Hydrogen metabolism and nitrogen fixation in wild type and Nif− mutants of Rhodopseudomonas acidophila

BIOCHIMIE, 1978, 60, 261-265.

Hydrogen metabolism and nitrogen fixation in wild type and Nif- mutants of Rhodopseudomonas acidophila. Eike S I E F E R T a n d N o r b e r t PFENNIG.

Institut [Or Mikrobiologie der Gesellschaft fiir Strahlen und Umweltforschung mbH, Grisebachstr. 8, D 3#00 G6ttingen - Federal Republic of Germany.

R6sum6.

Summary.

Les auteurs monirent que la fixation d'azote, la r6duction d'ac6tyl6ne et la production d'hydroq~ne chez Rhodopseudomonas acidophila DSM 137 sont dans les rapports stoechiom6triques de 1:2,8:2,8. La plus forte vitesse d'oxydation de l'hydroq6ne est, chez Rhodopseudomonas acidophila DSM 137, environ 6 lois plus qrande clue la vitesse maximale de production de H2. Des mutants nii- ont 6t6 isol6s et test6s ; tous avaient perdu leur facult6 de r6duire C~2H2 et de d6qaqer H2. Chez deux mutants n i f l'activit6 hydroq6nase et la facult6 de pousser de faqon autotrophe sur H2 6taient aussi fortement diminu6es. Les r6vertants nif + ont non seulement retrouv6 la facult6 de r6duire C2H~ et de d6qaqer H2, mais anssi la pleine capacit6 de pousser de faqon autotrophe sur H2.

N2 fixation, C~_H2reduction and H2 production in Rhodopseudomonas acidophila DSM 137 wore shown to be stoichiometrically related in ratios of 1:2.8:2.8. The hiqhest possible H2 oxidation rate has been calculated to be about 6 fold hiqher in Rhodopseudomonas acidophila DSM 137 than the maximum rate of H2 production. N i l mutants were isolated and tested ; all of them had lost their ability to reduce C~H~ and to produce H2. In two nif mutants hydroqenase activity and the capacity for autotrophic qrowth with H2 were also sironqly diminished. Nif + revertants not only regained their ability for C2H2 reduction and H2 production but also their fuU capacity for autotrophic qrowth with H2.

Phot(ytrophic bacteria are able ,to utilize H e for autotrophic C02 assimilation. U n d e r n i t r o g e n starvation a n a e r o b i c a l l y in the light, on the other h a n d , they evolve H e from organic or i n o r g a n i c substrates. It appears likely that different enzymes are r e s p o n s i b l e for these opposite reactions [17]. Nitrogenase catalyses the Jr.reversible, ATP dep e n d e n t H e p r o d u c t i o n [2] w h i c h is not i n h i b i t e d by CO, w h e r e a s the c o n v e n t i o n a l h y d r o g e n a s e catalyses in vitro the reversible reaction H 2 ~ 2H ÷ + 2e-, w h i c h is i n h i b i t e d by GO [13]. In vivo, however, h y d r o g e n a s e of photo.trophic b a c t e r i a appears to catalys, e only t h e uptake r e a c t i o n of H e w h e r e a s H e p r o d u c t i o n is forced by n i t r o g e n a s e in the absence of its substrate N2. Hydrogenase and n i t r o g e n ase are regulated differently. The latter is repressed as well as i n h i b i t e d by N H ( , w h e r e a s h y d r o g e n a s e is i n d u c e d in the presence of H 2 regardless of NH4+ or organic substrates b e i n g present or not [13]. The h y d r o g e n a s e m e d i a t e d assi-

m i l a t i o n of (502 wi4h H e is i n h i b i t e d by o r g a n i c substrates [8]. This clear-cut different regulation p a t t e r n of nitrogenase and h y d r o g e n a s e is confused by the fact, that in p h o t o t r o p h i c bacteria derep r e s s i o n of n i t r o g e n a s e is a c c o m p a n i e d by a partial i n d u c t i o n of hydrogenase. It has been suggested by Dixon [3] that h y d r o g e n a s e has the function to reutilize the H 2, w h i c h is evolved as a byp r o d u c t of the nitrogenase r e a c t i o n u n d e r physiological conditions, since atmospheric Ne does not saturate the enzyme [4, 10]. The e x p e r i m e n t s reported i n this communi~a~ion ~vith the w i l d type s t r a i n and nif- m u t a n t s of Rhodopseudomonas acidophila confirm on the one h a n d the results of Wall et at., 1975 [171, that H e p r o d u c t i o n a n d H e utilization are catalyzed by different enzymes, on the other h a n d they demonstrate that these two enzymes are possibly linked genetically or by regulation.

