Improved method for high-efficiency electrotransformation of Escherichia coli with the large BAC plasmids

High transformation competency of Escherichia coli is one of the critical factors in the bacterial artificial chromosome (BAC)-based DNA library construction. Many electroporation protocols have been published until now, but the majority of them was optimized for transformation of small plasmids. Large plasmids with a size above 50 kbp display reduced transformation efficiency and thereby require specific conditions in the preparation and electroporation of electrocompetent cells. In the present work, we have optimized the parameters critical to the application of BAC DNA electrotransformation into E. coli. Systematic evaluation of electroporation variables has revealed several key factors like temperature of growth, media supplements, washing buffer, and cell concentration. Improvements made in the transformation protocol have led to electrocompetent cells with transformation efficiency up to 7?×?10⁸ transformants per microgram of 120 kbp BAC plasmid DNA. We have successfully used in-house prepared competent cells, the quality of which is comparable with those produced by different companies, in the construction of metagenomic libraries from the soil. Our protocol can also be beneficial for other application with limited DNA source.     

Efficient transformation ofKlebsiella oxytoca by electroporation

A protocol for the transformation ofKlebsiella oxytoca by electroporation was developed. Preparation of competent cells at early exponential phase was most critical to obtain a high transformation efficiency. The highest efficiency of 1.6 × 10⁶ transformants per μg DNA (pBR 322) could be obtained by electroporation ofK. oxytoca cells prepared at the OD₆₀₀ of 0.2 with 1.25 μg DNA at the filed strength of 2.5 kV, the parallel resistance of 200 Ω and capacitance of 25 μF.     

Biomass relationship to growth and phosphate uptake ofPseudomonas fluorescens,Escherichia coli andAcinetobacter radioresistens in mixed liquor medium

The ability ofPseudomonas fluorescens, Escherichia coli andAcinetobacter radioresistenns to remove phosphate during growth was related to the initial biomass as well as to growth stages and bacterial species. Phosphate was removed by these bacteria under favourable conditions as well as under unfavourable conditions of growth. Experiments showed a relationship between a high initial cell density and phosphate uptake. More phosphate was released than removed when low initial cell densities (10²–10⁵ cells ml^–1) were used. At a high initial biomass concentration (10⁸ cells ml^–1), phosphate was removed during the lag phase and during logarthmic growth byP. fluorescens. Escherichia coli. at high initial biomass concentrations (10⁷ cells ml^–1), accumulated most of the phosphate during the first hour of the lag phase and/or during logarithmic growth and in some cases removed a small quantily of phosphate during the stationary growth phase.Acinetobacter radioresistens, at high initial cell densities (10⁶, 10⁷ cells ml^–1) removed most of phosphate during the first hour of the lag phase and some phosphate during the stationary growth phase.Pseudomonas fluorescens removed phosphate more thanA. radioresistens andE. coli with specific average ranges from 3.00–28.50 mg L^–1 compared to average ranges of 4.92–17.14 mg L^–1 forA. radioresistens and to average ranges of 0.50–8.50 mg L^–1 forE. coli.     

Preparation of highly efficient electrocompetent Escherichia coli using glycerol/mannitol density step centrifugation

Traditional protocols for preparing Escherichia coli for electroporation are laborious and often deliver highly variable transformation efficiencies. Many laboratories resort to purchasing expensive commercially prepared cells. This article describes a simple method for producing electrocompetent E. coli by centrifuging bacteria through a glycerol/mannitol density cushion. The method is rapid and replaces tedious multistep procedures with two 15-min centrifugations. Standard cloning strains consistently produce more than 8 × 10⁹ transformants/μg pUC18, whereas the strains TG1 and LE392 display efficiencies of more than 3 × 10¹⁰/μg DNA.     

