Genomic insights and biosynthetic gene cluster analysis of Streptomyces chrestomyceticus strain B5 showing antimicrobial activities

ABSTRACT

Streptomyces bacteria are known for their different biological activities and enormous biosynthetic potential. Fungal pathogens, especially Candida auris, have emerged as clinically important pathogens involved in causing systemic infections and high mortality. Therefore, antimicrobial compounds effective against C. auris are needed to tackle infections caused by these multidrug-resistant yeast pathogens. A soil bacterium was isolated from soil in Delhi, India, exhibited broad-spectrum antimicrobial activities against various pathogenic bacteria, such as Bacillus subtilis, Staphylococcus aureus, and Escherichia coli, and pathogenic fungi, including Candida albicans, Fusarium oxysporum, Aspergillus fumigatus, and Candida glabrata. It was subjected to whole-genome sequencing and resulted in a high-quality draft genome with a single contig of 9.61 Mb genome size and 72.1% GC content. Phylogenetic analysis and average nucleotide identity identified that the strain belongs to Streptomyces chrestomyceticus. The genome annotation predicted plant growth-promoting traits and stress-response genes. Genome mining revealed non-ribosomal peptide synthetase, polyketide synthase (PKS), and hybrid biosynthetic gene clusters (BGCs) synthesizing known and unknown metabolites. Most of the encoded compounds by the BGCs were reported for antibacterial and antitumor activities, and two BGCs were identified as antifungal clusters. A trans-acyltransferase polyketide synthase (transAT-PKS) cluster was identified with 27% and 19% similarity to cycloheximide and 9-methylstreptimidone clusters, respectively. The comparison of 9-methylstreptimidone and transAT-PKS cluster revealed differences in the organization of genes in closely related strains. The crude extract of S. chrestomyceticus B5 possessed strong antibacterial and antifungal activities. The bioassay-guided purification and LC-MS identified an antifungal compound (streptimidone derivative or isomer, molecular mass 293.1 Da), which specifically inhibited C. glabrata and C. auris. The transAT-PKS cluster might be responsible for encoding this streptimidone derivative or isomer.

IMPORTANCE

The study highlighted the biosynthetic potential of Streptomyces chrestomyceticus B5, which is a promising strain for producing various bioactive metabolites and is rich in biosynthetic gene clusters responsible for encoding multiple metabolites and producing an antifungal compound, a streptimidone derivative or isomer, active against drug-resistant Candida auris.

INTRODUCTION

Bacterial and fungal infections continue to remain among the foremost contributing causes of global mortality, posing a significant challenge to public health around the globe. The rising antimicrobial resistance (AMR) has resulted in limited treatment options due to the ineffectiveness of present antimicrobials against pathogens. A recent study on bacterial AMR has reported 1.27 million deaths annually caused by major pathogens such as Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and Streptococcus pneumoniae (1). Fungal infections kill more than 2 million people every year, with major pathogenic genera such as Cryptococcus, Candida, Aspergillus, and Pneumocystis. Individuals who are immunocompromised or have underlying health conditions are more vulnerable to severe fungal infections (2). Invasive infections caused by Candida species affect around 1.6 million people every year and cause nearly 1 million deaths. Candida auris, a recently identified multidrug-resistant fungal pathogen, represents a significant global risk to human health. It spreads easily in healthcare settings, is non-susceptible to multiple antifungal drugs, can make biofilms, and survives on medical surfaces for prolonged periods. It is usually misidentified with commercially available test kits (3, 4).

A limited number of antifungal drug classes are available for treating invasive fungal infections, which is comparatively few compared with antibiotic classes. The increasing resistance to one or two major antifungals is leading to untreatable invasive fungal infections and, ultimately, death (3, 5–7). Until now, fungal infections were not considered part of AMR initiatives; however, past outbreaks with secondary infections caused by fungal pathogens in the second COVID-19 wave and the growing resistance among fungal pathogens have prompted the WHO to issue its first fungal priority pathogen list in 2022, which includes C. auris, Candida albicans, and Aspergillus fumigatus in the critical group, whereas Nakaseomyces glabratus (Candida glabrata) is included in the high-priority group (8).

Natural products isolated from microorganisms are known for their diverse scaffolds and complex structures and are vital in drug discovery against infections caused by pathogens (9). In particular, Actinobacteria and the representative genus Streptomyces are known to produce various bioactive molecules with different biological activities (10). They are the source of more than 70% of clinically important antibiotics (11).

Over time, efforts to discover new antimicrobials decreased due to the limitations of classical screening methods and the increasing possibility of rediscovering known molecules (12). It was thought that Streptomyces’ potential to produce antimicrobial compounds had been exhausted; however, genome sequencing has revealed that these bacteria hold much greater biosynthetic potential to produce new antimicrobials. However, their metabolic diversity is still untapped (13, 14). Given the extensive genomic data availability, one can effectively mine the genomes of microbes for biosynthetic gene clusters (BGCs) potentially encoding known or novel molecules using bioinformatic tools such as BAGEL, CLUSEAN, and antiSMASH (15). antiSMASH is a commonly used tool for identifying and characterizing BGCs, which can detect up to 81 biosynthetic gene cluster types (16).

Streptomyces chrestomyceticus produces various bioactive metabolites, including aminoglycoside antibiotic paromomycin, which binds to the small ribosomal subunit and inhibits biosynthesis of proteins (17), polycyclic tetrahydroxyxanthones, chrestoxanthones, albofungin, chlorofungin, and streptimidone (18, 19). A novel compound, chresdihydrochalcone, was recently isolated from S. chrestomyceticus BCC 24,770 and demonstrated bioactivity against gram-positive bacteria (19). Another novel antifungal compound with activity against C. albicans and C. auris was isolated from S. chrestomyceticus ADP4 (20). Therefore, mining the genomes of these Streptomyces species is crucial for uncovering potential biosynthetic pathways in their genomes.

