Chapter 27
Methods
Used
Genetically
To
Track
Engineered
Introduced Organisms
1
2
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Philip C. Kearney and James M. Tiedje 1
Natural Resources Institute, U.S. Department of Agriculture, Beltsville, MD 20705 Departments of Crop and Soil Sciences and Departments of Microbiology and Public Health, Michigan State University, East Lansing, MI 48824
2
Cultural, metabolic, genetic and immunological techniques that could be used to trace genetically engineered microorganisms released into the environment are reviewed. The advantages, disadvantages, and in several cases the sensitivity are considered for each method. Special attention is focused on gene probe technology, since this technique appears to hold considerable promise for monitoring released microorganisms. Many parallels exist between the current concerns about the intentional release of genetically engineered microorganisms (GEMs) into the environment and concerns expressed about the release of massive amounts of organic pesticides in the environment during the early 1960 s. The issues, however, are essentially the same, i.e, the stability, movement, and the safety of altered products. Concurrent with the environmental pressures mounted against the older chlorinated hydrocarbon insecticides was the emerging development of gas chromagraphic (glc) systems that offered increasing levels of sensitivity with a growing number of sophisticated detectors that provided highly specific compound recognition. Unfortunately, as we see in the following section, glc does not offer the selective, automated systems for the rapid or sensitive detection of the altered DNA molecule or the microbial vehicle harboring that DNA. The chlorinated hydrocarbons are stable, lipophilic structures possessing one or more halogens that greatly facilitated detection in the parts per millon range, while modern instrumentation permits even greater sensitivity. By contrast, the DNA molecule is a liable, polar substance in a fragile carrier. More troublesome is the ability of the carrier microorganism to mutate, conjugate, and poliferate in a diverse community. We are faced with a far more complex problem in attempting to monitor the elusive gene in a complex matrix than the stable pesticide with many more easily detectable markers. By comparison, we have only a limited number of methods to detect the specific fragment of DNA of interest in a rather facile delivery system. The object of this section of the symposium is to examine f
0097-6156/88/0379-0352S06.00/0 • 1988 American Chemical Society
In Biotechnology for Crop Protection; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
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the methods now a v a i l a b l e t o us and the s u i t a b i l i t y o f those methods f o r p o t e n t i a l l y v a s t m o n i t o r i n g programs t o a l l a y c u r r e n t a p p r e h e n s i o n s about the r e a l o r p r e c e i v e d damages of i n t e n t i o n a l release.
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CURRENT DETECTION SYSTEMS A r e v i e w o f c u r r e n t l y a v a i l a b l e d e t e c t i o n methods f o r GEMs has been p r e p a r e d by T i e d j e (1) and McCormick ( 2 ) . T h i s o v e r v i e w f o l l o w s t h e s e r e v i e w s c l o s e l y . The methods can be c o n v e n i e n t l y grouped under t h r e e major headings o f c u l t u r e and m e t a b o l i c t e c h n i q u e s , g e n e t i c t e c h n i q u e s and i m m u n o l o g i c a l t e c h n i q u e s . A d e t a i l e d , i n - d e p t h d i s c u s s i o n of each o f t h e s e o p t i o n s i s beyond the scope o f t h i s o v e r v i e w paper a l t h o u g h gene probe t e c h n i q u e s a r e c o n s i d e r e d i n g r e a t e r d e t a i l due t o t h e i r p r o m i s i n g r o l e i n m o n i t o r i n g s t u d i e s . A few comments about the advantages and d i s a d v a n t a g e s o f t h e s e methods i s a p p r o p r i a t e . CULTURE AND METABOLIC TECHNIQUES Plating C o u n t i n g organisms on agar p l a t e s i s a c l a s s i c a l t e c h