Analytical Biochemistry 409 (2011) 130137

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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

Enhancing electro-transformation competency of recalcitrant

Bacillus amyloliquefaciens by combining cell-wall weakening
and cell-membrane uidity disturbing
Guo-qiang Zhang a,b, Peng Bao a,c, Yun Zhang a, Ai-hua Deng a, Ning Chen c, Ting-yi Wen a,
a
b
c

Key Laboratory of Systems Microbial Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, 1 West Beichen Road, Chaoyang District, Beijing 100101, China
Graduate University of Chinese Academy of Sciences, Beijing 100049, China
College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China

a r t i c l e

i n f o

Article history:
Received 24 August 2010
Received in revised form 8 October 2010
Accepted 9 October 2010
Available online 14 October 2010
Keywords:
Recalcitrant Bacillus
Electro-transformation
Cell-wall weakening
Cell-membrane uidity disturbance

a b s t r a c t
Bacillus amyloliquefaciens has been a major workhorse for the production of a variety of commercially
important enzymes and metabolites for the past decades. Some subspecies of this bacterium are recalcitrant to exogenous DNA, and transformation with plasmid DNA is usually less efcient, thereby limiting
the genetic manipulation of the recalcitrant species. In this work, a methodology based on electro-transformation has been developed, in which the cells were grown in a semicomplex hypertonic medium, cell
walls were weakened by adding glycine (Gly) and DL-threonine (DL-Thr), and the cell-membrane uidity
was elevated by supplementing Tween 80. After optimization of the cell-loosening recipe by response
surface methodology (RSM), the transformation efciency reached 1.13 0.34  107 cfu/lg syngeneic
pUB110 DNA in a low conductivity electroporation buffer. Moreover, by temporary heat inactivation of
the host restriction enzyme, a transformation efciency of 8.94 0.77  105 cfu/lg DNA was achieved
with xenogeneic shuttle plasmids, a 103-fold increase compared to that reported previously. The optimized protocol was also applicable to other recalcitrant B. amyloliquefaciens strains used in this study.
This work could shed light on the functional genomics and subsequent strain improvement of the recalcitrant Bacillus, which are difcult to be transformed using conventional methods.
2010 Elsevier Inc. All rights reserved.

Bacillus amyloliquefaciens is a rod-shaped, endospore-forming,

Gram-positive bacterium, which widely exists in soil. It has been
the commercial producer of various enzymes including a-amylase
[1], levansucrase [2], brinolytic enzymes [3], and others. Since the
Bacillus genus exhibits a high ux of the pentose phosphate
pathway, B. amyloliquefaciens and its close relatives have also been
stable producers of purine nucleosides [4] and riboavin [5].
B. amyloliquefaciens also synthesizes various secondary metabolites
via the nonribosomal pathway, such as bioactive lipopeptides,
which have been applied to promote plant growth and suppress
a broad spectrum of pathogens [6]. The exhaustive use of
B. amyloliquefaciens in industrial fermentation and plant growth
promotion generated the need for genetic manipulation. Recently,
with the completion of the genome sequencing of rhizobacteria
B. amyloliquefaciens FZB42 [7], the bacterium has been pushed into
an era of functional genomics.
To gain insight into the genetic signicance of various
phenotypes, and for the purpose of strain improvement, genetic
manipulation of bacteria is an essential tool [8], with genetic

Corresponding author. Fax: +86 10 62522397.

E-mail address: wenty@im.ac.cn (T.-y. Wen).
0003-2697/$ - see front matter 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2010.10.013

transformation as the crucial step. Some B. amyloliquefaciens

strains can develop natural competence as found in Bacillus subtilis
[7,9], allowing automatic incorporation of DNA and subsequent
integration into their chromosomes at a high frequency [10],
whereas others do not have this capability. In addition, some B.
amyloliquefaciens strains are extremely recalcitrant to exogenous
DNA.
Electroporation is a universal and convenient technique for
transforming various bacteria efciently [11]. Since its introduction
to the genetics of Bacillus [1214], various methods have been
developed and optimized with hypertonic agents, pulse voltage,
and electroporation buffers, yielding different transformation
efciencies. For example, Xue et al. [15] reported that B. subtilis
and Bacillus licheniformis grown in a medium of high osmolarity
were transformed at a frequency of 1.4  106 and 1.8  104 cfu/lg
of plasmid DNA, respectively; using early growing stage cultures
and a high voltage, the electro-transformation efciency was up to
2  109 cfu/lg/ml for Bacillus cereus [16]. However, these protocols
are highly species or strain specic, and the efciency is relatively
variable for different strains even using the same protocol.
Methodologies for recalcitrant B. amyloliquefaciens transformation have been extended to protoplast transformation [17], phage
transduction [18], and electroporation [19] for the purpose of

