Analytical Biochemistry 409 (2011) 130–137Contents lists available at ScienceDirect 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 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 LBBHIS (10 g tryptone. response surface methodology. The dam. After washing four times with ice-cold ETM buffer (0. and 91. amyloliquefaciens reached 1. and 91. and combining cellwall-weakening and cell-membrane fluidity-disturbing techniques to loosen the cells. and 91. and Tween 80 concentration (X3). BGSC BGSC BGSC BGSC Ciarán Condon BGSC TcR. isolated from soil in Tianjin. 2910 bp EmR. Furthermore. whereas Tween 80 enhances electro-competence by disturbing the cell-membrane fluidity. 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. coli–Bacillus shuttle plasmid. B. coli–Bacillus shuttle plasmid. Difco. and the previously reported electroporation technique is also less effective for the recalcitrant strains [19. pH 7. CmR E. The cell culture was cooled on ice for 20 min. rolling circle replicative. ANOVA. DES. [45]. pH 7. central composite design.2). 1. pH 7. 11. together with the effects of heat inactivation on a host restriction–modification system. Hepes. [44]. 0.5. according to xi ¼ ðX i X 0 =DXÞ. methionine sulfoxide. chemicals. 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. [42]. UK). amyloliquefaciens has been developed by growing the cells in semicomplex hypertonic medium first.13 ± 0. amyloliquefaciens B. the electro-competent cells were resuspended in 1/100 vol of the original culture.4 g K2HPO4. and 91. and BHIS [23] (34 g BHI. coli INV110 B. agents that could affect the efficiency were tested in the washing buffers. Tris. and 2.5 M sorbitol. 2 g glucose. genetic manipulation. amyloliquefaciens FZB42 and H were also used for method evaluations. N-(2-hydroxyethyl)-N0 -2piperazine-ethanesulfonic acid. erythromycin resistance. pH 7. Adenosine was supplemented at 50 mg/L for the adenine auxotrophic Bacillus. BGSCb [7]. tetracycline resistance. Effects of cell-wall-weakening and cell-membrane-disturbing combination were evaluated using RSM. [41].2). chloramphenicol. Central composite design (CCD) was used to investigate the effects of three independent variables on the transformation efficiency (Y).and MSO-resistant mutant. guanosine producing strain Wild type Wild type Invitrogen CGMCCa This study [25]. The optimal efficiency for recalcitrant B. 100 ll 1 M CaCl2. polyethylene glycol. which were coded at levels of 2. cell-wall weakening and/or cell-membrane fluidity disturbing was performed by adding Gly. In initial screening of the optimal medium for electro-competent cell preparation. 10 g Brain Heart Infusion (BHI. 5 g yeast extract. diethyl sulfate. B. ampicillin (Amp. In addition. including NCM [21] (17. CmR. 5 g glucose. coli–Bacillus shuttle plasmids. amyloliquefaciens B. China TA1001 derivate. a China General Microbiological Culture Collection Center. Basingstoke. kanamycine resistance. or erythromycin (5.05 g MgSO47H2O. ð1Þ . Hampshire. KanR E. When appropriate. DL-Thr. Wilmington. azaguanine. USA). respectively (Table 2).34 107 cfu/lg of plasmid DNA using this approach.1 g sorbitol in 1 L deionized water. 5 g tryptone (Oxoid. coli–Bacillus shuttle plasmid. The bacterial growth was monitored by measuring the optical density (OD) at 600 nm using a Nanodrop 2000C spectrophotometer (Thermo Scientific. results from this work could be applicable to functional genomics and strain improvement of refractory Bacillus. 5 g yeast extract.1 g sorbitol in 1 L deionized water. 10 g casein hydrolysate (Oxoid). BGSC KanR. Zhang et al. which was generated by iteratively UV and DES1 treatment of the wild-type strain TA1001 (CGMCC 4013). USA). coli). ultraviolet. The plasmids with different replicons and antibiotic resistance markers were used for evaluations. amyloliquefaciens TA208 was used for electroporation method development. DE.2). DL-Thr (X2). 