Transport of D- and L-Tryptophan in Bacillus megaterium by an Inducible Permeasel

Transcription

1 JOURNAL OF BACTEROLOGY, Jan. 1975, p Copyright i 1975 American Society for Microbiology Vol. 121, No. 1 Printed in U.S.A. Transport of D- and L-Tryptophan in Bacillus megaterium by an nducible Permeasel REYNARD R. BOUKNGHT AND HAROLD L. SADOFF* Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan Received for publication 2 October 1974 Tryptophan-grown cells of Bacillus megaterium ATCC contain a permease system that transports both D- and L-tryptophan and is inhibited by sodium aide. Arginine-grown cells contain little tryptophan permease activity, suggesting that the system is inducible. Arginine represses the tryptophan permease as well as the transport system for leucine and phenylalanine. Kynurenine was a more effective inducer of the tryptophan transport system than either D- or L-tryptophan. n bacteria, tryptophan transport is mediated by systems that require specific membrane proteins and the generation of energy (9). Escherichia coli has at least three tryptophan "permeases" (3, 5, 6). The general aromatic transport system is constitutive and has an affinity for tryptophan, phenylalanine, and tyrosine. There is also a specific constitutive transport system that transports only tryptophan. The third E. coli tryptophan permease is inducible and probably functions in tryptophan catabolism. Pseudomonas acidovorans also has an inducible tryptophan transport system (12) which is induced not by tryptophan but by kynurenine, a product of tryptophan breakdown. Konings and Freese (8) studied amino acid transport in Bacillus subtilis by using membrane vesicles. They observed energy-dependent radioactive uptake for all of the natural amino acids with the exception of L-tryptophan. However, competition studies demonstrated that tryptophan could be transported by a general aromatic amino acid permease. During studies of tryptophan catabolism in B. megaterium ATCC (4), we found that this organism produces a tryptophan permease that is induced by kynurenine and repressed by arginine. The induction of the permease by kynurenine is of considerable interest because this compound is an intermediate in tryptophan metabolism which proceeds in B. megaterium via the anthranilic acid pathway (4). Some properties of the transport system are presented in this paper. Journal Article no from the Michigan Agricultural Experiment Station. MATERALS AND METHODS Organism. B. megaterium ATCC was used throughout these studies. This aerobic sporeformer grows well in a simple, defined media and requires only a carbon source, a nitrogen source, salts, and trace elements. Media and cultivation. The growth medium used was a modified sucrose-salts (SS) broth (14), which contains sucrose as the carbon source. When sucrose was replaced by L-tryptophan or L-arginine, the media were called Trp-S broth or Arg-S broth, respectively. Tryptophan was filter-sterilied with a Nalgene filter unit (.2 gm, plain membrane; Nalge Sybron Corp., Rochester, N.Y.). Organisms were maintained at -2 C or 4 C as washed-spore suspensions. Spores were routinely heat-shocked in 1-ml amounts at 7 C for 3 min. A portion of the heat-shocked suspension was used to inoculate a 5-ml Erlenmeyer flask containing 1 ml of fresh, sterile broth. The culture was aerated at 3 C on a New Brunswick rotary platform shaker at 18 rpm. Starter cultures contained inosine and alanine (each at 1 Ag/ml) to initiate germination. Portions of exponential starter cultures were used to inoculate Trp-S broth for tryptophan permease induction or Arg-S broth for tryptophan permease repression. Scintillation counting. The uptake of DL- [14C ]tryptophan (29.1 mci/mmol; Calatomic), L- [14C]phenylalanine (47 mci/mmol; New England Nuclear), and L-[14C]leucine (312 mci/mmol; New England Nuclear) was assayed by scintillation counting of bacterial samples collected on filters. The scintillation fluid contained 6 g of 2,5-diphenyloxaole and.1 g of 1,4-bis-2-(5-phenyloxaolyl)benene per liter of toluene (7). Scintillation fluid (1 ml) was added to glass vials containing dried samples, and radioactivity was assayed with a model 332 Packard Tri-Carb scintillation spectrometer. Amino acid transport. The procedure used for monitoring the transport of DL- [14C]tryptophan, L- [14C]phenylalanine, and L- [14C Jleucine was a modifi- 65

