Identifying Neisseria species by use of the 50S ribosomal protein L6 (rplF) gene

Abstract

The comparison of 16S rRNA gene sequences is widely used to differentiate bacteria; however, this gene can lack resolution among closely related but distinct members of the same genus. This is a problem in clinical situations in those genera, such as Neisseria, where some species are associated with disease while others are not. Here, we identified and validated an alternative genetic target common to all Neisseria species which can be readily sequenced to provide an assay that rapidly and accurately discriminates among members of the genus. Ribosomal multilocus sequence typing (rMLST) using ribosomal protein genes has been shown to unambiguously identify these bacteria. The PubMLST Neisseria database (http://pubmlst.org/neisseria/) was queried to extract the 53 ribosomal protein gene sequences from 44 genomes from diverse species. Phylogenies reconstructed from these genes were examined, and a single 413-bp fragment of the 50S ribosomal protein L6 (rplF) gene was identified which produced a phylogeny that was congruent with the phylogeny reconstructed from concatenated ribosomal protein genes. Primers that enabled the amplification and direct sequencing of the rplF gene fragment were designed to validate the assay in vitro and in silico. Allele sequences were defined for the gene fragment, associated with particular species names, and stored on the PubMLST Neisseria database, providing a curated electronic resource. This approach provides an alternative to 16S rRNA gene sequencing, which can be readily replicated for other organisms for which more resolution is required, and it has potential applications in high-resolution metagenomic studies.

FIG 1

Evolutionary relationships among 44 Neisseria species based on concatenated sequences from 53 whole ribosomal protein genes and single rplF gene fragments. (a) Concatenated sequences from 53 ribosomal protein genes. (b) rplF gene fragments. Type strains of previous species: *, N. flavescens; **, N. macacae. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The unit of measure for the scale bars is the number of nucleotide substitutions per site.

FIG 2

Evolutionary relationships among Neisseria species based on fragments from 16S rRNA and rplF genes. (a) 16S rRNA gene fragments. (b) rplF gene fragments. ^T, type strain. Type strains of previous species: *, N. flavescens; **, N. macacae; ***, N. sicca; ****, N. mucosa var. heidelbergensis. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The unit of measure for the scale bars is the number of nucleotide substitutions per site.