T H E N IT R O G E N N U T R IT IO N A N D M E T A B O L IS M O F
E F F E C T IV E A N D IN E F F E C T IV E S T R A IN S O F
R H IZ O B IU M m e li l o t i

by
D A V ID C A R L Y L E J O R D A N

A TH E S IS
S u b m itte d to th e S ch o o l o f G ra d u a te S tu d ie s o f M ic h ig a n
S ta te C o lle g e o f A g r ic u lt u r e and A p p lie d S cie n ce
in p a r t ia l f u lf illm e n t o f th e re q u ir e m e n ts
f o r th e d e g re e o f
D O C T O R O F P H IL O S O P H Y

D e p a rtm e n t o f B a c te r io lo g y
1955

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ACKNOWLEDGMENTS

T h e a u th o r w is h e s to th a n k P r o fe s s o r E . H . G a r r a r d o f
th e D e p a rtm e n t o f B a c te r io lo g y , O n ta rio A g r ic u lt u r a l C o lle g e
f o r m a k in g a v a ila b le th e space and e q u ip m e n t f o r th is in v e s t i­
g a tio n .

T h e in t e r e s t and c o n s tr u c tiv e c r i t i c i s m o f D r . C , L ,

S a n C le m e n te o f th e D e p a rtm e n t o f M ic r o b io lo g y , M ic h ig a n State
C o lle g e , is a ls o g r a t e f u lly a c k n o w le d g e d .
The a u th o r is , in a d d itio n , in d e b te d to th e D e p a rtm e n t o f
E n g in e e rin g , O n ta r io A g r ic u lt u r a l C o lle g e , f o r th e c o n s tr u c tio n
o f c e r ta in s p e c ia liz e d a p p a ra tu s .

TABLE OF CONTENTS

G E N E R A L IN T R O D U C T IO N
PART I

N IT R O G E N N U T R IT IO N A N D M E T A B O L IS M
In tr o d u c tio n and L it e r a t u r e R e v ie w

A.

1

5

T h e u t iliz a t io n o f L - , D - 0 and D L - a m in o a c id s and
the e ffe c ts o f pH and te m p e r a tu re

18

M e th o d s
R e s u lts
D is c u s s io n
B . T h e u t iliz a t io n o f s y n th e tic d i - and t r i- p e p t id e s

39

M e th o d s
R e s u lts
D is c u s s io n
C . T h e u t iliz a t io n o f a m m o n iu m , n it r a t e and n i t r it e
s a lts in

th e p re s e n c e o f a m in o and c a r b o x y lic a c id s

D . G e n e ra l m e ta b o lis m o f c e r ta in a m in o a c id s
1.

D e c a rb o x y la tio n

49
55
59

M e th o d s
R e s u lts and D is c u s s io n
2,

O x id a tio n and D e a m in a tio n
M e th o d s
R e s u lts and D is c u s s io n

68

3,

T r a n s a m in a tio n

78

M e th o d s
R e s u lts and D is c u s s io n
4.

R a ce m ase a c t iv it y

84

M e th o d s
R e s u lts and D is c u s s io n
PART II

G R O W T H S T IM U L A T IO N IN A M IN O A C ID M E D IA
In tr o d u c tio n and L it e r a t u r e R e v ie w

91

M e th o d s
R e s u lts and D is c u s s io n
SUMMARY

100

REFERENCES

104

A P P E N D IX

113

- 1 -

G E N E R A L IN T R O D U C T IO N

M a n is dependent, e ith e r d ir e c t ly o r in d ir e c t ly , u p o n p la n t p r o te in
f o r the m a in te n a n c e o f h is l if e p ro c e s s e s .

T h e n itr o g e n n e c e s s a ry f o r

the s y n th e s is o f th is p r o te in is d e riv e d m a in ly f r o m the s o il and m u s t be
c o n s ta n tly re p le n is h e d .

In a d d itio n to lo s s e s o f s o il n itr o g e n v ia p la n t

u p ta ke th e p ro c e s s e s o f e ro s io n , le a c h in g , sew age lo s s and b io lo g ic a l
d e n it r if ic a t io n a n n u a lly re m o v e tre m e n d o u s q u a n titie s o f c o m b in e d n i t r o ­
gen f r o m the t e r r e s t r i a l c y c le .

F e r t iliz a t io n has be en e m p lo y e d f o r m a n y

y e a rs f o r re p le n is h m e n t p u rp o s e s , b u t is e x p e n s iv e , e s p e c ia lly the s y n ­
th e tic n itro g e n o u s f e r t i l i z e r s w h ic h depend up o n h ig h te m p e r a tu re and
p r e s s u r e f o r t h e ir p ro d u c tio n .

B y f a r the g re a te s t p o te n tia l s o u rc e o f

th is e le m e n t is the a tm o s p h e re and an e c o n o m ic a l and e ff ic ie n t m e a n s o f
c o n v e rtin g th is f r e e m o le c u la r n itr o g e n in to f o r m s w h ic h p la n ts ca n
a s s im ila te is th ro u g h b io lo g ic a l n itr o g e n f ix a t io n .

E v e r s in c e the c la s s i­

c a l e x p e rim e n ts o f H e llr ie g e l and W ilf a r t h i t has been r e a liz e d th a t a
m a jo r f a c t o r in b io lo g ic a l n itr o g e n fix a tio n is the s y m b io tic r e la tio n s h ip
b e tw e e n b a c te r ia o f th e genus R h iz o b iu m and c e r ta in le g u m in o u s p la n ts .
T h e s e b a c te r ia d w e ll w ith in r o o t n o d u le s and, in c o m b in a tio n w ith th e h o s t
p la n t, a re ca p a b le o f f ix in g la r g e q u a n titie s o f fr e e n itr o g e n .

N ot a ll

s tr a in s o f R h iz o b iu m a r e c a p a b le , h o w e v e r, o f f ix in g n itr o g e n .

S tra in s

e x is t w h ic h v a r y f r o m a c o n d itio n o f e ffe c tiv e n e s s , th ro u g h in e ffe c tiv e n e s s ,
to p a r a s itis m .

T h e s e tw o la t t e r s tr a in s a r e a m a t te r o f s o m e c o n c e rn as

th e y a r e p o te n tia lly a b le to cause c ro p f a ilu r e s in f ie ld s w h ic h a re lo w in

- 2 -

in o rg a n ic n itr o g e n .
In s p ite o f in te n s iv e in v e s tig a tio n , b e g in n in g in 1888 w h e n
B e ije r in c k f i r s t is o la te d th e n o d u le o rg a n is m , th e re is s t i l l m u c h to be
le a rn e d a b o u t th e se m ic r o o r g a n is m s f r o m the s ta n d p o in t o f m e c h a n is m
o f n itr o g e n fix a tio n , b io c h e m ic a l b a s is o f in e ffe c tiv e n e s s and p a r a s itis m ,
and m e ta b o lis m in g e n e ra l.

S ince the k e y in te r m e d ia te o f f ix a t io n is

b e lie v e d to be a m m o n ia and th e f i r s t p ro d u c t e a s ily d e te c te d is g lu ta m ic
a c id th e in t r a c e llu la r m e c h a n is m s in v o lv in g a m m o n ia and a m in o a c id s a re
o f p a r t ic u la r im p o rta n c e .
T he p re s e n t in v e s tig a tio n w as c o m m e n c e d u n d e r the a u s p ic e s o f the
D e p a rtm e n t o f B a c te r io lo g y , O n ta rio A g r ic u lt u r a l C o lle g e and the L e g u m e
R e s e a rc h C o m m itte e in O n ta r io .

I t s p u rp o s e w as to m a k e a s o m e w h a t

g e n e ra l s u r v e y o f the a lfa lfa - s w e e t c lo v e r o rg a n is m (R h iz o b iu m m e l i l o t i )
f r o m the s ta n d p o in t o f o rg a n ic and in o rg a n ic n itr o g e n m e ta b o lis m and
n u t r it io n .

F r o m a p r a c t ic a l v ie w p o in t i t w as hoped th a t in fo r m a tio n w o u ld

be g a in e d w h ic h w o u ld :
(a) h e lp in th e d e v e lo p m e n t o f a r a p id b io c h e m ic a l te s t f o r e ff e c tiv e ­
n e ss, in e ffe c tiv e n e s s , and p a r a s itis m a m ong th e le g u m e b a c te r ia ,
(b) a id in the f o r m u la tio n o f a s u p e r io r s y n th e tic m e d iu m f o r the
c u ltiv a tio n and s tu d y of th e se m ic r o o r g a n is m s , and
(c) c o n trib u te to an u n d e rs ta n d in g o f the e n z y m ic c a p a b ilitie s o f
r h iz o b ia so th a t f u r t h e r s tu d ie s on th e m e c h a n is m o f n itr o g e n
fix a tio n , in te r c o n v e r s io n o f c e llu la r n itr o g e n o r t r a n s f e r of

- 3 -

fix e d n itr o g e n to th e h o s t p la n t c o u ld be c a r r ie d o u t m o re
in t e llig e n t ly .
T h is s tu d y is c o m p lic a te d , as a re a l l in v e s tig a tio n s on liv in g c e lls
o r c ru d e c e llu la r p r e p a ra tio n s , b y the tre m e n d o u s in t e r p la y o f in t r a c e llu ­
la r enzym es.

In a d d itio n , l i t t l e is kn o w n o f the r a te s o f re a c tio n s w h ic h

o c c u r in th e liv in g c e ll.

A n y p a r t ic u la r r a te is n e c e s s a r ily d y n a m ic in

th a t i t is a c c e le ra te d o r re ta r d e d de p e n d in g up on the n a tu re o f the p h y s io c h e m ic a l e n v iro n m e n t, and upon the o p e ra tio n o f in te rc o n n e c te d p ro c e s s e s .
A l l th a t can be s a id a f t e r su ch a s tu d y is th a t th e o r g a n is m is p o te n tia lly
c a p a b le o f c a r r y in g out su ch and su ch a re a c tio n , b u t even th a t re p re s e n ts
a g l i t t e r o f lig h t in th e d a rk n e s s o f c e llu la r m e ta b o lis m .

- 4 -

PART I

N IT R O G E N N U T R IT IO N A N D M E T A B O L IS M

- 5 -

IN T R O D U C T IO N A N D L IT E R A T U R E R E V IE W

T he lit e r a t u r e d e a lin g w ith th e le g u m e n o d u le b a c te r ia is f i l l e d
w ith in n u m e ra b le r e p o r ts on th e n u t r it io n o f th e s e u n u s u a l m ic r o o r g a n ­
is m s .

M a n y o f th e se r e p o r ts a re c o n tr a d ic to r y and the r e s u ltin g c o n ­

fu s io n p ro b a b ly s te m s f r o m the v a r ie d e x p e r im e n ta l c o n d itio n s w h ic h the
d iffe r e n t in v e s tig a to r s u s e d .

In som e ca se s th e m e d ia u s e d as a ba se

c o n ta in e d s o il o r p la n t e x tr a c ts w hose e x a c t c o m p o s itio n a r e un know n ,
even to d a y .

M o s t o f th e o ld e r w o r k e r s e m p lo y e d u n w a sh e d c e lls as

in o c u la and the p re s y n th e s iz e d m a te r ia ls a d h e rin g to th e se c e lls w e re
n e g le c te d in the in te r p r e ta t io n o f the e x p e r im e n ta l fin d in g s .

N e v e r th e le s s

m a n y o f the r e p o r ts a re v a lu a b le in th a t th e y do in d ic a te w h e th e r o r n o t
th e r h iz o b ia l s tr a in s e m p lo y e d c o u ld u t iliz e th e v a r io u s te s t s u b s ta n c e s ,
but the a b ilit y o f su ch su b s ta n c e s to in it ia t e the g ro w th o f w a sh e d c e lls in a
c h e m ic a lly d e fin e d in o rg a n ic b a s a l m e d iu m c o u ld n o t be s ta te d w ith a n y
d e g re e o f c e r ta in ty .

S ta te d in a n o th e r fa s h io n the c o m p o u n d s u n d e r te s t

c o u ld be c la s s ifie d as s tim u la t o r y o r n o n - s tim u la to r y o n ly , and n o t as
e s s e n tia l o r n o n - e s s e n tia l.

A n o th e r im p o r ta n t fe a tu r e th a t m u s t n o t be

d is re g a rd e d in re a d in g th e a v a ila b le lit e r a t u r e is the in flu e n c e o f p h y s io
c h e m ic a l fa c t o r s on the n u t r it io n o f the b a c te r ia .

Such f a c t o r s a s pH , E h ,

te m p e r a tu r e e tc . a re e x tr e m e ly im p o r ta n t and i t is to be la m e n te d th a t
m u c h o f th is in fo r m a tio n has be en o m itte d f r o m e a r l i e r e x p e r im e n ta l
re p o rts .

S p e cie s o r s t r a in d iffe re n c e s a re , o f c o u rs e , a d d itio n a l m a jo r

- 6 -

fa c t o r s and in v e s tig a tio n s w h ic h a t f i r s t a p p e a r c o n tr a d ic t o r y a r e o fte n
a r e s u lt o f su ch in h e re n t d iffe re n c e s a m o n g m ic r o o r g a n is m s .
T h e q u e s tio n as to w h e th e r o r n o t r h iz o b ia a re c a p a b le o f i n i t i a t ­
in g g ro w th in c h e m ic a lly p u re in o r g a n ic s y n th e tic m e d ia is e x tr e m e ly
im p o r ta n t, b u t in a l l p r o b a b ility can n e v e r be a n s w e re d in e ith e r th e
a f f ir m a t iv e o r th e n e g a tiv e f o r a l l s t r a in s o f th e o r g a n is m .

E a c h s t r a in

w o u ld be e xp e cte d to be have in i t s ow n p e c u lia r fa s h io n in th is r e s p e c t.
W ils o n and W ils o n (101) fo u n d th a t c o n tin u o u s t r a n s f e r o f R h. m e li l o t i
and R h. t r i f o l i i w as p o s s ib le in a m e d iu m c o n ta in in g p u r if ie d s a lts p lu s
a lc o h o l e x tr a c te d g lu c o s e , s u c ro s e o r s y n th e tic g ly c e r o l.

T h e y c o n c lu d e d ,

h o w e v e r, th a t th e re w e re a t le a s t th r e e p r in c ip a l g ro u p s o f o rg a n is m s :
a) th o se th a t g re w v e r y p o o r ly in the ab se n ce o f b io t in and re a c h e d a p o p u ­
la tio n o f about one te n th the m a x im u m b ) th o s e th a t a tta in e d p r a c t ic a lly
m a x im u m g ro w th in the ab sence o f b io t in and c) th o s e s tr a in s th a t w e re
u n a b le to g ro w u n le s s b io tin w a s s u p p lie d .
m e d iu m w a s a m m o n iu m c h lo r id e .

T h e n itr o g e n s o u rc e in t h e ir

W e s t and W ils o n (98) s ta te d th a t

“ s m a ll in o c u la , w a sh e d f r e e o f m e ta b o lic p ro d u c ts o f the c u ltu r e f r o m
w h ic h th e y w e re ta k e n , p ro d u c e l i t t l e o r no g ro w th in the ab se n ce o f the
R h iz o b iu m f a c t o r '* .

A lth o u g h th e “ f a c t o r ” in th is ca se w a s a h e a t la b ile

su b s ta n c e p ro d u c e d b y a c tiv e ly g ro w in g c u ltu r e s o f th e o r g a n is m and
th o u g h t to c o n ta in th ia m in and r ib o f la v in the f a c t is e m p h a s iz e d in a m o re
re c e n t r e p o r t (48) th a t th e “ f a c t o r ” m a y a c tu a lly be a n y one o f a n u m b e r
o f a m in o a c id s w h ic h s u p p ly e s s e n tia l a m in o g ro u p s to th e o r g a n is m .

- 7 -

F o r th e p u rp o s e s o f c l a r i t y th is r e v ie w ha s be en d iv id e d in to s e v e r a l
s e c tio n s , a l l r e la te d to th e n itr o g e n n u t r it io n o f the n o d u le b a c te r ia .

The

f i r s t s e c tio n d e a ls w ith the u t iliz a t io n o f in o r g a n ic n itr o g e n w h ile th e

se­

cond has re fe re n c e to o rg a n ic n itro g e n o u s c o m p o u n d s .

T h e f in a l s e c tio n

b r ie f l y d is c u s s e s the e ffe c t o f pH and te m p e r a tu re on r h iz o b ia .

I . In o rg a n ic

A s f a r b a c k as 1890 P r a z m o w s k i (73) re c o m m e n d e d the u se o f a
n itr o g e n - p o o r m e d iu m f o r th e g ro w th o f r h iz o b ia .

T h is re c o m m e n d a tio n

w a s s u p p o rte d b y su ch m e n as Z in s s e r (107) and D a w so n (2 3).

H a r r is o n

and B a r lo w (39) and G r e ig - S m ith (36), a m ong o th e rs , a c tu a lly a d v o c a te d
the use o f m e d ia d e v o id o f an y added c o m b in e d n itr o g e n s o u rc e .

Of

c o u rs e tra c e s o f n itro g e n o u s co m p o u n d s in the m in e r a l s a lts u s e d in the
p r e p a r a tio n o f su c h m e d ia w o u ld have e n s u re d the p re s e n c e o f a t le a s t
som e c o m b in e d n itr o g e n , o th e rw is e the o rg a n is m s , w h ic h c a n n o t f i x fr e e
n itr o g e n in the a b sence o f th e h o s t p la n t, w o u ld have s u ffe re d f r o m n i t r o ­
gen s ta r v a tio n .

F r e d , B a ld w in , and M c C o y (26) r e p o r te d g r o w th s t im u ­

la tio n o f n o d u le o rg a n is m s b y th e a d d itio n o f n it r a t e to a m e d iu m s u c h as
A s h b y ’ s.

B e ije r in c k (8), M u lle r and Stapp (63), L e o n a rd (55), and

P o h lm a n (72) a l l g re w r h iz o b ia in n it r a t e m e d ia .

H o w e v e r, a l l th e s e

w o r k e r s u s e d u n w a sh e d in o c u la and in som e c a s e s (55), s o il and p la n te x tr a c t b a s a l m e d ia as w e ll.

M u lle r and Stapp (63) fo u n d th a t a m m o n iu m

s a lts c o u ld be u t iliz e d b y un w a sh e d c e lls o f th e s e b a c te r ia , b u t t r i e d

- 8 -

u n s u c c e s s fu lly to g ro w th e m in m e d ia c o n ta in in g n i t r i t e .

P o h lm a n ’ s

w o r k (72) e m p h a s iz e d s p e c ie s d iffe re n c e s w h e n he r e p o r te d th a t R h.
ja p o n ic u m u t iliz e d a m m o n iu m s u lp h a te in a m a n n ito l m in e r a l- s a lts
m e d iu m to b e t t e r a d vantage th a n R h . m e lilo t i, p o s s ib ly b e c a u s e th e
soybean o r g a n is m is ca p a b le o f n e u tr a liz in g th e s u lp h u r ic a c id l i b e r a ­
te d , th u s p ro lo n g in g th e p e rio d o f fa v o ra b le g r o w th .
A llis o n and H o o v e r (3), u s in g a y e a s t- e x t r a c t m in e r a l- s a lt s m e d iu m ,
p u b lis h e d a r e p o r t s ta tin g th a t p o ta s s iu m n it r a t e and a m m o n iu m c h lo r id e
w e re u t iliz e d , b u t la t e r (42) r e p o r te d th a t th e g ro w th o f c e r t a in s p e c ie s
in a s y n th e tic m e d iu m c o m p o s e d o f s u g a r, in o r g a n ic s a lts and n it r a t e
w as n e g lig ib le i f p u re in g re d ie n ts w e re u s e d .

A lth o u g h T h o rn e and

W a lk e r (90) a d vo ca te d th e a d d itio n o f c y s te in e to the above m e d iu m to
o v e rc o m e th e h ig h o x id a tio n -re d u c tio n p o te n tia l, N ils s o n , B ja lfv e , and
B u r s tr o m (66) o b s e rv e d th a t no g ro w th to o k p la c e in th is m e d iu m even
w hen re d u c in g s u b s ta n c e s w e re added o r w hen a m m o n iu m c h lo r id e w as
s u b s titu te d f o r th e n it r a t e .

A lly n and B a ld w in (5) in v e s tig a te d th e e ffe c t

o f the re d o x p o te n tia l on u n w ashe d c e lls o f n o d u le b a c te r ia and fo u n d
th a t th e y g re w in a s o lid m a n n ito l n it r a t e m e d iu m , b u t n o t in m a n n ito l
n it r a t e b r o th u n le s s c y s te in e w as added.

T h o rn e and W a lk e r (89) in

f u r t h e r s tu d ie s m a in ta in e d th a t in t h e i r w o r k s e v e r a l R h iz o b iu m s p e c ie s
w e re u n a b le to m a in ta in g r o w th in a c h e m ic a lly p u re m in e r a l- s a lt s
s u c ro s e m e d iu m w ith p o ta s s iu m n it r a te as th e n itr o g e n s o u rc e .

N e v e r­

th e le s s , th e se s tr a in s c o u ld be c u ltiv a te d in d e fin ite ly i f th e p o ta s s iu m

- 9 n it r a t e w a s re p la c e d b y N H ^ C l o r a s p a ra g in e ,

J o rd a n (48) u s in g w e ll

w a sh e d r h iz o b ia l c e lls s ta te d th a t th e s tr a in s o f a lfa lfa - s w e e t c lo v e r
b a c te r ia u s e d in h is s tu d ie s w o u ld n o t u t iliz e a m m o n iu m c h lo r id e as th e
s o le s o u rc e o f n itr o g e n in a c h e m ic a lly d e fin e d m e d iu m c o n ta in in g p u r i ­
fie d s u c ro s e and m in e r a l s a lt s ,

R ic h a rd s o n and J o rd a n (77) e xte n d e d

th e se r e s u lts to in c lu d e a m m o n iu m s u lp h a te , c a lc iu m n it r a t e , p o ta s s iu m
n itr a te , p o ta s s iu m n it r it e , s o d iu m n it r a t e and s o d iu m n i t r i t e .

Is h iz a w a

(43) a ls o re p o r te d th a t c e r ta in cow pea r h iz o b ia w e re u n a b le to u t iliz e
a m m o n iu m s a lts o r n it r a t e .
A n u m b e r o f w o r k e r s have s tu d ie d th e re d u c tio n o f n it r a t e b y
r h iz o b ia ,

P ru c h a (75), H il l s (40), and W ils o n (99) fo u n d no n it r a t e r e ­

d u c tio n f o r the f ie ld pea, a lfa lfa , and soyb ean o r g a n is m r e s p e c tiv e ly ,
Is h iz a w a (43) r e p o r te d th a t a fe w s tr a in s o f bean and cow pea r h iz o b ia
w e re u n a b le to re d u c e n it r a t e in n it r a t e y e a s t- w a te r m a n n ito l m e d iu m .
N e v e rth e le s s , m o re re c e n t w o r k e r s s u c h as W ils o n and C h in (102) a re
o f the o p in io n th a t a l l s tr a in s o f r h iz o b ia m a y re d u c e n it r a t e s to n i t r it e s
w hen a c e r ta in c o n c e n tra tio n o f n it r a t e is p ro v id e d .

W ils o n (100) c la im e d

th a t th e n i t r it e w i l l r e a c t in a c id ic m e d ia w ith a n u m b e r o f co m p o u n d s to
lib e r a te ga seous n itr o g e n .

P o h lm a n (72) fo u n d th a t n i t r it e s te n d e d to

a c c u m u la te in c u ltu r e s o f R h. m e li l o t i and R h. ja p o n ic u m and th e re w as
som e in d ic a tio n o f a s lig h t n i t r i t e u t iliz a t io n b y b o th o r g a n is m s .

Is h iz a w a

(43) show ed th a t d is tin c t d iffe re n c e s a re p re s e n t a m o n g s t d iffe r e n t s tr a in s
o f r h iz o b ia in t h e ir a b ilit y to u t iliz e a m m o n iu m s a lts , n it r a t e and n i t r i t e .
A lth o u g h the a lfa lfa o r g a n is m w a s u n a b le to u t iliz e n i t r i t e c e r ta in s p e c ie s

- 10 -

w e re ca p a b le o f d o in g so .
T h e re is l i t t l e e x p e r im e n ta l e v id e n c e c o n c e rn in g the a b ilit y o f
r h iz o b ia to f o r m a m m o n ia f r o m n it r a te s a lth o u g h Z ip f e l (108) n o te d
th a t th e o rg a n is m s w h ic h n o d u la te pea, r e d c lo v e r , h o rs e bean and g a r ­
den be an a l l re d u c e n it r a t e to n i t r it e , b u t n o t to a m m o n ia .

H o w e v e r,

th e se r e s u lts m a y be due to th e r a p id u t iliz a t io n b y the o rg a n is m s o f an y
a m m o n ia fo r m e d .
S e v e ra l in v e s tig a to r s have s tu d ie d th e s tim u la t o r y p o w e rs o f added
in o r g a n ic n itr o g e n s o u rc e s upon th e u p ta k e o f o xyg en b y c e lls susp end ed
in c a rb o h y d ra te m e d ia .

W a lk e r, A n d e rs o n , and B ro w n (97) in a s e r ie s

o f m ic r o r e s p ir o m e t e r s tu d ie s re p o r te d th a t un w ashed c e lls o f R h . le g u m in o s a ru m in a g lu c o s e m in e r a l's a lt s m e d iu m w e re n o t s tim u la te d in t h e ir
oxygen u p ta k e b y a m m o n iu m c h lo r id e o r s o d iu m n it r a t e o v e r a p e rio d o f
t h ir t y h o u rs .

N e a l and W a lk e r (64) in v e s tig a te d th e o xyg e n c o n s u m p tio n

b y tw ic e -w a s h e d a lfa lfa r h iz o b ia g ro w in g f o r tw e n ty - fo u r h o u rs in W a rb u rg
v e s s e ls c o n ta in in g e ith e r s o d iu m n it r a t e o r a m m o n iu m c h lo r id e m in e r a ls a lts b a s a l m e d iu m p lu s an y one o f s e v e r a l d iffe r e n t c a rb o n s o u rc e s .

The

u p ta k e of gaseous o xyg e n w as g r e a te r in the a m m o n iu m c h lo r id e m e d iu m
tha n in the n it r a t e b a se .
B u r r is an d W ils o n (15) e m p lo y e d “ r e s tin g c e l l ” te c h n iq u e s in a
s e r ie s o f m a n o m e tr ic e x p e rim e n ts d e s ig n e d to d e te rm in e the m a n n e r o f
u t iliz a t io n o f h e m o g lo b in - lik e co m p o u n d s b y r h iz o b ia .

T h e ir r e s u lts

- 11 -

show ed th a t c e r ta in in o r g a n ic n itr o g e n s a lts (as w e ll as m a n y a m in o
a c id s ) in c re a s e d th e r e s p ir a t io n o f w a sh e d s u s p e n s io n s o f f o u r s tr a in s
o f r h iz o b ia fu r n is h e d w ith g lu c o s e .

W ith c e r ta in s tr a in s C a (N 0 3 )2 ,

(N H 4 )2H P 0 4, (N H 4 )2 S 0 4> N a N 0 2 and N H 4N O s w e re , h o w e v e r, d e c id e d ly
in h ib it o r y to w a rd s oxyg e n u p ta k e .
A s a re s u m e i t m a y be s a id th a t c e r ta in s p e c ie s o r s tr a in s o f
n o d u le b a c te r ia a re c a p a b le o f in it ia t in g g ro w th in a c h e m ic a lly d e fin e d
m e d iu m c o n ta in in g a m m o n iu m s a lts o r n itr a te as th e s o le s o u rc e o f n i t ­
ro g e n ,

T o the m a jo r it y o f s tr a in s , h o w e v e r, th e s e in o rg a n ic n itr o g e n

s o u rc e s a re p u r e ly s tim u la to r y , n o t o n ly to w a rd s c e ll p r o lif e r a t io n , b u t a ls o
to w a rd s c e llu la r r e s p ir a t io n as ty p ifie d b y “ r e s tin g c e l l ” e x p e r im e n ts .
In m o s t ca se s th e n it r a t e is re d u c e d to n i t r it e and th e n c e to a m m o n ia ,
w h ic h , h o w e v e r, is p ro b a b ly r a p id ly m e ta b o liz e d and m a y n o t be d e te c t­
a b le in c u ltu r e m e d ia .

A lth o u g h n i t r i t e m a y be s tim u la t o r y to som e s p e c ie s

in m o s t c a s e s i t is n o t u t iliz e d to an y d e te c ta b le e x te n t.

2. O rg a n ic

D u rin g a s tu d y o f the e ffe c ts o f o rg a n ic n itr o g e n co m p o u n d s on
r h iz o b ia l g ro w th , M u lle r and Stapp (63) fo u n d th a t a s p a ra g in e , s o d iu m
a s p a ra g in a te , and u r ic a c id w o u ld s e rv e as n itr o g e n s o u rc e s f o r u n w a sh e d
c e lls w h ile g ly c in e and h ip p u r ic a c id w e re u n s u ita b le .
and u re a gave o n ly w e a k g r o w th .

A la n in e , le u c in e ,

P o h lm a n (72) r e p o r te d th a t u n w a sh e d

c e lls o f R h . m e li l o t i and R h. ja p o n ic u m u t iliz e d g ly c in e , d l- a la n in e ,

- 12 -

d l- a m in o - n - b u t y r ic a c id , d l- v a lin e , d - g lu ta m ic a c id , 1 -c y s tin e , 1ty r o s in e , d l-p h e n y la la n in e , u re a , a s p a ra g in e and p e p to n e , u s u a lly w ith
an a tta c k up o n the a m in o g ro u p and w ith th e lib e r a t io n o f fr e e a m m o n ia
in th e case o f g ly c in e and d l- a la n in e .

In c e r ta in c u ltu r e s a s lo w o x id a ­

tio n o f ty r o s in e to d ih y d ro x y p h e n y la la n in e and f i n a l ly to m e la n in to o k
p la c e .

W e s t and W ils o n (98) m e n tio n e d th a t J 3 -a la n in e w as e s s e n tia l f o r

the g ro w th o f R h. t r i f o l i i , b u t N ie ls e n and Jo h a n se n (65) d is a g re e d s in c e
th e y fo u n d th a t th is p a r t ic u la r a m in o a c id w as n e ith e r e s s e n tia l n o r
s tim u la to r y to t h e ir s tr a in s o f R h. m e li l o t i , R h. t r i f o l i i and R h . lu p in i.
A llis o n an d H o o v e r (3) u s e d b o th u r e a and a s p a ra g in e in t h e ir b a s a l
m e d iu m as the s o le s o u rc e o f n itr o g e n and d is c o v e re d th a t u r e a w as to x ic
to th e c lo v e r o r g a n is m b u t n o t to the a lf a lfa R h iz o b iu m .

T h is in h ib itio n

w as b e lie v e d to be due to an e x c e s s iv e r is e in p H .
R ic h a rd s o n and J o rd a n (77) in a re c e n t s tu d y fo u n d th a t w a sh e d
r h iz o b ia w e re u n a b le to u t iliz e a c e ta m id e , a m y la m in e , b u ty la m in e ,
b u ty ra m id e , fo r m a m id e , g u a n id in e h y d r o c h lo r id e , h e x y la m in e , o x a m id e ,
p ro p io n a m id e , p ro p y la m in e , s u c c in im id e , th io u re a , u re a , v a le ra m id e ,
h y d ra z in e o r h y d ro x y la m in e as th e s o le s o u rc e o f n itr o g e n in a c h e m ic a lly
d e fin e d m e d iu m .

G lu ta m in e w as u t iliz e d w ith the p ro d u c tio n o f c o n s id e r ­

a b le q u a n titie s of f r e e a m m o n ia .

H is ta m in e w as a ls o u tiliz e d , b u t to a

v e r y s m a ll e x te n t and no a m m o n ia w a s fo u n d , a lth o u g h th is c o u ld c o n c e iv ­
a b ly be due to a r a p id a n a b o lis m o f th e s m a ll a m o u n t of a m m o n ia fo r m e d .
W a lk e r, A n d e rs o n and B ro w n (97) r e p o r te d th a t a la n in e and u r e a

- 13 -

d id n o t in c re a s e th e oxyg e n u p ta k e b y u n w a sh e d c e lls o f R h . t r i f o l i i ,
R h. p h a s e o li, and R h . le g u m in o s a ru m i n a g lu c o s e m in e r a l- s a lts m e d ­
iu m w h e re a s R h . m e li l o t i w a s s tim u la te d b y th e s e c o m p o u n d s .

T h is

la t t e r s tim u la tio n w as o f th e sam e o r d e r a s th e s tim u la tio n o f a l l f o u r
s tr a in s b y y e a s t e x tr a c t, w h ic h gave in c re a s e d a m o u n ts o f o x y g e n c o n ­
s u m p tio n in p r o p o r tio n to the a m o u n t ad ded .

In th is r e g a r d A lle n and

A lle n (2) su g g e st s e v e r a l p la u s ib le e x p la n a tio n s f o r the g e n e ra l u t i l i z a ­
tio n o f y e a s t e x tr a c t b y r h iz o b ia s u c h as a) th e a v a ila b ilit y o f v a r io u s
e s s e n tia l p r o te in d e g ra d a tio n p ro d u c ts , b) th e p re s e n c e o f c e r ta in s u b ­
s tr a te s ca p a b le o f s tim u la tin g r e s p ir a t io n , c) the s tim u la to r y e ffe c t o f
v a r io u s v ita m in s and a c c e s s o ry g ro w th fa c t o r s , d) the p re s e n c e o f c e r ta in
tr a c e e le m e n ts , and e) th e p o is in g o f th e E h a t an o p tim u m v a lu e .
B u r r is and W ils o n (14) r e p o r te d th a t im p u r e , b u t not p u re fr a c t io n s
o f b io tin p re p a ra tio n s s tim u la te d r e s p ir a t io n .

N one o f a n u m b e r o f v i t a ­

m in s and a c c e s s o ry g ro w th f a c t o r s c o u ld r e p la c e th is im p u r e f r a c t io n .
O f a n u m b e r of a m in o a c id s e x a m in e d , a s p a r tic a c id , a s p a ra g in e , h i s t i ­
d in e , and a r g in in e w e re fo u n d to be v e r y s t im u la to r y .

C a s e in h y d ro ly s a te

c o m p le te ly re p la c e d th e s tim u la t o r y a c t iv it y o f the im p u r e b io t in f r a c t io n
and h is tid in e and a r g in in e c lo s e ly a p p ro a c h e d i t .

T h is a c tio n o f the n i t r o ­

genous co m p o u n d s a p p a re n tly d id n o t a r is e v ia a s tim u la tio n o f c e ll d iv is io n .
T h e s e sam e w o r k e r s (15) in a f u r t h e r s tu d y fo u n d th a t c e r t a in a m in o a c id s
in c re a s e d th e r e s p ir a t io n o f w a s h e d s u s p e n s io n s of f o u r s tr a in s o f r h iz o b ia
fu r n is h e d w ith g lu c o s e .

T h is s tim u la tio n w a s im m e d ia te and w as n o t due

- 14 -

to c e llu la r p r o lif e r a t io n .

T h e to t a l in c re a s e in o xyg e n u p ta k e w as d e p e n ­

d e n t up on the c o n c e n tra tio n o f the n itr o g e n

c o m p o u n d s u p p lie d and yo u n g

c e lls w e re s tim u la te d so m e w h a t m o re th a n o ld e r c e lls .

C y s te in e w as

in h ib it o r y to th re e o f th e f o u r s t r a in s in r e s p e c t to the u p ta k e o f ga seou s
o xyg e n .
J o rd a n (48), in a s tu d y in v o lv in g a la r g e n tim b e r o f a lfa lfa - s w e e t
c lo v e r r h iz o b ia and m u ta n ts d e riv e d f r o m th e m b y X - r a y ir r a d ia t io n ,
s ta te d th a t w a sh e d

c e lls r e q u ir e d a m in o a c id s f o r g ro w th in a s y n th e tic ,

p u r ifie d m in e r a l- s a lt s s u c ro s e m e d iu m a t pH 7 .0 .
c o u ld be

T h is re q u ir e m e n t

s a tis fie d b y a n y one o f a la r g e n u m b e r of a m in o a c id s , w h ic h

v a r ie d s lig h t ly f r o m c u ltu r e to c u ltu r e .

T h e s e a m in o a c id s w e re n o t

c o n c e n tra te d in t r a c e l lu l a r l y as in the case o f c e r ta in , i f n o t a ll, G ra m
p o s itiv e b a c te r ia and c o u ld n o t be re p la c e d b y in o rg a n ic n itro g e n o u s c o m ­
pounds e ith e r in the p re s e n c e o r a b sence o f a n y one o f a n u m b e r o f v i t a ­
m in s , p u rin e s o r p y r im id in e s .

T h e r e fo r e , th e s tim u la tio n o f th e s e o r ­

g a n is m s b y y e a s t e x tr a c t w as th o u g h t to be p r in c ip a lly due to th e h ig h
c o n c e n tra tio n o f a m in o a c id s c o n ta in e d in th is m a te r ia l, a lth o u g h th e b e n e ­
f i c i a l in flu e n c e o f o th e r m a t e r ia ls c o u ld not be e n t ir e ly n e g le c te d .
T h e s e r e p o r ts m a y se e m c o n tr a d ic to r y in m a n y c a s e s b u t, as s ta te d
b e fo re , m a y no t a c tu a lly be so s in c e s t r a in d iffe re n c e s and e n v ir o n m e n ta l
c o n d itio n s a r e a l l c a p a b le o f m o d ify in g r e s u lts .

