University of Groningen

Beer spoilage bacteria and hop resistance in Lactobacillus brevisSakamoto, Kanta

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Beer Spoilage Bacteriaand

Hop Resistancein

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 DevelopmentLaboratory, Asahi Breweries, Ltd., Japan and at the Department ofMicrobiology of the University of Groningen, The Netherlands.

RIJKSUNIVERSITEIT GRONINGEN

Beer spoilage bacteria and hop resistancein Lactobacillus brevis

Proefschrift

ter verkrijging van het doctoraat in deWiskunde en Natuurwetenschappenaan de Rijksuniversiteit Groningen

op gezag van deRector Magnificus, dr. F. Zwarts,in het openbaar te verdedigen op

dinsdag 8 oktober 2002om 13.15 uur

door

Kanta Sakamoto

geboren op 1 september 1968te Hyogo (Japan)

Promotor: Prof. dr. W. N. Konings

Beoordelingscommissie: Prof. dr. L. DijkhuizenProf. dr. A. J. M. DriessenProf. 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 greathonor for me to be granted the doctor’s degree from the University of Groningenunder your promotion. It was one of the most nervous moments in my life when wemet for the first time in May 1997 at your office in Haren, to discuss our study onthe 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 neverknew at that time that we would later have a very successful collaboration and thatyou would give me the precious opportunity of the Ph. D. promotion at thisuniversity.

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 greathonor 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. Mycuriosity 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 provenduring your stay in Japan.

I thank Sami-san, too. It was you that discovered this very interesting gene andthat opened my eyes to science. I can clearly remember that I was very excited toknow your study on hop-acclimatization in beer spoilage bacteria soon after Ientered Asahi Breweries. In the end I became mad for this gene and finally I gotthe protein.

Abelardo, thank you very much. You were always very kind to teach me theextraordinary complicated and sophisticated techniques for the expression,purification and reconstitution of membrane proteins even when you were verybusy. Without you, I could never have made the collaboration successful. I enjoyeddiscussing with you a lot, as well as the many “intelligent” Spanish words youtaught 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 alwaysbeen very kind to me. I clearly remember that your and Patricia’s eyes were full ofcuriosity to me, the first Japanese for you, when we met for the first time. Owing toyou, Mayu and I know about the elegant Polish culture. How nice it is that you aremy paranimf together with Eli!

Eli, my other paranimf, you are also kind to me and patient to translate my thesisinto Dutch. Thank you very much. I will never forget that we drank a lot with fununtill 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, andfrom 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 allthe 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 todisturb your busy job. Thank you very much.

I also thank Dr. J. Kok for the gift of pGK, Prof. H. Kobayashi and Dr. H. Saitofor the H+-ATPase, Prof. Atsushi Yokota, Dr. Y. Sasaki and Dr. T. Sasaki, forencouraging me, Dr. Sato, Prof. Sonomoto, Dr. Nakayama and other members ofJapanese Society for Lactic Acid Bacteria for nice discussions.

I appreciate my colleagues and bosses from Asahi Breweries, Ltd: Yamashita-san, 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, Takai-san, Yoshioka-san and all the others, thank you very much for the nice discussionsand kind support. I really appreciate Asahi Breweries Ltd., to give me the chance tostudy and finish my thesis at the University of Groningen.

Now, my family: Father, Mother and my sisters, thank you very much. I wouldalso like to thank Mayu’s father, mother and brother very much as well as mygrandmothers and all my relatives.

Finally, Mayu, my dearest lady and wife. Thank you very muuuuuch. I cannotlive without you any more. I would not be here if you had not said to me: “What anice 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 Iconsulted you about the invitation letter from Wil. It made me decide to come tothe Netherlands again. And finally I become a Ph. D. How happy I am togetherwith 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 RNAGene of Pectinatus sp. DSM20764 and Improvementof PCR Detection of Beer Spoilage Bacteria by theCombined Use of Specific and Universal Primers

27

CHAPTER 3 Electrotransformation of Lactobacillus brevis 37

CHAPTER 4 A Plasmid pRH45 of Lactobacillus brevis Confers HopResistance

41

CHAPTER 5 Hop Resistance in the Beer Spoilage BacteriumLactobacillus brevis Is Mediated by the ATP-BindingCassette Multidrug Transporter HorA

49

CHAPTER 6 The Membrane Bound ATPase Contributes to HopResistance of Lactobacillus brevis

61

SUMMARY 71

SAMENVATTING 75

総 括 79

REFERENCES 83

LIST OF PUBLICATIONS 97

Curriculum vitae 99

1

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 BACTERIA2.1. Gram-Positive Bacteria2.1.1. Lactobacillus2.1.2. Pediococcus2.1.3. Other Gram-Positive Bacteria2.2. Gram-Negative Bacteria2.2.1. Pectinatus2.2.2. Megasphaera2.2.3. Other Gram-Negative Bacteria

3. DETECTION OF BEER SPOILAGE BACTERIA3.1. Culture Media3.2. Identification Methods3.3. Discrimination of Beer Spoilage Bacteria from Non-Spoilers4. ANTIBACTERIAL ACITVITY OF HOP COMPOUNDS4.1. History of Hop Usage for Beer4.2. Hop Plant4.3. Antibacterial Compounds in Hops4.4. Antibacterial Mechanism of Hop Compounds5. HOP RESISTANCE IN LACTIC ACID BACTERIA5.1. Variation of Hop Resistance5.2. Features of Hop Resistance5.3. Mechanisms of Hop Resistance

6. BEER SPOILING ABILITY IN LACTIC ACID BACTERIA6.1. Factors Affecting Beer Spoiling Ability6.2. Prediction of Beer Spoilage by Lactic Acid Bacteria

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1. INTRODUCTIONBeer 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 anunfavorable 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 highcontent of carbondioxide (approx. 0.5% w/v), the low pH (3.8-4.7), the extremelyreduced content of oxygen (<0.1 ppm) and the presence of only traces of nutritivesubstances such as glucose, maltose and maltotriose. These latter carbon sourceshave been substrates for brewing yeast during fermentation. As a result pathogenssuch as Salmonellae typhimurium and Staphylococcus aureus do not grow orsurvive in beer (Bunker, 1955).

However, in spite of these unfavorable features a few micro-organisms stillmanage to grow in beer. These, so-called beer spoilage microorganisms, can causean increase of turbidity and unpleasant sensory changes of beer. Needless to saythat these changes can affect negatively not only the quality of final product butalso the financial gain of the brewing companies.

A number of micro-organisms have been reported to be beer spoilage micro-organisms, among which both Gram-positive and Gram-negative bacteria, as wellas so-called wild yeasts. Gram-positive beer spoilage bacteria include lactic acidbacteria belonging to the genera Lactobacillus and Pediococcus. They arerecognized as the most hazardous bacteria for breweries since these organisms areresponsible for approximately 70% of the microbial beer-spoilage incidents (Back,1994). The second group of beer spoilage bacteria is Gram-negative bacteria of thegenera Pectinatus and Megasphaera. The roles of these strictly anaerobic bacteriain beer spoilage have increased since the improved technology in modernbreweries has resulted in significant reduction of oxygen content in the finalproducts. Wild yeasts do cause less serious spoilage problems than bacteria but areconsidered a serious nuisance to brewers because of the difficulty to discriminatethem from brewing yeasts.

Considerable effort has been made by many microbiologists to control microbialcontamination in beer. The most commonly used method today for detecting beerspoilage micro-organisms in breweries is still traditional incubation on culturemedia. A number of selective media have been developed since Louis Pasteurpublished in 1876 ‘Études sur La Bière (Studies on beer)’. It usually takes a weekor even longer for bacteria to form visible colonies on plates or to increase theturbidity in nutrient broths. Consequently, the products are often already releasedfor sale before the microbiological results become available. If a beer spoilagemicro-organism is then detected and identified in the beer product it needs to berecalled from the market. This will cause serious commercial damages to thebrewery. Most microbiologists have focused on developing more specific and rapidmethods for the detection of beer spoilage micro-organisms than using the

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traditional culture methods. A number of advanced biotechnological techniques areemployed such as immunoassays with antibodies specific to beer spoilage bacteria.Recently polymerase chain reaction (PCR) technology, targeting specificnucleotide sequences of ribosomal RNA genes (rDNA), has been successfullyapplied for the rapid identification of both beer spoilage bacteria and wild yeasts.These methods can identify exactly the taxonomy of the micro-organism(s) foundin beer. However, not all beer spoilage bacteria can actually grow in beer. Amethod is needed to determine whether the detected bacterium is capable ofgrowing 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 inbeer. It usually takes one month or even longer to detect visible turbidity in theinoculated beer, meaning that this method is not very practical. A rapid method topredict beer spoiling ability is therefore urgently needed and the development ofsuch a method will be essential for understanding the nature of the beer-spoilingability.

Among the components of beer, hop compounds have received a lot of attentionfor reason of their preservative values and their bitterness. For centuries it wasgenerally 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 bacteriaand not of Gram-negative bacteria. His findings had a great impact because manypathogens such as Salmonella species are Gram-negative bacteria. Feature(s) suchas low pH and alcohol content undoubtedly have a negative effect on growth ofthese pathogens in beer. Among Gram-positive bacteria some species of lactic acidbacteria are less sensitive to hop compounds and are able to grow in beer. Insight inthe mechanism of resistance of lactic acid bacteria to hop compounds is crucial forunderstanding their beer spoiling ability. The antibacterial activity of hopcompounds and the hop-resistance of lactic acid bacteria have extensively beeninvestigated. On the other hand little studies have been done on the beer spoilingability of Gram-negative bacteria.

This chapter reviews the currently available information about beer spoilagebacteria, their growth and spoilage activity in beer.

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2. BEER SPOILAGE BACTERIABeer 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 lowerthan most bacteria can tolerate for growth. Furthermore, the high carbondioxideconcentration (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 ofnutritive substances, such as saccharides and amino acids, are very low since mosthave been consumed by brewing yeasts during fermentation.

Only a few bacteria are able to grow under such inhospitable conditions and areable to spoil beer (see Table 1). These bacteria include both Gram-positive and -negative species. Gram-positive beer spoilage bacteria belong almost always to thelactic acid bacteria. They are regarded as most harmful for brewing industry andare the cause of most of bacterial spoilage incidents. Only a few Gram-negativebacteria are known to cause beer spoilage. Some of these belong to the acetic acidbacteria and have received most attention. Today these aerobic bacteria do notpresent a serious problem in beer spoilage anymore, since improved brewingtechnology has led to a drastic reduction of the oxygen content in beer. Insteadstrictly anaerobic bacteria, typically Pectinatus spp. and Megasphaera cerevisiae,have become serious beer spoilage bacteria.

2.1. Gram-Positive BacteriaAlmost 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. Mosthazardous for the brewing industry are those belonging to the genera Lactobacillusand Pediococcus. In the period 1980-1990, 58-88% of the microbial beer-spoilageincidents in Germany were caused by lactobacilli and pediococci (Back et al.,1988; Back, 1994). Also in Czech all beer-spoilage bacteria detected in thebreweries belonged to lactic acid bacteria (Hollerová and Kubizniaková, 2001).The situation in other countries seems to be similar although for commercialreasons little statistical information has been supplied. These lactic acid bacteriaspoil beer by producing haze or rope and cause unpleasant flavor changes such assourness and atypical odor.

2.1.1. LactobacilliThe genus Lactobacillus is the largest genus of lactic acid bacteria and includes

numerous species. They are widely used in various fermentation processes,

CHAPTER 1

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including food products such as beer, wine, yoghurt and pickles. In contrast to thegeneral believe that all lactobacilli can grow in beer, only a few species have beenreported to be capable of beer spoilage (Rainbow, 1981; Priest, 1987, 1996;Jespersen and Jakobsen, 1996). Lb. brevis appears to be the most important beerspoiling Lactobacillus species and is detected at high frequency in beer andbreweries. More than half of the bacterial incidents were caused by this species(Back et al., 1988; Back, 1994; Hollerová and Kubizniaková, 2001). Lb. brevis isan obligate heterofermentative bacterium. It is one of the best-studied beer spoilagebacteria and grows optimally at 30°C and pH 4-6 and is generally resistant to hopcompounds. It is physiologically versatile and can cause various problems in beersuch as super-attenuation, due to the ability to ferment dextrins and starch(Lawrence, 1988). The antibacterial effects of hop compounds and themechanism(s) responsible for hop resistance have been studied in detail in thisspecies (Simpson, 1991, 1993a, 1993b; Simpson and Smith, 1992; Fernandez andSimpson, 1993; Simpson and Fernandez, 1994; Sami et al., 1997a, 1997b, 1998;Sakamoto et al., 2001, 2002; Suzuki et al., 2002). These studies will be describedin Chapter 4, 5 and 6.

The second most important beer spoiling lactobacillus, Lb. lindneri isresponsible 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 onlyrecently has Lb. lindneri been recognized on the basis of its 16S rRNA genesequence as a phylogenetically separate species in the genus Lactobacillus (Back etal., 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) butsurvives higher thermal treatments than other lactic acid bacteria (Back et al.,1992). All Lb. lindneri strains, tested so far, are capable of beer spoilage, whileother lactobacilli comprise both beer spoiling and non-spoiling strains (Rinck andWackerbauer, 1987a, 1987b; Storgårds et al., 1998). It is particularly problematicthat Lb. lindneri grows slowly on media commonly used in breweries while growthin beer can be very rapid.

Lb. buchneri, Lb. casei, Lb. coryneformis, Lb. curvatus and Lb. plantarum areless common beer spoiling bacteria than the two species described above (Priest,1996). Lb buchneri resembles closely Lb. brevis, but differs in its ability to fermentmelezitose. In addition, some Lb. buchneri strains require riboflavin in their growthmedium (Sharpe, 1979; Back, 1981). Lb. casei can produce diacetyl, which givesbeer an unacceptable buttery flavor. Diacetyl appears to be more potent in thatrespect than lactic acid, the major end-product of lactic acid bacteria. The thresholdvalue 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 byyeast and too high levels of diacetyl are produced when yeast is not properlyremoved from young beer (Inoue, 1981). Lb. casei is particularly problematic

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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 etal., 1988), Lb. collinoides and Lb. paracasei subsp. paracasei (Hollerová andKubizniaková, 2001) have also been reported to be beer spoilage species.

Recently few additional newly discovered species of lactobacilli have beenadded to the list of beer spoilers. According to its 16S rRNA gene sequence, astrain called BS-1 is related to Lb. coryneformis, but its narrow fermentationpattern (only limited to glucose, mannose and fructose) differs significantly notonly from that of Lb. coryneformis but also from other Lactobacillus spp.(Nakakita et al., 1998). Two other novel lactobacillus species were found inspoiled beer with significantly different taxonomical properties from those of otherLactobacillus 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 otherspecies, LA-6, has a weak beer spoiling ability and did not show any significanthomology to the other Lactobacillus spp. (Funahashi et al., 1998).

2.1.2. PediococciPediococci are homofermentative bacteria which grow in pairs and tetrads. They

were originally known as ‘sarcinae’ because their cell organization was confusedwith that of true sarcinae. Beer spoilage, caused by cocci and characterized by acidformation 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 Tetragenococcushalophilus), P. inopinatus, P. parvulus and P. pentosaceus (Back, 1978; Back andStackebrandt, 1978). Among them is P. damnosus the most common beer spoiler. Itwas 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). Theincidence of beer spoilage by pediococci has decreased most likely as a result ofimproved sanitation conditions in breweries. P. damnosus is generally resistant tohop compounds. It is interesting that P. damnosus is commonly found in beer andlate fermentations, but seldom in pitching yeast. In contrast P. inopinatus isfrequently detected in pitching yeast but rarely in the other stages of beerfermentations (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 ofethanol and hop compounds (Lawrence, 1988). P. pentsauces and P. acidilacticihave never been reported to cause any defect in finished beer (Simpson andTaguchi, 1995). The amount of diacetyl produced by pediococci varies from

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species to species. P. damnosus produces large amounts of diacetyl, P. inopinatusless and P. pentsauces not at all. Brewers therefore usually pay attention only to P.damnosus.

2.1.3. Other Gram-Positive BacteriaIn addition to Lactobacillus and Pediococcus species also a species from the

genus Microcococcus has been reported to be occasionally responsible for beerspoilage. M. kristinae can grow in beer with low ethanol and hop compounds at pHvalues above 4.5 (Back, 1981). Micrococci are usually strictly aerobic, but M.kristinae can grow also under anaerobic condition (Lawrence and Priest, 1981). Itproduces a fruity atypical aroma in beer (Back, 1981).

2.2. Gram-Negative BacteriaSeveral genera of Gram-negative bacteria are known to be involved in beer

spoilage. The presence of a hydrophobic outer membrane makes Gram-negativebacteria generally resistant to hop compounds. Aerobic acetic acid bacteria i.e.Gluconobacter and Acetobacter spp. were well-known as beer spoilage organismsin breweries but the role of these bacteria in beer spoilage has been reducedsignificantly due to the much lower oxygen content during the brewing processesand in packaged beer of modern breweries. Instead, the occurrence of strictlyanaerobic bacteria in beer spoilage incidents has increased. These include thegenera Pectinatus, Megaspahera, Selenomonas, Zymomonas and Zymophilus.Especially Pectinatus and Megasphaera species cause much more seriousproblems for breweries than lactobacilli and pediococci, mainly due to theproduction of the offensive ‘rotten egg’ odor in finished beer.

