ANTIMcItoBIAL AGENTS A"N CHEmOTHERPY, May 1977, p. 877-880 Copyright 0 1977 American Society for Microbiology
Vol. 11, No. 5
Printed in U.S.A.
D-Cycloserine-Induced Alterations in the Transport of DAlanine and Glycine in Bacillus subtilis 168 VIRGINIA L. CLARK'
AND
FRANK E. YOUNG*
Department ofMicrobiology, University ofRochester, School ofMedicine and Dentistry, Rochester, New York 14642
Received for publication 22 December 1976
D-Alanine, i-alanine, and glycine transport was investigated in Bacillus subtilis 168 cells that were phenotypically resistant to D-cycloserine. These cells showed enhanced rates of uptake as compared with that observed in sensitive cells. The usual enhancement in D-alanine and glycine transport resulting from treatment of the cells with D-cycloserine could be prevented by the addition of rifampin. Kinetic analyses of the initial rate of glycine transport indicated an increase in the Vma; for transport in resistant cells, with no alteration in the Km for glycine. Investigations of the net transport of glycine revealed that resistant cells maintained a higher gradient of glycine than did sensitive cells. Kinetic analyses of the net transport of glycine suggested that a new system for the accumulation of glycine was present in D-cycloserine-resistant cells.
The antibiotic D-cycloserine is actively transported, in various bacterial species, by the same system(s) responsible for the transport of D-alanine, L-alanine, and glycine (3, 5-7, 9, 11). Resistance to i-cycloserine has been reported to result from a decreased accumulation of the antibiotic (1, 4, 5, 12). In Escherichia coli there is a concomitant decrease in the transport Dalanine, L-alanine, and glycine in resistant cells (5, 12). We have reported an inducible icycloserine resistance in Bacillus subtilis 168 (4) that is also a result of a decreased accumulation of the antibiotic. In this paper we will present evidence that D-cycloserine-resistant cells of B. subtilis do not transport i-alanine, Lalanine, and glycine at reduced rates. In fact, the transport of n-alanine and glycine is enhanced by treatment of the cell with D-cycloserine. MATERIALS AND METHODS Materials. The L_[U-_4C]alanine (specific activity, 153 mCi/mmol), L_[U-14C]proline (specific activity, 233 mCi/mmol), and Omnifluor were purchased from New England Nuclear; the D[U-14C]alanine (specific activity, 37 mCi/mmol) was obtained from Amersham/Searle Corp. Rifampin and D-alanine were purchased from Sigma Chemical Co., and acidhydrolyzed casein was from Nutritional Biochemicals Corp. All other chemicals were of reagent-grade purity. Strains and growth conditions. The strains of B. subtilis 168 and the growth conditions used were described previously (4). X Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
Measurement of transport. The preparation of cells for transport studies and the procedures for analysis of transport were similar to those in the accompanying paper (4). Protein determination. Protein was determined by the method of Lowry et al. (8) for trichloroacetic acid precipitates, with bovine serum albumin as a standard. Determination of radioactivity. The radioactivity of nonaqueous samples was determined with use of a scintillation fluid consisting of 4 g of Omnifluor per liter of toluene. Calculation of intracellular substrate concentration. If it is assumed that B. subtilis protein constitutes 50% of the dry weight of the cell, and that the cell is 80% water throughout the growth cycle, then 1 mg of protein is equal to 2 mg of dry weight plus 8 mg of water. Therefore, since 1 mg of water is 1 .ld, 1 mg of cell protein is approximately equal to 8 p.l of water. To convert the uptake values that are expressed as nanomoles of substrate transported per milligram of protein to millimolar, divide by a factor of 8.
RESULTS Transport of D-alanine in D-cycloserine-resistant cells. Previous reports of D-cycloserine resistance in E. coli (12) indicated a reduction in the transport of the antibiotic as the mechanism of resistance. In this case there is a concomitant reduction in the transport of n-alanine, L-alanine, and glycine. We investigated the rate of transport of n-alanine and glycine in growing cells of B. subtilis treated with D-cycloserine (Fig. 1). Under conditions in which Dcycloserine is transported at only 10% of the original rate after 90 min of treatment (data not 877
878
ANTIMICtOB. AGZNTS CHMOTHtR.
