Legume root nodule nitrogenase

Biochimica et Biophysica Acta, 371 (1974) 337-351 © Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands

BBA 36888 LEGUME ROOT NODULE NITROGENASE P U R I F I C A T I O N , P R O P E R T I E S , A N D S T U D I E S O N ITS G E N E T I C C O N T R O L

M. J. WHITING* and M. J. DILWORTH Department of Soil Science and Plant Nutrition, Institute of Agriculture, University of Western Australia, Nedlands (Australia) (Received June 17th, 1974)

SUMMARY 1. Nitrogenase from Rh&obium lupini root nodule bacteroids has been resolved into two components, which have been purified by a simple method involving chromatography on DEAE-cellulose and Sephadex G-150. Some properties of the components have been determined and found overall to be very similar to those from free-living N2-fixing bacteria. 2. Anaerobic and sodium dodecylsulphate gel electrophoresis of each component produced only one band. Component 1 had a molecular weight around 200 000, and appeared to be a tetramer composed of identical subunits with N-terminal serine. It contained 1 atom Mo and 18-20 atoms Fe per molecule. 3. Component 2 had a molecular weight of 65 000, and was a dimer of identical subunits also with N-terminal serine. It was extremely 02 sensitive, with a half-life of 1 min in air, and contained 3-4 atoms Fe per molecule but no Mo. 4. An attempt was made to answer the question of whether the plant and/or the bacterium contributes the genetic information for nitrogenase in the legume root nodule. Two dissimilar legumes, yellow lupin and serradella, were nodulated by the same strain of R. lupini and yellow lupin was nodulated by a serologically different strain of R. lupini. Nitrogenase components were purified from the three nodule types and analysed by a number of criteria. All Component 1 and all Component 2 samples were indistinguishable by anaerobic or sodium dodecylsulphate gel electrophoresis. The Component 1 samples could not be distinguished immunologically. Amino acid analysis and limited peptide mapping also revealed no conclusive differences between components from the various plant-Rhizobium symbioses. 5. The results favour Rhizobium as having both nitrogenase genes, although the possibility of a conservative nitrogenase gene existing in the plant cannot be eliminated. INTRODUCTION Nitrogenase, the enzyme complex that catalyses the reduction of N 2 to NH3, * Present address: The Rockefeller University, New York, N.Y. 10021, U.S.A.

338 as well as the reduction of other compounds such as acetylene, C N - and N3-, has now been extensively purified from the free-living bacteria Clostridium pasteurianum [1, 2], Klebsiella pneumoniae [3] and Azotobacter vinelandii [4-6], and some of its properties have been determined (for recent reviews see refs 7-9). In all cases, purification leads to dissociation of the enzyme into two O2-sensitive components, which are both essential for enzyme activity. Component 1 has a molecular weight in excess of 200 000 and contains Mo and Fe, while Component 2 has a molecular weight of around 60 000 and contains Fe, but no Mo. Legume root nodules also contain nitrogenase as one of several proteins resuiting from the symbiosis between plant and Rhizobium. Other proteins known to be produced only after the establishment of this intimate relationship are leghemoglobin and several electron carriers responsible for transferring electrons to nitrogenase [10]. Although the enzyme is found within the Rhizobium bacteroids which are enclosed in membrane packets inside the plant cell [11], the location of the genes coding for the enzyme components has not been proven to be in the bacterial cell. This is because free-living rhizobia do not contain any measurable nitrogenase activity, and all attempts to isolate mutants capable of reducing N2 or acetylene [12], or to induce enzyme activity in laboratory-grown rhizobia with plant extracts [13], have failed. Even growth of R. japonicum under anaerobic conditions similar to those experienced by the bacteroid in the plant cell, does not apparently allow synthesis of nitrogenase components [12], although several factors capable of transferring electrons to R. japonicum nitrogenase, and not found in aerobically grown cells, are formed [14]. To explain the absence of nitrogenase in free-living Rhizobium, and the production of enzyme only after interaction between rhizobia and plant cells during the establishment of the legume root nodule, Dilworth and Parker [15] suggested that the genetic information for some part of the enzyme complex is divided between plant and bacterium. This division could have arisen during evolution of the symbiosis by gene transfer from a now non-existent free-living Rhizobium capable of fixing N2. An analogy may exist in the endosymbiotic theory of the origin of cell organelles [16] in that this theory implies gene transfer from prokaryote symbiont to host cell during evolution. An example of a dual gene location for an enzyme is that of the two subunits of Fraction 1 protein, which catalyses the first step in photosynthetic CO2 fixation in chloroplasts. The gene for the small subunit is in the plant nucleus, while the gene coding for the large subunit is in the chloroplast [17]. To test for a possible plant influence on the structure of the components of nitrogenase, the approach used in this work is similar to that used by Dilworth [18] in showing that the plant is the genetic determinant for leghemoglobin. Two generically different legumes, lupin and serradella, have been nodulated with the same strain of R. lupini and the nitrogenase components purified and examined by a number of criteria for any plant-determined molecular differences. A similar approach has been used by Phillips et al. [12] in studies on the genetic control of Component 1 from R. japonicum bacteroids. Electrophoretic methods failed to reveal any effect of plant or rhizobial differences, and amino acid analysis indicated significant differences in four amino acids due to rhizobial variation and in two due to plant variation. The conclusion, which was only tentative, was that Component 1 was a rhizobial gene product, but the close phylogenetic similarity of the two legumes used did not constitute a rigorous test for potential plant influences.

