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Characterization and kinetics of the biosynthesis of some nitrogen fixation (nif) gene products in Klebsiella pneumoniae
BIOCHIMIE, 1980, 62, 267-275.
Characterization and kinetics of the biosynthesis of some nitrogen fixation (nif) gene products in Klebsiella pneumoniae. Jean HOUMARD <>, Didier BOGUSZ, R6gis BIGAU~LT and Claudine ELMERICH. (21-12-1979).
R~sum~. Chez K l e b s i e l l a pneumoniae, une 6tude comparative des prot6ines synth6tis6es soft p a r une souche Nil ~, soft p a r des mutants nff::Mu, soft encore par des mutants portant une d616tion de q~nes nff a 6t6 r6alis6e p a r la technique des gels & deux dimensions. La comparaison des autoradiocjrammes obtenus avec les extraits des cellules incub&es 10 minutes en pr6sence d'acides amin6s 14C a permis de rep6rer six produits sp6cifiques des g~nes n i l En plus des produits des q~nes de structure du complexe nitrog6nase (nitK, nitD, nifH) et du produit de nil! pr6alablement caract6ris6s, un polypeptide de 10.000 daltons et de pI environ 4,5 a 6t6 identiff6 comme produit du g~ne nffF, et un polypeptide de 22.000 daltons et de pI 5 comme prc~ duit du q~ne niiU. La biosynth~se de ces deux derniers produits est sous le contr&le du q~ne de r6gulation n i t A , tout comme cela avait d6j& 6t6 montr6 pour les autres produits nil. Les r6sultats de cette ~tude confirment les donn6es pr6alahlement obtenues par l'analyse g6n6tique concernant l'organisation en unit6s de transcription des q~nes nit de K l e b s i e l l a pneumoniae. Une cin6tique de la biosynth~se des six produits nil identifi6s et de rapparition de l'activJt6 nitrog6nase, lors de la d6r6pression des g~nes n i l a 6t6 r6alis6e. Les six produits ne sont d6celables qu'apr~s 45 minutes et leur apparition est simultan6e, ce qui sugg~re une transcript/on coordonn6e des diff6rentes unit6s. Cependant, l'activit6 nitrog6nase n'est d6celable, rant in v i v o qu'in vitro, que 30 minutes plus tard. Une 6tude cin6tique analogue a 6t~ r6alis6e avec la souche Nif÷, cultiv6e en conditions de
0 To whom all correspondence should be addressed.
Unitd de Physiologie Cellulaire, D~partement de B i o c h i m i e el Gbn~tique Moldculaire, l n s t i t u t Pasteur, 28 rue du Docteur Roux, F-7579_~ Paris CEdex 15.
fixation, & laquelle des ions NH~ ont 6t6 aiout6s. Dans ces conditions, on observe un arr~t de la biosynth~se des diff6rents produits nil. Cependant, cet arr~t n'est pas synchrone pour les six produits car la synth~se du produit de nffU n'est plus d6celable 5 minutes apr~s l'addition d'ions NH., celle de n/if, K, D et ] ne l'est plus apr~s 30 minutes tandis qu'on observe une synth~se r6siduelle du polypeptide nitH apr~s 60 minutes. Lors de la r6pression par les ions NH4, il semble donc que la transcription des g~nes nil soft bloqu6e et que seuls les RNA messagers pr6existants continuent d'6tre traduffs. Mots-cl~s : Fixation de l'azote ; Klebsiella pneumon/ae ; produits des g~nes nil.
Summary. Analysis of 1~C pulse-labelled proteins, synthesized b y a Nil+ K l e b s i e l l a pneumon/ae strain and b y a number of qenetically m a p p e d nif::Mu and nil deletion mutants, was perform e d b y two-dimensional gel electrophoresis. By comparison of the autoradioqrams, s.~x nff-specific polypeptides were identified. In addition to the previously characterized nifK, nffD, nifH a n d nit] products, the product of nffF was identified as a polypeptide of 10,000 daltons a n d pI about 4.5 and the product of nifU as a polypeptide of 22,000 daltons and pI 5. Moreover, the biosynthesis of nifF and n i f U polypeptides was shown to be prevented in mutants affectinq the regulatory qene nitA, which is known to control the biosynthesis of the other nff genes products so far identified. In all cases, the biochemical phenotypes of the different polar mutants were in good agreement with those expected from
M. H o u m a r d
268
the transcriptional o r g a n i z a t i o n of the nff cluster p r e v i o u s l y established by genetic a n a l y s i s . Kinetic studies of both n i t r o g e n a s e activity a n d of the b i o s y n t h e s i s of the six nil-specific p o l y p e p t i d e s w e r e performed with the Nil + strain, i n c u b a t e d either under conditions of derepression or under conditions of repression b y NH4 + ions. Upon derepression, the b i o s y n t h e s i s of the six nit polypeptides, w h i c h b e l o n g to four different transcriptional units, s e e m s to b e coordinated since they a p p e a r s i m u l t a n e o u s l y after a lag of 45 minutes. Under those conditions, both in v i v o a n d in vitro n i t r o g e n a s e activities
Introduction. Genetic studies of n i t r o g e n fixation i n KlebsielIa p n e u m o n i a e led to the definition of 14 n i l cistrons organized i n seven t r a n s c r i p t i o n a l u n i t s [1-5]. Biochemical c h a r a c t e r i z a t i o n s of some n i l m u t a t i o n s have been r e p o r t e d [I, 2, 6, 7]. However, relatively few of the n i l gene p r o d u c t s and f u n c t i o n s have so far been identified u n a m b i g u o u s l y . The nitrogenase complex of Klebsiella p n e u m o n i a e consists of two p r o t e i n s Kp I a n d Kp~ [8] w h i c h , a c c o r d i n g to Hageman and Burris [9], are t e r m e d respectively nitrogenase (iKpl) a n d n i t r o g e n a s e reductase (Kp2). The nitrogenase is a t e t r a m e r m a d e up of two n o n - i d e n t i c a l s u b u n i t s %[~.) [10] ; the nitrogenase reductase is a d i m e r nmde up of i d e n t i c a l p r o t o m e r s [8]. The nifH, nifD a n d n i f K genes have been identified as the genes c o d i n g for KP2, Kpl(t a n d iKpl~ s u b u n i t s [1, 2, 6]. NifA was s h o w n to be involved in r e g u l a t i o n [1, 2, 6, 11], n i f B [7] a n d tentatively n i f N and n i l e [6] in the molybd e n u m - i r o n cofactor (FeMoco) synthesis, nifM in
a n d coll.
w e r e d e t e c t a b l e o n l y 30 m i n u t e s later. U p o n addition of NH4 + ions, the b i o s y n t h e s i s of the six nit p o l y p e p t i d e s w a s rapidly abolished. H o w e ver, the kinetics of residual biosynthesis, probab l y due to the transcription of preexisting m R N A s , w a s not similar for the six nil products. The nHU product w a s n o longer detectable after 5 minutes, the n / i f , K, D a n d I products w e r e not d e t e c t a b l e after 30 minutes, w h e r e a s s o m e nffH product w a s still slightly detectable after 60 minutes. Key.words:Nitrogen fixation ; Klebsiella pneumoniae ; rib"gene products.
but not in vitro [6]. I n a d d i t i o n to the p r o d u c t s of nifH, D, K a n d J, the p r o d u c t s of nifN, nilE, n i f S a n d n i f F have been identified as p o l y p e p t i d e s of 50,000, 46,000, 18,000 a n d 17,000 daltons molecular weight [6]. All these p r o d u c t s are p r e s e n t under a n a e r o b i c c o n d i t i o n s , only w h e n b a c t e r i a are g r o w n in the absence of NH4 + ions. We r e p o r t here the identification of two n e w products, w h i c h w e assigned to ni[F a n d ni[U genes, and the k i n e t i c s of derepression a n d repression by a m m o n i u m ions of the b i o s y n t h e s i s of the nifF, nifU, nifK, nifD, n i f t I and n i f J products.
Materials and Methods.
Bacteria and plasmids. The K. pneumoniae strains are listed in table I. The genotype of the different Nif- plasmids [2, 4] appears in table II. As a uniform nomenclature has been
"[[-'ABLEI. Klebsiella p n e u m o n i a e strains. Strain UNF107 KP52 UNFI31 UNF156 UNFI69 UNFI77
Geaotype or phenotype
A A A A A A
(gnd-his-niI) 107 rpsL (gnd-his-nil) 4648 rpsL (his-ni/l 2473 hsdRl rpsL ni1-2479 hsdRl rpsLl ni1-2482 hsdRl (his-r~i]) 2483 h~dRl
Reference Dixon el al. 1977 [11] Stretcher el al. 1972 [14] Merrick el al. 1978 [1] Merrick el al. 1978 ]1] Merrick et al. 1978 [1] Merrick el al. 1979 [4]
Genetic symbols are those of Bachman et al. [12].
the processing of KI)2 [6]. The p r o d u c t of ni[J has been identified as a p o l y p e p t i d e of 120,0'00 daltons molecular weight El, 2, 6] that would be r e q u i r e d for a c e t y l e n e - r e d u c i n g activity in vivo,
BIOCH1MIE, 1980, 62, n" 4.
recently set up, plasmids previously termed pLS1, pLS17, pNC172 for example [2] are now termed pPC1, pPC17 and pPC172, with allele numbers 8001, 8017 and 8177. All the plasmids are pCE1 derivatives. The pCEl plasmid derives from pRD1 (amp* tel + kan ~ find'
K. pneumoniae
nif
his ~ n i p shi ÷tra +incP) c o n s t r u c t e d b y D i x o n et at, [13JU n l i k e pRD1, pCEI does n o t s u p p r e s s t h e Mu s e n s i t i vity in E. colt s t r a i n s [2]. I n t h i s s t u d y , all t h e p l a s raids were c a r r i e d b y UNF107 K. pneumoniae nil total deletion strain,
gene products.
269
cell p e l l e t w a s r e s u s p e n d e d in 200 ~tl o f t h e <) d e s c r i b e d b y O ' F a r r e l l [161. T r i t o n X-100 w a s u s e d i n s t e a d of N o n i d e t P40. S a m p l e s were s u b j e c t e d to five cycles o f f r e e z i n g in l i q u i d n i t r o g e n a n d t h a wing. A f t e r c e n t r i f u g a t i o n at 4,500 g f o r 10 m i n u t e s ,
TABLE II.
Biosynthesis of nif-speci[ic polypeptides by n i l mutants. Gene products Genotype
Plasniids or strains {allele} Spott
Spot2
Spot3
Spot4
Spot5
Spot6
ni[B : : Mu
+-~~--~-
-+--+-~--~-
+-{+-~-
-~-~-~-~-
-+-+ -}-7t-
+-~-+--j-
niIA : : hill : : nijF : : nitM: :
. . ~---~-{--
. . -{---~-~---[-
. . -~---{-~-+
. . . . -[-7tq---~-
--J-+ -~--~-
--~--~-
++
++
++
-~-+
+-[+-~-
wild t y p e Mu Mu Mu Mu
ni]S : : Mu
++
ni]U : : Mu niJN : : Mu
. .
