- Email: [email protected]
[17] Isolation of aurovertin-resistant mutants from Escherichia coli
178
GENETIC APPROACHES
[17]
cells. Recently, the isolation of a h e m i n - p e r m e a b l e mutant o f Escherichia coli has been reported, and this m a y extend the range hem mutants available for biochemical and genetic analysis. 9 Mutant strains of E. coli and Salmonella typhimurium are being exploited for genetic studies; they have been characterized mainly by their ability to f o r m c y t o c h r o m e s when grown with A L A , and by the accumulation of P B G and porphyrins from A L A . t°-'2 Reconstruction studies have been made with such mutants of E. coli. As in the staphylococcal mutants, a p o - c y t o c h r o m e b is apparently formed by the coliforms when grown without A L A , but the conditions for reconstruction are complex. Formation of c y t o c h r o m e s b, o, and a2 a c c o m p a n y the dev e l o p m e n t of respiration in intact cells incubated with A L A and gluc o s e ) TM H e m i n is not effective with cells, but it restores b - t y p e cytoc h r o m e and respiratory activity to m e m b r a n e preparations, provided that ATP is present. The role of ATP is unknown.t4 C y t o c h r o m e - m e d i a t e d reduction of nitrate cannot be restored to either cells or m e m b r a n e s of the mutant E. coli, in contrast to the s y s t e m s f r o m S. aureus. '~ The relative simplicity of the latter is an advantage in studies of a s s e m b l y of respiratory s y s t e m s and of the role of h e m o p r o t e i n s in the process. M. McConville and H. P. Charles, Proc. Soc. Gen. Microbiol. 3, 14 (1976).
,o A. Sasarman, K. E. Sanderson, M. Surdeanu, and S. Sonea, J. Bacteriol. 102, 531 (1970). 1, R. Cox and H. P. Charles, J. Bacteriol. 113, 122 (1973). 1~A. Sasarman, P. Chartrand, R. Proschek, M. Desrochers, D. Tardif, and C. Lapointe, J. Bacteriol. 124, 1205 (1975). t3 B. A. Haddock and H. U. Schairer, Eur. J. Biochem. 35, 34 (1973). 14B. A. Haddock, Biochern. J. 136, 877 (1973). 15M. B. Kemp, B. A. Haddock, and P. B. Garland, Biochem. J. 148, 329 (1975).
[17] Isolation of Aurovertin-Resistant Escherichia
Mutants
from
coli
B y M I C H E L S A T R E , G E R A R D K L E I N , a n d P I E R R E V. V I G N A I S
A u r o v e r t i n is a potent inhibitor o f oxidative phosphorylation in mitochondria. It inhibits both ATPase activity and ATP synthesis coupled to electron transfer in the respiratory chain. 1-7 It is also an inhibitor of ' H. A. L a r d y , J. L. Connelly, and D. J o h n s o n , Biochemistry 3, 1961 (1964). 2 H. A. L a r d y a n d C. H. C. Lin, in "Inhibitors-Tools in Cell R e s e a r c h " (T. Biicher and H. Sies, eds.), p. 279. Springer-Verlag, Berlin and N e w York, 1969. 3 G. Lenaz, Biochem. Biophys. Res. Commun. 21, 170 (1965).
