Progress in Microbes and Molecular Biology Genome Report 1 Complete genome of mangrove-derived anti-MRSA streptomycete, Strep- tomyces pluripotens MUSC 135 T Hooi-Leng Ser 1,2,3,4, Kok-Gan Chan 5,6, Wen-Si Tan 5, Wai-Fong Yin 5, Bey-Hing Goh 2,3,4,8*, Nurul-Syakima Ab Mutalib 7, Learn-Han Lee 2,3,4,8* 1Institute of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou 510006, PR China. 2Novel Bacteria and Drug Discovery (NBDD) Research Group, Biomedicine Research Advancement Centre (BRAC), School of Pharmacy, Monash University Malaysia, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia 3Biofunctional Molecule Exploratory (BMEX) Research Group, Biomedicine Research Advancement Centre (BRAC), School of Pharmacy, Monash University Malaysia, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia 4Biomedical Research Laboratory, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia 5Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia 6International Genome Centre, Jiangsu University, Zhenjiang 212013, PR China 7UKM Medical Molecular Biology Institute (UMBI), UKM Medical Centre, University Kebangsaan Malaysia, Kuala Lum- pur, Malaysia 8Center of Health Outcomes Research and Therapeutic Safety (Cohorts), School of Pharmaceutical Sciences, University of Phayao, Phayao, Thailand Abstract : Microorganisms serve as attractive resources, owing to their ability to synthesize structurally-diverse substances with various bioactivities. Within the Bacteria domain, members of the genus Streptomyces have demonstrated remarkable ability to produce clinically useful, secondary metabolites such as anticancer, antioxidants, antivirals and antibacterials. Streptomyces pluripotens MUSC 135T was isolated as novel strain from mangrove forest in Malaysia. This strain exhibited broad spectrum bacteriocin against several pathogens including methicillin-resistant Staphylococcus aureus (MRSA) strain ATCC BAA-44, Salmonella typhi ATCC 19430T and Aeromonas hydrophila ATCC 7966T. Thus, the strain was selected for whole genome sequencing as an attempt to explore its bioactive potential. Here we report the first complete genome of S. pluripotens MUSC 135T genome which comprise of 7.35 Mbp with G+C content of 69.9 %. A total of 6,404 open reading frames (ORFs) were predicted, along with 18 rRNA and 69 tRNA genes. Using bacteriocin mining tool, BAGEL detected eights gene clusters associated with bacteriocin production including lanthipeptides and linear azol(in)e-containing peptides (LAPs). Members of Streptomyces have contributed greatly towards improving lives, particularly against deadly infections and chronic diseases. The availability of S. pluripotens MUSC 135T genome sequence has opened new window for drug discovery, particularly for effective drugs against harmful pathogens such as MRSA and certainly deserves further detailed study. Keywords: Streptomyces; MRSA; mangrove; bioinformatics; genome Received: 5th September 2018 Accepted: 20th September 2018 Published Online: 10th October 2018 *Correspondence to: Learn-Han Lee, School of Pharmacy, Monash University Malaysia, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia; lee.learn.han@monash.edu; leelearnhan@ yahoo.com; Bey-Hing Goh, School of Pharmacy, Monash University Malaysia, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia; goh.bey.hing@monash.edu Citation: Ser HL, Chan KG, Tan WS, et al. Complete genome of mangrove-derived anti-MRSA streptomycete, Streptomy- ces pluripotens MUSC 135T. Prog Micobes Mol Biol 2018; 1(1): a0000004. Short Introduction Members of genus Streptomyces are known to produce various types of compounds with bioactivities includ- ing anticancer, antioxidant, antifungal and antibacterial properties[1-7]. The unique life