MATERIALS AND METHODS

CHAPTER 3

RIATERIALS 4 N D hlETHODS

3.1. hlaterials

3.1.1. Chemicals

Llcd~a conlpunenrs, protcose pcptone. trjptoiic. )c3ht c\tract, heef extract.

ulkccrol, agar and othzi cheni~cals. , o l i ~ o ~ n cl1l1,ridr. ammonium cliloridc. d ~ s o d ~ u n i - .

l iyd~ogc~i phosphate. dlpowsilum hydrogen pIiospIint~' a11d n l a g n e ~ ~ ~ l n i s~ lp l ia tc were

piirchaszd frum HI-Mcd~a. India ' N lahelsli amnionium chloride \\as purchaacd from

C'ambridye isotopc lah<~rator~ur. 1IS:i. Organic salient\ such a\ bcltrcilc, ethyl acetate.

mcthenol, chloroibrn~. d~chloroniethanc, dlcthkl cthcr. acctonc arid ilcelon~tr~lc a r r u

porchascd from Sisc(1 Rcscarch Lnhorntories (Sit1 ) and Quol~gcn\. lndla All col\cnts

2nd reaycnth uccd a c r e anal>ticnl or I~quid chronintog~.aphic grade chemicals.

('lirumatographic materials such a\ prcpdr~t i \c ti1111 l a y chromatograph\. ( 1I.C) p l a t o

dild silica gel (100-200 mcsh) \\tic auppl~cd b! F~,chi.r S~icntil ir . . 1 S A and Sl) line.

Ind~a respectively. PCR reagents such as laq DNA p~l!mcrasc. lnagneslum chlor~dc. 10x

reaction buffer and dNTPs \\ere purchahed Tram Promcga. 1IS.A and the 1)yNAzyme

I)UA pol>merase and t X T b u f i r a a s purchaxd frr~ni I in/)mcs. Finland

3.1.2. Microorganisms

Fungi. Mucrophorninit phusrolino MI'S. !%fugnoporfhr grrreu MGS.

Collr~o~richurn ,/n/caturn C'1:l.. C, glcosporotilc~s ('(;I.. C (upsrci ('CL.. Rhi:ocfoniu

soiani KSRI. F u s n r i ~ i n ~ orrsporuttz f. sp. ~rihcrt.\(~ FOC. F o\lsporldm 1: sp. ~ ~ i i s i t ~ r ~ u n ~

1 OVS. Sirroclirdiiini o~?,:(!c SONS, Borr:i,~is cincru BCI 'NAI ' and Pcsriil~iiia f h m ~ PI'S

a z r e obta~ned finin the culture collecr~on of 1)epartmmt ol'f31otechnolog). Pondicherry

Ilni\crslt\.

3.1.3. Cancer cell lines

('olon canccr cclls. S\V4RO, lung cancer cell$ 4530, hrcnst cancer cclls. MCP7

and cer~ica l caiiccr cell\. Hei a ~ c r c obtn~ncd l i u l n tllc hatlondl rcnt re Ibr ( cll Scicncc.

I'une. Indla.

3.1.4. hledia

King's medium B (KB) (King 1951)

Protcusc peptone 20 g

Cilycerol 1.5 ml

K2HP04 1.5 g

MgS04.?1I?0 1.5 g

Uistillcd \\atcr 1000 ml

pll adjusted to 7.0 using NaOll

King's medium B agar (KBA)

1.5% of agar \rns added to KH hefore autocla\ing

Lh-anner medium (Kanner 1978)

NazHPO? 4 g

KzHPOl 1.5 g

NH4C-1 1 .O g

MgS04.7t IzO 0 . 2 g

Ferric citr-ate 0.005 g

pll was adjusted to 7.2 using NaOH

Distilled water- 1000ml

liutrient Broth (NB) (Atlas 1993)

Pcptonc 5 g

Na('1 5 g

Hoef'extrnct 1.5 g

Y cast extract 1.5 g

pi4 adjustcd to 7.4 with NaOH

Distilled water I000 nil

Potato dextrose agar (PDA) (Atlas 1993)

Potato infusion 200 g

Cilucosr 20 g

Agar 1.5 g

Distilled water I000 mL

Dworkin and Foster (DF) salt medium (Atlas 1993)

h19 minimal medium (Miller 1974)

Thiani~ne I IC I sol utlon ( 1 0 0 rng 1 I00 m l )

0 2 g

1 niy

1 0 PC

1 0 pg

70 Pf

5 0 Pf

1 0 pg

1000 nil.

