Purification and properties of the adenylate kinases from Rhodopseudomonas palustris,Rhodopseudomonas sphaeroides and Rhodopseudomonas rubrum

The adenylate kinases (EC 2.7.4.3) from photosynthetically grown Rhodopseudomonas palustris, Rhodopseudomonas sphaeroides and Rhodospirillum rubrum were purified to homogeneity by the same procedure. The purified enzymes showed optimal rates of activity with MgCl₂ at 25° C and pH 8.0. They were found to be heat labile and were characterized by pI-values of 4.5. Apparent molecular weights of 33 500 for R. palustris, 34 400 for R. sphaeroides and 32 100 for R. rubrum were determined by high performance liquid chromatography. No separation into subunits was observed by use of sodium dodecylsulfate polyacrylamide gel electrophoresis. The apparent K _m -values for ADP corresponded to 0.26 mM for R. palustris, 0.27 mM for R. sphaeroides and 0.24 mM for R. rubrum. ADP in excess had a strong inhibitory effect. Competitive product inhibition was found for AMP, with K _i-values of 0.017 mM for R. palustris, 0.018 mM for R. sphaeroides and 0.014 mM for R. rubrum. A competitive inhibitor likewise was P¹,P⁵-di(adenosine-5)pentaphosphate with K _i-values of 0.020 M for R. palustris and R. sphaeroides, and 0.017 M for R. rubrum. Sulfhydryl-reacting reagents like p-chloromercuribenzoate and iodoacetic acid were found to be non-inhibitory. All measurements of adenylate kinase activity were carried out with the stabilized and most sensitive luciferin-luciferase system.     

Mutants of Rhodopseudomonas sphaeroides useful in genetic analysis

Two kinds of mutants of Rhodopseudomonas sphaeroides that should be useful in extending genetic analysis of this organism have been isolated. One is deficient in recombination and has been used to isolate derivatives of the plasmid R 68.45 which incorporate chromosomal genes of R. sphaeroides. The other is apparently defective in a DNA restriction enzyme; transfer of plasmid borne chromosomal genes of R. sphaeroides from Escherichia coli back to R. sphaeroides is greatly enhanced in these mutants.In memory of R. Y. Stanier     

DNA-DNA hybridization of Rhodopseudomonas capsulata,Rhodopseudomonas sphaeroides and Rhodopseudomonas sulfidophila strains

The genetic relatedness of 21 Rhodopseudomonas strains has been studied by means of DNA-DNA hybridization. All strains included in the study belonged to the subgroup of the genus Rhodopseudomonas which is characterized by a short-rod to coccus morphology, a vesicular intracytoplasmic membrane system and carotenoids of the spheroidene group. Mol percentages guanine + cytosine ranged from 64 to 73, most strains having values between 68 and 72. With few exceptions, the hybridization data obtained were in agreement with the subdivision in three (or possibly four) species on the basis of classical taxonomy. Strain SCJ, formerly considered to be a somewhat atypical R. capsulata strain, is most probably a R. sphaeroides strain and two out of seven strains that were received as R. sulfidophila did not fit in this species on the basis of the hybridization data. The results also showed that two undesignated strains that were previously thought to be related to R. capsulata (Hansen et al. 1975) cannot be assigned to this species and may be representatives of another species. The seven strains that required approximately 2.5% NaCl in the medium and that had been designated R. sulfidophila were found to synthesize far higher levels of bacteriochlorophyll during fully aerobic growth in the dark than the purple bacteria studied thus far.Abbreviations GC guanosine + cytosine - SSC standard saline citrate buffer     

Inhibition of nitrogenase by nitrite and nitric oxide in Rhodopseudomonas sphaeroides f. sp. denitrificans

In cells of Rhodopseudomonas sphaeroides f. sp. denitrificans nitrite and nitric oxide, the products of denitrification, inhibit activity of nitrogenase enzyme.Ferredoxin-linked CO₂ fixation, with H₂ as a reductant, was also inhibited by nitrite and NO in denitrifying cells.EPR spectroscopy of cell preparations treated with NO showed that it reacts with non-haem iron-sulphur proteins to form iron-nitrosyl complexes. Nitrite also reacts with these iron-sulphur proteins, but the formation of ironnitrosyl complexes was dependent on the presence of dithionite. Since nitrite is reduced to NO by dithionite it is likely that nitrogenase and CO₂ fixation reactions are inhibited not only by nitrite itself, but also by nitric oxide.Abbreviation DPPH 1,1-diphenyl-2-picrylhydrazyl     

