Integrated genomics and proteomics analysis of Paenibacillus peoriae IBSD35 and insights into its antimicrobial characteristics

Antimicrobial resistance has been developing fast and incurring a loss of human life, and there is a need for new antimicrobial agents. Naturally occurring antimicrobial peptides offer the characteristics to counter AMR because the resistance development is low or no resistance. Antimicrobial peptides from Paenibacillus peoriae IBSD35 cell-free supernatant were salted out and purified using chromatography and characterized with liquid chromatography–tandem-mass spectrometry. The extract has shown a high and broad spectrum of antimicrobial activity. Combining the strain IBSD35 genome sequence with its proteomic data enabled the prediction of biosynthetic gene clusters by connecting the peptide from LC–MS/MS data to the gene that encode. Antimicrobial peptide databases offered a platform for the effective search, prediction, and design of AMPs and expanded the studies on their isolation, structure elucidation, biological evaluation, and pathway engineering. The genome-based taxonomy and comparisons have shown that P. peoriae IBSD35 is closely related to Paenibacillus peoriae FSL J3-0120. P. peoriae IBSD35 harbored endophytic trait genes and nonribosomal peptide synthases biosynthetic gene clusters. The comparative genomics revealed evolutionary insights and facilitated the discovery of novel SMs using proteomics from the extract of P. peoriae IBSD35. It will increase the potential to find novel bio-molecules to counter AMR.


Results
Genomic deoxyribonucleic acid (DNA) estimation and annotation. Paenibacillus peoriae IBSD35 inoculated on Luria Bertani (LB) agar plate was visible after 12 h of incubation at 38 °C. It is an endosporeforming and neutralophilic bacterium based on Integrated Microbial Genomes Annotation Pipeline (IMG AP) v.4.16.6 28,29 . The genomic deoxyribonucleic acid (gDNA) assessed with 0.8% agarose was estimated to be more than 50-kilo base pairs (kbp), and the concentration was 188.63 µg µl −1 (Supplementary Fig. S1). P. peoriae IBSD35 has a linear DNA topology with a total length of 5,862,582 bp. Its closest species reference genome is Paenibacillus peoriae KCTC 3763 with a symmetrical identity of 62.7495% and a symmetrical identity of 89.4884% with P. peoriae FSL J3-0120 in National Center for Biotechnology Information (NCBI) neighbor genome report 30 . Spectrum of antimicrobial activity of the P. peoriae IBSD35 inoculum. The CFS of P. peoriae Optimum growth conditions for antimicrobial metabolites production. The P. peoriae IBSD35 inoculum's optimum optical density (OD) was measured to be 0.65 at λ600 nm observed on the 6th day, coinciding with the exponential growth phase in the LB medium. The optimum growth was at pH 6.8-7. P. peoriae IBSD35 is a mesophile bacterium that grows best in moderate temperatures observed at 38 °C ( Fig. 2A). The supernatant antimicrobial activity was determined as an arbitrary unit (AU) per milliliter which is the reciprocal of the highest dilution (2 n ) that resulted in the inhibition of S. aureus ATCC 25923 indicator lawns 31 . The AU was measured to be 1600 AU ml −1 .
Salting out and antimicrobial activity retention test of dialysate. The fraction (F4) eluted out with the 700 milimolar (mM) sodium chloride (NaCl) at the flow rate of 2 ml min −1 has shown antimicrobial activity against S. aureus ATCC 25923. The fraction OD was measured to be 1.147 and 2.803 at wavelength (λ)214 and λ280, respectively (Table S1). The fraction (F4) was collected, lyophilized into powder form, and store at − 20  32 . The number of sequence protein entries predicted in the P. peoriae IBSD35 Uniprot database is 15 protein groups. Different peptide sequences have detected during the process (Table 1). An uncharacterized protein was predicted from the experimental LC-MS/MS with accession no. A0A2S6NUT6. The Uniprot search has shown that it is 94.87% similar to A0A509FZN2_PAEPO of Paenibacillus polymyxa (A0A509FZN2) (Bit score-296, E-value-3.6 e−89) 33 . Combining the genome mining and proteomics analysis has shown that the gene (ID 2816869382) is 97% similar to the gene (ID 2556935736) of Paenibacillus polymyxa CR1 (NC_023037) (Bit score-286; 1e−95) and it has 134 amino acid residues.
