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Carbohydrate transport in Staphylococcus aureus
BIOCHIMICA ET BIOPHYSICA ACTA
63
BBA 45 271 C A R B O H Y D R A T E T R A N S P O R T IN S T A P H Y L O C O C C U S A U R E U S I I I . S T U D I E S OF T H E T R A N S P O R T P R O C E S S J. BARRY EGAN* AND M. L. MORSE** Department of Biophysics, University of Colorado Medical Cenler, and The Webb-Waring Inslitute for Medical Research, Denver, Colo. (U.S.A.)
(Received April 2ist, 1965)
SUMMARY The u p t a k e of carbohydrates b y wild-type (car +) strains of Staphylococcus aureus was investigated to support the postulate t h a t c a r b o h y d r a t e transport is mediated b y specific permeases and a c o m m o n m e m b r a n e carrier. U p t a k e of c~-methylglucoside, glucose, lactose and maltose exhibited specificity and saturation kinetics, suggesting an enzymatic step which is presumed to reflect the function of specific permeases. The uptake of sucrose was anomalous in t h a t the rate did not saturate even at high concentrations. A second step, c o m m o n to the transport of carbohydrates, was indicated: (a) b y the ability of one c a r b o h y d r a t e to displace another accumulated within the cells (counterflow) ; and (b) b y the finding t h a t lactose inhibits the uptake of other carbohydrates only in cells previously induced to transport lactose. Lactose, ~-methylglucoside, sucrose, and maltose accumulate in cells partially or totally in the form of derivatives. Preliminary experiments on the characterization of these derivatives are discussed but the significance of the derivatives is as yet unknown. The present results are compatible with the essential features of the model proposed b y KEPES in 196o for the transport of ~-galactosides across the cell membrane. However, for carbohydrate transport in general in Staphylococcus, it appears necessary to invoke permeases specific for various carbohydrates but a carrier c o m m o n to all. I t is proposed t h a t the car locus is concerned with the formation of a functional carrier.
INTRODUCTION There are two facts 1 related to the transport of carbohydrates across the staphylococcal cell m e m b r a n e t h a t indicate the involvement of two functions (at least) in accomplishing such transport; (a) the isolation of a m u t a n t t h a t has simultaneously lost the ability to transport a n u m b e r of carbohydrates indicates the existence of some function in carbohydrate transport t h a t is c o m m o n to these carbo* Present address: Department of Biochemistry, Stanford University, Palo Alto, Calif. (U.S.A.). Request reprints from M. L. MORSB. "* Career Development Awardee, Public Health Service (U.S.A.). Biochim. Biophys. Acta, 112 (1966) 63-73
64
j.B.
E G A N , M. L. MORSE
hydrates; (b) the fact that only lactose and galactose uptake is inducible while that of the other carbohydrates is constitutive necessitates a second transport function peculiar in behavior to lactose and galactose transport. We have proposed 2 4 that the above two facts reflect the existence of two steps (at least) in membrane transport for the wild-type cell--the simultaneous loss of the ability to transport a number of carbohydrates represents defective membrane carrier function that is common to, and necessary in, "ferrying" each carbohydrate across the membrane, while the inducible nature of lactose uptake reflects permease function, specific for each carbohydrate and whose role is to catalyze the association of carbohydrate with carrier and so greatly increase the overall rate of uptake of a carbohydrate. This specific permease-common carrier model oI membrane transport is similar to that recently proposed by KOCH5. Genetic and biochemical characterization of the car- mutant 1,4 supports the concept that the car gene is concerned with the formation of a functional membrane carrier, which, if defective (as in tile car- mutant), will prevent transport whether "active" or by diffusion. It is the purpose of this paper to provide data to test for the existence in the wild-type (car +) Staphylococcus aureus cell of two steps in transport--one specific for each carbohydrate and saturable (the permease), and the second, a mobile membrane carrier common to the transport of each carbohydrate 6. MATERIALS AND METHODS
The following materials and methods supplement those previously described2,! Rate of uptake. Preliminary experiments indicated that the uptake of the various carbohydrates can be considered as linear over the first Io rain. Therefore, after IO rain incubation of the cell suspension in minimal succinate medium, 25 ttg chloramphenicol/ml, and increasing concentrations of the 14C-labeled carbohydrate, I.o-ml samples were filtered, washed, and counted in the usual manner. The accumulated radioactivity was taken as a measure of the initial rate of uptake. Collection of radioactive CO 2. Immediately after adding the radioactive substrate to a suspension of bacteria in minimal succinate medium, the tube was sealed with a rubber stopper holding appropriate glass fittings. After a given time of incubation at 37 °, the suspension was chilled, concentrated HC1 added, and the CO2 released was blown over with N~. These gases were slowly bubbled into a I-ml solution of 0.55 N Ba(OH)2 and 2 °4 KOH, contained in the barrel of a 2-ml Luer-Lok syringe with a Swinny adapter attached. After IO min of bubbling, the BaCO 3 precipitate waa collected on Whatman No. 50 filter paper fitted inside the Swinny adapter, and the precipitate was washed with IO ml of water. The filter was dried and counted in the scintillation counter. Competition experiments. 0.85 ml of a cell suspension (A ~ o.35) in minimal succinate medium plus 25/~g chloramphenicol/ml was added at time zero, to 0. 4 ml of an aqueous solution containing the 14C-labeled carbohydrate (2" IO GM) with and without the second unlabeled carbohydrate (2" lO-4 M). After IO rain at 37 °, I.o-ml samples were filtered and counted in the usual manner. The radioactivity accumulated by the bacteria in the absence of a second carbohydrate is taken as the standard uptake (lOO%) and the accumulations in the presence of the second carbohydrate are calculated as the percentage of this value. Cells used in lactose studies, when Biochim. Biophys. Acta, 112 (1966) 63-73
CARBOHYDRATE TRANSPORT IN S. a u r e u s
65
induced, had been previously grown in o.2% galactose, centrifuged, and suspended in minimal succinate medium. Counterflow experiments. In each case a mutant strain selected for inactivity of the appropriate metabolic enzyme was used as the strain capable of accumulating carbohydrate A. In general IO-5 M ~4C-labeled carbohydrate A was added to cells suspended to a density of 50 fig cell dry weight/ml in minimal succinate medium plus 25 fig chloramphenicol/ml. I.o-m] samples were taken with increasing time, filtered, washed and counted in the usual manner. After 12o rain unlabeled carbohydrate B was added to a final concentration oi IO - 3 M and further I . o - m l samples taken at later times. The efflux of radioactivity from the cells is taken as indicative of counterflow of carbohydrate A by carbohydrate B. Whenever lactose was tested in a combination the cells had been previously induced by growth in galactose. Intracellular state of accumulated carbohydrates. In general the cells were suspended in 0.2 ml 1% peptone and 14C-labeled carbohydrate added. After I h stationary incubation at 37 ° the cells were centrifuged and resuspended in 0.2 ml 5 % n-butanol in water. After approximately IO rain at 37 ° this suspension was transferred in toto to a chromatogram and developed with the sodium acetate-ethanol solvent. 200
- -
150
u
~ 10o
200
r
c 5(3 ©
~
o 150 ~/--
~-o
100 (a) Lactose
q
50
(b)Maltose
40 ~o ~o ~
5o
~oo
~5o
Curve1 E x
40 3O
_c
500
7
•
4OO
/o
0
300
/
200 / f ' ~ (c) G-Methyl Glucoside
;o io
•
Curve 20 o
16 100 ~ (d)Glucose
,0 ~ ~o ~oo
'~ ~'o ~'5 2'o
External concentration (mM)
Fig. i. R a t e of c a r b o h y d r a t e u p t a k e b y car + as a function of concentration. (a) ]3S 29o2 (lac-) was first induced in 0.2% galactose before the s t u d y w i t h [I-14C]lactose. The density of the cell suspension was 72 fig cell d r y weight/ml. (b) [I-14C]Maltose u p t a k e was studied with a IOI # g / m l suspension of ]3S 29o2 (real-). (c) U n i f o r m l y 14C-labeled ~-methylglucoside u p t a k e was studied w i t h a lO2 ffg/ml suspension of BS 2902. (d) [I-14C]Glucose u p t a k e was studied w i t h an 84 ffg/ml suspension of ]3S 5oo2 (glu +) and the a c c u m u l a t e d counts are plotted in Curve i. Curve 2 represents the 14CO2 released in io rain from I - m l suspensions (96 ffg/ml) of BS 5002 (glu+), with increasing concentrations of [I-14C~glucose. The counts as Ba14CO3 can be treated in the calculation as equal to c o u n t s of [I-14C]glucose a n d Curve 2 is d r a w n as the n u l n b e r of/*moles of [I-14C]glucose/g cell d r y weight t h a t yields the observed c o u n t s of ]3a14CO3.
BiGchim. Biophys. Acta, 112 (1966) 63-73
66
J.B.
