In vitro Screening and Genome-wide Analysis of Probiotic Bacillus spp.from Locally Fermented Yogurt for Preliminary Probiotic Potential

In vitro Screening and Genome-wide Analysis of Probiotic Bacillus spp.from Locally Fermented Yogurt for Preliminary Probiotic Potential | 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 Research Article In vitro Screening and Genome-wide Analysis of Probiotic Bacillus spp.from Locally Fermented Yogurt for Preliminary Probiotic Potential Janeeta Raheel, Zainab zia, Haris Owais, Sikandar Hayat, Sajid Iqbal, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8865594/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 The research was focused on extracting Bacillus spp. and genomic-wide analysis from locally fermented yogurt to assess their potential as probiotics through genetic characterization. Two bacterial strains, JF-5 and isolate JY-2 were isolated from total 25 yogurt samples. The samples were collected from markets in Rawalpindi and Islamabad. Isolation was carried out using BHI agar as culture medium. The tested isolates received morphological and biochemical examinations, biosafety testing and enzymatic evaluations to evaluate their preliminary probiotic-associated traits. The probiotic characteristics of both strains differed, although they shared Gram-positive morphology, together with rod-shaped features and exhibited catalase activity. Analyses demonstrated that isolate JF-5 possessed proteolytic and lipolytic capacities and showed resilience against bile and pH variations. However, isolate JY-2 displayed DNase activity in addition to hemolysis, making it unsuitable for use as a safe probiotic. Whole-genome sequencing was applied to the strain JF-5 as the next analysis step. The bacterial strain was defined as Bacillus altitudinis through genome sequencing, which showed a 3.77 Mbp genome size alongside 41.2% GC content and 3962 coding sequences. The antiSMASH analysis platform detected various biosynthetic gene clusters that can produce antibacterial and probiotic traits, such as lichenysin, bacilysin, fengycin and siderophore compounds. CRISPR -Cas systems and vancomycin resistance-related genes (vanG, vanY, vanT) were identified with analysis. These antimicrobial resistance determinants have important biosafety implications especially with regard to possible horizontal gene transfer. This is why the strain cannot be proposed to be implemented as probiotics without a careful study of its safety. Even though the genomic presence indicates environmental adaptability through the presence of stress response genes, sporulation genes, and nutrient assimilation genes, alone in the genome does not indicate functional probiotic efficacy. This investigation presents the first detailed genomic analysis of indigenous Pakistan-based probiotic Bacillus strains. Whole-genome sequencing were performed by MicrobesNG Lab, Birmingham, United Kingdom. General Microbiology Food Science & Technology Bacteriology Probiotics fermented yogurt biochemical characterization biosafety profiling whole-genome sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Probiotics are active microorganisms that enhance the health of the host once taken in adequate amounts. Even though probiotic preparations traditionally consisted of lactic acid bacteria such as Lactobacillus and Bifidobacterium, Bacillus spores have been of greater interest due to their outstanding environmental resistance and ability to withstand severe processing factors. ( 1 , 2 ). Bacillus spores are also good choices in industrial applications and oral delivery systems because they are resistant to heat, dryness and acidic environment ( 3 , 4 ). There are also these advantages, but the safety and functional effectiveness of the Bacillus- based probiotics should be evaluated. The recent discoveries in the field of whole-genome sequencing (WGS) have improved the process of the evaluation of the probiotic candidates significantly ( 4 ). Genome-wide analysis can predict genes associated with antimicrobial compound biosynthesis stress resistance, sporulation and metabolic functions; however, genomic prediction does not necessarily indicate functional expression or probiotic efficacy ( 5 , 6 ) Meanwhile, the potential presence of biosafety risks is identifiable through genomic screening (e.g., antibiotic resistance gene, virulence factor and pathogenicity-related determinants). Notably, the presence of probiotic-related genes in the genome does not determine the presence of probiotic activity, though, it provides the details of the genetic potential, which is to be substantiated with help of the phenotypic confirmation. The fermented foods are a nutritious source of potentially valuable microbes which are diverse. ( 7 , 8 ) Local isolates of such foods can have special adaptive properties, but systematic genomic characterization of locally isolated Bacillus strains has not been mostly undertaken. Moreover, genomic analysis of these isolates in terms of safety must come first, prior to their use in food or therapeutic preparations ( 9 , 10 ). Despite the availability of a number of commercial probiotic products across the world, little published information exists on genomic and biosafety profiling of native Bacillus isolates of fermented foods in Pakistan ( 11 ). An organized methodology that combines in vitro screening and genome-wide analysis can deliver background data about the traits associated with probiotics and the possible safety risks. This is preliminary characterization required as a first step before further functional, clinical or in vivo validation ( 12 ). Thus, this research was aimed at isolating Bacillus spp. in locally fermented yogurt and conducting initial in vitro screening along with whole-genome sequencing to assess the probiotic-related features and genomic biomarkers of biosafety. This paper does not identify functional probiotic efficacy, but it gives first phenotypic and genomic data that will be useful in future validation and safety evaluation investigations. ( 13 ) 2. Materials & Methods No in vivo experiments were performed in this research, all evaluations are preliminary in vitro and genomic analyses. 2.1. Sample Collection The current study was designed to isolate potential probiotic candidates from locally fermented yogurts. For this purpose, 5 g samples (n = 25) were collected from locally fermented yogurt from local units (small shops selling yogurt) in Rawalpindi and Islamabad capital territory. 2.2. Isolation, Purification and Identification of bacterial isolates All the samples were immediately transported to the BJ Micro Lab and processed for bacterial isolation. Two potential Bacillu s strains were isolated from yogurt and were evaluated for probiotic potential. After incubating samples at 37 o C for 24 hours, bacterial colonies appeared on the Brain Heart Infusion media petri plates. The purification of the colonies was done by using the steak plate method. Morphological identification was done using Gram staining, motility test and spore formation test. The isolated strains were preliminarily identified using the biochemical tests including catalase, oxidase, VP, TSI, and citrate tests ( 14 ). 2.3. Screening assays The screening for probiotic potential was determined by the Lipolytic test, amylolytic test, proteolytic test and bile tolerance test according to the procedures and precautions previously published( 15 ). Bacterial growth was observed by the formation of colonies on the agar plates. 2.4. Antibiotic susceptibility test (AST) Inoculum was prepared by using a sterile inoculating loop or needle; four or five isolated colonies of the organism to be tested were touched and suspended in 2 mL of normal saline. The saline tube was vortexed to create a smooth suspension. Next, the turbidity of this suspension was adjusted to a 0.5 McFarland standard by adding more culture suspension if the suspension was too light or diluting with sterile saline if the suspension was too heavy. The petri plates are ready with MHA agar (Accumix, India) and will solidify in the plates. A cotton swab was dipped in the cell suspension and streaked on the petri plates repeatedly. The antibiotic discs were firmly placed on the plate ( 16 ). 2.4.1. Tolerance to different pH Nutrient broth (HI, India) was prepared, and pH was adjusted to pH 2, pH 7, pH 9 using dilute HCL and NaOH. and inoculated with bacterial isolates JF-5 and JY-2, incubated at 37°C for 24 hrs. Bacterial growth was assessed by measuring it using a spectrophotometer at 600nm. 2.4.2. Tolerance to different temperatures Nutrient broth (HI, India) was prepared and inoculated with bacterial isolates JF-5 and JY-2 and were incubated at the different temperature (30°C, 37°C and 50°C) for 24 hours. 2.4.3. Hemolysis and DNase activity Brain Heart Infusion agar powder (HI, India) was dissolved in distilled water as per the manufacturer’s protocol. And the medium was autoclaved at 121°C for 15 minutes. After autoclaving, the medium was cooled to 55°C, and then 5% of the whole human blood was added to the agar. After it was mixed well and poured onto plates. The bacteria were streaked on the plates and then incubated at 37°C overnight. DNase agar powder (HI, India) was dissolved in distilled water, and the mixture was brought to a boil to ensure complete dissolution. The medium was then autoclaved at 121°C for 30 minutes for sterilization. After autoclaving, the agar was allowed to cool slightly, mixed thoroughly, and poured into sterile petri plates. Isolates were streaked onto the solidified agar, and the plates were incubated at 37°C overnight. 2.5. DNA extraction and next-generation sequencing 2.5.1. DNA extraction A total of 1 µL cell suspension was lysed with 120 l of TE buffer containing lysozyme (MPBio, USA), metapolyzyme (Sigma Aldrich, USA), and RNase A (ITW Reagents, Spain), and incubated for 25 minutes at 37°C. Proteinase K (VWR Chemicals, Ohio, USA) (final concentration 0.1 mg/mL) and SDS (Sigma-Aldrich, Missouri, USA) (final concentration 0.5% v/v) were added, and the mixture was incubated for 5 minutes at 65°C. Genomic DNA was purified using an equal volume of SPRI beads and resuspended in EB buffer (10 mM Tris-HCl, pH 8.0). The extracted DNA was quantified using the Quant-iT dsDNA HS assay (Thermo Fisher Scientific) in an Eppendorf AF2200 plate reader (Eppendorf UK Ltd, United Kingdom) and diluted as appropriate. 2.5.2. Next generation sequencing, assembly and annotation Genomic DNA libraries were prepared using the Nextera XT Library Prep Kit (Illumina, San Diego, USA) following the manufacturer’s protocol with the following modifications: input DNA was increased 2-fold, and PCR elongation time was increased to 45 seconds. DNA quantification and library preparation were carried out using a Hamilton Microlab STAR automated liquid handling system (Hamilton Bonaduz AG, Switzerland). Libraries were sequenced on an Illumina NovaSeq 6000 (Illumina, San Diego, USA) using a 250 bp paired- end protocol. Whole-genome sequencing and library preparation were performed by MicrobesNG Lab, Birmingham, United Kingdom. Reads were adapter-trimmed using Trimmomatic version 0.30 with a sliding window quality cutoff of Q15. De novo assembly was performed using SPAdes version 3.7, and contigs were annotated using Prokka 1.11 ( 17 ). 2.6. Phylogenetic analysis Phylogenetic Analysis of the isolated strain Bacillus. altitudinis was generated based on core- genome and whole-genome analysis ( 18 ). 2.7. Genome-wide analysis To visualize the genomic feature of the Bacillus. altitudinis strain JF-5, a web-based Proksee server https://proksee.ca/ was employed. While the CRISPR Finder was used to detect the presence of the CRISPR cas sequence. ( 19 ). 2.7.1. Genome mining for biosynthetic gene clusters (BGCs) and bacteriocins and probiotics The antiSMASH bacterial version was used to identify biosynthetic gene clusters BGCs in B. altitudinis. ( 20 ). Bacteriocin gene clusters were identified using the BAGEL (Bacteriocin Genome mining Tool) http://bagel4.molgenrug.nl/ platform, which specializes in detecting putative bacteriocin operons in bacterial genomes. Probiotic-associated genes involved in stress resistance, adhesion, immune modulation, and metabolic adaptation were identified through a whole-genome annotation process using Prokka v1.14.6 ( 21 ). 2.7.2. Comparative genome analysis The subsystem feature categories of B. altitudinis with a comparison of reference genome strain GR-8 were determined using a Rapid Annotation Subsystem Technology (RAST) server. Comparative proteome analysis of Bacillus altitudinis strain JF-5 with closely related. Genomes, including the reference Bacillus altitudinis GR-8 genome, was conducted using Ortho Venn analysis. ( https://orthovenn3.bioinfotoolkits.net/ ) 2.7.3. Core pan genome analysis To investigate genomic diversity and identify the core and accessory genome components, we performed a comprehensive core and pan-genome analysis using the Bacterial Pan Genome Analysis (BPGA) tool. (https://iicb.res.in/bpga/). 2.8. Average nucleotide identity ANI analysis was performed using the EZBioCloud online tool ( https://www.ezbiocloud.net/tools/ani ). Genomic FASTA files were uploaded, and the OrthoANI algorithm was used to calculate pairwise nucleotide identity and pairwise nucleotide was shown in heatmap. ( 12 ). 3. RESULTS A total of two bacterial strains were isolated from (n = 25) yogurt samples and showed their growth on Brain Heart Infusion media plates and were labelled as JF-5, JY-2. Out of 25 samples, 13 were found to be Gram-negative. Nine samples displayed cocci morphology under the microscope, while two samples were identified as Gram-positive rods. Spore staining test for both isolates (a) JF-5 and (b) JY-2 under the microscope, showing the presence of rod- shaped bacterial cells with visible endospores. Catalase and oxidase tests show positive reactions toward both strains; VP shows positive reaction toward both strains. Both strains were motile and indole negative, citrate test shows positive reaction toward both strains and for TSI results show that strain JF-5 showed a red slant and yellow bottom, indicating fermentation of glucose only, with no lactose or sucrose fermentation. Strains were evaluated for preliminary probiotic-associated traits in vitro, Functional probiotic activity was not tested in vivo. 3.1. Screening activity Both strains JF-5 and JY-2 showed positive amylolytic activity and zones appeared on the starch media plates. Strain JF-5 and JY-2 showed positive proteolytic activity, and clear zones appeared on the media. 