[15] Isolation of carbodiimide-resistant ATPase mutants from Escherichia coli

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by single deletion, it should correspond to the following restriction patterns. 1. If the deletion is small and does not affect a restriction site, one fragment should be reduced in size and the other unaffected. Since deletions are usually large, this is a rare pattern. 2. I f restriction sites are deleted, one or m o r e restriction bands should be missing, and a new band representing sequences adjoining the deleted sequence should appear. 3. The bands should be present in equimolar ratios, but it m a y be impossible to judge this accurately for large fragments, since yeast m t D N A is r a n d o m l y cleaved as isolated. The presence of multiple new bands indicates molecular complexities which m a y include inverted repeats, molecular heterogeneity, and multiple deletions. 4. Most importantly, the restriction bands present in the m t D N A of petite clones must c o r r e s p o n d to a contiguous region of the restriction m a p of the grande m t D N A .

[15]

Isolation of Carbodiimide-Resistant ATPase from Escherichia coli 1

Mutants

B y ROBERT H. FILLINGAME

This article s u m m a r i z e s a selection procedure for mutants of Escherichia coli which contain a dicyclohexylcarbodiimide (DCCD)-resistant energy-transducing ATPase complex. T h e s e mutants have been used to identify the D C C D - r e a c t i v e site of the ATPase complex, 2-~ and are proving useful in distinguishing between the specific effects o f D C C D , that are caused by covalent reaction with the carbodiimide-reactive protein of the ATPase complex, from nonspecific effects due to general chemical modification of other proteins. 4 With these mutants, H a r e 5 was able to show that the D C C D sensitivity of a detergent-solubilized ATPase complex reflected the D C C D sensitivity of the ATPase complex in native 1The work described was supported by Public Health Service Grant GM-23105-01, and grants from the University of Wisconsin Graduate School and the University of Wisconsin Medical School. The studies were initiated in the laboratory of Dr. Eugene P. Kennedy, Harvard Medical School, and supported by Grants GM-13952-09, GM-18731-03, and a Damon Runyon Cancer Research Fellowship (DRF-852). 2R. H. Fillingame, J. Bacteriol. 124, 870 (1975). a R. H. Fillingame, J. Biol. Chem. 251, 6630 (1976). 4A. P. Singh and P. D. Bragg, J. Bacteriol. 119, 129 (1974). 5j. F. Hare, Biochem. Biophys. Res. Commun. 66, 1329 (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

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membranes, and Patel and Kaback 8 demonstrated that DCCD reduced the proton permeability of chaotrope-treated membrane vesicles by reacting with the specific DCCD-reactive site of the ATPase complex. Further analysis of this class of mutants should yield information on the complexity of the energy-transducing reactions that are inhibited by DCCD. Selection of DCCD-Resistant Mutants Principle. The protocol developed requires that the cells grow on carbon sources, that must be metabolized via oxidative phosphorylation, in the presence of DCCD. In order for E. coli to grow on such carbon sources, e.g., citric acid cycle intermediates, the ATPase complex must have the capacity to function in the direction of ATP synthesis. DCCD will thus inhibit the growth of wild-type E. coli when only succinate, or an equivalent carbon source, is included in the growth medium. DCCDresistant strains have been isolated by selecting for mutants that are capable of growing on a succinate-acetate-malate medium containing DCCD. This selection strategy was based upon the assumption that some mutants would be found in which the ATPase complex retained full energytransducing capacity, but was minimally modified such that it was insensitive to inhibition by DCCD. It should be noted that E. coli can grow on glucose or equivalent carbon sources when the ATPase complex is mutationally inactivated, or when it is inhibited by the addition of DCCD to the growth medium. Selection Procedure. The medium 63 of Cohen and Rickenberg 7 [containing per liter: 13.6 g KH2PO4, 4 g KOH, 2 g (NH4)2SO4, 0.2 g MgSO4 • 71-120, 0.5 mg FeSO4, and adjusted to pH 7.0] is supplemented with the carbon sources: 0.6% disodium succinate hexahydrate, 0.3% potassium acetate, and 0.2% potassium L-malate. Thiamine is added at 2 mg/liter and L-arginine at 1 mM. Concentrated stocks of the basic medium, the carbon sources, and the other supplements are prepared separately and sterilized in an autoclave, except thiamine which is sterilized by filtration. The wild-type E. coli K-12 strain used in these studies is AN180 (argE3, thi-1, Strn). a The isogenic strain AN120 (argE3, thi-1, Str R" uncA401 ),s which lacks membrane ATPase activity, is used in the transduction mapping studies. Both strains require arginine and thiamine and are resistant to streptomycin.

