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University of Groningen
Beer spoilage bacteria and hop resistance in Lactobacillus brevis
Sakamoto, Kanta
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Beer Spoilage Bacteria
and
Hop Resistance
in
Lactobacillus brevis
Kanta Sakamoto
Cover design: Mayu Uei, 2002.
Printer: Ridderprint offsetdrukkerij b.v., Ridderkerk, The Netherlands
This study was carried out at the Brewing Research and Development
Laboratory, Asahi Breweries, Ltd., Japan and at the Department of
Microbiology of the University of Groningen, The Netherlands.
RIJKSUNIVERSITEIT GRONINGEN
Beer spoilage bacteria and hop resistance
in Lactobacillus brevis
Proefschrift
ter verkrijging van het doctoraat in de
Wiskunde en Natuurwetenschappen
aan de Rijksuniversiteit Groningen
op gezag van de
Rector Magnificus, dr. F. Zwarts,
in het openbaar te verdedigen op
dinsdag 8 oktober 2002
om 13.15 uur
door
Kanta Sakamoto
geboren op 1 september 1968
te Hyogo (Japan)
Promotor: Prof. dr. W. N. Konings
Beoordelingscommissie: Prof. dr. L. Dijkhuizen
Prof. dr. A. J. M. Driessen
Prof. dr. M. Veenhuis
ISBN: 90-367-1689-6
VOORWOORD
First of all, I would like to thank Prof. Wil N. Konings very much. It is a great
honor for me to be granted the doctor’s degree from the University of Groningen
under your promotion. It was one of the most nervous moments in my life when we
met for the first time in May 1997 at your office in Haren, to discuss our study on
the horA gene. I was very glad to see that you were interested in our research,
although my English was terrible and needed to be corrected by Atsushi. I never
knew at that time that we would later have a very successful collaboration and that
you would give me the precious opportunity of the Ph. D. promotion at this
university.
I would also like to thank the reading committee for my thesis very much: Prof.
Arnold J. M. Driessen, Prof. L. Dijkhuizen, and Prof. M. Veenhuis. It is a great
honor for me to present my study to you and have it evaluated by you.
Rik, thank you very much for supervising me during the collaboration. My
curiosity for science was stimulated much more by you than it had been before.
You are not only an excellent scientist but also a nice gourmet, which was proven
during your stay in Japan.
I thank Sami-san, too. It was you that discovered this very interesting gene and
that opened my eyes to science. I can clearly remember that I was very excited to
know your study on hop-acclimatization in beer spoilage bacteria soon after I
entered Asahi Breweries. In the end I became mad for this gene and finally I got
the protein.
Abelardo, thank you very much. You were always very kind to teach me the
extraordinary complicated and sophisticated techniques for the expression,
purification and reconstitution of membrane proteins even when you were very
busy. Without you, I could never have made the collaboration successful. I enjoyed
discussing with you a lot, as well as the many “intelligent” Spanish words you
taught me. I am now convinced to get along with any Spanish guys by these words.
Piotr, my Polish brother, thank you very much. You and Marjon have always
been very kind to me. I clearly remember that your and Patricia’s eyes were full of
curiosity to me, the first Japanese for you, when we met for the first time. Owing to
you, Mayu and I know about the elegant Polish culture. How nice it is that you are
my paranimf together with Eli!
Eli, my other paranimf, you are also kind to me and patient to translate my thesis
into Dutch. Thank you very much. I will never forget that we drank a lot with fun
untill early in the morning.
Other people from the MDR group: Monique, Wim, René (thank you for the car),
Marloes, Patricia, Robbert, Irene, Marjon, Gerrit, Jacek, Margreet and Ronald, and
from the Molecular Microbiology department: Juke, Chris, Sonja, Nico, Oscar,
Carmen, Paolo, Zalan, Jessica (thank you for the printer), Bastiaan, Titia, Hein,
Andreas, Trees, Danka, Asia, Monika, Tiemen, Brutus, Bert, Gert, Anna, and all
the others, thank you very much. And Jos and the nice secretaries: Petra, Bea,
Marga, and Theresia. You are always kind to me. I always felt sorry for you to
disturb your busy job. Thank you very much.
I also thank Dr. J. Kok for the gift of pGK, Prof. H. Kobayashi and Dr. H. Saito
for the H+-ATPase, Prof. Atsushi Yokota, Dr. Y. Sasaki and Dr. T. Sasaki, for
encouraging me, Dr. Sato, Prof. Sonomoto, Dr. Nakayama and other members of
Japanese Society for Lactic Acid Bacteria for nice discussions.
I appreciate my colleagues and bosses from Asahi Breweries, Ltd: Yamashitasan, Funahashi-san, Ishibiki-san, Suzuki-san, Jibiki-san, Yamagishi-san, Hatchan,
Motoyama-san, Miyamoto-san, Okazaki-san, Eto-san, Matsui-san, Kagami-san,
Nakagawa-san, Ohtake-san, Ozaki-san, Ikeda-san, Yuuki-san, Hirono-san, Takaisan, Yoshioka-san and all the others, thank you very much for the nice discussions
and kind support. I really appreciate Asahi Breweries Ltd., to give me the chance to
study and finish my thesis at the University of Groningen.
Now, my family: Father, Mother and my sisters, thank you very much. I would
also like to thank Mayu’s father, mother and brother very much as well as my
grandmothers and all my relatives.
Finally, Mayu, my dearest lady and wife. Thank you very muuuuuch. I cannot
live without you any more. I would not be here if you had not said to me: “What a
nice chance! You shouldn’t miss it. You can always have a chance to obtain a Ph.
D. from the University of Tokyo, but not always from the University of Groningen.
If you like, you can just obtain a Ph. D. from both universities, can’t you?” when I
consulted you about the invitation letter from Wil. It made me decide to come to
the Netherlands again. And finally I become a Ph. D. How happy I am together
with you! How nice it is that you painted the cover of my thesis! I love you.
‘Never give up. Dream comes true.’
Kanta
CONTENTS
CHAPTER 1 Beer Spoilage Bacteria and Hop Resistance
1
CHAPTER 2 The Nucleotide Sequence of the 16S Ribosomal RNA
Gene of Pectinatus sp. DSM20764 and Improvement
of PCR Detection of Beer Spoilage Bacteria by the
Combined Use of Specific and Universal Primers
27
CHAPTER 3 Electrotransformation of Lactobacillus brevis
37
CHAPTER 4 A Plasmid pRH45 of Lactobacillus brevis Confers Hop
Resistance
41
CHAPTER 5 Hop Resistance in the Beer Spoilage Bacterium
Lactobacillus brevis Is Mediated by the ATP-Binding
Cassette Multidrug Transporter HorA
49
CHAPTER 6 The Membrane Bound ATPase Contributes to Hop
Resistance of Lactobacillus brevis
61
SUMMARY
71
SAMENVATTING
75
総
79
括
REFERENCES
83
LIST OF PUBLICATIONS
97
Curriculum vitae
99
CHAPTER 1
-Review-
Beer Spoilage Bacteria and Hop Resistance
Kanta Sakamoto and Wil N. Konings
This chapter was submitted to Internatinal Journal of Food Microbiology.
1.
INTRODUCTION
2. BEER SPOILAGE BACTERIA
2.1. Gram-Positive Bacteria
2.1.1. Lactobacillus
2.1.2. Pediococcus
2.1.3. Other Gram-Positive Bacteria
2.2. Gram-Negative Bacteria
2.2.1. Pectinatus
2.2.2. Megasphaera
2.2.3. Other Gram-Negative Bacteria
3. DETECTION OF BEER SPOILAGE BACTERIA
3.1. Culture Media
3.2. Identification Methods
3.3. Discrimination of Beer Spoilage Bacteria from Non-Spoilers
4. ANTIBACTERIAL ACITVITY OF HOP COMPOUNDS
4.1. History of Hop Usage for Beer
4.2. Hop Plant
4.3. Antibacterial Compounds in Hops
4.4. Antibacterial Mechanism of Hop Compounds
5. HOP RESISTANCE IN LACTIC ACID BACTERIA
5.1. Variation of Hop Resistance
5.2. Features of Hop Resistance
5.3. Mechanisms of Hop Resistance
6. BEER SPOILING ABILITY IN LACTIC ACID BACTERIA
6.1. Factors Affecting Beer Spoiling Ability
6.2. Prediction of Beer Spoilage by Lactic Acid Bacteria
1
CHAPTER 1
1. INTRODUCTION
Beer has been recognized for hundreds of years as a safe beverage. It is hard to
spoil and has a remarkable microbiological stability. The reason is that beer is an
unfavorable medium for many micro-organisms due to the presence of ethanol
(0.5-10% w/w), hop bitter compounds (approx. 17-55 ppm of iso-α-acids), the high
content of carbondioxide (approx. 0.5% w/v), the low pH (3.8-4.7), the extremely
reduced content of oxygen (<0.1 ppm) and the presence of only traces of nutritive
substances such as glucose, maltose and maltotriose. These latter carbon sources
have been substrates for brewing yeast during fermentation. As a result pathogens
such as Salmonellae typhimurium and Staphylococcus aureus do not grow or
survive in beer (Bunker, 1955).
However, in spite of these unfavorable features a few micro-organisms still
manage to grow in beer. These, so-called beer spoilage microorganisms, can cause
an increase of turbidity and unpleasant sensory changes of beer. Needless to say
that these changes can affect negatively not only the quality of final product but
also the financial gain of the brewing companies.
A number of micro-organisms have been reported to be beer spoilage microorganisms, among which both Gram-positive and Gram-negative bacteria, as well
as so-called wild yeasts. Gram-positive beer spoilage bacteria include lactic acid
bacteria belonging to the genera Lactobacillus and Pediococcus. They are
recognized as the most hazardous bacteria for breweries since these organisms are
responsible for approximately 70% of the microbial beer-spoilage incidents (Back,
1994). The second group of beer spoilage bacteria is Gram-negative bacteria of the
genera Pectinatus and Megasphaera. The roles of these strictly anaerobic bacteria
in beer spoilage have increased since the improved technology in modern
breweries has resulted in significant reduction of oxygen content in the final
products. Wild yeasts do cause less serious spoilage problems than bacteria but are
considered a serious nuisance to brewers because of the difficulty to discriminate
them from brewing yeasts.
Considerable effort has been made by many microbiologists to control microbial
contamination in beer. The most commonly used method today for detecting beer
spoilage micro-organisms in breweries is still traditional incubation on culture
media. A number of selective media have been developed since Louis Pasteur
published in 1876 ‘Études sur La Bière (Studies on beer)’. It usually takes a week
or even longer for bacteria to form visible colonies on plates or to increase the
turbidity in nutrient broths. Consequently, the products are often already released
for sale before the microbiological results become available. If a beer spoilage
micro-organism is then detected and identified in the beer product it needs to be
recalled from the market. This will cause serious commercial damages to the
brewery. Most microbiologists have focused on developing more specific and rapid
methods for the detection of beer spoilage micro-organisms than using the
2
CHAPTER 1
traditional culture methods. A number of advanced biotechnological techniques are
employed such as immunoassays with antibodies specific to beer spoilage bacteria.
Recently polymerase chain reaction (PCR) technology, targeting specific
nucleotide sequences of ribosomal RNA genes (rDNA), has been successfully
applied for the rapid identification of both beer spoilage bacteria and wild yeasts.
These methods can identify exactly the taxonomy of the micro-organism(s) found
in beer. However, not all beer spoilage bacteria can actually grow in beer. A
method is needed to determine whether the detected bacterium is capable of
growing in beer or not. Up to date the only available method is the so-called
‘forcing test’ in which the detected bacterium is re-inoculated and incubated in
beer. It usually takes one month or even longer to detect visible turbidity in the
inoculated beer, meaning that this method is not very practical. A rapid method to
predict beer spoiling ability is therefore urgently needed and the development of
such a method will be essential for understanding the nature of the beer-spoiling
ability.
Among the components of beer, hop compounds have received a lot of attention
for reason of their preservative values and their bitterness. For centuries it was
generally believed that hops protect beer from infection by most organisms,
including pathogens, but it was only in the 20th century that Shimwell (1937a,
1937b) showed that hop compounds only inhibit growth of Gram-positive bacteria
and not of Gram-negative bacteria. His findings had a great impact because many
pathogens such as Salmonella species are Gram-negative bacteria. Feature(s) such
as low pH and alcohol content undoubtedly have a negative effect on growth of
these pathogens in beer. Among Gram-positive bacteria some species of lactic acid
bacteria are less sensitive to hop compounds and are able to grow in beer. Insight in
the mechanism of resistance of lactic acid bacteria to hop compounds is crucial for
understanding their beer spoiling ability. The antibacterial activity of hop
compounds and the hop-resistance of lactic acid bacteria have extensively been
investigated. On the other hand little studies have been done on the beer spoiling
ability of Gram-negative bacteria.
This chapter reviews the currently available information about beer spoilage
bacteria, their growth and spoilage activity in beer.
3
CHAPTER 1
2. BEER SPOILAGE BACTERIA
Beer is a poor and rather hostile environment for most micro-organisms. Its
ethanol concentration ranges from 0.5 to 10% (w/w) and is usually around 4 to 5%.
These concentrations are high enough to make beer bacteriostatic or bactericidal.
Beer is usually slightly acid with pH’s ranging from pH 3.8 to 4.7, which is lower
than most bacteria can tolerate for growth. Furthermore, the high carbondioxide
concentration (approx. 0.5% w/v) and extremely low oxygen content (<0.1 ppm)
makes beer a near to anaerobic medium.
Beer also contains bitter hop compounds (approx. 17-55 ppm of iso-α-acids),
which are toxic, especially for Gram-positive bacteria. The concentrations of
nutritive substances, such as saccharides and amino acids, are very low since most
have been consumed by brewing yeasts during fermentation.
Only a few bacteria are able to grow under such inhospitable conditions and are
able to spoil beer (see Table 1). These bacteria include both Gram-positive and negative species. Gram-positive beer spoilage bacteria belong almost always to the
lactic acid bacteria. They are regarded as most harmful for brewing industry and
are the cause of most of bacterial spoilage incidents. Only a few Gram-negative
bacteria are known to cause beer spoilage. Some of these belong to the acetic acid
bacteria and have received most attention. Today these aerobic bacteria do not
present a serious problem in beer spoilage anymore, since improved brewing
technology has led to a drastic reduction of the oxygen content in beer. Instead
strictly anaerobic bacteria, typically Pectinatus spp. and Megasphaera cerevisiae,
have become serious beer spoilage bacteria.
2.1. Gram-Positive Bacteria
Almost all the beer spoilage Gram-positive bacteria belong to lactic acid
bacteria. They are a large group of species and genera of Gram-positive bacteria.
Only a few of these lactic acid bacteria are beer-spoiling organisms. Most
hazardous for the brewing industry are those belonging to the genera Lactobacillus
and Pediococcus. In the period 1980-1990, 58-88% of the microbial beer-spoilage
incidents in Germany were caused by lactobacilli and pediococci (Back et al.,
1988; Back, 1994). Also in Czech all beer-spoilage bacteria detected in the
breweries belonged to lactic acid bacteria (Hollerová and Kubizniaková, 2001).
The situation in other countries seems to be similar although for commercial
reasons little statistical information has been supplied. These lactic acid bacteria
spoil beer by producing haze or rope and cause unpleasant flavor changes such as
sourness and atypical odor.
2.1.1. Lactobacilli
The genus Lactobacillus is the largest genus of lactic acid bacteria and includes
numerous species. They are widely used in various fermentation processes,
4
CHAPTER 1
including food products such as beer, wine, yoghurt and pickles. In contrast to the
general believe that all lactobacilli can grow in beer, only a few species have been
reported to be capable of beer spoilage (Rainbow, 1981; Priest, 1987, 1996;
Jespersen and Jakobsen, 1996). Lb. brevis appears to be the most important beer
spoiling Lactobacillus species and is detected at high frequency in beer and
breweries. More than half of the bacterial incidents were caused by this species
(Back et al., 1988; Back, 1994; Hollerová and Kubizniaková, 2001). Lb. brevis is
an obligate heterofermentative bacterium. It is one of the best-studied beer spoilage
bacteria and grows optimally at 30°C and pH 4-6 and is generally resistant to hop
compounds. It is physiologically versatile and can cause various problems in beer
such as super-attenuation, due to the ability to ferment dextrins and starch
(Lawrence, 1988). The antibacterial effects of hop compounds and the
mechanism(s) responsible for hop resistance have been studied in detail in this
species (Simpson, 1991, 1993a, 1993b; Simpson and Smith, 1992; Fernandez and
Simpson, 1993; Simpson and Fernandez, 1994; Sami et al., 1997a, 1997b, 1998;
Sakamoto et al., 2001, 2002; Suzuki et al., 2002). These studies will be described
in Chapter 4, 5 and 6.
The second most important beer spoiling lactobacillus, Lb. lindneri is
responsible for 15-25% of beer-spoilage incidents (Back et al., 1988; Back, 1994).
The physiology of Lb. lindneri is very similar to that of Lb. brevis and only
recently has Lb. lindneri been recognized on the basis of its 16S rRNA gene
sequence as a phylogenetically separate species in the genus Lactobacillus (Back et
al., 1996; Anon., 1997). Lb. lindneri is highly resistant to hop compounds (Back,
1981) and grows optimally at 19-23°C (Priest, 1987; Back et al., 1996) but
survives higher thermal treatments than other lactic acid bacteria (Back et al.,
1992). All Lb. lindneri strains, tested so far, are capable of beer spoilage, while
other lactobacilli comprise both beer spoiling and non-spoiling strains (Rinck and
Wackerbauer, 1987a, 1987b; Storgårds et al., 1998). It is particularly problematic
that Lb. lindneri grows slowly on media commonly used in breweries while growth
in beer can be very rapid.
Lb. buchneri, Lb. casei, Lb. coryneformis, Lb. curvatus and Lb. plantarum are
less common beer spoiling bacteria than the two species described above (Priest,
1996). Lb buchneri resembles closely Lb. brevis, but differs in its ability to ferment
melezitose. In addition, some Lb. buchneri strains require riboflavin in their growth
medium (Sharpe, 1979; Back, 1981). Lb. casei can produce diacetyl, which gives
beer an unacceptable buttery flavor. Diacetyl appears to be more potent in that
respect than lactic acid, the major end-product of lactic acid bacteria. The threshold
value of diacetyl in beer is much lower (0.15 ppm) than of lactic acid (300 ppm)
(Hough et al., 1982). Diacetyl is also produced during normal fermentation by
yeast and too high levels of diacetyl are produced when yeast is not properly
removed from young beer (Inoue, 1981). Lb. casei is particularly problematic
5
CHAPTER 1
because its contamination in finished beer can give rise to high levels of diacetyl.
The pathway of diacetyl formation by lactic acid bacteria has been studied in detail
(Speckman and Collins, 1968, 1973; Jönsson and Pettersson, 1977).
Lb. brevisimilis (Back, 1987), Lb. malefermentans, Lb. parabuchneri (Farrow et
al., 1988), Lb. collinoides and Lb. paracasei subsp. paracasei (Hollerová and
Kubizniaková, 2001) have also been reported to be beer spoilage species.
Recently few additional newly discovered species of lactobacilli have been
added to the list of beer spoilers. According to its 16S rRNA gene sequence, a
strain called BS-1 is related to Lb. coryneformis, but its narrow fermentation
pattern (only limited to glucose, mannose and fructose) differs significantly not
only from that of Lb. coryneformis but also from other Lactobacillus spp.
(Nakakita et al., 1998). Two other novel lactobacillus species were found in
spoiled beer with significantly different taxonomical properties from those of other
Lactobacillus spp. One species, LA-2, with a 16S rRNA gene sequence 99.5%
similar to that of Lb. collinoides, has a strong beer spoiling ability. The other
species, LA-6, has a weak beer spoiling ability and did not show any significant
homology to the other Lactobacillus spp. (Funahashi et al., 1998).
2.1.2. Pediococci
Pediococci are homofermentative bacteria which grow in pairs and tetrads. They
were originally known as ‘sarcinae’ because their cell organization was confused
with that of true sarcinae. Beer spoilage, caused by cocci and characterized by acid
formation and buttery aroma of diacetyl, was therefore called ‘sarcina sickness’.
Pediococcus spp. produce rope, and extensive amounts of diacetyl like Lb. casei.
They are found at many stages in the brewing process from wort till finished beer.
Several Pediococcus spp. have been found in breweries: P. acidilactici, P.
damnosus, P. dextrinicus, P. halophilus (recently classified as Tetragenococcus
halophilus), P. inopinatus, P. parvulus and P. pentosaceus (Back, 1978; Back and
Stackebrandt, 1978). Among them is P. damnosus the most common beer spoiler. It
was responsible for more than 20% of all bacterial incidents in the period 1980-87
(Back et al., 1980, 1988) but only for 3-4% in 1992-93 (Back, 1994). The
incidence of beer spoilage by pediococci has decreased most likely as a result of
improved sanitation conditions in breweries. P. damnosus is generally resistant to
hop compounds. It is interesting that P. damnosus is commonly found in beer and
late fermentations, but seldom in pitching yeast. In contrast P. inopinatus is
frequently detected in pitching yeast but rarely in the other stages of beer
fermentations (McCaig and Weaver, 1983; Priest, 1987). P. inopinatus and P.
dextrinicus can grow in beer at pH values above 4.2 and with low concentrations of
ethanol and hop compounds (Lawrence, 1988). P. pentsauces and P. acidilactici
have never been reported to cause any defect in finished beer (Simpson and
Taguchi, 1995). The amount of diacetyl produced by pediococci varies from
6
CHAPTER 1
species to species. P. damnosus produces large amounts of diacetyl, P. inopinatus
less and P. pentsauces not at all. Brewers therefore usually pay attention only to P.
damnosus.
2.1.3. Other Gram-Positive Bacteria
In addition to Lactobacillus and Pediococcus species also a species from the
genus Microcococcus has been reported to be occasionally responsible for beer
spoilage. M. kristinae can grow in beer with low ethanol and hop compounds at pH
values above 4.5 (Back, 1981). Micrococci are usually strictly aerobic, but M.
kristinae can grow also under anaerobic condition (Lawrence and Priest, 1981). It
produces a fruity atypical aroma in beer (Back, 1981).
