Vol. 130, No. 3 Printed in U.S.A.

JOURNAL OF BACTUROLOGY, June 1977, p. 1024-1029 Copyright 0 1977 American Society for Microbiology

Regulation of Amino Acid Transport in Escherichia coli by Transcription Termination Factor Rho STEVEN C. QUAY1 AND DALE L. OXENDER* Department ofBiological Chemistry, The University of Michigan Medical School, Ann Arbor, Michigan 48109 Received for publication 1 February 1977

Amino acid transport rates and amino acid binding proteins were examined in a strain containing the rho-120 mutation (formerly SuA), which has been shown to lower the rho-dependent, ribonucleic acid-activated adenosine triphosphatase activity to 9% of the rho activity in the isogenic wild-type strain. Tryptophan and proline transport, which occur by membrane-bound systems, were not altered. On the other hand, arginine, histidine, leucine, isoleucine, and valine transport were variably increased by a factor of 1.4 to 5.0. Kinetics of leucine transport showed that the LIV (leucine, isoleucine, and valine)-I (binding protein-associated) transport system is increased 8.5-fold, whereas the LIVII (membrane-bound) system is increased 1.5-fold in the rho mutant under leucine-limited growth conditions. The leucine binding protein is increased fourfold under the same growth conditions. The difference in leucine transport in these strains was greatest during leucine-limited growth; growth on complex media repressed both strains to the same transport activity. We propose that rho-dependent transcriptional termination is important for leucine-specific repression of branched-chain amino acid transport, although rho-independent regulation, presumably by a corepressor-aporepressor-type mechanism, must also occur. The regulation of branched-chain amino acid transport has been extensively studied recently (13-16). Our current understanding ofthis process indicates that leucine, but not isoleucine or valine, interacts with leucine transfer ribonucleic acid (RNA) and leucyl-transfer RNA synthetase in affecting repression. This regulation has been established as primarily changing the differential rate of synthesis of transport components relative to total cellular proteins, without changing the rate of turnover of the ratelimiting component(s) for transport (15). The recent genetic mapping of a trans-recessive transport regulatory gene (1) and the identification of the structural genes for the leucine, isoleucine, valine (LIV), and leucine-specific binding proteins (J. J. Anderson and D. L. Oxender, manuscript in preparation) complement these physiological studies. Significant advances in the understanding of the regulation of histidine and tryptophan biosynthetic operons have recently been made (2, 6). In addition to regulation by the classical mechanism of inhibition of RNA polymerase initiation by a corepressor-aporepressor comI Present address: Department of Biology and Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139.

plex (8), there appear to be methods for changing the frequency of RNA transcriptional termination, apparently at an attenuator site in the deoxyribonucleic acid proximal to the structural genes (the "leader region") (6). The protein factor rho, originally identified as important in the relief of mutational polarity (4), seems to be involved in transcriptional termination at the attenuator site in the trp leader region (9). The purpose of this communication is to determine if mutations of the rho factor affect leucine-dependent regulation of branchedchain amino acid transport. We report here that a strain containing an altered rho factor has greatly derepressed branched-chain amino acid transport and binding protein activity. The findings communicated here strongly imply that at least some aspects of transport regulation occur at the level of messenger RNA transcription, perhaps at an attenuator site proximal to the transport protein structural genes. MATERIALS AND METHODS Bacterial strains. The Escherichia coli K-12 strains used in this study were CU300 (W3110, trpE(Oc)9851 leu(Am)277 SuA+) and CU2054 (CU300 with SuA120), isolated-by Morse and Guer-

