logo logo
Transformation of Pseudomonas aeruginosa by electroporation. Diver J M,Bryan L E,Sokol P A Analytical biochemistry Optimum conditions were defined for the electrotransformation of Pseudomonas aeruginosa PAO1 with plasmid pLAFR1, resulting in a 1500-fold increase in transformation efficiency compared to conventional chemical transformation with MgCl2. In addition, PAO236 and two out of three recent clinical isolates of P. aeruginosa from the sputum of cystic fibrosis patients were successfully transformed with plasmid pUC19 1.8. The applied voltage and the electroporation buffer composition were shown to have the greatest effect on transformation efficiency. Freezing the cells and prolonged storage at -70 degrees C did not significantly affect the transformation efficiency. The clinical isolates tested had lower transformation efficiencies than PAO1. 10.1016/0003-2697(90)90046-c
Efficient transformation of pseudomonas strains with pNI vectors by electroporation. Itoh N,Kouzai T,Koide Y Bioscience, biotechnology, and biochemistry The optimum conditions for electro-transformation of some Pseudomonas strains including P. putida, P. fluorescens, and P. flavida, were defined using the pNI105 vector, resulting in 100-10,000 fold increases in transformation efficiency compared with conventional chemical transformation with MgCl2. The growth phase of the cultured cells and the field strength were important in obtaining high transformation efficiency. Under optimal conditions, 3.2 x 10(7) transformants per 1 microgram DNA were obtained using a combination of pNI105 and P. fluorescens IAM12022. This value is the highest ever obtained for a Pseudomonas species. In addition, the procedures were applicable to the effective transformation of fluorescent Pseudomonas strains with pNI vectors. 10.1271/bbb.58.1306
Transformation of Pseudomonas putida by electroporation. Iwasaki K,Uchiyama H,Yagi O,Kurabayashi T,Ishizuka K,Takamura Y Bioscience, biotechnology, and biochemistry The optimum electrotransformation conditions were determined for Pseudomonas putida PpY101 with plasmid pSUP104 (9.5 kb) and pSR134 (18.6 kb). Field strength was a very important parameter for electrotransformation efficiency. Optimum efficiencies (1.1 x 10(5) transformants/micrograms DNA) with pSUP104 and pSR134 were obtained at a field strength of 12.5 kV/cm, a time constant of about 4.5 ms (resistance setting of 200 ohms), a supercoiled DNA concentration of 100 ng/ml, and a cell concentration of 10(9)/ml. Because the efficiency obtained is high enough, electrotransformation is useful for the direct cloning of P. putida PpY101. No significant relationship between plasmid size and electrotransformation efficiency was observed. These efficiencies were about 4.5 times higher than those using the MgCl2 method. Under these conditions, electrotransformation efficiencies of relaxed plasmid DNA treated with topoisomerase I and that linearized by EcoRI digestion were high.
Establishment of a gene transfer system for Rhodococcus opacus PD630 based on electroporation and its application for recombinant biosynthesis of poly(3-hydroxyalkanoic acids). Kalscheuer R,Arenskötter M,Steinbüchel A Applied microbiology and biotechnology A gene transfer system for Rhodococcus opacus PD630 based on electroporation was established and optimized employing the Escherichia coli-Rhodococcus shuttle vectors pNC9501 and pNC9503 as well as the E. coli-Corynebacterium glutamicum shuttle vector pJC1 as suitable cloning vectors for R. opacus PD630, resulting in transformation efficiencies up to 1.5 x 10(5) CFUs/microgram plasmid DNA. Applying the optimized electroporation protocol to the pNC9501-derivatives pAK68 and pAK71 harboring the entire PHB synthesis operon from Ralstonia eutropha and the PHA synthase gene phaC1 from Pseudomonas aeruginosa, respectively, recombinant PHA biosynthesis was established in R. opacus PD630 and the TAG-negative mutant ROM34. Plasmid pAK68 enabled synthesis and accumulation of poly(3HB) in R. opacus PD630 and ROM34 during cultivation under storage conditions from 1% (w/v) gluconate, of poly(3HB-co-3HV) from 0.2% (w/v) propionate and of poly(3HV) from 0.1% (w/v) valerate. Under storage conditions, recombinant strains of PD630 and ROM34 harboring pAK71 were able to synthesize and accumulate PHA of the medium chain length hydroxyalkanoic acids 3HHx, 3HO, 3HD and 3HDD from 0.1% (w/v) hexadecane or octadecane and a copolyester composed of 3HHp, 3HN and 3HUD from 0.1% (w/v) pentadecane or heptadecane. In the recombinant strains of PD630 and ROM34, the thiostrepton-induced overexpression of a 20 kDa protein was observed with its N-terminus exhibiting a homology of 60% identical amino acids to TipA from Streptomyces lividans.