2.2. Gram-Negative Bacteria
Several genera of Gram-negative bacteria are known to be involved in beer
spoilage. The presence of a hydrophobic outer membrane makes Gram-negative
bacteria generally resistant to hop compounds. Aerobic acetic acid bacteria i.e.
Gluconobacter and Acetobacter spp. were well-known as beer spoilage organisms
in breweries but the role of these bacteria in beer spoilage has been reduced
significantly due to the much lower oxygen content during the brewing processes
and in packaged beer of modern breweries. Instead, the occurrence of strictly
anaerobic bacteria in beer spoilage incidents has increased. These include the
genera Pectinatus, Megaspahera, Selenomonas, Zymomonas and Zymophilus.
Especially Pectinatus and Megasphaera species cause much more serious
problems for breweries than lactobacilli and pediococci, mainly due to the
production of the offensive ‘rotten egg’ odor in finished beer.
2.2.1. Pectinatus
Pectinatus spp. are now recognized as one of the most dangerous beer spoilage
bacteria. They play a major role in 20 to 30% of bacterial incidents, mainly in nonpasteurized beer rather than in pasteurized beer (Back, 1994). Pectinatus species
were long thought to be Zymomonas spp. because of their phenotypical similarities.
The first isolate was obtained from breweries in 1971 (Lee et al., 1978) and so
were all subsequent isolates (Back et al., 1979; Haukeli, 1980; Kirchner et al.,
1980; Haikara et al., 1981; Takahashi, 1983, Soberka et al., 1988, 1989). The
natural habitat of the Pectinatus species are still unknown (Haikara, 1991). Two
species are found in this genus: P. cerevisiiphilus and P. frisingensis (Schleifer et
al., 1990). There is also one strain DSM20764 isolated from spoiled beer that
differs considerably in genotype from the two other species (Weiss, personal
communication, 1987). Its 16S rRNA gene sequence is distinctly different from
that of the other two species (Sakamoto, 1997). Pectinatus spp. are non-sporeforming motile rods with lateral flagella attached to the concave side of the cell
7
CHAPTER 1
body. They swim actively and appear X-shaped when cells are young and snakelike and longer when cells are older. They possess some features that are
characteristic for Gram-positive bacteria (Haikara et al., 1981) and are regarded as
being intermediate between Gram-positive and -negative bacteria. Growth takes
place between 15 and 40°C with an optimum around 32°C (Chelak and Ingledew,
1987), between pH 3.5 and 6 with an optimum at 4.5 (Chelak and Ingledew, 1987;
Watier et al., 1993) and in media containing up to 4.5% (w/v) of ethanol. During
growth considerable amounts of propionic and acetic acids are produced as well as
succinic and lactic acids and acetoin. Pectinatus spp. can also ferment lactic acid.
Since lactic acid is the sole source of carbon in SMMP medium (see section 3.1),
this medium is used for the selective isolation of Pectinatus spp. (and Megaspaera
spp.) (Lee, 1994). The most characteristic feature of spoilage caused by Pectinatus
spp. is extensive turbidity and an offensive ‘rotten egg’ smell brought by the
combination of various fatty acids, hydrogen sulfide and methyl mercaptan (Lee et
al., 1978, 1980; Haikara et al., 1981). This spoilage activity can cause serious
damages for breweries.
2.2.2. Megasphaera
Megasphaera has emerged in breweries along with Pectinatus and is responsible
for 3 to 7% of bacterial beer incidents (Back et al., 1988; Back, 1994). They are
non-spore-forming, nonmotile, mesophilic cocci that occur singly or in pairs and
occasionally as short chains. This genus includes two species, M. elsdeni and M.
cerevisiae. Since the first isolations in 1976 only M. cerevisiae has been blamed to
be responsible for beer spoilage (Weiss et al., 1979; Haikara and Lounatmaa, 1987;
Lee, 1994). M. cerevisiae grows between 15 to 37°C with an optimum at 28°C and
at pH values above 4.1. The growth is inhibited at ethanol concentrations above 2.8
(w/v) but is still possible up to 5.5 (w/v) (Haikara et al., 1987; Lawrence, 1988). It
is the most anaerobic species known to exist in the brewing environment (Seidel et
al., 1979). Beer spoilage caused by this organism results in a similar extreme
turbidity as Pectinatus and the production of considerable quantities of butyric acid
together with smaller amounts of acetic, isovaleric, valeric and caproic acids as
well as acetoin (Seidel et al., 1979). Like Pectinatus, the production of hydrogen
sulfide causes a fecal odor in beer (Lee, 1994), which makes this bacterium one of
the most feared organisms for brewers.
2.2.3. Other Gram-Negative Bacteria
In addition to the two genera described above, some other Gram-negative
bacteria have been found to cause problems in the brewing industry. Anaerobic
Zymomonas spp. have been found in primed beer to which sugar was added and in
ale beer. Zymomonas mobilis is an aerotolerant anaerobe and grows above pH 3.4
and at ethanol concentrations below 10% (w/v) (van Vuuren, 1996). There is no
8
CHAPTER 1
report of Zymomonas mobilis spoilage in lager beer, probably because of its
selective fermentation character (non-fermentative of maltose, maltotriose but
fermentative of glucose, fructose and sucrose). It produces high levels of
acetaldehydes and hydrogen sulfide. Also another Zymophilus spp., Z.
raffinosivorans has been reported as a beer spoiler (Schleifer et al., 1990; SeidelRüfer, 1990). The genus Zymophilus is phylogenetically close to the genus
Pectinatus. Zymophilus spp. can grow in beer, like Pectinatus spp., at pH values
above 4.3-4.6 and ethanol concentrations below 5% (w/v). Also their beer spoilage
activity is similar to that of Pectinatus spp. (Jespersen and Jakobsen, 1996).
Another Gram-negative bacterium Selenomonas lacitifex has also been reported
to play a role in certain beer spoilage incidents but this species has hardly been
studied. Historically a lot of attention of the brewing industry has been given to
aerobic Gram-negative bacteria. Acetic acid bacteria i.e. Gluconobacter and
Acetobacter used to be well-known to breweries. They convert ethanol into acetate,
which results in vinegary off-flavor of beer. For reasons explained above such
aerobes are no longer important in modern breweries.
Hafnia protea, formerly Obesumbacterium proteus, and Rahnella aquatilis,
formerly Enterobacter agglomerans, have been detected in pitching yeasts but
never in finished beer. They can retard the fermentation process. Beer produced
with yeasts contaminated with H. protea has a parsnip-like or fruity odor and flavor
(van Vuuren, 1996). Abnormally high levels of diacetyl and dimethyl sulfide were
detected in beer produced from wort contaminated by R. aquatilis (van Vuuren,
1996).
Recently a novel strictly anaerobic Gram-negative bacterium was isolated from a
brewery (Nakakita et al., 1998). It is a rod-shaped bacterium with no flagella that
can grow in beer at pH values above 4.3 and does not produce propionic acid.
Genetic and phenotipical studies indicated that this bacterium is different from
Pectinatus, Zymomonas and Selenomonas spp..
9
CHAPTER 1
Table 1. Beer Spoilage Bacteria
Gram-positive bacteria
Rod-shaped
Cocci
Lactobacillus spp.
Pediococcus spp.
Lb. brevis
P. damnosus
Lb. brevisimilis
P. dextrinicus
Lb. buchneri
P. inopinatus
Lb. casei
Lb. coryneformis
Micrococcus sp.
Lb. curvatus
M. kristinae
Lb. lindneri
Lb. malefermentans
Lb. parabuchneri
Lb. plantarum
Gram-negative bacteria
10
Rod-shaped
Pectinatus spp.
P. cerevisiiphilus
P. frisingensis
P. sp. DSM20764
Selenomonas sp.
S. lacticifex
Zymophilus sp.
Z. raffinosivorans
Cocci
Megasphaera sp.
M.cerevisiae
Zymomonas sp.
Z. mobilis
CHAPTER 1
3. DETECTION OF BEER SPOILAGE BACTERIA
We have seen above that only a limited number of bacterial species are
responsible for beer spoilage and that only a few species are major beer spoilage
bacteria. For quality assurance of finished beer it is usually sufficient to control
potential contaminations by Lactobacillus brevis, Lb. lindneri, Pediococcus
damnosus, and Pectinatus spp.. Most studies on beer spoilage bacteria have
focused on the taxonomical classification of these bacteria. These studies have
made it possible to identify the bacteria detected in beer and breweries and to take
the proper measures to control them.
3.1. Culture Media
Along with the taxonomical studies a number of selective culture media for beer
spoilage bacteria have been developed. European Brewery Convention
recommends three media for the detection of lactobacilli and pediococci: MRS (de
Man, Rogosa and Sharpe) agar supplemented with cycloheximide to prevent
growth of aerobes such as yeasts and moulds, Raka-Ray medium supplemented
with cycloheximide and VLB (Versuchs- und Lehranstalt für Brauerei in Berlin)
S7-S. Other optional media are UBA (Universal Beer Agar) supplemented with
cycloheximide, HLP (Hsu’s Lactobacillus and Pediococcus medium), NBB
(Nachweismedium für bierschädliche Bakterien), WLD (Wallerstein Differential),
Nakagawa, SDA (Schwarz Differential Agar) and MRS modified by addition of
maltose and yeast extract at pH 4.7. None of these media are suitable for detecting
all strains of lactobacilli and pediococci but a combination of some of these media
yields the best results. For the detection of Pectinatus and Megasphaera the
following media are recommended: Concentrated MRS broth, PYF (Peptone, Yeast
extract and Fructose) and Thioglycolate Medium for enrichment of beer, LL-Agar
for growth in Lee tube, and UBA, NBB and Raka-Ray for routine analysis at
breweries. For Zymomonas spp., Zymomonas Enrichment Medium is also
recommended (EBC Analytica Microbiologica II, 1992). American Society of
Brewing Chemists recommends UBA and Brewer’s Tomato Juice Medium for
general microbial detection and other media including LMDA (Lee’s MultiDifferential Agar), Raka-Ray, BMB (Barney-Miller Brewery Medium) and MRS
for the detection of lactic acid bacteria and SMMP (Selective Medium for
Megaspahera and Pectinatus) (Methods of Analysis of the American Society of
Brewing Chemists, Eighth Revised Edition, 1992). Brewery Convention of Japan
also recommends the use of some of those media (BCOJ Biseibutu Bunsekihou,
1999). These culture media are listed in Table 2.
11
CHAPTER 1
Table 2. Selective culture media for the detection of beer spoilage bacteria
Media
Bacteria
Recommended by3
MRS (de Man, Rogosa and Sharpe)
LAB1
EBC, ASBC, BCOJ
Raka-Ray
LAB, G(-)2 EBC, ASBC, BCOJ
VLB S7-S (Versuchs- und Lehranstalt für LAB
EBC, BCOJ
Brauerei in Berlin)
HLP (Hsu’s Lactobacillus and Pediococcus LAB
EBC, BCOJ
medium)
WLD (Wallerstein Differential)
LAB
EBC, BCOJ
Nakagawa
LAB
EBC, BCOJ
SDA (Schwarz Differential Agar)
LAB
EBC, BCOJ
Concentrated MRS
G(-)
EBC, BCOJ
PYF (Peptone, Yeast extract and Fructose) G(-)
EBC, BCOJ
Thioglycolate Medium
G(-)
EBC
LL-Agar
G(-)
EBC, BCOJ
UBA (Universal Beer Agar)
LAB, G(-) EBC, ASBC, BCOJ
NBB (Nachweismedium für
LAB, G(-) EBC, BCOJ
bierschädliche Bakteriën)
Brewer’s Tomato Juice Medium
LAB, G(-) ASBC
LMDA (Lee’s Multi-Differential Agar)
LAB
ASBC
BMB (Barney-Miller Brewery Medium)
LAB
ASBC
SMMP (Selective Medium for
G(-)
ASBC, BCOJ
Megasphaera and Pectinatus)
1
LAB, Lactic acid bacteria
G(-), Gram-negative bacteria
3
EBC, European Brewery Convention; ASBC, American Society of Brewing Chemists; BCOJ,
Brewery Convention of Japan
2
The conventional detection method based on culturing of the organisms in these
media has the significant disadvantage that is very time-consuming. One week or
even longer is needed to obtain visible colonies on plates or turbidity in broths.
Consequently, the products are often already released for sale before the
microbiological results become available. Another problem is that these media are
not species-specific. Media for the detection of beer spoiling lactic acid bacteria
allow also growth of non-beer-spoilage species such as Lactobacillus delbrueckii
and Pediococcus acidilactici. If the selectivity is increased by the addition of
specific chemicals to these media, even longer detection times might be required.
3.2. Identification Methods
Following the bacterial detection in these media, species identification is needed.
Besides the basic tests such as colony morphology, cell morphology, Gram-staining
and catalase assays, also biochemical tests such as sugar fermentation pattern and
chromatographic analysis of organic acids can be performed. Also specific
detection and identification methods are used such as immunoassays with
12
CHAPTER 1
polyclonal or monoclonal antibodies (Claussen et al., 1975, 1981; Dolezil and
Kirsop, 1976; Haikara, 1983; Gare, et al., 1993; Sato et al., 1994; Whiting et al.,
1992, 1999a, 1999b; Ziola et al., 1999, 2000a, 2000b), DNA-DNA hybridization,
DNA sequencing and PCR (Polymerase Chain Reaction) (Tsuchiya et al., 1992,
1993, 1994; DiMichele and Lewis, 1993; Thompson et al., 1994; Vogesor et al.,
1995a, 1995b; Yasui, 1995; Stewart and Dowhanick, 1996; Yasui et al., 1997;
Sakamoto, 1997; Sakamoto et al., 1997; Satokari et al., 1997, 1998; Juvonen and
Satokari, 1999; Motoyama and Ogata, 2000; Bischoff et al., 2001). These modern
methods have been reviewed (Berney and Kot, 1992; Schmidt, 1992; Dowhanick
and Russel, 1993; Dowhanick, 1995; Schofield, 1995; Hammond, 1996; Schmidt,
1999). Especially the application of PCR has recently significantly been improved
(see Chapter 2).
3.3. Descrimination of Beer Spoilage Bacteria from Non-Spoilers
After detection and identification it is for some species necessary to identify the
bacterium as an actual beer spoiler. While all strains belonging to Pectinatus spp.
and Megasphaera cerevisiae have been reported to be capable of spoiling beer
(Haikara, 1991), lactic acid bacteria include both beer spoilage and non-spoilage
strains. Among Lb. brevis and P. damnosus most of the strains are capable of
spoiling beer and only a few strains are not. On the other hand the number of beer
spoilage strains in Lb. casei, Lb. coryneformis and Lb. plantarum is limited.
Exceptionally, all strains of Lb. lindneri have been reported to be capable of
spoiling beer (Rinck and Wackerbauer, 1987a, 1987b; Storgårds et al., 1998).
Before the beginning of 1990s the only method was available for judging the
beer spoiling potential of a bacterium, the so-called ‘forcing test’. In this test the
bacterium was re-inoculated into beer or beer enriched with concentrated nutrient
medium. However, this test has proven to be far from practical for quality
assurance since a few months are needed to obtain conclusive results.
More rapid procedures have been developed. The identification at the strain level
can now be done at the genome level. Ribotyping, based on Southern hybridization
with a ribosomal gene as a probe, has been successfully introduced (Motoyama et
al., 1998, 2000; Satokari et al., 2000; Suihko and Haikara, 2000; Barney et al.,
2001). Fully automated ribotyping machines are now commercially available and
only eight hours are needed to obtain conclusive results. AFLP (Amplified
Fragment Length Polymorphisms) (Perpete et al., 2001) and RAPD-PCR (Random
Amplified Polymorphic DNA) (Savard et al., 1994; Tompkins et al., 1996) have
also successfully been applied for bacterial strain identification as well as for
identification of brewing yeasts. In these methods the genotype of each beer
spoilage strain is registered in a database. A comparison of the genotype of a newly
detected strain with registered genotypes will make a risk assessment possible.
Another approach is to determine the common physiological properties
13
CHAPTER 1
responsible for beer spoiling ability. For beer spoiling lactic acid bacteria the
common physiological denominator is hop resistance, which allows growth of
these bacteria in beer. However, measuring hop resistance by culturing in hop
containing medium, is too time-consuming. It will be much faster to detect the
physiological traits that cause hop resistance with immunoassays or PCR. Before
this can be done the cause of the antibacterial activity of hop compounds and the
mechanism(s) responsible for resistance towards hop compounds need to be
known.
14
CHAPTER 1
4. ANTIBACTERIAL ACTIVITY OF HOP COMPOUNDS
4.1. History of Hop Usage in Beer
The comfortable bitterness experienced in beer drinking is characteristic for and
is mainly caused by hop compounds. These hop compounds are present in the
flowers of the hop plant, Humulus lupulus, L., which are added to the wort. This
plant has been known for thousands of years. However, its use in beer is not as old
as the history of beer itself (5,000 to 7,000 years). A few descriptions of hops as a
beer additive as well as a decoration for gardens were found in documents of the
sixth century B.C. German monks in the 12th century often used hops in beer
making. In those days, as in ancient times, it was popular to use a variety of fruits,
herbs and spices to flavor beer (so-called gruit beer). Initially the bitter taste from
hops was not particularly appreciated. However, when in the 14th century beer
production increased and beer was exported, the importance of hops in beer was
gradually more and more appreciated, not only for its contribution to beer flavor
but also for its contribution to the stability. Hopped beer can be preserved
significantly longer than gruit beer. In 1516 Wilhelm IV, the lord of Bayern,
enacted the ‘Reinheitsgebot (Purity Law)’ which ordered that beer must be made
from barley, water and hops. Since then, the use of hops became more popular and
standard. Many aspects of this law were adopted by other countries, which made
hops indispensable for the brewing industry.
4.2. Hop Plant
The hop plant is a vine, belonging to the family of hemp (Fig. 1). It is dioecious
and blooms yearly. Nowadays it is mainly cultured for the brewing industry. Only
the female flowers, the so-called cones, are used for beer. The mature cones contain
golden resinous granules, the lupulin, which are the most important part of the
flower for the bitterness and preservation of beer. Hop resins are extracted and
fractionated as shown in Fig. 2.
Figure 1. Hop plant. (a) Hop
cones at the end of the vine. (b) A
vertical section of hop cone.
Lupuline glands locate on the
base of each bract. They contain
the hop resins and essential oils
which give beer a unique flavor.
Strig
B ract
Lupulin
glands
a
b
15
CHAPTER 1
Figure 2. Fractionation of hop resins (Hough et al., 1971)
Hop cones
soluble in ether and cold methanol
Total resins (15-30%)
hexane
soluble
insoluble
Total soft resins (10-25%)
Hard resin (3-5%)
Formation
as lead salt
α-acids (5-13%)
β-fraction (5-15%)
β-acids (3-8%)
Unknown soft resins (2-7%)
4.3. Antibacterial Compounds in Hops
Hop chemistry has been developed since 19th century and has been extensively
reviewed (Verzele, 1986; Moir, 2000). Research has especially been focused on the
antibacterial properties of hop compounds and the bitter substances derived from
hops. This research goes back to 1888 when Hayduck showed first that antiseptic
properties of the hops are due to the soft resins (Hayduck, 1888). In the Institute of
Brewing of the United Kingdom, Walker conducted from 1922 till 1941 a longterm investigation on ‘the preservative principles of hops’ (Pyman et al., 1922;
Walker, 1923a, 1923b, 1924a, 1924b, 1925, 1938, 1941; Hastings et al., 1926;
Hastings and Walker, 1928a, 1928b, 1929; Walker and Hastings, 1931, 1933a,
1933b; Walker et al., 1931, 1932, 1935, 1940; Walker and Parker, 1936, 1937a,
1938, 1940a, 1940b). The study focused on the antiseptic properties of α-acid
fraction (humulone) and β-acid fraction (lupulone).
The α-acid fraction is a mixture of homologous compounds, the α-acids, which
are not transferred as such to beer. During the wort boiling stage in the brewing
process, α-acids are converted by a rearrangement or isomerization to iso-α-acids,
which are much more soluble and bitterer than the original compounds. This
conversion, which is very important in hop chemistry, was advanced first in 1888
(Hayduck). Wieland et al. (1925) suggested that the hydrolysis of humulone to
humulinic acid proceeded via an intermediate. Windisch et al. (1927) investigated
the humulone boiling products under alkaline conditions and isolated a resinous
16
CHAPTER 1
and bitter oil termed “Resin A” with chemical properties similar to the intermediate
and isomeric with humulone. Around 1950, Rigby and Bethune showed that α-acid
fraction is a mixture of three major compounds; humulone, cohumulone and
adhumulone (Figure 3) (Rigby and Bethune, 1952, 1953). The bittering compounds
of beer were found to comprise three major analogues of these three α-acids, which
are now known as iso-α-acids; isohumulone, isocohumulone and isoadhumulone
(Figure 3). Stereoisomers (cis- and trans-) exist for each iso-α-acid. Finally the
chemical structure and configuration of naturally occurring (-)-humulone (De
Keukeleire and Verzele, 1970) and isohumulones (De Keukeleire and Verzele,
1971) were elucidated. The isomerization yield of α-acids during wort boiling
process is low [typically of the order of 30% (Hughes, 2000)] due to relatively
acidic condition of wort (ca. pH 5.2) and the adsorption to the wort coagulum
during boiling and fermentation.
β-acids or lupulones in hops are very poorly soluble in wort and beer and cannot
undergo the same isomerization processes as α-acids. Consequently, they are not
transferred to beer and have no direct value in brewing.