C e r t a in ly i t is a p p a re n t

th a t m o s t a m in o a c id s a re c a p a b le o f in it ia t in g g r o w th o f w a sh e d c e lls in
a s y n th e tic m e d iu m d e v o id o f an y o th e r n itr o g e n s o u rc e and th a t in a d d i-

- 15 -

tio n th e y can, f o r th e m o s t p a r t, s tim u la te th e c e llu la r r e s p ir a t io n o f
*‘ r e s tin g c e lls * * susp end ed in a c a rb o h y d ra te s u b s ta te .
T h e re a r e a m a z in g ly fe w r e p o r ts d e a lin g w ith th e m e c h a n is m s o f
a m in o a c id u t iliz a t io n b y th e n o dule b a c te r ia .

A s m e n tio n e d p r e v io u s ly

P o h lm a n (72) b e lie v e d th a t th e m ode o f a tta c k w a s d ir e c te d a g a in s t the
a m in o g ro u p and, in the case o f tw o a m in o a c id s , w ith th e fo r m a t io n o f
f r e e a m m o n ia .

W ils o n (103) in h is “ r e s tin g c e l l ” p ro c e d u re fo u n d th a t

a m m o n ia w as lib e r a te d b y endogenous r e s p ir a t o r y a c t iv it ie s , due p r o ­
b a b ly to d e a m in a tio n o f c e llu la r a m in o a c id s . V ir ta n e n et a l. (95) d e c id e d
th a t th e m o de o f a tta c k a g a in s t a s p a r tic and g lu ta m ic a c id s w as one o f
d e c a rb o x y la tio n , w ith the r e s u lt in g fo r m a tio n , in th e f o r m e r ca se , o f
b e ta -a la n in e .

B ille n and L ic h s te in (11) f u r t h e r s tu d ie d th e a s p a r tic a c id

d e c a rb o x y la s e o f R h . t r i f o l i i and e s tim a te d th e pH o p tim u m a t 5. 7.
J o rd a n (49) e m p h a s iz e d th e im p o rta n c e o f tra n s a m in a tio n r e a c tio n s in
R h iz o b iu m m e ta b o lis m w h e n he d e m o n s tra te d the fo r m a tio n o f g lu ta m ic
a c id f r o m a lp h a - k e to g lu ta r ic a c id w hen a s p a r tic a c id , a la n in e , le u c in e ,
and v a lin e a c te d a s a m in o d o n o rs .
In r e la t io n to th e a c t iv it y o f r h iz o b ia to w a rd s s y n th e tic p e p tid e s ,
B e r g e r , Jo h n so n , and P e te rs o n (9) c o m p a re d th e r e la t iv e p e p tid a s e c o n ­
te n ts o f a n u m b e r o f m ic r o - o r g a n is m s .

A n e x tr a c t f r o m te n g r a m s o f

w e t R h . t r i f o l i i c e lls p o s s e s s e d a c t iv it y a g a in s t d l- a la n y lg ly c in e , d l le u c y lg ly c in e , d ig ly c in e , d l- a la n y ld ig ly c in e , d l- le u c y ld ig ly c in e and t r i ­
g ly c in e .

T h e r e s u lt in g r a te s w e re o f a lo w o r d e r c o m p a re d to m a n y o f

\/l) v

-16 -

the o th e r o rg a n is m s te s te d .

B u r r is and W ils o n (15) show ed th a t g ly -

c y lg ly c in e w a s ca p a b le o f s tim u la tin g th e u p ta k e o f ga seou s o x y g e n b y
fo u r R h iz o b iu m s tr a in s s u p p lie d w ith g lu c o s e .
T h e r e have be e n no p u b lis h e d r e p o r ts to th e a u th o r ’ s k n o w le d g e
w h ic h d e a l p r i m a r i l y w ith th e u t iliz a t io n o f D - a m in o a c id s o r p u re D -a m in o
a c id p e p tid e s b y m e m b e rs o f th e genus R h iz o b iu m .

3. pH and T e m p e ra tu re

F r e d , B a ld w in and M c C o y (26) have s u m m a riz e d the to p ic c o n ­
c e rn in g th e e ffe c t o f h y d ro g e n io n c o n c e n tra tio n on th e g ro w th and lo n g e ­
v it y o f r h iz o b ia in liq u id m e d ia and in th e s o il.

O p in io n s v a r y b u t, in

g e n e ra l, th e v a r io u s s p e c ie s a r e a ffe c te d d if f e r e n t ly b y a c id it y and p o s s ­
ess a b out th e s a m e to le ra n c e to a lk a lin it y .

R h . m e li l o t i is the m o s t a c id

s e n s itiv e and is fa v o r e d b y s lig h t a lk a lin it y o r n e u t r a lit y ,
ic u m and R h. lu p in i a r e the m o s t a c id to le r a n t s p e c ie s .

R h . ja p o n ­
U s in g th e W a r ­

b u rg te c h n iq u e T h o rn e and W a lk e r (89) s tu d ie d th e g ro w th and r e s p i r a ­
tio n o f r e p r e s e n ta tiv e s tr a in s o f R h. m e li l o t i and R h . ja p o n ic u m a s a
fu n c tio n o f m e d iu m pH .

T h e g ro w th o f th e f o r m e r in a g lu c o s e y e a s t-

e x tr a c t m e d iu m w a s fo u n d to d e c re a s e f r o m a m a x im u m a t pH 7. 0 to
z e ro b e tw e e n pH 4 . 6 and 4. 9 on th e a c id s id e o f n e u t r a lit y and pH 9. 6 on
th e a lk a lin e s id e .

T h e g ro w th o f R h. ja p o n ic u m in a ra b in o s e y e a s t - e x ­

t r a c t m e d iu m d e c re a s e d f r o m a m a x im u m a t pH 6. 7 - 6. 9 to z e ro a t
pH 4. 2 and 9. 5.

T h e i n i t i a l r e s p ir a t io n r a te f o r the tw o s p e c ie s re a c h e d

- 17 -

a m a x im u m b e tw e e n pH 7, 2 and 8 . 0 and d e c re a s e d to a v e r y s m a ll v a lu e
a t th e c r i t i c a l p H .

D e h y d ro g e n a s e a c t iv it y o f th e n o d u le b a c te r ia o c c u rs

b e tw e e n pH 4 . 0 an d 10. 0 w ith an o p tim u m in th e a lk a lin e ra n g e o f pH
8 .0 to 8 .2 (8 6 ).

Is h iz a w a (44) d e c id e d th a t a n e u tr a l o r a lk a lin e pH

(7 -7 . 5) w as b e tte r f o r a lfa lfa , soyb e a n and cow pea b a c te r ia , w h ic h w e re
a l l a c id s e n s itiv e h a v in g a c r i t i c a l pH o f 5. 5 - 6. 0.
A llis o n and M in o r (4) s u m m e d up th e s ta tu s o f th e k n o w le d g e on
th e te m p e r a tu re r e q u ir e m e n ts o f th e n o d u le b a c te r ia in th e s ta te m e n t,
“ T h e lit e r a t u r e is s u r p r is in g ly d e fic ie n t in d a ta s h o w in g q u a n tita tiv e ly
th e e ffe c t of te m p e r a tu re on the g ro w th r a te s o f r h iz o b ia * * .

These

w o r k e r s u se d a b a s a l m in e r a l m e d iu m c o n ta in in g s u c ro s e , y e a s t w a te r
and a s p a ra g in e and c e ll n u m b e rs w e re co u n te d w ith a L e v y - H a u s s e r
c h a m b e r.

U s in g n in e R h iz o b iu m c u ltu r e s , r e p re s e n tin g n in e s p e c ie s

th e y show ed th a t th e o p tim u m g ro w th ra te f o r m o s t o f th e s e o rg a n is m s
la y b e tw e e n 29 and 3 1 °C a lth o u g h R h . m e li l o t i g re w b e s t a t 3 5 °C .

No

g ro w th o c c u r r e d w ith in 2 4 -4 8 h r s . w ith m o s t s p e c ie s h e ld a t 3 7 °C , b u t
a te m p e r a tu re o f 4 1 °C w a s r e q u ir e d to p re v e n t th e g ro w th o f R h. m e li l o t i .
T h e g r o w th r a te a t 15°C w as o n ly one q u a r te r to one h a lf as g re a t as a t
3 0 °C .

Is h iz a w a (45) c o n fir m e d A llis o n and M in o r ’ s w o r k w ith th e c o n ­

c lu s io n th a t the o p tim u m te m p e r a tu re f o r R h . m e li l o t i w as a b o u t 3 4 °C .
I t m u s t be r e a liz e d th a t a g r e a t d e a l o f w o r k m u s t s t i l l be done on
R h iz o b iu m p h y s io lo g y , n o t o n ly in th e p u r s u it o f new lin e s o f in v e s tig a tio n ,
b u t a ls o f o r th e p u rp o s e o f a r e a p p r a is a l o f f o r m e r w o r k in th e lig h t o f

- 18 -

n e w e r d is c o v e r ie s and te c h n iq u e s .

I t is o n ly th ro u g h an e x a c t u n d e r ­

s ta n d in g o f the b io c h e m ic a l a c t iv it ie s o f th e se m ic r o o r g a n is m s th a t one
can hope to d is c e r n d iffe r e n c e s b e tw e e n p a r a s itic , in e ffe c tiv e and
e ffe c tiv e r h iz o b ia and to g a in f u r t h e r in s ig h t in to th e a c tu a l m e c h a n is m
o f the p ro c e s s o f s y m b io tic fix a t io n ,

A,

T h e u t iliz a t io n o f L - , D - , and D L - a m in o a c id s and th e e ffe c ts
o f pH and te m p e r a tu r e .

In a lm o s t e v e r y te x t-b o o k o f b io c h e m is tr y a m in o -a c id s a r e sp o ke n
o f a s th e “ b u ild in g b lo c k s * ' f o r p r o te in s y n th e s is .

A lth o u g h th is s ta te ­

m e n t has n o t y e t been a b s o lu te ly p ro v e d ^ it is , n e v e rth e le s s , a lm o s t i m ­
p o s s ib le to o v e re m p h a s iz e th e v a lu e o f th e se co m p o u n d s to the liv in g c e ll.
A m in o a c id s a re u s u a lly d e s ig n a te d b y the p r e f ix L .- o r D - , d e n o tin g a
p a r t ic u la r o p tic a l is o m e r , s in c e a l l of th e se a c id s e x c e p t g ly c in e have an
a s y m m e tr ic a lp h a - c a rb o n a to m .

T h e c o n fig u r a tio n o f the n a t u r a lly p r e v a ­

le n t a m in o a c id s is s i m i l a r to th a t o f L - la c t ic a c id and hence th e s e c o m ­
pounds a re s a id to p o s s e s s th e L - c o n fig u r a tio n .

B y f a r the m a jo r it y

o f p ro b le m s in b a c t e r ia l p h y s io lo g y have d e a lt w ith L - a m in o a c id s , b u t
th e re is s u ffic ie n t in fo r m a tio n a v a ila b le to m a k e a s tu d y o f th e D - is o m e r s
e x tr e m e ly fa s c in a tin g .

(a) S tr a in d e s c r ip tio n
T h e s tr a in s o f R h iz o b iu m m e li l o t i u s e d in th is and s u b se q u e n t
in v e s tig a tio n s w e re d e s ig n a te d

-^ 2 0 anc* ^ 2 0 -2 7 A *

T h e ir e f f e c tiv e -

- 19 -

n e ss in n itr o g e n f ix a t io n and t h e ir c o lo n ia l d e s c r ip tio n s a r e g iv e n
in T A B L E I .

TABLE I
T A B L E IN D IC A T IN G T H E S O U R C E, E F F E C T IV E N E S S , A N D C O L O N ­
I A L D E S C R IP T IO N S O F E A C H O F T H E R H IZ O B IU M T E S T S T R A IN S

S tra in

S o u rce

E ffe c tiv e n e s s
on a lfa lf a

C o lo n ia l d e s c r ip tio n

R 21

O tta w a

e ffe c tiv e

c o n v e x , tra n s lu c e n t
w ith w h itis h c e n te r,
e n tir e m a r g in , e x ­
tr e m e ly m u c o id and
g lis te n in g

R 20

O tta w a

p a r a s itic to
in e ffe c tiv e

c o n v e x , tr a n s lu c e n t
w ith w h it is h c e n te r,
e n tir e m a r g in , s lig h t ­
l y m u c o id and
g lis te n in g

R 2 0 -2 7 A

X -ra y
m u ta n t
o f R 20

e ffe c tiv e

p u lv in a te , t r a n s ­
lu c e n t w ith w h itis h
c e n te r, e n tir e m a r g in ,
m u c o id a p p e a rin g b u t
“ ro u g h ’ * o r p e b b le d
on the s u r fa c e . I n ­
t e r i o r o f c o lo n y a p ­
p e a rs m a r b le d w ith
lig h t and dense a re a s

(47

)

On m o d ifie d L o c h h e a d ’ s m e d iu m s t r a in R 2 i (the m o s t c o p io u s g u m
p r o d u c e r) and R2 0 -2 7 A e x h ib ite < i a m o re p ro fu s e and r a p id g r o w th th a n
■^20*

R o th R 2 1 «md R 2 0 w e re fo u n d to ha ve th e sam e fe r m e n ta tiv e a b ilit y

- 20 -

on a ra b in o s e , g a la c to s e , g lu c o s e , g ly c e r o l, la c to s e , m a n n o s e , and
x y lo s e , f r o m w h ic h th e y p ro d u c e d a c id .

T h e y w e re a b le to u t iliz e ,

b u t p ro d u c e d no a c id f r o m , d e x tr in , d u lc it o l, in u lin , le v u lo s e , m a l­
to s e , m a n n ito l, s u c ro s e , fu m a ra te , s u c c in a te , o r p y ru v a te w he n th e s e
co m p o u n d s w e re u se d as th e s o le c a rb o n s o u rc e .

T h e y p ro d u c e d a m m o n ­

ia in m i l k and b ro u g h t a b out c o m p le te d ig e s tio n o f th e c a s e in .

A l l th r e e

s tr a in s w e re u n a b le to g ro w in a l i v e r in fu s io n b r o th , b u t u t iliz e d a
w id e v a r ie t y o f a m in o a c id s as th e s o le s o u rc e o f n itr o g e n in a c h e m i­
c a lly d e fin e d m e d iu m (48).

(b) M e d ia
T h e fo r m u la e f o r a m o d ifie d L o c h h e a d ’ s m e d iu m , M o d ifie d M e d iu m
“ 79” and a m o d ifie d W ils o n and W ils o n ’ s m e d iu m a re g iv e n in the a p p e n ­
d ix .

T h e la t t e r m e d iu m w a s c o m p o s e d o f s p e c ia lly p u r if ie d in g re d ie n ts

and c o n s titu te d the n itr o g e n - fr e e base e m p lo y e d in m u c h o f th e p re s e n t
w o rk .

T h e c h e m ic a ls u se d in the f o r m u la tio n of the se m e d ia w e re o f

“ A n a la r ” (C . P . a n a ly z e d ) g ra d e p u rc h a s e d f r o m B r i t i s h D ru g H o u se s,
L td .

(c) P u r if ic a t io n o f b a c t e r ia l s tr a in s
T h e o r g a n is m s w e re g ro w n a t 3 0 °C . f o r 4 8 h r. on s la n ts o f L o c h ­
h e a d ’ s m e d iu m and p o rtio n s o f th is g ro w th e m u ls ifie d in M o d ifie d M e d iu m
“ 79” .

T h e s e s u s p e n s io n s w e re each p a sse d th ro u g h a H a n d u ro y “ G e r m

S e p a ra to r”

(38) and th e a e r o s o l th u s fo r m e d a llo w e d to f a l l on open p la te s

- 21 -

o f L o c h h e a d 's m e d iu m , w h ic h w e re im m e d ia te ly c o v e re d and in c u b a te d
a t 3 0 °C . u n t i l c o lo n ie s d e v e lo p e d .

R e p re s e n ta tiv e c o lo n ie s w e re p ic k e d ,

e m u ls ifie d in liq u id m e d iu m and a g a in p a ssed th ro u g h the a e r o s o l d e v ic e .
S in g le c e ll is o la tio n s w e re m a de w ith a de F o n b ru n e p n e u m a tic m ic r o m a n ip u la to r , u s in g the g ro w th w h ic h d e v e lo p e d on th e s e seco nd p la te s .
T h e m o s t s u c c e s s fu l te c h n iq u e w as as fo llo w s :

a s t e r ile c o v e r s lip ,

h a v in g a th in la y e r o f s o lid ifie d c le a r a g a r on one s id e w as p la c e d , a g a r
s id e dow n, o v e r a s t e r ile m o is t c h a m b e r c o n ta in in g w a te r-s o a k e d f i l t e r
p a p e r.

T h e c o v e r s lip w a s s e a le d to the c h a m b e r w ith v a s e lin e and a

c ir c le d ra w n on the u p p e r s u r fa c e w ith a w a x p e n c il.

On the a g a r d i r e c t -

b e n e a th th is c i r c le a m in u te a m o u n t o f lo g phase b a c t e r ia l g ro w th w as
p la c e d b y m e a n s o f a s le n d e r p la tin u m n e e d le and s p re a d s lig h t ly so th a t
som e c e lls w o u ld be w e ll s e p a ra te d f r o m th e p a re n t m a s s .

T h e m o is t

c h a m b e r w as th e n p la c e d on the m e c h a n ic a l stage o f a b in o c u la r m i c r o ­
scop e, f r o m w h ic h the to p c o n d e n s e r le n s w as re m o v e d , and the b a c t e r ­
i a l c e lls b ro u g h t in to fo c u s .

R ig h t-a n g le d m ic r o p ip e tte s w ith a 4. 5 m ic r o n

lu m e n o p e n in g w e re m a n u fa c tu re d in a de F o n b ru n e m ic r o fo r g e , s t e r iliz e d
in in d iv id u a l g la s s tu b e s , p a r t ia lly f i l l e d w ith s t e r ile M o d ifie d M e d iu m
“ 79**, and a tta c h e d to a s y rin g e b y m e a n s of a f le x ib le r u b b e r tu b e .
T h e tip s o f th e s e in s tr u m e n ts w e re m o v e d im m e d ia te ly b e lo w in d iv id u a l
b a c t e r ia l c e lls w h ic h w e re th e n d ra w n in b y c a p i ll a r i t y o r b y th e a p p lic a ­
tio n o f v e r y s lig h t s u c tio n .

T h e c e lls and th e c o n ta in e d liq u id w e re e x ­

p e lle d on to p r e d e te rm in e d s p o ts on the s u rfa c e o f a p la te o f s o lid if ie d

- 22 -

L o c h h e a d ’ s m e d iu m , w h ic h w a s s u b s e q u e n tly in c u b a te d a t 3 0 ° C . u n t il
c o lo n ie s d e v e lo p e d .

U s u a lly , te n is o la tio n s w e re m a de f r o m ea ch b a c ­

t e r i a l s tr a in , w ith a p e rc e n ta g e o f s u c c e s s w h ic h v a r ie d b e tw e e n 7 0 -80 % .

(d) E f f e c t iv it y te s ts
T h e b a c t e r ia l c u ltu r e s w e re te s te d on O n ta rio V a rie g a te d a lf a lfa
u s in g the s t e r ile g la s s a s s e m b lie s o f L e o n a rd (56) and th e te c h n iq u e s
p r e v io u s ly d e s c rib e d b y J o rd a n and G a r r a r d (5 0 ).
in o c u la te d and in o c u la te d w ith R2

1

T h e tr e a tm e n ts (u n ­

, R2 0 and R 2 0 -2 7 A ) w e re r e p lic a te d

and th e v a r io u s a s s e m b lie s p la c e d on th e g re e n h o u s e b e n c h in a 4 x 4
L a t in S q u a re .
p h o to p e rio d .

S u p p le m e n ta ry lig h t in g w a s p ro v id e d to e n s u re a 14 h r .
T h e p la n ts w e re h a rv e s te d a f t e r a g ro w in g p e rio d o f fiv e

w e e ks and th e a e r ia l p o r tio n s e x c is e d and d r ie d a t 1 1 5 °C . to c o n s ta n t
w e ig h t.

T h e n itr o g e n c o n te n ts o f re p r e s e n ta tiv e p o rtio n s o f d r ie d m a t e r ­

i a l w e re d e te rm in e d u s in g a m ic r o - K je ld a h l p ro c e d u re w h ic h w i l l be
d e s c rib e d in a l a t e r s e c tio n o f the th e s is .

T h e r e s u lt s w e re s t a t is t ic a lly

a n a ly z e d f o r s ig n ific a n c e u s in g th e a n a ly s is o f v a r ia n c e (see a p p e n d ix ).
O n ly th e R 2 1 an d R 2 0 -2 7 A in o c u la te d p la n ts w e re s ig n if ic a n t ly b e tte r th a n
the u n in o c u la te d c o n tr o ls and hence o n ly th e s e tw o b a c t e r ia l s tr a in s can
be re g a rd e d as e ffe c tiv e .

(e) D e te r m in a tio n o f m a x im u m c u ltu r e g ro w th u s in g th e P h o to n e p h e lo m e te r.
T h e t e r m “ n e p h e lo s ” r e f e r s to th e a b ilit y o f a su sp end ed phase to
s c a tt e r lig h t b y v ir t u e o f o p tic a l d is c o n tin u ity and does n o t r e f e r to a t r a n s -

- 23 -

m is s io n p h eno m en on.

T h e lig h t s c a tte re d b y p a r tic le s ( T y n d a ll lig h t )

is p r o p o r tio n a l to the n u m b e r and s iz e o f th e s e s m a ll b o d ie s and, in
the ca se o f b a c t e r ia l c e lls , to th e to t a l a m o u n t o f p r o to p la s m .

I t is th u s

an e x c e lle n t in d ic a to r o f g ro w th , w h ic h in i t s tr u e sen se is d e fin e d as
an in c re a s e in p r o to p la s m and n o t m e r e ly an in c re a s e in c e ll n u m b e rs .
T h e d e te r m in a tio n o f c u ltu r e n e p h e lo s w as m ade w ith a C o le m a n M o d e l
7 P h o to n e p h e lo m e te r w h ic h w as c a lib r a te d w ith a c o m m e r c ia lly p re p a re d
s ta n d a rd ea ch tim e i t w a s u s e d .

T h e “ n u ll** m e th o d w a s e m p lo y e d , w h ic h

has an e s tim a te d in s tr u m e n t e r r o r o r 0 .2 % .

T h e c u v e tte s u s e d w e re o r i g ­

in a lly o p tic a lly m a tc h e d , b u t m in u te s c ra tc h e s o c c u r r e d w ith c o n s ta n t
h a n d lin g .

T o c o r r e c t f o r su ch o p tic a l im p e r fe c tio n s te n c u v e tte s w e re

cho sen a t ra n d o m and f i l l e d w ith a b a c t e r ia l s u s p e n s io n o f lo w , m e d iu m ,
o r h ig h

n e p h e lo s .

R e a d in g s w e re ta k e n on each c u v e tte w ith each s o lu tio n

and the s ta n d a rd d e v ia tio n s c a lc u la te d (see a p p e n d ix ).

T h e s e v a lu e s w e re

t 2 . 57, t 2 .9 8 and t 2 . 52 f o r the lo w , m id d le and h ig h ra n g e s r e s p e c tiv e ly
and in d ic a te d th a t d e v ia tio n s in n e p h e lo s f r o m th e tr u e v a lu e a r e n o t r e ­
la te d to th e n e p h e lo s m a g n itu d e .

S ince s ig n ific a n c e a t th e 5% p o in t r e q u ir e s

a d iffe re n c e e x c e e d in g ^ 1. 96 s ta n d a rd d e v ia tio n s , an a r b i t r a r y d iffe r e n c e
o f f o u r s ta n d a rd d e v ia tio n s b e tw e e n an y tw o n e p h e lo s re a d in g s has been
r e q u ir e d f o r s ig n ific a n c e .

T h is a m o u n ts to about 11 n e p h e lo s u n its (4 x th e

a v e . S .D . o f 2 .6 9 = 10. 76) and a n y re a d in g s d if f e r in g b y le s s th a n th is
a m o u n t a r e to be re g a rd e d as n o n - s ig n ific a n t.
T h e p h o to n e p h e lo m e tric m e th o d depends upon the a s s u m p tio n th a t th e re

- 24 -

is a lin e a r r e la tio n s h ip b e tw e e n c o n c e n tra tio n and in s tr u m e n t re s p o n s e .
T h e v a lid it y o f th is a s s u m p tio n f o r v ia b le c e lls w a s sh o w n b y a c tu a l
te s t.

D ilu tio n s o f a b r o th c u ltu r e o f R 2 1 w e re m a de and the n e p h e lo s

d e te rm in e d .

T h e s e d ilu tio n s w e re th e n p la te d out in t r ip lic a t e on L o c h -

h e a d ’ s m e d iu m u s in g a c o n v e n tio n a l b a c t e r io lo g ic a l te c h n iq u e .

The

p la te s w e re in c u b a te d a t 3 0 °C . f o r th r e e d a ys and the c o lo n y c o u n ts
w e re p lo tte d a g a in s t th e n e p h e lo s re a d in g s .

F IG U R E 1 illu s t r a t e s the

lin e a r re s p o n s e o b ta in e d .
W hen b a c t e r ia l g r o w th c u rv e s w e re p lo tte d in te r m s o f n e p h e lo s i t
w a s fo u n d th a t the v a lu e s re a c h e d a m a x im u m and th e n d e c re a s e d .

One

to tw o d a y s a f t e r th is i n i t i a l d e c re a s e the n e p h e lo s re a d in g s r o s e .

The

d e c re a s e w as a ttr ib u te d to a c e s s a tio n o f g ro w th fo llo w e d b y th e s e ttlin g
ou t o f th e b a c t e r ia l c e lls w h ile the seco nd in c re a s e a p p e a re d to c o in c id e
w ith th e o n s e t o f a u to ly s is .
m a w e re c o n s id e re d .

F o r c o m p a ra tiv e p u rp o s e s the p r im a r y m a x i­

5

io

Viable bacterial count XlO^/ml.
Pig. 1

Correlation between viable bacterial counts and
nephelos readings.

- 25 -

M e th o d s :
A n u m b e r o f 6 -3 0 4 B P h o to n e p h e lo m e te r c u v e tte s w e re c le a n e d
b y w a rm in g in 33% n i t r i c a c id and th o ro u g h ly r in s e d 10 tim e s in o r d in ­
a r y d is t ille d w a te r and tw ic e in d o u b le g la s s - d is t ille d w a te r*

They

w e re th e n p lu g g e d w ith c h e e s e c lo th -c o v e re d c o tto n and s t e r iliz e d b y
a u to c la v in g .

M o d ifie d W ils o n and W ils o n ’ s m e d iu m w as p re p a re d , a d ­

ju s te d to a pH o f 6* 0, 7. 0, o r 7. 5 and s t e r iliz e d b y S e itz f i l t r a t i o n , u s ­
in g a s p e c ia l d e v ic e in w h ic h th e s t e r iliz e d f lu id w as a llo w e d to c o lle c t
in a g ra d u a te d d is p e n s in g c o lu m n *

T h e f i r s t 2 0 m l. o f m e d iu m p a s s in g

th ro u g h the f i l t e r pad w e re d is c a rd e d and 9. 5 m l. a liq u o ts o f th e r e m a in ­
in g m e d iu m w e re a s e p tic a lly added to th e s t e r ile c u v e tte s .

A pH v a lu e

g r e a te r th a n 7. 5 w as n o t u s e d s in c e t u r b id it y re s u lte d in the m e d iu m *
S to c k a m in o a c id s o lu tio n s w e re p re p a re d w ith d o u b le g l a s s - d i s t i l l ­
ed w a te r, the c o n c e n tra tio n s b e in g s u c h th a t th e a d d itio n o f 0. 5 m l. to
9. 5 m l. o f b a s a l m e d iu m w o u ld y ie ld a s o lu tio n c o n ta in in g the e q u iv a le n t
o f 0 ,0 0 0 1 m o le s o f a m in o a c id p e r l i t e r .

T h is is th e o p tim u m m o la r it y

o f s e v e r a l a m in o a c id s in r e g a r d to u t iliz a t io n b y R h iz o b iu m s p e c ie s (76).
T h e pH o f the a c id ic and b a s ic a m in o a c id s w e re c a r e f u lly a d ju s te d to
7 .0 w ith a g la s s - e le c tr o d e p o te n tio m e te r b e fo re b e in g b ro u g h t to t h e ir
f in a l v o lu m e .

A f t e r s t e r iliz a t io n in an a u to c la v e a t 15 lb . p r e s s u r e f o r

6 m in . , 0 . 5 m l. a liq u o ts o f th e s e s o lu tio n s w e re added to t h e ir re s p e c tiv e
c u v e tte s .
T h e a m in o a c id s u se d c o n s is te d o f the n e u tr a l a lip h a tic a m in o a c id s

- 26 -

g ly c in e , le u c in e , and v a lin e , the n e u tr a l s u lp h u r c o n ta in in g a c id m e th ­
io n in e , th e a c id ic a m in o a c id g lu ta m ic and th e b a s ic a m in o a c id h is tid in e .
T h e y w e re a l l o b ta in e d f r o m N u t r it io n a l B io c h e m ic a ls C o rp . (C le v e la n d ,
O hio , U . S . A . ) and m e t the fo llo w in g s p e c ific a tio n s :
n itr o g e n

100% * 1% o f th e o ry

m o is tu r e

le s s th a n 0. 30%

ash

le s s th a n 0 .1 0 %

c h lo r id e

le s s th a n 0. 03%

a m m o n ia

le s s th a n 0 .0 3 %

h e a v y m e ta ls
ir o n

le s s tha n 0 .0 0 3 %
le s s th a n 0.0 05 %

s p e c ific r o ta tio n 100% t 2% o f th e o ry
T h e s to c k s o lu tio n s w e re a n a ly z e d b y p a p e r p a r t it io n c h ro m a to g ra p h y
u s in g w a te r - s a tu r a te d p h e n o l (104), and w e re fo u n d to be p u re , e a ch g iv ­
in g r is e to o n ly one n in h y d r in p o s itiv e s p o t o f a p p ro p ria te R f (12).
T h i r t y m i l l i l i t e r s o f a 24 h r . 3 0 °C . c u ltu r e o f the te s t o r g a n is m
in M o d ifie d M e d iu m **79** w e re c e n trifu g e d and th e s e d im e n te d c e lls
w a sh e d th re e tim e s in 0 .8 5 % s a lin e p re p a re d f r o m p u r if ie d N a C l (101)
and d o uble g la s s - d is t ille d w a te r .

T h e r e s u ltin g c e ll m a s s w a s b ro u g h t

u p tto a f i n a l v o lu m e o f 30 m l. w ith th e sam e f lu id and th e n e p h e lo s a s e p t ic a l l y a d ju s te d to 5 5 .0 (ca . 0. 1 m g m . b a c t e r ia l n itr o g e n / m l . ) u s in g
s a lin e as a b la n k .
a c id c u v e tte s .

A liq u o ts o f 0 . 2 m l. s e rv e d as the in o c u la f o r th e a m in o

T h e to t a l i n i t i a l v o lu m e o f each c u v e tte w a s 10, 2 m l. and

- 27 -

w as e x p e r im e n ta lly d e te rm in e d to ta k e in to c o n s id e r a tio n th e a m o u n t
o f e v a p o ra tio n d u rin g the in c u b a tio n p e r io d .

W ith a v e s s e l o f w a te r in

the in c u b a to r to s e rv e as a h u m id if ie r 0. 2 m l. o f c u ltu r e e v a p o ra te d in
fiv e to s ix d a y s .

T h is c o rre s p o n d e d to th e a p p ro x im a te m a x im u m in th e

g ro w th o f the te s t o rg a n is m s and a t th is p o in t the c o n c e n tra tio n o f th e
a m in o a c id w as m o re e x a c tly 0 .0 0 0 1 M .
‘ In each e x p e r im e n ta l s e r ie s one s t r a in o f o r g a n is m w as te s te d f o r
the a b ilit y to u t iliz e , as the s o le s o u rc e o f n itro g e n , g ly c in e and th e L .-,
D - , an d D L - f o r m s o f a l l the o th e r a m in o a c id s a t th e th re e d iffe r e n t p H 's .
In o c u la te d and u n in o c u la te d c o n tr o l c u v e tte s , c o n ta in in g no a m in o a c id s ,
w e re in c lu d e d in each ru n , the la t t e r s e r v in g as a b la n k f o r th e n e p h e lo s
re a d in g s w h ic h w e re ta k e n d a ily in th e P h o to n e p h e lo m e te r.

In c u b a tio n

w a s c a r r ie d out a t 30° C . u n t il the re a d in g s b e g a n to d e c re a s e in m a g n i­
tu d e .

A f t e r in c u b a tio n th e pH o f each c u ltu r e w as ta k e n w ith a p o te n tio ­

m e te r and G r a m s ta in s w e re m a de as an in d e x o f p u r it y .
T h e “ o p tim u m ” te m p e r a tu re f o r the g ro w th o f a l l th re e te s t o r g a n ­
is m s in a y e a s t- e x t r a c t m in e r a l- s a lt s m e d iu m w as d e te rm in e d b y in o c u ­
la tin g th re e s e r ie s o f c u v e tte s , ea ch c o n ta in in g 10 m l. o f M o d ifie d M e d iu m
“ 79” , w ith w a sh e d c e lls o f I ^ l ’ ^2 0 * anc^ ^ 2 0 - 2 7 A .
in c u b a te d a t 3 5 ° C ., 3 0 °C . and 2 7 °C .

s e r *es w e re

One c u v e tte in e a ch s e r ie s s e rv e d

as an u n in o c u la te d c o n tr o l and d a ily n e p h e lo s re a d in g s w e re m a de on
ea ch c u ltu r e .
T h e a c tio n o f a te m p e r a tu re lo w e r th a n 3 0 ° C . on the s p e c tru m o f

- 28 -

a m in o a c id s u t iliz e d b y r h iz o b ia w as s tu d ie d w ith a t r ip lic a t e s e r ie s o f
c u v e tte s c o n ta in in g 9, 5 m l. o f b a s a l m e d iu m , 0. 5 m l. o f a s to c k s o lu ­
tio n o f g ly c in e o r the L - o r D - is o m e r s o f th e o th e r a m in o a c id s , and
0 .2 m l. o f w a sh e d c e ll in o c u lu m (n eph elos 5 5 .0 ).

T h e pH o f th e b a s a l

m e d iu m in each c u v e tte w as th e o p tim u m f o r th e c o n ta in e d a m in o a c id ,
as d e te rm in e d in the p re v io u s e x p e rim e n t on pH .
g iv e n in T A B L E 2.

T h e s e v a lu e s a re

T h e in c u b a tio n te m p e r a tu re w as 2 7 ° C . and n e p h e lo s

re a d in g s w e re ta k e n ea ch da y u n t il th e m a x im a w e re re a c h e d .

TABLE 2
T A B L E IN D IC A T IN G T H E I N I T I A L pH V A L U E S O F T H E A M IN O
A C ID M E D IA IN T H E “ T E M P E R A T U R E E F F E C T ” C U V E T T E S

C u v e tte

A m in o A c id

O rg a n is m

R 21

R 2 0 -2 7 A

R 20

1

g ly c in e

7 .5

7 .5

7 .0

2

L - le u c in e

7 .5

7 .5

7 .0

3

D - le u c in e

7 .5

7 .5

7 .0

4

L - v a lin e

7 .5

7 .5

7 .0

5

D - v a lin e

7 .5

7 .0

7 .0

6

L - m e th io n in e

7 .5

7 .5

7 .0

7

D -m e th io n in e

7* 0

7 .0

7 .0

8

L - g lu t a m ic a c id

7 ,5

7 .5

7 .0

9

D - g lu ta m ic a c id

7 .5

7 .0

7 .0

- 29 -

T a b le 2 (c o n t’ d)

A m in o A c id

R 2 0 -2 7 A

0

R 21

5d

O rg a n is m
to

C u v e tte

10

L - h is t id in e

7 .5

7 .5

7 .5

11

D - h is tid in e

6 .0

7 .5

6 .0

12

in o c u la te d c o n tr o l

7 .0

7 .0

7 .0

13

u n in o c u la te d c o n tr o l

7 .0

7 .0

7 .0

A d u p lic a te s e r ie s o f c h e m ic a lly c le a n te s t-tu b e s w e re f i l l e d w ith
9 .5

m l. o f s u c r o s e - fr e e m o d ifie d W ils o n and W ils o n ’ s b a s e .

One s e r ie s

c o n ta in e d 0. 5 m l. o f L - h is t id in e s to c k s o lu tio n and the o th e r c o n ta in e d
0 . 5 m l. o f L - g lu t a m ic a c id s to c k s o lu tio n .
in o c u la te d w ith w a sh e d c e lls o f R 2

0

T h e tu b e s in each lo t w e re

R 21» R 2 0 -2 7 A anc* a nurrih e r o f o th e r

s tr a in s o f r h iz o b ia w h ic h a r e in d ic a te d in T A B L E 3 .

T h e p ro d u c tio n b y

X - r a y ir r a d ia t io n o f th e m u ta n ts lis te d has been p r e v io u s ly d e s c rib e d (47).
In c u b a tio n w as a t 2 7 °C f o r e ig h t d a ys and the p re s e n c e o r ab sence o f v i s ­
u a l g ro w th r e c o r d e d .

T h is e x p e rim e n t in d ic a te d the a b ilit y o f th e b a c t e r ­

i a l s tr a in s to u t iliz e th e s e tw o a m in o a c id s as s o le s o u rc e s o f b o th c a rb o n
and n itr o g e n .

