Optimization and Comparison of Different Methods and Factors for Efficient Transformation of Brucella abortus RB51strain

Gheibi, Azam; Khanahmad, Hossein; Ali Kardar, Gholam; Boshtam, Maryam; Rezaie, Sassan; Kazemi, Bahram; Khorramizadeh, Mohammad Reza

Optimization and Comparison of Different Methods and Factors for Efficient Transformation of Brucella abortus RB51strain

Document Type : Original Article

Authors

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

Abstract

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

References

1.	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.
2.	Gheibi A, Khanahmad H, Kashfi K, Sarmadi M, Khorramizadeh MR. Development of new generation of vaccines for Brucella abortus. Heliyon 2018;4:e01079.
3.	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.
4.	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.
5.	Smith LD, Heffron F. Transposon Tn5 mutagenesis of Brucella abortus. Infect Immun 1987;55:2774-6.
6.	Ficht TA, Pei J, Kahl-McDonagh M.In vitromutagenesis of Brucella species. Methods Mol Biol 2010;634:15-35.
7.	Steele C, Zhang S, Shillitoe EJ. Effect of different antibiotics on efficiency of transformation of bacteria by electroporation. Biotechniques 1994;17:360-5.
8.	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.
9.	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.
10.	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.
11.	Errington J. Cell wall-deficient, L-form bacteria in the 21^st century: A personal perspective. Biochem Soc Trans 2017;45:287-95.
12.	Gebicki JM, James AM. The preparation and properties of spheroplasts of Aerobacter aerogenes. J Gen Microbiol 1960;23:9-18.
13.	Spicer AB, Spooner DF. The inhibition of growth of Escherichia coli spheroplasts by antibacterial agents. J Gen Microbiol 1974;80:37-50.
14.	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.
15.	Hammes W, Schleifer KH, Kandler O. Mode of action of glycine on the biosynthesis of peptidoglycan. J Bacteriol 1973;116:1029-53.
16.	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.
17.	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.
18.	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.
19.	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.
20.	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.
21.	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.
22.	Baughn RE, Freeman BA. Antigenic structure of Brucella suis spheroplasts. J Bacteriol 1966;92:1298-303.
23.	Suzuki M, Szalay A. Recombinant DNA. In: Wu R, editor. Methods in Enzymology. Vol. 68. New York: Academic Press; 1979.
24.	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.
25.	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.
26.	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.
27.	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.
28.	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.
29.	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.
30.	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.
31.	Kawata Y, Yano S, Kojima H. Escherichia coli can be transformed by a liposome-mediated lipofection method. Biosci Biotechnol Biochem 2003;67:1179-81.
32.	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.
33.	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.
34.	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.
35.	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.
36.	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.
37.	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.

Advanced Biomedical Research