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Appl Environ Microbiol. Jan 2008; 74(1): 208–215.
Published online Nov 9, 2007. doi:  10.1128/AEM.01711-07
PMCID: PMC2223197

Lactobacillus Strain Diversity Based on Partial hsp60 Gene Sequences and Design of PCR-Restriction Fragment Length Polymorphism Assays for Species Identification and Differentiation[down-pointing small open triangle]

Abstract

A phylogenetic tree showing diversities among 116 partial (499-bp) Lactobacillus hsp60 (groEL, encoding a 60-kDa heat shock protein) nucleotide sequences was obtained and compared to those previously described for 16S rRNA and tuf gene sequences. The topology of the tree produced in this study showed a Lactobacillus species distribution similar, but not identical, to those previously reported. However, according to the most recent systematic studies, a clear differentiation of 43 single-species clusters was detected/identified among the sequences analyzed. The slightly higher variability of the hsp60 nucleotide sequences than of the 16S rRNA sequences offers better opportunities to design or develop molecular assays allowing identification and differentiation of either distant or very closely related Lactobacillus species. Therefore, our results suggest that hsp60 can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genus Lactobacillus and that the chosen primers can be used in a simple PCR procedure allowing the direct sequencing of the hsp60 fragments. Moreover, in this study we performed a computer-aided restriction endonuclease analysis of all 499-bp hsp60 partial sequences and we showed that the PCR-restriction fragment length polymorphism (RFLP) patterns obtainable by using both endonucleases AluI and TacI (in separate reactions) can allow identification and differentiation of all 43 Lactobacillus species considered, with the exception of the pair L. plantarum/L. pentosus. However, the latter species can be differentiated by further analysis with Sau3AI or MseI. The hsp60 PCR-RFLP approach was efficiently applied to identify and to differentiate a total of 110 wild Lactobacillus strains (including closely related species, such as L. casei and L. rhamnosus or L. plantarum and L. pentosus) isolated from cheese and dry-fermented sausages.

Lactobacilli are some of the most important taxa involved in food microbiology and human nutrition owing to their role in food and feed production and preservation, including the probiotic properties exhibited by some strains. These traits are of increasing importance and have received attention in the food and feed industry (2).

At the beginning of 2005, the genus Lactobacillus was reported to include about 100 validly described species (10). However, the number of species is continually changing due to the description of new species and/or reclassification of others: indeed, at the beginning of 2007 this genus had reached about 120 species (http://www.bacterio.cict.fr/).

Their classification into three groups based on metabolism and physiological characteristics (30) is not in agreement with the results of phylogenetic studies by sequencing of 16S rRNA (7). Schleifer and Ludwig (49) reviewed the phylogeny of the genus Lactobacillus based on the 16S rRNA sequences but did not clarify the taxonomy of either the L. casei group or the L. delbrueckii subspecies. Recently, several systematic studies of lactobacilli have been carried out (10, 25, 40, 46, 60). With 16S rRNA-based phylogenetic analysis, at least eight phylogenetic groups have been recognized (10). Investigations by many authors using several molecular approaches focused on closely related species, such as the L. casei-related taxa (6, 9, 12, 38, 44, 47, 57, 61), the L. acidophilus group (19, 32, 41, 45, 47), the L. delbrueckii subspecies (20, 35, 36, 56), and the L. plantarum-related species (54). Nevertheless, the discrimination of very closely related species, especially of subspecies, is often critical (10). Thus, the sequencing of several other genes, such as the tuf gene (encoding elongation factor Tu, involved in protein biosynthesis), mal (encoding malolactic enzyme), the S-layer gene (encoding surface layer proteins), pepC (encoding aminopeptidase C), pepN (encoding aminopeptidase N), htrA (encoding stress-inducible trypsin-like serine protease), recA (encoding recombinase A), and rpoB (encoding RNA polymerase beta subunit), has been used for the discrimination of lactobacilli (4, 16, 22, 24, 42, 55, 58, 59). However, in these last studies, a small number of different Lactobacillus species were analyzed.

In this study, we evaluated the polymorphism within the hsp60 (groEL, encoding a 60-kDa heat shock protein) genes of different Lactobacillus species, with a view to designing molecular systems allowing identification and differentiation of a broad number of species.

MATERIALS AND METHODS

Bacterial strains and culture media.

