Ice Nucleation-Deficient Bacteria as Frost ... - ACS Publications

Chapter

18

Ice Nucleation-Deficient Bacteria as Frost Protection Agents

Downloaded by UNIV OF MASSACHUSETTS AMHERST on November 6, 2017 | http://pubs.acs.org Publication Date: March 18, 1987 | doi: 10.1021/bk-1987-0334.ch018

G. J. Warren, J. Lindemann, T. V. Suslow, and R. L. Green Advanced Genetic Sciences, Inc., 6701 San Pablo Avenue, Oakland, CA 94608

Deletion mutants of Pseudomonas syringae and Pseudomonas fluorescens were developed for b i o l o g i c a l control of related, ice-nucleating s t r a i n s . The deletions, constructed i n v i t r o and introduced into the bacteria by marker exchange, ensured that the c o n t r o l l i n g strains were deficient in ice nucleation. In other diagnostic and colonization tests the mutant strains were indistinguishable from their wild-type progenitors. These data form a basis for assessing the potential benefits of using the constructed strains as frost protection agents, and for considering the issue of safety i n releasing the recombinant bacteria into the environment.

I n l i q u i d water between 0°C and - 4 0 ° C , i c e f o r m a t i o n r e q u i r e s a heterogeneous n u c l e a t i o n e v e n t : m o l e c u l e s o t h e r than H o must p a r t i c i p a t e i n f o r m i n g an i n i t i a l i c e t e m p l a t e . I c e c r y s t a l s o f the s m a l l s i z e s l i k e l y t o o c c u r s p o n t a n e o u s l y above - 4 0 ° C a r e inadequate as t e m p l a t e s , because the growth o f an i c e c r y s t a l i s t h e r m o d y n a m i c a l l y u n f a v o r a b l e when i t possesses a s u f f i c i e n t l y l a r g e s u r f a c e - t o volume r a t i o (Y). Thus an i c e n u c l e u s i s something which promotes the f o r m a t i o n o f a c r y s t a l l a r g e enough t h a t c r y s t a l growth, r a t h e r than d i s s o l u t i o n , becomes t h e r m o d y n a m i c a l l y f a v o r e d . At r e l a t i v e l y warm temperatures (above - 6 ° C ) , i t i s b e l i e v e d t h a t t h i s can o n l y be a c h i e v e d by p r o v i d i n g a t e m p l a t e w i t h the c a p a b i l i t y o f q u i t e a c c u r a t e l y l a t t i c e - m a t c h i n g w i t h a l a r g e number of water m o l e c u l e s i n an i c e - l i k e c o n f i g u r a t i o n . Ice n u c l e a t i o n i s important to a g r i c u l t u r a l biotechnology because i c e - n u c l e a t i n g b a c t e r i a i n c r e a s e the p r o b a b i l i t y o f f r o s t damage t o p l a n t s (2,3), and because i t may be p o s s i b l e t o a l t e r t h a t s i t u a t i o n through b a c t e r i a l e n g i n e e r i n g . F r o s t damage i s u s u a l l y caused by the m e c h a n i c a l d i s r u p t i o n r e s u l t i n g from i c e c r y s t a l l i z a t i o n , r a t h e r than by low temperatures p e r s e . The t i s s u e s o f most p l a n t s can s u p e r c o o l t o between -6 and -8°C, and t h e r e b y a v o i d such f r e e z i n g i n j u r y , u n l e s s f r e e z i n g i s i n i t i a t e d 0097-6156/87/0334-0215$06.00/0 © 1987 American Chemical Society

LeBaron et al.; Biotechnology in Agricultural Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


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e x o g e n o u s l y . U n f o r t u n a t e l y , exogenous i c e n u c l e i are p r o v i d e d i n abundance by b a c t e r i a o f e p i p h y t i c growth h a b i t : Pseudomonas s y r i n g a e , P. f l u o r e s c e n s , and E r w i n i a h e r b i c o l a (3>±>5). Most f i e l d c o n d i t i o n s i n temperate c l i m a t e s are conducive t o c o l o n i z a t i o n by such e p i p h y t e s , which w i l l then s h o r t - c i r c u i t a p l a n t ' s l a t e n t c a p a c i t y t o s u r v i v e low temperatures by f r e e z e - a v o i d a n c e . The e l i m i n a t i o n o f such b a c t e r i a would c l e a r l y p r o v i d e a l a r g e degree o f f r o s t p r o t e c t i o n - but complete e l i m i n a t i o n i s an u n r e a l i s t i c g o a l . However, thanks t o the spectrum o f i c e n u c l e a t i o n a c t i v i t y (INA) shown even by c l o n a l p o p u l a t i o n s ( F i g u r e 1), the r e d u c t i o n o f i c e n u c l e a t o r p o p u l a t i o n s w i l l p r o v i d e a s m a l l e r , but s t i l l s i g n i f i c a n t , degree o f f r o s t p r o t e c t i o n . How can the p o p u l a t i o n s o f these b a c t e r i a be reduced? Various b a c t e r i c i d e s and i c e n u c l e a t i o n - i n h i b i t i n g compounds may be e f f e c t i v e a t r e d u c i n g f r o s t i n j u r y t o p l a n t s (£,7,8). B i o l o g i c a l c o n t r o l i s a l s o an a t t r a c t i v e o p t i o n s i n c e b i o l o g i c a l agents can propagate and t h e r e f o r e they may r e q u i r e fewer a p p l i c a t i o n s than c h e m i c a l s ; they are a l s o l e s s l i k e l y t o be p h y t o t o x i c o r t o c o n t r i b u t e t o e n v i r o n m e n t a l p o l l u t i o n . B a c t e r i o p h a g e s can reduce the INA t i t r e i n v i t r o (£), and they are worth t e s t i n g i n a f i e l d s i t u a t i o n ; but i t must be noted t h a t a l t h o u g h b a c t e r i o p h a g e s have been known much l o n g e r than a n t i b i o t i c s , t h e r e are few i f any examples o f t h e i r e f f e c t i v e use i n p r a c t i c a l s i t u a t i o n s (JJ)). A t h i r d approach, t h a t o f u s i n g c o m p e t i t i v e e x c l u s i o n t o reduce t h e p o p u l a t i o n s o f i c e n u c l e a t o r s , i s the s u b j e c t o f t h i s c o n t r i b u t i o n . Construction