In the current study, a bacterium was isolated from soil in New Delhi that was potentially active against pathogenic bacteria and fungi, and it was identified as Streptomyces chrestomyceticus. Genome annotation identified plant-growth-promoting traits, stress-response pathways, and DNA repair pathways. BGC analysis with other S. chrestomyceticus genomes predicts potential non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) biosynthetic pathways and several unknown BGCs with different metabolic potential. A trans-acyltransferase polyketide synthase (transAT-PKS) cluster was identified as a putative antifungal cluster that exhibits 27% and 19% similarity to cycloheximide and 9-methylstreptimidone clusters. The bioassay-guided purification resulted in the identification of an antifungal compound (streptimidone derivative or isomer) with bioactivity against Candida species, including drug-resistant C. auris.

MATERIALS AND METHODS

Isolation and antimicrobial activities of the strain B5

The soil sample was collected from a garden in the New Delhi area, and serial dilutions were prepared to isolate bacteria from the soil in PBS buffer. The dilutions were plated on LB agar plates and incubated at 30°C for 6 days. Following incubation, colonies with morphological characteristics resembling Streptomyces were chosen and further restreaked to obtain pure cultures. A distinct soil bacterial culture, B5, was screened against E. coli W3110 ATCC 27325, P. aeruginosa MTCC 424, Bacillus subtilis IA748, S. aureus MTCC 96, and Mycobacterium smegmatis mc² 155 ATCC 700084 and fungi including C. albicans MTCC 227, Saccharomyces cerevisiae K616, C. glabrata MTCC 3019, Fusarium oxysporum MTCC 2773, and A. fumigatus MTCC 343 to evaluate its antimicrobial efficacy using the agar overlay method (21). A fresh culture of the strain B5 was grown on the center of an LB media plate and incubated at 30°C for 6 days. After incubation, plates were overlaid with soft agar mixed with test pathogens and then incubated overnight at 37°C (for bacterial pathogens) and 1 to 3 days at 30°C for fungal pathogens to form the zone of inhibition, and the diameter of the zone was measured in millimeters.

Whole-genome sequencing and assembly

S. chrestomyceticus B5 was grown in LB broth at 30°C for 72 h. Following incubation, genomic DNA was extracted using the DNeasy UltraClean Microbial Kit (Qiagen). The genomic DNA was quantified by 0.7% agarose gel electrophoresis and further purified using AMPure XP beads (Beckman Coulter). Whole-genome sequencing was performed on the MiSeq platform (Illumina) for short reads and the MinION (Oxford Nanopore Technologies) for long reads. The de novo assembly was carried out using Canu 1.8 (22) with nanopore-generated long reads. Following nanopore assembly, the short reads were aligned to the long-read assembly for correcting errors using Pilon v.1.8 (23), and genome completeness was checked by CheckM (24).

Phylogenetic analysis and whole genome-based comparison of S. chrestomyceticus B5

The taxonomy of the strain B5 was identified from the EzBioCloud database (https://www.ezbiocloud.net/). The 16S rRNA gene of the strain B5 was extracted using RNAmmer (25) and analyzed in the EzBioCloud database. The 16S rRNA gene sequences of the top hits were retrieved from this database and aligned with Clustal W integrated in MEGA11. The phylogenetic tree was constructed using the maximum composite likelihood model with the neighbor-joining method and 1,000 bootstrap values in MEGA11 (26). The in silico DNA-DNA hybridization (DDH) values were calculated using the genome-to-genome distance calculator 3.0 (https://ggdc.dsmz.de/ggdc.php) with closely related Streptomyces species. The comparative average nucleotide identity (ANI) values with related Streptomyces species were calculated using Pyani (27).

Genome annotation and BGC analysis of S. chrestomyceticus B5

Three reference genomes of S. chrestomyceticus were downloaded from NCBI (GCF_003865135.1, GCF_017168205.1, GCF_036562025.1) for comparative genome analysis available on NCBI. Functional genome annotation was carried out using Rapid Annotation using Subsystem Technology (RAST) (28), and functionally annotated genes were assigned to different subsystems. Orthologous protein clusters of S. chrestomyceticus B5 and other S. chrestomyceticus strains were predicted using OrthoVenn3 (29). The BGCs in S. chrestomyceticus B5 and other S. chrestomyceticus genomes were predicted using antiSMASH v.7.0 (16) with default settings. The predicted biosynthetic gene cluster pathways were manually analyzed in these strains.

Extraction and minimum inhibitory concentration of the S. chrestomyceticus B5 crude extract

To isolate the bioactive compounds, the S. chrestomyceticus B5 culture (1%) was inoculated into ISP2 media (30, 31) and incubated at 30°C for 72 h. The supernatant was collected by centrifuging at 7,500 rpm for 15 min. The cell-free supernatant was extracted with an equal volume of ethyl acetate. The organic layer was collected and evaporated to dryness in a vacuum concentrator to get the crude extract. It was resuspended in methanol and checked for antimicrobial efficacy against test microorganisms: C. glabrata, C. albicans, A. fumigatus, S. aureus, and E. coli through the agar well diffusion method. The minimum inhibitory concentration of the crude extract was also estimated against the above-mentioned pathogens. The MIC range of the crude extract was 0 μg–10 μg. The test bacterial cultures were grown at 37°C overnight and fungal pathogens at 30°C for 48–72 h. Following incubation, these cultures were diluted to an O.D. of 1 at 600 nm, and 1% of the culture was added to media tubes containing different concentrations of S. chrestomyceticus B5 crude extract for each test microorganism. The tubes were incubated for 18 h at 37°C, 200 rpm for test bacteria, 48 h for Candida species, and 72 h for A. fumigatus at 30°C, 200 rpm for fungi.

