Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Effects of phosphate starvation on Pseudomonas aeruginosa Hou, Cynthia Isobel 1965

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1965_A6_7 H6.pdf [ 4.74MB ]
Metadata
JSON: 831-1.0105499.json
JSON-LD: 831-1.0105499-ld.json
RDF/XML (Pretty): 831-1.0105499-rdf.xml
RDF/JSON: 831-1.0105499-rdf.json
Turtle: 831-1.0105499-turtle.txt
N-Triples: 831-1.0105499-rdf-ntriples.txt
Original Record: 831-1.0105499-source.json
Full Text
831-1.0105499-fulltext.txt
Citation
831-1.0105499.ris

Full Text

THE EFFECTS OF PHOSPHATE STARVATION ON PSEUDOMONAS AERUGINOSA t y CYNTHIA I . HOU B.S.A., U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1963 A THESIS SUBMITTED I N PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n A g r i c u l t u r a l M i c r o b i o l o g y i n t h e D i v i s i o n o f A n i m a l S c i e n c e We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA A p r i l , I965 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study* I f u r t h e r agree t h a t p e r -m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y purposes may be g r anted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i -c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my . w r i t t e n p e r m i s s i o n * Department of A n i m a l S c i e n c e The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada D a t e A p r i l , I965 A B S T R A C T T h e r e s p o n s e o f P s e u d o m o n a s a e r u g i n o s a t o p h o s p h a t e s t a r v a t i o n a n d s u b s e q u e n t r e f e e d i n g w a s s t u d i e d b y f o l l o w -i n g c h a n g e s i n t u r b i d i t y , c e l l c o u n t a n d c h e m i c a l c o m p o s i t i o n o n i n c u b a t i o n i n p h o s p h a t e d e f i c i e n t m e d i u m . I n s h a k e n , p h o s p h a t e d e f i c i e n t c u l t u r e s , t h e t u r b i d i t y a n d v i a b l e c e l l c o u n t w e r e s h o w n t o i n c r e a s e s i g n i f i c a n t l y , w i t h t h e l a t t e r r e a c h i n g a m a x i m u m l e v e l a t 2 4 h o u r s u n d e r t h e c o n d i t i o n s e m p l o y e d . A l i n e a r r e s p o n s e o f p h o s p h a t e s t a r v e d c e l l s t o l o w l e v e l s o f p h o s p h a t e s u p p l i e d e x o g e n o u s l y w a s e v i d e n t f r o m t u r b i d i t y m e a s u r e m e n t s , a n d a t h r e s h o l d r e q u i r e m e n t f o r p h o s p h a t e a n a l o g o u s t o t h e " e n e r g y o f m a i n t e n a n c e " ( M c G r e w a n d M a l l e t t e , 1962) w a s n o t d e t e c t e d . I n s t i l l , p h o s p h a t e d e f i c i e n t c u l t u r e s , t h e t u r b i d i t y a n d t o t a l c e l l c o u n t i n c r e a s e d a n d t h e v i a b l e c e l l c o u n t d e c r e a s e d s l i g h t l y a t 2 4 h o u r s . T h e l e v e l s o f p r o t e i n a n d d e o x y r i b o n u c l e i c a c i d (DNA) p e r m l o f c u l t u r e i n c r e a s e d d u r i n g t h i s p e r i o d , a n d t h e a m o u n t o f r i b o n u c l e i c a c i d ( R N A ) d e c r e a s e d . E x t e n s i v e r i b o s o m a l d e g r a d a t i o n w a s a p p a r e n t f r o m s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n p a t t e r n s . A n e n z y m e h a v i n g a n a l k a l i n e pH o p t i m u m a n d d i s p l a y i n g a c t i v i t y a g a i n s t a w i d e v a r i e t y o f p h o s p h o m o n o e s t e r s w a s - i i i -d e m o n s t r a t e d i n p h o s p h a t e - s t a r v e d c e l l s . T h e e n z y m e w a s i n h i b i t e d b y i n o r g a n i c p h o s p h a t e , a n d w a s c o n s i d e r e d t o b e t h e c o u n t e r p a r t o f t h e r e p r e s s i b l e p h o s p h o m o n o -e s t e r a s e r e p o r t e d i n o t h e r m i c r o o r g a n i s m s a n d s t u d i e d i n d e t a i l i n E s c h e r i c h i a c o l i ( T o r r i a n i , i 9 6 0 ; G a r e n a n d L e v i n t h a l , I960; H e p p e l , H a r k n e s s a n d H i l m o e , 1962). T h e e n z y m e a c t i v i t y o f c e l l f r e e e x t r a c t s o f P. a e r u g i n o s a w a s s h o w n t o b e a s s o c i a t e d m a i n l y w i t h t h e r i b o s o m a l f r a c t i o n . B l y t h e A. E a g l e s , Dean - F a c u l t y o f A g r i c u l t u r e - i v -T A B L E OF CONTENTS P a g e I N T R O D U C T I O N 1 R E V I E W OF THE L I T E R A T U R E 2 I . P h o s p h a t e S t a r v a t i o n o f M i c r o o r g a n i s m s 2 I I . S t a r v a t i o n o f O t h e r E s s e n t i a l N u t r i e n t s a n d t h e E f f e c t u p o n C e l l u l a r RNA 7 I I I . T h e F o r m a t i o n a n d P r o p e r t i e s o f R e p r e s s i b l e P h o s p h o m o n o e s t e r a s e s i n B a c t e r i a 13 1. T h e f o r m a t i o n o f t h e e n z y m e 13 2. T h e r e p r e s s i o n o f e n z y m e s y n t h e s i s 15 3 . G e n e t i c s t u d i e s 16 4 . M e c h a n i s m o f a c t i o n 17 5 . S p e c i f i c i t y 18 6. S t a b i l i t y 19 7. T h e e f f e c t o f v a r i o u s c o m p o u n d s o n a c t i v i t y 20 8. T r a n s p h o s p h o r y l a t i o n 20 9 . T h e l o c a t i o n o f a c t i v i t y 21 I V . T h e O c c u r r e n c e o f a R e p r e s s i b l e A c i d P h o s p h o m o n o e s t e r a s e i n Y e a s t s 25 -V-P a g e M A T E R I A L S AND METHODS 27 I . B a c t e r i o l o g i c a l M e t h o d s 27 I I . A n a l y t i c a l M e t h o d s 28 1. P r o t e i n 2 8 2. N u c l e i c a c i d s 2 8 3. I n o r g a n i c p h o s p h a t e 29 I I I . I n i t i a l S t u d i e s w i t h S h a k e n C e l l S u s p e n s i o n s 29 1. P r e p a r a t i o n o f c e l l s 29 2. T u r b i d i m e t r y a n d p l a t e c o u n t s 30 3 . P r e p a r a t i o n o f g l a s s w a r e 30 I V . S t u d i e s w i t h S t i l l C u l t u r e s 30 1. P r e p a r a t i o n o f c e l l s 30 2. T u r b i d i m e t r y a n d p l a t e c o u n t s 31 3 . C o u l t e r c o u n t s o n c e l l s u s p e n s i o n s 31 k. C h e m i c a l f r a c t i o n a t i o n o f c e l l s 32 V . S u c r o s e D e n s i t y G r a d i e n t P a t t e r n s 3^ 1. P r e p a r a t i o n o f c e l l f r e e e x t r a c t s 3^ 2. P r e p a r a t i o n o f g r a d i e n t s 3^ V I . D e m o n s t r a t i o n o f A l k a l i n e P h o s p h a t a s e A c t i v i t y i n E x t r a c t s o f S t a r v e d C e l l s 36 1. P r e p a r a t i o n o f c e l l f r e e e x t r a c t s 36 2 . A s s a y b y r e l e a s e o f £ - n i t r o p h e n o l f r o m p _ - n i t r o p h e n y l p h o s p h a t e 36 3 . A s s a y b y r e l e a s e o f i n o r g a n i c p h o s p h a t e f r o m s u b s t r a t e s 37 - v i -P a g e V I I . C h a r a c t e r i z a t i o n o f A l k a l i n e P h o s p h a t a s e A c t i v i t y 38 1. T h e e f f e c t o f b u f f e r c o m p o s i t i o n a n d c o n c e n t r a t i o n 38 2 . T h e e f f e c t o f E D T A a n d i n o r g a n i c i o n s 38 3 . D e t e r m i n a t i o n o f a c t i v i t y a t v a r i o u s pH v a l u e s 39 4 . D e t e r m i n a t i o n o f a c t i v i t y w i t h i n c r e a s i n g t e m p e r a t u r e 39 5. D e t e r m i n a t i o n o f a c t i v i t y a g a i n s t v a r i o u s s u b s t r a t e s 39 6. D e g r e e o f i n h i b i t i o n b y i n o r g a n i c p h o s p h a t e 4-0 V I I I . T h e L o c a t i o n o f A l k a l i n e P h o s p h a t a s e A c t i v i t y i n F r a c t i o n a t e d C e l l F r e e E x t r a c t s 40 1 . P r e p a r a t i o n o f c e l l f r e e e x t r a c t s 4 0 2 . F r a c t i o n a t i o n o f c e l l f r e e e x t r a c t s b y c e n t r i f u g a t i o n 4 1 3 . A s s a y o f f r a c t i o n s 4 2 R E S U L T S AND D I S C U S S I O N 4 3 I . I n i t i a l S t u d i e s w i t h S h a k e n C e l l S u s p e n s i o n s 4 3 1 . T h e c o u r s e o f s t a r v a t i o n i n p h o s p h a t e d e f i c i e n t m e d i u m 4 3 - v i i -Page 2. The r a t e of response of starved and nonstarved c e l l s to a f i x e d l e v e l of phosphate 47 3 . The response of starved and nonstarved c e l l s to graded l e v e l s of phosphate 51 I I . Studies w i t h S t i l l C u l t u r e s 56 1. Changes i n t u r b i d i t y , c e l l numbers and p r o t e i n on phosphate s t a r v a t i o n 56 2. Chemical changes i n c e l l u l a r components on s t a r v a t i o n 59 3. Sucrose d e n s i t y gradient p a t t e r n s 62 I I I . A l k a l i n e Phosphatase A c t i v i t y 67 1. The e f f e c t of b u f f e r composition and concen t r a t i o n 67 2. The e f f e c t of EDTA and i n o r g a n i c ions 69 3 . A c t i v i t y a t various pH values 73 4 . A c t i v i t y w i t h i n c r e a s i n g temperature 75 5 . A c t i v i t y w i t h v a r i o u s substrates 75 6. Degree of i n h i b i t i o n by i n o r g a n i c phosphate 80 IV. The L o c a t i o n of A l k a l i n e Phosphatase 81 1. A c t i v i t y i n whole c e l l s 81 2. A c t i v i t y i n c e l l f r e e e x t r a c t f r a c t i o n s 82 GENERAL DISCUSSION I . Ribosomal M a t e r i a l as an Endogenous Reserve 84 I I . A l k a l i n e Phosphatase 87 - v i i i -Page SUMMARY 91 BIBLIOGRAPHY 93 - i x -L I S T OF F I G U R E S F i g u r e P a g e 1. C h a n g e s i n v i a b l e c e l l c o u n t a n d t u r b i d i t y d u r i n g s t a r v a t i o n o f P. a e r u g i n o s a i n s h a k e c u l t u r e s i n p h o s p h a t e d e f i c i e n t m e d i u m f o r 30 h o u r s . 4 4 2. C h a n g e s i n v i a b l e c e l l c o u n t a n d t u r b i d i t y d u r i n g s t a r v a t i o n o f P. a e r u g i n o s a i n s h a k e c u l t u r e s i n p h o s p h a t e d e f i c i e n t m e d i u m f o r 6 d a y s . 4 6 3 . F i n a l t u r b i d i t y o f p h o s p h a t e - s t a r v e d s h a k e c u l t u r e s o f P. a e r u g i n o s a a s a f u n c t i o n o f i n o c u l u m s i z e . 4 8 4 . R a t e o f r e s p o n s e o f s h a k e c u l t u r e s o f P. a e r u g i n o s a t o 0.1 jam p h o s p h a t e p e r t u b e . 50 5 . F i n a l t u r b i d i t y o f s h a k e c u l t u r e s o f P. a e r u g i n o s a a s a f u n c t i o n o f a d d e d p h o s p h a t e i n t h e r a n g e o f 0 t o 0.1 ;um p e r t u b e . 52 - X -Figure Page 6. F i n a l t u r b i d i t y of shake c u l t u r e s of P. aeruginosa as a f u n c t i o n of added phosphate i n the range of 0 t o 1.0 )m per tube. 53 7. F i n a l t u r b i d i t y of shake c u l t u r e s of P. aeruginosa as a f u n c t i o n of added phosphate i n the range of 0 to 2.5 )xm per tube. 55 8 . Ribosome patterns of e x t r a c t s of nonstarved, phosphate-starved and r e f e d c e l l s . Gradients contained 10~ 2 M f i n a l c o n c e n t r a t i o n of MgCl£. 63 9 . Ribosome patterns of e x t r a c t s of nonstarved, phosphate-starved and r e f e d c e l l s . Gradients contained 10"^ M f i n a l c o n c e n t r a t i o n of MgClg. 64 10. A l k a l i n e phosphatase a c t i v i t y i n a c e l l f r e e e x t r a c t (CFX) of P. aeruginosa as a funct i o n of b u f f e r composition and c o n c e n t r a t i o n . 68 11. A l k a l i n e phosphatase a c t i v i t y i n a c e l l f r e e e x t r a c t of P. aeruginosa as a f u n c t i o n of pH. 74 12. A l k a l i n e phosphatase a c t i v i t y i n a c e l l f r e e e x t r a c t of P. aeruginosa as a f u n c t i o n of temperature. 76 - x i ACKNOWLEDGEMENT I w o u l d l i k e t o e x p r e s s my d e e p e s t a p p r e c i a t i o n t o D r . A . F. G r o n l u n d f o r h e r g u i d a n c e a n d e n c o u r a g e m e n t d u r i n g t h e c o u r s e o f t h i s s t u d y . I w o u l d a l s o l i k e t o e x p r e s s my g r a t i t u d e t o D r . J . J . R. C a m p b e l l a n d D r . M. F . M a l l e t t e f o r t h e i r v e r y w e l c o m e s u g g e s t i o n s a n d c r i t i c i s m s , a n d t o t h e N a t i o n a l R e s e a r c h C o u n c i l o f C a n a d a f o r t h e f i n a n c i a l a s s i s t a n c e g i v e n me. I N T R O D U C T I O N T h e d e g r a d a t i o n o f r i b o n u c l e i c a c i d a n d t h e b r e a k d o w n o f r i b o s o m a l s t r u c t u r e h a s b e e n r e p o r t e d b y a n u m b e r o f a u t h o r s t o b e a common o c c u r r e n c e i n b a c t e r i a w h i c h h a v e c e a s e d t o g r o w . T h i s e f f e c t h a s b e e n o b s e r v e d u n d e r c o n d i t i o n s w h e r e v a r i o u s n u t r i e n t s a r e l i m i t i n g t o g r o w t h ( M a n d e l s t a m , i 9 6 0 ) . I n t h e s p e c i f i c c a s e o f p h o s p h a t e s t a r v a t i o n t h e c o n c o m i t a n t s y n t h e s i s o f a r e p r e s s i b l e p h o s p h o m o n o e s t e r a s e h a s b e e n d e m o n s t r a t e d . T h i s e n z y m e w a s s h o w n i n E s c h e r i c h i a c o l i a n d o t h e r s p e c i e s t o b e s t r o n g l y i n h i b i t e d b y i n o r g a n i c p h o s p h a t e a n d w a s s u g g e s t e d t o r e p r e s e n t a n e x a m p l e o f t h e w e l l k n o w n p h e n o m e n o n o f f e e d b a c k i n h i b i t i o n ( H o r i u c h l , 1959; T o r r i a n i , i 9 6 0 ) . T h e p r e s e n t s t u d y w a s u n d e r t a k e n i n a n a t t e m p t t o d e t e r m i n e h o w P s e u d o m o n a s a e r u g i n o s a i s a f f e c t e d b y s u c h c o n d i t i o n s o f p h o s p h a t e d e p r i v a t i o n a n d h o w i t r e s p o n d s t o t h i s s i t u a t i o n . C h a n g e s i n t h e p o p u l a t i o n . o n s t a r v a t i o n a n d r e f e e d i n g o f p h o s p h a t e w e r e f o l l o w e d b y t u r b i d i m e t r y a n d p l a t e c o u n t d e t e r m i n a t i o n s o f v i a b i l i t y . C h a n g e s i n t h e c h e m i c a l c o m p o s i t i o n a n d r i b o s o m e p a t t e r n s o f t h e c e l l s w e r e a l s o f o l l o w e d , a n d t h e f o r m a t i o n , p r o p e r t i e s a n d l o c a t i o n o f a n e n z y m e c o m p a r a b l e t o t h e r e p r e s s i b l e p h o s p h o m o n o e s t e r a s e o f o t h e r m i c r o o r g a n i s m s w a s s t u d i e d . - 2 -R E V I E W OF THE L I T E R A T U R E T h e b r e a k d o w n a n d u t i l i z a t i o n o f e n d o g e n o u s m a t e r i a l s u n d e r c o n d i t i o n s o f n u t r i e n t d e p r i v a t i o n h a s r e c e n t l y b e e n t h e s u b j e c t o f a c o n s i d e r a b l e a m o u n t o f r e s e a r c h a n d o f a n u m b e r o f e x t e n s i v e r e v i e w s ( D a w e s a n d R i b b o n s , 1962 a n d 1964; L a m a n n a , 1 9 6 3 ) . T h e p r e s e n t s u r v e y w i l l a t t e m p t t o p o i n t o u t o n l y t h o s e r e p o r t s w h i c h a r e d i r e c t l y c o n c e r n e d w i t h t h e e f f e c t s o f p h o s p h a t e s t a r v a t i o n o n m i c r o o r g a n i s m s o r w h i c h p r e s e n t r e s u l t s w h i c h may b e r e l a t e d t o o r c o m p a r e d w i t h t h o s e s h o w n h e r e . T h e l a t t e r w i l l c o n t r i b u t e t o a b r i e f d i s c u s s i o n o f t h e e f f e c t s o f d e p r i v i n g c e l l s o f v a r i o u s o t h e r s p e c i f i c n u t r i e n t s . T h e f i n a l a n d m o s t e x t e n s i v e p o r t i o n o f t h i s r e v i e w w i l l d e a l w i t h t h e f o r m a t i o n a n d p r o p e r t i e s o f t h e r e p r e s s i b l e e n z y m e p h o s p h o m o n e s t e r a s e w h i c h h a s b e e n d e m o n s t r a t e d i n o t h e r m i c r o o r g a n i s m s d u r i n g p h o s p h a t e s t a r v a t i o n a n d w h i c h i s s h o w n i n t h i s p r e s e n t a t i o n t o o c c u r a l s o i n P s e u d o m o n a s a e r u g i n o s a . I . P h o s p h a t e S t a r v a t i o n o f M i c r o o r g a n i s m s T h e g r o w t h a n d s u r v i v a l o f E s c h e r i c h i a c o l i i n p h o s p h a t e - l i m i t i n g m e d i u m w a s s t u d i e d b y M a l l e t t e , C o w a n - 3 -a n d C a m p b e l l (1964) u s i n g t h e t e c h n i q u e s o f t u r b i d i m e t r y a n d p l a t e c o u n t i n g . T h i s w o r k a r o s e f r o m t h e p r e v i o u s s t u d i e s o f M c G r e w a n d M a l l e t t e (1962) o n t h e u t i l i z a t i o n o f s m a l l a m o u n t s o f g l u c o s e f o r t h e m a i n t e n a n c e o f v i a b i l i t y w i t h o u t c o n c o m i t a n t g r o w t h . A s m a l l b u t r e p r o d u c i b l e i n t e r c e p t i n a p l o t o f t u r b i d i t y i n c r e m e n t a g a i n s t t h e a m o u n t o f g l u c o s e a d d e d t o a n o t h e r w i s e c o m p l e t e m e d i u m p r o v i d e d e v i d e n c e f o r a n " e n e r g y o f m a i n t e n a n c e " n o t p r e v i o u s l y d e t e c t e d i n m i c r o o r g a n i s m s . T h e s e r e s u l t s r a i s e d t h e q u e s t i o n o f w h e t h e r o r n o t a s i m i l a r p h e n o m e n o n m i g h t o c c u r f o r o t h e r r e q u i r e d n u t r i e n t s s u c h a s p h o s p h o r u s . A s i n t h e s t u d i e s o f M c G r e w a n d M a l l e t t e , M a l l e t t e e t a l . (1964) u s e d a r e l a t i v e l y l a r g e , w a s h e d i n o c u l u m a n d l o w l e v e l s o f p h o s p h a t e t o i m p r o v e t h e s e n s i t i v i t y o f t h e e x t r a p o l a t i o n t h r o u g h t h e z e r o l e v e l o f p h o s p h a t e . T h e p r e l i m i n a r y s t u d i e s o f M a l l e t t e ejt a l . (1964) o n c e l l s u s p e n s i o n s s h a k e n a t 37 C i n a d e f i n e d m e d i u m w i t h n o a d d e d p h o s p h a t e i n d i c a t e d t h a t a n i n c r e a s e i n t u r b i d i t y a n d p l a t e c o u n t s d i d o c c u r . T h e n u m b e r o f v i a b l e c e l l s i n c r e a s e d m o r e r a p i d l y a n d t o a g r e a t e r e x t e n t t h a n d i d t h e t u r b i d i t y . A s u b s e q u e n t l o g a r i t h m i c d e c r e a s e i n t h e v i a b l e c o u n t w a s n o t p r e v e n t e d b y t h e a d d i t i o n o f .4-glucose, i n d i c a t i n g than an adequate supply of energy was present and thus was not the l i m i t i n g factor. The absence of contaminating exogenous phosphate i n the phosphate d e f i c i e n t medium was suggested by the f a i l u r e of phosphate starved c e l l s to increase i n t u r b i d i t y i n fresh, phosphate d e f i c i e n t medium, and also by the l i n e a r i t y and extrapolation through the o r i g i n of a plot of f i n a l t u r b i d i t y i n phosphate d e f i c i e n t medium against inoculum s i z e . Having determined the conditions required fo r maximum growth of a starving culture, Mallette et a l . determined the time required for f u l l turbidimetric response to a given l e v e l of phosphate, and measured the response of both starved and nonstarved c e l l s to phosphate when t h i s was added at d i f f e r e n t l e v e l s . Of i n t e r e s t was the f a i l u r e of the prestarved c e l l s to respond to higher l e v e l s of phosphate as f u l l y as did the nonstarved c e l l s . Plate count data indicated no loss of v i a b i l i t y of the prestarved c e l l s a f t e r incubation with phosphate, and thus excluded death as an explanation for the f a i l u r e of the prestarved c e l l s to grow normally. The observed extra-polation of both plots through the zero l e v e l of phosphate indicated the absence of a threshold requirement of E. c o l i -9 f o r phosphate (at l e a s t above 1 x 10 moles per ml) which might be considered analogous to the energy of maintenance previously discussed. - 5 -T h e p h y s i o l o g i c a l e f f e c t s o f s t a r v i n g E . c o l i i n a p h o s p h a t e d e f i c i e n t m e d i u m h a v e b e e n d e s c r i b e d b y H o r i u c h i ( 1 959 ) . He g r e w c e l l s i n s h a k e c u l t u r e i n a p e p t o n e m e d i u m l i m i t i n g w i t h r e s p e c t t o p h o s p h a t e a n d f o l l o w e d c h a n g e s i n t h e c u l t u r e o v e r a . p e r i o d o f f i v e t o s e v e n h o u r s a f t e r i n o c u l a t i o n . I n t h e p e r i o d f o l l o w i n g t h e p h o s p h a t e e x h a u s t i o n o f t h e m e d i u m ( a t a p p r o x i m a t e l y t h r e e h o u r s ) o p t i c a l d e n s i t y w a s n o t e d t o i n c r e a s e l i n e a r l y . C h e m i c a l a n a l y s e s p e r f o r m e d o n a l i q u o t s o f c u l t u r e r e v e a l e d a d o u b l i n g i n t h e l e v e l s o f p r o t e i n a n d d e o x y r i b o n u c l e i c a c i d (DNA) a n d a 20 t o 30 p e r c e n t d e c r e a s e i n t h e a m o u n t o f r i b o n u c l e i c a c i d ( R N A ) p e r m l o f c u l t u r e . V i a b l e c o u n t s w e r e s h o w n t o i n c r e a s e 5 t o 6 f o l d d u r i n g t h i s p e r i o d , a n d DNA, RNA a n d p r o t e i n n i t r o g e n p e r c e l l w e r e c a l c u l a t e d t o b e o n e t h i r d , o n e t e n t h a n d o n e t h i r