+
W
+-+~-~-
-{Jr--~
W +-~-
+-~-~-+
nile : : Mu nilK : : Mu
-{--~-~-
-~--
+-+--
--{--~- --~--+-~--~-~---~-
nilD : : Mu ni/H : : Mu niIJ : : Mu nilA (M-J) niIA (B-V) ni[A (B-M) ni[A (N-H) nilA (K.J) nilA (-B) n q A (B-J)
-~-~---+-. . . + . +% .
--+--J. . . . . . -. . -{-% . .
--~--~----~--j--J-+ . . . . . . . . --. -]--% -{---% . . .
+-{--~-~-
--J--+--{-+ --{--+-
-{---{Jr--}--J---{-[--~-
--J-+ --J---J%--{-
-J- --~+-{-]-+
pCEI pPC501
(niI-850I) pPC80 (ni]-8080) (ni[-8172) pPC141 (ni[-8141) p P C l (nil-8001) p P C l l 2 (ni[-8112) pPC503 (ni]-8503) pPC7 (ni[-8007) pPC52 (ni[-8052) p P C I 0 0 (ni[-8100) (ni1-8504) pPC31 (nip8031) pPC41 (nit-8041) (ni[-8557) pPC71 (nip8071) pPC560 (ni[-8560) pPC92 (nip8092) pPC569 (nip8569) (nil-8570) pPC40 (ni1-8040) pPC505 (nip8505) pPC16 (ni[-8016) pPC21 (ni/-8021) (nipS076) pPC47 (ni[-8047) p P C l l 7 (ni[-8117) p P C l l (ni]-8011) pPC27 (ni/-8027) pPC15 (nip8015) pPC45 (ni1-8045) p P C l l l . 1 (nip8760) KP52 (nit-d648) UNF131 (ni/-2473) U N F 1 5 6 (nil-2479) U N F 1 6 9 (ni[-2~[82) U N F 1 7 7 (ni[-2483) U N F 1 0 7 (nip107)
R e l a t i v e i n t e n s i t i e s w e r e e s t i m a t e d b y v i s u a l c o m p a r i s o n a n d n o t e d q- or + + c o r r e s p o n d i n g s p o t w a s n o t d e t e c t e d ; w i n d i c a t e s t h a t t h e s p o t w a s w e a k l y visible.
Culture media. N i t r o g e n f r e e (NFM) a n d L u r i a B r o t h (LB) m e d i a h a v e been p r e v i o u s l y d e s c r i b e d [2, 15].
Growth conditions for radioactive labelling of proteins. T h e f o l l o w i n g p r o c e d u r e h a s b e e n u s e d for t h e d e r e p r e s s i o n o f t h e n i t r o g e n a s e c o m p l e x . S t r a i n s were g r o w n o v e r n i g h t , u n d e r N2, in 10 m l of NFM cont a i n i n g 19 m M NI-LC1. A f t e r c e n t r i f u g a t i o n u n d e r argon, b a c t e r i a w e r e r e s u s p e n d e d , at 109 cells p e r ml in a n a e r o b i c NFM s u p p l e m e n t e d w i t h 100 t t g / m l a s p a r t a t e a n d w i t h o r w i t h o u t 19 m M NH4C1. Five m i l l i l i t e r s of culture were then incubated with shaking, under argon, at 30°C. Samples of 3 ml were withdrawn, usually after 3 h o u r s of i n c u b a t i o n , a n d a d d e d a n a e r o b i c a l l y to a c e n t r i f u g e t u b e w h i c h c o n t a i n e d 6 ~tCi of a a4C p r o tein h y d r o ] y s a t e s o l u t i o n (CEA, F r a n c e , ref. C4B). Ten m i n u t e s later, 4 m l o f NFM c o n t a i n i n g 2 m g / m l c a s a m i n o - a e i d s w e r e a d d e d a n d t h e cells w e r e c e n t r i f u g e d . Cells w e r e w a s h e d w i t h 5 m l o f t h e NFM e a s a m i n o acids a n d c e n t r i f u g e d f o r 10 m i n u t e s a t 6,500 g. E a c h
BIOCHIMIE, 1980, 62, n ° 4.
; --
pPC172
pPC504 pPC557 pPC570 pPC76
indicates that the
s u p e r n a t a n t s w e r e w i t h d r a w n , f r o z e n in l i q u i d n i t r o gen a n d k e p t at - - 2 0 ° C .
Assays for nitrogen fixation. N i t r o g e n a s e a c t i v i t y of w h o l e cells w a s e s t i m a t e d b y the a c e t y l e n e r e d u c t i o n test [17]. In vitro a c e t y l e n e r e d u c i n g a c t i v i t i e s w e r e m e a s u r e d as d e s c r i b e d b y E a d y et aL [8]. E t h y l e n e p r o d u c e d w a s m e a s u r e d w i t h a 204 P y e U n i c a m gas c h r o m a t o g r a p h , b y i n j e c t i n g 0.25 ml gas s a m p l e into a 40 X 0.2 c m c o l u m n filled w i t h P o r a p a k T a n d e q u i l i b r a t e d at 55°C.
Two-dzTnensional polyacrylamide gel electrophoresis. T h e i s o e l e c t r i c f o c u s i n g w a s p e r f o r m e d a c c o r d i n g to I b o r r a a n d B u h l e r [18] w i t h a LKB M u l t i p h o r a p p a r a tus. T h e gel c o n t a i n e d 5 p e r cent a e r y l a m i d e , 8.3 M u r e a a n d 2 p e r c e n t a m p h o l i n e s p H 3.5-10 (LKB). A f t e r 1 h o u r of p r e f o c u s i n g (20 mA, 1,000 v o l t s a n d 200 w a t t s w e r e set u p as m a x i m a on t h e LKB2103 p o w e r s u p p l y ) , a b o u t 0.1 ~tCi o f l a b e l l e d p r o t e i n s w e r e l o a d e d o n t o t h e gel a n d f o c u s i n g w a s a l l o w e d to r u n f o r 2 h o u r s at 12"C. Gel s t r i p s of a b o u t 1 eva w i d t h
270
M. H o u m a r d
a n d coll.
were then cut and equilibrated in SDS Ortee sample buffer for 15 m i n u t e s at 30°C. Samples were then frozen in a dry ice-acetone b a t h and kept at --20°C.
d r a w n and assayed f o r nitrogenase activity. Protein concentration was d e t e r m i n e d with Coomassie blue G250, using bovine s e r u m a l b u m i n as a s t a n d a r d [21].
The SDS gel electrophoresis, in the second dimension, was p e r f o r m e d as described by L a e m m l i [19]. Either 10 or 13 per cent a e r y l a m i d e r u n n i n g gels (about 10 em height) were used. The height of the stacking gel (4.5 per cent aerylamide) was 1 em. The focusing strip was set up on the top of the stacking gel and layered by a 2 per cent agarose solution (made up in 25 mM Tris-glyeine buffer pH 8.6, containing 0.1 per cent SDS).
Repression by ammonium ions. K. pneumoniae UNF107(pCE1) was grown in a 1.5
Molecular weights were d e t e r m i n e d using bovine serum a l b u m i n (67,000), egg a l b u m i n (45,000), a-ehym o t r y p s i n o g e n A (25,700), soybean t r y p s i n i n h i b i t o r (21,500), ribonuelease (14,700) and h o r s e h e a r t eytochrome e (13,700) as s t a n d a r d s in the second d i m e n sion. For pI d e t e r m i n a t i o n s , focusing strips were sliced and the profile of the g r a d i e n t was established by measuring the pH of each slice. In addition, the nifH, D, K and J products, of w h i c h m o l e c u l a r weights and pls have been p r e v i o u s l y reported [1, 2, 61, were used as i n t e r n a l controls. Scintillating a u t o r a d i o g r a p h y was p e r f o r m e d according to Bonner a n d Laskey [20]. A f t e r w a t e r precip i t a t i o n of 2,5-diphenyloxazole (PPO) and washings, the gel was soaked in a glycerol : acetic acid : w a t e r (1:10:89 v/v) solution f o r 1 h o u r at r o o m t e m p e r a t u r e and dried between two cellophane sheets, u n d e r a hood. W h e n e v e r necessary, p r o t e i n staining was achieved using Coomassie b r i l l i a n t blue R250.
liter f e r m e n t o r , u n d e r conditions of nitrogen fixation. W h e n cell c o n c e n t r a t i o n of about 109 cells per ml was reached, a m m o n i u m ions (19 mM final concentration) were added. At different times, 10 ml samples were w i t h d r a w n f o r in vivo assay of nitrogenase activity and 3 m l samples for radioactive pulse labelling.
Results.
I d e n t i f i c a t i o n of n i f gene products. The methodology was based on the comparison of a u t o r a d i o g r a m s o f t w o - d i m e n s i o n a l g e l s o f pulse-labelled proteins synthesized by the wild type strain and by nit mutants incubated under c o n d i t i o n s of d e r e p r e s s i o n or r e p r e s s i o n by a m m o n i u m ions of n i t r o g e n fixation.
A f e r m e n t o r containing 1.5 liter of NFM supplemented w i t h 19 mM NH+C1 was inoculated w i t h 7.5 ml of a LB grown UNFI07 (pCEI) exponential culture. Initial absorbanee was 0.02 at 570 nm. Cells were grown overnight at 23°C u n d e r N2. After e e n t r i f u g a t i o n u n d e r argon, bacteria were r e s u s p e n d e d at 109 cells per ml in a f e r m e n t o r containing 1.5 liter of anaerobic NFM s u p p l e m e n t e d w i t h 100 ~,g/ml aspartate. Cells were incubated u n d e r argon at 30°C. Aliquots were w i t h d r a w n at given t i m e s : 3 ml samples for radioactive labelling (as described above), 10 ml samples for in vivo assays of nitrogenase activity and samples of about 150 ml for in vitro assays of nitrogenase activity. The latter samples were centrifuged u n d e r argon for 10 minutes at 6,500 g. The cell pellets were frozen in liquid nitrogen and stored a t - - 2 0 ° C .
As s h o w n i n f i g u r e 1, w i t h t h e w i l d t y p e UNF107(pCE1), six spots, present when bacteria w e r e i n c u b a t e d u n d e r c o n d i t i o n s of n i t r o g e n fixation, were missing when bacteria were grown in t h e p r e s e n c e o f NH4 + i o n s . F i v e s p o t s c a n b e s e e n o n b o t h 10 p e r c e n t a n d 13 p e r c e n t a c r y l a m i d e gel a u t o r a d i o g r a m s , s p o t s 1 to 5 i n f i g u r e l e a n d s p o t s 2 t o 6 i n f i g u r e l d . N o n e of t h e s i x p o l y p e p t i d e s , c a n b e d e t e c t e d w i t h UNF107, a n i t l o t a l d,eletion, i n c u b a t e d e i t h e r w i t h ( d a t a n o t s h o w n ) o r w i t h o u t a m m o n i a (fig. l a a n d b). It c a n t h u s b e a s s u m e d t h a t t h e s i x s p o t s c o r r e s p o n d to s h o w n o r w i t h o u t a m m o n i a (fig. l a a n d b). It t h u s c a n h e a s s u m e d t h a t t h e s i x s p o t s c o r r e s p o n d to n i / - s p e c i f i c p o l y p e p t i d e s . C o e l e c t r o p h o r e s i s of pulse-labelled UNF107(pCE1) extracts with pure K p 1 a n d .Kp2 p r o t e i n s s h o w e d t h a t s p o t s 2 a n d 3 c o r r e s p o n d to Kt) 1 a a n d ~ s u b u n i t s a n d t h a t s p o t 4 c o r r e s p o n d s to ~Kp2 s u b u n i t .