1 nil

1,uria Bertani agar (LBA) (Atlas 1993)

Tryptone

Yeast extract

NaCl

Agar 15 g

p H adjusted to 7 2 with NdOII

D~stllled water 1000 rnL

plkovrkayd's agar m e d ~ u m (Plkovskayd 1948)

Yeaa extract

C alc~um phoiphdte

Ammon~um sulphatc

KC I

MgC l

M n \ 0 4

r ~ r r o u s sulpl~dte

Agdr

I I I \ ~ I I I L ~ WLLLLI.

3.1.5. Buffers and solutions

DNA extraction

Glucosc 0 99 g ( 5 0 m M )

Trls 0 3 g ( 2 5 m M )

FDTA O 7 7 g ( 1 0 mM)

Adlusted to pH X 0

Double dlbtlll~d w d t ~ r 1 00 m 1

Sodlum dodecyl ~ u l p h a t e (5DS) 20%

20 g of SDS was dlssolvcd In 100 ml o f douhlr d i s t ~ l l ~ d water

2.5 rng of KUase .4 u a s dlurol!ed in 1 ml ol' 10 mM I rih (p t l 7 . 0 ) dnd h[~tlcd at

7O"C l'or I0 rnln.

1X.i11 g o f b l 1 l . l u a \ d l s s o l i r d in 100 nil ul'douhlc dtstillcd i\atcr

0.12 g of Irir mas d!\\ulicd [ti 100 mL ol double disttllcd watcr and aillusted to

pH 7 h using I [('I.

Sodium acetate (3 \I)

10.2 g of codllim acetate was d~ssol \cd In 25 ml. ol'douhle d ~ s t ~ l l e d water

Buffered phenol

Distilled phenol was extracted once with eqlial volumv ol' 1 M Iris II('I hufl'cr.

'The upper aqueous phase u a a dlscardcd and the phenol u a s again extracted w ~ t h 0.1 M

Tris until pH 8. To thc phcnol. 0. 8-hydroxyquinoltnc (0.1'%1) u a s added.

Chloroform : lsoamyl alcohol

Chlomfom 24 ml

lsoamyl alcohol I mL

'TE buffer

'I Iis

I 1) 1.4

pH adjusted lo 8.0

D \ 4 loading d!e

Glycerol SOUb

k1)TA pH 8.0 0.2 M

Hromophcnol blur 0.0.5"/~,

Kthidium hrornide

10 mg of rthidium brom~de nab dlssol\'sd In 1 ml. ol'uarzr.

'T4E buffer 50 X

l r l r base 242 g

Glacial acetic acid 57.1 mL

O.iM E.1) I A. pH 8.0 1 O i l mL

Adjusted to pH 7.2 and final volurnc madc up to 1000 mi uvng dluilled watcr.

3.1.6. IAA estimation

~ a l b o n s l i i ' s reagent (Bric et al. 1491)

Conc. II-.SO? 150 mL

Diar~lled water 250 mL

0.5 M Tc('li.6H:O 7.5 mL

3.1.7. Cytotoxicity assay

I'BS

C r o n t h medium

T r y ~ s l n 0.25°;,

LDT.4 1 mh4

Thc a h o w component5 werc dissolbcd In PHSA

hlTT

5 mg of MTT was dlssol\ed in I mL of double dlatlllcd >+ater and filler stcrillred.

MTT lysis buffer

209, SIX In 50% d~mcthyl f o n a m i d c

3.2. Methods

3.2.1. Maintenance of bacteria

Rdctenal culturcs were maintained o n KBA plates in a rzfngerator fiir routine use.

I or lung lerm storage. the cells ucrc suspenrlcd In ster~le uatcr and htvicd at 4°C' or ucrc

prc\t.ncd in 40°0 glycerol at -7O'C.

3.2.2. Maintenance of fungi

rung" culturcs n r r r mnlnlaincd on I'I)A pldti.5 In a refr~gcrator fbr routlnc we.

I ~ i r long term storage. the culturcs were maintamed oil PDiI ~ l a n t s In a stenle test tuhc at

4'C

3.2.3. Sterilization

All ths m e d ~ a , h u f i r a and reagents used in )hi+ ?tidy ibsrc stcril~/ed st IS

Ih? Inch' for 20 min u n l e s ~ othcruise specified. Thc chemicals u111ch M C I ~ i"ound to he

h u t lab~le wcrc filter sterillrcd uslng 0.2 p filter (Milliporc. Molahclm. F r a n u ) .

