FEMS Microbiology Letters 181 (1999) 109^112
Evidence of a glucose proton motive force-dependent permease
and a fructose phosphoenolpyruvate:phosphotransferase transport
system in Lactobacillus reuteri CRL 1098
Maria Pia Taranto a , Graciela Font de Valdez a , Gaspar Perez-Martinez
a
b
b;
*
Centro de Referencia para Lactobacilos (CERELA), CONICET, Chacabuco 145, San Miguel de Tucumän, 4000 Tucumän, Argentina
Departamento de Biotecnolog|¨a, Instituto de Agroqu|¨mica y Tecnolog|¨a de Alimentos (CSIC), Pol|¨gono de la Coma s/n, Apartado de correos,
P.O. Box 73, Burjasot, 46100 Valencia, Spain
Received 14 June 1999; received in revised form 1 October 1999 ; accepted 4 October 1999
Abstract
Sugar uptake and phosphoenolpyruvate phosphorylation assays have shown that the heterofermentative strain Lactobacillus
reuteri CRL 1098, of likely probiotic value, can transport D-fructose through an inducible fructose-specific phosphotransferase
system (Km 95 WM) and D-glucose mainly through a proton motive force-driven permease. These data open new perspectives
for metabolic and regulatory studies in this bacterium. ß 1999 Federation of European Microbiological Societies. Published
by Elsevier Science B.V. All rights reserved.
Keywords : Fructose ; Glucose transport; Phosphoenolpyruvate-dependent phosphotransferase system ; Lactobacillus reuteri
1. Introduction
Lactic acid bacteria (LAB) are Gram-positive bacteria which obtain energy through substrate level
phosphorylation during sugar fermentation [1]. The
group is subdivided on the basis of the pathways
used for sugar catabolism in homofermentative bacteria, that use the Embden-Meyerhof pathway
(EMP) to generate mainly lactate, and heterofermentative bacteria, which use the pentose phosphoketolase pathway (PKP) to produce a mixture of CO2 ,
ethanol, acetate and lactate [2]. There is also an im-
* Corresponding author. Tel.: +34 (96) 390 0022; Fax:
+34 (96) 363 6301; E-mail: [email protected]
portant number of species that can use both pathways, as a function of the sugar available and the
environmental conditions. The use of a speci¢c metabolic route by those microorganisms will also have
other implications, such as the sugar transport mechanism used. It has been postulated that the phosphoenolpyruvate (PEP):phosphotransferase system
(PTS) would be operative for sugar transport in bacteria that use glycolysis, but it would be absent in
those using the PKP. PEP is the phosphate donor in
the phosphoryl transfer chain leading to sugar-speci¢c PTS transport and phosphorylation. Hence, according to the catabolic pathway used, a di¡erent
pool of PEP will be generated that would condition
the transport system [1,3].
The objective of this work was to determine the
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 5 2 1 - 2
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M.P. Taranto et al. / FEMS Microbiology Letters 181 (1999) 109^112
mechanism for glucose and fructose transport in
Lactobacillus reuteri, a heterofermentative LAB generally considered as a regular colonizer of the human
and animal gastrointestinal tract [4,5]. The study of
the mechanism of sugar transport would be of great
importance to understand the production of di¡erent metabolites by these bacteria, as its primary
and secondary metabolism could be controlled
through the known PTS/CcpA signal transduction
pathway, the main catabolite repression system in
Gram-positive bacteria which also operates in lactobacilli [6,7].
2. Materials and methods
2.3. PEP-dependent PTS activity and enzymatic
assays
2.1. Bacteria and culture conditions
The strain L. reuteri CRL 1098 used in this study
was obtained from the culture collection of CERELA (San Miguel de Tucumän, Argentina). The strain
was cultured in MRS broth [8] at 37³C for 16 h
before use.
For transport experiments and measurement of
PEP-dependent PTS activity, the strain was grown
at 37³C under static conditions on fermentation
MRS (ADSA-MICRO, Scharlau, S.A., Barcelona,
Spain), supplemented with 5 g l31 of the sugar to
be assayed.
