Streptococcus lactis

Transcription

1 JOURNAL OF BACTERIOLOGY, Aug. 1976, P Copyright C 1976 American Society for Microbiology Vol. 127, No. 2 Printed in U.S.A. Characteristics and Energy Requirements of an a-aminoisobutyric Acid Transport System in Streptococcus lactis JOHN THOMPSON New Zealand Dairy Research Institute, Palmerston North, New Zealand Received for publication 1 April 1976 Galactose-grown cells ofstreptococcus lactis ML3 accumulated a-aminoisobutyric acid (AMB) by using energy derived from glycolysis and arginine catabolism. The transport system displayed low-affinity Michaelis-Menten saturation kinetics. Using galactose or arginine as energy sources, similar Vmax and Km values for AMB entry were obtained, but on prolonged incubation the intracellular steady-state concentration of AIB in cells metabolizing arginine was only 65 to 70% that attained by glycolyzing cells. Efflux of AIB from preloaded cells was temperature dependent and exhibited the characteristics of a first-order reaction. The rate of AIfB exit was accelerated two- to threefold in the presence of metabolizable energy sources. Metabolic inhibitors including p-chloromercuribenzoate, dinitrophenol, azide, arsenate, and N,N'-dicyclohexylcarbodiimide either prevented or greatly reduced AIB uptake. Fluoride, iodoacetate and N- ethylmaleimide abolished galactose-dependent, but not arginine-energized, AIB uptake. K+ and Rb+ reduced the steady-state intracellular AIB concentration by approximately 40%, and these cations also induced rapid efflux of solute from actively transporting cells. Equivalent concentrations (10 mm) of Na+, Li+, or NH4+ were much less inhibitory. The proton-conducting ionophores tetrachlorosalicylanilide and carbonylcyanide m-chlorophenylhydrazone abolished uptake and induced AIB efflux even though glycolysis and arginine catabolism continued at 60 and 140%, respectively, of control rates. A proton motive force is most likely involved in the active transport of AMB, whereas data from efflux studies suggests that energy is coupled to AIB exit in cells of S. lactis ML3. Group N streptococci (Streptococcus lactis, 14, 15) and Kashket and Wilson (23, 24) in a S. cremoris, S. diacetylactis) are particularly series of elegant studies related to amino acid fastidious in their nutritional requirements uptake in S. faecalis 9790 and 83-galactoside and are able to use only a limited range of transport by S. lactis 7962, respectively. The carbohydrates as energy sources for growth. results of these investigations in conjunction The glycolytic pathway provides the main route with data obtained from Staphylococcus aureus for adenosine 5'-triphosphate (ATP) formation and E. coli (for review, see 12) have been instrumental in establishing the principles out- in S. lactis, but this strain can, under certain circumstances, generate ATP via the arginine lined in Mitchell's chemiosmotic hypothesis dihydrolase series of reactions (10, 35). Unlike (27, 28) as a basis for the active transport of Escherichia coli and many other organisms, solutes in a number of bacterial systems (for cells of S. lactis are essentially devoid of metabolizable intracellular energy reserves (36) Amino acid transport in S. faecalis (group D) reviews, see 5, 12, 14, 15, 34). and, lacking a functional electron transportrespiratory chain complex, are unable to gener- with the exception of a preliminary report of has been extensively studied (1, 2, 7, 29), but ate ATP by oxidative phosphorylation. valine uptake by S. diacetylactis (33) there is a The relative simplicity of the energy-generating systems of the streptococci has proved to be uptake by other species of the genus Streptococ- paucity of information concerning amino acid an extremely useful characteristic, particularly cus. In the present report, S. lactis ML3 has for the study of energy coupling to the active been used to extend and augment our knowledge of the characteristics and energy require- transport of solutes. The virtues of the streptococci have been exploited to considerable advantage, notably by Harold and co-workers; (1, grown cells of S. lactis ML3 can metabolize ments for amino acid transport. Galactose- 719

2 720 THOMPSON J. BACTERIOL. arginine, glucose, and lactose in addition to galactose, and such cells have been routinely used in this investigation. The observation that certain naturally occurring neutral amino acids were extensively metabolized by S. faecalis (1) prompted the use of a-aminoisobutyric acid (AIB), a non-metabolizable analogue of alanine, as the model amino acid in this investigation. MATERIALS AND METHODS Organisms. S. lactis ML3 and S. lactis 7962 were obtained from the culture collection of the New Zealand Dairy Research Institute. Culture maintenance. S. lactis ML3 was grown in chemically defined medium containing an appropriate carbohydrate as energy source. S. lactis 7962 was grown in defined medium supplemented with 0.5% Trypticase (Baltimore Biological Laboratories) and 1% tryptone (Oxoid). After attaining the mid-logarithmic phase of growth, 2- to 3-ml volumes of the culture were transferred aseptically to sterile glass vials. The cultures were rapidly frozen in an ethanol-dry ice mixture. Frozen cultures were stored at -80 C in an ultralow-temperature freezer. Thawed cultures contained 50 to 80% viable organisms after 5 months of storage (36). Composition of defined medium. The chemically defined medium contained the following, per liter, (i) amino