Journal of Applied Bacteriology 1992, 73, 349-354
MI3 DNA fingerprinting, a new tool for classification and identification of Lactobacillus spp. V.i. Miteva, A.N. Abadjieva and Tz. T. Stefanova'
'
Institute of Microbiology,Bulgarian Academy of Sciences,Sofia and ELBY Engineering,Sofia, Bulgaria 4085/01/92: accepted 27 April 1992
The optimal conditions for the application of M13 DNA fingerprinting to the genus Lactobacillus were determined. Comparative fingerprint analysis of representative strains of Lactobacillus delbrueckii subsp. delbrueckii, Lact. delbrueckii subsp. lactis, Lact. delbrueckii subsp. bulgaricus, Lact. helveticus and Lact. casei permitted t h e differentiation of species, subspecies a n d individual strains a n d the quantitative determination of their genetic relatedness. The results confirm the high specificity of M13 DNA fingerprinting and indicate that it might be used in the classification of Lactobacillus spp. V . I . M I T E V A , A . N . A B A D J I E V A A N D T Z . T. S T E F A N O V A . 1992.
INTRODUCTION
chromosome. Similar results are obtained with the whole M 13 molecule and with M 13-derived 15 bp oligonucleotide (Huey and Hall 1989). Certain advantages of M I 3 as a universal probe are its wide availability and easy application to different prokaryotes and eukaryotes. It is possible to compare closely related and taxonomically farstanding species and strains, while the species-specific probes are restricted to a definite species. Compared with REA, DNA fingerprinting also has the advantage that the whole genome is examined but the number of bands is smaller; this facilitates the quantitative interpretation of the results. In this paper we report the application of M13 DNA fingerprinting to lactobacilli and the development of optimal conditions for comparative analysis of different species and strains.
The genus Lactobacillus comprises more than 50 species. According to G C content it is heterogenous, with a wide range, 32-51 mol% (Kandler & Weiss 1986). The identification of new strains is often difficult and doubtful because of their similar nutritional and growth requirements. Recent studies emphasize that the present classification of lactobacilli is unsatisfactory and does not reflect the real phylogenetic relatedness of different strains and species (Stahl et al. 1990; Collins et al. 1991). Several new genetic and chemotaxonomic approaches have been used with the aim of improving this classification and for strain identification such as analysis of the plasmid content (Nes 1984) and total soluble cell protein SDSPAGE patterns (Dicks & van Vuuren 1987), sequencing of 16s rRNA (Collins et al. 1991), restriction endonuclease analysis (REA) (Manachini & Parini 1983; Stahl et al. 1990), and development of species-specific probes (Petrick et al. 1988; Pilloud & Mollet 1990; Delley et al. 1990). Each of these methods has specific applications and advantages. Recently we have successfully applied a new molecular approach, M13 DNA fingerprinting, to some Grampositive micro-organisms (Miteva et al. 1990, 1991). Phage M13 DNA is used as a probe for DNA fingerprinting due to the presence of a tandemly repeated 15 bp sequence in the protein I11 gene (Vassart et al. 1987; Ryskov et al. 1988). The hybridization patterns reflect the individual and strainspecific distribution of hypervariable sequences along the
isolation of chromosomal DNA
Correspondence to :Dr Vanya Miteva, Department of Genetics, Institute of Microbiology, Bulgarian Academy of Sciences, Acad. Bonchev Str. 26, I l l 3 Sofia, Bulgaria.
T h e method of Chater et al. (1982) was used with some modifications. T h e cells, obtained from 50 ml of an overnight MRS culture were harvested, washed in T E buffer
+
MATERIALS AND METHODS Strains and culture conditions
The Lactobacillus strains studied are listed in Table 1. T h e cultures were maintained in skim milk. Overnight milk cultures, incubated at 45°C were used to inoculate 10 ml of MRS broth (De Man et al. 1960). After static incubation at 37°C for 6-8 h the M R S cultures were used as an inoculum of a larger volume of M R S (MRS 1 : 10 v/v), which were cultivated overnight at 37°C.
350 V . I . MITEVA E r A L .
Species
Designation
G + C (mol%)
Lactobacillus delbrueckii subsp. delbrueckii subsp. lactic subsp. bulgaricus Lact. helveticus Lact. casei subsp. casei
ATCC 9649 ATCC 12315 ATCC 11842 ATCC 15009 IAM 1118 A 157 543
49-5 1 49-5 1 49-5 1 3WO 4547 4547 4547
b5 1,23374
49-5 1
Lact. delbrueckii subsp. bulgaricus local industrial strain locally-isolated strains (not identified)
Table 1 Lactobacillus strains studied
40
(10 mmol/l Tris, 1 mmol/l EDTA, pH 8), resuspended in 5 ml TE, containing 5 mg/ml lysozyme and incubated at 37°C for 60 min. RNAase was added to give a final concentration of 200 pg/ml and the mixtures were incubated for another 15 min. EDTA (1.2 ml of 0.5 mol/l p H 8) and 130 p1 of predigested proteinase K (10 mg/ml) were then added, followed by incubation at 37°C for 10-30 min. T h e cells were lysed with sodium lauryl sarkosinate (SLS) at a final concentration of 2% and incubated again with gentle shaking until the solution became clear. Deproteinization was performed by one extraction with phenol, two with phenokhloroform followed by one with chloroform. DNA was precipitated with isopropanol in the presence of 2.5 mol/l ammonium acetate. After washing in 70% ethanol and drying it was dissolved in T E .
solution (single strength Denhardt’s solution is 0.02% bovine serum albumin, 0.02% Ficoll, 0.02% polyvinylpyrrolidone), 0.1% SDS, 5 mmol/l EDTA at 57°C or 51°C for 18 h. T h e filters were washed in 2 x SSC and 0.1% SDS at the same temperature and exposed to X-ray films at - 70°C. Hybridization with digoxigenin-labelled M 13 was done overnight in 5 x SSC, 1% (w/v) blocking reagent (Boehringer), 0.1% SLS, 0.02% SDS at 51°C. T h e filters were washed in 2 x SSC, 0.1% SDS once for 15 min at room temperature and twice for 15 min at 51°C. T h e detection was performed according to the manufacturer’s instructions, with antidigoxigenin alkaline phosphatase conjugate, X-phosphate and N B T (nitroblue tetrazolinium salt). T h e colour was developed in the dark by layering the dye solution on the filter surface.
