Optimization and Comparison of Different Methods and Factors for Efficient Transformation of Brucella abortus RB51strain
Azam Gheibi1, Hossein Khanahmad2, Gholam Ali Kardar3, Maryam Boshtam4, Sassan Rezaie5, Bahram Kazemi6, Mohammad Reza Khorramizadeh7
1 Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
3 Department of Medical Biotechnology, School of Advanced Technologies in Medicine; Department of Immunology, Asthma and Allergy Research Institute, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran
4 Isfahan Cardiovascular Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran
5 Department of Mycology and Parasitology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
6 Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
7 Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
|Date of Web Publication||29-May-2019|
Prof. Mohammad Reza Khorramizadeh
Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, P.O. Box: 1411413137, Tehran
Source of Support: None, Conflict of Interest: None
Background: The development of protective vaccines for Brucella spp. has been hampered by the difficulty in transformation of Brucella cells with foreign DNA for genetic manipulation. It seems that the formation of Brucella spheroplasts would increase the efficiency of transformation. The aim of this study was to devise an efficient method for the transformation of Brucella spp. Materials and Methods: At first, spheroplast of Brucella was prepared by glycine and ampicillin induction and transformed using optimized protocols of CaCl2, electroporation, and lipofection methods. Then, the efficacy of transformation was compared between the three-mentioned methods. Results: Ampicillin-induced spheroplasts from early-log phase culture of brucella when incubated in a medium-containing 0.2 M sucrose during cell recovery had higher transformation efficiency in three different methods. Comparison of the transformation efficiency of Brucella abortus RB51 using the CaCl2, lipofection, and electroporation methods revealed that the transformation efficiency with the lipofection method was significantly higher than with other two methods (P < 0.05). Conclusions: Lipofection method by lipofectamine 2000 on ampicillin-induced spheroplasts can be a suitable approach for Brucella transformation.
Keywords: Ampicillin, Brucella, lipofectamine 2000, lipofection, spheroplasts, sucrose
|How to cite this article:|
Gheibi A, Khanahmad H, Kardar GA, Boshtam M, Rezaie S, Kazemi B, Khorramizadeh MR. Optimization and Comparison of Different Methods and Factors for Efficient Transformation of Brucella abortus RB51strain. Adv Biomed Res 2019;8:37
|How to cite this URL:|
Gheibi A, Khanahmad H, Kardar GA, Boshtam M, Rezaie S, Kazemi B, Khorramizadeh MR. Optimization and Comparison of Different Methods and Factors for Efficient Transformation of Brucella abortus RB51strain. Adv Biomed Res [serial online] 2019 [cited 2019 Dec 8];8:37. Available from: http://www.advbiores.net/text.asp?2019/8/1/37/259376
| Introduction|| |
Brucella More Details genus is Gram-negative intracellular microbes that cause Brucellosis More Details, which can result in abortion in domestic animals and undulant fever in human. Brucellosis is the most common zoonotic disease and causes considerable economic losses in the world livestock industry; a potential concern also exists about the use of Brucella as bioterrorism weapons., Hence, the development of sensitive and specific molecular diagnostic methods and effective vaccines to eradicate brucellosis are required. There are some limitations to develop recombinant Brucella, because of the difficulties associated through gene delivery and targeted gene deletion, insertion, or genetic manipulation of Brucella.,
No evidence has yet been presented for the existence of a natural genetic transformation system in Brucella spp.; hence, the transformation of these cells has to be induced by artificial methods. An electroporation protocol has been used for many years as a current approach to transform Brucella spp. Nevertheless, transformation yield of these methods is insufficient, especially when homologous or nonhomologous recombination of DNA is desired and selected by antibiotics.,, Optimization of a high-efficiency gene delivery system or method for Brucella spp. would facilitate genetic manipulation of these microbes.
