- Email: [email protected]
Uptake and transamination of amino acids by Streptococcus faecium
538
BIOCHIMICA ET BIOPHYSICA ACTA
U P T A K E AND T R A N S A M I N A T I O N OF AMINO ACIDS BY STREPTOCOCC US FA ECI UM* WILLIAM R. CHESBRO** AND JAMES B. EVANS***
Division of Bacteriology, American Meat Institute Foundation, University of Chicago, and Department of Biology, Illinois Institute of Technology, Chicago, Ill. (U.S.A .) (Received August 8th, 1961)
SUMMARY
A technique, which by first depleting the cellular freely extractable amino acids of
Streptococcus faecium rendered subsequent amino acid uptake readily demonstrable, was used to prepare cell suspensions with which the uptake of seven amino acid species was studied. All the amino acids studied, except arginine, required glucose for uptake (including lysine, contrary to previous reports). D-Amino acids were taken up with the same facility as their L-isomers. Uptake of exogenous amino acids was accompanied by (a) extensive transamination of the amino acid taken up, if it was a type not characteristically found in cell walls of this genus and (b) appearance of cellular freely extractable alanine, lysine, and glutamic and aspartic acids from a cellular component, whether or not they had been added to the suspension. The latter observation supports a previous postulate that S. faecium contains a heat-stable amino acid reservoir, noted in the present study to be connected to its amino acid transport system. Considered together, these observations suggest how transamination and the reservoir (or cell-wall components), without necessarily being part of the amino acid transport system, may be used by S. faecium to circumvent the thermodynamic limitation placed upon concentrative amino acid accumulation by the concentration gradient between these compounds within and without the cell: the cell, in effect, stores amino acids taken up both as their freely extractable ketonic precursors, readily regenerating the amino acid by transamination, and in reversible combination with the reservoir (or cell-wall components).
INTRODUCTION
A study of the ability of the enterococci, particularly Streptococcus faeciura, to survive and initiate growth in highly alkaline media 1 led to the finding that resting suspensions of S. faeciu~n, Streptococcus faecalis, and Staphylococcus aureus exhibit a hydroxyl-ion* Journal paper No. x99, American Meat Institute Foundation, Chicago, I11. (U.S.A.). ** Present address: Department of Bacteriology, University of New Hampshire, Durham, N.H. (U.S.A.). * ** Present address: Department of Botany and Bacteriology, North Carolina State College, Raleigh, N.C. (U.S.A.). Biochim. Biophys. A cta, 58 (1962) 538-549
AMINO ACID UPTAKE BY
Streptococcusfaecium
539
dependent release of amino acids to their exocellular environment ~. Occurring simultaneously with this release there is a disappearance of those amino acids, tile cellular freely extractable amino acids, that can be extracted from these organisms by boiling or by membrane-disrupting b u t a n o l - e t h a n o l mixtures; and although the processes of amino acid release to the exocellular environment and amino acid disappearance from the cellular freely extractable amino acids are coupled, their differences in kinetics and in the amino acids each process involves make them demonstrably distinct. Although an original purpose of our study had been to extend the observations of GALE3 upon uptake of amino acids into the cellular freely extractable amino acid pool of S. faecalis, we were unable at first to conclusively demonstrate the uptake of any amino acid tested, except" arginine, by resting suspensions of S. faecalis or S. faecium. Since the suspensions, when harvested from the growth medium, contained considerable freely extractable amino acid, it seemed likely that their capacity for further uptake might be limited. It then occurred to us that since adding glucose to a resting suspension of enterococci in alkaline media halted both the hydroxyl-ion-dependent release of amino acids and the concurrent disappearance of freely extractable amino acids, and since glucose is known to be required by these organisms for the uptake of most of the amino acids thus far studied3, 4, it was possible that the release and disappearance processes represented a reversal of the normal mechanisms whereby tile enterococci take up amino acids. Therefore, these processes might be used to deplete, or desaturate, the resting cell suspensions, thus placing them in a condition in which they would more readily exhibit amino acid uptake. Accordingly, cell suspensions were held at pH lO.5 for 45 min, after which they were centrifuged and resuspended in fresh buffer at p H 8.3. Such depleted cells, in the presence of glucose and any amino acids that were added to the cell suspension, exhibited a prompt increase in their freely extractable amino acid content. This paper describes some aspects of amino acid uptake by such cells in the p H range 9.5 to lO.5, together with a study of the relationship between the uptake process and tile appearance, during uptake, of: (a) cellular freely extractable amino acids which are synthesized, and (b) cellular freely extractable amino acids which are neither synthesized nor present in the exoceUular environment. MATERIALS AND METHODS
Cultural methods Growth and maintenance procedures for S. faecium strain HF8AG have been described elsewherO. Prior to use, cultures were transferred twice in a medium at p H 7.5 and once in a medium at p H IO.O. The last transfer was used to inoculate several liters of pH IO.O broth. After 14-16 h at 37 ° the cells were harvested by centrifugation, washed several times in the buffer previously described, and finally suspended in buffer in one tenth the volume in which they had grown.
Analytical procedures One- and two-dimensional chromatography and quantitation of amino acids was performed as described previously 2. The range of this method is 1-2o/,g of amino acid, with a precision of -4- 5 %. Keto acids were chromatographed as the 2,4-dinitroBiochim. Biophys. Acta, 58 (I962) 538-549
54 °
W. R. CHESBRO, J. B. EVANS
phenylhydrazones according to the methods described by BASSETT AND HARPER5. Spectral characteristics of the separated and eluted dinitrophenylhydrazones were determined on a Beckman DK-2 recording spectrophotometer. For two-dimensional chromatography of the dinitrophenylhydrazone derivatives, the solvent system described by BASSETT AND HARPER was used for development in the first direction. Development in the second direction was with a solvent system consisting of o . I M Tris neutralized with formic acid to p H 8.3-8. 4. Inulin concentration was measured with the anthrone method discribed by NEISH e.
Radiochemical procedures [14C]Glucose was obtained from the Volk Radiochemical Company, Chicago, Ill.; DL-[2-1~C]leucine, and DL-[2-1aC]alanine were obtained from Calbiochem, Los Angeles, Calif. (U.S.A.). To determine the radioactivity of amino acids and peptides extracted from the cells by boiling water, such extracts (freely extractable amino acids) were desalted and freed of neutral and anionic compounds as described previously 2. After the desalted extract was dried, an amount equal to 0.25-0.75 ml of the original extract was distributed onto 5 to 7 small disks which were fixed to the origin of a chromatogram at 1. 5 cm intervals. Two further disks containing the equivalent of 3o-80 ~1 of the extract were placed at either end of the series, 2.5-3.0 cm from the nearest disk. After solvent development, the tracks containing the contents of these guide disks were cut out and treated with ninhydrin. These ninhydrin-developed tracks were then used to locate amino acid bands in the body of the chromatogram. Bands containing amino acids were cut out, eluted with IO % isopropyl alcohol, and dried. The eluates were transferred to stainless-steel planchets as layers of infinite thinness and counted in the Geiger-Mfiller region of a windowless detection chamber.