EGAN,
M. L. M O R S E
RESULTS
Ki~etics of @take. The rate of uptake of various carbohydrates was determined as a function of concentration and the results are plotted in Fig. I. It can be seen that the rates of uptake of lactose, maltose, ~-methylglucoside and glucose saturate at higher carbohydrate concentrations, results compatible with either enzyme or adsorption kinetics. In contrast to tile study of lactose, maltose, and c~-methylglucoside uptake, in the glucose uptake experiment the substrate under transport can be metabolized with the ultimate yield of ~4CO2. In scoring uptake by the accumulation of counts within the cells, the possible loss of counts as ~4CO2 had to be considered in the interpretation of the plateau in Curve I of Fig. I(d). Therefore the evolution of 14CO., was determined with increasing concentration of ~I-l~C_~glucose taken up/Io min/g cell dry weight that yields the observed counts of BauCOa. This curve shows saturation kinetics with the plateau commencing at 4" I0-5 M, the parallel of Curve I. The true curve~of the rate of glucose uptake vs. concentration is therefore the sum of Curves I and 2, which itself is compatible with saturation kinetics.
104I
/
/
._c E
/."
O
E
/-
&
/O
0) c~
c
/ •
./ .......
./ i
10-5
t
e/°
, ,ht,,I
i
10-4
....
lit]
10-3
i
t t,HN]
10-2
External concentraUon(M)
I
I 1~1113J
10-~
F i g . 2. R a t e of s u c r o s e u p t a k e b y cat ".+ a s a f u n c t i o n of c o n c e n t r a t i o n . s u c r o s e u p t a k e w a s s t u d i e d w i t h a 6 7 / t g / m l s u s p e n s i o n of B S 5 o o 2 ( s u e ) .
Uniformly
l~C-labeled
Sucrose uptake was, however, dramatically different. It was impossible to saturate the system, and the rate of uptake continued to rise with increasing sucrose concentration over a concentration range of IO ~ M to lO-1 M. The results have been plotted on a log-log scale (Fig. 2), and from the gradient of the resulting line, one can calculate that the rate of uptake is proportional to the o.83 power of the initial extracellular concentration. The plot on linear axes would not therefore be linear but concave towards the concentration axis. B i o c h i m . B i o p h y s . A c t a , 1±2 ( I 9 6 6 ) 6 3 - 7 3
CARBOHYDRATE
TRANSPORT
67
IN S . aure~ts
The experiment was carried out with BS 5o02, a sucrase-less mutant. If all the the counts accumulated were indicative of free sucrose within the cell, then, at an extracellular concentration of lO-1 M, the intracellular concentration after IO rain would be 3.6 M. This is equivalent to i . I g of sucrose per 0.3 g cell dry weight (1.2 g cell wet weight). Competition between carbohydrates during uptake. The demonstration of saturation kinetics suggested the possibility of an enzymatic step in the transport of carbohydrates. The specificity of carbohydrate transport in S. aureus was therefore investigated. The rate of uptake of a particular carbohydrate was determined in the presence of a second carbohydrate at a Ioo-fold greater concentration, and compared to the rate of uptake in the absence of the second carbohydrate. TABLE
I
THE UPTAKE OF CERTAIN CARBOHYDRATES IN THE PRESENCE OF A SECOND CARBOHYDRATE AT A HIGHER CONCENTRATION T h e r a d i o a c t i v i t y a c c u m u l a t e d b y t h e b a c t e r i a i n t h e a b s e n c e of a s e c o n d c a r b o h y d r a t e is t a k e n a s t h e s t a n d a r d u p t a k e ( i o o % ) a n d t h e a c c u m u l a t i o n s i n t h e p r e s e n c e of t h e s e c o n d c a r b o h y d r a t e a r e c a l c u l a t e d a s t h e p e r c e n t a g e o f t h i s v a l u e . Ceils w e r e i n d u c e d f o r l a c t o s e u p t a k e s t u d i e s , b u t n o t f o r t h e s t u d y of g a l a c t o s e a n d l a c t o s e i n h i b i t i o n of t h e u p t a k e of o t h e r c a r b o h y d r a t e s .
Second carbohydrate (2.o'1o 4 M )
o~-Methylglucoside (z 4"IO -6 M )
None a-Methylglucoside Glucose Lactose Maltose Sucrose Fructose Galactose Mannitol
ioo 22 7 116 iio 80 161 14o 93
Uptake of 14C-labeled carbohydrate Glucose (2. 4 • Io -6 M )
Lactose (3.3" Io-6 M )
Maltose (2.z. i o -e M )
Sucrose (3.9" lO-6 M )
IOO 143 19 lO 3 lO 4 lO 4 lO9 ioo 82
ioo 90 86 7 iOl IO8 IOO lO 7 lO 7
IOO 12o 25 Ioo 14 3° 92 98 92
ioo lO8 18 97 61 64 79 lO6 97
As seen in Table I, 29 of 35 different carbohydrate combinations showed less than 20 % inhibition and, in general therefore, the uptake of one carbohydrate is relatively insensitive to the presence of a second carbohydrate, indicating that carbohydrate transport is a relatively specific process. The exceptions to this include glucose inhibition of the uptake of the other carbohydrates except possibly lactose, and maltose and sucrose, which retard the uptake of one another. The possible inhibitory effect of lactose on the uptake of carbohydrates by cells themselves induced to transport lactose, was also investigated (Table II). It was found that with cells previously induced in galactose, the uptake of c,-methylglucoside, maltose, sucrose, but not of glucose, was markedly inhibited by the presence of lactose. This same inhibitory effect of lactose was obtained wiLh induced cells that could metabolize the carbohydrate whose uptake was under study. Lactose therefore inhibits the uptake of other carbohydrates only when lactose itself is being transported. Whether the induced state per se is inhibitory to the transport of the other carbohydrates cannot be stated at this time. Biochim. Biophys. Acta, 112 ( 1 9 6 6 ) 6 3 - 7 3
68
j.B. EGAN, M, L. MORSE
T A B L E II UPTAKE
0F
CERTAIN
CARBOHYDRATI~S
IN
THE
PRESENCE
OF
LACTOSE
The radioactivity accumulated by the bacteria in the absence of a second carbohydrate is taken as the standard uptake (ioo %), and the accumulations in the presence of the second carbohydrate are calculated as the percentage of this value. The cells, when induced, had been previously grown in 0.2 % galaetose. Culture used
Uptake of 14C-labeled carbohydrate in the presence of 2. zo -a .,~I lactose o:-7Vlethylglucoside (L 4 • ZO-~ 3/I)
BS 2902 (lac-mal-)-non-induced BS 5002 (suc-)-non-induced BS 2902 (lac mal-)-induced B S 5002 (suc-)-induced BS 56oi (lac+mal+suc+)-induced
I26
Glucose (2.4" 7.0--6 igI)
Sucrose
(2.2" IO 6 J~I)
(3.9" ±o-S M)
lO 3
ioo
- -
18
Maltose
-
lO8
37 27
31
- -
13
- -
-
97 -39
-
C o u n t e r f l o w e x p e r i m e n t s . C o u n t e r f l o w is e x h i b i t e d w h e n t h e u p t a k e of c a r b o h y d r a t e B i n t o a cell d i s p l a c e s c a r b o h y d r a t e A p r e v i o u s l y a c c u m u l a t e d w i t h i n t h e celF. T h i s p h e n o m e n o n h a s b e e n c o n s i d e r e d as e v i d e n c e for t h e e x i s t e n c e of a m o b i l e m e m b r a n e c a r r i e r , c o m m o n t o t h e t r a n s p o r t of c a r b o h y d r a t e s A a n d B G-11. D e m o n s t r a t i o n s of c o u n t e r f l o w w e r e a t t e m p t e d w i t h p a i r s of c a r b o h y d r a t e s in t h i s s t a p h y l o c o c c a l s y s t e m . A cell s u s p e n s i o n in m i n i m a l s u c c i n a t e m e d i u m p l u s 25 t~g c h l o r a m p h e n i c o l / m l w a s p e r m i t t e d t o a c c u m u l a t e l ~ C - l a b e l e d c a r b o h y d r a t e A, and then unlabeled carbohydrate B was added at a concentration approximately I o o - f o l d t h e i n i t i a l c a r b o h y d r a t e A c o n c e n t r a t i o n . T h e r e s u l t s of t h e q u a n t i t a t i v e data s have been summarized and appear in Table Ill.
