25Phylogenetic diversity of cultivable actinobacteria isolated from sponges and associated 26 environments of intertidal zones, along the northern parts of west coast of India, were studied 27 using 16S rRNA gene sequences. A subset of actinobacteria were screened for three activities, 28 namely predatory behavior, antibacterial activity and enzyme inhibition. We recovered 207 29 isolates of actinobacteria belonging to 16 families and 25 genera, which could be attributed to 30 55 putative species using Poisson Tree Processes and 60 putative species based on Bayesian 31Poisson Tree Processes. Although the trends in the discovery of actinobacterial genera isolated 32 from sponges was consistent with previous studies from different study areas, we provide first 33 report of six actinobacterial species from sponges. We observed widespread non-obligate 34 epibiotic predatory behavior in eight actinobacterial genera and we provide first report of 35 predatory activity in Brevibacterium, Glutamicibacter, Micromonospora, Nocardiopsis, 36Rhodococcus and Rothia. Sponge associated actinobacteria showed significantly more 37 predatory behavior than environmental isolates. While antibacterial activity by actinobacterial 38 isolates mainly affected Gram-positive target bacteria with little to no effect on Gram-negative 39 bacteria, predation targeted both Gram-positive and Gram-negative prey with equal propensity. 40 Actinobacterial isolates from both sponge and associated environment produced inhibitors of 41 serine proteases and angiotensin converting enzyme. Predatory behavior was strongly 42 associated with inhibition of trypsin and chymotrypsin. Our study suggests that sponge and 43 associated environment of western coast of India are rich in actinobacterial diversity with 44 widespread predatory activity, antibacterial activity and production of enzyme inhibitors. 45

The phylogenetic diversity of cultivable actinobacteria isolated from sponges (Haliclona spp.) and associated intertidal zone environments along the northern parts of the western coast of India were studied using 16S rRNA gene sequences. A subset of randomly selected actinobacterial cultures were screened for three activities, namely predatory behaviour, antibacterial activity and enzyme inhibition. We recovered 237 isolates from the phylum Actinobacteria belonging to 19 families and 28 genera, which could be attributed to 95 putative species using maximum-likelihood partition and 100 putative species using Bayesian partition in Poisson tree processes. Although the trends in the discovery of actinobacterial genera isolated from sponges were consistent with previous studies from different study areas, we provide the first report of nine actinobacterial species from sponges. We observed widespread non-obligate epibiotic predatory behaviour in eight actinobacterial genera and we provide the first report of predatory activity in Brevibacterium , Glutamicibacter , Micromonospora , Nocardiopsis , Rhodococcus and Rothia . Sponge-associated actinobacteria showed significantly more predatory behaviour than environmental isolates. While antibacterial activity by actinobacterial isolates mainly affected Gram-positive target bacteria with little or no effect on Gram-negative bacteria, predation targeted both Gram-positive and Gram-negative prey with equal propensity. Actinobacterial isolates from both sponges and associated environments produced inhibitors of serine proteases and angiotensin-converting enzyme. Predatory behaviour was strongly associated with inhibition of trypsin and chymotrypsin. Our study suggests that the sponges and associated environments of the western coast of India are rich in actinobacterial diversity, with widespread predatory activity, antibacterial activity and production of enzyme inhibitors. Understanding the diversity and associations among various actinobacterial activities – with each other and the source of isolation – can provide new insights into marine microbial ecology and provide opportunities to isolate novel therapeutic agents.

21Phylogenetic diversity of culturable actinobacteria isolated from the intertidal regions of west 22 coast of Maharashtra, India was studied using 16S rRNA gene sequencing. Total of 140 23 actinobacterial isolates were obtained, which belonged to 14 genera, 10 families and 65 putative 24 species with Streptomyces being the most dominant (63%) genus followed by Nocardiopsis 25 and Micromonospora. They were screened for production of extracellular protease inhibitors 26 (PI) against three pure proteases viz. chymotrypsin, trypsin, subtilisin and one crude 27 extracellular protease from Pseudomonas aeruginosa. Eighty percent of the isolates showed PI 28 activity against at least one of the four proteases, majority of them belonged to genus 29 Streptomyes. Actinobacterial diversity from two sites Ade (17°52' N, 73°04' E) and Harnai 30 (17°48' N, 73°05' E) with varying degree of anthropological pressure showed that more putative 31 species diversity was obtained from site with lower human intervention i.e Ade (Shannon's H 32 3.45) than from Harnai (Shannon's H 2.83), a site with more human intervention. Further, in 33

Phylogenetic diversity of culturable actinobacteria isolated from the intertidal regions of west coast of Maharashtra, India was studied using 16S rRNA gene sequencing. Total of 140 actinobacterial isolates were obtained, which belonged to genera, 10 families and 65 putative species with Streptomyces being the most dominant (63%) genus followed by Nocardiopsis and Micromonospora. They were screened for production of extracellular protease inhibitors (PI) against three pure proteases viz. chymotrypsin, trypsin, subtilisin and one crude extracellular protease from Pseudomonas aeruginosa. Eighty percent of the isolates showed PI activity against at least one of the four proteases, majority of them belonged to genus Streptomyes. Actinobacterial diversity from two sites Ade (17°52' N, 73°04' E) and Harnai (17°48' N, 73°05' E) with varying degree of anthropological pressure showed that more putative species diversity was obtained from site with lower human intervention i.e Ade (Shannon's H 3.45) than from Harnai (Shannon's H 2.83), a site with more human intervention. Further, in Ade percentage of isolates not showing PI activity against any of the proteases was close to 21% and that in Harnai was close to 9%. Considering human activities in the coastal region might be contributing to increasing the organic load and in turn increasing the presence of extracellular enzymes in the intertidal environments it would be interesting to look at the association of PI production and organic load in these habitats.

Streptomyces have long been studied for their defense metabolites and Protease Inhibitors (PIs) they secrete for mediating biotic interactions. Current study focuses on diversity of extracellular PIs obtained from four marine Streptomyces species isolated from intertidal regions of West Coast of India. Streptomyces PIs showed strong inhibition of subtilisin followed by trypsin and chymotrypsin. Using shotgun proteomics approach, we identified 15 Streptomyces PIs belonging to 4 PI families namely Streptomyces subtilisin inhibitor (SSI), Potato peptidase inhibitor, Ovomucoid and Serpin. This is a first report of identification of Ovomucoid and Potato peptidase inhibitor families from Streptomyces. Amongst the 15 PIs, 12 were SSIs with 20-75% sequence similarity and variations in the conserved 73rd aa residue. Moreover, 10 SSI isoforms were co-expressed in a single species S. longispororuber. In-silico and in-vitro assays with proteases from microbial, bovine and insect sources suggested that the Streptomyces PIs had a broad-spectrum inhibitory activity. In co-culture of Streptomyces with other protease producing bacteria, cellular secretions differentially affected growth, sporulation and/or pigment production. Present study elucidates the diversity and plasticity of extracellular PIs from marine Streptomyces spp. Further, their role in chemical ecology of the bacterial communities as well as their potential applications in therapeutics are discussed.