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Journal articles on the topic 'Plasmid production'
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Trivedi, Ram Narayan, Parvez Akhtar, Jonathan Meade, Patrick Bartlow, Mohammad M. Ataai, Saleem A. Khan, and Michael M. Domach. "High-Level Production of Plasmid DNA by Escherichia coli DH5α ΩsacBby IntroducingincMutations." Applied and Environmental Microbiology 80, no. 23 (September 12, 2014): 7154–60. http://dx.doi.org/10.1128/aem.02445-14.
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ABSTRACTFor small-copy-number pUC-type plasmids, theinc1andinc2mutations, which deregulate replication, were previously found to increase the plasmid copy number 6- to 7-fold. Because plasmids can exert a growth burden, it was not clear if further amplification of copy number would occur due toincmutations when the starting point for plasmid copy number was orders of magnitude higher. To investigate further the effects of theincmutations and the possible limits of plasmid synthesis, the parent plasmid pNTC8485 was used as a starting point. It lacks an antibiotic resistance gene and has a copy number of ∼1,200 per chromosome. During early stationary-phase growth in LB broth at 37°C,inc2mutants of pNTC8485 exhibited a copy number of ∼7,000 per chromosome. In minimal medium at late log growth, the copy number was found to be significantly increased, to approximately 15,000. In an attempt to further increase the plasmid titer (plasmid mass/culture volume), enzymatic hydrolysis of the selection agent, sucrose, at late log growth extended growth and tripled the total plasmid amount such that an approximately 80-fold gain in total plasmid was obtained compared to the value for typical pUC-type vectors. Finally, when grown in minimal medium, no detectable impact on the exponential growth rate or the fidelity of genomic or plasmid DNA replication was found in cells with deregulated plasmid replication. The use ofincmutations and the sucrose degradation method presents a simplified way for attaining high titers of plasmid DNA for various applications.
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Kojic, Milan, Ivana Strahinic, Djordje Fira, Branko Jovcic, and Ljubisa Topisirovic. "Plasmid content and bacteriocin production by five strains ofLactococcus lactisisolated from semi-hard homemade cheese." Canadian Journal of Microbiology 52, no. 11 (November 1, 2006): 1110–20. http://dx.doi.org/10.1139/w06-072.
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In this study, the plasmid content and bacteriocin production of natural isolates of lactococci were investigated. Five bacteriocin producing lactococcal strains (Lactococcus lactis subsp. lactis BGMN1-2, BGMN1-3, BGMN1-5, BGMN1-6, and BGMN2-7) were isolated as nonstarter microflora of semi-hard homemade cheese and characterized. All isolates contained a number of plasmids. It was shown that lcnB structural genes for bacteriocin lactococcin B were located on large plasmids in all isolates. In the strains BGMN1-3 and BGMN1-5 proteinase prtP genes collocated with lcnB. Furthermore, these strains produced two additional bacteriocins (LsbA and LsbB) with genes responsible for their production and immunity located on the small rolling circle-replicating plasmid pMN5. Using deletion experiments of pMN5, minimal replicon of the plasmid and involvement of a bacteriocin locus in plasmid maintenance were identified. In addition, plasmid curing experiments showed that genes for catabolism or transport of 10 carbohydrates in the strain BGMN1-5 were plasmid located.Key words: lactococci, natural isolates, bacteriocin, plasmid curing.
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Münch, Karin M., Johannes Müller, Sarah Wienecke, Simone Bergmann, Steffi Heyber, Rebekka Biedendieck, Richard Münch, and Dieter Jahn. "Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity." Applied and Environmental Microbiology 81, no. 17 (June 26, 2015): 5976–86. http://dx.doi.org/10.1128/aem.00807-15.
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ABSTRACTDuring the past 2 decades,Bacillus megateriumhas been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single-cell level using green fluorescent protein as a model product revealed cell culture heterogeneity characterized by a significant proportion of less productive bacteria. Due to the enormous size ofB. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time lapse microscopy and flow cytometry. Cell culture heterogeneity was not caused simply by plasmid loss: instead, an asymmetric distribution of plasmids during cell division was detected during the exponential-growth phase. Multicopy plasmids are generally randomly distributed between daughter cells. However,in vivo andin vitroexperiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was achieved mainly by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multicopy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in aBacillusspecies.
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Fong, Ryan, Zhihao Hu, C. Richard Hutchinson, Jianqiang Huang, Stanley Cohen, and Camilla Kao. "Characterization of a Large, Stable, High-Copy-Number Streptomyces Plasmid That Requires Stability and Transfer Functions for Heterologous Polyketide Overproduction." Applied and Environmental Microbiology 73, no. 4 (December 1, 2006): 1296–307. http://dx.doi.org/10.1128/aem.01888-06.
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ABSTRACT A major limitation to improving small-molecule pharmaceutical production in streptomycetes is the inability of high-copy-number plasmids to tolerate large biosynthetic gene cluster inserts. A recent finding has overcome this barrier. In 2003, Hu et al. discovered a stable, high-copy-number, 81-kb plasmid that significantly elevated production of the polyketide precursor to the antibiotic erythromycin in a heterologous Streptomyces host (J. Ind. Microbiol. Biotechnol. 30:516-522, 2003). Here, we have identified mechanisms by which this SCP2*-derived plasmid achieves increased levels of metabolite production and examined how the 45-bp deletion mutation in the plasmid replication origin increased plasmid copy number. A plasmid intramycelial transfer gene, spd, and a partition gene, parAB, enhance metabolite production by increasing the stable inheritance of large plasmids containing biosynthetic genes. Additionally, high product titers required both activator (actII-ORF4) and biosynthetic genes (eryA) at high copy numbers. DNA gel shift experiments revealed that the 45-bp deletion abolished replication protein (RepI) binding to a plasmid site which, in part, supports an iteron model for plasmid replication and copy number control. Using the new information, we constructed a large high-copy-number plasmid capable of overproducing the polyketide 6-deoxyerythronolide B. However, this plasmid was unstable over multiple culture generations, suggesting that other SCP2* genes may be required for long-term, stable plasmid inheritance.
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Fridjonsson, Olafur, and Ralf Mattes. "Production of Recombinant α-Galactosidases inThermus thermophilus." Applied and Environmental Microbiology 67, no. 9 (September 1, 2001): 4192–98. http://dx.doi.org/10.1128/aem.67.9.4192-4198.2001.
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ABSTRACT A Thermus thermophilus selector strain for production of thermostable and thermoactive α-galactosidase was constructed. For this purpose, the native α-galactosidase gene (agaT) ofT. thermophilus TH125 was inactivated to prevent background activity. In our first attempt, insertional mutagenesis ofagaT by using a cassette carrying a kanamycin resistance gene led to bacterial inability to utilize melibiose (α-galactoside) and galactose as sole carbohydrate sources due to a polar effect of the insertional inactivation. A Gal+ phenotype was assumed to be essential for growth on melibiose. In a Gal−background, accumulation of galactose or its metabolite derivatives produced from melibiose hydrolysis could interfere with the growth of the host strain harboring recombinant α-galactosidase. Moreover, the AgaT− strain had to be Kms for establishment of the plasmids containing α-galactosidase genes and the kanamycin resistance marker. Therefore, a suitable selector strain (AgaT− Gal+ Kms) was generated by applying integration mutagenesis in combination with phenotypic selection. To produce heterologous α-galactosidase in T. thermophilus, the isogenes agaA and agaBof Bacillus stearothermophilus KVE36 were cloned into anEscherichia coli-Thermus shuttle vector. The region containing the E. coli plasmid sequence (pUC-derived vector) was deleted before transformation of T. thermophilus with the recombinant plasmids. As a result, transformation efficiency and plasmid stability were improved. However, growth on minimal agar medium containing melibiose was achieved only following random selection of the clones carrying a plasmid-based mutation that had promoted a higher copy number and greater stability of the plasmid.
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Chakrabarty, P. K., Sheo Raj, M. K. Meshram, A. Mahadevan, and D. W. Gabriel. "Plasmid-borne determinants of pigmentation, exopolysaccharide production, and virulence in Xanthomonas campestris pv. malvacearum." Canadian Journal of Microbiology 41, no. 8 (August 1, 1995): 740–45. http://dx.doi.org/10.1139/m95-101.
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Three plasmids of 55.0, 31.2, and 7.4 kb, designated as pXCM18-I, pXCM18-II, and pXCM18-III, respectively, were identified and isolated from strain HR13B/2 of Xanthomonas campestris pv. malvacearum. The bacterium was cured of all three plasmids in high frequency (3.1%) with heat (42 °C). The cured strains were nonmucoid and light yellow and were greatly impaired in exopolysaccharide production and virulence. Transformation of a cured strain using a mixture of purified plasmid DNA from HR13B/2 resulted in colonies carrying all six possible combinations of the three plasmids. Of the transformants, two morphologically distinct colony types were observed. The first type of transformants were mucoid and deep yellow, not distinguished from wild-type colonies in morphology, and fully complemented for exopolysaccharide production. These transformants invariably carried pXCM18-II. The second type of transformants were not distinguished from the untransformed strain in morphology but were found to be transformed after screening for plasmid content. These transformants carried either pXCM18-I, pXCM18-III, or both, but never pXCM18-II. In pathogenicity tests, each of the three plasmids restored virulence significantly, and additively, with plasmid pXCM18-I playing a predominant role.Key words: Xanthomonas campestris pv. malvacearum, cotton, plasmid, pigmentation, exopolysaccharide, virulence.