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Electroporation of recalcitrant B. amyloliquefaciens / G.-q. Zhang et al. / Anal. Biochem. 409 (2011) 130137
Table 1
Bacterial strains and plasmids used in this study.
Strains or plasmids
Strains
E. coli INV110
B. amyloliquefaciens
B. amyloliquefaciens
B. amyloliquefaciens
B. amyloliquefaciens

TA1001
TA208
H
FZB42

Plasmids
pUB110
pC194
pE194
pMK4
pDG148
pHCMC02

Relevant characteristics

References or sources

Genotype of restrictionmodication systems: dam dcm D(mcrC-mrr)102::Tn10 (TcR)

CGMCC 4013, Wild type, isolated from soil in Tianjin, China
TA1001 derivate, guanosine producing strain
Wild type
Wild type

Invitrogen
CGMCCa
This study
[25], BGSCb
[7], BGSC

KanR, 4548 bp
CmR, 2910 bp
EmR, 3728 bp
E. coliBacillus shuttle plasmid, rolling circle replicative, CmR
E. coliBacillus shuttle plasmid, rolling circle replicative, KanR
E. coliBacillus shuttle plasmid, theta replicative, CmR

[40],
[41],
[42],
[43],
[44],
[45],

BGSC
BGSC
BGSC
BGSC
Ciarn Condon
BGSC

TcR, tetracycline resistance; KanR, kanamycine resistance; CmR, chloramphenicol resistance; EmR, erythromycin resistance.
a
China General Microbiological Culture Collection Center.
b
Bacillus Genetic Stock Center.

genetic manipulation. However, the rst two techniques are

labor-intensive, and the previously reported electroporation
technique is also less effective for the recalcitrant strains [19,20].
In addition, these techniques are inefcient for direct mutagenesis
or construction of a mutant library. Based on these considerations,
a highly efcient method for electroporation of recalcitrant
B. amyloliquefaciens has been developed by growing the cells in
semicomplex hypertonic medium rst, and combining cellwall-weakening and cell-membrane uidity-disturbing techniques
to loosen the cells. Furthermore, agents that could affect the
efciency were tested in the washing buffers, together with the
effects of heat inactivation on a host restrictionmodication system. The optimal efciency for recalcitrant B. amyloliquefaciens
reached 1.13 0.34  107 cfu/lg of plasmid DNA using this approach, enabling an effective genetic modication for the strains.
Ultimately, results from this work could be applicable to functional
genomics and strain improvement of refractory Bacillus.
Materials and methods
Bacterial strains and plasmids
The bacterial strains and the plasmids used in this work are
listed in Table 1. B. amyloliquefaciens TA208 was used for electroporation method development, which was generated by iteratively
UV and DES1 treatment of the wild-type strain TA1001 (CGMCC
4013), and was an adenine auxotrophic, and 8-AG- and MSO-resistant mutant. The plasmids with different replicons and antibiotic
resistance markers were used for evaluations. B. amyloliquefaciens
FZB42 and H were also used for method evaluations.
The dam, dcm, and hsdRMS-decient Escherichia coli INV110 was
used to prepare the unmethylated E. coliBacillus shuttle plasmids.
Media, chemicals, and culture conditions
Escherichia coli and Bacillus were routinely cultured in LuriaBertani (LB) liquid medium at 37 C and 200 rpm or LB agar plate at
37 C. When appropriate, ampicillin (Amp; 100 lg/ml for E. coli),
chloramphenicol, kanamycin, or erythromycin (5, 10, and 5 lg/ml
for Bacillus, respectively) was added to the medium. Adenosine
was supplemented at 50 mg/L for the adenine auxotrophic Bacillus.

1
Abbreviations used: AG, azaguanine; MSO, methionine sulfoxide; UV, ultraviolet;
DES, diethyl sulfate; PEG, polyethylene glycol; Hepes, N-(2-hydroxyethyl)-N0 -2piperazine-ethanesulfonic acid; Tris, 2-amino-2-(hydroxymethyl)-1,3-propanediol;
RSM, response surface methodology; CCD, central composite design; ANOVA, analysis
of variance.

