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Escherichia coli: Reversion from streptomycin dependence, a mutation in a specific 30 s ribosomal protein
J. Mol. Biol. (1969) 43, 327-329
LETTERS TO THE EDITOR
Escherichia coli: Reversion from Streptomycin Dependence, a Mutation in a Specific 30 s Ribosomal Protein Ribosomes from strains sensitive to streptomycin, resistant to it, dependent on it, or revertant from dependence, can all be distinguished by functional tests in vitro (Flaks, Cox, Whitting & White, 1962; Speyer, Lengyel & Basilio, 1962; Likover & Kurland, 1967 ; Apirion C Schlessinger, 1967) ; therefore, we undertook to find structural differences between the ribosomal proteins of such strains. Here we report the correlation of a change in a protein of the 30 s ribosomal subunit with a mutation that produces reversion from streptomycin dependence, and show that this mutation is either in the str locus itself or in one very closely linked to it. The strains used in this analysis were AB774, a streptomycin-sensitive strain (for details about this and other strains, see Apirion & Schlessinger, 1968~) ; N522, a streptomycin-dependent derivative of AB774; and strain N537, a streptomycinindependent revertant isolated from strain N522. Electrophoretic analysis of the 30 and 50 s ribosomal proteins of these three strains was carried out in polyacrylamide gels. The patterns of the ribosomal proteins isolated from the 50 s ribosomes of all strains were identical. The patterns of the 30 a ribosomal proteins of strains AB774 and N522 were also equivalent; however, a difference could be detected in the pattern of 30 s proteins from strain N537. Plate I shows the characteristic band profile of 30 s ribosomal proteins. A group of five relevant bands have been marked 1 to 5 in Plate I and Figure 1. The second and third bands in the parental strain AB774, as in other Escherichia coli strains, are almost inseparable. In contrast, in strain N537, the third band of this group is modified, and migrates distinctly faster than the corresponding band in the parental strains. The fourth band of this group is the K12 band (cf. for example, Leboy, Cox Q Flaks, 1964; Mayuga, Meier & Wang, 1968). In Figure 1, superimposed densitometer tracings of the gels are shown and the difference in mobility induced in the third band of strain N537 can be seen. These differences in mobility were observed in all fifteen runs of proteins from three different batches of cells. A comparable change was observed when ribosomal proteins were extracted and subjected to electrophoresis under a different set of conditions (Fogel $ Sypherd, 1968). To check the location of the revertant mutation in relation to the streptomycin locus, strain N637 (arg -ilv -his -pro - thi -) was crossed to the streptomycin-sensitive Hfr strain JC12 (ade - met -), which transfers the str region of the chromosome early. To ensure that the zygotes included a sizeable portion of the Hfr chromosome including the str region, Arg+ Ilv+ Ade+ Met + Str-R recombinant8 were selected. (Like many revertants from streptomycin dependence, N537 is still resistant to the drug, and streptomycin-resistant recombinants could therefore be selected from the zygotes. However, because of the dominance of sensitivity to streptomycin (Lederberg, 1951), immediate plating of the mating mixture in the presence of 200 pg drug/ml. would 327
328
D.
(a)
APIRION
et uZ.
(b)
FIG. 1. Superimposed densitometer tracings of amido black-stained gel patterns of 30 s ribosomal proteins. Parental strain AB774 (-) compared to revertant from streptomycin-dependent N537 (---). The peek numbers correspond to the numbered band of Plate I. Scanning was done st 425 rnp on whole gels, using the Gilford linear transport attaohment (Gilford Instrument Company, Oberlin, Ohio). The peek heights of the bands are only approximate, since they can be modified along a gel by changing the angle of the scanning beam to the gel; however, the positions of the peaks are not thereby modified. In (a), the gels were run for 3 hr, as in Plate I. In (b), duplicate samples were electrophoresed for 6 hr, to increase the separation of bands in the region of interest.