acids (all 0.1 g)-l-asparagine, L-cystine, L-glutamine, glycine, L-histidine hydrochloride, L- isoleucine, L-leucine, L-methionine, L-proline, L- phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine (other amino acids included were DL-alanine, DL-aspartic acid, DL-glutamic acid (all 0.3 g); L-lysine hydrochloride, 0.2 g; and L-arginine hydrochloride, 0.5 g); (ii) vitamins-p-aminobenzoic acid, 0.2 mg; biotin, 0.1 mg; folic acid, 0.1 mg; nicotinic acid, 1 mg; pantothenic acid (calcium salt), 1 mg, pyridoxal hydrochloride, 0.2 mg; pyridoxine hydrochloride, 1 mg; riboflavin, 0.1 mg; and thiamine hydrochloride, 0.1 mg; and (iii) basesadenine, 5 mg, guanine, 1 mg; uracil, 5 mg; and xanthine, 5 mg. Other compounds present were: Tween 80, 1 g; acetate (sodium salt), 1 g, disodium ethylenediaminetetraacetate, 4 mg; FeSO4 * 7H20, 1 mg-, MnCl2 * 4H2O, 1 mg, MgSO4*7H2O, 2.5 g; Na2HPO4, 8.5 g; KH2PO4, 2.0 g; and carbohydrate, 5 g Ṫhe vitamins were dissolved in warm water, and the solution was filter sterilized and then kept frozen until required. Carbohydrate and MgSO4 solutions were prepared separately, and these, and other solutions, were sterilized by autoclaving at 121 C for 15 min. This medium had an initial ph of 7.2. Growth ceased due to carbohydrate limitation, and the final ph of the medium was ph 5.6. Growth of organisms. When required, a deep frozen culture was thawed, and 1 drop of the suspension was transferred to 10 ml of defined medium containing the appropriate carbohydrate as energy source. After overnight growth, 6 to 7 ml of culture was transferred to 300 ml of fresh medium. All cultures were grown without agitation at 320C. The approximate doubling times (in minutes) for S. lactis ML3 growing in the defined medium containing various sugars were: glucose, 54; galactose, 62; lactose, 54; and fructose, 105. Cells from 200 ml of growth medium were harvested during mid-logarithmic growth by centrifugation at 13,000 x g for 1 min. The supernatant fluid was removed, and, the cell pellet was washed twice by resuspension in, and centrifugation from, 200-ml volumes of 0.01 M MgSO4 solution. The cell pellet was resuspended with approximately 3 ml of 0.01 M MgSO4 solution to provide a homogeneous thick cell suspension containing 20 to 25 mg (dry weight) of cells per ml. Measurement of AIB uptake. The transport assay contained, in a final volume of 10 ml, 0.1 M tris- (hydroxymethyl)aminomethane(tris) - maleate buffer (ph 7.0), 0.2 mm [14C]AIB (0.4,uCi/,umol), and appropriate energy source (2 mm). To this system was added 80 to 100,ul of the previously prepared thick cell suspension to obtain a final cell density of 200,ug (dry weight) of cells per ml. The cell suspensions were gently agitated for 10 min at 30 C using a water bath shaker before addition of ['4C]AIB. At intervals, 1.0-ml samples were withdrawn and vacuum filtered through 0.45-Am membrane filters (type HA, Millipore Corp., Bedford, Mass.). The retained cells were washed quickly by drawing 5 ml of 0.1 M Tris-maleate (ph 7.0) buffer through the filters. In kinetic studies, the initial rates of AIB uptake were determined by rapid sampling of 1-ml volumes of cell suspension after 1 min of incubation. The velocity, v, of uptake was calculated as micromoles of AIB accumulated per gram (dry weight) of cells per minute. Preloading procedure and AIB efflux studies. Washed cells ofs. lactis ML3 (or S. lactis 7962) were suspended at a concentration of 1 mg (dry weight) of cells per ml in 20 ml of the transport medium containing 0.8 mm [14C]AIB (0.4,uCi/,umol) and 5 mm glucose. After 60 min at 30 C, the cell suspension was rapidly cooled to 00, followed by centrifugation at 12,000 x g for 1 min at 00C. The supernatant fluid was carefully removed by a vacuum aspirator, and the cell pellet was resuspended with a further 20 ml of ice-cold 0.1 M Tris-maleate buffer (ph 7.0) and centrifuged once more. After removal of the wash supernatant fluid, the ['4C]AIB-preloaded cell pellet was resuspended with the aid of two or three small glass beads and a Vortex mixer in 5 ml of icecold 0.1 M Tris-maleate buffer (ph 7.0). For studies of [14C]AIB efflux, 0.5 ml of the preloaded cell suspension was added to 9.5 ml of incubation medium at 30 C, giving a final cell density of approximately 200 Ag (dry weight) of cells per ml. Exit of the amino acid analogue was monitored by filtration of 1-ml volumes ofcell suspension at appropriate time intervals. The rate constants of efflux (kex) were determined from the slope of plots of ln [AIB]i, versus time. The half-time of the exit reaction was calculated from T1/2 = ln 2lkex. Radioactivity determination. The membrane filters plus adhering cells were placed in glass scintillation vials and dried under an infrared lamp. A 5-

