APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1986, p. 825-831 Vol. 51, No.

4
0099-2240/86/040825-07$02.00/0
Copyright © 1986, American Society for Microbiology

Transport and Metabolism of Lactose, Glucose, and Galactose in

Homofermentative Lactobacilli
MALCOLM W. HICKEY,'t ALAN J. HILLIER,2* AND G. RICHARD JAGO2
Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria 3052,1 and Dairy Research
Laboratory, Division of Food Research, Commonwealth Scientific and Industrial Research Organisation, Highett,
Victoria 3190,2 Australia
Received 14 August 1985/Accepted 30 December 1985

A number of species of lactobacilli were examined for their ability to ferment both the glucose and galactose
moieties of lactose. Lactobacillus helveticus strains metabolized both the glucose and galactose moieties,
whereas L. bulgpricus, L. lactis,iand L. acidophilus strains metabolized only the glucose moiety and released
galactose into the growth medium. All four species tested contained 3-galactosidase activity, and no significant
phospho-3-galactosidase actiyity was observed. L. bulgaricus and L. helveticus had a phosphoenolpyruvate
(PEP):glucose phosphotransferase system for the uptake of glucose, but no evidence for a PEP:lactose
phosphotransferase or PEP,galactose phosphotransferase system was obtained.

Lactobacilli are used extensively in the dairy industry for
the manufacture of Bulgarian buttermilk, yogurt, Kefir,
Koumiss, and Swiss, Emmental, and Italian cheese (13);
they are present in Cheddar cheese mostly as contaminants
(22).
The lactobacilli produce a greater range of end products
than do the lactic acid (group N) streptococci (traditional
starters for Cheddar cheese manufacture) and appear to be
less susceptible to attack by bacteriophages (14). The greater
diversity of metabolic activities within the lactobacilli sug-
gested that some strains might exist which possess all of the
desirable characteristics of the group N streptococci, as well
as greater phage resistance and the ability to grow and
produce acid at higher temperatures than do the group N
streptococci. The last characteristic would allow the use of
these organisms as starters in Cheddar cheese manufacturing
procedures which require a higher-than-normal cooking tem-
perature (42°C versus 37°C; e.g., the short method of Czulak
et al. [5]). Streptococcus thermophilus, the organism cur-
rently used in these procedures, ferments only the glucose
moiety of lactose, resulting in the accumulation of galactose
in the cheese (32). This could be undesirable because (i) the
fermentation of galactose by group N streptococci is more
heterolactic than that of glucose or lactose and (ii) the
residual galactose could change the microflora of the cheese
during cheese maturation.
Lactic acid bacteria contain two systems for the transport
of lactose into the cell: a phosphoenolpyruvate (PEP):lac-
tose phosphotransferase system and a lactose permease
system. Group N streptococci possess a PEP:sugar
phosphotransferase system for the transport of glucose,
galactose, and lactose into the cell (16, 25-28, 30, 31). The
sugars are translocated through the cell membrane to yield
glucose 6-phosphate, galactose 6-phosphate, and lactose
phosphate, respectively. The lactose phosphate is cleaved
by phospho-p-D-galactosidase to yield glucose and galactose
6-phosphate. Glucose is further metabolized through the
Embden-Meyerhof-Parnas (glycolytic) pathway, whereas
galactose 6-phosphate is metabolized through the D-tagatose
*
Corresponding author.
t Present address: Gilbert Chandler Institute of Dairy Technol-
ogy, Department of Agriculture, Werribee, Victoria 3030, Australia.
825
6-phosphate pathway to form dihydroxyacetone phosphate
and glyceraldehyde 3-phosphate, which are further metabo-
lized by the enzymes of the glycolytic pathway. Some S.
lactis strains have both P-galactosidase and phospho-p-
galactosidase activities (8, 9) which indicate, respectively,
the presence of a lactose permease system and a lactose
phosphotransferase system for the transport of lactose into
the cell. S. lactis can also transport galactose and glucose
into the cell by an ATP-driven permease system (28).
In contrast, the mode of transport of sugars, particularly
lactose, in the lactobacilli is less clearly defined. Romano et
al. (20) showed that the homofermentative organisms
Lactobacillus casei and L. plantarum, which utilize the
glycolytic pathway, exhibited PEP:glucose phosphotransfer-
ase and'glucokinase activities, whereas the heterofermenta-
tive organisms L. brevis and L. buchneri, which utilize the
pentose phosphoketolase pathway, had only glucokinase
activity. The presence of both lactose and galactose phos-
photransferase systems in L. casei is well established (2, 3),
and a gene encoding the enzyme phospho-p-galactosidase
has been cloned from L. casei plasmid DNA (15).
