Genomic Discovery of Robust Molecular Markers Differentiating Lactobacillaceae Genera and Providing Novel Tools for Functional Insights

1. Introduction

The family Lactobacillaceae [1,2,3] encompasses Gram-positive, non-spore-forming bacteria with diverse metabolic profiles, including obligate heterofermentative, facultative heterofermentative, or obligate homofermentative [4,5]. Recent taxonomic revisions, most notably the reclassification of Lactobacillus species into 23 new genera [3] and the merger of the former Leuconostocaceae family [6,7] with Lactobacillaceae, have expanded the family to 36 genera with validly published names. Many species within Lactobacillaceae, particularly those from genera such as Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Leuconostoc, Oenococcus, and Weissella, are widely utilized in food production and as probiotics due to their health promoting properties [8,9,10,11,12]. Currently, species from different Lactobacillaceae genera are primarily distinguished based on phylogenetic clustering, genomic similarity metrics such as average amino acid identity (AAI), and, in some cases, ecological characteristics [3,5,13,14,15].

However, despite extensive research, no consistent biochemical or molecular markers have been identified that reliably differentiate species from different Lactobacillaceae genera. The discovery of such markers would not only refine taxonomic boundaries but also enable novel diagnostic and functional studies. In our previous work on the former Leuconostocaceae family [7], which is now part of Lactobacillaceae, we identified 46 conserved signature indels (CSIs) in various proteins that were specific to the genera Convivina, Fructobacillus, Leuconostoc, Oenococcus, Periweissella, and Weissella [16]. These Taxon-specific Indels (TAXIs), which results from rare genetic changes in a common ancestor [17,18,19], serve as molecular synapomorphies, providing strong evidence for the evolutionary relatedness of species from these lineages [20,21,22].

Given the ecological diversity and biotechnological relevance of Lactobacillaceae species [8,9,10,13,23,24,25,26], new species belonging to this family are being rapidly discovered. Since the taxonomic overhaul by Zheng et al. [3], large numbers of new Lactobacillaceae species have been described [27], with genome sequences available in the NCBI database [28]. These genomic resources offer a valuable opportunity for identifying genus-specific molecular markers [28,29,30,31,32]. In this study, we analyzed 411 Lactobacillaceae genome sequences (available in the NCBI database as of March 1, 2024) using phylogenomic and comparative protein sequence analyses. In our comparative genomic analysis, we use the INDELIBLE (Indel-based Identification of Bacterial Lineages and Evolution) approach to identify molecular markers (i.e. CSIs) that are specific to various Lactobacillaceae genera.

In this work, we initially constructed a robust phylogenomic tree for all 411 genomes of Lactobacillaceae species to clarify their evolutionary relationships. Subsequently, we performed comparative analyses of protein sequences using the INDELIBLE method to identify CSIs unique to specific genera [16,21,33]. These analyses led to the discovery of 171 novel TAXIs in diverse proteins, each uniquely present in species from specific Lactobacillaceae genera. These TAXIs serve as reliable molecular markers for distinguishing between Lactobacillaceae genera. To demonstrate the practical utility of these TAXIs, we used these markers in conjunction with the AppIndels.com server [34] to predict the taxonomic affiliation of 113 previously uncharacterized Lactobacillus strains. Based on the presence or absence of specific TAXIs, we accurately assigned 112 of these 113 strains to 11 Lactobacillaceae genera.

Structural analyses of representative TAXIs identified in this work revealed that they are consistently located in surface-exposed loops of the proteins, suggesting their functional importance. Further genetic and biochemical studies on these TAXIs could contribute to the discovery of novel genus-specific functional traits, including those involved in metabolic innovations, host interactions or environmental adaptations [5,26]. Thus, the genus-specific molecular markers identified in this study not only enhance taxonomic resolution within Lactobacillaceae but also provide a foundation for exploring functional diversity and characteristics of its different genera. These findings have important implications for advancing our understanding of the Lactobacillaceae species and their possible applications in food technology, probiotics, and health sciences.