In initial screening of the optimal medium for electro-competent cell preparation, strain TA208 was grown in various hypertonic media with different nutritional ingredients and buffering
salts, including NCM [21] (17.4 g K2HPO4, 11.6 g NaCl, 5 g glucose,
5 g tryptone (Oxoid, Basingstoke, Hampshire, UK), 1 g yeast extract
(Oxoid), 0.3 g trisodium citrate, 0.05 g MgSO47H2O, and 91.1 g sorbitol in 1 L deionized water, pH 7.2), M9YE [22] (100 ml 10  M9
salts, 3 g yeast extract, 10 g casein hydrolysate (Oxoid), 2 g glucose,
2 ml 1 M MgSO4, 100 ll 1 M CaCl2, and 91.1 g sorbitol in 1 L deionized water, pH 7.2), LBSP [20] (10 g tryptone, 5 g yeast extract, 10 g
NaCl, 50 mM KH2PO4 and K2HPO4, and 91.1 g sorbitol in 1 L deionized water, pH 7.2), LBBHIS (10 g tryptone, 5 g yeast extract, 10 g
NaCl, 10 g Brain Heart Infusion (BHI; Difco, Detroit, MI, USA), and
91.1 g sorbitol in 1 L deionized water, pH 7.2), and BHIS [23]
(34 g BHI, and 91.1 g sorbitol in 1 L deionized water, pH 7.2).
Preparation of the electro-competent cells
An overnight LB culture of the Bacillus cells was diluted 100-fold
to fresh medium for preparation of the electro-competent cells.
The bacterial growth was monitored by measuring the optical density (OD) at 600 nm using a Nanodrop 2000C spectrophotometer
(Thermo Scientic, Wilmington, DE, USA). When an OD600 reading
reached 0.5, cell-wall weakening and/or cell-membrane uidity
disturbing was performed by adding Gly, DL-Thr, Amp, or Tween
80 into the culture and continued to shake for 1 h. The cell culture
was cooled on ice for 20 min, and collected by centrifugation at
4 C, 8000g for 5 min. After washing four times with ice-cold
ETM buffer (0.5 M sorbitol, 0.5 M mannitol, and 10% glycerol),
the electro-competent cells were resuspended in 1/100 vol of the
original culture.
Development of a combined cell-wall-weakening and cell-membrane
uidity-disturbing approach using response surface methodology
Preliminary one-way experiments indicated that Gly and DL-Thr
are effective for improving transformation efciency by weakening
the cell wall, whereas Tween 80 enhances electro-competence by
disturbing the cell-membrane uidity. Effects of cell-wall-weakening and cell-membrane-disturbing combination were evaluated
using RSM. Central composite design (CCD) was used to investigate
the effects of three independent variables on the transformation
efciency (Y), Gly (X1), DL-Thr (X2), and Tween 80 concentration
(X3), which were coded at levels of 2, 1, 0, 1, and 2, respectively
(Table 2), according to

xi X i  X 0 =DX;

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Electroporation of recalcitrant B. amyloliquefaciens / G.-q. Zhang et al. / Anal. Biochem. 409 (2011) 130137

Table 2
Independent variables involved in CCD trials shown in actual and coded levels.
Independent variables

Symbols

Gly concentration (%)

DL-Thr

concentration (%)
Tween 80 concentration (%)

Coded levels

Uncoded

Coded

2

1

X1
X2

x1
x2

2
0.8

2.5
0.9

3
1.0

3.5
1.1

4
1.2

X3

x3

0.01

0.02

0.03

0.04

0.05

where xi and Xi represent the coded and actual values of variables,

respectively, X0 represents the central points of the coded levels,
and DX is the step change between the actual neighboring levels.
All trials of the CCD experiments including eight 231 factorials
(18), six start points (914), and six central point runs (1520)
are listed in Table 3.
The relationship between transformation efciency (Y) and the
independent variables was analyzed using a quadratic polynomial
model, and data were t into the following equation

Y b0 b1 x1 b2 x2 b3 x3 b12 x1 x2 b23 x2 x3 b13 x1 x3

b11 x21 b22 x22 b33 x23

where b0 is a scaling constant; b1, b2, and b3 are linear coefcients;

b12, b23, and b13 are interactive coefcients; b11, b22, and b33 are
quadratic coefcients. Design Expert 8.0 (Stat-Ease, Minneapolis,
MN, USA) was used for design and data regression of the CCD
experiments.