339 A't the commencement of this work, no methods were available for the complete anaerobic purification of legume root nodule nitrogenase components. Earlier investigations of the partially purified enzyme from R.japonieum bacteroids had shown it was similar to the enzyme from free-living bacteria in consisting of two components [19, 20]. An anaerobic purification method was therefore developed and this enabled a number of properties of both nitrogenase components isolated from R. lupini bacteroids to be determined, and compared with those of the enzyme from other sources. Very recently, R. japonicum Component 1 has been obtained in an homogeneous form [21]. METHODS

Production of nodules Seeds of yellow lupin (Lupinus luteus L. cultivar Weico III) and serradella (Ornithopus sativus Brot.) were inoculated with either R. lupini Strain W U 8 or WU 425, and plants grown as described by Dilworth [18]. Nodules were picked before flowering and checked for rhizobial strain with specific antisera [22]. Lupin nodules were used fresh, while serradella nodules were sometimes snap-frozen in liquid N2 and stored at --15 °C for later use.

Isolation of bacteroids Because of the extreme 02 sensitivity of nitrogenase, all operations were carried out with particular care to exclude 02. Nodules (200 g) were homogenised under N 2 in 250 ml of de-aerated buffer containing 0.05 M potassium phosphate (pH 7.4), 0.2 M sucrose, 2 ~o (w/v) soluble polyvinylpyrollidone and 0.5 mM dithiothreitol, and bacteroids isolated as described by Kennedy [23]. The washed bacteroids were finally suspended in l0 ml of a buffer containing 0.05 M TES (pH 7.4), 2 mM MgCl2, 5 mM dithiothreitol and 5 mM Na28204 and sonicated under a stream of N2 for 15 min in a cooled Raytheon 9-kHz-type sonicator (Raytheon Co., Mass.) at full power. Cell debris was removed by centrifugation at 80 000 x g for 30 rain in Nz-flushed tubes. The supernatant, with a specific activity in the range of 20-30 nmoles acetylene reduced/min per mg protein, was stored in liquid N 2.

Separation of nitrogenase components by DEAE-ceHulose chromatography The following methods were developed using WU 8 bacteroid extracts prepared from lupin nodules. Dark-brown nitrogenase extracts were treated for around 60 min at room temperature with 2 mg ribonuclease A and 300 #g deoxyribonuclease I (Sigma Chemical Co., Mo.) before loading with a syringe on a DEAE-cellulose column (30 cm × 2.5 cm) previously equilibrated anaerobically at 15 °C at a flow rate of 55 ml/h with 0.05 M Tris-HC1 buffer (pH 7.4) containing 0.1 M NaCI, 2 mM NazSzO4 and 0.2 mM dithiothreitol. With this buffer, a pink-brown band of protein was eluted from the column, followed by a dark-brown, Fe-containing band which corresponded to nitrogenase Component 1. Further washing of the column with buffer plus 0.1 M MgC12 eluted three brown bands, with the second corresponding to nitrogenase Component 2. Fractions of 20 ml were collected manually in Nz-filled serum bottles.

