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. 2025 Apr 2;207(5):109. doi: 10.1007/s00203-025-04316-0

Genome mining reveals the biosynthetic potential of a novel Lysinibacillus zambalensis sp. nov., isolated from a hyperalkaline spring

Joyce Amarachi Aja 1, Lawrence Dave Llorin 1, Kenji Rowel Q Lim 2, Jade Joshua Teodosio 3, Erwin John Sioson 1,4, Ron L Dy 1, Carlo A Arcilla 4,5, Doralyn S Dalisay 3,6, Jose Enrico Hizon Lazaro 1,✉
PMCID: PMC11961540  PMID: 40169433

Abstract

A novel bacterium, designated as strain M3T, was isolated from a hyperalkaline spring in the Philippines and identified as a new species within the genus Lysinibacillus through 16 S rRNA gene sequence and genomic analyses. Although strain M3T shared a high 16 S rRNA gene sequence similarity (> 98.7%) with many Lysinibacillus species, the digital DNA-DNA hybridization and orthologous average nucleotide identity values between strain M3T and its closet relative, Lysinibacillus xylanilyticus DSM 23,493T, were 41.2% and 90.6%, respectively—both below the established threshold for prokaryotic species delineation. Genome mining of the 5.3 Mbp-draft genome of strain M3T revealed eight biosynthetic gene clusters, which shared little sequence similarity with characterized clusters, suggesting the potential for encoding novel specialized metabolites. The cells of strain M3T were Gram-stain-positive, aerobic, rod-shaped, non-motile, and capable of endospore formation. Optimum growth was observed at 30 °C, pH 8.0, and 0.5% (w/v) NaCl. The major respiratory quinone was menaquinone-7, and the predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, and two unknown phospholipids. Its fatty acid profile showed an elevated level of iso-C15:0, and the peptidoglycan type was determined to be A4α (L-Lys–D-Asp). This study contributes to the growing database and understanding of the genus and aims to help drive future research on the bioactive potential of the genus. Lysinibacillus zambalensis sp. nov. is proposed with strain M3T as the type strain (= TISTR 10640T = BIOTECH 10973T).

Supplementary Information

The online version contains supplementary material available at 10.1007/s00203-025-04316-0.

Keywords: Lysinibacillus, Hyperalkaline spring, Comparative genomics, Genome mining, Secondary metabolite biosynthetic gene clusters

Introduction

Alkaline springs may serve as a source of novel bacteria (Baculi et al. 2017). The Poon Bato Spring, Zambales province, Philippines, is situated in ophiolites; the weathering of mineral-rich ultramafic rocks results in waters with high pH of ~ 11.5 (Baculi et al. 2017; Monnin et al. 2021) and temperatures ranging from 27 to 32 °C (Cardace et al. 2015). The spring exhibited dissolved organic carbon (DOC) concentrations of less than 2 ppm and dissolved inorganic carbon (DIC) concentrations below 20 ppm (Cardace et al. 2015). Baculi et al. (2017) reported that the majority of bacteria isolated from the Poon Bato alkaline spring belonged to the phylum Bacillota, with many isolates being capable of producing alkaline enzymes including amylase, protease, lipase, and cellulase.

Members of the genus Lysinibacillus are Gram-stain-positive, rod-shaped cells that belong to the family Bacillaceae within the phylum Bacillota. They were classified as members of the genus Bacillus until 2007. Reclassification was based on the discovery that bacteria of the genus Lysinibacillus have lysine and aspartic acid in their cell wall peptidoglycan instead of meso-diaminopimelic acid present in Bacillus (Ahmed et al. 2007; Nam et al. 2012; Amaresan et al. 2020). The genus Lysinibacillus consisted of 22 species with validly published and correct names at the time of writing (Parte 2013, https://lpsn.dsmz.de/genus/lysinibacillus), with Lysinibacillus boronitolerans T-10aT as the type strain (Ahmed et al. 2007). Lysinibacillus species are ubiquitous in soil (Ahmed et al. 2007) and have been isolated from diverse environments including deep-sea sediments (Yu et al. 2019), and even pufferfish liver (Wang et al. 2010). Lysinibacillus species have also been isolated from alkaline-fermented leaves (Ouoba et al. 2015), saline-alkaline soils (Kong et al. 2014; Sun et al. 2017), and the alkaline Lonar Lake water in India (Tambekar et al. 2016).

Members of the genus may serve as a source of pharmacologically relevant compounds (Jamal and Ahmad 2022). Lysinibacillus spp. produce a variety of bacteriocins that are effective against foodborne bacterial and fungal pathogens (Ahmad et al. 2014; Ahmad and Khan 2015). Bacteriocins, ribosomally synthesized peptides that are often post-translationally modified, can kill or inhibit other bacterial strains but will not harm the producing strain (Yang et al. 2014; Dicks et al. 2018). A novel bacteriocin, produced by Lysinibacillus sp. strain JX416855, showed broad-spectrum antibacterial activity and strongly inhibited the fungi Aspergillus, Fusarium, and Trichoderma (Ahmad et al. 2014). L. sphaericus ZA9 produces 2-penthyl-4-quinolinecarboxylic acid, an antifungal compound, as well as binary toxin Cry48/Cry49 which possesses antimalarial and larvicidal properties (Naureen et al. 2017). L. odysseyi KC149512 produces compounds with antimicrobial, anticancer, antioxidant, and anti-inflammatory properties (Karthick and Mohanraju 2020). L. sp. AS-1 (Maela et al. 2022), L. sp. MK212927 (El-Sayed et al. 2022)d fusiformis S9 also produces bioactive compounds (Pradhan et al. 2014).