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McMillan, Elizabeth A., Jamie L. Wasilenko, Kaitlin A. Tagg, Jessica C. Chen, Mustafa Simmons, Sushim K. Gupta, Glenn E. Tillman, Jason Folster, Charlene R. Jackson, and Jonathan G. Frye. "Carriage and Gene Content Variability of the pESI-Like Plasmid Associated with Salmonella Infantis Recently Established in United States Poultry Production." Genes 11, no. 12 (December 18, 2020): 1516. http://dx.doi.org/10.3390/genes11121516.
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Salmonella Infantis carrying extended spectrum β-lactamase blaCTX-M-65 on a pESI-like megaplasmid has recently emerged in United States poultry. In order to determine the carriage rate and gene content variability of this plasmid in U.S. Salmonella Infantis, whole genome sequences of Salmonella isolates from humans and animals in the U.S. and internationally containing the pESI-like plasmid were analyzed. The U.S. Department of Agriculture Food Safety and Inspection Service (FSIS) identified 654 product sampling isolates containing pESI-like plasmids through hazard analysis and critical control point (HACCP) verification testing in 2017 and 2018. The Centers for Disease Control and Prevention identified 55 isolates with pESI-like plasmids in 2016–2018 through the National Antimicrobial Resistance Monitoring System. Approximately 49% of pESI-like plasmids from FSIS verification isolates and 71% from CDC NARMS contained blaCTX-M-65. Pan-plasmid genome analysis was also performed. All plasmids contained traN and more than 95% contained 172 other conserved genes; 61% contained blaCTX-M-65. In a hierarchical clustering analysis, some plasmids from U.S. animal sources clustered together and some plasmids from South America clustered together, possibly indicating multiple plasmid lineages. However, most plasmids contained similar genes regardless of origin. Carriage of the pESI-like plasmid in U.S. appears to be limited to Salmonella Infantis and carriage rates increased from 2017 to 2018.
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Ishibashi, Y. "Genetic Studies into Musty Odor Production by Actinomycetes." Water Science and Technology 25, no. 2 (January 1, 1992): 171–76. http://dx.doi.org/10.2166/wst.1992.0049.
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Musty taste and odor is a serious problem for drinking water. Much data regarding its occurrence are being accumulated. However, we may be able to solve this problem by inquiring into the nature of secondary metabolism through the mevalonic acid pathway in actinomycetes and/or cyanobacteria that yield musty smelling compounds. Therefore, genetic analysis is applied to look for new solutions. By investigating Streptomyces sp. with the protocol to recover plasmid DNA, it was difficult to get the infinitesimally small covalently closed circular (ccc) plasmid. On the other hand, Streptomyces strains which have been cured, which implicates plasmids, lost phenotypic characters such as musty odor production with aerial mycelium and pigment formation. DNA in Streptomyces are unstable and often cause amplification and deletion. The existence of giant linear plasmids was detected in musty smelling strains using pulsed – field gel electrophoresis.
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Hausjell, Johanna, Regina Kutscha, Jeannine D. Gesson, Daniela Reinisch, and Oliver Spadiut. "The Effects of Lactose Induction on a Plasmid-Free E. coli T7 Expression System." Bioengineering 7, no. 1 (January 6, 2020): 8. http://dx.doi.org/10.3390/bioengineering7010008.
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Recombinant production of pharmaceutical proteins like antigen binding fragments (Fabs) in the commonly-used production host Escherichia coli presents several challenges. The predominantly-used plasmid-based expression systems exhibit the drawback of either excessive plasmid amplification or plasmid loss over prolonged cultivations. To improve production, efforts are made to establish plasmid-free expression, ensuring more stable process conditions. Another strategy to stabilize production processes is lactose induction, leading to increased soluble product formation and cell fitness, as shown in several studies performed with plasmid-based expression systems. Within this study we wanted to investigate lactose induction for a strain with a genome-integrated gene of interest for the first time. We found unusually high specific lactose uptake rates, which we could attribute to the low levels of lac-repressor protein that is usually encoded not only on the genome but additionally on pET plasmids. We further show that these unusually high lactose uptake rates are toxic to the cells, leading to increased cell leakiness and lysis. Finally, we demonstrate that in contrast to plasmid-based T7 expression systems, IPTG induction is beneficial for genome-integrated T7 expression systems concerning cell fitness and productivity.
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Carnes,, Aaron E. "High-Yield Plasmid DNA Production." Genetic Engineering & Biotechnology News 32, no. 8 (April 15, 2012): 42–43. http://dx.doi.org/10.1089/gen.32.8.18.
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Fernández, Antonio, Nikki Horn, Michael J. Gasson, Helen M. Dodd, and Juan M. Rodríguez. "High-level coproduction of the bacteriocins nisin A and lactococcin A by Lactococcus lactis." Journal of Dairy Research 71, no. 2 (May 2004): 216–21. http://dx.doi.org/10.1017/s0022029904000123.
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In this study, a two-plasmid system for enhanced and consistent biosynthesis of the model lactococcal bacteriocin lactococcin A in non-producing Lactococcus lactis hosts was developed. The system comprised a plasmid carrying the genes lcnA and lciA under the control of the nisin-inducible nisA promoter, and a second plasmid harbouring the lcnC and lcnD genes. The introduction of both plasmids into two strains containing the nisRK genes required for nisin-controlled expression, Lc. lactis FI5876 (a nisin A-producer strain) and FI7847, resulted in production of extracellular lactococcin A at a higher level than that for the parental strain, Lc. lactis WM4. In addition, transformation of the nisin-producing host with both plasmids led to a high-level production of both lactococcal bacteriocins, which may provide a means to exploit their complementary properties in cheese ripening.
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Kojic, Milan, Ivana Strahinic, and Ljubisa Topisirovic. "Proteinase PI and lactococcin A genes are located on the largest plasmid inLactococcus lactissubsp.lactisbv. diacetylactis S50." Canadian Journal of Microbiology 51, no. 4 (April 1, 2005): 305–14. http://dx.doi.org/10.1139/w05-009.
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Lactococcus lactis subsp. lactis bv. diacetylactis S50 produces a lactococcin A-like bacteriocin named bacteriocin S50, and cell envelope-associated PI-type proteinase activity. This strain harbours 3 small size plasmids: pS6 (6.3 kb), pS7a (7.31 kb), and pS7b (7.27 kb). Plasmid curing using a combination of novobiocin treatment (10 µg·mL–1) and sublethal temperature (40 °C) resulted in a very low yield (0.17%) of Prt–, Bac–, Bacsderivatives, which retained all 3 small size resident plasmids. Pulsed-field gel electrophoresis of DNA isolated from the strain S50 and cured derivatives in combination with restriction enzyme analysis and DNA–DNA hybridization revealed that S50 contains 2 additional large plasmids: pS140 (140 kb) and pS80 (80 kb). Conjugation experiments using strain S50 as a donor and various lactococcal recipients resulted in Prt+, Bac+, Bacrtransconjugants. Analysis of these transconjugants strongly indicated that plasmid pS140 harbours the prt and bac genes encoding proteinase and bacteriocin production, and immunity to bacteriocin, since each Prt+, Bac+, Bacrtranconjugant contained pS140. Accordingly, none of the Prt–, Bac–, Bacstransconjugants contained this plasmid. pS140 was a self-transmissible conjugative plasmid regardless of the host lactococcal recipient used in the test. Frequency of conjugation of plasmid pS140 did not depend on either the donor or recipient strain.Key words: Lactococcus, plasmids, conjugation, bacteriocin, proteinase.
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ISOM, LOWELL L., ALI H. AHMED, and SCOTT E. MARTIN. "Influence of Plasmids on Catalase and Superoxide Dismutase Activities in Listeria monocytogenes." Journal of Food Protection 58, no. 9 (September 1, 1995): 955–59. http://dx.doi.org/10.4315/0362-028x-58.9.955.
