THE THIRD GENERAL TRANSPORT SYSTEM BRANCHED-CHAIN AMINO ACIDS IN SALMONELLA T YPHIMURI UM KEIKO MATSUBARA, KUNIHARU OHNISHI, AND KAZUYOSHI KIRITANI

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1 J. Gen. Appl. Microbiol., 34, (1988) THE THIRD GENERAL TRANSPORT SYSTEM BRANCHED-CHAIN AMINO ACIDS IN SALMONELLA T YPHIMURI UM FOR KEIKO MATSUBARA, KUNIHARU OHNISHI, AND KAZUYOSHI KIRITANI Department of ' Microbiology, School of ' Pharmacy, Hokuriku University, Kanazawa , Japan (Received March 15, 1988) Some properties of the LIV-III transport system, a common factor in the entry of branched-chain amino acids in Salmonella typhimurium, have been studied using a double mutant, KA266 (liva brnq), which is defective in transport via either the LIV-I or LIV-II system. The LIV-III system operates with a low affinity (Km: > 15 /2M) for L-isoleucine, L-leucine, and L-valine. Isoleucine transport via the LIV-III system is inhibited competitively by leucine or valine, but non-competitively by L-cysteine. Uptake of branched-chain amino acids into cells is partially repressed when the bacteria are grown in the presence of 2 mm glycyl-l-leucine or more. In the wild type, transport activity of the LIV-III system for isoleucine and leucine is not detected. The LIV-II system appears to show a low affinity for valine similar to the LIV-III system, hence the combined activity of these systems is expressed as the activity of the LIV-III system. The defect in transport via the LIV-II system leads to a great reduction in the transport of valine in the LIV-III system. Three distinct, general transport systems, high-affinity ( -LIV-I), low-affinity-(1) (LIV-II) and low-affinity-(2) (LIV-III) systems, participate in the uptake of isoleucine, leucine and valine in Salmonella typhimurium (1). Leucine may also enter cells through a leucine-specific (LS) system (2, 3). Among these systems, the LIV-II system plays a major role in taking up branched-chain amino acids into cells. When a defect in the LIV-II system is induced by the brnq mutation in an isoleucinevaline-requiring mutant, KA931 (ilvc), the resulting double mutant does not grow on minimal medium containing isoleucine and valine (4). The LIV-I system is associated with a periplasmic leucine-isoleucine-valine-threonine binding protein, Address reprint requests to: Dr. K. Kiritani, Department of Microbiology, School of Pharmacy, Hokuriku University, Kanazawa , Japan. 183

2 184 MATSUBARA, OHNISHI, and KIRITANI VOL. 34 and shows a low Km for the branched-chain amino acid transport (3). The ilvc mutant harboring an additional defect in the LIV-I system does not significantly influence growth of the bacteria on minimal medium containing isoleucine and valine (5). The transport activities of the LIV-I and LS systems are completely repressed when cells are grown with excess glycyl-l-leucine, whereas the activity of the LIV-II system is partially repressed (2). In the wild type strain, the activity of the LIV-III system for isoleucine is generally not detectable. But the LIV-III system is detectable in mutant strains that are defective in the transport via the LIV-II system, but not in a mutant strain lacking activity only in the LIV-I system (5). Thus there is a possibility that the LIV-III system is the one to transport mainly a substance other than branched-chain amino acids, and operates as an auxiliary system for branchedchain amino acid transport. In order to clarify this, we attempted to examine the LIV-III system using a double-transport mutant KA266, which is defective in the transport mediated by both the LIV-I and LIV-II systems. Here we show that the LIV-III system is specific to the transport of branchedchain amino acids, and the activity is repressible by glycyl-l-leucine. We also show that the LIV-II system operates with a low affinity for valine similar to the LIV-III system. MATERIALS AND METHODS Bacterial strains. The bacterial strains used were the wild-type of S. typhimurium LT2 and its derivatives, KA203 (brnq3) (6) and KA266 (brnq3 lival) (5). KA203 is defective in the LIV-II transport system for branched-chain amino acids, and KA266 has an additional defect in the LIV-I system. Media. Bacteria were grown in minimal medium (4), and the medium, when required, was supplemented with glycyl-l-leucine as indicated in the text. Transport assay. Transport assays were performed on exponentially growing cells as described previously (2). Apparent Km and Vmax values of the transport were calculated according to the formula of NEAL (7). Chemicals. Labeled amino acids were purchased from Amersham Co. All other chemicals were commercial materials of analytical grade. RESULTS Transport system (s) in KA266 Detailed kinetic analysis of branched-chain amino acid transport in KA266 was carried out, and the data were compared with those of the wild type and KA203. As reported previously, a steady-state level of accumulation of branchedchain amino acids into cells was reached in 2 to 5 min (1, 4). The initial rates of isoleucine uptake were measured in the concentration range of 0.12 to 115 /im, and double-reciprocal plots of the data are illustrated in Fig. 1. The biphasic kinetics of the isoleucine transport were observed in the wild type and KA203 (Fig. la and B),