CLARK AND YOUNG
4 w
w
I-4 I0
TIME (min) FIG. 1. Effect of D-cycloserine incubatioi s on the transport of D-alanine and glycine. 0.1 m AM Dcycloserine was added to growing BR151 cells a time, and the transport of D-alanine (0) and gily the (0) was measured in washed cell sampls a indicated times. The control uptake rate wasi that observed at zero time. The substrate concentrcztions were: 1 pM D-4CJalanine and 20 M[14CJglyci ,ne.
ycine
shown), the transport of D-alanine and glycine was substantially increased. The increase in alanine transport could also occur in the glucose-minimal salts medium used for the induction of -cycloserine resistance (Fig. 2). Uninduced cells were suspended in milimal salts with no addition or plus -cycloserine. The cells to which the antibiotic was added were initially slightly inhibited, but with time transport of alanine by these cells exceeded the level observed in untreated cells. This enhancement could be prevented by simultaneously treating the cells with rifampin (Fig. 2B). Induced cells accumulated D-alanine to a higher degree and showed no further enhancement in the presence of -cycloserine (Fig. 2C). Transport of glycine in D-cycloserine-resistant cells. We investigated the effects of -cycloserine incubation on the initial. rates of transport and on the net transport of -alanine, xalanine, glycine, and L-proline (Table 1). Transport. was investigated at two substrate concentrations, 1 and 20 MM, because of the two transport systems observed.in B. subtilis (3). IProline was included as a control substrate that
is not transported by the alanine systems. Incubation in D-cycloserine caused an increased ability by the cells t,o transport D- and L-alanine and glycine when initial rates were measured. When net transport was measured, however, enhancement was evident only with -alanine and glycine. The enhanced level of retention was especially pronounced with glycine. The effect of -cycloserine incubation on glycine transport was further investigated by determining the kinetic parameters for the initial rate of transport of glycine (Fig. 3). The initial rate of transport by induced cells had the same Km value for glycine, but a higher V.a. than was observed in uninduced cells. The net transport of glycine was investigated at various concentrations of glycine in uninduced and induced cells. The intracellular concentration of glycine was calculated from the values of nanomoles glycine transported per 15 min per milligram of protein as described in Materials and Methods and plotted versus the extracellular glycine concentration (Fig. 4). The gradient of glycine retained in induced cells was compared with that in uninduced cells (Fig. 4, insert). It can be seen that the increased retention of glycine by induced. cells was more pronounced at higher glycine concentrations. Although the Michaelis-Menten equation was devised to deal with initial rates of reaction, the use of the equation B.
A.
2o w
0.
0
IS
30
0
IS
30
0
Is
3o
TIME (min)
FIG. 2. Effect of D-cycloserine incubation on transport of D-alanine. (A) Uninduced RUB1402 cells were incubated in Spizizen minimal salts with no addition (-) or plus 0.1 pMD-cycloserine (0). (B) Uninduced RUB1402 cells were incubated in Spizizen minimal salts containing 5 pg ofrifampin per ml with no further addition (0) or plus 0.1 mM Dcycloserine (0). (C) RUB1402 cells induced by incubation in Spizizen minimal salts plus 01 mM Dcycloserine for 15 min were washed and incubated in Spizizen minimal salts with no addition (-) or plus 0.1 mM D-cycloserine (0). All samples had 1 M z [14C]alanine added at zero time. Uptake is expressed as nanomoles of D-alanine transported per milligram of protein.
V VOL. 1-ALANINE AND GLYCINE TRANSPORT IN B. SUBTILIS 11, 1977
879
TABLE 1. Enhanced uptake rates for D-alanine and glycine in D-cycloserine-resistant cellsa Uptake rate
Compound transported
Concn
(,M)
Initial rate n
Uninduced
Net transport
t
Induced
9 of unin-
duced
Uninduced
of unin-
Induced
duced
n-Alanine
1 0.770 1.03 134 5.44 6.19 114 20 8.67 9.73 112 46.2 49.2 106 L-Alanine 1 0.289 0.502 174 2.14 2.13 100 20 4.31 5.03 117 38.7 24.7 64 Glycine 1 0.521 0.712 137 1.77 2.32 131 20 4.64 8.74 188 6.62 240 15.9 L-Proline 1 0.349 0.384 110 2.71 2.09 77 20 1.32 0.632 48 16.2 11.6 72 a Cells were induced for D-cycloserine resistance as described in Materials and Methods. Initial rates of transport were measured for 30 s and net transport for 15 min as described in Materials and Methods. Rates are expressed as nanomoles transported per minute per milligram of protein and nanomoles transported per 15 minute per milligram of protein for initial rates and net transport, respectively.
