Uptake and Role of Molybdenum in Nitrogen-Fixing Bacteria

22 Uptake and Role of Molybdenum in Nitrogen-Fixing Bacteria

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PHILIP T. PIENKOS, V I N O D K. S H A H ,

and W I N S T O N J. B R I L L

Department of Bacteriology and Center for Studies of Nitrogen Fixation, University of Wisconsin, Madison, WI 53706

Mutant strains of nitrogen-fixing bacteria have been obtained with defects in their ability to synthesize an active molybdenum cofactor. Such strains are used to assay this cofactor. Ammonia in the medium represses the synthesis of the molybdenum cofactor. Molybdenum is transported by an energy-requiring reaction, and the metal becomes part of a molybdenum storage compound. Molybdenum in the medium affects the regulation of nitrogenase synthesis be cause Klebsiella pneumoniae does not synthesize either of the two nitrogenase components when it lacks sufficient molybdenum.

TT7e

s h a l l o u t l i n e a p p r o a c h e s u s e d to i n v e s t i g a t e t h e r o l e of m o l y b -

^ * d e n u m i n n i t r o g e n fixation b y b a c t e r i a . I t is h o p e d that this t y p e of w o r k w i l l m a k e i t easier f o r t h e c h e m i s t t o focus o n t h e m u l t i p l e roles t h a t m o l y b d e n u m p l a y s i n these b a c t e r i a . T h e a p p r o a c h that has g i v e n us most i n s i g h t i n t o t h e m e c h a n i s m of n i t r o g e n fixation i n v o l v e s t h e use of m u t a n t strains that s p e c i f i c a l l y a r e u n a b l e t o g r o w o n n i t r o g e n ( N i f " mutants).

S u c h studies h a v e y i e l d e d i n f o r m a t i o n o n t h e a c t i v e site o f

n i t r o g e n a s e ( J ) , h o w nitrogenase synthesis is r e g u l a t e d (2), t h e o r d e r of genes specific f o r n i t r o g e n fixation (nif g e n e s ) (3, 4), a n d t h e existence of factors other t h a n nitrogenase that are s p e c i f i c a l l y r e q u i r e d i f a n o r g a n i s m is to fix n i t r o g e n (4). vinehndii,

T w o organisms are discussed,

Azotobacter

a b a c t e r i u m t h a t o n l y fixes n i t r o g e n a e r o b i c a l l y , a n d

pneumoniae,

a n organism

that only

fixes

nitrogen under

Klebsiella anaerobic

conditions. T h e e n z y m e , nitrogenase, is c o m p o s e d of t w o p r o t e i n s — c o m p o n e n t I a n d c o m p o n e n t I I . C o m p o n e n t I has a m o l e c u l a r w e i g h t of a b o u t 220,000 a n d contains either o n e o r t w o m o l y b d e n u m a n d 24 i r o n atoms. 402

In Bioinorganic Chemistry—II; Raymond, K.; Advances in Chemistry; American Chemical Society: Washington, DC, 1977.

Compo-

22.

piENKOS E T AL.

403

Molybdenum in Nitrogen-Fixing Bacteria

n e n t I I has a m o l e c u l a r w e i g h t of 60,000 a n d has f o u r i r o n atoms. r e a c t i o n r e q u i r e s 1 2 - 2 4 A T P f o r e a c h n i t r o g e n fixed ( 5 ) .

The

W e were able

to p r e p a r e cell-free extracts of t h e N i f " m u t a n t strains a n d t i t r a t e e a c h extract (1)

w i t h purified components.

w h i c h of the c o m p o n e n t s

T h u s i t w a s p o s s i b l e to i d e n t i f y

w a s l a c k i n g i n a c t i v i t y . I t w a s i m p o r t a n t to

k n o w i f a m u t a n t l a c k i n g c o m p o n e n t I a c t i v i t y does n o t p r o d u c e

com-

p o n e n t I at a l l or w h e t h e r c o m p o n e n t I is s y n t h e s i z e d , b u t i n a n i n a c t i v e form.

S e r o l o g i c a l tests for t h e c o m p o n e n t s

were performed using anti-


s e r u m f r o m r a b b i t s i n j e c t e d w i t h e i t h e r of t h e t w o p u r i f i e d c o m p o n e n t s i s o l a t e d f r o m the w i l d t y p e .

