Vol. 122, No. 1 Printed in U.SA.

JOURNAL OF BACTERIOLOGY, Apr. 1975, p. 110-119 Copyright 0 1975 American Society for Microbiology

Regulation of Methionine Transport Activity in Escherichia coli ROBERT J. KADNER Department of Microbiology, The University of Virginia School of Medicine, Charlottesville, Virginia 22901

Received for publication 7 January 1975

Methionine transport activity in cells of Escherichia coli K-12 is regulated by the level of the internal methionine pool. Transport activity is depressed either in cells grown in the presence of methionine or in cells exposed to methionine immediately prior to harvest. a-Keto-y-methiol-butyrate, D-methionine, or methionine sulfoxide have little effect on the initial rate of uptake of L-methionine when they are added simultaneously with the substrate. However, methionine transport is markedly reduced in cells exposed to these sources of L-methionine before the addition of substrate. This reduction is prevented if the cells are treated with amino oxyacetic acid. The initial rate of uptake into L-methionine-loaded cells was lower than that into unloaded cells. This inhibition affected both methionine transport systems and the inhibition by the internal pool appeared to be non-competitive with the external methionine concentration. Two classes of mutants with increased methionine pools have decreased rates of uptake. Conversely, starvation for methionine in a methionine auxotroph with high rates of methionine degradation resulted in a substantial increase in the rate of methionine transport. Thus, these transport systems are subject to regulation by the internal pool size and possibly by repression. In most cases, studies of the regulation of bacterial transport systems have revealed that control is on the formation of the transport system rather than on its activity. Most of the sugar transport systems are induced along with the appropriate catabolic enzymes. There have been some interesting studies, reviewed by Kornberg (11), that show that the activity of a number of sugar transport systems is regulated by the level of cellular metabolites or by functioning of a different transport system. Unfortunately, little is known yet about the mechanism of this regulation. The kinetic and genetic characterization of the transport of most of the common amino acids into cells of Escherichia coli has been described (16). Repression processes have been inferred for the control of many of these amino acid transport systems. Thus, leucine transport, for example, is decreased coordinately with the level of the leucine binding protein in cells grown in the presence of leucine (14). Similar results in which transport activity is depressed in cells grown in the presence of the appropriate amino acid have been described in a number of other transport systems (2, 8, 15, 17). This regulation has been attribdted to a repression phenomenon, although in no case has the level of the transport system been found to be under

control of the regulatory gene controlling the appropriate biosynthetic enzymes. In contrast, a number of transport systems in several fungal species are modulated both by some type of genetic regulation requiring protein synthesis and by a direct inhibition of the rate of influx by the intracellular pool of the transported compound (1, 7, 12, 13). This latter form of regulation, termed transinhibition, has been predicted by Cuppoletti and Segel (3) to be a property of carrier-mediated transport systems, analogous to product inhibition of a soluble enzyme. However, this process is not usually observed in bacteria and, under certain circumstances, the reverse effect, counterflow, is observed. In our studies of methionine transport in E. coli (9), two unusual results were observed. On the one hand, cells grown with or without methionine in the growth medium had similar rates of methionine uptake only if the non-supplemented cells were exposed to methionine at the time of harvest. Otherwise, the non-supplemented cells had considerably higher rates of uptake. Therefore we stated that this transport system was not subject to repression control. Second, preloading experiments showed that the rate of uptake into loaded cells was somewhat lower than that into empty cells. These

