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[15] Mutations in the eup locus of Escherichia coli, energy uncoupled phenotype
[15]
eup MUTATIONSIN E. coli
187
[15] M u t a t i o n s in t h e e u p L o c u s o f E s c h e r i c h i a coli, E n e r g y Uncoupled Phenotype B y CHARLES A . PLATE
Three genetic loci have been identified in Escherichia coli in which mutations result in an energy uncoupled phenotype, i.e., lack of growth on nonfermentable carbon sources, reduced growth yields on limiting glucose, and normal electron transport. The best characterized of the three, the unc locus near minute 84 on the E. coli linkage map, ~ encodes the subunits of the BFoFrATPase. 2 Certain Unc mutants are abnormally permeable to protons resulting in a reduced protonmotive force (PMF), which in turn leads to defective proton/solute cotransport and a low level resistance to aminoglycoside antibiotics) The second locus, the ecfA locus near minute 65, is the subject of another chapter in this volume 4 and will not be dealt with further here. The third locus, independently identified by three laboratories, maps near minute 88 and has been variously designated ecfB, 5 ssd, 6 and e u p 7,8 (the latter designation to be used hereafter). 9 Mutations within the eup locus result in strains that grow on glucose, do not grow on succinate, have reduced growth yields on limiting glucose, have normal electron transport, have normal BFoFrATPase activity, and are defective in proton/solute cotransport. In addition, Eup mutants have decreased sensitivity to colicins K and A and they exhibit low level resistance to aminoglycoside antibiotics. Although Eup mutants phenotypically resemble the proton permeable Unc mutants, they differ in one significant respect. Eup mutants are not abnormally permeable to protons and they are capable of generating and maintaining a PMF of normal magnitude.8,1°,l~ The phenoi B. J. Bachmann, Microbiol. Rev. 47, 180 (1983). z j. A. Downie, F. Gibson, and G. B. Cox, Annu. Rev. Biochem. 48, 103 (1979). 3 B. P. Rosen, J. Bacteriol. 116, 1124 (1973). 4 j. S. Hong, this volume [14]. 5 S. H. Thorbjarnardottir, R. A. Magnusdottir, G. Eggertsson, S. A. Kagan, and O. S. Andresson, Mol. Gen. Genet. 161, 89 (1978). 6 E. B. Newman, N. Malik, and C. Walker, J. Bacteriol. 150, 710 (1982). 7 C. A. Plate, J. Bacteriol. 125, 467 (1976). 8 C. A. Plate and J. L. Suit, J. Biol. Chem. 256, 12974 (1981). 9 It should be noted that while it is likely that ecfB, ssd, and eup are alleles, complementation studies to definitively establish this have not yet been done. 10 E. R. Kashket, J. Bacteriol. 146, 377 (1981). H G. D. Hitchens, D. B. Kell, and J. G. Morris, J. Gen. Microbiol. 128, 2207 (1982).
METHODS IN ENZYMOLOGY, VOL. 125
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
188
GENERALAPPROACHES
[15]
typic similarities between Eup and Unc mutants suggests that a product of the eup locus is important to the process of energy coupling, but the precise nature of its involvement has yet to be determined. The purpose of this article is to describe the procedures for obtaining and identifying eup mutations in E. coli. Selection Procedure E. coli Strains and Growth Media. The media used in these studies are LB broth (containing per liter: tryptone, 10 g; yeast extract, 5 g; NaC1, 5 g; adjusted to pH 7.0 with 1.0 N NaOH) and Ozeki minimal base (containing per liter: K2HPO4, 10.5 g; KH2PO4, 4.5 g; (NH4)2SO4, 1.0 g; MgSO4, 0.05 g; sodium citrate, 0.47 g) supplemented with a carbon source (0.4%), required amino acids (50 ~g/ml), and thiamine (0.5 t~g/ml). Solid Ozeki minimal and LB media contain 1.5% agar and LB soft agar contains 0.65% agar. As previously stated, one characteristic of Eup mutants is their inability to grow on succinate as sole carbon source. We and others have found that this particular Eup trait is subject to extragenic suppression. 6,8 The following Eup mutant isolation protocol assumes that the E. coli strain to be employed does not harbor this ill-defined suppressor and is sensitive to colicins A and K and amino glycoside antibiotics such as neomycin. We have used two E. coli K12 strains that satisfy these conditions: strain A279a (HfrH3000) and strain M72 [lacZ(Am) trp(Am) thi]. Both of these strains are available from Dr. S. E. Luria (Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139). The required E. coli strains colicinogenic for colicins A, K, El, E2, and E3 can be obtained from this same source or from the E. coli Genetic Stock Center (Yale University School of Medicine, New Haven, CT 06510). Mutagenesis and Screening for Eup Mutants. Ethylmethane sulfonate mutagenesis is carried out essentially by the procedure of Miller. 12E. coli cells are grown in minimal medium with glucose as carbon source to a density of 2 x 108 cells/ml. The cells are pelleted, washed, and resuspended in one-half the original volume of the minimal medium base without carbon source. To 2.0 ml of this cell suspension add 0.03 ml of ethylmethane sulfonate, mix vigorously to dissolve, and incubate the cells at 37° with shaking for 2 hr (this results in approximately a 90% reduction in viable count). The cells are then pelleted and resuspended in 3.0 ml of unsupplemented minimal medium base. 12j. H. Miller, in "Experiments in Molecular Genetics," p. 138. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1972.