Changes in Rhodopseudomonas sphaeroides isoaccepting phenylalanyl-tRNA species during transitions from chemoheterotrophic to photoheterotrophic growth

A new iso-accepting tRNA^phe from extracts of chemoheterotrophic and photoheterotrophic cells of Rhodopseudomonas sphaeroides has been identified by both BDEAE cellulose and RPC-5 chromatography. Rechromatography of each of the tRNA^phe species in either the acylated or deacylated state shows that they migrate as single homogeneous peaks.In steady-state chemoheterotrophic cultures of R. sphaeroides tRNA _I–II ^phe account for 25–30% of the total phenylalanine accepting activity while in steadystate photoheterotrophic cultures tRNA _I–II ^phe account for no more than 10% of the total phenylalanine accepting activity.During the transition from chemoheterotrophic to photoheterotrophic growth conditions the levels of tRNA _I–II ^phe fall in an exponential manner during the first half of the intracytoplasmic membrane induction period. tRNA _I ^phe then remains at a level 10% that of its steady-state chemoheterotrophic level as long as photoheterotrophic growth conditions remain. tRNA _II ^phe , after dropping to 10% of its former chemoheterotrophic level then returns to a level 50% that of its chemoheterotrophic level as long as photoheterotrophic growth conditions remain.Abbreviations BDEAE benzoylated diethyl amino ethyl - RPC reversed phase chromatography - TCA tricholroacetic acid - ICM intracytoplasmic membrane Submitted by WDS in partial fullfilment of requirements for the M.S. degree     

Long-term effects of nitrate on lucerne (Medicago sativa L.) nitrogen fixation is not influenced by the denitrification status of the microsymbiont

Studies on the inhibitory effects of combined nitrogen on biological nitrogen fixation in legume crops have been usually carried out after short-term nitrate treatments at high concentrations. As these treatments are quite different from field conditions, a study was conducted to evaluate the effects of the continuous presence of nitrate (0, 1, 5 and 10 mM) throughout three months on lucerne (Medicago sativa L.). Plants were grown in a greenhouse with perlite as substrate and were inoculated with a denitrifying Sinorhizobium meliloti strain (102-F-51) and a non-denitrifying strain (102-F-65). During the first 60 days of growth, the highest nitrate treatment resulted in a complete inhibition of the main symbiotic parameters (nodule initiation and development and specific nitrogen fixation) in plants inoculated with either strain. However, after 3 months of growth in the presence of nitrate, this inhibition was partly abolished, with a high number of new functioning nodules being formed. Acetylene reduction activity (ARA) of these plants was 70% of the control plants. As this process was observed in plants nodulated with either strain, it is concluded that this was not related to the denitrifying ability of the strain, but is an intrinsic property of the lucerne nitrogen fixing system. As legume plants usually grow under natural field conditions in the continuous presence of nitrate, the ability to use simultaneously nitrate and atmospheric nitrogen could be of adaptive and agronomic importance. This revised version was published online in June 2006 with corrections to the Cover Date.     

Growth and adaptation to phototrophic conditions of Rhodospirillum rubrum and Rhodopseudomonas sphaeroides at different temperatures

The influence of temperature on yields of cell protein and bacteriochlorophyll as well as on the rates of growth and bacteriochlorophyll synthesis was studied with Rhodospirillum rubrum and Rhodopseudomonas sphaeroides. Under chemotrophic conditions net cell-protein production increased in cultures of both species along with temperature from 14°C up to the optimum at 33°C. Under phototrophic conditions cell-protein yields were largely constant within the range from 21°C to 33°C. At temperatures below 21°C and above 33°C yields decreased. These results are interpreted in terms of coupling between energy yielding or redox equivalent providing metabolisms and cell biosynthesis. Upon adaptation from chemotrophic to phototrophic conditions a direct relationship between temperature increase and bacteriochlorophyll level was observed. Arrhenius plots of both, specific growth rates and rates of bacteriochlorophyll synthesis, revealed discontinuities at about 20°C. Temperature coefficients either above or below those discontinuities were similar in both species. In R. rubrum temperature coefficients of the synthesis of total bacteriochlorophyll were also representative of the synthesis of photochemical reaction center and light harvesting bacteriochlorophylls. But in R. sphaeroides significant differences were observed between temperature coefficients of the syntheses of bacteriochlorophylls of the costantly composed reaction centerlight harvesting complex on one hand and of both, total and the quantitatively variable light harvesting bacteriochlorophylls on the other. The results are interpreted in light of hypotheses on the regulation (a) of cellular bacteriochlorophyll levels as well as (b) of the ratio of functionally different bacteriochlorophylls in the photosynthetic apparatus.Abbreviation Bchl bacteriochlorophyll     