L. Patel and H. R. Kaback,Biochemistry 15, 2741 (1976). r G. N. Cohen and H. V. Rickenberg,Ann. Inst. Pasteur, Paris 91,693 (1956). sj. D. Butlin, G. B. Cox, and F. Gibson,Biochem. J. 1/,4, 75 (1971).

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Selection plates for DCCD-resistant mutants are prepared by slowly adding 1 volume of 0.5 M DCCD in ethanol to a stirred suspension of 100 volumes of sterilized medium containing 1.8% agar (Difco) at 50°C. Plates are poured immediately. On solidification the plates are slightly turbid with a slippery, grease-like surface. This is probably due to precipitation of DCCD and dicyclohexylurea, the water adduct of DCCD which forms spontaneously in aqueous solution. The bacteria are plated on the DCCD-selection plates on the day of preparation, since the DCCD in the medium is continuously converted to dicyclohexylurea as the plates stand. Approximately l0 n cells of AN180, from an exponentially growing succinate-acetate-malate culture, are spread over the surface of a DCCD-selective plate. The plates are incubated in the upright position overnight at 37°, since the cellular suspension does not immediately wet the plate because of the greasy crust on the surface. On the following morning, a crystal of the mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (NTG), is placed with tweezers in the center of each plate. A light background of growth, with an inner clear circle of killing around the NTG crystal, appears on the plates between the fourth and seventh day of incubation at 37°C. Distinct colonies appear on the top of this background growth. These DCCD-resistant candidate colonies are picked with a sterile platinum loop or toothpick and transferred and spread as a small patch on a succinate-acetate-malate plate lacking DCCD. Casamino acids or other nutrient supplements should not be included in the DCCDselection plates, since they greatly increase the amount of background growth. Screening of DCCD-Resistant Candidates. The patches of the DCCDresistant candidates are allowed to grow up over a period of 2 days at 37°. The patches are then used as sources of single colonies, which are prepared by streaking samples of the patches on rich nutrient plates (Difco Antibiotic Medium 3 and 1.8% agar). A purified single colony from each DCCD-resistant candidate is sampled with the fiat blunt end of a toothpick, which is then stabbed into a sterile agar plate to remove excess bacteria, and finally streaked in a small patch on a succinate-acetatemalate plate containing 5 mM DCCD. The patches from DCCD-resistant mutants demonstrate heavy growth by the end of a 48-hr incubation at 37°. The growth of patches from other mutants that are only slightly resistant to DCCD is not detectable until the end of the third or fourth day of incubation at 37°. Detectable growth by patches of wild type (AN180) is often significant by the fourth day of incubation on such plates. It is, therefore, essential to compare the relative rate of appearance of growth of such patches when distinguishing between DCCD-resistant candidates.