An AMP predicted from the protein lists with accession no. A0A2S6NUT6 is designated peoriaerin IBSD35 based on its source name ( Table 2). Its sequence is AAGIQAQAGFGLSDSIQGTGKQKCSFCK. The N-terminal of the sequence considered is alanine (Ala). It has 28 amino acid residues with a molecular weight (MW) of 2802.17 Da (Supplementary Fig. S2). Its sequence backbone has a large portion of helix and residues embedded in the membrane. It suggested peoriaerin IBSD3 might insert the membrane for its antimicrobial activity (Fig. 3). The peptide sequence alignment indicated that it is 38.23% similar to fermentin 20 . The three-dimensional (3-D) model of peoriaerin IBSD35 was generated using an automated protein structure de novo modeling and deposited at ModelArchive with accession no. 10.5452ma-id7l4 (Fig. 3). The graphic diagram of the P. peoriae IBSD35 optimum growth curve in LB medium. The purple zone indicates the non-growth region, yellow zone represents the growth region, and the black dot indicates the optimum growth zone. (Left Y-axis is the optical density, Right Y-is the optimum temperature, and X-axis is the pH). (B) The chromatogram of the RP-HPLC. X-axis shows the retention time in min while Y-axis shows the absorbance. (The retention times indicate the peaks for practical purposes. 4.165 represents peak P4, and 4.567 represents peak P5). P4 retention time is 4.165 min. The P5 retention time is 4.567 min. Sharp peaks of P4 and P5 indicate they are sufficiently separated and have good biomolecule concentration. The P4 fractions exhibiting higher antimicrobial activity were pooled together and selected.  (Fig. 4). The OD at λ600 nm was 1.409 indicating the cell growth and debris turbidity. There was no  Figure 3. A 3-D model of peoriaerin IBSD35 predicted using de novo modeling. The amino acids composition of peptide is AAGIQAQAGFGLSDSIQGTGKQKCSFCK. Sequence contains high Gly content. It is indicated in black color. Glycine is integral to the formation of alpha-helices in secondary protein structure due to its compact form. The purple color indicated the helix structure property. It included the green color which intersects with the purple color. The green color indicated the residues embedded in membrane. The light orange color indicated the Trp which is the rings in the residues. The red color indicated the Ala. The whole structure represented the backbone and side chain of the AMP except the Gly indicated in black color. is related to the genetic information process (47%), environment information processing (45%), carbohydrate metabolism protein family (42%), and signaling process (31%) ( Supplementary Fig. S3) 37,38 . Membrane and cell contributed the highest percentage of genes in the cellular component category, while the catalytic activity, binding, and transportation account for the highest number in molecular function. The metabolic and cellular processes have the highest percentage within the biological processes category (Fig. 5B) 42,43 . The Joint Genome Institute-Integrated Microbial Genomes and Microbiomes System (JGI-IMG/M) analysis revealed a putative horizontally transferred genes count of 10 (0.19%) with the best hits to genes from Proteobacteria and Clostridia. The trans-membrane count is 1392 (26.28%) and might serve as a gateway to transport specific substances across the membrane 28,29 . The signal peptide is 303 (5.72%) of the total CDS identified. It might prompt the translocation of a cell protein secretion 44,45 .
Comparison of genomes characteristics. The selected Paenibacillus species have shown varying numbers of DNA scaffolds ranging from 1 to 56. Paenibacillus sp. JDR-2 has the largest genome size (7,184,930 bp), and B. subtilis subtilis AG1839 has the smallest genome size (4,193,640 bp) among the selected genomes ( Table 3). The dot plot homology revealed a low level of P. peoriae IBSD35 synteny with Paenibacillus peoriae KCTC 3763 but a higher synteny with Paenibacillus peoriae HS311, P. polymyxa SC2, and P. polymyxa E681 (Supplementary Fig. S4). The predicted numbers of nonribosomal peptide synthase (NRPS) BGCs ranged from 0 to 18 among the eleven genomes indicating their diverse and rich SM natural products (Table 3). P. peoriae IBSD35 harbored 18 NRPS (72% of the total 25 BGCs) and encompassed the highest number of NRPS BGCs among the selected genomes comparisons.
Antibiotics and other secondary metabolites (SMs) gene cluster of P. peoriae IBSD35. The CFS of P. peoriae IBSD35 has shown a high and broad-spectrum antimicrobial activity against S. aureus ATCC 25923, E. coli ATCC 25922, C. albicans ATCC 10231, Klebsiella pneumoniae ATCC 4352, and Salmonella typhimurium ATCC 14028 (Fig. 1A-E). The activity was comparable with ampicillin and nisin at a much lower dose. Antibiotics and Secondary Metabolite Analysis Shell (AntiSMASH) v5 using Integrated Microbial Genomes Atlas of Biosynthetic gene clusters (IMG-ABC) web server tools predicted P. peoriae IBSD35 harbored 25 BGCs 50,51 . LC-MS sequencing of RP-HPLC purified sample have predicted 15 protein lists (Table 3).
Paenibacillus peoriae IBSD35 harbored resistance genes for beta-lactam, vancomycin, cationic antimicrobial peptide (CAMP), platinum drug, and antifolate drug [52][53][54][55] . The Rapid Annotation of microbial genomes using Subsystems Technology (RAST) tool predicted that the P. peoriae IBSD35 genome harbored 29 compounds resistant www.nature.com/scientificreports/ to antibiotics and toxic compounds as compared to the metabolic reconstruction of P. polymyxa E681. They shared 23 compounds, while one is unique in the P. peoriae IBSD35 and nine are unique in P. polymyxa E681. Paenibacillus peoriae IBSD35 harboring 18 NRPS indicated that nonribosomal peptide compounds are the most abundant SMs of our understudy strain IBSD35 genome (Fig. 6A). The genome encompassed three gene clusters-one Trans-acyltransferase polyketide synthase (Trans AT-PKS) and two clusters of Trans AT-PKS. Two BGCs are associated with Butyrolactone, and one each for Type III-polyketide synthase (T3PKS), bacteriocin, phosphonate, lanthipeptide, NRPS-like, polyketide synthases-like, and lasso peptide. At least 12 BGCs start from a position, and 2 BGCs code rhizomides A, B, and C as predicted by the Antibiotics and Secondary Metabolite Analysis Shell-Minimum Information about a Biosynthetic Gene Cluster (AntiSMASH-MIBiG) comparison (Table 4).
