Genome-Scale Analysis Reveals Strain Kdesi as a Distinct Evolutionary Lineage and Extensive Cryptic Diversity in the Genus Bdellovibrio

Genome-Scale Analysis Reveals Strain Kdesi as a Distinct Evolutionary Lineage and Extensive Cryptic Diversity in the Genus Bdellovibrio | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Genome-Scale Analysis Reveals Strain Kdesi as a Distinct Evolutionary Lineage and Extensive Cryptic Diversity in the Genus Bdellovibrio Temidayo Oluyomi Elufisan, Isabel Cristina Rodríguez-Luna, Yewande Olajumoke Ajao, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8476110/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bdellovibrio are predatory bacteria with potential applications in treating multidrug-resistant bacterial infections. We isolated Bdellovibrio sp. kdesi from sewage in Reynosa, Mexico, and performed comprehensive genomic characterization. The complete genome (3,343,978bp; 48.5% GC content; 3,208 coding sequences) contains a single rRNA operon with two identical 16S rRNA genes at distinct loci. While 16S rRNA analysis showed 99.3% identity to B. bacteriovorus SSB218315, polyphasic genomic analysis revealed kdesi as a distinct species. Core-genome phylogeny, average nucleotide identity (ANI ~ 89%), average amino acid identity (AAI ~ 82%), and digital DNA-DNA hybridization (dDDH < 70%) clearly demonstrated that Kdesi along with several other strains currently classified as B. bacteriovorus represents novel genomospecies requiring formal taxonomic revision. Comparative genomic analysis of 29 Bdellovibrio genomes revealed an open pan-genome (~ 25,000 genes) with limited transposable elements, absence of SecB in the Sec secretion system, and strain-specific adaptations in Type IV secretion systems. These findings underscore the need for genome-based taxonomic revision of the genus and highlight the importance of using multiple molecular criteria beyond 16S rRNA for bacterial species delineation. Biological sciences/Genetics Biological sciences/Microbiology Bdellovibrio sp. Kdesi comparative genomics average nucleotide identity taxonomic revision predatory bacteria Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Bdellovibrio and like organisms (BALOs) are obligate predatory bacteria that prey on Gram-negative pathogens, making them promising alternatives to conventional antibiotics [ 1 , 2 ]. These bacteria invade the periplasmic space of prey cells, replicate within this protected niche (termed the bdelloplast), and emerge to attack new hosts [ 3 ]. This is particularly relevant given the global crisis of antibiotic resistance, and as a result, Bdellovibrio spp. have attracted significant research attention for their potential therapeutic and probiotic applications [ 4 , 5 ]. The taxonomic classification of BALOs has undergone several revisions. Initially divided into four genera ( Bdellovibrio , Bacteriovorax, Halobacteriovorax, and Peridibacter) based on habitat and GC content [ 6 ], more recent genomic analyses have identified epibiotic strains as a separate genus, Pseudo Bdellovibrio [ 7 , 8 ]. However, the genus Bdellovibrio itself contains only three formally described species (B. bacteriovorus, B. reynosensis, and B. svalbardensis), despite substantial genomic diversity among isolates labeled as B. bacteriovorus [ 1 , 9 , 10 ]. The proliferation of whole-genome sequencing has revealed that 16S rRNA gene-based identification often fails to capture species-level diversity in bacteria [ 11 ]. This limitation is particularly evident in Bdellovibrio , where strains sharing > 98% 16S rRNA identity exhibit average nucleotide identity (ANI) values well below the 95–96% species threshold [ 12 ]. Modern taxonomic frameworks increasingly rely on genome-scale metrics including ANI, average amino acid identity (AAI), digital DNA-DNA hybridization (dDDH), and core-genome phylogeny to delineate bacterial species [ 13 , 14 ]. In this study, we isolated Bdellovibrio sp. Kdesi from sewage in Reynosa, Mexico, and performed comprehensive genomic characterization. Our objectives were to (1) determine the precise taxonomic position of Kdesi using polyphasic genomic analysis, (2) perform comparative genomics to identify strain-specific adaptations, and (3) evaluate the taxonomic status of other Bdellovibrio isolates. Our findings reveal substantial cryptic diversity within Bdellovibrio and support the need for formal taxonomic revision of this ecologically and clinically important genus. Results Isolation and General Genomic Features of Kdesi Predatory plaques appeared on DNB agar after 48 hours of incubation. Partial 16S rRNA sequencing identified the isolate as Bdellovibrio with 99.3% identity to B. bacteriovorus SSB218315 (accession MG957118), suggesting initial classification as B. bacteriovorus (Fig. 1). Figure 1. Phylogenetic tree based on 16S rRNA sequences showing placement of Bdellovibrio sp. Kdesi within the B. bacteriovorus cluster. The complete genome of Kdesi comprises a single circular chromosome of 3,343,978 bp with 48.5% GC content. NCBI PGAP annotation identified 3,274 genes, including 3,208 protein-coding sequences, 3 rRNAs (single operon), 48 tRNAs, and 15 ncRNAs. Notably, the genome contains two identical 16S rRNA genes: one within the rRNA operon and a second copy at a distinct genomic location. The genome has been deposited in NCBI under accession CP102930 (BioProject PRJNA524134). General genomic features for all strains analyzed are summarized in Table 1 . Table 1 General genomic features of Bdellovibrio species and strains analyzed in this study. Organism Name Strain Genome Size (Mb) GC (%) Protein-coding Genes Pseudogenes Completeness (%) Institution Bdellovibrio bacteriovorus 109J 3.84 50.5 3628 97.72 Kurume University School of Medicine Bdellovibrio sp. 22V 22V 3.6 46.5 3437 6 95.81 CBG Instituto Politecnico Nacional Bdellovibrio bacteriovorus Bd10 3.87 50.0 3639 3 96.96 College of Veterinary Medicine Bdellovibrio bacteriovorus Bd16 3.43 43.0 3194 4 83.90 College of Veterinary Medicine Bdellovibrio bacteriovorus Bd3 4.09 45.0 3878 43 90.23 College of Veterinary Medicine Bdellovibrio bacteriovorus Bd4 3.74 45.5 3486 5 93.25 College of Veterinary Medicine Bdellovibrio bacteriovorus Bd5 3.77 48.5 3536 21 94.02 College of Veterinary Medicine Bdellovibrio sp. HCB-110 HCB-110 4.17 45.0 3933 4 96.37 Instituto Politecnico Nacional Bdellovibrio sp. HCB117 HCB117 3.73 45.5 3487 6 96.46 Huazhong Agricultural University Bdellovibrio sp. HCB-162 HCB-162 3.89 44.5 3705 6 96.69 Huazhong Agricultural University Bdellovibrio sp. HCB185ZH HCB185ZH 3.92 45.5 3745 7 95.53 Huazhong Agricultural University Bdellovibrio sp. HCB209 HCB209 3.86 45.0 3718 3 95.63 Huazhong Agricultural University Bdellovibrio sp. HCB274 HCB274 3.72 46.5 3567 4 96.32 Huazhong Agricultural University Bdellovibrio sp. HCB288 HCB288 3.65 46.5 3510 3 96.31 Huazhong Agricultural University Bdellovibrio sp. HCB290 HCB290 3.82 46.0 3680 5 95.63 Huazhong Agricultural University Bdellovibrio sp. HCB337 HCB337 4.28 45.0 4092 7 89.43 Huazhong Agricultural University Bdellovibrio bacteriovorus HD100 HD100 3.78 50.5 3583 98.31 Max-Planck-Institute Bdellovibrio bacteriovorus HK2 3.86 50.5 3870 159 88.61 College of Veterinary Medicine Bdellovibrio bacteriovorus kdesi 3.34 48.5 3208 18 89.04 CBG Instituto Politecnico Nacional Bdellovibrio sp. KM01 KM01 3.96 45.5 3752 17 95.02 Providence College Bdellovibrio reynosensis LBG001 3.58 43.0 3289 7 92.88 Tufts University Bdellovibrio sp. NC01 NC01 3.98 44.5 3773 23 95.51 Providence College Bdellovibrio sp. SKB1291214 SKB1291214 3.68 45.0 3570 13 94.90 Centro de Biotecnologia Genomica, Instituto Politecnico Nacional Bdellovibrio bacteriovorus SSB218315 3.77 50.5 3536 43 96.88 Centro de Biotecnologia Genomica, Instituto Politecnico Nacional Bdellovibrio bacteriovorus str. Tiberius Tiberius 3.99 50.0 3738 96.08 University of Nottingham Bdellovibrio bacteriovorus W W 3.01 43.5 2871 University of Oklahoma Health Sciences Center Bdellovibrio sp. ZAP7 ZAP7 4.12 45.5 3937 26 95.56 Providence College Phylogenomic Analysis Reveals Kdesi as a Distinct Lineage Core-genome phylogeny based on 760 concatenated single-copy genes (or 56 genes constituting a 50,201 bp alignment when using Roary analysis) resolved 29 Bdellovibrio strains into two major clades with several singleton lineages (Fig. 2 ). Kdesi clustered in Clade 1 but formed a distinct branch separate from the type strain B. bacteriovorus HD100 and its close relative 109J. Clade 1 also contained B. bacteriovorus strains Tiberius, SSB218315, EC13, BER2, Bd3, and Bd4, as well as several unclassified isolates (HCB110, HCB-162, HCB117, Bd16) and B. reynosensis LBG001. Clade 2 comprised primarily environmental isolates including NC01, ZAP7, KM01, and SKB1291214, with B. svalbardensis PAP01 branching basally. B. bacteriovorus W formed a singleton lineage, while PseudoBdellovibrio exovorus served as the outgroup. Core-SNP phylogeny generated by REALPHY produced a congruent topology (Fig. S1 ), supporting the robustness of these relationships In contrast, 16S rRNA phylogeny (Fig. 1) grouped Kdesi with SSB218315 within a well-supported B. bacteriovorus cluster, illustrating the limitations of 16S rRNA for species-level resolution in this genus. Genome-Based Taxonomic Metrics Support Novel Species Status ANI analysis revealed that Kdesi shares ~ 89% identity with SSB218315 and even lower values (~ 85–88%) with other Bdellovibrio strains (Fig. 3). These values fall well below the 95–96% species threshold, indicating that Kdesi represents a distinct species. Similarly, AAI values between Kdesi and other strains ranged from ~ 76% to ~ 82%, with SSB218315 showing the highest similarity at 82% (Fig. S2). All pairwise comparisons exceeded the ~ 65–70% genus-level threshold, confirming Kdesi's placement within Bdellovibrio. The percentage of conserved proteins (POCP) was 56%, well above the 50% genus threshold. Digital DNA-DNA hybridization (dDDH) provided additional support for species-level distinctness. Using GGDC formulas 1, 2, and 3, dDDH between Kdesi and SSB218315 was 40.2%, 21.9%, and 33.9%, respectively—all substantially below the 70% species cutoff. Similarly, dDDH between Kdesi and HD100 yielded values of 38.8%, 22.0%, and 33.0%. Notably, several strains currently classified as B. bacteriovorus (e.g., Tiberius, W, EC13, BER2) also showed dDDH 70% under all three formulas, confirming their conspecific status. SSB218315 showed formula-dependent relationships: formulas 1 and 3 suggested conspecificity with HD100/109J (dDDH ~ 72–75%), while formula 2 yielded values of ~ 68%, just below the threshold. Given the concordant low ANI (~ 89%) and AAI (~ 82%) values, we provisionally treat SSB218315 as a putative genomospecies pending further validation. Based on integrated ANI, AAI, dDDH, and phylogenomic data, we propose the existence of at least 12 distinct genomospecies within the genus Bdellovibrio , including Kdesi, substantially expanding the recognized diversity of this genus (Table 2 ). Figure 3. Heatmap of Average Nucleotide Identity (ANI) values among 29 Bdellovibrio genomes, highlighting < 95% identity thresholds supporting novel species status. Table 2 Genome-based species delineation metrics (ANI, AAI, dDDH) and proposed genomospecies classification among 29 Bdellovibrio strains. Cluster Member strains Interpretation Cluster 1 109J, HD100 B. bacteriovorus Cluster 2 BER2, Bd4 B. bacteriovorus -like (genomospecies) Cluster 3 Bd3, sp. 110 Novel genomospecies Cluster 4 reynosensis B. reynosensis Cluster 5 svalbardensis PAP01 B. svalbardensis Singletons Tiberius, Bd10, SSB219315, Bd5, Kdesi, Bd16, W, ZAP7 Putative novel species Other novel species HCB288, HCB274, HCB290, HCB209, SKB1291224, HCB185ZH, KM01, HCB337, 22V, NC01 Putative novel species Pan-Genome Structure and Functional Architecture Pan-genome analysis using BPGA identified approximately 25,000 genes across 29 Bdellovibrio genomes, with Heaps' law analysis (α < 1) indicating an open pan-genome (Fig. S3). The core genome comprised 760 genes present in all strains, representing fundamental cellular processes. Roary analysis with more stringent parameters (≥ 95% presence threshold) identified 56 core genes. The accessory genome varied substantially among strains, with strain-specific gene counts ranging from ~ 150 (Kdesi) to 2031 ( Bdellovibrio sp. HCB337; 47.6% of its CDS), reflecting diverse ecological adaptations. OrthoFinder identified 6,312 orthologous groups accounting for ~ 97% of all genes, with 1,425 core orthogroups shared by all strains (Table 2 ). Functional annotation using eggNOG-mapper assigned the core genome to 20 COG categories, 304 KEGG pathways, and 819 GO terms. Nearly all core proteins (> 95%) carried PFAM domains, indicating high structural conservation of essential functions (Fig. S4). COG category distribution in the core genome was dominated by housekeeping functions: translation (J), transcription (K), and replication/repair (L) collectively accounted for ~ 35% of core genes. Genes involved in amino acid metabolism (E), energy production (C), and cell envelope biogenesis (M) were also highly conserved. In contrast, categories related to signal transduction (T), motility (N), and secondary metabolism (Q) showed greater inter-strain variation, suggesting strain-specific adaptations in environmental sensing and predatory behavior. Kdesi-specific genes (n = ~ 150) are enriched for regulatory functions including multiple histidine kinases (e.g., RegB-like sensor kinases) and transcriptional regulators (LysR family), suggesting enhanced environmental sensing. Additionally, Kdesi possesses unique ABC transporters for osmoprotectants (glycine betaine/proline uptake systems OpuBA), a citrate transporter, and restriction-modification systems, potentially reflecting adaptation to sewage environments with high osmotic stress and frequent phage exposure. Approximately 26% of Kdesi-unique genes encode proteins with domains of unknown function (DUFs), representing candidates for novel predation-associated factors. Genomic Islands and Horizontal Gene Transfer Island Viewer 4 identified five genomic islands (GIs) in Kdesi totaling 63,559 bp and spanning positions 213,412 − 229,341; 1,033,588-1,041,791; 1,319,854-1,325,685; 1,493,316-1,503,22; and 1,876,696-1,882,115 (Fig. S5). These GIs are enriched for genes encoding surface structures, transporters, and regulatory factors. Notably, GI-1 contains the flagellar filament capping protein FliD and two flagellin genes, critical for motility and prey detection. Other GI-encoded functions include ABC transport systems, phosphoenolpyruvate carboxylase, transposases, ecdysteroid kinases, and SAM-dependent methyltransferases. Forty-two GI genes (66%) remain hypothetical, highlighting the potential for discovery of novel predation factors. The number of GIs varied across strains: HD100 (5), 109J (4), BER2 (3), EC13 (6), KM01 (4), Tiberius (13), SSB218315 (3), W (6). Interestingly, three GI genes—two flagellins and one acetyltransferase—are conserved across all strains and thus part of the core genome, suggesting ancient horizontal acquisition and subsequent vertical inheritance as part of a minimal predatory toolkit. Limited Transposable Elements in Bdellovibrio Genomes Bdellovibrio genomes harbor remarkably few insertion sequences (IS) compared to other bacteria. ISEScan detected only 41 IS elements across all 29 strains, belonging to families IS21, IS3, ISNCY, and ISL3 (Fig. 4). IS21 was present in all strains except one, suggesting a role in genome plasticity. IS3 occurred sporadically in Bd3, HCB274, Bd5, HCB117, W, and P. exovorus, while ISL3 was restricted to W, BER2, and P. exovorus. The paucity of IS elements may reflect genome streamlining associated with an obligate predatory lifestyle or strong purifying