3 1988 Branched-chain Amino Acid Transport in S. typhimurium 185 Fig. 1. Double-reciprocal plot of initial rate of uptake of L-isoleucine. grown in minimal medium. Bacteria were whereas the kinetics were monophasic in KA266 (Fig. 1C). The kinetics of leucine and valine uptake in these cells were analogous to those shown in Fig. 1. The apparent Km and Vmax values of isoleucine, leucine and valine for the transport systems of the wild type, KA203 and KA266 were calculated from the doublereciprocal plots (Table 1). In the wild type, the LIV-II system showed high activity for the entrance of isoleucine and leucine, but not valine. In contrast, in the LIV-III system the transport activity for valine was high, and no activity for either isoleucine or leucine was detected. When the transport activity of the LIV-II system was lost by brnq mutation as seen in KA203, the activity of the LIV-III system for isoleucine and leucine became measurable, and the activity for valine was greatly reduced. As discussed later, the apparent transport activity for valine, noted in the LIV-III system of the wild type, may exhibit a combined activity of both the LIV-II and LIV-III systems. In KA266, the branched-chain amino acids entered the cells mainly through the remaining LIV-III system, though a residual low activity was

4 186 MATSUBARA, OHNISHI, and KIRITANI VOL. 34 Table 1. Apparent Km and Vmax for the transport systems.a noted in the LIV-I system, which may be due to the activity of the leucine-specific system. Inhibition of isoleucine uptake To study the inhibition of labeled isoleucine uptake by an unlabeled amino acid, 6 mm unlabeled L-amino acid was generally added to the uptake mixture containing 60 µm L-(14C)isoleucine. Entry of the labeled isoleucine into KA266 cells was inhibited by cysteine, isoleucine, leucine or valine added to the mixture (Table 2). The inhibitory effect of these amino acids was not significantly altered when the concentration of labeled isoleucine was increased to 0.2 mm. The isoleucine uptake was competitively inhibited by leucine (Fig. 2A) or valine (data not shown), while the inhibition by cysteine was non-competitive (Fig. 2B). Repression of the LI V -Ill transport activity by glycyl-l-leucine As shown in Fig. 3, the transport activity of the LIV-III system for branched-

5 1988 Branched-chain Amino Acid Transport in S typhimurium 187 Table 2. Inhibition of L-(14C)isoleucine uptake by adding unlabeled amino acids in KA266. chain amino acids was partially repressed by adding glycyl-l-leucine to the bacterial growth medium. The maximum repression of the transport activity was attained with more than 2 mm glycyl-l-leucine. When labeled isoleucine, leucine or valine was used at a concentration of 60 µm, the extent of repression of the transport ranged from 40 to 70%; the repression patterns at 220 µm of the respective labeled amino acids were similar to those at 60RM. In order to study kinetic behavior of the transport systems in the wild type, KA203 and KA266, cells were grown with 5 mm glycyl-l-leucine, and the repressed rates of branched-chain amino acid transport were examined. As shown in Table 1, repression of the LIV-III activity in KA266 corresponded well with the results presented in Fig. 3. The transport activity of the LIV-I system was almost completely repressed, though the repression of activity