d , r e s p e c t i v e l y , o f t h e l e v e l s f o u n d i n c e l l s i n t h e l o g a r i t h m i c s t a g e o f g r o w t h . E l e c t r o n m i c r o s c o p y s h o w e d t h a t t h e c e l l s a t t h i s p o i n t h a d a s s u m e d a s m a l l e r , s p h e r i c a l s h a p e . B y u s i n g a n i n o c u l u m g r o w n i n t h e 32 p r e s e n c e o f P a n d b y f o l l o w i n g t h e l a b e l i n t h e v a r i o u s c e l l f r a c t i o n s , i t w a s s h o w n t h a t p h o s p h o r u s c o n t i n u e d t o b e i n c o r p o r a t e d i n t o DNA i n t h e p e r i o d f o l l o w i n g p h o s p h a t e e x h a u s t i o n o f t h e m e d i u m , e v i d e n t l y a t t h e e x p e n s e o f - 6 -RNA p h o s p h a t e . A s l i g h t i n c r e a s e i n a c i d s o l u b l e - P 3 2 a n d m e d i u m - P a l s o o c c u r r e d . U l t r a c e n t r i f u g a l a n d e l e c t r o p h o r e t i c a l p a t t e r n s o f c e l l f r e e e x t r a c t s ( t h e f o r m e r d e s c r i b e d b y H o r i u c h i , H o r i u c h i a n d M i z u n o , 1959) w e r e s a i d t o s u g g e s t t h e p r e f e r e n t i a l u t i l i z a t i o n o f RNA f r o m 4 0 S a n d 29S p a r t i c l e s r a t h e r t h a n t h a t o f t h e s m a l l e r 8 S a n d 5S p a r t i c l e s . C h l o r a m p h e n i c o l w a s f o u n d t o c o n s e r v e t h e RNA a n d a r s e n a t e t o s t i m u l a t e i t s d e g r a d a t i o n . I n v i e w o f t h e o b s e r v e d i n c r e a s e i n p r o t e i n d u r i n g t h e p h o s p h a t e d e f i c i e n t p h a s e , H o r i u c h i t e s t e d t h e a b i l i t y o f t h e s t a r v i n g c e l l s t o e l a b o r a t e n e w e n z y m e s a n d f o u n d t h a t t h e i n d u c t i o n ° f y S - g a l a c t o s i d a s e b y m e l i b i o s e o c c u r r e d , a l t h o u g h a t a g r e a t l y r e d u c e d r a t e . I n c r e a s e s w e r e a l s o n o t e d i n t h e s p e c i f i c a c t i v i t i e s o f r i b o n u c l e a s e ( 2 t o 8 f o l d ) , p h o s p h o d i e s t e r a s e ( 2 t o 3 f o l d ) a n d p h o s p h o m o n o e s t e r a s e ( 1 0 0 f o l d ) , t h e l a t t e r b e i n g o f p a r t i c u l a r i n t e r e s t i n r e l a t i o n t o t h e w o r k t o b e p r e s e n t e d h e r e . A f u r t h e r e f f e c t o f p h o s p h a t e s t a r v a t i o n o n m i c r o o r g a n i s m s i s t h e p h e n o m e n o n o f " p o l y p h o s p h a t e o v e r p l u s " , w h i c h h a s b e e n d e s c r i b e d i n v a r i o u s f u n g i a n d b a c t e r i a a n d i s t h e a c c u m u l a t i o n o f p o l y p h o s p h a t e u p o n a d d i t i o n o f o r t t o p h o s p h a t e t o p h o s p h a t e - s t a r v e d o r g a n i s m s . T h i s p r o b l e m h a s b e e n i n v e s t i g a t e d r e c e n t l y b y H a r o l d a n d S y l v a n ( 1 9 6 3 ) a n d H a r o l d ( 1 9 6 3 a n c ^ 1 9 6 * 0 , who h a v e s t u d i e d t h e r e l a t i o n s h i p -7-o f t h e p r o c e s s o f p o l y p h o s p h a t e a c c u m u l a t i o n i n A e r o b a c t e r a e r o g e n e s t o o t h e r c e l l u l a r f u n c t i o n s s u c h a s n u c l e i c a c i d s y n t h e s i s , a n d who h a v e a t t e m p t e d t o p r o v i d e a p h y s i o l o g i c a l e x p l a n a t i o n f o r t h e p h e n o m e n o n . T h e t h r e e e n z y m e s p o l y p h o s p h a t e k i n a s e , a l k a l i n e p h o s p h a t a s e a n d p o l y p h o s p h a t a s e ( t h e f i r s t t w o b e i n g d e f i n i t e l y r e p r e s s e d b y i n o r g a n i c p h o s p h a t e ) w e r e i n d i c a t e d t o f o r m a u n i t o f g e n e t i c r e g u l a t i o n , a n d s u g g e s t e d t o c o n s t i t u t e a p h y s i o l o g i c a l u n i t c o n c e r n e d f u n c t i o n a l l y w i t h p h o s p h a t e s t o r a g e . I I . S t a r v a t i o n o f O t h e r E s s e n t i a l N u t r i e n t s a n d t h e  E f f e c t u p o n C e l l u l a r RNA T h e b r e a k d o w n o f RNA d u r i n g p h o s p h a t e s t a r v a t i o n o f E . c o l i . a s o b s e r v e d b y H o r i u c h i (1959), i s n o t p e c u l i a r t o t h i s p a r t i c u l a r s i t u a t i o n , b u t may b e a s s o c i a t e d , i n b a c t e r i a , w i t h t h e c e s s a t i o n o f g r o w t h c a u s e d b y d i v e r s e f a c t o r s . C o n d i t i o n s w h i c h p e r m i t t h e r e s y n t h e s i s o f RNA f r o m t h e d e g r a d a t i o n p r o d u c t s , a n d w h i c h a l l o w t h e b a l a n c i n g o f t h e c a t a b o l i c a n d a n a b o l i c r e a c t i o n s , s u p p o r t " t u r n o v e r " , w h i c h i s c o n s i d e r e d t o b e a c h a r a c t e r i s t i c o f s t a t i o n a r y p h a s e p o p u l a t i o n s ( M a n d e l -s t a m , i 9 6 0 ) . M a n d e l s t a m a n d H a l v o r s o n ( i 9 6 0 ) , f o r e x a m p l e , r e p o r t e d a n e t t r a n s f e r o f RNA f r o m t h e r i b o s o m e s t o t h e s o l u b l e f r a c t i o n d u r i n g n i t r o g e n s t a r v a t i o n , a n d s u g g e s t e d - 8 -t h a t r i b o s o m a l d e g r a d a t i o n , n o t f u l l y b a l a n c e d b y r e -s y n t h e s i s , s u p p l i e d a l m o s t h a l f o f t h e a m i n o a c i d s a n d v i r t u a l l y a l l o f t h e r i b o n u c l e o t i d e s p a s s i n g t h r o u g h t h e f r e e p o o l s d u r i n g s t a r v a t i o n . T u r n o v e r o f t h e p r o t e i n i n b o t h r i b o s o m a l a n d s o l u b l e f r a c t i o n s w a s a l s o i n d i c a t e d . T h a t r i b o s o m a l m a t e r i a l may s e r v e a s a c a r b o n s o u r c e a s w e l l w a s s u g g e s t e d b y a d e c r e a s e i n p e n t o s e l e v e l c o n c u r r e n t l y w i t h t h e d e g r a d a t i o n o f e n d o g e n o u s RNA i n A . a e r o g e n e s ( S t r a n g e , D a r k a n d N e s s , 1961). G o l d s t e i n , B r o w n a n d G o l d s t e i n ( i 9 6 0 ) , s t u d y i n g t h e e f f e c t s o f l e u c i n e s t a r v a t i o n o n a n E . c o l i a u x o t r o p h , n o t e d t h e a p p e a r a n c e o f f r e e h y p o x a n t h i n e a n d u r a c i l a n d s ome n u c l e o s i d e d i - a n d t r i p h o s p h a t e s i n t h e a c i d s o l u b l e f r a c t i o n o f t h e c e l l s . T h e r e s u l t s w e r e s u g g e s t e d t o r e p r e s e n t g e n e r a l s e c o n d a r y e f f e c t s o f a b l o c k a g e o f RNA s y n t h e s i s . I n a s u b s e q u e n t p a p e r o n t h e s ame t o p i c , G o l d s t e i n a n d B r o w n ( i960) r e p o r t e d t h a t a l t h o u g h n e t s y n t h e s i s o f b o t h p r o t e i n a n d RNA h a d c e a s e d , i s o t o p i c 32 e x p e r i m e n t s w i t h P i n d i c a t e d t h a t a t u r n o v e r o f RNA n u c l e o t i d e s d i d i n f a c t t a k e p l a c e . I n a p y r i m i d i n e d e f i c i e n t m u t a n t s t u d i e d b y B a r n e r a n d C o h e n (1958)» t u r n o v e r w a s i n d i c a t e d b y t h e i s o l a t i o n o f u r i d i n e c o n t a i n i n g l a b e l l e d r i b o s e a f t e r 90 m i n u t e s o f - 9 -p y r i m i d i n e s t a r v a t i o n i n t h e p r e s e n c e o f G - g l u c o s e . A r e c y c l i n g o f u r a c i l w a s t h e r e f o r e i n d i c a t e d . E v i d e n c e f o r t h e s t a b i l i t y o f b o t h r i b o s o m a l p r o t e i n a n d r i b o s o m a l RNA d u r i n g u r a c i l s t a r v a t i o n w a s p r e s e n t e d b y N a k a d a a n d S m i t h (1962). U s i n g a u r a c i l -d e f i c i e n t m u t a n t o f E . c o l i , t h e s e w o r k e r s d e t e c t e d t h e 14 i n c o r p o r a t i o n o f l a b e l f r o m C - a d e n l n e i n t o m e s s e n g e r RNA a n d i n t o t h e s o l u b l e f r a c t i o n b y s u c r o s e g r a d i e n t c e n t r i f u g a t i o n o f c r u d e e x t r a c t s o f c e l l s e x p o s e d t o t h i s c o m p o u n d d u r i n g u r a c i l s t a r v a t i o n . L i t t l e , i f a n y , i n c o r p o r a t i o n i n t o r i b o s o m a l RNA w a s i n d i c a t e d . T u r n o v e r o f r i b o s o m a l p r o t e i n w a s a l s o s a i d t o b e a b s e n t , o n t h e 14 g r o u n d s t h a t 60 m i n u t e s ' e x p o s u r e t o L - l - C - l e u c i n e d u r i n g u r a c i l s t a r v a t i o n g a v e i n c o r p o r a t i o n o n l y i n t o p r o t e i n w h i c h c o u l d b e d e t a c h e d f r o m t h e r i b o s o m e s b y c e n t r i f u g a t i o n i n a c e s i u m c h l o r i d e g r a d i e n t , a n d w h i c h w a s t h e r e f o r e s u g g e s t e d t o b e n e w l y f o r m e d p r o t e i n a t t a c h e d l o o s e l y t o t h e r i b o s o m a l s u r f a c e . T h e same r e s u l t s w e r e o b t a i n e d w h e n a m e t h i o n i n e - d e f i c i e n t m u t a n t w a s s u p p l i e d w i t h l a b e l l e d l e u c i n e . T h e i n d i c a t e d s t a b i l i t y o f r i b o s o m a l m a t e r i a l w a s c o n t r a r y t o t h e r e s u l t s o f M a n d e l s t a m a n d H a l v o r s o n ( i960) c i t e d p r e v i o u s l y , a l t h o u g h i t w a s s u g g e s t e d t h a t t h e d i s c r e p a n c y m i g h t a r i s e f r o m t h e d i f f e r e n t c o n d i t i o n s -10-of s t a r v a t i o n used or from the p u r i f i c a t i o n of ribosomes i n the cesium c h l o r i d e g r a d i e n t . The general phenomenon of turnover of RNA and p r o t e i n has a l s o been described i n E. c o l i i n the s u l f u r -d e f i c i e n t s t a t e (Anraku, Andoh and Mizuno, 1963). This type of s t a r v a t i o n might be expected to have the same general e f f e c t s as amino a c i d or n i t r o g e n s t a r v a t i o n , i n t h a t net production of the s u l f u r - c o n t a i n i n g amino a c i d s i s prevented. In the three hours f o l l o w i n g s u l f u r exhaustion, RNA and p r o t e i n were found to be degraded by 10 and 5 per cent r e s p e c t i v e l y , although the continuous 14 14 i n c o r p o r a t i o n of 2- C - u r a c i l and 1- C-leucine i n d i c a t e d simultaneous syn t h e s i s of both to occur a t a l e v e l not b a l a n c i n g degradation. The RNA of sedimentation c o e f f i c i e n t 4S to 16S was s a i d to be most a c t i v e i n the observed t u r n -over. The l a s t example of a s p e c i f i c n u t r i e n t d e p r i v a t i o n t o be discussed here i s that of magnesium s t a r v a t i o n of E. c o l i described by McCarthy (1962). Incubation of washed 32 e x p o n e n t i a l l y growing c e l l s i n a g l u c o s e - s a l t s - P i medium l a c k i n g only magnesium r e s u l t e d i n an i n i t i a l l o s s of the 100S and 70S ribosomes, with only a small 70S peak remaining a f t e r s i x hours. By twenty hours, both 50S and 30S peaks -11-had disappeared, and i s o t o p i c d i l u t i o n with a J P - l a b e l l e d e x t r a c t of exponential c e l l s showed the ribosome content to be only 5 per cent of that of exponential c e l l s . The absorbancy of the magnesium starved c u l t u r e a t 650 mu and the number of v i a b l e counts were shown to increase during the f i r s t three hours of s t a r v a t i o n and then to remain constant f o r a t l e a s t 40 hours, i n d i c a t i n g the a b i l i t y of the c e l l s to s u r v i v e the almost t o t a l l o s s of ribosomal s t r u c t u r e . On r e a d d i t i o n of magnesium a f t e r 24 hours of s t a r v a t i o n , growth was shown to resume almost immediately a t a low but s t e a d i l y i n c r e a s i n g r a t e , reaching the exponen-t i a l . stage w i t h a normal generation time a f t e r f i v e to s i x hours. The number of ribosomes i n the c u l t u r e was shown to increase e x p o n e n t i a l l y w i t h the normal generation time a f t e r a l a g of only one hour and to reach the normal l e v e l a t the time that the increase i n absorbancy becomes e x p o n e n t i a l . S c h l i e r e n photographs showed an i n i t i a l b u ildup of 50S and 30S p a r t i c l e s , f o llowed by the appearance of 70S m a t e r i a l . P r o t e i n s y n t h e s i s was shown t o be a p p r o x i -mately p r o p o r t i o n a l t o the ribosomal content of the c e l l s (a r e l a t i o n s h i p demonstrated a l s o by K e n n e l l and Magasanik, 1962) and to be d i r e c t e d f o r the f i r s t two or three hours toward a b u i l d i n g up of ribosomal p r o t e i n . The d i s s o c i a t i o n -12-o f r i b o s o m a l p r o t e i n f r o m RNA d u r i n g s t a r v a t i o n w a s i n d i c a t e d b y t h e c h r o m a t o g r a p h i c b e h a v i o r o f t h e d e g e n e r a t e r i b o s o m a l m a t e r i a l . T h e RNA w a s a p p a r e n t l y d e g r a d e d v e r y s l o w l y ( w h i c h w a s s u r p r i s i n g i n t h a t l i b e r a t i o n o f r i b o n u c l e a s e f r o m r i b o s o m e s n o r m a l l y o c c u r s i n E . c o l i a t c o n c e n t r a t i o n s o f m a g n e s i u m b e l o w 3 x 10 ^M) a n d w a s i n d i c a t e d n o t t o b e r e u t i l i z e d w h e n m a g n e s i u m w a s a g a i n s u p p l i e d . T h e e n d o g e n o u s u t i l i z a t i o n o f RNA u n d e r c o n d i t i o n s o f s t a r v a t i o n i n b u f f e r i s a l s o w e l l d o c u m e n t e d . I n c e r t a i n o r g a n i s m s , i n c l u d i n g P . a e r u g i n o s a , r i b o s o m a l RNA i s i n d i -c a t e d t o b e o n e o f t h e p r i m a r y e n d o g e n o u s s u b s t r a t e s ( G r o n l u n d a n d C a m p b e l l , 1963). T h e m e c h a n i s m s b y w h i c h s u c h r i b o s o m a l d e g r a d a t i o n m i g h t o c c u r w e r e s t u d i e d b y Wade ( I96I a n d 1964) who d e s c r i b e d t w o p a t h w a y s o p e r a t i v e i n E . c o l i . O n e , t h e M r o u t e , w a s d e p e n d e n t u p o n t h e p r e s e n c e o f m a g n e s i u m i o n s a n d s t i m u l a t e d b y i n o r g a n i c p h o s p h a t e , a n d w a s f u r t h e r i n d i c a t e d b y t h e n u c l e o s i d e 5 * -p h o s p h a t e s f o r m e d t o r e s u l t f r o m p o l y n u c l e o t i d e p h o s p h o r y l a s e a c t i v i t y . T h e o t h e r , t h e V r o u t e , w a s o p e r a t i v e i n a n e x c e s s o f e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ( E D T A ) a n d g a v e n u c l e o s i d e 2 ' , 3 , - c y c l i c p h o s p h a t e s w h i c h c o u l d b e h y d r o l y z e d t o 3 ' - p h o s p h a t e s a n d w h i c h s u g g e s t e d t h e a c t i v i t y o f a r i b o -n u c l e a s e . I n t h e c a s e o f P. a e r u g i n o s a , G r o n l u n d a n d C a m p b e l l -13-(1963) noted an i n h i b i t o r y e f f e c t of EDTA on the release of 260-absorbing, cold perchloric a c i d (PCA) soluble material from ribosomes incubated i n tris(hydroxymethyl)-aminomethane ( t r i s ) buffer and ruled out the operation of a ribonuclease i n t h i s organism. A strong stimulatory e f f e c t of inorganic phosphate implicated polynucleotide phosphorylase, previously demonstrated i n the ribosomal f r a c t i o n by Strasdine, Hogg and Campbell (1962), to be the degradative enzyme involved. Ribosomal material would seem, i n fa c t , to be an i d e a l endogenous source of both energy and such es s e n t i a l nutrients as carbon, nitrogen, s u l f u r , phosphorus, nucleic a c i d bases and amino acids to enable the c e l l to withstand the lack of an exogenous supply of metabolites. I I I . The Formation and Properties of Repressible  Phosphomonoesterases i n Bacteria 1. The formation of the enzyme The formation of a repressible, non-specific phosphomonoesterase i n various strains of E. c o l i grown under conditions of phosphate l i m i t a t i o n was discovered independently by Horiuchi (1959) and T o r r i a n i ( i 9 6 0 ) . The enzyme was noted by both authors to be strongly i n h i b i t e d by inorganic phosphate, and was suggested to provide an example of the "negative feedback" phenomenon. The enzyme d i f f e r e d from the constitutive a c i d phosphatase - 1 4 -p r e s e n t i n t h a t t h e f o r m a t i o n o f t h e l a t t e r w a s n o t i n f l u e n c e d b y t h e l e v e l o f p h o s p h a t e p r e s e n t i n t h e m e d i u m . T h e a c i d p h o s p h a t a s e w a s a l s o l e s s s t r o n g l y i n h i b i t e d b y i n o r g a n i c p h o s p h a t e , b u t w a s s e n s i t i v e t o s o d i u m f l u o r i d e , w h i c h h a d n o e f f e c t o n t h e a c t i v i t y o f t h e a l k a l i n e e n z y m e ( T o r r i a n i , i 9 6 0 ) . B o t h H o r i u c h i (1959) a n d T o r r i a n i ( i960) s t u d i e d t h e k i n e t i c s o f f o r m a t i o n o f t h e a l k a l i n e p h o s p h a t a s e a n d f o u n d t h e e n z y m e t o a p p e a r a t t h e t i m e t h e p h o s p h a t e i n t h e m e d i u m w a s e x h a u s t e d . E n z y m e f o r m a t i o n q u i c k l y c e a s e d w h e n p h o s p h a t e w a s a d d e d t o t h e s t a r v i n g c u l t u r e , a n d a c t i v i t y p e r m l o f c u l t u r e r e m a i n e d c o n s t a n t t h e r e a f t e r . H o r i u c h i (1959) r e p o r t e d t h a t t h e f o r m a t i o n o f t h e e n z y m e w a s i n h i b i t e d b y c h l o r a m p h e n i c o l , i n d i c a t i n g t h a t a n e w p r o t e i n s y n t h e s i s w a s i n v o l v e d . G a r e n a n d L e v i n t h a l (1960) s t a r v e d c e l l s o f p h o s p h a t e i n a m e d i u m c o n t a i n i n g 1 4 C - p r o l i n e a n d d e m o n s t r a t e d t h e c o i n c i d e n c e o f p e a k s o f e n z y m e a c t i v i t y a n d r a d i o a c t i v i t y w h e n h i g h s p e e d s u p e r -n a t a n t s o f c r u s h e d c e l l s w e r e s u b j e c t e d t o c o l u m n c h r o m a -t o g r a p h y . T h e l a t t e r a u t h o r s c a l c u l a t e d t h a t a b o u t 6 p e r c e n t o f t h e t o t a l p r o t e i n s y n t h e s i z e d d u r i n g t h e s t a r v a t i o n p e r i o d w a s a l k a l i n e p h o s p h a t a s e p r o t e i n . T h e s y n t h e s i s o f t h e e n z y m e w a s a l s o d e m o n s t r a t e d t o o c c u r i n a n i n v i t r o s y s t e m d e r i v e d f r o m B a c i l l u s s u b t i l i s ( W h i t e l e y a n d O i s h i , 1 9 6 3 ) , -15-a n d w a s s h o w n t o b e p a r t i a l l y i n h i b i t e d b y c o m p o u n d s k n o w n t o i n t e r f e r e w i t h p r o t e i n s y n t h e s i s o r g e n e e x p r e s s i o n . R i b o n u c l e a s e w a s s a i d t o b e p a r t i c u l a r l y e f f e c t i v e i n b l o c k i n g s y n t h e s i s . 2. T h e r e p r e s s i o n o f e n z y m e s y n t h e s i s T h e n o r m a l r e p r e s s i o n o f a l k a l i n e p h o s p h a t a s e s y n t h e s i s i n E . c o l i w a s i n d i c a t e d b y E c h o l s , G a r e n , G a r e n a n d T o r r i a n i ( I96I) t o b e u n d e r t h e c o n t r o l o f a n o p e r a t o r g e n e a n d t w o r e g u l a t o r g e n e s . A c o n s t i t u t i v e m u t a t i o n i n e i t h e r w a s s a i d t o p r e v e n t r e p r e s s i o n , a l l o w i n g t h e c e l l t o p r o d u c e a l k a l i n e p h o s p h a t a s e i n t h e p r e s e n c e o f p h o s p h a t e . G a l l a n t a n d S t a p l e t o n (1964a), e m p l o y i n g a m u t a n t o f E . c o l i i n w h i c h r e p r e s s i o n v a r i e d i n v e r s e l y w i t h t e m p e r a t u r e , d e m o n s t r a t e d t h a t t h e s y n -t h e s i s o f t h e r e p r e s s o r s u b s t a n c e w a s d e p e n d e n t o n t h e p r e s e n c e o f a r e q u i r e d a m i n o a c i d , w a s b l o c k e d b y c h l o r a m p h e n i c o l a n d p r o c e e d e d i n t h e p r e s e n c e o f 5 - f l u o r o u r a c i l ( w h i c h i n h i b i t s RNA s y n t h e s i s ) a n d t h e a b s e n c e o f t h y m i n e ( n e e d e d f o r DNA s y n t h e s i s ) . T h e y . c o n c l u d e d t h a t t h e r e p r e s s o r w a s a p r o t e i n w h o s e r a t e o f s y n t h e s i s v a r i e d i n v e r s e l y w i t h t e m p e r a t u r e . T h e c o - r e p r e s s o r , p h o s p h a t e , w a s s u g g e s t e d t o a c t b y a l l o w i n g t h e s y n t h e s i s o f a m e t a b o l i c a l l y u n s t a b l e p r o t e i n r e p r e s s o r t o p r o c e e d . I n a n o t h e r p a p e r , G a l l a n t a n d S t a p l e t o n (1964b) -16-d e s c r i b e d t h e d e r e p r e s s i o n o f E . c o l i a l k a l i n e p h o s p h a t a s e b y g r o w t h i n a m e d i u m c o n t a i n i n g e x c e s s p h o s p h a t e p l u s c h l o r a m p h e n i c o l o r c a n a v a n i n e . T h e s e c o m p o u n d s i n h i b i t e d g e n e r a l p r o t e i n s y n t h e s i s b y 50 VeT c e n t a t t h e c o n c e n -t r a t i o n s u s e d , w h i l e a l k a l i n e p h o s p h a t a s e s y n t h e s i s i n c r e a s e d s e v e r a l f o l d . One o r b o t h o f t h e c o m p o u n d s w a s s u g g e s t e d t o i n h i b i t r e p r e s s o r s y n t h e s i s p r e f e r e n t i a l l y . C a n a v a n i n e w a s f o u n d t o b e t h r e e t i m e s m o r e e f f e c t i v e , a n d a s i t i s k n o w n t o i n h i b i t a r g i n i n e i n c o r p o r a t i o n i t w a s s u g g e s t e d t h a t t h e r e p r e s s o r p r o t e i n w a s r i c h i n t h i s a m i n o a c i d . 