Characterization of a gram-positive broad-host-range plasmid isolated from Lactobacillus hilgardii

PLASMID

21, g-20

(1989)

Characterization

of a Gram-Positive Broad-Host-Range Isolated from Lactobacillus hilgardii

Plasmid

KATTYJOSSON,' TREES SCHEIRLINCK,F~ANKMICHIELS,CHRISTPLATTEEUW, PATRICK STANSSENS,HENKJOOS,PATRICK DHAESE,* MARK ZABEAQ~ANDJACQUESMAHILLON Plant Genetic Systems, J. Plateaustraat 22, B-9000 State University of Ghent, Ledeganckstraat and fHELIX, Onajhankel~kheidslaan, Received

September

Ghent, Belgium; *Laboratorium 35, B-9000 Ghent, Belgium; 38, B-9000 Ghent, Belgium

22, 1988; revised

November

Genetica,

28, 1988

Two plasmids, pLABlOO0 and pLAB2000 (3.3 and 9.1 kb, respectively), have been isolated from a grass silage strain of LuctobaciIIus hilgardii. Both plasmids were cloned in Escherichia cofi and characterized through restriction mapping. A 1.6-kb XbaI-Sac1 fragment of pLABlOO0 appeared to be sufficient for autonomous replication in Lactobacillus plantarum and in Bacillus subtihs. Different shuttle vectors for E. coli and gram-positive bacteria were developed using the pLABloo0 plasmid. These could stably be maintained in LuctobaciIlus, Enterococcus, and Bacillus under selective conditions. Plasmids sharing DNA homologies with pLABlOO0 have been observed 0 1989 Academic Press. Inc. in different StraitIS of the related Spe&S I%. p/antUI’Um.

For centuries, lactobacilli have been essential in food and feed fermentation. In the dairy industry the most important species are Lactobacillus bulgaricus, L. helveticus, and L. casei, whereas in plant fermentation, L. plantarum and L. brevis play a prominent role (reviewed in Chassy, 1985). In addition, some lactobacilli (e.g., L. acidophilus) were found to be of prime interest as probiotics (Fernandes et al., 1987). Recently, new insights into Lactobacillus biochemistry coupled with the opportunity for genetic manipulation offered exciting prospects for strain improvement. Different genetic transfer systems have been established in lactic acid bacteria. Conjugative broad-host-range plasmids such as pVA797 can mobilize smaller vector plasmids from Streptococcus sanguis to different Lactobacillus strains (Shrago and Dobrogosz, 1988). Protoplast transformation (Lin and Savage, 1986), protoplast fusion (Iwata et al., 1986) ’ To whom

all correspondence

should

and electroporation (Chassy and Flickinger, 1987; Aukrust and Nes, 1988) have been applied successfully to introduce plasmid DNA in several Lactobacillus strains. Natural resident plasmids have been shown to be abundant in Lactobacillus and some have already been isolated from L. acidophilus (Damiani et al., 1987), L. plantarum (von Husby and Nes, 1986), and L. casei (LeeWickner and Chassy, 1985). Most of these plasmids are small and very little is known about their mode or replication, stability, or function(s), with the exception of some metabolic traits (Shimizu-Kadota, 1987) or drug resistances (Vescovo et al., 1982). In the present study we show that a strain of L. hilgardii, a lactic acid bacterium generally associated with alcoholic fermentation (Kandler and Weiss, 1986), harbors several cryptic plasmids. Two plasmids were characterized, and one of these (3.3 kb) appeared to replicate in a variety of gram-positive species. We describe the construction of gram-positive cloning vectors based on this replicon.

be addressed.

9

0147-619X/89 Copyrisht

0

1989

$3.00 by Academic

Press,

Inc.

10

JOSSON ET AL.

