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O ratio in Escherichiacoli
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The Effect of Colicin E1 on Proton Extrusion and the H+/O Ratio in Escherichia coli
J.
Michael Gould, W.A. Cramer, and G. van Thienen Dept. of Biological Sciences Purdue University West Lafayette, Indiana, U.S.A. 47907 and Laboratory for Biochemistry University of Amsterdam PlantageMuidergracht 12 Amsterdam, The Netherlands
Received
August
13,1976
Summary Tl%e addition of a small pulse of oxygen to an anaerobic suspension of colicin sensitive E. coli B/I,5 cells induces a relatively small, slowly reversible extrusion of protons by the cells into the medium. Pretreatment of the cells with colicins E1 and K greatly accelerates the rate of proton extrusion as well as the rate of decay of the measured pH change, while increasing the amplitude of the H+/O ratio+from <0.5 to >2.0. Colicin E1 had no effect on the proton extrusion or H /O ratio in the tolerant E. coli strain _.~ ~ A . These effects suggest that, in the presence of colicins E1 and K~8~he E. coli cell envelope becomes freely permeable to a counterion and is therefore no longer able to maintain a membrane electrical potential.
Introduction A class of colicins
including E1 and K can be defined by its
ability to inhibit macromolecular synthesis and the active transport of many metabolites, and to cause the leakage of K + ions and possibly other ions into the medium (e.g., ref. i).
The apparent deenergization of the
cell membrane, considered in the context of the chemiosmotic model (2) for energy coupling, may be attributed to depolarization of the cell membrane potential by colicin, although the o~ly experimental test of this hypothesis involves the effects of colicin K on the fluorescence
Abbreviation:
MOPS, morpholinopropane sulfonic acid
Copyright© 1976by AcademicPress, Inc. Allr~hts ~ reproductioninany/orm reserved.
1519
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
behavior of a cyanine dye (3).
It has been inferred from studies on other
membrane systems that such dyes can monitor a transmembrane electrical potential (e.g. ref. 4).
Although the pattern of cyanine probe fluores-
cence changes caused by colicin K is consistent with colicin causing membrane depolarization, it is not clear whether the probe is only responding to chan~es in transmembrane potential, since (a) the presence of the permeability barrier of the outer membrane of E. coli would tend to prevent the charged cyanine probe from binding to the inner membran~ and (b) the pattern of cyanine dye fluorescence changes induced by coliein K are very similar to those exhibited by the uncharged, lipophilic fluorescence probe, Nphenyl-l-naphthylamine in studies with colicin K or colicin E1 (5-7).
The
latter probe will not respond directly to changes in membrane potential, although the membrane structural changes it does monitor could be a consequence of inner membrane depolarization and deenergization. Despite the above reservation about the origin of the cyanine fluorescence changes observed with whole cells of E. coli the general inference that colicin dissipates the cell membrane potential is consistent with data presented in this paper on the effect of colicin E1 on the respirationdriven proton efflux from intact E. coli.
Experimental Methods Escherichia coli strain B/I,5 obtained from S. Silver was grown in Erlenmeyer culture flasks at 37°C in a minimal medium (pH 7.0) containing (per liter): i g (NH~)oS0~, 0.5 g sodium citrate, 0.I g MgSOA'7 HgO, 7 g KgHPO~, 3 g KHoPO h an~ I0 ~ suecinic acid. E. coli strain A.~. obtained f~om ~.E. Luri$ w~s grown on a medium (pH 7,5) c--~aining (p~°liter): i g (NHA)gSOA, 0.I g Mg2S04.7 H20, 10.5 g K2HPO ~, 4.5 g KH2PO 4, i mg thiamine, 20 ~g proline, 20-mg-leuclne, 20 mg tNreonine and 4 g glucose. Cultures were grown with vigorous shaking until they had reached mid-log phase growth (4.5-5 hr.), when the cells were harvested by centrifugation at 4°C. The harvested cells were washed twice in 150 mM KCI, i mM MOPSKOH (p~ 7.0) ~nd resuspended in this medium to a concentration of approximately 3 x i0 cells/ml. A 2 ml aliquot of the cell suspension was placed in a water-jacketed reaction chamber (3 ml capacity) which contained a small magnetic stirring bar. The opening to the reaction chamber was sealed with a rubber stopper through which passed a Sargent miniature combination pH electrode. Small teflon tubes also passing through the stopper allowed a continuous stream
1520
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of water-saturated N_ gas to pass through the sample chamber. Another small hole admittted the n~edle of a microliter syringe which was used to make additions to the anaerobic sample. The pR electrode was connected to a Corning Model 14 meter operated on the expanded (0.i pH) scale. The output from the pH meter was monitored on a strip chart recorder with a scale expansion of 0.02 pH units full scale ~6 in.). The sample chamber was maintained at 33°C by a constant temperature circulating water bath. Colicin was added in a small volume (2 ~i) and the cell suspension was incubated with stirring at 33°C for 5 min. in air before the initiation of anaerobiosis. After the cell suspension had become anaerobic (approx. 15 min) small pulses of oxygen were added in air-saturated double distilled water. The chart paper was calibrated in hydrogen ion equivalents by titration of the cell suspension with 0.001 M HCI in the presence of i0 ~M FCCP. Colicin E1 was prepared from the colicinogenic E. coli strain JC411 by the method of Schwartz and Helinski (8). Colicin K was a gift from Dr. M.A. Jesaitis of Rockefeller University.
