Bioac vity and gene c screening of marine ac nobacteria associated with red algae Gelidiella acerosa

Bacterial resistance to exis ng an bio cs has driven a search for new an bio cs from marine ac nobacteria. Bioac vity and gene c screening of ac nobacteria associated with red algae Gelidiella acerosa were conducted to discover new an bacterial compounds against Vibrio alginoly cus. A total of 14 ac nobacteria isolates were obtained from G. acerosa. The isolates were subjected to gene c screening for nrps (non-ribosomal pep de synthetase) and FADH2-dependent halogenase genes. The isolates’ ability to produce secondary metabolites was examined by fermenta on in various media in a six-well mini plate. The bioac vity of the secondary metabolites was screened using a micro ter assay and the agar overlay method. The results showed that all 14 isolates had the nrps gene, whereas none had the halogenase gene. Meanwhile, eight of the ac nobacteria isolates showed an bacterial ac vity against V. alginoly cus.

Antibiotics play a crucial role in treating bacterial infections in both humans and animals.Overuse and inappropriate prescribing of antibiotics contribute to problems antibiotic resistance and emergence of new pathogenic bacteria (Luyt et al. 2014).One of the pathogenic bacteria in marine aquaculture and human disease is Vibrio sp.(Austin 2010).Vibrio spp.are commonly present in seawater, sediments and occupy aquatic environments at temperatures ranging from 10 to 30°C and contaminate seafood.Available antibiotic was limited to combat the infection in human (Shaw et al. 2014).New antibiotics is necessary for those cases and due to limited shelf-life of antibiotics (Kasanah and Hamann 2004).In this study Vibrio alginolyticus was used as test strain because V. alginolyticus has high potential as reservoirs of disease-carrying genes in other Vibrio sp. and cause gastroenteritis and peritonitis due to consumption of seafood contaminated by these bacteria (Gómez-León et al. 2005;Mustapha et al. 2013).
The Marine environment is a complex and unique ecosystem characterized by high salinity and pressure, low temperatures and variable oxygen concentrations (Bull et al. 2000).A marine ecosystem consists of huge variety forms of life that allow interactions and associations between themselves.The most frequent associations are between eukaryotic cells and microorganisms (Egan et al. 2008).Many bioactive compounds such as antibiotics, antitumor, antifouling or antioxidant from the marine microorganism are due to the involvement of specific function in their respective hosts (El-Shatoury et al. 2009).
Red algae, the most potential sources of unique secondary metabolites compared with green and brown algae (Schwartsmann et al. 2001).More than 1400 secondary metabolites were successfully isolated from red algae from 1963-2016(Blunt et al. 2014)).Red algae live as host for diverse and abundant symbiotic microorganisms.These bacteria protect the host from a predator or others harmful things in pelagic areas (Singh and Reddy 2014) and produce important compounds for human and animal health (Kanagasabhapathy et al. 2006;Penesyan et al. 2010).Various novel compounds with biological activity have already been identified from seaweedassociated bacteria.Streptomyces sp.YM5-799 associated brown algae Analipus japonicus produced Streptobactin as an iron chelating activity for treatment human disease (Matsuo et al. 2011).Bacterial strain member of the genus Alteromonas was associated with the red algae Laurencia pacifica produced a high molecular weight of polysaccharide and used for industrial application (Soria-Mercado et al. 2012).Gelidiella acerosa is a potential source of bioactive compounds.The research about sec- ondary metabolites of associated microorganism of Gelidiella acerosa was unexplored.Some studies have demonstrated the presence of antibacterial activity from Gelidiella acerosa but not the associated microorganism.A study reported that ethanol extract of Gelidiella acerosa has antibacterial activity against Staphylococcus aureus (Elsie et al. 2011).
Marine bacteria as associated microorganisms are possible to participate in the biosynthesis of bioactive compound.The use of microorganism is more effective as a source of bioactive compound than its host due to the short life cycle of actinobacteria and the ability to be cultured in vitro (Piel et al. 2004).Approximately 80% of antibiotics have been found mainly produced by actinobacteria especially genus Streptomyces and Micromonospora (Naikpatil and Rathod 2011).Actinobateria continue to play a significant role in drug discovery and development.Bioactive compounds produced by marine actinobacteria were abysomicin C, diazepinomicin, and salinoporamide A (Lam 2006).Because of the excellent record of actinobacteria, a significant effort has been focused on the isolation of novel actinobacteria from marine for developing drug discovery especially antibiotics.
A new strategy for selecting potential actinobacteria is through genetic approach by detecting gene target involved in the biosynthesis of bioactive compounds.These bioactive compounds can be the result of the activity of non-ribosomal peptide synthetases (NRPS) and FADH 2dependent halogenase.NRPS is multimodular enzyme consist of three domains, A domain (Adenylation domain) serves as an introduction and activation substrate, domain PCP (Peptidyl Carrier Protein) for transfer to the center catalyst and the C domain (Condensation domain) is connecting peptide bonds to form the final product (Schwarzer and Marahiel 2001).FADH 2 -dependent halogenase as tailoring enzyme catalyzes genes encoding halogenated compounds, this gene is also part of the domain of NRPS gene cluster decisive one functional group of compound and determine the bioactivity (Hur et al. 2012).These gene encode a successful formation of antibiotics compounds such as chloramphenicol, vancomycin, and teicoplanin (Pée and Zehner 2003).Therefore, the objective of this study was to select the potential actinobacteria associated red algae Gelidiella acerosa through genetic screening of nrps and hal genes, and to examine antibacterial activity against V. alginolyticus.