3 VOL. 127, 1976 ml volume of scintillation cocktail containing 5 g of 2,5-diphenyloxazole (PPO) and 0.3 g of 1,4-bis-2-(5- phenyloxazolyl)benzene (POPOP) per liter of toluene was added per vial, and radioactivity was determined by using a Packard series 2002 liquid scintillation spectrometer (Packard Instrument Co. Inc., Downers Grove, Ill.). Calculation of intracellular AIB concentration. From radioactive measurements the intracellular AIB concentration was calculated knowing the specific activity of the amino acid, the counting efficiency of the spectrometer, and the intracellular (protoplast) fluid volume. Using previously described methods (3, 4, 8), it was determined that 1 g (dry weight) of cells was equivalent to 1.67 ml of intracellular (protoplast) fluid. Analytical methods. The effects of the inhibitors carbonylcyanide m-chlorophenylhydrazone (CCCP) and tetrachlorosalicylanilide (TCS) upon carbohydrate and arginine utilization were followed using the basal transport system in which the sugar or arginine concentrations were 10 and 5 mm, respectively, and the cell density was 500,ug (dry weight)/ ml. At intervals throughout a 60-min period, 1.0-ml volumes of suspension were removed and filtered through 0.45-,um filters. Filtrates were collected in small glass tubes, sealed, frozen in an ethanol-dry ice mixture, and retained for subsequent analysis. Glucose and galactose were assayed by using Glucostat and Galactostat reagents (Worthington Biochemicals Corp., Freehold, N.J.). Filtrates were assayed for arginine disappearance or ornithine and NH3 formation on the basic column of an amino acid analyzer (Locarte Co., London, England), using histidine as internal standard. Peak areas were integrated using a computing integrator (Spectraphysics, Mountain View, Calif.). Extraction and identification of radioactive material. Preloaded cells ofs. lactis ML:, were prepared using the method previously described. The washed cell pellet obtained from 20 ml of preloading medium was extracted with 15 ml ofboiling water for 15 min, and the suspension was clarified by centrifugation at 25,000 x g for 5 min. The supernatant fluid was removed by a Pasteur pipette, concentrated to 5 ml by rotary evaporation, and finally desalted (9). More than 93% of the radioactivity originally present in the cells was recovered after the extraction and desalting procedures. The solution was further concentrated to 0.6 ml, and 10-,ul samples were assayed for radioactivity using Bray reagent (6). Subsequently, 10,lj of this solution, containing a known amount of AMINO ACID TRANSPORT IN S. LACTIS 721 radioactivity, was applied to thin-layer plates together with a standard mixture of amino acids. The chromatographic technique was essentially that of Pillay and Mehdi (30) using cellulose MN300 layers and the solvent D systems described therein. Only one radioactive spot could be detected by autoradiography, and >90% of the radioactivity initially applied to the plate was found in that position. Comparison of the position of the radioactivity relative to authentic AIB and standard amino acid markers confirmed the material to be unchanged AIB. The identity of the radioactive material appearing in the incubation medium during efflux experiments was similarly identified qualitatively and quantitatively as AIB. Reagents. a-['4c]aib was purchased from Radiochemical Centre, Amersham, England. CCCP was obtained from the Sigma Chemical Co., St. Louis, Mo. TCS was generously donated by A. Hamilton, Department of Biochemistry, University of Aberdeen, and N,N'-dicyclohexylcarbodiimide was obtained from the Aldrich Chemical Co. For inhibitor studies, TCS, CCCP, and N,N'-dicyclohexylcarbodiimide were dissolved in 95% ethanol, such that on addition to the incubation system the final ethanol concentration did not exceed 0.1%. RESULTS Energy requirements for AIB accumulation. Galactose-grown cells ofs. lactis ML:, had the capacity to utilize galactose, glucose, lactose, and arginine as energy sources for AIB accumulation (Fig. 1A). The intracellular AIB concentration attained in glycolyzing cells exceeded that of the medium by 100-fold and bv approximately 65-fold when arginine was the energy donor. AIB transport by glycolyzing, galactose-grown cells of S. cremoris AM., was also similar to that found in S. lactis ML, but the former strain was unable to utilize arginine as an energy source for uptake. In the absence of an appropriate energy source, the concentration of analogue within cells of both strains exceeded that of the medium by approximately twofold. Optimum buffer and ph requirements for AIB transport. All buffers tested, with the exception of K+-maleate, were equally effective in the transport assay (Fig. 2A). Whereas perhaps not the optimum conditions for solute transport (Fig. 2B), it was felt that the choice of 0.1 M Tris-maleate buffer (ph 7.0) would permit comparison of the data obtained for AIB transport by S. lactis ML3 with data from other studies, particularly from S. faecalis (1). Tris-maleate buffer was also appropriate in experiments concerned with the effects of monovalent cations on the AIB transport system. Kinetics of AIB uptake by S. lactis ML3. The initial rate of AIB uptake was dependent upon the concentration of the analogue in the incubation medium. The transport system displayed low-affinity Michaelis-Menten saturation kinetics when three different energy sources were used (Fig. 3). Steady-state accumulation of AIB using galactose or arginine as energy sources. Data from kinetic experiments (Fig. 3) showed arginine to be as efficient an energy source for AIB transport as the sugars tested, and, importantly, the presence of arginine did not inhibit AIB entry. Since the kinetic parameters Vmax