cycle of streptomycetes have enhanced their ability to persist in nature and to survive in harsh environmental conditions[8-13]. Strepto- myces pluripotens MUSC 135T was firstly isolated as Copyright 2018 by Ser HL et al. and HH Publisher. This work under licensed under the Creative Commons Attribution-NonCommer- cial 4.0 International Lisence (CC-BY-NC 4.0) 2 novel strain from mangrove forest in Peninsular Malay- sia during a screening program for antibiotic produc- ers[14,15]. This strain exhibited broad spectrum bacteriocin against several pathogens including methicillin-resistant Staphylococcus aureus (MRSA) strain ATCC BAA-44, Salmonella typhi ATCC 19430T and Aeromonas hy- drophila ATCC 7966T. In order to gain insights on its bioactive potential, strain MUSC 135T was subjected to complete genome sequencing and annotation to identify presence of biosynthetic gene clusters. Data Description Streptomyces pluripotens MUSC 135T was isolated from Tanjung Lumpur mangrove forest located in the city of Kuantan, State of Pahang, in December of the year 2012[14,15]. Purified cultures were maintained on ISP me- dium 2 slants at room temperature for short-term stor- age and as glycerol suspensions (20%, v/v) at −80°C for long-term storage[16]. The general features of the strain are summarized in Table 1[17]. The strain is available from culture collection centers under accession number of MCCC 1K00252T = DSM 42140T. Table 1: General features of Streptomyces pluripotens MUSC 135T and MIGS mandatory information. Property Description Classification Domain Bacteria Phylum Actinobacteria Class Actinobacteria Order Actinomycetales Family Streptomycetaceae Genus Streptomyces Species pluripotens strain: MUSC 135T Gram stain Positive Cell shape Branched mycelia Motility Dispersion of spores Sporulation Yes Temperature range 20–40°C Optimum temperature 28–32°C pH range; Optimum 5.0–9.0; 5.0–8.0 Carbon source Varied Oxygen requirement Aerobic Pathogenicity Non-pathogenic Geographic location Tanjung Lumpur, Pahang, Malaysia Latitude 3° 48’ 3.2”N Longitude 103° 20’ 22.7”E Altitude 0 above sea level The genomic DNA of MUSC 135T was extracted with Wizard® Genomic DNA Purification Kit (Promega) pri- or to cleanup process using AMPure® PB beads. DNA quality was examined using NanoDrop spectrophotom- eter (Thermo Scientific, Waltham, MA, USA) and a Qu- bit version 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA)[18]. SMRTbell DNA libraries (Pacific Biosci- ences) were constructed according to the PacBio standard protocol with the BluePippin size-selection system (Sage Science). Whole genome sequencing of MUSC 135T was performed with PacBio RSII sequencing technology us- ing P6-C4 chemistry, yielding output data with an average genome coverage of 217.22-fold (Table 2). Table 2: Genome statistics of Streptomyces pluripotens MUSC 135T Sequencing platform PacBio RSII Assembly HGAP3 Number of replicon 1 Accession number CP021080 Genome size (bp) 7,346,075 G + C content % 69.9 Protein coding genes 6,404 tRNA 69 rRNA 6, 6, 6 (5S, 16S, 23S) Upon sequencing, the raw reads were assembled using HGAP3. De novo assembly of the insert reads was per- formed with the Hierarchical Genome Assembly Process (HGAP) algorithm. The genome sequence of MUSC 135T was assembled into a single GC-rich (69.9 %) contig with genome size of 7,346,075 bp. rRNA and tRNA predic- tions were performed using ARAGORN and RNAmmer, respectively[19,20]. Using these tools, a total of 77 tRNA genes and 18 rRNA operons was predicted in MUSC 135T genome. Gene prediction was conducted using Prodigal (version 1.20), which 6,404 open reading frames (ORFs) were predicted in MUSC 135T genome[21]. The whole genome project of MUSC 135T was deposited at DDBJ/ EMBL/GenBank under accession number CP021080. The assembly was uploaded for annotation to Rapid An- notation using Subsystem Technology (RAST)[22]. From RAST annotation system, most of the genes in MUSC 135T were involved in amino acids and derivatives me- tabolism, followed by carbohydrates metabolism and pro- tein metabolism subsystems (Figure 1). Interestingly, one of the genes belonging to virulence, disease and defense subsystem was predicted to encode for colicin E2 tolerance protein CbrC-like protein. The detection of this gene suggests ability of the strain to sur- vive against action of DNA endonuclease, colicin E2 and persist in the environment[23,24]. In addition to that, a web- based bacteriocin mining tool, BAGEL identified eights gene clusters associated with bacteriocin production[25]. Among these gene clusters, one of them was found to be responsible for production of linear azol(in)e-containing peptides (LAPs). The members of LAPs family, such as goadsporin and plantazolicin have previously been de- tected from microbes have shown potent antibacterial activities against pathogens including Bacillus anthracis and MRSA[26-28]. In conclusion, we report the complete Whole genome of Streptomyces pluripotens musc 135T... A sequence of S. pluripotens MUSC 135T. The availability of its genome sequence has suggested potential exploitation of the strain for potentially useful compounds, particularly against pathogens such as MRSA and certainly deserves further detailed study. Acknowledgement This work was supported by Industry Grant (Biotek Abadi Vote No. GBA-808813), MOSTI eScience funds (Project No. 06-02-10-SF0300) awarded to L.-H.L. and a Universi- ty of Malaya for High Impact Research Grant (UM-MOHE HIR Nature Microbiome Grant No. H- 50001-A000027 and No. A000001-50001) and PPP Grant (PG090-2015B) awarded to K.-G.C. Conflict of Interest The authours declared that there is no conflict of interest. Reference 1. Berdy J. Bioactive microbial metabolites. J Antibiotics 2005; 58: 1–26. 2. Kannan RR, Iniyan AM, Vincent SG. Production of a compound against methicillin resistant Staphylococcus aureus (MRSA) from Streptomyces rubrolavendulae ICN3 & its evaluation in zebrafish em- bryos. Indian J Med Res 2014; 139(6): 913. 3. Ser HL, Palanisamy UD, Yin WF, et al. Streptomyces malaysiense sp. nov.: A novel Malaysian mangrove soil actinobacterium with antioxida- tive activity and cytotoxic potential against human cancer cell lines. Sci Rep 2016; 6. doi: 10.3389/fmicb.2016.00899. 4. Ser HL, Tan LTH , Palanisamy UD, et al. Streptomyces antioxidans sp. nov., a novel mangrove soil actinobacterium with antioxidative and neuroprotective potentials. Front Microbiol 2016; 7: 899. doi: 10.3389/ fmicb.2016.00899. 5. Law JWF, Ser HL, Khan TM, et al. The potential of Streptomyces as biocontrol agents against the rice blast fungus, Magnaporthe oryzae (Pyricularia oryzae). Front Microbiol 2017; 8. 6. Kemung HM, Tan LTH, Khan TM, et al. Streptomyces as a prominent resource of future anti-MRSA drugs. Front Microbiol 2018; 9: 2221. 7. Kannan RR, Iniyan AM, Prakash VS. Isolation of a small molecule with anti-MRSA activity from a mangrove symbiont Streptomyces sp. PVRK-1 and its biomedical studies in Zebrafish embryos. Asian Pacific J Trop Biomed 2011; 1(5): 341. 8. Ser HL, Yin WF, Chan KG, et al. Antioxidant and cytotoxic poten- tials of Streptomyces gilvigriseus MUSC 26T isolated from man- grove soil in Malaysia. Progress Microbes Mol Biol 2018;1(1). url: http://www.journals.hh-publisher.com/index.php/pmmb/article/ view/3 9. Adam D, Maciejewska M, Naômé A, et al. Isolation, characteriza- tion, and antibacterial activity of hard-to-culture actinobacteria from cave moonmilk deposits. Antibiotics 2018; 7(2): 28. doi: 10.3390/antibiotics7020028. 