JOURNAL OF BACTERIOLOGY, Apr. 1975, p. 329-331 Copyright 0 1975 American Society for Microbiology
Vol. 122, No. 1 Printed in U.S.A.
Chromosomal Location of Mutations Affecting the Electrophoretic Mobility of Malate Dehydrogenase in Escherichia coli K-12 JOHN T. HEARD, JR., MARY ANN BUTLER, JAMES N. BAPTIST, AND THOMAS S. MATNEY* The University of Texas Health Science Center at Houston, Graduate School of Biomedical Sciences* and Biology Department, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77025 Received for publication 4 November 1974
The structural locus for a soluble malate dehydrogenase (L-malate:NAD oxidoreductase, EC 1.1.1.37), mdh, lies about 1.2 min from aspB on the Escherichia coli chromosome in the 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-methylN'-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 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'-nitroN-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
J. BACTERIOL.
NOTES
330
TABLE 1. Escherichia coli K-12 derivatives Source
Relevant markers"
UTH no.a
Mutational derivative of KL16-99 from B. Low Reference 3 JC-411 from A. J. Clark Reference 5 Reference 5
452
Hfr, thi-1, met-95, ilvA466, recAl Hfr, hisA323, ilvA467 F-, hisGI, argG6, leu-6, metBIF-, hisGI, argG6, leu-6, metBI, mdh-9 F-, hisGI, argG6, leu-6, metBI, tyr-8, mdh-10 F-, pro -49
6774 6674 6754
F-, thi-1, thr-1, leu-6, trp-59, mdh-2 F, argG53, aspB24 F, prototroph
Reference 2 WR 2045 from L. S. Baron Arg+ Asp+ P1 transductant of 6674
6241 672 475 6276 6406
Mutational derivative of W2979 from J. Leder-
berg -
UTH prefix designates Univ. of Texas at Houston stock collection. °Genotypic symbols indicate nutritional requirements for thiamine (thi), methionine (met), isoleucine (ilvA), histidine (his), arginine (arg), leucine (leu), tyrosine (tyr), proline (pro), tryptophan (trp), and aspartate (asp); structural locus for malate dehydrogenase (mdh); and recombination (rec). a
TABLE 2. Co-transduction frequencies involving the argG, aspB and mdh loci Crosses
P1 - 452 x 6276 P1 - 672 x 6276 P1 -6754 x 6674 P1 -6754 x 6674 P1 - 6774 x 6674 P1 6774 x 6674
Selected for:
Arg+ Arg+ Asp+ Arg+ Arg+ Asp+
involved Mutations in co-transduction argG6, mdh-9 argG6, mdh-9 argG53, aspB24 argG53, aspB24 argG53, mdh-2 aspB24, mdh-2-
carry the wild-type mdh+ allele. An additional 99 Arg+ transductants from the P1 * 672 X 6276 cross yielded an additional four colonies carrying mdh+. Co-transduction of argG+ mdh+ occurred at a frequency of 0.047, equivalent to a map distance of 1.3 min (8) (Table 2). It had previously been shown that the argG and aspB loci were separated by approximately 0.41 min (4). This distance was confirmed by P1 transduction experiments involving the UTH 6754 donor and UTH 6674 recipient, as noted in Table 2. The recipient strain (UTH 6674) was obtained from L. S. Baron as WR 2045 and contained mutations in both the argG53 and aspB24 loci. Many E. coli K-12 derivatives carry an active aspB24 suppressor (4). This problem was avoided by employing the argG+ -aspB+ contransductant (UTH 6754) of UTH 6674 as the donor. The data yield an average map distance of 0.40 min between argG and aspB. To position the mdh locus relative to these markers, P1 transductions were performed using a donor (UTH 6774) obtained from J. Courtright as W945T1-2, an mdh-2 bearing strain (2) which also contained the nutritional mutations thi-1, thr-1, leu-6, and trp-59. Unlike the mdh-9 and mdh-10 mutations which affect only the electrophoretic mobility, the mdh-2 mutation rendered
Co-transduction frequency
Distance (min)
0.055 (4/72) 0.040 (4/99) 0.53 (118/224) 0.46 (155/339) 0.019 (4/207) 0.066 (23/346)
1.25 1.30 0.36 0.45 1.45 1.20
the isolate incapable of producing an active malate dehydrogenase, as judged by the absence of a band on starch gel zymograms and by its inability to utilize malate as a carbon source. One may thus assume mdh-9, mdh-10, and mdh-2 to be allelic. Of 207 Arg+ transductants, four failed to grow on minimal media supplemented with aspartate and having malic acid as the sole carbon source. Of 346 Asp+ transductants, 23 failed to grow on minimal malate supplemental with arginine. These data position aspB between argG and mdh, 0.4 min from argG, and 1.2 min from mdh. This study was supported by the U. S. Atomic Energy Commission contract no. AT-(40-1)-4024 and Public Health Service research grant GM 15597 from the National Institute of General Medical Sciences. We gratefully acknowledge the technical assistance of Beulah S. Harriell and Diane J. Thomas. We also wish to thank J. Courtright who graciously supplied us with strain W945T1-2 and L. S. Baron for strain WR2045. LITERATURE CITED 1. Baptist, J. N., C. R. Shaw, and M. Mandel. 1969. Zone electrophoresis of enzymes in bacterial taxonomy. J.