3.3. Antibiotic susceptibility test (AST) Antibiotic susceptibility test was used to investigate the resistance or susceptibility profile of bacterial strains against multiple antibiotic drugs. JF-5 and JY-2 were susceptible to Amoxicillin and clavulanic acid, Amikacin, Chloramphenicol and Penicillin. Bar graphs showing the zones of inhibition (mm) of strains JF-5 and JY-2 against selected antibiotics, indicating their susceptibility profile. 3.4. Stress Tolerance The survival of Bacillus altitudinis strains JF-5 and JY-2 in various pH levels (pH 5, pH 7, and pH 9). Survival was highest at neutral pH (pH 7) for both strains, at acidic pH (pH 5), the survival rate was reduced dramatically, showing that they have low acid tolerance. Both strains exhibited no growth at alkaline conditions (pH 9). The findings indicated that Bacillus altitudinis strains grow best under neutral conditions. Growth rates of two strains of Bacillus altitudinis (JF-5 and JY-2) in various temperatures between 37 o C, 30 o C and 50 o C. The two strains also display the maximum growth rates at 30 o C. On further rise of the temperature to 37 o C, a slight decrease in growth is seen in both the strains. Both strains grow best at 30 o C. A significant reduction in growth at 50 o C indicates the inhibitory effect of high temperature on both strains. Both strains showed maximum growth at neutral pH (pH 7) and 30 o C and less at other acidic, alkaline and high temperatures. 3.5. DNase test and Hemolytic activity This test was used to determine the presence or absence of deoxyribonuclease enzyme in the bacterial Isolates. The enzymatic potential of this enzyme showed the hydrolysis of genomic DNA into multiple pieces. Results of this test were observed by the formation of clear zones around the bacterial colonies. Strain JF-5 showed negative DNase activity, and no clear zones appeared on the media, while strain JY-2 showed positive DNase activity. The hemolysis test was used to determine the ability of bacterial isolates to lyse red blood cells. Results of this test were observed by the formation of clear zones around the bacterial colonies. Strain JY-2 showed positive hemolytic activity, and clear zones appeared on the media and strain JF-5 showed no zone and was recorded as hemolysis-negative. 3.6. Whole genome sequencing and annotation The draft genome of Bacillus altitudinis JF-5 is 3,774,388 bp in size and contains 41.2% GC content. The genome comprises 36 contigs and harbors 324 subsystems, 3962 coding sequences, and 83 RNAs. RAST subsystem prediction shows the genome of B. altitudinis with comparison of B.altitudinis GR-8. B. altitudinis JF-5 contains 183 genes for protein metabolism while B. altitudinis GR-8 has 201 genes for protein metabolism. Genes for stress response in the genome of B. altitudinis JF-5 are 44, while B. altitudinis GR-8 has 43 genes related to stress response. The presence of stress-related genes leads to probiotic potential. Furthermore, genes for amino acid metabolism contain 286 genes in B. Altitudinis JF-5, while B. altitudinis GR-8 has 266 genes (Fig. 5). The greater number of genes 247 in the B.altitudinis strain JF-5 are specified for carbohydrate metabolism, and the B. altitudinis GR-8 contains 219 genes for carbohydrate metabolism. B. altitudinis JF-5 contains 55 genes for RNA metabolism, while B. altitudinis GR-8 contains 54 genes. B.altitudinis strain JF-5 and B.altitudinis GR-8 have equal to 60 genes for DNA metabolism. The draft genome of Bacillus altitudinis strain JF-5 was visualized using Proksee, an online genome visualization tool. This map displayed the overall genome size, GC content, and GC skew (both positive and negative), providing a clear representation of nucleotide composition. To show important genomic features, a circular map of the B. altitudinis JF-5 genome was constructed. The ORFs, which are the annotated coding parts of the genome, are seen on the outermost ring. A special CRISPR-Cas group was found inside the bacterium, showing it defends itself against attacks by bacteriophages with adaptive abilities. Approximately the region containing CRISPR, the following resistance genes were found vanG, vanT and vanY which may provide protection against vancomycin. This means that the genes confer defense from glycopeptides. More rings in the tracks display GC content and GC skew, revealing information about the genome’s nucleotides and possible replication origins. Using GC content variation and skewing helps determine gene stability and key genomic regions. CRISPR systems and resistance genes provide evidence of the strain’s strong ability to stand and adapt, suitable for probiotic usage. Resistance genes including vanG, vanY and vanT, associated with vancomycin resistance clusters, were identified in the genome. The presence of these genes represents a potential biosafety concern due to the possible risk of horizontal gene transfer within the gastrointestinal microbiota. Therefore, despite exhibiting stress tolerance and biosynthetic gene clusters, the strain should not be considered a probiotic candidate without extensive safety and pathogenicity assessments. Figure 3.6 . Circular graphical representation of Bacillus altitudinis strain JF-5genome. The first external ring of the circular graphic displays Bacillus altitudinis ORFs and all other circular levels start from the inside out. The third ring presents the CRISPR-Cas cluster, while the fourth ring shows AMR genes between the CRISPR-Cas rings. GC content variation data is visualized in the fifth ring followed by GC skew data (green and purple) in the inner ring. The genome displays the labeling of vanG, vanT and vanY vancomycin resistance genes in the clusters. 3.6.1 Genome mining for biosynthetic gene clusters (BGCs) AntiSMASH analysis confirms B. altitudinis JF-5 can synthesize 10 secondary metabolites encoded by biosynthetic gene clusters. The genome of Bacillus altitudinis contains biosynthetic gene clusters that produce terpene and beta lactone compounds as well as T3PKS and NRPS pathways and RiPP-like secondary metabolites. The T3PKS cluster shows similarity to known polyketides pathways that are associated with antibiotics and anticancer agents and signaling molecules, among other bioactive compounds, however functional metabolite production was not experimentally validated in this study. One of the β-lactone clusters showed 53% similarity to fengycin biosynthetic cluster suggesting potential antifungal biosynthetic capabilities that requires experimental confirmation. The strain harbors multiple biosynthetic gee cluster, indicating potential capacity for secondary metabolite production but functional validation is pending. The genome mining analysis demonstrates that B. altitudinis contain several biosynthetic gene clusters, suggesting that Bacllius altitudinis JF-5 may serve as candidate for further investigation in natural product research, subject to experimental validation. Table 3.6 Identified biosynthetic gene cluster Region Type From To Most similar known cluster Similarity Region 1.1 Beta lactone 86,900 115,302 Fengycin 53% Region 1.2 Terpene 183,385 205,262 No similarity No similarity Region 1.3 T3PKS 243,439 284,536 No similarity No similarity Region 1.4 RiPP-like 597,041 607,367 No similarity No similarity Region 1.5 Beta lactone 754,632 778,081 No similarity No similarity Region 2.1 RRE-containing 8,534 29,439 No similarity No similarity Region 2.2 Terpene, NI-Siderophore 178,336 216,092 Schizokinen 60% Region 3.1 Other 308,636 350,056 Bacilysin 85% Region 6.1 NRPS-like,Sactipeptide, Ranthipeptide, RiPP-like 26,439 69,441 Sporulation killing factor 100% Region 6.2 NRPS 216,965 289,436 Lichenysin 85% Subsystem Coverage Subsystem Category distribution Subsystem Feature Counts Figure 3.6 . A summary of the PATRIC annotated subsystem gene distribution. The bar diagram illustrates subsystem coverage, whereas the pie chart depicts the distribution of subsystem features Table 3.6 .1 Genome assembly statistics of B. altitudinis Attribute Value Size 3,774,388 GC content 41.2 Number of contigs 36 Number of Subsystems 324 Number of coding sequences 3962 Number of RNA 83 3.6.2 Identification of genes associated with probiotic potential The presence of stress resistance genes such as yock,cspD, yhbH, cspB , and dps enables B. altitudinis to tolerate multiple abiotic stresses, including bile salts, heat, acidic pH, and oxidative stress. Moreover, DNA protection genes like recN, recO, dnaJ, dnaK, radA , and clpP ensure genomic stability and protein quality control under stress conditions, enhancing bacterial survival. Adhesion associated genes such as cwlS and ItaS1_1 , where identified which may support colonization however adhesion assays were not performed in this study. Immune modulation is facilitated by genes like pdaC_1 , which modify cell wall components to aid immune evasion and require functional validation. Table 3.6 Probiotic genes identified in B. altitudinis Gene name Response Locus Tag Stress Resistance Genes yock General stress protein 297074_JF5_00173 cspD Cold shock protein 297074_JF5_00250 yhbH Stress response protein 297074_JF5_00862 cspB Cold shock protein 297074_JF5_00867 nhaX Stress response protein 297074_JF5_00918 yhaX Stress response protein 297074_JF5_00932 ydaD_1 General stress protein 297074_JF5_00978 ykoL Stress response protein 297074_JF5_01253 yug1 General stress protein 297074_JF5_01700 yceC Stress response protein 297074_JF5_02464 YceD_1 General stress protein 297074_JF5_02465 YceD_2 General stress protein 297074_JF5_02466 srkA Stress response Kinase A 297074_JF5_02627 yocM Salt stress-responsive protein 297074_JF5_02668 yfIT General stress protein 297074_JF5_02681 yfkM General stress protein 297074_JF5_02716 yvgO Stress response protein 297074_JF5_02736 farB Fatty acid resistance protein 297074_JF5_03078 ydaD_2 General stress protein 297074_JF5_03114 ydaG General stress protein 297074_JF5_03115 cspC Cold shock protein 297074_JF5_03218 yceD_3 General stress protein 297074_JF5_03666 Dps General stress protein 297074_JF5_03671 Ctc General stress protein 297074_JF5_03816 csaA Putative chaperone 297074_JF5_00153 ctpA Carboxy-terminal processing 297074_JF5_00202 bepA_1 Beta-barrel assembly-enhancing 297074_JF5_00309 prsW Protease 297074_JF5_00346 recN DNA repair protein 297074_JF5_00474 gluP-1 Rhomboid protease 297074_JF5_00536 recO DNA repair protein 297074_JF5_00578 dnaJ Chaperone protein 297074_JF5_00597 dnaK Chaperone protein 297074_JF5_00598 hemW Heme chaperone 297074_JF5_00601 Gpr Germination protease 297074_JF5_00605 lon1 Protease 297074_JF5_00797 lon2 Protease 297074_JF5_00798 Clpx Protease ATP-binding subunit 297074_JF5_00799 htpX_1 Protease 297074_JF5_00973 Vclp Petrobactin import ATP-binding protein 297074_JF5_00989 htpX_2 Protease 297074_JF5_00998 htrA_1 Seine Protease 297074_JF5_01209 Isp Major intracellular serine protease 297074_JF5_01240 htrA_2 Serine protease 297074_JF5_01321 prtV Pre-pro-metalloprotease 297074_JF5_01427 Vpr Minor extracellular protease 297074_JF5_01551 yraA Putative cysteine protease 297074_JF5_01681 htrB Serine protease 297074_JF5_01838 copZ Copper chaperone 297074_JF5_01903 clpP Protease proteolytic subunit 297074_JF5_01981 bepA_2 Beta-barrel assembly-enhancing protease 297074_JF5_01996 Fra Intracellular iron chaperone frataxin 297074_JF5_02009 aprX Serine protease 297074_JF5_02049 rasP Regular sigma-W protease 297074_JF5_02112 clpY Protease ATPase subunit 297074_JF5_02153 clpQ Protease subunit 297074_JF5_02154 surA Chaperone 297074_JF5_02370 epr_1 Minor extracellular protease 297074_JF5_02372 wprA Cell-wall associated protein 297074_JF5_02417 epr_2 Minor extracellular protease 297074_JF5_02429 stiP Cysteine protease 297074_JF5_02470 MroQ Membrane-embedded protease 297074_JF5_02558 paiB Protease synthase and sporulation protein 297074_JF5_02562 ctpB Carboxyl-terminal processing protease 297074_JF5_02856 fliS Flagellar secretion chaperone 297074_JF5_02871 yciC Putative metal chaperone 297074_JF5_02997 gluP_2 Rhomboid protease 297074_JF5_03153 htpX_3 Protease 297074_JF5_03454 clpE Protease ATP-binding subunit 297074_JF5_03476 radA DNA repair protein 297074_JF5_03778 clpC Protease regulator 297074_JF5_03779 ftsH Zinc metalloprotease 297074_JF5_03797 yabG Sporulation-specific protease 297074_JF5_03825 Adhesion and Colonization cwlS Cell wall hydrolase helps remodeling and colonization 297074 _JF5 _00188 ItaS1_1 Lipoteichoic acid synthesis facilitates adhesion and immune interaction 297074 _JF5 _00533 Immune Modulation pdaC_1 Cell wall component modification contributes to immune evasion 297074 _JF5 _00637 Antimicrobial Resistance Genes merR1_1 Metal resistance regulator supports detoxification 297074 _JF5 _00059 bmr3 Multidrug efflux transporter bile and drug resistance 297074 _JF5 _00086 norm Multidrug transporters help resist bile salts and antibiotics 297074 _JF5 _00191 Metabolic Adaptation Genes cdaR_1 Regulate carbohydrate metabolism and aids survival in gut environment 297074 _JF5 _00112 3.6.3. Average nucleotide identity OrthoANI heatmap analysis identified specific genomic similar clusters among the analyzed bacterial strains. The close genetic correlation (high ANI values marked in red) between GR- 8, ECC22, ZAP62, and OCA19 suggests that these should be species of the same or closely related species subtypes. LZP02 and F2 were also moderately to highly like this cluster, implying a distinction in the same genus. The JF-5 strain exhibited a high similarity especially to GR-8, ECC22, since these zones are light red, indicating that JF-5 and these strains are closely related strains and may share the same or very closely related species. JF-5 too was moderately like ZAP62 and OCA19 (yellow areas) but it had a lower similarity with LZP02 and F2. Strain 3P01AB and 9432a1 on the other hand, with all other strains including JF-5 displayed predominantly green patterns, suggesting ANI values (< 90%), indicating they are genomic wise distant and probably belong to other species or genera. These results emphasize that JF-5 is most genetically like GR-8 and ECC22, which places it within a genetically related cluster, but is in also marked distinction of the more peripheral strains. 