Fifty reference (type and previously identified) strains, representing 30 different Lactobacillus species, were used in this study (see Table S1 in the supplemental material). Moreover, to validate the implemented hsp60 PCR-restriction fragment length polymorphism (RFLP) approach, 110 wild lactobacilli isolated during previous research in our department were included in the analysis. Of these strains, 80 were isolated during manufacturing of Provolone del Monaco cheese (Table (Table1),1), while the remaining 30 were isolated from different samples of dry-fermented sausages produced in Vallo di Diano (southern Italy) (Table (Table2).2). Both fermented products were traditionally manufactured without starter addition. All isolates were stored in MRS broth (Oxoid) cultures with 20% glycerol at −25°C. Working cultures were prepared in 10 ml of MRS broth (Oxoid) incubated anaerobically for 24 h at either 30 or 37°C.

TABLE 1.
Identification of the wild Lactobacillus strains isolated during manufacturing of Provolone del Monaco cheese by application of the PCR-RFLP assay designed during this study
TABLE 2.
Identification of the wild Lactobacillus strains isolated from different samples of dry-fermented sausages produced in Vallo di Diano (southern Italy) by application of the PCR-RFLP assay designed during this study

DNA isolation.

DNA extraction was carried out from a single colony grown on MRS agar (Oxoid) plates by using InstaGene Matrix (Bio-Rad Laboratories, Hercules, CA), following the conditions described by the supplier.

Amplification and sequencing of the hsp60 gene.

Universal hsp60 oligonucleotide primers H279 and H280, previously described by Goh et al. (21), were used to amplify a 650-bp fragment internal to the hsp60 gene.

PCR amplifications were performed with a 50-μl total volume including 5 μl of the target DNA, 5.0 μl of Taq DNA polymerase 10× buffer (Invitrogen, SG Milanese, Italy), 2.5 μl of 50 mM MgCl2, 0.5 μl of a deoxynucleoside triphosphate mix (25 mM each), 0.125 μl of each primer (0.1 mM), and 0.5 μl of Taq DNA polymerase (5 U/μl) (Invitrogen). The PCR consisted of 40 cycles (30 s at 94°C, 30 s at 37°C, and 1 min at 72°C) and one additional, final cycle at 72°C for 5 min. The PCR amplification fragments were resolved by agarose (2%, wt/vol) gel electrophoresis at 100 V for 2 h. The gel was stained with ethidium bromide, and the bands were visualized under UV illumination at 254 nm.

After gel electrophoresis, the 650-bp PCR fragment was purified by using a QIAquick gel extraction kit (Qiagen S.p.A., Milan, Italy) and sequenced. In order to design a primer set for direct sequencing of the H279-H280 PCR products, the Lactobacillus hsp60 gene sequences found in GenBank (http://www.ncbi.nlm.nih.gov/) or in the cpnDB database (http://cpndb.cbr.nrc.ca/) were aligned. Two primers, LB308F (TGAAGAAYGTNRYNGCYGG) and LB806RM (AANGTNCCVCGVATCTTGTT), were chosen from highly similar regions. These primers target positions 308 to 328 and 806 to 787 of the hsp60 gene, respectively (the nucleotide numbering is based on the hsp60 gene of L. acidophilus CRL639, GenBank accession number AF300645).

The DNA sequences were determined by the dideoxy chain termination method by using a DNA sequencing kit (Perkin-Elmer Cetus, Emeryville, CA), according to the manufacturer's instructions. The sequences were analyzed by MacDNasis Pro v3.0.7 (Hitachi Software Engineering Europe S. A., Olivet Cedex, France), and research for DNA similarity was performed with GenBank and the EMBL database (1).

Phylogenetic analysis was performed using MEGA version 4.0 (50) after multiple alignment of data by ClustalW 1.8 (53). Distance matrix and neighbor-joining methods (48) were applied for tree construction.

PCR-RFLP of the 499-bp internal hsp60 fragments.

PCR-RFLP of 499-bp internal fragments of the hsp60 genes was performed by digesting the PCR product obtained using primers LB308F and LB806RM. For this last PCR amplification, we used the reaction conditions described above, while the thermal condition consisted of 40 amplification cycles (30 s at 95°C, 40 s at 52°C, and 40 s at 72°C) and one additional, final cycle at 72°C for 5 min. Restrictions were performed by digesting 35 μl of a PCR product with 20 U of a restriction enzyme (Promega) in a total volume of 50 μl at 37°C for 5 h. Digested fragments were separated by agarose (2%, wt/vol) gel electrophoresis at 100 V for 3 h.

RESULTS

Sequence analysis of hsp60 gene sequences.