Results and Discussion Mitchell and Moyle originally observed a stoichiometric, coupling sitedependent ejection of protons from anaerobic suspensions of mitochondria given a small pulse of oxygen (9,10).
According to the chemiosmotic theory,
this proton extrusion results from a vectorial topological arrangement of the electron transport carriers across the mitochondrial membrane (2).
A
similar proton efflux driven by an oxygen pulse delivered to anaerobic stationary phase E. eoli cells has also been observed (e.g., refs. 11,12). The values of the H+/O ratio reported here in the presence of colicin for E. coll B/I,5 grown on suecinate are very close to those obtained in ref. 12 in the presence of SCN- as permeant counterion using glycerol, lactate or succinate as carbon source.
It is important to point out that in order
to obtain maximal hydrogen ion extrusion and H+/O ratios in mitochondria? sub~itochondrial particles~ and bacteria, it is necessary to include a membrane permeant counter ion in the reaction mixture Ce.g., SCN- or K+ plus vallnomycin) so that the outward movement of H+ ions can be electrically compensated and a large membrane potential is not created (e.g., refs. II, 12). Figure I shows the response to an oxygen pulse of an anaerobic suspen-
1521
Vol. 72, No. 4, 1976
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
30 sec
F T oofo2pH 5 nequw
~
l cells H+/0=039
=2
B/I,5 t
H+/O=0 70
HYO= 0 73
A586
t
t control
+ cohcm E I
Figure i, Effect of colicin E1 on protsn extrusion from E. coli after an oxygen pulse [_upward arrows). The H /O ratio was ealculat~ed from the maximum extent of the pH change after the oxygen pulse. The pulses Consisted of I0 ~l air saturated double distilled water containing 5.5 ngm~ atoms+O. The concentration of colicin E1 was i ~g/ml. The difference in the H /O ratio (in the absence of colicin) between B/I,5 and A58 ~ cells is related to the carbon source used for growth (see Methods) and will be discussed in detail in a separate paper (13).
sion of logarithmic phase E. coli strain B/I,5 cells, which are sensitive to colicin El.
In the absence of colicin El, the proton efflux is small
and exhibits rather sluggish kinetics, with an apparent H+/O ratio of only 0.39.
Addition of colicin E1 causes a dramatic increase in both the ampli-
tude and the relaxation kinetics of the pH change, with an apparent H+/O ratio of more than 2.0,
Under the same conditions, there is no significant
change in the H+/O ratio when colicin E1 is added to cells of the colicin tolerant mutant A586 (which are not killed by the colicin) as shown in Figure I.
It is important to note that the H+/O ratio measured in the
presence of colicin is as large as that obtained in the presence of added SCN- as a permeant counterion (13). That this effect is in fact a consequence of the single-hit nature of colicin E1 action, and not of any mass or bulk protein effect on the membrane, is shown by the data in Table I,
1522
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I
Ef,feat of Colicin E1 Concentration on the Colicin-Dependent l~crease in eroton Extrusion [AH+)and th e ~+/0 Ratio
*
E1 (~g/ml)
1
Multiplicity
AH + ** (nequiv.)
i00
11.71
2.13
0,i
5
9.90
1.80
0.01
1
7.92
1.44
0
-
1.32
0.24
*E. coli strain B/I,5 cells at 2 x 109 cells/ml. Multiplicity was calculated from a parallel viability experiment according to the relation. -m shlp S=e where S is survival and m is the multiplicity. * * h H + w a s calculated from the maximum extent of the pH change after an oxygen pulse. The oxygen pulse contained 5.5 ngm-atoms O.
where the H+/0- ratio obtained in the presence of colicin is presented as a function of the amount of added colicin.