Purification of antimicrobial compound from S. chrestomyceticus B5

Upon bioactivity assay, the crude extract was purified through silica (SiO₂) gel column chromatography in mobile solvents, methanol:ethyl acetate (2:8), methanol, and methanol:water (8:2). Fractions, each of 2 mL, were collected, evaporated to dryness, and resuspended in 200 μL methanol, and 10 μL of each fraction was used for bioactivity screening using the agar well diffusion method.

Reverse-phase high-performance liquid chromatography and thin-layer chromatography to purify the antimicrobial compounds

Bioactive fractions were pooled to purify on reverse-phase high-performance liquid chromatography using an Agilent C18 column. An Agilent system with a photodiode array detector was used. HPLC separation was performed with the following conditions: 0–5 min 95% A, 5% B; 5–20 min 90% A, 10% B; 20–22 min 50% A, 50% B; 22–23 min 30% A, 70% B; 23–27 min 10% A, 90% B; 27–30 min 95% A, 5% B (A: water with 0.1% formic acid, B: acetonitrile with 0.1% formic acid), with flow rate of 0.5 mL/min, and UV absorption was taken at 254 nm, and bioactive fraction was collected. After HPLC purification, the bioactive fraction was further purified using thin-layer chromatography with mobile phase acetonitrile and water (9:1). The bioactive spot was visualized under UV at 254 nm, scraped, and pooled to get the purified compound.

Identification of bioactive compound with mass spectrometry and infrared spectroscopy

The purified bioactive antifungal compound was identified using a quadrupole time-of-flight mass spectrometer (TTOF 5600+, SCIEX) equipped with an electrospray ionization source and integrated with an Ultimate 3000 UHPLC system (Thermo Scientific). The bioactive compound was reconstituted in LC-MS grade methanol, and a 10 μL sample was used for injection. The sample was analyzed in reverse-phase mode using Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm, Waters). The mobile phases used for separation were Buffer A: LC-MS grade water with ammonium acetate (10 mM) and 0.1% formic acid (vol/vol), and Buffer B: LC-MS grade acetonitrile with 0.1% formic acid (vol/vol). A 30 min gradient was applied at a flow rate of 0.2 mL/min. Ion scanning was performed in a mass range of 50 to 1,000 m/z under both positive and negative ionization modes to achieve comprehensive metabolome coverage. During each duty cycle, ions with an intensity above 100 cps in the MS1 scan and a charge of +1 were selected for MS/MS (MS2) fragmentation. A collision energy of 30 eV was applied with an energy spread of 15 eV. The infrared spectrum was recorded on an FTIR spectrometer (Thermo Fisher Scientific) over the range of 4,000 to 400 cm⁻¹ using a potassium bromide pellet.

RESULTS

Strain B5 exhibits strong antimicrobial activities against clinically relevant pathogens

The strain B5 was filamentous in nature and formed white aerial hyphae, as observed from colony morphology. It exhibited medium inhibition toward B. subtilis (9 mm), followed by S. aureus (17 mm); however, it demonstrated strong activity against E. coli, with an inhibition zone of 24.2 mm. In addition, M. smegmatis showed complete inhibition around strain B5, forming a clear zone of inhibition, which indicates strong antibacterial activity, whereas P. aeruginosa showed no inhibition. (Fig. 1A). It exhibited weak antifungal activity against C. albicans (7 mm). It showed strong inhibition against A. fumigatus (20.5 mm), followed by F. oxysporum (21 mm), and C. glabrata (29 mm). The highest antifungal activity was observed against S. cerevisiae, with a zone of inhibition of 37.5 mm (Fig. 1B). The complete antimicrobial profile of strain B5 was summarized in Table S1.

Fig 1.

Antimicrobial potential of the B5 strain against test pathogens using the agar overlay method: (A) Antibacterial activity exerted by the B5 strain against gram-negative and gram-positive bacterial pathogens. (B) Antifungal activity displayed by the B5 strain against various fungal pathogens.

16S rRNA-based phylogeny and whole genome-based comparison of the strain B5

Based on the 16S rRNA sequence, strain B5 shared 99.93% sequence similarity with S. chrestomyceticus NBRC 13444 and Streptomyces paromomycinus NBRC 15454, and 99.52% with Streptomyces albofaciens JCM 4342. In the phylogenetic tree, strain B5 was grouped into a clade with S. chrestomyceticus NBRC 13444, S. albofaciens JCM 4342, and S. paromomycinus NBRC 15454 (Fig. 2A).

Fig 2.

Phylogenomic analysis of B5 strain: (A) phylogenetic analysis based on the 16S rRNA sequence. The phylogenetic tree indicated that the B5 strain is closely related to Streptomyces chrestomyceticus NBRC 13444, Streptomyces paromomycinus NBRC 15454, and Streptomyces albofaciens JCM 4342. Phylogenetic analysis was done by the maximum likelihood model using the neighbor-joining method in MEGA11 with 1,000 bootstraps. Nocardia asteroides NBRC 15531 was considered the outgroup for the phylogenetic tree. (B) Average nucleotide identity heatmap of S. chrestomyceticus B5 with closely related Streptomyces species. ANI values suggested that S. chrestomyceticus B5 shares high genome similarity with S. chrestomyceticus ADP4 compared to other strains.