2. Materials and Methods

2.1. Construction of Phylogenetic Trees

Genome sequences for the type strains and/or reference strains of 411 Lactobacillaceae species, whose annotated protein sequences were available in the NCBI database as of July 1, 2024, were downloaded. The genomes of 2 Bacillus species (Bacillus subtilis and B. cereus) were included for rooting purposes. Based on these genome sequences, a phylogenomic tree was constructed based on concatenated sequences of 87 conserved proteins, which constitute the phyloeco marker set for the phylum Bacillota [35]. The tree was constructed using an internally developed pipeline described in our earlier work [22,36]. Briefly, the CD-HIT program and the profile Hidden Markov Models (HMMs) [37] of the phyloeco set of proteins, were used to identify homologs of these proteins in the input genomes that were present in at least 80% of the input genomes and shared a minimum of 50% sequence identity and length. Multiple sequence alignments of these protein families were generated using the Clustal Omega algorithm [38]. After removing the poorly aligned regions using trimAl [39], the final concatenated sequence alignment for the 87 proteins consisted of 26527 aligned positions. A maximum-likelihood tree based on this sequence alignment was constructed based on the Whelan and Goldman model of protein sequence evolution [40] and formatted using MEGA X [41].

2.2. Identification of Conserved Signature Indels (CSIs)

Identification of CSIs for different Lactobacillaceae genera was carried out using the methods described in our earlier work [16,18,33]. Briefly, local BLASTp searches were carried out on protein sequences from the genomes of representative Lactobacillus species from different clades of interest and several outgroup species. Based on these BLASTp searches, sequences of high-scoring homologs (E value <1e-²⁰) of different proteins were retrieved for several species (generally between 4 to 10) from the group of interest, and 10-15 outgroup species. Multiple sequence alignments of different proteins were performed using Clustal X 2.1 [42]. The alignments were visually inspected for the presence of insertions or deletions (indels) of fixed lengths that were present in conserved regions (i.e. flanked on both sides by minimally 5-6 conserved aa residues in the neighboring 40-50 aa) and which were only found in the species from a specific Lactobacillaceae genus. The indels which were not present in conserved regions were not further considered. To confirm the specificity of these candidate CSIs, the query sequences containing the indel and 30–50 flanking amino acids on either side (typically beginning and ending with conserved residues) were used in a second, broader BLASTp search against the NCBI non-redundant (nr) database. The top 300–500 hits were examined to assess the taxonomic distribution of the indel. Indels found exclusively in species from a single Lactobacillaceae genus were identified as CSIs or TAXIs and formatted using the SIGCREATE and SIGSTYLE tools [18,33], available via the Gleans.net server (http://gleans.net/). Due to space constraints, representative sequence data are shown in the main figures for a limited number of species. However, unless otherwise noted, the CSIs (TAXIS) described are genus-specific and absent in all 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

The protein sequences of 113 uncharacterized Lactobacillus isolates analyzed in this study were obtained from the NCBI genome database in .faa format. These sequences were processed using the AppIndels.com server [34], which performs local BLASTp searches to identify TAXIs specific for different genera. If the number of TAXIs in the input protein file exceeds the threshold for identification of a given taxon, the server assigns the genome to that taxon. A detailed explanation of the server’s methodology is available in previous work [34].

