ANALYTICAL

BIOCHEMISTRY

189,

75-79

(1990)

Transformation of Pseudomonas aerughosa by Electroporation’ Jonathan

M. Diver,2

Department

of Microbiology

Received

January

Larry

E. Bryan,

and Infectious

and Pamela

A. Sokol

Diseases, University

of Calgary,

Calgary, Alberta,

T2N 4Nl

Canada

23,199O

Optimum conditions were defined for the electrotransformation of Pseudomonas aeruginosa PA01 with plasmid pLAFR1, resulting in a 1500-fold increase in transformation efficiency compared to conventional chemical transformation with MgCl,. In addition, PA0236 and two out of three recent clinical isolates of P. aeruginosa from the sputum of cystic fibrosis patients were successfully transformed with plasmid pUC 19 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% did not significantly affect the transformation efficiency. The clinical isolates tested had lower transformation efficiences than PA0 1. o ISSO Academic Press, IOC.

Transformation of bacteria with DNA is an important tool in molecular biology. In a few species (e.g., Haemophilus influenzae, Streptococcus pneumoniae, and Bacillus subtilis), natural competence has been described (1). In addition, laboratory techniques such as the CaCl, method for transforming Escherichia coli (2), and the PEG3 method for B. subtilis protoplasts (3) have been developed to induce competence. However, many bacterial species cannot be made competent. Recently the technique of electroporation has been used to induce competence in various bacterial species (4-11). This technique uses a pulse-treatment of cells with a high intensity electric field to reversibly generate pores in the membrane, allowing the entry of DNA. The

i We acknowledge the financial support of the Canadian Cystic brosis Foundation through Research and Development Program grant, and Grant 1568. J.M.D. is a CCFF Research Fellow. ’ To whom correspondence should be addressed. 3 Abbreviations used: PEG, polyethylene glycol; MIC, minimum hibitory concentration; HGEB, Hepes-glycerol electroporation buffer; SMEB, sucrose-magnesium electroporation buffer. 0003-2697/90 $3.00 Copyright 0 1990 hy Academic Press, Inc. All rights of reproduction in any form reserved.

Fi2

in-

equipment for the generation of such electric fields is commercially available. Pseudomonas aeruginosa is an important human pathogen causing infections of the urinary tract, burns, other wounds, and the chronic lung infections found in patients suffering from cystic fibrosis (12). The organism is difficult to eradicate from infected sites due mainly to its intrinsic resistance to antimicrobial agents (13). During the study of antibiotic resistance mechanisms in recent P. aeruginosa isolates from lung sputum of cystic fibrosis patients, it was decided to attempt transformation by electroporation after attempts at chemical treatments (MgCl,) had been unsuccessful. Electrotransformation has been previously described for laboratory strains of Pseudomonas putida (7,8), but to our knowledge not for either laboratory strains or recent clinical isolates of P. aeruginosa. In this study we investigated the ability of P. aeruginosa to be electrotransformed with plasmid DNA. Electroporation conditions were optimized for the standard laboratory strain PA01 and then applied to recent clinical isolates. Additional experiments were performed to measure the effect of storing the cells at -70°C prior to electroporation. MATERIALS

AND

METHODS

Bacterial strains and plasmiok P. aeruginosa PA01 (14) and its nalidixic acid-resistant derivative PA0236 (15) were used as standard laboratory strains. Strains U4973, X5111, and Y4492 were isolated from sputum samples from cystic fibrosis patients undergoing therapy with the fluoroquinolone antimicrobial agent, ciprofloxacin. These strains were of interest because they had become ciprofloxacin-resistant during therapy. The plasmids used for electrotransformation were the 4.6kb pUC19 1.8 (16) and the 21.6-kb pLAFR1 (17), expressing resistance to carbenicillin and tetracycline, respectively. Plasmid pUC19 1.8 consists of a 1.8-kb Pstl fragment of plasmid RSF 1010 inserted into pUC19, 75

