365
Biochimica et Biophysica Acta, 565 (1979)365--378 © Elsevier/North-Holland Biomedical Press
BBA 99562
THE BIOSYNTHESIS OF NITROGENASE MoFe PROTEIN POLYPEPTIDES IN FREE-LIVING CULTURES OF RHIZOBIUM.[APONICUM
D. B A R R Y SCOTT, H A U K E H E N N E C K E
and SOOT. LIM
Plant Growth Laboratory and Department of Agronomy and Range Science, Universityof California,Davis, CA 95616 (U.S.A.) (Received April 20th, 1979)
Key words: Nitrogen fixation; Nitrogenase polypeptide synthesis; (Rhizobium japonicum)
Summary The biosynthesis of the constituent polypeptides of nitrogenase component I (Rj 1) in free-living cultures of Rhizobium ]aponicum (strain 110) was investigated under different growth conditions. Cells were pulse~labelled and the proteins analysed by one and two-dimensional gel electrophoresis. The positions of the constituent Rj 1 polypeptides were identified by co-electrophoresis with purified Rj 1 isolated from bacteroids of soybean nodules, and by comparison with an immunoprecipitate from a culture induced for nitrogenase. The synthesis of the proteins preceded any detectable enzyme activity and increased with time, reaching a maximum after 3 days. At this time, between 6 and 8% of the total sodium dodecyl sulfate-soluble protein synthesized was Rj 1. Exposure to air led to a dramatic decrease in the rate of Rj 1 synthesis, with almost complete regression after 20 min. In the presence of KNO3, there was no nitrogenase activity, but the proteins were present in similar amounts (7%) as the control culture. When mannitol and glycerol were used as the sole carbon sources, the amount of Rj 1 synthesized was extremely low. Introduction The nitrogen-fixing symbiosis established between leguminous plants and Rhizobium spp. is a complex process involving biochemical, physiological and morphological changes in both the symbiont and the host. The development of a free-living nitrogen-fixing system [1--5] has enabled studies to be carried out Abbreviations: SDS, s o d i u m dodecyl sulfate; Mops, 3-(N-mor~holino)propane sulfonlc acid; Hepes, N-2hydroxyethylpiperazine-N'-2-ethanesul fonic acid.
366 on the regulation of nitrogen fixation in Rhizobium, separated from the host. Considerable progress has been made on the elucidation of the physiological requirements needed for the formation of whole-cell nitrogenase activity in this asymbiotic system [6--12]. The main requirements for maximum rates of nitrogen fixation are the use of an appropriate carbon (e.g. gluconate) and nitrogen (e.g. glutamate) source together with a very low dissolved oxygen concentration (1 gM, Ref. 6) in the media. Although some of the fixed nitrogen can be assimilated for growth [11,13,14] most of it is exported into the media as NH~ [11], a situation analogous to that found for isolated bacteroids
[151. At the molecular level, the establishment of nitrogenase activity involves a n u m b e r of steps including the biosynthesis of the constituent polypeptides of the enzyme, the incorporation of cofactors, the functioning of an electron transport system to nitrogenase, and the availability of a sufficient energy supply. As a first step in studying the regulation of nitrogen fixation (nil) gene expression in Rhizobium ]aponicum, we decided to investigate the biosynthesis of the nitrogenase polypeptides using the techniques of pulse-labelling [16,17] and polyacrylamide gel electrophoresis. The effect of oxygen, nitrate, and various carbon sources on nitrogenase c o m p o n e n t I (Rj 1) biosynthesis was investigated. Methods
Bacterial strains and growth conditions. R. japonicum strain 3Ilb 110 was obtained from D.F. Weber, US Department of Agriculture, Beltsville. A nitrate reductase mutant of R. japonicum strain 3Ilb 110 (R. ]aponicum strain C33) was obtained from S.H. Hua. This mutant was isolated as a chlorate-resistant derivative, following N-methyl-N'-nitro-N'-nitrosoguanidine (Aldrich Chemical Co.) mutagenesis. Stock cultures of Rhizobium were maintained on afar slants of mannitol/ salts/yeast extract medium [11]. The medium used for the induction of nitrogenase in free-living cultures of R. ]aponicum was as described previously [11]. The carbon sources used included both malate (1.5 g . 1-1) and gluconate (4 g • 1-1) and the nitrogen source was L-glutamate (1.0 g • 1-1). In one experim e n t (see Table II) several individual carbon sources, all at a concentration of 4 g • 1-1, were used. The medium was buffered with 50 mM morpholinopropane sulfonic acid (Mops), pH 6.8. Cultures (2 ml) were inoculated with a 2% (v/v) inoculum (A420 = 0.12, 1 cm light path) and the flasks (70 ml) evacuated and backfilled with argon four times. The gas phase was adjusted to 3% acetylene and 97% argon and the cultures incubated at 25°C with gentle shaking (100 rev./min). After 48 h oxygen was added to the gas phase (0.1% final concentration}. Subsequent additions of oxygen were made every 12 h to maintain a concentration of 0.1% in the gas phase. In experiments requiring large volumes of cells, 250-ml cultures were grown under identical conditions in 2.2-1 Buchner flasks. Growth was followed b y measuring the absorbance at 420 nm in a Gilford Model 300 N s p e c t r o p h o t o m e t e r (1 cm light path) and also by the increase in cell protein as previously described [11]. Protein was routinely assayed by the procedure of L o w r y et al. [18], using bovine serum albumin as a standard.