Despite the potential of numerous bacterial species to produce new bioactive compounds, genetically encoded secondary metabolites (SMs) often escape detection by conventional approaches (Monciardini et al. 2014). These constraints may be overcome by mining the genome for biosynthetic gene clusters (BGCs), including transcriptionally silent BGCs (Kalkreuter et al. 2020). Bioinformatic algorithms identify signatures from genomic information and help to prioritize strains or BGCs for investigation, potentially making the discovery of new compounds more efficient (Albarano et al. 2020; Kalkreuter et al. 2020). Using this approach, a previously uncharacterized bacteriocin gene cluster in L. boronitolerans was identified via genome sequencing and bioinformatic analysis, which led to its characterization via heterologous expression in Escherichia coli. The product of the BGC inhibited the growth of several pathogenic bacteria (Tang et al. 2023). In addition, genome mining and prioritizing of an orphan polyketide synthase-nonribosomal synthase (PKS-NRPS) hybrid gene cluster from the genus Salinispora led to the identification of thiotetronic acid natural products via heterologous expression (Tang et al. 2015). Chemical synthesis can be used to synthesize predicted compounds known as synthetic-bioinformatic natural products (syn-BNPs). The syn-BNPs may have only a resemblance to the original natural products because the post-assembly modifications of the original compound may not be accurately predicted (Scherlach and Hertweek 2021). A syn-BNP, humimycin A, which has high efficacy against methicillin-resistant Staphylococcus aureus (MRSA), was identified via chemical synthesis of an NRPS gene cluster from Rhodococcus equi (Chu et al. 2016).

This study describes the genomic, phenotypic, and chemotaxonomic analyses of strain M3T, isolated from the Poon Bato Spring in Botolan, Zambales, Philippines. Genome mining revealed a diverse array of BGC classes, including siderophores, polyketide synthases, nonribosomal peptide synthases, ribosomally synthesized and post-translationally modified peptides (RiPPs), opine-like metallophores, and betalactones, with low sequence similarities to experimentally characterized BGCs in the Minimum Information about a Biosynthetic Gene cluster (MIBiG) database. This unique genomic, phenotypic, and chemotaxonomic profile suggests that strain M3T represents a previously uncharacterized species with potentially novel biochemical and bioactive properties. We propose to assign strain M3T to a new species, Lysinibacillus zambalensis sp. nov.

Materials and methods

Strain isolation

Strain M3T was isolated from water samples collected from the Poon Bato, Zambales hyperalkaline spring in the Philippines (latitude 15.32214° N, longitude 120.07404° E). Samples were diluted and plated on tryptone soya agar (TSA) plates (pH 8.5), and incubated at 30 ˚C for 24 to 72 h. The culture medium was obtained from TM Media (Delhi, India), a manufacturer of microbiological media. Isolates that grew well in pH 8.5 media were selected and subcultured repeatedly to obtain pure cultures. The pure cultures were stored at -80 °C in 50% glycerol prepared by mixing equal volumes of glycerol and bacterial broth culture (TSB, pH 8.5) and used for 16 S rRNA identification and further physiological and biochemical characterization.

Whole genome sequencing, assembly, and annotation

Genomic DNA was extracted using the Qiagen QIAamp DNA Mini Kit according to the manufacturer’s instructions. Routine quality checks were performed via Qubit and TapeStation220. Library preparation was performed using the TruSeq DNA Nano Library Preparation Kit. DNA sequencing was performed using the NextSeq 500/550 High Output Kit (2 × 150 cycles) on the Illumina NextSeq™ 500 Sequencing system.

The online bioinformatics platform Bacterial Genome Assembly Service of the Bacterial and Viral Bioinformatics Resource Center (BV-BRC; https://www.bv-brc.org/; Olson et al. 2023) was used to trim and assemble raw sequencing read data. The sequences were trimmed via TrimGalore (v0.6.5, www.bioinformatics.babraham.ac.uk/projects/trim_galore/), and SPAdes v4.0.0 (Bankevich et al. 2012) was used for de novo assembly. The genome was annotated using both the BV-BRC platform with Lysinibacillus xylanilyticus DSM 23,493T as the reference strain, and the Rapid Annotations using Subsystems Technology (RAST) version 2 RASTtk pipeline (Aziz et al. 2008; Overbeek et al. 2014; Brettin et al. 2015). BUSCO version 5.6.1 was used to assess the completeness of the genome assembly (Manni et al. 2021). The BV-BRC algorithm, which employs EvalG, was also used to analyze the genome quality (Olson et al. 2023). EvalG implements the CheckM algorithm (Parks et al. 2015) to assess the genome’s completeness and contamination.

Phylogenetic analysis based on 16 S rRNA gene and whole genome

The 16 S rRNA gene of strain M3T was amplified by PCR with the universal 27F and 1492R primers as well as the 518 F and 800R primers (Senthilraj et al. 2016). The amplicons were sequenced using Sanger dideoxy sequencing at Macrogen, Korea. The contigs were assembled using CAP3 (Huang and Madan 1999). The sequences were assembled to generate the nearly complete 16 S rRNA gene sequence (1407 nucleotides) of strain M3T (GenBank accession number PQ061501). Additionally, the 16 S rRNA gene sequences of strain M3T and related Lysinibacillus species were extracted from the genome using the ContEst16S algorithm (Lee et al. 2017) available in the EzBioCloud server (Chalita et al. 2024). The 16 S rRNA gene sequences of L. mangiferihumi M-GX18T (JF731238), L. parviboronicapiens BAM-582T (AB333000598), L. fusiformis ATCC 7055T (AJ310083), L. odysseyi 34hs-1T (AF526913), and L. alkalisoli CGMCC 1.15760T (MT759970), on the other hand, were retrieved from the accession numbers listed in the LPSN database (Parte 2013, https://lpsn.dsmz.de/genus/lysinibacillus) due to the inability to extract their respective 16 S rRNA gene sequences using the ContEst16S algorithm. The 16 S rRNA gene sequence of strain M3T and other Lysinibacillus species were aligned using the ClustalW algorithm (Thompson et al. 1994) in MEGA 11 (Tamura et al. 2021). Phylogenetic trees were constructed using the maximum-likelihood (Felsenstein 1981), neighbor-joining (Saitou and Nei 1987), and maximum parsimony algorithms (Fitch 1971), with bootstrap analysis based on 1,000 replications (Felsenstein 1985); Bacillus subtilis DSM 10T was used as the outgroup. The percent identities of strain M3T compared to related type strains were determined using the NCBI BLAST algorithm (Altschul et al. 1997) (Table S1).

Phylogenomic analysis of strain M3T based on the whole genome sequence was carried out as described by Alam et al. (2022). Briefly, the assembled genome of strain M3T and related Lysinibacillus species from the LPSN database (Parte 2013) were run through the Type (Strain) Genome Server (TYGS) (available at https://tygs.dsmz.de/; Meier-Kolthoff and Göker 2019). The phylogenomic tree was inferred using FastME2.1.6.1 (Lefort et al. 2015) from Genome BLAST distance phylogeny (GBDP) distances calculated from the genomic sequences. The branch lengths were scaled in terms of GBDP formula d5. The trees were rooted at the outgroup. Additionally, the phylogenomic tree based on 81 core genes of strain M3T and related strains was constructed with the UBCG (Up-to-date bacterial core gene set) version 3.0 pipeline (Na et al. 2018).