Temperature dependence of membrane-bound enzymes of the energy metabolism in Rhodospirillum rubrum and Rhodopseudomonas sphaeroides

Rhodospirillum rubrum grown either chemotrophically or phototrophically at 14°C and 30°C, was employed to study the effect of temperature on fatty acid composition as well as on several membrane bound functions involved in energy metabolism. Upon growth at both temperatures the fatty acid composition of membranes showed differences, which could be attributed to an incomplete formation of photosynthetically active membranes rather than specifically to the growth temperature. Activities of NADH dependent respiration and light induced proton extrusion by cells did not show discontinuities in Arrhenius plots down to temperatures of 15°C and 5°C, respectively. In contrast, coupling factor Mg²⁺- and Ca²⁺-ATPase as well as succinate cytochrome c oxidoreductase showed significant breaks at 20°C and 18°C, respectively. Similarly, in Rhodopseudomonas sphaeroides. NADH dependent respiration and light induced proton extrusion by cells was continuous over the entire range of temperatures applied. ATPase as well as succinate cytochrome c oxidoreductase, on the other hand, featured discontinuities in Arrhenius plots at 20°C and 19°C. The implication of the data on growth rates and membrane structure are discussed.Abbreviation Bchl baceteriochlorophyll     

Denitrification in Rhodopseudomonas sphaeroides f. denitrificans

Rhodopseudomonas sphaeroides f. denitrificans grown photosynthetically with NO ₃ ^- under anaerobic conditions accumulated NO ₂ ^- in the culture medium. In washed cells succinate, lactate, fumarate, citrate and malate, were effective electron donors for the reduction of NO ₃ ^- , NO ₂ ^- and N₂O to N₂ gas. Nitrate reductase was inhibited by amytal and potassium thocyanate. Nitrite reductase activity was severely restricted by potassium cyanide, sodium diethyldithiocarbamate, Amytal and 2-n-heptyl-4-hydroxyquinoline-N-oxide whereas N₂O reductase was inhibited by NaN₃, C₂H₂ and KCNS. Cells incubated with either K¹⁵NO₃ or K¹⁵NO₂ produced ¹⁵N₂O and ¹⁵N₂. A stoichiometry of 2:1 was recorded for the reduction of either NO ₃ ^- or NO ₂ ^- to N₂O and N₂ and for N₂O to N₂ it was 1:1.Abbreviations BVH reduced benzyl viologen - MVH reduced methyl viologen - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - CCCP carbonyl cyanide-m-chlorophenyl-hydrazone - DIECA diethyl dithiocarbamate - KCN potassium cyanide     

The photosynthesis gene cluster of Rhodobacter sphaeroides

The photosynthetic bacteria are at the forefront of the study of many aspects of photosynthesis, including photopigment biosynthesis, photosynthetic-membrane assembly, light-harvesting, and reaction center photochemistry. The facultative growth of some photosynthetic bacteria, their simple photosystems, and their ease of genetic manipulation have all contributed to advances in these areas. Amongst these bacteria, the purple non-sulfur bacterium Rhodobacter sphaeroides has emerged as, arguably, the leading contender for a model system in which to integrate the studies of all the different aspects of the assembly and function of the photosynthetic apparatus. Many of the genes encoding photosynthesis-related proteins are known to be clustered within a small region of the genome in this organism. As a further aid to studying the assembly and function of the photosystem of Rb. sphaeroides, the DNA sequence for a genomic segment containing this photosynthesis gene cluster (PGC) has been assembled from previous EMBL submissions and formerly unpublished data. The Rb. sphaeroides PGC is 40.7 kb in length and consists of 38 open reading frames encoding the reaction center H, L and M subunits, the and polypeptides of the light-harvesting I (B875) complex, and the enzymes of bacteriochlorophyll and carotenoid biosynthesis. PGCs are a feature of gene organization in several photosynthetic bacteria, and the similarities between the clusters of Rb. sphaeroides and Rb. capsulatus have been apparent for some time. Here we present the first comprehensive analysis of the PGC of Rb. sphaeroides, as well as a comparison with that of Rb. capsulatus.     

