III. MATERIALS AND METHODS

3.1. Sample Collection

Specimens of three types of marine sponges namely Halichondria nigrocutis,

Prostylyssa foetida (Class: Demospongiae; Order: Halichondrida; Family:

Halichondriidae) and Suberites carnosus [(Class: Demospongiae; Subclass:

Tetractinomorpha; Order: Hadromerida; Family: Suberitidae) (Plate 1)] were collected

during lowest low tide from the intertidal zone of Majali beach (Karwar, Karnataka, India;

latitude: 14◦ 56' 23.74" N and longitude: 74◦ 04' 30.77"E) and Madh Island (Mumbai,

Maharastra, India; latitude: 19◦ 08' 02.88" N and Longitude: 72◦ 47' 43.82" E") (Plate 2).

Soon after the collection, samples were divided into two parts and one part was transferred

directly to sterile plastic bag containing seawater from the collection sites and another part

of was frozen in liquid nitrogen. The samples were transported to the laboratory within 2 h

and processed immediately for the isolation of culturable bacteria.

3.2. Isolation of marine bacteria

Sponge associated bacteria were isolated by following the method outlined by

Premanand et al., (2006). Initially, the sponge samples were washed with jets of filtered

and autoclaved seawater until they were visibly free of debris. Then the sponge surface was

sterilized by a rapid wash of 70 % ethanol and immediately immersed in autoclaved and

filtered seawater and then removed. One gram of sponge tissue was removed with a sterile

scalpel and the tissue was immediately transferred to 99 of ml sponge dissociation

medium. The samples were soaked for 20 min and then the tissue was homogenized and

the homogenate was suitable diluted and plated on Zobell marine agar 2216 (HiMedia,

Mumbai). The inoculated plates were incubated at room temperature (approx. 27-30ºC) for

Plate 1. Sponges from the Arabian sea analyzed in this study.

Halichondria nigrocutis

Prostylyssa foetida

Suberites carnosus

Plate 2. India map showing sample collection sites on west coast.

Mumbai

N

Karwar

Mumbai

7 days and isolation of bacteria with different colony characteristics was carried out from

the third day onwards up to the seventh day. Seventh day counts were used for the

calculation of colony forming units (CFU). The isolated colonies were purified by streak

plate method and stored in Zobell marine agar slants at 4◦C for further studies.

Sponge Dissociation Medium

Ingredients G/L

Sodium chloride 27.0

Potassium chloride 0.08

Sodium sulfate 0.10

pH 8.00

These ingredients were dissolved in 1000 ml distilled water and sterilized by

autoclaving at 121°C for 15 min.

Zobell Marine Agar 2216 (HiMedia, Mumbai)

Ingredients G/L

Peptic digest of animal tissue 5.00

Yeast extract 1.00

Ferric citrate 0.10

Sodium chloride 19.45

Magnesium chloride 8.80

Sodium sulphate 3.24

Calcium chloride 1.80

Potassium chloride 0.55

Sodium bicarbonate 0.16

Potassium bromide 0.08

Strontium chloride 0.034

Boric acid 0.022

Sodium silicate 0.004

Sodium fluorate 0.0024

Ammonium nitrate 0.0016

Disodium phosphate 0.008

Agar 15.00

pH 7.6+0.2

These ingredients were dissolved in 1000 ml distilled water and sterilized by

autoclaving at 121°C for 15 min.

3.3. Bacterial identification

A series of biochemical tests were performed to identify the bacterial isolates (Mac

Faddin, 1980). The bacterial identification was done according to the scheme for Gram

positive bacteria (Le Chevallier et al., 1980) and the Gram negative bacteria (Bain and

Shewan, 1968 and Le Chevallier et al., 1980). The identification schemes are

schematically represented in Fig. 6A, 6B, 6C.

3.3.1. Gram staining Hucker's modification of Gram staining procedure was followed. After staining, slides

were observed under oil immersion objective of the compound microscope to record the

Gram reaction of each isolate.

3.3.2. Motility test

16 to 18 h young cultures of bacteria, grown in TSB containing 1 % NaCl, were tested

for motility by hanging drop technique using cavity slide.

Figure 6A. Flow chart for identification of microorganisms

* Excludes catalase negative streptococci Source : Lechevallier et al. (1980)

Gram positive organism

+/+ + or -/- Staphylococcus Micrococcus

+ - Bacillus Cell morphology

Cocci Rods Branching filaments Catalase* + No spores Oxidase - Not acid fast O/F glucose Spores Colonies rubbery, firmly attached to agar medium Actinomycetes

Rods – Coccus Snapping division Transformation Corynebacterium Arthrobacter

Gram negative organisms

O/F Glucose -/- or +/- O/F Glucose +/+ Non fermentative bacteria Fig 1C Cocci or Coccobacilli Rods Oxidase (or sensitivity to penicillin) Motility + - + - Moraxella Acinetobacter Water soluble Indole

Blue green pigments + - + - Pseudomonas No acid or gas

from sugars No acid or gas Moraxella - like Alcaligenes pigments not O/F Glucose soluble Flavobacterium

Oxidation Alkaline No action

Green fluorescent No diffusible No diffusible no diffusible diffusible pigment pigment pigment pigment Pseudomonas Pseudomonas Pseudomonas Pseudomonas Group I Group II Group III Group IV Figure 6B. Flow chart for identification of microorganisms. Source: Modified from Bain and Shewan, 1968 and Le chavellier et al., 1980.