W i t h t e c h n i q u e s s u c h as these,

several

h u n d r e d m u t a n t strains w e r e classified. Molybdenum

Cofactor

K e t c h u m et a l . ( 6 ) s h o w e d t h a t c r u d e extracts of a m u t a n t s t r a i n of Neurospora

crassa, c a l l e d N i t - 1 , that w o u l d not r e d u c e n i t r a t e c o u l d b e

a c t i v a t e d b y a d d i n g a c i d - t r e a t e d m o l y b d o p r o t e i n to t h e extract.

The

a c t i v a t i n g f a c t o r c o u l d b e o b t a i n e d f r o m m o l y b d o p r o t e i n s s u c h as n i t r o genase c o m p o n e n t I, b o v i n e l i v e r sulfite oxidase, x a n t h i n e oxidase, a l d e h y d e oxidase, a n d n i t r a t e r e d u c t a s e f r o m v a r i o u s sources ( 7 ) . et a l . ( S ) o b t a i n e d m u t a n t strains of Rhizobium

Kondorosi

meliloti t h a t c o u l d n o t

r e d u c e n i t r a t e . S o m e of these strains also w e r e u n a b l e to p r o d u c e effect i v e ( n i t r o g e n fixing ) a l f a l f a n o d u l e s , a n d t h e y c o n c l u d e d t h a t a c o m m o n g e n e t i c d e t e r m i n a n t is r e q u i r e d f o r n i t r a t e reductase a n d n i t r o g e n a s e a c t i v i t y . T h i s d e t e r m i n a n t m i g h t b e the m o l y b d e n u m c o f a c t o r t h a t seems to b e c o m m o n to a l l m o l y b d o p r o t e i n s . T h e m o l y b d e n u m cofactor f r o m Rhodospirillum a n d is i n s e n s i t i v e to t r y p s i n ( 9 ) .

rubrum is d i a l y z a b l e

T h e cofactor c a n easily b e i n a c t i v a t e d

b y heat. O n e of the p r o b l e m s i n p u r i f y i n g this cofactor is the i n s t a b i l i t y a n d l o w y i e l d s f r o m p u r i f i e d e n z y m e s a n d c r u d e extracts. L e e et a l . s h o w e d that t h e m o l y b d e n u m cofactor

(10)

is s t a b i l i z e d b y 0 . 0 1 M s o d i u m

m o l y b d a t e a n d that the absence of a i r a d d s to the s t a b i l i t y . T h e s e w o r k e r s u s e d M o " l a b e l i n g to s h o w t h a t t h e m o l y b d e n u m f r o m the cofactor is f o u n d i n a c t i v a t e d n i t r a t e r e d u c t a s e f r o m the m u t a n t s t r a i n of N. crassa. G a n e l i n et a l . (11)

p r o v i d e d e v i d e n c e t h a t the m o l y b d e n u m cofactor

is a m o l y b d e n u m p e p t i d e w i t h a m o l e c u l a r w e i g h t of a b o u t 1000.

The

same p r o p e r t i e s h a v e b e e n c l a i m e d f o r the cofactor f r o m x a n t h i n e o x i dase

(12). I t was i m p o r t a n t to i d e n t i f y m u t a n t strains w i t h defects

i n nitro-

genase s i m i l a r to the d e f e c t o b s e r v e d i n n i t r a t e r e d u c t a s e i n the N i t - 1 m u t a n t strain of N. crassa (6).

It was p o s t u l a t e d t h a t s u c h strains w o u l d

b e a b l e to synthesize a c t i v e c o m p o n e n t I I a n d a n i n a c t i v e c o m p o n e n t that c o u l d b e a c t i v a t e d i n v i t r o b y the m o l y b d e n u m cofactor.


I

Cell-free

404

BIOINORGANIC C H E M I S T R Y

II

extracts f r o m strains p r o d u c i n g i n a c t i v e c o m p o n e n t I w e r e p r e p a r e d a n d tested w i t h m o l y b d e n u m cofactor m a d e b y a c i d - t r e a t i n g a n d n e u t r a l i z i n g p u r i f i e d c o m p o n e n t I f r o m the w i l d t y p e o r g a n i s m ( 1 3 ) . vinehndii

(14)

a n d K. pneumoniae (4)

Strains of A .

were found w i t h inactive com-

p o n e n t I that c o u l d b e a c t i v a t e d i n v i t r o w i t h the m o l y b d e n u m

cofactor.

T h e m u t a t i o n s that p r e v e n t t h e f o r m a t i o n of the m o l y b d e n u m h a v e b e e n l o c a t e d o n t h e c h r o m o s o m e i n b o t h organisms ( 3 , 4).

cofactor Figure 1

shows the l o c a t i o n (nif B ) of t h e g e n e ( s ) t h a t s p e c i f y t h e m o l y b d e n u m


cofactor i n K. pneumoniae.