3 . G e n e t i c s t u d i e s T h e p r o d u c t i o n o f a l k a l i n e p h o s p h a t a s e a c t i v i t y i n E . c o l i u n d e r f a v o r a b l e c o n d i t i o n s h a s a t t r a c t e d t h e i n t e r e s t o f g e n e t i c i s t s , who h a v e r e a l i z e d i t s i m p o r t a n c e a s " a n e x p r e s s i o n o f a r e p r e s s i b l e g e n e i n a g e n e t i c a l l y w e l l - m a p p e d o r g a n i s m " ( H u m m e l a n d K a l n i t s k y , 1964). C o m p l e m e n t a t i o n s t u d i e s h a v e d e m o n s t r a t e d t h e o c c u r r e n c e o f h y b r i d p r o t e i n f o r m a t i o n i n E . c o l i b o t h i n v i t r o ( S c h l e s i n g e r a n d L e v i n t h a l , 1963) a n d i n v i v o ( G a r e n a n d G a r e n , 1963). I n t e r g e n e r i c h y b r i d i z a t i o n o f a l k a l i n e p h o s p h a t a s e f r o m E . c o l i a n d S e r r a t i a m a r c e s c e n s h a s a l s o b e e n d e m o n s t r a t e d t o o c c u r i n v i v o a n d i n v i t r o ( L e v i n t h a l , S i g n e r a n d F e t h e r o l f , 1962). - 1 7 -4 . M e c h a n i s m o f a c t i o n T h e m e c h a n i s m o f a c t i o n o f t h e n o n s p e c i f i c p h o s p h o m o n o e s t e r a s e s i n g e n e r a l i s k n o w n t o p r o c e e d b y a c l e a v a g e o f t h e P - 0 b o n d w h i c h i s t h o u g h t t o f o l l o w a t t a c h m e n t o f t h e p h o s p h o r y l g r o u p o f t h e s u b s t r a t e t o t h e e n z y m e ( S c h m i d t a n d L a s k o w s k i , 1961). T h e i n c o r -p o r a t i o n o f p h o s p h a t e i n t o E . c o l i a l k a l i n e p h o s p h a t a s e a t pH 5 t o 6, w h e r e t h e e n z y m e i s i n a c t i v e , h a s b e e n d e m o n s t r a t e d b y v a r i o u s w o r k e r s ( A l d r i d g e , B a r m a n a n d G u t f r e u n d , 1964; P e g r e t t i a n d M i l s t e i n , I 9 6 5 ) . 3 2 P - l a b e l l e d p e p t i d e s h a v e b e e n i s o l a t e d b y i o n o p h o r e s i s o f e n z y m i c a n d a c i d h y d r o l y s a t e s ( M i l s t e i n , 1964). T h e i n c o r p o r a t i o n o f m o r e t h a n o n e m o l e o f p h o s p h a t e p e r m o l e o f e n z y m e , a n d t h e a p p a r e n t a t t a c h m e n t o f p h o s p h a t e t o s e r i n e r e s i d u e s w i t h a common s e q u e n c e , s u g g e s t e d t h e p r e s e n c e o f t w o i d e n t i c a l a c t i v e s e r i n e r e s i d u e s p e r m o l e c u l e o f a c t i v e e n z y m e ( P e g r e t t i a n d M i l s t e i n , 1965). T h e r a t e o f f o r m a t i o n o f t h e p h o s p h o r y l e n z y m e a n d o f i t s d e c o m p o s i t i o n a t a l k a l i n e p H v a l u e s w a s s u g g e s t e d t o s u p p o r t t h e i d e a o f a t r a n s i e n t p h o s p h o r y l a t i o n o f t h e e n z y m e ( A l d r i d g e , B a r m a n a n d G u t f r e u n d , 1964). A n a f f i n i t y o f o r t h o p h o s p h a t e f o r t h e a c t i v e s i t e h a d b e e n p o s t u l a t e d b y G a r e n a n d L e v i n t h a l ( i960) t o e x p l a i n a p r o t e c t i v e e f f e c t o f p h o s p h a t e a g a i n s t E DTA i n h i b i t i o n . -18-5. S p e c i f i c i t y T h e n o n s p e c i f i c n a t u r e o f t h e a l k a l i n e p h o s p h a t a s e a c t i v i t y i s i n d i c a t e d b y t h e w i d e v a r i e t y o f s u b s t r a t e s a t t a c k e d . T o r r i a n i ( i 9 6 0 ) c o m p a r e d t h e s p e c i f i c i t i e s o f t h e a c i d a n d a l k a l i n e p h o s p h a t a s e s o f E . c o l i a n d r e p o r t e d t h e f o r m e r t o d i s p l a y a f a i r l y h i g h s p e c i f i c i t y f o r t h e h e x o s e p h o s p h a t e s , w h i l e t h e l a t t e r w a s a c t i v e a g a i n s t a d e n o s i n e m o n o p h o s p h a t e (AMP) a n d ^ - g l y c e r o p h o s -p h a t e a s w e l l . No a c t i v i t y o f a l k a l i n e p h o s p h a t a s e w a s s e e n a g a i n s t t h e p h o s p h o d i e s t e r b i s - j p _ - n i t r o p h e n y l p h o s p h a t e ( b i s - N P P ) o r a g a i n s t a d e n o s i n e t r i p h o s p h a t e ( A T P ) . G a r e n a n d L e v i n t h a l ( i 9 6 0 ) i n v e s t i g a t e d a w i d e r r a n g e o f s u b s t r a t e s a n d r e p o r t e d t h e e n z y m e t o b e i n a c t i v e a g a i n s t A T P , s o d i u m p y r o p h o s p h a t e a n d c r e a t i n e p h o s p h a t e . I n a m o r e r e c e n t r e p o r t , H e p p e l , H a r k n e s s a n d H i l m o e (I962) r e p o r t e d t h a t n u c l e o s i d e p o l y p h o s p h a t e s , p y r o p h o s p h a t e a n d s h o r t c h a i n p o l y m e t a p h o s p h a t e s w e r e c l e a v e d b y t h e e n z y m e , a n d p r e s e n t e d e v i d e n c e t h a t AMP, p y r o p h o s p h a t e a n d A T P w e r e a c t e d u p o n b y a s i n g l e e n z y m e . T h e y s h o w e d s i m i l a r M i c h a e l i s c o n s t a n t s a n d pH o p t i m a f o r t h e a c t i v i t y a g a i n s t e a c h s u b s t r a t e , a n d d e m o n s t r a t e d t h a t t h e r e w a s n o s u m m a t i o n o f r a t e s o f p h o s p h a t e r e l e a s e w h e n s a t u r a t i n g c o n c e n t r a t i o n s o f AMP a n d A T P w e r e m i x e d . A d d i t i o n a l e v i d e n c e w a s p r o v i d e d b y t h e p a r a l l e l r e c o v e r y o f a c t i v i t y a g a i n s t AMP, p y r o p h o s p h a t e -19-a n d A T P . T h e a l k a l i n e p h o s p h a t a s e w a s a l s o e f f e c t i v e i n r e m o v i n g t h e t e r m i n a l p h o s p h a t e f r o m v a r i o u s o l i g o n u c l e o t i d e s , a n d h a s b e e n u s e d f o r t h i s p u r p o s e i n s t u d i e s o f n u c l e o t i d e s e q u e n c e . 6 . S t a b i l i t y T h e a l k a l i n e p h o s p h a t a s e a c t i v i t y o f E . c o l i i s k n o w n t o b e s t a b l e b o t h t o c o l d s t o r a g e a n d t o h e a t i n a c t i v a t i o n . T o r r i a n i ( i960) r e p o r t e d o n l y 50 p e r c e n t l o s s o f a c t i v i t y i n t o l u e n e - l y s e d c e l l s u s p e n s i o n s a f t e r 5 m i n u t e s a t 100 C, a n d G a r e n a n d L e v i n t h a l ( i 9 6 0 ) m a i n t a i n e d t h e c r u d e e n z y m e a t 85 C f o r a t l e a s t 30 m i n u t e s w i t h o u t l o s s o f a c t i v i t y . A l t h o u g h t h e p u r i f i e d e n z y m e i s u n s t a b l e a t h i g h t e m p e r a t u r e s , s t a b i l i t y c o m p a r a b l e t o t h a t o f t h e c r u d e e x t r a c t i s c o n f e r r e d b y t h e a d d i t i o n o f 0 . 0 1 M MgCl2 o r MgSO/j, t o t h e b u f f e r m e d i u m ( G a r e n a n d L e v i n t h a l , I 9 6 O ; H e p p e l e_t a l , 1962). T h e l a t t e r w o r k e r s r e p o r t e d a n a d d i t i o n a l p r o t e c t i v e e f f e c t w h e n 0 . 0 2 M p h o s p h a t e w a s a d d e d w i t h M g C l 2 , a n d a d e c r e a s e i n s t a b i l i t y i n t h e p r e s e n c e o f 0 . 1 o r 0.5M N a C l o r 0 . 8 M K C 1 . T h e r a t e o f r e c o v e r y f r o m h e a t d e n a t u r a t i o n w a s f o u n d t o v a r y w i t h t h e p e r i o d o f e x p o s u r e a n d w i t h r e c o v e r y t e m p e r a t u r e , r e a c h i n g a p l a t e a u a t 25 C. R e c o v e r y may b e e x p l a i n e d b y a r e u n i o n o f t h e t w o s u b u n i t s t o f o r m t h e e n z y m i c a l l y a c t i v e d i m e r . - 2 0 -7. T h e e f f e c t o f v a r i o u s c o m p o u n d s o n a c t i v i t y T h e e f f e c t o f v a r i o u s c o m p o u n d s o n t h e a c t i v i t y o f t h e e n z y m e was s t u d i e d b y H e p p e l e t a l (1962). R e v e r s i b l e i n a c t i v a t i o n w a s c a u s e d b y c a d m i u m , u r e a a n d c y a n i d e . S o d i u m f l u o r i d e was f o u n d t o s t i m u l a t e a c t i v i t y , a s d i d a n u m b e r o f o t h e r s a l t s ( N H ^ C l , N H ^ S O ^ , N a 2 S 0 ^ , M g S O ^ , M g C l 2 , M n C l 2 , N a C l ) a d d e d a t c o n c e n t r a t i o n s u p t o 0 . 5 M . W i l s o n , D a y a n a n d C y r (1964) a l s o o b s e r v e d a c t i v a t i o n b y s a l t s . 8 . T r a n s p h o s p h o r y l a t i o n T h e a c t i v i t y o f t h e e n z y m e i n E . c o l i i s k n o w n t o i n c r e a s e w i t h i n c r e a s i n g m o l a r i t y o f t r i s b u f f e r ( H e p p e l e t a l , 1962). T h e p h e n o m e n o n h a s b e e n s t u d i e d b y W i l s o n e_t a l (1964). T h e s e w o r k e r s s u p p o r t t h e t h e o r y t h a t a t r a n s p h o s p h o r y l a t i o n o f t r i s i s i n v o l v e d b y d a t a s h o w i n g t h e n o n - e q u i v a l e n c e o f p h o s p h a t e a n d p _ - n i t r o p h e n o l r e l e a s e d f r o m n i t r o p h e n y l p h o s p h a t e ( N P P ) , a n d b y c h r o m a -t o g r a p h i c e v i d e n c e f o r a n a n a l o g o u s p h o s p h o r y l a t i o n o f t h e s t r u c t u r a l l y - s i m i l a r c o m p o u n d e t h a n o l a m i n e . P h o s p h o -t r a n s f e r a s e a c t i v i t y i s k n o w n t o b e a r e a c t i o n w h i c h i s c a r r i e d o u t b y b o t h a c i d a n d a l k a l i n e p h o s p h o m o n o e s t e r a s e s i n t h e p r e s e n c e o f a s u i t a b l e h y d r o x y l a t e d a c c e p t o r a n d w h i c h may b e much m o r e s e l e c t i v e s t e r e o c h e m i c a l l y t h a n t h e h y d r o l a s e a c t i v i t y ( S c h m i d t a n d L a s k o w s k i , I 9 6 I ) . - 2 1 -T h e b i o l o g i c a l s i g n i f i c a n c e o f t h i s i s u n c e r t a i n i n v i e w o f t h e h i g h l e v e l o f a c c e p t o r n e e d e d ( S t a d t m a n , I 9 6 I ) . A s t i m u l a t i o n o f a l d o l a s e a c t i v i t y w a s a l s o r e p o r t e d b y d e J i m e n e z , L e e , T o r r e s a n d Sober6n (1964), who d i d n o t p r o p o s e a n y e x p l a n a t i o n f o r t h i s e f f e c t . 9. T h e l o c a t i o n o f a c t i v i t y T o r r i a n i ( i960) n o t e d t h a t w h o l e c e l l s u s p e n s i o n s o f E . c o l i d i s p l a y e d e q u a l a c t i v i t y w h e t h e r l i v i n g o r m e r t h i o l a t e - k i l l e d , a n d t h a t t h e s e g a v e t h e same a c t i v i t y a s t o l u e n e - l y s e d o r s o n i c a t e d s u s p e n s i o n s . O n l y 10 t o 15 p e r c e n t o f t h e a c t i v i t y w a s s a i d t o r e m a i n i n t h e s u p e r -n a t a n t f l u i d o f c e l l s u s p e n s i o n s a f t e r c e n t r i f u g a t i o n , i n d i c a t i n g a c l o s e a s s o c i a t i o n o f a c t i v i t y w i t h t h e c e l l s . C o m p l e m e n t a r y r e s u l t s w e r e p r e s e n t e d b y M a l a m y a n d H o r e c k e r ( I96I) who o b s e r v e d a r a p i d a n d q u a n t i t a t i v e u p t a k e o f r a d i o a c t i v i t y b y w h o l e c e l l s o f E . c o l i w i t h ^ 32 g l u c o s e - 6 - P a s a s u b s t r a t e . P h o s p h o r y l a t e d c o m p o u n d s a r e g e n e r a l l y b e l i e v e d n o t t o e n t e r t h e c e l l a s s u c h . M a l a m y a n d H o r e c k e r ( I96I) a l s o o b s e r v e d t h e q u a n t i t a t i v e l i b e r a t i o n o f a c t i v i t y i n t o t h e s u r r o u n d i n g m e d i u m w h e n t h e c e l l s w e r e c o n v e r t e d t o p r o t o p l a s t s b y l y s o z y m e t r e a t m e n t . T h i s w a s s a i d t o s u g g e s t e i t h e r a p e r i - p l a s m i c l o c a t i o n o f t h e e n z y m e o r a n a s s o c i a t i o n w i t h t h e c e l l w a l l . C a s h e l a n d F r e e s e (1964), who r e p o r t e d t h e e x c r e t i o n -22-of a l k a l i n e phosphatase by B. s u b t i l i s , suggested that the c e l l w a l l of E. c o l i was apparently capable of c o n f i n i n g p e r i - p l a s m i c enzymes, while that of B. s u b t i l i s was not. Neu and Heppel (1964a) reported the r e l e a s e of a l k a l i n e phosphatase on EDTA-lysozyme treatment of c o l i . as w e l l as on EDTA-water washing. The former treatment a l s o r e l e a s e d r i b o n u c l e a s e , while the l a t t e r r e l e a s e d only 10 per cent of t h i s a c t i v i t y . The c e l l s were shown to r e t a i n 60 to 90 per cent v i a b i l i t y during the l a t t e r treatment. Neither a l k a l i n e phosphatase nor RNA was relea s e d on i n j u r y to the c e l l w a l l , on prevention of c e l l w a l l s y n t h e s i s with p e n i c i l l i n , or on treatment of c e l l s with Dowex 50 plus lysozyme to give o s m o t i c a l l y f r a g i l e s pheroplasts. Because of c e r t a i n p a r a l l e l s between the study of the c e l l u l a r l o c a t i o n of a l k a l i n e phosphatase and r i b o -nuclease, i t i s of i n t e r e s t to consider the l a t t e r i n more d e t a i l . Neu and Heppel (1964b) reported t h a t although the ribonuclease of E. o o l i was r e l e a s e d from c e l l s under c e r t a i n c o n d i t i o n s , i t was a s s o c i a t e d s t r o n g l y w i t h the 30S ribosomes i n c e l l e x t r a c t s . Spahr and H o l l i n g w o r t h (1961) found that p u r i f i c a t i o n of a ribosomal p r e p a r a t i o n from E. c o l i by c e n t r i f u g a t i o n through a cesium c h l o r i d e s o l u t i o n d i d not remove ribonuclease a c t i v i t y from the - 2 3 -r i b o s o m e s , a n d c o n c l u d e d t h a t i t w a s n o t l i k e l y b o u n d d u r i n g i s o l a t i o n . N e u a n d H e p p e l (1964b), h o w e v e r , d e m o n s t r a t e d t h a t r i b o s o m e s c o u l d b i n d a n e x c e s s o f r i b o n u c l e a s e e v e n u n d e r d i s s o c i a t e d c o n d i t i o n s . T h e s e w o r k e r s s h o w e d t h e a p p a r e n t i n t e g r i t y o f t h e r i b o s o m e s ( a s g a u g e d b y t o t a l RNA c o n t e n t a n d b y s u c r o s e d e n s i t y -2 -4 g r a d i e n t p a t t e r n s i n 10 a n d 10 M m a g n e s i u m ) , b u t s t r e s s e d t h e n e e d f o r m o r e s e n s i t i v e c r i t e r i a o f i n t e g r i t y . T h e n e c e s s i t y o f r i b o s o m a l b r e a k d o w n o c c u r r i n g b e f o r e r e l e a s e o f t h e e n z y m e f r o m s p h e r o p l a s t s w a s r e f u t e d o n t h e g r o u n d s o f t h e r a p i d r e l e a s e s e e n a n d t h e s l o w e r , c o m p l e t e r e l e a s e f r o m i s o l a t e d r i b o s o m e s t r e a t e d w i t h E D T A . I n a n o t h e r p a p e r , N e u a n d H e p p e l (1964c) s p e c u l a t e d t h a t m o s t o f t h e e n z y m e o c c u r s i n a f r e e s t a t e i n l o g p h a s e c e l l s , a s s p h e r o p l a s t s p r e p a r e d f r o m s u c h c e l l s r e l e a s e d u p t o 90 p e r c e n t o f t h e i r t o t a l r i b o n u c l e a s e c o n t e n t . T h e y s u g g e s t e d f u r t h e r t h a t t h e f r e e e n z y m e w a s c o n c e n t r a t e d n e a r t h e c e l l s u r f a c e a n d t h a t a s m a l l f r a c t i o n o f t h e e n z y m e w a s b o u n d t o t h e r i b o s o m e s . T h e r e l e a s e o f o n l y 45 t o 60 p e r c e n t o f a c t i v i t y f r o m s t a t i o n a r y p h a s e c e l l s t r e a t e d w i t h E DTA a n d l y s o z y m e w a s s u g g e s t e d t o i n d i c a t e t h e e x i s t e n c e o f h a l f o f t h e r i b o n u c l e a s e i n a f r e e s t a t e a n d t h e a t t a c h m e n t o f t h e b a l a n c e t o t h e r i b o s o m e s . T h e e v i d e n c e o f v a r i o u s w o r k e r s -24-f o r t h e a t t a c h m e n t o f p o l y r i b o s o m e s t o t h e c y t o p l a s m i c m e m b r a n e i n v a r i o u s b a c t e r i a l s p e c i e s i s a l s o o f i n t e r e s t h e r e ( S c h l e s s i n g e r , 19&3; P f i s t e r a n d L u n d g r e n , 1964; H e n d l e r , B a n f i e l d , T a n i a n d K u f f , 1964). T h e h i g h p r o t e i n s y n t h e s i z i n g a b i l i t y o f t h e p o l y r i b o s o m e s o n t h e p r o t o p l a s t m e m b r a n e s o f B. m e g a t e r i u m w a s d i s c u s s e d b y S c h l e s s i n g e r (1963)> who p o i n t e d o u t t h a t t h e o b s e r v e d a t t a c h m e n t c o u l d b e a n a r t i f a c t o f a d s o r p t i o n o r i n c l u s i o n . W i t h r e g a r d t o t h e p h o s p h a t a s e s , i t h a s b e e n s h o w n b y S p a h r a n d H o l l i n g w o r t h (1961) t h a t t h e a c i d p h o s p h a t a s e o f E . c o l i i s a s s o c i a t e d w i t h t h e r i b o s o m a l f r a c t i o n i n c e l l f r e e e x t r a c t s . A s t h e e n z y m e i s d e s t r o y e d a t h i g h s a l t c o n c e n t r a t i o n s , i t c o u l d n o t b e s u b j e c t e d t o c e n t r i -f u g a t i o n t h r o u g h c e s i u m c h l o r i d e s o l u t i o n . T h e r i b o s o m e s w e r e , h o w e v e r , p u r i f i e d b y s i m i l a r t r e a t m e n t i n 50 p e r c e n t s u c r o s e a n d s h o w n t o r e t a i n t h e a c i d p h o s p h a t a s e a c t i v i t y ( S p a h r a n d H o l l i n g w o r t h , I 9 6 I ) . A r i b o s o m a l l o c a t i o n o f t h e a l k a l i n e p h o s p h a t a s e w o u l d a l s o b e o f i n t e r e s t i n v i e w o f t h e s u b s t r a t e s w h i c h w o u l d b e r e a d i l y a v a i l a b l e t o i t o n b r e a k d o w n o f RNA. T h e l o c a t i o n o f t h e a c t i v i t y i n a s o l i d p h a s e m i g h t a l s o b e a d v a n t a g e o u s a s a m e a n s o f p r e v e n t i n g u n n e c e s s a r y d e g r a d a t i o n o f o t h e r p h o s p h o r y l a t e d c o m p o u n d s s u c h a s h e x o s e p h o s p h a t e s . -25-I V . T h e O c c u r r e n c e o f a R e p r e s s i b l e A c i d P h o s p h o m o n o -e s t e r a s e i n Y e a s t s T h e o c c u r r e n c e o f a r e p r e s s i b l e a c i d p h o s p h o m o n o -e s t e r a s e i n y e a s t s h a s b e e n d i s c u s s e d b y a n u m b e r o f a u t h o r s . T h i s e n z y m e h a s b e e n s h o w n t o b e p r o d u c e d i n p h o s p h a t e d e f i c i e n t m e d i u m , a n d t o b e i n h i b i t e d b y p h o s p h a t e , u n l i k e t h e c o n s t i t u t i v e a l k a l i n e p h o s p h a t a s e ( W e i m b e r g a n d O r t o n , 1963). L i k e t h e r e p r e s s i b l e a l k a l i n e p h o s p h a t a s e o f b a c t e r i a , t h e e n z y m e w a s s h o w n t o p o s s e s s a l o w d e g r e e o f s u b s t r a t e s p e c i f i c i t y . A c t i v i t y o f t h e y e a s t a c i d p h o s p h a t a s e h a s b e e n s h o w n w i t h w h o l e c e l l s ( S c h m i d t e_t a l , 1963) a n d s u g g e s t e d t o b e l o c a t e d i n a c e l l c o m p a r t m e n t n e a r t h e c e l l s u r f a c e . W i t h i n t a c t c e l l s , t h e a b s o r p t i o n o f o r t h o p h o s p h a t e a n d i t s m e t a b o l i c u t i l i z a t i o n w e r e d e s c r i b e d a s o c c u r r i n g s i m u l t a n e o u s l y w i t h t h e c l e a v a g e o f t h e s u b s t r a t e . M c L e l l a n a n d L a m p e n (I963) r e p o r t e d a m u c h h i g h e r a c t i v i t y w i t h i n t a c t c e l l s t h a n w i t h p r o t o p l a s t s o r l y s e d p r o t o -p l a s t s ; t h e y a l s o d e m o n s t r a t e d t h a t w a s h e d p r o t o p l a s t s i n c u b a t e d i n a m e d i u m f r e e o f i n o r g a n i c p h o s p h a t e c o u l d p r o d u c e t h e e n z y m e , w h i c h w a s f o u n d i n t h e m e d i u m . F u r t h e r e v i d e n c e f o r a n e x o c e l l u l a r l o c a t i o n o f t h e e n z y m e w a s g i v e n b y t h e r e s u l t s o f W e i m b e r g a n d O r t o n (1964) who p r e p a r e d e n z y m i c a l l y a c t i v e c e l l w a l l s f r o m w h i c h t h e - 2 6 -enzyme could be removed by treatment with papain or s n a i l gut extract. The role of the enzyme was suggested by Weimberg and Orton (I963) to l i e i n providing a supply of phosphate to the c e l l from external sources i n the absence of inorganic phosphate. This theory was supported by Heredia, Yen and Sols ( 1 9 6 3)t who demon-strated the enzyme to be produced only i n the absence of inorganic phosphate and to allow growth at the expense of a phosphorylated carbon source. - 2 7 -M A T E R I A L S AND METHODS I . B a c t e r i o l o g i c a l M e t h o d s P s e u d o m o n a s a e r u g i n o s a A T C C 9027 w a s g r o w n i n R o u x f l a s k c u l t u r e i n a t r i s - b u f f e r e d g l u c o s e s a l t s m e d i u m o f t h e f o l l o w i n g f i n a l c o m p o s i t i o n , p r e p a r e d f r o m s e p a r a t e l y a u t o c l a v e d s o l u t i o n s a s d e s c r i b e d b y M a l l e t t e e t a l . (1964) a n d a d j u s t e d t o pH 7 .4 w i t h HC1 b e f o r e a u t o c l a v i n g : N H ^ C l 0 . 