aim Advances in Microbiology 2165-3410 2165-3402 Scientific Research Publishing 10.4236/aim.2026.164008 aim-150540 Article Biomedical Life Sciences Insights into the Genotypic and Phenotypic Features of Streptomyces sp. PSAA01: Exploring Its Vast Promising Potential 0000-0003-3367-3036 Das Prasenjit 1 † Sarkar Biraj 1 0009-0005-6776-6478 Patra Dipanwita 1 0000-0002-7752-0982 Mandal Sukhendu 1 † Laboratory of Molecular Bacteriology, Department of Microbiology, University of Calcutta, Kolkata, India

These authors contributed equally to this work.

The authors declare that there are no conflicts of interest.

 01 04 2026 04 2026 16 04 151 172 12 01 2026 28 03 2026 31 03 2026 © 2026 by the authors and Scientific Research Publishing Inc. 2026 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( https://creativecommons.org/licenses/by/4.0/ ). https://doi.org/10.4236/aim.2026.164008

Streptomycessp. are actinobacteria, which are generally known to be prolific producers of antibiotics and various antimicrobial compounds. More than 90% of the reported Streptomycessp. are known to be isolated from soil samples. Exploring the genomes of such actinobacteria has sparked interest in finding bioactive secondary metabolites after the remarkable breadth of biosynthetic gene clusters (BGCs) for antibacterial compounds was discovered. Streptomyces sp. PSAA01 was obtained from soil samples taken in Manas National Park, Assam, India. It was discovered that this strain and S.yatensis DSM 41771T are closely related. The strain PSAA01 has been found to harbour diverse biosynthetic gene clusters (BGCs), which might be responsible for the production of many significant bioactive secondary metabolites.

 Actinobacteria Antimicrobial Whole-Genome Genomics Biosynthetic Gene Clusters (BGCs) 1. Introduction

Actinobacteria are Gram-positive bacteria with characteristic high GC content and are known to constitute a cosmopolitan phylum that includes both rod-shaped and filamentous bacteria [1]-[3]. Actinobacteria are known to be ubiquitous in terrestrial, freshwater, and marine ecosystems and exhibit the ability for both solitary inhabitation and symbioses with other microbes and/or higher-order organisms [3][4]. The filamentous actinobacteria, particularly those which belong to the family Actinomycetaceae, are prolific producers of a wide range of secondary metabolites, which include around two-thirds of all known antibiotics, various antifungal, anticancer, and immunosuppressive agents, as well as numerous industrially relevant enzymes [3]. Among all the reported genera belonging to Actinomycetaceae, the most explored and well-studied genus is Streptomyces [3]. It is an incredibly diverse genus constituted of around 1231 species, which are included in the List of Prokaryotic names with Standing in Nomenclature, i.e., LPSN [5] with a validly published correct name under ICNP (International Code of Nomenclature of Prokaryotes), as accessed on 16th July 2024. Aerobic soil bacteria make up the majority of the Streptomyces species that have been reported [3] and are supposed to constitute almost 90% of all the actinobacteria found in soil [6]. Species belonging to the genus Streptomyces display a distinct filamentous life cycle and reproduce by producing spores [3]. The commencement of sporulation is believed to be associated with the production of numerous bioactive secondary metabolites like antibiotics, anti-fungals, antivirals, antitumor, and/or insecticidal compounds [3][7][8]. The genus Streptomyces produces around 50% - 55% of the total antibiotics produced by all microorganisms [9][10] and approximately 80% of all the antibiotics produced by actinobacteria [11]-[14].

Unfortunately, the rapid emergence of resistant bacteria, which has been occurring worldwide, compromises the effectiveness of once-significant antibiotics. This catastrophe is attributed to the overuse and misuse of antibiotics, along with the scarcity of new, efficient drug discoveries due to a lack of economic incentives and challenging regulatory requirements [15].

Amidst this crisis, there is a recent growing interest in finding new natural products (NPs) with diverse antimicrobial or antibiotic properties [3]. The discovery of an unforeseen number of biosynthetic gene clusters (BGCs) for antibiotic-like substances from the genomes of actinomycetes has acted as a stimulus among microbiologists to explore the genomes of actinomycetes to discover bioactive secondary metabolites [3][9][16]-[18].

This study delineates the polyphasic taxonomical identification of a Streptomyces sp. PSAA01, which was isolated from a soil sample of Manas National Park, Assam, India. The genome of strain PSAA01 was explored in search of various biosynthetic gene clusters (BGCs).

2. Materials and Methods 2.1. Isolation and Maintenance

The strain PSAA01 is a soil isolate obtained from a soil sample of Manas National Park, Assam, India. Initially, to selectively isolate actinobacteria from the soil sample, the sample was subjected to CaCO3 treatment at an ambient temperature for around 168 hours, followed by heating the soil sample at 65˚C for 2 hours [19]. Following the preparatory steps for the selective separation of actinobacteria from the soil sample, 1 g of the soil was dissolved in 1 ml of 0.9% NaCl solution and diluted up to 10−6 times. Later, 100 µL of the final dilution sample was spread on Starch casein medium [20] supplemented with 50 µg/mL of two antifungal compounds, i.e., nystatin and cycloheximide, and incubated for 3 to 4 days at 30˚C until single colonies appeared on the selection plate. Later, the colonies were individually picked with a sterile inoculation loop and streaked again on sterile Starch casein agar plates for purification.

