Differential Responses to Amino Acids in Bacterial Growth - Advances

9

Differential Responses to Amino Acids in Bacterial Growth GERRIT TOENNIES

Downloaded by PURDUE UNIV on June 7, 2016 | http://pubs.acs.org Publication Date: January 1, 1964 | doi: 10.1021/ba-1964-0044.ch009

The Institute for Cancer Research, Philadelphia 11, Pa.

The paper discusses the changes which result during the growth of Streptococcus faecalis, when different e s s e n t i a l a m i n o a c i d s a r e d e pleted while all other nutrients remain available in excess. Depletion of lysine, an amino acid which is a building block of cell wall glycopeptides as well as of protein, is followed by bacterial lysis, while depletion of other protein components is followed by further growth. The depletion of different amino acids elicits different changes in cellular composition, characterized by different proportions of protein, cell wall, membrane, DNA, RNA, etc. A tentative cytological interpretation of some of these changes is presented.

R i c h a r d B l o c k ' s " A m i n o A c i d H a n d b o o k " i s a c o m p e n d i u m of a n a l y t i c a l p r o c e d u r e s and a n a l y t i c a l r e s u l t s . A m o n g the a n a l y t i c a l p r o c e d u r e s a chapter i s devoted to the m i c r o b i o l o g i c a l methods, and i t i s f r o m a study of m i c r o b i o l o g i c a l methods f o r the d e t e r m i n a t i o n of a m i n o a c i d s that the studies h e r e s u m m a r i z e d have developed. M o r e d e t a i l e d accounts of v a r i o u s phases of the w o r k have been p u b l i s h e d (1-15). Principle

of Microbiological

Assay

The p r i n c i p l e of m i c r o b i o l o g i c a l a s s a y i s s i m p l e . F o r i n s t a n c e , i n o r d e r to d e t e r m i n e , say, p h e n y l a l a n i n e , one s e l e c t s a m i c r o o r g a n i s m that needs p h e n y l a l a n i n e . In a m e d i u m w h i c h contains an abundance of a l l other n u t r i e n t s , the extent of b a c t e r i a l g r o w t h w i l l be d e t e r m i n e d by the amount of phenylalanine added to the m e d i u m . The analyst w i l l set up a s e r i e s of tubes, s t a n d a r d s and unknowns, and after i n o c u l a t i o n put them i n the i n c u b a t o r . A f t e r a s p e c i f i e d period—overnight o r s e v e r a l

118

Stekol; Amino Acids and Serum Proteins Advances in Chemistry; American Chemical Society: Washington, DC, 1964.

9.

TOENNIES

119

Amino Acids in Bacterial Growth


days—he w i l L t a k e the tubes out and e i t h e r t i t r a t e the a c i d i t y f o r m e d as a r e s u l t of the g r o w t h , o r m e a s u r e i n the s p e c t r o p h o t o m e t e r the amount of t u r b i d i t y f o r m e d . H e i s not l i k e l y to look at the tubes before the method c a l l s f o r i t , e s p e c i a l l y i f he i s a c h e m i s t who knows l i t t l e about bacteria. W e happened to belong to t h i s c a t e g o r y , but just the s a m e we d i d look at o u r tubes before they w e r e r e a d y f o r analysis—the r e a s o n b e i n g that we w e r e unhappy about the l a c k of l i n e a r i t y of the r e s p o n s e s . The r e s p o n s e c u r v e s v a r i e d , not so m u c h f r o m one e x p e r i m e n t to another, but s t r a n g e l y f r o m one a m i n o a c i d to another. T h i s i s how we c a m e to study the g r o w t h c u r v e s . O n the left side of F i g u r e 1 w e see the t u r b i d i t y c u r v e w i t h t i m e

