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

Abstract

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.

Robust demarcation of the family Peptostreptococcaceae and its main genera based on phylogenomic studies and taxon-specific molecular markers

“…Earlier work on CSIs specific for different taxonomic groups has shown that these molecular characteristics exhibit a high degree of predictive ability to be found in other members (newly identified or sequenced) of these taxa [27,47,48,60]. This inference is also supported by the results presented here where several unnamed Peptostreptococcaceae species that shared CSIs specific for a given genus/clade were found to branch reliably with its members.…”

“…9), the strains of all uncharacterized species, which shared the CSIs specific for a given clade, branched reliably with the named species from that clade. These results provide further evidence supporting the contention that the identified CSIs are highly specific characteristics of the observed clades and their shared presence in an uncharacterized species is strongly indicative of a close relationship with that specific clade [47,48,53].…”

“…The lineages which contained only a single species also showed very low AAI values (generally <70 %) with the neighbouring genera/lineages, indicating that they are genetically distinct. Although the AAI values do not provide a consistent means for the demarcation of genus-level clades [21, 46, 47], results from these studies support the inferences that the different species clades marked in represent genus-level lineages.…”

“…To obtain further independent evidence to support these inferences, and to reliably demarcate the different family- and genus-level clades, we have carried out comparative analyses on the protein sequences from Peptostreptococcaceae genomes to identify molecular markers consisting of conserved signature indels (CSIs), which are specific for different clades. The CSIs which are exclusively found in members of specific clades, represent molecular synapomorphies, and they provide important means for the demarcation of taxonomic clades of different ranks in molecular terms [22, 27, 29, 47–49]. Our analyses of protein sequences from the Peptostreptococcaceae genomes have identified 75 novel CSIs, which are distinguishing characteristics of different Peptostreptococcaceae clades/lineages.…”

“…Similarly, the CSIs specific for the emended genus Clostridioides and Metaclostridioides gen, nov. are also shared by other uncharacterized strains related to them. Based on the demonstrated predictive abilities of the CSIs to be found in other members of specific taxa, we have recently developed a web-based tool/server (https://appindels.com/), which can predict taxonomic affiliation based on the presence of known taxon-specific CSIs in a genome sequence [47]. Thus, upon the addition of information for these newly identified CSIs to the AppIndels server, it should greatly facilitate the identification of both cultured and uncultured isolates/ species related to the described taxa.…”