- 30 -

TABLE 3
R H IZ O B IU M S T R A IN S . O T H E R T H A N R 21, R 2 0 - 2 7 A ' A N D R 20,
T E S T E D F O R T H E IR A B I L I T Y T O U T IL IZ E L - H IS T ID IN E O R
L - G L U T A M IC A C ID AS S O LE SOURCES O F B O T H
C A R B O N A N D N IT R O G E N

S tr a in

S o u rce

R 23

O. A . C . s to c k c u ltu r e

e ffe c tiv e

CO
CM
1
O
CM
«

X - r a y m u ta n t o f R 2 q

e ffe c tiv e

R 2 0 -3 1

X - r a y m u ta n t o f R 2 q

e ffe c tiv e

R 2 0 -1 6

X - r a y m u ta n t o f R 2 q

in e ffe c tiv e

r

2 0 -1 7

X - r a y m u ta n t o f R 2 q

in e ffe c tiv e

r

2 0 -1 9

X - r a y m u ta n t o f R 2 0

in e ffe c tiv e

r

2 0 -2 0

X - r a y m u ta n t o f R 2 0

in e ffe c tiv e

* te s te d on G r im m a lfa lfa .

E ffe c tiv e n e s s in
n itr o g e n f ix a t io n *

T h o s e s tr a in s d e s ig n a te d as “ in e ffe c tiv e ”

m a y a ls o ha ve p a r a s itic te n d e n c ie s (50).

R e s u lt s :* *
F IG U R E S 2, 3, and 4 g r a p h ic a lly re p re s e n t th e m a x im u m g ro w th
a tta in e d b y th e th re e R h iz o b iu m s tr a in s in the a m in o a c id m e d ia a t the
th r e e d iffe r e n t p H ’ s.

T h e f in a l pH v a lu e s w e re g e n e r a lly fo u n d to be s e v e r a l

© ©

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- 31 -

te n th s o f a u n it lo w e r th a n th e o r ig in a l (see a p p e n d ix ).
A l l the s tr a in s o f th e n o d u le b a c te r ia w e re r e a d ily ca p a b le o f u s in g
D -a m in o * a c id s as th e s o le n itr o g e n s o u rc e .

In s e v e r a l ca se s th e o r g a n ­

is m s m a d e b e tte r use o f th e se *‘u n n a tu ra l’ * is o m e r s th a n th e y d id o f the
c o rre s p o n d in g “ n a tu ra l* * ones.
W ith H 2 1 a pH o f 7. 5 w a s o p tim u m f o r u t iliz a t io n o f a l l the a m in o
a c id s w ith th e e x c e p tio n o f D - and D L - h is tid in e (pH 6. 0 b e s t) and D m e th io n in e (pH 7. 0 b e s t).

A l l th re e f o r m s o f h is tid in e s u p p o rte d e x c e p ­

t io n a l g ro w th a t a l l p H ’ s, in c lu d in g 6. 0 at w h ic h pH g ro w th d id n o t o c c u r
w ith th e m a jo r it y o f the a c id s e x c e p t to a v e r y lim it e d e x te n t w ith L - v a lin e ,
L - g lu t a m ic a c id and L , D - and D L - m e th io n in e .

T he d iffe r e n t is o m e r s

o f an y one a m in o a c id w e re fo u n d to v a r y in t h e ir a b ilit y to be u t iliz e d ,
d e p e n d in g on the p H .

T h e g ro w th o b s e rv e d in the ra c e m ic m ix tu r e s

in d ic a te d th a t b o th in h ib itio n and s tim u la tio n c o u ld o c c u r.

T h e s e la s t

e ffe c ts , to g e th e r w ith tho se fo u n d w ith R 2 0 and R 2 0 r2 7 A * a r e s u m m a riz e d
in T A B L E 4 .
F o r R2 Q

27A

a ^

^ w a s *-ounc* to

o p tim u m f o r th e u t i l i z a ­

tio n o f 10 o f the 16 d iffe r e n t fo r m s o f a m in o a c id s te s te d , w h ile a pH o f
7. 0 w as b e s t f o r 5 o f the r e m a in d e r .

* * N u m e r ic a l d a ta p e r ta in in g to m o s t o f th e c h a rts and g ra p h s p re s e n te d
in th is th e s is a r e g iv e n in th e a p p e n d ix .

- 32 -

M a x im u m g r o w th in D L - h is t id in e w as o b s e rv e d a t pH 6. 0.

TABLE 4
IN H IB IT IO N A N D S T IM U L A T IO N F O U N D IN T H E R A C E M IC M IX T U R E S O F
V A R IO U S A M IN O A C ID S W H E N T E S T E D A G A IN S T T H R E E S T R A IN S O F
R H IZ O B IU M

S tr a in

pH

In h ib itio n

R 21

7 .0

h is tid in e (7 7 .0 % )*

S tim u la tio n

g lu ta m ic (86. 5%)
7 .5

h is tid in e (84. 9%)
g lu ta m ic (74. 3%)

R 20 -2 7 A

6* °

m e th io n in e (64. 5%)
g lu ta m ic (55.9% )

7 .0

le u c in e (2 85.4 % )
v a lin e (272. 5%)
h is tid in e (1 27.7 % )

m e th io n in e (58. 6%)
g lu ta m ic (85.6% )

7 .5

m e th io n in e (53. 3%)
g lu ta m ic (67.5% )
le u c in e (72. 4%)
v a lin e (46. 5%)

R20

6* 0

h is tid in e (1 21.5 % )

7 .0

h is tid in e (1 1 1 .2 % )

^ n u m b e rs in b r a c k e ts r e f e r to the p e rc e n t o f th e th e o r e tic a l g r o w th .

T h is

- 33 -

th e o r e tic a l g ro w th is e q u a l to o n e -h a lf th e g ro w th on th e L - is o m e r + o n e h a lf th e g ro w th on th e D - is o m e r a t the sam e pH, a s s u m in g no in h ib itio n .
O n ly v a lu e s le s s th a n 90% o r m o re th a n 110% o f the th e o r e tic a l a re lis t e d .

H is tid in e in a l l it s fo r m s be have s in a fa s h io n s i m i l a r to th a t o b ­
s e rv e d w ith Rg j in th a t i t p ro m o te s e x c e lle n t g ro w th a t a l l p H ’ s .

R 2 0 -2 7 A

is d e c id e d ly m o re a c id to le r a n t th a n R 2 i> g ro w th o c c u r r in g a t 6 .0 w ith
e v e ry a m in o a c id .

T h is o rg a n is m , h o w e v e r, does n o t m a k e as good u se

o f th e a m in o a c id s (at t h e ir o p tim u m pH ) as does R 2 i* b u t due to in e v ita b le
v a r ia tio n s in in o c u lu m s iz e no c r i t i c a l c o m p a ris o n s w e re a tte m p te d .
R 2 0 is d e fin ite ly fa s tid io u s in it s pH r e q u ir e m e n ts .

W ith 13 out o f

the 16 a m in o a c id s a g a in s t w h ic h i t w as te s te d i t g re w o n ly a t pH 7 .0 , a l ­
th o u g h w ith h is tid in e i t g re w w e ll a t a l l th re e p H ’ s.
T h e a p p ro x im a te o p tim u m te m p e ra tu re s f o r the g ro w th o f R 2 D
R

2 0 - 27 A and R 2 0 in the y e a s t- e x tr a c t m in e r a l- s a lt s m e d iu m c o n ta in in g

no added c a rb o h y d ra te w e re 3 5 ° C ,, 3 0 - 3 5 ° C, and 2 7 °C ,

r e s p e c tiv e ly .

T h e o p tim u m f o r R 2 0 is s o m e w h a t lo w e r th a n th a t g iv e n in v a r io u s l i t e r a ­
tu r e r e p o r ts f o r R h iz o b iu m m e li l o t i .

T h e e ffe c ts o f a 2 7 °C . in c u b a tio n

te m p e r a tu re on a m in o a c id u t iliz a t io n a re show n in F IG U R E 5.

G e n e ra lly ,

w ith the e x c e p tio n o f L - g lu t a m ic a c id and R 2 0 -2 7 A * the b e s t g ro w th w as
a tta in e d a t 3 0 °C . r a t h e r th a n 2 7 ° C ., eve n w ith R 2 0 .

N o s ig n ific a n t

d iffe r e n c e w as n o tic e d , h o w e v e r, in th e u t iliz a t io n o f g ly c in e and L m e th io n in e b y R 20,-27A and gLy c in e and L - g lu t a m ic a c id b y R 2Q.

A

m o r e c a r e f u l c o n s id e r a tio n of the d a ta in d ic a te s th a t w ith c e r ta in a m in o

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- 34 -

a c id s th e r e is an “ is o m e r ic e ffe c t’ * w h ic h is te m p e r a tu re dependent
(see T A B L E 5).

T h a t is , one is o m e r is m o re e ffe c tiv e a t 2 7 ° C . w h ile

th e o th e r is m o re e ffe c tiv e a t 3 0 °C .
TABLE 5
T H E E F F E C T O F T E M P E R A T U R E O N T H E U T IL IZ A T IO N O F T H E L A N D D - IS O M E R S O F C E R T A IN A M IN O A C ID S B Y R H IZ O B IA

O rg a n is m

A m in o a c id

R 21

le u c in e

v a lin e

r 20

le u c in e

v a lin e

g lu ta m ic a c id

R 2 0 -2 7 A

le u c in e

v a lin e

T e m p e ra tu re

A c t iv it y

2 7 °C .

L*

3 0 °C .

D

2 7 °C .

D

3 0 °C .

L

2 7 °C .

N . S .D .

3 0 °C .

D

2 7 °C .

D

3 0 ° C,

L

2 7 °C .

L

3 0 °C .

D

2 7 °C .

N . S .D .

3 0 °C .

D

2 7 °C .

N .S .D .

3 0 °C .

L

* L - L - i s o m e r y ie ld s b e s t g ro w th
D = D - is o m e r y ie ld s b e s t g r o w th
N . S. D . =ho s ig n ific a n t d iffe re n c e b e tw e e n is o m e r s

- 35 L - h is t id in e w as fo u n d to be c a p a b le o f s e r v in g as th e s o le s o u rc e
o f n itr o g e n and c a rb o n f o r a l l th e s tr a in s te s te d w h ile L - g lu t a m ic w as a c ­
tiv e f o r a l l the s tr a in s e x c e p t R 2 0 -1 9 and R 20-20«
R e p e titio n o f the e x p e rim e n ts in th is s e c tio n gave e s s e n tia lly
the sam e o v e r a ll r e s u lts as th o se lis te d *
D is c u s s io n :
R ydo n (80) in a r e v ie w a r t ic le , l i s t s a n u m b e r o f m ic r o o r g a n is m s
w h ic h have be en r e p o r te d as u t iliz in g D - a m in o a c id s .

N e g a tiv e r e s u lts

w ith c e r ta in b a c te r ia can not a lw a y s be a tt r ib u te d to th e la c k o f a c a p a b le
e n z y m e w ith in the c e ll s in c e c e r ta in w o r k e r s (10) have in d ic a te d th a t the
f a ilu r e o f r e s tin g s u s p e n s io n s to m e ta b o liz e D -a m in o a c id s is o fte n due
to .an in a b ilit y o f th e se com pou nds to p e n e tra te the c e ll b a r r i e r .

T h is

fe a tu r e m a y a ls o e x p la in the r e la t iv e ly s lo w r a te o f D - is o m e r u t iliz a t io n
in s o m e in s ta n c e s .

In c o n s id e r a tio n o f the d a ta the R h iz o b iu m s tr a in s

te s te d in the p re s e n t in v e s tig a tio n can not be re g a rd e d a s u n u s u a l w ith
r e s p e c t to t h e ir a b ilit y to u se th e “ u n n a tu ra l** is o m e r s o f a m in o a c id s .
In c o n tra s t to in t e r n a l m a m m a lia n c e lls , w h ic h fu n c tio n u n d e r a
c o n d itio n o f h o m e o s ta s is , b a c t e r ia l c e lls a re c o n tin u a lly s u b je c te d to the
e ffe c ts o f t h e ir e x te r n a l e n v iro n m e n t.

A lth o u g h som e m ic r o o r g a n is m s

m a y c o n tr o l t h e ir in t r a c e llu la r pH to som e e x te n t b y d e a m in a tio n o f
a m in o a c id s b y h ig h pH and d e c a rb o x y la tio n a t lo w pH (30), i t is d i f f i ­
c u lt to b e lie v e th a t no pH s h ift a t aU o c c u rs .

T h e pH o p tim a o f e n z y m e s

a re s c a tte re d a ro u n d th e p h y s io lo g ic a l ra n g e , and s m a ll s h ifts in pH w i l l
in c re a s e the a c t iv it y o f som e e n z y m e s , d e c re a s e th a t o f o th e rs and le a v e
th a t o f th e r e s t u n ch a n g e d .

S m a ll changes in pH w i l l , th e r e fo r e , m a r k e d ly

- 36 -

in flu e n c e m e ta b o lic re a c tio n s dependent on the fu n c tio n in g o f s e r ie s o f
enzym es.

F is h e r (25) s ta te s a n o th e r p o s s ib le e ffe c t o f pH on c e llu la r fu n c ­

tio n in th e se w o rd s , “ i t is to be e x p e c te d th a t i t w i l l m o d ify the d e g re e
o f io n iz a tio n o f c o n s titu e n ts of the b o u n d a ry la y e r o f th e c e ll, w h ic h w i l l
cause changes in th e p a tte r n and the p r o p e r tie s o f th is la y e r

as a w h o le ,

a p o s s ib le con se q u e n ce b e in g a lte r a tio n s o f the p e r m e a b ility o f the la y e r ,
w h ic h , b y m o d ify in g exchange o f m e ta b o lite s b e tw e e n th e ceU and the
e n v iro n m e n t c o u ld m o d ify th e p a tte rn o f m e ta b o lis m ’ *.

T h e r e s u lts o f

m e d iu m pH on a m in o a c id u t iliz a t io n b y r h iz o b ia c o u ld , th e r e fo r e , be e x ­
p la in e d b y c o n s id e r a tio n o f th e e ffe c ts o f the pH on e ith e r th e e n z y m e s o r
the c e ll m e m b ra n e s .
I t is g e n e r a lly co n c lu d e d th a t b a c t e r ia l c e lls a re u n a b le to m a in ta in
th e m s e lv e s a t te m p e r a tu re s above o r b e lo w th a t o f t h e ir e n v iro n m e n t.

T he

h e a t p ro d u c e d d u rin g m e ta b o lis m , f o r e x a m p le , is lo s t b y c o n v e c tio n and
r a d ia tio n and is n o t u se d f o r te m p e r a tu re m a in te n a n c e .

T h e “ o p tim u m **

te m p e r a tu re f o r a b a c t e r ia l c e ll is o n ly a r e fle c tio n o f the e ffe c t o f th is
fa c t o r on e n z y m a tic a c t iv it y .

I f a te m p e r a tu re o f 3 0 °C . p ro d u c e s m o re

g ro w th w ith one p a r t ic u la r a m in o a c id th a n a te m p e r a tu re o f 2 7 °C i t m a y
be c o n c lu d e d th a t th e e n z y m e (s ) c o n c e rn e d a c ts a t an in c re a s e d r a te at
the h ig h e r te m p e r a tu re , th a t a s e c o n d a ry “ in h ib it in g ” r e a c tio n is s u p p ­
re s s e d a t the h ig h e r te m p e r a tu re o r th a t the r e a c tio n n o r m a lly o c c u r r in g
a t th e lo w e r te m p e r a tu re is s u p p re s s e d and re p la c e d b y a m o re e ff ic ie n t
p a th w a y o f u t iliz a t io n .

I f i t is a s s u m e d th a t a t le a s t tw o d iffe r e n t e n z y m e s

- 37 -

a re im p lic a te d in th e m e ta b o lis m o f the L - and D - is o m e r s o f a n y one o f
s e v e r a l a m in o a c id s th e n th e e ffe c ts n o te d in T A B L E 5 m a y be e x p la in e d
b y one o r m o re o f the a fo re m e n tio n e d m e c h a n is m s .

A s a g e n e ra l s ta t e ­

m e n t, the L - is o m e r o f v a lin e is p r e f e r e n t ia lly u t iliz e d a t 3 0 ° C , and the
D - f o r m a t 2 7 ° C #J w h ile th e c o n v e rs e is tr u e f o r L - and D - le u c in e .

S ince

i t w o u ld be e x p e c te d th a t a n u m b e r o f d iffe r e n t ro u te s o f a m in o a c id m e ta ­
b o lis m w o u ld e x is t in in ta c t R h iz o b iu m c e lls i t w o u ld be h a z a rd o u s to
m a ke a n y s p e c ific s ta te m e n t w ith re g a r d to a p a r t ic u la r a c id .
T h e r e p o r ts o f a n u m b e r o f p re v io u s w o r k e r s , as m e n tio n e d in the
l it e r a t u r e r e v ie w , have be en c o n firm e d in th a t R h iz o b iu m m e li l o t i is
a c id s e n s itiv e , s tim u la te d b y a n e u tr a l o r s lig h t ly a lk a lin e pH and h a s a
te m p e r a tu re o p tim u m w h ic h lie s a p p ro x im a te ly w ith in th e ra n g e 2 7 ° -3 5 ° C .
T h e a b ilit y o f h is tid in e to s e rv e as a n itr o g e n s o u rc e f o r th e r h iz o b ia
is o u ts ta n d in g and p o s s ib ly r e s u lts f r o m the fa c t th a t th is a m in o a c id is
so m e w h a t c o m p le x and m ig h t th e r e fo r e be c o m p a r a tiv e ly d if f ic u lt to
s y n th e s iz e b y th e o r g a n is m s .

I t w o u ld th e n a c t as a li m i t i n g f a c t o r in

g ro w th u n le s s s u p p lie d p r e fo r m e d in the m e d iu m .

T h is im p lie s th a t a l l

the o th e r e s s e n tia l a m in o a c id s can be r e a d ily fo r m e d f r o m h is tid in e , b u t
th a t th e c o n v e rs e is n o t t r u e .

T h is c o n c e p t is re a s o n a b le s in c e h is tid in e

is kn o w n to be c o n v e r tib le th ro u g h a s e r ie s o f un kn o w n in te r m e d ia te s in to
fo r m a te and g lu ta m ic a c id .

T r a n s a m in a tio n o f the la t t e r c o u ld th e n

“ fu n n e l** th e h is tid in e a m in o n itr o g e n in to o th e r a m in o a c id s .

L i t t l e is

kn o w n o f th e s y n th e s is o f h is tid in e e x c e p t th a t th e p y r im id in e - lik e c a rb o n

- 38 -

o f the im id a z o le r in g is d e riv e d f r o m fo r m a te .
T h e in h ib itio n o f a n y e n z y m e b y a s u b s tra te an a lo g u e and the a c ­
tio n o f an e n z y m e on a s u b s tra te a re c lo s e ly r e la te d , as b o th in v o lv e the
a tta c h m e n t o f th e s u b s tr a t e /in h ib ito r to c e r ta in re a c tiv e g ro u p s on the
e n z y m e m o le c u le .

A n in h ib it o r o f th is ty p e is s im p ly a p o te n tia l s u b s tra te

w h ic h , a lth o u g h i t can a tta c h i t s e l f to th e e n z y m e , is s u ff ic ie n t ly d is s im ila r
to the n o r m a l s u b s tra te as to be n o t a c te d upon b y the e n zym e a f t e r th e
a tta c h m e n t has o c c u r r e d .

W ith co m pou nds as s i m i l a r as th e L - and

D - is o m e r s o f a m in o a c id s i t is n o t s u r p r is in g th a t r e p o r ts o f D -a m in o
a c id in h ib it io n o f b a c t e r ia l g ro w th a re r e la t iv e ly p le n t ifu l.

Such i n h ib i­

tio n u s u a lly s te m s f r o m th e fa c t th a t th e m o le c u le s o f th e D - a m in o a c id
a r e n o t u t iliz e d and “ c lo g ” up re a c tiv e s ite s on th e L - a m in o a c id e n z y m e .
In th e r h iz o b ia e x a m in e d , h o w e v e r, the D - is o m e r is a ls o u t iliz e d f o r
g ro w th p u rp o s e s and in som e c a s e s b e tte r th a n th e c o rre s p o n d in g L - f o r m .
T h e r e fo r e , in o r d e r to a c c o u n t f o r the d e v ia tio n s in g ro w th as c o m p a re d
w ith th e th e o r e tic a l - see T A B L E 4 - a so m e w h a t h y p o th e tic a l e x p la n a tio n
m u s t be p o s tu la te d .

A s s u m in g th a t the e n z y m e s f i r s t a tta c k in g th e a m in o

a c id s in a r a c e m ic m ix t u r e a r e s te r e o s p e c ific and in n o n - lim it in g and
a p p r o x im a te ly e q u a l a m o u n ts and a s s ig n in g an a r b i t r a r y g ro w th v a lu e
o f 50 to an L - a m in o a c id m o le c u le and a v a lu e o f 25 to a D -a m in o a c id
m o le c u le a h y p o th e tic a l r a c e m ic m ix t u r e o f tw o L - and tw o D - m o le c u le s
w o u ld in v o k e an o v e r a ll g ro w th re s p o n s e o f 150.

I f a m u tu a l in h ib itio n

o c c u rs b e tw e e n th e se m o le c u le s , th a t is , i f one m o le c u le o f th e D - is o m e r

- 39 -

is a ffix e d to an L - a m in o a c id e n z y m e s ite and is no t u tiliz e d , and v ic e
v e rs a , th e n the g ro w th re s p o n s e w o u ld be 75 u n its ( = 1 /2 = 50% o f the
th e o r e tic a l, w h e re a l l th e m o le c u le s a r e u t iliz e d ) .

D e v ia tio n s in the

r e la t iv e a m o u n ts o f th e tw o e n z y m e s , su ch as m ig h t o c c u r w ith pH (o r
te m p e r a tu r e flu c tu a tio n s , w o u ld n e c e s s a r ily cause p ro p o rtio n a te d e v ia ­
tio n s in th e p e rc e n ta g e o f th e th e o r e tic a l th a t w o u ld be o b s e rv e d .

I f the

L - a m in o a c id e n z y m e m o le c u le s w e re n u m e r ic a lly re d u c e d le s s D -a m in o
a c id m o le c u le s w o u ld be a ffix e d to th e m and i t m ig h t a p p e a r th a t m o re
o f th e la t t e r s h o u ld be u t iliz e d as a r e s u lt o f D -a m in o a c id e n zym e a c t i ­
v it y .

A t the sa m e tim e , h o w e v e r, m o re L - a m in o a c id m o le c u le s w o u ld

be a v a ila b le f o r in h ib itio n o f th e D -e n z y m e .

T he n e t r e s u lt w o u ld v a r y

s t a t is t ic a lly d e p e n d in g on th e p r o p o r tio n o f c o m p e tin g m o le c u le s .

T h is

c o n c e p t m a y e x p la in th e v a r io u s d e g re e s o f in h ib itio n fo u n d in the r a c e m ic
m ix tu r e s lis t e d in T A B L E 4.
T h e s t im u la t o r y e ffe c ts b ro u g h t a b o u t b y the r a c e m ic m ix t u r e s o f
c e r ta in a m in o a c id s a t th e lo w e r pH *s is in e x p lic a b le a t p re s e n t.

B,

T h e U t iliz a t io n o f S y n th e tic D i- and T r i - p e p tid e s

In 1949 D unn and M c C lu r e (24) fo u n d th a t p a r t ia l h y d ro ly s a te s o f
p r o te in w e re m o re e ffe c tiv e s o u rc e s o f e s s e n tia l a m in o a c id s f o r
L a c to b a c illu s c a s e i th a n c o m p le te h y d ro ly s a te s .

P e te r s and S n e ll (70)

o b s e rv e d s i m i l a r r e s u lts w ith L a c to b a c illu s d e lb r u e c k ii and a tt r ib u t e d
the fin d in g s to the p re s e n c e of u n id e n tifie d p e p tid e s .

T h e re a r e now a n u m -

- 40 -

b e r o f r e p o r t s in th e lit e r a t u r e d e a lin g w ith the g ro w th s tim u la tin g
e ffe c ts o f p e p tid e s as c o m p a re d to th e e ffe c ts o f t h e ir c o n s titu e n t a m in o
a c id s .

I t w as f e lt th a t th e b e n e fic ia l r e s u lts o f a m in o a c id p e p tid e s

m ig h t a ls o be m a n ife s te d in r h iz o b ia and the fo llo w in g s e r ie s o f e x p e r i­
m e n ts w e re d e v is e d to a s c e r ta in the p re s e n c e and e x te n t o f su ch a c t iv it y .

M e th o d s :
T h e p e p tid e s te s te d w e re o b ta in e d f r o m N u t r it io n a l B io c h e m ic a ls
C o r p ., ( U . S . A . ) and c o n s is te d o f g ly c y lg ly c in e , g ly c y lg ly c y lg ly c in e ,
L - and D - le u c y lg ly c in e , L - and D - le u c y lg ly c y lg ly c in e , and g l y c y l - L try p to p h a n e .

S to c k s o lu tio n s w e re p re p a re d w ith d o uble g la s s - d is t ille d

w a te r so th a t th e a d d itio n o f 0. 5 m l. to 9. 5 m l. o f a f lu id w o u ld r e s u lt
in a f in a l p e p tid e c o n c e n tra tio n o f 0. 0 0 0 1 M .

T h e s e s o lu tio n s w e re

s t e r iliz e d a t 15 lb . p r e s s u r e f o r 8 m in . and 0 .0 3 m l. a liq u o ts s p o tte d
on a W h a tm a n #1 f i l t e r p a p e r sh e e t and c h ro m a to g ra p h e d in w a t e r - s a t u r ­
a te d p h e n o l a t 2 7 ° C . , a c c o rd in g to the m e th o d o f W o iw o d

(104).

A fte r

d r y in g ,th e sh e e t w a s s p ra y e d w ith 0 .1 % a lc o h o lic n in h y d r in s o lu tio n .

It

w as fo u n d th a t s tro n g h e a tin g o f th e a re a on the f i l t e r p a p e r to w h ic h the
p e p tid e s o lu tio n w a s b e in g t r a n s f e r r e d d u rin g th e “ s p o ttin g ” p ro c e d u re ,
r e s u lte d in a s p lit t in g o f th e p e p tid e .

T h e r e fo r e , h e a tin g w as e lim in a te d

in th is o p e ra tio n .
A t th e sam e tim e th a t an o rg a n is m w as te s te d f o r th e e ffe c t o f pH on
it s a m in o a c id u t iliz a t io n i t w as a ls o e x a m in e d f o r g ro w th in p e p tid e m e d ia .
T h u s , the g ro w th v a lu e s o b ta in e d on the v a r io u s a m in o a c id s can be a c c u r ­

- 41 -

a te ly c o m p a re d w ith th e p e p tid e g ro w th v a lu e s .

F o r ea ch o f the th r e e

te s t o rg a n is m s (R 21, R 2 0 -2 7 A * and R 20) 9* 5 ^

o f PH 7* ° m o d ifie d

W ils o n an d W ils o n ’ s m e d iu m and 0 . 5 m l. o f s t e r ile s to c k p e p tid e s o lu tio n
w e re added to ea ch o f se ve n s t e r ile , c h e m ic a lly c le a n c u v e tte s .

One

c u v e tte , w ith o u t a n y p e p tid e added, s e rv e d as an in o c u la te d c o n tr o l, and
a n o th e r, th e u n in o c u la te d c o n tr o l, w as u se d as a b la n k f o r th e n e p h e lo s
re a d in g s .

W ashed c e ll in o c u lu m w as p re p a re d , as p r e v io u s ly d e s c rib e d ,

and a 0 .2 m l. a liq u o t w as u s e d to in o c u la te the v a r io u s c u v e tte s .

The

tu b e s w e re th e n in c u b a te d a t 3 0 °C . and d a ily n e p h e lo s re a d in g s ta k e n
w ith the P h o to n e p h e lo m e te r.

F in a l pH v a lu e s w e re d e te rm in e d w ith a

B e c k m a n g la s s - e le c tr o d e p o te n tio m e te r.
A c e ll- f r e e e x tr a c t o f R 2 ^ w as p re p a re d f r o m a th re e l i t e r , 24 h r .
a e ra tio n c u ltu r e (M o d ifie d M e d iu m e*79” ) b y c e n tr ifu g a tio n in a S c h a rp le s
c e n tr ifu g e , w a s h in g th e s e d im e n te d c e lls in 0 . 85% s a lin e and g r in d in g
f o r 12 m in . w ith a lu m in u m o x id e u s in g a P o tte r h o m o g e n iz e r and an ic e b a th .

T h e c e llu la r e x tr a c t w as c e n trifu g e d a t 20, 000 x g in a S e r v a li

v a c u u m -ty p e c e n tr ifu g e in o r d e r to s e d im e n t the c e llu la r d e b r is and th e
tra n s lu c e n t s tr a w - c o lo r e d s u p e rn a ta n t d ilu te d w ith s a lin e to a f i n a l v o l­
u m e o f 5 m l. (0 .4 1 2 m g m . b a c t e r ia l n it r o g e n / m l. ).

A liq u o ts o f 0, 5 m l.

w e re added to p e p tid e s o lu tio n s c o n ta in in g 5 m g m . o f p e p tid e p e r 0 . 5 m l.
do u b le g la s s - d is t ille d w a te r .

T h e p e p tid e s use d w e re L - , and D - le u c y l-

g ly c in e , L - and D - le u c y lg ly c y lg ly c in e , and g ly c y l- L - tr y p to p h a n e .

In

a d d itio n L - and D - le u c y lg ly c in e s o lu tio n s w e re p re p a re d c o n ta in in g a

- 42- -

f i n a l c o n c e n tra tio n o f s o d iu m c y a n id e a m o u n tin g to 0 . 0 1M .

A fte r in ­

c u b a tio n f o r one h o u r a t 3 0 ° C ., 0. 04 m l. q u a n titie s o f each s o lu tio n
w e re s p o tte d on f i l t e r p a p e r sh e e ts and c h ro m a to g ra p h e d in w a t e r - s a t u r ­
a te d p h e n o l.

C o n tro ls in c lu d e d c e ll- f r e e e x tr a c t a lo n e , know n a m in o

a c id s and p e p tid e s , te s t s o lu tio n s s a m p le d at z e ro tim e and te s t s o lu tio n s
c o n ta in in g kn o w n a m in o a c id s (in o r d e r to d e m o n s tra te u n ifo r m m o v e m e n t
o f th e se a c id s in th e te s t m ix tu r e ) *

Spot te s ts f o r a m m o n ia w e re c a r r ie d

out u s in g N e s s le r ’ s re a g e n t.

R e s u lts :
C h ro m a to g ra p h ic a n a ly s is d e m o n s tra te d th e p u r it y o f the p e p tid e s
u se d and e n s u re d th a t th e s t e r iliz a t io n m e th o d d id n o t r e s u lt in a n y s p l i t ­
tin g o f th e p e p tid e b o n d s.

T h e R f v a lu e s fo u n d f o r the v a r io u s a m in n

a c id s and p e p tid e s a re g iv e n in T A B L E 6.

TABLE 6
T A B L E SHO W ING T H E R f V A L U E S O F A M IN O A C ID S A N D P E P T ID E S
O B T A IN E D D U R IN G P A P E R P A R T IO N C H R O M A T O G R A P H Y
IN W A T E R -S A T U R A T E D P H E N O L A T 2 7 °C .

C om pounds

R f v a lu e

g ly c in e

0 .4 1

le u c in e

0 .8 2

try p to p h a n e

0 .7 3

- 43 -

C om p ou nds

R f v a lu e

g ly c y lg ly c in e

0 .4 2

g ly c y lg ly c y lg ly c in e

0 .4 8

g ly c y l- L . -try p to p h a n e

0 .7 1

L - and D - le u c y lg ly c in e

0 .7 6

L - and D= le u c y lg ly c y lg ly c in e

0. 79

T h e u t iliz a t io n o f the d i - and t r i - p e p tid e s as s o le s o u rc e s o f n i t r o ­
gen b y R 2 P ■^20-27A* an<^ ^ 2 0

p ic t o r ia lly re p re s e n te d in F IG U R E

6.

E x c e p t f o r an o v e r a ll re d u c tio n in the m a g n itu d e of g ro w th (and in the
u t iliz a t io n o f L - le u c y lg ly c y lg ly c in e as c o m p a re d to L - le u c y lg ly c in e ) the
re s p o n s e p a tte r n o f ^ 2 0 - 2 7 A *s v e r y s i m i l a r to th a t o f R 2 i«
p a tte rn , h o w e v e r, is n o tic e a b ly d iff e r e n t .

The R 2 0

W ith th is o rg a n is m th e re w as

a re m a r k a b le g ro w th re s p o n s e b ro u g h t a b out b y g ly c y l- L - tr y p to p h a n e ,
b e tte r g ro w th on g ly c y lg ly c y lg ly c in e a s c o m p a re d w ith L - le u c y lg ly c in e
and a g r e a te r g ro w th a tta in e d on D - le u c y lg ly c y lg ly c in e th a n on L - le u c y l g l y ­
c y lg ly c in e .

In fa c t, th is p a r t ic u la r o rg a n is m m ade b e tte r use o f th o se p e p ­

tid e s w h ic h c o n ta in e d D -a m in o a c id s th a n the o th e r tw o s tr a in s o f r h iz o b ia .
W ith a l l th re e s tr a in s , h o w e v e r, L - le u c y lg ly c in e p ro m o te d b e tte r g ro w th
th a n D - le u c y lg ly c in e .

T he f in a l pH v a lu e s ran ged ir o m

6.

2 to

6. 8

.

T h e c h ro m a to g ra p h ic r e s u lts in d ic a te d th a t R 2 1 a t le a s t p a r t ia lly
h y d ro ly z e d a l l the p e p tid e s a g a in s t w h ic h i t w as te s te d .
c in e w a s fo u n d as th e fr e e a c id .

In a l l c a s e s g ly ­

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- 44 -

S in ce th e R f v a lu e o f le u c in e w as s i m i l a r to th a t o f the le u c in e p e p tid e s
no c r i t i c a l a tte m p t w a s m a de to id e n tify th is a m in o a c id , a lth o u g h s e v e r ­
a l d iffe r e n t s o lv e n t s y s te m s w e re t r ie d in an e f f o r t to o b ta in b e tte r r e s o lu ­
tio n ,

E lu t io n o f th e g ly c in e s p o ts in 10 m l, o f 50% e th a n o l: w a te r and

c o m p a ris o n o f the r e s u ltin g s o lu tio n s in a C o le m a n S p e c tro p h o to m e te r
a t 570 m illim ic r o n s (12) re v e a le d th a t th e a m o u n t o f g ly c in e p ro d u c e d
f r o m D - le u c y lg ly c y lg ly c in e w as o n ly about o n e -h a lf th a t p ro d u c e d f r o m
the o th e r p e p tid e s , w h ic h p ro d u c e d s i m i l a r a m o u n ts o f g ly c in e .

Am m o­

n ia w a s d e te c te d in e v e ry te s t s o lu tio n e x c e p t in th o se c o n ta in in g c y a n id e ,
and im p lie d th a t a c y a n id e s e n s itiv e d e a m in a tio n o f th e lib e r a te d a m in o
a c id s o c c u r r e d ,
T A B L E 7 in d ic a te s th e s tim u la tiv e and in h ib it o r y a c t iv it y re s id e n t
in c e r ta in o f the p e p tid e s , a s f a r as r h iz o b ia l g ro w th is c o n c e rn e d .

D is c u s s io n :
A lth o u g h h y d r o ly s is o f th e p e p tid e s b y the c e ll- f r e e b a c te r ia l e x ­
t r a c ts w as d e m o n s tra te d th e e x p e rim e n ta l r e s u lts a re n o t c o n s is te n t w ith
the co n ce p t o f c o m p le te h y d r o ly s is .

T h e d i- and t r i - p e p tid e s o f g ly c in e ,

and g ly c in e p lu s L - le u c in e , w ith tw o e x c e p tio n s , s tim u la te d th e g ro w th
o f th e th r e e R h iz o b iu m s tr a in s above th e v a lu e s th a t w o u ld be e x p e c te d
i f c o m p le te h y d r o ly s is w e re a s s u m e d .

T h e tw o e x c e p tio n s o c c u r r e d w ith

R 2 0 -2 7 A and th e PePtid e s g ly c y lg ly c y lg ly c in e and L - le u c y lg ly c in e , b o th
o f w h ic h y ie ld e d “ o b s e rv e d ** g ro w th v a lu e s w h ic h w e re n o t s ig n if ic a n tly

-

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-46d if f e r e n t f r o m
a tio n ,

v a lu e s c a lc u la te d on th e b a s is o f c o m p le te p e p tid e d e g ra d ­

T h is o r g a n is m , th e r e fo r e , a p p a re n tly has th e e n z y m ic c a p a c ity

to h y d ro ly z e th e s e tw o p e p tid e s c o m p le te ly .

W ith g ly c y lg ly c y lg ly c in e

th e e n zym e ( o r e n z y m e s ) c o n c e rn e d m a y a tta c k b o th ends o f th e t r ip e t id e
c h a in s im u lta n e o u s ly , th e re b y le a v in g no s tim u la tiv e d ip e p tid e f r a c tio n s .
I t c a n n o t be s a id w ith c e r t a in ty th a t th e u t iliz a t io n o f a m in o a c id s
f o r p r o te in s y n th e s is b y b a c te r ia in v o lv e s p r i o r in c o r p o r a tio n in to
s m a ll p e p tid e s , b u t c o n s id e ra b le e v id e n c e f o r th is co n c e p t h a s been ob• ta in e d f r o m o b s e rv a tio n s on b a c te r ia such as L a c to b a c illus c a s e i

w h ic h

r e q u ir e s , f o r o p tim a l g ro w th , th e p re s e n c e o f p r e fo r m e d p e p tid e s (2 9).
O th e r o r g a n is m s , as w e ll as r h iz o b ia , use p e p tid e s f o r g ro w th m o re
e f f ic ie n t ly th a n th e y do th e a m in o a c id s w h ic h c o m p o s e th e m .