Algae collec on and prepara on
Red algae was collected from Drini Gunungkidul Yogyakarta Indonesia on 26th January 2015.Sample was identified as Gelidiella acerosa.Sample was rinsed with sterile fresh water, transferred in a sterile zip-lock bag and transported to the laboratory.One g of Gelidiella acerosa sample was aseptically cut into small pieces and placed in a sterile mortar with 1 mL of sterile water.Decimal dilutions were made up to 10-3 and plated on different isolation media.

UV visualiza on of PCR products
The PCR products were observed on agarose gels, containing 30 mL TAE 1x and 0.75 μL RedSafe was added for electrophoresis staining then visualized on a UV transilluminator.This process was aimed to ensure that a fragment of the correct size had been amplified.

Fermenta on and extrac on of metabolites
Production of secondary metabolites of actinobacteria was assessed using 6 well plate.Actinobacteria were inoculated in various media and incubated at 28 °C for 6 days.

An bacterial bioassay of 96 well plate
The antimicrobial activity of actinobacteria was assessed using 96 well plate.V. alginolyticus was used as test strain for antibacterial activity.V. alginolyticus was incubated in TSB saline media for 18 h.A final concentration of 5 x 10 8 cfu/mL was adopted for this assay.A volume of 20 µL of fermentation product of actinobacteria, TSB saline media, and V. alginolyticus was pipetted into each well.The first row of the plate consist of positive control cyprofloxacin and amphicillin at a dose of 1mg/mL, TSB media and V. alginolyticus as negative control.The plate was incubated at 37°C for 16-20 h.After incubation, 10 µL resazurin was diluted as indicator then gently shake the plate.The color change was observed then assessed visually.Any color changes from purple to pink or colourless were recorded as negative bioactivity.

An bacterial overlay agar
In this study, the antimicrobial activity also conducted using overlayed agar in 6 well plate.V. alginolyticus as test strain with the OD of 0.3 was inoculated in TSA media (0.8% agar) and incubated at 37°C for 16-18 h.Total of 1 mL V. alginolyticus were overlayed on each plate of 7 days cultured actinobacteria and incubated for 16-18 h.After incubation, 25 mL resazurin was diluted as indicator then gently shake the plate until resazurin cover the entire surface.The color change was observed visually.

Quick detec on of pep de
Peptide as representative compound of product NRPS gene was detected using on TLC plate and visualization by ninhydrin reagent.Ninhydrin reagent consist of 0.2 g ninhydrin in 100 mL ethanol (Zarzycki 2015).A total of 10 μL product fermentation of actinobacteria was spotted on TLC plate and immersed in ninhydrin reagent.Plate of TLC was heated at 110°C and visually observed the appearance of spots.Peptide was characterized by the presence of pink, purple, until yellow spot on the TLC plate depends on variety aminoacids (Gibbons 2006).

Sequencing and BLAST sequence analysis
Product of PCR was cleaned using GenElute ™ Gel Extraction Kit (Sigma-Aldrich).Product were sequenced by 1 st BASE Laboratories Malaysia services.Sequencing was performed using BigDye® Terminator v3.1 Cycle Sequencing Kit protocol.Technically sequencing performed using the same forward and reverse primer during PCR.DNA sequencing was carried out to determine the percent genotype similarity of isolates by 16S rRNA and nrps gene.The sequences were compared to some available DNA sequences in GenBank (https://www.ncbi.nlm.nih.gov/genbank/).Sequences of nrps were translated using the Sequence Manipulation Suite (http://www.bioinformatics.org/sms2/)and compared to sequences within the NCBI protein database using BLASTp.Closely related 16S rRNA and nrps gene sequences were retrived from GenBank database to make phylogenetic trees.The phylogenetic trees were contructed using MEGA7 by neighbor-joining algorithm and a bootstrap consisting of 1,000 replications.

