Vol. 124, No. 1 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Oct. 1975, p. 296-306 Copyright ® 1975 American Society for Microbiology

Methionine Transport in Yersinia pestis DIANE B. MONTIE AND THOMAS C. MONTIE* Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37916

Received for publication 6 May 1975

Yersinia pestis TJW, an avirulent wild-type strain, requires phenylalanine and methionine for growth. It was of interest to examine and define the methionine transport system because of this requirement. The methionine system showed saturation kinetics with a Km for transport of approximately 9 x 10-' M. After 8 s of methionine transport, essentially all of the methionine label appeared in S-adenosyl-L-methionine (SAM) as detected in ethanol extracts. Small amounts of free methionine was detected intracellularly after 1 min of transport. Addition of glucose increased significantly the amount of intracellular methionine at 1 min. A series of SAM metabolic products was detected after 90 s to 5 min of transport including: 5'-thiomethyladenosine, homoserine lactone, S-adenosyl homoserine, and a fluorescent methyl receptor compound. Results from assays for SAM synthetase in spheroplast fractions showed a small (16%) but significant portion of synthetase associated with the membrane. However, most of the enzyme activity was associated with the cytoplasmic fraction. Methionine transport was characterized by a high degree of stereospecificity. No competition occurred from structurally unrelated amino acids. Although uptake was inhibited by uncoupling and sulfhydryl reagents, no efflux was observed. Results using energy inhibitors on unstarved and starved cells showed that respiratory inhibitors such as potassium cyanide (KCN) and amytal were most effective, and that arsenate was least effective. KCN plus arsenate completely blocked utilization of energy derived from glucose, and KCN completely blocked utilization of energy deived from D-lactate. The data indicate that methionine transport in Y. pestis is linked to the trapping of methionine in SAM. The results further suggest that this transport system can be classified as a permease-bound system where transport is coupled to an energized membrane state and to

respiration. Studies on bacterial transport systems have uncovered a variety of systems which impart to the cell a mechanism for concentrating necessary nutrients and balancing influx and efflux with metabolic requirements (5). Some of these transport systems are highly specific, whereas others may show overlap based on chemical analogy. In some cases metabolites are transported unmodified, but in others, such as the phosphotransferase system, metabolites are modified facilitating entry. Both amino acid and sugar transport have been studied in some detail with regard to energy couple for active transport. Attempts to explain energy couple by a general mechanism have so far been unsuccessful, although insights into possible modes of couple have been obtained. These include respiratory chain couple (9) and couple related to proton motive force (17). More recently, reports by others (3, 4, 12) have indicated the importance of differences in energy couple. In fact, it appears that metabo296

lite transport may be classified based on energy derived either from the respiratory-linked energized state (permease bound) or from adenosine 5'-triphosphate (ATP) (shockable systems) (4). Our interests have been centered on understanding the importance of two amino acid transport systems for growth and functioning of avirulent Yersinia pestis, a gram-negative, enteric-like bacterium (6). Methionine and phenylalanine are two amino acids essential for growth of avirulent and virulent Y. pestis. Thus, highaffinity transport systems for these amino acids would be selectively advantageous for these cells. Previously our studies on aromatic amino acid transport (20) uncovered marked differences between phenylalanine and tryptophan in response to sulfhydryl reagents and energy inhibitors. These results provoked our interest in further examining the mechanism of transport of methionine and comparing it to other amino acid systems. Our findings support the concept of heterogeneity among amino acid transport

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METHIONINE TRANSPORT IN Y. PESTIS

systems in regard to transport mechanisms and energy couple. Methionine transport systems have been characterized first in Escherichia coli by Piperno and Oxender (19) and more recently by Kadner (11). Ayling and Bridgeland (2) described a methionine system in Salmonella typhimurium which is probably linked to the control of methionine biosynthesis. All of these systems are highly specific for the L-isomer of methionine. Kadner also reported (11) the presence of a second low-affinity system (Km 4 x 10-I M). In all of these studies the rapid metabolism of transported metfiionine was noted, but did not seem significant to the authors. Our studies, reported here, have re-

vealed a high-affinity methionine transport system in Y. pestis that is directly coupled to metabolism through formation of S-adenosylmethionine (SAM). Data is also presented relating the methionine system to a mode of energy derived from a respiratory-linked energized state. Characteristics of the Y. pestis and E. coli methionine systems, as well as the methionine and phenylalanine transport systems of Y. pestis, are discussed.