Orthologous gene clusters of P. peoriae IBSD35 genome. OrthoVenn analysis has shown the selected Paenibacillus species (P. peoriae HS311, Paenibacillus sp. JDR-2, P. polymyxa E681, P. polymyxa SC2, P. terrae HPL-003, and P. peoriae IBSD35 formed 5615 clusters, 5296 orthologous clusters (at least contains two species), and 2125 single-copy gene clusters (E-value = 1e−2 and inflation value = 1.5) (Fig. 6B). The OrthoVenn diagram has shown that all 6 Paenibacillus species have shared 2241 gene clusters suggesting their conservation in the lineage. Additionally, it has 6-clusters specific to P. peoriae IBSD35 that might be lineage-specific gene clusters. The single-copy genes have suggested they maintained single-copy status through evolutionary time after the divergence of these species.
Central carbon metabolism (CCM) of P. peoriae IBSD35 genome. The metabolite precursors of P.
Commensal lifestyle of P. peoriae IBSD35. The genome was predicted to harbor sugar-specific component phenotypes, thus possibly utilizing the host plant trehalose, l-arabinose, sucrose, and maltose. It harbored   The branch lengths are scaled in terms of GBDP distance formula d5. (Leaf labels are annotated by affiliation to species (1) and subspecies (2) clusters, genomic G + C content (3), δ values (4), overall genome sequence length (5), number of proteins (6), and the kind of strain (7). User-provided GenBank accession IDs are shown in parentheses; master record accessions are truncated (Percent G + C-43.5-72.

Discussion
Antibiotics have revolutionized the treatment of tropical infections, communicable diseases, food-borne, and other poverty-related infectious diseases. However, the efficacy is short-lived and turned into a resilient clinical problem eliciting innumerable public health crises and causing economic burden 1,2,4 . Therefore, there is a continuous need for a new antimicrobial agent with high specificity, efficacy, efficiency, and anti-resistant to tackle the AMR challenge 2,64 . AMPs have prospects because of their antibacterial, anticancer, antiviral, antifungal, anti-aging, and anti-parasite, regulating cell proliferation, extracellular matrix production, cellular immune responses, and adjuvant characteristics 15,16,65 . Lately, many important SMs have reported from endophytes of medicinal plants from less explored sources which offered unprecedented opportunities to find novel antimicrobial agents 5,6 . Moreover, the availability of ever-increasing genome sequences, a comprehensive proteomics database, and advanced bioassay techniques offer a platform for the dereplication of old compounds, effective search, prediction, and design of novel AMP to counter AMR 19,20 . Paenibacillus peoriae IBSD35 from the stock solution has retained antimicrobial activity against S. aureus ATCC 25923, indicating its inherent antimicrobial potential 66 . Endospore forming traits may help its endophytic association with the host plant 67 . Its neutralophilic characters might help neutralize its cytoplasm relative to the plant cell to meet the pH challenges. It might be through direct active uptake or efflux of protons from the cells or tissues 29 . These indirectly suggested host-plant physiology as well. The optimum growth condition of the strain IBSD35 recorded at 38°, and pH 6.8-7 in LB medium indicated the mesophilic traits. The cultivable characters confirmed P. peoriae IBSD35's ability to live as a free-living bacterium in laboratory conditions. An antimicrobial protein mixture precipitated from LB fermentation broth with 70% ammonium sulfate suggests that at least one or more of the putative 25 BGCs of P. peoriae IBSD35 might express antimicrobial metabolites. The 700 mM NaCl eluent from DEAE-C column chromatography has retained activity against S. aureus ATCC 25923. The RP-HPLC peaks P4 and P5, absorption at λ205 and λ214, suggested the presence of amino acids and peptide bonds with other impurities. The absorbance at λ280 confirmed the presence of proteins 68,69 . Antimicrobial protein total yield was low at 0.05%, which can be enhanced using an advanced purification system or by up-regulating its BGC expression. MIC of peoriaerin IBSD35 at 0.04 µg µl −1 indicating its high specific activity and potency against S. aureus ATCC 25923 compared with ampicillin and nisin at 1 mg ml −1 .
The purified AMP has shown antimicrobial activity against S. aureus ATCC 25923, E. coli ATCC 25922, C. albicans ATCC 10231, K. pneumoniae ATCC 4352, and S. typhimurium ATCC 14028 connoted its broadspectrum antimicrobial character (Tables S6, S7). The sample has retained activity after prolonged storage, and its resistance to degradative enzymes indicated its potential for drug development against AMR. Antimicrobial activity was altering at different stages of purification. It might be due to the disintegration of proteins. The treatment of SDS and high-temperature slightly increased the activity. It might be from the disintegration or aggregation of the proteins and other biomolecule complexes (Table S3). The sample can withstand autoclaving at 121 °C for 15 min presenting a potential antimicrobial agent and food preservatives 17,21,70 .
