Enhancing electro-transformation competency of recalcitrant Bacillus amyloliquefaciens by combining cell-wall weakening and cell-membrane fluidity disturbing

Analytical Biochemistry 409 (2011) 130–137

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Enhancing electro-transformation competency of recalcitrant Bacillus amyloliquefaciens by combining cell-wall weakening and cell-membrane fluidity 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 fluidity 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 efficient, 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 fluidity was elevated by supplementing Tween 80. After optimization of the cell-loosening recipe by response surface methodology (RSM), the transformation efficiency 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 efficiency 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 difficult to be transformed using conventional methods. Ó 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], fibrinolytic enzymes [3], and others. Since the Bacillus genus exhibits a high flux of the pentose phosphate pathway, B. amyloliquefaciens and its close relatives have also been stable producers of purine nucleosides [4] and riboflavin [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 significance 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: [email protected] (T.-y. Wen). 0003-2697/$ - see front matter Ó 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 efficiently [11]. Since its introduction to the genetics of Bacillus [12–14], various methods have been developed and optimized with hypertonic agents, pulse voltage, and electroporation buffers, yielding different transformation efficiencies. 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 efficiency was up to 2  109 cfu/lg/ml for Bacillus cereus [16]. However, these protocols are highly species or strain specific, and the efficiency 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) 130–137 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 restriction–modification 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. coli–Bacillus shuttle plasmid, rolling circle replicative, CmR E. coli–Bacillus shuttle plasmid, rolling circle replicative, KanR E. coli–Bacillus shuttle plasmid, theta replicative, CmR

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

BGSC BGSC BGSC BGSC Ciarán 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 first 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 inefficient for direct mutagenesis or construction of a mutant library. Based on these considerations, a highly efficient method for electroporation of recalcitrant B. amyloliquefaciens has been developed by growing the cells in semicomplex hypertonic medium first, and combining cellwall-weakening and cell-membrane fluidity-disturbing techniques to loosen the cells. Furthermore, agents that could affect the efficiency were tested in the washing buffers, together with the effects of heat inactivation on a host restriction–modification system. The optimal efficiency for recalcitrant B. amyloliquefaciens reached 1.13 ± 0.34  107 cfu/lg of plasmid DNA using this approach, enabling an effective genetic modification 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-deficient Escherichia coli INV110 was used to prepare the unmethylated E. coli–Bacillus shuttle plasmids. Media, chemicals, and culture conditions Escherichia coli and Bacillus were routinely cultured in Luria–Bertani (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 Scientific, Wilmington, DE, USA). When an OD600 reading reached 0.5, cell-wall weakening and/or cell-membrane fluidity 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 fluidity-disturbing approach using response surface methodology Preliminary one-way experiments indicated that Gly and DL-Thr are effective for improving transformation efficiency by weakening the cell wall, whereas Tween 80 enhances electro-competence by disturbing the cell-membrane fluidity. 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 efficiency (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 ¼ ðX i  X 0 =DXÞ;

ð1Þ

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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

0

1

2

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 (1–8), six start points (9–14), and six central point runs (15–20) are listed in Table 3. The relationship between transformation efficiency (Y) and the independent variables was analyzed using a quadratic polynomial model, and data were fit into the following equation

Y ¼ b0 þ b1 x1 þ b2 x2 þ b3 x3 þ b12 x1 x2 þ b23 x2 x3 þ b13 x1 x3 þ b11 x21 þ b22 x22 þ b33 x23

ð2Þ

where b0 is a scaling constant; b1, b2, and b3 are linear coefficients; b12, b23, and b13 are interactive coefficients; b11, b22, and b33 are quadratic coefficients. 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-purified 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 efficiencies were calculated by counting the colonies on plates after incubation at 37 °C for 16 h. Some transformants were verified by plasmid extraction and restriction enzyme digestion. Heat inactivation of host restriction–modification 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 Tris–HCl (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 efficiency. a Trial number

Variable

Transformation efficiency (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– modification 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 modifications. When the OD600 reached 0.8, the competent cells were prepared and transformed with 100 ng pUB110 plasmid DNA. The efficiencies were calculated (Fig. 1). For the five hypertonic media tested, the semicomplex NCM medium gave the highest efficiency (1.98 ± 0.57  104 cfu/lg plasmid DNA). The efficiency 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 efficiency were investigated. Gly exhibited the most potent effect on transformation efficiency enhancement, with an efficiency 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) 130–137

