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The Rhizobium-legume symbiosis accounts for a significant proportion of nitrogen available to leguminous plants. Thus, there is a need to manipulate rhizobia to increase their symbiotic efficiency and host range. An important prerequisite for genetic improvement of any bacterial species is the availability of a highly efficient gene transfer system. Transformation systems developed for rhizobia (1, 14) are far less efficient than those for other bacteria. So, introduction of foreign DNA into rhizobia has been possible exclusively via conjugal matings with Escherichia coli (4); such procedures are time-consuming, however, and limited to special plasmids having the mob gene.

Electroporation involves the use of a high-intensity electric field of short duration to induce reversible permeabilization in the cell membrane to facilitate the entrance of macromolecules such as DNA (3). Electroporation was applied initially for transformation studies in mammalian cells (12) and was found to be effective with bacterial protoplasts as early as 1983 (16). Shortly thereafter, this technique was applied successfully to transform intact cells of both gram-positive and gram-negative bacterial species with plasmid DNA (5, 8). Now, electroporation is a novel approach for introduction of foreign DNA into bacterial species poorly transformable or for which transformation protocols have yet to be established. The electroporation conditions required for maximum transformation efficiency vary from cell to cell. This paper reports the various conditions (electric field strength, pulse length, cell concentration, and plasmid DNA concentration) required for efficient introduction of plasmid DNA into rhizobia by electroporation.

A plasmid-free, chloramphenicol-sensitive rhizobial strain, R. leguminosarum T-19 C (Department of Microbiology, CCS Haryana Agricultural University, Hisar, India), was used in the present study and was grown in yeast extract-mannitol (YEM) broth (7). E. coli S-17-1 (17) harboring plasmid pMP154 was grown at 37°C in Luria-Bertani (LB) broth (15) containing 20 µg of chloramphenicol per ml. Plasmid pMP154, used for electrotransformation study, is an IncQ transcriptional fusion plasmid (15 kb) containing the nodA promoter of R. leguminosarum Sym plasmid pRL1JI cloned as a 114-bp restriction fragment in front of the E. coli lacZ gene and also carries a chloramphenicol resistance marker (18).

Plasmid DNA was isolated by the alkaline lysis method and purified by Sephadex G-50 spun-column chromatography (15).

One loopful of rhizobial cells from a fresh culture was inoculated into YEM broth and grown for 72 h at 30°C with vigorous shaking to mid-logarithmic phase (absorbance at 600 nm of 0.4 to 0.6). Cells were prepared for electroporation by a modification of the procedure of Dower and coworkers (6). Cells were chilled for 15 to 30 min on ice and then harvested by centrifugation at 9,000 rpm for 10 min at 4°C. The cell pellet was washed four times with cold sterile deionized water and finally washed with 10% glycerol. The cells were resuspended in 10% glycerol to have an approximate concentration of 10¹⁰ to 10¹¹ CFU/ml and kept on ice.

The cell suspension was distributed in aliquots of 90 µl and mixed thoroughly with plasmid DNA (2 µg) by vortexing at high speed for 10 s and then kept on ice for 30 min. The cell-DNA mixture was loaded in a chilled electroporation cuvette with a 0.1-cm gap (BTX Inc., San Diego, Calif.) and was subjected to a single pulse of high voltage. For pulse generation, an electrocell manipulator, model 600 (BTX Inc.), was used that was capable of generating a field strength of up to 25 kV/cm with a 0.1-cm-gap cuvette. After the pulse was delivered, the cuvettes were kept on ice for 10 min. For expression, the electroporated cells were suspended in LB broth and incubated for 24 h at 30°C. The cell suspension was diluted and plated on nonselective medium (YEM agar) to calculate the number of survivors and selective medium (YEM agar plus 20 µg of chloramphenicol/ml) to calculate the number of transformants. The numbers of CFU were scored after 7 to 8 days of incubation at 30°C. The control consisted of cells from which either plasmid pMP154 or the pulse or both had been omitted.