o f Ice N u c l e a t i o n - D e f i c i e n t Competitor B a c t e r i a

Why use recombinant DNA t o c o n s t r u c t the c o m p e t i t o r s ? INA" e p i p h y t i c c o l o n i z e r s occur n a t u r a l l y , and can a l s o be d e r i v e d from I N A s t r a i n s by c h e m i c a l mutagenesis. S t r a i n s o f both o r i g i n s have been demonstrated t o be c a p a b l e o f p r o v i d i n g some degree o f f r o s t p r o t e c t i o n (J,JJ^) · Why then i s i t n e c e s s a r y t o c o n s t r u c t c o m p e t i t o r s by g e n e t i c e n g i n e e r i n g ? One r a t h e r c y n i c a l m i s c o n c e p t i o n i s t h a t g e n e t i c e n g i n e e r i n g can improve o n l y t h e p a t e n t a b i l i t y o f the m i c r o o r g a n i s m s . I t seems u n l i k e l y t h a t n a t u r a l l y - o c c u r r i n g i n a " b a c t e r i a would occupy the e x a c t same n i c h e s as I N A s t r a i n s , even though they may be e p i p h y t i c c o l o n i z e r s o f the same p l a n t s p e c i e s . ( T h i s argument i s based on the s u p p o s i t i o n t h a t e v o l u t i o n a r y maintenance o f the i c e n u c l e a t i o n gene has r e q u i r e d s e l e c t i o n f o r the i c e n u c l e a t i o n phenotype. There i s l i t t l e d i r e c t e v i d e n c e f o r t h i s i d e a , and one g o a l o f o u r r e s e a r c h i s t o t e s t i t . Another way t o examine t h i s view would be by d e t e r m i n i n g whether most n a t u r a l l y o c c u r r i n g INA" and I N A b a c t e r i a c o n s t i t u t e g e n e t i c a l l y d i s t i n c t p o p u l a t i o n s . ) Since n i c h e e x c l u s i o n i s p r o b a b l y e s s e n t i a l f o r e f f e c t i v e c o m p e t i t i o n , a n a t u r a l l y - o c c u r r i n g INA" b a c t e r i u m i s t h e r e f o r e l i k e l y t o be i n f e r i o r t o one t h a t i s i s o g e n i c w i t h an I N A s t r a i n , except a t t h e l o c u s c o n t r o l l i n g INA. Chemical mutagenesis i s n o t a s a t i s f a c t o r y way t o o b t a i n such i s o g e n i c s t r a i n s . For p r a c t i c a l reasons i t i s common t o use c h e m i c a l mutagens a t c o n c e n t r a t i o n s which are l i k e l y t o cause m u l t i p l e m u t a t i o n s ; the a d d i t i o n a l m u t a t i o n s which do not a f f e c t INA are l i k e l y t o be u n d e t e c t a b l e i n the l a b o r a t o r y , b u t a f f e c t f i t n e s s a d v e r s e l y i n t h e f i e l d . The +

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p o s s i b i l i t y o f unknown mutations b e i n g p r e s e n t w i l l always c o m p l i c a t e experiments w i t h c h e m i c a l l y - i n d u c e d m u t a n t s . Genetic e n g i n e e r i n g o f f e r s the means t o o b t a i n t r u l y i s o g e n i c INA" mutants and thus perform more s t r a i g h t f o r w a r d e x p e r i m e n t s , w i t h i n c r e a s e d chances o f s u c c e s s . In a d d i t i o n , we can e n g i n e e r d e l e t i o n s , which w i l l be n o n - r e v e r t a b l e ; t h i s i s c o n s i d e r e d a d e s i r a b l e t r a i t i n an organism which i s scheduled f o r d e l i b e r a t e r e l e a s e i n t o the environment. C l o n i n g o f the INA genes from Pseudomonas s y r i n g a e and Pseudomonas fluorescens. A gene c o n f e r r i n g i c e n u c l e a t i o n a c t i v i t y was f i r s t c l o n e d from P . s y r i n g a e s t r a i n C i t 7 (V2). We i d e n t i f i e d the c o r r e s p o n d i n g genes, by DNA h y b r i d i z a t i o n w i t h the c l o n e d C i t 7 gene of p l a s m i d p I C E 1 . 1 , i n n a t u r a l l y - o c c u r r i n g i s o l a t e s o f P . s y r i n g a e (S203) and P . f l u o r e s c e n s (MS1650). In each case the h y b r i d i z i n g sequence was shown to be p r e s e n t as a s i n g l e copy per genome. The INA genes were c l o n e d from these s t r a i n s by s i z e - s e l e c t i o n o f r e s t r i c t i o n - e n d o n u c l e a s e - d i g e s t e d genomic DNA, f o l l o w e d by c l o n i n g into c o l i p l a s m i d s and s c r e e n i n g f o r i c e n u c l e a t i o n a c t i v i t y . (The INA gene from S203 was o b t a i n e d on an 8.5 kb EcoR1 genomic fragment, c l o n e d i n t o E c o R 1 - l i n e a r i z e d v e c t o r pUC8 ( 1 3 ) » The INA gene from MSI650 was sublconed on a 9.0 kb EcoR1-Sal1 genomic fragment, p r e s e n t i n cosmid pJJINA ( 2 5 . ) · ) genes i s o l a t e d from the d i f f e r e n t s t r a i n s each possessed a d i s t i n c t d i s t r i b u t i o n o f r e s t r i c t i o n endonuclease s i t e s ( F i g u r e 2 ) . T