TABLE III DISPLACEMENT
OF
ACCUMULATED
CARBOHYDRATE
~Al B Y
CARBOHYDRATE
t~
In the table: + , indicates counterflow; --, its absence; + / - - , doubtful (less t h a n 2o o/o/ e f t l u x of carbohydrate A) ; o, combination not tested. The table summarizes the data recorded in EGANs. Carbohydrate B
c~-Methylglucoside Glucose Lactose Maltose Sucrose
Carbohydrate A o~-Methylglucoside
Lactose
7Vialtose
Sucrose
+ + o ---
o + + + / -+ / --
o + + + +
o + / + / -+/ + / --
T h e r e s u l t s i n d i c a t e t h e e x i s t e n c e of c o u n t e r f l o w w i t h c e r t a i n c a r b o h y d r a t e c o m b i n a t i o n s . I n o t h e r c a s e s t h e r e w a s n o n e . T h e a b s e n c e of c o u n t e r f l o w i n t h e s e c a s e s c a n b e e x p l a i n e d s a t i s f a c t o r i l y a f t e r c o n s i d e r a t i o n of t h e i n t r a c e l l u l a r s t a t e of t h e a c c u m u l a t e d c a r b o h y d r a t e , a n d t h e r e s u l t s will b e r e c o n s i d e r e d i n t h e DISCUSSION with the relevant data from experiments in the next section. Biochim. Biophys. Acta, 112 (1966) 63-73
CARBOHYDRATE TRANSPORT IN S. aureus
69
The intracellular state of the accumulated carbohydrates. In the study of the carbohydrate uptake, m u t a n t s of S. aureus, defective in the first enzyme of the p a t h w a y of the carbohydrates' ultilization, have been used. Thus BS 22Ol (lac-) and its derivatives are fi-galactosidaseless, BS 5002 (suc-) is sucraseless, and BS 2902 (mal-) is maltaseless. One would expect therefore that these carbohydrates, being nonmetabolizable, would accumulate as the free carbohydrate within the respective m u t a n t cells. A similar situation would be expected for the structural analogue, ~-methylglucoside. Such m u t a n t cells were permitted to accumulate the respective 14C-labeled carbohydrates, and the state of the accumulated material was examined by chromatography of the debris plus extract after treatment of the cells with 5 % butanol, using sodium acetate-ethanol as the developing solvent. Representative tracings appear in Fig. 3.
LACTOSE Fig. 3. T h e i n t r a c e l l u l a r s t a t e of [14C]lactose a c c u m u l a t e d d u r i n g t r a n s p o r t a n d i t s c ha s e b y u n l a b e l e d c a r b o h y d r a t e . T h e cells were s u s p e n d e d in o. 4 ml 1% p e p t o n e a n d lO -4 M 14C-labeled c a r b o h y d r a t e a d d e d . A f t e r i h a t 37 °, t h e s u s p e n s i o n w a s c e n t r i f u g e d a n d r e s u s p e n d e d in o. 4 ml of 1% p e p t o n e . The s u s p e n s i o n w a s d i v i d e d i n t o 2 × o.2-ml lots, o.o2 ml of w a t e r a d d e d t o one, a n d o.o2 m l of lO-1 M u n l a b e l e d c a r b o h y d r a t e to t h e second. A f t e r 2o m i n a t 37 °, o.oi m l n-butanol was added and the suspension chromatographed with the sodium acetate-ethanol s o l v e n t . T h e left c h r o m a t o g r a m is t h e i n t r a c e l l u l a r s t a t e p r i o r t o chase, t h e r i g h t , p o s t chase. T h e c h r o m a t o g r a p h i c l o c a t i o n of t h e p a r e n t c o m p o u n d is i n d i c a t e d b y a n arrow.
In lactose (Fig. 3a) and ~-methylglucoside uptake the radioactivity accumulated within the cell was found in the torm of an unknown compound, compound X*, that traveled as a discrete peak on the chromatogram more slowly than the carbohydrates themselves. In maltose and sucrose uptake (not shown), a large percentage of the radioactivity accumulated as the free carbohydrate, but there also was a peak of intermediate RE that seemed analogous to the peak of compound X in the chromatograms after lactose and a-methylglueoside uptake. In addition some radioactivity appeared in the debris at the origin with both sucrose and maltose. The peaks of intermediate RE values were investigated further. In the case of lactose uptake, the formation of compound X needs the integrity of the cell, as addition of the radioactive lactose after the cells had been treated with butanol did not yield the compound. Compound X is soluble in 5 % trichloroacetic acid and insoluble in chloroform. Acid hydrolysis (I N HC1, boiled for 2, 4, 6, 8 min) partially degraded the compound X to release radioactivity moving with the RE of glucose, but little, if any, free lactose was released. * T h e g e n e r a l use of t h e t e r m c o m p o u n d X for t h e a c c u m u l a t i o n of di ffe re nt c a r b o h y d r a t e s i m p l i e s t h a t , in each case, t h e c a r b o h y d r a t e a p p e a r s in a s i m i l a r l y d e r i v e d form. T h e r e is no e v i d e n c e for such a n i m p l i c a t i o n , a n d t h e g e n e r a l t e r m c o m p o u n d X is us e d s ol e l y for t h e s a k e of discussion.
Biochim. Biophys. Acta, 112 (1966) 63-73
7°
J . B . EGAN, M . L . MORSE
W h e n a chase of unlabeled lactose was added to a suspension of cells containing c o m p o u n d X, more than 5o % of the radioactivity m o v e d from the RE of the comp o u n d X to t h a t of free lactose (Fig. 3b). Once again the integrity of the cell was necessary to effect this chase, as cells containing 14C-compound X, but treated with butanol before the addition of unlabeled lactose, showed no shift of the radioactivity to the lactose peak. The radioactivity in the c o m p o u n d X for ~-methylglucoside uptake was completely chased with unlabeled e-methylglucoside to the RE of free c~-methylglucoside. Comparable results, but less dramatic, were seen in similar experiments with maltose and sucrose uptake. In the case of sucrose uptake, it was further shown t h a t unlabeled maltose chased radioactivity from the c o m p o u n d X peak to the free sucrose peak. DISCUSSION
The absence of o-nitrophenylgalactoside diffusion and active transport in the car- m u t a n t 4 is compatible with the postulate t h a t the m e m b r a n e carrier is defective in this m u t a n t . The transport of carbohydrates in car + strains has been investigated to test the postulate of carbohydrate-specific permeases and a carbohydrate c o m m o n carrier, as outlined in the INTRODUCTION. The specific saturable step in carbohydrate transport. The uptake of ~-methylglucoside, glucose, lactose and maltose b y care cells satisfies Michaelis-Menten kinetics, which could reflect saturation of either an enzyme (the permease) or of an adsorbing site (the carrier). The respective Michaelis constants related to saturation can be calculated from L i n e w e a v e r - B u r k plots of the data presented in Fig. I. T h e y are for lactose, K i n = 5"1o -6 M; for ~-methylglucoside, K m z 2 . I o - ~ M ; and for maltose, K i n - i. i o -~ M. If the saturable step in transport were also the step c o m m o n to the transport of carbohydrates, then one can estimate the inhibition (assumed competitive) expected for the u p t a k e of one carbohydrate in the presence of an excess of a second carbohydrate. These calculations are presented in Table IV along with the actually observed values. It is clear from a comparison of the expected and observed inhibition values t h a t the saturable step in transport is not a step c o m m o n to all the tested carbohydrates but is rather a step specific for each. In contrast to the uptake of ~-methylglucoside, glucose, lactose and maltose, the uptake of sucrose does not show saturation kinetics, and the initial rate of uptake continues to increase with increasing sucrose concentration over a five-log range of concentrations. Common step in carbohydrate transport. Evidence for a c o m m o n step in carboh y d r a t e transport comes from three sources: (I) the loss b y m u t a t i o n of the ability to transport as least 8 carbohydrates ; (2) competitive uptake studies ; and (3) counterflow experiments. Genetic evidence, indicating a c o m m o n step distinct from the specific (presumably permease) step has been presented in the previous publications in this series1, 4. Additional evidence for the existence of a c o m m o n carrier comes from the results of competition experiments. It was observed that, while lactose did not interfere with the uptake of other carbohydrates in non-induced cells, it did inhibit uptake when the cells had been previously induced with galactose. This finding Biochim. Biophys. Acla, 112 (1966) 63 73
CARBOHYDRATE TRANSPORT IN S. aureus
71
TABLE IV COMPARISON SATURABLE
OF
TIIE
EXPECTED
COMPONENT
Uptake o/
COMMON
AND TO
OBSERVED THE
In presence of
INHIBITION
TRANSPORT
Kin*
OF
OF
CARBOHYDRATE
Calculated
2" 1 0 - 4 M
rate**
FOR
A
Inhibition Expected Observed
(%)
Lactose 3.3" lO-6 M
UPTAKE
CARBOHYDRATES
(%)
Maltose ~-Methylglucoside
5" I O - 6 M i. lO_4 M 2. 5- lO-5 IV[
o.182 V 0.069 V
55 83
Maltose 2.2. io 6 M
c~-Methylglucoside
I. lo -4 M 2. 5" lO-5 M
o.o215 V 0.0024 V
-89
--20
~-Methylglucoside 1.4- lO-8 M
-Maltose
2.5" Io -5 M I. Io 4 M
o.o531 V o.o183 V
-66
--IO
0.40
V
--
-
I IO
* The Miehaelis constants Km were calculated from the Lineweaver-Burk plots of the data presented in Fig. i, and the data of Table I. ** Predicted rates were calculated from v
VS/Km and vt = VS/Km where I + S/Km I + I/K~ + S/KIn
V = maximum rate; v,vt -- predicted rates; S, I = substrate and inhibitor concentrations; and Kin, K i - Michaelis constants for the substrate and inhibitor. The inhibition is assumed to be competitive.
