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Plasmids of hiochi-bacteria
JOURNAL OF FERMENTATION AND BIOENGINEERING VOI. 72, NO. 6, 479-480. 1991
NOTES Plasmids of Hiochi-Bacteria K A Z U H I K O O H B U C H I , * A K I H I R O K A N D A , M A S A A K I H A M A C H I , AND Y A T A R O N U N O K A W A
General Research Institute, Ozeki Corporation, 4-9 Imazu-dezaike, Nishinomiya, Hyogo 663, Japan Received 1 July 1991/Accepted 2 September 1991 Hiochi-bacteriai plasmids were screened. Many hiochMactobacilli had plasmids of several molecular sizes, whereas only a few true hiochi-bacilli had just one or two plasmids. A 4.7 kb cryptic plasmid in hiochi-lactobacilli homo-fermenter A-1 strain was cloned into pBR322 vector for Escherichia coli; it has three unique cleavage sites for HpaI, BglII and M/uI.
C o m p a r i n g the 3 methods, K a d o and Liu's had high b a c k g r o u n d and smear bands, whereas the methods o f A n d e r s o n and McKay, and Klaenhammer were suitable for our research since the results of digestion by restriction endonucleases were satisfactory. As shown in Fig. 1, there were some strains had 5, 6 bands. Moreover, some bands which migrated with m o r e delay than the chromosomal bands were also detected. Delayed bands occasionally a p p e a r on lactic-bacteria (7-9). A l t h o u g h Vescovo et al. (10) found that the true hiochi-bacilli hetero-fermenter H-1 (the strain they used was ATCC15435) had a plasmid, it was not recognized in this experiment. Strains which were proven to have plasmids are summarized on Table 1. It is, however, possible that the indicated numbers o f bands are not identical to the numbers o f plasmids. The reason for this is discussed below. Plasmids existed in many strains, and especially in most hiochi-lactobacilli strains, where there were also various molecular sizes o f plasmids in a strain. On the other hand, on true hiochi-bacilli, strains containing multiple plasmids were scarce, and only
Hiochi-bacteria is a general name for lactobacilli which can grow even in alcohol at a concentration o f 15%o or more, such as "sake". F r o m the standpoint o f t a x o n o m y , they belong to Lactobacillus acidophilus, L. fermentum, L. plantarum, L. homohiochii, L. fructivorans and so on (1-3), but most strains have not been identified yet. Thus, hiochi-bacteria are customary classified into alcohol-tolerant hiochi-lactobacilli and alcophilic true hiochi-bacilli, and are divided into homo-fermenters and hetero-fermenters by the type o f sugar fermentation to lactic acid (4). They have the unique characteristic o f being able to grow under high alcoholic circumstances. In the case of general lactobacilli, there have been m a n y investigations of the plasmids and their functions, and also reports o f the construction o f vectors using them. In the hiochi-bacteria, however, there have been very few such investigations. In order to build host-vector systems to study the unique characteristics o f these bacteria, investigation o f plasmid D N A s which might become vector candidates was first o f all conducted. The 50 strains used were provided by the National Research Institute of Brewing (the initial letter o f these strains is S- or H-) or were stock cultures in our laboratory. The hiochi-bacteria to be examined were cultivated in an SI m e d i u m (5) at 30°C for 5 d statically. The c o m p o nents o f SI m e d i u m were following; 2.5% glucose, 0 . 5 ~ P o l y p e p t o n , 1.0% yeast extract, 1.0% sodium acetate, 0.01% MgSO4.7HzO, 0.00025% MnSO4.5H20, 0.00025/°i~ FeSO4-7H20, 0.0005% mevalolacton, 10% (v/v) ethanol, and adjusted to p H 5.2-5.3. Ethanol was a d d e d after autoclaving at 115°C for 5 min. The grown cells were harvested by centrifugation (10,000 × g, 10 min) and washed with TM buffer ( 1 0 m M Tris-maleate, 1 m M MgC12, p H 6.5). The cleared lysates were prepared according to the method o f Ohbuchi et al. (6) except for the following. The lysing reaction was done with 0.5 M E D T A on ice. Plasmids were prepared from the cleared lysates using 3 methods, i.e., those described by K a d o and Liu (7), Anderson and McKay (8) and K l a e n h a m m e r (9), and stained by ethidium b r o m i d e after developing with 0.8% agarose gel electrophoresis. The c h r o m o s o m a l D N A s o f hiochi-bacteria were j u d g e d by the bands showing a similar mobility to that o f E . coli HBI01 which did not include a plasmid, and plasmids were recognized as the bands besides these.