2 66 BOUKNGHT AND SADOFF J. BACTEROL. cation of the procedure used by Schlesinger and Magasanik (13). Bacteria were harvested from exponential cultures, washed with SS medium minus substrate (SS buffer) and resuspended to approximately 2. x 17 cells/ml (viable count). The incorporation medium (2 ml) contained approximately 4. x 18 cells,.5 ACi of radioactive amino acid per ml, and 1 ug of chloramphenicol per ml. Appropriate flasks contained 1 Mmol of sodium aide per ml. Reaction mixtures were incubated at 3 C on a New Brunswick rotary shaker. Radioactive compounds were added after 3 min of incubation. After specific time intervals, 1-ml samples were withdrawn and passed through a membrane filter (Gelman nstrument Co., Metricel GN-6, pore sie.45 gm, diameter 25 mm). The filter was washed with 1 ml of cold SS buffer (addition of unlabeled tryptophan did not alter the results) and dried in an oven, and its radioactivity was determined. Competition experiments. The ability of unlabeled amino acids to compete with the uptake of DL- [14C ]tryptophan was measured. Unlabeled D-tryptophan, L-tryptophan, L-leucine, L-arginine, and L- phenylalanine were added to reaction mixtures at various concentrations. Radioactive DL-tryptophan was always present at a concentration of 1.72 nmol/ ml. RESULTS nduction of tryptophan permease. When grown on arginine, B. megaterium had low tryptophan permease activity. However, when bacteria were grown on L-tryptophan, high levels of the tryptophan permease were induced (Fig. 1). The organism did not metabolie (4) or grow on D-tryptophan, thus providing a convenient assay of the purity of that stereoisomer. Sodium aide effectively prevented tryptophan transport, implying that the generation of energy was necessary for the process. Transport of D- and itryptophan. n some studies employing DL- [14C]tryptophan, cells transported all of the radioactive amino acid that had been added, suggesting that the permease transported both isomers. Competition experiments with unlabeled D- and L-tryptophan indicated that this was the case. When either isomer was added to the reaction mixture at a concentration that was 1 times the labeled tryptophan (2 nmol/ml), the uptake of the label was inhibited (Fig. 2). When the concentration of either unlabeled isomer was raised to 5 times the concentration of the [4C ]tryptophan, the transport of the radioactive amino acid was completely inhibited. Arginine, leucine, and phenylalanine competition. L-Arginine, L-leucine, and L- phenylalanine were tested to determine their abilities to compete with tryptophan in the transport reaction. Each of these amino acids E du o 7 w a _ a' FG. 1. nduction of the tryptophan permease. Bacteria were grown on Trp-S broth () or Arg-S broth (A) and harvested in the exponential phase. Cells were washed with SS buffer and resuspended to approximately 2. x 17 cells/ml (viable count). The reaction mixture (2 ml) contained approximately 4. x 18 cells,.5 MCi of DL-['4CJtryptophan per ml (1.72 nmol/ml), and 1 Mg of chloramphenicol per ml. Sodium aide (M), 1 ;tmol/ml, was added to cells grown on Trp-S broth. The uptake of DL- [14C ]tryptophan by cells was monitored by the procedure given in Materials and Methods. n this and subsequent figures, the picomoles of tryptophan taken up per 2 x 17 cells can be calculated by dividing the counts per minute (CPM) by This factor was calculated from the counting efficiency and specific activity of the [14C]tryptophan. has some relationship to tryptophan catabolism in B. megaterium. Arginine supports growth of the organism but does not affect catabolite repression of tryptophan metabolism (4); the leucine permease is derepressed when B. megaterium is grown on tryptophan; and both phenylalanine and tryptophan can be transported by a general aromatic transport system. Arginine, leucine, and phenylalanine at concentrations of 2 nmol/ml did not markedly inhibit the uptake of DL- [14C ]tryptophan (1.72 nmol/ ml) by cells of B. megaterium that had been grown in tryptophan. Even when the concentrations of L-leucine and L-arginine were raised to 1 mmol/ml (about 5 times that of ["4C ]tryptophan), the maximal inhibition of uptake of labeled tryptophan was only 4% (Fig. 3). On the other hand, phenylalanine at the high