3.6.4. Comparative proteome Analysis Comparison of genomes in this study consisted of 5 bacterial strains, NBTC-002, SGAir0031, GR-8, HM-7 and JF-5, to determine the core, accessory and unique gene content in the genome. It is obvious through the Venn diagram that the number of shared gene clusters across each strain is 3,345, which is the core genome; these gene compositions presumably encode some vital functions that have been conserved with rapid evolution. Conversely, the strains similarly each contain a unique set of gene clusters and gene families that are not found in any other strain, suggesting strain-specific adaptations or niche specialization. Of these, 138 unique gene clusters are in NBTC-002, 91 in JF-5, 25 in SGAir0031, 23 in GR-8, 5 in HM-7, which indicates different genomic diversity among strains. The studied diversification of distinctive genes can be asymmetrical with adaptation to various environmental conditions, resistance to antimicrobial agents, or distinctive metabolic processes. More details can be found in the bar graph demonstrating the total number of protein-coding genes, clusters of genes, and singletons (strain-specific genes) per strain. JF-5 exhibited the greatest gene clusters (3,686) and proteins (3,982), implying a more complex or expanded genome. On the other hand, HM-7 also has also had 3,601 gene clusters, but it has the greatest number of singletons (305), which shows that it has quite a large proportion of novel or divergent genes. The fact that NBTC-002 and GR-8 also have a rather high number of singletons (299 and 308, respectively) further highlights the uniqueness in the genomes of each of these strains compared to each other, despite the greater overall similarity. An Upset plot was applied as a more scalable representation of Venn diagrams to further break down the gene-sharing patterns. The plot shows the largest vertical bar at 3,345 shared clusters again validating the core genome. The smaller intersections reflect the gene groups that are common in two, three, or four strains. Such visualization is essential to learn about the conservation of genes, their distribution and uniqueness in strains. The bar plot below depicts the strain combinations present at each intersection of the gene clusters in a more detailed way due to the connected dots beneath the bars. Interestingly, several modest-to large intersection points are shared genes between subsets of strains, each of which may represent a potential shared ecological activity or horizontal gene transfer event. Together, this revaluation indicates that although a vast amount of the genome has been kept equal in these strains, each strain still contains distinctive genomic elements. Particularly help to understand the diversity of bacteria, phylogenetic relations and functional capacity, particularly about strain-specific roles in microbial communities, prospects of developing probiotics or living in environments. 3.6.5. Core pan genome analysis The core genome analysis reveals significant genetic diversity among the studied bacterial strains. The plot shows that the pan genome increases with the addition of more genomes, indicating an open genome structure where new genes continue to emerge. In contrast, the core genome decreases, suggesting that fewer genes are universally shared across all strains. This pattern reflects a high level of genomic variability and adaptability, likely due to horizontal gene transfer or ecological diversity. Overall, the results highlight the evolutionary flexibility and rich gene content of the bacterial species B. altitudinis . In Fig A presents the distribution of gene families across the 67 genomes. A bimodal pattern is evident: many gene families are either found in nearly all genomes (core genes) or in very few genomes (accessory or strain-specific genes). This reflects the genetic landscape where a flexible accessory genome complements a stable core genome. The presence of gene families unique to only one or a few strains, such as those specific to JF-5, underscores its genetic distinctiveness and possible ecological specialization. In Fig. B , the number of new genes per genome is shown. The graph demonstrates a steep decline in novel gene discovery as additional genomes are included in the analysis. This suggests that early genomes contribute a high number of unique genes to the pan-genome, while later genomes, including JF-5, add fewer. Such a pattern is indicative of an open pan- genome, which may be approaching saturation. The strain JF-5 contributes a moderate number of unique genes compared to other strains, indicating its potential for harboring novel functions or adaptations. 4. Phylogenetic analysis The core-genome phylogenetic tree was annotated using NCBI Prokaryotic Genome Annotation Pipeline. The genome was deposited in GenBank under accession numbers JBRIIV000000000. In this analysis, the strain of interest clustered within a distinct clade along with closely related B. altitudinis strains, including 40-NJ-V2.faa, 19-AJ-V2.faa, and 17- BC255T1.faa, indicating a high degree of genomic similarity. These strains formed a coherent group in one of the major color-coded clades in the circular phylogenetic layout, suggesting shared evolutionary lineage and conserved functional traits. The closest relative to the studied strain appears to be 40-NJ- V2.faa, which shares a highly similar core genome and may exhibit comparable ecological or antimicrobial properties. In contrast, more distantly related strains, such as 20-CES-OCA- 12.faa and 21-CGMC-3072.faa, were positioned in separate clades, reflecting greater genomic divergence. This phylogenetic placement highlights the evolutionary proximity of the studied strain to specific members of the B. altitudinis group and supports its classification within a genetically cohesive cluster. 5. Discussion Probiotics are described as living organisms which provide health benefits to the host when taken in sufficient quantity; but the classification of probiotics must be strictly evaluated in terms of safety, gastrointestinal survivability, functional activity and lack of transposable antibiotic resistance or virulence factors. ( 23 ). Fermented foods are known to be valuable sources of potentially useful bacteria, such as those of the genus Bacillus , which have been receiving growing interest because of their sporulating ability and ability to survive in the environment. Bacillus spp. endospores enable a considerable survival benefit in severe environments, including heat, desiccation and extreme pH, admittedly further increasing stability during food processing and possibly enabling potential survival via gastric transit. However, spores cannot be called probiotics and thorough phenotypic, genomic and safety validation is required before a single strain can be deemed appropriate to be used by humans. ( 24 ). The present work filtered 25 locally fermented yogurt samples to isolate Bacillus strains that had the potential of being used as a probiotic. It is not an epidemiological study and is a very initial exploratory sampling. In vitro screening assays, such as acid tolerance, bile salt tolerance, enzyme profiling, antimicrobial activity testing, hemolysis, DNase production, antibiotic susceptibility and the entire genome sequencing were performed on isolates ( 25 ). A number of the isolates were found to survive at acidic and bile salt conditions indicating that it was able to withstand simulated gastrointestinal stress. Enzyme activities of proteolytic, amylolytic and lipolytic activity in the selected strains were detected by enzymatic assays, which could play a role in supportive digestion and nutrient degradation. Desirable as these features are in probiotic candidates, it is important to note that the functionality of probiotics in vitro is not directly proportional to clinical efficacy ( 26 ). The most important determinant in the development of probiotics is safety assessment. The preliminary virulence indicators were hemolytic activity and DNase production. Strain JF-5 was negative in the hemolytic and DNase tests indicating a relatively good safety profile. Conversely, JY-2 strain was found to have hemolytic and DNase activity, which is a possible safety issue and eliminates it as a probiotic ( 27 ). One of the biosafety problems that were found during the research was the presence of vancomycin-associated resistance genes (vanG, vanY, vanT) in strain JF-5. Such antimicrobial resistance determinants show serious biosafety concern because of potential for horizontal gene transfer is a possible risk and cannot be viewed as positive. Thus, JF-5 cannot be considered a safe choice of probiotic. On the contrary, strain JF-5 was resistant to the majority of available antibiotics and lacked clinically significant resistance determinants, in favor of its relatively safe genomic profile ( 28 ). Whole-genome sequencing placed strain JF-5 as Bacillus altitudinis . The comparative genomic and pan-genome studies identified core genes that are conserved and are related to metabolism, stress response, DNA repair and sporulation. AntiSMASH and BAGEL in silico genome mining identified biosynthetic gene clusters that could produce antimicrobial compounds like bacillysin, fengycin, lichenysin, and siderophores with up to possible ecological competitiveness and antimicrobial activity ( 29 ). Genomic prediction on its own however does not establish active production of metabolites and must be proven by experiment to be functionally expressed under physiological conditions. The discovery of stress resistance genes, adhesion genes, and immune related genes hence should be understood as genetic possibilities but not proven probiotic features ( 30 ). It should be noted that some of the key probiotic validation assays were omitted in this study such as epithelial cell adhesion models (e.g., Caco-2 or HT-29 assays), immunomodulatory profiling, in vivo animal analysis, plasmid profiling, horizontal gene transfer analysis and detailed analysis of virulence factor and pathogenicity islands. In turn, although strain JF-5 exhibits potentially attractive in vitro properties and its genomic profile is relatively good, it can be viewed as a pre-probe probiotic strain that needs additional functional confirmation, as opposed to a proven probiotic strain. Conclusion The aim of the current research was to isolate and characterize Bacillus species of locally fermented yogurt and to assess their initial probiotic-related attributes by microbiological and genomic methodologies. Two of the 25 isolates (JF-5 and JY-2) were characterized in more detail. Strain JF-5 showed acid and bile tolerance, enzymatic activities, no hemolytic and DNase activity and sensitivity to the majority of the antibiotics which were tested. Whole- genome sequencing revealed the strain as belonging to a Bacillus altitudinis and genomic analysis showed that the strain had genes related to stress response, sporulation, adhesion- related systems and predicted antimicrobial biosynthetic clusters. Strain JY-2 on the other hand was hemolytic, DNase positive. These discoveries are also important biosafety issues, and thus JY-2 cannot be a probiotic contender. Genomic comparison of JF-5 showed that there are core genes, which are conserved and associated with metabolic stability and stress tolerance. The presence of probiotic associated genes however does not verify the functional probiotic efficacy. There are functional assays, which are epithelial adhesion, immunomodulation, in vivo safety validation and horizontal gene transfer risk assessment that are needed prior to absolute probiotic classification. This study is a genome-wide description of Bacillus altitudinis which was found in fermented yogurt in Pakistan. Instead of stating definite probiotic status we make a suggestion that Bacillus altitudinis JF-5 should be viewed as a promising native candidate that can be explored in the future. Future work should include : Cell line adhesion assays Animal model validation in vivo. Rigorous screening in areas of virulence and pathogenicity. Resistance genes transferability. Clinical and toxicological safety investigations. It is only upon these tests that the strain can be assured to be developed into nutraceutical or industrial probiotics. Declarations Conflicts of Interest: No conflict of interest is declared by the authors. Consent to Participate , Consent to Publish and Ethics : Human blood used in hemolysis assays were obtained from certified local blood bank. No human subjects were directly involved. Funding: This research received no external funding. Data Availability: Whole genome shotgun sequencing data of Bacillus altitudinis strain produced in this study were deposited in the NCBI GenBank database under accession number JBRIIV000000000. These BioProject and BioSample accession numbers are PRJNA1335593 and SAMN52016192, respectively. The NCBI Prokaryotic Genome Annotation Pipeline (PGAP) was used to conduct the genome annotation. The GenBank record is publicly available at https://www.ncbi.nlm.nih.gov/nuccore/JBRIIV000000000 . 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Int J Life Sci Biotechnol 7(2):120–132 Jaiswal P, Srivastav AK (2021) A study on the assessment of the antimicrobial susceptibility pattern of microorganisms present in yogurt. Annals Romanian Soc Cell Biology 25(6):15171–15183 Wu T, Guo S, Kwok L-Y, Wang J, Zhang H (2025) Characterization of Growth and Metabolomic Profiles of Lacticaseibacillus paracasei ProSci-92 as a Potential Probiotic Culture in Extended Milk Fermentation. J Dairy Sci Liu C, Xue W-j, Ding H, An C, Ma S-j, Liu Y (2022) Probiotic potential of Lactobacillus strains isolated from fermented vegetables in Shaanxi, China. Front Microbiol 12:774903 Hudson LK, Orellana LAG, Bryan DW, Moore A, Munafo JP, den Bakker HC et al (2021) Phylogeny of the Bacillus altitudinis Complex and Characterization of a Newly Isolated Strain with Antilisterial Activity. J Food Prot 84(8):1321–1332 Proal AD, Lindseth IA, Marshall TG (2017) Microbe-microbe and host-microbe interactions drive microbiome dysbiosis and inflammatory processes. Discov Med 23(124):51–60 Additional Declarations The authors declare no competing interests. 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-8865594","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":590504722,"identity":"c0dd17c9-8253-496a-a238-a6b86cc22032","order_by":0,"name":"Janeeta 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University","correspondingAuthor":false,"prefix":"","firstName":"Sajid","middleName":"","lastName":"Iqbal","suffix":""},{"id":590504727,"identity":"bb4a5820-36f3-44fc-b760-37274e77ad2a","order_by":5,"name":"Fasiha Haider","email":"","orcid":"","institution":"Abasyn university Islamsbad Campus","correspondingAuthor":false,"prefix":"","firstName":"Fasiha","middleName":"","lastName":"Haider","suffix":""},{"id":590504728,"identity":"e285b5c5-dcf6-4db2-926d-c8acdab394b8","order_by":6,"name":"Tashfeen Tariq","email":"","orcid":"","institution":"Abasyn university Islamabad Campus","correspondingAuthor":false,"prefix":"","firstName":"Tashfeen","middleName":"","lastName":"Tariq","suffix":""}],"badges":[],"createdAt":"2026-02-12 20:59:21","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-8865594/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8865594/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102744195,"identity":"75d7b0c6-9e55-409a-bd1a-9af725c89312","added_by":"auto","created_at":"2026-02-16 08:27:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":18397,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 3.3 \u003c/strong\u003eAntibiotic susceptibility test (AST) of \u003cem\u003eBacillus altitudinis strains \u003c/em\u003eJF-5 and JY-2.