50 mM KH2PO4 and K2HPO4. 10 g NaCl. or Tween 80 into the culture and continued to shake for 1 h. 1 Abbreviations used: AG. respectively) was added to the medium. and hsdRMS-deficient Escherichia coli INV110 was used to prepare the unmethylated E. 0. Based on these considerations. UV. LBSP [20] (10 g tryptone. 8000g for 5 min.6 g NaCl.3-propanediol. pH 7.3 g trisodium citrate.-q. Media. analysis of variance.2). 1. 10 g NaCl. and collected by centrifugation at 4 °C. M9YE [22] (100 ml 10 M9 salts. RSM. b Bacillus Genetic Stock Center. 2 ml 1 M MgSO4. Strains or plasmids Strains E. and 8-AG. Wild type.131 Electroporation of recalcitrant B. the first two techniques are labor-intensive. and 5 lg/ml for Bacillus. CmR [40]. kanamycin. MSO. Biochem. PEG. / Anal. CCD. a highly efficient method for electroporation of recalcitrant B. and 91. EmR. 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. 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. and 10% glycerol). [43].1 g sorbitol in 1 L deionized water.2). Amp. 100 lg/ml for E. strain TA208 was grown in various hypertonic media with different nutritional ingredients and buffering salts. 3728 bp E. KanR. 10. Detroit. 4548 bp CmR. rolling circle replicative. However. 0. 0. theta replicative. these techniques are inefficient for direct mutagenesis or construction of a mutant library. 2-amino-2-(hydroxymethyl)-1. chloramphenicol resistance. MI.5 M mannitol. and was an adenine auxotrophic.20]. 1 g yeast extract (Oxoid). Ultimately. Gly (X1). 409 (2011) 130–137 Table 1 Bacterial strains and plasmids used in this study. dcm. amyloliquefaciens / G. enabling an effective genetic modification for the strains. When an OD600 reading reached 0.1 g sorbitol in 1 L deionized water.1 g sorbitol in 1 L deionized water. Materials and methods Bacterial strains and plasmids The bacterial strains and the plasmids used in this work are listed in Table 1. amyloliquefaciens B. 3 g yeast extract. 1. respectively.03% of Tween 80 were washed four times with ice-cold ETM. such as potassium phosphates and trisodium citrate.03 106 1. b11. and Tween 80 added to the medium until OD600 reached 0. Minneapolis. b12. b23. DL-Thr. All trials of the CCD experiments including eight 231 factorials (1–8).Z.482 105 3. 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. Gly exhibited the most potent effect on transformation efficiency enhancement.0).285 105 4.04 0. Bacteria cultivated in the superrich BHIS medium yielded only 50 ± 10 cfu/lg plasmid DNA. MN.09 106 9.18 106 6.252 105 2.03 0.32 105 1.856 105 7. but it decreased with the increase of nutritional ingredients.519 105 9. 1 mM Tris–HCl (pH 7. the cell– DNA mixture was shocked by a single 2. b1.145 105 6. and 0.5 1. and transformed with 100 ng pUB110 DNA. b22. 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. and b3 are linear coefficients.2 X3 x3 0. OMEGA Bio-tek.23 105 8.017 106 1. amyloliquefaciens TA208 was cultured aerobically in NCM with various amounts of Gly. which is an increase of 32. Zhang et al. Media with different buffering salts and nutritional ingredients including NCM. coli INV110 were used for transformation.2-fold compared to the control of NCM blank (Fig.844 105 6. and Amp were added separately into NCM cultures for weakening the cell-wall synthesis.30 105 4.8 2. with an efficiency of 6. Design Expert 8. LBSP.0 3. When the OD600 reached 0. M9YE. X0 represents the central points of the coded levels.57 104 cfu/lg plasmid DNA).01 0. Results Initial screening of the optimal growth medium To evaluate the effects of ingredients in growth media on transformation. The efficiencies were calculated (Fig.04 106 1.200 106 8. After a brief incubation on ice. [24]. and loaded into a prechilled 1-mm gap electroporation cuvette. The efficiency was positively correlated to concentrations of the salts. Biochem. Electroporation Electro-competent cells (100 ll) were mixed with column-purified plasmid DNA (100 ng) with Plasmid Mini Kit (E.664 105 1.017 106 1. / Anal. or 1 mM MgCl2.9 3 1. amyloliquefaciens / G.0).056 106 1.06% of DL-Thr.64 105 1. USA).. and the effects of three agents on transformation efficiency were investigated. Heat inactivation of host restriction–modification systems Optimization of the electroporation buffer The strain TA208 cells treated with 3.02 0.5.046 106 1.01 106 7. 