Letters in Applied Microbiology 1990,11, 220-223
FLD/06
Characterization and molecular cloning of Bifidobacterium longum cryptic plasmid pMBl D. M A T T E U Z Z IP * ,A T R I Z B IA R I G I D IM, A D D A L E NROSSI A & D I A N DI A G I OAI Fermentation Chemistry and Industrial Microbiology, Department of Pharmaceutical Sciences, University of Bologna, via Belmeloro 6,40126 Bologna, Italy Received 2 July 1990 and accepted 3 July 1990
M A T T E U Z ZD., I , B R I G I D IP, . , Ross], M . & DI G I O I AD. , 1990. Characterization and molecular cloning of Bifidobacterium longum cryptic plasmid pMB1. Letters in Applied Microbiology 11, 220-223. The small cryptic plasmid pMBl (1.9 kb), previously isolated from BiJidobacterium longurn, has been characterized by physical mapping. Two cloning vectors, pMR3 and pDG7, carrying chloramphenicol and ampicillin resistances derived from pJH101, have been electroporated in Escherichia coli.
It is well known that some intestinal bacterial groups affect positively natural resistance to infection. The bifidobacteria, which constitute one of the major components of human and animal intestinal flora, play an important role in this respect for their biological activities (Rasic & Kurmann 1983). Recently tablets and food containing Bijdobacterium cells have been produced and used in dairy preparations and also clinically (Bezkorovainy & Miller-Catchpole 1989). The species B. longum, B. breve, B. bijidum and B. infuntis are those generally employed in these preparations. In view of the development of bifidobacteria probiotic strains with improved characteristics, molecular genetic studies are of crucial importance. Unfortunately, little is known about Bijidobacterium genetics; up to now only a few studies concerning protoplast formation and regeneration (Brigidi et a / . 1986) and plasmid screening (Sgorbati et a/. 1982, 1986a, b; Iwata & Morishita 1989) are reported. In order to develop a cloning vector for the application of recombinant DNA technology to the breeding of bifidobacteria, we report the restriction map and the molecular cloning in Escherichia coli of the small cryptic
* Corresponding author.
plasmid pMB1, previously isolated from B. longum B2577 (Sgorbati et a / . 1982). Materials and Methods BACTERIAL STRAINS A N D C U L T U R E CONDITIONS
Bijidobacterium iongum B2577 (Sgorbati et ai. 1982) was grown anaerobically at 37°C in PTY medium (Scardovi 1986). As recipient strains for transformation E . coli HBlOl (leu-, pro-) and Bacillus subtilis PB1424 (his-, met-, trp-), cultured at 37°C in LB medium (Maniatis et a/. 1982), and Lactobacillus reuteri DSM 20016, grown at 37°C on MRS medium (Merck) were used. Transformant colonies were selected on the appropriate medium plates supplemented with chloramphenicol (Cm) and ampicillin (Amp) (Sigma) at 30 and 50 pg/ml, respectively for E. coli and Cm at 10 pg/ml for the other strains. Escherichia coli V517 (Macrina et al. 1978) was used as plasmid size marker. P L A S M I D D N A ISOLATION
Plasmid DNA from B. longum B2577 was isolated according to LeBlanc & Lee (1979). For
Bifidobacterium longum plasmid characterization
22 1
Fig. 1. Agarose gel electrophoresis of the pMBl plasmid DNA and the relative two-dimensional banding pattern. (A) Lane 1, Escherichiu coli V517 size marker; lane 2, pMBl DNA. (B) pMBl electrophoretic migration in horizontal sense, and (C) corresponding second-dimensionelectrophoresis.
preparative purposes a scale up of this method was used. Escherichia coli transformants were screened for plasmid content employing the rapid alkaline procedure described by Maniatis et al. (1982). Plasmid topoisomers were identified by the method of Hintermann et ul. (1981). Between the two electrophoresis steps, made in perpendicular directions, the DNA was U.V.irradiated for 10 min on a Fotodyne transilluminator (254 nm).