Denitrification in lucerne nodules is not involved in nitrite detoxification

Dissimilatory reduction of ionic nitrogen oxides to gaseous forms such as nitrous oxide or nitrogen can be carried out by free living or symbiotic forms of some strains of Rhizobium meliloti. In this paper we investigate whether bacteroid denitrification plays a role in the alleviation of the inhibitory effects of nitrate on nitrogen fixation both in bacteroid incubations as in whole nodules. The presence of a constitutive nitrate reductase (NR) activity in isolated bacteroids caused nitrite accumulation in the incubation medium, and acetylene reduction activity in these bacteroids was progressively inhibited, since nitrite reductase (NiR) activity was unable to reduce all the nitrite produced by NR and denitrification occurred slowly. Even nodules infiltrated with nitrate and nitrite failed to increase gaseous forms of nitrogen substantially, indicating that nitrite availability was not limiting denitrification by bacteroids. In spite of the low rates of bacteroidal denitrification, the effect of nodule denitrification on the inhibition of nitrogen fixation by nitrate in whole plants was tested. For that purpose, lucerne plants (Medicago sativa L. cv. Aragon) were inoculated with two Rhizobium meliloti strains: 102-F-65 (non denitrifying) and 102-F-51 (a highly denitrifying strain). After a seven days nitrate treatment, both strains showed the same pattern of inhibition, and it occurred before any nitrate or nitrite accumulation within the nodules could be detected. This observation, together with the lack of alleviation of the ARA inhibition in the denitrifying strain, and the limited activity of dissimilatory nitrogen reduction present in these bacteroids, indicate a role other than nitrite detoxification for denitrification in nodules under natural conditions.     

Oxidation of cytochrome c 2 by photosynthetic reaction centers of Rhodospirillum rubrum and Rhodopseudomonas sphaeroides in vivo. Effect of viscosity on the rate of reaction

In Rhodospirillum rubrum and Rhodopseudomonas sphaeroides it is shown that the oxidation of cytochrome c ₂ involves a diffusion limited process. From analysis of the results it follows that the electron transfer probability must be very low. This is corroborated by in vitro studies using the isolated components.     

Periplasmic location of dissimilatory nitrate and nitrite reductases in a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroides forma sp. denitrificans

The localization of dissimilatory nitrate and nitrite reductasesof a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroidesforma sp. denitrificans, was investigated. Nitrate and nitritereductases were located in the periplasmic space of the bacteriumgrown anaerobically in the presence of nitrate either in lightor in darkness. Chromatophores showed nitrate and nitrite reductaseactivities when dithionite-reduced benzyl viologen was an electrondonor; this suggests that the enzymes were trapped inside thevesicles. ¹Present address: Japanese Red Cross Central Blood Center, Hiroo4-1-31, Shibuyaku, Tokyo 150, Japan. ²Present address: Plant Growth Laboratory, University of California,Davis, California 95616, U.S.A. (Received November 7, 1979; )     

Sporomusa,a new genus of gram-negative anaerobic bacteria including Sporomusa sphaeroides spec. nov. and Sporomusa ovata spec. nov.

A new genus of strictly anaerobic, gram-negative, banana-shaped bacteria is described. Cells formed spores and were motile by means of up to 15 laterally inserted flagella. Nitrate or sulfate were not used as electron acceptor. Organic substrates that were fermented included N-methyl compounds, such as betaine, N,N-dimethylglycine and sarcosine, primary alcohols, hydroxy fatty acids, and 2,3-butanediol. In addition, molecular hydrogen and carbon dioxide were fermented to acetate. The latter was the characteristic fermentation product in general. During growth on betaine, trimethylamine was formed in addition. The degradation of N,N-dimethylglycine yielded acetate, monomethylamine, and trimethylamine. The presence of cytochrome b and of ubiquinone in the cells was shown. The deoxyribonuleic acid base composition of the strains was between 41.3 and 47.4 mol% guanine plus cytosine. The name Sporomusa is proposed for this new genus. On the basis of the DNA-DNA homology values obtained, the shape of the spores and some other properties, the isolated strains were assigned to two species. Names proposed: Sporomusa sphaeroides and Sporomusa ovata. The type species is S. sphaeroides and the type strains are strain E, DSM 2875 (S. sphaeroides) and strain H1, DSM 2662 (S. ovata).Dedicated to Prof. H. G. Schlegel on the occasion of his 60th birthday     

