Abstract:
Strains of the Lactobacillus delbrueckii species are identified and distinguished from other species by a DNA probe containing an EcoRI fragment, or subfragment thereof, of the plasmid pY85, which hybridizes specifically to chromosomal DNA of strains of the L. delbrueckii species.

Description:
This application is a continuation of Ser. No. 07/494,139, filed Mar. 14, 1990 now U.S. Pat. No. 5,359,049. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates to a DNA probe for identifying bacterial strains of the Lactobacillus delbrueckii species, a process for producing such a probe and methods for identifying bacterial strains of this species with this DNA probe. 
     Lactobacillus delbrueckii subsp. bulgaricus, delbrueckii and lactis are very important bacteria for the fermentation of food. L.bulgaricus and L.lactis are predominantly used in fermentation of milk products and are therefore found in starter cultures for yoghurt and cheese production, whereas L.delbrueckii is mainly found in vegetable fermentations. Fermentation and maturation of these food products usually result from growth association and interaction of different bacteria, in most cases different Lactobacilli, Lactococci and other bacterial species. As most of these bacterial species have very similar nutritional requirements and grow under similar environmental conditions a clear identification within the Lactobacillus species is sometimes very difficult. So far the classification of these species is very tedious and involves many unreliable criteria like sugar fermentation patterns and acid production. Due to these tests, a differentiation of different species remains difficult, sometimes doubtful and often arbitrary. 
     DNA hybridisation techniques, using specific DNA probes, are a very valuable tool for the identification of bacterial and viral strains and have already found application in clinical diagnostics. Such DNA probes have already been used for the identification of Yersinia enterocolitica (J.Jagow et al., Appl. Environ. Microbiol. 51, 441-443, 1986), Plasmodium falciparum (R. H. Barker et al,. Science 231, 1434-1436, 1986), Salmonella typhi (F. A. Rubin et al., J.Clin.Microbiol. 22, 600-605, 1985), Bacillus subtilis (J.Krauss et al., FEMS Microbiol. Lett. 33, 89-93, 1986), Haemophilus influenzae (F. Malouin et al., J. Clin. Microbiol. 26, 2132-2138, 1988) and other bacterial species, of DNA viruses (J.Brandsma et al., Proc.Natl. Acad. Sci.USA 77, 6851-6855; P.Stolhandske et al., J.Clin.Microbiol. 15, 744-747, 1982; P.Stolhandske et al., J.Med.Virol. 12, 213-218,1985), as well as RNA viruses (J.Flores et al., Lanceti, 555-559, 1983; M. Lin et al., J. Virol. 55, 509-512, 1985). 
     In the species of Lactobacillus, only a probe for L.curvatus (H.A.R.Petrick et al, Appl.Environ.Microbiol. 54, 405-408, 1988), which is specifically associated with spoilage of vacuum-packed meats, has been isolated. It certainly would be of use in the dairy industry to have a quick and reliable method to identify and classify relevant strains of the lactic acid bacteria. 
     SUMMARY OF THE INVENTION 
     A first object of the present invention is to provide a specific DNA probe, which can be used in hybridisation procedures to specifically identify strains belonging to the Lactobacillus delbrueckii species, either in bacterial cultures, or as food constituents or during food processing, e.g. during industrial fermentation. 
     A second object of the present invention is to provide a process for producing such a specific DNA probe. 
     A third object of the present invention is to provide methods for specifically identifying strains belonging to the L. delbrueckii species with this DNA probe or with DNA parts thereof. To this end, firstly, the DNA probe according to the present invention comprises a DNA fragment lypridizes specifically to chromosomal DNA of strains of the L.delbrueckii species. This DNA fragment is preferably labeled by any suitable means, such as  32  p,  35   S  or biotin, for example. Preferably, this DNA fragment comprises an EcoRI fragment of chromosomal DNA from a strain of the L.delbrueckii species. Even more preferably this EcoRI fragment comprises a large open reading frame capable of complementation of a Leu minus lesion. Such a DNA fragment may be the 1633 base pair long EcoRI fragment of the plasmid pY85, for example. 
     Secondly, the process for producing a DNA probe according to the present invention comprises preparing an EcoRI clonebank from a strain of the L.delbrueckii species, transforming said EcoRI clonebank into a strain of E.coli having a Leu minus lesion, selecting for Leu plus clones, and isolating therefrom a clone of which an EcoRI DNA fragment is capable of hybridisation to chromosomal DNA of strains of the L.delbrueckii species. 
     Thirdly, a first method for identifying a bacterial strains of the L.delbrueckii species according to the present invention comprises preparing chromosomal DNA of a strain to be identified and checking whether this DNA hybridises to the present probe or to a probe produced by the present process. 