selection against disruptive transposition. Figure 4. Distribution of insertion sequence (IS) elements across Bdellovibrio genomes, showing limited transposable element abundance Protein Secretion Systems All Bdellovibrio genomes encode complete Sec, Tat, Type II secretion (T2SS), and Type IV secretion (T4SS) systems, consistent with their role in exporting hydrolytic enzymes and other effectors during prey invasion. Gene counts for Sec, Tat, and T2SS ranged from 17–20 per genome. Notably, SecB—a molecular chaperone that delivers unfolded substrates to the Sec translocon in E. coli was absent from all Bdellovibrio strains (Table S1 , Fig. S6), suggesting functional compensation by alternative chaperones (e.g., DnaK, GroEL) or distinct substrate-targeting mechanisms. T4SS components varied more extensively among strains, ranging from 15 proteins ( B. reynosensis ) to 21 ( B. bacteriovorus Tiberius). Core T4SS genes conserved across all strains included VirB11, VirB1, TrbN, TcpA, DotB, and TraJ, essential for pilus assembly and substrate translocation. Accessory T4SS components showed strain-specific distributions: TraF (only in NC01), TrwF (SKB1291214 and ZAP7), PrgC and TrwD (Tiberius), and VirD4 (RO). This modular T4SS architecture likely reflects diverse strategies for prey invasion, DNA uptake, or effector delivery. Synteny of Predatory Gene Clusters Bidirectional synteny analysis between Kdesi and HD100 revealed high conservation of gene order across the chromosome, with no major inversions or translocations detected (Fig. S6a). Kdesi and SSB218315 shared 2,852 orthologous genes, while Kdesi and HD100 shared 2,844 genes. Despite conserved synteny, only ~ 10% of orthologous protein pairs exhibited > 96% amino acid identity, whereas ~ 64% showed ≤ 85% identity consistent with substantial sequence divergence supporting species-level distinctness. Targeted synteny analysis of predation-associated loci revealed contrasting patterns. The Type IVb pilus (T4P) cluster essential for prey attachment and invasion showed near-perfect synteny across HD100, 109J, Tiberius, Kdesi, and SSB219315 (Fig. S7), indicating strong purifying selection on this critical apparatus. In contrast, an oligopeptide transport cluster exhibited profound divergence in Kdesi: the corresponding ~ 13.5 kb region contained no conserved synteny and harbored unique genes (KFHEMNGN_ome, GASZ_ome) not found in HD100, 109J, or Tiberius. SSB219315 showed intermediate architecture (~ 11.9 kb) with distinct genes (ca4A, gpp8_2), suggesting lineage-specific adaptation in peptide sensing or uptake that may influence prey range or environmental fitness. Discussion Kdesi Represents a Novel Species Revealing Evolutionary Dynamics Our polyphasic genomic analysis unequivocally demonstrates that Bdellovibrio sp. Kdesi represents a novel species. While 16S rRNA sequencing initially suggested classification as B. bacteriovorus (99.3% identity to SSB218315), genome-scale metrics reveal substantial divergence: ANI ~ 89%, AAI ~ 82%, and dDDH < 40%, all well below established species thresholds. These findings underscore a critical limitation of 16S rRNA-based taxonomy, where high rRNA similarity does not guarantee conspecificity, a phenomenon documented in genera such as Enterobacter , Corynebacterium , and Acinetobacter [ 11 , 15 ] The cgSNP phylogeny provides crucial insight into kdesi's evolutionary history, revealing it as a recently diverged lineage within the B. bacteriovorus complex that has undergone accelerated evolution. While cgSNP analysis places kdesi within the B. bacteriovorus radiation, its extended branch length (0.00693 substitutions/site) indicates rapid genetic changes following ecological specialization [ 16 ]. This apparent paradox of recent common ancestry but current genomic distinctness exemplifies ecological speciation in action, where environmental pressures drive rapid genomic reorganization that outpaces SNP accumulation in core genes. kdesi's unique genomic features, including strain-specific regulatory systems, osmoprotectant transporters, and remodeled oligopeptide uptake clusters, represent adaptive solutions to its sewage environment. The high osmotic conditions likely selected for unique transporter systems (OpuBA) (Wang et al., 20250), while diverse prey communities drove expansion of environmental sensing capabilities and predation machinery. This pattern of recent divergence followed by rapid niche adaptation represents a compelling model for bacterial speciation dynamics. The robust genomic evidence presented here, which includes phylogenomic placement, ANI ~ 89%, AAI ~ 82%, and dDDH < 40% unequivocally demonstrates that strain kdesi represents a novel gemospecies within the genus Bdellovibrio , distinct from all currently described species including B. bacteriovorus , B. reynosensis , and B. svalbardensis . The unique genomic features of strain kdesi particularly adaptations for osmotic stress tolerance (OpuBA transporters), enhanced environmental sensing (multiple histidine kinases), and modified oligopeptide transport suggest specialization to high-osmolarity wastewater environments and provide genomic predictions that could guide phenotypic characterization should conspecific strains be isolated [ 17 ]. Cryptic Diversity Necessitates Taxonomic Revision of Bdellovibrio Beyond kdesi, our analysis revealed extensive cryptic diversity within Bdellovibrio. Based on ANI, AAI, and dDDH thresholds, we identified at least 12 distinct genomospecies among the 29 strains examined (Table 2 ). Notably, strains labeled B. bacteriovorus exhibit substantial genomic heterogeneity: only HD100 and 109J consistently showed dDDH > 70%, confirming conspecificity. Other B. bacteriovorus isolates (e.g., Tiberius, W, EC13, BER2, SSB218315) represent putative novel species that warrant formal characterization. The cgSNP phylogeny further supports this taxonomic complexity, revealing multiple evolutionarily distinct lineages currently classified under single species designations. Strains such as W and Bd16 form long-branch lineages suggesting substantial evolutionary distance, while environmental isolates (HCB series) cluster separately from the core B. bacteriovorus group. This phylogenetic structure aligns with ecological origins, suggesting that habitat specialization has been a major driver of Bdellovibrio diversification [ 18 ]. The taxonomic confusion in Bdellovibrio mirrors broader challenges in microbial systematics. Reliance on single-gene markers (16S rRNA) and phenotypic traits has led to taxonomic oversimplification in many bacterial groups [ 54 , 55 ]. The advent of affordable whole-genome sequencing now enables rigorous species delineation using multiple independent criteria phylogenomic, AAI, and dDDH that collectively provide a robust taxonomic framework. Our findings support recent calls for comprehensive taxonomic revision of Bdellovibrio and highlight the genus as a model system for exploring bacterial diversity and evolution in predatory lifestyles [ 19 ]. Genomic Architecture Reflects Predatory Lifestyle Adaptations The Bdellovibrio core genome is enriched for housekeeping functions (COG categories J, K, L), consistent with essential cellular processes required across both attack and growth phases [ 20 ]. Accessory genomes vary substantially, reflecting niche-specific adaptations: Kdesi's unique genes for osmoprotection, environmental sensing, and oligopeptide transport likely facilitate survival in sewage habitats with high osmotic stress and diverse prey communities. The open pan-genome (~ 25,000 genes) indicates high genomic plasticity, where each new genome sequenced contributes novel genes. This plasticity enables Bdellovibrio to adapt to diverse ecological niches freshwater, marine, soil, and wastewater and to prey on a broad range of Gram-negative bacteria [ 3 , 21 ]. Genomic islands encoding motility factors, transporters, and hypothetical proteins suggest ongoing horizontal gene transfer, although limited IS element abundance (~ 41 total across 29 genomes) implies constrained transposition activity, possibly reflecting genome streamlining or strong purifying selection associated with an obligate predatory lifestyle. The universal absence of SecB is intriguing. In E. coli , SecB maintains substrates in unfolded states for Sec-mediated export [ 22 ]. Its absence in Bdellovibrio suggests either: (1) functional compensation by DnaK or GroEL chaperones, (2) Sec substrates adopt transport-competent conformations independently, or (3) Tat-mediated export of folded proteins predominates. Given that Bdellovibrio secretes numerous hydrolases during prey invasion, understanding its secretion pathway preferences is critical for elucidating its predatory mechanisms. Evolutionary and Ecological Perspectives Comparative analysis among B. bacteriovorus HD100, B. reynosensis , B. svalbardensis , and Bdellovibrio sp. Kdesi demonstrates a remarkable balance between genomic conservation and ecological innovation. The cgSNP phylogeny reveals a complex evolutionary history where recent radiation coexists with long-divergent lineages. The extremely short branches among HD100, 109J, and related strains (0.00000085 substitutions/site) indicate recent clonal expansion, while long-branch taxa like B. exovorus JSS (0.06083) and B. bacteriovorus W (0.02871) represent evolutionarily distinct lineages. The syntenic conservation of predation-associated operons, such as the Type IV pilus cluster, points to strong evolutionary constraints on the core predatory machinery. In contrast, divergence in oligopeptide transport and regulatory gene clusters suggests adaptive evolution to distinct environmental niches soil, and sewage [ 23 ]. Kdesi's evolutionary trajectory appears particularly dramatic: recent divergence from the B. bacteriovorus core group followed by rapid genomic changes, potentially driven by the unique selective pressures of sewage environments. The coexistence of conserved predatory frameworks and lineage-specific adaptations supports an eco-evolutionary model of Bdellovibrio diversification: a stable predatory core optimized for prey recognition and invasion, supplemented by flexible accessory genomes that enable niche specialization. This dual architecture likely underlies the genus's global ecological success and provides genomic flexibility for adapting to new environments and prey spectra. Implications for Functional Genomics and Applied Research The identification of 26% of Kdesi-unique genes as hypothetical proteins (DUFs) represents an exciting frontier for functional discovery. Predatory bacteria possess unique molecular machinery for prey recognition, invasion, bdelloplast formation, and resource extraction [ 24 ]. Characterizing these DUFs through transcriptomics, proteomics, and targeted mutagenesis may reveal novel antimicrobial mechanisms applicable to treating drug-resistant infections. Kdesi's evolutionary story offers particular promise for applied research. As a recently evolved sewage specialist, it may possess enhanced capabilities for targeting pathogens prevalent in wastewater environments, including multidrug-resistant strains. Its unique transporter systems and regulatory networks could be harnessed for improving predation efficiency in complex microbial communities. The modular T4SS architecture across Bdellovibrio strains suggests diverse strategies for effector delivery. Strain-specific components (e.g., TraF, TrwF, VirD4) may influence prey range, killing kinetics, or resistance to host defenses. Comparative functional studies could guide the rational selection of Bdellovibrio strains optimized for specific therapeutic applications, e.g., targeting biofilm-embedded pathogens or multidrug-resistant Pseudomonas aeruginosa or Acinetobacter baumannii . The syntenic conservation of the T4P operon across divergent lineages underscores its non-redundant role in prey attachment and invasion, making it an attractive target for synthetic biology applications. Engineering enhanced T4P variants could improve predation efficiency or expand host range, advancing Bdellovibrio as a next-generation antimicrobial platform. Conclusions We showed through comprehensive comparative genomic analysis that Bdellovibrio sp. kdesi is a distinct species within the genus Bdellovibrio , highlighting the limitations of 16S rRNA-based taxonomy and the necessity of polyphasic genomic approaches for accurate species delineation. The cgSNP phylogeny reveals kdesi as a compelling case of recent ecological speciation, where adaptation to sewage environments might have driven rapid genomic changes leading to novel species formation while maintaining phylogenetic signal of recent common ancestry with B. bacteriovorus . The comparative analysis of 29 genomes reveals substantial cryptic diversity within Bdellovibrio , with at least 12 genomospecies requiring formal taxonomic revision. The genus exhibits an open pan-genome, limited transposable elements, universal absence of SecB, and modular T4SS architecture—features reflecting adaptation to obligate predatory lifestyles across diverse environments. These findings provide a genomic foundation for future functional studies aimed at harnessing Bdellovibrio for antimicrobial therapy and offer a model system for understanding bacterial predation at the molecular level. The evolutionary dynamics revealed by kdesi's genome recent divergence followed by rapid adaptation provide insight into the process of bacterial speciation in real-time. These findings call for a systematic taxonomic revision of Bdellovibrio , integrating genome-based species delineation (ANI, AAI, dDDH) with ecological and phylogenetic criteria. Materials and Methods Bacterial Isolation and Preliminary Identification A 500 mL sewage sample was collected from Colonia Las Delicias, Reynosa, Tamaulipas, Mexico (26.099793°N, 98.981904°W), filtered through 0.45 µm membranes to remove protozoans and larger bacteria, and enriched using Klebsiella pneumoniae as prey following established protocols [ 25 ]. Briefly, overnight prey cultures were harvested by centrifugation (4,000 rpm, 20 min, 4°C), washed twice in HEPES buffer (pH 7.4), and co-incubated with HEPES buffer at 30°C with shaking. After visible lysis occurred, the supernatant was passed through two additional enrichment cycles to increase predator titer. Predatory bacteria were isolated using double-layer agar plating [ 25 ] Serial dilutions of enriched supernatants (10 − 1 to 10 − 8 ) were mixed with HEPES-suspended prey and overlaid on dilute nutrient broth (DNB) agar. Plaques appearing after 48 hours were isolated, and plaque-forming bacteria were co-cultured with fresh prey. Genomic DNA was extracted from predator-enriched lysates using the Promega Wizard Genomic DNA Extraction Kit following manufacturer instructions. Preliminary identification was performed by PCR amplification of partial 16S rRNA using Bdellovibrio -specific primers BdsF (5′-TCTGGCTCAGAACAAACGCT-3′) and BdsR (5′-GCTTCGTCACTGAAGGGGTC-3′), which amplify an ~ 818 bp fragment. PCR conditions were initially denatured at 95°C for 3 min; 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s; final extension at 72°C for 5 min. Amplicons were sequenced using Sanger sequencing (AB3130, Thermo Fisher Scientific) and queried against the NCBI nucleotide database using BLASTN. Genome Sequencing, Assembly, and Annotation Genomic DNA quality was assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Whole-genome sequencing was performed on the Illumina MiSeq platform (MyGenomics LLC, USA). Raw reads were quality-filtered using FastQC v0.11.9 [ 26 ], trimmed with Trim Galore v0.6.7 [ 27 ] and assembled using SPAdes v13.0.1 [ 28 ]. The assembly was further processed and consolidated using Unicycler v0.5.0 [ 29 ]. Assembly quality was evaluated with QUAST v5.0 [ 30 ]. To identify potential contaminants, assembled contigs were mapped against the B. bacteriovorus HD100 reference genome using NUCmer [ 31 ]. Contaminant sequences were removed using custom Perl scripts. The resulting four contigs were scaffolded into a single circular chromosome using MEDUSA [ 32 ] and NUCmer, with gap closure validated by manual inspection of read alignments. The closed genome was annotated using Prokka v1.14.6 [ 33 ] and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [ 34 ]. Phylogenomic and Taxonomic Analyses Twenty-nine Bdellovibrio genomes (27 complete, 2 draft) were retrieved from NCBI (Table 1 ). All genomes were re-annotated with Prokka v1.14.6 to ensure consistent gene calling. Pan-genome analysis was performed using Bacterial Pan Genome Analysis (BPGA) v1.3 [ 35 ] with default parameters and Roary v3.13 [ 36 ] with 90% BLASTP identity threshold and core genes defined as present in ≥ 95% of isolates. Core-genome phylogeny was reconstructed from 760 single-copy core genes extracted by BPGA. These core genes were concatenated and aligned using MAFFT v7.475 [ 37 ]. Maximum-likelihood phylogenetic trees were inferred using IQ-TREE v2.4.0[ 38 ] with automatic model selection (best model: GTR + F + I + G4) and 1,000 ultrafast bootstrap replicates. Core-genome SNP (cgSNP) analysis was performed using REALPHY ( http://realphy.unibas.ch/fcgi/realphy , last accessed November 5, 2025) [ 39 ], and trees were visualized using MEGA 12[ 40 ] and iTOL v6 [ 41 ]. Average nucleotide identity (ANI) was calculated using PyANI v0.2.10 (ANIb method) [ 42 ]. Average amino acid identity (AAI) was computed using COMPASS [ 43 ]. Digital DNA-DNA hybridization (dDDH) values were obtained from the Genome-to-Genome Distance Calculator (GGDC) v3.0[ 44 ] via the Type Strain Genome Server (TYGS) [ 14 ]. Percentage of conserved proteins (POCP) was calculated using Galaxy Proteinortho[ 45 , 46 ] to confirm genus-level assignment. Orthologous gene groups were identified using OrthoFinder v2.5.4[ 47 ] with default parameters. Functional annotation of core genes was performed using eggNOG-mapper v2.1.9 [ 48 ], and metabolic pathways were mapped to the KEGG database [ 49 ]. Clusters of Orthologous Groups (COG) distributions were analyzed using COGclassifier v1.0.5 [ 50 ] Comparative Genomic Analyses Synteny analysis between Kdesi and reference strain HD100 was performed using bidirectional best-hit comparisons in the RAST annotation server [ 51 ]. Genomic islands (GIs) were identified using IslandViewer 4 [ 52 ], which integrates predictions from IslandPath-DIMOB, SIGI-HMM, and Islander methods. Insertion sequences (IS) were detected using ISEScan v1.7.2.1 [ 53 ]. Protein secretion systems were annotated using KEGG automatic annotation [ 49 , 54 ] and the SecreT4/6 web server [ 55 , 56 ] with default parameters. Synteny of predatory gene clusters, including the Type IV pilus operon, was analyzed using SyntTax [ 57 ]. Statistical significance of COG category distributions was assessed using Fisher's exact test with Bonferroni correction for multiple comparisons (α = 0.05). Declarations Conflict of Interest: The authors declare no competing interests. Ethical Approval : Not applicable. Funding: This research was supported by grants (20230747, 20231050) from the Secretaría de Investigación y Posgrado del Instituto Politécnico Nacional. Author Contribution X.G., M.A.V.-L., and I.C.R.-L. designed the study. T.O.E. performed experiments, genome assembly, annotation, and comparative analyses, and wrote the first draft. A.S.-V. and I.C.R.-L. participated in experimental design. A.Y.O., I.J.A., O.O.O., and R.F.-C. reviewed the manuscript. X.G., M.A.V.-L., and T.O.E. supervised the study and edited the manuscript. Acknowledgement This research was supported by grants (20230747, 20231050) from “la Scretario de investigación y Posgrado del Instituto Politécnico Nacional. We thank the staff at MyGenomics LLC for sequencing services and the Centro de Biotecnología Genómica for laboratory support. Data Availability The complete genome sequence of Bdellovibrio sp. strain Kdesi has been deposited in GenBank (NCBI) under accession number CP102930 (BioProject PRJNA524134). Kdesi has been deposited as a reference research strain at the Centro Nacional de Recursos Genéticos (CNRG-INIFAP, Mexico) under accession CM-CNRG 961. This deposit is intended solely to ensure strain preservation and reproducibility of the genomic analyses reported in this study. Certificate of Deposit The following document represents the official deposit certificate of the strain Bdellovibrio sp. Kdesi (CM-CNRG 961) issued by the Centro Nacional de Recursos Genéticos (CNRG-INIFAP), confirming its registration and preservation as a microbial culture deposit. References Ajao, Y. O. et al. Bdellovibrio reynosensis sp. nov., from a Mexico soil sample. Int J. Syst. Evol. Microbiol 72 , (2022). Dashiff, A., Junka, R. A., Libera, M. & Kadouri, D. E. Predation of human pathogens by the predatory bacteria Micavibrio aeruginosavorus and Bdellovibrio bacteriovorus. J. Appl. Microbiol. 110 , 431–444 (2011). Ajao, Y. O. et al. Antibacterial activity of two newly isolated Bdellovibrio bacteriovorus strains on Salmonella enterica serovars of food safety concern. Microbiol Spectr 13 , (2025). Gophna, U., Charlebois, R. L. & Doolittle, W. F. Ancient lateral gene transfer in the evolution of Bdellovibrio bacteriovorus. Trends Microbiol. 14 , 64–69 (2006). Shemesh, Y. & Jurkevitch, E. Plastic phenotypic resistance to predation by Bdellovibrio and like organisms in bacterial prey. Environ. Microbiol. 6 , 12–18 (2004). Koval, S. F., Williams, H. N. & Colin Stine, O. Reclassification of bacteriovorax marinus as Halobacteriovorax marinus gen. Nov., comb. nov. and bacteriovorax litoralis as halobacteriovorax litoralis comb. nov.; description of halobacteriovoraceae fam. nov. in the class Deltaproteobacteria. Int. J. Syst. Evol. Microbiol. 65 , 593–597 (2015). Berleman, J. E. & Kirby, J. R. Deciphering the hunting strategy of a bacterial wolfpack. FEMS Microbiology Reviews vol. 33 Preprint at (2009). https://doi.org/10.1111/j.1574-6976.2009.00185.x Pasternak, Z., Sasson, T., Ben, Cohen, Y., Segev, E. & Jurkevitch, E. A new comparative-genomics approach for defining phenotype-specific indicators reveals specific genetic markers in predatory bacteria. PLoS One . 10 , 1–11 (2015). Choi, S. Y., Mun, W., Choi, S., Lee, K. & Mitchell, R. J. Bdellovibrio svalbardensis sp. nov., a newly described predator isolated from Svalbard, Norway. Int. J. Syst. Evol. Microbiol. 74 , 006248 (2024). Stolp, H. & Starr, M. P. Bdellovibrio bacteriovorus gen. et sp. n., a predatory, ectoparasitic, and bacteriolytic microorganism. Antonie Van Leeuwenhoek . 29 , 217–248 (1963). Elbir, H. Updating the Relationship Between the Threshold Value of Average Nucleotide Identity and Digital DNA–DNA Hybridization for Reliable Taxonomy of Corynebacterium. Vet. Sci. 11 , 661 (2024). Kim, M., Oh, H. S., Park, S. C. & Chun, J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int. J. Syst. Evol. Microbiol. 64 , 346–351 (2014). Davis, S. C., Cerra, J., Williams, L. E. & Campbell, B. J. Comparative genomics of obligate predatory bacteria belonging to phylum Bdellovibrionota highlights distribution and predicted functions of lineage-specific protein families. mSphere 9, (2024). Meier-Kolthoff, J. P. & Göker, M. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat. Commun. 10 , 1–10 (2019). Machado, R. A. R. et al. Acinetobacter nematophilus sp. nov., Alcaligenes nematophilus sp. nov., Enterobacter nematophilus sp. nov., and Kaistia nematophila sp. nov., Isolated from Soil-Borne Nematodes and Proposal for the Elevation of Alcaligenes faecalis subsp. faecalis, Alcaligenes faecalis subsp. parafaecalis, and Alcaligenes faecalis subsp. phenolicus to the Species Level. Taxonomy 3, (2023). Huang, Z. et al. Genome-wide expanding of genetic evolution and potential pathogenicity in Vibrio alginolyticus. Emerg Microbes Infect 13 , (2024). Wang, W., Lin, H. & Cao, Y. Effects of opuB on the growth and biofilm formation of Streptococcus mutans under acid stress. Microb. Pathog . 205 , 107674 (2025). Williams, H. N. & Chen, H. Environmental Regulation of the Distribution and Ecology of Bdellovibrio and Like Organisms. Front. Microbiol. 11 , 545070 (2020). Maher, R. L. et al. Comparative analysis of novel Pseudobdellovibrionaceae genera and species yields insights into the genomics and evolution of bacterial predation mode. bioRxiv 2025.02.19.638989 (2025). 10.1101/2025.02.19.638989 Deeg, C. M., Le, T. T., Zimmer, M. M. & Suttle, C. A. From the inside out: An epibiotic bdellovibrio predator with an expanded genomic complement. J Bacteriol 202 , (2020). Oyedara, O. O. et al. Isolation of Bdellovibrio sp. from soil samples in Mexico and their potential applications in control of pathogens. Microbiologyopen 5 , 992–1002 (2016). Collier, D. N. & SecB A Molecular Chaperone of Escherichia Coli Protein Secretion Pathway. Adv. Protein Chem. 44 , 151–193 (1993). Talwar, C. et al. Defining the Environmental Adaptations of Genus Devosia: Insights into its Expansive Short Peptide Transport System and Positively Selected Genes. Scientific Reports 10, 1151- (2020). (2020). Oyedara, O. O. et al. Whole-genome sequencing and comparative genome analysis provided insight into the predatory features and genetic diversity of two bdellovibrio species isolated from soil. Int J Genomics (2018). (2018). Jurkevitch, E. Isolation and classification of bdellovibrio and like organisms. Curr Protoc Microbiol Chap. 7 , Unit 7B.1 (2006). Andrews, S., FastQC & A Quality Control tool for High Throughput Sequence Data.. Http://Www.Bioinformatics.Babraham.Ac (2010). .Uk/Projects/Fastqc/ https://doi.org/citeulike-article-id:11583827 doi:citeulike-article-id:11583827 Krueger, F., Trim & Galore! A wrapper tool around Cutadapt and FastQC to consistently apply quality and adapter trimming to FastQ files. Babraham Institute (2015). Bankevich, A. et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19 , 455–477 (2012). Wick, R. R., Judd, L. M., Gorrie, C. L. & Holt, K. E. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput. Biol. 13 , e1005595 (2017). Gurevich, A., Saveliev, V., Vyahhi, N. & Tesler, G. QUAST: Quality assessment tool for genome assemblies. Bioinformatics 29, 1072–1075 (2013). Marçais, G. et al. MUMmer4: A fast and versatile genome alignment system. PLoS Comput. Biol 14 , (2018). Bosi, E. et al. MeDuSa: A multi-draft based scaffolder. Bioinformatics 31 , 2443–2451 (2015). Seemann, T. & Prokka Rapid prokaryotic genome annotation. Bioinformatics 30 , 2068–2069 (2014). Zhao, Y. et al. PGAP: pan-genomes analysis pipeline. Bioinformatics 28 , 416–418 (2012). Chaudhari, N. M., Gupta, V. K. & Dutta, C. BPGA-an ultra-fast pan-genome analysis pipeline. Sci Rep 6 , (2016). Page, A. J. et al. Roary: Rapid large-scale prokaryote pan genome analysis. Bioinformatics 31, (2015). Katoh, K. & Standley, D. M. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol. Biol. Evol. 30 , 772–780 (2013). Nguyen, L. T., Schmidt, H. A., Von Haeseler, A. & Minh, B. Q. IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Mol. Biol. Evol. 32 , 268–274 (2015). Bertels, F., Silander, O. K., Pachkov, M., Rainey, P. B. & Van Nimwegen, E. Automated Reconstruction of Whole-Genome Phylogenies from Short-Sequence Reads. Mol. Biol. Evol. 31 , 1077–1088 (2014). Kumar, S. et al. MEGA12: Molecular Evolutionary Genetic Analysis Version 12 for Adaptive and Green Computing. Mol. Biol. Evol. 41 , 1–9 (2024). Letunic, I. & Bork, P. Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Res. 52 , W78–W82 (2024). Pritchard, L. pyani. (2016). https://github.com/widdowquinn/pyani Preprint at. Sadreyev, R. & Grishin, N. C. O. M. P. A. S. S. A Tool for Comparison of Multiple Protein Alignments with Assessment of Statistical Significance. J. Mol. Biol. 326 , 317–336 (2003). Auch, A. F., von Jan, M., Klenk, H. P. & Göker, M. Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand. Genomic Sci. 2 , 117–134 (2010). Afgan, E. et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2022 update. Nucleic Acids Res. 50 , W345–W351 (2022). Holzer, M. POCP-nf: an automatic Nextflow pipeline for calculating the percentage of conserved proteins in bacterial taxonomy. Bioinformatics 40 , (2024). Emms, D. M., Kelly, S. & OrthoFinder Phylogenetic orthology inference for comparative genomics. Genome Biol 20 , (2019). Huerta-Cepas, J. et al. Fast Genome-Wide Functional Annotation through Orthology Assignment by eggNOG-Mapper. Mol. Biol. Evol. 34 , 2115–2122 (2017). Aramaki, T. et al. KofamKOALA: KEGG Ortholog assignment based on profile HMM and adaptive score threshold. Bioinformatics 36 , 2251–2252 (2020). ‎Shimoyama Yuki. COGclassifier: A tool for classifying prokaryote protein sequences into COG functional category. github. (2022). Aziz, R. K. et al. The RAST Server: Rapid annotations using subsystems technology. BMC Genomics 9 , (2008). Bertelli, C. et al. IslandViewer 4: Expanded prediction of genomic islands for larger-scale datasets. Nucleic Acids Res. 45 , W30–W35 (2017). Xie, Z. & Tang, H. ISEScan: automated identification of insertion sequence elements in prokaryotic genomes. Bioinformatics 33 , 3340–3347 (2017). Kanehisa, M., Furumichi, M., Sato, Y., Matsuura, Y. & Ishiguro-Watanabe, M. KEGG: biological systems database as a model of the real world. Nucleic Acids Res. 53 , D672–D677 (2025). Bi, D. et al. SecReT4: A web-based bacterial type IV secretion system resource. Nucleic Acids Res 41 , (2013). Li, J. et al. SecReT6: A web-based resource for type VI secretion systems found in bacteria. Environ. Microbiol. 