Vol. 130, No. 3 Printed in U.S.A.
JOURNAL OF BACTRIUOLOGY, June 1977, p. 1244-1252 Copyright © 1977 American Society for Microbiology
Independent Regulation of Transport and Biosynthesis of Arginine in Escherichia coli K-12 T. F. R. CELIS Department of Microbiology, New York University School of Medicine, New York, New York 10016
Received for publication 29 December 1976
From an arginine auxotrophic strain, a mutant was isolated which is able to utilize D-arginine as a source of L-arginine and shows a high sensitivity to inhibition of growth by canavanine. Transport studies revealed a four- to fivefold increased uptake of arginine and ornithine in cells from the mutant strain. The kinetics of entry of arginine and ornithine evidenced elevated maximal influx values for the arginine- and ornithine-specific transport systems. A close parallel between arginine transport activity and arginine binding activity with one arginine-specific binding periplasmic protein in the mutant strongly suggests that such binding protein is a component ofthe arginine-specific permease. The affinity between arginine and the binder, isolated from the mutant cells, as well as the electrophoretic mobility of the protein, remain unchanged. The enhanced transport activity of arginine and ornithine with mutant cells is insensitive to repression by arginine or ornithine, whereas the biosynthesis of arginine-forming enzymes is normally repressible. When transport activity was examined in strains with mutations leading to derepression of arginine biosynthesis, the regulation of arginine transport was found to be normal. These studies support the conclusion that arginine transport and arginine biosynthesis, in Escherichia coli K-12, are not regulated in a concerted manner, although both systems may have components in common.
The synthesis of arginine from glutamate in Escherichia coli K-12 is mediated by eight enzymes specified by nine structural genes, argA through argI, which are located at six different chromosomal sites (31). Four of these genes form a cluster, argECBH, and two separate genes, argF and argI, specify the synthesis of one enzyme (10). There is a single regulatory gene, argR, which codes for a cytoplasmic protein, the arginine repressor (12, 20, 21). In argR+ strains, the arginine repressor, together with arginine or with a derivative of arginine, is able to elicit repression of all nine structural genes in parallel but not coordinately. In argRmutants, high levels of the eight enzymes are formed even in the presence of arginine (9, 11, 19). Little information is available on the regulation of the transport of arginine. The level of arginine uptake can be lowered by growth of E. coli with exogenous arginine or ornithine, which represses the arginine-specific transport system. The general transport system for arginine, ornithine, and lysine is repressible only by lysine (7). This paper describes attempts to determine whether transport activity and biosynthetic activity for arginine are regulated in a concerted manner in E. coli K-12. Studies are reported on a mutant strain with derepressed 1244
levels of arginine and ornithine transport, and an elevated synthesis of one arginine-specific binding protein. The regulation of transport and biosynthesis of arginine was examined in strains of E. coli K-12 carrying mutations in the regulation of either transport or of biosynthesis. MATERIALS AND METHODS Bacterial strains and media. All strains used in these experiments were derived from E. coli K-12 and are listed in Table 1. Bacteria were grown on minimal medium A, on medium AF, or on neopeptone broth as described in the accompanying paper (6). Chemicals. L4[3-3Hlarginine, L_[U-_4C]arginine,
L43_3H]ornithine, i_[U-_4C]ornithine, L-[4,5_3H]lysine, L43-3H]histidine, [U-_4C]glutamine, and L-
[U-_4C]proline were purchased from New England Nuclear Corp. L-Canavanine, chloramphenicol, and aminooxyacetic acid hemi-hydrochloride were from Sigma Chemical Co. All amino acids were of Lform unless specified. Sodium dodecyl sulfate (BDH) was from Gallard-Schlesinger. Acrylamide, N,Nmethylenebisacrylamide, N,N,N',N'-tetramethylethylenediamine, and ammonium peroxydisulfate were purchased from Eastman-Kodak. Assay for transport activity. Amino acid uptake was measured as described in the accompanying paper (6).
