1 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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1
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
2
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
3
6. Discussion 49
7. Acknowledgments 59
8. References 60
4
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
5
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
7
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
8
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
9
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 .
10
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 .
11
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