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Growth and preparation of cells for transport studies. Cells were grown overnight in a minimal medium. Cells were adjusted with minimal medium to an optical density of 70 Klett units (no. 42 filter; 112 Ag of protein per ml) (18) and were grown to late logarithmic phase at 27 C with shaking. Cells were collected by centrifugation (12,000 x g for 10 min) at 4 C and washed twice in 80 ml of cold buffer containing either potassium phosphate (0.05 M), magnesium chloride (0.005 M), pH 7, or, when using sodium arsenate as an inhibitor, in tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (0.025 M) magnesium chloride (0.005 M) buffer, pH 7. The cells were finally suspended in buffer to an optical density of 135 Klett units (no. 62 filter; 330 gg of cell protein per ml). The cell suspension was incubated with either 200 ug of chloramphenicol (CAP) per ml alone or, when specified, CAP (200 ,g/ml) and D-glucose (2 mg/ml) or CAP (200 Ag/ml) and D-lactate (3.94 mg/ml) for 15 min with shaking. The cell suspension was then used immediately for individual experi-

ments. A modified method of Berger (3) was employed for preparation of starved cells. Cells were grown to late logarithmic phase, centrifuged (12,000 x g for 10 min), washed with phosphate suspension buffer, and then resuspended in minimal medium containing DNP (0.005 M) but no glucose. The cells were incubated for 1 h at 37 C, centrifuged, and washed with cold buffer three times and resuspended to the MATERIALS AND METHODS appropriate optical density as described above. Organism. Y. pestis TJW (previously named PasAssay of methionine transport. Cell suspensions teurella pestis) is the wild-type, methionine- and were incubated with shaking at 27 C on a Dubnoff phenylalanine-requiring, avirulent strain used in metabolic shaker (Precision Scientific, Chicago, Ill). these studies. Stock cultures of this organism were Labeled methionine was added to the cell suspension maintained on agar slants (4 C), containing en- to a final concentration of 3 x 10-1 M. Duplicate zymatic casein hydrolysate, glucose, and mineral salts samples were assayed for each time point. At specified medium (18). Cells were grown using a gyratory water times, 0.5 ml of cell suspension was transferred to a bath shaker in a glucose-salts minimal medium con- membrane filter (24 mm; Schleicher and Schuell, taining five amino acids without added vitamins (18). 0.45-Mm). Filtration of the samples took less than 1 s. These cells, stored at 4 C, were used for daily inocula- The cells on the filter were washed with 5 ml of tions. suspension buffer (27 C), and the filters were dried. Materials. L- [methyl-3H ]methionine (2.6 Ci/ The filters were placed in scintillation vials to which 5 mmol), L- 1 14C0]methionine (59 mCi/mmol), L- [U- ml of scintillation fluid, BBOT (2,5-bis-2- [-tert-butyl14C ]methionine (260 mCi/mmol), and L- [3H ]methio- benzoxazolyl -thiophene-toluene, 4 g/liter), was nine randomly labeled (0.5 Ci/mmol) were obtained added. Samples were counted in a Beckman LS-100 from Schwarz/Mann Co. L- [methyl-sH Imethionine liquid scintillation spectrometer. Background counts (11 Ci/mmol) and [8-140 ATP tetrasodium salt (50.3 on the filters were less than 5% of the total and were mCi/mmol) were obtained from New England Nu- subtracted from the total of each sample. Nanomoles clear Corp. DL-Cystathionine hemihydrate, L-cystine, of uptake were computed from the average of the and L-methionine were obtained from Cyclo Chemi- duplicate samples. cal. DL-Methionine sulfone, L-cysteine, and deoxyriboThe effect of energy poisons or sulfhydryl reagents nuclease (beef pancrease 1X crystallized) were ob- was measured by determining the rate of uptake by tained from Mann Research Laboratories. DL- cells after 10-min incubation in the presence of the Homocysteine, L-ethionine, D-methionine, DL-methyl- agent at 27 C before addition of the substrate, unless methionine sulfonium, a-methyl DL-methionine, S- otherwise noted. methyl L-cysteine, L-norleucine, L-methionine methyl In competition experiments, the competitor was ester hydrochloride, and a-methyl DL-methionine added concurrently with labeled methionine, and were purchased from Nutritional Biochemical Co. uptake was measured after 2 min. 2,4-Dinitrophenol (DNP), S-adenosyl-L-methionine, Possible metabolic alterations of transported meand S-adenosyl-L-ethionine were purchased from thionine were detected by repetition of uptake experiSigma Chemical Co. Sodium ampicillin USP (Princi- ments using methionine labeled differentially, either pen/N) was purchased from E.R. Squibb and Sons, carboxyl, methyl, randomly, or uniformly labeled New York. Dithiothreitol was purchased from Calbio- material. chem. Measurement of the methionine pool. A cell