     A second method comprises preparing chromosomal DNA of a strain to be identified and carrying out a polymerase chain reaction on this DNA with parts of DNA sequences identical with parts of DNA sequences of the present probe or of a probe produced by the present process. 
     In preferred embodiments of these methods the chromosomal DNA is prepared by growing cells of the strain to be identified on a culture medium supplemented with a fermentable carbon source, incubating them in the presence of proteinases, treating them with an N-acetyl-muramidase, further incubating them in the presence of an emulsifying agent, a chelating agent and a proteinase, phenol extracting DNA therefrom, ethanol precipitating the extracted DNA, treating this DNA with an RNaSe and chloroform extracting the RNase treated DNA. 
     The above second method, preferably including the above preferred embodiment, is especially provided for identifying strains the cells of which are only present in a very low concentration in some substrates. On the other hand, the above first method, which has proven to be of a very high sensitivity, may even be used for carrying out identification tests on chromosomal DNA prepared by simply lysing cells of a strain to be identified, either from cultures or on solid supports such as e.g. filter paper or nitrocellulose paper. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     FIG. 1 is a restriction map of the EcoRI DNA fragment of the plasmid pY85. 
     The scale is indicated in base pairs (bp). Stickyend or bluntend restriction sites are indicated for each enzyme. 
     Enzymes that do not cut are: 
     BcII, ClaI, EcoRV, KpnI, MluI, NdeI, NruI, PstI, and PvuII. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The plasmid pY85 was deposited pursuant to the Budapest Treaty on May 18, 1994, in the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France, where it was given the number CNCM I-1419. 
     By transforming a clonebank of L.bulqaricus into GE891, it was possible to isolate individual clones, which can complement the Leu minus lesion of this E.coli strain. One of these clones, pY85, proved to be very useful as genetic probe in hybridisation assays to specifically identify the species of Lactobacillus delbrueckii. In screening experiments applying dot-blot hybridisation, over 30 different Lactobacillus strains from different origins were tested. It could be shown that pY85 only hybridised specifically to the L.delbrueckii species, ie. its subspecies bulgaricus, delbrueckii and lactis. No hybridisation to any other of the tested bacteria (table I) could be detected. In order to test the specificity of the present probe, the washing conditions of the dot-blots were lowered to room temperature (20° C.) and the SSC concentration were increased to 2XSSC. Even under these very low stringency conditions, it was absolutely not possible to find hybridisation of pY85 with any non L.delbrueckii species. 
     The sensitivity of pY85 to detect L.delbrueckii DNA was tested by hybridising it to serial dilutions of appropriate chromosomal DNA&#39;s. It was noted that with an over-night exposure of an X-ray film to a filter, it was possible to easily detect hybridisation to as little as 2pg of target DNA. Furthermore, hybridisation of subfragments of pY85, a 200bp and 500bp long fragment, to Lactobacillus was tested and identical results with respect to the complete fragment were found. It can therefore be concluded that any part of the pY85 fragment may be sufficient for specifically identifying L.delbrueckii. From these data it can furthermore be assumed, that the PCR (polymerase chain reaction) (Saiki et al., Science 230, 1350-1354, 1985; Saiki et al., Science 239, 487-491, 1988), which makes use of part of the pY85 DNA sequence, may work as well in identifying this specific Lactobacillus species. Because of its great amplification effect, the PCR method may be regarded as a very interesting possibility in cases where minute amounts of Lactobacillus bacteria have to be traced as for example in food samples. 
     DNA sequence analysis and further genetic analysis showed that the pY85 fragment is coding for a structural gene. As essential gene products are usually subject to selection of their functions, these genes are mostly good conserved regions of DNA. It is therefore likely that because of the structural function of the pY85 fragment, this piece of DNA serves as such a good, highly specific probe for the Lactobacillus delbrueckii species. 
     EXAMPLES 
     MATERIALS AND METHODS 
     Bacteria and plasmids 
     Lactic acid bacterial strains used in the Examples hereafter are shown in table I. E.coli strains are HB101 (leuB6 proA2 recA13 thil ara14 lacY1 galK2 xy15 mtl1 rpsL20 supE44 hsdS20) (H. W. Boyer et al., J.Mol.Biol. 41, 459-472, 1969) and GE891 (F -  endA1 thi1 hsdR17 supE44 leu291 ilvD145) (G. Eggertsson, Institut of Biology, University of Iceland, Reykjavik, Iceland, unpublished). The plasmid used as vector was YRP17 (Hottinger et al., Mol.Gen. Genet. 188,219-224, 1982). 