T h is

a p p ro a c h to th e s tu d y o f p r o te in s y n th e s is is a t p re s e n t b e in g e x p lo re d by
F r u to n and S im m o n d s (2 8 ),

I f i t is a ssu m e d th a t th e s tim u la tiv e p e p tid e s

can be d ir e c t ly u t iliz e d f o r p r o te in s y n th e s is by r h iz o b ia th e a m o u n t o f
o b s e rv e d s tim u la tio n is p ro b a b ly r e p re s e n ta tiv e o f a b a la n c e b e tw e e n
the ra te o f in c o r p o r a tio n o f th e p e p tid e s in to c e llu la r p r o te in s and th e
r e la tiv e p e p tid a s e a c t iv it y o f th e s e m ic r o o r g a n is m s .

I f the p e p tid a s e

a c t iv it y o f a p a r t ic u la r s t r a in is g r e a te r th a n th e in c o r p o r a tio n ra te
th e n th e a m o u n t o f s tim u la tio n b y a p e p tid e w o u ld be m u c h lo w e r th a n i f
th e c o n v e rs e w e re t r u e .

T h is p ro b a b ly e x p la in s th e d iffe r e n t d e g re e s '

o f s tim u la tio n o b s e rv e d w ith th e b a c te r ia l s tr a in s u s e d in th e p re s e n t
w o rk .

- 47 -

A n in h ib it o r y e ffe c t w a s n o te d w hen R 2 1 and R 2 0 -2 7 A w e re g ro w n
in th e p re s e n c e o f th e D - le u c in e p e p tid e s .

T h is m a y p a r t ly be th e r e s u lt

o f an i n a b ilit y to c a r r y out tra n s p e p tid a tio n re a c tio n s in v o lv in g D - a m in o
a c id s .

I t is a ls o n e c e s s a ry , h o w e v e r, to p o s tu la te th a t an in h ib it o r y

a c tio n o c c u r r e d b e tw e e n g ly c in e and D - le u c in e .

I f in c o r p o r a tio n d id no t

o c c u r b y re a s o n o f th e c o n ta in e d D -a m in o a c id s i t w o u ld have be e n e x ­
p e cte d th a t h y d r o ly s is w o u ld have p ro c e e d e d to c o m p le tio n , the a m in o
r

a c id p ro d u c ts b e in g re m o v e d f r o m the s ite b y v a r io u s ty p e s o f s e c o n d a ry
re a c tio n s , su ch as d e a m in a tio n .

W ith su ch h y d r o ly s is th e “ o b se rve d **

g ro w th o f th e o r g a n is m s h o u ld have been e q u a l to th e “ c a lc u la te d ** g ro w th
w ith no e v id e n c e o f in h ib itio n .

T h e m e re fa c t th a t in h ib it io n w as p re s e n t

m a k e s i t a p p e a r th a t one o f th e lib e r a te d a m in o a c id s b lo c k e d th e u t i l i z a ­
tio n o f th e o th e r as e ls e th a t a m u tu a l in h ib itio n o c c u r r e d .

C o n fir m a tio n

o f su ch in h ib itio n w as o b ta in e d b y c o m p a rin g the g ro w th o f w ash ed R 2 1
c e lls in b a s a l m e d iu m c o n ta in in g g ly c in e , D -le u c in e , o r g ly c in e + D le u c in e .

A d e fin ite re d u c tio n in g ro w th w as fo u n d in th e s o lu tio n c o n ta in ­

in g b o th a m in o a c id s w hen it s m a x im u m n e p h e lo s w as c o m p a re d w ith the
th e o r e tic a l.

T h is a c tio n is p ro b a b ly s i m i l a r to th a t o b s e rv e d b y K ih a r a

and S n e ll (51), w ho show ed th a t h ig h le v e ls o f g ly c in e in h ib ite d th e u t i l i ­
z a tio n o f D -a la n in e in L a c to b a c illu s c a s e i, b u t d id n o t a ffe c t L - a la n in e .
O th e r re a s o n s f o r th e s tim u la tiv e r e s u lts o f c e r t a in p e p tid e s , as
su g g e ste d b y o th e r w o r k e r s , m a y o r m a y n o t a p p ly to r h iz o b ia .
re a s o n s in c lu d e :

These

- 48 -

(a) th e p r o te c tio n a ffo rd e d a g a in s t the d e s tr u c tio n o f th e in d iv id u a l
a m in o a c id s b y o th e r e n z y m a tic fo r c e s (51),
(b) th e p r o te c tio n a ffo rd e d a g a in s t a m in o a c id in h ib ito r s , as show n
b y s e v e r a l g ro u p s o f in v e s tig a to r s (41), (74),
and (c) the a b ilit y o f the o rg a n is m to c o n c e n tra te the p e p tid e , b u t n o t
th e a m in o a c id (71).
One o f th e m o re in te r e s tin g e ffe c ts n o tic e d w as th e s tim u la tio n o f
R h iz o b iu m R 2 q b y D - le u c y lg ly c in e .

One m ig h t p o s tu la te in c o r p o r a tio n

o f th is p e p tid e in to th e p r o te in o f the m ic r o o r g a n is m , b u t the p o s s ib lity
o f o th e r u n kn o w n m e c h a n is m s o c c u r r in g m u s t f i r s t be in v e s tig a te d .

At

p re s e n t th e e ffe c t is in e x p lic a b le on th e b a s is o f f o r m e r w o r k .
In the a b sence o f f u r t h e r data l i t t l e can be s a id a b out th e n a tu re o f
th e in t r a c e llu la r p e p tid e - s p littin g e n z y m e s p re s e n t in r h iz o b ia .

These

b a c te r ia p ro b a b ly c o n ta in a g ly c in e d ip e p tid a s e , w h ic h is a r a th e r s p e c i­
f i c e n zym e a c c o rd in g to S m ith (81).

In v ie w o f the fa c t th a t D - le u c y lg ly -

c in e is a tta c k e d as r e a d ily as L - le u c y lg ly c in e b y the se s tr a in s o f n o dule
b a c te r ia th e re s p o n s ib le e n zy m e (o r e n z y m e s ) m u s t be r a d ic a lly d iffe r e n t
f r o m m o s t o f the c la s s ic a l p e p tid a s e s , su ch as le u c in e a m in o p e p tid a s e ,
w h ic h e x h ib its a s te r e o c h e m ic a l s p e c ific ty f o r L - a m in o a c id p e p tid e s .
I t m a y be th a t th e L - le u c in e p e p tid e s a r e b ro k e n dow n in th e r h iz o b ia b y
le u c in e a m in o p e p tid a s e and th a t a s e p a ra te e n zym e is re s p o n s ib le f o r the
a tta c k up o n the D - f o r m s .

T h e p e p tid a s e a c t iv it y o f r h iz o b ia a p p e a rs

s o m e w h a t s i m i l a r to th a t o f L e u c o n o s to c m e s e n te ro id e s w h ic h S m ith ( 8 ),

-

49

-

in h is r e v ie w a r t ic le , s ta te s as h a v in g tw o d ip e p tid a s e s and a t le a s t tw o
tr ip e p tid a s e s .

In a d d itio n , th is o rg a n is m a ls o e x e rts a r a p id a c t iv it y

on th e D .-fo rm s o f a v a r ie t y o f p e p tid e s , a phenom enon w h ic h i t now
s h a re s w ith c e r ta in s tr a in s o f R h iz o b iu m .
T h e e n z y m e re s p o n s ib le f o r the b re a k d o w n o f g ly c y l- L - tr y to p h a n e
is u n kn o w n and r e p re s e n ts an in te r e s tin g p o s s ib ilit y f o r f u r t h e r r e s e a r c h .

C.

T h e U t iliz a t io n o f A m m o n iu m , N itr a t e and N it r it e S a lts in the
P re s e n c e o f A m in o and C a r b o x y lic A c id s .

A lth o u g h R 2 1 , R 2 0 -2 7 A * an<* ^ 2 0 w e re no^ c a Pa^ le o f u s in g a m m o n ­
iu m , n it r a t e o r n i t r it e s a lts “ p e r s e * * as s o le s o u rc e o f n itr o g e n (48)
th e re w as a p o s s ib ilit y th a t u t iliz a t io n m ig h t o c c u r in th e p re s e n c e o f
c e r ta in o th e r co m p o u n d s, p a r t ic u la r ly a m in o a c id s .

T h e s e e x p e rim e n ts

w e re d e v is e d to te s t th is h y p o th e s is .

M e th o d s :
S te r ile , c h e m ic a lly c le a n o p tic a l c u v e tte s w e re f i l l e d w ith 9. 0 m l.
a liq u o ts o f s t e r ile pH 7. 0 m o d ifie d W ils o n and W ils o n 's m e d iu m to g e th e r
w ith

0. 5

m l, o f s t e r ile a m m o n iu m c h lo r id e , s o d iu m n it r a t e , o r s o d iu m

n it r it e s to c k s o lu tio n s so th a t the f in a l c o n c e n tra tio n s w e re e q u iv a le n t to
0 .0 1 , 0 .0 0 1 and 0 .0 0 0 1 m o le s o f s a l t / l i t e r .

T h e se s to c k s o lu tio n s w e re p r e ­

p a re d f r o m C . P . g ra d e com p o u n d s ( B r it is h D ru g H ou ses L t d . - “ A n a la r* *
g ra d e )

and w e re s t e r iliz e d b y s in te re d g la s s f i l t r a t i o n .

T o each c u v e tte

- 50 -

w as added 0 . 5 m l. o f s to c k L - le u c in e s o lu tio n ( fin a l c o n c e n tra tio n =
0 . 00 0 1 M ) and 0 .2 m l. o f w a sh e d R20* -^21* o r -^2 0 -2 7 A c e ^ s » In o c u la t­
ed and u n in o c u la te d c o n tr o ls w e re p re p a re d w h ic h c o n ta in e d le u c in e b a s a l
m e d iu m and 0 . 5 m l. o f s t e r ile d is t ille d w a te r in p la c e o f the s a lt s o lu ­
tio n .

In c u b a tio n w a s c a r r ie d out a t 3 0 °C . and d a ily n e p h e lo s re a d in g s

re c o rd e d f o r ea ch c u v e tte .

A m m o n ia and n i t r it e te s ts (60) w e re m ade

a f t e r s e v e r a l da ys and a g a in a t th e p e rio d of m a x im u m g ro w th .
T o a s e r ie s o f n in e s t e r ile c u v e tte s 9 .0 m l. o f s t e r ile pH 7 .0
m o d ifie d W ils o n an d W ils o n 's m e d iu m w e re added.

T o c u v e tte s #1 to

#4 0. 5 m l. o f s to c k a m m o n iu m c h lo r id e s o lu tio n w as added (to g iv e a
f in a l c o n c e n tra tio n o f 0. 01M ) to g e th e r w ith 0. 5 m l. o f 0. 5M a lp h a k e to g lu ta r ic a c id ( N u t r itio n a l B io c h e m ic a ls L t d . ) o r s o d iu m fu m a ra te
( B r it is h D ru g H o u s e s L t d . ).

C u v e tte #5 c o n ta in e d a m m o n iu m c h lo r id e

b a s a l m e d iu m and 0. 5 m l. o f s t e r ile d is t ille d w a te r, w h ile th e r e m a in ­
in g tubes h e ld b a s a l m e d iu m p lu s the c a r b o x y lic a c id s and 0. 5 m l, o f
s t e r ile w a te r .

A f t e r in o c u la tio n w ith 0 .2 m l. o f w a sh e d R 2 1 c e lls these

c u v e tte s , to g e th e r w ith in o c u la te d and u n in o c u la te d b a s a l m e d iu m c o n ­
t r o ls w e re in c u b a te d a t 3 0 °C .

and n e p h e lo s re a d in g s m a de a t 24 h r .

in t e r v a ls .
A tte m p ts w e re m ade to re p la c e th e c a r b o x y lic a c id s b y gaseous
c a rb o n d io x id e .

B a s a l m e d iu m c o n ta in in g 0. 0 1 M a m m o n iu m c h lo r id e

w as in o c u la te d w ith w a sh e d R 2 1 c e lls and p la c e d in a la r g e d e s s ic a to r
to g e th e r w ith s u ita b le c o n tr o ls .

M e a s u re d a m o u n ts o f a i r w e re w ith d ra w n

- 51 -

f r o m th e c h a m b e r b y m e a n s o f a s y rin g e f it t e d w ith a tw o -w a y v a lv e
and re p la c e d w ith an e q u iv a le n t v o lu m e o f gaseous c a rb o n d io x id e (M a th is o n C o. U . S . A . ) .

T h e d e s s ic a to r w as p la c e d in a 3 0 °C . in c u b a to r f o r

seve n d a ys, a t w h ic h tim e th e tub es w e re re m o v e d and e x a m in e d .

C a r­

bon d io x id e c o n c e n tra tio n s o f 5-10% w e re e m p lo y e d .
W ashed c e lls o f R h iz o b iu m R 2 1 w e re added to c u v e tte s c o n ta in in g
9 .0 m l. o f m o d ifie d pH 7. 5 b a s a l m e d iu m , 0. 5 m l. o f s to c k L - h is t id in e
s o lu tio n ( f in a l c o n c e n tra tio n = 0. 0 0 0 1M) and 0. 5 m l. o f e ith e r w a te r o r
an a m m o n iu m c h lo r id e s o lu tio n .

U s in g a m m o n iu m c h lo r id e s o lu tio n s

o f d iffe r e n t c o n c e n tra tio n s f in a l c o n c e n tra tio n s ra n g in g f r o m O.to 0. 02M
w e re o b ta in e d .

A f t e r in c u b a tio n at 3 0 °C . the m a x im u m n e p h e lo s r e a d ­

in g s w e re p lo tte d a g a in s t th e m o la r it y of a m m o n iu m c h lo r id e to y ie ld
a g ro w th re s p o n s e c u r v e .

(F IG U R E 7)

R e s u lts :
F IG U R E

8

illu s t r a t e s the m a x im u m g ro w th in n e p h e lo s u n its fo u n d

d u rin g th e u t iliz a t io n o f a m m o n iu m , n itr a te , and n i t r it e s a lts b y n o d u le
b a c te r ia g ro w in g in the p re s e n c e o f a m in o a c id n itr o g e n .

N o fr e e a m m o n ­

ia w as lo c a te d in the n it r a t e o r n i t r it e tub es and no n i t r i t e w as fo u n d in
the n it r a t e tu b e s .

T h e re w as d e fin ite e v id e n c e th a t the in o rg a n ic n i t r o ­

gen s a lts (in c lu d in g n it r it e , w h ic h som e w o r k e r s re g a r d as b e in g n o n ­
u t iliz a b le b y a lfa lfa r h iz o b ia ) w e re u t iliz e d w hen a m in o a c id s w e re p r e ­
se n t to in it ia t e g ro w th .

T h e o p tim u m m o la r it y o f a m m o n iu m c h lo r id e in

o

.0 0 5

.0 1 0

.0 1 5

.0 2 0

Concentration of ammonium chloride (moles/liter)

Pig. 7

Growth response curve of Rhlzobium R21 in histidine
basal medium containing various concentrations of
ammonium chloride.

S
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- 52

h is tid in e m e d iu m w a s 0.

01

-

(see F IG U R E 7).

T a b le

8

g iv e s v a lu e s o b ­

ta in e d w ith the c a r b o x y lic a c id s and in d ic a te s th a t the a m in o a c id s can
be re p la c e d b y c e r ta in o rg a n ic a c id s p lu s in o rg a n ic n itro g e n o u s s a lts .
N o g ro w th w a s e v id e n t in a n y o f th e a m m o n iu m c h lo r id e tu b e s in c u b a te d
u n d e r in c re a s e d c a rb o n d io x id e te n s io n s .

TABLE

8

M A X IM U M N E P H E L O S R E A D IN G S A T T A IN E D B Y R 2 \ IN S Y N T H E T IC
B A S A L M E D IA C O N T A IN IN G A M M O N IU M C H L O R ID E A N D C E R T A IN
C A R B O X Y L IC A C ID S

C a r b o x y lic A c id

p lu s N H 4 C1

m in u s N H 4 C I

a lp h a - k e t o g lu ta ra te

2 0 4 .0

24,

p y ru v a te

15 8.0

15. 0 *

fu m a ra te

3 2 .0

none

0 .0

16.

0

0

*

*

0 .0

* n o t s ig n if ic a n t ly d iffe r e n t f r o m each o th e r

D is c u s s io n :
A lth o u g h a m m o n iu m io n s a r e n o t u t iliz e d b y the te s t R h iz o b iu m
s tr a in s a s th e s o le s o u rc e o f n itr o g e n in s u c ro s e b a s a l m e d iu m th e y a re
r e a d ily u t iliz e d , as a r e n it r a t e and n i t r it e io n s , w hen e ith e r a m in o o r
c e r ta in c a r b o x y lic a c id s a re p re s e n t.

T h e se r e s u lts m a y be in te r p r e te d

- 53 -

b y a s s u m in g th a t in “ r e s t in g ” r h iz o b ia in t r a c e llu la r c a rb o n a c e o u s a c id s
im p o r ta n t in th e s y n th e s is o f a m in o a c id s f r o m a m m o n ia do n o t a c c u m u ­
la te in q u a n titie s c o m p a tib le w ith g ro w th .

W hen c a r b o x y lic o r a m in o

a c id s a r e s u p p lie d in th e e x te r n a l m e d iu m s u ffic ie n t q u a n titie s o f th e se
e s s e n tia l “ a m m o n ia - fix in g ” a c id s b e co m e a v a ila b le f o r s y n th e tic p u r ­
p o se s.

T h e s e r e s u lts a re no t b y any m e a n s n o v e l s in c e V ir ta n e n and

R au ta n e n (96) have r e p o r te d th a t D as o b s e rv e d g ro w th o f A e r o b a c te r a e r o genes on a m m o n iu m n itr o g e n in the p re s e n c e o f fu m a r ic , p y r u v ic , a lp h a k e to g lu ta r ic and o x a la c e tic a c id s .

One o f the m o s t p ro m in e n t a c id s in

a m in o a c id s y n th e s is is a lp h a - k e to g lu ta r ic a c id f r o m w h ic h a m in o -a c id s
ca n be fo r m e d b y a r e v e r s a l o f L - g lu t a m ic a c id d e h y d ro g e n a s e a c t iv it y
(94).

T h is re a c tio n , to g e th e r w ith th a t o f a s p a rta s e , f o r m s one o f the

tw o kn o w n p a th w a y s o f a m in o a c id s y n th e s is v ia a m m o n ia u t iliz a t io n (67).
T h is m a y e x p la in th e h ig h a c t iv it y o f a lp h a - k e to g lu ta r ic a c id in the p r e ­
s e n t e x p e rim e n t.

T h e o th e r c a r b o x y lic a c id s , in c lu d in g th o s e p ro d u c e d

f r o m an y a m in o a c id p re s e n t, m a y n o t be in th e m s e lv e s re a c tiv e , b u t
a re ca p a b le o f fo r m in g a lp h a -k e to g lu ta ra te w ith in the c e ll, p e rh a p s b y a
c y c lic s y s te m o f r e a c tio n s su c h as th e K r e b ’ s c y c le .

,

T h e c o m p a r a tiv e ly

s m a ll a m o u n ts o f g ro w th o b s e rv e d in th e p re s e n c e o f the th re e o rg a n ic
a c id s , b u t in th e a b sence o f an y added a m m o n iu m s a lt p ro b a b ly in d ic a te s
th a t tr a c e a m o u n ts o f in o rg a n ic n itr o g e n p re s e n t in th e m e d iu m c o n s ti­
tu e n ts w e re u t iliz e d .

A m m o n iu m c h lo r id e w as fo u n d to be a b e tte r s o u rc e

o f n itr o g e n in th e o rg a n ic a c id m e d ia th a n e ith e r n it r a t e o r n i t r it e , w h ic h

- 54 -

p ro b a b ly

e m p h a s iz e s it s p r im e im p o rta n c e .

T h e tw o l a t t e r co m pou nds

a r e no d o ubt c o n v e rte d to a m m o n ia p r io r to u t iliz a t io n , s in c e b o th a re
r e a d ily r e d u c ib le in th e sequence: n it r a t e

^ n itr it e

^ a m m o n ia ( 9 4 ).

T h e o p tim u m c o n c e n tra tio n o f a m m o n iu m c h lo r id e and n itr a te w as
a p p r o x im a te ly 0 . 0 1M , w h ile a t th is c o n c e n tra tio n n i t r it e w a s m a r k e d ly
in h ib it o r y .

I t is in te r e s tin g to no te th a t in M e y e rh o f’ s w o rk , as r e p o r te d

b y S tephenson (82), the r a te o f n i t r it e o x id a tio n b y N itr o b a c te r w as g r e a t ­
e s t a t a n i t r i t e c o n c e n tra tio n o f
tio n in c re a s e d .

0

.

1%

and f e l l o ff r a p id ly as the c o n c e n tra ­

S tephenson b e lie v e s th a t th is c o u ld be e x p la in e d b y the

c o n s id e r a tio n th a t n i t r i t e a c ts as a c e ll p o is o n , p o s s ib ly b y re a c tin g w ith
the fr e e a m in o g ro u p s o f c e llu la r p r o te in s .

T he m a x im u m g ro w th on

n i t r it e in the p re s e n t w o r k w as a t a f in a l s o d iu m n i t r it e c o n c e n tra tio n o f
0 .0 0 1 M and the in h ib it o r y a c t iv it y o f g r e a te r a m o u n ts th a n th is m a y be a
r e s u lt o f th e above m e n tio n e d to x ic e ffe c ts .
C lifto n (1 9 ) m e n tio n s th a t the a s s im ila tio n o f a m m o n ia b y b a c te r ia
and y e a s t m a y be dependent upon e n e rg y y ie ld in g p ro c e s s e s .

I f th e a m in o

and c a r b o x y lic a c id s a re m o re e ff ic ie n t e n e rg y p ro d u c e rs f o r th is p u rp o s e
th a n th e s u c ro s e p re s e n t in th e b a s a l m e d iu m t h e ir in flu e n c e on a m m o n ia
a s s im ila tio n m a y be p a r t ia lly e x p la in e d in th is m a n n e r as w e ll.
S om e h e te r o tr o p h ic b a c te r ia a re kno w n to r e q u ir e the p re s e n c e o f
added c a rb o n d io x id e f o r o p tim u m g ro w th and the s u b s titu tio n o f s e v e r a l
d ic a r b o x y lic a c id s f o r th is gas can s a tis fy th e re q u ire m e n t (1 ).

The

u t iliz a t io n o f c a r b o x y lic a c id s b y r h iz o b ia is a p p a re n tly n o t due to a C O 2

r e q u ir e m e n t, s in c e th is gas c o u ld no t re p la c e th e a c id s in a m m o n iu m
io n u t iliz a t io n .
A lth o u g h no e v id e n c e w as fo u n d f o r the c o n v e rs io n o f n itr a te to
n i t r it e , o r o f n i t r i t e to a m m o n ia th e se re a c tio n s m a y a c tu a lly e x is t,
the end p ro d u c ts b e in g e ith e r v e r y t r a n s it o r y in n a tu re o r r a p id ly
u t iliz e d .
T h e u t iliz a t io n o f in o rg a n ic n itr o g e n s o u rc e s does n o t a p p e a r
p r o m is in g a s an in d ic a tio n o f e ffe c tiv e n e s s o r in e ffe c tiv e n e s s in the
n o d u le b a c te r ia , h o w e v e r the d a ta have re s u lte d in the fo r m u la tio n o f
a s y n th e tic m e d iu m f o r f u r t h e r g ro w th s tu d ie s on w ash ed c e lls of
R h iz o b iu m m e li l o t i .

I t is co m p o se d o f s u c ro s e , 2. 50 g m . ; a m m o n iu m

c h lo r id e , 0. 53 g m * ; d ip o ta s s iu m h y d ro g e n phosphate, 0 . 50 g m . ; s o d iu m
c h lo r id e ,

0 .1 0

g m . ; m a g n e s iu m s u lp h a te ,

0

.

20

g m . ; c a lc iu m s u lp h a te ,

0 .2 0 g m . ; f e r r i c c h lo r id e , 0. 10 m g m .; h is tid in e , 7 .5 0 m g m .; g la s s d is t ille d w a te r, 1000 m l.

T h e pH is a d ju s te d to 7. 5 and s t e r iliz a t io n

a c c o m p lis h e d b y S e itz f i l t r a t i o n .

T he p u r ific a tio n o f th e s u c ro s e and

m in e r a l s a lts is c a r r ie d ou t u s in g the m e th o d s o f W ils o n and W ils o n (101).

D.

T h e g e n e r a l m e ta b o lis m o f c e r t a in a m in o a c id s

r

T h e R h iz o b iu m s tr a in s h e r e in in v e s tig a te d a re a p p a re n tly d e p e n ­
dent up on th e p re s e n c e o f p r e fo r m e d a m in o a c id s ( o r a m m o n iu m io n s
p lu s c e r ta in o rg a n ic a c id s ) f o r g ro w th in a c h e m ic a H y d e fin e d m e d iu m a t le a s t so f a r a s s m a ll in o c u la a re c o n c e rn e d .

T h e a c tu a l e n z y m a tic

- 56 -

m e c h a n is m s b y w h ic h th e se o rg a n is m s m e ta b o liz e a m in o a c id s is s o m e ­
w h a t o b s c u re and f u r t h e r in fo r m a tio n is needed*

F o r th is re a s o n the

p re s e n t s e c tio n o f the w o r k w as c o m m e n c e d .
A n y liv in g c e ll p o s s e s s e s an a m a z in g a r r a y o f e n z y m ic p o te n tia l­
it ie s , s o m e o f w h ic h a re e x c e e d in g ly c o m p le x and, in m a n y c a s e s , n o t
as y e t v is u a liz e d b y th e c e llu la r p h y s io lo g is t*

T h e p ro c e s s e s w h ic h

o c c u r a r e e ve n m o re c o m p lic a te d in th a t th e y m a y be e a s ily m o d ifie d
b y cha nge s i n th e p h y s io c h e m ic a l e n v iro n m e n t.

In a d d itio n , a n u m b e r

o f m e ta b o lic p a th w a ys m a y e x is t f o r an y one com pound and th e i m p o r t ­
ance o f a n y p a r t ic u la r ro u te m a y be e a s ily u n d e r - o r o v e rra te d .

In

the w o rd s o f C ohen and M c G ilv e r y (21): “ I t is p la in th a t no d e ta ile d
p ic tu r e o f n itr o g e n m e ta b o lis m in te r m s o f s p e c ific e n z y m a tic re a c tio n s
can be d ra w n .

In the tis s u e s lic e , tis s u e b r e i, and c ru d e tis s u e e x tr a c t,

the b re a k d o w n o f a p a r t ic u la r a m in o a c id m a y in v o k e tra n s a m in a tio n ,
tr a n s im in a tio n , d e c a rb o x y la tio n , and p o s s ib ly o th e r y e t u n kn o w n n o n -o x id a tiv e re a c tio n s y ie ld in g d iffe r e n t n itr o g e n com pounds m o re s u s c e p tib le
to a tta c k , so th a t a d ir e c t in te r p r e ta t io n o f the r e s u lts o b ta in e d w ith such
s y s te m s is u s u a lly d i f f ic u lt and s u b je c t to m a n y d o u b ts ” .

In s p ite o f

th is a u ra o f p e s s im is m i t w a s f e lt th a t the la b y r in th o f in t r a c e llu la r r e ­
a c tio n s c o u ld a t le a s t be p a r t ia lly re s o lv e d b y a n u m b e r o f b io c h e m ic a l
p ro c e d u re s .

U t iliz in g som e o f th e se p ro c e d u re s a g e n e ra l s u r v e y o f the

a m in o a c id m e ta b o lis m o f th e e ffe c tiv e R h iz o b iu m s t r a in R 2 i w as m a d e .
I t w as hoped, th e re b y , to g a in an u n d e rs ta n d in g o f the “ n o r m a l” g e n e ra l

- 57 -

m e ta b o lic p a th s in h e re n t in r h iz o b ia so th a t an a p p ro a c h to su b sequ ent
s tu d ie s o f “ a b n o r m a l” m e ta b o lis m , as m a n ife s te d in the in e ffe c tiv e o r
p a r a s itic s tr a in s , c o u ld be m ade w ith m o re a s s u ra n c e .

N o a tte m p t w as

m a d e to e lu c id a te the tr a n s fo r m a tio n s s p e c ific f o r in d iv id u a l a m in o a c id s .
In o r d e r to e lim in a te n e e d le s s r e p e titio n s e v e r a l o f th e e x p e rim e n ­
t a l m e th o d s u s e d re p e a te d ly in th is s e c tio n w i l l be g iv e n b e lo w :

(a)

P r e p a r a tio n o f c e ll su sp e n s io n s and c e ll- f r e e e x tr a c ts .
T h re e l i t e r s o f M o d ifie d M e d iu m “ 79” w e re in o c u la te d w ith a 10 m l.

lo g a r ith m ic phase c u ltu r e o f

^ and in c u b a te d u n d e r c o n tin u o u s a e ra tio n

f r o m a s in te r e d g la s s s p a rg e r f o r 48 h r .

A f t e r th is p e rio d th e c e lls

w e re c h e cke d f o r p u r it y b y the G ra m s ta in , s e d im e n te d in a S h a rp ie s
c e n trifu g e , w a s h e d tw ic e w ith 0.85% s t e r ile s a lin e and b ro u g h t to the
d e s ire d v o lu m e w ith b u ffe r o f a p p ro p ria te pH .
s titu te d a “ w a sh e d ceU s u s p e n s io n ” .

T h is p r e p a ra tio n c o n ­

F o r th e p r e p a r a tio n o f c e ll- f r e e

e x tr a c ts the w a s h e d c e lls w e re s e d im e n te d , m ix e d w ith f in e ly p o w d e re d
a lu m in u m o x id e and g ro u n d f o r 12 m in . in a P o tte r h o m o g e n iz e r, u s in g
an ic e b a th to re d u c e f r i c t i o n a l h e a t.

T h e r e s u ltin g m ix tu r e w a s c e n t r i­

fu g e d in a S e r v a ll v a c u u m c e n trifu g e f o r 15 m in . a t 18, 000 x g in o r d e r
to s e d im e n t, the c e llu la r d e b r is .

T h e r e s u ltin g tra n s lu c e n t s u p e rn a ta n t

co m p o se d the “ c e U - fr e e e x tr a c t” .

- 58 -

(b) D e te r m in a tio n o f “ t o t a l c e ll n itro g e n **
A liq u o ts o f th e c e llu la r p re p a ra tio n s w e re h e a t-d ig e s te d w ith the
m ix tu r e o f C a m p b e ll and H anna (17), w h ic h e m p lo y s a c o p p e r -s e le n iu m
c a ta ly s t and b o th p h o s p h o ric and s u lp h u ric a c id s .

A f t e r d ig e s tio n the

m ix tu r e s w e re t r a n s f e r r e d q u a n tita tiv e ly to a M a rk h a m m ic r o - K je ld a h l
a p p a ra tu s and a f t e r th e a d d itio n o f 48% s o d iu m h y d ro x id e the a m m o n ia w as
s te a m d is t ille d in to 4% b o r ic a c id c o n ta in in g a m ix e d in d ic a to r (2 2 ).
a m m o n iu m b o ra te th u s fo r m e d w as t it r a t e d w ith s ta n d a rd a c id .

The

A re a g e n t

b la n k w a s r u n w ith ea ch d u p lic a te d e te rm in a tio n and the a m o u n t o f s ta n d a rd
a c id u se d s u b s tra c te d f r o m the t it r a t io n v a lu e s o b ta in e d f o r the te s t m ix ­
tu r e s ,

U s in g a s ta n d a rd s o lu tio n o f a m m o n iu m s u lp h a te th e r e c o v e r y b y

th is m e th o d w a s c a lc u la te d a t 99. 6 %.

(c)

P a p e r - p a r t it io n c h ro m a to g ra p h y
T he te c h n iq u e u se d w as e s s e n tia U y th a t o f W oiw od (104).

A liq u o ts

o f the m ix tu r e s to be a n a ly z e d w e re s p o tte d on W h a tm a n #1 f i l t e r p a p e r
sh e e ts w ith g e n tle w a rm in g and the she ets p la c e d in an e q u ilib r a te d
c h ro m a to g ra p h y ta n k f o r s e v e ra l h o u rs .

T he s o lv e n t w as th e n added and

d e s c e n d in g p a p e r c h ro m a to g ra p h y c a r r ie d out f o r 2 4 -2 8 h r . a t 2 7 °C .
sh e e ts w e re d r ie d in a w a rm oven, s p ra y e d w ith a
tio n o f n in h y d r in and h e a te d .

0

.

1%

The

a lc o h o lic s o lu ­

W a te r -s a tu ra te d p h e n o l w a s one o f th e b e tte r

s o lv e n t s y s te m s fo u n d f o r the d e te c tio n o f a m in o a c id s in v a r io u s te s t m i x ­
tu r e s .

B u ta n o l* a c e tic ten de d to p ro d u c e a “ ta ilin g * * e ffe c t.

- 59 -

(d) M a n o m e tric te c h n iq u e s
A W a rb u rg c o n s ta n t-v o lu m e m ic r o r e s p ir o m e te r w as u se d in a l l
cases.

A f t e r p r e p a r in g th e fla s k s and m a n o m e te rs th e y w e re p la c e d on

the w a te r b a th and sh a ke n c o n tin u o u s ly a t 96 o s c illa t io n s / m in .

A 10 m in .

p e rio d w a s a llo w e d f o r te m p e r a tu re e q u ilib r a tio n , a ft e r w h ic h tim e the
m a n o m e te rs w e re s e t a t 150 m m . , the s to p c o c k s c lo s e d , and re a d in g s
ta k e n e v e ry f iv e m in u te s u n t il the change in each f la s k w as a p p ro x im a te ly
th e s a m e .

T h e c o n te n ts o f fla s k s and s id e a rm s w e re th e n m ix e d and

re a d in g s m a d e p e r io d ic a lly u n t il the end o f the ru n .
T h e o rg a n ic c h e m ic a ls use d w e re a l l o b ta in e d f r o m N u t r it io n a l
B io c h e m ic a ls L t d . (O h io, U . S . A . ) .

1.

D e c a rb o x y la tio n

C e r ta in a m in o a c id s (a rg in in e , ly s in e , h is tid in e , o r n ith in e , t y r o ­
s in e , g lu ta m ic a c id , b e ta -h y d ro x y g lu ta m ic a c id , and a s p a r tic a c id ) a re
d e c a rb o x y la te d b y so m e b a c te r ia to f o r m c a rb o n d io x id e and th e c o r r e s ­
pondin g a m in e , a c c o rd in g to the re a c tio n :
r.c h (n h

2

)C O O h — » r . c h 2 n h

2

T h e c o e n z y m e in m o s t ca se s is p y r id o x a l p h osph ate .

+

co2
G a le (32) fo u n d

th a t th e e n z y m e s c o n c e rn e d w e re s p e c ific f o r one a m in o a c id and th a t the
a m in o a c id s u b s tra te s m u s t be o f the L - c o n f ig u r a tio n and p o s s e s s m o re

- 60 -

tha n tw o u n s u b s titu te d p o la r g ro u p s .

T h e s e e n z y m e s , w ith th e p o s s ib le

e x c e p tio n o f g lu ta m ic a c id d e c a rb o x y la s e , a re s t r i c t l y a d a p tiv e , a c t o n ly
a t an a c id ic pH and a re fo r m e d to a m a x im u m e x te n t a t about the tim e
a c tiv e c e l l d iv is io n c e a s e s .
A lth o u g h d e c a rb o x y la tio n o c c u rs th e re is som e doubt as to th e a c ­
tu a l m e ta b o lic s ig n ific a n c e o f the re a c tio n .

I t is a p p a re n tly n o t a ro u te

f o r a m in o a c id s y n th e s is be cause the e q u ilib r iu m lie s f a r to the r ig h t ,
b u t i t m a y s e rv e to lib e r a te c a rb o n d io x id e f o r the s y n th e s is o f o rg a n ic
a c id s v ia a W o o d -W e rk m a n ty p e re a c tio n .

T h e a m in e s p ro d u c e d a re

p ro b a b ly f u r t h e r m e ta b o liz e d w ith the lib e r a tio n o f a m m o n ia .
A n a s p a r tic d e c a rb o x y la s e has been s tu d ie d in som e R h iz o b iu m
s p e c ie s ( 1 1 ), b u t c o m p a r a tiv e ly l i t t l e w o r k has been done on the d e c a r b o x y la tio n o f h is tid in e and g lu ta m ic a c id .

In a s e r ie s o f u n p u b lis h e d

e x p e rim e n ts the a u th o r fo u n d th a t c a rb o n d io x id e i s a e r o b ic a lly lib e r a te d
f r o m th e se p a r t ic u la r a m in o a c id s b y th e a c t iv it y o f c e ll- f r e e e x tr a c ts
o f the n o d u le b a c te r ia .

T h e se r e s u lts w e re n o t in th e m s e lv e s to o s ig n i­

fic a n t, h o w e v e r, s in c e c a rb o n d io x id e e v o lu tio n w as a ls o o b s e rv e d f r o m
o th e r a m in o a c id s s u c h as v a lin e and m e th io n in e .