Gram negative organisms O/F Glucose

+/+ (Fermentative)

Oxidase test + - Sensitivity to 0/129 Enterobacteriaceae (C) + - - Vibrio Aeromonas Plesiomonas Escherichia Klebsiella Enterobacter Salmonella Hafnia Serratia Proteus Arginine - + + Catalase + + + + + + + Lysine + - + Ornithine + - + Oxidase - - - - - - - Motility + - + + + + + Indole + - - - - D D M.R. + - - + - D D V.P. - - + - D + - Citrate - + + + + + D Urease - + D - - - +

Figure 6C. Flow chart for identification of Microorganisms Note: D. some strains positive and some negative

Source: Carpenter (1966) (c) Cruickshank et al. (1978)

Tryptone Soya broth with 1% NaCl (TSBS)

Ingredients G/L

Tryptone 17.00

Soya Soya peptone 3.00

NaCl 10.00

K2HPO4 2.50

These ingredients were dissolved in 1000 ml distilled water and by autoclaving at

121C for 15 min.

3.3.3. Oxidation fermentation test (O/F test)

Hugh and Leifson's O/F medium was used to test whether the organism was

fermentative, oxidative or inert. About 3 ml each of media were poured into a set of two

tubes and autoclaved. Organisms were stabbed into the butt and one of the tubes was

overlaid with liquid paraffin. Fermentative organisms produced acid in both tubes and

oxidative organisms produced acid in the tube that is not overlaid liquid paraffin. Inert

organisms failed to produce acid and hence medium colour was unchanged.

Hugh Leifson O/F medium

Ingredients G/L

Soya peptone 2.00

Yeast extract 5.00

Sodium chloride 10.00

Glucose 10.00

Bromocresol purple 0.015

Agar 3.00 – 4.00

pH 7.2 + 0.1

The ingredients were added to 100 ml of distilled water and boiled to dissolve the agar.

The medium was then distributed in 2.5 ml volume in test tubes and sterilized at 110C for

15 min.

Liquid paraffin

This was sterilized at 160-180C for 11/2 - 2 h in a hot air oven. Liquid paraffin was

used to create anaerobic environment in the fermentative tubes.

3.3.4. Sensitivity to O/129 compound

A lawn culture o f test stra in was prepared on TSAS and O/129 disc (150

µg) was placed at the centre of the lawn. Development of a c lear zone of

inhib it ion around the disc was recorded as sensit ive and growth around the

disc indicated resistance to this compound . Only isloates sensit ive to this

compound were fu rther p rocessed. O/129 compound was prepared by

disso lving in acetone and 100 discs o f 6 .5 mm diameter each (pre-ster ilized

at 140 C for 1 h) were soaked in the so lution, dried gent ly to evaporate the

acetone and stored in a dark bottle at 4C. Each disc conta ined 150 g of

the p teridine compound .

3.3.5.Oxidase test

Cytochrome oxidase test was performed using moistened filter paper

str ips soaked with 1% oxidase reagent. Young co lonies o f bacter ia were

spotted on the oxidase paper using sterile toothpicks. Development o f dark

purple colour within 10 sec ind icated positive react ion.

Oxidase reagent

Oxidase reagent (N, N, N’, N’ – tetramethyl

p-phenylene diamine dihydrochloride) 1.0 g

Distilled water 100 ml

Whatman filter paper No.1 was cut into strips of 2.5 x 1.0 cm, sterilized in hot air oven

at 140C for 1h. The strips were later dipped in oxidase reagent, allowed to absorb and

then dried. The strips were stored in dark bottle at 4C.

O/129 reagent

O/129 (2 -4,Diamino 6-7 Di isopropyl

pterid ine)

15 mg

Acetone 1 ml

3.3.6. Amino acid decarboxylase test

Ability of the microorganism to decarboxylate aminoacids lysine, ornithine and arginine

was tested by inoculating test cultures into media containing amino acids in separate tubes

and to a tube with only basal medium which served as a control. After overlaying with

liquid paraffin, all the tubes were incubated at 30C. The change in the colour of the

indicator from purple to yellow and back to purple was considered as positive for

decarboxylation.

Basal medium for amino acid decarboxylase test

Ingredients G/L

Soya peptone 5.00

Yeast extract 3.00

Sodium chloride 10.00

Glucose 1.00

Bromocresol purple 0.016

pH 7.2 + 0.2

The basal medium was divided into four parts and aminoacid lysine, ornithine and

arginine were added individually to each quarter at a concentration of 0.5, 0.5 and 0.4%

respectively. The last quarter served as control. The media was distributed into tubes in 4

ml volumes and sterilized at 110C for 15 min.

3.3.7. Sugar fermentation test

Different sugars viz. glucose, sucrose, arabinose, mannose and m-inositol were used as

substrates to test the ability of the bacterial cultures to utilize them with resultant

production of acid. Cultures were inoculated into pre-sterilized individual sugar media in

tubes and colour change from purple to yellow indicated fermentation of the sugar.

