View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Frontiers - Publisher Connector ORIGINAL RESEARCH published: 16 June 2016 doi: 10.3389/fmicb.2016.00899 Streptomyces antioxidans sp. nov., a Novel Mangrove Soil Actinobacterium with Antioxidative and Neuroprotective Potentials Hooi-Leng Ser 1, 2, Loh Teng-Hern Tan 1, 2, Uma D. Palanisamy 2, Sri N. Abd Malek 3, Wai-Fong Yin 4, Kok-Gan Chan 4, Bey-Hing Goh 1, 5* and Learn-Han Lee 1, 5* 1 Novel Bacteria and Drug Discovery Research Group, School of Pharmacy, Monash University Malaysia, Bandar Sunway, Malaysia, 2 Biomedical Research Laboratory, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Bandar Sunway, Malaysia, 3 Biochemistry Program, Faculty of Science, Institute of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia, 4 Division of Genetics and Molecular Biology, Faculty of Science, Institute of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia, 5 Center of Health Outcomes Research and Therapeutic Safety (Cohorts), School of Pharmaceutical Sciences, University of Phayao, Phayao, Thailand Edited by: Yuji Morita, A novel strain, Streptomyces antioxidans MUSC 164T was recovered from mangrove Aichi Gakuin University, Japan forest soil located at Tanjung Lumpur, Malaysia. The Gram-positive bacterium forms Reviewed by: Jem Stach, yellowish-white aerial and brilliant greenish yellow substrate mycelium on ISP 2 University of Newcastle, UK agar. A polyphasic approach was used to determine the taxonomy status of strain Huda Mahmoud Mahmoud, T Kuwait University, Kuwait MUSC 164 . The strain showed a spectrum of phylogenetic and chemotaxonomic Janice Lorraine Strap, properties consistent with those of the members of the genus Streptomyces. University of Ontario Institute of The cell wall peptidoglycan was determined to contain LL-diaminopimelic acid. Technology, Canada The predominant menaquinones were identified as MK-9(H ) and MK-9(H ), *Correspondence: 6 8 Bey-Hing Goh while the identified polar lipids consisted of aminolipid, diphosphatidylglycerol, [email protected]; glycolipid, hydroxyphosphatidylethanolamine, phospholipid, phosphatidylinositol, Learn-Han Lee [email protected]; phosphatidylethanolamine, phosphatidylglycerol and lipid. The cell wall sugars consist [email protected] of galactose, glucose and ribose. The predominant cellular fatty acids (>10.0%) were identified as iso-C15:0 (34.8%) and anteiso-C15:0(14.0%). Phylogenetic analysis identified Specialty section: T T This article was submitted to that closely related strains for MUSC 164 as Streptomyces javensis NBRC 100777 T Antimicrobials, Resistance and (99.6% sequence similarity), Streptomyces yogyakartensis NBRC 100779 (99.6%) Chemotherapy, and Streptomyces violaceusniger NBRC 13459T (99.6%). The DNA–DNA relatedness a section of the journal T Frontiers in Microbiology values between MUSC 164 and closely related type strains ranged from 23.8 ± 0.3% T Received: 15 March 2016 to 53.1 ± 4.3%. BOX-PCR fingerprints comparison showed that MUSC 164 exhibits Accepted: 26 May 2016 a unique DNA profile, with DNA G + C content determined to be 71.6 mol%. Based on Published: 16 June 2016 the polyphasic study of MUSC 164T, it is concluded that this strain represents a novel Citation: species, for which the name Streptomyces antioxidans sp. nov. is proposed. The type Ser H-L, Tan LT-H, Palanisamy UD, T T T T Abd Malek SN, Yin W-F, Chan K-G, strain is MUSC 164 (=DSM 101523 = MCCC 1K01590 ). The extract of MUSC 164 Goh B-H and Lee L-H (2016) showed potent antioxidative and neuroprotective activities against hydrogen peroxide. Streptomyces antioxidans sp. nov., a Novel Mangrove Soil Actinobacterium The chemical analysis of the extract revealed that the strain produces pyrazines and with Antioxidative and Neuroprotective phenolic-related compounds that could explain for the observed bioactivities. Potentials. Front. Microbiol. 7:899. doi: 10.3389/fmicb.2016.00899 Keywords: Streptomyces antioxidans, actinobacteria, mangrove, neuroprotective, antioxidative Frontiers in Microbiology | www.frontiersin.org 1 June 2016 | Volume 7 | Article 899 Ser et al. Streptomyces antioxidans sp. nov. INTRODUCTION be potentially useful for discovery of new drugs or drug leads for neurodegenerative diseases which role of oxidative stress Many therapeutic agents, such as antibiotics, anti-inflammatory has been implicated, including Parkinson’s diseases, Alzheimer’s and antioxidant compounds have been isolated from disease and multiple sclerosis. microorganisms (Bérdy, 2005; Williams, 2009). Discovery In this study, a novel strain, MUSC 164T was discovered from of these bacterial-derived bioactive compounds has a major a mangrove soil located in east coast of Peninsular Malaysia. impact