T h is fa c t, co u p le d

w ith th e fin d in g th a t b o th the L - and D - is o m e r s w e re a tta c k e d c a s t som e
doubt as to w h e th e r o r n o t a g lu ta m ic o r h is tid in e d e c a rb o x y la s e e x is te d .
I t w as m o re l ik e l y th a t th e gas w as d e riv e d f r o m th e k e to a c id s p ro d u c e d
f r o m th e s e a m in o a c id s b y p r io r e n z y m a tic a c tiv ity , b u t f u r t h e r w o r k w as
r e q u ir e d f o r s u b s ta n tia tio n .

- 61 -

M e th o d s :
C e ll- f r e e e x tr a c ts and w ashed R 2 1 c e lls w e re p re p a re d as p r e ­
v io u s ly d e s c rib e d and d ilu te d w ith M /2 0 pH

6.

0 phosphate b u ffe r .

lic a te W a rb u rg fla s k s , w ith s id e a rm gas v e n ts , c o n ta in e d 0.

2

D up­

m l. of

w a te r in th e c e n te r w e lls , 0 . 5 m l. o f c e ll p r e p a ra tio n in the s id e a rm s
and 0 .8 m l. o f M /2 0 pH 6 .0 b u ffe r p lu s 0 .5 m l. 0 . 125M n e u tr a liz e d L o r D - g lu ta m ic a c id ( o r h is tid in e ) in th e m a in c o m p a rtm e n ts .

The a v e r­

age m ic r o - K je ld a h l n itr o g e n c o n te n t o f th e c e ll- f r e e e x tr a c t w as 0. 54
m g m . / m l.

T h e v e s s e ls w e re s e a te d on the m a n o m e te rs and jo in e d in

s e r ie s w ith s h o r t p ie c e s o f r u b b e r tu b in g ru n n in g f r o m the s id e a rm ve n t
o f one f la s k to th e open s to p c o c k a r m o f the m a n o m e te r b e lo n g in g to th e
a d ja c e n t fla s k .

P r e p u r ifie d n itr o g e n gas (M a th e s o n C o. U . S . A . ) w as th e n

s lo w ly p a sse d th ro u g h a l l the fla s k s so th a t the c o n ta in e d a i r w as flu s h e d
o u t.

A f t e r the passage o f s e v e r a l l i t e r s o f gas the p r e s s u re w ith in the

fla s k s w as r a is e d s lig h t ly b y p a r t ia lly c o m p re s s in g th e n itr o g e n o u tle t
tube ru n n in g f r o m th e end fla s k o f the s e r ie s .

T h e m a n o m e te r s to p c o c k s

and s id e a r m v e n ts w e r e im m e d ia te ly c lo s e d and th e m a n o m e te rs p la c e d on
the w a te r b a th .

A s th e fla s k s w a rm e d up th e s to p c o c k s w e re opened

m o m e n ta r ily to r e le a s e g a s .

T he b a th te m p e r a tu re w as 3 0 °C . and the

fla s k s w e re c o n tin u o u s ly a g ita te d .
A tw o - m a n o m e te r te c h n iq u e f o r the s im u lta n e o u s d e te rm in a tio n o f
o xyg e n u p ta k e and c a rb o n d io x id e e v o lu tio n w as c a r r ie d out w ith w ash ed c e lls
and L - g lu t a m ic a c id , w ith o u t u s in g an y n itr o g e n flu s h .

T h e b u ffe r pH w as

« 62 -

7. 0 and th e c a rb o n d io x id e fla s k - c o n s ta n ts w e re c a lc u la te d u s in g the
a lp h a p r im e c o r r e c t io n fa c t o r f o r r e te n tio n of c a rb o n d io x id e in the b u ffe r
(9 3).

T h e c o n te n ts o f th e fla s k s w e re th e sam e as p r e v io u s ly d e s c rib e d

e x c e p t th a t the o xyg e n fla s k s c o n ta in e d 0. 2 m l. o f 10% K O H in the c e n te r
w e lls w h ile th e c a rb o n d io x id e fla s k s c o n ta in e d

0 .2

m l, o f w a te r .

T h e a e ro b ic e v o lu tio n o f c a rb o n d io x id e f r o m R h iz o b iu m c e lls g r o w ­
in g in the p re s e n c e o f g ly c in e w as s tu d ie d b y m e ans o f ra d io a c tiv e t r a c e r
te c h n iq u e s .

T w o h u n d re d m i l l i l i t e r s o f m o d ifie d W ils o n and W ils o n 's

m e d iu m c o n ta in in g 15 m g m . o f n o n -la b e lle d g ly c in e and 5 m g m . o f g ly c in e
- 1 -

( s p e c ific a c t iv it y - 0 .0 2 5 m ic r o c u r ie s / m g m . * 925 d ,p . s . / m g m . )

w as a d ju s te d to pH 7. % s t e r iliz e d b y S e itz f i l t r a t i o n , and p la c e d in a s p e c ­
i a l l y d e s ig n e d f la s k h a v in g a te s t-tu b e s e a le d in to the b o tto m .
w e re w a sh e d and added to th is m e d iu m as the in o c u lu m .
e m p lo y e d is show n in F IG U R E 9.

R g j c e lls

T h e a p p a ra tu s

A i r w as b u b b le d th ro u g h a t a ra te o f

a p p r o x im a te ly 64 b u b b le s /m in . and d a ily n e p h e lo s re a d in g s w e re m ade
w ith th e P h o to n e p h e lo m e te r b y c u ttin g o ff th e a e ra tio n and u s in g the
a tta c h e d te s t-tu b e as a c u v e tte .

T he b a r iu m c a rb o n a te th a t a c c u m u la te d

in the tra p p in g v e s s e ls w as f ilt e r e d o ff e v e ry 24 h r . , c a r e f u lly d r ie d and
9 5 .0 m g m . a c c u r a te ly w e ig h e d o u t.

T h is a m o u n t w as e v e n ly s p re a d o v e r

th e b o tto m o f a s p e c ia l, s h a llo w a lu m in u m pan w hose a re a o f 4. 7 c m . w as
the sa m e as th a t o f th e end w in d o w o f the G e ig e r - M u lle r c o u n te r u s e d .
T h is pan w as in s e r te d in a p la s tic s a m p le h o ld e r w h ic h h e ld i t im m e d ia te ly
b e lo w the th in - m ic a end w in d o w o f the c o u n tin g tu b e .

A le a d c a s tle p r o -

to

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O rH *H «©
3 *H £ *H

TJ Fh W X

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3

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t>s

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CW^P

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{>»©
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•H F) O C f e
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fe

- 63 -

v id e d adequate s h ie ld in g .

T h e c o u n tin g tube w as o p e ra te d a t one k ilo v o lt ,

w h ic h re p re s e n te d the m id - p o in t o f the p la te a u on a c o u n tin g r a te - v o lta g e
c u rv e .

T h e decade s c a le r ( E le c tr o n ic A s s o c ia te s L t d , E A -S C 3 T ) w as

o p e ra te d f o r te n m in u te s and th e c o u n ts a v e ra g e d as “ c o u n ts p e r m in u te
(c. p. m . ) ” .

N o r e s o lv in g tim e c o r r e c tio n s w e re m a de s in c e the co u n ts

w e re no t th a t n u m e ro u s .
A f t e r th e f i r s t d a y s m a ll a liq u o ts o f th e c u ltu r e w e re re m o v e d
d a ily , c e n trifu g e d and 0. 1 m l. o f the s u p e rn a ta n ts s p o tte d on a W hatm an
#1

f i l t e r p a p e r sh e e t and c h ro m a to g ra p h e d in w a te r - s a tu r a te d p h e n o l.

A f t e r d e v e lo p m e n t th e c o lo re d n in h y d r in - p o s itiv e a re a s w e re c u t o u t,
i

e x tra c te d f o r 30 m in . b y s h re d d in g in 3 m l. o f 50% ( v /v ) aqueous e th y l
a lc o h o l and th e o p tic a l d e n s ity o f th e e x tr a c ts d e te rm in e d in a C o le m a n
J u n io r S p e c tro p h o to m e te r a t 570 m illim ic r o n s (12),
U pon the c o m p le tio n o f the e x p e rim e n t the c e lls w e re c e n tr ifu g a liz e d f r o m the m e d iu m , g ro u n d w ith a lu m in u m o x id e , c e n trifu g e d a t
20, 000

x g, and an a liq u o t o f th e r e s u ltin g c le a r e x tr a c t d r ie d and e x a m ­

in e d f o r r a d io a c t iv it y .

T h e p r o te in p re s e n t in the d r ie d a liq u o t and r e ­

m a in in g f lu id e x tr a c t w as p r e c ip ita te d w ith h e at and t r ic h lo r o a c e tic a c id
and a ft e r h ig h speed c e n tr ifu g a tio n and th o ro u g h w a s h in g o f the s o lid r e s i ­
due the r e s id u a l r a d io a c t iv it y o f th e “ p r o te in ” and e v a p o ra te d “ n o n - p r o ­
te in ” fr a c t io n s w e re d e te rm in e d .

V a lu e s w e re c o r r e c te d to a d r y w e ig h t

b a s is .
T h e a c t iv it y o f w a sh e d in ta c t K 2 1 c e lls on tw o o f the k e to a c id s of

- 64 -

im p o rta n c e in in te r m e d ia r y m e ta b o lis m w as in v e s tig a te d u s in g a d o u b le m a n o m e te r te c h n iq u e s i m i l a r to th a t e m p lo y e d f o r L - g lu t a m ic a c id .
a e ro b ic and a n a e ro b ic (n itro g e n flu s h )

B o th

c o n d itio n s w e re use d w ith 0 . 5M

a lp h a - k e to g lu ta r ic a c id and 0, 5M s o d iu m p y ru v a te as s u b s tra te s .

U nder

a n a e ro b ic c o n d itio n s th e b u ffe r pH w as 6 .0 (35), w h ile u n d e r a e ro b ic c o n ­
d itio n s th e b u ffe r w as o f pH 7 .0 .

T h e o p tim u m pH f o r the a e ro b ic d e ­

c a r b o x y la tio n o f th e s e k e to a c id s is 8. 0 (84), b u t a pH o f 7. 0 w as cho sen
s in c e th e a lp h a p r im e c o r r e c tio n fa c t o r f o r c a rb o n d io x id e r e te n tio n in the
b u ffe r is s u b je c t to c o n s id e ra b le e r r o r a t pH v a lu e s above 7 .0 .
gen co n te n t o f the c e ll s u s p e n s io n a m o u n te d to 4 .6 1 m g m .

The n itr o ­

N / m l.

A fte r

the r e a c tio n w a s c o m p le te d the c o n te n ts of th e a e ro b ic W a rb u rg v e s s e ls
w e re ch e cke d f o r the p re s e n c e o f a ld e h y d e s b y the S c h iff re a g e n t, w ith the
r e a liz a tio n th a t a n e g a tiv e r e s u lt w o u ld be o f l i t t l e va lu e s in c e any a ld e ­
hyde fo r m e d m ig h t be im m e d ia te ly re d u c e d o r d is m u ta te d .

R e s u lts and D is c u s s io n :
T he c e llu la r p re p a ra tio n s o f R h iz o b iu m R ^ d id no t f o r m any d e ­
te c ta b le a m o u n ts o f c a rb o n d io x id e a n a e r o b ic a lly f r o m h is tid in e , g lu ta m ic
a c id , s o d iu m p y ru v a te o r a lp h a - k e to g lu ta r ic a c id .

I t m a y be c o n c lu d e d ,

th e re fo re , th a t a m in o a c id d e c a rb o x y la s e s f o r h is tid in e and g lu ta m ic a c id
w e re a b se n t u n d e r the e x p e rim e n ta l c o n d itio n s d e s ig n a te d .

U n d e r a e ro b ic

c o n d itio n s , h o w e v e r, c a rb o n d io x id e w as r a p id ly e v o lv e d f r o m a t le a s t
th re e o f the f o u r co m p o u n d s .

T A B L E 9 lis t s e x p e r im e n ta lly d e te rm in e d

v a lu e s f o r p y ru v a te , a lp h a - k e to g lu ta r ic a c id and L - g lu t a m ic a c id .

- 65 -

TABLE 9
Q c o 2 (N )

V A L U E S , Q o 2 ( N ) V A L U E S A N D CO 2 R A T IO S
W

F O R T H E O X ID A T IV E P R O D U C T IO N O F C A R B O N D IO X ID E
F R O M S E V E R A L SUBSTRATES B Y R 2 1

Q c o 2 (N )*

Q o 2 ( N )* *

co2
02

s o d iu m p y ru v a te

1 9 6 .4

1 9 0.8

1.

a lp h a - k e to g lu ta r ic a c id

161.

7

1 5 0 .8

1. 13

L -g lu ta m ic

173.

6

1 7 0 .3

6 0 .4

79. 3

endogenous

*

16

CD
0
0

a c id

•0

S u b s tra te

0. 77

m ic r o U te r s o f C 0 2 e v o lv e d p e r h o u r p e r m g m , c e ll n itr o g e n endogenous n o t s u b tra c te d .

* * m ic r o l it e r s o f 0 2 ta k e n up p e r h o u r p e r m g m , c e ll n itr o g e n endogenous n o t s u b tra c te d .
T h e v a lu e s g iv e n a re a v e ra g e s o b ta in e d f r o m d u p lic a te fla s k s .

T h e en dogenous C 0 2 r a t io p ro b a b ly re p re s e n ts a r e s p ir a t o r y q u o -

W
tie n t in d ic a tiv e of p r o te in o x id a tio n , a c o n c lu s io n f u r t h e r s tre n g th e n e d
b y the fin d in g o f e n d o g e n o u sly p ro d u c e d a m m o n ia (see “ O x id a tio n and
D e a m in a tio n ** s e c tio n ).

T h e r a t io f o r

L -g l u t a m i c

a c id w as

a p p r o x im a te ly one and w as s i m i l a r to th a t fo u n d f o r b o t h

k e t o a c id s .

T h is r a t io is u s u a lly re p re s e n ta tiv e o f c a rb o h y d ra te o x id a tio n (7).

- 66 -

T he r e m o v a l o f the a m in o g ro u p s f r o m m o s t of the g lu ta m ic a c id m o le ­
c u le s b y tr a n s a m in a tio n w o u ld y ie ld k e to a c id s w h ic h c o u ld be m e ta b o l­
iz e d to g iv e s u c h a v a lu e .
F IG U R E 10 g r a p h ic a lly illu s t r a t e s the r e la tio n s h ip s b e tw e e n the
d is a p p e a ra n c e o f g ly c in e , th e p ro d u c tio n o f C
o f the te s t o r g a n is m .

^2

sind the g ro w th c u rv e

T h e re le a s e o f ra d io a c tiv e c a rb o n d io x id e is

d ir e c t ly c o r r e la t e d w ith the d is a p p e a ra n c e o f g ly c in e and the g ro w th o f
»

21

.

T h e c a rb o n d io x id e p ro d u c e d b y R 2

1

h i g ly c in e m e d iu m is , t h e r e ­

fo r e , p a r t ia lly d e riv e d f r o m th e c a r b o x y l g ro u p o f th is a m in o a c id .

T h is

fa c t, to g e th e r w ith th e o th e r r e s u lts , in d ic a te s th a t k e to a c id s a re p r o ­
b a b ly p ro d u c e d f r o m a t le a s t som e a m in o a c id s b y p ro c e s s e s su ch as
d e a m in a tio n and tra n s a m in a tio n and th a t the se k e to a c id s can be o x id a ­
t iv e ly d e c a rb o x y la te d .

I t is e x tr e m e ly u n lik e ly th a t g ly c in e d e c a rb o x y la ­

tio n can o c c u r s in c e no such d e c a rb o x y la s e is kno w n.

In a d d itio n g ly c in e

has o n ly tw o p o la r g ro u p s and the pH o f the m e d iu m used w as n o t c o n ­
d u c iv e to p o te n tia l d e c a rb o x y la s e a c t iv it y .
N o t a l l o f the r a d io a c tiv e c a rb o n escaped in to th e a tm o s p h e re as c a r ­
bon d io x id e .

P a r t o f i t w a s in c o rp o ra te d in to c e llu la r m a te r ia l, p a r t i ­

c u la r ly p r o te in (see T A B L E 10).

T h e m e c h a n is m o f in c o r p o r a tio n is u n kn o w n

b u t m a y have in v o lv e d a n u m b e r o f p ro c e s s e s such as d ir e c t a s s im ila tio n ,
o f g ly c in e a n d /o r a s s im ila tio n o f p a r t o f th e e v o lv e d c a rb o n d io x id e b y
re a s o n o f a W ood - W e rk m a n typ e re a c tio n w h ic h c o u ld r e s u lt in th e p r i ­
m a r y s y n th e s is o f a k e to a c id .

T h is la t t e r m e c h a n is m c o u ld a c c o u n t f o r the

Q
H

I V

O

O «3

00

r4

•

C O

O X >» fl

I-1

ao

Background

ij.00

300

200

o

C.P.M,

Above

r—) O

100

120
Incubation Time (Hours)
Pig, 10 Graphs illustrating the production of radio­
active carbon dioxide from glycine-l-C1^ by
^21* (1) the disappearance of added glycine
(2) the output of Cll+02 (3) the growth curve
of R21

- 67 -

r a d io a c t iv it y fo u n d in the “ n o n -p ro te in * * c e ll f r a c t io n .

I t is u n lik e ly

th a t th is r a d io a c t iv it y is d e riv e d f r o m u n a s s im ila te d in t r a c e llu la r g ly ­
c in e s in c e a m in o a c id s a re a p p a re n tly n o t a c c u m u la te d in t r a c e llu la r ly
to any d e te c ta b le e x te n t b y th is o rg a n is m (48).

T A B L E 10
R A D IO A C T IV IT Y P R E S E N T IN T H E “ P R O T E IN ** A N D “ N O N -P R O T E IN ”
F R A C T IO N S O F R H IZ O B IU M R 2 i A F T E R G R O W TH IN G L Y C IN E - 1 - C 1 4
M E D IU M

“ P ro te in * *
f r a c t io n

“ N o n -p ro te in * *
f r a c t io n

T o ta l co u n t (c . p . m . / m g m . d r y w t)

89. 3

59. 7

B a c k g ro u n d cou nt (c. p. m . )

15. 5

15. 5

C ou nt above b a c k g ro u n d (c. p . m . / m g m .
d ry w t.)

7 3 .8

4 4 .2

% o f o r ig in a l c o u n t*

62. 5

37. 5

* to t a l o r ig in a l co u n t (m in u s b a c k g ro u n d and b e fo re fr a c tio n a tio n
= 1 1 9 .0 c . p . m . / m g m , d r y w t.

T h e S c h iff te s t w as n e g a tiv e and no s ta te m e n t can be m ade as to the
in te r m e d ia te fo r m a tio n o f a ld e h y d e s d u rin g d e c a rb o x y la tio n o f th e k e to
a c id s , a lth o u g h i t w o u ld be a s s u m e d th a t ald ehyd e w o u ld o n ly be p ro d u c e d
b y a n a e ro b ic d e c a rb o x y la tio n .

- 68 -

2

.

O x id a tio n and d e a m in a tio n

In a d d itio n to th e p o s s ib ilit y o f h a v in g it s c a r b o x y l g ro u p re m o v e d
v ia d e c a rb o x y la tio n an a m in o a c id m a y lo s e it s a m in o g ro u p b y the p r o ­
ce ss o f o x id a tio n .

A lth o u g h the sam e o rg a n is m m a y c a r r y out b o th p r o ­

c e sse s a t one tim e o r a n o th e r th e y do n o t o c c u r s im u lta n e o u s ly .

D e a m in a ­

tio n o c c u rs a t an a lk a lin e pH w he n the a m in o a c id c a r b o x y l g ro u p is
io n iz e d (R . C H (N H 2) . C O O -), w h ile d e c a rb o x y la tio n ta k e s p la c e a t an
a c id ic pH w h e re th e a m in o g ro u p is io n iz e d (R . C H (N H g+ ). C O O H ).

The

g e n e ra l a m in o a c id o x id a s e s (d e a m in a s e s ) a re o f tw o m a jo r ty p e s th o se w h ic h a tta c k L - a m in o a c id s and th o se w h ic h a c t on the D - is o m e r s .
T h e se e n z y m e s e x h ib it d iffe re n c e s depen ding upon t h e ir s o u rc e , b u t th e y
a re e s s e n tia lly s te r e o s p e c ific .

G ly c in e , g lu ta m ic a c id , s e rin e , th r e o ­

n in e , c y s te in e and c y s tin e a re not a tta c k e d .

A s p e c ific g ly c in e o x id a s e e x ­

is t s , h o w e v e r, and L - g lu t a m ic a c id m a y be d e a m in a te d v ia a s te r e o s p e c ific L - g lu t a m ic a c id d e h y d ro g e n a s e .

L - a s p a r t ic a c id m a y have it s

a m in o g ro u p re m o v e d as a r e s u lt o f a s p a rta s e a c tiv ity , a re a c tio n o fte n
r e f e r r e d to as d e s a tu ra tio n d e a m in a tio n s in c e one o f th e end p ro d u c ts is
\

an u n s a tu ra te d a c id ( fu m a r ic ) .

T h is la t t e r re a c tio n can o n ly be d e m o n ­

s tra te d b y in h ib itin g s u c c in ic d e h yd ro g e n a se b y c y c lo h e x a n o l and fu m a rd s e
a c t iv it y b y to lu e n e .

S e rin e , th re o n in e , and c y s te in e m a y u n d e rg o d e h y \

d r a tio n d e a m in a tio n to y ie ld p y r u v ic a c id and a m m o n ia , th e r e a c tio n o c c u r r ­
in g in s e v e r a l s te p s .

69 -

W h e re a s the D~ and L - a m in o a c id o x id a s e s and g ly c in e o x id a s e
a p p e a r to be fla v o p r o te in e n z y m e s ca p a b le o f u s in g m o le c u la r oxyg en as
t h e ir a c c e p to r (a s lig h t a n a e ro b ic a c t iv it y is know n to o c c u r) L - g lu t a m ic
d e h yd ro g e n a se r e q u ir e s D . P . N . o r T . P . N . and can be te r m e d an an ae­
r o b ic d e h y d ro g e n a s e .

R e g a rd le s s o f w h ic h o f th e s e e n z y m e s b r in g s about

d e a m in a tio n the p ro c e s s i t s e l f is a lw a y s o x id a tiv e in n a tu re and o c c u rs
in tw o s te p s (7).

1.

R . C H (N H 2). COOH — * R . C * N H . COOH + 2H

2.

R . C * N H . COOH + H 20 — * R. C * O. COOH + N H 3

I t is p ro b a b le th a t the im in o a c id in te rm e d ia te is a ls o p ro d u c e d in the
d e a m in a tio n o f th e h y d ro x y a c id s , s e rin e and th re o n in e .
p e ro x id e fo r m e d w h e n th e h y d ro g e n o f re a c tio n

#1

T h e h y d ro g e n

is t r a n s fe r r e d to

o xyg en b r in g s a b o u t a d e c o m p o s itio n o f the k e to a c id fo r m e d in the seco nd
re a c tio n .

T he o v e r a ll r e a c tio n , th e re fo re , is :

R . C H (N H 2). COOH + 0

2

---- > R . COOH + C 0 2 + N H 3

S ince the d e a m in a tio n o f a m in o a c id s p la y s a v e r y im p o r ta n t m e ta b o lic
r o le it s p re s e n c e w as in v e s tig a te d in R h iz o b iu m R ^ .

M e th o d s :
W ashed R 2 1 c e lls w e re d ilu te d to 14 m l. w ith pH 8 .0 M /4 0 p h o s­
phate b u ffe r and a e ra te d a s e p tic a lly f o r 2 h r .

T w o 1, 2 m l. lo ts w e re

re c e n tr ifu g e d , th e s u p e rn a ta n t d is c a rd e d and the c e lls re s u s p e n d e d w ith

- 70 -

1 .2 m l, o f pH 6 ,0 and pH 7 ,0 b u ffe r .
1, 74 m g m , b a c t e r ia l n it r o g e n / m l.

T h e se s u s p e n s io n s c o n ta in e d

D u p lic a te W a rb u rg v e s s e ls w e re p r e ­

p a re d c o n ta in in g 0, 2 m l. 10% K O H in the c e n te r w e lls ,
w ash ed c e lls in the s id e a r m s and 0 ,

8

m l. pH

8,

0

. 5 m l. o f

0 M /4 0 phosphate b u f­

f e r p lu s 0 , 5 m l. 0 . 12 5M L - a m in o a c id s o lu tio n in the m a in c o m p a rtm e n ts .
T he g lu ta m ic a c id and h is tid in e s o lu tio n s w e re p r e v io u s ly n e u tra liz e d to
pH 7. 0 .
6,

W ith g ly c in e d u p lic a te fla s k s w e re p re p a re d u s in g b u ffe r s o f pH

0, 7, 0 and

8,

0 and c o n ta in in g w ashed c e lls re s u s p e n d e d in b u ffe r s o f

c o rre s p o n d in g p H .

E ndogenpus r e s p ir a t io n fla s k s c o n ta in e d d o u b le -d is ­

t ille d w a te r in p la c e o f the a m in o a c id s o lu tio n .
w as 3 0 °C ,

T h e b a th te m p e r a tu re

A f t e r tw o h o u rs the c o n te n ts o f the fla s k s w e re c e n trifu g e d

and the s u p e rn a ta n ts q u a n tita tiv e ly a n a ly z e d f o r a m m o n ia b y N e s s le r iz a tio n , as c a r r ie d out b y P e e c h (69),

The m e th o d w as as fo llo w s :

1 m l. o f the te s t s o lu tio n w as p ip e tte d in to a 125 m l. E r le n m e y e r fla s k
to w h ic h w as added 45 m l. d o uble g la s s - d is t ille d w a te r, 1 m l. 10% s o d iu m
t a r t r a t e and 2, 5 m l. o f N e s s le r 's re a g e n t, w ith s h a k in g a ft e r each a d d i­
tio n .

A f t e r 25 m in . th e tra n s m itta n c e w as m e a s u re d in a C o le m a n

J u n io r S p e c tro p h o to m e te r u s in g a 6 -3 0 4 B c u v e tte and a w a v e le n g th o f 450
m ill i m i c r o n s .

T h e p e rc e n t tr a n s m itta n c e was r e f e r r e d to a p r e v io u s ly

p re p a re d c o n c e n tra tio n - tr a n s m itta n c e (c a lib r a tio n ) g ra p h and the
a m m o n ia c o n c e n tra tio n d e te rm in e d as a m m o n iu m .
tio n th e en dog eno us a m m o n ia

F r o m th is c o n c e n tra ­

v a lu e w as s u b tra c te d and the r e s u ltin g

fig u r e w as u s e d to c a lc u la te the m illim o le s o f a m m o n ia fo u n d / f l a s k / 2 h r , /

- 71 -

ih g m . c e ll n itr o g e n .
Q o 2 (N) v a lu e s w e re c a lc u la te d f r o m the s lo p e s o f the lin e a r p o rtio n s
o f th e o xyg e n u p ta k e c u rv e s (0 -3 0 m i n . ) T h e endogenous r e s p ir a t io n w as
s u b tra c te d f r o m the to t a l oxyg en u p ta ke in th e se c a lc u la tio n s , a lth o u g h
B u r r is and W ils o n (15) o b s e rv e d th a t g ly c in e s tim u la te d th e endogenous
r e s p ir a t io n o f c e r ta in r h iz o b ia .
T h e endogenous oxyg e n u p take o f the te s t o rg a n is m w as q u ite h ig h
and w as no t a p p re c ia b ly re d u c e d b y d o u b lin g the a e ra tio n p e rio d a ft e r
w a s h in g .

L a t e r w o r k in d ic a te d th a t i t c o u ld be re d u c e d b y s to r in g the

w ashed c e lls f o r th re e days a t 5 °C .

in a s m a ll a m o u n t o f p h y s io lo g ic a l

s a lin e .
T h e above m a n o m e tric p ro c e d u re w as d u p lic a te d u s in g D -a m in o
a c id s and a w a sh e d and a e ra te d c e ll s u s p e n s io n c o n ta in in g
o f b a c te r ia l n itr o g e n p e r m l.

0

.

686

m gm .

In a d d itio n , the a c tio n o f c e r ta in in h ib ito r s

on g ly c in e o x id a tio n w as s tu d ie d .

T h e s e com pou nds (s o d iu m b e n zo a te ,

s o d iu m a z id e and s o d iu m c y a n id e ) w e re added so as to g iv e an i n i t i a l c o n c e n tra tio n . in th e fla s k s o f

0.01

m o le s / lit e r .

A n a tte m p t w a s m ade to is o la te the k e to a c id ( g ly o x y lic ) p ro d u c e d
b y the o x id a tiv e d e a m in a tio n o f g ly c in e .

T h e p ro c e d u re use d w as a c o m ­

b in a tio n o f the m e th o d s o f Janke and T a y e n th a l (46) and C a v a llin i and
F r o n t a li (18) and w as as fo llo w s :
5

m g m , o f a m in o a c i d / m l . ,

20

20 m l. o f a g ly c in e s o lu tio n c o n ta in in g

m l. o f a h e a v y s u s p e n s io n o f w a sh e d R 2 1

c e lls su sp e n d e d in pH 8 .0 M /4 0 phosphate b u ffe r (o r 5 m l. o f g ro u n d

- 72 -

c e lls , in c lu d in g c e ll - w a ll fra g m e n ts ) and
p e n s io n o f

2,

5

m l. o f a

5%

a lc o h o lic s u s ­

4 - d in itr o p h e n y lh y d r a z in e w e re added to g e th e r and in c u b a t­

ed o v e rn ig h t at 3 0 °C .

In s e v e r a l in s ta n c e s c a ta la s e , p re p a re d f r o m b e e f

l i v e r b y the p ro c e d u re o f S u m n e r and S o m e rs (85) w as u se d in p la c e o f
the d in itr o p h e n y lh y d r a z in e .

(In th is case the re a g e n t o f F rie d e m a n n and

H augen (27) w as added a f t e r the in c u b a tio n p e rio d and b e fo re e th e r e x tr a c ­
tio n . ) T h e s u s p e n s io n s w e re d e p ro te in iz e d w ith a 10% s o lu tio n o f t r i c h l o r ­
a c e tic a c id , c e n trifu g e d , and the s u p e rn a ta n ts e x tra c te d s e v e r a l tim e s
w ith e th y l e th e r .

T h e e th e r s o lu tio n s w e re th e n b ro u g h t to d ry n e s s in a

va cu u m d e s s ic a to r o v e r ^ 'D r ie r ite * * and K O H , one m i l l i l i t e r o f IN a m m o n iu m
h y d ro x id e and an e q u a l a m o u n t o f c h lo r o fo r m added, and the m ix tu r e s
shaken g e n tly .

A f t e r c e n trifu g a tio n the s u p e rn a ta n t a m m o n ia s o lu tio n s w e re

s p o tte d on a W h a tm a n #1 f i l t e r p a p e r she et and c h ro m a to g ra p h e d in a s o lu ­
tio n o f n -b u ta n o l, e th a n o l, and w a te r (4 0 /1 0 /5 0 b y v o lu m e .)

C o n tro ls

in c lu d e d the d in itro p h e n y lh y d ra z o n e s o f kno w n k e to a c id s and a p ro c e s s e d
r e a c tio n m ix t u r e w h ic h c o n ta in e d no added g ly c in e .
T h e m e th y le n e b lu e re d u c tio n te c h n iq u e w as e m p lo y e d f o r the d e te c ­
tio n o f an L - g lu t a m ic a c id d e h yd ro g e n a se in R 21.
v e n tio n a l d e s ig n c o n ta in e d 3 m l. o f pH

8.

T h u n b e rg tub es o f c o n ­

0, M /4 0 phosphate b u ffe r , 3 m l.

o f M /5 0 n e u tr a liz e d L - g lu t a m ic a c id , 1 m l. o f 1 :1 0 ,0 0 0 aqueous m e th y le n e
b lu e and, in th e ca p , 1 m l. o f c e ll s u s p e n s io n .

T h e c e lls w e re p re p a re d

b y c e n tr ifu g in g a 100 m l. M o d ifie d M e d iu m **79*'* c u ltu r e o f
been in c u b a te d 72 h r . a t 2 7 °C .

w h ic h had

T h e c e lls w e re th e n w a sh e d tw ic e in 0. 85%

- 73 -

s a lin e , re s u s p e n d e d in
5 °C .

0.2

m l. o f s a lin e and s to re d f o r th re e da ys at

J u s t p r io r to use th e y w e re d ilu te d w ith 1 m l. of pH

8

. 0 b u ffe r

and s ta n d a rd iz e d in th e s p e c tro p h o to m e te r to 18% T a t 420 m illim ic r o n s ,
u s in g th e sam e b u ffe r as a d ilu e n t.

E ndogenous tub es c o n ta in e d w a te r

in p la c e o f th e s u b s tra te and c o n tr o l tub es c o n ta in e d c e lls k ille d b y b o i l ­
in g and th e a d d itio n o f c h lo ro b u ta n o l.
m in . w ith a v a c u u m pu m p,

T h e tubes w e re e va cu a te d f o r 3

e q u ilib r a te d in a 42 ° C . w a te r b a th f o r

m in . and th e c e lls added f r o m the c a p s .

10

T he r a te o f dye re d u c tio n w as

fo llo w e d in a s p e c tro p h o to m e te r a t a w a v e le n g th o f 540 m illim ic r o n s ( 8 6 ).

R e s u lts and D is c u s s io n :
R h iz o b iu m R 2 1 w as fo u n d to con sum e oxyg en d u rin g a m in o a c id
m e ta b o lis m .

T h e u p ta ke c u rv e s a re show n in F IG U R E S 11 and 12.

The

g ra d u a l in c re a s e in ra te a ft e r the i n i t i a l 30 m in . p e rio d , w h ic h w as a ls o
n o tic e d b y B u r r is and W ils o n (15), is a ttr ib u te d to c e llu la r g ro w th and
is n o t to be co n fu se d w ith th e r a p id in c re a s e in ra te fo u n d im m e d ia te ly
upon the a d d itio n o f s u b s tra te .

T h e z e ro tim e p o in t on the se c u rv e s r e ­

p re s e n ts th e p o in t o f s u b s tra te a d d itio n .

T A B L E 11 in d ic a te s the Q og(N )

v a lu e s o b ta in e d w ith the v a r io u s a m in o a c id s , to g e th e r w ith th e m illim o le s
o f a m m o n ia (as N H ^ + ) re c o v e re d .

aoo

Microliters

oxygen

uptake

300

100

15

30

6o

Time (minutes)
Pig. 11 Oxygen uptake of Rhizobium R21 on glycine and L-amino
acid substrates.
(A) L-glutamic acid (B) glycine (c)
L-histidine (D) L-valine (E) L-methionine (F) L-leucine
(G) endogenous

A
B
C

Microliters

oxygen

uptake

D

20

30

15
Time

kS

60

(minutes)

Pig. 1 2 Oxygen uptake of Rhizobium R21 on D-amino acid substrates.
(A) D-glutamic acid and D-leuclne (B) D-methionine (C)
D-histidine (D) D-valine (E) endogenous

- 74 -

T A B L E 11

Q ° 2 (N) v a l u e s a n d m i l l i m o l e s o f a m m o n ia r e c o v e r e d d u r i n g
T H E A C T IV IT Y O F R H IZ O B IU M R 2 l ON L - A N D D - A M IN O A C ID S

A m in o a c id *

A v e ra g e n u m b e r o f m M
a m m o n ia r e c o v e r e d /f la s k /
2 h r . J m g m . ceH n itro g e n * *

Q o 2 (N) * *

L - g lu ta m ic a c id

1 .4

168.

1

g ly c in e

4 .8

128,

1

L - h is t id in e

3 .0

1 1 3.1

L - v a lin e

2.1

8 1 .9

L - m e th io n in e

1.8

6 8 .3

L - le u c in e
\

1 .4

3 3 .6

D -le u c in e

1 0 .5

7 8 .2

3 .8

7 7 .0

D -m e th io n in e

1 5 .2

72, 9

D - h is tid in e

14.3

7 0 .8

8 .4

68.8

D - g lu ta m ic a c id

D - v a lin e

*

35 m M / f la s k

* * endogenous s u b tra c te d

- 75 -

I t m u s t be e m p h a s iz e d th a t the se se ts o f v a lu e s a re o n ly o f a r e la t iv e
n a tu re .

T h e s m a ll a m o u n ts o f a m m o n ia re c o v e re d w ith the L - a m in o

a c id s m a y im p ly th a t tra n s a m in a tio n o c c u rre d and th a t the o xyg en u p ta k e
re s u lte d f r o m th e m e ta b o lis m o f the k e to a c id s fo r m e d .

T he la r g e r

a m o u n ts o f a m m o n ia fo u n d w ith the D -a m in o a c id s m a y in f e r a s lo w e r
r a te o f tr a n s a m in a tio n and a c o rre s p o n d in g ly h ig h e r ra te o f o x id a tiv e
d e a m in a tio n .

T h e s e ra te s w o u ld be exp e cte d to v a r y , d e pen ding on the

a m in o a c id s u b s tra te .

T h e h ig h oxyg en c o n s u m p tio n and lo w a m m o n ia

r e c o v e r y f r o m L - and D - g lu ta m ic a c id p ro b a b ly r e fle c t s the h ig h a c t iv it y
o f th is a m in o a c id in tra n s a m in a tio n (see s e c tio n on *<;T r a n s a m in a tio n , , ).
T h e a m m o n ia p ro d u c e d e n d o g e n o u sly (a ve ra g e o f 0 .7 m M / f la s k / 2 h r . /m g m ,
c e ll n itr o g e n ) m a y have re s u lte d f r o m d e a m in a tio n o f in t r a c e llu la r a m in o
-a cid s, as s u g g e ste d b y W ils o n (15).