ABCDE
F
a
b
DNA
c
'kb) ;kb) ;kb) ;6kb) tkb)
d e
f g
FIG. 1. Agarose gel electrophoresis of hiochi-bacterial plasmids. Lane A, hiochi-lactobacilli homo-fermenter A-I; lane B, hiochilactobacilli homo-fermenter S-4; lane C, hiochi-lactobacilli heterofermenter S-7; lane D, true hiochi-bacilli homo-fermenter S-24; lane E, true hiochi-bacilli homo-fermenter S-25; lane F, true hiochi-bacilli hetero-fermenter H-I; Bands a-g indicates bands of plasmids in A-I. Markers indicated the configurations of chromosomal DNA of E. coli HB101 and circular plasmid DNAs as references.
* Corresponding author.
479
480
OHBUCHI ET AL.
J. FERMENT. BIOENG., TABLE 1. Plasmids of hiochi-bacteria Strains (numbers of bands of plasmids)
Hiochi lactobacilli homo-fermenters
L. acidophilus H-7 (1), Lactobacillus sp. S-4 (4), Lactobacillus sp. S-8 (0), Lactobacillus sp. S-31 (4), Lactobacillus sp. S-32 (1), Lactobacillus sp. A-1 (7), Lactobacillus sp. A-4 (1), Lactobacillus sp. E-18 (3), Lactobacillus sp. AE-2 (5), Lactobacillus sp. TS-1 (0)
Hiochi lactobacilli hetero-fermenters
L. fermentum H-34 (1), L. fermentum H-50 (3), Lactobacillus sp. S-7 (5), Lactobacillus sp. A-2 (0), Lactobacillus sp. AD-1 (1), Lactobacillus sp. BK-1 (2), Lactobacillus sp. CE-I 1 (5), Lactobacillus sp. CE-12 (0), Lactobacillus sp. CK-1 (1), Lactobacillus sp. E-14 (1), Lactobacillus sp. OS-I (2), Lactobacillus sp. T-18 (4), Lactobacillus sp. JH-76 (1)
True hiochi-bacilli homo-fermenters
L. homohiochii S-24 (0), Lactobacillus sp. S-25 (2), Lactobacillus sp. S-26 (1), Lactobacillus sp. S-27 (0), Lactobacillus sp. S-35 (1), Lactobacillus sp. S-40 (0), Lactobacillus sp. S-46 (0)
True hiochi-bacilli hetero-fermenters
L. fructivorans H-I (0), L. fructivorans H-3 (1), L. fructivorans H-4 (1), L. fructivorans H-5 (1), L. fructivorans H-9 (1), L. fructivorans H-10 (2), L. fructivorans S-14 (0), Lactobacillus sp. S-17 (0), Lactobacillus sp. S-18 (1), Lactobacillus sp. S-19 (0), Lactobacillus sp. S-20 (0), Lactobacillus sp. S-29 (0), Lactobacillus sp. T-I (2), Lactobacillus sp. BA-2 (1), Lactobacillus sp. T-12 (2), Lactobacillus sp. CK-2 (1), Lactobacillus sp. M-4 (2), Lactobacillus sp. M-5 (0), Lactobacillus sp. M-9 (1), Lactobacillus sp. JH-42 (0)
£coRV
PstI
~
/2.