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Two of nine strains of Listeria monocytogenes examined were found to contain plasmid DNA. Strain 19112 contained a 31.1 kb plasmid and strain 7644 contained a 49.4 kb plasmid. Each of the strains was cured of its plasmid by heat treatment at 46°C. Both the wild-type and plasmid-negative forms of each strain were screened for cadmium resistance. The 31.1 kb plasmid of L. monocytogenes 19112 was shown to confer resistance to cadmium. Listeria monocytogenes 7644 did not show resistance to cadmium. The plasmids for both strains were isolated and purified by CsCl density-gradient centrifugation. Each plasmid was electroporated back into the respective plasmid-negative strain. The catalase (CA) and superoxide dismutase (SOD) activities were determined for the wild type, plasmid-negative and electroporated strains. There was a significant decrease in CA and SOD activities upon loss of the plasmid from each strain of L. monocytogenes. Strain 19112 showed a 36% decrease in CA activity and an 81% decrease in SOD activity as a result of plasmid removal. Strain 7644 showed a 22% decrease in both CA and SOD activities following plasmid loss. Catalase and SOD activity levels increased for both strains following reinsertion of the plasmid through electroporation. Catalase and SOD activity levels of L. monocytogenes 7644 were higher for the transformed strain than those of the wild type. Catalase and SOD activity levels of transformed L. monocytogenes 19112 were less than in the corresponding wild type. It appears that plasmids in L. monocytogenes strains 19112 and 7644 may be involved in influencing the regulation of the production of CA and SOD. Plasmid copy number may influence the level of activity of these enzymes.
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Rasko, David A., M. J. Rosovitz, Ole Andreas Økstad, Derrick E. Fouts, Lingxia Jiang, Regina Z. Cer, Anne-Brit Kolstø, Steven R. Gill, and Jacques Ravel. "Complete Sequence Analysis of Novel Plasmids from Emetic and Periodontal Bacillus cereus Isolates Reveals a Common Evolutionary History among the B. cereus-Group Plasmids, Including Bacillus anthracis pXO1." Journal of Bacteriology 189, no. 1 (October 13, 2006): 52–64. http://dx.doi.org/10.1128/jb.01313-06.
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ABSTRACTThe plasmids of the members of theBacillus cereussensu lato group of organisms are essential in defining the phenotypic traits associated with pathogenesis and ecology. For example,Bacillus anthraciscontains two plasmids, pXO1 and pXO2, encoding toxin production and encapsulation, respectively, that define this species pathogenic potential, whereas the presence of a Bt toxin-encoding plasmid definesBacillus thuringiensisisolates. In this study the plasmids fromB. cereusisolates that produce emetic toxin or are linked to periodontal disease were sequenced and analyzed. Two periodontal isolates examined contained almost identical ∼272-kb plasmids, named pPER272. The emetic toxin-producing isolate contained one ∼270-kb plasmid, named pCER270, encoding the cereulide biosynthesis gene cluster. Comparative sequence analyses of theseB. cereusplasmids revealed a high degree of sequence similarity to theB. anthracispXO1 plasmid, especially in a putative replication region. These plasmids form a newly defined group of pXO1-like plasmids. However, these novel plasmids do not contain the pXO1 pathogenicity island, which in each instance is replaced by plasmid specific DNA. Plasmids pCER270 and pPER272 share regions that are not found in any other pXO1-like plasmids. Evolutionary studies suggest that these plasmids are more closely related to each other than to other identifiedB. cereusplasmids. Screening of a population ofB. cereusgroup isolates revealed that pXO1-like plasmids are more often found in association with clinical isolates. This study demonstrates that the pXO1-like plasmids may define pathogenicB. cereusisolates in the same way that pXO1 and pXO2 define theB. anthracisspecies.
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Ito, Yoshiyuki, Yasushi Kawai, Kensuke Arakawa, Yoshiko Honme, Takashi Sasaki, and Tadao Saito. "Conjugative Plasmid from Lactobacillus gasseri LA39 That Carries Genes for Production of and Immunity to the Circular Bacteriocin Gassericin A." Applied and Environmental Microbiology 75, no. 19 (August 7, 2009): 6340–51. http://dx.doi.org/10.1128/aem.00195-09.
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ABSTRACT Gassericin A is a circular bacteriocin produced by Lactobacillus gasseri strain LA39. We found a 33,333-bp plasmid, designated pLgLA39, in this strain. pLgLA39 contained 44 open reading frames, including seven genes related to gassericin A production/immunity (gaa), as well as genes for replication, plasmid maintenance, and conjugative transfer. pLgLA39 was transferred from LA39 to the type strain of L. gasseri (JCM 1131) by filter mating. The transconjugant exhibited >30-fold-higher more resistance to gassericin A and produced antibacterial activity. Lactobacillus reuteri LA6, the producer of reutericin 6, was proved to harbor a plasmid indistinguishable from pLgLA39 and carrying seven genes 100% identical to gaa. This suggests that pLgLA39 might have been transferred naturally between L. gasseri LA39 and L. reuteri LA6. The seven gaa genes of pLgLA39 were cloned into a plasmid vector to construct pGAA. JCM 1131T transformed with pGAA expressed antibacterial activity and resistance to gassericin A. pGAA was segregationally more stable than a pGAA derivative plasmid from which gaaA was deleted and even was more stable than the vector. This suggests the occurrence of postsegregational host killing by the gaa genes. pLgLA39 carried a pemIK homolog, and segregational stabilization of a plasmid by the pLgLA39-type pemIK genes was also confirmed. Thus, pLgLA39 was proved to carry the genes for at least two plasmid maintenance mechanisms, i.e., gaa and pemIK. Plasmids containing a repA gene similar to pLgLA39 repA were distributed in several L. gasseri strains.
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Prather, Kristala Jones, Sangeetha Sagar, Jason Murphy, and Michel Chartrain. "Industrial scale production of plasmid DNA for vaccine and gene therapy: plasmid design, production, and purification." Enzyme and Microbial Technology 33, no. 7 (December 2003): 865–83. http://dx.doi.org/10.1016/s0141-0229(03)00205-9.
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Shubin, F. N., A. V. Rakov, N. A. Kuznetsova, T. V. Yakubich, and I. P. Snetkova. "FORMATION OF POPULATION MORBIDITY WITH SALMONELLOSIS CAUSED BY SALMONELLA ENTERITIDIS IN REGIONS WITH INCOMPLETE SUPPLY OF LOCAL POULTRY PRODUCTS." Journal of microbiology epidemiology immunobiology, no. 1 (February 28, 2017): 61–67. http://dx.doi.org/10.36233/0372-9311-2017-1-61-67.
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Aim. Study plasmid characteristics of S. enteritidis strains in patients and features of epidemiology of the infection in regions with incomplete supply of population with local poultry production. Materials and methods. Plasmid analysis of microbe strains isolated from 382 patients and 8 samples of products was carried out, and significance of plasmid types in population morbidity was evaluated. Identification of salmonella was carried out by conventional methods, plasmid specter - by Kado C.I. and Liu S.T. (1981) method. Results. 98.4% of strains contained virulence plasmid p38, and 80.1% of strains also had small plasmids. Sakhalin strains were divided into 16 plasmid types (D=0.794), and strains from Jewish AO - 10 (D=0.834). Uniformity of strains in patients during infection outbreaks and in transmission factors was detected. Conclusion. Features of salmonellosis in the studied subjects of Russian Federation are determined by higher risk of import of products containing salmonella. Monitoring based on plasmid analysis is an effective base for epidemiologic control.
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Soares, Alexandra, Luciana C. Gomes, Gabriel A. Monteiro, and Filipe J. Mergulhão. "The Influence of Nutrient Medium Composition on Escherichia coli Biofilm Development and Heterologous Protein Expression." Applied Sciences 11, no. 18 (September 17, 2021): 8667. http://dx.doi.org/10.3390/app11188667.
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In the present study, the effects of different nutrient media on the development of Escherichia coli biofilms and the production of a heterologous protein were examined. E. coli JM109(DE3) cells transformed with pFM23 plasmid carrying the gene for enhanced green fluorescent protein (eGFP) expression were used. Cells were grown in two different culture media, Lysogenic Broth (LB) and M9ZB, in a flow cell system for 10 days. Epifluorescence microscopy, fluorimetry, and a high-performance liquid chromatography (HPLC) method based on hydrophobic interaction chromatography (HIC) were used to assess bacterial growth, plasmid copy number (PCN), and eGFP production in both planktonic and biofilm cells. The results showed that biofilm development was favored in M9ZB medium when compared with LB. However, the number of eGFP-expressing cells was higher in LB for both planktonic and sessile states (two-fold and seven-fold, respectively). In addition, the PCN in biofilm cells was slightly higher when using LB medium (on average, 29 plasmids per cell versus 20 plasmids per cell in M9ZB), and higher plasmid stability was observed in biofilms formed in LB compared to their planktonic counterparts. Hence, E. coli biofilms grown in LB enhanced both plasmid stability and capacity to produce the model heterologous protein when compared to M9ZB.
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Santos, Saritza, Maité Ramírez, Eric Miranda, Nelson Reyes, Osmarie Martínez, Maxier Acosta-Santiago, José M. Rivera, and Miguel Otero. "Enhancement of Immune Responses by Guanosine-Based Particles in DNA Plasmid Formulations against Infectious Diseases." Journal of Immunology Research 2019 (May 22, 2019): 1–15. http://dx.doi.org/10.1155/2019/3409371.
