JOURNAL OF BACTRIUOLOGY, Mar. 1977, p. 1257-1265 Copyright C0 1977 American Society for Microbiology

Vol. 129, No. 3 Printed in U.S.A.

Role of Transport Systems in Amino Acid Metabolism: Leucine Toxicity and the Branched-Chain Amino Acid Transport Systems STEVEN C. QUAY,1 THOMAS E. DICK, AND DALE L. OXENDER* Department of Biological Chemistry, The University of Michigan Medical School, Ann Arbor, Michigan 48109 Received for publication 13 October 1976

The livR locus, which leads to a trans-recessive derepression of branchedchain amino acid transport and periplasmic branched-chain amino acid-binding proteins, is responsible for greatly increased sensitivity toward growth inhibition by leucine, valine, and serine and, as shown previously, for increased sensitivity toward toxicity by branched-chain amino acid analogues, such as 4azaleucine or 5',5',5'-triflvoroleucine. These phenotypes are similar to those of relA mutants; however, the livR mutants retain the stringent response of ribonucleic acid synthesis. However, an increase in the rate of transport or in the steady-state intracellular level of amino acids in the livR strain cannot completely account for this sensitivity. The ability of the LIV-I transport system to carry out exchange of pool amino acids for extracellular leucine is a major factor in leucine sensitivity. The previous finding that inhibition of threonine deaminase by leucine contributes to growth inhibition is confirmed by simulating the in vivo conditions using a toluene-treated cell preparation with added amino acids at levels corresponding to the internal pool. The relationship between transport systems and corresponding biosynthetic pathways is discussed and the general principle of a coordination in the regulation of transport and biosynthetic pathways is forwarded. The finding that the LIV-I transport system functions well for amino acid exchange in contrast to the LIV-Il system provides another feature that distinguishes these systems in addition to previously described differences in regulation and energetics.

Early experiments on the response of Escherichia coli to the transfer from rich to minimal medium (i.e., shift-down) indicated that regulatory mechanisms exist that control general ribonucleic acid (RNA) and protein synthesis (3, 17, 23). In addition, it was found that certain amino acids, such as leucine, could greatly lengthen the growth lag during a shift-down experiment (13, 23). The mechanism of growth inhibition by leucine was studied previously, and suggestions included effects on RNA synthesis (13), regulatory anomalies of acetohydroxy acid synthetase (30) and, more recently, inhibition of threonine deaminase (5, 33). The finding that mutants constitutively repressed for threonine deaminase were permanently leucine sensitive pointed to this enzyme as the site of leucine sensitivity (19). Our own interest in this area arose when we observed an increased leucine sensitivity of strains carrying the livR 1 Present address: Departments of Biology and Chemis-

locus, which codes for a trans-recessive element in branched-chain amino acid transport regulation and which leads to derepression of the LIVI and LIV-Il transport systems and corresponding binding proteins. The LIV-I uptake system involves the shockable binding proteins and has a high affinity for its substrates, while the LIV-II system is membrane bound and of a lower affinity (2). We will present evidence in this report that leucine sensitivity is greatly enhanced through isoleucine limitation by transport-coupled exchange of the intracellular isoleucine for extracellular leucine. (A preliminary report of portions of this work was presented at the annual meeting of the American Society of Biological Chemists [G. Cecchini, S. C. Quay, T. E. Dick, and D. L. Oxender, Fed. Proc. 35:1357, 19761.)

MATERIALS AND METHODS Bacterial strains. The strains from E. coli K-12 try, Massachusetts Institute of Technology, Cambridge, used in this study were strains E0300 (F-, wild-type MA 02139. ATCC 14948); E0317 (dlu livR; derived from strain 1257