Electroporation
Electro-competent cells (100 ll) were mixed with column-puried plasmid DNA (100 ng) with Plasmid Mini Kit (E.Z.N.A., OMEGA
Bio-tek, Duraville, USA), and loaded into a prechilled 1-mm gap
electroporation cuvette. After a brief incubation on ice, the cell
DNA mixture was shocked by a single 2.1 kV/cm pulse generated
by BTX ECM399 electroporator (BTX, Harvard Apparatus, Holliston,
MA, USA), with the resistance and capacitance set at 150 X and
36 lF, respectively. The cells were immediately diluted into 1 ml
of corresponding recovery medium (growth medium plus 0.38 M
mannitol [15]) and shaken gently at 37 C for 3 h to allow expression of the antibiotic resistant genes, and aliquots of the dilutions
were then spread onto LB agar plates supplemented with appropriate antibiotics. Transformation efciencies were calculated by
counting the colonies on plates after incubation at 37 C for 16 h.
Some transformants were veried by plasmid extraction and
restriction enzyme digestion.
Heat inactivation of host restrictionmodication systems

Optimization of the electroporation buffer

The strain TA208 cells treated with 3.89% of Gly, 1.06% of DL-Thr,
and 0.03% of Tween 80 were washed four times with ice-cold ETM,
and resuspended in 1/100 vol of the original culture in ETM buffers
containing 10% PEG 6000, 1 mM Hepes (pH 7.0), 1 mM TrisHCl
(pH 7.0), 1 mM KH2PO4 and K2HPO4 (pH 7.0), or 1 mM MgCl2,
and transformed with 100 ng pUB110 DNA.

Table 3
CCD matrix of three variables in coded units and the experimental and predicted
values of transformation efciency. a
Trial number

Variable

Transformation efciency
(cfu/lg of plasmid DNA)

x1

x2

x3

Observed

Predicted

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

1
1
1
1
1
1
1
1
2
0
0
2
0
0
0
0
0
0
0
0

1
1
1
1
1
1
1
1
0
2
0
0
2
0
0
0
0
0
0
0

1
1
1
1
1
1
1
1
0
0
2
0
0
2
0
0
0
0
0
0

1.18  106
6.23  105
8.32  105
1.01  106
7.63  105
5.21  105
2.82  105
1.12  105
6.63  105
3.30  105
4.04  105
1.09  106
9.30  105
7.64  105
1.034  106
1.056  106
1.04  106
1.022  106
1.046  106
1.03  106

1.139  106
7.989  105
8.519  105
9.856  105
7.145  105
6.285  105
4.337  105
2.799  105
4.252  105
2.482  105
3.664  105
1.200  106
8.844  105
6.742  105
1.017  106
1.017  106
1.017  106
1.017  106
1.017  106
1.017  106

a
B. amyloliquefaciens TA208 was cultured aerobically in NCM with various
amounts of Gly, DL-Thr, and Tween 80 added to the medium until OD600 reached 0.5.
The competent cells were prepared and transformed with 100 ng pUB110 DNA as
demonstrated under Materials and methods.

When the shuttle plasmids extracted from E. coli INV110 were

used for transformation, heat inactivation of the host restriction
modication systems was conducted. Cells resuspended in the
recovery medium were heated in a 46 C water bath for 6 min before plating as described by Van der Rest et al. [24].
Results
Initial screening of the optimal growth medium
To evaluate the effects of ingredients in growth media on transformation, strain TA208 was grown in various media for preparation of the electro-competent cells. Media with different
buffering salts and nutritional ingredients including NCM, M9YE,
LBSP, LBBHIS, and BHIS were selected by literature mining with
slight modications. When the OD600 reached 0.8, the competent
cells were prepared and transformed with 100 ng pUB110 plasmid
DNA. The efciencies were calculated (Fig. 1).
For the ve hypertonic media tested, the semicomplex NCM
medium gave the highest efciency (1.98 0.57  104 cfu/lg plasmid DNA). The efciency was positively correlated to concentrations of the salts, such as potassium phosphates and trisodium
citrate, but it decreased with the increase of nutritional ingredients. Bacteria cultivated in the superrich BHIS medium yielded
only 50 10 cfu/lg plasmid DNA.
Effects of cell-wall-weakening and membrane-disturbing agents
Gly, DL-Thr, and Amp were added separately into NCM cultures
for weakening the cell-wall synthesis, and the effects of three
agents on transformation efciency were investigated. Gly exhibited the most potent effect on transformation efciency enhancement, with an efciency of 6.38 0.92  105 cfu/lg plasmid DNA
achieved when tested at a concentration of 3%, which is an increase
of 32.2-fold compared to the control of NCM blank (Fig. 2a). DL-Thr