2.2. Biochemical Characterization

The carbon utilization behavior of the isolated strain PSAA01 was assessed using 1% (w/v) of different carbon sources, such as glucose, galactose, ribose, arabinose, maltose, xylose, rhamnose, melibiose, raffinose, adonitol, inositol, melizitose, pyruvate, and dulcitol in ISP-9 medium [21][22]. The tests performed were as previously reported for the hydrolysis of starch, cellulose breakdown, tributyrin hydrolysis, nitrate reduction, gelatin liquefaction, IMVIC test, and H2S generation [23]. Using conventional techniques, the production of urease, catalase, and indole acetic acid (IAA) was evaluated [23]. This isolate was shown to hydrolyze xanthine, hypoxanthine, xylan, tyrosine, casein, and arbutin, which was assessed by the standard method [23]. The standard Hicarbo Kit from Himedia, India, which has 35 distinct carbon sources, was used to ferment the carbohydrates.

2.3. Genomic DNA Isolation and Whole-Genome Sequencing

The genomic DNA of PSAA01 was isolated from freshly grown 5 mL of ISP-2 broth. The genomic DNA was extracted by the phenol: chloroform method according to the standard protocol [24]. Using the Illumina NovaSeq 6000 platform (Neuberg Diagnostics Pvt Ltd., Ahmedabad, India), the paired-end libraries were produced and sequenced.

The complete 16S rDNA sequence of the isolate PSA001 was obtained from the draft genome sequence of the strain and was compared with the 16S rDNA sequences of the closest type strains using EzBioCloud [25]. Using MEGA 6 software and the ClustalW program, multiple sequence alignment was carried out with the 16S rDNA sequences [26]. The phylogenetic tree based on the 16S rDNA sequences was constructed using the Maximum-Likelihood (ML) method [27]. Phylogenetic analysis was performed with the Jukes-Cantor model [28], gamma distributed with invariant sites (G + I) for the ML method [29]. Bootstrap analysis (1000 resampled datasets) was used to evaluate the topology of the trees.

For the multi-locus sequence analysis (MLSA) of PSAA01, four housekeeping genes, i.e., atpD (F0 - F1 ATP synthase subunit beta), gyrB (DNA gyrase subunit B), recA (recombinase RecA), and rpoB (DNA-directed RNA polymerase subunit beta) were obtained from its draft genome sequence. The four mentioned housekeeping gene sequences of the closest relatives (based on 16S rDNA nucleotide sequence data) were directly downloaded from GenBank or retrieved from respective draft or complete genome sequences. Before being subjected to further analysis, the relevant genes’ nucleotide sequences were manually cut at the same position after being aligned using MEGA6 software. The four housekeeping gene nucleotide sequences were then concatenated head to tail as atpD-gyrB-recA-rpoB (1074, 1724, 1000, and 2774 nucleotides, respectively) for PSAA01 and its closest relatives. Pairwise distance was calculated using the Kimura-2 parameter model [30]. MEGA 6 software was used to determine the optimal model for maximum likelihood phylogenetic analysis, which was subsequently applied to the analysis. The best model for maximum likelihood was found to be the General Time Reversible model with Gamma distributed with invariant sites (GTR + G + I) [31]. The bootstrap technique (using 1000 similar datasets) was employed to assess the tree topology.

The closest type strain genomes were aligned and compared using the BLAST Ring Image Generator (BRIG) to create a circular map of each genome [32], with PSAA01 as the reference strain. The National Center for Biotechnology Information (NCBI) and KEGG GENOME Database are public databases that provide access to and downloads of the genome sequences of all known type strains of Streptomyces (a total of 30 strains, including strain PSA001). Whole genome-based taxonomic analyses were conducted using the Type Strain Genome Server (TYGS) (https://tygs.dsmz.de) [33]. FastME was used to build the phylogenomic tree from the genome blast distance phylogeny (GBDP). First, pairwise genome comparisons were performed using the GBDP, and inter-genomic distances were inferred under the algorithm “trimming” and distance formula d5. The inference of branch supports was derived from 100 pseudo-bootstrap replicates [33]. The genome was annotated according to the RAST protocol [34].

The average nucleotide identity (ANI) values between strain PSAA01 and its closest type strains were calculated in EzGenome (https://ezbiocloud.net/tools/ani) [25]; the average amino acid identity (AAI) was calculated using the online calculator (http://enve-omics.ce.gatech.edu/aai/) [35], and the genome-to-genome distances (GGDs)/digital DNA-DNA hybridization (dDDH) values were calculated using the Genome-to-Genome Distance Calculator (GGDC; http://ggdc.dsmz.de) [36].