0
)I
a
0
._
S
X
c
-
0
I
a
c
la
._
Cs
E0 ._
._
S
t
Cs
w
c
.2 U
c 10
.' 2%
0
a
.5
0
VDAM
S
2
Glycine]
FIG. 3. Lineweaver-Burk plot of initial rates of glycine transport in uninduced and induced cells. Initial rates of transport (30 s) ofglycine were determined in cells unexposed to D-cycloserine (0) and cells induced for 30 min in Spizizen minimal salts plus 0.1 mM D-cycloserine (0). The kinetic parameters were: K. = 25 MM glycine, VmaZ = 11 nmol of glycine transported per min per mg of protein for uninduced cells, and Km = 25 pMglycine, Vma, = 16 nmol ofglycine transported per min per mg ofprotein for induced cells.
for measuring transport under other than initial rate conditions has been recognized as providing useful information (2). When this equation was applied to the net transport of glycine (Fig. 5), it appeared that there were two Km values (4 and 25 ,uM glycine) for induced cells, rather than the one Km value (4 ,uM glycine) observed in uninduced cells.
DISCUSSION D-Cycloserine resistance in E. coli (5, 12) is accompanied by a loss of transport of the antibiotic and a concomitant loss of transport of nalanine and glycine. We have described an inducible resistance to n-cycloserine in B. subtilis
-o
a
1100
50
Extrocellular glycine
0
25
50
75
(IjM)
100
Extracellular glycine (pM) FIG. 4. Ratio of intracellular to extracellular gly-
cine in uninduced and induced cells. Strain BR151 was induced for D-cycloserine resistance as described in Materials and Methods. Net transport (15 min) of [14C]glycine was measured in uninduced (0) and induced (0) cells, and the uptake was converted from nanomoles per minute per milligram of protein to millimolar as described in Materials and Methods. The extracellular concentration of glycine was corrected for uptake, and the ratios of intracellular to extracellular ['4C]glycine were determined. Insert: Ratio of intracellular to extracellular glycine in induced cells was divided by that in uninduced cells.
(4) that also results from a decreased accumulation of the antibiotic. Under conditions in which D-cycloserine resistance in B. subtilis is induced, however, we found that the transport
880
CLARK AND YOUNG
/ EM
Glycine]
ANTIMICROB. AGENTS CHEMOTHER.
We propose that i-cycloserine induces a new transport system in B. subtilis 168. This new system transports D-alanine and glycine, with glycine the preferred solute. D-Cycloserine-resistant cells would maintain a higher gradient of glycine as a result of the synthesis of this system. We further postulate that this new transport system also transports D-cycloserine, but that the rate of uptake is less than the rate of efflux of the antibiotic. This would cause a net efflux of D-cycloserine, which would render the cells resistant to the antibiotic. These observations suggest that an analysis of the transport of each antimicrobial agent used in combination with t-cycloserine might reveal patterns of illicit transport that could produce inhibition of microbial growth at lower concentrations of antibiotic and thereby reduce toxicity.
FIG. 5. Lineweaver-Burk plot of the net transport ofglycine in uninduced and induced cells. Net transport (30 min) of glycine determined in cells unexposed to D-cycloserine (0) and in cells induced for 30 min in Spizizen minimal salts plus 0.1 mM D-cycloserine (0). The kinetic parameters were: Km = 4 uM glycine for uninduced cells and Km = 4 juM and Km = 25 pM for induced cells .