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TABLE 1. List of E. coli K-12 strainsa
a
Strain
Genotypea
MA-5 RC-10 RC-13 RC-14 JC-182 JC-182-9 MA 5000
argA gal argA gal gal gal thi pur-1 thi pur-1 argA (brad) s, sensitive; s-s, super-sensitive
Abbreviations:
Canavanine
Source or comment
W. Mass s Canavanine super-sensitive of MA-5 S-s argA+ revertant of MA-5 s s-s argA+ revertant of RC-10 Reference 8 s ArgR of JC-182 (8) r W. Maas s (highly sensitive to canavanine inhibition); r, resistant;
brad, braditroph. b For gene symbols see Bachmann et al. (4). Isolation of the mutant strain RC-10. The parent strain MA5 (argA) cannot utilize D-arginine as a source of i-arginine; it can grow in the presence of acetyl-glutamate as a precursor of arginine. This strain was mutagenized with N-methyl-N'-nitro-nitrosoguanidine (1), and after the cells were washed 0.2 ml of the culture was suspended in 10 ml of neopeptone broth and allowed to grow for 15 h at 37°C. The culture was then washed and aliquots were plated on AF agar plates containing 200 ,ug of D-arginine per ml. Mutants able to utilize D-arginine appeared after 3 days of incubation at 37°C. Mutations occurred with a frequency of about 10-8. Prototrophic revertants of the arginine marker were discarded after streaking the cells on AF and AF-Darginine plates. Initial --rates of arginine uptake were measured on more than 100 mutants. None of them showed any significant difference with the parental strain in the concentrative ability of the labeled substrate. One of these D-arginine utilizers was mutagenized again with N-methyl-N'-nitro-nitrosoguanidine as described above and cells were plated on AF agar plates supplemented with 50 jig of D-arginine per ml. Mutants that were able to grow on this amount of D-arginine as a source of L-arginine were then tested for their sensitivity to inhibition of growth by canavanine. AF plates supplemented with acetyl-glutamate and AF plates with acetyl-glutamate and canavanine were used for restreaking the colonies. Only cells that did not grow in the presence of canavanine after 48 h of incubation at 37°C were used for uptake studies. Three percent of the D-arginine utilizer mutants obtained after the second cycle of mutagenesis showed increased levels of arginine and ornithine transport activity. One of them, RC-10, was selected for further studies. Osmotic shock, DEAE-cellulose chromatography, and assay for binding activity. Cold osmotic shock, diethylaminoethyl (DEAE)-cellulose chromatography of osmotic shock fluids, and assays for binding activity were performed as described in the accompanying paper (6). Enzyme assays. Biosynthetic arginine decarboxylase, biodegradative (inducible) arginine decarboxylase, and agmatine ureahydrolase were measured by the procedures reported in the accompanying paper (6). Biosynthetic ornithine decarboxylase and biodegradative ornithine decarboxylase activities were determined by the procedures described by Morris and Pardee (23).
Acid phosphatase was measured by the procedure reported by Neu and Heppel (24). Procedures for measuring ornithine transcarbamylase (OTC) and arginine-transfer ribonucleic acid (tRNA) synthetase were described in the accompanying paper (6). Transductions. Transductions using the P1-like bacteriophage 363 were performed as described by Glansdorff (8). Polyacrylamide gel electrophoresis. The discontinuous sodium dodecyl sulfate (SDS) buffer system of Laemmli (15) and the apparatus described by Studier (30) were used in polyacrylamide gel electrophoresis. Spacer gels contained 0.63 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 6.8), 0.1% SDS, 2 mM ethylenediaminetetraacetic acid (EDTA), and 5% acrylamide. Resolving gels contained 0.19 M Tris-hydrochloride (pH 8.8), 0.1% SDS, 2 mM EDTA, and 10% acrylamide. The acrylamide-bisacrylamide ratio was 30:0.8. Polymerization was initiated with N,N,N',N'-tetraethylmethylenediamine and ammonium persulfate. The electrode buffer contained 0.05 M Tris, 0.38 M glycine, 0.1% SDS, and 2 mM EDTA adjusted to pH 8.3. The periplasmic protein sample was prepared on a buffer containing 0.05 M Tris-hydrochloride (pH 6.8), 1% SDS, 10% glycerol, and 2 mM EDTA. The sample was boiled for 2 min, and approximately 15 jig of protein was added to each gel slot in the 1-mm gel slab. Electrophoresis was run at 15 mA, at room temperature for 6 h. At the end of the run, the gel slab was placed for 1 h in Coomassie blue stain (2.0 g of Coomassie blue per liter, 50% methanol, 7% acetic acid) (22). The gel was destained by repeated washings in 7.5% acetic acid and 5% methanol, by using slow agitation. Protein determinations. Protein determination followed the method of Lowry et al. (18) with crystalline bovine serum albumin as standard.
RESULTS Transport properties of the mutant strain RC-10. The mutant herein described was isolated as a strain able to utilize D-arginine as a source of L-arginine and highly sensitive to inhibition of growth by canavanine. Whereas wild-type E. coli is able to overcome canavanine toxicity and grows on analogue-containing plates after 24 h of incubation at 370C, no de-
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J. BACTERIOL.