76

DIVER,

BRYAN,

conferring plasmid stability in both E. coli and P. aeruginosa (16). Large-scale plasmid DNA preparations from E. coli hosts were performed with standard CsCl-ethidium bromide gradient centrifugation (18). As the final step, samples were dialyzed against 10 mM Tris-HCl, 1 mM EDTA (pH 8.0) buffer overnight. Some of this buffer was autoclaved and stored to be used as a control in cell samples electroporated without DNA. For rapid smallscale isolations of plasmid DNA from P. aeruginosa cultures, an alkaline-SDS lysis method was used (19). Electroporation apparatus, Electroporation was performed with a Gene Pulser (Bio-Rad Laboratories, Richmond, CA), set to discharge a 25-PF capacitor through a sample of cells at an initial voltage of between 1 and 12.5 kV/cm, in conjunction with a Pulse Controller unit (Bio-Rad) containing a high power, 20-Q resistor in series with the sample, and a selection of resistors of 100 to 1000 Q in parallel with the sample. Varying the parallel resistors allowed control of the “time constant” (time for a given pulse to decline to 37% from its initial setting). Preparation of cells and electroporation conditions. A 5-ml sample of an overnight culture of cells in L broth was inoculated into 100 ml of fresh L broth and incubated at 37°C with shaking until the late logarithmic phase (ABS,, = 0.5-1.0). Cells were immediately placed on ice and harvested by centrifugation. The pellet was then washed three times with either (a) 1 mM Hepes, pH 7.0, followed by a final resuspension in 1 mM Hepes containing 10% glycerol (HGEB); or (b) 1 mM Hepes, pH 7.0, containing 300 mM sucrose and 1 mM MgCl, (SMEB). All cell-washing procedures were performed on ice. A sample (50 ~1) of the final resuspension (volume, 500-800 ~1) was removed and used to determine the viable cell count (between lOa and lOlo cfu/ml, depending on the strain used). Aliquots of 100 ~1 were placed in 1.5-ml polypropylene tubes and between 1 and 10 ~1 of plasmid DNA added. The same volume of buffer was added to a control tube. Tubes were kept on ice for 1 min and then the cells were transferred to a prechilled, sterile electroporation cuvette (0.2-cm electrode gap) and placed in the electroporation apparatus. The electrical setting in all experiments other than initial optimization experiments were as follows: set voltage, 2.5 kV (12.5 kV/cm); discharge capacitor, 25 pF; and pulse controller parallel resistor, 200 or 400 Q (giving a theoretical time constant of 5 or 10 ms, respectively). The capacitor was then discharged through the sample. Immediately after discharge, 900 ~1 of ice-cold SOC medium (2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl,, 10 mM MgSO,, 20 mM glucose) was added directly to the cuvette and mixed carefully with the sample. Samples were transferred back to their polypropylene tubes and incubated

AND SOKOL

on ice for 30 min. In experiments with pLAFR1 (tet’), cells were incubated at 37°C for 2 h prior to plating onto L agar containing 100 pg/ml tetracycline. For pUC19 1.8, cells were plated immediately onto L agar containing 1000 pg/ml carbenicillin. In addition, aliquots were removed, diluted into PBS, and plated onto L agar to determine the viable cell count. All plates were incubated for up to 48 h at 37°C. As additional controls, DNA or buffer was added to two aliquots of cells which were not electroporated. These samples acted as controls for possible cell death, or spontaneous transformation, in the absence of electroporation. Results were expressed as either transformation efficiency (number of transformants/pg DNA) or as transformation frequency (percentage of post-pulse viable cells that were transformed). Transformation of PA01 by chemical treatment. To compare the difference in transformation efficiency using electrotransformation with a conventional chemical method, strain PA01 was transformed by treatment with MgCl, (17). Cells were grown as for electrotransformation, harvested, washed, and concentrated lo-fold in 0.15 M MgCl,. pLAFR1 (0.22 pg) was then added to 500-111aliquots of cells and incubated on ice for 1 h. The cells were then heat-pulsed at 37°C for 3 min, 500 ~1of L broth was added, and the resulting sample was incubated at 37°C for 2 h. Transformants were selected by plating on L agar containing 100 pg/ml tetracycline. RESULTS Optimization experiments for the electrotransformation of PA01 with pLAFR1. Initial experiments were performed to determine the optimum electroporation conditions for P. aeruginosa PA01 with plasmid pLAFR1. All possible permutations of the electrical variables (applied voltage, discharge capacitor size, and pulse controller resistance setting [time constant variation]) were not attempted due to the expense of the electroporation cuvettes. In particular, the discharge capacitor was set at the maximum 25 PF throughout, as little is known about altering this variable for other bacterial species. The effect of alteration of the applied voltage on transformation efficiency was examined. Applied voltage was varied between 0.4 and 2.5 kV (field strength, 2 to 12.5 kV/cm). The pulse controller resistor was set at 200 0, giving a predicted time constant of 5 ms. The actual time constant remained fairly stable for each voltage setting (mean = 4.8 f 0.75). Figure 1 shows that the transformation efficiency increased as voltage was increased up to the maximum of 12.5 kV. Below 6 kV/cm, no transformants were detected. It appears that a threshold field strength must be applied to the cells before electrotransformation occurs. Determination of the viable cell count after electroporation showed that