[15]
eup MUTATIONSIN E. coli
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Fifteen tubes, each containing 2.0 ml of LB broth supplemented with glucose (0.4%), are inoculated with 0.2 ml of the mutagenized cell suspension and incubated overnight at 37° with shaking. Dilute an aliquot of each culture 100-fold and spread 0.1 ml of each dilution onto a minimal glucose agar plate containing neomycin sulfate (20 pg/ml). The plates are incubated at 37 ° and, after 3 days, there should be approximately 100-200 colonies/plate. The neomycin-resistant colonies are replica plated onto minimal succinate, minimal glucose, minimal glucose-neomycin (10 /zg/ml), and LB agar plates supplemented with glucose (0.4%) and K C I O 3 (0.2%). ~3-15All plates are incubated at 37° for 2 days. Putative Eup mutants will grow on the glucose and glucose-neomycin plates, will grow aerobically in the presence of chlorate, but will not grow on the succinate plates. Colonies exhibiting a succinate-nonutilizing, neomycin-resistant, chlorate-resistant phenotype are then checked for colicin A and/or K sensitivity. Eup mutants exhibit a markedly reduced sensitivity to these colicins but exhibit normal sensitivity to colicins El, E2, and E3. 7 Colonies to be checked are inoculated into LB broth and grown overnight at 37°. LB agar plates are stabbed with cells colicinogenic for colicin A or K and these also are incubated overnight at 37°. The colicinogenic cell buttons are removed with filter paper and the plates sterilized by exposure to chloroform vapors for 30 min. Aliquots of the LB cultures to be tested are inoculated into LB soft agar (maintained at 45°), spotted over the colicinogenic stabs, and the plates incubated overnight at 37°. Colicin sensitivity is indicated by a clear zone in the soft agar overlay, and Eup mutants will give a much smaller zone of growth inhibition than the wild-type starting strain. Colonies exhibiting a reduced colicin K or A sensitivity can then be checked in the same manner for colicin El, E2, and E3 sensitivity, using E. coli strains colicinogenic for these colicins. M a p p i n g o f e u p Mutations. The eup locus maps near minute 88 on the E. coli linkage map and cotransduces with m e t B at a frequency of approximately 20%. 5,8 To determine if the mutation resulting in a Eup phenotype has occurred within the eup locus, bacteriphage PI lysates are prepared on the putative Eup mutants and used to transduce an E. coli m e t B 13 Selection for neomycin resistance can yield electron transport defective mutants, some of which are chlorate sensitive under aerobic conditions. ~4,~5 Nitrate reductase synthesis, which is repressed under aerobic conditions in wild-type E. coli, is derepressed in these mutants. The chlorate sensitivity is due to nitrate reductase reducing chlorate to chlorite which is toxic to E. coli. ~4R. D. Simoni and M. K. Shallenberger, Proc. Natl. Acad. Sci. U.S.A. 69, 2663 (1972). ~5G. Giordano, L. Grillet, R. Rosset, J. H. Dou, E. Azoulay, and B. A. Haddock, Biochem. J. 176, 553 (1978).
