Genome Mining and Characterization of Landfill-Derived Bacillus pretiosus Reveals Biosynthetic Gene Clusters and Candidate Plastic-Degrading Genes

Genome Mining and Characterization of Landfill-Derived Bacillus pretiosus Reveals Biosynthetic Gene Clusters and Candidate Plastic-Degrading Genes | 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 Genome Mining and Characterization of Landfill-Derived Bacillus pretiosus Reveals Biosynthetic Gene Clusters and Candidate Plastic-Degrading Genes Jasmine Velo, Albert Noblezada, Ma. Carmel Javier, Victor Marco Emmanuel Ferriols This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7818688/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 rapid escalation of plastic pollution necessitates sustainable microbial solutions, particularly from organisms inhabiting waste-rich environments. This study reports the draft genome sequence of Bacillus pretiosus , isolated from landfill soil in Iloilo City, Philippines, with predicted genetic potential for plastic degradation. Whole genome sequencing using an Illumina NextSeq 1000 platform yielded a 5.82 Mbp genome with a GC content of 35.12%, N50 of 1.61 Mbp, and 99.8% BUSCO completeness(bacillales_odb10, n = 450). Phylogenomic analysis via TYGS and ANI comparisons confirmed the isolate’s identity as B. pretiosus (dDDH: 73.2%, ANI: 96.93%). Annotation revealed 6,589 genes, including 6,092 protein-coding sequences. Notably, 13 genes encoding for enzymes associated with the degradation of 13 plastic types, including polyhydroxyalkanoates (PHB), polyethylene (PE), polyethylene terephthalate (PET), and polyethylene (PE) were identified using PlasticDB. In addition, biosynthetic gene clusters encoding siderophores, nonribosomal peptide synthetases (NRPS), β-lactones, RiPP-like compounds, terpenes, linear azol(in)e-containing peptides (LAPs), and lassopeptides were detected. These findings underscore the diverse potential of environmental Bacillus in both plastic biodegradation and discovery of secondary metabolites for biotechnological applications. biodegradation Bacillus protein coding genes plastics genome biosynthetic gene cluster Figures Figure 1 Figure 2 Figure 3 Introduction Bacillus is a diverse genus belonging to the Phylum Firmicutes family Bacillaceae , comprising of more than 60 species that are widely distributed in nature, from soil, water, plants, and animals (Shelef, 2003 ; Maughan and Van der Auwera, 2011 ; Økstad and Kolstø, 2011 ). They have been found to play a crucial role in pharmaceuticals, agriculture and industry (Sales and Rigobelo, 2024 ) due to their ability to produce a variety of beneficial substances and extracellular enzymes(Qian et al., 2023 ). In addition, Bacillus species are known to synthesize secondary metabolites such as surfactin(Benoit et al., 2015 ), plipastatin, bacillaene, fengycin, and macrolactin which play diverse biotechnological applications(De la Cruz-Rodríguez et al., 2023 ). Bacillus pretiosus , a novel species of Bacillus first isolated from the rhizosphere of Medicago sativa in the mining district of Almadén, Spain, is a Gram-positive, motile bacillus, with a size ranging from 2 ± 0.10 × 0.70 ± 0.15 µm, with creamy texture and dump color colonies visible when incubated at 37°C for 24 hours on nutrient agar(Mora et al., 2023 ). This species has been reported to have biotechnological applications, particularly as a plant growth-promoting bacterium (PGPB) (Penalba-Iglesias et al., 2025 ). With plastic pollution emerging as one of the most pressing environmental problems, microorganisms like Bacillus are gaining attention for their possible roles in providing cost effective and sustainable solutions to this global challenge (Singh et al., 2024a ). Various Bacillus strains have been reported to produce enzymes with biodegradative potential, having demonstrated the capability to degrade polymers, including polyvinyl chloride, polypropylene, polyurethane, polystyrene, and polyethylene (Singh et al., 2024b ). Nonetheless, there remains a limited understanding of the specific genetic determinants for plastic degradation, particularly in environmental isolates from waste-rich ecosystems. Addressing this gap is crucial for identifying biodegradation mechanisms and advancing microbial solutions to plastic pollution. In this study, a Bacillus sp. isolate was subjected to whole-genome sequencing to identify putative genes associated with plastic degradation, as well as biosynthetic gene clusters encoding secondary metabolites. These findings provide insights into the organism’s potential in plastic biodegradation and in the production of bioactive compounds with diverse biotechnological applications. Materials and Methods Bacterial Isolation and revival from glycerol stock A total of 40 bacterial isolates were previously obtained from a landfill in Iloilo City. Among these, 15 isolates exhibited biodegradation activity toward low-density polyethylene (LDPE) and were partially identified through 16S rRNA sequencing (Supplementary Fig. 1). Biodegradation potential was assessed using an in vitro weight loss assay with LDPE films. Of the 15 active isolates, most were identified as Bacillus spp., with one isolate showing the highest biodegradation capability against LDPE; this isolate was selected for further analysis. For long-term preservation, pure bacterial isolates were cultured in tryptic soy broth (TSB) broth at 37°C for 18–24 hours. Glycerol stocks were prepared by combining equal volumes (500 µL each) of sterile 50% (v/v) glycerol and bacterial culture in 2 mL cryotubes(Fadanka, 2022 ). The mixtures were snap-frozen in liquid nitrogen and stored at − 80°C freezer. Morphological, microscopic and biochemical testing of the isolate The glycerol stock culture stored in − 80°C freezer was revived in tryptic soy agar (TSA) and incubated at 37°C overnight. The isolate was subjected to Gram staining, and endospores were visualized using the Schaeffer-Fulton staining method. Microscopic observations were performed under oil immersion at 100× magnification with a 1.25 numerical aperture using an Olympus BX43 upright microscope. The size range of the samples was measured using the ImageJ software. Morphological, microscopic, and biochemical features were assessed for presumptive identification following Bergey’s Manual of Systematic Bacteriology(Vos et al., 2009 ). The isolated bacterium was then subjected to whole-genome sequencing for further analysis. DNA Extraction and Whole Genome Sequencing A single colony of the isolate from TSA plate was inoculated into 5 mL of TSB and incubated at 37°C for 18–24 h. Cells were harvested by centrifugation at 10,000 rpm for 10 minutes, and bacterial pellets were extracted using the Qiagen DNeasy Blood and Tissue Kit. DNA purity was assessed with a MultiSkan Sky™ spectrophotometer, and integrity was verified via gel electrophoresis and Bio-Rad XR + Gel Documentation System (Supplementary Fig. 2). DNA (100 ng/µL) was prepared using the Illumina DNA Prep Kit. Library concentration and size were measured using a Qubit™ Fluorometer and Agilent™ Bioanalyzer, respectively. Sequencing was performed on an Illumina NextSeq 1000 platform at 800 pM loading concentration. Genome Assembly FASTQ files were demultiplexed via Illumina BaseSpace. Quality control was done using FastQC(Andrews, 2010 ), followed by trimming with Trimmomatic v0.36(Bolger et al., 2014 ) (--phred33 SLIDINGWINDOW:4:20 LEADING:20 TRAILING:20 MINLEN:36), and de novo assembly using SPAdes v3.13(Bankevich et al., 2012 ) with --careful -k 21,33,55,77,99,127. Assembly quality was evaluated using QUAST v5.0.2 (Gurevich et al., 2013 ), and completeness was assessed with BUSCO v5.8.3(Manni et al., 2021 ) using the bacillales_odb10 database. The circular genome was visualized using Proksee web application ( https://proksee.ca/ ) (Grant et al., 2023 ). Paired-end reads were aligned to the reference genome using BWA-MEM(Li and Durbin, 2009 ) with default settings. SAM files were converted to BAM. The final BAM files mapping quality, coverage, and depth metrics were obtained using SAMtools v. 1.21(Li et al., 2009 ). Genome Annotation and Phylogenetic Analysis Annotation was performed using PGAP v6.10 (Tatusova et al., 2016 ) on NCBI. All tools were run with default parameters unless otherwise specified. The genome sequence was uploaded to the Type (Strain) Genome Server (TYGS) ( https://tygs.dsmz.de ) for whole genome-based taxonomic analysis using the d4 formula (sum of identities / HSP length) to delineate species similarity (70% dDDH (d4) cutoff). Creation of a phylogenomic tree by digital DNA-DNA hybridisation (dDDH) was inferred using FastME 2.1.6.1 (Lefort et al., 2015 ; Meier-Kolthoff and Göker, 2019 ). The files generated by TYGS (.nwk file) were viewed in Interactive tree of life (iTOL) (Letunic and Bork, 2007 ). The closely related genomes based on TYGS were compared using FastANI, with a 95% ANI threshold for species-level similarity (Jain et al., 2018 ). Genome Mining of proteins-coding genes for plastic degradation and identification of biosynthetic gene clusters (BGCs) The annotated protein-coding genes by PGAP were queried on the PlasticDB (E-value 1e-6, ≥ 30% identity) to identify plastic-degrading potential (Gambarini et al., 2022 ). Biosynthetic gene clusters (BGCs) were also predicted using antiSMASH v6.1. Databases such as eggNOG -mapper ( http://eggnog-mapper.embl.de/ ) (Cantalapiedra et al., 2021 ) and ShinyGO was used for functional annotation of COG categories and gene functions. Results Morphological, Microscopic, and Biochemical Characteristics of bacteria The bacterial isolate revived from glycerol stocks produced cream-colored, matte colonies on nutrient agar. Colonies were irregular in shape, with undulate margins, raised elevation, and a dry surface texture (Fig. 1a). Microscopic examination using an Olympus BX43 upright microscope at 100× magnification (1.25 numerical aperture) revealed Gram-positive rods arranged in pairs or in short chains with endospores (Fig. 1b). Endospore staining by the Schaeffer-Fulton method, which uses malachite green to penetrate the endospore wall, showed green ellipsoidal spores (Fig. 1c). Results of the biochemical testing shows that the isolate assimilates and acidifies several sugars, is catalase positive, Vogues Proskauer positive, amygdalin positive, gelatinase positive, urease negative, and reduces nitrates to nitrite among other traits (Table 1). A positive Vogues Proskauer (VP) test is a useful trait in differentiating Bacillus from other Gram-positive spore formers by producing fluorescent red coloration. Fermentation and utilization patterns of glucose and mannitol or assimilation of sucrose and