Figure

1. Growth in presence

5

of 5.3 χ ΙΟ" M

L-valine

of a c u l t u r e of S. f a e c a l i s w h i c h contains a l i m i t e d c o n c e n t r a t i o n of v a l i n e . A f t e r 24 hours the c u r v e has n i c e l y l e v e l e d off, and t h i s would s e e m to be the l o g i c a l t i m e to take the a n a l y t i c a l r e a d i n g s . E v e n t u a l l y we c a m e to d e t e r m i n e the fate of a l i m i t i n g a m i n o a c i d o v e r the whole c o u r s e of b a c t e r i a l g r o w t h . F o r i n s t a n c e , at v a r i o u s points a l o n g the g r o w t h c u r v e we d e t e r m i n e d how m u c h v a l i n e w a s p r e s e n t w i t h i n the o r g a n i s m and how m u c h was left i n the m e d i u m . The s u m of these two quantities r e m a i n e d constant, and e q u a l to the amount added i n the b e ginning—i.e., t h e r e w a s no m e t a b o l i c l o s s . It w a s a l i t t l e m o r e s u r p r i s i n g to find that the point at w h i c h a l l the v a l i n e had d i s a p p e a r e d f r o m the m e d i u m and w a s i n c o r p o r a t e d into the b a c t e r i a l c r o p w a s not up at the plateau, but s o m e w h e r e a l m o s t halfway down before the p l a teau w a s r e a c h e d .


ADVANCES IN CHEMISTRY SERIES

120

T h e r i g h t half of F i g u r e 1 shows the s a m e data as the left s i d e , but the t u r b i d i t y v a l u e s a r e plotted on a 1 g a r i t h m i c s c a l e i n s t e a d of the o r d i n a r y a r i t h m e t i c a l s c a l e . U n d e r these conditions the f i r s t p a r t of the g r o w t h c u r v e i s e s s e n t i a l l y a s i t should be i f the b a c t e r i a g r o w at a constant r a t e of d u p l i c a t i o n o r , as i t i s c a l l e d , a r e i n the exponential o r l o g phase. N o w the point at w h i c h a l l v a l i n e has been r e m o v e d f r o m the m e d i u m , w h i c h we c a l l the depletion point, i s c l o s e to the end of the exponential g r o w t h p h a s e . I n t h i s c a s e , as i n the other c a s e s d i s c u s s e d , after d e p l e t i o n of the l i m i t i n g a m i n o a c i d a l l other a m i n o a c i d s r e m a i n available i n large excess. F i g u r e 2 shows a d d i t i o n a l e x a m p l e s . T h e d e p l e t i o n point i s r e a c h e d


ISOLEUCINE β

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THREONINE

LEUCINE

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20

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in response acids

D. P. Depletion

10

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to different

limiting

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point

w e l l before g r o w t h attains i t s m a x i m u m , and i n e v e r y c a s e tends to c o i n c i d e w i t h the end of the e x p o n e n t i a l phase. L y s i n e i s an i n t e r e s t i n g e x c e p t i o n : T h e t h e o r e t i c a l depletion point i s not f o l l o w e d by f u r t h e r g r o w t h , but by p r o m p t l y s i s and eventual d i s appearance of the c u l t u r e . Bacterial

Cell

Walls

T h i s w a s the evidence s o m e y e a r s ago. A t that t i m e studies began to a p p e a r i n the l i t e r a t u r e about the c o m p o s i t i o n of b a c t e r i a l c e l l w a l l s . It b e c a m e evident that a m o n g the a m i n o a c i d s we had studied o n l y l y -



9.

TOENNIES

121


s i n e had been found as a m a j o r component of w a l l substance. T h e r e f o r e , one c o u l d tentatively say that l y s i n e i s e s s e n t i a l f o r the g r o w t h of the c e l l w a l l , but that i s o l e u c i n e , l e u c i n e , threonine, h i s t i d i n e , v a l i n e , and s o m e o t h e r s a r e not. A l l of t h e m a r e , of c o u r s e , e s s e n t i a l f o r c y toplasmic protein synthesis. One c o u l d f u r t h e r conclude that at the d e p l e t i o n point ( and the end of exponential growth) p r o t e i n s y n t h e s i s stops but w a l l s y n t h e s i s c o n t i n u e s . In the c a s e of l y s i n e d e p l e t i o n , h o w e v e r , both p r o t e i n s y n t h e s i s and w a l l s y n t h e s i s a r e i m p o s s i b l e , and i n the absence of continuous r e n e w a l through new g r o w t h the c e l l s f a l l v i c t i m to a u t o l y s i s . T h e shapes of the g r o w t h c u r v e s after the d e p l e t i o n point show c h a r a c t e r i s t i c and r e p r o d u c i b l e d i f f e r e n c e s f r o m one l i m i t i n g a m i n o a c i d to another. T h e m o s t s t r i k i n g e x a m p l e of these d i f f e r e n c e s i s i l l u s t r a t e d by F i g u r e 3, i n w h i c h the depletion points of a v a l i n e - l i m i t e d