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Making d e l e t i o n s w i t h i n the INA r e g i o n s i n s u i t a b l e m o b i l i z a t i o n vectors. Our method f o r marker exchange i n Pseudomonas r e q u i r e s the c o n j u g a l t r a n s m i s s i o n o f the i n t r o d u c e d marker from E . c o l i i n a n o n r e p l i c a t i n g p l a s m i d . The requirement f o r t r a n s m i s s i b i l i t y made n e c e s s a r y the r e c l o n i n g o f the i c e genes from the o r i g i n a l p l a s m i d s into different vectors. Then the d i s s i m i l a r r e s t r i c t i o n maps o f the two genes r e q u i r e d d i s t i n c t methods f o r i n t r o d u c i n g an I c e " d e l e t i o n i n t o each. These s t e p s are d e s c r i b e d b e l o w . (a) : Gene from S203. The gene was r e c l o n e d i n t o the EcoR1 s i t e o f v e c t o r pBR325 (V5) on the same 8.5 kb EcoR1 fragment. Two S a i l fragments were d e l e t e d ( c o o r d i n a t e s 1.3 t o 2.4 i n F i g u r e 2) by p a r t i a l d i g e s t i o n w i t h S a i l , f o l l o w e d by r e l i g a t i o n . T h i s removes 1.1 kb from the 3-6 kb gene which we know t o be e s s e n t i a l f o r i c e n u c l e a t i o n a c t i v i t y i n S203 The f i n a l d e l e t i o n c o n s t r u c t i o n i s c a l l e d pRLG13. (b) : Gene from MS1650. The gene was r e c l o n e d i n the same 9.0 kb EcoR1-Sal1 fragment i n t o v e c t o r PLVC18 (Vf). A d e l e t i o n was i n t r o d u c e d by Hpa1/Bal31 d i g e s t i o n , f o l l o w e d by r e l i g a t i o n . T h i s r e s u l t e d i n the l o s s o f the r e g i o n between c o o r d i n a t e s 6.0 and 7.5 i n F i g u r e 2 . Thus i t removes 1.5 kb from the 4 . 0 kb gene which we have mapped i n the r e g i o n from MS1650 ( J J p . The f i n a l d e l e t i o n c o n s t r u c t i s c a l l e d pLVC40. P l a s m i d m o b i l i z a t i o n and marker exchange. The r e s p e c t i v e d e l e t i o n c o n t a i n i n g sequence was s u b s t i t u t e d f o r the w i l d - t y p e gene i n each Pseudomonas s t r a i n by double r e c i p r o c a l r e c o m b i n a t i o n t o the u n a l t e r e d f l a n k i n g r e g i o n s immediately a d j a c e n t t o the d e l e t e d sequence. In t h i s way we avoided the p o s s i b i l i t y o f c r e a t i n g any



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F i g u r e 1. Spectrum o f i c e n u c l e a t i o n frequency d i s p l a y e d by a g e n e t i c a l l y homogeneous p o p u l a t i o n o f b a c t e r i a : coli strain JC10291 w i t h p l a s m i d pRLG12 (]6). The source o f the inaZ gene i n pRLG12 i s s y r i n g a e S203, which g i v e s an almost i d e n t i c a l spectrum: t h e r e f o r e the p h e n o t y p i c h e t e r o g e n e i t y i s u n l i k e l y t o be due t o g e n o t y p i c h e t e r o g e n e i t y . The graph i n d i c a t e s t h a t very few n u c l e i a r e a c t i v e a t the warmer t e m p e r a t u r e s ; n u c l e a t i o n frequency i n c r e a s e s by o r d e r s o f magnitude towards c o o l e r t e m p e r a t u r e s . T h e r e f o r e , i n s m a l l b a c t e r i a l p o p u l a t i o n s such as o c c u r on l e a v e s , the p r o b a b i l i t y o f n u c l e a t i o n a t a g i v e n temperature w i l l be reduced when the mean p o p u l a t i o n s i z e i s reduced. F r o s t p r o t e c t i o n may be assessed by the temperature d i f f e r e n c e between t h e "warmest" n u c l e i p r e s e n t w i t h and w i t h o u t the use o f the p r o t e c t i o n agent.