340 0

g 160

~

12C

5O u 8C

o z

5

10

15 Fraction number

20

25

3O

100

Fig. 1. Anaerobic chromatography of R. lupini WU 8 extract on DEAE-cellulose equilibrated with 0.2 mM dithiothreitol, 2 mM Na2S204 and 0.1 M NaCI in 0.05 M Tris-HCl buffer, pH 7.4. Details are described in the text. Aliquots (0.2 ml) from Fractions 1-24 were assayed for Component 1 activity by the acetylene reduction method after combination with 0.2 ml of Fraction 24 as a source of Component 2. Similarly, Fractions 20-28 (0.2 ml) were assayed for Component 2 activity after adding 0.2 ml of Fraction 10 as a source of Component 1. All fractions were assayed for Fe by atomic absorption spectrometry. Absorbance at 280 nm of the column eluate was monitored with an LKB Uvicord II continuous-flow spectrophotometer. , absorbance at 280 nm; O---Q, iron content; A--A, Component 1 activity; I - - I I , Component 2 activity. Fig. 1 shows the results o f Fe analyses and acetylene reduction assays on the eluted fractions. To detect acetylene reducing activity, it was necessary to mix fractions to ensure that both components were present in the assay (see Fig. 1). The profile obtained indicated that the nitrogenase complex had been separated into two components, coinciding with the two major Fe peaks. A small a m o u n t of C o m p o n e n t 1 (approx. 1 0 ~ ) was not removed f r o m the column with equilibrating buffer, but eluted when 0.1 M MgC12 was added. Fractions containing C o m p o n e n t 2 had measurable nitrogenase activity (spec. act. 30 units/mg) without added C o m p o n e n t 1.

Sephadex gel chromatography Active fractions f r o m the DEAE-cellulose column were concentrated anaerobically by ultrafiltration t h r o u g h an A m i c o n U M - 2 0 E membrane (Amicon Corp., Mass). Concentrated components (5 ml) were then separately run on a column of Sephadex G-150 (86 cm × 2.5 cm) previously equilibrated anaerobically with 0.05 M Tris-HC1 buffer (pH 7.4) containing 2 m M NazSzOa and 0.2 m M dithiothreitol at a flow rate of 20 ml/h. Fractions were again collected in N2-filled serum bottles. Elution profiles for C o m p o n e n t s 1 and 2 showed that acetylene reducing activity determined in the presence of excess of the complementary c o m p o n e n t coincided with the major peak o f 280-nm-absorbing material and protein-bound Fe. Yields and specific activities of the purified components at each stage o f the purification are shown in Table I.

341 TABLE I PURIFICATION OF THE COMPONENTS OF NITROGENASE FROM WU 8 BACTEROIDS OF LUPIN NODULES See the text for experimental details. A unit of activity is defined as the amount of enzyme catalysing the formation of 1 nmole ethylene/min. Fraction

Vol. Total activity (ml) (units)

1. Bacteroid extract 13 2. DEAE-cellulose fractions Component 1 40 Component 2 21 3. Sephadex G-150 fractions Component 1 25 Component 2 24

Total protein (mg)

31 720

1040

42 600 15 180

124 65

10 940 6 240

18.5 14.4

Spec.act. (units/mg) 30.5 344 233 704 434

Yield (~) 100 134 48 79* 25*

* Overall yield; not all of each component from Step 2 was chromatographed at one time.

Enzyme assays Nitrogenase activity was measured by the acetylene reduction method [19]. The assay mixture contained the following compounds adjusted to p H 7.4, in a final volume of 1 ml: TES buffer, 50 #moles; creatine phosphate, 25/~moles; ATP, 2.5 #moles; MgCI2, 5/~moles; and creatine phosphokinase, 0.2 mg. Serum bottles were evacuated and flushed three times with 10 ~o acetylene in Ar before injection of enzyme and pre-incubation at 30 °C for 3 min. Reactions were started with 20 #moles of Na2S204 taken from a neutral 200 m M solution prepared anaerobically. After 15 min shaking at 90 strokes/min at 30 °C, assays were terminated with 0.3 ml of 10 ~o (w/v) trichloroacetic acid. Ethylene formation was measured at 48 °C on a Pye model 104 gas chromatograph (W. G. Pye, Cambridge, U.K.) fitted with a Poropak R column (1.5 m × 4 mm) with N2 as carrier gas at a flow rate of 30 ml/min.

Acrylamide gel electrophoresis Anaerobic electrophoresis in 7 . 5 ~ gels was carried out in the Tris-glycine (pH 8.9) system of Davis [24], modified by including 1 m M NazSzO4 in the upper buffer reservoir and continually flushing the gas space above it with N2. Gels were pre-run at 2 mA/gel for 30 min before samples containing 10~o (w/v) sucrose were loaded anaerobically with a syringe on top of the gels. Proteins were stained with 1 (w/v) amido black in 7 ~ acetic acid, and Fe-containing proteins detected with 1 (w/v) a,a'-dipyridyl in 7 ~o acetic acid. Sodium dodecylsulphate gel electrophoresis was carried out by the method of Weber and Osborne [25] and gels stained with coomassie blue according to Fairbanks et al. [26].

