APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2002, p. 53745378 Vol. 68, No.

11
0099-2240/02/$04.000 DOI: 10.1128/AEM.68.11.53745378.2002
Copyright 2002, American Society for Microbiology. All Rights Reserved.
Membrane-Bound ATPase Contributes to Hop Resistance of
Lactobacillus brevis
Kanta Sakamoto,
1
* H. W. van Veen,
2
Hiromi Saito,
3
Hiroshi Kobayashi,
3
and Wil N. Konings
2
Fundamental Research Laboratory, Asahi Breweries, Ltd., Moriya-shi, Ibaraki 302-0106,
1
and Faculty of
Pharmaceutical Science, Chiba University, Inage-ku, Chiba 263-8522,
3
Japan, and Department of
Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University
of Groningen, 9751NN Haren, The Netherlands
2
Received 20 February 2002/Accepted 2 August 2002
The activity of the membrane-bound H

-ATPase of the beer spoilage bacterium Lactobacillus brevis ABBC45

increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6
and increased up to fourfold when L. brevis was exposed to 666 M hop compounds. The extent of activation
depended on the concentration of hop compounds and was maximal at the highest concentration tested. The
ATPase activity was strongly inhibited by N,N-dicyclohexylcarbodiimide, a known inhibitor of F
o
F
1
-ATPase.
Western blots of membrane proteins of L. brevis with antisera raised against the - and -subunits of
F
o
F
1
-ATPase from Enterococcus hirae showed that there was increased expression of the ATPase after hop
adaptation. The expression levels, as well as the ATPase activity, decreased to the initial nonadapted levels
when the hop-adapted cells were cultured further without hop compounds. These observations strongly
indicate that proton pumping by the membrane-bound ATPase contributes considerably to the resistance of L.
brevis to hop compounds.
The hop plant, Humulus lupulus L., is used in beer fermen-
tation because of its contribution to the bitter avor of beer.
Furthermore, the use of hops in the brewing industry is pre-
ferred because hops have antibacterial activity and prevent
beer from bacterial spoilage. Hop compounds are weak acids,
which can cross cytoplasmic membranes in undissociated form
in response to the transmembrane pH gradient (16). Due to
the higher internal pH, these compounds dissociate internally,
thereby dissipating the pH gradient across the membrane. As
a result of this protonophoric action of hop compounds, the
viability of the exposed bacteria decreases (1416). Some bac-
teria, however, are able to grow in beer in spite of the presence
of hop compounds. Sami et al. (12) reported that Lactobacillus
brevis strain ABBC45 could adapt to hop treatment and de-
velop a high level of resistance to hop compounds. During the
development of hop resistance the copy number of plasmid
pRH45 harboring the horA gene increased (12). Subsequent
studies revealed that horA encodes a bacterial ATP-binding
cassette (ABC) multidrug resistance transporter (MDR) which
can extrude hop compounds from the cell membranes upon
ATP hydrolysis (11). As a result of exogenous expression of
HorA in Lactococcus lactis, the resistance of this organism to
hop compounds increased up to twofold. Microorganisms have
been found to increase the proton motive force (PMF)-gener-
ating activities in their cytoplasmic membranes when they are
confronted with a high inux of protons (20). The thermophilic
bacterium Bacillus stearothermophilus (4) increases proton-
pumping respiratory chain activities when the proton perme-
ability of its cytoplasmic membrane increases drastically at
higher temperatures. In Enterococcus hirae (formerly Strepto-
coccus faecalis) (6, 7) and Saccharomyces cerevisiae (20) the
proton-translocating ATPase levels in the membranes were
found to increase upon exposure to protonophores such as
carbonyl cyanide-m-chlorophenylhydrazone or weak acids. Ob-
viously, the main reason for this increase in proton-pumping
activities is to maintain the PMF and the internal pH at viable
levels. In view of the protonophoric activities of hop com-
pounds, it was of interest to investigate whether the hop-resis-
tant organism L. brevis would respond in a similar way to the
action of hop compounds and whether functional expression of
its proton-translocating ATPase in addition to expression of
the MDR HorA would increase. In this study, we found that
this is indeed the case and that functional expression of the
proton-translocating ATPase of L. brevis increases during
growth in the presence of hop compounds.
MATERIALS AND METHODS