The peptide sequences generated using LC-MS/MS coupled to a quadrupole time-of-flight (Q-TOF) had aligned with P. peoriae IBSD35 proteome using proteomics tools 21,22,24,33,38 . The sequence alignment using the Antimicrobial Peptide Database (APD3) tool indicated that peoriaerin IBSD35 is 38.23% similar to fermentin from Lactobacillus fermentum 20,25 . Peoriaerin IBSD35 is a trans-membrane helix with an extended strand of 10.71%, beta-turn of 21.43%, and a random coil of 64.00% (Fig. 3, Supplementary Fig. S2). It has a high percentage of helix and residues embedded in the membrane, suggesting at least some part of the sequence may embed in the hydrophobic region of the membrane 20 . Peoriaerin IBSD35 has a total net charge of + 2 and a theoretical pI of 8.86. These properties indicated an antimicrobial peptide nature. The aliphatic index was 56.07, and the grand average of the hydropathicity index (GRAVY) was − 0.132 25 . Antimicrobial action was visible after 1 h of incubation. The slower kinetics of killing suggested that peoriaerin IBSD35 might penetrate the cell membrane and transport it inside the cell cytoplasm to cause the effect 18 . However, the leakage of DNA and proteins was lacking.
The draft genome sequencing and analysis of P. peoriae IBSD35 provided insights into its endophytic characters, antimicrobial traits, and interaction with the host plant 10,11,13 . The characteristics contributing to strain IBSD35 epiphytic fitness are iron acquisition, capacity to colonize plants, and stress-tolerant. Additional functional genes suggested P. peoriae IBSD35's close association with the environment through nitrogen fixation 14,37,40 . However, there are about 980 (18.50%) protein-coding genes without assigning any function, as per IMG AP v.4.16.6 (Table S4), and their relationship to molecular networks of the database is unknown 28,29,49 . Possibly, they may be endophytic specific, which needs further study to ascertain.
The genes encoding flagellins and flagellar motor proteins are visible in P. peoriae IBSD35. They helped bacterial motility in the plant-associated lifestyle 28,46,71 . The flagellin proteins (Flg) also facilitated the endophyte to pass through the first line of the plant immune defense system. Moreover, the presence of flagellin (fliC), glycerol kinase [EC: 2.7.1.30], elongation factor (Tu), and molecular chaperone (htpG) are crucial for plant-pathogen interaction indicating P. peoriae IBSD35 close association with the host plant 72 . Generally, motility enabled bacteria to move and colonize plants and systematically spread within the plant, promoting the endophytic lifestyle. The TCS genes reinforced traits needed for thriving in heterogeneous, highly competitive, and adverse environments 44,73 . The proteins or SMs might transport from the bacterial cytoplasm through its Tat and Sec secretion systems 61,74 .
The TCS represented a primary means by which an organism sensed and responded to stress and changing physiological environments 45,71 . The putative genes encoding small acid-soluble proteins (SASPs) annotated in P. peoriae IBSD35 generally helped the bacterial endospore DNA against heat, UV-radiation, and enzymic degradation 45 . They are responsible for sporulation septum, engulfment, spore morphogenesis, and germination in harsh environments. The genome ATP-binding cassette (ABC) transporter proteins are other plausible candidates for genes mediating the export of small molecules across the cells 44  www.nature.com/scientificreports/ genes conferring tolerance to oxidative stress in the strain IBSD35 genome supported the proposed importance of oxidative stress tolerance to fitness in symbiotic association. It is consistent with P. peoriae IBSD35 endophytic lifestyle 60,61 . However, there was no evidence of the biosynthesis of phytotoxins or cellulases. The KEGG pathway database analysis (www. kegg. jp/ kegg/ kegg1. html) indicated that carbohydrate and amino acid metabolism accounted for a large proportion of the P. peoriae IBSD35 genome 44,45,50 , and its CCM network analysis included GG, TCA, PPP metabolites, and pyruvate metabolism 57 . Most organisms have shared a common core to their metabolic networks 58,59 . The CCM pathways convert sugars into metabolic precursors and provide the entire cell biomass, including the SMs 57 . P. peoriae IBSD35 harbored a collection of metabolite and drug efflux systems and bioremediation gene clusters which indicated a need to protect against toxic concentrations of metabolites or metabolic analogs 54 . It has shown the strain's capability for bioremediation, survival defense against oxidative stress, and production of antimicrobial compounds and toxins. A repertoire of NRPS (72%) and their expressions might neutralize the competing microbes and antagonistic biomolecules. Gene resistance to β-lactam, cationic AMPs, and antifolate with the antibiotic BGCs in the strain IBSD35 genome supported that the clusters might involve in antibiotic production, which is vital to avoid self-toxicity from its own antibiotics production 54,55 .
The SMs gene clusters predicted from the P. peoriae IBSD35 genome showed low similarity with other BGCs. It exhibited 100% similarity for fusaricidin and polymyxin BGCs in the MIBiG cluster database (Table 4) 50,51,75 . A repertoire of NRPS SMs might help microbes in existence and communication, and their elucidation will facilitate the finding of novel bio-molecules 52 . They might also provide a competitive advantage in the battle for resources and adaptive mechanisms for survival in host plant cells or tissues 52,53,55 . The different number of BGCs in the analysis of the genomes reflected their habitats, genetics, and functional differences. Thus, a difference in genomes' SM is a difference in environment and resource competition 55 . Generally, two closely related strains share most of their genes and will not be unique to the individual species 76,77 . The in-silico identification of PKS, NRPS, or SM gene clusters revealed considerable differences between the closely related species supporting that P. peoriae IBSD35 is a unique species slightly different from free-living or symbiotic rhizobium bacteria.