indicates that lactose can only retard the uptake of another carbohydrate if lactose itself is being transported. The lactose inhibition observed could then be due either to competition for limited amounts of common carrier, or to the counterflow by lactose of other carbohydrates as soon as the latter is accumulated. The second possibility was examined by studying the uptake of a carbohydrate by ceils that could metabolize the carbohydrate, so making it unavailable for counterflow by lactose. However, no moderation of the inhibition by lactose was observed under these conditions, which lends support for the existence of a common carrier in limited amounts. T h e c o u n t e r f l o w e x p e r i m e n t s also p r o v i d e e v i d e n c e for a c o m m o n s t e p in t r a n s p o r t b u t t h e r e s u l t s a r e b e t t e r u n d e r s t o o d if t h e i n t r a c e l l u l a r s t a t e of t h e a c c u m u l a t e d c o m p o u n d s is c o n s i d e r e d . C h r o m a t o g r a p h i c a n a l y s i s of t h e c e l l u l a r c o n t e n t s a f t e r a c c u m u l a t i o n of t h e v a r i o u s c a r b o h y d r a t e s r e v e a l e d t h e p r e s e n c e of n e w c o m p o u n d s , g e n e r a l l y t e r m e d c o m p o u n d X , w h i c h in e a c h c a s e m o v e d a t v a l u e s i n t e r m e d i a t e r e l a t i v e t o t h e R F of t h e f r e e c a r b o h y d r a t e s . L a c t o s e a n d c ~ - m e t h y l g l u c o s i d e a c c u m u l a t e m a i n l y i n t h e f o r m of t h e i r X - d e r i v a t i v e s , w h i l e s u c r o s e a n d m a l t o s e a c c u m u l a t e as free c a r b o h y d r a t e , as t h e X - d e r i v a t i v e , a n d as a c h r o m a t o g r a p h i c a l l y i m m o b i l e c o m p o n e n t . S i m i l a r o b s e r v a t i o n s h a v e b e e n m a d e in o t h e r b a c t e r i a l s y s t e m s : g a l a c t o s e , ~- a n d f i - m e t h y l g l u c o s i d e a c c u m u l a t e as t h e free c a r b o h y d r a t e a n d t h e 6 - p h o s p h o r i c a c i d e s t e r in Eseherichia coli12,13; a n d m a l t o s e a c c u m u l a t e s as f r e e m a l t o s e a n d t w o o t h e r c o m p o n e n t s in E. col# 4. T h e a c c u m u l a t i o n of c a r b o h y d r a t e s in t h e f o r m of d e r i v a t i v e s is of i m p o r t a n c e t o t h e i n t e r p r e t a t i o n of s e v e r a l r e s u l t s . (a) The counterflow experiments. I t w a s o b s e r v e d t h a t w h i l e l a c t o s e c a n d i s p l a c e accumulated maltose, maltose cannot displace accumulated lactose. A similar lack
Biochim. Biophys. Acta, 112 (1966) 63-73
72
J.B.
EGAN,
M. L. M O R S E
of reciprocity was observed with certain other pairs of carboydrates. This situation probably results from differences in the chemical states of the accumulated material, and from the superposition of other equilibria with new orders of specificity. Maltose accumulates in the cell as free maltose, c o m p o u n d X, plus an immobile component. Lactose presumably can only displace free maltose, while sucrose, maltose and glucose m a y also release [14Clmaltose from c o m p o u n d X (Table III). With lactose accumulation, there is little or no free lactose within the cell but mostly lactose-compound X. While unlabeled lactose can release [HC~lactose from the c o m p o u n d X form, maltose is incapable of doing so (Table III). Similar reasoning can be used to explain the absence of lactose counterflow b y sucrose, and the absence of ~-methylglucoside counterflow b y maltose and sucrose. With sucrose counterflow by other carbohydrates a larger outflow of [14C~sucrose was expected than was observed. However, at the low extracellular concentration of E14C~sucrose used in the counterflow experiments (4' IO-8 M) it is considered t h a t most of the accumulated radioactivity has been lost, irreversibly, to the chromatographically immobile form s . This possibility is supported b y the fact t h a t unlabeled sucrose itself displaces Fl4C~sucrose from the cells no better than the other carbohydrates. TABLE
V
INTRACELLULAR ACCUMULATION OF CARBOHYDRATES T h e i n t r a c e l l u l a r c o n c e n t r a t i o n of e a c h s u b s t r a t e w a s c a l c u l a t e d f r o m t h e d a t a a p p e a r i n g in F i g . i a , b, c, d, of EGAN AND ~V[oRSE 1 a s s u m i n g t h a t a l l c o u n t s r e p r e s e n t u n a l t e r e d s u b s t r a t e w i t h i n t h e cell. T h e c o u n t i n g e f f i c i e n c y of t h e s c i n t i l l a t i o n c o u n t e r f o r l a c w a s a p p r o x i m a t e l y 5o°.o. S. aurevts h a s b e e n s h o w n t o c o n t a i n 7 5 % w a t e r b y w e i g h t (AKANO 15) a n d f o r t h e c a l c u l a t i o n s i t w a s a s s u m e d t h a t i g cell d r y w e i g h t is e q u i v a l e n t t o 3 m l o f i n t r a c e l l u l a r w a t e r . T h e l a s t c o l u n m g i v e s t h e r a t i o of i n t r a - t o e x t r a c e l l u l a r c o n c e n t r a t i o n . Car + strain
BS BS t3S t3S
2902 (lac) 2902 (real-) 5002 (suc) 56Ol
Carbohydrate
Lactose Maltose Sucrose c~-Methylglucoside
Extraeellular concentration
I~ztracelhdar conce~tratio~z
Factor
(M)
(M)
1.2- IO -~ 2. 3. l o -5 5 . 6 . IO 6
8 . 6 . lO -2 1.6- IO 2 2 . 9 ' I o -3
7200 700 520
1.5" 1 ° - 5
5-5" IO-S
37 °
(b) The active transport of carbolgvdrates in this bacterium. (See Table V). I n the case of maltose and sucrose a large fraction of the accumulated radioactivity is in the free carbohydrate, and maltose and sucrose are, therefore, being transported against a concentration gradient. With c~-methylglucoside uptake, the a m o u n t of free carbohydrate within the cell is smaller, but sufficient to give a considerable ratio of intra- to extracellular concentration, again suggesting active transport. Lactose, however, appears to be present within the cell solely as c o m p o u n d X, and therefore it cannot be considered to enter against a concentration gradient. (c) The anomalous uptake of sucrose. If all the radioactivity accumulated is indicative of free sucrose within the sucraseless cell then, after IO rain uptake from io 1 M sucrose, the intracellular sucrose concentration is 3.6 M. Although the present results indicate that "I14~Csucrose '' activity appears in two other peaks on the chromat o g r a m beside t h a t of sucrose, it was conservatively estimated t h a t the tree sucrose, B i o c h i m . ]Biophys. A c t a , 112 ( i 9 6 6 ) 63 73
CARBOHYDRATE TRANSPORT IN S. aureus
73
present would be 1.2 M under these circumstances s. Therefore, at lO -1 M, sucrose is being taken up against a concentration gradient, and the uptake of sucrose cannot be by simple diffusion. In conclusion the present evidence, which supports a specific permease-common carrier model of carbohydrate transport, further justifies our postulate that the c a r locus is concerned with the formation of a functional carrier common to the membrane transport of carbohydrates. ACKNOWLEDGEMENTS
From a thesis submitted to the University of Colorado in partial fulfillment of the requirement for the Ph. D. Degree. This investigation was supported by research grants from the National Science Foundation, G-I42IO and GB-276. Contribution No. 257 from the Department of Biophysics. REFERENCES i J. BARRY EGAN AND M. L. MORSE, Biochim. Biophys. Acta, 97 (1965) 3 lo. 2 J. BARRY EGAN AND M. L. MORSE, Abstract MCzo, Biophysical Society, 7th Ann. Meeting, New York, 1963 . 3 J. BARRY EGAN AND M. L. MORSE, Abstract, Bacteriol. Proc., (1962) 42. 4 J. BARRY EGAN AND M. L. MORSE, Biochim. Biophys. Acta, lO9 (1965) 172. 5 A. L. KocH, Biochim. Biophys. Acta, 79 (1964) 177. 6 W. WILBRANDT AND T. ROSENBERG, Pharmacol. Revs., 13 (1961) lO 9. 7 T. ROSENBERG AND W. WILBRANDT, J. GeE. Physiol., 41 (1957) 289. 8 J. BARRY EGAN, Genetic and Biochemical Evidence for a Common Step in Carbohydrate Transport in Staphylococcus aureus, Doctoral Dissertation, University of Colorado, Denver, Colo., 1964. 9 T. ROSENBERG AND W*. WILBRANDT, Exptl. Cell Res., 27 (1962) Ioo. IO T. ROSENBERG AND W. WILBRANDT, J. Theoret. Biol., 5 (1963) 288. I I W. WILBRANDT, Ann. Rev. Physiol., 25 (1963) 6Ol. 12 D. ROGERS AND S.-H. Y u , J, Bacteriol., 84 (1962) 877. 13 H. HAGIHARA, T. H. WILSON ANn E. C. C. LIN, Biochim. Biophys. Acta, 78 (1963) 505 . 14 I-I. \VIESMEYER AND M. COHN, Biochim. Biophys. Acta, 39 (196o) 44 o. 15 R. AKANO, t~fyoto Furitsu Ika Daigahu Zasshi, i o (1934) 464 .