Procedure for following amino acid uptake Depleted cells were prepared b y holding washed-cell suspensions in buffer at p H lO.5 for 45 rain at 37 °, after which they were centrifuged and resuspended in an equal volume of fresh buffer (pH 8.3-8.5). Cells which were to be used without depletion were suspended in p H 8.3-8. 5 buffer, after harvesting and washing, without further treatment. Cell suspensions thus prepared were brought to the desired p H levels at 37 ° and amino acid sufficient to produce the desired final concentration added. Ten millilitres of the suspension was then withdrawn. An amount of glucose calculated to yield a final concentration of 2.0 mg/ml was added, and a second Io-ml portion withdrawn. Both Io-ml portions were centrifuged immediately at IOOOO x g. The p H of the cell suspension was kept constant, and further Io-ml samples were withdrawn at intervals and centrifuged, centrifugation taking 7 to 9 min. The supernatant was decanted and the centrifuge-tube walls wiped dry. The pellet was taken up in 2 ml of deionized water and placedin a boiling-water bath for IO min. Previous trials had shown that a constant amount of alpha-amino nitrogen is released by boiling times of from 2 to 20 min. The boiled cells were centrifuged and the supernatant (cellular freely extractable amino acids) decanted. Biochim. Biophys. Act,~., 58 (1962) 538-549
AMINO ACID UPTAKE BY Streptococcusfaecium
541
At the termination of the experiment a further portion of the suspension was centrifuged at ioooo × g for io min to determine the packed-ceU volume. RESULTS
Uptake of L-amino acids by S. faecium in the pH range 9.5-Io. 5 When amino acids are added to a suspension of depleted cells there is an immediate increase in the apparent amount of amino acid that can be extracted from the cells with boiling water. Since this level does not increase further with time unless glucose is added to the suspension, it is attributable to amino acids passively carried with the cells during their centrifugal separation from the test medium: (a) in the intercellular space of the cell pellet, (b) in the cell-wan space of the cell pellet (since the bacterial cell wall is permeable to solutes with a molecular weight 7 less than IOOOO), and (c) diffused, or passively transported, within the plasma membrane of the cells. In his studies, GALEa accounted for amino acids present in the intercellular space of the cell pellet by adding specific decarboxylases, to which the intact cell is impermeable, so that any amino acids exterior to the cell were destroyed before the cellular amino acid pool was released for measurement; however, in the light of current knowledge ~ of the impermeability of the bacterial cell wall to molecules with the dimensions of enzymes, it is doubtful that amino acid decarboxylases, or Oxidative deaminases, can reach or act upon amino acids that have diffused into the cell-wall space. Therefore, an enzymic technique of this sort does not unambiguously distinguish amino acids actively taken up by the cells from those passively carried with the cells. Because of this, we chose not to employ an enzymic procedure similar to GALE'S to account for passively carried amino acids. Nor did we wish to wash the cell pellets by resuspension and recentrifugation, since our earlier study z had shown that S. faecium released amino acids into buffer solutions at any pH we employed, and such a release, occurring in this step, would obscure the amino acid uptake actually achieved by the cells. Consequently, cell pellets were extracted by boiling immediately after centrifugation and decantation, without further treatment. We have taken the criterion of active amino acid uptake in our experiments to be a positive difference when the level of freely extractable amino acids found in the cell pellet immediately after the addition of glucose to the suspension is deducted from that found some time after adding glucose to the suspension. This subtraction removes from the estimation of active amino acid uptake the three categories of amino acids, mentioned earlier, that are passively carried with the cells during their centrifugal separation from the medium. Three further observations supported the use of this criterion: (a) when a test amino acid mixture was added to the cell suspension, a sample withdrawn prior to the addition of glucose, and a second sample withdrawn immediately after the addition of glucose, the freely extractable amino acids of the cell pellets obtained from the two samples rarely differed by more than 5 % (within the precision of our method of measurement) ; (b) when glucose was absent from the cell suspension only arginine, of the amino acids tested, was taken up (although arginine was taken up from the exocellular environment, it was citruLline that appeared in the freely extractable amino acids, presumably due to the activity of arginine desiminase s) ; (c) under the alkaline conditions of our test media, unless glucose was present and
Biochim. Biophys. Acta, 58 (I962) 538-549
542
W. R. C H E S B R O , J . B. E V A N S
the cells actively took up amino acids, amino acids were released from the cells to the media. In the first experiment to be reported depleted cells of S. faecium were exposed to two different amino acid mixtures in the presence of glucose. The first mixture contained L-leucine, L-methionine, and L-serine; the second mixture contained Lalanine, L-aspartic acid, and L-lysine. Thus, the first mixture contained amino acids more typical of the cytoplasmic protein of Gram-positive bacteria, while the second contained amino acids typically found in the cell wall of Gram-positive bacteria as well as in the cytoplasmic protein. The changes in freely extractable amino acids of cells exposed to the first mixture at four pH levels between 9.5 and lO.5 are shown in Table L In Table II these changes are shown for cells exposed to the second amino acid mixture. Although the cells used at any given pH level are the same for both mixtures, three lots of cells depleted at different times were used during the tests. These lots exhibited differences in their degree of depletion and subsequent amino acid uptake, but were qualitatively similar in their behavour. The exocellular amino acid concentration remained essentially constant during the course of the experiments, since the 0.03-0.05 ml of packed cells present per ml of TABLE
I
CHANGES IN THE FREELY EXTRACTABLE AMINO ACIDS OF Streptococcus L-METHIONINE, L-LEUCINE AND L-SERINE
fagcium EXPOSED
TO
O.54 / * m o l e of L - m e t h i o n i n e , o . 7 3 # m o l e of L-leucine and o.61 / , m o l e of L-serine were added/ml o f cell suspension. The suspensions contained, per ml, o.o3-o.o 5 ml of packed cells depleted of freely extractable amino acids b y holding at p H lO. 5 f o r 45 min at 37 °. T w o m i l l i g r a m s of glucose/ m l of cell suspension was added after adding the amino acid mixture and adjusting the p H t o the
desired level, where it was maintained for the experiment's duration.
pH*
Time" (mini
Methionine
Leucine
Serine
Asparticacid
Alanine
Lysine
Ghaamicacid
( l~rnolesl ml packed cells)
9.5
o 7 15 25
0.03 o.19 0.25 0.20
0.02 0.48 o.6o 0.47
0.06 0.25 o.44 0.34
0.52 o.61 o.75 0.67
0.07 o.15 0.29 0.20
o.oo 0.06 o.II 0.22
1.9 2. 4 2. 4 2.1
9.9
o II 20 3°
o.29 0.48 0.65 o.71
o.24 I.O 1.4 1. 5
0.29 0.38 o.59 1.2o
0.97 1.2 1.5 2. 3
o.oo 0.50 o.94 0.85
0.o7 o.71 1.3 ° 1.9o
3.6 3.8 4.6 4.9
lO.2
o IO 20 3o
0.30 o.51 o.66 0.66
0.35 1.2 1. 3 1. 4
0.3o o.71 o.71 0.85
1.2 2.1 2.2 2.2
o.oo o.54 o.71 0.96
o.13 o.81 1.6o 2.20
2.9 3-5 3-9 4.9
lO. 5
o 12 23 34
o.21 o.45 0.47 0.37
o.19 o.9z o.88 o.88
o.36 o.46 o.38 o.36
1.2 1.4 1. 7 1.6
o.4o o.53 0.56 o.61
0.83 0.94 I.IO o.94
5.8 6.2 6.2 6.2
* The experiments at p H 9 , 9 a n d lO.2 were performed with the same lot of depleted cells; the experiments at 9-5 and lO.5 were performed with separately depleted lots of ceils. ** Time elapsed after the addition of glucose to the cell suspension.
Biochim. Biophys, Acta, 58 (1962) 5 3 8 - 5 4 9
AMINO ACID UPTAKE BY
Streptococcusfaecium
543
TABLE II ~HANGES IN THE FREELY EXTRACTABLEAMINO ACIDS OF Streptococcus faecium EXPOSED TO L-ALAHINE, L-LYSINE AND L-ASPARTICACID 0.72 /*mo]e of L-alanine, 0.53 /,mole of L-lysine and o.48/,mole of L-aspartic acid were sddedlml of cell suspension. The suspensions contained, per ml, 0.03-0.05 ml of packed cell depleted of ffreely extractable amino acids by holding at pH lO. 5 for 45 rain at 37 °. Two milligrams of glucose/ ml of cell suspension was added after adding the amino acid mixture and adjusting the pH to the desired level, where it was maintained for the experiment's duration.
pH*
Time** (rain)
Aspartic acid
Alanine
Lysine
Glutamic acid
O,mo~sl,~packedteas)
9-5
o 8 16 25
0.67 1.8 2.8 4-4
0.38 0.73 i.o 1.3
o.18 0.86 1.2 1.6
1.6 1.9 1.9 2.2
9.9
o 1o 20 3o
I.i 1.5 2.9 4.5
o.oo 0.79 1.3 1-7
o.I9 1.2 2.7 4.3
1.5 2.6 2.9 3-4
lO.2
o io 2o 3°
t.i 1.2 2.6 4.5
o.oo 0.97 1.8 1.7
o.25 i.o 2.2 4.3
1. 7 2. 5 3.o 3.4
Io.5
o ii 23 34
o.81 1.6 3.3 3 .8
0.53 o.69 0.92 o.92
0.66 i.o 1.4 1.2
4.8 5.6 6.2 6.2
* The experiments at pH 9.9 and lO.2 were performed with the same lot of depleted cells, the experiments at pH 9-5 and lO.5 were performed with separately depleted lots of cells. ** Time elapsed after the addition of glucose to the cell suspension. cell suspension did n o t r e m o v e e n o u g h a m i n o acid from t h e exocellular e n v i r o n m e n t to m a t e r i a l l y change its a m in o acid content. T h e freely e x t r a c t a b l e a m i n o acid c o n t e n t of the cell pellets usually reached a m a x i m u m 20 m i n after th e a d d i t i o n of glucose to t h e suspension an d t h e r e a f t e r rem a i n e d r e l a t i v e l y c o n s t a n t or fell somewhat. T h e m a x i m u m u p t a k e a c h i e v e d e x c e e d e d t he c o n c e n t r a t i o n in t h e exocellular e n v i r o n m e n t at p H 9.9 for sefine a n d at p H 9-9 a n d IO.2 for leucine, b u t did n o t do so at a n y p H for m e t h i o n i n e ; only in t h e former cases for sefine a n d leucine, then, was th e possibility definite t h a t c o n c e n t r a t i o n against t h e a m i n o acid g r a d i e n t h a d occurred. H o w e v e r , since t h e i n u l i n - i m p e r m e a n t v o l u m e of t h e cell P e l l e t - - t h e t r u e cell v o l u m e - - w a s a c t u a l l y o n l y 65-70 % of t h e t o t a l pellet v o l u m e , there is a possibility t h a t c o n c e n t r a t i o n against t h e g r a d i e n t also occurred in some of t h e o t h e r samples. B u t w h a t is e v i d e n t from th e d a t a in T a b l e I is t h a t w h e n e v e r u p t a k e of the ad d ed a m i n o acids occurred, w h e t h e r there was c o n c e n t r a t i o n against t h e g r a d i e n t or not, t he cellular c o n t e n t of freely e x t r a c t a b l e alanine, lysine, a n d aspartic a n d g l u t a m i c acids increased m a r k e d l y ; similarly, it is seen in T ab l e I I t h a t cellular freely e x t r a c t a b l e g l u t a m i c acid increased d u r i n g t h e experiments, a l t h o u g h this a m i n o acid was n o t a d d e d t o t h e cell suspension.
