Isolation , Identification and Molecular Screening of Pseudomonas sp . Metabolic Pathways NRPs and PKS Associated with the Red Sea Sponge , Hyrtios aff . Erectus , Egypt

Marine coastal areas of the Red sea in Egypt have great biodiversity of sponges which harbors a huge number of endosymbiotic bacteria which are the source of many potential bioactive metabolites and consider as an excellent source. The study focuses on the isolation of Pseudomonas sp associated with marine sponge Hyrtios aff. Erectus from the Red Sea in pure culture, phylogenetically identified and screening of their metabolic pathways polyketide synthases and Nonribosomal peptides (PSK and NRPs). Characterization of the bacterial produced bioactive metabolite was done through antioxidant and cytotoxicity assays using DPPH and cell line. The 16S rRNA gene sequencing of bacterial isolate revealed that the bacterial strain was closely related to Pseudomonas sp., and the active metabolic pathways genes in the Pseudomonas sp was PSK II. The antioxidants and cytotoxicity bioactivity of Pseudomonas sp as is most probably related to the presence of associated bioactive metabolic pathways genes related to polyketides family type II. Furthermore, the antioxidant activity of Pesudomance sp extract, performed by quantification of total phenolic content (TPC) and 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging activity antioxidant activities, were evaluated. The highest antioxidant activity was exhibited at different concentrations (50 mg-6.25 mg) which showed a potent antioxidant capacity by 100% at 50mg, 25 mg and 12.25 mg. From the cytotoxicity, antioxidant and bioactive metabolic screening pathways this bacterial strain was revealed to be an excellent source of natural antioxidant and cytotoxicity compounds against free radical and cancerous cell line.

Marine microbes are generally exposed to extremes conditions such pressure, temperature, salinity and nutrient availability (Kennedy et al., 2008, De Carvalho et al., 2010, Dalmaso et al., 2015).These harsh conditions possess highly diverse bacterial communities, raising the potentially unique biochemistry of marine microbial environmental habitats (Manivasagana, et al., 2013El Komy et al., 2015a,b, Abdel Monein et al., 2017).Marine invertebrates are known to be natural unique accumulators of specific microbial communities due to their filter-feeding habits.Additionally, they are an unusually rich source of bioactive natural products with medicinal and pharmaceutical potentials (Taylor et al., 2007, Hegazy et al., 2015 a,b).Furthermore, marine sponges harbor rich communities of symbiotic bacteria that have been reported as the true producers of sponge-derived compounds.Although these bacteria in most cases cannot be cultivated, culture-independent methods, such as isolating and analyzing biosynthetic gene clusters using metagenomic strategies, have recently provided the first insights into their chemical potential (Hochmuth and Piel, 2009).
A tremendous number of biologically active compounds were isolated from marine sponges and their associated microbes (Debbab et al., 2010, El Komy et al., 2015a,b, Abdel Monein et al., 2017).Blunt et al., (2013) reported that about 300 novel compounds were reported in 2011 from the phylum Porifera.Most of sponge-derived products are important with a unique chemically diverse entities and they possess different biological activity such ,as anti-inflammatory, antitumour, immunoor neurosuppressive, antiviral, antimalarial, antibiotic and antifouling (Imhoff et al., 2011).Which are, in fact, produced by their associated microbial communities (Wang, 2006;Khan et al., 2014).Marine bacteria, were under focuses by researchers as they are attractive because they can potentially produce compounds with unique biological properties (Maithili et al., 2014).Many bacterial isolates were obtained such as marine Streptomyces, Pseudomonas, Pseudoalteromonas, Bacillus, Vibrio, and Cytophaga were isolated from seawater, sediments, algae, and marine invertebrates are known to produce bioactive agents (Soliev et al., 2011).They are able to produce indole derivatives, alkaloids, polyenes, macrolides, peptides, and terpenoids.The marine world, due to its phenomenal biodiversity, is a rich natural resource of many biologically active compounds such as polyunsaturated fatty acids (PUFAs), sterols, proteins, polysaccharides, antioxidants and pigments ( Polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS) are multifunctional enzymes that are involved in the synthesis of a broad range of structurally diverse natural compounds, many of which are of medical importance (Hutchinson, 2003).Bacteria associated with marine sponge have been shown to produce a huge number of biologically active natural agents, including polyketides (Proksch et al., 2002).Also, many of bioactive compounds that have entered clinical and pre-clinical development are believed to be ultimately microbial in origin isolated from sponge (Kennedy et al., 2008).
Antioxidants are natural substances that may prevent or delay some types of cell damage.These molecules protect the cells from the damage caused by unstable molecules known as free radicals.Marine bacteria have not been examined for antioxidant compounds as extensively as terrestrial bacteria.It was reviewed that the bioactive metabolites extracted from seaweed as well as sponges and their associated bacteria had good antioxidant activity which needs extensive attention in terms of drug discovery (Balakrishnan et al., 2014).Marine sponges, seaweeds and their associated bacteria produce bioactive compounds which have been found to be important for health promotion and disease prevention.These beneficial effects can be attributed to the complex mixture of phytochemicals, which possess antioxidant, antimicrobial, anticancer and antiviral activity.The compounds responsible for these activities comprise phenolic compounds, sulphated polysaccharides and organic acids (Wijffels, 2008).The antioxidants generally produced by marine environment include polyphenolic compounds such as flavonoids, cinnamic acid, benzoic acid, gallic acid, phlorotannins and quercy (Al-Saif et al., 2014).
In the present study, bioactive strain (Pesudomance sp.) was isolated from a sponge specimen and identified by using a phylogenetic tree taxonomic approach.The bacterial strain was investigated for biosynthetic pathways of PSK and NRPs using PCR screening and sequencing.Moreover, the bioactivity of the bacterial strain was screened for their cytotoxicity and total antioxidant capacity using ethyl acetate extracts of bacterial strain against the hepatic cell line.