on human health, helping people to live longer and A polyphasic approach determined that MUSC 164T represents reducing the mortality rate due to infectious and/or chronic a novel species of the Streptomyces genus, for which the name diseases. In recent years, the accumulation of free radicals Streptomyces antioxidans sp. nov. is proposed. As a means to or oxidative stress has been identified as one of the major explore the bioactivities possessed by the strain, the extract of contribution to neuronal loss and occur early in all major MUSC 164T was subjected to several antioxidant assays prior neurodegenerative diseases (Lin and Beal, 2006; Fischer and to in vitro neuroprotective screening against hydrogen peroxide Maier, 2015; Leszek et al., 2016). By reducing the presence of free (H2O2). Gas chromatography-mass spectrometry (GC-MS) was radicals, increased intake of antioxidants is known to prevent used to perform chemical analysis for MUSC 164T extract in and decrease the risk of these chronic diseases (Devasagayam order to reveal the chemical constituents present in the extract. et al., 2004; Bonda et al., 2010). Thus, continuous efforts have Taken altogether, this study has implicated the potential of the been directed toward searching for potent, natural antioxidants mangrove-derived strain Streptomyces antioxidans sp. nov. in to prevent the deleterious effects of free radicals. producing bioactive compounds, specifically with antioxidative Over the years, exploring new taxa remains as one of the and neuroprotective activities. successful strategies which lead to discovery of therapeutic agents (Williams, 2009). As the most prolific producer of MATERIALS AND METHODS bioactive compounds, Streptomyces genus was initially proposed by Waksman and Henrici (1943) and metabolites isolated from Isolation and Maintenance of Isolate these organisms have been shown to possess pharmaceutically Strain MUSC 164T was recovered from a soil sample collected relevant activities such as anti-inflammatory, antimicrobial, at site MUSC-TLS4 (3◦ 48′ 21.3′′ N 103◦ 20′ 3.3′′ E), located antioxidant activities (Bérdy, 2005; Wang et al., 2013; Kumar in the mangrove forest of Tanjung Lumpur in the state of et al., 2014; Ser et al., 2015a, 2016a; Tan et al., 2016). Moreover, Pahang, Peninsular Malaysia in December 2012. Topsoil samples the metabolites derived from Streptomyces are described as of the upper 20cm layer (after removing the top 2–3cm) were potent protective agents in neuronal cells against oxidative stress collected and sampled into sterile plastic bags using an aseptic induced damage. In fact, a recent study by Leiros et al. (2013) has metal trowel, and stored at −20◦C. Air-dried soil samples were identified seven bioactive compounds produced by Streptomyces ground with a mortar and pestle. Selective pretreatment of soil sp. which protects against hydrogen peroxide (H2O2) challenge samples was performed using wet heat in sterilized water (15min in primary cortical neurons. Unfortunately, many previous at 50◦C; Takahashi et al., 1996). Five grams of the pretreated drug screening program focused on novel actinomycetes from air-dried soil was mixed with 45 mL sterilized water and mill terrestrial source, which in turn resulted in inefficient rediscovery ground, spread onto the isolation medium ISP 2 (Shirling and of known bioactive compounds. Thus, researchers began to divert Gottlieb, 1966) supplemented with cycloheximide (25 µg/mL) their attention to new or underexplored habitats, in hope to find and nystatin (10 µg/mL), and incubated at 28◦C for 14 days. Pure new species that may yield promising bioactive compounds. cultures of strain MUSC 164T were isolated and maintained on As one of the world’s most dynamic environments, the slants of ISP 2 agar and in glycerol suspensions (20% v/v). mangrove ecosystem yields commercial forest products, supports coastal fisheries and protects coastlines (Alongi, 2008). Genomic and Phylogenetic Analyses Recently, there has been a renewed interest in the mangrove Extraction of DNA was performed as previously described (Hong microorganisms’ resources, considering that the changes in et al., 2009), followed by 16S rRNA gene amplification carried salinity and tidal gradient in the mangrove can trigger metabolic out as stated by Lee et al. (2014b). Using CLUSTAL-X software, adaptations that could result in valuable metabolites production the 16S rRNA gene sequence of strain MUSC 164T was aligned (Hong et al., 2009; Lee et al., 2014a; Azman et al., 2015). Several with representative sequences of related type strains of the studies have discovered novel actinobacteria from the poorly genus Streptomyces retrieved from the GenBank/EMBL/DDBJ explored mangrove