The s e e m in g ly lo w o v e r a ll a m m o n ia

v a lu e s c o u ld be a r e s u lt o f p a r t ia l a s s im ila tio n o f th e e v o lv e d gas, o r
i t s escape in to th e a tm o s p h e re due to th e b a s ic n a tu re o f the b u ffe r u s e d .
In th e a b sence o f a n y c o n c re te kn o w le d g e o f the r a te s o f m e ta b o lic
re a c tio n s w ith in l iv in g R h iz o b iu m c e lls an y d e fin ite s ta te m e n ts c o n c e rn ­
in g the a m o u n ts o f a m m o n ia fo u n d o r the q u a n titie s o f o xyg e n co n sum e d
w o u ld be p re s u m p tu o u s .

F IG U R E 13 show s the e ffe c t o f pH on th e oxyg en u p ta k e in th e p r e ­
sence o f g ly c in e .

A pH o f

8.

0 w as d is t in c t ly s u p e r io r to m o re a c id ic pH*s

and th is is to be e x p e c te d i f the p ro c e s s in v o lv e d is one o f d e a m in a tio n ,

15

30

l|.$

60

Time (minutes)
Fig.

The effect of pH on the oxygen uptake of Rhizobium
on glycine substrate.
(A) pH 8.0 (B) pH V.u (C) pH o.O
(D) endogenous

- 76 -

s in c e th e pH o p tim u m f o r th is p a r t ic u la r re a c tio n lie s in the ra n g e

8

-

9 (31).
T h e e ffe c ts o f added in h ib ito r s a re in d ic a te d in F IG U R E 14.

C yan­

id e is u s u a lly c o n s id e re d to be an in h ib it o r o f m e ta llo p r o te in e n zym e s
su ch as c y to c h ro m e o x id a s e , a lth o u g h i t is known to in h ib it tr a n s a m in a ­
tio n (20) and c e r ta in o th e r re a c tio n s as w e ll.

O f a l l the e n z y m e s d e a m in -

a tin g a m in o a c id s o n ly th e L - a m in o a c id o x id a s e of P ro te u s v u lg a r is and
a s i m il a r e n zym e fo u n d b y K re b s in l i v e r and k id n e y b r e is a re s a id to
be in h ib ite d b y c y a n id e (54),

N e v e rth e le s s , g ly c in e o x id a tio n w as c o m ­

p le te ly in h ib ite d b y th is agent, a lth o u g h th e re is a p o s s ib lity th a t the
in h ib itio n is n o t p r im a r y , bu t th a t i t is d ire c te d a g a in s t a s e c o n d a ry
re a c tio n s u c h as th a t in v o lv e d in the tra n s p o rta tio n o f g ly c in e a c ro s s the
c e ll w a ll (34,
s io n .

68,

79), i f th is o c c u rs b y m e th o d s o th e r tha n s im p le d if f u ­

T h e c y a n id e cau sed an 85% re d u c tio n in a m m o n ia fo r m a tio n f r o m

g ly c in e , c o m p a re d w ith 63. 2% and 11. 3% re d u c tio n b y a z id e and b e nzoa te
re s p e c tiv e ly .
A z id e a p p e a re d to in h ib it the oxygen u p ta ke on g ly c in e
and to p a r t ia lly d e c re a s e the endogenous r e s p ir a t io n .

c o m p le te ly

A s u p p le m e n ta ry

e x p e rim e n t w a s c a r r ie d out to s tu d y the e ffe c t o f a z id e on the endogenous
r e s p ir a t io n a lo n e and it d e fin ite ly in d ic a te d th a t the oxyg en u p ta k e in the
ab sence o f s u b s tra te is o n ly s lig h t ly a ffe c te d b y th is in h ib it o r .
endogenous r e s p ir a t io n o f R 2 1

The

th e re fo re , s o m e w h a t s i m i l a r to th a t o f

A e r o b a c te r a e ro g e n e s , w h ic h T is s ie r e s (91) r e p o r ts to be a z id e - r e s is ta n t.

15

30

45

6o

Time (minutes)
Pig. 14 The effect of inhibitors on the oxygen uptake of
Rhizobium Ron on glycine substrate (A) glycine (b)
glycine + ,0X1/1 benzoate (C) endogenous (D) glycine +
,01M azide.
Complete inhibition observed with ,01M
cyanide

- 77 -

W ith b e nzoa te^a n in h ib itio n w as a p p a re n t d u rin g the i n i t i a l 30 m in .
p e rio d , b u t a f t e r th is tim e th e re w as a s tim u la tio n o f the oxyg en u p ta k e .
T h is ph eno m en on m a y be a ttr ib u te d to an a d a p tiv e m e c h a n is m ; the b e n ­
zoa te a c tin g as an in h ib it o r u n t il su ch tim e as a d a p tiv e e n z y m e s a re
p ro d u c e d w h ic h can o x id iz e i t .

On the o th e r hand i t m a y be the r e s u lt

o f s lo w e n tra n c e o f th is com pound in to the c e ll.
G ly o x y lic a c id , r e s u ltin g f r o m the o x id a tiv e d e a m in a tio n o f g ly ­
c in e , w as id e n tifie d as the

2,

4 -d in itro p h e n y lh y d ra z o n e , b u t o n ly in the

m ix tu r e s w h ic h o r ig in a lly c o n ta in e d d in itro p h e n y lh y d ra z in e .

N o a c id w as

is o la te d f r o m th o s e m ix tu r e s to w h ic h c a ta la s e had been added and the
im p lic a tio n is th a t th is a c id is m e ta b o liz e d b y a m e c h a n is m o th e r tha n
m e re h y d ro g e n p e ro x id e d e c a rb o x y la tio n .
T he a n a e ro b ic re d u c tio n o f m e th y le n e b lu e in the p re s e n c e o f i n ­
ta c t R 2 1 c e lls and L - g lu t a m ic a c id is show n in F IG U R E 15.

The i n i t i a l

la g m a y be due to the tim e r e q u ir e d f o r th e a c id to p e rm e a te th e c e ll
b a r r ie r .

A n L - g lu t a m ic a c id d e h yd ro g e n a se is th e re fo re p re s e n t and p o t­

e n tia lly e n a b le s th is o r g a n is m to

4 ‘f i x

’ ' a m m o n ia b y a r e v e r s a l o f it s a c t i ­

v it y .
A l l a v a ila b le e v id e n c e in d ic a te s the p re s e n c e o f an L - and D -a m in o
a c id o x id a s e , a g ly c in e o x id a s e and an L - g lu ta m ic a c id d e h y d ro g e n a se in
R h iz o b iu m R 2 1 .

T h e d e a m in a tio n o f D -a m in o a c id s w o u ld p ro v id e a re a d y

m e ans f o r th e u t iliz a t io n o f the se is o m e r s s in c e the r e s u ltin g a m m o n ia
c o u ld be u s e d to a m in a te c e r ta in o rg a n ic a c id s w ith the r e s u ltin g p ro d u c -

0.6

Millimicrons
Optical

Density

at 5^0

0 .8

o4

3

5

7

9

11

Time (minutes)
Pig. 15

The anaerobic reduction of methylene blue by R2 1 in
the absence (A) and presence (B) of L-glutamic acid.

- 78 -

tio n o f L - a m in o a c id s w h ic h c o u ld be m e ta b o liz e d in the n o r m a l w a y ,
L y m a n and K u ik e n (59) r e p o r t th a t la c to b a c ilL L can u t iliz e D -a m in o a c id s
i f th e m e d iu m c o n ta in s p y rid o x a m in e , the suggested m e c h a n is m b e in g
d e a m in a tio n o f the D - is o m e r s to the c o rre s p o n d in g k e to a c id s w h ic h a re
the n re a m in a te d and u t iliz e d as su ch ,

A so m e w h a t s i m i l a r m e c h a n is m

m a y o p e ra te in r h iz o b ia .

3,

T ra n s a m in a tio n

T r a n s a m in a tio n , w h ic h in v o lv e s the tr a n s fe r o f the a m in o g ro u p
o f an a lp h a -a m in o a c id to the a lp h a p o s itio n o f an a lp h a -k e to a c id w as
f i r s t d e s c rib e d b y B ra u n s te in and K r itz m a n (13),

Since th a t tim e a

n u m b e r o f su ch re a c tio n s have been found in v a rio u s tis s u e s and b a c te ria ,
p y r id o x a l phosph ate b e in g the coe nzym e in v o lv e d .

In a l l e x c e p t a r e l a ­

t iv e ly fe w c a s e s g lu ta m ic a c id is one of th e re a c ta n ts .

S tud ie s on the

u t iliz a t io n o f L - and D L - a m in o a c id s b y p a r a s itic and e ffe c tiv e s tr a in s
o f R h iz o b iu m s u g g e ste d th a t som e o f these com pounds m ig h t be lin k e d
b y tra n s a m in a tio n m e c h a n is m s (48), and th is s u p p o s itio n w as s u p p o rte d
b y the a u th o r ’ s fin d in g o f a n u m b e r o f su ch re a c tio n s in c e ll- f r e e e x tr a c ts
o f th e se m ic r o o r g a n is m s (49),

T h e fa c t th a t r h iz o b ia a re a ls o cap able

o f u t iliz in g D - a m in o a c id s su g g e ste d th a t tra n s a m in a tio n m ig h t be
in v o lv e d in th e s e ca se s as w e ll, a lth o u g h m o s t o f th e p re v io u s r e p o r ts
have in d ic a te d th a t in these re a c tio n s the o r ig in a l and s y n th e s iz e d a m in o
a c id s a r e of the L - c o n fig u r a tio n .

- 79 M e th o d s:
C e ll- f r e e e x tr a c ts o f R h iz o b iu m R 0 1 w e re p re p a re d in the u s u a l
m a n n e r and d ilu te d w ith pH 8 ,0 M /2 0 phosphate b u ffe r to a f in a l v o lu m e
o f 9 m l. ( 1 . 23 m g m . b a c t e r ia l n itr o g e n / m l. ) .
p b ta in e d b y m ix in g

10

A c e to n e - d rie d c e lls w e re

v o lu m e s o f ic e - c o ld acetone w ith w ashed c e lls and

r a p id ly e v a p o ra tin g o ff th e s o lv e n t in a v a cu u m d e s s ic a to r o v e r “ D r i e r i t e ” and g ly c e r o l.

A f t e r d ilu tio n to 9 m l. w ith pH 8 .0 b u ffe r a m ic r o -

K je ld a h l n itr o g e n d e te rm in a tio n re v e a le d an a v e ra g e o f 5 .2 7 m g m . N / m l.
R e a c tio n m ix tu r e s w e re th e n p re p a re d h a v in g the c o m p o s itio n g iv e n in
T A B L E 12.

T A B L E 12
C O M P O S IT IO N O F T R A N S A M IN A T IO N R E A C T IO N -M IX T U R E S

M ix tu r e s

1

0 .3 m l. M /2 0 pH 8 ,0 phosphate
b u ffe r

HTn

2

3

4

5

f

■t

"fc

+

-

KG

KG

DA

DV

DA
+

0 , 1 m l. 0. 5 M a lp h a -k e to a c id

P

KG

0 .2 m l, 0 . 125M D - a m in o a c id

DA

DA

0

. 1 m l. ( 1 . 0 m g m . ) adeno sine
trip h o s p h a te * *

+

+

+

+

0 .1 m l. (3 m ic r o g r a m s )

+

+

+

+

+

+

+

+

+

+*

p y r id o x a l HC1
0

.

8

m l, c e ll p r e p a r a tio n

- 80 -

P * s o d iu m p ry u v a te
KG » n e u tr a liz e d a lp h a - k e to g lu ta r ic a c id
D A * n e u tr a liz e d D - a s p a r t ic a c id
D V ss D - v a lin e
*

b o ile d
d is o d iu m s a lt - 95% p u re

T h ese m ix tu r e s w e re p la c e d in T h u n b e rg tub es w ith the c e llu la r p r e p a ra ­
tio n s in the c a p s .
5 m in .

T h e c o n tr o l m ix tu re * n u m b e r fiv e * w as h e a te d f o r

in a b o ilin g w a te r b a th and it s c e ll m a te r ia l s i m i l a r ly h e a t-

tre a te d .

A f t e r e v a c u a tio n w ith a va cu u m pum p f o r 3 m in . the tubes w e re

sealed* th e e n z y m e p re p a ra tio n s added and a n a e ro b ic in c u b a tio n c a r r ie d
out f o r

1

h r . a t 3 0 °C .

T h e re a c tio n w as then im m e d ia te ly stopped b y

p la c in g th e u n s e a le d tu b e s in a b o ilin g w a te r b a th f o r fiv e m in u te s and*
a ft e r c e n trifu g a tio n * 0. 03 m l. a liq u o ts o f the v a rio u s s u p e rn a ta n ts w e re
sp o tte d on W h a tm a n #1 f i l t e r p a p e r and c h ro m a to g ra p h e d in w a t e r - s a t u r ­
a te d p h e n o l.

C o n tr o ls in c lu d e d know n a m in o a c id s * c e llu la r p re p a ra tio n s

a lo n e , r e a c tio n m ix tu r e s s a m p le d at z e ro tim e and the c o m p le te m ix tu r e
p lu s e ith e r L - g lu t a m ic a c id o r L a la n in e .

The la t t e r w as in c lu d e d to

d e m o n s tra te u n ifo r m m o v e m e n t o f the g lu ta m ic a c id and a la n in e s p o ts .
In o r d e r to d e te c t an y a s p a rta s e a c t iv it y o r p o s s ib le a n a e ro b ic a m in a tio n
o f a lp h a - k e to g lu ta r a te th e e n tir e p ro c e d u re w as re p e a te d w ith re a c tio n
m ix tu r e s c o n ta in in g a m m o n iu m c h lo r id e and e ith e r s o d iu m fu m a ra te
(0. 5M ) o r n e u tr a liz e d a lp h a - k e to g lu ta r ic a c id (0. 5 M ).

- 81 -

M ix tu r e s c o n ta in in g D « a s p a rtic a c id w e re in c u b a te d f o r

3

h r . and

a n a ly z e d m a n o m e tr ic a lly u s in g a d o u b le -m a n o m e te r te c h n iq u e and an
L - g lu t a m ic a c id d e c a rb o x y la s e p re p a re d f r o m C lo s tr id iu m p e rfrin g e n s
(w e lc h ii) (3 3).

(T h e e x a c t te c h n iq u e w i l l be g iv e n in the fo llo w in g s e c ­

tio n on “ R a ce m a se A c tiv ity * * ) .

W a rb u rg v e s s e ls c o n ta in e d 1 m l. o f

te s t m ix tu r e * 0 .2 m l. o f 10% K O H o r w a te r in the c e n te r w e lls * and
0. 8

m l. o f d e c a rb o x y la s e p r e p a ra tio n in th e s id e a r m s .
S ince a l l o f th e L - a m in o a c id s use d in th is th e s is * w ith the e x c e p ­

tio n o f g ly c in e and L - h is t id in e a had been show n to a c t as a m in o d o n o rs
in the s y n th e s is o f g lu ta m ic a c id b y the te s t o rg a n is m (49), i t w as f e lt
th a t th e se tw o a m in o a c id s s h o u ld a ls o be te s te d f o r a c t iv it y .

C o n se ­

q u e n tly 0 , 125M g ly c in e and L - h is t id in e w e re in c u b a te d in re a c tio n m i x ­
tu r e s c o n ta in in g a lp h a - k e to g lu ta r ic a c id and a ft e r the re a c tio n p e rio d
the se m ix tu r e s w e re h e a t-tre a te d , c e n trifu g e d and a n a ly z e d c h ro m a to g r a p h ic a lly f o r g lu ta m ic a c id .

R e s u lts and D is c u s s io n :
T h e c e ll- f r e e e x tr a c t w as fo u n d to have s y n th e s iz e d g lu ta m ic
a c id in th o s e m ix tu r e s w h ic h c o n ta in e d a lp h a - k e to g lu ta r ic a c id and
e ith e r D - a s p a r t ic a c id o r D - v a lin e .

No a la n in e w as d e te c te d in the D -

a s p a r tic a c id - p y ru v a te m ix tu r e and no new a m in o a c id s w e re n o tic e d
in the z e ro tim e c o n tr o ls .

In a d d itio n * no n in h y d r in p o s itiv e s p o ts

re p re s e n tin g s y n th e s iz e d a m in o a c id s w e re fo u n d w ith c o n tr o l m ix tu r e s

_ - 82 -

th r e e and fiv e o r w ith the c e ll p re p a ra tio n s a lo n e .
W hen a c e to n e -d rie d c e lls w e re used m in u te a m o u n ts o f g lu ta m ic
a c id and a la n in e w e re fo u n d in a l l th e m ix tu r e s in c lu d in g the h e a ttre a te d c o n tr o l and w e re no t e n z y m a tic a lly p ro d u c e d d u rin g the in c u b a ­
tio n p e rio d .

S ubsequent e x p e rim e n ta tio n in d ic a te d th a t the se a m in o

a c id s d id n o t a r is e as a r e s u lt o f the h e at tre a tm e n t, b u t th a t th e y m a y
have be en a b s o rb e d onto the c e ll w a lls p r io r to d r y in g and lib e r a te d
upon th e a d d itio n o f b u ffe r .

In a d d itio n , tra c e a m o u n ts o f an unknow n

a m in o a c id w ith an R f s im ila r to s e rin e w as fou nd in a l l the m ix tu r e s
exce p t th e c o n tr o l and a p p e a re d to have been e n z y m a tic a lly fo r m e d .
L a rg e a m o u n ts o f g lu ta m ic a c id w e re fo u n d in those m ix tu r e s c o n ta in ­
in g a lp h a - k e to g lu ta r ic a c id and e ith e r D - a s p a r t ic a c id o r D -v a lin e ,
w h ile c o n s id e ra b le a m o u n ts of a la n in e w e re o b s e rv e d in the D - a s p a r tic
a c id - p y r u v a te m ix tu r e .

T h e a m oun t of a la n in e fo r m e d in the la t t e r case

w as c o n s id e r a b ly in e x ce ss o f th a t w h ic h c o u ld have been p ro d u c e d b y
a tra n s a m in a tio n re a c tio n in v o lv in g p y ru v a te and the tr a c e a m o u n t of
c o n ta m in a tin g g lu ta m ic a c id w h ic h w as p re s e n t.

S ince D - a s p a r t ic -

a la n in e tr a n s a m in a tio n a c t iv it y w as o n ly fo u n d to be a s s o c ia te d w ith a c e ­
to n e - d r ie d c e lls i t w as p o s s ib le th a t the m e th o d used in p r e p a rin g the
c e ll- f r e e e x tr a c ts had b ro u g h t about in a c tiv a tio n o f the e n zym e (s)
c o n c e rn e d .

H o w e v e r, the a m o u n t o f su ch a c t iv it y m ig h t have been s m a ll

enough so th a t i t c o u ld o n ly be d e te c te d in v e r y la rg e a m o u n ts o f c e llu la r
m a t e r ia l and in the

p re s e n t case m u c h m o re m a t e r ia l w as added as

- 83 -

a c e to n e -d rie d c e lls th a n as c e ll- f r e e e x tr a c t.

B o th c e ll p re p a ra tio n s

s y n th e s iz e d g lu ta m ic a c id f r o m g ly c in e and L - h is t id in e .
T h e m a n o m e tr ic p ro c e d u re re v e a le d th a t a t le a s t a s m a ll p a rt
o f the g lu ta m ic a c id s y n th e s iz e d f r o m D - a s p a r tic a c id w as o f the L c o n fig u r a tio n .

D u r in g the 3 h r . in c u b a tio n p e rio d the a m oun t o f L -

g lu ta m ic a c id p ro d u c e d w as c a lc u la te d at 0, 14 m g m .
A lth o u g h i t is te m p tin g to assu m e th a t a tr u e D - a s p a r tic
L - g lu t a m ic a c id tra n s a m in a tio n had take n p la ce (thus p o s tu la tin g a
new e n z y m e )

c e r ta in o th e r p o s s ib ilitie s m u s t be e x a m in e d .

O x id a tiv e

d e a m in a tio n , w h ic h c o u ld p o s s ib ly r e s u lt in the s y n th e s is of L - g lu ta m ic
a c id v ia a m in a tio n o f a lp h a - k e to g lu ta r ic a c id b y the lib e r a te d a m m o n ia ,
can be e lim in a te d as a f a c t o r in the p re s e n t w o r k .

N e v e rth e le s s , i t is

p o s s ib le th a t th e s y n th e s iz e d g lu ta m ic a c id w as o r ig in a lly o f the D - c o n ­
fig u r a tio n and w as s u b s e q u e n tly ra c e m iz e d .

T h e re is a ls o a p o s s ib ility

o f the o c c u rre n c e o f a s e r ie s o f re a c tio n s som ew ha t s i m i l a r to tho se
p o s tu la te d b y T h o rn e ( 8 8 ).

F o r e x a m p le , the fo llo w in g sequence c o u ld

have ta k e n p la c e :

(a) D - a s p a r t ic a c id + p y ru v a te -— ^ D -a la n in e + o x a la c e tic a c id
(b) D -a la n in e
(c)

ra c e m a s e

A D r a la n in e + L - a la n in e

L - a la n in e + a lp h a - k e to g lu ta r ic a c id „ ------ 1 L - g lu t a m ic a c id +
p y r u v ic a c id .

R e a c tio n (c) is kn o w n to o c c u r in R 2 1 (49) and the p ro c e s s w o u ld , th e r e -

- 84 -

fo r e , depend up on th e p re s e n c e of D - a s p a r t ic — D -a la n in e tr a n s a m in ­
ase and an a la n in e ra c e m a s e ,

A la n in e has been fo u n d to be s y n th e s iz e d

f r o m D - a s p a r t ic a c id and p y ru v a te in the a c e to n e -d rie d c e lls , b u t the
p re s e n c e

o f an a la n in e ra c e m a s e is de batable w ith o u t f u r t h e r w o rk *

T h o rn e (89)

in a m o re re c e n t r e p o r t s ta te d th a t s o n ic e x tr a c ts of

B a c illu s s u b t ilis c o n ta in e d tra n s a m in a s e s w h ic h re s u lte d in the s y n th e s is
of D - g lu ta m ic a c id f r o m e ith e r D - a s p a r tic a c id o r D -a la n in e , and le s s
a c tiv e ly f r o m D -m e th io n in e and D - s e r in e ,

Such re a c tio n s , in the p r e s ­

ence o f c e r ta in ra c e m a s e s w o u ld p ro v id e a m e c h a n is m f o r the c o n v e rs io n
of D -a m in o a c id s in to L - a m in o a c id s p r io r to f u r t h e r m e ta b o lis m .

4,

R acem ase a c tiv ity

T he nam e “ ra c e m a s e *’ has been p ro p o s e d f o r an enzym e c a ta ly z ­
in g the in t e r c o n v e rs io n o f D - and L - a m in o a c id s .
have been r e p o r te d .

T w o su ch e n zym e s

A la n in e ra c e m a s e is p re s e n t in m a n y m ic r o o r g a n ­

is m s and has been p a r t ia lly p u r ifie d b y Wood and G u n s a lu s (105) f r o m
S tre p to c o c c u s fa e c a lis e x tr a c ts .

I t a p p a re n tly has a re q u ire m e n t f o r

p y r id o x a l phosphate as a co e n z y m e .

A y e n g a r and R o b e rts ( 6 ) have

d e m o n s tra te d a g lu ta m ic a c id ra c e m a s e w ith an o p tim u m pH o f
acetone p o w d e rs o f L a c to b a c illu s a ra b in o s u s .

8.

0 in

The m e c h a n is m b y w h ic h

the se e n z y m e s o p e ra te is as y e t un know n .
In o r d e r to e lu c id a te as f u l l y as p o s s ib le the m e c h a n is m of D —a m in o
a c id u t iliz a t io n b y r h iz o b ia i t w as c o n s id e re d n e c e s s a ry to in v e s tig a te th e

- 85 “

o c c u rre n c e o f a ra c e m a s e w ith in the se m ic r o o r g a n is m s .

T h e r e s u lts

o b s e rv e d in the p re v io u s s e c tio n on D -a m in o a c id tra n s a m in a tio n m ig h t,
f o r e x a m p le , be e x p lic a b le on the b a s is o f ra c e m iz a tio n .

M e th o d s:
P r e lim in a r y w o r k on an “ a s p a r tic ra c e m a s e ” in v o lv e d a e ro b ic
in c u b a tio n o f in ta c t R 2 1 c e lls o r c e ll- f r e e e x tr a c ts in pH 8 .0 b u ffe r
c o n ta in in g e ith e r L - o r D - a s p a r t ic a c id .

A f t e r in h ib itio n o f the a c t iv it y

b y heat th e m ix tu r e s w e re c e n trifu g e d and the s u p e rn a ta n ts a n a ly z e d
m a n o m e tr ic a lly f o r L - and D - a s p a r tic a c id u s in g fr e s h ly p re p a re d
P ro te u s v u lg a r is L - a m in o a c id o x id a s e (83) and hog k id n e y D -a m in o
a c id o x id a s e .

A lth o u g h the da ta a p p e a re d to in d ic a te the absence o f any

a s p a r tic r a c e m iz a tio n th e re w as the in h e re n t p o s s ib lity th a t o x id a tiv e
d e a m in a tio n w o u ld in t e r f e r e in the in te r p r e ta tio n o f the r e s u lts .

C on­

s e q u e n tly th e p ro c e d u re w as m o d ifie d to e lim in a te d e a m in a tio n .
C e ll- f r e e R h iz o b iu m R g^ e x tr a c ts (0. 50 - 1. 30 m g m . b a c t e r ia l
n it r o g e n / m l. )
p re p a re d .

and a c e to n e -d rie d c e lls (1. 57 - 1 .8 5 m g m . N / m l . ) w e re

(In s o m e ca se s, d u rin g the g ro w th o f the c e lls the p a r t ic u la r

a m in o a c id u n d e r te s t w as added s e v e ra l h o u rs p r io r to h a rv e s tin g , bu t
th is p ro c e d u re w as la t e r d is c a r d e d .)

T h e re a c tio n m ix tu r e s use d had

the fo llo w in g c o m p o s itio n :
2 .0 m l. M /2 0 pH 8 .0 phosphate b u ffe r

- 86 -

0 . 8 m l. 0 . 125M n e u tra liz e d L - a s p a r t ic a c id o r L - a la n in e
0 ,4 m l, (12 m ic r o g r a m s ) p y r id o x a l h y d ro c h lo rid e
0 ,4 m l, (4 m g m , ) adeno sine trip h o s p h a te (d is o d iu m s a lt)
L a s p a r t i c a c id and L - a la n in e c o n tr o l s o lu tio n s and t h e ir enzym e p r e ­
p a ra tio n s w e re h e a t-tre a te d and the v a rio u s m ix tu r e s p la c e d in T h u n b e rg tu b e s w ith 1, 5 m l, o f c e llu la r m a t e r ia l and p ro c e s s e d in the
sam e fa s h io n as in the tra n s a m in a tio n e x p e rim e n ts .

A f t e r in c u b a tio n

a t 3 0 °C . f o r 1 h r , the r e a c tio n w as stopped b y im m e r s in g the u n s e a le d
tubes in b o ilin g w a te r f o r 5 m in .

T h e v a rio u s m ix tu r e s w e re th e n c e n ­

tr ifu g e d and 0 ,0 3 m l, a liq u o ts o f the s u p e rn a ta n ts a n a ly z e d b y p a p e rp a r titio n c h ro m a to g ra p h y in w a te r-s a tu ra te d ph eno l.

T e s ts f o r a m m ­

on ia w e re c a r r ie d out w ith N e s s le r ’ s re a g e n t and p ro v e d n e g a tiv e .
T h e m ix tu r e s w e re e x a m in e d m a n o m e tr ic a lly f o r the p re s e n c e
o f D -a m in o a c id s .

D u p lic a te W a rb u rg fla s k s c o n ta in e d 0 .2 m l. 10%

K O H in the c e n te r w e lls , 1 m l. o f te s t m ix tu r e in the m a in c h a m b e rs
and 0 .8 m l. o f D -a m in o a c id o x id a s e in the sid e a r m s .
p e ra tu re w as 3 7 °C .

T he b a th te m ­

In s e v e ra l e x p e rim e n ts the o x id a s e w as p re p a re d

fr o m g u in e a p ig k id n e y c o r te x b y the m e th o d of K re b s (52) b u t in la t e r
w o rk a c o m m e r c ia lly a v a ila b le hog k id n e y D -a m in o a c id o x id a s e w as
e m p lo y e d .
u n its ).

( N u t r itio n a l B io c h e m ic a ls C o r p ., O h io . 1 g m , = 150, 000

A s o lu tio n o f th is w as p re p a re d w h ic h c o n ta in e d 1500 u n it s / 0 . 8 m l.

T e s t m ix t u r e s w e re p re p a re d c o n ta in in g D - g lu ta m ic a c id in p la ce
o f L - a s p a r t ic a c id o r D -a la n in e .

M a n o m e tric a n a ly s e s w e re m ade on

- 87 -

th e se m ix tu r e s , a f t e r re a c tio n , u s in g a s ta n d a rd d o u b le -m a n o m e te r
te c h n iq u e f o r c a rb o n d io x id e (93), and an L - g lu ta m ic a c id d e c a rb o x y ­
la s e p r e p a r a tio n o b ta in e d f r o m C lo s tr id iu m per fr in g e n s (33).
p r e p a r a tio n o f th e e n zym e su s p e n s io n w as as fo llo w s :

The

a fla s k c o n ­

ta in in g 200 m l, o f C ooked M e a t M e d iu m (D ifc o ) w as in o c u la te d w ith a
lo g phase c u ltu r e o f C l. p e r fr in g e n s * and in c u b a te d a t 3 7 °C . f o r 24 h r .
T h is c u ltu r e w a s f ilt e r e d th ro u g h a B u c h n e r fu n n e l, c e n trifu g e d , and
the c e lls w a sh e d in 0 .8 5% s a lin e and d ilu te d w ith s a lin e to a f in a l v o l­
um e o f 6 .2 m l. (1 .8 3 m g m . c e llu la r N / m l . ).
im m e d ia te ly use d to o b v ia te any d e te r io r a tio n .

T h is s u s p e n s io n w as
T he d u p lic a te W a rb u rg

fla s k s c o n ta in e d 1. 0 m l. o f te s t m ix tu r e in the m a in c h a m b e r, 0. 2 m l.
10% K O H o r w a te r in the c e n te r w e lls and 0. 8 m l. of d e c a rb o x y la s e
p r e p a ra tio n in th e s id e a r m s .

T h e b a th te m p e ra tu re w as 3 7 °C .

In

a d d itio n to a b o ile d c o n tr o l m ix tu r e th is m e th o d r e q u ir e d an endogen-*
ous r e s p ir a t io n fla s k c o n ta in in g pH 8 .0 b u ffe r , K O H , and C l. p e rfrin g e n s
s u s p e n s io n .

R e s u lts and D is c u s s io n :
N o s ig n ific a n t d iffe re n c e in oxygen uptake w as fo u n d b e tw e e n the
re a c tio n m ix t u r e o r ig in a lly c o n ta in in g L - a s p a r t ic a c id and it s re s p e c -

*

T h is c u ltu r e had been c a r e f u lly che cked f o r a c t iv it y on b o th L - and
D - g lu ta m ic a c id and w as fo u n d to a ct o n ly on the L - is o m e r .

- 88 --

tiv e c o n tr o l,

w h e n e x a m in e d b y D -a m in o a c id o x id a s e .

The L - g lu t a m ­

ic a c id d e c a rb o x y la s e y ie ld e d no s ig n ific a n t a m oun ts o f c a rb o n d io x id e
in e xce ss o f th e c o n tr o l w hen added to fla s k s c o n ta in in g D - g lu ta m ic
a c id te s t m ix t u r e .

I t m a y , th e re fo re , be con clude d th a t no a s p a r tic

o r g lu ta m ic a c id r a c e m iz a tio n o c c u rre d u n d e r the e x p e rim e n ta l c o n d i­
tio n s u s e d .
T h e o xyg e n u p ta ke c u rv e , p lo tte d fr o m date o b taine d f r o m the
a c tio n o f D -a m in o a c id o x id a s e on an L - a la n in e m ix tu r e is g iv e n in
F IG U R E 16 and is in d ic a tiv e o f th e fo r m a tio n o f a D -a m in o a c id b y R h iz ■
o b iu m H21*

P a p e r - p a r titio n c h ro m a to g ra p h y fa ile d to r e v e a l the p r e ­

sence of a n y a m in o a c id no t p re s e n t in the c o n tr o l and, th e r e fo r e , the
D - is o m e r is co n c lu d e d to be th a t of a la n in e .

In th is p a r t ic u la r case

the a c e to n e -d rie d c e lls (1 .5 7 m g m . N / m l . ) p ro d u c e d 0 .3 5 m g m . D a la n in e f r o m 1. 7 m g m . L - a la n in e (in 1 m l. a liq u o t o f te s t m ix tu r e ) in
1 h r . a t 3 0 °C . - a 20, 5% c o n v e rs io n .

No oxygen uptake w as fo u n d

upon m a n o m e tric a n a ly s is o f the heated L - a la n in e c o n tr o l m ix tu r e .
T h e s y n th e s is o f s m a ll q u a n titie s o f L - g lu ta m ic a c id f r o m D a s p a rtic a c id , as o b s e rv e d w ith R 2 1 , c o u ld be e x p la in e d b y a s e r ie s o f
re a c tio n s in v o lv in g D -a la n in e ra c e m a s e , as p r e v io u s ly s u g g e s te d .

If

D -a m in o a c id s o th e r th a n D - a s p a r t ic c o u ld be c o n v e rte d to D -a la n in e
then r a c e m iz a tio n o f th is a m in o a c id w o u ld p ro v id e a p a th w a y f o r the
u t iliz a t io n o f D - is o m e r s .

N e v e rth e le s s , s in c e n o th in g is kno w n o f the

r e la tiv e r a te s o f D -a m in o a c id c o n v e rs io n v ia D -a m in o a c id tra n s a m in a *

Microliters

oxygen

uptake

iSoJ

100 ►

60

20

20
Time (minutes)
Pig. 16

Oxygen uptake curve of D-amino acid oxidase in a
racemase test mixture originally containing L-alanine
and acetone-dried Rhizobium Rgi cells.

- 89 -

tio n and r a c e m iz a tio n c a re m u s t be ta k e n no t to a s s ig n to the se p r o c e s s ­
es an y m a jo r p o s itio n in D -a m in o a c id m e ta b o lis m w ith o u t c o n s id e ra b le
r e s e r v a tio n .

T h e y m a y o n ly be o f m in o r im p o rta n c e , w ith the m a jo r

ro u te b e in g d e a m in a tio n and subsequent a m in a tio n o f one o r m o re k e to
a c id s w ith th e r e s u lta n t p ro d u c tio n o f L - a m in o a c id s .

A lth o u g h th e r e s u lts p re s e n te d in s e c tio n D a re no t in th e m s e lv e s
a b s o lu te ly c o n c lu s iv e th e y do sug gest a p o s s ib le in te g ra te d sche m e of
n o n - s p e c ific re a c tio n s f o r the u t iliz a t io n and c o n v e rs io n o f L - and D a m in o a c id s b y R h iz o b iu m R 21.

F IG U R E 17 illu s t r a t e s the p o s s ib le

re a c tio n s of su ch a sch e m e , w ith the e m p h a s is b e in g upon the p ro d u c ­
tio n o f L - g lu t a m ic a c id .

T h e im p o rta n c e o f th is p a r t ic u la r a m in o a c id

is c o n tin u a lly b e in g b ro u g h t to the fo re g ro u n d in re c e n t s c ie n tific l i t e r a ­
tu r e as i t p ro v id e s a co m m o n m e e tin g p la ce f o r n itro g e n s o u rc e s p r io r
to th e ir in c o r p o r a tio n in to p r o te in o r th e ir tr a n s fo r m a tio n in to a m in o
a c id s b y tr a n s a m in a tio n .

PART II

G R O W T H S T IM U L A T IO N IN A M IN O A C ID M E D IA

- 91 -

In tr o d u c tio n and L it e r a t u r e R e v ie w

A s in th e case o f in v e s tig a tio n s on the n itro g e n n u tr itio n , the
lit e r a t u r e d e a lin g w ith th e g ro w th fa c to r re q u ire m e n ts o f rh iz o b ia m ust
be c a r e f u lly in te r p r e te d (in th e p re s e n t stu d y the te r m " g r o w th fa c to rs * *
w i l l im p ly v ita m in s and n u c le ic a c id fr a c tio n s .

In som e cases a m in o

a c id s m a y be c la s s ifie d as g ro w th fa c to r s , b u t th e y w i l l be o m itte d
f r o m th is d is c u s s io n ).

T he sam e r e s t r ic t io n s a p p ly to th e se r e p o r ts

as to the n itr o g e n da ta in th a t, in m a ny in s ta n c e s , unw ashed c e lls and
c o m p le x m e d ia w e re u se d .

T h e s y n th e s is , e s s e n tia lity , o r n o n ­

e s s e n tia lity o f b io tin , th ia m in e , r ib o fla v in , p y rid o x in e , n ic o tin ic a c id ,
p -a m in o b e n z o ic a c id , b e ta -a la n in e , and p a n to th e n ic a c id in re g a r d to
the n o d u le b a c te r ia have been a d e q u a te ly re v ie w e d in ta b u la r f o r m by
A lle n and A lle n (2).

