Microbial Pathogenesis 1992 ; 12 : 63-68
Short communications Conditions for transformation of Pasteurella multocida by electroporation L . Jablonski, N . Sriranganathan,* S . M . Boyle and G . R . Carter Department of Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, U .S.A . (Received July 6, 1991 ; accepted in revised form September 4, 1991)
Jablonski, L . (Dept of Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, U .S .A .), N . Sriranganathan, S . M . Boyle and G . R . Carter . Conditions for transformation of Pasteurella multocida by electroporation . Microbial Pathogenesis 1992; 12 : 63-68 . Conditions for electroporation of plasmid DNA into Pasteurella multocida were determined for use in developing a cloning system to study virulence factors of P. multocida . The highest efficiency of transformation (1 .25x10' cfu/µg DNA) was obtained when 7 .6X10 10 cells of P . multocida strain R473 were electroporated at 12 .5 kV/cm (10 ms, 5 ng of pVM109) . Transformation efficiencies of cells prepared at mid-log-phase were approximately 0 .5 log 70 lower than early, late, or stationary phases . Neither pBR322 nor pUC-19 were able to transform strain R473 under these conditions, even when DNA concentrations were increased to 1 pg . When pBR322 was ligated with a Pasteurella plasmid, pLAR-1, the hybrid was able to transform strain R473 at an efficiency between 4 .5 x 10 2 and 8 x 10 4 cfu/µg DNA . Six strains of P. multocida including serotypes A, B, D, and E were transformed successfully . Key words: Pasteurella multocida ; electroporation ; plasmids.
Introduction Pasteurella multocida is the causative agent of fowl cholera, hemorraghic septicemia,
and snuffles in birds, cattle, and rabbits, respectively . Pasteurella multocida in association with Bordetella bronchiseptica causes atrophic rhinitis in swine .' Pasteurella multocida is a secondary pathogen associated with shipping fever, pneumonia, and abortion in cattle and sheep .2 In order to study the molecular basis of pathogenesis, an efficient means of transforming P. mu/tocida is necessary . Our laboratory has been unsuccessful using traditional methods 3 and therefore attempts to electroporate P. mu/tocida were undertaken . The results of this study describe optimum conditions for electroporation of P . multocida R473 (serotype B :2) with pVM109, a streptomycin, sulfonamideresistance plasmid isolated from P. multocida strain 1085 . In addition to strain R473, we were able to transform five strains of P. mu/tocida (strains 1062, P86-338, Bunia II, 3865 and 3867) . Lastly, we describe the successful transformation of P . * Author to whom correspondence should be addressed . 0882-4010/92/010063+06 $03 .00/0
© 1992 Academic Press Limited
L . Jablonski et al.
64
multocida with hybrid plasmids which replicate in both P . multocida and Escherichia coli .
Results Electric field strength To determine the optimal field strength, cells from late log-phase (260 Klett Units) were washed and concentrated to 10 10 colony forming units (cfu)/ml . Aliquots were electroporated between field strengths of 6 to 14 kV/cm (Fig . 1) . The maximum transformation frequency of 4 .5 x 10' transformants/µg of DNA was obtained at a field strength of 12 .5 kV/cm corresponding to 625 kV . Field strength is defined as the voltage applied multiplied by a factor of 0 .02 (BTX Operating Manual, p . 14) . Pulse time To determine the effect of pulse time on electroporation efficiency, 50 µl of cells (1 .9 x 1010 cfu/ml) were electroporated at a field strength of 12 .5 kV/cm while varying
0°
Z
10'
0 a
U
UJ
I I I , I I
lo, 4
6
8 10 12 Field strength (kV/cm)
14
16
Fig . 1 . Effect of field strength on electroporation efficiency . Cells were electroporated using the following modifications of Dower et al.' Overnight cultures were used to inoculate Brain Heart Infusion broth (BHI, Difco) to 1 per cent . Briefly, cultures grown to late log-phase, were incubated on ice for 15 min and then centrifuged for 15 min at 5000xg, 4°C . Cell pellets were rinsed twice in 1 mm Hepes buffer (pH 7 .0, 4°C) . After the final rinse, cell pellets were resuspended in 10% glycerol to 1/12 their original culture volume . The cell suspension was centrifuged at 5000xg for 10 min and resuspended in the remaining glycerol solution after the supernatant was decanted . Cells were aliquoted and frozen by immersion in dry ice and ethanol and then stored at -80'C . Prior to electroporation, 50 p1 of thawed cells were incubated with 5 ng of pVM109 plasmid for 5 min on ice . Cells were electroporated using a BTX Transfector (model 100, Biotechnologies and Experimental Research Inc ., San Diego, California; electrode no . 474, 0 .5 mm gap) while field strength was varied from 8 to 14 kV/cm . A pulse time of 10 ms was used throughout the experiment . As a control for the electroporation conditions, 50 pl of prepared cells were electroporated in the absence of pVM1 09 . Electroporated cells were allowed to recover in 950 µl of BHI for 2 h . Transformants were selected on BHI agar containing 35 pg/ml of streptomycin (Sigma Chemicals) . The efficiencies reported are the averages of three independent electroporations .
