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Novobiocin—A specific inhibitor of semiconservative DNA replication in permeabilized Escherichia coli cells
J. Mol. Biol. (1975) 96, 201-205
LETTERS
TO THE EDITOR
Novobiocin - A Specific Inhibitor of Semiconservative DNA Replication in Permeabilized Escherichia cob Cells on macromolecule synthesis was investigated in Escheriby treatment with toluene or 2 M-sucrose. It was found that (1) semiconservative DNA replication is strongly and immediately inhibited and (2) ATP-independent DNA repair as well as RNA and protein synthesis are not affected.
The effect of novobiocin
chia coli cells permeabilized
The mode of action of novobiocin, a clinically useful antibiotic, is not fully understood. Since it causes the accumulation of cell wall precursors in Staphylowwus aurew (Strominger & Threnn, 1959), inhibition of cell wall synthesis was considered to be the primary lethal effect of novobiocin. However, later +n viva studies have shown that in addition to inhibiting cell wall synthesis, novobiocin also leads to an inhibition of DNA replication and to a lesser extent of RNA and protein synthesis (Wishnow et al., 1965; Smith & Davis, 1967). Moreover, attempts to identify a specific novobiocin-sensitive step in peptidoglycan synthesis were inconclusive
30
60
90
120
Time (mid
FIO. 1. Effect of novobioain on DNA synthesis in toluene-treated aells. E. coli 169 (F+ thy- her-) was grown to a density of 6x 1Oa cells/ml and permeabilized by treatment with toluene or 2 M-sucrose &Bdescribed previously (Mazes, 1972; Wickner & Hurwitz, 1972). DNA synthesis was essayed in incubation mixtures containing 20 mM-HXPES (pH 8-O), 0.1 M-KC& 10 mM-magnesium acetate, 1 mM-dithiothreitol, 2 mu-ATP, 0.1 man-NAD, 0.6 mM each of dATP, dCTP, and dC+TP. 0.02 mx-[3aP]dTTP (600 ots/min per pmol), and 6x log cells/ml. Inoubations were performed at 30°C. At the times indicated assayed for &d-insoluble redioaotivity.
-O--O-, control
without
Untreated ATP.
oontrol; -@-a-,
novobiocin 201
samples (0.06 ml) were removed
and
(100 pg/ml) added; -- x -- x --,
W.
202
L.
STAUDENBAUER
(Staudenbauer & Strominger, unpublished observations). Since novobiocin can form a charge-transfer complex with magnesium ions, it has been suggested that it might interfere non-specifically with various enzymic reactions by inducing a magnesium deficiency (Brock, 1967). This letter describes the effect of novobiocin on macromolecule synthesis under controlled ionic conditions employing nucleotide-permeable E. co& cells. Bacterial cells permeabilized by treatment with toluene or high concentrations of sucrose have been used previously to study the effects of various inhibitors on DNA replication (Pisetsky et csl., 1972; Burger & Glaser, 1973; Masker & Hanawalt, 1974). Replicative DNA synthesis in these cells can be distinguished from repair synthesis since the former requires ATP and the latter is ATP-independent and markedly stimulated by endonucleases (Moses C Richardson, 1970; Moses, 1972). The effect of novobiocin on the incorporation of [3H]TMP in toluene-treated E. coli cells is shown in Figure 1. It ia evident that novobiocin (100 pg/ml) drastically reduces the amount of ATP-dependent replicative DNA synthesis. The residual incorporation observed in the presence of the drug is close to the background level of ATP-independent DNA synthesis. A similar effect is observed in plasmolysed cells, i.e. cells rendered permeable by 2 M-sucrose (data not shown). DNA replication in toluene-treated cells represents a continuation of the in viva replication process and proceeds only at the region of the genome about to be replicated before treatment (Burger, 1971). Novobiocin must therefore block the extension process at the growing forks and not just the initiation of replication at the chromosomal origin. It can be seen in Figure 2 that DNA replication is inhibited by more than 80% at drug concentrations as low as 2 pg/ml. On the other hand, even at the highest concentrations tested (200 pg/ml) novobiocin has no inhibitory effect on the repairtype synthesis stimulated by pancreatic DNAase in the absence of ATP. This is
1
0.1 Novobiocin (pg/mI)
2. Seleotive inhibition of ATP-dependent DNA syntheaia Incubation mixtures (O-l-ml) were aa described in Fig. 1. Repair synthesis was assayed in inoubation mixtures containing 0.6 @ panareetia DNAaae/ml and no ATP. Novobiocin was added at the oonoentretione indioated. Inoubation time, 30 min et 3O’C. DNA synthesis ia expressed as per oent of the inoorporation in the abeenoe of novobiooin. 100% corresponds to an inoorporation of 270 pmol [3aP]TMP (ATP-dependent synthesis), end 60 pmol [aaP]!i’MP (repair synthesis), respectively. -O-O-, ATP-dependent synthesis; -X -x -, repair synthesis. Pm.