2.2. Glucose and fructose transport assays
D-[
14
(CCCP). D-[14 C]Glucose (0.6 mCi mmol31 , 11.1
MBq mmol31 ) was added, 1-ml aliquots were withdrawn at time intervals of 0, 15, 30, 60 and 120 s and
were quickly cooled by mixing with 10 ml ice-cold
PMB. The samples were then ¢ltered through 0.45Wm membrane ¢lters (HAWP 02500, Millipore) and
washed with 10 ml ice-cold PMB. Filters were dried
and immersed in scintillation cocktail (Hisafe3,
LKB-Pharmacia, Uppsala, Sweden). Radioactivity
was quanti¢ed with a scintillation counter (Wallac,
LKB-Pharmacia, Uppsala, Sweden). The same procedure was used with D-[14 C]fructose (0.3 mCi
mmol31 , 11.1 MBq mmol31 ). Labelled sugars were
purchased from Amersham (UK).
C]Glucose uptake by whole cells of L. reuteri
was estimated according to Chassy and Thompson
[9], with some modi¢cations [10]. Cells grown in 250
ml fermentation MRS with 5 g l31 glucose to an
optical density at 560 nm of 0.6^0.8 were collected
by centrifugation, washed twice with phosphate bu¡er (pH 7.4) supplemented with 1 mM MgCl2 (PMB)
and ¢nally resuspended in 5 ml PMB. Keeping the
cells on ice at this stage was essential to maintain the
PEP potential. One ml of the previous suspension,
that contained 100 Wg dry weight ml31 , was used for
each assay. Four £asks containing 15 ml of the cell
suspension in PMB with 25, 75, 125 or 250 WM glucose at 37³C were used. Another set of four £asks,
with the same sugar concentrations, contained 0.2
mM carbonyl cyanide m-chlorophenyl hydrazone
PTS activity, estimated as the consumption of PEP
in the presence of glucose and fructose, was determined according to Chassy and Thompson [11].
3. Results and discussion
Previous studies in L. reuteri CRL 1098 showed
that it can use a very limited number of sugars,
such as arabinose, ribose, galactose, glucose, fructose, maltose, lactose, melibiose, sucrose and ra¢nose
(M.P. Taranto, unpublished). However, some strains
of L. reuteri have been shown not to metabolize
fructose [12]. Sugar-speci¢c PTS transporters concomitantly translocate and phosphorylate sugar molecules with the special feature that no ATP is consumed in the process because the system uses PEP as
phosphoryl donor. The phosphate group is transferred ¢rst to elements common to all PTS systems
(EI and HPr) and ¢nally to the sugar-speci¢c PTS
elements (EII), which comprise transmembrane proteins.
L. reuteri CRL 1098 was grown on glucose or
fructose-containing medium and PTS activity was
quanti¢ed measuring PEP consumption in the presence of fructose and glucose in permeabilized cells
(Table 1). Fructose PTS activity of fructose-grown
cells was ¢ve times greater than glucose PTS activity.
No fructose PTS activity could be detected in glucose-grown cells, indicating that the system could be
FEMSLE 9100 8-11-99
M.P. Taranto et al. / FEMS Microbiology Letters 181 (1999) 109^112
Table 1
Phosphorylation of fructose and glucose by permeabilized cells of
L. reuteri CRL 1098
Cells grown on
Sugar added to the assay
Fructose
Fructose
Glucose
Glucose
a
53.2 þ 1.1
0
11.3 þ 0.7
13.1 þ 0.9
Data are expressed as the consumption of PEP in nmol min31
(mg dry weight)31 .
a
S.D.
genetically induced by fructose, as it has been reported for other microorganisms [13^15].
A small but constant PEP consumption rate in the
presence of glucose was found in both glucose and
fructose-grown cells (Table 1). This activity could be
due to a slow-rate glucose PTS transport activity.
