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Among the epimerases specific to alginate, some of them in Azotobacter genera convert β-d-mannuronic acid to α-l-guluronic acid but also have lyase activity to degrade alginate. The remarkable characteristics of these epimerases make it a promising enzyme for tailoring alginates to meet specific demands. Here, we determined the structure of the bifunctional mannuronan C-5 epimerase AlgE3 from Azotobacter chroococcum (AcAlgE3) in complex with several mannuronic acid oligomers as well as in apo form, which allowed us to elucidate the binding manner of each mannuronic acid oligomer, and the structural plasticity, which is dependent on calcium ions. Moreover, a comprehensive analysis of the lyase activity profiles of AcAlgE3 combined with structural characteristics explained the preference for different chain length oligomers., (© 2024 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Prebiotics are known for their ability to modulate the composition of the human microbiome and mediate health-promoting benefits. Endo-levanases, which hydrolyze levan into short-chain FOS, could be used for the production of levan-based prebiotics. The novel endo-levanase (LevB 2286 ) from Azotobacter chroococcum DSM 2286, combines an exceptionally high specific activity with advantageous hydrolytic properties. Starting from levan isolated from Timothy grass, LevB 2286 produced FOS ranging from DP 2 - 8. In contrast to endo-levanases described in the literature, LevB 2286 formed minor amounts of fructose and levanbiose, even with greatly extended incubation. The combined activity of LevB 2286 and the levansucrase LevS 1417 from Gluconobacter japonicus LMG 1417 led to a one-step synthesis of levan-type FOS from sucrose. 387.4 ± 17.3 g L -1 FOS were produced within 48 h by the production strategy based on crude cell extract of recombinant Escherichia coli expressing levS1417 and levB2286 simultaneously., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Given the excellent characteristics of alginate, it is an industrially important polysaccharide. Mannuronan C5-epimerase (MC5E) is an alginate-modifying enzyme that catalyzes the conversion of β-D-mannuronate (M) to its C5 epimer α-L-guluronate (G) in alginate. Both the biological activities and physical properties of alginate are determined by M/G ratios and distribution patterns. Therefore, MC5E is regarded as a biotechnological tool for modifying and processing alginate. Various MC5Es derived from brown algae, Pseudomonas and Azotobacter have been isolated and characterized. With the rapid development of structural biology, the crystal structures and catalytic mechanisms of several MC5Es have been elucidated. It is necessary to comprehensively understand the research status of this alginate-modifying enzyme. In this review, the properties and potential applications of MC5Es isolated from different kinds of organisms are summarized and reviewed. Moreover, future research directions of MC5Es as well as strategies to enhance their properties are elucidated, highlighted, and prospected., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Bacterial alginate initially consists of 1-4-linked β-D-mannuronic acid residues (M) which can be later epimerized to α-L-guluronic acid (G). The family of AlgE mannuronan C-5-epimerases from Azotobacter vinelandii has been extensively studied, and three genes putatively encoding AlgE-type epimerases have recently been identified in the genome of Azotobacter chroococcum. The three A. chroococcum genes, here designated AcalgE1, AcalgE2 and AcalgE3, were recombinantly expressed in Escherichia coli and the gene products were partially purified. The catalytic activities of the enzymes were stimulated by the addition of calcium ions in vitro. AcAlgE1 displayed epimerase activity and was able to introduce long G-blocks in the alginate substrate, preferentially by attacking M residues next to pre-existing G residues. AcAlgE2 and AcAlgE3 were found to display lyase activities with a substrate preference toward M-alginate. AcAlgE2 solely accepted M residues in the positions - 1 and + 2 relative to the cleavage site, while AcAlgE3 could accept either M or G residues in these two positions. Both AcAlgE2 and AcAlgE3 were bifunctional and could also catalyze epimerization of M to G. Together, we demonstrate that A. chroococcum encodes three different AlgE-like alginate-modifying enzymes and the biotechnological and biological impact of these findings are discussed.

Thiol dioxygenases are non-heme mononuclear iron enzymes that catalyze the O 2 -dependent oxidation of free thiols (-SH) to produce the corresponding sulfinic acid (-SO 2 - ). Regardless of the phylogenic domain, the active site for this enzyme class is typically comprised of two major features: (1) a mononuclear ferrous iron coordinated by three protein-derived histidines and (2) a conserved sequence of outer Fe-coordination-sphere amino acids (Ser-His-Tyr) spatially adjacent to the iron site (∼3 Å). Here, we utilize a promiscuous 3-mercaptopropionic acid dioxygenase cloned from Azotobacter vinelandii ( Av MDO) to explore the function of the conserved S-H-Y motif. This enzyme exhibits activity with 3-mercaptopropionic acid ( 3mpa ), l-cysteine ( cys ), as well as several other thiol-bearing substrates, thus making it an ideal system to study the influence of residues within the highly conserved S-H-Y motif (H157 and Y159) on substrate specificity and reactivity. The p K a values for these residues were determined by pH-dependent steady-state kinetics, and their assignments verified by comparison to H157N and Y159F variants. Complementary electron paramagnetic resonance and Mössbauer studies demonstrate a network of hydrogen bonds connecting H157-Y159 and Fe-bound ligands within the enzymatic Fe site. Crucially, these experiments suggest that the hydroxyl group of Y159 hydrogen bonds to Fe-bound NO and, by extension, Fe-bound oxygen during native catalysis. This interaction alters both the NO binding affinity and rhombicity of the 3mpa -bound iron-nitrosyl site. In addition, Fe coordination of cys is switched from thiolate only to bidentate (thiolate/amine) for the Y159F variant, indicating that perturbations within the S-H-Y proton relay network also influence cys Fe binding denticity.