RESTRICTION ANALYSIS
The purified plasmid pMBl was digested by 33 different restriction endonucieases under the conditions specified by the suppliers (Boehringer GmbH and Bethesda Research Laboratories). Agarose gel electrophoresis, staining and visualization of gels were performed as described by Maniatis et a / . (1982). HindIIIIEcoRI digested
bacteriophage lambda DNA was used as molecular weight marker. PLASMID LIGATION A N D TRANSFORMATION
Plasmid pJHlOl (Ferrari et al. 1983) was used as cloning vector. After digestion with EcoRV and dephosphorylation, vector molecules were ligated with either EcoRV- or PuuII-linearized pMB1, using T4 DNA ligase (Boehringer GmbH). The ligation mixtures were incubated for about 16 h at 15°C and then used to transform by electroporation (Bio-Rad Gene PulserTM)intact cells of E. coli HBlOl (Dower et u1. 1988), €3. sublilis PB1424 (Brigidi ef al. 1990) and L. reuteri DSM 20016 (Chassy & Flickinger 1987).
Results and Discussion Up to now only the species B. longurn and B. breue among the bifidobacteria of human origin
D. Matteuzzi et al.
222
Pod, PuuII and RsaI each cleaved pMBl once. Two recognition sites for AuaI and three for Hue11 were found; the molecular weights of the fragments were 0.9, 1.0 and 1.2, 0.4, 0.3 kb, respectively. Numerous fragments very small in size were obtained with AluI, HeaIII, HhaI, Sau 961 and ToqI digestions; restriction size for these enzymes could not be assigned on the map. Furthermore, pMBl is not cleaved by AuaII, BamHI, BglI, BgIII, BstEII, ClaI, DraI, DraII, EcoRI, EcoRII, HindIII, HpaI, KpnI, Mbol, PstI, SalI, ScaI, StuI, XbaI and Xhol. To better characterize pMBl plasmid, in the attempt to locate the replication region, we employed the cloning strategy outlined in Fig. 2. Furthermore, in this way we succeeded in label-
were found to harbour plasmids. In order to construct cloning vectors based on endogenous replicon, we examined some strains of B. longurn with different plasmid profiles (Sgorbati et al. 1986a). Bijidohacterium longurn B2577 appeared to be the most interesting because we demonstrated that it exhibits the single small plasmid pMB1. Figure 1 shows that, out of the two bands of the electrophoretical profile, that of the major mobility contains CCC DNA (1.9 kb) and the other OC DNA. The plasmid pMBl was characterized by physical mapping (Fig. 2). The positions of the cleavage sites for the restriction enzymes were determined by analysis of singleand double-digestion products fractionated on agarose gels. The enzymes DdeI, EcoRV, Hinfl,
)RH/PvuK
w \ /
\\ 'h
// Avo1
PVUI
k
T4-DNA ligase
HinfI
RcoRP
pJH 101
5.4 kb
DdeI
T4-DNA ligase
pv"x
AvaI
HinfI.
EcoRQ
// //
\\
Avo1
\
pDG7 7 . 3 kb
G"
//
Fig. 2. Restriction map of pMBl plasmid and cloningstrategy
Bifidobacterium longum plasmid characterization ing the cryptic plasmid with two antibiotic resistances (AmpR, CmR). As a source of both antibiotic genes, we used pJH101, a hybrid plasmid containing pBR322, and the chloramphenicol acetyl transferase (CAT) gene from pC194; it replicates in E . coli but not in Grampositive bacteria. The construction of the recombinant plasmids pDG7 and pMR3 was made by digesting pMBl in two opposite unique sites with EcoRV and PuuII, respectively. Both digested DNAs were ligated with EcoRV-linearized pJHlOl and then used to transform by electroporation E. coli HB101, B. subtilis PB1424 and L. reuteri DSM 20016. AmpR and CmR transformant clones were selected only in E. coli. The size (7.3 kb) and the digestion patterns of both chimeras recovered from the transformants clearly demonstrated that the two hybrid plasmids pMR3 and pDG7 can replicate stably in E. coli. Both cloning vectors transformed E. coli at the same high eficiency (ca 1 x lo6 transformants/pg DNA). The restriction enzymes AuaI and PuuI were used to determine the pMBl orientation in these chimeras. Purified pMR3 and pDG7 DNAs, extracted from E. coli, were used to retransform E . coli HB101, B. subtilis PB1424 and L. reuteri DSM 20016. Once again, transformant colonies were isolated only in E. coli; no rearrangements had occurred in the chimeras after retransformation. Therefore our attempts to demonstrate replication of vectors based on bifidobacteria replicon in B. subtilis and L. reuteri have so far been unsuccessful. This may be explained by the disruption of the pMBl ori sequence in the construction of the two chimeras. Even if the two cleavage sites are opposite, the small size of pMBl may suggest that the replication region is inactivated. It can also be supposed that bifidobacteria origin region does not replicate in the two Grampositive bacteria tested. Studies concerning the construction of new chimeras and the transformation of bifidobacteria by electroporation or via protoplast are in progress. We wish to thank Mr Fabrizio Calanchi for his technical assistance.