 2.4. Biosynthetic Gene Clusters (BGCs): Characterization and Comparative Study of Their Distribution

Secondary metabolite biosynthetic gene clusters (BGCs) from the genome sequences of PSAA01 and its 10 closest type strains were analyzed using antiSMASH version 6.1.0 [37] to examine the distribution of BGCs among the studied organisms. The structures of the secondary metabolites encoded by the genome of PSAA01 were predicted using the PRISM 4.4.5 webserver [38].

 2.5. Identification of BGCs, PKS-KS Domains, and NRPS C Domains

Antibiotics and secondary metabolite analysis shells were used to identify the BGCs found in the genome of Streptomyces (antiSMASH v7.1.0) (https://antismash.secondarymetabolites.org) [39]. After retrieving the relevant sequences from each identified BGC, phylogenetic analyses were performed for the KS and C domains of the PKS and NRPS genes, respectively.

3. Results and Discussion

The strain PSAA01 was isolated from soil samples taken from the Manas National Park in Assam, India. From the biochemical analysis of strain PSAA01 (Table S1), it has been found that the strain is capable of utilizing L-rhamnose, D-sucrose, inositol, melezitose, maltose, D-galactose, raffinose, adonitol, pyruvate, and melibiose; but is unable to use D-ribose, glucose, arabinose, xylose, and dulcitol; whereas, S.yatensis is reported to utilize glucose and several pentose sugars such as ribose, arabinose, and xylose. These differences in pentose sugar utilization further distinguish PSAA01 from S.yatensis [40]. According to the species description of S.yatensis, the type strain shows positive reactions for gelatin liquefaction, casein hydrolysis, and tyrosine degradation while PSAA01 shows negative results in caseinase and gelatinase activity. Strain PSAA01 utilized L-alanine, L-arginine, and L-asparagine. These reactions are consistent with general metabolic characteristics of the genus Streptomyces and do not strongly differentiate PSAA01 from S.yatensis. The strain PSAA01 can hydrolyze xylan and hypoxanthine, but cannot hydrolyze tyrosine and xanthine. PSAA01 was catalase positive and capable of nitrate reduction, consistent with reported characteristics of S.yatensis. It was negative for H2S production, methyl red, Voges-Proskauer, and indole tests, which also aligns with general streptomycete properties.

Table 1. Shows the respective accession numbers, genome size (bp), and contigs used for the genomic analyses.

Strain Accession No. Genome size (bp) Contigs
Streptomyces sp. PSAA01 NZ_JAKKUU000000000 9,224,189 271
S. melanosporofaciens DSM 40318 T NZ_FNST00000000 10,769,732 2
S. antimycoticus NBRC 12839 T NZ_BJHV00000000 11,174,199 3
S. yatensis DSM 41771 T NZ_CP072941 10,360,357 1
S. rhizosphaericus DSM 41760 T NZ_JAGMTS000000000 11,044,339 5
S. indonesiensis DSM 41759 T NZ_JAGSHY000000000 11,666,822 2
S. cangkringensis DSM 41769 T NZ_JAGMTV000000000 11,723,082 4
S. hygroscopicus subsp. hygroscopicus NBRC 13472 T NZ_BBOX00000000 9,464,604 680
S. antioxidans MUSC 164 T NZ_LAKD00000000 9,118,065 282
S. sioyaensis DSM 40032 T NZ_SDIF00000000 7,847,945 289
S. decoyicus NRRL 2666 T NZ_CP082301 8,632,952 1
S. asiaticus DSM 41761 T NZ_JAGSHX000000000 11,877,923 6
S. rimosus subsp. rimosus ATCC 10970 T NZ_CP048261 9,643,891 2
S. himastatinicus ATCC 53653 T NZ_ACEX00000000 11,030,030 783
S. lydicus ATCC 25470 T NZ_RDTD00000000 7,935,716 20
S. chattanoogensis NRRL ISP-5002 T NZ_LGKG00000000 9,129,105 217
Kitasatospora setae KM-6054 T NC_016109 8,783,278 1

Figure 1. Shows the maximum-likelihood (ML) phylogenetic tree based on the 16S rRNA sequence of strain PSAA01 and its closest type strains. Only bootstrap values >95% (expressed as % of 1000 replications) are shown at the nodes. The bar at the bottom indicates the scale of the branch lengths.

The draft genome sequence of the strain PSAA01 was obtained in 271 contigs, with an estimated genome size of 9,224,189 bp and 71.2% GC content (Table 1). The draft sequence of the strain is submitted to the NCBI under the accession number NZ_JAKKUU000000000. The 1533 bp long complete 16S rRNA gene sequence of the strain PSAA01 showed the highest similarity of 99.72% with Streptomycesmelanosporofaciens DSM 40138T according to EZBioCloud. Yet, according to the maximum-likelihood (ML) tree based on the 16S rRNA gene sequences of strain PSAA01 and its closest type strains (Figure 1), it was found that strain PSAA01 was placed in a distinct clade. The whole genome sequences of the closest strains or type strains based on the 16S rDNA sequence similarities were obtained from the NCBI GenBank, along with an outgroup strain for phylogenetic analyses (Table 2).