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experiments involve exposure of cells to a rela- ity was determined in a Packard model 3320 scintillatively high level of methionine with the labeled tion counter after the solubilization of the filter. Initial rates are measured in duplicate after 0.20 min. tracer added at either zero time or later. Preloading and efflux experiments. The methodThis paper extends these findings to the and results from the usual type of preloading conclusion that methionine transport activity in ology been presented previously (9). In experiment E. coli is modulated by the level of the intracel- general, thesehave involve exposure of cells to a fairly high lular methionine pool. This conclusion is based concentration of unlabeled methionine and the addion transport rates into cells in which the methition of trace amounts of labeled methionine at either onine pool has been manipulated either by zero time or later. A modification of this protocol loading the cells prior to assay or through involves exposure of cells to [14C Imethionine (109 uM, mutations altering either methionine biosyn- 0.5 MCi/ml) in a volume of 250 Ml. At intervals, this is thesis or utilization. Some kinetic evidence as to diluted to varying final volumes with medium A the nature of the effect of the internal pool on containing glucose and chloramphenicol and [sH]methionine (1 lCi, 5.3 nmol present in the specithe rates of influx and efflux is presented. fied volume [5 to 100 mlD. For uptake into empty MATERIALS AND METHODS Bacterial strains and growth conditions. The genotype the E. coli K-12 strains employed are presented in Table 1. Wild-type strains possess two L-methionine transport systems; the high-affinity system is lacking in metD strains (10). These are also unable to transport D-methionine (R. Kadner, unpublished data). The metK and metJ strains were provided by R. Greene. Cells were grown in minimal medium with saltmedium A of Davis and Mingioli (4), supplemented with 0.5% glucose and any required amino acids (100 Ag/ml), adenine (40 ug/ml), and thiamine (1 Mg/ml). The cells were incubated at 35 C and harvested in the middle of the exponential growth phase by centrifugation. Unless specified, cells were washed twice in medium A containing glucose (0.5%) and chloramphenicol (100 Mg/ml). Transport assays. Transport of methionine is assayed by mixing washed cells with labeled methionine at room temperature (23 C). At intervals, samples (0.20 ml) were removed and transferred to the center of a membrane filter (Millipore Corp.), filtered, and washed with 5 ml of medium A. "4C-labeled radioactivity was measured in a gas-flow planchet counter, while 3H- or 3H- and "4C-labeled radioactiv-

cells, the [14H]methionine is added together with the ['HJmethionine in the dilution medium. Portions of the diluted mixture are withdrawn, filtered, and washed, such that the same number of cells are collected regardless of the extent of dilution. The 14Clabeled radioactivity allows determination of the rate of efflux and of the intracellular concentration of methionine, whereas the 3H label assays the rate of influx. The concentrations and specific activities of the isotopes were chosen

so

that both the 3H- and

"4C-labeled radioactivities were of the same order of magnitude (103 to 104 counts/min over background). Materials. The radioactive L-methionine was obtained from New England Nuclear. All other chemicals were obtained from Sigma, except aminooxy acetic acid which was obtained from Upjohn Co. but is now available from Sigma. RESULTS

Effect of growth and harvest conditions on transport activity. The data presented previously (9, 10) indicate that methionine is transported by two systems with a high degree of

substrate specificity. No evidence for the repression of these systems was observed, al-

TABLE 1. Bacterial strains Strain

Genotype

Reference or

E15 KBT001 RK4205 RK4211 K12 RG62 RG73 RG109 RG81 RG100 AB1932

Hfr C. thi phoA leu proC lysA trp purE metE strA as KBT001, but metE+ as RK4205, but metD F+, X lysogen as K-12, but metK84 as K-12, but metK85 as K-12, but metK86 as K-12, but metJ12 as K-12, but metJ31 argHl, metA28, thi-1, lac Yl, lacZ4,

E. Lin

RG326

as AB1932, but arg+ metJ31

R. Greene (5)

R. Greene (5) R. Greene (5) R. Greene (5) R. Greene (5) R. Greene (5) R. Greene (5) R. Greene (5)

tsx-6, xyl-4, galK2

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KADNER

though it was noted that cells grown in the presence of methionine had lower rates of transport than those grown without methionine supplementation. It was shown that cells grown under either condition had similar rates of uptake if methionine was added to the cells immediately before harvest. Since this suggested that methionine uptake was regulated by a type of feedback inhibition rather than the repression usually observed with other amino acid transport systems, further studies on this process were initiated. Table 2 presents the rates of methionine transport into cells exposed to methionine in the presence of chloramphenicol immediately prior to harvest and washing. There is a decrease in the uptake of either methionine isomer approaching the level observed in cells grown in the presence of methionine for many generations. This activity could be restored almost to the control level by storing the loaded cells at 4 or 23 C for 30 min. Similarly, data not presented showed that when cells were grown in the presence of methionine and then transferred to medium lacking methionine, uptake activity increased about fourfold over a 2-h period. This increase was considerably inhibited by the presence of chloramphenicol. This may indicate a derepression TABLE 2. Effect of exposure of cells to methionine Concn (mM) of L-methionine during:a