Crude extracts were obtained by d i s r u p t i n g the cells w i t h glass beads in a Mickle a p p a r a t u s (Gomshall, Surrey). Cell pellets were r e s u s p e n d e d in 4 ml of 100 mM Tris-HC1 pH 8.7 buffer, containing 1 mM sodium dithionite and 0.1 g/1 d i t h i o t h r e i t o l . After addition of 4 ml glass beads, the suspension was shaken three times for 1 m i n u t e u n d e r argon. After c e n t r i f u g a t i o n at 1,000 g for 5 minutes, crude extracts were w i t h -
A u t o r a d i o g r a m s o f gels o b t a i n e d -with e x t r a c t s f r o m ni[::Mu a n d n i t d e l e t i o n m u t a n t s w e r e e x a m i n e d f o r t h e p r e s e n c e of e a c h of t h e s i x nils p e c i f i c s p o t s d e f i n e d a b o v e . A c c o r d i n g to t h e r e s u l t s r e p o r t e d i n t a b l e II, it w a s p o s s i b l e to a s s i g n s p o t 1 to ni[J, s p o t s 2 a n d 3 to n i f K a n d D, s p o t 4 to nifH, s p o t 5 t o n i f U a n d s p o t 6 to nifF.
Kinetics of the derepression of nif genes.
Fro. 1. - - Two-dimensional gel autoradiograms of I+C proteins synthesized by the Nil- strain UNF107 and the Nil + strain UNFI07 (pCE1). a and b : UNF107 ; e, d, e and f : UNF107(pCE1). a, b, e and d : cells incubated u n d e r argon in NFM q- 100 jxg/m] aspartate. e and f : c e l l s incubated u n d e r argon in NFM -[- 100 ixg/ml a s p a r t a t e -1- 19 mM NH4CI. a, e and e : 10 per cent p o l y a e r y l a m i d e SDS gels. b, d and f : 13 per cent p o l y a e r y l a m i d e SDS gels. Arrows indicate the location of p r e s u m e d n/f-specific polypeptides.
BIOCHIMIE, 1980, 62, n ° 4.
K. pneumoniae nif gene products.
BIOCHIMIE, 1980, 62, n" 4.
271
18
M. Hounmrd and coll.
272
The phenotypes of nifB::Mu, nifM::Mu, ni[N::Mu a n d nilE: :Mu m u t a n t s c o u l d n o t b e dis-
--results c o n c e r n i n g Mu i n s e r t i o n s i n ni[K, ni[D, ni[H a n d ni[J w e r e i n a g r e e m e n t w i t h t h o s e
tinguished from the phenotype
previously
of t h e w i l d l y p e ;
reported
using
one-dimensional
gels
TABLE III.
Kinetics o[ the biosynthesis of nif-speci[ic polypeptides under conditions o[ derepression. Gene products
Time after NH4+ exhaustion (minutes)
nilF
ni[U
ni/K
ni/D
0
.
.
.
.
.
.
5
.
.
.
.
.
.
. . .
. . .
10 15 30
45 60
9o each hour from 120to360
W ++ +4-~-4"
. . .
. . .
4" ++ ++ 4-4-
4" ++ ++ 4-4-
. . .
nilH
nilJ
@ ++ ++ -~--~-
++ +4-~--1-
. . .
4" ++ ++ 4"-~-
-}-
Relative i n t e n s i t i e s were e s t i m a t e d by visual c o m p a r i s o n a n d noted + or + +
;
- - in,dicates t h a t the corresponding spot was not detected. NH~÷ repressed cells were r e s u s p e n d e d in NFM at zero time.
TABLE IV.
Kinetics of repression by NH~÷ ions of the biosynthesis o f nif-speci[ic polypeptides. Time after N[t4+ addition (mieutes)
o 2 5 10 15 30 60
Gear products
nilF ++ +44-+ ++ + . .
niCU ++ 4-4--. . . .
nilK
nilD
nilH
nilJ
++ +4++ 4. + . .
+4+4. +÷ 4. 4.
+44-44-4++ 4. 4. 4.
++ 4-4+44. + ---
Relative i n t e n s i t i e s were e s t i m a t e d by visual c o m p a r i s o n a n d noted + or + + - - indicates t h a t the corresponding spot was not detected.
;
-- an entirely negative phenotype was observ e d i n nifA::,~Iu,nifL::Mu m u t a n t s a n d i n m u t a n t s c a r r y i n g d e l e t i o n s t h a t c o v e r e d t h e nifA/L r e gion ;
[1, 2] o n w h i c h b a n d s c o r r e s p o n d i n g 1o s p o t s 1, 2, 3 a n d 4 h a d b e e n c h a r a c t e r i z e d . I n t h o s e m u t a n t s , t h e p o l y p e p t i d e s c o r r e s p o n d i n g to s p o t s 5 and 6 were present ;
ni[F::Mu
as e x p e c t e d , p h e n o t y p e s of t h e d e l e t i o n m u t a n t s w e r e i n a g r e e m e n t w i t h t h o s e of M u i n s e r tion mutants.
- - s p o t 6 is t h e o n l y m i s s i n g s p o t i n mutants ;
- - i n nifS a n d nifU m u t a n t s , lonv a m o u n t s of t h e p o l y p e p t i d e s c o r r e s p o n d i n g to s p o t s 2 a n d 3 w e r e p r e s e n t a n d , i n nifU m u t a n t s , s p o t 5 w a s totally missing ;
BIOCHIMIE, 1980, 62, n ° 4.
Molecular weight and pI determinations showe d t h a t t h e nifU p r o d u c t is a p o l y p e p t i d e of a b o u t 22,000 d a l t o n s , p I 5 a n d t h a t t h e nifF p r o d u c t is
K. p n e u m o n i a e a p o l y p e p t i d e of about 10,000 daltons and p I 4.5. Molecular w e i g h t a n d pI d e t e r m i n a t i o n s of the nifK, D, H a n d J p r o d u c t s w e r e s i m i l a r to those p r e v i o u s l y r e p o r t e d [1, 2, 6].
Kinetics of the derepression of nif genes. It can be seen in table I H that the spots corresp o n d i n g to the ,six identified p o l y p e p t i d e s appeared s i m u l t a n e o u s l y on the autoradiograms, w h e n bacteria had been i n c u b a t e d for 45 m i n u t e s u n d e r c o n d i t i o n s of d e r e p r e s s i o n in NFM. The i n t e n s i t y of the spots r e a c h e d a m a x i m u m after one h o u r and did not change up to 6 hours. This suggests that the rate of b i o s y n t h e s i s of the six polypeptides was m a x i m u m after 1 h o u r a n d r e m a i n e d c o n s t a n t afterwards. In the same e x p e r i m e n t , the a p p e a r a n c e of the a c e t y l e n e - r e d u c i n g activity was m e a s u r e d both in vivo a n d in vitro (data not shown). Activity was first detected after 75 m i n u t e s of derepression, i n c r e a s e d l i n e a r l y from 2 hours up to 4 hours a n d then r e m a i n e d constant. A lag between the a p p e a r a n c e of Kpl and Kp2 s u b u n i t s and the detection of a c e t y l e n e - r e d u c i n g activity was p r e v i o u s l y r e p o r t e d with intact cells [22]. In addition, we f o u n d that both in vivo and in vitro activities a p p e a r e d s i m u l t a n e o u s l y .
Repression by ammonium ions. A m m o n i u m chloride (19 mM) was added to the culture vessel of a N2-fixing culture of Klebsiella pneumoniae UNF1.07(pCE1) at zero lime. K i n e t i c studies of both the a c e t y l e n e - r e d u c i n g activity and of the b i o s y n t h e s i s of nil-specific polypeptides were performed. Addition of NiH4+ ions resulted in a slight increase of the total a c e t y l e n e - r e d u c i n g activity of the culture d u r i n g the first 10 minutes, followed by a plateau up to at least 3 hours. Decrease of the specific activity was directly related to dilution due to cell d i v i s i o n (generation time of about 2 hours). These results were in agreement w i t h data p r e v i o u s l y r e p o r t e d by T u b b and Postgate
[23]. The effect of NIH4+ a d d i t i o n on the b i o s y n t h e s i s of the six n/f-specific p o l y p e p t i d e s is s h o w n in table IV. The nifU p r o d u c t was no longer detectable on the a u t o r a d i o g r a m s 5 m i n u t e s after NH4 + addition. The ni[F, K, D and J products w e r e no more detectable after 30 minutes, while some nifH p r o d u c t was still synthesized after 60 minutes.
BIOCHIMIE, 1980, 62, n ° 4.
n i f gene products.
273
Discussion. By t w o - d i m e n s i o n a l electrophoresis of extracts of nif::Mu and nil deletion m u t a n t s of Klebsiella pneumoniae pulse-labelled w i t h 14C a m i n o a c i d s , the p r o d u c t s of nifF, U, K, D, H a n d J w e r e characterized. P r o d u c t s of nifK, D, H a n d J are n o w well i d e n t i f i e d [1, 2, 6]. In a d d i t i o n Roberts et al. [6] p r e v i o u s l y reported the c h a r a c t e r i z a t i o n , by t w o - d i m e n s i o n a l gel electrol)horesis, of the products of nifF, S, N and E. A c c o r d i n g to these authors, the nifF p r o d u c t w o u l d be a p o l y p e p t i d e of 17,000 daltons and of pI 5, w h e r e a s results reported here lead to the c o n c l u s i o n that the nifF p r o d u c t would have a m o l e c u l a r vceight of 10,000 daltons and a p I about 4.5. A n e w nil p r o d u c t , that of nifU, was i d e n t i f i e d as a p o l y p e p t i d e of 22,000 daltons and of pI 5. Our c h a r a c t e r i z a t i o n of ni[F p r o d u c t was based on the p h e n o t y p e s of pPC7 a n d p~PC503 m u t a n t s w h i c h w e r e i d e n t i f i e d as nifF by c o m p l e m e n t a tion a n a l y s i s a n d deletion m a p p i n g [4]. None of the m u t a n t s c o m p l e m e n t s the nifF4066 p o i n t mutatio,n d e s c r i b e d by St. J o h n et al. [7]. M.oreover c r u d e extracts of s t r a i n s c a r r y i n g pPC7 (nifF8OOT::Mu) e x h i b i t a h i g h a c e t y l e n e - r e d u c i n g activity in vitro ( u n p u b l i s h e d results). This is i n a g r e e m e n t w i t h the usual p h e n o t y p e of ni[F mutants [6, 7, 11]. No obvious e x p l a n a t i o n can therefore a c c o u n t for the d i s c r e p a n c i e s b e t w e e n the results of Roberts et al. and those r e p o r t e d here. As we did not show that p o i n t m u t a t i o n s in
nifF a n d nifU altered the electrophoretic m o b i l i t y of the c o r r e s p o n d i n g polypeptides, we c a n n o t rule out the possibility that nifF a n d nifU are regulatory genes involved in the b i o s y n t h e s i s of the i d e n t i f i e d polypeptides. A m o n g all the n i f : : M u insertions, only nifA:~:Mu in a d d i t i o n to nifF::Mu and nifU::Mu lead to the d i s a p p e a r a n c e of the p o l y p e p t i d e s assigned to nifF a n d ni[U. Conseq u e n t l y if the polypeptides w e r e not the p r o d u c t s of nifF a n d ni[U, they could only be the p r o d u c t s of u n i d e n t i f i e d nil genes. Genetic studies have s h o w n that the nil genes are organized in seven t r a n s c r i p t i o n a l u n i t s : nifQB, nifAL, nifF, nifMVSU, nifNE, nifKDH a n d nifJ [3, 4]. The first six u n i t s vrere s h o w n to be t r a n s c r i b e d in the same d i r e c t i o n from ni[H t o w a r d s nifQ [3, 4, 24]. The existence of nifKDH and nifJ as i n d e p e n d e n t units, as ~vell as the d i r e c t i o n of t r a n s c r i p t i o n w i t h i n the nifKDH operon, has been already c o n f i r m e d by biochemical studies [1, 2, 6]. Results r e p o r t e d here are in agreement w i t h ni[F b e l o n g i n g to a t r a n s c r i p t i o n a l