367
Enzyme assays. Whole cell nitrogenase activity was determined using the acetylene reduction procedure [19] using a Varian Aerograph Model 1400 equipped with a flame ionization detector and a Porapak R column. For the purification of Rj 1, in vitro nitrogenase activity was determined using a partially purified fraction of Rj 2. Assays were carried out essentially as described by Whiting and Dilworth [20]. The reaction mixture as modified contained in a final volume of 1.5 ml: 30 mM Hepes buffer (pH 7.5), 6.7 mM MgCI2, 26.8 mM creatine phosphate, 2.7 mM ATP, 4.4 units creatine phosphokinase (Sigma), 13.3 mM sodium dithionite, and a suitable amount of enzyme. Nitrate reductase activity in crude cell-free extracts was determined by a modification of the procedure described by Lowe and Evans [21]. The reaction mixture contained in a final volume of 1 ml: 50 mM potassium phosphate buffer (pH 7.0), 20 mM KNO3, 0.15 mM methyl viologen, 2 mM KCNO, 4.6 mM sodium dithionite, 9.5 mM NaHCO3, and an appropriate dilution of enzyme. The reaction was started by the addition of sodium dithionite and incubated at 30°C for 5 rain. Linear rates of reaction were maintained for this period only in the presence of KCNO [22]. Reactions were terminated by vortexing the mixture, and the concentration of nitrite was determined as previously described [23]. Purification of nitrogenase. Field-grown soybean plants (Glycine max cv. Evans) nodulated with a commercial inoculum of R. ]aponicum (Nitragin Co., Milwaukee) were supplied by D.A. Phillips. The enzyme was purified from bacteroids of fully developed nodules essentially according to Whiting and Dflworth [20] with modifications as described below. The diethyl aminoethyl cellulose (DEAE) column was equilibrated in the described buffer [20] conraining 0.05 M NaC1. Component I, or Rj 1, following the nomenclature of Kennedy et ai. [24] was eluted with 0.15 M NaC1. The gel filtration step was performed on a Biogel A-5m (Bio Rad) column (80 × 2.5 cm) equilibrated in 50 mM tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.4, containing 0.2 M NaC1, 4 mM sodium dithionite and 0.2 mM dithiothreitol. To remove further impurities it was necessary to repeat the gel filtration step. The enzyme is about 95% pure as determined by gel electrophoresis. It cross-reacts weakly with antibodies against purified component I from Klebsiella pneumoniae. The molecular weight is approximately 240 000 as determined on a calibrated gel filtration column (Biogel A-5m). Upon gel electrophoresis in the presence of sodium dodecyl sulfate (SDS; using BDH and Serva brands), Rj 1 separates into two polypeptide chains [24] of molecular weights 56 000 (a subunit) and 58 000 (~ subunit) as shown in Fig. la. Radioactive labelling of R. ]aponicum. Samples (0.5 ml) of cells were removed anaerobically from a nitrogenase-induced culture and injected into At-filled serum bottles (10 ml) containing 50/~Ci of L-[35S]methionine (561.21 Ci/mmol, New England Nuclear). The gas phase was adjusted to 0.1% (v/v) O2 and the samples then incubated for 20 rain at 28°C. The labelling was stopped by the addition of 1 mg of unlabelled L-methionine. After 5 min further incubation, the cells were chilled and collected by centrifugation (15 000 ×g, 3 rain). The pellet was then washed three times with 0.9% (w/v) NaC1 to remove exopolysaccharides [25]. The labelled cells were then frozen and stored at --20°C.
