Cultivable Bacterial Symbionts from the Marine Sponge with Biological Activity

Marine sponges are envisaged as microbial diversity hotspots,
as they harbor enormous diversity of microorganisms...


Introduction
Marine sponges are envisaged as microbial diversity hotspots, as they harbor enormous diversity of microorganisms. Symbiotic microbes have recently been demonstrated as the site of biosynthesis of sponge-derived metabolites with pharmaceutical activity. The diversity of microorganisms inside these evolutionarily oldest animals covers most bacterial phyla. Sponges maintain a unique relationship with the microbes which constitutes up to 40-60% of their biomass [1]. These diverse microbes enter the sponge body through their filter-feeding habit, and some of them are ingested while others reside inside the sponge mesohyl as symbionts. These symbionts play significant roles in the nutrition, health, and chemical defense of sponges, and the symbionts, in turn, benefit from nutrients and protection by the sponge host [2]. Approximately 0.1% of the bacterial species are known until now [3] and approximately 32 bacterial phyla and candidate phyla have been found in sponges [4]. This clearly illustrates the great microbial diversity left unexplored which may serve as a good source of bioactive compounds. The compounds from marine symbiotic microbes have been found to possess varied biological activities as several metabolites with antibacterial, antioxidant, immunosuppressive, antihypertensive, hypocholesterolemic,

Morphological, Biochemical and Physiological Characteristics of the Symbiotic Bacteria
The pure colonies of the two selected isolates were carefully observed for identifying their morphology. Standard protocols were used for determining motility, gram staining, and various biochemical characteristics such as catalase and oxidase activities, nitrate reduction, etc. An extensive range of biochemical properties was analyzed by using the VITEX2 system. Physiological characterization of the two strains was done after adjusting the absorbance of the broth cultures to 1OD and various parameters such as different concentrations of NaCl, various temperatures, pH, and varying seawater concentrations were evaluated.
PCR was carried out as described by Chun and Goodfellow [8].
The amplification program consisted of an initial denaturation step of 94ºC for 3 min, and 30 cycles of reaction with each cycle of 94ºC for 30s, 58ºC for 30s, 72ºC for 2min, and a final extension step of 72ºC for 7minutes. PCR product was checked by agarose gel electrophoresis, amplified DNA fragments were observed and sequencing was performed. The unknown bacterium was identified using the Genbank database. The obtained sequences were analyzed using the BLAST tool to get the relative identification of each bacterial species [9] and the culture sequences were deposited in Genbank through NCBI and obtained accession numbers.

Preparation of Microbial Extract
The isolates were inoculated into sterile Zobell marine broth and kept at 25°C for 3 days. Turbidity was seen during the incubation period. 1mL (~4%) of inoculum of three-day-old cultures were transferred to 250mL marine broth and kept at 25°C for 7 days. The cultures were then centrifuged at 10000 rpm for 30 minutes and the supernatant was collected. The collected supernatants were lyophilized and the solvent ethyl acetate was added in the ratio 1:1 to the supernatant taken in a separating funnel and extracted for three days. The extracts were then concentrated by evaporating the solvent in a rotary evaporator. The dried extracts were stored at -20°C.

Screening of Chemical Constituents
The ethyl acetate extracts of the symbiotic microbial isolates were screened for chemical constituents with slight modifications of the method (Table 1) of Harborne [10]. Purple color.

4.
Test for alkaloids: The cold extract was mixed with acetic acid (aqueous layer decanted) and followed by the addition of a few drops of Dragendofff's reagent.

5.
Test for phenolics: To the cold extract, neutral FeCl 3 was added.
An intense blue/ violet color 6.
Test for xanthoprotein: To the cold extract, 1N Con.HNO 3 was added followed by excess ammonia Red-orange precipitate 7.
Test for aromatic acids: To the cold extract, saturated NaHCO 3 was added.

Brisk effervescence
Test for flavonoids: 1-2mg of Mg 2+ was added to the cold extract, followed by 0.5mL of the con. HCl Red/orange color

Antioxidant Activity Assay
The organic extracts of the symbiotic microbial extracts were subjected to the following antioxidant assays.

DPPH Radical Scavenging Activity Assay
The stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the microbial extracts was determined using the method of Mensor et al. [11]. The samples and the reference at differing concentrations of 62.5µg/mL, 125µg/mL, 250µg/mL, 500µg/mL and 1000µg/mL were dissolved in methanol were then mixed with the DPPH solution. The remaining DPPH amount was measured at 517nm using a spectrophotometer. Ascorbic acid was employed as the reference standard. Inhibition of DPPH in percentage was calculated as given below: where A b is the absorbance of the control reaction and A s is the absorbance of the extracts.

Nitric Oxide Scavenging Assay
This assay was performed according to the protocol of Rana et al. [12]. 10mM Sodium nitroprusside (2mL) in phosphate buffer saline was incubated with the test compounds at different concentrations for 30 minutes at room temperature. 0.5mL of the incubated solution was then mixed with mL of Griess reagent and the absorbance was measured at 546nm. Ascorbic acid was used as the reference standard. The radical scavenging activity was calculated as per the equation: where A 0 is the blank and A 1 is the test sample.