However, previous data showed that ATP consumption by glucose-grown cells of L. reuteri CRL 1098
was signi¢cant, 38.0 þ 0.8 nmol min31 (mg dry
weight)31 (M.P. Taranto, unpublished). In the presence of glucose, an e¤cient glucokinase activity
could be consuming the ATP formed by pyruvate
kinase from the PEP present in the reaction.
The incorporation rate of D-[14 C]fructose showed
a certain saturation at 300 WM fructose (Fig. 1A),
with a Vmax of 40 nmol min31 mg31 dry weight and
111
a mean apparent Km of 95 WM (Fig. 1B). The addition of the ionophore CCCP had no e¡ect on fructose transport (Vmax 39 nmol D-[14 C]fructose min31
mg31 dry weight and Km 103 WM). Ionophores, such
as CCCP, are used to uncouple the membrane potential, thus suggesting that D-fructose transport
must be independent from the proton gradient.
These data indicate that the main functional fructose
transport system in L. reuteri CRL 1098 would be a
PTS.
The uptake rate of D-[14 C]glucose (Fig. 1A) and
the apparent Km (103 WM) were similar to those
obtained for fructose, although with a lower Vmax
(25 nmol D-[14 C]glucose min31 mg31 dry weight).
However, the glucose transport rate decreased 3.5
times in the presence of CCCP (Vmax 7 nmol D[14 C]glucose min31 mg31 dry weight and Km 330
WM), which strongly suggests that glucose translocation in L. reuteri CRL 1098 is mainly occurring
through a PMF-driven permease.
The PTS system o¡ers important advantages to
organisms carrying out anaerobic glycolysis, as
pointed out by Roseman [16]. First, the system provides a tight linkage between sugar transport and its
subsequent metabolism. Second, since the sugar is
transported and concomitantly phosphorylated, it
can directly enter catabolic and anabolic pathways,
Fig. 1. (A) Incorporation of D-[14 C]fructose (O) and D-[14 C]glucose (a) by L. reuteri CRL 1098. Closed symbols indicate the presence of
CCCP. (B) Lineweaver-Burk plot of D-[14 C]fructose (O) and D-[14 C]glucose (a) uptake. Closed symbols indicate the presence of CCCP.
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M.P. Taranto et al. / FEMS Microbiology Letters 181 (1999) 109^112
hence saving ATP and favoring growth under limited
energy supply conditions. The PTS was considered
to be only present in homofermentative LAB (fermenting glucose through the EMP pathway) but
not in heterofermentative species using the PKP
pathway [3]. Recently, Saier et al. [15] reported
that, while growing on fructose, the synthesis of a
fructose-speci¢c PTS and glycolytic enzymes is induced in Lactobacillus brevis, which allows for fructose to be metabolized via the EMP. The results
reported here support previous results in L. brevis,
as we showed that also another heterofermentative
bacterium, L. reuteri CRL 1098, could transport Dfructose via an inducible fructose PTS. In contrast,
accumulation of glucose would take place mainly
through a PMF-driven permease. These facts, and
in particular the PEP-dependent PTS activity, disclosed the great potential of L. reuteri CRL 1098,
especially regarding future studies on carbohydrate
metabolism and regulation of enzyme synthesis.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
Acknowledgements
This work was supported by the EU contract
BIO4-CT96-0380 and by a grant from CONICET
(Argentina).
[12]
[13]
References
[1] Chassy, B.M. and Murphy, C.M. (1993) Lactococcus and Lactobacillus. In : Bacillus subtilis and Other Gram-Positive Bacteria : Biochemistry, Physiology and Molecular Genetics (Sonenshein, A.L., Hoch, J.A. and Losick, R., Eds.), pp. 65^82.
American Society for Microbiology, Washington, DC.
[2] DeMoss, R.D., Bard, R.C. and Gunsalus, I.C. (1951) The
mechanism of the heterolactic fermentation : a new route of
ethanol formation. J. Bacteriol. 62, 499^511.
