Complete Genome Sequence of the Butirosin-Producing Bacillus vitellinus NBRC 13296 and Its Reclassification to Paenibacillus chitinolyticus

Abstract

Butirosins are naturally occurring aminoglycoside (AG) antibiotics featuring a 4,5-disubstituted 2-deoxystreptamine (2-DOS) with a (2S)-4-amino-2-hydroxybutyrate (AHBA) side chain. This side chain has been shown to confer resistance against AG-modifying enzymes, leading to ongoing studies on the butirosin biosynthetic pathway and the corresponding enzymes. Butirosin is produced by Niallia (formerly Bacillus) circulans and Bacillus vitellinus, with most research focused on the first strain. To date, no whole-genome analysis has been performed on B. vitellinus. In this study, we sequenced the complete genome of B. vitellinus NBRC 13296 and performed a comparative analysis of different butirosin biosyntheric gene clusters (BGCs), including those from N. circulans. The complete genome of B. vitellinus NBRC 13296 comprises a 6,331,192-base circular chromosome with GC content of 52.68%. The annotation revealed the presence of 5605 CDSs, 70 tRNA genes, 30 rRNA genes, and 3 ncRNA genes in NBRC 13296. The highest dDDH and ANI values between NBRC 13296 and the most closely related type strain, Paenibacillus chitinolyticus KCCM 41,400, were 97.8% and 98.66%, respectively. Based on these genome-based comparative analyses, we propose reclassifying B. vitellinus NBRC 13296 as P. chitinolyticus. Genome mining revealed 18 gene clusters encoding the biosynthesis of diverse secondary metabolites in the genome of B. vitellinus NBRC 13296, indicating the enormous biosynthetic potential of this strain. The predicted structural diversity of the secondary metabolites includes aminoglycosides, PKS, NRPS, PKS–NRPS hybrids, metallophores, phosphonates, terpenes, β-lactones, and RiPP peptides. We then comparatively characterized the butirosin BGCs previously studied in several N. circulans strains. Additionally, the comparative genome analysis revealed complete butirosin BGCs identified from P. chitinolyticus KCCM 41,400, P. chitinolyticus NRRL B-23119, P. chitinolyticus NRRL B-23120, P. chitinolyticus B-14908, P. chitinolyticus YSY-3.1, P. chitinolyticus JMW06, Paenibacillus sp. GbtcB18, Paenibacillus sp. HGH0039, and Paenibacillus sp. MZ04-78.2. Finally, we identified the core region consisting of BtrS, BtrN, BtrM, BtrL, BtrA, BtrB, BtrC, BtrD, BtrD, BtrE, BtrF, BtrG, BtrH, BtrI, BtrI, BtrJ, BtrK, BtrO, BtrP, and BtrV, followed by an upstream region organizing BtrQ, BtrW, BtrX, BtrY, and BtrZ in the same transcriptional direction and sequential genetic arrangement, and a downstream region organizing various proteins based on BtrT, BtrR2, BtrU, and BtrR1. Our study provides insights into the reclassification of B. vitellinus NBRC 13296 to P. chitinolyticus and suggests the need for continued studies on butirosin biosynthesis from an enzymatic perspective.

1. Introduction

Aminoglycosides are an important class of antibiotics used to treat serious infections caused by several Gram-negative and Gram-positive bacteria, despite their nephrotoxic and ototoxic side effects. The mechanism of action of aminoglycosides involves binding to the 30S ribosomal subunit, which interferes with protein synthesis by causing errors in translation—specifically in codon reading—thereby disrupting protein synthesis [1,2]. Aminoglycosides consist of highly functionalized aminosugars and deoxysugars linked by glycosidic bonds to an aminocyclitol aglycone, with most aglycones containing a 2-deoxystreptamine (2-DOS) moiety, to which other amino sugars are attached at the 4 and 5 or 4 and 6 positions of the 2-DOS carbons. Aminoglycoside producers primarily belong to the actinomycetes group of bacteria, but butirosin is one of the few aminoglycosides produced by a non-actinomycete bacterium [3,4].

