A REGULATORY TRANSPORT MUTANT FOR BRANCHED-CHAIN AMINO ACIDS IN SALMONELLA TYPHIMURIUM KUNIHARU OHNISHI, KEIKO MURATA AND KAZUYOSHI KIRITANI

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1 JAPAN. J. GENETICS Vol. 55, No. 5: (1980) A REGULATORY TRANSPORT MUTANT FOR BRANCHED-CHAIN AMINO ACIDS IN SALMONELLA TYPHIMURIUM KUNIHARU OHNISHI, KEIKO MURATA AND KAZUYOSHI KIRITANI Department of Microbiology, School of Pharmacy, Hokuriku University, Kanazawa Received May 27, 1980 A new regulatory mutant, KA231 (ilvc8 brnq4 liv-231), derepressed in the transport of branched-chain amino acids was isolated from a transport-defective mutant, CE5 (ilvc8 brnq4) of Salmonella typhimurium LT2. Kinetic analysis of isoleucine and leucine transports indicated that activities of the high-affinity and the low-affinity-(1) systems of KA2313, an Ilv+ transductant of KA231, were increased 1.3- to 2.5- fold over the levels of KA204 (brnq4). The activity of specific binding protein(s) found in the periplasmic space of KA2313 cells was also increased several-fold. Glycyl-L-leucine was able to repress both the transport and the binding activities of KA2313, but the extent of repression was weaker than that of repression induced in wild-type strain. Nature of liv-231 mutation distinct from another regulatory mutation, glen, is discussed. INTRODUCTION The transport of branched-chain amino acids in S. typhimurium is mediated by at least three kinetically distinct systems (high-affinity, low-affinity-(1) and low-affinity- (2)) (Kiritani and Ohnishi 1978). In the wild-type strain, the low-affinity-(1) system is the main route of cells for uptake of these amino acids. Activity of the low-affinity- (2) system is concealed by that of the other two systems, and becomes detectable in mutants defective in both the high-affinity and the low-affinity-(1) systems. The highaffinity and the low-affinity-(1) systems are repressible by L-leucine or several glycyldipeptides, especially by glycyl-l-leucine (Kiritani and Ohnishi 1977). When the strain KA931 (ilvc8), an isoleucine-valine requiring mutant, was cultivated in a minimal medium containing isoleucine, valine and a large amount of glycylleucine, the growth of cells was strongly inhibited due to repression of the transport activities for required substances caused by glycylleucine. A derepressed transport-mutant for branched-chain amino acids, KA224 (ilvc8 glerl), which grew well on isoleucine, valine and excess glycylleucine, was isolated from KA931 (Ohnishi and Kiritani 1978). The gler gene specifying the expression of glycylleucine-resistance in the mutant was located in a region near brnq (Ohnishi and Kiritani 1980), which is the structural gene for the low-

2 350 K. OHNISHI, K. MURATA AND K. KIRITANI affinity-(1) transport system (Kiritani and Ohnishi 1978). To conduct detailed studies on the regulatory systems for the transport of branched-chain amino acids, we have attempted to obtain new regulatory mutants by selecting for clones from Brn+ revertants of CE5 (ilvc8 brnq4), whose transport activity is insensitive to glycylleucine. We postulated that a regulatory mutation, if occurred in CE5, would enhance the transport activity for branched-chain amino acids, compensating for loss of the activity deleted by brnq mutation, and the transport activity in this mutant would not be susceptible to repression caused by glycylleucine. In this paper, we report the isolation and characterization of a regulatory transport-mutant, in which the transport activity for branched-chain amino acids and the level of periplasmic branched-chain amino acidbinding protein(s) seem to be constitutive. MATERIALS AND METHODS Bacteria and phage: The bacterial strains used were all derivatives of S. typhimurium LT2 and listed in Table 1. For transductions phage P22 was used. Media : Composition of minimal medium used in most experiments was described previously (Kiritani 1974). Unless otherwise noted, supplements, when required, were L-isoleucine (0.08,moles/ml), L-leucine (0.08,moles/ml), L-valine (0.17 ~imoles/ml), Capantothenate (0.1 ig/ml), glycyl-l-isoleucine (0.11 ~cmoles/ml), glycyl-l-valine (0.23 pmoles/ ml), and vitamin-free Casamino Acids (0.1%). Penassay broth (antibiotic medium 3, Difco) was used as nutrient broth. Isolation of regulatory mutants: Strain CE5 was mutagenized with ethyl methanesulfonate by the method described previously (Kiritani 1974). After the treatment, cells were grown overnight in Penassay broth, harvested by centrifugation, washed twice with minimal medium, and suspended in the same medium. One tenth ml of the sus- Table 1. Bacterial strains