274
M. Houmard and coll.
u n i t i n d e p e n d e n t of nifMVSU, as Mu inserts in nifF do not p r e v e n t the expression of ni[U, nor Mu inserts in a n y of the nifM, nifS or nifU genes the expression of nifF. Moreover, our results agree w i t h nifNE and nifMVSU b e i n g separate units a n d w i t h nifMVSU t r a n s c r i b e d from nifU to nifM as in both nifN::Mu a n d nifS::Mu m u t a n t s the nifU p r o d u c t is present. Identification of nifA as a r e g u l a t o r y gene was first based on results r e p o r t e d b y Dixon el el. who observed that p o i n t m u t a t i o n s i n nifA prevented the b i o s y n t h e s i s of both Kpl a n d Kp~ subu n i t s [11]. This o b s e r v a t i o n was c o n f i r m e d w i t h i n s e r t i o n m u t a n t s a n d it was s h o w n that insertions in nifA p r e v e n t e d not only the expression of ni[K, D a n d H, b u t also that of nifJ El, 2, 6] a n d of nifF, S, N a n d E E6]. Results r e p o r t e d here showed that n o n e of the nil-specific p o l y p e p t i d e s that we identified, i n c l u d i n g the nifF a n d ni[U products, w e r e synthesized b y nifA::Mu m u t a n t s n o r by m u t a n t s c a r r y i n g a deletion of the nffA/L region. It thus appears that the nifA p r o d u c t is r e q u i r e d for the b i o s y n t h e s i s of all the nif polypeptides i d e n t i f i e d so far. C o n c e r n i n g the o r g a n i z a t i o n of the t r a n s c r i p tional u n i t nifKDH, a difficulty p r e v i o u s l y m e n tioned [1, 2, 6, 11] was met once more. Indeed, i n s e r t i o n m u t a n t s i n nifK synthesized the nifH p r o d u c t b u t not the p r o d u c t of nifD. Two hypotheses w e r e proposed to a c c o u n t for this observation : either the absence of the nifD p r o d u c t is due to an a n t i p o l a r i t y effect [1], or the (~ s u b u n i t of the nitrogenase is u n s t a b l e in the absence of the ~ s u b u n i t [1, 2, 6]. F u r t h e r m o r e , Mu i n s e r t i o n s in nifU and nlfS were s h o w n here to interfere with the presence of Kp~ subunits. Such an effect has been reported by Roberts et ol. [6] w i t h nifS::Mu m u t a n t s , b u t ~xas not observed with insertions i n nlfM or nifV [6, this report~. It is not yet possible to decide w h e t h e r only the nifS product or both the nifU a n d nifS p r o d u c t s are req u i r e d for the p r e s e n c e of <> amounts of Kp 1 s u b u n i t s since i n s e r t i o n s in nifU have a polar effect on nifS. Among all the nif::Mu mutants studied, only i n s e r t i o n s in nlfA/L, nifS a n d nlfU displayed a p l e i o t r o p i c effect on the presence of IKp1 subunits. This raises the question of w h e t h e r nifS and nifU play a r e g u l a t o r y role sim i l a r to that of nifA in the expression of nifK and nifD. Since <> a m o u n t s of Kp=, s u b u n i t s are synthesized in nifS and nifU m u t a n t s , it is difficult to i m a g i n e such a regulatory role at the t r a n s c r i p t i o n a l level. A more likely e x p l a n a t i o n might be that either the nifS p r o d u c t or both the
BIOCHIMIE, 1980, 62, n ° 4.
nffU a n d nifS p r o d u c t s are i n v o l v e d in m a t u r a t i o n of the nifJ~ a n d nifD products. The k i n e t i c study of the a p p e a r a n c e of the nil p r o d u c t s a n d of n i t r o g e n a s e activity d u r i n g der e p r e s s i o n showed that the p r o d u c t s of ni[F, U, K, D, H a n d J w e r e detectable s i m u l t a n e o u s l y after 45 m i n u t e s of d e r e p r e s s i o n w h e r e a s nitrogenase activity w a s detectable only 30 m i n u t e s later. The six nil p o l y p e p t i d e s belong to four ind e p e n d e n t t r a n s c r i p t i o n a l u n i t s but nevertheless their b i o s y n t h e s i s a p p e a r to be coordinated. One m a y thus i m a g i n e that the first event of the der e p r e s s i o n is the s y n t h e s i s of a specific positive activator (the nifA p r o d u c t for example) a n d that once the effector is present, the t r a n s c r i p t i o n of all the other nil t r a n s c r i p t i o n a l units proceeds c o n c o m i t a n t l y . F l e m m i n g and H aselkorn w o r k i n g w i t h blue green algae [25] a n d Eady et al. w i t h Klebsiella pneumoniae E22] have s h o w n that, dur i n g the d e r e p r e s s i o n of n i t r o g e n fixation, the c o m p o n e n t s of the n i t r o g e n a s e complex are detectable long before the a p p e a r a n c e of e n z y m i c activity. Data reported here c o n f i r m this observation a n d show that the nifF, U and J products appear c o n c o m i t a n t l y w i t h the nifg, D and H polypeptides. It was suggested that the lag between the a p p e a r a n c e of Kpl a n d Kp., s u b u n i t s and that of nitrogenase activity m e a s u r e d in vivo might be due to the lack of the specific electron d o n o r [22]. Our results do not s u p p o r t this hypothesis as the b i o s y n t h e s i s of the nifF p r o d u c t is not particularly delayed and as the a p p e a r a n c e of nitrogenase activity m e a s u r e d in c r u d e extracts, u s i n g dithionite as the electron donor, is c o n c o m i t a n t w i t h the a p p e a r a n c e of the activity in whole cells. As m e n t i o n e d by E a d y el al. [22], the possibility that the lag is due to tile slow assembly of Kpl or Kpe from their respective s u b u n i t s c a n n o t be excluded. As p r e v i o u s l y reported [22, 23J, a d d i t i o n of NH~+ ions to a N2-fixing culture of Klebsiella pneumoniae was f o u n d to have no effect on the p r e e x i s t i n g n i t r o g e n a s e activity w h i c h r e m a i n s at a c o n s t a n t level. However, as s h o w n in table IV, tile b i o s y n t h e s i s of n/f-specific polypeptides is rap i d l y abolished. It thus seems that t r a n s c r i p t i o n of the nil genes is t u r n e d off w h e n NH4 + ions are added and that the observed biosyntheses correspond to the t r a n s l a t i o n of p r e e x i s t i n g mRNAs. Nevertheless, the k i n e t i c s of the residual b i o s y n theses is not s i m i l a r for all the nit p r o d u c t s studied. The nifU p r o d u c t is no longer synthesized 5 m i n u t e s after NH4 + ions a d d i t i o n w h e r e a s some nffH p r o d u c t is still synthesized at a low rate
K. pneumoniae nif gene products. after 60 minutes. All our results are in agreement with the h y p o t h e s i s of T u b b and Postgate E23] who postulate that the repression by a m m o n i u m is effective at the level of mRNA synthesis. However the m e c h a n i s m by w h i c h repression is mediated is not clearly understood. Acknowledgements.
The authors wish to thank Drs R. A. Dixon and M. Merrick for gift of bacterial strains, Dr. J.-P. Aubert for helpful discussions and Dr. J. Shapiro for critical reading of the manuscript. D. Bogusz was the recipient of an O.R.S.T.O.M. studentship. R. Biyault was on leave from the Entreprise Mini~re et Chimique (E.M.C.). This investigation was supported bg research funds from the University Paris VII and by a grant from the D.G.R~.T. (No. 78-7-0~0). REFERENCES. 1. Merriek, M., Filser, M., K e n n e d y , C. ,¢ Dixon, R. (1978) Molec. yen. Genel., 165, 103-111. 2. E l m e r i c h , C., H o u m a r d , J., Sibold, L., M a n h e i m e r , I. ~ C h a r p i n , N. (1978) Molec. gen. Genet., 165, 181-189. 3. MacNeil, T., MacNeil, D., Roberts, G. P., Supiano, M. ~ Brill, W. J. (1978) J. Bacteriol., 136, 253-266. 4. Merriek, M., Filser, M., Dixon, R., E l m e r i c h , C., Sibold, L. ~ H o u m a r d , J. (1980) J. Gen. Microbiol., 117, 509-520. 5. Elmerieh, C. (1979) Physiol. Vdff., 17, 883-906. 6. Roberts, G. P., MacNeil, T., MacNeil, D. ~ Brill, W. J. (1978) J. Baeteriol., 136, 267-279. 7. St. J o h n , R. T., J o h n s t o n , M. H., Seidman, C., Garfinkel, D., Gordon, J. K., Shah, V. K. ~ Brill, W. J. (1975) J. Bacteriol., 121, 759-765.