Sponge Sampling and Identification
Sponge samples were collected from the Red Sea at Hueghada City (Figure 1).The Red sea represents a lot of different environmental ecosystem with more than 10 thousands sea organisms.However, it started to suffer from several major threats (Shriadah et al., 2004 Shreadah et al., 2008aShreadah et al., , b,c,d, 2011Shreadah et al., , 2015)).Samples were collected by scuba diving, transferred directly to the laboratory in sterile polyethylene bags under reduced temperature (zero 0 C) for identification and microbial isolation.Voucher sample was preserved in 90% ethanol for taxonomic identification and deposited in Naturalis Biodiversity Center, Leiden using general external and internal morphological characters analysis, i.e.,shape, type, size and arrangement of skeletal Structures (spicules) following the System Porifera classification system.And further examination of additional samples, enable identification to species level this species was previously reported and identified as Hyrtios aff.Erectus (Figure 2) by Dr. Nicole Voogd, de with voucher number RMNH POR.8633 in the natural biodiversity center Leiden, Holland.

Isolation and maintaining culture media of bacteria from sponge
The isolation of bacteria from sponge has been done according to method of Santavy et al. (1990).Four different media were applied, namely marine agar media, nutrient agar, R2A and ISP2 agar ,all media were supplemented with 0.2 ìm pore size filtered cycloheximide (100 µg/mL), nystatin (25 ìg/mL) and nalidixic acid (25 ìg/mL) to facilitate the isolation of slow-growing strain.

Genomic DNA Extraction
Bacterial cells were collected by centrifugation at 20,000 rpm.Genomic DNA was extracted from 1.75ml of bacterial culture (QIAGEN, USA).

Phylogenetic analysis for bacterial strain
For bacterial identification, sequences were submitted to BLA-STN (National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov).The 16S rRNA gene sequences from each species and the matched sequences from GenBank were aligned using ClustalW2 similarity analysis of the nucleotides was performed by BLAST searches against sequences available in GenBank.For phylogenetic tree construction, multiple sequences were obtained from GenBank and the alignments were performed using MEGA 5 software version 5.1 (Dereeper et al, 2008, Tamura et al., 2011), also other analysis using geneious 7.0 were done for bacteria.
PCR products were amplified in each reaction and subjected to electrophoresis using 0.8% agarose gel.Then, each PCR fragment was purified using the QIAquick gel extraction kit (Qiagen).PCR fragments were sequenced by automated DNA sequencing based on enzymatic chain terminator technique developed by Sanger et al. (1977) using 3130X DNA Sequencer (Genetic Analyzer, Applied Biosystems, Hitachi, Japan).

Bioinformatics analysis
The draft genomes were submitted to secondary metabolite gene cluster analysis using PRISM (Skinnider et al.,2016) , Nap Dos (Ziemert etal.,2012) and for bioassay analysis we used pub chem Bioassay.