\u003c/p\u003e\n\u003cp\u003eBar graphs showing the zones of inhibition (mm) of strains JF-5 and JY-2 against selected antibiotics, indicating their susceptibility profile.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8865594/v1/e867f14fb6d69b8c3dc90680.png"},{"id":102749267,"identity":"461bb436-a78a-44a2-879a-483ccf8092b5","added_by":"auto","created_at":"2026-02-16 09:12:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":97257,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 3.4: (a)\u003c/strong\u003eGrowth behavior of \u003cem\u003eB. altitudinis J\u003c/em\u003eF-5 and JY-2 to varying pH (pH 5, pH 7 and pH 9) and \u003cstrong\u003e(b) \u003c/strong\u003etemperature (30 \u003csup\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/sup\u003eC, 37 \u003csup\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/sup\u003eC and 50 \u003csup\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/sup\u003eC).\u003c/p\u003e\n\u003cp\u003eBoth strains showed maximum growth at neutral pH (pH 7) and 30 \u003csup\u003eo\u003c/sup\u003eC and less at other acidic, alkaline and high temperatures.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8865594/v1/0e4cb34be8e65164320fc5ad.png"},{"id":102744201,"identity":"c2b7c7b8-7d20-4988-8880-a13a1a2ac8b2","added_by":"auto","created_at":"2026-02-16 08:27:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":592903,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 3.5 \u003c/strong\u003e(a) Negative hemolysis activity of strain JF-5. (b) Negative DNase activity of strain JF-5.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8865594/v1/d560c90bef8f7afe7eeae012.png"},{"id":102744197,"identity":"23b5c495-58d1-4707-a6a5-2bf67374ac76","added_by":"auto","created_at":"2026-02-16 08:27:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":709466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 3.6. \u003c/strong\u003eCircular graphical representation of \u003cem\u003eBacillus altitudinis \u003c/em\u003estrain JF-5genome. The first external ring of the circular graphic displays \u003cem\u003eBacillus altitudinis \u003c/em\u003eORFs and all other circular levels start from the inside out. The third ring presents the CRISPR-Cas cluster, while the fourth ring shows AMR genes between the CRISPR-Cas rings. GC content variation data is visualized in the fifth ring followed by GC skew data (green and purple) in the inner ring. The genome displays the labeling of vanG, vanT and vanY vancomycin resistance genes in the clusters.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8865594/v1/a02baf467f583c12a3a0c5cb.png"},{"id":102749410,"identity":"e79786ef-0b8c-42ae-91ea-2deaa346074f","added_by":"auto","created_at":"2026-02-16 09:12:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":138355,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 3.6. \u003c/strong\u003eA summary of the PATRIC annotated subsystem gene distribution. The bar diagram illustrates subsystem coverage, whereas the pie chart depicts the distribution of subsystem features\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8865594/v1/3a357071d2a6bbee61d75761.png"},{"id":102748775,"identity":"9080f791-313e-4a50-b446-a660c77cf2d7","added_by":"auto","created_at":"2026-02-16 09:11:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":235895,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 5.3 \u003c/strong\u003eOrthoANI heatmap indicating genomic relatedness between strains of bacteria. Red is used to show high ANI values (strong similarity), green is low ANI values (distant relationship), and yellow indicates moderate similarity.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8865594/v1/2e804749e9b0265f1ec63941.png"},{"id":102744199,"identity":"dc9f6b68-ed88-4e41-af2c-c1dec2f8d7ae","added_by":"auto","created_at":"2026-02-16 08:27:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":443490,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 6.0 \u003c/strong\u003eVenn diagram showing strain GR-8 \u003cem\u003eBacillus altitudinis \u003c/em\u003especific and shared orthologous and shared orthologous gene clusters with the genome of \u003cem\u003eBacillus altitudinis \u003c/em\u003estrains SGAir0031, NBTC-002, JF-5 and HM-7.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8865594/v1/f32f1387c6108eb8f94b05ab.png"},{"id":102751238,"identity":"f9941704-7f19-4d52-97bd-ce440d01cf0b","added_by":"auto","created_at":"2026-02-16 09:24:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4079024,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8865594/v1/8a565379-d926-4162-9d4b-6d27b6edc8aa.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eIn vitro Screening and Genome-wide Analysis of Probiotic Bacillus spp.from Locally Fermented Yogurt for Preliminary Probiotic Potential\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eProbiotics are active microorganisms that enhance the health of the host once taken in adequate amounts. Even though probiotic preparations traditionally consisted of lactic acid bacteria such as \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBifidobacterium, Bacillus\u003c/em\u003e spores have been of greater interest due to their outstanding environmental resistance and ability to withstand severe processing factors. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). \u003cem\u003eBacillus\u003c/em\u003e spores are also good choices in industrial applications and oral delivery systems because they are resistant to heat, dryness and acidic environment (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere are also these advantages, but the safety and functional effectiveness of the \u003cem\u003eBacillus-\u003c/em\u003e based probiotics should be evaluated. The recent discoveries in the field of whole-genome sequencing (WGS) have improved the process of the evaluation of the probiotic candidates significantly (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Genome-wide analysis can predict genes associated with antimicrobial compound biosynthesis stress resistance, sporulation and metabolic functions; however, genomic prediction does not necessarily indicate functional expression or probiotic efficacy (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eMeanwhile, the potential presence of biosafety risks is identifiable through genomic screening (e.g., antibiotic resistance gene, virulence factor and pathogenicity-related determinants). Notably, the presence of probiotic-related genes in the genome does not determine the presence of probiotic activity, though, it provides the details of the genetic potential, which is to be substantiated with help of the phenotypic confirmation. The fermented foods are a nutritious\u003c/p\u003e \u003cp\u003esource of potentially valuable microbes which are diverse. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eLocal isolates of such foods can have special adaptive properties, but systematic genomic characterization of locally isolated \u003cem\u003eBacillus\u003c/em\u003e strains has not been mostly undertaken. Moreover, genomic analysis of these isolates in terms of safety must come first, prior to their use in food or therapeutic preparations (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Despite the availability of a number of commercial probiotic products across the world, little published information exists on genomic and biosafety profiling of native \u003cem\u003eBacillus\u003c/em\u003e isolates of fermented foods in Pakistan (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). An organized methodology that combines in vitro screening and genome-wide analysis can deliver background data about the traits associated with probiotics and the possible safety risks. This is preliminary characterization required as a first step before further functional, clinical or in vivo validation (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThus, this research was aimed at isolating \u003cem\u003eBacillus\u003c/em\u003e spp. in locally fermented yogurt and conducting initial in vitro screening along with whole-genome sequencing to assess the probiotic-related features and genomic biomarkers of biosafety. This paper does not identify functional probiotic efficacy, but it gives first phenotypic and genomic data that will be useful in future validation and safety evaluation investigations. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Materials \u0026 Methods","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eNo in vivo experiments were performed in this research, all evaluations are preliminary in vitro and genomic analyses.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Sample Collection\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe current study was designed to isolate potential probiotic candidates from locally fermented yogurts. For this purpose, 5 g samples (n\u0026thinsp;=\u0026thinsp;25) were collected from locally fermented yogurt from local units (small shops selling yogurt) in Rawalpindi and Islamabad capital territory.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Isolation, Purification and Identification of bacterial isolates\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAll the samples were immediately transported to the BJ Micro Lab and processed for bacterial isolation. Two potential \u003cem\u003eBacillu\u003c/em\u003es strains were isolated from yogurt and were evaluated for probiotic potential. After incubating samples at 37\u003csup\u003eo\u003c/sup\u003eC for 24 hours, bacterial colonies appeared on the Brain Heart Infusion media petri plates. The purification of the colonies was done by using the steak plate method.\u003c/p\u003e \u003cp\u003eMorphological identification was done using Gram staining, motility test and spore formation test. The isolated strains were preliminarily identified using the biochemical tests including catalase, oxidase, VP, TSI, and citrate tests (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Screening assays\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe screening for probiotic potential was determined by the Lipolytic test, amylolytic test, proteolytic test and bile tolerance test according to the procedures and precautions previously published(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Bacterial growth was observed by the formation of colonies on the agar plates.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Antibiotic susceptibility test (AST)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eInoculum was prepared by using a sterile inoculating loop or needle; four or five isolated colonies of the organism to be tested were touched and suspended in 2 mL of normal saline. The saline tube was vortexed to create a smooth suspension. Next, the turbidity of this suspension was adjusted to a 0.5 McFarland standard by adding more culture suspension if the suspension was too light or diluting with sterile saline if the suspension was too heavy. The petri plates are ready with MHA agar (Accumix, India) and will solidify in the plates. A cotton swab was dipped in the cell suspension and streaked on the petri plates repeatedly. The antibiotic discs were firmly placed on the plate (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Tolerance to different pH\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eNutrient broth (HI, India) was prepared, and pH was adjusted to pH 2, pH 7, pH 9 using dilute HCL and NaOH. and inoculated with bacterial isolates JF-5 and JY-2, incubated at 37\u0026deg;C for 24 hrs. Bacterial growth was assessed by measuring it using a spectrophotometer at 600nm.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Tolerance to different temperatures\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eNutrient broth (HI, India) was prepared and inoculated with bacterial isolates JF-5 and JY-2 and were incubated at the different temperature (30\u0026deg;C, 37\u0026deg;C and 50\u0026deg;C) for 24 hours.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3. Hemolysis and DNase activity\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBrain Heart Infusion agar powder (HI, India) was dissolved in distilled water as per the manufacturer\u0026rsquo;s protocol. And the medium was autoclaved at 121\u0026deg;C for 15 minutes. After autoclaving, the medium was cooled to 55\u0026deg;C, and then 5% of the whole human blood was added to the agar. After it was mixed well and poured onto plates. The bacteria were streaked on the plates and then incubated at 37\u0026deg;C overnight.\u003c/p\u003e \u003cp\u003eDNase agar powder (HI, India) was dissolved in distilled water, and the mixture was brought to a boil to ensure complete dissolution. The medium was then autoclaved at 121\u0026deg;C for 30 minutes for sterilization. After autoclaving, the agar was allowed to cool slightly, mixed thoroughly, and poured into sterile petri plates. Isolates were streaked onto the solidified agar, and the plates were incubated at 37\u0026deg;C overnight.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.5. DNA extraction and next-generation sequencing\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. DNA extraction\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA total of 1 \u0026micro;L cell suspension was lysed with 120 l of TE buffer containing lysozyme (MPBio, USA), metapolyzyme (Sigma Aldrich, USA), and RNase A (ITW Reagents, Spain), and incubated for 25 minutes at 37\u0026deg;C. Proteinase K (VWR Chemicals, Ohio, USA) (final concentration 0.1 mg/mL) and SDS (Sigma-Aldrich, Missouri, USA) (final concentration 0.5% v/v) were added, and the mixture was incubated for 5 minutes at 65\u0026deg;C. Genomic DNA was purified using an equal volume of SPRI beads and resuspended in EB buffer (10 mM Tris-HCl, pH 8.0). The extracted DNA was quantified using the Quant-iT dsDNA HS assay (Thermo Fisher Scientific) in an Eppendorf AF2200 plate reader (Eppendorf UK Ltd, United Kingdom) and diluted as appropriate.