MATERIALS

AND METHODS

TABLE I

STRAINSUSEDINTHISSTUDY Bacterial strains and plasmids. Bacterial strains used in this study are listed in Table 1. Organism Strain source” Strains isolated from natural grass silages in Belgium were identified using standard mi- Escherichia coli K514 Zabeau and crobiological techniques (Kandler and Weiss, Stanley, 1986) and their identifications confirmed (1982) through the biochemical test API CHSOL (API Bacillus subtilis lA42 BGSC Systems S. A., France). Carbohydrate metabEnterococcus faecalis OGlX Ike et al. olism of L. hilgardii 67 corresponded to that (1983) DSM 4234 DSM of the L. hilgardii reference strain as described Lactobacillus plantarum 50 by Kandler and Weiss ( 1986). Gas formation Lactobaciks DSM 4229 DSM was detected upon fermentation of maltose plantarum 80 and gluconate. Detailed analysis revealed that Lactobacillus casei DSM 20020 DSM L-arabinose and D-xylose could be degraded Lactobacillus DSM 4233 DSM hilgardii 67 by this specific strain of L. hilgardii, but not D-arabinose, lactose, melezitose, sucrose, ga’ BGSC, Bacillus Genetic Stock Center (Columbus, lactose, and L-xylose. OH); ATCC, American Type Culture Collection (RockPlasmids pVA838 (Macrina et al., 1982), ville, MD); DSM, Deutsche Sammlung ftir MikroorganpLK58 (Botterman and Zabeau, 1987), and ismen (Gdttingen, Germany). L. plantarum 50, L. planpBR322 (Bolivar et al., 1977) used in the tarum 80, L. casei DSM 20020, and L. hilgardii 61 are cloning experiments have already been de- described for the first time in this manuscript. scribed. The pPGV 1LH plasmid, used as source for a chloramphenicol resistance gene cillus and Enterococcus. In electroporation under the control of a Lactobacillus promotor, experiments (see below), MLS selection for is described elsewhere (Platteeuw et al., Lactobacillus and Enterococcus was made on manuscript in preparation). pC 194 and plates containing both Erm and Lint at conpGKV2 have been described by Horinouchi centrations of 10 and 100 &ml, respectively. and Weisblum ( 1982) and van der Vossen et Plasmid and total DNA preparations. Total al. (1985), respectively. DNA was prepared from Lactobacillus acMedia and growth conditions. Lactobacillus cording to a protocol combining previously strains were grown in MRS (Oxoid), Enterodescribed methods based on mutanolysin-incoccus in Ml7 (Difco) supplemented with duced lysis (Klaenhammer, 1984). Cells were 0.5% glucose, and Bacillus and Escherichia grown overnight in MRS medium supplecoli in Luria broth (Miller, 1972). Cultures mented with 20 mM DL-threonine (Sigma) were incubated at 37”C, with rotary shaking (Chassy and Giuffrida, 1980). After centrifbaeration for Bacillus and E. coli strains. Cm2 gation, 200 mg of cells were washed twice with and Erm were used at concentrations of 20 20 ml of 10 IBM Tris-HCl, pH 7, and resusand 200 &ml, respectively, for E. coli, and pended in 4 ml of 10 I’IIM Tris-HCI, pH 7, at 20pg/ml for Bacillus; Cm was used at a 12% polyethylene glycol (PEG) 6000, and concentration of 10 &ml for both Lactobamutanolysin, 8.5 U/ml (Sigma). After 1 h incubation at 6O”C, proteinase K (Sigma) was added to a final concentration of 0.1 mg/ml 2 Abbreviations used: Amp, ampicillin; Erm, etythromycin; Cm, chloramphenicol; cat86, chloramphenicol and the sample was incubated for another hour acetyltransferase 86; Lint, lincomycin; CCC, covalently at 37°C on a rotary shaker. Spheroplasts were closed circular; GC, open circular, L, linear; PEG, polyrecovered and lysed in 4 ml of a lysis solution ethylene glycol; MLS, Macrolide Lincosamide and Strep(10 mM EDTA, 20 I’tIM Tris-HCl, pH 8, and togramin; CPU, colony forming unit; EtBr, ethidium bro3% SDS) for 30 min at room temperature. The mide; SDS, sodium dodecyl sulfate.

