(~) INSTITUTPASTEUR/ELsEVIER Paris 1991
Res. Microbiol. 1991, 142, 643-652
Single-stranded DNA plasmid, vector construction and cloning of Bacillus stearothermophilus .-amilase in Lactobacillus P.S. Cocconcelli (l), M.J. Gasson (2), L. Morelli (1) and V. Bottazzi O) m Istituto di Microbiologia, Universit?t Cattolica del Sacro Cuore, via Emilia Parmense 84, 29100 Piacenza (Italy) and ~2~AFRC, Institute o f Food Research, Dept. o f Genetics and Technology, Colney Lane, Norwich NR4 7UA (UK)
SUMMARY Vector plasmids were constructed by ligating chloramphenicol and erythromycin resistance genes to Taql-digested DNA of a cryptic plasmid from Lactobacillus plantarum. The minimal region of Lactobacillus plasmid DNA that was required for DNA replication was defined and a single-stranded DNA intermediate replication system was observed. Homologies with other origins of replication of plasmids from Gram-positive bacteria, replicating via rolling circle mechanism, were found. It was shown that the constructed vectors, named pPSC20 and pPSC22, were transformable into L. plantarum, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus helveticus, Lactococcus lactis subsp, lactis, Bacillus subtilis, and Escherichia coil Using plasmid pPSC22, the c(-amylase gene of Bacillus stearothermophilus was cloned and expressed in several Lactobacillus species.
Key-wolds: Replication, Lactobacillus, DNA, Plasmid, Amylase; ssDNA, Cloning, Vectors, B. stearothermophilus.
INTRODUCTION Due to the importance of the species of the genus Lactobacillus in the manufacturing of fermented food, in vegetable fermentation and in the ecology of the intestinal tract, the lactobaciUi are receiving increasing attention as organisms targeted for genetic manipulation (Chassy, 1987). Although different authors have recently reported the development of electrotransformation protocols for lactobacilli (Chassy and Flickinger, 1987; Luchansky et al., 1988), few Submitted October 24, 1990, accepted March 11, 1991.
reports exist on the construction of vectors and on cloning and expression of heterologous genes in Lactobacillus. Most of the work has been done with strains of Lactobacillusplantarum and Lactobacillus casei (Bates et al., 1989; Scheirlink et al., 1989) and no evidence is reported for other species of this economically important group of microbes. The primary objective of the work presented here was the development of a versatile vector-cloning system active in different species
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Erythromycin was added when necessary at a final concentration of 10 ttg/rnl for lactobacilli, lactococci and B. subtilis and 250 ttg/ml for E. coll. Chloramphenicol was used at a final concentration of I0 ttg/ml.
belonging to the three subgroups o f the genus Lactobacillus, the thermophilic homofermentative, facultative homofermentative and heterofermentative lactobacilli. In order to achieve this goal, we constructed shuttle vectors, based on the origin o f replication o f a cryptic plasmid o f L. plantarum, and we demonstrated the ability o f these vector plasmids to replicate and to be stably maintained in different hosts, such as several Lactobacillus species, in Lactococcus lactis subsp, lactis, Bacillus subtilis and Escherichia coil, crossing the barrier between Gram-positive and Gramnegative bacteria. In addition, we describe the cloning o f the Bacillus stearothermophilus ~-amylase gene, in Lactobacillus reuteri, L. plantarum and Lactobacillus helveticus and demonstrate the functional expression and secretion o f the encoded s-amylase in lactobacilli.
DNA manipulation General molecular biology techniques were as described by Manlatis et al. (1982). Restriction endonuclease and DNA ligase were purchased from Boehringer (Mannheim, Federal Republic of Germany) and Promega (Madison, WI, USA) and were used according to the suppliers' recommendation. Plasmid DNA purification from B. subtilis and for L. lactis subsp, lactis was as previously described (Gasson, 1986). The extraction of extrachromosomal DNA from lactobacilli was performed as already reported (Morelli et al., 1988). For hybridization purposes, the non-radioactive labelling of DNA with degoxigenin-dUTP and the detection of hybrids by enzyme immunoassay were used following the instruction of the supplier (Boehringer, Manheim, Federal Republic of Germany).
MATERIALS AND METHODS
Bacterial strains and plasmids The bacteri,~l strains used in this work are listed in table I. The L. plantarum strains L7, L38 and L40 were isolated from Italian corn silage and the taxonomic position was determinated by means of the API 50CHL (API system S.A., Montalie Vercieu, France), and by calculation of DNA-DNA hybridization homology determined according to the methodology of Deilaglio et al. (1973). Plasmid pCKI7 was previously described (Gasson and Anderson, 1985). The replicator probe plasmid pOC103 was obtained from Dr. M. Young (University College of Wales, Aberystwyth). Plasmids pE194 and pC194 have already been described (Horinouchi and Weisblum, 1982a,b). The m-amylasegene was present in the plasmid pAMY8, a 6.8-kh derivative of the pAMY7 (Thudt et al., 1985) obtained with a PvuII deletion.
Table I. Bacterial strains. Strains
Media Lactobacilli were routinely grown on MRS meditun (De Man et al., 1960), L. lactis subsp, lactis on M17 media (Terzaghi and Sandine, 1975), E. coli on L broth and L agar (Lennox, 1955) and B. subtilis on brain-heart infusion broth (Oxoid, UK).
Cm
= chloramphenicol.
Source or reference
L. acidophilus AMPI Morelli et al., 1987 L. helveticus $36-2 Smiley & Fryder, 1978 L. helveticus NP3 IMPC L. plantarum NCFB 1752 NCFB L. plantarum NCFB 1193 NCFB L. plantarum L7 This study L. plantanan L38 This study L. plantarum 1.40 This study L. fermentum F18 IMPC L. reuteri DSM20016 DSM L. reuteri D6 IMPC L. lactis subsp, lactis MOl~63Gasson & Anderson, 1985 B. subtilis 1280 Gasson & Anderson, 1985 E. coil 1106 Gasson & Anderson, 1985 DSM=Deutsche Sammlungyon Mikrooganismen,Braunschweig(FRI~). IMPC=Colleetion of the Istituto di Microbiologla,Piacenza 0~Y). NCFB= National Collectionof Food Bacteria, Reading(UK).
[
Em
= erythromycin.