Biochim. Biophys. Acta, 58 (1962) 538-549
544
w.R. CHESBRO, J. B. EVANS
The uptake calculated from the data in Table II for alanine, aspartic acid, and lysine indicates that these amino acids were concentrated against the gradient in every case, except that of alanine at p H lO.5. Thus, the amino acids characteristically found in the cell wall of this species were apparently concentrated to a greater extent in this pH range than were the amino acids characteristically found in its cytoplasmic proteins, although the contributions of synthesis to the levels of freely extractable amino acids during the uptake process were yet to be estimated. Because the p K values of the alpha amino groups of the amino acids shown in Tables I and II bore no direct correlation to their relative uptakes in the pH range 9-5 to lO.5, where these alpha amino groups underwent dissociation, the charge state of this group cannot be a determining factor in amino acid uptake by S. faecium.
Uptake of D-amino acids by S. faecium at pH 9.5 D-Leucine, D-methionine, D-serine, and glucose were added to a cell suspension at pH 9-5. The resultant changes in the freely extractable amino acids of the cell pellets obtained from these suspensions are shown in Table III. Leucine, methionine, TABLE III CHANGES
IN
THE
IrlLEELY EXTRACTABLE
AMINO
D-METHIONINE,
ACIDS
D-LI~UCINE
OF
AND
Streptococcus faecium
EXPOSED
TO
D-SERINE
0.54 # m o l e of n - m e t h i o n i n e , o.73 /zmole of D-leucine, a n d o.61 /zmole of D-serine were a d d e d / m l of cell s u s p e n s i o n . T h e s u s p e n s i o n contained, p e r ml, o.o 4 m l of p a c k e d ceils depleted of freely e x t r a c t a b l e a m i n o acids b y h o l d i n g a t p H lO.5 for 45 rain a t 37 °. After a d d i n g t h e a m i n o acid m i x t u r e , t h e p H of t h e s u s p e n s i o n w a s b r o u g h t to 9.5, 2 m g of glucose/ml s u s p e n s i o n w a s added, a n d t h e p H m a i n t a i n e d a t 9.5 for t h e b a l a n c e of t h e e x p e r i m e n t . Time* (rain)
Makionote
Leucine
Serine
Aspartic acid
A lanine
Lysine
Glutamic acid
0.28 o.73 o.8I o.7o
o.37 1.2 2.1 2.5
5.7 5-9 7.5 7.2
(Izmoles/ml packed cells)
o II 23 34
0.23 o.83 0.73 o.61
0.57 1. 7 2.0 1.6
0.36 o.82 0.97 o.84
i.I 2.2 3.1 3.2
* T i m e elapsed a f t e r t h e a d d i t i o n of glucose to t h e cell s u s p e n s i o n .
and serine appeared rapidly in the cell pellet, with only leucine being apparently concentrated against the gradient. Again, the uptake of these amino acids, even though as the D-isomer, was accompanied by an increase in the freely extractable alanine, lysine, and glutamic and aspartic acids of the cell pellet.
Labelling of amino acids and acid carbonyl compounds that are freely extractable from S. faecium exposed to [14C~glucose, DL- E2J*C]leucine, and DL- [2-1*C]alanine The appearance of freely extractable alanine, lysine, and glutamic and aspartic acids, concurrently with uptake of amino acids that had been added to the cell suspension, could be attributed either to their synthesis, by transamination, from other amino acids and glycolytically generated ketonic acceptors or to their release from cellular components. To estimate the extent to which each process contributed Biochim. Biophys. Acta, 58 (I962) 538-549
AMINO A c I D UPTAKE BY
Streptococcusfaecium
545
to the appearance of these amino acids, duplicate suspensions of S. faecium received equal specific activities of [x4C]glucose, together with L-leucine, L-serine, and Lmethionine for the first suspension, while the second suspension received, instead, L-aspartic acid, L-alanine, and L-lysine. After 25 min at pH 9-5 the cells were separated from the two media and extracted with boiling water. When the amino acids in the extract had been separated and counted, it was found that only alanine and aspartic acid were labelled to a significant extent. Some further radioactivity, however, was found in a faintly ninliydrin-positive band moving near lysine, but not corresponding to any of the major freely extractable amino acids. TABLE IV RADIOACTIVITY APPEARING IN THE FREELY EXTRACTABLE ALANINE AND ASPARTIC ACID OF Streptococcus fascium DURING AMINO ACID UPTAKE IN THE PRESENCE OF RADIOACTIVE GLUCOSE I n e x p e r i m e n t a l condition i t h e cells were exposed to 1.61/,moles of L-methionine, 2 . 2 2 / , m o l e s of L-leucine, and 2.26 p m o l e s of L-serine/ml cell suspension; in e x p e r i m e n t a l condition 2 t h e y were exposed to 2.25 /*moles of L-alanine, 1.6o/*moles of L-lysine, a n d 1.44 p m o l e s of L-aspartic acid; in b o t h cases [14C]glucose (3/*C//*mole) w a s added after the a m i n o acid m i x t u r e h a d been added a n d t h e p H of the suspension b r o u g h t to 9.5. The suspension was m a i n t a i n e d at p H 9.5 for 25 rain at 37 ° . Freely extractable amino acid
Radioactivity (counts/rain)
Amount Total amount syntlwsized* extracted (l~moles) (pmoles)
Percent synthesized
Aspartic acid Condition i Condition 2
167 67
0-05 0.02
1.35 6.03
3-7 0.3
Alanine Condition i Condition 2
59 67
o.o2 o.02
o.31 2.14
o.6 o.9
* Calculated on the a s s u m p t i o n t h a t each p m o l e of glucose could give rise to 2 /,moles of alanine or aspartic acid.
The amounts of radioactivity found in alanine and aspartic acid were used to estimate what percentage of these amino acids appearing in the cellular freely extractable amino acids had been synthesized. The estimations are shown in Table IV. When two further experiments of this nature were performed the results were essentially similar, with the highest percentage of aspartic acid synthesized being 8.8 and the highest percentage of alanine synthesized being 5.8. Duplicate cell suspensions at pH 9.5 received glucose plus DL-[234C]leucine and glucose plus 9L-[234C]alanine, respectively. After 25 min the cells were centrifuged, resuspended in 2 N HCI containing 2,4-dinitrophenylhydrazine, and held 16 h at 4 °. The suspension was then centrifuged, and the supernatant extracted with ethyl acetate to remove unreacted 2,4-dinitrophenylhydrazine and any neutral or acidic derivatives. The separated aqueous phase was neutralized and thereafter treated in the same manner as the boiling-water extracts had been; however, after passage of the neutralized solution over Dowex-5o, followed by elution with 2.5 N NH4OH, no trac~ of dinitrophenylhydrazones was found, indicating that neither basic nor amphoterJ derivatives had been formed during extraction of the cells. Biochim. Biophys. Acta, 58 (1962) 538-549
546
W.R. CHESBRO, J. B. EVANS
When the radioactivity of the amino acids extracted from the cells receiving DL-[2-1~C]leucine was examined it was found to be entirely in leucine, but the acid carbonyl derivatives extracted from these cells contained approximately twice as much radioactivity as that found in leucine. The acid carbonyl derivatives extracted from the cells receiving DL-E2-14C~alanine exhibited a different ratio of labelling when compared to the labelling of the extracted alanine, containing only 5 % as much radioactivity as did the extracted amino acid. Also different, in this case, was the distribution of radioactivity in the freely extractable amino acids, for, in addition to the label present in the freely extractable alanine, there was nearly twice as much present in a ninhydrin-positive compound that migrated near lysine on the chromatograms used to separate the extracted amino acids. Depleted cells were then exposed simultaneously to [14C~glucose, Dfi-[2J4C]leucine, DL-methionine, and DL-serine, at p H 9-5. Carbonyl compounds were extracted from the ce{1pellets with acid 2,4-dinitrophenylhydrazine , separated by two-dimensional chromatography, eluted, counted, and examined spectrophotometri~ally. Radioactivity was found in compounds corresponding to the derivatives of oxalacetic acid, pyruvic acid, and a-ketoisocaproic acid. In addition, a non-radioactive area was found which corresponded to the dinitrophenylhydrazone of fl-hydroxypyruvic acid. From these results, it seems likely that serine and leucine are extensively transaminated and 'the ketonic acids thus formed retained by the cell, in company with ketonic acids arising from glycolysis. The transamination of these amino a~ds would account, at least in part, for the decrease, observable in a number of instances in Tables I and III, in the amounts of serine and leucine taken up after 20-30 min. Such an expl.~ation seems inapplicable to the fall also occasionally observed with methionine, since no ketonic transamination product for methionine was found. Consequently, additional factors were operating in lowering the maximum uptake levels achieved: possibly some of the amino acid taken up was returned to the exocellular environment 4, but this possibility was not examined further. Since over half the leucine taken up, and probably a similar amount of the serine taken up, was transaminated, this might have been adequate to account for the freely extractable alanine, lysine, and glutamic and aspartic acids that appeared, had these latter compounds arisen as products of transamination. In fact, however, less than IO % of the alanine and aspartic acid that appeared was synthesized, and of the glutamic acid and lysine that appeared none at all was synthesized. Obviously, therefore, much of the amino acid that must have been synthesized when the amino acids taken up were transaminated (since the enterococci are not known to possess amino acid deaminases) had disappeared from the freely extractable amino acids of the cell pellet ; again, possibly, they were released to the medium, or, instead, they might have been combined with a cellular component. Turning to the observations made with E14C]glucose and DL-I2-14C]alanine, these results suggest that basic peptides were formed, containing alanine both from synthetic processes and from the exocellular medium. In this respect, IKAWA AND SNELL9 have reported extracting alanine peptides from S. faecalis with cold trichloroacetic acid; in their experiments the peptides contained only alanine. In addition, BROWN1° has extracted adenyl peptides from acetone-dried S. faecalis with cold trichloroacetic acid (any such carboxyl-activated pept)des present in the intact cells Biochim. Biophys. Attar, 58 (1962) 538-549
AMINO ACID UPTAKE BY
Streptococcusfaecium
547
used in our studies would probably be hydrolyzed to the free peptide by the extraction procedures we employed.).
