JouRNAL OF BACTERIOLOGY, Dec. 1975, p. 1269-1272 Copyright 0 1975 American Society for Microbiology
Vol. 124, No. 3 Printed in U.S.A.
Regulation of the hut Operons of Salmonella typhimurium and Klebsiella aerogenes by the Heterologous hut Repressors STANTON L. GERSON ANJD BORIS MAGASANIK* Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received for publication 11 August 1975
In merodiploid strains of Klebsiella aerogenes with chromosomal hut genes of K. aerogenes and episomal hut genes of Salmonella typhimurium, the repressor of either species can regulate the hut operons of the other species. The repression exerted by the homologous repressor on the left-hand hut operon is, in both organisms, stronger than that exerted by the heterologous repressor.
In the preceding paper (5) we showed that the hut (histidine utilization) operons of Salmonella typhimurium introduced on an episome into a strain of Klebsiella aerogenes whose hut genes have been deleted respond normally to regulation by the product of their repressor gene hutC. We shall now consider whether in merodiploid strains of K. aerogenes with chromosomal hut genes of K. aerogenes and episomal hut genes of S. typhimurium the repressors coded by the hutC gene of one species can regulate the expression of the hut genes of the other species. This study was prompted by the observation of marked differences in the expression of the hut operons of the two species, even when they are present in the same cytoplasm, i.e., that of K. aerogenes (6). The levels of the enzymes determined by the hut genes of K. aerogenes are 4- to 10-fold higher than those determined by the hut genes of S. typhimurium (6); induction brings about a more than 10-fold increase in the level of formiminoglutamate (FGA) hydrolase of K. aerogenes but only a 2- to 3-fold increase in the level of the enzyme of S. typhimurium (1, 6). (Our attempt to discover in extracts of K. aerogenes material that, like the hut repressor of S. typhimurium, could bind to hut deoxyribonucleic acid of S. typhimurium was unsuccessful [D. Hagen, S. L. Gerson, and B. Magasanik, unpublished data].) We find now that the repressor of either species can regulate the hut operons of the other species in the intact cell. However, we detect some differences between intra- and interspecies-specific regulation. MATERIALS AND METHODS Chemicals. The chemicals used were described
previously (5, 6). The hut and gal mutants were isolated and the merodiploid strains were constructed
by previously described methods (2, 6). Cultivation of bacteria. A 0.05-ml portion of a culture grown overnight at 37 C in LB broth was used as inoculum, in a test tube (2.5 by 20 cm), for 5 ml of minimal medium containing 0.4% citrate as the carbon source (6). The tubes were then shaken, slanted, at 37 C. Growth was allowed to continue until a cell density of 120 + 10 Klett units was reached. The cultures were then chilled on ice, and the cells were collected by centrifugation, washed with 2.5 ml of buffer (potassium phosphate [pH 7.4, 0.01 M] containing 5 mM 2-mercaptoethanol), and finally resuspended in 0.5 ml of the buffer. The suspensions were kept at 4 C; they were used for FGA hydrolase assay within 24 h and for histidase and urocanase assays within 48 h. Enzyme assays. The assay of histidase, urocanase, and FGA hydrolase in hexadecyl trimethylammonium bromide-treated cell suspensions was by previously described procedures (8, 9). Protein was determined by a modification of the method of Lowry et al. (4), with bovine serum albumin as the standard. Enzyme specific activities are given in nanomoles of substrate consumed or product formed per minute per
milligram of protein. The values given in the tables are the average of two or more assays on cells of the same strain grown as separate cultures in media of the same composition.
RESULTS We chose as our measure of operon expression in the absence of repressor the enzyme levels in mutants that are constitutive because of a mutation in the hutC gene. We previously found that this is a more reliable indication of derepression than the determination of enzyme levels in induced cells with a functioning hutC gene (3). This is probably due to the difficulty with which the true inducer, urocanate, enters the cell (1) and to an incompletely understood repressive effect exerted by the physiological
previously (1, 5, 6). Bacterial strains. The bacterial strains used are listed in Table 1. Some of the strains were described 1269