17 , 2196–2202 (2015). Oberto, J. & SyntTax A web server linking synteny to prokaryotic taxonomy. BMC Bioinformatics 14 , (2013). Additional Declarations No competing interests reported. Supplementary Files SupplementaryfigureKdesi1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8476110","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":571693240,"identity":"dea4ef96-2903-4b98-9d9e-e5c0dfb6b51f","order_by":0,"name":"Temidayo Oluyomi Elufisan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYLCCBDYgwd4AJAwsiFDOBtPCcwCkRYJILWBCIgHEIkKLuXzz0Q0PyuwS+yWfX93wo0CCgb+9OwGvFss2trQbCeeSE2fOzim72QN0mMSZsxvwajE4xmN2I7GNOXHD7Zy0GzxALQYSuYS08H8DaqlP3HDzTNrNP8Rp4WEDajmcuOEG+7HbRNli2ZZmBvTLceOZPTlst2UMJHgI+sWc+fCzmz/KqmX72Y8/u/nmj40cf3svAYdBaccGBh4wmwevcmQt9sAU84Cg6lEwCkbBKBiZAAAOk0v38+P5QAAAAABJRU5ErkJggg==","orcid":"","institution":"Oak Ridge Institute for Science and Education","correspondingAuthor":true,"prefix":"","firstName":"Temidayo","middleName":"Oluyomi","lastName":"Elufisan","suffix":""},{"id":571693241,"identity":"34437e5e-8fa8-401c-a39d-9ffdf968ef85","order_by":1,"name":"Isabel Cristina Rodríguez-Luna","email":"","orcid":"","institution":"Instituto Politécnico Nacional","correspondingAuthor":false,"prefix":"","firstName":"Isabel","middleName":"Cristina","lastName":"Rodríguez-Luna","suffix":""},{"id":571693243,"identity":"c71597cf-c9aa-48cb-9a4a-4dd9acb2f44f","order_by":2,"name":"Yewande Olajumoke Ajao","email":"","orcid":"","institution":"Instituto Politécnico Nacional","correspondingAuthor":false,"prefix":"","firstName":"Yewande","middleName":"Olajumoke","lastName":"Ajao","suffix":""},{"id":571693245,"identity":"ea84b3e3-c24f-4ded-aaf3-74ae6806c440","order_by":3,"name":"Ibukun John Abulude","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Ibukun","middleName":"John","lastName":"Abulude","suffix":""},{"id":571693249,"identity":"f6676766-6c07-4797-a49d-3593a083c0ee","order_by":4,"name":"Alejandro Sánchez-Varela","email":"","orcid":"","institution":"Instituto Politécnico Nacional","correspondingAuthor":false,"prefix":"","firstName":"Alejandro","middleName":"","lastName":"Sánchez-Varela","suffix":""},{"id":571693252,"identity":"2dba7145-6007-4976-b490-6677b5db97ea","order_by":5,"name":"Omotayo Opemipo Oyedara","email":"","orcid":"","institution":"Osun State University","correspondingAuthor":false,"prefix":"","firstName":"Omotayo","middleName":"Opemipo","lastName":"Oyedara","suffix":""},{"id":571693255,"identity":"d0b5821c-61c4-4531-963f-857808bf2b21","order_by":6,"name":"Ronald Ferrera-Cerrato","email":"","orcid":"","institution":"Collegio de Postgraduado","correspondingAuthor":false,"prefix":"","firstName":"Ronald","middleName":"","lastName":"Ferrera-Cerrato","suffix":""},{"id":571693256,"identity":"02b3d932-3d87-472d-8c13-fe8022f442a1","order_by":7,"name":"Miguel Angel Villaloboz_Lopez","email":"","orcid":"","institution":"Instituto Politécnico Nacional","correspondingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"Angel","lastName":"Villaloboz_Lopez","suffix":""},{"id":571693258,"identity":"02f7b66b-cec5-46d3-9f6d-8bd28585a7ed","order_by":8,"name":"Xianwu Guo","email":"","orcid":"","institution":"Instituto Politécnico Nacional","correspondingAuthor":false,"prefix":"","firstName":"Xianwu","middleName":"","lastName":"Guo","suffix":""}],"badges":[],"createdAt":"2025-12-29 23:08:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8476110/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8476110/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":99897236,"identity":"ba619e0c-7c61-4d79-b60f-3ddca1bcf021","added_by":"auto","created_at":"2026-01-09 15:05:05","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2116844,"visible":true,"origin":"","legend":"","description":"","filename":"AllvsAllANIHeatmap.tif","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/8f94b99bebb22f5081763653.tif"},{"id":100358862,"identity":"a78492b6-7a69-4f0a-9936-3d03595ac5b9","added_by":"auto","created_at":"2026-01-16 07:21:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2825693,"visible":true,"origin":"","legend":"","description":"","filename":"NewbdellovibriosubmissionfileScientificreportrevised.docx","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/eb43dc69be38753bbc2bb31c.docx"},{"id":99897238,"identity":"04b1151c-f8c0-4fe0-a540-4f428f32ef89","added_by":"auto","created_at":"2026-01-09 15:05:05","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":256510,"visible":true,"origin":"","legend":"","description":"","filename":"Kdesi16Sphylogenetictree.tif","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/522468c0067e7e7ef6b10e84.tif"},{"id":100359073,"identity":"3eacf0c9-64e2-4ed1-a430-177289b3c35f","added_by":"auto","created_at":"2026-01-16 07:21:41","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":95128,"visible":true,"origin":"","legend":"","description":"","filename":"figure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/8c2f2c290a72547d931eedc6.tif"},{"id":100359408,"identity":"803677bf-41c2-4626-bcae-86467542ee98","added_by":"auto","created_at":"2026-01-16 07:22:13","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":515270,"visible":true,"origin":"","legend":"","description":"","filename":"kdesicoregenomephylogeny.tif","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/d238fcc3b1aff0f847343a96.tif"},{"id":99897241,"identity":"3d6efaac-2b3c-4b40-9da0-9e14bcc07386","added_by":"auto","created_at":"2026-01-09 15:05:06","extension":"json","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10151,"visible":true,"origin":"","legend":"","description":"","filename":"f0e40cb9a89e410a8cd7e07cab328a5d.json","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/5c3f6f55ead47cec779ddfd9.json"},{"id":100358872,"identity":"d9680082-4b67-4acd-85e1-247e3502694f","added_by":"auto","created_at":"2026-01-16 07:21:31","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2968829,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryfigureKdesi1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/55e2689204379335db9ad4ba.docx"},{"id":100358921,"identity":"b8f0050f-6608-49f5-94e6-e658849aa093","added_by":"auto","created_at":"2026-01-16 07:21:34","extension":"xml","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":143522,"visible":true,"origin":"","legend":"","description":"","filename":"f0e40cb9a89e410a8cd7e07cab328a5d1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/43467488e320023b06d3edac.xml"},{"id":100358957,"identity":"2cead177-334f-40a3-bc71-57c8c9aca4ef","added_by":"auto","created_at":"2026-01-16 07:21:36","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2116844,"visible":true,"origin":"","legend":"","description":"","filename":"AllvsAllANIHeatmap.tif","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/3bcc0a21fc7bde4b2250ab14.tif"},{"id":99897250,"identity":"eaa33ba3-a3e0-4783-bfe6-4511692ba38a","added_by":"auto","created_at":"2026-01-09 15:05:06","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":256510,"visible":true,"origin":"","legend":"","description":"","filename":"Kdesi16Sphylogenetictree.tif","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/da4fe932fc3505c1ed723260.tif"},{"id":100358257,"identity":"7d53ff02-4f35-47c8-9dca-35306630e249","added_by":"auto","created_at":"2026-01-16 07:20:48","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":95128,"visible":true,"origin":"","legend":"","description":"","filename":"figure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/a9549865a029cd5522c88143.tif"},{"id":99897253,"identity":"9b51f957-f6c5-4cde-8352-fd875fbc369d","added_by":"auto","created_at":"2026-01-09 15:05:06","extension":"tif","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":515270,"visible":true,"origin":"","legend":"","description":"","filename":"kdesicoregenomephylogeny.tif","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/c19e6892dad77b5e3395fa56.tif"},{"id":100358371,"identity":"d24c0b0d-bf72-4cc2-bc20-c4bed36740dd","added_by":"auto","created_at":"2026-01-16 07:20:59","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":141538,"visible":true,"origin":"","legend":"","description":"","filename":"f0e40cb9a89e410a8cd7e07cab328a5d1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/e695157e9c40a506d840d8f5.xml"},{"id":100359390,"identity":"0e6eb53e-2394-4b7f-a799-60cfa356e032","added_by":"auto","created_at":"2026-01-16 07:22:12","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":160280,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/b7a3aaf1b2591c6393b5deca.html"},{"id":99897235,"identity":"eed26822-e46b-4679-b941-d257a8eb5231","added_by":"auto","created_at":"2026-01-09 15:05:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":83607,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree based on 16S rRNA sequences showing placement of \u003cem\u003eBdellovibrio \u003c/em\u003esp. Kdesi within the \u003cem\u003eB. bacteriovorus \u003c/em\u003ecluster.\u003c/p\u003e","description":"","filename":"OnlineKdesi16Sphylogenetictree.png","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/b8bdf8763a25c736a1f916f8.png"},{"id":100358626,"identity":"b2144a7a-da76-4336-b679-32752e7e87ba","added_by":"auto","created_at":"2026-01-16 07:21:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":189365,"visible":true,"origin":"","legend":"\u003cp\u003eCore genome-based phylogeny using 50 core genes generated by Roary pan genomic analysis tool\u003c/p\u003e","description":"","filename":"Onlinekdesicoregenomephylogeny.png","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/c91a31ad2548629c65c7528b.png"},{"id":100358908,"identity":"b6914554-8d05-4aa6-9007-2c5250f84b61","added_by":"auto","created_at":"2026-01-16 07:21:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":285328,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of Average Nucleotide Identity (ANI) values among 29 \u003cem\u003eBdellovibrio \u003c/em\u003egenomes, highlighting \u0026lt;95% identity thresholds supporting novel species status.\u003c/p\u003e","description":"","filename":"OnlineAllvsAllANIHeatmap.png","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/44d50361529c9320376e30a2.png"},{"id":100358916,"identity":"52a44c5e-ce1d-41ed-809c-4d6b8fdbd174","added_by":"auto","created_at":"2026-01-16 07:21:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":13807,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of insertion sequence (IS) elements across \u003cem\u003eBdellovibrio \u003c/em\u003egenomes, showing limited transposable element abundance\u003c/p\u003e","description":"","filename":"Onlinefigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/d7713ce4a11084c10f764211.png"},{"id":102529639,"identity":"3f8f7e80-44e9-4fcb-8786-edcc0e32292d","added_by":"auto","created_at":"2026-02-12 16:11:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2236875,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/44d930c7-ec61-42bf-93be-9879c801e1da.pdf"},{"id":99897247,"identity":"1a3c1d8a-49e9-4285-be79-027f6312c2b2","added_by":"auto","created_at":"2026-01-09 15:05:06","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2968829,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryfigureKdesi1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8476110/v1/2604677898b58d74921b0eee.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-Scale Analysis Reveals Strain Kdesi as a Distinct Evolutionary Lineage and Extensive Cryptic Diversity in the Genus Bdellovibrio","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eBdellovibrio\u003c/em\u003e and like organisms (BALOs) are obligate predatory bacteria that prey on Gram-negative pathogens, making them promising alternatives to conventional antibiotics [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These bacteria invade the periplasmic space of prey cells, replicate within this protected niche (termed the bdelloplast), and emerge to attack new hosts [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This is particularly relevant given the global crisis of antibiotic resistance, and as a result, \u003cem\u003eBdellovibrio\u003c/em\u003e spp. have attracted significant research attention for their potential therapeutic and probiotic applications [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe taxonomic classification of BALOs has undergone several revisions. Initially divided into four genera (\u003cem\u003eBdellovibrio\u003c/em\u003e, Bacteriovorax, Halobacteriovorax, and Peridibacter) based on habitat and GC content [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], more recent genomic analyses have identified epibiotic strains as a separate genus, Pseudo\u003cem\u003eBdellovibrio\u003c/em\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, the genus \u003cem\u003eBdellovibrio\u003c/em\u003e itself contains only three formally described species (B. bacteriovorus, B. reynosensis, and B. svalbardensis), despite substantial genomic diversity among isolates labeled as \u003cem\u003eB. bacteriovorus\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The proliferation of whole-genome sequencing has revealed that 16S rRNA gene-based identification often fails to capture species-level diversity in bacteria [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This limitation is particularly evident in \u003cem\u003eBdellovibrio\u003c/em\u003e, where strains sharing\u0026thinsp;\u0026gt;\u0026thinsp;98% 16S rRNA identity exhibit average nucleotide identity (ANI) values well below the 95\u0026ndash;96% species threshold [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Modern taxonomic frameworks increasingly rely on genome-scale metrics including ANI, average amino acid identity (AAI), digital DNA-DNA hybridization (dDDH), and core-genome phylogeny to delineate bacterial species [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In this study, we isolated \u003cem\u003eBdellovibrio\u003c/em\u003e sp. Kdesi from sewage in Reynosa, Mexico, and performed comprehensive genomic characterization. Our objectives were to (1) determine the precise taxonomic position of Kdesi using polyphasic genomic analysis, (2) perform comparative genomics to identify strain-specific adaptations, and (3) evaluate the taxonomic status of other \u003cem\u003eBdellovibrio\u003c/em\u003e isolates. Our findings reveal substantial cryptic diversity within \u003cem\u003eBdellovibrio\u003c/em\u003e and support the need for formal taxonomic revision of this ecologically and clinically important genus.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIsolation and General Genomic Features of Kdesi Predatory plaques appeared on DNB agar after 48 hours of incubation. Partial 16S rRNA sequencing identified the isolate as \u003cem\u003eBdellovibrio\u003c/em\u003e with 99.3% identity to \u003cem\u003eB. bacteriovorus\u003c/em\u003e SSB218315 (accession MG957118), suggesting initial classification as \u003cem\u003eB. bacteriovorus\u003c/em\u003e (Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eFigure 1. Phylogenetic tree based on 16S rRNA sequences showing placement of \u003cem\u003eBdellovibrio\u003c/em\u003e sp. Kdesi within the \u003cem\u003eB. bacteriovorus\u003c/em\u003e cluster.\u003c/p\u003e \u003cp\u003eThe complete genome of Kdesi comprises a single circular chromosome of 3,343,978 bp with 48.5% GC content. NCBI PGAP annotation identified 3,274 genes, including 3,208 protein-coding sequences, 3 rRNAs (single operon), 48 tRNAs, and 15 ncRNAs. Notably, the genome contains two identical 16S rRNA genes: one within the rRNA operon and a second copy at a distinct genomic location. The genome has been deposited in NCBI under accession CP102930 (BioProject PRJNA524134). General genomic features for all strains analyzed are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGeneral genomic features of \u003cem\u003eBdellovibrio\u003c/em\u003e species and strains analyzed in this study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrganism Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGenome Size (Mb)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGC (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProtein-coding Genes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePseudogenes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCompleteness (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eInstitution\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio bacteriovorus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e109J\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3628\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e97.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eKurume University School of Medicine\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio sp.\u003c/em\u003e 22V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e46.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3437\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e95.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCBG Instituto Politecnico Nacional\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio bacteriovorus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBd10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3639\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e96.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCollege of Veterinary Medicine\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio bacteriovorus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBd16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e43.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3194\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e83.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCollege of Veterinary Medicine\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio bacteriovorus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBd3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3878\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e90.