Toxin production in Alexandrium tamarense Investigating ... · Toxin production in Alexandrium tamarense: Investigating clonal variation and ... (Jakim and Gentil 1968 ... with artificial

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University of Bremen and

Max Planck International Research School for Marine

Microbiology

Toxin production in Alexandrium tamarense:

Investigating clonal variation and extra-cellular

bacterial effects

Chibola Chikwililwa

Master Thesis

First reviewer: Allan Cembella

Second reviewer: Uwe John

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Index

1 . Summary 4

2. Introduction 5 3. Thesis object ives 9

4 . Materials and methods 10

4 .1 Sampling s i te 11

4 .2 Strains and cul ture condi t ions 11

4 .2 .1 Axenic and xenic cul tures 11

4 .2 .2 Sub-clone cul tures 13

4 .3 Sampling procedure 14

4.3.1 Axenic and xenic cul ture 14

4.3.2 Sub-clone cul tures 14

4.4 Cel l and bacter ial counts 15

4.4.1Cel l counts 15

4.4.2 Bacterial counts 15

4.5 PSP extract ion and determinat ion 16

4.6Allelochemical t es ts 21

4 .7Amplif ied Fragment Length Polymorphism 22

4.7.1 DNA extract ion 22

4.7.2 DNA Restr ict ion 23

4.7.3 DNA Ligation 23

4.7.4 Preamplification 23

4.7.5 Amplification 24

4.7.6 Capillary electrophoresis 24

4.7.7 AFLP Analysis 23

5. Results 26

5.1 Axenicity of cultures 26

5.2Growth of Axenic and Xenic cultures 26

5. 3 Production of PSP toxins by axenic and xenic cultures 30

5.3.1 Toxin profiles 30

5.4 Toxin content verses cell growth in axenic and xenic cultures 34

5.5 Total toxin content 38

5.6 Allelochemical tests 39

5.7 Sub clones 39

5.7.1Toxin profiles 39

5.7.2 Variations in total toxicity 42

5.8 AFLP 45

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6. Discussion 49

7. Acknowledgments 59

8. References 60

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Su mma r y :

My thesis investigated the role of extra-cellular bacteria on the PSP toxin producing A

tamarense. Results showed that bacteria have no effect on the dinoflagellate growth rate.

The toxin profile remained relatively constant through out the experiment. Three

samples exhibited low production of GTX1/4. This could be further investigated to

determine if these cultures lose the ability to produce this STX derivative.

Bacteria have no effect allelochemical produced by A. tamarense. This effect was seen in

both lytic and non-lytic strains. The toxin profile remained constant for sub-clonal

cultures, signifying that PSP toxin production is in actual fact a genetically determined

characteristic. Variations in toxin content were observed, but they where not large

enough to claim that PSP toxigenesis was non chromosomally linked.

There is still

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1 ) In t r odu ct ion :

M a r i n e e c os ys t em s h os t a l ga e t h a t p l ay a c r i t i c a l r o l e a s t h e so u r c e

o f p r i m ar y p r o du c t io n . T h e y n o t on l y h a v e a b e ne f i c i a l ro l e , t h e y

c a n a l s o b e d e t r im en t a l . On e o f t h e w ays o f do in g t h i s i s b y c a u s in g

H a r m fu l Al ga l Bl oo ms (H A Bs) . H A Bs a r e a wid e sp r e ad m a r i n e

p h en om e na t h a t c on s i s t o f up t o m i l l i o ns o f c e l l s pe r l i t r e a nd c aus e

n um e ro us p r ob le ms to h um an h e a l th , f i sh e r i e s r es ou r c es a n d m ar in e

e c o s ys t e m s . Th e y o c c u r i n c o as t a l w a t e r s wo r l dw id e ( H a l l g r a e f f

1 9 93 ) a nd ex h ib i t va r yi n g e f f e c t s on th e a re a wh e r e t h e y a r e fo un d .

T h e i r e f f e c t r a n ge s f r om s t r i pp in g t h e w a t e r co lu mn o f oxyg e n ( d u e

t o t h e h i gh b iom a ss c a us i n g t h e d ea th o f mi c r o f lo r a a nd m a c ro f a un a )

t o p ro du c i n g p o t en t t ox i ns t h a t c au s e ga s t r o i n t es t i n a l an d

n e u ro l o g i c a l i l l n es s i n hu ma ns (H a l l gr a e f f 1 99 5 ) .

P a r a l yt i c sh e l l f i s h p o i so n i n g (P SP) i s o n e su c h n eu ro lo g ic a l i l l n es s .

I t i s s o c a l l e d du e to t h e s ym p t oms exh ib i t ed b y h i gh e r an im al s ,

p a r t i cu l a r l y m a mma l s an d b i r ds t h a t i n ge s t c on t am in a t e d sh e l l f i s h

( i . e . c on t ami n a t ed th r ou gh t h e a c cum ula t i on o f t he c au s a t iv e a l ga l

s p e c i es b y s u sp e ns io n f e e d in g in t o t h e t i s su e s o f s h e l l f i sh ) . T h es e

t ox in s wo r k b y b i nd in g to r e c e p to r s o f mu sc l e o r n e r ve ce l l s a nd

d i s r up t t he f l ow o f s od iu m i on s . Th i s i n t e r ru p t i on o f s i gna l

t r an smi s s io n i s w ha t ca us e s p a r a l ys i s . Du e to i t s s e r io us h um an

h e a l th an d e c on omic imp a c t i t h as b e en o f g r e a t im po r t a nc e t o s t ud y

t h e c au s a t i v e mi c roo r ga n i sm s .

T h e c a us a t iv e t ox i ns w er e fo un d t o s yn t h es i s ed b y a g r o up o f

m ic r o a l ga l sp e c i es , n am el y: G y mn o d ium c a t en a t um , P yr od in iu m

b a ha m en se an d th e m em b e rs o f t h e genu s Al e x an dr i u m (Ce m b el l a

1 9 98 )

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T h es e n e u r o t ox in s , i n c l ud in g s ax i tox in an d 20 o th e r c h em ic a l l y

r e l a t e d de r iv a t iv es d i f f e r i n ch em i c a l s t ru c t u re (G a l l a ch er & S mi t h

1 9 99 ) a nd v a r y i n t ox i c i t y .

T h e ge n us A l ex an dr iu m c on s i s t s o f 27 sp e c i e s , o f w h i c h 9 a r e kn ow n

t o p ro du c e PSP tox i ns i n va r yi n g a mou n t s . A l th ou gh i t i s a r gu ab l y

t h e b es t ch a r a c t e r i z e d h a rm f u l a l ga l gen us t o d a t e (C em b el l a 1 99 8 ,

S c ho l in 1 99 8) l i t t l e i s k no wn ab ou t t h e e nz ym e s a nd ge n es i n vo lv ed

i n t h e s yn t h e s i s o f PSP t ox ins (P l uml ey 1 9 9 7 ) .

S om e s c i en t i s t s h av e s u gge s t e d e ub a c t e r i a l i nv o l ve m en t i n t h i s

p r o c es s e i t h e r d i r ec t l y ( i . e . a s au t on om ou s PS T p ro du c e rs ) o r

i nd i r e c t l y ( i . e . b y a f f e c t in g th e g ro wt h r a t e , nu t r i en t u p t ak e , e t c . ) .

T h i s c on c ep t i s s t i l l s om e wh at co n t ro ve r s i a l . T h e on l y n on -

d in o f l a ge l l a t e sp p th a t a r e ge n e r a l l y a c c e p t e d to b e a b l e t o p r od u c e

PSP t ox ins au to nom ou s l y a r e s e v e ra l s p e c i es o f c ya n o b ac t e r i a ,

n a m el y; A p ha n i z o me n on f l o s - aq ua e ( J ak im a nd G en t i l 19 68 ; Mo ho od

a n d Ca r mi ch a e l 1 98 6 ) , A na ba e na c i r c i na l i s (H um p a ge e t a l . 1 99 4 ;

N e gr i e t a l . 1 99 7) a n d Ly ng b ya w o l l e i ( Ca rm i ch a e l e t a l . 1 9 97 ;

O n od e r a e t a l . 19 97 ) .

T h e no t i on o f b a c t e r i a e i t h e r p ro du c ing o r h a v i n g a n e f f ec t o n PS P

t ox i gen e s i s w as f i r s t h yp o t h es i z e d b y S i lv a ( 19 62 , 19 82 ) . I t s t em e d

f r om t h e f a c t t h a t sh e o bs e r ve d b a c t e r i a - l i k e s t ru c t u r es wi t h i n A.

t a mar e ns e ce l l s . Th i s w a s f o l l ow e d up b y N e l in d a (1 98 5) w ho

d e t ec t ed PSP tox i ns i n a n on tox i c d i no f l a ge l l a t e cu l tu r e a f t e r i t w as

t r an s f e r r e d i n t o m ed iu m f rom t ox ic cu l tu r es . Sh e a t t r i bu t e d t h i s t o

t h e ex t r a c e l l u l a r ba c t e r i a p r es en t i n t h e m e d iu m. Fu r th e r s t ud i e s

s ho w e d t h a t t h e r e w a s n o t a s i gn i f i c an t am ou n t o f t ox i ns t r a ns f e r r ed

b y e x t r a - ce l l u l a r ba c t e r i a . Th es e f i n d in gs d id n o t r e f u t e S i lv a ’ s

p os tu l a t i o n du e t o t h e f ac t t h a t h e r ob se r v a t io ns w e r e b ase d o n

i n t r a c e l l u l a r b ac t e r i a . Ko d am a ( 19 88 ; 1 9 90 ) f o cus e d on in t ra c e l lu l a r

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b a c t e r i a an d w as ab l e t o i so l a t e a b a c t e r iu m sp e c i e s c a l l ed

M o ra x e l la f r om a t ox i c s t r a i n o f A. t am a r ens e . I t s ho w ed th e ab i l i t y

t o a u to no mo us l y p r o d uc e PSP tox i ns . S e ve r a l p a p er s f o l l o w e d t h i s

b y c l a imi n g p r oo f o f t he ab i l i t y o f b ac t e r i a t o p r od u ce PSP t ox ins

( K o da m a e t a l . 19 88 , 19 90 ; O ga t a e t a l . 1 9 90 ; Do uc e t t e & T r i ck ,

1 9 95 ; Le v a s se u r e t a l . 19 96 ; S h imiz u e t a l . 1 99 6 ; Ko pp e t a l . 19 97 ;

G a l l a ch e r e t a l . 19 96 , 1 99 7) . M o re r e ce n t p ap e rs r e f u t i n g th e se

c l a i ms f o l l o w ed b y r e po r t i n g t h e i n co r r e c t i d e n t i f i c a t i o n o f b a c t e r i a l

m e t a bo l i t e s a s PSP tox i ns b y H P LC a na l ys i s ( N e gr i e t a l . 1 9 97 ,

O n od e r a e t a l . 19 97 , S a to e t a l . 1 99 8 , P e r e i r a e t a l . 2 00 0 a nd Ba ke r

e t a l . 2 00 3 ) . Th i s h a s l e a d to t h e co n t i nu a l co n t ro v e rs y s u r r ou nd i n g

a u to som a l o r a s s i s t e d b a c t e r i a l PSP t ox i ge n es i s . W i th i n t h i s wh o l e

d e b a t e , a n ew ap p ro a c h w as u s ed to de t e rmi n e th e e f f e c t o f

ex t r a c e l lu l a r b a c t e r i a o n d in o f l a ge l l a t e c u l t u r e t ox i c i t y; na m el y t h e

r e mo v a l o f t h e ex t ra c e l l u l a r mi c r o f l o ra w e r e us e d . T h e me t ho ds

e m plo ye d t o ob t a in ax en ic cu l tu r e s w er e ; s t e r i l e wa sh in g ( S i n gh e t

a l . 19 82 ; Kim e t a l . 1 99 3) an t ib io t i c s (D a n tz e r e t a l . 19 97 , Ho ld e t

a l . 2 00 1 ) an d l ys o z ym e / SDS t r e a tm e n t (D ou c e t t e an d Pow e l l 1 99 8 ) .

T h i s h as l e ad to an o n go in g d eb a t e o n th e ac tu a l e f f e c t s o f b a c t e r i a

o n PST t ox i gen e s i s w i th p a p er s p ro f es s in g a nd r e f u t i n g th i s a s pe c t .

A n o t he r u ns o lv e d as p e c t o f P SP t ox i gen e s i s i s t h e ge n e t i c c on t r o l o f

t ox in p ro du c t io n . Fe w s tu d i e s ha v e be e n co nd u c t ed t o de t e rmi n e th e

ge n e s i nv o lv e d i n t h i s . I t i s n o t c l e a r w h e t h e r ch r om os om al ,

m i to c ho nd r i a l , c h l o r o p l as t D NA o r n u c l e i c a c id s f ro m b a c t e r i a ,

p l asm id s o r v i rus e s a r e i n vo lv e d . On e o f t h e f i r s t s t ud i e s t o t a ck l e

t h i s w as c on du c t ed b y O ga t a e t a l . ( 198 7 ) . Th e y l o ok e d a t t he

t ox i c i t y o f n um e rou s su b - c lo n es o r i g in a t in g f r om on e c lon a l c u l t u r e

a n d f ou nd v a r i a t i o ns . Th e y a t t r i bu t ed t h i s t o t h e f a c t t h a t t ox i n

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p r od u c t i on i s no t he r e d i t a r y, t hu s l e a ds t o t h e v a r i a t i on ob s e rv e d .

K o d am a ( 19 90 ) ob se r v e d t h e s am e d i f fe r e n c es an d su gge s t e d t h a t

e n do nu c le a r b a c t e r i a w h er e as s o c i a t e d i n t ox in p r od u c t i on .

S oo n a f t e r , S ak o ( 19 9 5) an d Is h i d a (1 99 8 ) sh ow e d th a t t ox i ge n es i s

o f PSP tox i ns i s ch r o mo som al l y b a s e d , t hu s a h e r ed i t a r y c h a r a c t e r .

T h i s w as d on e b y c o n du c t i n g m at in g ex p e r im e n t s o n d i f f e r e n t

s t r a in s o f A. t a m are n s e a nd A . c a t en e l l a w i th d i f f e r in g PSP p r o f i l e s

a n d d e t e rmi n i n g wh e th e r t h e PSP p ro f i l e m e as u r ed w a s a

c o mbi n a t i on o f b o th pa r e n t s . T h e tox i n p ro f i l e wa s us e d a s a

p h en o t yp i c ch a r a c t e r i s t i c s i nc e t h e t ox in p r o f i l e i s t h ou gh t t o b e

r e a d i l y s t a b l e i n A le x a nd r i um s p ec i es ( C em b el l a . , e t a l 19 8 7 ; O ga t a

e t a l . , 1 98 7 ; Kim e t a l 19 93 ) . T he s e s t ud i e s sh o we d th a t t h e

p r o ge n y e x h ib i t ed a c om bin a t io n o f bo th p a r en t a l t ox in s a n d mo s t

s t r a in s sh ow e d a Me n d e l i a n p a t t e rn o f i n h er i t an c e . Th i s l e a d t o t h e

c o n c l us i on t ha t t h e ge n e s c od i n g fo r PS T p ro du c i n g enz ym e s wh e r e

o n n uc l ea r DN A. N o n e th e l e s s , no t a l l F1 p r o ge n y o n t h es e m at in g

ex p e r im e n t s ex h ib i t e d t h e M en d e l i a n p a t t e rn o f i n h er i t anc e . I t w a s

p r op os e d t h a t t h i s w a s d ue t o r e co mbin a t io n b e t w e en ge ne s .

O n e mo r e asp e c t o f PSP p ro du c t io n i s t h e r e l e va n c e o f t he s e t ox i ns

t o t h e o r ga n i s m. Mo s t s c i e n t i s t s v i ew t h es e t ox i ns a s s e co n da r y

m et a bo l i t e s (P l umle y 1 9 9 7 ) . Th i s t e rm wa s c o i ne d b y V i n i n g in

1 9 90 a nd d e f i n ed as “ com p ou nds t h a t d o n o t f u l f i l a ro l e i n

i n t e rm ed i a r y m e t abo l i s m ” L i t t l e i s kn o wn as t o t h e i mpo r t a n c e o f

t ox in p ro du c t io n wi th r e sp e c t t o t h e ca u s a t i v e o r ga n i sm . I t h a s b e en

s u gge s t e d th a t PSP tox i ns m a y p l a y a n in t r i n s i c r o l e i n DN A

m et a bo l i s m (M i ck e l so n & Y en t s ch 1 979 ; A nd e rs on & Ch en g 1 99 8)

o r a id i n n i t r o ge n s t o ra ge ( An d e rs on e t a l 19 90 ) o r a c t a s

p h e rom o ne s (W ya t t a nd J e nk ins on 1 997 ) t o p r om ot e m at in g o r a s

a n t i -p r ed a t io n / a l l e lo c h emi c a l co mp ou nd s (W hi t e & M a ra nd a 1 97 8 ;

H a n e y e t a l 19 95 ) . S i n c e t h e e f f e c t i v en e ss o f a n a l l e l oc he mi c a l i s

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i t s a b i l i t y t o b e r e l e a s e d i n t o t h e e nv i r o nm en t , PSP tox i ns d o no t f i t

i n t o t h i s de f in i t i o n ( i . e . PSP t ox ins a re k ep t i n t h e c e l l an d o n l y

r e l e as e d du r i n g s e ne s c e nc e ) . R e c en t s t ud i e s a r e i n d i c a t i ng t h es e

a l l e lo c he mi c a l s a l so c au s e an e f f e c t on s p e c i f i c mi c ro o r ga n i sm s .