Bacteriol. 99:180-188. 2. Courtright, J. B., and U. Henning. 1970. Malate dehydrogenase mutants in Escherichia coli K-12. J. Bacteriol. 102:722-728. 3. Matney, T. S., E. P. Goldschmidt, N. S. Erwin, and R. A.
VOL. 122, 1975
NOTES
Scroggp. 1964. A preliminary map of genomic sites for
F-attachment in Escherichia coli K-12. Biochem. Biophys. Res. Communication. 17:278-281. 4. Reiner, A. M. 1969. Isolation and mapping of polynucleotide phosphorylase mutants of Escherichia coli. J. Bacteriol. 97:1431-1436. 5. Shaw, C. R., J. N. Baptist, D. A. Wright, and T. S. Matney. 1973. Isolation of induced mutations in E. coli affecting the electrophoretic mobility of enzymes. Mut.
331
Res. 18:247-250. 6. Tritz, G. J., T. S. Matney, R. K. Gholson. 1970. Mapping of the nadB locus adjacent to a previously undescribed purine locus in Escherichia coli K-12. J. Bacteriol. 102:377-381. 7. Whitney, E. N. 1971. The toiC locus in Escherichia coli K-12. Genetics 67:39-53. 8. Wu, T. T. 1966. A model for 2-point analysis of random general transduction. Genetics 54:405-410.
Vol. 122, No. 1 Printed in U.S.A.
Chromosomal Location of Mutations Affecting the Electrophoretic Mobility of Malate Dehydrogenase in Escherichia coli K-12 JOHN T. HEARD, JR., MARY ANN BUTLER, JAMES N. BAPTIST, AND THOMAS S. MATNEY* The University of Texas Health Science Center at Houston, Graduate School of Biomedical Sciences* and Biology Department, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77025 Received for publication 4 November 1974
The structural locus for a soluble malate dehydrogenase (L-malate:NAD oxidoreductase, EC 1.1.1.37), mdh, lies about 1.2 min from aspB on the Escherichia coli chromosome in the 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-methylN'-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 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'-nitroN-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
J. BACTERIOL.
NOTES
330
TABLE 1. Escherichia coli K-12 derivatives Source
Relevant markers"
UTH no.a
Mutational derivative of KL16-99 from B. Low Reference 3 JC-411 from A. J. Clark Reference 5 Reference 5
452
Hfr, thi-1, met-95, ilvA466, recAl Hfr, hisA323, ilvA467 F-, hisGI, argG6, leu-6, metBIF-, hisGI, argG6, leu-6, metBI, mdh-9 F-, hisGI, argG6, leu-6, metBI, tyr-8, mdh-10 F-, pro -49
6774 6674 6754
F-, thi-1, thr-1, leu-6, trp-59, mdh-2 F, argG53, aspB24 F, prototroph
Reference 2 WR 2045 from L. S. Baron Arg+ Asp+ P1 transductant of 6674
6241 672 475 6276 6406
Mutational derivative of W2979 from J. Leder-
berg -
UTH prefix designates Univ. of Texas at Houston stock collection. °Genotypic symbols indicate nutritional requirements for thiamine (thi), methionine (met), isoleucine (ilvA), histidine (his), arginine (arg), leucine (leu), tyrosine (tyr), proline (pro), tryptophan (trp), and aspartate (asp); structural locus for malate dehydrogenase (mdh); and recombination (rec). a
TABLE 2. Co-transduction frequencies involving the argG, aspB and mdh loci Crosses
P1 - 452 x 6276 P1 - 672 x 6276 P1 -6754 x 6674 P1 -6754 x 6674 P1 - 6774 x 6674 P1 6774 x 6674
Selected for:
Arg+ Arg+ Asp+ Arg+ Arg+ Asp+
involved Mutations in co-transduction argG6, mdh-9 argG6, mdh-9 argG53, aspB24 argG53, aspB24 argG53, mdh-2 aspB24, mdh-2-