tectable growth was seen with cells from the mutant after 48 or even 72 h of incubation. It has been shown that canavanine inhibits the uptake of arginine and ornithine in E. coli by competing for the general transport system and the arginine- and ornithine-specific permeases (6, 7). Only the arginine- and ornithinespecific transport systems showed increased levels of substrate accumulation in the mutant (Fig. 1). The general transport system, explored with labeled lysine at 0.1 ,M concentration, and the lysine-specific system remain unaffected by the mutation. Inhibition of the highaffinity system for lysine uptake (LAO) by arginine or ornithine was found to be as efficient in the mutant as in the parent strain. Transport activity for histidine, proline, and glutamine (not shown) was also found to be unaltered in the mutant. Since the mutant was isolated in an arginine auxotroph which can grow on acetyl-glutamate as a precursor of arginine, cells were grown on medium supplemented with acetyl-glutamate to obviate the repression effect by arginine. Transport studies, carried out on prototrophic revertants (RC-13 and RC-14) of the arginine marker, showed the same differences in transport activities found with the original parent and mutant strains. The S/V
versus S plots of data for the kinetics of entry of arginine and ornithine are shown in Fig. 2 and Fig. 3. The constant values calculated from these plots are presented in Table 2. The analysis of these data bears resemblance to the situation found in the canavanine-resistant strain described in the accompanying paper (6). The two permeases for ornithine are functioning in the mutant with an elevated maximal influx of substrate through the specific system. The kinetics of entry of arginine in the mutant revealed the operation of only one system with kinetic values comparable to those that describe the arginine-specific system in the wild0.6 0.5 0.4 S V
0.3
0.2 0.1 0
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
[AM]
FIG. 2. Kinetics of L-arginine entry in parent and mutant strains. Cells were grown in medium A supZ ARGININE [1,MM] LYSINE [I10M] plemented with acetyl-glutamate and initial rates of w 1--8 ~~~~~~~~4 influx were measured as described in the text, by using L-[3H]arginine at different concentrations be0Z tween 0.5 x 10-8 and 10-4 M. The circles represent cr 6 ~~~~~~~~~~3 the experimental values; the line was drawn by using 4 the theoretical values calculated from the equation -J42 for two simultaneous reactions on the same substrate (6). Symbols: 0, parent strain; *, mutant strain. SI w V, Molarity per nanomole per milligram of cellular protein per minute. E CA
~~~~~~~LYSINE [0.1AM]
0
c8
12
1.0 10
w
0.
S
o4
ORNITHINE
1I0gmJ
0.5
0 2
V
6 4
z
2
2
6
10
2 MINUTES
6
10
FIG. 1. Time-dependence uptake of labeled amino acids in parent (MA-5) and mutant (RC-10). Transport was measured as outlined in the text. The initial concentration is indicated for each substrate. Symbols: *, uptake in parent strain; 0, uptake in the mutant.
A
%J
0
2
3
4
5
6
7
8
9
[ALM] FIG. 3. Same as Fig. 2, but with L-[3H]ornithine. Symbols: 0, parent strain; 0, mutant strain. SIV, Molarity per nanomole per milligram of cellular protein.
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ARGININE TRANSPORT AND BIOSYNTHESIS REGULATION
type strain a similar Km and a fivefold increased Vmax of influx. The S/V versus S plot of data for the kinetics of lysine entry (not shown) revealed the operation of the two lysine transport systems in the mutant with constant values similar to those that describe the corresponding systems in the parent strain. These results are taken to mean that the mutation does not affect the lysine transport, nor the LAO system, nor the lysine-specific system. -
Arginine biosynthesis and arginine and ornithine conversions on the mutant strain. The pathway of arginine biosynthesis in the mutant is repressible by arginine. The activity of the enzyme OTC depends upon the absence or presence of arginine in the culture media (see Table 5). The incorporation of arginine into tRNA, as well as the activity of the enzymes involved in the conversion of both arginine and ornithine into putrescine, is not affected by the mutation in strain RC-10 (Table 3).
1247
Binding proteins on the mutant. Binding protein assays with shock fluids from parent
and mutant strains appear in Table 4. There is a four- to fivefold increased binding activity with labeled arginine and the shock fluid released from the mutant. Since the amount of protein released after the osmotic shock is similar for cells from parent and mutant strains (4 to 5% of the total cellular protein), and since the levels of acid phosphatase activity are also comparable in both strains, it can be concluded that the mutation does not affect the release of particular components of the periplasmic area. Fractionation of the crude osmotic shock fluid on DEAE-cellulose chromatography (Fig. 4) showed that the arginine-specific binding protein II is the molecular compound responsible for the elevated binding activity with arginine in the crude shock fluid from the mutant. In Fig. 5, an SDS-polyacrylamide gel of periplasmic proteins is presented. Electrophoretic
TABLE 2. Kinetic values for uptake ofarginine and ornithine on strains MA-5 (parent) and RC-10 (mutant)a Substrate
Arginine
K,
=
Strain MA-5 (parent) 1.0 x 10-7 M, V1 = 1.66
K2 = 1.0 X 10-" M, V2 =0.04 K, = 6.0 x 10-6 M, VI = 1.0
Ornithine
Strain RC-10 (mutant)
K,
=1.5 x 10-7, V1 = 8.0
K, = 4.0 x 10-6, V1 = 4.0 K2 = 1.0 x 10- M, V2 = 0.28 K2 = 1.0 x 10-i, V2 = 0.35 a The constants were calculated from S/V versus S plots as described in the accompanying paper (6). V1 and V2 are measured in nanomoles per milligram of cellular protein per minute. Initial rates of uptake were measured as described in the text. TABLE 3. Activities of enzymes involved in the metabolism of argininea Enzyme sp act (U/mg of protein)' Enzyme
Product measured
Parent (MA-5)
Mutant (RC-10)
Arginine biosynthetic decarboxylase CO2 0.007 0.006 Arginine biodegradative decarboxylase CO2 0.060 0.040 Ornithine biosynthetic decarboxylase CO2 0.019 0.021 Ornithine biodegradative decarboxylase 0.007 0.008 C02. Agmatine ureahydrolase Urea 0.026 0.018 Arginine tRNA synthetase Arg-tRNA 2.05 2.66 a Enzyme assays are described in the text. b Units are micromoles of product formed per minute, except for arginine-tRNA synthetase where units are nanomoles of labeled arginine incorporated into tRNA per minute. TABLE 4. Binding activity on shock fluida Total unitsb
Sp act (U/mg)c
Strain
Arg
MA-5 (parent)
120
Lys 82
Orn
Arg
76
0.58
Lys 0.40
Acid phosphaOn
tase (U/mg)
0.37
6.96
RC-10 (mutant) 530 77 74 0.41 0.40 2.80 6.84 a Binding activity for each amino acid was measured at 10 MM initial concentration by equilibrium dialysis on Plexiglas chambers as described in the text. Units of acid phosphatase = micromoles of phosphate released per hour per milligram of shock fluid protein. b Nanomoles of amino acid bound to the shock fluid obtained from a 10-liter culture. c Nanomoles of amino acid bound per milligram of shock fluid protein.