The (S)-4-amino-2-hydroxybutyrate (AHBA) group attached to the C-1 amine of the 2-DOS moiety in butirosin has long been of interest because this AHBA moiety can prevent the action of several aminoglycoside-modifying enzymes (AMEs), such as aminoglycoside N-acetyltransferases (AACs), which can theoretically or practically cause aminoglycoside resistance. For example, amikacin is a semisynthetic aminoglycoside with an AHBA group attached to the C-1 of the 2-DOS aglycone of kanamycin and is considered one of the few antibiotics that can overcome various AME mechanisms [5,6,7]. Butirosin was identified as a pair of epimers, butirosins A and B, from Niallia (formerly Bacillus) circulans in 1971 and was subsequently discovered in Bacillus vitellinus [8,9]. B vitellinus Z-1159 was first isolated in 1971 by Takeda Chemical Industries, Ltd. (Osaka, Japan), where it was designated as FERM-P 1203. The strain was subsequently deposited under the accession number IFO 13296 at the Institute for Fermentation, Osaka (IFO), for the purpose of international patent registration. Following the transition of the IFO’s culture collection to the Biological Resource Center, NITE (NBRC), the strain was re-designated as NBRC 13296. The complete strain designation is as follows: B. vitellinus Z-1159 = FERM-P 1203 = IFO 13296 = NBRC 13296 = American Type Culture Collection (ATCC) 31,078 = Korean Collection for Type Cultures (KCTC) 3886. However, this strain is no longer available through the ATCC and is currently only accessible via the NBRC and KCTC.

To date, the whole-genome sequence (WGS) of N. circulans in the National Center for Biotechnology Information (NCBI) databases has been completed for the reference strain N. circulans NBRC 13626 (SAMD00046991), with additional WGSs reported for N. circulans DC10 (SAMN18824981), isolated from textile dye-contaminated soil sediments in India, and N. circulans GN 3 (SAMN14847575), isolated from purple rhizosphere soil growing the cabbage Brassica campestris in China. However, there are no reports of complete genome information for B. vitellinus, one of the main producer strains of butirosin. Here, we report the complete genome sequence of B. vitellinus NBRC 13296 to better understand the biosynthesis of butirosin and other bioactive secondary metabolites. The genome was sequenced using long-read PacBio technologies to obtain high-quality genomic sequences [10,11].

Meanwhile, bioinformatics software such as antiSMASH, a platform for the automated genome mining of secondary metabolite producers, can be used to rapidly identify, analyze, and annotate BGCs from genome-wide information and identify similarly characterized gene clusters in the MIBiG repository [12,13]. Finally, comparative genomics analysis using next-generation sequencing (NGS) techniques and bioinformatics software confirmed the presence of a butirosin BGC.

Interestingly, during the BLASTP search using antiSMASH 6.1.1, butirosin BGCs were identified as biosynthetic enzymes in the type strain Paenibacillus chitinolyticus KCCM 41,400 (SAMN08222605), as well as in P. chitinolyticus NRRL B-23119 (SAMN27675096), P. chitinolyticus NRRL B-23120 (SAMN27675097), P. chitinolyticus YSY-3.1 (SAMD00444452), P. chitinolyticus JMW06 (SAMN19998407), P. chitinolyticus B-14908 (SAMN33770086), Paenibacillus sp. GbtcB18 (SAMN18679176), Paenibacillus sp. MZ04-78.2 (SAMN28689874), Paenibacillus sp. HGH0039 (SAMN02596731).), N. circulans ATCC 21557 (AJ781030), N. circulans ATCC 21558 (LC571042.1), and N. circulans SANK 72073 (AB097196). The full genome-derived 16S rRNA gene sequences, average nucleotide identity (ANI), and digital DNA–DNA hybridization (dDDH) suggest that all strains mentioned above should be classified as members of the same species. We propose the reclassification of B. vitellinus NBRC 13296 as P. chitinolyticus NBRC 13296 and an emended description of P. chitinolyticus. Finally, in this article, we aim to provide scientists with a comparative analysis of butirosin BGCs to assist in the study of the butirosin biosynthetic pathway and corresponding enzymes.

