World Journal of Microbiology & Biotechnology 10, 265-289

L-glutamate transport in Lactobacillus helveticus G.S. de Giori* and G.F. de Valdez An energy source (glucose or lactose) was required for the transport of L-glutamic acid by Lactobacillus helveticus. Mg a+, K + and Li + increased its accumulation while Ca z+ and Na + decreased it. It was inhibited by NaF, indicating that ATP may be involved in uptake. Optimum transport was at pH 7.3 and 45°C. L-Glutamic acid transport showed a high degree of stereospecificity, as neither D-glutamate nor D-aspartate were active. Proton-conducting uncouplers, like carbonyl cyanide-m-chlorophenylhydrazone, and ionophores (nigericin, monensin and gramicidin) were strongly inhibitory, These results indicate that a proton motive force may be involved in the transport of L-glutamic acid. Key words: Amino acid, lactic acid bacteria, Lactobacillus, transport.

Lactic acid bacteria are a nutritionally fastidious group of microorganisms, the satisfactory growth of which depends on the availability of amino acids, peptides and vitamins in the culture medium. The amino acid requirements are considerably different for each species but glutamic acid is essential for the growth of lactococci, thermophiJic streptococci and lactobacilli (Ledesma et al. 1977). Among the latter, Lactobacillus helveticus is commonly used with Streptococcus thermophilus as a starter culture for the manufacture of Swiss and Italian cheeses. Amino acid uptake is an initial step in the metabolic pathway giving good cell growth in milk, a substrate which has only a limited amount of free glutamate. There must be membrane-associated enzymes with high protease and peptidase activities to release glutamate and so support the growth of the microorganisms. Amino acid uptake has been widely studied both in lactococci (Driessen el al. 1987; Poolman et al. 1987a or b) and in thermophilic streptococci (Asghar eta]. 1973; Bakker & Harold 1980), but little information is available on uptake in thermophilic lactobacilli. The present investigation was carried out in order to characterize the L-glutamic acid transport system in Lac~ob. helveticus.

The authors are with the Centro de Referencia para Lactobacilos, Chacabuco 145 4000 S.M. de Tucum:~n, Argentina (fax: 54 81 311462) and the C~,tedra de Microbiologia Superior, Universiclad Nacional de Tucum.~n, Argentina. *Corresponding author.

Materials and Methods Microorganism Lactobacillus helveticus G1 was from the stock collection of the Centro de Referencia para Lactobacilos (CERELA), being originally isolated from a Swiss-type cheese.

Preparation of the Cells Cultures were grown in MRS broth (De Man et al. 1960) at 37°C for 12 h, centrifuged (6000 x g, 10 rain, 4°C) and the cell pellet washed twice with 50 mM Hepes, pH 7.3, and resuspended in the same buffer.

Transport Assays The energy reserves of the ceils were depleted by shaking for 2 h in 50 mM Hepes buffer, pH 7.3, at 37°C and the rate of uptake was measured after addition of the energy source. The absorbance of the cell suspension was adjusted to give 1.2 mg dry wt/m[. Transport assays were carried out with freshly prepared ceils equilibrated on a shaker (120 rev/min) at 37°C for 10 rain unless indicated otherwise. Cells pre-incubated for I0 rain with 20 mM glucose were used in all transport assays. Reactions were started by adding I00 ,aM L-fI,4-14C]gIutamicacid (sp. act. 250 mCi/mmol) to the cell suspension. At intervals, 0.2-ml samples of the reaction mixture were removed and immediately filtered through 0.45-/tin pore filters (HA-type; Millipore). Filter residues were washed twice with 50 mM Hepes, pH 7.3, dried under an i.r. lamp and placed in scintillation vials containing 5.0ml of commercial scintillant (Ready-solv Hp/b; Beckmann). Radioactivity was measured by liquid scintillation with a counting efficiency 3-95% for [1,4-14C]glutamic acid. The initial rate of glutamic acid uptake was

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G.S. de Giori and G.F. de Valdez determined from the slopes of the kinetic curves and expressed as nmol L-glutamic acid/mg of dry wt per s. The uptake kinetic constants were determined by using L-glutamic acid from I to 2 raM. Kt is the amount of glutamate transported at half maximum velocity.