Premi et al. (18) and Hemme et al. (10) reported that both
phospho-p-galactosidase activity and P-galactosidase activ-
ity were present in several species of lactobacilli (including
L. bulgaricus and L. helveticus). This suggested that the
uptake and dissimilation of lactose may occur by two routes:
(i) a PEP:phosphotransferase system followed by phospho-
P-galactosidase activity and (ii) a permease-type system
followed by P-galactosidase activity. The aim of the present
investigation was to study the utilization and transport of
lactose, glucose, and galactose by lactobacilli and to deter-
mine the nature and properties of the enzymes involved in
the hydrolysis of lactose in these organisms.
MATERIALS AND METHODS
Bacteria. (i) Growth conditions. The lactobacilli used in this
investigation were L. bulgaricus 1243, 1373, 1489, and 1978;
L. helveticus 30, 261, 384, 766, 1829, and YB1; L. lactis 270;
and L. acidophilus 1748. L. helveticus YB1 was isolated from
yogurt culture YB (a mixed culture of L. helveticus and S.
thermophilus) obtained from the Commonwealth Scientific
and Industrial Research Organisation, Dairy Research
826 HICKEY ET AL.
Laboratory, Highett, Victoria, Australia, and all other
organisms were obtained from the National Collection of
Dairy Organisms, National Institute for Research in Dairying,
Reading, England.
Stock cultures were maintained at 5°C in sterile skim milk
containing littmus and calcium carbonate and were subcul-
tured at 3-month intervals. The cultures in use were subcul-
tured daily into sterile skim milk and inoculated, as required,
into MRS broth (7) containing the appropriate carbohydrate
(2%, wt/vol), which was autoclaved separately before addi-
tion to the growth medium. L. helveticus YB1, which did not
grow in MRS broth, was grown in M17 broth (24). All
bacteria were grown at 37°C. When large quantities of cells
were required for the preparation of cell extracts, the growth
medium was maintained at pH 6.2 by the addition of 10 M
NaOH, with the additions controlled by a magnetic valve
(Radiometer MNV1) connected to a titrator (Radiometer
TTT11b). The cells were harvested by centrifugation at 4,000
x g for 10 min at 4°C in a Sorvall RC2-B refrigerated
centrifuge, washed twice, and suspended in 0.9% (wt/vol)
NaCl for the preparation of cell extracts or 0.1 M KH2PO4
(adjusted to the appropriate pH with 1.0 M NaOH) for the
uptake of sugars and sugar analogs.
(ii) Preparation of cell extracts. Cell extracts were prepared
as described previously (11).
(iii) Toluene or detergent treatment of bacterial suspensions.
Bacterial cells were rendered permeable to extracellular
solutes to assay enzyme activities in situ. For studies of
3-galactosidase and phospho-p-galactosidase, the cells were
treated with Triton X-100 as described previously (17). For
studies of PEP:sugar phosphotransferase activity and
ATP:sugar kinase activity, which required concentrated cell
suspensions, toluene-acetone was used to increase the per-
meability of the cells (4).
(iv) Measurement of cell growth. Cell densities in the broth
media were determined by direct measurement of the A650 in
a Zeiss PMQII spectrophotometer.
Assays of enzyme activities. (i) j8-galactosidase and
phospho-,-galactosidase. P-Galactosidase and phospho-p-
galactosidase activities were estimated by measuring the rate
of hydrolysis of o-nitrophenyl-,-D-galactopyranoside
(ONPG) and its phosphorylated derivative o-nitrophenyl-p-
D-galactopyranoside 6-phosphate (ONPG-6-P), respectively.
For cell extracts, the reaction mixture (2.5 ml) contained 240
,umol of triethanolamine hydrochloride (pH 7.5), 10 ,umol of
either ONPG or ONPG-6-P, and 0.1 ml of cell extract. For
cells rendered permeable with Triton X-100, the reaction
mixture (1.0 ml) contained 60 ,umol of triethanolamine hy-
drochloride (pH 7.5), 4 ,umol of ONPG, and 20 ,ul of
permeabilized cells. The reactions were stopped by the
addition of an equal volume of 0.5 M Na2CO3. The amounts
of ONPG and ONPG-6-P hydrolyzed were determined by
measuring the A420 and using the molar extinction coefficient
for o-nitrophenol at pH 10.2 (E420) of 2.13 x 104 (6).
(ii) Alkaline phosphatase. Alkaline phosphatase activity
was determined by measuring the rate of hydrolysis of
p-nitrophenyl phosphate (1).