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suspension, without CAP, was incubated for 15 min and then Tris-hydrochloride (0.02 M, pH 7.3) buffer with glucose (0.2%) and L-phenylalanine (10-' M) or potassium phosphate (0.025 M)-magnesium chlowith shaking at 27 C. L-[methyl-8HJmethionine (3 x ride (0.0025 M) buffer (pH 7) was used for subsequent 10-6 M) was added, and at the indicated times two breaking of the spheroplasts. The washed spheroplasts 0.5-ml samples of the cell suspension were withdrawn were suspended in breaking buffer (4 ml) to which and filtered through a membrane filter (0.45-Am), dithiothreitol (10' M) and deoxyribonuclease (10 which was washed, dried, and counted as previously Ag/ml) were added and then homogenized with a described. At the same indicated times, 0.5 ml of cell motor-driven Teflon pestle. The resulting homogenate suspension was added to perchloric acid (0.5 ml, 6%, was centrifuged at 32,000 x g for 10 min. The 7 C) to stop the reaction. The lysed cell suspension supernatant was designated cytoplasmic fraction, and was extracted for 15 min at 7 C, followed by filtration the pellet was designated membrane fraction. The on a membrane filter (0.45-jum). The precipitate was membrane fraction was resuspended in the above washed with ethanol (70%, 10 ml). The filters were breaking buffer containing dithiothreitol (10-3 M) counted as described previously. The methionine pool and rehomogenized until no intact spheroplasts were was calculated by determining the difference in radio- detected by microscopy. The membrane fraction was activit.y between the cell suspension and the per- centrifuged at 32,000 x g, and the supernatant was chloric acid-insoluble fraction. added to the cytoplasmic fraction. The membrane Measurement of temperature effects on methio- pellet was washed once with the breaking buffer nine transport. Cell suspensions, CAP treated, were containing dithiothreitol (10-I M) and resuspended in preincubated at temperatures ranging from 2 to 35 C Tris-hydrochloride buffer (0.02 M, pH 7.3) for assay. for 10 min. L- [methyl-8H ]methionine (3 x 10' M) Excepting the omission of toluene in one experiment, was then added and transport was assayed at 3 min. a SAM synthetase (ATP-L-methionine S-adenosylSamples were removed with pipettes precooled to the transferase, EC 2.5.1.6) assay was performed on the desired temperature. two fractions according to Holloway et al. (8). The Identification of labeled methionine metabolites. identity and purity of labeled SAM was verified by Radioactive methionine was added to CAP-treated using thin-layer chromatography. cells, as described above, with or without glucose. The Assay of ATP. Cell suspensions were treated as cells (5 ml) were vacuum filtered on 47-mm mem- previously described with or without an inhibitor. The brane filters (0.5-m, Millipore Corp.) at indicated method of Stanley and Williams (22) was employed to times and washed with 50 ml of suspension buffer. measure ATP in cells lysed with PCA (3%). The filters were immediately removed and immersed in 15 ml of ethanol (70%, 4 C). After 30 min, the RESULTS extract was centrifuged (4 C) for 10 min (12,000 x g) and the supernatant was vacuum-dried. The remainCharacteristics of methionine uptake. The ing insoluble residue contained less than 8% of the uptake of labeled methionine as a function of total radioactivity. The dried material was resus- time into CAP cells is illustrated (see, for pended in N-butanol-acetic acid-water (2:1:4) and example, curve for control cells in Fig. 7). spotted on Whatman 1 MM paper (18.25- by 22.5- Determination was also made in incorporation inch [ca. 46- by 57-cm] sheet). Descending paper chromotography was employed using N-butanol- of label into the perchloric acid-soluble pool acetic acid-water (2:1:1). The chromatogram was without CAP in the presence of phenylalanine dried after 20 h, dipped in acetone-acetic acid and glucose. The uptake profile indicated satusolution (50:3, vol/vol) containing 100 mg of ninhy- ration of the pool label (Fig. 1). In minus CAP drin, dried by heating, and cut into 2-mm squares. cells without glucose or phenylalanine, almost Each square was placed in a scintillation vial to which all of the methionine label was associated with 10 ml of scintillation fluid (BBOT) was added. SAM, the acid-soluble pool (data not shown). boiled SAM, methionine, methionine sulfoxide, and The data in Fig. 2 and 3 show that methionine DL-homocysteine were routinely employed as standwas concentration dependent and satuards for chromatography or cochromatography for uptakewith an apparent Km (K, for transport) rable, identification of unknowns in the cell extracts. Preparation of spheroplasts and assay of frac- for uptake of approximately 9 x 10- M. The Kt tions for SAM synthetase activity. Cells grown determination gave the same value when meaovernight in minimal medium were adjusted, after sured at 2 min (Fig. 3) or 30 s. It is apparent centrifugation, with minimal medium to an optical from the data presented below that the Kt densitv of 70 Klett units (no. 42 filter). After 1 h of determinations include the equilibrium begrowth, sucrose to 20% (wt/vol) and ampicillin (500 tween methionine in transport and formation of U/ml) were added. The cells were incubated at 27 C SAM via the SAM synthetase reaction. A lowon a gvrotary shaker for 4 to 10 h until 95 to 98% of the affinity transport system also was identified organisms were converted to spheroplasts. Sphero- with a K, of approximately 2 x 10-4 M. The plasts were removed by centrifugation (10,000 x g) at 4 C for 10 min. Isolation of spheroplast fractions was latter system may be comparable to the isoleuperformed at 4 C. The spheroplasts were washed with cine-valine-threonine system of Templeton and potassium phosphate (0.05 M, pH 7.0)-magnesium Savageau (23). Measurement of the temperature optimum chloride (0.005 M) buffer containing sucrose (20%),

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VOL. VOL124,1975 METHIONINE TRANSPORT IN Y. PESTIS 124, 1975