2.2.1. PectinatusPectinatus 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 non-pasteurized beer rather than in pasteurized beer (Back, 1994). Pectinatus specieswere 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 sowere all subsequent isolates (Back et al., 1979; Haukeli, 1980; Kirchner et al.,1980; Haikara et al., 1981; Takahashi, 1983, Soberka et al., 1988, 1989). Thenatural habitat of the Pectinatus species are still unknown (Haikara, 1991). Twospecies are found in this genus: P. cerevisiiphilus and P. frisingensis (Schleifer etal., 1990). There is also one strain DSM20764 isolated from spoiled beer thatdiffers considerably in genotype from the two other species (Weiss, personalcommunication, 1987). Its 16S rRNA gene sequence is distinctly different fromthat of the other two species (Sakamoto, 1997). Pectinatus spp. are non-spore-forming motile rods with lateral flagella attached to the concave side of the cell

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body. They swim actively and appear X-shaped when cells are young and snake-like and longer when cells are older. They possess some features that arecharacteristic for Gram-positive bacteria (Haikara et al., 1981) and are regarded asbeing intermediate between Gram-positive and -negative bacteria. Growth takesplace 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. Duringgrowth considerable amounts of propionic and acetic acids are produced as well assuccinic 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 Megaspaeraspp.) (Lee, 1994). The most characteristic feature of spoilage caused by Pectinatusspp. is extensive turbidity and an offensive ‘rotten egg’ smell brought by thecombination of various fatty acids, hydrogen sulfide and methyl mercaptan (Lee etal., 1978, 1980; Haikara et al., 1981). This spoilage activity can cause seriousdamages for breweries.

2.2.2. MegasphaeraMegasphaera 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 arenon-spore-forming, nonmotile, mesophilic cocci that occur singly or in pairs andoccasionally 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 tobe 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 andat 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). Itis the most anaerobic species known to exist in the brewing environment (Seidel etal., 1979). Beer spoilage caused by this organism results in a similar extremeturbidity as Pectinatus and the production of considerable quantities of butyric acidtogether with smaller amounts of acetic, isovaleric, valeric and caproic acids aswell as acetoin (Seidel et al., 1979). Like Pectinatus, the production of hydrogensulfide causes a fecal odor in beer (Lee, 1994), which makes this bacterium one ofthe most feared organisms for brewers.

2.2.3. Other Gram-Negative BacteriaIn addition to the two genera described above, some other Gram-negative

bacteria have been found to cause problems in the brewing industry. AnaerobicZymomonas spp. have been found in primed beer to which sugar was added and inale beer. Zymomonas mobilis is an aerotolerant anaerobe and grows above pH 3.4and at ethanol concentrations below 10% (w/v) (van Vuuren, 1996). There is no

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report of Zymomonas mobilis spoilage in lager beer, probably because of itsselective fermentation character (non-fermentative of maltose, maltotriose butfermentative of glucose, fructose and sucrose). It produces high levels ofacetaldehydes and hydrogen sulfide. Also another Zymophilus spp., Z.raffinosivorans has been reported as a beer spoiler (Schleifer et al., 1990; Seidel-Rüfer, 1990). The genus Zymophilus is phylogenetically close to the genusPectinatus. Zymophilus spp. can grow in beer, like Pectinatus spp., at pH valuesabove 4.3-4.6 and ethanol concentrations below 5% (w/v). Also their beer spoilageactivity is similar to that of Pectinatus spp. (Jespersen and Jakobsen, 1996).

Another Gram-negative bacterium Selenomonas lacitifex has also been reportedto play a role in certain beer spoilage incidents but this species has hardly beenstudied. Historically a lot of attention of the brewing industry has been given toaerobic Gram-negative bacteria. Acetic acid bacteria i.e. Gluconobacter andAcetobacter used to be well-known to breweries. They convert ethanol into acetate,which results in vinegary off-flavor of beer. For reasons explained above suchaerobes are no longer important in modern breweries.

Hafnia protea, formerly Obesumbacterium proteus, and Rahnella aquatilis,formerly Enterobacter agglomerans, have been detected in pitching yeasts butnever in finished beer. They can retard the fermentation process. Beer producedwith 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 weredetected in beer produced from wort contaminated by R. aquatilis (van Vuuren,1996).

Recently a novel strictly anaerobic Gram-negative bacterium was isolated from abrewery (Nakakita et al., 1998). It is a rod-shaped bacterium with no flagella thatcan 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 fromPectinatus, Zymomonas and Selenomonas spp..

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Table 1. Beer Spoilage BacteriaGram-positive bacteria Rod-shaped Cocci

Lactobacillus spp. Pediococcus spp.Lb. brevis P. damnosusLb. brevisimilis P. dextrinicusLb. buchneri P. inopinatusLb. caseiLb. coryneformis Micrococcus sp.Lb. curvatus M. kristinaeLb. lindneriLb. malefermentansLb. parabuchneriLb. plantarum

Gram-negative bacteria Rod-shaped CocciPectinatus spp. Megasphaera sp.

P. cerevisiiphilus M.cerevisiaeP. frisingensisP. sp. DSM20764

Selenomonas sp. Zymomonas sp.S. lacticifex Z. mobilis

Zymophilus sp.Z. raffinosivorans

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11

3. DETECTION OF BEER SPOILAGE BACTERIAWe 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 spoilagebacteria. For quality assurance of finished beer it is usually sufficient to controlpotential contaminations by Lactobacillus brevis, Lb. lindneri, Pediococcusdamnosus, and Pectinatus spp.. Most studies on beer spoilage bacteria havefocused on the taxonomical classification of these bacteria. These studies havemade it possible to identify the bacteria detected in beer and breweries and to takethe 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 Conventionrecommends three media for the detection of lactobacilli and pediococci: MRS (deMan, Rogosa and Sharpe) agar supplemented with cycloheximide to preventgrowth of aerobes such as yeasts and moulds, Raka-Ray medium supplementedwith cycloheximide and VLB (Versuchs- und Lehranstalt für Brauerei in Berlin)S7-S. Other optional media are UBA (Universal Beer Agar) supplemented withcycloheximide, 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 ofmaltose and yeast extract at pH 4.7. None of these media are suitable for detectingall strains of lactobacilli and pediococci but a combination of some of these mediayields the best results. For the detection of Pectinatus and Megasphaera thefollowing media are recommended: Concentrated MRS broth, PYF (Peptone, Yeastextract and Fructose) and Thioglycolate Medium for enrichment of beer, LL-Agarfor growth in Lee tube, and UBA, NBB and Raka-Ray for routine analysis atbreweries. For Zymomonas spp., Zymomonas Enrichment Medium is alsorecommended (EBC Analytica Microbiologica II, 1992). American Society ofBrewing Chemists recommends UBA and Brewer’s Tomato Juice Medium forgeneral microbial detection and other media including LMDA (Lee’s Multi-Differential Agar), Raka-Ray, BMB (Barney-Miller Brewery Medium) and MRSfor the detection of lactic acid bacteria and SMMP (Selective Medium forMegaspahera and Pectinatus) (Methods of Analysis of the American Society ofBrewing Chemists, Eighth Revised Edition, 1992). Brewery Convention of Japanalso recommends the use of some of those media (BCOJ Biseibutu Bunsekihou,1999). These culture media are listed in Table 2.

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12

Table 2. Selective culture media for the detection of beer spoilage bacteriaMedia Bacteria Recommended by3

MRS (de Man, Rogosa and Sharpe) LAB1 EBC, ASBC, BCOJRaka-Ray LAB, G(-)2 EBC, ASBC, BCOJVLB 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, BCOJNakagawa LAB EBC, BCOJSDA (Schwarz Differential Agar) LAB EBC, BCOJConcentrated MRS G(-) EBC, BCOJPYF (Peptone, Yeast extract and Fructose) G(-) EBC, BCOJThioglycolate Medium G(-) EBCLL-Agar G(-) EBC, BCOJUBA (Universal Beer Agar) LAB, G(-) EBC, ASBC, BCOJNBB (Nachweismedium für LAB, G(-) EBC, BCOJ

bierschädliche Bakteriën)Brewer’s Tomato Juice Medium LAB, G(-) ASBCLMDA (Lee’s Multi-Differential Agar) LAB ASBCBMB (Barney-Miller Brewery Medium) LAB ASBCSMMP (Selective Medium for G(-) ASBC, BCOJ

Megasphaera and Pectinatus)1 LAB, Lactic acid bacteria2 G(-), Gram-negative bacteria3 EBC, European Brewery Convention; ASBC, American Society of Brewing Chemists; BCOJ,

Brewery Convention of Japan

The conventional detection method based on culturing of the organisms in thesemedia has the significant disadvantage that is very time-consuming. One week oreven longer is needed to obtain visible colonies on plates or turbidity in broths.Consequently, the products are often already released for sale before themicrobiological results become available. Another problem is that these media arenot species-specific. Media for the detection of beer spoiling lactic acid bacteriaallow also growth of non-beer-spoilage species such as Lactobacillus delbrueckiiand Pediococcus acidilactici. If the selectivity is increased by the addition ofspecific chemicals to these media, even longer detection times might be required.

3.2. Identification MethodsFollowing the bacterial detection in these media, species identification is needed.

Besides the basic tests such as colony morphology, cell morphology, Gram-stainingand catalase assays, also biochemical tests such as sugar fermentation pattern andchromatographic analysis of organic acids can be performed. Also specificdetection and identification methods are used such as immunoassays with

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polyclonal or monoclonal antibodies (Claussen et al., 1975, 1981; Dolezil andKirsop, 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 andSatokari, 1999; Motoyama and Ogata, 2000; Bischoff et al., 2001). These modernmethods have been reviewed (Berney and Kot, 1992; Schmidt, 1992; Dowhanickand 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-SpoilersAfter 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-spoilagestrains. Among Lb. brevis and P. damnosus most of the strains are capable ofspoiling beer and only a few strains are not. On the other hand the number of beerspoilage strains in Lb. casei, Lb. coryneformis and Lb. plantarum is limited.Exceptionally, all strains of Lb. lindneri have been reported to be capable ofspoiling beer (Rinck and Wackerbauer, 1987a, 1987b; Storgårds et al., 1998).

Before the beginning of 1990s the only method was available for judging thebeer spoiling potential of a bacterium, the so-called ‘forcing test’. In this test thebacterium was re-inoculated into beer or beer enriched with concentrated nutrientmedium. However, this test has proven to be far from practical for qualityassurance since a few months are needed to obtain conclusive results.

More rapid procedures have been developed. The identification at the strain levelcan now be done at the genome level. Ribotyping, based on Southern hybridizationwith a ribosomal gene as a probe, has been successfully introduced (Motoyama etal., 1998, 2000; Satokari et al., 2000; Suihko and Haikara, 2000; Barney et al.,2001). Fully automated ribotyping machines are now commercially available andonly eight hours are needed to obtain conclusive results. AFLP (AmplifiedFragment Length Polymorphisms) (Perpete et al., 2001) and RAPD-PCR (RandomAmplified Polymorphic DNA) (Savard et al., 1994; Tompkins et al., 1996) havealso successfully been applied for bacterial strain identification as well as foridentification of brewing yeasts. In these methods the genotype of each beerspoilage strain is registered in a database. A comparison of the genotype of a newlydetected strain with registered genotypes will make a risk assessment possible.

Another approach is to determine the common physiological properties

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responsible for beer spoiling ability. For beer spoiling lactic acid bacteria thecommon physiological denominator is hop resistance, which allows growth ofthese bacteria in beer. However, measuring hop resistance by culturing in hopcontaining medium, is too time-consuming. It will be much faster to detect thephysiological traits that cause hop resistance with immunoassays or PCR. Beforethis can be done the cause of the antibacterial activity of hop compounds and themechanism(s) responsible for resistance towards hop compounds need to beknown.

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4. ANTIBACTERIAL ACTIVITY OF HOP COMPOUNDS

4.1. History of Hop Usage in BeerThe comfortable bitterness experienced in beer drinking is characteristic for and

is mainly caused by hop compounds. These hop compounds are present in theflowers of the hop plant, Humulus lupulus, L., which are added to the wort. Thisplant has been known for thousands of years. However, its use in beer is not as oldas the history of beer itself (5,000 to 7,000 years). A few descriptions of hops as abeer additive as well as a decoration for gardens were found in documents of thesixth century B.C. German monks in the 12th century often used hops in beermaking. 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 fromhops was not particularly appreciated. However, when in the 14th century beerproduction increased and beer was exported, the importance of hops in beer wasgradually more and more appreciated, not only for its contribution to beer flavorbut also for its contribution to the stability. Hopped beer can be preservedsignificantly longer than gruit beer. In 1516 Wilhelm IV, the lord of Bayern,enacted the ‘Reinheitsgebot (Purity Law)’ which ordered that beer must be madefrom barley, water and hops. Since then, the use of hops became more popular andstandard. Many aspects of this law were adopted by other countries, which madehops indispensable for the brewing industry.

4.2. Hop PlantThe 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. Onlythe female flowers, the so-called cones, are used for beer. The mature cones containgolden resinous granules, the lupulin, which are the most important part of theflower for the bitterness and preservation of beer. Hop resins are extracted andfractionated as shown in Fig. 2.

Figure 1. Hop plant. (a) Hopcones at the end of the vine. (b) Avertical section of hop cone.Lupuline glands locate on thebase of each bract. They containthe hop resins and essential oilswhich give beer a unique flavor.

Strig

Bract

Lupulinglands

a b

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16

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%)

Formationas lead salt

α-acids (5-13%) β-fraction (5-15%)

β-acids (3-8%) Unknown soft resins (2-7%)

Hop cones

soluble in ether and cold methanol

Total resins (15-30%)

hexane

soluble insoluble

Total soft resins (10-25%) Hard resin (3-5%)

Formationas lead salt

α-acids (5-13%) β-fraction (5-15%)

β-acids (3-8%) Unknown soft resins (2-7%)

4.3. Antibacterial Compounds in HopsHop chemistry has been developed since 19th century and has been extensively

reviewed (Verzele, 1986; Moir, 2000). Research has especially been focused on theantibacterial properties of hop compounds and the bitter substances derived fromhops. This research goes back to 1888 when Hayduck showed first that antisepticproperties of the hops are due to the soft resins (Hayduck, 1888). In the Institute ofBrewing of the United Kingdom, Walker conducted from 1922 till 1941 a long-term 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-acidfraction (humulone) and β-acid fraction (lupulone).

The α-acid fraction is a mixture of homologous compounds, the α-acids, whichare not transferred as such to beer. During the wort boiling stage in the brewingprocess, α-acids are converted by a rearrangement or isomerization to iso-α-acids,which are much more soluble and bitterer than the original compounds. Thisconversion, which is very important in hop chemistry, was advanced first in 1888(Hayduck). Wieland et al. (1925) suggested that the hydrolysis of humulone tohumulinic acid proceeded via an intermediate. Windisch et al. (1927) investigatedthe humulone boiling products under alkaline conditions and isolated a resinous

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and bitter oil termed “Resin A” with chemical properties similar to the intermediateand isomeric with humulone. Around 1950, Rigby and Bethune showed that α-acidfraction is a mixture of three major compounds; humulone, cohumulone andadhumulone (Figure 3) (Rigby and Bethune, 1952, 1953). The bittering compoundsof beer were found to comprise three major analogues of these three α-acids, whichare now known as iso-α-acids; isohumulone, isocohumulone and isoadhumulone(Figure 3). Stereoisomers (cis- and trans-) exist for each iso-α-acid. Finally thechemical structure and configuration of naturally occurring (-)-humulone (DeKeukeleire and Verzele, 1970) and isohumulones (De Keukeleire and Verzele,1971) were elucidated. The isomerization yield of α-acids during wort boilingprocess is low [typically of the order of 30% (Hughes, 2000)] due to relativelyacidic condition of wort (ca. pH 5.2) and the adsorption to the wort coagulumduring 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 nottransferred to beer and have no direct value in brewing.

I II R α-acids β-acids-(CH) 2CH(CH3)2 humulone lupulone-CH(CH3) 2 cohumulone colupulone-CH(CH3)CH2CH3 adhumulone adlupulone

III IV V(-)-humulones trans-isohumulones cis-isohumulones

Figure 3. Chemical structures of hop coumpounds. The name of each α-acid (I) and β-acid (II) is dependent on its side chain. During wort boiling process, α-acids (naturally R-body; III) are isomerized to result in stereoisomers of trans-isohumulones (IV) and cis-isohumulones (V).

O

OH

OH

R

O

OH

O

O

O

OH

OH R

O

O

O

OH

OH R

OH

O

OOH

R

O OH

OH

R

O

O

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18

4.4. Antibacterial Mechanism of Hop CompoundsThe 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 butthey dissolve to less extent in beer and water. Studies of the antiseptic properties ofhopped wort and hop boiling product showed that they inhibit growth of Gram-positive bacteria but not of Gram-negative bacteria (Shimwell, 1937a; Walker andBlakebrough, 1952). It was first reported by Shimwell (1937a) that the antisepticpotency of hop increases at lower pH. Interestingly, he predicted that the antisepticpotency of hop is associated with permeability changes of the bacterial cell wall(1937b). The ‘bacteriostatic power’ was also studied of hop compounds, includinghumulone and the humulone boiling product (Walker and Blakebrough, 1952). Thehumulone 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 atpH 4.3, the pH of beer. The hop constituents (lupulone, humulone, isohumuloneand humulinic acid) were found to cause leakage of the cytoplasmic membrane ofBacillus subtilis, resulting in the inhibition of active transport of sugar and aminoacids (Teuber and Schmalreck, 1973). Subsequently inhibition of respiration andsynthesis of protein, RNA and DNA was also observed. Since the iso-α-acids aremainly present in beer among the hop resins and their derivatives, a preciseinvestigation of the antibacterial activity of iso-α-acids was needed forunderstanding the preservation or bacterial stability of beer. The molecularmechanism of antibacterial activity of iso-α-acids and the effects of pH of thegrowth medium and other variables on the antibacterial activity of hop compoundswere investigated after 1990 (Simpson and Smith, 1992). Hop compounds areweak acids and the undissociated forms are mainly responsible for inhibition ofbacterial growth (Fig. 4).