Application of R-plasmid DNA’s fromEscherichia coli minicells in genetic transformation

Components of minicell lyzatesof Escherichia coli P678-54 (Rldrd19) andEscherichia col P678-54 (R6K) were visualized in an electron microscope and used for the transformation ofEscherichia coli JC7623. The frequency of the resulting transformants (of the order of 10⁻⁶ %) was not appreciably influenced by the manner of lyzate preparation. The presence of covalently closed circular DNA was detected in two different transformants using radioisotopes, thus demonstrating an autonomous existence of Rldrd19 or R6K plasmids in tested transformants. This finding corresponds with the results of their genetic analysis.     

Study on the electro-transformation conditions of improving transformation efficiency for Bacillus subtilis

Aims: To optimize the transformation conditions and improve the transformation efficiency of Bacillus subtilis WB800 and DB104. Methods and Results: Trehalose, which could decrease the damage of electric shock to the cells, was added to the electroporation medium containing sorbitol and mannitol. The factors affecting the transformation efficiency, such as the growth phase of bacteria, cell concentration, electric field strength and plasmid variety, were examined and improved. The new method increased the transformation efficiency of B. subtilis by nearly 100‐fold compared with the conventional one. Conclusions: With the optimized method, the transformation efficiency came up to 3·64 × 10⁵ transformants μg^?1 DNA for WB800, and 2·10 × 10⁵ transformants μg^?1 DNA for DB104. Significance and Impact of the Study: This improvement in transformation efficiency will be largely attributed to the research of expression of exogenous genes in B. subtilis, gene library construction for directed evolution and transformation of wild‐type B. subtilis strains.     

Conditions for quantitative transformation inAcinetobacter calcoaceticus

A transformation procedure that yielded high efficiencies was developed forAcinetobacter calcoaceticus. Strain BD413 Ura⁻ trpE was transformed to tryptophan prototrophy using highly purified DNA. Experimental parameters studied were: (i) recipient cell concentration, (ii) DNA concentration, (iii) growth phase of the recipient cell population, (iv) composition of the growth and transforming medium, and (v) time of incubation of recipient cells with donor DNA. Strain BD413 was competent for transformation throught the growth cycle, with highest competence occurring early in the exponential phase of growth. Maximal transformation efficiencies of 0.5% to 0.7% were obtained in media supporting rapid growth. Recipient cell concentrations of 1×10⁶ to 6×10⁶ cells/ml yielded the highest transformation frequencies, regardless of DNA concentration.     

The inhibitory effect of albomycin (Grisein) on growth ofBacillus cereus andEscherichia coli

It was confirmed that albomycin inhibits the growth ofBacillus cereus andEscherichaa coli depending on a concentration of the antibiotic and physiological state of the culture. Salts of trivalen iron in a test medium reduce the time of the effect of albomycin, however, they also increase the growth of the control culture ofBacillus cereus and increase a value of maximal out-growth. An exponential growth of the culture is restored following a short lag when albomcycin is removed from a test medium. The antibiotic effect of albomycin A₁ onBacillus cereus andEscherichia coli is removed by adding a 10-fold concentration of albomycin A₃. The effect of Neilands ferrichrome is similar. On comparison withEscherichia coli, albomycin does no inhibit the growth ofBacillus cereus when cultivated at 28°C. The addition of CrCl₃ solution partially restored the antibiotic activity of albomycin deprived of iron. It was found by microbiological tests that a retored growth of the whole culture was a result of albomycin inactivation. However, we did not find what change in the albomycin molecule is responsible for this inactivation.     

Optimized transformation by electroporation ofLactobacillus plantarum strains with plasmid vectors

Summary Whole cell transformation ofLactobacillus plantarum CCM 1904 by electroporation was optimized. Pulse duration and electric field strength were shown to be important parameters: the optimum conditions were 12.5 kV/cm, a time-constant of 10 ms for an exponential decay waveform and 6.7 kV/cm applied during 2.5 ms for a square waveform. Transformation efficiency was increased if cells were cultivated on medium containing sorbitol and harvested during their early exponential growth phase: 8 × 10^–4 transformants/g pGK12 DNA per viable cell were obtained, with a survival rate of 10%–30% Cryotreatment by several freeze-and-thaw cycles decreased transformant yields. Transformation efficiency with different plasmids was studied and plasmid pGK12 was found to transformL. plantarum the most efficiently. Transformation by electroporation ofL. plantarum is strain dependent. The best results were obtained withL. plantarum NCIB 7220, giving 5 × 10⁶ transformants/gmg plasmid pGK12 DNA.     

