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Evidence of an active glucose uptake in Rhizobium meliloti
Ann. Inst. Pasteur/Microbiol. 1985, 136 A, 261-269
9 ELSEVIER Paris 1985
EVIDENCE
OF IN
AN
ACTIVE
RHIZOBIUM
GLUCOSE
UPTAKE
MELILOTI
by P. A. Theodoropoulos, J. P. Hornez, B. Courtois and J. C. Derieux
Laboraloire de Microbiologie, Universil~ des Sciences el Techniques de Lille, 59655 Villeneuve-d'Aseq Cedex (France)
SUMMAt/Y
Rhizobium meliloti M5N1 showed immediate uptake of glucose. Glucose accumulation was a saturable function of the glucose concentration, and km and Vmax values for uptake were determined. The glucose uptake system was found to be proteic with a turnover of about 6 h. This system was observed to be an active ATP-dependent process, since it was severely inhibited by uncouplers. Glucose analogues and others hexoses had some inhibitory effects on glucose uptake, but not polyols and lactose. KEY-WORDS; Rhizobium meliloli, Glucose; Uptake.
INTRODUCTION In Rhizobium/legume associations, the exchange of materials beetween the symbiotic partners is of utmost importance in the development and maintenance of symbiotic N2 fixation. Although hexoses were the most a b u n d a n t carbohydrates in nodules [1], their requirement for the establishment of efficient nodules is not clearly proved. Indeed, mutants of R. lri[olii [18] and R. leguminosarum [9] unable to utilize glucose and fructose were able to nodulate and fix N~, whereas a fructokinase m u t a n t of R. meliloli was reported to give inefficient nodules [7]. Growth of free-living rhizobia can occur with various carbon substrates [10]. In previous work, the decrease in pH was reported when R. meliloti was grown on glucose medium [2]. This was due to the production of 2-ketogluconic acid [13]. Its accumulation in the medium was increased when cells were grown with high concentrations of glucose [3]. Manuscri! requ le 12 octobre 1984, acceptd le 29 janvier 1985.
262
P.A.
T H E O D O R O P O U L O S AND COLL.
This p a p e r deals with t h e glucose u p t a k e s y s t e m a n d its specificity in
R. meliloti M5N1.
MATERIALS
AND
METHODS
Organism. Rhizobium meliloli M5N1 (Rm-M5N1) was isolated from a root nodule of alfalfa (Medicago saliva L.). Media and culture conditions. Bacteria were grown in RHB1 minimal medium [4]. Glucose was added at a concentration of 10 g/1. Cells were cultured in 10-1 ~ New-Brunswick ,~ fermentor vessels at 30 ~ C with pH control.
Preparation o[ cells/or uptake experimenls. Exponentially grown cells were harvested by centrifugation at 8,000 g for 20 rain at 4 ~ C, washed with T N P (tampon de non prolif~ration) [3] and resuspended in the same medium. Cells were diluted to yield a final optical density (OD) of 1 unit ( 1 0 D u n i t = l . 2 • 109 viable cells/ml) and were pre-incubated at 30 ~ C for 15 rain before being used in uptake experiments. The total assay volume was always 1 ml.
Uplake assays. 1'C-glucose (250 mCi/mmol) was obtained from the Radiochemical Centre, Amersham. Uptake was initiated by the addition of 0.1 mM l~C-glucose to the cells. Inhibitors and competitors were added 30 s prior to the radioactive solute. Samples (0.1 ml) were taken at appropriate times and were rapidly filtered through Millipore nitrocellulose filters (0.45 ~zm pore size) and washed with 2 ml of cold T N P plus 5 % NaC1. The filters were then dried under an IR lamp, placed into scintillation vials with 7.5 ml seintillant (Lipoluma) and analysed on a Beckman liquid scintillation spectrometer.
Protein determination. Protein was determined by the method of Lowry el at. [15] using bovine serum albumin as the standard.