n i q u e used e x t e n s i v e l y by the m i c r o b i o l o g i s t s . The advantages a r e t h a t the method i s e a s y , i n e x p e n s i v e , measures e x p r e s s i o n , i s s u b j e c t t o s t a t i s t i c a l a n a l y s i s and c a n be v e r y s e n s i t i v e ( 1 ) . The d i s a d v a n t a g e s a r e t h a t the organisms must be c u l t u r a b l e , i t must p o s s e s s an i n s e r t e d marker ( e . g . , a n t i b i o t i c r e s i s t a n c e ) , t h a t marker must be s t a b l e , and t h a t marker must not d i s r u p t the m e t a b o l i s m o r u l t i m a t e l y the s u r v i v a l o f t h a t m i c r o o r g a n i s m . F o r example, r i f a m p i n r e s i s t a n c e i n d e n i t r i f i e r s t r a i n s has been shown t o reduce maximum growth r a t e ( 3 ) . The l e v e l o f s e n s i t i v i t y o f p l a t i n g t e c h n i q u e s i s i n the range o f 10-100 c e l l s / g r a m o f s o i l . C o l o r m e t r i c Media T h i s method i s based on an o b v i o u s c o l o r change i n the t a r g e t o r g a n i s m . The advantages a r e t h a t i t i s v e r y s p e c i f i c and a good s c r e e n i n g t o o l . Drahos e t a l . (4) have used t h i s method f o r m o n i t o r i n g a recombinant Pseudomonas f l u o r e s c e n s e x p r e s s i n g the B a c i l l u s t h u r i n g e n i s i s endotoxin. JP. fluorescens i s a n a t u r a l l y o c c u r r i n g r o o t i n h a b i t i n g m i c r o o r g a n i s m t h a t makes i t an i d e a l c a n d i d a t e as a d e l i v e r y v e h i c l e f o r n a t u r a l p e s t i c i d e s . They developed a s e n s i t i v e , s e l e c t a b l e marker i n P . f l u o r e s c e n s based on the i n a b i l i t y o f most f l u o r e s c e n t Pseudomonas t o use l a c t o s e as a s o l e s o u r c e o f c a r b o n . The IS. c o l i l a c Y and Z genes were i n s e r t e d i n t o the e n g i n e e r e d P^. f l u o r e s c e n s chromosome. The Z gene produces B - g a l a c t o s i d a s e and the l a c Y gene encodes f o r l a c t o s e permease. Thus the t r a n s f o r m e d b a c t e r i a can grow on x - g a l , a chromogenic substrate. M e t a b o l i s m produced a c h a r a c t e r i s t i c b l u e - g r e e n c o l o n y , q u i t e d i s t i n c t from the p a l e c o l o r o f the untransformed c e l l s . In the f i n a l a n a l y s i s , t h r e e d i s c r e e t markers were used t o d e t e c t t h e t r a n s f o r m e d JP. f l u o r e s c e n s i n s o i l s , i . e . , B - g a l a c t o s i d a s e , f l u o r e s c e n t p i g m e n t s , and r i f a m p i c i n r e s i s t a n c e . These marker
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systems enabled the d e t e c t i o n o f l a c + t r a n s f o r m a n t s s e n s i t i v i t y of 10 c e l l s / g r a m o f s o i l .
at a
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Direct Staining T h i s i s a refinement o f t h e p l a t i n g t e c h n i q u e t h a t g i v e s a t o t a l count o f l i v i n g o r g a n i s m s . I t overcomes the problem o f n o n - c u l t u r a b i l i t y . The a d d i t i o n o f n a l d i x i c a c i d t o a y e a s t e x t r a c t f o r t i f i e d agar p l a t e s produces an e l o n g a t e d c e l l w h i c h c a n be s t a i n e d w i t h , f o r example, a c r i d i n e orange s t a i n , c o n c e n t r a t e d on a f i l t e r d i s k and c o u n t e d . F l u o r e s c e n t m o n o c l o n a l a n t i b o d i e s c a n a l s o be added t o the p l a t e t o g i v e a count o f v i a b l e organisms s p e c i f i c t o an e n g i n e e r e d subgroup. E n r i c h e d Media The a d d i t i o n of s o i l p a r t i c l e s d i r e c t l y i n the media has the advantage o f b e i n g a v e r y c l a s s i c a l method f o r w h i c h the s o i l microbiologist i s quite familiar. T h i s approach m a g n i f i e s the p o p u l a t i o n o f i n t e r e s t , but i t s u f f e r s from a q u a n t i t a t i v e s t a n d p o i n t , l a c k s a c c u r a c y , and r e q u i r e s a v e r y s e n s i t i v e s c r e e n t o be u s e f u l . Spent Media A n a l y s i s T h i s i s a system based on the i d e n t i f i c a t i o n o f m i c r o o r g a n i s m s by the a n a l y s i s of the c u l t u r e medium f o r unique b y p r o d u c t s of t h a t s p e c i f i c group o f o r g a n i s m s . The advantages of t h i s approach i s t h a t the s e n s i t i v e , automated c h e m i c a l s y s t e m s , l i k e g l c o r h p l c , c o u l d be u s e d . The d i s a d v a n t a g e s t o t h i s approach a r e o b v i o u s , i . e . , t h a t a tremendous d a t a b a s e on the q u a l i t a t i v e p r o f i l e of t h o s e m e t a b o l i c p r o d u c t s would have t o be a v a i l a b l e . G i v e n the complexity of that a n a l y s i s , i t i s u n l i k e l y that t h i s i s a practicable solution. GLC and HPLC As d i s c u s s e d i n the i n t r o d u c t i o n , t h e s e t o o l s have a v e r y l i m i t e d r o l e i n d i r e c t m i c r o o r g a n i s m t r a c k i n g . They a r e used i n s i t u a t i o n s where m i c r o o r g a n i s m s can be i d e n t i f i e d by d e t e c t i o n of c h a r a c t e r i s t i c p r o f i l e s o f f a t t y a c i d s , l i p i d s and s t e r o i d l i n k a g e s . T h i s would a l s o r e q u i r e a huge database t h a t does not exist. P r o t e i n Product F i n g e r p r i n t i n g T h i s method i s based on the i d e n t i f i c a t i o n o f m i c r o o r g a n i s m s by a n a l y z i n g p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s p a t t e r n s produced by r a d i o l a b e l e d p r o t e i n s . T h i s a g a i n would r e q u i r e a d e t a i l e d knowledge o f those p a t t e r n s . T h i s method i s b e s t used t o d i s t i n g u i s h between c l o s e l y r e l a t e d s t r a i n s .
In Biotechnology for Crop Protection; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
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GENETIC TECHNIQUES
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Gene Probes These t e c h n i q u e s r e l y on the s p e c i f i c i t y p r o v i d e d by t h e h y b r i d i z a t i o n between two complementary ( o r n e a r l y so) sequences of n u c l e i c a c i d s . Sequences c a n be s e l e c t e d f o r p r o b i n g t h a t a r e v e r y common among o r g a n i s m s , f o r f u n c t i o n a l o r p h y l o g e n i c g r o u p s , o r v e r y s p e c i f i c f o r o n l y one s t r a i n of an o r g a n i s m . The main advantages o f t h i s methodology i n c l u d e s : (1) t h e a b i l i t y t o d e t e c t organisms t h a t cannot be c u l t u r e d because they a r e s t r e s s e d o r f a s t i d i o u s , w h i c h i s a common l i m i t a t i o n i n the s t u d y o f the t o t a l m i c r o b i a l p o p u l a t i o n i n n a t u r e , (2) t h e a b i l i t y t o d e t e c t organisms w i t h o u t the n e c e s s i t y o f h a v i n g a s p e c i f i c s e l e c t a b l e m a r k e r , such as a n t i b i o t i c r e s i s t a n c e o r r e q u i r i n g the development o f o t h e r s e l e c t i v e m e d i a , (3) the a b i l i t y t o t r a c k the gene of i n t e r e s t r e g a r d l e s s o f whether i t i s e x p r e s s e d o r t r a n s f e r r e d t o a n o t h e r o r g a n i s m , and (4) the p o t e n t i a l f o r h i g h e r s e n s i t i v i t y t h a n i s p o s s i b l e by any o t h e r methods. The d i s a d v a n t a g e s o f the gene probe method a r e t h a t i t i s more complex w h i c h reduces the number o f samples t h a t can be p r o c e s s e d , q u a n t i t a t i o n o f t h e number o f gene c o p i e s per gram o f sample i s s t i l l d i f f i c u l t and i m p r e c i s e . The use o f the method i n m i c r o b i a l e c o l o g y i s s t i l l i n i t s i n f a n c y so t h a t problems w i t h r e p r o d u c i b i l i t y , a d a p t a b i l i t y to d i f f e r e n t s o i l composition, d i f f e r e n t organisms and probes w i l l t a k e t i m e t o r e s o l v e b e f o r e t h e method can be used e f f e c t i v e l y . A number o f r e s e a r c h groups a r e now a c t i v e l y p u r s u i