(iii) Glucokinase. Glucokinase (ATP:D-glucose 6-
phosphotransferase [EC 2.7.1.2]) activity was measured
either spectrophotometrically or by the ATP-dependent
phosphorylation of D-[U-'4C]glucose or 2-deoxy-D-[1-
14C]glucose ([14C]2DG).
In the spectrophotometric assay, the assay mixture (2.12
ml) contained 140 ,umol of sodium phosphate (pH 7.8), 8
,umol of MgSO4, 2.6 ,umol of NADP, 2.6 ,umol of ATP, 10
,umol of glucose or 2DG, 0.01 ml of glucose 6-phosphate
APPL. ENVIRON. MICROBIOL.
dehydrogenase (180 U/ml; Sigma Chemical Co.), and 0.01 ml
of cell extract (approximately 50 ,ug of protein). Initial
reaction rates were estimated from the change in the A340
immediately after the addition of the cell extract. One unit of
enzyme activity phosphorylates 1.0 xmol of glucose or 2DG
per min at 37°C. Specific activity was defined as the number
of glucokinase units per milligram of protein.
Glucokinase activity was also measured by the ATP-
dependent phosphorylation of D-[U-14C]glucose or [14C]2DG
by a modification of the method of Richey and Lin (19), in
which the phosphorylated products were adsorbed to DEAE
paper. The reaction mixture (0.5 ml) contained 30 jxmol of
Tris hydrochloride or KH2PO4 (pH 7.2), 7 ,umol of ATP, 0.01
,mol of MgCl2, 5 ,umol of NaF (where indicated), 0.6 ,umol
of D-[U-_4C]glucose or [14C]2DG (0.2 ,uCi/,umol), and 0.1 ml
of a toluene-treated cell suspension or of cell extract. A
control mixture without ATP was used to determine the
amount of endogenous phosphorylation of each labeled
substrate. Cell suspensions or cell extracts were preincu-
bated in buffer for 10 min at 37°C to reduce endogenous
levels of carbohydrate, and the reaction was started by the
addition of labeled substrate. Samples (50 ,ul) were removed
at regular time intervals and applied to Whatman DE 81 filter
paper (diameter, 2.5 cm) on a vacuum filter. The reaction
was stopped immediately by passing 10 ml of 80% (wt/vol)
ethanol through the filter. The filters were washed three
times with 10 ml of H20, dried, and placed into scintillation
vials for counting. The amount of endogenous phosphoryla-
tion of each labeled substrate (determined in the absence of
ATP) was subtracted from the corresponding amount of
phosphorylation in the presence of ATP to give the amount
of ATP-dependent phosphorylation.
(iv) Galactokinase. Galactokinase activity was measured
by the ATP-dependent phosphorylation of D-[U-14C]galac-
tose. The reaction mixture was that described above for
glucokinase activity, except that D-[U-14C]galactose re-
placed the D-[U-_4C]glucose or [14C]2DG.
(v) PEP:sugar phosphotransferase. PEP:sugar phospho-
transferase activities were measured by the PEP-dependent
phosphorylation of the labeled substrates [14C]2DG, [methyl-
14C]1-thio-3-D-galactopyranoside ([14C]TMG), or D-[U-
14C]galactose. The assay conditions were those described
above for glucokinase activity, except the PEP (7 pumol)
replaced ATP and toluene-treated cells were used as the
source of the enzyme.
Uptake of sugars and sugar analogs by starved cells. The
uptake of sugars and sugar analogs by starved cells was
determined as follows. The reaction mixture (5.0 ml) con-
tained 400 ,umol of KH2PO4 (at the appropriate pH), 5 ,mol
of either [14C]2DG, [14C]TMG, methyl-a-D-[U-14C]gluco-
pyranoside, D-[U-14C]glucose, D-glucose-[1-14C]lactose, or
D-[U-_4C]galactose (0.2 ,uCi/,umol), 50 ,umol of glucose
(where indicated), and 10 mg (dry weight) of cells. Bacterial
cell suspensions were preincubated with 0.1 M phosphate
buffer (at the appropriate pH) for 10 min at 37°C to remove
any endogenous energy sources. The reaction was started by
the addition of a 14C-labeled sugar or sugar analog, and
glucose, when included, was added 5 min before the addition
of the labeled compound. Samples (0.5 ml) were taken at
intervals, and the cells were harvested on membrane (nitro-
cellulose) filters (pore size, 0.45 ,um; diameter, 2.5 cm;
Millipore Corp.) under vacuum. After the cells were washed
three times (within 30 s of sampling) at room temperature
with 5 ml of the incubation buffer, the membrane filters were
partially dried by air drawn through the vacuum filter appa-
ratus and then completely dried under an infrared heat lamp
VOL. 51, 1986 SUGAR TRANSPORT AND METABOLISM IN LACTOBACILLI 827

(150 W) before they were placed in scintillation vials for TABLE 2. Utilization of lactose and galactose by L. bulgaricus,