have killed the sensitive zygotes. Therefore, the mating was first carried out for four hours (or for 1 hr followed by 2 hr of further growth) in absence of streptomycin, in order to permit segregation of resistant recombinants. Similar conditions were adequate for the transfer of resistant and dependent alleles (cf. Hashimoto, 1960; Luzzatto, Schlessinger & Apirion, 1968).) 295 streptomycin-resistant recombinants were analyzed; among these none was dependent. The failure to detect recombination between the original mutation to dependence and the second mutation to streptomycin independence indicates that they are very closely linked, as was also shown earlier for revertants from streptomycin dependenoe by Newcomb & Nyholm (1950) and Hashimoto (1960). The two mutations are probably more closely linked, for example, than are str and spc alleles (Davies, Anderson t Davis, 1965) ; str and spc alleles, which were up to 50% cotransducible in Pl-mediated transduction (Apirion & Schlessinger, 196&z), gave rise to 5 to 10% recombination in comparable mating experiments. Since mutations to streptomycin resistance and dependence occur in a single gene (Luzzatto, Schlessinger & Apirion, 1968) that specifies a 30 s ribosomal protein (Silengo, Schessinger, Mangiarotti & Apirion, 1967; Traub & Nomura, 1968; Apirion
Part of gels enlarged Total
gels
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2345-
LEl-‘-ERS
TO
THE
EDITOR
329
t Schlessinger, 1968b) and since the mutations to streptomycin dependence and its are closely linked, we conclude that the mutation to reversion in strain N537 is either in the str gene or in a closely linked gene that also specifies a 30 s ribosomal protein. revertant
Note added in proof In a transduction experiment the streptomycin dependent mutation in strain N537 was separated from its modifier in 37% of the Str-R transductants isolated, which suggests that the modifier mutation is in a different protein from the mutation to streptomycin dependence. Thus, strain N537 is another example for interactions between two ribosomal proteins. This investigation was supported by Public Health Service research grants and GM-10447, and by Training Grant 5T-Al-257, from the National Institutes and by the American Cancer Society Grant no. P-477. Department of Microbiology Washington University School St. Louis, Miss. 63110, U.S.A.
D. A~IRION D. SCHLESSINGER s. PHILLIPS
of Medicine
Department of Microbiology University of Illinois Urbana, Ill., U.S.A. Received
21 October
HD-01956 of Health,
P. SYPRERD
1968, and in revised
form
17 February
1969
REFERENCES Apirion, D. & Schlessinger, D. (1967). J. Bact. 94, 1275. Apirion, D. & Schlessinger, D. (196%). J. Bact. 96, 768. Apirion, D. & Schlessinger, D. (19683). J. Bact. 96, 1431. Davies, cJ., Anderson, P. & Davis, B. D. (1965). Science, 149, 1096. Flairs, J., Cox, E., Whitting, M. L. & White, J. R. (1962). Biochem. Biophys. Res. Comm. 7, 390. Fogel, S. & Sypherd, P. S. (1968). J. Bact. 96, 358. Hashimoto, K. (1960). Genetics, 45, 49. Krembel, J. & Apirion, D. (1968). J. Mol. Biol. 33, 363. Leboy, P. S., Cox, E. C. & Flaks, J. G. (1964). Proc. Nat. Acad. Sci. Wash. 52, 1367. Lederberg, J. (1951). J. Bact. 61, 549. Likover, T. E. & Kurland, C. G. (1967). J. Mol. Biol. 25, 497. Luzzatto, L., Schlessinger, D. & Apirion, D. (1968). Science, 161, 478. Mayuga, C., Moier, D. & Wang, T. (1968). Biochem. Biophys. Res. Gomm. 33, 203. Newcomb, H. B. & Nyholm, M. H. (1950). Genetics, 35, 603. Silengo, L., Schlessinger, D., Mangiarotti, G. & Apirion, D. (1967). Mutation Research, 4, 701. Speyer, J. F., Lengyel, P. & Basilio, C. (1962). Proc. Nat. Acad. Sci., Wash. 48, 684. Traub, P. & Nomura, M. (1968). Science, 160, 198.
