The alginate lyase-encoding gene (algL) of Azotobacter chroococcum was localized to a 3.1-kb EcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme.

Nitrogen-fixing Azotobacter chroococcum cells, but not ammonium- or nitrate-grown cells, exhibited two polypeptide components of 22 and 35 kDa, respectively, that we termed P22 and P35. Bidimensional polyacrylamide gel electrophoresis analysis of preparations from N2-fixing cells that had been transferred to nitrate medium and then incubated for 2 h revealed that P22 had shifted to a more acidic part of the gel while P35 did not change its electrophoretic pattern. Using [32P]orthophosphoric acid it could be demonstrated that the shift in mobility of P22 was due to the phosphorylation of the polypeptide dependent on nitrate (nitrite). The A. chroococcum TR1 strain, which is unable to use nitrate as a nitrogen source and displays activities of nitrogenase, nitrate reductase and nitrite reductase, exhibited both polypeptides. In contrast, P22 and P35 were absent from A. chroococcum MCD1, a mutant strain that cannot assimilate nitrate and lacks the nitrate-reducing enzymatic system. The results suggest that P22 could act as a sensor protein for nitrate in A. chroococcum.

Azotobacter chroococcum cells exhibiting the capacity to take up nitrate actively could transport [14C]cyanate. This activity was dependent on the nitrogen source present in the culture medium, ammonium acting as a repressor and nitrate as an inducer. The uptake of cyanate required metabolic energy and was absent from A. Chroococcum TR1, a mutant strain lacking the nitrate transport system, but was present at wild-type levels in A. chroococcum E4, a mutant strain deficient in nitrate reductase. These results show that cyanate is transported by the nitrate permease in A. chroococcum and therefore [14C]cyanate may be useful as a nitrate analogue for studies on nitrate transport.

Using anti-(Fe protein) antibody raised against the Fe protein of the photosynthetic bacterium Rhodospirillum rubrum, it was found that the Fe protein component of nitrogenase (EC 1.18.2.1) from Azotobacter chroococcum cells subjected to an ammonium shock, and hence with an inactive nitrogenase, appeared as a doublet in Western blot analysis of cell extracts. The Fe protein incorporated [32P]phosphate and [3H]adenine in response to ammonium treatment, and L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase (L-glutamate: ammonia ligase (ADP forming), EC 6.3.1.2), prevented Fe protein from inhibition and radioisotope labelling. These results support that A. chroococcum Fe protein is most likely ADP-ribosylated in response to ammonium. After ammonium treatment, when in vivo activity was completely inhibited, Fe-protein modification was still increasing. This suggests the existence of another mechanism of nitrogenase inhibition faster than Fe-protein modification. When ammonium was intracellularly generated instead of being externally added, as occurs with the short-term nitrate inhibition of nitrogenase activity observed in A. chroococcum cells simultaneously fixing molecular nitrogen and assimilating nitrate, a covalent modification of the Fe protein was likewise demonstrated.

Measurements of enzymes involved in alginate biosynthesis were straightforward in mucoid (alginate-positive)Azotobacter chroococcum ATCC 4412 crude extracts. At the stationary growth phase, where the production of the exopolysaccharide was greatest, the enzymes phosphomannose isomerase and GDP-mannose pyrophosphorylase increased markedly, whereas phosphomannomutase and GDP-mannose dehydrogenase kept the high activity levels measured in the acceleration growth phase. In nonmucoid (alginatenegative)A. chroococcum andA. vinelandii strains, the activities of phosphomannose isomerase and GDP-mannose pyrophosphorylase were rather low or, in some cases, undetectables. Except inA. chroococcum MCD1, which exhibited a low activity, phosphomanomutase was high in the nonmucoidAzotobacter strains, and GDP-mannose dehydrogenase reached a significant activity level in two out of four nonmucoid strains tested. The results suggest that derepression of phophomannose isomerase and GDP-mannose pyrophosphorylase is asine qua non condition for alginate formation byA. chroococcum.