3 REGULATORY TRANSPORT MUTANT 351 pension (about 1 X 109 cells/ml) was plated on minimal agar containing isoleucine and valine, where the parent CE5 could hardly grow due to a deficiency in transport of the amino acids. After 48 h incubation at 37 C, colonies appeared were further selected for isoleucine-valine requirers (Brn+ revertants), and purified. To analyze isoleucinetransport activity of the revertants, cells were grown for several hours in minimal medium supplemented with isoleucine and valine, and with or without glycyl-l-leucine (1 mm). These cultures were harvested by centrifugation, and the transport activity of cells for 14C-isoleucine was assayed. When the transport activity of a Brn+ clone grown in the presence of glycylleucine was over 70% of that of the same clone grown in its absence, it was saved for further studies. Three such mutants were obtained among 36 clones examined, and KA231 was chosen as a representative for the present study. The mutational site induced in KA231 was tentatively referred to as liv-231. Measurement of generation time : Overnight cultures were prepared in minimal medium containing following supplements; for KA931 and KA231, isoleucine, leucine, valine, pantothenate and Casamino Acids, and for CE5, glycylisoleucine, glycylvaline, pantothenate and Casamino Acids. Cells in these cultures were harvested by centrifugation, washed twice with minimal medium, and suspended in the same medium. Five-tenth ml aliquots of each suspension were added in minimal medium containing appropriate supplements as indicated in the text, and the cells were grown with shaking at 37 C for 4 h to minimize effects of extra nutrients carried over from the preculture. These cultures were then diluted 20-fold with the same media, respectively, and shaking was continued at 37 C. The optical density of these cultures was measured at 660 nm using Shimadzu-Bausch and Lomb Spectronic 20 colorimeter. Transport assay: Since we found that chloramphenicol inhibits entry of branchedchain amino acids into cells mediated by the high-affinity system (unpublished data), the method of transport assay (Kiritani 1974) was modified. Exponentially growing cells (about 3X 108 cells/ml) were harvested by centrifugation, washed twice with cold minimal medium, suspended in the same medium adjusting the optical density of cells to 0.15 at 660 nm, and the suspension was kept in ice till use. For transport assay, the suspension was warmed at 37 C for 5 min, and 0.5 ml was added to a tube containing 0.02 ml of L-(14C)-amino acid solution. The mixture was incubated for 20 sec at 37 C, and a 0.2 ml aliquot of the sample was quickly filtered through a Sartorius membrane filter (24-mm diameter, 0.45-nm pore size), followed by a 5 ml wash of minimal medium at 37 C. Radioactivity retained on the filter was determined in a Beckmann liquid scintillation counter. Cell suspensions for control experiments were prepared in the minimal medium lacking glucose. After adjusting the optical density to 0.15 at 660 nm, the suspension was incubated 40 min at 37 C in the presence of dinitrophenol (4 mm). A control value obtained with the dinitrophenol-treated cells was subtracted from the corresponding sample value. It should be noted that, when wild-type strain was used, an amount of 14C-labeled isoleucine incorporated in cellular proteins under the assay condition was about 20% of the total uptake. The Km values thus obtained in the absence of chloramphenicol were not significantly different from those obtained in its presence