2. Materials and Methods

2.1. Bacterial Strains and Growth Conditions

Bacillus vitellinus NBRC 13296 was obtained from the NITE Biological Research Center (NBRC, Tokyo, Japan). The cells were grown in a tryptic soy broth (TSB) medium containing 1.7% tryptone (pancreatic digest of casein), 0.3% soytone (peptic digest of soybean), 0.25% glucose, 0.5% sodium chloride, and 0.25% dipotassium phosphate (BD Biosciences, Franklin Lakes, NJ, USA) at 30 °C for 24 h.

2.2. Genome De Novo Sequencing, Assembly, and Annotation

The genomic DNA of B. vitellinus was extracted and purified using the Nanobind DNA extraction kit, which produces HiFi reads on the PacBio system (Pacific Biosciences, Menlo Park, CA, USA). The gDNA obtained from the extraction was first sheared using the g-TUBE (Covaris), following the manufacturer’s protocol. SMRTbell libraries were created using the PacBio SMRTbell Express template preparation kit (v1.0). SMRTbell templates were annealed using the PacBio DNA/Polymerase Binding Kit P6. The PacBio DNA Sequencing Kit 4.0 and 8 SMRT cells were used for sequencing. SMRT cells (Pacific Biosciences, Menlo Park, CA, USA) using C4 chemistry and 240 min movies were captured for each SMRT cell using the PacBio Sequel II (Pacific Biosciences) sequencing platform. These sequences were assembled de novo using the CANU ver. 2.2 workflow. The assembly was circularized by Circulator (v. 1.5.5) and rearranged to start at the dnaA gene [14]. Regarding gene prediction and annotation, protein coding, tRNA, rRNA genes, and repeat regions were predicted using the NCBI Prokaryotic Genome Annotation Process (PGAP). Gene annotation was performed using RefSeq and the HMM library (TIGRFAM, Pfam, PRK HMMs) database [15,16].

2.3. Phylogenetic Analysis

Phylogenetic trees were constructed based on the complete 16S rRNA gene sequence derived from the whole-genome information of B. vitellinus NBRC 13296. The evolutionary tree based on the 16S rRNA sequences was constructed with the MEGA 11 package [17], applying neighbor-joining and maximum-likelihood algorithms after multiple alignments of the sequence data by the ClustalW program. This process primarily used BlastN homology searches and referenced BacDive, the largest worldwide database for standardized bacterial information, for type strains. The confidence of the tree topologies was assessed by 1000 bootstrap replicates.

2.4. Comparative Genomic Studies and Whole-Genome Relatedness

The digital DNA–DNA hybridization (dDDH) values of the B. vitellinus NBRC 13296 genome and its neighbors were calculated using the Genome-to-Genome Distance Calculator (GGDC 4.0) within the Type (Strain) Genome Server (TYGS) of the Leibniz Institute DSMZ [18]. The average nucleotide identity (ANI) values between B. vitellinus NBRC 13296 and its nearest neighbors were computed using the ANI calculator of Ezbiocloud, an online tool used to compare two prokaryotic genome sequences [19].

2.5. Secondary Metabolite and Butirosin BGC Analysis

To discover BGCs involved in the production of secondary metabolites, including butirosin, various computational programs, such as Known ClusterBlast, ClusterBlast, SubClusterBlast, ActiveSiteFinder, and Cluster PFam analysis, were employed. The PRISM 4 and BAGEL 4 tools were implemented with the default settings. Anti-SMASH 7.0 facilitated the identification, annotation, and analysis of the secondary metabolite BGCs across the genome [12]. Ribosomally synthesized and post-translationally modified peptides (RiPPs) and bacteriocins were mined by BAGEL 4 [20], while PRISM 4 version 4.4.5 was used for the comprehensive analysis of secondary metabolite structures and biological activities [21]. These sophisticated computational tools provide accurate predictions of the microbial secondary metabolite encoding potential and putative structures.