3 VOL. 121, 1975 TRYPTOPHAN TRANSPORT N B. MEGATERUM 67 such a transport system. The experimental approach utilied in resolving this question was 18 the determination of the rates at which cells transported DL- [14C ]phenylalanine when the 1.6 Control tryptophan permease was induced or repressed. E / Arginine, which may produce general permease L_ 1A4 repression, decreased the levels in cells of trypu 1 tophan, phenylalanine, and leucine permeases o / (Fig. 5). The phenylalanine permease was re-, 1.2 pressed along with the tryptophan permease le but not to the same extent. cl 1._ / Product induction of the tryptophan O_ tpermease. n B. megaterium, tryptophan oxy genase.8 (L-tryptophan:oxygen oxidoreductase; <.8 / 2 M D trp EC ), the first enyme in the trypto- / Dtrp phan oxidative pathway, is induced by kyno.6 urenine, pha xdtv which is aha,ia pathway intermediate nue ykn (4). Q_ / ^/ Wa Therefore, kynurenine was tested for its ability > _.4 ; * /o to induce the tryptophan permease in B. 2 / megaterium growing in Arg-S medium. Kyn-.2F / X 2 A M L t rp urenine was an effective inducer of the per FG. 2. Competition of unlabeled D- and L-trypto- 4 phan for the tryptophan permease. Unlabeled D- and L-tryptophan were added to reaction mixtures at 2 1 mn nmol/ml (2 psm). Radioactive DL- ["C]tryptophan A 1 mole arg/ml was present in a concentration of 1.72 nmol/ml. / Symbols: *, no addition of unlabeled tryptophan; A, U 3. addition of D-tryptophan; *, addition of L-trypto- - phan. Values for [14Cltryptophan uptake in the pres-/ ence of 1 ;mol of sodium aide per ml have been L./ subtracted. concentration completely blocked the uptake of D 2. the labeled amino acid. The nature of the inhibition was investigated by studying the rate of [14C ]tryptophan uptake by cells over the range 2.5 x 1-6 M to 1-4 M x with and without 5 x 1-5 M phenylalanine. x 1.c< 1 mole phe/ml Samples of cells were taken at 1, 3, and 5 min after the addition of the labeled tryptophan, and the extent of the linear uptake per minute over the period of 1 to 5 min was taken as the velocity. Lineweaver-Burk analysis of the data shown in Fig. 4 revealed that the Km for tryptophan (the concentration corresponding to om N UnTlE 5% S of the maximal velocity rate) was 3.4 x FG. 3. Competition of unlabeled L-arginine, L- 1-6 M. Phenylalanine was a noncompetitive leucine, and L-phenylalanine for the tryptophan perinhibitor of tryptophan transport with a K1 of mease. Unlabeled amino acids were added to reaction 3.8 x 1' M. mixtures at 1 Mmol/ml. Radioactive DL-tryptophan was present in a concentration of 1.72 nmol/ml. General aromatic permeases have been re- Symbols:, no addition of unlabeled amino acids, A, ported which transport phenylalanine, tyrosine, addition of unlabeled L-arginine, 1 umol/ml;, and tryptophan (1-6). The question arose addition of unlabeled L-leucine, 1 gmol/ml; () whether the tryptophan permease was actually addition of phenylalanine, 1 MUmol/ml. g

4 68 1/i5 [s] FG. 4. Kinetics of DL-[14C]tryptophan uptake by B. megaterium cells and its inhibition by L- phenylalanine. 7 on w u D Q U BOUKNGHT AND SADOFF.L ~~~~~~p he(trp) U- Ceu (trp +arg) d/y 5/ t r p (trp. arg phe (trp-ars ~~~~~~~ FG. 5. Repression of tryptophan, phenylanine, and leucine permeases by arginine. Bacteria were grown in the presence of tryptophan (trp) or tryptophan plus arginine (trp + arg). Cells were washed with SS buffer and resuspended to approximately 2. x 17 cells/ml (viable count). The ability of these cells to transport DL-[14C ]tryptophan, L-['4C lphenylalanine, or L- [4C ]leucine was determined as described in Materials and Methods. The designation trp(trp) indicates the uptake of tryptophan by cells grown on that substrate. mease even at conditions where the transport system is normally repressed (Fig. 6). A study of the time course for the induction of the permease by kynurenine in washed, arginine-grown cells (Fig. 7) confirmed the data derived from the growth experiment. DSCUSSON B. megaterium synthesies an inducible tryptophan permease that probably functions in tryptophan catabolism. This transport system has a high affinity for tryptophan (Km of 3.4 x 1-6 M) and, in this respect, is similar to that reported for P. acidovorans (12). Competition studies showed that the permease transported L- and D-tryptophan but not L-arginine or L-leucine. The transport of the D isomer seems gratuitous since tryptophan oxygenase will not attack it and a tryptophan racemase is apparently absent from the cells (4). Phenylalanine is a noncompetitive inhibitor of tryptophan transport, and its permease is repressed and derepressed under the conditions that control synthesis of tryptophan permease. Therefore, the possibility was considered that the two per- U wllj J. BACTEROL FG. 6. nduction of tryptophan permease by L- kynurenine. Bacteria were grown in the presence of L-arginine (), L-tryptophan plus L-arginine (-), or L-kynurenine plus L-arginine (A). The ability of these cells to transport DL- [14C]tryptophan was determined as described in Materials and Methods. Values for tryptophan uptake in the presence of sodium aide have been subtracted.