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Scale [kb] ι • ι • ι , ι • ι , ι , ι , ι , ι , ι F i g u r e 2. R e s t r i c t i o n maps o f the r e g i o n s cloned from P. s y r i n g a e and P^ f l u o r e s c e n s . S o l i d bars i n d i c a t e the r e g i o n d e l e t e d f o r the marker exchange e x p e r i m e n t s . A: B a l l ; B: BamHI; C: B g l I I ; E: EcoRI; F: EcoRV; H: Hpal; K: K p n l ; L: SphI; P: P v u I I ; S: S a l i ; T: S e a l . An arrow i n d i c a t e s t h e r e g i o n e s s e n t i a l f o r i c e n u c l e a t i o n i n each c a s e , and i t s d i r e c t i o n o f transcription.


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new sequence arrangements by the r e c o m b i n a t i o n o f h e t e r o l o g o u s DNA, Our method a c h i e v e d t h e double r e c o m b i n a t i o n i n a two-step p r o c e s s , as d e t a i l e d below. The p r i n c i p l e o f the p r o c e s s i s i l l u s t r a t e d i n F i g u r e 3· F i g u r e 4 shows a Southern b l o t a n a l y s i s o f t h e Pseudomonas s t r a i n s b e f o r e m a n i p u l a t i o n , then a f t e r s t a g e a, and f i n a l l y a f t e r stage b. T h i s a n a l y s i s p r o v i d e s c o n f i r m a t i o n t h a t t h e marker exchange o c c u r s as d e s c r i b e d . (Stage a ) : I n t e g r a t i v e r e c o m b i n a t i o n . pRLG13 and pLVC40 were m o b i l i z e d i n t o S203 and MS1650 r e s p e c t i v e l y , w i t h s e l e c t i o n f o r t e t r a c y c l i n e r e s i s t a n c e . (The R64drd11/pGJ28 system 0 8 ) was used f o r pRLG13. P l a s m i d R751 (^9) was used t o m o b i l i z e pLVC40.) T c t r a n s c o n j u g a n t s were o b t a i n e d a t a frequency o f a p p r o x i m a t e l y 10 per r e c i p i e n t c e l l . S i n c e the t r a n s f e r r e d p l a s m i d s c o u l d n o t r e p l i c a t e i n Pseudomonas, these t r a n s c o n j u g a n t s arose from the c o m p a r a t i v e l y r a r e events o f p l a s m i d i n t e g r a t i o n i n t o t h e chromosome. Two k i n d s o f evidence i n d i c a t e t h a t the plasmid::chromosome c o i n t e g r a t e s occur by s i n g l e events o f homologous r e c o m b i n a t i o n : f i r s t l y , no T c t r a n s c o n j u g a n t s were o b t a i n e d when a c o n t r o l was performed i n which the t r a n s f e r r e d p l a s m i d l a c k e d homology, and s e c o n d l y , the Southern b l o t a n a l y s e s i n F i g u r e 4 a r e most r e a d i l y e x p l a i n e d by s i n g l e c r o s s o v e r s . (Stage b ) : E x c i s i v e r e c o m b i n a t i o n . The c o i n t e g r a t e s t r a i n s were grown w i t h o u t a n t i b i o t i c s e l e c t i o n f o r 30 g e n e r a t i o n s i n L - b r o t h , and then p l a t e d a t a d e n s i t y o f 400 CFU p e r p l a t e . A f t e r growth t o s m a l l c o l o n i e s , these were r e p l i c a - p l a t e d onto L-agar p l u s t e t r a c y c l i n e . Comparison o f the masters w i t h the r e p l i c a p l a t e s r e v e a l e d t e t r a c y c l i n e - s e n s i t i v e c o l o n i e s a t a frequency o f a p p r o x i m a t e l y 0.1$. From the S203::pRLG13 s t r a i n , a p p r o x i m a t e l y 5% of the T c c o l o n i e s were p h e n o t y p i c a l l y I c e " . For the MS1650::pLVC40 s t r a i n , the frequency was 20%· A l l the T c c l o n e s r e s u l t from a second event o f homologous r e c o m b i n a t i o n , which e x c i s e s the r e g i o n o f DNA c o n t a i n i n g v e c t o r sequences and p e r m i t s i t s l o s s . However, o n l y those c r o s s o v e r s which o c c u r on t h e o p p o s i t e s i d e o f the d e l e t i o n , r e l a t i v e t o the p o s i t i o n o f t h e i n t e g r a t i v e c r o s s o v e r , can l e a v e the d e l e t i o n behind as a chromosomal a l l e l e . A m a j o r i t y o f second c r o s s o v e r s a r e expected t o occur i n the same i n t e r v a l as the f i r s t : hence the c o m p a r a t i v e l y low r a t i o o f I c e " : T c . Southern b l o t s showed t h a t the I c e " , T c s t r a i n s had the expected chromosomal s t r u c t u r e , and r e t a i n e d no homology t o the t e m p o r a r i l y i n s e r t e d v e c t o r sequences.