A l l e l o ch e mi c a l a r e d e f i n ed as ch em ic a l s t h a t ca us e i nh i b i t i o n o r

s t im u l a t io n i n a no th e r o r ga n i sm du r ing t h e i r i n t e r a c t i on ( M ol i s ch

1 9 37 ) . A rz u l e t a l ( 1 99 8 ) t e s t ed t h e a l l e l o ch e mi c a l e f f e c t s o f t h r e e

t ox i c Al e xa nd r iu m s p e c i es an d f ou nd th a t t h e c e l l - f r e e f i l t r a t e s o f

t h es e c u l t u r es h ad a n a l l e lo ch e mi c a l e f f e c t on Ch a e t oc e ro s gr a c i l l e ,

G y m no d i n iu m m i k im o to i a nd S c r ip ps i e l l a t r o ch i od e a . Th e y

s u gge s t e d th a t t h i s e f f e c t w as d u e t o co mp ou nd s o t he r t han PSP

t ox in s . S ev e r a l p ape r s f ou nd t h a t t h i s w a s t ru e i n m or e r ec e n t c as e

s tu d i es . ( O ga ta & K o d am a 19 86 ; Lu s h & Ha l l e g r a e f f 199 6 ; T i l l ma nn

& J oh n 20 02 ) .

H o w ev e r n o th in g i s k no wn ab ou t t h e f a c t o r s a f f e c t i n g p ro d uc t io n o f

t h es e a l l e l o ch emi c a l b y A . t am ar e ns e . O n e o f t h e po ss i b i l i t i e s

c o u l d b e t h a t ex t r a - c e l l u l a r b ac t e r i a a r e i nv o l v ed in t h i s p r o c es s .

T h i s qu e s t i on w i l l b e ad d re s s e d in m y th e s i s .

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3 . M y t h e s i s p ro j e c t d e a l s wi th t h r e e as p e c t s r e l a t ed t o t ox in

p r od u c t i on i n A. t am a r ens e , n am el y:

1 ) D e t e rmi n in g th e e f f e c t s o f ex t r a - c e l l u l a r b a c t e r i a on t wo A .

t a mar e ns e s t a ins by o b s e r v in g th e g row t h r a t e , t ox in p ro f i l e

a n d ov e r a l l t ox in co n t e n t wi th in ax en ic a nd x e n i c c u l t u r es .

2 ) D e t e rmi n in g th e p os s i b l e ge n e t i c b i as o f t ox i gen e s i s b y

s tu d yi n g t h e ph e no t yp e ( t ox i n p r o f i l e ) o f su b -c lo n es .

D e t e rmi n in g o f t h i s v a r i a t i on c an b e s ee n a t a ge n e t i c l ev e l

u s i n g A FLP .

3 ) D e t e rmi n in g th e a l l e l o ch em ic a l e f fe c t s o f t h e f o r e m e n t i on e d

ax e n i c , x e n i c a nd cu l t u r e s us i n g a b i oa s s a y w i th t h e

C r yp t op h yt e R h od om o na s ba l t i ca t o de t e rmi n e th e b a c t e r i a l

i n f lu e n c e on th e u nk n ow n a l l e lo c h emic a l .

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4 ) M a te r ia l s and me t h od s :

4 . 1 Sa mpl e s i t e :

T h e Al e xa nd r iu m ta m ar e ns e s t r a in s us e d i n t he s e ex p er im e n t s w e r e

c o l l e c t ed f rom t wo s t a t i o ns o f f t h e c oa s t o f S co t l an d d eno t ed b y S 2

( 5 6° N , b e t w e en D un d e e an d Ed i nb u r gh ) a nd S6 ( 57 ° 4 0 ’ N , n o r th o f

A b e rd e e n ) . T h es e c u l t u r es w e r e i so l a t ed f r om n a t u ra l p l a nk to n

s a mpl e s a s s in g l e ce l l s .

4 . 2 S t ra ins and Cul tu r e c ond i t ion s :

4 . 2 . 1 Ax en ic a nd xe n i c cu l tu r es

T w o s t r a i ns o f A. t a m ar e ns e w he r e u se d fo r t h e b a c t e r i a l

ex p e r im e n t s : S 2 - P4- D 1 a nd S 2-P 4 - F2 .

K me d i um ( K el l e r e t a l 19 87 ) m ad e wi th N o r th S e a w a t e r a d j us t e d t o

p H 8 , wi th t h e ad d i t i o n o f s to c k s o l u t io ns ( s e en i n T ab le 1 ) . T h es e

c u l tu r e s wh e r e t h e n t r a ns f e r r ed to K me d ium co n t a i n i n g an

a n t ib io t i c c o ck t a i l ( i n T ab l e 2 ) . Th es e c u l t u r es w h e re m ain t a in e d a t

1 5 °C , w i t h a r t i f i c i a l l i gh t a t a ph o t on f l ux d e ns i t y o f 10 0µ Em- 2

s- 1

a n d a l i gh t :d a r k c yc l e o f 14 :1 0 . Th e x e n i c c u l tu r es w h e re

m ai n t a i n ed i n t h e sa m e f a sh i on wi t h t h e o mis s i on o f a n t ib io t i c

m e d iu m. To r ed uce th e a mo un t o f ex t r a c e l l u l a r ba c t e r i a w i t h i n t h e

c u l tu r e s , gauz e f i l t e r in g ( us in g a 20 um m es h ) wi th s t e r i l e - f i l t e r ed

s e a w at e r w as co ndu c t ed d u r in g t h e s ec o nd w ee k . T h i s en t a i l e d

ge n t l y p o u r i n g th e c u l tu r e t h r ou gh s t e r i l e ga uz e , a nd r i n s i n g t h r e e

t im es w i th s t e r i l e - f i l t e re d s e aw a te r . Th e s e c u l t u r e s w e r e t h en

t r an s f e r r e d i n t o K m e d iu m a nd m ai n t a i n ed as p r ev i ou s l y d e s c r ib e d .

A f t e r t h e s ec on d we e k , c u l t u re s w he r e t e s t e d f o r b a c t e r i a l

12

c o n t am in a t io n ( as d e s c r ib e d b e l l ow ) an d u s ed as i n o cu l a f o r g r o wth

b o t t l e s co n t a i n in g 5 L o f K m ed ium ( to en su r e e no u gh s am pl i n g

v o l um e ) . Th e i no cu l a wh e r e us e d to a l l o w f o r a f i n a l c onc e n t r a t i o n

o f ap p rox im at e l y 2 0 0 c e l l sml- 1

. E a ch t r e a tm e n t w as co ndu c t ed in

t r i p l i c a t e

T a b l e 1 : L i s t o f r ea ge n t s u s ed i n m a k i n g K m e d i um.

A mo un t add ed /L S to ck s o lu t i on

1 ml N a N O 3 ( 75 .0 g / L H 2 O )

1 ml N H 4 C l ( 2 . 68 g / L H 2 O )

1 ml N a H 2 P O 4 . H 2 O (5 g /L H 2 O )

0 . 1ml H 2 S eO 3 ( 1 . 29 m g/ L)

1 ml T r i s Bas e (p H 7 . 2 ) (1 2 1 . 1 g/ L H 2 O)

1 ml K T r ac e m et a l s o l u t io n

1 ml F / 2 Vi t ami n so l u t i on

13

T abl e 2 : A n t i b io t i c c o ck t a i l u s e d in ax e n i c m ed ium .

A n t ib i o t i c S to ck s o lu t i on A ddi t i on /L me d ium

A m pi c i l l i n

1 5m g/m l 3 . 33m l

G e n t a m yc i n e

1 m g/ml 3 . 33m l

S t r e p to m yc i n

2 5m g/m l 1 ml

C h l o r am ph e n i c o l

3 4m g/m l 3 0 u l

C i p ro f l ox a c i n

1 m g/ml 1 u l

4 . 2 . 2 Sub - c l on e cu l tu r es

Si n g l e c e l l s o f A. t a m ar e ns e f ro m th e s t r a in s : S2 -P4 - D1 , S 2- P4 - F2 ,

S 2 - P6 - G1 2 an d S6 -P 1- G 7 w e re p i ck e d f r om ex i s t i n g cu l t u r e s u s in g a

f i ne t i p pe d p l a s t i c p ip e t t e a t t ac h ed t o a r ub b er p ip e . Cu l t u re s w e r e

d i l u t ed an d p l a c ed i n a P e t r i d i sh . C e l l s t h a t w er e s us p en d ed wi t h in

t h e m e d i um o r mot i l e w e r e p i c k ed . T he y w e r e p i c k ed b y a p p l yi n g a

s l i gh t s u c t i on , wh ic h a l l o we d th e ce l l s t o b e su ck e d in t o t h e p i p e t t e

t i p a nd t r an s f e r r e d t o a w e l l o f a 9 6 we l l p l a t e c on ta in i n g 2 00 u l o f

d i l u t ed (1 : 10 ) K me d ium ( Ke l l e r e t a l . , 1 98 7) . Th e p l a t es w h er e t he n

c h e c k ed to co n f i rm th a t o n l y o n e ce l l p e r w e l l w as t r an s fe r r e d . T h e

p l a t es fo r e a c h s t r a i n w e r e i n cu b a t ed an d m ain t a in e d a t 15 °C ,

1 0 0µ E .m- 2

. s- 1

p ho to f l ux d e ns i t y a n d a l i gh t :d a rk c yc l e o f 14 :1 0 .

O n c e th e c lo n es w er e f o r me d th e y w e r e t r a ns f e r re d to a g l a s s c u l t u r e

14

v i a l c on t a in in g 10 m l o f u nd i lu t ed K m e d iu m. O nc e t he c l on es ha d

r e a c h ed s ub s t an t i a l c on c e n t r a t i on s t h ey w e r e t r an s fe r r ed i n t o 20 0 m l

f r e sh K m e d i um wi t h p en i c i l l i n ( 0 . 1 g /L) i n 25 0ml cu l tu r in g f l a sk s

a n d nu mb e r ed 1 to 20 wi t h du p l i c a t e s am pl es f rom t he cu l tu r e w h e r e

t h e s in g l e c e l l s whe r e p i ck e d ( i . e . p a re n t c l on e ) . T h e y w e r e

m ai n t a i n ed a t t h e s a m e g ro wt h c on d i t i o ns m e n t i on e d p r e v i ou s l y.

4 . 3 S a mpl in g p r o ce du r e :

4 . 3 . 1 Ax en ic a nd xe n i c cu l tu r es

S am pl es fo r c e l l c ou n t s a nd PSP m e asu r em e n t s w e re t a ken ev e r y

s e c on d d a y t i l l t h e c u l tu r e s r e a ch ed t he l a g -p h as e o f t h e i r g r o wt h

c u r v e ( i . e . 2 0 da ys i n t o t a l ) . Fu r th e r s a m pl es we r e t ak e n d u r i n g

t h re e s t a ge s o f t h e ex p e r im e n t f o r b a c t e r i a l d e t e rm in a t io n us in g

A c r id i n e o r an ge . Sa m pl es fo r d e t e rmin in g th e a l l e l o ch em ic a l e f f ec t

o f t he cu l tu r e s w e re t ak en o n d a ys 8 an d 1 0 .

4 . 3 . 2 Sub - c l on e cu l tu r es

C ul t u re s w e r e h a r ve s t e d w h en ce l l num b er r e ac h ed ap p rox im at e l y

3 0 00 c e l l s /m l . 1ml w a s us e d f o r c e l l co u n t s , 40 ml f o r PSP

d e t e r min a t io n , 2 0ml fo r a l l e l oc h emi c a l t e s t s a nd 10 0ml fo r DN A

ex t r a c t io n f o r A FLP an a l ys i s .

15

4 . 4 C e l l and b ac t er i a l c oun ts :

4 . 4 . 1C el l c oun ts

T h e Ut e rm oh l -m et ho d w a s us ed fo r t h e ax en ic , x en i c cu l tu r e s a nd

s ub - c l on e s am pl e s t o d e t e rmi n e c e l l co n c en t r a t i o n . Th e se r e su l t s

w e r e u s ed to d e t e rm in e th e g ro wt h r a t e o f e a ch s t r a in i n t h e ax e n i c

a n d x en ic cu l tu r e s a n d to ev a l u a t e P ST co n c en t r a t i o ns pe r c e l l fo r

b o t h ex p e r im e n t s . C ou n t i n g wa s do n e b y m ix in g 1ml o f s a m pl e wi th

2 0 u l Lu go l ’ s so lu t io n ( t o o b t a i n a f i n a l Lu go l ’ s s o l u t i on o f 2 % ) .

1 0 0u l o f t h i s s am ple w as t h en ad de d to a c ou n t i n g ch am be r

c o n t a i n i n g 2 00 u l o f K m ed ium . Th e to t a l n um b er o f c e l l s w a s

c o un t ed in t h e ch am b er wi t h a n i nv e r t e d mi c r os co p e ( Ze i s s Ax io v e r t

4 0 C ) an d th e n umbe r s w e r e c on v e r t ed t o c e l l s p e r ml . The g r o wt h

r a t e w as c a l cu l a t ed b y:

G r o wt h r a t e = ln ( N 1 / N 0 )

T 1 - t 0

W h e r e N r e f e r s t o t h e c e l l co n ce n t r a t i o n a t t im e t .

4 . 4 . 2 B a ct e r i a l coun ts :

St a in i n g wi t h A c r id in e o r an ge ( Ho bb ie e t a l , 19 77 ) w a s u s ed t o

d e t e r min e t h e am oun t o f b ac t e r i a i n t he Ax en i c an d x e n i c c u l t u r es .

T h i s w as d on e b y f i r s t a dd i n g 20 µ l Fo r ma ld e h yd e t o 1ml o f s am pl e .

T h es e s am pl es w ere s t o r e d in t h e f r i dge t i l l f u r th e r an a lys i s . In

o r d e r t o b e gi n s t a in in g , a c e l l u l os e n i t r a t e f i l t e r ( po r e s i z e 0 . 45 µ m ,

M i l l i p o r e ) w a s m oun t ed o n to t h e f i l t e r i n g u n i t fo l l o w ed by a b l a ck

N u c l e op o r e f i l t e r (p o r e s i z e 0 . 2 µ m) .

1 ml o f t h e f i x e d s am pl e w a s ad d ed t o t h e f i l t e r in g un i t w i th 1 00 µ l

o f Ac r id i n e O r an ge ( 1m g/ml ) an d i n cub a t ed f o r 3 m in u t es . Th en t h e

16

s a mpl e wa s f i l t e r ed o f f , t h e n u c l eo po re f i l t e r w as re mo v ed wi t h

p in c e r s , mo un t e d on to a s l i d e wi t h c i t i f l uo r a nd c ov e r e d w i th

c o v e r - s l i p s . T h e y w h e r e t h en s to r ed in t h e f r i d ge t i l l a na l ys i s . T he

b l an k w as p r ep a r e d in t he s a me m a nn er wi th f i l t e r ed M i l l i - Q w at e r

a s t he s a mpl e .

Ba c t e r i a l co un t s o f e a c h sa mp le w er e d o ne as fo l l o ws : 20 g r id s on

e a c h f i l t e r w h er e co u n t ed u nd e r a n e p i f l uo r e sc e n c e m i c ro s co p e wi th

1 0 0X o i l imm e rs ion ob j e c t iv e .

4 . 5 PS P ex t ra c t i on and d e te r mi na t i on :

T hi s me th od wa s ap p l i e d t o t he ax e n ic , x e n i c a nd s ub - c lo n e

c u l tu r e s . ST X a nd i t s d e r iv a t iv es ( F i gu r e 1 ) w er e d e t e rm ine d u s i n g

k n ow n s t an d a rds . T h e s am pl es w h e r e p r e p ar e d a s fo l l o w s :

V o lum e s r an g i n g f ro m 30 ml to 9 00m l ( i . e . t o o b t a i n a pp rox i ma t e l y

3 0 00 c e l l s /m l ) o f t h e s am pl e cu l tu r es w e r e c en t r i f u ge d a t 1 30 00

r p m, 40C f o r 15 min u t e s t o ob t a in a pe l l e t f ro m t h e c u l t u r e . T h e

p e l l e t w a s t h en f r oz en an d s to r e d a t -2 5 o

C p r io r t o ex t ra c t i on .

