Electrotransformation of whole cells of Brevibacterium sp. R312 a nitrile hydratase producing strain: Construction of a cloning vector

Abstract: A rapid and effective method is described for electroporation of Brevibacterium sp. R312, a coryneform strain producing nitrile hydratase and amidase. The transformation efficiency of the method is 10⁸ transformants per μg of plasmid under optimal conditions. Parameters optimised included field strength (11.8 kV cm⁻¹), pulse length (2.4 ms), plasmid DNA concentration (0.25 μg ml⁻¹ and cell density (10¹⁰ cells ml⁻¹). Surprisingly, the transformation efficiency did not vary with the growth stage, in contrast to results in the literature. A shuttle vector was constructed containing several unique cloning sites down-stream of the SP6 RNA polymerase promoter.     

Electroporation-mediated transient gene expression in isolated scutella of Hordeum vulgare

Electroporation was used to introduce DNA into excised scutella of immature embryos of Hordeum vulgare L. cvs Golden Promise and Delita. Using the firefly luciferase gene as reporter, parameters were analyzed for high transient gene expression while maintaining tissue viability. Enzymatic wounding was necessary for DNA uptake. The optimized protocol involves use of linearized DNA and addition of 15% (w/v) polyethylene glycol at a field strength of 950 V cm⁻¹ and approximately 56 ms pulse length. A one-day preculture was required for obtaining callus after electroporation. Transient gene expression was further demonstrated using the β-glucuronidase gene. Blue spots were detected at the abaxial scutellar surface, indicating that cells competent for somatic embryogenesis are also amenable to transfection by electroporation.     

Electrofusion of protoplasts from Aspergillus nidulans

Abstract Electrical parameters were determined and quantified for the stimulation of the optimum alignment and fusion of Aspergillus nidulans protoplasts. In a non-homogeneous alternating electrical field A. nidulans protoplasts aligned to form pearl chains associated with the electrodes of the fusion chamber. Most protoplasts were in pearl chains in an alignment field frequency of 3.0 MHz but maximum pair formation occurred at 1.0 MHz. At a field strength between 100 and 1000 V · cm⁻¹ pearl chain formation occurred with minimal protoplast rotation or lysis. The application of DC pulses resulted in protoplast fusion. Most fusion events were observed after two 500 V · cm⁻¹ DC pulses with a 0.5 s interpulse period. Using 1 × 10³ protoplasts · cm⁻³ in a 7 μm fusion chamber a maximum of 17.2 ± 2.0% fusion events were achieved.     

Physiological stress effects of continuous- and pulsed-DC electroshock on juvenile bull trout

Juvenile bull trout Salvelinus confluentus exposed to continuous- or pulsed-DC electroshock exhibited rapid elevations in plasma cortisol and glucose, but plasma chloride did not change. In a 1-h experiment using 240 V at 1·4 A of 60-Hz pulsed DC (voltage gradient 0·81 V cm⁻¹), which proved lethal, plasma cortisol and glucose rose significantly within 15 min of a 10-s electroshock. Plasma cortisol reached a peak level of 156 ± 18 ng ml⁻¹ at 45 min and then decreased, whereas plasma glucose reached its highest level of 179 ± 7·5mg dl⁻¹ at 1 h. In a 24-h experiment using lower dosages, plasma cortisol increased from 6·1-16 ng ml⁻¹ to peak levels of 155–161 ng ml⁻¹ in 1 h in response to a 10-s electroshock of continuous (130 V, 0·5 A, 1·45 V cm⁻¹) or pulsed (120 V, 0·5 A, 60 Hz, 0·55 V cm⁻¹) DC. Although plasma concentrations declined thereafter, levels remained above control values at 24 h. Plasma glucose was elevated from 60–65 to 120–134 mg dl⁻¹ after 1h by both electroshock treatments and remained near or above those levels for the 24-h duration. Plasma cortisol and glucose levels were much higher in electroshocked bull trout at 1 h compared with those in fish 1 h after receiving a 30-s handling stressor (cortisol, 90 ± 12 ng ml⁻¹; glucose, 82 ± 6·1 mg dl⁻¹). The results indicate that both continuous and pulsed DC were more stressful to juvenile bull trout than handling and that recovery, at least for pulsed DC, may take longer than 24 h.     

An efficient transformation system for Bifidobacterium spp.