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. the semicomplex NCM medium gave the highest efficiency (1. Harvard Apparatus. Duraville.92 105 cfu/lg plasmid DNA achieved when tested at a concentration of 3%. strain TA208 was grown in various media for preparation of the electro-competent cells.0 (Stat-Ease.017 106 1.017 106 1.022 106 1.337 105 2. The cells were immediately diluted into 1 ml of corresponding recovery medium (growth medium plus 0.30 105 7. the competent cells were prepared and transformed with 100 ng pUB110 plasmid DNA.5 0.04 105 1. heat inactivation of the host restriction– modification systems was conducted. MA. and b13 are interactive coefficients.017 106 1.989 105 8.742 105 1.034 106 1.98 ± 0.63 105 3. 1). LBBHIS. 409 (2011) 130–137 Table 2 Independent variables involved in CCD trials shown in actual and coded levels.21 105 2. six start points (9–14). Some transformants were verified by plasmid extraction and restriction enzyme digestion. with the resistance and capacitance set at 150 X and 36 lF. Effects of cell-wall-weakening and membrane-disturbing agents Gly.12 105 6.8. and resuspended in 1/100 vol of the original culture in ETM buffers containing 10% PEG 6000. 2a).-q.89% of Gly.017 106 a B.N.0). and six central point runs (15–20) are listed in Table 3.A.139 106 7. 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. and DX is the step change between the actual neighboring levels.63 105 5.38 M mannitol [15]) and shaken gently at 37 °C for 3 h to allow expression of the antibiotic resistant genes.1 kV/cm pulse generated by BTX ECM399 electroporator (BTX.132 Electroporation of recalcitrant B. DL-Thr. respectively. For the five hypertonic media tested. and b33 are quadratic coefficients.1 4 1.05 where xi and Xi represent the coded and actual values of variables. 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. Holliston. 1 mM Hepes (pH 7.38 ± 0.799 105 4. USA) was used for design and data regression of the CCD experiments. 1 mM KH2PO4 and K2HPO4 (pH 7. USA). b2.82 105 1. Transformation efficiencies were calculated by counting the colonies on plates after incubation at 37 °C for 16 h. and BHIS were selected by literature mining with slight modifications. DL-Thr . Table 3 CCD matrix of three variables in coded units and the experimental and predicted values of transformation efficiency. and aliquots of the dilutions were then spread onto LB agar plates supplemented with appropriate antibiotics. DL-Thr. A positive effect of Tween 80 was also observed on strain TA208 transformation. Combinatorial cell-wall-weakening and cell-membrane fluiditydisturbing strategy with RSM design Fig. Therefore Amp was no longer used in the following RSM designs. respectively.1.03%.10 ± 0. Transformation efficiencies shown are averages of at least three replicates with a ± SD. Strain TA208 was grown in various media until OD600 reached 0.50 105 and 3. 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. respectively. Plots of transformation efficiency against concentrations of the cell-wall-weakening or cell-membrane fluidity-disturbing agents.0%. 1. We also conducted the cell-wall-weakening experiments by adding two and three agents. DLThr.-q. in which the running concentrations of Gly.8. Data shown are averages of at least three independent experiments with a ± SD. Gradient concentrations of the cell-wall-weakening or cell-membrane fluidity-disturbing agents (0–6% of Gly (a). and Tween 80 were determined by preliminary one-way experiments. However. and Tween 80 were set at 3. Relationship between transformation efficiency of strain TA208 and culture media for the competent cells.36 105 cfu/lg plasmid DNA. To determine the optimal concentration of Gly. a CCD including 20 runs was conducted (Table 3). 