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8. Eady, R. R., Smith, B. E., Cook, K. A. ~ Postgate, J. R. (1972) Biochem. J., 128, 655-675. 9. H a g e m a n , R. V. & Burris, R. H. (1978) Proc.. Natl. Acad. Sci. USA, 75, 2699-2702. I0. Kennedy, C., Eady, R. R., Kondorosi, E. & K l a v a n s Rekosh, D. (1976) Biochem. J., 155, 383-389. 11. Dixon, R. A., K e n n e d y , C., Kondorosi, A., K r i s h n a pillai, V. ,¢ Merrick, M. (1977) Molec. gen. Genet., 157, 189-198. 12. B a c h m a n , B. J., Low, K. B. ,~ Taylor, A. L. (1976) Bacteriol. Rev., 4~), 116-167. 13. Dixon, R., Cannon, F. C. a Kondorosi, A. (1976) Nature, 260, 268-271. 14. Stretcher, S. L., Gurney, E. G. ~¢ Valentine, R. C. (1972) Nature, 239, 495-499. 15. C a n n o n , F. C., Dixon, R. A., Postgate, J. R. a P r i m rose, S. B. (1974) J. Gen. Microbiol., 89, 227-239. 16. O'Farrell, P. H. (1975) J. Biol. Chem., 250, 4007-4021. 17. Postgate, J. R. (1972) in (~Methods in Microbiology >> (Norris, J. R. a n d Ribbons, D. W., eds), vol. 6B, pp. 343-356, Academic Press, New York a n d London. 18. Iborra, F. ~ Buhler, J. M. (1976) Anal. Biochem., 74. 503-511. 19. L a e m m l i , U. K. (1970) Nature, 227, 680-685. 20. B o n n e r , W. M. a Laskey, R. A. (1974) Eur. J. Biochem., 46, 83-88. 21. S e d m a k , J. J. a Grossberg, S. E. (1977) Anal. Biochem., 79. 544-552. 22. Eady, R. R., Issack, R., K e n n e d y , C., Postgate, J. R. •~ Rateliffe, H. D. (1978) J. Gen. Microbiol., 104, 277-285. 23. Tubb, R. S. a Postgate, J. R. (1973) J. Gen. Microbiol., 79, 103-117. 24. J a n s s e n , K. A., Riedel, G. E., Ausubel, F. M. Cannon, F. C. (1980) in <(Nitrogen Fixation ~) (Newton, W. E. & O r m e - J o h n s o n , W. H., edsl, vol. 1, pp. 85-93, U n i v e r s i t y P a r k Press, Baltimore. 25. F l e m m i n g , H. a Haselkorn, R. (1974) Cell, 3, 159170.
Characterization and kinetics of the biosynthesis of some nitrogen fixation (nif) gene products in Klebsiella pneumoniae. Jean HOUMARD <>, Didier BOGUSZ, R6gis BIGAU~LT and Claudine ELMERICH. (21-12-1979).
R~sum~. Chez K l e b s i e l l a pneumoniae, une 6tude comparative des prot6ines synth6tis6es soft p a r une souche Nil ~, soft p a r des mutants nff::Mu, soft encore par des mutants portant une d616tion de q~nes nff a 6t6 r6alis6e p a r la technique des gels & deux dimensions. La comparaison des autoradiocjrammes obtenus avec les extraits des cellules incub&es 10 minutes en pr6sence d'acides amin6s 14C a permis de rep6rer six produits sp6cifiques des g~nes n i l En plus des produits des q~nes de structure du complexe nitrog6nase (nitK, nitD, nifH) et du produit de nil! pr6alablement caract6ris6s, un polypeptide de 10.000 daltons et de pI environ 4,5 a 6t6 identiff6 comme produit du g~ne nffF, et un polypeptide de 22.000 daltons et de pI 5 comme prc~ duit du q~ne niiU. La biosynth~se de ces deux derniers produits est sous le contr&le du q~ne de r6gulation n i t A , tout comme cela avait d6j& 6t6 montr6 pour les autres produits nil. Les r6sultats de cette ~tude confirment les donn6es pr6alahlement obtenues par l'analyse g6n6tique concernant l'organisation en unit6s de transcription des q~nes nit de K l e b s i e l l a pneumoniae. Une cin6tique de la biosynth~se des six produits nil identifi6s et de rapparition de l'activJt6 nitrog6nase, lors de la d6r6pression des g~nes n i l a 6t6 r6alis6e. Les six produits ne sont d6celables qu'apr~s 45 minutes et leur apparition est simultan6e, ce qui sugg~re une transcript/on coordonn6e des diff6rentes unit6s. Cependant, l'activit6 nitrog6nase n'est d6celable, rant in v i v o qu'in vitro, que 30 minutes plus tard. Une 6tude cin6tique analogue a 6t~ r6alis6e avec la souche Nif÷, cultiv6e en conditions de
0 To whom all correspondence should be addressed.
Unitd de Physiologie Cellulaire, D~partement de B i o c h i m i e el Gbn~tique Moldculaire, l n s t i t u t Pasteur, 28 rue du Docteur Roux, F-7579_~ Paris CEdex 15.
fixation, & laquelle des ions NH~ ont 6t6 aiout6s. Dans ces conditions, on observe un arr~t de la biosynth~se des diff6rents produits nil. Cependant, cet arr~t n'est pas synchrone pour les six produits car la synth~se du produit de nffU n'est plus d6celable 5 minutes apr~s l'addition d'ions NH., celle de n/if, K, D et ] ne l'est plus apr~s 30 minutes tandis qu'on observe une synth~se r6siduelle du polypeptide nitH apr~s 60 minutes. Lors de la r6pression par les ions NH4, il semble donc que la transcription des g~nes nil soft bloqu6e et que seuls les RNA messagers pr6existants continuent d'6tre traduffs. Mots-cl~s : Fixation de l'azote ; Klebsiella pneumon/ae ; produits des g~nes nil.
Summary. Analysis of 1~C pulse-labelled proteins, synthesized b y a Nil+ K l e b s i e l l a pneumon/ae strain and b y a number of qenetically m a p p e d nif::Mu and nil deletion mutants, was perform e d b y two-dimensional gel electrophoresis. By comparison of the autoradioqrams, s.~x nff-specific polypeptides were identified. In addition to the previously characterized nifK, nffD, nifH a n d nit] products, the product of nffF was identified as a polypeptide of 10,000 daltons a n d pI about 4.5 and the product of nifU as a polypeptide of 22,000 daltons and pI 5. Moreover, the biosynthesis of nifF and n i f U polypeptides was shown to be prevented in mutants affectinq the regulatory qene nitA, which is known to control the biosynthesis of the other nff genes products so far identified. In all cases, the biochemical phenotypes of the different polar mutants were in good agreement with those expected from
M. H o u m a r d
268
the transcriptional o r g a n i z a t i o n of the nff cluster p r e v i o u s l y established by genetic a n a l y s i s . Kinetic studies of both n i t r o g e n a s e activity a n d of the b i o s y n t h e s i s of the six nil-specific p o l y p e p t i d e s w e r e performed with the Nil + strain, i n c u b a t e d either under conditions of derepression or under conditions of repression b y NH4 + ions. Upon derepression, the b i o s y n t h e s i s of the six nit polypeptides, w h i c h b e l o n g to four different transcriptional units, s e e m s to b e coordinated since they a p p e a r s i m u l t a n e o u s l y after a lag of 45 minutes. Under those conditions, both in v i v o a n d in vitro n i t r o g e n a s e activities
Introduction. Genetic studies of n i t r o g e n fixation i n KlebsielIa p n e u m o n i a e led to the definition of 14 n i l cistrons organized i n seven t r a n s c r i p t i o n a l u n i t s [1-5]. Biochemical c h a r a c t e r i z a t i o n s of some n i l m u t a t i o n s have been r e p o r t e d [I, 2, 6, 7]. However, relatively few of the n i l gene p r o d u c t s and f u n c t i o n s have so far been identified u n a m b i g u o u s l y . The nitrogenase complex of Klebsiella p n e u m o n i a e consists of two p r o t e i n s Kp I a n d Kp~ [8] w h i c h , a c c o r d i n g to Hageman and Burris [9], are t e r m e d respectively nitrogenase (iKpl) a n d n i t r o g e n a s e reductase (Kp2). The nitrogenase is a t e t r a m e r m a d e up of two n o n - i d e n t i c a l s u b u n i t s %[~.) [10] ; the nitrogenase reductase is a d i m e r nmde up of i d e n t i c a l p r o t o m e r s [8]. The nifH, nifD a n d n i f K genes have been identified as the genes c o d i n g for KP2, Kpl(t a n d iKpl~ s u b u n i t s [1, 2, 6]. NifA was s h o w n to be involved in r e g u l a t i o n [1, 2, 6, 11], n i f B [7] a n d tentatively n i f N and n i l e [6] in the molybd e n u m - i r o n cofactor (FeMoco) synthesis, nifM in
a n d coll.
w e r e d e t e c t a b l e o n l y 30 m i n u t e s later. U p o n addition of NH4 + ions, the b i o s y n t h e s i s of the six nit p o l y p e p t i d e s w a s rapidly abolished. H o w e ver, the kinetics of residual biosynthesis, probab l y due to the transcription of preexisting m R N A s , w a s not similar for the six nil products. The nHU product w a s n o longer detectable after 5 minutes, the n / i f , K, D a n d I products w e r e not d e t e c t a b l e after 30 minutes, w h e r e a s s o m e nffH product w a s still slightly detectable after 60 minutes. Key.words:Nitrogen fixation ; Klebsiella pneumoniae ; rib"gene products.
but not in vitro [6]. I n a d d i t i o n to the p r o d u c t s of nifH, D, K a n d J, the p r o d u c t s of nifN, nilE, n i f S a n d n i f F have been identified as p o l y p e p t i d e s of 50,000, 46,000, 18,000 a n d 17,000 daltons molecular weight [6]. All these p r o d u c t s are p r e s e n t under a n a e r o b i c c o n d i t i o n s , only w h e n b a c t e r i a are g r o w n in the absence of NH4 + ions. We r e p o r t here the identification of two n e w products, w h i c h w e assigned to ni[F a n d ni[U genes, and the k i n e t i c s of derepression a n d repression by a m m o n i u m ions of the b i o s y n t h e s i s of the nifF, nifU, nifK, nifD, n i f t I and n i f J products.
Materials and Methods.
Bacteria and plasmids. The K. pneumoniae strains are listed in table I. The genotype of the different Nif- plasmids [2, 4] appears in table II. As a uniform nomenclature has been
"[[-'ABLEI. Klebsiella p n e u m o n i a e strains. Strain UNF107 KP52 UNFI31 UNF156 UNFI69 UNFI77
Geaotype or phenotype
A A A A A A
(gnd-his-niI) 107 rpsL (gnd-his-nil) 4648 rpsL (his-ni/l 2473 hsdRl rpsL ni1-2479 hsdRl rpsLl ni1-2482 hsdRl (his-r~i]) 2483 h~dRl
Reference Dixon el al. 1977 [11] Stretcher el al. 1972 [14] Merrick el al. 1978 [1] Merrick el al. 1978 ]1] Merrick et al. 1978 [1] Merrick el al. 1979 [4]
Genetic symbols are those of Bachman et al. [12].
the processing of KI)2 [6]. The p r o d u c t of ni[J has been identified as a p o l y p e p t i d e of 120,0'00 daltons molecular weight El, 2, 6] that would be r e q u i r e d for a c e t y l e n e - r e d u c i n g activity in vivo,
BIOCH1MIE, 1980, 62, n" 4.
recently set up, plasmids previously termed pLS1, pLS17, pNC172 for example [2] are now termed pPC1, pPC17 and pPC172, with allele numbers 8001, 8017 and 8177. All the plasmids are pCE1 derivatives. The pCEl plasmid derives from pRD1 (amp* tel + kan ~ find'
K. pneumoniae
nif
his ~ n i p shi ÷tra +incP) c o n s t r u c t e d b y D i x o n et at, [13JU n l i k e pRD1, pCEI does n o t s u p p r e s s t h e Mu s e n s i t i vity in E. colt s t r a i n s [2]. I n t h i s s t u d y , all t h e p l a s raids were c a r r i e d b y UNF107 K. pneumoniae nil total deletion strain,
gene products.