The cell wall and outer membrane of bacteria comprise the main barriers for DNA uptake and transformation. Using Brucella spheroplasts, which lack the cell wall and most of the outer membrane,, might be the best solution for the described transformation problems. At present, several cell-wall-weakening agents exerting different effects on the cell wall are widely used to enhance the transformation efficiency of bacteria.,,
Cationic liposomes are being widely used to transform eukaryotic cells (lipofection); this method is characterized by high efficiency and safety., Cationic lipid subunits through liposome formation entrap the negatively charged DNA molecules. The DNA-lipid complex overcomes the electrostatic repulsion of the cell membrane, and the cationic lipid fuses with the cell membrane lipids, and finally, the DNA molecule enters to the cell.,
We hypothesized that cationic liposomes could be used for the transformation of Brucella spheroplasts, that is why the inner membrane is exposed and might promote transformation efficiency. To test this, Brucella spheroplasts were formed and transformed with foreign DNA using three different methods, including chemical (CaCl2), physical (electroporation), and lipofection method. In an effort to increase the transformation efficiency, the impact of some parameters on the efficiency of the three methods was examined.
| Materials and Methods|| |
Bacterial strains, growth conditions, and plasmids
Escherichia More Details coli TOP10F' strain was grown at 37°C in Luria-Bertani (LB) broth or agar (Merck, Germany) containing 80 μg/mL of tetracycline. Brucella abortus RB51 strain was grown at 37°C in trypticase soy agar (TSA) or trypticase soy broth (TSB) (Quelab, Canada) supplemented with 50 μg/mL of rifampin and kanamycin or 50–100 μg/mL of ampicillin Plasmids used in this study are listed in [Table 1]. Plasmid pUC57/pBBR1ori (about 4 kb, bearing an ampicillin resistance gene) containing the origin of replication of pBBR1 plasmid and is able to replicate in Brucella spp.,, pBBR1ori fragment (1.7 kb) was designed and ordered to synthetize to Gene cust company (Gene cust, Luxembourg) and cloned in pUC57 using EcoRI and NdeI restriction enzymes. Furthermore, from plasmid pBGGT/ΔureC/kana/LLO which was prepared in another study (unpublished data), EcoRI and NdeI digested fragment-containing ΔureC/kana/LLO (5.3 kb) was separated and ligated to PBBR1ori fragment and pBBR1ori/ΔureC/kana/LLO (7 kb bearing a kanamycin resistance gene) was constructed.
The plasmids were extracted by Miniprep extraction kit (SolGent, Korea) and dissolved in ddH2O and quantified by nanodrop spectrophotometer (Nanolytik, Germany).
Some factors such as the concentrations of ampicillin, glycine, and sucrose solutions used for spheroplast induction, culture growth phase, and the time of spheroplast formation were optimized.
A single colony of B. abortus RB51 was grown in 5 mL of TSB supplemented with rifampin at 37°C and shaking at 200 rpm until the optical density at 600 nm (OD600) reached 0.2–0.4. Then, 200–300 mL of fresh TSB-containing rifampin was added, and the culture was incubated at 37°C with shaking for 24 h until OD600 intended for each transformation method was reached. The bacteria were pelleted by centrifugation, and the cells were added to TSB-containing ampicillin (10 μg/mL) with 0.2 M sucrose or TSB-containing 2% glycine and 0.2 M sucrose. Ampicillin and glycine were used as spheroplast induction agents. Glycine or ampicillin-containing media without sucrose were prepared and used as negative control treatments of the osmotic protection step. The treated cultures were incubated at 37°C with gentle rotation (80 rpm) for 24 h. Spheroplast formation was observed using phase contrast microscope at the end of the incubation period.
Transformation with cationic lipid method
Once the glycine- and ampicillin-induced spheroplasts (OD600 = 0.2–0.4) were prepared, the cells (50 mL) were chilled on ice for 30 min, centrifuged at 1000 ×g for 10 min, and washed 3–5 times with ice-cold DDW-containing 0.2 M sucrose. The spheroplasts were then suspended in 10 mL of ice-cold ddW-containing 0.2 M sucrose. Nearly 2 μL of each plasmid DNA (pUC57/pBBR1ori and pBBR1ori/ΔureC/kana/LLO, 1 μg/mL) and an optimized volume of 5 μL lipofectamine 2000 reagent (Invitrogen, USA) were mixed and incubated for 20 min at room temperature. A solution of each plasmid (1 μg/μg) without the cationic lipid was also prepared as a control. The prepared spheroplasts (100 μL) were added to the DNA-cationic lipid complex solutions and mixed by gently pipetting. The mixtures were then kept on ice for 1 h. Next, each mixture was split into two aliquots; one portion was transferred to 1 mL of TSB-containing 0.2 M sucrose (to continue the osmotic protection); the other portion was transferred to 1 mL TSB without sucrose. All tubes were incubated at 37°C for 6 h without shaking for cell recovery. Ampicillin or kanamycin (the related resistance genes were encoded by plasmids used in the study) were added to all tubes, to select transformants, and the incubation continued at 37°C for 24 h with gentle shaking. Finally, all cells were transferred to TSA plates-containing antibiotics, incubated at 37°C for 48 h, and the resulted colonies were counted. Transformation of nonspheroplast B. abortus cells was performed similarly.