0 2 0 0 M K C 1 . 0 0 1 0 t r i s .0500 K H g P O ^ . 0 0 4 5 N a 2 H P 0 ^ .0055 g l u c o s e . 0 1 1 0 MgSOi,, . 0 0 1 6 T h e f i n a l g l u c o s e c o n c e n t r a t i o n o f t h e m e d i u m w a s 0 . 2 $ , t o c o r r e s p o n d t o t h e m e d i u m u s e d b y W a r r e n , E l l s a n d C a m p b e l l ( i 9 6 0 ) . S t o c k c u l t u r e s w e r e s t o r e d a t 4-6 C a f t e r a t w e n t y - f o u r g r o w t h p e r i o d i n t u b e c u l t u r e i n t h e m e d i u m , a n d w e r e p e r i o d i c a l l y c h e c k e d f o r p u r i t y b y m i c r o s c o p i c e x a m i n a t i o n o f s t a i n e d s l i d e s a n d b y o b s e r -v a t i o n o f c o l o n i a l f o r m a n d p y o c y a n i n p r o d u c t i o n o n g l y c e r o l p e p t o n e a g a r . - 2 8 -T h e g l u c o s e - s a l t s m e d i u m , w i t h t h e p h o s p h a t e s a l t s o m i t t e d , w a s u s e d a s a s o l u t i o n f o r w a s h i n g t h e c e l l s f r e e o f e x o g e n o u s p h o s p h a t e a n d a s t h e s u s p e n d i n g f l u i d i n s t a r v a t i o n e x p e r i m e n t s . I t i s r e f e r r e d t o a s p h o s p h a t e d e f i c i e n t m e d i u m . C e l l s t o b e u s e d f o r s t a r v a t i o n s t u d i e s w e r e h a r v e s t e d a s e p t i c a l l y f r o m t h e l i q u i d m e d i u m a f t e r 8 h o u r s a t 37 C o r 18 h o u r s a t 30 C a s i n d i c a t e d , w a s h e d f o u r t i m e s w i t h c o l d , s t e r i l e p h o s p h a t e d e f i c i e n t m e d i u m , a n d r e s u s p e n d e d i n t h e same m e d i u m . I I . A n a l y t i c a l M e t h o d s 1. P r o t e i n P r o t e i n w a s d e t e r m i n e d b y t h e m e t h o d o f L o w r y e t a l . (195D» u s i n g c r y s t a l l i n e e g g a l b u m i n ( N u t r i t i o n a l B i o c h e m i c a l C o r p o r a t i o n ) a s a s t a n d a r d . 2 . N u c l e i c a c i d s RNA w a s d e t e r m i n e d b y a m o d i f i c a t i o n o f t h e S c h n e i d e r (1957) t e c h n i q u e , u s i n g r e a g e n t g r a d e RNA ( N u t r i t i o n a l B i o c h e m i c a l C o r p o r a t i o n ) o r y e a s t RNA a s s t a n d a r d s . RNA v a l u e s w e r e c o r r e c t e d f o r t h e DNA p r e s e n t i n t h e h o t a c i d s o l u b l e f r a c t i o n . DNA w a s d e t e r m i n e d b y t h e m e t h o d o f S c h n e i d e r (1957), u s i n g c a l f t h y m u s DNA ( W o r t h i n g t o n B i o c h e m i c a l C o r p o r a t i o n ) a s a s t a n d a r d . -29-3. Inorganic phosphate Inorganic phosphate (P^) was determined by a m o d i f i c a t i o n of the method of King (1932), or by the method of Chen, T o r i b a r a and Warner (1956), the l a t t e r being used where a h i g h l y s e n s i t i v e assay i n the range of 2-20 mum of phosphate was r e q u i r e d . In the procedure of Chen et a l . c o l o u r was developed by 20 minutes' h o l d i n g a t 4-5 C. A f u r t h e r m o d i f i c a t i o n of the King assay was a l s o employed, wi t h reagent volumes being s c a l e d down to enable determinations to be made i n the range of 1-10 ug of phosphorus. Standards were prepared from d e s i c c a t e d KHgPO^. I l l . I n i t i a l Studies with Shaken C e l l Suspensions 1. P r e p a r a t i o n of c e l l s I n i t i a l s t u d i e s , undertaken i n an attempt to f o l l o w p o p u l a t i o n changes by t u r b i d i m e t r y and p l a t e counts, were c a r r i e d out using washed c e l l suspensions prepared as p r e v i o u s l y described from c u l t u r e s grown a t 37 C f o r 8 hours. C e l l concentrations were adjusted to gi v e i n i t i a l t u r b i d i t i e s a t 400 mu of approximately 0.1 when 1.0 ml of suspension was added to a 4.8 ml system of phosphate d e f i c i e n t medium plus phosphate s o l u t i o n as r e q u i r e d i n the p a r t i c u l a r experiment. - 3 0 -2. T u r b i d i m e t r y a n d p l a t e c o u n t s T u r b i d i t y d a t a r e p o r t e d f o r t h e s e e x p e r i m e n t s w e r e r e c o r d e d a t 4 0 0 mu i n a c u v e t t e o f p a t h l e n g t h 1.0 cm, u s i n g a B e c k m a n m o d e l DU s p e c t r o p h o t o m e t e r s e t f o r m a x i m u m s e n s i t i v i t y . P l a t e c o u n t s r e p o r t e d w e r e made -2 i n q u a d r u p l i c a t e o n s u s p e n s i o n s d i l u t e d i n 10 M p h o s p h a t e b u f f e r ( p H 7.2) a n d f i n a l l y i n a d i l u t i n g a g a r c o n s i s t i n g o f o n e p a r t D i f c o p l a t e c o u n t a g a r t o t w o p a r t s b r o t h o f t h e s ame c o m p o s i t i o n w i t h a g a r o m i t t e d . A n a l i q u o t o f t h i s f i n a l d i l u t i o n w a s p i p e t t e d o n t o a p r e v i o u s l y p o u r e d a n d s o l i d i f i e d b a s e l a y e r o f p l a t e c o u n t a g a r i n a p e t r i d i s h . P l a t e s w e r e t h e n i n c u b a t e d 1 8 - 2 0 h o u r s a t 37 C, a n d c o l o n i e s c o u n t e d w i t h t h e a i d o f a Q u e b e c c o l o n y c o u n t e r . 3 . P r e p a r a t i o n o f g l a s s w a r e G l a s s w a r e u s e d i n t h e e x p e r i m e n t s d e s c r i b e d w a s w a s h e d , s o a k e d o v e r n i g h t i n 10% EBOj a n d r i n s e d s e v e r a l t i m e s w i t h d i s t i l l e d w a t e r t o r e m o v e a l l t r a c e s o f i n o r g a n i c p h o s p h a t e . I V . S t u d i e s w i t h S t i l l C u l t u r e s 1. P r e p a r a t i o n o f c e l l s I n t h e s e c o n d s e r i e s o f e x p e r i m e n t s , w a s h e d c e l l s u s p e n s i o n s w e r e p r e p a r e d a t 10 t i m e s ( 1 0 X ) g r o w t h -31-c o n c e n t r a t i o n f r o m R o u x f l a s k c u l t u r e s g r o w n a t 30 C f o r 18 h o u r s . T h e 1 0 X s u s p e n s i o n s w e r e t h e n u s e d t o i n o c u l a t e R o u x f l a s k c u l t u r e s i n p h o s p h a t e d e f i c i e n t m e d i u m a t t h e l e v e l o f Jfo, e s t a b l i s h i n g a n i n i t i a l a r b i t r a r y t u r b i d i t y o f a p p r o x i m a t e l y 30$ o f t h a t o f t h e 18 h o u r c u l t u r e . T h e s t a r v i n g c u l t u r e s w e r e t h e n i n c u -b a t e d a t 30 C f o r 2 4 h o u r s . I n c e r t a i n e x p e r i m e n t s , t h e c o n t e n t s o f t h e f l a s k s o f s t a r v i n g c e l l s w e r e m e a s u r e d a n d p o o l e d a s e p t i c a l l y a t 2 4 h o u r s , a n d b a t c h e s r e t u r n e d t o t h e R o u x f l a s k s . T h e s e w e r e t h e n s u p p l i e d w i t h p h o s p h a t e _2 a t t h e l e v e l o f 10 M , a s i n t h e c o m p l e t e g r o w t h m e d i u m , a n d i n c u b a t e d a f u r t h e r 6 h o u r s a t 30 C . 2 . T u r b i d i m e t r y a n d p l a t e c o u n t s T u r b i d i t y m e a s u r e m e n t s w e r e r e c o r d e d a t 6 6 0 mu i n a c u v e t t e o f p a t h l e n g t h 1 . 0 c m , u s i n g a B e c k m a n m o d e l B s p e c t r o p h o t o m e t e r . P l a t e c o u n t s w e r e d o n e i n q u a d r u p l i c a t e o n s u s p e n s i o n s d i l u t e d i n 10 M p h o s p h a t e b u f f e r ( p H 7 . 2 ) a n d w e r e p e r f o r m e d b y t h e p o u r p l a t e t e c h n i q u e , u s i n g p e t r i d i s h e s c o n t a i n i n g a p r e v i o u s l y p o u r e d a n d s o l i d i f i e d b a s e l a y e r o f p l a t e c o u n t a g a r . P l a t e s w e r e t h e n i n c u b a t e d 2 d a y s a t 30 G . 3. C o u l t e r c o u n t s o n c e l l s u s p e n s i o n s P a r t i c l e c o u n t s w e r e d e t e r m i n e d o n a l i q u o t s o f -32-s t a r v i n g c u l t u r e d i l u t e d i n f i l t e r e d a n d f o r m a l i z e d O.9/0 s a l i n e s o l u t i o n ( R o y a l J u b i l e e H o s p i t a l , V i c t o r i a , B r i t i s h C o l u m b i a ) i n t u b e s w h i c h h a d b e e n r i n s e d w i t h d i s t i l l e d w a t e r a n d a i r d r i e d . T h e i n s t r u m e n t u s e d w a s a C o u l t e r m o d e l B e l e c t r o n i c p a r t i c l e c o u n t e r , a n d s e t t i n g s u s e d f o r t o t a l c o u n t s w e r e a s f o l l o w s : _1 a p e r a t u r e c u r r e n t 0.707 _1 a m p l i f i c a t i o n 1 u p p e r t h r e s h o l d 100 l o w e r t h r e s h o l d 1 A s a m p l e o f p h o s p h a t e d e f i c i e n t m e d i u m a t t h e s ame d i l u t i o n w a s a l s o c o u n t e d t o g i v e a b a c k g r o u n d v a l u e w h i c h w a s t h e n s u b t r a c t e d . T o o b t a i n i n f o r m a t i o n o n t h e s i z e d i s t r i b u t i o n o f t h e c e l l s , t h e s e t t i n g f o r t h e l o w e r t h r e s h o l d w a s v a r i e d b e t w e e n 1 a n d 10. 4. C h e m i c a l f r a c t i o n a t i o n o f c e l l s C o n c e n t r a t e d c e l l s u s p e n s i o n s (1-4 mg d r y w e i g h t p e r m l ) w e r e p r e p a r e d f r o m m e a s u r e d v o l u m e s o f t h e 24 h o u r p h o s p h a t e s t a r v e d c u l t u r e s h a r v e s t e d b y c e n t r i f u g a t i o n a n d m ade u p t o k n o w n v o l u m e s w i t h d i s t i l l e d w a t e r o r p h o s p h a t e d e f i c i e n t m e d i u m . N o n s t a r v e d c o n t r o l s u s p e n s i o n s w e r e p r e p a r e d i n t h e same m a n n e r f r o m f r e s h l y - i n o c u l a t e d s t a r v a t i o n c u l t u r e s , o r , i n m o s t c a s e s , t a k e n a s a l i q u o t s o f t h e o r i g i n a l w a s h e d 10X s u s p e n s i o n s u s e d a s t h e i n o c u l a -33-f o r t h e s t a r v e d c u l t u r e s . D r y w e i g h t d e t e r m i n a t i o n s w e r e made o n t h e c o n c e n t r a t e d c e l l s u s p e n s i o n s u s i n g t a r e d a l u m i n u m f o i l c u p s a n d h o l d i n g a t 105 C f o r 48 h o u r s . W h e r e a p p l i c a b l e , t h e d r y w e i g h t o f a c o r r e s -p o n d i n g v o l u m e o f t h e s u s p e n d i n g f l u i d , s i m i l a r l y d e t e r m i n e d , w a s s u b t r a c t e d . A l i q u o t s o f - c o n c e n t r a t e d s u s p e n s i o n s i n d i s t i l l e d w a t e r w e r e f r a c t i o n a t e d d i r e c t l y ; i n c a s e s w h e r e t h e s u s p e n s i o n s h a d b e e n made i n p h o s p h a t e d e f i c i e n t m e d i u m , a l i q u o t s w e r e c e n t r i f u g e d a n d t h e p e l l e t s r e s u s p e n d e d i n a k n o w n v o l u m e o f d i s t i l l e d w a t e r . I n t h e f r a c t i o n a t i o n p r o c e d u r e ( a m o d i f i c a t i o n o f H u t c h i s o n , D o w n i e a n d M u n r o , 1962) a v o l u m e o f c o n c e n t r a t e d c e l l s u s p e n s i o n (1ml) i n w a t e r w a s m i x e d w i t h a n e q u a l v o l u m e o f c o l d 1.4 N P C A , h e l d i n i c e - f o r 20 m i n u t e s a n d c e n t r i f u g e d a t 4-6 C. T h e s u p e r n a t a n t f l u i d i s r e f e r r e d t o a s t h e c o l d a c i d s o l u b l e f r a c t i o n . T h e p e l l e t w a s r e s u s p e n d e d i n 2 m l o f 0.7 N P G A , e x t r a c t e d a t 90 C f o r 15 m i n u t e s a n d c e n t r i f u g e d . T h i s s u p e r n a t a n t f l u i d i s r e f e r r e d t o a s t h e h o t a c i d s o l u b l e f r a c t i o n . T h e p e l l e t w a s r e s u s p e n d e d i n 2 m l o f 0.1 N NaOH a n d i s r e f e r r e d t o a s t h e h o t a c i d i n s o l u b l e f r a c t i o n . -34-V . S u c r o s e D e n s i t y G r a d i e n t P a t t e r n s 1. P r e p a r a t i o n o f c e l l f r e e e x t r a c t s S a m p l e s o f s t a r v e d a n d n o n s t a r v e d c e l l s , a n d c e l l s r e s u p p l i e d w i t h p h o s p h a t e w e r e h a r v e s t e d a s d e s c r i b e d p r e v i o u s l y , w a s h e d t w i c e w i t h 0.85$ N a C l ( p H 7 .4) a n d _2 r e s u s p e n d e d i n 10 M M g C l 2 a t a c o n c e n t r a t i o n o f a p p r o x i m a t e l y 20 mg d r y w e i g h t (10,000 - 12,000 u g o f p r o t e i n ) p e r m l . D e o x y r i b o n u c l e a s e ( D N a s e , 0 .05 m l a t 1 mg p e r m l ) w a s a d d e d , a n d t h e s u s p e n s i o n c r u s h e d i m m e d i a t e l y u s i n g a c h i l l e d F r e n c h p r e s s u r e c e l l ( M i l n e r , L a w r e n c e a n d F r e n c h , 1950) ' T h e e x t r a c t s w e r e o b t a i n e d b y p a s s i n g t h e m a t e r i a l d r o p w i s e t h r o u g h t h e o r i f i c e o f t h e p r e s s u r e c e l l u n d e r a p r e s s u r e o f 10,000-15,000 l b s , m a i n t a i n e d w i t h a C a r v e r h y d r a u l i c p r e s s . E x t r a c t s w e r e t h e n c e n t r i f u g e d a t 8,000 x g f o r 10 m i n u t e s a t 4-6 C t o r e m o v e w h o l e c e l l s a n d c e l l w a l l s . 2. P r e p a r a t i o n o f g r a d i e n t s S u c r o s e d e n s i t y g r a d i e n t s w e r e p r e p a r e d f r o m 2 .5 m l e a c h o f 5 a n d 20$ s u c r o s e s o l u t i o n s i n 10~ 2 M t r i s - H C l b u f f e r ( p H 7.4) c o n t a i n i n g 1 0 " 2 o r 10"^ M M g C l 2 , W t h e u s e o f a P l e x i g l a s s m i x i n g a p p a r a t u s h a v i n g t w o c h a m b e r s 1 c o n n e c t e d b y a U - t u b e ( B r i t t e n a n d R o b e r t s , I960) . T T h e a p p a r a t u s f o r t h e p r e p a r a t i o n o f g r a d i e n t s a n d t h e c o l l e c t i o n o f f r a c t i o n s w e r e made b y D r . R. S t a c e - S m i t h , C a n a d a D e p a r t m e n t o f A g r i c u l t u r e . - 3 5 -T h e g r a d i e n t s w e r e p r e p a r e d i n L u s t e r o i d t u b e s o f 5 m l c a p a c i t y , a n d w e r e c h e c k e d f o r b a l a n c e b e f o r e a d d i t i o n o f t h e c e l l f r e e e x t r a c t . A s a m p l e o f 0.2 m l o f e x t r a c t w a s l a y e r e d o n t o p o f t h e g r a d i e n t b y u s e o f a 1 m l t u b e r c u l i n s y r i n g e f i t t e d w i t h a N o . 18 n e e d l e . G r a d i e n t s c o n t a i n i n g _2 10 M M g C l 2 w e r e c e n t r i f u g e d a t 36,000 r p m f o r 90 m i n u t e s u s i n g a SW 39 r o t o r i n a S p i n c o m o d e l L p r e p a r a t i v e u l t r a -c e n t r i f u g e . T h o s e c o n t a i n i n g 10"^ M M g C l 2 w e r e c e n t r i f u g e d a t t h e same s p e e d f o r 150 m i n u t e s . F o l l o w i n g c e n t r i f u g a t i o n , t h e t u b e s w e r e p l a c e d i n a s p e c i a l l y c o n s t r u c t e d h o l d e r , a n d f r a c t i o n s o f a p p r o x i m a t e l y 0.2 m l w e r e d i s p e n s e d d r o p w i s e f r o m t h e b o t t o m o f t h e L u s t e r o i d t u b e i n t o 1 m l c e n t r i f u g e t u b e s , u s i n g a N o . 22 h y p o d e r m i c n e e d l e . T e n -f o l d d i l u t i o n s o f t h e f r a c t i o n s w e r e made i n d i s t i l l e d w a t e r , a n d t h e o p t i c a l d e n s i t y (O.D.) o f e a c h m e a s u r e d a t 260 mu w i t h a B e c k m a n m o d e l DU s p e c t r o p h o t o m e t e r . P r o t e i n v a l u e s d e t e r m i n e d o n e a c h c e l l f r e e e x t r a c t a n d t o t a l c o u n t s d e t e r m i n e d o n a s t a r v i n g c u l t u r e a t 0 a n d 2k h o u r s w e r e u s e d t o c o r r e c t t h e o p t i c a l d e n s i t y r e a d i n g s t o c o r r e s p o n d o n a p r o t e i n b a s i s o r o n a t o t a l c o u n t b a s i s t o t h o s e o b t a i n e d f o r t h e e x t r a c t p r e p a r e d f r o m u n s t a r v e d c e l l s . -36-V I . Demonstration of A l k a l i n e Phosphatase A c t i v i t y i n  E x t r a c t s of Starved C e l l s 1. P r e p a r a t i o n of c e l l f ree e x t r a c t s Concentrated suspensions were prepared from samples of sta r v e d and nonstarved c e l l s which were harvested, washed twice i n I O - 1 M t r i s - H C l b u f f e r (pH ?.4) and resuspended i n the same b u f f e r to give approximately 10,000-12,000 ug of p r o t e i n per ml. The suspensions were crushed i n the French pressure c e l l and c e n t r i f u g e d as p r e v i o u s l y o u t l i n e d . 2. Assay by re l e a s e of p-nitrophenol from p - n i t r o p h e n y l phosphate The procedure used was based on s e v e r a l found i n the l i t e r a t u r e and i n v o l v e s the l i b e r a t i o n of £-nitrophenol, which i n a l k a l i n e s o l u t i o n has a yellow c o l o u r measurable at 410 mu (Stadtman, 1961). Reaction mixtures normally contained the f o l l o w i n g : t r i s - H C l b u f f e r (1.0 M, pH 7.5 or 8.0) 0.5 ml; p - n i t r o p h e n y l phosphate (disodium s a l t , N u t r i t i o n a l Biochemical Corporation, 2 mg per ml) 0.1 ml; c e l l f r e e e x t r a c t 10-25 u l ; water to 1.0 ml. Components e x c l u s i v e of substrate were brought to 35 C i n a 1 ml cuvette set i n a water bath, and were then placed i n the warmed chamber of the Beckman model DU or DB spectr o -photometer. The machine was set to zero on the cu v e t t e , and warmed substrate added with mixing a t zero time. O p t i c a l d e n s i t y was followed a t 410 mu f o r 3 minutes. - 3 7 -A c t i v i t i e s are expressed as AO. D. per minute per aliquo t of c e l l free extract. Modifications of t h i s basic procedure were employed i n certa i n experiments and w i l l be outlined under the appropriate headings. 3 . Assay by release of inorganic phosphate from substrates This method of determining a c t i v i t y was employed when substrates other than p,-nitrophenyl phosphate were used. In preliminary experiments, phosphate release was determined by a modification of the King (1932) procedure, while i n l a t e r experiments the highly sensitive assay of Chen et al_. (195&) was employed. In these, a l l tubes used for enzyme reactions and phosphate determinations and a l l glassware used f o r preparation of substrates and reagents was freed of contaminating phosphate by auto-claving i n d i s t i l l e d water, followed by three or four rinses i n cold d i s t i l l e d water. Reaction mixtures i n the experiments to be considered contained the following: t r i s - H C l buffer (1 .0 M, pH 8 .0 at 45 C) 0.1 ml; substrate (25 ;um per ml) 20 pi; water to 0 .2 ml; c e l l free extract 10 ; i l . The substrates used were always i n excess. Components exclusive of enzyme were brought to 45 C i n small test tubes i n a water bath, and cold enzyme preparation added with mixing at zero time. - 3 8 -A l i q u o t s o f 10 t o 50 , u l w e r e r e m o v e d a t z e r o t i m e a n d a t o n e o r m o r e i n t e r v a l s t h e r e a f t e r a n d p i p e t t e d i n t o 50 pi o f 5N H 2 S 0 4 t o s t o p t h e r e a c t i o n . A t t h e e n d o f t h e e x p e r i m e n t , t h e a c i d i f i e d s a m p l e s w e r e a s s a y e d f o r i n o r g a n i c p h o s p h a t e b y t h e m e t h o d o f C h e n e t a l . (1956) . A s a n i n i t i a l r a t e s t u d y w i t h a d e n o s i n e 5 ' - m o n o p h o s p h a t e (5*-AMP o r AMP) a s s u b s t r a t e h a d d e m o n s t r a t e d t h e l i n e a r i t y o f t h e r e a c t i o n o v e r a p e r i o d o f a t l e a s t 20 m i n u t e s , s u b s e q u e n t d e t e r m i n a t i o n s w e r e l i m i t e d t o a i m i n u t e a n d a 20f m i n u t e s a m p l e . T h e v a l u e s o f A O . D., 20' s o o b t a i n e d w e r e u s e d t o c a l c u l a t e r e l a t i v e a c t i v i t i e s w i t h d i f f e r e n t s u b s t r a t e s , b a s e d o n t h e a c t i v i t y w i t h 5 '-AMP d e s i g n a t e d a s 1 .00. V I I . C h a r a c t e r i z a t i o n o f A l k a l i n e P h o s p h a t a s e A c t i v i t y 1. T h e e f f e c t o f b u f f e r c o m p o s i t i o n a n d c o n c e n t r a t i o n A c t i v i t y w a s d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y a t pH 8 . 0 u s i n g t r i s - a c e t a t e a n d t r i s - H C l b u f f e r s a t v a r y i n g m o l a r i t i e s . 2 . T h e e f f e c t o f E D T A a n d i n o r g a n i c i o n s A c t i v i t y w a s d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y a t pH 8 .0 i n t h e p r e s e n c e o f v a r i o u s c o n c e n t r a t i o n s o f E D T A , M g C l 2 , N a C l , KC1 a n d K H g P O ^ . I n t h e c a s e o f E D T A , a 20 m i n u t e p e r i o d o f p r e i n c u b a t i o n w a s a l l o w e d b e f o r e a d d i t i o n o f s u b s t r a t e t o t h e c u v e t t e . - 3 9 -3 . D e t e r m i n a t i o n o f a c t i v i t y a t v a r i o u s pH v a l u e s A c t i v i t y w a s d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y , u s i n g t r i s - H C l " b u f f e r s a t p H 7 . 