Genomic Discovery of Taxon-Specific Molecular Markers for Lactobacillaceae Genera

1. Introduction

The family Lactobacillaceae [1,2,3] has undergone significant taxonomic revisions in recent years. Notably, Lactobacillus species were reclassified into 23 new genera [3] and the former Leuconostocaceae family [4,5] was merged into Lactobacillaceae [3]. The family Lactobacillaceae presently comprises 36 validly published genera [6]. Many Lactobacillaceae species from genera such as Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Leuconostoc, Oenococcus, and Weissella, are widely used in food production and as probiotics due to their health promoting properties [7,8,9,10].

Currently, species from different Lactobacillaceae genera are distinguished primarily their by clustering in phylogenetic trees based on 16S rRNA genes, core protein sequences, or genomic similarity metrics such as average amino acid identity (AAI), ecological traits, and relative evolutionary distances in the Genome Taxonomy Database (GTDB) [3,11,12,13,14,15]. However, despite extensive research, no consistent biochemical or molecular markers have been identified that reliably differentiate each of the 36 Lactobacillaceae genera. The discovery of such markers would not only more reliably differentiate species from each genus but would also provide novel and specific genetic tools for diagnostic and functional studies. Given the ecological diversity and biotechnological relevance of Lactobacillaceae species [7,8,9,11,16,17,18,19], new members of this family are being rapidly discovered [6], with genome sequences increasingly available in public databases such as the NCBI [20]. These resources offer a valuable opportunity for identifying genus-specific molecular markers [20,21,22,23,24].

In our previous work on the former Leuconostocaceae family, we identified 46 conserved signature indels (CSIs), or taxon-specific indels (TAXIs), specific to the genera Convivina, Fructobacillus, Leuconostoc, Oenococcus, Periweissella, and Weissella [25]. In our work, the term CSIs refer broadly to all conserved indels specific to different clades, whereas the term TAXIs refer to CSIs which are uniquely associated with a known taxon. These TAXIs, which results from rare genetic changes in a common ancestor of the specific taxa [26,27,28], serve as molecular synapomorphies, with strong predictive value, and they provide robust evidence of the evolutionary relatedness of species from these lineages [29,30,31]. Unlike other genus demarcation methods, such as phylogenetic clustering, relative evolutionary distances in the GTDB tree, or genomic similarity metrics like AAI [32], and percentage of conserved proteins (POCP) [21], which can be influenced by various parameters [12,27,33,34], and do not reveal any uniquely shared traits for the species of different genera , TAXIs offer a distinct advantage. These molecular markers are binary presence/absence traits, uniquely found in members of a specific taxon, which make them powerful tools for unambiguous genus-level differentiation [27,31].

Building on our earlier work identifying TAXIs for select Lactobacillaceae genera, this study expands the analysis to include additional genera within the family. We analyzed 410 publicly available Lactobacillaceae genome sequences (as of July 1, 2024) using phylogenomic and comparative protein sequence analyses. A robust phylogenomic tree was first constructed to delineate evolutionary relationships. We then applied the INDELIBLE (Indel-based Identification of Bacterial Lineages and Evolution) method to identify CSIs specific to individual genera [25,30,35,36]. This analysis led to the discovery of 167 novel TAXIs i.e., genus-specific CSIs in diverse proteins, providing reliable molecular markers for distinguishing between Lactobacillaceae genera. To demonstrate their practical utility, we used these TAXIs in conjunction with the AppIndels.com server [37] to predict the taxonomic affiliation of 113 previously uncharacterized Lactobacillus isolates. Based on the presence or absence of genus-specific TAXIs, 111 of these strains /isolates were accurately assigned to 11 Lactobacillaceae genera.

Structural analysis of representative TAXIs revealed their consistent location in surface-exposed protein loops, suggesting potential functional roles in genus-specific protein–protein or protein–ligand interactions. Further genetic and biochemical studies may uncover novel functional traits, such as metabolic adaptations or ecological specializations [12,19]. Overall, the genus-specific molecular markers identified here enhance taxonomic resolution and provide a foundation for exploring functional diversity across this ecologically and industrially important bacterial family.