Hpa I BamlfI £coRV t l[ind IIl EcoR
0.3
1_
p B B -
l[ i n d llI BgllI
0.70.6N~//ind
1 5
lll
~f__MluI Ba~HI
(bands c, e in Fig. 1) o f the d e v e l o p e d A-1 strain's total D N A in agarose gel electrophoresis (data not shown). It is likely that the p r e p a r e d plasmids in this e x p e r i m e n t cont a i n e d n o t o n l y c o v a l e n t l y closed circular D N A s but also o p e n circular D N A s in part. This result also suggests that the n u m b e r o f p l a s m i d species h a r b o r e d by one strain is not e q u a l to the n u m b e r o f b a n d s detected in agarose gel electrophoresis. W e are n o w g o i n g to clone s o m e small m o l e c u l a r weight plasmids o f o t h e r strains and e v a l u a t e t h e m as to their suitability as vectors. REFERENCES
Pst I FIG. 2. Restriction map of the plasmid pBB-15. Size (in kb) is indicated within the circle. The open box represents the 4.7 kb of plasmid from A-l, inserted into pBR322 which is represented by the thin line.
o n e or two p l a s m i d were f o u n d in the strains, if they c o n t a i n e d a p l a s m i d at all. In o r d e r to m a k e shuttle vectors for E. coli and hiochibacteria, at first a p l a s m i d in h i o c h i - l a c t o b a c i l l i h o m o f e r m e n t e r A-1 strain which had a relatively small m o l e c ular weight was c l o n e d into the B a m H I site o f pBR322. P l a s m i d s o f A-1 strain were purified t h r o u g h a P l a s m i d Q u i k TM p u s h c o l u m n (Stratagene) to r e m o v e c h r o m o s o m a l D N A . T h e B a m H I - d i g e s t e d p l a s m i d D N A m i x t u r e was ligated with B a m H I - d i g e s t e d and B A P - d e p h o s p h o l y l a t e d p B R 3 2 2 D N A . A r e c o m b i n a n t p l a s m i d designated as p B B 15 was constructed by cloning a 4.7 kb plasmid o f A-1 strain (indicated as b a n d c or e in lane A o f Fig. 1). Its restriction m a p is s h o w n in Fig. 2. pBB-15 has three u n i q u e cleavage sites for H p a I , BgllI a n d M l u I and is possibly used as a shuttle vector. S o u t h e r n h y b r i d i z a t i o n was d o n e using 32P-labeled p B B 15 as a p r o b e , pBB-15 was hybridized with two bands
1. Kitahara, K., Kaneko, T., and Goto, O.: Taxonomical studies on the hiochi-bacteria part I. Nippon Nogeikagaku Kaishi, 31, 556560 (1957). 2. Kilahara, K., Kaneko, T., and Goto, O.: Taxonomical studies on the hiochi-bacteria part II. Nippon Nogeikagaku Kaishi, 31,560564 (1957). 3. Momose, H., Yamagishi, T., and Nosiro, K.: Several characters of hiochi-bacteria. J. Soc. Brew. Jpn., 65, 799-803 (1970). 4. Mnmose, H., Yamanaka, E., Akiyama, H., and Nosiro, K.: Taxonomic study on hiochi-bacteria, with special reference to deoxyribonucleic acid base composition and chemical composition of bacterial cell wall. J. Gen. Appl. Microbiol., 20, 179-185 (1974). 5. Sugama, S. and Iguehi, T.: Seisyu no hiochi boushi ni kansuru kenkyu. J. Soc. Brew. Jpn., 65, 720-725 (1970). 6. Ohhuehi, K., Higashida, Y., Kanda, A., Hamaehi, M., and Honma, T.: Conditions of lysis and protoplast formation of hiochibacteria. Hakkokogaku, 67, 491-498 (1989). 7. Kado, C. I. and Liu, S. T.: Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol., 145, 1365-1373 (1981). 8. Anderson, D. G. and Mekay, L. L.: Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol., 46, 549-552 (1983). 9. Klaenhammer, T. R.: A general method for plasmid isolation in lactobacilli. Curr. Microbiol., 10, 23-28 (1984). 10. Vescovo, M., Bottazzi, V., Sarra, P. G., and Dellaglio, F.: Evidence of plasmid deoxyribonucleic acid in Lactobacillus. Microbiologica, 4, 413-419 (1981).