3.2.4. Isolation of fluorescent pseudomonad bacteria

A suspension of rlce rhirosphere soil was obta~ned by shaking 10 g of root plus

1~~1,tl) odhcnng soil in 90 ml 0.1 M 41gSO1.7I420 huiftr for 10 min at 180 rpm In a

lovary shaker. Hundrud ~nlcroliters oC the ruspenslon nay spread onto KHA (King et al.

1054) and ~ncuhatrd at 27°C for 2 days. Srnglc colonies that fluoresced undcr IrV hpht

,?oh nm) Mcrc s~raahed onto KHA tor ohtrlning purr cultures.

3.2.5. Screening of antagonistic fluorescent bacteria

Standard agar plate bioassay Mas h l l o a e d to acreen the antagonist~c fluorescent

p\cudomonads (Sakthivel and (~nnnaman~ckam 1987). Hr~etl), hoctrrial plugs o f agar

ucre r c n ~ o ~ e d from a 48-h cnlturc and transrerred to potato dcxtm\t: agar (1'I)A) plates

ah101 has hccn spray ~noculatcd prev~nuslj u ~ t t i a lilngal spore whpension (10"

sonidia niL). I he zone ol' i n h ~ h ~ t ~ o n around the hactrrial plug was n~rasurcd aiier 3-4

da!, lncuhat~on of assa) plates at 2SuC.

3.2.6. Experimental design, data collection and analysis

Experiments were set up in a completely randomized design with 3 replicat~ons

lor each treatment. Lkta on lone of inh~bition and cell ~ ~ a b ~ l ~ t y \\.ere recorded 3-4 days

afier incubation. Mean and standard error ( S t ) were calculated and differences hetueen

means were tested using Duncan's multiple range test at the level o f p = 0.05.

3.2.7. Taxonomic characterization of antagonistic strains

Taxonomic characteri~ation of the hroad-spectrum antagonistic hactena %ere

Ljollc on the h a i s of routine hlochemical tests ~ u c h as fluorescence on KHA. c!.tochrome

,,*idn~e, arglnine d~hldrolasc , nitrate rcduction. gelatin hydrolysis, levan production.

l~tiltiatlon oi' carhon source such as glucosc. I.-arahtnuse. werose. sorhilol, er>thrltol.

~n~ann~tol. nlaltose. adonitol, citratc, trehalosc. glycinc, phenyl acetate, hlppurate.

tnlcntinure and paraffins (Staniur n al. lY60: C'hamp~on st al. 1980: Barett ct al. 1986,

I r 0 ~ ~ 1 , ct al. 2000). Keaults of rhcsc teats \%ere acored aa cither p o s ~ t i w or negatibc. Cell

m(~rpholopy. (,ram statning. growth at .?"C and 4?"C %as determ~ned fo l lou~ng standard

nierhods.

3.2.8. Isolation of Cenomic DNA

Total genomlc DNA u a s extracted as described (Leach ct al 19'12: Sakthivel et

dl. 2001). Bacteria wcrc grown for 24 h 111 1.5 mL YB at 28°C on a rotary shaker at 200

rpm I he cells were pelieted at 10.000 g for 5 min In a mlcro centni'ugc tube. 1 he pcllet

\<as resuspended In 330 pL uf solution I and ~ncuhated for I0 nun at room temperature.

l a this, 8.3 pl. o f 2 0 % SOS mas added and incubated at 50°C for 10 min. Tu the mixture.

13 11. of Rhase A was added and after 1 h of incubal~on at 37°C. 17 pL of 0.5 M bD1 A

'+as added and incubated at 50UC for 10 min. ' lo \he lysdtc. 10 pL of pronase was addcd

and lncuhated at 37°C for 3 h. This was extracted ta ice with equal volume of buffered

phenul by centrifuging at 10,000 g for 10 min at room temperature. The aqueous layer

ohtJlnzd was extracted w ~ t h equal volume ufchlorof'olm 1soain)1 alcohol by centnfuglng

,t lo.(i00 g for 10 mln. l o thc resultant aqueous layer. 50 flL of 3 hl amrnonlum acetate

1,000 pL of 95"e ice-cold cthanol mas added and rn~xed uell to precipitate the DNA.

l h e 1)NA \\as pelleted at 10.000 g for 5 min and thc pellet u a s bvashcd in 70% icc-cold

C~IWIIOI. Thc pcllet was then dricd In a I)\A speed \ac system ('I henno Sabant 7hcrmo.

