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Noble K Kurian, Harisree P Nair and Sarita G Bhat, Melanin producing Pseudomonas stutzeri BTCZ10 from marinesediment at 96 m depth (Sagar Sampada cruise #305), Int.J.Curr.Biotechnol., 2014, 2(5):6-11.

Melanin producing Pseudomonas stutzeri BTCZ10 from marine sediment at 96 m depth (SagarSampada cruise #305)

Noble K Kurian, Harisree P Nair and Sarita G Bhat*

Department of Biotechnology, Cochin University of Science and Technology, Cochin-682022.

A R T I C L E I N F O A B S T R A C T

Article History:Received 14 May 2014Received in revised form 16 May 2014Accepted 20 May 2014Available online 26 May 2014

Key words:Melanin, Melanin Ghost, Marine, Pig-ment, Pseudomonas stutzeri, BTCZ10.

A melanin producing bacterial strain BTCZ10 was isolated from marine sedi-ments at more than 96 m depth. Phylogenetic analysis revealed 100% ho-mology of the 16S rDNA sequence with Pseudomonas stutzeri strains inNCBI database. BTCZ10 melanin polymerization occurred outside the cellwall, which could be visualized as melanin ghosts by light microscopy. Thepigment was soluble in alkali, but insoluble in water, acid and organic sol-vents. Melanin turned colorless by the treatment with oxidizing and reducingagents. UV visible spectrum revealed a higher absorption at the UV regionwhich decreased as it reached the visible region. Strain BTCZ10 produced47.47±0.2µg/mL of melanin.

*Corresponding author.Email address: saritagbhat@gmail.com

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IntroductionMelanins are one of the most inevitable and ubiquitouspigments which help in sustaining life on earth. In humansit is responsible for skin pigmentation as well as protectionfrom UV radiation, while in plants it acts as cell wallstrengtheners. Microbes too have the pigment, whichshields from thermal, chemical (heavy metal and oxidizingagent) and biochemical stresses (reactive oxygen speciesgenerated by the exposure of solar UV radiation)(Hamilton and Gomez, 2002). In fungi it is reported to actas a photosynthetic pigment which sequesters ã radiationin order to generate energy for growth (Dadachova etal., 2007). These diverse biological roles of melanin makeit suitable for the use in various medical, cosmetologicaland pharmacological applications.

Melanins are classified based on the sources as well asthe biochemical pathways that produce them. Black tobrown pigment produced by oxidative polymerization oftyrosine is eumelanin. Red pheomelanin is formed due toincorporation of cysteine in the polymer. The allomelaninsforming the third class are heterogeneous pigmentsformed from a variety of sources like dihydrofolate,homogentisic acid, ã-glutaminyl-4-hydroxybenzene,catechols, etc. (Plonka and Grabacka, 2006).

Several bacteria are reported to produce melanin, amongthem Pseudomonas aeruginosa (Yabuuchi and Omyama,1972), Shewanella. colwelliana, Vibrio cholerae ,Hyphomonas sp., Azotobacter chroococcum,Alcaligenes eutrophus, Aeromonas salmonicida,Burkholderia cepacia, E.coli, Bordetella pertusis,Campylobacter jejuni, Yersinia pestis (Tarangini andMishra, 2013) and also certain marine bacteria likeAlteromonas nigrifaciens, Pseudomonas sp,Cellulophaga tyrosinoxydans and Marinomonasmediterranea (Soo-Jin et al., 2009; Solanol and Sanchez-Amat, 1999; Ivanova et al., 1996; Kotob et al., 1995) etc.Most melanin producers are terrestrial while marine deepsea sediments are still widely unexplored.

There are few reports on melanin production among thePseudomonads that includes Pseudomonas aeruginosa(Yabuuchi and Ohyama, 1972), Pseudomonas stutzeri(Kumar et al., 2013), Pseudomonas maltophila (Wang etal., 2000) to name a few. This paper focuses on molecularand phylogenetic characterization of a melaninproducing Pseudomonas stutzeri BTCZ10 from marinesediment collected from 96 m depth in the Arabian Seaon the West coast of India. The work also discusses thechemical and spectroscopic characterization of themelanin produced.

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Figure 1: Screening for melanin production (a) tyrosine basal agar plates with Pseudomonas stutzeri BTCZ10showing clear zones around the colonies (b) Dark brown melanin produced in tyrosine basal broth.

Figure 2: Molecular Identification and Phylogenetic characterization of BTCZ10 (a) Agarose gel showing 16 rDNAamplicon of BTCZ10 in lane 1, lane 2 has the Thermo Scientific Gene Ruler 1Kb Ladder b) Phylogenetic treeshowing the position of the Pseudomonas stutzeri BTCZ10 with reference to the related strains.