STUDY OF Lactobacillus hilgardii

DNA was then first extracted with phenol (equilibrated with 100 mM Tris-HCl, pH 8) and then by chloroform/isoamyl alcohol (24/ 1 v/v) and finally precipitated by adding isopropanol(50%, v/v) and sodium perchlorate to a final concentration of 200 mM. The DNA was recovered in 500 ~1 of TE buffer (10 mM Tris-HCl, pH 8,0.1 mM EDTA). This protocol has been successfully applied for DNA preparation from L. hilgardii, L. plantarum (this work), L. casei, and L. xylosus (unpublished observation). B. subtilis plasmid DNA was prepared using the method described by Goldfarb et al. (1982). Plasmid DNA preparations from Enterococcus and Lactobacillus were made according to the method described by Anderson and McKay (1983). Identification ofplasmid topoisomers. Plasmid topoisomers were identified by the method of Hinterman et al. (198 1). Between the two electrophoresis steps (made in perpendicular directions) the DNA was uv-irradiated for 8 min on a Fotodyne transilluminator (254 nm). General cloning techiques. Digestions with restriction enzymes were carried out as recommended by the supplier (Biolabs). Plasmid recovery from 1% low melting temperature agarosc, colony and Southern hybridization, and other general cloning techniques were carried out as described by Maniatis et al. (1982). Transformation of B. subtilis competent cells was made according to the protocol of Saunders et al. (1984). Transformation of Lactobacillus and Enterococcus by electroporation. Lactic acid bacteria (Lactobacillus spp. and Enterococcus faecalis) were transformed by electroporation, using a Gene Pulser apparatus (Bio-Rad Laboratories, Richmond, CA). Originally, the 4mm-width plastic cuvettes (maximum capacity of 800 ~1) were used, but they were recently replaced by the new 2-mm-width cuvette (40400 ~1capacity) coupled to a parallel resistance selector (Bio-Rad pulse controller) set at 400 and 200 Ohm for Lactobacillus and Enterococcus, respectively. Electroporation condi-

PLASMID

11

tions have been optimized using L. plantarum 80 as reference strain together with pLAB 1000 derivatives, pGKV2 or pC194 plasmids. Although several other parameters remain to be tested, the current protocol used in this study could be described as follows: A l/50 dilution of an overnight culture was grown in liquid media at 37’C to an ODm of 0.5-1.0. For Enterococcus, the medium was supplemented with 20 I’nM DL-threonine. Cells were then harvested by centrifugation, washed twice with demineralized, sterile water at room temperature, and resuspended in 30% PEG 1000 to a final volume of 10 times the cell pellet. These bacterial suspensions, containing between 10” and 10” CFU/ml, were stored at -7O’C or used immediately. Plasmid DNA (0.5 pg) was added to 0.1 ml of cells and this suspension was subjected to a single electric pulse (25 PF at 8500 V/cm for L. plantarum 50, 80, and L. casei DSM 20020; 12,500 V/ cm for Enterococcus). The electroporated cells were kept on ice for 30 min, then diluted IO-fold in liquid medium, and incubated for 2 h to allow expression of the antibiotic resistance. Cells were subsequently plated on medium containing the appropriate antibiotics. Survival ratios were estimated from serial dilutions plated on media without antibiotic. During these experiments, the following observations have been made with L. plantarum 80: aeration ( 150- 180 rpm) during exponential growth increased the electroporation efficiency by a factor of 4, although the growth rate remained the same. Washing the cells with cold water reduced the electroporation efficiency five to six times. Although various salts and buffers failed to raise the efficiency, addition of PEG 1000 increased the efficiency lo- to 50-fold. This could be due to a volume exclusion effect, increasing the effective DNA and cell concentration, or to the effect of PEG on cell wall-DNA interactions (known in Bacillus protoplast transformation or cell fusion). PEG with molecular weight higher than 1000 gave lower efficiencies. The use of PEG also allowed cells to be frozen at -70°C. Finally, a direct correlation has been observed between

JOSSON ET AL.