LETTERS
TO
THE
EDITOR
203
further demonstrated by the density labelling experiment described in Figure 3. TTP was replaced by its analogue bromodeoxyuridine triphosphate and [3H]dCTP was used as radioactive label. The newly synthesized DNA was fragmented by shear and analysed by equilibrium centrifugation in neutral CsCI. Under conditions allowing replicative DNA synthesis most of the incorporated label is found in the hybrid density region of the gradient where the product of semiconservative DNA replication is expected to band. When novobiocin was added to the density labelling mixture, practically no semiconservative synthesis occurred (Fig. 3(a)). On the other hand,
-. -. P--.
20
,75 ,70 65
(b).
1430 I .75 I.70 I.65
I-
Fraction
number
Fro. 3. Buoyant density centrifugation of bromodeoxyuridine-labeled DNA. 5 X. log toluene-treated cells were incubated for 30 min at 30°C in l-ml incubation mixtures with [3aP]TTP replaced by 0.05 mM-bromodeoxyuridine triphosphate and dCTP replaced by 0.02 ma6-[3H]dCTP. Reaction mixtures contained either 2 mu-ATP (a) or 0.6 pg panoreatic DNAase/ ml and no ATP (b). Incorporation was stopped by the addition of l-ml cold 0.1 M-EDTA, the oells were pelleted, washed once with cold Tris/EDTA/NaCl buffer, resuspended in l-ml buffer and incubated with lysozyme (0.6 mg/ml) for 30 min at 0°C. Cells were lysed by the addition of 0.1 ml 5% sarkosyl and the viscosity of the lysata was reduced by repeated shearing with a syringe. The total lysates were then analysed, subjeated to equilibrium centrifugation in a Spinco fixed-angle Ti-60 rotor in polypropylene tubes containing 6.2 g CsCl and 4.0 g of sample plus Tris/EDTA/NaCl buffer. The tubes were tiled with light mineral oil and centrifuged at 40,000 revs/min for 36 h at 16°C. Densities (p) were determined from refractometer readings. -o--O-, Untreated control; -a-@--, novobiocin (100 pg/ml) added.
W. L.
204
STAUDENBAUER
the repair synthesis triggered by pancreatic DNAase in the absence of ATP is totally unaffected by novobiocin (Pig. 3(b)). Novobiocin resembles in this respect other inhibitors of semiconservative DNA replication like nalidixic acid, mitomycin C, and araCTP, all of which do not interfere with the repair synthesis performed by DNA polymerase I (Vosberg & Hoffmann-Berling, 1971; Pisetsky et cal., 1972; Masker & Hanawalt, 1974). Thus novobiocin, which is the most potent inhibitor of this group, permits a further distinction between these two types of DNA synthesis.
V
I
I
I
20
40
60
Time (min) (a)
Time (mid (b)
FIG. 4. Effeot of novobiooin on RNA and protein synthesis. (8) [sH]UMP inoorpomtion ~8s 8ssayed in in~ubetion mixtures oonteining 20 maa-HEPES (pH 8-O), 10 mna-KCl, 10 m8r-msgnesimn 8&.&e, 0.2 mn-M&l,, 06 mu eaoh of ATP, CTP 8nd GTP, 0.1 ~M-[~H]UTP (60 ct.s/min per pmol), and 6 x lo9 oells/ml. Incubation w8s performed at 30°C. At the times indicated s8mples (0.06ml) were removed and assayed for acid-insoluble radioactivity. -O-O-, Untreated oontrol; -@--•-, novobiocin (200 pg/ml) added; -A-A-, rifampioin (20 ~/ml) added. (b) [3H]leuoine incorpomtion was assayed in inaubetion mixtures oontcrining 20 mm-HEPES (pH 8*0), 40 mnr-KCl, 10 mnr-magnesium aoetate, 0.2 mn-M&l,, 0.6 mn eaoh of ATP, CTP, GTP, 8nd UTP, 6 maa-phosphoenolpyruvate, 60 pg pyruv8te kinase/ml, 0.1 mw-[3H]leuoine (100 cts/ min per pmol), 0.6 rnM of e8oh of the other 19 amino acids, and 6 x 10s oells/ml. Inoubation w&s performed 8t 30°C. At the times indioeted samples (0.06 ml) were removed and assayed for trichloroaoetio aoid-insoluble redioeativity. -O--O-, Untre8ted control; -O-a---, novobiooin (200 pg/ml) 8dded; -A-A---, ohlommpheniool(200 m/ml) added.