RESULTS
AND
DISCUSSION
Nature of the glucose uptake system. Rm-M5N1 g r o w n in R H B 1 plus glucose s h o w e d an i m m e d i a t e c a p a c i t y t o t a k e up l~C-glucose (fig. 1). T h e i n c u b a t i o n of cells with c h l o r a m p h e n i c o l
Ars EDTA IR NEM 01)
-= = = =
sodium arsenate. ethylene diaminetetraacelic infrared. N-ethyl-maleimide. optical densily.
acid.
PTS ~ phosphotransferase system. R m - M 5 N I = I ~ h i z o b i u m meliloli. TNP = ~ tampon de non proliferation Vi initial rate of transport.
,,.
ACTIVE GLUCOSE UPTAKE IN R. MELILOTI
263
200"
~. 15o. E
,Z E
9-~ 1 0 0 -
r
50-
io T ime I:IG.
1.
--
Uptake of l~C-glucose by H.
(m i n ) meliloti
grown on glucose.
(200 ~g/ml) prior to the addition of 14C-glucose inhibited the uptake system (fig. 2). The maximal inhibition was observed after an incubation of 6 h. This indicated that glucose was taken up via a proteic system which had a turnover of about 6 h.
Effect of metabolic inhibilors on glucose uptake. The effects of sodium azide (NAN3), N-ethyl-maleimide (NEM), sodium lluoride (NaF), potassium cyanide (KCN), sodium arsenate (Ars) and ethylene diaminetetraacetic acid (EDTA) were examined in cells of RmM5N1 (fig. 3). Glucose uptake was completely inhibited by inhibitors of respiration, like NaN3 and KCN, at 10 and 1 mM respectively. NaF even at high concentrations of 40 mM showed no effect on uptake and, similarly, sodium arsenate at 20 mM did not markedly reduce this uptake. These data provide evidence that glucose uptake in Rm-M5N1 is an active ATP and energized, membrane-dependent process.
264
P. A. THEODOROPOULOS AND COLL. 100-
O
5 O
50-
$
10o
Pre-iucubation Fro. 2. --- E[[ecl o[ chloramphenicol
Time
(hours)
(200 Dg/ml) on glucose uptake.
Cells were pre-ineubated with ehloramphenicol (luring different times (30 min, 1 h, 2 h, 4 h, 6 h. 7 h, 8 h) before the addition of l~C-glucose. U p t a k e was measured for 5 rain. The inhibition was determined by comparing to samples w i t h o u t chloramphenicol.
Similar results for glucose uptake have been reported in R. legumino-
sarum [14] and in cowpea rhizobia [19]. In other bacteria, hexoses may be transported by another type of transport: the phosphoenol pyruvate hexose phosphotransferase system (PTS) [6]. Hexoses transported by this system are phosphorylated and uncouplers are known to stimulate rather to inhibit this phosphorylative transport [12, 17]. The considerable inhibition of glucose uptake by uncouplers like azide suggest that, in Rm-M5N1, glucose is transported as free sugar. Glucose uptake was also inhibited by the thiol reactive agent NEM present at concentrations between 3 and 5 raM, suggesting t h a t there is an essential --SH group in the carrier system. The low inhibition caused by EDTA at 2 and 5 mM indicated that divalent cations were not essential for this uptake system.
Kinetics of glucose uptake and specificitg of the glucose uptake system. Glucose transport assays using different cell density suspensions (OD =1 ; O D = 0 . 5 ; OD=0.3) were carried out to specify the cell density, for
ACTIVE GLUCOSE UPTAKE IN R. MELILOTI
265
which glucose uptake was a linear function of time. Such an uptake for at least 1 rain was obtained with a cell Suspension of OD =0.3 (fig. 4). Initial rates of transport (Vi) were derived from measurements of the radioactivity accumulated after cells (0D----0.3) incubation with 14C-glucose at concentrations from 0.5 to 10 ~M during 10, 20, 40 and 60 s. A non-linear Lineweaver-Burk plot was obtained when V i values of glucose uptake were measured in Rm-M5N1 (fig. 5). The apparent K,, and V ..... were 0.68 ~M and 38.5 nmoles/min/mg protein, respectively. The specificity of the uptake system was examined by pre-incubating Rm-M5N1 with various compounds for 30 s prior to the addition of 1'Cglucose (table I). The uptake of glucose is glucose-specific: polyols and lactose did not show any effect on this uptake; while other hexoses and organic acids had o low affinity for this system; these compounds appear to possess their awn uptake system. Fructose was a competitive inhibitor of glucose 1
Ot : NaN 3
5
mM
~o
o~5 "~, --,.0
,.,r
~-
mM
2
NaF
Ars o
1'0
mM :]o
NEM
:]o
4'o mM
E DTA
Concentrations Fro. 3. - - Effect of metabolic inhibitors on glucose uptake. Inhibitors were added 30 s prior to the addition of 14C-glucose. Uptake was measured for 5 rain. The control rate of u p t a k e was 24.5 n m o l e s / m i n / m g protein.