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Figure 3: UV–Visible spectrum of melanin pigment (a) Pseudomonas stutzeri BTCZ10 melanin (b) synthetic mela-nin (c) sepia melanin

Figure 4: Melanin ghosts encircled under light microscope (40X).

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Materials and MethodsChemicals and Bacterial IsolatesSynthetic melanin and Sepia melanin (Sigma ChemicalsCo, St Louis, USA), L-tyrosine (Himedia chemicals,Mumbai, India) and all other chemicals used were ofanalytical reagent grade.

Marine sediment samples were collected from 96.47mdepth (9.59660 N, 75.39220 E) during the Sagar Sampadacruise no 305 in the Arabian Sea on the West coast ofIndia. Bacteria were isolated after serial dilution and pourplating, followed by quadrant streaking to purify them.

Screening for melanin producersPrimary screening involved spot inoculating on tyrosinebasal agar plates with 2g/L L-tyrosine added as the solesource of carbon and nitrogen (Yabuuchi and Ohyama,1972). Selection was based on clear zone formationaround the colonies.

Melanin production was quantitatively analyzed byseeding the isolates selected after the primary screeninginto the melanin production media containing KH2PO4,2.0 g: NaCl, 5.0 g: MgSO4.7H2O, 0.1g and distilled water,1000 mL with 2g/L L-tyrosine serving as carbon andnitrogen sole source (Yabuuchi and Ohyama, 1972).Melanin production was monitoredspectrophotometrically at 400nm (Turick et al., 2002)using synthetic melanin (Sigma, USA) as standard.

Molecular identification of melanin producing bacteriaGenomic DNA was isolated and purified (Sambrook etal., 2000); a portion of the 16S rDNA was amplified usinga primer pair for 16S rDNA (Reddy et al., 2002). Theproducts after PCR amplification were purified by geneclean kit (Bangalore Genei, Bangalore, India) and thenucleotide sequence was determined by the ABI Prism310 Genetic analyzer using the big dye terminator kit(Applied Biosystems, USA). The identity of thesequences was determined by comparing the 16S rDNAsequence with the sequences available in the publicnucleotide databases at the National Center forBiotechnology Information (NCBI) by using their WorldWide Web site (http://www.ncbi.nlm.nih.gov) and theBLAST (Basic Local Alignment Search Tool) algorithm(Altschul et al., 1990) and the nucleotide sequence wassubmitted to NCBI Genbank.

Phylogenetic characterization of the bacteriaThe nucleotide sequence were aligned with the mostsimilar sequences obtained by BLAST search usingBioEdit Sequence Alignment Editor (Hall, 1999) and thephylogenetic tree was constructed using the neighbor-joining method using MEGA5: Molecular EvolutionaryGenetics Analysis (MEGA) software version 5.0 (Tamuraet al., 2011).

Extraction and Purification of melaninMelanin was extracted from the cell free supernatant,following acidification with 1 N HCl to pH 2 and standingfor a week at room temperature. The resulting suspensionwas boiled for 1 h and then centrifuged (Sigma 3K30,Germany). The black pigment pellet thus formed waswashed three times with 15 mL of 0.1 N HCl, followed bywater. To this pellet, 10 mL of ethanol was added and themixture was incubated in a boiling water bath for 10 minand kept at room temperature for a day. The pellet waswashed with ethanol twice and then air dried (Sajjan etal., 2013) and this purified pigment was used for furtheranalysis.

Chemical analysis of the pigmentSolubility of melanin in deionized water, 1N HCl, 1N NaOH,1N KOH, ethanol, acetone, chloroform, benzene, xylene,hexane and acetonitrile was evaluated. Reaction withoxidizing agent hydrogen peroxide (H2O2) and reducingagent sodium sulfite (Na2SO3) was determined (Fava etal., 1993). All estimations were compared with syntheticmelanin as standard.

UV-Visible spectrum of purified melaninU V -Visible spectrum of the purified melanin at thewavelength ranging from 250nm to 800nm was generated.Melanin was dissolved in 0.1N NaOH, which served asthe blank for evaluation (Wenlin et al., 2007). Spectrumobtained was compared with synthetic and sepiamelanins.

Preparation of Melanin ghostsBTCZ10 was cultured in tyrosine basal broth at 37oC in ashaking incubator for 5 days. Melanin ghosts wereisolated from the spent medium by boiling in HCl andethanol extraction (Eisenman et al., 2007). Ghosts werefixed on glass slides by gently heating for 1-2 s andviewed under a light microscope (Radical, India) (40 X)without staining.