This study describes a broad host transformation protocol that enables the uptake of plasmid DNA into 10 different species of Bifidobacterium , some of which have never been transformed before. The vector pNC7 (4·9 kb) was used to optimize the electroporation protocol. Transformation efficiencies ranged from 3·6×10⁻¹ to 1·2×10⁵ transformations per μg DNA. The impact of growth medium composition and electric field strength on transformation efficiency were independently optimized. Electrocompetent cells were grown in Iwata medium broth enriched with Actilight^RP 16%, harvested during the early exponential growth phase, and pulsed at 12·5 kV cm⁻¹, 100 Ω and 25 μF.     

Arginine utilization by Mycoplasma fermentans is not regulated by glucose metabolism: A 13C-NMR study

Abstract Electrofusion of protoplasts of two mutant strains of Hansenula polymorpha resulted in high fusion and hybrid yields when the calcium ions present in the conventional fusion medium replaced by zinc ions. The optimal fusion conditions were an alignment field of 0.4 kV cm⁻¹ strength and 2 MHz frequency for 30 s, followed by two consecutive pulses of 12 kV cm⁻¹ strength and 15 μs duration. With 0.05–0.1 mM zinc ions in the fusion medium an average clone number of 10⁴–10⁵ clones per 10⁸ input cells was reached. The presence of about 0.6 mM magnesium ions in the zinc fusion medium was essential.     

Electrotransformation of Streptococcus pneumoniae

Conditions of electroporation to transform encapsulated strains of Streptococcus pneumoniae with plasmid DNA have been defined. For a heavily encapsulated strain, an electroporation solution of 10–20% (v/v) glycerol and 3.2 kV cm^-1 field strength, 1000 Ω resistance and 25 μF capacitance were optimal. For lightly encapsulated or non-encapsulated strains, optimal conditions were a sucrose-based electroporation solution and 12.5 kV cm^-1 field strength, 200 Ω resistance and 25 μF capacitance.     

Reduction of Brochothrix thermosphacta on beef surfaces following immobilization of nisin in calcium alginate gels

Lean and adipose beef carcass tissues inoculated with Brochothrix thermosphacta (BT) (approx. 4.50 log₁₀ cfu cm⁻²) were left untreated (U) or treated with 100 μg ml⁻¹ nisin (N), calcium alginate (A) or 100 μg ml⁻¹ nisin immobilized in a calcium alginate gel (AN). Tissue samples were refrigerated after treatments and bacterial populations and nisin activity were determined at 0, 1, 2 and 7 d. U, A and N treatments of lean and adipose tissues did not suppress bacterial growth (>6 log₁₀ cfu cm⁻² by day 7) while treatments of lean and adipose tissues with AN suppressed bacteria (>2.42 log₁₀ cfu cm⁻² by day 7). Bacteriocin titres from both tissues were higher in AN vs N samples after the 7 d incubation. This study demonstrates that immobilization of nisin in a gel may be a more effective delivery system of a bacteriocin to the carcass surface than direct application.     

Quantitative study of electroporation-mediated molecular uptake and cell viability

Electroporation's use for laboratory transfection and clinical chemotherapy is limited by an incomplete understanding of the effects of electroporation parameters on molecular uptake and cell viability. To address this need, uptake of calcein and viability of DU 145 prostate cancer cells were quantified using flow cytometry for more than 200 different combinations of experimental conditions. The experimental parameters included field strength (0.1-3.3 kV/cm), pulse length (0.05-20 ms), number of pulses (1-10), calcein concentration (10-100 microM), and cell concentration (0.6-23% by volume). These data indicate that neither electrical charge nor energy was a good predictor of electroporation's effects. Instead, both uptake and viability showed a complex dependence on field strength, pulse length, and number of pulses. The effect of cell concentration was explained quantitatively by electric field perturbations caused by neighboring cells. Uptake was shown to vary linearly with external calcein concentration. This large quantitative data set may be used to optimize electroporation protocols, test theoretical models, and guide mechanistic interpretations.     

The cellular location and effect on nisin immunity of the NisI protein from Lactococcus lactis N8 expressed in Escherichia coli and L. lactis

Abstract Vibrio anguillarum and Pasteurella piscicida are Gram-negative bacteria which are pathogenic for marine fish and we report here the first successful transformation of these two bacteria by electroporation. The optimal conditions for electroporation included a field strength of 12.5 kV cm^t-1 and a time constant of 5 ms using 0.2-cm cuvettes. With these parameters, three plasmids (pSU2718, pCML, pEV3) with molecular sizes of 2.6, 5 and 13.7 kb, respectively were successfully transformed into both pathogens. V. anguillarum isolates belonging to serotypes O1 and O2 were transformed with greatest efficiency, 2.5 × 10³ transformants per μg DNA, being achieved in the serotype O2 strains using plasmid pCML. Strains of serotype O3 were not transformed. In the case of P. piscicida the maximum efficiency achieved was 9.8 × 10² transformants per μg pCML plasmid DNA. This optimized system will allow development of procedures for the genetic manipulation of these pathogens.     