Sugar fermentation medium

Ingredients G/L

Soya peptone 10.00

Sodium chloride 10.00

Sugars 10.00

Bromocresol purple 0.016

pH 7.1 + 0.2

The ingredients were dissolved in distilled water and then dispensed in 4 ml volume

into test tubes containing Durham’s tubes and sterilized by autoclaving at 110C for 10

min.

3.3.8. Indole test

The cultures were grown in tryptophan broth for 24-48 h and then a few drops of

Kovac's reagent were added. Formation of a pink indole ring at the surface of culture was

recorded as a positive reaction.

Tryptone broth

Tryptone 10.00

Sodium chloride 10.00

pH 7.1 + 0.

This was distributed in 5 ml volumes into test tubes and autoclaved at 121C for 15 min.

Kovac's reagent

p-Dimethyl aminobenzaldehyde (DMAB) 5.0 g

Amyl alcohol 75 ml

Conc. Hydrochloric acid 25 ml

DMAB was dissolved in amyl alcohol and then the Conc. HCl was added slowly. The

solution was stored at 4C in a dark bottle and used to perform the test whenever required.

3.3.9. Salt tolerance test

Test cultures were inoculated into tryptone broth containing 0 %, 3 %, 8 % and 11 %

NaCl and incubated at room temperature for 18 – 24 h. Turbidity in the tubes was

considered positive.

Tryptone salt medium

Tryptone 10 g

NaCl 0 or 3 or 8 or 11g

Dist illed water 1000 ml

pH 7 .2 + 0.2

This med ium was d istr ibuted in 5 ml vo lumes into test tubes and

autoclaved at 121 oC for 15 min.

3.3.10. Voges Proskauer (VP) test

Test cultures were inoculated into Methyl red - Voges Proskauer (MR-

VP) broth and incubated for 48 h at 37C. To 1 ml of the culture, 0.6 ml of

-Naphthol and 0.2 ml of KOH reagent were added and mixed by shaking.

Development of port wine co lour indicated a posit ive react ion. VP negat ive

isolates were proceeded with.

MR-VP broth (HiMedia, Mumbai)

Ingredients G/L

Glucose 5.00 g

Peptone 7.00 g

K2HPO4 5.00 g

NaCl 5.00 g

The medium was dispensed in 5 ml volumes into tubes and autoclaved at 121oC for 15

min.

Voges - Proskauer reagent

Solution A

- naphthol 50.00 g

Absolute alcohol 1000 ml

Solution B

KOH 40.00 g

Dist illed water 100 ml

3.3.11. Citrate utilization test

Ability of bacteria to use citrate as the sole source of carbon for metabolism was tested

by growing the cultures in Simmon’s citrate agar slants. Change of colour from green to

prussian blue colour and growth of bacteria was recorded as a positive reaction.

Simmon’s citrate agar

Ingredients G/L

Sodium chloride 10.00

Magnesium sulphate 0.20

Ammonium dihydrogen phosphate 1.00

Potassium dihydrogen phosphate 1.00

Sodium citrate 2.00

Bromothymol blue 0.08

Agar 20.00

pH 6.8

The medium was boiled to dissolve, distributed into tubes in 3 ml volumes and

sterilized at 121 ºC for 15 min. Slants were prepared after it was autoclaved.

3.3.12. Urease test

Cultures were grown on urea slants to see their ability to break down urea. The reaction

was recorded as positive if the slants changed colour from yellow to pink after incubation

with the culture.

Basal medium

Ingredients G/L

Peptone 1.00

Dipotassium hydrogen phosphate 2.00

Glucose 1.00

Phenol red 0.012

Agar 15.00

pH 6.9

The basal medium was autoclaved at 110 ºC for 15 min. To 900 ml of this molten

medium 100ml of 20 % filter sterilized urea solution was added, distributed in 4 ml volume

in to sterile tubes and slants prepared.

3.4. Molecular methods

3.4.1. Molecular identification and phylogenetic analysis of the culturable bacteria

The cultivable bacteria were identified by sequencing 16s rDNA gene. Single isolated

colonies of the selected bacterial cultures were picked from Zobell Marine agar 2216

(HiMedia, Mumbai) plates and suspended in 50 μl of colony lysis solution (10 mM Tris–

HCl, pH 7.5, 10 mM EDTA and 50 μg/ml of proteinase-K). The reaction mixture was

incubated at 55°C for 15 min followed by proteinase-K inactivation at 80°C for 10 min.

The reaction mixture was centrifuged at 15,000 x g at 4°C for 15 min. The supernatant

containing genomic DNA was directly used as template for PCR amplification.