0 , 7 . 5 , 8 . 0 , 8.5 a n d 9 . 0 a t 35 C. A s t h e c o l o u r o f t h e p - n i t r o p h e n o l r e l e a s e d h a s a n a b s o r p t i o n m a x i m u m i n t h e a l k a l i n e r a n g e , a s t a n d a r d s o l u t i o n o f p - n i t r o p h e n o l a t 1 urn p e r m l w a s p r e p a r e d , a n d t h e o p t i c a l d e n s i t y o f 25 u l i n 1 m l o f t r i s - H C l b u f f e r d e t e r m i n e d a t e a c h o f t h e p H v a l u e s c o n s i d e r e d t o o b t a i n a c o r r e c t i o n f a c t o r f o r A O . D. p e r m i n u t e a t e a c h pH v a l u e u s e d . 4. D e t e r m i n a t i o n o f a c t i v i t y w i t h i n c r e a s i n g t e m p e r a t u r e A c t i v i t y w a s d e t e r m i n e d b y t h e s p e c t r o p h o t o m e t r i c m e t h o d , u s i n g t r i s - H C l b u f f e r s s e t t o pH 7.5 a t 35, 4-5, 55 * 65, 70 a n d 75 C. ( A d j u s t m e n t o f p H a t t h e t e m p e r a t u r e o f a s s a y i s n e c e s s i t a t e d b y t h e h i g h t e m p e r a t u r e - s e n s i t i v i t y o f t r i s b u f f e r . ) I n t h i s e x p e r i m e n t , c u v e t t e c o n t e n t s , e x c l u s i v e o f e n z y m e , w e r e b r o u g h t t o t e m p e r a t u r e a n d t h e r e a c t i o n s t a r t e d b y t h e a d d i t i o n o f c o l d c e l l f r e e e x t r a c t . 5 . D e t e r m i n a t i o n o f a c t i v i t y a g a i n s t v a r i o u s s u b s t r a t e s A c t i v i t y a g a i n s t t h e r i b o n u c l e o s i d e m o n o - , d i - a n d t r i p h o s p h a t e s a n d a g a i n s t a n u m b e r o f m i s c e l l a n e o u s s u b s t r a t e s i n c l u d i n g N P P a n d b i s - N P P w a s d e t e r m i n e d b y t h e r e l e a s e o f i n o r g a n i c p h o s p h a t e f r o m s u b s t r a t e . A s i t w a s t h o u g h t t h a t p o l y n u c l e o t i d e p h o s p h o r y l a s e m i g h t b e c o n t r i b u t i n g -ko-to t h e r e l e a s e o f I n o r g a n i c p h o s p h a t e f r o m t h e r i b o -n u c l e o s i d e d i p h o s p h a t e s , a c h e c k f o r t h i s a c t i v i t y w a s m a d e b y p i p e t t i n g a t i n t e r v a l s a l i q u o t s o f r e a c t i o n m i x t u r e c o n t a i n i n g a d e n o s i n e d i p h o s p h a t e ( A D P ) i n t o i c e -c o l d 1.5$ P C A , c e n t r i f u g l n g t o r e m o v e a n y a c i d i n s o l u b l e p o l y m e r f o r m e d , a n d r e a d i n g 0. D. a t 260 mu. A d e c r e a s e i n a b s o r p t i o n o v e r t h e t i m e i n t e r v a l c o n s i d e r e d w o u l d i n d i c a t e t h e r e m o v a l o f a d d e d A D P a s p o l y a d e n y l i c a c i d , a n d t h e r e f o r e i n d i c a t e p o s s i b l e i n t e r f e r e n c e b y p o l y n u c l e o t i d e p h o s p h o r y l a s e . 6. D e g r e e o f i n h i b i t i o n b y i n o r g a n i c p h o s p h a t e A s some o f t h e s u b s t r a t e s e m p l o y e d c o n t a i n e d c o n s i d e r a b l e a m o u n t s o f i n o r g a n i c p h o s p h a t e a s c o n t a m i n a t i o n , a n d a s some o f t h e s e g a v e l o w e r r a t e s o f a c t i v i t y r e l a t i v e t o t h a t w i t h AMP, t h e d e g r e e o f i n h i b i t i o n r e s u l t i n g f r o m t h e a d d i t i o n o f s i m i l a r c o n c e n t r a t i o n s o f p h o s p h a t e t o s y s t e m s c o n t a i n i n g 5 '-AMP w a s d e t e r m i n e d . A c c o r d i n g l y , a s s a y s w e r e r u n i n t h e p r e s e n c e o f 0.25 a n d 0.50 urn o f KH2PO4 p e r m l . V I I I . T h e L o c a t i o n o f A l k a l i n e P h o s p h a t a s e A c t i v i t y  i n F r a c t i o n a t e d C e l l F r e e E x t r a c t s 1. P r e p a r a t i o n o f c e l l f r e e e x t r a c t s C o n c e n t r a t e d c e l l s u s p e n s i o n s a p p r o x i m a t e l y 12,000 p.g p r o t e i n p e r m l ) w e r e p r e p a r e d f r o m s t a r v e d c e l l s w a s h e d o n c e -41-i n 0.05 M t r i s - H C l buffer (pH 7.4) containing -4 10 M MgCl2» and crushed i n the French pressure c e l l as previously described. 2. Fractionation of c e l l free extracts by c e n t r i f ligation A f t e r removal of whole c e l l s , a measured volume of extract (4 ml) was centrifuged at 25,000 x g for 30 minutes (Campbell, Hogg and Strasdine, 1962) i n a S e r v a l l r e f r i g e r a t e d centrifuge. The supernatant f l u i d was re-centrifuged, and the combined "membrane" p e l l e t s were washed twice with 0.05 M t r i s - H C l (pH 7.4) and resuspended i n the same buffer. The 25,000 x g supernatant f l u i d was brought to 1 0 ~ 2 M with respect to MgCl£ to allow the ribosomes to associate as 70S p a r t i c l e s , and then c e n t r i -fuged at approximately 110,000 x g (40,000 rpm) for 2 hours i n a Spinco model L preparative u l t r a c e n t r i f u g e . The resultant "ribosome" p e l l e t was transferred to a t e f l o n Potter homogenizer and resuspended i n 0.05 M t r i s - H C l (pH 7.4) containing 10"^ M MgClg. The supernatant f r a c t i o n of the l a s t centrifugation i s referred to as the "soluble cytoplasm". At each step, the volume of material centrifuged was noted and the p e l l e t of the succeeding run washed and resuspended i n the same volume of diluent to give f i n a l concentrations comparable to that of the o r i g i n a l c e l l free extract. -42-T o c h e c k t h e p u r i t y o f t h e i n d i v i d u a l f r a c t i o n s , u l t r a v i o l e t ( U V ) s p e c t r a i n t h e r a n g e 200-350 mu w e r e d e t e r m i n e d o n s u i t a b l e d i l u t i o n s i n d i s t i l l e d w a t e r u s i n g t h e B a u s c h a n d Lomb S p e c t r o n i c 505 r e c o r d i n g s p e c t r o -p h o t o m e t e r , a n d 280/260 r a t i o s o f o p t i c a l d e n s i t y w e r e d e t e r m i n e d . 3 . A s s a y o f f r a c t i o n s F r a c t i o n s w e r e a s s a y e d b y t h e r e l e a s e o f i n o r g a n i c p h o s p h a t e f r o m 5 '-AMP. -43-R E S U L T S AND D I S C U S S I O N I • I n i t i a l S t u d i e s w i t h S h a k e n C e l l S u s p e n s i o n s 1. T h e c o u r s e o f s t a r v a t i o n i n p h o s p h a t e d e f i c i e n t m e d i u m I n t h e f i r s t o f t h e i n i t i a l s t u d i e s , t h e c o u r s e o f s t a r v a t i o n i n p h o s p h a t e d e f i c i e n t m e d i u m w a s f o l l o w e d b y o b s e r v i n g c h a n g e s i n t u r b i d i t y a n d v i a b l e ( p l a t e ) c o u n t s o f c e l l s u s p e n s i o n s s h a k e n a t 37 C i n s t e r i l e 18 mm m e t a l - c a p p e d t u b e s o r s t e r i l e F r e u d e n r e i c h f l a s k s . T h e l a t t e r w e r e u s e d t o p r o v i d e a r e l a t i v e l y l a r g e s u r f a c e a r e a w i t h m i n i m u m e v a p o r a t i o n o f l i q u i d d u r i n g a l o n g e r t e r m e x p e r i m e n t . A s e p a r a t e t u b e o r f l a s k w a s u s e d f o r e a c h t i m e i n t e r v a l c o n s i d e r e d . A s i t w a s t h o u g h t t h a t g r o w t h o f t h e i n o c u l u m o n a l o w e r l e v e l o f p h o s p h a t e m i g h t a f f e c t t h e p a t t e r n o f r e s p o n s e s e e n , c e l l s g r o w n i n m e d i u m c o n t a i n i n g 1 0 " ^ M p h o s p h a t e w e r e s t u d i e d c o n c u r r e n t l y w i t h c e l l s g r o w n i n t h e n o r m a l m e d i u m , c o n t a i n i n g 1 0 ~ 2 M p h o s p h a t e , i n t h e l o n g e r t e r m e x p e r i m e n t . When c e l l s g r o w n o n 1 0 ~ 2 M p h o s p h a t e w e r e u s e d a n d s h a k e n i n m e t a l - c a p p e d t u b e s f o r 30 h o u r s t h e f o l l o w i n g o b s e r v a t i o n s w e r e made ( F i g . 1 ) : ( a ) T h e " v i a b l e " 1 c o u n t r o s e s h a r p l y a t f i r s t , b u t w i t h a d e c r e a s i n g r a t e o v e r t h e e n t i r e p e r i o d , r e a c h i n g -44-4x10 3x10' Hi CL CO -J -J UJ O 2x10" -JxlO cells per ml 6 12 18 24 HOURS INCUBATION 0.3 ' E O o I-•0.2 P I G . 1. Changes i n v i a b l e c e l l c o u n t a n d t u r b i d i t y d u r i n g s t a r v a t i o n o f P. a e r u g i n o s a i n s h a k e c u l t u r e s i n p h o s p h a t e d e f i c i e n t medium f o r 30 h o u r s . - 4 5 -an apparent p l a t e a u a t 24 to 30 hours. An e i g h t - f o l d i n c rease i n counts suggested that three doublings of po p u l a t i o n had occurred. (b) The t u r b i d i t y (T) a t 400 mu measured over the p e r i o d rose i n l i n e a r f a s h i o n , appearing to taper o f f somewhat between 24 and JO hours and reaching, a t t h i s p o i n t , a value more than double the i n i t i a l l e v e l . The f a c t that glucose was not l i m i t i n g was i n d i c a t e d by the f a i l u r e of glucose to increase t u r b i d i t y or v i a b l e counts a t 30 hours when added a t the l e v e l of a f u r t h e r 0.2$ a t 24 hours. The subsequent long-term experiment c a r r i e d out over a p e r i o d of 6 days confirmed and extended these observations ( F i g . 2 ) . T u r b i d i t y was found to continue to r i s e , although l e s s s t e e p l y , to a maximum between 48 and 72 hours, and to have decreased somewhat a t 144 hours (6 days). V i a b l e counts were found to drop o f f beyond the peak seen a t approximately 24 hours, and to approach the i n i t i a l value at 72 hours. L i t t l e f u r t h e r decrease was seen a t 144 hours, when a count i n the range of the zero time value was s t i l l obtained. The same behavior was d i s p l a y e d by c e l l s grown on medium co n t a i n i n g the normal l e v e l of 1Q~^ M phosphate or the - 4 6 -24 48 72 144 HOURS INCUBATION PIG. 2. Changes i n v i a b l e c e l l count and t u r b i d i t y d u r i n g s t a r v a t i o n of P. aeruginosa i n shake , c u l t u r e s i n phosphate • d e f i c i e n t medium f o r 6 days. Symbols: O and A , v i a b l e c e l l count and t u r b i d i t y r e s p e c t i v e l y f o r c e l l s grown i n i t i a l l y on 10 M phosphate; • and # , v i a b l e c e l l count and t u r b i d i t y r e s p e c t -i v e l y f o r c e l l s grown i n i t i a l l y on 10~^ M phosphate. -47-reduced l e v e l of IO"4" M phosphate, and the former was consequently used i n the experiments reported. An obvious a b i l i t y of the c e l l s to survive and i n fact p r o l i f e r a t e under conditions of phosphate starvation i s c l e a r l y demonstrated by these experiments. Similar r e s u l t s were obtained by Horiuchi (1959) who found that a marked increase i n viable counts of E. c o l i occurred within a few hours of phosphate exhaustion of the medium, and by Mallette et a l . (1964) who studied E. c o l i under conditions s i m i l a r to those i n the present experiments. The fact that exogenous phosphate was not introduced at an e f f e c t i v e concentration i n the phosphate d e f i c i e n t medium i s shown i n F i g . 3 hy the l i n e a r i t y , and extrapolation through the o r i g i n , of a plot of t u r b i d i t y attained i n 24 hours of starvation against inoculum s i z e . This c r i t e r i o n for the detection of exogenous phosphate was suggested by Mallette e_t aJL. (1964). Any phosphate contributing to the growth observed must therefore have been endogenous i n o r i g i n . 2. The rate of response of starved and nonstarved c e l l s to a fixed l e v e l of phosphate In the second type of experiment i n t h i s s e r i e s , the rate of response to a fixed l e v e l of phosphate -48-.25 .50 .75 INOCULUM ( ML) FIG. 3„ F i n a l t u r b i d i t y of phosphate-starved shake c u l t u r e s of P. aeruginosa as a f u n c t i o n of inoculum s i z e . - 4 9 -(0.1 ^m per 5 .8 ml system) was followed t u r b i d i m e t r i c a l l y on suspensions shaken at 37 C i n s t e r i l e , metal-capped tubes. In order to determine what ef f e c t p r i o r phosphate starvation might have upon the rate of response, a sample of washed suspension i n phosphate d e f i c i e n t medium was shaken at 37 C for 21-22 hours. This period of starvation was shown by the previous experiments to r e s u l t i n a maximum number of viable c e l l s . A s i m i l a r amount of each suspension was stored at 4-6 C for the same period of time, and the tubes for the starved and cold-stored c e l l s prepared at the same time. The r e s u l t s plotted i n F i g . 4 demonstrate that the responses of the starved and nonstarved (cold-stored) c e l l s were e s s e n t i a l l y i d e n t i c a l . An i n i t i a l , comparatively rapid response was observed i n each case u n t i l approximately 10 hours a f t e r the addition of phosphate, and t h i s was followed by a considerably slower rate of response. An incubation period of from 12 to 24 hours was therefore shown to be adequate to produce a near-maximum response to the 0.1 pm of phosphate, as gauged by t u r b i d i t y measurements, and consequently was used i n the following experiments. -50-0.4r l I  I  i i 0 6 12 18 24 HOURS INCUBATION PIG. 4. Rate of response of shake, c u l t u r e s of P. aeruginosa to 0.1 ;um phosphate per tube. -51-3. The response of starved and nonstarved c e l l s to graded l e v e l s of phosphate In the t h i r d type of experiment, the response of starved and nonstarved c e l l s to graded l e v e l s of phosphate (up to 2.5 prn. per 5.8 ml system, or 0.43 pM per ml) was determined t u r b i d i m e t r i c a l l y on c e l l suspensions i n phosphate d e f i c i e n t medium shaken i n metal-capped tubes at 37 C for 12 or 2k hours. In order to e s t a b l i s h that the response of the starved c e l l s was not l i m i t e d by nutrients other than phosphate or i n h i b i t e d by metabolic byproducts which had accumulated during phosphate starvation, aliquots of the starved c e l l s i n cer t a i n experiments were harvested by centrifugation and resuspended to the same volume i n fresh phosphate d e f i c i e n t medium before use i n the response experiments. As a control, a corresponding aliquot of starved c e l l s was centrifuged and resuspended i n i t s own supernatant. The r e s u l t s obtained i n an experiment using a 24 hour period of incubation are plotted i n Pig. 5 and F i g . 6. The response obtained was l i n e a r at low l e v e l s of phosphate i n the case of both starved and nonstarved c e l l s , with both displaying the same increment of t u r b i d i t y for a given amount of phosphate (Fig. 5). At higher l e v e l s of phosphate, however, the response of the - 5 2 -I I I I I 0 0.05 0.10 jum Pj PER TUBE FIG. 5 « F i n a l t u r b i d i t y of shake c u l t u r e s o.f P. aeruginosa as a f u n c t i o n of added phosphate i n the range of 0 to 0.1 ,um per tube.' - 5 3 -0.8 £ O o P: PER TUBE FIG. 6. F i n a l t u r b i d i t y of shake c u l t u r e s of P. aeruginosa as a f u n c t i o n of added phosphate i n the range of 0 to 1 . 0 um per tube. The,dotted p o r t i o n c o r r e s -ponds to F i g . 5 « - 5 4 -starved c e l l s was s i g n i f i c a n t l y l e s s than that of the nonstarved c e l l s (Fig. 6 ) . Similar r e s u l t s were obtained when a 1 2 hour period of incubation was employed. When starved c e l l s resuspended i n fresh phosphate d e f i c i e n t medium were studied, they were found to behave s i m i l a r l y to the starved c e l l s resuspended i n t h e i r own supernatant, giving a response lower than that of the nonstarved c e l l s (Fig. 7 ) . The observed f a i l u r e of starved c e l l s to respond as f u l l y as nonstarved c e l l s was observed also by Mallette et ajU ( 1 9 6 4 ) i n studies on E. c o l i , and was indicated by plate count data not to r e s u l t from a loss of v i a b i l i t y during the period of incubation with exogenous phosphate. These authors suggested that the i n a b i l i t y of the starved c e l l s to divide i n the glucose-s a l t s medium used may have resulted from the loss of one or more components which were necessary for c e l l d i v i s i o n and which were resupplied (or reformed?) i n the complex p l a t i n g medium used. Although plate counts were not performed i n the present experiments, such an explanation would seem to apply to the r e s u l t s obtained. This w i l l be discussed more f u l l y i n a l a t e r section of t h i s report. Another factor which also may be of considerable - 5 5 -pm Pj PER TUBE F I G , 7. F i n a l t u r b i d i t y o f s h a k e c u l t u r e s o f P. a e r u g i n o s a a s a f u n c t i o n o f a d d e d p h o s p h a t e i n t h e r a n g e o f 0 t o 2.5 ;m p e r t u b e . - 5 6 -i m p o r t a n c e i n t h e s e e x p e r i m e n t s i s t h e a d v e r s e e f f e c t o f v i g o r o u s a e r a t i o n o n g r o w i n g c u l t u r e s o f P. a e r u g i n o s a . T h e p h o s p h a t e s t a r v e d c e l l s , b e i n g i n a w e a k e n e d c o n d i t i o n , may b e m o r e s u s c e p t i b l e t o p o s s i b l e t o x i c e f f e c t s o f o x y g e n . 1 A l t h o u g h t h e t e n d e n c y o f P. a e r u g i n o s a t o w a r d s p e l l i c l e f o r m a t i o n a n d c l u m p i n g g a v e r i s e t o d i f f i c u l t y i n o b t a i n i n g h i g h l y a c c u r a t e t u r b i d i m e t r i c d a t a , t h e r e s u l t s o f t h e f o r e g o i n g e x p e r i m e n t s w o u l d s e e m t o p a r a l l e l t h o s e o b t a i n e d i n t h e s t u d y o f M a l l e t t e e_t a l . (1964) w i t h E . c o l i . T h e p l o t s f o r b o t h s t a r v e d a n d n o n s t a r v e d c e l l s i n t h e r e s p o n s e e x p e r i m e n t e x t r a p o l a t e d t h r o u g h t h e z e r o l e v e l o f p h o s p h a t e , i n d i c a t i n g t h a t t h e s m a l l e s t a m o u n t o f p h o s p h a t e s u p p l i e d e x o g e n o u s l y w a s c a p a b l e o f p r o d u c i n g a m e a s u r a b l e t u r b i d i m e t r i c r e s p o n s e , a n d t h u s r u l e d o u t t h e e x i s t e n c e o f a t h r e s h o l d r e q u i r e -m e n t f o r p h o s p h a t e d e t e c t a b l e b y t h i s m e t h o d o f i n v e s t i g a t i o n . I I . S t u d i e s w i t h S t i l l C u l t u r e s 1. C h a n g e s i n t u r b i d i t y , c e l l n u m b e r s a n d p r o t e i n o n p h o s p h a t e s t a r v a t i o n When R o u x f l a s k c u l t u r e s i n p h o s p h a t e d e f i c i e n t m e d i u m w e r e i n c u b a t e d a t 30 C f o r 24 h o u r s t h e f o l l o w i n g c h a n g e s w e r e n o t e d : a m a r k e d r i s e i n t u r b i d i t y , a s l i g h t d e c l i n e i n v i a b l e c o u n t a n d a s l i g h t i n c r e a s e i n 1 D r . J . J . R . C a m p b e l l , p e r s o n a l c o m m u n i c a t i o n -57-t o t a l c e l l n u m b e r s a s d e t e r m i n e d b y t h e u s e o f t h e C o u l t e r c o u n t e r . T h e r e s u l t s f o r o n e s u c h e x p e r i m e n t a r e g i v e n i n T a b l e I . T a b l e I . C h a n g e s i n t u r b i d i t y , v i a b l e c e l l c o u n t , t o t a l c e l l c o u n t a n d p r o t e i n p e r m l o f c u l t u r e o n p h o s p h a t e s t a r v a t i o n o f P. a e r u g i n o s a . V a l u e 0 24 24 h o u r v a l u e a s c o n s i d e r e d h o u r s h o u r s % o f 0 h o u r v a l u e t u r b i d i t y a t 660 mu 0.185 0 .350 189 — Pi v i a b l e c e l l s x 10~ 7 .3 6.6 90 t o t a l c e l l s x 10~ 8 7.12 8.58 120 p r o t e i n , >ug p e r m l o f c u l t u r e 45 80 178 p r o t e i n , ,ug p e r o t o t a l c e l l x 10 6 .3 9 .3 148 A s u b s t a n t i a l i n c r e a s e i n t o t a l p r o t e i n o f c e l l p e l l e t s f r o m a l i q u o t s o f s t a r v e d c u l t u r e s w a s a l s o s e e n . C a l c u l a t i o n o n a " p e r t o t a l c e l l " b a s i s i n d i c a t e d t h i s t o b e d u e m o r e t o a n i n c r e a s e I n t h e a m o u n t o f p r o t e i n p e r c e l l t h a n t o t h e o b s e r v e d i n c r e a s e i n t o t a l c e l l n u m b e r s . T h e m a r k e d i n c r e a s e i n p r o t e i n p e r c e l l w a s n o t -58-r e f l e c t e d I n any obvious increase i n c e l l s i z e on s t a i n e d s l i d e s of the c u l t u r e s , l i k e l y because the change i n o v e r a l l dimensions would be too small to be r e a d i l y detected m i c r o s c o p i c a l l y . S i z e d i s t r i b u t i o n p a t t e r n s obtained from C o u l t e r count data a l s o d i d not i n d i c a t e any s i g n i f i c a n t change i n c e l l s i z e . These r e s u l t s are contrary to those obtained by H o r i u c h i (1959) and suggested by the s t u d i e s on shaken c u l t u r e s , i n v o l v i n g a marked increase i n c e l l numbers and concomitant decrease i n c e l l s i z e . The d i f f e r e n t c o n d i t i o n s used i n the present experiments would th e r e f o r e seem to p r o h i b i t comparison on t h i s b a s i s , a t l e a s t , with r e s u l t s obtained u s i n g shaken c u l t u r e s . To determine i f a change i n r e f r a c t i v e index d i f f e r e n t i a l between c e l l s and suspending f l u i d was c o n t r i b u t i n g to the observed increase i n t u r b i d i t y of s t a r v e d c u l t u r e s , a l i q u o t s of starved and nonstarved c e l l s were c e n t r i f u g e d , and p e l l e t s of each type resuspended i n both uninoculated (fresh) phosphate d e f i c i e n t medium and 24-hour s t a r v a t i o n supernatant. O p t i c a l d e n s i t y was then read a t 660 mu a g a i n s t d i s t i l l e d water. Samples of each d i l u e n t were a l s o read a g a i n s t d i s t i l l e d water and readings subtracted from the sample value s . The r e s u l t s obtained are summarized In Table I I . - 5 9 -T a b l e I I . T u r b i d i t y a t 660 mu o f s u s p e n s i o n s o f s t a r v e d a n d n o n s t a r v e d c e l l s i n f r e s h a n d d e p l e t e d p h o s p h a t e d e f i c i e n t m e d i u m . T u r b i d i t y a t 660 mu i n T u r b i d i t y i n d e p l e t e d F r e s h D e p l e t e d m e d i u m a s % o f t h a t C e l l s m e d i u m m e d i u m i n f r e s h m e d i u m N o n s t a r v e d 0.282 0.323 1 1 4 S t a r v e d .280 .332 119 T h e f a c t t h a t a c h a n g e i n r e f r a c t i v e i n d e x o f t h e m e d i u m r e l a t i v e t o t h a t o f t h e c e l l s may c o n t r i b u t e t o t h e i n c r e a s e i n t u r b i d i t y w a s c o n f i r m e d b y t h e e f f e c t o f d e p l e t e d s u p e r n a t a n t o n t h e t u r b i d i t y o f b o t h s t a r v e d a n d n o n s t a r v e d c e l l s . T h a t a c h a n g e i n r e f r a c t i v e i n d e x o f t h e c e l l s t h e m s e l v e s may o c c u r i s a f u r t h e r p o s s i b i l i t y w h i c h i s n o t c o n f i r m e d b y t h e d a t a p r e s e n t e d . 