Digestion wlth restriction endonucieases and electrophoresis
Data analysis
Fifteen pg of DNA were digested overnight with 40-50 U of restriction endonuclease (Amersham). Electrophoresis was carried out in 25 cm 1% (w/v) agarose gels (Sigma) at 35 V for 18 h in Tris-acetate buffer, pH 7.6. M13 probe labelling
T h e approach of Jeffreys et al. (1985) and Nybom et al. (1990) was used. I n the comparative analysis several autoradiograms of different intensity were used to evaluate the number and intensity of the bands. T h e similarity index D was calculated for each pair of patterns A and B according to the equation D,, = 2 x No. of shared fragments/No. of fragments in A No. of fragments in B. It reflects the probability that a fragment in A is also present in B.
+
Single stranded M13mp8 DNA was labelled by the primer extension method using [32P] dCTP, sequencing primer and DNA Klenow polymerase (Amersham) at 37°C for 18 h. T h e non-radioactive labelling was performed in the same way but with digoxigenin-dUTP (Boehringer).
T h e reproducibility of the method was checked by repeated preparation and analysis of two or more blots from each digestion and with DNA samples from different isolations.
Hybridization
RESULTS
DNA fragments were transferred from the agarose gels to nitrocellulose filters (Hybond-C, Amersham), with 20 x SSC ( 1 x SSC is 0.15 mol/l NaCI, 0.015 mol/l sodium citrate pH 7.0) and fixed by baking at 80°C according to Southern (1975). Hybridization with [32P]-labelled M13 DNA was performed in 5 x SSC, 5 x Denhardt’s
Optimization of the procedures for DNA isolation, electrophoresis and preparation of the Southern blots
Reproducibility
T h e method of Chater et al. (1982), used for isolation of high molecular weight DNA, gave good results for most of the strains. Considerable difficulties appeared with strains
DNA FINGERPRINTING OF L A C T O B A C I L L U S 351
2
I
belonging to Lact. delbrueckii subsp. bulgaricus. A rapid DNA shearing and loss of viscosity was observed after lysing the cells. Some modifications of the procedure including the use of a combined lysing solution, containing RNAase, proteinase K and SLS allowed us to isolate chromosomal DNA only from two of six strains. Our experiments showed that reproducible fingerprints were obtained only after total cleavage of DNA and perfect resolution of the fragments in the agarose gels.
kb
Optimization of conditions for hybridization and probe labelling
23.1
-
9.4
-
6.5
-
Hybridization and washing of one and the same filter was performed at different stringency conditions - 57°C and 51". Figure 1 shows that in both cases the patterns were species-specific but the higher number of bands at lower stringency made the comparison more accurate as larger numbers of band position differences could be detected. Further experiments were therefore done out at 51°C. T h e results obtained with radioactive and non-radioactive labelling of the probe are shown in Fig. 2. T h e sensitivity was somewhat lower when digoxigenin was used but it overcame the problem of overexposure in cases with very strong bands.
-
4-3
2.3
-
2.0
-
Comparative analysis of LBCtOb8C///US species, subspecies and strains Fig. 1 Southern hybridization of [32P]-labelled MI3 DNA to Hind 111 and ~ ~ 1 digested 1 1 Lacrobac,&,s helveticus DNA at different stringencies.Lane 1, Temperature of hybridization and washing 57°C; lane 2, temperature of hybridization and washing 51°C. I? Hind I 1 1 is used as a size marker
Strains from several species including the closely related subspecies of the Lact. delbrueckii group and Some with a more distant taxonomic position such as Lact. helveticus and Lact. casei were studied (Table 1).
( b l
( 0 )
I
kb 23.1
Fig. 2 (a) Southern hybridization of [32P]-labelled M13 DNA to Hind 111
digested DNA from Lactobacillus. Lane 1, Lact. delbrueckii subsp. bulgaricus b5 ; lanes 2, 3, 5, 6, industrial strains of Lact. helveticus; lane 4, Lact. delbrueckii subsp. bulgaricus ATCC 11842; lane 7, Lact. helveticus ATCC 15009; lane 8, Lact. delbrueckii subsp. lactis ATCC 12315 ; lane 9, Lact. delbrueckii subsp. delbrueckii ATCC 9649. (b) The same nitrocellulose filter reprobed with digoxigenin-labelled MI3 DNA
-
6.5
-
4.3
-
9.4
2.3 2.0
-
2
3
4
5
6
7
8
9
I
2
3
4
5
6
7
8
9
352
V . I . MITEVA ET A L .
Table 2 Restriction endonucleases used
AT-rich recognition sites
I
GC-rich recognition sites
2
3
4
5
6
7
8
9
kb
9.4 6.5 -
23.1
Eco RI Hind 111 Bgl I1 EGORV Barn H I
G/AATTC A/AGCTT A/GATCT GAT/ATC G/GATCC
CTGCA/G CC(A)GC GG/CC C/CGG
Pst I
Mva I Hae I1
Msp I
4.3
We used single and double digestions with enzymes, recognizing 4, 5 and 6 nucleotides, GC- or AT-rich. (Table 2) Some interesting observations were made. As mentioned at the beginning the genus Lactobacillus covers a very wide range of G C content. This was expressed in quite different patterns, which were difficult to be compared in some cases. A correlation was observed between the G + C content of the strains and the profiles obtained after digestion with certain enzymes. When the DNA samples were double digested with Hind I11 amd Msp I, the latter one recognizing 4 GC-rich nucleotides, the profiles of the Lact. delbrueckii group gave patterns consisting mainly of low molecular size fragments (Fig. 3, lanes 1, 6, 8, 9). At the same time species with low G + C content performed good band distribution by size (Fig. 3, lanes 2, 3, 4, 5, 7). This finding was confirmed with other enzymes as wellMva I and Hue 111, recognizing 5 and 4 GC-rich nucleotides respectively (results not shown). The single and double digestions with the following endonucleases, recognizing six nucleotides: Hind 111, Barn HI, Hind I11 and Eco RI, Hind 111 and Bgl 11, EGORI and Pst I, Eco RV and Pst I produced comparable profiles in terms of number and distribution of hybridization bands observed (Fig. 2). The quantitative analysis of the patterns was performed by pairwise comparison and calculation of the similarity indexes. Table 3 presents the results from the comparison of the three subspecies of Lact. delbrueckii and Lact. helveticus. The values of the similarity indexes were relatively
-
2.0 -
2.3
+
Table 3 Comparison of Lactobacillus delbrueckii subspecies and Lactobacillus helveticus
Index of similarity ( D ) Enzymes
Hind 111
Hind I11 + Eco RI
Species Strain
Lact. delbrueckii
Lact. delbrueckii
11842
11842
12315 9649
12315
9649
Lact. delbrueckii 12315
0.56
0.36
Latt. delbrueckii 9649
0.57
0.51
0.26
040
0.27
0.38
0.11
0.18
Lact. helveticus 15009
0.35
0.28
Fig. 3 Southern hybridization of [32P]-labelled M13 DNA to Hind 111 and Msp I digested DNA from Lactobacillus. Lane 1, Lact. delbrueckii subsp. bulgaricus b5; lanes 2-5, industrial strains of Lact. helveticus; lane 6, Lact. delbrueckii subsp. bulgaricus ATCC 11842; lane 7, Lact. helveticus ATCC 15009; lane 8, Lact. delbrueckii subsp. l a d s ATCC 12315; lane 9, Lact. delbrueckii
subsp. delbrueckii ATCC 9649
high within the Lact. delbrueckii group when Hind I11 was used. They show that the genomes of the three subspecies are distinguishable but recognizably related. Lactobacillus helveticus gave lower values of D when compared with the three subspecies of Lact. delbrueckii. Similar correlations were obtained with Hind I11 and Eco RI (Table 3), Eco RI and Pst I, Hind I11 and Bgl I1 with the only difference that in general all the values were lower, which could be due to the larger number of fragments obtained after double digestion. The comparison of different strains, belonging to one and the same species, Lact. casei, with other species is presented in Table 4. Relatively lower were the values when Lact. casei was compared with Lact. helveticus and Lact. delbrueckii subsp. bulgaricus. Highest values of D were obtained when the three Lact. casei strains were compared in between. Obviously these strains possess certain genetic similarity but they could be differentiated as well. Similar
DNA F I N G E R P R I N T I N G OF LACTOBACILLUS
Table 4 Comparison of strains of Lactobacillus casei, Lactobacillus helveticus and Lactobacillus deibrueckii subsp. bulgaricus
353
revealed with some of the restriction enzymes M v a I (Fig. 4), Eco RV and Pst I.
Index of similarity ( D ) Enzyme
Hind I11 and Eco RI
Species
Lact. casei A 157 IAM 1118
Strain Lact. casei IAM 1118 543 Lact. delbrueckii 11849 Lact. helveticus 15009
DISCUSSION
543
0.59 0.55
0.48
0.33
0.22
0.32
0.22
0.32
0.25
Lact. delbrueckii 11842
0.21
results with high values of D were obtained when the two Lact. delbrueckii subsp. bulgaricus strains studied were compared (Fig. 2, lanes 1, 4). Finally, the high individual specificity of M I 3 DNA fingerprinting and the possibility of revealing minor genomic differences was shown when some locally isolated strains with similar cultural characteristics were studied. Slight differences in the position of one or two bands were
I
2
3
4
kb
23.1
-
9.4
-
6.5
-
4.3
-
2.0 -
2.3
Fig. 4 M13 DNA fingerprints of DNA from local industrial strains of Lactobacillus helveticus, digested with Mva I
T h e present study showed that optimization of the conditions at all stages of M13 DNA fingerprinting is of great importance for the achievement of reliable and reproducible results in the comparative analysis of strains and species of the genus Lactobacillus. T h e difficulties in the isolation of DNA from lactobacilli are well known and have led to the development of many different procedures aimed at improvement of the cell lysis and the production of high molecular DNA of enough purity. In this work the initial problem associated with DNA shearing, probably due to high intracellular nuclease activity, was partially solved by some modifications of the method used. An important point in obtaining reproducible patterns was the achievement of total cleavage and perfect resolution of the fragments in the agarose gels. T h e promising results with the digoxigenin labelling of M13, the lack of harmful effect and easier handling of nonradioactive probes are also very important especially if we bear in mind the perspective that DNA fingerprinting could become a widely used procedure for classification and identification of Lactobacillus spp. T h e successful application of DNA fingerprinting to the genus Lactobacillus confirmed our previous observations that Gram-positive bacteria need less stringent conditions for hybridization than the Gram-negatives (Miteva et al. 1990). T h e polymorphic fingerprint patterns obtained under the conditions used were unique for a given enzyme and genome. T h e correlation found between the G C content and the patterns observed could be used when unknown strains are studied in order to make a preliminary differentiation according to G + C content. If a wide variety of strains, covering the whole range of G C content, is examined it would be better to use single or double digestions with enzymes recognizing six nucleotides such as Hind 111, Eco RI, Bgl I1 and Pst I. These conclusions prove the importance of the appropriate choice of endonucleases used and the necessity of performing restriction with several different enzymes in parallel, especially when closely related strains are studied. Such an approach could greatly enhance the objectivity of the evaluation and interpretation of the fingerprints. T h e high specificity of the fingerprints permitted the differentiation of species, subspecies and individual Lactobacillus strains. In addition, the quantitative comparative analysis performed enabled us to make some conclusions
+
+
354 V . I . MITEVA ET AL
about the degree of genetic relatedness and the taxonomic position of the species studied. The relatively higher values of the similarity indexes, calculated in the pairwise comparisons of the three subspecies of Lact. delbrueckii confirmed their genetic relatedness and are consistent with their present classification. Collins et al. (1991) proved the close genetic relatedness of the subspecies of Lact. delbrueckii, which were considered in one phylogenetic cluster according to 16s rRNA sequence analysis. Obviously the investigation of different strains within a species by modern molecular methods facilitates the assessment of a more detailed picture of the intraspecific structure. The precise determination of the phylogenetic relatedness of different lactobacilli certainly needs the extension of our DNA fingerprint analysis to additional species and strains, careful choice of restriction endonucleases and reference strains and will be enhanced by the application of computer programs for cluster analysis. The results presented here, although performed with small numbers of strains, confirmed the high species and strain specificity of M13 DNA fingerprinting. This raises the possibility of using this new method for identification and differentiation of lactobacilli at the level of species, subspecies and strains. In combination with other molecular approaches this could lead to the development of new clatsificiation schemes.
ACKNOWLEDGEMENTS This work was supported by a Project grant from the Bulgarian National Foundation for Scientific Research and by ELBY Engineering. The authors express their gratitude to D r Bringle, Boehringer GmbH, for kindly supplying the D I G labelling kit.
REFERENCES C H A T E R ,K . F . , HOPWOOD,D.A., K E I S E R , T . & T O M P S O N C.J. , (1982) Gene cloning in Streptomyces. Current Topics in Microbiology and Immunology 96,69-95. C O L L I N SM.D., , R O D R I G U E SU., , ASH, C., A G U I R E ,M., FARROW J.A.E., , M A R T I N E Z - M U R CA., I A ,P H I L L I P S , B.A., W I L L I A M A.M. S, & WALLBANKS, S. (1991)Phylogenetic analysis of the genus Lactobacillus and related lactic acid bacteria as determined by reverse transcriptase sequencing of 16s rRNA. FEMS Microbiology Letters 77, 5-12. R , (1990) DNA DELLEY, M., MOLLET, B. & H O T T I N G EH. probes for Lactobacillus delbrueckii. Applied and Environmental Microbiology 56 (6), 1967-1970.
DE M A N ,J.C., ROGOSA,M. & S H A R P EM.E. , (1960) A medium for the cultivation of lactobacilli. Journal of Applied Bacteriology 23, 13&135. D I C K SL , . M . T . & V A N V U U R E N H.J.J. , (1987) Relatedness of heterofermentative Lactobacillus species revealed by numerical analysis of total cell protein patterns. International Journal of Systematic Bacteriology 37 (4), 4 3 7 4 0 . H U E Y ,B. & H A L L ,J . (1989) Hypervariable DNA fingerprinting in E. coli. Minisatellite probe from bacteriophage M13. Journal of Bacteriology 171, 252a2532. J E F F R E Y S , A., WILSON,V . & T H E I NS, . (1985) Individual specific fingerprints of human DNA. Nature (London) 316, 76-79. K A N D L E R0. , & WEISS, N. (1986) Regular, nonsporing Gram-positive rods. In Bergey's Manual of Systematic Bacteriology ed. Sneath, P.H.A., Mair, N.S., Sharpe, M.E. & Holt, J.G. Vol. 2, pp. 1209-1234. Baltimore: Williams & Wilkins Co. M A N A C H I NP.L. I, & P A R I N I C. , (1983) DNA restriction endonuclease cleavage patterns, DNA sequence similarity and phenotypical characteristics in some strains of Lactobacillus helveticus and Lactobacillus jugurti. Antonie van Leeuwenhoek 49, 143-152. M I T E V AV., , A B A D J I E V A ,A., I V A N O VP., & G R I G O R O V A , R. (1990) M13 bacteriophages DNA as a probe for DNA fingerprinting in Gram-positive microorganisms. Systematic and Applied Microbiology 13,35&353. M I T E V AV., , A B A D J I E V A ,A. & G R I G O R O V RA., (1991) Differentiation among strains and serotypes of Bacillus thuringiensis by MI3 DNA fingerprinting. Journal of General Microbiology 137, 593-600. NES, I . F . (1984) Plasmid profiles of ten strains of Lactobacillus plantarurn. FEMS Microbiology Letters 21, 35!+361. , (1990) NYBOM,H., ROGSTAD,S . H . & S C A A L B.A. Genetic variation detected by use of the M13 DNA fingerprinting probe in Malus, Prunus and Rubus (Rosaceae). Theoretical and Applied Genetics 79, 153-156. P E T R I C K ,H.A.P., AMBROSIO, R.E. 8i H O L Z A P F E L , W. H. (1988) Isolation of a DNA probe for Lactobacillus curvatus. Applied and Environmental Microbiology 54 (2), 405-408. P I L L O U DN. , & MOLLET,B. (1990) DNA probes for detection of Lactobacillus helveticus. Systematic and Applied Microbiology 13, 345-349. K, RYSKOV,A.P., J I N C H A R A D Z E , A.G., P R O S N Y A M.L., I V A N O VP.L. , & L I M B O R S K S.A. A , (1988) M13 phage DNA as a universal marker for DNA fingerprinting of animals, plants and microorganisms. FEBS Letters 233, 388-392. S O U T H E R E.M. N, (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98, 503-517. S T A H L ,M., MOLIN, G., PERSSON,A . , A H R N E , S. & ST A H L , S . ( 1990) Restriction endonuclease patterns and multivariable analysis as a classification tool for Lactobacillus spp. International Journal of Systematic Bacteriology 40 (2), 189-193. VASSART, G., G E O R G E SM., , M O N S I E U RR., , BROCAS, H., L E Q U A R RA. E , & CHRISTOPHE, D. (1987) A sequence in M13 phage DNA detects hypervariable minisatellites in human and animal DNA. Science Reports 235,683-686.
MI3 DNA fingerprinting, a new tool for classification and identification of Lactobacillus spp. V.i. Miteva, A.N. Abadjieva and Tz. T. Stefanova'
'
Institute of Microbiology,Bulgarian Academy of Sciences,Sofia and ELBY Engineering,Sofia, Bulgaria 4085/01/92: accepted 27 April 1992
The optimal conditions for the application of M13 DNA fingerprinting to the genus Lactobacillus were determined. Comparative fingerprint analysis of representative strains of Lactobacillus delbrueckii subsp. delbrueckii, Lact. delbrueckii subsp. lactis, Lact. delbrueckii subsp. bulgaricus, Lact. helveticus and Lact. casei permitted t h e differentiation of species, subspecies a n d individual strains a n d the quantitative determination of their genetic relatedness. The results confirm the high specificity of M13 DNA fingerprinting and indicate that it might be used in the classification of Lactobacillus spp. V . I . M I T E V A , A . N . A B A D J I E V A A N D T Z . T. S T E F A N O V A . 1992.
INTRODUCTION
chromosome. Similar results are obtained with the whole M 13 molecule and with M 13-derived 15 bp oligonucleotide (Huey and Hall 1989). Certain advantages of M I 3 as a universal probe are its wide availability and easy application to different prokaryotes and eukaryotes. It is possible to compare closely related and taxonomically farstanding species and strains, while the species-specific probes are restricted to a definite species. Compared with REA, DNA fingerprinting also has the advantage that the whole genome is examined but the number of bands is smaller; this facilitates the quantitative interpretation of the results. In this paper we report the application of M13 DNA fingerprinting to lactobacilli and the development of optimal conditions for comparative analysis of different species and strains.
The genus Lactobacillus comprises more than 50 species. According to G C content it is heterogenous, with a wide range, 32-51 mol% (Kandler & Weiss 1986). The identification of new strains is often difficult and doubtful because of their similar nutritional and growth requirements. Recent studies emphasize that the present classification of lactobacilli is unsatisfactory and does not reflect the real phylogenetic relatedness of different strains and species (Stahl et al. 1990; Collins et al. 1991). Several new genetic and chemotaxonomic approaches have been used with the aim of improving this classification and for strain identification such as analysis of the plasmid content (Nes 1984) and total soluble cell protein SDSPAGE patterns (Dicks & van Vuuren 1987), sequencing of 16s rRNA (Collins et al. 1991), restriction endonuclease analysis (REA) (Manachini & Parini 1983; Stahl et al. 1990), and development of species-specific probes (Petrick et al. 1988; Pilloud & Mollet 1990; Delley et al. 1990). Each of these methods has specific applications and advantages. Recently we have successfully applied a new molecular approach, M13 DNA fingerprinting, to some Grampositive micro-organisms (Miteva et al. 1990, 1991). Phage M13 DNA is used as a probe for DNA fingerprinting due to the presence of a tandemly repeated 15 bp sequence in the protein I11 gene (Vassart et al. 1987; Ryskov et al. 1988). The hybridization patterns reflect the individual and strainspecific distribution of hypervariable sequences along the
isolation of chromosomal DNA
Correspondence to :Dr Vanya Miteva, Department of Genetics, Institute of Microbiology, Bulgarian Academy of Sciences, Acad. Bonchev Str. 26, I l l 3 Sofia, Bulgaria.
T h e method of Chater et al. (1982) was used with some modifications. T h e cells, obtained from 50 ml of an overnight MRS culture were harvested, washed in T E buffer
+
MATERIALS AND METHODS Strains and culture conditions
The Lactobacillus strains studied are listed in Table 1. T h e cultures were maintained in skim milk. Overnight milk cultures, incubated at 45°C were used to inoculate 10 ml of MRS broth (De Man et al. 1960). After static incubation at 37°C for 6-8 h the M R S cultures were used as an inoculum of a larger volume of M R S (MRS 1 : 10 v/v), which were cultivated overnight at 37°C.
350 V . I . MITEVA E r A L .
Species
Designation
G + C (mol%)
Lactobacillus delbrueckii subsp. delbrueckii subsp. lactic subsp. bulgaricus Lact. helveticus Lact. casei subsp. casei
ATCC 9649 ATCC 12315 ATCC 11842 ATCC 15009 IAM 1118 A 157 543
49-5 1 49-5 1 49-5 1 3WO 4547 4547 4547
b5 1,23374
49-5 1
Lact. delbrueckii subsp. bulgaricus local industrial strain locally-isolated strains (not identified)
Table 1 Lactobacillus strains studied
40
(10 mmol/l Tris, 1 mmol/l EDTA, pH 8), resuspended in 5 ml TE, containing 5 mg/ml lysozyme and incubated at 37°C for 60 min. RNAase was added to give a final concentration of 200 pg/ml and the mixtures were incubated for another 15 min. EDTA (1.2 ml of 0.5 mol/l p H 8) and 130 p1 of predigested proteinase K (10 mg/ml) were then added, followed by incubation at 37°C for 10-30 min. T h e cells were lysed with sodium lauryl sarkosinate (SLS) at a final concentration of 2% and incubated again with gentle shaking until the solution became clear. Deproteinization was performed by one extraction with phenol, two with phenokhloroform followed by one with chloroform. DNA was precipitated with isopropanol in the presence of 2.5 mol/l ammonium acetate. After washing in 70% ethanol and drying it was dissolved in T E .
solution (single strength Denhardt’s solution is 0.02% bovine serum albumin, 0.02% Ficoll, 0.02% polyvinylpyrrolidone), 0.1% SDS, 5 mmol/l EDTA at 57°C or 51°C for 18 h. T h e filters were washed in 2 x SSC and 0.1% SDS at the same temperature and exposed to X-ray films at - 70°C. Hybridization with digoxigenin-labelled M 13 was done overnight in 5 x SSC, 1% (w/v) blocking reagent (Boehringer), 0.1% SLS, 0.02% SDS at 51°C. T h e filters were washed in 2 x SSC, 0.1% SDS once for 15 min at room temperature and twice for 15 min at 51°C. T h e detection was performed according to the manufacturer’s instructions, with antidigoxigenin alkaline phosphatase conjugate, X-phosphate and N B T (nitroblue tetrazolinium salt). T h e colour was developed in the dark by layering the dye solution on the filter surface.
