0021-9193/78/0133-0422$02.00/0 JOIJRNAI, OF BACTERIOLOGY, Jan. 1978, p. 422-423 Copyright © 1978 American Society for Microbiology
Vol. 133, No. 1 Printed in U.S.A.
Elevated Cyclic AMP Concentration in StreptomycinDependent Escherichia coli W. J. POLGLASE,* D. IWACHA, AND M. THOMSON Department of Biochemistry, University of British Columbia, Vancouver, British Columbia, Canada
Received for publication 2 August 1977
Streptomycin-dependent Escherichia coli B and K-12 cultures, which have relaxed catabolite repression when grown on glucose-salts medium, have an elevated concentration of cyclic AMP. Previous publications from this laboratory have demonstrated that catabolite repression is relaxed in streptomycin (SM)-dependent Escherichia coli mutants growing on glucose-salts medium containing excess antibiotic (4, 11). Since the degree of catabolite repression in E. coli is believed to be inversely proportional to the cellular content of cyclic AMP (cAMP) (10), it was of interest to determine the cAMP content of SM-dependent E. coli cultures. This report shows that these mutants produce sufficient cAMP to account for the observed relaxation of catabolite repression (4, 11). The SM-resistant and -dependent cultures used in this investigation have been characterized in earlier work (4, 11). Cultures were grown in minimal salts medium (5) supplemented as indicated. Growth was monitored by measuring optical densities at 420 nm with a Spectronic 20 spectrophotometer. Protein was determined by the method of Lowry et al. (9) and was found to be similar for all cultures (0.23 mg of protein per ml of culture at an optical density at 420 nm of 1. Plate counts of exponentially growing cells showed that, at an optical density at 420 nm of 1.0, E. coli B (wild type) had 5.8 x 108 cells per ml, whereas the SM-dependent mutant of E. coli B had 6.4 x 108 cells per ml, and the SMresistant mutant had 5.4 x 108 cells per ml. At an optical density at 420 nm of 1.0, E. coli K-12 (wild type) had 2.4 x 10' cells per ml, and the SM-dependent mutant of this strain had 1.6 x 109 cells per ml. For determination of intracellular and extracellular cAMP, samples (usually 5 ml of culture) were filtered rapidly on membrane cellulose ester filters (47-mm diameter, 0.45-,um pore size) (Millipore Corp., Bedford, Mass.) that had been boiled three times with distilled water. The filter and cells were either immediately extracted or were first washed with warm growth medium before extraction. The extraction was performned by heating the filter in a tube in a boiling-water
bath for 10 min with 2.5 ml of 0.1 N HCl. The acid extract was centrifuged (12,000 x g for 20 min). To the clear supernatant was added 0.2 ml of a 10% suspension of charcoal in 0.01 N HCl prepared as described by Epstein et al. (7). After the nucleotides were washed, they were extracted from the charcoal with 1 M ammonia in 50% ethanol as described by Epstein and coworkers (7). The extract and washings were centrifuged and filtered to remove charcoal. The eluates were then dried in a stream of air, and the resulting residues were dissolved in water for determination of cAMP. The cell filtrates were acidified to pH 4 with concentrated acetic acid, and samples were heated in tubes in a boilingwater bath for 10 min and then assayed to determine extracellular cAMP. Total cAMP was determined by acidifying and heating a sample of culture as described above for the determination of extracellular cAMP, followed by centrifugation and assay of the clear supernatant solution. cAMP was determined by the method of Gilman (8) modified by using binding protein isolated from beef kidney by the method of Cheung (3) and purified as described by Botsford (2). The standard binding reaction was carried out at 0°C (in triplicate) in a final volume of 200 pl that contained the sample and 0.8 pmol of [I:H]cAMP in 50 mM sodium acetate, pH 4. Reactions were initiated by addition of binding protein. At the end of 90 min, the reaction mixture was diluted with 1 ml of ice-cold phosphate buffer (0.02 M, pH 6). The diluted solution was quickly filtered through a membrane cellulose ester filter (25-mm diameter, 0.45-,Lm pore size) (Millipore Corp.). The filter was washed with 10 ml of buffer and 2 ml of ethanol. Filters were dried by incubating at 90°C for 30 min, and radioactivity was determined by conventional liquid scintillation counting techniques. The results shown in Tables 1 and 2 were obtained during the growth period in which the
422
NOTES
VOL. 33, 1978
TABLE 1. cAMP in E. coli B strains grown on glucose-salts mediuma cAMP (pmol/mg of protein)" Strain
Extracellular Intracellular 190 7.0 Wild type 11.0 330 SM resistant 800 34.0 SM dependent a Exponentially growing cultures were collected on membrane filters (Millipore Corp.). SM-resistant and SM-dependent cultures were grown on medium supplemented with 1 mg of dihydrostreptomycin sulfate per ml. Glucose was added to give an initial concentration of 0.5% (wt/vol). b Each result is an average of cAMP assays on six samples taken during logarithmic growth. TABLE 2. Total cAMP in exponential cultures of E. coli B and K-1? E. coli strain
Carbon source
Total cAMP (pmol/mg of protein)
B Wild type SM resistant
SM dependent
Glucose Glycerol Glucose Glycerol Glucose Glycerol
200 640 280 560 696 626
K-12 Wild type
Glucose 1,540 2,880 Glycerol Glucose SM dependent 4,400 a Exponentialy growing cultures were sampled in mid-log phase and assayed immediately for cAMP. SM-resistant and SM-dependent cultures were grown on medium supplemented with 1 mg of dihydrostreptomycin sulfate per ml. The carbon source (glucose or glycerol) was added to give an initial concentration of 0.5% (wt/vol).