+ E2 FIG. 1. Identification of L. hilgurdii plasmids. (A) Total DNA from L. hilgardii 67 was separated on a 0.7% agarose gel containing 0.5 fig/ml EtBr. The DNA bands formed spots due to the round shape of the slot. (B) After uv irradiation, the gel was subjected to a second electrophoresis (E2), perpendicular to the first one (El). The different plasmid conformations, the original open circular @Cl), the newly formed open circle (OC2), and the supercoil (CCC) of both plasmids, pLABlOO0 and pLAB2000, formed rectangular triangles after the second electrophoresis. The straight line corresponds to the migration of linear DNA molecules (due to degradation of chromosomal DNA).

increasing cell concentration ( lo9 to 10” CFU/ ml) and electroporation efficiency. Using the optimal conditions described above, electroporation efficiencies varying between lo4 and lo6 transformants/~g DNA (usually corresponding to 30% survival) were routinely obtained. It should be noted that, when electroporated with a Erm-resistance plasmid, the Lactobacillus strains were selected on both Erm (10 &ml) and Lint ( 100 &ml). Indeed, selection on Erm leads to the appearance of a background of Erm resistant colonies at frequencies of about 1O-‘- 1Oe8. As previously reported by Youngman (1987) for B. subtilis, combination of Erm with a similar antibiotic (lincomycin) turned out to fully eliminate this residual growth. RESULTS

L. hilgardii

Resident Plasmids

Total L. hilgardii 67 DNA showed a complex pattern of DNA bands in agarose electrophoresis. By the method of Hinterman et al.

(198 l), some of the bands could be assigned to open circular and supercoil forms of two plasmids designated pLAB1000 and pLAB2000 (Fig. 1). Larger plasmids hidden by chromosomal DNA or plasmids with low copy numbers might also be present but this issue was not further investigated. Gel-purified pLAB 1000 plasmid was digested with several restriction enzymes and analyzed on an agarose gel. For pLAB2000, samples of total DNA from L. hilgardii 67 were digested with 32 different enzymes, and the pLAB2OOOderived bands were subsequently visualized through Southern hybridization using this plasmid as probe. Through these experiments the size of pLABlOO0 and pLAB2000 was estimated to be 3.3 and 9.1 kb, respectively. Since pLABlOO0 and pLAB2000 contained a unique restriction site for XbaI, XbaI-linearized molecules of both plasmids were cloned into the XbaI site of pUCl8 (Norrander et al., 1983) giving rise to the recombinant plasmids pLABllO1 and pLAB2 10 1, respectively (see below). These E. coli chimaeric plasmids were used to achieve detailed restriction mapping of pLAB 1000 and pLAB2000, as shown in Fig. 2. Distribution of pL4BlOOO-Related Sequences among Diflerent GramPositive Bacteria In order to allow further use of pLABlOO0 as a possible Lactobacillus cloning vehicle, it was necessary to investigate the distribution of pLAB 1000-related DNA sequences among lactic acid bacteria or other potential grampositive hosts. Therefore, a collection of 72 different gram-positive bacteria were colonyhybridized with a pLAB 1000 32P-labeled DNA probe. As shown in Table 2, three strains of L. plantarum (L. plantarum 30, 183, 195) turned out to harbor pLABlOOO-related sequences, and no hybridization was observed with the strains belonging to other species. These DNA sequence relationships were further analyzed through Southern hybridizations using total DNA isolated from L. hilgardii 67 and L. plantarum strains 30, 183,

13

STUDY OF Lactobacillus hilgardii PLASMID

SQ

pLABlOO0 33kb

XMNI

loo0 -zYJ

6000

/

FIG. 2. Characterization of pLAB1000 and pLAB2000. Restriction maps of both L. hilgardii 67 plasmids were deduced from single and double digests. Restriction sites absent for pLABlOO0 are BarnHI, Bg/I, Bg&I, EcoRI, PstI, PvuI, PvuII, and Sa& and for pLAB2000, BglI, Kpnl, Pstl, AluI, PvuII, and SalI. The hatched region in pLAB 1000 corresponds to the minimal replication region as determined by subcloning experiments described in Fig. 5.

and 195 (Fig. 3). For L. hilgardii 67, pLABlOO0 showed no hybridization with pLAB2000, or with chromosomal DNA (Fig. 3A). This result indicated that pLAB 1000 and pLAB2000 not only showed different physical restriction maps (Fig. 2) but that they do not share common DNA sequences. Concerning the L. plantarum strains (Fig. 3B), pLAB1 000-related sequences are located exclusively on plasmid molecules, with sizes similar to that of pLAB1000.