METHODS IN ENZYMOLOGY, VOL. LVI
Copyright © 1979by AcademicPress, Inc. All rights of reproduction in any form reserved. 1SBN 0-12-181956-6
[1 7]
AUROVERTIN-RESISTANT MUTANTS
179
o x i d a t i v e p h o s p h o r y l a t i o n in a n u m b e r o f b a c t e r i a i n c l u d i n g Rhodospirillum rubrum, s'9 Alcaligenes faecalis, TM Paracoccus denitrificans, n a n d Escherichia coli. ~2 T h e f o l l o w i n g p r o c e d u r e h a s b e e n u s e d to i s o l a t e a u r o v e r t i n - r e s i s t a n t m u t a n t s o f E . coli, in w h i c h t h e A T P a s e is no l o n g e r s e n s i t i v e to a u r o v e r t i n , lz
Selection Procedure and Properties
Preparation o f A u r o v e r t i n D. A u r o v e r t i n is p r e p a r e d f r o m 2 - w e e k - o l d c u l t u r e s o f Calcarisporium arbusculurn. 13,14 T h e g r o w t h m e d i u m a n d t h e m y c e l i u m a r e s e p a r a t e l y e x t r a c t e d a n d a u r o v e r t i n D is p u r i f i e d b y prep a r a t i v e t h i n - l a y e r c h r o m a t o g r a p h y on s i l i c a gel p l a t e s in e t h y l a c e t a t e : b e n z e n e ( 7 : 3 ) . A u r o v e r t i n D is s t o r e d at - 2 0 ° C as an e t h a n o l i c s o l u t i o n p r o t e c t e d f r o m light. Its c o n c e n t r a t i o n is c a l c u l a t e d f r o m a m o l a r a b s o r p t i o n coefficient o f 42,700 at 367.5 nm. 15 Isolation o f Aurovertin-Resistant Mutants. T h e s t a n d a r d m e d i u m u s e d f o r t h e g r o w t h o f E . coli c o n s i s t s o f m e d i u m E 16,~7 d i l u t e d t w e n t y f o l d w i t h w a t e r a n d s u p p l e m e n t e d w i t h L - a r g i n i n e ( 4 0 / z g / m l ) , t h i a m i n e (1 /~g/ml), and a mixture of sodium succinate, sodium acetate, and sodium malate e a c h at a c o n c e n t r a t i o n o f 0 . 4 % as n o n f e r m e n t a b l e c a r b o n s o u r c e s . A g a r p l a t e s a r e p r e p a r e d f r o m 30 ml o f t h e s t a n d a r d m e d i u m solidified w i t h 1.5% agar. P l a t e s c o n t a i n i n g a u r o v e r t i n a r e o b t a i n e d b y s p r e a d i n g 1 ml o f 9 m M a u r o v e r t i n in e t h a n o l o v e r t h e s u r f a c e o f agar. T h e p l a t e s a r e m a i n t a i n e d at 37°C until t h e e t h a n o l h a s c o m p l e t e l y d i s a p p e a r e d .
4 A. M. Roberton, R. B. Beechey, C. T. Holloway, and I. C. Knight, Biochem. J. 104, 54 (1967). s R. M. Bertina, P. I. Schrier, and E. C. Slater, Biochim. Biophys. Acta 305, 503 (1973). ~R. J. Van de Stadt and K. Van Dam, Biochim. Biophys. Acta 347, 253 (1974). r R. J. Van de Stadt, K. Van Dam, and E. C. Slater, Biochim. Biophys. Acta 347,224 (1974). sZ. Gromet-Elhanan, Proc. Int. Congr. Photosynth., 3rd, 1974 Vol. 1, p. 791 (1975). 9R. A. Ravizzini, W. I. M. Lescano, and R. H. Vallejos, FEBS Lett. 58, 285 (1975). ~oR. Adolfsen and E. N. Moudrianakis, Biochemistry 15, 4163 (1976). 11D. A. Harris, P. John, and G. K. Radda, Biochirn. Biophys. Acta 459, 546 (1977). ~2M. Satre, G. Klein, and P. V. Vignais, J. Bacteriol. 134, 17 (1978). ~3Calcarisporium arbusculum was obtained courtesy of Dr. J. J. Ellis, Northern Regional Research Center, Peoria, Illinois 61604. 14M. D. Osselton, H. Baum, and R. B. Beechey, Biochem. Soc. Trans. 2, 200 (1974). ~sC. L. Baldwin, L. C. Weaver, R. M. Brooker, T. N. Jacobsen, C. E. Osborne, and H. A. Nash, Lloydia 27, 85 (1964). ~6L. Caro and C. M. Berg, Vol. 21, p. 444 (1971). ~7Medium E consists of 6 g MgSO4 " 7H20, 60 g citric acid • IH20, 300 g K2HPO4, 105 g NaNH4HPO4 - 41-I20 and 1330 mi H20.
