Figure 2

PCR and Southern blot experiments showed that group II intron-based vectors efficiently targeted the Clostridium cellulolyticum mdh and ldh genes in pure cultures. (A) Primers MdhF/intronR1 (5' junction) and intronF1/MdhR (3' junction) produced bands from the Ccel_0137 mutant cells (lanes 1 and 2) but not from wild-type (lanes 4 and 5). Primers MdhF/MdhR amplified a single band from the mutant (lane 3) that is 915 bp larger than the wild-type (lane 6). (B) Primers LdhF/intronR1 (5' junction) and intronF1/LdhR (3' junction) produced bands in the Ccel_2485 mutant cells (lanes 1 and 2) but not in the wild-type (lanes 4 and 5). Primers LdhF/LdhR, amplified a single band from the mutant (lane 3), which is 915 bp larger than the wild-type (lane 6). (C) Amplifications using plasmid-specific primers pWH199F2 and pintronR1 confirmed plasmid curing. Lane 1, positive control (plasmid); lane 2, Ccel_0137 mutant; lane 3, Ccel_2485 mutant; lane 4, negative control. (D) Amplification from wild-type DNA using primers LdhF-R (lane 1) and MdhF-R (lane 2) produced low molecular weight products. Genes containing insertions were amplified from the ldh mutant using primers LdhF-R (lane 3) and from the mdh mutant using primers MdhF-R (lane 4), producing larger products. The same size PCR products were obtained in amplifications from ldh mdh mutant DNA using primers LdhF-R (lane 5) and MdhF-R (lane 6). (E) A Southern blot using an intron-specific probe confirmed the intron insertions in DNA digested with Eco RI. No band was detected in the chromosomal DNA of wild-type cells (lane 1), while two bands in the ldh mdh mutant (lane 2) correspond to bands in the ldh mutant (lane 3) and the mdh mutant (lane 4). No band corresponding to the plasmid (lane 5) was identified in any of the plasmid-cured strains.