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCollege of Veterinary Medicine\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio bacteriovorus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBd4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3486\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e93.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCollege of Veterinary Medicine\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio bacteriovorus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBd5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e48.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3536\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e94.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCollege of Veterinary Medicine\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio sp.\u003c/em\u003e HCB-110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHCB-110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3933\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e96.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eInstituto Politecnico Nacional\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio sp.\u003c/em\u003e HCB117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHCB117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3487\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e96.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHuazhong Agricultural University\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio sp.\u003c/em\u003e HCB-162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHCB-162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e44.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3705\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e96.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHuazhong Agricultural University\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio sp.\u003c/em\u003e HCB185ZH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHCB185ZH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3745\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e95.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHuazhong Agricultural University\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio sp.\u003c/em\u003e HCB209\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHCB209\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3718\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e95.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHuazhong Agricultural University\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio sp.\u003c/em\u003e HCB274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHCB274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e46.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3567\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e96.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHuazhong Agricultural University\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio sp.\u003c/em\u003e HCB288\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHCB288\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e46.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3510\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e96.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHuazhong Agricultural University\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio sp.\u003c/em\u003e HCB290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHCB290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e46.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3680\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e95.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHuazhong Agricultural University\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio sp.\u003c/em\u003e HCB337\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHCB337\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4092\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e89.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHuazhong Agricultural University\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio bacteriovorus\u003c/em\u003e HD100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHD100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3583\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e98.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMax-Planck-Institute\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio bacteriovorus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHK2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3870\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e159\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e88.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCollege of Veterinary Medicine\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio bacteriovorus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ekdesi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e48.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3208\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e89.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCBG Instituto Politecnico Nacional\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio sp.\u003c/em\u003e KM01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKM01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3752\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e95.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eProvidence College\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio reynosensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLBG001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e43.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3289\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e92.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTufts University\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio sp.\u003c/em\u003e NC01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e44.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3773\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e95.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eProvidence College\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio sp.\u003c/em\u003e SKB1291214\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSKB1291214\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3570\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e94.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCentro de Biotecnologia Genomica, Instituto Politecnico Nacional\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio bacteriovorus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSSB218315\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3536\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e96.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCentro de Biotecnologia Genomica, Instituto Politecnico Nacional\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio bacteriovorus\u003c/em\u003e str. Tiberius\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTiberius\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3738\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e96.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUniversity of Nottingham\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio bacteriovorus\u003c/em\u003e W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e43.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2871\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUniversity of Oklahoma Health Sciences Center\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBdellovibrio sp.\u003c/em\u003e ZAP7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZAP7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3937\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e95.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eProvidence College\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenomic Analysis Reveals Kdesi as a Distinct Lineage\u003c/h2\u003e \u003cp\u003eCore-genome phylogeny based on 760 concatenated single-copy genes (or 56 genes constituting a 50,201 bp alignment when using Roary analysis) resolved 29 \u003cem\u003eBdellovibrio\u003c/em\u003e strains into two major clades with several singleton lineages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Kdesi clustered in Clade 1 but formed a distinct branch separate from the type strain \u003cem\u003eB. bacteriovorus\u003c/em\u003e HD100 and its close relative 109J. Clade 1 also contained \u003cem\u003eB. bacteriovorus\u003c/em\u003e strains Tiberius, SSB218315, EC13, BER2, Bd3, and Bd4, as well as several unclassified isolates (HCB110, HCB-162, HCB117, Bd16) and B. reynosensis LBG001. Clade 2 comprised primarily environmental isolates including NC01, ZAP7, KM01, and SKB1291214, with \u003cem\u003eB. svalbardensis\u003c/em\u003e PAP01 branching basally. \u003cem\u003eB. bacteriovorus\u003c/em\u003e W formed a singleton lineage, while \u003cem\u003ePseudoBdellovibrio\u003c/em\u003e exovorus served as the outgroup. Core-SNP phylogeny generated by REALPHY produced a congruent topology (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), supporting the robustness of these relationships In contrast, 16S rRNA phylogeny (Fig.\u0026nbsp;1) grouped Kdesi with SSB218315 within a well-supported \u003cem\u003eB. bacteriovorus\u003c/em\u003e cluster, illustrating the limitations of 16S rRNA for species-level resolution in this genus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenome-Based Taxonomic Metrics Support Novel Species Status\u003c/h3\u003e\n\u003cp\u003eANI analysis revealed that Kdesi shares\u0026thinsp;~\u0026thinsp;89% identity with SSB218315 and even lower values (~\u0026thinsp;85\u0026ndash;88%) with other \u003cem\u003eBdellovibrio\u003c/em\u003e strains (Fig.\u0026nbsp;3). These values fall well below the 95\u0026ndash;96% species threshold, indicating that Kdesi represents a distinct species. Similarly, AAI values between Kdesi and other strains ranged from ~\u0026thinsp;76% to ~\u0026thinsp;82%, with SSB218315 showing the highest similarity at 82% (Fig. S2). All pairwise comparisons exceeded the ~\u0026thinsp;65\u0026ndash;70% genus-level threshold, confirming Kdesi's placement within \u003cem\u003eBdellovibrio.\u003c/em\u003e The percentage of conserved proteins (POCP) was 56%, well above the 50% genus threshold.\u003c/p\u003e \u003cp\u003eDigital DNA-DNA hybridization (dDDH) provided additional support for species-level distinctness. Using GGDC formulas 1, 2, and 3, dDDH between Kdesi and SSB218315 was 40.2%, 21.9%, and 33.9%,\u003c/p\u003e \u003cp\u003erespectively\u0026mdash;all substantially below the 70% species cutoff. Similarly, dDDH between Kdesi and HD100 yielded values of 38.8%, 22.0%, and 33.0%. Notably, several strains currently classified as B.\u003c/p\u003e \u003cp\u003ebacteriovorus (e.g., Tiberius, W, EC13, BER2) also showed dDDH\u0026thinsp;\u0026lt;\u0026thinsp;70% relative to the typed strain HD100, suggesting they too represent undescribed species. Among analyzed strains, only HD100 and 109J consistently showed dDDH\u0026thinsp;\u0026gt;\u0026thinsp;70% under all three formulas, confirming their conspecific status.\u003c/p\u003e \u003cp\u003eSSB218315 showed formula-dependent relationships: formulas 1 and 3 suggested conspecificity with HD100/109J (dDDH\u0026thinsp;~\u0026thinsp;72\u0026ndash;75%), while formula 2 yielded values of ~\u0026thinsp;68%, just below the threshold. Given the concordant low ANI (~\u0026thinsp;89%) and AAI (~\u0026thinsp;82%) values, we provisionally treat SSB218315 as a putative genomospecies pending further validation.\u003c/p\u003e \u003cp\u003eBased on integrated ANI, AAI, dDDH, and phylogenomic data, we propose the existence of at least 12 distinct genomospecies within the genus \u003cem\u003eBdellovibrio\u003c/em\u003e, including Kdesi, substantially expanding the recognized diversity of this genus (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFigure 3. Heatmap of Average Nucleotide Identity (ANI) values among 29 \u003cem\u003eBdellovibrio\u003c/em\u003e genomes, highlighting\u0026thinsp;\u0026lt;\u0026thinsp;95% identity thresholds supporting novel species status.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenome-based species delineation metrics (ANI, AAI, dDDH) and proposed genomospecies classification among 29 \u003cem\u003eBdellovibrio\u003c/em\u003e strains.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMember strains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInterpretation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e109J, HD100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eB. bacteriovorus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBER2, Bd4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eB. bacteriovorus\u003c/em\u003e-like (genomospecies)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBd3, sp. 110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNovel genomospecies\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ereynosensis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eB. reynosensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCluster 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esvalbardensis PAP01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eB. svalbardensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSingletons\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTiberius, Bd10, SSB219315, Bd5, Kdesi, Bd16, W, ZAP7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePutative novel species\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOther novel species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHCB288, HCB274, HCB290, HCB209, SKB1291224, HCB185ZH, KM01, HCB337, 22V, NC01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePutative novel species\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003ePan-Genome Structure and Functional Architecture\u003c/h3\u003e\n\u003cp\u003ePan-genome analysis using BPGA identified approximately 25,000 genes across 29 \u003cem\u003eBdellovibrio\u003c/em\u003e genomes, with Heaps' law analysis (α\u0026thinsp;\u0026lt;\u0026thinsp;1) indicating an open pan-genome (Fig. S3). The core genome comprised 760 genes present in all strains, representing fundamental cellular processes. Roary analysis with more stringent parameters (\u0026ge;\u0026thinsp;95% presence threshold) identified 56 core genes. The accessory genome varied substantially among strains, with strain-specific gene counts ranging from ~\u0026thinsp;150 (Kdesi) to 2031 (\u003cem\u003eBdellovibrio\u003c/em\u003e sp. HCB337; 47.6% of its CDS), reflecting diverse ecological adaptations.\u003c/p\u003e \u003cp\u003eOrthoFinder identified 6,312 orthologous groups accounting for ~\u0026thinsp;97% of all genes, with 1,425 core orthogroups shared by all strains (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Functional annotation using eggNOG-mapper assigned the core genome to 20 COG categories, 304 KEGG pathways, and 819 GO terms. Nearly all core proteins (\u0026gt;\u0026thinsp;95%) carried PFAM domains, indicating high structural conservation of essential functions (Fig. S4).\u003c/p\u003e \u003cp\u003eCOG category distribution in the core genome was dominated by housekeeping functions: translation (J), transcription (K), and replication/repair (L) collectively accounted for ~\u0026thinsp;35% of core genes. Genes involved in amino acid metabolism (E), energy production (C), and cell envelope biogenesis (M) were also highly conserved. In contrast, categories related to signal transduction (T), motility (N), and secondary metabolism (Q) showed greater inter-strain variation, suggesting strain-specific adaptations in environmental sensing and predatory behavior.