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Immunogenicity of DNA vaccines can be efficiently improved by adding adjuvants into their formulations. In this regard, the application of nano- and microparticles as vaccines adjuvants, or delivery systems, provides a powerful tool in designing modern vaccines. In the present study, we examined the role of “Supramolecular Hacky Sacks” (SHS) particles, made via the hierarchical self-assembly of a guanosine derivative, as a novel immunomodulator for DNA plasmid preparations. These plasmids code for the proteins HIV-1 Gag (pGag), the wild-type vaccinia virus Western Reserve A27 (pA27L), or a codon-optimized version of the latter (pOD1A27Lopt), which is also linked to the sequence of the outer domain-1 (OD1) from HIV-1 gp120 protein. We evaluated the enhancement of the immune responses generated by our DNA plasmid formulations in a murine model through ELISpot and ELISA assays. The SHS particles increased the frequencies of IFN-γ-producing cells in mice independently immunized with pGag and pA27L plasmids. Moreover, the addition of SHS to pGag and pA27L DNA plasmid formulations enhanced the production of IFN-γ(Th1-type) over IL-4 (Th2-type) cellular immune responses. Furthermore, pGag and pA27L plasmids formulated with SHS, triggered the production of antigen-specific IgG in mice, especially the IgG2a isotype. However, no improvement of either of those adaptive immune responses was observed in mice receiving pOD1A27Lopt+SHS. Here, we demonstrated that SHS particles have the ability to improve both arms of adaptive immunity of plasmid coding “wild-type” antigens without additional strategies to boost their immunogenicity. To the best of our knowledge, this is the first report of SHS guanosine-based particles as DNA plasmid adjuvants.
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Daniel, Ebakota, Osarueme Osazee, Frances Olisaka, Jocelyn Aibangbee, Panmwa GALAU, and Joseph Osazee. "Antibiotic susceptibility, plasmid isolation and curing of some foodborne pathogens." International Journal of Biological Research 4, no. 2 (November 27, 2016): 321. http://dx.doi.org/10.14419/ijbr.v4i2.6779.
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The indiscriminate use of antibiotics by individuals as well as in food production has been tagged one of the major reasons for the spread of antibiotic resistance in pathogens. Thus, there is a concern that foodborne bacteria may act as reservoirs of antibiotic resistance genes when distributed in large doses with food. This study aimed at determining the antibiotic susceptibility, plasmid isolation and curing of foodborne bacteria isolated from ready to eat (RTE) foods and salads in eating centers at the Benson Idahosa University, Benin City. Isolates were Enterobacter aerogenes, Escherichia coli, Staphylococcus aureus, Bacillus spp., Micrococcus sp. and Salmonella sp with S. aureus occurring most frequently. Total resistance to cefuroxime and augmentin as well as considerable resistance to ceftazidime and cefixime were observed in all isolates in antimicrobial susceptibility tests were done on Mueller-Hinton agar. Relative sensitivity to gentamicin, ofloxacin, nitrofurantoin and ciprofloxacin were observed. Plasmid profiling indicated that all isolates possess plasmids ranging from 100 bp to 1 kbp. Plasmid curing using sodium dodecyl sulfate (SDS) improved the sensitivity of isolates to antibiotics they were previously sensitive to but most isolates remained resistance to ceftazidime, cefuroxime, cefixime, and augmentin. This study shows that foodborne bacteria can possess and possibly transfer persistent antibiotic resistance plasmids thus calling for more caution in the use of antibiotics in food production and reduced antibiotics abuse. Further research is currently ongoing to cure the isolates of all plasmids and to elucidate how these plasmids are being transferred.
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Bihannic, Morgan, Marisa Haenni, Eric Oswald, and Jean-Yves Madec. "Divergent Evolution of the repFII Replicon of IncF Plasmids Carrying Cytotoxic Necrotizing Factorcnf2, Cytolethal Distending ToxincdtIII, andf17AeFimbrial Variant Genes in Type 2 Necrotoxigenic Escherichia coli Isolates from Calves." Applied and Environmental Microbiology 82, no. 2 (November 6, 2015): 510–17. http://dx.doi.org/10.1128/aem.02641-15.
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ABSTRACTAmong the pathovars ofEscherichia coliin cattle, necrotoxigenicE. coli(NTEC) is defined by the production of cytotoxic necrotizing factors (CNFs). In particular, type 2 NTEC (NTEC2) strains are frequent in diarrheic and septicemic calves and usually coproduce CNF type 2 (CNF2), cytolethal distending toxin type III (CDTIII), and fimbrial adhesins of the F17 family, whose genetic determinants have frequently been reported on the same Vir-like plasmid. In this study, we investigated the genetic environment of thecnf2,f17Ae, andcdtIIIgenes in a collection of fecalE. coliisolates recovered from 484 French and 58 Iranian calves. In particular, we highlighted the spread ofcnf2,f17Ae, andcdtIIIon similar 150-kb IncF plasmids harboring the newly assigned repFII replicon allele F74 in NTEC2 isolates. Interestingly, this 150-kb IncF plasmid differed from the 140-kb IncF plasmid harboring the newly assigned repFII replicon allele F75 and carryingcnf2alone. These results suggest two divergent lineages ofcnf2-carrying IncF plasmids depending on the presence of thef17AeandcdtIIIgenes. This partition was observed inE. colistrains of unrelated backgrounds, suggesting two different evolutionary paths ofcnf2-carrying IncF plasmids rather than divergent evolutions of NTEC2 clones. The driving forces for such divergent evolutions are not known, and further studies are required to clarify the selection of plasmid subtypes spreading virulence determinants inE. coli, in particular, plasmids of the IncF family.
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McClure, Nicholas C., Ali-Reza Ahmadi, and Bruce G. Clare. "Construction of a Range of Derivatives of the Biological Control Strain Agrobacterium rhizogenes K84: a Study of Factors Involved in Biological Control of Crown Gall Disease." Applied and Environmental Microbiology 64, no. 10 (October 1, 1998): 3977–82. http://dx.doi.org/10.1128/aem.64.10.3977-3982.1998.
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ABSTRACT The biological control strain Agrobacterium rhizogenesK84 is an effective agent in the control of Agrobacteriumpathogens, the causative agents of crown gall disease. A number of factors are thought to play a role in the control process, including production of the specific agrocins 84 and 434, which differ in the spectra of pathogenic strains that they inhibit in vitro. A range of derivatives of strain K84 has been developed with every combination of the three resident plasmids, pAgK84, pAgK434, and pAtK84b, including a plasmid-free strain. These derivatives produced either both, one, or neither of the characterized agrocins 84 and 434 and were isolated by plasmid curing, conjugation, and Tn5 transposon mutagenesis. The ability of the derivative strains to inhibit gall formation on almond roots was compared to that of the wild-type K84 parent. Treatment with the plasmid-free derivative did not result in a significant level of control of an A. rhizogenes pathogen based on numbers or dry weight of galls formed on injured almond roots. The presence of plasmid pAgK84, pAgK434, or pAtK84b significantly enhanced the biological control efficacy of K84 derivatives, and the highest level of control was observed with strains harboring two or more plasmids. The results observed with strains deficient in agrocin 434 production suggest that this product may play an important role in the biological control of A. rhizogenes pathogens. The involvement of plasmid pAgK84b in biological control has not previously been reported. This study supports the conclusion that multiple factors are involved in the success of strain K84 as a biological control agent.
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Parente, Ana Flávia, Ildinete Silva-Pereira, José Ivo Baldani, Victor Hugo da Silva Tibúrcio, Sônia Nair Báo, and Marlene T. De-Souza. "Construction ofBacillus thuringiensiswild-type S76 and Cry–derivatives expressing a green fluorescent protein: two potential marker organisms to study bacteria–plant interactions." Canadian Journal of Microbiology 54, no. 9 (September 2008): 786–90. http://dx.doi.org/10.1139/w08-061.
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Collectively, the species Bacillus thuringiensis , Bacillus cereus , and Bacillus anthracis represent microorganisms of high economic, medical, and biodefense importance. Although the genetic correlation and pathogenic characteristics have been extensively dissected, the ecological properties of these three species in their natural environments remain poorly understood. Thus, a tractable marker for detecting these bacteria under specific environmental and physiological conditions is a valuable tool. With this purpose, a plasmid (pAD43-25) carrying a functional gfp gene sequence (gfpmut3A) was introduced into the wild-type strain Bacillus thuringiensis subsp. kurstaki S76, which bears approximately 11 plasmids, allowing constitutive synthesis of green fluorescent protein (GFP) during vegetative growth (strain S76GFP+). Additionally, this vector was transferred to a plasmid-cured (Cry–) B. thuringiensis host. Bright green cells were detected by fluorescence microscopy in both recombinants by 2 h after inoculation in liquid medium and could be seen throughout the remaining cultivation time until complete sporulation was accomplished. For strain S76GFP+protein profile and plasmid DNA analyses indicate, respectively, that this recombinant maintained Cry proteins expression and resident plasmid outline. Thus, in addition to the potential of strain S76GFP+as a marker organism in bacteria–plant interaction studies, the production and stability of active GFPmut3a make this unique expression system a useful experimental model to study adaptive changes of host–plasmid as well as plasmid–plasmid relationships in a population of cells stressed by the production of a recombinant protein.