1270
J. BACTERIOL.
GERSON AND MAGASANIK
is also strong repression of the FGA hydrolase from K. aerogenes; the level of the enzyme is lowered approximately 25-fold. On the other hand, there is much less repression of the FGA hydrolase from S. typhimurium; the level of this enzyme is lowered less than 2.5-fold. The effects of the two repressors on the FGA hydrolases of the two organisms are summarized in Table 5. It is apparent that the enzyme of K. aerogenes is more readily repressed than that of S. typhimurium by either repressor. In addition, the homologous repressor appears, in each case, to be more effective than the heterol-
inducer histidine, but which is not attributable to hutC (1). The constitutive levels of the enzymes coded by the chromosomal hutK genes of K. aerogenes and the episomal hut, genes of S. typhimurium are shown in Table 2. It is apparent that the levels of K. aerogenes enzymes are much higher than those of the S. typhimurium enzymes. The effects of the hut repressor from S. typhimurium on the hut operons of both species are shown in Table 3. The enzymes of the right-hand operon, histidase and urocanase, are almost completely repressed regardless of origin; their levels are lowered at least 50-fold. The repression of FGA hydrolase is less complete; the level of the enzyme from S. typhimurium is lowered approximately fourfold, and that of the K. aerogenes enzyme is lowered approximately eightfold. The effects of the hut repressor from K. aerogenes are shown in Table 4. In this case too there is essentially complete repression of histidase and urocanase, regardless of origin. There
ogous repressor.
DISCUSSION Our results show that homologous and heterologous repressors are equally potent in their ability to repress histidase and urocanase, whose structural genes comprise the right-hand hut operon. The right-hand hut operators of both K. aerogenes and S. typhimurium are very sensitive; the basal enzyme levels in uninduced cells with a nkrmal repressor (hutC+) are too TABLE 1. Bacterial strains low to be measured with confidence. The equal repression exerted by homologous and heteroloStraina Genotype" gous repressor, therefore, does not prove that they are equally effective, but rather that they Wild type MK1 MK6 hut*511, F' (gal+,hut,++,bio+) are present in amounts adequate for complete hutKC515 MK53 of this operon. repression MK1O1 hut*511, F' (gal+,hut,C7,bio+) whose structural gene beFGA hydrolase, MK2438 hut*511, F' (gal',hutsC7,hutsU25, bio +) longs to the left-hand hut operon, is not as MK5325 gal-911, hutKC515 MK5343 gal-911, hutKC515 (F' gal+,hut,G1O1,bio+) strongly repressed as histidase and urocanase. MK8331 gal-911, hutKG1007 (F' gal+,hut,C7,bio+) In the case of K. aerogenes, the homologous MK8405 gal-912, hutKH1003 repressor reduces the level of this enzyme by a MK8425 gal-913, hutKC515,hutKH1003 factor of 25, and in the case of S. typhimurium MK8443 gal-912, hut KH1003 (F' gal+,hut,C7,hut,U25, bio+) the homologous repressor reduces its level only MK8452 gal-913, hutKC515,hutsHl003, (F' gal, + ,hut,U165, by a factor of 5. It is likely that the left-hand bio+) operators do not bind the repressor as tightly as a All strains are K. aerogenes. do the right-hand operators; indeed, an altered shut*511 represents a deletion of gal, chIR, hut, and bio; repressor resulting from a mutation in hutC, the hutK represents hut genes of K. aerogenes; and huts repre- structural gene for the repressor, was more sents hut genes of S. typhimurium. hutH, hutU, and hutG are the structural genes for histidase, urocanase, and FGA effective in reducing the level of FGA hydrolase than normal repressor (3). hydrolase, respectively. TABLE 2. Constitutive expression of hut genes from S. typhimurium and from K. aerogenes in the cytoplasm of K. aerogenes Enzyme sp acta
hut gene origin
Strain H
MK101 MK2438 MK53 MK8425 MK5325 a
I
U
S t S t
S t None
K.a
K.a
None K a
K a K a
G
S S K K K
t t a a a
Histidase
Urocanase
FGA hydrolase
140 120 520 8 ND
17 1 ND 74 ND
320 NDb 2,300 ND 2,500
Nanomoles of product formed per minute per milligram of protein. Not determined.