4 722 THOMPSON J. BACTERIOL. mm 16 In 0 1v time (minutes) FIG. 1. (A) Energy requirement for AIB accumulation by galactose-grown cells ofs. lactis ML3. Cells were suspended at 200 pg (dry weight)lml in 0.1 M Tris-maleate buffer (ph 7.0) containing appropriate energy sources (2 mm). After 10 min ofpreincubation at 300C, 4 x 10-4 M ['4C]AIB (0.4 pci,umol) was added to the system. (B) Efflux of AIB from galactose-grown, ['4C]AIB-preloaded cells of S. lactis ML3. The cells were suspended at 200 pg (dry weight)/ml in 0.1 M Tris-maleate buffer (ph 7.0) containing various energy sources at 2 mm final concentration. Symbols: *, control (no exogenous energy source) (0.018, 38.7); A, arginine (0.036, 19.5); 0, galactose (0.025, 28.2); *, lactose (0.033, 21.3); and O, glucose (0.039, 17.9). Values in parentheses refer to experimentally determined rate constants ofexit (k, per minute) and half-time ofsolute minutes), respectively. exit (TI/2, tirne (minutes) FIG. 2. (A) Effect of different buffers on AIB uptake by galactose-grown cells of S. lactis ML3. Cells were suspended in appropriate buffer (0.1 M) containing 10 mm glucose as energy source, and other conditions were as described in Materials and Methods. Symbols: 0, Tris-hydrochloride; U, KOH-maleate; 0, Tris-maleate; A, NaOH-maleate; and A, phosphate. (B) Optimum ph for AIB uptake. AIB uptake by cells was determined after 10 min of incubation and buffer systems used were: 0, Tris-2-[Nmorpholinolethane sulfonic acid; 0, citrate-phosphate; A, Tris-maleate; *, Tris-hydrochloride and A, NaOH-succinate. and Km for AIB entry were similar when cells used arginine or galactose as energy sources, it was surprising to find upon prolonged incubation that the intracellular steady-state concentration of the analogue was considerably ph * _ m LAIB]0 greater (ca. 30 to 35%) in glycolyzing than in arginine-metabolizing cells (see Fig. 1A). This observation was confirmed over a wide range of extracellular AIB concentrations (Fig. 4A). In preliminary investigations using AIB-preloaded cells, it had been noted that the rate of exit of the intracellular solute was, to a marked degree, influenced by the nature and presence of metabolizable energy sources in the suspendmm1 FIG. 3. Kinetics of AIB transport by galactosegrown cells of S. lactis ML3 using various energy sources. Experimental conditions were as described in Materials and Methods. Energy sources used, at 2 mm final concentration, were: A, arginine; 0, galactose; O, glucose.

5 VOL. 127, 1976 ing medium. It seemed possible that the differences in intracellular steady-state concentrations of AIB might be due to variations in AIB efflux rates from cells utilizing the different energy sources. Using the simplest representation of a "pump-and-leak" system, the rate of change of intracellular solute concentration (21) would be expressed by: (entry) d[iaib]in = VmaAAIB]tx - k,.x[aib]in (1) dt Km + [AEB]e.x where [AIB]i0 and [AIBV., refer to the intracellular and medium concentrations of solute, respectively, k,.x is the appropriate rate constant of solute exit, and V,.,, and Km are constants and independent of the nature of the energy source used for transport. Under steady-state conditions, d[aib]jnidt = 0, and transposition and rearrangement of equation 1 yields 1 _ k,.>k,. 1 +±k.x (2) [AIBI,n VIPOUX AIOBPX VmflrX According to linear equation 2, plots of 1/ LAIB]i, versus 1/[AIB],.\ should yield the straight lines illustrated in Fig. 4B, having intercepts on the y axis equal to k,e\lvmax. From a knowledge of V,,,, (18.2,molIg [dry weight] per min; or mmol/liter per min) and intercept values for galactose (0.0116) and arginine (0.0175), the theoretical rate constants of AIB exit from cells utilizing the two energy sources were calculated to be: k,.x, 0.127/min (cells using galactose); and k,, 0.191/min (cells using arginine). AIB efflux from preloaded cells. Direct esti- AMINO ACID TRANSPORT IN S. LACTIS 723 mations of the rates of AIB efflux were conducted using preloaded cells, and their values were compared with the theoretical values previously calculated from the steady-state data. The rate of AIB exit was found to be temperature dependent and exhibited the characteristics of a first-order reaction. At 00C efflux was comparatively slow (k,. = 0.004/ min; r1/2, ca. 170 min). At 3000, in the absence of an energy source, a fourfold increase in exit rate (kex = 0.018; r,12, 38 min) was observed (Fig. 1B). At this temperature, the inclusion in the medium of those compounds that supported AIB accumulation (see Fig. 1A) further increased the rate of AIB exit by two to three times (Fig. 1B). Although AIB exit from preloaded cells was faster in the presence of arginine than in that of galactose, the experimentally determined rates of efflux (Fig. 1B legend) were four to five times slower than the rates predicted from steady-state data. Similar rates and half-times of AIB efflux were found when preloaded cells were incubated with lactose, glucose, or arginine (Fig. 1B), but from the previously discussed pump-andleak mechanism one would have anticipated AIB efflux to be slower in the presence of the sugars than in the presence of arginine. Effects of metabolizable energy sources on the rates of AIB exit. The above observations prompted the question as to whether acceleration of AIB exit in the presence of the exogenous substrates was a consequence of competitive inhibition by the compounds upon an AIB recapture system (31, 37) or, alternatively, whether the increased rate of exit was associated with energy generation from these com LAIB.. mm A nmm- FIG. 4. (A) Relationship between the extracellular AIB concentration versus intracellular steady-state concentrations, maintained by galactose-grown cells of S. lactis ML3 using galactose (0) or arginine (A) as energy source for transport. (B) Double reciprocal plot of data illustrated in (A).