Bacterial strains and growth conditions. L. brevis ABBC45 was grown anaer-
obically at 30C in MRS broth (Merck, Darmstadt, Germany). The initial pH of
the growth medium was adjusted to 5.5 with HCl. Hop resistance and expression
of HorA were achieved by growing L. brevis in the presence of hop compounds
at concentrations up to 666 M, as described previously (12). Cells grown in the
presence of 666 M hop compounds were subcultured without hop compounds
added in order to monitor the ATPase activity under these growth conditions.
Hop compounds. A concentrated isomerized hop extract (Hopsteiner GmbH,
Mainburg, Germany) was the hop compound preparation used. The iso--acid
contents were determined by high-performance liquid chromatography (10). The
concentration of hop compounds in the medium was expressed as the concen-
tration of iso--acids.
Preparation of the membrane. L. brevis was grown to the late exponential
phase in the absence and in the presence of 100 and 666 M hop compounds.
Cells of L. brevis were harvested by centrifugation at 7,000 g for 15 min and
washed twice at room temperature in 50 mM potassium HEPES (pH 7.4) con-
* Corresponding author. Mailing address: Fundamental Research
Laboratory, Asahi Breweries, Ltd., 1-21, Midori 1-chome, Moriya-shi,
Ibaraki 302-0106, Japan. Phone: 81 297 461504. Fax: 81 297 461506.
E-mail: kanta.sakamoto@asahibeer.co.jp.
Present address: Department of Pharmacology, University of
Cambridge, CB2 1QJ Cambridge, United Kingdom.
5374
taining 5 mM MgSO
4
. The cells, suspended in the same buffer, were lysed at 37C
by treatment for 1.5 h with 1 mg of lysozyme (Sigma Chemical Co., St. Louis,
Mo.) per ml and 50 g of mutanolysin (Sigma) per ml in the presence of a
cocktail of proteinase inhibitors (Complete; Boehringer, Mannheim, Germany).
After addition of DNase I (50 g/ml) and RNase (1 g/ml), the suspension was
passed three times through an ice-cold French pressure cell at 70 MPa. Unbro-
ken cells were subsequently removed by centrifugation at 7,000 g for 15 min at
room temperature. The supernatant was centrifuged at 200,000 g for 45 min at
4C, and the pellet was suspended in the same buffer. This membrane fraction
was used for ATPase assays and Western blot analysis. The concentration of the
membrane proteins was determined with a D
C
protein assay kit (Bio-Rad Lab-
oratories, Richmond, Calif.) by using bovine serum albumin as a quantitative
standard.
ATPase assay. ATPase activity was estimated from the release of inorganic
phosphate as measured by a modication of the method of Driessen et al. (5).
One or two micrograms of membrane protein was incubated at 30C for 10 min
in 50 mM potassium MES (morpholineethanesulfonic acid) buffer (usually at pH
5.5) containing 5 mM MgCl
2
. ATP (potassium salt) was added at a nal con-
centration of 2 mM to initiate the reaction. The reaction (total volume, 40 l)
was stopped after 5 min by immediately cooling the test tubes on ice. A malachite
green solution (200 l of a 0.034% solution) was added, and after 40 min color
development was terminated by adding 30 l of a citric acid solution (34%,
wt/vol). The absorbance at 660 nm was measured immediately with a multiscan
photometer (Multiskan MS; Labsystems, Vantaa, Finland). One unit of ATPase
activity was dened as the amount of enzyme that released 1 mol of inorganic
phosphate in 1 min. Calibration was done by using a series of P
i
standards
(Sigma). To determine the pH dependence of the ATPase activity, membranes
were incubated for 60 min on ice in 50 mM potassium MES buffer adjusted to
various pH values. The ATPase activity was assayed at the different pH values as
described above. To measure the effects of inhibitors on the ATPase activity, the
membranes were preincubated with N,N-dicyclohexylcarbodiimide (nal con-
centration, 0.2 mM), ortho-vanadate (nal concentration, 0.2 mM), or nitrate
(K
2
NO
3
) (nal concentration, 25 mM) for 10 min at 30C and subsequently for
60 min on ice. A membrane sample without inhibitor was used as the control.
Western blot analysis. The membrane protein of L. brevis, prepared as de-
scribed above, was solubilized in Laemmli sample buffer containing 2% sodium
dodecyl sulfate (9) and was separated by electrophoresis (20 g of protein/lane)
through a sodium dodecyl sulfate10% polyacrylamide gel by the method of
Laemmli (9). The protein bands were transferred to a polyvinylidene diuoride
lter membrane and detected with antisera raised against the F
1
complex of E.
hirae H