The digital DNA-DNA hybridization (dDDH) values were calculated using the Genome-to-Genome Distance Calculator (GGDC) v3.0 (Meier-Kolthoff et al. 2022) using the recommended formula 2 (identities / length of high-scoring segment pairs), and the corresponding heatmap was generated using MORPHEUS (https://software.broadinstitute.org/morpheus). The orthologous average nucleotide identity (orthoANI) values and the corresponding heatmap were generated using the OAT software v0.93.1 (Lee et al. 2016). The genome characteristics, 16 S rRNA gene percent similarity to strain M3T, size, G + C content, and accession numbers) of the Lysinibacillus species used in this study are found in Table S1.

Gram Staining, Endospore Staining, and Scanning Electron Microscopy.

Gram staining was carried out according to standard procedure (Froböse et al. 2020). Endospore staining was carried out on an 18–24 h-old TSB culture of strain M3T with the Schaeffer-Fulton method, using malachite green and safranin as dyes (Schaeffer and Fulton 1933). For scanning electron microscopy, a colony of strain M3T was fixed using 2.5% glutaraldehyde and incubated at 4 °C for 4 h. The fixed bacterial suspension was washed with phosphate-buffered saline (PBS). Then, the bacterial pellets were resuspended in 1 mL of 1% PBS to remove the insoluble salts. A 5 µL aliquot of the bacterial suspension was mounted onto a copper tape and allowed to air dry overnight. The dried suspension was sequentially dehydrated for approximately 10 min each with 20%, 40%, 60%, 80%, and 100% ethanol. The specimen was placed in an aluminum stub (JEOL Ø25 × 10 mm cylinder SEM sample stub TM, Tokyo, Japan), followed by sputter coating with gold for 30 s using the JEOL Smart Coater TM (Tokyo, Japan) to enhance sample conductivity. The morphology of the cells was observed using the JCM-7000 NeoScope™ Benchtop SEM (Tokyo, Japan) in high vacuum mode.

Phenotypic characterization

The optimum growth conditions of strain M3T were determined by growing the bacterium in TSB at temperatures ranging from 5 ˚C to 45 ˚C (5, 15, 25, 30, 37, 45) and pH ranging from 4 to 12 (4, 5, 6, 7, 8, 9, 10, 11, 12). A seed culture was prepared by inoculating a single colony of strain M3T into 5 mL of TSB at 37 ˚C overnight. The seed culture was then used to inoculate 100 mL of TSB and the broth cultures were incubated at the different temperatures for 7–8 h. To test for pH tolerance, 1 mL of a strain M3T seed culture was used to inoculate 100 mL of TSB which was adjusted to pH values ranging from 4 to 12. The pH of the media was adjusted using acetate buffer for pH 4 to 6, glycine-NaOH buffer for pH 9 to 12, and HCl or NaOH for pH 7 and 8. The final pH of each medium was verified using a pH meter before inoculation. NaCl tolerance was determined by inoculating 500 µL of the seed culture in 150 mL of TSB with NaCl at concentrations ranging from 0.5 to 18% (0.5, 3, 6, 9, 12, 15, 18). The cultures for the pH and NaCl tolerance tests were incubated at 30 ˚C for 7–8 h under aerobic conditions. The optical densities (OD) were measured at 600 nm using a NanoDrop™ 2000c spectrophotometer. The OD600 was measured to monitor cell growth. For antibiotic sensitivity testing, antibiotic-impregnated filter paper discs (ciprofloxacin and penicillin G) were placed on Muller-Hinton Agar plates inoculated with a strain M3T culture, which was evenly spread on the agar surface using a sterile swab. The plates were then incubated at 35 °C for 24 h to assess bacterial growth inhibition (Mounyr et al. 2016).

Cell growth under anaerobic conditions was determined by culturing strain M3T under a layer of sterile mineral oil (Umehara and Aoyagi 2023). The BIOLOG GEN III Microplate analysis of strain M3T was carried out according to the standard protocol (Ngalimat et al. 2019). This assay comprised 94 tests: 71 for carbon source utilization and 23 for chemical sensitivity. Motility was assessed by inoculating a soft TSA medium (0.4% agar) with strain M3T, followed by incubation for 7 days. Catalase activity was determined by adding 3% hydrogen peroxide to a single colony and observing bubble formation while oxidase activity was tested using 1% (v/v) tetramethyl p-phenylenediamine, with a color change indicating a positive result (Reiner 2010; Shields and Cathcart 2010). To test for nitrate reduction, sulfanilic acid and α-naphthylamine reagents were added to nitrate broth (Buxton 2011). Methyl Red and Voges-Proskauer (VP) tests were performed by adding methyl red (MR) reagent and α-naphthol/KOH, respectively, to MR-VP broth, according to McDevitt (2009). Lysine decarboxylase activity was evaluated using Moeller’s lysine decarboxylase agar (Lal and Cheeptham 2015) while the ability of strain M3T to use citrate as a carbon source was determined using Simmons citrate agar slants (MacWilliams 2009a, b). Urease activity was tested by inoculating urea broth and observing a color change from yellow to pink (Brink 2010), and indole production was detected by adding Kovac’s reagent to tryptone broth (MacWilliams 2009a, b).

Chemotaxonomic analysis

Analyses of cellular fatty acids, respiratory quinones, polar lipids, whole-cell sugars, and the peptidoglycan structure of strain M3T were carried out by DSMZ Services, Leibniz-Institut DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany. Briefly, the cellular fatty acids were evaluated using the Microbial Identification System (MIDI; microbial ID) protocols. Respiratory quinones were extracted with hexane from freeze-dried cell material and purified by silica-based solid-phase extraction, followed by analysis by HPLC analysis using a reverse phase column and recording absorption spectra (Vieira et al. 2021). Polar lipids were analyzed using a modified protocol by Bligh and Dyer (1959). Polar lipids were extracted from freeze-dried cells using chloroform: methanol:0.3% aqueous NaCl mixture and separated by two-dimensional silica gel thin layer chromatography (TLC). TLC on cellulose plates was used to determine the diagnostic sugars in whole-cell hydrolysates of strain M3T. Peptidoglycans were analyzed according to the protocol described by Schumann (2011). The identity of all amino acids was confirmed by agreement in the gas-chromatographic retention time with those of the standards and by characteristic mass spectrometric fragment ions of the derivatives.

