J. Microbiol. Biotechnol. (2019), 29(7), 1144–1154 https://doi.org/10.4014/jmb.1906.06037 Research Article Review jmb Draft Genome Analysis of Antimicrobial Streptomyces Isolated from Himalayan Lichen S Byeollee Kim1, So-Ra Han1, Janardan Lamichhane2, Hyun Park3, and Tae-Jin Oh1,4,5* 1Department of Life Science and Biochemical Engineering, SunMoon University, Asan 31460, Republic of Korea 2Department of Biotechnology, Kathmandu University, Kathmandu, Nepal 3Unit of Polar Genomics, Korea Polar Research Institute, Incheon 21990, Republic of Korea 4Genome-based BioIT Convergence Institute, Asan, Republic of Korea 5Department of Pharmaceutical Engineering and Biotechnology, SunMoon University, Asan 31460, Republic of Korea Received: June 17, 2019 Revised: July 5, 2019 There have been several studies regarding lichen-associated bacteria obtained from diverse Accepted: July 9, 2019 environments. Our screening process identified 49 bacterial species in two lichens from the First published online Himalayas: 17 species of Actinobacteria, 19 species of Firmicutes, and 13 species of July 10, 2019 Proteobacteria. We discovered five types of strong antimicrobial agent-producing bacteria. *Corresponding author Although some strains exhibited weak antimicrobial activity, NP088, NP131, NP132, NP134, Phone: +82-41-530-2677; and NP160 exhibited strong antimicrobial activity against all multidrug-resistant strains. Fax: +82-41-530-2279; E-mail: [email protected] Polyketide synthase (PKS) fingerprinting revealed results for 69 of 148 strains; these had similar genes, such as fatty acid-related PKS, adenylation domain genes, PfaA, and PksD. Although the association between antimicrobial activity and the PKS fingerprinting results is poorly resolved, NP160 had six types of PKS fingerprinting genes, as well as strong antimicrobial activity. Therefore, we sequenced the draft genome of strain NP160, and predicted its secondary metabolism using antiSMASH version 4.2. NP160 had 46 clusters and was predicted to produce similar secondary metabolites with similarities of 5–100%. Although NP160 had 100% similarity with the alkylresorcinol biosynthetic gene cluster, our results showed low similarity with existing members of this biosynthetic gene cluster, and most have S upplementary data for this paper are available on-line only at not yet been revealed. In conclusion, we expect that lichen-associated bacteria from the http://jmb.or.kr. Himalayas can produce new secondary metabolites, and we found several secondary metabolite-related biosynthetic gene clusters to support this hypothesis. pISSN 1017-7825, eISSN 1738-8872 Copyright© 2019 by Keywords: Antimicrobial activity, draft genome sequencing, fingerprinting, Himalayan lichen- The Korean Society for Microbiology and Biotechnology associated bacteria, polyketide synthase, secondary metabolites Introduction non-photosynthetic partner (bacterium). Before 2005, research regarding lichens was focused on lichenicolous Approximately 13,500 species of lichen are estimated to fungus [2] and cyanobacteria [3]; the word ‘lichen- inhabit 8% of the earth’s surface in various environments, associated bacteria’ began to appear in the literature, due to including low temperature, drought, and darkness [1]. To the work by Alexandra et al., beginning in 2011 [4]. It has survive in such environments, lichens produce unique recently been reported that millions of lichen-associated secondary metabolite-related compounds that have anti- bacteria greatly influence the survival of lichen through microbial, antitumoral, immunostimulating, and/or antiviral various roles such as resistance stress factor, detoxification activities. In addition, they contribute to symbiosis of the of metabolites, support for photosynthesis, and nutrient mycobiont (fungal partner), photobiont (photosynthetic supply [5]. In 2018, Delphine et al. reported using genome partner, usually a green algae or cyanobacterium), and analysis to identify a new metabolite from the alpha- J. Microbiol. Biotechnol. Draft Genome Analysis of Himalayan Lichen-Associated Bacteria 1145 proteobacterium strain MOLA1416, a marine type of of the washed, broken lichen solution were spread on replicate lichen-associated Proteobacteria [6]. Recent studies of lichen- plates containing MY media (3.0 g malt extract, 3.0 g yeast extract, associated bacteria have been performed to confirm activity 10.0g peptone, 10.0 g dextrose, and 20.0 g agar, pH 7.2), R2A and select cultivable bacteria for in-depth genome analysis. media (0.5 g proteose peptone, 0.5 g casamino acid, 0.5 g yeast Most research studies regarding lichen-associated bacteria extract, 0.5 g dextrose, 0.5 g soluble starch, 0.3 g sodium pyruvate, 0.3 g dipotassium phosphate, and 0.05 g magnesium sulfate, pH are