Further whole genome-based comparisons, like ANI and DDH, suggested the strain B5 belongs to S. chrestomyceticus, as it shared more genomic similarity with S. chrestomyceticus ADP4, with 98.9% ANI and 91.40% DDH (Fig. 2B; Table S2) and more than 95% ANI and 69% DDH values with S. chrestomyceticus NBRC 13444 and S. chrestomyceticus TBRC 1925 strains, respectively.

The strain B5 also formed a clade with S. paromomycinus NBRC 15454 and S. albofaciens JCM 4342 in the phylogenetic tree; however, the ANI values with these species were 94.7% and 87.8%, and the DDH values were 59.9% and 44.4%, respectively, which are less than the cutoff values to define a species (Fig. 2B; Table S2).

Therefore, based on the high ANI and DDH values of the strain B5, with S. chrestomyceticus strains, it was designated as S. chrestomyceticus B5. Three reference genomes of S. chrestomyceticus (NBRC 13444, TBRC 1925, and ADP4) were chosen for comparative study based on their genomic similarity.

General genomic characteristics of S. chrestomyceticus B5

The genome of S. chrestomyceticus B5 was assembled into a single contig, with a genome size of 9.61 Mb and 72.1% GC content. The number of coding sequences was 8,918, with 86 RNA and 98.75% genome completeness (Table S3).

Genome annotation revealed plant growth-promoting (PGP) and stress-response genes in S. chrestomyceticus B5

The comparative genome annotation analysis suggested S. chrestomyceticus strains had a high number of metabolism-related genes, with high-abundance genes for amino acid and derivatives, carbohydrate metabolism, followed by protein metabolism, fatty acids and lipids, cofactors and vitamin biosynthesis, and DNA metabolism, as compared to the other three S. chrestomyceticus strains (Fig. 3A). From the genome annotation analysis, it was predicted that S. chrestomyceticus B5 possesses PGP traits; it contains genes for siderophore production (desABCD), auxin biosynthesis, ammonia assimilation (glnE, glnD, amt), nitrate and nitrite ammonification, denitrification (narG, narH, narJ, narI), and phosphate-releasing genes such as exopolyphosphatase (ppx) and polyphosphate kinase (ppk). While many Streptomyces species have been reported for plant growth-promoting properties, such properties have not been described in S. chrestomyceticus strains. Therefore, the presence of these traits in S. chrestomyceticus B5 suggests a symbiotic relationship with plants and may be ecologically relevant. S. chrestomyceticus B5 also contained more gene copies for stress response as compared to the other three S. chrestomyceticus strains. It contains oxidative stress genes (nsrR, ahpC, soxR), osmotic stress genes (betB, opuD, opuA), and genes for heavy metal resistance, including cobalt-zinc-cadmium resistance (Transcriptional regulator, MerR family), copper homeostasis (copC, copD, clfA), and mercuric reductase (PF00070). Besides, S. chrestomyceticus B5 also possesses genes for DNA repair pathways such as non-homologous end joining, UvrABC system, base excision, and RecBCD pathway, containing genes for ATP-dependent DNA helicase SCO5183 and SCO5184. The S. chrestomyceticus B5 also possesses CRISPR-associated cas genes (cas1, cas2, cas3, cse1, cas5e).

Fig 3.

Functional annotation of S. chrestomyceticus strains: (A) RAST subsystem classification in S. chrestomyceticus strains. The strains contain more genes in amino acid and carbohydrate metabolism, and S. chrestomyceticus B5 contains more gene copies in the stress response category. (B) Distribution of orthologous clusters and strain-specific clusters in S. chrestomyceticus strains.

Comparative protein cluster analysis of S. chrestomyceticus strains

Comparative protein cluster analysis revealed that 6,325 clusters were shared by four S. chrestomyceticus strains, associated with various cellular and metabolic processes, which indicates a conserved core genome in these strains. S. chrestomyceticus B5 contains 7,424 clusters, which is comparable to S. chrestomyceticus ADP4 (7,489) and S. chrestomyceticus TBRC 1925 (7,319), but higher than S. chrestomyceticus NBRC 13444 (7,091) (Table S4). Beyond the core clusters, several groups of shared clusters highlighted functional differences among strains. S. chrestomyceticus B5, ADP4, and TBRC 1925 share 401 clusters, responsible for RNA and nitrogen compound metabolism, DNA replication and repair, and amino acid transport, suggesting similar stress response and nutrient processing in these strains. Additionally, S. chrestomyceticus B5 and ADP4, and S. chrestomyceticus B5 and NBRC 13444 share clusters responsible for ion and phosphorus transport and organic acid metabolism, thus suggesting a role for environmental adaptation in these strains. The presence of strain-specific clusters provides further insight into genomic differences. S. chrestomyceticus B5 possesses 33 clusters associated with nitrogen and organic acid metabolism and transposition, suggesting accessory functions that are not present in the other three strains. These results indicate that these strains share a conserved core genome, while B5 contains accessory genes which may contribute to strain-level variation (Fig. 3B).