ACKNOWLEDGMENTS This work was supported by Public Health Service grants 5TI-GM-00592 from the National Institute of General Medical Science and 5ROI-AI-10141 from the National Institute of Allergy and Infectious Diseases.
of D-alanine, L-alanine, and glycine is not decreased. When initial rates of transport are measured at 1 ,uM concentrations of the substrate, the transport of D- and L-alanine and glycine is increased. This may be due to an artifactual example of antiport, such that the transport substrate is pulled into the cell by the efflux of D-cycloserine. When the kinetic parameters for the initial rate of transpoqrt of glycine are determined, the Km for glycine remains constant while the Vmax for transport increases. This would be consistant with an antiportdriven transport of glycine. Net transport represents the balance between the uptake and the efflux of substrate (2). In D-cycloserine-resistant cells the net transport of D-alanine and glycine is higher than in sensitive cells. D-Cycloserine-resistant cells maintain a glycine gradient that is two to three times that observed in sensitive cells. This would indicate that in D-cycloserine-resistant cells either the glycine uptake rates is increased or the glycine efflux rate is decreased, causing the retention of a larger portion of the glycine. The kinetic parameters for the net transport of glycine indicate the presence of a high Km value for glycine transport induced by D-cycloserine which can be prevented by treatment with rifampin. Thus, protein synthesis is required for the alterations to occur.
LITERATURE CITED 1. Benveniste, R., and J. Davies. 1973. Mechanisms of antibiotic resistance. Annu. Rev. Biochem. 42:471506. 2. Christensen, H. N. 1975. Biological transport. W. A. Benjamin, Inc., Reading, Mass. 3. Clark, V. L., and F. E. Young. 1974. Active transport of n-alanine and related amino acids by whole cells of Bacillus subtilis. J. Bacteriol. 120:1085-1092. 4. Clark, V. L., and F. E. Young. 1977. Inducible resistance to D-cycloserine in Bacillus subtilis 168. Antimicrob. Agents Chemother. 11:871-876. 5. Franklin, T. J. 1973. Antibiotic transport in bacteria. Crit. Rev. Microbiol. 2:253-272. 6. Halpern, Y. S. 1974. Genetics of amino acid transport in bacteria. Annu. Rev. Genet. 8:103-133. 7. Leach, F. R., and E. E. Snell. 1960. The adsorption of glycine and alanine and their peptides by Lactobacillus casei. J. Biol. Chem. 235:3523-3531. 8. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 9. Robbins, J. C., and D. L. Oxender. 1973. Transport systems for alanine, serine, and glycine in Escherichia coli K-12. J. Bacteriol. 116:12-18. 10. Spizizen, J. 1958. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Natl. Acad. Sci. U.S.A. 44:1072-1078. 11. Wargel, R. J., C. A. Shadur, and F. C. Neuhaus. 1970. Mechanism of D-cycloserine action: transport systems for D-alanine, D-cycloserine, L-alanine, and glycine. J. Bacteriol. 103:778-788. 12. Wargel, R. J., C. A. Shadur, and F. C. Neuhaus. 1971. Mechanism of D-cycloserine action: transport mutants for o-alanine, D-cycloserine, and glycine. J. Bacteriol. 105:1028-1035.
Vol. 11, No. 5
Printed in U.S.A.
D-Cycloserine-Induced Alterations in the Transport of DAlanine and Glycine in Bacillus subtilis 168 VIRGINIA L. CLARK'
AND
FRANK E. YOUNG*
Department ofMicrobiology, University ofRochester, School ofMedicine and Dentistry, Rochester, New York 14642
Received for publication 22 December 1976
D-Alanine, i-alanine, and glycine transport was investigated in Bacillus subtilis 168 cells that were phenotypically resistant to D-cycloserine. These cells showed enhanced rates of uptake as compared with that observed in sensitive cells. The usual enhancement in D-alanine and glycine transport resulting from treatment of the cells with D-cycloserine could be prevented by the addition of rifampin. Kinetic analyses of the initial rate of glycine transport indicated an increase in the Vma; for transport in resistant cells, with no alteration in the Km for glycine. Investigations of the net transport of glycine revealed that resistant cells maintained a higher gradient of glycine than did sensitive cells. Kinetic analyses of the net transport of glycine suggested that a new system for the accumulation of glycine was present in D-cycloserine-resistant cells.