The CaCl2 method for transformation
At first optimization of the culture growth phase, CaCl2 concentration, heat-shock duration, and competent cell induction were performed. Once glycine- and ampicillin-induced spheroplast (50 mL) was formed (OD600 = 0.2–0.4), the bacteria were chilled on ice for 30 min, centrifuged at 1000 ×g for 10 min, and washed five times with ice-cold DDW-containing 0.2 M sucrose. The pelleted spheroplasts were resuspended in 5 mL of 0.1 M CaCl2 or 0.2 M CaCl2 supplemented with 0.2 M sucrose and incubated on ice for 30 min. The spheroplasts were centrifuged at 1000 ×g for 10 min, resuspended in 2 mL of 0.1 and 0.2 M CaCl2-containing 0.2 M sucrose, and incubated on ice for 30 min; the cells were pelleted again and gently resuspended in 1 mL of 0.1 or 0.2 M CaCl2 solution with 0.2 M sucrose. The competent spheroplasts (100 μL) were gently mixed with 1 μg/μg of each plasmid DNA solution (pUC57/ori or pBBR1ori/ΔureC/kana/LLO) and incubated on ice for 30 min. This was followed by a heat-shock, 2 min at 42°C; the cells were immediately incubated on ice for 2 min. Next, each batch of transformed competent cells was split into two portions. One portion was added to 1 mL of TSB-containing 0.2 M sucrose (to continue the osmotic protection), the other portion was added to 1 mL of TSB without sucrose. All tubes were incubated at 37°C for 6 h without shaking to allow the cells to recover. Ampicillin or kanamycin was added depending on the plasmid used for transformation; the incubation was continued at 37°C for 24 h with gentle shaking. Finally, all transformed cells were spread on TSA plates containing the appropriate antibiotics, incubated at 37°C for 48 h, and the transformants were counted. Spheroplasts that had not been treated by CaCl2 were used as a negative control. Transformation of nonspheroplast B. abortus cells was performed in the same condition.
Three 250 mL cell culture samples were grown to different OD600 values, i.e. early-log phase (OD600 = 0.1–0.4), mid-log phase (OD600 = 0.4–0.7), and late-log phase (OD600 = 0.7–1.0). The cultures were treated by glycine or ampicillin, with or without sucrose, to induce spheroplast formation as described above. To prepare electrocompetent cells, spheroplast and nonspheroplast cells were chilled on ice for 30 min. The spheroplasts were centrifuged at 1000 ×g for 10 min, washed three times with ice-cold ddH2O-containing 0.2 M sucrose, and two times with an ice-cold solution containing 10% glycerol 0.2 M sucrose. Finally, the cells were resuspended in 1 mL of 10% glycerol supplemented with 0.2 M sucrose. Electro-competent cells were prepared from nonspheroplast cells as above, except that sucrose was excluded from washing solutions. Immediately before electroporation, 100 μL of competent cells were mixed with 2 μL of two different plasmid solutions (pUC57/pBBR1ori or pBBR1ori/ΔureC/kana/LLO, 1 μg/mL). The mixtures were loaded into prechilled 2-mm gap width cuvettes, and electroporation was performed using a Gene Pulser (Bio-Rad, USA) set at 2.5 kV, 25 μF, and 600 W. After a single electrical pulse, the cells were immediately diluted with 1 mL of TSB with or without 0.2 M sucrose and incubated at 37°C for 24 h without shaking to allow cell recovery. The cells were then transferred to 1 mL of TSB (with or without sucrose, with an appropriate antibiotic) and incubated at 37°C by shaking for 24 h. Finally, the cells were harvested and plated on TSA plates supplemented with ampicillin or kanamycin. The transformants were counted following 48–72 h of incubation at 37°C.
The data were analyzed using the SPSS software (version 17.0, Chicago, IL, USA). The experiments were repeated five times, and the means and standard deviations of transformation efficiencies were calculated for each method. Independent t-test and one-way ANOVA were used for the comparison of transformation efficiencies with different treatments within and between the three different transformation methods. A value ofP < 0.05 was considered as statistically significant.
| Results|| |
Spheroplast formation was induced in a 24 h culture of B. abortus RB51 grown in TSB with shaking at 37°C, with either glycine or ampicillin and sucrose, as inducing agents. Microscopic observations revealed spherical morphology of >95% of cells treated with ampicillin and about 75% of cells treated with glycine were converted into spheroplasts. Complete lysis of bacterial cells was observed in the control groups, in the absence of sucrose.