409 (2011) 130–137 133 (data not shown).10 105 cfu/lg plasmid DNA (Fig. 2d). and x3 are concentrations of Gly. transformation was elevated to 1. When supplemented at a concentration of 0. when supplemented at 1. and the competent cells were prepared and transformed by pUB110 as described under Materials and methods. amyloliquefaciens / G. and x1.2. with the observed and predicted values of 20 trials listed in Table 3. 2b and c). These 20 trials include six central points where the concentrations of Gly.Electroporation of recalcitrant B. when Amp was cosupplemented with either Gly or DL-Thr. Fig. DL-Thr. Biochem. 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.5. and the cells were shaken for 1 h before the competent cells were prepared.2% and 50 lg/ml (Fig.03%. and Tween 80 in a combined cell-wall-weakening and cell-membrane fluidity-disturbing procedure. Zhang et al. The transformation efficiency was regressed to a second-order polynomial function. and Tween 80 in coded values. 0–100 lg/ml of Amp (c). / Anal.5% of DL-Thr (b). 0–2. Strain TA208 was cultured in NCM to OD600 reading of 0.88 ± 0. respectively. . DL-Thr. 0–1% of Tween 80 (d)) were supplemented to the culture. and 0. x2.0%. Heat inactivation of host restriction–modification system To investigate the effect of temporary inactivation of the host restriction–modification system on the transformation with .884 1011 7.34 107 cfu/lg Table 4 ANOVA of the regression model.938 104 4.0492 0. Gly and Tween 80.241 105 1 2. Such efficiency allows direct gene inactivation using an antibiotic resistant cassette. (3)) was also evaluated by ANOVA (Table 5).126 10 1 2.862 104 4. and Tween 80 were set at 3.48 0.84%) suggests a high precision with which the trials are compared. 409 (2011) 130–137 Furthermore. Fig. Optimization of the electroporation buffer The effects of electroporation buffers containing different ingredients on transformation were tested under the above optimized conditions. Meanwhile.125 103 4.876 1011 1. addition of 0.-q.0001 SS.438 104 4. and 0. respectively.742 104 20. Statistical significance of the coefficient values in the RSM model (Eq.13 ± 0. DLThr. with pUB110 under the same conditions also resulted in a similar efficiency (1. To validate the RSM model.938 105 1. and the quadratic coefficients of x22 and x23 are significant at the 95% confidence level (P < 0.125 103 5. which is in close agreement with the value predicted by the model. and x3.75 <0. Zhang et al. statistical significance of the RSM quadratic model was evaluated by analysis of variance (ANOVA) (Table 4). and Tween 80.438 104 3.06 106 cfu/lg plasmid DNA. amyloliquefaciens. degree of freedom.5 mM KH2PO4 and K2HPO4 together with 0. and transformed with pUB110. mean square.8086.878 1012 9 10 5 5 19 1.0021 1. respectively.03%.280 108 1. DF. These plots show the interactive effects of Gly and DL-Thr. ETM buffers containing one or two sorts of agents are shown in Table 6.9341 0.01 0.742 104 31.862 104 4. Model term Coefficient DF Standard error F value Prob > F Intercept x1 x2 x3 x1x2 x2x3 x1x3 x21 1. Three-dimensional response surface and contour plots of transformation efficiency against concentrations of Gly.2 103 7. x2. sum of squares. Biochem. DL-Thr.23 ± 0.099 104 1 1 1 1 1 1 1 1 5.456 108 9.694 104 1.5 mM MgCl2 improved the transformation efficiency to 1.07% at the 95% confidence level.92 0. The maximum transformation efficiency predicted by the model was 1. The fitness of the quadratic model is expressed by an adjusted determination coefficient (R2Adj ). a relatively low coefficient of variance (CV) in this model (17. The efficiency was calculated to be 1.41 5.06%.3. To better