Reproducibility All results presented in this paper are means of three replicate assays.

Results and Discussion An energy source was required for the transport of glutamic acid by Lactob. helveticus, glucose being more efficient than lactose (Figure 1). To verify the need for an energy source, a cell suspension was pre-incubated with glutamic acid, the uptake being found to start only 30 s after the addition of glucose (data not shown). This shows that the energy

reserves of the cells were completely depleted after shaking for 2 h in Hepes buffer at 37°C. These results were similar to those obtained for Lactococcus lactis subsp, lactis (Rice et al. 1978) and 5. thermophilus (Bracquart et al. 1984), but different from those obtained for S. faecalis and S. faecium (Asghar ef al. 1973; Brock & Moo-Penn 1962) for the uptake of glycine, alanine, serine and threonine. L-Glutamic acid uptake by Lac~ob. helveticus was temperature-dependent (Figure 2) with no transport below 30°C. Above this temperature, the transport rate increased and reached an optimum at 45°C which was also the optimum temperature for growth of the lactobacillus in a culture medium (data not shown). No uptake occurred above 50°C.

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Time (min) Figure 1. L-Glutamic acid uptake by Lactob. helveticus G1 in presence of lactose (/k) or glucose (O) as energy source or without an energy source (A)-

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Figure 2. Effect of temperature on the transport of L-glutamic acid by lactob, helveticu8 G1. Ceil suspensions were pre-incubated in 20 film glucose at 0 to 70°C. L-[1,4J4C]glutamic acid (100/IM)

was added. After 10 rain, the cells were processed and the initial rate of L-glutamic acid uptake calculated.

Amino acid transport in lactobacilli Glutamic acid transport was sensitive to pH and to the type of buffer used (Figure 3). Thus, in Hepes buffer, transport was maximal at pH 7.3, while it was five times lower in 50 mM Tris at the same pH. In both cases the uptake fell rapidly when the medium became acidic or alkaline. Similar patterns were obtained for pH and temperature in the transport of L-serine by Brevibacterium linens (Hamouy et al. 1985). Uptake kinetics constants were also calculated from Eadie-Hofstee plots (Dixon & Weeb 1979). A single straight line was obtained, indicating the presence of only one kinetically distinguishable glutamic acid transport system (Kt = 5.38 x I0 3 nM; Vma x ~- 27.3 x 103 nmol.mg-X.min). The low value of Kt indicates that the transport system has a high affinity for its substrate. Similar transport rate and Vmax values were obtained for the same amino acid in other lactic acid bacteria (Poolman et al. 1987b; Strobel et al. 1989). The influence of different ions on uptake is shown in Figure 4. Maximum stimulation was obtained with 5 mM Mg 2+, the transport rate being increased 17-fold with respect to the control. This ion may act as a specific cofactor for the membrane-bound enzymes, as was reported by Heckels et al. (1977), Heptinstal] et al. (1970) and Thompson (1976). When Ca 2+ or Na + was present, the intracellular amino acid concentration was decreased 0.5- and 0.7-fold, respectively, with respect to the control. However, K + and Li + increased the amino acid accumulation in the cell by

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Figure 4. Effect of metal ions on uptake of L-glutamate by Lactob. helveticus G1. Metal ion chlorides (10mM), added to the pre-incubation mixtures were Li + (A), Rb + (O), Cs + (O), NH+ (V), K + (V), Na + ( x ) , Cu 2+ (O), Ca 2+ (E]), Mg 2+ (r~), or Mn 2+ (0). The cells were then processed and the initial rate of L-glutamic acid uptake calculated. A control, without added ions (m) was also tested.

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Figure 3. Effect of pH and buffer on the transport of L-glutamate by Lactob. helveticus GI. The cell pellet was resuspended with 50 mM Mes (pH 4.0 to 6.0) (O), KH~POJK2HPO4 (pH 6.0 to 7.5) (A), Hepes (pH 6.0 to 8.5) (O), or Tris/HCI (pH 7.5 to 9.5) (/k), each containing 10 mM energy source. Uptake was started by adding 100/~M L-[1,4-14C]glutamic acid.