269
cell p e l l e t w a s r e s u s p e n d e d in 200 ~tl o f t h e <
TABLE II.
Biosynthesis of nif-speci[ic polypeptides by n i l mutants. Gene products Genotype
Plasniids or strains {allele} Spott
Spot2
Spot3
Spot4
Spot5
Spot6
ni[B : : Mu
+-~~--~-
-+--+-~--~-
+-{+-~-
-~-~-~-~-
-+-+ -}-7t-
+-~-+--j-
niIA : : hill : : nijF : : nitM: :
. . ~---~-{--
. . -{---~-~---[-
. . -~---{-~-+
. . . . -[-7tq---~-
--J-+ -~--~-
--~--~-
++
++
++
-~-+
+-[+-~-
wild t y p e Mu Mu Mu Mu
ni]S : : Mu
++
ni]U : : Mu niJN : : Mu
. .
+
W
+-+~-~-
-{Jr--~
W +-~-
+-~-~-+
nile : : Mu nilK : : Mu
-{--~-~-
-~--
+-+--
--{--~- --~--+-~--~-~---~-
nilD : : Mu ni/H : : Mu niIJ : : Mu nilA (M-J) niIA (B-V) ni[A (B-M) ni[A (N-H) nilA (K.J) nilA (-B) n q A (B-J)
-~-~---+-. . . + . +% .
--+--J. . . . . . -. . -{-% . .
--~--~----~--j--J-+ . . . . . . . . --. -]--% -{---% . . .
+-{--~-~-
--J--+--{-+ --{--+-
-{---{Jr--}--J---{-[--~-
--J-+ --J---J%--{-
-J- --~+-{-]-+
pCEI pPC501
(niI-850I) pPC80 (ni]-8080) (ni[-8172) pPC141 (ni[-8141) p P C l (nil-8001) p P C l l 2 (ni[-8112) pPC503 (ni]-8503) pPC7 (ni[-8007) pPC52 (ni[-8052) p P C I 0 0 (ni[-8100) (ni1-8504) pPC31 (nip8031) pPC41 (nit-8041) (ni[-8557) pPC71 (nip8071) pPC560 (ni[-8560) pPC92 (nip8092) pPC569 (nip8569) (nil-8570) pPC40 (ni1-8040) pPC505 (nip8505) pPC16 (ni[-8016) pPC21 (ni/-8021) (nipS076) pPC47 (ni[-8047) p P C l l 7 (ni[-8117) p P C l l (ni]-8011) pPC27 (ni/-8027) pPC15 (nip8015) pPC45 (ni1-8045) p P C l l l . 1 (nip8760) KP52 (nit-d648) UNF131 (ni/-2473) U N F 1 5 6 (nil-2479) U N F 1 6 9 (ni[-2~[82) U N F 1 7 7 (ni[-2483) U N F 1 0 7 (nip107)
R e l a t i v e i n t e n s i t i e s w e r e e s t i m a t e d b y v i s u a l c o m p a r i s o n a n d n o t e d q- or + + c o r r e s p o n d i n g s p o t w a s n o t d e t e c t e d ; w i n d i c a t e s t h a t t h e s p o t w a s w e a k l y visible.
Culture media. N i t r o g e n f r e e (NFM) a n d L u r i a B r o t h (LB) m e d i a h a v e been p r e v i o u s l y d e s c r i b e d [2, 15].
Growth conditions for radioactive labelling of proteins. T h e f o l l o w i n g p r o c e d u r e h a s b e e n u s e d for t h e d e r e p r e s s i o n o f t h e n i t r o g e n a s e c o m p l e x . S t r a i n s were g r o w n o v e r n i g h t , u n d e r N2, in 10 m l of NFM cont a i n i n g 19 m M NI-LC1. A f t e r c e n t r i f u g a t i o n u n d e r argon, b a c t e r i a w e r e r e s u s p e n d e d , at 109 cells p e r ml in a n a e r o b i c NFM s u p p l e m e n t e d w i t h 100 t t g / m l a s p a r t a t e a n d w i t h o r w i t h o u t 19 m M NH4C1. Five m i l l i l i t e r s of culture were then incubated with shaking, under argon, at 30°C. Samples of 3 ml were withdrawn, usually after 3 h o u r s of i n c u b a t i o n , a n d a d d e d a n a e r o b i c a l l y to a c e n t r i f u g e t u b e w h i c h c o n t a i n e d 6 ~tCi of a a4C p r o tein h y d r o ] y s a t e s o l u t i o n (CEA, F r a n c e , ref. C4B). Ten m i n u t e s later, 4 m l o f NFM c o n t a i n i n g 2 m g / m l c a s a m i n o - a e i d s w e r e a d d e d a n d t h e cells w e r e c e n t r i f u g e d . Cells w e r e w a s h e d w i t h 5 m l o f t h e NFM e a s a m i n o acids a n d c e n t r i f u g e d f o r 10 m i n u t e s a t 6,500 g. E a c h
BIOCHIMIE, 1980, 62, n ° 4.
; --
pPC172
pPC504 pPC557 pPC570 pPC76
indicates that the
s u p e r n a t a n t s w e r e w i t h d r a w n , f r o z e n in l i q u i d n i t r o gen a n d k e p t at - - 2 0 ° C .
Assays for nitrogen fixation. N i t r o g e n a s e a c t i v i t y of w h o l e cells w a s e s t i m a t e d b y the a c e t y l e n e r e d u c t i o n test [17]. In vitro a c e t y l e n e r e d u c i n g a c t i v i t i e s w e r e m e a s u r e d as d e s c r i b e d b y E a d y et aL [8]. E t h y l e n e p r o d u c e d w a s m e a s u r e d w i t h a 204 P y e U n i c a m gas c h r o m a t o g r a p h , b y i n j e c t i n g 0.25 ml gas s a m p l e into a 40 X 0.2 c m c o l u m n filled w i t h P o r a p a k T a n d e q u i l i b r a t e d at 55°C.
Two-dzTnensional polyacrylamide gel electrophoresis. T h e i s o e l e c t r i c f o c u s i n g w a s p e r f o r m e d a c c o r d i n g to I b o r r a a n d B u h l e r [18] w i t h a LKB M u l t i p h o r a p p a r a tus. T h e gel c o n t a i n e d 5 p e r cent a e r y l a m i d e , 8.3 M u r e a a n d 2 p e r c e n t a m p h o l i n e s p H 3.5-10 (LKB). A f t e r 1 h o u r of p r e f o c u s i n g (20 mA, 1,000 v o l t s a n d 200 w a t t s w e r e set u p as m a x i m a on t h e LKB2103 p o w e r s u p p l y ) , a b o u t 0.1 ~tCi o f l a b e l l e d p r o t e i n s w e r e l o a d e d o n t o t h e gel a n d f o c u s i n g w a s a l l o w e d to r u n f o r 2 h o u r s at 12"C. Gel s t r i p s of a b o u t 1 eva w i d t h
270
M. H o u m a r d
a n d coll.
were then cut and equilibrated in SDS Ortee sample buffer for 15 m i n u t e s at 30°C. Samples were then frozen in a dry ice-acetone b a t h and kept at --20°C.
d r a w n and assayed f o r nitrogenase activity. Protein concentration was d e t e r m i n e d with Coomassie blue G250, using bovine s e r u m a l b u m i n as a s t a n d a r d [21].
The SDS gel electrophoresis, in the second dimension, was p e r f o r m e d as described by L a e m m l i [19]. Either 10 or 13 per cent a e r y l a m i d e r u n n i n g gels (about 10 em height) were used. The height of the stacking gel (4.5 per cent aerylamide) was 1 em. The focusing strip was set up on the top of the stacking gel and layered by a 2 per cent agarose solution (made up in 25 mM Tris-glyeine buffer pH 8.6, containing 0.1 per cent SDS).
Repression by ammonium ions. K. pneumoniae UNF107(pCE1) was grown in a 1.5
Molecular weights were d e t e r m i n e d using bovine serum a l b u m i n (67,000), egg a l b u m i n (45,000), a-ehym o t r y p s i n o g e n A (25,700), soybean t r y p s i n i n h i b i t o r (21,500), ribonuelease (14,700) and h o r s e h e a r t eytochrome e (13,700) as s t a n d a r d s in the second d i m e n sion. For pI d e t e r m i n a t i o n s , focusing strips were sliced and the profile of the g r a d i e n t was established by measuring the pH of each slice. In addition, the nifH, D, K and J products, of w h i c h m o l e c u l a r weights and pls have been p r e v i o u s l y reported [1, 2, 61, were used as i n t e r n a l controls. Scintillating a u t o r a d i o g r a p h y was p e r f o r m e d according to Bonner a n d Laskey [20]. A f t e r w a t e r precip i t a t i o n of 2,5-diphenyloxazole (PPO) and washings, the gel was soaked in a glycerol : acetic acid : w a t e r (1:10:89 v/v) solution f o r 1 h o u r at r o o m t e m p e r a t u r e and dried between two cellophane sheets, u n d e r a hood. W h e n e v e r necessary, p r o t e i n staining was achieved using Coomassie b r i l l i a n t blue R250.
liter f e r m e n t o r , u n d e r conditions of nitrogen fixation. W h e n cell c o n c e n t r a t i o n of about 109 cells per ml was reached, a m m o n i u m ions (19 mM final concentration) were added. At different times, 10 ml samples were w i t h d r a w n f o r in vivo assay of nitrogenase activity and 3 m l samples for radioactive pulse labelling.
Results.
I d e n t i f i c a t i o n of n i f gene products. The methodology was based on the comparison of a u t o r a d i o g r a m s o f t w o - d i m e n s i o n a l g e l s o f pulse-labelled proteins synthesized by the wild type strain and by nit mutants incubated under c o n d i t i o n s of d e r e p r e s s i o n or r e p r e s s i o n by a m m o n i u m ions of n i t r o g e n fixation.
A f e r m e n t o r containing 1.5 liter of NFM supplemented w i t h 19 mM NH+C1 was inoculated w i t h 7.5 ml of a LB grown UNFI07 (pCEI) exponential culture. Initial absorbanee was 0.02 at 570 nm. Cells were grown overnight at 23°C u n d e r N2. After e e n t r i f u g a t i o n u n d e r argon, bacteria were r e s u s p e n d e d at 109 cells per ml in a f e r m e n t o r containing 1.5 liter of anaerobic NFM s u p p l e m e n t e d w i t h 100 ~,g/ml aspartate. Cells were incubated u n d e r argon at 30°C. Aliquots were w i t h d r a w n at given t i m e s : 3 ml samples for radioactive labelling (as described above), 10 ml samples for in vivo assays of nitrogenase activity and samples of about 150 ml for in vitro assays of nitrogenase activity. The latter samples were centrifuged u n d e r argon for 10 minutes at 6,500 g. The cell pellets were frozen in liquid nitrogen and stored a t - - 2 0 ° C .