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May, Thithiwat, and Satoshi Okabe. "Escherichia coli Harboring a Natural IncF Conjugative F Plasmid Develops Complex Mature Biofilms by Stimulating Synthesis of Colanic Acid and Curli." Journal of Bacteriology 190, no. 22 (September 12, 2008): 7479–90. http://dx.doi.org/10.1128/jb.00823-08.
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ABSTRACT It has been shown that Escherichia coli harboring the derepressed IncFI and IncFII conjugative F plasmids form complex mature biofilms by using their F-pilus connections, whereas a plasmid-free strain forms only patchy biofilms. Therefore, in this study we investigated the contribution of a natural IncF conjugative F plasmid to the formation of E. coli biofilms. Unlike the presence of a derepressed F plasmid, the presence of a natural IncF F plasmid promoted biofilm formation by generating the cell-to-cell mating F pili between pairs of F+ cells (approximately two to four pili per cell) and by stimulating the formation of colanic acid and curli meshwork. Formation of colanic acid and curli was required after the initial deposition of F-pilus connections to generate a three-dimensional mushroom-type biofilm. In addition, we demonstrated that the conjugative factor of F plasmid, rather than a pilus synthesis function, was involved in curli production during biofilm formation, which promoted cell-surface interactions. Curli played an important role in the maturation process. Microarray experiments were performed to identify the genes involved in curli biosynthesis and regulation. The results suggested that a natural F plasmid was more likely an external activator that indirectly promoted curli production via bacterial regulatory systems (the EnvZ/OmpR two-component regulators and the RpoS and HN-S global regulators). These data provided new insights into the role of a natural F plasmid during the development of E. coli biofilms.
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Mingyar, Erik, Lucas Mühling, Andreas Kulik, Anika Winkler, Daniel Wibberg, Jörn Kalinowski, Kai Blin, Tilmann Weber, Wolfgang Wohlleben, and Evi Stegmann. "A Regulator Based “Semi-Targeted” Approach to Activate Silent Biosynthetic Gene Clusters." International Journal of Molecular Sciences 22, no. 14 (July 15, 2021): 7567. http://dx.doi.org/10.3390/ijms22147567.
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By culturing microorganisms under standard laboratory conditions, most biosynthetic gene clusters (BGCs) are not expressed, and thus, the products are not produced. To explore this biosynthetic potential, we developed a novel “semi-targeted” approach focusing on activating “silent” BGCs by concurrently introducing a group of regulator genes into streptomycetes of the Tübingen strain collection. We constructed integrative plasmids containing two classes of regulatory genes under the control of the constitutive promoter ermE*p (cluster situated regulators (CSR) and Streptomyces antibiotic regulatory proteins (SARPs)). These plasmids were introduced into Streptomyces sp. TÜ17, Streptomyces sp. TÜ10 and Streptomyces sp. TÜ102. Introduction of the CSRs-plasmid into strain S. sp. TÜ17 activated the production of mayamycin A. By using the individual regulator genes, we proved that Aur1P, was responsible for the activation. In strain S. sp. TÜ102, the introduction of the SARP-plasmid triggered the production of a chartreusin-like compound. Insertion of the CSRs-plasmid into strain S. sp. TÜ10 resulted in activating the warkmycin-BGC. In both recombinants, activation of the BGCs was only possible through the simultaneous expression of aur1PR3 and griR in S. sp. TÜ102 and aur1P and pntR in of S. sp. TÜ10.
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Paula, Marcia O., Elerson GaettiI-Jardim Jr., and Mario J. Avilla-Campos. "Plasmid profile in oral Fusobacterium nucleatum from humans and Cebus apella monkeys." Revista do Instituto de Medicina Tropical de São Paulo 45, no. 1 (January 2003): 05–09. http://dx.doi.org/10.1590/s0036-46652003000100002.
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Fusobacterium nucleatum is a strict anaerobe and is indigenous of the human oral cavity. This organism is commonly recovered from different monomicrobial and mixed infections in humans and animals. In this study, the plasmid profile, the plasmid stability and the penicillin-resistance association in oral F. nucleatum isolated from periodontal patients, healthy subjects and Cebus apella monkeys were evaluated. Forty-five F. nucleatum strains from patients, 38 from healthy subjects and seven from C. apella were identified and analyzed. Plasmid extraction was performed in all the isolated strains. These elements were found in 26.7% strains from patients and one strain from C. apella. Strains from healthy subjects did not show any plasmid. Most of strains showed two plasmid bands ranging from 4 to 16 Kb, but digestions with endonucleases showed that they belonged to a single plasmid. The plasmid profile was similar and stable in human and monkey strains. Also, plasmids were classified into three groups according to size. Two strains were positive to beta-lactamase production and no plasmid DNA-hybridization with a beta-lactamase gene probe was observed, suggesting a chromosomal resistance.
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Kalousek, S., and W. Lubitz. "High-level poly(β-hydroxybutyrate) production in recombinantEscherichia coliin sugar-free, complex medium." Canadian Journal of Microbiology 41, no. 13 (December 15, 1995): 216–21. http://dx.doi.org/10.1139/m95-190.
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The poly(β-hydroxybutyrate) (PHB) biosynthetic genes of Alcaligenes eutrophus that are organized in a single operon (phbCAB) have been cloned in Escherichia coli, where the expression of the genes in the wild-type phb operon from plasmid p4A leads to the formation of 10 or 50–80% PHB/cell dry mass when the cells are grown in Luria–Bertani medium alone or supplemented with 1% glucose (w/v), respectively. To further stimulate PHB formation independent of additional carbon source in Luria–Bertani medium, molecular methods have been applied to provide efficient E. coli transcription and translation signals for the PHB synthase gene (phbC). The lac promoter present upstream of the phbC sequence allows its expression to be controlled depending on the LacI status of the chosen host strain. The T7 gene 10 ribosome binding site is utilized for translational initiation. PHB production in E. coli was compared in strains either harboring plasmid p4A containing the intact phbCAB operon or harboring two compatible plasmids carrying the β-ketothiolase (phbA) and acetoacetyl-CoA-reductase (phbB) genes under transcriptional control of the lac promoter–operator region and also carrying separately the phbC gene with its natural promoter sequence. In addition, plasmid pSYN allowing the phbC gene to be expressed under new transcription and translation conditions combined with plasmid pUMS gave rise to the same amount of PHB formation (70% PHB cell dry mass) in E. coli when grown in Luria–Bertani medium without glucose supplement.Key words: PHB production, engineered phbCAB operon.
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Hansen, Katrine Hartung, Valeria Bortolaia, Christine Ahl Nielsen, Jesper Boye Nielsen, Kristian Schønning, Yvonne Agersø, and Luca Guardabassi. "Host-Specific Patterns of Genetic Diversity among IncI1-Iγ and IncK Plasmids Encoding CMY-2 β-Lactamase in Escherichia coli Isolates from Humans, Poultry Meat, Poultry, and Dogs in Denmark." Applied and Environmental Microbiology 82, no. 15 (May 27, 2016): 4705–14. http://dx.doi.org/10.1128/aem.00495-16.
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ABSTRACTCMY-2 is the most common plasmid-mediated AmpC β-lactamase inEscherichia coliisolates of human and animal origin. The aim of this study was to elucidate the epidemiology of CMY-2-producingE. coliin Denmark. Strain and plasmid relatedness was studied in 93 CMY-2-producing clinical and commensalE. coliisolates collected from 2006 to 2012 from humans, retail poultry meat, broilers, and dogs. Multilocus sequence typing (MLST), antimicrobial susceptibility testing, and conjugation were performed in conjunction with plasmid replicon typing, plasmid multilocus sequence typing (pMLST), restriction fragment length polymorphism (RFLP), and sequencing of selectedblaCMY-2-harboring plasmids. MLST revealed high strain diversity, with fewE. colilineages occurring in multiple host species and sample types.blaCMY-2was detected on plasmids in 83 (89%) isolates. Most (75%) of the plasmids were conjugative and did not (96%) cotransfer resistance to antimicrobials other than cephalosporins. The main replicon types identified were IncI1-Iγ (55%) and IncK (39%). Isolates from different host species mainly carried distinct plasmid subtypes. Seven of the 18 human isolates harbored IncI1-Iγ/sequence type 2 (ST2), IncI1-Iγ/ST12, or IncK plasmids highly similar to those found among animal isolates, even though highly related human and animal plasmids differed by nonsynonymous single nucleotide polymorphisms (SNPs) or insertion sequence elements. This study clearly demonstrates that the epidemiology of CMY-2 can be understood only by thorough plasmid characterization. To date, the spread of this β-lactam resistance determinant in Denmark is mainly associated with IncK and IncI1-Iγ plasmids that are generally distributed according to host-specific patterns. These baseline data will be useful to assess the consequences of the increasing human exposure to CMY-2-producingE. colivia animal sources.IMPORTANCECMY-2 is the most common plasmid-mediated AmpC β-lactamase inEscherichia coli. This β-lactamase is poorly inhibited by clavulanic acid and confers resistance to cephamycins, third-generation cephalosporins, and aztreonam. Furthermore, resistance to carbapenems has been reported inE. colias a result of production of plasmid-encoded CMY-2 β-lactamase in combination with decreased outer membrane permeability. The gene encoding CMY-2 generally resides on transferable plasmids belonging to different incompatibility groups. The prevalence of CMY-2-mediated cephalosporin resistance inE. colivaries significantly depending on the geographical region and host. This study demonstrates that the epidemiology of CMY-2 can be understood only by thorough plasmid characterization. To date, the spread of this β-lactam resistance determinant in Denmark is mainly associated with IncK and IncI1-Iγ plasmids, which are generally distributed according to host-specific patterns. These data will be useful to assess the consequences of the increasing human exposure to CMY-2-producingE. colivia animal sources.