Fo r PSP t ox in ex t ra c t i on , 1ml o f a ce t i c a c i d ( 0 . 03 N ) w as a d d ed t o

t h e c e l l pe l l e t . T h i s w as t h e n hom o gen iz ed u s in g l ys i n g m at r ix D

b e a ds (T he r mo sa v an t ) i n a h om o gen iz e r ( Fas t P r e p FP 120 ,

T h e rm os av a n t ) f o r 4 0 s e co nd s fo l l ow ed b y c e n t r i fu ga t i on

( E pp e nd o r f 5 41 5R ) f o r 15 mi nu te s a t 1 3 00 0 rp m. 50 0 µ l o f t h e

s up e r n a t an t w as t he n p a s s e d t h ro u gh a 0 . 5ml s p i n f i l t e r ( M i l l i po r e )

a n d c e n t r i fu g i n g f o r 30 s e c a t 30 00 r pm . T h e f i l t r a t e w as co l l e c t ed

i n 2ml c r im p g l as s v i a l s (A g i l e n t T e c hn o l o g ie s ) a nd c a ppe d . A

p o r t i on o f t he f i l t r a t e w a s h yd r o l yz e d to d e t e rm in e th e N-

s u l f oc a r ba mo yl c on t e n t ( T ab le 3 ) . T h i s w as do n e b y a d d in g 7 5µ l

17

H Cl (1M ) t o 30 0µ l o f t he ex t r a c t e d s am pl e f o l l o we d b y 9 0 °C

i n cu b a t i on f o r 15 m in . 1 50µ l o f s od iu m a c e t a t e (1 M) wa s t he n

a d d ed a f t e r t h e s a mp l e c oo l e d to r oo m t em p e r a tu r e . T h e N -

s u l f oc a r ba mo yl c on t e n t w a s de t e r min ed i nd i r e c t l y b y c a l c u l a t i n g t he

c o n c en t r a t i o n d i f f e r e n c e b e t w e en th e h yd r o l yz e d a nd n on-

h yd r o l yz e d s am pl e . A l l s am pl e s w er e k e p t a t - 25 °C p r io r t o

a n a l ys i s .

T h e HP LC m et ho d b a s ed o n io n - p a i r ch r om at o gr ap h y w i th

o c t an es u l fo n i c ac id , pos t -c o l um n de r i va t i z a t i o n f o l l ow e d b y

f l uo r es c en c e d e t e c t i on (H umm e r t e t a l . , 1 99 7) wa s us e d to an a l yz e

t h e c on c en t r a t i o ns o f PSP d e r i v a t i v es . Th e m eth od u s ed t w o

a q u eo us e lu e n t s (Ta b l e 4A ) w i th d i f f e r e n t g r a d i en t s o ve r t im e

( T a b l e 4 B) . Th e ox id i z in g r e a ge n t f o r t h e p os t co l um n

d e r i v a t i z a t i o n c on s i s t ed o f a 10m M p er i od i c a c id , 55 0m M N H 3

s o l u t i on an d 0 . 75 M n i t r i c a c i d . F lu o re s c e n t t ox in d e r i v a t iv es w er e

d e t ec t ed a t ex c i t a t i o n a nd emi s s io n w av e l e n gt hs o f 3 33 nm an d

3 9 5nm r esp e c t iv e l y .

S t a nd a r ds f o r s ax i tox i n (S T X) , n eo s ax i tox in (N E O ) , gon ya u tox in s

( G T X 1 - 4 ) an d D ec a r b omo yl t ox i ns (d c ST X, d c G TX 2 an d d c GT X

3 ) p u r ch as e d b y t h e N a t io n a l Re s e a r c h C ou n c i l C an a d a , Ha l i f ax ,

N o v a S c o t i a , C a n ada , w er e u s ed . T h ese w e r e us e d t o c a l i b r a t e t h e

s a mpl e PSP tox i n co n c en t r a t i o ns .

18

Fi g ur e 1 : S T X an d i t s d e r iv a t iv e s d ep ic t i n g t h e fo u r ma jo r g r o up s

o f PSP tox i ns . (d e pe n d i n g o n t h e g r o up s ub s t i t u t e d on R 4 p os i t i o n )

n a m el y, C a r b amo yl , N -su l fo c a rb am o yl a nd D ec a r ba mo yl t ox i ns .

S ax i t ox in a nd Ne oS TX a r e t h e m os t po t en t o f t he s e t ox ins , w i th N -

s u l f oc a r ba mo yl b e in g th e l e as t t ox i c an d D e c a rb am o yl t ox in s h av i n g

i n t e rm ed i a t e t ox in c o n t e n t .

19

T abl e 3 : T ab le o f t h e N -s u l fo c a rb am oyl c o mp ou n ds a nd th e

h yd r o l yz e d d e r iv a t iv e s t h e y a r e h yd r o lyz e d t o i n d i r e c t l y d e t e r min e

t h e i r c on c en t ra t i on .

N - s u l f o c ar ba my l

H yd ro ly se d

c o mp ound f o r me d

B1 � S TX

B2 � N E O

C 1 � G T X 2

C 2 � G T X 3

C 3 � G T X 1

C 4 � G T X 4

20

T abl e 4 A : Elu e n t s u s ed i n l i q u id ch ro m ato gr a p h y

El u ent

A 6 mM O c t an es u l fo n ic a c i d

6 mM T et r a su l f on i c a c id

4 0m M

A mm on ium ph os ph a t e

0 . 75 % T e t r ah yd r o fu r a n e

B 1 3m M O ct a ne su l f on i c a c i d

5 0m M P ho sp ho r i c a c i d

a d ju s t ed t o ph 6 .9 w i t h

N H 3 .

1 5 % A c e t on i t r i l e

1 . 5% T e t r a su l f on ic a c id

T abl e 4B : E lu e n t g r a d i en t s u se d f o r c h r om at o gr ap h y. In j e c t i on :

5 µ l , f l ow 1 .0 ml min- 1

.

Ti me ( mi n ) Gr a d i en t

0 1 0 0% A

1 5 1 0 0% A

1 6 1 0 0% B

3 5 1 0 0% B

3 6 1 0 0% A

4 5 1 0 0% A

C on c en t ra t i ons o f G T X 1a nd GT X4 , GT X 2 a nd G T X3 , C 1 a nd C 2 a nd

C 3 a nd C4 we r e r ep o r t ed as t he s um o f t h e t wo in d i v id u a l s du e to

t h e po t e n t i a l o f ep im e r i z a t io n du r i n g th e ex t r a c t io n p ro c es s .

21

4 . 6 Al l e l o ch e mi ca l t e s t s :

T h e a l l e l o ch emi c a l e f f e c t s o f A l ex an dr iu m ta ma r en s e cu l t u re s w e r e

q u an t i f i ed wi t h a b i o as sa y u s in g t h e c r yp t o p h yt e R ho do mo n as

b a l t i ca . A f t e r ex p os in g R. b a l t i ca t o A. t a mar e ns e , t h e t a rge t c e l l s

i n c r e a s ed s l i gh t l y i n s i z e du e to s w e l l i n g , fo rm e d b l i s t e r s a nd

f i na l l y l ys e d . Co un t in g t h e nu mb e r o f i n t a c t R . ba l t i c a c e l l s ( wi th

n o rm al c e l l s ha p e s t i l l v i s i b l e ) a f t e r a c e r t a i n i n cu b a t i on p e r i od

a l l o w ed f o r t h e qu an t i f i c a t i o n o f t h e l y t i c e f f e c t .

T h e b i o as sa y w a s pe r f o rm ed as fo l l o ws : t h e c e l l d en s i t y o f an

ex po n en t i a l l y g r o w i n g cu l tu r e o f R. ba l t i c a ( cu l t u r e d a t 1 5 °C ,

u n de r a r t i f i c i a l l i gh t a t a ph o t on f l ux d e ns i t y o f 1 00 µ E m- 2

s- 1

o n a

1 6 : 8 h l i gh t -d a r k cyc l e ) w a s e s t i ma t ed b y m i c ro s co p e c e l l co un t s . A

d i l u t i on to a f i n a l c o n c en t r a t i o n o f abo u t 4 x 1 05 m l

- 1 c e l l s w as

p r e p ar e d . In t r i p l i ca t e , 3 . 9 m l o f a p r ed e f i n ed co n c en t r a t i o n o f A.

t a mar e ns e ce l l s w as p ip e t t ed i n to 6m l g l a s s s c in t i l l a t i o n v i a l s

( W h e a t on S c i n t i l l a t i on Vi a l s ) . F r esh K - m ed i um wa s us ed as

c o n t ro l . V i a l s w er e t h e n sp i k ed wi t h 0 . 1 ml o f t h e R . ba l t i c a s t o c k

c u l tu r e ( f i n a l co n ce n t r a t i o n : 1 0 x 103 m l

- 1) . Af t e r ad d i n g R . ba l t i ca ,

0 . 5 m l o f e a ch c on t r o l v i a l w a s f i x ed w i t h 2 % Lu go l ´ s so l u t i on i n

o r d e r t o d e t e rmi n e t h e a c t u a l i n i t i a l R . b a l t i c a c on c en t ra t i o n .

S am pl es w er e t he n i n cu b a t ed a t 15 ° C a t l o w l i gh t ( ab ou t 5 µ m ol m- 2

s- 1

) on a 16 : 8 h l i gh t -d a rk c yc l e . A f t e r 24 h , s amp l es w e re f i x ed

w i t h 2 % Lu go l ´ s so lu t io n a nd c e l l con c e n t r a t i on s o f bo th A.

t a mar e ns e an d R. ba l t i c a w e r e de t e r min e d . Al l co un t s o f w e r e

p e r f o r me d w i th an i nv e r t e d m i c r os c ope ( Ze i s s Ax io v e r t 40 C ) . Th e

v o l um e f o r c e l l co un t s w a s 0 . 5 ml . Fo r A . t a ma re ns e , wh o l e

c h a mb e rs w e r e c oun t ed , ex c e p t f o r t h e h i gh l y c o n c e n t r a t ed cu l tu r e

o f S2 -P 4 - F2 , w he r e r e p re s en t a t i v e s ub - a r e a s ( > 40 0 c e l l s p e r

s a mpl e ) w e r e c ou n te d . A s ub a r e a o f a t l e as t 80 0 c e l l s i n t h e c on t r o l

w a s c ou n t e d fo r R . b a l t i ca . In o rd e r t o qu an t i f y l yt i c e f fe c t s , o n l y

22

i n t a c t ce l l s o f t he t a r ge t s p e c i es w e r e s c o r ed . F i n a l c e l l co u n t s o f R .

b a l t i ca w e r e c on v er t ed t o bo th c on c e n t r a t i o n ( c e l l s m l- 1

) a n d

p e r c en t mo r t a l i t y ( t r e a tm e n t c om pa r e d t o c on t r o l ) .

In o r d e r t o qu a n t i t a t i v e l y c o mp a r e l yt i c po t en c y o f a n t ib io t i c -

t r e a t e d an d un t re a t e d cu l tu r es , do s e - re s po ns e cu rv e s c ove r in g a wid e

r a n ge o f d i f f e r en t A . t a m ar en s e c on c en t ra t i ons w er e p lo t t e d . Be fo r e

s t a r t i n g th e ex pe r im e n t , cu r r en t a l ga l c e l l c on c e n t r a t i ons o f t he A.

t a mar e ns e cu l tu r es ( 3 r ep l i c a t es a n t i b io t i c t r e a t ed , 3 r ep l i c a t es

u n t r e a t e d ) we r e d e t e r mi n ed b y a l ga l co u n t s an d a pp ro p r i a t e

d i l u t i on s w e re p r e pa r e d to ga in a s e r i e s o f s e ve n f i n a l ce l l

c o n c en t r a t i o ns r a n gi n g f r om 5 0 to 2 000 c e l l s ml- 1

. E a ch d i lu t i o n w as

s e t up in t r i p l i c a t e . In t h i s ex p e r im e n t , s i x v i a l s c o n t a in in g K -

m e d iu m s e r v ed as co n t r o l s . Th e R ho dom o na s b io a s s a y w a s t h en

p e r f o r me d a s d es c r i b ed ab ov e .

4 . 7 A mpl i f i ed Fr ag me n t L eng th Po ly mo r ph i s m:

4 . 7 . 1 DN A ex t ra c t io n

D N A w a s ex t r ac t ed f r om 10 0m l o f ex po n en t i a l l y g r o w i n g c u l tu r e s .

T h e c u l t u r es w h er e f i l t e r ed o n t o 3 µ m p or e s i z e p o l yc a r b o n a t e

f i l t e r s ( Mi l l i p o r e ) a n d w as h ed wi t h s t e r i l e - f i l t e r e d s ea w at e r t o

r e mo v e ex t r a -c e l lu l a r b ac t e r i a . Th e f i l t e r s c on t a in i n g t h e s am pl es

w e r e p l a c e d in 2m l r e a c t io n v i a l s (E pp e nd o rp h ) an d imme d i a t e l y

f r oz en i n l i q u i d n i t r o ge n an d s to r e d a t - 80oC u n t i l ex t r ac t i on . T h e

D N A ex t r a c t io n w as d on e fo l lo wi n g t he i n s t ru c t io ns i n t he DN e a s y

P l a n t Mi n i k i t ( Q IA G E N ) .

Be f o r e D N A ex t r a c t io n , t h e c e l l s w er e d i s ru p t ed u s in g a

t i s s u e l ys e r ( Q IA G E N ) . A 0 . 5 mm s t a in l es s s t e a l b e ad w as ad d ed t o

t h e Ep p en do r f t u be c on t a in in g t h e f roz en f i l t e r s . Th e s am pl es w er e

p u t i n l i q u id n i t ro ge n fo r 3 0 s e c an d p l a c ed i n t o t h e t i s sue l ys e r .

23

C e l l s w e r e b ro k en b y v i go r ou s l y s h a k i n g a t a n i n t e ns i t y o f 3 0 Hz fo r

o n e min u t e . T h i s w a s r e p e a t ed th r e e t im es , wi t h r e f r e ez i n g a f t e r

e v e r y c yc l e . Th e DN A p r e p ar a t i o n w as co nd u c t ed as p e r p r o t oc o l

w i t h a mi no r a d j us tm e n t . T he DN A wa s e lu t ed in a sm al l e r vo l um e

t h an s u gge s t e d t o ob t a in h i gh er co n c en t ra t ed D N A. T h e D N A w a s

s to r ed a t - 80 t i l l f u r t he r an a l ys i s .

4 . 7 . 2 DN A R e s tr i c t i on

T h e D N A w as d i lu t e d a c co rd in g l y t o ob t a in 2 50 n g /5 0µ L. T he DN A

w a s d i ge s t e d us in g a M as t e r c yc l e r ( E pp o nd or ph ) wi t h t h e f o l l o wi n g

c yc l e s : 16oC fo r 9 h o ur s , 2 0

oC f o r 6 ho u rs an d 65

oC f o r 10 m in ( i . e .

t o i n a c t i v a t e t h e enz ym e s ) . Th e enz ym e s us ed fo r t h e d ige s t i on

w h e r e 10 U E coR I a n d 10 U Ms e I ( N e w En g l a nd Bi o l ab s ) . S amp le s

w h e r e do n e in d up l i c a t e .

4 . 7 . 3 DN A Li ga t i on

1 7µ l o f t he l i ga t e d s a mpl e wa s a dd e d to a mix tu r e o f w a t e r ( 2 . 5µ l ) ,

1 0m M A TP( 1 . 7µ l ) , 1 0x T4 l i ga s e bu f f e r ( 0 . 8µ l ) , 1 0 pm ol µ l- 1

E c oR I a d a p te r (1µ l ) , 5 0 pm ol µ l- 1

M s eI a d a p t e r ( 1µ l ) a n d T 4 D NA

l i gas e (1µ l ) . T h e l i ga t i on w a s c a r r i ed o u t u s in g a M as t e rc yc l e r

g r a d i en t a t 1 6oC f o r 9h , 20

o C fo r 6 h ou r s a nd 6 5

oC fo r 1 0 mi nu t e s .

T h e l i ga t ed mix t u re w as t h en d i lu t e d 1 :5 wi t h 0 . 1 x TE bu f f e r a nd

s to r ed a t – 25oC .