LETTERS TO THE EDITOR
Escherichia coli: Reversion from Streptomycin Dependence, a Mutation in a Specific 30 s Ribosomal Protein Ribosomes from strains sensitive to streptomycin, resistant to it, dependent on it, or revertant from dependence, can all be distinguished by functional tests in vitro (Flaks, Cox, Whitting & White, 1962; Speyer, Lengyel & Basilio, 1962; Likover & Kurland, 1967 ; Apirion C Schlessinger, 1967) ; therefore, we undertook to find structural differences between the ribosomal proteins of such strains. Here we report the correlation of a change in a protein of the 30 s ribosomal subunit with a mutation that produces reversion from streptomycin dependence, and show that this mutation is either in the str locus itself or in one very closely linked to it. The strains used in this analysis were AB774, a streptomycin-sensitive strain (for details about this and other strains, see Apirion & Schlessinger, 1968~) ; N522, a streptomycin-dependent derivative of AB774; and strain N537, a streptomycinindependent revertant isolated from strain N522. Electrophoretic analysis of the 30 and 50 s ribosomal proteins of these three strains was carried out in polyacrylamide gels. The patterns of the ribosomal proteins isolated from the 50 s ribosomes of all strains were identical. The patterns of the 30 a ribosomal proteins of strains AB774 and N522 were also equivalent; however, a difference could be detected in the pattern of 30 s proteins from strain N537. Plate I shows the characteristic band profile of 30 s ribosomal proteins. A group of five relevant bands have been marked 1 to 5 in Plate I and Figure 1. The second and third bands in the parental strain AB774, as in other Escherichia coli strains, are almost inseparable. In contrast, in strain N537, the third band of this group is modified, and migrates distinctly faster than the corresponding band in the parental strains. The fourth band of this group is the K12 band (cf. for example, Leboy, Cox Q Flaks, 1964; Mayuga, Meier & Wang, 1968). In Figure 1, superimposed densitometer tracings of the gels are shown and the difference in mobility induced in the third band of strain N537 can be seen. These differences in mobility were observed in all fifteen runs of proteins from three different batches of cells. A comparable change was observed when ribosomal proteins were extracted and subjected to electrophoresis under a different set of conditions (Fogel $ Sypherd, 1968). To check the location of the revertant mutation in relation to the streptomycin locus, strain N637 (arg -ilv -his -pro - thi -) was crossed to the streptomycin-sensitive Hfr strain JC12 (ade - met -), which transfers the str region of the chromosome early. To ensure that the zygotes included a sizeable portion of the Hfr chromosome including the str region, Arg+ Ilv+ Ade+ Met + Str-R recombinant8 were selected. (Like many revertants from streptomycin dependence, N537 is still resistant to the drug, and streptomycin-resistant recombinants could therefore be selected from the zygotes. However, because of the dominance of sensitivity to streptomycin (Lederberg, 1951), immediate plating of the mating mixture in the presence of 200 pg drug/ml. would 327
328
D.
(a)
APIRION
et uZ.
(b)
FIG. 1. Superimposed densitometer tracings of amido black-stained gel patterns of 30 s ribosomal proteins. Parental strain AB774 (-) compared to revertant from streptomycin-dependent N537 (---). The peek numbers correspond to the numbered band of Plate I. Scanning was done st 425 rnp on whole gels, using the Gilford linear transport attaohment (Gilford Instrument Company, Oberlin, Ohio). The peek heights of the bands are only approximate, since they can be modified along a gel by changing the angle of the scanning beam to the gel; however, the positions of the peaks are not thereby modified. In (a), the gels were run for 3 hr, as in Plate I. In (b), duplicate samples were electrophoresed for 6 hr, to increase the separation of bands in the region of interest.