Next it was investigated whether novobiocin specifically blocks DNA replication in permeable cells or whether it inhibits RNA and protein synthesis as well. Figure 4(a) shows the kinetics of [3H]UMP incorporation into an acid-insoluble product by toluene-treated cells. It can be seen that this synthesis is sensitive to low concentrations of rifampicin whereas novobiocin (209 pg/ml) shows no inhibitory effect. Nearly identical results were obtained with plasmolysed cells (data not shown). As far as protein synthesis is concerned, it can be seen in F’igure 4(b) that [3H]leucine incorporation is only slightly impaired by high novobiocin concentrations (200 pg/ml). As expected, chloramphenicol reduces the incorporation to background
LETTERS
TO THE
“0.5
EDITOR
levels. Plasmolyaed cells were employed in this experiment since they gave a roughly tenfold higher rate of [3H]leucine incorporation as compared to toluene-treated cells. h has been reported previously (Ben-Hamida & Gros, 1971), this protein synthesis requires a complete amino acid mixture and nucleoside triphosphate-dependent RNA synthesis. The data presented in this letter taken together with the in vivo results of Rmit~h & Davis (1967) leave little doubt that the primary action of novobiocin in E. colt' is a specific inhibition of DNA replication. The inhibition of cell wall synthesis ohserved in growing cells (Wishnow et al., 1965) could thus merely be a secondary effect, of blocking DNA replication. However, the immediat,e and complete cessation of cell division in the presence of novobiocin (Smith & Davis. 1967) might point t(o an intimate connection between DNA replication and septum formation (Helmstrttrr. 1973). The author is indebted to Dr P. H. Hofsclmeider fnr helpful comments and critical reading of the manuscript. This work wm siupported hy H grant from thcb Dentsche Forst,hnngsgemeinsctl~ft . ~Iax-Planck-Institat fiir Riochemie Abteihmg Hofschneider 8033 Martins&d bei Miinchen, Germany
\\IAT.TRR
I,.
S;;TAITDENRAT;ER
REFERENCES Ben-Harnida, F. 8: Gros, F. (1971). Biochimie (Paris), 53, 71-80. Brock, T. D. (1967). In Antibiotica (Gottlieb, D. and Shaw, P. D., eds). vol. 1, pp. 651-665, Springer-Verlag, Berlin. Burger, R. M. (1971). Proc. Nat. AcadS&., U.S.A. 68,2124-2126. Burger, R. M. & Gleser, D. A. (1973). Proc. Nat. AcadS&, U.S.A. 70, 1955-1958. Helmstetter, C. E. (1974). J. Mol. Biol. 84, 21-36. Masker, W. E. & Hanawalt, P. C. (1974). Biochim. Biophya. Acta, 340, 229-236. Moses, R. E. (1972). J. BioZ. Chem. 247, 6031-6038. Moses, R. E. & Richardson, C. C. (1970). Proc. Nat. Acud. Sci., U.S.A. 67, 674-681. Pisetsky, D., Berkower, I., Wickner, R. & Hurwitz, J. (1972). J. &Jol. Biol. 71, 557-571. Smith, D. H. 8: Davis, B. D. (1967). J. Baderiol. 93, 71-79. Strominger, J. L. & Threnn, R. H. (1969). Biochim. Biophys. Acta, 33, 280-282. Vosberg, H. P. & Hoffmann-Berling, H. (1971). J. Mol. BioZ. 58, 739-753. Wickner, R. B. & Hurwitz, J. (1972). Biochem. Biophye. Res. Commun. 47, 202-211. Wishnow, R. M., Strominger, J. L., Birge, C. H. Br Threnn, R. H. (1965). J. Bacterial. 89, 1117-1123.