Digestion wlth restriction endonucieases and electrophoresis
Data analysis
Fifteen pg of DNA were digested overnight with 40-50 U of restriction endonuclease (Amersham). Electrophoresis was carried out in 25 cm 1% (w/v) agarose gels (Sigma) at 35 V for 18 h in Tris-acetate buffer, pH 7.6. M13 probe labelling
T h e approach of Jeffreys et al. (1985) and Nybom et al. (1990) was used. I n the comparative analysis several autoradiograms of different intensity were used to evaluate the number and intensity of the bands. T h e similarity index D was calculated for each pair of patterns A and B according to the equation D,, = 2 x No. of shared fragments/No. of fragments in A No. of fragments in B. It reflects the probability that a fragment in A is also present in B.
+
Single stranded M13mp8 DNA was labelled by the primer extension method using [32P] dCTP, sequencing primer and DNA Klenow polymerase (Amersham) at 37°C for 18 h. T h e non-radioactive labelling was performed in the same way but with digoxigenin-dUTP (Boehringer).
T h e reproducibility of the method was checked by repeated preparation and analysis of two or more blots from each digestion and with DNA samples from different isolations.
Hybridization
RESULTS
DNA fragments were transferred from the agarose gels to nitrocellulose filters (Hybond-C, Amersham), with 20 x SSC ( 1 x SSC is 0.15 mol/l NaCI, 0.015 mol/l sodium citrate pH 7.0) and fixed by baking at 80°C according to Southern (1975). Hybridization with [32P]-labelled M13 DNA was performed in 5 x SSC, 5 x Denhardt’s
Optimization of the procedures for DNA isolation, electrophoresis and preparation of the Southern blots
Reproducibility
T h e method of Chater et al. (1982), used for isolation of high molecular weight DNA, gave good results for most of the strains. Considerable difficulties appeared with strains
DNA FINGERPRINTING OF L A C T O B A C I L L U S 351
2
I
belonging to Lact. delbrueckii subsp. bulgaricus. A rapid DNA shearing and loss of viscosity was observed after lysing the cells. Some modifications of the procedure including the use of a combined lysing solution, containing RNAase, proteinase K and SLS allowed us to isolate chromosomal DNA only from two of six strains. Our experiments showed that reproducible fingerprints were obtained only after total cleavage of DNA and perfect resolution of the fragments in the agarose gels.
kb
Optimization of conditions for hybridization and probe labelling
23.1
-
9.4
-
6.5
-
Hybridization and washing of one and the same filter was performed at different stringency conditions - 57°C and 51". Figure 1 shows that in both cases the patterns were species-specific but the higher number of bands at lower stringency made the comparison more accurate as larger numbers of band position differences could be detected. Further experiments were therefore done out at 51°C. T h e results obtained with radioactive and non-radioactive labelling of the probe are shown in Fig. 2. T h e sensitivity was somewhat lower when digoxigenin was used but it overcame the problem of overexposure in cases with very strong bands.
-
4-3
2.3
-
2.0
-
Comparative analysis of LBCtOb8C///US species, subspecies and strains Fig. 1 Southern hybridization of [32P]-labelled MI3 DNA to Hind 111 and ~ ~ 1 digested 1 1 Lacrobac,&,s helveticus DNA at different stringencies.Lane 1, Temperature of hybridization and washing 57°C; lane 2, temperature of hybridization and washing 51°C. I? Hind I 1 1 is used as a size marker
Strains from several species including the closely related subspecies of the Lact. delbrueckii group and Some with a more distant taxonomic position such as Lact. helveticus and Lact. casei were studied (Table 1).
( b l
( 0 )
I
kb 23.1
Fig. 2 (a) Southern hybridization of [32P]-labelled M13 DNA to Hind 111
digested DNA from Lactobacillus. Lane 1, Lact. delbrueckii subsp. bulgaricus b5 ; lanes 2, 3, 5, 6, industrial strains of Lact. helveticus; lane 4, Lact. delbrueckii subsp. bulgaricus ATCC 11842; lane 7, Lact. helveticus ATCC 15009; lane 8, Lact. delbrueckii subsp. lactis ATCC 12315 ; lane 9, Lact. delbrueckii subsp. delbrueckii ATCC 9649. (b) The same nitrocellulose filter reprobed with digoxigenin-labelled MI3 DNA
-
6.5
-
4.3
-
9.4
2.3 2.0
-
2
3
4
5
6
7
8
9
I
2
3
4
5
6
7
8
9
352
V . I . MITEVA ET A L .