Cultivation, production and isolation of secondary metabolites from bacterial strain for screening purposes
The cultivation and production of secondary metabolites from Pseudomonas sp for in-vitro biochemical assays was done according to the methods of Al-Zereini et al., (2014).Liquid bacterial cultures were grown in 1 L erlenmeyer flasks containing 0.5L nutrient broth medium and incubated in shaking incubator at 102 rpm.Culture were homogenized and centrifuged by cooling centrifuge at 8000 rpm for 2 min at 4 o C. Resultant cell mass was extracted with EtOAC (3 x 50 ml), the organic fractions were combined, and the solvent removed at reduced pressure at 35 o C. Residues were re-dissolved in DMSO for further bioassay studies.

Prescreening bioassays using in-vitro cytotoxicity using cell lines
Different concentrations of Pesudomance marine extracts in ìg/ml were tested for hepatic cell line.Samples were dissolved in DMSO and further diluted with cell culture medium.The final DMSO concentration used was 1% of total volume of the medium in all treatments, including the control group.Cells with no treatment were examined as negative and positive controls, respectively (Kosaniae et al., 2015).

Determination of total phenolic content in pesudomance marine extracts
Total phenolic contents in the bacterial pesudomance extract was determined by the method of Taga et al. (1984).

The identification of bacterial strain associated with sponge using 16S rRNA Gene analysis identification
The identification of bacterial strain using 16SrRNA gene analysis using the BLAST search on NCBI resulted in Pseudomonas sp., based on the highest identy value, small gap value (0%), high score, bit and E-value (table 1).As the lower E-value provided the more similar the sequence and the more confidence the BLAST search result which are listed in tables 1 for bacterial strains and shown in phylogenetic tree of Figure ( 3 ) .

Polymerase chain reaction (PCR) screening and sequencing of nonribosomal peptide synthetases (NRPS) and polyketide synthases PKS-II gene fragments in bacteria and Bioinformatics bioactive screening
The Pseudomonas sp.screening to assess their biosynthetic potential pathways were listed and summarized in Table 2 & Fig. 4 and  5.The PCR screening was carried out for the presence of secondary metabolite genes encoding nonribosomal synthetase (NRPS) and polyketide synthases (PKSI&PSKII).The only primer give result was KSF& KSR primer for PKSII while other give -ve result under the same condition for PSK-I and NRPs.The bacterial strain studied was isolated from the Hyrtios aff sponge.Sponges were collected in the coastal area in the Red sea.The gene of 16S rRNA was amplified using the bacterial primers .The obtained sequences were translated in silico into protein sequences using the EMBL program (Hall.,1999) and compared to the published sequence data using the BLAST software of the NCBI site (Altschul et al.,1990).The nucleotide sequences of the 16S rRNA gene and the polyketide synthase KS genes were deposited in the GenBank database (KR269850.1,KU848194.1,AMQ36814).Based on the 16S rRNA gene sequence (~998 bp long), strain KR269850.1,was identified as a Pseudomonas strain; one sequence fragments of ketosynthase (KS) domain genes of the PKS gene cluster were identified in its genome.The amino acid sequences obtained by the in silico translation showed 39% homology.The BLAST analysis of these sequences revealed their similarity to modular KS domains of different bacteria, including known producers of biologically active metabolites and unidentified strains.Their closest homologues, which were identified by a BLAST search, are listed in the Table ( 2 ).It was found that KS1_PF and KS2_PF were similar to the KS domains of gene clusters of (uncultured bacterium) Sequence ID: AGG47970.1.the screening of bioactivity using secondary metaboliters softwear using PRISM , PubMed Chem Bioassay and NaPDoSR resulted in variety of bioactive domain are listed in Tables (3,4 and 5).

Bioactivity of the Pseudomonas Sp. extract
Cytotoxicity of Pseudomonas Sp. extracts against hepatocellular carcinoma cells Inhibitory activity against Hepatocellular carcinoma was detected and the IC 50 was 31.6 µg.

The total phenolic and total antioxidant capacity
The Pseudomonas extract using ethyl acetate showed higher total phenolic content than water extract as shown in Figure (7).where the levels of total antioxidant capacity in different Psudomance extract concentration have been done using DPPH inhibition.The data in Figure (8) showed the inhibitory effect of Pseudomonas extract at all concentration have been shown potent antioxidant.Where the total phenolic content in ethyl acetate extract showed higher content than water extract as shown in Figure (7).