VECTOR PLASMID CONSTRUCTION AND CLONING IN LACTOBACILLUS Detection of single-stranded plasmid DNA and copy number determination The detection of single stranded DNA (ssDNA) w~s performed following the technique based on accumala~ion of ssDNA induced by rifampin (Boe et aL, 1989) and the Sl-nuclease digestion as previously described by Te Riele et al. (1986). Plasmid DNA obtained from the L. reuteri cells harbouring the pPSC22 plasmid, grown to a mid-log phase and then incubated for 2 h in the presence of 100 t~g/rni of rifampin was digested with SI nuclease, electrophoresed through an 0.8 % agarose gel and stained with ethidium bromide. The gel was deaaturated, transferred to a nylon filter following standard methods (Maniatis et al., !982) and hybridized with the degoxigenin-dUTP-labelled DNA of pPSC22. The number of copies of pPSC22 plasmid per cell of L. reuteri was determined as previously described by Seheer-Abramowitz et al. (1981). Transformation techniques E. coil competent cell transformation was already described by Maniatis et aL (1982). Techniques for transformation of polyethylene-glycol-treated protoplasts were used as described for Laetobaeillus (Morelli et al., 1987), L. lactis subsp, lactis (Kondo and McKay, 1984) and B. subtilis (Chang and Cohen, 1979). The "Bio Rad Gene" pulser apparatus and the "Bio Rad Pulse Controller" system (Bin Rad Laboratories, USA) were used for electroporation experiments. For the electroporation of E. coli, the protocol described by Dower et al. (1988) was followed. Lactobacillus strains were transformed using the method described by Luchansky et al. (1988) modified as follows: the cells were collected in Iogaritmic phase and washed twice with the electroporation buffer (EB: 0.5 M sucrose, 10 mM K phosphate buffer, pH 7) and resuspended in 1/20 of the initial volume and chilled on ice. Plasmid DNA (1 izg in 2 lal of Tris-EDTA) was added to 200 Izl of cell suspension. The mixture DNA-celIs was transferred into a 0.2-era gap cuvette and subjected to a pulse in the "Gene Pulser" apparatus at 2,500 V, 25 I~F, 200 ft. After electroporation, the cell suspension was diluted with 1 ml of mouse reaginic serum MRS, placed at 37°C for 1 h and plated on MRS agar with l0 ~tg/ml of chloramphenicol or erythromycin. Detection of ~t-amylase expression In order to assay the a-amylase activity in agar plates, the colonies of lactobacilli were replicated on MRS containing 1 070 soluble starch and incubated at 37°C for 48 h. The plates were stained for a-
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amylase activity with iodine (I2/KI) solution and the size of halos around the colonies reflected the a-amylase activity. The activity of a-amylase in the supernatant of the overnight cultures of Lactobaeillus was detected following the method of Fuwa (1954). RESULTS Selection o f a L.
plantarum repUeon
A 7-kb plasmid present in L. plantarum NCFB 1752 was purified and partially digested with restriction endonucleas¢ TaqI. This D N A was ligated to TaqI-digested D N A of plasmid pOCl03, a derivative of the E. coli vector pAT153 that carried a chloramphenicol (Cm) resistance gene originally derived f r o m staphylococcal plasmid pC194. This gene is known to be functional in a variety of Grampositive bacteria and is present on a TaqI fragment. Ligated D N A was transformed into B. subtilis 1280 by polyethylene-glycol treatment of protoplasts and the selection of Cm-resistant protoplast regenerants. Under these conditions, only hybrid plasmids that combined the replication region o f Lactobaciilus plasmid with the Gram-positive Cm-resistance gene would generate transformants. All of the 45 transformants obtained were analysed for plasmid content and were found to carry small but variably sized molecules. One of the smallest plasmid constructs was retained for further characterization. This construct named pPSC1 was a 4.l-kb plasmid consisting of three TaqI fragments of 2.5, 1.0 and 0.6-kb (fig. 1). It was found that pPSC1 was transformable into protoplasts o f L. reuteri DSM 20016 and into E. coli 1106 and, because of the greater stability that was experienced when using E. coli, further vector construction was done in this host. Plasmid pPSC1 was digested to completion with restriction endonuclease TaqI, religated and transformed into E. coil 1106. Analysis of the transformants obtained revealed that, in all cases, the simplest construct recovered was a 4 . l - k b plasmid consisting of the same 3 TaqI fragments that make up pPSC1. This suggested that the minimal D N A for replication was present in pPSCI.
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T~I~.~Bcll Tacll
~ m r
cm r
T~ -I LH~pal
'
~l pPSCIO
1"aqlHpal
Hpal
pPSC15
T~t
Fig. 1. Physical maps of pPSC plasmids. Symbols and abbreviations: dotted areas represent the L. plantarum plasmid replication functions, solid axeas axe derived from pE194 and open areas from plasmid pOCl03 ; em r= erythromycin resistance; cm r = chloramphenicol resistance.
C o n s t r u c t i o n o f a L. plantarum v e c t o r
In order to provide a useful vector for lactobacilli, a second antibiotic resistance gene was introduced into plasmid pPSC1. The staphylococcal e r y t h r o m y c i n (Em) resistance gene present o n p l a s m i d pE194 is e n c o d e d by a
1.4-kb TaqI f r a g m e n t ( H o r i n o u c h i a n d Weisb l u m , 1982a). P l a s m i d pE194 D N A was purified f r o m B. subtilis a n d digested first with restriction endonuclease M b o I I to disrupt t h e additional u n w a n t e d TaqI f r a g m e n t s that were also present a n d t h e n with restriction e n d o n u c l e a s e TaqI. T h i s D N A was ligated with p l a s m i d
VECTOR PLASMID CONSTRUCTION AND CLONING IN LACTOBACILLUS pPSCI DNA that had been partially digested with restriction endonuclease i'aq! such that only one of the available TaqI recognition sites was cleaved. The ligated DNA was transformed into E. coil 1106 with selection for both Cm and Em resistances. Transformants were obtained that carried a 5.5-kb plasmid consisting of the three TaqI fragments from pPSCI and the 1.4-kb fragment that encoded the Em-resistance gene. One of the plasmids isolated was named pPSC20 and used for further work.
Transformation with pPSC20 A number of additional species of bacteria have been transformed with vector pPSC20. The ability of the vector to transform B. subtilis and E. coil was established during the construction of the vector. The plasmid-free L. lactis subsp. lactis strain MG1363 was transformed with pPSC20 using polyethylene-glycol-treated protoplasts at a frequency of 500 transformants per p.g of DNA. The electrotransformation technique was successfully used for the transformat i o n o f L. r e u t e r i D S M 2 0 0 1 6 a n d D6, L. p l a n t a r u m L38, L40 a n d N C F B l 1 9 3 , L. acidophilus AMP1, L. f e r m e n t u m F18 and L. helveticus $36-2 with pPSC20 plasmid. The frequencies of transformation observed ranged from 102 to 105 transformants/l~g of pPSC20 DNA.