During these trials, no chloramphenicol-resistant colony appeared when rhizobial cells subjected to electroporation in the absence of plasmid pMP154 or incubated with plasmid pMP154 without electric pulse treatment were plated on selective medium plates.

The electrotransformants were confirmed by studying qualitatively the appearance of blue or white colonies in the presence of the nod gene inducer, naringenin, by spot tests on YEM agar plates containing 5-bromo-4-chloro-3-indolyl--D-galactopyranoside as a substrate (2). The electrotransformants formed blue colonies, whereas nonelectroporated rhizobial cells formed white colonies.

The amplitude (electric field strength) and duration (pulse length) of discharge waveform are important effectors of electrotransformation. The electric field strength necessary for maximum transformation of bacterial cells ranges from 2 to 18 kV/cm (3, 10). The electric field strength (14 kV/cm) that we have optimized in R. leguminosarum is also in the upper range of bacterial transformation. An increase or decrease in the electric field strength beyond the standard electric field strength resulted in a 10-fold reduction in the total number of transformants (Table 1). The maximum number of transformants (8.8 × 10⁸) was obtained at a pulse length of 7.3 ms, while a further increase in the pulse length, to 10.1 ms, resulted in a 20-fold reduction in the number of transformants (Table 2). The results in Tables 1 and 2 also illustrate that survivability decreases with an increase in either the electric field strength or the pulse length. In the case of Bradyrhizobium japonicum, a member of the family Rhizobiaceae, the maximum transformation efficiency was obtained at an electric field strength of 12.5 kV/cm and a pulse length of 6.6 ms. These workers could not obtain peak transformation efficiency at the maximum available field strength of 12.5 kV/cm due to limitation of electroporation unit. The field strength optimized in rhizobia seems to be similar to that for a closely related member, Agrobacterium tumefaciens, which was transformed maximally at a field strength of 14.4 kV/cm (11).

Some loss of cell viability certainly occurs when any bacterial cell is electroporated. It means that pores formed during electroporation not only facilitate the entry of extracellular material but also result in the loss of intracellular components. In case of R. leguminosarum, the cell survivability was 63% under standard conditions (14-kV/cm field strength and 7.3-ms pulse length), which is similar to the survivability of B. japonicum, which has been reported to be 75% under standard electric parameters (9), whereas the survivability of E. coli under standard electric conditions is 30 to 40% (6). So, the organism under study seems to be more resistant to electroporation than E. coli. These results imply that different cell types vary in their responses to electric pulses due to difference in membrane makeup and cell wall thickness, structure, and density.

The rhizobial cell suspension (2.4 × 10¹⁰ cells/ml) was diluted (1:1, 1:3, 1:9, 1:27, 1:81, and 1:243) and electroporated in the presence of a fixed plasmid DNA concentration. In agreement with the findings of many workers (6, 13), our data also show that the number of transformants increases with increasing cell concentration with no sign of saturation, indicating that a greater number of transformants may be possible with a higher cell concentration (Table 3). This implies that DNA concentration may not be the limiting factor within the cell concentration range tested.

When the relationship between plasmid DNA concentration and transformation efficiency was studied, the transformation efficiency increased to a maximum value of 5.8 × 10⁸ transformants/µg of DNA with an increase in the plasmid DNA concentration of up to 0.5 µg/ml of cell suspension, while a greater DNA concentration resulted in less transformation efficiency. This decrease in transformation efficiency may be due to the presence of deleterious chemicals in the DNA preparation which could enter the cell during electroporation, since highly purified DNA was not used in the present study.

A major barrier to genetic studies with rhizobia seems to be the lack of efficient, reliable, and rapid gene exchange technologies. The present study demonstrates that electroporation is an efficient, reliable, rapid, and simple method for introducing plasmid DNA into R. leguminosarum. Since conjugation is frequently used for introducing foreign DNA into rhizobia but limited to special plasmids carrying gene transfer function, electroporation-induced transformation should become the method of choice to facilitate molecular genetic studies with rhizobia.