Uptake of lysine in the ~resence and absence of glucose GALEa has stated that lysine is concentratively accumulated by S. fa~calis in the absence of glucose. As part of the present studies L-lysine was added to depleted cells at pH 9-5 and after an interval glucose was added also. Similar additions were made to suspensions of undepleted cells, and with one of these suspensions of undepleted cells glucose addition preceded lysine addition. As revealed by the data in Fig. I, the question of whether or not glucose is required for lysine uptake by S. faecium is complicated by the findings, first, that the cellular freely extractable lysine of the depleted cells increased when glucose was present in the exocellular environment but lysine was not, and, second that the rate of this increase was not altered by the addition of lysine to the exocellular environment. Since lysine was shown not to be synthesized from precursors generated by glycolysis in this organism, it is assumed that here, too, lysine was being released from a cellular component. But however much release from a cellular component and uptake from the exocellular environment may each have contributed to the increase, during glucose metabolism, of freely extractable lysine, this increase did not occur when lysine was present in the exocellular environment but glucose was not; consequently, it is unlikely that lysine is taken up by this species in the absence of glucose. This conclusion is
3.[ /
LYSINE ADDED
:k. 2.4
~
\
O
e ~ ~
ttJ Z
2.0
=, II1
,.s
cc
1.2
6LUGOSE ADDED o
/
E ADDED
LU ADDED
LYSINE ADDED
~-=×
a. 0.4
0
0
/
f
0sE
ADDED
L
,
,
,
,
,
I0
20
30
40
50
60
I
7O
MINUTES
Fig. x. Changesinthe freely extractable lysine of depleted and non-depleted cells of Streptococcus faecium exposed to glucose (xo mg/m]) and L-lysine (o.o52 pmole/m]) singly and in combination; O - - O , depleted cells; O - - O , non-depleted cells. Biockim. Biophys. Acta, 58 (I962) 538-549
548
W. R. CHESBRO, J.B. EVANS
substantiated by tile observation that it required the addition of glucose to the undepleted cells to reverse the disappearance of cellular freely extractable lysine that normally occurs at this medium pH (9.5) (see ref. 2) ; lysine, by itself, did not halt or reverse the disappearance. Perhaps the disparity between our finding that lysine uptake does not take place in the absence of glucose, and the finding of GALE3 that it does, is explicable by reference to the report of BRITT AND GERHARDT11 that the greater portion of the freely extractable lysine of Micrococcus lysodeikticus is held by electrostatic interaction with the cell wall, external to the plasma membrane. If this finding is equally applicable to enterococci, then diffusion of lysine into the cell wall and its absorption there could occur independently of the presence of glucose. Failure to distinguish this from lysine taken up within the plasma m e m b r a n e - - a n d , as was pointed out earlier, it is unlikely that decarboxylases such as GALE employed can reach, or act upon, amino acids absorbed within the cell wall--would lead to the conclusion that lysine could be taken up by enterococci without the necessity for a concurrent metabolism of glucose. DISCUSSION
In a previous report S we noted that freely extractable alanine, lysine, and glutamic and aspartic acids appeared, after 25 rain, in S. faecium suspensions containing added glucose but no added amino acids. (DAWES AND HOLMES1. have observed apparently similar behavior in Sarcina lutea: in that organism the metabolism of glucose, in the absence of exogenously added amino acids, resulted in the appearence of freely extractable y-aminobutyric acid, alanine, and glutamic and aspartic acids). In the present study, freely extractable alanine, lysine, and glutamic and aspartic acids again a p p e a r e d - - b u t immediately, without any lag--concurrently with the uptake of exogenously added amino acids, whether or not they too had been supplied to the suspension. These four amino acids are identical to the freely extractable amino acids which disappear from the cell during depletion, and differ, therefore, from the amino acids which are released to the medium during this process 2. It was postulated previously 2 that because the amino acids involved in the coupled, OH--dependent amino acid disappearance-release processes (which occur during depletion of the cellular freely extractable amino acids) can differ in kind and in quantity, a heat-stable amino acid "reservoir", common to both processes, must exist. The existence of this "reservoir", which contains a limited number of kinds of amino acids; which combines with the cellular freely extractable amino acids during the depletion process; and which returns these combined amino acids to their freely extractable state, after a lag period, when glucose is metabolized, is reaffirmed by the present finding that freely extractable amino acids (which are of the same kinds as those that disappeared during the depletion process) appear immediately when exogenously added amino acids are taken up. The "reservoir" is thus intimately linked both with the processes involved in the amino acid disappearance-release that takes place when the cells are depleted and with the processes mediating amino acid uptake by the cells. It is possible that the two groups of processes may be one and the same. The amino acids released from the "reservoir" to the freely extractable pool during uptake are characteristically found as cell-wall components in this genera of Biochim. Biophys. Acta, 58 (1962) 538-549
AMINO ACID UPTAKE BY
Streptococcusfaecium
549
microorganisms TM. Thus, the reservoir seems related to the cell wall, perhaps being a cell-wall component; and because it is also coupled to amino acid transport, it constitutes a linkage--direct, or nearly so--between cell-wall components and amino acid transport, possibly important to the synthesis of cell wall. Such a linkage and the fact that when amino acids such as leucine and serine, characteristic of cell protein rather than cell wall, are taken up they can be transaminated, yielding their alpha-keto homologues, to twice the extent that they are retained as the amino acid would permit the cell to circumvent the thermodynamic limitation placed upon concentrative amino acid accumulation by the concentration gradient between these compounds within and without the cell. Since there is reason to believe that an alpha-keto acid does not compete with its homologous amino acid for the amino acid's transport system 14, transamination of a freely extractable amino acid acts to lower the concentration gradient between that amino acid and the same amino acid in the exocellular environment. In effect, the cell stores the amino acid as its readily convertible precursorl And further, since the products of transamination in S. faecium are alanine and aspartic acid, their combination with the "reservoir" (or with cell-wall components) provides a means for continually lowering the pool concentration of these amino acids; but since the flow of these amino acids into the "reservoir"-bound form is readily reversible, in effect, they too are stored. The foregoing hypothesis indicates, in corollary, how vitamin B 6 and the transaminases might contribute to the capacity of the cell for concentrative amino acid accumulation 15 without actually being part of the amino acid transport system. ACKNOWLEDGEMENT
This research was supported, in part, by a grant from the National Institutes of Health. REFERENCES t W . R. CNESBRO AND J. B. EVANS, J. Bacteriol., 78 (1959) 848. 2 W. R. CHESBRO AND J. B. EVANS, J. Bacteriol., 79 (196o) 682. s E. F. GALE, Advances in Protein Chem., 8 (1953) 285. 4 j . T. HOLDER AND J. HOLMAN, J. Biol. Chem., 234 (1959) 865. 5 E. W. BASSETT AND W. J. HARPER, J. Dairy Sci., 9 (1958) 12o6. 6 A. C. NEIsrI, Analytical Methods for Bacterial Fermentations, N a t i o n a l R e s e a r c h Council of Canada, Saskatoon. 7 p. MITCHELL, Ann. Rev. Microbiol., 13 (I959) 4o7 . 8 B. PETRACK, L. SULLIVAN AND S. RATNER, Arch. Biochem. Biophys., 69 (1957) 186. 9 M. IKAWA AND E. SNELL, Arch. Biochem. Biophys., 78 (1958) 338. x0 A. D. BROWN, Biochim. Biophys. Acta, 3 ° (1958) 447. 11 E. M. BR[TT AND P. GERHARDT, J. Bacteriol., 76 (I958) z88. 12 E. A. DAWES AND W. H. HOLMES, Biochim. Biophys. Acta, 3o (1958) 278. lZ G. D. SI-IOCKMAN,J. Biol. Chem., 234 (1959) 234 o. it E. BALL, J. HUMPHREYS AND W. SHIVE, Arch. Biochem. Biophys., 73 (1958) 4 lo. 15 j. T. HOLMAN,J. Biol. Chem., 234 (I959) 872.