2. Materials and Methods

2.1. Construction of Phylogenetic Trees

Genome sequences for type strains and/or reference strains of 410 Lactobacillaceae species, with annotated protein data available in the NCBI database as of July 1, 2024, were downloaded for analysis. To root the phylogenetic tree, genomes of two Bacillus species (Bacillus subtilis and B. cereus) were included as outgroups due to their phylogenetic proximity to Lactobacillaceae [38]. A phylogenomic tree was constructed for our genomes dataset based on concatenated sequences of 87 conserved proteins that comprise the “phyloeco” marker set (single-copy genes universally distributed across members of this phylum) for the phylum Bacillota [39]. The tree was generated using an internally developed pipeline described in our earlier work [31,40]. Briefly, homologs of the 87 phyloeco set of proteins were identified using the CD-HIT v4.6 program and profile Hidden Markov Models of these proteins [41], at default settings. Proteins present in at least 80% of the genomes and sharing a minimum of 50% sequence identity and length were retained. Multiple sequence alignments of these protein families were generated using the Clustal Omega v1.2.2 at default settings [42], and poorly aligned regions were removed using trimAl v1.4 [43]. The final concatenated sequence alignment consisted of 26527 aligned positions. A maximum-likelihood phylogenetic tree based on this alignment was constructed using the Whelan and Goldman model of protein evolution [44] and formatted using MEGA X [45].

2.2. Identification of Conserved Signature Indels (CSIs)

Identification of CSIs for different Lactobacillaceae genera was performed using methods described in our earlier studies [25,27,35]. Briefly, local BLASTp searches were conducted on protein sequences from genomes of representative Lactobacillus species across different clades of interest. Based on these searches, sequences of high-scoring homologs (E value <1e-²⁰) were retrieved for 4 to 10 within the target group and 10-15 outgroup species. Multiple sequence alignments were generated using Clustal X 2.1 [46] and visually inspected for the presence of insertions or deletions (indels) of fixed lengths located within conserved regions. Indels that were retained for further analysis met the following criteria [25,27,35]: (i) flanked on both sides by at least 5–6 conserved amino acid residues within a 40–50 residue window, and (ii) primarily found in species from a specific Lactobacillaceae genus but absent in other genera and outgroup species. Indels not located in conserved regions were excluded [25,27,35].

To confirm their taxon specificity, candidate CSIs, along with 30–50 flanking amino acids, were used in broader BLASTp searches against the NCBI non-redundant (nr) database. The top 300–500 hits were examined to assess the taxonomic distribution of each indel. Indels found exclusively in species from a single Lactobacillaceae genus were designated as CSIs and formatted using the SIGCREATE and SIGSTYLE tools [27,35], available via the Gleans.net server (http://gleans.net/). Due to space limitations, representative sequence data are shown in the main figures for selected species. Unless otherwise noted, all described CSIs are genus-specific and absent from other bacterial homologs among the top BLASTp hits. Additional sequence details for each CSI are provided in the Supplemental Figures.

2.3. Taxonomic Predictions of Lactobacillus Strains/Isolates Using AppIndels.com Sever

Protein sequences for 113 uncharacterized Lactobacillus isolates were retrieved from the NCBI genome database in .faa format. These sequences were analyzed using the AppIndels.com server [37] which performs local BLASTp searches to detect TAXIs that are specific for different genera. If the number of TAXIs identified in a genome exceeds the predefined threshold for a given taxon, the server assigns the genome to that taxon. A detailed description of the server’s methodology is available in previous work [37].

2.4.Determination of Protein Structures Using AlphaFold Model Generation to Map the Locations of CSIs

To investigate the structural context of CSIs, we analyzed four proteins containing genus-specific CSIs from Apilactobacillus, Lacticaseibacillus, Lactiplantibacillus, and Lactobacillus. FASTA sequences of both CSI-containing proteins and their homologs lacking the CSIs were retrieved from the NCBI protein database and submitted to the AlphaFold 3 server for structure prediction using default parameters [47]. The top-ranked predicted models were visualized and analyzed using PyMOL v2.5.5 [48]. Structural confidence was assessed using predicted Local Distance Difference Test (pLDDT) and predicted Template Modeling (pTM) scores [49,50]. Only models with high-confidence predictions (pLDDT > 50 and pTM > 0.8) were included in further analyses [47,51]. Final models were superimposed in PyMOL to localize the CSIs within the protein structures [48]. Structural similarity be-tween CSI-containing and CSI-lacking homologs was evaluated using root mean square deviation (RMSD) values.

