651 CHARACTERIZATION OF AN ANTIMICROBIAL AND ANTIOXIDANT COMPOUND FROM A MARINE BACTERIUM GSA10 ASSOCIATED WITH THE SPONGE HALICHONDRIA GLABRATA Rashmi Phadale, Maushmi S. Kumar* Address(es): Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, SVKM’S NMIMS, V.L. Mehta Road, Vile Parle (W), Mumbai- 400056, India. *Corresponding author: maushmiskumar@gmail.com ABSTRACT Keywords: Antimicrobial activity, Bioautography, Hopanoids, LC-MS INTRODUCTION The marine environment is one of the most complex ecosystems with an enormous diversity of marine organisms living in close associations. Among them, eukaryotic and microorganism’s association is understood to play an important role in mimicking the production of the secondary metabolites and thus these microbial associates serve as an alternative source of bioactive compounds (Egan et al., 2008). Marine eukaryotes have been explored sufficiently for their ability to produce bioactive compounds in large-scale production, but face difficulty due to their insufficient ability to survive in mass culture. This limits their availability for future use and research (Molinski et al., 2009). As an alternative, symbionts and marine eukaryotes associated microorganisms can be easily cultured and grown in fermenters and therefore represent the best sustainable source for biologically active compounds (Sarkar et al., 2008). Currently, when the exact nature of the relationship between the marine host and their microorganisms remain unclear, it has been hypothesized that the microbial partners build chemical microenvironments within the eukaryotic host and lives in syntrophy, participating in cycling of nutrients. They also prevent entering of predators in the host by producing bioactive molecules forming a defensive mechanism (Sharp et al., 2005). The close metabolic association between microorganism and their host makes it difficult to reveal the responsible mechanism for the production of a particular metabolite. Most of the marine sponge’s porous body harbor dense and highly diverse microbial communities. These bacteria generally belong to phyla - Proteobacteria, Actinobacteria, Nitrospira, Chloroflexi, Planctomycetes, Cyanobacteria and Acidobacteria as well as both major lineages of Archaea and a range of unicellular eukaryotes such as diatoms and dinoflagellates. These microbes are often specific to sponges, with many microbial phylotypes appearing to live exclusively within sponge hosts and not in the surrounding seawater (Schmitt et al., 2008). These microorganisms exhibit diverse metabolic traits useful to the host such as nitrification, photosynthesis, anaerobic metabolism and secondary metabolite production. The bioactive compounds from sponges in few cases, after investigation have shown to be produced by the microbes, rather than the sponge itself. In one of the studies, Zheng et al. (2005) isolated twenty-nine marine bacterial strains from the sponge Hymeniacidon perleve among which eight strains inhibited the growth of terrestrial microorganisms. Among them, the strain NJ6-3-1 was identified as Pseudoalteromonas piscicida with wide antimicrobial spectrum and the major antimicrobial metabolite norharman was identified (a beta- carboline alkaloid) by EI-MS and NMR. The role of these diverse microbes in sponge biology varies from source of nutrition to mutualistic symbiosis with the sponge (Kennedy et al., 2009). A developing sponge acquires bacterial symbionts mainly by two pathways, firstly by selective absorption of specific bacteria from the surrounding water that passes through and the second one is by vertical transmission of symbionts through the gametes of the sponge by inclusion of the bacteria in the oocytes or larvae (Radjasa et al., 2007).These pathways allow diverse microbial community to reside inside marine sponges due to its porous body. Several researchers have attempted to culture microorganisms from invertebrates with hope of obtaining bioactive invertebrate metabolites. These attempts have been successful in discovering novel natural products with rare demonstration of the presence of sponge metabolites in the microbial isolates. The suspicion about the origin of the cytotoxic compounds discovered in sponges were also established to be produced by their associated microorganisms which resulted into many other associated microorganisms to be the bioactive compounds producer (Konig et al., 2006; Penesyan et al., 2010). Different polyketides and nonribosomal proteins (that are generally associated with bacterial metabolism) with potent anti-cancer properties, such as halichondrin B, bryostatin and discodermolide have been also isolated from associated microorganisms of marine sponges. Specifically for bryostatin, the microbial origin was demonstrated by the identification of polyketide synthase genes involved in its biosynthesis in the genome of the bryozoan bacterial symbiont Candidatus endobugula sertula (Sudek et al., 2007). In another study, a peptide antibiotic andrimid was found in a Hyatella sp. sponge as in a Vibrio sp. cultured from that sponge (Haygood et al., 1999). Halichondria okadai and Halichondria melanodocia species of Halichondria contain the protein phosphatase inhibitor okadaic acid. It was first isolated from the sponge Halichondria okadai, but later was found out to be produced by dinoflagellate Prorocentrum lima (Kelecom, 2002). Two unidentified bacteria of the genera Pseudomonas and Alteromonas isolated from Halichondria okadai homogenates i.e. Pseudomonas sp. KK10206C also produced a novel C50- carotenoid, okadaxanthine and Alteromonas sp. was responsible for the production of a well-known lactam Alteramide A (Shigemori et al., 1992, Bhalla et al., 2002, Thomas et al., 2010). With the aim of finding new bioactive compounds from marine sponge associated microorganisms, we investigated Halichondria glabrata associated Three marine sponges- Halichondria glabrata, Sigmadocia petrosioides and Pseudosuberitus andrewsi were collected for isolation of sponge-associated bacteria from Mumbai coastal regions. Among 110 isolates, six strains were selected and studied for its antibacterial activity by disc-diffusion assay. TLC- Bioautography studies were performed. GSA10 isolate from Halichondria glabrata was selected for showing good antagonistic activity for E. coli MTCC-1687, P. aeruginosa MTCC- 1688, B. subtilis MTCC- 441 and S. aureus MTCC - 737. Various biochemical tests and 16S ribosomal RNA sequencing were performed for GSA10 for its identification which confirmed it to be a Bacillus species. To decipher the activity of GSA10 bacteria, we aligned the 16S rRNA sequence in NCBI database. It represented close relationship with pG1 Bacillus amyloliquefaciens and hypothetical protein ATP synthase from Staphylococcus aureus. TLC direct bioautography was developed and results were found positive for a specific band at Rf 6.8. This bioactive compound was isolated by preparative TLC and characterized by UV, FT-IR, and LC-MS. It was further evaluated for antioxidant activity. It showed good antioxidant activity in DPPH scavenging (99%) and TRAP assay (91%).The purified compound was characterized as a C-30 hopanoid from a marine- sponge associated bacteria. ARTICLE INFO Received 15. 11. 2017 Revised 8. 3. 2018 Accepted 16. 5. 2018 Published 1. 6. 2018 Regular article doi: 10.15414/jmbfs.2018.7.6.651-658