C o m p a ra tiv e ly fe w fa c ts can be u n c o v e re d c o n ­

c e rn in g th e a lfa lfa - s w e e t c lo v e r o rg a n is m (R h. m e li l o t i ).

Some s tr a in s

o f th is o rg a n is m r e q u i r e .b io tin w h ile o th e rs s y n th e s iz e i t (101),

T h ia ­

m in e (65), p a n to th e n ic a c id (62), and b e ta -a la n in e (65) a re re g a rd e d as
n o n -e s s e n tia l, as a r e p y r im id in e , th ia z o le , b e ta -o x y p ro p io n ic a c id
and p im e lic a c id (67).

B u rto p and L o ch h e a d (16) te s te d s e v e n ty s tr a in s

o f r h iz o b ia re p r e s e n tin g s ix s p e c ie s and fou nd th a t R h. m e lilo t i w as
s h a rp ly d is tin g u is h e d f r o m the o th e r s p e c ie s b y it s a b ilit y to p ro d u c e
s ig n if ic a n tly h ig h e r q u a n titie s o f v ita m in

L i l l y and L e o n ia n (57)

s tre s s e d the im p o rta n c e o f te m p e ra tu re and a e ra tio n up on the s tim u la -

« 92 t o r y a c t iv it y o f se ve n v ita m in s on the g ro w th o f R h. t r i f o l i i .

A n in ­

c re a s e in te m p e r a tu r e f r o m 2 5 °C to 30°C g r e a tly in c re a s e d the need o f
th is o rg a n is m f o r v ita m in s ,
J o rd a n (48) r e p o r te d th a t u n d e r the e x p e rim e n ta l c o n d itio n s he e m ­
p lo y e d none o f fif t e e n v ita m in s , p u rin e s , and p y r im id in e s w as a b le to i n i ­
tia te the g r o w th o f w ashed c e lls o f the a lfa lfa -s w e e t c lo v e r o rg a n is m in a
c h e m ic a lly d e fin e d a m m o n iu m c h lo r id e m e d iu m and hence the se c e lls
w e re a b le to s y n th e s iz e th e se com pounds w hen a u t iliz a b le n itro g e n
s o u rc e w as p re s e n t.

I t w as in an a tte m p t to e xte nd th is p a r t ic u la r in v e s ­

tig a tio n to a m in o a c id u t iliz a t io n th a t the p re s e n t s e c tio n o f the w o rk w as
com m enced.

A lth o u g h the g ro w th fa c to r s p r e v io u s ly e x a m in e d w e re app­

a r e n tly n o t u t iliz e d f o r the in it ia t io n o f c e llu la r p r o life r a t io n the fa c t r e ­
m a in s th a t th e y m a y be c o n c e rn e d in g ro w th s tim u la tio n .

Since a b s o lu te

d iffe re n c e s in g ro w th s tim u la tio n m a y r e s u lt in d e te c ta b le d iffe re n c e s
am ong p a r a s it ic , e ffe c tiv e , and in e ffe c tiv e rh iz o b ia th is p o rtio n o f the r e ­
s e a rc h is f u r t h e r e m p h a s iz e d .

A ls o , the kno w le dg e o f tho se com pounds

w h ic h a r e c a p a b le o f b r in g in g about la r g e in c re a s e s in g ro w th w h ile p r e ­
se n t in m in u te q u a n titie s w o u ld be in v a lu a b le in the fo r m u la tio n o f new
m e d ia f o r the p ro p a g a tio n o f la rg e n u m b e rs o f e ffe c tiv e , v ia b le b a c te r ia
f o r th e p ro d u c tio n o f h ig h g ra d e le g u m e in o c u la n ts .
M e th o d s :
A n u m b e r o f s to c k v ita m in s o lu tio n s w e re p re p a re d (T A B L E 13)
u s in g d o u b le g la s s - d is t ille d w a te r.

T h e v a rio u s c o n c e n tra tio n s w e re

those d e s ig n a te d b y R oepke, L ib b y and S m a ll (78) w ith the e x c e p tio n o f
p y r id o x a l h y d r o c h lo r id e , a s c o rb ic a c id , and v ita m in B - ^ ,

- 93 -

T A B L E 13
T A B L E SHOW ING T H E V IT A M IN S T E S T E D , T H E IR SOURCE A N D
S T O C K S O L U T IO N C O N C E N T R A T IO N

V ita m in

S o u rce

a s c o rb ic a c id

B r i t i s h D ru g House

1.0

M e r c k and C o.

0 .0 0 0 5

b io tin

N u tr it io n a l B io c h e m ic a ls

0 .0 0 5

c a lc iu m p a n to ­
thenate

B r it is h D ru g House

1.0

f o lic a c id

N u tr it io n a l B io c h e m ic a ls

0 .0 0 0 5

in o s it o l

B r it is h D ru g House

n ic o tin a m id e

N u t r it io n a l B io c h e m ic a ls

2.0

p a ra - a m in o b e n z o ic a c id

N u tr it io n a l B io c h e m ic a ls

0.2

p y r id o x a l H C I

N u tr it io n a l B io c h e m ic a ls

1.0

p y rid o x in e H C I

N u t r it io n a l B io c h e m ic a ls

1.0

r ib o fla v in

E a s tm a n -K o d a k Co.

1.0

th ia m in

E a s tm a n -K o d a k C o.

0

B 12

C o n c e n tra tio n
(m g m . / 2 5 m l. w a te r)

10.0

.

8

T e n m i l l i l i t e r s o f s t e r ile m o d ifie d W ils o n and W ils o n ’ s b a s a l
m e d iu m , 0 ,2 m l. o f a s t e r ile c y s te in e s o lu tio n (10 m g m . c y s te in e /2 5 m L
w a te r),

0.5

m l. o f v ita m in s o lu tio n , and

0,2

m l. o f w ash ed c e lls o f R 2 1 5

r 2 0 -2 7 A * an d R 20 w e re added a s e p tic a lly to a s e r ie s o f s t e r iliz e d o p tic a l

-

c u v e tte s .

-

T h e s e tu b e s , to g e th e r w ith in o c u la te d and u n in o c u la te d c o n ­

t r o ls , w e re in c u b a te d at 2 7 °C .
e lo m e te r .

94

and d a ily re a d in g s ta k e n w ith a P h o to n e p h -

A s p e c ia l u n in o c u la te d c o n tr o l c o n ta in in g c y s te in e b a s a l m e d ­

iu m , 0, 2 m l. o f w a te r and r ib o f la v in s o lu tio n w as p re p a re d to s e rv e as
a b la n k f o r the in o c u la te d r ib o fla v in tube w h ic h had a s lig h t y e llo w is h
c o lo r a tio n .

T h is e x p e rim e n t w as re p e a te d u s in g 0. 0 0 0 1M h is tid in e

b a s a l m e d iu m .
T h re e s e r ie s o f c u v e tte s c o n ta in in g 9. 5 m l. o f m o d ifie d W ils o n and
W ils o n ’ s m e d iu m , 0. 5 m l. o f le u c in e s o lu tio n ( fin a l c o n c e n tra tio n =
0 .0 0 0 5 M ) and 0 .5 m l. o f a p u rin e o r p y r im id in e s o lu tio n (fin a l c o n c e n tra ­
tio n 0. 0001M ) w e re in o c u la te d w ith 0 .2 m l. o f w ashed c e lls o f
R 2 0 -2 7 A and R 20*

^ h e te s t com pounds w e re a l l o b ta in e d fr o m N u t r it io n a l

B io c h e m ic a ls C o rp . ( U . S . A . ) and c o n s is te d o f adenine su lp h a te , guanine
H C I, x a n th in e , th y m in e , u r a c il, and c y to s in e .
in th e s e r ie s in o c u la te d w ith
in o c u la te d s e r ie s i t w as 7 .0 .

The b a s a l m e d iu m pH

and ^ 2 0 -2 7 A w as

w h ile f ° r the R 2 0

In c u b a tio n w as at 3 0 °C . and n e phe los r e a d ­

in g s w e re ta k e n a t d e fin ite in te r v a ls .
In a b r ie f s tu d y o f in h ib itio n b y s t r u c t u r a l an alogues o f p u rin e s and
p y r im id in e s tw o a d d itio n a l s e r ie s o f c u v e tte s c o n ta in in g 9. 0 m l. of pH
7. 5 m o d ifie d W ils o n and W ils o n ’ s m e d iu m , and 0. 5 m l. o f s to c k le u c in e
s o lu tio n ( fin a l c o n c e n tra tio n * 0 .0 0 0 1 M ) w e re se t up as show n in T A B L E
14.

T h e in h ib it o r , p u rin e and p y r im id in e s o lu tio n s c o n ta in e d 10 m g m .

o f e a ch c o m p o u n d / 10 m l.

double

g la s s - d is tille d w a te r.

T h e re fo re , the

- 95 -

0. 5 m l. a liq u o ts added to each c u v e tte c o n ta in e d 500 m ic r o g r a m s o f
in g re d ie n t (1 06).

T h e tubes w e re in o c u la te d w ith 0 .2 m l. o f w ash ed R 21

c e lls and in c u b a te d , w ith s u ita b le c o n tro ls a t 30®C.

T A B L E 14
S U P P L E M E N T S A D D E D T O L E U C IN E B A S A L M E D IU M F O R
D E T E R M IN A T IO N OF S T R U C T U R A L A N A L O G U E IN H IB IT IO N O F R 21

S e rie s A

C u v e tte #

B e n z im id a z o le * S o lu tio n

A d e n in e S o lu tio n

W a te r

1

0 m l.

0 m l.

1. 0 m l.

2

0 m l.

0. 5 m l.

0. 5 m L

3

0. 5 m l.

0 m l.

0. 5 m L

4

0. 5 m l.

0. 5 m l.

0 mL

S e rie s B

C u ve tte #

2 - a m in o - 4 - m e th y l
p y r im id in e ^ 's o lu t io n

T h y m in e S o lu tio n

W a te r

1

0 m l.

0 m l.

1. 0 m l.

2

0 m l.

0. 5 m l.

0. 5 m l.

3

0. 5 m l,

0 m l.

0. 5 m l.

4

0. 5 m l.

0. 5 m l.

0 m l.

-

*

HC

II

-

H
H
C

II

HC
C I
I
1
1
HC = c -

96

-

-**
H
N
N

H 2N

N = CH
I
I
- C
CH

\

II

■v,C H

N

II
— C -

CH,

R e s u lts and D is c u s s io n :
O f th e 12 v ita m in s te s te d o n ly n ic o tin a m id e y ie ld e d m a x im u m
g ro w th re s p o n s e s above th a t o f the in o c u la te d c y s te in e o r h is tid in e
b a s a l m a d ia a lo n e .
in F IG U R E 18,

T he g ro w th c u rv e s on c y s te in e m e d iu m a re show n

T h o se o b ta in e d on the h is tid in e m e d ia a re a lm o s t id e n t i­

c a l e x c e p t in th e o v e r a ll m a g n itu d e o f g ro w th .
fo u n d w ith R 2 ^ and ^ 2 0 -2 7 A

The typ e s of c u rv e s

Pr o ^ a^-*-y in d ic a te th a t the n ic o tin a m id e

m e r e ly s e rv e d as an a d d itio n a l n itro g e n s o u rc e f o r these o rg a n is m s .
W ith R h iz o b iu m R 2 0 the c u rv e w as m o re re p re s e n ta tiv e of an a c c e s s o ry
g ro w th f a c t o r re s p o n s e , the m a in e ffe c t, in a d d itio n to p ro v id in g m o re
g ro w th , b e in g to re d u c e the lo n g i n i t i a l la g p e rio d .

T h e g ro w th la g , as

c a lc u la te d a c c o rd in g to M onod (61), w as d e c re a s e d f r o m 4 3 ,0 ne p h e lo s
u n its to 22. 5.

T h e cause o f th is le n g th y la g p e rio d in R 2q is unknow n,

bu t m a y be the r e s u lt of an a d a p tiv e re s p o n s e .

T h e n ic o tin a m id e m a y

m e r e ly s e rv e a s a m o re r e a d ily a v a ila b le n itro g e n s o u rc e f o r R 2q th a n
the a m in o a c id p re s e n t and th e re fo re is u tiliz e d f i r s t .

W hen the a m in o

a c id b e g in s to be u t iliz e d the g e n e ra l e ffe c t w o u ld be a re d u c tio n in the
la g .

Units

20

Nephelos

20

60

*

20

Time (in days)

hhvee Hhizobium strains in cysteine
medium in the presence (XI and absence (B) of
added nicotinamide.
^

F 1 S -X

basal

- 97 -

F IG U R E 19 re p re s e n ts the m a x im u m g ro w th o b s e rv e d on the
p u r in e - and p y r im id in e - c o n ta in in g m e d ia .

R 2 0 w as in h ib ite d b y these

co m pou nds f o r som e unknow n re a s o n , but the o th e r tw o s tr a in s r e a d ily
u t iliz e d th e m .

T h e g ro w th c u rv e s w e re v e r y s im ila r to th o s e g iv e n in

F IG U R E 14 f o r R 2 2 and a re in d ic a tiv e of u t iliz a t io n as a d d itio n a l n i t ­
ro g e n s o u rc e s .

T he a u th o r has p r e v io u s ly show n th a t th e se com pounds

can not be u se d as s o le s o u rc e s of n itro g e n (48), h o w e v e r, f r o m the
p re s e n t w o r k i t a p p e a rs th a t u t iliz a t io n can o c c u r w hen a m in o a c id s a re
p re s e n t in the m e d iu m .

One o f the d e g ra d a tio n p ro d u c ts o f b o th p u rin e s

and p y r im id in e s is a m m o n ia (92) and th is com pound c o u ld be u tiliz e d b y
rh iz o b ia in the sam e m a n n e r as th a t d e s c rib e d in the s e c tio n d e a lin g
w ith g ro w th in the p re s e n c e of a m m o n iu m c h lo rid e and e ith e r k e to - o r
a m in o a c id s .

T ra n s a m in a s e s e x is ts in som e o rg a n is m s w h ic h c a ta ly z e

g lu ta m a te fo r m a tio n f r o m a lp h a -k e to g lu ta ra te w hen p y rid o x a m in e ,
ad e n in e , g u anin e o r c y s to s in e a c t as a m in o d o n o rs (37) and these e n zym e s
m a y fu n c tio n in r h iz o b ia once a s u p p ly o f a c c e p to r k e to a c id s (o r s u b s ta n ­
ces g iv in g r is e to su ch a c id s ) a re added.
Th e a c tio n o f the s t r u c t u r a l analog ues is show n in T A B L E 15.

T he

r e s u lts o f s e r ie s B a re c o m p lic a te d o w in g to the fa c t th a t th e “ in h ib it o r ”
use d w as a c tu a lly u t iliz e d b y the te s t o rg a n is m .

A c a lc u la te d in h ib itio n

w as o b s e rv e d in b o th s e r ie s o f c u v e tte s , bu t in s e r ie s B the added
th y m in e a p p e a re d to in te r fe r e w ith the u t iliz a t io n o f the 2 - a m in o - 4 m e th y l p y r im id in e r a t h e r than v ic e v e rs a .

The a c tio n o f b e n z im id a z o le

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-'98 -

m a y ha ve re s u lte d f r o m an in te rfe r e n c e w ith p u rin e u t iliz a t io n o r /a n d
to an in h ib itio n o f u n re la te d en zym es (53).

I t w as hoped th a t these

m e ta b o lic a n a lo g u e s w o u ld s e rv e as s p e c ific in h ib it o r s o f those e n z y m e s
c o n c e rn e d in th e im m e d ia te u tiliz a t io n o f adenine and th y m in e and w o u ld
hence be in v a lu a b le as an a id in e lu c id a tin g the type s o f re a c tio n s th a t
o c c u r.

I t is e v id e n t, h o w e v e r, th a t u n t il the in h ib ito r y a c t iv it y o f

b e n z im id a z o le in le u c in e base is e x p la in e d the use o f th is com pound
as a s p e c ific in h ib it o r o f “ p u rin e -a tta c k in g e n z y m e s ” is c o n tra in d ic a te d .
T he u t iliz a t io n o f 2 - a m in o - 4 - m e th y l p y r im id in e b y the te s t o rg a n is m
in d ic a te s th a t a tte m p ts to use th is com pound as a “ b lo c k in g a g e n t”
w o u ld be u s e le s s .

T A B L E 15
A C T IO N O F “ IN H IB IT O R S ” ON T H E U T IL IZ A T IO N OF A D E N IN E A N D
A N D T H Y M IN E IN L E U C IN E B A S A L M E D IU M

S e rie s
A

C u v e tte

S u p p le m e n t

M a x im u m nephelos

1

none

5 4 .0

2

b e n z im id a z o le

3 8 .0

3

adenine

3 0 4 .0

4

b o th

178.0

C a lc u la te d in h ib itio n
in c u v e tte #4

= 100 ~ ^ 8 .80+(30 4 .0 - 5 4 .0 )

x 100\

= 61- 8%

-

99

-

T a b le 15 (c o n t’ d)

S e rie s

C u v e tte

B

1

S upp lem e nt

none

M a x im u m n e p h e lo s

5 9 .0

2

2 - a m in o - 4 m e th y l p y r im id in e

3 4 0 .4

3

th y m in e

2 7 9 .0

4

b o th

3 0 2 .4

C a lc u la te d in h ib itio n
/ 3 0 2 .4
x 100)
in c u v e tte #4
= 100 — ( 3 4 0 .4 + (2 7 9 .0 -5 9 .0 )
°)

= 4 6 .0 %

In c o n c lu s io n i t m a y be s a id th a t, w ith th e e x c e p tio n o f n ic o tin a ­
m id e and
s tim u la n ts .

none

com pou nds te s te d show p r o m is e as g ro w th

T h e in c re a s e s b ro u g h t about b y the p u rin e s and p y r im id in e s

can be m o re e c o n o m ic a lly a tta in e d b y the use o f in o rg a n ic a m m o n iu m
s a lts and l i t t l e , i f a n y, b e n e fit w o u ld be o b ta in e d b y t h e ir in c lu s io n in
a s y n th e tic m e d iu m .

I t is d e b a ta b le w h e th e r o r n o t the a c tio n o f R 2 0 on

added n ic o tin a m id e is r e la te d to in e ffe c tiv e o r p a r a s itic te n d e n c ie s on
the p a r t o f th is n o d u le o rg a n is m , b u t i t does p ro v id e a le a d f o r f u r t h e r
re s e a rc h .

- 100 -

SUMMARY

One e ffe c tiv e and tw o in e ffe c tiv e s tr a in s o f R h iz o b iu m m e li l o t i
w e re e x a m in e d in r e g a r d to t h e ir a b ilit y to u t iliz e o rg a n ic and i n o r ­
g a n ic s o u rc e s o f n itr o g e n .

P re v io u s w o r k had show n th a t w a sh e d c e lls

o f the se o rg a n is m s w o u ld g ro w in a p u r ifie d s y n th e tic m e d iu m c o n ­
ta in in g a m in o a c id s , b u t th a t th e s e a c id s c o u ld n o t be re p la c e d b y o th e r
o rg a n ic o r in o r g a n ic n itro g e n o u s co m p o u n d s.
In th e p re s e n t in v e s tig a tio n a l l th re e s tr a in s w e re found- to u t iliz e
b o th th e L - and D - is o m e r s o f g ly c in e , le u c in e , v a lin e , m e th io n in e ,
g lu ta m ic a c id , and h is tid in e w h e n the se co m p o u n d s s e rv e d as th e s o le
s o u rc e s o f n itr o g e n in a p u r if ie d s u c r o s e - m in e r a l s a lts - m e d iu m .

The

m a x im u m g ro w th on an y one is o m e r w as a fu n c tio n o f pH and te m p e r a ­
tu r e , b u t g e n e r a lly a pH o f a b out 7. 5 and a te m p e r a tu re above 2 7 °C .
w e re b e s t.

I n h ib it o r y e ffe c ts w e re fo u n d in c e r ta in r a c e m ic m ix tu r e s

and e x p la in e d b y m u tu a l s te r e o c h e m ic a l in te r fe r e n c e o f th e e n z y m e s
a tta c k in g the L - and D - is o m e r s .

H is tid in e w as re m a r k a b le in th a t b o th

it s f o r m s y ie ld e d th e g re a te s t a m o u n t o f g ro w th a t a l l pH v a lu e s (6 ,0 ,
7. 0, 7. 5) w ith a l l th r e e o rg a n is m s , in c lu d in g the in e ffe c tiv e s t r a in
w h ic h d id n o t g ro w at pH 6. 0 on an y o th e r a m in o a c id .

T h is a b ilit y o f

h is tid in e is p ro b a b ly a r e s u lt o f it s r e la t iv e c o m p le x ity w h ic h m a k e s it
d if f i c u lt to s y n th e s iz e .

T h e th re e s tr a in s w e re fo u n d to be a c id s e n s itiv e ,

in h ib it io n o f g ro w th o c c u r r in g a t pH 6. 0.

- 101 -

In a d d itio n to a m in o a c id s th e o rg a n is m s w e re fo u n d to be c a p ­
a b le o f u s in g a v a r ie t y o f d i- and t r i- p e p tid e s o f g ly c in e and L - and D le u c in e as s o le n itr o g e n s o u rc e s .

P a r t ia l h y d r o ly s is o c c u rre d , b u t

s tim u la to r y e ffe c ts w e re a ls o o b s e rv e d and e x p la in e d on th e b a s is o f
d ir e c t in c o r p o r a tio n o f th e p e p tid e s in to p r o te in s .
T h e s e r h iz o b ia u t iliz e d a m m o n iu m , n it r a t e , and n i t r i t e s a lts p r o ­
v id in g th a t a m in o o r c e r ta in c a r b o x y lic a c id s w e re a ls o p re s e n t.
la t t e r c o u ld n o t be re p la c e d b y e x c e s s C 0 2.

The

T h e su g g e ste d m e c h a n is m

is the a c c u m u la tio n o f o rg a n ic a c id s w h ic h can be a m in a te d b y a m m o n ia
to f o r m a m in o a c id s .

C om pounds s u c h as p u rin e s and p y r im id in e s ,

w h ic h can be d e g ra d e d to a m m o n ia s u b s titu te d f o r the in o rg a n ic n i t r o ­
genous s a lts w it h tw o o f th e b a c t e r ia l s tr a in s .
No g lu ta m ic o r h is tid in e d e c a rb o x y la s e a c t iv it y w as lo c a te d , b u t
a m a n o m e tr ic e x a m in a tio n o f one of the e ffe c tiv e s tr a in s d e m o n s tra te d
an u p ta k e o f ga seous o xyg en on b o th L - and D -a m in o a c id s .

A m m o n ia w as

fo r m e d o x id a tiv e ly , b u t the a m o u n ts v a r ie d w ith in d iv id u a l a c id s , b e in g
p a r t ic u la r ly lo w w ith g lu ta m ic a c id w h ic h is a good a m in o g ro u p d o n a to r.
T h e k e to a n a lo g u e o f g ly c in e , fo r m e d as a r e s u lt o f o x id a tio n , w as is o la ­
te d .

T h e r e s p ir a t o r y q u o tie n t on g lu ta m ic a c id w a s a p p r o x im a te ly one,

the sa m e as th a t o b s e rv e d w ith s e v e r a l k e to a c id s , and is in d ic a tiv e o f
c a rb o h y d ra te m e ta b o lis m su ch as m ig h t o c c u r
k e to a c id s .

th ro u g h d e g ra d a tio n o f

T h e endogenous q u o tie n t w as s u g g e s tiv e o f p r o te in m e ta b o lis m .

- 102 -

S tu d ie s on g ly c in e o x id a tio n re v e a le d th a t a pH o f 8 .0 w as b e tte r th a n
lo w e r pH v a lu e s in r e g a r d to oxyg e n u p ta k e and th a t c y a n id e and a z id e
in h ib ite d th e re a c tio n in in ta c t c e lls .
T ra n s a m in a s e e x p e rim e n ts d e m o n s tra te d th a t c e U - fr e e e x tr a c ts
o f a t le a s t one o f th e K h iz o b iu m s tr a in s w e re ca p a b le o f s y n th e s iz in g
g lu ta m ic a c id f r o m a lp h a - k e to g lu ta r ic a c id w hen g ly c in e , L - h is t id in e ,
D ~ a s p a r tic a c id , o r D - v a lin e a c te d as a m in o g ro u p d o n a to rs .

A s m a ll

a m o u n t o f L - g lu t a m ic a c id w as lo c a te d in the case o f D - a s p a r t ic a c id ,
and c o u ld have been fo r m e d as a consequence o f a s e r ie s o f re a c tio n s
s i m i l a r to th o se p o s tu la te d b y T h o rn e *

In a d d itio n , a c e to n e -d rie d c e lls

s y n th e s iz e d a la n in e f r o m p y ru v a te and D - a s p a r t ic a c id .

A la n in e r a c e -

m a se a c t iv it y w as lo c a te d , b u t ra c e m iz a tio n o f added a s p a r tic o r g lu ta m ic
a c id s d id n o t o c c u r u n d e r the c o n d itio n s u s e d .
A n L - g lu t a m ic a c id d e h y d ro g e n a s e w as fo u n d u s in g the T h u n b e rg
te c h n iq u e and p r o v id e s a p o te n tia l m e c h a n is m f o r “ a m m o n ia f ix a tio n * *
b y a r e v e r s a l p f i t s a c t iv it y .
N one o f 12 v ita m in s w e re fo u n d to be s tim u la to r y to the g ro w th
o f th e th r e e s tr a in s in c y s te in e o r h is tid in e m e d ia , w ith the e x c e p tio n o f
n ic o tin a m id e .

T h is co m p o u n d a p p e a re d to s e rv e m e r e ly as an a d d i­

t io n a l s o u rc e o f n itr o g e n , a lth o u g h i t re s u lte d in a re d u c tio n o f a lo n g
i n i t i a l la g p e r io d w ith th e in e ffe c tiv e s tr a in .
T h e in e ffe c tiv e s t r a in v a r ie d f r o m the e ffe c tiv e s tr a in s in its
e x tr e m e a c id s e n s itiv it y , it s , b e t t e r u t iliz a t io n o f D -a m in o a c id p e p tid e s

- 103 -

p a r t ic u la r ly D - le u c y lg ly c in e , it s lo w e r o p tim u m te m p e r a tu re , i t s la g
phase r e d u c tio n b y n ic o tin a m id e , and it s in a b ilit y to u t iliz e p u rin e s and
p y r im id in e s .

I t is un kn o w n w h e th e r o r n o t a n y o f th e se fe a tu r e s a re

c o r r e la t e d w ith in e ffe c tiv e n e s s in n itr o g e n f ix a tio n .

S uch d iffe re n c e s

m a y o n ly be due to th e in h e re n t d iffe re n c e s n o r m a lly fo u n d am ong
d iffe r e n t s tr a in s o f th is o rg a n is m and m o re w o r k is r e q u ir e d a lo n g th is
lin e o f e n d e a v o u r,
A s y n th e tic m in e r a l s a lts - h is tid in e - s u c r o s e m e d iu m has been
fo r m u la te d f o r g ro w th s tu d ie s on w a sh e d R h iz o b iu m m e li l o t i c e lls .
T h e e x p e r im e n ta l r e s u lts s u g g e st th a t in r h iz o b ia m o s t a m in o
a c id s a r e p r i m a r i l y d e a m in a te d o r tra n s a m in a te d and th a t th e k e to a c id s p ro d u c e d a re o x id iz e d o r e ith e r d ir e c t ly o r in d ir e c t ly a m in a te d to
r e f o r m a m in o a c id s ,

R a c e m iz a tio n , c o u p le d w ith tra n s a m in a tio n a p p e a rs

to p la y a r o le in the u t iliz a t io n o f D -a m in o a c id s , b u t d e a m in a tio n and
su b se q u e n t a m in a tio n o f one o r m o re k e to -a c id s is p ro b a b ly o f g r e a te r
s ig n ific a n c e .

- 104 -

REFERENCES

1. A j l , S .J . and W e rk m a n , C .H . R e p la c e m e n t o f C O 2 in h e te r o tr o p h ic
m e ta b o lis m . A r c h . B io c h e m . 1 9 :4 8 3 -4 9 2 .1 9 4 8 .
2.

A lle n ,

E th y l K . and A lle n , O .N . B io c h e m ic a l and s y m b io tic
p e r tie s o f r h iz o b ia . B a c t. R e v . 1 4 :2 7 3 -3 3 0 . 1945.

p ro ­

3.

A llis o n , F . E . and H o o v e r, S .R . A n a c c e s s o ry f a c t o r f o r le g u m e
n o d u le b a c te r ia . J . B a c t. 2 7 :5 6 1 -5 8 1 . 1934.

4. A llis o n , F . E . and M in o r , F . W . T h e e ffe c ts o f te m p e r a tu re on th e
g r o w th r a te s o f r h iz o b ia . J . B a c t. 3 9 :3 6 5 -3 7 1 . 1940.
5. A lly n , W . P . and B a ld w in , I . L . T he e ffe c t o f the o x id a tio n re d u c tio n c h a r a c te r o f th e m e d iu m on th e g ro w th o f an a e ro b ic
f o r m o f b a c te r ia . J . B a c t. 2 0 :4 1 7 -4 3 8 . 1930.
6. A y e n g a r, P . and R o b e rts , E . U t iliz a t io n o f D - g lu ta m ic a c id b y
L a c to b a c illu s a ra b in o s u s : g lu ta m ic ra c e m a s e . J . B io l. C h e m .
1 9 7 :4 5 3 -4 6 0 .1 9 5 2 .
7. B a ld w in , E . D y n a m ic a s p e c ts o f b io c h e m is tr y .
U n iv . P r e s s , E n g la n d . 1952.

2nd. ed.

C a m b rid g e

8. B e ije r in c k , M . W . D ie B a c te r ie n d e r P a p ilio n a c e e n k n o llc h e n .
Z t g . 4 6 :7 2 6 -7 3 5 . 1888.

B o t.

9. B e r g e r , J . , Jo h n so n , M . J . , and P e te rs o n , W .H . T h e p r o te o ly tic
e n z y m e s o f b a c te r ia . I I . T h e p e p tid a s e s o f som e c o m m o n
b a c te r ia . J . B a c t. 3 6 :5 2 1 -5 4 5 . 1938.
10. B e rn h e im , F . , B e rn h e im , M a r y , and W e b s te r, M . D o ro th y . O x id a tio n
o f c e r ta in a m in o a c id s b y " r e s t in g " B a c illu s p ro te u s . J . B io l.
C h e m . 1 1 0 :1 6 5 -1 7 2 . 1935.
11. B ille n , D . and L ic h s te in , H . C . A s p a r tic a c id d e c a rb o x y la tio n b y
R h iz o b iu m t r i f o l i i . J . B a c t. 5 7 :2 6 7 -2 6 8 . 1949.
12. B lo c k , R . J . P a p e r G h r o m a t o g r a p h y .
Y o r k . 1952.

A c a d e m ic P r e s s , I n c . , N ew
if

13. B r a u n s te in , A . E . and K r itz m a n , M . G . U b e r den A b -u n d A u fb a u vo n
A m in o s a u r e n d u rc h U m a m in ie ru n g . E n z y m o lo g ia , 2 :1 2 9 -1 4 6 . 1937.

- 105 14. B u r r is , R ,H . and W ils o n , P . W . R e s p ir a to r y e n zym e s y s te m s in
s y m b io tic n itr o g e n fix a tio n . C o ld S p rin g H a rb o u r S y m p o s ia
Q u a n t. B io l. 7 :3 4 9 -3 6 1 . 1939.
15. B u r r is , R .H . and W ils o n , P . W . E ffe c ts o f h a e m o g lo b in and o th e r
n itro g e n o u s co m pou nds on th e r e s p ir a t io n o f th e r h iz o b ia .
B io c h e m . J . 5 1 :9 0 -9 6 . 1952.
16. B u rto n , M a r g . O . and L o c h h e a d , A . G . P ro d u c tio n o f v ita m in B ^
b y R h iz o b iu m s p e c ie s . C an . J . B o t. 3 0 :5 2 1 -5 2 4 . 1952.
17. C a m p b e ll, W . R . and H anna, M . I .
m o d ifie d K je ld a h l m e th o d s .

T h e d e te rm in a tio n o f n itr o g e n b y
J . B io l. C h e m . 1 1 9 :1 -7 . 1937.

18. C a v a llin i, D . and F r o n t a li, N . Q u a n tita tiv e d e te rm in a tio n o f k e to
a c id s b y p a p e r p a r t it io n c h ro m a to g ra p h y . B io c h e m . et
B io p h y s . A c ta , 1 3 :4 3 9 -4 4 5 .1 9 5 4 .
19. C lifto n , C . E . in B a c te r ia l p h y s io lo g y , ed. b y C . H . W e rk m a n and
P .W . W ils o n . A c a d e m ic P re s s , I n c . , N ew Y o r k . 1951.
20. C ohen, P . P . in S y m p o s iu m on r e s p ir a t o r y e n z y m e s . U n iv . o f W is ­
c o n s in P r e s s . M a d is o n . 1942.
21. C ohen, P . P . and M c G ilv e r y , R .W . in R e s p ir a to r y e n z y m e s , ed. b y
A . L a r d y . B u rg e s s P u b . C o ., M in n . 1949.
22. C o le , J .O . and P a rk s , C .R . S e m im ic r o - K je ld a h l p ro c e d u re f o r
c o n tr o l la b o r a to r ie s . In d . and E n g . C h e m . A n a ly t. E d . 18:
6 1 -6 2 .1 9 4 6 .
23. D aw son , M . F u r t h e r o b s e rv a tio n s on th e n a tu re and fu n c tio n s o f th e
n o d u le s o f le g u m in o u s p la n ts . R o y . Soc. (L o n d o n ) P h il. T r a n s . ,
S e r. B , 1 9 3 :5 1 -6 7 .1 9 0 0 .
24. D unn, M .S . and M c C lu r e , L . E . T h e re s p o n s e o f L a c to b a c illu s c a s e i
to p a r t ia l h y d ro ly s a te s o f p r o te in . J . B io l. C h e m . 1 8 4 :2 2 3 -2 3 3 .
1949.
25. F is h e r , R . B . Protein m e ta b o lis m . M e th u e n and C o . , L t d . L o n d o n .
1954.
26. F r e d , E . B . , B a ld w in , I. L . , and M c C o y , E liz a b e th . R o o t n o dule
b a c te r ia and le g u m in o u s p la n ts . U n iv . W is e . S tu d ie s in S c ie n c e .
5. 1932.

- 106 27. F re id e m a n n , F . E . and H augen, G . P y r u v ic a c id . I I . T h e d e te r ­
m in a tio n o f k e to a c id s in b lo o d and u r in e . J . B io l. C h e m .
1 4 7 :4 1 5 -4 4 1 .1 9 4 3 .
28. F r u to n , J .S . in S c ie n tific A m e r ic a n R e a d e r. S im o n and S c h u s te r,
N e w Y o r k . 1953.
29. F r u to n , J .S . and S im m o n d s . S o fia . G e n e ra l b io c h e m is tr y .
W ile y and Sons, I n c . , N ew Y o r k . 1953.

J.

30. G a le , E . F . and E p p s , H . M . R . T h e e ffe c t o f th e pH o f th e m e d iu m
d u rin g g ro w th on th e e n z y m ic a c tiv it ie s o f b a c te r ia (E . c o li
a n d M . ly s o d e ik tic u s ) and the b io lo g ic a l s ig n ific a n c e o f the
changes p ro d u c e d . B io c h e m . J . 3 6 :6 0 0 -6 1 8 . 1942.
31. G a le , E . F . F a c to r s in flu e n c in g the e n zym e a c tiv itie s o f b a c te ria .
B a c t. R e v. 7 :1 3 9 -1 7 3 . 1943.
32. G a le , E . F . T h e b a c te r ia l a m in o a c id d e c a rb o x y la s e s .
E n z y m o lo g y , 6 :1 -3 2 . 1946.

A d v . in

33. G a le , E . F . T h e use o f e n z y m e p re p a ra tio n s fr o m b a c te r ia f o r th e
e s tim a tio n o f a m in o a c id s . B io c h e m . J . 4 1 : v ii- ix . 1947.
34. G a le , E . F . T h e n itr o g e n m e ta b o lis m o f g r a m - p o s itiv e b a c te ria .
B u ll. Jo h n s H o p k in s H o s p . 8 3 :1 1 9 -1 7 5 . 1948.
35. G re e n , D . E . , H e r b e r t, D . , and S u b ra h m a n ya n , V .
J . B io l. C h e m . 1 3 8 :3 2 7 -3 3 9 . 1941.

C a rb o x y la s e .

36. G r e ig - S m ith , R . T h e d e te rm in a tio n o f r h iz o b ia in th e s o il.
B a k t. 2 A b t. 3 4 :2 2 7 -2 2 9 . 1912.

C e n tb l.

37. G u n s a lu s , C a ro ly n , F . , and T o n z e tic h . J . T ra n s a m in a s e s f o r
p y r id o x a m in e and p u rin e s . N a tu re , 170:162. 1952.
38. H a n d u ro y , P . L e " s e p a r a te u r de g e rm e s " . B u ll. A s s o c .
M ic r o b io l. F a c . P h a r m . N a n cy, 3 9 :5 -9 . 1950.

D ip lo m e s

39. H a r r is o n , F . G . and B a rlo w , B . T he no dule o rg a n is m o f th e
L e g u m in o s a e , it s is o la tio n ,c u ltiv a tio n , id e n tific a tio n , and
c o m m e r c ia l a p p lic a tio n . C e n tb l. B a k t. 2 A b t. 1 9 :2 6 4 -2 7 2 . 1907.
40. H ills , T . L .. In flu e n c e o f n itr a te s on n itr o g e n a s s im ila tin g b a c te r ia .
J . A g r . R e s . ( U . S . A . ) 1 2 :1 8 3 -2 3 0 . 1918.