5 VOL. 121, 1975 TRYPTOPHAN TRANSPORT N B. MEGATERUM 69 u2 Ld - D T4 CL H- 5 4 FG. 7. Time course for the induction of the tryptophan permease by L-kynurenine in arginine-grown B. megaterium (). The control () cells were exposed to kynurenine plus 1 ;&g of chloramphenicol per ml. Values for tryptophan uptake in the presence of sodium aide plus chloramphenicol have been subtracted. meases were, in fact, the expression of a general aromatic amino acid transport system. This does not appear to be the case since such general transport systems (2, 11) have almost equal affinities for the amino acids and since phenylalanine, at 1 times the tryptophan concentration, should have produced marked inhibition of the uptake of the labeled compound. n these studies arginine was a general repressor of amino acid permeases. The true inducer of the Bacillus tryptophan permease may be L-kynurenine. This has been shown to be the case for induction in P. acidovorans (12). As previously suggested by Pallermi and Stanier (1), with kynurenine as the inducer, the cell has two advantages. (i) Kynurenine is usually present only when tryptophan is being oxidied. Therefore, it could serve as a definite signal of tryptophan breakdown. On the other hand, the presence of tryptophan would be an ambiguous signal for tryptophan catabolism since this amino acid can be synthesied intracellularly for protein synthesis. (ii) The use of kynurenine by the cell ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant A-1863 from the National nstitute of Allergy and nfectious Diseases. R. R. Bouknight was a National Science Foundation predoctoral trainee on grant GZ-229. LTERATURE CTED as an inducer essentially eliminates the possibility of permease induction by D-tryptophan. This would conserve energy since the organism cannot derive any obvious benefits from the D isomer. 1. Ames, G. F Uptake of amino acids by Salmonella typhimurium. Arch. Biochem. Biophys. 14: Ames, G. F., and J. R. Roth Histidine and aromatic permeases of Salmonella typhimurium. J. Bacteriol. 96: Boei, J. A., and R. D. DeMoss Properties of a tryptophan transport system in Escherichia coli. Biochim. Biophys. Acta 49: Bouknight, R. R., and H. L. Sadoff Tryptophan catabolism in Bacillus megaterium. J. Bacteriol. 121: Brown, K. D Formation of amino acid pools in Escherichia coli K-12. J. Bacteriol. 14: Burrows, S. E., and R. D. DeMoss Studies on tryptophan permease in Escherichia coli. Biochim. Biophys. Acta 73: Byfield, J. E., and. H. Scherbaum A rapid radioassay technique for cellular suspensions. Anal. Biochem. 17: Konings, W. N., and E. Freese Amino acid transport in membrane vesicles of Bacillus subtilis. J. Biol. Chem. 247: Oxender, D. L Amino acid transport in microorganisms. Metabol. Pathol. 6: Pallermi, N. J., and R. Y. Stanier Regulatory mechanisms governing synthesis of the enymes for tryptophan oxidation in Pseudomonas fluorescens. J. Gen. Microbiol. 35: Piperno, J. R., and D. L. Oxender Amino acid transport systems in Escherichia coli K-12. J. Biol. Chem. 243: Rosenfeld, H., and P. Feigelson Product induction in Pseudomonas acidovorans of a permease which transports L-tryptophan. J. Bacteriol. 97: Schlesinger, S., and B. Magasanik midaole propionate, a nonmetaboliable inducer for the histidine-degrading enymes in Aerobacter aerogenes. J. Biol. Chem. 24: Slepecky, R., and J. W. Foster Alterations in metal content of spores of Bacillus megaterium and the effect on some spore properties. J. Bacteriol. 78: