FEMS Microbiology Letters Volume 81 issue 1 1991 [doi 10.1111%2Fj.1574-6968.1991.tb04707.x] D. Wu;...

8/13/2019 FEMS Microbiology Letters Volume 81 issue 1 1991 [doi 10.1111%2Fj.1574-6968.1991.tb04707.x] D. Wu; X.L. Cao; Y.Y. Bai; A.I. Aronson -- Sequence of a

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FEM S Microbiology Letters 81 (1991) 31-36 1991 Federation of E uropean M icrobiological Societies 0378-1097/91/ 03.50A D O N I S 037810979100283P31

FEM SLE 04479

S e q u e n c e o f a n o p e r o n c o n t a i n i n g a n o v e l 8 e n d o t o x i n g e n ef r o m a c i l l u s t h u r i n g i en si s

D . W u 1 X . L . C a o 2 y . y . B a i 2 a n d A .I . A r o n s o n a1 Dep artme nt of Biological Sciences, Purd ue Uniuersity, West Lafayette, IN, U.S.A . and 2 Sha ngh ai Institute o f Plant Physiology,

Academ ia Sinica, Shanghai, P.R . ChinaReceived 3 January 1991Revision received 5 M arch 1991Accepted 6 M arch 1991

K e y w o r d s : C r y l I e n d o t o x i n; L e p i d o p t e r a n a c t iv e ; A m i n o a c id r e p e a ts

1 . S U M M A R Y 2. I N T R O D U C T I O N

A n i n s e c t l a r v a l t o x i n d e s i g n a t e d C r y I I i s p r o -d u c e d b y s e v e r a l s u b s p e c i e s o f B a c i l l u s t h u r i n g i e n -s i s a n d d i f f e r s f r o m t h e o t h e r m a j o r 8 - e n d o t o x i n si n t h e s e b a c t e r i a i n i t s s i z e , t o x i c i t y p r o f i l e a n dp r e s e n c e a s p a r t o f a n o p e r o n w i t h t h r e e o p e nr e a d i n g f ra m e s ( O R F ) . S u c h a n o p e r o n f r o m an o v e l B . t h u r i n g i e n s i s i s o l a t e h a s b e e n c l o n e d a n dd i f f e rs f r o m o n e p r e v i o u s l y c h a r a c t e r i z e d i n t h ef o l l o w i n g w a y s : ( a ) t h e s i z e a n d n u m b e r o f a m i n oa c i d r e p e a t s in o n e o f t h e O R F s ; ( b ) th e s m a l l e rs iz e o f th e C r y I I p r o t o x i n a n d t h e p r e s e n c e o f au n i q u e l l 0 - k D a C r y I I - r e l a t e d a n t ig e n ; a n d ( c )h i g h l a r v i c i d a l a c t i v i t y f o r a p a r t i c u l a r L e p i -d o p t e r a n b u t l o w a c t i v i t y f o r a D i p t e r a n . V a r i o u ss u b c l o n e s o f t h i s o p e r o n w e r e i n t r o d u c e d i n t o ap l a s m i d - f r e e B . t h u r i n g i e n s i s s t r a i n a n d o n l y t h ec r y H g e n e w a s f o u n d t o b e n e c e s s a r y f o r p r o t o x i na c c u m u l a t i o n .

Correspondence to: D. Wu, D epartm ent of B iological Sciences,Purdue Unive rsity, W est Lafaye tte, IN 47907, U.S.A.

T h e m a j o r 8 - e n d o t o x i n s ( C r y I ) p r o d u c e d b y B .t h u r i n g i e n s i s a r e g e n e r a l l y p r e s e n t i n b i p y r a m i d a li n c l u s i o n s i n s p o r u l a t i n g c e l l s . S o m e s t r a i n s a l s op r o d u c e c u b o i d a l i n c l u s i o n s c o n t a i n i n g a n o t h e rp r o t o x i n d e s i g n a t e d C r y I I [ 1 ] . T h e l a t t e r d i f f e r sf r o m t h e C r y I t y p e s i n s i z e [ 2 ] , s o l u b i l i t y , i n s e c ts p e c i f ic i t y a n d p r e s e n c e a s p a r t o f a n o p e r o nc o n t a i n i n g t w o u p s t r e a m O R F s [ 3 ] . T h e s e q u e n c eo f s u c h a n o p e r o n f r o m B . t h u r i n g i e n s i s ssp .k u r s t a k i H D 1 h a s b e e n p u b l i s h e d [ 3 ] a n d h e r e w er e p o r t s o m e o f t h e u n i q u e f e a t u r e s o f s u c h a no p e r o n f r o m a n o v e l B . t h u r i n g i e n s i s i s o l a t e .