180
GENETIC APPROACHES
[17]
Aurovertin-resistant cells are generated by nitrosoguanidine mutagenesis of the parental strain AN 180,ls'~9 (E. coli K-12, arg-E3, thi-1, StrR). Strain AN 180 is grown overnight in 5 ml of LB medium~° consisting of 1% Difco tryptone, 0.5% Difco yeast extract, 1% NaCI, and supplemented with 0.2% glucose. The cells are washed with 5 ml of 0.85% NaC1 and resuspended in 2.5 ml of 0.85% NaCI. A sample of the suspension is mixed with 3 ml " F - t o p " agar~° to give a concentration of about 108 cells/ml, and the mixture is poured over the surface of the agar plates containing aurovertin. A small crystal (1-2 mm2) of N-methylN-nitrosoguanidine is placed in the center, and the plates are incubated at 37° for 3 days. Of the distinct colonies which are obtained, the largest ones are selected and grown at 37° in 2 ml of a standard liquid medium supplemented with streptomycin (110 /~g/ml) and 0.3 mM aurovertin. Aurovertin-resistant colonies are further purified by two successive transfers of single colonies on agar plates containing 0.2 mM aurovertin. Aurovertin-resistant strains are referred to as MA strains. Aurovertin-Resistant ATPase. Cell-free extracts for ATPase assays are prepared by lysozyme-EDTA treatment. 2~ Bacteria are harvested, washed twice with 0.85% NaC1, and resuspended in 25 mM Tris-sulfate 2.5 mM EDTA, and tysozyme (0.1 mg/ml), final pH 8.2. After incubation at 37°C for 10 min, the clarified suspension is briefly homogenized with a Potter type homogenizer. The lysate is kept at room temperature and immediately assayed for ATPase activity at 37°CY2 The reaction mixture contains 20 mM ATP, 10 mM MgSO4, 1.5 mM EDTA, 100 mM Tris sulfate, pH 9.0, in a final volume of 0.5 ml. After a 15-min incubation at 37°, the reaction is stopped by adding 0.1 ml of 2.5 N perchloric acid. Inorganic phosphate released from ATP is determined on 0.2-ml aliquots by the method of Fiske and Subbarow 23 using Elon (p-methylaminophenol sulfate) as the reducing agent. The ATPase activity in cell-free extracts of the parental strain AN 180 is highly sensitive to aurovertin, half-maximal inhibition being obtained with 1 /~M aurovertin. On the other hand, 15 MA mutants which have been assayed have been found to possess an ATPase activity totally resistant to 20/zM aurovertin. The ATPase of mutant MA1, which has been lsj. D. Butlin, G. B. Cox, and F. Gibson, Biochem. J. 124, 75 (1971). t9 Strains AN 180 and AN 120 were obtained courtesy of Dr. Barbara Bachman, Escherichia coli Genetic Stock Center, Yale University, New Haven, Connecticut 06510. 20j. H. Miller, in "Experiments in Molecular Genetics," p. 201. Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1972. 21 E. Hawrot and E. P. Kennedy, Proc. Natl. Acad. Sci. U.S.A. 72, 1112 (1975). 22 j. D. Butlin, G. B. Cox, and F. Gibson, Biochim. Biophys. Acta 292, 366 (1973). 2s C. A. Fiske and Y. Subbarow, J. Biol. Chem. 66, 375 (1925).
[17]
AUROVERTIN-RESISTANT MUTANTS
181
TABLE I EFFECT OF VARIOUS INH1BITORS ON ATPASE ACTIVITIES OF MEMBRANE-BOUND ATPAsE IN CELL-FREE EXTRACTS OF E. coli AN 180 AND THE AUROVERTIN-RESISTANT MUTANT MAI
Inhibition of ATPase activity (%)0 Compound
AN 180
MA1
Aurovertin (20/~M) Citreoviridin (60 ~M) DCCD ~ (100/zM) Nbf-CF (50/zM) Quercetin (0.8 mM) EEDQ a (0.4 raM) Sodium azide (10 mM)
81 60 93 91 11 90 91
6 0 89 89 10 92 92
The ATPase specific activities were 1.5 and 1.1 /zmoles phosphate released/min/mg protein for AN 180 and MAI, respectively. DCCD, dicyclohexylcarbodiimide. c Nbf-Cl, 7-chloro-4-nitrobenzofurazan. d EEDQ, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline.
further characterized, is also resistant to citreoviridin, an antibiotic structurally related to aurovertin, 24 but it retains its sensitivity to other ATPase inhibitors including azide, dicyclohexycarbodiimide, 7-chloro-4-nitrobenzofurazan, and N-ethoxycarboxyl-2-ethoxy-1,2-dihydroquinoline (see Table I). The energization ofE. coli membrane vesicles caused by ATP hydrolysis can be monitored by atebrin fluorescence quenching. "5 The atebrin assay shows that aurovertin reverses the ATP-dependent energization in the parental strain AN 180. In contrast, 20/zM aurovertin has no effect on the ATP-dependent energization in the MAI mutant. Mapping o f Mutation MA1 Mutation MA1 maps in the unc region 2s of the bacterial chromosome like other mutations of the ATPase complex as shown by transduction with phage Pl.le A phage (P1) lysate of the mutant MA1 was used to infect an ATPase negative mutant, strain AN 120 (unc A 401). TM Unc + recombinants were selected by growth on agar plates with succinate, acetate, and malate as nonfermentable carbon sources. To 24R. B. Beechey, D. O. Osselton, H. Baum, P. E. Linnett, and A, D. Mitchell, in "Membrane Proteins in Transport and Phosphorylation" (G. F. Azzone et al., eds.), p. 201. North-Holland Publ., Amsterdam, 1974. 2~F. Gibson, G. B. Cox, J. A. Downie, and J. Radik, Biochem. J. 162, 665 (1977). 26 B. J. Bachmann, K. B. Low, and A. L. Taylor, Bacteriol. Rev. 40, 116 (1976).