E. Siefert and N. Pfenniq.

262 Materials

and

Methods.

ORGANISM AND GROVCTH CONDITIONS.

Rhodopseudomonas acidophila DSM 137 w a s g r o w n in a m e d i u m c o n t a i n i n g p e r l i t r e : 1 g KH2PO4 ; 0.5 g NH,C1 ; 0.5 g MgSO~ • 7 H~O ; 0.4 g NaCl ; 0.05 g CaCl;- • 2 H.20 ; 1 g s o d i u m L - l a c t a t e ; 5 lnl i r o n ( I I I ) - e i t r a t e s o l u t i o n (1 g/l) ; a n d 10 m l t r a c e e l e m e n t s o l u t i o n SL~ [15]. T h e p ~ w a s a d j u s t e d w i t h s u l f u r i c acid to 5.6. NH4C1 w a s o m i t t e d if N.z s e r v e d as sole n i t r o g e n s o u r c e . F o r g r o w t h e x p e r i m e n t s a 500 m l fiat b o t t l e (Meplatz) w a s filled w i t h 4~00, m l of t h e m e d i u m w h i c h w a s c o n t i n o u s l y g a s s e d w i t h 95 p e r c e n t N: a n d 5 p e r c e n t CO._,. T h e b o t t l e w a s k e p t in a n a l l - g l a s s w a t e r b a t h at a c o n s t a n t t e m p e r a t u r e of 30°C u s i n g a K r y o t h e r m o s t a t e . To o b t a i n m a x i m a l g r o w t h r a t e s at s a t u r a t i n g l i g h t i n t e n s i t y , c u l t u r e s w e r e i l l u m i n a t e d w i t h 1 0 0 - w a t t A t r a l u x t u n g s t e n l a m p s f r o m a dist a n c e of 5 c m (30,0~60-50,00# l a x ) . S a m p l e s w e r e t a k e n w i t h s t e r i l e 10 m l s y r i n g e s t h r o u g h a l o n g n e e d l e i n s e r t e d i n t o t h e r u b b e r s t o p p e r of t h e flask. A u t o t r o p h i c g r o w t h w i t h H.., w a s t e s t e d u s i n g 100 m l c o t t o n - p l u g g e d E r l e n m e y e r flasks p l a c e d in a n a n a e r o b i c j a r c o n t a i n i n g 95 p e r c e n t H~ a n d 5 p e r c e n t CO~. ISOLATION OF NIF- MUTANTS.