based on the hypothesis that such bacteria will produce 7.2), or Bennett’s media (10.0 g glucose, 1.0 g yeast extract, 2.0 g one or more unusual secondary metabolites in the symbiotic Bacto-peptone, and 1.0 g beef extract, pH 7.2); the plates were relationship. The biosynthesis of secondary metabolites, incubated at different temperatures (8, 15, 25, and 37°C) for 3 to analyzed by biosynthetic gene cluster, is already well 30days. known; this includes multidomain enzymes such as polyketide synthase (PKS) and non-ribosomal peptide 16S rRNA Gene Sequence and Phylogenetic Analysis synthetase (NRPS) [7–10]. Although PKS and NRPS comprise All isolated bacteria were identified by 16S rRNA gene most of the metabolite class, knowledge regarding the sequence analysis. For 16S rRNA analysis, the genomic DNA of biological activities of PKS and NRPS genes remains each strain was extracted from a 100-μl culture sample from a 15– insufficient. Lichen-associated bacteria have been analyzed 30day pure culture of a single colony and centrifuged for 10 min from diverse environments, including thermal spots [5], at 10,000 ×g; extraction was performed using the DNeasy Blood sub-Arctic [11], and Arctic [12]. Thus far, 465 species of and Tissue Kit (Qiagen, Inc., USA). The 16S rRNA was amplified with two universal primers: 518F (5’-CCAGCAGCCGCGGTA lichen have been identified in Nepal [13]; nevertheless, ATACG-3’) and 800R (5’-TACCAGGGTATCTAATCC-3’). For there has been no study of the diversity of lichen- difficult to analyze sequences, other primers were used: 27F (5’- associated bacteria or their PKS and NRPS genes. Notably, AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-TACGGYTAC lichen flora, market potential, and various activities CTTGTTACGACTT-3’). Sequencing was performed by Genotech (antioxidant, antimicrobial, and toxicity) were reported Ltd. (Korea); the results were compared with the GenBank regarding lichens [14, 15]. Other lichen-associated bacteria database using BLAST to identify the most similar sequences. have been reported as examples of bacterial diversity [16], Closest relative strains were also identified using EzBioCloud as sources of novel metabolites [6], and as samples for (ChunLab, Inc., Korea), and the species of the isolates were genome analysis [17]. predicted. The sequences were pairwise aligned, and phylogenetic We undertook an extensive study regarding the anti- analysis was performed using a modified version of the method microbial potential of lichen-associated bacteria. We aimed described by Kimura et al. [19]. A phylogenetic tree was inferred to identify lichen-associated bacteria from Himalayan using the neighbor-joining method in MEGA X [20]. lichens and to analyze the genomes of selected bacteria that Extraction of Isolated Strains and Disk Diffusion Assay showed antimicrobial activities with secondary metabolite- For the antimicrobial assay, each of the 49 lichen-associated related biosynthetic gene clusters. Based on identification bacteria isolates was cultured in 200 ml liquid medium in of PKS and NRPS genes using fingerprinting polymerase conditions identical to those in which it was isolated. A double chain reaction (PCR), we propose the use of lichen- volume of analytical-grade ethyl acetate (Daejung, Korea) was associated bacteria to produce antimicrobial agents. This added into the grown culture fluid, and shaken using a funnel at study also includes draft genome analysis of lichen- room temperature for 2 h for extraction. The solvent layer was associated bacteria to understand the biosynthesis of concentrated using a rotary evaporator (EYELA A-1000S, Tokyo secondary metabolites. Rikakikai Co., Japan), and each extract was dissolved in 2.0 ml ethyl acetate. The antimicrobial activities of all extracts obtained from the 49 isolated strains were investigated using 16 multidrug- Materials and Methods resistant microorganisms, including anaerobic and aerobic bacteria, by the disc diffusion method. Anaerobic bacteria were Isolation of Lichen-Associated Bacteria Staphylococcus aureus (KCTC1927), Streptococcus mutans (KCTC3065), Two lichens from the Himalayas (Fig. S1 and Table S1) were Streptococcus sanguinis (KCTC3284), Streptococcus sobrinus (KCTC3308), collected (27.718805, 85.322705), categorized, and identified by Streptococcus cricetid (KCTC3640), Streptococcus ratti (KCTC3655), morphological analysis. To isolate associated bacteria from Aggregatibacter actinomycetemconitans (KCTC3698), Streptococcus lichens, a modified version of the method of Kim et al. [18] was anginosus (KCTC3983), Actinomyces viscosus (KCTC5531), and used. The thallus segments of the lichens were spread with Actinomyces israelii (KCTC9054); aerobic bacteria were Bacillus sterilized scissors, and 2.0 ml of 0.85% NaCl