The terpenoids and polyketides biosynthetic are the backbones of other SMs 78 . Pathwhiz has been used to illustrate the type II polyketide biosynthesis pathway of the P. peoriae IBSD35 genome predicting its proteins, enzymes, SMs, and other products 79 . Dihydrokalafungin, oxytetracycline, tetracycline, nogalavinone, elloramycin, and tetranomycin F are predicted to be derived from simple carbon building blocks of the pathway (Supplementary Fig. S7). The KEGG pathway analysis (www. kegg. jp/ kegg/ kegg1. html) indicated that terpenoids have derived from 2-C-methyl-d-erythritol 4-phosphate/1-deoxy-d-xylulose 5-phosphate (MEP/DOXP) pathway in P. peoriae IBSD35. The non-mevalonate Pathway of strain IBSD35 has the potential to produce isoprenoids, antibiotics, and anticancer alkaloids 80 . However, the expressions of SM clusters are typically under environmental and developmental control mediated by complex regulatory cascades that relay signals to the pathway-specific switches 53 .
The P. peoriae IBSD35 genome harbored the putative genes of the nitrogen cycle. Nitrogen fixation by nitrate to ammonia and other biologically reduced forms might incorporate into amino acids and other vital compounds through nitrogen metabolism 40 . Nitrogen fixation is energetically costly, and therefore, it is tightly regulated at the transcriptional level by sophisticated regulatory networks that respond to multiple environmental cues 40,41,63 . However, the nitrogen fixation regulator (nifL-nifA) was not visible, but it was difficult to discern how oxygen levels affect the regulation of nitrogen fixation in our present study (www. kegg. jp/ kegg/ kegg1. html). However, it is clear that the nitrogen fixation is different from those previously described for other γ-proteobacteria and possibly depends on the host plant for its nitrogen source 63 . It is one of the unique characteristics of P. peoriae IBSD35 endophytic traits.
The Type (Strain) Genome Server annotation (TYGS) and PGAP annotation predicted that the strain IBSD35 has a close species relation with other P. peoriae strains. Phylogenetically, P. peoriae IBSD35 is closely related to P. peoriae FSL J3-0120. However, the protein Basic Local Alignment Search Tool (BLAST) indicated P. peoriae IBSD35 close similarity with P. polymyxa SC2 (E-value: 0.0, Score: 2273, Ident.: 98.9%). Therefore, the understudy genome is within the species of P. peoriae and related to the P. polymyxa species. The genomic data quality and analysis need further improvement as some of the selected genomes are draft and limited for web server analysis. The variation in genome size suggested that different strains of the same species from different habitats potentially produced their own unique NRPS products. Several gene clusters are common in selected Paenibacillus species suggesting their conservation in the lineage 49 . Paenibacillus has only been designated a separate species since 1993 12,13 . Some lineage-specific clusters and the predicted unique clusters in our understudy genome could be involved in SMs biosynthesis 12 .
The unique genes or 2125 single-copy genes from the six Paenibacillus genomes might encode some of the functions in the cell, as they are found only in one organism. These genes might be involved in environmental adaptation, specific competition with other species, or speciation 55,76 . The synteny dot plot indicated P. peoriae IBSD35 resemblance to its genus. There is also evidence of gene sharing (Mitogen-activated protein kinase (MAPK) pathway from plant, nitrogen fixation gene cluster) and horizontal transfer of genes.
Given the P. peoriae IBSD35 potential applications in agriculture, medicine, industries, and bio-remedy 14 , its genome sequencing and its extracellular metabolite analysis are crucial to the basic and applied research to find novel antimicrobial agents. Genome mining has enabled the prediction of the putative BGC region in the P. peoriae IBSD35 by linking a protein group from LC-MS/MS data and connecting these natural products to the genes 24 . The genome information and the proteomics analysis provided here will further enhance the study of environmental information processing and its antimicrobial pathways 55,64,65,81,82 . It will also facilitate the understanding of evolutionary relationships among Paenibacillus species. Moreover, the less explored ecological niches provide new sources of an unprecedented opportunity to find novel microbes and metabolites to counter AMR to our already exhausted sources of bio-molecules.

Conclusions
Paenibacillus peoriae IBSD35 is an endophytic bacterium. It is a mesophile, endospore forming, and neutralophile bacterium. It has a linear DNA topology with a total length of 5,862,582 bp. It harbored 25 SMs BGCs, out of which 18 BGCs are NRPS. The P. peoriae IBSD35 closest species reference genome is P. peoriae KCTC 3763 with a symmetrical identity of 62.7495% and a symmetrical identity of 89.4884% with P. peoriae FSL J3-0120. The CFS of P. peoriae IBSD35 exhibited a broad spectrum of antimicrobial activity against S. aureus ATCC 25923, E. coli ATCC 25922, C. albicans ATCC 10231, K. pneumoniae ATCC 4352, and S. typhimurium ATCC 14028. P. peoriae IBSD35 inoculum's optimum growth pH was observed at 6.8-7. It is a mesophile bacterium that grows best at 38 °C. The supernatant antimicrobial activity arbitrary unit was 1600 AU ml −1 . The putative extracellular AMPs were purified using salting out and chromatography. A 15 proteins list and AMP sequences have predicted from LC-MS coupled to Q-TOF. An AMP from the protein lists with accession no. A0A2S6NUT6 was selected. It has been given the name peoriaerin IBSD35, based on its source name. The amino acid sequence of peoriaerin IBSD35 is AAGIQAQAGFGLSDSIQGTGKQKCSFCK with a MW of 2802.17 Da. Its sequence backbone has a large portion of helix and residues embedded in the membrane. It exhibited high antimicrobial activity against S. aureus ATCC 25923 at range of 0.04 µg µl −1 .