Replication function of pPSC20 and construction of other vectors During the construction of vector pPSC20, it was clearly established that the Cm-resistance gene was encoded by a 2.5-kb TaqI fragment. This observation implied that during the construction of p P S C I using B. subtilis as host, a complex DNA rearmngment occurred, since the source of Cm-resistance gene is known to be a 1.33-kb TaqI fragment originating from plasmid pOCl03. Rearrangment of DNA was shown to occur as a direct consequence of ssDNA plas-
647
mid replication (Gruss and Ehrlich, 1989) and has frequently been encountered during previous work with lactic acid bacteria DNA. In order to create the simplest vector plasmids possible, two additional derivatives of pPSCI and pPSC20 were made using E. coli as the cloning host. In pPSCI 1 and pPSC22, the enlarged 2.5-kb fragment that was formed during the construction of pPSCI has been replaced by the 1.33-kb TaqI fragment of pOCl03 that encodes the Cm-resistance gene. These plasmid vectors behave in an identical way to the original vector constructs pPSC1 and pPSC20. Plasmid p P S C I 0 of 3-kb, which encodes only an erythromycin-resistance gene, was made by the removal of the 2.5-kb TaqI fragment from pPSC20 by TaqI digestion and religation. The structure and the derivation of these various vectors are illustrated in figure 1. The minimal Lactobacillus plasmid DNA that is required for vector replication has been established as the two Taql fragments of 0.6 and l-kb that are present in all the constructs. During the derivation of pPSC 10 from pPSC20, some transformants, resistant only to Em, were found to carry p P S C I 0 and a smaller molecule of 2-kb. The smaller plasmid present in these transformants, named pPSCI5, was demonstrated to be composed of the 1.4-kb Taql fragment carrying the Em-resistance gene together with the 0.6-kb fragment from Lactobacillus plasmid replication origin. Transformation of E. coil and L. reuteri to Em resistance with pPSCI5 D N A proved to be unsuccessful, but in similar experiments using E. coli and L. reuteri already carrying the Cm-resistance plasmid pPSC1, Em-resistant transformants were readily obtained and were shown to carry two plasmids equivalent to pPSC 15 and pPSC1. These observations suggest that the origin of D N A replication is encoded by the 0.6-kb TaqI fragment but the replication depends on a transacting function encoded by the l-kb TaqI fragment present in all the constructs except pPSCI5. The number of copies of pPSC22 in L. reuteri, calculated with the method of ScheerAbramowitz et al. (1981), was 60 copies per cell.
P.S. COCCONCELLI ET AL.
648
Single-strand intermediate replication of pPSC plasmids Cell lysates obtained from L. reuteri DSM20016 harbouring pPSC22 and grown in the presence of 100 ~g/ml of rifampin, either treated or not with SI nuclease, were hybridized with the labelled 0.6 TaqI fragment of pPSC22. Figure 2 (lane A) shows the supercoiled and circuiar plasmid monomers as well as the fast migrating single-stranded DNA band. This band was absent in the DNA preparation digested with nuclease SI (lane B), confirming its single-
stranded nature. The accumulation of ssDNA in L. reuteri is rifampin-dependent and suggests the role of RNA polymerase in the conversion of ssDNA to dsDNA as reported for several ssDNA plasmids isolated from Gram-positive bacteria (Gruss and Ehrlich, 1989).
Homologies of pPSC origin of replication with other ssDNA plasmids Hybridization studies, shown in figure 3, demonstrated the presence of homology between the origin of replication of pPSC plasmids and the replication region of pE194 and with the replication function of pSH71 present on the vector pCKI7 (Gasson and Anderson, 1985). No homology was observed with the origin of replication of pC194.
Cloning and expression of B. stearothermophiius u-amylase gene in various Lactobacillus species
~D
A
~t~
-
CCC
B
Fig. 2. Detection of single-stranded pPSC22 DNA. Agarose gel electrophoresis of lysates prepared from L. reuteri DSM 20016 containing pPSC22 grown in presenceof rifampin. Sampleswerenot treated (A) or were treated (B) with nuclease$1 and hybridizedto the pPSC22 probe. CCC and SS refer to the position of supercoiled monomers and single-stranded forms of pPSC22.
The B. stearothermophilus u-amylase gene was located on the pAMY8, a PvuII deletion derivative of pAMY7 (Thudt et al., 1985). The pAMY8 plasmid was partially digested with BamHI and ligated with pPSC22 linearized with BclI and treated with bacterial alkaline phosphatase. The resulting plasmid D N A was transformed in L. reuteri using the electroporation technique. The transformants, selected on MRS without glucose and containing starch as sole carbon source and 10 ~g/ml of Cm were found to carry a 8.3-kb plasmid denominated pPSA10 (fig. 4). Expression of u-amylase gene was observed in L. reuteri DSM20016 harbouring plasmid pPSA10 in an iodine test. The recombinant plasmid pPSAI0 was electrotransformed in L. plantarum LT, L38 and L40 strains, in L. acidophilus AMP 1 and in L. helveticus $36-2 and NP3 and expression of the u-amylase gene was detected in all the Lactobacillus transformants (fig. 5). The m-amylase activities of L. reuteri DSM20016 and L. plantarum L38 and L40 strains, determined in MRS broth during early stationary growth phase following the method of
VECTOR PLASMID CONSTRUCTION AND CLONING IN LACTOBACILLUS
A
649
B
\
&
m
13
34
12
34
Fig. 3. Southern hybridizationof 0.6-kb Taql fragment of pPSC1, carrying the origin of replication, with the replication region of pE194 and pSH71. (A) Lanes 1 and 2:pPSC22 and 7-kb plasmia DNA of L. plantarum NCFBI752 respectively; lane 3 and 4: hybridization with the labelled 0.6-kb Taql fragment of pPSCI. (B) Lane 1: TaqI digestedpE194; lane 2:pCK17 digestedwith restriction endonueleasesBglII and EcoRI; lane 3 and 4: hybridizationof the labelled0.6-kb Taql of pPSCI to each of the DNA preparationsin lane 1 and 2. The origin of replicationof pPSC plasmidshybridizedwith the 1.3-kb fragment of pE194 and with the 3-kb EcoRI-Bglll fragment of pCKI7 harbouring the replication function of these two plasmids.
Fuwa (1954), were respectively 1,500 and 1,850 units par liter. Since both orientations o f the aamylase gene gave rise to comparable amounts of enzyme activity (data not shown) the transcription probably starts from the ct-arnylase promoter.
ing the L. reuteri DSM20016 for about 600 generations without starch and Cm-selective pressure, 95 % of the cell population had retained the ability to hydrolyse starch and the Cm resistance and the plasmid profile analysis demonstrated the presence o f pPSAI0.
Stability of pPSAIO in lactobacilli DISCUSSION Plasmid pPSAIO exhibited high structural and segregational stability in L. reuteri and in the other species o f lactobacilli. After cultivat-
In this study, we report the identification and the preliminary characterization o f the minimal
P.S. COCCONCELLI
650
bcll
ET AL.
hpal
smal psll
sail
pPSA10 8.3 kb pvul
hlndlll Fig. 4. Restriction map of the plasmid pPSAI0 carrying the ~-amylase gene of B. stearothermophilus.