M u t a t i o n of t h e w i l d t y p e Rhodopseudomonas acidophilo DSM 137 w a s i n d u c e d b y t r e a t m e n t w i t h t h e m u t a g e n i c a g e n t N-methyl-N'-nitro-N-nitrosoguanidine a t a final c o n c e n t r a t i o n of 2 I~tg p e r m l s t a n d a r d m e dium during the exponential growth phase. After 3 h r s d u r i n g w h i c h t h e v i a b l e cell n u m b e r d e c r e a s e d f r o m 1.~'108 to 1.0"10'6 t h e m u t a g e n w a s r e m o v e d b y c e n t r i f u g a t i o n a n d w a s h i n g . T h e cells w e r e r e s u s p e n d e d i n t h e fivefold v o l u m e of f r e s h m e d i u m a n d i n c u b a t e d a n a e r o b i c a l l y in t h e l i g h t to p e r m i t t w o d o u b l i n g s of t h e cell t u r b i d i t y f o r p h e n o t y p i c e x p r e s sion. T h e cells of t h e w h o l e c u l t u r e w e r e t r a n s f e r r e d to a m e d i u m w i t h o u t N H d w h i c h w a s g a s s e d w i t h 95 p e r c e n t No a n d 5 p e r cent C.%. A f t e r t o t a l u t i l i z a t i o n of t h e NH~+ w h i c h w a s i n t r o d u c e d w i t h t h e i n o c u l u m , a m p i c i l l i n w a s a d d e d to a final c o n c e n t r a t i o n of 2,0 m g / l f o r s e l e c t i o n of n i f - m u t a n t s . A f t e r 4 h r s t h e c u l t u r e w a s c e n t r i f u g e d a n d w a s h e d free of a m p i c i l l i n . A p a r t o f t h e cells w a s t r a n s f e r r e d i n t o f r e s h m e d i u m a n d i n c u b a t e d a n a e r o b i c a l l y in t h e l i g h t f o r 6 d a y s . A n a p p r o p r i a t e d i l u t i o n of t h e s e cells w a s s p r e a d o n a g a r p l a t e s to get a b o u t 300 c o l o n i e s p e r p l a t e a f t e r i n c u b a t i o n in i l l u m i n a t e d tall g l a s j a r s u n d e r 95 p e r c e n t N._, a n d 5 p e r c e n t CO._,. T h e a g a r m e d i u m cont a i n e d 2.7 m g NH~C1/1 i. e. 0.5 p e r c e n t of t h e n o r m a l c o n c e n t r a t i o n . T h i s s m a l l a m o u n t of NH,CI a l l o w e d nif- m u t a n t s to f o r m s m a l l p i n p o i n t colonies, w h e r e a s c o l o n i e s of No fixing cells were m u c h l a r g e r . P i n p o i n t c o l o n i e s w e r e t r a n s f e r r e d to p l a t e s w i t h a n d w i t h o u t NH~+. M u t a n t s w h i c h f a i l e d to g r o w on p l a t e s w i t h o a t NH~+ w e r e p u r i f i e d f r o m r e v e r t a n t s b y s t r e a k i n g t h e m on a g a r p l a t e s w i t h l o w NH~+ c o n c e n t r a t i o n .

d e r m i c needle. T h e b o t t l e s were s h a k e n in a w a t e r b a t h at 30°C a n d 7 000 l u x i n c a n d e s c e n t l i g h t . T h e r e a c t i o n w a s s t a r t e d b y i n j e c t i o n of 10 p e r c e n t acet y l e n e . 0.2 m l s a m p l e s w e r e t a k e n in i n t e r v a l s of 10 r a i n a n d t h e e t h y l e n e f o r m e d a n a l y s e d in a P e r k i n Elmer, Fll gasehromatograph equipped with a flame i o n i s a t i o n d e t e c t o r . E t h y l e n e w a s s e p a r a t e d f r o m acet y l e n e u s i n g a c o l u m n of 2 m l e n g t h a n d 1 / 8 i n c h d i a m e t e r p a c k e d w i t h P o r a p a k R (100-120 m e s h ) . T h e o v e n t e m p e r a t u r e w a s 44°C, t h e flow r a t e of t h e c a r r i e r gas, N~ w a s 30 m l / m i n . MANOMI~TEIC METHODS.

Hydrogenase activity was measured manometrically at 30°C w i t h m e t h y l e n e b l u e a s e l e c t r o n a c c e p t o r . T h e W a r b u r g v e s s e l c o n t a i n e d cells s u s p e n d e d in 2.0 nil g r o w t h m e d i u m of w h i c h t h e p h o s p h a t e c o n c e n t r a t i o n w a s i n c r e a s e d t w o f o l d a n d NH,,C1 a n d l a c t a t e w e r e o m i t t e d . T h e c e n t e r well c o n t a i n e d 0.2 m l 20 p e r c e n t N a O H a n d a f o l d e d filter p a p e r . A f t e r g a s s i n g 15 m i n w i t h H2, t h e r e a c t i o n w a s s t a r t e d b y t i p p i n g 10 ~ m o l c s m e t h y l e n e b l u e d i s s o l v e d in 0.~ m l w a t e r f r o m t h e side arm into the main compartment. F o r m a n o m e t r i c m e a s u r e m e n t of H~ p r o d u c t i o n p h o t o t r o p h i c cells w e r e s u s p e n d e d at a final c o n c e n t r a t i o n of 8.9 m g p r o t e i n / m l in g r o w t h m e d i u m c o n t a i n i n g t h e d o u b l e c o n c e n t r a t i o n of p h o s p h a t e , no NH,C1 a n d no l a c t a t e . T h e a t m o s p h e r e w a s a r g o n . To s t a r t t h e r e a c t i o n l i g h t (ca. 6000, l u x ) w a s s w i t c h e d on.