\u003c/p\u003e \u003cp\u003eKdesi-specific genes (n\u0026thinsp;=\u0026thinsp;~\u0026thinsp;150) are enriched for regulatory functions including multiple histidine kinases (e.g., RegB-like sensor kinases) and transcriptional regulators (LysR family), suggesting enhanced environmental sensing. Additionally, Kdesi possesses unique ABC transporters for osmoprotectants (glycine betaine/proline uptake systems OpuBA), a citrate transporter, and restriction-modification systems, potentially reflecting adaptation to sewage environments with high osmotic stress and frequent phage exposure. Approximately 26% of Kdesi-unique genes encode proteins with domains of unknown function (DUFs), representing candidates for novel predation-associated factors.\u003c/p\u003e\n\u003ch3\u003eGenomic Islands and Horizontal Gene Transfer\u003c/h3\u003e\n\u003cp\u003eIsland Viewer 4 identified five genomic islands (GIs) in Kdesi totaling 63,559 bp and spanning positions 213,412\u0026thinsp;\u0026minus;\u0026thinsp;229,341; 1,033,588-1,041,791; 1,319,854-1,325,685; 1,493,316-1,503,22; and 1,876,696-1,882,115 (Fig. S5). These GIs are enriched for genes encoding surface structures, transporters, and regulatory factors. Notably, GI-1 contains the flagellar filament capping protein FliD and two flagellin genes, critical for motility and prey detection. Other GI-encoded functions include ABC transport systems, phosphoenolpyruvate carboxylase, transposases, ecdysteroid kinases, and SAM-dependent methyltransferases. Forty-two GI genes (66%) remain hypothetical, highlighting the potential for discovery of novel predation factors.\u003c/p\u003e \u003cp\u003eThe number of GIs varied across strains: HD100 (5), 109J (4), BER2 (3), EC13 (6), KM01 (4), Tiberius (13), SSB218315 (3), W (6). Interestingly, three GI genes\u0026mdash;two flagellins and one acetyltransferase\u0026mdash;are\u003c/p\u003e \u003cp\u003econserved across all strains and thus part of the core genome, suggesting ancient horizontal acquisition and subsequent vertical inheritance as part of a minimal predatory toolkit.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLimited Transposable Elements in\u003c/b\u003e \u003cb\u003eBdellovibrio\u003c/b\u003e \u003cb\u003eGenomes\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eBdellovibrio\u003c/em\u003e genomes harbor remarkably few insertion sequences (IS) compared to other bacteria. ISEScan detected only 41 IS elements across all 29 strains, belonging to families IS21, IS3, ISNCY, and ISL3 (Fig.\u0026nbsp;4). IS21 was present in all strains except one, suggesting a role in genome plasticity. IS3 occurred sporadically in Bd3, HCB274, Bd5, HCB117, W, and P. exovorus, while ISL3 was restricted to W, BER2, and P. exovorus. The paucity of IS elements may reflect genome streamlining associated with an obligate predatory lifestyle or strong purifying selection against disruptive transposition.\u003c/p\u003e \u003cp\u003eFigure 4. Distribution of insertion sequence (IS) elements across \u003cem\u003eBdellovibrio\u003c/em\u003e genomes, showing limited transposable element abundance\u003c/p\u003e\n\u003ch3\u003eProtein Secretion Systems\u003c/h3\u003e\n\u003cp\u003eAll \u003cem\u003eBdellovibrio\u003c/em\u003e genomes encode complete Sec, Tat, Type II secretion (T2SS), and Type IV secretion (T4SS) systems, consistent with their role in exporting hydrolytic enzymes and other effectors during prey\u003c/p\u003e \u003cp\u003einvasion. Gene counts for Sec, Tat, and T2SS ranged from 17\u0026ndash;20 per genome. Notably, SecB\u0026mdash;a molecular chaperone that delivers unfolded substrates to the Sec translocon in \u003cem\u003eE. coli\u003c/em\u003e was absent from all \u003cem\u003eBdellovibrio\u003c/em\u003e strains (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Fig. S6), suggesting functional compensation by alternative chaperones (e.g., DnaK, GroEL) or distinct substrate-targeting mechanisms. T4SS components varied more extensively among strains, ranging from 15 proteins (\u003cem\u003eB. reynosensis\u003c/em\u003e) to 21 (\u003cem\u003eB. bacteriovorus\u003c/em\u003e Tiberius). Core T4SS genes conserved across all strains included VirB11, VirB1, TrbN, TcpA, DotB, and TraJ, essential for pilus assembly and substrate translocation. Accessory T4SS components showed strain-specific distributions: TraF (only in NC01), TrwF (SKB1291214 and ZAP7), PrgC and TrwD (Tiberius), and VirD4 (RO). This modular T4SS architecture likely reflects diverse strategies for prey invasion, DNA uptake, or effector delivery.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSynteny of Predatory Gene Clusters\u003c/h2\u003e \u003cp\u003eBidirectional synteny analysis between Kdesi and HD100 revealed high conservation of gene order across the chromosome, with no major inversions or translocations detected (Fig. S6a). Kdesi and SSB218315\u003c/p\u003e \u003cp\u003eshared 2,852 orthologous genes, while Kdesi and HD100 shared 2,844 genes. Despite conserved synteny, only\u0026thinsp;~\u0026thinsp;10% of orthologous protein pairs exhibited\u0026thinsp;\u0026gt;\u0026thinsp;96% amino acid identity, whereas ~\u0026thinsp;64% showed\u0026thinsp;\u0026le;\u0026thinsp;85% identity consistent with substantial sequence divergence supporting species-level distinctness.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTargeted synteny analysis of predation-associated loci revealed\u003c/h3\u003e\n\u003cp\u003econtrasting patterns. The Type IVb pilus (T4P) cluster essential for prey attachment and invasion showed near-perfect synteny across HD100, 109J, Tiberius, Kdesi, and SSB219315 (Fig. S7), indicating strong purifying selection on this critical apparatus. In contrast, an oligopeptide transport cluster exhibited profound divergence in Kdesi: the corresponding\u0026thinsp;~\u0026thinsp;13.5 kb region contained no conserved synteny and harbored unique genes (KFHEMNGN_ome, GASZ_ome) not found in HD100, 109J, or Tiberius. SSB219315 showed intermediate architecture (~\u0026thinsp;11.9 kb) with distinct genes (ca4A, gpp8_2), suggesting lineage-specific adaptation in peptide sensing or uptake that may influence prey range or environmental fitness.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eKdesi Represents a Novel Species Revealing Evolutionary Dynamics\u003c/h2\u003e \u003cp\u003eOur polyphasic genomic analysis unequivocally demonstrates that \u003cem\u003eBdellovibrio\u003c/em\u003e sp. Kdesi represents a novel species. While 16S rRNA sequencing initially suggested classification as \u003cem\u003eB. bacteriovorus\u003c/em\u003e (99.3% identity to SSB218315), genome-scale metrics reveal substantial divergence: ANI\u0026thinsp;~\u0026thinsp;89%, AAI\u0026thinsp;~\u0026thinsp;82%, and dDDH\u0026thinsp;\u0026lt;\u0026thinsp;40%, all well below established species thresholds. These findings underscore a critical limitation of 16S rRNA-based taxonomy, where high rRNA similarity does not guarantee conspecificity, a phenomenon documented in genera such as \u003cem\u003eEnterobacter\u003c/em\u003e, \u003cem\u003eCorynebacterium\u003c/em\u003e, and \u003cem\u003eAcinetobacter\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe cgSNP phylogeny provides crucial insight into kdesi's evolutionary history, revealing it as a recently diverged lineage within the \u003cem\u003eB. bacteriovorus\u003c/em\u003e complex that has undergone accelerated evolution. While cgSNP analysis places kdesi within the \u003cem\u003eB. bacteriovorus\u003c/em\u003e radiation, its extended branch length (0.00693 substitutions/site) indicates rapid genetic changes following ecological specialization [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This apparent paradox of recent common ancestry but current genomic distinctness exemplifies ecological speciation in action, where environmental pressures drive rapid genomic reorganization that outpaces SNP accumulation in core genes.\u003c/p\u003e \u003cp\u003ekdesi's unique genomic features, including strain-specific regulatory systems, osmoprotectant transporters, and remodeled oligopeptide uptake clusters, represent adaptive solutions to its sewage environment. The high osmotic conditions likely selected for unique transporter systems (OpuBA) (Wang et al., 20250), while diverse prey communities drove expansion of environmental sensing capabilities and predation machinery. This pattern of recent divergence followed by rapid niche adaptation represents a compelling model for bacterial speciation dynamics.\u003c/p\u003e \u003cp\u003eThe robust genomic evidence presented here, which includes phylogenomic placement, ANI\u0026thinsp;~\u0026thinsp;89%, AAI\u0026thinsp;~\u0026thinsp;82%, and dDDH\u0026thinsp;\u0026lt;\u0026thinsp;40% unequivocally demonstrates that strain kdesi represents a novel gemospecies within the genus \u003cem\u003eBdellovibrio\u003c/em\u003e, distinct from all currently described species including \u003cem\u003eB. bacteriovorus\u003c/em\u003e, \u003cem\u003eB. reynosensis\u003c/em\u003e, and \u003cem\u003eB. svalbardensis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe unique genomic features of strain kdesi particularly adaptations for osmotic stress tolerance (OpuBA transporters), enhanced environmental sensing (multiple histidine kinases), and modified oligopeptide transport suggest specialization to high-osmolarity wastewater environments and provide genomic predictions that could guide phenotypic characterization should conspecific strains be isolated [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eCryptic Diversity Necessitates Taxonomic Revision of\u003c/b\u003e \u003cb\u003eBdellovibrio\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBeyond kdesi, our analysis revealed extensive cryptic diversity within \u003cem\u003eBdellovibrio.\u003c/em\u003e Based on ANI, AAI, and dDDH thresholds, we identified at least 12 distinct genomospecies among the 29 strains examined (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Notably, strains labeled \u003cem\u003eB. bacteriovorus\u003c/em\u003e exhibit substantial genomic heterogeneity: only HD100 and 109J consistently showed dDDH\u0026thinsp;\u0026gt;\u0026thinsp;70%, confirming conspecificity. Other \u003cem\u003eB. bacteriovorus\u003c/em\u003e isolates (e.g., Tiberius, W, EC13, BER2, SSB218315) represent putative novel species that warrant formal characterization.\u003c/p\u003e \u003cp\u003eThe cgSNP phylogeny further supports this taxonomic complexity, revealing multiple evolutionarily distinct lineages currently classified under single species designations. Strains such as W and Bd16 form long-branch lineages suggesting substantial evolutionary distance, while environmental isolates (HCB series) cluster separately from the core \u003cem\u003eB. bacteriovorus\u003c/em\u003e group. This phylogenetic structure aligns with ecological origins, suggesting that habitat specialization has been a major driver of \u003cem\u003eBdellovibrio\u003c/em\u003e diversification [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe taxonomic confusion in \u003cem\u003eBdellovibrio\u003c/em\u003e mirrors broader challenges in microbial systematics. Reliance on single-gene markers (16S rRNA) and phenotypic traits has led to taxonomic oversimplification in many bacterial groups [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The advent of affordable whole-genome sequencing now enables rigorous species delineation using multiple independent criteria phylogenomic, AAI, and dDDH that collectively provide a robust taxonomic framework. Our findings support recent calls for comprehensive taxonomic revision of \u003cem\u003eBdellovibrio\u003c/em\u003e and highlight the genus as a model system for exploring bacterial diversity and evolution in predatory lifestyles [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGenomic Architecture Reflects Predatory Lifestyle Adaptations\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eBdellovibrio\u003c/em\u003e core genome is enriched for housekeeping functions (COG categories J, K, L), consistent with essential cellular processes required across both attack and growth phases [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Accessory genomes vary substantially, reflecting niche-specific adaptations: Kdesi's unique genes for osmoprotection, environmental sensing, and oligopeptide transport likely facilitate survival in sewage habitats with high osmotic stress and diverse prey communities.\u003c/p\u003e \u003cp\u003eThe open pan-genome (~\u0026thinsp;25,000 genes) indicates high genomic plasticity, where each new genome sequenced contributes novel genes. This plasticity enables \u003cem\u003eBdellovibrio\u003c/em\u003e to adapt to diverse ecological niches freshwater, marine, soil, and wastewater and to prey on a broad range of Gram-negative bacteria [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Genomic islands encoding motility factors, transporters, and hypothetical proteins suggest ongoing horizontal gene transfer, although limited IS element abundance (~\u0026thinsp;41 total across 29 genomes) implies constrained transposition activity, possibly reflecting genome streamlining or strong purifying selection associated with an obligate predatory lifestyle.\u003c/p\u003e \u003cp\u003eThe universal absence of SecB is intriguing. In \u003cem\u003eE. coli\u003c/em\u003e, SecB maintains substrates in unfolded states for Sec-mediated export [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Its absence in \u003cem\u003eBdellovibrio\u003c/em\u003e suggests either: (1) functional compensation by DnaK or GroEL chaperones, (2) Sec substrates adopt transport-competent conformations independently, or (3) Tat-mediated export of folded proteins predominates. Given that \u003cem\u003eBdellovibrio\u003c/em\u003e secretes numerous hydrolases during prey invasion, understanding its secretion pathway preferences is critical for elucidating its predatory mechanisms.\u003c/p\u003e \u003cp\u003eEvolutionary and Ecological Perspectives\u003c/p\u003e \u003cp\u003eComparative analysis among \u003cem\u003eB. bacteriovorus\u003c/em\u003e HD100, \u003cem\u003eB. reynosensis\u003c/em\u003e, \u003cem\u003eB. svalbardensis\u003c/em\u003e, and \u003cem\u003eBdellovibrio\u003c/em\u003e sp. Kdesi demonstrates a remarkable balance between genomic conservation and ecological innovation. The cgSNP phylogeny reveals a complex evolutionary history where recent radiation coexists with long-divergent lineages. The extremely short branches among HD100, 109J, and related strains (0.00000085 substitutions/site) indicate recent clonal expansion, while long-branch taxa like \u003cem\u003eB. exovorus\u003c/em\u003e JSS (0.06083) and \u003cem\u003eB. bacteriovorus\u003c/em\u003e W (0.02871) represent evolutionarily distinct lineages.\u003c/p\u003e \u003cp\u003eThe syntenic conservation of predation-associated operons, such as the Type IV pilus cluster, points to strong evolutionary constraints on the core predatory machinery. In contrast, divergence in oligopeptide transport and regulatory gene clusters suggests adaptive evolution to distinct environmental niches soil, and sewage [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Kdesi's evolutionary trajectory appears particularly dramatic: recent divergence from the \u003cem\u003eB. bacteriovorus\u003c/em\u003e core group followed by rapid genomic changes, potentially driven by the unique selective pressures of sewage environments.\u003c/p\u003e \u003cp\u003eThe coexistence of conserved predatory frameworks and lineage-specific adaptations supports an eco-evolutionary model of \u003cem\u003eBdellovibrio\u003c/em\u003e diversification: a stable predatory core optimized for prey recognition and invasion, supplemented by flexible accessory genomes that enable niche specialization. This dual architecture likely underlies the genus's global ecological success and provides genomic flexibility for adapting to new environments and prey spectra.