14
LETTERS
TO THE EDITOR
Novobiocin - A Specific Inhibitor of Semiconservative DNA Replication in Permeabilized Escherichia cob Cells on macromolecule synthesis was investigated in Escheriby treatment with toluene or 2 M-sucrose. It was found that (1) semiconservative DNA replication is strongly and immediately inhibited and (2) ATP-independent DNA repair as well as RNA and protein synthesis are not affected.
The effect of novobiocin
chia coli cells permeabilized
The mode of action of novobiocin, a clinically useful antibiotic, is not fully understood. Since it causes the accumulation of cell wall precursors in Staphylowwus aurew (Strominger & Threnn, 1959), inhibition of cell wall synthesis was considered to be the primary lethal effect of novobiocin. However, later +n viva studies have shown that in addition to inhibiting cell wall synthesis, novobiocin also leads to an inhibition of DNA replication and to a lesser extent of RNA and protein synthesis (Wishnow et al., 1965; Smith & Davis, 1967). Moreover, attempts to identify a specific novobiocin-sensitive step in peptidoglycan synthesis were inconclusive
30
60
90
120
Time (mid
FIO. 1. Effect of novobioain on DNA synthesis in toluene-treated aells. E. coli 169 (F+ thy- her-) was grown to a density of 6x 1Oa cells/ml and permeabilized by treatment with toluene or 2 M-sucrose &Bdescribed previously (Mazes, 1972; Wickner & Hurwitz, 1972). DNA synthesis was essayed in incubation mixtures containing 20 mM-HXPES (pH 8-O), 0.1 M-KC& 10 mM-magnesium acetate, 1 mM-dithiothreitol, 2 mu-ATP, 0.1 man-NAD, 0.6 mM each of dATP, dCTP, and dC+TP. 0.02 mx-[3aP]dTTP (600 ots/min per pmol), and 6x log cells/ml. Inoubations were performed at 30°C. At the times indicated assayed for &d-insoluble redioaotivity.
-O--O-, control
without
Untreated ATP.
oontrol; -@-a-,
novobiocin 201
samples (0.06 ml) were removed
and
(100 pg/ml) added; -- x -- x --,
W.
202
L.
STAUDENBAUER
(Staudenbauer & Strominger, unpublished observations). Since novobiocin can form a charge-transfer complex with magnesium ions, it has been suggested that it might interfere non-specifically with various enzymic reactions by inducing a magnesium deficiency (Brock, 1967). This letter describes the effect of novobiocin on macromolecule synthesis under controlled ionic conditions employing nucleotide-permeable E. co& cells. Bacterial cells permeabilized by treatment with toluene or high concentrations of sucrose have been used previously to study the effects of various inhibitors on DNA replication (Pisetsky et csl., 1972; Burger & Glaser, 1973; Masker & Hanawalt, 1974). Replicative DNA synthesis in these cells can be distinguished from repair synthesis since the former requires ATP and the latter is ATP-independent and markedly stimulated by endonucleases (Moses C Richardson, 1970; Moses, 1972). The effect of novobiocin on the incorporation of [3H]TMP in toluene-treated E. coli cells is shown in Figure 1. It ia evident that novobiocin (100 pg/ml) drastically reduces the amount of ATP-dependent replicative DNA synthesis. The residual incorporation observed in the presence of the drug is close to the background level of ATP-independent DNA synthesis. A similar effect is observed in plasmolysed cells, i.e. cells rendered permeable by 2 M-sucrose (data not shown). DNA replication in toluene-treated cells represents a continuation of the in viva replication process and proceeds only at the region of the genome about to be replicated before treatment (Burger, 1971). Novobiocin must therefore block the extension process at the growing forks and not just the initiation of replication at the chromosomal origin. It can be seen in Figure 2 that DNA replication is inhibited by more than 80% at drug concentrations as low as 2 pg/ml. On the other hand, even at the highest concentrations tested (200 pg/ml) novobiocin has no inhibitory effect on the repairtype synthesis stimulated by pancreatic DNAase in the absence of ATP. This is
1
0.1 Novobiocin (pg/mI)
2. Seleotive inhibition of ATP-dependent DNA syntheaia Incubation mixtures (O-l-ml) were aa described in Fig. 1. Repair synthesis was assayed in inoubation mixtures containing 0.6 @ panareetia DNAaae/ml and no ATP. Novobiocin was added at the oonoentretione indioated. Inoubation time, 30 min et 3O’C. DNA synthesis ia expressed as per oent of the inoorporation in the abeenoe of novobiooin. 100% corresponds to an inoorporation of 270 pmol [3aP]TMP (ATP-dependent synthesis), end 60 pmol [aaP]!i’MP (repair synthesis), respectively. -O-O-, ATP-dependent synthesis; -X -x -, repair synthesis. Pm.