NITROGEN

AND P R O T E I N DETERMINATION.

Total nitrogen was determined using a modified K j e l d a h l m e t h o d a c c o r d i n g to H u m p h r i e s , 1956 [9]P r o t e i n w a s d e t e r m i n e d b y t h e m e t h o d of L o w r y el al. [12].

Results

and

Stoichiometric relationship between N 2 [ixation, CzH 2 reduction and H 2 production rate. Nitrogenase c a t a l y s e s N 2 f i x a t i o n , G,H., r e d u c tion and H 2 production in stoichiometric ratios c l o s e to 1 : 3 : 3 [5]. T h e s e r e a c t i o n s a r e e x p e c t e d to be catalysed in similar ratios by whole cells of phototrophic bacteria provided H._, i s p r o d u c e d exclusively by nitrogenase. The rate of N 2 fixation o f R h o d o p s e u d o m o n a s acidophila w a s c a l c u l a t e d from a growth experiment (fig. 1) u s i n g t h e e q u a tion :

(1) ACETYLENI~ REDUCTION TEST.

1 inl s a m p l e s of a g r o w i n g or r e s t i n g c u l t u r e w e r e i n j e c t e d in 20 m l b o t t l e s closed w i t h s e r u m s t o p p e r s . h n m e d i a t e l y a f t e r w a r d s t h e b o t t l e s were e v a c u a t e d and flushed several times with argon through a hypo-

BIOCHIMIE, 1978, 60, n ° 3.

Discussion.

ds

1

- 4~ • (6) dl • x y (~ ---- s p e c i f i c g r o w t h r a t e , y = g d r y w e i g h t p e r ds

mole N 2 fixed,

dt " x unit of dry weight).

- - m o l e N,2 r e d u c e d / h

per

263

N, f i x a t i o n , C,H, r e d u c t i o n , H, p r o d u c t i o n : Rps. a c i d o p h i l a . y and ~ have been d e t e r m i n e d to be 271 nag dry w e i g h t / n m m l e N2 a n d 0.193 h -1 r e s p e c t i v e l y . Thus a specific N 2 fixation rate of 11.8 nlnoles N 2 / m i n p e r ing d r y w e i g h t w a s obtained. The e x p e r i m e n tally d e t e r m i n e d C..,H2 r e d u c t i o n rate almost parallels g r o w t h (fig. 1). On the average 33.0 nmoles CeH4/min p e r mg dry w e i g h t was formed. The range was 17.2 to 45.8 nmoles CeH4/min p e r mg d r y w e i g h t d e p e n d i n g on the g r o w t h phase. The ratio of 2.8 Cu.H~ f o r m e d to 1 N 2 r e d u c e d is fairly close to the t h e o r e t i c a l value of 3. The stoichio-

Rhodopseudomonas

acidophila w o u l d theoretically p r o d u c e at best 37 nmoles H ~ / m i n per mg dry weight. F o r R h o d o p s e n d o m o n a s capsulata strain Z-l, in c o m p a r i s o n , the l n a x i m u m rate of

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40

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Fro. 2. - - H~ production and (:~H~ reduction rate of Rhodopseudomonas acidophila. The reaction vessels contained each 8.9 mg cells (protein) suspended in 1 ml of growth medium of which the phosphate concentration was increased twofold, the pH was adjusted to 6.5 (pH' optimum of the CzH2 reduction), and NH4CI was omitted. Lactate concentration was 10 mmole per litre. (O) Photoproduction of H_~ under an atmosphere of argon at 6 000 lux, (O) C,,H_o-reduction at 6 000 lux.