3. M A T E R I A L S A N D M E T H O D S

3 . 1 . C l o n i n gP l a s m i d s w e r e i s o l a t e d [4 ] f r o m a n o n - m o t i l e B .

t h u r i n g i e n s i s s t r a i n B . t h u r i n g i e n s i s S a ) f r o m ac o l l e c ti o n a t t h e S h a n g h a i I n s t i t u t e o f P l a n t P h y s i -o l o g y . T h i s s t r a i n p r o d u c e d a t l e a s t f o u r s i ze c la s s e so f C r y I - t y p e p r o t o x i n s p l u s a C r y I I - t y p e w h i c h


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32w a s a l i t t l e s m a l l e r t h a n C r y I I A . T h e i n c l u s i o n sw e r e t o x i c t o a v a r i e t y o f L e p i d o p t e r a a n d t oAedes aegypti i l a r v a e ( u n p u b l i s h e d d a t a ) . P l a s m i dD N A w a s p a r t i a l l y d i g e s t e d w i t h Bgl I I , l i g a t e di n t o t h e B a m H I , s it e o f p U C 1 8 a n d t r a n s f o r m e di n t o E. col i D H 5 a . T r a n s f o r m a n t s w e r e sc r e en e dw i t h r a b b i t a n t i b o d y t o B. thuringiensis S~ inc lu-s i o n s . P o s i t iv e c l o n e s w e r e b i o a s s a y e d o n Pierisrapea l a r v a e a n d a n a c t i v e c l o n e d e s i g n a t e dB T 7 3 2 2 w a s f u r t h e r a n a l y s e d . T h e 8 . 3 - k b i n s e r tw a s s u b c l o n e d a n d a 5 .3 - k b f ra g m e n t f r o m a nE c o R I s i t e w i t h i n t h e i n s e r t t o a H i n d I I I s i te inp U C 1 8 ( p B T 5 3 ) w a s s t il l t o x i c .3.2. SequencingT h e 5 . 3 - k b f r a g m e n t f r o m p B T 5 3 w a s c l o n e di n t o M 1 3 m p 1 8 f o r s e q u e n c i n g ( C y c l o n e I B i o s y s -t er n , I B I) . T h r e e O R F s w e r e f o u n d ( F ig . 1 ). O R F 2c o n t a i n e d m a n y r e p e a t s a n d t h e n u m b e r a n d s e -q u e n c e o f e a c h w a s e s t a b l i s h e d b y d i g e s t i n g v a r i -o u s s e q u e n c e d d e l e t i o n c l o n e s g e n e r a t e d t h r o u g ht h e C y c l o n e s y t e m w i t h P vu I I ( r e s t r i c t i o n s i t e so n l y w i t h i n t h e M 1 3 v e c t o r ) . T h e s i z e s o f t h e s ef r a g m e n t s w e r e m e a s u r e d b y t h e i r m o b i l i t i e s i na g a r o s e g e l s r e l a t i v e t o s t a n d a r d s a n d f o u r w h i c hd i f f e r e d , b y a b o u t 4 0 0 b p e a c h , c o u l d b e m e r g e do n t h e b a s i s o f u n i q u e o v e r l a p s .3.3. Construction of operon deletions

O R F 1 w a s e x c i se d b y d i g e s t io n w i t h K p n I a t t h e2 0 t h c o d o n o f orfl a n d a t a u n i q u e N s i I s i t e i n t her e g i o n b e t w e e n orfa a n d orf2 ( F ig . 1 ) . T h i s 568- bpf r a g m e n t w a s r e p l a c e d i n p B T 5 3 b y a 2 3 - b pK p n I - P s t I f r a g m e n t f r o m t h e p o l y l i n k e r r e g i o n o fp U C 1 8 . T h e r e s u l t i n g p l a s m i d ( p B T 5 3 - A O R F 1 )c o n t a i n e d t h e u p s t r e a m p r o m o t e r , t h e f i r s t 2 0c o d o n s o f orfl a n d t h e orf2 p l u s c r y H C g e n e s(Fig. 1) .

