Isolation of strains SBTS01T and W18L9T
Strain SBTS01T was isolated from Jasmine rice (Oryza sativa KDML 105) and arose as a dark green colony on leaf sheath tissue after incubation for nine weeks on Humic acid vitamin B agar (HVA) (Hayakawa and Nonomura 1987). Strain W18L9T was also isolated from Jasmine rice and emerged as a white colony on leaf tissue after incubation for two weeks on Tap Water Yeast extract agar (TWYA) (Coombs and Franco 2003).
Phylogenetic analysis of the 16S rRNA gene
The phylogenetic evaluation of strains SBTS01T and W18L9T with the closely related members of the genus Streptomyces showed that both strains belonged to genus Streptomyces and were encompassed by other members of this genus (Fig 1 and Fig S1). Strain SBTS01T shared 16S rRNA gene similarity at 98.6% with strain W18L9T. Both strains were placed in different clusters in both trees. Based on these results, both strains were considered as different species.
The closest type strains which shared the highest 16S rRNA gene sequence similarity with strain SBTS01T were S. rochei NRRL B-2410T and S. naganishii NBRC 12892T, both at 99.0%. However, strain SBTS01T formed a different cluster with these type strains and formed a single phylogenic lineage in both trees (Fig 1 and Fig S1). The type strains which shared the highest 16S rRNA gene sequence similarity with strain W18L9T were S. shenzhenensis DSM 42034T, S. graminisoli NBRC 108883T, S. jiujiangensis DSM 42115T, and S. hyaluromycini DSM 100105T at 99.7, 99.2, 98.9, and 98.9%, respectively. The closest neighbor of strain W18L9T positioning in the same clade was S. shenzhenensis DSM 42034T in both trees with high bootstrap support at 81 and 67, respectively (Fig. 1 and Fig. S1).
Based on the percentage of 16S rRNA gene similarity and phylogenetic position of strains SBTS01T and W18L9T, two closely related type strains of strain SBTS01T and four closely related type strains of strain W18L9T as mentioned above were selected for a side-by-side comparison study.
Genome comparison between strains SBTS01T and W18L9T
The phylogenetic tree based on TYGS revealed that strain SBTS01T was placed in the different species cluster with strain W18L9T (Fig S2). The genome analysis showed the dDDH, ANIb and ANIm values of the draft genome between strain SBTS01T and strain W18L9T being 28.3%, 81.6% and 87.1%, respectively which were well below the threshold for species delineation (Meier-Kolthoff et al. 2013; Chun et al. 2018). Based on a genome comparison study, both strains were in different species. Genome features of strains SBTS01T, W18L9T and their closest type strains were shown on Table S1.
Genome comparison of strain SBTS01T
The draft genome sequence of strain SBTS01T was 9.17 Mb with DNA G+C content determined by in silico genome sequence as 72.5 mol%. The phylogenetic tree based on TYGS revealed the relationship between strain SBTS01T and the related type strains. The phylogenomic tree showed that strain SBTS01T was placed in the different species cluster from all related type strains (Fig. S2). Strain SBTS01T formed a single phylogenetic cluster with the closest neighbour, Streptomyces corchorusii DSM 40340T, while it positioned away from the closest type strains, S. rochei NRRL B-2410T and S. naganishii NBRC 12892T.