A monospecific anti-(glutamine synthetase) antibody raised against glutamine synthetase of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 immunoreacted with glutamine synthetase from the N2-fixing heterotrophic bacterium Azotobacter chroococcum. In Western-blotting experiments this antibody recognized a single protein of a molecular mass of 59 kDa corresponding to glutamine synthetase subunit. This protein was in vivo-labelled in response to addition of ammonium, both [3H]adenine and H(3)32PO4 preincubation of the cells being equally effective. Nevertheless, the amount of glutamine synthetase present in A. chroococcum was independent of the available nitrogen source. Modified, inactive glutamine synthetase was re-activated by treatment with snake-venom phosphodiesterase but not by alkaline phosphatase. L-Methionine-DL-sulphoximine, an inhibitor of glutamine synthetase, prevented the enzyme from being covalently modified. We conclude that, in A. chroococcum, glutamine synthetase is adenylylated in response to ammonium and that for the modification to take place ammonium must be metabolized.

A slab gel electrophoretic method for the study of bacterial alginate lyase has been developed. By incorporating alginate in acrylamide gels, the method is based on renaturation of the enzyme after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, subsequent staining with cetylpyridinium chloride, and quantification of the spot by densitometric scanning. The molecular mass of alginate lyase can be determined from its position in the gel.

Active transport systems in bacteria can be divided into two groups: those that are osmotic shock‐resistant with one single membrane protein, and those that are shock‐sensitive and have a membrane‐bound protein complex plus a soluble periplasmic protein. Whether the bacterial assimilatory nitrate transport falls into the one or the other of these two groups has not been studied before. We report that nitrate uptake by the strictly aerobic, N2‐fixing heterotrophic bacterium Azotobacter chroococcum is sensitive to osmotic shock. The polypeptide composition of cytoplasmic membranes changes in response to the nitrogen source available to the cells. Incorporation of [35S]‐methionine into proteins as well as use of the A. chroococcum TRI mutant, which is defective in nitrate transport, and the A. choococcum MCD1 strain, a mutant unable to use nitrate as a nitrogen source, suggest that nitrate transport into A. chroococcum cells is mediated by a multicomponent system tightly bound to the cytoplasmic membrane.

Nitrate uptake by Azotobacter chroococcum ATCC 4412 was sensitive to osmotic shock. Cytoplasmic membrane preparations from nitrate-grown cells exhibited three polypeptide components of 52, 49 and 44 kDa, respectively, that were missing from, or very much decreased in, N2-fixing or ammonium-grown cells. The A. chroococcum TRI strain, which is deficient in nitrate uptake but exhibits normal levels of nitrate reductase (EC 1.6.6.1) and nitrite reductase (EC 1.6.6.4), lacked the 44 kDa membrane-bound protein while exhibiting the other two polypeptide components. Transfer of ammonium-grown A. chroococcum cells to a medium containing nitrate as the sole nitrogen source and [S-35]methionine resulted in parallel development of nitrate uptake activity and the above mentioned 52, 49, and 44 kDa polypeptides radioactively labelled. A. chroococcum MCD1, a pleiotropic mutant unable to use nitrate as a nitrogen source, had none of the membrane proteins inducible by nitrate in the wild-type cells. The results strongly suggest that a multicomponent system transports nitrate in A. chroococcum.

Analysis of distribution of invertase (EC 3.2.1.26) activity in Azotobacter chroococcum ATCC 4412 and A. vinelandii UW cells revealed that A. chroococcum invertase is an exocellular protein, whereas the A. vinelandii enzyme is located inside the cell. Invertase has been purified by a simple and rapid procedure from cell-free supernatants of A. chroococcum batch cultures. The enzyme preparation appeared homogeneous on SDS-PAGE (SDS-polyacrylamide gel electrophoresis) and was identified as a glycoprotein. The exoinvertase was freed of its carbohydrate component, without loss of catalytic activity, upon treatment with β-N-acetylglucosaminidase (EC 3.2.1.30). Invertase molecular weight was estimated to be 57 kDa by analytical gel filtration and 59 KDa by SDS-PAGE. It appears, therefore, to be composed of a single subunit. The amino acid analysis of A. chroococcum invertase revealed a high proportion of acidic residues and lack of arginine and histidine. 5 mm pyridoxal-5′-phosphate or pyridoxamine nearly abolished the invertase activity. Activation energy of the enzyme for sucrose hydrolysis was 18 kcal mol−1. The specific activity of A. chroococcum invertase, expressed as turnover number, was 100 s−1.