     Media 
     For growth of the different Lactobacilli, Lactococci and Propionibacteria MRS broth (Difco Laboratories) was used, supplemented with 1% lactose. E.coli strains were grown in LB medium. 
     Preparation of DNA 
     i) Chromosomal DNA from Lactobacillus, Lactococcus and Propionibacteria. Cells were diluted from over-night cultures into 10ml MRS, supplemented with 1% lactose and grown to mid-log phase at 43° C. The cells were then harvested by centrifugation, washed once in cold 1M NaCl, and incubated for lh at 37° C. in the presence of Proteinase K (250 μg/ml) and Pronase E (500 μg/ml). The cells were washed in TE (10 mM Tris hydrochloride pH 7.4; lmM EDTA) and treated with Mutanolysin (200 μg/ml) in the presence of TE for 1h at 37° C. SDS, EDTA and Proteinase K were added to a final concentration of 0.1%, 75 mM and 200μg/ml, resp., and incubated for 4h at 65° C. The DNA was then phenol extracted, ethanol precipitated and spooled onto a sterile toothpick. The DNA was resuspended in TE in the presence of RNase A (200 μg/ml), chloroform extracted, reprecipitated in ethanol and spooled again onto a toothpick. The DNA was then resuspended in 100 l of TE and stored at 4° C. 
     ii) Plasmid DNA from E.coli. Plasmid DNA from E.coli was isolated and as needed purified on CsC1 gradient according to Maniatis et al. (Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982). Chemicals were purchased from E.Merck Chemicals Inc. and the enzymes from Sigma Chemical Co.. 
     Construction of an EcoRI clonebank from Lactobacillus bulgaricus in YRP17 
     Chromosomal DNA from L.bulgaricus type was digested to completion with EcoRI and ligated into the vector YRP17,  which had been previously linearised by cleavage at its unique EcoRI site and phosphatase treated. The ligation mixture was then transformed into HB101 and a representative number of about 20&#39;000 colonies, 66% of which had insertions of a mean size of about 1.9 kb, were collected and amplified. 
     The enzymes were used according to the suppliers and transformation of E.coli was done according to Maniatis et al. 
     Selection for Leu -   Complementing Clones 
     The EcoRI clonebank had been transformed into GE891. In order to enrich for transformants, the culture had been first grown in LB medium containing 70 g/ml ampicillin over night at 37° C. The cells were then washed in minimal medium and appropriate dilutions were plated onto minimalagar plates containing isoleucine, valine and glucose in 20 μg/ml, 20 μg/ml and 1 mg/ml, respectively. The incubation was at 37° C. over night. 
     Labeling of DNA Fragments 
     To radioactively label the DNA, we used a fill-in-replacement DNA synthesis (Maniatis et al. ). The T4 DNA polymerase was purchased from Boehringer- Mannheim, [ - 32  P]dATP from Amersham Co. 
     Hybridisation Procedures 
     i) Dotblot hybridisation. Aliquots of 200ng of chromosomal DNA in TE were denatured by heating for 5 minutes at 95° C. SSC was added to the aliquots to give a final concentration of 16X SSC and then the mixture was spoted onto 20XSSC wetted GeneScreen paper and rinsed once with 20XSSC. A Bio-Rad dotblot apparatus was used. The filter was then ready for DNA hybridisation, applying standard procedures with hybridisation at 65° C. 6XSSC and a subsequent wash at 65° C. and 0,1XSSC (E. Southern, J. Mol. Biol. 98, 503-517, 1975). To detect the hybridisation signals, the filters were used to expose X-ray films. 
     ii) Southern-blot hybridisation. Southern-blot hybridisations were carried out according to standard procedures (E.Southern). Hybridisations and washes were at 65° C. 
     Restriction Mappiny and DNA Sequencing 
     Restriction enzymes were purchased from Boehringer-Mannheim Co. and New England Biolabs Co. and were used as recommended by the suppliers. DNA sequencing was performed by the chain termination reaction method using M13phage derivates (F.Sanger et al., J.Mol.Biol. 94, 441-448, 1975; F.Sanger et al., Proc.Natl.Acad. Sci. 74, 5463-5467, 1977; J.Messing et al., Gene 19, 269-276, 1982). 
     EXAMPLE 1 
     1.1. 
     Isolation of GE891 Leu -   Complementing Clones from Lactobacillus 
     EcoRI digested chromosomal DNA of L.bulgaricus NCDO 1489 has been used to establish a clonebank in vector YRP17. This clonebank served to transform GE891, as described above. Several individual colonies which were growing on the minimalagar plates were isolated and analysed. In order to exclude spontaneous leu291 revertants from the screening, plasmid DNA of these colonies was isolated and used for retransforming GE891, with subsequent selection on the aminoacid substituted minimal agar plates. A high frequency of complementation of the leu291 lesion of GE891 in this second transformation indicated that the complementing factor is located on the plasmid. One of the clones isolated in this way is pY85. 