368
Electrophoresis and autoradiography. The labelled cells were analyzed by both one
Biochimica et Biophysica Acta, 565 (1979)365--378 © Elsevier/North-Holland Biomedical Press
BBA 99562
THE BIOSYNTHESIS OF NITROGENASE MoFe PROTEIN POLYPEPTIDES IN FREE-LIVING CULTURES OF RHIZOBIUM.[APONICUM
D. B A R R Y SCOTT, H A U K E H E N N E C K E
and SOOT. LIM
Plant Growth Laboratory and Department of Agronomy and Range Science, Universityof California,Davis, CA 95616 (U.S.A.) (Received April 20th, 1979)
Key words: Nitrogen fixation; Nitrogenase polypeptide synthesis; (Rhizobium japonicum)
Summary The biosynthesis of the constituent polypeptides of nitrogenase component I (Rj 1) in free-living cultures of Rhizobium ]aponicum (strain 110) was investigated under different growth conditions. Cells were pulse~labelled and the proteins analysed by one and two-dimensional gel electrophoresis. The positions of the constituent Rj 1 polypeptides were identified by co-electrophoresis with purified Rj 1 isolated from bacteroids of soybean nodules, and by comparison with an immunoprecipitate from a culture induced for nitrogenase. The synthesis of the proteins preceded any detectable enzyme activity and increased with time, reaching a maximum after 3 days. At this time, between 6 and 8% of the total sodium dodecyl sulfate-soluble protein synthesized was Rj 1. Exposure to air led to a dramatic decrease in the rate of Rj 1 synthesis, with almost complete regression after 20 min. In the presence of KNO3, there was no nitrogenase activity, but the proteins were present in similar amounts (7%) as the control culture. When mannitol and glycerol were used as the sole carbon sources, the amount of Rj 1 synthesized was extremely low. Introduction The nitrogen-fixing symbiosis established between leguminous plants and Rhizobium spp. is a complex process involving biochemical, physiological and morphological changes in both the symbiont and the host. The development of a free-living nitrogen-fixing system [1--5] has enabled studies to be carried out Abbreviations: SDS, s o d i u m dodecyl sulfate; Mops, 3-(N-mor~holino)propane sulfonlc acid; Hepes, N-2hydroxyethylpiperazine-N'-2-ethanesul fonic acid.
366 on the regulation of nitrogen fixation in Rhizobium, separated from the host. Considerable progress has been made on the elucidation of the physiological requirements needed for the formation of whole-cell nitrogenase activity in this asymbiotic system [6--12]. The main requirements for maximum rates of nitrogen fixation are the use of an appropriate carbon (e.g. gluconate) and nitrogen (e.g. glutamate) source together with a very low dissolved oxygen concentration (1 gM, Ref. 6) in the media. Although some of the fixed nitrogen can be assimilated for growth [11,13,14] most of it is exported into the media as NH~ [11], a situation analogous to that found for isolated bacteroids
[151. At the molecular level, the establishment of nitrogenase activity involves a n u m b e r of steps including the biosynthesis of the constituent polypeptides of the enzyme, the incorporation of cofactors, the functioning of an electron transport system to nitrogenase, and the availability of a sufficient energy supply. As a first step in studying the regulation of nitrogen fixation (nil) gene expression in Rhizobium ]aponicum, we decided to investigate the biosynthesis of the nitrogenase polypeptides using the techniques of pulse-labelling [16,17] and polyacrylamide gel electrophoresis. The effect of oxygen, nitrate, and various carbon sources on nitrogenase c o m p o n e n t I (Rj 1) biosynthesis was investigated. Methods
Bacterial strains and growth conditions. R. japonicum strain 3Ilb 110 was obtained from D.F. Weber, US Department of Agriculture, Beltsville. A nitrate reductase mutant of R. japonicum strain 3Ilb 110 (R. ]aponicum strain C33) was obtained from S.H. Hua. This mutant was isolated as a chlorate-resistant derivative, following N-methyl-N'-nitro-N'-nitrosoguanidine (Aldrich Chemical Co.) mutagenesis. Stock cultures of Rhizobium were maintained on afar slants of mannitol/ salts/yeast extract medium [11]. The medium used for the induction of nitrogenase in free-living cultures of R. ]aponicum was as described previously [11]. The carbon sources used included both malate (1.5 g . 1-1) and gluconate (4 g • 1-1) and the nitrogen source was L-glutamate (1.0 g • 1-1). In one experim e n t (see Table II) several individual carbon sources, all at a concentration of 4 g • 1-1, were used. The medium was buffered with 50 mM morpholinopropane sulfonic acid (Mops), pH 6.8. Cultures (2 ml) were inoculated with a 2% (v/v) inoculum (A420 = 0.12, 1 cm light path) and the flasks (70 ml) evacuated and backfilled with argon four times. The gas phase was adjusted to 3% acetylene and 97% argon and the cultures incubated at 25°C with gentle shaking (100 rev./min). After 48 h oxygen was added to the gas phase (0.1% final concentration}. Subsequent additions of oxygen were made every 12 h to maintain a concentration of 0.1% in the gas phase. In experiments requiring large volumes of cells, 250-ml cultures were grown under identical conditions in 2.2-1 Buchner flasks. Growth was followed b y measuring the absorbance at 420 nm in a Gilford Model 300 N s p e c t r o p h o t o m e t e r (1 cm light path) and also by the increase in cell protein as previously described [11]. Protein was routinely assayed by the procedure of L o w r y et al. [18], using bovine serum albumin as a standard.