Metal determinations The Fe and Mo content of protein solutions was determined directly by spraying into the flame of a Perkin-Elmer 403 atomic absorption spectrophotometer (Perkin-Elmer, Norwalk, Conn.). Appropriate standards were prepared in enzyme buffer. In addition, known amounts of standard were added to protein solutions as a check on the revovery of metal in the presence of protein.

342

S-carboxymethylation of purified components Reduction and S-carboxymethylation of freeze-dried protein was carried out essentially as described by Harrap and Woods [27] except that conditions for reduction were altered to 3 h at 37 °C in a solution at pH 10.5 containing 8 M urea, 0.5 M ethanolamine, and 0.1 M 2-mercaptoethanol. Amino acid analysis Samples of S-carboxymethylated nitrogenase components (1 mg) were hydrolysed in 1 ml of 6 M HC1 plus one drop of 5 ~ phenol at 110 °C for 24 and 72 h [28]. Hydrolysates were analysed by the method of Piez and Morris [29] on a Beckman 120C analyser (Beckman Instruments Inc., Calif.). N-terminal analysis The S-carboxymethylated components were dansylated as described by Thompson et al. [30] and N-terminal amino acids identified by chromatography on polyamide sheets [31], using the solvent systems of Ramshaw et al. [32]. Immunological techniques Antiserum to Component 1 from R. lupini WU 8 bacteroids was prepared in rabbits by subcutaneous injection of 1 ml of purified component (1 mg) emulsified in an equal volume of complete Freund's adjuvant (Commonwealth Serum Laboratories, Victoria). 2 weeks later, a further I mg of antigen was injected intraperitoneally, followed a further 2 weeks later by 0.5 mg of protein (0.5 ml) in adjuvant. Blood samples were collected from the marginal ear vein, and tested for cross-reacting material by the Ouchterlony double-diffusion method in agar [33]. A strong precipitin line was obtained from sera collected 6-10 days after the third series of injections. Protein estimations For crude bacteroid extracts, the biuret reagent [34] was used, while dilute solutions of purified components were assayed by the method of Lowry et al. [35]. Bovine serum albumin (Sigma Chemical Co., Mo.) was used as standard. RESULTS

Properties of purified R. lupini WU 8 nitrogenase components Behaviour upon anaerobic disc gel electrophoresis. When samples of Component 1 or Component 2 obtained from the final Sephadex step of the purification procedure were electrophoresed anaerobically at pH 9.5, only one band was observed after staining with amido black (see Fig. 2B). These single bands were visible prior to staining, since both components were coloured brown, and they also stained pink with a,a'-dipyridyl, indicating the presence of Fe. Electrophoresis of samples from the DEAE-cellulose step prior to Sephadex chromatography (Fig. 2A) showed that even at this stage of the purification method, the nitrogenase components were the major proteins present in their respective fractions. Molecular weights of the native components. The Sephadex G-150 column used in the purification procedure was calibrated with proteins of known molecular weight,

34J

IA

Fig. 2. Acrylamide gel electrophoresis of purified nitrogenase Components 1 and 2. Anzerobic 7.5 acrylamide gels were loaded with approx. 50 pg of protein and stained with amido black. (A) Fractions from DEAE-cellulose chromatography. (B) Fractions from Sephadex chromatography. Sodium dodecylsulphate acrylamide gels loaded with approx. 25 pg of S-carboxymethylated protein were stained with coomassie blue. (C) Fractions from Sephadex chromatography.

and the molecular weights of the nitrogenase components calculated from their elution volumes, according to Andrews [36]. Values obtained were 194 000 for Component 1 and 65 000 for Component 2, as shown in Fig. 3. Molecular weights of the subunits of the components. Nitrogenase components were denatured by incubation with sodium dodecylsulphate, and electrophoresed on 1 0 ~ sodium dodecylsulphate acrylamide gels [25]. Both Component 1 and Component 2 appeared to be composed of one type of subunit (see Fig. 2C). By comparison of electrophoretic mobilities with protein standards, as seen in Fig. 4, molecular weights of 57 000 and 32 000, respectively were calculated. Densitometric scanning of the gels showed both components were at least 90 ~ pure. Amino acid composition and N-terminal analysis. Duplicate samples of S-carboxymethylated components were hydrolysed for 24 and 72 h, and the relative amounts ot each amino acid present were calculated. The amino acid compositions of Componenl 1 and Component 2 are shown in Table II.