Identification of Putative Biosynthetic Gene Clusters (BGCs).

Putative secondary metabolite BGCs were predicted using antiSMASH v7.0 (Blin et al. 2023). For antiSMASH, the detection strictness was set to “relaxed”, and all the extra features were selected: KnownClusterBlast, ClusterBlast, SubClusterBlast, MIBiG cluster comparison, ActiveSiteFinder, RREFinder, Cluster Pfam analysis, Pfam-based GO term annotation, and TIGRFam analysis. BAGEL4 was used to mine the genome for RiPPS and bacteriocins (van Heel et al. 2018). RiPPMiner was used for sequence comparison of RiPPs (Agrawal et al. 2017).

Results and discussion

Genomic and phylogenetic analyses

The draft genome of strain M3T had a size of 5,298,580 bp (~ 5.3 Mbp) within 129 contigs; the mean DNA G + C content was 36.5%. The draft genome consisted of 5,175 coding sequences, 16 rRNAs, and 73 tRNAs (Table S2). The draft genome of strain M3T has been deposited in the GenBank database under the accession number JBEGDG000000000. The BV-BRC algorithm determined that the completeness and contamination scores of the draft genome were 99.9% and 1.9%, respectively (Table S2), while BUSCO analysis indicated a completeness score of 99.1% (data not shown). According to the RAST annotation, 322 subsystems were identified with the majority of the genes being associated with amino acid and derivative metabolism and carbohydrate metabolism (Fig. S1). No plasmid DNA was detected in the genome, according to the BV-BRC and RAST annotations.

Strain M3T shared high 16 S rRNA gene sequence similarity (> 98.7%) with several Lysinibacillus species, having the highest similarity (99.7%) with the type strain L. xylanilyticus DSM 23,493T (Table S1). In the maximum likelihood and maximum parsimony phylogenetic trees, strain M3T formed a monophyletic lineage with L. xylanilyticus DSM 23,493T, and their closest relative was “L. agricola” FJAT-51,161T (Fig. 1 and Fig. S2). However, in the phylogenetic tree analyzed using the neighbor-joining algorithm, strain M3T formed a monophyletic lineage with “L. agricola” FJAT-51,161T, and their closest relative was L. xylanilyticus DSM 23,493T (Fig. S2). Although listed as a child taxon in the LPSN database, the name “L. agricola” has not been validly published (Parte 2013).

Fig. 1.

Fig. 1

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Maximum-likelihood phylogenetic tree based on the 16 S rRNA gene sequence of strain M3T and closely related Lysinibacillus species. Bootstrap values are expressed as percentages of 1000 replications. The GenBank accession numbers of the genomes, from which the 16 S rRNA gene sequences were extracted using the ContEst16S algorithm on the EzBioCloud server, or the accession numbers of the 16 S rRNA gene sequences, are specified in parentheses. Names not validly published are indicated in quotation marks. Asterisks and crosses denote branches identified through the maximum parsimony and neighbor-joining methods, respectively

Several studies indicated that species with high 16 S rRNA gene sequence similarity may still be classified as distinct (Stackebrandt and Goebel 1994). For example, the Bacillus subtilis group comprises eight closely related species that cannot be differentiated by 16 S rRNA gene sequence analysis, exhibiting similarities of 98.1-99.8% (Wang et al. 2007). This applies to other genera, including Mycobacterium (Blackwood et al. 2000) and Streptomyces (Komaki 2023). This issue highlights the potential challenges with 16 S rRNA gene-based classification, particularly in the misclassification of novel species.

To further confirm species delineation of strain M3T, digital DNA-DNA hybridization (dDDH) and orthologous average nucleotide identity (orthoANI) values were calculated. In contrast to 16 S rRNA gene similarities, dDDH and ANI analyses provide a comprehensive evaluation of genetic relatedness and allow for more accurate species distinctions (Goris et al. 2007). Strain M3T and its closest relative, L. xylanilyticus DSM 23,493T, showed dDDH and orthoANI values of 41.2% and 90.6%. respectively (Fig. 2A and B). The dDDH and orthoANI values of strain M3T and “L. agricola” FJAT-51,161T were 40.1% and 89.8%, respectively. The recognized cutoff values for prokaryotic species delineation are 70% for dDDH and 95% for ANI (Goris et al. 2007), suggesting strain M3T represents a novel species within the genus Lysinibacillus.

Fig. 2.

Fig. 2

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Pairwise comparisons of (A) digital DNA-DNA hybridization (dDDH) and (B) orthologous average nucleotide (orthoANI) values between strain M3T and its closely related type strains. Values in the dark box show the comparisons between strain M3T and related type strains. The color bar indicates the similarity between the compared strains, ranging from (A) light orange to red or from (B) blue to red. As the color intensity transitions towards red, it indicates a closer relationship between the strains. Names not validly published are indicated in quotation marks

The phylogenomic tree based on whole genome sequences further confirmed the close relationship between strain M3T and L. xylanilyticus DSM 23,493T (Fig. 3A). This topology was consistent with the phylogenomic tree based on the concatenation of 81 core genes in strain M3T and several Lysinibacillus species (Fig. 3B).

Fig. 3.

Fig. 3

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(A) GBDP phylogenomic tree of strain M3T compared to closest relatives based on whole genome sequences and (B) phylogenomic tree based on 81 aligned UBCG sequences of strain M3T and related Lysinibacillus species. Bootstrap values are given at the nodes; only values above 70% are indicated. Bacillus subtilis DSM 10T was used as the outgroup species. Names not validly published are indicated in quotation marks

Phenotypic characterization of strain M3T

The colonies of strain M3T appeared round, creamy white, with an entire margin, slightly raised elevation, and an opaque appearance on tryptone soya agar (TSA) after 18 h of incubation (Fig. S3A). The cells were found to be Gram-stain-positive and formed ellipsoidal, terminal endospores (Fig. S3B). Strain M3T grew within a temperature range of 25–37 °C, with optimum growth at 30 °C (Fig. S4A). It demonstrated a pH tolerance at a range of 6–10, with optimum growth at pH 8 (Fig. S4B). Growth at pH 10 suggests its tolerance for alkaline conditions. Strain M3T exhibited growth in media containing 0.5–3% NaCl, with optimal growth at 0.5% (Fig. S4C). The growth characteristics of strain M3T were slightly different compared to its closest relatives with L. xylanilyticus DSM 23,493T growing in pH 5–9 and “L. agricola” FJAT-51,161T growing in pH 7–9 (Table 1). Like strain M3T, strains L. xylanilyticus DSM 23,493T and “L. agricola” FJAT-51,161T grow optimally at 30 °C (Lu and Liu 2021).