The antibiotic D-cycloserine is actively transported, in various bacterial species, by the same system(s) responsible for the transport of D-alanine, L-alanine, and glycine (3, 5-7, 9, 11). Resistance to i-cycloserine has been reported to result from a decreased accumulation of the antibiotic (1, 4, 5, 12). In Escherichia coli there is a concomitant decrease in the transport Dalanine, L-alanine, and glycine in resistant cells (5, 12). We have reported an inducible icycloserine resistance in Bacillus subtilis 168 (4) that is also a result of a decreased accumulation of the antibiotic. In this paper we will present evidence that D-cycloserine-resistant cells of B. subtilis do not transport i-alanine, Lalanine, and glycine at reduced rates. In fact, the transport of n-alanine and glycine is enhanced by treatment of the cell with D-cycloserine. MATERIALS AND METHODS Materials. The L_[U-_4C]alanine (specific activity, 153 mCi/mmol), L_[U-14C]proline (specific activity, 233 mCi/mmol), and Omnifluor were purchased from New England Nuclear; the D[U-14C]alanine (specific activity, 37 mCi/mmol) was obtained from Amersham/Searle Corp. Rifampin and D-alanine were purchased from Sigma Chemical Co., and acidhydrolyzed casein was from Nutritional Biochemicals Corp. All other chemicals were of reagent-grade purity. Strains and growth conditions. The strains of B. subtilis 168 and the growth conditions used were described previously (4). X Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
Measurement of transport. The preparation of cells for transport studies and the procedures for analysis of transport were similar to those in the accompanying paper (4). Protein determination. Protein was determined by the method of Lowry et al. (8) for trichloroacetic acid precipitates, with bovine serum albumin as a standard. Determination of radioactivity. The radioactivity of nonaqueous samples was determined with use of a scintillation fluid consisting of 4 g of Omnifluor per liter of toluene. Calculation of intracellular substrate concentration. If it is assumed that B. subtilis protein constitutes 50% of the dry weight of the cell, and that the cell is 80% water throughout the growth cycle, then 1 mg of protein is equal to 2 mg of dry weight plus 8 mg of water. Therefore, since 1 mg of water is 1 .ld, 1 mg of cell protein is approximately equal to 8 p.l of water. To convert the uptake values that are expressed as nanomoles of substrate transported per milligram of protein to millimolar, divide by a factor of 8.
RESULTS Transport of D-alanine in D-cycloserine-resistant cells. Previous reports of D-cycloserine resistance in E. coli (12) indicated a reduction in the transport of the antibiotic as the mechanism of resistance. In this case there is a concomitant reduction in the transport of n-alanine, L-alanine, and glycine. We investigated the rate of transport of n-alanine and glycine in growing cells of B. subtilis treated with D-cycloserine (Fig. 1). Under conditions in which Dcycloserine is transported at only 10% of the original rate after 90 min of treatment (data not 877
878
ANTIMICtOB. AGZNTS CHMOTHtR.
CLARK AND YOUNG
4 w
w
I-4 I0
TIME (min) FIG. 1. Effect of D-cycloserine incubatioi s on the transport of D-alanine and glycine. 0.1 m AM Dcycloserine was added to growing BR151 cells a time, and the transport of D-alanine (0) and gily the (0) was measured in washed cell sampls a indicated times. The control uptake rate wasi that observed at zero time. The substrate concentrcztions were: 1 pM D-4CJalanine and 20 M[14CJglyci ,ne.