VOL. 124, 1975
REGULATION OF hut OPERONS
1271
TABLE 3. Effect of hut repressor from S. typhimurium on the expression of hut genes from S. typhimurium and K. aerogenes Enzyme sp acta
hut gene origin
Strain
MK6 MK8452 MK5343
H
U
G
Histidase
Urocanase
FGA hydrolase
S-t S*t Both
S-t K*a Both
S-t Both K*a
3 4 ND
1 2 ND
70 NDb 310
Nanomoles of product formed per minute per milligram of protein. b Not determined.
a
TABLE 4. Effect of hut repressor from K. aerogenes on the expression of hut genes from K. aerogenes and S. typhimurium Enzyme sp act"
hut gene origin
Strain
MK1 MK8405 MK8443 MK8331
H
U
G
Histidase
Urocanase
FGA hydrolase
Ka None S t Both
K-a K*a K*a Both
K-a Ka Both S t
6 4 5 ND
1 2 2 ND
90 NDb ND 140
a Nanomoles
of product formed per minute per milligram of protein. & Not determined. TABiL 5. Comparison of repressor effects on FGA account for our failure to detect the hut rehydrolase pressor in cell extracts of K. aerogenes by its
ability to bind hut-specific deoxyribonucleic acid from S. typhimurium. In conclusion, we must stress the great simi(%)a Enzyme Repressor larity of hut repressors of the two organisms, as S t 22 S-t revealed by the heterologous interactions. This Ka 4 K-a similarity is perhaps unexpected since the two S-t K-a 13 enteric organisms are rather distantly related Ka 43 S-t by deoxyribonucleic acid composition: 58 and aThe specific activity of FGA hydrolase in strain 51% guanine plus cytosine for K. aerogenes and MK101 (Table 2) for the enzyme from S. S. typhimurium, respectively (7). Origin
Activity remaining
typhimurium and in strain MK5325 (Table 2) for the enzyme from K. aerogenes was set at 100.
We now find that the left-hand operons of both organisms respond to heterologous repressors. The repressor of S. typhimurium represses the FGA hydrolase of K. aerogenes more strongly than its own. Conversely, the repressor of K. aerogenes repressers the FGA hydrolase of S. typhimurium less strongly than its own (Table 5). These observations indicate that the stronger homologous repression in K. aerogenes, when compared with that in S. typhimurium, reflects a greater sensitivity of the K. aerogenes operator. We also find that the repression exerted by the homologous repressor on the FGA hydrolase of either organism is somewhat stronger than that exerted by the heterologous repressor (Table 5), suggesting that the two repressors are not structurally identical. This difference may
ACKNOWLEDGMENTS We are indebted to D. C. Hagen for helpful discussion. This study was supported by Public Health Service research grants GM 07446 from the National Institute of General Medical Sciences and AM 13894 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, and by grant GB 03398 from the National Science Foundation.
LITERATURE CITED 1. Brill, W. J., and B. Magasanik. 1969. Genetic and metabolic control of histidase and urocanase in Salmonella typhimurium strain 15-59. J. Biol. Chem. 244:5392-5402. 2. Goldberg, R. B., and B. Magasanik. 1975. Gene order of the histidine utilization (hut) operons in Klebsiella aerogenes. J. Bacteriol. 122:1025-1031. 3. Hagen, D. C., S. L. Gerson, and B. Magasanik. 1975. Isolation of super-repressor mutants in the histidine utilization system of Salmonella typhimurium. J. Bacteriol. 121:583-593. 4. Layne, E. 1957. Spectrophotometric and turbidimetric methods for measuring proteins, p. 447-454. In S. P.
1272
GERSON AND MAGASANIK
Colowick and N. 0. Kaplan (ed.), Methods in enzymolvol. 3. Academic Press Inc., New York. 5. Parada, J. L., and B. Magasanik. 1975. Expression of the hut operons of Salmonella typhimarium in Klebsiella aerogenes and Escherichia coli. J. Bacteriol. 124:1263-1268. 6. Prival, M. J., and B. Magasanik. 1971. Resistance to catabolite repression of histidase and proline oxidase during nitrogen-limited growth of Klebsiella aerogenes. J. Biol. Chem. 246:6288-6296. 7. Shapiro, M. 1970. Distribution of purines and pyrimidines in deoxyribonucleic acid, p. M80-M1l1. In H. A. Sober ogy,
J. BACTERIOL. (ed.), Handbook of biochemistry, Chemical Rubber Co., Cleveland. 8. Smith, G. R., Y. S. Halpern, and B. Magasanik. 1971. Genetic and metabolic control of enzymes responsible for histidine degradation in Salmonella typhimurium. 4-Imidazole-5-propionate amidohydrolase and N-formimino-L-glutamate formiminohydrolase. J. Biol. Chem. 246:3320-3329. 9. Smith, G. R., and B. Magasanik. 1971. Nature and self-regulated synthesis of the repressor of the hut operons in Salmonella typhimurium. Proc. Natl. Acad. Sci. U.S.A. 68:1493-1497.