T h i s g e n e ( s ) is l o c a t e d close to the s t r u c t u r a l

genes for the t w o n i t r o g e n a s e c o m p o n e n t s .

Figure 1. Map of nif genes in Klebsiella pneumonia. The designation, (act), represents the molybdenum cofactor. Component II (II ) is reduced by the nif electron transfer system. II then binds and reduces l (act), and the resulting complex with ATF DNA can reduce nitrogen to ammonia. ox

rcd

ox

his D

nif B

nif F

It is p o s s i b l e to m a k e the w i l d t y p e A . vinelandii

nif D, H

produce

active

component II a n d an inactive component I b y substituting tungsten for m o l y b d e n u m i n the m e d i u m (14). cofactor is not p r o d u c e d .

I n this w a y , a n a c t i v e m o l y b d e n u m

However, if acid-treated component

I from

cells g r o w n o n m o l y b d e n u m is a d d e d to extracts f r o m t u n g s t e n - g r o w n cells, a c t i v a t i o n ocurs. A d d i t i o n of m o l y b d a t e to s u c h extracts does n o t reactivate component

I.

W e are u s i n g m u t a n t strains d e f e c t i v e i n the

m o l y b d e n u m cofactor as w e l l as t u n g s t e n - g r o w n cells as assays to p u r i f y the m o l y b d e n u m cofactor. Regulation N i t r o g e n - f i x i n g cells h a v e a n o b v i o u s r e q u i r e m e n t f o r m o l y b d e n u m ; h o w e v e r , n o m o l y b d e n u m is r e q u i r e d w h e n these cells g r o w i n a m e d i u m c o n t a i n i n g excess N H nitrogenase ( 1 5 ) .

4

+

.

NH

4

+

completely

represses

t h e synthesis

of

N i t r o g e n fixation is q u i t e a n e n e r g y - d e m a n d i n g p r o c -

ess, a n d so i t m a k e s sense f o r t h e o r g a n i s m not to p r o d u c e n i t r o g e n a s e w h e n i t is n o t n e e d e d . is r e p r e s s e d b y N H

4

+

T h e synthesis of t h e m o l y b d e n u m cofactor also (14).

C e r t a i n m u t a n t strains h a v e b e e n i s o l a t e d

that c o n t i n u e to s y n t h e s i z e a c t i v e nitrogenase i n t h e p r e s e n c e of (2,16).

NH

4

+

T h e r e f o r e , i t seems that the factors r e s p o n s i b l e f o r t h e r e g u l a t i o n

of the n i t r o g e n a s e s t r u c t u r a l genes are also r e s p o n s i b l e f o r r e g u l a t i n g t h e


22.

405


piENKOS E T AL.

synthesis of a c t i v e m o l y b d e n u m cofactor. also h a v e b e e n m a p p e d o n the

S o m e of these r e g u l a t o r y genes

chromosome.

T h e r e are m a n y reports (e.g., R e f s . 17,18) i n w h i c h n i t r o g e n

fixation

is l i m i t e d i n a s o i l b e c a u s e m o l y b d e n u m is deficient. A d d i t i o n of m o l y b d e n u m to s u c h soils a l l o w s n i t r o g e n denum

deficiency

fixation

to p r o c e e d .

This molyb-

is e x t r e m e l y i m p o r t a n t i f a c r o p of n i t r o g e n - f i x i n g

l e g u m e s is d e s i r e d . I n fact, f a r m e r s c a n b u y m o l y b d a t e s to s p r e a d o n t h e field or to a p p l y to the seed b e f o r e s o w i n g . W e w o n d e r e d w h a t h a p p e n s


to a n i t r o g e n - f i x i n g b a c t e r i u m t h a t is deficient i n fixed n i t r o g e n , b u t is u n a b l e to fix n i t r o g e n b e c a u s e m o l y b d e n u m is u n a v a i l a b l e . S u c h a s i t u a t i o n m i g h t cause the b a c t e r i u m to synthesize a c t i v e c o m p o n e n t I I a n d a n inactive component

I. T h i s c o n d i t i o n is d e t r i m e n t a l to the c e l l since i t

wastes e n e r g y s y n t h e s i z i n g proteins that h a v e n o benefit to i t . H o w e v e r , K. pneumoniae has a r e g u l a t o r y m e c h a n i s m that p r e v e n t s

nitrogenase

synthesis w h e n there is not e n o u g h m o l y b d e n u m a v a i l a b l e . W h e n derepress

we

this o r g a n i s m i n the absence of m o l y b d e n u m , n e i t h e r

com-

p o n e n t I nor c o m p o n e n t I I is s y n t h e s i z e d , e v e n i n a n i n a c t i v e f o r m

(19).