n g the gene probe method f o r use lit the e n v i r o n m e n t , and improvements i n i t s s e n s i t i v i t y and ease o f use a r e expected i n the near f u t u r e . The d i r e c t i o n s t a k e n by many o f the U . S . w o r k e r s have been reviewed by H o l b e n and T i e d j e ( 5 ) , and the r e c e n t REGEM Conference P r o c e e d i n g s r e p o r t s t h e work o f many i n t e r n a t i o n a l w o r k e r s on t h i s s u b j e c t ( 6 ) . The f o l l o w i n g i s a b r i e f s u m m a r i z a t i o n o f i m p o r t a n t f e a t u r e s and advances i n the use of gene probes f o r d e t e c t i n g m i c r o o r g a n i s m s i n nature. S e l e c t i o n o f Probes There a r e t h r e e b a s i c c l a s s e s o f genes w h i c h have been used as p r o b e s . Each has been used s u c c e s s f u l l y , and a t t h i s s t a g e o f knowledge, none can be argued t o be g e n e r a l l y p r e f e r r e d . These c l a s s e s a r e probes t a r g e t e d f o r r i b o s o m a l RNA ( r R N A ) , f o r randomly c l o n e d sequences o f DNA unique t o the o r g a n i s m o f i n t e r e s t , and f o r t h e e n g i n e e r e d sequence i t s e l f . I f one i s t r a c k i n g a g e n e t i c a l l y e n g i n e e r e d m i c r o o r g a n i s m (GEM) the l a t t e r i s u s u a l l y used because the probe i s a l r e a d y a v a i l a b l e and i t i s t h i s gene t h a t i s o f g r e a t e s t i n t e r e s t f o r GEMs. F o r non-GEMs, the f i r s t two probe t y p e s a r e more l i k e l y t o be u s e d . F o c u s i n g on rRNA has the advantage o f p o t e n t i a l l y p r o v i d i n g probes common f o r s e v e r a l groups o r s p e c i f i c f o r one s t r a i n depending on whether one s e l e c t s a c o n s e r v e d o r v a r i a b l e r e g i o n of the 16S rRNA. O b t a i n i n g Sample DNA Three approaches have been used t o r e c o v e r DNA from n a t u r a l
In Biotechnology for Crop Protection; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
samples
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f o r p r o b i n g . The f i r s t r e p o r t e d i n v o l v e d c e l l l y s i s and e x t r a c t i o n of t o t a l DNA i n sediment by SDS and a l k a l i n e t r e a t m e n t ( 7 ) . This method has the advantage o f a more q u a n t i t a t i v e r e c o v e r y o f DNA from the s o i l but has the d i s a d v a n t a g e o f r e q u i r i n g more c l e a n - u p of the DNA s i n c e o t h e r problem c o n t a m i n a n t s a r e a l s o e x t r a c t e d . A second approach i s t o f i r s t e l u t e the m i c r o b i a l c e l l s from s o i l b e f o r e l y s i s t h e r e b y p r o t e c t i n g t h e DNA from s o i l c o n t a m i n a n t s . T h i s method a l s o has the advantage i n t h a t the DNA i s r e t a i n e d i n l a r g e m o l e c u l a r weight fragments ( a p p r o x i m a t e l y 50 kb) so t h a t r e s t r i c t i o n d i g e s t i o n , and g e l s e p a r a t i o n y i e l d s d i s c r e t e fragments t h a t a l l o w a n a l y s i s by S o u t h e r n b l o t s ( 8 ) . B o t h o f t h e above methods g i v e more DNA t h a n c a n be accounted f o r i n t h e p o p u l a t i o n d e t e c t e d by p l a t e c o u n t i n g i m p l y i n g t h a t DNA from n o n - c u l t u r a b l e organisms i s a l s o b e i n g r e c o v e r e d . The t h i r d method i s t o f i r s t f o s t e r growth o f t h e p o p u l a t i o n o f i n t e r e s t , but a f t e r d i l u t i o n and s e p a r a t i o n by the most p r o b a b l e number (MPN) p r o t o c o l . The c u l t u r e d c e l l s i n w e l l s a r e then l y s e d and probed w i t h the gene ( 9 ) . I n t h i s case the presence o r absence of t h e t a r g e t gene i s used w i t h a MPN t a b l e t o e s t i m a t e the o r i g i n a l p o p u l a t i o n d e n s i t y o f the o r g a n i s m . The advantage o f t h i s method i s t h a t the t a r g e t DNA i s a m p l i f i e d and p u r i f i e d by g r o w t h . D e t e c t i o n l i m i t s o f 10 t o 100 o r g a n i s m s / g , depending on the p a r t i c u l