BGC analysis reveals strong biosynthetic potential of S. chrestomyceticus strains

The antimicrobial activities of S. chrestomyceticus B5 suggested a wide antimicrobial potential toward bacteria and fungi. Therefore, we investigated the BGCs present in this strain, which could be responsible for the synthesis of various metabolites with different bioactivities. The analysis predicted 48 BGC regions with 72 BGCs in S. chrestomyceticus B5, with many pathways encoding metabolites with putative antibacterial, antitumor, and a few clusters with putative antifungal bioactivities. S. chrestomyceticus NBRC 13444 possesses 69 BGCs, S. chrestomyceticus TBRC 1925 has 71 BGCs, and S. chrestomyceticus ADP4 contains 76 BGCs. S. chrestomyceticus B5 contains 25 BGC classes or types, and reference strains possess 24 BGC types, with predominant BGC types including NRPS, NRPS-like (NRPS-like fragment), terpene, chemical hybrid BGCs, and PKS, and rare BGC types, such as lassopeptide, transAT-PKS, triceptide, CDPS, arylpolyene, phosphonates, linaridin, indole, and 2-deoxy-streptamine aminoglycoside (Fig. 4A), which possibly encode unknown or novel metabolites.

Fig 4.

Biosynthetic diversity in S. chrestomyceticus strains and identification of putative antifungal cluster: (A) BGC classes or types predicted in S. chrestomyceticus strains. A few BGCs were unique to each strain. (B) Principal component analysis of BGC types in S. chrestomyceticus strains based on Euclidean distance. (C) Gene synteny between the BGC of 9-methylstreptidmidone from Streptomyces himastatinicus ATCC 53653 and the transAT-PKS cluster with putative smd genes in S. chrestomyceticus B5.

Several BGC types were unique to individual strains, which suggests that these strains may possess varying biosynthetic potential. S. chrestomyceticus B5 specifically contains the lanthipeptide-class-V BGC type, while reference strains do not. The PCA plot based on BGC types also suggested biosynthetic diversity in these strains (Fig. 4B).

Putative secondary metabolites predicted in S. chrestomyceticus strains

Many of the BGCs present in these strains have high similarity to their corresponding known clusters, which suggests the possibility of encoding identical or similar metabolites. The conserved BGCs for known metabolites identified in these strains were desferrioxamine B, hopene, griseobactin, geosmin, ectoine, terpenes, and isorenieratene. The presence of these metabolites is characteristic of Streptomyces, as they are involved in the adaptation of these microbes in soil. The other common BGCs present in these strains possibly encode known molecules with 75 to 100% similarity to antipain, thiazostatin/watasemycin, sapB, purincyclamide, paromomycin, rimomycin, cyclothiazomycin C, and tyrobetaine clusters. Many of these BGCs are reported for bioactivity against bacteria, including B. subtilis and S. aureus (32, 33). The possible bioactivity of S. chrestomyceticus B5 against these bacteria could be due to any of these BGC clusters. A biosynthetic pathway shows similarity to the paromomycin cluster, which is reported for activity against gram-negative bacterial pathogens (34). S. chrestomyceticus B5 strongly inhibited E. coli that could be due to either the paromomycin pathway or some other unknown biosynthetic pathway with low similarity to known clusters.

Half of the BGCs identified in these strains share low similarity with the known clusters (less than 30% similarity), indicating the potential of these strains for novel BGCs, some of which might be involved in encoding novel or new secondary metabolites.

Different biosynthetic capacity of S. chrestomyceticus strains

Few of the BGCs in these strains were encoding different metabolites specific to each strain, for example, BGC cluster for bafilomycin B1 (55%), marinoterpin A/B (12%), pristinin (23%), and hygrosin A/B (19%) were specific to S. chrestomyceticus B5 only (Table S5). Likewise, in reference strains, S. chrestomyceticus NBRC 13444 contains known BGCs for ebelactone A/B, alkylresorcinol, and cattlecin clusters, and unknown BGCs for acyldepsipeptide 1, dudomycin, and olimycin A/B clusters, which might produce new compounds (Table S6). S. chrestomyceticus TBRC 15454 contains BGCs for quinolidomicin A, phosphinothricin-triceptide, formicamycins A–M, chlorothricin/deschlorothricin clusters (Table S7). Although S. chrestomyceticus ADP4 shared high genomic similarity with S. chrestomyceticus B5, it also possesses specific BGCs for lysolipin I catenulisporolides, SCH-47554/46555, and lobosamide A/B/C (Table S8), suggesting differences between strains. Therefore, the high number of BGCs and their variation in encoding metabolites in these strains suggest differences in their biosynthetic potential, thus indicating the unique secondary metabolite biosynthetic potential of each strain.