6 724 THOMPSON pounds. Evidence in support of the latter possibility was obtained from studies using glucose-grown cells of S. lactis ML3. During growth on glucose both the arginine dihydrolase complex and galactose transport systems are apparently repressed, since neither compound supported significant AIB accumulation by the cells. Lactose was also a poor energy source for AIB transport in glucosegrown cells (Fig. 5A). Data obtained from efflux experiments (Fig. 5B) showed clearly that galactose, arginine, and lactose failed to enhance the rate of solute loss from preloaded cells. However, glucose, which is a highly efficient energy source for uptake, increased the rate of AIB exit by almost threefold. Acceleration of AIB efflux by metabolizable energy sources was also found using cells ofs. cremoris AM2 and S. lactis Galactosegrown cells of S. lactis 7962 were unable to metabolize lactose, but glucose and galactose were equally effective energy sources for AIB uptake (Fig. 6A). Arginine metabolism supported accumulation of the amino acid analogue to a steady-state concentration approximately 40% that attained by glycolyzing cells. Efflux of AIB from these cells in the absence of an energy source was comparatively slow, and the rate of exit was not increased when lactose was present in the incubation medium. However, glucose, galactose, and particularly arginine (ca. fourfold) increased the rate of exit of the intracellular solute (Fig. 6B). Possibility of AIB catabolism by S. lactis ML3. The radioactivity lost from preloaded J. BACTERIOL. cells could be quantitatively recovered in the filtrate (Fig. 7). That this material represented efflux of AIB per se and not possible metabolic products was evidenced by the findings that (i) no loss of radioactivity occurred upon acidification and nitrogen sparging of the filtrate, indicating no 14CO., liberation from [1-'4C]AIB, and (ii) after thin-layer chromatography using standard amino acid markers (Fig. 8A), essentially all the radioactive material applied to the layer could be quantitatively recovered as a single spot, which co-chromatographed with authentic AIB (Fig. 8B). Effects of inhibitors on AIB accumulation. It was of interest to compare the effects of various metabolic inhibitors upon AIB accumulation when cells utilized alternative substrates, either arginine or galactose, as energy sources for AIB transport. Both azide and the adenosine triphosphatase inhibitor N,N'-dicyclohexylcarbodiimide drastically reduced, and p-chloromercuribenzoate completely prevented, AIB accumulation by the cells when galactose (or arginine, data not shown) was used as the energy source (Fig. 9). Arsenate and dinitrophenol caused, respectively, 70 and 45% inhibition of AIB uptake. N-ethylmaleimide, iodoacetate, and fluoride (the classic inhibitors of glyceraldehyde-3-phosphate dehydrogenase and 2-phospho-D-glycerate hydrolase, respectively) abolished AIB uptake by glycolyzing cells. These three inhibitors did not prevent AIB uptake when arginine was utilized as the source of energy for transport. time (minutes) FIG. 5. (A) Energy requirements for AIB uptake by glucose-grown cells ofs. lactis ML3. (B) Efflux ofaib from glucose-grown, AIB-loaded cells of S. lactis ML3. Experimental conditions were as described in Fig. 1. Symbols: *, control (no added energy source, 0.017, 42.0); A, arginine (0.019, 36.7); 0, galactose (0.016, 43.0); M, lactose (0.017, 41.0); 0, glucose (0.045, 15.4). See legend to Fig. 1 for values in parentheses.