~- 15-
/
E
"z E
10-
5
,'o
2'0 Time
4'0
6'0
(set'ands) H. meliloti density suspensions.
Fro. 4. - - Uptake o~ 14C-glucose (3 ~ M ) by different
OD = 1 (i) ; OD = 0.5 (A) ; OD = 0.3 (e).
E E
t,
"~ E
0.1-
Fro. 5. -- Lineweaver-Burk plot of the initial rate of glucose uptake (e) and of the inhibition of glucose uptake by fructose 2 m M (A) and 4 m M (").
ACTIVE GLUCOSE UPTAKE IN R. MELILOTI TABLE
I.
--
267
Specificity of glucose uptake.
Competitors
Inhibition of uptake (%)
None Glucose 3-O-Methyl-~-D-glucopyranoside a-Methyl-D-glucoside 2-Deoxy+D-glucose Fructose Galactose Mannose Sodium gluconate Sodium succinate Lactose Sorbitol Mannitol
0 97 69 53.2 36.8 40.5 39 39.1 17.3 28.8 3 0 0
Competitors were added at the concentration of 20 mM 30 s prior to the addition of 0.1 mM 14C-glucose. Uptake was measured for 5 rain. The control rate of uptake was 24 n m o l e s / m i n / m g of protein.
uptake (fig. 2). The Ki values for 2 and 4 mM fructose were 1.5 and 3.5 raM, respectively. Glucose uptake was partially inhibited by structural analogues of glucose, such as 3-O-methyl-~-D-glucopyranoside (3-0-MeGlc), ~-methylD-glucoside-(=MeG) and 2-deoxy-D-glucose (2DG), indicating that these compounds and glucose do not have identical affinity for the carrier. Nevertheless, the uptake system has more affinity for 3-O-MeGlc than for ~MeG or 2DG; change at the C3 position does not appear to induce essential modifications in glucose stereospecificity. An active transport system for glucose has been described in Pseudomonas aeruginosa [8]. In this organism, glucose analogues such as ~MeG and 2DG were also taken up. Nevertheless, the carrier system appeared to have a much lower affinity for these analogues than for glucose [11, 16]. A multiple transport system has been described for the glucose transport in R. leguminosarum RBL1 [5]. In this organism, glucose, ~MG and 2DG were transported by two (a high-affinity and a low-affinity) transport systems. Nevertheless, glucose possesses a higher affinity than analogues for this multiple transport system. The data reported here for Bm-M5N1 provide a first examination of the glucose transport system. A more detailed analysis, especially concerning its regulation, is in progress in our laboratory.
RESUME MISE
EN
]~VIDENCE D ' U N T R A N S P O R T A C T I F D U GLUCOSE CHEZ {( R I - I I Z O B I U M M E L I L O T I ))
Rhizobium meliloti M5N1 incorpore rapidement le glucose par un syst~me de transport prot6ique ayant un (, turnover ,) d'environ 6 h. Ce
268
P. A. THEODOROPOULOS AND COLL.
syst~me est ATP-d~pendant, puisqu'il est inhib~ fortement par les d~couplants ~nerg~tiques et non par le fluorure. Les analogues structuraux du gh|cose ainsi que d'autres hexoses ont un effet inhibiteur sur le transport du glucose contrairement aux polyols et au lactose. MOTS-CLI~S : Rhizobium meliloli, Glucose; Transport.