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

These analyses were performed on four proteins containing CSIs specific to the genera Apilactobacillus, Lacticaseibacillus, Lactiplantibacillus, and Lactobacillus. FASTA sequences of both the CSI-containing proteins and their homologs lacking the CSIs were retrieved from the NCBI protein database and they were used as input for structural prediction. Protein structures were predicted using the AlphaFold 3 server with default parameters [43]. The top-ranked predicted models were selected for visualization and analysis using PyMOL v2.5.5 [44]. To assess the confidence of the predicted structures, we used the predicted Local Distance Difference Test (pLDDT) and predicted Template Modeling (pTM) scores [45,46]. Only models with high-confidence predictions (pLDDT > 50 and pTM > 0.8) were included in subsequent analyses [43,47]. The final models were superimposed in PyMOL using default settings to localize the CSIs within the protein structures. Structural similarity between CSI-containing and CSI-lacking proteins 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 generic affiliations of all 411 Lactobacillaceae species with available genomes as of July 1, 2024, a maximum likelihood phylogenomic tree was constructed using concatenated sequences of 87 conserved proteins. The resulting tree is presented in a compressed form in Figure 1, where the species clades from different genera are coalesced. However, an uncompressed form of this tree is provided in Figure S1. In this tree (Figure 1 and Figure S1), which we will be referring 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 [3,5]. Species from several Lactobacillaceae genera (viz. Acetilactobacillus, 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 Figure S1) provides strong support for the monophyly of different Lactobacillaceae genera. This tree serves as the framework for the central focus of this study, which is the identification of molecular markers, referred to as taxon-specific conserved signature indels (TAXIs) that are unique to different genera. Previous research on other prokaryotic taxa has demonstrated that TAXIs provide valuable molecular markers for evolutionary and taxonomic studies [16,18,22,33,36,48,49,50]. Building on this foundation, we conducted detailed analyses of protein sequences from Lactobacillaceae species using the INDELIBLE approach to identify CSIs specific to individual genera. These analyses have led to the discovery of 171 novel CSIs in diverse proteins, each uniquely present in species from specific Lactobacillaceae genera. 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 [3,4]. Prior to 2020, the genus included over 260 species, which exhibited polyphyletic branching alongside species from the family Leuconostocaceae and displayed substantial phenotypic and ecological diversity [5,14,23,51,52]. However, because of the recent reclassification of Lactobacillus species, which led to their division into 23 distinct genera [3], Lactobacillus species now form a monophyletic clade in phylogenetic analyses. The composition and branching of species from the clade corresponding to genus Lactobacillus in our phylogenetic tree are shown in Figure 2A.

Despite the division of this genus into multiple genera, the genus Lactobacillus still comprises more than 46 named species [27], which exhibit considerable genetic diversity (Figure 2A). Some species from this genus are widely used as probiotics [25,53,54], and some, such as Lactobacillus delbrueckii subsp. bulgaricus, play key roles in dairy fermentation processes like yogurt production [55,56]. However, despite the industrial and scientific importance of species from this genus, no molecular characteristics have previously been identified that are specific to Lactobacillus species.

Our analyses have 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 2 aa insertion within a conserved region of the 50S ribosomal protein subunit L10 is found exclusively in all 46 genome-sequenced Lactobacillus species. The ribosomal protein L10 is a component of the L7/L12 stalk of the 50S ribosomal subunit and it plays a central role in protein synthesis by recruitment of translation factors to the ribosome and stimulation of GTP hydrolysis [57]. 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 information for only a subset of Lactobacillus and other Lactobacillaceae species. More comprehensive sequence data for this and the remaining 15 CSIs specific to Lactobacillus are provided in Figures S2–S17, with key features summarized in Table 1. Given their specificity for the Lactobacillus species, these TAXIs likely originated in a common ancestor of the genus Lactobacillus and serve as reliable molecular markers for distinguishing its species from those of other Lactobacillaceae genera.

The genus Lacticaseibacillus includes species that have been extensively studied for their probiotic properties, their role in dental caries, and their increasing association with cases of bacteremia [58,59]. 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 shown in Figure 3A. Using the INDELIBLE approach, we have 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 is absent in all other Lactobacillaceae species. Detailed sequence information for this CSI, along with the other eight other TAXIs for this genus, is provided in Figures S18–S26. Key characteristics of these CSIs are summarized in Table 1. Collectively, these markers offer reliable molecular tools for distinguishing Lacticaseibacillus species from those of other Lactobacillaceae genera.

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. Species from this genus are predominantly associated with fructose-rich environments, such as the guts of bees and flowers, reflecting their ecological link to insects [3,5,60,61]. Our analysis has 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 all other Lactobacillaceae species. Detailed sequence information for this CSI and the other three Apilactobacillus-specific CSIs is provided in Figures S27–S30, and key characteristics are summarized in Table 1. These CSIs serve as reliable molecular markers for distinguishing Apilactobacillus species from all other genera within the Lactobacillaceae family.

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, owing to its ability to ferment plant-derived and phenolic compounds, its antioxidant properties, and its antimicrobial activity through bacteriocin production [62,63].The genus Lactiplantibacillus currently includes 20 validly published species, which form a well-supported clade in the phylogenomic tree constructed in this study. Branching of the species from this genus in our phylogenetic tree is shown in Figure 4C. Our comparative genomic analysis has identified eight CSIs in various proteins that are uniquely present in species from this genus. One representative CSI is shown in Figure 4D, where a one amino acid insert in a highly conserved region of the protein pyridoxal phosphate-dependent aminotransferase is shared exclusively by all Lactiplantibacillus species and absent in all other Lactobacillaceae species. Detailed sequence information for this CSI, along with the other seven Lactiplantibacillus-specific CSIs, is provided in Figures S31–S38. Key characteristics of these CSIs are summarized in Table 1.