arabinose, provide insights into the organism’s metabolic activity. A study of the characterization of Bacillus sp. from rice also generated similar positive results on catalase, Vogues Proskauer, nitrate reduction, utilization of arginine, sucrose, mannitol, glucose, arabinose(Sawant et al., 2022). This suggests consistency on the identification of the isolate being a member of the genus Bacillus . Table 1 Biochemical profiling of the Bacillus sp. isolate using acidification, fermentation, and other standard tests. REACTIONS/ENZYMES RESULTS REACTIONS/ENZYMES RESULTS catalase + oxidase - Tryptophane Deaminase - Voges Proskauer + acidification(D-fructose) + Arginine Dihydrolase - acidification(D-mannose) + Urease - Glucose + inositol - acidification(maltose) + ß-galactosidase (Ortho NitroPhenyl-ßD- Galactopyranosidase) - acidification(lactose) + Arginine Dihydrolase - acidification(D-trehalose) + Lysine Decarboxylase - acidification(D-mannitol) + Ornithine Decarboxylase - acidification(xylitol) + trisodium citrate - acidification(D-melibiose) + H2S production - acidification (raffinose) + indole production - acidification (xylose) + Tryptophane Deaminase - acidification (saccharose) + gelatinase + acidification (Methyl-αD- Glucopyranoside) + reduction of nitrates to nitrites + acidification (N-Acetyl-Glucosamine) + oxidation/fermentation (amygdalin) + Assembly, Mapping Statistics and Phylogenetic Analysis The assembled genome of Bacillus sp. isolate had a total length of 5,823,789 bp (Fig. 2 a), with an N50 value of 1,606,001 bp exhibiting a high degree of contiguity, GC content of 35.12%, and low ambiguity with 11.59 Ns per 100 kbp. A BUSCO completeness score of 99.8%, also indicates high assembly quality, with no fragmented BUSCOs. A high mapping rate (99.55%), coverage depth of 1192.6x, and proper pairing of reads (97.63%) indicate accurate alignment and good genome quality (Table 2 ). PGAP annotation in NCBI revealed a total of 6,589 genes, of which 6,092 were protein-coding. Table 2 Genome information using QUAST for assembly quality, BUSCO to evaluate completeness of the genome assembly, and BWA to map raw sequencing reads and calculate genome coverage. Genome Information Value Total length (bp) 5,823,789 Number of contigs 717 Largest contig 1,612,162 N50 1,606,001 N75 599,804 L50 2 L75 4 GC (%) 35.12 # N's per 100 kbp 11.59 Complete BUSCOs (%) 99.8 Complete and single-copy BUSCOs (%) 98.9 Complete and duplicated BUSCOs (%) 0.9 Fragmented BUSCOs (%) 0 Missing BUSCOs (%) 0.2 Total BUSCO groups searched 450 Average coverage 1192.6 The Bacillus isolate was found to be closely related to Bacillus pretiosus with a dDDH of 73.2, indicating that the two strains belong to the same species (value ≥ 70%) based on TYGS analysis (Table 3 ). FastANI results confirmed close relatedness with 96.93% average nucleotide identity and phylogenomic tree with 99% bootstrap support (Fig. 2 b). Table 3 Pairwise dDDH values between Bacillus sp. isolate and the selected typestrain genomes from TYGS. The dDDH values are provided along with their confidence intervals (C.I.). Subject strain Size (Mb) % GC dDDH C.I. (d4, in %) ANI(%) (d4, in %) Bacillus pretiosus SAICEU11t 5.4 35.3 73.2 [70.2–76.0] 96.93 Bacillus wiedmannii FSL W8-0169 5.3 35.2 68.7 [65.7–71.5] 96.14 Bacillus mobilis MCCC 1A05942 5.7 35.3 58.9 [56.1–61.7] 94.63 Bacillus albus N35-10-2 5.8 34.9 55.1 [52.4–57.8] 93.93 Bacillus basilensis 403507-21 5.8 35.2 53.2 [50.5–55.8] 93.56 Bacillus fungorum 17-SMS-01 5.7 35 52.3 [49.6–55.0] 93.23 Bacillus tropicus N24 5.2 35.2 51.8 [49.1–54.5] 93.34 Bacillus pacificus MCCC 1A06182 5.4 35.2 51.5 [48.9–54.2] 93.17 Bacillus dicomae MHSD28T 5.6 35.2 51.4 [48.7–54.0] 93.17 Bacillus paranthracis MCCC 1A00395 5.5 35.2 50.8 [61.2–68.6] 93.04 Bacillus anthracis ATCC 14578 5.5 35.2 50.3 [47.7–52.9] 93.07 Bacillus luti MCCC 1A00359 5.1 35.4 44.6 [42.1–47.2] 91.55 Bacillus cereus ATCC 14579 5.4 35.3 44 [41.5–46.6] 91.33 Bacillus toyonensis NCIMB 14858 5 35.6 43.8 [41.2–46.6] 91.42 Bacillus thuringiensis ATCC 10792 6.3 34.8 43.6 [41.1–46.2] 91.8 Bacillus proteolyticus MCCC 1A00365 5.9 35.1 40.6 [38.1–43.1] 90.24 Bacillus velezensis NRRL B-41580 4 46.3 40.2 [37.8–42.8] below 80% Bacillus amyloliquefaciens DSM 7 4 46.1 32.9 [30.5–35.5] below 80% Bacillus subtilis ATCC 6051 4.2 43.5 28.7 [26.3–31.1] below 80% Bacillus thermotolerans SgZ-8T 3.8 44.1 24.2 [21.9–26.7] below 80% Bacillus safensis FO-36b 3.7 41.6 20.7 [18.5–23.1] below 80% Functional Annotation of Genes The genome of the Bacillus sp. isolate was annotated using the EggNOG-mapper platform to predict the functional roles of its genes. The majority of the annotated genes (a total of 1,528 genes) were associated with energy production and conversion, as well as the transport and metabolism of various nutrients (COG categories C, G, E, F, H, I, P, and Q). A considerable proportion of genes (1,363) were also assigned to functions of unknown characterization (category S). A total of 896 genes were found to be related to information storage and processing, including those involved in translation, transcription, and replication (categories J, A, K, L, and B) and 829 genes annotated corresponded to functions associated with cellular processes and signaling, including cell motility, and membrane biogenesis(categories D, Y, V, T, M, N, Z, W, U, and O) (Fig. 3 a). Gene Ontology (GO) annotation further highlighted the top functional roles (top 10) of the Bacillus sp. isolate across the three GO domains: molecular function, cellular component, and biological process. At the molecular function level, the predominant activities included DNA helicase activity, DNA-dependent ATPase activity, purine nucleoside binding, nucleoside triphosphatase activity, hydrolase activity, and protein binding. For the cellular component domain, the top functions were associated with the cytoplasm, nucleoid, DNA helicase complex, cell septum, cell projection, and intracellular structures, reflecting roles in cellular organization and motility. At the biological process level, enriched terms included cellular component assembly and biogenesis, anatomical structure formation, sporulation, cell differentiation, and developmental processes (Fig. 3 b). These GO terms collectively emphasize the genetic potential of the isolate to support growth, metabolism, adaptation, and survival of the isolate in the environment. Mining of biosynthetic gene clusters and functional genes for plastic degradation Analysis with PlasticDB predicted a total of 13 protein-coding genes associated with enzymes putatively involved in the degradation of 13 types of plastics, including polyhydroxyalkanoates (PHB), polyethylene (PE), and polyethylene terephthalate (PET), polybutylene succinate (PBS), polybutylene succinate-co-adipate (PBSA), polycaprolactone (PCL), polyethylene succinate (PES), polylactic acid (PLA), polyhydroxyalkanoates (PHA), polyethylene glycol (PEG), poly (3-Hydroxyvalerate) (P3HV), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and polybutylene adipate terephthalate (PBAT). The predicted enzymes comprised proteases, lipases, esterases, copper oxidase, PEG aldehyde dehydrogenase, PLA depolymerase, PETase, hydrolase, 3HV dehydrogenase, carboxylesterase, PHB depolymerase, and chitinase (Table 4 ). In addition, genome mining revealed the presence of multiple biosynthetic gene clusters (BGCs) potentially responsible for the production of secondary metabolites. These included clusters encoding siderophores, nonribosomal peptide synthetases (NRPS), β-lactones, RiPP-like compounds, terpenes, linear azol(in)e-containing peptides (LAPs), and lassopeptides(Table 5 ). These findings highlight the metabolic versatility of the landfill-derived Bacillus isolate and its potential applications in both bioremediation and natural product discovery. Table 4 List of protein sequence hits associated with plastic degradation identified through PlasticDB analysis. qseqid Protein Microorganism Plastic Degraded Reference MGK0531714.1 PLA depolymerase Uncultured bacterium PBS PBSA PCL PES PLA PHA (Mayumi et al., 2008 ) MGK0531752.1 Copper oxidase Rhodococcus opacus PE (Zampolli et al., 2023 ) MGK0531764.1 Protease Parengyodontium album PLA (Yamashita et al., 2005 ) MGK0531871.1 Protease Bacillus licheniformis PLA (Oda et al., 2000 ) MGK0532142.1 PEG aldehyde dehydrogenase Streptomyces sp. PEG (Ohta et al., 2005 ) MGK0532414.1 Lipase Pelosinus fermentans PBAT (Biundo et al., 2016 ) MGK0532414.1 esterase Clostridium botulinum PBAT (Perz et al., 2016 ) MGK0532538.1 PETase Uncultured bacterium PET (Erickson et al., 2022 ) MGK0532551.1 hydrolase Alcanivorax borkumensis PLA,PHBV,PCL,PBSA,PES, PHA (Hajighasemi et al., 2016 ) MGK0533196.1 3HV dehydrogenase Paracoccus denitrificans P3HV,PHBV,PHA (Lu et al., 2014a ) MGK0533320.1 Carboxylesterase Uncultured bacterium PBAT (Müller et al., 2017 ) MGK0534488.1 PHB depolymerase Bacillus thuringiensis PHB,PHA (Wang et al., 2014 ) MGK0536372.1 Chitinase Geomyces sp. PBS,PBSA,PCL (Urbanek et al., 2018 ) Table 5 Biosynthetic gene clusters associated with Bacillus sp. isolate predicted using antiSMASH. Region Type Cluster Position Most similar known cluster Similarity Region 1.1 siderophore 458580–472285 petrobactin Other 100% Region 1.2 NRPS 800359-845,491 bacillibactin NRP 46% Region 1.3 NRPS 922164-985,427 zwittermicin A NRP + Polyketide 18% Region 1.4 betalactone 994745-1,019,983 fengycin NRP 40% Region 1.5 RiPP-like 1070295-1,080,456 Region 1.6 RiPP-like 1145680-1,155,919 Region 1.7 NRPS 1172906-1,219,916 Region 2.1 terpene 1257805-1,279,658 molybdenum cofactor Other 17% Region 3.1 LAP,RiPP-like 463115-486,622 Region 4.1 NRPS 316547-385,057 Region 5.1 NRPS-like,NRPS 90813-151,456 cereulide NRP:Cyclic depsipeptide 21% Region 13.1 lassopeptide 5757-27,984 Discussion The bacterial isolate, originally obtained from landfill soil, was identified as Bacillus pretiosus based on an ANI of 96.93% and a dDDH value of 73.2%, which together indicate that it belongs to the same species. In general, an ANI of ~ 96% combined with a dDDH of ≥ 70% demonstrates that the genomes share both a high degree of orthologous gene similarity and overall whole-genome relatedness. Although Bacillus weidmannii exhibited a comparable ANI of 96.14%, its dDDH value was below 70% (68.7%) (Table 3 ), suggesting that it does not belong to the same species. As proposed by Moore et al. ( 1987 ), species can be delineated using a 70% dDDH threshold(Moore et al., 1987 ), which remains more widely accepted than ANI alone because dDDH accounts for whole-genome sequence similarity rather than only orthologous regions (Goris et al., 2007 ). Landfills serve as primary repositories of global plastic wastes, with polyethylene (PE), polystyrene(PS), and polypropylene(PP) as the most prevalent polymers(Kabir et al., 2023 ). In a typical household setting where most waste end up and accumulate in landfills, common plastic wastes include, low- density polyethylene (LDPE), high density polyethylene (HDPE), polyvinyl chloride (PVC), and polyethylene terephthalate (PET) (Gwada et al., 2019 ). Given these patterns, exploring the biodegradation potential of landfill-derived microorganisms, such as the Bacillus sp. isolate in this study, is critical for developing effective waste management strategies. Consistent with the diversity of plastics that accumulate in landfills, genome mining of the Bacillus sp. isolate revealed multiple putative genes linked to plastic biodegradation. PlasticDB homolog searches revealed that the Bacillus sp. isolate harbors putative genes encoding for proteases and hydrolases, including lipases and esterases, which act extracellularly to cleave the stable carbon skeleton of plastic polymers, breaking them into smaller molecules that can be assimilated by microorganisms(Han et al., 2024 ). These enzymes are particularly associated with the degradation of polylactides (PLA), where proteolytic activity is known to accelerate the breakdown process. In addition, copper oxidases are involved in the oxidation of the hydrocarbon backbone of polyethylene (Sivan, 2011 ). Similar enzymes have been reported in Rhodococcus (Zampolli et al., 2022 ) and Streptomyces( Osma et al., 2010 ) where they were identified and have been implicated in polyethylene (PE) degradation. Copper oxidases have also been associated with high density polyethylene degradation (HDPE) in Bjerkandera adusta ( Kang et al., 2019 ). Importantly, the genome also harbored a PETase homolog, an enzyme initially characterized from Ideonella sakaiensis that is well known for hydrolyzing polyethylene terephthalate (PET) (Palm et al., 2019 ), thereby suggesting the current isolate’s potential to contributing to PET degradation. Additional genes were also identified including those encoding for PEG aldehyde dehydrogenase (previously reported to degrade polyethylene in Rhodococcus) (Tao et al., 2023 ), PHB depolymerase (enzyme found in a soil thermophile Lihuaxuella thermophila degrading plastics such as polylactic acid (PLA) and polycaprolactone (PCL)) (Thomas et al., 2022 ). Similar PHB depolymerases was also found and characterized Diaphorobacter sp. which has shown PHB/PHBV-degrading ability(Zhang et al., 2010 ). In addition to depolymerases, 3HV dehydrogenase has also been found in the Bacillus sp. isolate, an enzyme involved in the degradation of polyhydroxybutyrate and polyhydroxyvalerate(Lu et al., 2014b ). Collectively, these genes further support the organism’s capacity to degrade a broad spectrum of plastics including PLA, PHBV, PCL, PBSA, PES, PHA, and P3HV suggesting that the organism may have acquired enzymatic systems enabling survival in plastic-contaminated environments. Importantly, the ability of a single microbial isolate to target not just one type of polymer but also complex mixtures of plastic waste highlights its environmental relevance since mixed plastic waste poses a major challenge to conventional waste management strategies where single-polymer degraders are often insufficient, this finding opens new avenues for microbial-based solutions. Furthermore, the predicted enzymes were cross-validated against the PAZy database, a curated repository of biochemically characterized plastic-active enzymes (36), confirming their potential roles in diverse plastic degradation pathways. The genomic predictions were also found consistent with the biochemical assay results. The isolate demonstrated positive reactions for extracellular enzyme production, including gelatinase activity, which reflects protease secretion. Proteases and hydrolases detected in biochemical tests not only validate the genomic findings but also provide functional evidence of active enzyme production. These in silico predictions and biochemical tests underscores the metabolic versatility of the isolate and highlights its potential for biotechnological applications. Gelatinase, in particular, is an industrially relevant protease widely used in pharmaceuticals, and large-scale enzyme production. Its co-occurrence with plastic-active enzymes suggests that the isolate could serve as a dual-purpose organism, capable of both conventional industrial enzyme production and bioplastic degradation(Balan et al., 2012 ). Analysis of the genome of the Bacillus sp isolate also revealed genes with functional roles linked to its biotechnological potential, particularly in the production of functional enzymes and biosynthetic gene clusters. Annotation using eggNOG mapping showed that most genes were assigned to COG categories related to energy production and conversion, as well as lipid transport and metabolism(1,580 genes). Protein-coding genes for enzymes such as α-hydrolases, PETase, carboxylesterase, and PHV depolymerase were classified within these categories. These assignments align with the top 10 Gene Ontology (GO) terms (Fig. 3 b), where hydrolase activity emerged as one of the predominant molecular functions. 3HV dehydrogenase, PEG aldehyde dehydrogenase, and copper oxidases also fall within these COG categories under secondary metabolites biosynthesis, transport, and catabolism. These functions are consistent with the organism’s potential role in plastic degradation, where microorganisms secrete enzymes that depolymerize plastics into shorter chains, allowing the recovery of energy and resources needed for cellular processes and survival. The detection of BGCs encoding siderophores (petrobactin), nonribosomal peptide synthetases (NRPS) (bacillibactin, zwittermicin, A, cereulides, and fengicyn), β-lactones, RiPP-like compounds, terpenes (molybdenum cofactor), linear azol(in)e-containing peptides (LAPs), and lassopeptides underscores the metabolic versatility of this isolate. Siderophores are organic compounds produced by microorganisms that chelate metals thereby facilitating the bioremediation in heavy metals(Roskova et al., 2022 ). NRPS are enzymes that synthesize a wide variety of peptides(Iacovelli et al., 2021 ) and has been reported to be function as biocontrol agent and in bioremediation of contaminated soils (Ranjan et al., 2023 ). Additionally, terpenes are secreted by microorganisms to the extracellular environment where they facilitate monomer uptake through the cellular membrane for metabolic utilization(Rana, 2019 ). In parallel, 75 genes were assigned to COG functional category Q, which is associated with secondary metabolite biosynthesis, transport, and catabolism, thereby providing functional support for the antiSMASH-predicted pathways. These secondary metabolite systems are known to contribute to diverse applications in biotechnology, industry, and pharmaceuticals, including antimicrobial, cytotoxic, and anticancer activities, emphasizing the potential of Bacillus sp. isolate identified as B. pretiosus as a valuable resource for metabolomics-driven discovery. The findings of this study lay the foundation for understanding the key genes and enzymes underlying the versatile roles of this Bacillus sp. isolate , providing valuable insights into its potential in plastic biodegradation and secondary metabolite production. Future work should focus on the functional characterization of the predicted plastic-degrading enzymes, validation of biosynthetic gene clusters, and exploration of their bioactive products. Integrating omics-driven studies such as genomics with proteomics, metabolomics, and experimental assays will be crucial to establishing the organism’s ecological role and harnessing its capabilities for sustainable bioremediation and biotechnological applications. Declarations Acknowledgments The authors would like to thank the Regional Research Center for the assistance on the use of equipment during the conduct of the study. Data availability The sequencing reads were deposited in the National Center for Biotechnology Information (NCBI) under the Bioproject number PRJNA1285566, accession number SRR34411118.The Whole Genome Shotgun project has been deposited in GenBank under the accession no. JBPPHF000000000. Conflict Of Interest The authors declare no conflict of interest. Funding Declaration This research received in house funding from Philippine Genome Center Visayas. References Andrews, S., 2010. FastQC: a quality control tool for high throughput sequence data. [WWW Document]. URL http://www.bioinformatics.babraham.ac.uk/projects/fastqc (accessed 8.12.23). Balan, S.S., Nethaji, R., Sankar, S., Jayalakshmi, S., 2012. Production of gelatinase enzyme from Bacillus spp isolated from the sediment sample of Porto Novo Coastal sites. 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15:48:47","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":356376,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7818688/v1/cf76a6be8be61527e6fad8ec.png"},{"id":94474281,"identity":"c7229d0a-d32e-48f1-9d9a-289aee63f7d8","added_by":"auto","created_at":"2025-10-27 15:48:06","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":103500,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7818688/v1/6da9e4f026dd580ba38ca013.png"},{"id":94474240,"identity":"00c1720e-1051-4f31-b238-2a454fe1aad6","added_by":"auto","created_at":"2025-10-27 15:48:01","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":108895,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7818688/v1/b4d2c1405cd12314b542319e.png"},{"id":94473954,"identity":"b3637afc-8cda-403d-8c69-77801d3d2160","added_by":"auto","created_at":"2025-10-27 15:46:21","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":66222,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7818688/v1/5b86ec8fb362dde254bdc15f.png"},{"id":94474024,"identity":"eb0b673a-11b9-4802-b97d-dbb62d97ae43","added_by":"auto","created_at":"2025-10-27 15:46:51","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":171107,"visible":true,"origin":"","legend":"","description":"","filename":"86bf0f397640489c8e20877d0d9acc641structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7818688/v1/bb14ff41bb1ba4cc36e6b813.xml"},{"id":94473868,"identity":"8d59511b-1c82-46da-b602-b1a70598ccd4","added_by":"auto","created_at":"2025-10-27 15:46:03","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":177456,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7818688/v1/13bd93407746aedab2ed1969.html"},{"id":94474162,"identity":"0da44b84-fe0b-4c1a-ac05-09a7b00a9b58","added_by":"auto","created_at":"2025-10-27 15:47:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":614064,"visible":true,"origin":"","legend":"\u003cp\u003e(a)Petri plates showing colonies of \u003cem\u003eBacillus\u003c/em\u003e sp. isolated from landfill soil grown on tryptic soy agar and incubated at 37 °C for 24–48 h. (b) Gram-stained cells of the isolate observed under oil immersion (100×/1.25 NA) using a light microscope, and (c) Endospores stained with 1% malachite green and 0.5% safranin following the Schaeffer-Fulton method, showing green endospores and red vegetative cells under oil immersion (100×/1.25 NA). A scale bar (upper left) indicates the size range of the cells.