[3] Romano, A.H., Trifone, J.D. and Brustolon, M. (1979) Distribution of the phosphoenolpyruvate :glucose phosphotransferase system in fermentative bacteria. J. Bacteriol. 139, 93^97.
[4] Salminen, S., Deighton, M.A., Benno, Y. and Gorbach, S.L.
(1998) Lactic acid bacteria in health and disease. In : Lactic
Acid Bacteria : Microbiology and Functional Aspects (Salmi-
[14]
[15]
[16]
nen, S. and von Wright, A., Eds.), pp. 211^224. Marcel Dekker, New York.
Taranto, M.P., Medici, M., Perdigön, G., Ruiz Holgado, A.P.
and Valdez, G.F. (1998) Evidence for hypocholesterolemic
e¡ect of Lactobacillus reuteri in hypercholesterolemic mice.
J. Dairy Sci. 81, 2336^2340.
Monedero, V., Gosalbes, M.J. and Përez-Mart|¨nez, G. (1997)
Catabolite repression in Lactobacillus casei ATCC 393 is
mediated by CcpA. J. Bacteriol. 179, 6657^6664.
Lokman, B.C., Heerikhuisen, M., Leer, R.J., van den Broek,
A., Borsboom, Y., Chaillou, S., Postma, P.W. and Pouwels,
P.H.J. (1997) Regulation of expression of the Lactobacillus
pentosus xylAB operon. J. Bacteriol. 179, 5391^5397.
De Man, J.C., Rogosa, M. and Sharpe, M.E. (1960) A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23,
130^135.
Chassy, B.M. and Thompson, J. (1983) Regulation and characterization of the galactose phosphoenolpyruvate-dependentphosphotransferase system in Lactobacillus casei. J. Bacteriol.
154, 1204^1214.
Veyrat, A., Monedero, V. and Përez-Mart|¨nez, G. (1994) Glucose transport by the phosphoenolpyruvate :mannose phosphotransferase system in Lactobacillus casei ATCC 393 and
its role in carbon catabolite repression. Microbiology 140,
1141^1149.
Chassy, B.M. and Thompson, J. (1983) Regulation of lactose
phosphoenolpyruvate-dependent-phosphotransferase system
and L-D-phosphogalactosidase galactohydrolase activities in
Lactobacillus casei. J. Bacteriol. 154, 1195^1203.
Vogel, R.F., Bocker, G., Stoltz, P., Ehrmann, M., Fanta, D.,
Ludwig, W., Pot, B., Kersters, K., Schleifer, K.H. and
Hammes, W.P. (1994) Identi¢caction of lactobacilli from
sourdough and description of Lactobacillus pontis sp. nov..
Int. J. Syst. Bacteriol. 44, 223^229.
Titgemeyer, F.J., Walkenhorst, J., Reizer, J., Stuiver, M., Cui,
X. and Saier Jr., M.H. (1995) Identi¢cation and characterization of phosphoenolpyruvate:fructose phosphotransferase system in three Streptomyces species. Microbiology 141, 51^58.
Mitchell, W., Reizer, J., Herring, C., Hoischen, C. and Saier
Jr., M.H. (1995) Identi¢cation of phosphoenolpyruvate :fructose phosphotransferase system (fructose-1-phosphate forming) in Listeria monocytogenes. J. Bacteriol. 175, 2758^2761.
Saier Jr., M.H., Ye, J.J., Klinke, S. and Nino, E. (1996) Identi¢cation of an anaerobically induced phosphoenolpyruvatedependent fructose-speci¢c phosphotransferase system and
evidence for the Embden-Meyerhof glycolytic pathway in
the heterofermentative bacterium Lactobacillus brevis. J. Bacteriol. 178, 314^316.
Roseman, S. (1969) The transport of carbohydrates by a bacterial phosphotransferase system. J. Gen. Physiol. 54, 138^
184.
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