O
OH
O
O
R
R
O
HO
HO
OH
O
I
R
-(CH) 2CH(CH3)2
-CH(CH3) 2
-CH(CH3)CH2CH3
OH
O
O
R
HO
HO
II
α-acids
humulone
cohumulone
adhumulone
O
III
(-)-humulones
β-acids
lupulone
colupulone
adlupulone
O
O
R
HO
O
OH
IV
trans-isohumulones
O
R
HO
O
OH
V
cis-isohumulones
Figure 3. Chemical structures of hop coumpounds. The name of each α-acid (I) and βacid (II) is dependent on its side chain. During wort boiling process, α-acids (naturally Rbody; III) are isomerized to result in stereoisomers of trans-isohumulones (IV) and cisisohumulones (V).
17
CHAPTER 1
4.4. Antibacterial Mechanism of Hop Compounds
The antibacterial activities of α-acid (humulone) and β-acid (lupulone) have been
studied before 1950. Their antibacterial activities are higher than of iso-α-acids but
they dissolve to less extent in beer and water. Studies of the antiseptic properties of
hopped wort and hop boiling product showed that they inhibit growth of Grampositive bacteria but not of Gram-negative bacteria (Shimwell, 1937a; Walker and
Blakebrough, 1952). It was first reported by Shimwell (1937a) that the antiseptic
potency of hop increases at lower pH. Interestingly, he predicted that the antiseptic
potency of hop is associated with permeability changes of the bacterial cell wall
(1937b). The ‘bacteriostatic power’ was also studied of hop compounds, including
humulone and the humulone boiling product (Walker and Blakebrough, 1952). The
humulone boiling product had less bacteriostatic potency in malt extract (pH 5.5)
and wort (pH 5.2) than the original humulone, while its potency was the same at
pH 4.3, the pH of beer. The hop constituents (lupulone, humulone, isohumulone
and humulinic acid) were found to cause leakage of the cytoplasmic membrane of
Bacillus subtilis, resulting in the inhibition of active transport of sugar and amino
acids (Teuber and Schmalreck, 1973). Subsequently inhibition of respiration and
synthesis of protein, RNA and DNA was also observed. Since the iso-α-acids are
mainly present in beer among the hop resins and their derivatives, a precise
investigation of the antibacterial activity of iso-α-acids was needed for
understanding the preservation or bacterial stability of beer. The molecular
mechanism of antibacterial activity of iso-α-acids and the effects of pH of the
growth medium and other variables on the antibacterial activity of hop compounds
were investigated after 1990 (Simpson and Smith, 1992). Hop compounds are
weak acids and the undissociated forms are mainly responsible for inhibition of
bacterial growth (Fig. 4).
O
O
O
HO
O
O
HO
O
OH
pKa = 3.1
Antibacterial form
O
+ H+
Inactive form
Figure 4. Dissociation of trans-isohumulone. (Fernandez and Simpson, 1995b)
In Lb. brevis (Simpson, 1993b) trans-isohumulone reduces the uptake of leucine
and causes slow leakage of accumulated leucine. trans-Isohumulone dissipates
18
CHAPTER 1
effectively the transmembrane pH gradient (∆pH) of the proton motive force but
not the transmembrane electrical potential (∆ψ). Inhibition of H+-ATPase activity
was not observed. Potentiometric studies revealed that undissociated transisohumulone acts most likely as an ionophore, catalyzing electroneutral influx of
undissociated isohumulone, internal dissociation of (H+)-isohumulone and efflux of
the complex of isohumulone with divalent cations such as Mn2+. This cation is
known to be present at high concentrations in lactobacillus cells (Archibald and
Fridovich, 1981a, 1981b; Archibald and Duong, 1984). The result of this activity is
a decrease of the pH gradient across the membrane. It was reported that the
antibacterial activity of trans-isohumulone can be influenced by the presence of
cations in the medium. Protonophoric activity of trans-isohumulone requires the
presence of monovalent cations such as K+, Na+ or Rb+ and increases with the
concentration of these monovalent cations (Simpson and Smith, 1992). transIsohumulone cannot bind K+ unless a divalent cation, such as Mn2+, Mg2+, Ni2+ and
Ca2+ or a trivalent cation, such as Li3+ and Al3+, is present in the medium (Simpson
et al., 1993; Simpson and Hughes, 1993). Thus, the ability of hop compounds to
bind simultaneously two or more cations may be crucial for their antibacterial
action but the reason has been still unclear.
The properties of other hop acids are similar to those of trans-isohumulone and it
is likely that the mechanism of their antibacterial activities is also similar. Some
strains of lactic acid bacteria, which are sensitive to trans-isohumulone, are also
sensitive to (-)-humulone and colupulone and other strains resistant to transisohumulone are also resistant to the related compounds (Fernandez and Simpson,
1993). The antibacterial activities of 6 naturally occurring iso-α-acids, 5 chemically
reduced iso-α-acids and a reduced iso-α-acids mixture were higher at lower pHvalues while more hydrophobic reduced iso-α-acids were found to be far more
antibacterial than their naturally occurring analogues (Price and Stapely, 2001).
19
CHAPTER 1
5. HOP RESISTANCE IN LACTIC ACID BACTERIA
Beer spoiling lactic acid bacteria have to be hop resistant in order to grow in
beer. The understanding and elucidation of the mechanism of hop resistance is not
only of scientific interest but is also important for the microbial control in brewing
industry to predict the beer spoiling ability of lactic acid bacteria.
5.1. Variation of Hop Resistance
The extent of hop resistance varies between bacteria. Among beer spoiling lactic
acid bacteria Lb. brevis is so far the most resistant to hop compounds. The degree
of hop resistance varies among the different strains of Lb. brevis (Hough et al.,
1957; Harris and Watson, 1960; Simpson and Fernandez, 1992; Fernandez and
Simpson, 1993). Hop resistance of lactobacilli decreases upon prolonged serial
subculturing in the absence of hop compounds (Shimwell, 1936c; Yamamoto,
1958; Richards and Macrae, 1964). Hop resistance was thought to be caused by
immunity acquired by prolonged contact with hop compounds under brewing
conditions (Shimwell, 1937a). The necessity of beer spoiling lactic acid bacteria to
acclimatize to beer or hop compounds in order to reproduce in beer (Yamamoto,
1958) was solidly documented by Richards and Macrae in 1964. Hop resistance
increased 8 to 20 fold in strains of lactobacilli upon serial subculturing in media
containing increasing concentrations of hop compounds, while subculturing of
resistant populations in the absence of hop compounds resulted gradually in
decreased hop resistance. It took about one year of subculturing in unhopped beer
to maximally reduce hop resistance of lactobacilli, indicating that the acquired hop
resistance can be a very stable property (Shimwell, 1936c). However, organisms
isolated from spoiled beer frequently fail to grow upon reinoculation in beer.
Preculturing in the presence of sub-inhibitory concentrations of isohumulone is
needed in order to make growth in beer possible (Simpson and Fernandez, 1992).
The stability of hop resistance in Lb. brevis appears to vary from strain to strain.
The hop resistance in Lb. brevis strain BSO310 could not be altered by plasmid
curing or mutation induced with ultraviolet light (Fernandez and Simpson, 1993),
suggesting that it may be generally a stable character, both phenotypically and
genetically. The Lb. brevis strain ABBC45 can develop hop resistance in the same
way as observed by Richards and Macrea (Sami et al., 1997a). Hop resistance
increased with the copy number of plasmid pRH45. When Lb. brevis ABBC45 was
cured from this plasmid by serial subculturing in the absence of hop compounds,
the degree of hop resistance decreased (Sami et al., 1998; Suzuki et al., 2002). This
plasmid contains the horA gene that codes for a polypeptide that is 53% identical to
LmrA, the lactococcal ATP-binding cassette (ABC) multidrug transporter (van
Veen et al., 1996). HorA protein was expressed heterologously in Lactococcus
lactis and found to function as an ABC-type multidrug transporter and to excrete
hop compounds (Sakamoto et al., 2001).
20
CHAPTER 1
5.2. Features of Hop Resistance
The pattern of resistance or sensitivity of several species of lactobacilli to
isohumulone is similar to that of the related hop acids humulone and lupulone
(Richards and Macrea, 1964; Fernandez and Simpson, 1993). Fernandez and
Simpson (1993) compared the properties of hop resistant strains of lactobacilli and
pediococci with those of sensitive strains. No obvious correlation was found
between hop resistance and cell morphology, colony morphology, pH range for
growth, sugar utilization profile, products of metabolism, manganese requirement
and sensitivity to superoxide radicals, expression of cellular proteins, and
resistance to various antibacterial agents. However, differences were found in the
transmembrane pH gradient (∆pH) and the cellular ATP pool. Because hop
compounds act as protonophores that dissipate the ∆pH across the cellular
membrane (Simpson, 1993a, 1993b), these differences are of great importance for
understanding the mechanism of hop resistance.
5.3. Mechanisms of Hop Resistance
The molecular structure of antibacterial agents might supply an insight in the
mechanism of resistance. Resistance to trans-isohumulone also results in resistance
to (-)-humulone and colupulone (Fernandez and Simpson, 1993), suggesting a
common mechanism of resistance against a broad range of hop acids. transIsohumulone and (-)-humulone have three hydrophobic side chains while
colupulone has four. The side chains are attached to a five-membered ring of transisohumulone, but to a six-membered ring of (-)-humulone and colupulone (Fig. 3).
On the other hand resistance was not observed to other ionophores (nigericin,
A23187, CCCP, monesin), weak acid food preservatives (sorbic acid, benzoic
acid), solvents (ethanol), or antibiotics (ampicillin, cefamandole, vancomycin)
(Fernandez and Simpson, 1993). The resistance mechanism might be specific for
the β-triketone group of the hop acids, which plays an essential role in the
antibacterial action (Simpson, 1991).
Micro-organisms have developed various ways to resist the toxicity of
antibacterial agents:
(i) enzymatic drug inactivation. A well known example is β-lactamase which
hydrolyzes the β-lactam ring into innocuous substrates. In hop resistant strains of
Lb. brevis, neither conversion nor inactivation of trans-isohumulone was found
(Simpson and Fernandez, 1994).
(ii) target alteration. Cellular targets can be altered by mutation or enzymatic
modification in such a way that the affinity of the target for the antibiotics is
reduced. In the case of trans-isohumulone, the target site is the cell membrane
(Teuber and Schmalreck, 1973; Schmalreck et al., 1975; Simpson, 1993a, 1993b).
A ‘sake (Japanese rice wine)’ spoilage bacterium, Lb. heterohiochii, contains
extremely long chain fatty acids in its membrane (Uchida, 1974), which may play a
21
CHAPTER 1
role in ethanol resistance (Ingram, 1984). It is possible but not yet investigated that
hop resistant Lb. brevis has also a changed lipid composition of its membrane to
lower the permeability to hop compounds.
(iii) inhibition of drug influx. The outer membrane of Gram-negative bacteria
restricts the permeation of lipophilic drugs, while the cell wall of the Gram-positive
mycobacteria has been found to be an exceptionally efficient barrier. The
permeation of hop compounds might also be affected by the presence of a
galactosylated glycerol teichoic acid in beer spoilage lactic acid bacteria (Yasui and
Yoda, 1997b).
(iv) active extrusion of drugs. The presence of (multi)drug resistance pump in the
cytoplasmic membrane of many bacteria has been extensively documented
(Putman et al., 2000a). A multidrug resistance pump HorA (Sakamoto et al., 2001)
has been found in Lb. brevis ABBC45, which is overexpressed when exposed to
hop compounds (Sami, 1999). In addition a proton motive force dependent hop
excretion transporter was suggested in this strain (Suzuki et al., 2002).
(v) other mechanisms to tolerate the toxic effects of drugs. Since hop compounds
act as protonophores and dissipate the transmembrane pH gradient (∆pH), the cells
could respond by increasing the rate at which protons are expelled. The hop
resistant strains maintain a larger ∆pH than hop sensitive strains (Simpson and
Fernandez, 1993) and Lb. brevis ABBC45 increases its H+-ATPase activity upon
acclimatization to hop compounds (Sakamoto et al., 2002). The ATP pool in hop
resistant strains was also found to be larger than in hop sensitive strains (Simpson
and Fernandez, 1993; Okazaki et al., 1997). The ability of hop resistant strains to
produce large amounts of ATP in the cell is needed for the increased activity of the
H+-ATPase and for the hop extruding activity of HorA. It is interesting that these
responses occurred also in hop sensitive strains at sub-inhibitory concentration of
trans-isohumulone (Simpson, 1993a; Simpson and Fernandez, 1993). However, at
higher concentrations both the ∆pH and the ATP pool decreased in the hop
sensitive strains, but not in the hop resistant strains. A role of HitA in hop
resistance was suggested (Hayashi et al., 2001). It is about 30% identical to the
natural resistance-associated macrophage proteins (Nramp), which function as
divalent-cation transporters in many prokaryotic and eukaryotic organisms. HitA
could play a role in transport of divalent cations while isohumulone has been
claimed to exchange protons for cellar divalent cations such as Mn2+. Thus a
simple mechanism of hop resistance does not appear to exist. The resistance
mechanisms found so far in Lb. brevis are illustrated in Fig. 5.
22
CHAPTER 1
Hop-H
Hop-H
Hop-H
a
ATP
ATP
b
Hop-H
ADP
H+
Hop- + H+
ATP
H+
Mn2+
H+
H+
ADP
c
Hop-Mn-Hop
Cytoplasmic membrane
Cell wall
Hop-Mn-Hop
Figure 5. Mechanisms of hop resistance. Resistance to hop compounds is conferred by a
number of processes. Hop compounds (Hop-H) are expelled from the cytoplasmic
membrane in hop resistant cells by HorA (a) (Sakamoto et al., 2001) and probably also by a
pmf-dependent transporter (b) (Suzuki et al., 2002). When hop compounds enter the
cytoplasm they dissociate due to the higher internal pH into hop anions and protons.
Overexpression of H+-ATPase (c) results in increased proton pumping and pmf generation
(Sakamoto et al., 2002). Hop anions can trap divalent cations such as Mn2+ and diffuse out
of the cell. The hop resistant strains can generate more ATP than hop sensitive strains
(whirlpool).
23
CHAPTER 1
6. BEER SPOILING ABILITY IN LACTIC ACID BACTERIA
Hop resistance is crucial to beer spoiling ability of lactic acid bacteria. In many
bacteria hop resistance mechanisms need to be induced before growth in beer is
possible. For this induction some bacteria need to be exposed to sub-inhibitory
concentrations of hop compounds (Simpson, 1993).
6.1. Factors Affecting the Growth in Beer
Beer spoilage and bacterial growth depend on the strain and the type of beer. The
ability of 14 hop-resistant lactic acid bacterial strains, including Lb. brevis and P.
damnosus strains, was investigated for their capacity to grow in 17 different lager
beers with the biological challenge test (Fernandez and Simpson, 1995a). A
statistical analysis of the relationship between spoilage potential and 56 parameters
of beer composition revealed a correlation with eight parameters: pH, beer color,
the content of free amino nitrogen, total soluble nitrogen content and the
concentrations of a range of individual amino acids, maltotriose, undissociated SO2
and hop compounds. The effects of dissolved carbon dioxide (CO2) and phenolic
compounds including catechin, gallic, phytic and ferulic acids on beer spoilage
were also investigated (Hammond et al., 1999). CO2 was found to inhibit the
growth of lactobacilli at the concentrations present in typical beer but stimulate at
the lower concentrations. Among the phenolic compounds, ferulic acid, a
component of barley cell wall and hence present in all beers, exerted a stronger
antibacterial activity after enzymatic conversion into 4-vinyl guaiacol. Organic
acids in beer may also influence bacterial growth but this aspect has hardly been
studied.
6.2. Prediction of Beer Spoilage by Lactic Acid Bacteria
A number of attempts have been made to develop methods to predict beer
spoiling ability. For lactic acid bacteria hop resistance is the key factor. As
described above, some factors have been identified to cause hop resistance. Rapid
procedures for detecting these factors would be very beneficial for microbial
control in breweries. A set of PCR primers have been made that can specifically
detect the horA gene or its homologues in a wide range of lactobacilli (Sami et al.,
1997b). Most horA positive strains were found to have beer spoiling ability,
indicating that this is a very useful prediction method. Another prediction method
is based on ATP pool measurements in lactobacillus cells (Okazaki et al., 1997).
Polyclonal and monoclonal antibodies specific only for beer spoilage strains
have been reported. A series of antisera were made against the Lactobacillus group
E antigen, a cell wall glycerol teichoic acid beneath the S-layer protein (Yasui et
al., 1992, 1995; Yasui and Yoda, 1997a) and known to be present in Lb. brevis, Lb.
buchneri, Lb. delbrueckii subsp. lactis and subsp. bulgaricus (Sharpe, 1955; Sharpe
et al., 1964). When the beer spoilage strain Lb. brevis 578 was used as an antigen,
24
CHAPTER 1
the resulting antiserum reacted specifically with other beer spoilage strains of Lb.
brevis although they had been cultured in modified NBB medium (Nishikawa et
al., 1985) which does not contain any hop compounds or beer. Surprisingly, this
antiserum also reacted with beer spoilage strains of P. damnosus, but not with any
strains of Lb. lindneri (Yasui, and Yoda, 1997a). Galactosylated glycerol teichoic
acid was found to be the most likely epitope that presumably selectively increases
the cell barrier to hop compounds (Yasui and Yoda, 1997b). Three monoclonal
antibodies specific for beer spoilage ability of lactic acid bacteria were obtained by
immunizing mice with cells cultured in beer (Tsuchiya et al., 2000). The
monoclonal antibody raised against Lb. brevis reacted with all beer spoilage strains
of Lb. brevis and several beer spoilage strains of P. damnosus, but not with nonspoilage strains of Lb. brevis, P. damnosus and other lactic acid bacteria. The
monoclonal antibody raised against P. damnosus reacted significantly with all beer
spoilage strains of P. damnosus and weakly with many of the beer spoilage strains
of Lb. brevis. On the other hand the monoclonal antibody raised against Lb.
lindneri reacted specifically only with Lb. lindneri. The reactivity of the still
unknown antigens did not change regardless of the presence of hop compounds in
their culture media.
D-lactate dehydrogenase (LDH) of 60 strains of Lb. brevis, including 44 beer
spoiling strains and 16 non-spoiling strains, was also investigated. The strains
could be divided in five groups (A, B, C, D and E) on the basis of the mobility of
their D-LDH in native polyacrylamide gels (Takahashi et al., 1999). Forty out of 44
beer spoilage strains were classified to the group B suggesting a relationship
between the D-LDH profile and beer spoiling ability. The purified D-LDH of those
groups had different pH and temperature optima and isoelectric points. Especially
the temperature optimum of 50°C of the Group B D-LDH is significantly lower
than that of the other D-LDHs (60°C).
Except for the forcing test, none of the other methods so far available can predict
beer spoiling ability of all strains. Since hop resistance in lactic acid bacteria does
not appear to be a general feature of all beer spoiling bacteria, combination of
several methods will be required to detect all potential beer spoiling strains.
25
26
CHAPTER 2
The Nucleotide Sequence of the 16S Ribosomal RNA
Gene of Pectinatus sp. DSM20764 and Improvement
of PCR Detection of Beer Spoilage Bacteria by the
Combined Use of Specific and Universal Primers
Kanta Sakamoto, Wataru Funahashi, Hiroshi Yamashita, and Masakazu Eto.
This chapter is a combination of the article ‘A reliable method for detection and
identification of beer-spoilage bacteria with internal positive control PCR (IPCPCR)’ published in the Proceeding of European Brewery Convention Congress,
Maastricht, 1997, pp.631-638, and the patent (JP09-520359, WO97/20071 or
USP5869642: ‘Detection of genus Pectinatus’).
SUMMARY
A set of highly species-specific primers of beer spoilage bacteria were made on
the basis of their 16S ribosomal RNA gene (16S rDNA) sequences. These primers
are used for the rapid detection and identification of these beer spoilage bacteria
with the PCR method. The 16S rDNA sequences were derived from GENBANK,
except for Pectinatus sp. DSM20764, which is a unique strain with properties
different from those of Pectinatus cerevisiiphilus and P. frisingensis. Its 16S rDNA
sequence was determined in this study. To prevent that the PCR method yields
false negative results, a set of universal primers targeting consensus sequences in
16S rDNA was employed. The combined use of this set of primers with a set of
species-specific primers makes it possible to successfully perform PCR and DNA
extraction and to identify beer spoilage bacteria. This procedure improves
considerably the reliability of PCR detection of beer spoilage bacteria.
27
CHAPTER 2
INTRODUCTION
In recent years, Japanese consumers have shown preference for non-pasteurized
beer (so-called ‘Nama’ beer in Japan) over pasteurized beer. Consequently
microbiological quality assurance has become more and more important in the
breweries. A number of rapid microbiological detection and identification methods
have been developed to replace the time-consuming conventional analysis based on
the culture media (Barney and Kot, 1992). Among these methods, the polymerase
chain reaction (PCR) method is one of the most powerful tools because of its
rapidity and high sensitivity (Tshuchiya et al., 1992, 1993, 1994; DiMichel and
Lewis, 1993; Savard et al., 1994; Schofield, 1995; Taguchi et al., 1995; Vogessor
et al., 1995a, 1995b; Stewart and Dowhanick, 1996; Thomas et al., 1996).
However, several problems have been encountered in using this method for
practical analysis: (i) PCR primers specific for Gram-negative beer spoilage
bacteria, such as Pectinatus and Megasphaera have not yet been developed. From
the Pectinatus species, a DNA sequence was not available for P. sp. DSM20764,
which is a unique strain, different from either P. cerevisiiphilus or P. Firisingensis,
(ii) Some of PCR primers reported so far are not sufficiently specific. They detect
several species simultaneously and do not discriminate between beer spoiling and
non-spoiling bacteria, (iii) False negative results are sometimes obtained which
makes the procedure not very reliable. When PCR products are not found after
reaction with specific primers, it is not clear whether this represents indeed a
negative result or whether it is due to a failure of the reaction and/or the DNA
extraction.
In this study, we have solved these problems by (i) determining the sequence of
16S rDNA of Pectinatus sp. DSM20764, (2) designing more specific primers for
all the major beer spoilage bacteria and (3) employing a set of universal primers
targeting consensus sequences in 16S rDNA (Lane, 1991) as a probe for the
internal positive control.