Fig. 5. or-Amylaseexpression in Lactobacillus. The MRS-starch agar plates were inoculated with (A) L. acidophilus AMP1 (pPSAI0), (B) L. reuteri DSM20016 (pPSAI0), (C) L. plantarum L40 (pPSAI0), (D) L. helveticus $36-2 (pPSAI0), (E) L. helveticus NP3 (pPSAI0). The plates were incubated in anaerobic conditions for 48 h and then stained with iodine solution (12/KI). The halos around the colonies reflect the ~-amylase production.
VECTOR PLASMID CONSTRUCTION AND CLONING IN LACTOBACILLUS
replicon of the cryptic plasmid of L. plantarum NCFB1752 and its use for the construction of pPSC20 and pPSC22 plasmid vectors and for the cloning of B. stearothermophilus a-amylase gene in several Lactobacillus strains. The replicon of pPSC plasmids was demonstrated to be functional in a variety of Lactobacillus species, in other Gram-positive bacteria, such as L. lactis subsp, lactis and B. subtilis, and in E. coli. This feature is particularly useful for employing pPSC22 as an E. coli-Lactobacillus shuttle vector. The promiscuous replication property of these vectors is typical of others derived from cryptic plasmids present in lactococci (Kok et al., 1984; De Vos, 1987; Gasson and Anderson, 1985). Complementation studies suggest the separation of the replication region of pPSC plasmids into an origin-containing domain and a region functional in trans as observed in other replicons deriving from lactic acid bacteria (De Vos, 1987; Hayes et aL, 1990). The classification of replicon of pPSC vectors to the family of highly interrelated singlestranded plasmids of Gram-positive bacteria (Gruss and Erhlich, 1989) was supported by: (1) the presence of the ssDNA intermediate of replication, (2) the D N A homologies with the replication regions of the staphylococcal plasmid pE 194 and the lactococcal plasmid pSH71 and (3) the structural instability and D N A rearrangements observed during the construction of pPSC1. In order to evaluate the efficiency of the cloning system, plasmid pPSC22 was used for cloning and expression of the a-amylase gene of B. stearothermophilus in L. /~',~ntarum, in L. reuteri, in L. acidophilus and ~ ~elveticus. The a-amylase gene confers upon lactobacilli improved fermentative properties and, due to the simplified system of detection, may be used as a reporter gene in monitoring this group of bacteria. The constructed plasmid pPSA10, carrying the a-amylase gene, is characterized by high segregational stability not observed in other recombinant plasmids used for cloning in
651
L. plantarum (Scheirlink et al., 1989; Bates et aL, 1989).
The construction of stable cloning vectors has proven to be valuable in cloning heterologous genes in several Lactobacillus species, including the thermophilic species L. helveticus used in dairy production, L. plantarum and the members of intestinal microflora L. acidophilus and L. reuteri. In addition, the development of this stable vector system may provide a versatile alternative to the chromosomal integration in the genetic engineering of lactobacilli.
Plasmides ~t simple brin d'ADN vecteurs de construction et de clonage de I'a-amylase de Bacillus stearothermophilus chez Lactobacillus
Des plasmides vecteurs ont &6 construits par liaison des g6nes de la r6sistance au chloramph~nicol et l'6rythromycine/l I'ADN d'un plasmide cryptique de Lactobacillus p!antarum restreim par TaqI. La r6gion minimale de I'ADN plasmidique de Lactobacillus qui est n6cessaire pour la r6plication de I'ADN a ~t6 d6finie, et un syst~meinterm6diaire d'ADN simple brin a 6t6 exantin6. Nous avons trouv6 des homologies avec les points de d~part de la replication des plasmides de bact~ries Gram-positives se repliquant via un m~eanisme cireulaire. Nous montrons que |es vecteurs construits pPSC20 et pPSC22 sont transformables dans L. plantarum, Lactobacillus acidophilus, Lactobacillus renteri, Lactobacillus fermentum, Lactobacillus helveticum, Lactococcu lactis subsp, lactis, Bacillus subtilis et Escherichia coli. A l'aide du !flasmide pPSC22, le g/me de l'ctamylase de Bacillus stearotkermophilus a 6t~ clon6 et exprim6 chez plusieurs esp~ces de Lactobacillus. Mots-cl~s: R~plication, Lactobacillus, ADN, Plasmide, Amylase; ADN monocat6naire, Clonage, Vecteurs, B. stearothermophilus.
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P.S. C O C C O N C E L L I E T A L .
Boe, L., Gros, M.F., Te Riele, H., Ehrlich, S.D. & Gruss, A. (1989), Replication origins of single-stranded-DNA plasmid pUBII0. J. Bact., 171, 3366-3372. Chassy, B.M. (1987), Prospects for the genetic manipulation of lactobacilli. FEMS MicrobioL Rev., 46, 297-312. Chassy, B.M. & Flickinger, J.L. (1987) Transformation of Lactobacillus casei by electroporation. FEMS Microbiol. Letters, 44, 173-t77. Chang, S. & Cohen, S.N. (1979), High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol. gen. Genetic, 168, 111-115. Dellaglio, F., Bottazzi, V. & Trovatelli, L.D. (1973), Deoxyribonucleic acid homology and base composition in some thermophilic lactobacilli. J. gen. MicrobioL, 74, 289-297. De Man, J.C., Rogosa, H. & Sharpe, M.E. (1960), A medium for the cultivation of lactobacilli. J. Appl. Bact., 23, 130-135. De Vos, W.M. (1987), Gene cloning and expressionin lactic streptococci. FEMS Microbiol. Rev., 46, 281-295. Dower, W.J., Miller, J.F. & Rangsdale, C.W. (1988), High efficiency transformation of Escherichia coli by high voltage electroporation. Nucl. Acids Res., 16, 6127-6145. Fuwa, H. (1954), A new method for microdetermination of amylase activity by the use of amylose as the substrate. J. Biochem., 41,583-603. Gasson, M.J. (1986), in "Biomolecular engineering in the European Community" (Magnien E.) (pp. 489-495). Maninus Nijhoff Publ. Dordrecht. Gasson, M.J. & Anderson, P.H. (1985), High copy number plasmid vectors for use in lactic streptococci. FEMS Microbiol. Letters, 30, 193-196. Gruss, A. & Ehrlich, S.D. (1989), The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbiol. Rev., 53, 231-241. Hayes, F., Daly, C. & Fitzgerald, G.F. (1990), Identification of the minimal replicon of Lactobacillus lactis subsp, lactis UC317 plasmid pCI305. Appl. Environm. Microbiol., 56, 202-209. Horinouchi, S. & Weisblum, B. (1982a), Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibiotics. J. Bact., 150, 804-814. Horinouchi, S. & Weisblum, B. (1982b), Nucleotide sequence and functional map of pC194, a plasmid that specifies chloramphenicoi resistance. J. Bact., 150, 8t5-825.
