JOURNAL OF BACTERIOLOGY, Mar. 1975, p. 1216-1218 Copyright i 1975 American Society for Microbiology
Vol. 121, No. 3 Printed in U.S.A.
Procedure for Isolating Mutants Defective in Metabolite Transport or Utilization CLAIRE M. BERG,* JOHN J. ROSSI, JOYCE V. COLEMAN, AND MICHELE CONLON Biological Sciences Group, The University of Connecticut, Storrs, Connecticut 06268
Received for publication 27 August 1974
Mutants defective in uptake or utilization of a given metabolite can readily be obtained from facultative auxotrophs (for that metabolite) by penicillin enrichment under nonpermissive conditions in the presence of a low level of the required metabolite.
Mutants defective in the active transport of various metabolites have been isolated using procedures such as analogue resistance and the requirement for a high level of the metabolite (6, 8, 9). In this paper we describe a new procedure which has several advantages over those previously employed. Transport mutants as well as other classes of mutants unable to utilize low levels of a given metabolite are recovered. The procedure involves the use of facultative auxotrophs-strains which are auxotrophic under one set of conditions but prototrophic under another. Facultative auxotrophs may, for example, have temperature-sensitive (3) or osmotic-remedial auxotrophic lesions (2), or auxotrophy may be induced by nutritional manipulations. A well-known nutritionally inducible facultative auxotroph is wild-type Escherichia coli K-12, which requires neither isoleucine nor valine. However, the addition of valine to the medium results in a reversible isoleucine requirement, because isoleucine and valine share common biosynthetic enzymes, subject to regulation by valine (10). A similar situation is observed with indirectly suppressed Pro+ revertants of proA or proB (herein designated proAB-) auxotrophs which, because of mutation at argD, synthesize proline via N-
acetylglutamate--y-semialdehyde, an arginine
precursor (D. F. Bacon and H. J. Vogel, Fed. Proc. 22:476, 1963; 4). In these indirectly suppressed proAB- argD- mutants, addition of arginine to the medium inhibits the synthesis of N-acetylglutamate--y-semialdehyde and, as a conFequence, of proline (1, 4, 5). The new isolation procedure was tested using the facultative isoleucine requirement of wildtype E. coli K-12 and the facultative proline requirement of a proAB - argD - double mutant. The procedure involved a penicillin enrichment cycle for mutants unable to grow on low levels of the required metabolite when the inhibiting metabolite was present, but able to grow in the
absence of the required metabolite when the inhibiting metabolite was absent. The following is the protocol followed for penicillin enrichment for proline uptake mutants using CB0436, a facultative proline auxotroph. (i) Mutagenize cells. (ii) Grow cells with aeration in glucose minimal medium (11) plus 0.1% arginine assay medium (Difco) (growth medium). This contains 2 x 106- M proline plus other metabolites, but no arginine. (Minimal medium supplemented with proline alone would suffice, but cells grow faster in this medium.) (iii) When the cell titer reaches about 107/ml, "starve" for proline by adding arginine (22 gg/ml) and aerate. (Even higher cell titers can be used since this procedure is not sensitive to
cross-feeding.) (iv) After 3 h add penicillin (1,000 to 2,000 U/ml). (v) After 3 h wash and plate on growth medium (no arginine). (vi) After 48 h replica plate to growth medium plus arginine. (vii) After 48 h screen for colonies which did not grow on the replica plate and test.
For isoleucine uptake mutants use wild-type E. coli K-12. The growth medium is minimal medium plus 10-6 M isoleucine, and the "starvation" and replica media have 5 x 10- 4 M valine added. Using this procedure we have isolated a number of mutants of a proAB -, argD- strain, CB0436 (1), which have lost the capacity to grow on a low level (2 x 10- 6 M) of proline when arginine is present, and a number of mutants of W3110 which have lost the capacity to grow on a low level (10-6 M) of isoleucine when valine is present. The isoleucine uptake mutants have not been characterized further, but the proline uptake mutants fall into several classes. Most mutants have defects in the active transport of proline, whereas one exhibits enhanced trans-
1216
port but
may have a defect in proline activation (Fig. 1; Table 1). The unique aspect of the isolation procedure described here involves the use of facultative auxotrophy, either genetic or nutritionally induced. In part, this procedure is similar to one previously described (6) in that mutants unable to grow on a low level of a required metabolite are selected during penicillin enrichment. However, rather than permitting the mutants to grow by providing a high exogeneous level of the required metabolite, we remove the block to its endogeneous synthesis (this can be an important consideration if the required metabolite is costly or in short supply). It is probable that this procedure can be extended to the isolation of uptake mutants in
1200 0
-
~
_
800
O
600 0
4600 0
E
200 0 0
2
3
4
5
10
MINUTES
of proline uptake. Cells 37 C in medium E (11) plus glucose (0.5%), thymine (10 gg/ml), thiamine (20 Ag/m0, L-arginine (22 sg/mo), and L-proline (60 ,g/ml). Log-phase cells were harvested by centrifugation, washed twice with buffer (7), and resuspended at 109 cells/ml in medium E plus glucose (0.5%) and chloramphenicol (40 ug! ml). A 5-ml amount of cells was incubated with -shaking at 30 C for 5 min prior to addition of the substrate, L-['4CJproline (10-1 M, final concentration). At intervals, 0.2-ml samples were collected on filters (Millipore Corp.; type HA, 0.45-Mim pore size) and washed twice with buffer. The filters were dried at room temperature, placed in scintillation vials containing Econofluor (New England Nuclear) counting fluid, and counted in a Beckman liquid scintillation counter. Proline concentration in the medium is 10-" mmol/ml. Symbols: *, CB0436; 0, CB0501; A, CB0503; A, CB0504. FIG. 1.