counting. L. lactis, and L. acidophilus grown on lintiting lactose'
In competition experiments, a mixture of a "4C-labeled Amt of carbohydrate utilized or produced
substrate and an unlabeled potential inhibitor, equilibrated at (I,mol/ml) with:
37°C, was added at zero time, and uptake was compared
with that in the controls, which did not contain a potential Species and Limiting lactose Limiting lactose (4.0
strain (4.6 mM) tnM) plusmM)
galactose
inhibitor. mM) ~~~~(20
In kinetic studies, the initial rate of sugar or sugar analog Lactose Galactose Lactose Galactose
accumulation (in nanomoles per minute per milligram [dry utilized released utilized utilizedb
weight] of cells) was determined after 30 s of incubation. L. bulgaricus
The radioactivity in samples on solid supports (DE 81 1243 4.6 1.0 3.6 22.5
paper or nitrocellulose filters) was counted in 10 ml of a 1373 4.6 4.9 3.5 0.4
scintillation mixture containing 4.0 g of PPO (2,5- 1489 4.6 4.5 4.0 0.5
diphenyloxazole) and 0.1 g of dimethyl POPOP [1,4-bis(5- 1978 4.6 4.6 4.0 0.2
phenyloxazolyl)benzene] in 1 liter of a toluene-Triton X-100
mixture (2:1, vol/vol). Vials were counted in an LKB-Wallac L. lactis 270 4.6 1.1 3.8 9.5
liquid scintillation spectrometer (model 1215 Rackbeta) for
10 min on solid supports with 75% efficiency. L. acidophilus 4.6 4.8 3.9 <0.1
Utilization of carbohydrates and control of ,-galactosidase 1748
and phospho-o-galactosidase activities. Carbohydrate utiliza- aCells were grown in MRS broth, containing carbohydrate(s) at the
tion and the control of ,-galactosidase and phospho-,- specified concentration(s), for 72 h at 37°C. The lactose and galactose
contents of the medium were determined as described in Materials and
galactosidase activities were studied during cell growth at Methods.
37°C in MRS broth (M17 broth for strain YB1) containing 1% b The total available galactose in the growth medium was the galactose
(wt/vol) carbohydrate (glucose, galactose, or lactose). The added (20 mM) plus the galactose derived from the lactose utilized.
organism to be studied was inoculated (a 16-h culture at 10%,
vol/vol) into broth and incubated until the bacterial cells
reached the exponential phase of growth, at which point a
different carbohydrate was added to give a final concentra- centrifugation, stored at -20°C until assayed. Glucose,
tion of approximately 1% (wt/vol). The carbohydrate con- galactose, and lactose concentrations were estimated by
centration in the medium and the growth and P-galactosidase using kits prepared by Boehringer Mannheim Biochemicals.
activity of the organism were determined at intervals (ii) Protein. Protein content was estimated by the Lowry
throughout the incubation period. method. The protein content of whole-cell suspensions (0.1
Analytical methods. (i) Carbohydrates. The carbohydrate ml) was determined after extraction with 1 ml of 1 M NaOH
content of the medium was determined after the cells were at 100°C for 15 min and neutralization with 1 M HCl.
removed by centrifugation at 3,000 x g for 10 min at 4°C.
The supernatant was deproteinized by the addition of 0.66 N RESULTS
perchloric acid (1:1), neutralized with 2 N KOH, and, after Utilization of sugars. In the presence of excess lactose, L.
helveticus strains metabolized the galactose moiety of lac-
tose, whereas L. bulgaricus, L. lactis, and L. acidophilus
strains released galactose into the growth medium (Table 1).
TABLE 1. Utilization of the glucose and galactose moieties of No glucose was detected in the medium after growth of the
lactose by lactobacilli grown on lactose" organisms, and for L. bulgaricus, L. lactis, and L. aci-
Amt of lactose Amt of galactose dophilus, approximately 1 mol of galactose was released into
Sugar utilization Species and utilized released the medium for each mole of lactose utilized. When L.
(,umol/ml) (,umol/ml)
bulgaricus, L. lactis, and L. acidophilus strains (which did
Glucose but not L. bulgaricus not metabolize the galactose moiety of lactose when grown
galactose 1243 28.1 26.5 in the presence of excess lactose) were grown in a medium
1373 33.4 33.6 containing limiting lactose, only two strains (L. bulgaricus
1489 27.9 27.1 1243 and L. lactis 270) were able to metabolize the galactose
1978 34.8 33.0 moiety of lactose (Table 2). These two strains also utilized
L. lactis 270 20.2 19.6 significant amounts of galactose when galactose was added
to a growth medium containing limiting lactose (Table 2).