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s

s

C h a r a c t e r i z a t i o n o f the INA-Deficient Competitor B a c t e r i a Testing i n v i t r o . The INA" s t r a i n s were e x t e n s i v e l y c h a r a c t e r i z e d i n o r d e r t o v e r i f y t h a t they were i d e n t i c a l t o t h e i r I N A p r o g e n i t o r s i n every t r a i t except i c e n u c l e a t i o n a c t i v i t y . Eighteen s t a n d a r d t a x o n o m i c a l l y s i g n i f i c a n t t e s t s were performed as d e s c r i b e d p r e v i o u s l y (20*21^22). The s t r a i n s were a l s o screened f o r u t i l i z a t i o n o f 31 s u b s t r a t e s as s o l e sources o f c a r b o n , p r o d u c t i o n of b a c t e r i o c i n s and the p h y t o t o x i n s y r i n g o m y c i n and s e n s i t i v i t y t o 19 a n t i b i o t i c s . Each INA" s t r a i n was i d e n t i c a l t o the I N A s t r a i n from which i t was d e r i v e d i n a l l t r a i t s except i c e n u c l e a t i o n a c t i v i t y ( T a b l e I ) . Growth r a t e s o f t h e p a r e n t a l s t r a i n s and t h e i r d e r i v a t i v e s i n b r o t h media were i n d i s t i n g u i s h a b l e . +

+



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F i g u r e 3. S t r a t e g y f o r marker exchange. Z i g - z a g l i n e s denote the v e c t o r p o r t i o n of the s u i c i d e p l a s m i d . ( i ) A narrow-host range ( s u i c i d e ) p l a s m i d , c a r r y i n g a d e l e t i o n a l l e l e of the i n a gene, i s t r a n s f e r r e d from E. c o l i to Pseudomonas (where i t cannot replicate). ( i i ) Selection for Tc s e l e c t s c e l l s where the T c marker has been r e s c u e d by a s i n g l e c r o s s o v e r between p l a s m i d and chromosome. Stage " a " i s now complete. ( i i i ) Growth w i t h o u t a n t i b i o t i c s e l e c t i o n p e r m i t s the appearance of c e l l s i n which the p l a s m i d was e x c i s e d by a second, s i n g l e c r o s s o v e r , and ( i v ) was subsequently l o s t . There are two p o s s i b l e outcomes: e i t h e r the d e l e t i o n a l l e l e s u b s t i t u t e s the w i l d type a l l e l e , or the w i l d type a l l e l e i s r e s t o r e d . Stage "b" i s now complete. r


r


18.

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221

F i g u r e 4. Evidence f o r the chromosomal rearrangements. Above: Southern b l o t s probed w i t h pRLG13 ( l e f t ) and pLVC40 ( r i g h t ) . Below: r e l a t i o n s h i p o f observed fragments t o p r e d i c t e d s t r u c t u r e a t each s t a g e . Lower case l e t t e r s l a b e l c o r r e s p o n d i n g fragments above and below. E: EcoRI s i t e . S: S a i l s i t e . *: s t r o n g EcoRI* s i t e (note t h a t o n l y about 10$ o f m o l e c u l e s i n a p o p u l a t i o n a r e c u t a t t h i s p a r t i c u l a r s i t e ) . pLVCDI c a r r i e s a d e l e t i o n a l l e l e analogous t o t h a t i n pLVC40; i t i s shown here f o r comparison only.


222

BIOTECHNOLOGY IN AGRICULTURAL CHEMISTRY +

Table I . R e a c t i o n o f W i l d - t y p e I N A and INA" D e l e t i o n Mutant S t r a i n s o f Pseudomonas s y r i n g a e and P^ f l u o r e s c e n s B i o v a r I i n D i a g n o s t i c Tests P. s y r i n g a e S203 RGP36


Characteristic Gram r e a c t i o n C e l l shape Glucose f e r m e n t a t i o n F l u o r e s c e n t pigments Pyocyanine Growth a t 41°C No. o f f l a g e l l a Oxidase r e a c t i o n Arginine dihydrolase Potato r o t Levan from s u c r o s e Tobacco HR Denitrification Gelatin hydrolysis P e c t o l y t i c enzymes Syringomycin Ice n u c l e a t i o n Growth f a c t o r s r e q u i r e d Catalase Indole

P. f l u o r e s c e n s MS1650 GJP17B

rod + -

rod + -

rod + -

>1 + + + -

>1 +

>1 + + + +

>1 + + + +

+ + + -

+ + -

+ -

rod + -

T e s t i n g i n v i v o . The t r a i t o f g r e a t e s t i n t e r e s t t o us i s t h e a b i l i t y t o colonize plant surfaces, p a r t i c u l a r l y strawberry b l o s s o m s . The I N A and INA" s t r a i n s demonstrated g e n e r a t i o n times o f c a . 2 hours when sprayed onto newly opened s t r a w b e r r y blossoms and i n c u b a t e d under i n t e r m i t t e n t m i s t i n a greenhouse a t 21 * 3 C ( F i g u r e 5 ) . Mutant and p a r e n t a l s t r a i n s a l s o were a b l e t o c o l o n i z e blossoms o f almond, c h e r r y , pear and b l a c k b e r r y and l e a v e s o f s t r a w b e r y , bindweed and p u r s l a n e . None o f the s t r a i n s c o l o n i z e d l e a v e s o f tomato, p o t a t o , t o b a c c o , c o t t o n , l a m b s q u a r t e r s , y e l l o w s w e e t c l o v e r o r annual b l u e g r a s s . I n s o i l and s t r a w b e r r y r o o t environments the INA" b a c t e r i a e x h i b i t e d poor f i t n e s s compared t o o t h e r s o i l i n h a b i t i n g b a c t e r i a , as d i d t h e i r p a r e n t a l s t r a i n s . A l l o f the above evidence suggests t h a t the INA" s t r a i n s may be as e c o l o g i c a l l y f i t as t h e i r p a r e n t s , b u t p r o b a b l y are not more s o . F u r t h e r , t h e I N A and INA" s t r a i n s e x h i b i t e d i d e n t i c a l p r o p e r t i e s when grown on s t r a w b e r r y blossoms i n c o m p e t i t i v e s i t u a t i o n s w i t h o t h e r b a c t e r i a l s t r a i n s . C o m p e t i t i o n between t h e i s o g e n i c s t r a i n s was r e c i p r o c a l i n n a t u r e , and t h e r e f o r e t h e i n t e r a c t i o n was dose dependent ( 2 3 ) . Thus, i c e n u c l e a t i o n a c t i v i t y appears t o have no i n f l u e n c e on e i t h e r a x e n i c growth o r c o m p e t i t i o n between s t r a i n s under o p t i m a l c o n d i t i o n s . These d a t a , combined w i t h t h e p h y s i o l o g i c a l d a t a from d i a g n o s t i c t e s t s have l e d us t o conclude t h a t t h e INA" s t r a i n s indeed are not capable o f p r o p a g a t i n g an imbalance i n the ecosystem because they a c t e x a c t l y l i k e w i l d - t y p e s t r a i n s . We expect the INA" s t r a i n s w i l l be e f f e c t i v e c o m p e t i t o r s a t +