Localization of the Minimal Region of pLABlOO0

Replication

To allow selection upon reintroduction in different Lactobacillus strains, the chimaeric plasmid pLABllO1 (pLAB1000 + pUC18) (Fig. 4) was provided with the gram-positive selectable gene cat86 of B. pumilis (Williams et al., 198 1). Preliminary experiments indeed revealed that most of the strains from L. plantarum, L. casei, or L. hilgardii were naturally

TABLE 2 SCREENING

FOR pLABlOOO-RELATED

SEQUENCES

AMONG

DIFFERENT

GRAM-POSITIVE

BACTERIA Number

Bacterial strain

Origin

Number of strains tested

L. plantarum L. casei ssp. casei Pediococcus pentosaceus B. thuringiensis B. cereus

NSIb NSI NSI IPC Bed

53 5 2 11 1

of strains

sharing DNA homologies” 3 0 0 0 0

’ These numbers correspond to the number of isolates giving a positive reaction in a colony-hybridization experiment using pLAB 1000 as DNA probe. b NSI, Natural grass silage isolates. ‘The 11 B. thuringiensis strains were provided by Prof. H. de Batjac, Institut Pasteur, Paris, and belong to four different gagellar serotypes, namely, HI (6 strains), H3a3b (2), H7a (2), and H14 (1). d The B. cereus strain BCT4 was received from Dr Bruce Carlton, Ecogen, Langhome, USA.

14

JOSSON ET AL.

pLAB 1105 and pLAB 1108, respectively. After electroporation in L. plantarum 80 and selection on Cm, pLAB 1105 was shown to replicate in this organism, whereas no transformants were obtained in the case of pLABllO8 (Fig. 5B). This result indicated that the pLABlOO0 chr. DNApLABZOOOreplication origin was located within the 2.4kb HindIII-XbaI fragment. pLABlOOOTo further delineate the pLABlOO0 repli- ccc cation region, a new replicon-probe vector, pGI4010, was constructed (Fig. 5A). The pAMj31 Erm resistance gene was cloned by inserting the HindIII-ClaI fragment of pVA838 (Macrina et al., 1982) into the HindIII-ClaI restriction sites of pBR322 (Boet al. 1977), giving rise to pERM2. An livar FIG. 3. Identification of pLABlOOO-related sequences EcoRI-DdeI fragment, containing the ErmR in three L. plantarum strains. Total DNA from L. hilgardii 67 (A) and strains 30, 183, and 195 from L. plantarum gene, was made blunt and cloned in the (B) have been separated through agarose gel electrophoresis EcoRV site of the pLK58 vector (Botterman (I), transferred to nitrocellulose membranes and hybridized and Zabeau, 1987). From the resulting pGI40 1 to “*P-labeled pLABlOO0 DNA (II). Note that for L. hilvector, the P,-promotor was removed through gardii 67 (lane A-I) pLAB2000, that migrates between the a SalI-StuI cleavage and fill-in reaction, genlinear chromosomal DNA and the OC form of pLABlOO0 (Fig. 1), cannot be easily observed in this preparation. chr. erating pGI4010 (Fig. 5A). DNA, linear chromosomal DNA, OC, L, and CCC, open Various parts of the large pLAB 1000 XbaIcircular, linear, and covalently closed circular plasmid Hind111 fragment were subcloned in the poDNA, respectively. lylinker region of pG140 10 (Fig. 5B). A XbaISac1 pLAB 1000 fragment was cloned between resistant to aminoglycoside antibiotics (up to the XbaI-BarnHI sites of the cloning vector, more than 100 &ml) but sensitive to Cm and after filling in both Sac1 and BamHI sites by a Klenow reaction, yielding pLAB1301. Erm, at concentrations higher than 5 &ml. Therefore, an EcoRI-XhoII restriction fmg- pLAB 1302 was obtained by cloning the XbaIment from pPGV lLH, containing the cat86 ClaI fragment within the same pGI40 10 sites, coding region preceded by a L. hilgardii pro- ClaI and BamHI sites being previously filled motor sequence (Platteeuw et al., manuscript in. In the recombinant plasmid pLAB1303, in preparation), was inserted between the the blunt EagI-Sac1 fragment has been cloned sites of EcoRI-BamHI restriction sites of pLAB 110 1. within the filled-in XbaI-BarnHI The resulting plasmid, named pLAB 1102 (Fig. pGI40 10. All these pGI40 1O-derivatives were 4), conferred Cm resistance in E. cob. Upon then electroporated in L. plantarum 80. As shown in Fig. 5B, the 1.6-kb XbaI-Sac1 electroporation of pLABllO2 into L. plantarum 80, Cm-resistant transformants were fragment (pLAB 130 1) was shown to replicate shown to contain plasmids whose size and re- in L. plantarum 80, whereas deletions of 0.6 striction patterns were identical to those of kb at the XbaI site (pLAB 1303) or 0.2 kb at the Sac1 site (pLAB1302) abolished the reppLAB 1102. lication ability of the pLABlOO0 derivatives. In a second step, the two HindIII-XbaI subfragments of pLAB 1000 (Fig. 2) were in- All replicating constructs could be transformed serted in the HindIII-XbaI sites of the pUC 18 with the same efficiency and showed similar copy number in L. plantawm 80. These results polylinker to give the recombinant plasmids pLABllO4 and pLAB 1107 (Fig. 4). In these, indicated that the functions required for the replication of the 3.3-kb pLAB 1000 plasmid the cat86 gene was inserted, generating