MATERIALS AND METHODS
Bacterial strains
The bacteria used in this study are listed in Table 1. These bacterial strains,
including type strains, were obtained from the following public culture collections:
ATCC (American Type Culture Collection, USA), DSM (Deutsche Sammlung von
Mikroorganismen und Zellkulturen, Germany), IAM (Institute of Applied
Microbiology, Japan), IFO (Institute for Fermentation, Japan), JCM (Japan
Collection of Micoroorganisms) and from our own laboratory culture collection.
Lactic acid bacteria and Gram-negative bacteria were incubated anaerobically at
28
CHAPTER 2
30°C in MRS broth (Merck, Germany) or in TGC broth (Nissui, Japan),
respectively. Enscherichia coli was cultured in LB broth aerobically at 37°C.
Genera
Lactobacillus
Pediocossus
Lactococcus
Leuconostoc
Pectinatus
Megasphaera
Selenomonus
Zymomonus
Escherichia
unknownb
Table 1. Bacteria used in this study
species
number of strains
brevis
casei
collinoides
coryneformis
delburuekii
fermentum
lindneri
plantarum
rhamnosus
spp.
damnosus
lactis
lactis
cerevisiae
frisingensis
sp.
cerevisiae
lacticifex
pausivorans
raffinosivorans
coli
G(+)c
G(-)d
48
11
2
1
1
1
3
5
1
22
1
1
1
5
28
1a
2
1
1
1
1
6
2
a
DSM20764
non beer-spoilage bacteria isolated from breweries.
c
Gram-positive bacteria
d
Gram-negative bacteria
b
DNA extraction and purification
Chromosomal DNA was extracted from the bacterial cells using the
cetyltrimethylammonium bromide (CTAB) procedure (Olsen et al., 1991). One ml
of the bacterial culture in the late exponential phase was centrifuged and the
resulting cell pellet was washed once in sterile de-ionized water. The cells were
resuspended in 0.3 ml of suspension buffer (10 mM Tris-HCl [pH 8.0], 1 mM
EDTA, 0.35 M sucrose) containing 1 mg/ml lysozyme (Sigma, USA) and 50 µg/ml
N-Acetylmuramidase (Seikagaku Co., Japan) and incubated at 37°C for 30 min.
Subsequently, 0.3 ml of two times concentrated CTAB lysing buffer (100 mM
Tris-HCl [pH 8.0], 1.5 M NaCl, 20 mM EDTA, 2% [w/v] CTAB, 2% [v/v] 2Mercaptoethanol) containing 160 µg/ml Protease K (Nakarai Tesque, Inc., Japan)
29
CHAPTER 2
was added and the mixture was incubated for 1 h at 50°C. Phenol-chloroform (1:1
v/v) (0.6 ml) was added and vigorously mixed with a vortex. After centrifugation at
12,000 × g for 10 min, the aqueous phase (about 0.5 ml) was transferred carefully
to a new sterile test tube to avoid inclusion of debris. After addition of an equal
volume of isopropanol the mixture was centrifuged at 12,000 × g for 10 min. The
precipitated DNA was washed with 70% (v/v) ethanol, and purified by gel
filtration chromatography using CHROMA SPIN-1000 column (Clontech
Laboratories, Inc., USA).
For evaluation of the specifity of the species-specific primers, DNA was
extracted from pure cultures of each bacterium. For evaluation of the combined use
of the universal primers and the specific primers, DNA was extracted from a
mixture of cultures of a beer spoilage bacterium and non beer-spoilage bacteria
isolated from breweries (their species are not identified) to imitate a bacterial
contaminated sample from a brewery.
16S rDNA sequencing
The 16S rDNA sequence of P. sp. DSM20764 was determined separately by the
direct sequencing technique. Several parts of the 16S rDNA were amplified by
PCR with chromosomal DNA as template by employing universal primers (Lane,
1991). Table 2 lists the sequences of the primers used. The location and the pairs of
these primers in the gene are shown in Fig. 1.
Primer
Table 2. The primers used for DNA Sequencing
Sequencea (5’ > 3’)
27f
518r
530f
907r
926f
1114f
1392r
1525r
a
AGAGTTTGATCMTGGCTCAG
GWATTACCGCGGCKGCTGGCAC
GTGCCAGCMGCCGCGG
CCGTCAATTCMTTTRAGTTT
AAACTYAAAKGAATTGACGG
GCAACGAGCGCAACCC
ACGGGCGGTGTGTRC
AAGGAGGTGWTCCARCC
M=C:A, Y=C:T, K=G:T, R=A:G, W=A:T; all 1:1.
16S rDNA
27f
518r
530f
1114f
907r
926f
27f
1525r
1392r
907r
530f
1392r
Figure 1. The location of
primer sequences in 16S
rDNA and the pairs of
primers used for DNA
sequencing.
M13 sequence (5’-CACGACTTGTAAAACGAC-3’) was attached to the 5’ end
of each primer set to initiate sequencing of the DNA. PCR was carried out with Ex30
CHAPTER 2
Taq polymerase (Takara Shuzo, Ltd., Japan) in a GeneAmp 2400 PCR System
(Perkin-Elmer Corp., USA). The thermal cycling program consisted of four
continuous stages: (1) 94°C for 5 min. (2) five amplification cycles of [94°C for 1
min, 55°C for 2 min and 74°C for 2 min.] (3) 25 amplification cycles of [98°C for
30 sec. and 68°C for 2 min.] (4) 72°C for 10 min. After PCR, the amplified DNA
fragments were separated through agarose gel electrophoresis and eluted from the
gel with a SUPREC-01 spin column (Takara Shuzo, Ltd., Japan). The resulting
DNA fragment was used as a template in the following DNA sequencing
procedures using a SequiTherm Long-Read Cycle Sequencing Kit-LC (Epicentre
Technologies, USA) and a LI-COR 4000L automatic sequencing system (LI-COR,
USA) according to the manufacturer’s instructions. The infrared dye labeled IRDM13 forward (-29) primer (Aloka Co., Ltd., Tokyo, Japan) was used as the
sequencing primer. The DNA sequence was confirmed by both sense and antisense
sequencing.
Specific primers
A number of primers were designed and synthesized according to the speciesspecific sequence regions in the 16S rDNA of beer spoilage bacteria. Comparative
analysis of the sequences of the 16S rDNA was performed with DNASIS software
(Hitachi Software Engineering Co., Japan). The reactivity of each primer with
various bacteria, including both beer spoiling and non-spoiling bacteria, was
examined. The PCR was done in 30 cycles consisting of 1 min denaturalizing at
94°C, 1 min annealing at 55°C and 1 min elongation at 74°C. The PCR products
were separated through an agarose gel at 5 V/cm for 30 min. As the size standard, λ
DNA digested by Hind III or ΦX174 DNA digested by Hinc II was used. The
DNA-bands in the gel were visualized by ethidium bromide staining.
Simultaneous PCR
A set of universal primers (1114f-1392r) was employed as probe for an internal
positive control, because its target region is outside that of the specific primers in
16S rDNA. It was used together with each specific primer set in the same reaction
tube. PCR was performed as described above, but the concentration of universal
primers was 50 times more diluted than that of the specific primers (final
concentration: 15 pM).
31
CHAPTER 2
RESULTS
16S rDNA sequence of Pectinatus sp. DSM20764
The oligonucleotide sequence of 16S rDNA of Pectinatus sp. DSM20764 was
determined and is shown in Fig. 2. A comparison with DNASIS software of this
16S rDNA with that of other Gram-negative beer spoilage bacteria, showed 90.7%
similarity of the 16S rDNA sequences of Pectinatus sp. DSM20764 with P.
frisingensis, 89.0% with P. cerevisiiphillus, 82.4% with Zymophillus pausivorans
and 80.2% with Megasphaera cerevisiae.
AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAAC
GGGACTTTTATTTCGGTAAAAGTCTAGTGGCAAACGGGTGAGTAACGCGTAGGCAACCTA
CCTTCAAGATGGGGACAACATCCCGAAAGGGGTGCTAATACCGAATGTTGTAAGAGTACT
GCATGGTACTTTTACCAAAGGCGGCTTTTAGCTGTTACTTGGAGATGGGCCTGCGTCTGA
TTAGCTAGTTGGTGACGGTAATGGCGCACCAAGGCAACGATCAGTAGCCGGTCTGAGAGG
ATGGACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGG
AATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAACGAGGAAGGTCTTC
GGATCGTAAAGTTCTGTTGCAGGGGACGAATGGCATTAGTGCTAATACCACTAATGAATG
ACGGTACCCTGTTAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGG
CGGCAAGCGTTGTCCGGAATCATTGGGCGTAAAGGGAGCGCAGGCGGACATTTAAGCGGA
TCTTAAAAGTGCGGGGCTCAACCCCGTGATGGGGTCCGAACTGAATGTCTTGAGTGCAGG
AGAGGAAAGCGGAATTCCCAGTGTAGCGGTGAAATGCGTAGATATTGGGAAGAACACCAG
TGGCGAAGGCGGCTTTCTGGACTGTAACTGACGCTGAGGCTCGAAAGCCAGGGTAGCGAA
CGGGATTAGATACCCCGGTAGTCCTGGCCGTAAACGATGGATACTAGGTGTAGGGGGTAT
CGACCCCCCCTGTGCCGGAGTTAACGCAATAAGTATCCCGCCTGGGGAGTACGGCCGCAA
GGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATT
CGACGCAACGCGAAGAACCTTACCAGGGCTTGACATTGATTGACGCATTCAGAGATGGAT
GCTTCCTCTTCGGAGGACAAGAAAACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTG
AGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCATTTGTTGCCAGCACGTAAC
GGTGGGAACTCAAATGAGACTGCCGCGGACAACGCGGAGGAAGGCGGGGATGACGTCAAG
TCATCATGCCCCTTACGTCCTGGGCTACACACGTACTACAATGGGATACACAGAGGGAAG
CAAAGGAGCGATCCGGAGCGGAACCCAAAAAATATCCCCCAGTTCGGATTGCAGGCTGCA
ACTCGCCTGCATGAAGTCGGAATCGCTAGTAATCGCAGGTCAGCATACTGCGGTGAATAC
GTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAAAGTCATTCACACCCGAAGCCG
GCTAAGGGCCTTATGGAACCGACCGTCTAAGGTGGGGGCGATGATTGGGGTGAAGTCGTA
ACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTT
60
120
180
240
300
360
420
480
540
600
660
720
780
840
900
960
1020
1080
1140
1200
1260
1320
1380
1440
1500
1542
Figure 2. Sequence of 16S rDNA of Pectinatus sp. DSM20764. The number on the right
indicates the last nucleotide of each row. (Genbank accession number AR034890, Patent
No; JP09-520359, WO97/20071 or USP5869642).
Specificity of the primers
A number of 16S rDNA primers of beer spoilage bacteria were tested, including
of Lb brevis, Lb. casei, Lb. coryniformis, Lb. plantarum, Pectinatus cerevisiiphilus,
P. frisingensis, P. sp. DSM20764, and of Megasphaera cerevisiae. The most
specific primers for each bacterium were obtained by screening for specificity and
sensitivity, which allowed the construction of highly specific primer sets (Table 3).
32
CHAPTER 2
The specificity of those primers is shown in Fig. 3. No reactivity was observed of
these primers with DNA from the other bacteria or brewing yeast strains (data not
shown).
Primer set
Table 3. The Specific primers
Specific to
Sequencea (5’ > 3’)
Lb
Lb. brevis
Lca
Lb. casei
Lco
Lb. coryneformis
Lp
Lb. plantarum
Pc
P.cerevisiiphilus
Pf
P. frisingensis
Psp
P. sp. DSM20764
Mc
M. cerevisiae
Sizeb
c
CTGATTTCAACAATGAAGC
CCGTCAATTCCTTTGAGTTTd
ATCCAAGAACCGCATGGTTCTTGGCc
CCGTCAATTCCTTTGAGTTTd
GGGACTAGAGTAACTGTTAGTCC c
CCGTCAATTCCTTTGAGTTTd
TGGACCGCATGGTCCGAGCc
CCGTCAATTCCTTTGAGTTTd
CAGGCGGATGACTAAGCGe
AATATGCATCTCTGCATACGe
CAGGCGGAACATTAAGCG e
CTCAAGAACCTCAGTTCGe
TGGGGTCCGAACTGAATGe
GCATCCATCTCTGAATGCGe
CTGCCGGACTGGAGTGTC
CAGGATATCTCTATCCCTGG
861
735
453
731
443
74
393
385
a
Upper sequence: forward primer, lower sequence: reverse primer.
The size of PCR products is shown in base pairs.
c
Patent pending (JP10-210980).
d
The reverse primer for Lactobacillus spp. is the same as the universal primer 907r in Table 2.
e
Patent pending (JP09-520359, WO97/20071), or registered (USP5869642).
b
Simultaneous PCR
Two separate PCR products were obtained from a sample containing a beer
spoilage bacterium when a set of universal primers was used together with the
specific primer set. An example is shown in Fig. 4. Two bands were detected from
a sample containing M. cerevisiae (lane 1). The band of 308bp was amplified by
the universal primers and the band of 385bp was amplified by the primers specific
for M. cerevisiae. Only one PCR product of 308bp, amplified only by the universal
primers, was obtained from the samples containing the other bacteria (lane 2-4).
The absence of the 308bp band (lane 5-7) reveals that unsuccessful PCR is
responsible for the failure of the test procedure.
33
CHAPTER 2
1 2 3 4 5 6 M1
M1 1 2 3 4 5 6
a
M2 7 8 9 10 11 12
b
1 2 3 4 5 6 M1 M1 1 2 3 4 5 6
c
M2 7 8 9 10 11 12 M2 7 8 9 10 11 12
e
f
g
d
7 8 9 10 11 12 M2
h
Figure 3. Specific detection of beer spoilage bacteria. Reaction of specific primers with
DNA from various bacteria. The following specific primers were used: Lb (a), Lca (b), Lco
(c), Lp (d), Pc (e), Pf (f), Psp (g), and Mc (h) (See Table 3). The number above each lane
represents the bacterial species from which the total DNA was used as template. Lane 1:
Lb. brevis, 2: Lb. casei, 3: Lb. plantarum, 4: Lb. coryneformis, 5: Lb. lindneri, 6: Lb. sp. (a
non beer-spoilage strain), 7: P. cerevisiiphilus, 8: P. frisingensis, 9: P. sp. DSM20764, 10:
M. cerevisiae, 11: S. lacticifex, 12: Z. pausivorans. M1: λ DNA/Hind III DNA size marker,
M2: ΦX174/Hinc II DNA size marker.
1 2 3 4
34
5 6 7 M
Figure 4. Application of universal primers as an
internal positive control. PCR was performed with
the primer set specific for M. cerevisiae (Mc in Table
3) and the set of universal primers (1114f-1392r in
Table 2). Successful DNA extraction and PCR is
indicated by the appearance of a 308bp band amplified
by the universal primers (lane 1-4), while the absence
of this band indicates the failure of these procedures
(lane 5-7). The band of 385 bp in the lane 1 indicates
the presence of M. cerevisiae in the sample. Lane 1: a
bacterial mixture including both M. cerevisiae and non
beer-spoilage bacteria isolated from breweries. Lane 27: bacterial mixtures of non beer-spoilage bacteria
from breweries (different batches of DNA extraction).
M: ΦX174/Hinc II DNA size marker.
CHAPTER 2
DISCUSSION
This chapter presents the sequence of 16S rDNA of Pectinatus sp. DSM20764.
This gene is 89 to 91% similar to genes of other anaerobic Gram-negative beer
spoilage bacteria, indicating that this bacterium belongs to the genus Pectinatus but
differs from the other known Pectinatus species. The taxonomical information of
this strain is so far only available from DSMZ catalogue
(http://www.gbf.de/dsmz/strains/no020764.htm, personal communication from Dr.
N. Weiss). Since this strain has also been isolated from spoiled beer (Seidel, H.,
personal communication), the sequence of the gene is of great importance for
microbial quality control in breweries.
Here we report the first specific primers for P. cerevisiiphilus, P. frisingensis, P.
sp. DSM20764 and M. cerevisiae. In contrast to the previously reported primers for
lactic acid bacteria (Tsuchiya et al., 1992; Thompson et al., 1994) the primers
developed in this study are sufficiently specific to identify the bacteria at the
species level. Thus specific primers for the identification of all major beer spoilage
bacteria are now available.
The PCR method is very rapid, specific and highly sensitive but since it is an
enzymatic reaction it is subject to experimental problems. Failure of PCR can cause
failure to detect beer spoilage bacteria in samples from breweries (Di Michele,
1993). Several factors have been reported to inhibit the PCR reaction (Rossen et
al., 1992). For some strains belonging to lactic acid bacteria it is known that
extraction of DNA is difficult due to the presence of thick cell wall and/or
extracellular slime (Anderson and McKay, 1983). If DNA extraction fails,
detection of spoilage bacteria by PCR fails, which can lead to false negative
results. Cone et al. (1992) used a positive control template, specially designed and
constructed to be amplified with the same primers of the targeted gene to reveal
that PCR worked properly. To apply their method for a large variety of bacterial
species, special templates for each species had also to be developed. In stead of
constructing these special templates the consensus sequences in 16S rDNA were
used here as a positive control template. The universal primer amplifies a 308 bp
fragment and reveals successful extraction of DNA and PCR. In this way false
negative results can be eliminated. The combined use of specific and universal
primers in one reaction has improved considerably the reliability of PCR detection
of beer spoilage bacteria and the quality assurance by PCR in breweries.
35
36
CHAPTER 3
Electrotransformation of Lactobacillus brevis
Kanta Sakamoto
This chapter was submitted to Applied and Environmental Microbiolgy.
SUMMARY
The conditions for electrotransformation of five Lactobacillus brevis strains
lacking horA or its homologue were investigated. Two of them were successfully
transformed. The highest efficiency was 2.5 × 103 transformants per µg of DNA
(T/µg) for JCM1059 and 5.5 × 102 T/µg for ABBC45C, a segregant strain lacking
pRH45. No transformants were obtained from the other strains.
37
CHAPTER 3
INTRODUCTION
The horA gene was isolated from a hop resistant strain of Lb. brevis ABBC45
(Sami et al., 1997a). This gene is present on plasmid pRH45, which is amplified
during subculturing of the strain in medium containing increasing concentrations of
hop compounds. Most beer-spoilage lactobacilli strains harbor the horA gene or its
homologue (Sami, 1997b). The deduced amino acid sequence of HorA is very
homologous to that of LmrA, a lactococcal ABC-type multidrug transporter (van
Veen et al., 1996) which makes HorA a potential player in the hop resistance
mechanism.
Attempts to express HorA in Escherichia coli failed due to cell lysis soon after
the expression of this protein (Sami, 1999). Also the introduction of a marker gene
into pRH45 was unsuccessful possibly due to the instability of plasmid DNA
fragments in E. coli (Sami et al., 1997a). Recently Lb. brevis ABBC45C, which
spontaneously had lost pRH45, was segregated from the original strain ABBC45
by continuous culturing in the absence of hop compounds (Sami et al., 1998;
Suzuki et al., 2002). Re-introduction of pRH45 in ABBC45C or the other strains
lacking horA would be extremely helpful for studying the role of this protein in hop
resistance.
Successful gene transformation of Lactobacillus spp. has been developed
(Chassy and Flicklinger, 1987; Luchansky et al., 1988; Hashiba et al., 1990;
Aukrust and Blom, 1992; Bhowmik and Steele, 1993; Sasaki et al., 1993; Aukrust
et al., 1995; Klein et al., 1995; Berthier et al., 1996; Serror et al., 2002) but
successful transformation of Lb. brevis has not yet been reported. In this study the
conditions for electrotransformation of Lb. brevis were determined. The developed
transformation procedure was used for the re-introduction of pRH45 into
ABBC45C (Sami et al., 1998: Chapter 3).
MATERIALS AND METHODS
Bacteria and a plasmid
Lactobacillus brevis type strain JCM1059 was obtained from JCM (Japan
Collection of Microorganisms, Saitama, Japan). Lb. brevis ABBC45 and horAlacking strains 45C, 216, 218 and 241 (Sami et al., 1997b) were from our laboratory
culture collection.
The plasmid pGK13, which has both a chloramphenicol resistance gene and an
erythromycin resistance gene, was a gift from Dr. J. Kok (University of Groningen,
The Netherlands) and prepared from Esherichia coli DH5α.
Preparation of competent cells
The method previously described by Aukrust et al. (1995) was used with some
38
CHAPTER 3
modification. Cells were cultured anaerobically at 30°C in 100 ml of MRS medium
supplemented with 1% (w/v) glycine. At mid-exponential phase (A600= ~0.6), cells
were chilled on ice for 10 min and harvested by centrifugation at 1,500 × g for 5
min, washed once with 100 ml of cold 10 mM MgCl2 and collected by
centrifugation at 1,500 × g for 5 min. Subsequently, cells were washed with 100 ml
of cold SM solution (952 mM sucrose and 3.5 mM MgCl2) and harvested by
centrifugation at 5,000 × g for 10 min. After repeating this step twice, cells were
suspended gently in 1 ml of cold SM solution and used for electroporation.
Electroporation
Competent cells (40 µl) prepared as described above were mixed with 0.2 µg of
pGK13 and subjected to an electric pulse in a 0.1 cm cuvette by using a Gene
Pulser and a Pulse Controller apparatus (Bio-Rad, USA). Immediately MRS-SM
medium (MRS containing 0.5 M sucrose and 0.1 M MgCl2) was added and cells
were incubated for 2 h at 30°C before plating on MRS containing 15 µg/ml of
chloramphenicol and 5 µg/ml of erythromycin.
RESULTS
Among the Lb. brevis strains of our bacterial collection, five (ABBC45C, 216,
218, 241, and the type strain JCM1059) were found to lack horA or its homologue
(Sami et al., 1997b). In order to transform successfully pRH45 in these strains, the
electroporation procedure for Lb. brevis was optimized.