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REGULATION OF AMINO ACID TRANSPORT BY RHO

tin (11). The SuA120 allele is a missense mutation in the rho factor (17), which has 9% of the wild-type rho activity as measured by RNA-activated adenosine 5'-triphosphate hydrolysis (18). To conform to current nomenclature (3, 9), the SuA locus will be referred to as rho. Materials. MOPS (morpholinopropane sulfonic acid), tricine [N-tris(hydroxymethyl)methyl glycine], and nutritional supplements were obtained from Sigma Chemical Co. (St. Louis, Mo.). The radioisotopes used were: L-[4,5-3H]leucine (53 Ci/ mmol; Amersham); L-[4,5-3H]isoleucine (30 Ci/ mmol; Amersham); i[G-3H]tryptophan (2 to 10 Ci/ mmol; New England Nuclear Corp.); L-[G3H]proline (5 to 10 Ci/mmol; New England Nuclear Corp.); i[2,3-3H]valine (17.4 Ci/mmol; Amersham); _[U-14C]arginine (>270 mCi/mmol; New England Nuclear Corp.); L-[2,3-3H]histidine (55 Ci/mmol; Amersham); _[U-14C]glutamine (>200 mCi/mmol; New England Nuclear Corp.). All other chemicals were obtained from commercial sources and were of reagent grade or better. Growth conditions. Cells were grown with shaking at 37°C in MOPS salts supplemented with 0.02 M n-glucose (MOPS-G) (12) as described previously (15). Cell growth was followed by measuring the absorbancy at 420 nm at room temperature, using a GCA-McPherson model EU-707-12 spectrophotometer. The specific first-order growth constant (k = ln 2/mass doubling time [hours]) was determined as described by Quay and Oxender (15). Cells were harvested by centrifugation at 4°C and were washed with MOPS salts. Transport assays. Transport assays were performed exactly as described elsewhere (15). The kinetics of leucine transport were analyzed using an iterative procedure, as described by Quay and Oxender (15). One unit of activity is taken as the uptake of 1 ,umol of substrate in 1 min under the standardized conditions. Previous work (15) indicates the standard deviation for the determination of transport capacity of a given culture varies from +5 to

represent the mean of three determinations and vary with a standard deviation of 12%.

RESULTS

Growth properties. Preliminary experiments were performed to examine the growth characteristics of the isogenic CU300 (rho+) and CU2054 (rho-120) strains. As reported previously (9), in MOPS-G supplemented with 0.4 mM leucine and 25 mg of tryptophan per liter, the strains grew with nearly identical firstorder growth rate constants (0.55 and 0.65 h-1 for strains CU300 and CU2054, respectively). The strains were distinctive, however, in their response to a sudden reduction in the supply of leucine. Logarithmically growing cultures in MOPS-G medium containing 0.6 mM leucine and 25 mg of tryptophan per liter were diluted with identical medium without leucine to give a final concentration of leucine of 0.09 mM. This particular dilution was chosen because it produced a transient growth limitation in both strains after about 40 min. The delay in the onset of growth limitation corresponds to the time required for the cells to deplete the level of leucine in the medium below a level that can support growth without derepressing the highaffinity transport system for leucine. After growth limitation occurred, the rather long lag (68 min) in growth experienced by the wild-type strain was much shorter (22 min) in the rho-120 strain (Fig. 1). Since the leu(Am) locus precludes derepression of leucine biosyn0.30

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Preparation of shock fluid. After harvesting and washing appropriate cultures, the cell pellets were shocked as described previously (13). The shock fluid was concentrated to a convenient volume using an Amicon B15 Minicon macrosolute concentrator with a molecular weight cutoff of 15,000. By this method, a 1-liter culture, containing 50 to 60 mg (dry weight) of cells, was shocked in 15 to 20 ml of doubly distilled water and concentrated to 0.1 to 0.2 ml for binding protein assays. Amino acid binding assays. The binding assay for amino acids was modified (13) by usiIng cellulose dialysis tubing (1-cm flat diameter), which contained 0.1 to 0.2 mg of protein in 50 to 100 ,ul of buffer, and dialyzing this solution at 4°C against 400 ml of 0.01 M potassium phosphate, pH 7.2, containing 10-4 M MgSO4 and 10 AtM radiolabeled amino acids for 24 to 32 h. Samples were removed for protein determination (10) and for liquid scintillation spectrometry (15). One unit of activity represents the binding of 1 ,umol of amino acid under these assay conditions. The values reported in this paper