1248
J. BACTERIOL.
CELIS z 0 U
4
2.5
CD 15 N.
a
z
2.0
0 CD
0
10 1.5 a
4
0
co
o
0
1.0
0
0.5
"')5 w C
0
0
50
100 150 200 FRACTION NUMBER FIG. 4. Elution profile of the DEAE-cellulose fractionation of the osmotic shock fluid from parent (MA5) and mutant (RC-10) strains. Two columns (0.9 by 45 cm) with equilibrated DEAE-cellulose were loaded with 280 mg ofshock fluid protein. Conditions for fractionation have been described (6). Arrow 1 shows where the gradient (0 to 0.1 M NaCI) was begun. Arrow 2 shows where the columns were washed with 2.0 M NaCl. Arginine binding activity was measured by equilibrium dialysis on Plexiglas chambers (6). Symbols: *, [3H]arginine binding with fractions eluted from the column loaded with shock fluid released from the parent (MA-5) strain; 0, [PHlarginine binding with fractionated shock fluid from the mutant (RC-10). 0
patterns are shown of the proteins contained in the shock fluids and the arginine-specific binding protein II after DEAE fractionation from parent and mutant strains. The electrophoretic bands, corresponding to the partially pure binding protein preparations from both strains, have similar mobilities. The high binding activity with the arginine-specific binding protein II after DEAE fractionation is confirmed in the stained slab gel. A heavier band of such periplasmic component can be seen in the shock fluid, subjected to electrophoresis, from the mutant. Scatchard plots of data of the binding activity between labeled arginine and the arginine binding protein II showed that the dissociation constant for the isolated protein from the mutant is similar (about 10-7 M) to that found for the molecule from the parent strain. The implication is that the mutation on strain RC-10 affects the synthesis of the arginine-specific binding protein II, but not its physical properties or affinity constant. Mapping of strain RC-10. Genetic linkage between the mutation responsible for the elevated transport of arginine and ornithine and the serA marker was explored by transduction studies using the mutant strain RC-10 as donor and strain AB-856 (6) as recipient. Of more
than 200 serA + transductants analyzed, none of them revealed a high sensitivity to inhibition of growth by canavanine. Conjugation experiments, carried out by the procedure of Low (17), using 15 different Hfr strains, indicated that the mutation is located between the dsdA marker (point of entry of Hfr strain KL983) (16) and the lysA marker (point of entry of Hfr strain KL16) (16) on the linkage map of E. coli K-12. The Hfr strains were used as donors. The recipient strain was a streptomycin-resistant, argA+ prototrophic derivative of the original mutant RC-10. Recombinants were selected for the ability to grow on canavanine-supplemented plates after 48 h of incubation at 370C. Further transduction experiments indicated that the locus responsible for the increased transport and sensitivity to canavanine inhibition is closely linked (more than 90% cotransducible) to the argA marker. Details of this mapping will be published elsewhere (T.F.R. Celis, manuscript in preparation). Regulation of transport and biosynthesis of arginine and ornithine. If wild-type E. coli K12 cells are grown in the presence of 50 mg of Larginine or L-ornithine per liter, the initial rates of uptake of both amino acids are reduced about 70% (Table 5). The repression of arginine
ARGININE TRANSPORT AND BIOSYNTHESIS REGULATION
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1249
~~~~transport by either arginine or ornithine is substantially decreased in the mutant, and the / / formation of the omnithine transport system seems to be no longer repressible. If cells from the mutant are grown in medium supple- ~~~~ mented with a higher amount of arginine (500 mg/liter), more than 50% of arginine transport -~~~~~ ~can be repressed. However, the level of argihnine uptake is never lowered to the amount l l taken up by cells of the wild-type strain at gmaximum repression. In contrast, the biosynm S cthesis of arginine, measured by the level of w m s m OTC activity, is repressed as completely in the mutant as it is in the parent strain. These /f) g -
....Oexperiments indicate that the increased trans" in the and .. u activity of arginine )port It less sensitive ornithine to repression by is much mmutant s i a D 4tsgrowth in media containing arginine or oni( strain thine than in the parental strain. Furthermore, .inu-
JOURNAL OF BACTRIUOLOGY, June 1977, p. 1244-1252 Copyright © 1977 American Society for Microbiology
Independent Regulation of Transport and Biosynthesis of Arginine in Escherichia coli K-12 T. F. R. CELIS Department of Microbiology, New York University School of Medicine, New York, New York 10016
Received for publication 29 December 1976
From an arginine auxotrophic strain, a mutant was isolated which is able to utilize D-arginine as a source of L-arginine and shows a high sensitivity to inhibition of growth by canavanine. Transport studies revealed a four- to fivefold increased uptake of arginine and ornithine in cells from the mutant strain. The kinetics of entry of arginine and ornithine evidenced elevated maximal influx values for the arginine- and ornithine-specific transport systems. A close parallel between arginine transport activity and arginine binding activity with one arginine-specific binding periplasmic protein in the mutant strongly suggests that such binding protein is a component ofthe arginine-specific permease. The affinity between arginine and the binder, isolated from the mutant cells, as well as the electrophoretic mobility of the protein, remain unchanged. The enhanced transport activity of arginine and ornithine with mutant cells is insensitive to repression by arginine or ornithine, whereas the biosynthesis of arginine-forming enzymes is normally repressible. When transport activity was examined in strains with mutations leading to derepression of arginine biosynthesis, the regulation of arginine transport was found to be normal. These studies support the conclusion that arginine transport and arginine biosynthesis, in Escherichia coli K-12, are not regulated in a concerted manner, although both systems may have components in common.