Electroporation of recalcitrant B. amyloliquefaciens / G.-q. Zhang et al. / Anal. Biochem. 409 (2011) 130137

133

(data not shown). Therefore Amp was no longer used in the following RSM designs.
A positive effect of Tween 80 was also observed on strain TA208
transformation. When supplemented at a concentration of 0.03%,
transformation was elevated to 1.10  105 cfu/lg plasmid DNA
(Fig. 2d).
Combinatorial cell-wall-weakening and cell-membrane uiditydisturbing strategy with RSM design

Fig.1. Relationship between transformation efciency of strain TA208 and culture

media for the competent cells. Strain TA208 was grown in various media until
OD600 reached 0.8, and the competent cells were prepared and transformed by
pUB110 as described under Materials and methods. Transformation efciencies
shown are averages of at least three replicates with a SD.

To determine the optimal concentration of Gly, DL-Thr, and

Tween 80 in a combined cell-wall-weakening and cell-membrane
uidity-disturbing procedure, a CCD including 20 runs was conducted (Table 3), in which the running concentrations of Gly, DLThr, and Tween 80 were determined by preliminary one-way
experiments. These 20 trials include six central points where the
concentrations of Gly, DL-Thr, and Tween 80 were set at 3.0%,
1.0%, and 0.03%, respectively, with the observed and predicted values of 20 trials listed in Table 3. The transformation efciency was
regressed to a second-order polynomial function, and the mathematical model is shown in

Y 1:017  106 1:938  105 x1 1:591  105 x2 7:694

and Amp improved the efciencies to 2.88 0.50  105 and
3.10 0.36  105 cfu/lg plasmid DNA, respectively, when supplemented at 1.2% and 50 lg/ml (Fig. 2b and c). We also conducted
the cell-wall-weakening experiments by adding two and three
agents. However, when Amp was cosupplemented with either
Gly or DL-Thr, transformation efciencies decreased dramatically

 104 x3  1:938  104 x1 x2 4:125  103 x2 x3  4:125

 103 x1 x3  5:099  104 x21  1:126  105 x22  1:241
 105 x23

Y is the transformation efciency, and x1, x2, and x3 are concentrations of Gly, DL-Thr, and Tween 80 in coded values, respectively.

Fig.2. Plots of transformation efciency against concentrations of the cell-wall-weakening or cell-membrane uidity-disturbing agents. Strain TA208 was cultured in NCM to
OD600 reading of 0.5. Gradient concentrations of the cell-wall-weakening or cell-membrane uidity-disturbing agents (06% of Gly (a); 02.5% of DL-Thr (b); 0100 lg/ml of
Amp (c); 01% of Tween 80 (d)) were supplemented to the culture, and the cells were shaken for 1 h before the competent cells were prepared. Data shown are averages of at
least three independent experiments with a SD.

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Electroporation of recalcitrant B. amyloliquefaciens / G.-q. Zhang et al. / Anal. Biochem. 409 (2011) 130137