Figure

HOURS 3. Comparison of valinelimited growths

and

threonine

-

and a t h r e o n i n e - l i m i t e d c u l t u r e a r e both shown as 100 t u r b i d i t y u n i t s . In the c a s e of v a l i n e l i m i t a t i o n postexponential g r o w t h amounts to about 40% and attains i t s m a x i m u m after 20 h o u r s ; i n the c a s e of threonine l i m i t a t i o n the postexponential g r o w t h i s t w i c e as l a r g e and r e q u i r e s about t w i c e as long. T o evaluate the p o s s i b i l i t y of postexponential c e l l w a l l s y n t h e s i s o c c u r r i n g without p r o t e i n s y n t h e s i s , w e d i s r u p t e d b a c t e r i a by s h a k i n g w i t h g l a s s beads. U n d e r p r o p e r conditions t h i s y i e l d s a w a t e r - s o l u b l e and a w a t e r - i n s o l u b l e f r a c t i o n . A s a f i r s t a p p r o x i m a t i o n the s o l u b l e f r a c t i o n m a y be c a l l e d the c y t o p l a s m i c f r a c t i o n and the i n s o l u b l e p a r t the w a l l f r a c t i o n . F i g u r e 4 shows a study b y the g l a s s bead p r o c e d u r e of c e l l s f r o m the exponential g r o w t h phase and of 2 0 - h o u r c e l l s r e s u l t -




122

Figure 4. Mechanical disruption valine - and threonine-limited

of exponential postexponential

cells and cells

i n g f r o m v a l i n e depletion, as w e l l as 4 0 - h o u r c e l l s r e s u l t i n g f r o m t h r e onine d e p l e t i o n . The g r a p h shows the p a r t i t i o n of the total n i t r o g e n b e tween the s o l u b l e and the i n s o l u b l e f r a c t i o n s after different p e r i o d s of m e c h a n i c a l d i s r u p t i o n . The i n s o l u b l e n i t r o g e n f r a c t i o n i s 11% of the t o t a l f o r exponential c e l l s , 22% f o r s o - c a l l e d v a l i n e c e l l s , and 28% f o r threonine c e l l s . T h i s a g r e e s w e l l w i t h o u r expectation that the v a l i n e c e l l s a r e r i c h e r i n w a l l substance than the l o g c e l l s , and shows that the i n s o l u b l e n i t r o g e n f r a c t i o n i s l a r g e s t i n the threonine c e l l s , w h e r e p o s t exponential growth i s t w i c e as m u c h as i n v a l i n e c e l l s . T h e i n s o l u b l e substance i n a l l t h r e e c a s e s turned out to be i d e n t i c a l o r v e r y s i m i l a r i n c o m p o s i t i o n and to r e p r e s e n t n e a r l y p u r e w a l l substance. N o t h i n g i n these r e s u l t s g i v e s any i n d i c a t i o n as to why the p o s t e x ponential g r o w t h i s t w i c e a s l a r g e i n the c a s e of threonine depletion as i n v a l i n e d e p l e t i o n . It b e c a m e obvious that i n our a n a t o m i c a l a n a l y s i s of different b a c t e r i a l c r o p s we had to go beyond the two c o m p a r t m e n t s : s o l u b l e and i n s o l u b l e f r a c t i o n s . Bacterial

Membrane

P a r t i c u l a r l y i t b e c a m e n e c e s s a r y to c o n s i d e r the b a c t e r i a l m e m b r a n e . A s things stand now, the b a c t e r i a l c e l l w a l l i s thought of as an outer skeleton w h i c h c a n be f r e e l y t r a v e r s e d by m a c r o m o l e c u l e s and i s i m p o r t a n t f o r the m e c h a n i c a l s t r u c t u r e and p r o t e c t i o n of the c e l l . It i s made up of p o l y s a c c h a r i d e s and c a r b o h y d r a t e - d e r i v e d substances, w h i c h i n c l u d e some peptide g r o u p s . Underneath the c e l l w a l l t h e r e i s the c e l l m e m b r a n e , w h i c h a p p e a r s to be the seat of the i m p o r t a n t p h y s i o l o g i c a l functions of a s s i m i l a t i o n and e x c r e t i o n . Some findings suggest



9.