REFERENCES
[1] ANTONIW, L. D. & SPRENT, J. J., Primary metabolites of Phaseolus vulgaris nodules. Phglochemistry, 1978, 17, 675-678. [2] COURTOIS,B., HORNEZ, J. P., COURTOIS,J. & DERIEUX, J. C., Mise en 6vidence d'une propri6t6 mdtabolique de Rhizobium meliloti utilisable pour sa classification. Ann. Microbiol. (Inst. Pasteur), 1983, 134 A, 141-147. [31 CouaTois, B., HORNEZ, J. P. & DEmEUX, J. C., Effet de la synth~se d'acide 2 cdto-gluconique sur la production d'exopolysaccharides par une souchede Rhizobium meliloti. Canad. J. Microbiol., 1979, 25, 1191-1196. [41 CounToIs, B., DEmEUX, J. C. & HOaNEZ, J. P., t~tude des polyosides de Rhizobium ~t croissance rapide. Ann. Microbiol. (Inst. Pasteur), 1975, 126 B, 3-15. [5] DE VRIES, G. E., Van BRUSSEL, A. A. N. & QUISPEr., A., Mechanism and regulation of glucose transport in Rhizobium leguminosarum. J. Bael., 1982, 149, 872-879. [6] DILLS, S. S., APPERSON,A., SCHMIDT,M. R. & SAIEa, M. H. Jr, Carbohydrate transport in bacteria. Mierobiol. Reo., 1980, 44, 385-418. [7] DUNCAN, M. J., Properties of Tn5-induced carbohydrate mutants in Rhizobium meliloti. J. gen. Mierobiol., 1981, 122, 61-67. [8] EAGON, R. G. & PHmBS, P. V. Jr, Kinetics of transport of glucose, fructose and mannitol by Pseudomonas aeruqinosa. Canad. J. Biochem., 1971, 49, 1031-1041. [91 GLENN, A. R., ARWAS, R., McKAY, I. A. & DILWORTH, M. J., Sugar metabolism and the symbiotic properties of carbohydrate mutants of Rhizobium leguminosarum. J. gen. Microbiol., 1984, 130, 239-245. [101 G~AHAM,P. H., Selective medium for growth of Rhizobium. Appl. Microbiol., 1969, 17, 769-770. [11] GuY~ON, L. F. & EAGON, R. G., Transport of glucose, gluconate and methyl ~-D-glucoside by Pseudomonas aeruginosa. J. Bad., 1974, 117, 1261-1269. [121 HA~UENAUER,R. & KEPES, A., The cycle of renewal of intracellular ~-methyl glucoside accumulated by the glucose permease of E. coll. Biochimie, 1971, 53, 99-105. [13] HOUNEZ, J. P., COURTOIS,B. & DEmEUX, J. C., Mise en 6videuce de voies de mdtabolisme diff6rentes ~ partir du glucose ou du fructose chez Rhizobium meliloti. C. R. Acad. Sei. (Paris) (Sdr. D), 1976, 283, 1559-1562. [141 HUDMAN,J. F. & GLENN, A. R., Glucose uptake by free living and bacteroids forms of Rhizobium leguminosarum. Arch. Microbiol., 1980, 128, 72-77. [15] LOWRY, O. H., ROSEBROUGM,N. J., FARR, A. L. A RANDALL, R. J., Protein measurement with the Folin phenol reagent. J. biol. Chem., 1951, 193, 265-275. [161 MUKKADA,A. J., LONG, G. L. & ROMANO, A. H., The uptake of 2-deoxy-Dglucose by Pseudomonas aeruginosa and its regulation. Biochem. J., 1973, 132, 155-162.
ACTIVE GLUCOSE UPTAKE INR. M E L I L O T I
269
[171 REIDER, E., WAGNER, F. & SCHWEIGER, M., Control of phosphoenol-pyruvate-
dependent phosphotransferase-mediated sugar transport in Escherichia coli by energization of the cell membrane. Proc. nat. Acad. Sci. (Wash.), 1979, 76, 5529-5533. [18] RONSON, C. W. & PRIMROSE, S. B., Carbohydrate metabolism in Rhizobium lri/olii: identification and symbiotic properties of mutants. J. gen. Microbiol., 1979, 112, 77-88. [19] STOWERS, M. D. & ELKAN, G. H., The transport and metabolism of glucose in cowpea rhizobia. Canad. J. Microbiol., 1983, 29, 398-406.