The genome analysis showed the dDDH, ANIb and ANIm values between strain SBTS01T and two related species which shared 16S rRNA gene similarity at 99.0%; S. rochei NRRL B-2410T were 29.4, 81.1, 86.5%, and S. naganishii NBRC 12892T were 26.9, 82.1, 86.9%, respectively. Strain SBTS01T and its closest neighbour on the phylogenomic tree; S. corchorusii DSM 40340T had the highest dDDH, ANIb and ANIm values at 54.1, 92.6, 94.3%, respectively. However, strain SBTS01T and S. corchorusii DSM 40340T shared a 16S rRNA gene similarity of only 97.0%. Based on the previous report, the cutoff value at the species level was evaluated at 98.7% (Stackebrandt and Ebers 2006). Therefore, strain SBTS01T and this type strain belonged to different species. Hence, there is no requirement to compare physiological and biochemical characteristics between strain SBTS01T and this type strain. According to Richter and Rosselló-Móra (2019), the species delineation should have ANI values cut off lower than 95-96% and dDDH values were lower than the threshold of 70% used to define species level (Meier-Kolthoff et al. 2013; Chun et al. 2018). Based on the genome comparison, strain SBTS01T is a novel species of the genus Streptomyces.
Genome comparison of strain W18L9T
The draft genome sequence of strain W18L9T was 10.13 Mb with DNA G+C content determined by in silico genome sequence as 70.9 mol%. The phylogenetic tree based on TYGS revealed that strain W18L9T was placed in the same species cluster as its closest neighbour, S. shenzhenensis DSM 42034T but in a different subspecies cluster (Fig. S2). The genome analysis showed the dDDH, ANIb and ANIm values of the draft genome between strain W18L9T and its related species, S. shenzhenensis DSM 42034T being 72.5, 95.1, 97.0% and S. hyaluromycini DSM 100105T being 44.1, 88.8, 92.4%, respectively. The ANIb, ANIm and dDDH between strain W18L9T and the closest type strain S. shenzhenensis DSM 42034T were higher than 95-96% for ANI values (Richter and Rosselló-Móra 2009) and dDDH value was higher than the threshold of 70% used to define species delineation (Meier-Kolthoff et al. 2013; Chun et al. 2018). Based on these data, strain W18L9T belonged to the same species of S. shenzhenensis DSM 42034T. According to Nouioui et al. (2018), dDDH values between 70 and 79% defined subspecies delineation. Based on the dDDH value between strain W18L9 T and S. shenzhenensis DSM 42034T at 72.5% and the position at different subspecies clusters on the phylogenomic tree, strain W18L9T belonged to the same species of S. shenzhenensis DSM 42034T but it was defined as a novel subspecies.
The LL-isomer of DAP was detected in both strains SBTS01T and W18L9T. The whole-cell sugars of strain SBTS01T contained galactose, glucose and mannose and strain W18L9T contained galactose, glucose, mannose and xylose. The polar lipids of strain SBTS01T contained phosphatidylethanolamine (PE), phosphatidylinositol (PI), three aminoglycolipids and one glycolipid and strain W18L9T contained phosphatidylethanolamine (PE), phosphatidylinositol (PI), six glycolipids, one aminolipid, and four unknown lipids which correspond to phospholipid type II (Lechevalier et al. 1977) (Fig. S3).
The major menaquinones of strains SBTS01T and W18L9T were MK-9(H6) and MK-9(H8). The major cellular fatty acids (more than 10%) of strain SBTS01T were identified as anteiso-C15:0 (26.6%), iso-C16:0 (19.1%) and anteiso-C17:0 (19.1%) which were the same pattern with the closest type strains; S. rochei NRRL B-2410T (Table 1). The major fatty acids of strain W18L9T (more than 10%) were anteiso-C15:0 (21.8%), anteiso-C17:0 (20.9%) and iso-C16:0 (18.5%)
and which were the same pattern with the closest type strain; S. shenzhenensis DSM 42034T (Table S2).
Phenotypic and physiological characterization
The colony morphology of strain SBTS01T is described in Table S3. Strain SBTS01T showed morphological characteristics with well-developed substrate mycelium and aerial mycelia on most media used. Melanin pigment was produced on ISP 7. Electron micrographs showed that strain SBTS01T formed round rod-shaped spores with spiny surface and spore chains in loop forms (approximately 1 micron length x 1 micron diameter) (Fig. 2A). The physiological properties of strain SBTS01T and its closest type strain; S. rochei NRRL B-2410T were different. Strain SBTS01T could assimilate benzoate and tartrate and grow at NaCl 5% (w/v) but the closest type strain could not assimilate these compounds and could grow weakly at this salt concentration. Also, strain SBTS01T could grow weakly at 45 oC and produce melanin pigment on ISP 7 but the type strain could not. On the other hand, the type strain could hydrolyze L-tyrosine and xanthine but strain SBTS01T could not (Table 1).