Paenibacillus peoriae IBSD35 exhibited a close phylogenetic relationship with P. peoriae FSL J3-0120 and P. polymyxa CR1. The genome analysis indicated that it harbored nitrogen fixation genes and other putative nitrogen-fixing proteins but without a nitrogen fixation regulator (nifL/A). Its genome has a repertoire of 18 NRPS BGCs. The type II polyketide biosynthesis pathway revealed that terpenoids might be exclusively through (MEP/DOXP) pathway in P. peoriae IBSD35. Combining the genomics, mass spectrometry, and proteomics results obtained from P. peoriae IBSD35 through its extracellular biomolecules will enhance our search for AMPs that are stable, specific, and effective to counter AMR. The potential for applying comparative genomics to get evolutionary insights and discover novel AMPs from less explored regions will increase the probability of finding novel bio-molecules. P. peoriae IBSD35 culture and its morphology and genomic characters. An endophytic bacterium P. peoriae IBSD35 isolated from the stem of M. pachycarpa in our previous experiment was picked in our present context [7][8][9]66 . It was sub-cultured from 15% phosphate buffer saline (PBS) glycerol stock preserved in the Institute of Bioresources and Sustainable Development-Microbial Resources Division (IBSD-MRC) repository (Accession No. MRC-75001) by thawing for 5 min at room temperature (27 °C) 83 . A 100 µl of stock culture was pipetted using a micropipette and spread on a 25 ml LB agar plate. It was incubated at 38 °C to check the contamination and colony purity 84 . A single colony was streaked and sub-cultured repeatedly to maintain its growth curve character, colony purity, viability, and productivity 83,85 . The cut-well agar diffusion antimicrobial assay against S. aureus ATCC 25923 was performed 86 . Chemicals were added to the inoculum to avoid bacterial contamination in the fermentation system from instruments, flasks, nutrients, and pipetting.

Materials and methods
The strain IBSD35 pure colony was smeared on a glass slide and observed under the phase-contrast microscope (Zeiss Imager. Z2) 85 . The gDNA was extracted from an overnight grown culture using the cetyl trimethyl ammonium bromide (CTAB) method 40 . The quality and quantity of the gDNA were determined by A260/A280 ratio using UV-spectrophotometer (Nanodrop, Shimadzu Biotech Bio Spec-nano) and assessed in a 0.8% agarose gel electrophoresis. The genomic and molecular characters of the genome were analyzed by draft genome sequencing using Illumina HiSeq 2500 platform 87 .
Screening of antimicrobial activity. A 200 ml of P. peoriae IBSD35 inoculums was harvested by centrifugation (Sorvall ST 16R Centrifuge) at 3200×g for 15 min at 4 °C, and the supernatant was collected in an Eppendorf tube 88 . The spectrum of antimicrobial activity was tested using cut-well agar diffusion bioassay against the representative Gram-positive, Gram-negative, and fungal test pathogens (S. aureus ATCC 25923, E. coli ATCC 25922, C. albicans ATCC 10231) with a slight modification of Clinical and Laboratory Standard Institute (CSLI) methods [89][90][91] . The test pathogen lawn was prepared by spreading 20 µl of pathogen inoculums (0.5 Macfarland standard) over the Muller Hilton Agar (MHA) plate surface. A diameter of 3-5 mm was punched on the MHA surface with a sterile tip, and the wells were loaded with the test solution (20-100 µl) at desired concentration 86 . The MHA plates were incubated at 37 °C overnight as per the test pathogens' best growth condition. The activity zones of inhibition diameter were measured to quantify the antimicrobial activity.