223
References BEZKOROVAINY, A. & MILLER-CATCHPOLE, R. 1989 Biochemistry and Physiology of Bijidohacteria. Boca Raton: CRC Press. BRIGIDI,P., DE ROW, E., BERTARINI, M.L., RICCARDI, G. & MATTEUZZI, D. 1990 Genetic transformation of intact cells of Bacillus subtilis by electroporation. FEMS Microbiology Letters 67, 135-138. BRItiIUi, P., MATTEUZZI, D. & CROCIANI, F. 1986 Protoplast formation and regeneration in Bijidohacterium. Microbiologica 9, 243-248. CHASSY,B.M. & FLICKINGER, J.L. 1987 Transformation of Lactobacillus casei by electroporation. FEMS Microbiology Letters 44, 173-177. DOWER,W.J., MILLER, J.F. & RAGSDALE, W. 1988 High efficiency transformation of Escherichia coli by high voltage electroporation. Nucleic Acid Research 16,6127-6145. FERRARI, F.A., NGUYEN,A,, LANC,D. & HOCH,J.A. 1983 Construction and properties of an integrable plasmid for Bacillus suhtilis. Journal of Bacteriology 170,1182-1 190. HINTERMANN, G., FISCHER,H.M., CRAMERI,R. & HUTTER, R. 1981 Simple procedure for distinguishing CCC,OC and L forms of plasmid DNA by agarose gel electrophoresis. Plasmid 5, 371-373. IWATA,M. & MORISHITA, T. 1989 The presence of plasmids in Bifdobacterium breve. Letters in Applied Microbiology 9, 165-168. LEBLANC, D.J. & LEE,L.N. 1979 Rapid screening procedure for detection of plasmids in streptococci. Journal ofBacteriology 140, I 1 12- 1 115. MACRINA, F.L.,KOPECKO,D.J., JONES, K.R., AYEKS, D.J. & MCCOWEN, S.M. 1978 A multiple plasmid containing Escherichia coli strain: convenient source of size reference plasmid molecules. Plasmid 1,417420. MANIATIS, T., FRITSCH,E.F. & SAMBROOK, J. 1982 Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. RASIC,J.L. & KURMANN, J.A. 1983 Bifdobacteria and their Role. Basel: Birkhauser verlaf. SCARDOVI, V. 1986 The genus Bi$dobacterium. In Bergey's Manual of Systematic Bacteriology ed. Sneath, M.E. & Holt, J.G. Vol. 2, pp. 1418- 1434. Baltimore: William & Wilkins. SGORBATI, B., SCARDOVI, V. & LEBLANC, D.J. 1982 Plasmids in the genus Bifdobacterium. Journal o j General Microbiology 128,2121-2131. SGORBATI, B., SCARDOVI, V. & LEBLANC, D.J. 1986a Related structures in the plasmid profiles of Bifidobacterium longum. Microbiologica 9,415422. SGORBATI, B., SCARWVI,V. & LEBLANC, D.J. 1986b Related structures in the plasmid profiles of BiJidobacterium asteroides, B. indicum, and B. globosum. Microbiologica 9, 443456.