Biochim. Biophys. Acta, 58 (1962) 538-549
BIOCHIMICA ET BIOPHYSICA ACTA
U P T A K E AND T R A N S A M I N A T I O N OF AMINO ACIDS BY STREPTOCOCC US FA ECI UM* WILLIAM R. CHESBRO** AND JAMES B. EVANS***
Division of Bacteriology, American Meat Institute Foundation, University of Chicago, and Department of Biology, Illinois Institute of Technology, Chicago, Ill. (U.S.A .) (Received August 8th, 1961)
SUMMARY
A technique, which by first depleting the cellular freely extractable amino acids of
Streptococcus faecium rendered subsequent amino acid uptake readily demonstrable, was used to prepare cell suspensions with which the uptake of seven amino acid species was studied. All the amino acids studied, except arginine, required glucose for uptake (including lysine, contrary to previous reports). D-Amino acids were taken up with the same facility as their L-isomers. Uptake of exogenous amino acids was accompanied by (a) extensive transamination of the amino acid taken up, if it was a type not characteristically found in cell walls of this genus and (b) appearance of cellular freely extractable alanine, lysine, and glutamic and aspartic acids from a cellular component, whether or not they had been added to the suspension. The latter observation supports a previous postulate that S. faecium contains a heat-stable amino acid reservoir, noted in the present study to be connected to its amino acid transport system. Considered together, these observations suggest how transamination and the reservoir (or cell-wall components), without necessarily being part of the amino acid transport system, may be used by S. faecium to circumvent the thermodynamic limitation placed upon concentrative amino acid accumulation by the concentration gradient between these compounds within and without the cell: the cell, in effect, stores amino acids taken up both as their freely extractable ketonic precursors, readily regenerating the amino acid by transamination, and in reversible combination with the reservoir (or cell-wall components).
INTRODUCTION
A study of the ability of the enterococci, particularly Streptococcus faeciura, to survive and initiate growth in highly alkaline media 1 led to the finding that resting suspensions of S. faeciu~n, Streptococcus faecalis, and Staphylococcus aureus exhibit a hydroxyl-ion* Journal paper No. x99, American Meat Institute Foundation, Chicago, I11. (U.S.A.). ** Present address: Department of Bacteriology, University of New Hampshire, Durham, N.H. (U.S.A.). * ** Present address: Department of Botany and Bacteriology, North Carolina State College, Raleigh, N.C. (U.S.A.). Biochim. Biophys. A cta, 58 (1962) 538-549
AMINO ACID UPTAKE BY
Streptococcusfaecium
539
dependent release of amino acids to their exocellular environment ~. Occurring simultaneously with this release there is a disappearance of those amino acids, tile cellular freely extractable amino acids, that can be extracted from these organisms by boiling or by membrane-disrupting b u t a n o l - e t h a n o l mixtures; and although the processes of amino acid release to the exocellular environment and amino acid disappearance from the cellular freely extractable amino acids are coupled, their differences in kinetics and in the amino acids each process involves make them demonstrably distinct. Although an original purpose of our study had been to extend the observations of GALE3 upon uptake of amino acids into the cellular freely extractable amino acid pool of S. faecalis, we were unable at first to conclusively demonstrate the uptake of any amino acid tested, except" arginine, by resting suspensions of S. faecalis or S. faecium. Since the suspensions, when harvested from the growth medium, contained considerable freely extractable amino acid, it seemed likely that their capacity for further uptake might be limited. It then occurred to us that since adding glucose to a resting suspension of enterococci in alkaline media halted both the hydroxyl-ion-dependent release of amino acids and the concurrent disappearance of freely extractable amino acids, and since glucose is known to be required by these organisms for the uptake of most of the amino acids thus far studied3, 4, it was possible that the release and disappearance processes represented a reversal of the normal mechanisms whereby tile enterococci take up amino acids. Therefore, these processes might be used to deplete, or desaturate, the resting cell suspensions, thus placing them in a condition in which they would more readily exhibit amino acid uptake. Accordingly, cell suspensions were held at pH lO.5 for 45 min, after which they were centrifuged and resuspended in fresh buffer at p H 8.3. Such depleted cells, in the presence of glucose and any amino acids that were added to the cell suspension, exhibited a prompt increase in their freely extractable amino acid content. This paper describes some aspects of amino acid uptake by such cells in the p H range 9.5 to lO.5, together with a study of the relationship between the uptake process and tile appearance, during uptake, of: (a) cellular freely extractable amino acids which are synthesized, and (b) cellular freely extractable amino acids which are neither synthesized nor present in the exoceUular environment. MATERIALS AND METHODS
Cultural methods Growth and maintenance procedures for S. faecium strain HF8AG have been described elsewherO. Prior to use, cultures were transferred twice in a medium at p H 7.5 and once in a medium at p H IO.O. The last transfer was used to inoculate several liters of pH IO.O broth. After 14-16 h at 37 ° the cells were harvested by centrifugation, washed several times in the buffer previously described, and finally suspended in buffer in one tenth the volume in which they had grown.
Analytical procedures One- and two-dimensional chromatography and quantitation of amino acids was performed as described previously 2. The range of this method is 1-2o/,g of amino acid, with a precision of -4- 5 %. Keto acids were chromatographed as the 2,4-dinitroBiochim. Biophys. Acta, 58 (I962) 538-549
54 °
W. R. CHESBRO, J. B. EVANS
phenylhydrazones according to the methods described by BASSETT AND HARPER5. Spectral characteristics of the separated and eluted dinitrophenylhydrazones were determined on a Beckman DK-2 recording spectrophotometer. For two-dimensional chromatography of the dinitrophenylhydrazone derivatives, the solvent system described by BASSETT AND HARPER was used for development in the first direction. Development in the second direction was with a solvent system consisting of o . I M Tris neutralized with formic acid to p H 8.3-8. 4. Inulin concentration was measured with the anthrone method discribed by NEISH e.
Radiochemical procedures [14C]Glucose was obtained from the Volk Radiochemical Company, Chicago, Ill.; DL-[2-1~C]leucine, and DL-[2-1aC]alanine were obtained from Calbiochem, Los Angeles, Calif. (U.S.A.). To determine the radioactivity of amino acids and peptides extracted from the cells by boiling water, such extracts (freely extractable amino acids) were desalted and freed of neutral and anionic compounds as described previously 2. After the desalted extract was dried, an amount equal to 0.25-0.75 ml of the original extract was distributed onto 5 to 7 small disks which were fixed to the origin of a chromatogram at 1. 5 cm intervals. Two further disks containing the equivalent of 3o-80 ~1 of the extract were placed at either end of the series, 2.5-3.0 cm from the nearest disk. After solvent development, the tracks containing the contents of these guide disks were cut out and treated with ninhydrin. These ninhydrin-developed tracks were then used to locate amino acid bands in the body of the chromatogram. Bands containing amino acids were cut out, eluted with IO % isopropyl alcohol, and dried. The eluates were transferred to stainless-steel planchets as layers of infinite thinness and counted in the Geiger-Mfiller region of a windowless detection chamber.