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TIME, MIN FIG. 1. Growth curves of strains CU300 and CU2054. Cells were grown on MOPS-G supplemented with 0.6 mM leucine and 25 mg of tryptophan per liter. At the arrow, the cells were rapidly harvested by centrifugation and suspended in the same medium except the leucine level was lowered to 0.09 mM. Cell density was estimated by measuring absorbance at 420 nm in a GCA-McPherson spectrophotometer at 37°C and using a cuvette with a 1-cm light path. Symbols: (O) strain CU300; (0) strain CU2054.

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QUAY AND OXENDER

J. BACTZRIOL.

thesis after this leucine limitation, we tenta- media containing from 0.05 to 0.6 mM leucine tively conclude that some other leucine-procur- and measuring leucine transport after steadying mechanism, i.e., transport, is more rapidly state growth was established (defined as a conderepressed in response to leucine limitation in stant first-order growth rate constant for at least three generations). Only in the medium the rho-120 strain. Amino acid transport capacity in the rho containing the lowest leucine levels was care mutant. The uptake of a number of amino acids required to assure logarithmic growth, i.e., sevwas examined in the wild-type strain CU300 eral transfers to keep the cell density below and in CU2054, the rho-120 mutant. These about 0.015 mg/ml (dry weight). Figure 2 prestrains were grown in MOPS-G medium or in sents the results of the experiment and indiMOPS-Casamino Acids, and transport of eight cates the dramatic effect leucine limitation has substrates was measured (Table 1). Examining on the rho-associated increase in leucine transthe MOPS-G-grown cells first, one can see a port. Whereas the wild-type strain undergoes a number of differences in transport capacity as a 3.9-fold derepression during leucine limitation, result of the rho mutation. The uptakes of argi- the rho-120 strain shows a 15-fold increase nine, histidine, leucine, isoleucine, and valine upon shifting from 0.6 to 0.05 mM leucine. are increased from 32 to 94%. Although these Whereas the wild-type derepression of 3.8-fold increases are small, they are highly reproduci- is somewhat lower than that usually seen forE. ble from three to eight standard deviations coli K-12 (five- to eightfold is more typical [15]), above the wild-type level. Glutamine transport, it is not unusual to observe such variations in on the other hand, was decreased to one-third different strain backgrounds. The derepression the wild-type capacity. No change was seen in in the rho-120 strain is much larger and is the uptake of proline or tryptophan. In con- reminiscent of the derepression seen in trast, growth in Casamino Acids had two ef- leuS(Ts) mutants (14, 15). fects: a tendency to negate the transport differLeucine transport kinetics in the rho-120 ences in the rho-120 strain and a generalized strain. Since the experiment depicted in Fig. 2 decrease in all transport capacities. was done at a single leucine level, we wished to Specificity of the increase in branched- examine in a more quantitative manner the chain amino acid transport. Since the differ- derepression of transport in the presence of a ence in leucine, isoleucine, and valine trans- defective rho factor. For this purpose cells were port in strain CU2054 was small, the possibility grown in MOPS-G containing 0.1 mM leucine, existed that this increase could result from and the transport of leucine was examined over some rather nonspecific alteration in the a 58-fold range, from 8.5 x 10-8 to 5.0 x 10- M. growth properties of the rho-120 strain. The Figure 3 contains a Lineweaver-Burk plot of leu(Am) locus in these strains pernitted a test for leucine-linked derepression of transport, as .0.30 I I has been described in detail elsewhere (15). The experiment consisted of transferring logarithmically growing cultures in 0.6 mM leucine to TABLE 1. Effect of a mutation in termination factor rho on amino acid transporta

° 0.20 a

Z

Sp act (U/g of cells, dry wt)

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Substrate

Arginine, 3 AM Glutamine, 1 EiM Histidine, 1 ,uM Isoleucine, 1 ,uM Leucine, 1 MLM Proline, 3 izM Tryptophan, 3 MM Valine, 3 MM