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. Next generation sequencing, assembly and annotation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGenomic DNA libraries were prepared using the Nextera XT Library Prep Kit (Illumina, San Diego, USA) following the manufacturer\u0026rsquo;s protocol with the following modifications: input DNA was increased 2-fold, and PCR elongation time was increased to 45 seconds. DNA quantification and library preparation were carried out using a Hamilton Microlab STAR automated liquid handling system (Hamilton Bonaduz AG, Switzerland). Libraries were sequenced on an Illumina NovaSeq 6000 (Illumina, San Diego, USA) using a 250 bp paired- end protocol. Whole-genome sequencing and library preparation were performed by MicrobesNG Lab, Birmingham, United Kingdom. Reads were adapter-trimmed using Trimmomatic version 0.30 with a sliding window quality cutoff of Q15. De novo assembly was performed using SPAdes version 3.7, and contigs were annotated using Prokka 1.11 (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Phylogenetic analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePhylogenetic Analysis of the isolated strain \u003cem\u003eBacillus. altitudinis\u003c/em\u003e was generated based on core- genome and whole-genome analysis (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Genome-wide analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo visualize the genomic feature of the \u003cem\u003eBacillus. altitudinis\u003c/em\u003e strain JF-5, a web-based Proksee server \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://proksee.ca/\u003c/span\u003e\u003cspan address=\"https://proksee.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e was employed. While the CRISPR Finder was used to detect the presence of the CRISPR cas sequence. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1. Genome mining for biosynthetic gene clusters (BGCs) and bacteriocins and probiotics\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe antiSMASH bacterial version was used to identify biosynthetic gene clusters BGCs in \u003cem\u003eB. altitudinis.\u003c/em\u003e (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Bacteriocin gene clusters were identified using the BAGEL (Bacteriocin Genome mining Tool) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bagel4.molgenrug.nl/\u003c/span\u003e\u003cspan address=\"http://bagel4.molgenrug.nl/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e platform, which specializes in detecting putative bacteriocin operons in bacterial genomes. Probiotic-associated genes involved in stress resistance, adhesion, immune modulation, and metabolic adaptation were identified through a whole-genome annotation process using Prokka v1.14.6 (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2. Comparative genome analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe subsystem feature categories of \u003cem\u003eB. altitudinis\u003c/em\u003e with a comparison of reference genome strain GR-8 were determined using a Rapid Annotation Subsystem Technology (RAST) server. Comparative proteome analysis of \u003cem\u003eBacillus altitudinis\u003c/em\u003e strain JF-5 with closely related. Genomes, including the reference \u003cem\u003eBacillus altitudinis\u003c/em\u003e GR-8 genome, was conducted using Ortho Venn analysis. (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://orthovenn3.bioinfotoolkits.net/\u003c/span\u003e\u003cspan address=\"https://orthovenn3.bioinfotoolkits.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.7.3. Core pan genome analysis\u003c/h2\u003e \u003cp\u003eTo investigate genomic diversity and identify the core and accessory genome components, we performed a comprehensive core and pan-genome analysis using the Bacterial Pan Genome Analysis (BPGA) tool. (https://iicb.res.in/bpga/). \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Average nucleotide identity\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eANI analysis was performed using the EZBioCloud online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ezbiocloud.net/tools/ani\u003c/span\u003e\u003cspan address=\"https://www.ezbiocloud.net/tools/ani\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e Genomic FASTA files were uploaded, and the OrthoANI algorithm was used to calculate pairwise nucleotide identity and pairwise nucleotide was shown in heatmap. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA total of two bacterial strains were isolated from (n\u0026thinsp;=\u0026thinsp;25) yogurt samples and showed their growth on Brain Heart Infusion media plates and were labelled as JF-5, JY-2. Out of 25 samples, 13 were found to be Gram-negative. Nine samples displayed cocci morphology under the microscope, while two samples were identified as Gram-positive rods. Spore staining test for both isolates (a) JF-5 and (b) JY-2 under the microscope, showing the presence of rod- shaped bacterial cells with visible endospores.\u003c/p\u003e \u003cp\u003eCatalase and oxidase tests show positive reactions toward both strains; VP shows positive reaction toward both strains. Both strains were motile and indole negative, citrate test shows positive reaction toward both strains and for TSI results show that strain JF-5 showed a red slant and yellow bottom, indicating fermentation of glucose only, with no lactose or sucrose fermentation. Strains were evaluated for preliminary probiotic-associated traits in vitro, Functional probiotic activity was not tested in vivo.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Screening activity\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBoth strains JF-5 and JY-2 showed positive amylolytic activity and zones appeared on the starch media plates. Strain JF-5 and JY-2 showed positive proteolytic activity, and clear zones appeared on the media.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Antibiotic susceptibility test (AST)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAntibiotic susceptibility test was used to investigate the resistance or susceptibility profile of bacterial strains against multiple antibiotic drugs. JF-5 and JY-2 were susceptible to Amoxicillin and clavulanic acid, Amikacin, Chloramphenicol and Penicillin.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBar graphs showing the zones of inhibition (mm) of strains JF-5 and JY-2 against selected antibiotics, indicating their susceptibility profile.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Stress Tolerance\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe survival of \u003cem\u003eBacillus altitudinis\u003c/em\u003e strains JF-5 and JY-2 in various pH levels (pH 5, pH 7, and pH 9). Survival was highest at neutral pH (pH 7) for both strains, at acidic pH (pH 5), the survival rate was reduced dramatically, showing that they have low acid tolerance. Both strains exhibited no growth at alkaline conditions (pH 9). The findings indicated that \u003cem\u003eBacillus altitudinis\u003c/em\u003e strains grow best under neutral conditions. Growth rates of two strains of \u003cem\u003eBacillus altitudinis\u003c/em\u003e (JF-5 and JY-2) in various temperatures between 37 \u003csup\u003eo\u003c/sup\u003eC, 30 \u003csup\u003eo\u003c/sup\u003eC and 50 \u003csup\u003eo\u003c/sup\u003eC. The two strains also display the maximum growth rates at 30 \u003csup\u003eo\u003c/sup\u003eC. On further rise of the temperature to 37 \u003csup\u003eo\u003c/sup\u003eC, a slight decrease in growth is seen in both the strains. Both strains grow best at 30 \u003csup\u003eo\u003c/sup\u003eC. A significant reduction in growth at 50 \u003csup\u003eo\u003c/sup\u003eC indicates the inhibitory effect of high temperature on both strains.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBoth strains showed maximum growth at neutral pH (pH 7) and 30 \u003csup\u003eo\u003c/sup\u003eC and less at other acidic, alkaline and high temperatures.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5. DNase test and Hemolytic activity\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThis test was used to determine the presence or absence of deoxyribonuclease enzyme in the bacterial Isolates. The enzymatic potential of this enzyme showed the hydrolysis of genomic DNA into multiple pieces. Results of this test were observed by the formation of clear zones around the bacterial colonies. Strain JF-5 showed negative DNase activity, and no clear zones appeared on the media, while strain JY-2 showed positive DNase activity. The hemolysis test was used to determine the ability of bacterial isolates to lyse red blood cells. Results of this test were observed by the formation of clear zones around the bacterial colonies. Strain JY-2 showed positive hemolytic activity, and clear zones appeared on the media and strain JF-5 showed no zone and was recorded as hemolysis-negative.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Whole genome sequencing and annotation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe draft genome of \u003cem\u003eBacillus altitudinis\u003c/em\u003e JF-5 is 3,774,388 bp in size and contains 41.2% GC content. The genome comprises 36 contigs and harbors 324 subsystems, 3962 coding sequences, and 83 RNAs.\u003c/p\u003e \u003cp\u003eRAST subsystem prediction shows the genome of \u003cem\u003eB. altitudinis\u003c/em\u003e with comparison of \u003cem\u003eB.altitudinis\u003c/em\u003e GR-8. \u003cem\u003eB. altitudinis\u003c/em\u003e JF-5 contains 183 genes for protein metabolism while \u003cem\u003eB. altitudinis\u003c/em\u003e GR-8 has 201 genes for protein metabolism. Genes for stress response in the genome of \u003cem\u003eB. altitudinis\u003c/em\u003e JF-5 are 44, while \u003cem\u003eB. altitudinis\u003c/em\u003e GR-8 has 43 genes related to stress response. The presence of stress-related genes leads to probiotic potential. Furthermore, genes for amino acid metabolism contain 286 genes in \u003cem\u003eB. Altitudinis\u003c/em\u003e JF-5, while \u003cem\u003eB. altitudinis\u003c/em\u003e GR-8 has 266 genes (Fig.\u0026nbsp;5). The greater number of genes 247 in the \u003cem\u003eB.altitudinis\u003c/em\u003e strain JF-5 are specified for carbohydrate metabolism, and the \u003cem\u003eB. altitudinis\u003c/em\u003e GR-8 contains 219 genes for carbohydrate metabolism. \u003cem\u003eB. altitudinis\u003c/em\u003e JF-5 contains 55 genes for RNA metabolism, while \u003cem\u003eB. altitudinis\u003c/em\u003e GR-8 contains 54 genes. \u003cem\u003eB.altitudinis\u003c/em\u003e strain JF-5 and \u003cem\u003eB.altitudinis\u003c/em\u003e GR-8 have equal to 60 genes for DNA metabolism.\u003c/p\u003e \u003cp\u003eThe draft genome of \u003cem\u003eBacillus altitudinis\u003c/em\u003e strain JF-5 was visualized using Proksee, an online genome visualization tool. This map displayed the overall genome size, GC content, and GC skew (both positive and negative), providing a clear representation of nucleotide composition.\u003c/p\u003e \u003cp\u003eTo show important genomic features, a circular map of the \u003cem\u003eB. altitudinis\u003c/em\u003e JF-5 genome was constructed. The ORFs, which are the annotated coding parts of the genome, are seen on the outermost ring. A special CRISPR-Cas group was found inside the bacterium, showing it defends itself against attacks by bacteriophages with adaptive abilities.\u003c/p\u003e \u003cp\u003eApproximately the region containing CRISPR, the following resistance genes were found vanG, vanT and vanY which may provide protection against vancomycin. This means that the genes confer defense from glycopeptides. More rings in the tracks display GC content and GC skew, revealing information about the genome\u0026rsquo;s nucleotides and possible replication origins. Using GC content variation and skewing helps determine gene stability and key genomic regions. CRISPR systems and resistance genes provide evidence of the strain\u0026rsquo;s strong ability to stand and adapt, suitable for probiotic usage.\u003c/p\u003e \u003cp\u003eResistance genes including vanG, vanY and vanT, associated with vancomycin resistance clusters, were identified in the genome. The presence of these genes represents a potential biosafety concern due to the possible risk of horizontal gene transfer within the gastrointestinal microbiota. Therefore, despite exhibiting stress tolerance and biosynthetic gene clusters, the strain should not be considered a probiotic candidate without extensive safety and pathogenicity assessments.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3.6\u003c/span\u003e. Circular graphical representation of \u003cem\u003eBacillus altitudinis\u003c/em\u003e strain JF-5genome. The first external ring of the circular graphic displays \u003cem\u003eBacillus altitudinis\u003c/em\u003e ORFs and all other circular levels start from the inside out. The third ring presents the CRISPR-Cas cluster, while the fourth ring shows AMR genes between the CRISPR-Cas rings. GC content variation data is visualized in the fifth ring followed by GC skew data (green and purple) in the inner ring. The genome displays the labeling of vanG, vanT and vanY vancomycin resistance genes in the clusters.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.6.1 Genome mining for biosynthetic gene clusters (BGCs)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAntiSMASH analysis confirms \u003cem\u003eB. altitudinis\u003c/em\u003e JF-5 can synthesize 10 secondary metabolites encoded by biosynthetic gene clusters. The genome of \u003cem\u003eBacillus altitudinis\u003c/em\u003e contains biosynthetic gene clusters that produce terpene and beta lactone compounds as well as T3PKS and NRPS pathways and RiPP-like secondary metabolites. The T3PKS cluster shows similarity to known polyketides pathways that are associated with antibiotics and anticancer agents and signaling molecules, among other bioactive compounds, however functional metabolite production was not experimentally validated in this study. One of the β-lactone clusters showed 53% similarity to fengycin biosynthetic cluster suggesting potential antifungal biosynthetic capabilities that requires experimental confirmation. The strain harbors multiple biosynthetic gee cluster, indicating potential capacity for secondary metabolite production but functional validation is pending. The genome mining analysis demonstrates that \u003cem\u003eB. altitudinis\u003c/em\u003e contain several biosynthetic gene clusters, suggesting that \u003cem\u003eBacllius altitudinis\u003c/em\u003e JF-5 may serve as candidate for further investigation in natural product research, subject to experimental validation.