Putative antifungal clusters in S. chrestomyceticus B5

Since most of the BGC-encoded metabolites are reported to have antibacterial and antitumor activities. Surprisingly, the antimicrobial assay suggested that S. chrestomyceticus B5 possesses strong antifungal activity against different fungal pathogens. Therefore, BGCs were analyzed for antifungal clusters in its genome. A biosynthetic cluster showed 55% similarity with the bafilomycin B1 cluster. Bafilomycin has been reported to exhibit bioactivity against A. fumigatus (35). However, S. chrestomyceticus B5 also showed activity against Candida species, indicating the possible presence of additional or distinct bioactive metabolites. Therefore, BGCs with low similarity to known clusters were also examined. Among them, a transAT-PKS cluster with 57.9 kb size showed 27% and 19% similarity to cycloheximide and 9-methylstreptimidone clusters, respectively (Fig. S1). These compounds belong to polyketide glutarimide antibiotics, reported for antifungal activities (36, 37). This transAT-PKS cluster was conserved in all S. chrestomyceticus genomes; three BGCs were present together in a BGC region (aminopolycarboxylic-acid, transAT-PKS, and NAPAA clusters) in the other reference genomes, whereas in the S. chrestomyceticus B5 genome, two BGCs (NAPAA and transAT-PKS clusters) were present. In the transAT-PKS cluster of S. chrestomyceticus strains, variation in the gene organization was observed. The core biosynthetic gene was split into four ORFs in S. chrestomyceticus B5, whereas it was present as a single ORF in the other three strains (Fig. S1). This cluster comprises a modular PKS and tailoring genes. It contains a standalone acyltransferase (AT) gene, instead of an integrated AT domain within PKS modules, thus supporting transAT biosynthesis. The core megasynthetases consist of modules predicted to incorporate malonyl-CoA (mal), methylmalonyl-CoA (Me-mmal), and cyclohexyl-malonyl (ccmal) extender units, suggesting the assembly of a structurally diverse and functional polyketide. These modules harbor ketosynthase (KS), acyl carrier protein (ACP), ketoreductase (KR), dehydratase (DH), β-branching domain (B), and C-methyltransferase (cMT), which incorporates methyl group. A terminal thioesterase (TE) domain is present downstream of the modules, which is responsible for polyketide chain release. In addition to that, the cluster encodes tailoring enzymes, including alcohol dehydrogenase (AbH) and O-methyltransferase (oMT), which catalyze oxidation and methylation steps (Fig. 5). These features suggest that this BGC is responsible for the synthesis of a bioactive polyketide in strain S. chrestomyceticus B5. Given its intriguing architecture, the cluster was further analyzed and compared with the 9-methylstreptimidone biosynthetic cluster that could be responsible for the possible bioactivity against yeasts. Five core genes (smdF, smdG, smdH, smdI, and smdE) are required for the synthesis of 9-methylstreptimidone, as reported from previous literature (38) (Fig. S2). The gene-to-gene alignment between the transAT-PKS and 9-methylstreptimidone cluster suggested similarity in the smdF, smdG, smdH, smdJ, and smdK genes. However, there is no significant similarity present between the genes (smdE and smdI) of both pathways. The sequence homology also suggested similarity between genes of 9-methylstreptimidone (smdH, smdI, smdJ, and smdK) and the transAT-PKS cluster (Fig. 4C). However, the smdI gene in the transATPKS cluster was present in four ORFs in S. chrestomyceticus B5, and all of them showed more than 75% similarity with the smdI gene of the 9-methylstreptimidone cluster (Table S9), suggesting the possibility of some derivative of 9-methylstreptimidone based on sequence homology of the genes.

Fig 5.

In silico analysis of putative transAT-PKS cluster with predicted structure linked to antifungal activity: (A) gene cluster organization with ORFs of the transAT-PKS biosynthetic pathway. (B) Domain architecture of the encoded PKS enzymes. Each module is represented by a box with individual domains labeled. (C) Predicted core chemical structure from the transAT-PKS cluster.

Therefore, based on genome mining, we hypothesized that this pathway might be responsible for the bioactivity against Candida species and might synthesize some derivative of these antifungal compounds. S. chrestomyceticus B5 was subjected to bioactivity-guided purification to identify the antifungal compound.

Bioactivity assay and minimum inhibitory concentration of S. chrestomyceticus B5 crude extract

The fermented S. chrestomyceticus B5 supernatant was extracted using ethyl acetate to get a yellow crude extract, and it was estimated for bioactivity against bacteria and fungi. The bioactivity assay suggested that the crude extract possesses both antibacterial and antifungal activity against test pathogens. The extract showed activity against fungi A. fumigatus, C. glabrata, C. albicans, and against bacteria S. aureus. However, it showed weak activity against E. coli, as it formed a small zone of inhibition (Fig. S3). The results indicated that the extract exhibits a notable antibacterial effect against S. aureus, indicated by an MIC of 1 μg/mL, whereas MIC was not detected for E. coli. The crude extract was also potentially active against yeasts C. glabrata and C. albicans. The MICs were 5 μg/mL and 2 μg/mL, respectively, as well as 1 μg/mL for A. fumigatus (Table S10).

Purification of antimicrobial compounds by chromatography

The column-purified fractions were screened against C. albicans, A. fumigatus, C. glabrata, E. coli, and S. aureus. Two different bioactivities were observed: an antifungal activity against C. glabrata in ethyl acetate:methanol (8:2) separated fractions and an antibacterial activity against S. aureus in methanol:water (8:2) separated fractions. However, these fractions were not active against E. coli, C. albicans, and A. fumigatus. Furthermore, the bioactive fractions active against C. glabrata were separated using reverse-phase HPLC. The bioactive peak from HPLC separation was obtained at 24 min retention time (RT), detected at a wavelength of 254 nm (Fig. 6A). After HPLC purification, the bioactive antifungal compound was further purified by thin-layer chromatography to get a single purified spot of antifungal compound at R_f 0.75 (Fig. 6B) as a yellow solid, and it was soluble in methanol and purity was confirmed on HPLC as a single peak (Fig. 6C).

Fig 6.

Purification of antifungal compound through HPLC and TLC: (A) high-performance liquid chromatography to purify bioactive fractions collected from column purification. The antifungal compound was identified at a retention time of 24 min with UV absorption at 254 nm, expressed in milli-Absorbance Units (mAU). (B) The purified antifungal compound was visualized on TLC using anisaldehyde-H₂SO₄ reagent. The bioactive compound was identified as a purple spot present at R_f 0.75. (C) The purity of the antifungal compound was checked on HPLC, confirming its purity with a single resolved peak.