3. Results

3.1. Sequencing, Assembly, Phylogenetic Analysis, and Genomic Characteristics

In the whole-genome sequencing based on the PacBio Sequel IIe platform, the complete genome sequence of B. vitellinus NBRC 13296 was composed of one contig with a total length of 6,331,192 bps and average G + C content of 50.98%. A total of 5731 genes were identified in its genome, including 5605 annotated protein-coding genes and 92 tRNA, 30 rRNA, 3 ncRNA, and 1 tmRNA genes. Ten copies of 16SrRNAs were identified in the genome of B. vitellinus NBRC 13296, and variations at seven positions were also identified by alignment between the 16S rRNA sequences, totaling 1544 bp, using ClustalW. No plasmid was detected. In the 16S rRNA gene sequence analysis, B. vitellinus NBRC 13296 showed sequentially 99.66%, 95.71%, 95.60%, and 94.96% similarity to the type strains Paenibacillus chitinolyticus HSCC 596 (NR_040854.1), Paenibacillus gansuensis B518 (NR_043219.1), Paenibacillus lutrae N10 (NR_173496.1), and Paenibacillus favisporus GMP01 (NR_029071.1), respectively (Table S1). The phylogenetic tree constructed from the BLASTN, BacDive, and EzBioCloud 16S database [22] using the maximum-likelihood and neighbor-joining methods in the MEGA 11 application with 1000 bootstrap values showed B. vitellinus NBRC 13296 forming a clade with P. chitinolyticus, as shown in Figure 1 and Figure S1.

Comparative genomic analysis revealed that the digital DNA–DNA hybridization (dDDH) values between B. vitellinus NBRC 13296 and the closely related strain P. chitinolyticus KCCM 41400 were far above the 70% threshold value for species evaluation [23]. The average nucleotide identity (ANI) values were also within the threshold range (94–96%) for species identification [24]. The dDDH and ANI values based on the genome sequences between B. vitellinus NBRC 13296 and P. chitinolyticus KCCM 41400 were 97.8% and 98.66%, respectively, providing strong evidence that these strains may belong to the same taxonomic species.

3.2. Prediction of Secondary Metabolite Biosynthetic Gene Clusters (BGCs)

Using the standard cluster rule-based approach in antiSMASH analysis, a variety of natural product classes were identified in B. vitellinus NBRC 13296. Approximately 12.4% of the B. vitellinus NBRC 13296 genome is dedicated to secondary metabolism. The secondary metabolite gene clusters are concentrated in the center of the chromosome, spanning regions 5 to 16 (Figure S2). In total, 18 putative biosynthetic gene clusters were identified (

Table 1. Putative gene clusters coding for secondary metabolites in B. vitellinus NBRC 13296.

Region	Type	From	To	Most Similar Known Cluster	Similarity
Region 1	NRPS, LAP	305,619	356,240
Region 2	RRE-containing	747,520	767,798
Region 3	RiPP-like	851,869	864,055
Region 4	Crocagin, HR-T2PKS	945,752	1,014,754
Region 5	TransAT-PKS, NRPS	2,461,872	2,549,008
Region 6	TransAT-PKS, NRPS	2,563,881	2,640,650	Pelgipeptin	25%
Region 7	Cyclic-lactone-autoinducer	2,734,390	2,754,992
Region 8	Terpene	2,821,778	2,843,727
Region 9	Betalactone	3,032,661	3,062,918
Region 10	TransAT-PKS, NRPS	3,175,761	3,243,813
Region 11	Phosphonate	3,441,175	3,454,343
Region 12	Aminoglycosides	3,489,373	3,517,357	Butirosin A/B	84%
Region 13	NRPS	3,540,249	3,611,046	Octapeptin C4	29%
Region 14	NRP-metallophore, NRPS	3,614,412	3,666,638	Bacillibactin	100%
Region 15	Opine-like metallophore	3,687,196	3,729,044	Bacillopaline	100%
Region 16	T3PKS	3,837,645	3,878,793
Region 17	Proteusin	5,176,800	5,197,111
Region 18	TransAT-PKS-like, NRPS	5,276,428	5,336,092