I n o r d e r t o r e m o v e t h e r e p e a t s p r e s e n t i n orf2,t h e 2 . 4 - k b E c o R I - S t y I f r a g m e n t f r o m p B T 5 3 w a sc l o n e d i n t o p B R 3 2 2 . T h i s c l o n e w a s d i g e s t e d w i t hS n a B I s i n c e t h e r e s t r i c t i o n e n z y m e s i t e s f o r t h i se n z y m e w e r e p r e s e n t o n l y in e a c h r e p e a t . F o l l o w -i n g r e - l i g a t i o n , t h e d e l e t e d E c o R I - S t y I f r a g m e n tw a s i n s e r t e d b a c k i n t o t h e o r i g i n a l E c o R I - S t y I -d i g e s t e d p B T 5 3 t o g e n e r a t e p B T 5 3 - A O R F 2 . T h i ss u b c l o n e r e t a i n e d t h e p r o m o t e r , orf~, t he f i r s t 67c o d o n s o f orf2 a n d t h e c r y H C ge ne ( F ig . 1 ) .

T h e K p n I - N s i I f r a g m e n t f r o m p B T 5 3 - Z l O R F2w a s r e p t a c e d b y t h e K p n I - P s t I f r a g m e n t f r o m t h ep o l y l i n k e r o f p U C 1 8 ( p B T 5 3 -A O R F a , 2 ) . T h i s s u b -c l o n e r e t a i n e d o n l y t h e p r o m o t e r , t h e f i r s t 2 0c o d o n s o f orfl 6 7 c o d o n s f r o m orf2 a n d t h e c r y l I Cg e n e .

F o l l o w i n g p la s m i d p r e p a r a t i o n i n E. coli, t h e s es u b c l o n e d f r a g m e n t s w e r e i n t r o d u c e d i n t o t h e B .thuringiensis-E, coli s h u t t l e v e c t o r P X I 6 1 [ 5 ] , a n de l e c t r o p o r a t e d i n t o t h e p l a s m i d - c u r e d , a c r y s t a l l i -f e r o u s C r y B s t r a i n d e r i v e d f r o m B. thuringiensisssp. kurstaki H D 1 [ 6 ] . C e l l s w e r e g r o w n a s d e -s c r ib e d [5 ] b u t w a s h e d w i t h 1 m M N - 2 - h y d r o x y -e t h y l p i p e r a z i n e - N ' - 2 - e t h a n e s u l f o n i c a c id( H E P E S ; S i g m a ) p H 7 . 0 a n d 10 g l y c e r o l a n ds u s p e n d e d i n 1 0 g l y c e r o l [7 ] a t 1 0 0 - f o l d c o n -c e n t r a t io n f o r e le c t r o p o r a t io n i n a B i o R a d G e n eP u l s e r a t 1 . 7 5 k V a n d 4 0 0 ~2. I n c l u s i o n s f r o m t h eC r y B t r a n s f o r m a n t s w e r e p u r i f i e d a s p r e v i o u s l yd e s c r i b e d [8 ]. M o r e t h a n 9 0 o f t h e p r o t e i ns o l u b i l i z e d f r o m t h e s e i n c l u s i o n s m i g r a t e d a s a na p p r o x . 6 0 - k D a p o l y p e p t i d e i n 1 0 S D S - P A G E .3.4. Toxicity assays

P u r i f i e d C r y I I C i n c l u s i o n s w e r e s u s p e n d e d i nw a t e r , h o m o g e n i z e d b y a s h o r t p u l s e o f s o n i c a t i o na n d s e r i a ll y d i l u t e d f o r b i o a s s a y s . P r o t e i n c o n -c e n t r a t i o n s o f t h e s u s p e n s i o n s w e r e m e a s u r e d b yt h e m o d i f i e d B r a d f o r d m e t h o d [ 9 ] .