-ATPase (3), which can also bind with F

o
F
1
-ATPase from L. lactis (1).
Membranes from E. hirae prepared as previously described (3) were used as a
control. The antibody-bound proteins were visualized with nitroblue tetrazolium
and 5-bromo-4-chloro-3-indolylphosphate (Gibco BRL, Gaithersburg, Md.). The
intensities of the bands were measured by densitometric analysis with NIH Image
software, version 1.61 (National Institutes of Health).
RESULTS
Effect of hops on ATPase activity. Previously, it has been
demonstrated that under the conditions described above L.
brevis develops hop resistance by overexpressing the MDR
HorA (13). The cytoplasmic membranes of cells were isolated
as described in Materials and Methods, and the ATPase activ-
ities in these membranes were determined as a function of pH
at pH values ranging from 4.4 to 7.0. All membranes of L.
brevis grown in the presence of different levels of hop com-
pounds showed maximum ATPase activity at around pH 5.6
(Fig. 1). At pH 5.6 membranes from the cells adapted to 666
M hop compounds had the highest activity, which was about
fourfold greater than the ATPase activity of membranes from
nonadapted cells. The ATPase activities of membranes from
cells adapted to 100 M hop compounds were between these
extremes and were about 1.7-fold greater than the activity of
the membranes from the nonadapted cells. Once the cells
adapted to hop compounds (666 M) were subcultured in
medium without hop compounds, the ATPase activities of
their membranes decreased rapidly (Fig. 1).
Effects of inhibitors on the ATPase activity. To characterize
the type of ATPase present in the membrane of L. brevis, the
effects of several kinds of inhibitors on the ATPase activity
were studied (Fig. 2). The ATPase activities of membranes
from nonadapted cells and from cells adapted to different
concentrations of hop compounds were all signicantly inhib-
FIG. 1. pH prole of the ATPase activity in membranes of L.
brevis. The ATPase activities at pH values ranging from 4.4 to 7.0 were
measured for membranes prepared from cells grown without hop com-
pounds (W0) (E), from cells adapted to 100 M hop compounds
(W100) () or 666 M hop compounds (R666) (I), and from cells
deadapted by growth in the presence of 666 M hop compounds and
then growth for 2 days in the absence of hop compounds (R0) (). The
ATPase activity is expressed as the amount of inorganic phosphate (Pi)
released per minute per milligram of protein.
FIG. 2. Effects of inhibitors on the ATPase activity of L. brevis. The
ATPase activities of the membranes of W0, W100, and R666 (see the
legend to Fig. 1) were measured at pH 5.6 in the presence of 0.2 mM
N,N-dicyclohexylcarbodiimide (DCCD) (solid bars), 0.2 mM ortho-
vanadate (vertically striped bars), or 25 mM nitrate (horizontally
striped bars). The activity without any inhibitor was also measured as
a control (open bars).
VOL. 68, 2002 HOP RESISTANCE OF L. BREVIS 5375
ited by the F
o
F
1
-type inhibitor N,N-dicyclohexylcarbodiimide.
Moderate inhibition was observed with the P-type inhibitor
ortho-vanadate, while the V-type inhibitor K
2
NO
3
showed the
least inhibition or even activation with membranes from cells
grown in the presence of 100 M hop compounds (Fig. 2,
W100). These results correspond to the observations made for
the enterococcal F
o
F
1
-type ATPase activity, which is slightly
inhibited by ortho-vanadate and slightly enhanced by K
2
NO
3
(Y. Kakinuma, personal communication), indicating that F
o
F
1
-
type ATPase is the major ATPase in the membranes of L.
brevis.
Western blot analysis. Two strong bands were detected with
the membranes of L. brevis with the antisera against the - and
-subunits of the F
1
-ATPase complex from E. hirae, which
strongly indicates the F
o
F
1
-type nature of the ATPase of L.
brevis. The apparent molecular weights of these bands were
slightly higher than those of the - and -subunits of F
1
from
E. hirae (Fig. 3A). The intensities of both bands were higher in
membranes isolated from cells grown in the presence of higher
concentrations of hop compounds and were lower in mem-
branes from cells adapted to hop compounds (666 M) and
subcultured in medium without hop compounds (Fig. 3B.).
The intensities of both bands correlated well (correlation co-
efcient, 0.990) with the ATPase activities of the different
membranes. The rate and extent of growth in MRS broth of
hop-adapted cells were less than the rate and extent of growth
of nonadapted cells (12). Also, hop-adapted cells were smaller
than cells grown in the absence of hop compounds (data not
shown).
DISCUSSION
The beer spoilage bacterium L. brevis ABBC45 develops hop
resistance upon growth in hop-containing media (12). This
resistance was found to be mediated by the functionally ex-
pressed multidrug resistance ABC transporter HorA (11, 13).
Studies of HorA, functionally expressed in L. lactis, revealed
that HorA can excrete the lipophilic hop compounds and sev-
eral other MDR substrates from the membrane into the exter-
nal medium (11). Recently, a second PMF-driven MDR with
afnity for hop compounds has been found in L. brevis
ABBC45 lacking HorA (19). The activity of HorA and this
PMF-driven MDR results in a reduced inux of the undisso-
ciated and membrane-permeable iso--acids into the cyto-
plasm and thereby limits the antibacterial PMF-dissipating ef-
fect of hop compounds. Since L. brevis develops resistance
against rather high concentrations of hop compounds, the
question arose whether functional expression of HorA and the
PMF-driven MDR was sufcient to confer this resistance or
whether additional activities could contribute to hop resis-
tance. Anaerobic gram-positive lactic acid bacteria such as L.
brevis depend strongly on their membrane-bound H