2.6- and 3.6-fold, respectively. Similar results were reported by Rayman et al. (1972) for Escherichia coli K12. The specificity of the transport was investigated by studying the inhibitory effect of unlabelled amino acids, analogues, and peptides competing with the uptake of a

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G.S. de Giori and G.F. de Valdez Table 1. Effect of unlabelled competitors on uptake of L-[1,4-++C]glutamic acid by Lactob, helvetlcus.*

Table 2. Elfecl of inhlbltors on L-glutamlc acid transport by

Competitor (1 r a M )

Uptake of L-[1,4-14C]glutamlc acid (%)1"

Treatment1"

None L-Asp D-Asp L-Ash o-Glu L-Gin L-His Gly L-Arg L-Leu L-Ser L-Met L-Phe L-Lys L-Trp L-Ala L-Val Acetyl-L-Glu ?-Methylester-L-Glu L-Glu-¢-anilide Carbobenzoxy-L-Glu Glu-Val VaI-Glu Glu-Ala VaI-Gly-Ser-Glu Glu-Thr-Tyr

100 6 95 87 97 89 80 92 93 98 95 100 96 93 92 98 89 85 75 13 12 18 90 90 70 70

*Unlabelled amino acid, analogue or peptide and L-[1,414C]glutamic acid were added together to the cell suspension with 20 mM glucose. Cells were filtered after 2 min incubation, and treated. 1"Relative to competitor-free control.

labelled substrate (Table 1). The accumulation of the amino acid in the cell was not inhibited by L-histidine, glycine or L-arginine. In contrast, L-aspartic acid and certain L-glutamic acid analogues, such as L-glutamic acid 0¢-anilide and carbobenzoxy-L-glutarnic acid, were powerful uptake inhibitors, showing the lack of specificity of the transport system. These results show that the ~-carbon amino group is essential for binding to take place and that the chain length and the spatial distribution of the compounds used in the assays might play a significant role in the specificity of the uptake. L-Glutamic acid transport has a high degree of stereospecificity for L isomers, since neither D-glutamic acid nor D-aspartic forms were active (Table 1). Similar results were found with S. faecalis (Reid et al. 1970) and S. fhermophilus (Noji et aI. 1988). The effect of various metabolic inhibitors on the uptake of L-glutamic acid was tested to study the relationships between energy and transport (Table 2). In the presence of 10 mM NaF (an inhibitor of enolase) the uptake was completely inhibited, indicating that ATP generated by the

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Lactob. helveticus.*

COOP (50/IM)* N-Ethylmaleimide (1 mM) Gramicidin (1/~M) Monensin (3 #M) Nigericin (10/~M) Valinomycin (2 #M) IAA (10 mM) NaF (10 raM) Chlorhexidine digluconate (0.1 riM) DCCD (0.1 nM)

Uptake of L-[1,4-1+C]glutamlc acid (%) 1.3 39 8.2 2.3 2.1 1.6 14.5 2.8 1.9 1.2

* Inhibitor was added to the cell suspension with 20 mM glucose. After 10 rain pre-incubation, the uptake was started by adding 200 #M L-[1,4-1+C]glutamic acid. 1"Values in parenthesis are final concentrations. :1:Relative to inhibitor-free control.

fermentation process served as an energy source for L-glutamic acid transport. DCCD also stopped the uptake, the same effect being observed with the proton-conducting uncouplers CCCP, nigericin, monensin and valinomycin. These results appear to indicate that ATPase and a proton-motive force may play an essential role in the glutamate transport system; these findings are very similar to those reported for Lactob. casei (Strobel et al. 1989).

Acknowledgements This research was partially supported by the International Foundation for Science (IFS) grant E/1474-1.