Furthermore, statistical signicance of the RSM quadratic model

was evaluated by analysis of variance (ANOVA) (Table 4). The
Prob > F value of the model indicates that the probability of a
noise-caused F value is only 0.07% at the 95% condence level.
The tness of the quadratic model is expressed by an adjusted
determination coefcient (R2Adj ), which is found to be 0.8086, indicating that 80.86% of the variance of response can be explained by
this model. Meanwhile, a relatively low coefcient of variance (CV)
in this model (17.84%) suggests a high precision with which the trials are compared. Statistical signicance of the coefcient values in
the RSM model (Eq. (3)) was also evaluated by ANOVA (Table 5).
The Prob>F values indicate that the linear coefcients of x1, x2,
and x3, and the quadratic coefcients of x22 and x23 are signicant
at the 95% condence level (P < 0.05).
To better understand the relationship between transformation
efciency and the three independent variables, the threedimensional response surface and the two-dimensional contour
are plotted in Fig. 3. These plots show the interactive effects of
Gly and DL-Thr, Gly and Tween 80, and DL-Thr and Tween 80 on
the transformation efciency of strain TA208 using pUB110
plasmids.
The maximum transformation efciency predicted by the model
was 1.245  106 cfu/lg DNA, when the concentrations of Gly, DLThr, and Tween 80 were set at 3.89%, 1.06%, and 0.03%, respectively.
To validate the RSM model, four batches of the competent cells were
prepared under optimal conditions, and transformed with pUB110.
The efciency was calculated to be 1.23 0.06  106 cfu/lg plasmid
DNA, which is in close agreement with the value predicted by the
model.
Optimization of the electroporation buffer
The effects of electroporation buffers containing different ingredients on transformation were tested under the above optimized
conditions. ETM buffers containing one or two sorts of agents are
shown in Table 6. These agents exhibited positive effects on the
transformation efciency except for PEG 6000; moreover, addition
of 0.5 mM KH2PO4 and K2HPO4 together with 0.5 mM MgCl2 improved the transformation efciency to 1.13 0.34  107 cfu/lg

Table 4
ANOVA of the regression model.

a
b
c

Resource

SSa

DFb

MSc

F value

Prob > F

Model
Residual
Lack of t
Pure error
Total

1.688  1012
1.891  1011
1.884  1011
7.280  108
1.878  1012

9
10
5
5
19

1.876  1011
1.891  1010
3.767  1010
1.456  108

9.92

0.0007

258.75

<0.0001

SS, sum of squares.

DF, degree of freedom.
MS, mean square.

Fig.3. Three-dimensional response surface and contour plots of transformation

efciency against concentrations of Gly, DL-Thr, and Tween 80. Actual factors of (a
c) are set at 0 in coded levels, respectively.

Table 5
Signicance of the regression coefcients for the response surface model.
Model term

Coefcient

DF

Standard error

F value

Prob > F

Intercept
x1
x2
x3
x1x2
x2x3
x1x3
x21

1.017  106
1.938  105
1.591  105
7.694  104
1.938  104
4.125  103
4.125  103
5.099  104

1
1
1
1
1
1
1
1

5.485  104
3.438  104
3.438  104
3.438  104
4.862  104
4.862  104
4.862  104
2.742  104

31.78
21.41
5.01
0.16
7.2  103
7.2  103
3.46

0.0002
0.0009
0.0492
0.6986
0.9341
0.9341
0.0926

x22
x23

1.126  10

2.742  10

16.86

0.0021

1.241  105

2.742  104

20.48

0.0011

plasmid DNA. Such efciency allows direct gene inactivation using

an antibiotic resistant cassette, and mutant library construction in
recalcitrant B. amyloliquefaciens.
Transformation of strain TA1001, the parental strain of TA208,
with pUB110 under the same conditions also resulted in a similar
efciency (1.56 0.30  107 cfu/lg plasmid DNA).

Heat inactivation of host restrictionmodication system

To investigate the effect of temporary inactivation of the host
restrictionmodication system on the transformation with

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Electroporation of recalcitrant B. amyloliquefaciens / G.-q. Zhang et al. / Anal. Biochem. 409 (2011) 130137
Table 6
Transformation efciency of strain TA208 in various ETM buffers under optimal conditions.
Additives in ETM buffer

Concentration

Transformation efciency
(cfu/lg of plasmid DNA)

Reference

PEG 6000
Hepes
TrisHCl
KH2PO4 and K2HPO4
MgCl2
KH2PO4, K2HPO4, and MgCl2

10%
1 mM (pH
1 mM (pH
1 mM (pH
1 mM
0.25, 0.25,

1.02 0.35  106

2.83 0.45  106
3.43 0.28  106
5.78 0.56  106
7.35 0.92  106
1.13 0.34  107

[19]
[17]
[32]
[32]
[32]
[32]

7.0)
7.0)
7.0)
and 0.5 mM (pH 7.0)

Table 7
Effects of heat inactivation of host-restriction systems on transformation efciency (cfu/lg of plasmid DNA).
pMK4

Unheated
Heated
a

Syngeneic plasmids

Unmethylated plasmids treated with M. BamHI

Unmethylated

M. BamHI methylated

pC194

pE194

pHCMC02

pDG148

2.67 0.65  102

1.15 0.14  103

2.64 0.17  104

2.42 0.31  105

1.79 0.55  107

a

5.09 0.45  106

8.94 0.77  105

5.97 0.31  105

Not determined.

xenogeneic plasmids, cells were heated after electroporation.