344

ochrome c 300

~Qe- chymotrypsin 13~ochrome c

oxidase

E

v 25C bovine ~ik~OserumX ~-o\ -(5 >

g 5Ixl

dimer

mponent 2

albumin

hexokinase-~ "~actate

dehydrogenase

2oc

human ,v -globulinoe n"~c' t la"Pt°r~a ~'i 4.0

415 log

510 %5 (moleculor weight)

Fig. 3. Molecular weight estimations of nitrogenase components by gel filtration on a calibrated column of Sephadex G-150.

Both Components 1 and 2 clearly showed serine as the only N-terminal amino acid. Metal content. Fe and Mo determinations by atomic absorption spectrophotometry showed Component 1 to contain 18-20 g atoms Fe/mole and 1.0 g atoms Mo/ mole, while Component 2 contained 3.1-3.2 g atoms Fe/mole and no Mo.

• ,~bovine serum albumin 4.EJCo bQcatalase mpoT . \ nent 10~-globuiin (H)chain \

45

O~reatine phosphokinase

Component 2

pepsin

%

\

E

•-~ 4.4 ~:

~ n

(L)chain

8 4.2

ribonuclease 4.C

0.2

Q4

0'6

08 --

mobility

Fig. 4. Molecular weight determination of the subunits of nitrogenase components from R. lupim

bacteroids by sodium dodecylsulphate acrylamide electrophoresis. The conditions and molecular weights of protein standards were as described by Weber and Osborne [25].

345 TABLE II A M I N O ACID COMPOSITIONS OF NITROGENASE COMPONENTS F R O M R. L U P I N I WU 8 BACTEROIDS ISOLATED F R O M LUPIN ROOT NODULES Values for Ser, Thr, Tyr and Met were extrapolated to zero hydrolysis time, while values for Scarboxymethylated Cys, Val and Ile are 72-h recoveries. Other figures represent the average of 24and 72-h hydrolyses. Amino acid

Component l*

Component 2**

Ala Arg Asx S-carboxymethylated Cys Glx Gly His Ile Leu Lys Met Phe Pro Ser Thr Tyr Val Trp***

137 89 178

63 29 64

30 168 156 67 107 124 119 50 85 77 99 93 73 105 (29)

l0 72 59 14 45 57 30 14 9 19 35 23 22 36

* Residues per 200 000 molecular weight. ** Residues per 65 000 molecular weight. *** Not determined; value assumed as for R. japonieum Component 1 [21].

02 sensitivity. Both components were rapidly inactivated upon exposure to air, as seen in Fig. 5. As noted for other nitrogenases, Component 2 was especially labile with a half-life of only 1 rain. lO0

50

"6

5

10 1'5 Time exposed to oir (rain)

Fig. 5. Inactivation of purified nitrogenase components from R. lupini bacteroids by air. Components obtained by Sephadex gel filtration were exposed to air and shaken in a water bath at 30 °C over 2.5 cm at a rate of 80 strokes/rain. At various times, samples were taken and assayed for residual acetylene-reducing activity in the presence of excess complementary component. 0 - - 0 , residual activity of Component 1 ; O - - O , residual activity of Component 2.

346

Comparison of nitrogenase components from different plant-rhizobium symbioses The development of a relatively rapid method for purifying both nitrogenase Components 1 and 2 from lupin nodules, and the finding that the method was applicable to serradella nodules, made it possible to compare the properties of the enzyme isolated from different plant-Rhizobium combinations. The combinations used were (a) lupin-R, lupini WU 8 (L-8); (b) lupin-R, lupini WU 425 (L-425); and (c) serradella -R. lupini WU 425 (S-425). These combinations were chosen because lupin and serradella belong to different tribes of Leguminales and therefore have considerable genetic differences. Nevertheless, both these legumes can be effectively nodulated by R. lupini WU 425. If the plant does contain part of the genetic information for nitrogenase as postulated by Dilworth and Parker [l 5], comparison of the properties of nitrogenase components from combinations L-425 and S-425 might reveal a difference. A complication in this work arose, however, due to problems in growing the required large amounts of serradella under conditions where only one infective rhizobial strain was present. Lupin nodules showed only the inoculant strain when tested serologically. However serradella nodules tested serologically were found to be only 70-75 ~ type WU 425, the remainder being WU 8. In spite of this difficulty, nitrogenase components were purified by the described methods from the three nodule types, and their properties compared by the following techniques. Anaerobic aerylamide gel electrophoresis. Individual components and mixtures of components containing (L-8 + L-425) and (L-425 -? S-425) were electrophoresed anaerobically on 7.5 ~ acrylamide gels and stained with amido black. All Component 1 samples showed one identical band similar to that in Fig. 2. The Component 2 samples also showed a single identical band, with a trace of diffuse material running behind the band occasionally appearing in some gels. This probably represents an Oz denaturation product since the same samples when run on sodium dodecylsulphate acrylamide gels gave only one band. Ouchterlony immunodiffusion. An antiserum was prepared in rabbits against purified L-8 Component l, and tested for cross-reaction against other purified Component 1 fractions by the Ouchterlony double-diffusion method [33]. As seen in Fig. 6, all fractions cross-reacted identically, giving a single continuous precipitin line with no sign of spur formation. A crude extract of laboratory-grown R. lupini WU 8 did not show any cross-reaction, indicating for the first time by immunological means that nitrogenase Component l protein is not produced in the non-symbiotic form of