\u003c/p\u003e \u003c/div\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 3.6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIdentified biosynthetic gene cluster\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eType\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFrom\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMost similar\u003c/p\u003e \u003cp\u003eknown cluster\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSimilarity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegion 1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBeta lactone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e86,900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e115,302\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFengycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e53%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegion 1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTerpene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e183,385\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e205,262\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo similarity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo similarity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegion 1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT3PKS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e243,439\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e284,536\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo similarity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo similarity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegion 1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRiPP-like\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e597,041\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e607,367\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo similarity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo similarity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegion 1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBeta lactone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e754,632\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e778,081\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo similarity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo similarity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegion 2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRE-containing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8,534\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e29,439\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo similarity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo similarity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegion 2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTerpene, NI-Siderophore\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e178,336\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e216,092\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSchizokinen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e60%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegion 3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOther\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e308,636\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e350,056\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBacilysin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e85%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegion 6.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNRPS-like,Sactipeptide,\u003c/p\u003e \u003cp\u003eRanthipeptide, RiPP-like\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e26,439\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e69,441\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSporulation\u003c/p\u003e \u003cp\u003ekilling factor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegion 6.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNRPS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e216,965\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e289,436\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLichenysin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e85%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003eSubsystem Coverage\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003eSubsystem Category distribution\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSubsystem Feature Counts\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3.6\u003c/span\u003e. A summary of the PATRIC annotated subsystem gene distribution. The bar diagram illustrates subsystem coverage, whereas the pie chart depicts the distribution of subsystem features\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 \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3.6\u003c/span\u003e.1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenome assembly statistics of \u003cem\u003eB. altitudinis\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAttribute\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eValue\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSize\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3,774,388\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGC content\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of contigs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of Subsystems\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e324\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of coding sequences\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3962\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of RNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.6.2 Identification of genes associated with probiotic potential\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe presence of stress resistance genes such as \u003cem\u003eyock,cspD, yhbH, cspB\u003c/em\u003e, and \u003cem\u003edps\u003c/em\u003e enables \u003cem\u003eB. altitudinis\u003c/em\u003e to tolerate multiple abiotic stresses, including bile salts, heat, acidic pH, and oxidative stress. Moreover, DNA protection genes like \u003cem\u003erecN, recO, dnaJ, dnaK, radA\u003c/em\u003e, and \u003cem\u003eclpP\u003c/em\u003e ensure genomic stability and protein quality control under stress conditions, enhancing bacterial survival. Adhesion associated genes such as \u003cem\u003ecwlS\u003c/em\u003e and \u003cem\u003eItaS1_1\u003c/em\u003e, where identified \u003cem\u003ewhich\u003c/em\u003e may support colonization however adhesion assays were not performed in this study. Immune modulation is facilitated by genes like \u003cem\u003epdaC_1\u003c/em\u003e, which modify cell wall components to aid immune evasion and require functional validation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3.6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProbiotic genes identified in \u003cem\u003eB. altitudinis\u003c/em\u003e\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\u003eGene name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResponse\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLocus Tag\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eStress Resistance Genes\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\u003eyock\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeneral stress protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00173\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ecspD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCold shock protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00250\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eyhbH\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStress response protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00862\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ecspB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCold shock protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00867\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003enhaX\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStress response protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00918\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eyhaX\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStress response protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00932\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eydaD_1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeneral stress protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00978\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eykoL\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStress response protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_01253\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eyug1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeneral stress protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_01700\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eyceC\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStress response protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02464\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYceD_1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeneral stress protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02465\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYceD_2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeneral stress protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02466\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003esrkA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStress response Kinase A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02627\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eyocM\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSalt stress-responsive protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02668\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eyfIT\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeneral stress protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02681\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eyfkM\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeneral stress protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02716\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eyvgO\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStress response protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02736\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003efarB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFatty acid resistance protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_03078\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eydaD_2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeneral stress protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_03114\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eydaG\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeneral stress protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_03115\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ecspC\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCold shock protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_03218\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eyceD_3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeneral stress protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_03666\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDps\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeneral stress protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_03671\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCtc\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeneral stress protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_03816\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\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\u003e\u003cem\u003ecsaA\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePutative chaperone\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00153\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\u003ectpA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarboxy-terminal processing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00202\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ebepA_1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBeta-barrel assembly-enhancing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00309\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eprsW\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00346\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erecN\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDNA repair protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00474\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003egluP-1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRhomboid protease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00536\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erecO\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDNA repair protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00578\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ednaJ\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChaperone protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00597\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ednaK\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChaperone protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00598\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ehemW\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHeme chaperone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00601\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGpr\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGermination protease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00605\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003elon1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00797\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003elon2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00798\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eClpx\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease ATP-binding subunit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00799\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ehtpX_1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00973\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eVclp\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePetrobactin import ATP-binding protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00989\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ehtpX_2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_00998\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ehtrA_1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeine Protease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_01209\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIsp\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMajor intracellular serine protease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_01240\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ehtrA_2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSerine protease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_01321\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eprtV\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePre-pro-metalloprotease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_01427\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eVpr\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMinor extracellular protease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_01551\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eyraA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePutative cysteine protease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_01681\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ehtrB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSerine protease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_01838\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ecopZ\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCopper chaperone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_01903\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eclpP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease proteolytic subunit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_01981\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ebepA_2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBeta-barrel assembly-enhancing protease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_01996\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFra\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntracellular iron chaperone frataxin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eaprX\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSerine protease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02049\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003erasP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRegular sigma-W protease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02112\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eclpY\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease ATPase subunit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02153\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eclpQ\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease subunit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02154\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003esurA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChaperone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02370\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eepr_1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMinor extracellular protease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02372\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ewprA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell-wall associated protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02417\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eepr_2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMinor extracellular protease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02429\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003estiP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCysteine protease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02470\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\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\u003e\u003cem\u003eMroQ\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMembrane-embedded protease\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02558\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\u003epaiB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease synthase and sporulation protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02562\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabc\" border=\"1\"\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\u003e\u003cem\u003ectpB\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarboxyl-terminal processing protease\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02856\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\u003efliS\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlagellar secretion chaperone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02871\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eyciC\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePutative metal chaperone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_02997\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003egluP_2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRhomboid protease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_03153\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ehtpX_3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_03454\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eclpE\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease ATP-binding subunit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_03476\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eradA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDNA repair protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_03778\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eclpC\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtease regulator\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_03779\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eftsH\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZinc metalloprotease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_03797\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eyabG\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSporulation-specific protease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074_JF5_03825\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAdhesion and Colonization\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ecwlS\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell wall hydrolase helps remodeling and colonization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074 _JF5 _00188\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eItaS1_1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLipoteichoic acid synthesis facilitates adhesion and immune\u003c/p\u003e \u003cp\u003einteraction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074 _JF5 _00533\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eImmune Modulation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003epdaC_1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell wall component modification contributes to immune\u003c/p\u003e \u003cp\u003eevasion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074 _JF5 _00637\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAntimicrobial Resistance Genes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003emerR1_1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMetal resistance regulator supports detoxification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074 _JF5 _00059\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ebmr3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMultidrug efflux transporter bile and drug resistance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074 _JF5 _00086\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003enorm\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMultidrug transporters help resist bile salts and antibiotics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074 _JF5 _00191\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMetabolic Adaptation Genes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ecdaR_1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRegulate carbohydrate metabolism and aids survival in gut\u003c/p\u003e \u003cp\u003eenvironment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e297074 _JF5 _00112\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003e3.6.3. Average nucleotide identity\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eOrthoANI heatmap analysis identified specific genomic similar clusters among the analyzed bacterial strains. The close genetic correlation (high ANI values marked in red) between GR- 8, ECC22, ZAP62, and OCA19 suggests that these should be species of the same or closely related species subtypes. LZP02 and F2 were also moderately to highly like this cluster, implying a distinction in the same genus. The JF-5 strain exhibited a high similarity especially to GR-8, ECC22, since these zones are light red, indicating that JF-5 and these strains are closely related strains and may share the same or very closely related species. JF-5 too was moderately like ZAP62 and OCA19 (yellow areas) but it had a lower similarity with LZP02\u003c/p\u003e \u003cp\u003eand F2. Strain 3P01AB and 9432a1 on the other hand, with all other strains including JF-5 displayed predominantly green patterns, suggesting ANI values (\u0026lt;\u0026thinsp;90%), indicating they are genomic wise distant and probably belong to other species or genera. These results emphasize that JF-5 is most genetically like GR-8 and ECC22, which places it within a genetically related cluster, but is in also marked distinction of the more peripheral strains.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e \u003ch2\u003e3.6.4. Comparative proteome Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eComparison of genomes in this study consisted of 5 bacterial strains, NBTC-002, SGAir0031, GR-8, HM-7 and JF-5, to determine the core, accessory and unique gene content in the genome. It is obvious through the Venn diagram that the number of shared gene clusters across each strain is 3,345, which is the core genome; these gene compositions presumably encode some vital functions that have been conserved with rapid evolution. Conversely, the strains similarly each contain a unique set of gene clusters and gene families that are not found in any other strain, suggesting strain-specific adaptations or niche specialization. Of these, 138 unique gene clusters are in NBTC-002, 91 in JF-5, 25 in SGAir0031, 23 in GR-8, 5 in HM-7, which\u003c/p\u003e \u003cp\u003eindicates different genomic diversity among strains. The studied diversification of distinctive genes can be asymmetrical with adaptation to various environmental conditions, resistance to antimicrobial agents, or distinctive metabolic processes.\u003c/p\u003e \u003cp\u003eMore details can be found in the bar graph demonstrating the total number of protein-coding genes, clusters of genes, and singletons (strain-specific genes) per strain. JF-5 exhibited the greatest gene clusters (3,686) and proteins (3,982), implying a more complex or expanded genome. On the other hand, HM-7 also has also had 3,601 gene clusters, but it has the greatest number of singletons (305), which shows that it has quite a large proportion of novel or divergent genes. The fact that NBTC-002 and GR-8 also have a rather high number of singletons (299 and 308, respectively) further highlights the uniqueness in the genomes of each of these strains compared to each other, despite the greater overall similarity.\u003c/p\u003e \u003cp\u003eAn Upset plot was applied as a more scalable representation of Venn diagrams to further break down the gene-sharing patterns. The plot shows the largest vertical bar at 3,345 shared clusters again validating the core genome. The smaller intersections reflect the gene groups that are common in two, three, or four strains. Such visualization is essential to learn about the conservation of genes, their distribution and uniqueness in strains. The bar plot below depicts the strain combinations present at each intersection of the gene clusters in a more detailed way due to the connected dots beneath the bars. Interestingly, several modest-to large intersection points are shared genes between subsets of strains, each of which may represent a potential shared ecological activity or horizontal gene transfer event.\u003c/p\u003e \u003cp\u003eTogether, this revaluation indicates that although a vast amount of the genome has been kept equal in these strains, each strain still contains distinctive genomic elements. Particularly help to understand the diversity of bacteria, phylogenetic relations and functional capacity, particularly about strain-specific roles in microbial communities, prospects of developing probiotics or living in environments.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003e3.6.5. Core pan genome analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe core genome analysis reveals significant genetic diversity among the studied bacterial strains. The plot shows that the pan genome increases with the addition of more genomes, indicating an open genome structure where new genes continue to emerge. In contrast, the core genome decreases, suggesting that fewer genes are universally shared across all strains. This pattern reflects a high level of genomic variability and adaptability, likely due to horizontal gene transfer or ecological diversity. Overall, the results highlight the evolutionary flexibility and rich gene content of the bacterial species \u003cem\u003eB. altitudinis\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn \u003cb\u003eFig A\u003c/b\u003e presents the distribution of gene families across the 67 genomes. A bimodal pattern is evident: many gene families are either found in nearly all genomes (core genes) or in very few genomes (accessory or strain-specific genes). This reflects the genetic landscape where a flexible accessory genome complements a stable core genome. The presence of gene families unique to only one or a few strains, such as those specific to JF-5, underscores its genetic distinctiveness and possible ecological specialization.