10. Ser HL, Tan LT, Law JW, et al. Focused review: cytotoxic and an- tioxidant potentials of mangrove-derived Streptomyces. Front Mi- crobiol 2017; 8: 2065. doi: 10.3389/fmicb.2017.02065. 11. Padmanaban VP, Verma P, Venkatabaskaran S, et al. Antimicrobial potential and taxonomic investigation of piezotolerant Streptomy- ces sp. NIOT-Ch-40 isolated from deep-sea sediment. World J Mi- crobiol Biotech 2017; 33(2): 27. doi: 10.1007/s11274-016-2193-2. 12. Al-Dhabi NA, Esmail GA, Duraipandiyan V, et al. Isolation, iden- tification and screening of antimicrobial thermophilic Streptomyces sp. Al-Dhabi-1 isolated from Tharban hot spring, Saudi Arabia. Ex- tremophiles 2016; 20(1): 79-90. doi: 10.1007/s00792-015-0799-1. 13. Lee LH, Zainal N, Azman AS, et al. Diversity and antimicrobial ac- tivities of actinobacteria isolated from tropical mangrove sediments in Malaysia. Sci World J 2014; 2014. doi: 10.1155/2014/698178. 14. Lee LH, Zainal N, Azman AS, et al. Streptomyces pluripotens sp. nov., a bacteriocin-producing streptomycete that inhibits meticillin- resistant Staphylococcus aureus. Int J Syst Evol Microbiol 2014; 64: 3297–3306. doi: 10.1099/ijs.0.065045-0. 15. Ser HL, Ab Mutalib NS, Yin WF, et al. Genome sequence of Strep- tomyces antioxidans MUSC 164T isolated from mangrove forest. Progress Microbes Mol Biol 2018; 1(1). url: http://www.journals. hh-publisher.com/index.php/pmmb/article/view/1. 16. Ser HL, Tan WS, Ab Mutalib NS, et al. Genome sequence of Streptomyces pluripotens MUSC 135T exhibiting antibacterial and antioxidant activity. Mar Gen 2015; 24: 281–283. doi: 10.1016/j. margen.2015.09.010. 17. Ser HL, Tan WS, Ab Mutalib NS, et al. Draft genome sequence of mangrove-derived Streptomyces sp. MUSC 125 with antioxidant Ser HL, et al. 3 Figure 1. Subsystem category distribution of MUSC 135T based on RAST server. 4 potential. Front Microbiol 2016; 7: 1470. 18. Lowe TM, Eddy SR. tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. Nuc Acids Res 1997; 25: 955–964. 19. Lagesen K, Hallin P, Rodland EA, et al. RNAmmer: Consistent and rapid annotation of ribosomal RNA genes. Nuc Acids Res 2007; 35: 3100–3108. 20. Hyatt D, Chen GL, Locascio PF, et al. Prodigal: Prokaryotic gene recognition and translation initiation site identification. BMC Bioinfo 2010; 11: 119. 21. Aziz RK, Bartels D, Best AA, et al. The RAST Server: Rapid annota- tions using subsystems technology. BMC Genomics 2008; 9: 75. 22. Pugsley AP, Goldzahl N, Barker RM. Colicin E2 production and re- lease by Escherichia coli K12 and other Enterobacteriaceae. Microbiol 1985; 131(10): 2673–2686 23. Trivedi D, Jena PK, Seshadri S. Colicin E2 Expression in Lactoba- cillus brevis DT24, a vaginal probiotic isolate, against uropatho- genic Escherichia coli. ISRN Urology 2014; 2014: 869610. doi: 10.1155/2014/869610. 24. van Heel AJ, de Jong A, Montalban-Lopez M, et al. BAGEL3: Au- tomated identification of genes encoding bacteriocins and (non-) bactericidal posttranslationally modified peptides. Nucleic Acids Res 2013; 41(W1): W448–W453. 25. Arnison PG, Bibb MJ, Bierbaum G, et al. Ribosomally synthesized and post-translationally modified peptide natural products: over- view and recommendations for a universal nomenclature. Nat Prod Rep 2013; 30(1): 108–160. 26. Hao Y, Blair PM, Sharma, A, et al. Insights into methyltransferase specificity and bioactivity of derivatives of the antibiotic plantazo- licin. ACS chemical biology 2015; 10(5): 1209–1216. 27. Molohon KJ, Blair PM, Park S, et al. Plantazolicin is an ultra- narrow-spectrum antibiotic that targets the Bacillus anthracis membrane. ACS Infect Dis 2016; 2(3): 207–220. doi: 10.1021/ acsinfecdis.5b00115. Whole genome of Streptomyces pluripotens musc 135T...