Mol Gen Genet (1990) 221:287-290
© Springer-Verlag 1990
Electroporation of Bradyrhizobiumjaponicum
Mary Lou Guerinot, Barbara Anne Morisseau, and Taryn Klapatcb
Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA
Summary. Electroporation offers a fast, efficient and repro-
ducible way to introduce D N A into bacteria. We have suc-
cessfully used this technique to transform two commercially
important strains of Bradyrhizobium japonicum, the nitro-
gen-fixing soybean symbiont. Initially, electroporation con-
ditions were optimized using plasmid D N A which had been
prepared from the same B. japonicum strain into which the
D N A was to be transformed. Efficiencies of 105--106 trans-
formants/gg D N A were obtained for strains USDA 110
and 61A152 with ready-to-use frozen cells. Successful elec-
troporation of B. japonicum with plasmid D N A prepared
from Escheriehia coli varied with the E. coli strain from
which the plasmid was purified. The highest transformation
efficiencies (104 transformantsAtg DNA) were obtained us-
ing D N A prepared from a dcm-dam- strain ofE. coli. This
suggests that routine isolation of D N A from an E. coli
strain incapable of D N A modification should help in in-
creasing transformation efficiencies for other strains of bac-
teria where D N A restriction appears to be a significant
obstacle to successful transformation. We have also moni-
tored the rate of spontaneous mutation in electroporated
cells and saw no significant difference in the frequency of
streptomycin resistance for electroporated cells compared
to control cells.
Key words: Bradyrhizobium japonicum Transformation -
Electroporation - DNA modification
Introduction
Bradyrhizobium japonicum, the nitrogen-fixing symbiont of
soybeans, has been refractory to genetic transformation by
classical methods. Introduction of D N A into this species
has had to rely solely on conjugal matings with Escherichia
coli (e.g. Ditta et al. 1980). Using conjugation, researchers
have developed successful protocols for transposon muta-
genesis (Horn et al. 1984) and gene replacements (Hahn and
Hennecke 1984; Guerinot and Chelm 1986) but such proce-
dures are time-consuming and require the use of fresh cul-
tures of both B. japonicum and various E. coli strains. Elec-
troporation, which uses high intensity electric fields to re-
versibly permeabilize membranes, has now been reported
as a method for transforming a number of bacterial species,
including Agrobacterium tumefaciens which is closely re-
Offprint requests to: M.L. Guerinot
lated to B. japonicum (Mattanovich et al. 1989; Wirth et al.
1989). Although many of the reports involve the use of
freshly prepared cells (Chassy and Flickinger 1987; Allen
and Blaschek 1988; Aukrust and Nes 1988; Calvin and
Hanawalt 1988; Somkuti and Steinberg 1988; Miller et al.
1988; Taketo 1988; Luchansky et al. 1988; Belliveau and
Trevors 1989; David et al. 1989; Kim and Blaschek 1989;
McIntyre and Harlander 1989), protocols involving ready-
to-use frozen cells have been developed for several species
including E. coli (Dower et al. 1988), Bacillus thuringiensis
(Lereclus et al. 1989; Masson et al. 1989; Mahillon et al.
1989) and Agrobacterium spp. (Mattanovich et al. 1989).
Because electroporation offers a fast, reliable method for
D N A transformation, we set out to optimize conditions
for the introduction of plasmid D N A into two different
strains of B. japonicum via electroporation. In this report,
we document the conditions for electroporation resulting
in efficiencies as high as 1 x 106 transformantsAtg D N A
using frozen cells of B. japonicum. We also present evidence
that successful electroporation of B. japonicum is dependent
on the E. coli strain from which the plasmid D N A is puri-
fied and that rates of spontaneous mutation are not altered
by electroporation.
Materials and methods
Bacterial strains and plasmids. B. japonicurn strain
U S D A l l 0 is the small colony derivative as described pre-
viously (Guerinot and Chelm 1986) and B. japonieum
61A152 was generously provided by Nitragin Co. (Mil-
waukee, Wis.). E. coli strains HB101 (pro leu thi lacY hsd20
endA recA rpsL20 ara-14 galK2 xyl-5 rntl-1 supE44),
ED8654 (lacY1 galK2 galT22 metB1 trpR55 hsdR514
supE44 supF58) and GM2163 (F- 2-ara-I4 leuB6 tonA31
lacY1 tsx-78 supE44 galK2 galT22 hisG4 rpsL136 xyl-5 mtl-
1 thi-I daml3::Tn9 dcm-6 hsdR2 mcrB1 mcrA) were used
as hosts for various plasmids. Plasmid pRK290 is a 20 kb
broad host range cloning vector carrying a tetracycline re-
sistance determinant (Ditta etal. 1980). pLAFR1 is a
21.6 kb cosmid derivative of pRK290 (Friedman et al.