‘.,, ‘.., % ‘l, -80 1 ,o. : “.j,, -60 1 ‘... F I ‘.A. ; xELECTROTRANSFORMATION

lost

77

aeruginosa

-4Og

.s

103

'..

fc

Pseudomonas

1100

'..,

i

OF

or0 0

20

c 1

2

4 Field

6 Strength

8 (KV/an)

10

10'

12

1

IO21 0

5

10 DNA @g/ml

I 15

IlO4 20

x IO“)

FIG. 1. Effect of variation in field strength on transformation efficiency (A) and post-pulse cell survival (A) of PA01 with plasmid pLAFR1 in HGEB. Parallel resistor setting on Pulse Controller, 200 R.

FIG. 3. Effect of variation in sample DNA concentration on transformation efficiency (A) and transformation frequency (A) of PA01 with plasmid pLAFR1 in HGEB. Field strength setting, 12.5 kV/cm; parallel resistor setting, 200 a.

cell viability was unaffected by field strengths below the 6 kV/cm threshold, but rapidly decreased at higher field strengths, leaving only 15% of cells viable at 12.5 kV/ cm. For all other experiments, the applied voltage was set at the maximum of 2.5 kV (12.5 kV/cm). We next examined the effect of alteration in pulse length (time constant) by altering the size of the resistor placed in parallel with the sample in the Pulse Controller. Figure 2 shows that between resistance settings of 100 and 400 R (giving measured time constants of 3.3 and 12.1 ms, respectively), there was little effect on the transformation efficiency. At the maximum resistance setting of 1000 Q, arcing occurred in the cuvette, resulting in a time constant much shorter than predicted. Cell viability decreased as the time constant increased, only 4% of the cells being viable at 20.7 ms (800 a). It seems likely that the drop in transformation efficiency above

the time constant of 12.1 ms (400 Q) is due to excessive numbers of cells being killed by the pulse. Further optimization experiments were performed at a resistance setting of 200 Q (predicted time constant = 10 ms); however, subsequent experiments using different buffers, bacterial strains, and plasmids were performed at 400 Q (predicted time constant = 10 ms), as it was noted that the actual time constant was often lower than predicted. Figure 3 shows the effect of varying the DNA concentration in the cell suspension over a lo-fold range. A preparation of pLAFR1 was added to 100 ~1 of cells in volumes ranging from 1 to 10 ~1. The transformation efficiency remained relatively constant while the transformation frequency (percentage of post-pulse viable cells transformed) increased roughly in proportion to the DNA concentration. These data indicate that the amount of DNA added to the cells determines the probability of a cell becoming transformed. The increased volume of buffer added along with the DNA did not significantly affect the time constant (data not shown). As a final optimization experiment, the influence of a different electroporation and washing buffer was examined. In addition, the transformation efficiency obtained with plasmid pLAFR1 was compared with pUC19 1.8. Table 1 shows that the use of SMEB instead of HGEB resulted in a g-fold increase in transformation efficiency for pLAFR1 and a 38-fold increase for pUC19 1.8. Examination of viable cell counts showed that this effect was not due to increased survival of the cells in SMEB (data not shown). The use of SMEB resulted in a decrease in the measured time constant compared to HGEB, most likely due to the 1 mM MgCl, increasing the conductivity of the cell suspension. As a result of optimization of electroporation conditions and buffers, transformation efficiences for PA01 up to 1.5 X lo5 were achieved. This compares with a

Parallel

Flesktor

nma

Constant

setting

(n)

(ms)

FIG. 2. Effect of variation of time constant on transformation efficiency (A) and post-pulse survival (A) of PA01 with plasmid pLAFR1 in HGEB. Field strength setting, 12.5 kV/cm (2.5 kV applied voltage).