Colicin K, which bears many simi-
larities to colicin E1 in the manner in which it affects energy metabolism (I), also causes a large increase in the extent of H+ ion extrusion and the H+/O ratio after an oxygen pulse (Table II). The ability of colicins E1 and K to cause a large increase in the H+/O ratio in sensitive cells can best be explained by assuming that these colicins cause a significant increase in the permeability of the cell membrane to an electrically compensatory counter ion.
Indeed, the enhance-
ment of the H+/O ratio by colicin E1 is completely dependent upon the presence of potassium in the external medium (13). The effect of colicin
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Vol. 72, No. 4, 1 9 7 6
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE II Similar Effects of Colieins El an d K on Proton Extrusion (~+) and th~ H*/O Ratio
~H+ , (nequiv.)
~+/O
1.32
0.24
E1 (i ~g/ml)
11.71
2.13
K (i ~g/ml)
14.52
2.64
Colicin
None
Oxygen pulses consisted of i0 ~i additions of air-saturated distilled water containing 5.5 ngm-atoms 0. Cells ~ere E. coli strain B/I,5 at 2 x i0~ cel~s/m~.
E1 on the ~+/O ratio in E. coli is thus formally similar to the effect of added valinomycin plus potassium on the ~+/O ratio observed in submitochondrial particles (NMP, e.g., ref. 14).
In SMP the membrane potential is
dissipated in the presence of valinomycin and potassium, since in the presence of valinomycin, K+ can function as a freely permeable cation. Similarly, the demonstration of a large increase in the bacterial H+/O ratio caused by colicin E1 in the presence of potassium leads to the conclusion that the bacterial cell membrane is also freely permeable to potassium under these conditions.
It therefore follows that the cell
membrane potential, negative inside, which is present under energized conditions should be dissipated in the presence of colicin E1 and potassium in the external medium.
Acknowledgements The work was supported by a grant from the National Institutes of Health (GM 18457). J.M.G. is the recipient of a National Research Service
1524
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
Award (IF32GMOO913-01) from the National Institute of General Medical Sciences, National Institutes of Health. We thank ~rof. F.L. Crane for his critical reading of the manuscript and Pam Hurey for her help assembling it. Dr. K. van Dam and Dr. P.W. Postma provided helpful discussions during the initial stage of this work, at which time ~.A.C. was the recipient of a Senior Fellowship from the European Molecular Biology Organization. G.V.T. is supported by the Netherlands Organization for the Advancement of Pure Research (Z.W,O.) under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.).
References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14.
Reeves, P. (1972) The Bacteriocins pp. 60-61, Springer-Verlag, New York. Mitchell, P. (1966) Biol. Revs. 41,455-502. Brewer, G. (1976) Biochemistry 15,1387-1392. Sims, P.T., Waggoner, A.S., Wang, C-H. and Hoffman, J.F. (1974) Biochemistry 13,3315-3330. Phillips, S.K. and Cramer, W.A. (1973) Biochemistry, 12,1170-1176. Nivea-Gomez, D., Konisky, J. and Gennis, R.B. (1976) Biochemistry, 15,2747-2753. Cramer, W.A, Postma, P.W. and Helgerson, S.L. (1976) Biochim. Biophys. Acta, in press. Schwartz, S.A. and Helinski, D.R. (1971) J. Biol. Chem. 246,6318-6329. Mitchell, P. and Moyle, J. (1965) Nature, 208,147-151. Mitchell, P. and Moyle, J. (1967) Biochem. J. 105,1147-1162. West, I. and Mitchell, P. (1972) J. Bioenergetics, 3,445-462. Lawford, H.G. and Haddock, G.A. (~973) Biochem. J. 136,217-220. Gould, J.M. and Cramer, W.A. (1976) Manuscript in preparation. Hinkle, P.C. and Horstman, L.I° (1971) J. Biol. Chem. 246,6024-6028.