Ann. Inst. Pasteur/Microbiol., 136A,
n~ 2, 1985.
I~
9 ELSEVIER Paris 1985
EVIDENCE
OF IN
AN
ACTIVE
RHIZOBIUM
GLUCOSE
UPTAKE
MELILOTI
by P. A. Theodoropoulos, J. P. Hornez, B. Courtois and J. C. Derieux
Laboraloire de Microbiologie, Universil~ des Sciences el Techniques de Lille, 59655 Villeneuve-d'Aseq Cedex (France)
SUMMAt/Y
Rhizobium meliloti M5N1 showed immediate uptake of glucose. Glucose accumulation was a saturable function of the glucose concentration, and km and Vmax values for uptake were determined. The glucose uptake system was found to be proteic with a turnover of about 6 h. This system was observed to be an active ATP-dependent process, since it was severely inhibited by uncouplers. Glucose analogues and others hexoses had some inhibitory effects on glucose uptake, but not polyols and lactose. KEY-WORDS; Rhizobium meliloli, Glucose; Uptake.
INTRODUCTION In Rhizobium/legume associations, the exchange of materials beetween the symbiotic partners is of utmost importance in the development and maintenance of symbiotic N2 fixation. Although hexoses were the most a b u n d a n t carbohydrates in nodules [1], their requirement for the establishment of efficient nodules is not clearly proved. Indeed, mutants of R. lri[olii [18] and R. leguminosarum [9] unable to utilize glucose and fructose were able to nodulate and fix N~, whereas a fructokinase m u t a n t of R. meliloli was reported to give inefficient nodules [7]. Growth of free-living rhizobia can occur with various carbon substrates [10]. In previous work, the decrease in pH was reported when R. meliloti was grown on glucose medium [2]. This was due to the production of 2-ketogluconic acid [13]. Its accumulation in the medium was increased when cells were grown with high concentrations of glucose [3]. Manuscri! requ le 12 octobre 1984, acceptd le 29 janvier 1985.
262
P.A.
T H E O D O R O P O U L O S AND COLL.
This p a p e r deals with t h e glucose u p t a k e s y s t e m a n d its specificity in
R. meliloti M5N1.
MATERIALS
AND
METHODS
Organism. Rhizobium meliloli M5N1 (Rm-M5N1) was isolated from a root nodule of alfalfa (Medicago saliva L.). Media and culture conditions. Bacteria were grown in RHB1 minimal medium [4]. Glucose was added at a concentration of 10 g/1. Cells were cultured in 10-1 ~ New-Brunswick ,~ fermentor vessels at 30 ~ C with pH control.
Preparation o[ cells/or uptake experimenls. Exponentially grown cells were harvested by centrifugation at 8,000 g for 20 rain at 4 ~ C, washed with T N P (tampon de non prolif~ration) [3] and resuspended in the same medium. Cells were diluted to yield a final optical density (OD) of 1 unit ( 1 0 D u n i t = l . 2 • 109 viable cells/ml) and were pre-incubated at 30 ~ C for 15 rain before being used in uptake experiments. The total assay volume was always 1 ml.
Uplake assays. 1'C-glucose (250 mCi/mmol) was obtained from the Radiochemical Centre, Amersham. Uptake was initiated by the addition of 0.1 mM l~C-glucose to the cells. Inhibitors and competitors were added 30 s prior to the radioactive solute. Samples (0.1 ml) were taken at appropriate times and were rapidly filtered through Millipore nitrocellulose filters (0.45 ~zm pore size) and washed with 2 ml of cold T N P plus 5 % NaC1. The filters were then dried under an IR lamp, placed into scintillation vials with 7.5 ml seintillant (Lipoluma) and analysed on a Beckman liquid scintillation spectrometer.
Protein determination. Protein was determined by the method of Lowry el at. [15] using bovine serum albumin as the standard.