NOTES Plasmids of Hiochi-Bacteria K A Z U H I K O O H B U C H I , * A K I H I R O K A N D A , M A S A A K I H A M A C H I , AND Y A T A R O N U N O K A W A
General Research Institute, Ozeki Corporation, 4-9 Imazu-dezaike, Nishinomiya, Hyogo 663, Japan Received 1 July 1991/Accepted 2 September 1991 Hiochi-bacteriai plasmids were screened. Many hiochMactobacilli had plasmids of several molecular sizes, whereas only a few true hiochi-bacilli had just one or two plasmids. A 4.7 kb cryptic plasmid in hiochi-lactobacilli homo-fermenter A-1 strain was cloned into pBR322 vector for Escherichia coli; it has three unique cleavage sites for HpaI, BglII and M/uI.
C o m p a r i n g the 3 methods, K a d o and Liu's had high b a c k g r o u n d and smear bands, whereas the methods o f A n d e r s o n and McKay, and Klaenhammer were suitable for our research since the results of digestion by restriction endonucleases were satisfactory. As shown in Fig. 1, there were some strains had 5, 6 bands. Moreover, some bands which migrated with m o r e delay than the chromosomal bands were also detected. Delayed bands occasionally a p p e a r on lactic-bacteria (7-9). A l t h o u g h Vescovo et al. (10) found that the true hiochi-bacilli hetero-fermenter H-1 (the strain they used was ATCC15435) had a plasmid, it was not recognized in this experiment. Strains which were proven to have plasmids are summarized on Table 1. It is, however, possible that the indicated numbers o f bands are not identical to the numbers o f plasmids. The reason for this is discussed below. Plasmids existed in many strains, and especially in most hiochi-lactobacilli strains, where there were also various molecular sizes o f plasmids in a strain. On the other hand, on true hiochi-bacilli, strains containing multiple plasmids were scarce, and only
Hiochi-bacteria is a general name for lactobacilli which can grow even in alcohol at a concentration o f 15%o or more, such as "sake". F r o m the standpoint o f t a x o n o m y , they belong to Lactobacillus acidophilus, L. fermentum, L. plantarum, L. homohiochii, L. fructivorans and so on (1-3), but most strains have not been identified yet. Thus, hiochi-bacteria are customary classified into alcohol-tolerant hiochi-lactobacilli and alcophilic true hiochi-bacilli, and are divided into homo-fermenters and hetero-fermenters by the type o f sugar fermentation to lactic acid (4). They have the unique characteristic o f being able to grow under high alcoholic circumstances. In the case of general lactobacilli, there have been m a n y investigations of the plasmids and their functions, and also reports o f the construction o f vectors using them. In the hiochi-bacteria, however, there have been very few such investigations. In order to build host-vector systems to study the unique characteristics o f these bacteria, investigation o f plasmid D N A s which might become vector candidates was first o f all conducted. The 50 strains used were provided by the National Research Institute of Brewing (the initial letter o f these strains is S- or H-) or were stock cultures in our laboratory. The hiochi-bacteria to be examined were cultivated in an SI m e d i u m (5) at 30°C for 5 d statically. The c o m p o nents o f SI m e d i u m were following; 2.5% glucose, 0 . 5 ~ P o l y p e p t o n , 1.0% yeast extract, 1.0% sodium acetate, 0.01% MgSO4.7HzO, 0.00025% MnSO4.5H20, 0.00025/°i~ FeSO4-7H20, 0.0005% mevalolacton, 10% (v/v) ethanol, and adjusted to p H 5.2-5.3. Ethanol was a d d e d after autoclaving at 115°C for 5 min. The grown cells were harvested by centrifugation (10,000 × g, 10 min) and washed with TM buffer ( 1 0 m M Tris-maleate, 1 m M MgC12, p H 6.5). The cleared lysates were prepared according to the method o f Ohbuchi et al. (6) except for the following. The lysing reaction was done with 0.5 M E D T A on ice. Plasmids were prepared from the cleared lysates using 3 methods, i.e., those described by K a d o and Liu (7), Anderson and McKay (8) and K l a e n h a m m e r (9), and stained by ethidium b r o m i d e after developing with 0.8% agarose gel electrophoresis. The c h r o m o s o m a l D N A s o f hiochi-bacteria were j u d g e d by the bands showing a similar mobility to that o f E . coli HBI01 which did not include a plasmid, and plasmids were recognized as the bands besides these.