For the visualization of circular genome comparison, a BLASTN-based ring image was generated by BRIG version 0.95 [32] for available genomes of the closest type strains with Streptomyces sp. PSAA01 as the reference strain (Figure 2).

Table 2. Comparative genotypic analysis of Streptomyces sp. PSAA01 and its closest related type strains. aANIvalues (determined on https://ezbiocloud.net/tools/ani); bAAIvalues (determined on http://enve-omics.ce.gatech.edu/aai/); cDigitalDNA-DNA hybridization (dDDH) calculated with the Genome-to-Genome Distance Calculator 3.0 (GGDC 3.0) (available at https://ggdc.dsmz.de/); dMLSA distances (i.e., the pairwise distances of the housekeeping genes concatenated in the order atpD-gyrB-recA-rpoB) calculated in MEGA 6 software using the Kimura-2 parameter model for distance calculation.

Strains ANI value a AAI value b dDDH c MLSA distance d
S. melanosporofaciens DSM 40318 T 95.75 94.93 63.90 0.009
S. antimycoticus NBRC 12839 T 95.52 94.41 62.90 0.009
S. yatensis DSM 41771 T 97.21 96.53 73.80 0.005
S. rhizosphaericus DSM 41760 T 91.42 90.07 44.00 0.029
S. indonesiensis DSM 41759 T 91.41 90.01 43.80 0.028
S. cangkringensis DSM 41769 T 91.32 90.01 43.70 0.029
S. hygroscopicus subsp. hygroscopicus NBRC 13472 T 89.90 87.13 40.40 0.045
S. antioxidans MUSC 164 T 90.44 88.91 50.30 0.031
S. sioyaensis DSM 40032 T 78.76 71.20 22.60 0.094
S. decoyicus NRRL 2666 T 78.76 71.15 23.10 0.096
S. asiaticus DSM 41761 T 91.33 89.95 43.70 0.028
S. rimosus subsp. rimosus ATCC 10970 T 78.61 70.66 23.00 0.099
S. himastatinicus ATCC 53653 T 84.79 80.64 29.10 0.052
S. lydicus ATCC 25470 T 78.81 71.54 23.10 0.097
S. chattanoogensis NRRL ISP-5002 T 78.75 70.47 22.70 0.098

Figure 2. Comparative genomic map of the Streptomyces sp. PSA001 genome and the 15 other Streptomyces genomes. The BLASTN-based ring image was generated by the BLAST Ring Image Generator (BRIG) version 0.95 [32]. The innermost two rings show GC content (black) and GC skew (purple/green). The remaining 15 rings (from inside to outside) represent a BLASTN comparison with S.melanosporofaciens DSM 40318T; S.antimycoticus NBRC 12839T; S.yatensis DSM 41771T; S.rhizosphaericus DSM 41760T; S.indonesiensis DSM 41759T; S.cangkringensis DSM 41769T; S.hygroscopicus subsp. hygroscopicus NBRC 13472T; S.antioxidans MUSC 164T; S.sioyaensis DSM 40032T; S.decoyicus NRRL 2666T; S. asiaticusDSM 41761T; S.rimosus subsp. rimosus ATCC 10970T; S.himastatinicus ATCC 53653T; S.lydicus ATCC 25470T; S.chattanoogensis NRRL ISP-5002T, respectively.

In the maximum-likelihood (ML) tree of PSAA01 and its related strains based on the concatenated housekeeping gene sequences (i.e., atpD-gyrB-recA-rpoB), PSAA01 is found to share the same clade with S.yatensis DSM 41771T, indicating that they might have descended from the same ancestor (Figure 3). The MLSA pairwise distances between strain PSAA01 and its closely related type strains, except S.yatensis DSM 41771T (0.005), were all found to be above the cut-off point of 0.007, as recommended by [41] for novel species demarcation (Table 2 and Table S2). Furthermore, from the evaluation of the ANI values, it has been found that the values for S.melanosporofaciens DSM 40318T (95.75%), S.antimycoticus NBRC 12839T (95.52%), and S.yatensis DSM 41771T (97.21%) are above the cut-off level (95% - 96%) recommended as the average nucleotide identity (ANI) criterion for interspecies identity [42] (Table 2). Additionally, the AAI values of PSAA01 and related type strains were significantly lower than the recommended cut-off point (95% - 96%) for species delineation [43], except for S.yatensis DSM 41771T, which had an AAI value of 96.53%, which is above the range of the threshold (<95% - 96%) (Table 2). Similarly, except forS.yatensis DSM 41771T (73.80%), the dDDH values for all the related strains were found to be lower than the recommended threshold of 70% for species delineation. The PSAA01 strain was also found to be closely related to Streptomycesyatensis by the TYGS-based phylogenomic analysis [37] (Table 2; Figure 4). From all these, we can conclude that the strain PSAA01 is no longer distinguishable fromS.yatensis DSM 41771T, but rather appears to be very closely related.