I'orL, l lSA) and dissohed in 5 0 fiI. of I L.

3.2.9. Quantification of DNA

l r ~ i n~icrol~tcrs of DNA sample mas nilxcd a i t h 990 gL of' TE buffer and was

read at 260 nm in a spectrophotomckr iBecknian Cuulter. ITS:\). An O D balue of one

correrpond to approximately 50 pg.rnL double strandcd D S A (Sanibmok et a1 IOX'I).

l ioxd on the 01) \slue, DNA samples ucrc quantified.

3.2.10. Purity of DNA

Purity of 1)YA samples \ l a$ e~timated bawd on ratio between OD at 260 and 280

nm. I'ure Oh'A sample has an OD value 2601280 hetween 1.8 to 2.0. Contamination r i t h

Protrln or phmol reduces the value (Sambrook ct al. 1989).

3.2.1 1 . 16s rRNA gene sequencing

,Amplification of 16s rRNA gene from the genomlc DNA of bactcria was done as

Je,crlbed carlier using unt\ersal pnmers. fI)l (5'-ACil i'T(iATC'CTGCiC7C.A-3') and

(5'-:\C'GOCTACCI'I(iT~IACGACTT-3') (Weisburg ct al. 1991). The reacclon

I , I I \ I U I ~ (50 PI.) consisted o f 5 pL IOx I'CR reaction buffer, I U L of' 10 mM dNI'P mi\. I

1 01 T(1i1 IIN.4 polymerax. 3 )11 or25 mM MgCI:, 50 pmol of each pnnier and 50 ng of

~cmpldtc DNA. l l i r final \olume u a s made up to 5n 111 u\iny sterile double dlst~lled

uslcr The program conslhlzd of Initial dcnaturatlon at '11°C h r 1 min. 30 cyclca of

ilcnaturation at 94°C ibt. I min, annualing at 46°C' [or 3 0 sec and extension at 72°C for 4

in1111 n ~ t h a final cxlcnsion at 72°C' ror I 0 min. Ihc ampl~fted DNA was purificd using

tillcn~con" PC'R purificduon ds i ice (Villiporr Corporation. Bedford. MA. I I S A ) , diluted

ti1 LOO ng'pL, and \,as sequenced uslng the hcilit) ac Microsynrh Inc.. Balgach.

5alt7erland. Similarity searchc\ of ' th r qrquencc ohtamed u a s done using the BLAST

i.-\l~achul et al. 1990) function of CienBank and S~niilarity Rank of the Ribosomal

I)atabasc I'roject (KIIP) (hlaidack et al. 1997).

3.2.1 2. Production of extracellular fungal cell wall degrading enzymes

Production of cxtracellular fungal cell u!all degrad~ng enzymes, cellulase and

Pcctlnase was determined ar described e a r l ~ r r (Cattelan et al. 1999). M9 medium agar

illlller 1971) amended with 10 g of cellulose and 1.2 g of yeast extract per liter of

dl5tlllrd watcr was used to test the cellulase activity. iit w h ~ c h clear halo after 8 days of

of the colonies at 28°C was considered as pohlt~ve for cellulase production.

I detenninlng the pectinase activity. I0 g pcctln plu? 1.2 g yeast extract was amcndcd

~1') nled~um agar and the plates wcre floodcd w t h 2M IICI after 2 days uf ~ncuhatlon

28'C. Clear halos around the colonies uerc cons~derzd as pos l t~ce for pectinase

p~.o(Iuctlon.

3.2.13. Determination of plant growth influencing metabolites,

hormones and enzymes.

3.2.13.1. Hydrogen cyanide (HCS)

I'roductlon of tICN wah carr~rd nut ibllouiny standard method (HaLLer and

Sch~pper\ 1987). Singlc colony n a s strcahud onio KBA amcndcd with 4.4 g:L glycine. .4

'1 cni d~amctcr &'hatman No. I filter papcr disc soaked in 0.5'X picric a c ~ d in ?'%I sodium

i~rhonatc waz placed onto thc lid of the I'etn plate. The Petri platc n a s sealed \\it11

pardiilm and incubated for 4 d a y at 28°C. The change of coiour of' filter paper from deep

!~llou to orange indicated the product~ori of H('S by the bacterium 4 n unlnoculated

plate u a s used as control.