190
GENERALAPPROACHES
[16]
strain to MetB. +. The MetB + transductants are picked onto minimal glucose, minimal succinate, and LB-neomycin (10/zg/ml) plates to score for the Eup- phenotype. Measurements of Transport and the PMF. Eup mutants are defective in the PMF-coupled transport of lactose, proline, and alanine but are normal for the ATP-dependent transport of glutamine and arginine.7.8 Eup mutants are also normal in their ability to generate and maintain a pH gradient (inside alkaline) and a membrane potential (inside negative), the components of the PMF. 8,1°,H Procedures for making transport measurements and measuring the components of the PMF are given in detail elsewhere in this series.16,17 Storage of Eup Mutants. Eup mutants readily revert to Eup + and are difficult to maintain. The most successful method that we have found for preserving Eup stocks is to make liquid cultures 20% in glycerol and store aliquots frozen at - 8 0 °. Eup stocks cannot be maintained as stabs. Eup Null Phenotype. Results obtained with an E. coli strain deleted of the eup locus have shown that the eup null phenotype is quasi Eup+. TM The eup deletion strain grows on nonfermentable carbon sources, although not as well as its Eup + counterpart, has normal proton/solute cotransport activities, and is hypersensitive to the aminoglycoside amikacin. Apart from the constraints that this finding places on possible modes of eup function, it is significant in that it limits the types of mutations that might be expected to give rise to the Eup phenotype. Thus the Eupphenotype is not likely to result from insertion or early nonsense mutations that occur within the eup locus. 16 G. F. Ames, this series, Vol. 32, p. 843. 17 S. Ramos, S. Schuldiner, and H. R. Kaback, this series, Vol. 55, p. 680. J8 C. A. Plate, unpublished observation (1984).
[16] M e t h o d s for M u t a g e n e s i s o f t h e B a c t e r i o o p s i n G e n e
By MARIE A. GILLES-GONZALEZ, NEIL R. HACKETT, SIMON J. JONES, H . G O B I N D K H O R A N A , D A E - S I L L E E , K I N - M I N G Lo, a n d JOHN M. McCoY
Proton translocation is important in a number of biological systems and bacteriorhodopsin, an integral membrane protein, offers a simple model to study its mechanism. 1 However, investigations of the structure l W. Stoeckenius and R. A. Bogomolni, A n n u . Rev. Biochem. 52, 587 (1982).
METHODS IN ENZYMOLOGY, VOL. 125
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
eup MUTATIONSIN E. coli
187
[15] M u t a t i o n s in t h e e u p L o c u s o f E s c h e r i c h i a coli, E n e r g y Uncoupled Phenotype B y CHARLES A . PLATE
Three genetic loci have been identified in Escherichia coli in which mutations result in an energy uncoupled phenotype, i.e., lack of growth on nonfermentable carbon sources, reduced growth yields on limiting glucose, and normal electron transport. The best characterized of the three, the unc locus near minute 84 on the E. coli linkage map, ~ encodes the subunits of the BFoFrATPase. 2 Certain Unc mutants are abnormally permeable to protons resulting in a reduced protonmotive force (PMF), which in turn leads to defective proton/solute cotransport and a low level resistance to aminoglycoside antibiotics) The second locus, the ecfA locus near minute 65, is the subject of another chapter in this volume 4 and will not be dealt with further here. The third locus, independently identified by three laboratories, maps near minute 88 and has been variously designated ecfB, 5 ssd, 6 and e u p 7,8 (the latter designation to be used hereafter). 9 Mutations within the eup locus result in strains that grow on glucose, do not grow on succinate, have reduced growth yields on limiting glucose, have normal electron transport, have normal BFoFrATPase activity, and are defective in proton/solute cotransport. In addition, Eup mutants have decreased sensitivity to colicins K and A and they exhibit low level resistance to aminoglycoside antibiotics. Although Eup mutants phenotypically resemble the proton permeable Unc mutants, they differ in one significant respect. Eup mutants are not abnormally permeable to protons and they are capable of generating and maintaining a PMF of normal magnitude.8,1°,l~ The phenoi B. J. Bachmann, Microbiol. Rev. 47, 180 (1983). z j. A. Downie, F. Gibson, and G. B. Cox, Annu. Rev. Biochem. 48, 103 (1979). 3 B. P. Rosen, J. Bacteriol. 116, 1124 (1973). 4 j. S. Hong, this volume [14]. 5 S. H. Thorbjarnardottir, R. A. Magnusdottir, G. Eggertsson, S. A. Kagan, and O. S. Andresson, Mol. Gen. Genet. 161, 89 (1978). 6 E. B. Newman, N. Malik, and C. Walker, J. Bacteriol. 150, 710 (1982). 7 C. A. Plate, J. Bacteriol. 125, 467 (1976). 8 C. A. Plate and J. L. Suit, J. Biol. Chem. 256, 12974 (1981). 9 It should be noted that while it is likely that ecfB, ssd, and eup are alleles, complementation studies to definitively establish this have not yet been done. 10 E. R. Kashket, J. Bacteriol. 146, 377 (1981). H G. D. Hitchens, D. B. Kell, and J. G. Morris, J. Gen. Microbiol. 128, 2207 (1982).