O

O

O

OH

OH

O

O

O

O

OH

+ H+pKa = 3.1

Antibacterial form Inactive form

Figure 4. Dissociation of trans-isohumulone. (Fernandez and Simpson, 1995b)

In Lb. brevis (Simpson, 1993b) trans-isohumulone reduces the uptake of leucineand causes slow leakage of accumulated leucine. trans-Isohumulone dissipates

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19

effectively the transmembrane pH gradient (∆pH) of the proton motive force butnot the transmembrane electrical potential (∆ψ). Inhibition of H+-ATPase activitywas not observed. Potentiometric studies revealed that undissociated trans-isohumulone acts most likely as an ionophore, catalyzing electroneutral influx ofundissociated isohumulone, internal dissociation of (H+)-isohumulone and efflux ofthe complex of isohumulone with divalent cations such as Mn2+. This cation isknown to be present at high concentrations in lactobacillus cells (Archibald andFridovich, 1981a, 1981b; Archibald and Duong, 1984). The result of this activity isa decrease of the pH gradient across the membrane. It was reported that theantibacterial activity of trans-isohumulone can be influenced by the presence ofcations in the medium. Protonophoric activity of trans-isohumulone requires thepresence of monovalent cations such as K+, Na+ or Rb+ and increases with theconcentration of these monovalent cations (Simpson and Smith, 1992). trans-Isohumulone cannot bind K+ unless a divalent cation, such as Mn2+, Mg2+, Ni2+ andCa2+ or a trivalent cation, such as Li3+ and Al3+, is present in the medium (Simpsonet al., 1993; Simpson and Hughes, 1993). Thus, the ability of hop compounds tobind simultaneously two or more cations may be crucial for their antibacterialaction but the reason has been still unclear.

The properties of other hop acids are similar to those of trans-isohumulone and itis likely that the mechanism of their antibacterial activities is also similar. Somestrains of lactic acid bacteria, which are sensitive to trans-isohumulone, are alsosensitive to (-)-humulone and colupulone and other strains resistant to trans-isohumulone are also resistant to the related compounds (Fernandez and Simpson,1993). The antibacterial activities of 6 naturally occurring iso-α-acids, 5 chemicallyreduced iso-α-acids and a reduced iso-α-acids mixture were higher at lower pH-values while more hydrophobic reduced iso-α-acids were found to be far moreantibacterial than their naturally occurring analogues (Price and Stapely, 2001).

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20

5. HOP RESISTANCE IN LACTIC ACID BACTERIABeer 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 notonly of scientific interest but is also important for the microbial control in brewingindustry to predict the beer spoiling ability of lactic acid bacteria.

5.1. Variation of Hop ResistanceThe 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 degreeof hop resistance varies among the different strains of Lb. brevis (Hough et al.,1957; Harris and Watson, 1960; Simpson and Fernandez, 1992; Fernandez andSimpson, 1993). Hop resistance of lactobacilli decreases upon prolonged serialsubculturing in the absence of hop compounds (Shimwell, 1936c; Yamamoto,1958; Richards and Macrae, 1964). Hop resistance was thought to be caused byimmunity acquired by prolonged contact with hop compounds under brewingconditions (Shimwell, 1937a). The necessity of beer spoiling lactic acid bacteria toacclimatize to beer or hop compounds in order to reproduce in beer (Yamamoto,1958) was solidly documented by Richards and Macrae in 1964. Hop resistanceincreased 8 to 20 fold in strains of lactobacilli upon serial subculturing in mediacontaining increasing concentrations of hop compounds, while subculturing ofresistant populations in the absence of hop compounds resulted gradually indecreased hop resistance. It took about one year of subculturing in unhopped beerto maximally reduce hop resistance of lactobacilli, indicating that the acquired hopresistance can be a very stable property (Shimwell, 1936c). However, organismsisolated from spoiled beer frequently fail to grow upon reinoculation in beer.Preculturing in the presence of sub-inhibitory concentrations of isohumulone isneeded 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 plasmidcuring or mutation induced with ultraviolet light (Fernandez and Simpson, 1993),suggesting that it may be generally a stable character, both phenotypically andgenetically. The Lb. brevis strain ABBC45 can develop hop resistance in the sameway as observed by Richards and Macrea (Sami et al., 1997a). Hop resistanceincreased with the copy number of plasmid pRH45. When Lb. brevis ABBC45 wascured 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). Thisplasmid contains the horA gene that codes for a polypeptide that is 53% identical toLmrA, the lactococcal ATP-binding cassette (ABC) multidrug transporter (vanVeen et al., 1996). HorA protein was expressed heterologously in Lactococcuslactis and found to function as an ABC-type multidrug transporter and to excretehop compounds (Sakamoto et al., 2001).

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21

5.2. Features of Hop ResistanceThe 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 andSimpson (1993) compared the properties of hop resistant strains of lactobacilli andpediococci with those of sensitive strains. No obvious correlation was foundbetween hop resistance and cell morphology, colony morphology, pH range forgrowth, sugar utilization profile, products of metabolism, manganese requirementand sensitivity to superoxide radicals, expression of cellular proteins, andresistance to various antibacterial agents. However, differences were found in thetransmembrane pH gradient (∆pH) and the cellular ATP pool. Because hopcompounds act as protonophores that dissipate the ∆pH across the cellularmembrane (Simpson, 1993a, 1993b), these differences are of great importance forunderstanding the mechanism of hop resistance.

5.3. Mechanisms of Hop ResistanceThe molecular structure of antibacterial agents might supply an insight in the

mechanism of resistance. Resistance to trans-isohumulone also results in resistanceto (-)-humulone and colupulone (Fernandez and Simpson, 1993), suggesting acommon mechanism of resistance against a broad range of hop acids. trans-Isohumulone and (-)-humulone have three hydrophobic side chains whilecolupulone has four. The side chains are attached to a five-membered ring of trans-isohumulone, 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, benzoicacid), solvents (ethanol), or antibiotics (ampicillin, cefamandole, vancomycin)(Fernandez and Simpson, 1993). The resistance mechanism might be specific forthe β-triketone group of the hop acids, which plays an essential role in theantibacterial action (Simpson, 1991).

Micro-organisms have developed various ways to resist the toxicity ofantibacterial agents:(i) enzymatic drug inactivation. A well known example is β-lactamase whichhydrolyzes the β-lactam ring into innocuous substrates. In hop resistant strains ofLb. 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 enzymaticmodification in such a way that the affinity of the target for the antibiotics isreduced. 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, containsextremely long chain fatty acids in its membrane (Uchida, 1974), which may play a

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22

role in ethanol resistance (Ingram, 1984). It is possible but not yet investigated thathop resistant Lb. brevis has also a changed lipid composition of its membrane tolower the permeability to hop compounds.(iii) inhibition of drug influx. The outer membrane of Gram-negative bacteriarestricts the permeation of lipophilic drugs, while the cell wall of the Gram-positivemycobacteria has been found to be an exceptionally efficient barrier. Thepermeation of hop compounds might also be affected by the presence of agalactosylated glycerol teichoic acid in beer spoilage lactic acid bacteria (Yasui andYoda, 1997b).(iv) active extrusion of drugs. The presence of (multi)drug resistance pump in thecytoplasmic 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 tohop compounds (Sami, 1999). In addition a proton motive force dependent hopexcretion transporter was suggested in this strain (Suzuki et al., 2002). (v) other mechanisms to tolerate the toxic effects of drugs. Since hop compoundsact as protonophores and dissipate the transmembrane pH gradient (∆pH), the cellscould respond by increasing the rate at which protons are expelled. The hopresistant strains maintain a larger ∆pH than hop sensitive strains (Simpson andFernandez, 1993) and Lb. brevis ABBC45 increases its H+-ATPase activity uponacclimatization to hop compounds (Sakamoto et al., 2002). The ATP pool in hopresistant strains was also found to be larger than in hop sensitive strains (Simpsonand Fernandez, 1993; Okazaki et al., 1997). The ability of hop resistant strains toproduce large amounts of ATP in the cell is needed for the increased activity of theH+-ATPase and for the hop extruding activity of HorA. It is interesting that theseresponses occurred also in hop sensitive strains at sub-inhibitory concentration oftrans-isohumulone (Simpson, 1993a; Simpson and Fernandez, 1993). However, athigher concentrations both the ∆pH and the ATP pool decreased in the hopsensitive strains, but not in the hop resistant strains. A role of HitA in hopresistance was suggested (Hayashi et al., 2001). It is about 30% identical to thenatural resistance-associated macrophage proteins (Nramp), which function asdivalent-cation transporters in many prokaryotic and eukaryotic organisms. HitAcould play a role in transport of divalent cations while isohumulone has beenclaimed to exchange protons for cellar divalent cations such as Mn2+. Thus asimple mechanism of hop resistance does not appear to exist. The resistancemechanisms found so far in Lb. brevis are illustrated in Fig. 5.

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23

Cytoplasmic membrane

Cell wall

Hop-Mn-Hop

Hop-Mn-Hop

Hop-H

Hop- + H+

Hop-H

H+

Hop-H

b

c

Hop-H

ATP ADP

a

ATP ADP

ATPcH+

H+

H+

Mn2+

Cytoplasmic membrane

Cell wall

Hop-Mn-Hop

Hop-Mn-Hop

Hop-H

Hop- + H+

Hop-H

H+

Hop-H

b

c

Hop-H

ATP ADP

a

ATP ADP

ATPccH+

H+

H+

Mn2+

Figure 5. Mechanisms of hop resistance. Resistance to hop compounds is conferred by anumber of processes. Hop compounds (Hop-H) are expelled from the cytoplasmicmembrane in hop resistant cells by HorA (a) (Sakamoto et al., 2001) and probably also by apmf-dependent transporter (b) (Suzuki et al., 2002). When hop compounds enter thecytoplasm 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 outof the cell. The hop resistant strains can generate more ATP than hop sensitive strains(whirlpool).

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24

6. BEER SPOILING ABILITY IN LACTIC ACID BACTERIAHop 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 ispossible. For this induction some bacteria need to be exposed to sub-inhibitoryconcentrations of hop compounds (Simpson, 1993).

6.1. Factors Affecting the Growth in BeerBeer 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 lagerbeers with the biological challenge test (Fernandez and Simpson, 1995a). Astatistical analysis of the relationship between spoilage potential and 56 parametersof beer composition revealed a correlation with eight parameters: pH, beer color,the content of free amino nitrogen, total soluble nitrogen content and theconcentrations of a range of individual amino acids, maltotriose, undissociated SO2and hop compounds. The effects of dissolved carbon dioxide (CO2) and phenoliccompounds including catechin, gallic, phytic and ferulic acids on beer spoilagewere also investigated (Hammond et al., 1999). CO2 was found to inhibit thegrowth of lactobacilli at the concentrations present in typical beer but stimulate atthe lower concentrations. Among the phenolic compounds, ferulic acid, acomponent of barley cell wall and hence present in all beers, exerted a strongerantibacterial activity after enzymatic conversion into 4-vinyl guaiacol. Organicacids in beer may also influence bacterial growth but this aspect has hardly beenstudied.

6.2. Prediction of Beer Spoilage by Lactic Acid BacteriaA number of attempts have been made to develop methods to predict beer

spoiling ability. For lactic acid bacteria hop resistance is the key factor. Asdescribed above, some factors have been identified to cause hop resistance. Rapidprocedures for detecting these factors would be very beneficial for microbialcontrol in breweries. A set of PCR primers have been made that can specificallydetect 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 methodis based on ATP pool measurements in lactobacillus cells (Okazaki et al., 1997).

Polyclonal and monoclonal antibodies specific only for beer spoilage strainshave been reported. A series of antisera were made against the Lactobacillus groupE antigen, a cell wall glycerol teichoic acid beneath the S-layer protein (Yasui etal., 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; Sharpeet al., 1964). When the beer spoilage strain Lb. brevis 578 was used as an antigen,

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the resulting antiserum reacted specifically with other beer spoilage strains of Lb.brevis although they had been cultured in modified NBB medium (Nishikawa etal., 1985) which does not contain any hop compounds or beer. Surprisingly, thisantiserum also reacted with beer spoilage strains of P. damnosus, but not with anystrains of Lb. lindneri (Yasui, and Yoda, 1997a). Galactosylated glycerol teichoicacid was found to be the most likely epitope that presumably selectively increasesthe cell barrier to hop compounds (Yasui and Yoda, 1997b). Three monoclonalantibodies specific for beer spoilage ability of lactic acid bacteria were obtained byimmunizing mice with cells cultured in beer (Tsuchiya et al., 2000). Themonoclonal antibody raised against Lb. brevis reacted with all beer spoilage strainsof Lb. brevis and several beer spoilage strains of P. damnosus, but not with non-spoilage strains of Lb. brevis, P. damnosus and other lactic acid bacteria. Themonoclonal antibody raised against P. damnosus reacted significantly with all beerspoilage strains of P. damnosus and weakly with many of the beer spoilage strainsof Lb. brevis. On the other hand the monoclonal antibody raised against Lb.lindneri reacted specifically only with Lb. lindneri. The reactivity of the stillunknown antigens did not change regardless of the presence of hop compounds intheir culture media.

D-lactate dehydrogenase (LDH) of 60 strains of Lb. brevis, including 44 beerspoiling strains and 16 non-spoiling strains, was also investigated. The strainscould be divided in five groups (A, B, C, D and E) on the basis of the mobility oftheir D-LDH in native polyacrylamide gels (Takahashi et al., 1999). Forty out of 44beer spoilage strains were classified to the group B suggesting a relationshipbetween the D-LDH profile and beer spoiling ability. The purified D-LDH of thosegroups had different pH and temperature optima and isoelectric points. Especiallythe temperature optimum of 50°C of the Group B D-LDH is significantly lowerthan that of the other D-LDHs (60°C).

Except for the forcing test, none of the other methods so far available can predictbeer spoiling ability of all strains. Since hop resistance in lactic acid bacteria doesnot appear to be a general feature of all beer spoiling bacteria, combination ofseveral methods will be required to detect all potential beer spoiling strains.

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CHAPTER 2The Nucleotide Sequence of the 16S Ribosomal RNAGene of Pectinatus sp. DSM20764 and Improvementof PCR Detection of Beer Spoilage Bacteria by theCombined 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 andidentification of beer-spoilage bacteria with internal positive control PCR (IPC-PCR)’ published in the Proceeding of European Brewery Convention Congress,Maastricht, 1997, pp.631-638, and the patent (JP09-520359, WO97/20071 orUSP5869642: ‘Detection of genus Pectinatus’).

SUMMARYA 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 primersare used for the rapid detection and identification of these beer spoilage bacteriawith the PCR method. The 16S rDNA sequences were derived from GENBANK,except for Pectinatus sp. DSM20764, which is a unique strain with propertiesdifferent from those of Pectinatus cerevisiiphilus and P. frisingensis. Its 16S rDNAsequence was determined in this study. To prevent that the PCR method yieldsfalse negative results, a set of universal primers targeting consensus sequences in16S rDNA was employed. The combined use of this set of primers with a set ofspecies-specific primers makes it possible to successfully perform PCR and DNAextraction and to identify beer spoilage bacteria. This procedure improvesconsiderably the reliability of PCR detection of beer spoilage bacteria.

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INTRODUCTIONIn recent years, Japanese consumers have shown preference for non-pasteurized

beer (so-called ‘Nama’ beer in Japan) over pasteurized beer. Consequentlymicrobiological quality assurance has become more and more important in thebreweries. A number of rapid microbiological detection and identification methodshave been developed to replace the time-consuming conventional analysis based onthe culture media (Barney and Kot, 1992). Among these methods, the polymerasechain reaction (PCR) method is one of the most powerful tools because of itsrapidity and high sensitivity (Tshuchiya et al., 1992, 1993, 1994; DiMichel andLewis, 1993; Savard et al., 1994; Schofield, 1995; Taguchi et al., 1995; Vogessoret al., 1995a, 1995b; Stewart and Dowhanick, 1996; Thomas et al., 1996).

However, several problems have been encountered in using this method forpractical analysis: (i) PCR primers specific for Gram-negative beer spoilagebacteria, such as Pectinatus and Megasphaera have not yet been developed. Fromthe 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 detectseveral species simultaneously and do not discriminate between beer spoiling andnon-spoiling bacteria, (iii) False negative results are sometimes obtained whichmakes the procedure not very reliable. When PCR products are not found afterreaction with specific primers, it is not clear whether this represents indeed anegative result or whether it is due to a failure of the reaction and/or the DNAextraction.

In this study, we have solved these problems by (i) determining the sequence of16S rDNA of Pectinatus sp. DSM20764, (2) designing more specific primers forall the major beer spoilage bacteria and (3) employing a set of universal primerstargeting consensus sequences in 16S rDNA (Lane, 1991) as a probe for theinternal positive control.