The colony morphology of strain W18L9T is described in Table S3. Strain W18L9T showed morphological characteristics with well-developed substrate mycelia and aerial mycelia on most media used. No melanin pigment was observed on ISP 7. Electron micrographs showed that strain W18L9T formed oval rod-shaped spores (approximately 1.5 micron length x 1 microns diameter) with warty surface and formed spore chains in loops (Fig. 2B). The physiological properties of strain W18L9T and its closest type strain, S. shenzhenensis DSM 42034T were different. Strain W18L9T could assimilate maltose, hydrolyze urea, assimilate tartrate and could grow at 15 oC and pH 10 but the type strain could not. On the other hand, the type strain could hydrolyze hippurate and grow weakly at 5% NaCl (w/v) but strain W18L9T could not (Table 2).
Based on the results of polyphasic study, strain SBTS01T is proposed as a representative of a novel species of the genus Streptomyces named Streptomyces spinosus sp. nov. and strain W18L9T is proposed as a novel subspecies of Streptomyces shenzenensis DSM 42034T named Streptomyces shenzenensis subsp. endophyticus subsp. nov.
In vitro antibacterial and antifungal activities
Strains SBTS01T and W18L9T showed strong and moderate activity against a bacterial rice pathogen; Xoo PXO 86, respectively. Strains SBTS01T and W18L9T showed moderate and strong activity against P. grisea BRIP 61689, respectively. These results were obtained by the dual culture method. Both strains showed strong activity against S. aureus ATCC 29213 and MRSA 03120385 on both dual culture and agar extraction methods. The antifungal assay against other fungal pathogens was tested by using only a dual culture method. Strain SBTS01T showed weak activity against R. solani AG8 while strain W18L9T had strong activity against this fungus. Both strains showed moderate activity against a rice fungus; C. lunata BCC 15558. Strains SBTS01T and W18L9T showed moderate and weak activity against H. oryzae DOAC 1570, respectively (Table 3). HPDA was the best medium for antimicrobial activity of both strains tested by dual culture and agar extraction methods. HPDA and ISP 2 contained 10 and 4 g of dextrose, respectively. Then, it was not an effect of carbon catabolite regulation (CCR) which is a regulatory mechanism that limits production due to a negative effect applied by high concentrations of dextrose (Sanchez and Demain 2002). ISP 2 contained malt extract and yeast extract at 10 and 4 g/L while HPDA comprised potato infusion. It was reported that some nitrogen sources may decrease antibiotic production in different microorganisms including Streptomyces (Rafieenia 2013). Based on the result of antibiotic production, it is necessary to select at least two media with different compositions to screen for antimicrobial activity.
Plant growth promoting traits
Only strain W18L9T could grow on DF agar with ACC deaminase as a nitrogen source, fix nitrogen on NFB medium, solubilize inorganic phosphate and produce cellulase enzyme (Table 4). Strain SBTS01T had negative results for these tests. Both strains could not produce HCN but they could produce siderophore (hydroxymate type). Strains SBTS01T and W18L9T produced IAA at 71 and 150 ug/mL and produced cytokinins at 0.165 and 0.169 mg/L, respectively (Table 4).