“Similarity” refers to the ratio of homologous genes in the query and hit clusters. As defined by antiSMASH, homologous genes were selected based on high sequence identity (>30%) and short BLAST alignments (>25%). Abbreviations used in the table but not defined within the main text are as follows: PKS (polyketide synthase), HR-T2PKS (highly reducing type II PKS), and RRE (RiPP recognition element).

). Interestingly, region 15 contained two copies of an opine-like metallophore gene cluster, consisting of opine metallophore biosynthesis dehydrogenase, nicotianamine synthase family protein, and nickel/cobalt ABC transporter substrate-binding protein/permease (Figure S3). Thus, a total of 19 BGCs were identified through antiSMASH analysis.

Next, we used BAGEL to analyze the genome sequence of NBRC 13296 and found a total of two BGCs for different non-ribosomal peptides (NRPs)/linear azole-containing peptides (LAP) and sactipeptides. The former cluster identified by BAGEL overlaps with region 1 of antiSMASH, while the latter sactipeptides cluster is novel (Table S2 and Table S3). Using the PRISM algorithm, a total of 11 clusters were identified. Ten of these clusters overlapped with the AntiSMASH results, but an additional polyketide cluster, consisting of acyl carrier protein (ACP), beta-ketoacyl-ACP synthase II, ACP S-malonyltransferase, etc., was identified in cluster 10 (Figure S3 and Table S4). Based on the BGCs identified through the three approaches, we were ultimately able to identify 22 BGCs, including 19 from antiSMASH and an additional one each from BAGEL 4 and PRISM 4.4.5.

3.3. Comparative Characterization of Butorosin BGCs

Enzymatic studies of the butirosin biosynthetic pathway have led to remarkable scientific advances in Niallia circulans, with BGCs for N. circulans SANK 72073, N. circulans ATCC 21557, and N. circulans ATCC 21558 [4,25,26] identified in the NCBI database. First, we obtained a large gene cluster from BtrA to BtrZ based on the butirosin BGC of N. circulans SANK 72073 and the BGC of N. circulans ATCC 21,558, extended by genomic walking. We then identified additional genes in the upstream and downstream regions of the genome of B. vitellinus NBRC 13296, in addition to the tentative butirosin BGCs in region 12 analyzed by antiSMASH. As shown in Figure 2 and Table S5, B. vitellinus NBRC 13296 and the butirosin BGC from N. circulans share 67.81% to 92.87% high homology. Interestingly, during a BLASTP search for region 12 derived by antiSMASH, putative butirosine BGCs were identified in the type strain P. chitinolyticus KCCM 41,400, as well as in P. chitinolyticus NRRL B-23119, P. chitinolyticus NRRL B-23120, P. chitinolyticus B-14908, P. chitinolyticus YSY-3.1, P. chitinolyticus JMW06, Paenibacillus sp. GbtcB18, Paenibacillus sp. HGH0039, and Paenibacillus sp. MZ04-78.2. Next, we analyzed the genetic similarities and differences between B. vitellinus NBRC 13296, N. circulans, and the putative butirosin BGCs identified in Paenibacillus sp. The results showed that all BGCs had the following genes in common: BtrT, BtrS, BtrN, BtrM, BtrL, BtrA, BtrB, BtrC, BtrD, BtrD, BtrE, BtrF, BtrG, BtrH, BtrI, BtrI, BtrJ, BtrK, BtrO, BtrP, and BtrV were located in the same transcriptional orientation, followed by the BtrQ, BtrW, BtrX, BtrY, and BtrZ genes upstream of these core regions in the same transcriptional orientation and consecutive order. In contrast, the enzymes BtrT, BtrR2, BtrU, and BtrR1 are commonly found downstream of the core region, but, beyond this, the genes are arranged in a strain-specific manner. Specifically, B. vitellinus NBRC 13296, P. chitinolyticus KCCM 41400, P. chitinolyticus NRRL B-23119, P. chitinolyticus NRRL B-23120, and Paenibacillus sp. GbtcB18 B18 show the same genetic organization from BtrT2 to BtrT7, while P. chitinolyticus YSY-3.1 is similar only up to BtrT2, and the remaining strains lack some of the BtrT3 to BtrT7 genes. Furthermore, Paenibacillus sp. MZ04-78.2 contained five completely unrelated ORFs instead of the BtrT, BtrR2, BtrU, and BtrR1 genes. Finally, we sought to deduce the minimal unit of the butirosin BGC. Many classes of secondary metabolite BGCs, such as aminoglycosides, type I polyketides, type II polyketides, indolocarbazoles, and enediynes, are often accompanied by regulatory and resistance genes [27,28,29,30,31]. Therefore, it is likely that these genes are also present in the butirosin BGC. Based on these findings, it is highly likely that the regulatory genes BtrR1 and BurR2 are included in the butirosin BGC, as are all or part of the transporter genes BtrT2, BtrW, BtrX, and BtrY, which may act as resistance genes. However, further studies, such as heterologous expression or enzymatic approaches, are required.