\u003c/p\u003e \u003cp\u003eImplications for Functional Genomics and Applied Research\u003c/p\u003e \u003cp\u003eThe identification of 26% of Kdesi-unique genes as hypothetical proteins (DUFs) represents an exciting frontier for functional discovery. Predatory bacteria possess unique molecular machinery for prey recognition, invasion, bdelloplast formation, and resource extraction [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Characterizing these DUFs through transcriptomics, proteomics, and targeted mutagenesis may reveal novel antimicrobial mechanisms applicable to treating drug-resistant infections.\u003c/p\u003e \u003cp\u003eKdesi's evolutionary story offers particular promise for applied research. As a recently evolved sewage specialist, it may possess enhanced capabilities for targeting pathogens prevalent in wastewater environments, including multidrug-resistant strains. Its unique transporter systems and regulatory networks could be harnessed for improving predation efficiency in complex microbial communities.\u003c/p\u003e \u003cp\u003eThe modular T4SS architecture across \u003cem\u003eBdellovibrio\u003c/em\u003e strains suggests diverse strategies for effector delivery. Strain-specific components (e.g., TraF, TrwF, VirD4) may influence prey range, killing kinetics, or resistance to host defenses. Comparative functional studies could guide the rational selection of \u003cem\u003eBdellovibrio\u003c/em\u003e strains optimized for specific therapeutic applications, e.g., targeting biofilm-embedded pathogens or multidrug-resistant \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e or \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe syntenic conservation of the T4P operon across divergent lineages underscores its non-redundant role in prey attachment and invasion, making it an attractive target for synthetic biology applications. Engineering enhanced T4P variants could improve predation efficiency or expand host range, advancing \u003cem\u003eBdellovibrio\u003c/em\u003e as a next-generation antimicrobial platform.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe showed through comprehensive comparative genomic analysis that \u003cem\u003eBdellovibrio\u003c/em\u003e sp. kdesi is a distinct species within the genus \u003cem\u003eBdellovibrio\u003c/em\u003e, highlighting the limitations of 16S rRNA-based taxonomy and the necessity of polyphasic genomic approaches for accurate species delineation. The cgSNP phylogeny reveals kdesi as a compelling case of recent ecological speciation, where adaptation to sewage environments might have driven rapid genomic changes leading to novel species formation while maintaining phylogenetic signal of recent common ancestry with \u003cem\u003eB. bacteriovorus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe comparative analysis of 29 genomes reveals substantial cryptic diversity within \u003cem\u003eBdellovibrio\u003c/em\u003e, with at least 12 genomospecies requiring formal taxonomic revision. The genus exhibits an open pan-genome, limited transposable elements, universal absence of SecB, and modular T4SS architecture\u0026mdash;features reflecting adaptation to obligate predatory lifestyles across diverse environments.\u003c/p\u003e \u003cp\u003eThese findings provide a genomic foundation for future functional studies aimed at harnessing \u003cem\u003eBdellovibrio\u003c/em\u003e for antimicrobial therapy and offer a model system for understanding bacterial predation at the molecular level. The evolutionary dynamics revealed by kdesi's genome recent divergence followed by rapid adaptation provide insight into the process of bacterial speciation in real-time. These findings call for a systematic taxonomic revision of \u003cem\u003eBdellovibrio\u003c/em\u003e, integrating genome-based species delineation (ANI, AAI, dDDH) with ecological and phylogenetic criteria.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBacterial Isolation and Preliminary Identification\u003c/h2\u003e \u003cp\u003eA 500 mL sewage sample was collected from Colonia Las Delicias, Reynosa, Tamaulipas, Mexico (26.099793\u0026deg;N, 98.981904\u0026deg;W), filtered through 0.45 \u0026micro;m membranes to remove protozoans and larger bacteria, and enriched using Klebsiella pneumoniae as prey following established protocols [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Briefly, overnight prey cultures were harvested by centrifugation (4,000 rpm, 20 min, 4\u0026deg;C), washed twice in HEPES buffer (pH 7.4), and co-incubated with HEPES buffer at 30\u0026deg;C with shaking. After visible lysis occurred, the supernatant was passed through two additional enrichment cycles to increase predator titer.\u003c/p\u003e \u003cp\u003ePredatory bacteria were isolated using double-layer agar plating [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] Serial dilutions of enriched supernatants (10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e) were mixed with HEPES-suspended prey and overlaid on dilute nutrient broth (DNB) agar. Plaques appearing after 48 hours were isolated, and plaque-forming bacteria were co-cultured with fresh prey. Genomic DNA was extracted from predator-enriched lysates using the Promega Wizard Genomic DNA Extraction Kit following manufacturer instructions.\u003c/p\u003e \u003cp\u003ePreliminary identification was performed by PCR amplification of partial 16S rRNA using \u003cem\u003eBdellovibrio\u003c/em\u003e -specific primers BdsF (5\u0026prime;-TCTGGCTCAGAACAAACGCT-3\u0026prime;) and BdsR (5\u0026prime;-GCTTCGTCACTGAAGGGGTC-3\u0026prime;), which amplify an ~\u0026thinsp;818 bp fragment. PCR conditions were initially denatured at 95\u0026deg;C for 3 min; 35 cycles of 95\u0026deg;C for 30 s, 60\u0026deg;C for 30 s, and 72\u0026deg;C for 30 s; final extension at 72\u0026deg;C for 5 min. Amplicons were sequenced using Sanger sequencing (AB3130, Thermo Fisher Scientific) and queried against the NCBI nucleotide database using BLASTN.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGenome Sequencing, Assembly, and Annotation\u003c/h2\u003e \u003cp\u003eGenomic DNA quality was assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Whole-genome sequencing was performed on the Illumina MiSeq platform (MyGenomics LLC, USA). Raw reads were quality-filtered using FastQC v0.11.9 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], trimmed with Trim Galore\u003c/p\u003e \u003cp\u003ev0.6.7 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and assembled using SPAdes v13.0.1 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The assembly was further processed and consolidated using Unicycler v0.5.0 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Assembly quality was evaluated with QUAST v5.0 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. To identify potential contaminants, assembled contigs were mapped against the \u003cem\u003eB. bacteriovorus\u003c/em\u003e HD100 reference genome using NUCmer [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Contaminant sequences were removed using custom Perl scripts. The resulting four contigs were scaffolded into a single circular chromosome using MEDUSA [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] and NUCmer, with gap closure validated by manual inspection of read alignments. The closed genome was annotated using Prokka v1.14.6 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenomic and Taxonomic Analyses\u003c/h2\u003e \u003cp\u003eTwenty-nine \u003cem\u003eBdellovibrio\u003c/em\u003e genomes (27 complete, 2 draft) were retrieved from NCBI (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All genomes were re-annotated with Prokka v1.14.6 to ensure consistent gene calling. Pan-genome analysis was performed using Bacterial Pan Genome Analysis (BPGA) v1.3 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] with default parameters and Roary v3.13 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] with 90% BLASTP identity threshold and core genes defined as present in \u0026ge;\u0026thinsp;95% of isolates.\u003c/p\u003e \u003cp\u003eCore-genome phylogeny was reconstructed from 760 single-copy core genes extracted by BPGA. These core genes were concatenated and aligned using MAFFT v7.475 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Maximum-likelihood phylogenetic trees were inferred using IQ-TREE v2.4.0[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] with automatic model selection (best model: GTR\u0026thinsp;+\u0026thinsp;F\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G4) and 1,000 ultrafast bootstrap replicates. Core-genome SNP (cgSNP) analysis was performed using REALPHY (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://realphy.unibas.ch/fcgi/realphy\u003c/span\u003e\u003cspan address=\"http://realphy.unibas.ch/fcgi/realphy\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, last accessed November 5, 2025) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and trees were visualized using MEGA 12[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and iTOL v6 [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Average nucleotide identity (ANI) was calculated using PyANI v0.2.10 (ANIb method) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Average amino acid identity (AAI) was computed using COMPASS [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Digital DNA-DNA hybridization (dDDH) values were obtained from the Genome-to-Genome Distance Calculator (GGDC) v3.0[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] via the Type Strain Genome Server (TYGS) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Percentage of conserved proteins (POCP) was calculated using Galaxy Proteinortho[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] to confirm genus-level assignment. Orthologous gene groups were identified using OrthoFinder v2.5.4[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] with default parameters. Functional annotation of core genes was performed using eggNOG-mapper v2.1.9 [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], and metabolic pathways were mapped to the KEGG database [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Clusters of Orthologous Groups (COG) distributions were analyzed using COGclassifier v1.0.5 [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eComparative Genomic Analyses\u003c/h2\u003e \u003cp\u003eSynteny analysis between Kdesi and reference strain HD100 was performed using bidirectional best-hit comparisons in the RAST annotation server [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Genomic islands (GIs) were identified using IslandViewer 4 [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], which integrates predictions from IslandPath-DIMOB, SIGI-HMM, and Islander methods. Insertion sequences (IS) were detected using ISEScan v1.7.2.1 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Protein secretion systems were annotated using KEGG automatic annotation [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] and the SecreT4/6 web server [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] with default parameters. Synteny of predatory gene clusters, including the Type IV pilus operon, was analyzed using SyntTax [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Statistical significance of COG category distributions was assessed using Fisher's exact test with Bonferroni correction for multiple comparisons (α\u0026thinsp;=\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eConflict of Interest:\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e \u003cb\u003eEthical Approval\u003c/b\u003e:\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research was supported by grants (20230747, 20231050) from the Secretar\u0026iacute;a de Investigaci\u0026oacute;n y Posgrado del Instituto Polit\u0026eacute;cnico Nacional.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eX.G., M.A.V.-L., and I.C.R.-L. designed the study. T.O.E. performed experiments, genome assembly, annotation, and comparative analyses, and wrote the first draft. A.S.-V. and I.C.R.-L. participated in experimental design. A.Y.O., I.J.A., O.O.O., and R.F.-C. reviewed the manuscript. X.G., M.A.V.-L., and T.O.E. supervised the study and edited the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis research was supported by grants (20230747, 20231050) from \u0026ldquo;la Scretario de investigaci\u0026oacute;n y Posgrado del Instituto Polit\u0026eacute;cnico Nacional. We thank the staff at MyGenomics LLC for sequencing services and the Centro de Biotecnolog\u0026iacute;a Gen\u0026oacute;mica for laboratory support.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe complete genome sequence of Bdellovibrio sp. strain Kdesi has been deposited in GenBank (NCBI) under accession number CP102930 (BioProject PRJNA524134). Kdesi has been deposited as a reference research strain at the Centro Nacional de Recursos Gen\u0026eacute;ticos (CNRG-INIFAP, Mexico) under accession CM-CNRG 961. This deposit is intended solely to ensure strain preservation and reproducibility of the genomic analyses reported in this study.\u003c/p\u003e\u003cp\u003eCertificate of Deposit\u003c/p\u003e\n\u003cp\u003eThe following document represents the official deposit certificate of the strain \u003cem\u003eBdellovibrio\u0026nbsp;\u003c/em\u003esp. Kdesi (CM-CNRG 961) issued by the Centro Nacional de Recursos Gen\u0026eacute;ticos (CNRG-INIFAP), confirming its registration and preservation as a microbial culture deposit.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAjao, Y. O. et al. Bdellovibrio reynosensis sp. nov., from a Mexico soil sample. \u003cem\u003eInt J. Syst. Evol. Microbiol\u003c/em\u003e \u003cb\u003e72\u003c/b\u003e, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDashiff, A., Junka, R. A., Libera, M. \u0026amp; Kadouri, D. E. Predation of human pathogens by the predatory bacteria Micavibrio aeruginosavorus and Bdellovibrio bacteriovorus. \u003cem\u003eJ. Appl. Microbiol.\u003c/em\u003e \u003cb\u003e110\u003c/b\u003e, 431\u0026ndash;444 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAjao, Y. O. et al. Antibacterial activity of two newly isolated Bdellovibrio bacteriovorus strains on Salmonella enterica serovars of food safety concern. \u003cem\u003eMicrobiol Spectr\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGophna, U., Charlebois, R. L. \u0026amp; Doolittle, W. F. Ancient lateral gene transfer in the evolution of Bdellovibrio bacteriovorus. \u003cem\u003eTrends Microbiol.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 64\u0026ndash;69 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShemesh, Y. \u0026amp; Jurkevitch, E. Plastic phenotypic resistance to predation by Bdellovibrio and like organisms in bacterial prey. \u003cem\u003eEnviron. Microbiol.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 12\u0026ndash;18 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoval, S. F., Williams, H. N. \u0026amp; Colin Stine, O. Reclassification of bacteriovorax marinus as Halobacteriovorax marinus gen. Nov., comb. nov. and bacteriovorax litoralis as halobacteriovorax litoralis comb. nov.; description of halobacteriovoraceae fam. nov. in the class Deltaproteobacteria. \u003cem\u003eInt. J. Syst. Evol. Microbiol.\u003c/em\u003e \u003cb\u003e65\u003c/b\u003e, 593\u0026ndash;597 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerleman, J. E. \u0026amp; Kirby, J. R. Deciphering the hunting strategy of a bacterial wolfpack. \u003cem\u003eFEMS Microbiology Reviews\u003c/em\u003e vol. 33 Preprint at (2009). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1574-6976.2009.00185.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1574-6976.2009.00185.