Transport activity" Immediate

Growth Harvest

0 0 0 0 0.61 0.61

0 0.11 0.61 6.1 0 0.61

441 (100) 267 (61) 226 (51) 130 (30) 137 (31) 75 (17)

After 30 min (4 C)

After 30 min (23 C)

489 (111) 378 (86) 447 (101) 479 (109) 188 (43) 166 (38)

494 (112) 454 (103) 374 (85) 197 (45)

a Cells of strain E15 were grown in minimal growth medium containing the indicated concentration of L-methionine. While in the middle of the logarithmic growth phase, the cells were exposed to the indicated concentration of methionine and immediately centrifuged at 23 C for 7 min. The cells were washed twice with medium A containing glucose and chloramphenicol. 'The initial rate (0.2 min) of L-methionine uptake was measured in duplicate at 23 C, using washed cells either immediately after harvest or after they had been incubated for 30 min at either 4 or 23 C, then centrifuged, and resuspended in new media. Activity is expressed as picomoles of L-methionine accumulated per microliter of cell water times minutes. The external methionine concentration was 1.724 SM. (The values in parentheses represent percentage of control uptake.)

J. BACTERIOL.

process and/or a decrease in the methionine pool provided by protein synthesis. In the converse experiment in which cells were transferred from minimal to methionine-containing medium, transport activity decreased quite rapidly (within 5 min) to the level seen in methioninegrown cells. All of these results suggest the regulation of transport activity by the size of the intracellular methionine pool. These data do not rule out the existence of a repression process, since those processes that restore most of the activity to methionineloaded cells do not fully restore activity to methionine-grown cells. It appears from our data (not shown) that the high-affinity transport system may be subject to repression; however, it is difficult to document this owing to the feedback inhibition control that is present. Specificity of the regulatory process. aKeto-y-methiol-butyrate (the a-keto analogue of methionine) serves as a very good methionine source for Met- auxotrophs. It enters the cell by a system other than the methionine transport systems, as judged by its failure to affect the initial rate of methionine uptake and by the fact that it is a very good methionine source in methionine transport mutants (9, 10). The time course of L-methionine uptake in its presence showed that, although it did not affect the initial portion of the process (up to 0.25 min), it subsequently strongly inhibited uptake even to the extent of promoting efflux (Fig. 1). Incubation of the cells with a-keto-y-methiol-butyrate for 0.5 to 2 min before addition of methionine led to a progressive decrease of transport activity down to 10% of the control level. This decrease followed exposure to concentrations of the a-keto analogue ranging from 10 to 800 1AM. The relationship between the initial rate of uptake and the concentration of L-methionine was determined both in a strain with normal methionine transport activity and in a metD mutant lacking the high-affinity transport systems, under conditions in which the a-keto analogue was added either simultaneously with or 2 min before the labeled L-methionine (Fig. 2). The activity of both the high- and low-affinity systems was inhibited by prior incubation with this compound. For both systems, the inhibition by the a-keto analogue appeared to be non-competitive with the substrate L-methionine. The value of K,, the inhibition constant, is in the range of 1 mM but is unimportant because it presumably is not the keto acid itself which is inhibitory, but rather the L-methionine derived from it. The inhibtory action of a-keto-y-methiolbutyrate on uptake was depressed in cells

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FEEDBACK INHIBmON OF TRANSPORT

treated with the pyridoxal-enzyme inhibitor, aminooxy acetic acid (AOA). Table 3 shows that AOA at 1 mg/ml only slightly decreased the rate of L-methionine uptake but almost completely eliminated the inhibition by the a-keto analogue. D-Methionine uptake was even somewhat increased over the control rate in AOA-treated cells, and again the inhibition by keto-methionine was elimnated. Higher concentrations (5 mg/ml) of AOA did depress transport activity. The mode of action of AOA is presum-

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Regulation of methionine transport activity in Escherichia coli.

Methionine transport activity in cells of Escherichia coli K-12 is regulated by the level of the internal methionine pool. Transport activity is depre...
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