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of 8- and 15-s cell extracts from cells incubated with or without glucose. The data show that no methionine appeared in the ethanol extracts. Essentially the entire amount of label was identified as SAM in the 8-s extracts. After 15 s of exposure, incorporation of methionine into SAM increased concomitant with formation of SAH (Rf 0.21), an immediate product of SAM following methyl transfer. The presence of glucose caused some stimulation of label incorporation into SAH. A 1-min pool profile (Fig. 5, minus glucose) showed a profile similar to those of the shorter time periods, with label largely concentrated in the SAM fractions, although some label appeared in methionine (R, 0.61) and in SAH (R, 0.19). However, in the presence of glucose a significant portion of label appeared in methionine (Rf 0.64), and a lesser amount appeared in a fluorescent compound (R, 0.86). After 5 min of incubation (Fig. 6) extract profiles showed the 0.40

25

MINUTES FIG. 1. Kinetics of methionine uptake into intact cells, pool, and protein fractions.

was carried out further to characterize methionine uptake. Transport measurements showed a temperature optimum of 29 to 37 C. Arrhenius plot determinations revealed a transition temperature of 16 C for methionine uptake. Identification of methionine metabolic products. Methionine is rapidly converted into numerous metabolic products. The rapid formation of compounds derived from methionine has been inferred from several transport studies 19). Consequently, (2, experiments were pr per. (2,19 Conseqently, ,ex mets were formed to determine the metabolic fate of methionine as it entered the cell. Cells were incubated briefly with uniformly labeled methionine and then rapidly filtered, followed by extraction with 700% ethanol and chromatographic analyses. Cells incubated from 90 s to 5 min exhibited label predominantly in SAM (Rf 0.076), methionine (R, 0.60 [cells minus glucose] or R, 0.64 [cells plus glucose] as identified by cochromatography with unlabeled methionine), and S-adenosyl homocysteine (SAH) (Rt 0.19). The latter compound was identified as SAH by ultraviolet absorption, cochromatography, and by its absence in experiments where methyl-labeled methionine was employed. The high proportion of label appearing in SAM was seen in a series of time course experiments and prompted us to examine the pool after only a brief exposure to uniformly labeled methionine. Figure 4 illustrates the chromatographic profile

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32

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FIG. 4. Chromatographic profile of 8- and 15-s, intracellular-labeled methionine metabolites. Cell suspensions were preincubated with CAP with or without glucose (0.2%) and then incubated with L- ['4C]methionine, uniformly labeled for either 8 or 15 s, rapidly filtered and treated as described in Materials and Methods. L- [U- 4C]methionine was used as a standard (STD) marker. Methionine sulfoxide, MSO; methionine, MET.

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FIG. 5. Chromatographic profile of 1-min, intracellular-labeled methionine metabolites. Cell suspensions were preincubated with CAP with or without glucose (0.2%) and then incubated with L- [U- 'CJmethionine for 1 min, rapidly filtered, and treated as described in Materials and Methods. Methionine sulfoxide, MSO; methionine, MET; 5'-thiomethyladenosine, THIOMETAD; unidentified fluorescent compound, FLUOR.

following: (i) apparent maximum level of SAM, (ii) an increase in methionine in minus glucose cells, (iii) increased amounts of SAH (R, 0.19) and the appearance of two highly mobile compounds (Rf 0.71 and 0.86). The evidence indicated that the radioactive compound migrating at Rt 0.71 is 5'-thiomethyladenosine, because in separate experiments 5'-thiomethyladenosine appeared as an ultraviolet-absorbing spot (R, 0.71) following thermal decomposition of unla-

beled SAM. To accomplish thermal decomposition, SAM or cell extracts were boiled in water for 5 min (7). In further confirmation, 5'-thiomethyladenosine represented the labeled thermal decomposition product from [methyl'H ]SAM in boiled cell extracts. Homoserine (or the lactone) was the labeled thermal decomposition product from carboxyl-labeled SAM in boiled cell extracts. The appearance of 5'-thiomethyladenosine

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METHIONINE TRANSPORT IN Y. PESTIS

after 5 min with glucose is not surprising, since it is a first-step metabolite of SAM (16). In this reaction homoserine (or homoserine lactone) is also formed. The expected R, for the latter compound corresponded to the peak moving slightly ahead of methionine sulfoxide (Fig. 6). From studies with methyl- or carboxyl-labeled methionine it was apparent that the methionine fragment of SAM turned over rapidly, and that, in particular, methyl group transfer was increased by the addition of glucose. Chromatographic analyses of products in extracts from experiments using methyl-labeled methionine indicate that the methyl group was transferred to the unidentified fluorescent compound (R, 0.86). The indicated rapid turnover of carboxyl and methyl carbons of SAM was verified further in a preliminary chase experiment using differentially labeled methionine. The latter experiment also indicated that methionine accumulated after SAM levels reached a specific pool concentration. SAM was also identified as a major metabolite in perchloric acid cell ex-