RESULTS
AND
DISCUSSION
Nature of the glucose uptake system. Rm-M5N1 g r o w n in R H B 1 plus glucose s h o w e d an i m m e d i a t e c a p a c i t y t o t a k e up l~C-glucose (fig. 1). T h e i n c u b a t i o n of cells with c h l o r a m p h e n i c o l
Ars EDTA IR NEM 01)
-= = = =
sodium arsenate. ethylene diaminetetraacelic infrared. N-ethyl-maleimide. optical densily.
acid.
PTS ~ phosphotransferase system. R m - M 5 N I = I ~ h i z o b i u m meliloli. TNP = ~ tampon de non proliferation Vi initial rate of transport.
,,.
ACTIVE GLUCOSE UPTAKE IN R. MELILOTI
263
200"
~. 15o. E
,Z E
9-~ 1 0 0 -
r
50-
io T ime I:IG.
1.
--
Uptake of l~C-glucose by H.
(m i n ) meliloti
grown on glucose.
(200 ~g/ml) prior to the addition of 14C-glucose inhibited the uptake system (fig. 2). The maximal inhibition was observed after an incubation of 6 h. This indicated that glucose was taken up via a proteic system which had a turnover of about 6 h.
Effect of metabolic inhibilors on glucose uptake. The effects of sodium azide (NAN3), N-ethyl-maleimide (NEM), sodium lluoride (NaF), potassium cyanide (KCN), sodium arsenate (Ars) and ethylene diaminetetraacetic acid (EDTA) were examined in cells of RmM5N1 (fig. 3). Glucose uptake was completely inhibited by inhibitors of respiration, like NaN3 and KCN, at 10 and 1 mM respectively. NaF even at high concentrations of 40 mM showed no effect on uptake and, similarly, sodium arsenate at 20 mM did not markedly reduce this uptake. These data provide evidence that glucose uptake in Rm-M5N1 is an active ATP and energized, membrane-dependent process.
264
P. A. THEODOROPOULOS AND COLL. 100-
O
5 O
50-
$
10o
Pre-iucubation Fro. 2. --- E[[ecl o[ chloramphenicol
Time
(hours)
(200 Dg/ml) on glucose uptake.
Cells were pre-ineubated with ehloramphenicol (luring different times (30 min, 1 h, 2 h, 4 h, 6 h. 7 h, 8 h) before the addition of l~C-glucose. U p t a k e was measured for 5 rain. The inhibition was determined by comparing to samples w i t h o u t chloramphenicol.
Similar results for glucose uptake have been reported in R. legumino-
sarum [14] and in cowpea rhizobia [19]. In other bacteria, hexoses may be transported by another type of transport: the phosphoenol pyruvate hexose phosphotransferase system (PTS) [6]. Hexoses transported by this system are phosphorylated and uncouplers are known to stimulate rather to inhibit this phosphorylative transport [12, 17]. The considerable inhibition of glucose uptake by uncouplers like azide suggest that, in Rm-M5N1, glucose is transported as free sugar. Glucose uptake was also inhibited by the thiol reactive agent NEM present at concentrations between 3 and 5 raM, suggesting t h a t there is an essential --SH group in the carrier system. The low inhibition caused by EDTA at 2 and 5 mM indicated that divalent cations were not essential for this uptake system.
Kinetics of glucose uptake and specificitg of the glucose uptake system. Glucose transport assays using different cell density suspensions (OD =1 ; O D = 0 . 5 ; OD=0.3) were carried out to specify the cell density, for
ACTIVE GLUCOSE UPTAKE IN R. MELILOTI
265
which glucose uptake was a linear function of time. Such an uptake for at least 1 rain was obtained with a cell Suspension of OD =0.3 (fig. 4). Initial rates of transport (Vi) were derived from measurements of the radioactivity accumulated after cells (0D----0.3) incubation with 14C-glucose at concentrations from 0.5 to 10 ~M during 10, 20, 40 and 60 s. A non-linear Lineweaver-Burk plot was obtained when V i values of glucose uptake were measured in Rm-M5N1 (fig. 5). The apparent K,, and V ..... were 0.68 ~M and 38.5 nmoles/min/mg protein, respectively. The specificity of the uptake system was examined by pre-incubating Rm-M5N1 with various compounds for 30 s prior to the addition of 1'Cglucose (table I). The uptake of glucose is glucose-specific: polyols and lactose did not show any effect on this uptake; while other hexoses and organic acids had o low affinity for this system; these compounds appear to possess their awn uptake system. Fructose was a competitive inhibitor of glucose 1
Ot : NaN 3
5
mM
~o
o~5 "~, --,.0
,.,r
~-
mM
2
NaF
Ars o
1'0
mM :]o
NEM
:]o
4'o mM
E DTA
Concentrations Fro. 3. - - Effect of metabolic inhibitors on glucose uptake. Inhibitors were added 30 s prior to the addition of 14C-glucose. Uptake was measured for 5 rain. The control rate of u p t a k e was 24.5 n m o l e s / m i n / m g protein.