METHODS IN ENZYMOLOGY, VOL. 125
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
188
GENERALAPPROACHES
[15]
typic similarities between Eup and Unc mutants suggests that a product of the eup locus is important to the process of energy coupling, but the precise nature of its involvement has yet to be determined. The purpose of this article is to describe the procedures for obtaining and identifying eup mutations in E. coli. Selection Procedure E. coli Strains and Growth Media. The media used in these studies are LB broth (containing per liter: tryptone, 10 g; yeast extract, 5 g; NaC1, 5 g; adjusted to pH 7.0 with 1.0 N NaOH) and Ozeki minimal base (containing per liter: K2HPO4, 10.5 g; KH2PO4, 4.5 g; (NH4)2SO4, 1.0 g; MgSO4, 0.05 g; sodium citrate, 0.47 g) supplemented with a carbon source (0.4%), required amino acids (50 ~g/ml), and thiamine (0.5 t~g/ml). Solid Ozeki minimal and LB media contain 1.5% agar and LB soft agar contains 0.65% agar. As previously stated, one characteristic of Eup mutants is their inability to grow on succinate as sole carbon source. We and others have found that this particular Eup trait is subject to extragenic suppression. 6,8 The following Eup mutant isolation protocol assumes that the E. coli strain to be employed does not harbor this ill-defined suppressor and is sensitive to colicins A and K and amino glycoside antibiotics such as neomycin. We have used two E. coli K12 strains that satisfy these conditions: strain A279a (HfrH3000) and strain M72 [lacZ(Am) trp(Am) thi]. Both of these strains are available from Dr. S. E. Luria (Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139). The required E. coli strains colicinogenic for colicins A, K, El, E2, and E3 can be obtained from this same source or from the E. coli Genetic Stock Center (Yale University School of Medicine, New Haven, CT 06510). Mutagenesis and Screening for Eup Mutants. Ethylmethane sulfonate mutagenesis is carried out essentially by the procedure of Miller. 12E. coli cells are grown in minimal medium with glucose as carbon source to a density of 2 x 108 cells/ml. The cells are pelleted, washed, and resuspended in one-half the original volume of the minimal medium base without carbon source. To 2.0 ml of this cell suspension add 0.03 ml of ethylmethane sulfonate, mix vigorously to dissolve, and incubate the cells at 37° with shaking for 2 hr (this results in approximately a 90% reduction in viable count). The cells are then pelleted and resuspended in 3.0 ml of unsupplemented minimal medium base. 12j. H. Miller, in "Experiments in Molecular Genetics," p. 138. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1972.