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EO300 as described elsewhere [2, 28]); and CP79 (Arg- His- Leu- Tyr- Thi- relA [10]) obtained from M. J. Fournier. Materials. Morpholinopropane sulfonic acid (MOPS), tricine [N-tris(hydroxymethyl)methyl glycine], and nutritional supplements were obtained from Sigma Chemical Co., St. Louis, Mo. [63H]uridine was the generous gift of G. R. Greenberg, who obtained it from Amersham/Searle Corp. and who purified it by ascending chromatography with Whatman 3MM filter paper and the solvent isobutyric acid-concentrated NH4OH-water (66:1:33 by volume). This purification removes 3HOH and other non-charcoal-absorbable contaminants (32). All other chemicals were obtained from commercial sources and were of reagent grade. Growth conditions. Cells were grown with shaking at 37°C in MOPS salts supplemented with 0.02 M )-glucose (MOPS-G) as described previously (24). Culture density was followed spectrophotometrically as described previously (26). Cells were harvested by centrifugation at 4°C, except where indicated in the text, and were washed with MOPS salts. Threonine deaminase assay. Threonine deaminase (EC 4.2.1.6; L-threonine hydrolyase [deaminating]:threonine dehydratase) was assayed in toluene-treated cells by the method of Burns (4), except the absorbance of the 2,4-dinitrophenylhydrazone derivative of a-ketobutyrate was measured at 530 nm in a Zeiss PMQ2 spectrophotometer. The 2,4dinitrophenylhydrazine reagent was prepared fresh every 2 weeks as a 0.025% solution in 0.5 N HCI and stored in a light-proof bottle at 8°C. The toluenetreated cells were prepared by suspending washed cell pellets with 1 ml of 0.01 M tris(hydroxymethyl)amino methane (Tris)-hydrochloride (pH 8), and 0.05 ml of toluene and vortexing the suspension for 30 s. Specific activity is expressed as units per gram of protein, where a unit is 1 ,umol of a-ketobutyrate formed per min under the above conditions. Protein was determined by the method of Lowry et al. (21). Crystalline bovine albumin was used as a standard. Corrections were made for Tris buffer. Exodus experiments. The exodus of radiolabeled amino acids from cells was performed as described previously (12). Cells were grown, harvested, and washed thrice as described above. The cells were resuspended in MOPS salts to a density of 0.03 to 0.07 mg/ml and allowed to take up [3H]valine or [3H]isoleucine at 37°C to steady state, which occurred after 8 to 11 min for both substrates. The cultures were centrifuged at 15°C and resuspended in MOPS salts at 37°C. The zero time was taken at the instant the cultures were resuspended. The earliest time points represent a 6-s incubation. The time course of exodus was measured by removing 0.5-ml samples at appropriate intervals, filtering these through a 24-mm membrane filter (Millipore Corp.; 0.45-j,m pore size), and washing with 5 ml of MOPS salts at 37°C. Radioactivity was counted in a Packard liquid scintillation spectrometer with a standard scintillator in Triton X-100-toluene solution (26). Measurements of intracellular amino acids. Cul-

J. BACTERIOL. tures (100 ml) of appropriate strains were grown in MOPS-glucose in 500-ml Erlenmeyer flasks. At densities of 0.07 to 0.11 mg/ml (dry weight) the cells were harvested by centrifugation (at 20°C) for 10 min at 3,000 x g. The pellets were rapidly suspended in 1 ml of 3% sulfosalicylic acid with 0.1 ,umol of Lnorleucine and incubated at 100°C for 10 min. After centrifugation for 10 min at 4°C, the acidic and neutral amino acids were determined with a Beckman model 120C amino acid analyzer (22), using Lnorleucine as an internal control. The recovery of amino acids averaged 80% of theoretical, based on norleucine recovery. The pellet was resuspended in 0.2 N NaOH, and the protein was estimated by the method of Lowry et al. (21). The pool amino acids are expressed as millimoles per liter of cellular water. Measurement of RNA. RNA was measured by the uptake of [6-3H]uridine into trichloroacetic acid-insoluble material. Strains were grown at 37°C in MOPS-glucose with 20 amino acids (at 0.2 mM each) containing 0.10 mM [6-3H]uridine. At appropriate intervals, 0.5-ml samples were removed, filtered on 0.45-,um pore size nitrocellulose filters, and washed with 5 ml of 40C 10% trichloroacetic acid, and the retained radioactivity was measured by liquid scintillation as described above. To test for a relaxed phenotype, cultures growing in MOPS-glucose with 20 amino acids were harvested and resuspended with 0.10 mM of [6-3H]uracil in MOPS-glucose containing 1 mM valine to impose an isoleucine deprivation (18). This culture was shaken at 370C and periodically assayed for [6-3H]uracil incorporation into trichloroacetic acid-insoluble material as described above.