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7818688/v1/cf323a3e3c81c102cc3218c7.png"},{"id":94474242,"identity":"a9658024-c685-4763-9051-66a4adf94c1c","added_by":"auto","created_at":"2025-10-27 15:48:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":680437,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Map of Bacillus sp. with genomic features; (b) Phylogenomic tree of \u003cem\u003eBacillus \u003c/em\u003edataset in TYGS platform. The tree was inferred with FastME 2.1.6.1 from GBDP distances calculated from genome sequences. The numbers above branches are GBDP pseudo-bootstrap support values \u0026gt; 60 % from 100 replications. The \u003cem\u003eBacillus sp.\u003c/em\u003e isolate in ths study is highlighted in red.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7818688/v1/7b1dd12b81cd90a4c980c2f7.png"},{"id":94474163,"identity":"dcbae541-870b-4e05-bef2-94437b49b722","added_by":"auto","created_at":"2025-10-27 15:47:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":377074,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Number of Predicted Genes Assigned to COG Functional Categories mapped using eggNOG, and (b)Top 10 most abundant GO terms within the major categories.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7818688/v1/eb08a1b9f5882d0abffbe999.png"},{"id":97715931,"identity":"697ed579-5171-4a36-81e0-13c9f78f2019","added_by":"auto","created_at":"2025-12-08 14:39:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2914538,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7818688/v1/4104e0eb-58e0-48fb-b5d6-2ad2f0e5d803.pdf"},{"id":94473911,"identity":"cc274ea6-79ef-490f-af1d-2d94b4bd5518","added_by":"auto","created_at":"2025-10-27 15:46:15","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19149,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7818688/v1/de190469dbde3301202501fb.docx"},{"id":94474308,"identity":"9a25a6f3-1f27-4fd3-8f5d-e1f0df44d83c","added_by":"auto","created_at":"2025-10-27 15:48:16","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":92017,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7818688/v1/23f14ac9e3e3694f137bc3e8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome Mining and Characterization of Landfill-Derived Bacillus pretiosus Reveals Biosynthetic Gene Clusters and Candidate Plastic-Degrading Genes","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eBacillus\u003c/em\u003e is a diverse genus belonging to the Phylum Firmicutes family \u003cem\u003eBacillaceae\u003c/em\u003e, comprising of more than 60 species that are widely distributed in nature, from soil, water, plants, and animals (Shelef, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Maughan and Van der Auwera, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; \u0026Oslash;kstad and Kolst\u0026oslash;, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). They have been found to play a crucial role in pharmaceuticals, agriculture and industry (Sales and Rigobelo, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) due to their ability to produce a variety of beneficial substances and extracellular enzymes(Qian et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition, \u003cem\u003eBacillus\u003c/em\u003e species are known to synthesize secondary metabolites such as surfactin(Benoit et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), plipastatin, bacillaene, fengycin, and macrolactin which play diverse biotechnological applications(De la Cruz-Rodr\u0026iacute;guez et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eBacillus pretiosus\u003c/em\u003e, a novel species of Bacillus first isolated from the rhizosphere of \u003cem\u003eMedicago sativa\u003c/em\u003e in the mining district of Almad\u0026eacute;n, Spain, is a Gram-positive, motile bacillus, with a size ranging from 2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 \u0026times; 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 \u0026micro;m, with creamy texture and dump color colonies visible when incubated at 37\u0026deg;C for 24 hours on nutrient agar(Mora et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This species has been reported to have biotechnological applications, particularly as a plant growth-promoting bacterium (PGPB) (Penalba-Iglesias et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWith plastic pollution emerging as one of the most pressing environmental problems, microorganisms like \u003cem\u003eBacillus\u003c/em\u003e are gaining attention for their possible roles in providing cost effective and sustainable solutions to this global challenge (Singh et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e). Various Bacillus strains have been reported to produce enzymes with biodegradative potential, having demonstrated the capability to degrade polymers, including polyvinyl chloride, polypropylene, polyurethane, polystyrene, and polyethylene (Singh et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). Nonetheless, there remains a limited understanding of the specific genetic determinants for plastic degradation, particularly in environmental isolates from waste-rich ecosystems. Addressing this gap is crucial for identifying biodegradation mechanisms and advancing microbial solutions to plastic pollution.\u003c/p\u003e\u003cp\u003eIn this study, a \u003cem\u003eBacillus\u003c/em\u003e sp. isolate was subjected to whole-genome sequencing to identify putative genes associated with plastic degradation, as well as biosynthetic gene clusters encoding secondary metabolites. These findings provide insights into the organism\u0026rsquo;s potential in plastic biodegradation and in the production of bioactive compounds with diverse biotechnological applications.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eBacterial Isolation and revival from glycerol stock\u003c/h2\u003e\u003cp\u003eA total of 40 bacterial isolates were previously obtained from a landfill in Iloilo City. Among these, 15 isolates exhibited biodegradation activity toward low-density polyethylene (LDPE) and were partially identified through 16S rRNA sequencing (Supplementary Fig.\u0026nbsp;1). Biodegradation potential was assessed using an in vitro weight loss assay with LDPE films. Of the 15 active isolates, most were identified as \u003cem\u003eBacillus\u003c/em\u003e spp., with one isolate showing the highest biodegradation capability against LDPE; this isolate was selected for further analysis.\u003c/p\u003e\u003cp\u003eFor long-term preservation, pure bacterial isolates were cultured in tryptic soy broth (TSB) broth at 37\u0026deg;C for 18\u0026ndash;24 hours. Glycerol stocks were prepared by combining equal volumes (500 \u0026micro;L each) of sterile 50% (v/v) glycerol and bacterial culture in 2 mL cryotubes(Fadanka, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The mixtures were snap-frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C freezer.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMorphological, microscopic and biochemical testing of the isolate\u003c/h3\u003e\n\u003cp\u003eThe glycerol stock culture stored in \u0026minus;\u0026thinsp;80\u0026deg;C freezer was revived in tryptic soy agar (TSA) and incubated at 37\u0026deg;C overnight. The isolate was subjected to Gram staining, and endospores were visualized using the Schaeffer-Fulton staining method. Microscopic observations were performed under oil immersion at 100\u0026times; magnification with a 1.25 numerical aperture using an Olympus BX43 upright microscope. The size range of the samples was measured using the ImageJ software. Morphological, microscopic, and biochemical features were assessed for presumptive identification following Bergey\u0026rsquo;s Manual of Systematic Bacteriology(Vos et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The isolated bacterium was then subjected to whole-genome sequencing for further analysis.\u003c/p\u003e\n\u003ch3\u003eDNA Extraction and Whole Genome Sequencing\u003c/h3\u003e\n\u003cp\u003eA single colony of the isolate from TSA plate was inoculated into 5 mL of TSB and incubated at 37\u0026deg;C for 18\u0026ndash;24 h. Cells were harvested by centrifugation at 10,000 rpm for 10 minutes, and bacterial pellets were extracted using the Qiagen DNeasy Blood and Tissue Kit. DNA purity was assessed with a MultiSkan Sky\u0026trade; spectrophotometer, and integrity was verified via gel electrophoresis and Bio-Rad XR\u0026thinsp;+\u0026thinsp;Gel Documentation System (Supplementary Fig.\u0026nbsp;2). DNA (100 ng/\u0026micro;L) was prepared using the Illumina DNA Prep Kit. Library concentration and size were measured using a Qubit\u0026trade; Fluorometer and Agilent\u0026trade; Bioanalyzer, respectively. Sequencing was performed on an Illumina NextSeq 1000 platform at 800 pM loading concentration.\u003c/p\u003e\n\u003ch3\u003eGenome Assembly\u003c/h3\u003e\n\u003cp\u003eFASTQ files were demultiplexed via Illumina BaseSpace. Quality control was done using FastQC(Andrews, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), followed by trimming with Trimmomatic v0.36(Bolger et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) (--phred33 SLIDINGWINDOW:4:20 LEADING:20 TRAILING:20 MINLEN:36), and de novo assembly using SPAdes v3.13(Bankevich et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) with --careful -k 21,33,55,77,99,127. Assembly quality was evaluated using QUAST v5.0.2 (Gurevich et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and completeness was assessed with BUSCO v5.8.3(Manni et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) using the bacillales_odb10 database. The circular genome was visualized using Proksee web application (\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) (Grant et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Paired-end reads were aligned to the reference genome using BWA-MEM(Li and Durbin, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) with default settings. SAM files were converted to BAM. The final BAM files mapping quality, coverage, and depth metrics were obtained using SAMtools v. 1.21(Li et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eGenome Annotation and Phylogenetic Analysis\u003c/h3\u003e\n\u003cp\u003eAnnotation was performed using PGAP v6.10 (Tatusova et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) on NCBI. All tools were run with default parameters unless otherwise specified. The genome sequence was uploaded to the Type (Strain) Genome Server (TYGS) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tygs.dsmz.de\u003c/span\u003e\u003cspan address=\"https://tygs.dsmz.