Heavy metals are one of the major abiotic stresses that adversely affect the quantity and nutritive value of maize. Microbial management involving the use of plant growth promoting rhizobacteria (PGPR) is a promising inexpensive strategy for metal clean up from polluted soils. Considering these, metal tolerant plant growth promoting nitrogen fixing rhizobacterial strain CAZ3 identified by 16SrRNA gene sequence analysis as Azotobacter chroococcum was recovered from metal polluted chilli rhizosphere. When exposed to varying levels of metals, A. chroococcum survived up to 1400 and 2000 µg mL -1 of Cu and Pb, respectively and expressed numerous plant growth promoting activities even under metal stress. Strain CAZ3 secreted 65.5 and 60.8 µg mL -1 IAA at 400 µg mL -1 each of Cu and Pb, respectively and produced siderophores, ammonia and ACC deaminase under metal pressure. The melanin extracted from A. chroococcum revealed metal chelating ability under EDX. Following application, strain CAZ3 enhanced growth and yield of maize grown both in the presence of Cu and Pb. The dry biomass of roots of inoculated plants grown with 2007 mg Cu kg -1 and 585 mg Pb kg -1 was increased by 28% and 20%, respectively. At 585 mg Pb kg -1 , the bioinoculant also increased the kernel attributes. At 2007 mg Cu kg -1 strain CAZ3 enhanced the number, yield and protein of kernels by 10%, 45% and 6%, respectively. Interestingly, strain CAZ3 significantly reduced the levels of proline, malondialdehyde and antioxidant enzymes in foliage. The roots of inoculated plants accumulated greatest amounts of metals compared to other organs. In kernels, the concentration of Pb was more as compared to Cu. The metal concentrations in roots, shoots and kernels, however, declined following CAZ3 inoculation. Copper and lead had substantial distortive impact on root and leaf morphology while cell death were visible under CLSM and SEM. Conclusively, A. chroococcum CAZ3 could be a most suitable and promising option to increase maize production in metal polluted soils despite the soils being contaminated with heavy metals., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A family of seven mannuronan C5-epimerases (AlgE1-AlgE7) produced by Azotobacter vinelandii is able to convert β-d-mannuronate (M) to its epimer α-l-guluronate (G) in alginates. Even sharing high sequence homology at the amino acid level, they produce distinctive epimerization patterns. The introduction of new G-blocks into the polymer by in vitro epimerization is a strategy to improve the mechanical properties of alginates as biomaterial. However, epimerization is hampered when the substrate is modified or in the gelled state. Here it is presented how native and engineered epimerases of varying size perform on steric hindered alginate substrates (modified or as hydrogels). Reducing the size of the epimerases enables the epimerization of otherwise inaccessible regions in the alginate polymer. Even though the reduction of the size affects the productive binding of epimerases to the substrate, and hence their activity, the smaller epimerases could more freely diffuse into calcium-alginate hydrogel and epimerize it., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Previously we have reported that the Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 uses the 4,6-α-glucanotransferase GtfD to convert maltodextrins and starch into a reuteran-like polymer consisting of (α1→4) glucan chains connected by alternating (α1→4)/(α1→6) linkages and (α1→4,6) branching points. This enzyme constituted the single evidence for this reaction and product specificity in the GH70 family, mostly containing glucansucrases encoded by lactic acid bacteria (http://www.CAZy.org). In this work, 4 additional GtfD-like proteins were identified in taxonomically diverse plant-associated bacteria forming a new GH70 subfamily with intermediate characteristics between the evolutionary related GH13 and GH70 families. The GtfD enzyme encoded by Paenibacillus beijingensis DSM 24997 was characterized providing the first example of a reuteran-like polymer synthesizing 4,6-α-glucanotransferase in a Gram-positive bacterium. Whereas the A. chroococcum GtfD activity on amylose resulted in the synthesis of a high molecular polymer, in addition to maltose and other small oligosaccharides, two reuteran-like polymer distributions are produced by P. beijingensis GtfD: a high-molecular mass polymer and a low-molecular mass polymer with an average Mw of 27 MDa and 19 kDa, respectively. Compared to the A. chroooccum GtfD product, both P. beijingensis GtfD polymers contain longer linear (α1→4) sequences in their structure reflecting a preference for transfer of even longer glucan chains by this enzyme. Overall, this study provides new insights into the evolutionary history of GH70 enzymes, and enlarges the diversity of natural enzymes that can be applied for modification of the starch present in food into less and/or more slowly digestible carbohydrate structures.

Background: Originally the glycoside hydrolase (GH) family 70 only comprised glucansucrases of lactic acid bacteria which synthesize α-glucan polymers from sucrose. Recently we have identified 2 novel subfamilies of GH70 enzymes represented by the Lactobacillus reuteri 121 GtfB and the Exiguobacterium sibiricum 255-15 GtfC enzymes. Both enzymes catalyze the cleavage of (α1→4) linkages in maltodextrin/starch and the synthesis of consecutive (α1→6) linkages. Here we describe a novel GH70 enzyme from the nitrogen-fixing Gram-negative bacterium Azotobacter chroococcum, designated as GtfD., Methods: The purified recombinant GtfD enzyme was biochemically characterized using the amylose-staining assay and its products were identified using profiling chromatographic techniques (TLC and HPAEC-PAD). Glucans produced by the GtfD enzyme were analyzed by HPSEC-MALLS-RI, methylation analysis, 1D/2D [1]H/[13]C NMR spectroscopy and enzymatic degradation studies., Results: The A. chroococcum GtfD is closely related to GtfC enzymes, sharing the same non-permuted domain organization also found in GH13 enzymes and displaying 4,6-α-glucanotransferase activity. However, the GtfD enzyme is unable to synthesize consecutive (α1→6) glucosidic bonds. Instead, it forms a high molecular mass and branched α-glucan with alternating (α1→4) and (α1→6) linkages from amylose/starch, highly similar to the reuteran polymer synthesized by the L. reuteri GtfA glucansucrase from sucrose., Conclusions: In view of its origin and specificity, the GtfD enzyme represents a unique evolutionary intermediate between family GH13 (α-amylase) and GH70 (glucansucrase) enzymes., General Significance: This study expands the natural repertoire of starch-converting enzymes providing the first characterization of an enzyme that converts starch into a reuteran-like α-glucan polymer, regarded as a health promoting food ingredient., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Strains of Azotobacter mediate in the nitrogen fixation process by reducing of N2 to ammonia. In this study, 50 strains were isolated from different rhizospheric soil in central Iran, by using soil paste-plate method. These strains were biochemically identified and characterized on differential LG medium based on morphological and physiological properties. Results obtained showed that identified strains were belonging to three species, namely A. chroococcum, A. vinelandii and A. beijernckii. In order to molecular analysis, the 16S rRNA gene was amplified using 27f and 1495r primers and PCR products were subsequently digested with RsaI, HpaII and HhaI. Cluster analysis based on amplified ribosomal DNA restriction analysis were revealed intraspecific polymorphism and differentiated strains into two mains clusters, clusters A and B. Cluster A strains were related to the A. vinelandii, whereas cluster B strains were related to the A. chroococcum and A. beijerinckii. The results show that amplified ribosomal DNA restriction analysis is a powerful and discriminatory tool for the identification of members of the genus Azotobacter.