RESULTS Transitions in steady states of growth. During experiments to genetically map loci involved in the regulation of branched-chain amino acid transport proteins (2), it became clear that mutants derepressed for transport showed a long lag in the resumption of growth after certain shifts in medium composition. Preliminary work indicated that the growth lag was related to the presence of leucine in the growth media. Figure 1 shows growth curves of strains E0300 (wild type) and E0317 (livR) in rich and minimal media and the effect of shifting these cultures to minimal media plus 0.4 mM leucine. Figure 1A shows that both strains grew at the same rate in minimal media and that the addition of leucine to 0.4 mM caused a rapid and transient slowing of growth. The time required for establishment of a new steady state of growth with leucine was 16 and 70 min for strains E0300 and E0317, respectively. The addition of isoleucine and valine or isoleucine alone completely prevented the growth lag. In rich media (Fig. 1B) the strains grew more rapidly and the response of the cultures to a "classical nutritional shift-down" (17)

VOL. 129, 1977

LEUCINE TOXICITY AND TRANSPORT

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FIG. 1. Effect of leucine addition on the growth of strains E0300 and E0317. Solid symbols, Strain E0300; open symbols, strain E0317. (A) Cultures were established in steady-state growth in MOPS-G and, at the time indicated, either 0.4 mM leucine (squares) or 0.4 mM leucine, 0.4 mM isoleucine, and 1.0 mM valine (triangles) were added. (B) Cultures were established in nutrient broth and, at the time indicated, were washed and transferred to MOPS-G medium. (C) Cultures were established in nutrient broth and, at the time indicated, were washed and transferred to MOPS-G medium containing 0.4 mM leucine. Growth was followed as described in Materials and Methods. The data have been corrected for the small (1 to 2%) dilution that occurred when the additions were made.

was identical. The downshift from rich media to minimal media containing 0.4 mM leucine caused a decrease in the growth rate for both strains (Fig. 10). Although strain E0300 returned to a faster growth rate in about 6 h, strain E0317 remained at the slower growth rate for over 16 h. This transient inhibition of growth by leucine was also seen in relA mutants (13, 30). The relA and livR loci were shown to be distinct by measuring the rate of uracil incorporation into RNA during an isoleucine limitation, imposed by the addition of valine. The response of strain CP79, the archetypal relaxed mutant (10), was characteristic, and the stringent response of the livR strain indicates this strain contained an relA+ allele (data not shown). Amino acid pools during growth transitions. The decrease in growth associated with leucine supplementation was prevented by the addition of isoleucine and valine to the medium. The sparing effect of those amino acids could result if: (i) leucine interfered with the level of one or both of these amino acids either by inhibiting biosynthesis or by stimulating "utilization" (for example, by increasing losses via exchange transport), or (ii) if the site of leucine toxicity was unrelated to isoleucinevaline metabolism, but these amino acids could block the action of leucine. One possible site where isoleucine and valine could compete with leucine is at the level of transport (29). If the sparing effect of isoleucine and valine results from an inhibition of leucine uptake, glycylisoleucine and glycylvaline should not prevent leucine toxicity, since they are substrates of the

dipeptide uptake system (7). In fact, these dipeptides at 0.06 mM completely prevent growth inhibition by 0.4 mM leucine. To test if the inhibitory action of leucine is related to lowering the intracellular level of isoleucine or valine, the sulfosalicylate-extractable pool amino acids were measured under various growth conditions. Table 1 contains the acidic and neutral amino acid levels in strains E0300 and E0317 grown in minimal medium and 10 min after the addition of 0.4 mM leucine. The important features of these data are: (i) the threefold-higher steady-state leucine level in strain E0300 relative to strain E0317; (ii) the striking increase in intracellular leucine after the addition of extracellular leucine, even though the level of leucine reached in both strains was the same; (iii) the intracellular valine levels were not lowered and, in fact, increased slightly after leucine addition; (iv) the intracellular isoleucine level decreased dramatically after leucine addition (47% in strain E0300 and 93% in strain E0317); (v) alanine, the source of cellular pyruvate and ultimately valine, decreased 46% in strain E0300 and 88% in strain E0317 after leucine addition; and (vi) threonine, the first compound in isoleucine biosynthesis, increased after leucine supplementation. At 180 min after leucine addition, when strain E0300 had resumed uninhibited growth, the isoleucine and threonine levels returned to minimal medium levels. At the same time, strain E0317 had very low isoleucine levels and was still growing at a greatly reduced rate. The decrease in methionine levels that occurred after 10 min in strain E0317 was restored to

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TABLE 1. Amino acid pools under various growth conditionsa Strain E0317

Strain E0300 With Leu- With leu- Steady cine cine state with medium ___________(10 min) (180 min) leucine Amino acid

Alanine Aspartamine(ate) Cysteine |

Minimal 5.80

3.15

1.93

3.60 0.03 29.85 1.95 0.09 1.70 0.17

4.35