The result of cationic lipid method for bacterial transformation
The following parameters were optimized for this transformation method: mixing temperature and incubation time of the spheroplast and DNA-cationic lipid complex; the concentration of the cationic lipid used during transformation; the number of spheroplast washes before transformation; and the recovery time after transformation.
In the current study, nonspheroplast and glycine- and ampicillin-induced spheroplasts of B. abortus RB51 were transformed with two different plasmid-cationic liposome complexes. We attempted at least ten times transformations of nonspheroplast B. abortus cells by the lipofection method, but no transformants were obtained. Moreover, no transformants were observed when DNA was mixed with spheroplast cells without the cationic lipid as a control. In [Figure 1], the details of the transformation efficiency (cfu/μg of DNA) of spheroplasts transformed using the lipofection method in different treatment conditions were shown. All spheroplast groups were successfully transformed with a reasonable efficiency compared with control and nonspheroplast cells (P < 0.05 for all treated groups). The transformation efficiency of ampicillin-induced spheroplasts transformed with 4- or 7-kb plasmid-liposome complexes and incubated during cell recovery in a medium-containing 0.2 M sucrose were significantly higher than that of glycine-induced spheroplasts treated similarly (P < 0.001). The transformation efficiency of ampicillin-induced spheroplasts treated with a 4-kb plasmid-liposome complex and incubated in a medium without sucrose was significantly higher than glycine-induced spheroplasts that underwent the same treatment (P = 0.035). The transformation efficiency of ampicillin-induced spheroplasts transformed with a 7-kb DNA-liposome complex and incubated in the absence of sucrose was significantly higher than glycine-induced spheroplasts treated in the same manner (P < 0.001). The transformation efficiency of ampicillin-induced spheroplasts transformed by a 4- or 7-kb plasmid-liposome complex was significantly higher when sucrose was included in the recovery medium than in the absence of sucrose (P < 0.001). The same was true for glycine-induced spheroplasts (P = 0.008 for the 4-kb plasmid-liposome complex, andP = 0.001 for the 7-kb plasmid-liposome complex). For both ampicillin- and glycine-induced spheroplasts, transformation with a 4-kb plasmid-liposome complex showed higher efficiency than transformation with a 7-kb plasmid-liposome complex, regardless of the presence of sucrose in the recovery medium (P < 0.001). These data clearly indicated that spheroplast formation is prerequisite for the transformation of B. abortus RB51 by this method.
|Figure 1: The effect of different treatments of Brucella abortus transformation efficiency using the lipofection method. Results are mean ± standard error of the mean. *P < 0.05 compared to the ampicillin/sucrose (+)/plasmid 4 kb, †P < 0.05 compared to the ampicillin/sucrose (+)/plasmid 7 kb, ‡P < 0.05 compared to the ampicillin/sucrose (-)/plasmid 4 kb, #P < 0.05 compared to the ampicillin/sucrose (-)/plasmid 7 kb. Plasmids: 4 kb, pUC57/pBBR1ori; 7 kb, pBBR1ori/ΔureC/kana/LLO|
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The result of CaCl2 method
Transformation results of spheroplasts that had not been treated by CaCl2 as a control and ampicillin or glycine-induced spheroplasts of B. abortus transformed by 0.1 M CaCl2 were negative. The transformation efficiency of B. abortus RB51 spheroplasts (by both ampicillin and glycine methods) using the 0.2 M CaCl2 method is presented in [Figure 2]. There were no significant differences between the performances of ampicillin- or glycine-induced spheroplasts in the 0.2 M CaCl2 transformation method, even though the average efficiency of transformation was somewhat higher for ampicillin-induced spheroplasts (P > 0.05). The transformation efficiency of ampicillin-induced spheroplasts treated with 0.2 M CaCl2 transformed by a 4-kb plasmid and recovered in a medium-containing sucrose was not significantly different than that for glycine-induced spheroplasts treated in an analogous manner (P = 0.17). The transformation efficiency, of ampicillin-induced spheroplast transformed by a 4-kb plasmid incubated in a medium without sucrose, was not significantly different than that for glycine-induced spheroplasts that were submitted to the same treatment (P = 0.17). The transformation efficiencies, of both ampicillin- and glycine-induced spheroplasts transformed by the 4-kb plasmid, were significantly higher in sucrose supplemented recovery medium than in sucrose devoid recovery medium (P = 0.037). The transformation efficiencies of both ampicillin- and glycine-induced spheroplasts transformed by the 4-kb plasmid were higher for transformation with the 7-kb plasmid, regardless of the presence of sucrose in the recovery medium (P < 0.001). Transformation efficacy of both ampicillin and glycine spheroplasts treated 0.2 M CaCl2 and 7-kb plasmid was negative.