182
GENETIC
APPROACHES
[18]
s p a r e a u r o v e r t i n , the s c r e e n i n g of r e c o m b i n a n t s b y r e p l i c a t i o n o n aur o v e r t i n plates was a v o i d e d . I n s t e a d , single c o l o n i e s c o r r e s p o n d i n g to the unc÷ r e c o m b i n a n t s w e r e g r o w n in the s t a n d a r d liquid n o n f e r m e n t a b l e med i u m . O f 43 c l o n e s w h i c h h a v e b e e n a s s a y e d for A T P a s e a c t i v i t y , 40 w e r e f o u n d to h a v e a n A T P a s e totally r e s i s t a n t to 2 0 / ~ M a u r o v e r t i n , w h i c h suggests t h a t m u t a t i o n M A 1 m a p s in the unc region. Aurovertin-Resistant Mutants in Other Organisms. Yeast m u t a n t s with a m i t o c h o n d r i a l A T P a s e r e s i s t a n t to a u r o v e r t i n h a v e b e e n r e c e n t l y isolated in the y e a s t Saccharomyces cerevisiae. 27 T h e a u r o v e r t i n - r e s i s t a n c e has b e e n a s c r i b e d to a d e f e c t i v e fl s u b u n i t o f F1 A T P a s e . 2s 2r E. Agsterribbe, M. Douglas, E. Ebner, T. Y. Koh, and G. Schatz, in "Genetics and Biogenesis of Chloroplasts and Mitochondria" (T. Biicher et al., eds.), p. 135. NorthHolland Publ., Amsterdam, 1976. ~sM. G. Douglas, Y. Koh, M. E. Dockter, and G. Schatz, J. Biol. Chem. 252, 8333 (1977).
[18] B i o c h e m i c a l M e t h o d s to Locate G e n e s on t h e P h y s i c a l M a p of Yeast Mitochondrial D N A 1
By PIET BORST, JOHAN P. M. SANDERS, and CHRISTA HEYTING I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Procedure of Gene Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Identification of the Segment of Wild-Type mtDNA Present in Petite mtDNA by Restriction Endonuclease Fragmentation . . . . . . . . . . . . . . . . B. Identification of the Segment of Wild-Type mtDNA Present in Petite mtDNA by Hybridization Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Mapping of Genetic Markers by DNA-DNA Plateau Hybridization . . . . . . . . III. Pitfalls and Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The Yeast mtDNA Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. The Use of mtDNA Mapping in Mitochondrial Membrane Research . . . . . . . . . . .
182 185 185 191 192 192 194 194
I. I n t r o d u c t i o n
T h e m t D N A o f the y e a s t s Saccharomyces carlsbergensis a n d Saccharomyces cerevisiae is a c i r c u l a r d u p l e x D N A w i t h a m o l e c u l a r w e i g h t o f a b o u t 50 x 10 ° ( H o l l e n b e r g et al. la). Specific f r a g m e n t s o f this D N A h a v e b e e n m a d e with r e s t r i c t i o n e n d o n u c l e a s e s , z-7 a n d the f r a g m e n t s h a v e b e e n S u b m i t t e d for p u b l i c a t i o n in D e c e m b e r , 1976. S e e P. B o r s t a n d L . A. G r i v e l i , Cell 15, 705
(1978) for a more up to date account. ~a C. P. Hollenberg, P. Borst, and E. F. J. Van Bruggen, Biochim. Biophys. Acta 209, 1 (1970). J. P. M. Sanders, C. Heyting, and P. Borst, Biochem. Biophys. Re#. Commun. 65, 699 (1975). :~J. P. M. Sanders, P. Borst, and P. J. Weijers, Mol. Gen. Genet. 143, 53 (1975).