Figure 3. Multi-locus maximum likelihood (ML) phylogenetic tree based on concatenated housekeeping nucleotide sequences (atpD-gyrB-recA-rpoB) of strain PSAA01 and its closest type strains. Only bootstrap values >50% (expressed as % of 1000 replications) are shown at the nodes.

According to the results from antiSMASH version 6.0, the genome sequence of PSAA01 harbors 53 BGCs, of which 8 clusters code for NRPS, 25 clusters for PKS, 5 clusters for NRPS-like, 2 clusters for RiPP-like, 3 clusters for siderophore, 6 clusters for terpene, 2 clusters for ladderane, and 1 cluster each for PKS-like, arylpolyene, RRE-containing, hserlactone, ectoine, redox-cofactor, hglE-KS, lanthipeptide-class-ii, indole, and butyrolactone, respectively (Table S3). A high number of contigs in a draft genome can significantly affect the integrity, detection, and interpretation of biosynthetic gene clusters (BGCs), especially large modular systems such as polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). Below is a structured discussion of the key impacts. A comparative distribution of BGCs present in the genomes of strain PSAA01 and the 10 closest studied strains is shown in Figure 5.

Classic RAST predicted a total of 8418 protein-encoding genes (PEGs), of which 2179 with known functions were classified into 22 groups, each associated with distinct biological roles. Out of these 2179 genes, 54 were related to the categories of virulence, disease, and defense; 8 were related to dormancy and sporulation; 7 were related to secondary metabolism; 413 were related to the synthesis of amino acids and their derivatives; 200 were related to cofactors, vitamins, prosthetic groups, and pigment synthesis (Figure 6).

Figure 4. Phylogenetic tree based on genome sequences of the representativeStreptomyces strains in the TYGS tree inferred with FastME 2.1.6.1 [33] from the Genome BLAST Distance Phylogeny approach (GBDP); distances were calculated from genome sequences. The branch lengths are scaled in terms of the GBDP distance formula d5. The numbers above the branches are GBDP pseudo-bootstrap support values >60% from 100 replications. The tree was rooted at the midpoint [36].

Figure 5. BGCs distribution determined by antiSMASH version 6.0 [37] in the genomes of PSAA01 and 10 related Streptomyces sp.

Figure 6. Number of identified protein-encoding genes of known function in Streptomyces sp. PSAA01 that are present in different subsystems according to the RAST server subsystem classification. Numbers are represented as: 1: Respiration; 2: Potassium metabolism; 3: Virulence, Disease and Defense; 4: Regulation and Cell signaling; 5: Protein metabolism; 6: Cell Wall and Capsule; 7: DNA Metabolism; 8: Membrane Transport; 9: Sulfur Metabolism; 10: Iron acquisition and metabolism; 11: Amino Acids and Derivatives; 12: Dormancy and Sporulation; 13: Stress Response; 14: RNA Metabolism; 15: Nitrogen Metabolism; 16: Metabolism of Aromatic Compounds; 17: Secondary Metabolism; 18: Carbohydrates; 19: Phosphorus Metabolism; 20: Nucleosides and Nucleotides; 21: Fatty Acids, Lipids, and Isoprenoids; 22: Cofactors, Vitamins, Prosthetic Groups, Pigments.

All of the highly similar compounds were chemically characterized and were present in the Minimum Information about a Biosynthetic Gene cluster (MIBIG) database, which was determined directly through antiSMASH [44]. From the most common BGCs, one showed the antimicrobial compound hygrocin A (NRPS/PKS-like), which exhibited 70% gene similarity with Streptomyces sp. LZ35, 51% gene similarity with S. olivaceus,and 58% gene similarity withStreptomyces sp. HK10576. The gene (hygrocin) cluster contains five core biosynthetic genes, some additional biosynthetic genes, and other regulatory genes. The structure of these core genes (1 - 5) is made up of three modules with the domains KS-AT-DH-KR-CP, KS-AT-CP, and KS-AT-CP_Docking_C term; one module with the domain KS-AT-DH-ER-KR-CP_Docking_C term; one module with the domain KS-AT-DH-KR-CP_Docking_C term; one module with the domain KS-AT-DH-ER-KR-CP_Docking_C term, and two modules with KS-AT-CP, KS-AT-DH-KR-CP for the 1, 2, 3, 4, and 5 core genes, respectively. Polymer prediction by the aforementioned cluster is—(Me - ccmal - mal - ohmal) + (redemal) + (Me-redmal) + (Me - mal - ccmal), and the putative structure is given in Figure 7(e). The biosynthetic gene cluster contains five core genes: hgcA, hgcB, hgcC, hgcD, and hgcE. Another antimicrobial compound, curamycin (T2PKS), has been identified in three species: S.avermitilis (shows 100% similarity), S. cyaneus (shows 100% similarity), and S.collinus (shows 85% similarity) (Figure 7(b)). Two other important biosynthetic gene clusters produce the antimicrobial compounds nigericin and mediomycin A, which show very high structural similarity to those from Streptomyces sp. PSAA01. This compound is a natural by-product and can be used as a potent antimicrobial agent [45] (Figure 7).