Amplification of 16S rDNA gene was carried out with eubacteria specific primers 27F

(5’- AGA GTT TGA TCC TGG CTC AG -3’) and 1492R (5’- GGT TAC CTT GTT ACG

ACT T- 3’) (Lane, 1991). The PCR reactions were performed in 30 μl reaction mixture

consisting of a 10x PCR buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl; 2.5 mM MgCl2;

0.01% gelatin) (Bangalore GeNei, Bangalore), 10 pico mol each of forward and reverse

primers, 0.2 mM dNTP mix, 1.5 U of Taq polymerase and 500 ng of genomic DNA. The

PCR was performed in a DNA Engine DYADTM (M. J. Research Inc., U.S.A). The

amplification conditions consisted of an initial denaturation at 94°C for 5 min followed by

35 cycles of 94°C for 1 min, 54°C for 2 min and 72°C for 2 min with a final delay of 72°C

for 10 min for the post amplification synthesis of flush ends. Expected PCR product of

around 1.5 Kb was checked by electrophoresis of 5 μl of the PCR product on 1% agarose

gel in 1 x TAE buffer followed by staining with ethidium bromide (0.5 µg/ml) and bands

were visualized under UV light

PCR products were purified by using QIAquick PCR purification kit (QIAGEN,

Germany) and checked by electrophoresis in a 1% (wt/vol) agarose gel, and bands of

approximately 1.5 kb were confirmed. Sequencing of purified PCR product performed

using ABI Big Dye Terminator Kit Version 3.1 and 3100 Genetic Analyser (PE Applied

Biosystems, Foster City, California.) by M/s. Bangalore GeNei, Bangalore. Both forward

and reverse primers were used for sequencing (Table 4).

3.4.2. Extraction of DNA from sponge tissue

DNA was extracted from freeze-dried tissue of sponges using protocol described by

Webster et al. (2001a). Freeze dried tissue (1.5g) was ground in liquid nitrogen and

suspended in 5 ml of TE buffer (10 mM Tris-Cl and 1 mM EDTA, pH 8.0) containing 50

mg/ml lysozyme and incubated at 30ºC for 30 min. Guanidinium thiocyanate buffer (60%

guanidinium thiocyanate, 20 ml of 100 mM EDTA and 5 ml of 10% Sarkosyl) was added

and vortexed for 5 min. Samples were transferred to ice, and ammonium acetate was

added to a final concentration of 2.5 M. The supernatant was transferred to a fresh tube and

an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added, vortexed and

centrifuged at 10,000 × g for 10 min. The aqueous phase containing DNA was transferred

to a fresh tube and precipitated by adding 2-3 volumes of isopropanol followed by

centrifugation at 14,000 × g for 10 min. The pellet was washed with 1 ml of 95% ethanol

and then vacuum dried. Finally, the dried pellet was dissolved in 100 l of 1× TE buffer

(pH 8.0) and stored at -20oC until further use.

Table 4. Primers used for Sequencing

Primer Name Primer Sequence Product Length (bp)

Reference

M13F

M13R

5’ –AGT CAC GAC GTT GTA AAA CG -3’ 5’ –CGG ATA ACA ATT TCA CAC AG -3’

Variable -

27F

1492R

5’- AGA GTT TGA TCC TGG CTC AG -3’

5’- GGT TAC CTT GTT ACG ACT T- 3’

1500 Lane, 1991

3.4.3. Quantification of DNA

The total DNA extracted from each sponge sample was pooled and 2µl of the DNA

sample was used to determine the concentration using NanoDrop ND-1000

spectrophotometer (NanoDrop, USA). Genomic DNA from sponges was further confirmed

by electrophoresis in a 1.2% (wt/vol) agarose gel (Plate 3).

3.4.4. Primers and PCR protocol for 16S rRNA library construction

PCR was performed in a 50 µl reaction mixture consisting of 100 ng of template

DNA, 1x PCR buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl; 2.5 mM MgCl2; 0.01%

gelatin), 0.2 mM of dNTP mix, 10 pmol of each Universal Eubacterial primers [U1 5’ –

CCA GCA GCC GCG GTA ATA CG -3’, corresponding to nucleotides 518 to 537 of the

E.coli 16S rRNA gene, and U2 5’ –ATC GG (C/T) TAC CTT GTT ACG ACT TC -3’

corresponding to nucleotides 1513 to 1491 of the same gene (Lu et al., 2000)] designed to

amplify all bacterial 16S rDNA fragments, and 1.5 U of Taq DNA polymerase (Bangalore

GeNei, Bangalore). The PCR cycling conditions consisted of an initial delay at 94◦C for 10

min, followed by 35 cycles of 94◦C for 1 min, 55◦C for 1 min, and 72◦C for 2 min. A final

delay of 72°C for 10 min was given for the post amplification synthesis of flush ends. The

PCR products were electrophoresed on 1.5-2% agarose gel, stained with 0.5 g/ml

ethidium bromide and visualized by UV-transilluminator (Gel documentation system,

Herolab, Germany) (Plate 4).

3.4.5. Agarose gel electrophoresis

All the PCR products were analyzed in agarose gel of appropriate

percentage (w/v). The agarose gels were prepared in 1X Tris acetate EDTA

(TAE) buffer . When the molten agarose had coo led to below 65 oC, the gel

was cast. Twent y microliter s of the amplicons were mixed with 4 l of 6x

load ing buffer and loaded into the wells . 100 bp DNA ladder (Bangalore

GeNei, Bangalore) was used as a molecu lar weight marker. Electrophoresis

was carried out at 80-120 vo lts, sta ined with 0 .5 g/ml ethidium bromide

and the bands were visua lized under UV light.