Kok, J., Van der Vossen, J.M.B.M. & Venema, G. (1984), Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Edcherichia coil Appl. Environm. Microbiol., 48, 726-731. Kondo, J.K. & McKay, L.L. (1984), Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning. AppL Environm. Microbiol., 48, 252-259. Lennox, E.S. (1955), Transduction of linked characters of the host by bacteriophage P1. Virology, 1, 190-206.
Luchansky, J.B., Muriana, P.M. & Klaenhammer, T.R. (1988), Application of electroporation for transfer plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus and Propionibacterium. MoL Microbiol., 2, 637-646. Maniatis, T., Fritsch, E.F. & Sambrook, J. (1982), Molecular cloning. A laboratory manual. Cold Spring Harbout Laboratory, New York. Morelli, L., Cocconcelli, P.S., Bottazzi, V., Damiani, G., Ferretti, L. & Sgaramella, V. (1987), Lactobacillus protoplast transformation. Plasmid, 17, 73-75. Morelli, L., Sarra, P.G. & Bottazzi, V. (1988), In vivo transfer of pAM[31 from Lactobacillus reuteri to Enterococcus faecalis. J. Appl. Bact., 65, 371-375. Scheer-Abramowitz, J., Gryczan, T.J. & Dubnau, D. (1981), Origin and mode of replication of plasmids pEI94 and pUBII0. Plasmid, 6, 67-77. Scheirlink, T., Mahillon, J., Joos, H., Dhaese, P. & Michiels, F. (1989), Integration and expression of =amylase and endoglucanase genes in the Lactobacillus plantarum chromosome. Appl. Environm. Microbiol., 55, 2130-2137. Smiley, M.B. & Fryder, V. (1978), Plasmids, lactic acid production and N-acetyI-D-glucosaminefermentation in Lactobacillus helveticus subsp, yogurti. Appl. Environm. MicrobioL, 35,777-781. Te Riele, H., Michel, B. & Erlich, S.D. (1986), Singlestranded plasmid DNA in Bacillus subtilis and Staphylococcus aureus. Proc. nat. Acad. Sci. (Wash.), 83, 2541-2545. Terzaghi, B.K. & Sandine, N.E. (1975), Improved medium for lactic streptococci and their bacteriophages. Appl. Environm. Microbiol., 29, 807-813. Thudt, K., Schleifer, K.H. & Gotz, F. (1985), Cloning and expression of the ~-amylase gene from Bacillus stearothermophilus in several staphylococcal species. Gene, 37, 163-169.
Res. Microbiol. 1991, 142, 643-652
Single-stranded DNA plasmid, vector construction and cloning of Bacillus stearothermophilus .-amilase in Lactobacillus P.S. Cocconcelli (l), M.J. Gasson (2), L. Morelli (1) and V. Bottazzi O) m Istituto di Microbiologia, Universit?t Cattolica del Sacro Cuore, via Emilia Parmense 84, 29100 Piacenza (Italy) and ~2~AFRC, Institute o f Food Research, Dept. o f Genetics and Technology, Colney Lane, Norwich NR4 7UA (UK)
SUMMARY Vector plasmids were constructed by ligating chloramphenicol and erythromycin resistance genes to Taql-digested DNA of a cryptic plasmid from Lactobacillus plantarum. The minimal region of Lactobacillus plasmid DNA that was required for DNA replication was defined and a single-stranded DNA intermediate replication system was observed. Homologies with other origins of replication of plasmids from Gram-positive bacteria, replicating via rolling circle mechanism, were found. It was shown that the constructed vectors, named pPSC20 and pPSC22, were transformable into L. plantarum, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus helveticus, Lactococcus lactis subsp, lactis, Bacillus subtilis, and Escherichia coil Using plasmid pPSC22, the c(-amylase gene of Bacillus stearothermophilus was cloned and expressed in several Lactobacillus species.
Key-wolds: Replication, Lactobacillus, DNA, Plasmid, Amylase; ssDNA, Cloning, Vectors, B. stearothermophilus.
INTRODUCTION Due to the importance of the species of the genus Lactobacillus in the manufacturing of fermented food, in vegetable fermentation and in the ecology of the intestinal tract, the lactobaciUi are receiving increasing attention as organisms targeted for genetic manipulation (Chassy, 1987). Although different authors have recently reported the development of electrotransformation protocols for lactobacilli (Chassy and Flickinger, 1987; Luchansky et al., 1988), few Submitted October 24, 1990, accepted March 11, 1991.
reports exist on the construction of vectors and on cloning and expression of heterologous genes in Lactobacillus. Most of the work has been done with strains of Lactobacillusplantarum and Lactobacillus casei (Bates et al., 1989; Scheirlink et al., 1989) and no evidence is reported for other species of this economically important group of microbes. The primary objective of the work presented here was the development of a versatile vector-cloning system active in different species
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Erythromycin was added when necessary at a final concentration of 10 ttg/rnl for lactobacilli, lactococci and B. subtilis and 250 ttg/ml for E. coll. Chloramphenicol was used at a final concentration of I0 ttg/ml.
belonging to the three subgroups o f the genus Lactobacillus, the thermophilic homofermentative, facultative homofermentative and heterofermentative lactobacilli. In order to achieve this goal, we constructed shuttle vectors, based on the origin o f replication o f a cryptic plasmid o f L. plantarum, and we demonstrated the ability o f these vector plasmids to replicate and to be stably maintained in different hosts, such as several Lactobacillus species, in Lactococcus lactis subsp, lactis, Bacillus subtilis and Escherichia coil, crossing the barrier between Gram-positive and Gramnegative bacteria. In addition, we describe the cloning o f the Bacillus stearothermophilus ~-amylase gene, in Lactobacillus reuteri, L. plantarum and Lactobacillus helveticus and demonstrate the functional expression and secretion o f the encoded s-amylase in lactobacilli.
DNA manipulation General molecular biology techniques were as described by Manlatis et al. (1982). Restriction endonuclease and DNA ligase were purchased from Boehringer (Mannheim, Federal Republic of Germany) and Promega (Madison, WI, USA) and were used according to the suppliers' recommendation. Plasmid DNA purification from B. subtilis and for L. lactis subsp, lactis was as previously described (Gasson, 1986). The extraction of extrachromosomal DNA from lactobacilli was performed as already reported (Morelli et al., 1988). For hybridization purposes, the non-radioactive labelling of DNA with degoxigenin-dUTP and the detection of hybrids by enzyme immunoassay were used following the instruction of the supplier (Boehringer, Manheim, Federal Republic of Germany).
