Chromosomal Location of Mutations Affecting the Electrophoretic Mobility of Malate Dehydrogenase in Escherichia coli K-12

John T. Heard, Mary Ann Butler, James N. Baptist, Thomas S. Matney
1975 Journal of Bacteriology  
sequence argG, aspB, mdh. We have reported (5) the isolation of five mutants in Escherichia coli K-12 having active enzymes with altered electophoretic mobility from 1,400 clones mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine. Two mutations affected malate dehydrogenase, the others produced variant forms of 6-phosphogluconate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and esterase. Variants in nine other enzymes were not detected. The mutations were induced in an F-strain
more » ... ed in an F-strain of E. coli K-12 (UTH 475) obtained from A. J. Clark as JC-411 (Table 1) . This strain was selected since it carries four nutritional mutations invoking requirements for histidine (hisGl), arginine (argG6), leucine (leu-6), and methionine (metBI) which are evenly distributed around the chromosome. It was reasoned that electrophoretic variants could be grossly positioned by linkage to one of the four nutritional markers after matings with the contra-directional Hfr donors: KL16 (UTH 6241) o-recA, his, leu, metB, argG, and G6 (UTH 672) o-argG, metB, leu, his, recA. UTH 6241 was received from Brooks Low as KL16-99. It carried a growth requirement for thiamine (thi-1) and a recombinationless mutation in the recA13 locus. Additional requirements for methionine (met-95) and isoleucine (ilvA466) were introduced into KL16-99 by treatment with N-methyl-N'-nitro-N-nitrosoguanidine in this laboratory. The UTH 672 version of Hfr G6 (3) carries the hisA323 and ilvA467 nutritional mutations. Matings between the KL16 donor and the Fcarrying the mdh-9 mutation were interrupted by agitation after 40, 45, and 50 min to time the entrance of his+ and after 80, 85, 90, and 100 min for leu+ The his+ marker entered at 38 min and the leu+ at 74 min. Twenty recombinant clones were picked from each time interval and grown up collectively in nutritionally supple-mented minimal broth; the cells were harvested, washed, and disrupted in a sonifier, and the supematant was examined for the presence of wild-type enzyme by starch gel electrophoresis as previously described (1). Since all of the recombinants of both classes were found to produce only mutant enzyme, it was concluded that the mdh locus did not reside between recA and leu and was not closely linked to his as previously suspected (2). Interrupted matings were performed with the second Hfr donor, UTH 672, and the populations scored for Arg+, Met+, and Leu+ recombinants. Since only Arg+ clones were obtained with high frequency, the donor strain was suspected to have a large portion of cells which had converted from the Hfr to F' state in which the F-merogenote now carried the argG+ allele. Gel electrophoresis analysis indicated that 80% of the Arg+ colonies generated three bands for malate dehydrogenase and were, therefore, diploid (mdh+/mdh-9); one band corresponded to the mutant form of the enzyme, the second corresponded to wild type, whereas the third migrated to an intermediate position, thus confirming that the active form of the enzyme was a dimer and that the F-merogenote carried both the mdh and the argG loci. P1 transducing phage was prepared as previously described (6) on two donors which carried the wild-type alleles, argG+ and mdh+: UTH 452, an F-prolineless derivative, and the Hfr donor, UTH 672, previously described. Seventytwo Arg+ transductants obtained from the P1 -452 X 6276 (argG6, mdh-9) cross were analysed by the starch gel electrophoresis procedures previously described (5). In these experiments when an extract obtained from a mixture of 12 transductant cultures showed the presence of wild-type enzyme, the 12 cultures were examined in pairs. Four transductants were found to 329 on May 10, 2020 by guest
doi:10.1128/jb.122.1.329-331.1975 fatcat:4ljoiwtlxvecbdkfq3xhptkcyy