Biochim. Biophys. Acta, 112 (1966) 63-73
63
BBA 45 271 C A R B O H Y D R A T E T R A N S P O R T IN S T A P H Y L O C O C C U S A U R E U S I I I . S T U D I E S OF T H E T R A N S P O R T P R O C E S S J. BARRY EGAN* AND M. L. MORSE** Department of Biophysics, University of Colorado Medical Cenler, and The Webb-Waring Inslitute for Medical Research, Denver, Colo. (U.S.A.)
(Received April 2ist, 1965)
SUMMARY The u p t a k e of carbohydrates b y wild-type (car +) strains of Staphylococcus aureus was investigated to support the postulate t h a t c a r b o h y d r a t e transport is mediated b y specific permeases and a c o m m o n m e m b r a n e carrier. U p t a k e of c~-methylglucoside, glucose, lactose and maltose exhibited specificity and saturation kinetics, suggesting an enzymatic step which is presumed to reflect the function of specific permeases. The uptake of sucrose was anomalous in t h a t the rate did not saturate even at high concentrations. A second step, c o m m o n to the transport of carbohydrates, was indicated: (a) b y the ability of one c a r b o h y d r a t e to displace another accumulated within the cells (counterflow) ; and (b) b y the finding t h a t lactose inhibits the uptake of other carbohydrates only in cells previously induced to transport lactose. Lactose, ~-methylglucoside, sucrose, and maltose accumulate in cells partially or totally in the form of derivatives. Preliminary experiments on the characterization of these derivatives are discussed but the significance of the derivatives is as yet unknown. The present results are compatible with the essential features of the model proposed b y KEPES in 196o for the transport of ~-galactosides across the cell membrane. However, for carbohydrate transport in general in Staphylococcus, it appears necessary to invoke permeases specific for various carbohydrates but a carrier c o m m o n to all. I t is proposed t h a t the car locus is concerned with the formation of a functional carrier.
INTRODUCTION There are two facts 1 related to the transport of carbohydrates across the staphylococcal cell m e m b r a n e t h a t indicate the involvement of two functions (at least) in accomplishing such transport; (a) the isolation of a m u t a n t t h a t has simultaneously lost the ability to transport a n u m b e r of carbohydrates indicates the existence of some function in carbohydrate transport t h a t is c o m m o n to these carbo* Present address: Department of Biochemistry, Stanford University, Palo Alto, Calif. (U.S.A.). Request reprints from M. L. MORSB. "* Career Development Awardee, Public Health Service (U.S.A.). Biochim. Biophys. Acta, 112 (1966) 63-73
64
j.B.
E G A N , M. L. MORSE
hydrates; (b) the fact that only lactose and galactose uptake is inducible while that of the other carbohydrates is constitutive necessitates a second transport function peculiar in behavior to lactose and galactose transport. We have proposed 2 4 that the above two facts reflect the existence of two steps (at least) in membrane transport for the wild-type cell--the simultaneous loss of the ability to transport a number of carbohydrates represents defective membrane carrier function that is common to, and necessary in, "ferrying" each carbohydrate across the membrane, while the inducible nature of lactose uptake reflects permease function, specific for each carbohydrate and whose role is to catalyze the association of carbohydrate with carrier and so greatly increase the overall rate of uptake of a carbohydrate. This specific permease-common carrier model oI membrane transport is similar to that recently proposed by KOCH5. Genetic and biochemical characterization of the car- mutant 1,4 supports the concept that the car gene is concerned with the formation of a functional membrane carrier, which, if defective (as in tile car- mutant), will prevent transport whether "active" or by diffusion. It is the purpose of this paper to provide data to test for the existence in the wild-type (car +) Staphylococcus aureus cell of two steps in transport--one specific for each carbohydrate and saturable (the permease), and the second, a mobile membrane carrier common to the transport of each carbohydrate 6. MATERIALS AND METHODS
The following materials and methods supplement those previously described2,! Rate of uptake. Preliminary experiments indicated that the uptake of the various carbohydrates can be considered as linear over the first Io rain. Therefore, after IO rain incubation of the cell suspension in minimal succinate medium, 25 ttg chloramphenicol/ml, and increasing concentrations of the 14C-labeled carbohydrate, I.o-ml samples were filtered, washed, and counted in the usual manner. The accumulated radioactivity was taken as a measure of the initial rate of uptake. Collection of radioactive CO 2. Immediately after adding the radioactive substrate to a suspension of bacteria in minimal succinate medium, the tube was sealed with a rubber stopper holding appropriate glass fittings. After a given time of incubation at 37 °, the suspension was chilled, concentrated HC1 added, and the CO2 released was blown over with N~. These gases were slowly bubbled into a I-ml solution of 0.55 N Ba(OH)2 and 2 °4 KOH, contained in the barrel of a 2-ml Luer-Lok syringe with a Swinny adapter attached. After IO min of bubbling, the BaCO 3 precipitate waa collected on Whatman No. 50 filter paper fitted inside the Swinny adapter, and the precipitate was washed with IO ml of water. The filter was dried and counted in the scintillation counter. Competition experiments. 0.85 ml of a cell suspension (A ~ o.35) in minimal succinate medium plus 25/~g chloramphenicol/ml was added at time zero, to 0. 4 ml of an aqueous solution containing the 14C-labeled carbohydrate (2" IO GM) with and without the second unlabeled carbohydrate (2" lO-4 M). After IO rain at 37 °, I.o-ml samples were filtered and counted in the usual manner. The radioactivity accumulated by the bacteria in the absence of a second carbohydrate is taken as the standard uptake (lOO%) and the accumulations in the presence of the second carbohydrate are calculated as the percentage of this value. Cells used in lactose studies, when Biochim. Biophys. Acta, 112 (1966) 63-73
CARBOHYDRATE TRANSPORT IN S. a u r e u s
65
induced, had been previously grown in o.2% galactose, centrifuged, and suspended in minimal succinate medium. Counterflow experiments. In each case a mutant strain selected for inactivity of the appropriate metabolic enzyme was used as the strain capable of accumulating carbohydrate A. In general IO-5 M ~4C-labeled carbohydrate A was added to cells suspended to a density of 50 fig cell dry weight/ml in minimal succinate medium plus 25 fig chloramphenicol/ml. I.o-m] samples were taken with increasing time, filtered, washed and counted in the usual manner. After 12o rain unlabeled carbohydrate B was added to a final concentration oi IO - 3 M and further I . o - m l samples taken at later times. The efflux of radioactivity from the cells is taken as indicative of counterflow of carbohydrate A by carbohydrate B. Whenever lactose was tested in a combination the cells had been previously induced by growth in galactose. Intracellular state of accumulated carbohydrates. In general the cells were suspended in 0.2 ml 1% peptone and 14C-labeled carbohydrate added. After I h stationary incubation at 37 ° the cells were centrifuged and resuspended in 0.2 ml 5 % n-butanol in water. After approximately IO rain at 37 ° this suspension was transferred in toto to a chromatogram and developed with the sodium acetate-ethanol solvent. 200
- -
150
u
~ 10o
200
r
c 5(3 ©
~
o 150 ~/--
~-o
100 (a) Lactose
q
50
(b)Maltose
40 ~o ~o ~
5o
~oo
~5o
Curve1 E x
40 3O
_c
500
7
•
4OO
/o
0
300
/
200 / f ' ~ (c) G-Methyl Glucoside
;o io
•
Curve 20 o
16 100 ~ (d)Glucose
,0 ~ ~o ~oo
'~ ~'o ~'5 2'o
External concentration (mM)
Fig. i. R a t e of c a r b o h y d r a t e u p t a k e b y car + as a function of concentration. (a) ]3S 29o2 (lac-) was first induced in 0.2% galactose before the s t u d y w i t h [I-14C]lactose. The density of the cell suspension was 72 fig cell d r y weight/ml. (b) [I-14C]Maltose u p t a k e was studied with a IOI # g / m l suspension of ]3S 29o2 (real-). (c) U n i f o r m l y 14C-labeled ~-methylglucoside u p t a k e was studied w i t h a lO2 ffg/ml suspension of BS 2902. (d) [I-14C]Glucose u p t a k e was studied w i t h an 84 ffg/ml suspension of ]3S 5oo2 (glu +) and the a c c u m u l a t e d counts are plotted in Curve i. Curve 2 represents the 14CO2 released in io rain from I - m l suspensions (96 ffg/ml) of BS 5002 (glu+), with increasing concentrations of [I-14C~glucose. The counts as Ba14CO3 can be treated in the calculation as equal to c o u n t s of [I-14C]glucose a n d Curve 2 is d r a w n as the n u l n b e r of/*moles of [I-14C]glucose/g cell d r y weight t h a t yields the observed c o u n t s of ]3a14CO3.
BiGchim. Biophys. Acta, 112 (1966) 63-73
66
J.B.