STUDY OF Lactobacillus hiigardii

PLASMID

EC& / BamHl

/

FIG. 4. Construction of shuttle vectors derived from pLABlOO0 and pLAB2000. A detailed analysis of the constructions is described in the text. Hatched box, pLABlOO0 and pLAB2000 plasmid DNA; solid tine, pIJC18 vector; CM and AMP, chloramphenicol and ampicillin resistance genes; P, L. hilgardii promotor controlling the expression of the CM gene; ORI, gram-negative replication region.

15

0 I

0.6 I

IA I

1.6 ,

2.4 I

3.3 I

Kb

FIG. 5. Identification of the minimal replication region of pLABlOO0. (A) Constructions of the pGI401derivatives are described in the text. Resistance genes [ampicillin (AMP), erythromycin (ERM)] are indicated by open regions and vector fragments by solid lines. The arrow shows the polylinker into which the different pLABlOO0 restriction fragments (B) have been inserted. (B) Physical map of pLABIoo0 (shown as a unique XbuI fragment) together with subcloned fragments tested for replication in L. plantarum 80 via the electroporation-transformation method. The left column corresponds to the recombinant plasmids bearing the different inserts and the right column indicates their replication ability in Lmtobacillus. 16

STUDY OF Lactobacillus hilgardii

are located within a 1.6-kb (or slightly smaller) fragment.