Ultrasound-mediated DNA transfer for bacteria. Song Yizhi,Hahn Thomas,Thompson Ian P,Mason Timothy J,Preston Gail M,Li Guanghe,Paniwnyk Larysa,Huang Wei E Nucleic acids research In environmental microbiology, the most commonly used methods of bacterial DNA transfer are conjugation and electroporation. However, conjugation requires physical contact and cell-pilus-cell interactions; electroporation requires low-ionic strength medium and high voltage. These limitations have hampered broad applications of bacterial DNA delivery. We have employed a standard low frequency 40 kHz ultrasound bath to successfully transfer plasmid pBBR1MCS2 into Pseudomonas putida UWC1, Escherichia coli DH5alpha and Pseudomonas fluorescens SBW25 with high efficiency. Under optimal conditions: ultrasound exposure time of 10 s, 50 mM CaCl(2), temperature of 22 degrees C, plasmid concentration of 0.8 ng/microl, P. putida UWC1 cell concentration of 2.5 x 10(9) CFU (colony forming unit)/ml and reaction volume of 500 microl, the efficiency of ultrasound DNA delivery (UDD) was 9.8 +/- 2.3 x 10(-6) transformants per cell, which was nine times more efficient than conjugation, and even four times greater than electroporation. We have also transferred pBBR1MCS2 into E. coli DH5alpha and P. fluorescens SBW25 with efficiencies of 1.16 +/- 0.13 x 10(-6) and 4.33 +/- 0.78 x 10(-6) transformants per cell, respectively. Low frequency UDD can be readily scaled up, allowing for the application of UDD not only in laboratory conditions but also on an industrial scale. 10.1093/nar/gkm710
[Optimization of the conditions for electroporation and the addition nisin for Pseudomonas aeruginosa inhibition]. Santi L,Cerrutti P,Pilosof A M,de Huergo M S Revista Argentina de microbiologia A mathematical approach was applied in order to optimize the effect of electroporation by application of pulsed electric fields (PEF) and nisin addition on the inhibition of a strain of Pseudomonas aeruginosa isolated from river sediments. This strain showed to be highly resistant to nisin as only two log cycles reduction of viable cells were obtained in the presence of 84,000 IU/ml nisin. But when a combination of bacteriocin and selected PEF treatment conditions were applied, 4.4-decimal log cycle reduction could be achieved. PEF and nisin interaction seems to be complex, as at lower electric field intensities (i.e., 5 kV/cm) an increment in the number of pulses applied clearly induced a lower inhibitory effect of nisin. At higher PEF intensities (i.e., 11 kV/cm), the inhibitory effect of nisin increased with the number of pulses applied. Results overall, the obtained indicate the possibility of combining PEF and nisin treatments in order to improve the inhibition of resistant microorganisms. The Doehlert experimental design and surface response methodology was an interesting tool to obtain or predict the optimal combination of the stress factors applied.
[The study of optimal conditions of electroporation in Pseudomonas aeruginosa]. Shan Zhi-Ying,Xu Hai-Jin,Shi Xing-Qi,Nie Zhou,Yu Yan,Zhang Xiu-Ming,Bai Yan-Ling,Qiao Ming-Qiang,Gao Cai-Chang Yi chuan xue bao = Acta genetica Sinica A P. aeruginosa strain PA68 isolated from the sputum of a patient suffering from bronchiectasis was used as the recipient strain. Optimum conditions including growth stage of the strain, electroshock voltage, concentration and preservation of competent cell were defined for the electroporation of PA68 with plasmid pSMC28. It was showed that the highest transformation efficiency was up to 1.68 x 10(3) CFU/microgram DNA under the optimum conditions in which the competent cells were collected at logarithmic growth phase (OD(540) = 0.7-0.8) and concentrated to about 10(11) cells/ml, the mixture of the competent cells and plasmid pSMC28 was eletroporated at 2.6 kV. With this optimal condition, Mu transponson complexes have been successfully transformed into P. aeruginosa strain PA68 and the obtained efficiency was 2.47 x 10(4) CFU/microgram DNA. This is the first time to electroporate Mu transposon complexes into Pseudomonas spp. The artificial Mu transposons could integrate into bacterial genomes at a single site randomly. Then the phenotype change was the result of the gene inactivation caused by Mu transposon insertion. That will be very helpful for the study of genomic function of Pseudomonas spp.