MATERIALS AND METHODS

Bacterial strainsThe 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 vonMikroorganismen und Zellkulturen, Germany), IAM (Institute of AppliedMicrobiology, Japan), IFO (Institute for Fermentation, Japan), JCM (JapanCollection of Micoroorganisms) and from our own laboratory culture collection.Lactic acid bacteria and Gram-negative bacteria were incubated anaerobically at

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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.

Table 1. Bacteria used in this studyGenera species number of strainsLactobacillus brevis 48

casei 11collinoides 2coryneformis 1delburuekii 1fermentum 1lindneri 3plantarum 5rhamnosus 1spp. 22

Pediocossus damnosus 1Lactococcus lactis 1Leuconostoc lactis 1Pectinatus cerevisiae 5

frisingensis 28sp. 1a

Megasphaera cerevisiae 2Selenomonus lacticifex 1Zymomonus pausivorans 1

raffinosivorans 1Escherichia coli 1unknownb G(+)c 6

G(-)d 2a DSM20764b non beer-spoilage bacteria isolated from breweries.c Gram-positive bacteriad Gram-negative bacteria

DNA extraction and purification Chromosomal DNA was extracted from the bacterial cells using the

cetyltrimethylammonium bromide (CTAB) procedure (Olsen et al., 1991). One mlof the bacterial culture in the late exponential phase was centrifuged and theresulting cell pellet was washed once in sterile de-ionized water. The cells wereresuspended in 0.3 ml of suspension buffer (10 mM Tris-HCl [pH 8.0], 1 mMEDTA, 0.35 M sucrose) containing 1 mg/ml lysozyme (Sigma, USA) and 50 µg/mlN-Acetylmuramidase (Seikagaku Co., Japan) and incubated at 37°C for 30 min.Subsequently, 0.3 ml of two times concentrated CTAB lysing buffer (100 mMTris-HCl [pH 8.0], 1.5 M NaCl, 20 mM EDTA, 2% [w/v] CTAB, 2% [v/v] 2-Mercaptoethanol) containing 160 µg/ml Protease K (Nakarai Tesque, Inc., Japan)

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was added and the mixture was incubated for 1 h at 50°C. Phenol-chloroform (1:1v/v) (0.6 ml) was added and vigorously mixed with a vortex. After centrifugation at12,000 × g for 10 min, the aqueous phase (about 0.5 ml) was transferred carefullyto a new sterile test tube to avoid inclusion of debris. After addition of an equalvolume of isopropanol the mixture was centrifuged at 12,000 × g for 10 min. Theprecipitated DNA was washed with 70% (v/v) ethanol, and purified by gelfiltration chromatography using CHROMA SPIN-1000 column (ClontechLaboratories, Inc., USA).

For evaluation of the specifity of the species-specific primers, DNA wasextracted from pure cultures of each bacterium. For evaluation of the combined useof the universal primers and the specific primers, DNA was extracted from amixture of cultures of a beer spoilage bacterium and non beer-spoilage bacteriaisolated from breweries (their species are not identified) to imitate a bacterialcontaminated sample from a brewery.

16S rDNA sequencingThe 16S rDNA sequence of P. sp. DSM20764 was determined separately by the

direct sequencing technique. Several parts of the 16S rDNA were amplified byPCR 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 ofthese primers in the gene are shown in Fig. 1.

Table 2. The primers used for DNA SequencingPrimer Sequencea (5’ > 3’)27f AGAGTTTGATCMTGGCTCAG518r GWATTACCGCGGCKGCTGGCAC530f GTGCCAGCMGCCGCGG907r CCGTCAATTCMTTTRAGTTT926f AAACTYAAAKGAATTGACGG1114f GCAACGAGCGCAACCC1392r ACGGGCGGTGTGTRC1525r AAGGAGGTGWTCCARCCa M=C:A, Y=C:T, K=G:T, R=A:G, W=A:T; all 1:1.

Figure 1. The location ofprimer sequences in 16SrDNA and the pairs ofprimers used for DNAsequencing.

M13 sequence (5’-CACGACTTGTAAAACGAC-3’) was attached to the 5’ endof each primer set to initiate sequencing of the DNA. PCR was carried out with Ex-

16S rDNA518r27f

530f

27f

530f

1525r907r

1392r

1392r

926f907r

1114f

16S rDNA518r27f

530f

27f

530f

1525r907r

1392r

1392r

926f907r

1114f

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Taq polymerase (Takara Shuzo, Ltd., Japan) in a GeneAmp 2400 PCR System(Perkin-Elmer Corp., USA). The thermal cycling program consisted of fourcontinuous stages: (1) 94°C for 5 min. (2) five amplification cycles of [94°C for 1min, 55°C for 2 min and 74°C for 2 min.] (3) 25 amplification cycles of [98°C for30 sec. and 68°C for 2 min.] (4) 72°C for 10 min. After PCR, the amplified DNAfragments were separated through agarose gel electrophoresis and eluted from thegel with a SUPREC-01 spin column (Takara Shuzo, Ltd., Japan). The resultingDNA fragment was used as a template in the following DNA sequencingprocedures using a SequiTherm Long-Read Cycle Sequencing Kit-LC (EpicentreTechnologies, USA) and a LI-COR 4000L automatic sequencing system (LI-COR,USA) according to the manufacturer’s instructions. The infrared dye labeled IRD-M13 forward (-29) primer (Aloka Co., Ltd., Tokyo, Japan) was used as thesequencing primer. The DNA sequence was confirmed by both sense and antisensesequencing.

Specific primersA number of primers were designed and synthesized according to the species-

specific sequence regions in the 16S rDNA of beer spoilage bacteria. Comparativeanalysis of the sequences of the 16S rDNA was performed with DNASIS software(Hitachi Software Engineering Co., Japan). The reactivity of each primer withvarious bacteria, including both beer spoiling and non-spoiling bacteria, wasexamined. The PCR was done in 30 cycles consisting of 1 min denaturalizing at94°C, 1 min annealing at 55°C and 1 min elongation at 74°C. The PCR productswere separated through an agarose gel at 5 V/cm for 30 min. As the size standard, λDNA digested by Hind III or ΦX174 DNA digested by Hinc II was used. TheDNA-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 internalpositive control, because its target region is outside that of the specific primers in16S rDNA. It was used together with each specific primer set in the same reactiontube. PCR was performed as described above, but the concentration of universalprimers was 50 times more diluted than that of the specific primers (finalconcentration: 15 pM).

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RESULTS

16S rDNA sequence of Pectinatus sp. DSM20764 The oligonucleotide sequence of 16S rDNA of Pectinatus sp. DSM20764 wasdetermined and is shown in Fig. 2. A comparison with DNASIS software of this16S 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 pausivoransand 80.2% with Megasphaera cerevisiae.

AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAAC 60GGGACTTTTATTTCGGTAAAAGTCTAGTGGCAAACGGGTGAGTAACGCGTAGGCAACCTA 120CCTTCAAGATGGGGACAACATCCCGAAAGGGGTGCTAATACCGAATGTTGTAAGAGTACT 180GCATGGTACTTTTACCAAAGGCGGCTTTTAGCTGTTACTTGGAGATGGGCCTGCGTCTGA 240TTAGCTAGTTGGTGACGGTAATGGCGCACCAAGGCAACGATCAGTAGCCGGTCTGAGAGG 300ATGGACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGG 360AATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAACGAGGAAGGTCTTC 420GGATCGTAAAGTTCTGTTGCAGGGGACGAATGGCATTAGTGCTAATACCACTAATGAATG 480ACGGTACCCTGTTAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGG 540CGGCAAGCGTTGTCCGGAATCATTGGGCGTAAAGGGAGCGCAGGCGGACATTTAAGCGGA 600TCTTAAAAGTGCGGGGCTCAACCCCGTGATGGGGTCCGAACTGAATGTCTTGAGTGCAGG 660AGAGGAAAGCGGAATTCCCAGTGTAGCGGTGAAATGCGTAGATATTGGGAAGAACACCAG 720TGGCGAAGGCGGCTTTCTGGACTGTAACTGACGCTGAGGCTCGAAAGCCAGGGTAGCGAA 780CGGGATTAGATACCCCGGTAGTCCTGGCCGTAAACGATGGATACTAGGTGTAGGGGGTAT 840CGACCCCCCCTGTGCCGGAGTTAACGCAATAAGTATCCCGCCTGGGGAGTACGGCCGCAA 900GGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATT 960CGACGCAACGCGAAGAACCTTACCAGGGCTTGACATTGATTGACGCATTCAGAGATGGAT 1020GCTTCCTCTTCGGAGGACAAGAAAACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTG 1080AGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCATTTGTTGCCAGCACGTAAC 1140GGTGGGAACTCAAATGAGACTGCCGCGGACAACGCGGAGGAAGGCGGGGATGACGTCAAG 1200TCATCATGCCCCTTACGTCCTGGGCTACACACGTACTACAATGGGATACACAGAGGGAAG 1260CAAAGGAGCGATCCGGAGCGGAACCCAAAAAATATCCCCCAGTTCGGATTGCAGGCTGCA 1320ACTCGCCTGCATGAAGTCGGAATCGCTAGTAATCGCAGGTCAGCATACTGCGGTGAATAC 1380GTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAAAGTCATTCACACCCGAAGCCG 1440GCTAAGGGCCTTATGGAACCGACCGTCTAAGGTGGGGGCGATGATTGGGGTGAAGTCGTA 1500ACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTT 1542

Figure 2. Sequence of 16S rDNA of Pectinatus sp. DSM20764. The number on the rightindicates the last nucleotide of each row. (Genbank accession number AR034890, PatentNo; JP09-520359, WO97/20071 or USP5869642).

Specificity of the primersA 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 mostspecific primers for each bacterium were obtained by screening for specificity andsensitivity, which allowed the construction of highly specific primer sets (Table 3).

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The specificity of those primers is shown in Fig. 3. No reactivity was observed ofthese primers with DNA from the other bacteria or brewing yeast strains (data notshown).

Table 3. The Specific primersPrimer set Specific to Sequencea (5’ > 3’) Sizeb

Lb Lb. brevis CTGATTTCAACAATGAAGCc 861CCGTCAATTCCTTTGAGTTTd

Lca Lb. casei ATCCAAGAACCGCATGGTTCTTGGCc 735CCGTCAATTCCTTTGAGTTTd

Lco Lb. coryneformis GGGACTAGAGTAACTGTTAGTCC c 453CCGTCAATTCCTTTGAGTTTd

Lp Lb. plantarum TGGACCGCATGGTCCGAGCc 731CCGTCAATTCCTTTGAGTTTd

Pc P.cerevisiiphilus CAGGCGGATGACTAAGCGe 443AATATGCATCTCTGCATACGe

Pf P. frisingensis CAGGCGGAACATTAAGCG e 74CTCAAGAACCTCAGTTCGe

Psp P. sp. DSM20764 TGGGGTCCGAACTGAATGe 393GCATCCATCTCTGAATGCGe

Mc M. cerevisiae CTGCCGGACTGGAGTGTC 385CAGGATATCTCTATCCCTGG

a Upper sequence: forward primer, lower sequence: reverse primer.b 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).

Simultaneous PCR Two separate PCR products were obtained from a sample containing a beerspoilage bacterium when a set of universal primers was used together with thespecific primer set. An example is shown in Fig. 4. Two bands were detected froma sample containing M. cerevisiae (lane 1). The band of 308bp was amplified bythe universal primers and the band of 385bp was amplified by the primers specificfor M. cerevisiae. Only one PCR product of 308bp, amplified only by the universalprimers, 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 isresponsible for the failure of the test procedure.

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Figure 3. Specific detection of beer spoilage bacteria. Reaction of specific primers withDNA 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 lanerepresents 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. (anon 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.

Figure 4. Application of universal primers as aninternal positive control. PCR was performed withthe primer set specific for M. cerevisiae (Mc in Table3) and the set of universal primers (1114f-1392r inTable 2). Successful DNA extraction and PCR isindicated by the appearance of a 308bp band amplifiedby the universal primers (lane 1-4), while the absenceof this band indicates the failure of these procedures(lane 5-7). The band of 385 bp in the lane 1 indicatesthe presence of M. cerevisiae in the sample. Lane 1: abacterial mixture including both M. cerevisiae and nonbeer-spoilage bacteria isolated from breweries. Lane 2-7: bacterial mixtures of non beer-spoilage bacteriafrom breweries (different batches of DNA extraction).M: ΦX174/Hinc II DNA size marker.

e f g h

a b c d

1 2 3 4 5 6 M1 M1 1 2 3 4 5 6 M1 1 2 3 4 5 61 2 3 4 5 6 M1

M2 7 8 9 10 11 12 M2 7 8 9 10 11 12M2 7 8 9 10 11 12 7 8 9 10 11 12 M2

1 2 3 4 5 6 7 M1 2 3 4 5 6 7 M

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DISCUSSIONThis 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 beerspoilage bacteria, indicating that this bacterium belongs to the genus Pectinatus butdiffers from the other known Pectinatus species. The taxonomical information ofthis 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 formicrobial 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 forlactic acid bacteria (Tsuchiya et al., 1992; Thompson et al., 1994) the primersdeveloped in this study are sufficiently specific to identify the bacteria at thespecies level. Thus specific primers for the identification of all major beer spoilagebacteria are now available.

The PCR method is very rapid, specific and highly sensitive but since it is anenzymatic reaction it is subject to experimental problems. Failure of PCR can causefailure to detect beer spoilage bacteria in samples from breweries (Di Michele,1993). Several factors have been reported to inhibit the PCR reaction (Rossen etal., 1992). For some strains belonging to lactic acid bacteria it is known thatextraction of DNA is difficult due to the presence of thick cell wall and/orextracellular slime (Anderson and McKay, 1983). If DNA extraction fails,detection of spoilage bacteria by PCR fails, which can lead to false negativeresults. Cone et al. (1992) used a positive control template, specially designed andconstructed to be amplified with the same primers of the targeted gene to revealthat PCR worked properly. To apply their method for a large variety of bacterialspecies, special templates for each species had also to be developed. In stead ofconstructing these special templates the consensus sequences in 16S rDNA wereused here as a positive control template. The universal primer amplifies a 308 bpfragment and reveals successful extraction of DNA and PCR. In this way falsenegative results can be eliminated. The combined use of specific and universalprimers in one reaction has improved considerably the reliability of PCR detectionof beer spoilage bacteria and the quality assurance by PCR in breweries.

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CHAPTER 3Electrotransformation of Lactobacillus brevis

Kanta Sakamoto

This chapter was submitted to Applied and Environmental Microbiolgy.

SUMMARYThe conditions for electrotransformation of five Lactobacillus brevis strains

lacking horA or its homologue were investigated. Two of them were successfullytransformed. 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 lackingpRH45. No transformants were obtained from the other strains.

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INTRODUCTIONThe 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 amplifiedduring subculturing of the strain in medium containing increasing concentrations ofhop compounds. Most beer-spoilage lactobacilli strains harbor the horA gene or itshomologue (Sami, 1997b). The deduced amino acid sequence of HorA is veryhomologous to that of LmrA, a lactococcal ABC-type multidrug transporter (vanVeen et al., 1996) which makes HorA a potential player in the hop resistancemechanism.

Attempts to express HorA in Escherichia coli failed due to cell lysis soon afterthe expression of this protein (Sami, 1999). Also the introduction of a marker geneinto pRH45 was unsuccessful possibly due to the instability of plasmid DNAfragments in E. coli (Sami et al., 1997a). Recently Lb. brevis ABBC45C, whichspontaneously had lost pRH45, was segregated from the original strain ABBC45by 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 strainslacking horA would be extremely helpful for studying the role of this protein in hopresistance.

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; Aukrustet al., 1995; Klein et al., 1995; Berthier et al., 1996; Serror et al., 2002) butsuccessful transformation of Lb. brevis has not yet been reported. In this study theconditions for electrotransformation of Lb. brevis were determined. The developedtransformation procedure was used for the re-introduction of pRH45 intoABBC45C (Sami et al., 1998: Chapter 3).

MATERIALS AND METHODS

Bacteria and a plasmidLactobacillus brevis type strain JCM1059 was obtained from JCM (Japan

Collection of Microorganisms, Saitama, Japan). Lb. brevis ABBC45 and horA-lacking strains 45C, 216, 218 and 241 (Sami et al., 1997b) were from our laboratoryculture collection.

The plasmid pGK13, which has both a chloramphenicol resistance gene and anerythromycin resistance gene, was a gift from Dr. J. Kok (University of Groningen,The Netherlands) and prepared from Esherichia coli DH5α.

Preparation of competent cellsThe method previously described by Aukrust et al. (1995) was used with some

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modification. Cells were cultured anaerobically at 30°C in 100 ml of MRS mediumsupplemented with 1% (w/v) glycine. At mid-exponential phase (A600= ~0.6), cellswere chilled on ice for 10 min and harvested by centrifugation at 1,500 × g for 5min, washed once with 100 ml of cold 10 mM MgCl2 and collected bycentrifugation at 1,500 × g for 5 min. Subsequently, cells were washed with 100 mlof cold SM solution (952 mM sucrose and 3.5 mM MgCl2) and harvested bycentrifugation at 5,000 × g for 10 min. After repeating this step twice, cells weresuspended gently in 1 ml of cold SM solution and used for electroporation.

