A phylogenomic and molecular signature based approach for characterization of the phylum Spirochaetes and its major clades: proposal for a taxonomic revision of the phylum

Abstract

The Spirochaetes species cause many important diseases including syphilis and Lyme disease. Except for their containing a distinctive endoflagella, no other molecular or biochemical characteristics are presently known that are specific for either all Spirochaetes or its different families. We report detailed comparative and phylogenomic analyses of protein sequences from Spirochaetes genomes to understand their evolutionary relationships and to identify molecular signatures for this group. These studies have identified 38 conserved signature indels (CSIs) that are specific for either all members of the phylum Spirochaetes or its different main clades. Of these CSIs, a 3 aa insert in the FlgC protein is uniquely shared by all sequenced Spirochaetes providing a molecular marker for this phylum. Seven, six, and five CSIs in different proteins are specific for members of the families Spirochaetaceae, Brachyspiraceae, and Leptospiraceae, respectively. Of the 19 other identified CSIs, 3 are uniquely shared by members of the genera Sphaerochaeta, Spirochaeta, and Treponema, whereas 16 others are specific for the genus Borrelia. A monophyletic grouping of the genera Sphaerochaeta, Spirochaeta, and Treponema distinct from the genus Borrelia is also strongly supported by phylogenetic trees based upon concatenated sequences of 22 conserved proteins. The molecular markers described here provide novel and more definitive means for identification and demarcation of different main groups of Spirochaetes. To accommodate the extensive genetic diversity of the Spirochaetes as revealed by different CSIs and phylogenetic analyses, it is proposed that the four families of this phylum should be elevated to the order level taxonomic ranks (viz. Spirochaetales, Brevinematales ord. nov., Brachyspiriales ord. nov., and Leptospiriales ord. nov.). It is further proposed that the genera Borrelia and Cristispira be transferred to a new family Borreliaceae fam. nov. within the order Spirochaetales.

Keywords: Borreliaceae; Brachyspiriales; Leptospiriales; Spirochaetaceae; Spirochaetes; Spirochaetes phylogeny and taxonomy; conserved signature indels; molecular signatures.

Figure 1

A phylogenetic tree of genome sequenced members of the phylum Spirochaetes based on the concatenated amino acid sequences of 22 conserved proteins. The tree shown is a maximum-likelihood (ML) distance tree. Bootstrap values are shown at branch nodes for both maximum-likelihood and neighbor-joining tree construction methods as ML/NJ. The different sequenced families and two main clades of the family Spirochaetaceae supported by the tree are marked. The letter ^T refers to the type strain of the species.

Figure 2

A ML tree based on the 16S rRNA gene sequences of representative species from cultured genera within the phylum Spirochaetes. Bootstrap values are shown at branch nodes. The different families of the phylum Spirochaetes are marked. The letter^T refers to the type strain of the species. The accession numbers of the 16S rRNA gene sequences used in this analysis are provided in Supplemental Table 1.

Figure 3

A partial sequence alignment of the flagellar basal-body rod protein FlgC, showing a CSI (boxed) that is uniquely present in all members of the phylum Spirochaetes. Sequence information for only a limited number of species from the Spirochaetes and other bacteria is shown here, but unless otherwise indicated similar CSIs were detected in all members of the indicated group and not detected in any other bacterial species in the top 250 Blastp hits. The dashes (−) in the alignments indicate identity with the residue in the top sequence. GenBank identification (GI) numbers for each sequence are indicated in the second column. Sequence homologs for this protein were not identified from members of the genus Sphaerochaeta.

Figure 4

A partial sequence alignment of the protein alanyl-tRNA synthetase showing a two amino acid insertion (boxed) identified in homologs from the family Spirochaetaceae, but not found in the sequence homologs of any other sequenced bacteria. Sequence information for other Spirochaetaceae specific CSIs is presented in Supplemental Figures 3–6 and summarized in Table 2.

Figure 5

Partial sequence alignments of (A) Flagellar hook-associated protein FlgK and (B) DNA polymerase I, showing two CSIs that are specific for the family Brachyspiraceae, but not found in the sequence homologs of any other sequenced bacteria. Sequence homologs for flagellar hook-associated protein FlgK were not identified from members of the genus Sphaerochaeta. Sequence information for other Brachyspiraceae specific CSIs is presented in Supplemental Figures 7–10 and summarized in Table 3.

Figure 6

Partial sequence alignments of (A) 50S Ribosomal protein L14 and (B) Alanyl-tRNA synthetase, showing two CSIs that are specific for the family Leptospiraceae, but not found in the sequence homologs of any other sequenced bacteria. Sequence information for other Leptospiraceae specific CSIs is presented in Supplemental Figures 11–13 and summarized in Table 4.

Figure 7

(A) Partial sequence alignment of the protein 6-phosphofructokinase (pyrophosphate) containing a 1 amino acid insert in a conserved region that is specifically present in the species from the genera Treponema, Spirochaeta, and Sphaerochaeta, but not found in any other sequenced bacteria. (B) Partial sequence alignment of phosphofructokinase containing a 6 amino acid insert that is specific for the genera Borrelia. Sequence information for other CSIs showing similar specificities is provided in Table 5 and in Supplemental Figures 14–30.

Figure 8

A summary diagram depicting the distribution of identified CSIs and the proposed reclassification of the groups within the phylum Spirochaetes. A representative strain is listed for each genome sequenced species. The letter ^T refers to the type strain of the species.