L. acidophilus 12.2 12.3 ,I-Galactosidase and phospho-f8-galactosidase activities.
1748 Cell extracts of a number of species of lactobacilli were
assayed for P-galactosidase and phospho-p-galactosidase
activities. The activities of lactose-grown and glucose-grown
Glucose and L. helveticus S. faecium SD1 cells were included for comparison. This
galactose 30 16.8 1.2 organism has a lactose phosphotransferase system which is
261 10.2 1.5 fully repressed by growth on glucose (M. J. Coventry, Ph.D.
384 9.7 0.3
766 13.6 1.8 thesis, University of Melbourne, Parkville, Victoria, Austra-
1829 11.1 0.5 lia). Under the conditions used in this investigation, all of the
YB1 18.4 0.3 lactobacilli tested had 3-galactosidase activity, but none of
the species had significant phospho-3-galactosidase activity
"Cells were grown for 24 h at 37°C in MRS broth containing 42 mM (Table 3). The low levels of phospho-3-galactosidase activity
(nonlimiting) lactose. Strain YB1 was grown in M17 broth. The lactose and
galactose contents of the medium were determined as described in Materials observed could have been due to the hydrolysis of ONPG-
and Methods. 6-P to ONPG by alkaline phosphatase and the subsequent
828 HICKEY ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 3. 1-Galactosidase, phospho-p-galactosidase, and analogs 2DG and a-methylglucoside (21). However, neither
alkaline phosphatase activities in cell extracts of lactobacillia L. bulgaricus nor L. helveticus transported a-methylglu-
Activityb of: coside (results not shown), and therefore only 2DG was used
Species and strain Phospho-p- Alkaline to study the uptake and phosphorylation stages of glucose
Galactosidase galactosidase phosphatase transport.
(i) Uptake of the glucose analog 2DG. Resting cells of L.
L. bulgaricus helveticus 766 and 1829 accumulated [14C]2DG in the ab-
1243 367.8 1.8 2.1 sence of an exogenous energy source, and maximum uptake
1373 358.6 0.8 1.0
1489 396.3 0.3 0.4 occurred within 2 min of the addition of the substrate (Fig.
2). Thereafter, an efflux of labeled material from the cells
L. helveticus took place. A similar efflux from S. lactis cells has been
766 558.1 4.0 8.4 observed (31) and is probably due to the intracellular hydro-
1829 467.8 4.4 7.2 lysis of 2DG-6-phosphate followed by the expulsion of 2DG
YB1 415.1 0.8 2.4 (29). In the absence of a fermentable sugar, the intracellular
PEP potential is exhausted and 2DG uptake is prevented. L.
L. lactis 270 287.4 0.2 0.3 bulgaricus 1373, which grew poorly on glucose, was unable
L. acidophilus 1748 242.9 2.4 0.9
to accumulate 2DG, whereas L. bulgaricus 1243 and 1489
accumulated 2DG more slowly than did the L. helveticus
S. faecium SD1 47.0 303.0 strains (Fig. 2). The rapid accumulation of a sugar analog in
(lactose grown) the absence of an added energy source is typical of a
phosphotransferase system (26) and suggests that both L.
S. faecium SD1 2.0 12.0 bulgaricus and L. helveticus possess a glucose phospho-
(glucose grown) transferase system.
a Cells were grown in MRS broth (M17 broth for strain YB1) containing 2% (ii) Phosphorylation of 2DG and glucose. If L. bulgaricus
(wt/vol) lactose; the cells were harvested and cell extracts prepared as and L. helveticus do possess a glucose phosphotransferase
described in Materials and Methods. P-Galactosidase, phospho-P-galacto- system, as suggested by the uptake studies, they should also
sidase, and alkaline phosphatase activities were assayed as described in have PEP:glucose phosphotransferase activity. The PEP:glu-
Materials and Methods.
b Nanomoles of chromogenic substrate hydrolyzed per minute per milli- cose phosphotransferase activities of toluene-treated L.
gram of protein. bulgaricus and L. helveticus cells are presented in Table 4.
The activity was always greater in cells grown on glucose
than in those grown on lactose, and the activities of the L.
bulgaricus strains were greater than those of the L.
hydrolysis of ONPG by P-galactosidase. The levels of alka- helveticus strains. ATP was not able to substitute for PEP as
line phosphatase activity in the cell extracts were found to be the phosphate donor in the L. bulgaricus strains, but some
of the same order as that of the levels of apparent phospho- activity with ATP was observed in the L. helveticus strains.