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Zhao, Jing-yi, Li Zhong, Mei-juan Shen, Zhi-jie Xia, Qiu-xiang Cheng, Xia Sun, Guo-ping Zhao, Yue-zhong Li, and Zhong-jun Qin. "Discovery of the Autonomously Replicating Plasmid pMF1 from Myxococcus fulvus and Development of a Gene Cloning System in Myxococcus xanthus." Applied and Environmental Microbiology 74, no. 7 (February 1, 2008): 1980–87. http://dx.doi.org/10.1128/aem.02143-07.
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ABSTRACT Myxobacteria are very important due to their unique characteristics, such as multicellular social behavior and the production of diverse and novel bioactive secondary metabolites. However, the lack of autonomously replicating plasmids has hindered genetic manipulation of myxobacteria for decades. To determine whether indigenous plasmids are present, we screened about 150 myxobacterial strains, and a circular plasmid designated pMF1 was isolated from Myxococcus fulvus 124B02. Sequence analysis showed that this plasmid was 18,634 bp long and had a G+C content of 68.7%. Twenty-three open reading frames were found in the plasmid, and 14 of them were not homologous to any known sequence. Plasmids containing the gene designated pMF1.14, which encodes a large unknown protein, were shown to transform Myxococcus xanthus DZ1 and DK1622 at high frequencies (∼105 CFU/μg DNA), suggesting that the locus is responsible for the autonomous replication of pMF1. Shuttle vectors were constructed for both M. xanthus and Escherichia coli. The pilA gene, which is essential for pilus formation and social motility in M. xanthus, was cloned into the shuttle vectors and introduced into the pilA-deficient mutant DK10410. The transformants subsequently exhibited the ability to form pili and social motility. Autonomously replicating plasmid pMF1 provides a new tool for genetic manipulation in Myxococcus.
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Steinmetz, Eric. "Low Endotoxin Plasmid Production from E. coli." Genetic Engineering & Biotechnology News 34, no. 4 (February 15, 2014): 16–17. http://dx.doi.org/10.1089/gen.34.04.10.
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San Millán, J. L., R. Kolter, and F. Moreno. "Plasmid genes required for microcin B17 production." Journal of Bacteriology 163, no. 3 (1985): 1016–20. http://dx.doi.org/10.1128/jb.163.3.1016-1020.1985.
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Kanatani, Kazuo, and Masao Oshimura. "Plasmid-associated Bacteriocin Production by aLactobacillus plantarumStrain." Bioscience, Biotechnology, and Biochemistry 58, no. 11 (January 1994): 2084–86. http://dx.doi.org/10.1271/bbb.58.2084.
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Vescovo, Marisa, G. L. Scolari, and V. Bottazzi. "Plasmid-encoded ropiness production inLactobacillus casei SSP.casei." Biotechnology Letters 11, no. 10 (October 1989): 709–12. http://dx.doi.org/10.1007/bf01044102.
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Bergman, L. W., M. C. Stranathan, and L. H. Preis. "Structure of the transcriptionally repressed phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae." Molecular and Cellular Biology 6, no. 1 (January 1986): 38–46. http://dx.doi.org/10.1128/mcb.6.1.38.
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We developed a high-copy-number plasmid system containing the entire structural and regulatory sequences of the phosphate-repressible acid phosphatase (PHO5) gene and the TRP1/ARS1 replicator sequences of the yeast Saccharomyces cerevisiae to investigate the mechanism of repression-derepression of transcription. The resulting plasmid was used to transform either wild-type cells or a number of strains which contain mutations in various trans-acting regulatory loci for the production of acid phosphatase. Results of analysis of mRNA levels isolated from the transformed strains grown under repressed or derepressed conditions suggested that normal transcriptional regulation of the gene persisted, although gene copy number was significantly increased. Analysis of changes in linking number (i.e., the number of negative supercoils) of the plasmid isolated under repressed and derepressed growth conditions revealed that the transcriptionally inactive plasmid contained approximately three more negative supercoils than the transcriptionally active plasmid. This difference in topological state was similarly seen in a plasmid containing a sequence-related acid phosphatase gene (PHO11) under the same regulatory control system, but it was not seen in plasmids isolated from some strains containing mutations which caused either fully constitutive or nonderepressible production of acid phosphatase. Finally, analysis of the nucleosome positioning along the inactive gene sequence revealed that an abnormally broad internucleosomal spacer is present in a region presumed to function in the regulation of transcription by the level of Pi in the growth media.
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Bergman, L. W., M. C. Stranathan, and L. H. Preis. "Structure of the transcriptionally repressed phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae." Molecular and Cellular Biology 6, no. 1 (January 1986): 38–46. http://dx.doi.org/10.1128/mcb.6.1.38-46.1986.
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We developed a high-copy-number plasmid system containing the entire structural and regulatory sequences of the phosphate-repressible acid phosphatase (PHO5) gene and the TRP1/ARS1 replicator sequences of the yeast Saccharomyces cerevisiae to investigate the mechanism of repression-derepression of transcription. The resulting plasmid was used to transform either wild-type cells or a number of strains which contain mutations in various trans-acting regulatory loci for the production of acid phosphatase. Results of analysis of mRNA levels isolated from the transformed strains grown under repressed or derepressed conditions suggested that normal transcriptional regulation of the gene persisted, although gene copy number was significantly increased. Analysis of changes in linking number (i.e., the number of negative supercoils) of the plasmid isolated under repressed and derepressed growth conditions revealed that the transcriptionally inactive plasmid contained approximately three more negative supercoils than the transcriptionally active plasmid. This difference in topological state was similarly seen in a plasmid containing a sequence-related acid phosphatase gene (PHO11) under the same regulatory control system, but it was not seen in plasmids isolated from some strains containing mutations which caused either fully constitutive or nonderepressible production of acid phosphatase. Finally, analysis of the nucleosome positioning along the inactive gene sequence revealed that an abnormally broad internucleosomal spacer is present in a region presumed to function in the regulation of transcription by the level of Pi in the growth media.
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Wang, Dongguo, Wei Hou, Jiayu Chen, Yonghua Mou, Linjun Yang, Liqin Yang, Xiulian Sun, and Meiyun Chen. "Characterization of the bla KPC-2 and bla KPC-3 genes and the novel bla KPC-15 gene in Klebsiella pneumoniae." Journal of Medical Microbiology 63, no. 7 (July 1, 2014): 981–87. http://dx.doi.org/10.1099/jmm.0.073841-0.
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Three Klebsiella pneumoniae isolates exhibiting high-level resistance to carbapenem were analysed by PCR, PFGE, gene mapping, plasmid conjugation and Southern blot hybridization using a bla KPC probe. In addition to the frequently reported bla KPC-2 and bla KPC-3 genes, a novel bla KPC-15 gene was identified in one of the isolates. The results of plasmid analysis and Southern blot hybridization revealed that the three bla KPC genes were located on transferable plasmids exhibiting three different patterns. The patterns A, B and C were observed in the genetic makeup of each individual plasmid, and all three structures contained ISKpn6-like and ISKpn8 transposons. The results of the gene mapping and hybridization experiments performed with the bla KPC probe demonstrated that the plasmids harboured the three genes at approximately the 85.0, 54.0 and 73.0 kb positions. The study concluded that carbapenem resistance in the three isolates was primarily due to the production of carbapenem-hydrolysing β-lactamase.
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Urthaler, Jochen, Wolfgang Buchinger, and Roman Necina. "Improved downstream process for the production of plasmid DNA for gene therapy." Acta Biochimica Polonica 52, no. 3 (September 30, 2005): 703–11. http://dx.doi.org/10.18388/abp.2005_3434.
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Gene therapy and genetic vaccines promise to revolutionize the treatment of inherited and acquired diseases. Since viral vectors are generally associated with numerous disadvantages when applied to humans, the administration of naked DNA, or DNA packed into lipo- or polyplexes emerge as viable alternatives. To satisfy the increasing demand for pharmaceutical grade plasmids we developed a novel economic downstream process which overcomes the bottlenecks of common lab-scale techniques and meets all regulatory requirements. After cell lysis by an in-house developed gentle, automated continuous system the sequence of hydrophobic interaction, anion exchange and size exclusion chromatography guarantees the separation of impurities as well as undesired plasmid isoforms. After the consecutive chromatography steps, adjustment of concentration and final filtration are carried out. The final process was proven to be generally applicable and can be used from early clinical phases to market-supply. It is scaleable and free of animal-derived substances, detergents (except lysis) and organic solvents. The process delivers high-purity plasmid DNA of homogeneities up to 98% supercoiled form at a high yield in any desired final buffer.
