AppIndels.com server: a web-based tool for the identification of known taxon-specific conserved signature indels in genome sequences. Validation of its usefulness by predicting the taxonomic affiliation of >700 unclassified strains of Bacillus species.

Taxon-specific conserved signature indels (CSIs) in genes/proteins provide reliable molecular markers (synapomorphies) for unambiguous demarcation of taxa of different ranks in molecular terms and for genetic, biochemical and diagnostic studies. Because of their predictive abilities, the shared presence of known taxon-specific CSIs in genome sequences has proven useful for taxonomic purposes. However, the lack of a convenient method for identifying the presence of known CSIs in genome sequences has limited their utility for taxonomic and other studies. We describe here a web-based tool/server (AppIndels.com) that identifies the presence of known and validated CSIs in genome sequences and uses this information for predicting taxonomic affiliation. The utility of this server was tested by using a database of 585 validated CSIs, which included 350 CSIs specific for ≈45 Bacillales genera, with the remaining CSIs being specific for members of the orders Neisseriales, Legionellales and Chlorobiales, family Borreliaceae, and some Pseudomonadaceae species/genera. Using this server, genome sequences were analysed for 721 Bacillus strains of unknown taxonomic affiliation. Results obtained showed that 651 of these genomes contained significant numbers of CSIs specific for the following Bacillales genera/families: Alkalicoccus, 'Alkalihalobacillaceae', Alteribacter, Bacillus Cereus clade, Bacillus Subtilis clade, Caldalkalibacillus, Caldibacillus, Cytobacillus, Ferdinandcohnia, Gottfriedia, Heyndrickxia, Lederbergia, Litchfieldia, Margalitia, Mesobacillus, Metabacillus, Neobacillus, Niallia, Peribacillus, Priestia, Pseudalkalibacillus, Robertmurraya, Rossellomorea, Schinkia, Siminovitchia, Sporosarcina, Sutcliffiella, Weizmannia and Caryophanaceae. Validity of the taxon assignment made by the server was examined by reconstructing phylogenomic trees. In these trees, all Bacillus strains for which taxonomic predictions were made correctly branched with the indicated taxa. The unassigned strains likely correspond to taxa for which CSIs are lacking in our database. Results presented here show that the AppIndels server provides a useful new tool for predicting taxonomic affiliation based on shared presence of the taxon-specific CSIs. Some caveats in using this server are discussed.

Characterization of the family-level Borreliaceae pan-genome and development of an episomal typing protocol.

Background: The Borreliaceae family includes many obligate parasitic bacterial species which are etiologically associated with a myriad of zoonotic borrelioses including Lyme disease and vector-borne relapsing fevers. Infections by the Borreliaceae are difficult to detect by both direct and indirect methods, often leading to delayed and missed diagnoses. Efforts to improve diagnoses center around the development of molecular diagnostics (MDx), but due to deep tissue sequestration of the causative spirochaetes and the lack of persistent bacteremias, even MDx assays suffer from a lack of sensitivity. Additionally, the highly extensive genomic heterogeneity among isolates, even within the same species, contributes to the lack of assay sensitivity as single target assays cannot provide universal coverage. This within-species heterogeneity is partly due to differences in replicon repertoires and genomic structures that have likely arisen to support the complex Borreliaceae lifecycle in which these parasites have to survive in multiple hosts each with unique immune responses. Results: We constructed a Borreliaceae family-level pangenome and characterized the phylogenetic relationships among the constituent taxa which supports the recent taxonomy of splitting the family into at least two genera. Gene content pro les were created for the majority of the Borreliaceae replicons, providing for the first time their unambiguous molecular typing. Conclusion: Our characterization of the Borreliaceae pan-genome supports the splitting of the former Borrelia genus into two genera and provides for the phylogenetic placement of several non-species designated isolates. Mining this family-level pangenome will enable precision diagnostics corresponding to gene content-driven clinical outcomes while also providing targets for interventions.

Genetic Diversity of Borreliaceae Species Detected in Natural Populations of Ixodes ricinus Ticks in Northern Poland.

In Europe, Ixodes ricinus tick is the vector of Lyme disease spirochetes and their relatives (Borreliella genus) and Borrelia miyamotoi. However, a newly described tick I. inopinatus with similar biological features and separated from I. ricinus may act as a vector for different Borrelia species. To date, eleven Borreliella species were detected in the natural populations of I. ricinus. Recently, two North American species have been detected in ticks parasitizing bats and red foxes in Europe, i.e., B. lanei and B. californiensis pointing to the necessity for searching for them in natural tick populations. In this study, using the coxI molecular marker only I. ricinus was identified in field-collected ticks with the exception of individual specimens of Haemaphysalis concinna. Using the flaB gene and mag-trnI intergenic spacer as molecular markers 14 Borreliaceae species have been detected with various frequencies in different parts of northern Poland. Among infected ticks, the most frequent were Borreliella (Bl.) afzelii (29.4%) and Bl. garinii (20.0%), followed by Bl. spielmanii, Bl. valaisiana, Bl. lanei, Bl. californiensis, B. miyamotoi, Bl. burgdorferi, Bl. carolinensis, Bl. americana, B. turcica, Bl. lusitaniae, Bl. bissettiae and Bl. finlandensis. Three of the above-mentioned species, i.e., Bl. lanei, Bl. californiensis and B. turcica were detected in this study for the first time in the natural ixodid tick population in Europe. The existence of the newly detected spirochetes increases their total diversity in Europe and points to the necessity of careful identification and establishment of the actual distribution of all Borreliaceae species transmitted by I. ricinus.

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.

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.

Erratum: 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.

[This corrects the article on p. 217 in vol. 4, PMID: 23908650.].