ABCDE
F
a
b
DNA
c
'kb) ;kb) ;kb) ;6kb) tkb)
d e
f g
FIG. 1. Agarose gel electrophoresis of hiochi-bacterial plasmids. Lane A, hiochi-lactobacilli homo-fermenter A-I; lane B, hiochilactobacilli homo-fermenter S-4; lane C, hiochi-lactobacilli heterofermenter S-7; lane D, true hiochi-bacilli homo-fermenter S-24; lane E, true hiochi-bacilli homo-fermenter S-25; lane F, true hiochi-bacilli hetero-fermenter H-I; Bands a-g indicates bands of plasmids in A-I. Markers indicated the configurations of chromosomal DNA of E. coli HB101 and circular plasmid DNAs as references.
* Corresponding author.
479
480
OHBUCHI ET AL.
J. FERMENT. BIOENG., TABLE 1. Plasmids of hiochi-bacteria Strains (numbers of bands of plasmids)
Hiochi lactobacilli homo-fermenters
L. acidophilus H-7 (1), Lactobacillus sp. S-4 (4), Lactobacillus sp. S-8 (0), Lactobacillus sp. S-31 (4), Lactobacillus sp. S-32 (1), Lactobacillus sp. A-1 (7), Lactobacillus sp. A-4 (1), Lactobacillus sp. E-18 (3), Lactobacillus sp. AE-2 (5), Lactobacillus sp. TS-1 (0)
Hiochi lactobacilli hetero-fermenters
L. fermentum H-34 (1), L. fermentum H-50 (3), Lactobacillus sp. S-7 (5), Lactobacillus sp. A-2 (0), Lactobacillus sp. AD-1 (1), Lactobacillus sp. BK-1 (2), Lactobacillus sp. CE-I 1 (5), Lactobacillus sp. CE-12 (0), Lactobacillus sp. CK-1 (1), Lactobacillus sp. E-14 (1), Lactobacillus sp. OS-I (2), Lactobacillus sp. T-18 (4), Lactobacillus sp. JH-76 (1)
True hiochi-bacilli homo-fermenters
L. homohiochii S-24 (0), Lactobacillus sp. S-25 (2), Lactobacillus sp. S-26 (1), Lactobacillus sp. S-27 (0), Lactobacillus sp. S-35 (1), Lactobacillus sp. S-40 (0), Lactobacillus sp. S-46 (0)
True hiochi-bacilli hetero-fermenters
L. fructivorans H-I (0), L. fructivorans H-3 (1), L. fructivorans H-4 (1), L. fructivorans H-5 (1), L. fructivorans H-9 (1), L. fructivorans H-10 (2), L. fructivorans S-14 (0), Lactobacillus sp. S-17 (0), Lactobacillus sp. S-18 (1), Lactobacillus sp. S-19 (0), Lactobacillus sp. S-20 (0), Lactobacillus sp. S-29 (0), Lactobacillus sp. T-I (2), Lactobacillus sp. BA-2 (1), Lactobacillus sp. T-12 (2), Lactobacillus sp. CK-2 (1), Lactobacillus sp. M-4 (2), Lactobacillus sp. M-5 (0), Lactobacillus sp. M-9 (1), Lactobacillus sp. JH-42 (0)
£coRV
PstI
~
/2.