65
Electroporation of P. multocida
1 0,
1e z 0
Y T 1 C
108
Uu W 0
. .
0
1 .
. . .
10
1 .
20
. . . 1 . . .
. 1
. . . .
30 40 Pulse time (ms)
1 . . . .
50
60
Fig . 2 . Effect of pulsed time on the electroporation efficiency . Cells were prepared as described in Fig . 1 and electroporated at 625 kV for the pulse times indicated .
the pulse time . The highest transformation efficiency of 5 .25 x 10' transformants/µg of DNA was achieved at 10 ms (Fig . 2) . Cell concentration There was a direct correlation between cell concentration and electroporation efficiency (Table 1) . The highest efficiency of 1 .25x10' transformants/µg DNA was achieved at a cell concentration of 7 .6x10 10 cfu/ml . Transformants were not detected when cell concentrations were below 9 .8x 10' cfu/ml .
Table 1
Effect of cell concentration on trans-
formation efficiency Cell concentration (cfu/ml)
Transformation efficiency' (cfu/µg DNA)
7 .6x1010 7 .2x109 5 .7x108 9 .8x10' 2 .5 x 105
1 .25x10' 1 .56x10 6 1 .01 x10"
Short communications Conditions for transformation of Pasteurella multocida by electroporation L . Jablonski, N . Sriranganathan,* S . M . Boyle and G . R . Carter Department of Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, U .S.A . (Received July 6, 1991 ; accepted in revised form September 4, 1991)
Jablonski, L . (Dept of Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, U .S .A .), N . Sriranganathan, S . M . Boyle and G . R . Carter . Conditions for transformation of Pasteurella multocida by electroporation . Microbial Pathogenesis 1992; 12 : 63-68 . Conditions for electroporation of plasmid DNA into Pasteurella multocida were determined for use in developing a cloning system to study virulence factors of P. multocida . The highest efficiency of transformation (1 .25x10' cfu/µg DNA) was obtained when 7 .6X10 10 cells of P . multocida strain R473 were electroporated at 12 .5 kV/cm (10 ms, 5 ng of pVM109) . Transformation efficiencies of cells prepared at mid-log-phase were approximately 0 .5 log 70 lower than early, late, or stationary phases . Neither pBR322 nor pUC-19 were able to transform strain R473 under these conditions, even when DNA concentrations were increased to 1 pg . When pBR322 was ligated with a Pasteurella plasmid, pLAR-1, the hybrid was able to transform strain R473 at an efficiency between 4 .5 x 10 2 and 8 x 10 4 cfu/µg DNA . Six strains of P. multocida including serotypes A, B, D, and E were transformed successfully . Key words: Pasteurella multocida ; electroporation ; plasmids.
Introduction Pasteurella multocida is the causative agent of fowl cholera, hemorraghic septicemia,
and snuffles in birds, cattle, and rabbits, respectively . Pasteurella multocida in association with Bordetella bronchiseptica causes atrophic rhinitis in swine .' Pasteurella multocida is a secondary pathogen associated with shipping fever, pneumonia, and abortion in cattle and sheep .2 In order to study the molecular basis of pathogenesis, an efficient means of transforming P. mu/tocida is necessary . Our laboratory has been unsuccessful using traditional methods 3 and therefore attempts to electroporate P. mu/tocida were undertaken . The results of this study describe optimum conditions for electroporation of P . multocida R473 (serotype B :2) with pVM109, a streptomycin, sulfonamideresistance plasmid isolated from P. multocida strain 1085 . In addition to strain R473, we were able to transform five strains of P. mu/tocida (strains 1062, P86-338, Bunia II, 3865 and 3867) . Lastly, we describe the successful transformation of P . * Author to whom correspondence should be addressed . 0882-4010/92/010063+06 $03 .00/0
© 1992 Academic Press Limited
L . Jablonski et al.