3. Results

3.1. Phylogenomic Tree for the Lactobacillaceae Species

To determine the evolutionary relationships and genus-level affiliations of all 410 Lactobacillaceae species with available genomes as of July 1, 2024, we constructed a maximum likelihood phylogenomic tree using concatenated sequences of 87 conserved proteins. The resulting tree is shown in a compressed form in Figure 1, where species clades from different genera are coalesced for clarity. However, an expanded, uncompressed version of this tree is provided in Figure S1. In this tree (Figure 1 and Figure S1), which we will be referred to as the phyloeco tree, nearly all nodes exhibit 100% statistical support, indicating strong confidence in the inferred relationships. All examined Lactobacillaceae species clustered within clades corresponding to their respective genera, with branching patterns consistent with previous studies [3,12]. Species from several Lactobacillaceae genera (viz. Acetilactobacillus, Amylolactobacillus, Philodulcilactobacillus, Holzapfeliella, Nicoliella, Paralactobacillus), which contain only a single species also showed distinct branching, supporting their classification as separate genera [3].

3.2. Conserved Signature Indels Specific for Different Lactobacillaceae Genera

The phylogenetic tree shown in Figure 1 (and expanded in Figure S1) provides strong support for the monophyly of the various Lactobacillaceae genera. This tree forms the foundation for the central focus of this study, i.e. the identification of TAXIs unique to individual genera. Previous research across diverse prokaryotic taxa has demonstrated the value of TAXIs as reliable molecular markers for evolutionary and taxonomic studies [25,27,31,35,40,52,53,54]. Building on this foundation, we conducted detailed analyses of protein sequences from Lactobacillaceae species using the INDELIBLE approach. These analyses have identified167 novel CSIs in diverse proteins. Each of these CSI is uniquely present in species from a specific Lactobacillaceae genus. The results of these analyses are summarized and discussed below.

3.3. Molecular Markers Specific for the Genera Lactobacillus, Lacticaseibacillus Lactiplantibacillus and Apilactobacillus

The genus Lactobacillus is the type genus of the family Lactobacillaceae and remains its most populous and extensively studied member [3,55]. Prior to its reclassification in 2020, the genus included over 260 species, many of which exhibited polyphyletic branching alongside species from the former Leuconostocaceae family, and displayed substantial phenotypic and ecological diversity [12,13,16,56,57]. However, following the taxonomic revision by Zhang et al. [3], species from the genus Lactobacillus were redistributed into 23 distinct genera, resulting in a monophyletic grouping of different proposed genera in phylogenetic analyses. The composition and branching of species within the genus Lactobacillus, as observed in our phylogenetic tree, are shown in Figure 2A.

Despite its division multiple genera, Lactobacillus still comprises over 46 named species [6], which exhibit considerable genetic diversity (Figure 2A). Several species from this genus are widely used as probiotics [18,58,59], while others such as Lactobacillus delbrueckii subsp. bulgaricus, play key roles in dairy fermentation including yogurt production [60,61]. However, despite the industrial and scientific significance, no molecular characteristics have previously been identified that are uniquely specific to Lactobacillus species.

Our analyses identified 16 novel CSIs in diverse proteins, most of which are uniquely shared by all or most Lactobacillus species. One example is shown in Figure 2B, where a two aa insertion within a conserved region of the 50S ribosomal protein subunit L10 is found exclusively in all 46 genome-sequenced Lactobacillus species. This protein is part of the L7/L12 stalk of the 50S ribosomal subunit and plays a key role in protein synthesis by recruiting translation factors and stimulating GTP hydrolysis [62]. In this and other sequence alignments, dashes (–) indicate identity with the amino acid shown on the top line. This CSI is absent in all other Lactobacillaceae species and in other examined bacteria. Due to space constraints, Figure 2B displays sequence data for a subset of Lactobacillus and other Lactobacillaceae species. Comprehensive sequence data for this and the remaining 15 CSIs, each found in different proteins, are provided in Figures S2–S17, with key features summarized in Table 1. Given their specificity, these TAXIs likely originated in a common ancestor of the Lactobacillus genus and serve as reliable molecular markers for distinguishing its species from those of other Lactobacillaceae genera.