301

Rt value (11), was detected in the water extracts. The latter compound presumably was

not readily extracted with 70% ethanol, since it appeared as a major component of 15-min hot water extracts and was not detected in 15-min ethanol extracts. The formation of spermidine occurs from SAM and 5'-thiomethyladenosine in two enzymatic steps (16). Therefore, it is not unusual to find spermidine (and probably spermine) accumulating after 15 min of metabolism. Distribution of SAM synthetase in the cell. Since methionine was immediately converted to SAM in Y. pestis cells, it was of some interest to evaluate the possibility that SAM synthetase was a membrane component functioning directly or indirectly in methionine transport. Spheroplasts were prepared, broken, and fractionated into cytoplasm and membranes. Results showed that 16% of the total enzyme activity resided in the membrane fraction. This calculation was based on distribution of total counts in SAM after incubation of each fraction for 30 min with labeled ATP. These data tracts. Cells incubated with labeled methionine for indicate that SAM synthetase is located in the 15 s also were extracted with boiling water to cytoplasm or is loosely bound to the membrane compare results with those using ethanol ex- envelope. tracts. The hot water procedure has been emEffect of analogues on methionine uptake. ployed in some laboratories (11) to obtain the The uptake of methionine showed a high degree amino acid pool. Results with 15-s extracts of specificity, since 14 unrelated amino acids showed, as has been documented by Greene et tested gave no competition at 100-fold excess al. (7), that SAM was decomposed completely to concentration over methionine. The amino 5'-thiomethyladenosine and homoserine lactone acids tested included representatives from the during the hot water extraction process. A small major groups of amino acids, dicarboxylic, diaamount of spermidine, which was identified by mino, and aromatic, as well as alanine, leucine, 14

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FIG. 6. Chromatography of 5-min, intracellular-labeled methionine metabolites. Cell suspensions were preincubated with CAP with or without glucose (0.2%) and then incubated with L- [U- 14C ]methionine for 5 min, rapidly filtered, and treated as described in Materials and Methods. Methionine sulfoxide, MSO; methionine, MET; 5'-thiomethyladenosine, THIOMETAD; unidentified fluorescent compound, FLUOR.

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isoleucine, and valine. Table 1 lists a series of methionine analogues which were competitive or noncompetitive when present at 10- and 100-fold in excess to transport substrate. Esterification of the carboxyl group with a methyl group only partially altered specificity. Apparently, other less consequential modifications included addition of the methyl group in methionine or elimination of a carbon atom at the sulfur atom in homocysteine. The importance of stereochemistry for recognition was emphasized by the result showing 30% inhibition by only 10-fold excess norleucine. Thus, a minimum of a 4-carbon chain may be needed to express the three-dimensional configuration, because cysteine showed no competition in contrast to competition by homocysteine. Modification of the sulfur group in methionine sulfone or methyl methionine sulfonium severely decreased recognition. The D-isomers had no effect on uptake, indicating that uptake is specific for the L-isomers. Effect of inhibitors on transport. A series of inhibitors were employed to characterize the methionine transport system. The sulfhydryl inhibitor p-chloromercuribenzoate and the uncouplers sodium azide (NaN) or DNP were added 12 min after addition of methionine label to test the possibility of efflux of pool methioTABLE 1. Inhibition of methionine transport by analoguesa

nine (Fig. 7). Uptake was significantly inhibited by these uncouplers and p-chloromercuribenzoate. Similar results were obtained if these inhibitors were added only 4 min into the experiment, as exemplified by DNP effects (Fig. 8). Almost identical results as with DNP were obtained with carbonyl cyanide-m-chlorophenylhydrazone. When CAP-treated cells were preincubated for 5 min with N-ethylmaleimide (5 x 10-' M) or p-chloromercuribenzoate (5 x 10' M) uptake was inhibited more than 80%. Thiosalicylate, a weak uncoupler, was completely ineffective. Unlike the phenylalanine transport system of Y. pestis (21), sulfhydryl reagents and uncouplers did not cause efflux of accumulated methionine or its products. Also, in counterflow experiments, cells preloaded with uniformly labeled methionine for 2 min, or 2 min and 45 s, did not exhibit efflux when excess cold methionine was added immediately at the latter two time points (data not shown). These data are consistent with the previous results showing that the majority of the methionine is in the form of SAM and its metabolites, which would not be expected to exit readily from the cell. It is also possible, but unlikely, that efflux systems are inhibited by all the inhibitors tested. Characteristics of energy couple. The addition of glucose to cells markedly stimulated the rate of methionine uptake, approximately threeI

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and labeled L-methionine (3 x 10' M) were added together at zero time. The incubation period for the inhibition assay was 2 min. b The following analogues at 3 x 10- or 3 x 10- M gave inhibition below 20% at lOOx concentration: D-methionine, L-cystine, L-cysteine, DL-methionine sulfonium, a-methyl DL-methionine, S-adenosyl-Lmethionine, S-adenosyl-L-ethionine, and DL-cystathionine hemihydrate. c Uptake is the average of two or more experiments. a Analogues

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FIG. 7. Effect of selected inhibitors on the rate of uptake and accumulation of methionine. Cell suspensions were incubated with labeled methionine over a 12-min period. At 12 min DNP (0.001 M), NaN, (0.01 Al), and p-chloromercuribenzoate (CMB) (8 x 10M) were added to the indicated final concentrations.