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for whole genome-based taxonomic analysis using the d4 formula (sum of identities / HSP length) to delineate species similarity (70% dDDH (d4) cutoff). Creation of a phylogenomic tree by digital DNA-DNA hybridisation (dDDH) was inferred using FastME 2.1.6.1 (Lefort et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Meier-Kolthoff and G\u0026ouml;ker, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The files generated by TYGS (.nwk file) were viewed in Interactive tree of life (iTOL) (Letunic and Bork, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The closely related genomes based on TYGS were compared using FastANI, with a 95% ANI threshold for species-level similarity (Jain et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eGenome Mining of proteins-coding genes for plastic degradation and identification of biosynthetic gene clusters (BGCs)\u003c/h2\u003e\u003cp\u003eThe annotated protein-coding genes by PGAP were queried on the PlasticDB (E-value 1e-6, \u0026ge;\u0026thinsp;30% identity) to identify plastic-degrading potential (Gambarini et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Biosynthetic gene clusters (BGCs) were also predicted using antiSMASH v6.1. Databases such as eggNOG -mapper (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://eggnog-mapper.embl.de/\u003c/span\u003e\u003cspan address=\"http://eggnog-mapper.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Cantalapiedra et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and ShinyGO was used for functional annotation of COG categories and gene functions.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003eMorphological, Microscopic, and Biochemical Characteristics of bacteria\u003c/h2\u003e\n \u003cp\u003eThe bacterial isolate revived from glycerol stocks produced cream-colored, matte colonies on nutrient agar. Colonies were irregular in shape, with undulate margins, raised elevation, and a dry surface texture (Fig. 1a). Microscopic examination using an Olympus BX43 upright microscope at 100\u0026times; magnification (1.25 numerical aperture) revealed Gram-positive rods arranged in pairs or in short chains with endospores (Fig. 1b). Endospore staining by the Schaeffer-Fulton method, which uses malachite green to penetrate the endospore wall, showed green ellipsoidal spores (Fig. 1c).\u003c/p\u003e\n \u003cp\u003eResults of the biochemical testing shows that the isolate assimilates and acidifies several sugars, is catalase positive, Vogues Proskauer positive, amygdalin positive, gelatinase positive, urease negative, and reduces nitrates to nitrite among other traits (Table 1). A positive Vogues Proskauer (VP) test is a useful trait in differentiating Bacillus from other Gram-positive spore formers by producing fluorescent red coloration. Fermentation and utilization patterns of glucose and mannitol or assimilation of sucrose and arabinose, provide insights into the organism\u0026rsquo;s metabolic activity. A study of the characterization of Bacillus sp. from rice also generated similar positive results on catalase, Vogues Proskauer, nitrate reduction, utilization of arginine, sucrose, mannitol, glucose, arabinose(Sawant et al., 2022). This suggests consistency on the identification of the isolate being a member of the genus \u003cem\u003eBacillus\u003c/em\u003e.\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eBiochemical profiling of the \u003cem\u003eBacillus sp. isolate\u003c/em\u003e using acidification, fermentation, and other standard tests.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eREACTIONS/ENZYMES\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRESULTS\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eREACTIONS/ENZYMES\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRESULTS\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecatalase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eoxidase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTryptophane Deaminase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVoges Proskauer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacidification(D-fructose)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eArginine Dihydrolase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacidification(D-mannose)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUrease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlucose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003einositol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacidification(maltose)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026szlig;-galactosidase (Ortho NitroPhenyl-\u0026szlig;D- Galactopyranosidase)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacidification(lactose)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eArginine Dihydrolase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacidification(D-trehalose)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLysine Decarboxylase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacidification(D-mannitol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOrnithine Decarboxylase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacidification(xylitol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etrisodium citrate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacidification(D-melibiose)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH2S production\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacidification (raffinose)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cdiv\u003e\n \u003cdiv align=\"left\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eindole production\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacidification (xylose)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTryptophane Deaminase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacidification (saccharose)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egelatinase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacidification (Methyl-\u0026alpha;D- Glucopyranoside)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ereduction of nitrates to nitrites\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacidification (N-Acetyl-Glucosamine)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eoxidation/fermentation (amygdalin)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAssembly, Mapping Statistics and Phylogenetic Analysis\u003c/h2\u003e\u003cp\u003eThe assembled genome of \u003cem\u003eBacillus sp. isolate\u003c/em\u003e had a total length of 5,823,789 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), with an N50 value of 1,606,001 bp exhibiting a high degree of contiguity, GC content of 35.12%, and low ambiguity with 11.59 Ns per 100 kbp. A BUSCO completeness score of 99.8%, also indicates high assembly quality, with no fragmented BUSCOs. A high mapping rate (99.55%), coverage depth of 1192.6x, and proper pairing of reads (97.63%) indicate accurate alignment and good genome quality (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). PGAP annotation in NCBI revealed a total of 6,589 genes, of which 6,092 were protein-coding.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGenome information using QUAST for assembly quality, BUSCO to evaluate completeness of the genome assembly, and BWA to map raw sequencing reads and calculate genome coverage.\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\u003eGenome Information\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\u003eTotal length (bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5,823,789\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\u003e717\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLargest contig\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1,612,162\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1,606,001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e599,804\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eL50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eL75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGC (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e35.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e# N's per 100 kbp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.59\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eComplete BUSCOs (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e99.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eComplete and single-copy BUSCOs (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e98.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eComplete and duplicated BUSCOs (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFragmented BUSCOs (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMissing BUSCOs (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal BUSCO groups searched\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e450\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAverage coverage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1192.6\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\u003eThe \u003cem\u003eBacillus\u003c/em\u003e isolate was found to be closely related to \u003cem\u003eBacillus pretiosus\u003c/em\u003e with a dDDH of 73.2, indicating that the two strains belong to the same species (value\u0026thinsp;\u0026ge;\u0026thinsp;70%) based on TYGS analysis (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). FastANI results confirmed close relatedness with 96.93% average nucleotide identity and phylogenomic tree with 99% bootstrap support (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\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\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePairwise dDDH values between \u003cem\u003eBacillus sp. isolate\u003c/em\u003e and the selected typestrain genomes from TYGS. The dDDH values are provided along with their confidence intervals (C.I.).\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSubject strain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSize (Mb)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e% GC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003edDDH\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eC.I. (d4, in %)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eANI(%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(d4, in %)\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\u003eBacillus pretiosus SAICEU11t\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e73.