Partial sequences (499 bp) of the hsp60 genes of 52 Lactobacillus strains were determined during this study (see Table S1 in the supplemental material). Both strands of an hsp60 PCR fragment of about 650 bp, obtained by using primers H279 and H280, which were previously described (21), were sequenced by primers LB308F and LB806RM, designed during this study. A total of 116 (including 64 sequences found in the chaperonin database [http://cpndb.cbr.nrc.ca]) Lactobacillus hsp60 partial sequences, representing 50 different species, were compared. The results of neighbor-joining analysis of hsp60 sequences of the 116 strains are shown in the dendrogram depicted in Fig. Fig.11.

FIG. 1.
Neighbor-joining tree based on comparison of 499-bp hsp60 gene sequences showing the phylogenetic relationships between Lactobacillus species. Bootstrap values (expressed as percentages of 1,000 replications) greater than 50% are given at the ...

A total of 43 single species clusters were identified/detected. Due to their high sequence similarity (Fig. (Fig.1)1) and according to some recent systematic studies (11, 17, 31, 39), the pairs L. cypricasei and L. acidipiscis (cluster 27), L. ingluviei and L. thermotolerans (cluster 42), L. vaccinostercus and L. durianis (cluster 39), and L. plantarum and L. arizonensis (cluster 31) were considered synonyms. All strains of L. casei and L. paracasei showed very closely matched hsp60 nucleotide sequences, with the exception of strain L. casei subsp. casei ATCC 4913, which displayed about 4% nucleotide divergence from members of the same cluster (cluster 19). These results are in agreement with those obtained by other studies (4, 9, 12, 13) that considered L. casei and L. paracasei members of the same species.

The topology of the diversity tree from hsp60 nucleotide sequences showed a Lactobacillus species distribution similar, but not identical, to that based on the 16S rRNA gene sequence analysis reported by Dellaglio and Felis (10). In Fig. Fig.1,1, the respective 16S rRNA affiliation group according to Dellaglio and Felis (10) is reported for each species. Our results slightly change the picture presented by Dellaglio and Felis (10) regarding phylogenetic groups. In particular, L. homohiochii (16S rRNA L. buchneri group) showed high levels of similarity to strains of L. zeae (16S rRNA L. casei group), L. kefiranofaciens (16S rRNA L. delbrueckii group) showed high levels of similarity to strains of L. mali (16S rRNA L. salivarius group), and strains of species of the 16S rRNA L. salivarius group were divided into two groups according to hsp60 analysis (clusters 3 and 4 and clusters 26 to 30). Moreover, according to our results strains of the 16S rRNA L. plantarum group were divided into two groups (clusters 23 to 25 and clusters 31 to 33). These two groups correspond to 16S rRNA L. plantarum groups A (including L. plantarum, L. pentosus, and L. paraplantarum) and B (comprising L. alimentarius, L. farciminis, and L. kimchii) designed by Dellaglio and Felis (10). However, our results suggested that these two subgroups are very distant.

Computer-aided (in silico) restriction endonuclease analysis of all Lactobacillus hsp60 partial sequences was also performed by MacDNasis Pro (v3.0.7.) software so as to evaluate their restriction polymorphisms. The endonucleases AluI, BamHI, CfoI, HaeIII, HincII, HindIII, MseI, Sau3AI, and TacI were considered. Since in 115 of the 116 Lactobacillus hsp60 partial sequences analyzed, at least one AluI restriction site exists, this enzyme was initially selected (Table (Table3).3). In theory, on analysis of the AluI PCR-RFLP in silico patterns of the 499-bp hsp60 LB308F-LB806RM PCR fragment, fewer than half of the species considered were differentiated. However, on coupling of the TacI PCR-RFLP in silico patterns with the AluI RFLP in silico pattern, all species were differentiated, with the exception of the L. plantarum/L. pentosus pair (Table (Table3).3). Finally, the latter species were differentiated by digesting the 499-bp hsp60 LB308F-LB806RM PCR fragment with MseI or Sau3AI restriction enzymes (Table (Table44).

TABLE 3.
Positions of AluI and TacI restriction sites in the 499-bp internal hsp60 fragments of the different Lactobacillus species
TABLE 4.
Positions of the MseI and Sau3AI restriction sites in the 499-bp internal hsp60 fragment of the L. pentosus and L. plantarum/arizonensis species

Identification of wild isolates by hsp60 PCR-RFLP analysis.

Two groups of wild isolates, from Provolone del Monaco cheese manufacturing and from dry-fermented sausages, were identified by analyzing their AluI and TacI hsp60 PCR-RFLPs (Tables (Tables11 and and22).