Cells of Azotobacter chroococcum MAL-201 (MTCC 3853) are capable of accumulating the intracellular poly(3-hydroxybutyric acid) [P(3HB)], accounting for 65-71 % of its cell dry weight and also capable of synthesizing the enzyme alkaline phosphatase (APase), when grown in glucose and tricalcium phosphate containing nitrogen-free modified Stockdale medium. The concentration of insoluble phosphate in broth medium was optimized as 0.25 % (w/v) for growth and biosynthesis of APase. However, the suboptimal concentration of phosphate (0.1 %, w/v) appeared as the best suited for accumulation of P(3HB) by the strain. The significant differences were observed in biosynthesis of polymer and APase enzyme under variable phosphate concentrations. Glucose, 3.0 % (w/v) was recorded as the optimum concentration for all of the three parameters. The continuation of APase biosynthesis was observed during the period of significant decline in the cellular content of the polymer in the late phase of growth. In order to study the role of P(3HB), the rate of autodigestion of biopolymer and phosphate solubilization rate (k, mineralization constant) were determined in carbon-free medium under batch cultivation process and the parameters were found to be positively correlated. The maximum phosphate solubilization rate (k = 0.0154) by the strain MAL-201 timed at the 10th hour of incubation when the rate of polymer degradation concomitantly attained its peak corresponding to 87 mg/l/h and then declined gradually. Only a negligible amount of residual polymer remained undigested. These data strongly support the functional role of P(3HB) in response to multinutritional stress condition.

The enzyme nitrogenase, when reducing natural and unnatural substrates, requires large numbers of protons per chemical catalytic cycle. The active face of the catalytic site (the FeMo-cofactor, FeMo-co) is situated in a protein domain which is largely hydrophobic and anhydrous, and incapable of serial provision of multiple protons. Through detailed analysis of the high quality protein crystal structures available the characteristics of a chain of water molecules leading from the protein surface to a key sulfur atom (S3B) of FeMo-co are described. The first half of the water chain from the surface inwards is branched, slightly variable, and able to accommodate exogenous small molecules: this is dubbed the proton bay. The second half, from the proton bay to S3B, is comprised of a single chain of eight hydrogen bonded water molecules. This section is strictly conserved, and is intimately involved in hydrogen bonds with homocitrate, an essential component that chelates Mo. This is the proton wire, and a detailed Grotthuss mechanism for serial translocation of protons through this proton wire to S3B is proposed. This controlled serial proton relay from the protein surface to S3B is an essential component of the intramolecular hydrogenation paradigm for the complete chemical mechanisms of nitrogenase. Each proton reaching S3B, instigated by electron transfer to FeMo-co, becomes a hydrogen atom that migrates to other components of the active face of FeMo-co and to bound substrates and intermediates, allowing subsequent multiple proton transfers along the proton wire. Experiments to test the proposed mechanism of proton supply are suggested. The water chain in nitrogenase is comparable with the purported proton pumping pathway of cytochrome c oxidase.

Cold-adapted monomeric isocitrate dehydrogenase of a psychrophilic bacterium, Colwellia maris, (CmIDH) showed a high degree of amino acid sequential identity (69.5%) to a mesophilic nitrogen-fixing bacterium, Azotobacter vinelandii, (AvIDH). In this study, three Ala residues of CmIDH and the corresponding Pro residues of AvIDH were exchanged by site-directed mutagenesis, and several properties of single, double, and triple mutants of the two enzymes were investigated. The mutated CmIDHs, which replaced Ala719 with Pro, showed increased activity and elevation of the optimum temperature and thermostability for activity. In contrast, mutants of AvIDH, in which Pro717 was replaced by Ala, decreased the thermostability for activity. These results indicate that Ala719 of CmIDH and Pro717 of AvIDH are involved in thermostability. On the other hand, mutated CmIDH, in which Ala710 was replaced by Pro, and the corresponding AvIDH mutant, which replaced Pro708 with Ala, showed higher and lower specific activity than the corresponding wild-type enzymes, suggesting that Pro708 of AvIDH is involved in its high catalytic ability. Furthermore, the exchange mutations between Ala740 in CmIDH and the corresponding Pro738 in AvIDH resulted in decreased and increased thermostability for CmIDH and AvIDH activity respectively, suggesting that the native Ala740 and Pro738 residues make the enzymes thermostable and thermolabile.