367
Enzyme assays. Whole cell nitrogenase activity was determined using the acetylene reduction procedure [19] using a Varian Aerograph Model 1400 equipped with a flame ionization detector and a Porapak R column. For the purification of Rj 1, in vitro nitrogenase activity was determined using a partially purified fraction of Rj 2. Assays were carried out essentially as described by Whiting and Dilworth [20]. The reaction mixture as modified contained in a final volume of 1.5 ml: 30 mM Hepes buffer (pH 7.5), 6.7 mM MgCI2, 26.8 mM creatine phosphate, 2.7 mM ATP, 4.4 units creatine phosphokinase (Sigma), 13.3 mM sodium dithionite, and a suitable amount of enzyme. Nitrate reductase activity in crude cell-free extracts was determined by a modification of the procedure described by Lowe and Evans [21]. The reaction mixture contained in a final volume of 1 ml: 50 mM potassium phosphate buffer (pH 7.0), 20 mM KNO3, 0.15 mM methyl viologen, 2 mM KCNO, 4.6 mM sodium dithionite, 9.5 mM NaHCO3, and an appropriate dilution of enzyme. The reaction was started by the addition of sodium dithionite and incubated at 30°C for 5 rain. Linear rates of reaction were maintained for this period only in the presence of KCNO [22]. Reactions were terminated by vortexing the mixture, and the concentration of nitrite was determined as previously described [23]. Purification of nitrogenase. Field-grown soybean plants (Glycine max cv. Evans) nodulated with a commercial inoculum of R. ]aponicum (Nitragin Co., Milwaukee) were supplied by D.A. Phillips. The enzyme was purified from bacteroids of fully developed nodules essentially according to Whiting and Dflworth [20] with modifications as described below. The diethyl aminoethyl cellulose (DEAE) column was equilibrated in the described buffer [20] conraining 0.05 M NaC1. Component I, or Rj 1, following the nomenclature of Kennedy et ai. [24] was eluted with 0.15 M NaC1. The gel filtration step was performed on a Biogel A-5m (Bio Rad) column (80 × 2.5 cm) equilibrated in 50 mM tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.4, containing 0.2 M NaC1, 4 mM sodium dithionite and 0.2 mM dithiothreitol. To remove further impurities it was necessary to repeat the gel filtration step. The enzyme is about 95% pure as determined by gel electrophoresis. It cross-reacts weakly with antibodies against purified component I from Klebsiella pneumoniae. The molecular weight is approximately 240 000 as determined on a calibrated gel filtration column (Biogel A-5m). Upon gel electrophoresis in the presence of sodium dodecyl sulfate (SDS; using BDH and Serva brands), Rj 1 separates into two polypeptide chains [24] of molecular weights 56 000 (a subunit) and 58 000 (~ subunit) as shown in Fig. la. Radioactive labelling of R. ]aponicum. Samples (0.5 ml) of cells were removed anaerobically from a nitrogenase-induced culture and injected into At-filled serum bottles (10 ml) containing 50/~Ci of L-[35S]methionine (561.21 Ci/mmol, New England Nuclear). The gas phase was adjusted to 0.1% (v/v) O2 and the samples then incubated for 20 rain at 28°C. The labelling was stopped by the addition of 1 mg of unlabelled L-methionine. After 5 min further incubation, the cells were chilled and collected by centrifugation (15 000 ×g, 3 rain). The pellet was then washed three times with 0.9% (w/v) NaC1 to remove exopolysaccharides [25]. The labelled cells were then frozen and stored at --20°C.
368
Electrophoresis and autoradiography. The labelled cells were analyzed by both one