A p o s s i b l e m e c h a n i s m for c o n t r o l of nitrogenase synthesis m i g h t i n v o l v e a p r o t e i n that activates t r a n s c r i p t i o n of the nif genes. T h i s a c t i v a t o r is inactivated when N H

4

+

a c c u m u l a t e s . It is easy to i n t r o d u c e a m e c h a n i s m

f o r c o n t r o l b y m o l y b d e n u m b y h y p o t h e s i z i n g that this a c t i v a t o r r e q u i r e s molybdenum.

M u t a n t strains t h a t are d e f e c t i v e i n n i t r o g e n a s e r e g u l a t i o n

b y m o l y b d e n u m s h o u l d be u s e f u l for t e s t i n g this h y p o t h e s i s .

Molybdenum

Storage

W e are n o w t r y i n g to u n d e r s t a n d h o w the m o l y b d a t e that is a d d e d to the m e d i u m u l t i m a t e l y b e c o m e s a p a r t of nitrogenase c o m p o n e n t

I.

Studies w i t h m e t a b o l i c i n h i b i t o r s i n d i c a t e that m o l y b d a t e is t a k e n u p b y a n e n e r g y - d e m a n d i n g process i n A . vinelandii.

W e w e r e s u r p r i s e d to find

that this o r g a n i s m a c c u m u l a t e s m o r e t h a n 20 times m o r e

molybdenum

t h a n i t a c t u a l l y r e q u i r e s f o r m a x i m u m nitrogenase a c t i v i t y . C e l l s t h a t grow on N H

4

+

do not r e q u i r e m o l y b d e n u m .

Azotobacter vinelandii

g r o w n to m i d - l o g phase i n a m e d i u m c o n t a i n i n g N H num.

T h e cells w e r e w a s h e d free of N H

4

+

4

+

b u t no

was

molybde-

a n d t h e n a l l o w e d to derepress

f o r nitrogenase synthesis i n t h e presence a n d absence of c h l o r a m p h e n i c o l w h e n molybdate was added.

T a b l e I presents e v i d e n c e t h a t t h e p r o t e i n

synthesis is not r e q u i r e d for m o l y b d e n u m u p t a k e b u t is r e q u i r e d f o r synthesis of nitrogenase.

T h e m o l y b d e n u m u p t a k e a n d storage

factors

t h e n are n o t i n d u c i b l e b y m o l y b d e n u m . W h e n a c r u d e extract w a s f r a c t i o n a t e d o n a n i o n e x c h a n g e c o l u m n , most of the m o l y b d e n u m w a s associated w i t h a f r a c t i o n t h a t is n o t c o m p o n e n t I (20).

T h i s f r a c t i o n contains w h a t w e h a v e n a m e d the m o l y b -


406

BIOINORGANIC

CHEMISTRY

II

d e n u m - s t o r a g e c o m p o u n d , since m o l y b d e n u m c a n b e r e m o v e d f r o m this c o m p o u n d a n d t r a n s p o s e d t o c o m p o n e n t I . I n fact, cells g r o w n i n t h e presence of m o l y b d e n u m a n d N H

4

+

, w h e n transferred to m e d i u m l a c k i n g

b o t h , c a n synthesize a c t i v e nitrogenase f o r m a n y generations u s i n g t h e molybdenum i n the molybdenum-storage m e c h a n i s m b y w h i c h A. vinehndii

compound.

T h i s is a u s e f u l

stores m o l y b d e n u m w h e n excess is

a v a i l a b l e , b u t t h e n uses this excess w h e n i t finds itself i n a n e n v i r o n m e n t lacking molybdenum.

T u n g s t e n also c a n b e stored b y t h e m o l y b d e n u m -


storage p r o t e i n . W e are n o w s t u d y i n g m u t a n t strains t h a t seem t o l a c k the m o l y b d e n u m - s t o r a g e p r o t e i n . Table I. Effects of Chloramphenicol on Molybdenum Accumulation in Azotobacter vinelandii

CAM

Nitrogenase Specific Activity (nmoles acetylene reduced/min χ 10 cells)

+

0.59 0.00

7

Organism A. A.

vinelandii vinelandii

Mo (ng/lO 11

cells)

5.70 6.80

T h e r e a r e m a n y questions t h a t m u s t s t i l l b e a n s w e r e d . H o w a n d i n w h a t f o r m does m o l y b d e n u m enter t h e cell? H o w a n d i n w h a t f o r m does m o l y b d e n u m get into molybdenum-storage f o r m does m o l y b d e n u m

protein?