7 VOL. 127, 1976 AMINO ACID TRANSPORT IN S. LACTIS 725 mma ffn 6 - A{l[AIBI n1- A C mm time (minutes) FIG. 6. (A) AIB uptake bygalactose-grown cells ofs. lactis 7962 in the presence ofvarious potential energy sources. (B) Effect ofmetabolizable energy sources on the rate ofaib exit from galactose-grown AIB-preloaded cells ofs. lactis Symbols: 0, control (no energy source, 0.023,30.4); A, arginine (0.098, 7.1); 0, galactose (0.035, 19.7); U, lactose (0.016, 44.0) and 0, glucose (0.043, 15.9). See legend to Fig. 1 for values in parentheses. 14 c C.P.M ( pletely prevented AIB accumulation, still pero0 *,..._ mitted the cells to metabolize galactose at 60% of the control rate, whereas TCS and CCCP actually increased the rate of arginine utilization by 40% (Table 1). 0 The addition of TCS, CCCP, and N,N'-dicyclohexylcarbodiimide to actively transporting cell suspensions under steady-state conditions o0- / induced a rapid loss of the intracellular AIB (Fig. 10). 0o / Specificity of the AIB transport system. Unlabeled AIB as well as structurally similar 0, 3 neutral amino acids, including serine, ala nine, and glycine, markedly reduced [14C]AIB tirne (minutes) uptake by the cells (Fig. 11A), and these FIG. 7. Quantitative recovery of radioactive mate- amino acids also induced rapid efflux of the rial during efflux studies using [14C]AIB-preloaded amino acid analogue when added to suspen- ML3. Cells,preloaded as described in sino aciv transportin cel (i lib)n cells ofs. Xlactis Materials and Methods, were suspended at a concentration of, 200 pg (dry weight)/ml in 0.1 M phosphate Aromatic (e.g., proline, phenylalanine), buffer (pli1 7.0) containing 2 mm glucose as energy branched-chain, and basic (e.g., arginine) source. ALt appropriate intervals, 1.0-ml volumes amino acids caused little or no inhibition of were filte? red and radioactivity was determined in AIB accumulation. cells (U) atnd in the filtrate (0). The symbol * repre- Monovalent cations and AIB accumulation. sents total recovery ofradioactive material from cells The steady-state level of [14C]AIB accumuplus filtralte throughout the experiment. lated by cells suspended in K+-maleatebuffered medium was approximately 40% Effect of proton conductors on AIB accu- lower than the concentration attained when mulatiorn. The capacity of TCS and CCCP to Na+-maleate buffer was used (Fig. 2A). The facilitateathe transfer ofprotons across biologi- possible inhibitory effects of K+ and other cal memibranes is now well established (13, 17, monovalent cations upon AIB accumulation 19). Bot;h compounds at low concentration were investigated accordingly. When K+ or abolished AIB uptake by S. lactis ML3 when Rb+ (10 mm) was present in Tris-maleatee (Fig. 9) and arginine were used as buffered systems, the steady-state intracellu- galactos energy s;ources. It is significant that the pro- lar amino acid concentrations were reduced by ton condluctors, at concentrations that com- 36 and 46%, respectively. Equivalent concen- -1 -

8 726 THOMPSON A J.- BAcTzjuoL. 4' 'M X", me a B I1t 0 FIG. 8. Identification of radioactive material appearing in the incubation medium during efflux studies with ['4CMB-preloaded cells of S. lactis ML,3. (A) Two-dimensional thin-layer separation of a mixture of standard amino acids plus authentic AIB; (B) autoradiogram ofmaterial appearing in medium during efflux. trations of other monovalent cations caused little or no reduction in the steady-state level (Li+, 1%; Na+, 6%; and NH4+, 11%). Similar results were obtained when arginine was used as the energy source for transport. When K+ or Rb+ was added to media containing cells at steady-state conditions with respect to AIB accumulation, a rapid decrease in the intracellular concentration of the analogue was observed. Addition of equivalent concentrations of Na+, Li+, or NH4+ ions was essentially without effect (Fig. 12B).

9 VOL. 127, 1976 GBlin 10 mm AMINO ACID TRANSPORT IN S. LACTIS 727 source, but only p-chloromercuribenzoate and CCCP caused inhibition of uptake by cells metabolizing arginine. Fluoride, an inhibitor of glycolysis, also prevented accumulation of AIB when carbohydrate, but not arginine, was used as the energy source. The proton-conducting uncouplers (13, 16, 4-2 O S5 15 O time (minutes) FIG. 9. Effects of various inhibitors upon AIB accumulation by galactose-grown cells ofs. lactis ML3. The cells were preincubated for 10 min in the presence of galactose and appropriate inhibitor before addition of ['C]AIB (4 x 10-4 M, 0.4,uCil/pmol) to the system. Inhibitors: (A)-*, control (no inhibitor); 0, sodium azide (102 M); U, CCCP (5 x 10-5 M); +, TCS (10-5 M); 0, p-chloromercuribenzoate (10-3 M); A, no inhibitor, no energy sotsreg, and (B)-_, control (no inhibitor); 0, 2,4-dinitrophenot (10-3 M); U, sodium arsenate (10-2 M); CJ, N-ethylmaleimide (10-3 M); A, iodoacetate (10-2 M); and A, sodium fluoride (10-2 M). TABLE 1. Effect ofthe proton-conducting ionophores TCS and CCCP upon the rates of arginine and galactose utilization by galactose-grown cells of S. lactis ML3a S.mol of substrate used/mg (dry wt) of cells/h Substrate No inhibitor TCS (10-5 M) CC (5 x 10-' M) Arginine Galactose 5.41 t t t t t t 0.36 a Experimental and analytical procedures were as described in Materials and Methods. Utilization of arginine and galactose by the cells proceeded at linear rates for at least 60 min. DISCUSSION Galactose-grown cells of S. lactis ML3 have the capacity to accumulate AIB, a non-metabolizable amino acid analogue of alanine, against considerable concentration gradients when supplied with a suitable energy source such as arginine or carbohydrate. Inhibition of uptake and counterflow of AIB from preloaded cells in the presence of alanine, glycine, and serine suggest that this group of amino acids comprises the natural substrates for the carrier system under study. A number of sulfhydryl group inhibitors (32), including iodoacetate, N-ethylmaleimide, p-chloromercuribenzoate, and CCCP (22), prevented AIB accumulation when galactose was used as the energy time (minutes) FIG. 10. Induced efflux of AIB from actively transporting, galactose-grown cells of S. lactis ML:i. Symbols: 0, TCS (10 o- M); U, CCCP (5 x 10-' M); and 0, DCCD (10-4 M). The plot designated byrepresents the control system, and the arrow (at 60 min) indicates time of addition of inhibitors to the appropriate incubation systems. 5 r = 5, time (minutes) FIG. 11. (A) Effects of various amino acids upon AIB accumulation by galactose-grown cells ofs. lactis ML3. Cells were incubated for 10 min in 01 M Tris-maleate buffer (ph 7.0) containing glucose as energy source. Subsequently, [14CJAIB (2 x 10-4 M, 0.4 AiCU/Amol) plus appropriate amino acid solution (102M final concentration) was added to the system. (B) Induced efflux of AIB after the addition of various amino acids to actively transporting cell suspensions of S. lactis ML3. Amino acids were introduced after 40 min of incubation (arrow) to give a final medium concentration of 10-2 M. Symbols: *, control (no additional amino acid); +, proline; 0, phenylalanine; *, serine; 0, glycine; A, ["2C]AIB; and A, alanine.