3.2.13.2. Phosphate solubilizing enzyme

In ordcr lo dctcrmine phoqpharaic cnzlme that minerail7es organlc phosphate,

p~hin\ka)n ' s agar medium conta~niny tlicalciunl phosphate was used. Agar plates were

.pof innoculated a ~ t h the test hactcnal strains and incubated for 2 - 5 days at 28°C. The

,Is\c.lopmcnt of clear 70nc around inociilatlon site naa considered as an index of

soluhillration (Pikonkoya 1048).

3.2.13.3. Indole-3-acetic acid (IAA)

The production of I 4 A %as dctcmincd by using standard mcthod (Bnc el al

lC)OI) Single colony isas streaked onto I HA amended uith 5 mhl L-1r)ptophan. 0.06'%,

,udiorn dodccyl suiphatc and I n u glycerol. Plates here o\erlaid uilh Whatman no. I tiller

pdprr ( 8 2 mni diameter) ~ n d thc backr id alrain \%as alloi\cd lo gro\b fur a pcriod of 3

(ldys. i\ftcr the lncubat l~n pcriod, thc pdper u a s remo\ed and 1redtt.d nith Salkowski's

lcnpent (Gordon and Wchcr I q 5 l ) u11h rhe forn~ulation 2Do of 0.5 M ferric chlor~de In

ji'% perchloric acid. Membranes here saturetcd in a Pctn dlsh hy soaking dircctly In

Salkouski's rcagcnt and the production of 14A h a s ~dcntificd hy thc formation of o

characteristic red halo nithin the memhrenc immediately surrounding the colony.

Quantification of IhA u a s done folloning colorimetric method described earlier

(Pattzn and Glick 2002). A single colony of the bacterium was propagated overnight in 5

mL of I)F minimal salt medium and 20 $L of al~quot was transferred Into 5 ml. of L)k

ll,,nlmal salt medium amended with 500 fi&'mL of L-trqptophan. Alicr 40 h of growth at

1 5 , ' ~ in a rotary shaker at 180 rpm, the cells uerc pcllctcd at 5,000 g for 10 min. 70 1 ml.

,I the supernatant, 4 mI. of S a i k o ~ s k ~ ' ~ reagent was added, mlxed well and allowed to

, t~nd at room temperature for 20 mrn. The absorbance u a s measured immediately at 535

nl~,. An uninoculated control uith S a l k o u s k i ' ~ reagent was used as referalcc. I h e

iolicetltrotioll of IAA \%a> determined on comparing with the standard c u n c .

3.2.14. Extraction and purification of antibiotics by the antagonistic

strains. PUPa3 and PL23

3.2.14.1 Fermentation Media

I.or the product~i~n of anlibiotics by the atrarna. PCPa3 and P1123. PPM (Rosalcs

cl 41. 1995) and Kanner medium (Kanncr et al 1978) was uscd rcspcclr\ely. In order to

ohtsln ' N labeled antibiotic horn strain 1'1123. amm<)nium chloride in Kannsr medrum

iias suhsrltuted a l t h "N ammonlum chloride.

3.2.14.2. Extraction and purification of' antibiotic by the strain PLPa3

I h e strain PUPa3 was &Town in PPM for 5 days a1 28°C. The fermentat~on broth

"as then centrifuged at 10,000 g for 10 mln in order to remove thc cells and the

Yupernatanl (10 1,) was extracted into equal lolurne of ethyl acetate. The comhlned

,,li.nnic layer u a s concentrated under reduced prcssure and the crude extract u a s tested

hroad-spectrum actlblty against the f u n g ~ listcd carl~er. I h c crude extract uras

,l,romato&~aphed over slllca gel eluting with chlorofbrm. chiorofcirm-methanol mlxtures

,s I ) and finally with methanol and was further punfied using chromatotron. u ~ t h j0/,,

nlcrhanol in chlororonn as sol\ent slsteni. 7liese purified fractions were tested for

,il!t~lbnpal acti\lt) using .if. pha.seolina as the rest fungi following agar diffusion method

~ ( ~ i ~ r u s l d d a ~ a l ~ et 81. 1986). Actlve finct~ons \\ere poolcd trigcthcr and conccnaatcd under

icdu~cd nressurc.