Vol. 124, No. 3 Printed in U.S.A.
Regulation of the hut Operons of Salmonella typhimurium and Klebsiella aerogenes by the Heterologous hut Repressors STANTON L. GERSON ANJD BORIS MAGASANIK* Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received for publication 11 August 1975
In merodiploid strains of Klebsiella aerogenes with chromosomal hut genes of K. aerogenes and episomal hut genes of Salmonella typhimurium, the repressor of either species can regulate the hut operons of the other species. The repression exerted by the homologous repressor on the left-hand hut operon is, in both organisms, stronger than that exerted by the heterologous repressor.
In the preceding paper (5) we showed that the hut (histidine utilization) operons of Salmonella typhimurium introduced on an episome into a strain of Klebsiella aerogenes whose hut genes have been deleted respond normally to regulation by the product of their repressor gene hutC. We shall now consider whether in merodiploid strains of K. aerogenes with chromosomal hut genes of K. aerogenes and episomal hut genes of S. typhimurium the repressors coded by the hutC gene of one species can regulate the expression of the hut genes of the other species. This study was prompted by the observation of marked differences in the expression of the hut operons of the two species, even when they are present in the same cytoplasm, i.e., that of K. aerogenes (6). The levels of the enzymes determined by the hut genes of K. aerogenes are 4- to 10-fold higher than those determined by the hut genes of S. typhimurium (6); induction brings about a more than 10-fold increase in the level of formiminoglutamate (FGA) hydrolase of K. aerogenes but only a 2- to 3-fold increase in the level of the enzyme of S. typhimurium (1, 6). (Our attempt to discover in extracts of K. aerogenes material that, like the hut repressor of S. typhimurium, could bind to hut deoxyribonucleic acid of S. typhimurium was unsuccessful [D. Hagen, S. L. Gerson, and B. Magasanik, unpublished data].) We find now that the repressor of either species can regulate the hut operons of the other species in the intact cell. However, we detect some differences between intra- and interspecies-specific regulation. MATERIALS AND METHODS Chemicals. The chemicals used were described
previously (5, 6). The hut and gal mutants were isolated and the merodiploid strains were constructed
by previously described methods (2, 6). Cultivation of bacteria. A 0.05-ml portion of a culture grown overnight at 37 C in LB broth was used as inoculum, in a test tube (2.5 by 20 cm), for 5 ml of minimal medium containing 0.4% citrate as the carbon source (6). The tubes were then shaken, slanted, at 37 C. Growth was allowed to continue until a cell density of 120 + 10 Klett units was reached. The cultures were then chilled on ice, and the cells were collected by centrifugation, washed with 2.5 ml of buffer (potassium phosphate [pH 7.4, 0.01 M] containing 5 mM 2-mercaptoethanol), and finally resuspended in 0.5 ml of the buffer. The suspensions were kept at 4 C; they were used for FGA hydrolase assay within 24 h and for histidase and urocanase assays within 48 h. Enzyme assays. The assay of histidase, urocanase, and FGA hydrolase in hexadecyl trimethylammonium bromide-treated cell suspensions was by previously described procedures (8, 9). Protein was determined by a modification of the method of Lowry et al. (4), with bovine serum albumin as the standard. Enzyme specific activities are given in nanomoles of substrate consumed or product formed per minute per
milligram of protein. The values given in the tables are the average of two or more assays on cells of the same strain grown as separate cultures in media of the same composition.
RESULTS We chose as our measure of operon expression in the absence of repressor the enzyme levels in mutants that are constitutive because of a mutation in the hutC gene. We previously found that this is a more reliable indication of derepression than the determination of enzyme levels in induced cells with a functioning hutC gene (3). This is probably due to the difficulty with which the true inducer, urocanate, enters the cell (1) and to an incompletely understood repressive effect exerted by the physiological
previously (1, 5, 6). Bacterial strains. The bacterial strains used are listed in Table 1. Some of the strains were described 1269