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Zingali, Tiziana, Toni A. Chapman, John Webster, Piklu Roy Chowdhury, and Steven P. Djordjevic. "Genomic Characterisation of a Multiple Drug Resistant IncHI2 ST4 Plasmid in Escherichia coli ST744 in Australia." Microorganisms 8, no. 6 (June 14, 2020): 896. http://dx.doi.org/10.3390/microorganisms8060896.
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Antibiotic resistance genes (ARGs) including those from the blaCTX-M family and mcr-1 that encode resistance to extended spectrum β–lactams and colistin, respectively, have been linked with IncHI2 plasmids isolated from swine production facilities globally but not in IncHI2 plasmids from Australia. Here we describe the first complete sequence of a multiple drug resistance Australian IncHI2-ST4 plasmid, pTZ41_1P, from a commensal E. coli from a healthy piglet. pTZ41_1P carries genes conferring resistance to heavy-metals (copper, silver, tellurium and arsenic), β-lactams, aminoglycosides and sulphonamides. The ARGs reside within a complex resistance locus (CRL) that shows considerable sequence identity to a CRL in pSDE_SvHI2, an IncHI2:ST3 plasmid from an enterotoxigenic E. coli with serotype O157:H19 of porcine origin that caused substantial losses to swine production operations in Australia in 2007. pTZ41_1P is closely related to IncHI2 plasmids found in E. coli and Salmonella enterica from porcine, avian and human sources in Europe and China but it does not carry genes encoding resistance to clinically-important antibiotics. We identified regions of IncHI2 plasmids that contribute to the genetic plasticity of this group of plasmids and highlight how they may readily acquire new resistance gene cargo. Genomic surveillance should be improved to monitor IncHI2 plasmids.
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Silva, Filomena, João A. Queiroz, and Fernanda C. Domingues. "Evaluating metabolic stress and plasmid stability in plasmid DNA production by Escherichia coli." Biotechnology Advances 30, no. 3 (May 2012): 691–708. http://dx.doi.org/10.1016/j.biotechadv.2011.12.005.
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Zhao, Shuang-Xia, Shanli Tsui, Anthony Cheung, Raymond S. Douglas, Terry J. Smith, and J. Paul Banga. "Orbital fibrosis in a mouse model of Graves' disease induced by genetic immunization of thyrotropin receptor cDNA." Journal of Endocrinology 210, no. 3 (June 29, 2011): 369–77. http://dx.doi.org/10.1530/joe-11-0162.
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The TSH receptor (TSHR) is the critical target for antibody production in Graves' disease (GD). Insulin-like growth factor 1 receptor (IGF1R) has been proposed as a second autoantigen in complications of GD such as orbitopathy. We attempted to induce orbital tissue remodeling in mice undergoing immunizations with plasmids encoding TSHR and IGF1R delivered byin vivoskeletal muscle electroporation, a procedure known to give a sustained, long-term antibody response. FemaleBALB/cmice were challenged with TSHR A-subunit or IGF1Rα subunit plasmid by injection and electroporation. Mice challenged with TSHR A-subunit plasmid resulted in high frequency (75%) of hyperthyroidism and thyroid-stimulating antibodies. But strikingly, immunization with TSHR A-subunit plasmid also elicited antibody to IGF1Rα subunit. Mice challenged in the same manner with IGF1Rα subunit plasmid produced strong antibody responses to IGF1R, but did not undergo any changes in phenotype. Simultaneous challenge by double antigen immunization with the two plasmids in distant anatomical sites reduced the incidence of hyperthyroidism, potentially as a consequence of antigenic competition. Thyroid glands from the TSHR A-subunit plasmid-challenged group were enlarged with patchy microscopic infiltrates. Histological analysis of the orbital tissues demonstrated moderate connective tissue fibrosis and deposition of Masson's trichrome staining material. Our findings imply that immunization with TSHR A-subunit plasmid leads to generation of IGF1R antibodies, which together with thyroid-stimulating antibodies may precipitate remodeling of orbital tissue, raising our understanding of its close association with GD.
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Aqua, Mfon S., David Svinarich, Anju Dhar, and Sunil Palchaudhuri. "The stability of O-antigen plasmid is determined by a chromosomal region of Shigella dysenteriae." Canadian Journal of Microbiology 34, no. 1 (January 1, 1988): 58–62. http://dx.doi.org/10.1139/m88-010.
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It is well established that plasmids are involved in the expression of lipopolysaccharide in certain species of Shigella. In Shigella sonnei, both the biosynthesis of oligosaccharide side chains (O antigen), and cell invasiveness are controlled exclusively by a 120 megadalton (MDa) plasmid. In Shigella dysenteriae 1, a 10 kilobase (kb) plasmid is required for O-antigen production. Shigella dysenteriae 1 strains devoid of this plasmid lose the ability to synthesize O antigen. Interestingly, this 10-kb plasmid is not stably maintained in Escherichia coli K-12 strains, where it is lost spontaneously at a high frequency. Our genetic analyses of Shigella dysenteriae 1 strain IDBM11 and its derivatives indicate that the stability of this plasmid is associated with the histidine region of the chromosome which is unique to Shigella dysenteriae. Furthermore, the 10-kb plasmid is stably maintained in wild-type IDBM11 with an intact histidine locus. However, this plasmid is not stable in IDBM11 derivatives (e.g., IDBM11-1 and IDBM11-2), in which the his locus has been substituted with the histidine region of an E. coli K-12 chromosome. The S. dysenteriae IDBM11 strain, and its derivatives (lacking a 10-kb plasmid), displayed an invasive property as demonstrated by their internalization by HeLa cells in an in vitro assay. Thus the 10-kb plasmid of Shigella dysenteriae 1 is required for O-antigen synthesis but not for cell invasion.
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Yamaguchi, Takayuki, Tetsuya Hayashi, Hideto Takami, Makoto Ohnishi, Takahiro Murata, Keisuke Nakayama, Kayo Asakawa, Masaru Ohara, Hitoshi Komatsuzawa, and Motoyuki Sugai. "Complete Nucleotide Sequence of aStaphylococcus aureus Exfoliative Toxin B Plasmid and Identification of a Novel ADP-Ribosyltransferase, EDIN-C." Infection and Immunity 69, no. 12 (December 1, 2001): 7760–71. http://dx.doi.org/10.1128/iai.69.12.7760-7771.2001.
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ABSTRACT The complete nucleotide sequence of pETB, a 38.2-kbStaphylococcus aureus plasmid encoding the exfoliative toxin B (ETB), was determined. A total of 50 open reading frames were identified on the plasmid genome and, among these, 32 showed sequence similarity to known proteins. pETB contains three copies of IS257, which divide the pETB genome into three regions: (i) a cadmium resistance operon-containing region, (ii) a lantibiotic production gene-containing region, and (iii) the remaining part where genes for plasmid replication and/or maintenance are dispersed. In the third region, genes of various kinds of functions are present among the replication- and maintenance-related genes. They include two virulence-related genes, the etb gene and a gene encoding a novel ADP-ribosyltransferase closely related to EDIN, which belongs to the C3 family of ADP-ribosyltransferases modifying Rho GTPases. They also include genes for a cell wall-anchoring surface protein and a phage resistance protein. Based on the determined sequence of pETB, the genome structures of etb-bearing plasmids (ETB plasmids) from various clinical isolates were analyzed by the PCR scanning method. The data indicate that, although the ETB plasmids are highly heterogeneous in genome size, the fundamental genome organization is well conserved. The size variation of the plasmid is mainly attributed to defined regions which may be hot spots for gene shuffling.
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Deneke, Jan, and George Chaconas. "Purification and Properties of the Plasmid Maintenance Proteins from the Borrelia burgdorferi Linear Plasmid lp17." Journal of Bacteriology 190, no. 11 (March 28, 2008): 3992–4000. http://dx.doi.org/10.1128/jb.00057-08.
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ABSTRACT The Lyme disease spirochete Borrelia burgdorferi carries more plasmids than any other bacterium, many of which are linear with covalently closed hairpin ends. These plasmids have also been referred to as mini-chromosomes and essential genetic elements and are integral components of its segmented genome. We have investigated two plasmid maintenance proteins, BBD14 (the replication initiator) and BBD21 (a presumptive ParA orthologue), encoded by the linear plasmid lp17; these proteins are representatives of paralogous families 62 and 32, respectively. We have purified recombinant 6-his-BBD21 and shown it possesses an ATPase activity. 6-his-BBD14 initially could not be overexpressed in Escherichia coli by itself. It was only effectively overproduced in recombinant form through coexpression with other B. burgdorferi proteins and codon optimization. Although the mechanism for increased production through coexpression is not clear, this method holds promise for expression and purification of other B. burgdorferi proteins, a number of which have remained recalcitrant to purification from E. coli. Finally, we present evidence for the physical interaction of BBD14 and BBD21, a feature suggesting that BBD21 and the paralogous family 32 proteins are more likely involved in DNA replication than functioning as simple ParA orthologues as previously surmised based upon sequence homology. Such a role would not preclude a function in plasmid partitioning through interaction with the replication initiator.