Rhizobium. Amino acid analysis. Purified Components 1 and 2 from L-8, L-425 and S-425 were compared by amino acid analysis. All samples used gave one band on anaerobic acrylamide gel electrophoresis. Sodium dodecylsulphate gel electrophoresis loading 50/~g of protein per gel did show minor bands in L-425 and S-425 gels. However, scanning of these gels with a densitometer indicated that the major nitrogenase band accounted for at least 85-90 ~ of the protein sample. Since the molecular weights of the appropriate components from the various legume-Rhizobium systems were essentially identical, the amino acid analyses were converted to numbers of residues for molecular weights of 200 000 and 65 000 for Components 1 and 2, respectively. The results are shown in Table III. Allowing for experimental variation, determined to be around 5 ~ from

347

Fig. 6. Ouchterlony immunodiffusion of various nitrogenase Component 1 fractions against antibodies prepared against L-8 Component 1. Outside wells contained 20/4 of purified fractions (500 /~g/ml) under test. They were (1) S-425 Component 1, (2) soluble extract of laboratory-grown R. lupini W U 8, (3) saline, (4) L-425 Component 1, (5) L-8 Component 1, (6) L-8 Component 1. The centre well contained the ~-globulin fraction prepared from antiserum to L-8 Component l by treatment with an equal volume of cold saturated (NH4)2SO4, and dissolving the precipitated protein in 0.15 M NaC1 in 0.01 M sodium phosphate buffer, pH 7.2. TABLE lII C O M P A R I S O N OF A M I N O ACID COMPOSITIONS O F N I T R O G E N A S E C O M P O N E N T S F R O M D I F F E R E N T LEGUME-RHIZOB1UM SYMBIOSES Amino acid

Ala Arg Asx S-carboxymethylated Cys Glx Gly His Ile Leu Lys Met Phe Pro Ser Thr Tyr Val Trp***

Component 1"

Component 2**

L-425

L-8

S-425

L-425

L-8

S-425

157 91 178

137 89 178

178 91 177

66 30 63

63 29 64

67 31 66

23 162 170 63 114 130 118 0 86 82 116 107 66 118 (29)

30 168 156 67 107 124 119 50 85 77 99 93 73 105 (29)

20 170 164 59 109 137 121 18 77 80 105 109 59 123 (29)

9 65 62 13 45 56 29 13 11 21 38 26 20 40

10 72 59 14 45 57 30 14 9 19 35 23 22 36

10 69 61 11 48 57 30 11 10 20 34 23 22 39

* Residues per 200 000 molecular weight. ** Residues per 65 000 molecular weight. *** Not determined; value assumed as for R. japonicum Component 1 [21].