METHODS IN ENZYMOLOGY, VOL. LVI
Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181956-6
GENETIC APPROACHES
[17]
cells. Recently, the isolation of a h e m i n - p e r m e a b l e mutant o f Escherichia coli has been reported, and this m a y extend the range hem mutants available for biochemical and genetic analysis. 9 Mutant strains of E. coli and Salmonella typhimurium are being exploited for genetic studies; they have been characterized mainly by their ability to f o r m c y t o c h r o m e s when grown with A L A , and by the accumulation of P B G and porphyrins from A L A . t°-'2 Reconstruction studies have been made with such mutants of E. coli. As in the staphylococcal mutants, a p o - c y t o c h r o m e b is apparently formed by the coliforms when grown without A L A , but the conditions for reconstruction are complex. Formation of c y t o c h r o m e s b, o, and a2 a c c o m p a n y the dev e l o p m e n t of respiration in intact cells incubated with A L A and gluc o s e ) TM H e m i n is not effective with cells, but it restores b - t y p e cytoc h r o m e and respiratory activity to m e m b r a n e preparations, provided that ATP is present. The role of ATP is unknown.t4 C y t o c h r o m e - m e d i a t e d reduction of nitrate cannot be restored to either cells or m e m b r a n e s of the mutant E. coli, in contrast to the s y s t e m s f r o m S. aureus. '~ The relative simplicity of the latter is an advantage in studies of a s s e m b l y of respiratory s y s t e m s and of the role of h e m o p r o t e i n s in the process. M. McConville and H. P. Charles, Proc. Soc. Gen. Microbiol. 3, 14 (1976).
,o A. Sasarman, K. E. Sanderson, M. Surdeanu, and S. Sonea, J. Bacteriol. 102, 531 (1970). 1, R. Cox and H. P. Charles, J. Bacteriol. 113, 122 (1973). 1~A. Sasarman, P. Chartrand, R. Proschek, M. Desrochers, D. Tardif, and C. Lapointe, J. Bacteriol. 124, 1205 (1975). t3 B. A. Haddock and H. U. Schairer, Eur. J. Biochem. 35, 34 (1973). 14B. A. Haddock, Biochern. J. 136, 877 (1973). 15M. B. Kemp, B. A. Haddock, and P. B. Garland, Biochem. J. 148, 329 (1975).
[17] Isolation of Aurovertin-Resistant Escherichia
Mutants
from
coli
B y M I C H E L S A T R E , G E R A R D K L E I N , a n d P I E R R E V. V I G N A I S
A u r o v e r t i n is a potent inhibitor o f oxidative phosphorylation in mitochondria. It inhibits both ATPase activity and ATP synthesis coupled to electron transfer in the respiratory chain. 1-7 It is also an inhibitor of ' H. A. L a r d y , J. L. Connelly, and D. J o h n s o n , Biochemistry 3, 1961 (1964). 2 H. A. L a r d y a n d C. H. C. Lin, in "Inhibitors-Tools in Cell R e s e a r c h " (T. Biicher and H. Sies, eds.), p. 279. Springer-Verlag, Berlin and N e w York, 1969. 3 G. Lenaz, Biochem. Biophys. Res. Commun. 21, 170 (1965).