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Schmidt-Eisenlohr, Heike, and Christian Baron. "The Competitiveness of Pseudomonas chlororaphis Carrying pJP4 Is Reduced in the Arabidopsis thaliana Rhizosphere." Applied and Environmental Microbiology 69, no. 3 (March 2003): 1827–31. http://dx.doi.org/10.1128/aem.69.3.1827-1831.2003.
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ABSTRACT The effect of the large catabolic IncP plasmid pJP4 on the competitiveness of Pseudomonas chlororaphis SPR044 and on its derivatives SPR244 (GacS deficient), SPR344 (phenazine-1-carboxamide overproducer), and SPR644 (phenazine-1-carboxamide deficient) in the Arabidopsis thaliana rhizosphere was assessed. Solitary rhizosphere colonization by the wild type, SPR244, and SPR644 was not affected by the plasmid. The size of the population of SPR344 carrying pJP4, however, was significantly reduced compared to the size of the population of the plasmid-free derivative. The abiotic stress caused by phenazine-1-carboxamide overproduction probably resulted in a selective disadvantage for cells carrying pJP4. Next, the effect of biotic stress caused by coinoculation of other bacteria was analyzed. Cells carrying pJP4 had a selective disadvantage compared to plasmid-free cells in the presence of the efficient colonizer Pseudomonas fluorescens WCS417r. This effect was not observed after coinoculation with a variety of other bacteria, and it was independent of quorum sensing and phenazine-1-carboxamide production. Thus, the presence of large catabolic plasmids imposes a detectable metabolic burden in the presence of biotic stress. Plasmid transfer in the A. thaliana rhizosphere from P. chlororaphis and its derivatives to Ralstonia eutropha was determined by using culture-dependent and culture-independent techniques. With the cultivation-independent technique we detected a significantly higher portion of exconjugants, but pJP4 transfer was independent of the quorum-sensing system and of phenazine-1-carboxamide production.
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Draths, K. M., and John W. Frost. "Synthesis using plasmid-based biocatalysis: plasmid assembly and 3-deoxy-D-arabino-heptulosonate production." Journal of the American Chemical Society 112, no. 4 (February 1990): 1657–59. http://dx.doi.org/10.1021/ja00160a071.
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Agodi, A., C. Jones, E. J. Threlfall, M. D'Angelo, and M. Marranzano. "Molecular characterization of trimethoprim resistance inShigella sonneiin Sicily." Epidemiology and Infection 105, no. 1 (August 1990): 29–40. http://dx.doi.org/10.1017/s0950268800047610.
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SUMMARYDuring the 3-year period 1985–7, all strains ofShigella sonneiisolated in Catania, Sicily, showed a high level of resistance to trimethoprim (Tp) which was invariably associated with resistance to other antibiotics.Plasmid analysis showed 18 different electropherotypes: 35 of 37 strains harboured a plasmid of 70 Megadaltons (MDa), and 29 of 37 strains a plasmid of 130 MDa.Restriction endonuclease fingerprinting of purified 70 MDa plasmid DNA from different strains demonstrated that these plasmids were similar but not identical.In some strains with transferable Tp resistance, DNA hybridization analysis demonstrated the presence of genes coding for the production of dihydrofolate reductase (DHFR) type V. In contrast, there was no detectable hybridization with DNA probes specific for genes coding for DHFR types I, II and IV. This is the first report of the DHFR type V gene outside Sri Lanka.
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Li, Huaiyu, Saumya Bhaduri, and Wayne E. Magee. "Maximizing Plasmid Stability and Production of Released Proteins in Yersinia enterocolitica." Applied and Environmental Microbiology 64, no. 5 (May 1, 1998): 1812–15. http://dx.doi.org/10.1128/aem.64.5.1812-1815.1998.
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ABSTRACT Virulent serotypes of Yersinia enterocolitica carry a plasmid (pYV) encoding a family of proteins that are released into the medium and whose expression is temperature and calcium regulated. The plasmid is easily lost from cells during their growth in the laboratory. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with a monoclonal antibody (3.2C) that is specific for a 25-kDa released protein to show that 32�C is the lowest temperature at which plasmid-encoded proteins are expressed in quantity. The highest calcium concentration allowing full expression of these proteins was 445 to 545 μM at 32�C. Calcium concentrations of 745 μM and above at 37�C completely prevented the loss of pYV during multiple subcultures, while at 32�C, calcium concentrations of 245 μM and greater were sufficient to stabilize the plasmid. Growth of Y. enterocolitica at pH 5.5 was slower than at neutral pH values, but it also resulted in greatly increased stability of pYV. These studies showed that bacterial growth, retention of pYV, and expression of plasmid-encoded proteins may be maximized at 32�C with 445 μM calcium and that pYV stability is enhanced by growth at low pH. These observations suggest new approaches for isolation of plasmid-bearing virulent strains of Y. enterocolitica from samples contaminated with this organism and also may improve our understanding of pYV retention in vivo.
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Lee, Sang Yup, and Young Lee. "Metabolic Engineering of Escherichia coli for Production of Enantiomerically Pure (R)-(−)-Hydroxycarboxylic Acids." Applied and Environmental Microbiology 69, no. 6 (June 2003): 3421–26. http://dx.doi.org/10.1128/aem.69.6.3421-3426.2003.
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ABSTRACT A heterologous metabolism of polyhydroxyalkanoate (PHA) biosynthesis and degradation was established in Escherichia coli by introducing the Ralstonia eutropha PHA biosynthesis operon along with the R. eutropha intracellular PHA depolymerase gene. By with this metabolically engineered E. coli, enantiomerically pure (R)-3-hydroxybutyric acid (R3HB) could be efficiently produced from glucose. By employing a two-plasmid system, developed as the PHA biosynthesis operon on a medium-copy-number plasmid and the PHA depolymerase gene on a high-copy-number plasmid, R3HB could be produced with a yield of 49.5% (85.6% of the maximum theoretical yield) from glucose. By integration of the PHA biosynthesis genes into the chromosome of E. coli and by introducing a plasmid containing the PHA depolymerase gene, R3HB could be produced without plasmid instability in the absence of antibiotics. This strategy can be used for the production of various enantiomerically pure (R)-hydroxycarboxylic acids from renewable resources.
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Inglis, R. Fredrik, Bihter Bayramoglu, Osnat Gillor, and Martin Ackermann. "The role of bacteriocins as selfish genetic elements." Biology Letters 9, no. 3 (June 23, 2013): 20121173. http://dx.doi.org/10.1098/rsbl.2012.1173.
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Bacteria produce a wide arsenal of toxic compounds in order to kill competing species. Bacteriocins, protein-based toxins produced by nearly all bacteria, have generally been considered a ubiquitous anti-competitor strategy, used to kill competing bacterial strains. Some of these bacteriocins are encoded on plasmids, which also code for closely linked immunity compounds (thereby rendering toxin producing cells immune to their own toxin). However, the production of bacteriocins can also be interpreted as a means to promote plasmid stability by preferentially selecting for cells carrying the plasmid. If, for example, a cell were to lose the plasmid, it would no longer produce the immunity compound and would be killed by its bacteriocin-producing clone mates. In this respect, bacteriocins can be regarded as similar to previously described toxin–antitoxin systems that are able promote the stable transmission of plasmids to daughter cells. In order to test this prediction, we carried out an experimental evolution study using the bacterium Escherichia coli , finding that bacteriocins can indeed select for the stable maintenance of plasmids. This suggests that bacteriocins can act primarily as selfish genetic elements promoting their own transmission in the population, which may help explain their unique ecology and evolution.
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de la Cruz, Mitzi, Elisa A. Ramírez, Juan-Carlos Sigala, José Utrilla, and Alvaro R. Lara. "Plasmid DNA Production in Proteome-Reduced Escherichia coli." Microorganisms 8, no. 9 (September 21, 2020): 1444. http://dx.doi.org/10.3390/microorganisms8091444.
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The design of optimal cell factories requires engineering resource allocation for maximizing product synthesis. A recently developed method to maximize the saving in cell resources released 0.5% of the proteome of Escherichia coli by deleting only three transcription factors. We assessed the capacity for plasmid DNA (pDNA) production in the proteome-reduced strain in a mineral medium, lysogeny, and terrific broths. In all three cases, the pDNA yield from biomass was between 33 and 53% higher in the proteome-reduced than in its wild type strain. When cultured in fed-batch mode in shake-flask, the proteome-reduced strain produced 74.8 mg L−1 pDNA, which was four times greater than its wild-type strain. Nevertheless, the pDNA supercoiled fraction was less than 60% in all cases. Deletion of recA increased the pDNA yields in the wild type, but not in the proteome-reduced strain. Furthermore, recA mutants produced a higher fraction of supercoiled pDNA, compared to their parents. These results show that the novel proteome reduction approach is a promising starting point for the design of improved pDNA production hosts.