Lactobacillus strain diversity based on partial hsp60 gene sequences and design of PCR-restriction fragment length polymorphism assays for species identification and differentiation.
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.
Blaiotta
Giuseppe
G
Department of Food Science, School of Agriculture, The University of Naples Federico II, via Università 100, 80055 Portici, Italy. blaiotta@unina.it
Fusco
Vincenzina
V
Ercolini
Danilo
D
Aponte
Maria
M
Pepe
Olimpia
O
Villani
Francesco
F
D001426:Bacterial Proteins / Q000235:genetics*; D018834:Chaperonin 60 / Q000235:genetics*; D002611:Cheese / Q000382:microbiology; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism; D005516:Food Microbiology; D007778:Lactobacillus / Q000145:classification* / Q000235:genetics*; D008461:Meat Products / Q000382:microbiology; D008969:Molecular Sequence Data; D010444:Peptide Elongation Factor Tu / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D018834:Chaperonin 60; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; C024669:endodeoxyribonuclease AluI; C055927:endodeoxyribonuclease MseI; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases; D010444:Peptide Elongation Factor Tu