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[70.2\u0026ndash;76.0]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e96.93\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus wiedmannii FSL W8-0169\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e68.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[65.7\u0026ndash;71.5]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e96.14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus mobilis MCCC 1A05942\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e58.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[56.1\u0026ndash;61.7]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e94.63\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus albus N35-10-2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[52.4\u0026ndash;57.8]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e93.93\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus basilensis 403507-21\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e53.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[50.5\u0026ndash;55.8]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e93.56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus fungorum 17-SMS-01\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e52.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[49.6\u0026ndash;55.0]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e93.23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus tropicus N24\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e51.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[49.1\u0026ndash;54.5]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e93.34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus pacificus MCCC 1A06182\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e51.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[48.9\u0026ndash;54.2]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e93.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus dicomae MHSD28T\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e51.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[48.7\u0026ndash;54.0]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e93.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus paranthracis MCCC 1A00395\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[61.2\u0026ndash;68.6]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e93.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus anthracis ATCC 14578\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[47.7\u0026ndash;52.9]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e93.07\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus luti MCCC 1A00359\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e44.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[42.1\u0026ndash;47.2]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e91.55\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus cereus ATCC 14579\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[41.5\u0026ndash;46.6]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e91.33\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus toyonensis NCIMB 14858\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e43.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[41.2\u0026ndash;46.6]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e91.42\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus thuringiensis ATCC 10792\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e43.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[41.1\u0026ndash;46.2]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e91.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus proteolyticus MCCC 1A00365\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[38.1\u0026ndash;43.1]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e90.24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus velezensis NRRL B-41580\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[37.8\u0026ndash;42.8]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ebelow 80%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus amyloliquefaciens DSM 7\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[30.5\u0026ndash;35.5]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ebelow 80%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus subtilis ATCC 6051\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e43.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[26.3\u0026ndash;31.1]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ebelow 80%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus thermotolerans SgZ-8T\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e44.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[21.9\u0026ndash;26.7]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ebelow 80%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBacillus safensis FO-36b\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e41.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e[18.5\u0026ndash;23.1]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ebelow 80%\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=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eFunctional Annotation of Genes\u003c/h2\u003e\u003cp\u003eThe genome of the \u003cem\u003eBacillus\u003c/em\u003e sp. isolate was annotated using the EggNOG-mapper platform to predict the functional roles of its genes. The majority of the annotated genes (a total of 1,528 genes) were associated with energy production and conversion, as well as the transport and metabolism of various nutrients (COG categories C, G, E, F, H, I, P, and Q). A considerable proportion of genes (1,363) were also assigned to functions of unknown characterization (category S). A total of 896 genes were found to be related to information storage and processing, including those involved in translation, transcription, and replication (categories J, A, K, L, and B) and 829 genes annotated corresponded to functions associated with cellular processes and signaling, including cell motility, and membrane biogenesis(categories D, Y, V, T, M, N, Z, W, U, and O) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGene Ontology (GO) annotation further highlighted the top functional roles (top 10) of the \u003cem\u003eBacillus sp.\u003c/em\u003e isolate across the three GO domains: molecular function, cellular component, and biological process. At the molecular function level, the predominant activities included DNA helicase activity, DNA-dependent ATPase activity, purine nucleoside binding, nucleoside triphosphatase activity, hydrolase activity, and protein binding. For the cellular component domain, the top functions were associated with the cytoplasm, nucleoid, DNA helicase complex, cell septum, cell projection, and intracellular structures, reflecting roles in cellular organization and motility. At the biological process level, enriched terms included cellular component assembly and biogenesis, anatomical structure formation, sporulation, cell differentiation, and developmental processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). These GO terms collectively emphasize the genetic potential of the isolate to support growth, metabolism, adaptation, and survival of the isolate in the environment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eMining of biosynthetic gene clusters and functional genes for plastic degradation\u003c/h2\u003e\u003cp\u003eAnalysis with PlasticDB predicted a total of 13 protein-coding genes associated with enzymes putatively involved in the degradation of 13 types of plastics, including polyhydroxyalkanoates (PHB), polyethylene (PE), and polyethylene terephthalate (PET), polybutylene succinate (PBS), polybutylene succinate-co-adipate (PBSA), polycaprolactone (PCL), polyethylene succinate (PES), polylactic acid (PLA), polyhydroxyalkanoates (PHA), polyethylene glycol (PEG), poly (3-Hydroxyvalerate) (P3HV), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and polybutylene adipate terephthalate (PBAT). The predicted enzymes comprised proteases, lipases, esterases, copper oxidase, PEG aldehyde dehydrogenase, PLA depolymerase, PETase, hydrolase, 3HV dehydrogenase, carboxylesterase, PHB depolymerase, and chitinase (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In addition, genome mining revealed the presence of multiple biosynthetic gene clusters (BGCs) potentially responsible for the production of secondary metabolites. These included clusters encoding siderophores, nonribosomal peptide synthetases (NRPS), β-lactones, RiPP-like compounds, terpenes, linear azol(in)e-containing peptides (LAPs), and lassopeptides(Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These findings highlight the metabolic versatility of the landfill-derived \u003cem\u003eBacillus\u003c/em\u003e isolate and its potential applications in both bioremediation and natural product discovery.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eList of protein sequence hits associated with plastic degradation identified through PlasticDB analysis.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eqseqid\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProtein\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMicroorganism\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePlastic Degraded\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMGK0531714.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePLA depolymerase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eUncultured bacterium\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePBS PBSA PCL PES PLA PHA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Mayumi et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2008\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMGK0531752.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCopper oxidase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eRhodococcus opacus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Zampolli et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMGK0531764.1\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\u003e\u003cem\u003eParengyodontium album\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePLA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Yamashita et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2005\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMGK0531871.1\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\u003e\u003cem\u003eBacillus licheniformis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePLA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Oda et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2000\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMGK0532142.