Table 2 Restriction endonucleases used
AT-rich recognition sites
I
GC-rich recognition sites
2
3
4
5
6
7
8
9
kb
9.4 6.5 -
23.1
Eco RI Hind 111 Bgl I1 EGORV Barn H I
G/AATTC A/AGCTT A/GATCT GAT/ATC G/GATCC
CTGCA/G CC(A)GC GG/CC C/CGG
Pst I
Mva I Hae I1
Msp I
4.3
We used single and double digestions with enzymes, recognizing 4, 5 and 6 nucleotides, GC- or AT-rich. (Table 2) Some interesting observations were made. As mentioned at the beginning the genus Lactobacillus covers a very wide range of G C content. This was expressed in quite different patterns, which were difficult to be compared in some cases. A correlation was observed between the G + C content of the strains and the profiles obtained after digestion with certain enzymes. When the DNA samples were double digested with Hind I11 amd Msp I, the latter one recognizing 4 GC-rich nucleotides, the profiles of the Lact. delbrueckii group gave patterns consisting mainly of low molecular size fragments (Fig. 3, lanes 1, 6, 8, 9). At the same time species with low G + C content performed good band distribution by size (Fig. 3, lanes 2, 3, 4, 5, 7). This finding was confirmed with other enzymes as wellMva I and Hue 111, recognizing 5 and 4 GC-rich nucleotides respectively (results not shown). The single and double digestions with the following endonucleases, recognizing six nucleotides: Hind 111, Barn HI, Hind I11 and Eco RI, Hind 111 and Bgl 11, EGORI and Pst I, Eco RV and Pst I produced comparable profiles in terms of number and distribution of hybridization bands observed (Fig. 2). The quantitative analysis of the patterns was performed by pairwise comparison and calculation of the similarity indexes. Table 3 presents the results from the comparison of the three subspecies of Lact. delbrueckii and Lact. helveticus. The values of the similarity indexes were relatively
-
2.0 -
2.3
+
Table 3 Comparison of Lactobacillus delbrueckii subspecies and Lactobacillus helveticus
Index of similarity ( D ) Enzymes
Hind 111
Hind I11 + Eco RI
Species Strain
Lact. delbrueckii
Lact. delbrueckii
11842
11842
12315 9649
12315
9649
Lact. delbrueckii 12315
0.56
0.36
Latt. delbrueckii 9649
0.57
0.51
0.26
040
0.27
0.38
0.11
0.18
Lact. helveticus 15009
0.35
0.28
Fig. 3 Southern hybridization of [32P]-labelled M13 DNA to Hind 111 and Msp I digested DNA from Lactobacillus. Lane 1, Lact. delbrueckii subsp. bulgaricus b5; lanes 2-5, industrial strains of Lact. helveticus; lane 6, Lact. delbrueckii subsp. bulgaricus ATCC 11842; lane 7, Lact. helveticus ATCC 15009; lane 8, Lact. delbrueckii subsp. l a d s ATCC 12315; lane 9, Lact. delbrueckii
subsp. delbrueckii ATCC 9649
high within the Lact. delbrueckii group when Hind I11 was used. They show that the genomes of the three subspecies are distinguishable but recognizably related. Lactobacillus helveticus gave lower values of D when compared with the three subspecies of Lact. delbrueckii. Similar correlations were obtained with Hind I11 and Eco RI (Table 3), Eco RI and Pst I, Hind I11 and Bgl I1 with the only difference that in general all the values were lower, which could be due to the larger number of fragments obtained after double digestion. The comparison of different strains, belonging to one and the same species, Lact. casei, with other species is presented in Table 4. Relatively lower were the values when Lact. casei was compared with Lact. helveticus and Lact. delbrueckii subsp. bulgaricus. Highest values of D were obtained when the three Lact. casei strains were compared in between. Obviously these strains possess certain genetic similarity but they could be differentiated as well. Similar
DNA F I N G E R P R I N T I N G OF LACTOBACILLUS
Table 4 Comparison of strains of Lactobacillus casei, Lactobacillus helveticus and Lactobacillus deibrueckii subsp. bulgaricus
353
revealed with some of the restriction enzymes M v a I (Fig. 4), Eco RV and Pst I.
Index of similarity ( D ) Enzyme
Hind I11 and Eco RI
Species
Lact. casei A 157 IAM 1118
Strain Lact. casei IAM 1118 543 Lact. delbrueckii 11849 Lact. helveticus 15009
DISCUSSION
543
0.59 0.55
0.48
0.33
0.22
0.32
0.22
0.32
0.25
Lact. delbrueckii 11842
0.21
results with high values of D were obtained when the two Lact. delbrueckii subsp. bulgaricus strains studied were compared (Fig. 2, lanes 1, 4). Finally, the high individual specificity of M I 3 DNA fingerprinting and the possibility of revealing minor genomic differences was shown when some locally isolated strains with similar cultural characteristics were studied. Slight differences in the position of one or two bands were
I
2
3
4
kb
23.1
-
9.4
-
6.5
-
4.3
-
2.0 -
2.3
Fig. 4 M13 DNA fingerprints of DNA from local industrial strains of Lactobacillus helveticus, digested with Mva I
T h e present study showed that optimization of the conditions at all stages of M13 DNA fingerprinting is of great importance for the achievement of reliable and reproducible results in the comparative analysis of strains and species of the genus Lactobacillus. T h e difficulties in the isolation of DNA from lactobacilli are well known and have led to the development of many different procedures aimed at improvement of the cell lysis and the production of high molecular DNA of enough purity. In this work the initial problem associated with DNA shearing, probably due to high intracellular nuclease activity, was partially solved by some modifications of the method used. An important point in obtaining reproducible patterns was the achievement of total cleavage and perfect resolution of the fragments in the agarose gels. T h e promising results with the digoxigenin labelling of M13, the lack of harmful effect and easier handling of nonradioactive probes are also very important especially if we bear in mind the perspective that DNA fingerprinting could become a widely used procedure for classification and identification of Lactobacillus spp. T h e successful application of DNA fingerprinting to the genus Lactobacillus confirmed our previous observations that Gram-positive bacteria need less stringent conditions for hybridization than the Gram-negatives (Miteva et al. 1990). T h e polymorphic fingerprint patterns obtained under the conditions used were unique for a given enzyme and genome. T h e correlation found between the G C content and the patterns observed could be used when unknown strains are studied in order to make a preliminary differentiation according to G + C content. If a wide variety of strains, covering the whole range of G C content, is examined it would be better to use single or double digestions with enzymes recognizing six nucleotides such as Hind 111, Eco RI, Bgl I1 and Pst I. These conclusions prove the importance of the appropriate choice of endonucleases used and the necessity of performing restriction with several different enzymes in parallel, especially when closely related strains are studied. Such an approach could greatly enhance the objectivity of the evaluation and interpretation of the fingerprints. T h e high specificity of the fingerprints permitted the differentiation of species, subspecies and individual Lactobacillus strains. In addition, the quantitative comparative analysis performed enabled us to make some conclusions
+
+
354 V . I . MITEVA ET AL
about the degree of genetic relatedness and the taxonomic position of the species studied. The relatively higher values of the similarity indexes, calculated in the pairwise comparisons of the three subspecies of Lact. delbrueckii confirmed their genetic relatedness and are consistent with their present classification. Collins et al. (1991) proved the close genetic relatedness of the subspecies of Lact. delbrueckii, which were considered in one phylogenetic cluster according to 16s rRNA sequence analysis. Obviously the investigation of different strains within a species by modern molecular methods facilitates the assessment of a more detailed picture of the intraspecific structure. The precise determination of the phylogenetic relatedness of different lactobacilli certainly needs the extension of our DNA fingerprint analysis to additional species and strains, careful choice of restriction endonucleases and reference strains and will be enhanced by the application of computer programs for cluster analysis. The results presented here, although performed with small numbers of strains, confirmed the high species and strain specificity of M13 DNA fingerprinting. This raises the possibility of using this new method for identification and differentiation of lactobacilli at the level of species, subspecies and strains. In combination with other molecular approaches this could lead to the development of new clatsificiation schemes.