2 . C h e m i c a l c h a n g e s i n c e l l u l a r c o m p o n e n t s o n s t a r v a t i o n C h e m i c a l a n a l y s e s p e r f o r m e d o n c e l l s a t z e r o h o u r a n d a f t e r 2 4 h o u r s o f p h o s p h a t e s t a r v a t i o n d e m o n s t r a t e d t h a t b o t h p r o t e i n a n d DNA i n c r e a s e d a n d RNA d e c r e a s e d o n t h e b a s i s o f ;ug p e r m l o f c u l t u r e d u r i n g t h e s t a r v a t i o n p e r i o d . A l t h o u g h c o n s i d e r a b l e v a r i a t i o n w a s s e e n i n t h e - 6 0 -a b s o l u t e changes i n v o l v e d , the same t r e n d was observed i n each of a number of separate experiments. The r e s u l t s of a r e p r e s e n t a t i v e experiment are presented i n Table I I I . RNA v a l u e s g i v e n r e p r e s e n t the sum of the RNA i n the hot and c o l d a c i d s o l u b l e f r a c t i o n s . Table I I I . Changes o c c u r r i n g on s t a r v a t i o n of P. aeruginosa i n phosphate d e f i c i e n t medium. Value c o n s i d e r e d ^ 0 hour 24 hours 24 hour value as % of 0 hour value t u r b i d i t y a t 660 mu 0.115 0.155 135 —8 v i a b l e c e l l s x 10~ 4 .50 4.18 93 dry weight, p.g 29.6 44.4 150 p r o t e i n , ,ug 17.5 22 .8 130 DNA, Mg 2 .04 2 .46 121 RNA, p.g 6 .4 4 .6 72 per ml of c u l t u r e where a p p l i c a b l e Although the C o u l t e r counter was not a v a i l a b l e a t the time t h i s experiment was done, and t o t a l counts t h e r e f o r e not obtained, i t i s p o s s i b l e to i n f e r from the r e s u l t s presented i n Tables I and I I I that the amount of - 6 1 -p r o t e i n p e r c e l l i n c r e a s e d a n d t h a t t h e a m o u n t o f DNA p e r c e l l i n c r e a s e d s l i g h t l y o r r e m a i n e d c o n s t a n t . T h e o b v i o u s d e c r e a s e i n t h e a m o u n t o f RNA p e r c e l l w o u l d b e i n t e n s i f i e d b y a n i n c r e a s e i n t o t a l c e l l n u m b e r s . T h e o v e r a l l r e s u l t s a r e c o n s i s t e n t w i t h t h o s e o f H o r i u c h i ( 1 9 5 9 ) » who o b s e r v e d a s i m i l a r s y n t h e s i s o f p r o t e i n a n d DNA a n d d e g r a d a t i o n o f RNA t o o c c u r o n p h o s p h a t e s t a r v a t i o n o f E . c o l i , a n d who d e m o n s t r a t e d t h a t a l a r g e p o r t i o n o f t h e p h o s p h a t e r e l e a s e d f r o m RNA w a s u t i l i z e d f o r t h e s y n t h e s i s o f DNA. A s n o t e d i n t h e p r e v i o u s s e c t i o n , h o w e v e r , t h e p r o n o u n c e d d e c r e a s e i n c e l l s i z e s e e n b y H o r i u c h i (1959) w a s n o t e v i d e n t h e r e , a n d t h e r e p o r t e d n e t r e d u c t i o n o f a l l t h r e e c o m p o n e n t s o n a " p e r c e l l " b a s i s w a s n o t s h o w n t o o c c u r u n d e r t h e s e c o n d i t i o n s . T h e a d d i t i o n o f p h o s p h a t e t o t h e s t a r v e d c e l l s a t 2 4 h o u r s w a s s h o w n t o r e s u l t i n i n c r e a s e s i n t u r b i d i t y , p l a t e c o u n t a n d d r y w e i g h t , a n d i n t h e a m o u n t s o f p r o t e i n , DNA a n d RNA p e r m l o f c u l t u r e m e a s u r e d 6 h o u r s l a t e r ( T a b l e I V ) . T h e p e r c e n t i n c r e a s e o f RNA o v e r t h e 2 4 h o u r l e v e l w a s p a r t i c u l a r l y h i g h , s u g g e s t i n g a p r e f e r e n t i a l r e s y n t h e s i s o f t h e c o m p o u n d m o s t e x t e n s i v e l y d e g r a d e d d u r i n g s t a r v a t i o n . - 6 2 -Table IV. Changes o c c u r r i n g on r e a d d i t i o n of phosphate to phosphate-starved c u l t u r e s of P. aeruginosa. Value considered^ 24 hours 30 hours 30 hour value as % of 24 hour value t u r b i d i t y a t 660 mp. 0.155 0.340 219 v i a b l e c e l l s x 10" 8 4.18 7.68 184 dry weight, p.g 44.4 182 410 p r o t e i n , p.g 22.8 75.1 330 DNA, p.g 2.46 6.88 280 RNA, ^ ig 4.6 24.6 535 i per ml of c u l t u r e where a p p l i c a b l e 3 . Sucrose d e n s i t y gradient patterns Patterns obtained on u l t r a c e n t r i f u g a t i o n of c e l l _ p f r e e e x t r a c t s through sucrose gradients c o n t a i n i n g 10 -4 ; and 10 M MgCl 2 are i l l u s t r a t e d i n F i g . 8 and 9 . The p l o t t e d readings f o r 0 . D. a t 260 mu f o r the 24 hour samples have been c o r r e c t e d to correspond to the zero hour values on a t o t a l c e l l b a s i s , and those f o r the 30 hour samples have been c o r r e c t e d to correspond to the zero hour value on a p r o t e i n b a s i s . -63-0.8 0.6 E O CO CM O 0.4 0.2 70S • I0" 2M MgCI 2 * A i f 3 I fl I s i 20 TUBE NO. FIG. 8. Ribosome p a t t e r n s of e x t r a c t s of nonstarved, phosphate-starved and r e f e d c e l l s . G r a d i e n t s c o n t a i n e d 1 0 ~ 2 M f i n a l c o n c e n t r a t i o n of MgCl2» Symbols; 0 0 nonstarved; A — A s t a r v e d ; • — • incubated a f u r t h e r 6 hours with added phosphate. -6k-• 0 I I I 1 ! 0 |0 20 TUBE NO. F I G . 9« R i b o s o m e p a t t e r n s o f e x t r a c t s o f n o n s t a r v e d , p h o s p h a t e - s t a r v e d a n d r e f e d c e l l s . G r a d i e n t s c o n t a i n e d . 1.0"^ M f i n a l c o n c e n t r a t i o n o f M g C l £ . S y m b o l s : 0 0 n o n s t a r v e d ; A — A s t a r v e d ; • — o i n c u b a t e d a f u r t h e r 6 h o u r s w i t h a d d e d p h o s p h a t e . Figure 8 represents the p a t t e r n of ribosomal m a t e r i a l obtained i n a gradient IO""2 M with respect to magnesium. At t h i s c o n c e n t r a t i o n , free ribosomes are known t o e x i s t mainly as 70S p a r t i c l e s h e l d together by magnesium i o n s . The major peak i n the r e g i o n of tube number 10 represents 70S m a t e r i a l , while the shoulder at tube number 12 or 13 and the l e s s e r peak a t tube number 17 represent 50S and 30S m a t e r i a l r e s p e c t i v e l y . A s i g n i f i c a n t decrease i n the amount of 70S m a t e r i a l i s i n d i c a t e d i n the 24 hour sample, c l e a r l y i l l u s t r a t i n g t hat a degradation of ribosomal m a t e r i a l occurred during phosphate s t a r v a t i o n . Figure 9 represents the patterns obtained i n a grad i e n t c o n t a i n i n g 10"^ M magnesium, i n which the 70S p a r t i c l e s are known to d i s s o c i a t e to give 5 0 S and 3 0 S s p e c i e s . A marked decrease i n the amount of both 5 0 S and 3 0 S m a t e r i a l i s evident. The o v e r a l l r e s u l t s obtained were c o n s i s t e n t with those of va r i o u s other workers (McCarthy, 1962; Mandelstam and Halvorson, i960) who have reported ribosomal degradation on the c e s s a t i o n of growth due to various n u t r i t i o n a l d e f i c i e n c i e s . The a d d i t i o n of phosphate to the starved c e l l s a t 2k hours was again shown to promote recovery, as i t had i n the experiments of the preceding section,. As t o t a l - 6 6 -c e l l c o u n t s a t 30 h o u r s w e r e n o t o b t a i n e d , t h e r e s u l t s i n F i g . 8 a n d F i g . 9 a r e p l o t t e d r e l a t i v e t o t h e z e r o h o u r v a l u e s o n a p r o t e i n b a s i s , o n t h e a s s u m p t i o n t h a t t h e p r o t e i n c o n t e n t p e r c e l l h a s r e a t t a i n e d a n o r m a l v a l u e a f t e r 6 h o u r s o f r e c o v e r y . T h e h e i g h t o f t h e p e a k s , a s c o m p a r e d t o t h o s e o f t h e z e r o h o u r s a m p l e , may r e f l e c t t h e f a c t t h a t t h e g r o w i n g c e l l s a t 30 h o u r s a r e p h y s i o -l o g i c a l l y y o u n g e r t h a n t h o s e u s e d a s t h e i n o c u l u m a t z e r o h o u r , a s a h i g h RNA c o n t e n t i s k n o w n t o b e c h a r a c t e r -i s t i c o f l o g p h a s e c e l l s ( H e r b e r t , I 9 6 I ) . T h e r e s u l t s o f t h i s s e c t i o n c l e a r l y s u p p o r t t h e p r e v i o u s e v i d e n c e o f G r o n l u n d a n d C a m p b e l l (1963) f o r t h e r o l e o f r i b o s o m a l RNA a s a p r i m a r y e n d o g e n o u s r e s e r v e i n P. a e r u g i n o s a . T h e i n c r e a s e i n c e l l u l a r p r o t e i n a n d DNA o b s e r v e d c o n c u r r e n t l y w i t h a d e c r e a s e i n c e l l u l a r RNA i s i n a g r e e m e n t w i t h t h e r e s u l t s o f H o r i u c h i (1959) a n d s u g g e s t s t h a t i n t h e p r e s e n t s t u d i e s , a l s o , a s y n t h e s i s o f DNA o c c u r r e d a t t h e e x p e n s e o f R N A - p h o s p h o r u s . T h e g r a d i e n t p a t t e r n s , t o o , s h o w t h a t a d e c r e a s e i n r i b o s o m e c o n t e n t o f t h e c e l l s o c c u r r e d o n p h o s p h a t e s t a r v a t i o n , a n d s u p p o r t t h e c o n t e n t i o n t h a t t h e r i b o s o m e s , n o l o n g e r n e e d e d i n a n o n g r o w i n g c e l l , may a c t a s a s o u r c e o f n e e d e d m e t a b o l i t e s s u c h a s p h o s p h a t e . -67-I I I . A l k aline Phosphatase A c t i v i t y Because of reports of a phosphate-repressible phosphomonoesterase i n other organisms, i t was f e l t of i n t e r e s t to determine whether an increase i n such a c t i v i t y occurred on phosphate starvation of P. aeruginosa. Preliminary experiments showed that a 5 0 0 - f o l d increase i n s p e c i f i c a c t i v i t y of the enzyme did occur on 24 hours of starvation, and could he demonstrated r e a d i l y by the release of p_-nitrophenol from p_-nitrophenyl phosphate. The a c t i v i t y i n extracts of starved c e l l s was accordingly studied with regard to a number of features. 1. The e f f e c t of buffer composition and concentration As i t had been decided to determine the pH spectrum of phosphatase a c t i v i t y , a series of t r i s - a c e t a t e buffers ranging from pH 3 . 0 to 10.0 was prepared to give a f i n a l t r i s concentration of 0.04 M i n the assay system to be used. The a c t i v i t y seen i n the i n i t i a l assay at pH 8.0 was s i g n i f i c a n t l y lower than that obtained with t r i s - H C l buffer at a f i n a l t r i s concentration of 0 . 5 M. The e f f e c t of d i f f e r e n t concentrations of the two buffers was therefore studied by the spectrophotometry method, using NPP as a substrate. The r e s u l t s obtained are plotted i n F i g . 10. - 6 8 -TRIS (M) FIG, 10. Alkaline phosphatase a c t i v i t y i n a c e l l free extract (CFX) of P. aeruginosa as a function of buffer composition and concentration. - 6 9 -Two p e c u l i a r i t i e s may b e n o t e d i n t h e g r a p h s o b t a i n e d . T h e f i r s t o f t h e s e i s t h e p r o n o u n c e d r e l a t i o n s h i p o f a c t i v i t y t o t r i s c o n c e n t r a t i o n , w h i c h a p p e a r s l i n e a r u p t o a f i n a l c o n c e n t r a t i o n s o m e w h a t l e s s t h a n 0.5 M. A l t h o u g h t h e b u f f e r i n g c a p a c i t y a t s u c h l e v e l s f a r e x c e e d s t h e m a x i m u m d e m a n d s o f t h e s y s t e m , f i n a l t r i s c o n c e n t r a t i o n s o f 0.5 t o 1.0 M w e r e u s e d w i t h o u t e x p l a n a t i o n i n m o s t o f t h e r e f e r e n c e s c i t e d . T h i s p h e n o m e n o n o f a c t i v a t i o n b y t r i s h a s b e e n r e f e r r e d t o b y W i l s o n e t a l . (1964), who s u g g e s t e d t h a t t r i s a c t e d a s a p h o s p h a t e a c c e p t o r . T h e s e c o n d f e a t u r e t o b e n o t e d i n F i g . 10 i s t h e l o w e r a c t i v i t y o b t a i n e d i n t h e p r e s e n c e o f t r i s - a c e t a t e b u f f e r . P r e s u m a b l y t h i s m i g h t a r i s e f r o m a n i n h i b i t o r y e f f e c t o f a c e t a t e ( t o w h i c h l i t e r a r y r e f e r e n c e s c o u l d n o t b e f o u n d ) o r f r o m a s t i m u l a t o r y e f f e c t o f c h l o r i d e i n t h e t r i s - H C l b u f f e r . T h e l a t t e r s e e m s m o r e l i k e l y i n t h e l i g h t o f t h e s a l t e f f e c t n o t e d b y W i l s o n e_t a l . (1964) a n d H e p p e l e t a l . (1962) a n d i n d i c a t e d b y t h e r e s u l t s t o b e p r e s e n t e d i n t h e n e x t s e c t i o n o f t h i s r e p o r t . 2. T h e e f f e c t o f E D T A a n d i n o r g a n i c i o n s B e c a u s e o f t h e s u g g e s t i o n t h a t a d i v a l e n t c a t i o n , p o s s i b l y z i n c , i s i n v o l v e d a t t h e a c t i v e s i t e o f t h e -70-e n z y m e i n E . c o l i . a c t i v i t y w a s t e s t e d i n t h e p r e s e n c e o f E D T A t o d e t e r m i n e i f a n y i n h i b i t i o n w o u l d r e s u l t . A s t h e e x t e n t o f i n h i b i t i o n b y E D T A d e p e n d s o n t h e t i m e o f e q u i l i b r a t i o n , a 2 0 m i n u t e p e r i o d o f p r e i n c u b a t i o n w i t h E D T A w a s e m p l o y e d w h e n t h i s c o m p o u n d w a s p r e s e n t . T h e r e s u l t s o b t a i n e d a r e g i v e n i n T a b l e V . T a b l e V . T h e e f f e c t o f E D T A o n t h e a c t i v i t y o f P. a e r u g i n o s a a l k a l i n e p h o s p h a t a s e . F i n a l c o n c e n t r a t i o n o f b u f f e r E D T A pxa p e r m l A c t i v i t y a s % o f c o n t r o l t r i s - H C l , 0 . 5 M - 1 0 0 10 8 4 1 0 0 18 t r i s - a c e t a t e , 0 . 5 M - 1 0 0 1 0 0 18 E D T A a t t h e l e v e l o f 10 pm p e r m l c a u s e d o n l y a 1 6 p e r c e n t d e c r e a s e i n a c t i v i t y i n a s y s t e m c o n t a i n i n g 0 . 5 M t r i s - H C l , w h i l e 1 0 0 ^um p e r m l r e s u l t e d i n a n 8 2 p e r c e n t d e c r e a s e i n b o t h t r i s - H C l a n d t r i s - a c e t a t e b u f f e r s . T h e h i g h l e v e l o f E D T A r e q u i r e d t o p r o d u c e a m a r k e d e f f e c t w a s n o t i n l i n e w i t h t h e r e s u l t s o f G a r e n a n d L e v i n t h a l ( I 9 6 0 ) who o b t a i n e d a n 8 0 p e r c e n t r e d u c t i o n w i t h 2 . 5 ^un -71-of EDTA per ml i n 1 M t r i s buffer (pH 8 .0) at 23 C. The reason for t h i s difference i s not known, although the fact that Garen and Levinthal were working with p u r i f i e d enzyme may provide an explanation. The e f f e c t which various ions might have on enzyme a c t i v i t y was also of i n t e r e s t , from a general point of view and because of the s a l t e f f e c t suggested by the r e s u l t s of the previous section. Magnesium i s required for the a c t i v i t y of various other phosphatases (Morton, 1955) a*id has been reported by Garen and Levinthal ( i 9 6 0 ) to be required for f u l l a c t i v i t y of E. c o l i a l k a l i n e phosphatase at t r i s concentrations below 0.01 M. A c t i v i t y was therefore tested i n the presence of varying concentrations of t h i s ion. As a stimulation of a c t i v i t y was seen at high l e v e l s of magnesium, s i m i l a r and higher concentrations of mono-valent cations were also tested. The r e s u l t s obtained are summarized i n Table VI. That there might be some e f f e c t due s p e c i f i c a l l y to the divalent cation, magnesium, was suggested by the fact that the stimulation by 100 pm of MgClg per ml was greater than that given by 200 urn of either NaCl or KC1. An e f f e c t of i o n i c strength was also suggested by - 7 2 -the data i n Table VI. Table VI. The e f f e c t of inorganic ions on the a c t i v i t y of P. aeruginosa a l k a l i n e phosphatase. F i n a l concentra- Concentration A c t i v i t y as t i o n of buffer Addition ;j.m per ml % of control t r i s - H C l , 0.5 M - - 100 MgCl 2 5 100 10 107 20 120 100 144 NaCl 100 112 200 127 K C 1 100 120 200 126 KH 2 P0^ 10 3 t r i s acetate,0 . 5 M - - 100 MgCl 2 100 158 - 7 3 -T h a t t h e r e m i g h t b e some e f f e c t d u e s p e c i f i c a l l y t o t h e d i v a l e n t c a t i o n , m a g n e s i u m , w a s s u g g e s t e d b y t h e f a c t t h a t t h e s t i m u l a t i o n b y 1 0 0 pm o f M g C l 2 p e r m l w a s g r e a t e r t h a n t h a t g i v e n b y 2 0 0 j i m o f e i t h e r N a C l o r K C 1 . A n e f f e c t o f i o n i c s t r e n g t h w a s a l s o s u g g e s t e d b y t h e d a t a i n T a b l e V I . T h a t t h i s may h a v e b e e n m a s k e d t o some d e g r e e b y t h e c h l o r i d e i o n a d d e d i n p r e p a r i n g t h e t r i s - H C l b u f f e r i s o b v i o u s , a n d may s e r v e t o e x p l a i n t h e s o m e w h a t h i g h e r p e r c e n t a c t i v a t i o n g i v e n b y 1 0 0 p.m M g C l 2 i n t r i s - a c e t a t e b u f f e r . T h e g e n e r a l s t i m u l a t o r y e f f e c t o f i o n i c s t r e n g t h o n t h e a c t i v i t y o f t h e e n z y m e i n E . c o l i h a s b e e n n o t e d b y H e p p e l e t a l . ( 1 9 6 2 ) , who t e s t e d t h e e f f e c t o f v a r i o u s c o n c e n t r a t i o n s o f a n u m b e r o f s a l t s . A v e r y s t r o n g i n h i b i t i o n o f t h e e n z y m e b y i n o r g a n i c p h o s p h a t e w a s a l s o n o t e d a n d w i l l b e d i s c u s s e d s e p a r a t e l y . 3. A c t i v i t y a t v a r i o u s pH v a l u e s D e t e r m i n a t i o n o f a c t i v i t y a t v a r y i n g p H v a l u e s g a v e t h e c u r v e o f F i g . 1 1 . T h e v a l u e s p l o t t e d h a v e b e e n c o r r e c t e d f o r t h e c o l o u r o f p _ - n i t r o p h e n o l a t e a c h p H , a n d a r e e x p r e s s e d r e l a t i v e t o a n a r b i t r a r y v a l u e o f 1.00 a t t h e o b s e r v e d o p t i m u m o f p H 7.5* A s s a y s r u n s u b s e q u e n t l y w e r e c a r r i e d o u t a t t h i s p H . -74-0.5 L i i » » L . 7.0 7.5 8.0 8.5 9.0 pH FIG. 1 1 . Alkaline phosphatase a c t i v i t y i n a c e l l free extract of P. aeruginosa as a function of p H . -75-4. A c t i v i t y w i t h i n c r e a s i n g t e m p e r a t u r e D e t e r m i n a t i o n o f a c t i v i t y i n b u f f e r s p r e p a r e d a t p H 7-5 a t t h e i n d i c a t e d t e m p e r a t u r e s y i e l d e d t h e r e s u l t s p l o t t e d i n F i g . 12. A l o g a r i t h m i c i n c r e a s e i n a c t i v i t y w a s s e e n u p t o 70 C, w i t h a 10° r i s e i n t e m p e r a t u r e i n c r e a s i n g a c t i v i t y b y a f a c t o r o f 1.3* T h e c r u d e e n z y m e f r o m E . c o l i i s k n o w n t o b e r e m a r k a b l y s t a b l e t o t h e r m a l i n a c t i v a t i o n , a n d h a s b e e n r e p o r t e d t o w i t h s t a n d a t e m p e r a t u r e o f 85 C f o r a t l e a s t 30 m i n u t e s w i t h n o l o s s o f a c t i v i t y ( G a r e n a n d L e v i n t h a l , i960) o r 100 C f o r 5 m i n u t e s w i t h o n l y 50 p e r c e n t l o s s o f a c t i v i t y ( T o r r i a n i , I 9 6 0 ) . 