Table 1.

Comparative features of strain M3T and its closely related species

Characteristics 1 2 a 3 b 4 c 5 d
Cell size (µm) 0.6 × 3.0 0.8–1.0 × 3.0–5.0 0.8-1.1 × 2.0-3.4 0.9-1.1 × 3.0-5.0 0.8-1.5 × 3.0-5.0
Spore shape R/O, T R, T R, T R/O, T R/O, T
Motility Non-motile Motile Motile Motile Motile
pH range 6–10 5–9 7–9 7–9 5.5–9.5
pH optimal 7 NA 7 8 7
Temp range (°C) 15–45 10–45 25–40 10–45 16–45
Temp optimal (°C) 30 30 30 30 37
Oxidase - + + - +
Indole - - NA - -
Urease - NA - - +
Voges-Proskauer - - + + +
Hydrolysis of gelatin +/w + + +/w -
Arginine dihydrolase - - - - +
Lysine decarboxylase + - + - -
Utilization of: *
Citrate - + NA - +
Maltose - + NA NA -
Trehalose - - NA NA -
Sucrose - + NA NA -
Melibiose +/w + NA NA -
Mannose - - NA NA -
Rhamnose - + NA NA -
Sorbitol - + NA NA -
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1, Strain M3T; 2, L. xylanilyticus DSM 23,493T; 3, “L. agricola” FJAT-51,161T; 4, L. macroides DSM 54T; 5, L. boronitolerans T-10aT. All strains are Gram-stain-positive and endospore forming. All strains are catalase positive and negative for nitrate reduction. Strain M3T and related strains exhibit differences in motility, oxidase activity, lysine decarboxylation, and the utilization of various carbon sources including citrate, maltose, sucrose, melibiose, rhamnose, and sorbitol. All data was from this study unless indicated otherwise. O, oval or slightly oval; R, round; T, terminal; +, positive; -, negative; +/w, weakly positive; NA, no data available

a Data from Lee et al. (2010), b Data from Lu and Liu (2021), c Data from Coorevits et al. (2012), d Data from Ahmed et al. (2007), * Data were taken from Lee et al. (2010) for the reference species L. xylanilyticus DSM 23,493T and L. boronitolerans T-10aT

Scanning electron microscopy revealed a rod-shaped cellular structure measuring up to 3 μm in length and 0.6 μm in width, with flagella-like structures (Fig. 4). The presence of flagella-like structures is further supported by the identification of 43 flagella-related genes in the RAST annotation (Fig. S1). However, agar motility assays demonstrated that strain M3T was non-motile. While many species of Lysinibacillus are motile, L. fusiformis has been reported as non-motile (Ahmed et al. 2007). The flagella-like structures observed in strain M3T may be influenced by regulatory or environmental factors affecting flagellar gene expression, as seen in enteroinvasive Escherichia coli (EIEC) strains, which are typically non-motile but can produce functional flagella under specific conditions (Andrade et al. 2002). Additionally, flagella may have functions other than motility, including secretion, adhesion, and biofilm formation (Chaban et al. 2015; Akahoshi and Bevins 2022). Future studies are needed to elucidate the role and regulation of flagella in strain M3T.

Fig. 4.

Fig. 4

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Scanning electron micrographs of strain M3T using JCM-7000 NeoScope™ Benchtop scanning electron microscope (Tokyo, Japan). (A) Strain M3T cells have a rod-shaped morphology. (B) The white arrow shows a single flagella-like structure

Strain M3T exhibited no growth under anaerobic conditions. Biochemical analysis of strain M3T showed that it was catalase positive, lysine decarboxylase positive, citrate negative, indole negative, methyl red negative, nitrate reduction negative, oxidase negative, urease negative, and Voges-Proskauer negative. It displayed susceptibility (inhibition zone diameter greater than 25 mm) to ciprofloxacin (5 µg/disc) and penicillin G (10 µg/disc). Analysis for carbon utilization and chemical sensitivity using the BIOLOG GEN III Microbial Identification system showed that strain M3T did not match with any known species’ profile in the BIOLOG database; it shared low similarity scores of 0.455 and 0.115 with the closest reference organisms Bacillus decisifrondis and L. boronitolerans, respectively (Table S3). B. decisifrondis was identified in 2007 (Zhang et al. 2007)—the same year that a new taxonomic classification parameter split Lysinibacillus species from the Bacillus genus (Ahmed et al. 2007). It has since been reclassified as Lysinibacillus sp. FJAT-14,222 according to the data from the NCBI BioProject accession number PRJNA294250 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA294250). Lysinibacillus sp. FJAT-14,222 clustered with Lysinibacillus species in phylogenomic analyses (Fig. 3).

In the BIOLOG assay, strain M3T exhibited the ability to utilize D-gentibiose, D-turanose, glycerol, L-fucose, and N-acetyl-D-glucosamine, similar to its closest relative L. xylanilyticus DSM 23,493T (Lee et al. 2010). However, strain M3T could be differentiated from L. xylanilyticus DSM 23,493T by its inability to utilize maltose, sucrose, rhamnose, and sorbitol (Table 1). Additional distinct characteristics of strain M3T in relation to its close phylogenetic neighbors are displayed in Table 1.

Chemotaxonomic analysis

Chemotaxonomic analysis examines macromolecule distribution to assess the relatedness of bacterial strains to known species (Gokdemіr and Aras 2019). The chemotaxonomic characteristics of strain M3T were compared to those of L. xylanilyticus DSM 23,493T, “L. agricola” FJAT-51,161T, L. macroides DSM 54T, and L. boronitolerans T-10aT (Table 2). The two respiratory quinones of strain M3T were MK-7 (94.2%) and MK-6 (5.8%), with MK-7 having the highest percentage, a characteristic shared by many species of Lysinibacillus. The analysis of enantiomers of the total hydrolysate showed that the peptidoglycan type of strain M3T was L-Lys–D-Asp (type A4α), characteristic of all Lysinibacillus species (Ahmed et al. 2007). Whole-cell sugar analysis revealed the presence of ribose and glucose.