1525
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The Effect of Colicin E1 on Proton Extrusion and the H+/O Ratio in Escherichia coli
J.
Michael Gould, W.A. Cramer, and G. van Thienen Dept. of Biological Sciences Purdue University West Lafayette, Indiana, U.S.A. 47907 and Laboratory for Biochemistry University of Amsterdam PlantageMuidergracht 12 Amsterdam, The Netherlands
Received
August
13,1976
Summary Tl%e addition of a small pulse of oxygen to an anaerobic suspension of colicin sensitive E. coli B/I,5 cells induces a relatively small, slowly reversible extrusion of protons by the cells into the medium. Pretreatment of the cells with colicins E1 and K greatly accelerates the rate of proton extrusion as well as the rate of decay of the measured pH change, while increasing the amplitude of the H+/O ratio+from <0.5 to >2.0. Colicin E1 had no effect on the proton extrusion or H /O ratio in the tolerant E. coli strain _.~ ~ A . These effects suggest that, in the presence of colicins E1 and K~8~he E. coli cell envelope becomes freely permeable to a counterion and is therefore no longer able to maintain a membrane electrical potential.
Introduction A class of colicins
including E1 and K can be defined by its
ability to inhibit macromolecular synthesis and the active transport of many metabolites, and to cause the leakage of K + ions and possibly other ions into the medium (e.g., ref. i).
The apparent deenergization of the
cell membrane, considered in the context of the chemiosmotic model (2) for energy coupling, may be attributed to depolarization of the cell membrane potential by colicin, although the o~ly experimental test of this hypothesis involves the effects of colicin K on the fluorescence
Abbreviation:
MOPS, morpholinopropane sulfonic acid
Copyright© 1976by AcademicPress, Inc. Allr~hts ~ reproductioninany/orm reserved.
1519
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
behavior of a cyanine dye (3).
It has been inferred from studies on other
membrane systems that such dyes can monitor a transmembrane electrical potential (e.g. ref. 4).
Although the pattern of cyanine probe fluores-
cence changes caused by colicin K is consistent with colicin causing membrane depolarization, it is not clear whether the probe is only responding to chan~es in transmembrane potential, since (a) the presence of the permeability barrier of the outer membrane of E. coli would tend to prevent the charged cyanine probe from binding to the inner membran~ and (b) the pattern of cyanine dye fluorescence changes induced by coliein K are very similar to those exhibited by the uncharged, lipophilic fluorescence probe, Nphenyl-l-naphthylamine in studies with colicin K or colicin E1 (5-7).
The
latter probe will not respond directly to changes in membrane potential, although the membrane structural changes it does monitor could be a consequence of inner membrane depolarization and deenergization. Despite the above reservation about the origin of the cyanine fluorescence changes observed with whole cells of E. coli the general inference that colicin dissipates the cell membrane potential is consistent with data presented in this paper on the effect of colicin E1 on the respirationdriven proton efflux from intact E. coli.
Experimental Methods Escherichia coli strain B/I,5 obtained from S. Silver was grown in Erlenmeyer culture flasks at 37°C in a minimal medium (pH 7.0) containing (per liter): i g (NH~)oS0~, 0.5 g sodium citrate, 0.I g MgSOA'7 HgO, 7 g KgHPO~, 3 g KHoPO h an~ I0 ~ suecinic acid. E. coli strain A.~. obtained f~om ~.E. Luri$ w~s grown on a medium (pH 7,5) c--~aining (p~°liter): i g (NHA)gSOA, 0.I g Mg2S04.7 H20, 10.5 g K2HPO ~, 4.5 g KH2PO 4, i mg thiamine, 20 ~g proline, 20-mg-leuclne, 20 mg tNreonine and 4 g glucose. Cultures were grown with vigorous shaking until they had reached mid-log phase growth (4.5-5 hr.), when the cells were harvested by centrifugation at 4°C. The harvested cells were washed twice in 150 mM KCI, i mM MOPSKOH (p~ 7.0) ~nd resuspended in this medium to a concentration of approximately 3 x i0 cells/ml. A 2 ml aliquot of the cell suspension was placed in a water-jacketed reaction chamber (3 ml capacity) which contained a small magnetic stirring bar. The opening to the reaction chamber was sealed with a rubber stopper through which passed a Sargent miniature combination pH electrode. Small teflon tubes also passing through the stopper allowed a continuous stream
1520
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of water-saturated N_ gas to pass through the sample chamber. Another small hole admittted the n~edle of a microliter syringe which was used to make additions to the anaerobic sample. The pR electrode was connected to a Corning Model 14 meter operated on the expanded (0.i pH) scale. The output from the pH meter was monitored on a strip chart recorder with a scale expansion of 0.02 pH units full scale ~6 in.). The sample chamber was maintained at 33°C by a constant temperature circulating water bath. Colicin was added in a small volume (2 ~i) and the cell suspension was incubated with stirring at 33°C for 5 min. in air before the initiation of anaerobiosis. After the cell suspension had become anaerobic (approx. 15 min) small pulses of oxygen were added in air-saturated double distilled water. The chart paper was calibrated in hydrogen ion equivalents by titration of the cell suspension with 0.001 M HCI in the presence of i0 ~M FCCP. Colicin E1 was prepared from the colicinogenic E. coli strain JC411 by the method of Schwartz and Helinski (8). Colicin K was a gift from Dr. M.A. Jesaitis of Rockefeller University.