1982). pLAFR3 is a derivative of pLAFR1 containing the
polylinker region from pUC8 (Staskawicz et al. 1987).
Preparation ofDNA. Plasmids were mobilized via conjuga-
tion from E. coli into B. japonicum strains U S D A I I 0 and
61A152 using pRK2013 as a helper plasmid (Guerinot and
Chelm 1986) or were transformed into E. coli strains using
288
a standard CaClz technique. Plasmid D N A was then puri-
fied from both E. coli and B. japonicum strains by CsC1
gradient centrifugation using an alkaline lysis procedure
(Garger et al. 1983).
Preparation of cells for electroporation. Cells of USDA 110
were grown in yeast extract xylose (YEX) medium (Adams
et al. /984). For strain 61A152, cells were grown in yeast
extract-mannitol (YEM) medium (Vincent 1970). The pH
of both media was adjusted to 6.8 before autoclaving. Cells
were incubated at 30 ° C with shaking. Cells at various opti-
cal densities (monitored at 600 nm) were chilled on ice and
harvested by centrifugation at 4000 x g for 15 rain at 4 ° C.
The pellet from 1 1 of cells was resuspended in 1 1 of cold,
sterile distilled water, centrifuged as above and resuspended
in 0.5 1 of cold, sterile distilled water. The cells were centri-
fuged, resuspended in 20 ml of cold, sterile 10% glycerol,
centrifuged and finally resuspended in 2 ml of cold, sterile
10% glycerol. This concentrated suspension of cells was
distributed in 40 gl aliquots, frozen in liquid nitrogen and
stored at - 7 0 ° C. Each aliquot contained 109-10 l° colony
forming units/ml.
Transformation protocol. Cells prepared as described above
were thawed on ice. D N A was added (final concentration
of D N A varied from 12 ng/ml to 4 gg/ml depending on
the experiment) and the mixture was vortexed. After 5 min,
the mixture was transferred to a sterile, prechilled cuvette
(inter-electrode distance of 0.2 cm) and placed in the Gene
Pulser apparatus equipped with a Pulse Controller to vary
the resistance (Bio-Rad Laboratories, Richmond, Calif.).
Varying voltages were applied at a constant capacitance
setting of 25 gF. The resistance was set to generate theoreti-
cal time constants of 5 msec or 20 msec. Following the
pulse, the cells were immediately diluted with 1 ml of either
YEX for USDA 110 or yeast extract glutamate-gluconate
(YEGG) medium (Guerinot and Chelm 1986) for 61A152.
The cells were then transferred to a tube and incubated
at 30 ° C with shaking for 20 h, approximately two cell dou-
blings for these slow-growing bacteria (in keeping with the
manufacturer's recommendations). At the end of the recov-
ery period, the cells were diluted as necessary in 0.0/%
Tween 80 and plated onto selective plates containing tetra-
cycline at 100 ~tg/ml for USDA 110 or at 200 gg/ml for
61A152. Dilutions were also plated onto non-selective
plates to assess cell survival. Colony hybridizations verified
that antibiotic resistant colonies represented transformed
cells. Controls where either the electric pulse or D N A was
omitted resulted in no transformants. To determine whether
electroporation increased the rate of spontaneous mutation,
cells were plated onto Y E G G plates containing streptomy-
cin (1 mg/ml) or rifampicin (0.5 mg/ml).
Results and discussion
Optimization of protocol
Plasmid D N A which had been prepared from the same
B. japonicum strain into which it was to be transformed
was used to optimize conditions for electroporation. This
avoided any possible problems with D N A restriction of
transforming D N A isolated from a heterologous source,
which had previously been reported to have a dramatic
effect on the efficiency of transformation by electroporation
in Campylobacter jejuni (Miller et al. 1988). As discussed
below, the use of plasmid D N A prepared from various
E. coli strains did result in lower transformation efficiencies
in B. japonieum. Using pLAFR1 D N A prepared from B.
japonieum, we compared the effectiveness of electric pulses
over a wide range of field strengths at two time constants.