EGAN,
M. L. M O R S E
RESULTS
Ki~etics of @take. The rate of uptake of various carbohydrates was determined as a function of concentration and the results are plotted in Fig. I. It can be seen that the rates of uptake of lactose, maltose, ~-methylglucoside and glucose saturate at higher carbohydrate concentrations, results compatible with either enzyme or adsorption kinetics. In contrast to tile study of lactose, maltose, and c~-methylglucoside uptake, in the glucose uptake experiment the substrate under transport can be metabolized with the ultimate yield of ~4CO2. In scoring uptake by the accumulation of counts within the cells, the possible loss of counts as ~4CO2 had to be considered in the interpretation of the plateau in Curve I of Fig. I(d). Therefore the evolution of 14CO., was determined with increasing concentration of ~I-l~C_~glucose taken up/Io min/g cell dry weight that yields the observed counts of BauCOa. This curve shows saturation kinetics with the plateau commencing at 4" I0-5 M, the parallel of Curve I. The true curve~of the rate of glucose uptake vs. concentration is therefore the sum of Curves I and 2, which itself is compatible with saturation kinetics.
104I
/
/
._c E
/."
O
E
/-
&
/O
0) c~
c
/ •
./ .......
./ i
10-5
t
e/°
, ,ht,,I
i
10-4
....
lit]
10-3
i
t t,HN]
10-2
External concentraUon(M)
I
I 1~1113J
10-~
F i g . 2. R a t e of s u c r o s e u p t a k e b y cat ".+ a s a f u n c t i o n of c o n c e n t r a t i o n . s u c r o s e u p t a k e w a s s t u d i e d w i t h a 6 7 / t g / m l s u s p e n s i o n of B S 5 o o 2 ( s u e ) .
Uniformly
l~C-labeled
Sucrose uptake was, however, dramatically different. It was impossible to saturate the system, and the rate of uptake continued to rise with increasing sucrose concentration over a concentration range of IO ~ M to lO-1 M. The results have been plotted on a log-log scale (Fig. 2), and from the gradient of the resulting line, one can calculate that the rate of uptake is proportional to the o.83 power of the initial extracellular concentration. The plot on linear axes would not therefore be linear but concave towards the concentration axis. B i o c h i m . B i o p h y s . A c t a , 1±2 ( I 9 6 6 ) 6 3 - 7 3
CARBOHYDRATE
TRANSPORT
67
IN S . aure~ts
The experiment was carried out with BS 5o02, a sucrase-less mutant. If all the the counts accumulated were indicative of free sucrose within the cell, then, at an extracellular concentration of lO-1 M, the intracellular concentration after IO rain would be 3.6 M. This is equivalent to i . I g of sucrose per 0.3 g cell dry weight (1.2 g cell wet weight). Competition between carbohydrates during uptake. The demonstration of saturation kinetics suggested the possibility of an enzymatic step in the transport of carbohydrates. The specificity of carbohydrate transport in S. aureus was therefore investigated. The rate of uptake of a particular carbohydrate was determined in the presence of a second carbohydrate at a Ioo-fold greater concentration, and compared to the rate of uptake in the absence of the second carbohydrate. TABLE
I
THE UPTAKE OF CERTAIN CARBOHYDRATES IN THE PRESENCE OF A SECOND CARBOHYDRATE AT A HIGHER CONCENTRATION T h e r a d i o a c t i v i t y a c c u m u l a t e d b y t h e b a c t e r i a i n t h e a b s e n c e of a s e c o n d c a r b o h y d r a t e is t a k e n a s t h e s t a n d a r d u p t a k e ( i o o % ) a n d t h e a c c u m u l a t i o n s i n t h e p r e s e n c e of t h e s e c o n d c a r b o h y d r a t e a r e c a l c u l a t e d a s t h e p e r c e n t a g e o f t h i s v a l u e . Ceils w e r e i n d u c e d f o r l a c t o s e u p t a k e s t u d i e s , b u t n o t f o r t h e s t u d y of g a l a c t o s e a n d l a c t o s e i n h i b i t i o n of t h e u p t a k e of o t h e r c a r b o h y d r a t e s .
Second carbohydrate (2.o'1o 4 M )
o~-Methylglucoside (z 4"IO -6 M )
None a-Methylglucoside Glucose Lactose Maltose Sucrose Fructose Galactose Mannitol
ioo 22 7 116 iio 80 161 14o 93
Uptake of 14C-labeled carbohydrate Glucose (2. 4 • Io -6 M )
Lactose (3.3" Io-6 M )
Maltose (2.z. i o -e M )
Sucrose (3.9" lO-6 M )
IOO 143 19 lO 3 lO 4 lO 4 lO9 ioo 82
ioo 90 86 7 iOl IO8 IOO lO 7 lO 7
IOO 12o 25 Ioo 14 3° 92 98 92
ioo lO8 18 97 61 64 79 lO6 97
As seen in Table I, 29 of 35 different carbohydrate combinations showed less than 20 % inhibition and, in general therefore, the uptake of one carbohydrate is relatively insensitive to the presence of a second carbohydrate, indicating that carbohydrate transport is a relatively specific process. The exceptions to this include glucose inhibition of the uptake of the other carbohydrates except possibly lactose, and maltose and sucrose, which retard the uptake of one another. The possible inhibitory effect of lactose on the uptake of carbohydrates by cells themselves induced to transport lactose, was also investigated (Table II). It was found that with cells previously induced in galactose, the uptake of c,-methylglucoside, maltose, sucrose, but not of glucose, was markedly inhibited by the presence of lactose. This same inhibitory effect of lactose was obtained wiLh induced cells that could metabolize the carbohydrate whose uptake was under study. Lactose therefore inhibits the uptake of other carbohydrates only when lactose itself is being transported. Whether the induced state per se is inhibitory to the transport of the other carbohydrates cannot be stated at this time. Biochim. Biophys. Acta, 112 ( 1 9 6 6 ) 6 3 - 7 3
68
j.B. EGAN, M, L. MORSE
T A B L E II UPTAKE
0F
CERTAIN
CARBOHYDRATI~S
IN
THE
PRESENCE
OF
LACTOSE
The radioactivity accumulated by the bacteria in the absence of a second carbohydrate is taken as the standard uptake (ioo %), and the accumulations in the presence of the second carbohydrate are calculated as the percentage of this value. The cells, when induced, had been previously grown in 0.2 % galaetose. Culture used
Uptake of 14C-labeled carbohydrate in the presence of 2. zo -a .,~I lactose o:-7Vlethylglucoside (L 4 • ZO-~ 3/I)
BS 2902 (lac-mal-)-non-induced BS 5002 (suc-)-non-induced BS 2902 (lac mal-)-induced B S 5002 (suc-)-induced BS 56oi (lac+mal+suc+)-induced
I26
Glucose (2.4" 7.0--6 igI)
Sucrose
(2.2" IO 6 J~I)
(3.9" ±o-S M)
lO 3
ioo
- -
18
Maltose
-
lO8
37 27
31
- -
13
- -
-
97 -39
-
C o u n t e r f l o w e x p e r i m e n t s . C o u n t e r f l o w is e x h i b i t e d w h e n t h e u p t a k e of c a r b o h y d r a t e B i n t o a cell d i s p l a c e s c a r b o h y d r a t e A p r e v i o u s l y a c c u m u l a t e d w i t h i n t h e celF. T h i s p h e n o m e n o n h a s b e e n c o n s i d e r e d as e v i d e n c e for t h e e x i s t e n c e of a m o b i l e m e m b r a n e c a r r i e r , c o m m o n t o t h e t r a n s p o r t of c a r b o h y d r a t e s A a n d B G-11. D e m o n s t r a t i o n s of c o u n t e r f l o w w e r e a t t e m p t e d w i t h p a i r s of c a r b o h y d r a t e s in t h i s s t a p h y l o c o c c a l s y s t e m . A cell s u s p e n s i o n in m i n i m a l s u c c i n a t e m e d i u m p l u s 25 t~g c h l o r a m p h e n i c o l / m l w a s p e r m i t t e d t o a c c u m u l a t e l ~ C - l a b e l e d c a r b o h y d r a t e A, and then unlabeled carbohydrate B was added at a concentration approximately I o o - f o l d t h e i n i t i a l c a r b o h y d r a t e A c o n c e n t r a t i o n . T h e r e s u l t s of t h e q u a n t i t a t i v e data s have been summarized and appear in Table Ill.