a r organisms growth r e s p o n s e , were o b t a i n e d w i t h t h i s method. The C h o i c e o f D e t e c t i o n Method Once the probe and sample have been o b t a i n e d t h e r e a r e s e v e r a l c h o i c e s f o r d e t e c t i o n based on the e x p e r i m e n t a l g o a l s . The d e t e c t i o n methods v a r y i n s e n s i t i v i t y , ease of u s e , s t a b i l i t y o f p r o b e , and whether i n f o r m a t i o n on gene l o c a t i o n i s d e s i r e d . The most common a n a l y s i s format i s by dot o r s l o t b l o t i n w h i c h the DNA i s f i x e d t o a f i l t e r , the ^ ^ P - l a b e l e d probe added, n o n - h y b r i d i z e d DNA washed away and t h e amount of r a d i o a c t i v i t y r e m a i n i n g a n a l y z e d u s u a l l y by a u t o r a d i o g r a p h y . U s i n g t h e format and the M13/**2p probe systems o f Holben e t . a l . ( 8 ) , 1000 o r g a n i s m s / g s o i l were detected. More i n f o r m a t i o n i s o b t a i n e d , however, i f the DNA i s c u t by r e s t r i c t i o n enzymes, s e p a r a t e d a c c o r d i n g t o l e n g t h i n g e l s , and the l o c a t i o n o f the d i f f e r e n t s i z e d fragments d e t e c t e d by autoradiography a f t e r Southern t r a n s f e r to b l o t s . Multiple p o p u l a t i o n s o f organisms w i t h t h e same t a r g e t can a l l be d e t e c t e d i n one sample and any change i n gene p o s i t i o n e i t h e r w i t h i n i t s h o s t o r i f t r a n s f e r r e d t o o t h e r organisms can be d e t e c t e d because the t a r g e t gene would have d i f f e r e n t f l a n k i n g r e g i o n s and t h u s be i n d i f f e r e n t s i z e d fragments. U s i n g the M13 p r o b e , 10^ o r g a n i s m s / g c o u l d be d e t e c t e d i n S o u t h e r n b l o t s ( 8 ) . I f t h e g o a l f o r d e t e c t i o n i s s e n s i t i v i t y , the polymerase c h a i n r e a c t i o n (PCR) methodology o f f e r s e x c i t i n g new p o s s i b i l i t i e s . W i t h t h i s method a p r i m e r unique t o the t a r g e t i s added t o the e n v i r o n m e n t a l DNA sample, and t h r o u g h r e p e a t e d c y c l e s o f s y n t h e s i s o f complementary s t r a n d s and d e n a t u r a t i o n , the t a r g e t i s g r e a t l y m a g n i f i e d t h e r e b y i n c r e a s i n g s e n s i t i v i t y . When S t e f f a n and A t l a s (10) added a known organism (and t a r g e t gene) t o s e d i m e n t , t h e y were a b l e t o d e t e c t one o r g a n i s m / g . A v e r y d i f f e r e n t approach i s
In Biotechnology for Crop Protection; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
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to d e t e c t the m i c r o b i a l c e l l i t s e l f i n a m i c r o s c o p i c f i e l d by u s i n g gene probes t o i d e n t i f y the p a r t i c u l a r c e l l s of i n t e r e s t . This method, developed by P a c e ' s group ( 1 1 ) , i s based on probes t o the 16S rRNA. Because c e l l s have many r i b o s o m e s , t h e r e i s s u f f i c i e n t t a r g e t i n an i n d i v i d u a l c e l l so t h a t the s i g n a l c a n be e a s i l y r e c o g n i z e d by m i c r o a u t o r a d i o g r a p h y (3^S r 32p) probes o r v i s u a l l y by f l u o r e s c e n t p r o b e s . T h i s method p r e s e r v e s the i n f o r m a t i o n on c e l l morphology and perhaps l o c a t i o n i n a c o n s o r t i u m o r on s u r f a c e s i f t h a t i n f o r m a t i o n i s i m p o r t a n t . A l l d e t e c t i o n so f a r i n m i c r o b i a l e c o l o g y has been w i t h 32p-labeled probes. Because o f the r a d i a t i o n exposure and s h o r t s h e l f - l i f e of t h e s e p r o b e s , n o n - r a d i o a c t i v e probes would be of g r e a t value< Such probes have been d e v e l o p e d , e . g . , the b l o t i n - l a b e l e d r e s i d u e s w h i c h y i e l d a c o l o r r e a c t i o n , but they have not y e t proved s e n s i t i v e o r s p e c i f i c enough t o g e n e r a t e any i n t e r e s t by m i c r o b i a l e c o l o g i s t s .