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All mutants that we have selected have retained the auxotrophic requirements for arginine and thiamine, and remained streptomycin resistant. Reproducibility of the DCCD-Plating Procedure. Considerable variability is observed between the DCCD-containing plates which are used for the initial mutagenesis and selection of mutants. It is extremely difficult to spread the cellular suspension evenly on these plates because the surface does not wet. It is much like trying to spread a thin even coat of water on a surface of paraffin or wax. The background growth is consequently not always uniform over the surface of the plate. Colonies (false-resistant) appear in areas of the plate where background growth is light because there are fewer cells to compete for nutrients. It is also possible that the concentration of DCCD is not uniform throughout the plate. The colonies that have been selected from such plates, which proved to be DCCDresistant for growth, invaribly came from areas that are close to the circle of NTG killing, that is from areas of high but nonlethal concentrations of mutagen. Colonies picked from the outer periphery of the plate have proved to not be DCCD-resistant on further plating. In a typical experiment with 20 mutagenesis plates, perhaps a third of the plates will not show distinct background growth with a clear circle of killing. The plates that do show clear background growth vary greatly in the degree of uniformity, but from these plates several dozen distinct colonies found near the circle of killing can be isolated. We have not been successful in selecting spontaneous DCCD-resistant mutants from plates to which mutagen was not added. Any colonies that were observed proved to be falseresistant colonies. Verification of DCCD-Resistant Growth. The patching technique described above has proved to be the most convenient and most reliable. Since the rate of appearance of detectable growth does vary from one batch of DCCD plates to the next, we routinely patch AN180 and a well-characterized DCCD-resistant mutant,'-' RF-7, on each plate to aid in scoring. DCCD-resistant growth can also be scored by measuring growth in liquid culture via light scattering measurements (absorbance at 550 nm). The precipitation of DCCD does interfere with these turbidometric measurements. An approximate correction for precipitation of DCCD and dicyclohexylurea can be made by subtracting the change in turbidity caused by the addition of DCCD from subsequent readings of the same culture. This correction value does vary significantly from experiment to experiment and must be estimated each time for each culture. Finally, the change in turbidity due to DCCD does not remain constant with time. Therefore, the changes in turbidity due to bacterial gr-wth must be significantly greater than the changes due to DCCD precipitation if they are to be considered valid.

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Selection of EDC-Resistqnt Mutants. We have isolated a large number of ethyldimethylaminopropyl carbodiimide (EDC) growth-resistant mutants by using EDC in place of DCCD in the above selection procedure. EDC is added to the plates at a final concentration of 20 mM. EDC and the corresponding urea, which is formed as a water adduct, are both soluble in aqueous solution. Thus many of the problems of the DCCD-selection procedure are not encountered. However, the rate of growth of wild-type E. coli on EDC plates is greater than on DCCD plates and any resistant colonies must be scored within 2 or 3 days, while they are still distinct from the background growth. EDC growth-resistant candidates are most conveniently further screened by measuring the rate of growth in liquid culture in the presence and absence of 20 mM EDC. Since EDC does not precipitate, the problems inherent in measuring growth turbidometrically in the presence of DCCD are not encountered. The patching technique does not work well for EDC-resistant mutants because the rate of growth of wild-type strains on these plates is rapid enough to obscure distinct differences. Biochemical Analysis of DCCD-Resistant Mutants

Preparation of Membranes. When many mutant strains are being screened it is most convenient to prepare membranes by sonication, since only small quantities of cells are required. The more defined studies of mutants have been carried out with membranes prepared with a French pressure cell, because these membrane preparations are reproducibly uniform. In either method, cells are harvested in the late exponential or early stationary phase of growth and washed once with 50 mM tris(hydroxymethyl)aminomethane (Tris)-H2SO4 buffer, pH 7.8, containing 10 mM MgSO4. The washed cellular pellet is resuspended in the above buffer containing 1 mM dithiothreitol, 0.1 mM disodium ethylenediaminetetraacetic acid (EDTA), and 0.1 mg/ml deoxyribonuclease I (Worthington). If the membranes are to be prepared by sonication, 5-10 ml of a 10% (wet weight per volume) suspension are disrupted at full power with a Sonifier Cell Disruptor for 1.5 min with intermittent cooling in an ice bath. When the cells are to be disrupted with a French pressure cell, a 20% (wet weight per volume) suspension is passed through the cell at 2-3 ml/min, while maintaining a constant pressure of 18,000 psi. After removal of whole cells and debris by low-speed centrifugation (two sequential centrifugations at 5000 g for 15 min), the membranes are collected by centrifugation at 145,000 g (maximum) for 75 min. The membranes are resuspended and washed once with the above buffer and finally resuspended at