ResultsSelection of strain and Pigment productionBacteria (N=40) isolated from the deep sea sedimentsamples were spotted on tyrosine agar medium to selectfor tyrosinase producers. Strain BTCZ10 which formedclear zones (Fig 1a) around their colony was selected.Secondary screening showed significant pigmentproduction by strain BTCZ10 from the 20th hour itself.From 146th hour the production medium turn dark brown(Fig 1b) and the production entered the stationary phase,with 47.47±0.2µg/mL of melanin.

After DNA isolation from strain BTCZ10, the 16S rRNAgene was amplified (Fig 2a). The 1.5 kbp gene wassequenced, submitted to NCBI database and accessionnumber was obtained (KF909003).

Phylogenetic characterization of BTCZ1016S rDNA sequence of strain BTCZ10 showed 100%similarity with Pseudomonas stutzeri sequences from theNCBI database. Phylogenetic tree is shown in Figure 2 b.The sequence also showed 99% similarity with otherPseudomonas species like Ps. fulva, Ps. aeruginosa andPs. putida. Bacillus subtilis M55008 served as the out-group in the phylogenetic analysis.

The evolutionary history was inferred using theNeighbor-Joining method (Saitou and Nei, 1987). Theoptimal tree with the sum of branch length = 1.42112006is shown. The tree was drawn to scale, with branchlengths in the same units as those of the evolutionarydistances used to infer the phylogenetic tree. Theevolutionary distances were computed using theMaximum Composite Likelihood method (Tamura et al.,2004) and are in the units of the number of basesubstitutions per site. The analysis involved 11nucleotide sequences. All positions containing gaps andmissing data were eliminated. There were a total of 442positions in the final dataset. Evolutionary analyses wereconducted in MEGA5 (Tamura et al., 2011).

Chemical characterization of purified melaninThe dark brown melanin was purified and characterized.It was insoluble in water and most of the organic solventstested. It could be precipitated by addition of acids. Only

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alkaline solvents like sodium hydroxide (NaOH) andpotassium hydroxide (KOH) solubilized the pigment. Thepigment turned colorless on reacting with oxidizing andreducing agents like hydrogen peroxide (H2O2) andsodium sulfite (Na2SO3) respectively.

Spectroscopic Analysis of the PigmentUV Visible spectrum of melanin from 250-800nm wasgenerated and is as shown in Fig 3. Higher absorptionwas observed at the UV region (200-300nm) which thendecreased towards the visible region. It was noted thatthe melanin spectrum of BTCZ10 was similar to that ofthe synthetic and sepia melanin, and also to thosepreviously reported (Rosas et al., 2000).

Melanin GhostsMelanin ghosts obtained after the removal of unwantedcellular material (Fig 4) were visualized under a lightmicroscope (40X) as dark spots.

DiscussionMarine sediments from approximately 97 m depth in theArabian Sea were screened for melanin producers ontyrosine basal agar plates. Bacteria forming clear zoneson utilizing tyrosine was selected as a novel screeningmethod, as not only was it fast and effective, but evencolorless bacteria that formed clear zones on the agarplates produced melanin in the production media. StrainBTCZ10 secreted an extracellular black pigment.Production medium which was initially white in colorturned dark brown by the 6th day, and stabilized thereafter.

Strain BTCZ10 was identified based on the homologymatch of the 16S rDNA sequence with the referencestrains from NCBI database as Pseudomonas stutzeri.Pigment produced by strain BTCZ10 was identified asmelanin by physical and chemical characterization.Presence of conjugated complex structures in pigmentmakes its absorbance maxima fall in far UV region. Thisproperty reflects the ability of melanin in protecting theskin from harmful UV radiation. BTCZ10 melanin alsoshowed typical melanin like chemical properties,especially in their solubility. Melanins are soluble inalkaline solvents only. This limitation makes it difficult inthe elucidation of chemical structure of the pigment.

ConclusionMarine melanin producing sources are little explored. Amelanin producer Pseudomonas stutzeri BTCZ10 wasidentified from the depths of the sea. The purified BTCZ10melanin exhibited the physical and chemical propertiesof typical melanin. With its high UV absorption property,it can be utilized further in optical lenses and in sunscreenlotions. More studies are required to understand the otherproperties of melanin.

AcknowledgementsFirst author acknowledges DST INSPIRE for his JuniorResearch Fellowship and the authors acknowledgeCMLRE-MoES for providing facilities in sample collection.

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