,-galactosidase activity for all of the strains tested (Table 3). The fact that ATP stimulated the phosphorylation of 2DG
The ,-galactosidase activity in cell extracts, prepared from in toluene-treated L. helveticus cells suggested that the
cells grown on lactose, was reduced by approximately 95% glucokinase in this species may be able to phosphorylate
(results not shown) when the cells were grown on glucose or 2DG. This was confirmed when it was shown by spectro-
galactose. photometric assay that 2DG was a substrate for glucokinase
Induction and repression of 3-galactosidase activity. The in L. helveticus 1829 but not in L. bulgaricus 1243. The
effects of various sugars on the induction and repression of possibility existed, therefore, that at least some of the
P-galactosidase in L. helveticus 766 are shown in Fig. 1. The phosphorylation of 2DG by PEP in toluene-treated L.
results are typical of those for most strains of L. bulgaricus helveticus cells (Table 4) could have been a result of the
and L. helveticus tested, although some minor strain differ- formation of ATP by pyruvate kinase (equation 1) and the
ences were observed. subsequent glucokinase-catalyzed phosphorylation of 2DG
Low levels of P-galactosidase activity were observed by ATP (equation 2).
during the growth of L. helveticus on glucose, and these
levels were unchanged by the addition of galactose to the PEP + ADP -- ATP + pyruvate (1)
growth medium. However, there was a 10-fold increase in 2DG + ATP -* 2-deoxy-6-phosphoglucose + ADP (2)
the rate of ,-galactosidase synthesis after the addition of
lactose to the growth medium (Fig. la). This increase If this was the case, the phosphorylation of 2DG by PEP
occurred even when glucose was still present in excess. would be expected to occur in cell extracts because both
The ,-galactosidase activity of cells grown on lactose was pyruvate kinase (data not shown) and glucokinase (Table 2)
much higher than that of cells grown on glucose (Fig. la and are active in cell extracts. However, cell extracts of L.
b). The addition of glucose or galactose to cells growing on helveticus 1829 did not carry out the PEP-dependent phos-
lactose caused an immediate repression of 3-galactosidase phorylation of 2DG. (PEP:glucose phosphotransferase activ-
synthesis, the repression being slightly greater with glucose ity was destroyed in the preparation of cell extracts.)
than with galactose (Fig. lb). Mechanism of transport for lactose. (i) Uptake of the lactose
Cells growing on galactose showed moderate levels of analog TMG. L. bulgaricus 1243 and L. helveticus 1829 took
13-galactosidase activity, and the levels increased markedly up only low levels of [14C]TMG compared with the levels of
after the addition of lactose to the medium (Fig. lc). The uptake of 2DG. The uptake of TMG by both organisms was
addition of glucose to cells growing on galactose repressed stimulated by the presence of glucose, suggesting that the
,-galactosidase synthesis. uptake was dependent on an energy source, and the uptake
Mechanism of transport for glucose. Escherichia coli has appeared to follow Michaelis-Menten kinetics in both the
separate transport mechanisms for the uptake of the glucose presence and absence of glucose (data not shown). The
VOL. 51, 1986 SUGAR TRANSPORT AND METABOLISM IN LACTOBACILLI 829

0.4 0.6 0.8 1.0

CELL GROWTH (OD650)
FIG. 1. Induction and repression of P-galactosidase synthesis in L. helveticus 766. L. helveticus 766 was inoculated (a 16-h culture at 10%,
vol/vol) into MRS broth containing glucose (a), lactose (b), or galactose (c). When the cells reached the exponential phase of growth, an
additional sugar was added as indicated to give a final concentration of 50 mM for glucose and galactose and 25 mM for lactose. Samples were
taken at intervals, the A6S was measured, and bacterial extracts were assayed for P-galactosidase activity as described in Materials and
Methods. (a) Symbols: 0, glucose; A, glucose plus galactose; O, glucose plus lactose. (b) Symbols: 0, lactose plus glucose; A, lactose plus
galactose; O, lactose. (c) Symbols: 0, galactose plus glucose; A, galactose; 0, galactose plus lactose.

apparent Km values for TMG of 2.5 mM (strain 1243) and 5.0
mM (strain 1829) were not changed by the presence of
glucose, but the Vma, (6.3 nmol/min per mg of cells for strain
1243 and 4.3 nmol/min per mg of cells for strain 1829)
increased by 93 and 53%, respectively, when glucose was
present. Thus, glucose increased the rate of uptake but did
not affect the affinity of the carrier for TMG. This was
consistent with the presence of a permease-type system for
lactose transport in these organisms.