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10-30 mg protein per milliliter in the 50 mM Tris sulfate, pH 7.8, 10 mM MgSO4, 1 mM dithiothreitol buffer. ATPase Assay. To 0.9 ml of 55.5 mM Tris sulfate, pH 7.8, containing 0.222 mM MgSO4 in a test tube are added 20-50/zg of membrane protein, and the tube is incubated for 5 min at 30°. The assay is initiated by the addition of 0.1 ml of 4 mM tetrasodium ATP containing approximately 10~ cpm of [y-3~P]ATP. The assay is terminated after 2-5 min by the addition of 1.0 ml 10% trichloroacetic acid. The 32P-labeled inorganic phosphate liberated is quantitated after extraction of the molybydate complex into isobutanol-benzene (1 : 1). To 0.6 ml of the trichloroacetic acid supernatant solution of the assay mixture are added 0.1 ml of 5 M H2SO4, 0.2 ml of 5% ammonium molybydate, and 1.0 ml of isobutanol-benzene (1:1). After thorough mixing and phase separation, an aliquot of the upper phase is analyzed for radioactivity by liquid scintillation counting. The amount of membrane protein and the time of the assay is varied so that less than 10% of the total ATP is hydrolyzed, since the assay is linear over this range. Routine Screening for DCCD-Resistant ATPase Mutants. DCCD, dissolved in ethanol, is added to the assay mixture at a final concentration of 20 p2kt after addition of the membrane preparation but prior to the [3,-32P]ATP substrate. The tube is mixed thoroughly and incubated for 15 rain at 30°. The assay is then initiated by the addition of the [y-3Z]ATP substrate. The assay is terminated after an additional 5 min by the addition of trichloroacetic acid. Wild-type (AN180) ATPase activity is inhibited 70-80% by DCCD under these conditions, whereas the ATPase activity of DCCD-insensitive mutants is inhibited by only 10-20%. Factors Affecting Determination of the DCCD-Sensitivity of ATPase Activity. It is extremely important that the assay conditions used for comparison of the DCCD sensitivity of mutants be identical. The concentration of membrane protein and the temperature and time of incubation with DCCD are of particular importance. At low concentrations of membrane protein, maximal inhibition is observed with 20/xM DCCD after a 15-min incubation at 30°C. As the concentration of membrane protein is increased above 50/zg/ml, higher concentrations of DCCD are required for equivalent degrees of inhibition. However, the relationship between the concentration of membrane protein and the amount of DCCD required for a given degree of inhibition is not a simple linear function. At concentrations of membrane protein less than 50/~g/ml, the degree of inhibition at a given concentration of DCCD is not dependent upon the protein concentration. It is, therefore, extremely misleading to express inhibition as a function of the "nanomoles DCCD added per milligram of membrane protein." Inhibition by DCCD is irreversible. This can be shown by incubating a

[15]

D C C D - R E S I S T A N T A T P A S E MUTANTS OF E. coli

ioo-

169

I00.~ _ ~

>, <~

50-

50"

• RF-7

• KW79

o ANI80

oo

,b

o ANI80

2'0

0o

I00-

I00-

so"

50-

2'o

• KW91 o A N 180

• KW82

0o

,b

o ANI80 i

IO 20 DCCD(~M)

0

o

,b OCCO

2'o

(juMl

FIG. 1. Comparison of the DCCD sensitivity of membrane ATPase in wild-type and DCCD-resistant ATPase mutants ofE. coli. Each ATPase assay contained between 20 and 30/zg of membrane protein per milliliter. Preincubation with DCCD was for 15 min at 30°C. The assay was terminated 5 min after addition of [7-3~P]ATP. Wild-type strain is ANI80; DCCD-resistant ATPase strains are RF-7, KW79, KW82, and KW91.

concentrated suspension of membranes with DCCD and diluting the membrane suspension many-fold into the assay mixture. For example, if a 20 mg/ml suspension of membranes is incubated with 100/~M DCCD at 30°C for 1 hr and then diluted 1 : 1000 into the assay mixture, greater than 75% inhibition of ATPase activity is observed despite the fact that the final concentration of DCCD is 0.1 ~M. The rate of onset of DCCD inhibition is also temperature dependent. The rate at 30° is about 20 times faster than the rate at 0°. However, the rate of inactivation does not faithfully follow first-order kinetics under any conditions. Properties of DCCD-Resistant-ATPase Mutants. Four independently isolated DCCD growth-resistant mutants have been found which contain a DCCD-insensitive ATPase complex. 9 Each was selected from the wildtype strain AN180. The susceptibility of the membrane ATPase of these mutants to inhibition by DCCD is shown in Fig. 1. The ATPase activities of all four mutants are remarkably similar in their lack of sensitivity to inhibition by DCCD. The growth of each mutant was also assessed as 9 R. H. Fillingame and K. Knoebel, unpublished data (1976).