Results and Discussion Mitchell and Moyle originally observed a stoichiometric, coupling sitedependent ejection of protons from anaerobic suspensions of mitochondria given a small pulse of oxygen (9,10).
According to the chemiosmotic theory,
this proton extrusion results from a vectorial topological arrangement of the electron transport carriers across the mitochondrial membrane (2).
A
similar proton efflux driven by an oxygen pulse delivered to anaerobic stationary phase E. eoli cells has also been observed (e.g., refs. 11,12). The values of the H+/O ratio reported here in the presence of colicin for E. coll B/I,5 grown on suecinate are very close to those obtained in ref. 12 in the presence of SCN- as permeant counterion using glycerol, lactate or succinate as carbon source.
It is important to point out that in order
to obtain maximal hydrogen ion extrusion and H+/O ratios in mitochondria? sub~itochondrial particles~ and bacteria, it is necessary to include a membrane permeant counter ion in the reaction mixture Ce.g., SCN- or K+ plus vallnomycin) so that the outward movement of H+ ions can be electrically compensated and a large membrane potential is not created (e.g., refs. II, 12). Figure I shows the response to an oxygen pulse of an anaerobic suspen-
1521
Vol. 72, No. 4, 1976
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
30 sec
F T oofo2pH 5 nequw
~
l cells H+/0=039
=2
B/I,5 t
H+/O=0 70
HYO= 0 73
A586
t
t control
+ cohcm E I
Figure i, Effect of colicin E1 on protsn extrusion from E. coli after an oxygen pulse [_upward arrows). The H /O ratio was ealculat~ed from the maximum extent of the pH change after the oxygen pulse. The pulses Consisted of I0 ~l air saturated double distilled water containing 5.5 ngm~ atoms+O. The concentration of colicin E1 was i ~g/ml. The difference in the H /O ratio (in the absence of colicin) between B/I,5 and A58 ~ cells is related to the carbon source used for growth (see Methods) and will be discussed in detail in a separate paper (13).
sion of logarithmic phase E. coli strain B/I,5 cells, which are sensitive to colicin El.
In the absence of colicin El, the proton efflux is small
and exhibits rather sluggish kinetics, with an apparent H+/O ratio of only 0.39.
Addition of colicin E1 causes a dramatic increase in both the ampli-
tude and the relaxation kinetics of the pH change, with an apparent H+/O ratio of more than 2.0,
Under the same conditions, there is no significant
change in the H+/O ratio when colicin E1 is added to cells of the colicin tolerant mutant A586 (which are not killed by the colicin) as shown in Figure I.
It is important to note that the H+/O ratio measured in the
presence of colicin is as large as that obtained in the presence of added SCN- as a permeant counterion (13). That this effect is in fact a consequence of the single-hit nature of colicin E1 action, and not of any mass or bulk protein effect on the membrane, is shown by the data in Table I,
1522
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I
Ef,feat of Colicin E1 Concentration on the Colicin-Dependent l~crease in eroton Extrusion [AH+)and th e ~+/0 Ratio
*
E1 (~g/ml)
1
Multiplicity
AH + ** (nequiv.)
i00
11.71
2.13
0,i
5
9.90
1.80
0.01
1
7.92
1.44
0
-
1.32
0.24
*E. coli strain B/I,5 cells at 2 x 109 cells/ml. Multiplicity was calculated from a parallel viability experiment according to the relation. -m shlp S=e where S is survival and m is the multiplicity. * * h H + w a s calculated from the maximum extent of the pH change after an oxygen pulse. The oxygen pulse contained 5.5 ngm-atoms O.
where the H+/0- ratio obtained in the presence of colicin is presented as a function of the amount of added colicin.