ACKNOWLEDGEMENTS This work was supported by a Project grant from the Bulgarian National Foundation for Scientific Research and by ELBY Engineering. The authors express their gratitude to D r Bringle, Boehringer GmbH, for kindly supplying the D I G labelling kit.
REFERENCES C H A T E R ,K . F . , HOPWOOD,D.A., K E I S E R , T . & T O M P S O N C.J. , (1982) Gene cloning in Streptomyces. Current Topics in Microbiology and Immunology 96,69-95. C O L L I N SM.D., , R O D R I G U E SU., , ASH, C., A G U I R E ,M., FARROW J.A.E., , M A R T I N E Z - M U R CA., I A ,P H I L L I P S , B.A., W I L L I A M A.M. S, & WALLBANKS, S. (1991)Phylogenetic analysis of the genus Lactobacillus and related lactic acid bacteria as determined by reverse transcriptase sequencing of 16s rRNA. FEMS Microbiology Letters 77, 5-12. R , (1990) DNA DELLEY, M., MOLLET, B. & H O T T I N G EH. probes for Lactobacillus delbrueckii. Applied and Environmental Microbiology 56 (6), 1967-1970.
DE M A N ,J.C., ROGOSA,M. & S H A R P EM.E. , (1960) A medium for the cultivation of lactobacilli. Journal of Applied Bacteriology 23, 13&135. D I C K SL , . M . T . & V A N V U U R E N H.J.J. , (1987) Relatedness of heterofermentative Lactobacillus species revealed by numerical analysis of total cell protein patterns. International Journal of Systematic Bacteriology 37 (4), 4 3 7 4 0 . H U E Y ,B. & H A L L ,J . (1989) Hypervariable DNA fingerprinting in E. coli. Minisatellite probe from bacteriophage M13. Journal of Bacteriology 171, 252a2532. J E F F R E Y S , A., WILSON,V . & T H E I NS, . (1985) Individual specific fingerprints of human DNA. Nature (London) 316, 76-79. K A N D L E R0. , & WEISS, N. (1986) Regular, nonsporing Gram-positive rods. In Bergey's Manual of Systematic Bacteriology ed. Sneath, P.H.A., Mair, N.S., Sharpe, M.E. & Holt, J.G. Vol. 2, pp. 1209-1234. Baltimore: Williams & Wilkins Co. M A N A C H I NP.L. I, & P A R I N I C. , (1983) DNA restriction endonuclease cleavage patterns, DNA sequence similarity and phenotypical characteristics in some strains of Lactobacillus helveticus and Lactobacillus jugurti. Antonie van Leeuwenhoek 49, 143-152. M I T E V AV., , A B A D J I E V A ,A., I V A N O VP., & G R I G O R O V A , R. (1990) M13 bacteriophages DNA as a probe for DNA fingerprinting in Gram-positive microorganisms. Systematic and Applied Microbiology 13,35&353. M I T E V AV., , A B A D J I E V A ,A. & G R I G O R O V RA., (1991) Differentiation among strains and serotypes of Bacillus thuringiensis by MI3 DNA fingerprinting. Journal of General Microbiology 137, 593-600. NES, I . F . (1984) Plasmid profiles of ten strains of Lactobacillus plantarurn. FEMS Microbiology Letters 21, 35!+361. , (1990) NYBOM,H., ROGSTAD,S . H . & S C A A L B.A. Genetic variation detected by use of the M13 DNA fingerprinting probe in Malus, Prunus and Rubus (Rosaceae). Theoretical and Applied Genetics 79, 153-156. P E T R I C K ,H.A.P., AMBROSIO, R.E. 8i H O L Z A P F E L , W. H. (1988) Isolation of a DNA probe for Lactobacillus curvatus. Applied and Environmental Microbiology 54 (2), 405-408. P I L L O U DN. , & MOLLET,B. (1990) DNA probes for detection of Lactobacillus helveticus. Systematic and Applied Microbiology 13, 345-349. K, RYSKOV,A.P., J I N C H A R A D Z E , A.G., P R O S N Y A M.L., I V A N O VP.L. , & L I M B O R S K S.A. A , (1988) M13 phage DNA as a universal marker for DNA fingerprinting of animals, plants and microorganisms. FEBS Letters 233, 388-392. S O U T H E R E.M. N, (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98, 503-517. S T A H L ,M., MOLIN, G., PERSSON,A . , A H R N E , S. & ST A H L , S . ( 1990) Restriction endonuclease patterns and multivariable analysis as a classification tool for Lactobacillus spp. International Journal of Systematic Bacteriology 40 (2), 189-193. VASSART, G., G E O R G E SM., , M O N S I E U RR., , BROCAS, H., L E Q U A R RA. E , & CHRISTOPHE, D. (1987) A sequence in M13 phage DNA detects hypervariable minisatellites in human and animal DNA. Science Reports 235,683-686.