MATERIALS AND METHODS
Bacterial strains and plasmids The bacteri,~l strains used in this work are listed in table I. The L. plantarum strains L7, L38 and L40 were isolated from Italian corn silage and the taxonomic position was determinated by means of the API 50CHL (API system S.A., Montalie Vercieu, France), and by calculation of DNA-DNA hybridization homology determined according to the methodology of Deilaglio et al. (1973). Plasmid pCKI7 was previously described (Gasson and Anderson, 1985). The replicator probe plasmid pOC103 was obtained from Dr. M. Young (University College of Wales, Aberystwyth). Plasmids pE194 and pC194 have already been described (Horinouchi and Weisblum, 1982a,b). The m-amylasegene was present in the plasmid pAMY8, a 6.8-kh derivative of the pAMY7 (Thudt et al., 1985) obtained with a PvuII deletion.
Table I. Bacterial strains. Strains
Media Lactobacilli were routinely grown on MRS meditun (De Man et al., 1960), L. lactis subsp, lactis on M17 media (Terzaghi and Sandine, 1975), E. coli on L broth and L agar (Lennox, 1955) and B. subtilis on brain-heart infusion broth (Oxoid, UK).
Cm
= chloramphenicol.
Source or reference
L. acidophilus AMPI Morelli et al., 1987 L. helveticus $36-2 Smiley & Fryder, 1978 L. helveticus NP3 IMPC L. plantarum NCFB 1752 NCFB L. plantarum NCFB 1193 NCFB L. plantarum L7 This study L. plantanan L38 This study L. plantarum 1.40 This study L. fermentum F18 IMPC L. reuteri DSM20016 DSM L. reuteri D6 IMPC L. lactis subsp, lactis MOl~63Gasson & Anderson, 1985 B. subtilis 1280 Gasson & Anderson, 1985 E. coil 1106 Gasson & Anderson, 1985 DSM=Deutsche Sammlungyon Mikrooganismen,Braunschweig(FRI~). IMPC=Colleetion of the Istituto di Microbiologla,Piacenza 0~Y). NCFB= National Collectionof Food Bacteria, Reading(UK).
[
Em
= erythromycin.
VECTOR PLASMID CONSTRUCTION AND CLONING IN LACTOBACILLUS Detection of single-stranded plasmid DNA and copy number determination The detection of single stranded DNA (ssDNA) w~s performed following the technique based on accumala~ion of ssDNA induced by rifampin (Boe et aL, 1989) and the Sl-nuclease digestion as previously described by Te Riele et al. (1986). Plasmid DNA obtained from the L. reuteri cells harbouring the pPSC22 plasmid, grown to a mid-log phase and then incubated for 2 h in the presence of 100 t~g/rni of rifampin was digested with SI nuclease, electrophoresed through an 0.8 % agarose gel and stained with ethidium bromide. The gel was deaaturated, transferred to a nylon filter following standard methods (Maniatis et al., !982) and hybridized with the degoxigenin-dUTP-labelled DNA of pPSC22. The number of copies of pPSC22 plasmid per cell of L. reuteri was determined as previously described by Seheer-Abramowitz et al. (1981). Transformation techniques E. coil competent cell transformation was already described by Maniatis et aL (1982). Techniques for transformation of polyethylene-glycol-treated protoplasts were used as described for Laetobaeillus (Morelli et al., 1987), L. lactis subsp, lactis (Kondo and McKay, 1984) and B. subtilis (Chang and Cohen, 1979). The "Bio Rad Gene" pulser apparatus and the "Bio Rad Pulse Controller" system (Bin Rad Laboratories, USA) were used for electroporation experiments. For the electroporation of E. coli, the protocol described by Dower et al. (1988) was followed. Lactobacillus strains were transformed using the method described by Luchansky et al. (1988) modified as follows: the cells were collected in Iogaritmic phase and washed twice with the electroporation buffer (EB: 0.5 M sucrose, 10 mM K phosphate buffer, pH 7) and resuspended in 1/20 of the initial volume and chilled on ice. Plasmid DNA (1 izg in 2 lal of Tris-EDTA) was added to 200 Izl of cell suspension. The mixture DNA-celIs was transferred into a 0.2-era gap cuvette and subjected to a pulse in the "Gene Pulser" apparatus at 2,500 V, 25 I~F, 200 ft. After electroporation, the cell suspension was diluted with 1 ml of mouse reaginic serum MRS, placed at 37°C for 1 h and plated on MRS agar with l0 ~tg/ml of chloramphenicol or erythromycin. Detection of ~t-amylase expression In order to assay the a-amylase activity in agar plates, the colonies of lactobacilli were replicated on MRS containing 1 070 soluble starch and incubated at 37°C for 48 h. The plates were stained for a-
645
amylase activity with iodine (I2/KI) solution and the size of halos around the colonies reflected the a-amylase activity. The activity of a-amylase in the supernatant of the overnight cultures of Lactobaeillus was detected following the method of Fuwa (1954). RESULTS Selection o f a L.
plantarum repUeon
A 7-kb plasmid present in L. plantarum NCFB 1752 was purified and partially digested with restriction endonucleas¢ TaqI. This D N A was ligated to TaqI-digested D N A of plasmid pOCl03, a derivative of the E. coli vector pAT153 that carried a chloramphenicol (Cm) resistance gene originally derived f r o m staphylococcal plasmid pC194. This gene is known to be functional in a variety of Grampositive bacteria and is present on a TaqI fragment. Ligated D N A was transformed into B. subtilis 1280 by polyethylene-glycol treatment of protoplasts and the selection of Cm-resistant protoplast regenerants. Under these conditions, only hybrid plasmids that combined the replication region o f Lactobaciilus plasmid with the Gram-positive Cm-resistance gene would generate transformants. All of the 45 transformants obtained were analysed for plasmid content and were found to carry small but variably sized molecules. One of the smallest plasmid constructs was retained for further characterization. This construct named pPSC1 was a 4.l-kb plasmid consisting of three TaqI fragments of 2.5, 1.0 and 0.6-kb (fig. 1). It was found that pPSC1 was transformable into protoplasts o f L. reuteri DSM 20016 and into E. coli 1106 and, because of the greater stability that was experienced when using E. coli, further vector construction was done in this host. Plasmid pPSC1 was digested to completion with restriction endonuclease TaqI, religated and transformed into E. coil 1106. Analysis of the transformants obtained revealed that, in all cases, the simplest construct recovered was a 4 . l - k b plasmid consisting of the same 3 TaqI fragments that make up pPSC1. This suggested that the minimal D N A for replication was present in pPSCI.
P.S. COCCONCELLI E T AL.
646
T~I~.~Bcll Tacll
~ m r
cm r
T~ -I LH~pal
'
~l pPSCIO
1"aqlHpal
Hpal
pPSC15
T~t
Fig. 1. Physical maps of pPSC plasmids. Symbols and abbreviations: dotted areas represent the L. plantarum plasmid replication functions, solid axeas axe derived from pE194 and open areas from plasmid pOCl03 ; em r= erythromycin resistance; cm r = chloramphenicol resistance.