relationship between growth and cAMP concentration was constant, i.e., during exponential growth. The cAMP content of SM-dependent E. coli B was more than four times that of the parent wild-type culture, whereas cAMP was only slightly elevated in the SM-resistant mutant. In experiments in which cells were washed, the intracellular cAMP content was in all cases decreased, but that of the SM-dependent strain remained proportionately greater than that of the other strains. Extracellular cAMP was also higher for SM-dependent E. coli B cultures. In confirmation of other reports (10), the ratio of extracellular to intracellular cAMP remained constant during exponential growth. The determination of total cAMP is, therefore, adequate for the purpose of comparison of strains.
423
The results given for total cAMP in Table 2 show that the cAMP level of SM-dependent E. coli B cultures was about the same with either glucose or glycerol as the carbon source. E. coli K-12 cultures contained considerably more cAMP than those of E. coli B (Table 2). Although the occurrence of higher levels of cAMP in SM-dependent E. coli is consistent with previous observations of derepressed catabolitesensitive enzymes in these mutants (4, 11), there is no adequate explanation at the molecular level for these results. The only molecular modification known to result from spontaneous mutation to SM-dependence is in the S-12 ribosomal protein (1), the strA gene product. However, pleiotropic effects have been observed repeatedly by investigators of streptomycin action (6). Not only is ribosomal function affected by the antibiotic, but membrane damage and many other effects have been reported (6). These observations might be explained if one postulated an additional function for the ribosomal S-12 protein (for example, in the cell membrane) so that in the SM-dependent mutant the rate of glucose transport was decreased, resulting in increased cAMP fornation. This proposal is currently being investigated. LITERATURE CITED 1. Birge, E. A., and C. G. Kurland. 1969. Altered ribosomal proteins in streptomycin-dependent Escherichia coli. Science 166:1282. 2. Botsford, J. L. 1975. Metabolism of cyclic adenosine 3',5'-monophosphate and induction by tryptophanase in Escherichia coli. J. Bacteriol. 124:380-390. 3. Cheung, Y. 1972. Cyclic 3',5'-nucleotide phosphodiesterase. Effect of binding protein on the hydrolysis of cyclic AMP. Biochem. Biophys. Res. Commun. 46:99-105. 4. Coukell, M. B., and W. J. Polglase. 1969. Relaxation of catabolite repression in streptomycin-dependent Escherichia coli. Biochem. J. 111:279-285. 5. Davis, B. D., and E. S. Mingioli. 1950. Mutants of Escherichia coli requiring methionine or vitamin B12. J. Bacteriol. 60:17-28. 6. Dubin, D. T., R. Hancock, and B. D. Davis. 1963. The sequence of some effects of streptomycin in Escherichia coli. Biochim. Biophys. Acta 74:476-489. 7. Epstein, W., L B. Rothman-Denes, and J. Hesse. 1975. Adenosine 3',5'-cyclic monophosphate as mediator of catabolite repression in Escherichia coli. Proc. Nati. Acad. Sci. U.S.A. 72:2300-2304. 8. Gilman, A. G. 1970. A protein binding assay for adenosine 3',5'-cyclic monophosphate. Proc. Natl. Acad. Sci. U.S.A. 67:305-312. 9. 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. 10. Pastan, I., and S. Adhya. 1976. Cyclic adenosine 5'monophosphate in Escherichia coli. Bacteriol. Rev. 40:527-551. 11. Whitlow, K. J., and W. J. Polglase. 1975. Regulation of acetohydroxy acid synthase in streptomycin-dependent Escherichia coli. J. Bacteriol. 121:9-12.
