Open AccessLactobacillus Strain Diversity Based on Partial hsp60 Gene Sequences and Design of PCR-Restriction Fragment Length Polymorphism Assays for Species Identification and Differentiation
Author(s) -
Giuseppe Blaiotta,
Vincenzina Fusco,
Danilo Ercolini,
Maria Aponte,
Olimpia Pepe,
Francesco Villani
Publication year - 2008
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.01711-07
Subject(s) - biology , genetics , phylogenetic tree , restriction enzyme , restriction fragment length polymorphism , hsp60 , 16s ribosomal rna , lactobacillus , gene , ribosomal rna , polymerase chain reaction , computational biology , microbiology and biotechnology , heat shock protein , bacteria , hsp70
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 andtuf gene sequences. The topology of the tree produced in this study showed aLactobacillus 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 thehsp60 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 relatedLactobacillus species. Therefore, our results suggest thathsp60 can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genusLactobacillus and that the chosen primers can be used in a simple PCR procedure allowing the direct sequencing of thehsp60 fragments. Moreover, in this study we performed a computer-aided restriction endonuclease analysis of all 499-bphsp60 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 43Lactobacillus species considered, with the exception of the pairL. plantarum /L. pentosus . However, the latter species can be differentiated by further analysis with Sau3AI or MseI. Thehsp60 PCR-RFLP approach was efficiently applied to identify and to differentiate a total of 110 wildLactobacillus strains (including closely related species, such asL. casei andL. rhamnosus orL. plantarum andL. pentosus ) isolated from cheese and dry-fermented sausages.