cockles (Anadara granosa). Thirty-six isolates were analyzed. The prevalent biotypes were 1 (72.2% of the isolates) and 2 (27.8%). Among these, 21 strains of biotype 1 and two strains of biotype 2 contained plasmid DNA bands ranging in size from 1.4 to 9.7 MDa. Thirty-one (83.3%) were found to be resistant to one or more of the antimicrobial agents tested, however no specific correlation between antimicrobial resistance patterns and a single biotype was found. In addition, no particular plasmid profile was predictive of a particular pattern of antibiotic susceptibility. Two primers produced polymorphisms in all strains tested, producing bands ranging from 0.25 to 2.7 kb, indicating a high variability among both biotype 1 and biotype 2 of the V. vulnificus strains investigated. RAPD identity across biotypes was also observed among Vibrio vulnificus strains.
    Matched MeSH terms: Plasmids/genetics*
  20. Fulltext Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme
    Hancock SJ, Phan MD, Peters KM, Forde BM, Chong TM, Yin WF, et al.
    Antimicrob Agents Chemother, 2017 02;61(2).
    PMID: 27872077 DOI: 10.1128/AAC.01740-16
    Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae They are associated with the dissemination of multiple clinically relevant resistance genes, including blaCMY and blaNDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a blaNDM-1-positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053 Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of blaNDM-positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this blaNDM-containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies.
    Matched MeSH terms: Plasmids/genetics*
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