4 . 7 . 4 Pr e a mpl i f i c a t i on

1 0µ l o f t he d i l u t ed l i ga t e d D NA wa s ad d ed t o a r e a c t io n m ix tu r e o f

w a t e r (1 2 . 5µ l ) , 10x T aq DN A p o l ym e r a s e b u f f e r ( 3µ l ) , 10 pmo l /µ l

E c oR I+ A p r im e r ( 0 . 5µ l ) , 10 p mol /µ l M s e I+ C p r im e r ( 0 .5 µ l ) , 1 mM

d N TPs (3µ l ) an d Ta q DN A p l o ym e r a s e . T h e PCR r e a c t i on w as

c a r r i ed o u t i n a m as t e r c yc l e r g r a d i e n t ( E pp e nd o r f , ) w i th 2 2 c yc l e s

24

o f : 9 4oC fo r 1 mi nu t e (d e n a t u r a t i o n ) , 5 6

oC f o r 30 se c on ds

( a n n ea l i n g ) a nd 7 0oC fo r 1 min u t e ( ex t e n s i on ) . Fo l lo w ed b y 7 0

oC

f o r 5 mi nu t e s . Th e p ro du c t w as d i lu t ed 1 :1 0 wi th 0 .1x TE bu f f e r

a n d s t o r e d a t -2 5oC.

4 . 7 . 5 A mpl i f i c a t ion

5 µ l o f t h e p r e amp l i f i ed m ix tu r e w as ad d ed t o a mix tu r e o f : w a t e r

( 9 .9µ l ) , PCR - Buf f e r ( 2µ l ) , 1 mM d NTPs (2µ l ) , P r im e rs Mse I ( 0 . 6µ l ) ,

P r i me r E c oR I ( 0 . 1µ l ) a nd Ta q ( 0 . 4µ l )

T w o p r im e r c om bina t i on s w h er e u s ed : M s e I+ C C , E co R I+ A A ;

E c oR I+ A G a n d Ms eI+ C T . Th e a mpl i f i c a t i on wa s c a r r i ed o u t i n a

M a s t e r c yc l e r g r a d i e n t ( Ep pe nd o r f ) wi t h 12 c yc l e s o f : 94oC fo r 1 min

( d e n a tu r a t i o n ) , 65oC fo r 30 s ( an n e a l i ng ) a n d 70

oC fo r 1 m in

( ex t e ns io n ) an d 22 c yc l e s o f 94oC fo r 3 0s , 65

oC fo r 3 0s e co n ds a nd

7 0oC f o r 1 m in . T he p ro du c t w as s t o r ed a t - 2 5

oC.

4.7.6 Capillary electrophoresis

1 µ l o f t h e a mpl i f i ed p r od u c t w a s m ix ed wi t h 15 µ l o f H iD i

f o rm a mid e (A pp l i ed Bi os ) an d 0 . 5 µ l G e n e -S c an 5 00 m a rk e r (A BI) .

T h e mix tu r e w a s den a tu r ed a t 9 00C fo r 3 mi nu t e s t o ob t a in s in g l e

s t r a nd e d D NA . The s amp l es w e r e a n a l yz e d u s in g c a p i l l a r y

e l e c t r op ho r es i s .

4 . 7 . 7 A FL P An al ys i s :

G e n eM a pp e r ( A pp l i e d Bi os ys t e ms ) w as us e d to an a l ys e t he d a t a w as

a n a l ys e d us in g BIO N U ME R IC S so f t w ar e . Th i s p ro gr a m ca l c u l a t ed

t h e d i s t an c e m at r ix b y t h e f o r mul a fo r t h e “s i mpl e m at c h

c o e f f i c i e n t ” (K osm a n & Le o n a r d 20 05 ) Th e d e nd o gr ams t h r es h -

h o l d o f 40 0 . Th e co mp a r i so n w as d e t e r mi n ed b y l o ok i n g a t a l l e l e

25

s imi l a r i t i e s b e tw e en s ub c l on es . Th e a l l e l e s i z e s t a r t ed a t 7 5b p a nd

w a s c om p ar e d f o r sm al l es t a l l e l e s .

26

5 R esu l t s :

5 . 1 Ax en ic i ty o f cu l tu r e s :

Ba c t e r i a l co n t am ina t i on w as d e t e rmi ne d u s i n g A c r i d i n e o r a n ge

d u r i n g 4 s am pl i n g p e r i od s t h r ou gh ou t t h e wh o l e ex p e r i m en t . Th e s e

v a lu es a r e sum ma r i s e d in T a b l e 6 . T h e ax en ic cu l tu r e s bec a m e

c o n t am in a t e d wi th b a c t e r i a b e tw e e n da ys 1 5 an d 20 .

5 . 2 Grow th o f Ax en i c an d X en i c cu l tu r es :

T h e g r o wt h o f t h e x e n i c a nd ax e n i c cu l t u r e s w as m on i to r e d b y c e l l

c o un t s o v er t he 20 d a y p e r io d .

St r a i n S2 -P4 - D1 ( F i gu r e2 ) s ho w ed a s l i gh t d i f f e r e nc e i n t h e g r ow th

c u r v es b e t w e en th e x en i c an d ax en ic cu l t u r e s . Th e ax en ic c u l t u r es

r e a c h ed a h i gh e r c e l l co n c en t r a t i o n wi t h t h e i r h i gh e s t c e l l

c o n c en t r a t i o n a t d ay 2 0 w h i ch co r r es po n de d to a c e l l co nc e n t r a t i o n

o f 6 x 1 04 c e l l sm l

- 1. T he x e n i c c u l tu re r e a ch e d i t s h i ghes t

c o n c en t r a t i o n on da y 1 0 w h i ch co r r esp o nd ed t o a c e l l c on c e n t r a t i on

o f 4 .2 x 1 04 c e l l sml

- 1.

S t r a i n S2 -P4 - F2 ( F i gu r e 3 ) sh ow e d a s i mi l a r t r e nd , wi th t h e ax e n i c

c u l tu r e r e a c h in g h igh e r c e l l co n c en t r a t i o n t h an t he x e n i c c u l tu r e .

T h e h i gh e s t c on c e n t r a t i o n r e a ch ed b y t h e ax e n i c cu l tu r e w a s

a t t a in e d on D a y 1 6 . Th i s c o r r e sp on d ed t o a ce l l c on c e n t ra t i on o f

6 . 2 x 103c e l l sml

- 1. T h i s t he n l e ad to a sh a r p d ec r e as e i n c e l l

c o n c en t r a t i o n . Th e x en i c cu l t u r e s r e ac h e d a m ax imu m c e l l

c o n c en t r a t i o n o f 5 .4 x 103 c e l l sm l

- 1 on da y 1 8 wh e r e i t su r p as se d th e

c e l l co n c en t r a t i o n o f t he ax e n i c c u l t u r e s t o r ea c h a m ax im um o f 5 . 8

x 103 c e l l sm l

- 1.

27

0.00E+00

1.00E+03

2.00E+03

3.00E+03

4.00E+03

5.00E+03

6.00E+03

7.00E+03

0 2 4 6 8 10 12 14 16 18 20 22

Time (days)

Ce

lls

ml -1

axenic

xenic

Fi g ur e 2 : G ro wt h o f t h e c u l t u r e s t r a in S 2- P4 - D1 o ve r 2 0 d a ys .

0.00E+00

2.00E+03

4.00E+03

6.00E+03

8.00E+03

0 2 4 6 8 10 12 14 16 18 20 22

Time (days)

ce

lls

/m

l

axenic

xenic

Fi g ur e 3 : G r ow th o f t he cu l tu r e s t r a in S 2- P4 - F2 ov e r t he 20 d a y

ex p e r im e n t i n ax en i c an d x e n i c cu l tu re s .

T h e g r o wt h r a t e v a r i ed s l i gh t l y b e t w ee n t h e t w o S 2 -P4 - D1 cu l tu r e s

( F i gu r e 4 ) . Th e axe n i c cu l t u r e s h ad a g r o w th r a t e o f 0 . 42 , wh i l e t h e

28

x e n i c c u l tu r es h a d o n e o f 0 . 41 . T h e ax e n i c c u l tu r es h a d a 2 d a y l a g

p h as e an d w en t i n to ex po n en t i a l g ro wt h ( sum ma r i s ed i n t a b l e 6 ) .

T h e x en i c cu l t u r e s f o l l o w e d ro u gh l y t h e s am e p a t t e rn , w i t h l o w e r

c e l l co n c en t r a t i o ns . T he l a g ph as e l a s t e d t i l l t h e fo u r th da y, a n d

t h en th e cu l tu r es we n t i n t o ex p on e n t i a l g r ow th . T h e s t a t i o n a r y

p h as e w a s s im i l a r fo r b o t h c u l t u r e s a nd l a s t ed fo r t h e r em ai nd e r o f

t h e ex p e r im e n t .

T h e g r o wt h r a t e s o f b o th ax e n i c a nd x en i c c u l t u r e s o f S 2 -P 4- F2

( F i gu r e 5 ) w e r e v i r t u a l l y t h e s am e , n am el y 0 . 31 an d 0 . 33

r e s p ec t iv e l y. T h e d e f i n ed g r ow th p h as e s a r e su mm a r i s e d in T a b l e 6 .

Bo t h cu l tu r e s r e a che d t h e s t a t i on a r y p h a s e a t Da y 1 4 an d r e m ai n ed

t h e r e wi t h no s i gn i f i c an t c e l l co n c en t ra t i on ch a n ge s t i l l t h e

r e m ai nd e r o f t h e ex p e r i m en t .

2.00E+00

2.50E+00

3.00E+00

3.50E+00

4.00E+00

4.50E+00

0 2 4 6 8 10 12 14 16 18 20 22

Time (Days)

log

cell

s/m

l-1

axenic

xenic

Fi g ur e 4 : G r ow th c u r v e o f cu l tu r e o f s t r a in S2 -P 4 -D 1 d ep i c t i n g t he

l a g , ex po ne n t i a l a nd s t a t i on a r y p h a s e s .

29

2.00E+00

2.50E+00

3.00E+00

3.50E+00

4.00E+00

4.50E+00

0 2 4 6 8 10 12 14 16 18 20 22

Time (Days)

log c

ellsm

l -1

axenic

xenic

Fi g ur e 5 : G ro wt h cu r v e o f S 2- P4 - F2 sh o wi n g th e l a g , ex p o ne n t i a l

a n d s t a t i o na r y p h a se s d e f i n ed in t a b l e 6 .

T abl e 5 : Ba c t e r i a l c ou n t s fo r ax e n i c a n d x en ic cu l tu r e s u s i n g

A c r id i n e o r an ge o n f ou r s am pl i n g d a ys .

B a ct e r i a l coun t s ml- 1

S 2 - P4 - D 1 D a y 0 D a y 8 D a y1 4 D a y 2 0

Ax e n i c 0 0 0 5 . 8 x 1 02

X e n i c 9 . 4 x 105 9 . 2 x 10

5 8 . 4 x 10

5 7 . 1 x 10

5

S 2- P4 - F2

Ax e n i c 0 0 0 6 . 2 x 102

X e n i c 9 . 1 x 105 8 . 8 x 10

5 8 . 6 x 10

5 7 . 3 x 10

5

30

T abl e 6 : Th e g r owt h ph a s es a s d e f in ed . Th e g ro wt h r a t e w a s

c a l cu l a t ed u s i n g the d e f in e d ex p on e n t i a l p h as e s .

S t ra in L ag

ph as e ( Da ys )

E xpon en t ia l

ph as e ( D ay s )

S ta t ion a ry

ph as e ( D ay s )

Ax e n i c S 2 -P4 - D1 0 - 2 2 - 8 8 - 20

X e n i c S2 -P 4 -D 1 0 - 4 4 - 10 1 0 -2 0

Ax e n i c S 2 -P4 - F2 0 - 4 4 - 10 1 0 -2 0

X e n i c S2 -P 4 - F2 0 - 6 6 - 12 1 2 -2 0

5 . 3 Pr odu ct ion o f PS P t ox i ns by a xen i c an d xe n i c cu l tu r es :

5 . 3 . 1 T ox in p r of i l e s

In o r d e r t o d e t e r min e t h e PSP t ox in c on t en t , s am pl es w e re t ak en

e v e r y s e c on d d a y f o r a p e r io d o f 20 da ys .

T h e ax en ic an d x en i c cu l tu r es o f s t r a in S 2- P4 - D1 ( F i gu r e 6 A )

p r od u c ed th e fo l l ow i n g PSP tox in s : S TX , N e oST X, GTX 1 /4 , dc

S TX , C1 / 2 a nd C 3 /4 . ST X w as t h e mo s t ab un d an t , a c cou n t in g fo r

r o u gh l y 4 8 – 5 0% of t h e mo la r p e r c en t i n t he ax e n i c c u l t u r e s , wh i l e

i t a c co un te d f o r s l i gh t l y m o r e i n t h e x e n i c cu l t u r e s (5 3 -54 mo l% ) .

T h e s e co nd l a r ges t f r a c t i o n w a s f rom N e oS T X wh ic h a c co u n t ed fo r

3 1 % in t h e x e n i c an d 2 9% i n ax e n i c cu l t u r e s . Th e C1 /2 t ox in s

a c c o un t e d f o r 1 0 – 1 3 % i n bo t h c u l t u re s . G TX 2/ 3 , d c GT X 2/ 3 , d c

S TX an d C3 / 4 w e r e t h e l e as t ab un d an t a n d on l y a c c o u n t e d f o r 1 ,

0 . 5 , 0 .0 6 an d 0 . 04 % r es p e c t i v e l y.

T h e ax en ic an d x en i c cu l tu r es o f s t r a in S 2- P4 - F2 ’ s ( Fi gur e s 7 A &

B ) h ad a s im i l a r t ox in p r o f i l e t o t h a t o f S2 -P 4 -D 1 . ST X a c c o un t e d

f o r r ou gh l y 3 3 - 39 % in b o th t r e a tm e n t s . N eoS T X f o l l owe d wi t h a n

a b un d an c e o f 32 - 35% in b o th cu l tu r es . C 1 / 2 w as t h e t h i rd m os t

31

a b un d an t t ox in , a c co u n t i n g f o r 16 - 19 % o f t h e t o t a l mo l a r

c o n c en t r a t i o n / pe r ce n t . G TX 1/ 4 , G T X2 /3 , d cG T X2 /3 , dcS TX w er e

t h e l e a s t a bu nd a n t w i t h a v er a ge v a lu es o f 9 , 2 , 1 an d 0 . 05

r e s p ec t iv e l y.

T h es e p r o f i l e s r e ma i n ed c on s t a n t t i l l t h e c u l t u r e s h ad r ea c h e d t h e

s t a t i on a r y p h a s e . T h e 20t h

d a y o f s am pl i n g s ho w ed a s l i gh t

v a r i a t i o n in b o t h t he ax en i c an d x en i c t r i p l i c a t e s . Th e v a r i a t i o n w as

i n t h e f o rm o f a m ar k e d r ed u c t i on i n GT X 1/ 4 p r od u c t i on (F i gu r e

6 B) . Th es e l ev e l s w h e r e so l ow t ha t t h e y c o u ld n o t b e qu a n t i f i e d .

S 2 - P4 - F2 h a s s aw va r i a t i on i n t h e x en ic t r i p l i c a t e . I t s how e d a h i gh

d e c r e as e i n S TX p ro d uc t io n ( F i gu re 6 ) . T he m ol a r p e r c e n t s a nd

t o t a l t ox i c i t y d i f f e re d o v e r t h e c ou rs e o f t he ex p e r im e n t . S TX an d

N e oS T X s ho w e d t he g r e a t es t v a r i a t i o n w h er e b y t h e 2 0t h

d a y t h e

m ol a r r a t i o o f S T X h ad d e c r e as e d f rom 5 0 to 2 0% i n bo th t h e x en i c

a n d ax en i c cu l tu r es o f S 2 -P4 - D1 . Th e s am e w a s obs e r v ed f o r S 2 -

P 4- F2 w hi ch s a w the ST X mol a r r a t i o d e c r e as e f r om 3 5 to 16 %.