METHODS IN ENZYMOLOGY, VOL. LVI
Copyright © 1979by AcademicPress, Inc. All rights of reproduction in any form reserved. 1SBN 0-12-181956-6
[1 7]
AUROVERTIN-RESISTANT MUTANTS
179
o x i d a t i v e p h o s p h o r y l a t i o n in a n u m b e r o f b a c t e r i a i n c l u d i n g Rhodospirillum rubrum, s'9 Alcaligenes faecalis, TM Paracoccus denitrificans, n a n d Escherichia coli. ~2 T h e f o l l o w i n g p r o c e d u r e h a s b e e n u s e d to i s o l a t e a u r o v e r t i n - r e s i s t a n t m u t a n t s o f E . coli, in w h i c h t h e A T P a s e is no l o n g e r s e n s i t i v e to a u r o v e r t i n , lz
Selection Procedure and Properties
Preparation o f A u r o v e r t i n D. A u r o v e r t i n is p r e p a r e d f r o m 2 - w e e k - o l d c u l t u r e s o f Calcarisporium arbusculurn. 13,14 T h e g r o w t h m e d i u m a n d t h e m y c e l i u m a r e s e p a r a t e l y e x t r a c t e d a n d a u r o v e r t i n D is p u r i f i e d b y prep a r a t i v e t h i n - l a y e r c h r o m a t o g r a p h y on s i l i c a gel p l a t e s in e t h y l a c e t a t e : b e n z e n e ( 7 : 3 ) . A u r o v e r t i n D is s t o r e d at - 2 0 ° C as an e t h a n o l i c s o l u t i o n p r o t e c t e d f r o m light. Its c o n c e n t r a t i o n is c a l c u l a t e d f r o m a m o l a r a b s o r p t i o n coefficient o f 42,700 at 367.5 nm. 15 Isolation o f Aurovertin-Resistant Mutants. T h e s t a n d a r d m e d i u m u s e d f o r t h e g r o w t h o f E . coli c o n s i s t s o f m e d i u m E 16,~7 d i l u t e d t w e n t y f o l d w i t h w a t e r a n d s u p p l e m e n t e d w i t h L - a r g i n i n e ( 4 0 / z g / m l ) , t h i a m i n e (1 /~g/ml), and a mixture of sodium succinate, sodium acetate, and sodium malate e a c h at a c o n c e n t r a t i o n o f 0 . 4 % as n o n f e r m e n t a b l e c a r b o n s o u r c e s . A g a r p l a t e s a r e p r e p a r e d f r o m 30 ml o f t h e s t a n d a r d m e d i u m solidified w i t h 1.5% agar. P l a t e s c o n t a i n i n g a u r o v e r t i n a r e o b t a i n e d b y s p r e a d i n g 1 ml o f 9 m M a u r o v e r t i n in e t h a n o l o v e r t h e s u r f a c e o f agar. T h e p l a t e s a r e m a i n t a i n e d at 37°C until t h e e t h a n o l h a s c o m p l e t e l y d i s a p p e a r e d .
4 A. M. Roberton, R. B. Beechey, C. T. Holloway, and I. C. Knight, Biochem. J. 104, 54 (1967). s R. M. Bertina, P. I. Schrier, and E. C. Slater, Biochim. Biophys. Acta 305, 503 (1973). ~R. J. Van de Stadt and K. Van Dam, Biochim. Biophys. Acta 347, 253 (1974). r R. J. Van de Stadt, K. Van Dam, and E. C. Slater, Biochim. Biophys. Acta 347,224 (1974). sZ. Gromet-Elhanan, Proc. Int. Congr. Photosynth., 3rd, 1974 Vol. 1, p. 791 (1975). 9R. A. Ravizzini, W. I. M. Lescano, and R. H. Vallejos, FEBS Lett. 58, 285 (1975). ~oR. Adolfsen and E. N. Moudrianakis, Biochemistry 15, 4163 (1976). 11D. A. Harris, P. John, and G. K. Radda, Biochirn. Biophys. Acta 459, 546 (1977). ~2M. Satre, G. Klein, and P. V. Vignais, J. Bacteriol. 134, 17 (1978). ~3Calcarisporium arbusculum was obtained courtesy of Dr. J. J. Ellis, Northern Regional Research Center, Peoria, Illinois 61604. 14M. D. Osselton, H. Baum, and R. B. Beechey, Biochem. Soc. Trans. 2, 200 (1974). ~sC. L. Baldwin, L. C. Weaver, R. M. Brooker, T. N. Jacobsen, C. E. Osborne, and H. A. Nash, Lloydia 27, 85 (1964). ~6L. Caro and C. M. Berg, Vol. 21, p. 444 (1971). ~7Medium E consists of 6 g MgSO4 " 7H20, 60 g citric acid • IH20, 300 g K2HPO4, 105 g NaNH4HPO4 - 41-I20 and 1330 mi H20.