Environmental conditions affect the production of extracellular polysaccharide by Azotobacter chroococcum ATCC 4412. Production of exocellular polymer from a variety of carbon sources depended on the air flow rate. A high sucrose concentration in medium (8%) markedly favored exopolysaccharide production, which reached 14 g/L in about 72 h. In cell suspensions incubated in the presence of 8% sucrose in a nitrogen-free medium, biopolymer final concentration of 9 g L corresponds to 68 g/g biomass. Maximum efficiency of sucrose conversion into exopolysaccharide peaked at 70% for initial disaccharide concentration of 6%. High performance liquid chromatography and gas liquid chromatography of acid hydrolysates of the exopolymer revealed the presence of mannuronosyl, guluronosyl, and acetyl residues, but not neutral sugars. The infrared spectrum corroborated the presence of carboxylate anions and O-acetyl groups in the exopolymer. Though the presence of more than one kind of polysaccharide cannot be ruled out, these data suggest that, under the experimental conditions used in this work, only a type of alginate-like exopolysaccharide is produced by A. chroococcum ATCC 4412.

A linear correlation has been found between experimental heteroatom bond angles in saturated heterocycles of formulas X (CH2)n where XNH, O or S, and n = 2,3,4,5, and molecular ionization potentials. Theoretical models of the same molecules have been calculated by the semi-empirical SCFMNDO method and ab initio SCF with minimal basis STO-3G and contracted MINIl, with full optimization of the molecular geometry in all cases. Similar correlations have been found in the theoretical models which confirm the heteroatom electronic state origin of such effects, which are very useful for experimental structural chemists. Theoretical models can prove a good tool for establishing other empirical relationships which could be useful in the same way.

Nitrogen fixation and nitrate assimilation usually exclude each other because nitrate, through some product(s) generated in the assimilation of the ammonia resulting from its intracellular reduction, represses nitrogenase synthesis (Tubb and Postgate 1973; Postgate 1982). Besides this effect, nitrate exerts a rapid and reversible inhibition of nitrogenase activity that involves also nitrogen assimilation beyond ammonia (Cejudo and Paneque 1986, 1987; Paneque et al. 1987). Moreover, addition of ammonia to Azotobacter chroococcum cells either actively taking up nitrate (Revilla et al. 1986) or fixing nitrogen (Cejudo et al. 1984) causes an immediate inhibition of these activities. Whereas nitrogenase activity is the target for the effect of ammonia on nitrogen fixation, the inhibition of nitrate uptake by ammonia is attributed to a direct effect on the nitrate transport system. This transport system acts before the nitrate reduction step and is closely linked to the cell energy metabolism. Thus ammonia, through some compound(s) derived from its metabolism, blocks the first energy-dependent step in the sequence of events involved in either nitrogen fixation or nitrate uptake.

A chlorate-resistant Azotobacter chroococcum strain, which we designate TR1, has been isolated after mutagenesis of the parent strain with ICR-191. After growth in a medium containing nitrate, the mutant strain exhibited activities of nitrogenase, nitrate reductase and nitrise reductase, and it did not take up nitrate efficiently. Induction of nitrite reductase in the mutant required a nitrate concentration higher than in the wild type. The results suggest that TR1 is not a regulatory mutant but a strain deficient in the process of nitrate transport into the cell.