Table 2.

Chemotaxonomic characteristics of strain M3T and closely related species

Chemotaxonomic characteristic 1 2 a 3 a 4 a 5 b
Major menaquinones (%)
MK-5 - NA 6.3 NA NA
MK-6 5.8 NA 29.1 NA 13
MK-7 94.2 NA 58.3 NA 87
MK-8 - NA 6.3 NA NA
Major fatty acids (%)
iso-C14:0 3.0 1.6 4.7 2.6 1.7
C14:0 0.5 0.9 0.6 0.4 0.4
iso-C15:1ω10c 1.1 NA NA NA NA
iso-C15:0 41.6 58.2 46.7 45.9 31.8
anteiso-C15:0 1.6 8.0 6.9 7.4 21.4
C15:0 1.0 0 0 0 0.5
C16:1ω7c 20.9 7.0 14.0 10.1 7.6
iso-C16:0 19.0 1.8 15.8 12.2 11.2
C16:1ω11c 2.3 2.7 2.3 5.3 2.7
C16:0 1.1 1.9 1.9 2.7 1.8
iso-C17:1ω10c 2.2 6.3 0.6 2.0 1.3
iso-C17:1ω5c 0.4 NA NA NA NA
iso-C17:0 4.3 3.4 3.3 6.1 5.5
anteiso-C17:0 0.5 2.7 1.8 3.1 11.1
iso-C18:0 0.1 NA NA NA NA
Major polar lipids DPG, PE, PG, and two unknown phospholipids DPG, PE, PG

DPG, PE, one unknown aminolipid, two unknown aminophospholipids,

two unknown phospholipids

 DPG, PE, PG DPG, PG
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1, Strain M3T; 2, L. xylanilyticus DSM 23,493T; 3, “L. agricola” FJAT-51,161T; 4, L. macroides DSM 54T; 5, L. boronitolerans T-10aT. All data was from this study unless indicated otherwise. -, no detected value; NA, no data available

aData from Lu and Liu (2021), b Data from Ahmed et al. (2007)

DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol

The major polar lipids in strain M3T were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and two unknown phospholipids (Fig. S5). The cellular fatty acid profile contained high proportions of iso-C15:0, C16:1ω7c alcohol, and iso-C16:0. The chemotaxonomic characteristics of strain M3T are consistent with those expected of the genus (Ahmed et al. 2007). However, considerable differences in the proportions of the major menaquinones and fatty acids, and the composition of polar lipids were observed. These differences indicate the divergence of strain M3T from its closest relatives.

Secondary metabolite biosynthetic gene clusters in strain M3T

The biosynthetic potential of strain M3T was assessed using antiSMASH v7.0 (Blin et al. 2023). This web server enables the identification, annotation, and analysis of gene clusters related to secondary metabolite production in bacterial genomes. Eight (8) secondary metabolite-producing BGCs belonging to different classes were predicted (Table 3). The number is comparable to those of L. xylanilyticus DSM 23,493T and “L. agricola” FJAT-51,161T, for which antiSMASH predicted 9 and 7 BGCs, respectively (Table S4).

Table 3.

List of putative secondary metabolite-producing biosynthetic clusters as predicted by antiSMASH

S/N Region Type From To Most similar known cluster % Similarity
1 2.1 NRPS-independent siderophore 150,898 164,459 Petrobactin 33
2 2.2 Terpene 506,205 527,026
3 5.1 T3PKS 59,197 100,279 Bacillibactin/bacillibactin E and F (NRP) 30
4 5.2 Lanthipeptide class II 118,248 145,823 Gramicidin S (NRP) 6
5 7.1 RiPP-like 175,172 187,106 NA NA
6 13.1 NRPS, T1PKS 14,770 85,865 NA NA
7 15.1 Opine-like metallophore 25,102 47,240 Bacillopaline (Metallophore) 25
8 33.1 Betalactone 12,482 36,136 Fengycin (NRP) 46
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NA, not reported by antiSMASH

BGCs involving PKS and PKS/NRPS hybrids

Non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) BGCs can produce a variety of antibiotics, anticancer agents, and immunosuppressants (Chen et al. 2019). In these clusters, synthesis is carried out by large modular enzymes. The structural and catalytic similarities between polyketides and non-ribosomal peptides allow for hybrid clusters that include components from both classes to introduce structural modifications in the major classes of BGCs and increase secondary metabolite variety (Mizuno et al. 2013; Belknap et al. 2020).

A type III PKS BGC and a hybrid PKS/NPRS BGC were identified by antiSMASH in the genome of strain M3T. The 41-kb type III PKS BGC in region 5.1 comprises 46 genes. Comparisons within the MIBiG database (Terlouw et al. 2023) showed that the BGC was most similar to an alkylresorcinol BGC in Streptomyces griseus subsp. griseus NBRC 13,350, a type III PKS in Candidatus entotheonella serta, and hierridin B and C BGCs in Cyanobium sp. LEGE 06113 (Fig. S6). Alkylresorcinols have bioactivities that include antimicrobial, anticancer, lipid-lowering, and antioxidant properties (Zabolotneva et al. 2022).

A 71-kb PKS/NRPS cluster in region 13.1 was identified, comprising 37 putative ORFs consisting of biosynthesis, regulatory, and transporter genes (Fig. 5A and B). The cluster contained one type I PKS (ABNX05_16725), three NRPSs (ABNX05_16700, ABNX05_16705, and ABNX05_16710), a thioesterase (ABNX05_16675), an oxidoreductase (ABNX05_16725), and a 4′-phosphopantetheinyl transferase (PPT; ABNX05_16670). Gene ABNX05_16675 was predicted to encode a 4′-PPT, a member of a family of enzymes involved in the synthesis of a wide range of compounds, such as fatty acid, polyketide, and non-ribosomal peptide metabolites (Copp and Neilan 2006; Beld et al. 2014). ClusterBlast revealed BGCs with low gene sequence similarities (31% being the highest) to those found within region 13.1 in different bacteria, including those belonging to the genera Bacillus, Paenibacillus, Tumebacillus, Aquimarina, Myxococcus, and Kurthia (Fig. S7). However, no similar gene clusters were identified using the KnownClusterBlast feature of antiSMASH. MIBiG comparison revealed that the PKS gene (ABNX05_16725) module had a 0.69 similarity score with the cryB gene of the hybrid NRPS/PKS cylindrospermospin BGC in Aphanizomenon sp. strains 10E9 and 22D11. The ABNX05_16700, ABNX05_16705 and ABNX05_16710 NRPS modules shared some similarity with NRPS genes involved in the biosynthesis of compounds with varying biological activities (Fig. S8). The observed MIBiG similarities result from the general organization and domains present in NRPS, PKS, and hybrid NRPS/PKS BGCs.

