Restriction Endonuclease Patterns and Multivariate Analysis as a Classification Tool for Lactobacillus spp.

Abstract

Three Lactobacillus plantarum and seven Lactobacillus reuteri strains were studied by using restriction endonuclease analysis (REA) combined with principal-component analysis (PCA) and soft independent modeling of class analogy (SIMCA). Chromosomal DNAs from the strains were extracted and cleaved with restriction enzymes, and the DNA fragments were separated according to size by agarose gel electrophoresis. Band patterns were read by using a laser densitometer. The procedure to obtain reproducible digestion patterns with a suitable number of DNA fragments was optimized. Asp 718, ClaI and EcoRI were the most suitable of the 17 restriction enzymes tested, each giving 30 to 50 DNA fragments down to a molecular weight of 4.2 X lo6. The digestion patterns of all three enzymes, which gave a data set for 10 strains and 80 variables, were used for classification by PCA and SIMCA. All strains were clearly separated, and the separation within each species was in general accordance with data on DNA-DNA homology found in the literature. We concluded that restriction endonuclease analysis, combined with PCA and SIMCA, can be used for the classification of Lactobacillus spp.The genus Lactobacillus comprises about 50 validly described species; for historical reasons, in this genus circumscription of taxa has been based mainly on phenotypic features (e.g., the ability to ferment carbohydrates). Furthermore, the members of the genus Lactobacillus are formally differentiated from members of the genera Leuconostoc, Lactococcus, and Pediococcus on morphological grounds; in several cases this differentiation has caused systematic problems, as it can be diacult to distinguish between rods and cocci (23). Numerical studies have revealed the weakness in the phenotypic foundation (5,19). The systematics of this group have been improved by chemotaxonomy and DNA-DNA homology studies. Thus, the genus consists of deterministically defined taxa, but the relationships among the taxa are vague.Examples of characteristics that indicate the weakness in the present definition of the genus Lactobacillus are (i) the wide range in guanine-plus-cytosine contents of DNAs from different Lactobacillus species (32 to 53 mol%), (ii) the different kinds of cross-linkage of the peptidoglycans in cell walls, and (iii) the mingling in a phylogenetic tree of Lactobacillus, Leuconostoc, and Pediococcus strains as indicated by rRNA sequencing data (25). Thus, there seems to be a need to study the genus Lactobacillus with reclassification in mind. What method should be applied? Sequencing of rRNA is technically difficult and labor intensive. The accuracy of using enzymes as genetic markers (15) can be questioned as long as the majority of the information in the DNA remains unknown. And DNA-DNA homology is, unfortunately, not suitable outside the constraints of highly related species. An alternative method is restriction endonuclease analysis (REA), which, compared with rRNA sequencing, is technically easy to handle and may, in contrast to DNA-DNA homology, have g...

Classification of Erysipelothrix Strains on the Basis of Restriction Fragment Length Polymorphisms

“…The strains were grown in brain heart infusion broth supplemented with horse serum (5%). Chromosomal DNA was extracted by the method of Stdhl et al (15). One microgram of the DNAs was digested with 10 U of EcoRI (Boehringer Mannheim, Scandinavia AB, Bromma, Sweden) for 4 h at 37°C in the buffer supplied by the manufacturer.…”

“…RFLPs. Gels were run for 18 h as described earlier (15,16). Phage A DNA, cleaved with Hind111 and digoxigenin labeled (Boehringer Mannheim), was used as a fragment size marker.…”

Characterisation of lactic acid bacteria from spoiled, vacuum-packaged, cold-smoked rainbow trout using ribotyping

“…Paludan-Müller et al (1998), studying the role of Carnobacterium species in the spoilage of vacuum-packaged, cold-smoked salmon, used whole-cell-protein patterns for identification of the carnobacteria. Simple restriction of DNA (Manchini and Parini, 1983;Ståhl et al, 1990) has been used as a tool for separating different species of lactobacilli, and ribotyping (Grimont and Grimont, 1986), which is based on patterns generated from the ribosomal RNA genes only, has also been applied with success for LAB (Rodtong and Tannock, 1993;Korkeala, 1996a, 1996b;.…”

rRNA gene restriction patterns as a characterization tool for Lactobacillus sake strains producing ropy slime

“…restriction endonuclease analyses without probing 13 the fragments have previously been used as a tool for separating different species (Ståhl et al 1990). …”