+



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F i g u r e 5. Comparison o f the c o l o n i z a t i o n p o t e n t i a l (on s t r a w b e r r y blossoms) o f t h e s y r i n g a e w i l d - t y p e s t r a i n S203 and i t s INA" d e l e t i o n mutant RGP36.


224


s i t e s where we a p p l y them i n l a r g e numbers, but n o t a t d i s t a n t l o c a t i o n s t o which they w i l l be d i s s e m i n a t e d i n low c o n c e n t r a t i o n . Future D i r e c t i o n s P r o t e c t i o n o f s t r a w b e r r y blossoms from f r o s t i n j u r y a f t e r exposure t o a temperature o f -3·5 C has been a c h i e v e d under l a b o r a t o r y c o n d i t i o n s when the INA" b a c t e r i a were c h a l l e n g e d i n d i v i d u a l l y w i t h s e v e r a l d i f f e r e n t s t r a i n s o f I N A b a c t e r i a . S i m i l a r r e s u l t s were p r e v i o u s l y r e p o r t e d w i t h both c h e m i c a l l y - d e r i v e d INA" and d e l e t i o n mutant s t r a i n s o f P^ s y r i n g a e on crops such as c o r n , pear and tomato (£,24,25). The n e x t n e c e s s a r y s t e p i s t o conduct f i e l d e f f i c a c y trials. I n i t i a l l y , a s i n g l e t r i a l w i l l be conducted p r i m a r i l y t o e v a l u a t e t h e e n v i r o n m e n t a l f a t e o f the i n t r o d u c e d INA" s t r a i n s . The l o c a t i o n and s m a l l s i z e o f t h i s p l o t w i l l l i m i t i t s u s e f u l n e s s as an e f f i c a c y t r i a l p e r s e . However, a f t e r the e n v i r o n m e n t a l f a t e i s s u e o f t h i s r e l e a s e has been e v a l u a t e d under f i e l d c o n d i t i o n s , l a r g e r t r i a l s w i l l a l l o w us t o examine the e f f e c t i v e n e s s o f the INA" s t r a i n s a p p l i e d t o s t r a w b e r r y p l a n t s i n commercial f i e l d s a t s e v e r a l l o c a t i o n s . F i n a l l y , e f f i c a c y t r i a l s c o u l d be expanded t o i n c l u d e o t h e r major rosaceous f r u i t crops whose acreages are exposed t o f r o s t hazards.


+

F o l l o w i n g t h e t r i a l s i n commercial s t r a w b e r r y f i e l d s , we may wish t o i n i t i a t e l a b o r a t o r y e f f o r t s toward s t r a i n improvement f o r i n c r e a s e d e f f i c a c y . This would be e x p e d i t e d by an u n d e r s t a n d i n g o f the mechanism(s) o f growth i n h i b i t i o n o f the I N A by the INA" strains. Research aimed a t e l u c i d a t i n g such a mechanism i s c u r r e n t l y i n progress. I n the f u t u r e , e f f o r t s t o improve e f f i c a c y might i n v o l v e 1) s c r e e n i n g f o r v a r i a n t s w i t h f a s t e r growth r a t e s a t c o l d t e m p e r a t u r e s , 2) i n t r o d u c i n g an a d d i t i o n a l i n h i b i t o r y c h a r a c t e r i s t i c (eg. a n t i b i o t i c p r o d u c t i o n ) o r 3) i n t r o d u c i n g a s e l e c t a b l e t r a i t (eg. copper r e s i s t a n c e ) so t h a t b i o l o g i c a l and c h e m i c a l c o n t r o l s c o u l d be combined i n an i n t e g r a t e d p e s t management program. +