1270
J. BACTERIOL.
GERSON AND MAGASANIK
is also strong repression of the FGA hydrolase from K. aerogenes; the level of the enzyme is lowered approximately 25-fold. On the other hand, there is much less repression of the FGA hydrolase from S. typhimurium; the level of this enzyme is lowered less than 2.5-fold. The effects of the two repressors on the FGA hydrolases of the two organisms are summarized in Table 5. It is apparent that the enzyme of K. aerogenes is more readily repressed than that of S. typhimurium by either repressor. In addition, the homologous repressor appears, in each case, to be more effective than the heterol-
inducer histidine, but which is not attributable to hutC (1). The constitutive levels of the enzymes coded by the chromosomal hutK genes of K. aerogenes and the episomal hut, genes of S. typhimurium are shown in Table 2. It is apparent that the levels of K. aerogenes enzymes are much higher than those of the S. typhimurium enzymes. The effects of the hut repressor from S. typhimurium on the hut operons of both species are shown in Table 3. The enzymes of the right-hand operon, histidase and urocanase, are almost completely repressed regardless of origin; their levels are lowered at least 50-fold. The repression of FGA hydrolase is less complete; the level of the enzyme from S. typhimurium is lowered approximately fourfold, and that of the K. aerogenes enzyme is lowered approximately eightfold. The effects of the hut repressor from K. aerogenes are shown in Table 4. In this case too there is essentially complete repression of histidase and urocanase, regardless of origin. There
ogous repressor.
DISCUSSION Our results show that homologous and heterologous repressors are equally potent in their ability to repress histidase and urocanase, whose structural genes comprise the right-hand hut operon. The right-hand hut operators of both K. aerogenes and S. typhimurium are very sensitive; the basal enzyme levels in uninduced cells with a nkrmal repressor (hutC+) are too TABLE 1. Bacterial strains low to be measured with confidence. The equal repression exerted by homologous and heteroloStraina Genotype" gous repressor, therefore, does not prove that they are equally effective, but rather that they Wild type MK1 MK6 hut*511, F' (gal+,hut,++,bio+) are present in amounts adequate for complete hutKC515 MK53 of this operon. repression MK1O1 hut*511, F' (gal+,hut,C7,bio+) whose structural gene beFGA hydrolase, MK2438 hut*511, F' (gal',hutsC7,hutsU25, bio +) longs to the left-hand hut operon, is not as MK5325 gal-911, hutKC515 MK5343 gal-911, hutKC515 (F' gal+,hut,G1O1,bio+) strongly repressed as histidase and urocanase. MK8331 gal-911, hutKG1007 (F' gal+,hut,C7,bio+) In the case of K. aerogenes, the homologous MK8405 gal-912, hutKH1003 repressor reduces the level of this enzyme by a MK8425 gal-913, hutKC515,hutKH1003 factor of 25, and in the case of S. typhimurium MK8443 gal-912, hut KH1003 (F' gal+,hut,C7,hut,U25, bio+) the homologous repressor reduces its level only MK8452 gal-913, hutKC515,hutsHl003, (F' gal, + ,hut,U165, by a factor of 5. It is likely that the left-hand bio+) operators do not bind the repressor as tightly as a All strains are K. aerogenes. do the right-hand operators; indeed, an altered shut*511 represents a deletion of gal, chIR, hut, and bio; repressor resulting from a mutation in hutC, the hutK represents hut genes of K. aerogenes; and huts repre- structural gene for the repressor, was more sents hut genes of S. typhimurium. hutH, hutU, and hutG are the structural genes for histidase, urocanase, and FGA effective in reducing the level of FGA hydrolase than normal repressor (3). hydrolase, respectively. TABLE 2. Constitutive expression of hut genes from S. typhimurium and from K. aerogenes in the cytoplasm of K. aerogenes Enzyme sp acta
hut gene origin
Strain H
MK101 MK2438 MK53 MK8425 MK5325 a
I
U
S t S t
S t None
K.a
K.a
None K a
K a K a
G
S S K K K
t t a a a
Histidase
Urocanase
FGA hydrolase
140 120 520 8 ND
17 1 ND 74 ND
320 NDb 2,300 ND 2,500
Nanomoles of product formed per minute per milligram of protein. Not determined.