6 188 MATSUBARA, OHNISHI, and KIRITANI VOL. 34 Fig. 2. Inhibition of L-isoleucine uptake into KA266 by adding unlabeled amino acids. The initial uptake of L-(14C)isoleucine by bacteria in 15 sec was measured; the specific activity of L-(14C)isoleucine was 2.6 x 108 cpm/ imol in the concentration range of 0.7 to 4.4/1M, and 2.2 x 10' cpm/ imol at 9.3 to µm. The uptake was measured in the presence (~) or absence (0) of 6mM L-leucine (A), or 6mM L-cysteine (B). Fig. 3. Repression of the transport activity in the LIV-III system of KA266. Bacteria were grown in the presence of the indicated concentration of glycyl-l-leucine. The initial uptake of L-(14C)isoleucine (S), L-(14C)leucine (Q) and L-(14C)valine (p) by bacteria in 15 sec was measured; the concentration of each labeled amino acid was 60 µm, and the specific activities of labeled isoleucine, leucine and valine were 2.2 x 10', 1.7 x 10', and 2.5 x 10' cpm/[imol, respectively. for valine was incomplete in the wild type. The repression in the LIV-II system as well as in the LIV-III system was partial. DISCUSSION As we reported previously, there are three distinct, common transport systems for branched-chain amino acids in S. typhimurium (1). Among these systems, the LIV-III system operates with a very low affinity for isoleucine, leucine and valine, and its transport activity for isoleucine and leucine is usually not detected in the wild

7 1988 Branched-chain Amino Acid Transport in S. typhimurium 189 type (Table 1). It has not yet been determined whether the LIV-III system is truely specific for branched-chain amino acids. To examine the LIV-III transport system, a double transport mutant, KA266 (liva brnq), harboring only the active LIV-III system was used. Since isoleucine uptake into the cells is competitively inhibited only by the cognate leucine or valine (Table 2 and Fig. 2), the LIV-III system should be a member of the general transport systems for branched-chain amino acids. Transport activity of the LIV-III system is partially repressed when excess glycyl-lleucine is added to the bacterial culture (Fig. 3 and Table 1); the extent of repression is analogous to that found in the LIV-II system. The presence of a leucine-specific transport system has been predicted in S. typhimurium (2, 3), like that found in Escherichia coli (8) and Pseudomonas aeruginosa (9). So the residual activity for leucine noted in the LIV-I system of KA266 may represent activity due to the leucine-specific system. Peculiar results with regard to valine transport were obtained with the wild type; the transport activity is very high in the LIV-III system, and does not appear in the LIV-II system, though the LIV-II system plays a major role in the transport of isoleucine and leucine (Table 1). This suggests that either the LIV-II system has no affinity for valine, or the system operates with a low affinity for valine similar to the LIV-III system. As found in KA203, the brnq mutation leading to a defect in transport mediated by the LIV-II system greatly reduced the valine-transport activity in the LIV-III system. This evidence supports the second hypothesis. In E. coli and P. aeruginosa, two independent, general transport systems, LIV-I and LIV-II, for branched-chain amino acids have been reported (10, 11). The properties of these two systems in E. coli are comparable to those in S. typhimurium in respect to kinetic data and genetic regions controlling these systems (12, 13). But it is not known whether E. coli and P. aeruginosa, like S. typhimurium, have the LIV-III system, though an isoleucine-specific system with a very low affinity has been described in E. coli (14). REFERENCES 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) K. KIRITANI and K. OHNISHI, Jpn. J. Genet., 53, 265 (1978). K. OHNIsHI, M. MURATA, and K. KIRITANI, Jpn. J. Genet., 55, 349 (1980). K. OHNIsHI and K. KIRITANI, J. Biachem., 94, 433 (1983). K. KIRITANI, J. Bacterial., 120, 1093 (1974). K. MATSUBARA, K. OHNISHI, and K. KIRITANI, Jpn. J. Genet., 62, 189 (1987). K. KIRITANI and K. OHNISHI, J. Bacterial., 129, 589 (1977). J. L. NEAL, J. Thear. Biol., 35, 113 (1972). D. L. OXENDER and S. C. QUAY, Ann. N. Y. Acad. Sci., 264, 358 (1976). T. HosHINo and M. KAGEYAMA, J. Bacterial., 141, 1055 (1980). M. RAHMANIAN, D. R. CLAUS, and D. L. OXENDER, J. Bacterial., 116, 1258 (1973). T. HoSHINO, J. Bacterial., 139, 705 (1979). J. J. ANDERSON and D. L. OXENDER, J. Bacterial., 136, 168 (1976). J. GUARDIOLA and M. IACCARINO, J. Bacterial., 108, 1034 (1971). M. IACCARINO, J. GUARDIOLA, and M. DE FELICE, J. Membr. Sci., 3, 287 (1978).