Secondary metabolites and biosynthesis gene cluster prediction
There were many BGCs of strains SBTS01T and W18L9T derived from “antiSMASH” database. There were six groups of metabolites; terpene, type 1 polyketide synthase (T1PKS), type2 PKS (T2PKS), type3 PKS (T3PKS), Non-ribosomal peptide synthetase cluster (NRPS), lanthipeptide cluster and other compounds (Table 5). Both strains contained terpene clusters; geosmin and albaflavenone (100% similarity). Geosmin is recognized as an earthy odor that was generally produced by many genera of actinobacteria including Streptomyces species (Jiang et al. 2007). Albaflavenone, a tricyclic sesquiterpene antibiotic, which was produced by many Streptomyces sp. possessed an antibacterial activity (Moody et al. 2011). Also, strains SBTS01T and W18L9T contained gene clusters encoding hopene at 76 and 96% similarity, respectively. Hopene is a precursor for producing bioactive hopanoids, a group of pentacyclic triterpenoids such as diploterol which is toxic to mouse leukemia cells (Schmerk et al. 2015; Sohlenkamp and Geiger 2016). Strain SBTS01T contained isorenieratene (62% similarity) which is a carotenoid light- harvesting pigment produced by green sulfur bacteria; Chlorobium spp. There were some reports of isorenieratene biosynthesis by actinobacteria but the function of this compound is still unknown and it may relate to blue or UV light resistance (Maresca et al. 2008). Strain SBTS01T comprised T1PKS cluster; sceliphrolactam and antimycin (92 and 93% similarity). Sceliphrolactam, a macrocyclic lactam exhibited antifungal activity against amphotericin B-resistant Candida albicans (Oh et al. 2011). Antimycin is produced by many strains of Streptomyces sp. and it was reported that the antimycin A family possessed effective antifungal, insecticidal, nematocidal and anticancer activities (Ondrejíčková et al. 2016). Also, Nakayama et al. (1956) reported the activity of this compound against rice blast fungus which correlated to this study in which strain SBTS01T showed moderate activity against this pathogen in vitro. Moreover, strain SBTS01T contains T2PKS cluster; lugdunomycin (40% similarity). Lugdunomycin is an angucycline-derived molecule containing a heptacyclic ring system and represents a novel subclass of aromatic polyketides. It was reported that this compound had antimicrobial activity against the Gram-positive Bacillus subtilis 168 (Wu et al. 2019). As strain SBTS01T had low similarity at 40% with lugdunomycin gene cluster, it has a possibility to produce a novel compound of aromatic polyketides. Furthermore, strain SBTS01T contained a RiPP: Lanthipeptide cluster;
informatipeptin (57% similarity). It was stated that microorganisms containing a RiPP: Lanthipeptide clusters are likely to produce novel bioactive compounds (Repka et al. 2017). Strain SBTS01T comprised NRPS, ladderane cluster; ishigamide (100% similarity), a polyene-containing amide (Du et al. 2016). Furthermore, strain SBTS01T comprised gene cluster, alkaloid; anisomycin (61% similarity), a pyrrolidine antibiotic containing a variety of activities. It was reported that anisomycin can block peptide bond formation and showed potential against pathogenic protozoa and fungi. This compound can induce cell cycle apoptosis and activate the c-Jun N-terminal kinases (JNKs) signal pathway to exhibit potential antitumor activity (Shen et al. 2019).
In correlation with the genome data mining, strain SBTS01T also showed good activity against Xoo PXO 86, S. aureus ATCC 29213 and MRSA 03120385 in which this strain may be likely to produce novel antibiotics. Finally, strain SBTS01T contained a gene cluster, melanin (71% similarity). Melanin pigment produced from microorganisms could help to resist chemicals and oxidizing agents, thermal, and biochemical stresses (El-Naggar and El-Ewasy, 2017). This gene finding correlated with in vitro study in which strain SBTS01T produced melanin pigment on ISP 7. In addition, this strain contained a gene cluster encoding spore pigment which benefits bacteria to tolerate UV and other environmental stresses (El-Naggar and El-Ewasy, 2017). As strain SBTS01T was isolated from a rice paddy field which applied many pesticides such as fungicide, herbicide, and insecticide, this strain tended to adapt for resisting to survive from chemicals (personal communication with Dr. Ronnachai Changsri, a researcher at rice department, Thailand).