Lane M: 1kb DNA Ladder Lane1, 2: Genomic DNA

Plate 3. Genomic DNA of sponge

Lane M : 1kb DNA Ladder Lane1-6: 16S rRNA gene positive clones

Plate 4. PCR amplification of 16S rDNA gene by Universal eubacterial primers.

M 1 2 3 4 5 6

M 1 2

996bp

>2kb

Tris-acetate-EDTA (TAE) buffer (50×)

Ingredients

Tris base 242 g

Glacial acetic acid 57 ml

0.5 M EDTA (pH 8.0) 100 ml

The solution was made up to a final volume of 1 litre with distilled water. The working

solution (1000 ml of 1× TAE) was prepared by diluting 20 ml of 50× stock solution to

1000 ml with distilled water.

Sample loading buffer (6×)

Bromophenol blue 0.300 g

Sucrose 40.00 g

Distilled water 100 ml

Ethidium bromide (Sigma, U.S.A.)

A stock solution was prepared by dissolving 5 mg of ethidium bromide in 1 ml of

distilled water. The solution was either added to agarose gel during preparation or to the

appropriate buffer (1TAE buffer) to yield a final concentration of 0.5 g/ml. In the latter

case, the gel was allowed to stain in the solution for 20 min and destained in distilled water

for 10 min to remove excess stain.

3.5. Cloning of PCR products

3.5.1. Purification of PCR products

The PCR products were purified before ligation to remove contaminants like Taq DNA

polymerase, primer dimers, unutilized dNTPs which may interfere with subsequent

processes, using QIAquick PCR purification kit (QIAGEN, Germany).

To one volume of the PCR product, 5 volume of PB buffer was added, mixed and then

transferred to QIAquick spin column placed in a 2 ml collection tube. The flow through

was discarded after centrifugation at 10,000 g for 1 min, washed by adding 0.75 ml

buffer PE containing ethanol followed by centrifugation at 10,000 g for 1 min. The DNA

was eluted by adding 50 l of elution buffer (10 mM Tris-Cl, pH 8.5) QIAquick column

placed in a fresh microcentrifuge tube and collected by centrifuging the column at

10,000 g for 1 min (Fig.7).

3.5.2. Cloning of purified PCR products

Purified products were subsequently cloned into pSC-A cloning vector (Strataclone,

USA). This vector contained two DNA arms, each charged with topoisomerase I, on one

end and a loxP recognition sequence on the other end. The topoisomerase-charged ends

had a modified uridine (U*) overhang. The kit is based on UA cloning principle and the

vector map of cloning vector is give in Fig. 8. The overview of the strataclone™ cloning

protocol is depicted in Fig. 9.

3.5.3. Ligation of PCR products into the cloning vector

The ligation reaction mixture was prepared by adding 3 μl StrataClone cloning Buffer, 2

μl of PCR product (5–50 ng) and 1 μl StrataClone Vector Mix in a 200 μl microcentrifuge

Figure 7. Purification of PCR products using QIAquick® spin column.

Figure 8. Vector Map for the StrataClone™ PCR Cloning Vector pSC-A (Stratagene,

USA).

Figure 9. Overview of the StrataClone™ PCR cloning method.

tube and the reaction mixture was mixed gently by repeated pipetting. The reaction mixture

was then incubated at room temperature for 5 min. When the incubation was completed the

mixture was placed on the ice.

3.5.4. Transformation

For each ligation reaction a tube of StrataClone SoloPack competent cells was thawed

on ice to which 2 µl of the ligation reaction mixture was added. This was then mixed

gently by tapping and incubated on ice for 20 min. Heat-shock was given to the

transformation mixture at 42°C for 45 seconds by placing the tube in water bath set at

42°C. This mixture was then incubated on ice for 2 min. To this transformation reaction

mixture, 250 μl of pre-warmed (42° C) SOC medium was added. The competent cells were

allowed to recover for 90 min at 37°C with agitation on a horizontal shaker. Hundred

microliter of the transformation mixture was then plated on LB–ampicillin–X-gal plates

and incubated at 37°C for overnight.

LB Agar

Ingredients G/L

Tryptone 10.00

Yeast extract 5.00

NaCl 10.00

Agar 20.00

Add deionized H2O to a final volume of 1 liter adjust pH to 7.0 with 5 N NaOH

Autoclave pour into petri dishes (~25 ml/100 mm plate).

Glycerol broth

Ingredients

Tryptone 10 g

Yeast extract 5 g

Sodium chloride 10 g

Glycerol 30 ml

Distilled water 70 ml

The medium was autoclaved at 121°C for 15 min.

Ampicillin stock solution (100 mg/ml)

Ampicillin stock solution was prepared by dissolving 0.1089 g of ampicillin powder

[(having 98% purity)(Calbiochem, USA)] in 1 ml distilled water. The solution was filter

sterilized using 0.22 μm syringe filter (Pall Corporation, USA) and stored in aliquots at -

20°C.

LB–Ampicillin Agar (per Liter)

1 Lit of Luria Bertani agar was sterilized and cooled to 55°C. Add 10 ml of 10 mg/ml

filter-sterilized ampicillin, pour into petri dishes (~25 ml/100 mm plate)

SOC Broth (per 100 ml)

2 ml of filter-sterilized 20% (w/v) glucose or 1 ml of filter-sterilized 2 M glucose SOB

medium (autoclaved) to a final volume of 100 ml. This medium was prepared just before

use.