grown at
TABLE 1. Uptake of [14Cjproline Mutanta
CB0502 CB0504 CB0503 CB0506 CB0500 CB0501 CB0505
Uptake (% of CB0436)b
5.5 12.1 17.1 23.0 27.0 69.9c 175.0d
a Isolated after nitrosoguanidine mutagenesis and penicillin enrichment. bSee Fig. 1 legend. c The initial kinetics are the same as in wild type, but this strain is unable to concentrate proline to the same extent (see Fig. 1). d The initial kinetics are faster and the steady-state level of proline accumulation is higher than in wild type. Nonetheless, this mutant is no longer able to grow in the starvation medium which contains 2 x 10- 6 M proline (plus arginine). It may have a defect in proline activation.
-
°eJ~~ 1000
1217
NOTES
VOL. 121, 1975
Time course
were
which the facultative auxotrophy is due to a temperature-sensitive or a physiologically remedial auxotrophic lesion 'or to analoguemediated inhibition of metabolite synthesis. The procedure is applicable to obtaining uptake mutants for any metabolite for which a facultative requirement may be induced, as long as the auxotroph retains viability during several hours of starvation and penicillin (or similar enrichment) treatment under the nonpermissive conditions. The observation that this procedure is applicable to nutritionally induced facultative auxotrophs should make it particularly useful for obtaining uptake mutants in cultured cells of higher plants or animals in which auxotrophs are frequently unavailable. This investigation was supported by Public Health Service grant no. AI-10862 from the National Institute of Allergy and Infectious Diseases to C. M. Berg. J. J. Rossi was supported by an NDEA fellowship. We thank F. Vasington for the use of his facilities and for helpful discussions. LITERATURE CITED 1. Berg, C. M., and J. J. Rossi. 1974. Proline excretion and
indirect suppression in Escherichia coli and Salmonella typhimurium. J. Bacteriol. 118:928-939. 2. Hawthorne, D. C., and J. Friis. 1964. Osmotic-remedial mutants. A new classification for nutritional mutants in yeast. Genetics 50:829-839. 3. Horowitz, N. H., and U. Leupold. 1951. Some recent studies bearing on the one gene-one enzyme hypothesis. Cold Spring Harbor Symp. Quant. Biol. 16:65-74. 4. Itikawa, H., S. Baumberg, and H. J. Vogel. 1968. Enzymatic basis for a genetic suppression: accumulation and deacylation of N-acetylglutamic -y-semialdehyde in enterobacterial mutants. Biochim. Biophys. Acta 159:547-550.
1218
NOTES
5. Kuo, T., and B. A. D. Stocker. 1969. Suppression of proline requirement of proA and proAB deletion mutants in Salmonella typhimurium by mutation to arginine requirement. J. Bacteriol. 98:593-598. 6. Lubin, M., D. H. Kessel, A. Budreau, and J. D. Gross. 1960. The isolation of bacterial mutants defective in amino acid transport. Biochim. Biophys. Acta 42:535-538. 7. Rosen, B. P., and F. D. Vasington. 1971. Purification and characterization of a histidine-binding protein from Salmonella typhimurium LT-2 and its relationship to the histidine permease system. J. Biol. Chem. 246:5351-5360.
J. BACTERIOL.
8. Schwartz, J. H., W. K. Maas, and E. J. Simon. 1959. An impaired concentrating mechanism for amino acids in mutants of Escherichia coli resistant to L-canavanine and D-serine. Biochim. Biophys. Acta 32:582-583. 9. Tristam. H., and S. Neale. 1968. The activity and specificity of the proline permease in wild-type and analogue-resistant strains of Escherichia coli. J. Gen. Microbiol. 50:121-137. 10. Umbarger, H. E. 1969. Regulation of amino acid metabolism. Annu. Rev. Biochem. 38:323-370. 11. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 218:97-106.