4. Discussion

Over the past three decades, the 2-deoxystreptamine (2-DOS) aminoglycoside antibiotic butirosin has been extensively studied in N. circulans to develop semisynthetic antibiotics using the (2S)-4-amino-2-hydroxybutyrate (AHBA) side chain. Nearly all biosynthetic enzymes and biosynthetic gene clusters (BGCs) involved in this pathway have been characterized. This study was initiated following the identification of the complete genome of another butirosin producer, B. vitellinus NBRC 13296, with the aim of exploring a new approach to the butirosin biosynthetic pathway.

Through the comprehensive genome analysis of B. vitellinus NBRC 13296, we compared its digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values with those of P. chitinolyticus, the species most closely related based on 16S rRNA phylogenetic analysis, including N. circulans. Based on these genome-based comparative analyses, we propose reclassifying B. vitellinus NBRC 13296 as P. chitinolyticus.

We sought to obtain additional information regarding the type strain of B. vitellinus from the Bacterial Diversity Metadatabase (BacDive). However, upon a review of the available data, we were unable to locate any specific information pertaining to B. vitellinus. This absence of data supports the conclusion that B. vitellinus is best classified as P. chitinolyticus to maintain taxonomic clarity.

In this study, we conducted a comparative analysis of the butirosin BGCs between B. vitellinus, N. circulans, and P. chitinolyticus, identifying the minimal region of the butirosin BGC, spanning from BtrA to BtrZ. Previous biochemical investigations into the unique chemical structure of butirosin have primarily been conducted by a Japanese research team focusing on N. circulans SANK 72073, with additional studies on N. circulans ATCC 21557 and N. circulans ATCC 21558 carried out by Professor Pipersberg’s group in Germany. In light of the findings related to B. vitellinus, we hypothesized that the strains N. circulans SANK 72073, N. circulans ATCC 21557, and N. circulans ATCC 21558 may warrant reclassification from the genus Niallia to Paenibacillus. However, efforts to locate comprehensive genome sequences or 16S rRNA sequence data for these three strains within the NCBI database were unsuccessful. Attempts to retrieve strain histories from BacDive were similarly unfruitful. Notably, the butirosin BGCs were absent in the N. circulans NBRC 13626 reference strain, as well as in the genomes of N. circulans DC10 and other N. circulans strains, all of which were fully sequenced during our study of B. vitellinus-derived butirosin BGCs. Consequently, the taxonomic reclassification of N. circulans SANK 72073, N. circulans ATCC 21557, and N. circulans ATCC 21558 to the genus Paenibacillus appears to be well supported. However, this reclassification will necessitate the direct and comprehensive analysis of the 16S rRNA sequences and whole-genome data from these strains.