Vol. 133, No. 1 Printed in U.S.A.
Elevated Cyclic AMP Concentration in StreptomycinDependent Escherichia coli W. J. POLGLASE,* D. IWACHA, AND M. THOMSON Department of Biochemistry, University of British Columbia, Vancouver, British Columbia, Canada
Received for publication 2 August 1977
Streptomycin-dependent Escherichia coli B and K-12 cultures, which have relaxed catabolite repression when grown on glucose-salts medium, have an elevated concentration of cyclic AMP. Previous publications from this laboratory have demonstrated that catabolite repression is relaxed in streptomycin (SM)-dependent Escherichia coli mutants growing on glucose-salts medium containing excess antibiotic (4, 11). Since the degree of catabolite repression in E. coli is believed to be inversely proportional to the cellular content of cyclic AMP (cAMP) (10), it was of interest to determine the cAMP content of SM-dependent E. coli cultures. This report shows that these mutants produce sufficient cAMP to account for the observed relaxation of catabolite repression (4, 11). The SM-resistant and -dependent cultures used in this investigation have been characterized in earlier work (4, 11). Cultures were grown in minimal salts medium (5) supplemented as indicated. Growth was monitored by measuring optical densities at 420 nm with a Spectronic 20 spectrophotometer. Protein was determined by the method of Lowry et al. (9) and was found to be similar for all cultures (0.23 mg of protein per ml of culture at an optical density at 420 nm of 1. Plate counts of exponentially growing cells showed that, at an optical density at 420 nm of 1.0, E. coli B (wild type) had 5.8 x 108 cells per ml, whereas the SM-dependent mutant of E. coli B had 6.4 x 108 cells per ml, and the SMresistant mutant had 5.4 x 108 cells per ml. At an optical density at 420 nm of 1.0, E. coli K-12 (wild type) had 2.4 x 10' cells per ml, and the SM-dependent mutant of this strain had 1.6 x 109 cells per ml. For determination of intracellular and extracellular cAMP, samples (usually 5 ml of culture) were filtered rapidly on membrane cellulose ester filters (47-mm diameter, 0.45-,um pore size) (Millipore Corp., Bedford, Mass.) that had been boiled three times with distilled water. The filter and cells were either immediately extracted or were first washed with warm growth medium before extraction. The extraction was performned by heating the filter in a tube in a boiling-water
bath for 10 min with 2.5 ml of 0.1 N HCl. The acid extract was centrifuged (12,000 x g for 20 min). To the clear supernatant was added 0.2 ml of a 10% suspension of charcoal in 0.01 N HCl prepared as described by Epstein et al. (7). After the nucleotides were washed, they were extracted from the charcoal with 1 M ammonia in 50% ethanol as described by Epstein and coworkers (7). The extract and washings were centrifuged and filtered to remove charcoal. The eluates were then dried in a stream of air, and the resulting residues were dissolved in water for determination of cAMP. The cell filtrates were acidified to pH 4 with concentrated acetic acid, and samples were heated in tubes in a boilingwater bath for 10 min and then assayed to determine extracellular cAMP. Total cAMP was determined by acidifying and heating a sample of culture as described above for the determination of extracellular cAMP, followed by centrifugation and assay of the clear supernatant solution. cAMP was determined by the method of Gilman (8) modified by using binding protein isolated from beef kidney by the method of Cheung (3) and purified as described by Botsford (2). The standard binding reaction was carried out at 0°C (in triplicate) in a final volume of 200 pl that contained the sample and 0.8 pmol of [I:H]cAMP in 50 mM sodium acetate, pH 4. Reactions were initiated by addition of binding protein. At the end of 90 min, the reaction mixture was diluted with 1 ml of ice-cold phosphate buffer (0.02 M, pH 6). The diluted solution was quickly filtered through a membrane cellulose ester filter (25-mm diameter, 0.45-,Lm pore size) (Millipore Corp.). The filter was washed with 10 ml of buffer and 2 ml of ethanol. Filters were dried by incubating at 90°C for 30 min, and radioactivity was determined by conventional liquid scintillation counting techniques. The results shown in Tables 1 and 2 were obtained during the growth period in which the
422
NOTES
VOL. 33, 1978
TABLE 1. cAMP in E. coli B strains grown on glucose-salts mediuma cAMP (pmol/mg of protein)" Strain
Extracellular Intracellular 190 7.0 Wild type 11.0 330 SM resistant 800 34.0 SM dependent a Exponentially growing cultures were collected on membrane filters (Millipore Corp.). SM-resistant and SM-dependent cultures were grown on medium supplemented with 1 mg of dihydrostreptomycin sulfate per ml. Glucose was added to give an initial concentration of 0.5% (wt/vol). b Each result is an average of cAMP assays on six samples taken during logarithmic growth. TABLE 2. Total cAMP in exponential cultures of E. coli B and K-1? E. coli strain
Carbon source
Total cAMP (pmol/mg of protein)
B Wild type SM resistant
SM dependent
Glucose Glycerol Glucose Glycerol Glucose Glycerol
200 640 280 560 696 626
K-12 Wild type
Glucose 1,540 2,880 Glycerol Glucose SM dependent 4,400 a Exponentialy growing cultures were sampled in mid-log phase and assayed immediately for cAMP. SM-resistant and SM-dependent cultures were grown on medium supplemented with 1 mg of dihydrostreptomycin sulfate per ml. The carbon source (glucose or glycerol) was added to give an initial concentration of 0.5% (wt/vol).