10 728 THOMPSON mm time (minutes) FIG. 12. (A) Effect of monovalent cations on AIB uptake by galactose-grown cells of S. lactis ML3. Cells were suspended at a density of 200 pg (dry weight)iml in 0.1 M Tris-maleate (ph 7.0) containing 2 mm glucose as energy source, ['4C]AIB (2 x 10-4 M, 0.4 juci/p.mol), and monovalent cations (chloride form; 10-2 M). (B) Efflux ofaib induced by monovalent cations. Cells were incubated under standard transport conditions, and at 60 min (arrow) monovalent cations (10-2 M) were added to the appropriate system. Symbols: *, control (no added cation); 0, Li', A, Na+; A, NH4+; 0, K+; and M, Rb+. 19) TCS and CCCP abolished AIB uptake and induced rapid efflux of the analogue from actively transporting cells, even though glycolysis and arginine catabolism still continued (Table 1). ATP is the sole high-energy compound common to both the glycolytic and arginine dihydrolase pathways and is, a priori, the ultimate energy donor. However, the dissociation between ATP formation and active transport of AIB by cells exposed to TCS and CCCP suggests that ATP per se may not participate directly in the accumulation process. Energy coupling to AIB accumulation by S. lactis ML3, like active transport of neutral amino acids by S. faecalis (1) and thiomethyl-3-galactoside accumulation by S. lactis 7962 (23-25), can be most readily interpreted in terms of Mitchell's chemiosmotic hypothesis (27, 28). According to this scheme, ATP derived from substrate-level phosphorylation undergoes hydrolysis via the Mg2+-dependent, membranebound adenosine triphosphatase (14), with concomitant extrusion of protons across an essentially proton-impermeable cytoplasmic membrane. A proton motive force comprising an electrical potential and a ph gradient is thus established. Accumulation of AIB by S. lactis could be attributed to the carrier-mediated symport of the solute together with one or more protons in response to the proton motive force. Monovalent cations, including Na+ and K+, J. BACTERIIOL. affected neither the initial rate nor the extent of neutral amino acid uptake by S. faecalis (1, 29). However, in the case ofs. lactis ML, both K+ and Rb+ ions at low concentration (<10 mm) were found to inhibit AIB uptake by approximately 40%. The presence of the cations also induced rapid efflux of the analogue from actively transporting cells of S. lactis ML3. Inhibition of AIB transport by K+ and Rb+ would appear to be quite specific since other monovalent 'cations, including Na+, Li+, and NH4+, were essentially ineffective. It is not yet known whether inhibition by K+ and Rb+ is a consequence of an overall change in membrane potential or due to a more direct effect upon the AIB carrier system. Kinetic studies showed the initial rates of AIB entry to be similar when the energy for transport was derived from arginine metabolism or from glycolysis. However, upon prolonged incubation to steady-state conditions, arginine metabolism supported an internal AIB concentration of only 65% of that attained by glycolyzing cells (Fig. 1A). Comparable observations of the relative efficacy of arginine versus sugar as the energy source for solute accumulation were made by Kashket and Wilson (24, 25) during a study of the energetic aspects of thiomethyl-,8-galactoside transport by S. lactis The concentration of thiomethyl-,8-galactoside accumulated by glycolyzing cells was considerably greater than the concentration determined in cells metabolizing arginine as the energy source. Furthermore, in S. lactis 7962 and S. faecalis (18, 19), glycolyzing cells were found to maintain both electrical potential and ph components of the proton motive force, but in arginine-energized cells a ph gradient could not be detected. In agreement with chemiosmotic predictions, Kashket and Wilson (25) were able to demonstrate a direct correlation between the overall magnitude of the proton motive force and the capacity of the cells to accumulate the galactoside. AIB, like thiomethyl-,b-galactoside, is an electrically neutral molecule at ph 7.0, and a similar pattern of AMB accumulation, dependent upon the magnitude of the proton motive force, might also be expected. In S. faecalis, arginine is a poor energy source for amino acid transport (1). The rate of AIB exit from preloaded cells was found to increase in the presence of metabolizable energy sources. The possibility was also considered that the lower steady-state concentration of solute maintained by arginine-energized cells might be due to a greater rate of AIB exit from these, as compared with