180
GENETIC APPROACHES
[17]
Aurovertin-resistant cells are generated by nitrosoguanidine mutagenesis of the parental strain AN 180,ls'~9 (E. coli K-12, arg-E3, thi-1, StrR). Strain AN 180 is grown overnight in 5 ml of LB medium~° consisting of 1% Difco tryptone, 0.5% Difco yeast extract, 1% NaCI, and supplemented with 0.2% glucose. The cells are washed with 5 ml of 0.85% NaC1 and resuspended in 2.5 ml of 0.85% NaCI. A sample of the suspension is mixed with 3 ml " F - t o p " agar~° to give a concentration of about 108 cells/ml, and the mixture is poured over the surface of the agar plates containing aurovertin. A small crystal (1-2 mm2) of N-methylN-nitrosoguanidine is placed in the center, and the plates are incubated at 37° for 3 days. Of the distinct colonies which are obtained, the largest ones are selected and grown at 37° in 2 ml of a standard liquid medium supplemented with streptomycin (110 /~g/ml) and 0.3 mM aurovertin. Aurovertin-resistant colonies are further purified by two successive transfers of single colonies on agar plates containing 0.2 mM aurovertin. Aurovertin-resistant strains are referred to as MA strains. Aurovertin-Resistant ATPase. Cell-free extracts for ATPase assays are prepared by lysozyme-EDTA treatment. 2~ Bacteria are harvested, washed twice with 0.85% NaC1, and resuspended in 25 mM Tris-sulfate 2.5 mM EDTA, and tysozyme (0.1 mg/ml), final pH 8.2. After incubation at 37°C for 10 min, the clarified suspension is briefly homogenized with a Potter type homogenizer. The lysate is kept at room temperature and immediately assayed for ATPase activity at 37°CY2 The reaction mixture contains 20 mM ATP, 10 mM MgSO4, 1.5 mM EDTA, 100 mM Tris sulfate, pH 9.0, in a final volume of 0.5 ml. After a 15-min incubation at 37°, the reaction is stopped by adding 0.1 ml of 2.5 N perchloric acid. Inorganic phosphate released from ATP is determined on 0.2-ml aliquots by the method of Fiske and Subbarow 23 using Elon (p-methylaminophenol sulfate) as the reducing agent. The ATPase activity in cell-free extracts of the parental strain AN 180 is highly sensitive to aurovertin, half-maximal inhibition being obtained with 1 /~M aurovertin. On the other hand, 15 MA mutants which have been assayed have been found to possess an ATPase activity totally resistant to 20/zM aurovertin. The ATPase of mutant MA1, which has been lsj. D. Butlin, G. B. Cox, and F. Gibson, Biochem. J. 124, 75 (1971). t9 Strains AN 180 and AN 120 were obtained courtesy of Dr. Barbara Bachman, Escherichia coli Genetic Stock Center, Yale University, New Haven, Connecticut 06510. 20j. H. Miller, in "Experiments in Molecular Genetics," p. 201. Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1972. 21 E. Hawrot and E. P. Kennedy, Proc. Natl. Acad. Sci. U.S.A. 72, 1112 (1975). 22 j. D. Butlin, G. B. Cox, and F. Gibson, Biochim. Biophys. Acta 292, 366 (1973). 2s C. A. Fiske and Y. Subbarow, J. Biol. Chem. 66, 375 (1925).
[17]
AUROVERTIN-RESISTANT MUTANTS
181
TABLE I EFFECT OF VARIOUS INH1BITORS ON ATPASE ACTIVITIES OF MEMBRANE-BOUND ATPAsE IN CELL-FREE EXTRACTS OF E. coli AN 180 AND THE AUROVERTIN-RESISTANT MUTANT MAI
Inhibition of ATPase activity (%)0 Compound
AN 180
MA1
Aurovertin (20/~M) Citreoviridin (60 ~M) DCCD ~ (100/zM) Nbf-CF (50/zM) Quercetin (0.8 mM) EEDQ a (0.4 raM) Sodium azide (10 mM)
81 60 93 91 11 90 91
6 0 89 89 10 92 92
The ATPase specific activities were 1.5 and 1.1 /zmoles phosphate released/min/mg protein for AN 180 and MAI, respectively. DCCD, dicyclohexylcarbodiimide. c Nbf-Cl, 7-chloro-4-nitrobenzofurazan. d EEDQ, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline.
further characterized, is also resistant to citreoviridin, an antibiotic structurally related to aurovertin, 24 but it retains its sensitivity to other ATPase inhibitors including azide, dicyclohexycarbodiimide, 7-chloro-4-nitrobenzofurazan, and N-ethoxycarboxyl-2-ethoxy-1,2-dihydroquinoline (see Table I). The energization ofE. coli membrane vesicles caused by ATP hydrolysis can be monitored by atebrin fluorescence quenching. "5 The atebrin assay shows that aurovertin reverses the ATP-dependent energization in the parental strain AN 180. In contrast, 20/zM aurovertin has no effect on the ATP-dependent energization in the MAI mutant. Mapping o f Mutation MA1 Mutation MA1 maps in the unc region 2s of the bacterial chromosome like other mutations of the ATPase complex as shown by transduction with phage Pl.le A phage (P1) lysate of the mutant MA1 was used to infect an ATPase negative mutant, strain AN 120 (unc A 401). TM Unc + recombinants were selected by growth on agar plates with succinate, acetate, and malate as nonfermentable carbon sources. To 24R. B. Beechey, D. O. Osselton, H. Baum, P. E. Linnett, and A, D. Mitchell, in "Membrane Proteins in Transport and Phosphorylation" (G. F. Azzone et al., eds.), p. 201. North-Holland Publ., Amsterdam, 1974. 2~F. Gibson, G. B. Cox, J. A. Downie, and J. Radik, Biochem. J. 162, 665 (1977). 26 B. J. Bachmann, K. B. Low, and A. L. Taylor, Bacteriol. Rev. 40, 116 (1976).