(ii) Phosphorylation of TMG. Phosphorylation of
[14C]TMG was not observed when toluene-treated cells or
cell extracts of lactose-grown L. bulgaricus 1243 and 1489
and L. helveticus 766 and 1829 were incubated with either
PEP or ATP. Phosphorylation of [14C]lactose under the same
conditions could not be evaluated because of the presence of
P-galactosidase activity (Table 3). This was consistent with
the absence of a PEP:phosphotransferase system for the
uptake of lactose in these organisms.
Mechanism of transport for galactose. (i) Phosphorylation
of galactose. No PEP:dependent phosphorylation of D-[U-
14C]galactose was observed with cell extracts or toluene-
treated cells of L. helveticus 766 and 1829 grown on ga-
lactose (Table 5). This suggested that galactose was not
transported by a PEP:phosphotransferase system in these
organisms. L. bulgaricus 1243 did not grow sufficiently well
on galactose to be assayed.
(ii) Galactokinase activity. Because lactose was not trans-
ported by the PEP:phosphotransferase system (with the
subsequent formation of galactose 6-phosphate), it would be
necessary for lactose-grown cells to possess galactokinase
activity (galactose + ATP -* galactose 1-phosphate + ADP)
to phosphorylate the galactose moiety of lactose. Significant
levels of galactokinase activity were detected (Table 5 ) in L.
helveticus 766 and 1829 and in L. bulgaricus 1243 (the only
L. bulgaricus strain which was able to utilize the galactose
moiety of lactose). L. bulgaricus 1489, grown on glucose or
lactose, did not possess galactokinase activity. The results
suggested that L. helveticus 1829 and L. bulgaricus 1243
had a constitutive galactokinase but that the enzyme in
L. helveticus 766 was induced in the presence of galac-
tose.
Effect of glucose and glucose analogs on the uptake of lactose
and lactose analogs. The presence of glucose in the growth
medium excludes the uptake of other sugars by a number of
organisms (21). To determine whether glucose excluded the
uptake of lactose by L. bulgaricus 1243 and L. helveticus
1829, a number of competition experiments were carried out.
As expected, lactose and its analog TMG competed with
each other for uptake, and glucose stimulated the uptake of
TMG, presumably by providing the necessary membrane
potential (Fig. 2). However, 2DG inhibited TMG uptake, and
both 2DG and glucose inhibited the lactose uptake. Because
TMG did not inhibit the uptake of glucose or 2DG, even
when a 50-fold excess of TMG was present, it seems unlikely
that glucose and lactose were transported by the same
carrier. Therefore, the inhibition of the lactose uptake by
glucose and 2DG may be the result of an exclusion effect
exerted by glucose, similar to that found in E. coli (21).
DISCUSSION
The use of cheese starter bacteria which are unable to
ferment the galactose moiety of lactose may lead to atypical
cheese maturation (23, 32, 34). This inability appears to be
due, at least in part, to the accumulation and subsequent
metabolism of the galactose released from lactose (32, 34). In
some cases this problem can be overcome by including
galactose-fermenting strains in the starter (23, 34). Turner
and Martley (33) recently characterized the ability of
thermophilic lactobacilli to ferment galactose and used this
ability to differentiate L. helveticus (galactose positive) from
L. bulgaricus and L. lactis (galactose negative); their results
were confirmed in the present investigation. The difference
in the ability to ferment galactose was the only major
difference observed in the uptake and metabolism of lactose,
glucose, and galactose by L. helveticus and L. bulgaricus.
These data suggest, therefore, that in terms of galactose
utilization, L. helveticus is preferable to L. bulgaricus for
use in dairy fermentations. This is of course only one of the
830 HICKEY ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 4. Phosphorylation of [14C]2DG by PEP and ATP in

permeabilized L. bulgaricus and L. helveticus cellsa
* *
Species and strain ~~~~Growth Sp actb
Groth
substrate PEP ATP
L. bulgaricus
1243 Lactose 1.1 <0.2
Glucose 9.8 <0.2
1489 Lactose 6.3 <0.2
Glucose 18.3 <0.2
1373 Glucose 11.1 <0.2
L. helveticus
766 Lactose 1.5 0.8
Glucose 2.9 1.1
1829 Lactose 2.7 1.6
Glucose 3.5 1.3
S. faecium SD1 Lactose 3.0
Glucose 3.1
a Cells were grown for 16 h at 37°C in MRS broth containing 2% glucose or
2% lactose, harvested by centrifugation, washed, and suspended in 0.1 M
phosphate buffer (pH 7.2) as described in Materials and Methods. Cell
suspensions were preincubated for 10 min at 37°C, and the reaction was
started by the simultaneous addition of ['4C]2DG and either PEP or ATP.
