Brenda K. Scholz-Schroeder. Department of Plant Pathology, Washington State University, Pullman, WA 99163-6430. email@example.com
Scholz-Schroeder, B.K. 2001. Electroporation and marker exchange mutagenesis of Pseudomonas syringae pv. syringae. The Plant Health Instructor. DOI: 10.1094/PHI-I-2001-0424-01
transformation, plant-pathogenic bacteria, homologous recombination
To become familiar with the technique of electroporation for the introduction of plasmids into P. syringae
To become familiar with the important parameters of electroporation and how they affect transformation efficiency of various strains of P. syringae pv. syringae.
To become familiar with how electroporation can be used to introduce mutated genes into the genome of P. syringae pv. syringae via homologous recombination.
Electroporation is a method of transformation that allows the introduction of foreign DNA into host cells (prokaryotic or eukaryotic) via the application of high-voltage electric pulses. The electric field induces pore formation in the cell wall and increases the permeability of the host cells to macromolecules, which allows for the uptake of DNA (8). Electroporation is a common method for the introduction of foreign DNA, such as plasmids, into Escherichia coli,
and electroporation is being used increasingly for transformation of plant-pathogenic bacteria (2,4,8,11).
The ability to introduce foreign DNA into plant-pathogenic bacteria by electroporation is a powerful tool for studying pathogenicity at the molecular level (2,15). The first step in resolving the function of a gene is to mutate or disrupt the gene. The gene of interest can be disrupted by transposon mutagenesis or by site-directed insertional mutagenesis. The disrupted gene can then be introduced by transformation into the wild type organism by electroporation. Through the process of homologous recombination, the mutated gene can be recombined into the genome to disrupt the function of the wild-type gene (7). The resulting mutant can then be evaluated for a specific phenotype due to the presence of the nonfunctional gene.
Frequently, a biochemical assay is required to demonstrate activity for a specific protein. This usually can be accomplished by the overexpression of cloned genes in E. coli for protein isolation. There are some proteins, however, that must be overexpressed in the bacterium of origin to be active and functional, making it necessary to introduce overexpression vectors carrying cloned genes into bacterial strains other than E. coli (9). Previously, triparental mating was used for the introduction of plasmids into plant-pathogenic bacteria. Triparental mating requires a helper strain, carrying the genes that code for conjugation and DNA transfer, and a donor strain, carrying the plasmid to be introduced into the new bacterial strain. At least five to seven days are required in order to determine if the plasmid was successfully introduced into the new bacterial strain and confirm that there is no carryover of the helper or donor strain. Electroporation does not require a helper or donor strain. This helps avoid possible contamination with other strains. In addition, introduction of the plasmid can be verified in the recipient strain within two days, making electroporation a faster and more efficient method of transformation
Electroporation is being used for transformation of both Gram-positive and Gram-negative species of plant-pathogenic and plant-associated bacteria. The electroporation conditions can differ not only for bacterial species but also strains within a species. Consequently, it is important to define the optimal parameters of electroporation to ensure that each bacterial strain of interest is transformed efficiently. These parameters include the time constant and the field strength applied to the sample (8). The time constant is dependent on the total resistance of the sample and the capacitance of the pulse circuit for the electroporation device. The field strength is dependent on the initial voltage delivered by the electroporator and the distance between the electrodes of the cuvette (8). These parameters are discussed in detail by Lurquin (8). The optimal time constant for prokaryotic cells should range from 5 to 10 ms and the field strength should be 16 to 19 kV/cm (8). Although this laboratory exercise focuses on electroporation of P. syringae pv. syringae, many other plant-pathogenic bacteria can be transformed by electroporation.
This laboratory exercise is designed to be adapted to the instructor's available equipment as well as the goals of the instructor. It is divided into three sections with separate protocols, and the instructor can choose which sections will be most effective in their respective courses. They are listed in order of difficulty.
I. Introduction of broad host range plasmids into P. syringae pv. syringae strain B301D.
II. Introduction of broad host range plasmids into various strains of P. syringae pv. syringae.
III. Introduction of mutated genes into P. syringae pv. syringae strain B301D, selection for marker exchange, and screening of mutant strains for phenotypic changes.
Click any of the links above to go to a particular section.
LITERATURE CITED:Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. 1990. Current protocols in molecular biology. Green Publishing Associates / Wiley-Interscience, New York. Dennis, J. J. and Sokol, P. A. 1995. Electrotransformation of Pseudomonas. Methods Mol. Biol. 47:125-133. Dower, W. J. 1990. Electroporation of bacteria: A general approach to genetic transformation. Genetic Engineering 12:275-295. Grewal, S. I., Johnstone, K., and Hutchison, M. L. 1993. Transformation of Pseudomonas tolaasii by electroporation. Biomedical Letters 48:177-183. Gross, D. C. and DeVay, J. E. 1977. Population dynamics and pathogenesis of Pseudomonas syringae in maize and cowpea in relation to the in vitro production of syringomycin. Phytopathology 67:475-483. Keen, N. T., Tamaki, S., Kobayashi, D., and Trollinger, D. 1988. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70:191-197. Lewin, B. 1994. Genes V. Oxford University Press, Inc., New York. Lurquin, P. F. 1997. Gene transfer by electroporation. Mol. Biotechnol. 7:5-35. Rangaswamy, V., Ullrich, M., Jones, W., Mitchell, R., Parry, R., Reynolds, P., and Bender, C. L. 1997. Expression and analysis of coronafacate ligase, a thermoregulated gene required for production of the phytotoxin coronatine in Pseudomonas syringae. FEMS Microbiol. Lett. 154:65-72. Sambrook J., Frisch, E. F., and Maniatis, T. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Smith, A. W. and Iglewski, B. H.. 1989. Transformation of Pseudomonas aeruginosa by electroporation. Nucleic Acids Res. 17:10509. Vidaver, A. K. 1967. Synthetic and complex media for the rapid detection of fluorescence of phytopathogenic pseudomonads: Effect of the carbon source. Appl. Microbiol. 15:1523-1524. West, S. E. H., Schweizer, H. P., Dall, C., Sample, A. K., and Runyen-Janecky, L. J. 1994. Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18 19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148:81-86. Zhang, J. H., Quigley, N. B., and Gross, D. C. 1995. Analysis of the syrB and syrC genes of Pseudomonas syringae pv. syringae indicates that syringomycin is synthesized by a thiotemplate mechanism. J. Bacteriol. 177:4009-4020. Zhang, J. H., Quigley, N. B. and Gross, D. C. 1997. Analysis of the syrP gene, which regulates syringomycin synthesis by Pseudomonas syringae pv. syringae. Appl. Environ. Microbiol. 63:2771-2778.
This work was supported by grant 97-35303-4460 from the National Research Initiative Competitive Grants Program of the U.S. Department of Agriculture, Science and Education Administration. Special thanks to Jonathan D. Soule for critical review of this manuscript.