H o w and i n what

get t r a n s f e r r e d t o t h e m o l y b d e n u m

cofactor?

H o w a n d i n w h a t f o r m does t h e m o l y b d e n u m c o f a c t o r g e t i n t o n i t r o genase c o m p o n e n t I ? H o w a n d i n w h a t f o r m does m o l y b d e n u m r e g u l a t e the synthesis o f nitrogenase?

T h e s e questions r e q u i r e i n t e g r a t e d effort

b y chemists, e n z y m o l o g i s t s , geneticists, a n d b a c t e r i a l p h y s i o l o g i s t s .

Hope

fully, such work w i l l ultimately have an impact o n agriculture. Acknowledgments A p a r t o f this w o r k was s u p p o r t e d b y t h e C o l l e g e o f A g r i c u l t u r a l a n d L i f e Sciences, U n i v e r s i t y of W i s c o n s i n , M a d i s o n , b y N I H G r a n t G M 2 2 1 3 0 , a n d b y t h e C e l l u l a r a n d M o l e c u l a r B i o l o g y T r a i n i n g G r a n t G M 07215.

Literature Cited 1. Shah, V. K., Davis, L. C., Gordon, J. K., Orme-Johnson, W. H., Brill, W. J., Biochim. Biophys. Acta (1973) 292, 246-255. 2. Gordon, J. K., Brill, W. J.,Proc.Natl. Acad. Sci. USA (1972) 69, 35013503. 3. Bishop, P. E., Brill, W. J., Abstracts Annual Meeting American Society Microbiology, p. 163, 1976.



22.

PIENKOS E T A L .


407

4. St. John, R. T., Johnston, H. M., Seidman, C., Garfinkel, D., Gordon, J. K., Shah, V. K., Brill, W. J., J. Bacteriol. (1975) 121, 759-765. 5. Eady, R. R., Postgate, J. R., Nature (1974) 249, 805-810. 6. Ketchum, P. Α., Cambier, H. Y., Frazier, W. Α., Madansky, C. H., Nason, Α.,Proc.Natl. Acad. Sci. USA (1970) 66, 1016-1023. 7. Nason, Α., Lee, Κ. Y., Pan, S. S., Ketchum, P. Α., Lamberti, Α., De Vries, J.,Proc.Natl. Acad. Sci. USA (1972) 68, 3242-3246. 8. Kondorosi, Α., Barabas, I., Svab, Z., Orosz, L., Sik, T., Hotchkiss, R. D., Nature (1973) 246, 153-154. 9. Ketchum, P. Α., Sevilla, C. L., J. Bacteriol. (1973) 116, 600-609. 10. Lee, Κ. Y., Pan, S. S., Erickson, R., Nason, Α., J. Biol. Chem. (1974) 249, 3941-3952. 11. Ganelin, V. L., L'vov, N. P., Sergeev, N. S., Shaposhnikov, G. L., Kreto vich, V. L., Dokl. Akad. Nauk SSSR (1972) 206, 1236-1238. 12. McKenna, C., L'vov, N. P., Ganelin, V. L., Sergeev, N. S., Kretovich, V. L., Dokl. Akad. Nauk SSSR (1974) 217, 228-231. 13. Shah, V. K., Brill, W. J., Biochim. Biophys. Acta (1973) 305, 445-454. 14. Nagatani, H. H., Shah, V. K., Brill, W. J., J. Bacteriol (1974) 120, 697701. 15. Shah, V. K., Davis, L. C., Brill, W. J., Biochim. Biophys. Acta (1972) 256, 498-511. 16. Gordon, J. K., Garfinkel, D., Brill, W. J., Abstracts Annual Meeting Ameri can Society Microbiology, p. 175, 1975. 17. Evans, H. J., Purvis, E. R., Bear, F. E., Soil Sci. (1951) 71, 117-124. 18. Lobb, W. R., N. Z. Soil News (1953) 3, 9-16. 19. Brill, W. J., Steiner, A. L., Shah, V. K., J. Bacteriol. (1974) 118, 986-989. 20. Pienkos, P. T., Brill, W. J., Abstracts Annual Meeting American Society Microbiology, p. 164, 1976. RECEIVED July 26, 1976.


Uptake and Role of Molybdenum in Nitrogen-Fixing Bacteria

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