Strain W18L9T comprised a variety of BGCs. This strain contained T1PKS clusters; rhizomide A, B and C (100% similarity). Rhizomide A, B and C showed weak antitumor activity against several human tumor cells lines (IC50 34 μM to 96 μM). It was reported that rhizomide A showed protective activity against cucumber downy mildew (Pseudoperonospora cubenis), Staphylococcus aureus and Bacillus subtilis (Wang et al. 2018). Strain W18L9T contained T2PKS cluster; curamycin (57% similarity), a polyketide antibiotic produced by Streptomyces curacoi which could inhibit S. aureus (Galmarini and Deulofeu 1961). Strain W18L9T contained T3PKS clusters; germicidin and feglymycin (100 and 47% similarity). Germicidin was the auto regulative inhibitor of spore germination in the genus Streptomyces(Petersen et al. 1993).
Feglymycin, a novel peptide antibiotics was reported to inhibit the replication of the human immunodeficiency virus (HIV) in vitro (Vértesy et al. 1999). Furthermore, strain W18L9T comprised NRPS/NRPS like clusters; xenotetrapeptide, tolaasin I / tolaasin F (100 and 50% similarity). The novel xenotetrapeptides were nonribosomally synthesized peptides which have been still unknown function produced from Xenorhabdus nematophila, bacteria reside as endosymbionts in the foreguts of soil nematodes in genus Steinernema (Khandelwal and Banerjee-Bhatnagar 2003). In correlation with the genome data mining, strain W18L9T showed good activity against S. aureus ATCC 29213, MRSA 03120385, P. grisea BRIP 61689 and R. solani AG8 in vitro. The properties of strain W18L9T to inhibit rice pathogens are correspondent with its ecophysiology inside rice tissue which benefits plant to inhibit pathogens.
In addition, the result showed that both strains contained gene cluster to synthesize siderophore, desferrioxamine E which plays an important role in the growth and development of Streptomyces (Codd et al. 2018). It was correspondent with the result of PGP traits of both strains which could produce siderophore (hydroxymate type) in vitro.
In silico gene prediction
Other genes of strains SBTS01T and W18L9T encoding other bioactive compounds relating to plant growth promoting, degradable enzymes and bioremediation were scanned. The result showed that these strains contained a variety of genes encoding drought tolerant and stress response proteins and iron uptake protein relating to plant growth promoting properties (Table S4 and S5). For drought and salt tolerant proteins, both strains contained genes encoding ectoine production, glycine betaine/L-proline transporter, glycine, betaine and proline production, Ca2+: H+ antiporter and chloride channel protein. Only strain SBTS01T contained sodium solute symporter and potassium channel family protein which can support plants to tolerate salt stress (Horn et al. 2006).
Only strains W18L9T contained the gene encoding ACC deaminase which supports plants in stress condition such as drought, flooding, nutrient starvation, temperature, salt and oxidative stresses including toxic agents (Gupta and Pandey 2019). Strain W18L9T contained genes relating to nitrogen fixation; ADP-ribosyl-dinitrogen reductase. These genes correlated with the results in which this strain could produce ACC deaminase and had ability to fix nitrogen in vitro. Also, the gene encoding indole-3-glycerol phosphate synthase relating to indole acetic acid (IAA) production was detected in the genome of strain SBTS01T. IAA is a derivative of a plant hormone, auxin which promotes plant growth and support plant growth in salt stress condition (Keswani et al. 2020). In correlation with this gene finding, strain SBTS01T and W18L9 were able to produce IAA in vitro. Furthermore, there were many genes encoding universal stress proteins (usp) which were detected in both genomes. It was reported that bacteria containing usp genes play a role in maintaining internal and external protection mechanisms in biotic and abiotic stresses (Kim et al. 2012). Strain W18L9T and strain SBTS01T comprised genes encoding exopolyphosphatase and inorganic pyrophosphatase while strain SBTS01T also contained phosphatase and alkaline phosphatase. However, only strain W18L9T showed an ability to solubilize inorganic phosphate in vitro. From this result, it can conclude that strain SBTS01T contained cryptic genes which required gene induction to express phosphate solubilization. The rice paddy field which plant source collected is in Suvannabhum district, Roiet province. This area is known as Thug Gura Ronghai which contains poor soil, fine–loamy soil type and moderate salt (EC 4-8 dS/m). The subsoil contains a lot of rock salt and potash (Sahunalu 2003). In correlation with the ecophysiology, strains SBTS01T and W18L9T contained genes encoding plant growth promoting traits to promote rice growth in poor soil and salinity condition.