78

DIVER, TABLE

BRYAN,

1

Influence of Electroporation Buffer Composition on Transformation of PseudomonasaeruginosaPA01 with Plasmids pLAFR1 and pUC19 1.8 Transformants/pg Buffer used HGEB” SMEB b

Time constant” (ms) 11.9 8.3

pLAFR1 1.3 x 10” 1.2 x lo5

DNAd puc19

1.8

3.9 x lo3 1.5 x lo5

’ Hepes-glycerol electroporation buffer. b Hepes-sucrose-magnesium electroporation buffer. ’ Parallel resistor setting on Pulse Controller, 400 R. d 2 pg of 0.022 pg//.d pLAFR1 and 2 pl of 0.66 &g/p1 pUC19 1.8 added to 100 ~1 cells.

transformation transformation transformation

efficiency of 1.0 X 10’ when chemical using MgCl, was performed. The mean

efficiency for PA01 with pLAFR1 (using HGEB and a 200-Q parallel resistor setting) was 2.9 X lo4 (SD = 2.5 X 104) using cells prepared on different days.

The effect of freezing and storing the cells concentrated in SMEB prior to electroporation was examined. A 200-ml sample of PA01 cells was washed and concentrated in SMEB as usual. The transformation efficiency with pUC19 1.8 was then determined on a portion of unfrozen cells. The remainder of cells were divided and stored at -70°C for periods up to 4 weeks. The transformation efficiency dropped from 1.5 x lo5 to 4.7 X lo4 transformants/pg DNA after storage for 4 weeks at -7O”C, indicating that it would be possible to prepare a large batch of cells and store for later use. Electrotransformation of different strains of P. aeruginosa with pUCl9 1.8. Three recent isolates of P. aeruginosa, U4973, X5111, and Y4492, were chosen to test the applicability of the electrotransformation protocol optimized for PAOl. In addition, PA0236 was tested as another representative of the PA0 series. Electrotransformation was performed with HGEB and SMEB at settings of 400 D resistance, 25 PF capacitance, and 2.5 kV applied voltage. The results are shown in Table 2. Transformation was successful in U4973, X5111, and PA0236, but the transformation efficiency was less than PAOl. The use of SMEB was found to give a higher transformation efficiency in X5111 but not in U4973. Analysis of viable count data (not shown) showed that when using HGEB, cells of both X5111 and Y4492 underwent spontaneous loss of viability likely due to cell lysis in the low ionic strength medium. This problem was not encountered with PA01 or U4973. Use of SMEB reduced the problem of cell lysis, resulting in higher transformation efficiency for X5111. Y4492

AND SOKOL

could not be transformed conditions tried.

with pUC19 1.8 under any

DISCUSSION

Transformation of P. aeruginosa is possible by treating cells with CaCl, or MgCl,, and with PA02, transformation efficiencies of 1.3 X lo5 transformants/pg DNA have been reported (17). We successfully transformed PA01 by the MgCl, method, but transformation efficiencies were lower than those reported for PA02. With recent clinical isolates from sputum of cystic fibrosis patients, however, transformation was unsuccessful. The reason for this is unknown. It was decided to attempt electrotransformation on these strains. Transformation efficiencies by electroporation of between 10’ and lOlo transformants/pg DNA have been reported for E. coli (10). For other gram-negative bacteria, transformation efficiencies have been much lower and over a wider range. Enterobacteriaceae other than E. coli were reported to be transformed at transformation efficiencies ranging from 5 X lo1 for Enterobacter aerogenes to 6 X lo3 for Proteus mirabilis (7). Campylobatter jejuni was reported to transform at transformation efficiencies of 1.2 X lo6 transformants/pg DNA (6). Some species including Salmonella typhimurium, Klebsiella oxytoca, and Hafnia aluei were reported as untransformable, although conditions were not optimized for each species (7). For Pseudomonas species including P. putida, transformation efficiencies ranged from 7.6 X 10’ to 9.0 X lo4 transformants/pg DNA (7). The transformation efficiencies obtained in this study using the procedure optimized for P. aeruginosa PA01 are of this magnitude. In our hands, the use of electrotransformation compared to MgCl,-induced transformation resulted in a 1500-fold increase in transformation effi-

TABLE

Strain u4973 x5111

Y4492 PA0236

2

of Three Clinical aeruginosaand PA0236

Isolates of Pseudomonas with pUCl9 1.8

Buffer

Transformation efficiency” (transformants/pg DNA)

Transformation

HGEB SMEB HGEB SMEB HGEB SMEB HGEB SMEB

4.7 x lo2 1.9 x lo* 7.0 x 10’ 2.8 X 10%

0 0 7.6 x lOa NDb

DParallel resistor setting, 400 Q. For U4973 and PA0236, 2 pl of 0.66 rg/Nl pUC19 1.8 added, for X5111 and Y4492, 10 ~1 of same preparation added. * ND, not done.