ycine
shown), the transport of D-alanine and glycine was substantially increased. The increase in alanine transport could also occur in the glucose-minimal salts medium used for the induction of -cycloserine resistance (Fig. 2). Uninduced cells were suspended in milimal salts with no addition or plus -cycloserine. The cells to which the antibiotic was added were initially slightly inhibited, but with time transport of alanine by these cells exceeded the level observed in untreated cells. This enhancement could be prevented by simultaneously treating the cells with rifampin (Fig. 2B). Induced cells accumulated D-alanine to a higher degree and showed no further enhancement in the presence of -cycloserine (Fig. 2C). Transport of glycine in D-cycloserine-resistant cells. We investigated the effects of -cycloserine incubation on the initial. rates of transport and on the net transport of -alanine, xalanine, glycine, and L-proline (Table 1). Transport. was investigated at two substrate concentrations, 1 and 20 MM, because of the two transport systems observed.in B. subtilis (3). IProline was included as a control substrate that
is not transported by the alanine systems. Incubation in D-cycloserine caused an increased ability by the cells t,o transport D- and L-alanine and glycine when initial rates were measured. When net transport was measured, however, enhancement was evident only with -alanine and glycine. The enhanced level of retention was especially pronounced with glycine. The effect of -cycloserine incubation on glycine transport was further investigated by determining the kinetic parameters for the initial rate of transport of glycine (Fig. 3). The initial rate of transport by induced cells had the same Km value for glycine, but a higher V.a. than was observed in uninduced cells. The net transport of glycine was investigated at various concentrations of glycine in uninduced and induced cells. The intracellular concentration of glycine was calculated from the values of nanomoles glycine transported per 15 min per milligram of protein as described in Materials and Methods and plotted versus the extracellular glycine concentration (Fig. 4). The gradient of glycine retained in induced cells was compared with that in uninduced cells (Fig. 4, insert). It can be seen that the increased retention of glycine by induced. cells was more pronounced at higher glycine concentrations. Although the Michaelis-Menten equation was devised to deal with initial rates of reaction, the use of the equation B.
A.
2o w
0.
0
IS
30
0
IS
30
0
Is
3o
TIME (min)
FIG. 2. Effect of D-cycloserine incubation on transport of D-alanine. (A) Uninduced RUB1402 cells were incubated in Spizizen minimal salts with no addition (-) or plus 0.1 pMD-cycloserine (0). (B) Uninduced RUB1402 cells were incubated in Spizizen minimal salts containing 5 pg ofrifampin per ml with no further addition (0) or plus 0.1 mM Dcycloserine (0). (C) RUB1402 cells induced by incubation in Spizizen minimal salts plus 01 mM Dcycloserine for 15 min were washed and incubated in Spizizen minimal salts with no addition (-) or plus 0.1 mM D-cycloserine (0). All samples had 1 M z [14C]alanine added at zero time. Uptake is expressed as nanomoles of D-alanine transported per milligram of protein.
V VOL. 1-ALANINE AND GLYCINE TRANSPORT IN B. SUBTILIS 11, 1977
879
TABLE 1. Enhanced uptake rates for D-alanine and glycine in D-cycloserine-resistant cellsa Uptake rate
Compound transported
Concn
(,M)
Initial rate n
Uninduced
Net transport
t
Induced
9 of unin-
duced
Uninduced
of unin-
Induced
duced
n-Alanine
1 0.770 1.03 134 5.44 6.19 114 20 8.67 9.73 112 46.2 49.2 106 L-Alanine 1 0.289 0.502 174 2.14 2.13 100 20 4.31 5.03 117 38.7 24.7 64 Glycine 1 0.521 0.712 137 1.77 2.32 131 20 4.64 8.74 188 6.62 240 15.9 L-Proline 1 0.349 0.384 110 2.71 2.09 77 20 1.32 0.632 48 16.2 11.6 72 a Cells were induced for D-cycloserine resistance as described in Materials and Methods. Initial rates of transport were measured for 30 s and net transport for 15 min as described in Materials and Methods. Rates are expressed as nanomoles transported per minute per milligram of protein and nanomoles transported per 15 minute per milligram of protein for initial rates and net transport, respectively.
0
)I
a
0
._
S
X
c
-
0
I
a
c
la
._
Cs
E0 ._
._
S
t
Cs
w
c
.2 U
c 10
.' 2%
0
a
.5
0
VDAM
S
2
Glycine]
FIG. 3. Lineweaver-Burk plot of initial rates of glycine transport in uninduced and induced cells. Initial rates of transport (30 s) ofglycine were determined in cells unexposed to D-cycloserine (0) and cells induced for 30 min in Spizizen minimal salts plus 0.1 mM D-cycloserine (0). The kinetic parameters were: K. = 25 MM glycine, VmaZ = 11 nmol of glycine transported per min per mg of protein for uninduced cells, and Km = 25 pMglycine, Vma, = 16 nmol ofglycine transported per min per mg ofprotein for induced cells.
for measuring transport under other than initial rate conditions has been recognized as providing useful information (2). When this equation was applied to the net transport of glycine (Fig. 5), it appeared that there were two Km values (4 and 25 ,uM glycine) for induced cells, rather than the one Km value (4 ,uM glycine) observed in uninduced cells.