A maximum transformation efficiency of 1.8 x 10 s transfor-
mants/gg D N A occurred at a field strength of 11.25 kV/cm
for USDA 110 with a pulse length of 20 msec. The maxi-
mum transformation efficiency (1.1 x 106/gg DNA) for
strain 61A152 occurred at a field strength of 12.5 kV/cm
with a pulse length of 5 msec. Mattanovich et al. (1989)
have reported a similar transformation efficiency (1 x/06/
ggDNA) for A. tumefaciens at the same field strength
(12.5 kV/cm) which is the highest obtainable with the Gene
Pulser unit. Other researchers have also reported maximum
transformation efficiencies at the highest voltage setting;
however many have used cuvettes with a 0.4 cm electrode
distance which gives a maximum field strength of 6.25 kV/
cm (e.g. Allen and Blaschek /988). In the experiments to
optimize electroporation conditions, 12 ng of D N A was
used with cells whose concentration was 109-101° cells/ml.
Both the D N A and cell concentration determine the fre-
quency of transformation. Thus, with the conditions stated,
the frequency of transformation ranged from 10- 6 to 10 - 7.
The mortality associated with the high field strengths neces-
sary for transformation of B. japonicum ranged from
20%-95%, with a greater percentage of the cells surviving
the shorter pulse (data not shown). The efficiency of trans-
formation was similar for cells in early to mid log phase
of growth for both strains and, as has been reported by
others, we saw a linear relationship between the amount
of D N A added and the total number of transformants (data
not shown).
Colonies were visible on the selective plates 5-7 days
after plating. This compares quite favorably to conjugal
mating procedures which take over 2 weeks to complete.
Clearly the ability to have frozen competent cells on hand
greatly facilitates molecular genetic studies of B. japonicum.
Frequency of spontaneous mutation
Because of the high mortalities associated with electropora-
tion of B. japonicum, we investigated whether the surviving
Table 1. Effect of electroporation on the frequency of spontaneous
mutation to streptomycin resistance in Bradyrhizobiumjaponicum
strains USDA lt0 and 61A152
Strain Frequency of spontaneous mutation a
(colony forming units x 10 - s)
Mean ± 95% Confidence interval
USDA tl0
Electroporated cells 48 32
Control cells 18 20
61A152
Electroporated cells 5.0 0.8
Control cells 5.1 1.6
a All the values in the table are averages of five independent trials.
Cells were electroporated at a field strength of 12.5 kV/cm, with
a pulse length of 5 msec

cells had a higher frequency of spontaneous mutation than
control cells which had not been electroporated (Table 1).
There is no difference in the frequency of mutation to strep-
tomycin resistance for either USDA 110 or 61A152, as can
be seen by a comparison of the 95% confidence intervals.
There was also no difference in the frequency of rifampicin
resistance (data not shown), Therefore, although electro-
potation results in lethality, it does not appear to be muta-
genic.
Use of DNA prepared in E. coli for electroporation of
B. japonicum
After optimizing the conditions for electroporation, we in-
vestigated the transformation of B. japonicum using D N A
which had been prepared from a variety of E. coli strains.
Table 2 allows a comparison of transformation efficiencies
for strains USDA 110 and 61A152 when transformed with
D N A prepared from different hosts. Results from trials
which gave less than 30 total transformants are reported
but it should be noted that these values are not as statisti-
cally reliable as those from more successful trials. Plasmid
D N A prepared from E, coli strain GM2163 gave transfor-
mation efficiencies 1-3 orders of magnitude greater than
those observed with D N A prepared from E. coli strains
ED8654 or HB101. These same D N A preparations were
all capable of efficiently transforming E. coli strains via
electroporation (data not shown). In C. jejuni, transforma-
tion was also decreased by at least 4 orders of magnitude
when the transforming D N A was prepared from E. coli
strain HB101 (Miller et al. 1988). This is in contrast to
reports by others that D N A prepared from E. coli strains
HBI01 (Kim and Blaschek 1989) or MC1061 (David et al.