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(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 fluiditydisturbing strategy with RSM design

Fig.1. Relationship between transformation efficiency 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 efficiencies 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 fluidity-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 efficiency was regressed to a second-order polynomial function, and the mathematical model is shown in

Y ¼ 1:017  106 þ 1:938  105 x1 þ 1:591  105 x2 þ 7:694 and Amp improved the efficiencies 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 efficiencies decreased dramatically

 104 x3  1:938  104 x1 x2 þ 4:125  103 x2 x3  4:125  103 x1 x3  5:099  104 x21  1:126  105 x22  1:241  105 x23

ð3Þ

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

Fig.2. Plots of transformation efficiency against concentrations of the cell-wall-weakening or cell-membrane fluidity-disturbing agents. Strain TA208 was cultured in NCM to OD600 reading of 0.5. Gradient concentrations of the cell-wall-weakening or cell-membrane fluidity-disturbing agents (0–6% of Gly (a); 0–2.5% of DL-Thr (b); 0–100 lg/ml of Amp (c); 0–1% 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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Furthermore, statistical significance 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% confidence level. The fitness of the quadratic model is expressed by an adjusted determination coefficient (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 coefficient of variance (CV) in this model (17.84%) suggests a high precision with which the trials are compared. Statistical significance of the coefficient values in the RSM model (Eq. (3)) was also evaluated by ANOVA (Table 5). The Prob>F values indicate that the linear coefficients of x1, x2, and x3, and the quadratic coefficients of x22 and x23 are significant at the 95% confidence level (P < 0.05). To better understand the relationship between transformation efficiency 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 efficiency of strain TA208 using pUB110 plasmids. The maximum transformation efficiency 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 efficiency 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 efficiency except for PEG 6000; moreover, addition of 0.5 mM KH2PO4 and K2HPO4 together with 0.5 mM MgCl2 improved the transformation efficiency 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 fit 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 efficiency against concentrations of Gly, DL-Thr, and Tween 80. Actual factors of (a– c) are set at 0 in coded levels, respectively.

Table 5 Significance of the regression coefficients for the response surface model. Model term

Coefficient

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

5

1.126  10

1

2.742  10

4

16.86

0.0021

1.241  105

1

2.742  104

20.48

0.0011

plasmid DNA. Such efficiency 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 efficiency (1.56 ± 0.30  107 cfu/lg plasmid DNA).

Heat inactivation of host restriction–modification system To investigate the effect of temporary inactivation of the host restriction–modification system on the transformation with

135

Electroporation of recalcitrant B. amyloliquefaciens / G.-q. Zhang et al. / Anal. Biochem. 409 (2011) 130–137 Table 6 Transformation efficiency of strain TA208 in various ETM buffers under optimal conditions. Additives in ETM buffer

Concentration

Transformation efficiency (cfu/lg of plasmid DNA)

Reference

PEG 6000 Hepes Tris–HCl 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 efficiency (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 efficiencies with pMK4 plasmids are shown in Table 7. Heating increased the transformation efficiency 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 efficiency 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 efficiencies 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 efficiencies 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 efficiencies 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 fluidity-disturbing agents, electroporation buffer, and heat inactivation of host restriction enzyme were all investigated to achieve a high transformation efficiency. The improvement of transformation efficiency 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 efficiencies of bacteria. E. coli cells grown in SOB medium usually yield higher transformation efficiencies 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 efficiency 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 efficiency 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 efficiency than those grown in Gly-poor medium, and DL-Thr enhanced the transformation efficiency 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 efficiency. Similar observations have been

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reported by McDonald [30] and Zakataeva et al. [19]. However, Turgeon et al. [16] suggested that the cell-wall-weakening agents decreased the transformation efficiency of B. cereus under high electric field. 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 fluidity-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 identified by the second-order function model, and the actual transformation efficiency 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 significantly lower efficiencies 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 efficiency 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 efficiency of the restriction– modification system harboring bacteria including E. coli [38], Salmonella typhimurium [38], and C. glutamicum [24]. Finally, to overcome the restriction–modification 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 efficiency with xenogeneic plasmids was increased by 103-fold compared to previous reports [19]. However, the efficiency is still lower than that of syngeneic pUB110 DNA by a magnitude of two orders, suggesting that the restriction–modification enzymes other than the BamHI system exist in strain TA208. It was reported that the restriction–modification barrier could be overcome completely by artificially 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 efficiencies 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. Ciarán 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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