2% X-Gal (per 10 ml)

0.2 g of 5-bromo-4-chloro-3-inodlyl-β-D-galactopyranoside (X-Gal), 10 ml of

dimethylformamide (DMF), Stored at –20°C. 40 μl was spread per LB-agar plate.

3.5.5. Screening of transformants

Transformants were selected by picking white colonies and screened them for the

presence of insert by preparing crude lysate of DNA. Briefly, each of the selected colonies

were inoculated to 2 ml of LB broth and incubated overnight at 37C. To 50 µl of the

culture, 450 µl of TE buffer was added and cells were lysed at 100C for 10 min, snap

chilled, centrifuged briefly for 5 min and 2 µl of supernatant was used as template for PCR

using gene specific primers. Glycerol stock (30%) were made for all the positive clones

and stored at -80C. A negative-control cloning reaction was performed in which no

sponge-extract DNA was added.

3.6. Sequencing, sequencing analysis and GenBank submission

Sequencing was performed with the ABI Prism Big Dye Terminator Kit Version 3.1

and 3100 Genetic Analyser (PE Applied Biosystems, Foster City, California) by

M/s. Bangalore GeNei, Bangalore. M13 forward and M13 reverse primers were used for

sequencing (Table 4).

The partial 16S rDNA sequences were initially de-replicated by comparing all the

sequences in a data set to each other, using the ALIGN program

(http://blast.ncbi.nlm.nih.gov/bl2seq/wblast2.cgi) of NCBI. Only one representative

sequence having >98% similarity was taken for further analysis. Sequences were also

checked for the formation of chimeras (hybrid sequences) using the program CHECK

CHIMERA of the Ribosomal Database Project-II (RDP-II) website (Cole et al., 2003).

Sequences that showed formation of chimeras were not included in analysis. To identify

known sequences with a high degree of similarity, the clone sequences were matched to

sequences in the RDP-II database by using the SEQMATCH program and with sequences

in GenBank database using Basic Local Alignment Search Tool (BLAST) searches. Based

on the percentage similarity of unknown clone sequences to representative bacterial

sequences in RDP–II and GenBank databases, the sequences were further identified as

belonging to either species (97-100% match) or genus (93-96% match) levels or

considered as belonging to a related type (86-92% match) (Stackebrandt and Goebel,

1994). Phylogenetic trees were constructed using the MEGA4 (Molecular Evolutionary

Genetic Analysis version 4.0) software (Tamura et al., 2007). The phylogenetic tree was

inferred based on the neighbour joining method (Saitou and Nei, 1987) and evolutionary

distances were computed using the Maximum Composite Likelihood method (Tamura et

al., 2004). The robustness of inferred tree topologies was analysed by bootstrapping 1,000

replicates of the neighbor-joining data (Felsenstein, 1985). The 16S rDNA sequence of

Fusobacterium sp. (AY953256) was included as an outgroup in all phylogenetic analysis.

The partial clone sequences determined in this study have been deposited in the

GenBank database and assigned accession numbers.

3.7. Detection of antimicrobial activity of the culturable bacteria from sponges

Each bacteria culture were grown in 250 ml Erlenmeyer flasks containing 100 ml of

Luria Bertani broth and incubated for 24 h at 31 ± 1ºC in shaker incubator at 125 rpm.

After growth, culture media were centrifuged at 10,000 × g for 15 min, and the

supernatants were filtered through 0.22 μm membranes (Sartorius Stedim Biotech,

Germany). The resulting filtrate with a pH valve of 7.0 to 8.0 was used to evaluate

antimicrobial activity.

Antibacterial activity was of bacterial filtrate determined essentially as described

elsewhere with modifications (Motta et al., 2004). An aliquot of 20 μl cell-free culture

supernatant was applied into the well (6 mm) on Tryptone Soya agar plates previously

inoculated (the lawns were prepared by propagating fresh cultures to an OD600=0.5) with

each indicator strain suspension, such as Vibrio harveyi, Listeria monocytogenes,

Staphylococcus aureus, and Escherichia coli. Plates were incubated for 24 h at optimal

temperature for the test organism and inhibition zones around the well were measured.

Tryptone Soya agar (TSA)

Ingredients G/L

Tryptone type I 15.00

Yeast Extract powder 5.00

Sodium chloride 10.00

Agar 25.00

The ingredients were dissolved in 100 ml of distilled water, sterilized at 121C for 15

min and poured into sterile Petri plates.

3.7.1. Effect of heat and enzymes on antimicrobial substance

Effect of proteolytic enzymes on the antimicrobial activity of cell-free supernatant

was tested. One milliliter aliquot of each sample was treated with 2 mg ml–1 of Trypsin

(HiMedia, Mumbai) and Pepsin (HiMedia, Mumbai) at 37ºC for 1 h. Samples were then

boiled for 2 min to inactivate the enzyme. To analyze thermal stability, the samples were

exposed to temperatures ranging from 40 ºC to 100 ºC for 15 min and 121ºC for 15 min

before testing for antimicrobial activity. Trichloroacetic acid (TCA) was added to the

filtrates to reach a working concentration of 100 mg ml-1 and the samples were incubated

for 2 h at 4ºC. After treatment with TCA, samples were centrifuged at 10,000 x g for 5 min

and the supernatant was neutralized to pH 7.0 before testing for antimicrobial activity. The

resulting supernatant was then tested for antimicrobial activity against Vibrio harveyi,

Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli.