1989) transformed cells of Clostridium petfringens or Leu-
conostoc paramesenteroides, respectively, just as efficiently
as D N A prepared from the strain to be transformed. There
is also a recent report by Langella and Chopin (1989) that
plasmid D N A escapes restriction by two different restric-
tion/modification systems following its transfer by electro-
Table2. Electroporation of Bradyrhizobium japonicum strains
USDA 110 and 61A152
Source" Relevant genotype Transformation
efficiencyb
USDA 110c 61A152°
GM2163 dam dcm- hsdM + 2.7 x 104 4.9 x 103
HB101 dam+dem + hsdM- 1.3 x 103 9× 102.
ED8654 dam+dcm+hsdM + 5x 10 o * 9× 101 *
U S D A 110 1.1 × 105 8× 102*
61A152 5x 102* 1.1 × 106
a Strain from which donor plasmid was extracted and subsequently
CsC1purified prior to electroporation. Plasmids pRK290, pLAFRI
and pLAFR3 all gave equivalent results
b Expressed as antibiotic resistant colonies recovered per gg plas-
mid DNA. Each value is an average of at least two trials. Values
from trials which gave less than 30 total transformants are indi-
cated by an asterisk (*)
Recipient strain for eleetroporation experiments. Cells of strain
USDA 110 were electroporated at a field strength of 11.25 kV/cm
with a pulse length of 20 msec and cells of strain 61A152 were
electroporated at a field strength of 12.5 kV/cm with a pulse length
of 5msec
289
poration into Lactococcus lactis subsp, lactis. Clearly, each
species to be transformed via electroporation should first
be transformed with D N A prepared from itself and then
tested for its receptivity to E. coli prepared DNA. Table 2
also shows that fewer transformants were detected when
plasmid D N A prepared from B. japonicum strain 61A152
was used to transform B. japonicum strain USDA 110 and
vice versa, relative to the numbers obtained with homolo-
gous DNA. These results taken together indicate that these
B. japonicum strains probably have D N A restriction/modi-
fication systems. The other two published reports on elec-
troporation of members of the Rhizobiaceae did not ad-
dress D N A restriction (Wirth et al. 1989; Mattanovich
et al. 1989).
At the present time we know very little about restriction/
modification systems in B. japonicum. Introduction of for-
eign D N A into bacterial cells frequently leads to restriction,
which results either from the absence or presence of modi-
fied bases in DNA. Because E. eoli has at least three systems
(mcrA, mcrB and mrr) which degrade D N A specifically
when D N A is methylated either at cytosine residues (Ra-
leigh and Wilson 1986) or at adenine residues (Heitman
and Model 1987), we reasoned that B. japonicum might
have similar systems. Therefore, the use of unmodified
D N A might increase transformation frequencies. In E. coli,
there are three different systems which are known to modify
DNA: the dam system (Geier and Modrich 1979), the dcm
system (May and Hattman 1975) and the EcoK or EcoB
system (Bickle 1982). As noted above, D N A prepared from
strain GM2163, which is deficient in both the dam and
dcm systems, gave the best transformation efficiencies with
both strains of B. japonicum (Table 2). Presumably an
E. coli strain deficient in all three known modification sys-
tems would be the best host for preparation of D N A to
be used in electroporation. Interestingly, Luchansky et al.
(1988) also reported successful electroporation of a number
of bacterial species using D N A prepared from a d a m - d c m -
strain of E. coli (JM110). However, they presented no data
for D N A prepared from other E. eoli strains and did not
discuss whether they felt the genotype of the E. eoli strain
used contributed to successful transformation.
In summary, electroporation offers a fast, efficient
method for introduction of D N A into B. japonicum. The
major advantages offered by the procedure presented here
are speed and simplicity. Our results suggest that routine
isolation of D N A from an E. coli strain incapable of D N A
modification might help in increasing electroporation effi-
ciencies for other strains of bacteria for which D N A restric-
tion appears to be a significant obstacle to successful trans-
formation.
Acknowledgements. We thank Elizabeth Raleigh for providing
E. coli strain GM2163, Marina Abate and Erin Connolly for help
in the beginning stages of this project and Rob McClung for criti-
cally reviewing the manuscript. This research was supported by
NSF grant DMB 8615190 to MLG. Friends of the Norris Cotton
Cancer Center generously provided funds for the purchase of the
Gene Pulser Apparatus.
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C o m m u n i c a t e d by H. Hennecke
Received November 8, 1989