understand the relationship between transformation efficiency and the three independent variables.591 105 7. Actual factors of (a– c) are set at 0 in coded levels.2 103 3.0009 0. The Prob > F value of the model indicates that the probability of a noise-caused F value is only 0. The Prob>F values indicate that the linear coefficients of x1. These agents exhibited positive effects on the transformation efficiency except for PEG 6000.9341 0.0011 plasmid DNA.0007 258.30 107 cfu/lg plasmid DNA).862 104 2.46 0.78 21.891 1011 1. indicating that 80. Transformation of strain TA1001.0926 x22 x23 5 1. the parental strain of TA208.245 106 cfu/lg DNA. a b c Resource SSa DFb MSc F value Prob > F Model Residual Lack of fit Pure error Total 1.6986 0.56 ± 0.017 106 1. / Anal.16 7.485 104 3. Table 5 Significance of the regression coefficients for the response surface model. when the concentrations of Gly. amyloliquefaciens / G.688 1012 1. 3. 1. and DL-Thr and Tween 80 on the transformation efficiency of strain TA208 using pUB110 plasmids.742 10 4 16.86 0. and mutant library construction in recalcitrant B.767 1010 1.89%.86% of the variance of response can be explained by this model.0002 0. MS.891 1010 3. moreover. which is found to be 0. four batches of the competent cells were prepared under optimal conditions.05).438 104 3. the threedimensional response surface and the two-dimensional contour are plotted in Fig.134 Electroporation of recalcitrant B. xenogeneic plasmids.0) 7. Moreover. Nevertheless. pE194. In addition. Lactococcus lactis [31]. respectively. were also tested.31 105 1.62 ± 0.42 ± 0. Heating increased the transformation efficiency of strain TA208 with plasmids propagated in E. BamHI methylated pC194 pE194 pHCMC02 pDG148 2. 1. which are acceptable for the purpose of genetic manipulation. buffering salts in NCM. we found that the nutritional ingredients in growth medium exerted a negative effect on the transformation efficiency in B. Zhang et al. coli cells grown in SOB medium usually yield higher transformation efficiencies than those grown in LB and 2 YT media [26]. dipicolinic acid. BamHI Unmethylated M. coli INV110 by about 10-fold (plasmids treated with BamHI methyltransferase). The improvement of transformation efficiency after each optimization step and the proposed electroporation method for recalcitrant B.5 mM (pH 7. and protein.0) Table 7 Effects of heat inactivation of host-restriction systems on transformation efficiency (cfu/lg of plasmid DNA). probably caused by the difference in heat sensitivity between restriction enzymes from the two species. Transformation with various replicative plasmids To further evaluate the optimized protocol. the spores are hardly accessible by plasmid DNA [29]. cereus [32]. Unmethylated pMK4 plasmids were extracted from E. the competent cells of strains H and FZB42 were prepared by adding 3.03% Tween 80 to NCM culture.0) and 0. resulting in higher transformation efficiencies than pMK4 (Table 7). coli INV110 and treated with BamHI methyltransferase were subjected to heat inactivation of host restriction on transformation of strain TA208. By replacing the L. K2HPO4. cell-wall-weakening and cell-membrane fluidity-disturbing agents. Cells were transformed with syngeneic pUB110 DNA as described under Materials and methods. 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. MA.79 ± 0.65 102 1. which is the linker of N-acetylmuramic acid in the cell wall. subtilis [30] and Pediococcus acidilactici [34].55 107 –a 5.35 106 2.89% Gly.34 107 [19] [17] [32] [32] [32] [32] 7. and pDG148. However. Culture medium is a key factor for determining the transformation efficiencies of bacteria.0) 7. other replicative plasmids including pC194.94 ± 0.13 ± 0.and D-alanine. / Anal. B. and other extreme environments. Gly and DL-Thr can integrate into the tetrapeptide. respectively. and heat inactivation of host restriction enzyme were all