The genus Lacticaseibacillus comprises species that have been extensively studied for their probiotic properties, their involvement in dental caries, and their growing association with bacteremia [63,64]. In our phylogenetic analysis (Figure S1), members of this genus form a well-supported, deeply branching monophyletic clade. The species composition and branching pattern within this clade are illustrated in Figure 3A. Using the INDELIBLE approach, we identified nine novel conserved CSIs in various proteins that are uniquely present in Lacticaseibacillus species. One representative example is shown in Figure 3B, where a one amino acid insertion in a conserved region of the manganese-dependent inorganic pyrophosphatase protein is found in all 30 genome-sequenced Lacticaseibacillus species but absent in other Lactobacillaceae genera. Detailed sequence information for this CSI, along with the eight additional TAXIs identified for this genus, is provided in Figures S18–S26. Key features of these CSIs are summarized in Table 1. Together, these molecular markers offer reliable tools for distinguishing Lacticaseibacillus species from other members of the Lactobacillaceae family.

The genus Apilactobacillus comprises 12 validly published species, which form a distinct clade in our phylogenomic tree (Figure 1). The species composition and branching pattern within this clade are shown in Figure 4A. Members of this genus are predominantly associated with fructose-rich environments, such as the guts of bees and flowers, highlighting their ecological link to insects [3,12,65,66]. Our analysis identified four CSIs that are uniquely present in Apilactobacillus species. One representative example is shown in Figure 4B, where a two–amino acid insertion in the cyclopropane-fatty-acyl-phospholipid synthase family protein is uniquely shared by all 12 Apilactobacillus species but absent in other Lactobacillaceae genera. Detailed sequence information for this CSI along with the other three Apilactobacillus-specific CSIs is provided in Figures S27–S30. Key characteristics of these CSIs/TAXIs are summarized in Table 1. These TAXIS offer reliable tools for distinguishing Apilactobacillus species from other Lactobacillaceae genera.

Species of the genus Lactiplantibacillus inhabit a wide range of environments, including fermented foods (e.g., sauerkraut, kimchi), plant material, and the human gastrointestinal tract [3]. Among them, Lactiplantibacillus plantarum has been extensively studied for its probiotic benefits, including its ability to ferment plant-derived and phenolic compounds, its antioxidant properties, and its antimicrobial activity through bacteriocin production [67,68]. The genus Lactiplantibacillus currently includes 20 validly published species, which form a well-supported clade in the phylogenomic tree constructed in this study. The branching pattern of species from this genus is shown in Figure 4C.

Our comparative genomic analysis identified eight CSIs in various proteins that are uniquely present in species of the genus Lactiplantibacillus. One representative CSI is shown in Figure 4D, where a single amino acid insertion in a highly conserved region of the pyridoxal phosphate-dependent aminotransferase protein is uniquely shared by all Lactiplantibacillus species and absent in all other Lactobacillaceae genera. Detailed sequence information for this CSI, along with the other seven Lactiplantibacillus-specific CSIs, is provided in Figures S31–S38. Key characteristics of these markers are summarized in Table 1. These TAXIS provide reliable molecular tools for distinguishing Lactiplantibacillus species from all other Lactobacillaceae genera.

Figure 2, Figure 3 and Figure 4 present selected examples of TAXIs identified for some Lactobacillaceae genera. However, in addition to the results shown for Lactobacillus, Lacticaseibacillus, Apilactobacillus, and Lactiplantibacillus, our comprehensive protein sequence analyses across other Lactobacillaceae genera have revealed an additional 130 novel CSIs. Most of these CSIs are uniquely shared by species within a single genus, making them reliable molecular signatures for genus-level identification.

The numbers of CSIs that we identified for the other Lactobacillaceae genera in this study are as follows: Agrilactobacillus (4), Amylolactobacillus (4), Bombilactobacillus (7), Companilactobacillus (10), Dellaglioa (6), Fructilactobacillus (8), Furfurilactobacillus (19), Lapidilactobacillus (4), Latilactobacillus (8), Lentilactobacillus (3), Levilactobacillus (4), Ligilactobacillus-Liquorilactobacillus cluster (7) Limosilactobacillus (8), Loigolactobacillus (6), Paucilactobacillus (3), Pediococcus (10), Schleiferilactobacillus (15), Secundilactobacillus (4), and Xylocopilactobacillus (4).