3.7.2. Partial purification of the antimicrobial compound

About 5 ml of bacterial cultures was inoculated to 1000 ml of Luria Bertani broth and

incubated in a shaker incubator (at 150 rpm) at 30ºC for 24 h. The antimicrobial compound

was extracted from a cell-free solution by centrifugation at 10,000 rpm for 20 min at 40C.

Concentration of the antimicrobial compound

Although many protocols have been described for the concentration of antimicrobial

compound (Fig.10), the most common methods used are ammonium sulfate concentration,

adsorption-desorption and organic solvent extraction.

Ammonium sulphate precipitation

Extraction of antimicrobial substance from the marine sponge associated bacteria was

carried out using the protocol described by Barja et al. (1989) with some modification. The

antimicrobial compound being proteinaceous in nature was concentrated through the

application of salting-out methods with most commonly used salt, ammonium sulphate. In

this procedure the solid salt was added to the sample slowly until the desired saturation

percentage of ammonium sulphate was reached. A 24-h-old culture of the TSNC63 and

SBCC5 strain was centrifuged (8000×g, 10 min, 4ºC) and the cell free supernatant was

used for precipitation of antimicrobial compound. The peptidic fraction from the cell-free

supernatant was precipitated following gradient precipitation method using 20 -70 %

saturated ammonium sulphate as some antimicrobial compounds can precipitate at lower

ammonium sulphate concentrations, or even in a small range of saturation. The suspension

was incubated overnight at 4°C and agitated with a magnetic stirrer. Salted-out proteins

were precipitated by centrifugation (10,000 × g for 20 min) and dissolved in a small

volume of 0.01 M phosphate buffer (pH 7.5).

Phosphate buffer (pH 7.5)

Ingredients G/L

Monopotassium phosphate 136.09

Disodium phosphate 141.96

pp11

Figure 10. Methods used for the concentration of antimicrobial compound.

Screening Bacteriocin producer (Supernatant)

Indicator (growth culture)

-spot-on-lawn assay, -disc diffusion, -microtitre plate assay, -agar well diffusion assay,

Adsorption-desorption method Organic solvent extraction Ammonium sulphate precipitation

Anionic / Cationic exchange

Preparative isoelectric focusing

Active fraction concentration Solid phase extraction Dialysis

Active fraction concentration

Reverse-phase HPLC

Electrophoresis

Bioassay Silver stain

Sypro-rubi stain

Sequencing

Ultrafilteration

Purification

Detection and recovery

These ingredients were weighed and dissolved in 1000 ml distilled water, pH adjusted

to 7.5 and sterilized by autoclaving at 121°C for 15 min.

Dialysis

The suspension containing antimicrobial substance was desalted by dialysis with 0.01

M phosphate buffer (pH 7.5) at 4°C for 12 h by using benzoylated membranes (molecular

weight cut off 1200; Sigma-Aldrich) and the buffer change was done at every one hour.

Upon dialysis the residual sample was scraped off from the sides of the dialysis bag and

stored at -20º C until further use. A well diffusion assay was carried out to check the

antimicrobial activity of the protein precipitate obtained. The samples were tested for

antimicrobial activity against Vibrio harveyi, Listeria monocytogenes, Staphylococcus

aureus, and Escherichia coli.

3.8. Extraction of antimicrobial compounds with organic solvents

The antimicrobial compound present in cell free supernatant was extracted with organic

solvent, ethyl acetate (Abraham, 2004). Extraction from supernatant was carried out with 3

volumes of solvent for 2 h in a rotary shaker and the supernatant fraction was flash

evaporated using Rotary Vacuum Flash Evaporator at 45o C (Superfit Continental Pvt.

Ltd., Mumbai) to ensure complete removal of solvent and the extracts were evaporated to

dryness. The resulting residue was dissolved in 0.01 M Phosphate buffer (pH 7.5) and

stored at –20°C until further use.

Antimicrobial assay

The test strains were grown in Luria Bertani broth to an of OD600=0.5 and a lawn was

made on Tryptone soy agar plates. Wells were made with a sterile well borer (6mm) and

20μl of the samples (cell supernatants/purified extracts) to be assayed were pipetted into

the wells and incubated overnightat 37° C in upright position and the inhibition zones were

measured (Tagg and McGiven, 1971).

3.9. Protein profile on SDS-polyacrylamide gel electrophoresis

Sample showing antimicrobial activity were further characterized by SDS-PAGE

(Laemmli, 1970) with minor modifications.