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being highly resistant to inhibition by DCCD. By the patching technique on DCCD plates, heavy growth of RF-7 and KW-82 was observed after 1 day of incubation, and heavy growth of KW-79 and KW-91 by the second day. The rate of growth of RF-7 in liquid culture is only slightly affected by DCCD as reported previously." Several dozen other mutants have been isolated which are as DCCD growth resistant as these four aforementioned mutants. However, the membrane ATPase activity of these mutants retains the same sensitivity to inhibition by DCCD as wild type. Heavy growth on DCCD plates by these DCCD growth-resistant, but DCCD-inhibitable ATPase mutants, was detected on the first or second day by the patch test, and the growth of many of them in liquid culture was not reduced by addition of DCCD. In summary, we have isolated 27 mutants which were judged highly DCCD growth resistant. Of these, only four have proved to have a DCCD-insensitive membrane ATPase. None of the mutants which have been judged to be moderately DCCD growth resistant have proved to have a DCCD-insensitive ATPase, although the membrane ATPase of only ten of these mutants has been examined. The membrane ATPase activity in the four DCCD-insensitive ATPase mutants which have been isolated to date is as susceptible to inhibition by EDC as the membrane ATPase activity of wild-type AN180. The equal sensitivity of RF-7 and AN180 to inhibition by EDC and another polar carbodiimide was initially reported by Patel and Kaback. 6 A mutant of Streptococcus faecalis that was resistant to hydrophobic carbodiimides, but sensitive to more polar carbodiimides has been isolated. '° With the isolation of four mutants of E. coli, all of which demonstrate DCCDresistant but EDC-sensitive ATPases, these findings can no longer be regarded as merely fortuitous circumstance. We have also isolated several dozen mutants demonstrating a high degree of EDC-resistant growth. Of the sixteen EDC growth-resistant mutants analyzed to date, none has shown a membrane ATPase with a decreased susceptibility to inhibition by EDC (or DCCD). Strain RF-7, A Characterized DCCD-Resistant ATPase Mutant The following properties of RF-7, which is presently the most thoroughly characterized DCCD-resistant ATPase mutant, should be considered when the isolation and characterization of other carbodiimideresistant mutants is contemplated. To date, these properties have not been examined in the other DCCD-resistant ATPase mutants discussed above. I°A. Abrams, J. B. Smith, and C. Baron,J. Biol. Chem. 247, 1484(1972).