\u003c/p\u003e \u003cp\u003eIn \u003cb\u003eFig. B\u003c/b\u003e, the number of new genes per genome is shown. The graph demonstrates a steep decline in novel gene discovery as additional genomes are included in the analysis. This\u003c/p\u003e \u003cp\u003e suggests that early genomes contribute a high number of unique genes to the pan-genome, while later genomes, including JF-5, add fewer. Such a pattern is indicative of an open pan- genome, which may be approaching saturation. The strain JF-5 contributes a moderate number of unique genes compared to other strains, indicating its potential for harboring novel functions or adaptations.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Phylogenetic analysis","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe core-genome phylogenetic tree was annotated using NCBI Prokaryotic Genome Annotation Pipeline. The genome was deposited in GenBank under accession numbers JBRIIV000000000. In this analysis, the strain of interest clustered within a distinct clade along with closely related \u003cem\u003eB. altitudinis\u003c/em\u003e strains, including 40-NJ-V2.faa, 19-AJ-V2.faa, and 17- BC255T1.faa, indicating a high degree of genomic similarity. These strains formed a coherent group in one of the major color-coded clades in the circular phylogenetic layout, suggesting shared evolutionary lineage and conserved functional traits. The closest relative to the studied strain appears to be 40-NJ- V2.faa, which shares a highly similar core genome and may exhibit comparable ecological or antimicrobial properties. In contrast, more distantly related strains, such as 20-CES-OCA-\u003c/p\u003e \u003cp\u003e12.faa and 21-CGMC-3072.faa, were positioned in separate clades, reflecting greater genomic divergence. This phylogenetic placement highlights the evolutionary proximity of the studied strain to specific members of the \u003cem\u003eB. altitudinis\u003c/em\u003e group and supports its classification within a genetically cohesive cluster.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"5. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eProbiotics are described as living organisms which provide health benefits to the host when taken in sufficient quantity; but the classification of probiotics must be strictly evaluated in terms of safety, gastrointestinal survivability, functional activity and lack of transposable antibiotic resistance or virulence factors. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Fermented foods are known to be valuable sources of potentially useful bacteria, such as those of the genus \u003cem\u003eBacillus\u003c/em\u003e, which have been receiving growing interest because of their sporulating ability and ability to survive in the environment. \u003cem\u003eBacillus\u003c/em\u003e spp. endospores enable a considerable survival benefit in severe environments, including heat, desiccation and extreme pH, admittedly further increasing stability during food processing and possibly enabling potential survival via gastric transit. However, spores cannot be called probiotics and thorough phenotypic, genomic and safety validation is required before a single strain can be deemed appropriate to be used by humans. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe present work filtered 25 locally fermented yogurt samples to isolate \u003cem\u003eBacillus\u003c/em\u003e strains that had the potential of being used as a probiotic. It is not an epidemiological study and is a very initial exploratory sampling. In vitro screening assays, such as acid tolerance, bile salt tolerance, enzyme profiling, antimicrobial activity testing, hemolysis, DNase production, antibiotic susceptibility and the entire genome sequencing were performed on isolates (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). A number of the isolates were found to survive at acidic and bile salt conditions indicating that it was able to withstand simulated gastrointestinal stress. Enzyme activities of proteolytic, amylolytic and lipolytic activity in the selected strains were detected by enzymatic assays, which could play a role in supportive digestion and nutrient degradation. Desirable as these features are in probiotic candidates, it is important to note that the functionality of probiotics in vitro is not directly proportional to clinical efficacy (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe most important determinant in the development of probiotics is safety assessment. The preliminary virulence indicators were hemolytic activity and DNase production. Strain JF-5 was negative in the hemolytic and DNase tests indicating a relatively good safety profile. Conversely, JY-2 strain was found to have hemolytic and DNase activity, which is a possible safety issue and eliminates it as a probiotic (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). One of the biosafety problems that were found during the research was the presence of vancomycin-associated resistance genes (vanG, vanY, vanT) in strain JF-5. Such antimicrobial resistance determinants show serious biosafety concern because of potential for horizontal gene transfer is a possible risk and cannot be viewed as positive. Thus, JF-5 cannot be considered a safe choice of probiotic. On the contrary, strain JF-5 was resistant to the majority of available antibiotics and lacked clinically significant resistance determinants, in favor of its relatively safe genomic profile (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhole-genome sequencing placed strain JF-5 as \u003cem\u003eBacillus altitudinis\u003c/em\u003e. The comparative genomic and pan-genome studies identified core genes that are conserved and are related to metabolism, stress response, DNA repair and sporulation. AntiSMASH and BAGEL in silico genome mining identified biosynthetic gene clusters that could produce antimicrobial compounds like bacillysin, fengycin, lichenysin, and siderophores with up to possible ecological competitiveness and antimicrobial activity (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Genomic prediction on its own however does not establish active production of metabolites and must be proven by experiment to be functionally expressed under physiological conditions. The discovery of stress resistance genes, adhesion genes, and immune related genes hence should be understood as genetic possibilities but not proven probiotic features (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt should be noted that some of the key probiotic validation assays were omitted in this study such as epithelial cell adhesion models (e.g., Caco-2 or HT-29 assays), immunomodulatory profiling, in vivo animal analysis, plasmid profiling, horizontal gene transfer analysis and detailed analysis of virulence factor and pathogenicity islands. In turn, although strain JF-5 exhibits potentially attractive in vitro properties and its genomic profile is relatively good, it can be viewed as a pre-probe probiotic strain that needs additional functional confirmation, as opposed to a proven probiotic strain.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe aim of the current research was to isolate and characterize \u003cem\u003eBacillus\u003c/em\u003e species of locally fermented yogurt and to assess their initial probiotic-related attributes by microbiological and genomic methodologies. Two of the 25 isolates (JF-5 and JY-2) were characterized in more detail. Strain JF-5 showed acid and bile tolerance, enzymatic activities, no hemolytic and DNase activity and sensitivity to the majority of the antibiotics which were tested. Whole- genome sequencing revealed the strain as belonging to a \u003cem\u003eBacillus altitudinis\u003c/em\u003e and genomic analysis showed that the strain had genes related to stress response, sporulation, adhesion- related systems and predicted antimicrobial biosynthetic clusters. Strain JY-2 on the other hand was hemolytic, DNase positive. These discoveries are also important biosafety issues, and thus JY-2 cannot be a probiotic contender.\u003c/p\u003e \u003cp\u003eGenomic comparison of JF-5 showed that there are core genes, which are conserved and associated with metabolic stability and stress tolerance. The presence of probiotic associated genes however does not verify the functional probiotic efficacy. There are functional assays, which are epithelial adhesion, immunomodulation, in vivo safety validation and horizontal gene transfer risk assessment that are needed prior to absolute probiotic classification.\u003c/p\u003e \u003cp\u003eThis study is a genome-wide description of \u003cem\u003eBacillus altitudinis\u003c/em\u003e which was found in fermented yogurt in Pakistan. Instead of stating definite probiotic status we make a suggestion that \u003cem\u003eBacillus altitudinis\u003c/em\u003e JF-5 should be viewed as a promising native candidate that can be explored in the future.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFuture work should include\u003c/b\u003e:\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eCell line adhesion assays\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eAnimal model validation in vivo.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eRigorous screening in areas of virulence and pathogenicity.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eResistance genes transferability.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eClinical and toxicological safety investigations.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIt is only upon these tests that the strain can be assured to be developed into nutraceutical or\u003c/p\u003e \u003cp\u003eindustrial probiotics.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest:\u003c/h2\u003e \u003cp\u003eNo conflict of interest is declared by the authors.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to Participate\u003c/strong\u003e \u003cp\u003e, \u003cb\u003eConsent to Publish and Ethics\u003c/b\u003e: Human blood used in hemolysis assays were obtained from certified local blood bank. No human subjects were directly involved.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research received no external funding.\u003c/p\u003e\u003ch2\u003eData Availability:\u003c/h2\u003e \u003cp\u003eWhole genome shotgun sequencing data of \u003cem\u003eBacillus altitudinis\u003c/em\u003e strain produced\u003c/p\u003e \u003cp\u003ein this study were deposited in the NCBI GenBank database under accession number JBRIIV000000000. These BioProject and BioSample accession numbers are PRJNA1335593 and\u003c/p\u003e \u003cp\u003eSAMN52016192, respectively. The NCBI Prokaryotic Genome Annotation Pipeline (PGAP) was used to conduct the genome annotation.\u003c/p\u003e \u003cp\u003eThe GenBank record is publicly available \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eat\u003c/span\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/nuccore/JBRIIV000000000\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/nuccore/JBRIIV000000000\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDimidi E, Cox SR, Rossi M, Whelan K (2019) Fermented foods: definitions and characteristics, impact on the gut microbiota and effects on gastrointestinal health and disease. 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Discov Med 23(124):51\u0026ndash;60\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"Probiotics, fermented yogurt, biochemical characterization, biosafety profiling, whole-genome sequencing","lastPublishedDoi":"10.21203/rs.3.rs-8865594/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8865594/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe research was focused on extracting \u003cem\u003eBacillus\u003c/em\u003e spp. and genomic-wide analysis from locally fermented yogurt to assess their potential as probiotics through genetic characterization. Two bacterial strains, JF-5 and isolate JY-2 were isolated from total 25 yogurt samples. The samples were collected from markets in Rawalpindi and Islamabad. Isolation was carried out using BHI agar as culture medium. The tested isolates received morphological and biochemical examinations, biosafety testing and enzymatic evaluations to evaluate their preliminary probiotic-associated traits. The probiotic characteristics of both strains differed, although they shared Gram-positive morphology, together with rod-shaped features and exhibited catalase activity. Analyses demonstrated that isolate JF-5 possessed proteolytic and lipolytic capacities and showed resilience against bile and pH variations. However, isolate JY-2 displayed DNase activity in addition to hemolysis, making it unsuitable for use as a safe probiotic.\u003c/p\u003e \u003cp\u003eWhole-genome sequencing was applied to the strain JF-5 as the next analysis step. The bacterial strain was defined as \u003cem\u003eBacillus altitudinis\u003c/em\u003e through genome sequencing, which showed\u003c/p\u003e \u003cp\u003ea 3.77 Mbp genome size alongside 41.2% GC content and 3962 coding sequences. The antiSMASH analysis platform detected various biosynthetic gene clusters that can produce antibacterial and probiotic traits, such as lichenysin, bacilysin, fengycin and siderophore compounds. CRISPR -Cas systems and vancomycin resistance-related genes (vanG, vanY, vanT) were identified with analysis. These antimicrobial resistance determinants have important biosafety implications especially with regard to possible horizontal gene transfer. This is why the strain cannot be proposed to be implemented as probiotics without a careful study of its safety. Even though the genomic presence indicates environmental adaptability through the presence of stress response genes, sporulation genes, and nutrient assimilation genes, alone in the genome does not indicate functional probiotic efficacy.\u003c/p\u003e \u003cp\u003eThis investigation presents the first detailed genomic analysis of indigenous Pakistan-based probiotic \u003cem\u003eBacillus\u003c/em\u003e strains. Whole-genome sequencing were performed by MicrobesNG Lab, Birmingham, United Kingdom.\u003c/p\u003e","manuscriptTitle":"In vitro Screening and Genome-wide Analysis of Probiotic Bacillus spp.from Locally Fermented Yogurt for Preliminary Probiotic Potential","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-16 08:27:10","doi":"10.21203/rs.3.rs-8865594/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":"99f7618b-4828-493e-95ce-c9330e619b4a","owner":[],"postedDate":"February 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":62840059,"name":"General Microbiology"},{"id":62840060,"name":"Food Science \u0026 Technology"},{"id":62840061,"name":"Bacteriology"}],"tags":[],"updatedAt":"2026-02-16T08:27:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-16 08:27:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8865594","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8865594","identity":"rs-8865594","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}