348 separate analyses of duplicate samples, examination of the figures for Component 1 shows that differences do apparently exist, especially in alanine, S-carboxymethylcysteine, methionine, serine, tyrosine and valine. However, the values for S-carboxymethylcysteine, methionine and possibly tyrosine may not be reliable due to the variable recovery characteristic of these amino acids after protein hydrolysis. Since in this work it is assumed only one type of Component 1 is produced in the nodule, and that the genetic information for this component must either be in the plant or the Rhizobium, it would be expected that the figures for either L-425 and S-425 (plant variation), or L-425 and L-8 (rhizobial variation) would be the same. In fact, the results show that the variations that do exist between L-425 and S-425 are of the same magnitude as those between L-425 and L-8. From these figures then, no good evidence is obtained favouring the plant or the Rhizobium as having the genetic information for Component 1. The data for Component 2 show that within experimental error no differences can be detected between any of the different plant-Rhizobium combinations. Peptide mapping. Comparitive peptide mapping of tryptic digests of S-carboxymethylated components did not resolve the total number of peptides expected from the amino acid compositions of the components, although some separation of peptides was obtained. Nevertheless, no differences were observed in the overall fingerprint patterns from Component 1 samples, or Component 2 samples, although the peptide patterns for Component 1 and Component 2 were different. DISCUSSION The molecular properties of highly purified nitrogenase components isolated from R. lupini bacteroids have been found to be very similar to those of the enzyme from free-living Nz-fixing bacteria and R. japonicum bacteroids. Component 1 has a molecular weight of 194 000 by gel chromatography and is separated into subunits of molecular weight 57 000 by sodium dodecylsulphate. Unlike those from C. pasteurianum [37] and K. pneumoniae Component 1 [3], the subunits appear to be identical and have an N-terminal serine. Similar molecular weights have recently been reported for R. japonicum Component 1 [21]. A value of 200 000 was obtained for the native protein, while the subunit size was estimated at 55 000 on sodium dodecylsulphate acrylamide gels, and 50 000 by sedimentation analysis in guanidine. HC1 [21]. The tetrameric structure of Component 1 suggested by the molecular weight data was confirmed by electron microscopy of negatively stained preparations (Whiting, M. J., Armitage, T. and Dilworth, M. J., unpublished). Particles of the expected size composed of four subunits in a square planar arrangement were seen, along with other aggregated material which probably resulted from 02 denaturation of the protein during sample preparation. An electron microscopy study of Azotobacter Component 1 has also shown a tetrad arrangement of subunits [38]. The measured amounts of 1.0 g atom Mo/mole and 18-20 g atoms Fe/mole are lower than the values of !.3 g atoms Mo/mole and 29 g atoms Fe/mole reported for R. japonicum Component I [21 ], although the final specific activities of the purified proteins are the same. It seems likely that 10ss of Mo has occurred during the purifica-

349 tion procedure, as has been shown to occur for C. pasteurianum Component 1 [37], which has 2 g atoms Mo/mole in its highest specific activity form of around 2500 nmoles acetylene reduced/rain per mg. Component 2 has not previously been highly purified from Rhizobium bacteroids. The method described yields a preparation which shows only one band on anaerobic and sodium dodecylsulphate acrylamide gels. Although the final specific activity of the preparation was much lower than that reported for purified Component 2 from either C. pasteurianum [1] or A. vinelandii [4, 6], this is probably due to the extreme 02 sensitivity of this component, leading to rapid inactivation by traces of 02 present during the purification procedure. The Fe content of 3.1-3.2 g atoms/mole is less than the value of 4.0 reported for Component 2 from C. pasteurianum [39] and K. pneumoniae [3]. The molecular weight of 65 000 for the native component and subunit molecular weight of 32 000, with single N-terminal serine, indicate R. lupini Component 2 is a dimeric molecule similar to Component 2 from C. pasteurianum [2] and K. pneumoniae [3]. If indeed both Component 1 and Component 2 are homopolymers, only two genes need be involved in nitrogenase structure specification. Accordingly, plantRhizobium complementation between dissimilar subunits within Component 1 would need to be rejected. The amino acid compositions of both R. lupini nitrogenase components show a very similar pattern to the enzyme from other bacteria. Indeed, a statistical comparison of available amino acid composition data has already been carried out, confirming that all Component 1 and all Component 2 molecules form very closely related groups [9]. This unexpected conservatism of the nitrogenase enzyme through evolution has made it difficult to determine conclusively the location of legume root nodule nitrogenase genes by the experimental approach used in this work. No conclusive differences have been detected between nitrogenase components from lupin nodules produced by R. lupini WU 8 or lupin or serradella nodules produced by R. lupini WU 425. Even comparison of the amino acid compositions of Component 1 from L-8, L-425 and S-425 with Component 1 from soybean nodules produced by R. japonicum reveals that the values for this different plant and different Rhizobium combination are very similar to the figures in Table III. In a similar study of Component 1 fractions, Phillips et al. [12] tentatively concluded on the basis of very small differences in amino acid compositions that Rhizobium carries the genetic information for this component. However, the plants used were both from the tribe Phaseolae of the Leguminales and may not have been of sufficient genetic diversity for differences to be detected in any putative plant nitrogenase gene. Since no one symbiont has been clearly shown to define the structure of either nitrogenase component, it is still possible that a nitrogenase gene could reside in legumes. However, this gene, unlike the leghemoglobin genes, must be very similar in both lupin and serradella to enable differences in the gene product to have escaped detection by the electrophoretic, immunological and amino acid composition criteria used. On the other hand, both nitrogenase genes may in fact be present in the Rhizobium in a very tightly repressed state. It follows that since growth in almost N2-free conditions at low pO2 does not induce nitrogenase synthesis, the repression is not the