Unmethylated pMK4 plasmids were extracted from E. coli INV110
and treated with BamHI methyltransferase (New England Biolabs,
Ipswich, MA, USA) as described by the manufacturer.
Transformation efciencies with pMK4 plasmids are shown in
Table 7. Heating increased the transformation efciency of strain
TA208 with plasmids propagated in E. coli INV110 by about 10-fold
(plasmids treated with BamHI methyltransferase). However, this
ratio is lower than that reported in C. glutamicum, probably caused
by the difference in heat sensitivity between restriction enzymes
from the two species. Nevertheless, the transformation efciency
of xenogeneic plasmids was increased by three orders of magnitude compared to that reported for naturally nontransformable B.
amyloliquefaciens [19].
Transformation with various replicative plasmids
To further evaluate the optimized protocol, other replicative
plasmids including pC194, pE194, pHCMC02, and pDG148, bearing
divergent replicons and conferring different antibiotic resistance in
Bacillus other than pUB110, were also tested. pC194 and pE194
were pretransferred into strain TA208 to attenuate the host restriction effect, and the syngeneic plasmids also resulted in the transformation efciencies at the same magnitude as pUB110.
pHCMC02 and pDG148 shuttle plasmids extracted from E. coli
INV110 and treated with BamHI methyltransferase were subjected
to heat inactivation of host restriction on transformation of strain
TA208, resulting in higher transformation efciencies than pMK4
(Table 7).
Application of the protocol to referenced B. amyloliquefaciens strains
To test the applicability of the current protocol to other B. amyloliquefaciens strains, naturally transformable FZB42 [6,7] and nontransformable H [19,25] strains reported previously were used.
Unfortunately, the cells of strains FZB42 and H lysed on addition
of 3.89% Gly, 1.06% DL-Thr, and 0.03% Tween 80 to NCM culture.
Subsequently, the competent cells of strains H and FZB42 were
prepared by adding 3.89% Gly and 1.06% DL-Thr to loosen the cell
wall. Cells were transformed with syngeneic pUB110 DNA as described under Materials and methods. The transformation efciencies were 9.62 0.57  106 and 8.65 0.97  106 cfu/lg plasmids
DNA for strains H and FZB42, respectively, which are acceptable
for the purpose of genetic manipulation.

Discussion
In the current work, an electroporation protocol to transform
recalcitrant B. amyloliquefaciens has been systematically optimized,
in which the culture medium, cell-wall-weakening and cell-membrane uidity-disturbing agents, electroporation buffer, and heat
inactivation of host restriction enzyme were all investigated to
achieve a high transformation efciency. The improvement of
transformation efciency after each optimization step and the proposed electroporation method for recalcitrant B. amyloliquefaciens
are shown in Supplementary Table 1 and Supplementary Table 2,
respectively.
Culture medium is a key factor for determining the transformation efciencies of bacteria. E. coli cells grown in SOB medium usually yield higher transformation efciencies than those grown in LB
and 2 YT media [26]. In addition, the nutrient BHIS is regarded as
an appropriate medium for preparation of the competent cells in C.
glutamicum [27]. Nevertheless, we found that the nutritional ingredients in growth medium exerted a negative effect on the transformation efciency in B. amyloliquefaciens, possibly due to the high
sporulation rate of Bacillus in a rich medium [28]. Since the Bacillus
endospores are surrounded by a morphologically complex coat,
consisting of peptidoglycan, dipicolinic acid, and protein, and conferring the spores resistant to heat, UV, and other extreme environments, the spores are hardly accessible by plasmid DNA [29];
whereas the walls of vegetative cells grown in semicomplex NCM
medium are relatively loose and easy to form pore during electroporation, and subsequently more accessible by exogenous plasmid
DNA. Moreover, buffering salts in NCM, such as potassium phosphate and sodium citrate, enhance the transformation efciency
by providing the cells appropriate ions and pH.
Gly and DL-Thr have been used as cell-wall-weakening agents in
preparation of the electro-competent cells in various Gram-positive
bacteria. By replacing the L- and D-alanine, Gly and DL-Thr can integrate into the tetrapeptide, which is the linker of N-acetylmuramic
acid in the cell wall, reduce the cross-linking of the peptidoglycan
layer, and thereby make the cell wall more accessible by exogenous
DNA [30]. Lactococcus lactis [31], B. cereus [32], and C. glutamicum
[33] grown in Gly-rich medium were reported to show a higher
transformation efciency than those grown in Gly-poor medium,
and DL-Thr enhanced the transformation efciency of B. subtilis
[30] and Pediococcus acidilactici [34]. In this study, we also found that
Gly and DL-Thr loosen the cell wall of B. amyloliquefaciens and improve the transformation efciency. Similar observations have been