ElectroporationCompetent 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 GenePulser and a Pulse Controller apparatus (Bio-Rad, USA). Immediately MRS-SMmedium (MRS containing 0.5 M sucrose and 0.1 M MgCl2) was added and cellswere incubated for 2 h at 30°C before plating on MRS containing 15 µg/ml ofchloramphenicol and 5 µg/ml of erythromycin.

RESULTSAmong 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, theelectroporation procedure for Lb. brevis was optimized.

Aukrust et al. (1995) reported two different methods for preparing competentcells of lactobacilli. One of them includes a SM solution to wash cells (SMmethod), while the other includes a polyethylene glycol solution (PEG method).Both methods were applied for Lb. brevis ABBC45. The SM method was found toyield 2 to 40 times higher transformation efficiencies than the PEG method (datanot shown). The SM method was further optimized in this study. Some strains haveextracellular polysaccharides, which can be removed by a washing step with 10mM MgCl2. (Aukrust et al., 1992; Berthier et al., 1996). To improveelectroporation of competent cells MgCl2 was therefore included. Due to the highosmolarity of the SM solution some strains did not pellet at 1,500 or 3,000 × gcentrifugation for 10 min and centrifugation at 5,000 × g for 10 min was needed forsuccessful pelleting. Parameters of the electrical pulse (capacitance, resistance andvoltage) were varied. The highest transformation efficiency of 2.5 × 103 T/µg forJCM1059 was obtained at 25 µF, 100 or 200 Ω and 2.0 kV (Fig. 1A). Thetransformation efficiency increased with the capacitance from 0.25 to 25 µF whenthe other parameters were fixed at 400 Ω and 1.5 kV (data not shown). ForABBC45C 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.

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Figure 1. Transformation efficiency of Lb. brevis JCM1059 (A) and ABBC45C (B). Thecompetent cells were electroporated with pGK13 at various resistance (R) and voltage (V)values. The capacitance was fixed at 25 µF.

DISCUSSIONIn this study we demonstrated for the first time successful electrotransfomation

of Lb. brevis strains. The transformation efficiency varied strongly between thedifferent strains. The highest transformation efficiency was in the order of 103 T/µgfor JCM1059 and of 102 T/µg for ABBC45C. These values are comparable to thoseof other Lactobacillus spp. reported so far. The type strain of Lb. brevis JCM1059has no plasmid, which makes this a very useful strain for genetic and molecularstudies for Lb. brevis. However several attempts to transform Lb. brevis JCM1059with pRH45 failed (data not shown). The main reason for this failure can be thelarge size of plasmid pRH45 (15 kb). Successful electroporation of pRH45 into Lb.brevis ABBC45C was achieved and resulted in restoration of hop resistance (Samiet al., 1998, Chapter 3).

ACKNOWLEDGEMENTThe writer thanks Dr. J. Kok (University of Groningen, The Netherlands) for the

gift of pGK13.

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CHAPTER 4A 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 ofGeneral Applied Microbiology, 44:361-363 (1998) with some additionalinformation.

SUMMARYLactobacillus brevis ABBC45C was segregated from the original strain ABBC45

after repeating subculturing in the absence of hop compounds. Lb. brevisABBC45C lacks the hop-resistance related plasmid pRH45 that contains horA ofwhich the deduced amino acid sequence is 53% identical to LmrA, a lactococcalATP-binding cassette (ABC) multidrug transporter. Lb. brevis ABBC45C is lessresistant than ABBC45 to hop compounds and ethidium bromide (EtBr). WhenpRH45 was re-introduced into Lb. brevis ABBC45C by electroporation, the degreeof 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 ofnigericin, a higher rate of ethidium accumulation than cells of ABBC45. Theseresults indicate that pRH45 confers hop resistance in Lb. brevis ABBC45 byexcreting hop compounds by the multidrug ABC-type transporter HorA.

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INTRODUCTIONThe 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., exhibitresistance to hop compounds and grow in beer, thereby causing serious problemsfor the brewing industry. The mechanism(s) of hop resistance of lactobacilli ispoorly understood. A biochemical study suggested that unidentified components ofthe plasma membrane are responsible for the resistance (Simpson and Fernandez,1994).

In our previous work, we obtained a hop-resistant mutant from Lb. brevisABBC45 (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 openreading frame of 1749 nucleotides (DDBJ accession no. AB005752). HorA has sixputative transmembrane domains and an ATP-binding motif (Sami et al., 1997a).The deduced amino acid sequence of HorA shows significant similarity with thebacterial multidrug transporter LmrA (van Veen et al., 1996) and the mammalianmultidrug transporter MDR1 (Chen et al., 1986). LmrA has been identified as anATP-binding cassette (ABC) transporter, which confers resistance of Lactococcuslactis to various lipophilic toxic compounds, including ethidium bromide (Bolhuiset al., 1995; van Veen et al., 1996). Almost all lactobacilli isolated as beer-spoilagestrains 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 hopresistance is of crucial importance for the brewing industry, we investigated thecontribution of pRH45 in this hop resistance. In this study we obtained a segregantstrain ABBC45C, which had spontaneously lost the plasmid pRH45 and whichallowed us to demonstrate that horA on pRH45 is responsible for hop resistance.

MATERIALS AND METHODS

Bacterial cultureLactobacillus brevis strains were grown anaerobically at 30ºC in MRS broth

(Merck, Darmstadt, Germany, initial pH adjusted to 5.5 with HCl). Anaerobicconditions 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 byinoculating 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

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Anderson and McKay (1983). Plasmid profiles of the isolates and ABBC45 wereinvestigated by 0.75% agarose gel electrophoresis in TAE buffer (0.04 M Tris-acetate, 0.01 M EDTA, pH 8.0). A polymerase chain reaction (PCR) wasperformed using the total DNA extracted from Lb. brevis ABBC45C as a templateand two specific primer sets based on the ori and the horA sequences of pRH45. ASouthern blot analysis was also done for the plasmid DNAs of Lb. brevisABBC45C, using horA-specific DNA as a probe.

Re-introduction of pRH45Re-introduction of pRH45 into Lb. brevis ABBC45C was done by

cotransformation by electroporation with a generalized plasmid of lactic acidbacteria, 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 cold3.5 mM MgCl2 and twice with cold SM (925 mM sucrose, 3.5 mM MgCl2) andfinally suspended in 500 µl SM. Plasmid DNAs extracted from Lb. brevisABBC45, containing 1 µg of pRH45, were added to 40 µl of competent cellsuspension together with 10 ng of pGK13. Electroporation was done at 200 Ω, 2.0kV 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 0.5M sucrose and 0.1 M MgCl2) was added, and the cell suspension was incubated at30ºC for 2 h. Cells were harvested by centrifugation (5,500 × g, 4ºC, 5 min), spreadon the MRS agar plate containing 15 µg/ml chloramphenicol and 50 µM hopcompounds, and incubated anaerobically at 30ºC for 4 days.

Drug resistanceExponentially growing cells were diluted with sterile deionized water to a

concentration of 106 cells/ml. These cell suspensions (5 µl) were spotted on MRSagar plates containing various concentrations of hop compounds or EtBr, and theminimum inhibitory concentrations (MICs) were determined.

Ethidium acculmulationA 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 of0.7 was incubated with 10 mM EtBr after the preincubation of cells with 10 mM L-arginine and 4 µM nigericin at 30ºC for 10 min. L-arginine was added to generateATP by the arginine deiminase pathway (Cunnin et al., 1986) and nigericin fordissipating the transmembrane pH-gradient to prevent the action of pmf-driventransporters. Fluorescence was measured with an F-2000 fluorometer (Hitachi,Tokyo, Japan) for 20 min, using excitation and emission wavelengths of 500 and

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580 nm, respectively.

RESULTS

Lb. brevis ABBC45C lacks both pRH45 and horAA comparison of the plasmid profiles of Lb. brevis ABBC45 and of the segregant

ABBC45C obtained after repeated subcultering of ABBC45 in the absence of hopcompounds shows that ABBC45C

has lost pRH45, while the otherplasmids remained unchanged(Fig. 1). With the specific primersets for pRH45 and horA no PCRproducts were recognized in thetotal DNA of Lb. brevis ABBC45C

(data not shown). Also with thehorA-specific DNA probe nodiscrete band could be detected bya Southern blot analysis with theplasmid DNAs of Lb. brevisABBC45C (data not shown).

Re-introduction of pRH45Genetic engineering of lactic acid bacteria is not always possible. Attempts to

introduce a marker gene in pRH45 were unsuccessful, possibly due to theinstability of several DNA fragments of pRH45 in E. coli (Sami et al., 1997a).Cotransformation of Lb. brevis ABBC45C with pGK13 and pRH45 resulted inseven colonies. Examination of the plasmid profiles of these colonies revealed thatfour colonies had been successfully transformed with both pRH45 and pGK13(ABBC45C[pRH45, pGK13]; Fig. 2, lane 7, 8, 11, 12), while the other threecolonies 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 thetransformants was small, significant higher resistance to chloramphenicol wasrealized. Of the four transformants of Lb. brevis ABBC45C[pRH45, pGK13], onlyone 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 µMwhile the other transformants were only resistant hop compounds up to 300 µM(data not shown). This hop resistant transformant was used in the followingexperiments.

1 2 31 2 3Figure 1. Plasmid profiles ofLb. brevis ABBC45.Plasmids extracted from wildtype Lb. brevis ABBC45 (lane2) and ABBC45C (lane 3),were subjected to agarose gelelectrophoresis. Lane 1contains molecular weightstandards (λ DNA digestedwith Hind III). The position ofpRH45 is indicated by anarrow at the right side of thefigure.

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2.32.0

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pGK13pGK13

10 11 12 13(kb)

Figure 2. The plasmid profiles of wild type Lb. brevis ABBC45, pRH45-free segregantABBC45C and transformants of Lb. brevis ABBC45C. Plasmids extracted from ABBC45(lane 1), the pRH45-free segregant ABBC45C (lanes 2, 3; different amounts of DNA fromthe 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 seventransformants of ABBC45C electroporated with pGK13 and pRH45 (lanes 7-13) weresubjected to agarose gel electrophoresis. Lane 4 contains pGK13 extracted from the hoststrain of E. coli. The bands around 2.0, 3.0, and 5.0 kb in lane 4 correspond to the closedcircular DNA, the open circular DNA, and the linear DNA of pGK13, respectively. Thepositions of pRH45 and the open circular DNA of pGK13 are indicated by arrows on theright. M: molecular weight standards (λ DNA digested with Hind III) and their molecularweight (kb) are indicated on the left.

Drug resistanceUpon 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 re-introduction of pRH45 (Table 1). These results indicate that pRH45 contributes toresistance of Lb. brevis ABBC45 to hop compounds. Similar results were obtainedfor the resistance to EtBr. These differences in resistance were reproduciblyobserved in many experiments.

Table 1. Drug resistance of Lb. brevis ABBC45 MICa

Strains Hop compounds (µM) EtBr (µg/ml) ABBC45 (wild type) 200 30 ABBC45C 100 15 ABBC45C[pRH45, pGK13] 200 30 ABBC45C[pGK13] 100 15aMinimum inhibitory concentrations were determined from cell growth on MRS agar platescontaining various concentrations of hop compounds or ethidium bromide. MICs of hop compoundsare expressed as iso-α-acids concentrations (Simpson, 1993).

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Accumulation of EthidiumEthidium readily diffuses across the cell membrane and enters the cytoplasm.

Upon intercalation with DNA or RNA its fluorescence increases approximately 10-fold (Le Pecq and Paoletti, 1967). The amount of the intracellular ethidium cantherefore be followed fluorometrically. The rate of ethidium accumulation wassignificantly faster for cells of Lb. brevis ABBC45C than for cells of ABBC45 (Fig.3).

Figure 3. Accumulation of ethidium. The ethidium fluorescencedevelopment of cells of a pRH45-free segregant Lb. brevisABBC45C () and the wild-type ABBC45 () was shown. Cellswere preincubated with 10 mM L-arginine and 4 µM nigericin inHEPES (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 of10 µM ethidium bromide to the cell suspension. The fluorescence(arbitrary units, a.u.) was measured for 20 min by using theexcitation wavelength of 500 nm and the emission wavelength of580 nm.

DISCUSSIONThe resistance to hop compounds and EtBr of Lb. brevis ABBC45 decreased

when pRH45 was lost and completely recovered upon re-introduction of pRH45. Inprevious studies was found that the copy number of pRH45 increased with theresistance to EtBr and novobiocin when Lb. brevis ABBC45 was acclimatized tohigher concentrations of hop compounds (Sami et al., 1997a). The excellentcorrelation of the level of resistance to hop compounds and other drugs with thecopy number of pRH45 indicates a crucial role of pRH45 in conferring multidrugresistance.

Ethidium is a substrate of many bacterial multidrug resistant transporters,including BmrB of Bacillus subtilis (Neyfakh et al., 1991), QacA ofStaphylococcus aureus (Tennet et al., 1989), and LmrP (Bolhuis et al., 1995) andLmrA (van Veen et al., 1996) of Lactococcus lactis. The rate of ethidiumaccumulation in an MDR containing bacterium is determined by the rates ofdiffusion into and the pumping out of the cell (Bolhuis et al., 1994). Lb. brevisABBC45C was found to accumulate ethidium faster than ABBC45 in the presenceof nigericin. In these experiments nigericin was used to collapse thetransmembrane pH-gradient in order to inhibit proton-motive-force dependenttransporters. These results suggest therefore that the activity of the ATP-driven

Time (min)0 5 10 15 20

Ethi

dium

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extrusion system for ethidium is lower in the pRH45-free segregant Lb. brevisABBC45C than in the wild type ABBC45. Among the four transformants ofABBC45C[pRH45, pGK13] only the one, which contained pRH45 and pGK13 andhad retained the original plasmid profile of ABBC45C, showed the wild strainABBC45 level of hop resistance. It is concluded that the multidrug ABC-transporter HorA, encoded by horA on pRH45, is responsible for the increasedresistance to hop compounds and ethidum.

ACKNOWLEDGEMENTWe thank Dr. M. Yamasaki (Nihon University, Japan), Dr. T. Sasaki (Meiji Milk

Products Co. Ltd., Japan), and Dr. K. Abe (Kikkoman Co. Japan) for helpfulsuggestions, and Dr. J. Kok (University of Groningen, The Netherlands) for the giftof pGK13.

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CHAPTER 5Hop Resistance in the Beer Spoilage Bacterium

Lactobacillus brevis Is Mediated by the ATP-BindingCassette 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 withsome correction.

SUMMARYLactobacillus 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 thebeer a bitter taste. The hop resistance in Lb. brevis is, at least in part, dependent onthe 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 inLactococcus lactis. To study the role of HorA in hop resistance, HorA wasfunctionally expressed in L. lactis as a hexa-histidine-tagged protein using thenisin-controlled gene expression system. HorA expression increased the resistanceof L. lactis to hop compounds and cytotoxic drugs. Drug transport studies withL. lactis cells and membrane vesicles and with proteoliposomes containing purifiedHorA protein identified HorA as a new member of the ABC family of multidrugtransporters.

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INTRODUCTIONBacterial spoilage of beer products causes a serious problem in the brewing

industry. The iso-α-acids, derived from the flowers of the hop plant (Humuluslupulus L.), give beer a bitter taste and exert bacteriostatic effects on most Gram-positive bacteria due to their ability to dissipate the proton motive force (Simpsonand Smith, 1992; Simpson, 1993b; Simpson and Fernandez, 1994). A few lacticacid bacteria, such as Lactobacillus spp., are tolerant towards iso-α-acids and areable to grow in hopped beer (Simpson and Fernandez, 1992; Simpson, 1993a). Atpresent, the molecular mechanisms that underlie the hop resistance in lactic acidbacteria are not well understood.

Previously, Sami and colleagues have isolated a hop-tolerant Lactobacillus brevisstrain, ABBC45, in which the plasmid pRH45 confers hop resistance on the cells(1998). pRH45 harbors the horA gene, whose corresponding deduced amino acidsequence is 53% identical to that of the multidrug transporter LmrA in Lactococcuslactis (van Veen et al., 1996; Sami et al., 1997a). LmrA is a multidrug transporterable to transport a wide variety of amphiphilic compounds, including antibioticsand anticancer drugs, from the inner leaflet of the cytoplasmic membrane (Bolhuiset al., 1996; Shapiro and Ling, 1999; Putman et al., 2000b). Unlike other knownbacterial multidrug resistance proteins, LmrA is an ATP-binding cassette (ABC)transporter (Higgins, 1992; van Veen and Konings, 1998). The protein contains anN-terminal membrane domain with six transmembrane segments followed by theABC domain. Surprisingly, LmrA is a structural and functional homologue of thehuman multidrug resistance P-glycoprotein, overexpression of which is one of theprincipal causes of resistance of human cancer cells to chemotherapy, and can evencomplement P-glycoprotein in human lung fibroblast cells (van Veen et al., 1998a).

In this work, HorA was functionally overexpressed in L. lactis as a hexa-histidine-tagged protein. The hop resistance of L. lactis cells was increasedsignificantly as a result of HorA expression. The protein was purified by Ni2+-nitrilotriacetic acid (NTA) affinity chromatography and functionally reconstitutedinto proteoliposomes prepared from L. lactis lipids. Transport studies with cells,membrane vesicles, and proteoliposomes identified HorA as a multidrugtransporter which mediates the extrusion of structurally unrelated compounds,including iso-α-acids.