LETTERS
TO
THE
EDITOR
203
further demonstrated by the density labelling experiment described in Figure 3. TTP was replaced by its analogue bromodeoxyuridine triphosphate and [3H]dCTP was used as radioactive label. The newly synthesized DNA was fragmented by shear and analysed by equilibrium centrifugation in neutral CsCI. Under conditions allowing replicative DNA synthesis most of the incorporated label is found in the hybrid density region of the gradient where the product of semiconservative DNA replication is expected to band. When novobiocin was added to the density labelling mixture, practically no semiconservative synthesis occurred (Fig. 3(a)). On the other hand,
-. -. P--.
20
,75 ,70 65
(b).
1430 I .75 I.70 I.65
I-
Fraction
number
Fro. 3. Buoyant density centrifugation of bromodeoxyuridine-labeled DNA. 5 X. log toluene-treated cells were incubated for 30 min at 30°C in l-ml incubation mixtures with [3aP]TTP replaced by 0.05 mM-bromodeoxyuridine triphosphate and dCTP replaced by 0.02 ma6-[3H]dCTP. Reaction mixtures contained either 2 mu-ATP (a) or 0.6 pg panoreatic DNAase/ ml and no ATP (b). Incorporation was stopped by the addition of l-ml cold 0.1 M-EDTA, the oells were pelleted, washed once with cold Tris/EDTA/NaCl buffer, resuspended in l-ml buffer and incubated with lysozyme (0.6 mg/ml) for 30 min at 0°C. Cells were lysed by the addition of 0.1 ml 5% sarkosyl and the viscosity of the lysata was reduced by repeated shearing with a syringe. The total lysates were then analysed, subjeated to equilibrium centrifugation in a Spinco fixed-angle Ti-60 rotor in polypropylene tubes containing 6.2 g CsCl and 4.0 g of sample plus Tris/EDTA/NaCl buffer. The tubes were tiled with light mineral oil and centrifuged at 40,000 revs/min for 36 h at 16°C. Densities (p) were determined from refractometer readings. -o--O-, Untreated control; -a-@--, novobiocin (100 pg/ml) added.
W. L.
204
STAUDENBAUER
the repair synthesis triggered by pancreatic DNAase in the absence of ATP is totally unaffected by novobiocin (Pig. 3(b)). Novobiocin resembles in this respect other inhibitors of semiconservative DNA replication like nalidixic acid, mitomycin C, and araCTP, all of which do not interfere with the repair synthesis performed by DNA polymerase I (Vosberg & Hoffmann-Berling, 1971; Pisetsky et cal., 1972; Masker & Hanawalt, 1974). Thus novobiocin, which is the most potent inhibitor of this group, permits a further distinction between these two types of DNA synthesis.
V
I
I
I
20
40
60
Time (min) (a)
Time (mid (b)
FIG. 4. Effeot of novobiooin on RNA and protein synthesis. (8) [sH]UMP inoorpomtion ~8s 8ssayed in in~ubetion mixtures oonteining 20 maa-HEPES (pH 8-O), 10 mna-KCl, 10 m8r-msgnesimn 8&.&e, 0.2 mn-M&l,, 06 mu eaoh of ATP, CTP 8nd GTP, 0.1 ~M-[~H]UTP (60 ct.s/min per pmol), and 6 x lo9 oells/ml. Incubation w8s performed at 30°C. At the times indicated s8mples (0.06ml) were removed and assayed for acid-insoluble radioactivity. -O-O-, Untreated oontrol; -@--•-, novobiocin (200 pg/ml) added; -A-A-, rifampioin (20 ~/ml) added. (b) [3H]leuoine incorpomtion was assayed in inaubetion mixtures oontcrining 20 mm-HEPES (pH 8*0), 40 mnr-KCl, 10 mnr-magnesium aoetate, 0.2 mn-M&l,, 0.6 mn eaoh of ATP, CTP, GTP, 8nd UTP, 6 maa-phosphoenolpyruvate, 60 pg pyruv8te kinase/ml, 0.1 mw-[3H]leuoine (100 cts/ min per pmol), 0.6 rnM of e8oh of the other 19 amino acids, and 6 x 10s oells/ml. Inoubation w&s performed 8t 30°C. At the times indioeted samples (0.06 ml) were removed and assayed for trichloroaoetio aoid-insoluble redioeativity. -O--O-, Untre8ted control; -O-a---, novobiooin (200 pg/ml) 8dded; -A-A---, ohlommpheniool(200 m/ml) added.