Figure 2, Figure 3 and Figure 4 present selected examples of TAXIs specific to a few Lactobacillaceae genera. However, similar to the results shown for Lactobacillus, Lacticaseibacillus, Apilactobacillus, and Lactiplantibacillus, our comprehensive protein sequence analyses across other Lactobacillaceae genera have identified an additional 134 novel CSIs. Most of these CSIs are uniquely shared by species from a specific genus, serving as reliable molecular signatures.

The numbers of CSIs that we have identified for the other Lactobacillaceae genera in the present 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).

Detailed sequence information for these CSIs is provided in Figures S39–S172, and their key characteristics are summarized in Tables S1–S4. In addition to these CSIs, our previous work [16] has identified multiple CSIs specific for several other Lactobacillaceae genera, which were formerly part of the family Leuconostocaceae [7]. The names of these genera and the numbers of identified CSIs specific for them are as follows: Fructobacillus (5) Leuconostoc (5), Oenococcus (13), Periweissella (5), and Weissella (6) [16]. A summary of the species compositions of different Lactobacillaceae genera and the numbers of identified CSIs (TAXIs) that are specific for these genera is presented in Figure 5.

Based on these findings, all Lactobacillaceae genera that include two or more species can now be reliably distinguished from one another based on multiple, genus-specific TAXIs.

3.4. Predictive Ability of the CSIs and Their Application in Predicting the Taxonomic Affiliations of other Lactobacillus spp./Isolates

Previous studies on CSIs specific to various taxa and genera have demonstrated their strong predictive value i.e., TAXIs identified in known members of a group are often found in newly discovered or sequenced members of the same group [22,34,64,65,66]. The predictive ability of the CSIs is also demonstrated by the results presented in Figure 6 for two CSIs specific for Leuconostocaceae genera identified in our previous work [16]. Figure 6A shows a CSI consisting of an 8 aa insertion in the protein phospho-N-acetylmuramoyl-pentapeptide-transferases which was identified as specific for the genus Weissella [16], whose members exhibit probiotic and biotechnological potential [12]. When this CSI was identified, sequence information was available for 18 Weissella species. However, since then sequence information has become available for four additional species (shown in bold in Figure 6A), and this CSIs is present in all of them demonstrating its predictive ability. Figure 6B shows another example of a CSI identified in our previous work, specific for the genus Fructobacillus [67], which consists of fructose-fermenting microorganisms [26,68]. When this CSI was identified in 2022, sequence information was available for 5 Fructobacillus species. However, since then, eight new species belonging to this genus have been described [27], and this CSI is present in all of them. These results provide compelling evidence highlighting the long-term stability and predictive abilities of the CSIs (TAXIs).

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 [34]. To evaluate the utility of the TAXIs for Lactobacillaceae genera identified in this study, we have added the sequence information for these CSIs to the AppIndels.com server, and then used this server to analyze 113 uncharacterized Lactobacillus isolates whose genome sequence information was available in the NCBI database. In Figure 7, we show the results obtained from the AppIndels.com server for two representative Lactobacillus isolates. The server indicates that the strain Lactobacillus sp. CBA3605 is related to the genus Lactiplantibacillus, and its genome contains eight CSIs specific for this genus (Figure 7A). In contrast, the server predicted that the genome of Lactobacillus sp. UW_DM_LACCAS1_1 is related to the genus Lacticaseibacillus, as it contained nine CSIs specific for this genus (Figure 7B). In addition to indicating the numbers of CSIs specific to the predicted taxon, the server also provides the sequence information for the matching CSIs. Due to space considerations, the results of these analyses for all other analyzed strains are presented in Table S5 and they include, the accession numbers of the analyzed genomes, the predictions made by the AppIndels.com regarding the taxonomic affiliations of these genomes, and the number of TAXIs identified matching a specific genus in these genomes. As seen from Figure 7 and Table S5, the server successfully predicted the genus-level affiliation for 112 out of 113 isolates based on the presence of multiple genus-specific TAXIs in their genome sequences. These isolates were assigned to the following 11 genera: Agrilactobacillus (1), Apilactobacillus (1), Bombilactobacillus (7), Fructilactobacillus (1), Lacticaseibacillus (8), Lactiplantibacillus (2), Lactobacillus (81), Lentilactobacillus (1), Levilactobacillus (2), Limosilactobacillus (7), and Ligilactobacillus-Liquorilactobacillus cluster (1). One genome (accession number GCA_014796685.1) was not assigned to any genus, as it likely belongs to a taxon for which no TAXIs are currently available in the server’s database.