The synthesis of arginine from glutamate in Escherichia coli K-12 is mediated by eight enzymes specified by nine structural genes, argA through argI, which are located at six different chromosomal sites (31). Four of these genes form a cluster, argECBH, and two separate genes, argF and argI, specify the synthesis of one enzyme (10). There is a single regulatory gene, argR, which codes for a cytoplasmic protein, the arginine repressor (12, 20, 21). In argR+ strains, the arginine repressor, together with arginine or with a derivative of arginine, is able to elicit repression of all nine structural genes in parallel but not coordinately. In argRmutants, high levels of the eight enzymes are formed even in the presence of arginine (9, 11, 19). Little information is available on the regulation of the transport of arginine. The level of arginine uptake can be lowered by growth of E. coli with exogenous arginine or ornithine, which represses the arginine-specific transport system. The general transport system for arginine, ornithine, and lysine is repressible only by lysine (7). This paper describes attempts to determine whether transport activity and biosynthetic activity for arginine are regulated in a concerted manner in E. coli K-12. Studies are reported on a mutant strain with derepressed 1244
levels of arginine and ornithine transport, and an elevated synthesis of one arginine-specific binding protein. The regulation of transport and biosynthesis of arginine was examined in strains of E. coli K-12 carrying mutations in the regulation of either transport or of biosynthesis. MATERIALS AND METHODS Bacterial strains and media. All strains used in these experiments were derived from E. coli K-12 and are listed in Table 1. Bacteria were grown on minimal medium A, on medium AF, or on neopeptone broth as described in the accompanying paper (6). Chemicals. L4[3-3Hlarginine, L_[U-_4C]arginine,
L43_3H]ornithine, i_[U-_4C]ornithine, L-[4,5_3H]lysine, L43-3H]histidine, [U-_4C]glutamine, and L-
[U-_4C]proline were purchased from New England Nuclear Corp. L-Canavanine, chloramphenicol, and aminooxyacetic acid hemi-hydrochloride were from Sigma Chemical Co. All amino acids were of Lform unless specified. Sodium dodecyl sulfate (BDH) was from Gallard-Schlesinger. Acrylamide, N,Nmethylenebisacrylamide, N,N,N',N'-tetramethylethylenediamine, and ammonium peroxydisulfate were purchased from Eastman-Kodak. Assay for transport activity. Amino acid uptake was measured as described in the accompanying paper (6).