Phasins (PhaP) are proteins normally associated with granules of poly(3-hydroxybutyrate) (PHB), a biodegradable polymer accumulated by many bacteria as a reserve molecule. These proteins enhance growth and polymer production in natural and recombinant PHB producers. It has been shown that the production of PHB causes stress in recombinant Escherichia coli, revealed by an increase in the concentrations of several heat stress proteins. In this work, quantitative reverse transcription (qRT)-PCR analysis was used to study the effect of PHB accumulation, and that of PhaP from Azotobacter sp. strain FA8, on the expression of stress-related genes in PHB-producing E. coli. While PHB accumulation was found to increase the transcription of dnaK and ibpA, the expression of these genes and of groES, groEL, rpoH, dps, and yfiD was reduced, when PhaP was coexpressed, to levels even lower than those detected in the non-PHB-accumulating control. These results demonstrated the protective role of PhaP in PHB-synthesizing E. coli and linked the effects of the protein to the expression of stress-related genes, especially ibpA. The effect of PhaP was also analyzed in non-PHB-synthesizing strains, showing that expression of this heterologous protein has an unexpected protective effect in E. coli, under both normal and stress conditions, resulting in increased growth and higher resistance to both heat shock and superoxide stress by paraquat. In addition, PhaP expression was shown to reduce RpoH protein levels during heat shock, probably by reducing or titrating the levels of misfolded proteins.

Laccases are copper-containing phenol oxidases that are commonly found in many types of plant, insect, fungi and bacteria. Whilst phenol oxidases have been well characterized in fungal species, laccase-type enzymes originating from bacteria have been much less well defined. Bacteria belonging to the family Azotobacteraceae share many morphological characteristics with strains already known to exhibit polyphenol and phenol oxidase activity; and hence the aim of this work was to identify and characterize a novel laccase from the isolated strain Azotobacter chroococcum SBUG 1484 in an attempt to provide further understanding of the roles such enzymes play in physiological development. Laccase activity was clearly observed through oxidation of 2,6-dimethoxyphenol, other typical substrates including: methoxy-monophenols, ortho- and para-diphenols, 4-hydroxyindole, and the non-phenolic compound para-phenylenediamine. A. chroococcum SBUG 1484 showed production of a cell-associated phenol oxidase when grown under nitrogen-fixing conditions, and was also observed when cells enter the melanogenic and encystment stages of growth. Catechol which is structurally related to melanin compounds was also released from Azotobacter cells into the surrounding culture medium during nitrogen-fixing growth. From our results we propose that a membrane-bound laccase plays an important role in the formation of melanin, which was monitored to correlate with progression of A. chroococcum SBUG 1484 cells into the encystment stage of growth.

The cofactors of the Mo- and V-nitrogenases (i.e., FeMoco and FeVco) are homologous metal centers with distinct catalytic properties. So far, there has been only one report on the isolation of FeVco from Azotobacter chroococcum. However, this isolated FeVco species did not carry the full substrate-reducing capacity, as it is unable to restore the N(2)-reducing ability of the cofactor-deficient MoFe protein. Here, we report the isolation and characterization of a fully active species of FeVco from A. vinelandii. Our metal and activity analyses show that FeVco has been extracted intact, carrying with it the characteristic capacity to reduce C(2)H(2) to C(2)H(6) and, perhaps even more importantly, the ability to reduce N(2) to NH(3). Moreover, our EPR and XAS/EXAFS investigations indicate that FeVco is similar to, yet distinct from FeMoco in electronic properties and structural topology, which could account for the differences in the reactivity of the two cofactors. The outcome of this study not only permits the proposal of the first EXAFS-based structural model of the isolated FeVco but also lays a foundation for future catalytic and structural investigations of this unique metallocluster.

Polyhydroxyalkanoate (PHA) synthases catalyze chain transfer (CT) reaction after polymerization reaction of PHA by transferring PHA chain from PHA synthase to a CT agent, resulting in covalent bonding of CT agent to PHA chain at the carboxyl end. Previous studies have shown that poly(ethylene glycol) (PEG) is an effective exogenous CT agent. This study aimed to compare the effects of PEG on CT reaction during poly[(R)-3-hydroxybutyrate] [P(3HB)] synthesis by using six PHA synthases in Escherichia coli JM109. The synthesized P(3HB) polymers were characterized in terms of molecular weight and end-group structure. Supplementation of PEG to the culture medium reduced P(3HB) molecular weights by up to 96% due to PEG-induced CT reaction. The P(3HB) polymers were subjected to (1)H NMR analysis to confirm the formation of a covalent bond between PEG and P(3HB) chain at the carboxyl end. This study revealed the reactivity of PHA synthases to PEG with respect to CT reaction in E. coli.

Ten strains of Azotobacter chroococcum were studied for their ability to invade the endorhizosphere of wheat. Strain W-5 exhibited ability to invade endorhizosphere as shown in the microscopic observations. This strain was compared with the strain OA-3 which did not invade the endorhizosphere zone. Strain W-5 showed higher production of cellulase and pectinase than OA-3. Both the strains induced defense enzymes in the host plant. However, induction of peroxidase and phenylalanine ammonia lyase activities (PAL) was higher in OA-3 than W-5. Quantitative differences in flavonoid like compounds obtained from root extracts and root exudates of plants inoculated with these strains were observed., (((c) 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).)

Polyhydroxyalkanoates (PHAs) are accumulated as intracellular granules by many bacteria under unfavorable conditions, enhancing their fitness and stress resistance. Poly(3-hydroxybutyrate) (PHB) is the most widespread and best-known PHA. Apart from the genes that catalyze polymer biosynthesis, natural PHA producers have several genes for proteins involved in granule formation and/or with regulatory functions, such as phasins, that have been shown to affect polymer synthesis. This study evaluates the effect of PhaP, a phasin, on bacterial growth and PHB accumulation from glycerol in bioreactor cultures of recombinant Escherichia coli carrying phaBAC from Azotobacter sp. strain FA8. Cells expressing phaP grew more, and accumulated more PHB, both using glucose and using glycerol as carbon sources. When cultures were grown in a bioreactor using glycerol, PhaP-bearing cells produced more polymer (2.6 times) and more biomass (1.9 times) than did those without the phasin. The effect of this protein on growth promotion and polymer accumulation is expected to be even greater in high-density cultures, such as those used in the industrial production of the polymer. The recombinant strain presented in this work has been successfully used for the production of PHB from glycerol in bioreactor studies, allowing the production of 7.9 g/liter of the polymer in a semisynthetic medium in 48-h batch cultures. The development of bacterial strains that can efficiently use this substrate can help to make the industrial production of PHAs economically feasible.