Several biosynthetic gene clusters (BGCs) have been identified that encode putative high-value metabolites, many of which appear to be uniquely associated with specific species. Although many similar products have already been characterized from different sources, there are some compounds that are specific to Streptomyces species. Here, we have considered the clusters that have more than 50% similarity. The pentamycin gene cluster has a single T1PKS and five domains. Although the pentamycin gene sequence is between Streptomyces sp. S816 and S.chattanoogensis has 86% similarity, the domain and structural organization are completely different (Figure 8(a)). The domain of Streptomyces sp. S816 is composed of five modules (KS-AT-CP, KS-AT-KR-CP, KS-AT-DH-KR-CP, KS-AT-DH-KR-CP, KS-AT-DH-KR-CP) + (KS-AT-KR-CP, KS-AT-KR-CP, KS-AT-KR-CP, KS-AT-KR-CP) + (KS-AT-DH-KR-CP) + (KS-AT-DH-KR-CP, KS-AT-DH-KR-CP) + (KS-AT-KR-CP, KS-AT-KR-CP-TE) and the domain of S.chattanoogensis also has five modules but a different composition (KS-AT-KR-CP, KS-AT-DH-KR-CP, KS-AT-DH-KR-CP, KS-AT-DH-KR-CP) + (A-CP-KS-AT-CP) + (KS-AT-DH-KR-CP, KS-AT-KR-CP, KS-AT-KR-CP, KS-AT-KR-CP, KS-AT-CP, KS-AT-KR-CP) + (KS-AT-DH-KR-CP) + (KS-AT-DH-KR-CP-TE). The macrolide group of antibiotics meridamycin isolated from Streptomyces sp. NRRL 30748 and from S.arenicola CNS-205 shows 80% gene sequence similarity, but they are structurally and module-wise very different (Figure 8(b)). Monensin is an ionophoric antibiotic used to treat bacterial, fungal, and parasitic infections. In addition to Monensin, Herboxidiene represents another class of antimicrobial compounds. Although they share more than 50% sequence similarity with PSAA01, their structural features are markedly different (Figure 8(c), Figure 8(d)). Secondary metabolites identified in this study were predicted through genome mining based on biosynthetic gene cluster homology to characterized pathways. These compounds were not chemically extracted or structurally validated and therefore remain bioinformatically inferred products.

Figure 7. Highly similar antimicrobial gene clusters of Streptomyces species compared with known clusters in the antiSMASH database. Gene clusters for Hygrocin A (a), Curamycin (b), Nigericin (c), Mediomycin A (d); and the putative compounds produced by these clusters: Hygrocin A (e), Curamycin (f), Nigericin (g), Mediomycin A (h).

Figure 8. Species-specific clusters and their putative products from Streptomyces compared to the known clusters and their products from the antiSMASH database. (a) The biosynthetic cluster of pentamycin, a macrolide group of antibiotics, shows 86% gene similarity with S. chattanoogensis. (b) Another macrolide group of antibiotic, the meridamycin cluster, shows 80% similarity with S. arenicola CNS-205. (c) The monensin, an ionophoric group of antibiotic cluster, shows 61% gene similarity with Streptomyces sp. CS684. (d) The herboxidiene cluster shows 57% gene similarity with S. chromofuscus.

 4. Conclusion

The strain Streptomyces sp. PSAA01 is a soil isolate from the Manas National Park, Assam, India, and has been found to be very closely related to S.yatensis DSM 41771T. The strain exhibits amylase, catalase, lipase, cellulase, and urease activity. It can hydrolyze xylan and hypoxanthine. Strain PSAA01 can utilize L-rhamnose, D-sucrose, inositol, melezitose, maltose, D-galactose, raffinose, adonitol, pyruvate, and melibiose as carbon sources; it can also utilize amino acids like L-alanine, L-arginine, and L-asparagine. The strain has also been found to harbor diverse BGCs encoding various secondary metabolites like NRPS, PKS, NRPS-like, RiPP-like, siderophore, terpene, ladderane, PKS-like, arylpolyene, RRE-containing, hserlactone, ectoine, redox-cofactor, hglE-KS, lanthipeptide-class-ii, indole, and butyrolactone. According to the RAST server, 8418 protein-encoding genes (PEGs) were predicted in total, and among these, 2179 genes with known functions were divided into 22 categories, each with a unique biological role. The antibacterial chemical hygrocin A (similar to NRPS/PKS) exhibits gene similarity with Streptomyces sp. LZ35 of 70%, S.olivaceus of 51%, and Streptomycessp. HK10576 of 58%, as determined by antiSMASH. Two further types of antibacterial compounds with sequences longer than 50% but extremely different structures are monensin and herboxidiene, which have also been found by the server.