Optimum growth pH and temperature for antimicrobial production. 2 l of the production medium (pH 7 ± 2) was inoculated with an overnight grown culture of P. peoriae IBSD35 and incubated in a shaker incubator at 80 rpm (Eppendorf innova R42 Rotary shaker). Its optimum pH, OD, and temperature concerning antimicrobial activity were determined by aseptically aspirating out the inoculum from the fermentation broth 66 . The production medium was harvested by centrifugation at 3200×g for 15  www.nature.com/scientificreports/ antimicrobial activity was tested by serial dilution, and its arbitrary unit was calculated against S. aureus ATCC 25923 31 . To rule out any false-positive inhibition caused by acid production and catalytic enzyme, 0.6% of calcium carbonate and a unit (10 mg/ml) of 0.01% catalase were added to the production medium 84 . Toluene was added to avoid contamination from other microbes 88 . Precipitation and purification of the antimicrobial protein mixture. The 6-day-old inoculum of 2 l of CFS was harvested from the 6-day-old inoculums by centrifugation at 3200×g for 15 min at 4 °C. Its pH was adjusted to 3 with 2 N hydrochloric acids (HCl) to kill the experimental strain and re-adjusted to the original harvesting state (pH 6.8) after 4 h by adding 2 N sodium hydroxide (NaOH) 88 . The supernatant pH was readjusted to the original harvesting state (pH 6.8) by adding 2 N NaOH. The CFS was filtered through a 0.2 µm filter (Avixa) and precipitated in 70% ammonium sulfate (NH 4 ) 2 SO 4 ) followed by centrifugation (3200×g, 35 min at 4 °C) 92 . The precipitated protein from centrifugation was collected. The resulting pellet was re-suspended in 30 ml distilled water and dialyzed using PBS in a dialysis membrane (12.4 kDa cut-off) with intermittent buffer changing 36 .
The pellet was re-dissolved in 30 ml of distilled water and loaded in diethylaminoethyl cellulose (DEAE-C (Sigma Aldrich). A weakly positive resin, DEAE-cellulose was loaded for anion exchange column chromatography 93 . Sample was eluted out using 0.1, 0.2, 0.3, 0.4, 0.5 and 0.7 M or 100-700 mM NaCl gradient elution and the OD were recorded at selected wavelength of λ205, λ214, and λ280 15,16 . The fractions were pooled together and lyophilized into powder form and tested for retention of antimicrobial activity according to the CLSI assay 89,90 . Fractions showing antimicrobial activity were pooled together for lyophilization and further purified with RP-HPLC 94 .
RP-HPLC purification of protein mixture. The partially purified sample was again purified using ZOR-BAX 300 SB-C-18 semi-preparative (9.4 × 250 mm) 5 microns 91,94 . The column was irrigated with 0.1% TFA in 20% ACN for 45 min at an isocratic flow rate. The sample (150 µl per experiment) was loaded on the HPLC (UFLC Shimazdu) with semi-preparative Agilent ZORBAX 300SB reverse-phase C-18 of 5 µm and 9.4 × 250 mm column and run with 0.1% ion-pairing reagent, trifluoroacetic acid (TFA) 68,94 . The RP-HPLC mobile phase consisted of (i) HPLC-grade water containing 0.1% TFA and (ii) 20% acetonitrile (ACN) in an isocratic system at a flow rate of 2.5 ml min −193 . Eluents were monitored at a wavelength of λ205, λ214, and λ280 using a UV-spectrometer (Eppendorf, Biospectrometer) and collected manually. The fractions which exhibited higher antimicrobial activity were pooled together and collected separately for further analysis. The total protein concentration and activity, specific activity, yield percentage, and purification fold of the extract compared to the initial starting crude extract were calculated using UV-Spectroscopy 15,16 . The UV-spectrophotometric method for antimicrobial protein concentration was calculated as: concentration (mg/ml) = (1.55 × A280) (0.76 × A260) × dilution factor 69 .
The effect of external factors on sample antimicrobial activity. The sample was evaluated for sensitivity to degradative enzymes, detergents, organic solvents, temperature, and pH changes 78,84 . The retention of antimicrobial activity after the treatment was qualitatively determined through cut-well agar diffusion bioassay against the reference pathogen, S. aureus ATCC 25923 as described earlier 91 . For the pH stability test, the sample dissolved in dist. water was pre-adjusted to pH 2.0-8.9, followed by incubation at 38 °C for 12 h. The sample temperature sensitivity was tested by exposing to temperatures ranging from 20 to 100 °C at the interval of 10 °C for 2-3 h, and additionally at 121 °C for 15 min by autoclaving 86,91 .
The samples were treated with different degradative enzymes at the concentration of 1 mg ml −1 for 2-3 h at room temperature to test the stability against enzymes 84 . The organic solvent and test sample were mixed in a 1:1 ratio and incubated at 27 °C to evaporate all the organic solvent, and the samples were assessed for retention of antimicrobial activity. The samples were treated with detergents in a 1:1 ratio and checked for retention of antimicrobial activity 86,90 . Negative control consists of PBS (pH 7) and the untreated sample solution was used as a positive control.
Mass spectrometric analysis of peptide mixture. The experiment was performed using EASY-nLC 1000 system (Thermo Fisher Scientific) coupled to a Q-Exactive mass spectrometer (Thermo Fisher Scientific) equipped with a nano-electrospray ion source 23,24 . 1.0 µg of the RP-HPLC purified peptide mixture was resolved using a 15 cm PicoFrit column (360 µm outer diameter, 75 µm inner diameter, 10 µm tip) filled with 1.9 µm of C18-resin (Dr. Maeisch, Germany). The peptides were loaded with buffer A and eluted with a 0-40% gradient of buffer B (95% ACN, 0.1% formic acid) at a flow rate of 300 nl/min for 90 min 24 . The source heater was set at 300 °C to turn the sample into a vapor for analysis. The UPLC-separated samples were then analyzed by electrospray ionization at 4.5 kV and the peptides were dissociated with collision induced dissociation (CID) for MS/MS spectra 95 . MS data was acquired using a data-dependent top10 method dynamically choosing the most abundant precursor ions from the survey scan.
