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Plasmid transformation and replica filter plating of Acholeplasma laidlawii.

The restriction deficient mutant 8195 of Acholeplasma laidlawii strain JA1 was transformed by the promiscuous streptococcal plasmid vector pNZ18 at a frequency of 4 x 10(-4)/cfu. The plasmid was maintained without structural rearrangements but was lost in the absence of a selection pressure, i.e. kanamycin or neomycin. Transformed primary colonies were easily recognized due to a different colony morphology. Replica filter plating, previously not obtained with mycoplasmas, was achieved using pNZ18 as a marker by incubating the replica filters with the cell side down on the new agar plates. These findings should greatly facilitate the genetic and functional analysis of A. laidlawii.