Government R e g u l a t i o n

o f the Research

The l e g a l a s p e c t s o f u s i n g i c e n u c l e a t i o n d e f i c i e n t b a c t e r i a f o r f r o s t p r o t e c t i o n are r e s p o n s i b l e f o r the a t t e n t i o n accorded t o t h i s r e s e a r c h by the p o p u l a r media. T h e r e f o r e i t seems a p p r o p r i a t e t o p r o v i d e a s c i e n t i s t s ' p e r s p e c t i v e on these i s s u e s and on t h e r e g u l a t o r y p r o c e s s as i t a p p l i e s t o d e l i b e r a t e r e l e a s e e x p e r i m e n t s . The n o v e l i n t e r e s t h e r e was t h a t the f i e l d t e s t s proposed would have i n v o l v e d t h e f i r s t d e l i b e r a t e r e l e a s e o f g e n e t i c a l l y e n g i n e e r e d bacteria. I n the U n i t e d S t a t e s , the N a t i o n a l I n s t i t u t e s o f H e a l t h (NIH) a l r e a d y had e s t a b l i s h e d a Recombinant A d v i s o r y Committee (RAC) t o c o n s i d e r the h a z a r d s o f a c c i d e n t a l r e l e a s e ; i t was n a t u r a l t h a t RAC s h o u l d assume r e s p o n s i b i l i t y f o r r e g u l a t i n g the new c l a s s o f e x p e r i m e n t s . The RAC g u i d e l i n e s show t h a t the a c c i d e n t a l r e l e a s e o f e n g i n e e r e d P\ s y r i n g a e i s n o t c o n s i d e r e d a s e r i o u s problem, s i n c e the c o n s t r u c t i o n o f such s t r a i n s i s p e r m i t t e d under l o w l e v e l s o f p h y s i c a l containment. The key d i f f e r e n c e between a c c i d e n t a l and d e l i b e r a t e r e l e a s e i s i n the number o f organisms l i k e l y t o be i n t r o d u c e d i n t o the environment: thus the main q u e s t i o n was whether



18.

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the d i s p e r s a l o f l a r g e numbers o f engineered b a c t e r i a c o u l d cause s i g n i f i c a n t e n v i r o n m e n t a l impact beyond the f i e l d p l o t where they were t o be a p p l i e d ( i n the case o f our e x p e r i m e n t , an area o f l e s s than 0.5 a c r e ) . In 1982, S. Lindow and N. Panopoulos a p p l i e d t o RAC f o r permission to release i c e nucleation d e f i c i e n t b a c t e r i a ; permission was denied because o f t h e i r i n i t i a l p l a n t o r e p l a c e t h e i c e n u c l e a t i o n gene w i t h an element c o n f e r r i n g r e s i s t a n c e t o t h e a n t i b i o t i c kanamycin. T h e i r second a p p l i c a t i o n was approved b u t l e g a l c o n s t r a i n t s prevented e x e c u t i o n o f t h e f i e l d t e s t . Similarly, l e g a l c o n s i d e r a t i o n s a t f i r s t delayed the r e v i e w o f o u r own 1983 a p p l i c a t i o n , and subsequently prevented t h e NIH d i r e c t o r from a c t i n g on the RAC recommendation t o c o n f i r m i t s a p p r o v a l . I n 1984, t h e Environmental P r o t e c t i o n Agency (EPA) determined t h a t i c e n u c l e a t i o n d e f i c i e n t b a c t e r i a should be c o n s i d e r e d a form o f p e s t i c i d e . This c l a s s i f i c a t i o n w i l l p r o b a b l y a p p l y t o most b a c t e r i a l p r o d u c t s w i t h a g r i c u l t u r a l a p p l i c a t i o n s , b u t not t o o t h e r types o f recombinant organisms. The e x i s t i n g procedures f o r r e g u l a t i o n o f p e s t i c i d e s d i c t a t e d t h a t an e x p e r i m e n t a l use permit (EUP) would be r e q u i r e d f o r f i e l d t e s t i n g o f t h e b a c t e r i a . The EPA i s r e s p o n s i b l e f o r g r a n t i n g an EUP, b a s i n g t h e i r d e c i s i o n on a r e v i e w o f submitted d a t a . A p p r o v a l i s a l s o r e q u i r e d from the a p p r o p r i a t e S t a t e a u t h o r i t i e s . At the time o f w r i t i n g , i t i s r e p o r t e d t h a t NIH-RAC w i l l withdraw from the r o l e o f r e g u l a t i n g d e l i b e r a t e - r e l e a s e experiments. Implications for Agriculture The p r i n c i p l e u n d e r l y i n g our approach t o f r o s t p r o t e c t i o n i s t o use c l o s e l y - r e l a t e d organisms as c o m p e t i t o r s a g a i n s t p a t h o g e n i c m i c r o b e s . This i d e a i s not n o v e l but as e x p l a i n e d above, i t s most s a t i s f a c t o r y t e s t i n g requires genetic engineering of the competitors. T h e r e f o r e t h i s approach i s l i a b l e t o y i e l d some new i n f o r m a t i o n w i t h w i d e r i m p l i c a t i o n s f o r a g r i c u l t u r e . Much has a l r e a d y been w r i t t e n , both i n enthusiasm and i n c a u t i o n , about the d i r e c t i o n s i n which d e l i b e r a t e r e l e a s e experiments a r e l e a d i n g . We w i l l confine the d i s c u s s i o n t o i t s d e f i n a b l e , s c i e n t i f i c i m p l i c a t i o n s . These w i l l be l i m i t e d t o t h e o t h e r cases i n which t h e " s i b l i n g c o m p e t i t i o n " p r i n c i p l e c o u l d be expected t o o p e r a t e . Therefore: 1) A p a r t i a l , r a t h e r than a complete, r e d u c t i o n i n t h e p o p u l a t i o n o f the pathogen must be capable o f y i e l d i n g s i g n i f i c a n t r e s u l t s . 2) The c o l o n i z a t i o n and c o m p e t i t i o n a b i l i t y o f the pathogen must be independent o f i t s a b i l i t y t o cause d i s e a s e ( e l s e non-pathogenic d e r i v a t i v e s w i l l not be e f f e c t i v e c o m p e t i t o r s ) . 3) A l t e r a t i o n o f o n l y one o r a few genes must be capable o f eliminating pathogenicity. 4) The pathogen must e x i s t a t s i t e s which a r e a c c e s s i b l e t o some form o f d e l i v e r y system f o r t h e c o m p e t i t o r . 5) The pathogen must e x i s t a t p o p u l a t i o n l e v e l s where i n t e r c e l l u l a r c o m p e t i t i o n i s the main o r o n l y f a c t o r l i m i t i n g i t s population. E p i p h y t i c i c e n u c l e a t i n g b a c t e r i a p r o v i d e an i d e a l system f o r s t u d y i n g the f e a s i b i l i t y o f " s i b l i n g c o m p e t i t i o n " . A l l the above c o n d i t i o n s a r e thought t o be met, except t h a t no. 5 i s demonstrated