Aukrust et al. (1995) reported two different methods for preparing competent
cells of lactobacilli. One of them includes a SM solution to wash cells (SM
method), while the other includes a polyethylene glycol solution (PEG method).
Both methods were applied for Lb. brevis ABBC45. The SM method was found to
yield 2 to 40 times higher transformation efficiencies than the PEG method (data
not shown). The SM method was further optimized in this study. Some strains have
extracellular polysaccharides, which can be removed by a washing step with 10
mM MgCl2. (Aukrust et al., 1992; Berthier et al., 1996). To improve
electroporation of competent cells MgCl2 was therefore included. Due to the high
osmolarity of the SM solution some strains did not pellet at 1,500 or 3,000 × g
centrifugation for 10 min and centrifugation at 5,000 × g for 10 min was needed for
successful pelleting. Parameters of the electrical pulse (capacitance, resistance and
voltage) were varied. The highest transformation efficiency of 2.5 × 103 T/µg for
JCM1059 was obtained at 25 µF, 100 or 200 Ω and 2.0 kV (Fig. 1A). The
transformation efficiency increased with the capacitance from 0.25 to 25 µF when
the other parameters were fixed at 400 Ω and 1.5 kV (data not shown). For
ABBC45C the highest efficiency was 2.9 × 102 T/µg at 25 µF, 200 Ω and 1.5 kV
(Fig. 1B). No transformants were obtained for ABBC216, 218 and 241.
39
3000
2000
2.5
2.0
1000
1.5
0
1.0
V (kV)
Transformation efficiency (T/µg)
A
10
0
20
0
40
0
60
0
80
0
10
00
Transformation efficiency (T/µg)
CHAPTER 3
B
300
200
100
0
100 200
400 600
800
1.5
2.0
2.5 V
(kV)
R (Ω)
R (Ω)
Figure 1. Transformation efficiency of Lb. brevis JCM1059 (A) and ABBC45C (B). The
competent cells were electroporated with pGK13 at various resistance (R) and voltage (V)
values. The capacitance was fixed at 25 µF.
DISCUSSION
In this study we demonstrated for the first time successful electrotransfomation
of Lb. brevis strains. The transformation efficiency varied strongly between the
different strains. The highest transformation efficiency was in the order of 103 T/µg
for JCM1059 and of 102 T/µg for ABBC45C. These values are comparable to those
of other Lactobacillus spp. reported so far. The type strain of Lb. brevis JCM1059
has no plasmid, which makes this a very useful strain for genetic and molecular
studies for Lb. brevis. However several attempts to transform Lb. brevis JCM1059
with pRH45 failed (data not shown). The main reason for this failure can be the
large size of plasmid pRH45 (15 kb). Successful electroporation of pRH45 into Lb.
brevis ABBC45C was achieved and resulted in restoration of hop resistance (Sami
et al., 1998, Chapter 3).
ACKNOWLEDGEMENT
The writer thanks Dr. J. Kok (University of Groningen, The Netherlands) for the
gift of pGK13.
40
CHAPTER 4
A Plasmid pRH45 of Lactobacillus brevis Confers
Hop Resistance
Manabu Sami, Koji Suzuki, Kanta Sakamoto, Hiroshi Kadokura,
Katsuhiko Kitamoto and Koji Yoda.
This chapter is a modified version of the manuscript published in Journal of
General Applied Microbiology, 44:361-363 (1998) with some additional
information.
SUMMARY
Lactobacillus brevis ABBC45C was segregated from the original strain ABBC45
after repeating subculturing in the absence of hop compounds. Lb. brevis
ABBC45C lacks the hop-resistance related plasmid pRH45 that contains horA of
which the deduced amino acid sequence is 53% identical to LmrA, a lactococcal
ATP-binding cassette (ABC) multidrug transporter. Lb. brevis ABBC45C is less
resistant than ABBC45 to hop compounds and ethidium bromide (EtBr). When
pRH45 was re-introduced into Lb. brevis ABBC45C by electroporation, the degree
of resistance to hop compounds and EtBr was restored to the resistance level of Lb.
brevis ABBC45. Energized cells of Lb. brevis ABBC45C show, in the presence of
nigericin, a higher rate of ethidium accumulation than cells of ABBC45. These
results indicate that pRH45 confers hop resistance in Lb. brevis ABBC45 by
excreting hop compounds by the multidrug ABC-type transporter HorA.
41
CHAPTER 4
INTRODUCTION
The bitter compounds in beer derived from the hop plant are important for the
protection of beer from bacterial spoiling (Simpson and Smith, 1992). However,
some lactic acid bacteria, especially those belong to Lactobacillus spp., exhibit
resistance to hop compounds and grow in beer, thereby causing serious problems
for the brewing industry. The mechanism(s) of hop resistance of lactobacilli is
poorly understood. A biochemical study suggested that unidentified components of
the plasma membrane are responsible for the resistance (Simpson and Fernandez,
1994).
In our previous work, we obtained a hop-resistant mutant from Lb. brevis
ABBC45 (Sami et al., 1997a). This mutant was found to carry a plasmid, pRH45,
at a higher copy number than the wild type. Plasmid pRH45 contains horA, an open
reading frame of 1749 nucleotides (DDBJ accession no. AB005752). HorA has six
putative transmembrane domains and an ATP-binding motif (Sami et al., 1997a).
The deduced amino acid sequence of HorA shows significant similarity with the
bacterial multidrug transporter LmrA (van Veen et al., 1996) and the mammalian
multidrug transporter MDR1 (Chen et al., 1986). LmrA has been identified as an
ATP-binding cassette (ABC) transporter, which confers resistance of Lactococcus
lactis to various lipophilic toxic compounds, including ethidium bromide (Bolhuis
et al., 1995; van Veen et al., 1996). Almost all lactobacilli isolated as beer-spoilage
strains were found to possess a horA-like gene (Sami et al., 1997b). However,
direct evidence for the involvement of plasmid pRH45 in hop resistance of Lb.
brevis has not yet been provided. Since elucidation of the mechanism(s) of hop
resistance is of crucial importance for the brewing industry, we investigated the
contribution of pRH45 in this hop resistance. In this study we obtained a segregant
strain ABBC45C, which had spontaneously lost the plasmid pRH45 and which
allowed us to demonstrate that horA on pRH45 is responsible for hop resistance.
MATERIALS AND METHODS
Bacterial culture
Lactobacillus brevis strains were grown anaerobically at 30ºC in MRS broth
(Merck, Darmstadt, Germany, initial pH adjusted to 5.5 with HCl). Anaerobic
conditions were generated by AnaeroPack (Mitsubishi Gas Chemical, Tokyo,
Japan). Cells were stored in MRS broth containing 20% glycerol at –80ºC.
Segregation of ABBC45C
The wild-type Lb. brevis strain, ABBC45, was repeatedly subcultured by
inoculating 105 cells in 5 ml of MRS broth every 2 to 3 days. After 15 subcultures,
single colonies were isolated and plasmid DNAs were purified by the method of
42
CHAPTER 4
Anderson and McKay (1983). Plasmid profiles of the isolates and ABBC45 were
investigated by 0.75% agarose gel electrophoresis in TAE buffer (0.04 M Trisacetate, 0.01 M EDTA, pH 8.0). A polymerase chain reaction (PCR) was
performed using the total DNA extracted from Lb. brevis ABBC45C as a template
and two specific primer sets based on the ori and the horA sequences of pRH45. A
Southern blot analysis was also done for the plasmid DNAs of Lb. brevis
ABBC45C, using horA-specific DNA as a probe.
Re-introduction of pRH45
Re-introduction of pRH45 into Lb. brevis ABBC45C was done by
cotransformation by electroporation with a generalized plasmid of lactic acid
bacteria, pGK13 (Kok et al., 1984), containing a chloramphenicol-resistant gene.
The competent cells were prepared by the method of Sakamoto (See Chapter 3).
Cells of Lb. brevis ABBC45C, grown in 50 ml of MRS containing 1% glycine,
were harvested by centrifugation at early exponential phase, washed once with cold
3.5 mM MgCl2 and twice with cold SM (925 mM sucrose, 3.5 mM MgCl2) and
finally suspended in 500 µl SM. Plasmid DNAs extracted from Lb. brevis
ABBC45, containing 1 µg of pRH45, were added to 40 µl of competent cell
suspension together with 10 ng of pGK13. Electroporation was done at 200 Ω, 2.0
kV and 0.25 µF by using Gene Pulser (Bio-Rad, Hercules, CA, USA), as described
(See Chapter 3). Filtered MRSM medium (960 µl total) (MRS broth containing 0.5
M sucrose and 0.1 M MgCl2) was added, and the cell suspension was incubated at
30ºC for 2 h. Cells were harvested by centrifugation (5,500 × g, 4ºC, 5 min), spread
on the MRS agar plate containing 15 µg/ml chloramphenicol and 50 µM hop
compounds, and incubated anaerobically at 30ºC for 4 days.
Drug resistance
Exponentially growing cells were diluted with sterile deionized water to a
concentration of 106 cells/ml. These cell suspensions (5 µl) were spotted on MRS
agar plates containing various concentrations of hop compounds or EtBr, and the
minimum inhibitory concentrations (MICs) were determined.
Ethidium acculmulation
A washed cell suspension of Lb. brevis ABBC45C or ABBC45 in HEPES (50
mM potassium-HEPES supplemented with 3 mM MgSO4, pH 7.5) with an A600 of
0.7 was incubated with 10 mM EtBr after the preincubation of cells with 10 mM Larginine and 4 µM nigericin at 30ºC for 10 min. L-arginine was added to generate
ATP by the arginine deiminase pathway (Cunnin et al., 1986) and nigericin for
dissipating the transmembrane pH-gradient to prevent the action of pmf-driven
transporters. Fluorescence was measured with an F-2000 fluorometer (Hitachi,
Tokyo, Japan) for 20 min, using excitation and emission wavelengths of 500 and
43
CHAPTER 4
580 nm, respectively.
RESULTS
Lb. brevis ABBC45C lacks both pRH45 and horA
A comparison of the plasmid profiles of Lb. brevis ABBC45 and of the segregant
ABBC45C obtained after repeated subcultering of ABBC45 in the absence of hop
compounds shows that ABBC45C
Figure 1. Plasmid profiles of 1 2 3
has lost pRH45, while the other
Lb. brevis ABBC45.
plasmids remained unchanged
Plasmids extracted from wild
(Fig. 1). With the specific primer
type Lb. brevis ABBC45 (lane
sets for pRH45 and horA no PCR
2) and ABBC45C (lane 3),
were subjected to agarose gel
products were recognized in the
electrophoresis.
Lane
1
total DNA of Lb. brevis ABBC45C
contains
molecular
weight
(data not shown). Also with the
standards (λ DNA digested
horA-specific DNA probe no
with Hind III). The position of
discrete band could be detected by
pRH45 is indicated by an
a Southern blot analysis with the
arrow at the right side of the
plasmid DNAs of Lb. brevis
figure.
ABBC45C (data not shown).
Re-introduction of pRH45
Genetic engineering of lactic acid bacteria is not always possible. Attempts to
introduce a marker gene in pRH45 were unsuccessful, possibly due to the
instability of several DNA fragments of pRH45 in E. coli (Sami et al., 1997a).
Cotransformation of Lb. brevis ABBC45C with pGK13 and pRH45 resulted in
seven colonies. Examination of the plasmid profiles of these colonies revealed that
four colonies had been successfully transformed with both pRH45 and pGK13
(ABBC45C[pRH45, pGK13]; Fig. 2, lane 7, 8, 11, 12), while the other three
colonies contained only pGK13 but not pRH45 (Fig. 2, lane 9, 10, 13).
Transformation of Lb. brevis ABBC45C was also done with pGK13 alone
(ABBC45C[pGK13]; Fig. 2, lane 5, 6). Although the copy number of pGK13 in the
transformants was small, significant higher resistance to chloramphenicol was
realized. Of the four transformants of Lb. brevis ABBC45C[pRH45, pGK13], only
one had retained the original plasmid profile of ABBC45C and pRH45 and pGK13
(Fig. 2, lane 11). This transformant was resistant to hop compounds up to 600 µM
while the other transformants were only resistant hop compounds up to 300 µM
(data not shown). This hop resistant transformant was used in the following
experiments.
44
CHAPTER 4
(kb)
M
1
2
3
4
5
6
7
8
9 10 11 12 13
23.1
9.4
pRH45
6.6
4.4
pGK13
2.3
2.0
Figure 2. The plasmid profiles of wild type Lb. brevis ABBC45, pRH45-free segregant
ABBC45C and transformants of Lb. brevis ABBC45C. Plasmids extracted from ABBC45
(lane 1), the pRH45-free segregant ABBC45C (lanes 2, 3; different amounts of DNA from
the same sample were applied on the gel), ABBC45C transformed with pGK13 (lanes 5, 6;
different amounts of DNA from the same sample were applied on the gel), and seven
transformants of ABBC45C electroporated with pGK13 and pRH45 (lanes 7-13) were
subjected to agarose gel electrophoresis. Lane 4 contains pGK13 extracted from the host
strain of E. coli. The bands around 2.0, 3.0, and 5.0 kb in lane 4 correspond to the closed
circular DNA, the open circular DNA, and the linear DNA of pGK13, respectively. The
positions of pRH45 and the open circular DNA of pGK13 are indicated by arrows on the
right. M: molecular weight standards (λ DNA digested with Hind III) and their molecular
weight (kb) are indicated on the left.
Drug resistance
Upon loss of pRH45 the MIC to hop compounds of Lb. brevis ABBC45
decreased by a factor of 2 and this MIC was completely restored upon the reintroduction of pRH45 (Table 1). These results indicate that pRH45 contributes to
resistance of Lb. brevis ABBC45 to hop compounds. Similar results were obtained
for the resistance to EtBr. These differences in resistance were reproducibly
observed in many experiments.
Strains
Table 1. Drug resistance of Lb. brevis ABBC45
MICa
Hop compounds (µM)
EtBr (µg/ml)
ABBC45 (wild type)
ABBC45C
ABBC45C[pRH45, pGK13]
ABBC45C[pGK13]
200
100
200
100
30
15
30
15
a
Minimum inhibitory concentrations were determined from cell growth on MRS agar plates
containing various concentrations of hop compounds or ethidium bromide. MICs of hop compounds
are expressed as iso-α-acids concentrations (Simpson, 1993).
45
CHAPTER 4
Ethidium Fluorescence (a.u.)
Accumulation of Ethidium
Ethidium readily diffuses across the cell membrane and enters the cytoplasm.
Upon intercalation with DNA or RNA its fluorescence increases approximately 10fold (Le Pecq and Paoletti, 1967). The amount of the intracellular ethidium can
therefore be followed fluorometrically. The rate of ethidium accumulation was
significantly faster for cells of Lb. brevis ABBC45C than for cells of ABBC45 (Fig.
3).
Figure 3. Accumulation of ethidium. The ethidium fluorescence
development of cells of a pRH45-free segregant Lb. brevis
ABBC45C (●) and the wild-type ABBC45 (■) was shown. Cells
were preincubated with 10 mM L-arginine and 4 µM nigericin in
HEPES (50 mM HEPES, 25 mM K2SO4, 5 mM MgSO4, pH 7.5)
for 10 min. At zero time the assay was started by the addition of
10 µM ethidium bromide to the cell suspension. The fluorescence
(arbitrary units, a.u.) was measured for 20 min by using the
excitation wavelength of 500 nm and the emission wavelength of
580 nm.
0
5
10
15
20
Time (min)
DISCUSSION
The resistance to hop compounds and EtBr of Lb. brevis ABBC45 decreased
when pRH45 was lost and completely recovered upon re-introduction of pRH45. In
previous studies was found that the copy number of pRH45 increased with the
resistance to EtBr and novobiocin when Lb. brevis ABBC45 was acclimatized to
higher concentrations of hop compounds (Sami et al., 1997a). The excellent
correlation of the level of resistance to hop compounds and other drugs with the
copy number of pRH45 indicates a crucial role of pRH45 in conferring multidrug
resistance.
Ethidium is a substrate of many bacterial multidrug resistant transporters,
including BmrB of Bacillus subtilis (Neyfakh et al., 1991), QacA of
Staphylococcus aureus (Tennet et al., 1989), and LmrP (Bolhuis et al., 1995) and
LmrA (van Veen et al., 1996) of Lactococcus lactis. The rate of ethidium
accumulation in an MDR containing bacterium is determined by the rates of
diffusion into and the pumping out of the cell (Bolhuis et al., 1994). Lb. brevis
ABBC45C was found to accumulate ethidium faster than ABBC45 in the presence
of nigericin. In these experiments nigericin was used to collapse the
transmembrane pH-gradient in order to inhibit proton-motive-force dependent
transporters. These results suggest therefore that the activity of the ATP-driven
46
CHAPTER 4
extrusion system for ethidium is lower in the pRH45-free segregant Lb. brevis
ABBC45C than in the wild type ABBC45. Among the four transformants of
ABBC45C[pRH45, pGK13] only the one, which contained pRH45 and pGK13 and
had retained the original plasmid profile of ABBC45C, showed the wild strain
ABBC45 level of hop resistance. It is concluded that the multidrug ABCtransporter HorA, encoded by horA on pRH45, is responsible for the increased
resistance to hop compounds and ethidum.
ACKNOWLEDGEMENT
We thank Dr. M. Yamasaki (Nihon University, Japan), Dr. T. Sasaki (Meiji Milk
Products Co. Ltd., Japan), and Dr. K. Abe (Kikkoman Co. Japan) for helpful
suggestions, and Dr. J. Kok (University of Groningen, The Netherlands) for the gift
of pGK13.
47
48
CHAPTER 5
Hop Resistance in the Beer Spoilage Bacterium
Lactobacillus brevis Is Mediated by the ATP-Binding
Cassette Multidrug Transporter HorA
Kanta Sakamoto, Abelardo Margolles, Hendrik W. van Veen and Wil N. Konings.
This chapter was published in Journal of Bocteriology (2001) 183:5371-5375 with
some correction.
SUMMARY
Lactobacillus brevis is a major contaminant of spoiled beer. The organism can
grow in beer in spite of the presence of antibacterial hop compounds that give the
beer a bitter taste. The hop resistance in Lb. brevis is, at least in part, dependent on
the expression of the horA gene. The deduced amino acid sequence of HorA is 53%
identical to that of LmrA, an ATP-binding cassette multidrug transporter in
Lactococcus lactis. To study the role of HorA in hop resistance, HorA was
functionally expressed in L. lactis as a hexa-histidine-tagged protein using the
nisin-controlled gene expression system. HorA expression increased the resistance
of L. lactis to hop compounds and cytotoxic drugs. Drug transport studies with
L. lactis cells and membrane vesicles and with proteoliposomes containing purified
HorA protein identified HorA as a new member of the ABC family of multidrug
transporters.
49
CHAPTER 5
INTRODUCTION
Bacterial spoilage of beer products causes a serious problem in the brewing
industry. The iso-α-acids, derived from the flowers of the hop plant (Humulus
lupulus L.), give beer a bitter taste and exert bacteriostatic effects on most Grampositive bacteria due to their ability to dissipate the proton motive force (Simpson
and Smith, 1992; Simpson, 1993b; Simpson and Fernandez, 1994). A few lactic
acid bacteria, such as Lactobacillus spp., are tolerant towards iso-α-acids and are
able to grow in hopped beer (Simpson and Fernandez, 1992; Simpson, 1993a). At
present, the molecular mechanisms that underlie the hop resistance in lactic acid
bacteria are not well understood.
Previously, Sami and colleagues have isolated a hop-tolerant Lactobacillus brevis
strain, ABBC45, in which the plasmid pRH45 confers hop resistance on the cells
(1998). pRH45 harbors the horA gene, whose corresponding deduced amino acid
sequence is 53% identical to that of the multidrug transporter LmrA in Lactococcus
lactis (van Veen et al., 1996; Sami et al., 1997a). LmrA is a multidrug transporter
able to transport a wide variety of amphiphilic compounds, including antibiotics
and anticancer drugs, from the inner leaflet of the cytoplasmic membrane (Bolhuis
et al., 1996; Shapiro and Ling, 1999; Putman et al., 2000b). Unlike other known
bacterial multidrug resistance proteins, LmrA is an ATP-binding cassette (ABC)
transporter (Higgins, 1992; van Veen and Konings, 1998). The protein contains an
N-terminal membrane domain with six transmembrane segments followed by the
ABC domain. Surprisingly, LmrA is a structural and functional homologue of the
human multidrug resistance P-glycoprotein, overexpression of which is one of the
principal causes of resistance of human cancer cells to chemotherapy, and can even
complement P-glycoprotein in human lung fibroblast cells (van Veen et al., 1998a).
In this work, HorA was functionally overexpressed in L. lactis as a hexahistidine-tagged protein. The hop resistance of L. lactis cells was increased
significantly as a result of HorA expression. The protein was purified by Ni2+nitrilotriacetic acid (NTA) affinity chromatography and functionally reconstituted
into proteoliposomes prepared from L. lactis lipids. Transport studies with cells,
membrane vesicles, and proteoliposomes identified HorA as a multidrug
transporter which mediates the extrusion of structurally unrelated compounds,
including iso-α-acids.
MATERIALS AND METHODS
Bacterial strains and growth conditions
Lactobacillus brevis ABBC45 (Sami et al., 1998) was grown anaerobically at
30°C in MRS broth (Merck). Lactococcus lactis subsp. lactis NZ9000 was used as
a host for the nisin-controlled gene expression (NICE) vector pNZ8048 (de Ruyter
50
CHAPTER 5
et al., 1996) and its horA-containing derivatives. L. lactis was grown at 30°C in
M17 broth (Difco) supplemented with 5 µg of chloramphenicol/ml and with 0.5%
glucose (wt/vol) when appropriate.