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePasternak, Z., Sasson, T., Ben, Cohen, Y., Segev, E. \u0026amp; Jurkevitch, E. A new comparative-genomics approach for defining phenotype-specific indicators reveals specific genetic markers in predatory bacteria. \u003cem\u003ePLoS One\u003c/em\u003e. \u003cb\u003e10\u003c/b\u003e, 1\u0026ndash;11 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi, S. Y., Mun, W., Choi, S., Lee, K. \u0026amp; Mitchell, R. J. Bdellovibrio svalbardensis sp. nov., a newly described predator isolated from Svalbard, Norway. \u003cem\u003eInt. J. Syst. Evol. Microbiol.\u003c/em\u003e \u003cb\u003e74\u003c/b\u003e, 006248 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStolp, H. \u0026amp; Starr, M. P. Bdellovibrio bacteriovorus gen. et sp. n., a predatory, ectoparasitic, and bacteriolytic microorganism. \u003cem\u003eAntonie Van Leeuwenhoek\u003c/em\u003e. \u003cb\u003e29\u003c/b\u003e, 217\u0026ndash;248 (1963).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElbir, H. Updating the Relationship Between the Threshold Value of Average Nucleotide Identity and Digital DNA\u0026ndash;DNA Hybridization for Reliable Taxonomy of Corynebacterium. \u003cem\u003eVet. Sci.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 661 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, M., Oh, H. S., Park, S. C. \u0026amp; Chun, J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. \u003cem\u003eInt. J. Syst. Evol. Microbiol.\u003c/em\u003e \u003cb\u003e64\u003c/b\u003e, 346\u0026ndash;351 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavis, S. C., Cerra, J., Williams, L. E. \u0026amp; Campbell, B. J. Comparative genomics of obligate predatory bacteria belonging to phylum Bdellovibrionota highlights distribution and predicted functions of lineage-specific protein families. \u003cem\u003emSphere\u003c/em\u003e 9, (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeier-Kolthoff, J. P. \u0026amp; G\u0026ouml;ker, M. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 1\u0026ndash;10 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMachado, R. A. R. et al. Acinetobacter nematophilus sp. nov., Alcaligenes nematophilus sp. nov., Enterobacter nematophilus sp. nov., and Kaistia nematophila sp. nov., Isolated from Soil-Borne Nematodes and Proposal for the Elevation of Alcaligenes faecalis subsp. faecalis, Alcaligenes faecalis subsp. parafaecalis, and Alcaligenes faecalis subsp. phenolicus to the Species Level. \u003cem\u003eTaxonomy\u003c/em\u003e 3, (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang, Z. et al. Genome-wide expanding of genetic evolution and potential pathogenicity in Vibrio alginolyticus. \u003cem\u003eEmerg Microbes Infect\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, W., Lin, H. \u0026amp; Cao, Y. Effects of opuB on the growth and biofilm formation of Streptococcus mutans under acid stress. \u003cem\u003eMicrob. Pathog\u003c/em\u003e. \u003cb\u003e205\u003c/b\u003e, 107674 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams, H. N. \u0026amp; Chen, H. Environmental Regulation of the Distribution and Ecology of Bdellovibrio and Like Organisms. \u003cem\u003eFront. Microbiol.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 545070 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaher, R. L. et al. Comparative analysis of novel Pseudobdellovibrionaceae genera and species yields insights into the genomics and evolution of bacterial predation mode. \u003cem\u003ebioRxiv\u003c/em\u003e 2025.02.19.638989 (2025). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/2025.02.19.638989\u003c/span\u003e\u003cspan address=\"10.1101/2025.02.19.638989\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeeg, C. M., Le, T. T., Zimmer, M. M. \u0026amp; Suttle, C. A. From the inside out: An epibiotic bdellovibrio predator with an expanded genomic complement. \u003cem\u003eJ Bacteriol\u003c/em\u003e \u003cb\u003e202\u003c/b\u003e, (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOyedara, O. O. et al. Isolation of Bdellovibrio sp. from soil samples in Mexico and their potential applications in control of pathogens. \u003cem\u003eMicrobiologyopen\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e, 992\u0026ndash;1002 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCollier, D. N. \u0026amp; SecB A Molecular Chaperone of Escherichia Coli Protein Secretion Pathway. \u003cem\u003eAdv. Protein Chem.\u003c/em\u003e \u003cb\u003e44\u003c/b\u003e, 151\u0026ndash;193 (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalwar, C. et al. Defining the Environmental Adaptations of Genus Devosia: Insights into its Expansive Short Peptide Transport System and Positively Selected Genes. \u003cem\u003eScientific Reports\u003c/em\u003e 10, 1151- (2020). (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOyedara, O. O. et al. Whole-genome sequencing and comparative genome analysis provided insight into the predatory features and genetic diversity of two bdellovibrio species isolated from soil. \u003cem\u003eInt J Genomics\u003c/em\u003e (2018). (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJurkevitch, E. Isolation and classification of bdellovibrio and like organisms. \u003cem\u003eCurr Protoc Microbiol Chap. 7\u003c/em\u003e, Unit 7B.1 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndrews, S., FastQC \u0026amp; A Quality Control tool for High Throughput Sequence Data.. \u003cem\u003eHttp://Www.Bioinformatics.Babraham.Ac\u003c/em\u003e (2010). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e.Uk/Projects/Fastqc/ https://doi.org/citeulike-article-id:11583827\u003c/span\u003e\u003cspan address=\"http://.Uk/Projects/Fastqc/ https://doi.org/citeulike-article-id:11583827\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e doi:citeulike-article-id:11583827\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrueger, F., Trim \u0026amp; Galore! A wrapper tool around Cutadapt and FastQC to consistently apply quality and adapter trimming to FastQ files. \u003cem\u003eBabraham Institute\u003c/em\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBankevich, A. et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. \u003cem\u003eJ. Comput. Biol.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, 455\u0026ndash;477 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWick, R. R., Judd, L. M., Gorrie, C. L. \u0026amp; Holt, K. E. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. \u003cem\u003ePLoS Comput. Biol.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, e1005595 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGurevich, A., Saveliev, V., Vyahhi, N. \u0026amp; Tesler, G. QUAST: Quality assessment tool for genome assemblies. \u003cem\u003eBioinformatics\u003c/em\u003e 29, 1072\u0026ndash;1075 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMar\u0026ccedil;ais, G. et al. MUMmer4: A fast and versatile genome alignment system. \u003cem\u003ePLoS Comput. Biol\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBosi, E. et al. MeDuSa: A multi-draft based scaffolder. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e, 2443\u0026ndash;2451 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeemann, T. \u0026amp; Prokka Rapid prokaryotic genome annotation. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e, 2068\u0026ndash;2069 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao, Y. et al. PGAP: pan-genomes analysis pipeline. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e, 416\u0026ndash;418 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChaudhari, N. M., Gupta, V. K. \u0026amp; Dutta, C. BPGA-an ultra-fast pan-genome analysis pipeline. \u003cem\u003eSci Rep\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePage, A. J. et al. Roary: Rapid large-scale prokaryote pan genome analysis. \u003cem\u003eBioinformatics\u003c/em\u003e 31, (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatoh, K. \u0026amp; Standley, D. M. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. \u003cem\u003eMol. Biol. Evol.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e, 772\u0026ndash;780 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen, L. T., Schmidt, H. A., Von Haeseler, A. \u0026amp; Minh, B. Q. IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. \u003cem\u003eMol. Biol. Evol.\u003c/em\u003e \u003cb\u003e32\u003c/b\u003e, 268\u0026ndash;274 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBertels, F., Silander, O. K., Pachkov, M., Rainey, P. B. \u0026amp; Van Nimwegen, E. Automated Reconstruction of Whole-Genome Phylogenies from Short-Sequence Reads. \u003cem\u003eMol. Biol. Evol.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e, 1077\u0026ndash;1088 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar, S. et al. MEGA12: Molecular Evolutionary Genetic Analysis Version 12 for Adaptive and Green Computing. \u003cem\u003eMol. Biol. Evol.\u003c/em\u003e \u003cb\u003e41\u003c/b\u003e, 1\u0026ndash;9 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLetunic, I. \u0026amp; Bork, P. Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cb\u003e52\u003c/b\u003e, W78\u0026ndash;W82 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePritchard, L. pyani. (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/widdowquinn/pyani Preprint\u003c/span\u003e\u003cspan address=\"https://github.com/widdowquinn/pyani Preprint\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e at.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSadreyev, R. \u0026amp; Grishin, N. C. O. M. P. A. S. S. A Tool for Comparison of Multiple Protein Alignments with Assessment of Statistical Significance. \u003cem\u003eJ. Mol. Biol.\u003c/em\u003e \u003cb\u003e326\u003c/b\u003e, 317\u0026ndash;336 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAuch, A. F., von Jan, M., Klenk, H. P. \u0026amp; G\u0026ouml;ker, M. Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. \u003cem\u003eStand. Genomic Sci.\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e, 117\u0026ndash;134 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAfgan, E. et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2022 update. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cb\u003e50\u003c/b\u003e, W345\u0026ndash;W351 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolzer, M. POCP-nf: an automatic Nextflow pipeline for calculating the percentage of conserved proteins in bacterial taxonomy. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cb\u003e40\u003c/b\u003e, (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEmms, D. M., Kelly, S. \u0026amp; OrthoFinder Phylogenetic orthology inference for comparative genomics. \u003cem\u003eGenome Biol\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e, (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuerta-Cepas, J. et al. Fast Genome-Wide Functional Annotation through Orthology Assignment by eggNOG-Mapper. \u003cem\u003eMol. Biol. Evol.\u003c/em\u003e \u003cb\u003e34\u003c/b\u003e, 2115\u0026ndash;2122 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAramaki, T. et al. KofamKOALA: KEGG Ortholog assignment based on profile HMM and adaptive score threshold. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cb\u003e36\u003c/b\u003e, 2251\u0026ndash;2252 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026lrm;Shimoyama Yuki. COGclassifier: A tool for classifying prokaryote protein sequences into COG functional category. github. (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAziz, R. K. et al. The RAST Server: Rapid annotations using subsystems technology. \u003cem\u003eBMC Genomics\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBertelli, C. et al. IslandViewer 4: Expanded prediction of genomic islands for larger-scale datasets. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cb\u003e45\u003c/b\u003e, W30\u0026ndash;W35 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie, Z. \u0026amp; Tang, H. ISEScan: automated identification of insertion sequence elements in prokaryotic genomes. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cb\u003e33\u003c/b\u003e, 3340\u0026ndash;3347 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKanehisa, M., Furumichi, M., Sato, Y., Matsuura, Y. \u0026amp; Ishiguro-Watanabe, M. KEGG: biological systems database as a model of the real world. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cb\u003e53\u003c/b\u003e, D672\u0026ndash;D677 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBi, D. et al. SecReT4: A web-based bacterial type IV secretion system resource. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cb\u003e41\u003c/b\u003e, (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, J. et al. SecReT6: A web-based resource for type VI secretion systems found in bacteria. \u003cem\u003eEnviron. Microbiol.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 2196\u0026ndash;2202 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOberto, J. \u0026amp; SyntTax A web server linking synteny to prokaryotic taxonomy. \u003cem\u003eBMC Bioinformatics\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, (2013).\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bdellovibrio sp. Kdesi, comparative genomics, average nucleotide identity, taxonomic revision, predatory bacteria","lastPublishedDoi":"10.21203/rs.3.rs-8476110/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8476110/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eBdellovibrio\u003c/em\u003e are predatory bacteria with potential applications in treating multidrug-resistant bacterial infections. We isolated \u003cem\u003eBdellovibrio\u003c/em\u003e sp. kdesi from sewage in Reynosa, Mexico, and performed comprehensive genomic characterization. The complete genome (3,343,978bp; 48.5% GC content; 3,208 coding sequences) contains a single rRNA operon with two identical 16S rRNA genes at distinct loci. While 16S rRNA analysis showed 99.3% identity to \u003cem\u003eB. bacteriovorus\u003c/em\u003e SSB218315, polyphasic genomic analysis revealed kdesi as a distinct species. Core-genome phylogeny, average nucleotide identity (ANI\u0026thinsp;~\u0026thinsp;89%), average amino acid identity (AAI\u0026thinsp;~\u0026thinsp;82%), and digital DNA-DNA hybridization (dDDH\u0026thinsp;\u0026lt;\u0026thinsp;70%) clearly demonstrated that Kdesi along with several other strains currently classified as \u003cem\u003eB. bacteriovorus\u003c/em\u003e represents novel genomospecies requiring formal taxonomic revision. Comparative genomic analysis of 29 \u003cem\u003eBdellovibrio\u003c/em\u003e genomes revealed an open pan-genome (~\u0026thinsp;25,000 genes) with limited transposable elements, absence of SecB in the Sec secretion system, and strain-specific adaptations in Type IV secretion systems. These findings underscore the need for genome-based taxonomic revision of the genus and highlight the importance of using multiple molecular criteria beyond 16S rRNA for bacterial species delineation.\u003c/p\u003e","manuscriptTitle":"Genome-Scale Analysis Reveals Strain Kdesi as a Distinct Evolutionary Lineage and Extensive Cryptic Diversity in the Genus Bdellovibrio","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-09 15:05:01","doi":"10.21203/rs.3.rs-8476110/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d58064f6-b733-442d-ae2b-578c21e314b5","owner":[],"postedDate":"January 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":60839401,"name":"Biological sciences/Genetics"},{"id":60839402,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-02-12T16:09:00+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-09 15:05:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8476110","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8476110","identity":"rs-8476110","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}