MATERIALS AND METHODS

Bacterial strains and growth conditionsLactobacillus brevis ABBC45 (Sami et al., 1998) was grown anaerobically at

30°C in MRS broth (Merck). Lactococcus lactis subsp. lactis NZ9000 was used asa host for the nisin-controlled gene expression (NICE) vector pNZ8048 (de Ruyter

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et al., 1996) and its horA-containing derivatives. L. lactis was grown at 30°C inM17 broth (Difco) supplemented with 5 µg of chloramphenicol/ml and with 0.5%glucose (wt/vol) when appropriate.

Genetic manipulationsThe 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 ATGACG ATG ACA AAG CCC AAG CTC AGT CCA AGA ACA ATA CCA AG-3' tointroduce a PstI site, NcoI site, and hexa-histidine tag at the 5' end of horA and theoligonucleotide 5'-GTA CCC TTA TCT AGA TTA TCA CCC GTT GCT C-3' tointroduce an XbaI site at the 3' end of horA. The PCR product was cloned as a PstI-XbaI fragment into pAlter-1 (Promega) using Escherichia coli JM109 as a host.After the internal NcoI site in horA was removed silently by site-directedmutagenesis using the Altered Sites II in vitro Mutagenesis System (Promega) andthe mutagenic oligonucleotide 5'-CCA GGA CCA TCG CCA TCA TGA CC-3', thehorA gene was cloned as an NcoI-XbaI fragment into pNZ8048, givingpNZHHorA. Finally, horA was sequenced to ensure that only the intended changeshad been introduced.

Hop resistanceTo test the hop resistance of L. lactis NZ9000 harboring pNZ8048 or

pNZHHorA, overnight cultures were diluted into fresh medium and grown to mid-exponential growth phase. Subsequently the cells were diluted to an optical densityat 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 thenisin A-producing L. lactis strain NZ9700 [de Ruyter et al., 1996]), and hopcompounds (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-protein-binding microplate (Greiner). Sterile silicon oil (50 µl) was pipetted on top of thesample to prevent evaporation. Growth was monitored at 15°C by measuring theOD690

every 10 min with a multiscan photometer (Titertek multiskan MCC/340MKII; Flow Laboratories).

Solubilization, purification, and reconstitution of histidine-tagged HorAL. 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 theculture through a 1-in-1,000 dilution of the supernatant of the nisin A-producingL. lactis strain NZ9700 (de Ruyter et al., 1996). The cell suspensions were furtherincubated 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 preparedusing a French pressure cell, as described (Margolles et al., 1999; Putman et al.,

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1999; van Veen et al., 2000), and stored in liquid nitrogen. His-tagged HorA wassolubilized with 1% n-dodecyl-β-maltoside as described previously (Margolles etal., 1999). Insoluble components were removed by ultracentrifugation at280,000 × g for 15 min at 4°C. The soluble fraction was mixed with Ni-NTA-agarose (Qiagen Inc.) (20 µl of resin/mg of protein) which was equilibrated withbuffer 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 shakengently at 4°C for 1 h. The resin was transferred to a Bio-spin column (Bio-Rad) andwashed with 20 column volumes of buffer A containing 20 mM imidazole andsubsequently 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 with250 mM imidazole. For reconstitition of purified HorA in proteoliposomes ofL. lactis lipids, total lipids of L. lactis were extracted and purified as describedpreviously (Margolles et al., 1999). Unilamellar liposomes with a relativelyhomogeneous size were prepared by freezing in liquid nitrogen, slow thawing atroom temperature, and extrusion of the liposomes 11 times through a 400-nm-pore-size 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 todestabilize the liposomes. The purified HorA was mixed with dodecyl maltoside-destabilized liposomes at a protein/lipid ratio of 1:100 (wt/wt), after which thesuspension 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, theproteoliposomes were collected by ultracentrifugation at 280,000 × g for 15 min at10°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 pNZHhorAwere grown at 30°C to an OD690 of about 0.5. Subsequently, about 10 ng of nisinA/ml was added to the culture through a 1-in-1,000 dilution of the supernatant ofthe nisin A-producing L. lactis strain NZ9700 (de Ruyter et al., 1996). The cellsuspensions were further incubated at 30°C for 2 h, after which the cells werecollected by centrifugation at 4°C at 8,000 × g for 10 min. The cells were washedwith 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 µMethidium bromide to allow the diffusion of ethidium bromide into the cells. Theethidium bromide efflux was initiated by the addition of 25 mM glucose. Thefluorescence of ethidium bromide was monitored at 20°C with a Perkin-Elmer LS50B 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 HorA-

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expressing and nonexpressing L. lactis cells, cells were grown in mediumsupplemented with 30 mM arginine rather than glucose (Poolman, at al., 1987).After the induction of HorA expression as described above, cells were washed with50 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 thepresence or absence of 0.5 mM ortho-vanadate. Finally, 10 µM ethidium bromidewas added to the cell suspensions, and the fluorescence of ethidium bromide wasmeasured at 20°C as described above.(ii) Hoechst 33342 transport. For the transport of Hoechst 33342 in inside-outmembrane vesicles, membrane vesicles were diluted to a final concentration of0.5 mg of membrane protein/ml in KPi (pH 7.5) containing 2 mM MgSO4, 5 mMphosphocreatine, and 0.1 mg of creatine kinase/ml. Valinomycin and nigericin wereadded to a final concentration of 0.4 µM each, to dissipate the membrane potentialand transmembrane pH gradient, respectively. After an incubation for 1 min at20°C, 2.3 µM Hoechst 33342 was added. The fluorescence of Hoechst 33342 wasmeasured at 20°C using a Perkin-Elmer LS 50B fluorimeter with excitation andemission wavelengths of 355 and 457 nm, respectively, and slit widths of 3 nmeach. After the Hoechst 33342 fluorescence had reached a steady state, Hoechst33342 transport was initiated by the addition of 2 mM Mg-ATP. In controlexperiments, Mg-ATPγS was used rather than Mg-ATP. For the transport ofHoechst 33342, HorA-containing proteoliposomes were thawed slowly andextruded 11 times through a 400-nm-pore-size polycarbonate filter. Subsequentlyproteoliposomes 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 asdescribed above in the absence of ionophores, using proteoliposomes at a finalconcentration of 0.01 mg of protein/ml.

RESULTS

Overexpression of hexa-histidine-tagged HorAUsing the polymerase chain reaction, the horA gene on plasmid pRH45 of

Lb. brevis ABBC45 was cloned into the lactococcal NICE expression vectorpNZ8048 under the control of the nisin-inducible nisA promoter. A hexa-histidinetag was added to the amino terminus of HorA to facilitate the purification of theprotein by Ni2+-NTA affinity chromatography. Induction of HorA expression inL. lactis NZ9000 by the addition of nisin A to exponentially growing cells resultedin the expression of a plasma membrane-associated 66-kDa polypeptide, whichcould be detected on a Western blot by using an anti-hexa-histidine-tag monoclonalantibody (Fig.1). HorA expression in cells was maximal after an induction time of

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2 h. Quantitative immunoblotting and densitometry analysis revealed a HorAexpression level of about 30% of the total membrane protein under these conditions(data not shown). Densitometric analysis of Coomassie-stained sodium dodecylsulfate-polyacrylamide gels of the membrane fraction of HorA-expressing cells andthe purified HorA indicated a purity of HorA of more than 95% (data not shown).

Figure1.Expression, purification,and functional reconstitution ofhexa-histidine-tagged HorA. TheHorA protein was overexpressedin L. lactis as a hexa-histidine-tagged protein using the NICEsystem. A silver-stained sodiumdodecyl sulfate-polyacrylamide gelis shown. Lane 1, total membrane protein (20 µg) of L. lactis harboring pNZHHorA; lane2, soluble fraction (20 µg of protein) of a lysate of HorA-expressing cells; lane 3, Westernblot of the membrane fraction (20 µg of protein) of HorA-expressing cells, with anti-hexa-histidine antibody; lane 4, flowthrough fraction of membrane proteins (20 µl of the totalfraction of 2 ml) eluted from the Ni2+-NTA resin; lanes 5, 6, and 7, histidine-tagged HorAeluted from the NTA resin (20 µl out of the total fraction of 2 ml) in three consecutive stepswith buffer supplemented with 250 mM imidazole; lane 8, molecular mass markers; lane9, HorA reconstituted into proteoliposomes. Lanes 3 and 9 are Western blots; the otherlanes are silver-stained gels. The arrow indicates the position of hexa-histidine-taggedHorA protein.

HorA overexpression confers hop resistance on L. lactis cellsThe hop resistance of L. lactis NZ9000 cells harboring pNZHHorA was

compared with the hop resistance of cells harboring the pNZ8048 control vector. Inthe absence of iso-α-acids the HorA-expressing cells grew slightly more slowly andreached a slightly lower cell density than control cells (Fig. 2A). A similar effect onthe 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-α-acid compounds on the growth of HorA-expressing cells. The inhibition of growthby 100, 200, and 300 µM hop compounds is significantly higher for control cellsthan for HorA-expressing cells, indicating that HorA expression in L. lactis resultsin an increased hop resistance of the organism.

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Figure 2. (A) Growth of control L. lactis harboring pNZ8048 (triangles) and of HorA-expressing 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 HorA-expressing L. lactis (squares). Cells were grown at 15°C in the absence or presence of a50, 100, 200, or 300 µM concentration of iso-α-acids. The OD690 was measured every10 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 multidrugtransporter LmrA in L. lactis (Sami et al., 1997a; van Veen and Konings, 1998).Fluorimetric ethidium transport assays were performed to test if HorA can mediatethe transport of ethidium, a typical substrate for LmrA. Washed cell suspensions ofL. lactis NZ9000 containing pNZHHorA or pNZ8048 were pre-equilibrated in thepresence of 10 µM ethidium bromide. Subsequently the cells were energizedthrough the addition of 20 mM glucose. The energization of cells resulted in anincreased rate of ethidium extrusion for the HorA-expressing cells compared to therate observed for nonexpressing control cells, suggesting that HorA is able tomediate the active extrusion of ethidium (Fig. 3A). HorA is a member of the ABCsuperfamily and should display an ATP-dependent extrusion activity. To analyzewhether ethidium efflux was coupled to ATP hydrolysis, the effect of the ABCtransporter inhibitor ortho-vanadate was examined. For this purpose, cells werepreenergized with 30 mM L-arginine and preincubated in the presence of 0.5 mMortho-vanadate. In this way, cells could generate metabolic energy by metabolizingarginine via the arginine-deiminase pathway (Poolman et al., 1987). In contrast toglycolysis, which is inhibited by ortho-vanadate, the arginine-deiminase pathway isnot affected by this inhibitor. ortho-Vanadate increased the level of ethidium uptakein HorA-expressing cells, while no increase was observed in control cells. Theseobservations indicate inhibition by ortho-vanadate of HorA-mediated efflux ofethidium (Fig. 3B and C).

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Figure 3. Ethidium transport in HorA-expressing cells and nonexpressing cells ofL. 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 developmentof fluorescence of the DNA-ethidium complex in the cell suspension was monitored at20°C over time. At the arrow, 25 mM glucose was added. (B) Effect of ortho-vanadate onthe accumulation of ethidium in control cells. Cells were energized with arginine andincubated for 7.5 min in the presence or absence of 0.5 mM ortho-vanadate prior to theaddition of 10 µM ethidium (at the arrow). (C) Effect of ortho-vanadate on theaccumulation of ethidium in HorA-expressing cells. Cells were treated as described forpanel B.

(ii) Hoechst 33342 transport in membrane vesicles. In previous studies, thepositively charged bisbenzimide dye Hoechst 33342 proved to be a useful probe tostudy the activity of multidrug transporters such as LmrA and the human multidrugresistance P-glycoprotein (Margolles et al., 1999; Putman et al., 1999; van Veen etal., 2000). Hoechst 33342 is highly fluorescent when it is present in thehydrophobic environment of the phospholipid bilayer. The transport of Hoechst33342 from the membrane into the aqueous phase can be followed as a decrease ofHoechst 33342 fluorescence over time. The ionophores valinomycin and nigericinwere included in this fluorescence assay at a concentration of 0.4 µM to dissipate

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the membrane potential and transmembrane pH gradient, respectively, generatedthrough proton pumping by the F1F0 H+-ATPase. In the presence of ATP, Hoechst33342 fluorescence decreased in HorA-containing membrane vesicles five-foldfaster than in membrane vesicles from control cells. In the presence of the slowlyhydrolyzable ATP analog ATPγS, no significant decrease of Hoechst33342 fluorescence was observed in both types of membrane vesicles (Fig. 4). TheATP-dependent Hoechst 33342 transport in the control cells is most likely due tothe presence of low levels of endogenous LmrA, since under the experimentalconditions employed the secondary multidrug-transporter LmrP cannot workbecause a proton motive force is absent. The results demonstrate that in thepresence of Mg-ATP, HorA efficiently transports Hoechst 33342 from themembrane into the lumen of inside-out membrane vesicles prepared from HorA-expressing L. lactis.

Figure 4. Hoechst 33342 transport in inside-out membrane vesicles of HorA-expressingcells and nonexpressing cells of L. lactis.Membrane vesicles prepared from HorA-expressing cells (H) and control cells (C) werediluted to a concentration of 0.5 mg ofmembrane protein/ml in buffer containing theATP regenerating system (see Materials andMethods) and 0.4 µM of each of the ionophoresvalinomycin and nigericin to dissipate themembrane potential and transmembrane pHgradient, respectively. After incubation for1 min at 20°C, 2.3 µM Hoechst 33342 wasadded to the assay mixture. At the arrow, 2 mMMg-ATP or 2 mM Mg-ATPγS was added.Hoechst 33342 transport was measured at 20°Cby fluorimetry.

(iii) Hoechst 33342 transport in proteoliposomes. HorA-mediated transport ofHoechst 33342 was also studied using purified and functionally reconstitutedprotein. The protein was solubilized using 0.05% dodecyl maltoside and purified bynickel chelate affinity chromatography to a high degree of purity (Fig. 1). HorAwas reconstituted by mixing the purified protein with preformed dodecylmaltoside-destabilized liposomes, composed of L. lactis lipids, after which thedetergent was removed by extraction with polystyrene beads. Transport studiesrevealed that purified HorA was able to transport Hoechst 33342 intoproteoliposomes in the presence of ATP (Fig. 5).

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Figure 5. Transport of Hoechst 33342 in proteoliposomes. Liposomes withoutreconstituted HorA protein (A) and proteoliposomes containing reconstituted HorA protein(B) were diluted in buffer containing the ATP regenerating system. After incubation for1 min at 20°C, 2.3 µM Hoechst 33342 was added to the assay mixture. At the arrow 2 mMMg-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 mayalso be able to extrude hop compounds. The specificity of HorA for hopcompounds was analyzed in Hoechst 33342 transport assays in which hopcompounds were included as competing substrates (Fig. 6). Because hopcompounds are protonophores that act upon the proton motive force, hopcompounds may also indirectly affect Hoechst 33342 partitioning in the membrane.Therefore, the ionophores valinomycin and nigericin were included in the Hoechst33342 transport assays at final concentrations of 0.4 µM. The HorA-mediatedtransport of Hoechst 33342 in the presence of ionophores was inhibited by hopcompounds (Fig. 6). The degree of inhibition was proportional to the concentrationof hop compounds used, indicating that hop compounds are transport substrates forHorA.

Figure 6. HorA displays specificity for hopcompounds. The ATP-dependent transport ofHoechst 33342 in HorA-containing inside-outmembrane vesicles was measured as described inthe legend to Fig. 4. Hop compounds at indicatedconcentrations were added to the assay mixtureprior to the addition of Hoechst 33342. The hopcompounds did not affect the fluorescence ofHoechst 33342 in control membrane vesicleswithout HorA (data not shown). To dissipate aproton motive force generated by F1F0-ATPase, theionophores valinomycin (0.4 µM) and nigericin(0.4 µM) were included in the assay medium.

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DISCUSSIONAlthough 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), themechanism of hop resistance in this organism has not been studied previously. Toanalyze the function of HorA in greater detail, hexa-histidine-tagged HorA wasexpressed in L. lactis. By employing the NICE system (de Ruyter et al., 1996), highexpression levels were obtained of up to 30% of total membrane protein. Cellfractionation studies indicated that the overexpressed HorA protein was associatedwith the plasma membrane in L. lactis. HorA is a member of the ABC superfamilyand is a structural homologue of the multidrug transporter LmrA in L. lactis (vanVeen and Konings, 1998). Therefore, the ability of HorA to act as a drug pump wasinvestigated. Transport experiments with HorA-expressing L. lactis cells, HorA-containing inside-out membrane vesicles, and proteoliposomes containing purifiedand functionally reconstituted HorA demonstrated that HorA mediated the transportof 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 expressedHorA to act as an extrusion system for hop compounds: (i) in vivo resistance togrowth inhibition by hop compounds and (ii) the competitive inhibition of drugtransport by hop compounds. The increased hop resistance in HorA-expressingL. lactis cells and the inhibition of Hoechst 33342 transport by hop compounds bothindicate that hop compounds are transport substrates of HorA. Hop compounds areable to dissipate the proton motive force in Gram-positive bacteria through acycling mechanism in which the undissociated iso-α-acids enter the cell bydiffusion through the phospholipid bilayer and, after dissociation of a proton,diffuse back to the extracellular environment as complex of the anionic species anddivalent cation such as Mn2+ (Simpson and Smith, 1992; Simpson, 1993a, 1993b;Simpson and Fernandez, 1994). The HorA-mediated resistance of cells to hopcompounds 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 drivethe extrusion of drugs (Putman et al., 2000a). LmrA and HorA representprokaryotic ABC multidrug transporters that share significant sequence similaritywith ABC proteins in Bacillus subtilis, Staphylococcus aureus, Escherichia coli,Helicobacter pylori, Haemophilus influenzae, and Mycoplasma genitalium (vanVeen and Konings, 1998). Studies on the origin of multidrug resistance genesdemonstrate the importance of transfer of genetic information betweenmicroorganisms 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 aplasmid. Hence, prokaryotic members of the ABC transporter family can

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potentially be exchanged between pathogenic microorganisms and may beresponsible for acquired multidrug resistance in these organisms (Putman et al.,2000b).