Isola on ac nobacteria from Gelidiella acerosa
A total of 15 actinobacteria were isolated from the red algae Gelidiella acerosa collected from Drini Gunungkidul Yogyakarta.Three isolates were grown in AIA media, four isolates in SCSW media, three isolates in NB media, five isolates in MA media, and none of isolate grow in WAT-SW media.
Actinobacteria grow as branching hyphae on the agar surface, and mycelium form is prominent.Colonies grown on each media has a different morphology.Actinobacteria is able to producing brown, pink, gray, orange, purple, red, or white pigment (Holt 1994).Figure 1 showed an example one of isolate actinobacteria morphology white morphology spore-forming.

Amplifica on of 16S rRNA gene
The 16S rRNA gene was amplified using special set primers for actinobacteria ActF/ActR (640bp) for amplifying the 16S rRNA gene in actinobacteria (Stach et al. 2003).Based on 16S rRNA actinobacteria gene amplification, 14 isolates confirmed as actinobacteria as presented in Figure 2.

Screening of FADH 2 -dependent halogenase gene
Genetic screening of FADH 2 -dependent halogenase gene was carried out using primers Halo-B4-FW and Halo-B7-RV (Hornung et al. 2007).Figure 3 shows four isolates had 1000 bp as PCR product, while the correct size of FADH 2 -dependent halogenase gene targeted was 550 bp.Based on BLAST analysis in NCBI database revealed the 1000 bp sequences has no similarity with any halogenase genes.This concludes that none of actinobacteria in this study contain FADH 2 -dependent halogenase.

Screening of NRPS gene (non-ribosomal pep de synthase)
Detection of NRPS gene was carried out using set primers A3F/A7R.All of the templates were amplified at 700bp (100%) as represent in Figure 4.This results show presence of NRPS gene is dominant.Ayuso-Sacido and Genilloud ( 2005) screened 210 isolates of actinobacteria and obtained 167 have NRPS gene (79.5%).

Bioassay an vibrio
Bioassay anti-vibrio was performed using 96 well plate diluted bioassay.This assay was adopted from Gibbons (2006) with slight modifications.Test material used for this assay was fermentation extract of actinobacteria.The changing color from purple to pink or colourless is due to metabolism of bacteria means that the metabolites were inactive.While the absence of color changing means that no bacterial metabolism because the compounds killed the bacteria (Figure 5).About eight of isolates actinobaceria showed inhibition against V. alginolyticus.Bioassay antivibrio also performed in overlay agar using 6 well plate in 6 different media fermentation.Actinobacteria produced secondary metabolites intracellular within cells while extracellular secondary metabolites directly secreted out of the cell and diffused to media.It will determine the purple pattern on antibacterial bioassay.Intracellular secondary metabolites was showed on purple FIGURE 4 Visualiza on of NRPS gene representa ve selec ve amplifica on of 700 bp fragments using primers A3F and A7R in 1% (w/v) agarose gel stained with RedSafe.pattern at isolates grown area while the purple pattern of extracellular secondary metabolites was showed around media as represented in Figure 6.

Quick detec on of pep de
Based on detection with the use of ninhydrin as respective visualizing agent showed a total 54 of 84 product fermentation of actinobacteria were contained peptide as a bioactive compound.The appearance of pink, purple, or yellow spot on plate showed the compound contained peptide with different aminoacid (Figure 7).The pink spot presented the presence of phenylalanine, the purple spot showed the presence of tryptophan, and the yellow spot showed the presence of proline (Gibbons 2006;Zarzycki 2015).