METHIONINE TRANSPORT IN Y. PESTIS

VOL. 124, 1975

303

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accumulation of methionine. Cell suspensions were incubated with labeled methionine and allowed to accumulate methionine over a 4-min period. At 4 min DNP (0.001 M) was added. to fourfold. Under these conditions, partial efflux was observed after 4 min or more with methyl- or carboxyl-labeled methionine, but not uniformly labeled methionine, suggesting that metabolic products of methionine were subject to efflux. Efflux was never observed in the absence of glucose. It was demonstrated that D-lactate and L-lactate stimulated uptake in starved cells. The effects of various energy inhibitors were tested in unstarved and starved cells in an effort to identify energy requirements for transport. Figure 9 illustrates the effect of glucose in partially reversing the inhibition by potassium cyanide (KCN) in minus glucose cells. A summary of similar experiments is presented in Table 2 (unstarved cells). It is evident from these data that arsenate, an inhibitor of phosphorylation, was ineffective in blocking energy couple from glucose. A chromatographic profile of cell extracts from arseuateinhibited cells showed no shift in the ratio of SAM to methionine, but only a reduction in total counts associated with each peak. Levels of ATP were reduced approximately 50% in arsenate plus glucose cells, as judged by the luciferase assay of Stanley and Williams (22). The appearance of the unaltered profile in the presence of arsenate indicated that arsenate was working at the transport level rather than affecting the formation of SAM. Uncouplers, DNP and azide (also a respiratory inhibitor),

MINUTES

FIG. 9. Effect of KCN on the kinetics of methionine uptake. KCN (0.01 M) was incubated with CAP-treated cells plus or minus glucose (0.2%) for 10 min before addition of L-(methyl-3H)methionine (3 x 10- 6 M). Control cells were incubated with glucose (0.2%) but no KCN.

TABLE 2. Effect of glucose on the action of energy inhibitors Expt no.

1 2

3c

4

AdditionG

Methionine

Control plus glucose KCN plus glucose KCN minus glucose Control plus glucose DNP plus glucose NaN, plus KF plus glucose Control minus glucose Arsenate minus glucose Control plus glucose Arsenate plus glucose Control minus glucose Amytal minus glucose Control plus glucose Amytal plus glucose

1.24 0.34 0.07 1.69 0.71 0.75

uptakeb

0.26 0.10 0.47 0.40 0.60 0.21 0.88 0.33

% of control

27 6

42 44 48 85 34 37

aInhibitor concentrations: KCN (0.01 M), NaN, (0.01 M), DNP (0.001 M), KF (0.01 M), sodium arsenate (0.01 M), sodium amytal (0.005 M). b Nanomoles of uptake per milligram of cell protein per 2 min. cTris buffer (0.025 M) + MgCl, (0.005 M), pH 7, was used in this experiment.

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were more effective than arsenate in inhibiting transport. Potassium fluoride gave no inhibition of transport either in the presence or in the absence of glucose. Electron transport inhibitors such as KCN and amytal were the most effective inhibitors. KCN and azide have been shown to be effective inhibitors of respiration under similar conditions to those used for these transport studies (21). Similar results with respiratory inhibitors were obtained with DNPstarved cells using either D-glucose or D-lactate as substrates (Table 3). Arsenate was somewhat more effective with )-lactate as substrate, probably because ATP could only be generated via oxidative phosphorylation. Potassium cyanide completely blocked energy couple via Dlactate to transport, emphasizing the importance of respiratory-derived energy. Arsenate plus KCN with glucose as substrate also gave complete inhibition of energy transfer. DNP was moderately inhibitory of glucose-derived energy. These data taken together indicate the importance of respiratory energy, probably directly linked to an energized state necessary for energy couple.