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Site of Inhibition by DCCD. DCCD reacts covalently with a specific proteolipid (chloroform-methanol-soluble protein) of the ATPase complex. This has been shown by labeling studies with '4C-DCCD.2 A single protein, that was labeled by 14C-DCCD in wild type but not in RF-7, was identified by sodium dodecyl sulfate-acrylamide gel electrophoresis. This protein was shown to be extracted into chloroform-methanol and was subsequently purified, a The DCCD-reactive protein is a component of the intrinsic membrane sector of the ATPase complex and was found in a partially purified, deoxycholate-solubilized, DCCD-sensitive ATPase complex.5 It has not yet been established whether the DCCD-resistance of RF-7 is due to modification of this protein, rather than some other component of the membrane sector. Hybridization-reconstitution experiments have established that the genetically modified protein is in the membrane sector, rather than the extrinsic (F,) ATPase which can be removed from the membrane with EDTA." Genetic Mapping. The mutation causing DCCD resistance in RF-7 has been shown to be cotransducible with the uncA locus at 73 min on the E. coli K-12 linkage map." The transducing phage, Plkc, was grown on strain RF-7 and a lysate prepared. Strain AN120 (uncA401; ATPase negative), which cannot grow on succinate, was infected with this lysate. Transductants that were capable of growth on succinate were isolated. Approximately 90% of these transductants also proved to be DCCD growth resistant on the succinate-acetate-malate medium. Three of these transductants were further analyzed and shown to have a DCCD-resistant membrane ATPase and a proteolipid that was less susceptible to reaction with 14C-DCCD. Active Transport. Membrane vesicles isolated from wild type (AN180) and RF-7 exhibit identical respiration-dependent L-proline transport activities. ~ Transport in vesicleS from both strains is abolished by treatment with chaotropes, 6 a treatment which makes the vesicles permeable to hydrogen ion." Subsequent treatment with DCCD reactivates transport in wild-type vesicles, but does not reactivate transport in vesicles from RF-7." This finding correlates well with the observation that DCCD reacts with the proteolipid of wild-type vesicles, but not with the proteolipid in RF-7 vesicles. 6 Suppression of Carbodiimide-Resistance by Growth on Glucose and Glycerol. When strain RF-7 is grown on glycerol or glucose, the ATPase activity of membranes is much more susceptible to inhibition by DCCD than is the membrane ATPase of RF-7 cells grown on succinate" L. Patel, S. Schuldiner, and H. R. Kaback, Proc. Natl. Acad. Sci. U.S.A. 72, 3387 (1975).

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acetate-malate. 12 The proteolipid in the membranes prepared from RF-7 cells grown on glycerol or glucose is also susceptible to rapid labeling by 14C-DCCD, whereas the proteolipid in succinate-acetate-malate grown cells reacts much more slowly (vide supra). Although an explanation for this suppression of DCCD-resistance is not yet at hand, it is clear that conclusions from studies of these DCCD-resistant mutants should be drawn cautiously when the mutants are grown on carbon sources other than Kreb's cycle intermediates. It should be recalled that the mutants were isolated by selecting for resistant growth on succinate-acetatemalate medium; it may prove that these mutants will be fully resistant only when grown on these carbon sources. 12 R. H. Fillingame and A. E. Wopat, J. Bacteriol. 134, 687 (1978).

[16] H e i n e - D e f i c i e n t M u t a n t s o f S t a p h y l o c o c c u s a u r e u s

By JUNE LASCELLES Heme-deficient (hem) mutants of facultative fermentative bacteria are convenient tools to manipulate cytochrome synthesis and cytochromelinked electron transport systems. Mutants with defects in the respiratory system are readily isolable on the basis of resistance to antibiotics, in particular the aminoglycosides. Respiratory mutants can be distinguished from the normal phenotype by their inability to grow on nonfermentable substrates, and by their small colonies on fermentable energy sources. The first hem mutant of Staphylococcus aureus was discovered by Jensen and Thofern 1 in a study of the nutritional requirements of streptomycin-resistant isolates. Unlike the parent organism (SG-511A), the mutant failed to grow on nutrient agar unless supplemented with hemin. Later work with this mutant showed that growth without hemin occurred provided that the medium contained fermentable carbohydrate, uracil, and pyruvate or acetate. 2'3 These supplements are needed by wildtype staphylococci for growth under strictly anaerobic conditions, and are partly attributable to the dependance of the organism upon a functional respiratory chain for certain dehydrogenations; for example, dihydroorotare dehydrogenase is obligatorily coupled to the cytochrome system. '~ Kanamycin was used by Tien and White 4 in the isolation of hem mutants of S. aureus U-71. About 20% of the drug-resistant isolates were 1 j. Jensen and E. Thofern, Z. Naturforsch., Teil B 8, 599 (1953). 2 j. F. G a r d n e r and J. L a s c e l l e s , J. Gen. Microbiol. 29, 157 (1962). 3 j. L a s c e l l e s , Ann. N.Y. Acad. Sci. 236, 96 (1974). 4 W. Tien and D. C. White, Proc. Natl. Acad. Sci. U.S.A. 61, 1392 (1968).

METHODS IN ENZYMOLOGY, VOL. LVI

Copyright© 1979by AcademicPress, Inc. All fights of reproduction in any form reserved. ISBN 0-12-181956-6