FLD/06
Characterization and molecular cloning of Bifidobacterium longum cryptic plasmid pMBl D. M A T T E U Z Z IP * ,A T R I Z B IA R I G I D IM, A D D A L E NROSSI A & D I A N DI A G I OAI Fermentation Chemistry and Industrial Microbiology, Department of Pharmaceutical Sciences, University of Bologna, via Belmeloro 6,40126 Bologna, Italy Received 2 July 1990 and accepted 3 July 1990
M A T T E U Z ZD., I , B R I G I D IP, . , Ross], M . & DI G I O I AD. , 1990. Characterization and molecular cloning of Bifidobacterium longum cryptic plasmid pMB1. Letters in Applied Microbiology 11, 220-223. The small cryptic plasmid pMBl (1.9 kb), previously isolated from BiJidobacterium longurn, has been characterized by physical mapping. Two cloning vectors, pMR3 and pDG7, carrying chloramphenicol and ampicillin resistances derived from pJH101, have been electroporated in Escherichia coli.
It is well known that some intestinal bacterial groups affect positively natural resistance to infection. The bifidobacteria, which constitute one of the major components of human and animal intestinal flora, play an important role in this respect for their biological activities (Rasic & Kurmann 1983). Recently tablets and food containing Bijdobacterium cells have been produced and used in dairy preparations and also clinically (Bezkorovainy & Miller-Catchpole 1989). The species B. longum, B. breve, B. bijidum and B. infuntis are those generally employed in these preparations. In view of the development of bifidobacteria probiotic strains with improved characteristics, molecular genetic studies are of crucial importance. Unfortunately, little is known about Bijidobacterium genetics; up to now only a few studies concerning protoplast formation and regeneration (Brigidi et a / . 1986) and plasmid screening (Sgorbati et a/. 1982, 1986a, b; Iwata & Morishita 1989) are reported. In order to develop a cloning vector for the application of recombinant DNA technology to the breeding of bifidobacteria, we report the restriction map and the molecular cloning in Escherichia coli of the small cryptic
* Corresponding author.
plasmid pMB1, previously isolated from B. longum B2577 (Sgorbati et a / . 1982). Materials and Methods BACTERIAL STRAINS A N D C U L T U R E CONDITIONS
Bijidobacterium iongum B2577 (Sgorbati et ai. 1982) was grown anaerobically at 37°C in PTY medium (Scardovi 1986). As recipient strains for transformation E . coli HBlOl (leu-, pro-) and Bacillus subtilis PB1424 (his-, met-, trp-), cultured at 37°C in LB medium (Maniatis et a/. 1982), and Lactobacillus reuteri DSM 20016, grown at 37°C on MRS medium (Merck) were used. Transformant colonies were selected on the appropriate medium plates supplemented with chloramphenicol (Cm) and ampicillin (Amp) (Sigma) at 30 and 50 pg/ml, respectively for E. coli and Cm at 10 pg/ml for the other strains. Escherichia coli V517 (Macrina et al. 1978) was used as plasmid size marker. P L A S M I D D N A ISOLATION
Plasmid DNA from B. longum B2577 was isolated according to LeBlanc & Lee (1979). For
Bifidobacterium longum plasmid characterization
22 1
Fig. 1. Agarose gel electrophoresis of the pMBl plasmid DNA and the relative two-dimensional banding pattern. (A) Lane 1, Escherichiu coli V517 size marker; lane 2, pMBl DNA. (B) pMBl electrophoretic migration in horizontal sense, and (C) corresponding second-dimensionelectrophoresis.
preparative purposes a scale up of this method was used. Escherichia coli transformants were screened for plasmid content employing the rapid alkaline procedure described by Maniatis et al. (1982). Plasmid topoisomers were identified by the method of Hintermann et ul. (1981). Between the two electrophoresis steps, made in perpendicular directions, the DNA was U.V.irradiated for 10 min on a Fotodyne transilluminator (254 nm).