ELECTROTRANSFORMATION

ciency for PAOl, and successful transformation of two out of three clinical isolates untransformable with MgCl,. Optimization experiments with PA01 showed that the most important electrical variable in terms of increasing the transformation efficiency was the applied voltage. Altering the length of the pulse (time constant) had less effect on transformation efficiency. As reported by others (5,8,10), some loss of cell viability was necessary for optimal transformation. Our data suggest that it may be necessary to kill between 80 and 95% of the cells for optimum transformation efficiency. These results indicate that increasing the initial concentration of cells in the sample should increase the probability of detecting transformants. Increasing the concentration of DNA in the sample should also increase the number of cells transformed (i.e., increase the transformation frequency) but have little effect on transformation efficiency. This prediction was confirmed by our data over the narrow DNA concentration range tested. The composition of electroporation buffer also had a large effect on transformation efficiency for PA01 and X5111. The use of SMEB instead of HGEB increased transformation efficiencies for pUC19 1.8 by 38- and 40-fold, respectively. It is unknown whether the sucrose or the magnesium ions (or both) were responsible for these increases in transformation efficiency. It is possible that the magnesium ions acted to destablize the cross-linking between adjacent lipopolysaccharide molecules in the outer membrane (the basis of the MgCl, method of transformation), thus allowing enhanced entry of DNA. However, the presence of metal ions (including magnesium) during the electroporation of C. iejuni was shown to reduce transformation efficiency (6). The use of SMEB reduced the problem of spontaneous lysis encountered when X5111 and Y4492 were washed in HGEB, offering the alternative explanation of increased cell survival as the possible reason for the increased transformation efficiency in X51 11. In summary, the optimized electrical parameters for electrotransformation of P. aeruginosa PA01 were very

OF

Pseudomonas

79

aeruginosa

similar to those reported for other bacterial species (611). The use of electroporation buffers containing additional ellectrically conducting materials such as MgCl, is not recommended by the manufacturers when the Pulse Controller unit is used. However, this report shows that significant improvements in transformation efficiencies can be made by addition of MgCl, up to 1 mM without affecting measured electrical parameters such as the time constant. REFERENCES 1. Goodgal, 2. Mandel,

S. H. (1982) Annu. Reu. Genet. M., and Higa, A. (1970) J. Mol.

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S., and Cohen,

S. N. (1979)

4. Chassey, B. M., and Flickinger, 44,173-177. 5. Calvin, B. M., and Hanawalt, 2801. 6. Miller, Acad.

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9. McIntyre, D., and Harlander, S. (1989) Appl. Enuiron. Microbial. 55,604-610. 10. Dower, W. J., Miller, J. F., and Ragsdale, C. W. (1988) Nucleic Acids Res. 16,6127-6145. 11. Wen-jun,

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12. George, R. H. (1987) Arch. Dis. Child. 62,438-439. 13. Hancock, R. E. W. (1986) J. Antimicrob. Chemother. l&653-659. 14. Holloway, B. W., Krishnapillai, V., and Morgan, A. F. (1979) Microbiol. Rev. 43, 73-102. 15. Haas, D., and Holloway, B. W. (1976) Mol. Gen. Genet. 144,243251. 16. Frank, 4483.

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17. Friedman, A. M., Long, S. R., Brown, S. E., Buikema, W. J., and Ausubel, F. M. (1982) Gene l&289-296. 18. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, Cold Spring Harbor, NY. 19. Takahashi, 613.

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Transformation of Pseudomonas aeruginosa by electroporation.

Optimum conditions were defined for the electrotransformation of Pseudomonas aeruginosa PAO1 with plasmid pLAFR1, resulting in a 1500-fold increase in...
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