Transformation in Micrococcus radiodurans: Measurement of Various Parameters and Evidence for Multiple, Independently Segregating Genomes per Cell

Department of Microbiology, University of Edinburgh, School of Agriculture, Edinburgh EH9 3JG ABSTRACT Summary: Transformation frequencies greater than 1 % for some single markers were obtained in Micrococcus radiodurans when bacteria in the exponential phase of growth were resuspended in fresh growth medium containing 0.03 M-Ca 2+ before being incubated with transforming DNA. Mg 2+, Sr 2+ or Zn 2+ could not replace Ca 2- in giving high frequencies of transformation. The time required for the maximum expression of transformed markers was 2 to 3 h for resistance to rifampicin and acriflavin and 6 to 8 h for resistance to erythromycin, kanamycin and streptomycin. The comparative frequency of transformants at maximum expression for each resistance marker was: kanamycin, 1; streptomycin, 1; acriflavin, 4; erythromycin, 25; rifampicin, 64. Cultures were competent during all stages of exponential growth, the frequency of transformants only falling during stationary phase. The minimum time for DNA to be taken up by M. radiodurans into a DNAase-resistant form was between 3 and 6 s. From 6 s to 10 min exposure to DNA, the number of transformants increased non-linearly with time as though the process was inducible. The transformation frequency was directly proportional to the DNA concentration up to 1 µg ml –1, although even at 88 µg ml –1 the bacteria were not saturated. Attempts to measure the fraction of cells which were competent, using the unlinked marker technique, gave values well in excess of one. These were interpreted in terms of multiple genome copies and approximate values of between 0.25 and 0.72 were derived for the competent fractions. Department of Biology, University of Teacher Training, 49 Roosvelt Avenue, Tehran, Iran.