PLASMID

17

other bearing the Amp resistance gene and the gram-negative origin of pLK58. After Klenow polymerase treatment, both hagments were ligated at low DNA concentrations so as Host Range of the pLABlOO0 Replicon to favor self-annealing ligations. This DNA, Since pLAB1000, originating from L. hil- together with control plasmids (intact gardii, was shown to replicate in L. plantarum pLABl30 1 DNA, or restricted fragments 80 (see previous section) it was worth inves- without ligation) was transformed in E. coli tigating the possible use of other lactic acid and selection was made on Amp, Erm, or both bacteria or other gram-positive organisms as Amp + Erm. No E. coli colonies were obreplication host for that plasmid. served on the Erm nor on Amp-Erm plates, First, strains L. plantarum (50), L. casei although selection on Amp showed high (DSM20020), and E. faecalis (OGl X) were transformation efficiencies. To verify whether transformed by electroporation with pLA- the pLAB 1000 cassette was actually able to B1102 and pLABl301 (Fig. 5B). In all cases, work as a true replicon, the same ligation was Cm or Erm resistant transformants were also electroporated into L. plantarum 80, using shown to contain plasmid DNA with an aga- Erm-Lint antibiotic selection. Analysis of the rose gel mobility and restriction pattern in- plasmid DNA preparations conhrmed that the distinguishable from that of the starting transformants contained plasmids correplasmid molecules. Moreover, Southern sponding to the expected pLAB lOOO-Erm hybridization experiments confirmed that cassette. Therefore the above results argue that pLAB 1000-derivatives were present as plasmid pLAB 1000 cannot replicate in E. coli. DNA, and that no DNA rearrangement had DISCUSSION occurred with other resident plasmids, nor This paper describes two cryptic plasmids, with the chromosomal DNA (data not shown). designated pLAB 1000 (3.3 kb) and pLAB2000 Second, competent cells from B. subtilis 1A42 were transformed with both pLAB 1102 (9.1 kb), originating from a bacterial silage and pLAB1301 plasmids. Again, Cm or Erm isolate, L. hilgardii 67. Both plasmids were resistant colonies were obtained, but with re- isolated and characterized by restriction analysis. duced growth rate, as compared to control The minimal replication region of plasmids such as PC194 (CmR) or pGKV2 (ErmR). In the absence of antibiotic selection, pLAB 1000 in L. plantarum could be located however, the growth rate of these transforwithin a 1.6-kb XbaI-Sac1 fragment. In several mants was similar to that of the parental strain. other gram-positive replicons such as pSL1 Although the pLAB 1OOO-derivatives were (Shindoh et al., 1987) pFTB14 (Murai et al., hardly seen on a standard B. subtilis plasmid 1987), pE194 (Villafane et al., 1987), pC194 DNA preparation, their presence as CCC (Alonso and Tailor, 1987), and PUB 110 (Maciag et al., 1988), all information necessary for molecules was confirmed through hybridization experiments (data not shown). This low replication is located in fragments of compacopy number of pLAB 1000 in B. subtilis may rable sizes. Typically, these fragments harbor be the cause of (or at least contribute to) the a rep gene, encoding a protein essential for the reduced growth of the recombinant host upon initiation of the replication, and a correantibiotic selection. sponding target site. Sequence analysis of the In order to test whether pLAB 1000 has the minimal replication region of pLAB 1000 will ability to replicate in E. coli, the pLAB 130 1 enable us to define its structural organization plasmid (Fig. 5) was cleaved with EcoRI and and possible DNA or protein sequence simiBamHI. These cleavages generated two “cas- larities with other known replication systems. settes,” one containing the pLAB 1000 replicon After electroporation or transformation, associated with the Erm resistance gene, the pLAB 1000 derivatives were shown to replicate

18

JOSSON ET AL.