4 352 K. OHNISHI, K. MURATA AND K. KIRITANI (Kiritani and Ohnishi 1978). However, the Vmax values of the high-affinity system were about two-fold higher in the absence of chloramphenicol, while those of the lowaffinity-(1) system were not affected. Determination of kinetic constants: Apparent Km and Vmax values were calculated by double-reciprocal plotting, 1/V and 1/S, where S is concentration of the substrate expressed in micromoles per liter and V is micromoles of 14C-labeled amino acid incorporated per min per gram of cells (dry weight). Since biphasic kinetics was generally observed in the plotted data, the calculation was performed following the formula of Neal (1972). Preparation of osmotic-shock fluid: The method of Nossal and Heppel (1966) to release binding proteins from cells by osmotic shock was slightly modified. Exponentially growing cells (about 5 x 108 cells/ml) in minimal medium were harvested by centrifugation, and washed once with the same volume of minimal medium lacking glucose. One gram (wet weight) of cells was suspended in 80 ml of 30 mm Tris-HC1 buffer, ph 7.8, containing 40% sucrose and 2 mm Na2-ethylenediaminetetraacetate (EDTA). The suspension was gently stirred for 10 min at room temperature and centrifuged for 10 min at 13,000 g. The well drained pellet was rapidly dispersed in 80 ml of cold distilled water in an ice bath, stirred for 5 min and centrifuged. The supernatant fluid was collected and mixed with one-fiftieth volume of 0.5 M Tris-HC1 buffer, ph 7.8, and then concentrated 20-fold by means of ultrafiltration in an Amicon model 202 cell fitted with a UM-10 filter at a pressure of 2 kg/cm2 with a stream of nitrogen gas. The concentrated fluid was centrifuged to remove insoluble substances, and the supernatant was stored at-80 C. Assay for binding activity: Binding activities in osmotic-shock fluids were determined by equilibrium dialysis. Three tenth ml of a sample (about 0.5 mg of protein per ml) was dialyzed in a 6.4-mm diameter dialysis bag (Visking) for about 16 h at 4 C against 400 ml of 10 mm potassium phosphate buffer, ph 7.0, containing 0.1 pm L- (14C)-isoleucine or L-(14C)-leucine, and 0.1 pm L-(3H)-histidine. The specific activity of labeled isoleucine or leucine was 4 to 5 x 108 cpm/,mole, and of histidine was 2 to 7 X 108 cpm/pmole. After the dialysis, 200 pl aliquots of inside and outside solutions were pipetted on glass filters (1.5 X 2 cm; Whatman GF/B), respectively, and dried. The radioactivity of the filter was measured with a Beckmann liquid scintillation counter. The amount of substrate bound to protein was calculated by subtracting the radioactivity of the outside solution from that of the inside solution. Binding activity is expressed as pmoles of the labeled substrate bound per mg of protein. Linearity of the binding assay was attained with protein concentrations upto 0.6 mg per ml. Amounts of protein were determined by the method of Lowry et al. (1951). Chemicals : Glycyl-dipeptides were obtained from Tokyo Kasei Industries, Ltd., L-amino acids, Ca-pantothenate, Tris(hydroxymethyl)aminomethane (a component of Tris-HC1 buffer), and EDTA from Wako Pure Chemical Ind., ethyl methanesulf onate from Sigma Chemical Co., and vitamin-free Casamino Acids from Dif co Lab. Uniformly 14C-labeled L-amino acids and 3H-labeled L-histidine were obtained from the Radiochemical Centre,

