rpoB gene sequencing for identification of Corynebacterium species

Abstract

The genus Corynebacterium is a heterogeneous group of species comprising human and animal pathogens and environmental bacteria. It is defined on the basis of several phenotypic characters and the results of DNA-DNA relatedness and, more recently, 16S rRNA gene sequencing. However, the 16S rRNA gene is not polymorphic enough to ensure reliable phylogenetic studies and needs to be completely sequenced for accurate identification. The almost complete rpoB sequences of 56 Corynebacterium species were determined by both PCR and genome walking methods. In all cases the percent similarities between different species were lower than those observed by 16S rRNA gene sequencing, even for those species with degrees of high similarity. Several clusters supported by high bootstrap values were identified. In order to propose a method for strain identification which does not require sequencing of the complete rpoB sequence (approximately 3,500 bp), we identified an area with a high degree of polymorphism, bordered by conserved sequences that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (434 to 452 bp) allows accurate species identification and may be used in the future for routine sequence-based identification of Corynebacterium species.

FIG. 1.

Graphical representation of RSVs (y axis) in the rpoB and 16S rRNA gene sequences of Corynebacterium species studied by the use of windows of 50 nucleotides (the x axis indicates the nucleotide position). The hypervariable region bordered by conserved regions used for species identification with primers C2700F and C3130R is framed.

FIG. 2.

Dendrogram representing the phylogenetic relationships of Corynebacterium species obtained by the neighbor-joining method. The tree was derived from the alignments of rpoB gene sequences. The support of each branch, as determined from 1,000 bootstrap samples, is indicated by the value at each node (in percent).

FIG. 3.

Dendrogram representing the phylogenetic relationships of Corynebacterium species obtained by the neighbor-joining method. The tree was derived from alignment of 16S rRNA gene sequences. The support of each branch, as determined from 1,000 bootstrap samples, is indicated by the value at each node (in percent).