not only in different Lactobacillus but also in Enterococcus and Bacillus strains. Plasmid analysis revealed that no apparent rearrangements of the replicons had occurred in these organisms. We noticed that under selective conditions Enterococcus and Lactobacillus transformants had the same growth rate as the parental cells, whereas Bacillus transformants grew slower. The reduced growth rate of the transformed bacilli is apparently not due to a poor expression of the resistance genes (Cm and Erm), since the same genes, on other replicons, allowed normal cell growth on selective media (data not shown). On the other hand, it is more likely that the growth retardation is due to the low copy number observed for the pLAB 1OOO-derivatives in B. subtilis. Similar observation of copy number variability among different bacterial hosts has also been made for another lactic acid bacteria replicon, pSH7 1 (De Vos, 1986). Indeed, this LactoCOCCUSplasmid showed high copy number when introduced into Lactococcus lactis MG 1363, but low copy number in B. subtilis. Some gram-positive replicons such as pC194, pMV158, pSA5700, pSH71, and pWVO1 have been shown to replicate in E. coli (Goze and Ehrlich, 1980; Barany et al., 1982; de1 Solar et al., 1987; De Vos, 1987). Concerning pLAB 1000, attempts to demonstrate autonomous replication in E. coli have been so far unsuccessful. Therefore, we assume that the pLAB 1000 replication region is not functional in E. coli. In the course of our experiments, we found that two other gram-positive vectors, pGKV2 from Lactococcus lactis ssp. cremoris and pC 194 from Staphylococcus aureus, could also replicate and express selectable markers in L. plantarum 80. However, preliminary experiments revealed different segregational stability for these three vectors in L. plantarum 80. After 13 generations in the absence of antibiotic selection, pLAB 1000 derivatives were still present in more than 30% of the L. plantarum 80, whereas both pC194 and pGKV2 were observed in less than 10% of the cells. The maintenance of a cloning vector in a host organism depends on the structural and

segregational stability of this replicon. Among the factors affecting this stability, the presence of another plasmid in the same host might lead to DNA rearrangements (deletion, insertion, fusion) of the cloning vehicle or to incompatibility phenomena. From a collection of 72 gram-positive strains screened for pLAB 1000 homology, three Lactobacillus plantarum strains, all isolated from different sources than the one of L. hilgardii 67, harbored pLAB 1000-related plasmids. These homologous L. plantarum plasmids exhibited small differences in length and in restriction map as compared to pLAB 1000 (Josson et al., manuscript in preparation). Preliminary experiments also indicated that at least part of the homology is located outside the minimal replicon of pLAB 1000. Although L. hilgardii and L. plantarum are considered to belong to two quite different taxonomic groups (Priest and Barbour, 1985), the fact that both organisms contained related plasmid sequences is not surprising. Indeed, plasmids sharing DNA homology in hybridization have previously been described in L. plantarum (Von Husby and Nes, 1986) and L. casei (Lee-Wickner and Chassy, 1985). Moreover, replicons with virtually identical restriction map have been found in different genera such as Staphylococcus, Enterococcus, and Bacillus (Perkins and Youngman, 1983; Projan et al., 1987; Gilmore et al., 1982). Some metabolic functions such as the fermentation of lactose (Shimizu-Kadota, 1987), sucrose, mannose, galactose, glucose, and xylose (Sandine, 1987) are plasmid encoded. In order to determine whether pLABlOO0 harbored metabolic traits, a pLAB 1000~cured L. hilgardii 67 strain was isolated and analyzed for the fermentation of different sugars by the API CHSOL test. Results suggested that there is no correlation between the fermentation of these specific substrates and the presence of pLAB 1000 (K. Josson, unpublished results). Since we did not succeed in curing pLAB2000 from L. hilgardii, no function could be assigned to this plasmid yet. The different shuttle vectors described in this work are presently developed as promotor-

STUDY OF Lactobacillus

probe vectors or expression-secretion vectors for Lactobacillus. These new vectors will enable us to express different heterologous genes in lactic acid bacteria and possibly to improve Lactobacillus strains used in silage or dairy fermentations. Finally, it might be interesting to further analyze the different structural and functional characteristics of pLAB2000 in order to develop compatible cloning vehicles, complementary to those derived from pLAB1000. ACKNOWLEDGMENTS We thank B. Bruyneel (Radar, Belgium) for the isolation of L. hilgardii, F. Leyns for the detailed taxonomic analysis of the different lactobacilli, and J. Decock, C. Opsomer, and R. Verbeke for technical assistance. We are also indebted to G. Simons and W. de Vos for useful discussions and to J. Kok for providing pGKV2. K.J. and C.P. were supported by an IWONL fellowship (Institute for Scientific Research in Industry and Agriculture).

REFERENCES ALONSO, J. C., AND TAILOR, R. H. (1987). Initiation of plasmid pC194 replication and its control in Bacillus subtilis.

Mol.

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