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-

m e t r i c r e l a t i o n s h i p b e t w e e n C._,Hu r e d u c t i o n and H 2 f o r m a t i o n can be seen from figure 2 to be almost 1:1, as one w o u l d expect by t h e o r e t i c a l reasoning.

Comparison of m a x i m u m h y d r o g e n and hydrogen c o n s u m p t i o n rate.

production

On a c c o u n t of the s t o i c h i o m e t r i c r e l a t i o n s h i p b e t w e e n h y d r o g e n p r o d u c t i o n , C2H2 r e d u c t i o n and N 2 fixation in R h o d o p s e u d o m o n a s acidophila, one is able to calculate a m a x i m u m specific h y d r o g e n p r o d u c t i o n rate f r o m m a x i m u m n i t r o g e n ase activity m e a s u r e d w i t h the acetylene r e d u c t i o n assay.

BIOCHIMIE, 1978, 60, n ° 3.

H 2 p r o d u c t i o n has been r e p o r t e d to be 130 ~1 H e / h p e r mg d r y w e i g h t at 35°C [7] w h i c h is equivalent to 8.5.7 nmoles H 2 / m i n p e r mg d r y weight.

0.5 z

i

FIG. 1. Nitrogenase activity and growth of Rhodopseudomonas acidophila on N~ as sole nitrogen source. Growth conditions are described in Materials and Methods section. (O) Acetylene reduction rate/ml, (@) turbidity. -

°

20 30 Time (min)

In contrast to H 2 p r o d u c t i o n , H 2 c o n s u l n p t i o n is c o n s i d e r e d to be c o r r e l a t e d to CO 2 assimilation r a t h e r than to N.~ fixation. If one assumes that about t w o H 2 are o x i d i z e d for one CO 2 assimilated by a process w h i c h a p p r o x i m a t e s to (2) 21 H._, "4- 2 NH:~ -4- 10 (;02 ~ 2 (C:,HsO2N) + 16 H20, one is able to calculate the highest specific rate of h y d r o g e n o x i d a t i o n from m a x i m u m g r o w t h rate using f o r m u l a I l l . The m a x i m u m specific g r o w t h rate (,.~..... ) of R h o d o p s e u d o m o n a s acidophila, g r o w i n g a u t o t r o p h i c a l l y at the e x p e n s e of H 2, has been d e t e r m i n e d to be 0.155 h-~ (GSbel, p e r s o n a l c o m m u n i c a t i o n ) . A y i e l d of 10.8 g d r y w e i g h t / mole H 2 is o b t a i n e d f r o m equation (2). These t w o values c o r r e s p o n d to a m a x i m u m rate of 239 nmoles H 2 o x i d i z e d / m i n p e r nag d r y weight. The c a p a c i t y for H 2 o x i d a t i o n u n d e r a u t o t r o p h i c g r o w t h c o n d i t i o n s appears to be 3-6 fold h i g h e r than the m a x i m u m c a p a c i t y for H 2 p r o d u c t i o n . This is a f u r t h e r i n d i c a t i o n tba~ H~ o x i d a t i o n is closer c o r r e l a t e d to CO,, assimilation than to N:~ fixation.

264

E. S i e f e r t a n d N. P f e n n i g .

The effect of nif- mutations on H 2 metabolism.

s e e n f r o m f i g u r e 3 five s e l e c t e d m u t a n t s w i t h a low revertation rate have been tested by growth e x p e r i m e n t s f o r n i t r o g e n f i x a t i o n . Of t h e s e H32

T h e r e s u l t s d e s c r i b e d a r e i,n a g r e e m e n t w i t h t h e p o i n t of v i e w t h a t H.~ p r o d u c t i o n is c a t a l y s e d b y

2.0 _.e

H32

1.0'

_...%.z • . m =

t"1..---"-'"" - . . . .

_A . . . .