Ultrastructure of carotenoid mutant strain R-26 of Rhodopseudomonas sphaeroides

Stained thin-sections and freeze-fractured preparations of the carotenoid-less mutant strain R-26 of Rhodopseudomonas sphaeroides grown photosynthetically revealed 2 morphological kinds of intracellular membrane systems- spherical vesicles distributed throughout the cytoplasm and lamellae confined to the periphery of the cell. The lamellar membranes appeared to be large, flattened vesicles.Non-Standard Abbreviations R Rhodopseudomonas - E exoplasmic-half - P protoplasmic-half     

Sites of phospholipid biosynthesis during induction of intracytoplasmic membrane formation in Rhodopseudomonas sphaeroides

A rapid, gratuitous and cell-division uncoupled induction of intracytoplasmic photosynthetic membrane formation was demonstrated in low-aeration suspensions of chemotrophically grown Rhodopseudomonas sphaeroides. Despite a nearly 2-fold increase in phospholipid levels, no significant increases were detected in the specific activities of CDP-1,2-diacyl-sn-glycerol:sn-glycerol-3-phosphate phosphatidyltransferase (phosphatidylglycerophosphate synthase, EC 2.7.8.5) and CDP-1,2-diacyl-sn-glycerol:L-serine O-phosphatidyltransferase (phosphatidylserine synthase, EC 2.7.8.8), the first committed enzymes of anionic and zwitterionic phospholipid biosyntheses, respectively. The distribution of phosphatidylglycerophosphate and phosphatidylserine synthase activities after rate-zone sedimentation of cell-free extracts indicated that intracytoplasmic membrane phospholipids were synthesized mainly within distinct domains of the conserved cytoplasmic membrane. Labeling studies with ³²P_i and L-[³H]phenylalanine suggested that preexisting phospholipid was utilized initially as the matrix for insertion of intracytoplasmic membrane protein that was synthesized and assembled de novo during induction.Abbreviations BChl bacteriochlorophyll a - B800-850, B875 peripheral and core light-harvesting BChl-protein complexes, respectively, identified by near-IR absorption maxima This paper is dedicated to Professor Gerhart Drews on the occasion of his sixtieth birthday     

Homologies in processing and sequence between the 23S ribosomal ribonucleic acids of Paracoccus denitrificans and Rhodopseudomonas sphaeroides

We have shown that mature 50S ribosomal subunits of Paracoccus denitrificans lack intact 23S rRNA, containing instead rRNAs of 0.56 (16S) and 0.37 (14S)x10⁶ molecular weight. Kinetic labelling studies showed these to be derived from a 1.02x10⁶ dalton precursor, which may itself derive from a larger and very transient 23S species. A similar pattern of rRNA processing has been previously described for Rhodopseudomonas sphaeroides, and we have compared, by Tl oligonucleotide catalog analysis, the smaller (14S) fragments of P. denitrificans and R. sphaeroides 23S rRNAs. These were shown to exhibit strong sequence homology, and comparisons of 14S-derived oligonucleotides to oligonucleotides from an in vitro-generated 13S fragment of Escherichia coli 23S rRNA suggest that P. denitrificans and R. sphaeroides 14S rRNAs arise from the 5-terminal portions of their respective 23S precursors. Results are considered to be consistent with the claim that P. denitrificans arose, by loss of photophosphorylation, from a member of the Rhodospirillaceae.Abbreviations E buffer 60 ml 2 M Tris base, 20 ml 3 M sodium acetate, 15 ml 0.2M disodium EDTA, 6 ml glacial acetic acid, 900 ml distilled water - HEPES N-2-hydroxymethyl piperazine-N-2-ethanesulfonic acid - TMK 5 mM Tris-Cl, 0.1 mM MgSO₄, 60 mM KCl. pH 7.3 - TM3 10 mM Tris-Cl, 1 mM MgCl₂, pH 7.3 - Tris tris (hydroxymethyl) aminomethane - SDS sodium dodecyl sulphate     