As s h o w n i n f i g u r e 1, w i t h t h e w i l d t y p e UNF107(pCE1), six spots, present when bacteria w e r e i n c u b a t e d u n d e r c o n d i t i o n s of n i t r o g e n fixation, were missing when bacteria were grown in t h e p r e s e n c e o f NH4 + i o n s . F i v e s p o t s c a n b e s e e n o n b o t h 10 p e r c e n t a n d 13 p e r c e n t a c r y l a m i d e gel a u t o r a d i o g r a m s , s p o t s 1 to 5 i n f i g u r e l e a n d s p o t s 2 t o 6 i n f i g u r e l d . N o n e of t h e s i x p o l y p e p t i d e s , c a n b e d e t e c t e d w i t h UNF107, a n i t l o t a l d,eletion, i n c u b a t e d e i t h e r w i t h ( d a t a n o t s h o w n ) o r w i t h o u t a m m o n i a (fig. l a a n d b). It c a n t h u s b e a s s u m e d t h a t t h e s i x s p o t s c o r r e s p o n d to s h o w n o r w i t h o u t a m m o n i a (fig. l a a n d b). It t h u s c a n h e a s s u m e d t h a t t h e s i x s p o t s c o r r e s p o n d to n i / - s p e c i f i c p o l y p e p t i d e s . C o e l e c t r o p h o r e s i s of pulse-labelled UNF107(pCE1) extracts with pure K p 1 a n d .Kp2 p r o t e i n s s h o w e d t h a t s p o t s 2 a n d 3 c o r r e s p o n d to Kt) 1 a a n d ~ s u b u n i t s a n d t h a t s p o t 4 c o r r e s p o n d s to ~Kp2 s u b u n i t .
Crude extracts were obtained by d i s r u p t i n g the cells w i t h glass beads in a Mickle a p p a r a t u s (Gomshall, Surrey). Cell pellets were r e s u s p e n d e d in 4 ml of 100 mM Tris-HC1 pH 8.7 buffer, containing 1 mM sodium dithionite and 0.1 g/1 d i t h i o t h r e i t o l . After addition of 4 ml glass beads, the suspension was shaken three times for 1 m i n u t e u n d e r argon. After c e n t r i f u g a t i o n at 1,000 g for 5 minutes, crude extracts were w i t h -
A u t o r a d i o g r a m s o f gels o b t a i n e d -with e x t r a c t s f r o m ni[::Mu a n d n i t d e l e t i o n m u t a n t s w e r e e x a m i n e d f o r t h e p r e s e n c e of e a c h of t h e s i x nils p e c i f i c s p o t s d e f i n e d a b o v e . A c c o r d i n g to t h e r e s u l t s r e p o r t e d i n t a b l e II, it w a s p o s s i b l e to a s s i g n s p o t 1 to ni[J, s p o t s 2 a n d 3 to n i f K a n d D, s p o t 4 to nifH, s p o t 5 t o n i f U a n d s p o t 6 to nifF.
Kinetics of the derepression of nif genes.
Fro. 1. - - Two-dimensional gel autoradiograms of I+C proteins synthesized by the Nil- strain UNF107 and the Nil + strain UNFI07 (pCE1). a and b : UNF107 ; e, d, e and f : UNF107(pCE1). a, b, e and d : cells incubated u n d e r argon in NFM q- 100 jxg/m] aspartate. e and f : c e l l s incubated u n d e r argon in NFM -[- 100 ixg/ml a s p a r t a t e -1- 19 mM NH4CI. a, e and e : 10 per cent p o l y a e r y l a m i d e SDS gels. b, d and f : 13 per cent p o l y a e r y l a m i d e SDS gels. Arrows indicate the location of p r e s u m e d n/f-specific polypeptides.
BIOCHIMIE, 1980, 62, n ° 4.
K. pneumoniae nif gene products.
BIOCHIMIE, 1980, 62, n" 4.
271
18
M. Hounmrd and coll.
272
The phenotypes of nifB::Mu, nifM::Mu, ni[N::Mu a n d nilE: :Mu m u t a n t s c o u l d n o t b e dis-
--results c o n c e r n i n g Mu i n s e r t i o n s i n ni[K, ni[D, ni[H a n d ni[J w e r e i n a g r e e m e n t w i t h t h o s e
tinguished from the phenotype
previously
of t h e w i l d l y p e ;
reported
using
one-dimensional
gels
TABLE III.
Kinetics o[ the biosynthesis of nif-speci[ic polypeptides under conditions o[ derepression. Gene products
Time after NH4+ exhaustion (minutes)
nilF
ni[U
ni/K
ni/D
0
.
.
.
.
.
.
5
.
.
.
.
.
.
. . .
. . .
10 15 30
45 60
9o each hour from 120to360
W ++ +4-~-4"
. . .
. . .
4" ++ ++ 4-4-
4" ++ ++ 4-4-
. . .
nilH
nilJ
@ ++ ++ -~--~-
++ +4-~--1-
. . .
4" ++ ++ 4"-~-
-}-
Relative i n t e n s i t i e s were e s t i m a t e d by visual c o m p a r i s o n a n d noted + or + +
;
- - in,dicates t h a t the corresponding spot was not detected. NH~÷ repressed cells were r e s u s p e n d e d in NFM at zero time.
TABLE IV.
Kinetics of repression by NH~÷ ions of the biosynthesis o f nif-speci[ic polypeptides. Time after N[t4+ addition (mieutes)
o 2 5 10 15 30 60
Gear products
nilF ++ +44-+ ++ + . .
niCU ++ 4-4--. . . .
nilK
nilD
nilH
nilJ
++ +4++ 4. + . .
+4+4. +÷ 4. 4.
+44-44-4++ 4. 4. 4.
++ 4-4+44. + ---
Relative i n t e n s i t i e s were e s t i m a t e d by visual c o m p a r i s o n a n d noted + or + + - - indicates t h a t the corresponding spot was not detected.
;
-- an entirely negative phenotype was observ e d i n nifA::,~Iu,nifL::Mu m u t a n t s a n d i n m u t a n t s c a r r y i n g d e l e t i o n s t h a t c o v e r e d t h e nifA/L r e gion ;
[1, 2] o n w h i c h b a n d s c o r r e s p o n d i n g 1o s p o t s 1, 2, 3 a n d 4 h a d b e e n c h a r a c t e r i z e d . I n t h o s e m u t a n t s , t h e p o l y p e p t i d e s c o r r e s p o n d i n g to s p o t s 5 and 6 were present ;
ni[F::Mu
as e x p e c t e d , p h e n o t y p e s of t h e d e l e t i o n m u t a n t s w e r e i n a g r e e m e n t w i t h t h o s e of M u i n s e r tion mutants.
- - s p o t 6 is t h e o n l y m i s s i n g s p o t i n mutants ;
- - i n nifS a n d nifU m u t a n t s , lonv a m o u n t s of t h e p o l y p e p t i d e s c o r r e s p o n d i n g to s p o t s 2 a n d 3 w e r e p r e s e n t a n d , i n nifU m u t a n t s , s p o t 5 w a s totally missing ;
BIOCHIMIE, 1980, 62, n ° 4.
Molecular weight and pI determinations showe d t h a t t h e nifU p r o d u c t is a p o l y p e p t i d e of a b o u t 22,000 d a l t o n s , p I 5 a n d t h a t t h e nifF p r o d u c t is
K. p n e u m o n i a e a p o l y p e p t i d e of about 10,000 daltons and p I 4.5. Molecular w e i g h t a n d pI d e t e r m i n a t i o n s of the nifK, D, H a n d J p r o d u c t s w e r e s i m i l a r to those p r e v i o u s l y r e p o r t e d [1, 2, 6].
Kinetics of the derepression of nif genes. It can be seen in table I H that the spots corresp o n d i n g to the ,six identified p o l y p e p t i d e s appeared s i m u l t a n e o u s l y on the autoradiograms, w h e n bacteria had been i n c u b a t e d for 45 m i n u t e s u n d e r c o n d i t i o n s of d e r e p r e s s i o n in NFM. The i n t e n s i t y of the spots r e a c h e d a m a x i m u m after one h o u r and did not change up to 6 hours. This suggests that the rate of b i o s y n t h e s i s of the six polypeptides was m a x i m u m after 1 h o u r a n d r e m a i n e d c o n s t a n t afterwards. In the same e x p e r i m e n t , the a p p e a r a n c e of the a c e t y l e n e - r e d u c i n g activity was m e a s u r e d both in vivo a n d in vitro (data not shown). Activity was first detected after 75 m i n u t e s of derepression, i n c r e a s e d l i n e a r l y from 2 hours up to 4 hours a n d then r e m a i n e d constant. A lag between the a p p e a r a n c e of Kpl and Kp2 s u b u n i t s and the detection of a c e t y l e n e - r e d u c i n g activity was p r e v i o u s l y r e p o r t e d with intact cells [22]. In addition, we f o u n d that both in vivo and in vitro activities a p p e a r e d s i m u l t a n e o u s l y .
Repression by ammonium ions. A m m o n i u m chloride (19 mM) was added to the culture vessel of a N2-fixing culture of Klebsiella pneumoniae UNF1.07(pCE1) at zero lime. K i n e t i c studies of both the a c e t y l e n e - r e d u c i n g activity and of the b i o s y n t h e s i s of nil-specific polypeptides were performed. Addition of NiH4+ ions resulted in a slight increase of the total a c e t y l e n e - r e d u c i n g activity of the culture d u r i n g the first 10 minutes, followed by a plateau up to at least 3 hours. Decrease of the specific activity was directly related to dilution due to cell d i v i s i o n (generation time of about 2 hours). These results were in agreement w i t h data p r e v i o u s l y r e p o r t e d by T u b b and Postgate
[23]. The effect of NIH4+ a d d i t i o n on the b i o s y n t h e s i s of the six n/f-specific p o l y p e p t i d e s is s h o w n in table IV. The nifU p r o d u c t was no longer detectable on the a u t o r a d i o g r a m s 5 m i n u t e s after NH4 + addition. The ni[F, K, D and J products w e r e no more detectable after 30 minutes, while some nifH p r o d u c t was still synthesized after 60 minutes.
BIOCHIMIE, 1980, 62, n ° 4.
n i f gene products.