Discussion
NRPS are large multimodular enzymes involved in biosynthesis of peptide secondary metabolites produced by microorganisms, such as bacteria (Felnagle et al. 2008).Some of antibiotics such as vancomycin and teicoplanin have exquisitely complex structures catalysed by NRPS (Pace and Yang 2006).Indeed, almost all antibiotic peptide are catalysed by NRPS.The presence of nrps gene in isolates can be proved by molecular screening and the expression of this gene in producing peptide should be checked through chemical screening.
NRPS and FADH 2 -dependent halogenase are enzymes involved in the biosynthesis of important biological compounds produced by microorganism including acti-  nobacteria.A band about 700 bp length was detected in 14 isolates by PCR screening of NRPS gene.The high detection levels of NRPS biosynthetic systems observed in our isolates confirmed the wide distribution of these sequences in this bacterial group.While detection of FADH 2 -dependent halogenase gene was obtained a band about 1000 bp length.Hornung et al. (2007) mentioned the precise length of this gene was about 550 bp.Analysis of this 1000 bp sequences in NCBI database confirmed this sequences has no similarity with any halogenase genes.In this research none of FADH 2 -dependent halogenase gene was detected.Existence of this gene as auxiliary gene of secondary metabolites biosynthesis is rare or even absent.The presence of FADH 2 -dependent halogenase gene in all observed isolates was only 4% compared with existence of NRPS and PKS gene as the backbone of biosynthetis secondary metabolites (Hur et al. 2012).
Bioactivity screening was conducted to the fermentation product of actinobacteria that active as antibacterial agent.Fermentation was asessed by growing actinobacteria in various media to creating diverse environmental conditions.One strain many compound (OSMAC) show one strain of bacteria can produce diverse compounds depending on environmental conditions (Bode et al. 2002).A variety compounds are produced during actinobacteria fermentation and due to the nutrient limitation for the defense mechanisms.
Resazurin is an oxidation-reduction indicator used for the evaluation of bacterial growth (McNicholl et al. 2007).It is a blue dye that becomes pink when reduced to resofurin by oxidoreductases Gram negative bacteria (Leonard et al. 2008).Resofurin is further reduced to hydroresofurin (uncolored).The absence of indicator change color indicate that the fermentation product of actinobacteria was able to inhibit the growth of V. alginolyticus.The change color become pink or colorless showed that the fermentation product of actinobacteria failure to inhibit the test strain and the cells of V. alginolyticus still remain on the well.
Identification of peptide is important to assure that nrps gene was expressed under certain condition.Chemical screening on TLC plate was an easy and rapid approach that require specific reagents to visualize the isolates capability to produce active secondary metabolites.Due to the existence of -NH-CO-groups in peptides, ninhydrin has greater ability to detect peptides.The reaction between amino acids and ninhydrin produces reddish color as represented of most amino acids except proline and hydroxyproline produce yellow color.The result of chemical screening revealed that almost all isolates produced secondary metabolites containing peptide.The genetic, chemical, and bioactivity result of 14 isolates actinobacteria was showed in Figure 8.There are 47% isolates have nrps gene, produce peptide and active against V. alginolyticus, while the same number of isolates have nrps genes, produce peptide but inactive against V. alginolyticus.Only 6% isolates have nrps gene, no peptide but active against V. alginolyticus.
In this study, we identify one potential isolate of actinobacteria.Based on sequences analysis of 16S rRNA gene sequences of isolates DR-2S-115-5 has similarity to Nocardiopsis alba PCM 2702 (97%).Nocardiopsis species are able to prevail under different environmental conditions mainly because of their versatile genetic make-up, secretion of enzymes, production of compatible solutes and surfactants (Li et al. 2013;Bennur et    2014).In addition to these features, they produce an array of bioactive compounds that may help their survival under these conditions.Nocardiopsis has been isolated from marine sediments, marine invertebrates association, and hyper saline environments (He et al. 2015).Nocardiopsis produce bioactive compounds with various activities such as antibiotics, anticancer, and immunomodulator agent (Bennur et al. 2016)

Conclusions
Our result revealed that actinobacteria associated red algae Gelidiella acerosa are potential as producer of antibacterial compound.Genetic based screening yielded that 14 actinobacteria contained nrps gene associated with marine peptide.The bioactivity assay showed that eight isolates of actinobacteria were active against V. alginolyticus.In conclusion, isolate actinobacteria are wealth sources of bioactive compounds.

FIGURE 1
FIGURE 1 Morphology of one of the ac nobacteria isolated from Gelidiella acerosa.

FIGURE 2
FIGURE 2 Visualiza on of 16S rRNA gene representa ve selec ve amplifica on of 640 bp fragments using primers ActF/ActR in 1% (w/v) agarose gel stained with RedSafe.

FIGURE 6
FIGURE 6 Results of bioassay an vibrio before (le ) and a er indicator (right).

FIGURE 7
FIGURE 7 TLC visualiza on of secondary metabolites of ac nobactera with ninhydrin reagent.

FIGURE 8
FIGURE 8 Chart of gene c, bioac vity, and pep de screening of ac nobacteria associated red algae Gelidiella acerosa.

FIGURE 9
FIGURE 9 Chemical structure of macrocyclic thiopep de an bio cs TP-1161 produced by Nocardiopsis.

FIGURE 10
FIGURE 10 Phylogene c tree of isolates DR-2S-115-5 based on 16S rRNA gene sequence by neighbour joining method with 1000x bootsrap replica on.