One sample was processed and 1 RAW file generated was analyzed with a Proteome Discoverer against P. peoriae IBSD35 Uniprot reference proteome database 33 . For the Sequest search, the precursor and fragment mass tolerances were set at 10 ppm and 0.5 Da, respectively. Trypsin was used to generate peptides, carbamidomethyl on cysteine as fixed modification, and oxidation of methionine, and N-terminal acetylation was considered as variable modifications for database search. Both peptide spectrum match (PSM) and protein false discovery rate (FDR) were set to 0.01 FDR.
Physiochemical properties of the protein and AMPs were analyzed using the ExPASy-ProtParam tool (http:// web. expasy. org/ protp aram) and APD3 Antimicrobial Peptide Calculator 23 www.nature.com/scientificreports/ analyzed using SOPMA 96 . The 3-D conformation structure of the peptide was predicted using PEP-FOLD 3 de novo modeling and deposited in ModelArchive 26,27 . The model was built based on the target-template alignment using ProMod3 97 .
Peoraerin IBSD35 mode of action imaging through scanning electron microscopy. The AMP mode of action against the test pathogens was checked by the absorbance at λ260 and λ280 with UV-spectrometry. A direct visualization of peptide-induced S. aureus cellular damage and morphological changes were evaluated by SEM 98 . The samples were prepared by incubating S. aureus ATTC 25923 with the test sample as described earlier 89,90 . S. aureus were treated with antimicrobial agent at 1× MIC for 2-3 h and incubated at 30 °C ± 0.2. The inoculants were harvested by centrifugation as described earlier 87 . The pellet was washed with 0.1 M PBS (pH 7.2) and fixed in 2.5% glutaraldehyde and 2% paraformaldehyde made in PBS 97 . The fixed sample was further processed for coating and imaging 99 . The test pathogen without any treatment served as the experimental control.
Genome sequencing and assembly. The gDNA integrity was confirmed using a 0.8% agarose gel electrophoresis 97 . The draft genome sequencing was performed through Illumina HiSeq 2500 system 34 . The cleaned reads were subjected to the Kmer genie to predict the optimal k value and assembly size 100 . De novo assembly was performed using ABySS v.2.0 42 . The predicted genes were matched with bacterial proteins from the Uniprot database using the BLASTX program 101 . Genes that could establish homology relationships (E-value ≤ 10 −5 and similarity score ≥ 40%) were retained in the annotation pipeline for further annotation while the others remain unannotated 28-30 . Genome annotation and bioinformatics analysis. JGI-IMG Annotation Pipeline v.4.16.6 28,29 and the NCBI PGAP were used for gene annotation 30 . The gene functional prediction was performed by BLAST against the databases 101 .
Comparative genomic analysis. The close strains of P. peoriae IBSD35 (NZ_PTJM01000000) 7,66 were selected from the NCBI database (P. peoriae KCTC 3763 102 , P. polymyxa E681(NZ_CP048793) 75 , P. polymyxa SC2 (NC_014622) 103,104 , P. vortex V453 (NZ_ADHJ01000000) 105 , P. terrae HPL-003(NC_016641) 106 , Paenibacillus sp. JDR-2 (NC_012914) 107 , P. polymyxa CR1(NC_023037) 108 , B. subtilis subtilis AG1839 (NZ_CP008698) 109 , S. coelicolor A3(2) (AL645882) 110 , and P. peoriae HS311 (NZ_CP011512)). Bacillus subtilis subtilis AG1839 is selected because Paenibacillus represents a new genus from Bacillus 12,13 . S. coelicolor A3(2) is the model organism for SM BGC studies. P. peoriae HS311 represents the completely sequenced genomes of Paenibacillus and serves as the reference for the natural variation within the genus. P. peoriae KCTC 3763 represents the closest species reference genome with a symmetrical identity of 62.7495%. OrthoVenn was used for Venn diagram drawing which is based on the protein sequences clusters (E-value = 1e−2 and inflation value = 1.5) 76 . Web Gene Ontology Annotation Plot (WEGO) was used to compare the provided gene datasets and plot the distribution of GO annotation into a histogram 38 . SMs biosynthesis gene clusters were analyzed with AntiSMASH V.5 and JGI-IMG/ABC, and the type II polyketide biosynthesis pathway was drawn using KEGG and PathWhiz tool 43,50,51,79 . All comparative analyses were performed using IMG/M and RAST tools 29,37,39 . Genome-based taxonomy and phylogenetic tree. Genome-based taxonomy was determined using TYGS which infers genome-scale phylogenies and estimates for species and subspecies boundaries and automatically determined closest type genome sequences 111,112 . The pair-wise digital DNA:DNA hybridization (dDDH) values between user genomes and the selected types-strain genomes were performed 77 . The dDDH values are provided along with their confidence intervals (C.I.) and difference of G + C% using the Genome BLAST Distance Phylogeny (GBDP) formula d5 77,78 . The average nucleotide identity with the closely related species was determined using the OAT tool 113 . The phylogenetic tree was inferred with FastME 2.1.6.1 from GBDP distances calculated from genome sequences 114 . The AMP model was built based on the target-template alignment using ProMod3 97 .

Data availability
The datasets generated or analyzed during the current study are given in the