It should be noted that although our analysis identified multiple CSIs for all Lactobacillaceae genera containing two or more species, no genus-specific CSIs were found for Ligilactobacillus and Liquorilactobacillus. Previous studies indicate that species from these two genera, comprising primarily of free-living bacteria and vertebrate-associated species, respectively, are closely related [3,69]. Supporting this inference, our analysis has also identified eight CSIs that are uniquely shared between species of both genera, suggesting they shared a common ancestor distinct from other Lactobacillaceae.

Detailed sequence information for CSIs specific for the other Lactobacillaceae genera is provided in Figures S39–S168, and key characteristics are summarized in Tables S1–S4. In addition to these findings, our previous work identified multiple CSIs specific for several other Lactobacillaceae genera[25], which were formerly classified under the family Leuconostocaceae [5]. The numbers of CSIs identified for these genera are as follows: Fructobacillus (5), Leuconostoc (5), Oenococcus (13), Periweissella (5), and Weissella (6) [25]. A summary of the species compositions of the various Lactobacillaceae genera, including those formerly classified under the family Leuconostocaceae, and the number of genus-specific CSIs (TAXIs) identified for each is presented in Figure 5. Based on these findings, all Lactobacillaceae genera containing two or more species, except Ligilactobacillus and Liquorilactobacillus, can now be reliably distinguished from one another using multiple, genus-specific TAXIs.

3.4. Predictive Ability of Previously Identified CSIs to be Found in Newly Described Species

Previous studies on CSIs specific to various taxa and genera have demonstrated that they exhibit strong predictive value i.e., once identified in known members of a group, these markers are often consistently found in newly sequenced or discovered members of the same lineage [31,37,70,71,72]. This predictive capability is further illustrated in Figure 6, which presents updated sequence data for two CSIs specific to Leuconostocaceae genera identified in our previous work [25].

Figure 6A shows an eight aa insertion in the protein phospho-N-acetylmuramoyl-pentapeptide-transferases, originally identified as specific to the genus Weissella [25], whose members are known for their probiotic and biotechnological potential [73]. At the time of its discovery, sequence data were available for 14 Weissella species. Since then, genome sequences for four additional species have become available, and all of them contain this CSI, highlighting its stability and predictive value.

Similarly, Figure 6B shows a CSI specific to the genus Fructobacillus, a group of fructose-fermenting microorganisms [74]. When this CSI was first reported in 2022, it was found in five species. Since then, eight new Fructobacillus species have been described [6], and this CSI is present in all of them. These results provide compelling evidence highlighting the long-term stability and genus-specific conservation of CSIs, reinforcing their utility as reliable molecular markers for taxonomic classification and evolutionary studies.

3.5. Application of the Identified CSIs for Taxonomic Prediction of Uncharacterized Lactobacillus isolates

Based on the predictive capability of the TAXIs, we have recently developed a web-based tool, AppIndels.com, which uses the presence of known TAXIs in genome sequences to predict taxonomic affiliations [37]. To evaluate the utility of the TAXIs identified in this study for Lactobacillaceae genera, we added the corresponding CSI sequence data to the AppIndels.com server and used it to analyze 113 uncharacterized Lactobacillus isolates with available genome sequences in the NCBI database. In Figure S169, results from the server are shown for two representative Lactobacillus isolates. For Lactobacillus sp. CBA3605, the server predicted affiliation with the genus Lactiplantibacillus, identifying eight CSIs specific to this clade (Figure S169A). In contrast, Lactobacillus sp. UWDMLACCAS1_1 was predicted to belong to the genus Lacticaseibacillus, with nine genus-specific CSIs detected in its genome (Figure S169B). In addition to reporting the number of CSIs matching the predicted genus, the server provides access to the corresponding sequence data[37], which can be viewed by clicking the arrow next to the CSI count.

The taxonomic predictions made by the AppIndels.com for the genome sequences of all examined Lactobacillus isolates are summarized in Table S5. This table includes the accession numbers of the genomes, the predicted taxonomic affiliations from AppIndels.com, and the number of TAXIs identified for each genome.