Sec-independent translocation systems have been characterised in Escherichia coli and other bacteria and differ from the Sec-dependent system by transporting fully folded proteins using the transmembrane proton electrochemical gradient. Proteins transported by this system bear a twin-arginine motif (tat) in the N-terminal signal peptide and include several cofactor-containing proteins. Azotobacter chroococcum strain (MCD124) has a soluble hydrogenase, which exhibited low O(2)-dependent H(2) uptake, and a shift in the pH of the culture to a more alkaline range during growth. We show that the DNA region capable of complementing this strain contains the tatABC genes and that mutations in the tatA gene reproduced the soluble hydrogenase and the culture pH shift phenotypes. We also show that insertional mutation in the tatC gene at a position corresponding to its C-terminal region had no effect on hydrogenase activity, but induced the pH shift of the culture. Sequence and mutagenesis analyses of this genomic region suggest that these genes form an operon that does not contain a tatD-like gene. A mutation in hupZ of the main hup gene region, coding for a possible b-type cytochrome also yielded a soluble hydrogenase, but not the pH-shift phenotype.

Azotobacter chroococcum and Azotobacter vinelandii grown in Burk medium with 1% mannitol (BM) or in BM supplemented with 2.2 mg/mL ammonium acetate (BM+N) were found to have only iron-containing and CuZn-containing superoxide dismutase. Furthermore, genomic DNA from A. chroococcum and A. vinelandii were subjected to polymerase chain reaction analysis using sodA- and sodB-specific primers and yielded only a sodB product. These results dispute the assertion by Buchanan and Lees (Can. J. Microbiol. 26: 441-447, 1980) that A. chroococcum contains Mn-superoxide dismutase.

Bradyrhizobium japonicum transports oligopeptides and the heme precursor delta-aminolevulinic acid (ALA) by a common mechanism. Two Tn5-induced mutants disrupted in the lysC and ptsP genes were identified based on the inability to use prolyl-glycyl-glycine as a proline source and were defective in [(14)C]ALA uptake activity. lysC and ptsP were shown to be proximal genes in the B. japonicum genome. However, RNase protection and in trans complementation analysis showed that lysC and ptsP are transcribed separately, and that both genes are involved in oligopeptide transport. Aspartokinase, encoded by lysC, catalyzes the phosphorylation of aspartate for synthesis of three amino acids, but the lysC strain is not an amino acid auxotroph. The ptsP gene encodes Enzyme I(Ntr) (EI(Ntr)), a paralogue of Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase (PTS) system. In vitro pull-down experiments indicated that purified recombinant aspartokinase and EI(Ntr) interact directly with each other. Expression of ptsP in trans from a multicopy plasmid complemented the lysC mutant, suggesting that aspartokinase normally affects Enzyme I(Ntr) in a manner that can be compensated for by increasing the copy number of the ptsP gene. ATP was not a phosphoryl donor to purified EI(Ntr), but it was phosphorylated by ATP in the presence of cell extracts. This phosphorylation was inhibited in the presence of aspartokinase. The findings demonstrate a role for a PTS protein in the transport of a non-sugar solute and suggest an unusual regulatory function for aspartokinase in regulating the phosphorylation state of EI(Ntr).

The immunological relationship of the hydrogenase in Frankia KB5 to hydrogenases in other microorganisms was investigated using antisera raised against holo-[NiFe]-hydrogenases isolated from Alcaligenes latus, Azotobacter vinelandii, Ralstonia eutropha, and the small and large hydrogenase subunits from Bradyrhizobium japonicum. The antisera raised against the A. latus, R. eutropha, and B. japonicum (large subunit) polypeptides were found to recognize two polypeptides, corresponding to the unprocessed and processed forms of the hydrogenase subunit in Frankia KB5. None of the antisera, including the antibodies produced against the small hydrogenase subunit isolated from B. japonicum, recognized any polypeptide related to the small hydrogenase subunit in Frankia KB5. An immunogold localization study of the intracellular distribution of hydrogenase in Frankia KB5, with the cryo-section technique, showed that labeling in the membrane of both hyphae and vesicles was positively correlated with hydrogenase activity.

A new on-line method for measuring acetylene reduction is described. It consists of a gas-flow cell connected to an electronic gas-mixing system and an automatic sample loop in the gas chromatograph. Alternatively, ethylene can be determined by using laser-based trace gas detection. The laser-based trace gas detection technique achieves a detection limit that is three orders of magnitude better than gas chromatography. We have applied the on-line method to the measurement of nitrogen fixation in a culture of the heterocystous cyanobacterium Nodularia spumigena and compared it with conventional batch-type incubations. Incubation of N. spumigena in the gas-flow cell resulted in very short response times with a steady-state flux of ethylene obtained within 2 min. Nitrogenase was shown to respond immediately to changes in light and oxygen. Monitoring of nitrogenase activity could be continued for several hours without having a negative impact on nitrogen fixation rates in N. spumigena. This was not the case in batch incubations, in which changes in nitrogenase activities were recorded during incubations, probably as a result of varying oxygen concentrations. It was therefore concluded that the on-line method is superior to batch incubations when rates of nitrogenase activity are to be measured. The method is suitable for natural samples (water or sediment).

Chemical reconstitution of recombinant bovine adrenal mitochondrial apoadrenodoxin was carried out in the presence of the nonhomologous chaperone protein GroEL and of the cochaperone GroES, both in the presence and in the absence of ATP. The approach used here was different from the one characterizing studies on chaperone activity, as we used an adrenodoxin apoprotein, devoid of the cluster iron and sulfide, rather than a denaturant-unfolded form of the protein, and catalytic amounts of the chaperone proteins. A possible scaffolding role for two bacterial sulfur transferases, namely, rhodanese from Azotobacter vinelandii and a rhodanese-like sulfurtransferase from Escherichia coli, was also investigated in the absence of the enzyme substrates. The extent and the rate of adrenodoxin refolding following cluster insertion was measured by spectroscopy and by monitoring the activity recovery in a NADPH-cytochrome c reduction assay. These measurements were carried out on the unresolved reaction mixture and on the adrenodoxin-containing fraction obtained by HPLC fractionation of the reconstitution mixture at different reaction times. The rate and extent of cluster insertion and activity recovery were substantially improved by addition of GroEL and increased with increasing the GroEL/apoadrenodoxin ratio. GroES and ATP had no effect by themselves, and did not enhance the effect of GroEL. A. vinelandii rhodanese, the E. coli sulfurtransferase, and bovine serum albumin had no effect on the rate and yield of chemical reconstitution. The accelerated chemical reconstitution of apoadrenoxin in the presence of GroEL is therefore attributable to a scaffolding effect of this protein.