~- 15-
/
E
"z E
10-
5
,'o
2'0 Time
4'0
6'0
(set'ands) H. meliloti density suspensions.
Fro. 4. - - Uptake o~ 14C-glucose (3 ~ M ) by different
OD = 1 (i) ; OD = 0.5 (A) ; OD = 0.3 (e).
E E
t,
"~ E
0.1-
Fro. 5. -- Lineweaver-Burk plot of the initial rate of glucose uptake (e) and of the inhibition of glucose uptake by fructose 2 m M (A) and 4 m M (").
ACTIVE GLUCOSE UPTAKE IN R. MELILOTI TABLE
I.
--
267
Specificity of glucose uptake.
Competitors
Inhibition of uptake (%)
None Glucose 3-O-Methyl-~-D-glucopyranoside a-Methyl-D-glucoside 2-Deoxy+D-glucose Fructose Galactose Mannose Sodium gluconate Sodium succinate Lactose Sorbitol Mannitol
0 97 69 53.2 36.8 40.5 39 39.1 17.3 28.8 3 0 0
Competitors were added at the concentration of 20 mM 30 s prior to the addition of 0.1 mM 14C-glucose. Uptake was measured for 5 rain. The control rate of uptake was 24 n m o l e s / m i n / m g of protein.
uptake (fig. 2). The Ki values for 2 and 4 mM fructose were 1.5 and 3.5 raM, respectively. Glucose uptake was partially inhibited by structural analogues of glucose, such as 3-O-methyl-~-D-glucopyranoside (3-0-MeGlc), ~-methylD-glucoside-(=MeG) and 2-deoxy-D-glucose (2DG), indicating that these compounds and glucose do not have identical affinity for the carrier. Nevertheless, the uptake system has more affinity for 3-O-MeGlc than for ~MeG or 2DG; change at the C3 position does not appear to induce essential modifications in glucose stereospecificity. An active transport system for glucose has been described in Pseudomonas aeruginosa [8]. In this organism, glucose analogues such as ~MeG and 2DG were also taken up. Nevertheless, the carrier system appeared to have a much lower affinity for these analogues than for glucose [11, 16]. A multiple transport system has been described for the glucose transport in R. leguminosarum RBL1 [5]. In this organism, glucose, ~MG and 2DG were transported by two (a high-affinity and a low-affinity) transport systems. Nevertheless, glucose possesses a higher affinity than analogues for this multiple transport system. The data reported here for Bm-M5N1 provide a first examination of the glucose transport system. A more detailed analysis, especially concerning its regulation, is in progress in our laboratory.
RESUME MISE
EN
]~VIDENCE D ' U N T R A N S P O R T A C T I F D U GLUCOSE CHEZ {( R I - I I Z O B I U M M E L I L O T I ))
Rhizobium meliloti M5N1 incorpore rapidement le glucose par un syst~me de transport prot6ique ayant un (, turnover ,) d'environ 6 h. Ce
268
P. A. THEODOROPOULOS AND COLL.
syst~me est ATP-d~pendant, puisqu'il est inhib~ fortement par les d~couplants ~nerg~tiques et non par le fluorure. Les analogues structuraux du gh|cose ainsi que d'autres hexoses ont un effet inhibiteur sur le transport du glucose contrairement aux polyols et au lactose. MOTS-CLI~S : Rhizobium meliloli, Glucose; Transport.
REFERENCES
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