32

A)

0%

20%

40%

60%

80%

100%

D+1 D+2 D+3 D-1 D-2 D-3

% composition

C3/4

C1/2

B2

B1

dcGTX2/3

dcSTX

GTX 2/3

GTX 1/4

NEO

STX

B)

0%

20%

40%

60%

80%

100%

D+1 D+2 D+3 D-1 D-2 D-3

% composition

C3/4

C1/2

B2

B1

dcGTX2/3

dcSTX

GTX 2/3

GTX 1/4

NEO

STX

Fi g ur e 6 A and B : PST p r o f i l e o f S 2 -P 4- D 1 sh ow in g t h e m ol a r

p e r c en t c omp os i t i on o f e ac h PST a t a ) t = 0 ( s am pl i n g t im e z e r o )

a n d b ) T =2 0 ( en d o f t h e ex p e r i m en t )

33

A)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

F+1 F+2 F+3 F-1 F-2 F-3

% composition

C3/4

C1/2

B2

B1

dcGTX2/3

dcSTX

GTX 2/3

GTX 1/4

NEO

STX

B)

0%

20%

40%

60%

80%

100%

F+1 F+2 F+3 F-1 F-2 F-3

% composition

C3/4

C1/2

B2

B1

dcGTX2/3

dcSTX

GTX 2/3

GTX 1/4

NEO

STX

Fi g ur e s 7 A and B : P ST p ro f i l e o f S2 -P 4 - F2 i n m ol a r pe r c e n t a t a )

t = 0 ( s a mpl in g t ime z e ro ) an d b ) T= 20 ( en d o f t h e ex p e r i me n t )

34

5 . 4 T ox in c ont en t v e r s es c e l l g row th in a x en i c and x en ic cu l t u re s :

T ot a l t ox in co n t en t w a s p lo t t ed a ga i ns t t he g r o wth cu r ve t o

d e t e r min e t h e am oun t o f t ox in p r od uc t i on a t e a c h g r o wth p h as e .

T h e ax en ic cu l tu r e s o f S 2 -P4 - D1 ( F i gur e 8 a ) s ho w ed a d ec r e a s e i n

t ox i c i t y d u r i n g th e l a g ph a se ( f r om 9 0 f m ol / c e l l t o 60 fmol / c e l l ) .

T h e PST co n ce n t r a t i on f l u c tu a t e d du r in g th e ex po ne n t i a l p h as e ,

r e a c h i n g a h i gh o f 7 0 f mol / ce l l . T h e s t a t i on a r y p h a s e s ho w e d a

s h a rp d e c r ea s e i n t ox i n co n t en t ( f rom 7 0 – 49 f mol / ce l l ) f o l l o w e d

b y a n in c r e as e t o 60 fm ol / c e l l , w h i ch w a s t h e n f o l l ow e d b y a

p e rm a n en t de c r e as e f o r t h e r em ai nd e r o f t he ex p e r im e n t t o 25

f m ol / c e l l . T h e va r i a t i on s i n t ox i n c on te n t t h a t w h e re s t a t i s t i c a l l y

s i gn i f i c an t w as d u r i n g t he l a g / ex po n en t i a l p h as e a nd t h e s t a t i on a r y

p h as e . S2 -P 4 -D 1 x e n i c cu l t u r e h a d a h i gh er t ox in c on t en t t ha n th e

ax e n i c c u l t u r e . The l a g p ha s e s aw a s l i gh t i n c r e as e i n c e l l t ox in

c o n t e n t ( i . e . f r om 10 0 -1 10 fmo l / c e l l ) . T he ex po n en t i a l ph a s e

ge n e r a l l y s a w a n inc r e a s e i n t ox i c i t y . T h i s c o r r e sp on d ed t o t h e

h i gh es t t ox in c on t en t r e a ch e d b y t h e cu l t u r e o f 2 10 f mol / c e l l a t d a y

8 . Th e s t a t i o n ar y p h as e s a w a co n t i nu o us d e c re a s e i n t ox in co n t en t .

T h e r e w as a s t a t i s t i c a l l y s i gn i f i c a n t d i f f e r e n ce b e tw e e n th e t h r e e

g r o w th p h as es .

T h e ax en ic cu l tu r e s o f S 2 -P4 - F2 ( F i gu r e 8 B) sh ow e d a n in c r e as e i n

t ox i c i t y f r om t h e l ag t o ex p on e n t i a l p ha s e t h a t m imi c k ed th e g r ow th

c u r v e . I t i n c r e as e d s t e ad i l y t o 15 0 fmo l / c e l l t i l l t h e mi d

ex po n en t i a l p ha s e w h e r e i t d ro pp e d o f f s l i gh t l y t o 13 0 fmo l / c e l l . I t

i n c r e a s ed to 1 50 fm ol / c e l l a t t h e be g in n i n g o f t h e s t a t i on a r y p h a se

a n d s t e ad i l y d e c r e a s e d th e r e a f t e r t i l l i t r e a ch ed a v a l ue o f 5 0

f m ol / c e l l . Th e tox in co n t e n t o f t h e x en i c c u l t u r e s ho w ed l e s s

v a r i a t i o n . I t i n c r e as e d e v en l y f r o m the l a g p ha s e t o t h e ex p on en t i a l

p h as e t o i t s m ax imu m v a lu e o f 4 00 fmo l / c e l l . As t h e cu l tu r e mo v ed

35

i n t o i t s s t a t i o n ar y p h as e , t h e t ox i c i t y d r op p ed o f f s t e ad i ly t i l l i t

r e a c h ed t h e va lu e o f 70 fm ol / c e l l .

0.00E+00

1.00E+00

2.00E+00

3.00E+00

4.00E+00

0 2 4 6 8 10121416182022

Time (Days)

log c

ellsm

l-1

0

50

100

150

200

250

300

Toxin

conte

nt

(fm

olc

ell-1

)

log cells/ml

Toxincontent

A )

0

50

100

150

200

250

300

0 2 4 6 8 10121416182022

Time (Days)

To

xin

co

nte

nt

(fm

olc

ell-1

)

0.00E+00

1.00E+00

2.00E+00

3.00E+00

4.00E+00

log

ce

lls

ml-1

toxincontentlog cellsml-1

B)

Fi g ur e 8 A & B : Ch a n ge s i n t o t a l P SP tox i n co n c en t r a t i on an d c e l l

c o n c en t r a t i o n a s a f u n c t i on o f c u l tu r e a ge o f A ) ax en i c and B)x e n i c

S 2- P4 - D1 .

36

0

100

200

300

400

500

0 2 4 6 8 10121416182022

Time (days)

To

xin

co

nte

nt

(fm

ol/

ce

ll)

0.E+005.E-011.E+002.E+00

2.E+003.E+003.E+004.E+004.E+00

log

ce

llm

l-1

Toxin content

Log cell/ml

A )

0

100

200

300

400

500

0 2 4 6 810121416182022

Time (days)

To

xin

co

nte

nt

(fm

ol/

ce

ll)

0.E+00

5.E-01

1.E+00

2.E+00

2.E+00

3.E+00

3.E+00

4.E+00

4.E+00

log

ce

lls

ml-1

toxin content

log cells/ml

B )

Fi g ur e 9 A & B: Ch a n ge s i n t o t a l P ST a nd c e l l co n ce n t r a t i o n as a

f u n c t i on o f c u l tu r e a ge o f A ) ax en i c and B) x en i c S 2 -P4 - F2 .

37

C om pa r i n g t h e t w o s t r a in s i n t ox in p r od u c t i on a t t h e d i f f e r e n t

g r o w th p h as es yi e l d e d s im i l a r r e s u l t s ( F i gu r es 1 0 & 11 ) . T he x e n i c

c u l tu r e s sh ow e d a m a rk e d in c r e as e i n t ox i c i t y d u r i n g th e l a g a nd

ex po n en t i a l p ha s es . T he s t a t i on a r y p h a s e s a w th e t o t a l t ox i n

p r od u c t i on o f t h e ax e n i c c u l tu r es s u r pa s s t h a t o f t h e x e n ic c u l t u r es

i n bo th s t r a i ns . Th es e d i f f e r en c es wh e re on l y s t a t i s t i c a l l y

s i gn i f i c an t i n t h e ex p on en t i a l p h as e o f t h e x e n i c c u l t u re s o f b o t h

s t r a in s .

0

100

200

300

400

500

600

lag exponential stationary

Growth Phase

To

xin

co

nte

nt

(fm

ol/

cell

)

axenic

xenic

F i g ur e 1 0 : T ot a l t ox i n c on t e n t w i th r e s p e c t t o g ro wt h ph a s e o f

s t r a in S2 -P 4 -D 1 wi t h a s t a nd a rd d e v i a t i o n o f 6 .4 x 1 02(n= 3 ) .

0

200

400

600

800

1000

1200

lag exponential stationary

Growth phase

Toxin

conte

nt

(fm

ol/cell)

axenic

xenic

F i g ur e 1 1 : T ot a l t ox i n c on t e n t a s wi th r es p ec t t o g r o wt h p ha s e o f

s t r a in S2 -P 4 - F2 w i th a s t a nd a rd d e v i a t i on o f 1 . 2 x 102 ( n= 3 ) .

38

T o ta l to x in c on t en t :

T h e to t a l t ox i c i t y f o r t he ax e n i c a nd xe n i c cu l t u r e s o f s t r a i n S 2 -P4 -

D 1 ( f i gu r e 1 2 ) wh er e 6 . 4 x 102 a nd 1 .3 x 10

3 fm o l / c e l l r e s p e c t i v e l y.

T h e x en i c cu l t u r e s p r od u c ed t wi c e as m u ch PSP tox i n as t h e ax e n i c

c u l tu r e s . Th e ax en i c an d x e n i c cu l tu re s o f S2 -P 4- F2 ( F igu r e 11 )

w h e r e mo r e t ox i c t h a n S2 -P4 - D1 ( F i gu r e 1 0 ) , w i th a t o t a l t ox i c i t y o f

1 . 2x 1 03 an d 1 . 6x 10

3 fmo l / c e l l r e sp e c t iv e l y. Th e x e n i c cu l t u r e s

w h e r e 1 . 3 t im e s m or e t ox i c t h an t h e axe n i c cu l t u r e s . T h i s v a r i a t i o n

i n t ox in co n t en t w as s t a t i s t i c a l l y s i gn i f i c an t

0

500

1000

1500

2000

axenic xenic

Strain

To

tal

toxin

co

nte

nt

(fm

ol/

cell

)

S2-P4-D1

S2-P4-F2

F i gu r e 1 2 : To t a l t ox i c i t y o f b o th s t r a i ns o v er t h e 20 d a y p e r io d .

39

A l l e c l o ch e mi ca l t e s t s :

St r a i n S2 -P4 - D1 w as s ho w n t o h av e l yt i c p o t en t i a l . C e l l

c o n c en t r a t i o ns o v er 4 x 1 03

s a w no s u rv iv in g Rh od om on as c e l l s .

T h i s w as s e e n i n bo th cu l tu r es .

0

10000

20000

30000

40000

50000

0 2000 4000 6000 8000

A. tamarense concentration (ml-1)

Su

rviv

ing

Rh

od

om

on

as

(cell

sm

l-1)

axenic

xenic

Fi g ur e 1 3 : D os e - re s po ns e cu rv e d e p ic t i n g R ho dom on a s s u rv iv a l

w i t h r es p e c t t o ch an ge s i n A. t am ar e ns e c e l l co nc e n t r a t i on s .

5 . 7 Sub c l on es

5 . 7 . 1To x in p rof i l e s :

T h e tox i n p r o f i l e o f 4 s t r a i ns o f A. t am a r ens e wa s ob s e rv e d . A l l

s t r a in s h ad d i f f e re n t t ox in p ro f i l e s .

A l l 2 0 su b -c lo n es o f S 2 -P6 - G1 2 ( F i gu re 14 ) h a d s imi l a r t ox i n

p r o f i l e s t o t h a t o f t h e p a r en t c l on e s (PC 1 a nd PC2 ) . T h i s c on s i s t ed

o f B1 ( GT X 5 ) , C1 /2 , G T X1/ 4 , Ne oS TX , ST X , C 3 / 4 ( no t p r e s e n t i n

a l l s a mpl e s ) an d GT X 2/ 3 . Th e mo l a r r a t i o s w h e r e as fo l l o w s : B1

( G T X 5 ) : 32 - 54 %, C 1 / 2 : 20 – 3 0 % , GT X 1/ 4 : 3 -9 % , N eoST X : 8 - 20 ,

S TX : 4 -9 %, C 3 /4 : 1 - 8 % an d GT X 2/3 : 1 -7 % .

40

S 2- P1 - G7 ( F i gu r e ) s ho w e d t h e s am e t r e n d o f t h e s am e tox i n p ro f i l e

b e tw e e n P C’ s a nd th e 2 0 su b c lo ne s . T h e tox i n p r o f i l e s c o ns i s t e d o f

t h e fo l l ow in g ( wi t h t h e i r av e r a ge mo l a r r a t i o i n b r a ck e t s ) : GT X

5 ( 17 - 40 % ) , C1 /2 (30 - 49 ) , GT X 1/4 (5 - 12 ) , G TX 2 /3 ( 4 -1 0 ) , N eoS T X

( 2 - 11 ) a nd S TX (4 -7 ) . Lo w v a lu e s o f d c ST X a nd d c GT X2 /3 wh e r e

a l so o bs e rv e s an d ac c o un te d f o r a mo la r p e r c e n t r a n gi n g f r om 0 .0 1

t o 0 . 1 .

S e ve n o f t h e tw e n ty S 2 - P4 - D1 ( F i gu r e ) su b c l on e s sh ow ed

d i f f e r en c e s i n t ox in p r o f i l e s co mp a r ed t o P C1 an d 2 . T he tox in

p r o f i l e s co ns i s t e d o f t he fo l l o wi n g (wi th t h e i r av e r a ge mo l a r r a t i o i n

b r a c ke t s ) : GT X 2 / 3 ( 3 3 -4 0) , N e oST X (2 5 -3 5 ) , ST X ( 20 - 28) , GT X1 /4

( 2 - 6 ) , w i th l ow l eve l s o f d cS T X a nd dc G TX 2 /3

0%

20%

40%

60%

80%

100%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 PC1 PC 2

% composition

subclone S2-P6-G12

C3/4

C1/2

B2

B1

dcGTX2/3

dcSTX

GTX 2/3

GTX 1/4

NEO

STX

Fi g ur e 1 4 : Tox in p r o f i l e s h ow in g t h e m ol a r p e r c e n t o f a l l t he t ox ins

p r e s en t i n a l l s ub co n es o f S2 -P 6- G 12 .

41

0%

20%

40%

60%

80%

100%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 PC1 PC 2

% composition

subclone S6-P1-G7

C3/4

C1/2

B2

B1

dcGTX2/3

dcSTX

GTX 2/3

GTX 1/4

NEO

STX

Fi g ur e 1 5 : Tox in p r o f i l e s h ow in g t h e m ol a r p e r c e n t o f a l l t he t ox ins

p r e s en t i n a l l s ub co n es o f S1 -P 6- G 7 .

0%

20%

40%

60%

80%

100%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 PC1 PC 2

% composition

subclone S2-P4-D1

C3/4

C1/2

B2

B1

dcGTX2/3

dcSTX

GTX 2/3

GTX 1/4

NEO

STX

F i g ur e 1 6 : Tox in p r o f i l e s h ow in g t h e m ol a r p e r c e n t o f a l l t he t ox ins

p r e s en t i n a l l s ub co n es o f S2 -P 4- D 1 .

42

0%

20%

40%

60%

80%

100%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 PC1 PC 2

% composition

subclone S2-P4-F2

C3/4

C1/2

B2

B1

dcGTX2/3

dcSTX

GTX 2/3

GTX 1/4

NEO

STX

F i g ur e 1 7 : Tox in p r o f i l e s h ow in g t h e m ol a r p e r c e n t o f a l l t he t ox ins

p r e s en t i n a l l s ub co n es o f S2 -P 4- F2 .

5 . 7 . 2 Va r i a t i ons in t o ta l to x i c i ty :

T h e su b c lo ne s h ad v a r yi n g t ox i c i t y ( f i gu r e 1 8 ) wi t h t h e h i gh es t an d

l o we s t v a l u es o f 115 an d 34 fm ol / c e l l r e s p ec t iv e l y. T h i s a c c o un t s

f o r a 3 fo ld d i f f e r en c e i n t ox i c i t y . 10 o f t h e 2 2 s ub c l one s h a d

t ox i c i t y l e s s t h a n th a t o f t h e a ve r a ge tox i c i t y o f a l l t h e su b c lo n es

( F i gu r e 21 ) .

43

0

20

40

60

80

100

120

140

160

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20PC

1

PC

2

Sub clone

Tota

l to

xin

conte

nt (f

mol/cell)

Fi g ur e 1 8 : T o t a l t ox i c i t y o f s ub c lo ne s S2 -P 6 -G 12 . P a re n t c l on e s

d e no t ed a s PC1 an d PC2 wi t h a s t a nd a rd de v i a t i o n o f 2 5 . 25 (n = 20 )

-60

-40

-20

0

20

40

60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20PC

1

PC

2

Sub clones

To

tal

toxin

co

nte

nt

(fm

ol/

cell

)

Fi g ur e 1 9 : V a r i a t i o n in t ox in c on te n t o f S 2 -P6 - G1 2 a s a f un c t io n o f

t h e d i f fe r e n ce b e twe e n t he av e r a ge PSP tox i n c on t e n t o f t h e p a r en t

c l on es an d th e 2 0 su b c lo n es .

