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. 2003 Nov;69(11):6908-22.
doi: 10.1128/AEM.69.11.6908-6922.2003.

Analysis, characterization, and loci of the tuf genes in lactobacillus and bifidobacterium species and their direct application for species identification

Marco Ventura  1 , Carlos Canchaya, Valèrie Meylan, Todd R Klaenhammer, Ralf Zink
Affiliations

Analysis, characterization, and loci of the tuf genes in lactobacillus and bifidobacterium species and their direct application for species identification

Marco Ventura et al. Appl Environ Microbiol. 2003 Nov.

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.

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Figures

FIG. 1.
FIG. 1.
Phylogenetic tree of Lactobacillus EF-Tu and Bifidobacterium EF-Tu based on nucleotide sequence homology. The phylogenetic tree was constructed by the neighbor-joining method. The scale bar represents sequence divergence. In this tree topology, the phylogenetic distance between organisms is the sum of the horizontal segments. L. acidophilus group A includes the species L. acidophilus, L. amylovorus, L. gallinarum, and L. crispatus; L. acidophilus group B includes only L. gasseri and L. johnsonii. Bootstrap values are reported for a total of 1,000 replicates. tufA and tufB indicate the tufA and tufB genes, respectively.
FIG. 2.
FIG. 2.
Comparison between genetic distances estimated from 16S rRNA and synonymous distances estimated from tuf genes. The synonymous distances were obtained from the nucleotide sequences of the tuf genes by applying the method of Nei and Gojobori (29). r, coefficient of correlation.
FIG. 3.
FIG. 3.
Comparison of the organization of the tuf loci in bifidobacteria (a) and in lactobacilli (b). (a) B. longum NCC 2705. The DNA sequence fragments of Bifidobacterium represented here are 4.8 kb. (b) L. casei ATCC 334, L. gasseri ATCC 33323, L. johnsonii NCC 533, L. delbrueckii subsp. bulgaricus ATCC BAA-365, and L. plantarum WCFS1. The putative function of a protein is indicated above or below each arrow. Related proteins are linked by blue shading (≥80%) and pink shading (<80%) according to different amino acid similarities; percent amino acid identities are shown. The length of an arrow is proportional to the length of the predicted open reading frame. Corresponding genes are indicated by the same colors of arrows.
FIG.4.
FIG.4.
Southern and Northern hybridization analyses. (a) Southern hybridization analysis of HindIII-digested genomic DNAs of 13 Lactobacillus and 8 Bifidobacterium species with the tuf gene fragment as a probe. Northern hybridization analysis of Lactobacillus RNA and transcription unit mapping of the tuf locus. In panel a, the sizes of hybridizing fragments are shown. The tested strain was the type strain (Table 1). In the top portion of panel b, all predicted open reading frames are indicated and are annotated with their database matches. The positions of the probes used in Northern blot experiments are indicated under the gene map. The transcripts are depicted as arrows; the arrows point to the 3′ end of the mRNA. The length of the arrow is proportional to the length of the mRNA derived from the Northern blots. The estimated sizes of the mRNAs are indicated. Hairpins indicate possible rho-independent terminators. The transcripts are positioned with respect to the gene map. The width of an arrow indicates the relative abundance of the mRNA species. bind., binding. The bottom portion of panel b shows Northern blot hybridization of RNA isolated from lactobacilli with probes corresponding to the open reading frames in the gene map. The sizes calculated for the hybridization signals are provided.
FIG. 5.
FIG. 5.
Primer extension analysis and comparison of the putative promoter sequences of the tuf and trigger factor genes. (a and b) Results of primer extension experiments with oligonucleotides targeting the 5′ end of the tuf gene. P1, putative promoter P1; P2, putative promoter P2. (c) Results of primer extension experiments with an oligonucleotide targeting the 5′ end of the trigger factor gene. The lanes in panels a, b, and c indicate the sequencing products for the tuf and trigger factor genes. (d) Overview of experimentally determined 5′ ends of the tuf gene (tuf) and the trigger factor gene (trf). The endpoints of the primer extension products are underlined. Underlined bold type indicates the −10 and −35 boxes and direct repeats (REP); RBS, Shine-Dalgarno sequences. The start codon is given at the right end of the sequence.
FIG. 6.
FIG. 6.
Amplification products obtained from the tuf multiplex assay. Lane M, 50-bp DNA molecular marker (Sigma); lane m, 1-kb DNA ladder (Gibco BRL). Lane 1, L. casei NCC 2508; lane 2, L. paracasei subsp. paracasei NCC 989; lane 3, L. rhamnosus NCC 2504; lane 4, L. paracasei subsp. paracasei NCC 2461; lane 5, L. rhamnosus ATCC 11443; lane 6, L. reuteri DSM 2006; lane 7, L. fermentum ATCC 14931; lane 8, L. casei NCC 617; lane 9, L. paracasei subsp. paracasei NCC 438; lane 10, L. paracasei subsp. paracasei NCC 476; lane 11, L. paracasei subsp. paracasei NCC 400; lane 12, L. rhamnosus NCC 596; lane 13, L. rhamnosus NCC 587; lane 14, L. rhamnosus NCC 546; lane 15, L. rhamnosus NCC 2488; lane 16, L. rhamnosus NCC 2455; lane 17, L. paracasei subsp. paracasei NCC 1002; lane 18, L. paracasei subsp. paracasei NCC 2548; lane 19, L. paracasei subsp. paracasei NCC 2556; lane 20, L. paracasei subsp. paracasei NCC 171; lane 21, negative control.
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