Among strains isolated during manufacturing of Provolone del Monaco cheese, three different AluI hsp60 PCR-RFLPs were obtained: those showing fragments of 330 and 169 bp (pattern A); those displaying fragments of 215, 187, 63, and 34 bp (pattern B); and those showing fragments of 327, 138, and 34 bp (pattern C). Seventy-six strains showed pattern A, 2 strains pattern B, and 2 pattern C (Table (Table1).1). By analysis of the TacI hsp60 PCR-RFLPs of the same strains, three different patterns were also obtained (Table (Table1):1): those showing only one fragment, of 499 bp (pattern a; undigested PCR product); those displaying fragments of 439, 56, and 4 bp (pattern b); and those showing fragments of 278 and 221 bp (pattern c). Considering the molecular weights of the fragments of both RFLPs and considering the results reported in Table Table3,3, strains should be identified as follows (Table (Table1):1): L. casei, combined pattern A-a; L. rhamnosus, A-b; L. plantarum/L. pentosus, B-c; and L. helveticus, C-a. The two strains showing combined pattern B-c and displaying a Sau3AI hsp60 PCR-RFLP containing fragments of 334, 104, and 61 bp were identified as L. plantarum. Further confirmation of hsp60 PCR-RFLP-based identification was obtained, for some strains (Table (Table1),1), by performing hsp60 partial sequence analysis and species-specific PCR assays (18, 55) (data not shown). Finally, from the identification reported in Table Table1,1, it is evident that the Lactobacillus population occurring during the fermentation and ripening processes of the dairy product analyzed is mainly represented by strains of L. casei and L. rhamnosus. Moreover, the isolation medium also has an evident influence on the detection and enumeration of different species: all but one strain isolated on Rogosa agar were referable to L. rhamnosus, while strains isolated on Isolini agar medium were identified as L. casei, L. rhamnosus, L. plantarum, and L. helveticus species.

A single AluI hsp60 PCR-RFLP was obtained from 30 strains isolated from ready-to-eat samples of dry-fermented sausages produced in Vallo di Diano. On the basis of data reported in Table Table3,3, this pattern, showing fragments of 75, 78, 170, and 177 bp, was related to L. curvatus/L. sakei strains. Moreover, these strains, after TacI hsp60 PCR-RFLP analysis, were differentiated as follows: pattern a, L. sakei; pattern d, L. curvatus. Furthermore, also in this case, identification was confirmed by applying species-specific molecular assays (3) (data not shown).

DISCUSSION

Unambiguous identification of lactobacilli is a prerequisite for risk assessment (2). Identification of microbial species by use of phenotypic methods may sometimes be uncertain, complicated, and time-consuming. The use of molecular methods has revolutionized identification, improving its quality and efficacy (9). The techniques used for polyphasic analysis of lactobacilli have been reviewed elsewhere (5, 8). New, simple, and rapid procedures using genus- and species-specific primers for accurate identification of lactobacilli have been proposed. These molecular methods target mainly the rRNA genes or their spacer regions (5, 8). These approaches are particularly useful for rapid and reliable detection, identification, and/or monitoring of one or few Lactobacillus species in a particular environment. However, in the case of environments potentially hosting large numbers of Lactobacillus species, in order to choose the number of species-specific primers/probes to use, the preventive knowledge of the Lactobacillus species potentially occurring is needed. In these cases, more-general molecular approaches should be applied. In fact, many researchers compared partial or full 16S rRNA sequences, 16S rRNA gene PCR-RFLP patterns (43, 51, 59), or intergenic 16S-23S rRNA spacer region PCR-RFLP patterns (37) for the identification of Lactobacillus strains isolated from different environments. However, the 16S rRNA gene shows discrimination pitfalls in the identification of closely related species (10). Other target genes, present in only a single copy, such as the elongation factor Tu gene (tuf) (4, 58), the DNA repair recombinase gene (recA) (10), the RNA polymerase B subunit gene (rpoB) (42), and the chaperonin HSP60 gene (cpn60) (13), have been exploited for the differentiation of Lactobacillus species.

Recently, sequence analysis of hsp60 (encoding a 60-kDa heat shock protein) genes allowed differentiation of species and/or subspecies in different taxa, such as Staphylococcus, Macrococcus, Streptococcus, Lactococcus, Enterococcus, Pediococcus, Vagococcus, Bifidobacterium, Campylobacter, Yersinia, Vibrio, Salmonella, and Shigella species and Escherichia coli (14, 22, 23, 33, 34, 52, 62, 63). These studies highlight the significant advantage of this gene over 16S rRNA due to its higher level of species discrimination. In 2004, a new database based on chaperonin sequences from bacterial and eukaryotic species was established (http://cpndb.cbr.nrc.ca/) (26).