|Figure 2: The effect of different treatments of Brucella abortus on transformation efficiency using the 0.2 M CaCl2 method. Results are mean ± standard error of the mean. *P < 0.05 compared to the ampicillin/sucrose (+)/plasmid 4 kb, ‡P < 0.05 compared to the ampicillin/sucrose (-)/plasmid 4 kb. Plasmids: 4 kb, pUC57/pBBR1ori; 7 kb, pBBR1ori/ΔureC/kana/LLO|
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The result of electroporation
Nonspheroplast cells and glycine- and ampicillin-induced spheroplast of B. abortus RB51 prepared from three cultures with different OD600 values were electroporated in the presence of two different plasmids. The results are shown in [Figure 3] indicate that the electroporation of spheroplasts was more efficient than electroporation of nonspheroplast cells (P < 0.05). The electroporation efficiencies of ampicillin- and glycine-induced spheroplasts prepared from cultures at OD600 of 0.1–0.4 or 0.4–0.7 were significantly higher than those of nonspheroplast cells (P < 0.001). The electroporation efficiency of ampicillin- and glycine-induced spheroplasts induced from cultures at OD600 of 0.7–1.0 was significantly higher than those of nonspheroplast cells (P = 0.004). In addition, the use of early-log phase cultures (OD600 = 0.1–0.4) resulted in higher electroporation efficiency than when mid- and late-log phase cultures were used, for both spheroplast and nonspheroplast cells (P < 0.001 for ampicillin- or glycine-induced spheroplasts, andP = 0.008 for nonspheroplast cells). The independent t-test revealed no significant differences between the use of mid- and late-log phase cultures for nonspheroplasts and glycine-induced spheroplasts despite a high average electroporation efficiency when mid-log phase cultures were used (P = 0.24 for glycine spheroplast, andP = 0.056 for nonspheroplast cells). On the other hand, the electroporation efficiency of ampicillin-induced spheroplasts from mid-log phase cultures was significantly higher than for late-log phase culture spheroplasts (P = 0.044).
|Figure 3: Comparison of electroporation efficiencies of nonspheroplast and spheroplasts of Brucella abortus prepared from cultures grown to three different OD600 values. Nonspheroplast and glycine and ampicillin-induced spheroplasts were prepared and electroporated. The results are mean ± standard error of the mean and &P < 0.05. The same symbols indicate statistically significant differences (P < 0.05) between the groups|
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As shown in [Figure 4], ampicillin induction resulted in higher electroporation efficiency than glycine treatment (P < 0.05); the only exception was when ampicillin-induced spheroplasts were electroporated with a 7-kb plasmid and incubated in a medium without sucrose: compared with glycine-induced spheroplasts that were submitted to the same treatment, no statistically significant differences were observed, despite a higher average electroporation efficiency of ampicillin-induced spheroplasts (P = 0.35). The electroporation efficiency of ampicillin-induced spheroplasts electroporated with a 4-kb plasmid and incubated in a medium-containing sucrose was enhanced compared with glycine-induced spheroplasts that underwent the same treatment (P < 0.001); the same was observed when no sucrose was present in the recovery medium (P = 0.002). The electroporation efficiency of ampicillin-induced spheroplasts electroporated with a 7-kb plasmid and incubated in a medium-containing sucrose was significantly higher than for glycine-induced spheroplasts treated in an analogous manner (P = 0.001). In all treatment groups, the inclusion of sucrose in the recovery medium after electroporation significantly increased the efficiency of electroporation compared with medium lacking sucrose (P < 0.001). Furthermore, in all treatment groups, the electroporation efficiency was also significantly increased when the 4-kb plasmid was used rather than the 7-kb plasmid (P < 0.001).