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TABLE 1. List of E. coli K-12 strainsa
a
Strain
Genotypea
MA-5 RC-10 RC-13 RC-14 JC-182 JC-182-9 MA 5000
argA gal argA gal gal gal thi pur-1 thi pur-1 argA (brad) s, sensitive; s-s, super-sensitive
Abbreviations:
Canavanine
Source or comment
W. Mass s Canavanine super-sensitive of MA-5 S-s argA+ revertant of MA-5 s s-s argA+ revertant of RC-10 Reference 8 s ArgR of JC-182 (8) r W. Maas s (highly sensitive to canavanine inhibition); r, resistant;
brad, braditroph. b For gene symbols see Bachmann et al. (4). Isolation of the mutant strain RC-10. The parent strain MA5 (argA) cannot utilize D-arginine as a source of i-arginine; it can grow in the presence of acetyl-glutamate as a precursor of arginine. This strain was mutagenized with N-methyl-N'-nitro-nitrosoguanidine (1), and after the cells were washed 0.2 ml of the culture was suspended in 10 ml of neopeptone broth and allowed to grow for 15 h at 37°C. The culture was then washed and aliquots were plated on AF agar plates containing 200 ,ug of D-arginine per ml. Mutants able to utilize D-arginine appeared after 3 days of incubation at 37°C. Mutations occurred with a frequency of about 10-8. Prototrophic revertants of the arginine marker were discarded after streaking the cells on AF and AF-Darginine plates. Initial --rates of arginine uptake were measured on more than 100 mutants. None of them showed any significant difference with the parental strain in the concentrative ability of the labeled substrate. One of these D-arginine utilizers was mutagenized again with N-methyl-N'-nitro-nitrosoguanidine as described above and cells were plated on AF agar plates supplemented with 50 jig of D-arginine per ml. Mutants that were able to grow on this amount of D-arginine as a source of L-arginine were then tested for their sensitivity to inhibition of growth by canavanine. AF plates supplemented with acetyl-glutamate and AF plates with acetyl-glutamate and canavanine were used for restreaking the colonies. Only cells that did not grow in the presence of canavanine after 48 h of incubation at 37°C were used for uptake studies. Three percent of the D-arginine utilizer mutants obtained after the second cycle of mutagenesis showed increased levels of arginine and ornithine transport activity. One of them, RC-10, was selected for further studies. Osmotic shock, DEAE-cellulose chromatography, and assay for binding activity. Cold osmotic shock, diethylaminoethyl (DEAE)-cellulose chromatography of osmotic shock fluids, and assays for binding activity were performed as described in the accompanying paper (6). Enzyme assays. Biosynthetic arginine decarboxylase, biodegradative (inducible) arginine decarboxylase, and agmatine ureahydrolase were measured by the procedures reported in the accompanying paper (6). Biosynthetic ornithine decarboxylase and biodegradative ornithine decarboxylase activities were determined by the procedures described by Morris and Pardee (23).
Acid phosphatase was measured by the procedure reported by Neu and Heppel (24). Procedures for measuring ornithine transcarbamylase (OTC) and arginine-transfer ribonucleic acid (tRNA) synthetase were described in the accompanying paper (6). Transductions. Transductions using the P1-like bacteriophage 363 were performed as described by Glansdorff (8). Polyacrylamide gel electrophoresis. The discontinuous sodium dodecyl sulfate (SDS) buffer system of Laemmli (15) and the apparatus described by Studier (30) were used in polyacrylamide gel electrophoresis. Spacer gels contained 0.63 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 6.8), 0.1% SDS, 2 mM ethylenediaminetetraacetic acid (EDTA), and 5% acrylamide. Resolving gels contained 0.19 M Tris-hydrochloride (pH 8.8), 0.1% SDS, 2 mM EDTA, and 10% acrylamide. The acrylamide-bisacrylamide ratio was 30:0.8. Polymerization was initiated with N,N,N',N'-tetraethylmethylenediamine and ammonium persulfate. The electrode buffer contained 0.05 M Tris, 0.38 M glycine, 0.1% SDS, and 2 mM EDTA adjusted to pH 8.3. The periplasmic protein sample was prepared on a buffer containing 0.05 M Tris-hydrochloride (pH 6.8), 1% SDS, 10% glycerol, and 2 mM EDTA. The sample was boiled for 2 min, and approximately 15 jig of protein was added to each gel slot in the 1-mm gel slab. Electrophoresis was run at 15 mA, at room temperature for 6 h. At the end of the run, the gel slab was placed for 1 h in Coomassie blue stain (2.0 g of Coomassie blue per liter, 50% methanol, 7% acetic acid) (22). The gel was destained by repeated washings in 7.5% acetic acid and 5% methanol, by using slow agitation. Protein determinations. Protein determination followed the method of Lowry et al. (18) with crystalline bovine serum albumin as standard.
RESULTS Transport properties of the mutant strain RC-10. The mutant herein described was isolated as a strain able to utilize D-arginine as a source of L-arginine and highly sensitive to inhibition of growth by canavanine. Whereas wild-type E. coli is able to overcome canavanine toxicity and grows on analogue-containing plates after 24 h of incubation at 370C, no de-
1246
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tectable growth was seen with cells from the mutant after 48 or even 72 h of incubation. It has been shown that canavanine inhibits the uptake of arginine and ornithine in E. coli by competing for the general transport system and the arginine- and ornithine-specific permeases (6, 7). Only the arginine- and ornithinespecific transport systems showed increased levels of substrate accumulation in the mutant (Fig. 1). The general transport system, explored with labeled lysine at 0.1 ,M concentration, and the lysine-specific system remain unaffected by the mutation. Inhibition of the highaffinity system for lysine uptake (LAO) by arginine or ornithine was found to be as efficient in the mutant as in the parent strain. Transport activity for histidine, proline, and glutamine (not shown) was also found to be unaltered in the mutant. Since the mutant was isolated in an arginine auxotroph which can grow on acetyl-glutamate as a precursor of arginine, cells were grown on medium supplemented with acetyl-glutamate to obviate the repression effect by arginine. Transport studies, carried out on prototrophic revertants (RC-13 and RC-14) of the arginine marker, showed the same differences in transport activities found with the original parent and mutant strains. The S/V
versus S plots of data for the kinetics of entry of arginine and ornithine are shown in Fig. 2 and Fig. 3. The constant values calculated from these plots are presented in Table 2. The analysis of these data bears resemblance to the situation found in the canavanine-resistant strain described in the accompanying paper (6). The two permeases for ornithine are functioning in the mutant with an elevated maximal influx of substrate through the specific system. The kinetics of entry of arginine in the mutant revealed the operation of only one system with kinetic values comparable to those that describe the arginine-specific system in the wild0.6 0.5 0.4 S V
0.3
0.2 0.1 0
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
[AM]
FIG. 2. Kinetics of L-arginine entry in parent and mutant strains. Cells were grown in medium A supZ ARGININE [1,MM] LYSINE [I10M] plemented with acetyl-glutamate and initial rates of w 1--8 ~~~~~~~~4 influx were measured as described in the text, by using L-[3H]arginine at different concentrations be0Z tween 0.5 x 10-8 and 10-4 M. The circles represent cr 6 ~~~~~~~~~~3 the experimental values; the line was drawn by using 4 the theoretical values calculated from the equation -J42 for two simultaneous reactions on the same substrate (6). Symbols: 0, parent strain; *, mutant strain. SI w V, Molarity per nanomole per milligram of cellular protein per minute. E CA
~~~~~~~LYSINE [0.1AM]
0
c8
12
1.0 10
w
0.