Nitrate respiration,denitrification, and utilization of nitrogen sources by aerobic carbon monoxide-oxidizing bacteria

We describe the ability of carboxydotrophic bacteria for nitrate respiration or denitrification. Four out of fourteen strains examined could denitrify heterotrophically forming N₂ (Pseudomonas carboxydoflava) or N₂O (Pseudomonas carboxydohydrogena, Pseudomonas compransoris, and Pseudomonas gazotropha). Three carried out a heterotrophic nitrate respiration (Arthrobacter 11/x, Azomonas B1, and Azomonas C2). P. carboxydohydrogena could use H₂ as electron donor for nitrate respiration under chemolithoautotrophic growth conditions. CO did not support denitrification or nitrate respiration of carboxydotrophic bacteria, although the free energy changes of the reactions would be sufficiently negative to allow growth. CO at 50 kPa was a weak inhibitor of N₂O-reduction in carboxydotrophic and non-carboxydotrophic bacteria and decelerated denitrifying growth. Carboxydotrophic bacteria could utilize a wide range of N-sources. Results obtained with a plasmid-cured mutant of Pseudomonas carboxydovorans OM5 showed, that genes involved in nitrogen assimilation entirely reside on the chromosome. In the presence of an suitable electron donor, most carboxydotrophic bacteria could carry out a reduction of nitrate to nitrite that did not support growth and did not lead to the formation of ammonia.This article is dedicated to Professor Hans G. Schlegel on the occasion of his 65th birthday and in admiration for his élan and eternal idealism     

Regulatory properties of the ADPglucose pyrophosphorylase from Rhodopseudomonas sphaeroides and from Rhodopseudomonas gelatinosa

Highly purified fractions of chlorosomes and cytoplasmic membranes were isolated from Chloroflexus aurantiacus Ok-70-fl and Chlorobium limicola 6230. These fractions were comparatively analyzed for their pigmentation, phospholipid, glycolipid, and cytochrome c content as well as for their specific activities of succinate dehydrogenase and NADH-oxidase. The data showed that there are some differences in pigmentation and phospholipid content between the isolated fractions of Chloroflexus and Chlorobium. Chlorosomes of Chloroflexus contained a specific BChl a-complex with a characteristic absorption maximum at about 790 nm. This BChl a-complex could not be detected in spectra of chlorosomes from Chlorobium. The near infrared region of the spectra of the isolated cytoplasmic membranes of both organisms revealed considerable differences: The BChl a-complexes of Chloroflexus membranes exhibited peaks at 806 and 868 nm whereas the membranes of Chlorobium had a single BChl a-peak at 710 nm. In contrast to the findings with Chlorobium the chlorosomes of Chloroflexus contained at least twice as much phospholipids as did the cytoplasmic membranes. In Chlorobium the phospholipid content of cytoplasmic membranes is three times that of their chlorosomes. The distribution of all other components (carotenoid composition, enzyme activities, cytochrome c content, and glycolipids) was about the same in both strains. From the data it was concluded that differences in the organization of the photosynthetic apparatus are mainly based on differences of the organization of the photosynthetic units in the cytoplasmic membrane and probably the kind of linkage of the light harvesting system in the chlorosomes with the reaction center in the cytoplasmic membranes.Abbreviations BChl c bacteriochlorophyll c - BChl a bacteriochlorophyll a - DSM Deutsche Sammlung von Mikrorganismen     

Contribution of dissolved dinitrogen in culture solutions to growth of Rhodopseudomonas capsulata with various sources of combined nitrogen

Fixation of dissolved dinitrogen in culture solutions by the photosynthetic bacterium Rhodopseudomonas capsulata, strain B10, reduced the lag phase associated with growth with glutamate. A comparable effect was not observed with ammonium chloride. This strain assimilated nitrate but nitrogen fixation was depressed during early growth on nitrate. It is shown that nitrite, the first product of nitrate assimilation, inhibits nitrogen fixation during the early stages of cell growth.