DISCUSSION D-Cycloserine resistance in E. coli (5, 12) is accompanied by a loss of transport of the antibiotic and a concomitant loss of transport of nalanine and glycine. We have described an inducible resistance to n-cycloserine in B. subtilis
-o
a
1100
50
Extrocellular glycine
0
25
50
75
(IjM)
100
Extracellular glycine (pM) FIG. 4. Ratio of intracellular to extracellular gly-
cine in uninduced and induced cells. Strain BR151 was induced for D-cycloserine resistance as described in Materials and Methods. Net transport (15 min) of [14C]glycine was measured in uninduced (0) and induced (0) cells, and the uptake was converted from nanomoles per minute per milligram of protein to millimolar as described in Materials and Methods. The extracellular concentration of glycine was corrected for uptake, and the ratios of intracellular to extracellular ['4C]glycine were determined. Insert: Ratio of intracellular to extracellular glycine in induced cells was divided by that in uninduced cells.
(4) that also results from a decreased accumulation of the antibiotic. Under conditions in which D-cycloserine resistance in B. subtilis is induced, however, we found that the transport
880
CLARK AND YOUNG
/ EM
Glycine]
ANTIMICROB. AGENTS CHEMOTHER.
We propose that i-cycloserine induces a new transport system in B. subtilis 168. This new system transports D-alanine and glycine, with glycine the preferred solute. D-Cycloserine-resistant cells would maintain a higher gradient of glycine as a result of the synthesis of this system. We further postulate that this new transport system also transports D-cycloserine, but that the rate of uptake is less than the rate of efflux of the antibiotic. This would cause a net efflux of D-cycloserine, which would render the cells resistant to the antibiotic. These observations suggest that an analysis of the transport of each antimicrobial agent used in combination with t-cycloserine might reveal patterns of illicit transport that could produce inhibition of microbial growth at lower concentrations of antibiotic and thereby reduce toxicity.
FIG. 5. Lineweaver-Burk plot of the net transport ofglycine in uninduced and induced cells. Net transport (30 min) of glycine determined in cells unexposed to D-cycloserine (0) and in cells induced for 30 min in Spizizen minimal salts plus 0.1 mM D-cycloserine (0). The kinetic parameters were: Km = 4 uM glycine for uninduced cells and Km = 4 juM and Km = 25 pM for induced cells .
ACKNOWLEDGMENTS This work was supported by Public Health Service grants 5TI-GM-00592 from the National Institute of General Medical Science and 5ROI-AI-10141 from the National Institute of Allergy and Infectious Diseases.
of D-alanine, L-alanine, and glycine is not decreased. When initial rates of transport are measured at 1 ,uM concentrations of the substrate, the transport of D- and L-alanine and glycine is increased. This may be due to an artifactual example of antiport, such that the transport substrate is pulled into the cell by the efflux of D-cycloserine. When the kinetic parameters for the initial rate of transpoqrt of glycine are determined, the Km for glycine remains constant while the Vmax for transport increases. This would be consistant with an antiportdriven transport of glycine. Net transport represents the balance between the uptake and the efflux of substrate (2). In D-cycloserine-resistant cells the net transport of D-alanine and glycine is higher than in sensitive cells. D-Cycloserine-resistant cells maintain a glycine gradient that is two to three times that observed in sensitive cells. This would indicate that in D-cycloserine-resistant cells either the glycine uptake rates is increased or the glycine efflux rate is decreased, causing the retention of a larger portion of the glycine. The kinetic parameters for the net transport of glycine indicate the presence of a high Km value for glycine transport induced by D-cycloserine which can be prevented by treatment with rifampin. Thus, protein synthesis is required for the alterations to occur.
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