TOENNIES

123


that r i b o s o m a l p r o t e i n s y n t h e s i s i s a l s o c l o s e l y a s s o c i a t e d w i t h the m e m b r a n e s t r u c t u r e . L i p o p r o t e i n s e e m s to be the m a j o r component o r component c l a s s of the c e l l m e m b r a n e s w i t h w h i c h we a r e c o n c e r n e d . S p e c i m e n s of m e m b r a n e substance have been obtained b y m a k i n g b a c t e r i a l c e l l s l y s e t h r o u g h v a r i o u s m e a n s , p r e f e r a b l y p e r h a p s after the c e l l w a l l has been d i g e s t e d away by l y s o z y m e , and then a p p l y i n g c e n t r i fugation at i n t e r m e d i a t e speeds. T h i s r e s u l t s i n a f r a c t i o n of r e p r o d u c i b l e c o m p o s i t i o n w h i c h i s c h a r a c t e r i z e d by a high content of l i p o p r o t e i n and of l i p i d e phosphorus. T h e y i e l d i s v a r i a b l e and, u n l i k e the p r o c e d u r e f o r w a l l substance, f a r f r o m quantitative. A p p a r e n t l y s u b s t a n t i a l p o r t i o n s of the m e m b r a n e substance a r e s o l u b i l i z e d o r f r a g m e n t i z e d to a d e g r e e not a c c e s s i b l e to i n t e r m e d i a t e speed c e n t r i f u g a t i o n . T h e r e f o r e , other w a y s had to be found f o r m e a s u r i n g i n q u a n t i t a t i v e t e r m s the m e m b r a n e content i n a g i v e n c r o p . O u r s o l u t i o n of t h i s p r o b l e m f o r the o r g a n i s m of o u r studies, S t r e p t o c o c c u s f a e c a l i s , i s based on the l i p o p r o t e i n nature of the m e m b r a n e . A c c o r d i n g to e s t a b l i s h e d p r o c e d u r e s , the l i p i d e m o i e t y of l i p o p r o t e i n i s d e t e r m i n e d b y b r e a k i n g the l i p i d e - p r o t e i n l i n k a g e w i t h b o i l i n g m e t h a n o l , and then i s o l a t i n g the l i p i d e component i n p e t r o l e u m ether. T h e evaporation r e s i d u e of the p e t r o l e u m ether e x t r a c t can be weighed to give a m e a s u r e of the l i p i d e component. B e s i d e s , the p e t r o l e u m ether e x t r a c t can be used f o r phosphorus d e t e r m i n a t i o n , and t h i s gives a m e a s u r e ( c o l o r i m e t r i c ) of the l i p o p r o t e i n p h o s p h o r u s . W e found that both l i p o p r o t e i n l i p i d e and l i p i d e phosphorus o c c u r only i n the m e m b r a n e substance—i.e., they a r e absent f r o m c e l l w a l l substance as w e l l as the c y t o p l a s m i c substance. That h a v i n g been e s t a b l i s h e d , we c o u l d obtain r e p r o d u c i b l e quantitative v a l u e s f o r the m e m b r a n e content of different b a c t e r i a l c r o p s . W e p r e p a r e d s p e c i m e n s of the m e m b r a n e f r a c t i o n f r o m e a c h c e l l type and d e t e r m i n e d two p a r a m e t e r s : the l i p i d e content g r a v i m e t r i c a l l y , and the l i p i d e phosphorus c o l o r i m e t r i c a l l y (Table I). The l i p i d e phosphorus i s i n a l l three c a s e s of the o r d e r of 3% of the l i p i d e . T h e n we d e t e r m i n e d the s a m e two p a r a m e t e r s , l i p i d e g r a v i m e t r i c a l l y and l i p i d e phosphorus c o l o r i m e t r i c a l l y , on the w h o l e c e l l substance. A g a i n the l i p i d e phosphorus i s of the o r d e r of 3% of the l i p i d e . If both p a r a m e t e r s belong e x c l u s i v e l y to the m e m b r a n e substance, m e m b r a n e p e r centage can be c a l c u l a t e d independently by means of the two p a r a m e Table I. Lipide and Lipide Phosphorus in Different Cell Products

Cell Type

Log

Val

Thr

40.2 10.96 27.3

36.0 4.22 11.7

1.17 0.331 26.6

1.00 0.111 11.1

Lipide % of membrane % of cell Membrane, % of cell calcd.

28.3 4.93 17.4

Lipide Phosphorus % of membrane % of cell Membrane, % of cell calcd.

