Search

SUMMARY: In an attempt to isolate a strain of Rhizobium trifolii which could be highly mutated by ultraviolet light (u.v.), a u.v.-sensitive mutant was isolated using a semi-selective procedure. The mutant was not only 85 times more sensitive than the wild-type to the lethal effects of u.v., but was mutated at u.v. doses which had little mutagenic effect on the wild-type. Its sensitivity to the mutagenic agents methyl methanesulphonate and gamma rays was unaltered, but its spontaneous mutation frequencies for two antibiotic resistances were increased. The mutation conferring u.v. sensitivity was mapped on the chromosome of Rhizobium leguminosarum 300 in a position between the markers ser-2 and ade-88. Unsuccessful attempts were made to transfer into the u.v.-sensitive mutant any one of a number of plasmids known to decrease the lethality of u.v. and enhance its mutagenicity.

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-Ca2+ before being incubated with transforming DNA. Mg2+, Sr2+ or Zn2+ could not replace Ca2- 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.

SUMMARY: The efficiency of a variety of common mutagens in producing mutation in Rhizobium trifolii P3 was examined. Ethyl methanesulphonate, methyl methanesulphonate, decarbamoyl mitomycin C, nitrous acid and gamma radiation did not mutate R. trifolii P3. N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) and ultraviolet radiation were both mutagenic, the former being the more effective. Transposon mutagenesis with Tn5 yielded the same frequency and range of auxotrophs as did MNNG.