The various buffers and reagents used are as follows:

a) Acrylamide-bisacrylamide mixture

Twenty nine grams of acrylamide and 1.0 g of bisacrylamide (N, N’-methylene

bisacrylamide) were dissolved in 80 ml of distilled water. The volume was made up to

100 ml and stored at 4 ºC.

b) Separating gel buffer (1.5 M Tris-Cl, pH-8.8)

181.7 g of Tris base was dissolved in 800 ml of distilled water and pH was

adjusted using concentrated HCl. Then final volume was made up to 1 liter with

distilled water and stored at 4 ºC.

c) Stacking gel buffer (1 M Tris-HCl, pH-6.8)

121.1 g of Tris base was dissolved in 800 ml of distilled water and pH was

adjusted using concentrated HCl. Then final volume was made up to 1 liter with

distilled water and stored at 4 ºC.

d) 10 % sodium dodecyl sulphate (SDS) solution

10 g of SDS was dissolved in 100 ml of distilled water and stored at room

temperature.

e) 10% ammonium persulfate (APS) solution

1 g of APS was dissolved in 10 ml of the distilled water and stored at 4 ºC for 1

week.

f) N,N,N’,N’-tetramethylethylenediamine (TEMED)

Electrophoresis grade TEMED (Bangalore GeNei, Bangalore) stored at 4oC was

used.

g) Electrode buffer ( 5 solution)

5 stock solution was prepared by dissolving 15.10 g of Tris base and 94 g of

glycine (electrophoresis grade) in 900 ml distilled water and to this 50 ml of 10 %

SDS solution of electrophoresis grade was added and the final volume was made up to

1 liter with distilled water and stored at room temperature. For running the gel, 1X

buffer was prepared using 5 buffer.

h) SDS gel loading buffer (2)

2 gel loading buffer contained 100 mM Tris-Cl (pH 6.8); 4% (w/v) SDS; 20%

(v/v) glycerol; 0.1% (w/v) bromophenol blue and 200 mM β-mercaptoethanol. Gel

loading buffer lacking thiol reagent was stored at room temperature. 200 mM β-

mercaptoethanol was added to buffer from 14 M stock mercaptoethanol just before

use.

i) Staining solution

2.5 g of Coomassie Brilliant blue R250 (HiMedia, Mumbai), 450 ml of methanol

and 100 ml of acetic acid were mixed, filtered and volume was made up to 1 liter

with distilled water and stored at room temperature.

j) Destaining solution

300 ml of methanol and 100 ml of acetic acid were mixed with distilled water and

made up to 1 liter with distilled water and stored at room temperature.

k) Standard protein molecular weight marker

A broad-range protein molecular weight marker [(PMW-B) Bangalore GeNei,

Bangalore)] was used for determination of molecular weights of antimicrobial

proteins.

Preparation of SDS-PAGE apparatus (Sambrook et al., 1989)

Depending on the proteins to be separated, 10-15% resolving gel and 5% stacking gels

were used in this study. Glass plates were rinsed with absolute alcohol and cleaned using

tissue paper. Spacers (1 mm) were placed on both sides between the two plates in such a

way that any bubbles could not move through then and seated in a stand and checked by

water. A 10-15% resolving gel was prepared and poured up to 3\4 portion and allowed to

solidify. After solidification, 5% stacking gel was prepared, poured and 1 mm comb was

inserted into the staking gel and allowed to solidify. Comb was gently removed and the

solidified gel with plates was fixed out to a gel running apparatus. Gel running tank was

filled with 1 x electrode buffer and prepared samples were loaded into the wells. Samples

were resolved by applying constant current of 20 mA for 2 h. After electrophoresis, the gel

was transferred to a clean container and stained overnight at room temperature with

shaking. After staining, the gel was destained using destaining solution until a clear

background was obtained. Photographs of the gels were taken with gel documentation

system (Herolab, Germany).

Resolving gel mixture was prepared in a small beaker with a magnetic stirrer by mixing

the components for a desired concentration of acrylamide according to the following chart

(Harlow and Lane, 1998).

Composition for resolving gels (pH 8.8)

12% gel Solution component

Component volumes (ml)

Total volume 5.0 10.0

Deionised water 1.7 3.3

30% acrylamide mix 2.0 4.0

1.5 M Tris-HCl pH 8.8 1.3 2.5

10% (w/v) SDS 0.05 0.1

10 % (w/v) APS 0.05 0.1

TEMED 0.002 0.004

Immediately upon addition of APS and TEMED, the solution was poured into the

assembled plates and overlaid with iso-butanol to prevent diffusion of oxygen into the gel

and for obtaining a uniform margin of the gel. It was allowed to polymerize for about 15

min.

In a similar manner, stacking gel mixture with 5% acrylamide mix was prepared by

mixing the components as below (Sambrook et al., 1989).

Composition for stacking gels (pH 6.8)

Component 1 ml 3 ml 5 ml 8 ml 10 ml

H20 0.68 2.1 3.4 5.5 6.8

30% Acrylamide Mix 0.17 0.5 0.83 1.3 1.7

1.0 M Tris (pH-6.8) 0.13 0.38 0.63 1.0 1.25

10% SDS 0.01 0.03 0.05 0.08 0.1

10% APS 0.01 0.03 0.05 0.08 0.1

TEMED 0.001 0.003 0.005 0.008 0.01

Sample preparation for SDS-PAGE

Aliquots of 1 ml of freezed dried culture filtrates were suspended in 0.1 ml of 125 mM

tris pH 6.8 containing 0.1 % SDS, 20 % glycerol, and then applied to 12 % polyacrylamide

gels.