273
Discussion. By t w o - d i m e n s i o n a l electrophoresis of extracts of nif::Mu and nil deletion m u t a n t s of Klebsiella pneumoniae pulse-labelled w i t h 14C a m i n o a c i d s , the p r o d u c t s of nifF, U, K, D, H a n d J w e r e characterized. P r o d u c t s of nifK, D, H a n d J are n o w well i d e n t i f i e d [1, 2, 6]. In a d d i t i o n Roberts et al. [6] p r e v i o u s l y reported the c h a r a c t e r i z a t i o n , by t w o - d i m e n s i o n a l gel electrol)horesis, of the products of nifF, S, N and E. A c c o r d i n g to these authors, the nifF p r o d u c t w o u l d be a p o l y p e p t i d e of 17,000 daltons and of pI 5, w h e r e a s results reported here lead to the c o n c l u s i o n that the nifF p r o d u c t would have a m o l e c u l a r vceight of 10,000 daltons and a p I about 4.5. A n e w nil p r o d u c t , that of nifU, was i d e n t i f i e d as a p o l y p e p t i d e of 22,000 daltons and of pI 5. Our c h a r a c t e r i z a t i o n of ni[F p r o d u c t was based on the p h e n o t y p e s of pPC7 a n d p~PC503 m u t a n t s w h i c h w e r e i d e n t i f i e d as nifF by c o m p l e m e n t a tion a n a l y s i s a n d deletion m a p p i n g [4]. None of the m u t a n t s c o m p l e m e n t s the nifF4066 p o i n t mutatio,n d e s c r i b e d by St. J o h n et al. [7]. M.oreover c r u d e extracts of s t r a i n s c a r r y i n g pPC7 (nifF8OOT::Mu) e x h i b i t a h i g h a c e t y l e n e - r e d u c i n g activity in vitro ( u n p u b l i s h e d results). This is i n a g r e e m e n t w i t h the usual p h e n o t y p e of ni[F mutants [6, 7, 11]. No obvious e x p l a n a t i o n can therefore a c c o u n t for the d i s c r e p a n c i e s b e t w e e n the results of Roberts et al. and those r e p o r t e d here. As we did not show that p o i n t m u t a t i o n s in
nifF a n d nifU altered the electrophoretic m o b i l i t y of the c o r r e s p o n d i n g polypeptides, we c a n n o t rule out the possibility that nifF a n d nifU are regulatory genes involved in the b i o s y n t h e s i s of the i d e n t i f i e d polypeptides. A m o n g all the n i f : : M u insertions, only nifA:~:Mu in a d d i t i o n to nifF::Mu and nifU::Mu lead to the d i s a p p e a r a n c e of the p o l y p e p t i d e s assigned to nifF a n d ni[U. Conseq u e n t l y if the polypeptides w e r e not the p r o d u c t s of nifF a n d ni[U, they could only be the p r o d u c t s of u n i d e n t i f i e d nil genes. Genetic studies have s h o w n that the nil genes are organized in seven t r a n s c r i p t i o n a l u n i t s : nifQB, nifAL, nifF, nifMVSU, nifNE, nifKDH a n d nifJ [3, 4]. The first six u n i t s vrere s h o w n to be t r a n s c r i b e d in the same d i r e c t i o n from ni[H t o w a r d s nifQ [3, 4, 24]. The existence of nifKDH and nifJ as i n d e p e n d e n t units, as ~vell as the d i r e c t i o n of t r a n s c r i p t i o n w i t h i n the nifKDH operon, has been already c o n f i r m e d by biochemical studies [1, 2, 6]. Results r e p o r t e d here are in agreement w i t h ni[F b e l o n g i n g to a t r a n s c r i p t i o n a l
274
M. Houmard and coll.
u n i t i n d e p e n d e n t of nifMVSU, as Mu inserts in nifF do not p r e v e n t the expression of ni[U, nor Mu inserts in a n y of the nifM, nifS or nifU genes the expression of nifF. Moreover, our results agree w i t h nifNE and nifMVSU b e i n g separate units a n d w i t h nifMVSU t r a n s c r i b e d from nifU to nifM as in both nifN::Mu a n d nifS::Mu m u t a n t s the nifU p r o d u c t is present. Identification of nifA as a r e g u l a t o r y gene was first based on results r e p o r t e d b y Dixon el el. who observed that p o i n t m u t a t i o n s i n nifA prevented the b i o s y n t h e s i s of both Kpl a n d Kp~ subu n i t s [11]. This o b s e r v a t i o n was c o n f i r m e d w i t h i n s e r t i o n m u t a n t s a n d it was s h o w n that insertions in nifA p r e v e n t e d not only the expression of ni[K, D a n d H, b u t also that of nifJ El, 2, 6] a n d of nifF, S, N a n d E E6]. Results r e p o r t e d here showed that n o n e of the nil-specific p o l y p e p t i d e s that we identified, i n c l u d i n g the nifF a n d ni[U products, w e r e synthesized b y nifA::Mu m u t a n t s n o r by m u t a n t s c a r r y i n g a deletion of the nffA/L region. It thus appears that the nifA p r o d u c t is r e q u i r e d for the b i o s y n t h e s i s of all the nif polypeptides i d e n t i f i e d so far. C o n c e r n i n g the o r g a n i z a t i o n of the t r a n s c r i p tional u n i t nifKDH, a difficulty p r e v i o u s l y m e n tioned [1, 2, 6, 11] was met once more. Indeed, i n s e r t i o n m u t a n t s i n nifK synthesized the nifH p r o d u c t b u t not the p r o d u c t of nifD. Two hypotheses w e r e proposed to a c c o u n t for this observation : either the absence of the nifD p r o d u c t is due to an a n t i p o l a r i t y effect [1], or the (~ s u b u n i t of the nitrogenase is u n s t a b l e in the absence of the ~ s u b u n i t [1, 2, 6]. F u r t h e r m o r e , Mu i n s e r t i o n s in nifU and nlfS were s h o w n here to interfere with the presence of Kp~ subunits. Such an effect has been reported by Roberts et ol. [6] w i t h nifS::Mu m u t a n t s , b u t ~xas not observed with insertions i n nlfM or nifV [6, this report~. It is not yet possible to decide w h e t h e r only the nifS product or both the nifU a n d nifS p r o d u c t s are req u i r e d for the p r e s e n c e of <
BIOCHIMIE, 1980, 62, n ° 4.
nffU a n d nifS p r o d u c t s are i n v o l v e d in m a t u r a t i o n of the nifJ~ a n d nifD products. The k i n e t i c study of the a p p e a r a n c e of the nil p r o d u c t s a n d of n i t r o g e n a s e activity d u r i n g der e p r e s s i o n showed that the p r o d u c t s of ni[F, U, K, D, H a n d J w e r e detectable s i m u l t a n e o u s l y after 45 m i n u t e s of d e r e p r e s s i o n w h e r e a s nitrogenase activity w a s detectable only 30 m i n u t e s later. The six nil p o l y p e p t i d e s belong to four ind e p e n d e n t t r a n s c r i p t i o n a l u n i t s but nevertheless their b i o s y n t h e s i s a p p e a r to be coordinated. One m a y thus i m a g i n e that the first event of the der e p r e s s i o n is the s y n t h e s i s of a specific positive activator (the nifA p r o d u c t for example) a n d that once the effector is present, the t r a n s c r i p t i o n of all the other nil t r a n s c r i p t i o n a l units proceeds c o n c o m i t a n t l y . F l e m m i n g and H aselkorn w o r k i n g w i t h blue green algae [25] a n d Eady et al. w i t h Klebsiella pneumoniae E22] have s h o w n that, dur i n g the d e r e p r e s s i o n of n i t r o g e n fixation, the c o m p o n e n t s of the n i t r o g e n a s e complex are detectable long before the a p p e a r a n c e of e n z y m i c activity. Data reported here c o n f i r m this observation a n d show that the nifF, U and J products appear c o n c o m i t a n t l y w i t h the nifg, D and H polypeptides. It was suggested that the lag between the a p p e a r a n c e of Kpl a n d Kp., s u b u n i t s and that of nitrogenase activity m e a s u r e d in vivo might be due to the lack of the specific electron d o n o r [22]. Our results do not s u p p o r t this hypothesis as the b i o s y n t h e s i s of the nifF p r o d u c t is not particularly delayed and as the a p p e a r a n c e of nitrogenase activity m e a s u r e d in c r u d e extracts, u s i n g dithionite as the electron donor, is c o n c o m i t a n t w i t h the a p p e a r a n c e of the activity in whole cells. As m e n t i o n e d by E a d y el al. [22], the possibility that the lag is due to tile slow assembly of Kpl or Kpe from their respective s u b u n i t s c a n n o t be excluded. As p r e v i o u s l y reported [22, 23J, a d d i t i o n of NH~+ ions to a N2-fixing culture of Klebsiella pneumoniae was f o u n d to have no effect on the p r e e x i s t i n g n i t r o g e n a s e activity w h i c h r e m a i n s at a c o n s t a n t level. However, as s h o w n in table IV, tile b i o s y n t h e s i s of n/f-specific polypeptides is rap i d l y abolished. It thus seems that t r a n s c r i p t i o n of the nil genes is t u r n e d off w h e n NH4 + ions are added and that the observed biosyntheses correspond to the t r a n s l a t i o n of p r e e x i s t i n g mRNAs. Nevertheless, the k i n e t i c s of the residual b i o s y n theses is not s i m i l a r for all the nit p r o d u c t s studied. The nifU p r o d u c t is no longer synthesized 5 m i n u t e s after NH4 + ions a d d i t i o n w h e r e a s some nffH p r o d u c t is still synthesized at a low rate
K. pneumoniae nif gene products. after 60 minutes. All our results are in agreement with the h y p o t h e s i s of T u b b and Postgate E23] who postulate that the repression by a m m o n i u m is effective at the level of mRNA synthesis. However the m e c h a n i s m by w h i c h repression is mediated is not clearly understood. Acknowledgements.
The authors wish to thank Drs R. A. Dixon and M. Merrick for gift of bacterial strains, Dr. J.-P. Aubert for helpful discussions and Dr. J. Shapiro for critical reading of the manuscript. D. Bogusz was the recipient of an O.R.S.T.O.M. studentship. R. Biyault was on leave from the Entreprise Mini~re et Chimique (E.M.C.). This investigation was supported bg research funds from the University Paris VII and by a grant from the D.G.R~.T. (No. 78-7-0~0). REFERENCES. 1. Merriek, M., Filser, M., K e n n e d y , C. ,¢ Dixon, R. (1978) Molec. yen. Genel., 165, 103-111. 2. E l m e r i c h , C., H o u m a r d , J., Sibold, L., M a n h e i m e r , I. ~ C h a r p i n , N. (1978) Molec. gen. Genet., 165, 181-189. 3. MacNeil, T., MacNeil, D., Roberts, G. P., Supiano, M. ~ Brill, W. J. (1978) J. Bacteriol., 136, 253-266. 4. Merriek, M., Filser, M., Dixon, R., E l m e r i c h , C., Sibold, L. ~ H o u m a r d , J. (1980) J. Gen. Microbiol., 117, 509-520. 5. Elmerieh, C. (1979) Physiol. Vdff., 17, 883-906. 6. Roberts, G. P., MacNeil, T., MacNeil, D. ~ Brill, W. J. (1978) J. Baeteriol., 136, 267-279. 7. St. J o h n , R. T., J o h n s t o n , M. H., Seidman, C., Garfinkel, D., Gordon, J. K., Shah, V. K. ~ Brill, W. J. (1975) J. Bacteriol., 121, 759-765.
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