64
multocida with hybrid plasmids which replicate in both P . multocida and Escherichia coli .
Results Electric field strength To determine the optimal field strength, cells from late log-phase (260 Klett Units) were washed and concentrated to 10 10 colony forming units (cfu)/ml . Aliquots were electroporated between field strengths of 6 to 14 kV/cm (Fig . 1) . The maximum transformation frequency of 4 .5 x 10' transformants/µg of DNA was obtained at a field strength of 12 .5 kV/cm corresponding to 625 kV . Field strength is defined as the voltage applied multiplied by a factor of 0 .02 (BTX Operating Manual, p . 14) . Pulse time To determine the effect of pulse time on electroporation efficiency, 50 µl of cells (1 .9 x 1010 cfu/ml) were electroporated at a field strength of 12 .5 kV/cm while varying
0°
Z
10'
0 a
U
UJ
I I I , I I
lo, 4
6
8 10 12 Field strength (kV/cm)
14
16
Fig . 1 . Effect of field strength on electroporation efficiency . Cells were electroporated using the following modifications of Dower et al.' Overnight cultures were used to inoculate Brain Heart Infusion broth (BHI, Difco) to 1 per cent . Briefly, cultures grown to late log-phase, were incubated on ice for 15 min and then centrifuged for 15 min at 5000xg, 4°C . Cell pellets were rinsed twice in 1 mm Hepes buffer (pH 7 .0, 4°C) . After the final rinse, cell pellets were resuspended in 10% glycerol to 1/12 their original culture volume . The cell suspension was centrifuged at 5000xg for 10 min and resuspended in the remaining glycerol solution after the supernatant was decanted . Cells were aliquoted and frozen by immersion in dry ice and ethanol and then stored at -80'C . Prior to electroporation, 50 p1 of thawed cells were incubated with 5 ng of pVM109 plasmid for 5 min on ice . Cells were electroporated using a BTX Transfector (model 100, Biotechnologies and Experimental Research Inc ., San Diego, California; electrode no . 474, 0 .5 mm gap) while field strength was varied from 8 to 14 kV/cm . A pulse time of 10 ms was used throughout the experiment . As a control for the electroporation conditions, 50 pl of prepared cells were electroporated in the absence of pVM1 09 . Electroporated cells were allowed to recover in 950 µl of BHI for 2 h . Transformants were selected on BHI agar containing 35 pg/ml of streptomycin (Sigma Chemicals) . The efficiencies reported are the averages of three independent electroporations .
65
Electroporation of P. multocida
1 0,
1e z 0
Y T 1 C
108
Uu W 0
. .
0
1 .
. . .
10
1 .
20
. . . 1 . . .
. 1
. . . .
30 40 Pulse time (ms)
1 . . . .
50
60
Fig . 2 . Effect of pulsed time on the electroporation efficiency . Cells were prepared as described in Fig . 1 and electroporated at 625 kV for the pulse times indicated .
the pulse time . The highest transformation efficiency of 5 .25 x 10' transformants/µg of DNA was achieved at 10 ms (Fig . 2) . Cell concentration There was a direct correlation between cell concentration and electroporation efficiency (Table 1) . The highest efficiency of 1 .25x10' transformants/µg DNA was achieved at a cell concentration of 7 .6x10 10 cfu/ml . Transformants were not detected when cell concentrations were below 9 .8x 10' cfu/ml .
Table 1
Effect of cell concentration on trans-
formation efficiency Cell concentration (cfu/ml)
Transformation efficiency' (cfu/µg DNA)
7 .6x1010 7 .2x109 5 .7x108 9 .8x10' 2 .5 x 105
1 .25x10' 1 .56x10 6 1 .01 x10"