ACKNOWLEDGEMENTWe thank Dr. M. Sami and M. Nakagawa for valuable discussions and

G. Poelarends for drawing some of the figures. A.M. received a TMR fellowshipfrom the European Community, and H.W.V.V. was a Fellow of the RoyalNetherlands Academy of Sciences (KNAW).

61

CHAPTER 6The Membrane Bound ATPase Contributes to

Hop Resistance of Lactobacillus brevis

Kanta Sakamoto, H. W. van Veen, Hiromi Saito, Hiroshi Kobayashiand Wil N. Konings

This chapter was accepted to Applied and Environmental Microbiology.

SUMMARYThe activity of the membrane bound H+-ATPase of the beer-spoilage bacterium

Lactobacillus brevis ABBC45 increases upon adaptation to bacteriostatic hopcompounds. The ATPase activity is optimal around pH 5.6 and increases up to fourfold when Lb. brevis was exposed to 666 µM of hop compounds. The extent ofactivation depends on the concentration of hop compounds and is maximal at thehighest concentration tested. The ATPase activity is strongly inhibited by DCCD, aknown inhibitor of F0F1-ATPase. Western blots of membrane proteins of Lb. breviswith the antisera raised against the α- and β-subunits of F0F1-ATPase fromEnterococcus hirae show increased expression of the ATPase after hop adaptation.The expression levels as well as the ATPase-activity decreased to the initial non-adapted levels when the hop-adapted cells were cultured further without hopcompounds. These observations strongly indicate that proton pumping by themembrane-bound ATPase contributes considerably to the resistance of Lb. brevisto hop compounds.

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INTRODUCTIONThe 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 thebrewing industry is preferred because hop has antibacterial activity and preventsbeer from bacterial spoilage. Hop compounds are weak acids, which can crosscytoplasmic membranes in undissociated form in response to the transmembranepH-gradient (Simpson and Smith, 1992). Due to the higher internal pH thesecompounds dissociate internally thereby dissipating the pH gradient across themembrane. As a result of this protonophoric action of hop compounds the viabilityof 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 ofhop compounds. Sami et al. (1997a) reported that Lactobacillus brevis ABBC45strain could adapt to hop treatment and develop a high resistance to hopcompounds. During hop resistance development the copy number of plasmidpRH45 harboring horA gene increased (Sami et al., 1997a). Subsequent studiesrevealed that horA encodes a bacterial ATP-biding cassette (ABC) multidrugtransporter (MDR) which can extrude hop compounds from the cell membranesupon ATP hydrolysis (Sakamoto et al., 2001). As a result of this exogenousexpression of HorA in Lactococcus lactis, its resistance to hop compoundsincreased up to two fold. Micro-organisms have been found to increase the protonmotive force (pmf)-generating activities in their cytoplasmic membranes whenconfronted with a high influx of protons (Viegas et al., 1998). The thermophilicbacterium Bacillus stearothermophilus (De Vrij et al., 1998) increases protonpumping respiratory chain activities when the proton permeability of itscytoplasmic membrane increases drastically at higher temperatures. InEnterococcus hirae (formerly Streptococcus faecalis) (Kobayashi et al., 1984,1986) and Saccharomyces cerevisiae (Viegas et al., 1998) the proton translocatingATPase levels in their membranes were found to increase upon exposure toprotonophores such as carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) orweak acids. Obviously, the main reason for this increase of the proton pumpingactivities is to maintain the pmf and the internal pH at viable levels. In view of theprotonophoric activities of hop compounds it was of interest to investigate whetherthe hop-resistant Lb. brevis would respond in a similar way to the action of hopcompounds and would increase the functional expression of its proton translocatingATPase in addition to the expression of the MDR HorA. In this study, wedemonstrate that this is indeed the case and that Lb. brevis increases the functionalexpression of the proton translocationg ATPase during growth in the presence ofhop compounds.

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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 adjustedto 5.5 with HCl. Hop resistance and expression of HorA was achieved by growthof Lb. brevis in the presence of hop compounds, up to 666 µM, as describedpreviously (Sami et al., 1997a). Cells grown in the presence of 666 µM of hopcompounds were subcultured without hop compounds added in order to follow theATPase 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 usinghigh-performance liquid chromatography (HPLC) (Rode et al., 1990). Theconcentration of hop compounds in the medium was expressed as the concentrationof iso-α-acids.

Preparation of the membrane Lb. brevis was grown to late exponential phase in the absence and in the presenceof 100 µM and 666 µM hop compounds. Cells of Lb. brevis were harvested bycentrifugation at 7,000 × g for 15 min and washed twice at room temperature in 50mM (K) HEPES (pH 7.4) containing 5 mM MgSO4. The cells, suspended in thesame 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 acocktail of proteinase inhibitors (Complete [Boehringer Mannheim, Germany] ).After the addition of DNase I (50 µg/ml) and RNase (1 µg/ml), the suspension waspassed three times through an ice-cold French pressure cell at 70 MPa. Unbrokencells were subsequently removed by centrifugation at 7,000 × g for 15 min at roomtemperature. The supernatant was centrifuged at 200,000 × g for 45 min at 4°C andthe pellet was suspended in the same buffer. This membrane fraction was used forATPase assays and Western Blot analysis. The concentration of the membraneproteins was determined with DC protein assay kit (BioRad, USA) with bovineserum albumin as a quantitative standard.

ATPase Assay ATPase activity was estimated from the release of inorganic phosphate measuredby a modification of the method of Driessen et al. (1991). 1 or 2 µg of membraneprotein was incubated at 30°C for 10 min in 50 mM (K) Mes buffer (usually at pH5.5) containing 5 mM MgCl2. ATP [Potassium salt] was added at a final

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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. Malachitegreen solution (200 µl of 0.034%) was added, and after 40 min the colordevelopment was terminated by the addition of 30 µl citric acid solution (34%[w/v]). Immediately, the absorbance at 660 nm was measured with a multiscanphotometer (Multiskan MS; Labsystems, Finland). One unit ATPase-activity wasdefined as the release of 1 µmole of inorganic phosphate in 1 min. Calibration wasdone by using a series of Pi standards (Sigma, USA). For the determination of pHdependency of the ATPase activity, membranes were incubated for 60 min on icein 50 mM (K) Mes buffer, adjusted to various pH values. The ATPase activity wasassayed at those different pH values as described above. To measure the effects ofinhibitors 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 10min at 30°C, and subsequently for 60 min on ice. The membrane sample withoutinhibitor was used as the control.

Western Blotting Analysis The membrane protein of Lb. brevis, prepared as described above, wassolubilised in Laemmli sample buffer containing 2% SDS (Laemmli, 1970) andseparated by electrophoresis (20 µg of protein / lane) through 10% SDS-polyacrylamide gel by the method of Laemmli (1970). The protein bands weretransferred to a polyvinilidene difluoride (PVDF) filter membrane and detectedwith the antisera raised against the F1 complex of Enterococcus hirae H+-ATPase(Arikado et al., 1999) which can also bind with F0F1-ATPase from Lactococcuslactis (Amachi et al., 1998). Membranes from E. hirae prepared as previouslydescribed (Arikado et al., 1999) were used as control. The antibody-bound proteinswere made visible with nitrobluetertazorium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Gibco BRL. USA). The intensities of the bands weremeasured by densitometric analysis with NIH Image software v.1.61 (NIH, USA).

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RESULTS

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 afunction of the pH ranging from pH 4.4 to 7.0. All membranes of Lb. brevis grownin the presence of different levels of hop compounds showed maximum ATPaseactivity at around pH 5.6 (Fig. 1). At pH 5.6 membranes from the cells adapted to666 µM of hop compounds had the highest activity, which was about 4-times theATPase activity of membranes from non-adapted cells. The ATPase activities ofmembranes from cells adapted to 100 µM of hop compounds were in between theseextremes and about 1.7 times the activity of the membranes from the non-adaptedcells. Once the hop-adapted cells (666 µM) were subcultured in medium withouthop compounds the ATPase activity of their membranes decreased rapidly (Fig. 1).

Figure 1. The pH profile of theATPase activity in membranes of Lb.brevis. The ATPase activity at pHvalues ranging from 4.4 to 7.0 wasmeasured of membranes prepared fromcells grown without hop compounds(W0, ) and of cells adapted to 100 µM(W100, ×) and to 666 µM of hopcompounds (R666, ), and of cells de-adapted by growth first in the presenceof 666 µM of hop compounds followedby growth for two days in the absenceof hop compounds (R0, ). TheATPase activity was shown as theamount 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, theeffect of several kinds of inhibitors on the ATPase activity was studied (Fig. 2).The ATPase activities of membranes from non-adapted cells and from cellsadapted to different concentrations of hop compounds were all significantlyinhibited by the F0F1-type inhibitor N, N’-dicyclohexylcarbodiimide (DCCD).Moderate inhibition was observed with the P-type inhibitor ortho-vanadate, whilethe V-type inhibitor K2NO3 showed the least inhibition or even activation in

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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-typeATPase, which is slightly inhibited by ortho-vanadate and slightly enhanced byK2NO3 (Y. Kakinuma, personal communication), indicating that F0F1-type ATPaseis the major ATPase in membranes from Lb. brevis.

Figure 2. The effect of inhibitors onthe ATPase activity of Lb. brevis. TheATPase activity of the membranes ofW0, W100 and R666 (See the legend ofFig. 1) was measured at pH 5.6 in thepresence of 0.2 mM of N, N’ -dicyclohexylcarbodiimide (DCCD)(closed bars), 0.2 mM of ortho-vanadate(vertically striped bars) or 25 mM ofnitrate (horizontally striped bars). Theactivity without any inhibitor was alsomeasured as control (open bar).

Western Blot Analysis Two bands were strongly detected from the membranes of Lb. brevis with theantisera 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. Theapparent molecular weights of these bands are slightly higher than those of the α-and β- subunits of F1 from E. hirae (Fig. 3A). The intensities of both bands arehigher in membranes isolated from cells grown at higher concentrations of hopcompounds and decrease again in membranes from hop-adapted cells (666 µM)subcultured in medium without hop compounds (Fig. 3B.). The intensities of bothbands correlated well (r = 0.990; r = correlation coefficient) with the ATPaseactivities of the different membranes. The rate and extent of growth in MRS brothof hop adapted cells are slower than of non-adapted cells (Sami et al., 1997a). Alsohop-adapted cells are smaller than cells grown in the absence of hop compounds(data not shown).

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Figure 3. Western blot analysis ofmembranes of Lb. brevis and E. hiraewith antisera against F1 of E. hirae.Membranes of Lb. brevis were solubilizedand separated by electrophoresis through10% polyacrylamide gel (lanes 2-5). Forcomparison the results with membranesfrom E. hirae are shown in lane 1. Theproteins were transferred to a PVDF filtermembrane and reacted with the antiseraraised against the F1 complex of E. hiraeH+-ATPase. (A) The result of WesternBlotting: Lane1, E. hirae cultured at pH6.0; Lane 2, Lb. brevis grown without hop(W0); Lane 3, Lb. brevis adapted to 100µM of hop compounds (W100); Lane 4,Lb. brevis adapted to 666 µM of hopcompounds (R666); Lane 5, Lb. brevis de-adapted from 666 µM to 0 µM of hopcompounds (R0). The arrows indicated theposition of the α- or β-subunit of H+-ATPase from E. hirae. (B) The intensityof the lower bands of the ATPase fromLb. brevis. The intensities of these bandswere measured with NIH Image softwareand presented in arbitrary units.

DISCUSSIONThe beer spoilage bacterium Lb. brevis ABBC45 develops hop resistance upon

growth in hop-containing media (Sami et al., 1997a). This resistance was found tobe mediated by the functionally expressed multidrug resistance ABC transporterHorA (Sami, 1999; Sakamoto et al., 2001). Studies of HorA, functionallyexpressed in Lactococcus lactis, revealed that HorA can excrete the lipophilic hopcompounds and several other MDR substrates from the membrane into the externalmedium (Sakamoto et al., 2001). Recently a second proton motive force-drivenMDR with affinity for hop compounds has been found in Lb. brevis ABBC45lacking HorA (Suzuki et al., 2002). The activity of HorA and this pmf-drivenMDR thus results in a reduced influx of the undissociated and membranepermeable iso-α-acids into the cytoplasm and thereby limits the anti-bacterial pmf-dissipating effect of hop compounds. Since Lb. brevis develops resistance againstrather high concentrations of hop compounds, the question arose whether

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functional expression of HorA and the pmf-driven MDR was sufficient to conferthis resistance or whether additional activities could contribute to hop resistance.Anaerobic Gram-positive lactic acid bacteria such as Lb. brevis depend for thegeneration of their proton motive force strongly on their membrane bound H+-F0F1-ATPase (Kobayashi et al., 1986; Konings et al., 1995). In this study, wedemonstrated that the functional expression of a membrane-bound H+-F0F1-ATPaseincreased during hop-resistance development and decreased again when theexposure to hop compounds was stopped. Previously, it was demonstrated that alsothe expression of the HorA transporter increased during hop-resistancedevelopment (Sami, 1999). The H+-F0F1-type nature of the ATPase was confirmedby H+-F0F1-ATPase effectors and especially by immunologial studies with theantisera against α- and β-subunits of H+-F0F1-ATPase from E. hirae. In accordancewith the observations of Kobayashi et al. (Kobayashi et al., 1984, 1986) made inthe anaerobic Gram-positive bacterium E. hirae, the increased functionalexpression of H+-F0F1-ATPase most likely allows Lb. brevis to maintain a viablepmf 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 becomesresistant 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), anda pmf-driven MDR (Suzuki et al., 2002). HorA and the pmf-driven MDR reducethe influx of the weak acidic hop compounds by pumping the undissociated hopcompounds from the membrane environment into the external medium. The H+-ATPase compensates for the pmf-dissipating and internal pH-decreasing effects ofhop compounds, which have escaped the MDR activities, by pumping moreprotons from the cytoplasm across the membrane. As a result of the higherexpression of ATPase and of HorA and the energy dissipation by hop compoundsthe rate and extent of growth in MRS broth of hop adapted cells are slower than ofnon-adapted cells (Sami et al., 1997a). Those various hop resistance mechanisms(Fig. 4) are another demonstration of the versatility of bacteria and their capacity torecruit a variety of mechanisms to cope with toxic compounds in theirenvironments.

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H+Hop- + H+

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Figure 4. Proposed mechanisms of hop resistance in Lb. brevis ABBC45 by thecombined action of two ATP-driven systems and one proton motive force-drivenMDR. The undissociated hop compounds (Hop-H) intercalate into the cytoplasmicmembrane 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). Afraction of Hop-H escapes the pumping activity of the transporters and enters thecytoplasm. 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 secondarytransporter. Hop– may bind to cations such as Mn2+ (Archibalt and Fridovich, 1981;Simpson, 1993a, 1993b; Simpson et al., 1993: Simpson and Hughes, 1993), while theincreased H+-ATPase activity excretes H+ across the membrane (c) (This work).