Addition of MnCl2 but not of CaCl2, MgCl2 or ZnCl2, at low concentrations to nitrate-grown Azotobacter chroococcum cells enhanced the rate of assimilatory nitrate uptake. In the presence of Mn(II), however, the newly-reduced anion was excreted into the medium in the form of ammonium. When Mn(II) was added to a cell suspension in which the nitrate uptake activity had been inactivated by prior incubation with NH4Cl, stopping of assimilation of the ammonium present and resumption of nitrate uptake took place. If Mg(II) at a 100-fold concentration with respect to Mn(II) was then added, both NH4+ assimilation and NH4+ inhibition of nitrate uptake resumed. Cell suspensions that were preincubated with Mn(II) and assayed for glutamine synthetase (l-glutamate: ammonia ligase (ADP forming), EC 6.3.1.2) activity in situ, showed an increase in the Mg(II)-dependent biosynthetic activity and a simultaneous decrease in the Mn(II)-dependent biosynthetic activity. It could be shown, however, that the Mg(II)-dependent glutamine synthetase biosynthetic activity, as assayed in situ, was significantly inhibited by Mn(II). These results suggest that Mn(II) exerts an external control on nitrate assimilation in Azotobacter, glutamine synthetase being the target for the effect of Mn(II) on this biological process.

In Azotobacter chroococcum cells exhibiting both nitrate (nitrite) assimilation ability and nitrogen fixation capability, the extent of nitrogenase activity inhibition by nitrate or nitrite positively correlated (r = 0.922) with the rate of nitrate (nitrite) taken up by the cells. These results corroborate our previous proposal that the anion must be assimilated to exert its inhibitory effect, and indicate that the inhibition is a graded rather than an all-or-none process.

Nitrate assimilation by suspensions of Azotobacter chroococcum, as determined by the disappearance of the ion from the external medium, displayed saturation kinetics, was inhibited by nitrite, and exhibited an affinity for nitrate higher than that of nitrate reductase. This suggests that the entry of nitrate into the cell is mediated by a specific transporter. Nitrate assimilation required a readily utilizable carbon source and aerobic conditions and was blocked by the uncouplers carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol (DNP) but not by N, N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of ATPases. The inhibition of nitrate assimilation in the absence of an appropriate carbon source was overcome by the non-physiological energy source ascorbate plus N-methylphenazinium methylsulphate (PMS), a substrate combination that allowed respiration. Though an ATP-dependent nitrate uptake mechanism cannot be ruled out, these data suggest that transport of nitrate into the cell is directly dependent on the proton electrochemical gradient across the cytoplasmic membrane.

Nitrate-grown Azotobacter chroococcum ATCC 4412 cells lack the ability to fix N2. Nitrogenase activity developed after the cells were suspended in a combined nitrogen-free medium and was paralleled by a concomitant decrease in nitrate assimilation capacity. In such treated cells exhibiting transitory nitrate assimilation and N2-fixation capacity, nitrate or nitrite caused a short-term inhibitory effect on nitrogenase activity which ceased once the anion was exhausted from the medium. The analog L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase, prevented inhibition of nitrogenase activity by nitrate or nitrite without affecting the uptake of these antions, which were reduced and stoichiometrically released into the external medium as ammonium. Inhibition of nitrogenase by nitrate (nitrite) did not take place in A. chroococcum MCD1, which is unable to assimilate either. We conclude that the short-term inhibitory effect of nitrate (nitrite) on nitrogenase activity is due to some organic product(s) formed during the assimilation of the ammonium resulting from nitrate (nitrite) reduction.

Induction of the assimilatory nitrate uptake in the strictly aerobic, nitrogen-fixing chemi-autrophic bacterium Azotobacter chroococcum is strictly dependent on the presence of nitrate or nitrite in the extracellular medium; induction requires an energy source and is inhibited by chloramphenicol. Cyanate was found to act as a gratuitous inducer of the assimilatory nitrate uptake and as an inhibitor of nitrate transport into the bacterial cells.