To validate the accuracy of these taxonomic predictions, we constructed a phylogenetic tree which included the information for most uncharacterized isolates and representative species from various Lactobacillaceae genera (Figure 8). As seen from this tree, it showed 100% concordance between the predicted genus assignments by the AppIndels. com server and the phylogenetic placements of the isolates. These results demonstrate that the TAXIs for the Lactobacillaceae genera identified in this study provide a robust and practical tool for the taxonomic classification of novel or uncharacterized isolates from this family.

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

Earlier studies on CSIs specific for other prokaryotic taxa have shown that genetic changes are frequently located in surface-exposed loop regions of proteins, which are flexible, unstructured areas often involved in mediating novel protein-protein or protein-ligand interactions [69,70,71,72,73,74]. In view of these findings, we have also investigated the location of some representative CSIs specific for the Lactobacillaceae genera, for whom sequence information is presented in Figure 2, Figure 3 and Figure 4 in protein structures.

As described in the Methods section, AlphaFold was used to predict the structures of selected proteins containing conserved signature inserts (CSIs) (Figure 2, Figure 3 and Figure 4), as well as homologous proteins lacking the CSIs. To determine the structural localization of each CSI, we superimposed the predicted structures of the protein with and without the CSI. Figure 9 presents the results for four representative CSIs. In each case, the figure shows the structural alignment of the two protein models, with the CSI regions highlighted in red. These overlays clearly illustrate the position of the CSIs within the protein structures, and they show that all four examined CSIs viz. (i) a 2 aa insertion in the 50S ribosomal protein L10 specific for Lactobacillus (Figure 2B); (ii) 1 aa insertion in the protein manganese-dependent inorganic pyrophosphatase specific for the genus Lacticaseibacillus (Figure 3B); (iii) a 2 aa insertion in the protein cyclopropane-fatty-acyl-phospholipid synthase family specific for Apilactobacillus (Figure 4B), and (iv) a 1 aa insertion in the protein pyridoxal phosphate-dependent aminotransferase specific for Lactiplantibacillus (Figure 4D), are localized in surface-exposed loop regions of the proteins (Figure 9A–D). These findings demonstrate that like the structural locations of CSIs in previous studies [69,73,75,76], the identified CSIs specific for the Lactobacillaceae genera are also structurally localized to surface-exposed loops of the proteins and may contribute to genus-specific functional traits by mediating novel biologically important interactions.

4. Discussion

The family Lactobacillaceae, comprising a diverse group of lactic acid bacteria, plays a central role in food production, probiotic development, and human health due to its metabolic versatility and beneficial properties [1,2,3,7]. This study presents comprehensive phylogenomic and comparative genomic analyses of 411 Lactobacillaceae genomes, leading to the identification of 171 novel molecular markers in the form of conserved signature indels (CSIs) or TAXIs in diverse proteins, each specific to various Lactobacillaceae genera. These findings represent a significant advancement in our understanding of this family. By integrating molecular markers with a robust phylogenomic framework, this work provides powerful tools for taxonomic delineation, the development of reliable diagnostic strategies, and insights into genus-specific functional traits, thereby enhancing both scientific understanding and practical applications of Lactobacillaceae species.

Prior to 2020, the genus Lactobacillus, which encompassed the majority of the family Lactobacillaceae, was taxonomically problematic due to its genetic heterogeneity and polyphyletic placement in phylogenetic trees [5,11,14,23,51,52]. The landmark reclassification by Zheng et al. [3] addressed this issue by dividing Lactobacillus into 23 new genera and merging the former Leuconostocaceae family [7] into Lactobacillaceae, resulting in a more coherent and phylogenetically consistent taxonomy [3,13]. However, the lack of reliable molecular or biochemical markers to distinguish among the newly defined genera has limited the precision of taxonomic assignments, and further advancements in understanding unique aspects of the species from different genera.