5. A c t i v i t y w i t h v a r i o u s s u b s t r a t e s T h e a c t i v i t y o f t h e e n z y m e w i t h t h e r i b o n u c l e o s i d e 5'-mono-, d i - a n d t r i p h o s p h a t e s , a s w e l l a s t h e n o n s p e c i f i c s u b s t r a t e s p _ - n i t r o p h e n y l p h o s p h a t e ( N P P ) a n d b i s - N P P i s s h o w n i n T a b l e V I I . A c t i v i t i e s w e r e m e a s u r e d b y P^ r e l e a s e a n d a r e e x p r e s s e d r e l a t i v e t o a n a c t i v i t y o f 1.00 w i t h 5'-AMP. M o s t o f t h e v a l u e s o b t a i n e d f e l l w i t h i n a f a i r l y n a r r o w r a n g e , a s r e p o r t e d f o r t h e E . c o l i e n z y m e , i n d i c a t i n g i n t h i s c a s e a l s o a n o n s p e c i f i c a l k a l i n e p h o s p h a t a s e a c t i v i t y . N o n s p e c i f i c d i e s t e r a s e a c t i v i t y a g a i n b i s - N P P w a s n o t o b s e r v e d . C o n t r a r y t o t h e r e s u l t s o f e a r l y s t u d i e s w i t h E . c o l i ( T o r r i a n i , i960; G a r e n a n d -76-1.0 > > - 0 . 8 o UJ 0.6 UJ K 0.4 0.2 J L 35 45 5 5 65 TEMPERATURE ( C ) 75 PIG. 12. Alkaline phosphatase a c t i v i t y i n a c e l l free extract of P. aeruginosa as a function of temperature. i -77-T a b l e V I I . R e l a t i v e a c t i v i t y o f P. a e r u g i n o s a a l k a l i n e p h o s p h a t a s e a g a i n s t r i b o n u c l e o s i d e 5 ' - p h o s p h a t e s , N P P a n d b i s - N P P . R e l a t i v e D a t a r e p o r t e d f o r E . c o l i S u b s t r a t e A c t i v i t y V a l u e R e f e r e n c e 5'-AMP 1.00 0 . 8 , 1.0 1,2 GMP 1.00 1.00 1 CMP .92 1.2 1 UMP .73 1 . 3 , 0 . 8 5 1,2 A D P 1.09 GDP .97 CDP .86 UDP .92 1.0 2 A T P .51 1.02 2 G T P .55 1 . 0 5 2 C T P .53 1 . 0 5 2 UTP .41 _ -N P P 1.10 1 . 0 , 1.42 2 , 3 b i s - N P P 0 t r a c e 3 1 G a r e n a n d L e v i n t h a l ( i960) 2 H e p p e l e t a l . (1962) 3 T o r r i a n i ( i960) - 7 8 -Levinthal, i 9 6 0 ) , but consistent with those of more recent workers (Heppel et a l . , 1962), was the observed a c t i v i t y against ATP. That one enzyme i s active against both ATP and AMP has been indicated i n the case of E. c o l i by various data presented by Heppel et a l . (1962). The most marked deviation from the a c t i v i t y against 5'-AMP was seen here with the four triphosphates, which were, however, shown to contain contaminating phosphate at a l e v e l causing some i n h i b i t i o n of enzyme a c t i v i t y . This, and the further accumulation of inorganic phosphate during the i n i t i a l stages of the reaction, would act to prevent the hydrolysis from proceeding further towards completion with the l i b e r a t i o n of three moles of phosphate per mole of substrate. Heppel et a l . (1962) noted, i n fac t , that very l i t t l e adenosine was formed from ATP under t h e i r conditions of assay. The fact that polynucleotide phosphorylase a c t i v i t y did not contribute to the release of inorganic phosphate from the four ribonucleoside diphosphates i n the system used was indicated by the absence of a decrease i n 260 mu absorption i n the assay outlined i n Materials and Methods. This was not surprising i n view of the low substrate concentration employed and the very high phosphatase - 7 9 -a c t i v i t y o b s e r v e d i n c e l l f r e e e x t r a c t s . T h e a c t i v i t y o f t h e e n z y m e p r e p a r a t i o n w a s a l s o t e s t e d a g a i n s t a v a r i e t y o f p h o s p h o r y l a t e d c a r b o h y d r a t e s . R e l a t i v e a c t i v i t i e s a r e g i v e n i n T a b l e V I I I a n d a r e a g a i n e x p r e s s e d r e l a t i v e t o t h e a c t i v i t y o b t a i n e d w i t h 5 '-AMP a s s u b s t r a t e . T a b l e V I I I . R e l a t i v e a c t i v i t y o f P. a e r u g i n o s a a l k a l i n e p h o s p h a t a s e a g a i n s t m i s c e l l a n e o u s s u b s t r a t e s . S u b s t r a t e R e l a t i v e A c t i v i t y D a t a r e p o r t e d V a l u e f o r E . c o l i R e f e r e n c e 5'-AMP 1.00 g l u c o s e - 6 - P .86 0 . 9 , 0.93 1,2 6 - P - g l u c o n a t e 1.15 f r u c t o s e - 6 - P .49 f r u c t o s e - 1 , 6 - P .44 0.90 2 r i b o s e - 5 - P .94 0.7 1 3 - P - g l y c e r a t e .71 3 - P - g l y c e r a l d e h y d e 1.10 ^ - g l y c e r o p h o s p h a t e .94 H e p p e l e t a l . (1962) T o r r i a n i ( i 9 6 0 ) -80-As phosphatases f o r several of the substrates used are known to occur i n P. aeruginosa, control assays using extracts of unstarved c e l l s were performed f o r each compound. A s i g n i f i c a n t l e v e l of a c t i v i t y was not observed under the conditions of assay, and that seen with the extracts of starved c e l l s was therefore presumed to be due to the nonspecific a l k a l i n e phosphatase. The lower a c t i v i t y seen with fructose-1, 6-diphosphate was l i k e l y due to the observed contamination of t h i s substrate with inorganic phosphate, while that seen with f r u c t o s e s -phosphate i s unexplained. The r e s u l t s obtained here further indicate the nonspecific nature of the phospho-monoesterase a c t i v i t y , which seems to depend very l i t t l e on the structure of the substrate molecule. 6. Degree of i n h i b i t i o n by inorganic phosphate Inorganic phosphate was indicated i n a previous section to be a strong i n h i b i t o r of phosphatase a c t i v i t y . As lower r e l a t i v e a c t i v i t i e s were obtained f o r the ribonucleoside triphosphates, f o r example, which contributed an i n i t i a l l e v e l of phosphate contamination of approximately 0.5 to 0.6 _um per ml of reaction system, assays were carr i e d out, with 5'-AMP as substrate, i n the presence of 0, 0.25 and 0.50 urn of phosphate per ml to - 8 1 -d e t e r m i n e t h e d e g r e e o f i n h i b i t i o n w h i c h w o u l d r e s u l t . T h e r e s u l t s o b t a i n e d a r e g i v e n i n T a b l e I X . T h e d a t a i n d i c a t e t h a t t h e l o w e r a c t i v i t i e s s e e n i n s y s t e m s c o n t a i n i n g s i m i l a r l e v e l s o f p h o s p h a t e may b e a t t r i b u t e d l a r g e l y t o p h o s p h a t e i n h i b i t i o n . T A B L E I X . T h e i n h i b i t i o n o f P. a e r u g i n o s a a l k a l i n e p h o s p h a t a s e b y l o w l e v e l s o f i n o r g a n i c p h o s p h a t e . P h o s p h a t e R e l a t i v e ^m p e r m l A c t i v i t y 0 1.00 0.25 .79 0.50 .64 I V . T h e L o c a t i o n o f A l k a l i n e P h o s p h a t a s e 1. A c t i v i t y i n w h o l e c e l l s A s a m p l e o f w h o l e c e l l s s t o r e d o n i c e o v e r n i g h t w a s a s s a y e d a l o n g w i t h t h e c o r r e s p o n d i n g c e l l f r e e e x t r a c t a n d t h e e x t r a c t f r a c t i o n s o b t a i n e d b y d i f f e r e n t i a l c e n t r i f u g a t i o n . T h e a c t i v i t y o b s e r v e d w i t h w h o l e c e l l s w a s a l m o s t i d e n t i c a l t o t h a t o b t a i n e d w i t h t h e c e l l f r e e e x t r a c t ( T a b l e X ) . - 8 2 -T h i s w a s c o n s i s t e n t w i t h t h e r e s u l t s o b t a i n e d b y T o r r i a n i ( i960) w i t h E . c o l i . A l t h o u g h l y s i s may h a v e o c c u r r e d t o some d e g r e e o n s t o r a g e , i t s e e m s u n l i k e l y t h a t l y s i s w o u l d h a v e b e e n s u f f i c i e n t l y c o m p l e t e t o a c c o u n t f o r f u l l a c t i v i t y . T h i s o b s e r v a t i o n , t a k e n w i t h t h e e v i d e n c e o f t h e f o l l o w i n g s e c t i o n f o r a n a s s o c i a t i o n o f t h e p h o s p h a t a s e w i t h t h e r i b o s o m e s , i n d i c a t e s a s i t u a t i o n c o m p a r a b l e t o t h a t e x i s t i n g w i t h E . c o l i r i b o n u c l e a s e , w h o s e l o c a t i o n i n t h e i n t a c t c e l l i s s i m i l a r l y a m a t t e r o f c o n j e c t u r e ( N e u a n d H e p p e l , 1964a a n d 1964b). 2. A c t i v i t y i n c e l l f r e e e x t r a c t f r a c t i o n s C e n t r i f u g a l f r a c t i o n a t i o n o f a c e l l f r e e e x t r a c t , f o l l o w e d b y a s s a y o f e a c h o f t h e f r a c t i o n s , g a v e t h e r e s u l t s p r e s e n t e d i n T a b l e X. A c t i v i t i e s a r e e x p r e s s e d r e l a t i v e t o t h a t o f t h e u n f r a c t i o n a t e d c e l l f r e e e x t r a c t a r b i t r a r i l y s e t a t 1 .00. T h e a c t i v i t y w a s a s s o c i a t e d m a i n l y w i t h t h e r i b o s o m e f r a c t i o n , a l t h o u g h a s i g n i f i c a n t a m o u n t w a s p r e s e n t i n t h e m e m b r a n e f r a c t i o n a n d i n t h e f i r s t m e m b r a n e w a s h i n g s . - 8 2 a -T a b l e X. R e l a t i v e a l k a l i n e p h o s p h a t a s e a c t i v i t i e s o f c e l l s a n d c e l l e x t r a c t f r a c t i o n s o f P. a e r u g i n o s a , a n d r a t i o s o f 280/260 a b s o r p t i o n f o r e x t r a c t f r a c t i o n s . R a t i o s o f 280/260  a b s o r p t i o n R e l a t i v e R e f e r e n c e F r a c t i o n A c t i v i t y O b s e r v e d V a l u e ^ u n f r a c t i o n a t e d e x t r a c t 1.00 w h o l e c e l l s 1.06 w a s h e d m e m b r a n e s .15 .837 .89 f i r s t m e m b r a n e w a s h i n g s .17 .605 s e c o n d m e m b r a n e w a s h i n g s .03 .706 r i b o s o m e s .94 .655 .515 s o l u b l e c y t o p l a s m .06 .613 .626 t o t a l a c t i v i t y i n f r a c t i o n s o f e x t r a c t 1*35 G r o n l u n d (1964) -83-That the membrane f r a c t i o n was contaminated by ribosomes, and. v i c e versa, was suggested by the 280/260 r a t i o s c a l c u l a t e d from UV spect r a of the i n d i v i d u a l f r a c t i o n s . These are a l s o given i n Table X. The r a t i o f o r the f i r s t membrane washings, however, i n d i c a t e s the l o s s of 260-absorbing m a t e r i a l to the low-magnesium washing medium, and t h i s suggests the l o s s of ribosomal contamination from the membranes. Although the high percentage of a c t i v i t y a s s o c i a t e d w i t h the ribosome f r a c t i o n v i r t u a l l y r u l e s out the p o s s i b i l i t y of a c t i v i t y r e s u l t i n g mainly from contamination, a check was made f o r a c t i v i t y i n p u r i f i e d ribosomes obtained from another experiment. A c t i v i t y remained high i n the p u r i f i e d ribosomes, which had a 280/260 r a t i o of 0 .53 com-par i n g favorably with the suggested r a t i o of 0 .515« The i n d i c a t e d a s s o c i a t i o n of the phosphatase with the ribosomal f r a c t i o n could be of considerable advantage to a s t a r v i n g c e l l r e l y i n g on ribosomal RNA as a source of phosphorus f o r the synt h e s i s of DNA. - 8 4 -GENERAL DISCUSSION I. Ribosomal Material as an Endogenous Reserve The role of ribosomal material as a primary endogenous reserve i n P. aeruginosa has been indicated by the work of Campbell, Gronlund and Duncan (1963) and Gronlund and Campbell ( 1 9 6 3 ) , who have provided evidence for a decrease i n ribosomal RNA and protein and a release of ammonia and 260 absorbing material when c e l l s respire i n buffer. The presence of constitutive enzymes capable of oxidizing various nucleosides, purines and pyrimidines and degradation products of these compounds was also demonstrated. That ribosomal material i s a * l o g i c a l substrate capable of s a t i s f y i n g both the maintenance energy requirements of the c e l l and the need for free amino acids for new enzyme synthesis i s evident, when i t i s considered that protein synthesis i s proportional to the number of ribosomes present i n the c e l l (Kennell and Magasanik, 1962; McCarthy, 1962) and that the cessation of growth removes the need for a large complement of protein-synthesizing s i t e s . Ribosomal degradation under conditions where only one nutrient i s lacking seems equally reasonable, and has i n fact been evidenced by the work of -85-s e v e r a l a u t h o r s c i t e d p r e v i o u s l y ( M a n d e l s t a m a n d H a l v o r s o n , I960; A n r a k u , A n d o h a n d M i z u n o , 1963). T h a t a t l e a s t t w o e n z y m e s a r e a v a i l a b l e f o r t h e a u t o -d e g r a d a t i o n o f r i b o s o m e s i n E . c o l i h a s b e e n d e m o n s t r a t e d b y Wade (I96I) a n d Wade e t a l . (1964), a n d e v i d e n c e h a s b e e n p r o v i d e d i m p l i c a t i n g o n e , p o l y n u c l e o t i d e p h o s p h o -r y l a s e , a s t h e d e g r a d a t i v e e n z y m e a s s o c i a t e d w i t h t h e r i b o s o m a l f r a c t i o n i n P. a e r u g i n o s a ( S t r a s d i n e , H o g g a n d C a m p b e l l , 1962; G r o n l u n d a n d C a m p b e l l , 1963). I n t h e p r e s e n t s t u d y , e v i d e n c e f o r a s i m i l a r d e g r a d a t i o n o f r i b o s o m a l RNA u n d e r c o n d i t i o n s o f p h o s p h a t e s t a r v a t i o n w a s p r o v i d e d b y c h e m i c a l d a t a a n d b y r i b o s o m e p a t t e r n s o b t a i n e d b y s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . T h e f o r m e r i n d i c a t e d a n i n c r e a s e i n c e l l u l a r p r o t e i n a n d DNA a n d a d e c r e a s e i n t o t a l RNA o n s t a r v a t i o n . T h e o b s e r v e d i n c r e a s e i n c e l l u l a r p r o t e i n i s r e a s o n a b l e i n t h a t a l l r e q u i r e m e n t s f o r i t s s y n t h e s i s a r e s a t i s f i e d a s l o n g a s t h e r i b o s o m a l s t r u c t u r e r e m a i n s s u f f i c i e n t l y i n t a c t . T h e i n c r e a s e i n DNA i s s i m i l a r l y u n d e r s t a n d a b l e , w i t h t h e b r e a k d o w n p r o d u c t s o f t h e RNA c o n c e i v a b l y s e r v i n g a s s o u r c e s o f e n e r g y , b u i l d i n g b l o c k s a n d p h o s p h a t e . S u c h a s y n t h e s i s o f DNA u n d e r a d v e r s e c o n d i t i o n s h a s b e e n o b s e r v e d i n v a r i o u s o r g a n i s m s b y a n u m b e r o f a u t h o r s ( B o r e k , - 8 6 -R y a n a n d R o c f c e n b a c h , 1955; C a m p b e l l , G r o n l u n d a n d D u n c a n , 1963; H o r l u c h i , 1959) a n d w a s s u g g e s t e d b y C a m p b e l l e t a l . t o b e a m a n i f e s t a t i o n o f t h e o r g a n i s m ' s p r o g r e s s t o w a r d r e p r o d u c t i o n . T h e d i s p r o p o r t i o n a t e l y h i g h i n c r e a s e i n RNA w h i c h w a s i n d i c a t e d t o o c c u r o n r e a d d i t i o n o f p h o s p h a t e t o t h e s t a r v e d c e l l s i s a l s o r e a d i l y r a t i o n a l i z e d a s a p r e f e r e n t i a l r e s y n t h e s i s o f t h e c o m p o u n d m o s t e x t e n s i v e l y d e g r a d e d d u r i n g s t a r v a t i o n . T h i s i s i n a c c o r d w i t h t h e r e s u l t s o f M c C a r t h y ( 1962 ) , ' who r e p o r t e d a n i m m e d i a t e a n d r a p i d i n c r e a s e i n t h e n u m b e r o f r i b o s o m e s w h e n m a g n e s i u m w a s a d d e d t o a c u l t u r e o f E . c o l i s t a r v e d o f t h i s e l e m e n t . E v i d e n c e f o r t h e l o s s o f r i b o s o m a l s t r u c t u r e d u r i n g p h o s p h a t e s t a r v a t i o n w a s a l s o p r o v i d e d b y t h e s u c r o s e d e n s i t y g r a d i e n t p a t t e r n s o b t a i n e d , a n d b y t h e r e c o v e r y o b s e r v e d o n t h e r e a d d i t i o n o f p h o s p h a t e . T h e p a t t e r n s f o r s a m p l e s r u n i n a c o n c e n t r a t i o n o f m a g n e s i u m c a u s i n g d i s s o c i a t i o n o f 70S p a r t i c l e s t o 50S a n d 30S s p e c i e s i n d i c a t e d a d e c r e a s e i n b o t h c o m p o n e n t s ; t h e e n z y m e p o l y n u c l e o t i d e p h o s p h o r y l a s e i s k n o w n t o b e a t t a c h e d 1 f i r m l y t o b o t h t h e 50S a n d 30S s u b u n i t s . T h e r e s u l t s o u t l i n e d h e r e a l s o c o r r e l a t e w i t h t h o s e G r o n l u n d a n d C a m p b e l l ( J . B a c t . 1965* i n p r e s s ) - 8 7 -o b t a i n e d i n t h e i n i t i a l s t u d i e s o n t h e r e s p o n s e o f s h a k e n c u l t u r e s t o p h o s p h a t e s t a r v a t i o n . T h e p r o l i f e r a t i o n o f t h e c e l l s o b s e r v e d d u r i n g t h e f i r s t 2 4 h o u r s i n d i c a t e d a c o n s i d e r a b l e a b i l i t y o f t h e c e l l s t o w i t h s t a n d s t a r v a t i o n , a n d i n t h e l i g h t o f t h e c h e m i c a l : a n d g r a d i e n t d a t a i t i s s u g g e s t e d t h a t t h e a v a i l a b i l i t y o f e x p e n d a b l e r i b o s o m a l m a t e r i a l , i n c l u d i n g p h o s p h a t e , w a s l a r g e l y r e s p o n s i b l e f o r s u r v i v a l a n d g r o w t h . T h e r e s u l t s o b t a i n e d o n t h e a d d i t i o n o f p h o s p h a t e t o s t a r v e d a n d n o n s t a r v e d c e l l s c a n a l s o b e t e n t a t i v e l y e x p l a i n e d b y t h i s d a t a . W h i l e v e r y l o w l e v e l s o f p h o s p h a t e c o u l d p r o d u c e a t u r b i d i m e t r i c r e s p o n s e i n s t a r v e d c e l l s e q u a l t o t h a t g i v e n b y t h e n o n s t a r v e d c e l l s , t h e h i g h e r l e v e l s t e s t e d w e r e a p p a r e n t l y n o t s u f f i c i e n t t o e n a b l e r e b u i l d i n g o f t h e r i b o s o m a l c o n t e n t t o a l e v e l a t w h i c h c e l l d i v i s i o n c o u l d o c c u r . T h e n o n s t a r v e d c e l l s , w h i c h h a d a h i g h e r r i b o s o m a l c o n t e n t t o s t a r t w i t h , w e r e n o t s o h a n d i c a p p e d a n d w e r e a b l e t o u t i l i z e a l l o r m o s t o f t h e a v a i l a b l e p h o s p h a t e f o r g r o w t h o f t h e i n d i v i d u a l c e l l s a n d , p r e s u m a b l y , f o r c e l l d i v i s i o n . I I . A l k a l i n e P h o s p h a t a s e I n v i e w o f t h e f o r e g o i n g r e s u l t s , t h e a b i l i t y o f t h e c e l l t o p r o d u c e a n o n s p e c i f i c a l k a l i n e p h o s p h a t a s e u n d e r c o n d i t i o n s o f p h o s p h a t e s t a r v a t i o n w o u l d a p p e a r t o -88-b e a v a l u a b l e a t t r i b u t e . T h e a c t i v i t y o b s e r v e d a g a i n s t t h e r i b o n u c l e o s i d e p h o s p h a t e s , w h i c h m i g h t b e e x p e c t e d t o b e p r e s e n t e n d o g e n o u s l y i n c e l l s u n d e r g o i n g d e m o n s t r a b l e r i b o s o m a l d e g r a d a t i o n , i s o f i n t e r e s t a s a p l a u s i b l e m e a n s o f e n s u r i n g a s u p p l y o f i n o r g a n i c p h o s p h a t e f o r s u c h e s s e n t i a l p r o c e s s e s a s DNA s y n t h e s i s a n d c o e n z y m e r e g e n e r a t i o n . S u c h p h o s p h o r y l a t e d d e g r a d a t i o n p r o d u c t s o f RNA c o u l d v e r y w e l l a r i s e b y t h e a c t i o n o f p o l y n u c l e o t i d e p h o s p h o r y l a s e , k n o w n t o b e a s s o c i a t e d w i t h t h e 50S a * i d 30S r i b o s o m e s i n t h i s o r g a n i s m a n d t o b e a c t i v e u n d e r c o n d i t i o n s f a v o r i n g r i b o s o m a l d i s s o c i a t i o n . T h i s e n z y m e r e q u i r e s p h o s p h a t e f o r i t s d e g r a d a t i v e a c t i o n , w h i c h p r o c e e d s a c c o r d i n g t o t h e f o l l o w i n g e q u a t i o n : nXMP + ? ± ^ ( n - l ) X M P + X D P O n c e t h e r e a c t i o n w e r e i n i t i a t e d , t h e i n o r g a n i c p h o s p h a t e r e l e a s e d b y t h e a c t i o n o f t h e n o n s p e c i f i c p h o s p h a t a s e o n s u c h c o m p o u n d s a s X D P w o u l d s e r v e t o s t i m u l a t e f u r t h e r b r e a k d o w n . T h e c o m p l e t e r e l e a s e o f i n o r g a n i c p h o s p h a t e f r o m t h e s u b s t r a t e X D P w o u l d p r o v i d e a s e c o n d m o l e o f i n o r g a n i c p h o s p h a t e , r e s u l t i n g i n a n e t g a i n o f o n e m o l e . T h e s t r o n g i n h i b i t i o n o f p h o s p h a t a s e a c t i v i t y b y i n o r g a n i c -89-phosphate would act as a control on the system to hold i n check the degradation of ribosomal material. The ribosomal association of the enzyme apparent i n c e l l free extracts would, i f i t exists i n vivo, contribute to the e f f i c i e n c y of the system as a whole and might serve to prevent the random degradation of phosphorylated compounds which ought to be preserved (e.g. phosphorylated carbohydrate intermediates). The p o s s i b i l i t y that the association observed i s an a r t i f a c t of i s o l