136

Electroporation of recalcitrant B. amyloliquefaciens / G.-q. Zhang et al. / Anal. Biochem. 409 (2011) 130137

reported by McDonald [30] and Zakataeva et al. [19]. However,

Turgeon et al. [16] suggested that the cell-wall-weakening agents
decreased the transformation efciency of B. cereus under high electric eld. The different effects of Gly and DL-Thr on transformability
could be attributed to diverse composition between the cell walls of
B. amyloliquefaciens and B. cereus. Results from recent studies have
demonstrated that even the same B. cereus species of different
subgroups showed the cell-wall carbohydrates variations in their
glycosyl composition [35].
Transformation protocols for bacteria were usually optimized in
one-way experiments, reckoning without the interaction of different factors. However, it has been suggested in the literature that
those factors, such as growth and recovery media, and electrical
and physical parameters were virtually interactive [36], and such
interactions could dramatically affect the transformation. As a tool
of multifactor experiment design, RSM can empirically reveal the
response of the products or process on several input variables,
and has played a predominant role in various industrial process designs, such as optimization of fermentation conditions, extraction
of microbiological secondary metabolites, and optimization of
reaction conditions of chemical processes, etc. [37]. Hence a RSM
design regarding the linear, interactive, and quadratic effects of
factors was implemented to optimize the combined cell-wallweakening and cell-membrane uidity-disturbing experiments in
the current research. A 20-run CCD was used and a secondary polynomial function was deduced based on the trials. Finally, the optimal recipe was identied by the second-order function model, and
the actual transformation efciency was in close agreement with
the model prediction. Olivier et al. used multifactorial experiment
design to optimize eight quantitative factors in Thermophilus transformation [36], whereas in this study, we found that RSM is a reliable approach to optimize the transformation in recalcitrant B.
amyloliquefaciens.
Transformations of bacteria by DNA extracted from other species usually yield signicantly lower efciencies than syngeneic
DNA, since the xenogeneic DNA is sensitive to host restriction systems. For gene inactivation and overexpression purpose, integration and shuttle plasmids are usually constructed in E. coli. The
restriction enzymes in Bacillus will digest and degrade xenogeneic
DNA from a foreign source; hence the transformation efciency of
E. coli-propagated plasmids will be dramatically lower than that of
xenogeneic plasmids. Heat shock after transformation has been reported to increase the transformation efciency of the restriction
modication system harboring bacteria including E. coli [38], Salmonella typhimurium [38], and C. glutamicum [24]. Finally, to overcome the restrictionmodication barrier for interspecies DNA
transfer between E. coli and Bacillus, a previously described heat
treatment after transformation was performed in B. amyloliquefaciens, and the transformation efciency with xenogeneic plasmids
was increased by 103-fold compared to previous reports [19]. However, the efciency is still lower than that of syngeneic pUB110
DNA by a magnitude of two orders, suggesting that the restrictionmodication enzymes other than the BamHI system exist in
strain TA208. It was reported that the restrictionmodication
barrier could be overcome completely by articially modifying
shuttle plasmids in the E. coli host expressing multiple DNA
methyltransferases [39], on which we are currently working in
recalcitrant B. amyloliquefaciens.
In conclusion, this paper describes an electro-transformation
protocol for recalcitrant B. amyloliquefaciens, which is reproducible,
convenient, and applicable to many strains. The highest transformation efciencies achieved using this protocol was up to 1.13 0.34 
107 cfu/lg syngeneic pUB110 plasmids and 8.94 0.77  105 cfu/lg
xenogeneic pHCMC02 shuttle plasmids, respectively. We expect
that the protocol will be applicable to other Bacillus strains refractory to electroporation.

Acknowledgments
We are grateful to BGSC and Prof. Ciarn Condon for providing
the plasmids and strains used in this study. We thank Prof. Yongsheng Che for English revision. This research was funded by the
National Drug Discovery Program of China (2008ZX09401-05),
and the National Key Program of Research and Development of
China (2008BAI63B01).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ab.2010.10.013.

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