|Figure 4: The effect of different treatments of Brucella abortus on the electroporation efficiency. Results are mean ± standard error of the mean. *P < 0.05 compared to the ampicillin/sucrose (+)/plasmid 4 kb, †P < 0.05 compared to the ampicillin/sucrose (+)/plasmid 7 kb, ‡P < 0.05 compared to the ampicillin/sucrose (-)/plasmid 4 kb. Plasmids: 4 kb, pUC57/pBBR1ori; 7 kb, pBBR1ori/ΔureC/kana/LLO|
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Comparison of the different cell treatments in the three optimized transformation methods revealed that the lipofectamine method resulted in the highest transformation efficiency of all treatment groups (P < 0.001) [Figure 5].
|Figure 5: Comparison of the efficiency mean value of 10-time transformation of Brucella abortus RB51 using the CaCl2, lipofectamine, and electroporation methods. Results are mean ± standard error of the mean and *P < 0.05. Am: Ampicillin, Su: Sucrose, P: Plasmid|
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| Discussion|| |
Because of the difficulty in transforming Brucella spp. with foreign DNA, optimization of an efficient DNA transfer method for the introduction of exogenous genes into Brucella cells is the first step for carrying out genetic manipulation. Hence, we optimized and compared three different transformation methods and the effect of several parameters that may have potentially improved the transformation. In the current study, the induction of spheroplasts was achieved by cell-wall-weakening and osmotic protection agent. The transformation efficiencies of the three-mentioned methods were significantly higher with spheroplasts than with nonspheroplast cells. The obtained results supported our initial hypothesis that the formation of spheroplasts as an approach to overcome the cell wall as a barrier would improve the exogenous DNA uptake by B. abortus. Similar results have been obtained by Liu et al. in a study of the transformation ofE. colispheroplasts through the chemical CaCl2 method and in several other studies with different bacteria.,,,,, However, some researchers reported that cell-wall-weakening either had no effect on the transformation frequency. It seems that type of bacteria, cell-wall-weakening agents for spheroplast formation, and the transformation method used should be affected on the result. Based on our findings, the use of ampicillin for spheroplast induction resulted in a higher percentage of Brucella cells converting to spheroplasts than glycine induction; further, the efficiency of transformation of ampicillin-induced spheroplasts was significantly higher than for glycine-induced spheroplast after lipofection or electroporation. The same was observed by Zhang et al. who investigated the transformation of Bacillus subtilis ZK. Ampicillin induces Brucella spheroplast formation via the degradation of the peptidoglycan structure; while glycine by replacing alanine in the peptidoglycan of cell wall causes peptidoglycan cross-linking and susceptibility to spheroplast formation.,
Another important finding of the current study is the notion that the cationic liposome method resulted in the highest transformation efficiency of Brucella spheroplasts while no effect was observed for the nonspheroplast Brucella cells. Transformation of E. coli and the archaeon Methanosarcina was also successfully achieved with this method. In the current study, the transformation efficiency of ampicillin-induced spheroplasts transformed by the lipofection method was significantly higher than that of glycine-induced spheroplasts.
In contrast with the lipofection method, both nonspheroplast and spheroplast forms of B. abortus could be transformed by electroporation. However, the efficiency of electroporation was higher for spheroplasts than for nonspheroplast cells. Several previous studies documented that the use of a cell-wall weakening agent in culture before electroporation improves the transformation efficiency of cells.,, The growth phase of the bacteria was another factor examined in the current study; the most electroporation efficiency was observed in the early-log phase and the least one in the late-log phase. It seems that during the early-log phase, the cells are growing most rapidly, with the most dynamic cell wall and poorer cell-wall intensity that render them competent for a physical or chemical transformation. Previous studies have reported conflicting observations for different bacteria., In Bacillus thuringiensis and Bacillus cereus, similarly to our result, the use of early-log phase (OD600 = 0.1–0.4) cultures resulted in the highest electroporation efficiency;, in Bacillus amyloliquefaciens and B. subtilis IH6140, the late-log phase (OD600 = 0.7–1.0) culture cells had the highest electroporation efficiency., In the current study, comparison of the electroporation and lipofection revealed that the efficiency of lipofection was higher than electroporation. In addition, the incubation and recovery time of cells after transformation using the optimized electroporation method were longer than in the cationic lipid method. A probable explanation for these results may be the electrical shock of cells submitted in electroporation which cause more injury in the cell wall.
We also observed that using a high concentration of CaCl2 had no significant effect on transformation efficiency of B. abortus. In a report by Li et al., transformation efficiency ofE. coliDH5α significantly decreased when the concentration of CaCl2 exceeded 200 mM.