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePEG aldehyde dehydrogenase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eStreptomyces sp.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePEG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Ohta et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2005\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMGK0532414.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLipase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePelosinus fermentans\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePBAT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Biundo et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMGK0532414.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eesterase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eClostridium botulinum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePBAT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Perz et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2016\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMGK0532538.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePETase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eUncultured bacterium\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePET\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Erickson et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMGK0532551.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ehydrolase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eAlcanivorax borkumensis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePLA,PHBV,PCL,PBSA,PES, PHA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Hajighasemi et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMGK0533196.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3HV dehydrogenase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eParacoccus denitrificans\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eP3HV,PHBV,PHA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Lu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014a\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMGK0533320.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCarboxylesterase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eUncultured bacterium\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePBAT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(M\u0026uuml;ller et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMGK0534488.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePHB depolymerase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eBacillus thuringiensis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePHB,PHA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Wang et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2014\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMGK0536372.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eChitinase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eGeomyces sp.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePBS,PBSA,PCL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(Urbanek et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)\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=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBiosynthetic gene clusters associated with \u003cem\u003eBacillus sp.\u003c/em\u003e isolate predicted using antiSMASH.\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" 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\u003eCluster Position\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eMost similar known 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\u0026nbsp;1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003esiderophore\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e458580\u0026ndash;472285\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003epetrobactin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOther\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\u0026nbsp;1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNRPS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e800359-845,491\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ebacillibactin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNRP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e46%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRegion\u0026nbsp;1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNRPS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e922164-985,427\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ezwittermicin A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNRP\u0026thinsp;+\u0026thinsp;Polyketide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e18%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRegion\u0026nbsp;1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ebetalactone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e994745-1,019,983\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003efengycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNRP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e40%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRegion\u0026nbsp;1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRiPP-like\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1070295-1,080,456\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRegion\u0026nbsp;1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRiPP-like\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1145680-1,155,919\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRegion\u0026nbsp;1.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNRPS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1172906-1,219,916\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRegion\u0026nbsp;2.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eterpene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1257805-1,279,658\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003emolybdenum cofactor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOther\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e17%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRegion\u0026nbsp;3.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLAP,RiPP-like\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e463115-486,622\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRegion\u0026nbsp;4.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNRPS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e316547-385,057\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRegion\u0026nbsp;5.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNRPS-like,NRPS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e90813-151,456\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ecereulide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNRP:Cyclic depsipeptide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e21%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRegion\u0026nbsp;13.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003elassopeptide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5757-27,984\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe bacterial isolate, originally obtained from landfill soil, was identified as \u003cem\u003eBacillus pretiosus\u003c/em\u003e based on an ANI of 96.93% and a dDDH value of 73.2%, which together indicate that it belongs to the same species. In general, an ANI of ~\u0026thinsp;96% combined with a dDDH of \u0026ge;\u0026thinsp;70% demonstrates that the genomes share both a high degree of orthologous gene similarity and overall whole-genome relatedness. Although \u003cem\u003eBacillus weidmannii\u003c/em\u003e exhibited a comparable ANI of 96.14%, its dDDH value was below 70% (68.7%) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), suggesting that it does not belong to the same species. As proposed by Moore et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1987\u003c/span\u003e), species can be delineated using a 70% dDDH threshold(Moore et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1987\u003c/span\u003e), which remains more widely accepted than ANI alone because dDDH accounts for whole-genome sequence similarity rather than only orthologous regions (Goris et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLandfills serve as primary repositories of global plastic wastes, with polyethylene (PE), polystyrene(PS), and polypropylene(PP) as the most prevalent polymers(Kabir et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In a typical household setting where most waste end up and accumulate in landfills, common plastic wastes include, low- density polyethylene (LDPE), high density polyethylene (HDPE), polyvinyl chloride (PVC), and polyethylene terephthalate (PET) (Gwada et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Given these patterns, exploring the biodegradation potential of landfill-derived microorganisms, such as the \u003cem\u003eBacillus sp.\u003c/em\u003e isolate in this study, is critical for developing effective waste management strategies.\u003c/p\u003e\u003cp\u003eConsistent with the diversity of plastics that accumulate in landfills, genome mining of the \u003cem\u003eBacillus\u003c/em\u003e sp. isolate revealed multiple putative genes linked to plastic biodegradation. PlasticDB homolog searches revealed that the \u003cem\u003eBacillus sp.\u003c/em\u003e isolate harbors putative genes encoding for proteases and hydrolases, including lipases and esterases, which act extracellularly to cleave the stable carbon skeleton of plastic polymers, breaking them into smaller molecules that can be assimilated by microorganisms(Han et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These enzymes are particularly associated with the degradation of polylactides (PLA), where proteolytic activity is known to accelerate the breakdown process. In addition, copper oxidases are involved in the oxidation of the hydrocarbon backbone of polyethylene (Sivan, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Similar enzymes have been reported in \u003cem\u003eRhodococcus\u003c/em\u003e(Zampolli et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and \u003cem\u003eStreptomyces(\u003c/em\u003eOsma et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) where they were identified and have been implicated in polyethylene (PE) degradation. Copper oxidases have also been associated with high density polyethylene degradation (HDPE) in \u003cem\u003eBjerkandera adusta (\u003c/em\u003eKang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Importantly, the genome also harbored a PETase homolog, an enzyme initially characterized from \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e that is well known for hydrolyzing polyethylene terephthalate (PET) (Palm et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), thereby suggesting the current isolate\u0026rsquo;s potential to contributing to PET degradation. Additional genes were also identified including those encoding for PEG aldehyde dehydrogenase (previously reported to degrade polyethylene in Rhodococcus) (Tao et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), PHB depolymerase (enzyme found in a soil thermophile \u003cem\u003eLihuaxuella thermophila\u003c/em\u003e degrading plastics such as polylactic acid (PLA) and polycaprolactone (PCL)) (Thomas et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Similar PHB depolymerases was also found and characterized \u003cem\u003eDiaphorobacter sp.