DISCUSSION
M a r i n e n a t u r a l p r o d u c t s a r e valuable sources that could produce potential chemotherapeutic agents; marine derived organisms are widely recognized as important sources of these natural products with interesting biological activities (Graça et al., 2015).Red sea was recognized as a rich source of microbial diversity with unique metabolites that can be of pharmaceutical and medicinal importance.The discovery of novel active metabolites provides an intellectual challenge to examine new products for medicinal purposes (Nadeem et al., 2015).
The sponge-associated microorganisms have revealed high densities, amounting for some sponge species up to 37 % of the sponge biomass, which exceeded seawater concentrations by twothird orders of magnitude.More than 30 different phyla have so far been recognized in the domain Bacteria and Archaea as being associated with sponges ( Thomas et al.,2010).Environmental stressors such as metal pollution, elevated seawater temperature or other climate changes and/or diseases are speculated as having a significant impact both on the symbiotic microbial community and on the health of sponges (Olson and Kellogg, 2010) .Some of isolated bacteria have been specifically associated with marine sponges and are absent from the immediate surrounding seawater, reinforcing the concept of sponge specific microbes (Donia et al., 2003).
In the present study, bacterial strain was identified with potent antioxidant bioactivity as well as high total phenolic content (Figs. 7 and 8) and cytotoxicity against hepatocellular carcinoma cells (Fig. 6).Our result with agreement with many previous studies (Balakrishnan et al.,20104, Horta, et al., 2014, Velho-Pereira et al., 2015, Shindo and Misawa, 2014) which confirmed that the sponge associated bacteria have high bioactive compounds with potent antioxidant and anticancer activity, the bacterial strain which isolated from sponge in our study identified as Psudomance sp in agreement with previous studies (Graça et al.,2015) ,the molecular identification of Pseudomonas sp (GeneBankKR269850) was applied using the partial sequence of 16S rRNA.The bacterial strain was alignment with 23 Pseudomonas sp strains (Fig4).The tree topologies were evaluated by bootstrap analyses (Felsenstein, 1985) and phylogenetic trees were inferred using the neighbor-joining method (Saitou and Nei., 1987).The molecular techniques identification depend on the application of PCR that allowed the analysis of small numbers or even single spores and the selection of universal oligonucleotide primers specific to fungi and bacteria.This is facilitated and made the access to nucleotide sequences much easy (Guarro et al., 1999).In the bacterial strain Pseudomonas sp, Gram-negative, aerobic, and non-pigmented,using partial sequence analysis of the 16S rRNA gene showed a high percentage of similarity (96%) to the corresponding sequences of the species Pseudomonas sp.The lack of amplification of NRPs and PSK I genes in bacterial strain pesudomance might indicate their absence or that they were less conserved, hence low homology with the primers.On the other hand, the results of the present study revealing the negative result for NPRS and PKS I genes, although the bacterial strain showed highly bioactivity, suggests that the activities detected possibility suggests that these detected genes may be silent, i.e. nonfunctional (Hutchinson, 2003) and/or the products of these genes may be involved in primary metabolism (Pathom-Aree et al., 2006).Another possible explanation is the fermentation conditions used were not optimal for natural products production.
The present result of the identification of gene clusters potentially encoding secondary metabolites mined using bioinformatics tools, namely PRISM, NapDos, and pub chem Bioassay [Ziemert .,2012, Prlic et al.,2012 ].We found a genetic potential of secondary metabolite production in our sequence as many paper reported that the genome Bioactivity potential NRPS and or /PKS is richer and more spread among Gram-negative marine bacteria with genome Bioactivity potential -NRPS/PKS than Alphaand Gammaproteobacteria (Machado et al.,2015) .Although our collection of secondary metabolites screening using bioinformatics tools is limited in number.Here, we show that using bioinformatics tools a few clusters could be identified, but still the bioactive potential harbored in the genome of these genera appears much lower than that observed in Gammaproteobacteria ( Machado et al.,2015).
The bioactivity of Psudomance sp in the present study as potent antioxidant and cytotoxicity against cancerous cell line can be attributed to the high diversity of marine environmental which lead to great chemidoversty resulted in potent drug candidate (Fenical, 1988).The use of marine natural products as therapeutic and cure tools has been started since 1997 with many discoveries (Fautin, 1988, Cragg and Newman, 2013, Lahlou, 2013).The Psudomance extract was screened against human cancer cell line using the MTT cytotoxicity assay (Fig. 6).The study revealed that marine derived bacteria from the Red sea, especially those belong to genus Psudomance are rich source of structurally novel and biologically potent natural products (Nadeem et al., 2016).In agreement with recent studies which have shown that many microbial extracts from the Red sea displayed significant cytotoxic and apoptotic activity (Blunt et al., 2013, Cragg and Newman, 2013, Chen et al., 2014, Nadeem et al.,2016).Also many paper reported that marine bacteria develop unique metabolic and physiological capabilities, which enable them to survive in extreme habitats and to produce compounds that might not be produced by their terrestrial counterparts which explained potent antioxidant capacity of marine bacteria (Debbab et al.,2010, Kijjoa et al.,2004) another explanation for the highly and unique bioactive compounds from marine bacteria associated with sponge isolated from Red sea was the highly biodiversity of organisms habitat Red sea (Mohamed et al., 2015).As the deep-sea anoxic brines of the Red sea are one of the most extreme environments on earth in comparison to overlying seawater (Elica et al., 2012, Adelmoniem et al., 2013, Mohamed et al., 2015).The anoxic brines are characterized by high salinity (increased from 4% up to 26%), high temperature (up to 70°C), increased concentration of heavy metals, and decrease in O2 levels.Therefore, marine-derived microorganisms have the ability to tolerate and survive in these extreme conditions, since they are uniquely equipped with some self-defence systems.Red Sea microbial diversity (bacteria/fungi) is interesting objects for further studies to discover novel metabolites and new chemotherapeutic agents ( Nadeem et al.,2016).