Genetic manipulations
The horA gene was amplified from pRH45 by PCR using the oligonucleotide 5'GGG ATA CTG CAG CCA TGG GGC ATC ACC ATC ACC ATC ACG ATG
ACG ATG ACA AAG CCC AAG CTC AGT CCA AGA ACA ATA CCA AG-3' to
introduce a PstI site, NcoI site, and hexa-histidine tag at the 5' end of horA and the
oligonucleotide 5'-GTA CCC TTA TCT AGA TTA TCA CCC GTT GCT C-3' to
introduce an XbaI site at the 3' end of horA. The PCR product was cloned as a PstIXbaI fragment into pAlter-1 (Promega) using Escherichia coli JM109 as a host.
After the internal NcoI site in horA was removed silently by site-directed
mutagenesis using the Altered Sites II in vitro Mutagenesis System (Promega) and
the mutagenic oligonucleotide 5'-CCA GGA CCA TCG CCA TCA TGA CC-3', the
horA gene was cloned as an NcoI-XbaI fragment into pNZ8048, giving
pNZHHorA. Finally, horA was sequenced to ensure that only the intended changes
had been introduced.
Hop resistance
To test the hop resistance of L. lactis NZ9000 harboring pNZ8048 or
pNZHHorA, overnight cultures were diluted into fresh medium and grown to midexponential growth phase. Subsequently the cells were diluted to an optical density
at 690 nm (OD690) of 0.1 in M17 medium containing 5 µg of chloramphenicol/ml,
about 63 pg of nisin A/ml (through a 1-in-160,000 dilution of the supernatant of the
nisin A-producing L. lactis strain NZ9700 [de Ruyter et al., 1996]), and hop
compounds (Sami et al., 1997a) at various final concentrations (see Fig. 2).
Aliquots of 200 µl of the cell suspensions were dispensed into a sterile low-proteinbinding microplate (Greiner). Sterile silicon oil (50 µl) was pipetted on top of the
sample to prevent evaporation. Growth was monitored at 15°C by measuring the
OD690 every 10 min with a multiscan photometer (Titertek multiskan MCC/340
MKII; Flow Laboratories).
Solubilization, purification, and reconstitution of histidine-tagged HorA
L. lactis NZ9000 cells harboring pNZ8048 or pNZHhorA were grown at 30°C to
an OD690 of about 0.5. Subsequently, about 10 ng of nisin A/ml was added to the
culture through a 1-in-1,000 dilution of the supernatant of the nisin A-producing
L. lactis strain NZ9700 (de Ruyter et al., 1996). The cell suspensions were further
incubated at 30°C for 2 h, after which the cells were collected by centrifugation.
The inside-out membrane vesicles of HorA-expressing L. lactis cells were prepared
using a French pressure cell, as described (Margolles et al., 1999; Putman et al.,
51
CHAPTER 5
1999; van Veen et al., 2000), and stored in liquid nitrogen. His-tagged HorA was
solubilized with 1% n-dodecyl-β-maltoside as described previously (Margolles et
al., 1999). Insoluble components were removed by ultracentrifugation at
280,000 × g for 15 min at 4°C. The soluble fraction was mixed with Ni-NTAagarose (Qiagen Inc.) (20 µl of resin/mg of protein) which was equilibrated with
buffer A (50 mM KPi [pH 8.0] supplemented with 100 mM NaCl, 10% [vol/vol]
glycerol and 0.05% n-dodecyl-β-maltoside). The agarose suspension was shaken
gently at 4°C for 1 h. The resin was transferred to a Bio-spin column (Bio-Rad) and
washed with 20 column volumes of buffer A containing 20 mM imidazole and
subsequently with 10 column volumes of buffer A containing 40 mM imidazole.
The protein was eluted with buffer A, adjusted to pH 7.0, and supplemented with
250 mM imidazole. For reconstitition of purified HorA in proteoliposomes of
L. lactis lipids, total lipids of L. lactis were extracted and purified as described
previously (Margolles et al., 1999). Unilamellar liposomes with a relatively
homogeneous size were prepared by freezing in liquid nitrogen, slow thawing at
room temperature, and extrusion of the liposomes 11 times through a 400-nm-poresize polycarbonate filter. The liposomes were diluted to 1 mg of phospholipid/ml,
and dodecyl maltoside was added to a final concentration of 4 µmol/ml to
destabilize the liposomes. The purified HorA was mixed with dodecyl maltosidedestabilized liposomes at a protein/lipid ratio of 1:100 (wt/wt), after which the
suspension was incubated for 30 min at room temperature under gentle agitation.
The detergent was removed by absorption to polystyrene beads (Bio-Beads SM-2;
Bio-Rad) as described previously (Margolles et al., 1999). Finally, the
proteoliposomes were collected by ultracentrifugation at 280,000 × g for 15 min at
10°C, resuspended in 50 mM KPi (pH 7.0), and stored in liquid nitrogen.
Transport assays
(i) Ethidium transport. L. lactis NZ9000 cells harboring pNZ8048 or pNZHhorA
were grown at 30°C to an OD690 of about 0.5. Subsequently, about 10 ng of nisin
A/ml was added to the culture through a 1-in-1,000 dilution of the supernatant of
the nisin A-producing L. lactis strain NZ9700 (de Ruyter et al., 1996). The cell
suspensions were further incubated at 30°C for 2 h, after which the cells were
collected by centrifugation at 4°C at 8,000 × g for 10 min. The cells were washed
with 50 mM KPi (pH 7.0) containing 5 mM MgSO4. The washed cell suspensions
(OD690 of 0.5) were incubated for 10 min at 30°C in the presence of 10 µM
ethidium bromide to allow the diffusion of ethidium bromide into the cells. The
ethidium bromide efflux was initiated by the addition of 25 mM glucose. The
fluorescence of ethidium bromide was monitored at 20°C with a Perkin-Elmer LS
50B fluorimeter using excitation and emission wavelengths of 500 and 580 nm,
respectively, and slit widths of 5 and 15 nm, respectively (van Veen et al., 1996).
To study the effect of ortho-vanadate on the accumulation of ethidium in HorA52
CHAPTER 5
expressing and nonexpressing L. lactis cells, cells were grown in medium
supplemented with 30 mM arginine rather than glucose (Poolman, at al., 1987).
After the induction of HorA expression as described above, cells were washed with
50 mM (K)HEPES (pH 7.4) supplemented with 2 mM MgSO4. Washed cells
(OD690 of 0.5) were de-energized by incubation for 40 min at 30°C. Subsequently,
cells were reenergized for 7.5 min by the addition of 30 mM arginine, in the
presence or absence of 0.5 mM ortho-vanadate. Finally, 10 µM ethidium bromide
was added to the cell suspensions, and the fluorescence of ethidium bromide was
measured at 20°C as described above.
(ii) Hoechst 33342 transport. For the transport of Hoechst 33342 in inside-out
membrane vesicles, membrane vesicles were diluted to a final concentration of
0.5 mg of membrane protein/ml in KPi (pH 7.5) containing 2 mM MgSO4, 5 mM
phosphocreatine, and 0.1 mg of creatine kinase/ml. Valinomycin and nigericin were
added to a final concentration of 0.4 µM each, to dissipate the membrane potential
and transmembrane pH gradient, respectively. After an incubation for 1 min at
20°C, 2.3 µM Hoechst 33342 was added. The fluorescence of Hoechst 33342 was
measured at 20°C using a Perkin-Elmer LS 50B fluorimeter with excitation and
emission wavelengths of 355 and 457 nm, respectively, and slit widths of 3 nm
each. After the Hoechst 33342 fluorescence had reached a steady state, Hoechst
33342 transport was initiated by the addition of 2 mM Mg-ATP. In control
experiments, Mg-ATPγS was used rather than Mg-ATP. For the transport of
Hoechst 33342, HorA-containing proteoliposomes were thawed slowly and
extruded 11 times through a 400-nm-pore-size polycarbonate filter. Subsequently
proteoliposomes were washed twice and resuspended in 50 mM KPi (pH 7.5) or
(K)HEPES (pH 7.5). The Hoechst 33342 transport assay was performed as
described above in the absence of ionophores, using proteoliposomes at a final
concentration of 0.01 mg of protein/ml.
RESULTS
Overexpression of hexa-histidine-tagged HorA
Using the polymerase chain reaction, the horA gene on plasmid pRH45 of
Lb. brevis ABBC45 was cloned into the lactococcal NICE expression vector
pNZ8048 under the control of the nisin-inducible nisA promoter. A hexa-histidine
tag was added to the amino terminus of HorA to facilitate the purification of the
protein by Ni2+-NTA affinity chromatography. Induction of HorA expression in
L. lactis NZ9000 by the addition of nisin A to exponentially growing cells resulted
in the expression of a plasma membrane-associated 66-kDa polypeptide, which
could be detected on a Western blot by using an anti-hexa-histidine-tag monoclonal
antibody (Fig.1). HorA expression in cells was maximal after an induction time of
53
CHAPTER 5
2 h. Quantitative immunoblotting and densitometry analysis revealed a HorA
expression level of about 30% of the total membrane protein under these conditions
(data not shown). Densitometric analysis of Coomassie-stained sodium dodecyl
sulfate-polyacrylamide gels of the membrane fraction of HorA-expressing cells and
the purified HorA indicated a purity of HorA of more than 95% (data not shown).
Figure1.Expression, purification,
and functional reconstitution of
hexa-histidine-tagged HorA. The
HorA protein was overexpressed
in L. lactis as a hexa-histidinetagged protein using the NICE
system. A silver-stained sodium
dodecyl sulfate-polyacrylamide gel
is shown. Lane 1, total membrane protein (20 µg) of L. lactis harboring pNZHHorA; lane
2, soluble fraction (20 µg of protein) of a lysate of HorA-expressing cells; lane 3, Western
blot of the membrane fraction (20 µg of protein) of HorA-expressing cells, with anti-hexahistidine antibody; lane 4, flowthrough fraction of membrane proteins (20 µl of the total
fraction of 2 ml) eluted from the Ni2+-NTA resin; lanes 5, 6, and 7, histidine-tagged HorA
eluted from the NTA resin (20 µl out of the total fraction of 2 ml) in three consecutive steps
with buffer supplemented with 250 mM imidazole; lane 8, molecular mass markers; lane
9, HorA reconstituted into proteoliposomes. Lanes 3 and 9 are Western blots; the other
lanes are silver-stained gels. The arrow indicates the position of hexa-histidine-tagged
HorA protein.
HorA overexpression confers hop resistance on L. lactis cells
The hop resistance of L. lactis NZ9000 cells harboring pNZHHorA was
compared with the hop resistance of cells harboring the pNZ8048 control vector. In
the absence of iso-α-acids the HorA-expressing cells grew slightly more slowly and
reached a slightly lower cell density than control cells (Fig. 2A). A similar effect on
the growth of L. lactis was observed for LmrA-expressing cells (Margolles et al.,
1999). Fig. 2B shows the inhibitory effects of various concentrations of the iso-αacid compounds on the growth of HorA-expressing cells. The inhibition of growth
by 100, 200, and 300 µM hop compounds is significantly higher for control cells
than for HorA-expressing cells, indicating that HorA expression in L. lactis results
in an increased hop resistance of the organism.
54
CHAPTER 5
Figure 2. (A) Growth of control L. lactis harboring pNZ8048 (triangles) and of HorAexpressing L. lactis harboring pNZHHorA (squares) in the absence of iso-α-acids. (B)
Inhibition of growth by iso-α-acids of control L. lactis (triangles) and of HorAexpressing L. lactis (squares). Cells were grown at 15°C in the absence or presence of a
50, 100, 200, or 300 µM concentration of iso-α-acids. The OD690 was measured every
10 min. The growth rates were determined at the mid-exponential phase.
HorA is active as a multidrug transporter
(i) Ethidium transport in cells. HorA is a homologue of the ABC multidrug
transporter LmrA in L. lactis (Sami et al., 1997a; van Veen and Konings, 1998).
Fluorimetric ethidium transport assays were performed to test if HorA can mediate
the transport of ethidium, a typical substrate for LmrA. Washed cell suspensions of
L. lactis NZ9000 containing pNZHHorA or pNZ8048 were pre-equilibrated in the
presence of 10 µM ethidium bromide. Subsequently the cells were energized
through the addition of 20 mM glucose. The energization of cells resulted in an
increased rate of ethidium extrusion for the HorA-expressing cells compared to the
rate observed for nonexpressing control cells, suggesting that HorA is able to
mediate the active extrusion of ethidium (Fig. 3A). HorA is a member of the ABC
superfamily and should display an ATP-dependent extrusion activity. To analyze
whether ethidium efflux was coupled to ATP hydrolysis, the effect of the ABC
transporter inhibitor ortho-vanadate was examined. For this purpose, cells were
preenergized with 30 mM L-arginine and preincubated in the presence of 0.5 mM
ortho-vanadate. In this way, cells could generate metabolic energy by metabolizing
arginine via the arginine-deiminase pathway (Poolman et al., 1987). In contrast to
glycolysis, which is inhibited by ortho-vanadate, the arginine-deiminase pathway is
not affected by this inhibitor. ortho-Vanadate increased the level of ethidium uptake
in HorA-expressing cells, while no increase was observed in control cells. These
observations indicate inhibition by ortho-vanadate of HorA-mediated efflux of
ethidium (Fig. 3B and C).
55
CHAPTER 5
Figure 3. Ethidium transport in HorA-expressing cells and nonexpressing cells of
L. lactis. Panel (A) De-energized HorA-expressing and control cells (0.2 mg of protein/ml;
OD690, 0.5) were preequilibrated with 10 µM ethidium bromide at 30°C. The development
of fluorescence of the DNA-ethidium complex in the cell suspension was monitored at
20°C over time. At the arrow, 25 mM glucose was added. (B) Effect of ortho-vanadate on
the accumulation of ethidium in control cells. Cells were energized with arginine and
incubated for 7.5 min in the presence or absence of 0.5 mM ortho-vanadate prior to the
addition of 10 µM ethidium (at the arrow). (C) Effect of ortho-vanadate on the
accumulation of ethidium in HorA-expressing cells. Cells were treated as described for
panel B.
(ii) Hoechst 33342 transport in membrane vesicles. In previous studies, the
positively charged bisbenzimide dye Hoechst 33342 proved to be a useful probe to
study the activity of multidrug transporters such as LmrA and the human multidrug
resistance P-glycoprotein (Margolles et al., 1999; Putman et al., 1999; van Veen et
al., 2000). Hoechst 33342 is highly fluorescent when it is present in the
hydrophobic environment of the phospholipid bilayer. The transport of Hoechst
33342 from the membrane into the aqueous phase can be followed as a decrease of
Hoechst 33342 fluorescence over time. The ionophores valinomycin and nigericin
were included in this fluorescence assay at a concentration of 0.4 µM to dissipate
56
CHAPTER 5
the membrane potential and transmembrane pH gradient, respectively, generated
through proton pumping by the F1F0 H+-ATPase. In the presence of ATP, Hoechst
33342 fluorescence decreased in HorA-containing membrane vesicles five-fold
faster than in membrane vesicles from control cells. In the presence of the slowly
hydrolyzable ATP analog ATPγS, no significant decrease of Hoechst
33342 fluorescence was observed in both types of membrane vesicles (Fig. 4). The
ATP-dependent Hoechst 33342 transport in the control cells is most likely due to
the presence of low levels of endogenous LmrA, since under the experimental
conditions employed the secondary multidrug-transporter LmrP cannot work
because a proton motive force is absent. The results demonstrate that in the
presence of Mg-ATP, HorA efficiently transports Hoechst 33342 from the
membrane into the lumen of inside-out membrane vesicles prepared from HorAexpressing L. lactis.
Figure 4. Hoechst 33342 transport in insideout membrane vesicles of HorA-expressing
cells and nonexpressing cells of L. lactis.
Membrane vesicles prepared from HorAexpressing cells (H) and control cells (C) were
diluted to a concentration of 0.5 mg of
membrane protein/ml in buffer containing the
ATP regenerating system (see Materials and
Methods) and 0.4 µM of each of the ionophores
valinomycin and nigericin to dissipate the
membrane potential and transmembrane pH
gradient, respectively. After incubation for
1 min at 20°C, 2.3 µM Hoechst 33342 was
added to the assay mixture. At the arrow, 2 mM
Mg-ATP or 2 mM Mg-ATPγS was added.
Hoechst 33342 transport was measured at 20°C
by fluorimetry.
(iii) Hoechst 33342 transport in proteoliposomes. HorA-mediated transport of
Hoechst 33342 was also studied using purified and functionally reconstituted
protein. The protein was solubilized using 0.05% dodecyl maltoside and purified by
nickel chelate affinity chromatography to a high degree of purity (Fig. 1). HorA
was reconstituted by mixing the purified protein with preformed dodecyl
maltoside-destabilized liposomes, composed of L. lactis lipids, after which the
detergent was removed by extraction with polystyrene beads. Transport studies
revealed that purified HorA was able to transport Hoechst 33342 into
proteoliposomes in the presence of ATP (Fig. 5).
57
CHAPTER 5
Figure 5. Transport of Hoechst 33342 in proteoliposomes. Liposomes without
reconstituted HorA protein (A) and proteoliposomes containing reconstituted HorA protein
(B) were diluted in buffer containing the ATP regenerating system. After incubation for
1 min at 20°C, 2.3 µM Hoechst 33342 was added to the assay mixture. At the arrow 2 mM
Mg-ATP or 2 mM Mg-ATPγS was added.
(iv) Transport of hop compounds by HorA. If, as indicated by the above data,
HorA functions as a drug transporter with broad drug specificity, then HorA may
also be able to extrude hop compounds. The specificity of HorA for hop
compounds was analyzed in Hoechst 33342 transport assays in which hop
compounds were included as competing substrates (Fig. 6). Because hop
compounds are protonophores that act upon the proton motive force, hop
compounds may also indirectly affect Hoechst 33342 partitioning in the membrane.
Therefore, the ionophores valinomycin and nigericin were included in the Hoechst
33342 transport assays at final concentrations of 0.4 µM. The HorA-mediated
transport of Hoechst 33342 in the presence of ionophores was inhibited by hop
compounds (Fig. 6). The degree of inhibition was proportional to the concentration
of hop compounds used, indicating that hop compounds are transport substrates for
HorA.
Figure 6. HorA displays specificity for hop
compounds. The ATP-dependent transport of
Hoechst 33342 in HorA-containing inside-out
membrane vesicles was measured as described in
the legend to Fig. 4. Hop compounds at indicated
concentrations were added to the assay mixture
prior to the addition of Hoechst 33342. The hop
compounds did not affect the fluorescence of
Hoechst 33342 in control membrane vesicles
without HorA (data not shown). To dissipate a
proton motive force generated by F1F0-ATPase, the
ionophores valinomycin (0.4 µM) and nigericin
(0.4 µM) were included in the assay medium.
58
CHAPTER 5
DISCUSSION
Although hop resistance in Lb. brevis is known to be linked to the increased copy
number of the horA-containing plasmid pRH45 (Sami et al., 1997a, 1998), the
mechanism of hop resistance in this organism has not been studied previously. To
analyze the function of HorA in greater detail, hexa-histidine-tagged HorA was
expressed in L. lactis. By employing the NICE system (de Ruyter et al., 1996), high
expression levels were obtained of up to 30% of total membrane protein. Cell
fractionation studies indicated that the overexpressed HorA protein was associated
with the plasma membrane in L. lactis. HorA is a member of the ABC superfamily
and is a structural homologue of the multidrug transporter LmrA in L. lactis (van
Veen and Konings, 1998). Therefore, the ability of HorA to act as a drug pump was
investigated. Transport experiments with HorA-expressing L. lactis cells, HorAcontaining inside-out membrane vesicles, and proteoliposomes containing purified
and functionally reconstituted HorA demonstrated that HorA mediated the transport
of typical LmrA substrates, such as ethidium bromide and Hoechst 33342. Hence,
HorA and LmrA may be functionally equivalent proteins.
Two approaches were used to assess the ability of heterologously expressed
HorA to act as an extrusion system for hop compounds: (i) in vivo resistance to
growth inhibition by hop compounds and (ii) the competitive inhibition of drug
transport by hop compounds. The increased hop resistance in HorA-expressing
L. lactis cells and the inhibition of Hoechst 33342 transport by hop compounds both
indicate that hop compounds are transport substrates of HorA. Hop compounds are
able to dissipate the proton motive force in Gram-positive bacteria through a
cycling mechanism in which the undissociated iso-α-acids enter the cell by
diffusion through the phospholipid bilayer and, after dissociation of a proton,
diffuse back to the extracellular environment as complex of the anionic species and
divalent cation such as Mn2+ (Simpson and Smith, 1992; Simpson, 1993a, 1993b;
Simpson and Fernandez, 1994). The HorA-mediated resistance of cells to hop
compounds suggests that HorA mediates the extrusion of undissociated iso-α-acids,
by analogy with LmrA and the human multidrug resistance P-glycoprotein,
possibly from the phospholipid bilayer.
Most known bacterial multidrug transporters use the proton motive force to drive
the extrusion of drugs (Putman et al., 2000a). LmrA and HorA represent
prokaryotic ABC multidrug transporters that share significant sequence similarity
with ABC proteins in Bacillus subtilis, Staphylococcus aureus, Escherichia coli,
Helicobacter pylori, Haemophilus influenzae, and Mycoplasma genitalium (van
Veen and Konings, 1998). Studies on the origin of multidrug resistance genes
demonstrate the importance of transfer of genetic information between
microorganisms in the emergence and spread of multidrug resistance (Davies,
1994). Although the lmrA gene is on the genome of L. lactis, horA is carried by a
plasmid. Hence, prokaryotic members of the ABC transporter family can
59
CHAPTER 5
potentially be exchanged between pathogenic microorganisms and may be
responsible for acquired multidrug resistance in these organisms (Putman et al.,
2000b).
ACKNOWLEDGEMENT
We thank Dr. M. Sami and M. Nakagawa for valuable discussions and
G. Poelarends for drawing some of the figures. A.M. received a TMR fellowship
from the European Community, and H.W.V.V. was a Fellow of the Royal
Netherlands Academy of Sciences (KNAW).
60
CHAPTER 6
The Membrane Bound ATPase Contributes to
Hop Resistance of Lactobacillus brevis
Kanta Sakamoto, H. W. van Veen, Hiromi Saito, Hiroshi Kobayashi
and Wil N. Konings
This chapter was accepted to Applied and Environmental Microbiology.