     1.2 
     Restriction Mapping and DNA Sequencing 
     A restriction map of pY85 was determined using several restriction enzymes. For a more detailed investigation, however, the entire DNA sequence of the EcoRI fragment of pY85 has been determined. An analysis of the DNA sequence showed that the fragment contains one large open reading frame which is responsible for the complementation of the Leu minus mutation. By means of the DNA sequence, a more detailed restriction map of the 1633 basepair long EcoRI fragment of pY85 could be generated. It is shown in FIG. 1. 
     1.3 
     Specificity and Sensitivity of the Probe pY85 
     Hybridisation of the EcoRI fragment of pY85 as probe on Southern-blots against genomic DNA of the L.bulgaricus type strain NCDO 1489 showed that a single allelic gene had been isolated. However, it was observed that not all Lactobacillus strains hybridised to the probe. Therefore pY85 was tested against different representatives of the Lactobacillus genus and some other lactic acid bacteria with dot-blot hybridisations. As probes for these tests the  32  p labeled EcoRI fragment of pY85 was used. A summary is presented in Table I. 
     
                       TABLE I______________________________________Bacterial strainsCRN.sup.a)                         signal.sup.b)code  source      species          with pY85______________________________________LB1   CRN collection              L. bulgaricus   +LB2     &#34;           &#34;              +LB6     &#34;           &#34;              +LB9     &#34;           &#34;              +LB12    &#34;           &#34;              +LB32    &#34;           &#34;              +LB34    &#34;           &#34;              +LB57.1   &#34;           &#34;              +LB81.4   &#34;           &#34;              +LB92.9   &#34;           &#34;              +N52   NCDO 1006     &#34;              +N95   NCDO B 15     &#34;              +N123  NCDO 1489   L. bulgaricus type                              +N124  ATCC 21815  L. bulgaricus    +N141  Piacenza CO 14               &#34;              +N5    ATCC 12315  L. lactis type   +N9    Liebefeld 125               &#34;              +N62   NCDO 270      &#34;              +N8    NCIB 8130   L. delbrueckii type                              +N187  ATCC 9649     &#34;              +LD1   CRN collection               &#34;              +LB14    &#34;         L. helveticus    -LB15    &#34;           &#34;              -LB20    &#34;           &#34;              -N2    ATCC 15009  L. helveticus type                              -N6    NCDO 87       &#34;              -N106  NCDO 2395     &#34;              -N213  Piacenza b 50               &#34;              -N7    NCDO 1750   L. fermentum     -N27   ATCC 393    L. casei type    -N25   ATCC 4005   L. buchneri      -N24   ATCC 8041   L. plantarum     -N207  ATCC 27865  L. maltaromicus  -N26   ATCC 14869  L. brevis        -N12   ATCC 4356   L. acidophilus   -N211  DSM 20016   L. reuteri type  -N50   ATCC 12278  L. sp.           -N51   ATCC 13866  L. sp.           -SL9   CRN collection             Lactococcus lactis                              -ST1     &#34;         Lactococcus thermophilus                              -PP13     &#34;        Propionibac.shermanii                              -PP21    &#34;         Propionibac freudenreichii                              -HB101   &#34;         E. coli          -______________________________________ .sup.a) NestleResearch Centre .sup.b) with dotblot hybridisation 
    
     The hybridisation results show that pY85 specifically only lights up DNA from the Lactobacillus delbrueckii strains, subspecies bulgaricus, delbrueckii and lactis. All other tested strains of different Lactobacillus species, of Lactococcus and Propionibacteria were negative. 
     EXAMPLE 2 
     The two NcoI fragments of 200 bp and 500 bp length from the pY85 clone (FIG. 1) were isolated and used in the way disclosed in Example 1 as probes for the hybridisation. It was observed that the same high specific hybridisation was obtained with these shorter probes. 
     EXAMPLE 3 
     In order to test the sensitivity of the dot-blot test carried out in Examples 1 and 2, serial dilutions of L.lactis type chromosomal DNA were made and the diluted DNA was then hybridised with the pY85 EcoRI fragment. A positive signal could easily be detected at 1 pg of target DNA. Furthermore, hybridisation was tested under less stringent washing conditions using the different Lactobacillus strains against the pY85 probe. After washing in 2XSSC at 20° C., it was only possible to detect a signal of hybridisation with the DNA of the Lactobacillus delbrueckii species. For the Lactobacillus screening according to Examples 1 and 2, however, at least 200 ng of DNA per sample and stringent washing conditions were used.