B i o a s s a y s w i t h l a r v a e o f Manduca sexta, Helio-this virescens a n d Trichoplusia ni w e r e d o n e a sp r e v i o u s l y d e s c r i b e d [ 8 ] . A s s a y s w i t h A. aegyptiil a r v a e w e r e a s d e s c r i b e d b y S c h n e l l e t a l . [ 1 0 ] . A l la s s a y s w e r e r e p e a t e d a t l e a s t t w i c e w i t h d i f f e r e n tp r e p a r a t i o n s a n d t h e r a n g e s o f t h e L D s 0 v a l u e s a rer e p o r t e d (9 5 c o n f i d e n c e l im i t s w i t h i n t w o - f o l d o fthe L Ds0 va lue s ) .4 . R E S U L T S A N D D I S C U S S I O N

O n l y t h e s e q u e n c e o f O R F 3 c r y l I C g e n e ) h a sb e e n i n c l u d e d ( F i g . 2 ) s i n c e t h e r e s t o f t h e o p e r o ns e q u e n c e w a s v e r y s i m i l a r t o t h a t o f t h e c r y l I Ao p e r o n f r o m B. thuringiensis ssp. kurstaki H D 1[ 3 ] . T h e e n t i r e s e q u e n c e o f t h e c r y H C o p e r o n h a sb e e n s u b m i t te d t o th e E M B L D a t a L i b r a r y (a c ce s -s i o n n u m b e r X 5 7 2 5 2 ; B. thuringiensis p l a s m i d


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Fig. 1. Restnctlon enzyme maps ot the cryIlL operon m plasmld pItI and of deletions constructed as described in the text. E,EcoRI; H, HindIII; K, KpnI; N NsiI; P, PsrI; S, SnaBI; St, StyI. There are 21 SnsBI sites in orjz 21s). each 48 bp apart. Only4.0 kb of the 4.5kb portion of pTB53 which was sequenced is drawn to scale. The Hind111 site H) is within pUC18.


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34b a s e p a i r

c r y l l C o p e r o n

1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0I I I

o d lI I I

K N

o r f 2 c r y l l C] 1 I / / ~\ 2 1 s - J s t s

z ~ o r f lo r f 2 c r y l l C

I j I II IE P / 2 1 S 1 S H

, ~ , e r f 2o r f l c r y l l Ci i n l i /I I I I I | I

E K N S S t S H

c r y l l Co 1 0 2 1 / /

E K P / N S S t S HFig. 2. Nucleotide and deduced amino acid sequences of thecryllC gene portion of the operon from B. thuringiensis S1.The potential ribosome binding site (RBS) and terminator(inverted arrows) are based on homologies with comparableregions of the operon from B. thuringiensis ssp. kurstaki HD1[3]. Nucleotide numbering reflects a continuation of the se-quence of the entire operon. Amino acid differences for eitherthe CrylIA or liB protoxins are indicated below the sequenceof the CrylIC protein.

crylIC DNA). The upstream promoter region ofthis operon was virtually identical to that from thec r y l I A operon from subspecies k u r s t a k i HD1 [3].OR F 1 was 92% h omolo gous but appears to bemissing the first eight amino acids present inORF] from subspecies k u r s t a k i HD1. The first 67residues of the ORF2s were 82% homologous. Theamino acid repeats which followed differed some-what in that there were 21 (rather than 15) repeatsof 16 (rather than 15) amino acids in ORF2 fromB. thuringiensis S 1. The sequence at b oth ends ofthe repeats were very similar with a few substitu-tions in both cases:

Y YT Ik u r s t a k i HDI: T Y N Q S Q N V C P Q D L V DB. thuringiensis . S]: T Y N Q S Q N W L Y Q D L V D

G N M N

The third ORF encoded a CrylI protoxin(CrylIC) which was 82% homologous to CrylIAbut was shorter by 11 residues due to several small

deletions (Fig. 2). There were many scattered dif-ferences among the CrylIA, B and C protoxinswith the most extensive differences among thethree between residues 300-400. There was nomajor concentration of non-conserved differenceswhich could help to define a specificity region incontrast to the cluster of such differences betweenthe CrylIA and CrylIB sequences [11]. In thislatter case, however, it was noted that the recom-binants generated in E. col i produced protoxinsall of which had lower toxicities than the parentalprotoxins [11]. The formation of specificity do-mains in these CrylI protoxins may depend uponthe interaction of several regions of the toxin.