Another surprising observation was that repeated washing and addition of 0.2 M sucrose to the washing solution and recovery medium significantly enhanced the transformation efficiency in the three transformation methods. One possible explanation is that slow-growing Brucella cells, especially in spheroplast form, require a long time to grow and convert to the normal, nonspheroplast form. Osmotic protection afforded by sucrose, therefore, aids this process. Two previous studies examined the effect of osmotic protection of sucrose in the culture medium and treatment solutions before transformation and evidenced increased transformation efficiency in the presence of sucrose., The effect of sucrose in media after the transformation has not been examined. Finally, we observed that the transformation efficiency significantly decreased with increased plasmid size. A similar observation was reported by other studies.
| Conclusions|| |
The current study showed that employment of optimized lipofectamine protocol using ampicillin-induced spheroplasts form of Brucella could be the best alternative for the electroporation method in Brucella transformation.
We are grateful to the deputy of research at Tehran University of Medical Sciences for their generous support.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Smith GP, Jain-Gupta N, Alqublan H, Dorneles EM, Boyle SM, Sriranganathan N, et al.
Development of an auxotrophic, live-attenuated Brucella suis
vaccine strain capable of expressing multimeric GnRH. Vaccine 2019;37:910-4.
Gheibi A, Khanahmad H, Kashfi K, Sarmadi M, Khorramizadeh MR. Development of new generation of vaccines for Brucella abortus
. Heliyon 2018;4:e01079.
Kornspan D, Salmon-Divon M, Zahavi T. Transcriptomic analysis of the Brucella melitensis
Rev. 1 vaccine strain in an acidic environment: Insights into virulence attenuation. Front Microbiol 2019;10:250.
Halling SM, Detilleux PG, Tatum FM, Judge BA, Mayfield JE. Deletion of the BCSP31 gene of Brucella abortus
by replacement. Infect Immun 1991;59:3863-8.
Smith LD, Heffron F. Transposon Tn5 mutagenesis of Brucella abortus
. Infect Immun 1987;55:2774-6.
Ficht TA, Pei J, Kahl-McDonagh M.In vitro
mutagenesis of Brucella
species. Methods Mol Biol 2010;634:15-35.
Steele C, Zhang S, Shillitoe EJ. Effect of different antibiotics on efficiency of transformation of bacteria by electroporation. Biotechniques 1994;17:360-5.
Lee MH, Pascopella L, Jacobs WR Jr., Hatfull GF. Site-specific integration of mycobacteriophage L5: Integration-proficient vectors for Mycobacterium smegmatis
, Mycobacterium tuberculosis
, and bacille calmette-guérin. Proc Natl Acad Sci U S A 1991;88:3111-5.
Pelicic V, Jackson M, Reyrat JM, Jacobs WR Jr., Gicquel B, Guilhot C, et al.
Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis
. Proc Natl Acad Sci U S A 1997;94:10955-60.
Holo H, Nes IF. High-frequency transformation, by electroporation, of Lactococcus lactis
subsp. Cremoris grown with glycine in osmotically stabilized media. Appl Environ Microbiol 1989;55:3119-23.
Errington J. Cell wall-deficient, L-form bacteria in the 21st
century: A personal perspective. Biochem Soc Trans 2017;45:287-95.
Gebicki JM, James AM. The preparation and properties of spheroplasts of Aerobacter aerogenes
. J Gen Microbiol 1960;23:9-18.
Spicer AB, Spooner DF. The inhibition of growth of Escherichia coli
spheroplasts by antibacterial agents. J Gen Microbiol 1974;80:37-50.
Hermans J, Boschloo J, De Bont J. Transformation of Mycobacterium aurum
by electroporation: The use of glycine, lysozyme and isonicotinic acid hydrazide in enhancing transformation efficiency. FEMS Microbiol Lett 1990;72:221-4.
Hammes W, Schleifer KH, Kandler O. Mode of action of glycine on the biosynthesis of peptidoglycan. J Bacteriol 1973;116:1029-53.
Zhao M, Yang H, Jiang X, Zhou W, Zhu B, Zeng Y, et al.
Lipofectamine RNAiMAX: An efficient siRNA transfection reagent in human embryonic stem cells. Mol Biotechnol 2008;40:19-26.
Muripiti V, Brijesh L, Rachamalla HK, Marepally SK, Banerjee R, Patri SV. α -Tocopherol-ascorbic acid hybrid antioxidant based cationic amphiphile for gene delivery: Design, synthesis and transfection. Bioorg Chem 2019;82:178-91.