Procedure for following amino acid uptake Depleted cells were prepared b y holding washed-cell suspensions in buffer at p H lO.5 for 45 rain at 37 °, after which they were centrifuged and resuspended in an equal volume of fresh buffer (pH 8.3-8.5). Cells which were to be used without depletion were suspended in p H 8.3-8. 5 buffer, after harvesting and washing, without further treatment. Cell suspensions thus prepared were brought to the desired p H levels at 37 ° and amino acid sufficient to produce the desired final concentration added. Ten millilitres of the suspension was then withdrawn. An amount of glucose calculated to yield a final concentration of 2.0 mg/ml was added, and a second Io-ml portion withdrawn. Both Io-ml portions were centrifuged immediately at IOOOO x g. The p H of the cell suspension was kept constant, and further Io-ml samples were withdrawn at intervals and centrifuged, centrifugation taking 7 to 9 min. The supernatant was decanted and the centrifuge-tube walls wiped dry. The pellet was taken up in 2 ml of deionized water and placedin a boiling-water bath for IO min. Previous trials had shown that a constant amount of alpha-amino nitrogen is released by boiling times of from 2 to 20 min. The boiled cells were centrifuged and the supernatant (cellular freely extractable amino acids) decanted. Biochim. Biophys. Act,~., 58 (1962) 538-549
AMINO ACID UPTAKE BY Streptococcusfaecium
541
At the termination of the experiment a further portion of the suspension was centrifuged at ioooo × g for io min to determine the packed-ceU volume. RESULTS
Uptake of L-amino acids by S. faecium in the pH range 9.5-Io. 5 When amino acids are added to a suspension of depleted cells there is an immediate increase in the apparent amount of amino acid that can be extracted from the cells with boiling water. Since this level does not increase further with time unless glucose is added to the suspension, it is attributable to amino acids passively carried with the cells during their centrifugal separation from the test medium: (a) in the intercellular space of the cell pellet, (b) in the cell-wan space of the cell pellet (since the bacterial cell wall is permeable to solutes with a molecular weight 7 less than IOOOO), and (c) diffused, or passively transported, within the plasma membrane of the cells. In his studies, GALEa accounted for amino acids present in the intercellular space of the cell pellet by adding specific decarboxylases, to which the intact cell is impermeable, so that any amino acids exterior to the cell were destroyed before the cellular amino acid pool was released for measurement; however, in the light of current knowledge ~ of the impermeability of the bacterial cell wall to molecules with the dimensions of enzymes, it is doubtful that amino acid decarboxylases, or Oxidative deaminases, can reach or act upon amino acids that have diffused into the cell-wall space. Therefore, an enzymic technique of this sort does not unambiguously distinguish amino acids actively taken up by the cells from those passively carried with the cells. Because of this, we chose not to employ an enzymic procedure similar to GALE'S to account for passively carried amino acids. Nor did we wish to wash the cell pellets by resuspension and recentrifugation, since our earlier study z had shown that S. faecium released amino acids into buffer solutions at any pH we employed, and such a release, occurring in this step, would obscure the amino acid uptake actually achieved by the cells. Consequently, cell pellets were extracted by boiling immediately after centrifugation and decantation, without further treatment. We have taken the criterion of active amino acid uptake in our experiments to be a positive difference when the level of freely extractable amino acids found in the cell pellet immediately after the addition of glucose to the suspension is deducted from that found some time after adding glucose to the suspension. This subtraction removes from the estimation of active amino acid uptake the three categories of amino acids, mentioned earlier, that are passively carried with the cells during their centrifugal separation from the medium. Three further observations supported the use of this criterion: (a) when a test amino acid mixture was added to the cell suspension, a sample withdrawn prior to the addition of glucose, and a second sample withdrawn immediately after the addition of glucose, the freely extractable amino acids of the cell pellets obtained from the two samples rarely differed by more than 5 % (within the precision of our method of measurement) ; (b) when glucose was absent from the cell suspension only arginine, of the amino acids tested, was taken up (although arginine was taken up from the exocellular environment, it was citruLline that appeared in the freely extractable amino acids, presumably due to the activity of arginine desiminase s) ; (c) under the alkaline conditions of our test media, unless glucose was present and
Biochim. Biophys. Acta, 58 (I962) 538-549
542
W. R. C H E S B R O , J . B. E V A N S
the cells actively took up amino acids, amino acids were released from the cells to the media. In the first experiment to be reported depleted cells of S. faecium were exposed to two different amino acid mixtures in the presence of glucose. The first mixture contained L-leucine, L-methionine, and L-serine; the second mixture contained Lalanine, L-aspartic acid, and L-lysine. Thus, the first mixture contained amino acids more typical of the cytoplasmic protein of Gram-positive bacteria, while the second contained amino acids typically found in the cell wall of Gram-positive bacteria as well as in the cytoplasmic protein. The changes in freely extractable amino acids of cells exposed to the first mixture at four pH levels between 9.5 and lO.5 are shown in Table L In Table II these changes are shown for cells exposed to the second amino acid mixture. Although the cells used at any given pH level are the same for both mixtures, three lots of cells depleted at different times were used during the tests. These lots exhibited differences in their degree of depletion and subsequent amino acid uptake, but were qualitatively similar in their behavour. The exocellular amino acid concentration remained essentially constant during the course of the experiments, since the 0.03-0.05 ml of packed cells present per ml of TABLE
I
CHANGES IN THE FREELY EXTRACTABLE AMINO ACIDS OF Streptococcus L-METHIONINE, L-LEUCINE AND L-SERINE
fagcium EXPOSED
TO
O.54 / * m o l e of L - m e t h i o n i n e , o . 7 3 # m o l e of L-leucine and o.61 / , m o l e of L-serine were added/ml o f cell suspension. The suspensions contained, per ml, o.o3-o.o 5 ml of packed cells depleted of freely extractable amino acids b y holding at p H lO. 5 f o r 45 min at 37 °. T w o m i l l i g r a m s of glucose/ m l of cell suspension was added after adding the amino acid mixture and adjusting the p H t o the
desired level, where it was maintained for the experiment's duration.
pH*
Time" (mini
Methionine
Leucine
Serine
Asparticacid
Alanine
Lysine
Ghaamicacid
( l~rnolesl ml packed cells)
9.5
o 7 15 25
0.03 o.19 0.25 0.20
0.02 0.48 o.6o 0.47
0.06 0.25 o.44 0.34
0.52 o.61 o.75 0.67
0.07 o.15 0.29 0.20
o.oo 0.06 o.II 0.22
1.9 2. 4 2. 4 2.1
9.9
o II 20 3°
o.29 0.48 0.65 o.71
o.24 I.O 1.4 1. 5
0.29 0.38 o.59 1.2o
0.97 1.2 1.5 2. 3
o.oo 0.50 o.94 0.85
0.o7 o.71 1.3 ° 1.9o
3.6 3.8 4.6 4.9
lO.2
o IO 20 3o
0.30 o.51 o.66 0.66
0.35 1.2 1. 3 1. 4
0.3o o.71 o.71 0.85
1.2 2.1 2.2 2.2
o.oo o.54 o.71 0.96
o.13 o.81 1.6o 2.20
2.9 3-5 3-9 4.9
lO. 5
o 12 23 34
o.21 o.45 0.47 0.37
o.19 o.9z o.88 o.88
o.36 o.46 o.38 o.36
1.2 1.4 1. 7 1.6
o.4o o.53 0.56 o.61
0.83 0.94 I.IO o.94
5.8 6.2 6.2 6.2
* The experiments at p H 9 , 9 a n d lO.2 were performed with the same lot of depleted cells; the experiments at 9-5 and lO.5 were performed with separately depleted lots of ceils. ** Time elapsed after the addition of glucose to the cell suspension.
Biochim. Biophys, Acta, 58 (1962) 5 3 8 - 5 4 9
AMINO ACID UPTAKE BY
Streptococcusfaecium
543
TABLE II ~HANGES IN THE FREELY EXTRACTABLEAMINO ACIDS OF Streptococcus faecium EXPOSED TO L-ALAHINE, L-LYSINE AND L-ASPARTICACID 0.72 /*mo]e of L-alanine, 0.53 /,mole of L-lysine and o.48/,mole of L-aspartic acid were sddedlml of cell suspension. The suspensions contained, per ml, 0.03-0.05 ml of packed cell depleted of ffreely extractable amino acids by holding at pH lO. 5 for 45 rain at 37 °. Two milligrams of glucose/ ml of cell suspension was added after adding the amino acid mixture and adjusting the pH to the desired level, where it was maintained for the experiment's duration.
pH*
Time** (rain)
Aspartic acid
Alanine
Lysine
Glutamic acid
O,mo~sl,~packedteas)
9-5
o 8 16 25
0.67 1.8 2.8 4-4
0.38 0.73 i.o 1.3
o.18 0.86 1.2 1.6
1.6 1.9 1.9 2.2
9.9
o 1o 20 3o
I.i 1.5 2.9 4.5
o.oo 0.79 1.3 1-7
o.I9 1.2 2.7 4.3
1.5 2.6 2.9 3-4
lO.2
o io 2o 3°
t.i 1.2 2.6 4.5
o.oo 0.97 1.8 1.7
o.25 i.o 2.2 4.3
1. 7 2. 5 3.o 3.4
Io.5
o ii 23 34
o.81 1.6 3.3 3 .8
0.53 o.69 0.92 o.92
0.66 i.o 1.4 1.2
4.8 5.6 6.2 6.2
* The experiments at pH 9.9 and lO.2 were performed with the same lot of depleted cells, the experiments at pH 9-5 and lO.5 were performed with separately depleted lots of cells. ** Time elapsed after the addition of glucose to the cell suspension. cell suspension did n o t r e m o v e e n o u g h a m i n o acid from t h e exocellular e n v i r o n m e n t to m a t e r i a l l y change its a m in o acid content. T h e freely e x t r a c t a b l e a m i n o acid c o n t e n t of the cell pellets usually reached a m a x i m u m 20 m i n after th e a d d i t i o n of glucose to t h e suspension an d t h e r e a f t e r rem a i n e d r e l a t i v e l y c o n s t a n t or fell somewhat. T h e m a x i m u m u p t a k e a c h i e v e d e x c e e d e d t he c o n c e n t r a t i o n in t h e exocellular e n v i r o n m e n t at p H 9.9 for sefine a n d at p H 9-9 a n d IO.2 for leucine, b u t did n o t do so at a n y p H for m e t h i o n i n e ; only in t h e former cases for sefine a n d leucine, then, was th e possibility definite t h a t c o n c e n t r a t i o n against t h e a m i n o acid g r a d i e n t h a d occurred. H o w e v e r , since t h e i n u l i n - i m p e r m e a n t v o l u m e of t h e cell P e l l e t - - t h e t r u e cell v o l u m e - - w a s a c t u a l l y o n l y 65-70 % of t h e t o t a l pellet v o l u m e , there is a possibility t h a t c o n c e n t r a t i o n against t h e g r a d i e n t also occurred in some of t h e o t h e r samples. B u t w h a t is e v i d e n t from th e d a t a in T a b l e I is t h a t w h e n e v e r u p t a k e of the ad d ed a m i n o acids occurred, w h e t h e r there was c o n c e n t r a t i o n against t h e g r a d i e n t or not, t he cellular c o n t e n t of freely e x t r a c t a b l e alanine, lysine, a n d aspartic a n d g l u t a m i c acids increased m a r k e d l y ; similarly, it is seen in T ab l e I I t h a t cellular freely e x t r a c t a b l e g l u t a m i c acid increased d u r i n g t h e experiments, a l t h o u g h this a m i n o acid was n o t a d d e d t o t h e cell suspension.