11 VOL. 127, 1976 glycolyzing cells. However, such a simple pump-and-leak mechanism appears unlikely in view of the fivefold difference in value of the rate constants of exit, calculated from steadystate data, when compared with the constants obtained from direct efflux rate determinations using preloaded cells. Furthermore, the experimentally determined rate of AIB exit in the presence of arginine, although faster than the rate found from cells metabolizing galactose, was not significantly different from the efflux rates determined in the presence of lactose or glucose (Fig. 1B). The most interesting and unusual finding to emerge from this study was the observation that the rate of AIB exit from S. lactis ML,, S. lactis 7962, and S. cremoris AM., could be increased two- to fourfold in the presence of a metabolizable energy source (Fig. 1B, 5B, and 6B). These observations contrast with data obtained by Asghar et al. (1) from a study of amino acid exit from preloaded cells of S. faecalis. The rapid exit of intracellular glycine or threonine that took place when cells of S. faecalis were suspended in the absence of an energy source could be completely prevented in the presence of glucose. Unfortunately, attempts to repeat the AIB efflux studies using S. faecalis (strain 581) were precluded by the inability of the organism to accumulate significant concentrations of the amino acid analogue. The increased rate of loss of radioactivity from S. lactis ML.. in the presence of the energy sources did not reflect exit of products derived from the energy-dependent catabolism of AIB. Moreover, the addition of ['2C]AIB alone to the incubation medium did not accelerate the rate of ['4CIAIB exit (Thompson, unpublished data), and it is therefore unlikely that the response elicited by the presence of sugars and arginine can be attributed to competitive inhibition by these compounds upon a ['4CIAIB recapture system (31, 37) located externally to the cytoplasmic membrane. The results of numerous experiments using cells of S. lactis ML3 adapted to utilize specific energy sources have shown clearly that only those substrates that supported AIB accumulation could subsequently increase the rate of AIB exit from preloaded cells. Whereas uptake of AIB by S. lactis is compatible with a chemiosmotic mechanism, the exit of this molecule is not. At 300C (in the absence of an energy source) the rate of solute exit is fourfold greater than at 0 C. This temperature dependence of rate of exit suggests, but is not proof, that AIB efflux occurs by a carrier-mediated process. If exit and entry of AMINO ACID TRANSPORT IN S. LACTIS 729 AIB occur by the same carrier, it is difficult to understand how, and in what form, energy could be coupled so as to accelerate exit. Alternatively, if one considers exit to take place via a non-carrier-mediated "diffusion" pathway, it is again not apparent why the presence of an energy-yielding substrate should affect the rate of diffusion. The reviewer of this manuscript has pointed out the possibilities that (i) exit of AIB might normally occur by both carrier-mediated and simple diffusion pathways as found for galactosides in E. coli (26), or (ii) the carrier may conceivably be energized to a more active form, perhaps by phosphorylation, thereby effecting AIB translocation down a concentration gradient. It is of interest to note, in view of the latter suggestion, that Hoffee et al. (21) observed a reduction in the steady-state level of a-methylglucoside accumulated by E. coli and Salmonella typhimurium when the original growth substrate was added to the incubation medium. It was suggested that ATP, derived from metabolism of the growth substrate, might be coupled to an energy-requiring a-methylglucoside exit system. Halpern and Lupo (11) also studied this exit reaction in E. coli and arrived at similar conclusions. However, alternative explanations have recently been advanced for the earlier findings (20). In terms of requirements for exogenous energy sources, specificity toward structurally similar amino acids, mode of action of inhibitors, and effects of proton conductors upon solute accumulation and energy metabolism, the AIB transport system in S. lactis ML, has many of the characteristics of neutral amino acid transport systems in S. faecalis (1, 7, 29). However, some notable differences do exist, particularly with reference to effects of energy sources on amino acid efflux, capacity of arginine catabolism to support solute accumulation, and the inhibitory effects of K+ and Rb+ ions upon the respective transport systems. ACKNOWLEDGMENTS I would like to acknowledge the discussions and advice of T. D. Thomas and the expert technical assistance of A. R. Matheson. Appreciation is accorded to the reviewer of this manuscript for the helpful, constructive criticisms and for the interesting suggestions for future experiments. LITERATURE CITED 1. Asghar, S. S., E. Levin, and F. M. Harold Accumulation of neutral amino acids by Streptococcus faecalis. Energy coupling by a proton motive force. J. Biol. Chem. 248: Bibb, W. R., and W. R. Straughn Inducible transport system for citrulline in Streptococcus faecalis. J. Bacteriol. 87: Black, S. H., and P. Gerhardt Permeability of

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