investigated to achieve a high transformation efficiency.25. reduce the cross-linking of the peptidoglycan layer. amyloliquefaciens / G. coli INV110 and treated with BamHI methyltransferase (New England Biolabs. amyloliquefaciens.06% DL-Thr to loosen the cell wall. USA) as described by the manufacturer. and thereby make the cell wall more accessible by exogenous DNA [30]. pHCMC02. Transformation efficiencies with pMK4 plasmids are shown in Table 7.56 106 7.57 106 and 8. whereas the walls of vegetative cells grown in semicomplex NCM medium are relatively loose and easy to form pore during electroporation. Biochem.09 ± 0. Ipswich.83 ± 0. The transformation efficiencies were 9.02 ± 0. and DL-Thr enhanced the transformation efficiency of B.64 ± 0. possibly due to the high sporulation rate of Bacillus in a rich medium [28]. Unfortunately.89% Gly and 1. the transformation efficiency of xenogeneic plasmids was increased by three orders of magnitude compared to that reported for naturally nontransformable B.92 106 1.14 103 2.45 106 3. Nevertheless.-q. Similar observations have been .77 105 – 5. amyloliquefaciens has been systematically optimized.97 106 cfu/lg plasmids DNA for strains H and FZB42. 1. the cells of strains FZB42 and H lysed on addition of 3. Subsequently. this ratio is lower than that reported in C. amyloliquefaciens and improve the transformation efficiency. consisting of peptidoglycan.78 ± 0. Since the Bacillus endospores are surrounded by a morphologically complex coat.97 ± 0. and C. bearing divergent replicons and conferring different antibiotic resistance in Bacillus other than pUB110. an electroporation protocol to transform recalcitrant B. Application of the protocol to referenced B. glutamicum [27].7] and nontransformable H [19. electroporation buffer.15 ± 0. naturally transformable FZB42 [6.25] strains reported previously were used.06% DL-Thr. 409 (2011) 130–137 Table 6 Transformation efficiency of strain TA208 in various ETM buffers under optimal conditions.31 105 Not determined. UV. the nutrient BHIS is regarded as an appropriate medium for preparation of the competent cells in C. In this study.45 106 – – 8.43 ± 0. E. cells were heated after electroporation. and the syngeneic plasmids also resulted in the transformation efficiencies at the same magnitude as pUB110.25.35 ± 0. amyloliquefaciens are shown in Supplementary Table 1 and Supplementary Table 2. Discussion In the current work. and MgCl2 10% 1 mM (pH 1 mM (pH 1 mM (pH 1 mM 0. such as potassium phosphate and sodium citrate. we also found that Gly and DL-Thr loosen the cell wall of B. amyloliquefaciens [19]. glutamicum [33] grown in Gly-rich medium were reported to show a higher transformation efficiency than those grown in Gly-poor medium. and subsequently more accessible by exogenous plasmid DNA. pMK4 Unheated Heated a Syngeneic plasmids Unmethylated plasmids treated with M. Additives in ETM buffer Concentration Transformation efficiency (cfu/lg of plasmid DNA) Reference PEG 6000 Hepes Tris–HCl KH2PO4 and K2HPO4 MgCl2 KH2PO4. amyloliquefaciens strains. glutamicum.65 ± 0.28 106 5. 0. and 0.67 ± 0. pHCMC02 and pDG148 shuttle plasmids extracted from E. amyloliquefaciens strains To test the applicability of the current protocol to other B. and conferring the spores resistant to heat. in which the culture medium. pC194 and pE194 were pretransferred into strain TA208 to attenuate the host restriction effect.135 Electroporation of recalcitrant B.17 104 2. Res. Biotechnol. Supplementary data Supplementary data associated with this article can be found. J. E. A simple method to introduce marker-free genetic modifications into the chromosome of naturally nontransformable Bacillus amyloliquefaciens strains. Sukumaran. A. Biotechnol. [6] A. suggesting that the restriction–modification enzymes other than the BamHI system exist in strain TA208. J. Biochem. 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