The 171 CSIs identified in this study, each uniquely shared by species within a specific genus, offer a molecular solution to this challenge. These TAXIs, which result from rare genetic changes in conserved protein regions, serve as molecular synapomorphies providing independent evidence of evolutionary relationships [18,20,33]. The phylogenomic tree constructed from 411 genomes, based on 87 conserved proteins, further supports the monophyly of these genera and reinforces the revised taxonomic framework proposed by Zheng et al. [3].

Unlike traditional metrics such as average amino acid identity (AAI) or 16S rRNA sequence similarity, which often lack resolution at the genus level [77,78,79], CSIs provide highly specific and predictive genus-level molecular signatures [64]. Their reliability is also demonstrated in the present work by their consistent presence in newly sequenced species from the genera such as Fructobacillus and Weissella. This INDELIBLE (Indel-based Identification of Bacterial Lineages and Evolution) approach, which uses taxon-specific CSIs (TAXIs), has proven effective in clarifying the taxonomy of several other microbial groups, including Bacillus [22,80], Burkholderia [81,82], Enterobacterales [36], and Pseudomonas [83,84], and now establishes a robust molecular basis for Lactobacillaceae taxonomy. The predictive power of these TAXIs is further demonstrated by their successful application in classifying 112 out of 113 uncharacterized Lactobacillus isolates into 11 Lactobacillaceae genera using the AppIndels.com server [34]. This tool, developed to predict taxonomic affiliations based on the presence of known CSIs in genome sequences, enhances diagnostic efficiency, particularly in high-throughput genomic analyses. Given the rapid pace of discovery of Lactobacillaceae species, with over 150 new species described since 2020 [27], tools such as the Appindels.com server are invaluable for the accurate and scalable classification of novel or uncultured isolates as genomic databases continue to expand [34].

The absence of reliable diagnostic markers also hinders the identification of Lactobacillaceae species in clinical, industrial, and environmental settings. The TAXIs identified in this study, located in conserved regions of diverse proteins, offer a novel and highly specific diagnostic toolkit. The conserved flanking regions of these CSIs can be used to design PCR primers, or probes for qPCR and pyrosequencing, enabling selective amplification or detection of CSI-containing organisms. Similar TAXIs-based diagnostic assays have been successfully developed for Bacillus anthracis [85] and Escherichia coli O157:H7 [86], and primers targeting TAXIs have also been used to detect species from Actinobacteria [87] and Chlamydiae [88].

Beyond taxonomy and diagnostics, CSIs provide a gateway to exploring genus-specific functional traits. Located in conserved regions of proteins involved in essential cellular processes, these indels are likely to be functionally significant [18,72,89]. Prior studies have shown that such genetic changes often underlie unique biochemical or phenotypic characteristics [69,75,76,89,90,91,92]. Structural predictions using AlphaFold 3 revealed that all examined CSIs are located in surface-exposed loops, suggesting roles in host interactions, environmental adaptation, or substrate specificity. Such traits are likely to be critical to the ecological and industrial relevance of Lactobacillaceae species [13,68]. For example, Apilactobacillus species contain a surface-exposed CSI in the protein cyclopropane-fatty-acyl-phospholipid synthase (Figure 9C), which is known to facilitate acid stress resistance in gastric bacteria [93,94] and is predicted to enhance bile salt resistance through lipid synthesis [95]. Given that Apilactobacillus species thrive in highly acidic environments (pH < 3) [3], further investigation into this CSI may provide insights into their acidophilic nature and probiotic potential [60,96]. Similarly, Lactiplantibacillus species are distinguished by a CSI in a surface loop of pyridoxal phosphate-dependent aminotransferase (Figure 9D), an enzyme involved in amino acid metabolism [97], making it a promising marker for studying metabolic adaptations in this genus.

In summary, biochemical and functional studies of these genus-specific CSIs are likely to uncover novel traits unique to each Lactobacillaceae genus. Given the widespread industrial use of Lactobacillaceae species in food-related applications [24,25,26,98], the insights gained from such studies could have significant implications for biotechnology, health, and microbial ecology.