Minimal

medium

Complex

medium

CU300 CU2054 CU300 CU2054 2.92 3.61 0.60 1.09 1.90 0.63 _b 0.20 0.38 0.12 0.24 0.06 0.05 0.10 0.19 0.030 0.035 0.17 0.17 0.090 0.100 0.51 0.55 0.78 0.84 0.31 0.61

a Cells were grown in MOPS-G, 0.2 mM leucine, and 25 mg of tryptophan per liter for the minimal medium. Complex medium consisted of MOPS-G plus 0.2 mM of all 20 amino acids. Cells were harvested and transport was assayed as described in the text. b-, Not determined.

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.-n .k mM ACIDS LLEUCINEJ, -

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FIG. 2. Leucine-dependent derepression of transport in wild-type and mutant rho strains. Cells were grown as described in the text in MOPS-G supplemented with 25 mg of tryptophan per ml and 0.3% Casamino Acids. After steady-state growth had been established, the cultures were harvested by centrifugation, and the uptake of 0.1 pM leucine was measured as described in the text. Symbols: (O), strain CU300; (0), strain CU2054.

REGULATION OF AMINO ACID TRANSPORT BY RHO

VOL. 130, 1977 -

'

'

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8

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3: ac

3

12

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0 -4

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/S . MM 3. Kinetics of leucine transport in leucined cultures of strains CU300 and CU2054. Cells rrown in MOPS-G supplemented with 25 mg of tryphc:phan per liter and 0.1 mM leucine. Cells were harveQsted and transport capacity was measured as descricbed in the text. The solid lines represent theoreticaiI curves obtained by applying the kinetic paramet 'ers to the equation for two saturable systems, as CU2054. thehe text.ext given inin (0) CU0s4 (0) C300 CU300; (O () Symbols: Sybol:

these data with the solid lines drawn assuming the following equation describing the relationship of the observed velocity to the leucine concentration: (leucine) = Vmax, (leucine) vOb, Km, + (leucine) + Vmax2+ (leucine) The kinetic parameters for strain CU300, which were calculated as described previously (15), were: K,, = 0.15 ,uM, Viax, = 0.11 U/g, Km, = 3.0 uM, and Vmax, = 0.89 U/g. In a similar fashion, for strain CU2054 Km, = 0.20 ,uM, Vmax, = 0.85 U/g, Km, = 3.0 iM, and Vmax, = 1.4 U/g. As described previously, Km, and Vma,, are associated with the high-affinity LIVI uptake system, and Km, and Vma.r, are related to the low-affinity LIV-Il system. The kinetic parameters for strain CU300 are very similar to those expected for repressed cells, which was initially unexpected since these cultures had been grown in 0.1 mM leucine, a level normally causing transport derepression in batch culture (15). It should be noted, however, that our present method of multiple-culture transfers at low total cell density is unlike our previously used method of allowing maximum growth at low leucine levels and may prevent complete derepression. The 0.1 mM cultures were limited for leucine in some sense, at least, since the first-order growth rate constant was reduced from 0.55 + 0.01 h-' in high-leucine MOPS-G to 0.42 + 0.02 h-l for strain CU300. Amino acid binding proteins in the rho mutant. Periplasmic amino acid binding proteins have been proposed as components of several of

K+,

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the amino acid transport systems studied here (for review, see 13). One obvious correlate of this hypothesis is the coordinate regulation of transport capacity in whole cells and binding protein activity, as measured in vitro. The specific activity of leucine, histidine, arginine, and glutamine binding proteins in concentrated osmotic shock fluids was examined from MOPS-G cultures of strains CU300 and CU2054 (Table 2). To begin with, there are no major differences in the efficiency of the osmotic shock procedure for these strains. On examination of the specific activities of the binding proteins, it can be seen that the leucine binding activity showed a fourfold increase in the rho-120 strain. This compares favorably with the derepression of the