118

..A

~Q5 oKll

0 0.2 >, 0'11 -(3

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1"5 2"0 Time (h)

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3"0

35

Fie,. 3. - - Growth of nil- mutants on limited amounts of NH~+. G r o w t h conditions as in figure 1, except t h a t NH4C1 was present at a c o n c e n t r a t i o n of 2.5 mmoles/1 and lactate at a c o n c e n t r a t i o n of 15 mmoles/1. Solid line : NH, ~ is present in the medium, dotted line : NH~+ is not detectable in the m e d i u m using nesslers reagent. Nitrogen analyses revealed t h a t H~2 and E~ were leaky in respect to N2-fixation, w h e r e a s the cultures of the other m u t a n t s did not increase in total n i t r o g e n a f t e r e x h a u s t i o n of NHJ.

n i t r o g e n ase, w h e r e a s a d i f f e r e n t e n z y m e is resp o n s i b l . e f o r Hu u p t a k e . A m u t a n t d e f e c t i v e i n nit r o g e n f i x a t i o n , t h e r e f o r e , w a s e x p e c t e d to c o n s u m e H_o b u t n o t t o b e a b l e to p r o d u c e H._,. As c a n b e

a n d E 6 s h o w e d to b e l e a k y w i t h r e g a r d to N 2 fixat i o n , C~zHz r e d u c t i o n a n d H 2 p r o d u c t i o n , w h e r e a s t h e o t h e r t h r e e m u t a n t s s h o w e d n e i t h e r N 2 fixat i o n n o r C,2H 2 r e d u c t i o n . H 2 p r o d u c t i o n c o u l d also

TABLE, I.

A u t o t r o p h i c g r o w l h and hydrogcnase a c t i v i t y ot two nil- lnulanls in comparison to the w i l d t y p e strain of R h o d o p s e u d o m o n a s a c i d o p h i l a . H2 uptake of resting cells in the presence of methyleneblue (~l/min" mg protein)

Strains

Wildtype Nif- m u t a n t s 11~ K11 Nil + revertant of Its

Autotrophic growth with H~ and CO.~

-}---~-[~-~-~--~---~-

after growth on limited amount of NH4+ (2.5 mmoles/l)

alter an additional incubation period of 5 hrs under a H2 atmosphere to induce hydrogenase

0 . 5 8 (*)

n.d.

O. 06 0.06

O. 2 0.15

n.d.

n.d.

+ + + good g r o w t h ; 4- poor growth ; n.d., not d e t e r m i n e d , * P h o t o p r o d u c t i o n of h y d r o g e n was observed i n t h i s culture a f t e r u t i l i z a t i o n of NH4÷.

BIOCHIMIE, 1978, 60, n ° 3.