44

0

20

40

60

80

100

120

1 2 3 4 5 6 7 8 9 1011121314151617181920PC

1

PC

2

Sub clone

To

tal to

xin

co

nte

nt

(fm

ol/cell)

Fi g ur e 2 0 : To t a l t ox i n c on t e n t o f 20 s ub c l on es o f S2 -P1 - G 7 .

P a r en t c l on es d e no te d as PC 1 an d PC2 , w i th a s t an d ar d d ev i a t i on o f

1 0 .4 8 .

-20

-10

0

10

20

30

40

50

1 2 3 4 5 6 7 8 9 1011 1213 1415 1617 1819 20PC

1

PC

2

Sub-clone

To

tal

toxin

co

nte

nt

(fm

ol/

cell

)

Fi g ur e 2 1 : V a r i a t i o n in t ox in c on te n t o f S 2 -P1 - G7 as a fu n c t i on o f

t h e d i f fe r e n ce b e twe e n t he av e r a ge PSP tox i n c on t e n t o f t h e p a r en t

c l on es an d th e 2 0 su b c lo n es .

45

Fi g ur e 2 2 : To t a l PS T c on c en t r a t i o n o f su b c l on e s S 2 -P4 -D 1 .

P a r en t c l on es d e no te d as PC 1 an d PC2 . W i th a S T DE V o f 3 8 .1 6

-100

-50

0

50

100

150

1 2 3 4 5 6 7 8 9 10111213141516 17181920PC1PC2

Subclones

To

tal to

xin

co

nte

nt

(fm

ol/cell)

Fi g ur e 2 3 : V a r i a t i o n in t ox i c i t y o f S2 - P4 - D1 as a fu n c t i on o f t h e

d i f f e r en c e b e t w e en th e t o t a l t ox i c i t y o f t h e i n d i v id u a l s and av e r a ge

t ox i c i t y o f t h e g r oup .

5 . 6 A FL P:

T w o p r im e r c om bina t i on s w e r e us e d . T h e r e su l t s o f t h e d i f f e re n t

p r im e r co mb in a t io ns a r e s um ma r i s ed in Ta b l e 6 . T h e p r im e r

c o mbi n a t i on o f E _A G _M _C T w as u s ed du e to t h e h i gh er v a l u es

o b t a i n ed wh en co mp a r i n g t h e s imi l a r i t y o f t h e s ub - c l on e s

T h e d en do gr a m o f S 2- P4 - D1 ( F i gu r e ) s ho w e d t h e mos t v a r i a t i o n i n

s ub c l on e g ro up in g . I t ha d a l ow s imi l a r i t y o f 37 %. S ub - c l on e 5

w a s t h e m os t o bv iou s ou t l i e r . T he r em ai n i n g s ub - c l on es h ad a

s imi l a r i t y v a l u e o f 8 2 %. Th e s e wh e r e f u r t h e r d iv id e d i n to t h re e

46

d i s t i n c t g r ou ps w i th a s im i l a r i t y o v e r 9 0 %. T h e S 2 -P4 - F2 s ub -

c l on es s ho w e d l e s s v a r i a t i on an d h ad a h i gh e r bo o t s t r ap v a lu e o f

8 6 % wi th o n e ou t l i e r su b - c lo n e . Th e r e m ai n in g su b - c lo ne s wh e r e

d iv id ed i n t o t w o d i s t i n c t g r ou ps wi t h a bo o t s t r ap v a l u e o f 91 % .

S 6 - P1 - G7 s ho w ed a h i gh e r l ev e l o f s im i l a r i t y w i t h a b oo t s t r ap v a l u e

o f 8 3 % . T h e y w h e r e f u r th e r d i v id e d in to 2 d i s t i n c t g r oup s wi th

b o o t s t r a p v a lu e s o f 8 9 an d 97 % r es p ec t i v e l y. S2 -P 6 -G 12 ha d a

b o o t s t r a p v a lu e o f 8 9 %. Th e s ub c lo ne s wh e r e d iv id e d i n t o 2

d i f f e r en t g r o up s wi t h bo o t s t r a p v a l u es o f 97 % . D u e to t im e

c o ns t r a in t s t h i s d a t a c ou ld n o t b e a n a lys e d f u r t h e r t o d e t e r mi n e

w h e t h e r t h e d i f f e r en c e s s ee n w h er e s t a t i s t i c a l l y s i gn i f i c an t .

47

T ab l e 6 : C omp a r i so n o f p r im e r c om bin a t io ns u s ed i n A FLP .

Primer combination

E_AA_CC_

S2-P4-D1 TH75 TH100 TH350 TH400

av similarity 89.23867 89.23867 88.30536 88.22725 av sim duplicates 92.96455 92.85091 88.52773 91.05638

S2-P4-F2 TH75 TH100 TH350 TH400


S6-P1-G7 TH75 TH100 TH350 TH400


S2-P6-G12 TH75 TH100 TH350 TH400


Primer combination

E_AG_CT_

S2-P4-D1 TH75 TH100 TH350 TH400


S2-P4-F2 TH75 TH100 TH350 TH400


S6-P1-G7 TH75 TH100 TH350 TH400


S2-P6-G12 TH75 TH100 TH350 TH400

av similarity 93.82936 91.8222 94.43128 95.84905

av sim 96.03182 95.91727 95.345 96.36773

48

duplicates

S im pl e m atch in g (> 50 % ME AN )

A G _C T _ D u p T H _4 00

100

95

90

85

8075

70

6560

55

5045

40

D 1_ 03 _P la teA

D 1_ 04 _P la teA

D 1_ 11 _P la teA

D 1_ 12 _P la teA

D 1_ PC 1_ Pla teA

D 1_ 08 _P la teA

D 1_ 06 _P la teA

D 1_ 19 _P la teA

D 1_ 20 _P la teA

D 1_ 02 _P la teA

D 1_ PC _2 _P la te A

D 1_ 01 _P la teA

D 1_ 09 _P la teA

D 1_ 13 _P la teA

D 1_ 14 _P la teA

D 1_ 15 _P la teA

D 1_ 17 _P la teA

D 1_ 18 _P la teA

D 1_ 07 _P la teA

D 1_ 10 _P la teA

D 1_ 16 _P la teA

D 1_ 05 _P la teA

F i g ur e 2 4 : Dendogram depicting similarity between sub-clones of strain S2-P4-D1


A G _C T _ D u p T H _4 00

100

999897

96

95

94

93

92

91

90

89

88

8786

F 2_ 01 _P la te A

F 2_ 19 _P la te A

F 2_ 03 _P la te A

F 2_ 02 _P la te A

F 2_ 17 _P la te A

F 2_ 18 _P la te A

F 2_ 06 _P la te A

F 2_ PC _ 1 _P la te A

F 2_ PC _ 2 _P la te A

F 2_ 04 _P la te A

F 2_ 07 _P la te A

F 2_ 09 _P la te A

F 2_ 10 _P la te A

F 2_ 11 _P la te A

F 2_ 12 _P la te A

F 2_ 13 _P la te A

F 2_ 14 _P la te A

F 2_ 15 _P la te A

F 2_ 20 _P la te A

F 2_ 05 _P la te A

F 2_ 16 _P la te A

F 2_ 08 _P la te A

49

Figure 25: Dendogram depicting similarity between sub-clones of strain S2-P4-F2

S im ple m atch in g (> 50 % ME AN)

A G _C T _ D u p T H _4 00

100

99

98

97

96

95

94

93

929190

89

88

87

86

85

84

83

G 7 _0 8_ P late A

G 7 _0 9_ P late A

G 7 _0 4_ P late A

G 7 _0 5_ P late A

G 7 _0 6_ P late A

G 7 _1 6_ P late A

G 7 _0 2_ P late A

G 7 _0 3_ P late A

G 7 _0 7_ P late A

G 7 _1 1_ P late A

G 7 _1 2_ P late A

G 7 _1 3_ P late A

G 7 _1 4_ P late A

G 7 _1 5_ P late A

G 7 _1 7_ P late A

G 7 _1 8_ P late A

G 7 _1 9_ P late A

G 7 _2 0_ P late A

G 7 _P C_ 1_ Plate A

G 7 _P C_ 2_ Plate A

G 7 _0 1_ P late A

G 7 _1 0_ P late A

Figure 26: Dendogram depicting similarity between sub-clones of strain S6-P1-G7.


A G _C T _ D u p T H _4 00

100

99

98

97

96

959493

92

91

90

89

G 1 2_ 13 _ Pla teA

G 1 2_ 15 _ Pla teA

G 1 2_ 14 _ Pla teA

G 1 2_ 20 _ Pla teA

G 1 2_ 02 _ Pla teA

G 1 2_ 16 _ Pla teA

G 1 2_ 01 _ Pla teA

G 1 2_ 03 _ Pla teA

G 1 2_ 04 _ Pla teA

G 1 2_ 05 _ Pla teA

G 1 2_ 06 _ Pla teA

G 1 2_ 07 _ Pla teA

G 1 2_ 08 _ Pla teA

G 1 2_ 09 _ Pla teA

G 1 2_ 10 _ Pla teA

G 1 2_ 11 _ Pla teA

G 1 2_ 12 _ Pla teA

G 1 2_ 17 _ Pla teA

G 1 2_ 18 _ Pla teA

G 1 2_ 19 _ Pla teA

G 1 2_ P C _1 _P la teA

G 1 2_ P C _2 _P la teA

F i g ur e 2 7 : Dendogram depicting similarity between sub-clones of strain S 2 -P6 -

G 1 2 .

50

6 . D i s cus s i on :

T h e ex t e n t o f b ac t e r i a ’ s i n v o lv em e n t i n PSP t ox in p ro du c t io n i s s t i l l

c o n t i nu in g . I t h a s b e e n su gge s t ed t ha t b a c t e r i a c om pl em en t t h e

d in o f l a ge l l a t e P SP p r od u c er s b y a f f e c t i n g t he g r o wth r a t e o r t h e

a c t ua l PS P t ox in p ro d uc t io n . In o r d e r t o a s s es t h i s e f f e c t ,

ex p e r im e n t s w h e r e c o nd u c t ed t o d e t e rm in e w h i ch p a r am et e r i s

a f f e c t e d b y b a c t e r i a .

Gr ow th ra t e :

T h e g r o wt h r a t e s o f b o th s t r a i ns o f A . t a mar e ns e w h e re ve r y s i mi l a r .

T h e s im i l a r i t i e s i n g r o w th r a t e a r e c ons i s t e n t w i th Ho l d e t a l ( 20 01 )

f i nd i n gs on A . t a ma r en s e . T h e i r ex p e r i me n t s u s ed A . l u s t i t a n i cu m

a n d A. t a mar e ns e an d c om p ar e d th e g r o w th r a t e an d tox in p ro du c t io n

b e tw e e n ax e n i c an d x en i c cu l t u r e s . A nt ib i o t i c s wh e r e us ed t o ob t a in

ax e n i c c u l t u r es . Th e ax e n i c a nd x e n ic A. t a mar e ns e cu l tu r e s

s ho w e d eq u a l g ro wt h r a t e s . Th e a u t h en t i c i t y o f t h e ax en ic c u l t u r es

w a s t h o ro u gh l y i n ve s t i ga t e d ( so l id and l i q u id m e d i a ,

e p i f l u o re s c en c e mic r o s co p y w i th S yb r g r e e n 1 a n d PC R us in g

u n i v e r s a l e ub a c t e r i a l p r i me r s t a r ge t i ng 1 6S r RN A , ) .

E v en t ho u gh th e x en i c c u l t u r e s o f bo th s t r a in s t oo k 4 d ays t o r e a c h

t h e i r ex p on e n t i a l g r o wt h ph a s e ; t h i s d i d no t s e e m t o a f f ec t t h e

o v e r a l l g ro wt h r a t e . T he d i f f e r en c e i n c e l l c on c e n t r a t i ons w as

p r ob a b l y d u e to t h e d i f f e re n c es i n i n i t i a l i n o cu l a . W h e n th e ax e n i c

c u l tu r e s wh e r e t r a ns f e r r ed in to n e w me d ium , t h e x e n i c c u l tu r es

w h e r e a l so t r an s f e r r e d . T h i s i d en t i c a l t r e a tm en t r ed u c ed t h e c ha n c e

o f h a v i n g c u l t u r es t h a t w h er e n o t i n t h e s am e g r ow th p ha s e . T h e

ex t en de d l a g p h as e o bs e r v ed in t he x e n i c cu l tu r es s u gge s t s t h a t t h e

b a c t e r i a l m i c r o f l o ra i n t h e m e d iu m m igh t a f f e c t n u t r i e n t u p t a k e

i n i t i a l l y d u e to t he f a c t t h a t t h e o th e r f a c t o r s t h a t co u ld a f f e c t

51

g r o w th wh e r e c ons t a n t ( i . e . l i gh t i n t e ns i t y , t e mp e r a t u r e , s a l i n i t y ,

p H ) . Th ou gh th e b ac t e r i a a f f e c t e d t h e l a g ph a se i n i t i a l l y , t h e y s e e m

t o h av e n o e f fe c t on t h e g r o wt h r a t e o f c u l t u re s a f t e r . Th i s

d i f f e r en c e co u ld a l s o h av e b e e n du e to t h e l o w er c e l l con c e n t r a t i on s

u s ed i n i t i a l l y i n t he no n - ax en i c c u l t u re s .

T o ta l to x in c on t en t :

O n e o f t h e f i r s t s t ud i es t h a t co mp a r ed ax e n i c a nd x e n i c cu l t u r e s

w i t h r es p e c t t o g r ow t h an d tox i c i t y w e r e r ep o r t ed b y D a n tz e r &

Le v i n ( 19 97 ) . T hey u s e d A . t a ma r en se c u l t u r es a nd fo und t h a t t h e r e

w e r e n o d i f f e r e n c es i n t ox i c i t y b e t w ee n t h e ax e n i c a nd xe n i c

c u l tu r e s . Th e y t h en co n c l ud ed t h a t b ac t e r i a h ad n o e f f e c t o n

t ox i c i t y . Th i s i s co n t r ad i c to r y t o m y r e s u l t s b ec a us e , ove r a l l t h e

x e n i c c u l tu r es o f bo th s t r a i ns wh e r e m o re t ox i c t h an th e ax en ic

c u l tu r e s . X e n i c S2 -P 4- D 1 w as t wi c e a s t ox i c , wh i l e x en i c S 2- P4 - F2

w a s 1 .3 t im e s a s t ox i c . Th i s i s co ns i s t e n t wi th f i n d i n gs b y D o u c e t t e

& Po w el l (1 99 8 ) a nd Ho ld ( 20 01 ) .

T h e un i fo rmi t y o f t ox i n c on t e n t s e en by D a n tz e r & Le v i n ( 1 99 7 )

c o u l d h av e b e en d ue to t h e m et ho ds u se d t o d e t e rmi n e ba c t e r i a l

c o n t am in a t io n . T h ey g r e w th e i r cu l tu re s i n p en i c i l l i n - r i ch m e d iu m

a n d t e s t e d t h e ax en i c i t y o f t h e cu l tu r es b y u s in g m a r i n e aga r . T h i s

m et ho d w a s no t v e ry e f f e c t iv e d u e t o t h e f ac t t h a t 9 5 % o f ma r in e

b a c t e r i a a r e un cu l tu r a b l e (S c hu t e t a l 1 9 93 ) . S o l a ck o f b a c t e r i a l

g r o w th co u l d s im ply h a v e b e en d ue t o t h e un su i t ab i l i t y o f t h e m ed i a

u s ed . D ou c e t t e an d Po w el l (1 99 8) u s ed l ys o z ym e / SD S t o r e mo v e

b a c t e r i a f rom t h e i r A . l u s t i t a n i cu m c u l t u re s . T he y f o u nd t h a t t h e

ax e n i c c u l t u r es w he r e l e s s t ox i c t h a n t h e x en i c o n es a nd t h a t t ox i n

p r od u c t i on co u ld be r e s to r ed b y t h e ad d i t i o n o f b ac t e r i a b e l i ev e d to

b e PS T p ro du c e rs . T he y c o n c lu de d tha t t h e i n t e r ac t io n be t w e en A .

l u s t i t an i c um an d b ac t e r i a a c c ou n t ed fo r 50 % o f t h e ce l l u l a r t ox in s

52

p r od u c ed . T h i s i s c o ns i s t e n t w i th t he s t r a i n S 2 -P4 - D1 wh i ch

s ho w e d a tw o f o l d i n c r e a s e i n t h e t o t a l t ox in p ro du c t io n . So th i s

c o u l d po i n t t o t h e i d e a o f b a c t e r i a e nha n c in g t ox in p ro duc t i on .

H o w ev e r t h e ax e n ic n a t u r e o f D a n tz e r & Le v i ns (1 99 7 ) cu l t u r e s i s

q u es t i o n ab le d u e to t h e m et ho ds u se d fo r b a c t e r i a l co n t ami n a t i on .