High frequency transformation of the industrial erythromycin-producing bacterium Saccharopolyspora erythraea

The DNA transformation in the industrial erythromycin-producing Saccharopolyspora erythraea was investigated as standard protoplast transformation methods are ineffective. Intergeneric conjugal transfer of DNA from E. coli demonstrated transformation efficiencies from 0.05 × 10⁻⁸ to 7.2 × 10⁻⁸ exconjugants generated per recipient. Electroporation-mediated methodologies were also established. More than 10⁵ transformants were acquired per μg DNA. The proposed protocol provides an alternative route for the introduction of DNA into industrial strains.     

High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation

Summary Salmonella typhimurium and S. typhi were transformd with high efficiency by electroporation. Transformation efficiencies of up to 10¹⁰ transformants per g of pBR322 were obtained. In contrast to chemical transformation methods, neither the smooth lipopolysaccharide of S. typhimurium nor the Vi capsular polysaccharide of S. typhi greatly affected transformation efficiency. The introduction of a galE mutation slightly improved transformation efficiency in S. typhimurium (< tenfold) while the Vi antigen of S. typhi had no detectable effect. The transformation efficiency of S. typhimurium with DNA derived from Escherichia coli was increased greatly by the removal of the hsd restriction system (100-fold). Under these conditions electroporation can be used for the routine and direct transformation of Salmonella strains with partially purified (alkaline lysis) plasmid DNA from E. coli.     

Characterization of the submerged growth ofMoraxella bovis

A submerged batch cultivation ofMoraxella bovis in a medium containing enzymic casein hydrolyzate and supplemented with dialyzed ram blood was described. Up to the growth limitation the bacteria grew exponentially without a lag phase and with a doubling period of 64 min. During the exponential phase no significant decrease of viable cells and cell autolysis were observed. Amino acids were the limiting factor. At the end of growth glutamie acid, threonine and serine were detected at the lowest concentrations. Substrate limitation led to an irreversible decrease of the metabolic activity. Growth yield on oxygen was 2.3 × 10⁹ cells per mg O₂. The fraction of respiration required for growth-uncoupled processes was negligible. During the cultivation ammonia was produced and the pH increased but it was not the inhibitory factor. Respiration was not limited by oxygen at concentrations higher than 0.63 μmol O₂ per L. Sufficient pili were produced.     

Enhancing hypericin production of Hypericum perforatum cell suspension culture by ozone exposure

Accumulation of secondary metabolites is one of the common reactions of plants to ozone exposure in nature. To investigate the effect of ozone on the production of desired compounds of plant cell cultures, we assayed hypericin production of Hypericum perforatum suspension cell cultures treated with different doses of ozone at different culture phases. The results show that hypericin contents of the cells treated with 60 to 180 nL L^?1 ozone are significantly higher than those of the control, showing that ozone exposure may stimulate hypericin synthesis. Hypericin production of the cells treated with ozone at exponential phase is higher than that of lag and stationary phase, which suggests that exponential phase cell cultures are more responsive to ozone exposure than lag and stationary phase cells. The highest hypericin production is obtained by the cells exposed to 90 nL L^?1 ozone at late exponential phase for 3 h, being about fourfold of the control. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Thiosulfate oxidation by obligately heterotrophic bacteria