182
GENETIC
APPROACHES
[18]
s p a r e a u r o v e r t i n , the s c r e e n i n g of r e c o m b i n a n t s b y r e p l i c a t i o n o n aur o v e r t i n plates was a v o i d e d . I n s t e a d , single c o l o n i e s c o r r e s p o n d i n g to the unc÷ r e c o m b i n a n t s w e r e g r o w n in the s t a n d a r d liquid n o n f e r m e n t a b l e med i u m . O f 43 c l o n e s w h i c h h a v e b e e n a s s a y e d for A T P a s e a c t i v i t y , 40 w e r e f o u n d to h a v e a n A T P a s e totally r e s i s t a n t to 2 0 / ~ M a u r o v e r t i n , w h i c h suggests t h a t m u t a t i o n M A 1 m a p s in the unc region. Aurovertin-Resistant Mutants in Other Organisms. Yeast m u t a n t s with a m i t o c h o n d r i a l A T P a s e r e s i s t a n t to a u r o v e r t i n h a v e b e e n r e c e n t l y isolated in the y e a s t Saccharomyces cerevisiae. 27 T h e a u r o v e r t i n - r e s i s t a n c e has b e e n a s c r i b e d to a d e f e c t i v e fl s u b u n i t o f F1 A T P a s e . 2s 2r E. Agsterribbe, M. Douglas, E. Ebner, T. Y. Koh, and G. Schatz, in "Genetics and Biogenesis of Chloroplasts and Mitochondria" (T. Biicher et al., eds.), p. 135. NorthHolland Publ., Amsterdam, 1976. ~sM. G. Douglas, Y. Koh, M. E. Dockter, and G. Schatz, J. Biol. Chem. 252, 8333 (1977).
[18] B i o c h e m i c a l M e t h o d s to Locate G e n e s on t h e P h y s i c a l M a p of Yeast Mitochondrial D N A 1
By PIET BORST, JOHAN P. M. SANDERS, and CHRISTA HEYTING I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Procedure of Gene Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Identification of the Segment of Wild-Type mtDNA Present in Petite mtDNA by Restriction Endonuclease Fragmentation . . . . . . . . . . . . . . . . B. Identification of the Segment of Wild-Type mtDNA Present in Petite mtDNA by Hybridization Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Mapping of Genetic Markers by DNA-DNA Plateau Hybridization . . . . . . . . III. Pitfalls and Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The Yeast mtDNA Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. The Use of mtDNA Mapping in Mitochondrial Membrane Research . . . . . . . . . . .
182 185 185 191 192 192 194 194
I. I n t r o d u c t i o n
T h e m t D N A o f the y e a s t s Saccharomyces carlsbergensis a n d Saccharomyces cerevisiae is a c i r c u l a r d u p l e x D N A w i t h a m o l e c u l a r w e i g h t o f a b o u t 50 x 10 ° ( H o l l e n b e r g et al. la). Specific f r a g m e n t s o f this D N A h a v e b e e n m a d e with r e s t r i c t i o n e n d o n u c l e a s e s , z-7 a n d the f r a g m e n t s h a v e b e e n S u b m i t t e d for p u b l i c a t i o n in D e c e m b e r , 1976. S e e P. B o r s t a n d L . A. G r i v e l i , Cell 15, 705
(1978) for a more up to date account. ~a C. P. Hollenberg, P. Borst, and E. F. J. Van Bruggen, Biochim. Biophys. Acta 209, 1 (1970). J. P. M. Sanders, C. Heyting, and P. Borst, Biochem. Biophys. Re#. Commun. 65, 699 (1975). :~J. P. M. Sanders, P. Borst, and P. J. Weijers, Mol. Gen. Genet. 143, 53 (1975).
METHODS IN ENZYMOLOGY, VOL. LVI
Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181956-6