relationship between growth and cAMP concentration was constant, i.e., during exponential growth. The cAMP content of SM-dependent E. coli B was more than four times that of the parent wild-type culture, whereas cAMP was only slightly elevated in the SM-resistant mutant. In experiments in which cells were washed, the intracellular cAMP content was in all cases decreased, but that of the SM-dependent strain remained proportionately greater than that of the other strains. Extracellular cAMP was also higher for SM-dependent E. coli B cultures. In confirmation of other reports (10), the ratio of extracellular to intracellular cAMP remained constant during exponential growth. The determination of total cAMP is, therefore, adequate for the purpose of comparison of strains.
423
The results given for total cAMP in Table 2 show that the cAMP level of SM-dependent E. coli B cultures was about the same with either glucose or glycerol as the carbon source. E. coli K-12 cultures contained considerably more cAMP than those of E. coli B (Table 2). Although the occurrence of higher levels of cAMP in SM-dependent E. coli is consistent with previous observations of derepressed catabolitesensitive enzymes in these mutants (4, 11), there is no adequate explanation at the molecular level for these results. The only molecular modification known to result from spontaneous mutation to SM-dependence is in the S-12 ribosomal protein (1), the strA gene product. However, pleiotropic effects have been observed repeatedly by investigators of streptomycin action (6). Not only is ribosomal function affected by the antibiotic, but membrane damage and many other effects have been reported (6). These observations might be explained if one postulated an additional function for the ribosomal S-12 protein (for example, in the cell membrane) so that in the SM-dependent mutant the rate of glucose transport was decreased, resulting in increased cAMP fornation. This proposal is currently being investigated. LITERATURE CITED 1. Birge, E. A., and C. G. Kurland. 1969. Altered ribosomal proteins in streptomycin-dependent Escherichia coli. Science 166:1282. 2. Botsford, J. L. 1975. Metabolism of cyclic adenosine 3',5'-monophosphate and induction by tryptophanase in Escherichia coli. J. Bacteriol. 124:380-390. 3. Cheung, Y. 1972. Cyclic 3',5'-nucleotide phosphodiesterase. Effect of binding protein on the hydrolysis of cyclic AMP. Biochem. Biophys. Res. Commun. 46:99-105. 4. Coukell, M. B., and W. J. Polglase. 1969. Relaxation of catabolite repression in streptomycin-dependent Escherichia coli. Biochem. J. 111:279-285. 5. Davis, B. D., and E. S. Mingioli. 1950. Mutants of Escherichia coli requiring methionine or vitamin B12. J. Bacteriol. 60:17-28. 6. Dubin, D. T., R. Hancock, and B. D. Davis. 1963. The sequence of some effects of streptomycin in Escherichia coli. Biochim. Biophys. Acta 74:476-489. 7. Epstein, W., L B. Rothman-Denes, and J. Hesse. 1975. Adenosine 3',5'-cyclic monophosphate as mediator of catabolite repression in Escherichia coli. Proc. Nati. Acad. Sci. U.S.A. 72:2300-2304. 8. Gilman, A. G. 1970. A protein binding assay for adenosine 3',5'-cyclic monophosphate. Proc. Natl. Acad. Sci. U.S.A. 67:305-312. 9. 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. 10. Pastan, I., and S. Adhya. 1976. Cyclic adenosine 5'monophosphate in Escherichia coli. Bacteriol. Rev. 40:527-551. 11. Whitlow, K. J., and W. J. Polglase. 1975. Regulation of acetohydroxy acid synthase in streptomycin-dependent Escherichia coli. J. Bacteriol. 121:9-12.