J. BACTERIOL.

and methionine. In the methionine system, transport is directly linked to SAM formation, apparently unlike the methionine system reported for E. coli (11). In the latter case, however, SAM formation might have gone undetected, because SAM would have been destroyed in the boiling water extraction method employed (11). Also, only one of a series of mutants used for transport studies was used for detection of methionine metabolic products (11). Since the formation of SAM in Y. pestis is immediate, as demonstrated by the presence of methionine label in SAM at 8 s, Kt for transport reflects both the equilibria for transport and SAM formation. Methionine did not immediately accumulate, and SAM synthetase activity, therefore, is not a limiting factor. The lack of methionine counterflux is in agreement with the data concerning formation of pool metabolites. The fact that the transport system is saturable, and the energy is required, indicates that active transport occurs, although it may be possibly considered downhill active transport (13). Examination of the pool at various times showed chromatographic profiles indicating that SAM reaches saturation levels quickly, and DISCUSSION that at least the methionine portion of SAM is Y. pestis, which requires phenylalanine and rapidly turning over. Hence, saturation of the methionine for growth of the wild-type strain, perchloric acid-soluble pool probably reflects to contains high specificity and high-affinity some extent saturation of the SAM level in the transport systems for both phenylalanine (20) cell. After SAM reaches peak levels, free methionine begins to accumulate as a pool amino TABLE 3. Effect of inhibitors on DNP-starved cells acid. Thus, SAM synthetase acts as a trapping agent by rapidly forming the sulfonium derivaExpt Methionine % of tive in the cell, which in turn cannot exit from Additiona no. uptake" control the cell. Although most of SAM synthetase activity, as assayed by product formation, was as1c Control plus D-lactate 0.172 sociated with the cytoplasmic fraction, the Arsenate plus D-lactate 0.065 38 conversion of methionine to SAM is so rapid KCN plus D-lactate 0.033 19 2 Control plus D-lactate that it is interesting to speculate that (i) SAM 0.496 KCN plus D-lactate 0.016 3 enzymes are loosely bound to the membrane, Minus D-lactate 0.063 13 (ii) synthetase is in equilibrium between memKCN minus D-lactate 0.026 5 brane and cytoplasm, or (iii) two enzymes 3c Control plus glucose 0.741 exist, one cytoplasmic and one membranous. Arsenate plus glucose 60 0.448 The methionine transport system of Y. pestis KCN plus glucose 0.151 20 shows a high specificity for L-methionine. It is KCN plus arsenate and 0.016 2 very unlikely that the analogues tested are glucose working at the level of SAM synthetase, beControl minus glucose 5 0.038 4 cause ileucine is as good or better an inhibitor Control plus glucose 1.089 DNP plus glucose 48 0.522 than norleucine of SAM synthetase from either 5 Control plus D-lactate 0.639 E. coli, yeast, or rat liver (15). In Y. pestis, LDNP plus D-lactate 0.283 44 leucine does not compete with methionine for a Inhibitor concentrations: arsenate (0.01 M), KCN transport, whereas norleucine is a fairly good inhibitor (66% at 100-fold excess). (0.01 M), DNP (0.001 M). The data reported here support the concept of °Nanomoles of uptake per milligram of cell protein heterogeneity in the mode of amino acid transper 2 min. cTris buffer (0.025 M) + MgCl, (0.005 M), pH 7, port, particularly with respect to energy couple was used in these experiments. as observed in E. coli (3, 4). Similar results were

VOL. 124, 1975

observed by us in early studies of aromatic amino acid transport in Y. pestis (20). Results have shown that phenylalanine and methionine transport are inhibited by sulfhydryl reagents, but tryptoph4n transport is not inhibited. These experiments suggest that the action of thiol inhibitors may occur at the carrier level. More striking has been the selective effect of energy inhibitors. Thus, respiratory inhibitors were ineffective in preventing initial uptake of phenylalanine, and particularly accumulation of tryptophan, but KCN and amytal were strong inhibitors of methionine transport. Uncouplers of oxidative phosphorylation such as azide, DNP, and carbonyl cyanide-m-chlorophenylhydrazone partially blocked the uptake of methionine. The uncouplers seemed to be more effective when added a few minutes into the experiment (see Fig. 8), suggesting that an accumulated form of energy was dissipated. These compounds, whether preincubated or added sometime after labeled amino acid, immediately and completely blocked energy couple to phenylalanine transport, causing rapid efflux of phenylalanine (21). Thiosalicylate, a weak uncoupler, gave partial inhibition of phenylalanine transport but had no effect on methionine transport. It is also interesting that at 5 C, DNP is not effective in blocking transport of phenylalanine (21), whereas methionine uptake shows the same amount of inhibition at 28 or 5 C. We have proposed that DNP is altering membrane conformation which in turn affects phenylalanine transport selectively (21). Another contrast is seen in the 5- to 20-fold stimulation of methionine transport by D-lactate or glucose, whereas phenylalanine transport is not stimulated under the same conditions by exogenous energy sources. A comparison of Y. pestis methionine transport with an E. coli methionine transport system (11) shows some interesting variations also. The most significant difference is the lack of inhibition by fluoride in Y. pestis, whereas E. coli (11) was significantly inhibited by fluoride. In Y. pestis, methionine transport was more sensitive to respiratory inhibitors than E. coli methionine transport. Also, _omplete elimination of transport was seen in the presence of azide plus fluoride in E. coli, in contrast to the effectiveness of KCN plus arsenate in Y. pestis DNP-starved cells. Therefore, energy for the E. coli system seems to be linked to phosphorylation and in general is comparable to the glutamine-like system (4, 11). This might explain the low level of methionine transport in E. coli vesicles (10), since this system seems to require a periplasmic binding protein which is sensitive