Thiosulfate was oxidized stoichiometrically to tetrathionate during growth on glucose byKlebsiella aerogenes, Bacillus globigii, B. megaterium, Pseudomonas putida, two strains each ofP. fluorescens andP. aeruginosa, and anAeromonas sp. A gram-negative, rod-shaped soil isolate, Pseudomonad H_w, converted thiosulfate to tetrathionate during growth on acetate. None of the organisms could use thiosulfate as sole energy source. The quantitative recovery of all the thiosulfate supplied to heterotrophic cultures either as tetrathionate alone or as tetrathionate and unused thiosulfate demonstrated that no oxidation to sulfate occurred with any of the strains tested. Two strains ofEscherichia coli did not oxidize thiosulfate. Thiosulfate oxidation in batch culture occurred at different stages of the growth cycle for different organisms:P. putida oxidized thiosulfate during lag and early exponential phase,K. aerogenes oxidized thiosulfate at all stages of growth, andB. megaterium andAeromonas oxidized thiosulfate during late exponential phase. The relative rates of oxidation byP. putida andK. aerogenes were apparently determined by different concentrations of thiosulfate oxidizing enzyme. Thiosulfate oxidation byP. aeruginosa grown in chemostat culture was inducible, since organisms pregrown on thiosulfate-containing media oxidized thiosulfate, but those pregrown on glucose only could not oxidize thiosulfate. Steady state growth yield ofP. aeruginosa in glucose-limited chemostat culture increased about 23% in the presence of 5–22 mM thiosulfate, with complete or partial concomitant oxidation to tetrathionate. The reasons for this stimulation are unclear. The results suggest that heterotrophic oxidation of thiosulfate to tetrathionate is widespread across several genera and may even stimulate bacterial growth in some organisms.     

Optimum conditions for efficient transformation ofStreptomyces venezuelae protoplasts

Summary Optimum conditions for protoplast regeneration and transformation ofStreptomyces venezuelae ETH 14630 have been established. Protoplasts from mycelium grown to the stationary phase and treated with lysozyme in P medium under mild conditions gave the best regeneration frequency. Transformation of protoplasts with naked DNA was very efficient using either polyethylene glycol of mol. wt. 4000 or 6000, at concentrations of 28.5% or 36% (w/v) respectively. About 10⁵ transformants/g DNA could be isolated using protoplasts derived from cells cultivated to the early exponential growth phase in LB medium containing 0.2%-0.6% glycine and subsequently treated at 30°–32°C with 20 mg lysozyme/ml in P medium for 30 min. Selection of the transformants occurred on MRYE plates containing less than 10⁵ regenerating protoplasts per plate. Higher protoplast densities considerably decreased the regeneration frequency of the transformants.     

Assessing the Contamination Potential of Freshly Extracted Escherichia coli Biofilm Cells by Impedancemetry

Planktonic bacteria passing to a sessile state during the formation of a biofilm undergo many gene expression and phenotypic changes. These transformations require a significant time to establish. Inversely, cells extracted from a biofilm should also require a significant time before acquiring the same physiological characteristics as planktonic cells. Relatively few studies have addressed the kinetics of this inverse transformation process. We tested one aspect, namely, the contamination potential of freshly extracted Escherichia coli biofilm cells, precultured in a synthetic medium, in a rich liquid growth medium. We compared the time between inoculation and the beginning of the growth phase of freshly extracted biofilm cells, and suspended exponential and suspended stationary phase cells precultured in the same synthetic medium. Unexpectedly, the lag time for the extracted biofilm cells was the same as the lag time of the suspended exponential phase cells and significantly less than the lag time of the suspended stationary phase cells. The lag times were determined by an impedance technique. Cells extracted from biofilms, i.e., biofilms formed in canalizations and broken up by hydrodynamic forces, are an important source of contamination. Our work shows, in the case of E. coli, the high potential of freshly extracted biofilm cells to reinfect a new medium.     

Intensive DNA Replication and Metabolism during the Lag Phase in Cyanobacteria

Unlike bacteria such as Escherichia coli and Bacillus subtilis, several species of freshwater cyanobacteria are known to contain multiple chromosomal copies per cell, at all stages of their cell cycle. We have characterized the replication of multi-copy chromosomes in the cyanobacterium Synechococcus elongatus PCC 7942 (hereafter Synechococcus 7942). In Synechococcus 7942, the replication of multi-copy chromosome is asynchronous, not only among cells but also among multi-copy chromosomes. This suggests that DNA replication is not tightly coupled to cell division in Synechococcus 7942. To address this hypothesis, we analysed the relationship between DNA replication and cell doubling at various growth phases of Synechococcus 7942 cell culture. Three distinct growth phases were characterised in Synechococcus 7942 batch culture: lag phase, exponential phase, and arithmetic (linear) phase. The chromosomal copy number was significantly higher during the lag phase than during the exponential and linear phases. Likewise, DNA replication activity was higher in the lag phase cells than in the exponential and linear phase cells, and the lag phase cells were more sensitive to nalidixic acid, a DNA gyrase inhibitor, than cells in other growth phases. To elucidate physiological differences in Synechococcus 7942 during the lag phase, we analysed the metabolome at each growth phase. In addition, we assessed the accumulation of central carbon metabolites, amino acids, and DNA precursors at each phase. The results of these analyses suggest that Synechococcus 7942 cells prepare for cell division during the lag phase by initiating intensive chromosomal DNA replication and accumulating metabolites necessary for the subsequent cell division and elongation steps that occur during the exponential growth and linear phases.     

Interaction of lead nitrate and cadmium chloride withEscherichia coli K-12 andSalmonella typhimurium global regulatory mutants

Summary To investigate the interactions of heavy metals with cells, a minimal medium for the growth of enteric bacteria using glycerol-2-phosphate as the sole phosphorus source was developed that avoided precipitation of Pb²⁺ with inorganic phosphate. Using this medium, spontaneous mutants ofEscherichia coli resistant to addition of Pb(NO₃)₂ were isolated. Thirty-five independent mutants all conferred a low level of resistance. Disk diffusion assays on solid medium were used to survey the response ofE. coli andSalmonella typhimurium mutants altered in global regulatory networks to Pb(NO₃)₂) and CdCl₂. Strains bearing mutations inoxyR andrpoH were the most hypersensitive to these compounds. Based upon the response of strains completely devoid of isozymes needed to inactivate reactive oxygen species, this hypersensitity to lead and cadmium is attributable to alteration in superoxide dismutase rather than catalase levels. Similar analysis of chaperonedefective mutants suggests that these metals damage proteins in vivo.     

Transformation of Bacillus Protoplasts by Plasmid pTP4 DNA

Polyethylene glycol (PEG)-induced protoplast transformation by plasmid pTP4 DNA encoded chloramphenicol resistance determinant was developed for Bacillus subtilis, B. amyloliquefaciens, B. licheniformis, B. megaterium and B. pumilus. Protoplasts were formed by treatment of cells with lysozyme and the transformation frequencies (transformants per regenerants) were in the range of 1.3 × 10^?2 to 7.1 × 10^?1. Reisolated plasmid DNA prepared from transformants exhibited covalently closed and open circular forms similar to those of the donor DNA. These results indicate that PEG-induced protoplast transformation is an adequate method for plasmid transformation and pTP4 is a useful plasmid as a cloning vector in a wide range of varieties of the genus Bacillus.     

Optimum Conditions for Electric Pulse-mediated Gene Transfer to Bacillus subtilis Cells

It was found that plasmid DNA (pUB 110) can be introduced into not only protoplasts but also intact cells of Bacillus subtilis by electric field pulses. The transformation of, B. subtilis using protoplasts results in an efficiency of 2.5 × 10⁴ transformants per μg of DNA, with a single pulse of 50 jisec with an initial electric field strength of 7kV/cm. Even transformation of intact B. subtilis cells results in a maximum efficiency of 1.5 × 10³ transformants per μg DNA, with a single pulse of 400 μsec with an initial electric field strength of 16kV/cm. The cell survival of protoplasts and intact cells was approximately 100% and 30%, respectively, under the conditions found to be optimal for the transformation process. Plasmid DNA isolated from pUB 110 containing transformants was indistinguishable from authentic preparations of pBU 110 on gel electrophoretic analysis.