Colicin K, which bears many simi-
larities to colicin E1 in the manner in which it affects energy metabolism (I), also causes a large increase in the extent of H+ ion extrusion and the H+/O ratio after an oxygen pulse (Table II). The ability of colicins E1 and K to cause a large increase in the H+/O ratio in sensitive cells can best be explained by assuming that these colicins cause a significant increase in the permeability of the cell membrane to an electrically compensatory counter ion.
Indeed, the enhance-
ment of the H+/O ratio by colicin E1 is completely dependent upon the presence of potassium in the external medium (13). The effect of colicin
1523
Vol. 72, No. 4, 1 9 7 6
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE II Similar Effects of Colieins El an d K on Proton Extrusion (~+) and th~ H*/O Ratio
~H+ , (nequiv.)
~+/O
1.32
0.24
E1 (i ~g/ml)
11.71
2.13
K (i ~g/ml)
14.52
2.64
Colicin
None
Oxygen pulses consisted of i0 ~i additions of air-saturated distilled water containing 5.5 ngm-atoms 0. Cells ~ere E. coli strain B/I,5 at 2 x i0~ cel~s/m~.
E1 on the ~+/O ratio in E. coli is thus formally similar to the effect of added valinomycin plus potassium on the ~+/O ratio observed in submitochondrial particles (NMP, e.g., ref. 14).
In SMP the membrane potential is
dissipated in the presence of valinomycin and potassium, since in the presence of valinomycin, K+ can function as a freely permeable cation. Similarly, the demonstration of a large increase in the bacterial H+/O ratio caused by colicin E1 in the presence of potassium leads to the conclusion that the bacterial cell membrane is also freely permeable to potassium under these conditions.
It therefore follows that the cell
membrane potential, negative inside, which is present under energized conditions should be dissipated in the presence of colicin E1 and potassium in the external medium.
Acknowledgements The work was supported by a grant from the National Institutes of Health (GM 18457). J.M.G. is the recipient of a National Research Service
1524
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
Award (IF32GMOO913-01) from the National Institute of General Medical Sciences, National Institutes of Health. We thank ~rof. F.L. Crane for his critical reading of the manuscript and Pam Hurey for her help assembling it. Dr. K. van Dam and Dr. P.W. Postma provided helpful discussions during the initial stage of this work, at which time ~.A.C. was the recipient of a Senior Fellowship from the European Molecular Biology Organization. G.V.T. is supported by the Netherlands Organization for the Advancement of Pure Research (Z.W,O.) under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.).
References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14.
Reeves, P. (1972) The Bacteriocins pp. 60-61, Springer-Verlag, New York. Mitchell, P. (1966) Biol. Revs. 41,455-502. Brewer, G. (1976) Biochemistry 15,1387-1392. Sims, P.T., Waggoner, A.S., Wang, C-H. and Hoffman, J.F. (1974) Biochemistry 13,3315-3330. Phillips, S.K. and Cramer, W.A. (1973) Biochemistry, 12,1170-1176. Nivea-Gomez, D., Konisky, J. and Gennis, R.B. (1976) Biochemistry, 15,2747-2753. Cramer, W.A, Postma, P.W. and Helgerson, S.L. (1976) Biochim. Biophys. Acta, in press. Schwartz, S.A. and Helinski, D.R. (1971) J. Biol. Chem. 246,6318-6329. Mitchell, P. and Moyle, J. (1965) Nature, 208,147-151. Mitchell, P. and Moyle, J. (1967) Biochem. J. 105,1147-1162. West, I. and Mitchell, P. (1972) J. Bioenergetics, 3,445-462. Lawford, H.G. and Haddock, G.A. (~973) Biochem. J. 136,217-220. Gould, J.M. and Cramer, W.A. (1976) Manuscript in preparation. Hinkle, P.C. and Horstman, L.I° (1971) J. Biol. Chem. 246,6024-6028.
1525