TABLE III DISPLACEMENT
OF
ACCUMULATED
CARBOHYDRATE
~Al B Y
CARBOHYDRATE
t~
In the table: + , indicates counterflow; --, its absence; + / - - , doubtful (less t h a n 2o o/o/ e f t l u x of carbohydrate A) ; o, combination not tested. The table summarizes the data recorded in EGANs. Carbohydrate B
c~-Methylglucoside Glucose Lactose Maltose Sucrose
Carbohydrate A o~-Methylglucoside
Lactose
7Vialtose
Sucrose
+ + o ---
o + + + / -+ / --
o + + + +
o + / + / -+/ + / --
T h e r e s u l t s i n d i c a t e t h e e x i s t e n c e of c o u n t e r f l o w w i t h c e r t a i n c a r b o h y d r a t e c o m b i n a t i o n s . I n o t h e r c a s e s t h e r e w a s n o n e . T h e a b s e n c e of c o u n t e r f l o w i n t h e s e c a s e s c a n b e e x p l a i n e d s a t i s f a c t o r i l y a f t e r c o n s i d e r a t i o n of t h e i n t r a c e l l u l a r s t a t e of t h e a c c u m u l a t e d c a r b o h y d r a t e , a n d t h e r e s u l t s will b e r e c o n s i d e r e d i n t h e DISCUSSION with the relevant data from experiments in the next section. Biochim. Biophys. Acta, 112 (1966) 63-73
CARBOHYDRATE TRANSPORT IN S. aureus
69
The intracellular state of the accumulated carbohydrates. In the study of the carbohydrate uptake, m u t a n t s of S. aureus, defective in the first enzyme of the p a t h w a y of the carbohydrates' ultilization, have been used. Thus BS 22Ol (lac-) and its derivatives are fi-galactosidaseless, BS 5002 (suc-) is sucraseless, and BS 2902 (mal-) is maltaseless. One would expect therefore that these carbohydrates, being nonmetabolizable, would accumulate as the free carbohydrate within the respective m u t a n t cells. A similar situation would be expected for the structural analogue, ~-methylglucoside. Such m u t a n t cells were permitted to accumulate the respective 14C-labeled carbohydrates, and the state of the accumulated material was examined by chromatography of the debris plus extract after treatment of the cells with 5 % butanol, using sodium acetate-ethanol as the developing solvent. Representative tracings appear in Fig. 3.
LACTOSE Fig. 3. T h e i n t r a c e l l u l a r s t a t e of [14C]lactose a c c u m u l a t e d d u r i n g t r a n s p o r t a n d i t s c ha s e b y u n l a b e l e d c a r b o h y d r a t e . T h e cells were s u s p e n d e d in o. 4 ml 1% p e p t o n e a n d lO -4 M 14C-labeled c a r b o h y d r a t e a d d e d . A f t e r i h a t 37 °, t h e s u s p e n s i o n w a s c e n t r i f u g e d a n d r e s u s p e n d e d in o. 4 ml of 1% p e p t o n e . The s u s p e n s i o n w a s d i v i d e d i n t o 2 × o.2-ml lots, o.o2 ml of w a t e r a d d e d t o one, a n d o.o2 m l of lO-1 M u n l a b e l e d c a r b o h y d r a t e to t h e second. A f t e r 2o m i n a t 37 °, o.oi m l n-butanol was added and the suspension chromatographed with the sodium acetate-ethanol s o l v e n t . T h e left c h r o m a t o g r a m is t h e i n t r a c e l l u l a r s t a t e p r i o r t o chase, t h e r i g h t , p o s t chase. T h e c h r o m a t o g r a p h i c l o c a t i o n of t h e p a r e n t c o m p o u n d is i n d i c a t e d b y a n arrow.
In lactose (Fig. 3a) and ~-methylglucoside uptake the radioactivity accumulated within the cell was found in the torm of an unknown compound, compound X*, that traveled as a discrete peak on the chromatogram more slowly than the carbohydrates themselves. In maltose and sucrose uptake (not shown), a large percentage of the radioactivity accumulated as the free carbohydrate, but there also was a peak of intermediate RE that seemed analogous to the peak of compound X in the chromatograms after lactose and a-methylglueoside uptake. In addition some radioactivity appeared in the debris at the origin with both sucrose and maltose. The peaks of intermediate RE values were investigated further. In the case of lactose uptake, the formation of compound X needs the integrity of the cell, as addition of the radioactive lactose after the cells had been treated with butanol did not yield the compound. Compound X is soluble in 5 % trichloroacetic acid and insoluble in chloroform. Acid hydrolysis (I N HC1, boiled for 2, 4, 6, 8 min) partially degraded the compound X to release radioactivity moving with the RE of glucose, but little, if any, free lactose was released. * T h e g e n e r a l use of t h e t e r m c o m p o u n d X for t h e a c c u m u l a t i o n of di ffe re nt c a r b o h y d r a t e s i m p l i e s t h a t , in each case, t h e c a r b o h y d r a t e a p p e a r s in a s i m i l a r l y d e r i v e d form. T h e r e is no e v i d e n c e for such a n i m p l i c a t i o n , a n d t h e g e n e r a l t e r m c o m p o u n d X is us e d s ol e l y for t h e s a k e of discussion.
Biochim. Biophys. Acta, 112 (1966) 63-73
7°
J . B . EGAN, M . L . MORSE
W h e n a chase of unlabeled lactose was added to a suspension of cells containing c o m p o u n d X, more than 5o % of the radioactivity m o v e d from the RE of the comp o u n d X to t h a t of free lactose (Fig. 3b). Once again the integrity of the cell was necessary to effect this chase, as cells containing 14C-compound X, but treated with butanol before the addition of unlabeled lactose, showed no shift of the radioactivity to the lactose peak. The radioactivity in the c o m p o u n d X for ~-methylglucoside uptake was completely chased with unlabeled e-methylglucoside to the RE of free c~-methylglucoside. Comparable results, but less dramatic, were seen in similar experiments with maltose and sucrose uptake. In the case of sucrose uptake, it was further shown t h a t unlabeled maltose chased radioactivity from the c o m p o u n d X peak to the free sucrose peak. DISCUSSION
The absence of o-nitrophenylgalactoside diffusion and active transport in the car- m u t a n t 4 is compatible with the postulate t h a t the m e m b r a n e carrier is defective in this m u t a n t . The transport of carbohydrates in car + strains has been investigated to test the postulate of carbohydrate-specific permeases and a carbohydrate c o m m o n carrier, as outlined in the INTRODUCTION. The specific saturable step in carbohydrate transport. The uptake of ~-methylglucoside, glucose, lactose and maltose b y care cells satisfies Michaelis-Menten kinetics, which could reflect saturation of either an enzyme (the permease) or of an adsorbing site (the carrier). The respective Michaelis constants related to saturation can be calculated from L i n e w e a v e r - B u r k plots of the data presented in Fig. I. T h e y are for lactose, K i n = 5"1o -6 M; for ~-methylglucoside, K m z 2 . I o - ~ M ; and for maltose, K i n - i. i o -~ M. If the saturable step in transport were also the step c o m m o n to the transport of carbohydrates, then one can estimate the inhibition (assumed competitive) expected for the u p t a k e of one carbohydrate in the presence of an excess of a second carbohydrate. These calculations are presented in Table IV along with the actually observed values. It is clear from a comparison of the expected and observed inhibition values t h a t the saturable step in transport is not a step c o m m o n to all the tested carbohydrates but is rather a step specific for each. In contrast to the uptake of ~-methylglucoside, glucose, lactose and maltose, the uptake of sucrose does not show saturation kinetics, and the initial rate of uptake continues to increase with increasing sucrose concentration over a five-log range of concentrations. Common step in carbohydrate transport. Evidence for a c o m m o n step in carboh y d r a t e transport comes from three sources: (I) the loss b y m u t a t i o n of the ability to transport as least 8 carbohydrates ; (2) competitive uptake studies ; and (3) counterflow experiments. Genetic evidence, indicating a c o m m o n step distinct from the specific (presumably permease) step has been presented in the previous publications in this series1, 4. Additional evidence for the existence of a c o m m o n carrier comes from the results of competition experiments. It was observed that, while lactose did not interfere with the uptake of other carbohydrates in non-induced cells, it did inhibit uptake when the cells had been previously induced with galactose. This finding Biochim. Biophys. Acla, 112 (1966) 63 73
CARBOHYDRATE TRANSPORT IN S. aureus
71
TABLE IV COMPARISON SATURABLE
OF
TIIE
EXPECTED
COMPONENT
Uptake o/
COMMON
AND TO
OBSERVED THE
In presence of
INHIBITION
TRANSPORT
Kin*
OF
OF
CARBOHYDRATE
Calculated
2" 1 0 - 4 M
rate**
FOR
A
Inhibition Expected Observed
(%)
Lactose 3.3" lO-6 M
UPTAKE
CARBOHYDRATES
(%)
Maltose ~-Methylglucoside
5" I O - 6 M i. lO_4 M 2. 5- lO-5 IV[
o.182 V 0.069 V
55 83
Maltose 2.2. io 6 M
c~-Methylglucoside
I. lo -4 M 2. 5" lO-5 M
o.o215 V 0.0024 V
-89
--20
~-Methylglucoside 1.4- lO-8 M
-Maltose
2.5" Io -5 M I. Io 4 M
o.o531 V o.o183 V
-66
--IO
0.40
V
--
-
I IO
* The Miehaelis constants Km were calculated from the Lineweaver-Burk plots of the data presented in Fig. i, and the data of Table I. ** Predicted rates were calculated from v
VS/Km and vt = VS/Km where I + S/Km I + I/K~ + S/KIn
V = maximum rate; v,vt -- predicted rates; S, I = substrate and inhibitor concentrations; and Kin, K i - Michaelis constants for the substrate and inhibitor. The inhibition is assumed to be competitive.