226


i n t h e l a b o r a t o r y o n l y . I t i s the major unknown i n t h e f i e l d experiments s t i l l t o be done. I f i t h o l d s t r u e i n t h e f i e l d , many o t h e r t y p e s o f b a c t e r i a l d i s e a s e may be c o n t r o l l a b l e i n analogous ways. Other d i s e a s e s caused by e p i p h y t i c b a c t e r i a w i l l be t h e b e s t t a r g e t s (26), a l t h o u g h t h e same p r i n c i p l e i s p o t e n t i a l l y a p p l i c a b l e to b a c t e r i a t h a t l i v e i n s i d e the p l a n t o r i n i t s r h i z o s p h e r e .


Acknowledgments C. Orser p r o v i d e d p l a s m i d pICE1.1 p r i o r t o p u b l i c a t i o n . We thank L. C o r o t t o , L. Joe, and A. M o a y e r i f o r t e c h n i c a l a s s i s t a n c e . Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

18. 19. 20. 21. 22. 23.

Fletcher, Ν. H. In "The Chemical Physics of Ice"; Cambridge University Press: Cambridge, U.K., 1970; pp. 73-103. Amy, D. C.; Lindow, S. E.; Upper, C. D. Nature 1976, 262, 282-4. Lindow, S. E.; Amy, D. C.; Upper, C. D. Phytopathology 1978, 68, 523-527. Maki, L. R.; Galyan, E. L.; Chang-Chien, M.; Caldwell, D. R. Appl. Microbiol. 1974, 28, 456-9. Maki, L. R.; Willoughby, K. J . J . Appl. Meteorol. 1978, 252, 1049-53. Lindow, S. E. Ann. Rev. Phytopathol. 1983, 21, 363-84. Lindow, S. E. Plant Disease 1983, 67, 327-33. Lindow, S. E., Connell, J . H. J . Amer. Soc. Hort. Sci. 1984, 109, 48-53. Kozloff, L. M.; Schofield, Μ. Α.; Lute, M. J . B a c t e r i o l . 1983, 153, 222-31. Vidaver, A. K. Ann. Rev. Phytopathol. 1976, 14, 451-65. Lindow, S. E. Proc. 4th Int. Conçr. Plant Pathol., 1983. Orser, C.; Staskawicz, B. J . ; Panopoulos, N. J . ; Dahlbeck, D.; Lindow, S.E. J . B a c t e r i o l . 1985, 164, 359-66. V i e i r a , J . ; Messing, J . Gene 1982, 19, 259-68. Corotto, L. V.; Wolber, P. K.; Warren, G. J . EMBO Journal 1985, 5, 231-6. Bolivar, F. Gene 1978, 4, 121-36. Green, R. L.; Warren, G. J . Nature 1985, 317, 645-8. Warren, G. J . ; Corotto, L. V.; Green, R. L. In "Advances i n the Molecular Genetics of the Bacteria-Plant Interaction"; Szalay, Α.; Legocki, R., Eds.; 1985; pp. 212-4. Van Haute, E.; Joos, H.; Maes, M.; Warren, G.; Van Montagu, M.; Schell, J . EMBO Journal 1983, 2, 411-7. Jobanputra, R. S.; Datta, N. J . Med. Microbiol. 1974, 7, 169-72. Lindemann, J . ; Suslow, T. V. Proc. 6th Int. Conf. Plant Pathol. Bact., 1985. Stanier, R. Y.; P a l l e r o n i , N.J.; Douderoff, M. J . Gen. Microbiol. 1966, 43, 159-271. L e l l i o t t , R. Α.; B i l l i n g , E.; Hayward, A.C. J . Appl. B a c t e r i o l . 1966, 29, 470-89, Lindemann, J . ; Joe, L. K.; Moayeri, A. Phytopathology 1985, 75.


18.

24. 25. 26.

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Lindow, S. E.; Papp, J . ; Andersen, G. Proc. 2nd Am. Conf. INA Bact., 1984. Lindow, S. E. Proc. Conf. "Engineered Organisms i n the Environment: S c i e n t i f i c Issues", 1985. Lindemann, J. In "Biological Control on the Phylloplane:; Windels, C. E.; Lindow, S. E., Eds.; American Phytopathological Society: St. Paul, MN, 1985; pp. 116-30.


RECEIVED October 1, 1986


Ice Nucleation-Deficient Bacteria as Frost ... - ACS Publications

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