3.2.14.3. Extraction and purification of antibiotic by the strain PC23

Ihc strain PL23 \%as grown in Kanner m e d ~ u m for 120 h at 25°C. The

lzrrnentation broth Mas centnfugzd at 5.000 g ibr i nun 111 order to remo\c cells. To the

\ilpcrnatant (4 1) equal iolume of ethyl acetate war addcd and mincd in a rotarq ahakcr

lor 1-2 11 The resultant emulsion war then filtered in cheesecloth and ih i aqucoua layer

1\35 separated in a separating funnel. The crude ant~biotlc was rzco\crcd from organlc

Idler hq cvaporauon in a rotary evaporator. I hc antibiotic a a r adsorbed on a sillca gel.

dpplied to a previously packed silica column and eluted with chlorulom. Thc a c t h e

fructlons h e r e collected, applicd onto the prc-coated preparative thin layer

chroma[ogaphy (I1.C) plates and developed with sulvent system of chloroform-acetone

('1 1). I he plates were examined under LIV at 254 and 365 nm. ?he active greenish

!tlloa spot was scraped and extracted from s~l ica gel using chloroform. The purity of the

~ltlhiotic was confirmed by high-performance liquid chromatography (HPLC). .A slngle

\\,Ic detected in a Phenomenex Luna (2) ('18 reverse phased column (250 mm X 4.6

,,1111) "hen aceton~trile and uater (both containing 0.1% tntluoroacetic acid) in a 30 to

- r r o o Ilnenr gradlent u a s uszd as thc solvent qyqtem with a flow rate of 0.7 mlimin

(Thrrrnashow el al. 1990). The chromatogram was detected at 254 nm.

3.2.15. Structural elucidation of the antibiotics by the strains, PUPa3

and PL23

3.2.15.1. Fourier transform infrared (FT-IR)

To analyze tlic functional groups of lhc purllied antibiotic, FT-IR spectrum \\.a7

rzcorded using the Shlmndru 1'1-II~-1(300~8700 (Shimadlu, lokyo. Japan). with a

rcholi~tion of 4 cm, auto g a ~ n and at1 average o f 4 0 scans in the frequency range of 4,000

'00 cm I . L)ncd sample was loaded d~rectly and thu spectra Rere rrcordcd at room

temperature.

3.2.15.2. Mass spectral analyses

Electron Ionization Mass spectroscopy (El-%IS). The El-MS datd waa recorded

using Finn~gan MAT 1020 (Thermo Electron Corporation. San Josc, CA. LS.4).

Fast atom bombardment mass spectra (FAB-MS). The FAB-MS Fpcctra ucre

rrcllrded on a JOEL SX 102lDA-6000 mass spectron~eter uslng argon (6 kV. IOmA) as

tile FAB gas. The accelerating voltage \>as 10 kV and the spectra were recorded at room

tc"lpcrature, m-n~trobenryi alcohol (UBA) u a s used as the matrix.

Electrosprag mass spectra (ESI-\IS). 'I hc tSI-MS spcctra were recorded on a

\iI('KOM:ZSS QL:ATTRO I1 triple quadrupole mass 5pcctromctr.r. Ihe sample was

,ii,sol\ed in aceton~trile and introduced into 1:SI source through a syringe pump at the

~ ~ t e oESp1. min. The ESI capillary n a s set at 3.5 kV and the cone \ulidgr waa 10 V. Thc

q c c a a were collected in 6 s scana and rcwlt uos an abcragc spcctrum of 6-8 scans ['or

rllc \ I S M S spectra of EST-MS the cap~llary was sct at 3.5 k\' and the cone bollape way

511 \ , Argon u a s used as the collis~on gas at a prcsaurc such that the parent ion heam

~ntcns~ty was reduccd to 50''. The collision energy u,as 5-30 c\' u a s rccorded In the

spectra. The spectra a c r c collected in 2s scans and thc rchults wcrc axraged spectra of

25 rcana.

3.2.15.3. Nuclear magnetic resonance (NMR) analyses

One and two d~mensional NMR spcctra of the pur~fied antibiotics were recorded

on a 'state of art' 600 MHz Varian Lnity-Plus spectromcter (Vanan. Pal0 Alto C'A.

1 SA), operating at 499.96 MHz for proton and 50.68 MHz for ' N respectively, with a 5-

1nM trlple resonance Inverse detect~on probe. NMK data set was acquired at 27°C uvng

( DC1, as sol,ent. 'Two-dimensional l ~ - l l i 2D doublc quantum filtered correlat~on

,pcctroscopy (LIQF-COSY) (Piantini ct al. 1982) and the total correlation spectroscopy

( r ( K 5 Y ) methods were employed to resolve the complex ' H K41R signals. 'The

,~li.mical ~ h i f t assignments were confim~ed by a comparison of NMR slgnals In I D

jpcztrum w ~ t h those in 21) L)QI,-COSY and 7 0 T S Y spectra under sltnilar expenmental

,,,nd~tiona. .I11 2D NMR data sets a e r e collected In hyper complex phase acnsilice modc.