Strain SBTS01T and W18L9T comprised many genes encoding beneficial enzymes which can be applied in a variety of industries. There were several types of enzymes; alpha-N-arabinofuranosidase, amylase, β-glucosidase,β-xylosidase, and β-xylanase which could degrade hemicellulose, polysaccharide, or plant residue to obtain fermentable sugars for applying in bioethanol production (Podkaminer et al. 2012). Genes encoding chitinase were also detected in both strains. It was reported that bacteria producing chitinolytic enzymes could inhibit fungal pathogens by degrading fungal cell walls and degrading waste containing chitin (Brzezinska et al. 2014). Also, these two strains contained genes encoding lipase and various types of proteases which can be applied in many industries such as detergent, food and feed additive, leather and textile industries.
Genes encoding proteins in bioremediation were also mined. Strain W18L9T contained five genes encoding xenobiotic degradable enzymes; alkanesulfonate monooxygenase, nitroalkane oxidase, nitroreductase, epoxide hydrolase and phenol 2-monooxygenase. Alkanesulfonate monooxygenase participates in alkanesulfonate degradation. Nitroalkane oxidase relates to nitroalkane degradation in which nitroalkane compounds are used as solvents and explosive compounds and as herbicide and pesticide agents in agriculture and 2-nitropropane was reported as a carcinogen (Li et al. 2008). Nitroreductase can reduce pollutants such as 2,4‐dinitrotoluene and 2,4,6‐trinitrotoluene, as well as key chemical intermediates, 3‐nitrophthalimide, 4‐nitrophthalimide and 4‐nitro‐1,8‐naphthalic anhydride. Also, strain W18L9T contained gene encoding tellurium resistance protein. Gene encoding a latex clearing protein was detected in this strain which can apply to degrade waste containing rubber such as used gloves and tires. This will be valuable to degrade xenobiotic waste in the environment. As stated above, the rice paddy field was contaminated with many pesticides applied in the area for many years. Strain W18L9T may adapt to survive in the environment contaminated with these compounds and contains genes encoding enzymes for degrading pesticides.
Strain SBTS01T contained genes encoding aldehyde dehydrogenase family protein, 2-nitropropane dioxygenase and haloacid dehalogenase type II. This strain also contained genes encoding ion detoxification protein; selenium-binding protein, tellurium resistance protein,
and arsenate reduction. Gene encoding mycoredoxin which plays an important role in arsenic resistance was also detected in the genome of strain SBTS01T (Mourenza et al. 2019).
For anticancer compound, strain W18L9T comprised gene encoding immune checkpoint inhibitors (ICIs) which are monoclonal antibodies that block inhibitors of T-cell activation and function. The ICIs have potential effects on more than 14 different cancers which were reported to have effects in some patients for more than 5 years (20% - 40%; 5-year survival rate in patients with melanoma) (Johnson et al. 2018). Based on the genome data mining, strain W18L9T has the potential to produce anti-HIV and anticancer compounds which will be fruitful to study this strain in-depth for novel bioactive compounds in the future.