Acetylcholinesterase Inhibition Assay
The acetylcholinesterase inhibition activity was performed for the extracts of the sponge symbiotic bacteria at varying  The time course of the enzymatic reaction was monitored for 12 minutes at 412nm at 25°C.

Antiproliferative Activity
The ethyl acetate extracts of the two isolates were subjected where Ac is OD of Control and At is OD of Test.

Statistical Analysis
The

GC-MS Analysis
The ethyl acetate extracts of the two isolates were subjected to GCMS analysis. Analyses were carried out on an Agilent GC 7890A MS575C fitted with DB-SMS 30x0.25mmx0.25µm using Helium as the carrier gas. The injective temperature was set at 250°C. The column temperature was initially kept at 40°C for 5 min, then gradually increased to 250°C at a rate of 5°C/min and finally raised to 280°C hold for 10 minutes. The identification of the compounds was done with the help of the NIST library.

Morphological, Biochemical and Physiological Characteristics of the Symbiotic Bacteria
The two isolates-MBTU SOLY1 and MBTU SOBOP1, obtained from the sponge host Spongia officinalis var ceylonensis were identified by observing their culture characteristics, microscopic observation, biochemical identification, and molecular identification. MBTU SOLY1 was a yellow, small, entire, opaque, round, and smooth colony whereas MBTU SOBOP1 was an orange, small, round, entire, flat, and smooth colony. Both the isolates were gram-positive, non-motile cocci. The biochemical characterization of the isolates obtained by both manual and The physiological characterization of the isolates revealed that the optimum temperature for both the cultures was at 25°C. They were capable of surviving till 55°C which indicates the thermophilic nature of the isolates, but the growth was found to decrease with temperature. The optimum pH was 8 for both the cultures, but the growth of both the isolates got inhibited at a lower pH range.
The optimum NaCl concentration was 7% for MBTU SOLY1 and 4% for MBTU SOBOP1 and these isolates could survive up to 30% NaCl concentration. The Zobell medium in 50% seawater and 2% NaCl concentration was found to be the suitable medium for the optimum growth of the selected isolates on monitoring the growth of these isolates in different media such as Zobell media in 50% seawater, Zobell media in 100% seawater, Zobell media in distilled water, Zobell media in 50% seawater and 2% NaCl concentration Zobell media in distilled water and 2% NaCl concentration.

Phylogenetic Identification of the Isolates
The sequencing results confirmed that MBTU SOLY1 belonged to Micrococcus luteus sp. (Figure 1) and MBTU SOBOP1 belonged to Planococcus sp. (Figure 2). Both the sequences were deposited in Genbank through NCBI and obtained accession numbers as KT734853 and KX236450 respectively.

Screening of Chemical Constituents
The supernatants of both the isolates were lyophilized and were subjected to extraction using the solvent ethyl acetate. The chemical constituent analysis (Table 3) (Figure 3).

Acetylcholinesterase Inhibitory Activity
Anticholinesterases cause the accumulation of acetylcholine at their sites of action thus stimulating the parasympathetic nervous system-slowing heart action, lowering blood pressure, increasing secretion, and inducing contraction of smooth muscles. In this study, AChE inhibitory activity ( Figure 5) was greater for MBTU SOLY1 extracts with an IC 50 value of 530µg (Table 5).

Figure 5:
Acetylcholinesterase inhibitory activity of microbial extracts-Error bars in graphs represent standard error of means per triplicate samples.

Antiproliferative Activity
MTT assay for ethyl acetate extracts of microbes was performed against three cancer cell lines-HT-29, MCF-7, and HeLa cell lines.
Their activities were concentration-dependent and the viability of the cells decreased with the increasing concentrations of both the extracts. Against all the three tested cell lines, the extract of MBTU SOLY1 was more active ( Figure 6 and Table 6). Among these three   These compounds are reported to have antibacterial, antitumor, antioxidant activities (Table 9). This proves the symbiotic association of sponge with these microbes.     Table 9: The activity of chemicals in the extract.
These microbes produce various secondary metabolites with the prospect of developing into useful drugs. Numerous compounds have been reported from marine sponges and many of them are believed to be the products of symbiotic microbes in them. In this study, the two gram-positive halophiles-MBTU SOLY1 and MBTU SOBOP1 isolated from the marine sponge Spongia officinalis var.
ceylonensis were identified as Micrococcus luteus and Planococcus sp. respectively. This is the first report of isolating Micrococcus and Planococcus from the marine sponge Spongia officinalis var. ceylonensis from the Indian coast. The Micrococcus was isolated from various other marine sponges [14][15][16][17] and the Planococcus sp.
was isolated from the marine sponge, Plakortis simplex [18]. Li [19] reviewed that the Micrococcus sp. has been previously isolated from were reported to produce the bioactive triterpene, squalene whose primary source is liver oil from deep-sea sharks and whales [37].
The symbiotic microbes produce these bioactive compounds inside the sponges as it is in the favorable physical environment for the induction of its secondary metabolism. But, the metabolic profile of these microbes changes rapidly once they are devoid of studies of these isolates will yield potential drug candidates [38][39][40][41][42][43][44][45].