LIV-I transport system under the same growth conditions. In a similar manner, the histidine and arginine binding proteins are slightly increased in strain CU2054. The correlation between transport and binding protein activity is also seen for glutamine, which shows a threefold decrease in transport (Table 1) and a twofold decrease in binding protein specific activity. DISCUSSION The experiments reported in this paper were made to determine if rho was involved in regulation of branched-chain amino acid transport. Previous work had shown that transcriptional termination was important as a regulatory mechanism for late gene expression in bacteriophage lambda (19) and for the trp, his, and probably ilv biosynthetic operons in E. coli (2, 6, 20). In the biosynthetic operons, this regulation is apparently affected by rho (9), a protein originally identified as a suppressor of mutational polarity (4). Our data indicate that derepression of branched-chain amino acid transport in the wild-type strain is strongly limited by rho-dependent transcriptional termination. TABLE 2. Effect of a mutation in termination factor rho on amino acid binding proteinsa Binding activity (U/g of Substrate

protein)b CU300

CU2054

0.69 0.54 Arginine 0.20 0.35 Glutamine 0.58 0.41 Histidine 1.23 0.33 Leucine 0.041 0.050c Total shock protein a Cells were grown in minimal medium as described in Table 1. b Binding activity was assayed as described in the text. c Milligrams of protein released by osmotic shock per milligram of total cellular mass (dry weight).

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QUAY AND OXENDER

This is the first evidence we have obtained that indicates that transport regulation occurs, at least in part, at the level of messenger RNA transcription. The possibility that the effects of the rho-120 mutation are the result of nonspecific "read through" from some adjacent operon seems unlikely since the derepression seen was regulated by leucine. At the present time, numerous individual mutants exist at the rho locus (7, 17, 18). These differ greatly in their ability to suppress polarity in vivo or their RNA-dependent adenosine triphosphatase activity. The most severe rho mutant at present appears to be the temperature-sensitive strains isolated by Das et al. (7). These strains show no transcription termination 'activity at any temperature in vitro. In contrast, the SuA locus studied in the present work is a much weaker polarity suppressor (6, 18). Transport assays with a strain containing a rho polypeptide of slower polyacrylamide gel electrophoretic mobility indicate this strain is derepressed to a greater extent than rho-120 (unpublished observation). Although the significance is not known, neither "membrane-bound" transport system studied (proline or tryptophan) was derepressed. On the other hand, all binding protein-related systems were altered by the rho-120 locus. In the branched-chain amino acid transport systems, although the membrane-bound LIV-I system was slightly increased, the major effect was upon the binding protein-related LIV-I system. The observation that the major effect of a mutation in rho is on the binding protein uptake systems must be correlated with the recent evidence that the component of the membrane-bound Ca2+-, Mg2+-adenosine triphosphatase is altered in rho mutants (S. Adhya, personal communication). Previous work has indicated that the binding protein uptake systems receive energy from adenosine 5'-triphosphate, whereas the membrane-bound systems use electron transport energy more directly (5). We are examining the possible relationship of this recent finding to leucine transport regulation. The effect of rho mutations on the glutamine transport system and binding protein may be less related to a direct regulatory effect of rho on that uptake system than to an indirect action on the intracellular level of energy or some other "effector" of glutamine transport capacity. Evidence has been presented that cyclic adenosine 3',5'-monophosphate, NH4+, or some other compound may be a regulator of glutamine transport (21). The finding that growth in Casamino Acids negates the increase in transport components