C o n s t r u c t i o n o f a L. plantarum v e c t o r
In order to provide a useful vector for lactobacilli, a second antibiotic resistance gene was introduced into plasmid pPSC1. The staphylococcal e r y t h r o m y c i n (Em) resistance gene present o n p l a s m i d pE194 is e n c o d e d by a
1.4-kb TaqI f r a g m e n t ( H o r i n o u c h i a n d Weisb l u m , 1982a). P l a s m i d pE194 D N A was purified f r o m B. subtilis a n d digested first with restriction endonuclease M b o I I to disrupt t h e additional u n w a n t e d TaqI f r a g m e n t s that were also present a n d t h e n with restriction e n d o n u c l e a s e TaqI. T h i s D N A was ligated with p l a s m i d
VECTOR PLASMID CONSTRUCTION AND CLONING IN LACTOBACILLUS pPSCI DNA that had been partially digested with restriction endonuclease i'aq! such that only one of the available TaqI recognition sites was cleaved. The ligated DNA was transformed into E. coil 1106 with selection for both Cm and Em resistances. Transformants were obtained that carried a 5.5-kb plasmid consisting of the three TaqI fragments from pPSCI and the 1.4-kb fragment that encoded the Em-resistance gene. One of the plasmids isolated was named pPSC20 and used for further work.
Transformation with pPSC20 A number of additional species of bacteria have been transformed with vector pPSC20. The ability of the vector to transform B. subtilis and E. coil was established during the construction of the vector. The plasmid-free L. lactis subsp. lactis strain MG1363 was transformed with pPSC20 using polyethylene-glycol-treated protoplasts at a frequency of 500 transformants per p.g of DNA. The electrotransformation technique was successfully used for the transformat i o n o f L. r e u t e r i D S M 2 0 0 1 6 a n d D6, L. p l a n t a r u m L38, L40 a n d N C F B l 1 9 3 , L. acidophilus AMP1, L. f e r m e n t u m F18 and L. helveticus $36-2 with pPSC20 plasmid. The frequencies of transformation observed ranged from 102 to 105 transformants/l~g of pPSC20 DNA.
Replication function of pPSC20 and construction of other vectors During the construction of vector pPSC20, it was clearly established that the Cm-resistance gene was encoded by a 2.5-kb TaqI fragment. This observation implied that during the construction of p P S C I using B. subtilis as host, a complex DNA rearmngment occurred, since the source of Cm-resistance gene is known to be a 1.33-kb TaqI fragment originating from plasmid pOCl03. Rearrangment of DNA was shown to occur as a direct consequence of ssDNA plas-
647
mid replication (Gruss and Ehrlich, 1989) and has frequently been encountered during previous work with lactic acid bacteria DNA. In order to create the simplest vector plasmids possible, two additional derivatives of pPSCI and pPSC20 were made using E. coli as the cloning host. In pPSCI 1 and pPSC22, the enlarged 2.5-kb fragment that was formed during the construction of pPSCI has been replaced by the 1.33-kb TaqI fragment of pOCl03 that encodes the Cm-resistance gene. These plasmid vectors behave in an identical way to the original vector constructs pPSC1 and pPSC20. Plasmid p P S C I 0 of 3-kb, which encodes only an erythromycin-resistance gene, was made by the removal of the 2.5-kb TaqI fragment from pPSC20 by TaqI digestion and religation. The structure and the derivation of these various vectors are illustrated in figure 1. The minimal Lactobacillus plasmid DNA that is required for vector replication has been established as the two Taql fragments of 0.6 and l-kb that are present in all the constructs. During the derivation of pPSC 10 from pPSC20, some transformants, resistant only to Em, were found to carry p P S C I 0 and a smaller molecule of 2-kb. The smaller plasmid present in these transformants, named pPSCI5, was demonstrated to be composed of the 1.4-kb Taql fragment carrying the Em-resistance gene together with the 0.6-kb fragment from Lactobacillus plasmid replication origin. Transformation of E. coil and L. reuteri to Em resistance with pPSCI5 D N A proved to be unsuccessful, but in similar experiments using E. coli and L. reuteri already carrying the Cm-resistance plasmid pPSC1, Em-resistant transformants were readily obtained and were shown to carry two plasmids equivalent to pPSC 15 and pPSC1. These observations suggest that the origin of D N A replication is encoded by the 0.6-kb TaqI fragment but the replication depends on a transacting function encoded by the l-kb TaqI fragment present in all the constructs except pPSCI5. The number of copies of pPSC22 in L. reuteri, calculated with the method of ScheerAbramowitz et al. (1981), was 60 copies per cell.
P.S. COCCONCELLI ET AL.
648
Single-strand intermediate replication of pPSC plasmids Cell lysates obtained from L. reuteri DSM20016 harbouring pPSC22 and grown in the presence of 100 ~g/ml of rifampin, either treated or not with SI nuclease, were hybridized with the labelled 0.6 TaqI fragment of pPSC22. Figure 2 (lane A) shows the supercoiled and circuiar plasmid monomers as well as the fast migrating single-stranded DNA band. This band was absent in the DNA preparation digested with nuclease SI (lane B), confirming its single-
stranded nature. The accumulation of ssDNA in L. reuteri is rifampin-dependent and suggests the role of RNA polymerase in the conversion of ssDNA to dsDNA as reported for several ssDNA plasmids isolated from Gram-positive bacteria (Gruss and Ehrlich, 1989).
Homologies of pPSC origin of replication with other ssDNA plasmids Hybridization studies, shown in figure 3, demonstrated the presence of homology between the origin of replication of pPSC plasmids and the replication region of pE194 and with the replication function of pSH71 present on the vector pCKI7 (Gasson and Anderson, 1985). No homology was observed with the origin of replication of pC194.
Cloning and expression of B. stearothermophiius u-amylase gene in various Lactobacillus species
~D
A
~t~
-
CCC
B
Fig. 2. Detection of single-stranded pPSC22 DNA. Agarose gel electrophoresis of lysates prepared from L. reuteri DSM 20016 containing pPSC22 grown in presenceof rifampin. Sampleswerenot treated (A) or were treated (B) with nuclease$1 and hybridizedto the pPSC22 probe. CCC and SS refer to the position of supercoiled monomers and single-stranded forms of pPSC22.