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SUMMARYBeer 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 compoundsfrom the hop plant, Humulus Lupulus, L., into beer in the 12th to 13th century wasa major breakthrough due to the strong preservative value of hops. Neverthelessseveral microorganisms can still grow in beer. These beer spoilage microorganismsinclude a few lactic acid bacteria, a few Gram-negative bacteria and wild yeasts.Beer spoilage lactic acid bacteria include Lactobacillus and Pediococcus speciesand are the major contaminants in the brewing industry. They spoil beer byproducing acidity, turbidity and, in some species, ropiness and a buttery off-odor ofdiacetyl. Aerobic acetic acid bacteria such as Acetobacter and Gluconobacterspecies were well-known beer spoilage Gram-negative bacteria. However, theseaerobes have been replaced in the past decades by strictly anaerobic Gram-negativebacteria such as Pectinatus and Megasphaera species, due to the drasticallyreduced oxygen content in beer by the improved brewing technology. Theseanaerobic Gram-negatives cause more serious spoilage than lactic acid bacteria byproducing an offensive rotten egg smell of hydrogen sulfide. Wild yeasts aremainly 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 importantfor the quality assurance in breweries. The most popular method today is still theconventional culture method. It can take a week or even longer to detectmicroorganisms and consequently the products are often already released for salebefore the microbiological results become available. Hence more rapid detectionmethods for beer spoilage bacteria are required. A very powerful tool is thepolymerase chain reaction (PCR) since it enables detection and identification ofmicro-organisms in a very short period of time. Sets of PCR primers targetingspecies-specific regions in the bacterial 16S ribosomal RNA gene (rDNA) havebeen developed for each of the known beer spoilage bacteria (Chapter 2). Howeverfalse negative results were sometimes obtained as PCR can be prevented by thepresence of factors such as polyphenols in beer samples. These false negatives canbe eliminated by the use of the primers targeting to consensus sequences inbacterial 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 strainsfrom non-spoilers is therefore necessary. The most crucial feature in beer spoilagestrains of lactic acid bacteria is resistance to hop compounds present in beer as iso-α-acids. These hop compounds can inhibit the growth of Gram-positive bacteria.The mechanism of the antibacterial action of hop compounds was studied. The

SUMMARY

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major component of iso-α-acids, trans-isohumulone, was found to function as anionophore in a hop sensitive strain of Lb. brevis and to catalyze electroneutralexchange across the cytoplasmic membrane of protons for intracellular cationssuch as Mn2+ (Simpson, 1993b). Consequently the electrochemical proton gradientacross the cytoplasmic membrane is dissipated, resulting in the decrease of protonmotive force (pmf). The uptake of nutrients by pmf-driven uptake systems willthen also be decreased. Resistance to hop compounds in Lb. brevis has been studiedat the molecular level. The horA gene, encoding a polypeptide that is 53% identicalto 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. brevisABBC45 (Sami et al., 1997a). Amplification of pRH45 occurs when this strain isgrown in a continuous culture with increasing concentration of iso-α-acids. On theother hand, hop resistance decreased significantly when Lb. brevis ABBC45 wascured from pRH45, and resistance was regained when this plasmid was re-introduced by electrotransformation (Chapter 3 and 4). HorA was successfullyexpressed in Lactococcus lactis under control of the nicin-inducible expressionsystem (Chapter 5). Studies in cells, membrane vesicles and proteoliposomes,reconstituted with solubilized and purified HorA, revealed that this protein confershop resistance by excreting hop compounds in an ATP-dependent manner from thecell membrane to outer medium. In addition to the activity of HorA also increasedproton pumping by the membrane bound H+-ATPase contributes to increased hopresistance (Chapter 6). To energize such ATP-dependent transporters hop resistantcells contain larger ATP pools than hop sensitive cells (Simpson and Fernandez,1994). Furthermore evidence for the presence of a proton motive force dependenthop transporter was recently presented (Suzuki et al., 2002). The potentialmolecular mechanisms of hop resistance in Lb. brevis are shown in Fig. 1.

Understanding the mechanisms of hop resistance has enabled the development ofrapid methods to discriminate beer spoilage strains from non-spoilers. The horA-PCR method in which a set of specific primers detects the horA gene or itshomologues has been applied for bacterial control in breweries (Sami et al.,1997b). Most horA positive strains of Lactobacillus spp. were found to have beerspoiling ability. Also a discrimination method was developed based on ATP poolmeasurement in lactobacillus cells (Okazaki et al., 1997). However, some potentialhop resistant strain cannot grow in beer when they have not first been exposed tosub-inhibitory concentration of hop compounds (Simpson and Fernandez, 1992).

The beer spoilage ability of Pectinatus spp. and Megasphaera cerevisiae hasbeen poorly studied. Since all strains have been reported to be capable of beerspoiling, species identification is sufficient for the brewing industry. However, withthe current trend of beer flavor (lower alcohol and bitterness), there is the potentialrisk that not yet reported bacteria will contribute to beer spoilage. Investigation ofthe beer spoilage ability of especially Gram-negative bacteria may be useful to

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73

reduce this risk.

Figure 1. Mechanisms of hop resistance. Hop compounds act as ionophores thatexchange 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 higherinternal pH. Hop anions trap divalent cations such as Mn2+ and diffuse out of the cell. Theionophoric action together with the diffusion of the hop-metal complex results in anelectroneutral exchange of cations. Release of protons from hop compounds decreases theintracellular 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 bedecreased. In hop resistant cells hop compounds can be expelled from the cytoplasmicmembrane by HorA (a) (Sakamoto et al., 2001) and probably also by a pmf-dependenttransporter (b) (Suzuki et al., 2002). Furthermore, overexpressed H+-ATPase increases thepumping of protons released from the hop compounds (c) (Sakamoto et al., 2002). MoreATP 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 achanged lipid composition of the cytoplasmic membrane of beer spoilage lactic acidbacteria may increase the barrier to hop compounds.

Cytoplasmic membrane

Cell wall

Hop-H

H+ + Hop-

H+

Hop-Mn-Hop

Mn2+

Hop-Mn-Hop

Mn2+

Hop-Mn-Hop

Hop-H

H+

H+

H+

Hop-Mn-Hop

H+

Hop-sensitive cell

Hop-resistant cell

Hop-H

Hop- + H+

Hop-H

H+

Hop-H

b

cATP

ADP

ccH+

ATP

ADP

c

Hop-H

ATP ADP

a

H+

ATP

ADP

c

H+

ATP

ADP

c

ATP

74

75

SAMENVATTINGDe 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 ontdekkingvan de sterke preservatieve werking van hop-componenten, afkomstig uit dehopplant Humulus lupulus L, betekende dan ook een grote doorbraak in de strijdtegen het bierbederf. Rond de 12e/13e eeuw werd hop voor het eerst aan biertoegevoegd. Desondanks kunnen er nog steeds verschillende micro-organismengroeien in bier, waaronder gisten, melkzuur bacteriën en enkele Gram-negatievebacteriën. De voornaamste bedervers in de bier-industrie zijn melkzuur bacteriënvan de Lactobacillus en de Pediococcus familie. Deze bederven het bier doorvertroebeling, verhoging van de viscositeit, verlaging van de zuurgraad en somszelfs het veroorzaken van een schrale, boterige geur van diacetyl. De aërobe Gram-negatieve azijnzuur bacteriën zoals Acetobacter en Gluconobacter waren voorheenook beruchte bier-bedervers, maar dankzij het door verbeterde brouw-technologienverlaagde zuurstofgehalte in het bier zijn deze in de laatste decennia grotendeelsvervangen door de strikt anaërobe Gram-negatieve soorten Pectinatus enMegasphaera. Deze soorten veroorzaken echter een ernstiger vorm van bierbederfdan de melkzuur bacteriën omdat ze de afstotelijke “rotte eieren lucht” vanwaterstofsulfide (H2S) produceren. Gisten worden voornamelijk gedetecteerd in deleidingen van biertaps in café’s maar zelden in verpakt bier, en daarom vormen zeeen minder groot probleem dan de bacteriën.

Detectie en identificatie van bier bedervende micro-organismen is van grootbelang voor de kwaliteitswaarborging van brouwerijen en de conventionele kweekmethode is hiervoor nog altijd populair. Vaak zijn producten echter al in de verkoopvoordat alle microbiologische resultaten binnen zijn en dientengevolge is er veelinteresse in snellere detectie- en identificatie-methoden voor bier bedervendemicro-organismen. De “polymerase ketting reactie” (polymerase chain reaction:PCR) is een krachtige techniek voor het vermenigvuldigen van specifieke DNA ofRNA sequenties. PCR wordt dan ook veel gebruikt om micro-organismen in kortetijd te identificeren. In hoofdstuk 2 wordt de ontwikkeling besproken van soort-specifieke PCR primers die genen herkennen die coderen voor 16S ribosomaalRNA (16S rDNA) van alle bekende bier-bedervende bacteriën. Vals negatieveresulaten werden echter behaald doordat de PCR reactie geremd kan worden doorbepaalde componenten in bier, zoals polyfenolen. Deze vals negatieve resultatenkunnen opgespoord worden door gebruik te maken van interne positieve controlePCR reacties, die consensus sequenties in 16S rDNA herkennen (Hoofdstuk 2).

De meeste, maar niet alle stammen van Lactobacillus brevis en Pediococcusdamnosus kunnen groeien in bier. Het onderscheiden van bier-bedervende en niet-bier-bedervende stammen is derhalve essentiëel voor de kwaliteitswaarborging van

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het bier. De cruciale eigenschap van bier-bedervende melkzuurbacteriën is hunresistentie tegen hop-componenten, zoals iso-α-zuren, die in bier voorkomen. Dezehop-componenten kunnen de groei van Gram-positieve bacteriën remmen en deantibacteriële werking van deze stoffen is onderzocht. trans-Isohumulon, debelangrijkste component van iso-α-zuren, bleek te werken als een ionofoor in eenhopgevoelige Lactobacillus brevis stam. trans-Isohumulon katalyseert deelectroneutrale uitwisseling over de cytoplasmatische membraan van protonentegen intracellulaire kationen zoals mangaan (Mn2+). Zodoende wordt de protonengradiënt (pH-gradiënt) over de cytoplasma membraan verlaagd hetgeen resulteertin een verlaagde protonen drijvende kracht (proton motive force: PMF). Het gevolgvan een verlaagde PMF is een verlaagde opname van diverse nutriënten door PMFgedreven opname systemen.

Het resistentie mechanisme van de hop-resistente Lb. brevis stam ABBC45 tegenhop-componenten is op moleculair niveau onderzocht (Hoofdstuk 3 en 4). Groeivan deze stam in een continu culture met toenemende concentraties iso-α-zurenresulteert in amplificatie van het plasmide pRH45. Verwijdering van dit plasmideuit Lb. brevis ABBC45 gaat gepaard met een afname van hop resistentie en dit kanweer worden hersteld door herintroductie van het plasmide. Plasmide pRH45 bevathet horA gen, dat codeert voor een eiwit dat voor 53% identiek is aan LmrA, eenABC-type multidrug transporter uit Lactococcus lactis. Het HorA eiwit werd ondercontrole van het nisine induceerbare expressie systeem tot overexpressie gebrachtin L. lactis (Hoofdstuk 5). Experimenten met hele cellen, membraan vesikels enproteoliposomen met gezuiverd en gereconstitueerd HorA toonden aan dat dit eiwithop-resistentie realiseert door ATP-afhankelijke excretie van hop-componentenvanuit de cel membraan naar het externe medium. Een additioneel mechanisme datbijdraagt aan hop-resistentie is de verhoogde activiteit van de protonen pompendeH+-ATPase, hetgeen mogelijk gemaakt wordt door de hogere ATP concentraties inhop-resistente cellen in vergelijking met hop-gevoelige cellen (Simpson enFernandez, 1994). Evidentie voor de aanwezigheid van een PMF gedreven hop-resistentie transporter in Lb. brevis is onlangs gepresenteerd (Suzuki et al., 2002).De potentiële moleculaire mechanismen voor hop-resistentie zijn samengevat inFig. 1.

Het inzicht in de mechanismen van hop-resistentie heeft geleid tot deontwikkeling van een methode voor het onderscheiden van bier-bedervende enniet-bier-bedervende stammen op basis van het horA gen. De horA-PCR methodemaakt gebruik van primers specifiek voor horA of een homoloog gen, entoepassing van deze methode bij bacteriële controles in een brouwerij toonde aandat de meeste horA positieve stammen inderdaad bier-bedervend zijn.. Sommigepotentiëel hop-resistente stammen kunnen echter pas groeien in bier als ze eerst aanlage concentraties van hop-componenten zijn blootgesteld.

De bier-bedervings-capaciteit van Pectinatus spp. en Megasphaera cerevisiae is

SAMENVATTING

77

tot nu toe nauwelijks onderzocht, maar aangezien alle stammen in staat zijn bier tebederven volstaat het voor de bier-industrie om de soorten te identificeren. Dehuidige trends in de ontwikkeling van bieren (minder alcohol en minder bitterheid)brengt het risico mee dat nog niet eerder gerapporteerde bacteriën bij zullen dragenaan bierbederf. Dit risico zou verkleind kunnen worden door onderzoek naar debier-bedervings-capaciteit van vooral Gram-negatieve bacteriën.

Figuur 1. Mechanismen van hop-resistentie. Hop-componenten werken als ionoforen dieprotonen uitwisselen tegen intracellulaire divalente kationen. In een hop-gevoelige celdringen hop-componenten (Hop-H) de cel binnen om vervolgens te dissocieren in hop-anionen (Hop-) en protonen (H+) door de hogere interne pH. Hop anionen complexerendivalente kationen zoals Mn2+ en diffunderen de cel weer uit (Hop-Mn-Hop). De ionoforewerking van hop samen met de diffusie van van de hop-metaal complexen resulteert in eenelectroneutrale uitwisseling van kationen. Het afstaan van protonen door hop-componentenbinnen 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 opnamevan nutriënten wordt dientengevolge ook verlaagd. In hop-resistente cellen kunnen hop-componenten 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 nadissociatie van hop-componenten (c) (Sakamoto et al., 2002). In hop-resistente cellen wordtmeer ATP gegenereerd dan in hop-gevoelige cellen (Simpson en Fernandez, 1994).Gegalactosyleerd glycerol teichoine zuur in de celwand (Yasui et al., 1997) en eenveranderde lipidensamenstelling van de cytoplasmatische membraan van bierbedervendemelkzuurbacteriën kan de barrière tegen hop-componenten verhogen.

Cytoplasmatische membraan

Cel wand

Hop-H

H+ + Hop-

H+

Hop-Mn-Hop

Mn2+

Hop-Mn-Hop

Mn2+

Hop-Mn-Hop

Hop-H

H+

H+

H+

Hop-Mn-Hop

H+

Hop-gevoelige cel

Hop-resistente cel

Hop-H

Hop- + H+

Hop-H

H+

Hop-H

b

cATP

ADP

ccH+

ATP

ADP

c

Hop-H

ATP ADP

a

H+

ATP

ADP

c

H+

ATP

ADP

c

ATP

78

79

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RNA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 dam-

nosus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

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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ATPBinding 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pRH45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, 1997b）。 horA陽性となる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

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81

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。

Hop-H

H+ + Hop-

H+

Hop-Mn-Hop

Mn2+

Hop-Mn-Hop

Mn2+

Hop-Mn-Hop

Hop-H

H+

H+

H+

Hop-Mn-Hop

H+

Hop-H

Hop-+ H+

Hop-H

H+

Hop-H

b

cATP

ADP

ccH+

ATP

ADP

c

Hop-H

ATP ADP

a

H+

ATP

ADP

c

H+

ATP

ADP

c

ATP

Hop-H

H+ + Hop-

H+

Hop-Mn-Hop

Mn2+

Hop-Mn-Hop

Mn2+

Hop-Mn-Hop

Hop-H

H+

H+

H+

Hop-Mn-Hop

H+

Hop-H

Hop-+ H+

Hop-H

H+

Hop-H

b

cATP

ADP

cccH+

ATP

ADP

c H+

ATP

ADP

ATP

ADP

cc

Hop-H

ATP ADP

a

H+

ATP

ADP

c H+

ATP

ADP

ATP

ADP

cc

H+

ATP

ADP

c H+

ATP

ADP

ATP

ADP

cc

ATP

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。

ホップ感受性株

ホップ耐性株

細胞壁

細胞膜

82

83

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LIST OF PUBLICATONSSakamoto, K. (1997) Detection of the genus Pectinatus. Japanese Patent Application JP09-

520359, World Patent Application WO97/20071, US Patent 5869642.Sakamoto, K., Funahashi, W., Yamashita, H. and Eto, M. (1997) A reliable method for

detection and identification of beer-spoilage bacteria with internal positivecontrol PCR (IPC-PCR). Proc. Eur. Brew. Conv. Maastricht. 631-638.

Sami, M., Suzuki, K., Sakamoto, K., Kadokura, H., Kitamoto, K., and Yoda, K. (1998) Aplasmid pRH45 of Lactobacillus brevis confers hop resistance. J. Gen. Appl.Microbiol. 44:361-363.

van Veen, H. W., Margolles, A., Putman, M., Sakamoto, K., and Konings, W. N. (1999)Multidrug resistance in lactic acid bacteria: molecular mechanism and clinicalrelevance. Antonie van Leeuwenhoek 76:347-352.

van Veen, H. K., Putman, M., Margolles, A., Sakamoto, K., and Konings, W. N. (1999)Structure-function analysis of multidrug transporters in Lactococcus lactis.Biochimica et Biophysica Acta 1461:201-206.

van Veen, H. W., Putman, M., Margolles, A., Sakamoto, K., and Konings, W. N. (2000)Molecular pharmacological characterization of two multidrug transporters inLactococcus lactis. Pharmacology and Therapeutics 85:245-249.

Sakamoto, K., Margolles, A., van Veen, H. W., and Konings, W. N. (2001) Hop resistancein the beer-spoilage bacterium Lactobacillus brevis is mediated by the ATP-binding cassette multidrug transporter HorA. J. Bacteriol. 183:5371-5375.

Sakamoto, K., van Veen, H. W., Saito, H., Kobayashi, H., and Konings, W. N. Membranebound ATPase contributes to hop resistance of Lactobacillus brevis. Accepted toAppl. Environ. Microbiol.

Sakamoto, K. and Konings, W. N. Beer spoilage bacteria and hop resistance. Submitted toInt. J. Food Microbiology.

Sakamoto, K. Electrotransformation of Lactobacillus brevis. Submitted to Appl. Environ.Microbiol.

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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 ofAgriculture, the University of Tokyo, and granted the Bachelor’s degree ofAgricultural Science.Graduated in 1994 from the Graduate School of Agriculture, the University ofTokyo, 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 theGroningen Biomolecular Sciences and Biotechnology Institute (GBB) of theUniversity of Groningen for the collaboration between Asahi Breweries, Ltd. andthe University of Groningen.Ph. D. student in 2002 at the University of Groningen.