350 same as in other Nz-fixing bacteria where r e m o v a l o f fixed N2 from the environment induces nitrogenase. I f the nitrogenase genes are repressed in Rhizobium, it seems genetic techniques will be required to d e m o n s t r a t e their presence. This could be a c c o m p l i s h e d directly by the isolation o f a m u t a n t capable o f reducing N2 u n d e r a n a e r o b i c conditions, or by transfer ofRhizobium D N A to an e n v i r o n m e n t where nitrogenase genes can be transcribed and translated, leading to the f o r m a t i o n o f active enzyme. Using this latter a p p r o a c h , D u n i c a n a n d Tierney [40] have claimed that rhizobial D N A transferred to a m u t a n t Klebsiella that could n o t fix N2 resulted in the isolation o f h y b r i d Klebsiella c a p a b l e o f reducing acetylene. However, whether this enzyme activity resulted from expression o f Rhizobium genes, Klebsiella genes, o r both, with c o m p l e m e n t a t i o n of nitrogenase c o m p o n e n t s , remains to be proven. The c o n t r o l system in the hybrids was also one of NH3 repression, which a p p e a r s unlikely to be the case in Rhizobium, a n d m a y indicate t h a t the genetic material transferred to K. aerogenes m a y not have contained nitrogenase genes but some other necessary gene for Klebsiella nitrogenase expression. ACKNOWLEDGEMENTS The a u t h o r s w o u l d like to t h a n k M r s M. G r o u n d s a n d Miss J. S p a c k m a n for technical assistance, D r H. H a r d i n g (University o f A d e l a i d e ) for c o n d u c t i n g the a m i n o acid analyses, a n d D r M. R i c h a r d s o n (University o f D u r h a m ) for carrying out the N - t e r m i n a l analyses. This research was s u p p o r t e d in p a r t by a g r a n t f r o m the Australian Research G r a n t s C o m m i t t e e , a n d in p a r t by funds f r o m the Soil Fertility Research F u n d o f Western Australia. REFERENCES 1 Tso, M.-Y. W.. Ljones, T. and Burris, R. H. (1972) Biochim. Biophys. Acta 267, 600-604 2 Mortensen, L. E. (1972) Methods in Enzymology Vol. XXIV, pp. 446-456 Academic Press, New York 3 Eady, R. R., Smith, B. E., Cook, K. A. and Postgate, J. R. (1972) Biochem. J. 128, 655-675 4 Burns, R. C. and Hardy, R. W. F. (1972) Methods in Enzymology Vol. XXIV, pp. 480-496, Academic Press, New York 5 Bulen, W. A. and Le Comte, J. R. (1972) Methods in Enzymology Vol. XXIV, pp. 456-470, Academic Press, New York 6 Shah, V. K. and Brill, W. J. (1973) Biochim. Biophys. Acta 305, 445-454 7 Postgate, J. R. (1971) The Chemistry and Biochemistry of Nitrogen Fixation, Plenum Press, London 8 Dalton, H. and Mortenson, L. E. (1972) Bacteriol. Rev. 36, 231-260 9 Dilworth, M. J. (1974) Ann. Rev. Plant Physiol., in the press 10 Evans, H. J. and Russell, S. A. (1971) The Chemistry and Biochemistry of Nitrogen Fixation, (Posgate, J. R., ed.), pp. 191-244, Plenum Press, London 11 Koch, B., Evans, H. J. and Russell, S. A. (1967) Proc. Natl. Acad. Sci. U.S. 58, 1343-1350 12 Phillips, D. A., Howard, R. L. and Evans, H. J. (1973) Physiol. Plant 28, 248-253 13 Bergersen, F. J. (1971) Ann. Rev. Plant Physiol. 22, 121-140 14 Phillips, D. A., Daniel, R. M., Appleby, C. A. and Evans, H. J. (1973) Plant Physiol. 51,136-138 15 Dilworth, M. J. and Parker, C. A. (1969) J. Theor. Biol. 25, 208-218 16 Margulis, L. (1970) Origin of Eukaryotic Cells, Yale University Press, New Haven 17 Wildman, S. G., Kawashima, N., Bourgue, D. P., Wong, F., Singh, S., Chan, P. H., Kwok, S. Y., Sakano, K., Kung, S. D. and Thornber, J. P. (1972) The Biochemistry of Gene Expression in

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