As seen from the results presented in Table S5, the server successfully predicted genus-level affiliations for 111 out of 113 isolates based on the presence of multiple genus-specific TAXIs. These isolates were assigned to the following 10 genera: Agrilactobacillus (1), Bombilactobacillus (7), Fructilactobacillus (1), Lacticaseibacillus (8), Lactiplantibacillus (2), Lactobacillus (81), Lentilactobacillus (1), Levilactobacillus (2), Limosilactobacillus (7), and Ligilactobacillus-Liquorilactobacillus cluster (1). For two genomes (GCA_014796685.1 and GCA_019303535.1) no taxonomic predictions were made by the server. One of these genomes (accession number GCA_014796685.1) is indicated as contaminated in its NCBI record.

To validate the accuracy of these predictions, we constructed a phylogenetic tree that includes the 111 uncharacterized isolates along with representative species from various Lactobacillaceae genera (Figure 7). As seen from the tree in Figure 7, there was 100% concordance between the genus assignments predicted by the AppIndels.com server and the phylogenetic placements of the isolates. It should be noted that the high accuracy of the AppIndels.com server in predicting taxonomic affiliation results from its requirement that it predicts a positive taxonomic affiliation only when multiple CSIs specific for a particular genus are present in the analyzed genome [75]. Since each TAXI represents a rare genetic change [27,28,35], the likelihood of multiple CSIs from the same genus appearing in a genome by chance is extremely low. This feature of the AppIndels.com server ensures that the taxonomic predictions made by it are accurate. These results demonstrate that the TAXIs identified in this study provide a robust and practical tool for determining the taxonomic affiliation of novel or uncharacterized isolates from this family.

3.6. Taxon-specific CSIs are Localized in Surface Exposed Loops of Proteins

Previous studies on CSIs specific to various prokaryotic taxa have shown that these genetic changes are frequently located in surface-exposed loop regions of proteins, which are flexible, unstructured areas on solvent-accessible surfaces that often mediate novel protein–protein or protein–ligand interactions [76,77,78,79,80,81]. Considering these findings, we investigated the structural localization of selected CSIs specific to Lactobacillaceae genera that shown in Figure 2, Figure 3 and Figure 4. Results of these analyses are presented in Figure 8.

We used AlphaFold [47] to predict the structures of proteins containing conserved signature inserts (CSIs), along with homologous proteins lacking these inserts. To determine the structural localization of each CSI, we superimposed the predicted structures of the protein homologs with and without the CSI. Figure 8 shows the results for four representative CSIs, with the CSI regions highlighted in red. In all cases, the CSIs were localized to surface-exposed loop regions of the proteins. The RMSD values for three of the studied proteins viz. manganese-dependent inorganic pyrophosphatase (Figure 8B; RMSD = 1.1 Å), cyclopropane-fatty-acyl-phospholipid synthase (Figure 8C; RMSD = 0.3 Å), and pyridoxal phosphate-dependent aminotransferase (Figure 8D; RMSD = 1.0 Å), indicate minimal structural differences between the CSI-lacking and -containing proteins. In contrast, the 50S ribosomal protein L10, which contains a 2-amino acid insertion specific to Lactobacillus (Figure 8A), showed a higher RMSD (5.4 Å), suggesting a potential conformational change induced by the CSI.

These findings on the CSIs specific for Lactobacillaceae genera are consistent with earlier studies that the conserved indels in protein sequences are structurally localized to surface loops [76,80,82,83], and may facilitate novel genus-specific functional interactions.

4. Discussion

Lactobacillaceae species play crucial roles in food production, probiotic development, and human health due to their metabolic versatility [1,2,3,5]. Given their importance, it is essential to understand the unique characteristics shared by species across different Lactobacillaceae genera. Traditional phylogenetic and genomic similarity-based approaches used for genus-level classification often fail to reveal genus-specific traits [3,15,32,84]. This study presents a comprehensive phylogenomic and comparative genomic analysis of 410 Lactobacillaceae genomes, leading to the identification of 167 novel molecular markers, termed CSIs or TAXIs, found in diverse proteins. Each CSI is uniquely associated with a specific Lactobacillaceae genus. The discovery of these genus-specific molecular markers represents a significant advancement in our understanding of the Lactobacillaceae family. These TAXIs not only enable more precise molecular delineation of genera [25,31], but also offer new tools for diagnostic development [85,86], and for genetic and biochemical studies aimed at uncovering genus-specific functional traits [78,80,82,87]. This enhances both scientific insight and practical applications of Lactobacillaceae species.