A new exocellular polysaccharide (P2) has been produced by the manipulation of a glycosyl transferase gene (aceP) involved in the biosynthesis of the polysaccharide acetan by the bacterium Acetobacter xylinum strain CKE5. The P2 polysaccharide has been studied by methylation analysis, reductive cleavage, and 1H and 13C NMR spectroscopy. The data are consistent with the structure predicted when the aceP gene is deactivated: [Molecular structure: see text]. The effect of cooling on proton NMR line width indicates a coil-helix transition in P2 at about 70 degrees C.

We report the cloning and analysis of a gene and its cognate regulatory element from a member of the Azotobacteriaceae which are involved in the breakdown of an aromatic compound. The genes from Azotobacter chroococcum encoding p-hydroxybenzoate hydroxylase (pobA) and its regulatory protein (pobR) were cloned from a genomic library and sequenced. Sequence analysis of pobA revealed homology with other bacterial p-hydroxybenzoate hydroxylase enzymes. Residues essential to the structure and function of the enzyme have been conserved. The pobR gene encodes a DNA binding regulatory protein with similarity to proteins from the AraC/XylS family of transcriptional activators. A fragment containing both pobA and pobR was cloned into pUC19 and p-hydroxybenzoate hydroxylase activity was induced in Escherichia coli by the addition of p-hydroxybenzoate. A frame-shift mutation introduced into the pobR gene prevented expression of p-hydroxybenzoate hydroxylase, indicating that PobR is the protein required for transcription of pobA. Interestingly, A. chroococcum PobR has no homology to the PobR protein that is the transcriptional activator of pobA in Acinetobacter strain ADP1, a protein that is homologous to the IclR family of transcriptional regulators. However, PobR from A. chroococcum is homologous to several other proteins, suggesting that these proteins will also function as transcriptional activators of pobA.

The hypothesis of respiratory protection, originally formulated on the basis of results obtained with Azotobacter species, postulates that consumption of O(2) at the surface of diazotrophic prokaryotes protects nitrogenase from inactivation by O(2). Accordingly, it is assumed that, at increased ambient O(2) concentrations, nitrogenase activity depends on increased activities of a largely uncoupled respiratory electron transport system. The present review compiles evidence indicating that cellular O(2) consumption as well as both the activity and the formation of the respiratory system of Azotobacter vinelandii are controlled by the C/N ratio, that is to say the ratio at which the organism consumes the substrate (i.e. the source of carbon, reducing equivalents and ATP) per source of compound nitrogen. The maximal respiratory capacity which can be attained at increased C/N ratios, however, is controlled, within limits, by the ambient O(2) concentration. When growth becomes N-limited at increased C/N ratios, cells synthesize nitrogenase and fix N(2). Under these diazotrophic conditions, cellular O(2) consumption remains constant at a level controlled by the O(2) concentration. Control by O(2) has been studied on the basis of both whole cell respiration and defined segments of the respiratory electron transport chain. The results demonstrate that the effect of O(2) on the respiratory system is restricted to the lower range of O(2) concentrations up to about 70 microM. Nevertheless, azotobacters are able to grow diazotrophically at dissolved O(2) concentrations of up to about 230 microM indicating that respiratory protection is not warranted at increased ambient O(2) concentrations. This conclusion is supported and extended by a number of results largely excluding an obvious relationship between nitrogenase activity and the actual rate of cellular O(2) consumption. On the basis of theoretical calculations, it is assumed that the rate of O(2) diffusion into the cells is not significantly affected by respiration. All of these results lead to the conclusion that, in the protection of nitrogenase from O(2) damage, O(2) consumption at the cell surface is less effective than generally assumed. It is proposed that alternative factors like the supply of ATP and reducing equivalents are more important.

Spontaneous mutants resistant to methionine sulfoximine (Msx), methyl alanine (Mal) and methyl ammonium chloride (Mac) were derived from A. chroococcum strain A103. Msx and Mal-resistant mutants expressed 1.73 to 10.98% of the fully derepressed nitrogenase activity when grown in Burk's medium containing ammonium acetate. Mac-resistant mutants did not express nitrogenase activity in ammonium acetate supplemented medium. The mutants excreted ammonia even after 2 days of growth and some mutants excreted more ammonia as compared to the parent. Selected mutants were inoculated on wheat (Triticum aestivum) and barley (Hordeum vulgare) under field conditions. Majority of the derepressed mutants increased grain yield of wheat and barley varying from 1.2 to 33.3%. However, host-dependent effects on grain yield were observed with different mutants. Two mutants, Mal 27 and Mac 19 showed significant increase in grain yields of both the crops. The results suggest that metabolic analogue-resistant mutants of Azotobacter have potential for use as a biofertilizer for cereal crops.