J. BACTERIOL.

found in the rho mutant indicates that additional regulatory controls exist under these growth conditions. One could imagine that the major regulatory control in this situation might be prevention of RNA polymerase initiation, as in the classic model of Jacob and Monod (8). ACKNOWLEDGMENTS We thank H. E. Umbarger of Purdue University for supplying the bacterial strains used in this study. This investigation was supported by Public Health Service grant GM11024 to D. L. 0. from the National Institute of General Medical Sciences. LITERATURE CITED 1. Anderson, J. J., S. C. Quay, and D. L. Oxender. 1976. Mapping of two loci affecting the regulation of branched-chain amino acid transport in Escherichia coli K-12. J. Bacteriol. 126:80-90. 2. Artz, S. W., and J. R. Broach. 1976. Histidine regulation in Salmonella typhimurium: an activator-attenuator model of gene regulation. Proc. Natl. Acad. Sci. U.S.A. 72:3453-3458. 3. Bachmann, B. J., K. B. Low, and A. L. Taylor. 1976. Recalibrated linkage map of Escherichia coli K-12. Bacteriol. Rev. 40:116-167. 4. Beckwith, J. 1963. Restoration of operon activity by suppressors. Biochim. Biophys. Acta 76:162-164. 5. Berger, E. A., and L. A. Heppel. 1974. Different mechanisms of energy coupling for the shock-sensitive and shock-resistant amino acid permeases of Escherichia coli. J. Biol. Chem. 249:7747-7755. 6. Bertrand, K., L. Korn, F. Lee, T. Platt, C. L. Squires, C. Squires, and C. Yanofsky. 1975. New features of the regulation of the tryptophan operon. Science 189:22-26. 7. Das, A., D. Court, and S. Adhya. 1976. Isolation and characterization of conditional lethal mutants of Escherichia coli defective in transcription termination factor rho. Proc. Natl. Acad. Sci. U.S.A. 73:19591963. 8. Jacob, F., and J. Monod. 1961. Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3:318-356. 9. Korn, L. J., and C. Yanofsky. 1976. Polarity suppressors defective in transcription termination at the attepuator of the tryptophan operon of Escherichia coli have altered rho factor. J. Mol. Biol. 106:231-241. 10. 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. 11. Morse, D. E., and M. Guertin. 1972. Amber suA mutations which relieve polarity. J. Mol. Biol. 63:605-608. 12. Neidhardt, F. C;, P. K. Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. J. Bacteriol. 119:736-747. 13. Oxender, D. L., and S. C. Quay. 1976. Isolation and characterization of membrane binding proteins, p. 183-242. In E. D. Korn (ed.), Methods in membrane biology, vol. 6. Plenum Press, New York. 14. Quay, S. C., E. L. Kline, and D. L. Oxender. 1975. Role of leucyl-tRNA synthetase in regulation of branchedchain amino-acid transport. Proc. Natl. Acad. Sci. U.S.A. 72:3921-3924. 15. Quay, S. C., and D. L. Oxender. 1976. Regulation of branched-chain amino acid transport in Escherichia coli. J. Bacteriol. 127:1225-1238. 16. Quay, S. C., D. L. Oxender, S. Tsuyumu, and H. E. Umbarger. 1975. Separate regulation oftransport and biosynthesis of leucine, isoleucine, and valine in bac-

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teria. J. Bacteriol. 122:994-1000. 17. Ratner, D. 1976. Evidence that mutations in the suA polarity suppressing gene directly affect termination factor rho. Nature (London) 259:151-153. 18. Richardson, J. P., C. Grimley, and C. Lowery. 1975. Transcription termination factor rho activity is altered in Escherichia coli with suA gene mutations. Proc. Natl. Acad. Sci. U.S.A. 72:1725-1728. 19. Roberts, J. W. 1975. Transcription termination and late

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control in phage lambda. Proc. Natl. Acad. Sci. U.S.A. 72:3300-3304. 20. Wasmuth, J. J., and H. E. Umbarger. 1973. Effect of isoleucine, valine, or leucine starvation on the potential for formation of the branched-chain amino acid biosynthetic enzymes. J. Bacteriol. 116:548-561. 21. Willis, R. C., K. K. Iwata, and C. E. Furlong. 1975. Regulation of glutamine transport in Escherichia coli. J. Bacteriol. 122:1032-1037.

Regulation of amino acid transport in Escherichia coli by transcription termination factor rho.

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