 Data Availability

The 16S rRNA gene sequence and the genome of Streptomyces sp. PSAA01 were deposited in GenBank (NCBI) under the accession numbers MT829328 and JAKKUU000000000, respectively. Other data are available in this manuscript.

 Author Contributions

Prasenjit Das: Conceptualization, performed bioinformatic and formal analysis, writing review; Biraj Sarkar: Performed bioinformatic analysis and writing-original draft; Dipanwita Patra: Performed partial bioinformatic data analysis; Sukhendu Mandal: Conceptualization, editing and supervision. All authors have read and approved the manuscript.

 Consent for Publication

All the authors agree to submit the manuscript for publication.

 Acknowledgements

P.D. is appreciative of financial support in the form of a scholarship from the University Grants Commission (UGC), Government of India. B.S. is appreciative of the University of Calcutta’s financial support in the form of a fellowship.

 Supplementary

Table S1. Showing the biochemical test results of Strain PSAA01.

Biochemical analyses of strain PSAA01
Carbon Utilization
L-Rhamnose +
D-Ribose −
D-Sucrose +
Inositol +
Melezitose +
Glucose −
Maltose +
Arabinose −
Xylose −
D-Galactose +
Dulcitol −
Raffinose +
Adonitol +
Pyruvic acid +
Melibiose +
Degradation/hydrolysis
Gelatin −
Xanthine −
Hypoxanthine +
Starch +
Cellulose +
Urea +
Xylan +
Tyrosine −
Casein −
Amino acid utilization
L-Alanine +
L-Arginine +
L-Asperagine +
Other biochemical tests
H 2 S production −
Nitrate reduction +
Catalase +
Methyl red test −
Voges-Proskauer test −
Indole test −
Lipase test +

Table S2. Showing the pairwise distance of PSAA01 and other related strains based on concatenated sequences of the house-keeping gene sequences (atpD-gyrB-recA-rpoB). The MLSA pairwise distance was calculated on MEGA 6 by Kimura parameter 2 model. 1, PSAA01; 2, S.melanosporofaciens DSM 40318T; 3, S.antimycoticus NBRC 12839T; 4, S.yatensis DSM 41771T; 5, S.rhizosphaericus DSM 41760T; 6, S.indonesiensis DSM 41759T; 7, S.cangkringensis DSM 41769T; 8, S.hygroscopicus subsp. hygroscopicus NBRC 13472T; 9, S.antioxidans MUSC 164T; 10, S.sioyaensis DSM 40032T; 11, S.decoyicus NRRL 2666T; 12, S. asiaticusDSM 41761T; 13, S.rimosus subsp. rimosus ATCC 10970T; 14, S.himastatinicus ATCC 53653T; 15, S.lydicus ATCC 25470T; 16, S.chattanoogensis NRRL ISP-5002T; 17, Kitasatosporasetae KM-6054T.

Strains 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1
2 0.009
3 0.009 0.006
4 0.005 0.008 0.009
5 0.029 0.028 0.027 0.027
6 0.028 0.028 0.027 0.027 0.000
7 0.029 0.028 0.027 0.027 0.000 0.000
8 0.045 0.044 0.044 0.044 0.044 0.044 0.044
9 0.031 0.030 0.030 0.030 0.018 0.018 0.018 0.046
10 0.094 0.093 0.092 0.093 0.096 0.096 0.097 0.095 0.099
11 0.096 0.095 0.093 0.095 0.098 0.098 0.098 0.095 0.103 0.040
12 0.028 0.028 0.027 0.027 0.000 0.000 0.000 0.044 0.018 0.096 0.098
13 0.099 0.097 0.096 0.098 0.098 0.098 0.098 0.098 0.100 0.068 0.068 0.098
14 0.052 0.050 0.049 0.051 0.046 0.046 0.046 0.046 0.051 0.094 0.093 0.046 0.093
15 0.097 0.096 0.096 0.097 0.098 0.099 0.099 0.097 0.103 0.044 0.036 0.099 0.068 0.095
16 0.098 0.096 0.095 0.097 0.100 0.101 0.101 0.095 0.104 0.043 0.044 0.101 0.071 0.096 0.047
17 0.139 0.139 0.138 0.140 0.136 0.137 0.137 0.138 0.141 0.134 0.135 0.137 0.135 0.140 0.135 0.132

Table S3. Showing the diverse BGCs present in the genome of PSAA01 as predicted by antiSMASH 6.0.