In this study, genome mining using the antiSMASH, BAGEL 4, and PRISM programs revealed 22 gene clusters encoding the biosynthesis of diverse secondary metabolites in the genome of B. vitellinus NBRC 13296. The secondary metabolite analysis using antiSMASH, as detailed in Table 1, confirmed the presence of BGCs in the B. vitellinus genome that exhibit 100% homology to previously characterized gene clusters involved in the biosynthesis of bacillibactin and bacillopaline. Bacillibactin is a cyclic trimeric ester composed of 2,3-dihydroxybenzoic acid (DHB) and glycine-threonine, forming a macrolactone ring structure. Bacillibactin functions as a siderophore—a specialized molecule synthesized by certain bacteria to sequester iron from the environment, which is crucial for survival under iron-limited conditions. The genus Bacillus, particularly Bacillus subtilis, is well known for producing bacillibactin. This model organism for Gram-positive bacteria synthesizes bacillibactin as part of its iron acquisition strategy, particularly when the iron availability is low [32]. Bacillibactin BGCs have been identified not only in Bacillus species but also in a variety of other bacteria [33]. These BGCs typically encode key enzymes such as non-ribosomal peptide synthetase (NRPS), isochorismatase, (2,3-dihydroxybenzoyl) adenylate synthase, isochorismate synthase, 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, and bacillibactin hydrolase. In the B. vitellinus BGCs, these genes are organized similarly, with an additional 65-amino-acid MbtH family protein, forming an operon-like BGC in the same transcriptional orientation (Figure 3). Understanding bacillibactin’s structure and function provides crucial insights into microbial iron acquisition strategies and offers potential avenues for the development of antimicrobial therapies that target siderophore-mediated iron uptake in pathogenic bacteria.

Bacillopaline, on the other hand, is classified as a metallophore, a class of small molecules that bind metal ions and facilitate their transport within biological systems. Structurally, bacillopaline is derived from a nicotianamine-like framework. This structure includes various functional groups, such as carboxyl and amine groups, which are essential for its ability to chelate metal ions like zinc, iron, and nickel. The multiple hydroxyl (-OH) and amine (-NH₂) groups in its structure further enhance its metal-binding capacity, making it an effective agent for metal acquisition by the bacteria that produce it. The bacillopaline BGC was characterized in Paenibacillus mucilaginosus KNP414, a known major producer of bacillopaline, a novel opine-type metallophore [34]. In B. vitellinus, the bacillopaline BGC includes core regions encoding opine metallophore biosynthesis dehydrogenase, a nicotianamine synthase family protein, and diaminopimelate epimerase. Interestingly, six nickel/cobalt ABC transporter permease-like proteins are located consecutively downstream of the core region. Genome mining in B. vitellinus NBRC 13296, P. mucilaginosus, and other related strains within the Paenibacillus genus has revealed additional opine-type metallophore BGCs, suggesting that this genus harbors significant untapped biosynthetic potential. These findings indicate the potential discovery of numerous novel compounds similar to bacillopaline in related strains.

In conclusion, our study demonstrates that B. vitellinus NBRC 13296 provides a robust scientific foundation for its reclassification as P. chitinolyticus. Additionally, the comparative analysis of butirosin BGCs underscores the necessity for ongoing research into the biosynthetic pathways and further taxonomic investigations of other producing strains, namely N. circulans SANK 72073, N. circulans ATCC 21557, and N. circulans ATCC 21558. Finally, the genus Paenibaciilus also suggests the possibility of another treasure trove of secondary metabolites, including bacillibactin and bacillopaline, which need to be mined and studied.