VOL. 124, 1975
REGULATION OF hut OPERONS
1271
TABLE 3. Effect of hut repressor from S. typhimurium on the expression of hut genes from S. typhimurium and K. aerogenes Enzyme sp acta
hut gene origin
Strain
MK6 MK8452 MK5343
H
U
G
Histidase
Urocanase
FGA hydrolase
S-t S*t Both
S-t K*a Both
S-t Both K*a
3 4 ND
1 2 ND
70 NDb 310
Nanomoles of product formed per minute per milligram of protein. b Not determined.
a
TABLE 4. Effect of hut repressor from K. aerogenes on the expression of hut genes from K. aerogenes and S. typhimurium Enzyme sp act"
hut gene origin
Strain
MK1 MK8405 MK8443 MK8331
H
U
G
Histidase
Urocanase
FGA hydrolase
Ka None S t Both
K-a K*a K*a Both
K-a Ka Both S t
6 4 5 ND
1 2 2 ND
90 NDb ND 140
a Nanomoles
of product formed per minute per milligram of protein. & Not determined. TABiL 5. Comparison of repressor effects on FGA account for our failure to detect the hut rehydrolase pressor in cell extracts of K. aerogenes by its
ability to bind hut-specific deoxyribonucleic acid from S. typhimurium. In conclusion, we must stress the great simi(%)a Enzyme Repressor larity of hut repressors of the two organisms, as S t 22 S-t revealed by the heterologous interactions. This Ka 4 K-a similarity is perhaps unexpected since the two S-t K-a 13 enteric organisms are rather distantly related Ka 43 S-t by deoxyribonucleic acid composition: 58 and aThe specific activity of FGA hydrolase in strain 51% guanine plus cytosine for K. aerogenes and MK101 (Table 2) for the enzyme from S. S. typhimurium, respectively (7). Origin
Activity remaining
typhimurium and in strain MK5325 (Table 2) for the enzyme from K. aerogenes was set at 100.
We now find that the left-hand operons of both organisms respond to heterologous repressors. The repressor of S. typhimurium represses the FGA hydrolase of K. aerogenes more strongly than its own. Conversely, the repressor of K. aerogenes repressers the FGA hydrolase of S. typhimurium less strongly than its own (Table 5). These observations indicate that the stronger homologous repression in K. aerogenes, when compared with that in S. typhimurium, reflects a greater sensitivity of the K. aerogenes operator. We also find that the repression exerted by the homologous repressor on the FGA hydrolase of either organism is somewhat stronger than that exerted by the heterologous repressor (Table 5), suggesting that the two repressors are not structurally identical. This difference may
ACKNOWLEDGMENTS We are indebted to D. C. Hagen for helpful discussion. This study was supported by Public Health Service research grants GM 07446 from the National Institute of General Medical Sciences and AM 13894 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, and by grant GB 03398 from the National Science Foundation.
LITERATURE CITED 1. Brill, W. J., and B. Magasanik. 1969. Genetic and metabolic control of histidase and urocanase in Salmonella typhimurium strain 15-59. J. Biol. Chem. 244:5392-5402. 2. Goldberg, R. B., and B. Magasanik. 1975. Gene order of the histidine utilization (hut) operons in Klebsiella aerogenes. J. Bacteriol. 122:1025-1031. 3. Hagen, D. C., S. L. Gerson, and B. Magasanik. 1975. Isolation of super-repressor mutants in the histidine utilization system of Salmonella typhimurium. J. Bacteriol. 121:583-593. 4. Layne, E. 1957. Spectrophotometric and turbidimetric methods for measuring proteins, p. 447-454. In S. P.
1272
GERSON AND MAGASANIK
Colowick and N. 0. Kaplan (ed.), Methods in enzymolvol. 3. Academic Press Inc., New York. 5. Parada, J. L., and B. Magasanik. 1975. Expression of the hut operons of Salmonella typhimarium in Klebsiella aerogenes and Escherichia coli. J. Bacteriol. 124:1263-1268. 6. Prival, M. J., and B. Magasanik. 1971. Resistance to catabolite repression of histidase and proline oxidase during nitrogen-limited growth of Klebsiella aerogenes. J. Biol. Chem. 246:6288-6296. 7. Shapiro, M. 1970. Distribution of purines and pyrimidines in deoxyribonucleic acid, p. M80-M1l1. In H. A. Sober ogy,
J. BACTERIOL. (ed.), Handbook of biochemistry, Chemical Rubber Co., Cleveland. 8. Smith, G. R., Y. S. Halpern, and B. Magasanik. 1971. Genetic and metabolic control of enzymes responsible for histidine degradation in Salmonella typhimurium. 4-Imidazole-5-propionate amidohydrolase and N-formimino-L-glutamate formiminohydrolase. J. Biol. Chem. 246:3320-3329. 9. Smith, G. R., and B. Magasanik. 1971. Nature and self-regulated synthesis of the repressor of the hut operons in Salmonella typhimurium. Proc. Natl. Acad. Sci. U.S.A. 68:1493-1497.
