Electrofusion of cells of Acetogen Clostridium sp. MT 351 with erm(B) or cat in the chromosome

We report electrofusion (EF) of untreated cells of the Acetogen Clostridium sp. MT 351 as a step in developing tools for metabolic engineering in this group of microorganisms. This obligate anaerobe strain was isolated from the agricultural lagoon using the enrichment procedure in the presence of carbon monoxide and hydrogen. This strain reduced carbon monoxide to acetate, produces spores and stained Gram (+). Electrofusion was proven efficient for chromosomal recombination using this model system utilizing integration suicidal vectors comprising either erm(B) or cat. At first, we have obtained recombinants resistant to either Erythomycin or Chloramphenicol. Such recombinants were electrofused and selected as resistant to both Erytromycin and Chloramphenicol. The double antibiotic resistant clones were selected at the frequency 5.5 + 0.3 x 10-5 calculated per the number of either cell partner at the cell viability 15 + 2%. The erm(B) and cat were amplified from the total DNA preps of such recombinants using primers specific for each antibiotic resistance gene. The electrofusion technique is a fast and reliable alternative to the established procedures of protoplast fusion developed for both, Gram-negative and Gram-positive bacteria. This is the first report on electrofusion of Gram (+) microorganisms.