Bioactivities and genome insights of a thermotolerant antibiotics‐producing Streptomyces sp. TM32 reveal its potentials for novel drug discovery

Abstract A way to defeat antimicrobial resistance (AMR) crisis is to supply novel drugs to the pharmaceutical industry. This effort leads to a global call for seeking the beneficial microbes from underexplored habitats. To support this call, we isolated Streptomyces sp. TM32 from the rhizosphere soil of a medicinal plant, turmeric (Curcuma longa L.). TM32 exhibited strong antimicrobial activities against both human and plant pathogens, including an AMR pathogen, Staphylococcus haemolyticus MR‐CoNS. Surprisingly, such antimicrobial results of TM32's autoclaved crude extract remained the same. Based on the genome data analysis, TM32 belongs to the same genomic species with Streptomyces sioyaensis DSM 40032T, supported by the relatively high‐average nucleotide identity values (ANIb: 96.80% and OrthoANIu: 97.14%) and in silico DNA–DNA relatedness value of 75.40%. Importantly, the gene annotation analyses revealed that TM32's genome contains various genes encoding the biosynthesis of either known or unknown antibiotics and some metabolites involved in plant growth‐promoting traits. However, bioactivities and genome data comparison of TM32 and DSM 40032T showed a set of apparent differences, for example, antimicrobial potentials, genome size, number, and occurrence of coding DNA sequences in the chromosomes. These findings suggest that TM32 is a new strain of S. sioyaensis and serves as an emerging source for further discovery of valuable and novel bioactive compounds.

species, referring to the list of prokaryotic names with standing in nomenclature (http://www.bacterio.net/streptomyces.html) during the time of writing this article. Regarding these high numbers of published Streptomyces members, the recent trend of drug discovery is more focusing on other microbial resources, such as non-Streptomyces actinobacteria, so-called "rare actinomycetes" with the aim to avoid the repetitive finding of the formerly found bioactive metabolites (Tiwari & Gupta, 2012). However, hitherto, it is just a minority of microbes discovered, while the majority of them is hidden in nature and waiting for exploration.
Streptomyces sp. TM32 was isolated based on the concept of plant-microbe interactions from the rhizosphere soil of turmeric (Curcuma longa L.), a rhizomatous and herbaceous plant, often used in cooking (Nakaew, Rangjaroen, & Sungthong, 2015). A preliminary identification of TM32 using its 16S rRNA gene sequence revealed the closest phylogenetic relation to Streptomyces sioyaensis DSM 40032 T , a producer of a peptide thiazole antibiotic, siomycin A, possessing both antibacterial and anticancer functions (Gartel, 2013;Tori et al., 1979). TM32 showed strong antifungal activity and was proven for its plant growth-promoting (PGP) potentials in the suppression of phytopathogenic fungus, Rigidoporus sp. (Nakaew et al., 2015).
In this study, we aim to report some interesting bioactivities and the draft genomes of the type strain, S. sioyaensis DSM 40032 T , and our emerging strain, TM32. We also unveil the biotechnological treasures in both genomes by employing some bioinformatics tools and discuss the novelty of TM32 based on a set of comparative genome data.

| MATERIAL S AND ME THODS
The antimicrobial activities of crude extracts derived from DSM 40032 T (type strain) or TM32 were tested against either bacteria or fungi and some of which are pathogens of human and plant (Table 1). Each Streptomyces strain was grown in Hickey-Tresner agar at 30°C for 7 days, which was transferred to grow further in 200 ml of International Streptomyces Project medium II (ISP2) broth under shaking conditions at 150 rpm, 30°C for 14 days (Nakaew et al., 2015). The whole liquid culture was extracted with ethyl acetate at a solute and solvent ratio of 1:2 (v/v) for one night, in which the separated supernatant was collected and evaporated at 45°C using a rotary evaporator (Büchi, Switzerland). The crude extracts obtained TA B L E 1 Some antimicrobial activities of crude extracts derived from Streptomyces sioyaensis DSM 40032 T and Streptomyces sp. TM32 The antimicrobial activity was determined into five levels (excellent ++++, very good +++, good ++, fair +, and no activity −). The value in parenthesis refers to the average size (ø mm) ± standard deviation of the inhibitory zones derived from the duplicate antagonism assays.
were divided into two proportions and one of which was heated by autoclaving at 121°C, 15 psi for 15 min.
Both nonheated and heated crude extracts were dissolved with dimethyl sulfoxide (DMSO) and tested for their antimicrobial activities by the paper disk-diffusion method described elsewhere (Nakaew, Sungthong, Ortega-Calvo, & Lumyong, 2012). The overnight culture of test bacteria in nutrient broth (Himedia, India) and 2-day-old culture of test fungi in potato dextrose broth (Himedia, India) were prepared by shaking incubation at 150 rpm and 30°C.
The culture of each test microbe was swabbed over its corresponding agar medium mentioned before. Paper disks were pasted on the test agar medium, in which 30 μL of the crude extracts, 2 mg/ml nalidixic acid (positive antibacterial), 1 mg/ml cycloheximide (positive antifungal), or DMSO (negative control) was dropped. The antimicrobial activity was reported with the diameter size of the inhibitory zone appeared on the assayed plates.
The biomass of DSM 40032 T or TM32 was prepared after cultivation in ISP2 broth under shaking conditions at 150 rpm, 30°C for 5 days. Total genomic DNA of both strains was extracted and used for sequencing with the Illumina HiSeq2500 system, following the services provided by BaseClear BV (Leiden, the Netherlands). The reads generated from the sequencing were assembled using SPAdes version 3.9 (Bankevich et al., 2012) and evaluated by QUAST version 3.2 (Gurevich, Saveliev, Vyahhi, & Tesler, 2013). The gene annotation analysis was carried out based on all contigs of each genome using Prokka (Seemann, 2014). The secondary metabolite-related genes and gene clusters present in the genomes were predicted using antiSMASH 3.0 (Weber et al., 2015).
Identification of TM32 using its 16S rRNA gene sequence

| RE SULTS
The crude extracts provided by ethyl acetate extraction of both Streptomyces strains did not show antimicrobial activity against the test Gram-negative bacterium, Escherichia coli TISTR 887 (Table 1).  (Table 3). TM32 possesses an extra chitinase gene (chiD), which is absent in the chromosome of DSM 40032 T . This finding may result in the stronger antifungal activity of TM32 compared to DSM 40032 T (Table 1). However, TM32 lacks the exo-β-1,3-glucanase gene, which is present in DSM 40032 T 's chromosome.
The absence of the glucanase gene was concordant to the phenotype of TM32 previously observed by an in vitro enzymatic assay (Nakaew et al., 2015).
Both strains have a siomycin-encoding gene cluster in their genomes, which is a unique characteristic of S. sioyaensis (Gartel, 2013;Tori et al., 1979 a MIBiG refers to "Minimum Information about a Biosynthetic Gene cluster" in the antiSMASH 3.0 (Weber et al., 2015). b The presence (+) or absence (−) of each gene cluster is determined, while the value indicated in the parenthesis is the similarity percentage to the known cluster or the number of cluster found.