Identification of the compound from LC-MS and FTIR analysis

The purified compound was analyzed on LC-MS, which exhibited an m/z of 294.1 [M+H]⁺ in positive ionization mode at the retention time of 8.6 min, and an m/z of 292.1 [M−H]⁻ in negative ionization mode at the same RT, which provided the molecular mass of the antifungal compound as 293.1 (Fig. S4 and S5). Furthermore, the functional groups present in the compound were confirmed from FTIR analysis. The peaks present in region 1259.14, 1403.08, 1436.41, 1452.72, 1566.58, and 1570.01 suggested C−N stretching and N−H deformation, whereas the peaks at 1606.64, 1609.12, 1613.17, 1695.69, 1701.55 suggested C=C−C aromatic ring stretch, N−H, and C=O stretching. The peak at 2355.73 suggested the presence of a −CH2 group (alkyl group), and the major wide peaks in the region of 2926.68, 2969.44, 3088.58, 3324.01, 3363.31, and 3548.62 suggested C−H stretching in aromatic ring and O−H stretching (Fig. S6).

For the structural confirmation of the compound, the fragmentation pattern of the proposed compound was matched with that of the standard streptimidone (Cayman Chemicals). This resulted in confirmation of the purified compound as a streptimidone derivative or isomer since the fragmentation spectra of the purified compound matched with the fragmented ion spectra of streptimidone in positive ion scan mode (fragmented peaks were at m/z 198.07, 276.16, 180.06, 156.06, 138.05, 95.08, 67.05, and 60.04) (Fig. 7).

Fig 7.

Confirmation of purified compound as streptimidone derivative or isomer: (A) MS/MS of standard streptimidone. (B) MS/MS of purified B5 antifungal compound.

The synthetic streptimidone and purified compound were assayed for bioactivity against fungal pathogens, showing a similar pattern of bioactivity against different species of Candida and F. oxysporum, except C. albicans. The streptimidone and purified compound were also bioactive against C. glabrata and drug-resistant C. auris (Fig. 8; Fig. S7).

Fig 8.

Spot-on-lawn assay of purified streptimidone derivative or isomer against fungal pathogens: the purified compound and synthetic streptimidone were dissolved in AMQ, and 5 µL of the streptimidone and the purified compound was put on culture plates of different fungal pathogens. The streptimidone and purified compound showed a similar pattern of bioactivity against C. glabrata, C. krusei, C. tropicalis, S. cerevisiae, F. oxysporum, drug-resistant C. auris and drug-resistancet C. glabrata, except C. albicans.

DISCUSSION

Infectious diseases caused by bacterial and fungal pathogens are emerging as a major global health threat, and the rise of drug resistance in these pathogens would cause further complications in treating infections caused by them (39, 40). Therefore, finding bacteria with potential antimicrobial activities can be one way to find molecules that can be effective against them. Streptomyces are major producers of antimicrobial compounds with known greater potential (11). In this study, a Streptomyces species isolated from garden soil possesses wide antimicrobial activity against bacterial and fungal pathogens. Previous studies have isolated Streptomyces species from garden soil, which reported antimicrobial activities against E. coli, S. aureus, Bacillus species, Candida species, Fusarium, and multidrug-resistant bacteria (31). A new antibiotic, lariocidin, with broad-spectrum activity against different bacterial pathogens, has been isolated from a garden soil bacterium (41). Our isolate was also potentially active against these pathogens, suggesting garden soil contains Streptomyces species that can provide a diverse range of antimicrobial metabolites, possibly due to the competitive soil environment present in garden soil.

The strain B5 was identified as S. chrestomyceticus. The genome annotation with other reference strains suggested that the strains contain a high number of genes in metabolism, especially carbohydrates and amino acids. The primary source of energy for any soil bacteria is amino acids and carbohydrates (42). Therefore, the carbohydrates and amino acids might act as the source of carbon and nitrogen and might be helpful in the utilization of nutrients released from plant biomass (43, 44). S. chrestomyceticus B5 possesses genes for stress response, including DNA repair pathways, oxidative stress, osmotic stress, and heavy metal resistance genes, suggesting a role in the protection of bacteria from such stressors. Previously, a study has identified heavy metals including Pb, Mn, Ni, Zn, Fe, Cd, and Cr in soil from the national capital region (45). Another study has documented heavy metal contamination in the groundwater of the Delhi region (46). The exposure to heavy metal stress results in the production of reactive oxygen species and may also result in DNA damage (47). Besides, plants also produce reactive oxygen species as byproducts in soil, which can be harmful to the bacteria living in soil (48). Therefore, these implications might indicate stress response by soil bacteria. The genome annotation suggested that S. chrestomyceticus B5 possesses PGP traits, including genes for auxin biosynthesis, siderophore production, nitrite and nitrate ammonification, denitrification, and phosphate solubilization. These traits suggest that S. chrestomyceticus B5 might be in a symbiotic relationship with plants, thus might be helpful for plant growth (49, 50).

Since S. chrestomyceticus B5 possesses wide antimicrobial activity, the genome mining approach was employed to compare the BGCs with other reference genomes of this strain. The core BGCs present across all strains were desferroxamine B, hopene, griseobactin, geosmin, ectoine, terpenes, and isorenieratene. These essential metabolites are mostly useful for Streptomyces present in soil due to their role in iron acquisition from soil, mitigation of osmotic stress, stabilizing the plasma membrane, and protection of microbial cells from oxidative stress (51–54). Besides these metabolites, these strains also possess BGCs encoding various antibacterial and antitumor metabolites. All the strains contain BGC for paromomycin, suggesting this BGC is conserved in all strains of S. chrestomyceticus. Paromomycin is an aminoglycoside antibiotic reported for broad-spectrum activity, targeting both gram-negative and gram-positive bacterial species (55). The antibacterial effect against E. coli might be due to the paromomycin cluster, since paromomycin is reported to inhibit E. coli (56).