\cqulsltlon parameters includcd n 7iiOO Hz spectrum w d t h (SW). (1.4 s acquisition time

,.\TI. 9.Ous as 90" proton pulac a ld th and 2.0s pulse delaj and 16 transients per

~nclznicnt 'H- 'H 2D- TOCSY spectra ucrc recordcd urlng M I 1.V-I7 (Hal and Davis

IOhO fur so tropic miring for 70 ma at a HI field strength o f 7 KHz. Behre processing.

IIIC I? d~menslon ofl)Ql'-C'OSY darn sets were zero-filled to 8K and thc 11 dimension of

1)01 ( 'OSY data sets to 4K. All other evpenments s e r e zero-filled to 4K. When

ncczsiary. spectral rcsolut~on was enhanced hy I.orenztan-(jaussian apodlration and

( ' l ) i l : pcak at 7.26 ppm, \%as used as a chemical shift referencc. Proton decoupled '"4

\ h l K rpectrum u a s recorded using Cl)C'I, as the s o l ~ c n t .

3.2.15.4. Molecular modeling of antibiotic by the strain PLl23

For molecular modeling studies of thc antibiotic by thc strain P1J23. Ah rnilio

calculations were performed using Juguar-v4 ? from Schroed~nger Inc. and Gaussian98

ILaursian Inc., Wallingford. Cl'. LSA) . Geometry optimizations and energy

nlinlm~zations for monomer as well as the three dimcr conformations were performed

UUng B31.YP.6-31G* basis sets. All the energyminimized structures were further

charactcr17cd to be stable molecules by computing the second deri\atives of the energy.

3.2.16. Isolation and characterization of genes involved in the

11ios)nthesis of a novel dimer antibiotic by strain P.Juorescen.s PU23

3.2.16.1. Amplification of phenazine gene cluster and agarose gel

electrophoresis

The phrnarlne b~oaynthct~c gcnc cluster uns nnipl~ficd l i o n the yenomic DN.4 of

,[rain PL'2.i using the gene-rpecllic prlmcrs. P h ~ r (5'-TA:IGGII I CC'CiCi IAGTTCC'AA

~,CC'CACiAMC'-3') and l'hzI< I~'-C~CTTCTAC~AATCC~.-~.~C'AGCG~~AAC:~C~GCACA

( ~ , - 3 ' ) The reactlon mixturc (25 p1.I c i~ns~sted of Ix LYT bulTcr (f'in/ymes. Finland). 2

mM MgC12. !'V,, IIMSO. I 0 mhl cacb d N I P, 2(1 pniol of each primer. I 11 of D y N A / y i e

i l ~n/)mca. Finland) and 20 ng of ~ c n ~ p l a t e DNA. The nmpl~fication Mas performed on a

I'erkin Elmer GeneAmp PC'R ayslem 2400 (I'crkin Elmer. Rothrcu/, Si+it7erland) using a

30 cyclc program of denaturatlon at 04°C tor 30 scconds, anneal~ng at 64°C for 30

\cconda and extcnrion at 72'C for 7 niin with an inltial denaturation at 94"(' tbr 1 min

dnd a final extension at 72'C for 10 min. A 3-pL al~quot of ampllficatlon product was

s lcc t r~~horesed on a 0.7OLl agarose gel In I x 7.4E buffer at 50 V for 45 mln, stained w ~ t h

ctli~d~urn bromide, and the PC'R product was viauali7ed under UV.

3.2.16.2. Sequence analyses of phenazine biosynthetic gene cluster

I'he amplified DhA uras puntied using ~ l c r o c o n " PCR purlficatlon d e v ~ c e

i\lillipore Corporation. Bedfurd. M A . IlS.4). diluted to 200 n g p L , and was sequenced

ublng the facility at Micros)nth Inc.. lialgach. S\\itscrland. ' lhe ssquences were

ds,crnhled using the program "Seqald". Sim~larity searches of the sequence were

p ~ ~ f o r n i e d uzlng HI.AS I (Altschul et al. 1990) funcilon ofC;unHank.

