Phylogeny of γ-proteobacteria inferred from comparisons of 3’ end 16S rRNA gene and 5’ end 16S-23S ITS nucleotide sequences

Abstract

The phylogeny of γ-proteobacteria was inferred from nucleotide sequence comparisons of a short 232 nucleotide sequence marker. A total of 64 γ-proteobacterial strains from 13 Orders, 22 families, 40 genera and 59 species were analyzed. The short 232 nucleotide sequence marker used here was a combination of a 157 nucleotide sequence at the 3' end of the 16S rRNA gene and a 75 nucleotide sequence at the 5' end of the 16S-23S Internal Transcribed Spacer (ITS) sequence. Comparative analyses of the 3' end of the 16S rRNA gene nucleotide sequence showed that the last 157 bp were conserved among strains from same species and less conserved in more distantly related species. This 157 bp sequence was selected as the first part in the construction of our nucleotide sequence marker. A bootstrapped neighbor-joining tree based on the alignment of this 157 bp was constructed. This 157 bp could distinguish γ-proteobacterial species from different genera from same family. Closely related species could not be distinguished. Next, an alignment of the 16S-23S ITS nucleotide sequences of alleles from same bacterial strain was performed. The first 75 bp at the 5' end of the 16S-23S ITS was highly conserved at the intra-strain level. It was selected as the second part in the construction of our nucleotide sequence marker. Finally, a bootstrapped neighbor-joining tree based on the alignment of this 232 bp sequence was constructed. Based on the topology of the neighbour-joining tree, four major Groups, Group I to IV, were revealed with several sub-groups and clusters. Our results, based on the 232 bp sequence were, in general, in agreement with the phylogeny of γ-proteobacteria based on the 16S rRNA gene. The use of this 232 bp sequence as a phylogenetic marker presents several advantages over the use of the entire 16S rRNA gene or the generation of extensive phenotypic and genotypic data in phylogenetic analyses. First, this marker is not allele-dependant. Second, this 232 bp marker contains 157 bp from the 3' end of the 16S rRNA gene and 75 bp from the 5' end of the 16S-23S ITS. The 157 bp allows discrimination among distantly related species. Owing to its higher rate of nucleotide substitutions, the 75 bp adds discriminating power among closely related species from same genus and closely related genera from same family. Because of its higher percentage of nucleotide sequence divergence than the 16S rRNA gene, the 232 bp marker can better discriminate among closely related γ-proteobacterial species. Third, the method is simple, rapid, suited to large screening programs and easily accessible to most laboratories. Fourth, this marker can also reveal γ-proteobacterial species which may appear misassigned and for which additional characterization appear warranted.

Assessment of a short phylogenetic marker based on comparisons of 3' end 16S rDNA and 5' end 16S-23S ITS nucleotide sequences on the genus Xanthomonas

“…Owing to its higher rate of nucleotide substitutions, the 67-75 bp from the 5' end of the 16S-23S ITS added resolving power among closely related species from same genus. This marker had proven useful in reconstructing the phylogenies of the genus Bacillus and closely-related genera [32], the Order Bacillales [33] and the Class γ-proteobacteria [26] in accordance with accepted phylogenies inferred from much more comprehensive datasets. This marker presented several advantages over the use of the entire 16S rRNA gene or the 16S-23S ITS or the generation of extensive phenotypic and genotypic data in phylogenetic analyses.…”

“…In previous studies, we showed that a DNA sequence from 3' end 16S rRNA gene and 5' end 16S-23S ITS could be used as a marker in the reconstruction of phylogenies in the Gram-positive genus Bacillus and closely-related genera [32], the Gram-positive Order Bacillales [33], and the Gram-negative Class γ-proteobacteria [26]. This maker ranged in size from 220 bp to 232 bp.…”

“…In a recent study [26], a short 232 bp nucleotide sequence "marker" was used to reconstruct the phylogeny of the Class γ-proteobacteria. This 232 bp marker was a combination of the last 157 bp at the 3' end of the 16S rRNA gene and the first 75 bp at the 5' end of the 16S-23S rRNA internal transcribed spacer (ITS).…”

Assessment of a short phylogenetic marker based on comparisons of 3’ end 16S rDNA and 5’ end 16S-23S ITS nucleotide sequences of the Bacillus cereus group

“…More recently, we assessed the usefulness of the 220 bp marker by extending its analyses at a higher taxonomic level, the Gram-positive Order Bacillales [29]. In parallel, a similar marker was used to infer the phylogeny of the Gram-negative Class γ-proteobacteria [33]. In the current study, we further analyze the resolving power of this marker in inferring the phylogeny at a much lower taxa level: the Bacillus cereus group.…”

Phylogeny of the Order Bacillales inferred from 3’ 16S rDNA and 5’ 16S-23S ITS nucleotide sequences

“…Recently, in parallel, a similar maker was tested and shown to be able to reconstruct the phylogeny of the Class γ-proteobacteria [19].…”