5 REGULATORY TRANSPORT MUTANT 353 Amersham, England. RESULTS Uptake of labeled isoleucine A regulatory mutant for the transport of branched chain amino acids, KA231, was selected for from Ilv-Brn+ revertants of CE5, and the transport activity was compared with that of CE5 and KA931. Table 2 shows uptake of 14C-labeled isoleucine by CE5, KA231 and KA931 grown in the presence or absence of excess glycylleucine. Under the unrepressing growth condition, amount of isoleucine incorporated in KA231 and KA931 cells was about 11-times as large as that in CE5. The transport activity of KA931 and CE5 was repressed strongly by glycylleucine, while that of KA231 weakly. Growth properties Generation times of KA931 and transport mutants in various media are presented in Table 3. As reported previously (Kiritani 1974), CE5 could not grow in minimal medium unless large amounts of isoleucine and valine were supplemented. KA231 could grow in minimal medium containing small amounts of isoleucine and valine, at a rate comparable to that of KA931. However, addition of excess glycylleucine to the medium inhibited growth of KA931 markedly, but that of KA231 only slightly. Large amounts of isoleucine and valine could support normal growth of all strains regardless of presence or absence of glycylleucine. Repression of transport activity Since the transport activity of brnq mutants was repressed by branched-chain Table 2. Repression of transport activity for entry of isoleucine by glycyl-l-leucine

6 354 K. OHNISHI, K. MURATA AND K. KIRITANI Table 3. Generation time (min) of KA931 and transport mutants in various media Fig. 1. Repression of L-(14C)-isoleucine uptake by glycyl- L-leucine. Values of labeled-isoleucine uptake by cells of KA204 (A), KA2241 (s), KA2313 (0), and wild-type (s) strains grown in the absence of glycyl-l-leucine were 5.5, 49.1, 9.2, and 14.8,moles per min per g of cells, respectively. The concentration of L-(14C)-isoleucine was 75 pm and the specific activity was 2.3x 10~ cpm per,mole. amino acids (Kiritani and Ohnishi 1977, 1978), an Ilv+ transductant, KA2313 (brnq4 liv-231), was isolated from KA231 and used for transport assays. For comparative studies, Ilv+ transductants, KA2241 (glerl) and KA204 (brnq4), isolated from KA224 and CE5, respectively, were also used. The transport of branched-chain amino acids is derepressed in KA2241, and defective in KA204. KA2313 grew in minimal medium at a generation time of 90 min, which was longer than that of KA2241, KA204 and wild-type (60 mm). Figure 1 illustrates glycylleucine-repressed transport activity of KA2313 for isoleucine as well as that of KA204, KA2241 and wild-type. The degree of repression was weaker in KA2313 than in KA204, KA2241 and wild-type. Similar repression patterns of leucine uptake were also obtained with these strains (data not shown). Figure 2 shows the kinetic behavior of isoleucine uptake in transport mutant and wild-type strains.

7 REGULATORY TRANSPORT MUTANT 355 Fig. 2. Double-reciprocal plots of initial rate of L-isoleucine uptake. Bacteria were grown in the absence (A) and presence (B) of 5 mm glycyl-l-leucine. Uptake of L-(14C)-isoleucine (specific activities: 2.5 X 108 cpm/,umole in the concentration range of 0.1 to 2.3 MM, and 2.2 x 10~ cpm/mole in that over 2.3,uM) by KA204 (A), KA2241 (LX), KA2313 (0), and wild-type (I) cells was measured at 20 sec. The uptake kinetics of unrepressed cells showed biphasic curves (Fig. 2A) with two apparent Km values (Table 4). when the transport systems of cells were repressed, the curve was still biphasic in KA2313, while it became a straight line in KA2241 and wild-type (Fig. 2B). Because the transport activity of repressed KA204 was extremely low, kinetic analysis of the uptake was not possible with this strain. Apparent Km and Vmax values of transport mutants thus obtained are presented in Table 4. In the strain KA204, the low-affinity-(1) transport system was markedly impaired, similarly to the case found in KA203 (brnq3) (Kiritani and Ohnishi 1978). In the low-affinity-(1) transport system of KA2313, its activity for isoleucine was less than half of that in the wild-type strain, and the activity for leucine was undetectable. However, activities of the high-affinity and the low-affinity-(1) systems of KA2313 were increased 2- to 3- fold over the levels of KA204. In KA204 and wild-type strains, 5 mm glycylleucine strongly repressed both the high-affinity and the low-affinity-(1) systems. Under the same repressing condition, the transport activity of the high-affinity system in KA2313 was reduced by 30 to 40%, but that of the low-affinity-(1) system was not affected. Under unrepressing conditions, KA2241 possessed about 2- to 3-times higher activity