Fig. 5.

Fig. 5

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Genetic organization of the hybrid PKS/NRPS BGC in region 13.1 of strain M3T. (A) Genetic organization of the cluster, as annotated by antiSMASH. (B) Domain organization of the PKS and NRPS genes and the downstream thioesterase (TE) domain. The type I PKS consists of a ketosynthase (KS), an acyltransferase (AT), a dehydratase (DH), a ketoreductase, and an acyl carrier protein (ACP) domain. The NPRSs consist of condensation (C), adenylation (A), peptidyl carrier protein (PCP), epimerization (E), and ketosynthase (KS) domains

BGCs involving RiPPs

Ribosomally synthesized and post-translationally modified peptides (RiPPs) exhibit great structural and functional diversity (Zhong et al. 2020). Lanthipeptides, for example, are a class of RiPPs that prevent cell wall biosynthesis and disrupt cell membrane integrity (Li et al. 2021). A RiPP BGC encodes one or more precursor peptides and posttranslational maturation enzymes. The maturation enzymes introduce modifications to the core peptide before proteolytic release from the leader peptide, increasing structural diversity (Repka et al. 2017; Mordhorst et al. 2023).

BAGEL analysis (van Heel et al. 2018) revealed three RiPPs (Table S5). Contig_20.12 and Contig_3.4 were predicted to contain RiPP-like BGCs; however, certain core genes, including core peptides, were not predicted (Fig. S9). Of particular interest was Contig_5.42.AO1_01 (region 5.2 in antiSMASH), which was predicted to encode a class II lanthipeptide (Table S5 and Fig. 6). In class II lanthipeptides, a single bi-functional enzyme, LanM, catalyzes the dehydration and cyclization processes of a precursor peptide(s). LanM contains an N-terminal dehydratase domain with minimal sequence similarity to other known enzymes, and the C-terminal cyclization domain shares significant sequence similarity with class I LanC cyclases (Blin et al. 2014). Following modification, LanT, an ABC transporter with an ATP-binding domain, cleaves a leader peptide at a highly conserved double glycine motif associated with the export of the mature peptides outside of the cell (Nishie et al. 2009, 2011). The predicted class II lanthipeptide BGC identified in the genome of strain M3T included 4 predicted core peptides (ABNX05_09170, ABNX05_09175, ABNX05_09180, and ABNX05_09185), 2 modification enzymes (ABNX05_09165 and ABNX05_09190), 1 transport and leader cleavage gene (ABNX05_09160), 2 immunity/transport genes (ABNX05_09130 and ABNX05_09200), and 4 regulatory genes (ABNX05_09135, ABNX05_09140, ABNX05_09150, and ABNX05_09155); it appears complete (Fig. 6).

Fig. 6.

Fig. 6

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Genetic organization of the lanthipeptide II BGC in strain M3T, as predicted by BAGEL4

Using RiPPMiner (Agrawal et al. 2017), we found that core peptide genes (ABNX05_09170 to ABNX05_09185) shared a sequence similarity ranging from 35.42 to 46.67% with the core peptides of some class II bioactive lanthipeptides including haloduracin α (Bacillus halodurans C-125), geobacillin II (Geobacillus thermodenitrificans NG80-2, and lichenicidin VK21A2 (Bacillus licheniformis).

Other BGCs in strain M3T

Terpenes and terpenoids exhibit an array of biological activities including antimicrobial, antitumor, anti-inflammatory, and antioxidant activities (Masyita et al. 2022). A terpene BGC was identified in region 2.2. In the MIBiG database, the core biosynthetic gene, a predicted squalene/phytoene synthase, shared the highest similarity score (0.32) with the core biosynthetic gene involved in the biosynthesis of squalestatin S1 by Aspergillus sp. Z5 (Fig. S10A). Squalestatins are a class of potent squalene synthase inhibitors mostly found in various fungal taxa (Lebe and Cox 2019).

The predicted betalactone BGC in region 33.1 had the highest similarity to a gene in a saccharide BGC involved in the production of mid-chain acyl sugars in the genome of the plant Solanum lycopersicum (Fig. S10B). The BGC had many biosynthetic enzymes that may be involved in other pathways in strain M3T.

Region 2.1 was predicted to encode an NRPS-independent siderophore BGC involved in the biosynthesis of petrobactin, a siderophore produced by Bacillus anthracis Ames, according to MIBiG comparisons (Fig. S11A). Siderophores increase the uptake of iron (Ahmed and Holmström 2014). Their applications include enhancing the growth of unculturable microorganisms and promoting the growth and yield of plants; some have been shown to possess antimalarial and anticancer properties (Saha et al. 2016).

The opine-like metallophore BGC in region 15.1 has a low similarity score with the bacillopaline BGC in Paenibacillus mucilaginosus KNP414 (Fig. S11B). Opine metallophores are produced by two core enzymes: a nicotianamine synthase and an opine dehydrogenase, both detected in strain M3T. Although their functions have not been well-established, they were found to be associated with virulence in Staphylococcus aureus and Pseudomonas aeruginosa (McFarlane and Lamb 2017).

In summary, strain M3T is a novel species based on 16 S rRNA gene sequence similarity, dDDH and ANI values, phenotypic characterization, and chemotaxonomic analyses. Strain M3T contains putative BGCs similar to active ones found in other organisms but differing in DNA sequence. Owing to the low similarity scores and support from related studies, the secondary metabolite BGCs of the isolate require further investigation.

Description of Lysinibacillus zambalensis sp. nov.

Lysinibacillus zambalensis (zam.ba.len’sis. N.L. masc. adj. zambalensis, pertaining to Zambales, where the type strain was isolated).

