Electrotransformation of Clostridium thermocellum

Abstract

Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 +/- 0.5) x 10(5) transformants per micro g of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 +/- 1.8) x 10(4) transformants per micro g of plasmid DNA for strain ATCC 27405 and approximately 1 x 10(3) transformants per micro g of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was approximately 50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in C. thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.

FIG. 1.

Schematic diagram of custom-built generator.

FIG. 2.

Custom cuvette design.

FIG. 3.

Detection of mls gene in Em^r Lm^r clones of C. thermocellum strains by using PCR with various DNA templates. Lanes 1 and 11, 1-kb ladder; lanes 2 to 4, total DNAs from nontransformed cells of C. thermocellum ATCC 27405 (lane 2), DSM 1313 (lane 3), and DSM 4150 (lane 4); lane 5, pUC18; lane 6, no template; lane 7, pIKm1; lanes 8 to 10, total DNAs of Em^r Lm^r clones of C. thermocellum ATCC 27405 (lane 8), DSM 1313 (lane 9), and DSM 4150 (lane 10).

FIG. 4.

pIKm1 in E. coli DH5α after rescue from total DNA of Em^r Lm^r clones of C. thermocellum. Lanes 1 and 10, 1-kb ladder; lane 2, pIKm1 used for ET of C. thermocellum; lanes 3 to 5, pIKm1 isolated from E. coli electrotransformed with total DNAs of C. thermocellum ATCC 27405 (lane 3), DSM 1313 (lane 4), and DSM 4150 (lane 5); lanes 6 to 9, PstI digests of the DNA preparations shown in lanes 2 to 5.

FIG. 5.

ET yield (▴) and cell viability (•) in relation to DNA load for C. thermocellum DSM 1313. All experiments were carried out with pIKm1 DNA isolated from Dam⁺ E. coli cells. A square 10-ms pulse and an electric-field strength of 25 kV/cm were used. The values of other variables corresponded to the preferred conditions identified in Table 2. The cells were subcultured in the presence of 9 μg of isoniacin/ml. The error bars indicate standard deviations.

FIG. 6.

ET of C. thermocellum DSM 1313 and ATCC 27405 subcultured in the presence of isoniacin as a function of field strength. Shown are the ET efficiencies for the cells of DSM 1313 (▪) (20 μg of isoniacin/ml) and ATCC 27405 (□) (9 μg of isoniacin/ml) and the viabilities for the same cells of DSM 1313 (▴) and ATCC 27405 (•). The experiments were carried out with pIKm1 isolated from Dam⁺ E. coli cells (2.5 μg/sample; 10-ms single square pulse; the values of other variables corresponded to the preferred conditions identified in Table 2). The error bars indicate standard deviations.