carry the wild-type mdh+ allele. An additional 99 Arg+ transductants from the P1 * 672 X 6276 cross yielded an additional four colonies carrying mdh+. Co-transduction of argG+ mdh+ occurred at a frequency of 0.047, equivalent to a map distance of 1.3 min (8) (Table 2). It had previously been shown that the argG and aspB loci were separated by approximately 0.41 min (4). This distance was confirmed by P1 transduction experiments involving the UTH 6754 donor and UTH 6674 recipient, as noted in Table 2. The recipient strain (UTH 6674) was obtained from L. S. Baron as WR 2045 and contained mutations in both the argG53 and aspB24 loci. Many E. coli K-12 derivatives carry an active aspB24 suppressor (4). This problem was avoided by employing the argG+ -aspB+ contransductant (UTH 6754) of UTH 6674 as the donor. The data yield an average map distance of 0.40 min between argG and aspB. To position the mdh locus relative to these markers, P1 transductions were performed using a donor (UTH 6774) obtained from J. Courtright as W945T1-2, an mdh-2 bearing strain (2) which also contained the nutritional mutations thi-1, thr-1, leu-6, and trp-59. Unlike the mdh-9 and mdh-10 mutations which affect only the electrophoretic mobility, the mdh-2 mutation rendered
Co-transduction frequency
Distance (min)
0.055 (4/72) 0.040 (4/99) 0.53 (118/224) 0.46 (155/339) 0.019 (4/207) 0.066 (23/346)
1.25 1.30 0.36 0.45 1.45 1.20
the isolate incapable of producing an active malate dehydrogenase, as judged by the absence of a band on starch gel zymograms and by its inability to utilize malate as a carbon source. One may thus assume mdh-9, mdh-10, and mdh-2 to be allelic. Of 207 Arg+ transductants, four failed to grow on minimal media supplemented with aspartate and having malic acid as the sole carbon source. Of 346 Asp+ transductants, 23 failed to grow on minimal malate supplemental with arginine. These data position aspB between argG and mdh, 0.4 min from argG, and 1.2 min from mdh. This study was supported by the U. S. Atomic Energy Commission contract no. AT-(40-1)-4024 and Public Health Service research grant GM 15597 from the National Institute of General Medical Sciences. We gratefully acknowledge the technical assistance of Beulah S. Harriell and Diane J. Thomas. We also wish to thank J. Courtright who graciously supplied us with strain W945T1-2 and L. S. Baron for strain WR2045. LITERATURE CITED 1. Baptist, J. N., C. R. Shaw, and M. Mandel. 1969. Zone electrophoresis of enzymes in bacterial taxonomy. J.
Bacteriol. 99:180-188. 2. Courtright, J. B., and U. Henning. 1970. Malate dehydrogenase mutants in Escherichia coli K-12. J. Bacteriol. 102:722-728. 3. Matney, T. S., E. P. Goldschmidt, N. S. Erwin, and R. A.
VOL. 122, 1975
NOTES
Scroggp. 1964. A preliminary map of genomic sites for
F-attachment in Escherichia coli K-12. Biochem. Biophys. Res. Communication. 17:278-281. 4. Reiner, A. M. 1969. Isolation and mapping of polynucleotide phosphorylase mutants of Escherichia coli. J. Bacteriol. 97:1431-1436. 5. Shaw, C. R., J. N. Baptist, D. A. Wright, and T. S. Matney. 1973. Isolation of induced mutations in E. coli affecting the electrophoretic mobility of enzymes. Mut.
331
Res. 18:247-250. 6. Tritz, G. J., T. S. Matney, R. K. Gholson. 1970. Mapping of the nadB locus adjacent to a previously undescribed purine locus in Escherichia coli K-12. J. Bacteriol. 102:377-381. 7. Whitney, E. N. 1971. The toiC locus in Escherichia coli K-12. Genetics 67:39-53. 8. Wu, T. T. 1966. A model for 2-point analysis of random general transduction. Genetics 54:405-410.