CONCLUSION
In conclusion the present study confirmed the highly significance of bacterial associated sponges as reservoir of potent therapeutic and tremendous agents especially as antioxidant .it is consider as the first study in Egypt for our knowledge combine the bioactivity and screening of bioactive metabolic pathway gene as today sponge-associated microbial communities are considered to be responsible for the biosynthesis of many of these agents (anticancer, antioxidant, ant diabetic).

Fig. 1 .
Fig. 1.The Red Sea map showing the sampling area

Fig. 3 . 1 20
Fig. 3.The tree showing the phylogenetic diversity of pseudomonas sp with accession number KR269850 Neighbor-Joining (NJ) tree built with MEGA 5.1 based on partial 16S rRNA gene sequences of 23 strains belonging to the pseudomonas sp and the type strains of closely related genera.The numbers at the nodes indicate bootstrap support (%) based on a NJ analysis of 1.000 replicates; only values e"75% are given.The scale bar indicates 0.5

Fig. 4 .Fig. 5 .
Fig. 4. Phylogenetic diversity Pseudomonas Sp.Neighbor-Joining (NJ) tree based on partial 16S rRNA gene sequences of 17 sequence belonging Biosynthetic gene sequences detected: polyketide synthases PKS-II.The numbers at the nodes indicate bootstrap support (%) based on a NJ analysis of 1.000 replicates; only values e"75% are given.The scale bar indicates 0.003 substitutions per nucleotide position

Fig. 6 .Fig. 7 .
Fig. 6.The Inhibitory activity of Pseudomonas sp extracts against hepatocellular carcinoma cells (Fig 4 ) in agreement with many papers isolated Pseudomonas sp from sponge (Wang et al., 2014, Ye et al., 2015).Marine environments and their organisms represent an important source for discovery of new potential active and biologically functional natural products, such as compounds that exhibit anticancer, anti-inflammatory, antiviral and ant-toxic activities (Newman and Cragg, 2010).The purpose of genomic screening work was to screen bacterial isolate Pesudomance sp. which isolated from marine sponge, collected from Red Sea, for potential secondary metabolic production (Ehrenreich et al., 2005).The amplification of genes of PKS-I, PKS-II and NRPS from bacteria were detected using primer (K1f, M6R), (KSá, KSâ) and (A3F, A7R) for PKS-I, PKS-II and NRPS; respectively, only PKSII displayed a positive result.The present results are in agreement with Schirmer et al., 2005 who reported that sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides.The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules).According to Schirmer et al., (2005) report, the PSK is the most abundant large modular encoded by symbiotic bacteria and that indicate as well as with agreement with our result which confirm this finding as the screening for genomic biosynthetic pathways PKS and NRPs, resulting in a positive result with PSK.

Table 1 .
The identification of Pseudomonas sp using BLAST

Table 2 .
The BLAST result for Pseudomonas bacterial PSK II gene

Table 3 .
The Bioinformatics analysis of bioactive screening using PRISM program for DNA nucleotide sequence of KU848194.1|Pseudomonas sp.Nabil

Table 5 .
The result of screening of secondary metabolites using NaPDoSR program