Hill et al. (27) compared ileum microbiota of pigs fed corn-, wheat-, or barley-based diets by chaperonin 60 sequencing and quantitative PCR. More recently, Dumonceaux et al. (15) described PCR primers specifically amplifying the cpn60 genes of six individual species: Clostridium perfringens, Enterococcus faecalis, Enterococcus cecorum, L. amylovorus, L. johnsonii, and Escherichia coli. These data confirm the usefulness of conserved genes for comparing and differentiating related species. hsp60 (groEL) sequence databases cover a considerable number of sequences from Lactobacillus species, and for this reason, these genes can be exploited as targets for detection and identification of organisms, providing a valuable resource in microbial ecology studies as well as in phylogenetic analyses. However, the real intra- and interspecific polymorphism of the hsp60 gene in the Lactobacillus genus has not yet been evaluated. In this study, we designed a set of PCR primers for direct sequencing of the amplified hsp60 gene sequences of Lactobacillus spp. The method described overcomes limitations of the existing PCR-based method applied by Hill et al. (27), which requires time-consuming and complex postamplification steps, such as the cloning of amplification products. A similar strategy was recently applied by Hill and coworkers (28) for direct sequencing of Campylobacter species. The results obtained by analyzing the Lactobacillus hsp60 gene sequences demonstrate the potential utility of this tool for identifying Lactobacillus isolates. Moreover, a robust and rapid approach was applied for the identification and differentiation of Lactobacillus species. The protocol is based on restriction endonuclease analysis of the PCR-amplified hsp60 gene sequences (hsp60 PCR-RFLP). A sequential use of three endonucleases is proposed to simplify Lactobacillus strain identification: first, AluI; then, TacI; and finally, if necessary, Sau3AI (or MseI). Our strategy is similar to the 16S rRNA PCR-RFLP and 16S-23S rRNA ITS PCR-RFLP methods. However, the latter methods require the use of more than three endonucleases (59) and are often unable to differentiate among closely related species, such as L. casei and L. rhamnosus; L. acidophilus and L. crispatus (51); L. acidophilus, L. helveticus, and L. amylovorus (37); and L. plantarum and L. pentosus (43). Moreover, it must be remembered that identification based on 16S rRNA genes may be misleading if closely related species are analyzed (10). 16S rRNA gene sequence analysis is not able to reveal significant differences between recently diverged species, such as L. plantarum, L. paraplantarum, and L. pentosus or L. casei, L. rhamnosus, and L. zeae (10). The hsp60 PCR-RFLP technique is able to differentiate 45 Lactobacillus species. The amenability of this approach has been validated by the unambiguous identification of 110 wild Lactobacillus strains belonging to L. casei, L. rhamnosus, L. plantarum, L. pentosus, L. helveticus, L. curvatus, and L. sakei.

In conclusion, the high variability of the hsp60 nucleotide sequences allows the discrimination of very closely related species, opens new possibilities for a rapid and reliable identification of lactobacilli, and offers good opportunities for development of assays based on hybridization (probe design), PCR (primer design), or DNA chip technologies (microarray design). The results herein obtained clearly highlight the higher usefulness of hsp60 gene analysis than 16S rRNA or 16S-23S rRNA ITS analysis. In fact, hsp60 sequence analysis has confirmed most of the recent systematic studies concerning the taxonomy of Lactobacillus species (10, 11, 17, 31, 39). Therefore, it can be utilized as a reliable marker for identification of either distant or very closely related taxa of the Lactobacillus genus. Moreover, in several species of bacteria, nucleotide sequence analysis of multiple protein-encoding loci has led to reliable phylogenies that have improved our understanding of population structure. A multigenic approach fulfills the recent recommendations of the ad hoc committee for the reevaluation of the definition of the bacterial species. The hsp60 gene can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genus Lactobacillus and may represent a good candidate in multilocus schemes designed for the identification and characterization of lactobacilli. Owing to its specificity, manageability, and rapidity, the hsp60-based PCR-RFLP approach proposed in this study can be considered a valid strategy for typing at the species level Lactobacillus strains isolated from food samples.

Supplementary Material

[Supplemental material]

Acknowledgments

This work was supported by a grant from the Italian Ministry of Higher Education (action PRIN 2004) and by the Italian Ministry of Agriculture (Programme Valorizzazione e salvaguardia della microflora autoctona caratteristica delle produzioni casearie Italiane).

Footnotes

[down-pointing small open triangle]Published ahead of print on 9 November 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

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