-F
o
F
1
-
ATPase for generation of their PMF (6, 8). In this study, we
demonstrated that functional expression of a membrane-
bound H

-F
o
F
1
-ATPase increased during development of hop
resistance and decreased again when exposure to hop com-
pounds was stopped. Previously, it was demonstrated that ex-
pression of the HorA transporter increased during develop-
ment of hop resistance (13). The H

-F
o
F
1
nature of the
ATPase was conrmed by H

-F
o
F
1
-ATPase effectors and es-
pecially by immunological studies with the antisera against the
- and -subunits of H

-F
o
F
1
-ATPase from E. hirae. In accor-
dance with the observations of Kobayashi et al. (6, 7) made
with the anaerobic gram-positive bacterium E. hirae, the in-
creased functional expression of H

-F
o
F
1
-ATPase most likely
allows L. brevis to maintain a viable PMF and intracellular pH
in the presence of the protonophoric hop compounds.
The results of this study, together with those of previous
studies (11, 13, 19), indicate that L. brevis becomes resistant to
hop compounds due to the combined action of two ATP-driven
FIG. 3. Western blot analysis of membranes of L. brevis and E.
hirae with antisera against F
1
of E. hirae. (A) Membranes of L. brevis
were solubilized and separated by electrophoresis through a 10% poly-
acrylamide gel (lanes 2 to 5). For comparison the results obtained with
membranes from E. hirae are shown in lane 1. The proteins were
transferred to a polyvinylidene diuoride lter membrane and reacted
with the antisera raised against the F
1
complex of E. hirae H

-ATPase.
Lane 1, E. hirae cultured at pH 6.0; lane 2, L. brevis grown without hop
compounds (W0); lane 3, L. brevis adapted to 100 M hop compounds
(W100); lane 4, L. brevis adapted to 666 M hop compounds (R666);
lane 5, L. brevis deadapted from 666 to 0 M hop compounds (R0).
The arrows indicate the positions of the - and -subunits of H

-
ATPase from E. hirae. (B) Intensities of the lower bands of the ATPase
from L. brevis. The intensities of the bands were measured with the
NIH Image software and are expressed in arbitrary units (a.u.).
5376 SAKAMOTO ET AL. APPL. ENVIRON. MICROBIOL.
systems, the H

-ATPase and MDR pump HorA (11, 13) and

a PMF-driven MDR (19). HorA and the PMF-driven MDR
reduce the inux of the weakly acidic hop compounds by
pumping undissociated hop compounds from the membrane
environment into the external medium. The H

-ATPase com-
pensates for the PMF-dissipating and internal pH-decreasing
effects of hop compounds which have escaped the MDR ac-
tivities by pumping more protons from the cytoplasm across
the membrane. As a result of the higher expression of ATPase
and HorA and the energy dissipation by hop compounds, the
rate and extent of growth in MRS broth of hop-adapted cells
are less than the rate and extent of growth in MRS broth of
nonadapted cells (12). The various hop resistance mechanisms
(Fig. 4) provide another demonstration of the versatility of
bacteria and their capacity to develop a variety of mechanisms
to cope with toxic compounds in their environments.
ACKNOWLEGMENT
We thank Asahi Breweries, Ltd., for its support during this study.
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