8 356 K. OHNISHI, K. MURATA AND K. KIRITANI Table 4. Apparent Km and Vmax for the transport systems Table 5. Binding activity for branched-chain amino acids found in osmotic-shock fluids in the low-affinity-(1) system than the wild-type strain did, and the activity of this mutant was repressed with glycylleucine as efficiently as that of the wild-type strain. With regard to activity of the higy-affinity system of KA2241, complicated results were obtained. Under unrepressing conditions, its activities of KA2241 for isoleucine and leucine were not significantly different from those found in the wild-type strain, respectively. Upon repressing the transport systems in KA2241 with glycylleucine, activity of the high-affinity system for isoleucine disappeared almost completely, but that for leucine decreased only slightly. Binding protein Table 5 shows activities of binding proteins for labeled histidine, isoleucine or

9 REGULATORY TRANSPORT MUTANT 357 leucine, which were released by osmotic-shock from various strains grown under unrepressing and repressing conditions. Since specific activities of the binding protein(s) for isoleucine and leucine varied from preparation to preparation, the activities in each preparation were normalized with the binding activity for histidine. Under unrepressing growth condition, KA2313 possessed 5- to 6-fold higher binding activities for isoleucine and leucine than those found in wild-type strain. While, these activities of KA204 were slightly lower than those of wild-type strain. When the transport system was repressed, the binding activities were reduced in all strains, but the extent of this reduction was weak in KA2313 (15 to 23%), and strong in both KA204 and wild-type strains (more than 95%). Thus binding protein(s) of KA2313 for branched-chain amino acids is increased in the activity, and its biosynthesis is not effectively repressed by glycylleucine. Since the level of the binding activities for branched-chain amino acids in a preparation of KA2241 was not significantly different from that found in a corresponding preparation of wild-type strain, the function of gler may differ from that of liv-231. DISCUSSION We have attempted to isolate a new regulatory mutant concerning with the transport of branched-chain amino acids from a transport-defective mutant, CE5 (ilvc8 bynq4), as a colony former on minimal agar medium containing a low concentration of isoleucine and valine. Among clones of Ilv-Brn+ revertants isolated, clones, whose transport activity for isoleucine is relatively insensitive to repression caused by glycylleucine, have been selected for. A regulatory mutant, KA231 (ilvc8 bynq4 liv-231), thus isolated was chosen for present studies. Judging from results obtained with KA231 and its Ilv+ transductant, KA2313 (bynq4 liv-231), we consider that liv-231 mutation has occurred in a regulatory locus other than brnq (structural gene for the low-affinity-(1) transport system), and has derepressed the transport systems of the mutant for branched-chain amino acids. The evidences to support this notion are i) increment in the transport activity for branched-chain amino acids under both repressing and unrepressing growth conditions (Table 4), ii) increment in the specific binding activity released in the osmotic-shock fluid (Table 5), and iii) apparent insensitivity in the growth response to a high concentration of glycylleucine in the presence of limited amounts of isoleucine and valine (Table 3). Derepression of the transport of KA231 is considered to be manifested in both the high-affinity and the low-affinity-(1) systems, though the low-affinity-(1) systems appears to be not participating in the leucine transport (Table 4). The high-affinity system of S. typhimurium appears to be mediated by the specific binding protein(s), but the low-affinity-(1) system does not, because the protein(s) can bind with labeled isoleucine or leucine even at a low concentration of 0.1 pm. This notion is supported by the fact that, KA2313 (bynq4 liv-231), both the binding protein(s) and the high-affinity transport system were partially corepressed by glycylleucine, whereas the low-affinity- (1) system was not affected. The low-affinity-(1) transport system was derepressed in strain KA2241 (glerl)