T h e y u t i l i s ed ep i f l u o r es c en c e mi c ro s co p y a n d l i q u i d me d i um to

d e t e r min e t h e ex t en t o f b ac t e r i a l c on ta mi n a t i on . Th e y d i d no t

r e p o r t t h e f r eq u en cy o f t h e i r m on i to r in g o f b a c t e r i a l g row t h .

A n o t he r f a c to r w as t h a t t h e ob se r v a t i on s d i d no t t a k e i n t o a c c ou n t

t h e e f f ec t l ys o z ym e /S DS t r e a tm e n t mi gh t h av e h ad o n A .

l u s t i t an i c um ’ s g r ow t h r a t e .

T h e h i gh e s t t ox in p r o du c t io n o c cu r r ed d u r i n g th e ex po n en t i a l ph a se

f o r a l l t h e cu l tu r es . Th i s i s c on s i s t en t w i th r esu l t s b y Bo ye r e t a l

( 1 98 7 ) a nd A nd e rso n e t a l (1 99 0) . SX T an d N eoS T X in b o th x e n i c

a n d ax en i c cu l tu r es i n c r e as e d du r i n g th e ex po n en t i a l ph as e an d

s ho w e d a c l ea r d e c r e a s e du r i n g t h e s t a t i on a r y p h as e .

W h e n c omp a r i n g t he t o t a l t ox in p r od uc t i on a t t h e d i f f e r en t g r o wt h

p h as e s , m a rk e d ch an ge s a re o bs e r ve d . T he d i f f e r en c e s i n t h e

ex po n en t i a l g ro wt h p ha s e o f t w o da ys i n t h e ax en ic an d x e n i c

c u l tu r e s co u l d h av e l e ad to t h e d i f f e r en c e i n t o t a l t ox in co n t e n t .

T h e x en i c cu l t u r e s s p en t l on ge r i n t h e ex po n en t i a l g ro wt h p ha s e

w h e r e t ox in p r od u c t io n i s a t i t s h i gh e s t a l l ow in g th em t o p r od u c e

m o re t ox ins . Th i s o bs e r v a t i on w a s c on s i s t en t wi t h bo th s t r a in s . T h e

ax e n i c c u l t u r es i n S 2- P4 - F2 s ho w e d a d i f f e re n c e i n t h e s t a t i o n ar y

p h as e w h e r e m o r e t ox i ns w h e r e p r od u ce d t h an in t h e x e n ic c u l t u r es .

T h i s w as p r ob a b l y d u e to t h e f ac t t h a t t h e y h a d a l on ge r s t a t i on a r y

p h as e . E v en th o u gh t ox in p ro du c t io n d e c r e as e s i n t h i s g r o w th

p h as e , i t do e s so s t e a d i l y ( i . e . no s h a rp de c r e as e s ) . Th i s e n a b l ed

t h e ax e n i c cu l tu r es t o p r od u ce m o re t ox in s bu t t h i s d i f f e re n c e w a s

n o t s t a t i s t i c a l l y s i gn i f i c an t .

53

Bo ye r e t a l (1 98 7) c o nd u c t ed n u t r i e n t l im i t a t i o n ex p e r i men t s w i t h

n i t ro ge n an d ph os ph o ru s on A . t a ma r en s e . Th e y f o u nd tha t t h e

t ox i c i t y d e c r e as e d i n n i t r o ge n l imi t e d c u l tu r e s , w h i l e an i n c r e as e

w a s f ou nd i n ph os ph o ru s l im i t ed c u l tu r e s . T h e i n c re a s e i n PS T

p r od u c t i v i t y w a s du e t o t h e f a c t t ha t t h e c e l l s wh e r e un ab l e t o

d iv id e , c au s in g c e l l s t o ge t b i gge r . PS T p ro du c t i o n d id n o t s to p .

S o t h e P ST co nc e n t r a t i on i nc r e as e s p e r c e l l , b u t t h e ce l l

c o n c en t r a t i o n r em ai ns co ns t a n t l e ad i ng t o a n ob s e r v ed PS T i n c r ea s e .

S o i t c ou l d b e su gge s t e d th a t t h e b ac t e r i a mi gh t a f f e c t t h e n u t r i en t

u p t a k e o f t h e d in o f l a ge l l a t e , n am el y p h os ph o ru s s in c e t he x en ic

c u l tu r e s wh e r e mo re t ox i c . Th i s i s u n l ik e l y s in c e K m e d iu m i s n o t

n i t ro ge n o r p ho sp ho r us l i m i t e d an d the c e l l s i z e b e t w ee n th e ax en i c

a n d x en ic cu l tu r e s d id n o t v a r y e x t e ns i v e l y.

T h e d i f f e re n c es i n t ox i c i t y m i gh t no t h a v e b e en d ue t o t he b a c t e r i a ,

b u t d u e t o t h e e f f e c t o f t h e an t ib io t i c s u s ed . S t ud ie s on th e e f f e c t s

o f s t r e p t om yc i n a nd ch lo r am ph e n i co l ( w h i c h wh e r e b o th u s ed i n t h e

a n t ib io t i c c o ck t a i l ) sh o w ed h a rm fu l e f f e c t s o n so me a l ga l c e l l s

( M a r t i n s e t a l 20 04) . S t re p t om yc i n cau s ed l o s s o f p i gm en t a t i on an d

c h a n ge s i n c h l o r op la s t m o rp ho l o g y i n C hl or e l l a . In b a c t e r i a i t

i nh ib i t s p ro t e in s yn t h es i s i n g r am n e ga t iv e b a c t e r i a ( M ck an e an d

K a n d e l , 1 99 6 ) . W hi l e ch lo r am ph e n i co l c aus e d in h ib i t i on o f g r ow th

i n Eu gl e na a s w e l l a s a f f e c t i n g c h lo r op h yl l f o rm at i on . Th e r e i s n o

d o cu m en t a t io n o f t h e e f f e c t s o f a n t i b io t i c s o n d i no f l a ge l l a t es

( M a r t i n s e t a l 20 04) . T h e po ss i b l e e f f e c t s o n d i no f l a ge l l a t e c ou ld

b e d i f f e r en t i n t h e s e ns e o f a f f e c t i n g tox i n p ro du c t io n wh i c h wo u l d

l e a d t o a d e c r e as e i n t o t a l t ox i c i t y . O r i t c ou ld a f f e c t t h e c e l l w a l l

b y p e r f o r a t i n g i t , ca u s i n g l e ak a ge o f so m e PS P t ox in s . S i n c e t h e

PS T m ea su r e d i s f ro m in t a c t c e l l s , t he p oss ib l e PSP tox i ns i n t h e

m e d iu m o f t h e ax en i c cu l tu r es w ou ld no t b e d e t e c t ed . O r i t co u ld

a t t a c k th e o r ga n e l l e s o f en dos ym b i o t i c o r i g in ( “ p ro k ar yo t i c

54

c o mp on e n t s ” T h es e a r e b e l i e v ed t o h av e b e e n e n gu l f ed du r in g t h e

e v o l u t i on o f t h e euk a r yo t i c c e l l . ) , n am el y t h e mi to ch ond r i a an d

c h lo ro p l a s t . Th e s e v es t i g i a l o r ga ne l l e s co u l d b e a f fe c t e d b y t h e

a n t ib io t i c s t h a t t a rge t t h e p ro k ar yo t i c c e l l s o f b a c t e r i a ca u s i n g an

e f f e c t .

A n d er so n e t a l (1 99 0 ) c on du c t e d ex p er im e n t s wh e r e t h e y lo ok e d a t

t h e e f f ec t o f N a nd P l imi t a t i ons as we l l a s d e t e rm in in g th e r a t e o f

t ox in p ro du c t io n . T h e y f o u n d th a t c e l l s p ro du c ed t ox ins a t r a t es

e q u a l t o t ho se n e e de d t o r ep l ac e l o s s es t o d au gh t e r c e l l s d u r i n g ce l l

d iv i s i on ( i . e . t h e “g r o w th d i lu t i o n ” e f f e c t ) . I d o no t b e l i e v e t ha t

t h i s ha s a n e f f ec t on t h e s t r a in s us e d du e t o t h e f a c t t ha t i f t h i s l o s s

w a s su bs t an t i a l , t he g r e a t es t l o s s wo u l d b e du r i n g t he exp o ne n t i a l

p h as e s i n c e t h a t i s w h e n t h e mo s t d augh t e r c e l l s a r e p r odu c e d , s o

PSP t ox in c on t en t w o u l d de c r e as e . Bu t t h e h i gh e s t a mou n t o f PSP

t ox in p ro du c e d i s i n t h e ex p on en t i a l ph a s e . Th e y s u gge s t e d t h a t

t ox in p ro du c t io n wa s com pl ex a nd c oup l ed to c e l l g r ow th a t c e r t a i n

c o nd i t i on s a nd u n re l a t e d i n o t h e r co nd i t i on s .

T h i s c ou l d b e du e to t h e f a c t t h a t t h e b a c t e r i a co u l d p l a y a ro l e i n

r e mi n er a l i s a t i o n o f n u t r i en t s . T he y c o u l d a l so p r od u c e m et a bo l i t e s

t h a t a c t a s co - f a c to r s an d s t im u l a t e PSP tox i n p ro du c t i o n i n t h a t

m a nn e r .

T h e PSP tox in p r o f i l e r em ai n ed c on s t an t t h ro u gh ou t t h e 2 0 d a y

ex p e r im e n t a l t h ou gh on e o f t h e t r i p l i ca t e s am pl es o f e a c h s t r a i n

s ho w e d t h e l o s s o f a PSP tox in . T h i s w a s s e en i n on e t r i p l i c a t e o f

t h e s a mp le s : aux e n i c an d x e n i c S2 -P 4- D 1 a nd x e n i c S2 -P4 - F2 .

S i n c e t h i s d i f f e r e nc e w as s e e n i n bo th a ux e n i c a nd x e n i c c u l tu r e s ,

t h i s e f f e c t c an no t be a t t r i b u t e d t o b a c t e r i a l i n f lu en c e .

55

E n do nu c l e a r b a c t e r i a co u ld s t i l l h a v e a n e f f e c t o n tox i n p r od u c t i on .

T h i s c a nn o t b e d e t e r mi n ed wi t h t h e met ho d us e d s in c e w e w h e r e

f o c us i n g o n ex t r a - ce l l u l a r b a c t e r i a .

M y d a t a su gge s t s t h a t t h e e f f e c t o f b ac t e r i a i n PSP p r od uc t i on i s

s i gn i f i c an t . Th e y p r ob a b l y i n f lu e n c e PSP t ox in p ro du c t io n b y

p r od u c i n g c om po und s t h a t s t im u l a t e PSP t ox in p ro du c t ion .

A l l e l o ch e mi ca l t e s t s on a x en i c and xe n i c cu l tu r es

N u me r ou s s tu d i es ha v e b e e n co nd u c t e d in v es t i ga t in g t h e

a l l e lo c he mi c a l e f f ec t o f A . t a ma re ns e o n v a r i ous p h yt o p la n k t on

s p e c i es (O ga t a & Ko d om a 19 86 , Lu s h & H a l l e g r a e f f 19 96 , T i l l m an n

& J oh n 20 02 ) a nd ha v e s ho wn t h a t t h es e l yt i c c om po un ds a r e n o t

r e l a t e d t o P SP t ox in s . T h i s l e ad t o sugge s t io ns o f o t h e r f a c to r s

i n f lu e n c in g th e o bse r v e d a l l e lo p a th i c e f f e c t s . Co l e (1 982 )

s u gge s t e d th a t b a c t e r i a m a y m e d ia t e t h i s b y a f f e c t i n g th e

m et a bo l i s m o f t h e c a u s a t i v e s p e c i es , o r a l t e rn a t iv e l y t h ey c o u l d

r e l e as e a l l e l o ch em ic a l o f t h e i r o wn . Th e r es u l t s s ho w th a t t h i s i s

n o t t ru e i n t h e c as e o f s t r a i n S 2- P4 - D1 . T he s e r es u l t s a r e c on s i s t en t

w i t h t h os e r e po r t e d b y T i l l m an n & J ohn (2 00 2 ) a nd Su i kka n e n e t a l

( 2 00 4 ) . S 2 -P4 - D1 s ho w e d no d i f f e r enc e i n l yt i c e f f e c t on

R h od om on us c e l l s b e tw e e n t h e ax en i c a nd n on - ax en i c cu l t u re s . T h i s

s u gge s t s t h a t b a c t e r i a h av e n o e f f e c t on t h e a l l e lo c h emi c a l s

r e l e as e d . Th e m ed iu m i s t h ou gh t t o h av e b e e n ax e n i c d u r in g th e

s a mpl in g t im e b y. T h i s me a ns t ha t t h e a l l e l o ch em ic a l s a re p ro du c ed

b y t h e d i no f l a ge l l a t e . T h e p lo t o f dose c u rv e sh o w ed th a t t h i s e f f e c t

i s d ep e nd an t on th e c e l l c on c e n t r a t i ons . Th e no n - l yt i c S2 -P 4 - F2

s t r a in s ho w ed n o d i f f e r e n ce i n l yt i c e f f e c t b e t w e en ax en ic a nd n on -

ax e n i c c u l t u r es . Th i s g iv es fu r t h e r p ro o f t o t h e f a c t t h a t b a c t e r i a d o

n o t p r od u ce t h es e a l l e lo c h emi c a l s .

56

Sub c l on es

T ox in p r of i l e :

T h e tox i n p r o f i l e o f f ou r s t r a i ns o f A. t a mar e ns e w h e re an a l ys e d to

s e e i f t h e r e i s a n y v a r i a t i o n in t he t ox i ns p r od uc e d . S ome a u t ho rs

h a v e r ep o r t ed t h a t t h es e p ro f i l e s r em ai n c on s t an t ( Bo ye r e t a l 19 86 )

w h i l e o th e r s ha v e re p o r t ed v a r i a t i o ns ( Bo cz a r e t a l . 19 88 , An d er so n

e t a l 19 90 ) . Bo cz ar e t a l ( 19 86 ) s u gge s t e d th a t t h e p r o f i l e co u ld b e

m od i f i ed s i gn i f i ca n t l y w i th en v i ro nm en t a l c h an ge s . W hi l e Sa ko e t

a l ( 19 9 2) an d Ki m e t a l ( 19 93 ) f ou nd th a t t h e i r t ox i n p r o f i l e s d i d

n o t ch a n ge s i gn i f i ca n t l y u n d e r v a r i ou s g r o wt h c on d i t i o ns . Th e y a l s o

c o nd u c t ed m at in g ex p e r im e n t s an d f oun d th a t t ox in p ro f i l e s wh e r e

i nh e r i t e d i n a 1 :1 M e nd e l i an r a t i o . Th i s a l l ow e d th em to co n c l ud e

t h a t t ox i n co mp os i t i on i s de t e r min e d on a ge n e t i c b as i s . I s h i d a e t a l

( 1 99 8 ) c on du c t e d m at in g ex p e r im e n t s a s w e l l w i t h bo th A .

t a mar e ns e an d A. ca n t e n e l l a . Th e y t oo k s t r a i ns w i th d i f fe r e n t t ox in

p r o f i l e s an d m at ed t h em. T he y a n a l yz e d t h e t ox in co n t en t t o s e e i f

i t wo u l d be a mix tu r e o f bo t h p a re n t s t r a in s a nd fo un d t h a t m os t o f

t h e i r s t r a in s ex h ib i t e d a t ox i n p r o f i l e o f t he co mbi n ed p a r e n t

s t r a in s . T h e y s u gge s t ed th a t

I d e t e r mi n ed th e t ox in p r o f i l e s o f s i n g l e ce l l s p i ck e d f rom fo ur

P a r en t s t r a in s . Th i s wo u ld s u gge s t t ha t t h e y w h e r e ge ne t i c a l l y

i d en t i c a l du e t o t h e i r o r i g in . Tw o s t r a i ns n am e l y, S 2 -P6 -G 1 2 a nd

S 6- P1 - G7 s ho w ed so m e v ar i a t i on s i n t ox i n p ro f i l e . On c lo s e r

i n s p ec t i on i t w a s fo u nd th a t t h es e v a r i a t i o ns w h e re d u e to t h e f a c t

t h a t so m e o f t h e t ox in s wh e r e p ro du c ed i n su c h l o w am oun t s t ha t

t h e y w h e r e un qu an t i f i a b l e ( i . e . d cS TX ) . T he i n t ro du c t io n o f C 3 /4 i n

8 o f t h e 40 su b c lon e s w h i ch w a s no t p r e s en t i n t h e p a r en t c l on e s

c o u l d b e ex p l a i n ed a s an e r r o r du r i n g h yd r o l ys i s . S t r a i ns S 2- P4 - D1

a n d S2 -P4 - F2 sh o we d m or e d i f f e r en c es i n t h e i r t ox i n p r o f i l e s .