Downloaded by STANFORD UNIV GREEN LIBR on February 25, 2013 | http://pubs.acs.org Publication Date: November 22, 1988 | doi: 10.1021/bk-1988-0379.ch027
Q
Gene Sequencing At t h i s t i m e , gene sequencing c a n p r o b a b l y be d i s m i s s e d as a r e a s o n a b l e m o n i t o r i n g t o o l because of the h i g h l a b o r i n p u t , c o s t and l a r g e database t o i n t e r p r e t the r e s u l t s . IMMUNOLOGICAL TECHNIQUES Immunofluorescence T h i s p r o c e d u r e u t i l i z e s a s p e c i e s - s p e c i f i c a n t i s e r i u m and f l u o r e s c e n t d y e . On e x a m i n a t i o n w i t h an e p i f l u o r e s c e n e m i c r o s c o p e , s t a i n e d c e l l s appear as a p e r i p h e r a l g r e e n band v i s i b l e beneath the cell wall. The method has been used t o s t u d y Rhizobium e c o l o g y and t o m o n i t o r the s u r v i v a l o f pathogens i n an a q u a t i c environment ( 1 2 ) . The advantages a r e speed, and i t d e t e c t s organisms t h a t do not grow. The d i s a d v a n t a g e s a r e the s m a l l s i z e o f the sample a n a l y z e d , l a c k of s p e c i f i c i t y , and unknown g e n e t i c c o m p o s i t i o n . The s e n s i t i v i t y i s about 1000 c e l l s / g r a m o f s o i l . I t i s not l i k e l y t o be g e n e r a l l y u s e f u l f o r m o n i t o r i n g GEMs because i t cannot d i s t i n g u i s h the GEM from i t s p a r e n t o r c l o s e r e l a t i v e s i n n a t u r e . Immunoradiography The same p r i n c i p l e a p p l i e s here as w i t h immunofluorescence, t h a t the a n t i s e r u m i s c o u p l e d w i t h a l a b e l e d compound.
except
Flow Cytometry T h i s i s a c e l l s o r t i n g t e c h n i q u e t h a t uses a l a s e r beam and f l u o r g e n i c s t a i n i n g t e c h n i q u e s t o measure c e l l s i z e , and c e l l u l a r components i n a s i n g l e c e l l i n a l a r g e p o p u l a t i o n . T h i s automated c e l l - b y - c e l l a n a l y t i c a l t o o l has had l i m i t e d e n v i r o n m e n t a l m o n i t o r i n g a p p l i c a t i o n . I t ' s advantages a r e speed o f a n a l y s i s and sensitivity. The d i s a d v a n t a g e s a r e expense and ease o f o p e r a t i o n .
In Biotechnology for Crop Protection; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
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Affinity Concentration This is a method of concentrating low populations of released microorganisms from field samples. One option being considered is a selective immunobiological fixation on polystyrene beads or other supports coated with a suitable monoclonal antibody. Literature Cited Downloaded by STANFORD UNIV GREEN LIBR on February 25, 2013 | http://pubs.acs.org Publication Date: November 22, 1988 | doi: 10.1021/bk-1988-0379.ch027
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Tiedje, J . M. Proc. Pros. Phys. Biol. Contain. Genet. Engin. Organ. ERC-114 1985, 115. 2. McCormick, D. Biotechnology. 1986, 4, 419. 3. Smith, M. S.; Tiedje, J . M. Can. J . Microbiol. 1980, 26, 851. 4. Drahos, D. J.; Hemming, B. C.; McPherson, S. Biotechnology. 1986, 4, 439. 5. Holben, W. E.; Tiedje, J. M. Ecology. 1988, 69, 561. 6. REGEM 1. First International Conference on the Release of Genetically Engineered Microorganisms. Programme and Information. Cardiff Wales, UK. April 5th-8th, 1988. 7. Ogram, A.; Sayler, G. S.; Barkay, T. J . Microbiol. Methods. 1987, 7, 57. 8. Holben, W. E.; Jansson, J . K.; Chelm, B. K.; Tiedje, J . M. Appl. Environ. Microbiol. 1988, 54, 703. 9. Fredrickson, J . K.; Bezdicek, D. F.; Brockman, F. J.; L i , S. W. Appl. Environ. Microbiol. 1988, 54, 446. 10. Steffan, R.; Atlas, R. Appl. Environ. Microbiol. 1988, in press. 11. Giovannoni, S. J.; DeLong, E. F.; Olsen, G. J.; Pace, N. R. J. Bacteriol. 1988, 170, 720. 12. Colwell, R. R.; Brayton, P. R.; Grimes, D. B.; Roszak, S. A.; Huq, S. A.; Palmer, L. M. Biotechnology 1985, 3, 817. RECEIVED June 19, 1988
In Biotechnology for Crop Protection; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