10 358 K. OHNISHI, K. MURATA AND K. KIRITANI (Table 4), as found in KA224 (ilvc8 glerl) (Ohnishi and Kiritani 1978). However, the high-affinity system of this strain gave complicated results again. Under repressing growth conditions, the high-affinity system behaved heterogeneously, that is, the activity for isoleucine was almost completely repressed, but that for leucine only slightly in spite of marked reduction in the specific binding activity (Table 5). Based on these results, we assume that wild-type strain of S. typhimurium harbors a leucine-specific, repressible transport system, which has a similar Km value to the high-affinity system, and this leucine-specific system has aquired a constitutive state in KA2241 by gler mutation. It should be noted that kinetic data of transport by the high-affinity system of KA2241 do not correspond well with those of KA224, which were obtained from assays in the presence of chloramphenicol (Ohnishi and Kiritani 1978). The regulatory gene, liv-231, for transport of branched-chain amino acids may be distinct from the one, glen, which is closely linked to brnq (Ohnishi and Kiritani 1980), because i) the transport systems of KA2313 are relatively resistant to a repressor, glycylleucine, but those of KA2241 are sensitive, and ii) binding activity for branchedchain amino acids found in KA2313 is high and resistant to glycylleucine, whereas that in KA2241 is low and sensitive. It has been designated in E. coli that strains carrying livr or lstr mutation are increased not only in leucine-transport activity, but also in the level of leucine-binding proteins. These regulatory loci are located at 20 min on the E. coli genetic map (Anderson et al. 1976). It is as yet unknown, however, whether the liv-231 gene of S. typhimurium corresponds to either livr or lstr of E. coli, or not. ACKNOWLEDGMENTS We thank Prof. Sigeru Kuno at Kanazawa University for many useful discussions and suggestions. LITERATURE CITED Anderson, J. J., S. C. Quay, and D. L. Oxender, 1976 Mapping of two loci affecting the regulation of branched-chain amino acid transport in Escherichia coli K-12. J. Bacteriol. 126: Kiritani, K., 1974 Mutants of Salmonella typhimurium defective in transport of branched-chain amino acids. J. Bacteriol. 120: Kiritani, K., and K. Ohnishi, 1977 Repression and inhibition of transport systems for branchedchain amino acids in Salmonella typhimurium. J. Bacteriol. 129: Kiritani, K., and K. Ohnishi, 1978 Multiple transport systems for branched-chain amino acids as studied by mutants of Salmonella typhimurium. Japan. J. Genetics 53: Lowry, 0., N. J. Rosebrough, A. L. Farr, and R. J. Randall, 1951 Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Neal, J. L., 1972 Analysis of Michaelis Kinetics for two independent saturable membrane transport functions. J. Theor. Biol. 35: Nossal, N. G., and L. A. Heppel, 1966 The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J. Biol. Chem. 241: Ohnishi, K., and K. Kiritani, 1978 Glycyl-L-leucine-resistance mutation affecting transport of branched-chain amino acids in Salmonella typhimurium. Japan. J. Genetics 53:

11 REGULATORY TRANSPORT MUTANT 359 Ohnishi, K., and K. Kiritani Close linkage relationship between Salmonella typhimurium. Japan. J. Genetics 55: Sanderson, K. E., and P. E. Hartman, 1978 Linkage map of Salmonella Microbiol. Rev. 42: gler and brnq loci typhimurium, edition in V.