\u003c/em\u003e which has shown PHB/PHBV-degrading ability(Zhang et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In addition to depolymerases, 3HV dehydrogenase has also been found in the \u003cem\u003eBacillus sp.\u003c/em\u003e isolate, an enzyme involved in the degradation of polyhydroxybutyrate and polyhydroxyvalerate(Lu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014b\u003c/span\u003e). Collectively, these genes further support the organism\u0026rsquo;s capacity to degrade a broad spectrum of plastics including PLA, PHBV, PCL, PBSA, PES, PHA, and P3HV suggesting that the organism may have acquired enzymatic systems enabling survival in plastic-contaminated environments. Importantly, the ability of a single microbial isolate to target not just one type of polymer but also complex mixtures of plastic waste highlights its environmental relevance since mixed plastic waste poses a major challenge to conventional waste management strategies where single-polymer degraders are often insufficient, this finding opens new avenues for microbial-based solutions. Furthermore, the predicted enzymes were cross-validated against the PAZy database, a curated repository of biochemically characterized plastic-active enzymes (36), confirming their potential roles in diverse plastic degradation pathways.\u003c/p\u003e\u003cp\u003eThe genomic predictions were also found consistent with the biochemical assay results. The isolate demonstrated positive reactions for extracellular enzyme production, including gelatinase activity, which reflects protease secretion. Proteases and hydrolases detected in biochemical tests not only validate the genomic findings but also provide functional evidence of active enzyme production. These in silico predictions and biochemical tests underscores the metabolic versatility of the isolate and highlights its potential for biotechnological applications. Gelatinase, in particular, is an industrially relevant protease widely used in pharmaceuticals, and large-scale enzyme production. Its co-occurrence with plastic-active enzymes suggests that the isolate could serve as a dual-purpose organism, capable of both conventional industrial enzyme production and bioplastic degradation(Balan et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAnalysis of the genome of the \u003cem\u003eBacillus sp\u003c/em\u003e isolate also revealed genes with functional roles linked to its biotechnological potential, particularly in the production of functional enzymes and biosynthetic gene clusters. Annotation using eggNOG mapping showed that most genes were assigned to COG categories related to energy production and conversion, as well as lipid transport and metabolism(1,580 genes). Protein-coding genes for enzymes such as α-hydrolases, PETase, carboxylesterase, and PHV depolymerase were classified within these categories. These assignments align with the top 10 Gene Ontology (GO) terms (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), where hydrolase activity emerged as one of the predominant molecular functions. 3HV dehydrogenase, PEG aldehyde dehydrogenase, and copper oxidases also fall within these COG categories under secondary metabolites biosynthesis, transport, and catabolism. These functions are consistent with the organism\u0026rsquo;s potential role in plastic degradation, where microorganisms secrete enzymes that depolymerize plastics into shorter chains, allowing the recovery of energy and resources needed for cellular processes and survival.\u003c/p\u003e\u003cp\u003eThe detection of BGCs encoding siderophores (petrobactin), nonribosomal peptide synthetases (NRPS) (bacillibactin, zwittermicin, A, cereulides, and fengicyn), β-lactones, RiPP-like compounds, terpenes (molybdenum cofactor), linear azol(in)e-containing peptides (LAPs), and lassopeptides underscores the metabolic versatility of this isolate. Siderophores are organic compounds produced by microorganisms that chelate metals thereby facilitating the bioremediation in heavy metals(Roskova et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). NRPS are enzymes that synthesize a wide variety of peptides(Iacovelli et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and has been reported to be function as biocontrol agent and in bioremediation of contaminated soils (Ranjan et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additionally, terpenes are secreted by microorganisms to the extracellular environment where they facilitate monomer uptake through the cellular membrane for metabolic utilization(Rana, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In parallel, 75 genes were assigned to COG functional category Q, which is associated with secondary metabolite biosynthesis, transport, and catabolism, thereby providing functional support for the antiSMASH-predicted pathways. These secondary metabolite systems are known to contribute to diverse applications in biotechnology, industry, and pharmaceuticals, including antimicrobial, cytotoxic, and anticancer activities, emphasizing the potential of \u003cem\u003eBacillus sp. isolate\u003c/em\u003e identified as \u003cem\u003eB. pretiosus\u003c/em\u003e as a valuable resource for metabolomics-driven discovery.\u003c/p\u003e\u003cp\u003eThe findings of this study lay the foundation for understanding the key genes and enzymes underlying the versatile roles of this \u003cem\u003eBacillus sp. isolate\u003c/em\u003e, providing valuable insights into its potential in plastic biodegradation and secondary metabolite production. Future work should focus on the functional characterization of the predicted plastic-degrading enzymes, validation of biosynthetic gene clusters, and exploration of their bioactive products. Integrating omics-driven studies such as genomics with proteomics, metabolomics, and experimental assays will be crucial to establishing the organism\u0026rsquo;s ecological role and harnessing its capabilities for sustainable bioremediation and biotechnological applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Regional Research Center for the assistance on the use of equipment during the conduct of the study.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequencing reads were deposited in the National Center for Biotechnology Information (NCBI) under the Bioproject number PRJNA1285566, accession number SRR34411118.The Whole Genome Shotgun project has been deposited in GenBank under the accession no. JBPPHF000000000.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict Of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received in house funding from Philippine Genome Center Visayas.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAndrews, S., 2010. FastQC: a quality control tool for high throughput sequence data. [WWW Document]. URL http://www.bioinformatics.babraham.ac.uk/projects/fastqc (accessed 8.12.23).\u003c/li\u003e\n\u003cli\u003eBalan, S.S., Nethaji, R., Sankar, S., Jayalakshmi, S., 2012. Production of gelatinase enzyme from Bacillus spp isolated from the sediment sample of Porto Novo Coastal sites. 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World J Microbiol Biotechnol 26, 1803\u0026ndash;1811. https://doi.org/10.1007/s11274-010-0361-3\u003c/li\u003e\n\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"biodegradation, Bacillus, protein coding genes, plastics, genome, biosynthetic gene cluster","lastPublishedDoi":"10.21203/rs.3.rs-7818688/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7818688/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe rapid escalation of plastic pollution necessitates sustainable microbial solutions, particularly from organisms inhabiting waste-rich environments. This study reports the draft genome sequence of \u003cem\u003eBacillus pretiosus\u003c/em\u003e, isolated from landfill soil in Iloilo City, Philippines, with predicted genetic potential for plastic degradation. Whole genome sequencing using an Illumina NextSeq 1000 platform yielded a 5.82 Mbp genome with a GC content of 35.12%, N50 of 1.61 Mbp, and 99.8% BUSCO completeness(bacillales_odb10, n\u0026thinsp;=\u0026thinsp;450). Phylogenomic analysis via TYGS and ANI comparisons confirmed the isolate\u0026rsquo;s identity as \u003cem\u003eB. pretiosus\u003c/em\u003e (dDDH: 73.2%, ANI: 96.93%). Annotation revealed 6,589 genes, including 6,092 protein-coding sequences. Notably, 13 genes encoding for enzymes associated with the degradation of 13 plastic types, including polyhydroxyalkanoates (PHB), polyethylene (PE), polyethylene terephthalate (PET), and polyethylene (PE) were identified using PlasticDB. In addition, biosynthetic gene clusters encoding siderophores, nonribosomal peptide synthetases (NRPS), β-lactones, RiPP-like compounds, terpenes, linear azol(in)e-containing peptides (LAPs), and lassopeptides were detected. These findings underscore the diverse potential of environmental \u003cem\u003eBacillus\u003c/em\u003e in both plastic biodegradation and discovery of secondary metabolites for biotechnological applications.\u003c/p\u003e","manuscriptTitle":"Genome Mining and Characterization of Landfill-Derived Bacillus pretiosus Reveals Biosynthetic Gene Clusters and Candidate Plastic-Degrading Genes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-27 14:32:24","doi":"10.21203/rs.3.rs-7818688/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":"a1e5fa57-285f-4c0a-b489-ed315a513828","owner":[],"postedDate":"October 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T14:39:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-27 14:32:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7818688","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7818688","identity":"rs-7818688","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}