0.85 0.151 17.8



124


Table II. Estimated Composition of Three Cell Types

Fraction

100-Mg. Log Cells, %

145-Mg Val Cells, %

190-Mg. Thr Cells, %

Wall Membrane Cytoplasmic protein RNA DNA

25.5 17.6 30.5 30.2 2.84

38.0 27.0 13.4 23.8 2.18

44.0 11.4 16.5 20.0 4.05

t e r s . W e see that, i n d e e d , w e have r e a s o n a b l e a g r e e m e n t and, m o r e i n t e r e s t i n g l y p e r h a p s , that the t h r e e c u l t u r e types v a r y g r e a t l y i n the amount of m e m b r a n e substance w h i c h they c o n t a i n . T a b l e Π shows the changes i n the c o m p o s i t i o n of the b a c t e r i a l s u b stance w h i c h o c c u r when 100 m g . of l o g c e l l s at the depletion point g r o w i n t o v a l i n e c e l l s (if that i s the l i m i t i n g a m i n o a c i d ) , o r i n t o threonine c e l l s i f threonine i s l i m i t e d . T h e amounts f o r m e d f r o m 100 m g . at the point of d e p l e t i o n a v e r a g e 145 m g . i n the c a s e of v a l i n e and 190 m g . i n the c a s e of t h r e o n i n e . T h e d e t e r m i n a t i o n of w a l l substance by m e c h a n i c a l d i s r u p t i o n and the d e t e r m i n a t i o n of m e m b r a n e substance by l i p i d e a n a l y s i s have been o u t l i n e d . T h e other data a r e obtained by w a y of c o n v e n t i o n a l p r o c e d u r e s : D N A ( d e o x y r i b o n u c l e i c a c i d ) by d i p h e n y l a m i n e , and c o m p l e t e l y independently by t h y m i n e ; R N A ( r i b o n u c l e i c acid) by u l t r a v i o l e t e x t i n c t i o n ; and c y t o p l a s m i c p r o t e i n f r o m n i t r o g e n d e t e r m i nations c o r r e c t e d f o r the n i t r o g e n content of the other components. It i s i m p o r t a n t to r e m e m b e r that the data i n T a b l e Π a r e p e r c e n tage v a l u e s . F o r i n s t a n c e , s i n c e the weight of a c u l t u r e n e a r l y doubles i n the c a s e of threonine d e p l e t i o n , the 25 m g . of w a l l substance p r e s e n t at the d e p l e t i o n point w i l l i n c r e a s e to m o r e than 80 m g . , o r m o r e than t h r e e f o l d , i n the threonine d e p l e t i o n c e l l s . S i m i l a r l y , i n the c a s e of D N A , 2.8 m g . of D N A at the d e p l e t i o n point w i l l i n c r e a s e to about 8 m g . o r about t h r e e f o l d i n the threonine c e l l s , w h i l e i n the v a l i n e c e l l s — w h e r e the t o t a l m a s s goes f r o m 100 to 145 mg.—the t o t a l amount of D N A r e m a i n s n e a r l y constant. T h e r e f o r e , to see what a c t u a l l y happens d u r i n g the postexponential events, the v a l u e s i n the v a l i n e c o l u m n have to be m u l t i p l i e d by 1.45, and the v a l u e s i n the t h r e o n i n e c o l u m n b y 1.9. T h e r e s u l t s a r e shown d i a g r a m a t i c a l l y i n F i g u r e 5. T h e left b a r shows the a n a l y t i c a l b r e a k d o w n of 100 m g . of exponential c e l l s u b s t a n c e . P r o t e i n of the c y t o p l a s m i s shown i n white and the a r e a c o r r e sponding to the p r o t e i n m o i e t y of the m e m b r a n e l i p o p r o t e i n i s m a r k e d by v e r t i c a l dashed l i n e s . T h e s u m of these two p r o t e i n f r a c t i o n s i s n e a r l y the s a m e i n a l l t h r e e i n s t a n c e s . T h i s i s to be expected, s i n c e the t o t a l amount of b a c t e r i a l p r o t e i n cannot i n c r e a s e f u r t h e r a f t e r one of the p r o t e i n - e s s e n t i a l n u t r i e n t s has been u s e d up. T h e left b a r shows the c a l c u l a t e d c o m p o s i t i o n of an exponentially g r o w i n g c u l t u r e at the depletion point, whether the depleted n u t r i e n t i s v a l i n e , t h r e o n i n e , o r any other n u t r i e n t . T h e c e n t e r b a r shows the t o t a l amount of b a c t e r i a l


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Differential Responses to Amino Acids in Bacterial Growth - Advances

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