The predictive power of the identified TAXIs was demonstrated in this study through their successful use in classifying 111 out of 113 uncharacterized Lactobacillus isolates using the AppIndels.com server. This tool matches genome sequences against a curated database of known TAXIs, allowing rapid and accurate genus-level identification [37,71]. Such capabilities are especially valuable as genomic databases expand and new Lactobacillaceae species continue to be discovered. With the integration of TAXIs into the AppIndels.com server, it can also detect the presence of these genera in high-throughput genomic datasets and help resolve taxonomic ambiguities. For the genomes of two Lactobacillus isolates (GCA_014796685.1 and GCA_019303535.1) no taxonomic prediction was made by the server. The NCBI record of the genome GCA_014796685.1 indicates that it is contaminated, whereas the other genome (viz. GCA_019303535.1) could correspond to a Lactobacillaceae genus (viz. monotypic genera) for which no CSIs were identified in this study. While the AppIndels server provide a reliable tool for predicting taxonomic affiliation and supporting diagnostic applications based on genome sequences, its one key limitation is that it can only assign taxa to genera for which validated CSIs are present in its database [37]. Additionally, the server may fail to make a prediction or produce an incorrect result, if the genome sequence analyzed is contaminated or partial [37].

Beyond in silico detection, the identified TAXIs are also ideally suited for developing novel diagnostic assays. Because these CSIs are located within conserved regions of genes/proteins, sequences from their flanking regions can be used to design PCR primers or probes for qPCR and pyrosequencing, enabling selective amplification or detection of CSI-containing organisms [88]. Similar TAXI-based diagnostic assays have been successfully developed for other taxa, such as Bacillus anthracis [86] and Escherichia coli O157:H7 [85], demonstrating the broader applicability of this approach.

In addition to the utility of TAXIs for taxonomy and diagnostics studies, these taxon-specific molecular markers also provide a gateway to exploring genus-specific functional traits. Structural mapping of CSIs, including those identified in this study, shows that they are consistently located in surface-exposed loop regions of proteins [76,80,83]. These surface-exposed loops are flexible, solvent-accessible, and often involved in protein–protein or protein–ligand interactions [76,79]. Earlier experimental studies on selected CSIs have shown the functional importance of these CSIs for the group of organisms for which they are specific [89]. This suggests that the rare genetic changes represented by CSIs may underlie unique biochemical or phenotypic traits [76,82,83,89,90,91,92]. Therefore, further genetic and biochemical studies on the identified TAXIs could uncover novel metabolic or adaptive traits uniquely shared by species within individual genera.

It should be noted that although CSIs are powerful genus-specific molecular markers, determining their functional significance remains challenging. These indels are often small and located in conserved protein regions, making it difficult to directly link them to phenotypic traits or biochemical functions [79]. Many CSIs occur in proteins with poorly characterized roles, and their subtle structural effects may not lead to easily observable changes in cellular behavior [27,80]. To investigate the functional relevance of CSIs, several approaches can be employed. Structural modeling tools like AlphaFold can help localize CSIs within protein structures [47], while molecular dynamics simulations and docking studies may reveal how CSIs affect protein flexibility, stability, or binding interactions [78,80,82]. Experimental techniques such as site-directed mutagenesis and functional assays can validate the impact of specific indels [89,93,94]. Additionally, protein interaction studies and omics-based profiling (e.g., transcriptomics, proteomics) may uncover downstream effects [95,96]. Correlating the presence of a CSI with specific ecological or phenotypic traits of the species group [78,87] may also provide insights into its functional role.

In summary, this study identifies 167 genus-specific conserved signature indels (CSIs), or TAXIs, across Lactobacillaceae genomes, offering powerful molecular markers for taxonomic studies, diagnostic assay development, and functional trait discovery. Given the widespread industrial use of Lactobacillaceae species in food, health, and biotechnology, further biochemical and functional characterization of these TAXIs could uncover novel genus-specific traits with significant implications for microbial ecology and applied microbiology.