Dalby B, Cates S, Harris A, Ohki EC, Tilkins ML, Price PJ, et al.
Advanced transfection with lipofectamine 2000 reagent: Primary neurons, siRNA, and high-throughput applications. Methods 2004;33:95-103.
Felgner JH, Kumar R, Sridhar CN, Wheeler CJ, Tsai YJ, Border R, et al.
Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J Biol Chem 1994;269:2550-61.
Obranić‡ S, Babić‡ F, Maravić‡-Vlahoviček. Improvement of pBBR1MCS plasmids, a very useful series of broad-host-range cloning vectors. Plasmid 2013;70:263-7.
Ouahrani-Bettache S, Porte F, Teyssier J, Liautard JP, Köhler S. PBBR1-GFP: A broad-host-range vector for prokaryotic promoter studies. Biotechniques 1999;26:620-2.
Baughn RE, Freeman BA. Antigenic structure of Brucella suis
spheroplasts. J Bacteriol 1966;92:1298-303.
Suzuki M, Szalay A. Recombinant DNA. In: Wu R, editor. Methods in Enzymology. Vol. 68. New York: Academic Press; 1979.
Lee SH, Cheung M, Irani V, Carroll JD, Inamine JM, Howe WR, et al.
Optimization of electroporation conditions for Mycobacterium avium
. Tuberculosis (Edinb) 2002;82:167-74.
Zhang Z, Ding ZT, Shu D, Luo D, Tan H. Development of an efficient electroporation method for iturin A-producing Bacillus subtilis
ZK. Int J Mol Sci 2015;16:7334-51.
Dorella FA, Estevam EM, Cardoso PG, Savassi BM, Oliveira SC, Azevedo V, et al.
An improved protocol for electrotransformation of Corynebacterium pseudotuberculosis
. Vet Microbiol 2006;114:298-303.
Zhang GQ, Bao P, Zhang Y, Deng AH, Chen N, Wen TY, et al.
Enhancing electro-transformation competency of recalcitrant Bacillus
amyloliquefaciens by combining cell-wall weakening and cell-membrane fluidity disturbing. Anal Biochem 2011;409:130-7.
Peng D, Luo Y, Guo S, Zeng H, Ju S, Yu Z, et al.
Elaboration of an electroporation protocol for large plasmids and wild-type strains of Bacillus thuringiensis
. J Appl Microbiol 2009;106:1849-58.
Liu I, Liu M, Shergill K. The effect of spheroplast formation on the transformation efficiency in Escherichia coli
DH5. J Exp Microbiol Immunol 2006;9:81-5.
Katayama N, Takano H, Sugiyama M, Takio S, Sakai A, Tanaka K, et al.
Effects of antibiotics that inhibit the bacterial peptidoglycan synthesis pathway on moss chloroplast division. Plant Cell Physiol 2003;44:776-81.
Kawata Y, Yano S, Kojima H. Escherichia coli
can be transformed by a liposome-mediated lipofection method. Biosci Biotechnol Biochem 2003;67:1179-81.
Metcalf WW, Zhang JK, Apolinario E, Sowers KR, Wolfe RS. A genetic system for Archaea
of the genus Methanosarcina
: Liposome-mediated transformation and construction of shuttle vectors. Proc Natl Acad Sci U S A 1997;94:2626-31.
Kim YH, Kwon SG, Bae SJ, Park SJ, Im DJ. Optimization of the droplet electroporation method for wild type Chlamydomonas reinhardtii
transformation. Bioelectrochemistry 2019;126:29-37.
Xue GP, Johnson JS, Dalrymple BP. High osmolarity improves the electro-transformation efficiency of the Gram-positive bacteria Bacillus subtilis
and Bacillus licheniformis
. J Microbiol Methods 1999;34:183-91.
Turgeon N, Laflamme C, Ho J, Duchaine C. Elaboration of an electroporation protocol for Bacillus cereus
ATCC 14579. J Microbiol Methods 2006;67:543-8.
Zhang H, Li Y, Chen X, Sheng H, An L. Optimization of electroporation conditions for Arthrobacter
with plasmid PART 2. J Microbiol Methods 2011;84:114-20.
Li X, Sui X, Zhang Y, Sun Y, Zhao Y, Zhai Y, et al
. An improved calcium chloride method preparation and transformation of competent cells. Afr J Biotechnol 2010;9:8549-54.
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