Vol. 121, No. 3 Printed in U.S.A.
Procedure for Isolating Mutants Defective in Metabolite Transport or Utilization CLAIRE M. BERG,* JOHN J. ROSSI, JOYCE V. COLEMAN, AND MICHELE CONLON Biological Sciences Group, The University of Connecticut, Storrs, Connecticut 06268
Received for publication 27 August 1974
Mutants defective in uptake or utilization of a given metabolite can readily be obtained from facultative auxotrophs (for that metabolite) by penicillin enrichment under nonpermissive conditions in the presence of a low level of the required metabolite.
Mutants defective in the active transport of various metabolites have been isolated using procedures such as analogue resistance and the requirement for a high level of the metabolite (6, 8, 9). In this paper we describe a new procedure which has several advantages over those previously employed. Transport mutants as well as other classes of mutants unable to utilize low levels of a given metabolite are recovered. The procedure involves the use of facultative auxotrophs-strains which are auxotrophic under one set of conditions but prototrophic under another. Facultative auxotrophs may, for example, have temperature-sensitive (3) or osmotic-remedial auxotrophic lesions (2), or auxotrophy may be induced by nutritional manipulations. A well-known nutritionally inducible facultative auxotroph is wild-type Escherichia coli K-12, which requires neither isoleucine nor valine. However, the addition of valine to the medium results in a reversible isoleucine requirement, because isoleucine and valine share common biosynthetic enzymes, subject to regulation by valine (10). A similar situation is observed with indirectly suppressed Pro+ revertants of proA or proB (herein designated proAB-) auxotrophs which, because of mutation at argD, synthesize proline via N-
acetylglutamate--y-semialdehyde, an arginine
precursor (D. F. Bacon and H. J. Vogel, Fed. Proc. 22:476, 1963; 4). In these indirectly suppressed proAB- argD- mutants, addition of arginine to the medium inhibits the synthesis of N-acetylglutamate--y-semialdehyde and, as a conFequence, of proline (1, 4, 5). The new isolation procedure was tested using the facultative isoleucine requirement of wildtype E. coli K-12 and the facultative proline requirement of a proAB - argD - double mutant. The procedure involved a penicillin enrichment cycle for mutants unable to grow on low levels of the required metabolite when the inhibiting metabolite was present, but able to grow in the
absence of the required metabolite when the inhibiting metabolite was absent. The following is the protocol followed for penicillin enrichment for proline uptake mutants using CB0436, a facultative proline auxotroph. (i) Mutagenize cells. (ii) Grow cells with aeration in glucose minimal medium (11) plus 0.1% arginine assay medium (Difco) (growth medium). This contains 2 x 106- M proline plus other metabolites, but no arginine. (Minimal medium supplemented with proline alone would suffice, but cells grow faster in this medium.) (iii) When the cell titer reaches about 107/ml, "starve" for proline by adding arginine (22 gg/ml) and aerate. (Even higher cell titers can be used since this procedure is not sensitive to
cross-feeding.) (iv) After 3 h add penicillin (1,000 to 2,000 U/ml). (v) After 3 h wash and plate on growth medium (no arginine). (vi) After 48 h replica plate to growth medium plus arginine. (vii) After 48 h screen for colonies which did not grow on the replica plate and test.
For isoleucine uptake mutants use wild-type E. coli K-12. The growth medium is minimal medium plus 10-6 M isoleucine, and the "starvation" and replica media have 5 x 10- 4 M valine added. Using this procedure we have isolated a number of mutants of a proAB -, argD- strain, CB0436 (1), which have lost the capacity to grow on a low level (2 x 10- 6 M) of proline when arginine is present, and a number of mutants of W3110 which have lost the capacity to grow on a low level (10-6 M) of isoleucine when valine is present. The isoleucine uptake mutants have not been characterized further, but the proline uptake mutants fall into several classes. Most mutants have defects in the active transport of proline, whereas one exhibits enhanced trans-
1216
port but
may have a defect in proline activation (Fig. 1; Table 1). The unique aspect of the isolation procedure described here involves the use of facultative auxotrophy, either genetic or nutritionally induced. In part, this procedure is similar to one previously described (6) in that mutants unable to grow on a low level of a required metabolite are selected during penicillin enrichment. However, rather than permitting the mutants to grow by providing a high exogeneous level of the required metabolite, we remove the block to its endogeneous synthesis (this can be an important consideration if the required metabolite is costly or in short supply). It is probable that this procedure can be extended to the isolation of uptake mutants in
1200 0
-
~
_
800
O
600 0
4600 0
E
200 0 0
2
3
4
5
10
MINUTES
of proline uptake. Cells 37 C in medium E (11) plus glucose (0.5%), thymine (10 gg/ml), thiamine (20 Ag/m0, L-arginine (22 sg/mo), and L-proline (60 ,g/ml). Log-phase cells were harvested by centrifugation, washed twice with buffer (7), and resuspended at 109 cells/ml in medium E plus glucose (0.5%) and chloramphenicol (40 ug! ml). A 5-ml amount of cells was incubated with -shaking at 30 C for 5 min prior to addition of the substrate, L-['4CJproline (10-1 M, final concentration). At intervals, 0.2-ml samples were collected on filters (Millipore Corp.; type HA, 0.45-Mim pore size) and washed twice with buffer. The filters were dried at room temperature, placed in scintillation vials containing Econofluor (New England Nuclear) counting fluid, and counted in a Beckman liquid scintillation counter. Proline concentration in the medium is 10-" mmol/ml. Symbols: *, CB0436; 0, CB0501; A, CB0503; A, CB0504. FIG. 1.