Another cluster that showed similarity to the bafilomycin B1 cluster, possibly encoding a similar compound, might be responsible for bioactivity against A. fumigatus, as this compound is also reported for inhibiting filamentous fungi (35). Likewise, these bacteria also possess BGCs that could be involved in the synthesis of other bioactive molecules. However, in-depth BGC analysis indicated that different metabolites are produced by each strain, thus indicating differential biosynthetic diversity in these strains. They could be involved in the biosynthesis of some novel molecules, as these strains exhibited very low similarity to the known BGC clusters. Previous studies have also reported strain-level diversity in Streptomyces species (57, 58).

A BGC with low homology to an antifungal cluster was observed in all strains, identified as a transAT-PKS cluster, and showed very low similarity to cycloheximide and 9-methylstreptimidone clusters. These compounds belong to glutarimide-containing polyketides, comprising over 50 bioactive molecules with diverse activities that have been isolated from Streptomyces species (59). Cycloheximide and 9-methylstreptimidone are inhibitors of protein biosynthesis targeting the 60S subunit of the ribosome, thus inhibiting various phytopathogenic fungi (60). The 9-methylstreptimidone is one of the derivatives of the streptimidone compound reported from Streptomyces species; it contains a terminal alkene and possesses antifungal activities (61, 62). The cluster for biosynthesis of streptimidone is not known; however, a previous study has reported a putative BGC for streptimidone based on the similarity of the cluster with the 9-methylstreptimidone cluster. The biosynthesis of these compounds proceeds by extension of polyketide using trans-acyltransferase, which contains the domain for the synthesis of glutarimide ring and the incorporation of other −CH₃ substituents (38, 62).

This transAT-PKS cluster predicted through genome mining, which showed low homology to the 9-methylstreptimidone cluster, could be involved in the biosynthesis of streptimidone derivative or isomer, which consists of four core polyketide genes, along with two methyltransferases, putative acyl carrier, amidotransferase, malonyl CoA-acyl carrier protein transacylase, putative thioesterase, flavoprotein, and decarboxylase, which might be required for biosynthesis of streptimidone scaffold, similar to the genes for biosynthesis of 9-methylstreptimidone (38) (Fig. 5). Other than the required genes in 9-methylstreptimidone, the transAT-PKS cluster’s core gene was present in multiple ORFs in S. chrestomyceticus B5. This cluster also contains additional genes, which may further enhance the structure of the existing molecule and might give rise to some novel compound. Therefore, the genome mining approach can be used to guide and identify the putative bioactive compounds.

The S. chrestomyceticus B5 crude extract exerted antibacterial and antifungal activities against S. aureus, C. glabrata, C. albicans, and A. fumigatus. With bioactivity-guided fractionation, an antifungal fraction active against C. glabrata and another fraction active against S. aureus were separated. However, these fractions showed no activity against A. fumigatus, C. albicans, and E. coli, suggesting five different bioactive molecules are produced by S. chrestomyceticus B5 that could be responsible for these different bioactivities observed in this strain. The bioassay-guided purification of the antifungal fraction active against C. glabrata resulted in the identification of a streptimidone derivative or isomer, which exhibited fragmented peaks similar to those reported in an earlier study (63). The purified antifungal compound (streptimidone derivative or isomer) and synthetic streptimidone inhibited F. oxysporum and Candida species, including S. cerevisiae and C. glabrata; however, the purified compound did not inhibit the growth of C. albicans. Previous studies have also reported that the naturally isolated streptimidone exhibits activity against yeasts; however, it is ineffective against C. albicans even up to a concentration of 50 μg/mL (61, 62). The synthetic streptimidone exerted bioactivity against C. albicans, possibly because the streptimidone stereoisomers are known to exert differential bioactivity against S. cerevisiae (64); this different bioactivity could be due to a different stereoisomer of synthetic streptimidone. The purified antifungal compound was also active against drug-resistant C. auris, which is a multidrug-resistant pathogen responsible for bloodstream infections and associated with 60% mortalities (4).

Therefore, screening of pre-existing antifungal compounds can be one of the effective ways of fighting against these leading pathogens. Genome mining can predict the known molecules based on the similarity to known clusters, but the prediction of BGCs with minimal similarity to known reference clusters does not rule out the possibility of encoding the known compound, since the streptimidone pathway does not exist in the MIBiG database. Due to this limitation, the possible compound was not predicted from genome mining. This study highlighted the antimicrobial potential of S. chrestomyceticus through bioactivity assay and also its possession of multiple biosynthetic pathways, with unknown clusters as well, suggesting the possibility of encoding other bioactive metabolites, which could be responsible for other observed bioactivities.

Conclusion

S. chrestomyceticus B5 holds potential against a wide range of pathogens with antibacterial and antifungal activities. It contains plant growth-promoting traits, stress response and DNA repair genes, and NRPS and PKS pathways with antibacterial and antitumor activities, along with two putative antifungal clusters. The crude extract exerted strong antimicrobial activities, and bioassay-guided purification identified an antifungal compound active against Candida species and drug-resistant C. auris. S. chrestomyceticus B5 also possesses different bioactivities against other potential pathogens; therefore, identifying such antimicrobial compounds using genome mining approaches and bioassay-guided purification can help in the fight against antimicrobial-resistant pathogens.

DATA AVAILABILITY

The genome sequence of the strain in this study has been deposited in the National Center for Biotechnology Information (NCBI) under the bioproject accession number PRJNA1295772.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/spectrum.02688-25.

Supplemental figures. spectrum.02688-25-s0001.pdf.

Fig. S1 to S7.

Supplemental tables. spectrum.02688-25-s0002.xlsx.

Tables S1 to S10.

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