References Asghar, S,S., Levin, E. & Harold, F.M. 1973 Accumulation of neutral amino acid by S. faecalis. Journal of Biological Chemistry 248, 5225-5233. Bakker, E.P. & Harold, F.M. 1980 Energy coupling to potassium transport in S. faecalis. Interplay of ATP and the proton-motive force. Journal of Biological Chemistry 255, 433-440. Bracquart, P., Le Deaut, J.Y. & Linden, G. 1984 Uptake of glutamic acid by S. thermophilus. Journal of Dairy Research $6, 107-116. Brock, T.D. & Moo-Penn, G, 1962 An amino acid transport system in S. faecium. Archives of Biochemistry and Biophysics 98, 183-190. De Man, J.C., Rogosa, M. & Sharpe, M.E. 1960 A medium for the cultivation of lactobacilli. Journal of Applied Bacteriology 23, 130-155. Dixon, M. & Weeb, E.C. 1979 Enzymes, 3rd edn. London: Longman. Driessen, A.J.M., Kodde, J., De Jong, S. & Konings, W.N. 1987 Neutral amino acid transport by membrane vesicles of S. cremoris is subject to regulation by intemal pH. Journal of Bacteriology 169, 2748-2754.

Amino acid transport in lactobacilli Hamouy, D., Boyaval, E. & Desmazeaud, M.J. 1985 Active transport of L-serine in Brevibacterium linens ATCC 9175. Milchwissenshaft 40, 133-136. Heckels, J.E., Lambert, P.A. & Baddiley, J. 1977 Binding of magnesium and cell walls of Bacillus subtilis W 23 containing teichoic acid or teichuronic acid. Biochemical Journal 162, 359-362. Heptinstall, S., Archibald, A.R. & Baddiley, J. 1970 Teichoic acid and membrane function in bacteria. Nature, 225, 519-521. Ledesma, O.V., Ruiz Holgado, A.P., Oliver, G., Giori, G.S., Raibaud, P. & Galpin, J.V. I977 A synthetic medium for comparative nutritional studies of lactobacilli. Journal of Applied Bacteriology 42, 123-133. Noji, S., Sato, Y., Suzuki, R. & Taniguchi, S. 1988 Effect of intracellular pH and potassium ions on a primary transport system for glutamate/aspartate in Streptococcus mutans. European Journal of Biochemistry 175, 491-495. Poolman, B., Hellingwerf, K.J. & Konings, W.N. 1987a Regulation of the glutamate-glutamine transport system by intracellular pH in S. lactis. Journal of Bacteriology 169, 2272-2276. Poolman, B., Staid, E. & Konings, W.N. 1987b Kinetic properties of a phosphate-bond-driven gtutamate-glutamine transport system

in S. lactis and S. cremoris.Journal of Bacteriology 169, 2755-2761. Rayman, M.K., Lo, T.C.Y. & Sansal, B.D. 1972 Transport of succinate in E. coli II. Characteristics of uptake and energy coupling with transport in membrane preparations. Journal of Biological Chemistry 247, 6332-6339. Reid, K.G., Utech, N.M. & Holden, J.T. 1970 Multiple transport components for dicarboxylic amino acids in S. faecalis. Journal of Biological Chemistry 245, 5261-5272. Rice, G.H., Stewart, F.H.C., Hillier, A.J. & Jago, G.R. 1978 The uptake of amino acids and peptides by S. lactis. Journal of Dairy Research 45, 93-107. Strobel, J.H., Russel, J.B., Driessen, A.J.M. & Konings, W.N. 1989 Transport of amino acids in L. casei by proton-motive-forcedependent and non-proton-motive-force-dependent mechanisms. Journal of Bacteriology 171, 280-284. Thompson, J. 1976 Characteristics and energy requirements of an aminobutyric acid transport system in S. lactis. Journal of Bacteriology 127, 719-730.

(Received in revised form 7 October 1993; accepted 1I October I993)

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L-glutamate transport in Lactobacillus helveticus.

An energy source (glucose or lactose) was required for the transport of L-glutamic acid by Lactobacillus helveticus. Mg(2+), K(+) and Li(+) increased ...
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