SUMMARY
The activity of the membrane bound H+-ATPase of the beer-spoilage bacterium
Lactobacillus brevis ABBC45 increases upon adaptation to bacteriostatic hop
compounds. The ATPase activity is optimal around pH 5.6 and increases up to four
fold when Lb. brevis was exposed to 666 µM of hop compounds. The extent of
activation depends on the concentration of hop compounds and is maximal at the
highest concentration tested. The ATPase activity is strongly inhibited by DCCD, a
known inhibitor of F0F1-ATPase. Western blots of membrane proteins of Lb. brevis
with the antisera raised against the α- and β-subunits of F0F1-ATPase from
Enterococcus hirae show 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 Lb. brevis
to hop compounds.
61
CHAPTER 6
INTRODUCTION
The hop plant, Humulus lupulus, L. is used in beer fermentation for its
contribution to the bitter flavor of beer. Furthermore, the usage of hop in the
brewing industry is preferred because hop has antibacterial activity and prevents
beer from bacterial spoilage. Hop compounds are weak acids, which can cross
cytoplasmic membranes in undissociated form in response to the transmembrane
pH-gradient (Simpson and Smith, 1992). 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 (Simpson, 1993a, 1993b; Simpson and Smith,
1992). Some bacteria, however, are able to grow in beer in spite of the presence of
hop compounds. Sami et al. (1997a) reported that Lactobacillus brevis ABBC45
strain could adapt to hop treatment and develop a high resistance to hop
compounds. During hop resistance development the copy number of plasmid
pRH45 harboring horA gene increased (Sami et al., 1997a). Subsequent studies
revealed that horA encodes a bacterial ATP-biding cassette (ABC) multidrug
transporter (MDR) which can extrude hop compounds from the cell membranes
upon ATP hydrolysis (Sakamoto et al., 2001). As a result of this exogenous
expression of HorA in Lactococcus lactis, its resistance to hop compounds
increased up to two fold. Micro-organisms have been found to increase the proton
motive force (pmf)-generating activities in their cytoplasmic membranes when
confronted with a high influx of protons (Viegas et al., 1998). The thermophilic
bacterium Bacillus stearothermophilus (De Vrij et al., 1998) increases proton
pumping respiratory chain activities when the proton permeability of its
cytoplasmic membrane increases drastically at higher temperatures. In
Enterococcus hirae (formerly Streptococcus faecalis) (Kobayashi et al., 1984,
1986) and Saccharomyces cerevisiae (Viegas et al., 1998) the proton translocating
ATPase levels in their membranes were found to increase upon exposure to
protonophores such as carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) or
weak acids. Obviously, the main reason for this increase of the proton pumping
activities is to maintain the pmf and the internal pH at viable levels. In view of the
protonophoric activities of hop compounds it was of interest to investigate whether
the hop-resistant Lb. brevis would respond in a similar way to the action of hop
compounds and would increase the functional expression of its proton translocating
ATPase in addition to the expression of the MDR HorA. In this study, we
demonstrate that this is indeed the case and that Lb. brevis increases the functional
expression of the proton translocationg ATPase during growth in the presence of
hop compounds.
62
CHAPTER 6
MATERIALS AND METHODS
Bacterial strains and growth conditions
Lactobacillus brevis ABBC45 was grown anaerobically at 30°C 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 was achieved by growth
of Lb. brevis in the presence of hop compounds, up to 666 µM, as described
previously (Sami et al., 1997a). Cells grown in the presence of 666 µM of hop
compounds were subcultured without hop compounds added in order to follow the
ATPase activity under these growth conditions.
Hop compounds
A concentrated isomerized hop extract (Hopsteiner GmbH, Mainburg, Germany)
was used as hop compounds. The iso-α-acid contents were determined, by using
high-performance liquid chromatography (HPLC) (Rode et al., 1990). The
concentration of hop compounds in the medium was expressed as the concentration
of iso-α-acids.
Preparation of the membrane
Lb. brevis was grown to late exponential phase in the absence and in the presence
of 100 µM and 666 µM hop compounds. Cells of Lb. brevis were harvested by
centrifugation at 7,000 × g for 15 min and washed twice at room temperature in 50
mM (K) HEPES (pH 7.4) containing 5 mM MgSO4. The cells, suspended in the
same buffer, were lysed at 37°C by treatment for 1.5 h with 1 mg/ml lysozyme
(Sigma, USA) and 50 µg/ml mutanolysin (Sigma, USA) in the presence of a
cocktail of proteinase inhibitors (Complete [Boehringer Mannheim, Germany] ).
After the addition of DNase I (50 µg/ml) and RNase (1 µg/ml), the suspension was
passed three times through an ice-cold French pressure cell at 70 MPa. Unbroken
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 4°C 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 DC protein assay kit (BioRad, USA) with bovine
serum albumin as a quantitative standard.
ATPase Assay
ATPase activity was estimated from the release of inorganic phosphate measured
by a modification of the method of Driessen et al. (1991). 1 or 2 µg of membrane
protein was incubated at 30°C for 10 min in 50 mM (K) Mes buffer (usually at pH
5.5) containing 5 mM MgCl2. ATP [Potassium salt] was added at a final
63
CHAPTER 6
concentration of 2 mM to initiate the reaction. The reaction (total volume of 40 µl)
was stopped after 5 min by immediately cooling the test tubes on ice. Malachite
green solution (200 µl of 0.034%) was added, and after 40 min the color
development was terminated by the addition of 30 µl citric acid solution (34%
[w/v]). Immediately, the absorbance at 660 nm was measured with a multiscan
photometer (Multiskan MS; Labsystems, Finland). One unit ATPase-activity was
defined as the release of 1 µmole of inorganic phosphate in 1 min. Calibration was
done by using a series of Pi standards (Sigma, USA). For the determination of pH
dependency of the ATPase activity, membranes were incubated for 60 min on ice
in 50 mM (K) Mes buffer, adjusted to various pH values. The ATPase activity was
assayed at those different pH values as described above. To measure the effects of
inhibitors on the ATPase activity, the membranes were pre-incubated with N, N’ dicyclohexylcarbodiimide (DCCD; final concentration 0.2 mM), ortho-vanadate
(final concentration 0.2 mM) or nitrate (K2NO3; final concentration 25 mM) for 10
min at 30°C, and subsequently for 60 min on ice. The membrane sample without
inhibitor was used as the control.
Western Blotting Analysis
The membrane protein of Lb. brevis, prepared as described above, was
solubilised in Laemmli sample buffer containing 2% SDS (Laemmli, 1970) and
separated by electrophoresis (20 µg of protein / lane) through 10% SDSpolyacrylamide gel by the method of Laemmli (1970). The protein bands were
transferred to a polyvinilidene difluoride (PVDF) filter membrane and detected
with the antisera raised against the F1 complex of Enterococcus hirae H+-ATPase
(Arikado et al., 1999) which can also bind with F0F1-ATPase from Lactococcus
lactis (Amachi et al., 1998). Membranes from E. hirae prepared as previously
described (Arikado et al., 1999) were used as control. The antibody-bound proteins
were made visible with nitrobluetertazorium (NBT) and 5-bromo-4-chloro-3indolyl phosphate (BCIP) (Gibco BRL. USA). The intensities of the bands were
measured by densitometric analysis with NIH Image software v.1.61 (NIH, USA).
64
CHAPTER 6
RESULTS
ATPase activity
(Pi µmol/min/mg protein)
Effect of hop on the ATPase activity
In a previous publication it has been demonstrated that under these conditions Lb.
brevis develops hop resistance by overexpressing the MDR HorA (Sami, 1999).
The cytoplasmic membranes of cells were isolated as described in Materials &
Methods and the ATPase activities in these membranes were determined as a
function of the pH ranging from pH 4.4 to 7.0. All membranes of Lb. brevis grown
in the presence of different levels of hop compounds showed maximum ATPase
activity at around pH 5.6 (Fig. 1). At pH 5.6 membranes from the cells adapted to
666 µM of hop compounds had the highest activity, which was about 4-times the
ATPase activity of membranes from non-adapted cells. The ATPase activities of
membranes from cells adapted to 100 µM of hop compounds were in between these
extremes and about 1.7 times the activity of the membranes from the non-adapted
cells. Once the hop-adapted cells (666 µM) were subcultured in medium without
hop compounds the ATPase activity of their membranes decreased rapidly (Fig. 1).
1.0
0.8
0.6
0.4
0.2
0.0
4.4
4.8
5.2
5.6
pH
6
6.4
6.8
Figure 1. The pH profile of the
ATPase activity in membranes of Lb.
brevis. The ATPase activity at pH
values ranging from 4.4 to 7.0 was
measured of membranes prepared from
cells grown without hop compounds
(W0, ○) and of cells adapted to 100 µM
(W100, ×) and to 666 µM of hop
compounds (R666, ■), and of cells deadapted by growth first in the presence
of 666 µM of hop compounds followed
by growth for two days in the absence
of hop compounds (R0, ▲). The
ATPase activity was shown as the
amount of released inorganic phosphate
(Pi) per min / mg protein.
Effect of inhibitors on the ATPase activity
To characterize the type of ATPase present in the membrane of Lb. brevis, the
effect of several kinds of inhibitors on the ATPase activity was studied (Fig. 2).
The ATPase activities of membranes from non-adapted cells and from cells
adapted to different concentrations of hop compounds were all significantly
inhibited by the F0F1-type inhibitor N, N’-dicyclohexylcarbodiimide (DCCD).
Moderate inhibition was observed with the P-type inhibitor ortho-vanadate, while
the V-type inhibitor K2NO3 showed the least inhibition or even activation in
65
CHAPTER 6
ATPase activity
(Pi µmol/min/mg protein)
membranes from cells grown at 100 µM of hop compounds (W100 in Fig. 2).
These results correspond to the observations made for the enterococcal F0F1-type
ATPase, which is slightly inhibited by ortho-vanadate and slightly enhanced by
K2NO3 (Y. Kakinuma, personal communication), indicating that F0F1-type ATPase
is the major ATPase in membranes from Lb. brevis.
0.4
0.3
control
DCCD
vanadate
nitrate
0.2
0.1
0
W0
W100
R666
Figure 2. The effect of inhibitors on
the ATPase activity of Lb. brevis. The
ATPase activity of the membranes of
W0, W100 and R666 (See the legend of
Fig. 1) was measured at pH 5.6 in the
presence of 0.2 mM of N, N’ dicyclohexylcarbodiimide
(DCCD)
(closed bars), 0.2 mM of ortho-vanadate
(vertically striped bars) or 25 mM of
nitrate (horizontally striped bars). The
activity without any inhibitor was also
measured as control (open bar).
Western Blot Analysis
Two bands were strongly detected from the membranes of Lb. brevis with the
antisera against the α- and β- subunits of the F1 ATPase complex from E. hirae,
which strongly indicates the F0F1-type nature of the ATPase of Lb. brevis. The
apparent molecular weights of these bands are slightly higher than those of the αand β- subunits of F1 from E. hirae (Fig. 3A). The intensities of both bands are
higher in membranes isolated from cells grown at higher concentrations of hop
compounds and decrease again in membranes from hop-adapted cells (666 µM)
subcultured in medium without hop compounds (Fig. 3B.). The intensities of both
bands correlated well (r = 0.990; r = correlation coefficient) with the ATPase
activities of the different membranes. The rate and extent of growth in MRS broth
of hop adapted cells are slower than of non-adapted cells (Sami et al., 1997a). Also
hop-adapted cells are smaller than cells grown in the absence of hop compounds
(data not shown).
66
CHAPTER 6
1
A
2
3
4
5
(kDa)
97.4
66.2
55.0
42.7
40.0
α
β
31.0
100
Band Intensity (a.u.)
Figure 3. Western blot analysis of
membranes of Lb. brevis and E. hirae
with antisera against F1 of E. hirae.
Membranes of Lb. brevis were solubilized
and separated by electrophoresis through
10% polyacrylamide gel (lanes 2-5). For
comparison the results with membranes
from E. hirae are shown in lane 1. The
proteins were transferred to a PVDF filter
membrane and reacted with the antisera
raised against the F1 complex of E. hirae
H+-ATPase. (A) The result of Western
Blotting: Lane1, E. hirae cultured at pH
6.0; Lane 2, Lb. brevis grown without hop
(W0); Lane 3, Lb. brevis adapted to 100
µM of hop compounds (W100); Lane 4,
Lb. brevis adapted to 666 µM of hop
compounds (R666); Lane 5, Lb. brevis deadapted from 666 µM to 0 µM of hop
compounds (R0). The arrows indicated the
position of the α- or β-subunit of H+ATPase from E. hirae. (B) The intensity
of the lower bands of the ATPase from
Lb. brevis. The intensities of these bands
were measured with NIH Image software
and presented in arbitrary units.
B
75
50
25
0
W0
W 100 R666
R0
DISCUSSION
The beer spoilage bacterium Lb. brevis ABBC45 develops hop resistance upon
growth in hop-containing media (Sami et al., 1997a). This resistance was found to
be mediated by the functionally expressed multidrug resistance ABC transporter
HorA (Sami, 1999; Sakamoto et al., 2001). Studies of HorA, functionally
expressed in Lactococcus lactis, revealed that HorA can excrete the lipophilic hop
compounds and several other MDR substrates from the membrane into the external
medium (Sakamoto et al., 2001). Recently a second proton motive force-driven
MDR with affinity for hop compounds has been found in Lb. brevis ABBC45
lacking HorA (Suzuki et al., 2002). The activity of HorA and this pmf-driven
MDR thus results in a reduced influx of the undissociated and membrane
permeable iso-α-acids into the cytoplasm and thereby limits the anti-bacterial pmfdissipating effect of hop compounds. Since Lb. brevis develops resistance against
rather high concentrations of hop compounds, the question arose whether
67
CHAPTER 6
functional expression of HorA and the pmf-driven MDR was sufficient to confer
this resistance or whether additional activities could contribute to hop resistance.
Anaerobic Gram-positive lactic acid bacteria such as Lb. brevis depend for the
generation of their proton motive force strongly on their membrane bound H+-F0F1ATPase (Kobayashi et al., 1986; Konings et al., 1995). In this study, we
demonstrated that the functional expression of a membrane-bound H+-F0F1-ATPase
increased during hop-resistance development and decreased again when the
exposure to hop compounds was stopped. Previously, it was demonstrated that also
the expression of the HorA transporter increased during hop-resistance
development (Sami, 1999). The H+-F0F1-type nature of the ATPase was confirmed
by H+-F0F1-ATPase effectors and especially by immunologial studies with the
antisera against α- and β-subunits of H+-F0F1-ATPase from E. hirae. In accordance
with the observations of Kobayashi et al. (Kobayashi et al., 1984, 1986) made in
the anaerobic Gram-positive bacterium E. hirae, the increased functional
expression of H+-F0F1-ATPase most likely allows Lb. 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 reports (Sami, 1999;
Sakamoto et al., 2001; Suzuki et al., 2002) indicate that Lb. brevis becomes
resistant to hop compounds by the combined action of two ATP-driven systems:
the H+-ATPase and the MDR pump HorA (Sami, 1999; Sakamoto et al., 2001), and
a pmf-driven MDR (Suzuki et al., 2002). HorA and the pmf-driven MDR reduce
the influx of the weak acidic hop compounds by pumping the undissociated hop
compounds from the membrane environment into the external medium. The H+ATPase compensates for the pmf-dissipating and internal pH-decreasing effects of
hop compounds, which have escaped the MDR activities, by pumping more
protons from the cytoplasm across the membrane. As a result of the higher
expression of ATPase and of HorA and the energy dissipation by hop compounds
the rate and extent of growth in MRS broth of hop adapted cells are slower than of
non-adapted cells (Sami et al., 1997a). Those various hop resistance mechanisms
(Fig. 4) are another demonstration of the versatility of bacteria and their capacity to
recruit a variety of mechanisms to cope with toxic compounds in their
environments.
68
CHAPTER 6
Hop-H
Hop-H
Hop-H
a
ATP
Hop-H
b
ADP
Hop-
Hop- + H+
Mn2+
Hop-Mn-Hop
ATP
H+
H+
c
H+
ADP
Cytoplasmic membrane
Cell wall
Figure 4. Proposed mechanisms of hop resistance in Lb. brevis ABBC45 by the
combined action of two ATP-driven systems and one proton motive force-driven
MDR. The undissociated hop compounds (Hop-H) intercalate into the cytoplasmic
membrane and are pumped out by the multidrug resistant ABC-type transporter HorA (a)
(Sami, 1999; Sakamoto et al., 2001) and by a secondary MDR (b) (Suzuki et al., 2002). A
fraction of Hop-H escapes the pumping activity of the transporters and enters the
cytoplasm. In the cytoplasm Hop-H dissociates due to the higher internal pH into the anion
(Hop–) and H+. H+ also enters the cytoplasm in antiport with Hop-H by the secondary
transporter. Hop– may bind to cations such as Mn2+ (Archibalt and Fridovich, 1981;
Simpson, 1993a, 1993b; Simpson et al., 1993: Simpson and Hughes, 1993), while the
increased H+-ATPase activity excretes H+ across the membrane (c) (This work).
69
70
70
SUMMARY
Beer has a long history of 5,000-7,000 years. Since the start of beer production,
brewers have been bothered by beer spoilage. The introduction of hop compounds
from the hop plant, Humulus Lupulus, L., into beer in the 12th to 13th century was
a major breakthrough due to the strong preservative value of hops. Nevertheless
several microorganisms can still grow in beer. These beer spoilage microorganisms
include a few lactic acid bacteria, a few Gram-negative bacteria and wild yeasts.
Beer spoilage lactic acid bacteria include Lactobacillus and Pediococcus species
and are the major contaminants in the brewing industry. They spoil beer by
producing acidity, turbidity and, in some species, ropiness and a buttery off-odor of
diacetyl. Aerobic acetic acid bacteria such as Acetobacter and Gluconobacter
species were well-known beer spoilage Gram-negative bacteria. However, these
aerobes have been replaced in the past decades by strictly anaerobic Gram-negative
bacteria such as Pectinatus and Megasphaera species, due to the drastically
reduced oxygen content in beer by the improved brewing technology. These
anaerobic Gram-negatives cause more serious spoilage than lactic acid bacteria by
producing an offensive rotten egg smell of hydrogen sulfide. Wild yeasts are
mainly detected at taps and dispense lines at pubs but rarely in packaged beer.
These yeasts cause less serious spoilage problems than bacteria.
Detection and identification of beer spoilage micro-organisms are very important
for the quality assurance in breweries. The most popular method today is still the
conventional culture method. It can take a week or even longer to detect
microorganisms and consequently the products are often already released for sale
before the microbiological results become available. Hence more rapid detection
methods for beer spoilage bacteria are required. A very powerful tool is the
polymerase chain reaction (PCR) since it enables detection and identification of
micro-organisms in a very short period of time. Sets of PCR primers targeting
species-specific regions in the bacterial 16S ribosomal RNA gene (rDNA) have
been developed for each of the known beer spoilage bacteria (Chapter 2). However
false negative results were sometimes obtained as PCR can be prevented by the
presence of factors such as polyphenols in beer samples. These false negatives can
be eliminated by the use of the primers targeting to consensus sequences in
bacterial 16S rDNA as an internal positive control for proper PCR (Chapter 2).
Most strains in Lactobacillus brevis and Pediocuccus damnosus can grow in beer,
but some can not. For the quality assurance discrimination of beer spoiling strains
from non-spoilers is therefore necessary. The most crucial feature in beer spoilage
strains of lactic acid bacteria is resistance to hop compounds present in beer as isoα-acids. These hop compounds can inhibit the growth of Gram-positive bacteria.
The mechanism of the antibacterial action of hop compounds was studied. The
71
SUMMARY
major component of iso-α-acids, trans-isohumulone, was found to function as an
ionophore in a hop sensitive strain of Lb. brevis and to catalyze electroneutral
exchange across the cytoplasmic membrane of protons for intracellular cations
such as Mn2+ (Simpson, 1993b). Consequently the electrochemical proton gradient
across the cytoplasmic membrane is dissipated, resulting in the decrease of proton
motive force (pmf). The uptake of nutrients by pmf-driven uptake systems will
then also be decreased. Resistance to hop compounds in Lb. brevis has been studied
at the molecular level. The horA gene, encoding a polypeptide that is 53% identical
to LmrA, a lactococcal ABC-type multidrug transporter (van Veen et al., 1996),
was discovered in a plasmid pRH45 of the hop-resistant strain of Lb. brevis
ABBC45 (Sami et al., 1997a). Amplification of pRH45 occurs when this strain is
grown in a continuous culture with increasing concentration of iso-α-acids. On the
other hand, hop resistance decreased significantly when Lb. brevis ABBC45 was
cured from pRH45, and resistance was regained when this plasmid was reintroduced by electrotransformation (Chapter 3 and 4). HorA was successfully
expressed in Lactococcus lactis under control of the nicin-inducible expression
system (Chapter 5). Studies in cells, membrane vesicles and proteoliposomes,
reconstituted with solubilized and purified HorA, revealed that this protein confers
hop resistance by excreting hop compounds in an ATP-dependent manner from the
cell membrane to outer medium. In addition to the activity of HorA also increased
proton pumping by the membrane bound H+-ATPase contributes to increased hop
resistance (Chapter 6). To energize such ATP-dependent transporters hop resistant
cells contain larger ATP pools than hop sensitive cells (Simpson and Fernandez,
1994). Furthermore evidence for the presence of a proton motive force dependent
hop transporter was recently presented (Suzuki et al., 2002). The potential
molecular mechanisms of hop resistance in Lb. brevis are shown in Fig. 1.
Understanding the mechanisms of hop resistance has enabled the development of
rapid methods to discriminate beer spoilage strains from non-spoilers. The horAPCR method in which a set of specific primers detects the horA gene or its
homologues has been applied for bacterial control in breweries (Sami et al.,
1997b). Most horA positive strains of Lactobacillus spp. were found to have beer
spoiling ability. Also a discrimination method was developed based on ATP pool
measurement in lactobacillus cells (Okazaki et al., 1997). However, some potential
hop resistant strain cannot grow in beer when they have not first been exposed to
sub-inhibitory concentration of hop compounds (Simpson and Fernandez, 1992).