indicates that lactose can only retard the uptake of another carbohydrate if lactose itself is being transported. The lactose inhibition observed could then be due either to competition for limited amounts of common carrier, or to the counterflow by lactose of other carbohydrates as soon as the latter is accumulated. The second possibility was examined by studying the uptake of a carbohydrate by ceils that could metabolize the carbohydrate, so making it unavailable for counterflow by lactose. However, no moderation of the inhibition by lactose was observed under these conditions, which lends support for the existence of a common carrier in limited amounts. T h e c o u n t e r f l o w e x p e r i m e n t s also p r o v i d e e v i d e n c e for a c o m m o n s t e p in t r a n s p o r t b u t t h e r e s u l t s a r e b e t t e r u n d e r s t o o d if t h e i n t r a c e l l u l a r s t a t e of t h e a c c u m u l a t e d c o m p o u n d s is c o n s i d e r e d . C h r o m a t o g r a p h i c a n a l y s i s of t h e c e l l u l a r c o n t e n t s a f t e r a c c u m u l a t i o n of t h e v a r i o u s c a r b o h y d r a t e s r e v e a l e d t h e p r e s e n c e of n e w c o m p o u n d s , g e n e r a l l y t e r m e d c o m p o u n d X , w h i c h in e a c h c a s e m o v e d a t v a l u e s i n t e r m e d i a t e r e l a t i v e t o t h e R F of t h e f r e e c a r b o h y d r a t e s . L a c t o s e a n d c ~ - m e t h y l g l u c o s i d e a c c u m u l a t e m a i n l y i n t h e f o r m of t h e i r X - d e r i v a t i v e s , w h i l e s u c r o s e a n d m a l t o s e a c c u m u l a t e as free c a r b o h y d r a t e , as t h e X - d e r i v a t i v e , a n d as a c h r o m a t o g r a p h i c a l l y i m m o b i l e c o m p o n e n t . S i m i l a r o b s e r v a t i o n s h a v e b e e n m a d e in o t h e r b a c t e r i a l s y s t e m s : g a l a c t o s e , ~- a n d f i - m e t h y l g l u c o s i d e a c c u m u l a t e as t h e free c a r b o h y d r a t e a n d t h e 6 - p h o s p h o r i c a c i d e s t e r in Eseherichia coli12,13; a n d m a l t o s e a c c u m u l a t e s as f r e e m a l t o s e a n d t w o o t h e r c o m p o n e n t s in E. col# 4. T h e a c c u m u l a t i o n of c a r b o h y d r a t e s in t h e f o r m of d e r i v a t i v e s is of i m p o r t a n c e t o t h e i n t e r p r e t a t i o n of s e v e r a l r e s u l t s . (a) The counterflow experiments. I t w a s o b s e r v e d t h a t w h i l e l a c t o s e c a n d i s p l a c e accumulated maltose, maltose cannot displace accumulated lactose. A similar lack
Biochim. Biophys. Acta, 112 (1966) 63-73
72
J.B.
EGAN,
M. L. M O R S E
of reciprocity was observed with certain other pairs of carboydrates. This situation probably results from differences in the chemical states of the accumulated material, and from the superposition of other equilibria with new orders of specificity. Maltose accumulates in the cell as free maltose, c o m p o u n d X, plus an immobile component. Lactose presumably can only displace free maltose, while sucrose, maltose and glucose m a y also release [14Clmaltose from c o m p o u n d X (Table III). With lactose accumulation, there is little or no free lactose within the cell but mostly lactose-compound X. While unlabeled lactose can release [HC~lactose from the c o m p o u n d X form, maltose is incapable of doing so (Table III). Similar reasoning can be used to explain the absence of lactose counterflow b y sucrose, and the absence of ~-methylglucoside counterflow b y maltose and sucrose. With sucrose counterflow by other carbohydrates a larger outflow of [14C~sucrose was expected than was observed. However, at the low extracellular concentration of E14C~sucrose used in the counterflow experiments (4' IO-8 M) it is considered t h a t most of the accumulated radioactivity has been lost, irreversibly, to the chromatographically immobile form s . This possibility is supported b y the fact t h a t unlabeled sucrose itself displaces Fl4C~sucrose from the cells no better than the other carbohydrates. TABLE
V
INTRACELLULAR ACCUMULATION OF CARBOHYDRATES T h e i n t r a c e l l u l a r c o n c e n t r a t i o n of e a c h s u b s t r a t e w a s c a l c u l a t e d f r o m t h e d a t a a p p e a r i n g in F i g . i a , b, c, d, of EGAN AND ~V[oRSE 1 a s s u m i n g t h a t a l l c o u n t s r e p r e s e n t u n a l t e r e d s u b s t r a t e w i t h i n t h e cell. T h e c o u n t i n g e f f i c i e n c y of t h e s c i n t i l l a t i o n c o u n t e r f o r l a c w a s a p p r o x i m a t e l y 5o°.o. S. aurevts h a s b e e n s h o w n t o c o n t a i n 7 5 % w a t e r b y w e i g h t (AKANO 15) a n d f o r t h e c a l c u l a t i o n s i t w a s a s s u m e d t h a t i g cell d r y w e i g h t is e q u i v a l e n t t o 3 m l o f i n t r a c e l l u l a r w a t e r . T h e l a s t c o l u n m g i v e s t h e r a t i o of i n t r a - t o e x t r a c e l l u l a r c o n c e n t r a t i o n . Car + strain
BS BS t3S t3S
2902 (lac) 2902 (real-) 5002 (suc) 56Ol
Carbohydrate
Lactose Maltose Sucrose c~-Methylglucoside
Extraeellular concentration
I~ztracelhdar conce~tratio~z
Factor
(M)
(M)
1.2- IO -~ 2. 3. l o -5 5 . 6 . IO 6
8 . 6 . lO -2 1.6- IO 2 2 . 9 ' I o -3
7200 700 520
1.5" 1 ° - 5
5-5" IO-S
37 °
(b) The active transport of carbolgvdrates in this bacterium. (See Table V). I n the case of maltose and sucrose a large fraction of the accumulated radioactivity is in the free carbohydrate, and maltose and sucrose are, therefore, being transported against a concentration gradient. With c~-methylglucoside uptake, the a m o u n t of free carbohydrate within the cell is smaller, but sufficient to give a considerable ratio of intra- to extracellular concentration, again suggesting active transport. Lactose, however, appears to be present within the cell solely as c o m p o u n d X, and therefore it cannot be considered to enter against a concentration gradient. (c) The anomalous uptake of sucrose. If all the radioactivity accumulated is indicative of free sucrose within the sucraseless cell then, after IO rain uptake from io 1 M sucrose, the intracellular sucrose concentration is 3.6 M. Although the present results indicate that "I14~Csucrose '' activity appears in two other peaks on the chromat o g r a m beside t h a t of sucrose, it was conservatively estimated t h a t the tree sucrose, B i o c h i m . ]Biophys. A c t a , 112 ( i 9 6 6 ) 63 73
CARBOHYDRATE TRANSPORT IN S. aureus
73
present would be 1.2 M under these circumstances s. Therefore, at lO -1 M, sucrose is being taken up against a concentration gradient, and the uptake of sucrose cannot be by simple diffusion. In conclusion the present evidence, which supports a specific permease-common carrier model of carbohydrate transport, further justifies our postulate that the c a r locus is concerned with the formation of a functional carrier common to the membrane transport of carbohydrates. ACKNOWLEDGEMENTS
From a thesis submitted to the University of Colorado in partial fulfillment of the requirement for the Ph. D. Degree. This investigation was supported by research grants from the National Science Foundation, G-I42IO and GB-276. Contribution No. 257 from the Department of Biophysics. REFERENCES i J. BARRY EGAN AND M. L. MORSE, Biochim. Biophys. Acta, 97 (1965) 3 lo. 2 J. BARRY EGAN AND M. L. MORSE, Abstract MCzo, Biophysical Society, 7th Ann. Meeting, New York, 1963 . 3 J. BARRY EGAN AND M. L. MORSE, Abstract, Bacteriol. Proc., (1962) 42. 4 J. BARRY EGAN AND M. L. MORSE, Biochim. Biophys. Acta, lO9 (1965) 172. 5 A. L. KocH, Biochim. Biophys. Acta, 79 (1964) 177. 6 W. WILBRANDT AND T. ROSENBERG, Pharmacol. Revs., 13 (1961) lO 9. 7 T. ROSENBERG AND W. WILBRANDT, J. GeE. Physiol., 41 (1957) 289. 8 J. BARRY EGAN, Genetic and Biochemical Evidence for a Common Step in Carbohydrate Transport in Staphylococcus aureus, Doctoral Dissertation, University of Colorado, Denver, Colo., 1964. 9 T. ROSENBERG AND W*. WILBRANDT, Exptl. Cell Res., 27 (1962) Ioo. IO T. ROSENBERG AND W. WILBRANDT, J. Theoret. Biol., 5 (1963) 288. I I W. WILBRANDT, Ann. Rev. Physiol., 25 (1963) 6Ol. 12 D. ROGERS AND S.-H. Y u , J, Bacteriol., 84 (1962) 877. 13 H. HAGIHARA, T. H. WILSON ANn E. C. C. LIN, Biochim. Biophys. Acta, 78 (1963) 505 . 14 I-I. \VIESMEYER AND M. COHN, Biochim. Biophys. Acta, 39 (196o) 44 o. 15 R. AKANO, t~fyoto Furitsu Ika Daigahu Zasshi, i o (1934) 464 .
Biochim. Biophys. Acta, 112 (1966) 63-73