Biochim. Biophys. Acta, 58 (1962) 538-549
544
w.R. CHESBRO, J. B. EVANS
The uptake calculated from the data in Table II for alanine, aspartic acid, and lysine indicates that these amino acids were concentrated against the gradient in every case, except that of alanine at p H lO.5. Thus, the amino acids characteristically found in the cell wall of this species were apparently concentrated to a greater extent in this pH range than were the amino acids characteristically found in its cytoplasmic proteins, although the contributions of synthesis to the levels of freely extractable amino acids during the uptake process were yet to be estimated. Because the p K values of the alpha amino groups of the amino acids shown in Tables I and II bore no direct correlation to their relative uptakes in the pH range 9-5 to lO.5, where these alpha amino groups underwent dissociation, the charge state of this group cannot be a determining factor in amino acid uptake by S. faecium.
Uptake of D-amino acids by S. faecium at pH 9.5 D-Leucine, D-methionine, D-serine, and glucose were added to a cell suspension at pH 9-5. The resultant changes in the freely extractable amino acids of the cell pellets obtained from these suspensions are shown in Table III. Leucine, methionine, TABLE III CHANGES
IN
THE
IrlLEELY EXTRACTABLE
AMINO
D-METHIONINE,
ACIDS
D-LI~UCINE
OF
AND
Streptococcus faecium
EXPOSED
TO
D-SERINE
0.54 # m o l e of n - m e t h i o n i n e , o.73 /zmole of D-leucine, a n d o.61 /zmole of D-serine were a d d e d / m l of cell s u s p e n s i o n . T h e s u s p e n s i o n contained, p e r ml, o.o 4 m l of p a c k e d ceils depleted of freely e x t r a c t a b l e a m i n o acids b y h o l d i n g a t p H lO.5 for 45 rain a t 37 °. After a d d i n g t h e a m i n o acid m i x t u r e , t h e p H of t h e s u s p e n s i o n w a s b r o u g h t to 9.5, 2 m g of glucose/ml s u s p e n s i o n w a s added, a n d t h e p H m a i n t a i n e d a t 9.5 for t h e b a l a n c e of t h e e x p e r i m e n t . Time* (rain)
Makionote
Leucine
Serine
Aspartic acid
A lanine
Lysine
Glutamic acid
0.28 o.73 o.8I o.7o
o.37 1.2 2.1 2.5
5.7 5-9 7.5 7.2
(Izmoles/ml packed cells)
o II 23 34
0.23 o.83 0.73 o.61
0.57 1. 7 2.0 1.6
0.36 o.82 0.97 o.84
i.I 2.2 3.1 3.2
* T i m e elapsed a f t e r t h e a d d i t i o n of glucose to t h e cell s u s p e n s i o n .
and serine appeared rapidly in the cell pellet, with only leucine being apparently concentrated against the gradient. Again, the uptake of these amino acids, even though as the D-isomer, was accompanied by an increase in the freely extractable alanine, lysine, and glutamic and aspartic acids of the cell pellet.
Labelling of amino acids and acid carbonyl compounds that are freely extractable from S. faecium exposed to [14C~glucose, DL- E2J*C]leucine, and DL- [2-1*C]alanine The appearance of freely extractable alanine, lysine, and glutamic and aspartic acids, concurrently with uptake of amino acids that had been added to the cell suspension, could be attributed either to their synthesis, by transamination, from other amino acids and glycolytically generated ketonic acceptors or to their release from cellular components. To estimate the extent to which each process contributed Biochim. Biophys. Acta, 58 (I962) 538-549
AMINO A c I D UPTAKE BY
Streptococcusfaecium
545
to the appearance of these amino acids, duplicate suspensions of S. faecium received equal specific activities of [x4C]glucose, together with L-leucine, L-serine, and Lmethionine for the first suspension, while the second suspension received, instead, L-aspartic acid, L-alanine, and L-lysine. After 25 min at pH 9-5 the cells were separated from the two media and extracted with boiling water. When the amino acids in the extract had been separated and counted, it was found that only alanine and aspartic acid were labelled to a significant extent. Some further radioactivity, however, was found in a faintly ninliydrin-positive band moving near lysine, but not corresponding to any of the major freely extractable amino acids. TABLE IV RADIOACTIVITY APPEARING IN THE FREELY EXTRACTABLE ALANINE AND ASPARTIC ACID OF Streptococcus fascium DURING AMINO ACID UPTAKE IN THE PRESENCE OF RADIOACTIVE GLUCOSE I n e x p e r i m e n t a l condition i t h e cells were exposed to 1.61/,moles of L-methionine, 2 . 2 2 / , m o l e s of L-leucine, and 2.26 p m o l e s of L-serine/ml cell suspension; in e x p e r i m e n t a l condition 2 t h e y were exposed to 2.25 /*moles of L-alanine, 1.6o/*moles of L-lysine, a n d 1.44 p m o l e s of L-aspartic acid; in b o t h cases [14C]glucose (3/*C//*mole) w a s added after the a m i n o acid m i x t u r e h a d been added a n d t h e p H of the suspension b r o u g h t to 9.5. The suspension was m a i n t a i n e d at p H 9.5 for 25 rain at 37 ° . Freely extractable amino acid
Radioactivity (counts/rain)
Amount Total amount syntlwsized* extracted (l~moles) (pmoles)
Percent synthesized
Aspartic acid Condition i Condition 2
167 67
0-05 0.02
1.35 6.03
3-7 0.3
Alanine Condition i Condition 2
59 67
o.o2 o.02
o.31 2.14
o.6 o.9
* Calculated on the a s s u m p t i o n t h a t each p m o l e of glucose could give rise to 2 /,moles of alanine or aspartic acid.
The amounts of radioactivity found in alanine and aspartic acid were used to estimate what percentage of these amino acids appearing in the cellular freely extractable amino acids had been synthesized. The estimations are shown in Table IV. When two further experiments of this nature were performed the results were essentially similar, with the highest percentage of aspartic acid synthesized being 8.8 and the highest percentage of alanine synthesized being 5.8. Duplicate cell suspensions at pH 9.5 received glucose plus DL-[234C]leucine and glucose plus 9L-[234C]alanine, respectively. After 25 min the cells were centrifuged, resuspended in 2 N HCI containing 2,4-dinitrophenylhydrazine, and held 16 h at 4 °. The suspension was then centrifuged, and the supernatant extracted with ethyl acetate to remove unreacted 2,4-dinitrophenylhydrazine and any neutral or acidic derivatives. The separated aqueous phase was neutralized and thereafter treated in the same manner as the boiling-water extracts had been; however, after passage of the neutralized solution over Dowex-5o, followed by elution with 2.5 N NH4OH, no trac~ of dinitrophenylhydrazones was found, indicating that neither basic nor amphoterJ derivatives had been formed during extraction of the cells. Biochim. Biophys. Acta, 58 (1962) 538-549
546
W.R. CHESBRO, J. B. EVANS
When the radioactivity of the amino acids extracted from the cells receiving DL-[2-1~C]leucine was examined it was found to be entirely in leucine, but the acid carbonyl derivatives extracted from these cells contained approximately twice as much radioactivity as that found in leucine. The acid carbonyl derivatives extracted from the cells receiving DL-E2-14C~alanine exhibited a different ratio of labelling when compared to the labelling of the extracted alanine, containing only 5 % as much radioactivity as did the extracted amino acid. Also different, in this case, was the distribution of radioactivity in the freely extractable amino acids, for, in addition to the label present in the freely extractable alanine, there was nearly twice as much present in a ninhydrin-positive compound that migrated near lysine on the chromatograms used to separate the extracted amino acids. Depleted cells were then exposed simultaneously to [14C~glucose, Dfi-[2J4C]leucine, DL-methionine, and DL-serine, at p H 9-5. Carbonyl compounds were extracted from the ce{1pellets with acid 2,4-dinitrophenylhydrazine , separated by two-dimensional chromatography, eluted, counted, and examined spectrophotometri~ally. Radioactivity was found in compounds corresponding to the derivatives of oxalacetic acid, pyruvic acid, and a-ketoisocaproic acid. In addition, a non-radioactive area was found which corresponded to the dinitrophenylhydrazone of fl-hydroxypyruvic acid. From these results, it seems likely that serine and leucine are extensively transaminated and 'the ketonic acids thus formed retained by the cell, in company with ketonic acids arising from glycolysis. The transamination of these amino a~ds would account, at least in part, for the decrease, observable in a number of instances in Tables I and III, in the amounts of serine and leucine taken up after 20-30 min. Such an expl.~ation seems inapplicable to the fall also occasionally observed with methionine, since no ketonic transamination product for methionine was found. Consequently, additional factors were operating in lowering the maximum uptake levels achieved: possibly some of the amino acid taken up was returned to the exocellular environment 4, but this possibility was not examined further. Since over half the leucine taken up, and probably a similar amount of the serine taken up, was transaminated, this might have been adequate to account for the freely extractable alanine, lysine, and glutamic and aspartic acids that appeared, had these latter compounds arisen as products of transamination. In fact, however, less than IO % of the alanine and aspartic acid that appeared was synthesized, and of the glutamic acid and lysine that appeared none at all was synthesized. Obviously, therefore, much of the amino acid that must have been synthesized when the amino acids taken up were transaminated (since the enterococci are not known to possess amino acid deaminases) had disappeared from the freely extractable amino acids of the cell pellet ; again, possibly, they were released to the medium, or, instead, they might have been combined with a cellular component. Turning to the observations made with E14C]glucose and DL-I2-14C]alanine, these results suggest that basic peptides were formed, containing alanine both from synthetic processes and from the exocellular medium. In this respect, IKAWA AND SNELL9 have reported extracting alanine peptides from S. faecalis with cold trichloroacetic acid; in their experiments the peptides contained only alanine. In addition, BROWN1° has extracted adenyl peptides from acetone-dried S. faecalis with cold trichloroacetic acid (any such carboxyl-activated pept)des present in the intact cells Biochim. Biophys. Attar, 58 (1962) 538-549
AMINO ACID UPTAKE BY
Streptococcusfaecium
547
used in our studies would probably be hydrolyzed to the free peptide by the extraction procedures we employed.).