S
o4
ORNITHINE
1I0gmJ
0.5
0 2
V
6 4
z
2
2
6
10
2 MINUTES
6
10
FIG. 1. Time-dependence uptake of labeled amino acids in parent (MA-5) and mutant (RC-10). Transport was measured as outlined in the text. The initial concentration is indicated for each substrate. Symbols: *, uptake in parent strain; 0, uptake in the mutant.
A
%J
0
2
3
4
5
6
7
8
9
[ALM] FIG. 3. Same as Fig. 2, but with L-[3H]ornithine. Symbols: 0, parent strain; 0, mutant strain. SIV, Molarity per nanomole per milligram of cellular protein.
VOL. 130, 1977
ARGININE TRANSPORT AND BIOSYNTHESIS REGULATION
type strain a similar Km and a fivefold increased Vmax of influx. The S/V versus S plot of data for the kinetics of lysine entry (not shown) revealed the operation of the two lysine transport systems in the mutant with constant values similar to those that describe the corresponding systems in the parent strain. These results are taken to mean that the mutation does not affect the lysine transport, nor the LAO system, nor the lysine-specific system. -
Arginine biosynthesis and arginine and ornithine conversions on the mutant strain. The pathway of arginine biosynthesis in the mutant is repressible by arginine. The activity of the enzyme OTC depends upon the absence or presence of arginine in the culture media (see Table 5). The incorporation of arginine into tRNA, as well as the activity of the enzymes involved in the conversion of both arginine and ornithine into putrescine, is not affected by the mutation in strain RC-10 (Table 3).
1247
Binding proteins on the mutant. Binding protein assays with shock fluids from parent
and mutant strains appear in Table 4. There is a four- to fivefold increased binding activity with labeled arginine and the shock fluid released from the mutant. Since the amount of protein released after the osmotic shock is similar for cells from parent and mutant strains (4 to 5% of the total cellular protein), and since the levels of acid phosphatase activity are also comparable in both strains, it can be concluded that the mutation does not affect the release of particular components of the periplasmic area. Fractionation of the crude osmotic shock fluid on DEAE-cellulose chromatography (Fig. 4) showed that the arginine-specific binding protein II is the molecular compound responsible for the elevated binding activity with arginine in the crude shock fluid from the mutant. In Fig. 5, an SDS-polyacrylamide gel of periplasmic proteins is presented. Electrophoretic
TABLE 2. Kinetic values for uptake ofarginine and ornithine on strains MA-5 (parent) and RC-10 (mutant)a Substrate
Arginine
K,
=
Strain MA-5 (parent) 1.0 x 10-7 M, V1 = 1.66
K2 = 1.0 X 10-" M, V2 =0.04 K, = 6.0 x 10-6 M, VI = 1.0
Ornithine
Strain RC-10 (mutant)
K,
=1.5 x 10-7, V1 = 8.0
K, = 4.0 x 10-6, V1 = 4.0 K2 = 1.0 x 10- M, V2 = 0.28 K2 = 1.0 x 10-i, V2 = 0.35 a The constants were calculated from S/V versus S plots as described in the accompanying paper (6). V1 and V2 are measured in nanomoles per milligram of cellular protein per minute. Initial rates of uptake were measured as described in the text. TABLE 3. Activities of enzymes involved in the metabolism of argininea Enzyme sp act (U/mg of protein)' Enzyme
Product measured
Parent (MA-5)
Mutant (RC-10)
Arginine biosynthetic decarboxylase CO2 0.007 0.006 Arginine biodegradative decarboxylase CO2 0.060 0.040 Ornithine biosynthetic decarboxylase CO2 0.019 0.021 Ornithine biodegradative decarboxylase 0.007 0.008 C02. Agmatine ureahydrolase Urea 0.026 0.018 Arginine tRNA synthetase Arg-tRNA 2.05 2.66 a Enzyme assays are described in the text. b Units are micromoles of product formed per minute, except for arginine-tRNA synthetase where units are nanomoles of labeled arginine incorporated into tRNA per minute. TABLE 4. Binding activity on shock fluida Total unitsb
Sp act (U/mg)c
Strain
Arg
MA-5 (parent)
120
Lys 82
Orn
Arg
76
0.58
Lys 0.40
Acid phosphaOn
tase (U/mg)
0.37
6.96
RC-10 (mutant) 530 77 74 0.41 0.40 2.80 6.84 a Binding activity for each amino acid was measured at 10 MM initial concentration by equilibrium dialysis on Plexiglas chambers as described in the text. Units of acid phosphatase = micromoles of phosphate released per hour per milligram of shock fluid protein. b Nanomoles of amino acid bound to the shock fluid obtained from a 10-liter culture. c Nanomoles of amino acid bound per milligram of shock fluid protein.