Cluster Type From To Most similar known cluster Similarity (%) MIBiG accession
Cluster 1 NRPS 350,607 376,969 polyoxypeptin 10 BGC0000457
Cluster 2 T1PKS 56,760 148,040 salinomycin 28 BGC0001244
Cluster 3 T1PKS 1 26,477 nigericin 77 BGC0000041
Cluster 4 Terpene 2118 20,604 hopene 61 BGC0000663
Cluster 5 NRPS, T1PKS 1 34,169 meridamycin 28 BGC0000457
Cluster 6 T1PKS 1 17,440 laidlomycin 34 BGC0000041
Cluster 7 T2PKS 1 17,197 Spore pigment 83 BGC0000215
Cluster 8 T1PKS 1 13,922 nigericin 61 BGC0001068
Cluster 9 Siderophore, T1PKS 1 56,211 apoptolidin 23 BGC0000946
Cluster 10 terpene 71,496 92,569 BE-43547A1, BE-43547A2, BE-43547B1, BE-43547B2B3, BE-43547C1, BE-43547C2 20 BGC0000649
Cluster 11 Ladderane, arylpolyene 1 11,894 atratumycin 39 BGC0001444
Cluster 12 T1PKS 1 10,771 lydicamycin 32 BGC0000056
Cluster 13 T1PKS 1 10,646 Divergolide A/B/C/D 17 BGC0000041
Cluster 14 Siderophore 20,354 32,267 ficellomycin 3 BGC0000946
Cluster 15 T1PKS 1 8384 primycin 21 BGC0000041
Cluster 16 T1PKS 1 6974 - - BGC0001273
Cluster 17 T1PKS 1 6377 - - BGC0000056
Cluster 18 terpene 1 6024 carotenoid 36 BGC0000633
Cluster 19 NRPS 1 5825 telomycin 8 BGC0000396
Cluster 20 RRE-containing 1 5631 Chromomycin A3 5 BGC0001295
Cluster 21 T1PKS 1 4037 - - BGC0000056
Cluster 22 Hserlactone 96,510 109,829 Heronamide A/B/C/D/E/F 8 BGC0001682
Cluster 23 T1PKS 22,648 70,204 Argimycin PI/argimycin PII, nigrifactin/argimycin PIV/ argimycin PV/argimycin PVI/argimycin PIX 43 BGC0000675
Cluster 24 terpene 6349 28,676 geosmin 100 BGC0001181
Cluster 25 NRPS-like, NRPS, T1PKS 48,196 107,409 Griseoviridin, fijimycin A 8 BGC0000417
Cluster 26 NRPS 1 1310 Rhizomide A/B/C 100 BGC0001833
Cluster 27 T1PKS 80,398 106,590 Hygrocin A/B 70 BGC0001858
Cluster 28 T1PKS 1 1004 - - BGC0000260
Cluster 29 NRPS 1 24,103 Ochronotic pigment 75 BGC0002075
Cluster 30 ladderane 68,783 104,375 atratumycin 31 BGC0000056
Cluster 31 siderophore 22,749 34,539 Desferrioxamin B 100 BGC0001478
Cluster 32 ectoine 7021 17,425 ectoine 100 BGC0002052
Cluster 33 terpene 33,884 54,897 2-methylisoborneol 100 BGC0000658
Cluster 34 Redox-cofactor 7722 29,861 Lankacidin C 13 BGC0001484
Cluster 35 NRPS 56,402 90,716 RP-1776 18 BGC0000389
Cluster 36 hglE-KS, T1PKS, RiPP-like 13,146 67,557 - - BGC0002031
Cluster 37 T3PKS 25,793 66,968 7-deoxypactamycin 16 BGC0000280
Cluster 38 NRPS 26,563 86,830 phthoxazolin 4 BGC0000389
Cluster 39 T1PKS 22,650 85,901 ECO-02301 57 BGC0000041
Cluster 40 T1PKS, lanthipeptide-class-ii 61,498 83,906 actagardine 9 BGC0001909
Cluster 41 RiPP-like 13,217 24,524 - - BGC0001803
Cluster 42 PKS-like 124,492 165,511 rustmicin 20 BGC0001911
Cluster 43 T1PKS, NRPS-like 2 77,306 meridamycin 36 BGC0000041
Cluster 44 other 1 26,299 mitomycin 20 BGC0000719
Cluster 45 NRPS-like 91,444 134,371 Echoside A/B/C/D/E 100 BGC0000340
Cluster 46 NRPS, T1PKS, other, NRPS-like 1 64,442 polyoxypeptin 32 BGC0000427
Cluster 47 indole 12,079 33,224 5-isoprenylindole-3-carboxylate β -D-glycosyl ester 61 BGC0001483
Cluster 48 T1PKS 1 45,723 Mediomycin A 46 BGC0001164
Cluster 49 terpene 5686 26,642 pristinol 100 BGC0001746
Cluster 50 T1PKS 1 31,609 fostriecin 23 BGC0000041
Cluster 51 T1PKS 1 30,407 apoptolidin 12 BGC000067
Cluster 52 butyrolactone 18,184 29,116 - - BGC0000848
Cluster 53 NRPS-like 1 29,431 Echoside A/B/C/D/E 11 BGC0000888
NOTES

*These authors have contributed equally to this work.

#Corresponding author.

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