W h e r e mo r e t h a t h a l f o f t h e s ub c lo n es h ad a d i f f e r in g t ox i n p ro f i l e

57

t o t h a t o f t h e p a r en t c lo n es . S ev e n o f S 2- P4 - D1 s s ub c l on e s

d i f f e r ed t o t h a t o f t h e p a r en t c l on e . Th e s e d i f fe r e n ce s wh e r e d ue t o

t h e i n t r od uc t i on o f b o t h C3 / 4 a nd B2 . B2 w as n o t o bs e rv ed i n t h e

o r i g i n a l c h ro ma to gr a m. T he r e w a s no B2 s t a nd a r d s o i t s

c o n c en t r a t i o n w as m e as u r ed l i k e t h e o t h e r s u l f oc a r ba mo yl t ox ins b y

d e t e r min in g th e d i f f e r e n c e b e t we e n the no n h yd r o l ys e d an d

h yd r o l ys e d p e a ks o f N eo ST X. Th e h a r s h c on d i t i o ns o f h yd r o l ys i s

( a d d i t i o n o f HCl a nd t h e i n cu b a t i on a t 9 0oC ) su c h as an ex t en d ed

a m ou n t o f h e a t o r t h e v a r i a t i o ns i n pH mo s t l i ke l y f u r t h e r c on v e r t ed

C 3 / 4 i n t o N eoS T X. Th i s c ou l d h av e be e n d on e b y c l e a v in g th e

s u l f a t e g ro up p r e sen t on p os i t i on s R 2 a n d R3 r e sp e c t iv e ly . A s a

r e s u l t t h e d i f f e r en ce s e en w ou ld b e a t t r i bu t e d to t h e p r es en c e o f B2 .

I b e l i e v e t h a t t h e va r i a t i on s e e n i n t h es e s t r a i ns c an b e a cc o un t ed

f o r b y m e th od o lo g ic a l e r r o r s , s o no v a r i a t i o n i n t ox in p r o f i l e s i s

a c t ua l l y o bs e r v ed . T h i s d a t a s up po r t s t h e n o t i on o f t ox i n p ro du c t io n

b e in g a gen e t i c a l l y d e t e rmi n ed s t a b l e c h a r a c t e r i s t i c .

T h e ex t e n t o f t h i s h yd r o l ys i n g e r r o r ca n b e d e t e rm in e d by

c o nd u c t i n g ex p er ime n t s w i th v a r yi n g ac i d c on c en t r a t i o ns a n d t im e a t

9 0oC to es t a b l i sh t h e o p t i m al c on d i t i on s f o r t h i s m e th od . Th e

s a mpl e s c ou l d a l so b e ru n on c e mo r e t o s e e o f t h e r e i s a ny

d i f f e r en c e i n ch r om at o gr am p e a ks . Th e mi n i ma l p ro du c t i on o f s om e

PSP t ox ins i n t h e su b c lo n es c ou ld a l so be i nv e s t i ga t ed to s e e o f t h i s

w i l l ch a n ge o v er t he t im e p e r i od . A re c e n t p a p e r (M a r t in s e t a l

2 0 04 ) r e po r t e d t h e l o s s o f t ox in p ro duc t i on i n a o n ce t ox i c s t r a i n o f

A . l u s t an i c u m . Th ey a t t r i b u t ed t h i s t o e i t h e r ge n e t i c m ut a t io ns o r

e f f e c t s f r om th e p ro lo n ge d t r e a t m en t o f t he cu l tu r e s b y a n t i b io t i c s .

I t w o u ld b e i n t e r e s t i n g t o s e e o f t h es e c u l t u r es w i l l f o l l ow th e s am e

t r en d b y n o t p r od uc i n g s om e P SP t ox in s o r b y l o os in g the i r PSP

t ox in p ro du c t io n c om pl e t e l y.

58

V a ri a t i ons in t ox in con t en t :

T h e to t a l t ox in co n t e n t w a s de t e r min ed fo r t h e fo u r s t r a in s . T o t a l

t ox i c i t y w a s d e t e rm in ed an d c on t r a s t e d b e tw e e n t h e p a re n t c l on e s

a n d th e s ub - c lo n es .

O ga t a e t a l (1 98 7 ) c o nd u c t ed a s im i l a r ex p e r i m en t , u s in g

m on o c lo n a l cu l tu r es o b t a i n ed f r om th e s am e a r e a du r in g th e s am e

s e a so n . Th e se c l one s an d su b c l on e s w h e r e c u l t u re d un de r t h e s am e

c o nd i t i on s a nd th e i r g r o wt h r a t e a nd PSP t ox in p ro du c i n g

c a p a b i l i t i e s wh e r e d e t e r min e d a nd co n t r as t ed . T h e y f o u nd

s imi l a r i t i e s i n g r ow t h r a t es an d t ox in p ro f i l e s . In a dd i t i o n , g r e a t

d i f f e r en c e s i n t ox i c i t y b e t w e en t he v a r i ou s su b c l on e s whe r e

o bs e r v ed . T h i s m ax im um d i f f e r e n c e i n t ox i c i t y w a s 2 0 fo ld b e t w e en

s om e s am pl es an d 1 0 0 fo ld b e t w e en c l on e s . Th e y a t t r i bu t ed th i s

d i f f e r en c e t o t h e f ac t t h a t t ox in p r od uc t i on w as n o t a h e re d i t a r y

c h a r a c t e r i s t i c . M y c u l tu r e s d id n o t exh ib i t su c h a h i gh de gr e e o f

v a r i a t i o n . T he h i gh e s t PSP tox i n va r i a t i on s e e n w as 9 fo l d . Th i s

w a s o n l y s e e n i n 5 o f t he 22 su b c lo n es o bs e r ve d . Th i s i s n o w h er e

n e a r w h a t O ga t a e t a l ( 19 8 7) r ep or t e d , b u t t he s ou r c e o f t h i s

v a r i a t i o n i s s t i l l s om et h i n g t o de l i be r a t e d u e t o t h e f a c t t h a t t h es e

p r e sum e d ge n e t i ca l l y s im i l a r s t r a in s wh e r e c u l tu r ed u nd e r i d e n t i c a l

c o nd i t i on s , s o i t f o l l o ws re a so n to ex pe c t mo d es t v a r i a t i on s b e t w e en

s ub c l on es .

T h es e v a r i a t i o ns i n t o t a l c e l l t ox in con t en t co u l d s t em f ro m th e f a c t

t h a t d i no f l a ge l l a t e D N A i s n o t eq ua l l y sh a r e d /d iv i d ed b e tw e e n

d a u gh t e r c e l l s d u r in g a s ex u a l r e p r od uc t i on . Ev en t ho u gh th e y

p r ob a b l y h a v e mul t i p l e c op i es o f t h e s a m e gen e , so me m ec h a n i s m

c a u s es t h e p r od uc t io n o f P SP t ox in s t o b e no n un i fo rm .

59

T h i s l e a ds t o qu es t i on s a s t o w ha t c on t r o l s t he PS T p ro duc t i on .

S om e p ro du c e PST i n su c h l o w l e v e l s t h a t t h e y c o u l d no t b e

q u an t i f i ed . W h a t ca u s es t h e r ed uc t io n in t he p r od u c t i on o f s p e c i f i c

t ox in s? I t w ou ld b e i n t e r e s t i n g to an a lys e t h e t ox i n p r o f i l e o f t h e

s a mpl e s t h a t s ho w ed t h e l o w p ro du c t i on i n so me PSP t ox in s t o s e e i f

t h es e t ox i ns a r e p ro d uc e d in g r e a t e r qu a n t i t i e s , o r i f t he y a r e l o s t

e n t i r e l y .

D u e to t i m e c ons t ra i n t s , t h e A FLP d a t a c ou ld n o t b e su f f i c i e n t l y

a n a l ys e d to d e t e rm in e wh e t h er t h e r e wh e r e a c tu a l ge n e t i c

d i f f e r en c e s wi t h i n s ub - c l on e s . Th e p re l i mi na r y d a t a su gge s t s t h a t

t h e r e i s co n gr u en c e w i th - i n su b c lo ne s o f t h e s am e s t r a i n .

60

A c know l ed g me n ts :

I w o u l d l i k e t o t h an k th e fo l l o wi n g f o r a l l t h e i r mu c h n ee d e d h e lp

a n d su pp o r t :

P ro f . D r . A l l an C em b el l a

D r Uw e J oh n

D r U rb an T i l l m an n

D r Be rn d K ro c k

T i lm a n A pp e rm an

A n n e- g r e t Mü l l e r

In e s J u n g

61

R ef e r en c es :

A nd e rs on , D . M . , J . J . Su l l i van , and B . R e gu e ra . 19 89 .

P a r a l yt i c sh e l l f i s h p o i so n i n g in No r t hw e s t Sp a in : t h e t ox i c i t y o f t h e

d in o f l a ge l l a t e G y mn o d in iu m c a t en a t um . T ox i co n 27 :6 65– 6 74 .

A nd e rs on , D . M . , D . M. Kul l i s , J . J . Su l l i van , S . Ha l l , a nd

C . L e e . 1 99 0 . D yn a mi c s a nd p h ys i o log y o f s ax i tox i n p r od u c t i on b y

t h e d in o f l a ge l l a t es A l e xa nd r iu m s pp . M a r . B io l . 10 4 :5 11– 5 24 .

B o cz a r , B . A . , M . K . Be i t l e r , J . L i s to n , J . J . Su l l i v an , a nd

R . A . C at t o l i c o . 19 8 8 . P a r a l yt i c s h e l l f i sh t ox i ns i n P ro to g on ya u l a x

t a mar e ns i s a nd P ro t og on y au l a x c a t en e l la i n ax e n i c c u l t u re . P l a n t

P h ys i o l . 8 8: 12 85 –12 9 0 .

Carmichael W.W., Evans W.R., Yin Q.Q., Bell P .and E. Moczydlowski,

Evidence for paralytic shellfish poisons in the freshwater cyanobacterium Lyngbya wollei

(Farlow ex Gomont) comb. nov. Appl. Environ. Microbiol. 63 (1997), pp. 3104–3110

C e mb e l l a , A . D . , J . J . Su l l i v an , G . L . Bo y er , F . J . R .

T ay lo r , a nd R. J . A nd e rs en . 19 87 . Va r i a t i on o n p a r a l yt i c sh e l l f i s h

t ox in c om po s i t i o n w i t h i n t h e Pr o t og on y au la x t a m ar en s i s / c a t en e l la

s p e c i es com pl ex : r e d t i d e d i no f l a ge l l a t e s . Bio ch e m. S ys t . Ec o l .

1 5 :1 71 –1 86 .

C e mb e l l a , A . D . , J . C . Th e r r i au l t , and P . B e land . 19 88 .

T ox i c i t y o f c u l t u r ed i so l a t es a nd n a tu r a l po p u l a t i on s o f

P r o to go n ya u l ax t am a r ens i s f r om th e S t . La w r e n c e e s t u a ry . J .

S h e l l f i sh R es . 7: 611 – 62 1 .

C o le , J . J . , 19 82 . In t e r a c t io ns b e t w e en ba c t e r i a an d a l gae i n

a q u a t i c e cos ys t e ms . A nn . R e v . E c o l og . S ys t . 1 3 , 2 91 - 314 .

62

D an t z e r , W . R . , and R . E . L ev i n . 1 997 . Bac t e r i a l i n f l u enc e

o n th e p r od uc t io n o f p a ra l yt i c sh e l l f i sh t ox in s b y d i no f l age l l a t e d

a l ga e . J . Ap p l . M i c r o b i o l . 8 3 :4 64 - 46 9 .

D ou c e t t e , G. J . , and C . G . T r i ck . 199 5 . C h ar a c t e r i s a t i on o f

b a c t e r i a a s so c i a t e d w i th d i f f e r en t i so l a t es o f A l ex an dr ium

t a mar e ns e , p . 3 3– 38 . I n P . La s s u s ,

Ga l l a ch e r , S . , and E . S mi th . 1 99 9 . Ba c t e r i a a nd p a r a l yt i c

s h e l l f i sh t ox ins . P ro t i s t 15 0 :2 45 -2 55

G . A r zu l , E . E ra rd -L e D enn , P . Ge n t i en , and C .

M a r c a i l l ou -L e Bau t ( e d . ) , H a rm fu l ma r in e a l ga l b l oom s . La v o i s i e r ,

P a r i s , F r a n c e .

H al l e g ra ef f , G. M. 1 99 5 . H a rm f u l a l ga l b l oo ms : a g l ob a l

o v e rv i e w , p . 1 -2 2 . I n In G . M . H a l l e g r a e f f , D . M. A nd e rso n , an d A .

D . C em b el l a ( e d . ) , M a nu a l on h a rm fu l m a r i n e m i c r o a l ga e . IO C

m a nu a l s a nd gu id e s n o . 3 3 . In t e r gov e rn m en t a l O c e an o gr ap h i c

C omm iss io n o f UNE SC O, P ar i s , F r an ce .

D ou c e t t e . 1 99 5 . P r o k a r yo t e s i n as so c i a t i o n wi th a t ox i c

A l e xa nd r iu m lu s i t an i cu m i n cu l tu r e , p . 4 5– 51 . In P . La s s us , G . A rz u l ,

E . E r ad - Le D e n n , P . G e n t i e n , a nd C . Ma r c a i l l ou - Le Ba u t ( e d . ) ,

H a r m fu l m a r i ne a l ga l b l oo ms . La v o i s i e r , P a r i s , F r a nc e .

Fr a n ca , S . , L . P in to , P . Al v i to , I . Sous a , a nd V.

V a s con c e l os . 19 96 . S tu d i e s on p ro k a r yo t es a s s o c i a t e d wi th PSP

p r od u c i n g d i no f l a ge l l a t es . In

Fi s t ar o l , G .O . , L eg r and , C . , Gr an e ´ l i , E . , 20 03 .

A l l e l op a th i c e f f e c t o f P r ym n e s ium p a rv um o n a n a t u r a l p l a n k t on

c o mmu ni t y. M a r . Ec o l . P ro g . S e r . 25 5 , 1 15 – 12 5 .

63

Fi s t ar o l , G .O . , L eg r and , C . , S e land er , E . , Hu mme r t , C . ,

S to l te , W. , Gr an e ´ l i , E . , 20 04 M a r . Ec o l . P ro g . S e r . A l l e l op a t h y i n

A l ex a nd r i um s pp . : e f f e c t on a n a tu r a l p l an k to n c omm un i ty a n d on

a l ga l mo no c u l t u r e s . Aq u a t . M i c r ob . Ec o l .

H obb i e , J .E . . Da l ey R. J and J asp e r S . , U s e o f n u c l ep o r e

f i l t e r s f o r co un t in g b a c t e r i a b y f l u o r esc e n c e mi c ro s co p y. A p pl .

E n v i ro n . Mi cr ob i o l . 33 ( 19 77 ) , pp . 122 5 –1 22 8

H o ld , G . L . , E . A . S mi th , T . H . B i rkb e rk , and S . Ga l lac h e r .

2 0 01 . C om pa r i so n o f p a r a l yt i c s he l l f i s h t ox i n (PS T) p r od u c t i on b y

t h e d in o f l a ge l l a t es A l e xa nd r iu m lu s i t an i cu m Al ex a nd r i um

l us i t an i c um N EPCC 2 53 an d Al e xa ndr iu m ta ma r en s eA lex a nd r i um

t am a r e ns e N EPCC 4 0 7 i n t h e p r es e nc e a nd ab s en c e o f b ac t e r i a .

FE MS Mi c r ob i o l . Ec o l . 3 6 :2 23 -2 34 .

I sh id a , Y . , C . H . Ki m, Y . S ako , N . H ir oo ka , and A . Uc h id a .

1 9 93 . PSP t ox in i s c h r omo so m e de p end e n t i n Al e xa nd r ium sp p . , p .

8 8 1– 88 7 . I n T. J . Sm a yd a a nd Y. Sh imiz u ( ed . ) , T ox ic

p h yt o p l an k t on b lo om s i n t h e s e a . E l s ev i e r , A ms t e rd a m, Th e

N e th e r l a nd s .

Ki m, C . H . , Y . Sako , and Y . I sh ida . 19 9 3 . V a r i a t i o n o f t ox i n

p r od u c t i on an d c om po s i t i o n i n ax en ic c u l t u r es o f Al e xan dr iu m

c a t en e l la a nd A. t am a r ens e .

64

Ko da ma , M. , T . Og a ta , and S . Sa t o . 1 9 88 . Ba c t e r i a l

p r od u c t i on o f s ax i tox i n . A gr i c . B io l . C h em. 5 2 :1 07 5– 107 7 .

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