1248
J. BACTERIOL.
CELIS z 0 U
4
2.5
CD 15 N.
a
z
2.0
0 CD
0
10 1.5 a
4
0
co
o
0
1.0
0
0.5
"')5 w C
0
0
50
100 150 200 FRACTION NUMBER FIG. 4. Elution profile of the DEAE-cellulose fractionation of the osmotic shock fluid from parent (MA5) and mutant (RC-10) strains. Two columns (0.9 by 45 cm) with equilibrated DEAE-cellulose were loaded with 280 mg ofshock fluid protein. Conditions for fractionation have been described (6). Arrow 1 shows where the gradient (0 to 0.1 M NaCI) was begun. Arrow 2 shows where the columns were washed with 2.0 M NaCl. Arginine binding activity was measured by equilibrium dialysis on Plexiglas chambers (6). Symbols: *, [3H]arginine binding with fractions eluted from the column loaded with shock fluid released from the parent (MA-5) strain; 0, [PHlarginine binding with fractionated shock fluid from the mutant (RC-10). 0
patterns are shown of the proteins contained in the shock fluids and the arginine-specific binding protein II after DEAE fractionation from parent and mutant strains. The electrophoretic bands, corresponding to the partially pure binding protein preparations from both strains, have similar mobilities. The high binding activity with the arginine-specific binding protein II after DEAE fractionation is confirmed in the stained slab gel. A heavier band of such periplasmic component can be seen in the shock fluid, subjected to electrophoresis, from the mutant. Scatchard plots of data of the binding activity between labeled arginine and the arginine binding protein II showed that the dissociation constant for the isolated protein from the mutant is similar (about 10-7 M) to that found for the molecule from the parent strain. The implication is that the mutation on strain RC-10 affects the synthesis of the arginine-specific binding protein II, but not its physical properties or affinity constant. Mapping of strain RC-10. Genetic linkage between the mutation responsible for the elevated transport of arginine and ornithine and the serA marker was explored by transduction studies using the mutant strain RC-10 as donor and strain AB-856 (6) as recipient. Of more
than 200 serA + transductants analyzed, none of them revealed a high sensitivity to inhibition of growth by canavanine. Conjugation experiments, carried out by the procedure of Low (17), using 15 different Hfr strains, indicated that the mutation is located between the dsdA marker (point of entry of Hfr strain KL983) (16) and the lysA marker (point of entry of Hfr strain KL16) (16) on the linkage map of E. coli K-12. The Hfr strains were used as donors. The recipient strain was a streptomycin-resistant, argA+ prototrophic derivative of the original mutant RC-10. Recombinants were selected for the ability to grow on canavanine-supplemented plates after 48 h of incubation at 370C. Further transduction experiments indicated that the locus responsible for the increased transport and sensitivity to canavanine inhibition is closely linked (more than 90% cotransducible) to the argA marker. Details of this mapping will be published elsewhere (T.F.R. Celis, manuscript in preparation). Regulation of transport and biosynthesis of arginine and ornithine. If wild-type E. coli K12 cells are grown in the presence of 50 mg of Larginine or L-ornithine per liter, the initial rates of uptake of both amino acids are reduced about 70% (Table 5). The repression of arginine
ARGININE TRANSPORT AND BIOSYNTHESIS REGULATION
VOL. 130, 1977
1249
~~~~transport by either arginine or ornithine is substantially decreased in the mutant, and the / / formation of the omnithine transport system seems to be no longer repressible. If cells from the mutant are grown in medium supple- ~~~~ mented with a higher amount of arginine (500 mg/liter), more than 50% of arginine transport -~~~~~ ~can be repressed. However, the level of argihnine uptake is never lowered to the amount l l taken up by cells of the wild-type strain at gmaximum repression. In contrast, the biosynm S cthesis of arginine, measured by the level of w m s m OTC activity, is repressed as completely in the mutant as it is in the parent strain. These /f) g -
....Oexperiments indicate that the increased trans" in the and .. u activity of arginine )port It less sensitive ornithine to repression by is much mmutant s i a D 4tsgrowth in media containing arginine or oni( strain thine than in the parental strain. Furthermore, .inu-