A number of nitrogen-fixing bacteria were screened using PCR for genes (vnfG and anfG) unique to the V-containing nitrogenase (vnf) and the Fe-only nitrogenase (anf) systems. Products with sequences similar to that of vnfG were obtained from Azotobacter paspali and Azotobacter salinestris genomic DNAs, and products with sequences similar to that of anfG were obtained from Azomonas macrocytogenes, Rhodospirillum rubrum, and Azotobacter paspali DNAs. Phylogenetic analysis of the deduced amino acid sequences of anfG and vnfG genes shows that each gene product forms a distinct cluster. Furthermore, amplification of an internal 839-bp region in anfD and vnfD yielded a product similar to anfD from Heliobacterium gestii and a product similar to vnfD from Azotobacter paspali and Azotobacter salinestris. Phylogenetic analysis of NifD, VnfD, and AnfD amino acid sequences indicates that AnfD and VnfD sequences are more closely related to each other than either is to NifD. The results of this study suggest that Azotobacter salinestris possesses the potential to express the vanadium (V)-containing nitrogenase (nitrogenase 2) and that R. rubrum, Azomonas macrocytogenes, and H. gestii possess the potential to express the Fe-only nitrogenase (nitrogenase 3). Like Azotobacter vinelandii, Azotobacter paspali appears to have the potential to express both the V-containing nitrogenase and the Fe-only nitrogenase.

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.

A new restriction endonuclease Abe I has beenisolated from Azotobacter beijerinckii. This enzymerecognizes the asymmetric heptanucleotide sequence 5'-CCTCAGC-3' and cleaves within it symmetrically at positions -5/-2 in the opposing strands, producing three base protruding 5'-ends.

New type II restriction endonucleases AsiI and Bsp40091 are detected in Azotobacter species N55 and Bacillus species 4009, respectively. Purified preparations of the restriction enzymes free from interfering nucleases and phosphatases were obtained by column chromatography on phosphocellulose and heparin-sepharose (Asil) and phosphocellulose and DEAE-cellulose (Bsp40091). The yield of purified AsiI and Bsp40091 was 16 x 10(3) and 8 x 10(3) units per g of wet cells, respectively. The above restriction endonucleases recognize the 5'-G decreases GATCC-3' sequence on double-stranded DNA and cleave it as shown, thus being true isoschizomers of BamHI restriction endonuclease.

We have cloned and sequenced the gene encoding the homodimeric pyruvate dehydrogenase component (E1p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii and expressed and purified the E1p component in Escherichia coli. Cloned E1p can be used to fully reconstitute complex activity. The enzyme was stable in high ionic strength buffers, but was irreversibly inactivated when incubated at high pH, which presumably was caused by its inability to redimerize correctly. This explains the previously found low stability of the wild-type E1p component after resolution from the complex at high pH. Cloned E1p showed a kinetic behaviour exactly like the wild-type complex-bound enzyme with respect to its substrate (pyruvate), its allosteric properties, and its effectors. These experiments show that acetyl coenzyme A acts as a feedback inhibitor by binding to the E1p component. Limited proteolysis experiments showed that the N-terminal region of E1p was easily removed. The resulting protein fragment was still active with artificial electron acceptors but had lost its ability to bind to the core component (E2p) and thus reconstitute complex activity. E1p was protected against proteolysis by E2p. The allosteric effector pyruvate changed E1p into a conformation that is more resistant to proteolysis.

The enzyme which catalyzes the dehalogenation of 2,4,6-trichlorophenol (TCP) was purified to apparent homogeneity from an extract of TCP-induced cells of Azotobacter sp. strain GP1. The initial step of TCP degradation in this bacterium is inducible by TCP; no activity was found in succinate-grown cells or in phenol-induced cells. NADH, flavin adenine dinucleotide, and O2 are required as cofactors. As reaction products, 2,6-dichlorohydroquinone and Cl- ions were identified. Studies of the stoichiometry revealed the consumption of 2 mol of NADH plus 1 mol of O2 per mol of TCP and the formation of 1 mol of Cl- ions. No evidence for membrane association or for a multicomponent system was obtained. Molecular masses of 240 kDa for the native enzyme and 60 kDa for the subunit were determined, indicating a homotetrameric structure. Cross-linking studies with dimethylsuberimidate were consistent with this finding. TCP was the best substrate for 2,4,6-trichlorophenol-4-monooxygenase (TCP-4-monooxygenase). The majority of other chlorophenols converted by the enzyme bear a chloro substituent in the 4-position. 2,6-Dichlorophenol, also accepted as a substrate, was hydroxylated in the 4-position to 2,6-dichlorohydroquinone in a nondehalogenating reaction. NADH and O2 were consumed by the pure enzyme also in the absence of TCP with simultaneous production of H2O2. The NH2-terminal amino acid sequence of TCP-4-monooxygenase from Azotobacter sp. strain GP1 revealed complete identity with the nucleotide-derived sequence from the analogous enzyme from Pseudomonas pickettii and a high degree of homology with two nondehalogenating monooxygenases. The similarity in enzyme properties and the possible evolutionary relatedness of dehalogenating and nondehalogenating monooxygenases are discussed.

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.

The stability and catalytic efficacy of the L-2-haloacid dehalogenase isolated from Azotobacter sp. RC26 were studied after immobilisation on a DEAE Sephacel solid matrix. While the optimum temperature for the soluble dehalogenase falls in the range of 30-40 degrees C, the activity of the immobilised enzyme shows a four-fold increase at 60 degree C. Immobilisation on a plug-flow bioreactor extends the range of usable substrate concentration. The improved catalytic characteristics after immobilisation of the haloacid dehalogenase may be relevant for its possible utilization in biotechnological applications ranging from waste treatment to synthesis of stereoisomers.

The mercuric ion reduction system encoded by the Hg2+ inducible mer operon confers bacterial resistance to mercuric ion. The mer A gene product which is a FAD-containing enzyme catalyzes the reduction of Hg2+ to volatile elemental mercury with the help of intracellular thiols and NADPH as a cofactor (Schottel 1974; Summers and Silver 1978; Fox and Walsh 1982; Misra 1992). Our earlier studies have shown that growing cells of different mercury-resistant bacteria reduce Hg2+ compounds to Hg(O) (Ray et al. 1989; Pahan et al. 1990a; Gachhui et al. 1989). We have also shown the effect of thiol compounds and flavins on mercury-degrading enzyme activities in mercury-resistant bacteria (Pahan et al. 1990b). Here we report that resting cells of mercury-resistant bacteria survive in a buffer system for several hours, synthesize inducible mercury-degrading enzymes and volatilize mercury from a mercury-containing buffer system. We know of no information regarding studies of mercury-degrading enzymes in resting mercury-resistant bacterial cells.