[15]
eup MUTATIONSIN E. coli
189
Fifteen tubes, each containing 2.0 ml of LB broth supplemented with glucose (0.4%), are inoculated with 0.2 ml of the mutagenized cell suspension and incubated overnight at 37° with shaking. Dilute an aliquot of each culture 100-fold and spread 0.1 ml of each dilution onto a minimal glucose agar plate containing neomycin sulfate (20 pg/ml). The plates are incubated at 37 ° and, after 3 days, there should be approximately 100-200 colonies/plate. The neomycin-resistant colonies are replica plated onto minimal succinate, minimal glucose, minimal glucose-neomycin (10 /zg/ml), and LB agar plates supplemented with glucose (0.4%) and K C I O 3 (0.2%). ~3-15All plates are incubated at 37° for 2 days. Putative Eup mutants will grow on the glucose and glucose-neomycin plates, will grow aerobically in the presence of chlorate, but will not grow on the succinate plates. Colonies exhibiting a succinate-nonutilizing, neomycin-resistant, chlorate-resistant phenotype are then checked for colicin A and/or K sensitivity. Eup mutants exhibit a markedly reduced sensitivity to these colicins but exhibit normal sensitivity to colicins El, E2, and E3. 7 Colonies to be checked are inoculated into LB broth and grown overnight at 37°. LB agar plates are stabbed with cells colicinogenic for colicin A or K and these also are incubated overnight at 37°. The colicinogenic cell buttons are removed with filter paper and the plates sterilized by exposure to chloroform vapors for 30 min. Aliquots of the LB cultures to be tested are inoculated into LB soft agar (maintained at 45°), spotted over the colicinogenic stabs, and the plates incubated overnight at 37°. Colicin sensitivity is indicated by a clear zone in the soft agar overlay, and Eup mutants will give a much smaller zone of growth inhibition than the wild-type starting strain. Colonies exhibiting a reduced colicin K or A sensitivity can then be checked in the same manner for colicin El, E2, and E3 sensitivity, using E. coli strains colicinogenic for these colicins. M a p p i n g o f e u p Mutations. The eup locus maps near minute 88 on the E. coli linkage map and cotransduces with m e t B at a frequency of approximately 20%. 5,8 To determine if the mutation resulting in a Eup phenotype has occurred within the eup locus, bacteriphage PI lysates are prepared on the putative Eup mutants and used to transduce an E. coli m e t B 13 Selection for neomycin resistance can yield electron transport defective mutants, some of which are chlorate sensitive under aerobic conditions. ~4,~5 Nitrate reductase synthesis, which is repressed under aerobic conditions in wild-type E. coli, is derepressed in these mutants. The chlorate sensitivity is due to nitrate reductase reducing chlorate to chlorite which is toxic to E. coli. ~4R. D. Simoni and M. K. Shallenberger, Proc. Natl. Acad. Sci. U.S.A. 69, 2663 (1972). ~5G. Giordano, L. Grillet, R. Rosset, J. H. Dou, E. Azoulay, and B. A. Haddock, Biochem. J. 176, 553 (1978).
190
GENERALAPPROACHES
[16]
strain to MetB. +. The MetB + transductants are picked onto minimal glucose, minimal succinate, and LB-neomycin (10/zg/ml) plates to score for the Eup- phenotype. Measurements of Transport and the PMF. Eup mutants are defective in the PMF-coupled transport of lactose, proline, and alanine but are normal for the ATP-dependent transport of glutamine and arginine.7.8 Eup mutants are also normal in their ability to generate and maintain a pH gradient (inside alkaline) and a membrane potential (inside negative), the components of the PMF. 8,1°,H Procedures for making transport measurements and measuring the components of the PMF are given in detail elsewhere in this series.16,17 Storage of Eup Mutants. Eup mutants readily revert to Eup + and are difficult to maintain. The most successful method that we have found for preserving Eup stocks is to make liquid cultures 20% in glycerol and store aliquots frozen at - 8 0 °. Eup stocks cannot be maintained as stabs. Eup Null Phenotype. Results obtained with an E. coli strain deleted of the eup locus have shown that the eup null phenotype is quasi Eup+. TM The eup deletion strain grows on nonfermentable carbon sources, although not as well as its Eup + counterpart, has normal proton/solute cotransport activities, and is hypersensitive to the aminoglycoside amikacin. Apart from the constraints that this finding places on possible modes of eup function, it is significant in that it limits the types of mutations that might be expected to give rise to the Eup phenotype. Thus the Eupphenotype is not likely to result from insertion or early nonsense mutations that occur within the eup locus. 16 G. F. Ames, this series, Vol. 32, p. 843. 17 S. Ramos, S. Schuldiner, and H. R. Kaback, this series, Vol. 55, p. 680. J8 C. A. Plate, unpublished observation (1984).
[16] M e t h o d s for M u t a g e n e s i s o f t h e B a c t e r i o o p s i n G e n e
By MARIE A. GILLES-GONZALEZ, NEIL R. HACKETT, SIMON J. JONES, H . G O B I N D K H O R A N A , D A E - S I L L E E , K I N - M I N G Lo, a n d JOHN M. McCoY
Proton translocation is important in a number of biological systems and bacteriorhodopsin, an integral membrane protein, offers a simple model to study its mechanism. 1 However, investigations of the structure l W. Stoeckenius and R. A. Bogomolni, A n n u . Rev. Biochem. 52, 587 (1982).
METHODS IN ENZYMOLOGY, VOL. 125
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.