grown at
TABLE 1. Uptake of [14Cjproline Mutanta
CB0502 CB0504 CB0503 CB0506 CB0500 CB0501 CB0505
Uptake (% of CB0436)b
5.5 12.1 17.1 23.0 27.0 69.9c 175.0d
a Isolated after nitrosoguanidine mutagenesis and penicillin enrichment. bSee Fig. 1 legend. c The initial kinetics are the same as in wild type, but this strain is unable to concentrate proline to the same extent (see Fig. 1). d The initial kinetics are faster and the steady-state level of proline accumulation is higher than in wild type. Nonetheless, this mutant is no longer able to grow in the starvation medium which contains 2 x 10- 6 M proline (plus arginine). It may have a defect in proline activation.
-
°eJ~~ 1000
1217
NOTES
VOL. 121, 1975
Time course
were
which the facultative auxotrophy is due to a temperature-sensitive or a physiologically remedial auxotrophic lesion 'or to analoguemediated inhibition of metabolite synthesis. The procedure is applicable to obtaining uptake mutants for any metabolite for which a facultative requirement may be induced, as long as the auxotroph retains viability during several hours of starvation and penicillin (or similar enrichment) treatment under the nonpermissive conditions. The observation that this procedure is applicable to nutritionally induced facultative auxotrophs should make it particularly useful for obtaining uptake mutants in cultured cells of higher plants or animals in which auxotrophs are frequently unavailable. This investigation was supported by Public Health Service grant no. AI-10862 from the National Institute of Allergy and Infectious Diseases to C. M. Berg. J. J. Rossi was supported by an NDEA fellowship. We thank F. Vasington for the use of his facilities and for helpful discussions. LITERATURE CITED 1. Berg, C. M., and J. J. Rossi. 1974. Proline excretion and
indirect suppression in Escherichia coli and Salmonella typhimurium. J. Bacteriol. 118:928-939. 2. Hawthorne, D. C., and J. Friis. 1964. Osmotic-remedial mutants. A new classification for nutritional mutants in yeast. Genetics 50:829-839. 3. Horowitz, N. H., and U. Leupold. 1951. Some recent studies bearing on the one gene-one enzyme hypothesis. Cold Spring Harbor Symp. Quant. Biol. 16:65-74. 4. Itikawa, H., S. Baumberg, and H. J. Vogel. 1968. Enzymatic basis for a genetic suppression: accumulation and deacylation of N-acetylglutamic -y-semialdehyde in enterobacterial mutants. Biochim. Biophys. Acta 159:547-550.
1218
NOTES
5. Kuo, T., and B. A. D. Stocker. 1969. Suppression of proline requirement of proA and proAB deletion mutants in Salmonella typhimurium by mutation to arginine requirement. J. Bacteriol. 98:593-598. 6. Lubin, M., D. H. Kessel, A. Budreau, and J. D. Gross. 1960. The isolation of bacterial mutants defective in amino acid transport. Biochim. Biophys. Acta 42:535-538. 7. Rosen, B. P., and F. D. Vasington. 1971. Purification and characterization of a histidine-binding protein from Salmonella typhimurium LT-2 and its relationship to the histidine permease system. J. Biol. Chem. 246:5351-5360.
J. BACTERIOL.
8. Schwartz, J. H., W. K. Maas, and E. J. Simon. 1959. An impaired concentrating mechanism for amino acids in mutants of Escherichia coli resistant to L-canavanine and D-serine. Biochim. Biophys. Acta 32:582-583. 9. Tristam. H., and S. Neale. 1968. The activity and specificity of the proline permease in wild-type and analogue-resistant strains of Escherichia coli. J. Gen. Microbiol. 50:121-137. 10. Umbarger, H. E. 1969. Regulation of amino acid metabolism. Annu. Rev. Biochem. 38:323-370. 11. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 218:97-106.