The B. stearothermophilus u-amylase gene was located on the pAMY8, a PvuII deletion derivative of pAMY7 (Thudt et al., 1985). The pAMY8 plasmid was partially digested with BamHI and ligated with pPSC22 linearized with BclI and treated with bacterial alkaline phosphatase. The resulting plasmid D N A was transformed in L. reuteri using the electroporation technique. The transformants, selected on MRS without glucose and containing starch as sole carbon source and 10 ~g/ml of Cm were found to carry a 8.3-kb plasmid denominated pPSA10 (fig. 4). Expression of u-amylase gene was observed in L. reuteri DSM20016 harbouring plasmid pPSA10 in an iodine test. The recombinant plasmid pPSAI0 was electrotransformed in L. plantarum LT, L38 and L40 strains, in L. acidophilus AMP 1 and in L. helveticus $36-2 and NP3 and expression of the u-amylase gene was detected in all the Lactobacillus transformants (fig. 5). The m-amylase activities of L. reuteri DSM20016 and L. plantarum L38 and L40 strains, determined in MRS broth during early stationary growth phase following the method of
VECTOR PLASMID CONSTRUCTION AND CLONING IN LACTOBACILLUS
A
649
B
\
&
m
13
34
12
34
Fig. 3. Southern hybridizationof 0.6-kb Taql fragment of pPSC1, carrying the origin of replication, with the replication region of pE194 and pSH71. (A) Lanes 1 and 2:pPSC22 and 7-kb plasmia DNA of L. plantarum NCFBI752 respectively; lane 3 and 4: hybridization with the labelled 0.6-kb Taql fragment of pPSCI. (B) Lane 1: TaqI digestedpE194; lane 2:pCK17 digestedwith restriction endonueleasesBglII and EcoRI; lane 3 and 4: hybridizationof the labelled0.6-kb Taql of pPSCI to each of the DNA preparationsin lane 1 and 2. The origin of replicationof pPSC plasmidshybridizedwith the 1.3-kb fragment of pE194 and with the 3-kb EcoRI-Bglll fragment of pCKI7 harbouring the replication function of these two plasmids.
Fuwa (1954), were respectively 1,500 and 1,850 units par liter. Since both orientations o f the aamylase gene gave rise to comparable amounts of enzyme activity (data not shown) the transcription probably starts from the ct-arnylase promoter.
ing the L. reuteri DSM20016 for about 600 generations without starch and Cm-selective pressure, 95 % of the cell population had retained the ability to hydrolyse starch and the Cm resistance and the plasmid profile analysis demonstrated the presence o f pPSAI0.
Stability of pPSAIO in lactobacilli DISCUSSION Plasmid pPSAIO exhibited high structural and segregational stability in L. reuteri and in the other species o f lactobacilli. After cultivat-
In this study, we report the identification and the preliminary characterization o f the minimal
P.S. COCCONCELLI
650
bcll
ET AL.
hpal
smal psll
sail
pPSA10 8.3 kb pvul
hlndlll Fig. 4. Restriction map of the plasmid pPSAI0 carrying the ~-amylase gene of B. stearothermophilus.
Fig. 5. or-Amylaseexpression in Lactobacillus. The MRS-starch agar plates were inoculated with (A) L. acidophilus AMP1 (pPSAI0), (B) L. reuteri DSM20016 (pPSAI0), (C) L. plantarum L40 (pPSAI0), (D) L. helveticus $36-2 (pPSAI0), (E) L. helveticus NP3 (pPSAI0). The plates were incubated in anaerobic conditions for 48 h and then stained with iodine solution (12/KI). The halos around the colonies reflect the ~-amylase production.
VECTOR PLASMID CONSTRUCTION AND CLONING IN LACTOBACILLUS
replicon of the cryptic plasmid of L. plantarum NCFB1752 and its use for the construction of pPSC20 and pPSC22 plasmid vectors and for the cloning of B. stearothermophilus a-amylase gene in several Lactobacillus strains. The replicon of pPSC plasmids was demonstrated to be functional in a variety of Lactobacillus species, in other Gram-positive bacteria, such as L. lactis subsp, lactis and B. subtilis, and in E. coli. This feature is particularly useful for employing pPSC22 as an E. coli-Lactobacillus shuttle vector. The promiscuous replication property of these vectors is typical of others derived from cryptic plasmids present in lactococci (Kok et al., 1984; De Vos, 1987; Gasson and Anderson, 1985). Complementation studies suggest the separation of the replication region of pPSC plasmids into an origin-containing domain and a region functional in trans as observed in other replicons deriving from lactic acid bacteria (De Vos, 1987; Hayes et aL, 1990). The classification of replicon of pPSC vectors to the family of highly interrelated singlestranded plasmids of Gram-positive bacteria (Gruss and Erhlich, 1989) was supported by: (1) the presence of the ssDNA intermediate of replication, (2) the D N A homologies with the replication regions of the staphylococcal plasmid pE 194 and the lactococcal plasmid pSH71 and (3) the structural instability and D N A rearrangements observed during the construction of pPSC1. In order to evaluate the efficiency of the cloning system, plasmid pPSC22 was used for cloning and expression of the a-amylase gene of B. stearothermophilus in L. /~',~ntarum, in L. reuteri, in L. acidophilus and ~ ~elveticus. The a-amylase gene confers upon lactobacilli improved fermentative properties and, due to the simplified system of detection, may be used as a reporter gene in monitoring this group of bacteria. The constructed plasmid pPSA10, carrying the a-amylase gene, is characterized by high segregational stability not observed in other recombinant plasmids used for cloning in
651
L. plantarum (Scheirlink et al., 1989; Bates et aL, 1989).
The construction of stable cloning vectors has proven to be valuable in cloning heterologous genes in several Lactobacillus species, including the thermophilic species L. helveticus used in dairy production, L. plantarum and the members of intestinal microflora L. acidophilus and L. reuteri. In addition, the development of this stable vector system may provide a versatile alternative to the chromosomal integration in the genetic engineering of lactobacilli.
Plasmides ~t simple brin d'ADN vecteurs de construction et de clonage de I'a-amylase de Bacillus stearothermophilus chez Lactobacillus
Des plasmides vecteurs ont &6 construits par liaison des g6nes de la r6sistance au chloramph~nicol et l'6rythromycine/l I'ADN d'un plasmide cryptique de Lactobacillus p!antarum restreim par TaqI. La r6gion minimale de I'ADN plasmidique de Lactobacillus qui est n6cessaire pour la r6plication de I'ADN a ~t6 d6finie, et un syst~meinterm6diaire d'ADN simple brin a 6t6 exantin6. Nous avons trouv6 des homologies avec les points de d~part de la replication des plasmides de bact~ries Gram-positives se repliquant via un m~eanisme cireulaire. Nous montrons que |es vecteurs construits pPSC20 et pPSC22 sont transformables dans L. plantarum, Lactobacillus acidophilus, Lactobacillus renteri, Lactobacillus fermentum, Lactobacillus helveticum, Lactococcu lactis subsp, lactis, Bacillus subtilis et Escherichia coli. A l'aide du !flasmide pPSC22, le g/me de l'ctamylase de Bacillus stearotkermophilus a 6t~ clon6 et exprim6 chez plusieurs esp~ces de Lactobacillus. Mots-cl~s: R~plication, Lactobacillus, ADN, Plasmide, Amylase; ADN monocat6naire, Clonage, Vecteurs, B. stearothermophilus.
References
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