Samples (50 ,ul) were taken at intervals, and the amount of 2DG phosphoryla-
tion was determined on DEAE paper as described in Materials and Methods.
b Nanomoles of [14C]2DG phosphorylated per milligram of cells per
minute.
activity was always greater than phospho-,-galactosidase
activity. In fact, the level of phospho-,-galactosidase activ-
ity observed was probably a result of the hydrolysis of
0 2 4 6 8 10 12 ONPG-6-P to ONPG by alkaline phosphatase (Table 3).. The
Time min ) ONPG formed could then be cleaved by the ,-galactosidase
FIG. 2. Uptake of [14C]2DG by resting cells of L. bulgaricus present in the cell. This suggested that lactose was trans-
1243, 1373, and 1489 and L. helveticus 766 and 1829. The strains ported into the cell via a permease system and subsequently
were grown for 16 h at 37°C in MRS broth containing 2% glucose,
harvested by centrifugation, washed, and suspended in 0.1 M
phosphate buffer as described in Materials and Methods. Whole-cell
suspensions were preincubated for 10 min at 37°C to remove TABLE 5. Phosphorylation of D-[U-14C]galactose in cell extracts
endogenous energy sources, and the reaction was started by the and toluene-treated cellsa
addition of [14C]2DG. Samples (0.5 ml) were taken at intervals, and
the amount of radioactivity incorporated by the cells was deter- Prepn Species and Growth
ATP
Sp actbPEP
mined as described in Materials and Methods. Symbols: 0, L. strain substrate
helveticus 766; 0, L. helveticus 1829; A, L. bulgaricus 1243; A, L.
bulgaricus 1489; O, L. bulgaricus 1373. Cell extracts L. bulgaricus
1243 Lactose 11.3
Glucose 12.5
factors to be considered when choosing starter bacteria for 1489 Lactose 0.0
dairy fermentations. L. helveticus strains are more proteo- Glucose 0.0
lytic than group N streptococci (12) and differ from a number L. helveticus
of other species of lactobacilli in apparently lacking the 766 Lactose 23.8
enzyme pyruvate oxidase (11). Glucose 0.0
L. casei possesses a phosphotransferase system for the Galactose 21.3 <0.2
transport of lactose (2), and a gene encoding phospho-p- 1829 Lactose 32.5
galactosidase activity has been cloned from plasmid DNA Glucose 33.8
isolated from this organism (15). The situation for L. Galactose 15.0 <0.2
bulgaricus and L. helveticus is less clear. Hemme et al. (10)
reported similar levels of ,-galactosidase and phospho-p- Toluene-treated cells L. helveticus
galactosidase in L. helveticus and L. bulgaricus, but the 766 Galactose 0.8 <0.2
units of activities were not defined. Premi et al. (18) reported 1829 Galactose 0.8 <0.2
that L. helveticus and L. bulgaricus contained both ,3-
galactosidase and phospho-p-galactosidase activities and a Cells were grown for 16 h at 37°C in MRS broth containing the specified
that the activity of ,-galactosidase was always greater than carbohydrate (2%, wt/vol), and cell extracts and toluene-treated cells were
prepared as described in Materials and Methods. The mixtures were preincu-
that of phospho-p-galactosidase. The specific activity of the bated in buffer for 10 min at 37°C, and the reaction was started by the
3-galactosidase reported by Premi et al. was approximately simultaneous addition of D-[U-14C]galactose and either ATP or PEP. Samples
1,000 times greater than that observed in this investigation. (50 pL.) were taken at intervals, and the amount of galactose phosphorylation
The reason for this is not known. was determined on DEAE paper as described in Materials and Methods.
b Nanomoles of D-[U-14Cjgalactose phosphorylated per milligram of protein
Under the conditions used in this study, P-galactosidase (cell extracts) or per milligram of cells (permeabilized cells) per minute.
VOL. 51, 1986 SUGAR TRANSPORT AND METABOLISM IN LACTOBACILLI
hydrolyzed by P-galactosidase. Although the presence of a
glucose phosphotransferase system was detected, no evi-
dence for the presence of a lactose or galactose phospho-
transferase system was observed under the conditions used
in this investigation. Furthermore, the galactokinase levels
in lactose-grown cells were always equal to or greater than
the levels in galactose-grown cells (Table 2). This further
suggests that the galactose moiety of lactose was metabo-
lized via the Leloir pathway, rather than via the tagatose
6-phosphate pathway as would be the case if lactose was
transported by a phosphotransferase system (14).
These data suggest that in L. bulgaricus and L. helveticus,
lactose and galactose are transported into the cell via a
permease system rather than by a PEP-dependent phospho-
transferase system.
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