Uptake of lysine in the ~resence and absence of glucose GALEa has stated that lysine is concentratively accumulated by S. fa~calis in the absence of glucose. As part of the present studies L-lysine was added to depleted cells at pH 9-5 and after an interval glucose was added also. Similar additions were made to suspensions of undepleted cells, and with one of these suspensions of undepleted cells glucose addition preceded lysine addition. As revealed by the data in Fig. I, the question of whether or not glucose is required for lysine uptake by S. faecium is complicated by the findings, first, that the cellular freely extractable lysine of the depleted cells increased when glucose was present in the exocellular environment but lysine was not, and, second that the rate of this increase was not altered by the addition of lysine to the exocellular environment. Since lysine was shown not to be synthesized from precursors generated by glycolysis in this organism, it is assumed that here, too, lysine was being released from a cellular component. But however much release from a cellular component and uptake from the exocellular environment may each have contributed to the increase, during glucose metabolism, of freely extractable lysine, this increase did not occur when lysine was present in the exocellular environment but glucose was not; consequently, it is unlikely that lysine is taken up by this species in the absence of glucose. This conclusion is
3.[ /
LYSINE ADDED
:k. 2.4
~
\
O
e ~ ~
ttJ Z
2.0
=, II1
,.s
cc
1.2
6LUGOSE ADDED o
/
E ADDED
LU ADDED
LYSINE ADDED
~-=×
a. 0.4
0
0
/
f
0sE
ADDED
L
,
,
,
,
,
I0
20
30
40
50
60
I
7O
MINUTES
Fig. x. Changesinthe freely extractable lysine of depleted and non-depleted cells of Streptococcus faecium exposed to glucose (xo mg/m]) and L-lysine (o.o52 pmole/m]) singly and in combination; O - - O , depleted cells; O - - O , non-depleted cells. Biockim. Biophys. Acta, 58 (I962) 538-549
548
W. R. CHESBRO, J.B. EVANS
substantiated by tile observation that it required the addition of glucose to the undepleted cells to reverse the disappearance of cellular freely extractable lysine that normally occurs at this medium pH (9.5) (see ref. 2) ; lysine, by itself, did not halt or reverse the disappearance. Perhaps the disparity between our finding that lysine uptake does not take place in the absence of glucose, and the finding of GALE3 that it does, is explicable by reference to the report of BRITT AND GERHARDT11 that the greater portion of the freely extractable lysine of Micrococcus lysodeikticus is held by electrostatic interaction with the cell wall, external to the plasma membrane. If this finding is equally applicable to enterococci, then diffusion of lysine into the cell wall and its absorption there could occur independently of the presence of glucose. Failure to distinguish this from lysine taken up within the plasma m e m b r a n e - - a n d , as was pointed out earlier, it is unlikely that decarboxylases such as GALE employed can reach, or act upon, amino acids absorbed within the cell wall--would lead to the conclusion that lysine could be taken up by enterococci without the necessity for a concurrent metabolism of glucose. DISCUSSION
In a previous report S we noted that freely extractable alanine, lysine, and glutamic and aspartic acids appeared, after 25 rain, in S. faecium suspensions containing added glucose but no added amino acids. (DAWES AND HOLMES1. have observed apparently similar behavior in Sarcina lutea: in that organism the metabolism of glucose, in the absence of exogenously added amino acids, resulted in the appearence of freely extractable y-aminobutyric acid, alanine, and glutamic and aspartic acids). In the present study, freely extractable alanine, lysine, and glutamic and aspartic acids again a p p e a r e d - - b u t immediately, without any lag--concurrently with the uptake of exogenously added amino acids, whether or not they too had been supplied to the suspension. These four amino acids are identical to the freely extractable amino acids which disappear from the cell during depletion, and differ, therefore, from the amino acids which are released to the medium during this process 2. It was postulated previously 2 that because the amino acids involved in the coupled, OH--dependent amino acid disappearance-release processes (which occur during depletion of the cellular freely extractable amino acids) can differ in kind and in quantity, a heat-stable amino acid "reservoir", common to both processes, must exist. The existence of this "reservoir", which contains a limited number of kinds of amino acids; which combines with the cellular freely extractable amino acids during the depletion process; and which returns these combined amino acids to their freely extractable state, after a lag period, when glucose is metabolized, is reaffirmed by the present finding that freely extractable amino acids (which are of the same kinds as those that disappeared during the depletion process) appear immediately when exogenously added amino acids are taken up. The "reservoir" is thus intimately linked both with the processes involved in the amino acid disappearance-release that takes place when the cells are depleted and with the processes mediating amino acid uptake by the cells. It is possible that the two groups of processes may be one and the same. The amino acids released from the "reservoir" to the freely extractable pool during uptake are characteristically found as cell-wall components in this genera of Biochim. Biophys. Acta, 58 (1962) 538-549
AMINO ACID UPTAKE BY
Streptococcusfaecium
549
microorganisms TM. Thus, the reservoir seems related to the cell wall, perhaps being a cell-wall component; and because it is also coupled to amino acid transport, it constitutes a linkage--direct, or nearly so--between cell-wall components and amino acid transport, possibly important to the synthesis of cell wall. Such a linkage and the fact that when amino acids such as leucine and serine, characteristic of cell protein rather than cell wall, are taken up they can be transaminated, yielding their alpha-keto homologues, to twice the extent that they are retained as the amino acid would permit the cell to circumvent the thermodynamic limitation placed upon concentrative amino acid accumulation by the concentration gradient between these compounds within and without the cell. Since there is reason to believe that an alpha-keto acid does not compete with its homologous amino acid for the amino acid's transport system 14, transamination of a freely extractable amino acid acts to lower the concentration gradient between that amino acid and the same amino acid in the exocellular environment. In effect, the cell stores the amino acid as its readily convertible precursorl And further, since the products of transamination in S. faecium are alanine and aspartic acid, their combination with the "reservoir" (or with cell-wall components) provides a means for continually lowering the pool concentration of these amino acids; but since the flow of these amino acids into the "reservoir"-bound form is readily reversible, in effect, they too are stored. The foregoing hypothesis indicates, in corollary, how vitamin B 6 and the transaminases might contribute to the capacity of the cell for concentrative amino acid accumulation 15 without actually being part of the amino acid transport system. ACKNOWLEDGEMENT
This research was supported, in part, by a grant from the National Institutes of Health. REFERENCES t W . R. CNESBRO AND J. B. EVANS, J. Bacteriol., 78 (1959) 848. 2 W. R. CHESBRO AND J. B. EVANS, J. Bacteriol., 79 (196o) 682. s E. F. GALE, Advances in Protein Chem., 8 (1953) 285. 4 j . T. HOLDER AND J. HOLMAN, J. Biol. Chem., 234 (1959) 865. 5 E. W. BASSETT AND W. J. HARPER, J. Dairy Sci., 9 (1958) 12o6. 6 A. C. NEIsrI, Analytical Methods for Bacterial Fermentations, N a t i o n a l R e s e a r c h Council of Canada, Saskatoon. 7 p. MITCHELL, Ann. Rev. Microbiol., 13 (I959) 4o7 . 8 B. PETRACK, L. SULLIVAN AND S. RATNER, Arch. Biochem. Biophys., 69 (1957) 186. 9 M. IKAWA AND E. SNELL, Arch. Biochem. Biophys., 78 (1958) 338. x0 A. D. BROWN, Biochim. Biophys. Acta, 3 ° (1958) 447. 11 E. M. BR[TT AND P. GERHARDT, J. Bacteriol., 76 (I958) z88. 12 E. A. DAWES AND W. H. HOLMES, Biochim. Biophys. Acta, 3o (1958) 278. lZ G. D. SI-IOCKMAN,J. Biol. Chem., 234 (1959) 234 o. it E. BALL, J. HUMPHREYS AND W. SHIVE, Arch. Biochem. Biophys., 73 (1958) 4 lo. 15 j. T. HOLMAN,J. Biol. Chem., 234 (I959) 872.
Biochim. Biophys. Acta, 58 (1962) 538-549