The beer spoilage ability of Pectinatus spp. and Megasphaera cerevisiae has
been poorly studied. Since all strains have been reported to be capable of beer
spoiling, species identification is sufficient for the brewing industry. However, with
the current trend of beer flavor (lower alcohol and bitterness), there is the potential
risk that not yet reported bacteria will contribute to beer spoilage. Investigation of
the beer spoilage ability of especially Gram-negative bacteria may be useful to
72
SUMMARY
reduce this risk.
Hop-sensitive cell
Hop-H
Hop-Mn-Hop
Cell wall
Hop-H
Cytoplasmic membrane
ADP
H+
c
ADP
H+ + Hop-
H+
H+
ATP
Mn2+
ATP
H+
Hop-Mn-Hop
ADP
H+
ATP
Mn2+
Hop-Mn-Hop
ATP
Hop- + H+
ATP
ADP
H+
ATP
ADP
a
Hop-H
Hop-Mn-Hop
Hop-H
c
H+
c
H+
c
H+
b
Hop-H
Hop-resistant cell
Hop-H
Figure 1. Mechanisms of hop resistance. Hop compounds act as ionophores that
exchange protons for cellular divalent cations. In a hop-sensitive cell, hop compounds
(Hop-H) invade the cell and dissociate into hop anions and protons due to the higher
internal pH. Hop anions trap divalent cations such as Mn2+ and diffuse out of the cell. The
ionophoric action together with the diffusion of the hop-metal complex results in an
electroneutral exchange of cations. Release of protons from hop compounds decreases the
intracellular pH and results in a dissipation of the transmembrane proton gradient (∆pH)
and the proton motive force (pmf). Consequently, pmf-driven uptake of nutrients will be
decreased. In hop resistant cells hop compounds can be expelled from the cytoplasmic
membrane by HorA (a) (Sakamoto et al., 2001) and probably also by a pmf-dependent
transporter (b) (Suzuki et al., 2002). Furthermore, overexpressed H+-ATPase increases the
pumping of protons released from the hop compounds (c) (Sakamoto et al., 2002). More
ATP is generated in hop-resistant cells than in hop-sensitive cells (Simpson and Fernandez,
1994). Galactosylated glycerol teichoic acid in the cell wall (Yasui et al., 1997) and a
changed lipid composition of the cytoplasmic membrane of beer spoilage lactic acid
bacteria may increase the barrier to hop compounds.
73
74
SAMENVATTING
De geschiedenis van het bier telt al zo’n 5000 tot 7000 jaar, en sinds het begin
van de bierproductie hebben brouwers te kampen met bierbederf. De ontdekking
van de sterke preservatieve werking van hop-componenten, afkomstig uit de
hopplant Humulus lupulus L, betekende dan ook een grote doorbraak in de strijd
tegen het bierbederf. Rond de 12e/13e eeuw werd hop voor het eerst aan bier
toegevoegd. Desondanks kunnen er nog steeds verschillende micro-organismen
groeien in bier, waaronder gisten, melkzuur bacteriën en enkele Gram-negatieve
bacteriën. De voornaamste bedervers in de bier-industrie zijn melkzuur bacteriën
van de Lactobacillus en de Pediococcus familie. Deze bederven het bier door
vertroebeling, verhoging van de viscositeit, verlaging van de zuurgraad en soms
zelfs het veroorzaken van een schrale, boterige geur van diacetyl. De aërobe Gramnegatieve azijnzuur bacteriën zoals Acetobacter en Gluconobacter waren voorheen
ook beruchte bier-bedervers, maar dankzij het door verbeterde brouw-technologien
verlaagde zuurstofgehalte in het bier zijn deze in de laatste decennia grotendeels
vervangen door de strikt anaërobe Gram-negatieve soorten Pectinatus en
Megasphaera. Deze soorten veroorzaken echter een ernstiger vorm van bierbederf
dan de melkzuur bacteriën omdat ze de afstotelijke “rotte eieren lucht” van
waterstofsulfide (H2S) produceren. Gisten worden voornamelijk gedetecteerd in de
leidingen van biertaps in café’s maar zelden in verpakt bier, en daarom vormen ze
een minder groot probleem dan de bacteriën.
Detectie en identificatie van bier bedervende micro-organismen is van groot
belang voor de kwaliteitswaarborging van brouwerijen en de conventionele kweek
methode is hiervoor nog altijd populair. Vaak zijn producten echter al in de verkoop
voordat alle microbiologische resultaten binnen zijn en dientengevolge is er veel
interesse in snellere detectie- en identificatie-methoden voor bier bedervende
micro-organismen. De “polymerase ketting reactie” (polymerase chain reaction:
PCR) is een krachtige techniek voor het vermenigvuldigen van specifieke DNA of
RNA sequenties. PCR wordt dan ook veel gebruikt om micro-organismen in korte
tijd te identificeren. In hoofdstuk 2 wordt de ontwikkeling besproken van soortspecifieke PCR primers die genen herkennen die coderen voor 16S ribosomaal
RNA (16S rDNA) van alle bekende bier-bedervende bacteriën. Vals negatieve
resulaten werden echter behaald doordat de PCR reactie geremd kan worden door
bepaalde componenten in bier, zoals polyfenolen. Deze vals negatieve resultaten
kunnen opgespoord worden door gebruik te maken van interne positieve controle
PCR reacties, die consensus sequenties in 16S rDNA herkennen (Hoofdstuk 2).
De meeste, maar niet alle stammen van Lactobacillus brevis en Pediococcus
damnosus kunnen groeien in bier. Het onderscheiden van bier-bedervende en nietbier-bedervende stammen is derhalve essentiëel voor de kwaliteitswaarborging van
75
SAMENVATTING
het bier. De cruciale eigenschap van bier-bedervende melkzuurbacteriën is hun
resistentie tegen hop-componenten, zoals iso-α-zuren, die in bier voorkomen. Deze
hop-componenten kunnen de groei van Gram-positieve bacteriën remmen en de
antibacteriële werking van deze stoffen is onderzocht. trans-Isohumulon, de
belangrijkste component van iso-α-zuren, bleek te werken als een ionofoor in een
hopgevoelige Lactobacillus brevis stam. trans-Isohumulon katalyseert de
electroneutrale uitwisseling over de cytoplasmatische membraan van protonen
tegen intracellulaire kationen zoals mangaan (Mn2+). Zodoende wordt de protonen
gradiënt (pH-gradiënt) over de cytoplasma membraan verlaagd hetgeen resulteert
in een verlaagde protonen drijvende kracht (proton motive force: PMF). Het gevolg
van een verlaagde PMF is een verlaagde opname van diverse nutriënten door PMF
gedreven opname systemen.
Het resistentie mechanisme van de hop-resistente Lb. brevis stam ABBC45 tegen
hop-componenten is op moleculair niveau onderzocht (Hoofdstuk 3 en 4). Groei
van deze stam in een continu culture met toenemende concentraties iso-α-zuren
resulteert in amplificatie van het plasmide pRH45. Verwijdering van dit plasmide
uit Lb. brevis ABBC45 gaat gepaard met een afname van hop resistentie en dit kan
weer worden hersteld door herintroductie van het plasmide. Plasmide pRH45 bevat
het horA gen, dat codeert voor een eiwit dat voor 53% identiek is aan LmrA, een
ABC-type multidrug transporter uit Lactococcus lactis. Het HorA eiwit werd onder
controle van het nisine induceerbare expressie systeem tot overexpressie gebracht
in L. lactis (Hoofdstuk 5). Experimenten met hele cellen, membraan vesikels en
proteoliposomen met gezuiverd en gereconstitueerd HorA toonden aan dat dit eiwit
hop-resistentie realiseert door ATP-afhankelijke excretie van hop-componenten
vanuit de cel membraan naar het externe medium. Een additioneel mechanisme dat
bijdraagt aan hop-resistentie is de verhoogde activiteit van de protonen pompende
H+-ATPase, hetgeen mogelijk gemaakt wordt door de hogere ATP concentraties in
hop-resistente cellen in vergelijking met hop-gevoelige cellen (Simpson en
Fernandez, 1994). Evidentie voor de aanwezigheid van een PMF gedreven hopresistentie transporter in Lb. brevis is onlangs gepresenteerd (Suzuki et al., 2002).
De potentiële moleculaire mechanismen voor hop-resistentie zijn samengevat in
Fig. 1.
Het inzicht in de mechanismen van hop-resistentie heeft geleid tot de
ontwikkeling van een methode voor het onderscheiden van bier-bedervende en
niet-bier-bedervende stammen op basis van het horA gen. De horA-PCR methode
maakt gebruik van primers specifiek voor horA of een homoloog gen, en
toepassing van deze methode bij bacteriële controles in een brouwerij toonde aan
dat de meeste horA positieve stammen inderdaad bier-bedervend zijn.. Sommige
potentiëel hop-resistente stammen kunnen echter pas groeien in bier als ze eerst aan
lage concentraties van hop-componenten zijn blootgesteld.
De bier-bedervings-capaciteit van Pectinatus spp. en Megasphaera cerevisiae is
76
SAMENVATTING
tot nu toe nauwelijks onderzocht, maar aangezien alle stammen in staat zijn bier te
bederven volstaat het voor de bier-industrie om de soorten te identificeren. De
huidige trends in de ontwikkeling van bieren (minder alcohol en minder bitterheid)
brengt het risico mee dat nog niet eerder gerapporteerde bacteriën bij zullen dragen
aan bierbederf. Dit risico zou verkleind kunnen worden door onderzoek naar de
bier-bedervings-capaciteit van vooral Gram-negatieve bacteriën.
Hop-gevoelige cel
Hop-Mn-Hop
Hop-H
Cel wand
Hop-H
Cytoplasmatische membraan
ADP
H+
c
ADP
H+ + Hop-
H+
H+
ATP
Mn2+
ATP
H+
Hop-Mn-Hop
ADP
H+
ATP
Mn2+
ATP
ADP
Hop-Mn-Hop
ATP
Hop- + H+
H+
ATP
ADP
a
Hop-H
Hop-Mn-Hop
Hop-H
c
H+
c
H+
c
H+
b
Hop-H
Hop-resistente cel
Hop-H
Figuur 1. Mechanismen van hop-resistentie. Hop-componenten werken als ionoforen die
protonen uitwisselen tegen intracellulaire divalente kationen. In een hop-gevoelige cel
dringen hop-componenten (Hop-H) de cel binnen om vervolgens te dissocieren in hopanionen (Hop-) en protonen (H+) door de hogere interne pH. Hop anionen complexeren
divalente kationen zoals Mn2+ en diffunderen de cel weer uit (Hop-Mn-Hop). De ionofore
werking van hop samen met de diffusie van van de hop-metaal complexen resulteert in een
electroneutrale uitwisseling van kationen. Het afstaan van protonen door hop-componenten
binnen de cel verlaagt de pH en resulteert in een verlaging van de protonen gradient (∆pH)
en dus de protonen drijvende kracht (proton motive force: PMF). PMF gedreven opname
van nutriënten wordt dientengevolge ook verlaagd. In hop-resistente cellen kunnen hopcomponenten uit de cytoplasmatische membraan worden verwijderd door HorA (a)
(Sakamoto et al., 2001) of door een PMF afhankelijke transporter (b) (Suzuki et al. 2002).
Verhoogde expressie van H+-ATPase verhoogt de gepompte hoeveelheid protonen na
dissociatie van hop-componenten (c) (Sakamoto et al., 2002). In hop-resistente cellen wordt
meer ATP gegenereerd dan in hop-gevoelige cellen (Simpson en Fernandez, 1994).
Gegalactosyleerd glycerol teichoine zuur in de celwand (Yasui et al., 1997) en een
veranderde lipidensamenstelling van de cytoplasmatische membraan van bierbedervende
melkzuurbacteriën kan de barrière tegen hop-componenten verhogen.
77
78
総　括
ビールは5千～7千年の歴史を有するが、それは同時に微生物混濁との戦
いの歴史でもあったと思われる。12-13世紀にホップ(学名 Humulus Lupulus,
L.)が使われるようになると、ビールの微生物耐久性は飛躍的に向上した。
にもかかわらず、ある種の微生物はビール中で生育することができ、ビー
ル業者の悩みの種となっている。これらの微生物はビール混濁菌と呼ばれ
、数種の乳酸菌、数種のグラム陰性菌、および野生酵母からなる。ビール
混濁性乳酸菌にはLactobacillus属菌やPediococcus属菌が含まれ、それらは主
たるビール混濁菌である。これらの乳酸菌はビールを酸っぱくしたり、濁
らせたり、またある菌種は粘性物質を生産したり、ヂアセチルによるバタ
ー様臭を発生したりして、ビール品質を著しく低下させる。ビール混濁性
グラム陰性菌においては、かつてはAcetobacter属菌やGluconobacter属菌な
どの酢酸菌が有名であったが、醸造技術の進歩に伴いビール中の溶存酸素
量が激減したことにより、これらの好気性菌は姿を消した。しかし一方で
、Pectinatus属菌やMegasphaera cerevisiae菌などの偏性嫌気性グラム陰性菌
がそれらにとって代わった。これらの偏性嫌気性グラム陰性菌は、ビール
を混濁させるだけでなく硫化水素等の強い腐卵臭を発生するため、乳酸菌
による汚染よりも深刻な問題を引き起こす。野生酵母は、主に酒場でのビ
ール注ぎ口や配管から検出され、瓶・缶ビールからは殆ど検出されない。
野生酵母による汚染は上述のバクテリアほど深刻ではない。
ビール混濁微生物の検出および同定は、ビール品質管理にとって非常に
重要である。今日でも最も汎用されている検出・同定法は昔ながらの培養
法である。この方法では微生物を検出するまでに通常1週間からそれ以上を
必要とする。その結果、微生物検査の結果が出るまでに製品は既に出荷さ
れているケースが多い（その意味では微生物検査は品質管理よりも品質保
証として行われている）。それ故に、もっと迅速にビール混濁菌を検出・
同定する方法が待ち望まれてきた。PCR法は、非常に短時間に微生物の検
出・同定を行うことができるので、非常に有力な手法である。既知のビー
ル混濁菌種それぞれに対して特異的なPCRプライマーを、16SリボゾームR
NA遺伝子 (16S rDNA) 配列に基づいて設計することにより開発することが
できた（第2章）。しかしながら、PCR反応はビール中のポリフェノールな
どにより阻害されることがあり得るため、結果が偽陰性となる場合がある
。この問題は、16S rDNA上に存在するほぼ全ての菌種に共通する配列に対
してPCRプライマーを作製し、それを内部陽性標準として菌種特異的プラ
イマーと同時に利用することにより解決することができた（第2章）。
主たるビール混濁性乳酸菌であるLactobacillus brevis菌やPediococcus damnosus菌の殆どの菌株はビール中で生育できるが、生育できない菌株も存在
する。そのため、品質保証の観点からビール混濁性株と非混濁性株の判別
が必要となる。ビール混濁性乳酸菌を混濁菌ならしめている最も決定的な
形質は、ビール中にイソα酸として存在するホップ化合物に対する耐性（
ホップ耐性）である。ホップ化合物はグラム陽性菌全般の生育を阻害し、
その抗菌メカニズムについて研究がなされた。主要なイソα酸の一種、
trans-isohumuloneについて研究が進められた結果、本化合物はホップ感受
79
総　括
性のLb. brevis菌に対して、細胞内にプロトンを持ち込むのと引換えに細胞
内に存在する陽イオン（Mn2+など）を細胞外に持ち出すことにより、電気
的に中性なイオン交換を行うイオノフォアとして作用することが解明され
た（Simpson, 1992）。その結果、細胞膜を隔てた電気化学的プロトン勾配
が打ち消されプロトン駆動力（proton motive force: pmf）が減少し、pmf依
存的な栄養素取り込みが減少する。そのため、細胞は生育阻害もしくは死
に至る。一方で、ホップ耐性についてもLb. brevis菌を用いて分子レベルま
で研究が進められた。horAという遺伝子がホップ耐性株Lb. brevis ABBC45
が有するプラスミドpRH45から見つけられた（Samiら, 1997a）。本遺伝子
がコードするタンパク質は乳酸球菌Lactococcus lactis から見出されたATP
Binding Cassette (ABC) 型多剤排出ポンプLmrA (van Veenら, 1996) と53%の
相同性を有する。Lb. brevis ABBC45株を段階的に高濃度のイソα酸に馴化
培養するとpRH45の増幅が観察された（Samiら, 1997a）。一方、pRH45を
除去するとホップ耐性は喪失し、さらにエレクトロポレーション法によりp
RH45を再導入するとホップ耐性は回復した（第3章、第4章）。ナイシン誘
導性発現システムを用いてLactococcus lactis菌にHorAタンパク質を発現さ
せたところ、このタンパク質はATP依存的にイソα酸を細胞膜から細胞外
に排出することによってL. lactis菌にホップ耐性を与えていることが、細胞
、反転膜小胞、精製したHorAを用いて再構成されたプロテオリポソームを
用いた一連の研究から明らかにされた（第5章）。さらにHorAに加えて、
膜結合型プロトンATPアーゼが高発現することによって、イソα酸と同時
に細胞内に侵入したプロトンの排出が促進され、ホップ耐性に寄与してい
ることも見出された（第6章）。これらのATP依存的分子に十分なエネルギ
ーを供給するため、ホップ耐性株は感受性株より多くのATPを生産できる
（Simpson and Fernandez, 1994）。さらには、pmf依存的ホップトランスポ
ーターの存在も最近示された（Suzukiら, 2002）。現在までに考えられる
Lb. brevis菌のホップ耐性の分子メカニズムを図１に示した。
ホップ耐性メカニズムを解明することにより、ビール混濁性株と非混濁
性株とを迅速に判別する方法を開発することができた。horA遺伝子および
その相同遺伝子を検出することができるhorA-PCR法はビール工場における
微生物管理に実用化されている（Samiら,
horA陽性となる
1997b）。
Lactobacillus属の菌株の殆ど全てがビール混濁能を有していた。また、菌体
内ATPプールを測定することによりLactobacillus 属菌のビール混濁能を判別
する方法も開発されている（Okazakiら, 1995）。しかしながら、いくつか
の菌株は、潜在的にはホップ耐性ながら、準阻害的濃度のホップ化合物に
曝されない限りビール中に生育できないことも観察されている（Simpson
ら, 1994）。
Pectinatus属菌およびMegasphaera cerevisiae菌のビール混濁能については
殆ど研究がなされていない。これらの菌種に属する菌株は、今まで報告さ
れている限り全てビール混濁能を有することから、ビール業者にとっては
菌種同定だけで十分である。しかしながら、昨今のビール香味の傾向（低
アルコール化、低苦味化）を考慮すると、今まで報告されなかったバクテ
80
総　括
リアが新たにビール混濁を引き起こす危険性も潜在している。今後、特に
グラム陰性菌のビール混濁能を研究することにより、この危険性を低減さ
せることができるかもしれない。
ホップ感受性株
Hop-H
Hop-Mn-Hop
細胞壁
Hop-H
細胞膜
ADP
H+
c
ADP
H+ + Hop-
H+
H+
ATP
Mn2+
ATP
H+
Hop-Mn-Hop
ADP
H+
ATP
Mn2+
ATP
ADP
Hop-Mn-Hop
ATP
Hop- + H+
H+
ATP
ADP
a
Hop-H
Hop-Mn-Hop
Hop-H
c
H+
c
H+
c
H+
b
Hop-H
ホップ耐性株
Hop-H
図１．ホップ耐性メカニズム。　ホップ化合物は分子中のプロトンを細胞内に存
在する陽イオンと交換するイオノフォアとして作用する。ホップ感受性株では、
ホップ化合物（Hop-H）が細胞内に侵入すると、細胞外より高いpHためにホップ
陰イオンとプロトンに解離する。ホップ陰イオンはMn2+などの2価陽イオンと捕捉
し、細胞外に拡散する。イオノフォア作用は、ホップ-金属イオン複合体の拡散と
共に、電気的には中性な陽イオン交換を成立させる。ホップ化合物からのプロト
ンの放出は細胞内pHを低下させ、その結果、細胞膜を隔てたプロトン勾配（∆pH
）を打ち消し、そのためプロトン駆動力（pmf）も打ち消される。その結果、pmf
依存的な栄養素取り込みも減少する。ホップ耐性株では、ホップ化合物はHorA
(a)（Sakamotoら, 2001）および、おそらくpmf依存的な別のトランスポーター (b)
（Suzukiら, 2002）によって細胞膜から排出される。さらに、高発現したプロトン
ATPアーゼ(c)の働きにより、ホップ化合物から放出されたプロトンが排出される
（Sakamotoら, 2002）。 ホップ耐性株ではATP生産量がホップ感受性株より多い
(Simpson and Fernandez, 1994)。その他に、細胞壁に存在するガラクトシルグ
リセロールテイコ酸（Yasuiら, 1997）や、細胞膜の脂質組成の変化がビール混濁
性乳酸菌のホップ化合物に対するバリアー能を向上させているかもしれない。
81
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98
Curriculum vitae
Kanta Sakamoto
Born in 1968 in Hyogo-ken, Japan.
Graduated in 1987 from Nada high school (Kobe).
Graduated in 1992 from the Department of Agricultural Chemistry, the Faculty of
Agriculture, the University of Tokyo, and granted the Bachelor’s degree of
Agricultural Science.
Graduated in 1994 from the Graduate School of Agriculture, the University of
Tokyo, and granted the Master’s degree of Agricultural Science.
Entered in 1994 Asahi Breweries, Ltd. (Tokyo).
Studied from 1998 till 1999 at the Molecular Microbiology Group of the
Groningen Biomolecular Sciences and Biotechnology Institute (GBB) of the
University of Groningen for the collaboration between Asahi Breweries, Ltd. and
the University of Groningen.
Ph. D. student in 2002 at the University of Groningen.
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