RESTRICTION ANALYSIS
The purified plasmid pMBl was digested by 33 different restriction endonucieases under the conditions specified by the suppliers (Boehringer GmbH and Bethesda Research Laboratories). Agarose gel electrophoresis, staining and visualization of gels were performed as described by Maniatis et a / . (1982). HindIIIIEcoRI digested
bacteriophage lambda DNA was used as molecular weight marker. PLASMID LIGATION A N D TRANSFORMATION
Plasmid pJHlOl (Ferrari et al. 1983) was used as cloning vector. After digestion with EcoRV and dephosphorylation, vector molecules were ligated with either EcoRV- or PuuII-linearized pMB1, using T4 DNA ligase (Boehringer GmbH). The ligation mixtures were incubated for about 16 h at 15°C and then used to transform by electroporation (Bio-Rad Gene PulserTM)intact cells of E. coli HBlOl (Dower et u1. 1988), €3. sublilis PB1424 (Brigidi ef al. 1990) and L. reuteri DSM 20016 (Chassy & Flickinger 1987).
Results and Discussion Up to now only the species B. longurn and B. breue among the bifidobacteria of human origin
D. Matteuzzi et al.
222
Pod, PuuII and RsaI each cleaved pMBl once. Two recognition sites for AuaI and three for Hue11 were found; the molecular weights of the fragments were 0.9, 1.0 and 1.2, 0.4, 0.3 kb, respectively. Numerous fragments very small in size were obtained with AluI, HeaIII, HhaI, Sau 961 and ToqI digestions; restriction size for these enzymes could not be assigned on the map. Furthermore, pMBl is not cleaved by AuaII, BamHI, BglI, BgIII, BstEII, ClaI, DraI, DraII, EcoRI, EcoRII, HindIII, HpaI, KpnI, Mbol, PstI, SalI, ScaI, StuI, XbaI and Xhol. To better characterize pMBl plasmid, in the attempt to locate the replication region, we employed the cloning strategy outlined in Fig. 2. Furthermore, in this way we succeeded in label-
were found to harbour plasmids. In order to construct cloning vectors based on endogenous replicon, we examined some strains of B. longurn with different plasmid profiles (Sgorbati et al. 1986a). Bijidohacterium longurn B2577 appeared to be the most interesting because we demonstrated that it exhibits the single small plasmid pMB1. Figure 1 shows that, out of the two bands of the electrophoretical profile, that of the major mobility contains CCC DNA (1.9 kb) and the other OC DNA. The plasmid pMBl was characterized by physical mapping (Fig. 2). The positions of the cleavage sites for the restriction enzymes were determined by analysis of singleand double-digestion products fractionated on agarose gels. The enzymes DdeI, EcoRV, Hinfl,
)RH/PvuK
w \ /
\\ 'h
// Avo1
PVUI
k
T4-DNA ligase
HinfI
RcoRP
pJH 101
5.4 kb
DdeI
T4-DNA ligase
pv"x
AvaI
HinfI.
EcoRQ
// //
\\
Avo1
\
pDG7 7 . 3 kb
G"
//
Fig. 2. Restriction map of pMBl plasmid and cloningstrategy
Bifidobacterium longum plasmid characterization ing the cryptic plasmid with two antibiotic resistances (AmpR, CmR). As a source of both antibiotic genes, we used pJH101, a hybrid plasmid containing pBR322, and the chloramphenicol acetyl transferase (CAT) gene from pC194; it replicates in E . coli but not in Grampositive bacteria. The construction of the recombinant plasmids pDG7 and pMR3 was made by digesting pMBl in two opposite unique sites with EcoRV and PuuII, respectively. Both digested DNAs were ligated with EcoRV-linearized pJHlOl and then used to transform by electroporation E. coli HB101, B. subtilis PB1424 and L. reuteri DSM 20016. AmpR and CmR transformant clones were selected only in E. coli. The size (7.3 kb) and the digestion patterns of both chimeras recovered from the transformants clearly demonstrated that the two hybrid plasmids pMR3 and pDG7 can replicate stably in E. coli. Both cloning vectors transformed E. coli at the same high eficiency (ca 1 x lo6 transformants/pg DNA). The restriction enzymes AuaI and PuuI were used to determine the pMBl orientation in these chimeras. Purified pMR3 and pDG7 DNAs, extracted from E. coli, were used to retransform E . coli HB101, B. subtilis PB1424 and L. reuteri DSM 20016. Once again, transformant colonies were isolated only in E. coli; no rearrangements had occurred in the chimeras after retransformation. Therefore our attempts to demonstrate replication of vectors based on bifidobacteria replicon in B. subtilis and L. reuteri have so far been unsuccessful. This may be explained by the disruption of the pMBl ori sequence in the construction of the two chimeras. Even if the two cleavage sites are opposite, the small size of pMBl may suggest that the replication region is inactivated. It can also be supposed that bifidobacteria origin region does not replicate in the two Grampositive bacteria tested. Studies concerning the construction of new chimeras and the transformation of bifidobacteria by electroporation or via protoplast are in progress. We wish to thank Mr Fabrizio Calanchi for his technical assistance.