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to osmotic shock (11). The data on the Y. pestis methionine transport suggests a permease-like system (3, 10, 14), where transport is linked to an energized membrane state and to respiration. Although some energy can be derived from ATP as indicated by the partial effect of arsenate or DNP, the respiratory inhibitors were completely effective, particularly when employing DNP-starved cells with D-lactate as the substrate. In the presence of respiratory and phosphorylation inhibitors, energy couple from glucose is blocked completely. The inhibition of transport by sulfhydryl inhibitors together with the data discussed above tends to classify the methionine system as a permease-bound system. Such a system in Y. pestis would contribute toward physiological stability and would serve as a link between the external milieux and a closely associated trapping system in SAM synthetase. Consequently, the constitutive requirement for methionine would be advantageously served in wild-type Y. pestis. In motile organisms such as E. coli, SAM formation, and perhaps methionine transport, seem to be intimately linked with chemotaxis (1), where a periplasmic protein might be beneficially multifunctional. The existence of such relationships is currently under investigation. ADDENDUM IN PROOF

Preliminary experiments with E. coli K-12 wild type indicate that the majority of the methionine label taken up during 5 to 30 s appeared in SAM in ethanol extracts. LITERATURE CITED 1. Armstrong, J. B. 1972. An S-adenosylmethionine requirement for chemotaxis in Escherichia coli. Oan. J. Microbiol. 18:1695-1701. 2. Ayling, P. O., and E. S. Bridgeland. 1972. Methionine transport in wild-type and transport-defective mutants of Salmonella typhimurium. J. Gen. Microbiol. 73:127-141. 3. Berger, E. A. 1973. Different mechanisms of energy coupling for the active transport of proline and glutamine in Escherichia coli. Proc. Natl. Acad. Aci. U.S.A. 70:1514-1518. 4. 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. 5. Boos, W. 1974. Bacterial transport. Annu. Rev. Biochem. 43:123-146. 6. Brubaker, R-% R. 1972. The genus Yersinia: biochemistry and genetics of virulence, p. 109-158. In W. Arber et al. (ed.), Current topics in microbiology and immunology. vol. 57. Springer-Verlag. New York. 7. Greene, R. C., J. S. Hunter, and E. H. Coch. 197:3. Properties of metK mutants of Escherichia coli K-12. .J. Bacteriol. 115:57-67. 8. Holloway, C. T., R. C. Greene, and C. H. Su. 1970. Regulation of S-adenosylmethionine svnthetase in Escherichia coli. .J. Bacteriol. 104:734-747.

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9. Kaback, H. R. 1974. Transport studies in bacterial membrane vesicles. Science 186:882-892. 10. Kaback. H. R., and L. S. Milner. 1970. Relationship of a

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membrane bound D-(-)-lactic dehydrogenase to amino acid transport in isolated bacterial membrane preparations. Proc. Natl. Acad. Sci. U.S.A. 66:1008-1015. Kadner, R. J. 1974. Transport systems for L-methionine in Escherichia coli. J. Bacteriol. 117:232-241. Klein, W. L., and P. D. Boyer. 1972. Energization of active transport by Escherichia coli. J. Biol. Chem. 247:7257-7265. Koch, A. L. 1971. Energy expenditure is obligatory for the downhill transport of galactosides. J. Mol. Biol. 59:447-459. Lombardi. F. J., and H. R. Kaback. 1972. Mechanisms of active transport in isolated bacterial membrane vesicles. VIII. The transport of amino acids by membranes prepared from Escherichia coli. J. Biol. Chem. 247:7844-7857. Lombardini, J. B., and P. Talalay. 1970. Formation, functions, and regulatory importance of S-adenosyl-Lmethionine, p. 349-384. In G. Weber (ed.), Advances in enzyme regulation, vol. 9. Pergamon Press, New York. Meister, A. 1965. Biochemistry of the amino acids, vol 2. Academic Press, Inc., New York. Mitchell, P. 1970. Membranes of cells and organ-

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elles: morphology, transport and metabolism, p. 121166. In H. P. Charles and B. C. J. G. Knight (ed.), Organization and control in prokaryotic and eukaryotic cells. XXth Symp. Soc. Gen. Microbiol. Cambridge University Press, London. Montie, T. C., and S. J. Ajil. 1964. The anatomical distribution of murine toxin in spheroplasts of Pasteurella pestis. J. Gen. Microbiol. 34:249-258. Piperno, J. R., and D. L. Oxender. 1968. Amino acid transport systems in Escherichia coli K-12. J. Biol. Chem. 243:5914-5920. Smith, P. B., and T. C. Montie. 1975. Aromatic amino acid transport in Yersina pestis. J. Bacteriol. 122:1045-1052. Smith, P. B., and T. C. Montie. 1975. Separation of phenylalanine transport events by using selective inhibitors, and identification of specific uncoupler activity in Yersinia pestis. J. Bacteriol. 122:1053a

1061. 22. Stanley, P. E., and S. G. Williams. 1969. Use of the liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme. Anal. Biochem. 29:381-392. 23. Templeton, B. A., and M. A. Savageau. 1974. Transport of biosynthetic intermediates: homoserine and theonine uptake in Escherichia coli. J. Bacteriol. 117:1002-1009.

Methionine transport in Yersinia pestis.

Yersinia pestis TJW, an avirulent wild-type strain, requires phenylalanine and methionine for growth. It was of interest to examine and define the met...
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