Transient expression of electroporated gene in leaf protoplasts of indica rice and influence of template topology and vector sequences

Electroporation was used for gene delivery and evaluation of various parameters affecting transient expression of a gene for β-glucuronidase ( gus ) in leaf protoplasts of Oryza sativa var. Basmati-370. Transient expression was found to be dependent on voltage, capacitance, amount of plasmid and carrier DNA as well as period of culture. Maximum GUS activity was obtained when a 150 ms pulse at 300 V cm^-1 and 200 μF was applied to the protoplasts (l–2×10⁶ml⁻¹) in an electroporation buffer containing 20 μg of plasmid and 30 μg of calf thymus DNA, assayed 48 h after electroporation. DNA topology did not influence expression of the gene as both linear and supercoiled templates resulted in similar activities, but a 4-fold decrease in expression was observed if the gene was excised, reflecting the positive influence of vector sequences on gene expression.     

Inactivation of bacteria and yeasts on agar surfaces with high power Nd: YAG laser light

G.D. WARD, I.A. WATSON, D.E.S. STEWART-TULL, A.C. WARDLAW AND C.R. CHATWIN. 1996. Near infrared light from a high-powered, 1064 nm, Neodymium : Yttrium Aluminium Garnet (Nd : YAG) laser killed a variety of Gram-positive and Gramnegative bacteria and two yeasts, lawned on nutrient agar plates. A beam (crosssectional area, 1.65 cm²) of laser light was delivered in 10 J, 8 ms pulses at 10 Hz, in a series of exposure times. For each microbial species, a dose/response curve was obtained of area of inactivation vs energy density (J cm⁻²). The energy density that gave an inactivation area (IA) equal to 50% of the beam area was designated the IA₅₀-value and was plotted together with its 95% confidence limits. Average IA₅₀-values were all within a threefold range and varied from 1768 J cm⁻² for Serratia marcescens to 4489 J cm⁻² for vegetative cells of Bacillus stearothermophilus. There were no systematic differences in sensitivity attributable to cell shape, size, pigmentation or Gram reaction. At the lowest energy densities where inactivation was achieved for the majority of organisms (around 2000 J cm⁻²), no effect was observed on the nutrient agar surface, but as the energy density was increased, a depression in the agar surface was formed, followed by localized melting of the agar.     

Effects of osmolality and ions on the motility of stripped and testicular sperm of freshwater-and seawater-acclimated tilapia, Oreochromis mossambicus

A significantly higher concentration of testicular spermatozoa was obtained from freshwater Oreochromis mossambicus (9·9×10⁹ spermatozoa ml⁻¹) than seawater O. mossambicus (4·6×10⁹ spermatozoa ml⁻¹). The mean osmolality of the urine of freshwater fish (78·5 mOsmol kg⁻¹) was significantly different from that of seawater fish (304·8 mOsmol kg⁻¹). The mean length of the mid-piece of the spermatozoa together with the tail was more variable in freshwater O. mossambicus (8·80±0·23μm) than in seawater specimens (8·27±0·18 μm). Stripped sperm of freshwater O. mossambicus was highly contaminated by urine which was a good activator of sperm motility in O. mossambicus held in both fresh and sea water. The osmolality for initiation of motility in freshwater O. mossambicus spermatozoa was from 0 to 333 mOsmol kg⁻¹ while for seawater O. mossambicus spermatozoa it was from 0 to 1022 mOsmol kg⁻¹. The optimum osmolality for motility was from 70 to 333 mOsmol kg⁻¹ for freshwater O. mossambicus spermatozoa and from 333 to 645 mOsmol kg⁻¹ for seawater fish. In freshwater O. mossambicus spermatozoa, the presence of 20 mM CaCl₂ increased the permissive osmolality of NaCl from 184 to 645 mOsmol kg⁻¹. For seawater O. mossambicus spermatozoa, solutions of NaCl devoid of CaCl₂ were unable initiate motility, but the addition of 1·5 to 30 mM CaCl₂ to the NaCl solution (0–934 mOsmol kg₁) had a full motility initiating effect.     

In vitro susceptibility of Clostridium perfringens isolated from farm animals to growth-enhancing antibiotics

Minimal inhibitory concentration (MIC) determinations were carried out with seven growth-enhancing antibiotics against 95 Clostridium perfringens field isolates obtained during 1991 and 1992 from poultry, pigs and calves. All were resistant to 64 μg ml⁻¹ of the bambermycin antibiotic, flavomycin (flavophospholipol) and susceptible to avoparcin (MIC₉₀ 0.25 μg ml⁻¹), avilamycin (MIC₉₀ 0.5 μg ml⁻¹) and salinomycin (MIC₉₀≤ 0.12 μg ml⁻¹). Acquired resistance against bacitracin was detected in some isolates from poultry and bovines and resistance to tylosin and virginiamycin in some strains from all species investigated. Overall, the prevalence of resistance was comparable to the low levels recorded in 1979 in Cl. perfringens isolates from the same animal host species.     

Improved cloning vectors and transformation procedure for Lactococcus lactis

Four shuttle vectors (pMIG 1, 2, 2H and 3) have been constructed based on the broad host-range plasmid pCK1. All the pMIG vectors possess a multiple cloning site containing 12 or more unique restriction enzyme sites, and are stably maintained at either high or low copy number in Lactococcus lactis and in Escherichia coli. By cloning the E. coli pUC replicon into one of these vectors a plasmid was constructed which can replicate to high copy number in recA strains of E. coli. The broad host-range of the pCK1 replicon may enable these cloning vectors to be used in a number of Gram-positive bacteria. One of these vectors was used to optimize an electroporation procedure for transformation of a commonly used plasmid-cured strain MGI363 of L. lactis which routinely yielded 1 times 10⁷ to 5 times 10⁷ transformants μg^-1 supercoiled DNA using stored, snap-frozen cells. This transformation efficiency was obtained by growing the cells in medium containing the cell wall weakening agent glycine, to an upper limit of 2·5% w.v. Although growth of L. lactis strain MGI363 was inhibited by the use of 0·5 mol 1^-1 sucrose as an osmotic stabilizer, the presence of sucrose in the electroporation buffer was critical for high transformation efficiency. Other variables which were tested for their effect on the efficiency of transformation were cell concentration, DNA concentration, pulse time and field strength. These results provide a model procedure which can be followed to optimize conditions for the genetic transformation of various strains of L. lactis.     

Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis

The efficacy of high-temperature, short-time (HTST) pasteurization (72 °C/15 s) when low numbers (≤ 10³ cfu ml ⁻¹ ) of Mycobacterium paratuberculosis are present in milk was investigated. Raw cows' milk spiked with Myco. paratuberculosis (10³ cfu ml⁻¹, 10² cfu ml⁻¹, 10 cfu ml⁻¹, and 10 cfu 50 ml⁻¹) was subjected to HTST pasteurization using laboratory pasteurizing units. Ten bovine strains of Myco. paratuberculosis were tested in triplicate. Culture in BACTEC Middlebrook 12B radiometric medium detected acid-fast survivors in 14·8% and 10% of HTST-pasteurized milk samples at the 10³ and 10² cfu ml⁻¹ inoculum levels, respectively, whereas conventional culture on Herrold's egg yolk medium containing mycobactin J detected acid-fast survivors in only 3·7% and 6·7% of the same milk samples. IS900-based PCR confirmed that these acid-fast survivors were Myco. paratuberculosis . No viable Myco. paratuberculosis were isolated from HTST-pasteurized milk initially containing either 10 cfu ml⁻¹ or 10 cfu 50 ml⁻¹.     

Sequence of gonadal events and oestradiol levels in Sparus aurata (L.) under two photoperiod regimes

Individually tagged Sparus aurata were kept in tanks with running sea water (21°± 2°C) during their second and third years of life. Gonads were biopsied and blood was sampled at monthly intervals. Thirteen of 50 fish in the second year and 24 of 35 fish in the third year were phenotypically females. Fish kept under 16L/8D photoperiod from July 1981 to August 1982 did not reach maturation; waves of initial ovarian growth alternated with waves of atresia. When photoperiod was shortened as from August 1982, gonadal development commenced within a month and proceeded at a rate higher than that of the control fish reared under natural photoperiod. Under natural photoperiod oestradiol serum levels (E₂) were relatively low during the resting phase, May-October (108 ± 11 pg ml⁻¹), and during the late vitellogenic phase (502 ± 76 pg ml⁻¹). High levels (1669 ± 312 and 1240 ± 172pg ml⁻¹) occurred during the early vitellogenic and the maturational phases. The high level of E₂ during the spawning season of S. aurata is explained by earlier reports indicating a prolonged breeding season with daily release of eggs, and alternating daily surges of E₂ and 17α, 20β, dihydroxy-4-pregnen-3-one, possibly a 'maturational progestin' in this fish. In phenotypic males, E₂ was highest during early spermatogenic phases (745 ± 142pg ml⁻¹) but was low (155 ± 11 pg ml⁻¹) in males with running sperm.     

Transmission of stridulatory signals of the burrower bugs, Scaptocoris castanea and Scaptocoris carvalhoi (Heteroptera: Cydnidae) through the soil and soybean

Abstract.  Males and females of the burrower bug species Scaptocoris castanea Perty and Scaptocoris carvalhoi Becker emit stridulatory signals when on the roots of soybean. The substrate-borne components of the signal can be recorded on the plant but not on the surrounding soil surface. The stridulatory apparatus is composed of the tergal plectrum (lima) and the stridulitrum (stridulatory vein) on the underside of the hind wings. The male plectrum has one ridge and the female lima has 13 ridges. Stridulitra of different species differ in the length and in the number of teeth. Rubbing of plectrum (lima) ridges over the stridulitrum in one or both directions produces pulse trains. The velocity of signals that are recorded less than 0.5 cm from the bug is below 0.013 mm s⁻¹ on the soil and below 0.066 mm s⁻¹ on the leaf surface. Broadband spectra have a dominant frequency of less than 1 kHz and subdominant peaks extending up to 7 kHz. The dominant frequency of the stridulatory signal transmitted through a plant decreases together with the proportion of its higher frequency spectral components. Signals are attenuated for 3–9 dB cm⁻¹ when transmitted through the soil or soybean leaf and for approximately 1 dB cm⁻¹ when transmitted through soybean stem.     

Chemical and electroporated transformation of Edwardsiella ictaluri using three different plasmids

Transfer of DNA by conjugation has been the method generally used for genetic manipulation of Edwardsiella ictaluri because, previously, attempts to transform E. ictaluri by the uptake of naked DNA have apparently failed. We report here the successful transformation of seven strains of E. ictaluri using electroporation and two different chemical procedures [conventional calcium chloride (CaCl₂) and 'one-step' (polyethylene glycol, dimethyl sulfoxide and MgSO₄) protocols]. Seven strains of E. ictaluri were transformed using three different plasmids [pZsGreen, pUC18 and pET-30a(+)]. The highest transformation efficiency was achieved by electroporation (5.5±0.2 × 10⁴ transformants ng⁻¹ plasmid DNA) than with the CaCl₂ (8.1±6.1 × 10⁻¹ transformants ng⁻¹ plasmid) and the 'one-step transformation' protocol (2.5±2.7 transformants ng⁻¹ plasmid). An efficient transformation by electroporation required only 0.2 ng of plasmid compared with 200 ng required for the CaCl₂ and one-step protocols. The plasmids were stably maintained in E. ictaluri grown in the presence of antibiotic for 12 or more passages. The results of this study show that transformation of E. ictaluri by electroporation can be routinely used for the molecular genetic manipulation of this organism, and is a quicker and easier method than transformation performed by conjugation.     

Real-time quantification of Pseudomonas fluorescens cell removal from glass surfaces due to bacteriophage varphiS1 application

Aims:  To study the efficacy of the lytic phage φS1 in eliminating Pseudomonas fluorescens in the early stage of biofilm formation, using an in situ and real time methodology for cell quantification.
Methods and Results:  Cell adhesion and phage infection studies were carried out in a parallel plate flow chamber under laminar conditions. Cells were allowed to adhere until reaching 1·7–1·8 × 10⁶ cells cm⁻² and phage infection was performed with two different phage concentrations (2 × 10⁹ PFU ml⁻¹ and 1 × 10¹⁰ PFU ml⁻¹). Phage concentration clearly affects the speed of infection. The less concentrated phage solution promoted a three times slower rate of cell removal but did not affect the overall percentage of cell removal. In fact, after a longer infection period the less concentrated phage solution reached the same 93% cell removal value.
Conclusions:  Phages are efficient in the eradication of bacterial cells at the early stage of biofilm formation and their presence at the surface did not allow bacterial recolonization of the surface.
Significance and Impact of the Study:  To date, no published studies have been made concerning in situ and real time quantification of cell removal from surfaces due to phage action.