- 107 -

41. H ir s c h , M . L . and C ohen, G .N . A m in o a c id u t iliz a t io n in b a c te r ia l
g ro w th . I . P e p tid e u t iliz a t io n b y a le u c in e - r e q u ir in g m u ta n t
o f E s c h e r ic h ia c o li. B io c h e m . J . 5 3 :2 5 -3 0 .1 9 5 3 .
42. H o o v e r, S .R . and A llis o n , F . E . A g ro w th and r e s p ir a t o r y fa c to r
f o r c e r ta in r h iz o b ia . T r a n s . 3 rd . I n t e r n a t 'l. C o n g re s s
S o il Sc. 1 :1 5 8 -1 6 0 . 1935.
43. Is h iz a w a , S. S tu d ie s on th e ro o t-n o d u le b a c te r ia o f le g u m in o u s p la n ts .
I . C h a r a c te r s in a r t i f i c a l m e d ia . ( P a r t 3) P h y s io lo g ic a l
c h a r a c te r s (d) n itr o g e n s o u rc e . J . S c i. S o il and M a n u re . (Japan)
2 4 :2 9 -3 5 .1 9 5 3 .
44. Is h iz a w a , S. S tu d ie s on th e ro o t-n o d u le b a c te r ia o f L e g u m in o u s p la n ts .
I. C h a r a c te r s in a r t i f i c a l m e d ia . ( P a r t 4) T h e e ffe c t o f pH
on th e g ro w th o f r h iz o b ia . J . S c i. S o il and M a n u re (Japan)
2 4 :1 6 9 -1 7 2 .1 9 5 3 .
45. Is h iz a w a , S. S tu d ie s on th e ro o t-n o d u le b a c te r ia o f L e g u m in o u s p la n ts .
I . C h a r a c te r s in a r t i f i c a l m e d ia . ( P a r t 5) E ffe c ts o f te m p e r a tu r e
on th e g ro w th o f r h iz o b ia . J . S c i. S o il and M a n u re . (Japan)
2 4 :2 2 7 -2 3 0 .1 9 5 3 .
46. Ja n ke , A . and T a y e n th a l, W ilh e lm in e . P ro b le m e des S tic k s to ffk r e is la u fe s . I I I . M itte ilu n g . U b e r den A b b a u des G ly k o k o lls
d u rc h B a k te r ie n . B io c h e m . Z . 2 8 9 :7 6 -8 6 . 1937,
47. J o rd a n , D .C . S tu d ie s on th e le g u m e r o o t n o d u le b a c te r ia . I I . T he
p r o d u c tio n and b e h a v io r o f c o lo n ia l m u ta n ts p ro d u c e d b y X - r a y
ir r a d ia t io n . C an . J . B o t. 3 0 :1 2 5 -1 3 0 . 1952.
48. J o rd a n , D .C . S tu d ie s on th e le g u m e ro o t n o d u le b a c te ria . I I I . G ro w th
fa c t o r r e q u ir e m e n ts f o r e ffe c tiv e , in e ffe c tiv e , and p a r a s itic
s tr a in s . C an . J . B o t. 3 0 :6 9 3 -7 0 0 . 1952.
49. J o rd a n , D .C . T r a n s a m in a tio n in c e ll- f r e e e x tr a c ts o f e ffe c tiv e and
p a r a s it ic r h iz o b ia . J . B a c t. 6 5 :2 2 0 -2 2 1 .1 9 5 3 .
50. J o rd a n , D .C . and G a r r a r d , E .H . S tu d ie s on the le g u m e ro o t-n o d u le
b a c te r ia . I. D e te c tio n o f e ffe c tiv e and in e ffe c tiv e s tr a in s .
C an. J . B o t. 2 9 :3 6 0 -3 7 2 . 1951.
51. K ih a r a , H . and S n e ll, E . E . P e p tid e s and b a c t e r ia l g ro w th . I I . L - a la n in e
p e o tid e s and g ro w th o f L a c to b a c illu s c a s e i. J . B io l. C h e m . 197:
7 9 1 -8 0 0 .1 9 5 2 .

-108 -

52. K re b s , H . A . M e ta b o lis m o f a m in o a c id s . I I I . D e a m in a tio n o f a m in o
a c id s . B io c h e m . J . 2 9 :1 6 2 0 -1 6 4 4 . 1935.
53. K u s h n e r, D . J . and P a sco e , E n id . T h e a c tio n o f b e n z im id a z o le on
e n z y m a tic a d a p ta tio n . C an. J . B io c h e m . and P h y s . 3 3 :1 -9 . 1955.
54. L a r d y , H . A .
1949.

R e s p ir a to r y e n z y m e s .

B u rg e s s P u b lis h in g C o . , M in n .

55. L e o n a rd , L . T . N i t r i t e p ro d u c tio n b y som e s tr a in s o f cow pea and
so yb e a n o rg a n is m s . J . B a c t. 17:19. 1929.
56. L e o n a rd , L . T . A s im p le a s s e m b ly f o r use in the te s tin g o f c u ltu r e s
o f r h iz o b ia . J . B a c t. 4 5 :5 2 3 -5 2 5 . 1943.
57. L i l l y , V .G . and L e o n ia n , L . H . T he in te r r e la tio n s h ip s o f ir o n and
c e r ta in a c c e s s o ry fa c to r s in the g ro w th o f R h iz o b iu m t r i f o l i i .
s t r a in 205. J . B a c t. 5 0 :3 8 3 -3 9 5 . 1945.
58. L o c h h e a d , A .G .

p e rs o n a l c o m m u n ic a tio n v ia M r . F . N e w b o u ld .

59. L y m a n , C . M . and K u ik e n , K . A . T he e ffe c t o f v ita m in Bg on the
u t iliz a t io n o f D -a m in o a c id s b y la c t ic a c id b a c te r ia . F e d e r a tio n
P r o c . 7:1 70 . 1948.
60. M a n u a l o f m e th o d s f o r p u re c u ltu r e s tu d y o f b a c te ria , ed. b y C o m ­
m itte e on B a c t. T e c h . Soc. A m . B a c t. B io te c h P u b lis h in g C o . ,
G eneva, N e w Y o r k . 1946.
61. M o n o d , J . T h e g ro w th o f b a c te r ia l c u ltu r e s . A n n . R e v . M ic r o b io l.
3 7 1 -3 9 4 . 1949.
62. M c B u rn e y , C . H . , B o lle n , W . B . , and W illia m s , R . J . P a n to th e n ic
a c id and th e n o d u le b a c te ria -le g u m e s y m b io s is . P r o c . N a t’ l
A c a d . S c i. U .S . 2 1 :3 0 1 -3 0 4 .1 9 3 5 .
63. M u lle r , A . and Stapp, C . B e itra g e z u r B io lo g ie d e r L e g u m in o s e n k n o llc h e n b a k te rie n m it b e s o n d e re r B e ru c k s ic h tig u n g
i h r e r A r tv e r s c h ie d e n h e it. A r b . B io l. R e ic h s a n s t. L a n d u.
F o r s t w . 1 4 :4 5 5 -5 5 4 . 1925.
64. N e a l, O. R . and W a lk e r, R .H . P h y s io lo g ic a l s tu d ie s on R h iz o b iu m .
IV . U t iliz a t io n o f c a rb o n a c e o u s m a t e r ia ls . J . B a c t. 3 0 :1 7 3 -1 8 7 .
1935.

- 109 -

65. N ie ls e n , N . and Jo h a n se n , G . U n te rs u c h u n g e n u b e r b io lo g is c h e
S tic k s to ffb in d u n g . V . U n te rs u c h u n g e n u b e r d ie W irk u n g v e r s c h ie d e n e r W u c h s s ta ffe a u f 4 S ta m m e v o n B . r a d ic ic o la .
C o m p t. re n d . T r a v . L a b . C a r ls b e r g . S e r, P h y s io l. 23:
1 7 3 -1 9 3 .1 9 4 1 .
66

II
. N ils s o n , R . , B ja lfv e , G ., and B u r s tr o m , D . U b e r Z u w a c h s fa k to re n
b e i B a c t. .r a d ic io la . L a n tb ru k s - H o g s k o l. A n n . 5 :2 9 1 -3 2 2 .
1938.

67. O g in s k y , E v e ly n , L . and U m b r ie t, W .W . A n in tr o d u c tio n to b a c te r ia l
p h y s io lo g y . W . H . F r e e m a n and C o . , San F r a n c is c o . 1954.
68

. O s te rh o u t, W . J . V . T h e m e c h a n is m o f a c c u m u la tio n in liv in g c e lls .
J . G en. P h y s io l. 3 5 :5 7 9 -5 9 4 . 1952.

69. P e e ch , M . D e te r m in a tio n o f exch ang eab le c a tio n s and exchange
c a p a c ity o f s o ils . R a p id m ic ro m e th o d s u t iliz in g c e n trifu g e and
s p e c tro p h o to m e te r. S o il Sc. 5 9 :2 5 -3 8 . 1945.
70. P e te r s , V . J . and S n e ll, E . E . P e p tid e s and b a c te r ia l g ro w th . V I.
T h e n u t r it io n a l r e q u ire m e n ts o f L a c to b a c illu s d e lb r u e c k ii.
J . B a c t. 6 7 :6 9 -7 0 . 1954.
71. P e te r s . V . J . , P r e s c o tt, J . M . , and S n e ll, E . E . P e p tid e s and b a c t e r ia l
g ro w th , IV . H is tid in e p e p tid e s as g ro w th fa c to r s f o r L a c to ­
b a c illu s d e lb r u e c k ii 9649. J . B io l. C h e m . 2 0 2 :5 2 1 -5 3 2 . 1953.
72. P o h lm a n , G .G . C hanges p ro d u c e d in n itro g e n o u s com pou nds b y
R h . m e l i l o t i and R h . ja p o n ic u m . S o il Sc. 3 1 :3 8 5 -4 0 6 .1 9 3 1 .
73. P ra z m o w s k i, A . D ie W u rz e lk n o llc h e n d e r E rb s e . L a n d w . V e r s . Sta.
3 7 :1 6 1 -2 3 8 .1 8 9 0 .
74. P r e s c o tt, J . M . , P e te rs , V . J . , and S n e ll, E . E . P e p tid e s and b a c t e r ia l
g ro w th . V . S e rin e p e p tid e s and g ro w th o f L a c to b a c illu s d e l­
b r u e c k ii 9649. J . B io l. C h e m . 2 0 2 :5 3 3 -5 4 0 . 1953.
75. P ru c h a , M . J . P h y s io lo g ic a l s tu d ie s o f B a c illu s r a d ic ic o la o f C anada
f ie ld pe as. N . Y . ( C o r n e ll) A g r . C o lle g e M e m . 5 :1 -8 3 .1 9 1 5 .
76. R ic h a rd s o n , D . A . M . S . A . th e s is . U n iv e r s ity o f T o ro n to . 1954.
77. R ic h a rd s o n , D . A . and J o rd a n , D .C .

u n p u b lis h e d data.

-

110

*

78. R o e p ke , R . R . , L ib b y , R . L . , and S m a ll, M a r g a r e t, H . M u ta tio n o r
v a r ia t io n in E s c h e r ic h ia c o li w ith re s p e c t to g ro w th r e q u ir e ­
m e n ts . J . B a c t. 4 8 :4 0 1 -4 1 2 .1 9 4 4 .
79. R o s e n b e rg , T . and W ilb r a n d t, W . E n z y m a tic p ro c e s s e s in c e ll
m e m b ra n e p e n e tra tio n . In te r n . R e v. o f C y to l. 1 :6 5 -9 2 . 1952.
80. R yd o n , H . N . D -a m in o a c id s in m ic r o b io lo g ic a l c h e m is tr y . B io c h e m .
S oc. S y m p o s ia , 1 :4 0 -6 0 . 1948.
81. S m ith , E . L . in T h e e n zym e s , ed. b y J . B . S u m n e r and K . M y r b a c k .
A c a d e m ic P re s s , I n c . , N ew Y o r k . 1951.
82. S tephenson, M a r jo r y . B a c t e r ia l m e ta b o lis m . 3 rd . ed. L o n g m a n s ,
G re e n and C o ., L o n d o n . 1949.
83. S tu m p f, P . K . and G re e n , D . E . L - a m in o a c id o x id a s e o f P ro te u s
v u lg a r is . J . B io l. C h e m . 1 5 3 :3 8 7 -3 9 9 . 1944.
84. S tu m p f, P . K . , Z a ru d n a y a , K . , and G re e n , D .E . P y r u v ic and
a lp h a - k e to g lu ta r ic o x id a s e o f a n im a l tis s u e . J . B io l. C h e m .
1 6 7 :8 1 7 -8 2 5 . 1947.
85. S u m n e r, J . B . and S o m e rs , G. F . C h e m is tr y and m e th o d s o f e n z y m e s .
2nd. ed. A c a d e m ic P re s s , I n c . , N ew Y o r k . 1947.
86

. T a m , R . K . and W ils o n , P .W . R e s p ir a to r y e n zym e s y s te m s in s y m ­
b io tic n itr o g e n fix a tio n . I I I . T h e d e h yd ro g e n a se s y s te m s o f
R h iz o b iu m t r i f o l i i and R h iz o b iu m le g u m in o s a ru m . J . B a c t.
4 1 :5 2 9 -5 4 6 .1 9 4 1 .

87. T h o rn e , C . B . in S y m p o s iu m on a m in o a c id m e ta b o lis m . M c C o llu m P r a t t In s t it u te . T h e Joh ns H o p k in s U n iv e r s ity , B a ltim o r e . 1954.
88

. T h o rn e , C . B . T r a n s a m in a tio n o f D -a m in o a c id s . B a c t. P ro c e e d .
p. 1 0 4 .1 9 5 4 .

89. T h o rn e , D . W . and W a lk e r, R .H . P h y s io lo g ic a l s tu d ie s on R h iz o b iu m .
I I I . R e s p ir a tio n and g ro w th as in flu e n c e d b y th e r e a c tio n o f the
m e d iu m . J . B a c t. 3 0 :3 3 -4 3 . 1935.
90. T h o rn e , D . W . and W a lk e r, R .H . P h y s io lo g ic a l s tu d ie s on R h iz o b iu m .
V I A c c e s s o r y g ro w th fa c t o r s . S o il Sc. 4 2 :2 3 1 -2 4 0 .1 9 3 6 .

- Ill -

91. T is s ie r e s , A . E ffe c t o f a z id e on the r e s p ir a t o r y s y s te m o f A e r o b a c te r a e ro g e n e s . N a tu re , 166:1115. 1950.
92. U m b r e it, W .W . M e ta b o lic m a p s .
1952.

B u rg e s s P u b lis h in g C o . , M in n .

93. U m b r e it, W . W . , B u r r is , R . H . , and S ta u ffe r, J . F . M a n o m e tr ic
te c h n iq u e s and tis s u e m e ta b o lis m . B u rg e s s P u b lis h in g C o . ,
M in n . 1949.
94. V ita n e n , A . I . in S y m p o s iu m on m ic r o b ia l m e ta b o lis m . V I th .
I n t e r n a t 'l C o n g re s s M ic r o b io l. R o m e , 1953.
95. V ir ta n e n , A . I . and L a in e , T . The d e c a rb o x y la tio n o f d - ly s in e
and 1 - a s p a r tic a c id . E n z y m o lo g ia , 3 :2 6 6 -2 7 0 . 1936.
96. V ita n e n , A . I . and R autanen, N . in T h e e n z y m e s , ed. b y J . B .
S u m n e r and K . M y r b a c k . A c a d e m ic P re s s , I n c . , N e w Y o r k .
1952.
97. W a lk e r, R . H . , A n d e rs o n , D . A . , and B ro w n , P . E . P h y s io lo g ic a l
s tu d ie s on R h iz o b iu m . I I . T h e e ffe c t o f n itr o g e n s o u rc e on
o xyg e n c o n s u m p tio n o f R h. m e li l o t i , R h. t r i f o l i i , and R h.
p h a s e o li. S o il Sc. 3 8 :2 0 7 -2 1 7 .1 9 3 4 .
98. W e st, P . M . and W ils o n , P .W . G ro w th fa c t o r re q u ire m e n ts o f th e
r o o t n o d u le b a c te r ia . J . B a c t. 3 7 :1 6 1 -1 8 5 . 1939.
99. W ils o n , J . B . P h y s io lo g ic a l s tu d ie s o f th e B a c illu s r a d ic ic o la o f th e
s o yb e a n and o f th e fa c t o r s in flu e n c in g n o d u le p ro d u c tio n .
N . Y . ( C o r n e ll) A g r . E x p t. Sta. B u ll. 3 8 6 :3 6 9 -4 1 3 . 1917.
100. W ils o n , J . B . T h e le g u m e b a c te r ia lib e r a te gaseous n itr o g e n fr o m
n it r a t e . S o il. S c i. Soc. o f A m e r . P r o c . 1 2 :2 1 5 -2 1 6 . 1947.
101. W ils o n , J . B . and W ils o n , P .W . B io tin as a g ro w th fa c t o r f o r r h iz o b ia .
J . B a c t. 4 3 :3 2 9 -3 4 1 .1 9 4 2 .
102. W ils o n , J . K . and C h in , C . S y m b io tic s tu d ie s w ith is o la te s fr o m
n o d u le s o f A s tr a g a lu s . S o il Sc. 6 3 :1 1 9 -1 2 7 . 1947.
103. W ils o n , P .W . R e s p ir a to r y e n zym e s y s te m s in s y m b io tic n itr o g e n
fix a tio n . I . T h e ’’ r e s tin g c e ll" te c h n iq u e as a m e th o d f o r
s tu d y o f b a c te r ia l m e ta b o lis m . J . B a c t. 3 5 :6 0 1 -6 2 3 . 1938.

- 112 -

104. W o iw o d , A . J . A te c h n iq u e f o r e x a m in g la r g e n u m b e rs o f b a c te r ia l
c u ltu r e f il t r a t e s b y p a r t it io n c h ro m a to g ra p h y . J . G en.
M ic r o b io l. 3 :3 1 2 -3 1 8 .1 9 4 9 .
105. W ood, W . A . and G u n s a lu s , I. C . D -a la n in e fo r m a tio n : a ra c e m a s e
in S tre p to c o c c u s fa e c a lis . J . B io l. C h e m . 19 0 r4 0 3 -4 1 6 . 1951.
106. W o o lle y , D . W . Som e b io lo g ic a l e ffe c ts p ro d u c e d b y b e n z im id a z o le
and t h e ir r e v e r s a l b y p u rin e s . J . B io l. C h e m . 1 5 2 :2 2 5 -2 3 2 .
1944.
n

107. Z in s s e r , O . U b e r das V e rh a lte n v o n B a k te r ie n in s b e s o n d e re von
K n o llc h e n b a k te r ie n in leb e n d e n p fla n z lic h e n G ew eben. J a h rb .
W is s . B o t. 3 0 :4 2 3 -4 5 2 .1 8 9 7 .
108. Z ip f e l, H . B e itra g e z u r M o rp h o lo g ie und B io lo g ie d e r K n o ll­
c h e n b a k te rie n des L e g u m in o s e m . C e n tb l. B a k t. 2 A b t. 32:
9 7 -1 3 7 .1 9 1 1 .

- 113 A P P E N D IX

PART I

M e d ia

(1 ) W ils o n and W ils o n 1s M e d iu m m o d ifie d by the o m is s io n o f a m m o n iu m
c h lo r id e .
Ref.:

101
k

2h p o 4

0. 5 g m .

N aC l

0 .1 g m .

s u c ro s e

2 .5 g m .

M gS04 . 7 H2 O

0. 2 g m .

Ca S 0 4

0. 2 g m .

F e C l3

e x tra c te d w ith a b s o lu te e th y l
a lc h o l f o r 24 h r .

ig n ite d in oven f o r 2 h r .

. 001 g m . - s u b lim e d

S p e c ia l w a te r to 1000 m l. - d is t ille d , tre a te d w ith K M n 0 4 and AgNC >3
and r e d is t ille d in an a ll- g la s s a p p a ra tu s .

p H a d ju s te d w ith C. P . K O H s o lu tio n .

- 114 -

(2 )
R e f:

M o d ifie d M e d iu m " 7 9 M
103
R e m o d ifie d b y th e o m is s io n o f CaCC >3
K 2 HPO4
M gS 0

4

.

0. 5 g m .
7

H2

0

0,2

gm .

N aC l

0.1

gm .

Y e a s t E x tr a c t (D ifc o )

5 .0 gm .

D is t ille d w a te r to 1000 m l.
p H s h o u ld be 7. 0

\(3)
R e f:

M o d ifie d L o c h e a d 's M e d iu m
58
(a ) a g a r
ta p w a te r

600 m l.

po4

0

.4 g m .

M g S 0 4 . 7H 20

0

.

N aC l

0.1

C aS 0 4

0. 05 g m .

ta p w a te r

200

(b) k h

(c )

1 5 .0 g m .

2

s u c ro s e
y e a s t e x tr a c t (D ifc o )
ta p w a te r

1

gm .
gm .

m l.

lO .O g m .
1 .0 g m .
200

m l.

T h e th r e e s o lu tio n s a r e m ade up s e p a ra te ly , d is s o lv e d in th e s te a m e r and
m ix e d to g e th e r.

- 115 -

TABLE 1
S T A N D A R D D E V IA T IO N S O F N E P H E L O S R E A D IN G S

N e p h e lo s re a d in g s
S e rie s A (lo w )

S e rie s B (m e d iu m )

S e rie s C (h ig h )

3 6 .5

63. 0

107. 5

40. 2

6 6 .7

1 0 3 .4

4 0 .2

69. 0

101

4Q.G

62. 5

1 0 6 .9

40. 0

6 3 .5

108. 5

4 4 .0

70. 0

1 0 8 .5

4 1 .5

67. 2

107. 5

4 1 .5

6 9 .0

1 0 4 .2

4 5 .0

6 4 .0

100

.

0

4 5 .0

69. 0

106.

0

4 1 3 .9

663. 9
M ean =

Z d x 2 = 6 3 .8

Z d x 2 = 6 6 .3

Zdx2 =

S .D .

S .D .

S. D.

= 2 .5 2

.4

= 2 .5 7

0

1 0 5 3 .5

M e a n = 4 1 .4

66

.

M e an = 105. 3
88.8

= 2 .9 8

TABLE 2
V IA B L E B A C T E R IA L C O U N T V S. N E P H E L O S R E A D IN G S - R E S P O N S E C U R V E
N e p h e lo s
4 .0
5 0 .3
7 8 .4
1 1 2 .2

V ia b le cou nt X
1. 52
6 .8 5
9 .6 2
1 2 .7 3

106

- 116 -

TABLE 3
D R Y W E IG H T A N D N IT R O G E N C O N T E N T O F O N T A R IO V A R IE G A T E D
A L F A L F A IN O C U L A T E D W IT H T H R E E R H IZ O B IU M S T R A IN S .
F E B . - M A R C H . 1955

B a c t e r ia l
s t r a in

R 21

R 2 0 -2 7 A

R 20

P o s itio n in la t in s q u a re
R ow
C o lu m n

D r y w t. /
p la n t ( g m . )

1

4

.

0100

3 0 .3 5

2

1

. 0070

33. 16

3

3

.

0110

25. 36

4

2

.

0111

29. 44

1

1

.0102

27. 27

2

2

. 0125

25. 34

3

4

. 0107

2 3 .9 5

4

3

. 0135

24. 28

1

3

. 0059

1 3 .0 7

2

4

. 0059

21. 99

3

2

. 0043

23. 36

4

1

. 0061

1 9 .2 5

\
C o n tr o l
(n o t in o c . )

N itro g e n
c o n te n t
(m g m . /g m .
d ry w t . )

1

2

. 0065

18. 60

2

3

. 0059

1 8 .4 0

3

1

. 0059

18. 39

4

1

. 0059

1 9 .3 3

- 117 -

TABLE 4
A N A L Y S IS O F V A R IA N C E F O R M G M , N /G M , D R Y W T , O F P L A N T
T IS S U E - L A T IN S Q U AR E - A L F A L F A IN O C U L A T E D
W IT H R 2 1 ' R 20 -27 A » A N D

R 20

Sum o f s q u a re s f o r t o t a l = 8 9 9 9 .4 6
c o r r e c t io n = ( Z x ) 2 = 8580. 32

~W r
8 9 9 9 .4 6 - 8 5 8 0 .3 2 = 4 1 9 .1 4
Sum o f s q u a re s f o r tr e a tm e n ts = 8903. 63
8 9 0 3 .6 3 - 8 5 8 0 .3 2 = 32 3.3 1
Sum o f s q u a re s f o r ro w s = 8594. 60
8 5 9 4 .6 0 - 8 5 8 0 .3 2 = 1 4 .2 8
Sum o f s q u a re s f o r c o lu m n s = 8625. 51
8 6 2 5 .5 1 - 8 5 8 0 .3 2 = 4 5 .1 9
Sum o f s q u a re s f o r ra n d o m e r r o r = 3 6 .3 6

D iffe r e n c e
b e tw e e n

D e g re e s o f
F re e d o m

S um o f
S qua res

V a ria n c e

T r e a tm e n ts

3

3 2 3 .3 1

10 7 .7 7

R ow s

3

1 4 .2 8

4 .7 6

C o lu m n s

3

4 5 .1 9

1 5 .0 6

E rro r

6

3 6 .3 6

1 2 .1 2

T o ta l

15

_

4 1 9 .1 4

F fo r
s ig n ific a n c e
5%
6 .8 7

1%
9 .7 8

- 118 -

A N A L Y S IS O F V A R IA N C E (C O N T .)
S ig n ific a n c e b e tw e e n tre a tm e n ts
E r r o r (ra n d o m v a r ia n c e ) f o r each tr e a tm e n ts = 1 2 .1 2
E r r o r (ra n d o m v a r ia n c e ) f o r each m e a n = 1 2 .1 2 = 3 .3
4
R a n d o m v a r ia n c e b e tw e e n m e ans = 3 . 3 x 2 =

6.6

S ta n d a rd d e v ia tio n o f d iffe re n c e be tw e e n m e a n s due to
ra n d o m e r r o r =
t = 2.45

^

6.6

=

2.6

n e c e s s a ry m e a n d iffe re n c e = 2 . 4 5 x 2 . 6 =
6. 37

T a b le o f m e a n s o f tre a tm e n ts in o r d e r o f m a g n itu d e
T r e a tm e n t

M eans

R22

2 9 . 57

R 2 0 -2 7 A

25.20

R 20

19' 17

C o n tr o l

18. 68

R ^ and R 2 Q 21A a re s iS n ^ c a n t^ b e tte r th a n the c o n tr o l,
but R 2o is n o t*

- 119 -

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TABLE 7
IN F L U E N C E O F 2 7 °C . IN C U B A T IO N T E M P E R A T U R E ON T H E
U T IL IZ A T IO N O F L - A N D D - A M IN O A C ID S B Y T H R E E R H IZ O B IU M
S TR A IN S

A m in o a c id

M a x im u m g ro w th in n e phe los u n its

R 21

R 20

R 20-27A

g ly c in e

1 3 4 .0

36. 0

49. 0

L - le u c in e

1 6 5 .2

40. 0

84. 0

D -le u c in e

146. 0

40. 0

80.

L - v a lin e

100

D - v a lin e

125. 0

.0

.

0

0

44. 0

25. 0

36. 0

8

L - m e th io n in e

86

0

144. 0

71. 0

D -m e th io n in e

69. 0

54. 0

46. 0

L - g lu t a m ic a c id

1 9 1 .4

1 4 1 .0

D - g lu ta m ic a c id

122

.

0

L - h is t id in e

321.

6

D - h is tid in e

337. 0

.

66.0

226.

151.

0

33. 0

0

195. 0

241. 0

173. 0

- 1?2 -

TABLE

8

O P T IM U M T E M P E R A T U R E F O R G R O W TH IN M O D IF IE D M E D IU M " 7 9 "

T e m p e ra tu re

2 7 °C .

O rg a n is m

^20

R21
r 2 0 -2 7 A
3 0 °C .

^20
^21

R 2 0 -2 7 A
3 5 °C .

^20
^21

R 2 0 -2 7 A

M a x im u m ne p h e lo s v a lu e s

73.4
1 3 9 .0
86.0

7 1 .0
438. 0
110. 0
55. 0
494. 0
1 1 6 .0

- 123 -

- 124 -

T A B L E 10
G R O W T H R E S P O N S E O F R 2 1 ON H IS T ID IN E M E D IU M C O N T A IN IN G
i
V A R IO U S C O N C E N T R A T IO N S O F N H 4 C I.

M o la r it y o f N H 4 C I

M a x im u m g ro w th (n eph elos u n its )

0

400. 2

0. 005

5 0 0 .4

.

0 010

563. 0

0. 015

540. 3

0 . 020

463. 1

T A B L E 11
R E S P IR A T IO N O F R 2 i ON G L U T A M IC A C ID , P Y R U V IC A C ID
A N D A L P H A -K E T O G L U T A R IC A C ID

T im e
( m in . )

G lu ta m ic a c id

0*2

C0

2

T"'

C 02

p y r u v ic a c id

O2 *

CO2 ?*

_C ° 2

02

5
10

15
20

25

42. 9
87. 4
- 1 3 3 .6
176. 5
209. 9

50. 3
83. 6
121. 5
158. 9
193. 0

1.20

0.
0.
0.
0.

96
91
90
92

ave. = 0. 98

° 2

28. 6
73. 1

31. 0
83. 0

119. 2
1 6 7 .0
206. 7

137. 5
203. 0
252. 6
ave. =

1.

08
1. 13
1. 15
1 . 21
1 . 22
1

. 16

- 125 -

TABLE

T im e
( m in . )

11

(C O N T .)

a lp h a - k e to g lu ta r ic a c id

o 2*

c o 2* *

co2

endogenous

O2*

c o 2* *

34. 1
62. 0
91. 4
122. 4
14 1.0

24.
45.
69.
94.
114.

^2
5
10
15
20
25

29.0
65. 8
105. 6
145.3
179.0

38. 9
75. 0
11 3.2
155. 7
1 9 2 .0

1.
1.
1.
1.
1.

34
12
07
07
07

ave. = 1. 13

*

m i c r o l i t e r s O 2 ta k e n up

* * m i c r o l i t e r s COg e v o lv e d
C>2 and C O 2 v a lu e s a re a v e ra g e s o f d u p lic a te fla s k s .

7
0
7
5
7

c ° 2
o2

0.
0.
0.
0.
0.

79
73
76
77
81

ave. = 0. 77

- 126 -

T A B L E 12
D A T A R E G A R D IN G T H E G R O W TH O F R 2 1 ON G L Y C IN E - 1 - C

In c u b a tio n
tim e (d ays)

*
**

14

B ack-:
g ro u n d
(c .p .m .)

S am ple
count
(c .p .m .)

1

1 9 .1

7 1 .0

2

1 4 .3

158. 9

144.

6

297. 5

. 076

3

1 4 .4

204. 0

189.

6

487.

0

. 052

4

16.

6

282. 5

265. 9

999. 0

. 039

5

18.

2

310. 7

292. 5

1 4 15.0

. 018

6

17. 2

159.

6

1 4 2 .4

1 7 3 0 .0

.

012

7

1 7 .2

29. 9

12. 7

1 6 9 0 .0

.

000

c. p .m .
above
back­
g ro u n d

51. 9

N e p h e lo s *

60.

8

O. D . * *

-

c o r r e c te d f o r im p e r fe c tio n s in th e re a d in g tube
O. D . = o p tic a l d e n s ity o f s o lu tio n s o b ta in e d b y e x tr a c tin g c h ro m a to g ra m
a re a s .

- 127 -

i

TABLE 13
O X ID A T IO N O F L - A N D D -A M IN O A C ID S B Y R H IZ O B IU M R 2 1

A m in o a c id

O xyge n u p ta ke in m ic r o l i t e r s *
T im e ( m in . )
15

L - v a lin e

30

62.0

L - h is t id in e

70. 5

L - m e th io n in e

55.

45

1 2 3 .2

5

278.5

8

209. 0

2 9 1 .7

117.3

1 7 2 .3

239.0

136.
8

187.

60

L - le u c in e

50.0

102. 2

159. 7

220.4

L - g lu t a m ic a c id

84.

160,

7

230.4

326. 1

g ly c in e pH

2

.0

72.0

143. 3

212. 3

285.0

g ly c in e pH 7. 0

56. 3

1 1 0.3

150.

8

202.1

g ly c in e pH 6 .0

45.0

93.0

1 3 4 .2

182. 9

endogenous

40. 0

87. 0

127. 0

1 7 9 .4

D - v a lin e

1 0 .0

21

.

33. 9

46.2

22.4

32. 2

8

D - h is tid in e

6

7 .2

1 4 .0

D -m e th io n in e

12. 7

22.3

38.

2

57. 2

D - le u c in e

1 2 .5

23 . 2

41.

8

60.4

D - g lu ta m ic a c id

12. 7

23.0

4 1 .8

59.0

5 .7

9 .9

18. 5

28.0

endogenous
g ly c in e
g ly c in e + a z id e

13.5
4 .7

19 . 5
6 .3

34.5
12. 6

v-«62. 5
22.1

- 138 -

TABLE 13 (CONT.)
A m in o a c id

O xyg e n u p ta ke in m i c r o l i t e r s *
T im e ( m in . )
15

30

45

60

g ly c in e + b e nzoa te

1 .4

5 .8

1 4 .5

31. 9

g ly c in e + N aC N

0.0

0 .0

0.0

0

.

0

* a v e ra g e s o f d u p lic a te fla s k s

T A B L E 14
A N A E R O B IC R E D U C T IO N O F M E T H Y L E N E B L U E B Y R 2 1 IN T H E
P R E S E N C E O F L - G L U T A M IC A C ID

T im e ( m in . )

O p tic a l d e n s ity *
L - g lu t a m ic a c id

endogenous

0

0. 690

0. 710

3

0. 690

0. 710

5

0. 590

0. 720

7

0 .4 8 5

0. 710

9

0.390

0. 690

11

0. 325

0. 670

13

0.285

0. 650

14

0. 275

0. 640

15

0. 275

0. 630

* a v e ra g e o f t r ip lic a t e d e te rm in a tio n s

- 129 -

T A B L E 15
I

I

A C T IO N O F L - G L U T A M IC A C ID D E C A R B O X Y L A S E ON T R A N S A M IN A T IO N
T E S T M IX T U R E S O R G IN A L L Y C O N T A IN IN G A L P H A -K E T O G L U T A R IC
A C ID A N D D -A S P A R T IC A C ID

M ic r o lite r s

C02

e v o lu tio n

T im e (m in u te s )

S ta n d a rd * *
T e s t m ix t u r e
*
**

10

20

30

40

50

36. 0
10. 0

70. 1
21. 0

85.0
30 . 1

93. 0
37.6

101. 5
46. 0

a v e ra g e s o f d u p lic a te fla s k s - endogenous s u b tra c te d
c o n ta in in g

0

. 5 m g m . L - g lu t a m ic a c id /fla s k

T A B L E 16
A N A L Y S IS O F L - A L A N IN E R E A C T IO N M IX T U R E B Y
D -A M IN O A C ID O X ID A S E -('A L A N IN E R A C E M A S e )

F la s k c o n te n ts

O xyg e n u p ta k e in m i c r o l i t e r s *
T im e ( m in . )

*

40

50

29. 3

56. 2

84. 0

95.8

110. 9

122

.

2

132. 9

1.0

0

.

0

1.0

o -o

o
o

30

•

c o n tr o l

20

o
•
o

L - a la n in e
m ix t u r e

10

a v e ra g e s o f d u p lic a te fla s k s .

0

.

0

60

70

- 130 A P P E N D IX

PA R T II

T A B L E 17
D A T A P E R T A IN IN G T O T H E G R O W TH O F T H R E E R H IZ O B IU M S TR A IN S
IN C Y S T E IN E M E D IU M W IT H OR W IT H O U T A D D E D N IC O T IN A M ID E

T im e (d a ys)

N e p h e lo s re a d in g s

R 2 0 -2 7 A

R 21

A

B

A

B

.7

7 .9

2

30.4

4 1 .0

37.0

3

47.2

54.7

4

47. 1

61.

5

16.

R 20

.

A
3.

B
6

14. 0

44. 0

4.0

54. 3

39. 5

54. 3

25. 2

63. 5

2

35. 7

55. 2

49. 0

65. 5

42. 1

58.7

33.0

5 1 .0

53. 1

58. 1

6

4 0 .2

47.0

30.0

4 7 .0

53. 9

54.8

7

32.0

38.0

29.5

4 7 .0

5 1 .4

5 1 .3

1

8

A = w ith o u t added n ic o tin a m id e

0

10

0

B = w ith added n ic o tin a m id e

- 131 -

T A B L E 18
U T IL IZ A T IO N O F P U R IN E S A N D P Y R IM ID IN E S (0. 0001M ) B Y T H R E E
R H IZ O B IU M S T R A IN S G R O W IN G IN L E U C IN E B A S A L M E D IU M

C om pound

M a x im u m g ro w th in n e phe los u n its

r 20

adenin e s u lp h a te
g u anin e HC1
x a n th in e
th y m in e
u r a c il
c y to s in e
le u c in e o n ly

53.
36.
71.
51.
74.
96.
113.

R 2 1

0
0
0
0
0
0
0

438. 0
296. 6
375. 2
276. 6
354. 0
358. 0
222. 0

R 20-27A
377. 0
275. 0
242. 0
239. 0
224. 0
271. 0
182. 0