have killed the sensitive zygotes. Therefore, the mating was first carried out for four hours (or for 1 hr followed by 2 hr of further growth) in absence of streptomycin, in order to permit segregation of resistant recombinants. Similar conditions were adequate for the transfer of resistant and dependent alleles (cf. Hashimoto, 1960; Luzzatto, Schlessinger & Apirion, 1968).) 295 streptomycin-resistant recombinants were analyzed; among these none was dependent. The failure to detect recombination between the original mutation to dependence and the second mutation to streptomycin independence indicates that they are very closely linked, as was also shown earlier for revertants from streptomycin dependenoe by Newcomb & Nyholm (1950) and Hashimoto (1960). The two mutations are probably more closely linked, for example, than are str and spc alleles (Davies, Anderson t Davis, 1965) ; str and spc alleles, which were up to 50% cotransducible in Pl-mediated transduction (Apirion & Schlessinger, 196&z), gave rise to 5 to 10% recombination in comparable mating experiments. Since mutations to streptomycin resistance and dependence occur in a single gene (Luzzatto, Schlessinger & Apirion, 1968) that specifies a 30 s ribosomal protein (Silengo, Schessinger, Mangiarotti & Apirion, 1967; Traub & Nomura, 1968; Apirion
Part of gels enlarged Total
gels
. /_
I-
2345-
LEl-‘-ERS
TO
THE
EDITOR
329
t Schlessinger, 1968b) and since the mutations to streptomycin dependence and its are closely linked, we conclude that the mutation to reversion in strain N537 is either in the str gene or in a closely linked gene that also specifies a 30 s ribosomal protein. revertant
Note added in proof In a transduction experiment the streptomycin dependent mutation in strain N537 was separated from its modifier in 37% of the Str-R transductants isolated, which suggests that the modifier mutation is in a different protein from the mutation to streptomycin dependence. Thus, strain N537 is another example for interactions between two ribosomal proteins. This investigation was supported by Public Health Service research grants and GM-10447, and by Training Grant 5T-Al-257, from the National Institutes and by the American Cancer Society Grant no. P-477. Department of Microbiology Washington University School St. Louis, Miss. 63110, U.S.A.
D. A~IRION D. SCHLESSINGER s. PHILLIPS
of Medicine
Department of Microbiology University of Illinois Urbana, Ill., U.S.A. Received
21 October
HD-01956 of Health,
P. SYPRERD
1968, and in revised
form
17 February
1969
REFERENCES Apirion, D. & Schlessinger, D. (1967). J. Bact. 94, 1275. Apirion, D. & Schlessinger, D. (196%). J. Bact. 96, 768. Apirion, D. & Schlessinger, D. (19683). J. Bact. 96, 1431. Davies, cJ., Anderson, P. & Davis, B. D. (1965). Science, 149, 1096. Flairs, J., Cox, E., Whitting, M. L. & White, J. R. (1962). Biochem. Biophys. Res. Comm. 7, 390. Fogel, S. & Sypherd, P. S. (1968). J. Bact. 96, 358. Hashimoto, K. (1960). Genetics, 45, 49. Krembel, J. & Apirion, D. (1968). J. Mol. Biol. 33, 363. Leboy, P. S., Cox, E. C. & Flaks, J. G. (1964). Proc. Nat. Acad. Sci. Wash. 52, 1367. Lederberg, J. (1951). J. Bact. 61, 549. Likover, T. E. & Kurland, C. G. (1967). J. Mol. Biol. 25, 497. Luzzatto, L., Schlessinger, D. & Apirion, D. (1968). Science, 161, 478. Mayuga, C., Moier, D. & Wang, T. (1968). Biochem. Biophys. Res. Gomm. 33, 203. Newcomb, H. B. & Nyholm, M. H. (1950). Genetics, 35, 603. Silengo, L., Schlessinger, D., Mangiarotti, G. & Apirion, D. (1967). Mutation Research, 4, 701. Speyer, J. F., Lengyel, P. & Basilio, C. (1962). Proc. Nat. Acad. Sci., Wash. 48, 684. Traub, P. & Nomura, M. (1968). Science, 160, 198.