Discordant 16S and 23S rRNA gene phylogenies for the genus Helicobacter: implications for phylogenetic inference and systematics

Abstract

Analysis of 16S rRNA gene sequences has become the primary method for determining prokaryotic phylogeny. Phylogeny is currently the basis for prokaryotic systematics. Therefore, the validity of 16S rRNA gene-based phylogenetic analyses is of fundamental importance for prokaryotic systematics. Discrepancies between 16S rRNA gene analyses and DNA-DNA hybridization and phenotypic analyses have been noted in the genus Helicobacter. To clarify these discrepancies, we sequenced the 23S rRNA genes for 55 helicobacter strains representing 41 taxa (>2,700 bases per sequence). Phylogenetic-tree construction using neighbor-joining, parsimony, and maximum likelihood methods for 23S rRNA gene sequence data yielded stable trees which were consistent with other phenotypic and genotypic methods. The 16S rRNA gene sequence-derived trees were discordant with the 23S rRNA gene trees and other data. Discrepant 16S rRNA gene sequence data for the helicobacters are consistent with the horizontal transfer of 16S rRNA gene fragments and the creation of mosaic molecules with loss of phylogenetic information. These results suggest that taxonomic decisions must be supported by other phylogenetically informative macromolecules, such as the 23S rRNA gene, when 16S rRNA gene-derived phylogeny is discordant with other credible phenotypic and genotypic methods. This study found Wolinella succinogenes to branch with the unsheathed-flagellum cluster of helicobacters by 23S rRNA gene analyses and whole-genome comparisons. This study also found intervening sequences (IVSs) in the 23S rRNA genes of strains of 12 Helicobacter species. IVSs were found in helices 10, 25, and 45, as well as between helices 31' and 27'. Simultaneous insertion of IVSs at three sites was found in H. mesocricetorum.

FIG. 1.

Map of primer and IVS locations in helicobacter 23S rRNA gene. The positions of PCR and sequencing primers are shown by arrows with sequence designations above. The positions of four known intervening sequences are indicated by pairs of curly brackets.

FIG. 2.

Neighbor-joining tree based on comparison of 23S rRNA gene sequences. The tree shown is the simple neighbor-joining tree. All sequences are labeled by species, strain number, GenBank accession number in curly brackets, and the species from which it was isolated. The consensus bootstrap neighbor-joining tree had identical topology. The numbers immediately to the left of branches indicate the number of times out of 100 the clade was recovered by bootstrap resampling. The tree is divided into eight numbered clusters.

FIG. 3.

Neighbor-joining tree based on comparison of 16S rRNA gene sequences. The tree shown is the simple neighbor-joining tree. All sequences are labeled as in Fig. 2. Nodes marked with Xs were not shared between the simple neighbor-joining tree and the consensus bootstrapping tree. The tree is divided into clusters. Clusters 1 to 3 correspond to those in the 23S rRNA gene tree, whereas clusters A to E differ from clusters 4 to 8 of the 23S rRNA gene tree.

FIG. 4.

Parsimony trees based on comparison of 23S rRNA gene and 16S rRNA gene sequences. The trees shown are consensus bootstrap trees based on 100 resampled parsimony trees. The numbers at the nodes are the number of times out of 100 resamplings that the node was present. Sequences are labeled as in Fig. 2 with GenBank numbers omitted. The clusters are labeled as in Fig. 2 and Fig. 3.

FIG. 5.

Comparison of 870 H. hepaticus protein sequences to those from H. pylori (Hp), W. succinogenes (Ws), and C. jejuni (Cj). The sequences of 870 proteins shared by the four species were compared by BLASTP analysis. These 870 proteins were selected from all proteins found in these organisms based on reciprocal BLASTP matches with similarity values of E less than e⁻⁵ for all comparisons. The S value is a measure of similarity based on percent identity and length of comparison. The E value is the probability of obtaining an S value by chance based on comparison of the sequence to those in GenBank. The error bars represent standard deviations.

FIG. 6.

Phylogeny of H. hepaticus (Hh), H. pylori (Hp), W. succinogenes (Ws), and C. jejuni (Cj) based on comparison of 870 protein trees. There are six possible trees for a four-taxon comparison. The number of proteins supporting each topology is shown above the respective tree.