3.2.17. Antimicrobial acthi@ of the purified antibiotics

I o test the hroad-spectrum antlmicmhial activlt! of the purltied antibiotics, agar

citnilcion mcthod (Ciurusiddaiah er al. 1979) was I'ollo\~cd. Stenle paper discs ih mm)

\*err separately treated wlth diffcrcni concentration of nntihiot~c (2.14 pgniL) and

ylaccd iin the surface of P D A agar plater thar u e r r sprcad-~noculatcd with conidial spores

I 10'' mL) of test fungl, Assily plates \\ere ~ilcuhated at 28°C' fur 3 ddyi and the 7one

po\vth lnh~bition mas measured or hllC \%as iietemiincd in cazc of thc metahol~te from

\trnni 1'1123.

To evaluate the pH dependent activity of the antlhiotic by the strain P1123, the

.issays Here done on PI)!\ plates with pll range of 5 . 5 , 6 . 5 . 7.5. 8.5 and 9.5, uslng M.

l~~l~iic~olinrr M P S as the test ftmgus. hntimicrohlal acti\ity of monomeric phenazlne-1-

"rhoxyic acid n a s compared with antibiotic by the stratn PU23 usmg .S. onzue SONS.

3.2.17.1. Scanning electron microscope (SEM) analyses

In order to nna1)ze the morphological changes in the niycclial grouth and

spon~lat~on. SEM analy~es were camcd out. For SPM analyses. mycelial hamples from

111c peripher) of the rone of inhibition and control plates were mounted directly on

sciricli douhle-adhewe tape, coated w ~ t h gold to a thickness of 100 A" using Lacuum

cloporator ( H ~ t a c h ~ . 1ILS 5 GH, IoLyo. Japan). ('oated aaniples a e r e nnnljred In a S1.M

( 1 l ~ t ~ i l i l . S-40 . l ohyo. .lapan) operatcd at 15 L\' and at a resolution of 1000x.

3.2.18. Anticancer activity of the antibiotic by the strain PU23

3.2.1 8.1. Cell viabilic assays

Cellc line. SIV480. Ai.49, MC'l:7 and HeLa ~verc lcbtcd Car ccll biahility follouing

tlir modified method of Anto et al. (2000). Brlefly, cclls grown in 96-well microtitre

pldlex (5.000 celis'well) \\ere incubated for 18-h uith or w~thout different concentrations

ol' the alilihiotlc b! the saain PI123 (0.5 5 pM). \licortitrc paltcs (5000 cells'uell

uilhout a n t i b ~ o t ~ c sewed as control. Then the medium was removed and tiesh medlum

aaF added along with 20 pl- of 3-(4-5 dimethylthioiol-2-41) 2-5 d~phenyl-tetrazolium

bromide (MTT) (5 mg mL) to each hell. I he platcs uere incubated for another 3 h and

the formalan crystals formed were solubilized with MTT lysis buffer. The plates uere

placed protected from light. overnight at 37°C in on incubator. The color developed was

quantitated (measuring wabclcngth: 570 nm, reference wavelength: 630 nm) a ~ t h a 96-

n ~ l l $ate rcader (Bio Rad, USA). 7 he cell v~ablllty was expressed as percentage over the

~ i ~ n t r o l .

3.2.18.2. Acridine orangelethidium bromide and annexinlpropidium

iodide staining methods

Lung cancer cell?. 4549 (5.000 cclls:wcll) Rere cultllred In a 96-\bell plate and

~rc,itrd irlth the antihiot~c hy the strain 1'1123 in dirrcrcnt conccntratlon\ fur 24 h.

Il~cottitru p a l t e ~ (5000 cells uell uithour antlhlotlc acned as control. iifrcr \ \ash~ng once

uith pliosphate-bufkrcd snllne (PBS), the cells were stained u ith 100 pL of a mixture

11.1) of ncndine orange-cth~dium bromide (4 p g m L i solutions. The cclls uerc

immcdlarcly washcd once \ \ ~ r h PHS and vicwtd using an i n \ t n c d fluorescent microscope

I t,clip\c rl:!00. \ikon. 1 oi\)o. Japan).

I.or anncxlnpropldiuni iodide stalnlng, the A549 ccllr were seeded In a 96-\\ell

pldte (5.000 cclls'well) and treated with different concentrations of the ant~hiotic hy the

rtrdln PC23 for I h h. The cells were ujashed once with PBS and subsequentlq trealcd

~ l t h I X assay huiier, annexin-fluorescein isothiocyanatc and prop~dlum ~ o d l d e as per the

protocol described In the annexln V apoptosis detection kit (sc-4252 AK) (Santa CNZ

Hiotechnulogy, Santa C'ruz, CA. liS.4). After 10 niin in dark, the cells uere washed with

PBS and the green~sh apoptotic cells were observed uqing an invened fluorescent

nllcroscope and photogaphed.