For bioassays, inclusions were purified fromthe CryB transformants containing the entire op-eron and the deletion of orf l plus orf2. Bothpreparations were quite active on 7 . n i larvae(LDs0 of 2- 5 ng pr ot ein /c m 2) and less active onH. v irescens (LDs0 of 1 20-150 ng p ro tei n/ cm 2) orM . s e x t a (LDs0 of 1 2-16 ng pr ot ein /cm 2) larvaeamong the Lepidoptera tested. The activity for T.n i was comparable to that obtained with inclu-sions containing only the CrylA(b) protoxin (un-published data). There was no toxicity for A.ae gy p t i i larvae up to a concentration of 50 #gprotein/ml even though B. thuringien sis S 1 inclu-sions had mosquitocidal activity similar to thatfound with inclusions from B. thuringiensis ssp.k u r s t a k i HD1.

It is difficult to compare these values to thoserepo rted fo r othe r Cry lI protoxin s [2,3,11,12] sinceeither extracts of E. coli clones, clones in B.m e g a t e r i u m [12] or protoxin solubilized from B.t hur ing ie ns i s inclusions by differential pH extrac-tion [2] were used. CrylIB protoxin, however, wasalso inactive on A . ae gy p t i i larvae although thisgene appeared to be cryptic in B. thuringiensis ssp.k u r s t a k i HD1 [1t].

There were also some nucleotide sequence dif-ferences in the region between the or f s especiallybetween the orf2 and c r y l I C genes. As anti-cipated, antibody to the CrylIA protoxin cross-re-acted with the CrylIC protoxin as well as to all0-kDa antigen found only in inclusion extractsfrom B . t hur ing ie ns i s S 1 (unpublish ed data). Clonesof the entire operon, of o r f l - c r y H C o r f 2 - c r y H Cor c r y H C alone in the CryB strain each produced


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i n cl u si o ns c o n t a i n in g a p p r o x . 6 5 -k D a a n d l l 0 - k D aa n t i g e n s s o t h e l a t t e r w a s u n l i k e l y t o b e a c r o s s - r e -a c t i n g p r o t e i n p r e s e n t i n t h e b i p y r a m i d a l i n c l u -s i o n s a l t h o u g h i t s o r i g i n i s n o t k n o w n .M a j o r q u a n t i t i e s o f p o l y p e p t i d e s o f t h e e x -p e c t e d s iz es f o r O R F 1 o r O R F 2 w e r e n o t f o u n d i ni n c l u s i o n s f r o m B t h u r i n g i en s i s 1 o r t h e v a r i o u sc l o n e s i n C r y B s o t h e r e i s n o o b v i o u s f u n c t i o n f o rt h e s e O R F s i n p r o t o x i n a c c u m u l a t i o n , s o l u b i l i z a -t i o n o r t o x i c i t y . T h e y m a y h a v e a m o r e s u b t l ef u n c t i o n ( s ) i n i n c l u s i o n s t r u c t u r e o r d e p o s i t i o n o rp e r h a p s i n t h e i n t e r a c t i o n b e t w e e n t h e d i f f e r e n ti n c l u s i o n s p r o d u c e d b y B t h u r i n g i en s i s ssp .k u r s t a k i H D 1 o r B t h u r i n g i en s i s S t . B i p y r a m i d a li n c l u s i o n s c o n s i s t i n g o f C r y I p r o t o x i n s a r e f o u n da s s o c i a t e d w i t h c u b o i d a l i n c l u s i o n s c o n t a i n i n gC r y I I p r o t o x i n s [ 1 2 ] . I n t h i s r e s p e c t , t h e r e c o u l db e a c r u d e a n a l o g y t o t h e m u l t i c o m p o n e n t i n c l u -s i o n s u r r o u n d e d b y a n e t - l i k e s t r u c t u r e p r o d u c e db y B t h u r i n g i en s i s ssp . i s r a e l en s i s [13].

A C K N O W L E D G E M E N T SR e s e a r ch s u p p o r t e d b y a n N I H R e s e a r ch G r a n t

( G M 3 4 0 3 5 ) . D r . P . C . F i tz J a m e s , U n i v e r s i t y o fW e s t e r n O n t a r i o p r o v i d e d t h e C r y l I A a n t i b o d ya n d D r . G . C r a i g , N o t r e D a m e U n i v e r s i t y , s u p -p l i e d A a e g y p t i i eggs.

R E F E R E N E S

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FEMS Microbiology Letters Volume 81 issue 1 1991 [doi 10.1111%2Fj.1574-6968.1991.tb04707.x] D. Wu;...

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Transcript of FEMS Microbiology Letters Volume 81 issue 1 1991 [doi 10.1111%2Fj.1574-6968.1991.tb04707.x] D. Wu;...