Distinction between Borrelia and Borreliella is more robustly supported by molecular and phenotypic characteristics than all other neighbouring prokaryotic genera: Response to Margos' et al. "The genus Borrelia reloaded" (PLoS ONE 13(12): e0208432)

Abstract

In a recent publication in PLOS ONE, Gabriele Margos and colleagues have questioned the division of the genus Borrelia into two genera on the basis that the differences in percentage of conserved proteins (POCP) between these two groups is >50%, which an earlier study has suggested as the threshold for differentiating prokaryotic genera. However, the POCP threshold is a poorly characterized and rarely used criterion for establishing distinction among prokaryotic genera. Detailed evaluation of the intergeneric POCP values for 37 genera from 3 different families (viz. Enterobacteriaceae- 24 genera, Morganellaceae-8 genera and Cystobacteraceae-5 genera) presented here shows that the POCP values for all genera within each of these families exceeded >58%. Thus, the suggested POCP threshold is not a useful criterion for delimitation of genus boundary and the objection by Margos et al. on this ground is invalid. Additionally, Margos et al. have questioned the specificities of ~15-20% of the conserved signature indels (CSIs) described in our work. However, as shown here, this concern is due to misunderstanding of the results and the CSIs in question are still highly-specific characteristics of the members of these genera and they provide important information regarding the evolutionary relationships of two new reptiles-echidna-related species viz. Borrelia turcica and Candidatus Borrelia tachyglossi to other Borrelia species. Results presented here show that both these species are deeper-branching members of the genus Borrelia and their placement within this genus is strongly supported by phylogenetic analyses and multiple uniquely shared CSIs with the other Borrelia species. Based on the large body of evidence derived from phylogenetic, genomic, molecular, phenotypic and clinical features, it is contended that the characteristics clearly distinguishing the Borrelia and Borreliella genera are far more numerous and of different kinds than those discerning most (all) other neighbouring genera of prokaryotes. Thus, the placement of these two groups of microorganisms into distinct genera, Borrelia and Borreliella, which clearly recognizes the differences among them, is highly appropriate and it should lead to a better understanding of the clinical, molecular and biological differences between these two important groups of microbes.

Fig 1. A comparison matrix showing the averages of the percentage of conserved proteins (POCP) within and between different genera of the family Enterobacteriaceae.

POCP was determined for all genome sequenced species from the family Enterobacteriaceae detailed in our earlier work [26]. The values along the diagonal shows the average POCP values for different species within a given genus (i.e. interspecies values), whereas all other values represent average intergeneric POCP values for different genera within this family. The blank cells indicate that only a single species was available for these genera and hence their interspecies values could not be calculated.

Fig 2. A pair-wise comparison matrix based on percentage of conserved proteins (POCP) in chromosomal genes from different genome sequenced Borreliaceae species.

The matrix was constructed using an internally developed pipeline [13,14]. Genome pairs sharing higher POCP are shaded more darkly (red). Based on their POCP values, species belonging to the family Borreliaceae form two main groups, with one group containing all of the LD and related species (or Borreliella), and the other encompassing RF group of species together with the reptile-and echidna- associated species B. turcica and Candidatus Borrelia tachyglossi (genus Borrelia).

Fig 3. Phylogenetic trees showing the branching of Borreliaceae species.

(A) A maximum-likelihood (ML) tree based on concatenated sequences of 703 core proteins found in the genomes of Borreliaceae species; (B) A tree based on sequence alignment for the RNA polymerase β’- subunit (RpoC protein). (C) A ML tree for Borreliaceae species based on 16S rRNA gene sequences.

Fig 4. A summary diagram showing the species specificities of different CSIs reported in our earlier work [6].

The CSIs described in our earlier work were of two kinds. Panels (A) and (B) present the results for CSIs, where sequence information for outgroup species was available, whereas panels (C) and (D) show results for CSIs which are found in proteins that are limited to the Borreliaceae species (i.e. no homologs in any outgroup species). Panels (A) and (C) show the results as reported earlier [6], whereas panels (B) and (D) show how the observed specificities of the CSIs have been affected upon inclusion of sequences for B. turcica and Candidatus Borrelia tachyglossi. The asterisks (*) marks the CSIs whose specificities have been questioned by Margos et al. [10]. As shown here and as discussed in the text, these CSIs remain specific for the RF group (genus Borrelia) in addition to providing important information regarding the branching or phylogenetic placement of B. turcica and Candidatus Borrelia tachyglossi within the genus Borrelia and family Borreliaceae.

Fig 5. Partial sequence alignments of two CSIs in proteins with outgroup species that were previously reported as specific for the RF clade.

Panel (A) shows a 6 aa insert in a hypothetical protein BDU327 (BB_0326) that is specifically found in all members of the genus Borrelia including B. turcica and Candidatus Borrelia tachyglossi. (B) This panel shows a 1 aa insert in the L-lactate permease protein, which is only shared by all RF clade species but is absent in the B. turcica and Candidatus Borrelia tachyglossi homologs, which are deeper branching members of the genus Borrelia (see Figs 3 and 4). Dashes (-) in all alignments shows sequence identity with the amino acids on the top line.

Fig 6. Partial sequence alignments of three CSIs in proteins found only in the Borreliaceae species providing differentiation among members of the genera Borrelia and Borreliella.

(A) This panel shows a 2 aa CSI in a hypothetical protein BT0110 that differentiates the members of the genera Borrelia and Borreliella. Twenty nine other CSIs also show a similar species distribution (Table 3). Due to the absence of outgroup species it is difficult to infer whether this CSI is an insert in the genus Borrelia or a deletion in the genus Borreliella. (B) A 3 aa CSI in a putative lipoprotein that is specific for the RF clade of species. Due to the absence of this CSI in the LD clade as well as in B. turcica and Candidatus Borrelia tachyglossi homologs this CSI is an insert in the RF clade of species (see Fig 4). (C) A 2 aa CSI in DNA polymerase III subunit delta, which is commonly shared by the LD clade of species and Cand. Borrelia tachyglossi, but absent in B. turcica and the RF group of species. Based on its species distribution, this CSI is inferred to be an insert in a common ancestor of the RF clade and B. turcica (see Fig 4 for additional information).