Analysis, Characterization, and Loci of the tuf Genes in Lactobacillus and Bifidobacterium Species and Their Direct Application for Species Identification

Abstract

We analyzed the tuf gene, encoding elongation factor Tu, from 33 strains representing 17 Lactobacillus species and 8 Bifidobacterium species. The tuf sequences were aligned and used to infer phylogenesis among species of lactobacilli and bifidobacteria. We demonstrated that the synonymous substitution affecting this gene renders elongation factor Tu a reliable molecular clock for investigating evolutionary distances of lactobacilli and bifidobacteria. In fact, the phylogeny generated by these tuf sequences is consistent with that derived from 16S rRNA analysis. The investigation of a multiple alignment of tuf sequences revealed regions conserved among strains belonging to the same species but distinct from those of other species. PCR primers complementary to these regions allowed species-specific identification of closely related species, such as Lactobacillus casei group members. These tuf gene-based assays developed in this study provide an alternative to present methods for the identification for lactic acid bacterial species. Since a variable number of tuf genes have been described for bacteria, the presence of multiple genes was examined. Southern analysis revealed one tuf gene in the genomes of lactobacilli and bifidobacteria, but the tuf gene was arranged differently in the genomes of these two taxa. Our results revealed that the tuf gene in bifidobacteria is flanked by the same gene constellation as the str operon, as originally reported for Escherichia coli. In contrast, bioinformatic and transcriptional analyses of the DNA region flanking the tuf gene in four Lactobacillus species indicated the same four-gene unit and suggested a novel tuf operon specific for the genus Lactobacillus.

Labeling quality and molecular characterization studies of products containing Lactobacillus spp. strains.

“…As suggested by Ventura et al, 16 the low rate of 16S rDNA gene evolution is often responsible for the failure in identification of highly related bacterial species. In this scenario, the polyphasic taxonomy suggested by Vandamme et al 31 and by other authors, 17,18,32 is the better choice for bacterial identification.…”

“…The probiotic strain used in product III is indicated by depositor as L. casei DG (CNCM I-1572) and is considered sufficiently characterized by EFSA Panel on Dietic Products, Nutrition and Allergies (NDA). 27 However, our study, using multiplex PCR 15 and tuf gene amplification, 16 not used by depositor, identified the strain as L. paracasei. In fact, tuf gene is an important molecular marker to distinguish so related taxa as L. casei group.…”

“…In fact, tuf gene is an important molecular marker to distinguish so related taxa as L. casei group. [16][17][18] The strains of L. rhamnosus GC, L. reuteri DSM 17938, and L. plantarum P 17630 were identified as the species indicated on the label, even if genotypic techniques different from our methods were used by depositor. EFSA Panel on NDA retains L. rhamnosus GG sufficiently characterized at species level, 28 but not L. reuteri DSM 17938.…”

“…Primers used were designed from nucleotide sequences of the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA gene of members of the genus Lactobacillus proposed by Song et al 15 For the specific detection of L. paracasei, L. casei, and L. rhamnosus the strains of Lactobacillus spp. isolated were discriminated by tuf gene amplification described by Ventura et al 16 A detailed description of these procedures was recently reported by two of us. 17,18 …”

Pseudoscardovia radai sp. nov., a representative of the family Bifidobacteriaceae isolated from the digestive tract of a wild pig (Sus scrofa scrofa)

“…PCR conditions and primers for amplification of fusA, rplB and gyrB gene sequences were taken from Delétoile et al (2010). Similarly, conditions for amplification of xfp, tuf, rpoC and dnaK are mentioned in previous studies (Ventura et al, 2003(Ventura et al, , 2005(Ventura et al, , 2006Berthoud et al, 2005). PCR products were then checked, purified and sequenced as described above.…”