a t i o n r e s u l t i n g from adsorption of enzyme to ribosomes cannot be ruled out on the basis of the data av a i l a b l e , and should be checked by attempting further p u r i f i c a t i o n of the ribosomes i n a cesium chloride gradient (see Spahr and Hollingworth, I 9 6 I ) . I f the enzyme i s able by some means to act on substrates outside the c e l l , as i n the case of E. c o l i (Malamy and Horecker, 1961; T o r r i a n i , i960) and as suggested by the a c t i v i t y obtained here with whole c e l l s stored on ice overnight, the c e l l would also be able to u t i l i z e any exogenous phosphorylated compounds as a source of phosphate, and thus be able to supplement or spare i t s endogenous reserves. Although P. aeruginosa has not been demonstrated to contain a ribonuclease, i t might be of i n t e r e s t to r e -d e t e r m i n e i f s u c h a n e n z y m e c o u l d a l s o b e i n d u c e d b y p h o s p h a t e s t a r v a t i o n t o c o m p l e m e n t t h e p r o p o s e d a c t i v i t y o f p o l y n u c l e o t i d e p h o s p h o r y l a s e i n p r o v i d i n g s u b s t r a t e s f o r t h e p h o s p h a t a s e . I t m i g h t a l s o b e o f i n t e r e s t t o c h e c k f o r t h e p r e s e n c e o f a p h o s p h o d i e s t e r a s e a n a l o g o u s t o t h e s o l u b l e , p o t a s s i u m - a c t i v a t e d e n z y m e r e p o r t e d b y S p a h r a n d S c h l e s s i n g e r (1963) t o o c c u r i n E . c o l i a n d t o b r e a k d o w n m e s s e n g e r RNA i n t h e a b s e n c e o f i n o r g a n i c p h o s p h a t e t o g i v e n u c l e o s i d e 5 ' - m o n o p h o s p h a t e s . A s i m i l a r p o t a s s i u m - a c t i v a t e d p h o s p h o d i e s t e r a s e h a s b e e n r e p o r t e d b y K e i r , M a t h o g a n d C a r t e r (1964) t o b e i s o l a t e d f r o m t h e r i b o s o m a l f r a c t i o n o f L a c t o b a c i l l u s  c a s e i a n d t o p r o d u c e 5 ' - m o n o p h o s p h a t e s f r o m RNA. - 9 1 -SUMMARY Suspensions of P. aeruginosa starved of phosphate i n an otherwise complete s y n t h e t i c medium increased markedly i n t u r b i d i t y and v i a b l e c e l l count when shaken at 37 C f o r 24 hours. The l i n e a r t u r b i d i m e t r i c response obtained when suspensions of starved and nonstarved c e l l s were s u p p l i e d w i t h graded amounts of phosphate r u l e d out the existence of a detectable t h r e s h o l d requirement f o r phosphate analogous to tha t of E. c o l i f o r glucose (McGrew and M a l l e t t e , 1962). A lower response to added phosphate was seen i n the case of the starved c e l l s . Chemical analyses and sucrose d e n s i t y gradient pa t t e r n s of c e l l s starved f o r 24 hours i n s t i l l c u l t u r e a t 30 C i n d i c a t e d an increase i n p r o t e i n and DNA and a decrease i n RNA per ml of c u l t u r e , as w e l l as a breakdown of ribosomal s t r u c t u r e ; recovery of ribosomal s t r u c t u r e was seen on the r e a d d i t i o n of phosphate. The p e r i o d of s t a r v a t i o n employed was noted to r e s u l t i n a s l i g h t decrease i n v i a b l e c e l l count and a s l i g h t increase i n t o t a l c e l l count. Phosphate s t a r v a t i o n was a l s o found to r e s u l t i n the i n d u c t i o n of a n o n s p e c i f i c a l k a l i n e phosphomonoesterase, - 9 2 -w h i c h h a d a p H o p t i m u m o f 7 . 5 a t 35 C u n d e r t h e c o n d i t i o n s o f a s s a y , a n d w h i c h w a s r e m a r k a b l y a c t i v e a t t e m p e r a t u r e s u p t o 75 C. I t w a s a c t i v a t e d b y h i g h c o n c e n t r a t i o n s o f t r i s , p o s s i b l y b e c a u s e o f t h e a b i l i t y o f t h e l a t t e r t o s e r v e a s a n a c c e p t o r i n a t r a n s p h o s p h o r y l a t i o n r e a c t i o n . A s t i m u l a t o r y e f f e c t o f s a l t c o n c e n t r a t i o n o n a c t i v i t y w a s n o t e d , a s w e l l a s a h i g h d e g r e e o f i n h i b i t i o n b y i n o r g a n i c p h o s p h a t e . A c t i v i t y a g a i n s t a w i d e v a r i e t y o f c o m p o u n d s , i n c l u d i n g t h e r i b o n u c l e o s i d e 5 ' - p h o s p h a t e s , i n d i c a t e d t h e n o n s p e c i f i c n a t u r e o f t h e e n z y m e . E n z y m e a c t i v i t y w a s o b s e r v e d i n w h o l e c e l l s , a n d i n a f r a c t i o n a t e d c e l l f r e e e x t r a c t w a s s h o w n t o b e a s s o c i a t e d w i t h t h e r i b o s o m e s . I t i s s u g g e s t e d t h a t t h e a b i l i t y o f P. a e r u g i n o s a t o p r o d u c e a n e n z y m e c a p a b l e o f h y d r o l y z i n g d e g r a d e d r i b o s o m a l m a t e r i a l t o r e l e a s e i n o r g a n i c p h o s p h a t e c o n t r i b u t e s t o t h e d e m o n s t r a t e d c a p a c i t y o f t h e o r g a n i s m t o s u r v i v e p h o s p h a t e d e p r i v a t i o n . - 9 3 -B I B L I O G R A P H Y A l d r i d g e , W.N., T . E . B a r m a n , a n d H. G u t f r e u n d . 1964. T h e r a t e o f f o r m a t i o n a n d d e c o m p o s i t i o n o f p h o s p h o r y l -p h o s p h a t a s e ( E s c h e r i c h i a c o l i ) . B i o c h e m . J . 92:23C-25C A n r a k u , U., T. A n d o h , a n d D. M i z u n o . I963. T h e t u r n o v e r o f RNA a n d o f p r o t e i n o f E . c o l i i n t h e s u l f u r - d e f i c i e n t s t a t e . J . B i o c h e m . ( T o k y o ) ^4:246-252. B a r n e r , H.D., a n d S . S . C o h e n . 1958. P r o t e i n s y n t h e s i s a n d RNA t u r n o v e r i n a p y r l m i d i n e - d e f i c i e n t b a c t e r i u m . B i o c h i m . B i o p h y s . A c t a 30:12-20. B o r e k , E . , A. R y a n , a n d J . R o c k e n b a c h . 1955* N u c l e i c a c i d m e t a b o l i s m i n r e l a t i o n t o t h e l y s o g e n i c p h e n o m e n o n . J . B a c t e r i o l . 6£:460-467. B r i t t e n , R . J . , a n d R.B. R o b e r t s , i 9 6 0 . H i g h r e s o l u t i o n d e n s i t y g r a d i e n t s e d i m e n t a t i o n a n a l y s i s . S c i e n c e 131:32-33. C a m p b e l l , J . J . R . , A . F . G r o n l u n d , a n d M.G. D u n c a n . I 9 6 3 . E n d o g e n o u s m e t a b o l i s m o f P s e u d o m o n a s . A n n . N.Y. A c a d . S c i . 102:669-677. C a m p b e l l , J . J . R . , L . A . H o g g , a n d G.A. S t r a s d i n e . 1962. E n z y m e d i s t r i b u t i o n i n P s e u d o m o n a s a e r u g i n o s a . J . B a c t e r i o l . 8J;1155-I1ZQ". C a s h e l , M., a n d E . F r e e s e . 1964. E x c r e t i o n o f a l k a l i n e p h o s p h a t a s e b y B a c i l l u s s u b t i l i s . B i o c h e m . B i o p h y s . R e s . Commun. 16:541-544. C h e n , P . S . , T.Y. T o r i b a r a , a n d H. W a r n e r . 1956. M i e r o d e t e r m i n a t i o n o f p h o s p h o r u s . A n a l . Chem. 28:1756-1758. D a w e s , E . A . , a n d D.W. R i b b o n s . I962. T h e e n d o g e n o u s m e t a b o l i s m o f m i c r o o r g a n i s m s . A n n . R e v . M i c r o b i o l . 16:241-264. D a w e s , E . A . , a n d D.W. R i b b o n s . 1964. Some a s p e c t s o f t h e e n d o g e n o u s m e t a b o l i s m o f b a c t e r i a . B a c t e r i o l . R e v . 28:126-149. _94-E c h o l s , H., A. G a r e n , S. G a r e n , a n d A . T o r r i a n i . I 9 6 I . G e n e t i c c o n t r o l o f r e p r e s s i o n o f a l k a l i n e p h o s p h a t a s e i n E s c h e r i c h i a c o l i . J . M o l . B i o l . 2*. 425-438. G a l l a n t , J . , a n d R. S t a p l e t o n . 1964a. P h y s i o l o g i c a l e v i d e n c e o n t h e n a t u r e o f t h e r e p r e s s o r o f a l k a l i n e p h o s p h a t a s e s y n t h e s i s i n E s c h e r i c h i a c o l i . J . M o l . B i o l . 8:431-441. G a l l a n t , J . , a n d R. S t a p l e t o n . 1964b. D e r e p r e s s i o n o f a l k a l i n e p h o s p h a t a s e s y n t h e s i s b y c h l o r a m p h e n i c o l a n d c a n a v a n i n e i n h i b i t i o n . J. M o l . B i o l . 8:442-451. G a r e n , A . , a n d S. G a r e n , 1963* C o m p l e m e n t a t i o n i n v i v o b e t w e e n s t r u c t u r a l m u t a n t s o f a l k a l i n e p h o s p h a t a s e f r o m E s c h e r i c h i a c o l i . J . M o l . B i o l . £ : 13-22. G a r e n , A . , a n d C. L e v i n t h a l . i 9 6 0 . A f i n e - s t r u c t u r e g e n e t i c a n d c h e m i c a l s t u d y o f t h e e n z y m e a l k a l i n e p h o s p h a t a s e o f E . c o l i . I . P u r i f i c a t i o n a n d c h a r a c t e r i z a t i o n o f a l k a l i n e p h o s p h a t a s e . B i o c h i m . B i o p h y s . A c t a 38:470-483. G o l d s t e i n , A . , a n d B . J . B r o w n . i 9 6 0 . A m i n o a c i d s t a r v a t i o n i n a n E s c h e r i c h i a c o l i a u x o t r o p h . I I I . I n c o r p o r a t i o n o f 3 2 - p i n t o r i b o n u c l e i c a c i d a n d o t h e r c e l l c o m p o n e n t s . B i o c h i m . B i o p h y s . A c t a 4 4 : 4 9 1 - 5 0 0 . G o l d s t e i n , D.B., B . J . B r o w n , a n d A . G o l d s t e i n , i 9 6 0 . A m i n o a c i d s t a r v a t i o n i n a n E s c h e r i c h i a c o l i a u x o t r o p h . I I . A c i d s o l u b l e p u r i n e a n d p y r i m i d i n e d e r i v a t i v e s i n n o r m a l a n d s t a r v e d c e l l s . B i o c h i m . B i o p h y s . A c t a 4 3 : 5 5 - 6 1 . G r o n l u n d , A . F . 1964. A s t u d y o f e n d o g e n o u s r e s p i r a t i o n i n P s e u d o m o n a s a e r u g i n o s a . P h . D. t h e s i s . T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r , B r i t i s h C o l u m b i a . G r o n l u n d , A . F . , a n d J . J . R . C a m p b e l l . I 9 6 3 . N i t r o g e n o u s s u b s t r a t e s o f e n d o g e n o u s r e s p i r a t i o n i n P s e u d o m o n a s  a e r u g i n o s a . J . B a c t e r i o l . 8 6 : 5 8 - 6 6 . - 9 5 -Harold, F.M. I963. Enzymic and genetic c o n t r o l of polyphosphate accumulation i n Aerobacter aerogenes. J . Gen. M i c r o b i o l . 2 £ : 8 l - 9 0 . Harold, P.M. 1964. Accumulation of i n o r g a n i c polyphosphate i n Aerobacter aerogenes. I . R e l a t i o n s h i p to growth and n u c l e i c a c i d s y n t h e s i s . J . B a c t e r i o l . 86:216-221. Harold, F.M., and S. Sylvan. 1963. Accumulation of i n o r g a n i c polyphosphate i n Aerobacter aerogenes. I I . Environmental c o n t r o l and the r o l e of s u l f u r compounds. J . B a c t e r i o l . 86:222-231 . Hendler, R.W., W.G. B a n f i e l d , J . T a n i , and E.L. K u f f . 1964. On the c y t o l o g i c a l u n i t f o r p r o t e i n s y n t h e s i s i n v i v o i n E. c o l i . I I I . E l e c t r o n microscopic and u l t r a c e n t r i f u g a l examination of i n t a c t c e l l s and f r a c t i o n s . Biochim. Biophys. Acta 80:307-314. Heppel, L.A., D.R. Harkness, and R.J. Hilmoe. 1962. A study of the substrate s p e c i f i c i t y and other p r o p e r t i e s of the a l k a l i n e phosphatase of E s c h e r i c h i a c o l i . J . B i o l . Chem. 237:841-846. Herbert, D. 1961. The chemical composition of microorganisms as a f u n c t i o n of t h e i r environment, p. 391-416. In G.G. Meynell and H. Gooder (ed.), The Eleventh Symposium Soc. Gen. M i c r o b i o l . U n i v e r s i t y Press, Cambridge, England. Heredia, C.F., F. Yen, and A. S o l s . I963. Role and formation of the a c i d phosphatase i n yeast. Biochem. Biophys. Res. Commun. 10:14-18. H o r i u c h i , T. 1959. RNA degradation and DNA and p r o t e i n s y n t h e s i s of E. c o l i B. i n a phosphate d e f i c i e n t medium. J . Biochem. (TokyoT"46:1467-1480. H o r i u c h i , T., S. H o r i u c h i , and D. Mizuno. 1959. A p o s s i b l e negative feedback phenomenon c o n t r o l l i n g formation of a l k a l i n e phosphomonoesterase i n E s c h e r i c h i a c o l i . Nature 183:1520-1530. - 9 6 -H u m m e l , J . P . , a n d G. K a l n i t s k y . 1964. M e c h a n i s m s o f c e r t a i n p h o s p h o t r a n s f e r a s e r e a c t i o n s : c o r r e l a t i o n o f s t r u c t u r e a n d c a t a l y s i s i n some s e l e c t e d e n z y m e s . A n n . R e v . B i o c h e m . 33:15-50. H u t c h i s o n , W.C., E.D. D o w n i e , a n d H.N. M u n r o . 1962. F a c t o r s a f f e c t i n g t h e S c h n e i d e r p r o c e d u r e f o r e s t i m a t i o n o f n u c l e i c a c i d s . B i o c h i m . B i o p h y s . A c t a 55:561-570. d e J i m e n e z , E . S . , E . L e e , J . T o r r e s , a n d G. S o b e r o n . 1964. On t h e m e c h a n i s m o f t h e e f f e c t o f i o n i c s t r e n g t h o n c r y s t a l l i n e a l d o l a s e a c t i v i t y . J . B i o l . C hem. 239:4l54-4l58. K e i r , H.M., R.H. M a t h o g , a n d C . E . C a r t e r . P u r i f i c a t i o n o f a p o t a s s i u m i o n - a c t i v a t e d 5 ' - p h o s p h o d i e s t e r a s e f r o m L a c t o b a c i l l u s c a s e i . B i o c h e m i s t r y ,3_: 1188-1193 • K e n n e l l , D., a n d B. M a g a s a n i k . 1962. T h e r e l a t i o n o f r i b o s o m e c o n t e n t t o t h e r a t e o f e n z y m e s y n t h e s i s i n A e r o b a c t e r  a e r o g e n e s . B i o c h i m . B i o p h y s . A c t a 55:139-151. K i n g , E . J . 1932. T h e c o l o r i m e t r i c d e t e r m i n a t i o n o f p h o s p h o r u s . B i o c h e m . J . 26:292-297. L a m a n n a , C. 1963. E n d o g e n o u s m e t a b o l i s m w i t h s p e c i a l r e f e r e n c e t o b a c t e r i a . A n n . N.Y. A c a d . S c i . 102:515-793. L e v i n t h a l , C , E.R. S i g n e r , a n d K. F e t h e r o l f . 1962. R e a c t i v a t i o n a n d h y b r i d i z a t i o n o f r e d u c e d a l k a l i n e p h o s p h a t a s e . P r o c . N a t l . A c a d . S c i . U.S. 48:1230-1237. L o ? j r y , O.H. , N . F . R o s e b r o u g h , A . L . F a r r , a n d R . J . R a n d a l l . 1951. P r o t e i n m e a s u r e m e n t w i t h t h e F o l i n p h e n o l r e a g e n t . J . B i o l . C hem. 193:265-275. M c C a r t h y , B . J . I962. T h e e f f e c t s o f m a g n e s i u m s t a r v a t i o n o n t h e r i b o s o m e c o n t e n t o f E s c h e r i c h i a c o l i . B i o c h i m . B i o p h y s . A c t a ^:880-888. - 9 7 -McGrew, S.B., and M.P. M a l l e t t e . 1 9 6 2 . Energy of maintenance i n E s c h e r i c h i a c o l i . J . B a c t e r i o l . 83:844-850. McLellan, W.L., J r . , and J.O. Lampen. I 9 6 3 . The a c i d phosphatase of yeast. L o c a l i z a t i o n and s e c r e t i o n by p r o t o p l a s t s . Biochim. Biophys. Acta 67:324-326. Malamy, M., and B. Horecker. I 9 6 I . The l o c a l i z a t i o n of a l k a l i n e phosphatase i n E s c h e r i c h i a c o l i K12. Biochem. Biophys. Res. Commun. £:104-108. M a l l e t t e , M.F., C.I. Cowan, and J.J.R. Campbell. 1964. Growth and s u r v i v a l of E s c h e r i c h i a c o l i i n medium l i m i t e d i n phosphate. J . B a c t e r i o l . 87:779-785. Mandelstam, J . i 9 6 0 . The i n t r a c e l l u l a r turnover of p r o t e i n and n u c l e i c a c i d s and i t s r o l e i n biochemical d i f f e r e n t i a t i o n . B a c t e r i o l . Rev. 24:289-308. Mandelstam, J . , and H. Halvorson. i 9 6 0 . Turnover of p r o t e i n and n u c l e i c a c i d i n s o l u b l e and ribosome f r a c t i o n s of nongrowing E s c h e r i c h i a c o l i . Biochim. Biophys. Acta 40 : 4 3 - 4 9 . M i l n e r , H.W., N.S. Lawrence, and C.S. French. 1950. C o l l o i d a l d i s p e r s i o n of c h l o r o p h y l l m a t e r i a l . Science 111:633-635. M i l s t e i n , C. 1964. The amino a c i d sequence around the r e a c t i v e s e r i n e residue i n a l k a l i n e phosphatase from E s c h e r i c h i a c o l i . Biochem. J . 22:410-421. Morton, R.K. 1955. Phosphomonoesterase of m i l k , p.533-539* In S.P. Colowick and N.O. Kaplan (ed.), Methods i n Enzymology, v o l . 2 . Academic Press, Inc., New York. Nakada, D., and I . Smith. 1962. The s t a b i l i t y of ribosomes i n u r a c i l - s t a r v e d E s c h e r i c h i a c o l i . Biochim. Biophys. Acta 61:414-420. - 9 8 -Neu, H.C., and L.A. Heppel. 1964a. The surface l o c a l i z a t i o n of enzymes i n E s c h e r i c h i a c o l i . Biochem. Biophys. Res. Commun. 17:215-219. Neu, H.C., and L.A. Heppel. 1964b. Some observations on the " l a t e n t " ribonuclease of E s c h e r i c h i a c o l i . Proc. N a t l . Acad. S c i . U.S. $1: 1Z67-1ZW. Neu, H.C., and L.A. Heppel. 1964c. The r e l e a s e of ribonuclease i n t o the medium when E s c h e r i c h i a c o l i c e l l s are converted to spheroplasts. J . B i o l . Chem. 239:3893-3900. P e g r e t t i , M.M., and C. M i l s t e i n . I965 . A c i d i n a c t i v a t i o n and i n c o r p o r a t i o n of phosphate i n t o a l k a l i n e phosphatase from E s c h e r i c h i a c o l i . Biochem. J. 94:106-113. P f i s t e r , R.M., and D.G. Lundgren. 1964. E l e c t r o n microscopy of polyribosomes w i t h i n B a c i l l u s cereus. J . B a c t e r i o l . 88:1119-1129. S c h l e s s i n g e r , D. 1963. P r o t e i n s y n t h e s i s by polyribosomes on p r o t o p l a s t membranes of B. megaterium. J. Mol. B i o l . 2 :569-582. Schlesinger, M.J., and C. Levinthal. I963. Hybrid protein formation of Escherichia c o l i a l k a l i n e phosphatase leading to i n v i t r o complementation. J. Mol. B i o l . 7_:1-12. Schmidt, G., G. B a r t s c h , M. Laumont, T. Herman, and M. L i s s . I 9 6 3 . A c i d phosphatase of Bakers yeast: an enzyme of the e x t e r n a l c e l l s urface. Biochemistry 2:126-131. Schmidt, G., and M. Laskowski. 1961. Phosphate e s t e r cleavage (a survey), p. 3-35* J i l H. Lardy and K. Myrback (ed.), The Enzymes, v o l . 5» 2nd ed. Academic Press, Inc. New York. - 9 9 -Schneider, W.C. 195?• Determination of n u c l e i c a c i d s i n t i s s u e s by pentose a n a l y s i s , p. 680. In S.P. Colowick and N.O. Kaplan (ed.), Methods i n Enzymology, v o l . 3 » Academic Press Inc., New York. Spahr, P.F., and B.R. H o l l i n g w o r t h . 1961. P u r i f i c a t i o n and mechanism of a c t i o n of ribonuclease from E s c h e r i c h i a c o l i ribosomes. J . B i o l . Chem. 236:823-831. Spahr, P.F., and D. S c h l e s s i n g e r . 1963. Breakdown of messenger r i b o n u c l e i c a c i d by a potassium-activated phosphodiesterase from E s c h e r i c h i a c o l i . J . B i o l . Chem. 238:PC2251. Stadtman, T.C. 1961. A l k a l i n e phosphatases, p.55-71. In H. Lardy and K. Myrback (ed.), The Enzymes, v o l . 5 , 2nd ed. Academic Press, Inc., New York. Strange, R.E., F.A. Dark, and A.G. Ness. 1961. The s u r v i v a l of s t a t i o n a r y phase Aerobacter aerogenes st o r e d i n aqueous suspension. J . Gen. M i c r o b i o l . 25:61-76. S t r a s d i n e , G.A., L.A. Hogg, and J.J.R. Campbell. 1 9 6 2 . A ribosomal p o l y n u c l e o t i d e phosphorylase i n Pseudomonas  aeruginosa. Biochim. Biophys. Acta 5 5 : 2 3 1 - 2 3 2 . T o r r i a n i , A. i 9 6 0 . Influence of in o r g a n i c phosphate i n the formation of phosphatases by E s c h e r i c h i a c o l i . Biochim. Biophys. Acta 38:460-469. Wade, H.E. I 9 6 I . The autodegradation of r i b o n u c l e o p r o t e i n i n E s c h e r i c h i a c o l i . Biochem. J . 7 8 : 4 5 7 - 4 7 2 . Wade, H.E., S. L o v e t t , and H.K. Robinson. 1 9 6 4 . The auto-degradation of 3 2 P - l a b e l l e d ribosomes from E s c h e r i c h i a c o l i . Biochem. J . 9 3 : 1 2 1 - 1 2 8 . -100-W a r r e n , R . A . J . , A . F . E l l s , a n d J . J . R . C a m p b e l l , i960. E n d o g e n o u s r e s p i r a t i o n o f P s e u d o m o n a s a e r u g i n o s a . J . B a c t e r i o l . 7_£:875-879 • W e i m b e r g , R., a n d W.L. O r t o n . 1963. R e p r e s s i b l e a c i d p h o s p h o m o n o e s t e r a s e a n d c o n s t i t u t i v e p y r o p h o s p h a t a s e o f S a c c h a r o m y c e s m e l l i s . J . B a c t e r i o l . 86:805-813. W e i m b e r g , R., a n d W.L. O r t o n . 1964. E v i d e n c e f o r a n e x o c e l l u l a r s i t e f o r t h e a c i d p h o s p h a t a s e o f S a c c h a r o m y c e s m e l l i s . J . B a c t e r i o l . 88:1743-1754. W h i t e l y , H.R., a n d M. O i s h i . 1963* A n i n c r e a s e i n a l k a l i n e p h o s p h a t a s e i n a n i n v i t r o s y s t e m d e r i v e d f r o m B a c i l l u s s u b t i l i s . B i o c h e m . B i o p h y s . R e s . Commun. 13:6-11. W i l s o n , I . B . , J . D a y a n , a n d K. C y r . 1964. Some p r o p e r t i e s o f a l k a l i n e p h o s p h a t a s e f r o m E s c h e r i c h i a c o l i . T r a n s p h o s p h o r y l a t i o n . J . B i o l . Chem. 239:4182-4185. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0105499/manifest

Comment

Related Items