Next it was investigated whether novobiocin specifically blocks DNA replication in permeable cells or whether it inhibits RNA and protein synthesis as well. Figure 4(a) shows the kinetics of [3H]UMP incorporation into an acid-insoluble product by toluene-treated cells. It can be seen that this synthesis is sensitive to low concentrations of rifampicin whereas novobiocin (209 pg/ml) shows no inhibitory effect. Nearly identical results were obtained with plasmolysed cells (data not shown). As far as protein synthesis is concerned, it can be seen in F’igure 4(b) that [3H]leucine incorporation is only slightly impaired by high novobiocin concentrations (200 pg/ml). As expected, chloramphenicol reduces the incorporation to background
LETTERS
TO THE
“0.5
EDITOR
levels. Plasmolyaed cells were employed in this experiment since they gave a roughly tenfold higher rate of [3H]leucine incorporation as compared to toluene-treated cells. h has been reported previously (Ben-Hamida & Gros, 1971), this protein synthesis requires a complete amino acid mixture and nucleoside triphosphate-dependent RNA synthesis. The data presented in this letter taken together with the in vivo results of Rmit~h & Davis (1967) leave little doubt that the primary action of novobiocin in E. colt' is a specific inhibition of DNA replication. The inhibition of cell wall synthesis ohserved in growing cells (Wishnow et al., 1965) could thus merely be a secondary effect, of blocking DNA replication. However, the immediat,e and complete cessation of cell division in the presence of novobiocin (Smith & Davis. 1967) might point t(o an intimate connection between DNA replication and septum formation (Helmstrttrr. 1973). The author is indebted to Dr P. H. Hofsclmeider fnr helpful comments and critical reading of the manuscript. This work wm siupported hy H grant from thcb Dentsche Forst,hnngsgemeinsctl~ft . ~Iax-Planck-Institat fiir Riochemie Abteihmg Hofschneider 8033 Martins&d bei Miinchen, Germany
\\IAT.TRR
I,.
S;;TAITDENRAT;ER
REFERENCES Ben-Harnida, F. 8: Gros, F. (1971). Biochimie (Paris), 53, 71-80. Brock, T. D. (1967). In Antibiotica (Gottlieb, D. and Shaw, P. D., eds). vol. 1, pp. 651-665, Springer-Verlag, Berlin. Burger, R. M. (1971). Proc. Nat. AcadS&., U.S.A. 68,2124-2126. Burger, R. M. & Gleser, D. A. (1973). Proc. Nat. AcadS&, U.S.A. 70, 1955-1958. Helmstetter, C. E. (1974). J. Mol. Biol. 84, 21-36. Masker, W. E. & Hanawalt, P. C. (1974). Biochim. Biophya. Acta, 340, 229-236. Moses, R. E. (1972). J. BioZ. Chem. 247, 6031-6038. Moses, R. E. & Richardson, C. C. (1970). Proc. Nat. Acud. Sci., U.S.A. 67, 674-681. Pisetsky, D., Berkower, I., Wickner, R. & Hurwitz, J. (1972). J. &Jol. Biol. 71, 557-571. Smith, D. H. 8: Davis, B. D. (1967). J. Baderiol. 93, 71-79. Strominger, J. L. & Threnn, R. H. (1969). Biochim. Biophys. Acta, 33, 280-282. Vosberg, H. P. & Hoffmann-Berling, H. (1971). J. Mol. BioZ. 58, 739-753. Wickner, R. B. & Hurwitz, J. (1972). Biochem. Biophye. Res. Commun. 47, 202-211. Wishnow, R. M., Strominger, J. L., Birge, C. H. Br Threnn, R. H. (1965). J. Bacterial. 89, 1117-1123.
14