N~ f i x a t i o n , C~H~ r e d u c t i o n , H2 p r o d u c t i o n : R p s . a c i d o p h i l a . not be observed, although the mutants K l l and Ils w e r e s h o w n to h a v e an active h y d r o g e n a s e (table I). Nif + r e v e r t a n t s r e g a i n e d all t h r e e catalytic activities simultaneously. S i m i l a r results h a v e been o b t a i n e d w i t h nif- mutants of Rhodopseudomonas capsulala [17]. Different f r o m the latter report w e found that the n m t a t i o n not only affected N2 fixation but also H~ oxidation, as can be seen f r o m table I. In contrast to our e x p e c t a t i o n that a nil- m u t a n t could c o n s u m e H2, h y d r o g e n a s e activities of t w o mutants w e r e about ~enfold less t h a n that of the w i l d type g r o w n u n d e r s i m i l a r conditions. T h e p r e s e n c e of H, i n d u c e d the specific hydrogenase a c t i v i t y s o m e w h a t , but it did not r e a c h t h e level of the w i l d t y p e . No~ only h y d r o g e n a s e a c t i v i t y of the m u t a n t but also its c a p a c i t y f o r a u t o t r o p h i c g r o w t h ~vas greatly d i m i n i s h e d . A n i p r e v e r t a n t of K H r e g a i n e d its ability to gro~v ~vith H._, a u t o l r o p h i c a l l y . The gene~ic defect is likely to be a single p o i n t mutation judging from spontaneous revertations. One, t h e r e f o r e , m a y c o n c l u d e that a genetic or regul, atory linkage exists b e t w e e n n i t r o g e n a s e and h y d r o g e n a s e in Rhodopseudomonas acidophila. It w o u l d be of interest, therefore, to e x a m i n e the effect of a m u t a t i o n in H e m e t a b o l i s m on N 2 fixation. It has been p r o p o s e d that the p h y s i o l o g i c a l role of h y d r o g e n a s e in N 2 fixing o r g a n i s m s is to r e c y c l e H 2 [3], w h i c h is e v o l v e d by the nitrogenase, since N,, at a t m o s p h e r i c p r e s s u r e does not saturate the enzyme [4, 10]. This c o n c e p t has been strongly s u p p o r t e d by the findings that H 2 is prod u c e d both in a e r o b i c o r g a n i s m s [16] and in blue g r e e n algae [1] w h e n h y d r o g e n a s e in these organisms is i n h i b i t e d by acetylene and CO. The role of h y d r o g e n a s e to r e c y c l e H e m a y apply also for p h o t o t r o p h i c b a c t e r i a and m a y e x p l a i n the phen o m e n o n that n i t r o g e n a s e and h y d r o g e n a s e activity in p h o t o t r o p h i c b a c t e r i a i n c r e a s e simultaneously. Different to a e r o b i c b a c t e r i a and cyanobacteria, h y d r o g e n a s e in p h o t o t r o p h i c bacteria, w h i c h g r o w a n a e r o b i c a l l y in the light, c a n n o t

BIOCHIMIE, 1978, 60, n ° 3.

265

t r a n s f e r electrons to oxygen. Thus, two f u n c t i o n s of h y d r o g e n a s e do not a p p l y for p h o t o t r o p h i c bacteria : 1. to c o n s e r v e e n e r g y in a Knallgas r e a c t i o n or 2. to act as an o x y g e n s c a v e n g e r to p r e v e n t damage of nitrogenase. A p p a r e n t l y , r e c y c l i n g of H e in p h o t o t r o p h i c b a c t e r i a has e x c l u s i v e l y the function to retain r e d u c i n g e q u i v a l e n t s for N, or CO, r e d u c t i o n . Although r e d u c t i o n of f e r r e d o x i n or NAD by H e in p h o t o t r o p h i c b a c t e r i a is e n e r g y dep e n d e n t [11], the c a p a c i t y of r e c y c l i n g H2 must be of selective a d v a n t a g e for these bacteria, since most Ne-fixing species so far k n o w n possess a hydrogenase.

REFERENCES. 1. Bathe, H., Tennigkeit, J. • Eisbrenner, G. (1977) Arch. Microbial., 114, 43-49. 2. Burns, R. C. ~ Bulen, W. A. (1966) Arch. Biochem. Biophys., 113, 461-463. 3. Dixon, R. O. D. (1972) Arch. Microbial., 85, 19'3-201. 4. Hadfield, K. L. ~ Bulen, W. A. (1969) Biochemistry, 8, 5103-5108. 5. Hardy, R. W. F., Burns. R. C. ~ Holstein, R. D.

(19'73) Soil Biol. Biochem., 5, 47-88. 6. Herbert, D., Elsworth, R. -~ Telling, R. C. (1956) J. Gen. Microbial., 14, 601-622. 7. Hilhner, P. ~ Gest, H. (1977) J. Bacterial., 129, 724-731. 8. Hillmer, P. ~ Gest, H. (1977) J. Bacterial., 129, 732-739. 9. Hnmphries, E. C. (195.6) in <> (Neilands, J. B. ed.) pp. 2~1-282, Academic Press, New York and London. 15. Pfennig, N. (1974) Arch. Microbial., 100, 197-206. 16. Smith, L. A., Hill, S. ,~ Yates, M. G. (1976) Nature, 262, 209-210. 17. Wall, J. D., Weaver, P. F. • Gest, H. (1975) Nature, 258, 630-631.

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