Aerobic, Gram-stain-positive, non-motile, rod-shaped bacterium. On TSB plate, colonies are round, creamy white, with an entire margin, raised elevation, and opaque. The cell size is approximately 3 μm in length and 0.6 μm in width. Growth occurs at 25–37 °C (optimal 30 °C), and a pH range of 6–10 (optimal 8), and 0.5–3% (w/v) NaCl (optimal 0.5%). The cells form ellipsoidal terminal endospores. Catalase positive, citrate negative, indole negative, lysine decarboxylase positive, methyl red negative, nitrate reduction negative, oxidase negative, urease negative, and Voges-Proskauer negative. Exhibits sensitivity to ciprofloxacin and penicillin G. The result from the BIOLOG GEN III assay indicates the utilization of acetic acid, acetoacetic acid, D-fructose-6-PO4, D-gentibiose, D-lactic acid methyl ester, D-turanose, formic acid, glycerol, inosine, L-alanine, L-aspartic acid, L-fucose, L-glutamic acid, L-lactic acid, N-acetyl-D-glucosamine, N-acetyl-β-D-mannosamine, propionic acid, tween 40, α-hydroxy-butyric acid, β-hydroxy-D, L-butyric acid, and α-keto-butyric acid. Results indicate partial utilization of D-fucose, D-fructose, D-galactose, D-glucose-6-PO4, D-melibiose, dextrin, β-methyl-D-glucoside, glucuronamide, gelatin, L-malic acid, N-acetyl-D-galactosamine, and N-acetyl neuraminic acid. Showed tolerance to 1% sodium lactate, guanidine HCl, lithium chloride, potassium tellurite, sodium bromate, and sodium butyrate. Negative results are recorded for 3-methyl glucose, α-D-Glucose, D-maltose, α-D-lactose, α-keto-glutaric acid, bromo-succinic acid, citric acid, D-arabitol, D-aspartic acid, D-cellobiose, D-galacturonic acid, D-gluconic acid, D-glucuronic acid, D-mannose, D-mannitol, D-malic acid, D-raffinose, D-salicin, D-serine, D-sorbitol, D-saccharic acid, D-trehalose, γ-amino-butyric acid, glycyl-L-proline, L-arginine, L-histidine, L-pyroglutamic acid, L-rhamnose, L-serine, L-galactonic acid lactone, methyl pyruvate, mucic acid, myo-inositol, p-hydroxy-phenylacetic acid, pectin, quinic acid, stachyose, and sucrose. Negative results for the chemical sensitivity tests include 8% NaCl, fusidic acid, tetrazolium blue, and tetrazolium violet. The major respiratory quinone is MK-7 and the major polar lipids are diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and two unknown phospholipids. The predominant cellular fatty acid is iso-C15:0 and the peptidoglycan type is L-Lys–D-Asp (type A4α). The G + C content of the genome is 36.5%.

The type strain, designated M3T (= TISTR 10640T = BIOTECH 10973T), is isolated from water collected from the Poon Bato hyperalkaline spring in Zambales province, Philippines. The GenBank accession numbers for the 16 S rRNA gene and the genome of the type strain are PQ061501 and JBEGDG000000000, respectively.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (3MB, docx)

Acknowledgements

We would like to acknowledge Gamaliel Cabria (University of Waikato), Vina Argayosa, Camille Flores, and Rosalyn Diaz (Microbiological Research and Services Laboratory, Natural Sciences Research Institute, University of the Philippines Diliman) for their invaluable help in the microbiology portion of the work. We gratefully acknowledge the University of San Agustin, Center for Advanced New Materials, Engineering, and Emerging Technologies (CANMEET) in Iloilo City for their invaluable assistance in SEM data acquisition and analysis.

Abbreviations

DOC

Dissolved organic carbon

DIC

Dissolved inorganic carbon

LPSN

List of prokaryotic species with standing in nomenclature

dDDH

Digital DNA-DNA hybridization

orthoANI

Orthologous average nucleotide identity

TSA or TSB

Tryptone soya agar or tryptone soya broth

GBDP

Genome BLAST distance phylogeny

UBCG

Up-to-date bacterial core gene set

DPG

Diphosphatidylglycerol

PE

Phosphatidylethanolamine

PG

Phosphatidylglycerol

BGC

Biosynthetic gene cluster

NRPS

Non-ribosomal peptide synthetase

PKS

Polyketide synthase

RiPPs

Ribosomally synthesized and post-translationally modified peptides

Author contributions

Author Contributions: Conceptualization: J.A.A., K.R.Q.L., J.E.H.L., D.S.D., R.L.D.; Methodology, J.A.A, L.D.L. K.R.Q.L., J.E.H.L., J.J.T.; Validation: J.A.A, L.D.L., J.E.H.L., E.J.S.; Formal analysis: J.A.A; Investigation: J.A.A., K.R.Q.L., J.J.T., E.J.S., C.A.A.; Resources: J.E.H.L. and D.S.D.; Data curation: J.A.A., L.D.L., J.J.T., J.E.H.L.; Writing—original draft preparation: J.A.A; Writing—review and editing: All authors; Supervision: J.E.H.L., D.S.D., R.L.D., C.A.A.; Funding acquisition: J.E.H.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the in-house funds of the National Institute of Molecular Biology and Biotechnology, College of Science, University of the Philippines Diliman.

Data availability

Strain M3T was isolated from the Poon Bato Spring, Botolan, Zambales, Philippines. The isolate has been deposited in the Philippine National Collection of Microorganisms (UP Los Banos, Laguna, Philippines) under the accession number BIOTECH 10973T, and in the Thailand Institute of Scientific and Technological Research (TISTR) Culture Collection (Pathum Thani, Thailand) under the accession number TISTR 10640T. The draft genome sequence of strain M3T has been deposited in the GenBank database under the accession number JBEGDG000000000. The GenBank accession number of the 16 S rRNA gene sequence of strain M3T is PQ061501.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (3MB, docx)

Data Availability Statement

Strain M3T was isolated from the Poon Bato Spring, Botolan, Zambales, Philippines. The isolate has been deposited in the Philippine National Collection of Microorganisms (UP Los Banos, Laguna, Philippines) under the accession number BIOTECH 10973T, and in the Thailand Institute of Scientific and Technological Research (TISTR) Culture Collection (Pathum Thani, Thailand) under the accession number TISTR 10640T. The draft genome sequence of strain M3T has been deposited in the GenBank database under the accession number JBEGDG000000000. The GenBank accession number of the 16 S rRNA gene sequence of strain M3T is PQ061501.

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