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.

Aggregation-promoting factor (APF) was originally described as a protein involved in the conjugation and autoaggregation of Lactobacillus gasseri 4B2, whose corresponding apf gene was cloned and sequenced. In this report, we identified and sequenced an additional apf gene located in the region upstream of the previously published one. Inactivation of both apf genes was unsuccessful, indicating that APF function may be essential for the cell. Overproduction of APF proteins caused drastic alteration in the cell shape of this strain. These cells were irregular, twisted, enlarged, and tightly bound in unbreakable clumps of chains. Down-regulation of APF synthesis was achieved by cloning of the apf2 promoter region on a high-copy-number plasmid, which recruited a putative apf activator. As a consequence, the shape of the corresponding recombinant cells was elongated (filamentous) and cell division sites were no longer visible. None of the induced changes in APF production levels was clearly correlated with modifications of the aggregation phenotype. This report shows, for the first time, that APF proteins are mainly critical for L. gasseri 4B2 cell shape maintenance.Lactic acid bacteria are normal inhabitants of the human oral cavity and gastrointestinal (GI) and urogenital tracts. Interest in the health-promoting effects of specific lactic acid bacterial strains has increased in recent years (10, 12). Survival and persistence of probiotic bacteria in the human GI tract is often reported to provide them a competitive advantage. One of the proposed mechanisms that could increase the potential of bacteria to survive and persist in the GI tract is their ability to aggregate (13). However, this hypothesis has never been confirmed.To study the importance of aggregation, we chose an aggregating strain, Lactobacillus gasseri 4B2 (previously classified as L. plantarum 4B2), whose 32-kDa aggregation-promoting factor (APF) has already been described (25). This protein was purified from the culture supernatant as one of the most abundant proteins. The function of APF as a factor of aggregation was shown in vitro. After three subsequent washing steps, L. gasseri 4B2 lost its ability to aggregate. Addition of purified APF or of filtered supernatant reaggregated the washed cells. Furthermore, the role of APF in conjugation was also demonstrated (25). The frequency of pAM␤1 conjugal transfer (among Lactobacillus species whose aggregation is APF dependent) was increased upon addition of L. gasseri 4B2 filtered supernatant to the mating mixture. Since neither the apf gene nor any apf mutant was available, the exact physiological role of APF could not be demonstrated. Subsequent N-terminal sequencing of the L. gasseri 4B2 APF protein enabled the cloning and sequencing of the apf gene (accession no. Y08498; L. Morelli et al., direct submission).Recently, we showed that four L. johnsonii and two L. gasseri strains contain two apf genes in their genomes (34). Analysis of the gene organization, amino acid composition, and physical propert...

Eighty-nine Bifidobacterium strains from 26 species were identified and classified to the species level with an enterobacterial repetitive intergenic consensus (ERIC)-PCR approach. We demonstrated that ERIC-PCR is useful for a phylogenetic and taxonomical analysis but as well as for a species composition analysis of mixed bifidobacterial cultures isolated from dairy products and other environments.