Hpa I BamlfI £coRV t l[ind IIl EcoR
0.3
1_
p B B -
l[ i n d llI BgllI
0.70.6N~//ind
1 5
lll
~f__MluI Ba~HI
(bands c, e in Fig. 1) o f the d e v e l o p e d A-1 strain's total D N A in agarose gel electrophoresis (data not shown). It is likely that the p r e p a r e d plasmids in this e x p e r i m e n t cont a i n e d n o t o n l y c o v a l e n t l y closed circular D N A s but also o p e n circular D N A s in part. This result also suggests that the n u m b e r o f p l a s m i d species h a r b o r e d by one strain is not e q u a l to the n u m b e r o f b a n d s detected in agarose gel electrophoresis. W e are n o w g o i n g to clone s o m e small m o l e c u l a r weight plasmids o f o t h e r strains and e v a l u a t e t h e m as to their suitability as vectors. REFERENCES
Pst I FIG. 2. Restriction map of the plasmid pBB-15. Size (in kb) is indicated within the circle. The open box represents the 4.7 kb of plasmid from A-l, inserted into pBR322 which is represented by the thin line.
o n e or two p l a s m i d were f o u n d in the strains, if they c o n t a i n e d a p l a s m i d at all. In o r d e r to m a k e shuttle vectors for E. coli and hiochibacteria, at first a p l a s m i d in h i o c h i - l a c t o b a c i l l i h o m o f e r m e n t e r A-1 strain which had a relatively small m o l e c ular weight was c l o n e d into the B a m H I site o f pBR322. P l a s m i d s o f A-1 strain were purified t h r o u g h a P l a s m i d Q u i k TM p u s h c o l u m n (Stratagene) to r e m o v e c h r o m o s o m a l D N A . T h e B a m H I - d i g e s t e d p l a s m i d D N A m i x t u r e was ligated with B a m H I - d i g e s t e d and B A P - d e p h o s p h o l y l a t e d p B R 3 2 2 D N A . A r e c o m b i n a n t p l a s m i d designated as p B B 15 was constructed by cloning a 4.7 kb plasmid o f A-1 strain (indicated as b a n d c or e in lane A o f Fig. 1). Its restriction m a p is s h o w n in Fig. 2. pBB-15 has three u n i q u e cleavage sites for H p a I , BgllI a n d M l u I and is possibly used as a shuttle vector. S o u t h e r n h y b r i d i z a t i o n was d o n e using 32P-labeled p B B 15 as a p r o b e , pBB-15 was hybridized with two bands
1. Kitahara, K., Kaneko, T., and Goto, O.: Taxonomical studies on the hiochi-bacteria part I. Nippon Nogeikagaku Kaishi, 31, 556560 (1957). 2. Kilahara, K., Kaneko, T., and Goto, O.: Taxonomical studies on the hiochi-bacteria part II. Nippon Nogeikagaku Kaishi, 31,560564 (1957). 3. Momose, H., Yamagishi, T., and Nosiro, K.: Several characters of hiochi-bacteria. J. Soc. Brew. Jpn., 65, 799-803 (1970). 4. Mnmose, H., Yamanaka, E., Akiyama, H., and Nosiro, K.: Taxonomic study on hiochi-bacteria, with special reference to deoxyribonucleic acid base composition and chemical composition of bacterial cell wall. J. Gen. Appl. Microbiol., 20, 179-185 (1974). 5. Sugama, S. and Iguehi, T.: Seisyu no hiochi boushi ni kansuru kenkyu. J. Soc. Brew. Jpn., 65, 720-725 (1970). 6. Ohhuehi, K., Higashida, Y., Kanda, A., Hamaehi, M., and Honma, T.: Conditions of lysis and protoplast formation of hiochibacteria. Hakkokogaku, 67, 491-498 (1989). 7. Kado, C. I. and Liu, S. T.: Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol., 145, 1365-1373 (1981). 8. Anderson, D. G. and Mekay, L. L.: Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol., 46, 549-552 (1983). 9. Klaenhammer, T. R.: A general method for plasmid isolation in lactobacilli. Curr. Microbiol., 10, 23-28 (1984). 10. Vescovo, M., Bottazzi, V., Sarra, P. G., and Dellaglio, F.: Evidence of plasmid deoxyribonucleic acid in Lactobacillus. Microbiologica, 4, 413-419 (1981).