Intra-chromosomal heterogeneity between the four 16S rRNA gene copies in the genus Veillonella: implications for phylogeny and taxonomy

Abstract

Among the seven species characterized within the genus Veillonella, three (Veillonella dispar, Veillonella parvula and Veillonella atypica) have so far been isolated from human flora and during infectious processes. Sequencing and analysis of 16S rDNA (rrs) has been described as the best method for identification of Veillonella strains at the species level since phenotypic characteristics are unable to differentiate between species. rrs sequencing for the three species isolated from humans showed more than 98 % identity between them. Four rrs copies were found in the reference strains and in all the clinical isolates studied. The sequences of each rrs were determined for the clinical strain ADV 360.1, and they showed a relatively high level of heterogeneity (1?43 %). In the majority of cases, polymorphic positions corresponded to nucleotides allowing differentiation between the three species isolated from humans. Moreover, variability observed between rrs copies was higher than that between 16S rDNA sequences of V. parvula and V. dispar. Phylogenetic analysis showed that polymorphism between rrs copies affected the position of strain ADV 360.1 in the tree. Variable positions occurred in stems and loops belonging to variable and hypervariable regions of the 16S rRNA secondary structure but did not change the overall structure of the 16S rRNA. PCR-RFLP experiments performed on 27 clinical isolates of Veillonella sp. suggested that inter-rrs heterogeneity occurs widely among the members of the genus Veillonella. These results, together with the lack of phenotypic criteria for species differentiation, give preliminary arguments for unification of V. dispar and V. parvula.

Oligonucleotide microarray methodology for taxonomic and functional monitoringof microbial community composition

“…sequencing errors, wrong taxonomic affiliation of targets, under-representation of certain groups) or the targeted gene itself. In the case of rrs, this includes (in addition to its limited taxonomic resolution, as mentioned above) different copy numbers of ribosomal operons in bacterial genomes (up to 15, Klappenbach et al 2001), rrs gene polymorphism within a single species (Marchandin et al 2003, González-Escalona et al 2005) and (rarely) horizontal transfer (Schouls et al 2003), which may affect identification and abundance assessment based on rrs probes.…”

Veillonella denticariosi sp. nov., isolated from human carious dentine

“…nov., from carious dentine DNA was extracted from cells collected from Veillonella agar plates resuspended in 50 mM Tris/HCl (pH 8.0) using the QIAamp DNA Mini kit (Qiagen) as described by the manufacturer. PCR and sequencing of the 16S rRNA and dnaK genes was performed using primers described previously (Marchandin et al, 2003;Paster et al, 2001). Sequences were aligned using the program CLUSTAL_X (version 1.83) and phylogenetic analysis was performed using MEGA (version 3.1).…”

Veillonella magna sp. nov., isolated from the jejunal mucosa of a healthy pig, and emended description of Veillonella ratti

“…Phylogenetic analysis with MEGA software (version 4.0) (Tamura et al, 2007) was conducted to infer an evolutionary tree using the neighbour-joining method (Saitou & Nei, 1987) based on the Kimura two-parameter model (Kimura, 1980). The taxonomic position of strain lac18 T in the genus Veillonella was further resolved by means of partial sequence (703 bp) analysis of the 70 kDa heat-shock-protein gene (dnaK) as proposed by Marchandin et al (2003). Phylogenetic analysis in BLAST was carried out as described for the 16S rRNA gene.…”