223
References BEZKOROVAINY, A. & MILLER-CATCHPOLE, R. 1989 Biochemistry and Physiology of Bijidohacteria. Boca Raton: CRC Press. BRIGIDI,P., DE ROW, E., BERTARINI, M.L., RICCARDI, G. & MATTEUZZI, D. 1990 Genetic transformation of intact cells of Bacillus subtilis by electroporation. FEMS Microbiology Letters 67, 135-138. BRItiIUi, P., MATTEUZZI, D. & CROCIANI, F. 1986 Protoplast formation and regeneration in Bijidohacterium. Microbiologica 9, 243-248. CHASSY,B.M. & FLICKINGER, J.L. 1987 Transformation of Lactobacillus casei by electroporation. FEMS Microbiology Letters 44, 173-177. DOWER,W.J., MILLER, J.F. & RAGSDALE, W. 1988 High efficiency transformation of Escherichia coli by high voltage electroporation. Nucleic Acid Research 16,6127-6145. FERRARI, F.A., NGUYEN,A,, LANC,D. & HOCH,J.A. 1983 Construction and properties of an integrable plasmid for Bacillus suhtilis. Journal of Bacteriology 170,1182-1 190. HINTERMANN, G., FISCHER,H.M., CRAMERI,R. & HUTTER, R. 1981 Simple procedure for distinguishing CCC,OC and L forms of plasmid DNA by agarose gel electrophoresis. Plasmid 5, 371-373. IWATA,M. & MORISHITA, T. 1989 The presence of plasmids in Bifdobacterium breve. Letters in Applied Microbiology 9, 165-168. LEBLANC, D.J. & LEE,L.N. 1979 Rapid screening procedure for detection of plasmids in streptococci. Journal ofBacteriology 140, I 1 12- 1 115. MACRINA, F.L.,KOPECKO,D.J., JONES, K.R., AYEKS, D.J. & MCCOWEN, S.M. 1978 A multiple plasmid containing Escherichia coli strain: convenient source of size reference plasmid molecules. Plasmid 1,417420. MANIATIS, T., FRITSCH,E.F. & SAMBROOK, J. 1982 Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. RASIC,J.L. & KURMANN, J.A. 1983 Bifdobacteria and their Role. Basel: Birkhauser verlaf. SCARDOVI, V. 1986 The genus Bi$dobacterium. In Bergey's Manual of Systematic Bacteriology ed. Sneath, M.E. & Holt, J.G. Vol. 2, pp. 1418- 1434. Baltimore: William & Wilkins. SGORBATI, B., SCARDOVI, V. & LEBLANC, D.J. 1982 Plasmids in the genus Bifdobacterium. Journal o j General Microbiology 128,2121-2131. SGORBATI, B., SCARDOVI, V. & LEBLANC, D.J. 1986a Related structures in the plasmid profiles of Bifidobacterium longum. Microbiologica 9,415422. SGORBATI, B., SCARWVI,V. & LEBLANC, D.J. 1986b Related structures in the plasmid profiles of BiJidobacterium asteroides, B. indicum, and B. globosum. Microbiologica 9, 443456.