The enzyme which cleaves the benzene ring of 6-chlorohydroxyquinol was purified to apparent homogeneity from an extract of 2,4,6-trichlorophenol-grown cells of Streptomyces rochei 303. Like the analogous enzyme from Azotobacter sp. strain GP1, it exhibited a highly restricted substrate specificity and was able to cleave only 6-chlorohydroxyquinol and hydroxyquinol and not catechol, chlorinated catechols, or pyrogallol. No extradiol-cleaving activity was observed. In contrast to 6-chlorohydroxyquinol 1,2-dioxygenase from Azotobacter sp. strain GP1, the S. rochei enzyme had a distinct preference for 6-chlorohydroxyquinol over hydroxyquinol (kcat/Km = 1.2 and 0.57 s-1.microM-1, respectively). The enzyme from S. rochei appears to be a dimer of two identical 31-kDa subunits. It is a colored protein and was found to contain 1 mol of iron per mol of enzyme. The NH2-terminal amino acid sequences of 6-chlorohydroxyquinol 1,2-dioxygenase from S. rochei 303 and from Azotobacter sp. strain GP1 showed a high degree of similarity.

A Mo(V) electron paramagnetic resonance (EPR) study of the periplasmic respiratory nitrate reductase of the denitrifying bacterium Thiosphaera pantotropha has revealed that the molybdenum centre of this enzyme is very similar to that in the assimilatory nitrate reductase of Azotobacter vinelandii but is somewhat different from that of the membrane-bound bacterial respiratory nitrate reductases such as those of Escherichia coli and Paracoccus denitrificans. We have identified the Mo(V) species most likely to be the catalytically relevant one and characterised two other sets of Mo(V) EPR signals. As well as exhibiting EPR signals with g values typical of bacterial molybdenum-containing reductases, molybdenum-hydroxylase-like EPR signals can be elicited in the nitrate reductase of T. pantotropha upon treatment with excess dithionite. The only other enzyme known to display this phenomenon is the periplasmic dimethylsulphoxide reductase of Rhodobacter capsulatus. A mechanism for the generation of these signals is proposed which invokes reduction of the pterin ring of the molybdenum cofactor linked to GMP from the dihydro to the tetrahydro state. The possibilities and implications of there being cysteine ligands to the molybdenum centres of these two enzymes are discussed.

Hydrogen-uptake (Hup) activity in Azotobacter chroococcum depends upon a cluster of genes spread over 13,687 bp of the chromosome. Six accessory genes of the cluster, hupABYCDE, begin 4.8 kb downstream of the structural genes, hupSL, and are required for the formation of a functional [NiFe] hydrogenase. The sequencing of the intervening 4.8 kb of hup-specific DNA has now been completed. This revealed eight additional closely linked ORFs, which we designated hupZ, hupM, hupN, hupO, hupQ, hupR, hupT and hupV. These genes potentially encode polypeptides with predicted masses of 27.7, 22.3, 11.4, 16.2, 31.3, 8.1, 16.2 and 36.7 kDa, respectively. All eight genes are transcribed from the same strand as hupSL and hupABYCDE. A chroococcum, therefore, has a total of 16 contiguous genes affecting hydrogenase activity beginning with hupS and ending with hupE. The amino acid sequence deduced from hupZ has the characteristics of a b-type cytochrome. Insertion mutagenesis of hupZ resulted in a mutant incapable of supporting O2-dependent H2 oxidation. The deduced amino acid sequence of hupR shares high homology with bacterial rubredoxins. HupZ and HupR may both be involved in transferring electrons from hydrogenase to the electron transport chain. A mutation in hupV knocked out hydrogenase activity entirely; this gene may be involved in processing the large subunit of hydrogenase. It is now clear that the genes controlling [NiFe] hydrogenase activity in many bacteria including Azotobacter chroococcum, Alcaligenes eutrophus, Rhizobium leguminosarum, Rhodobacter capsulatus and Escherichia coli are highly conserved, organized in much the same manner, and likely derived from a common ancestor.

AnfA, an activator required for transcription of the structural genes encoding nitrogenase 3 (anfHDGK) in Azotobacter vinelandii, has a potential metal-binding site [(S19)H(C21)FTGE(C26)R] in its N terminus. Growth studies and expression of an anfH-lacZ fusion in mutants containing amino acid substitutions in this site indicate that Ser-19 is not required for AnfA activity whereas Cys-21 and Cys-26 are required. Residual expression of the anfH-lacZ fusion in AnfA- mutants was found to be due to activation by VnfA, the activator required for expression of genes encoding nitrogenase 2.

Azotobacter chroococcum MH1 was grown in a mannitol and nitrogen free medium supplemented with p-hydroxybenzoic acid, resorcinol, catechol or vanillic acid as a sole carbon source. Growth and nitrogenase activity of p-hydroxybenzoic acid were supported by 8, 6 and 4 mM of p-hydroxybenzoic acid, resorcinol and catechol, respectively. The generation time of 1.71 h in p-hydroxybenzoic acid did not differ from a generation time of 1.64 h, when grown in mannitol. The compound p-hydroxybenzoic acid was utilized rapidly. However, the decomposition of other phenolic compounds tested proceeded slowly. These results suggested that phenolic compounds released during biodegradation of plant wastes could be utilized as carbon sources for both growth and nitrogen fixation of Azotobacter 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.

The Azotobacter chroococcum chromosome contains a region spanning about 14 kb associated with hydrogen-uptake (Hup) activity. The small and large subunits of the hydrogenase are encoded by the structural genes hupS and hupL. Two other genes, hupD and hupE, are located 8.9 kb downstream from hupL and are required for the formation of a catalytically active hydrogenase. In this study, we determined the nucleotide sequence of a 3.8-kb region immediately upstream from hupD. This revealed four additional closely linked ORFs which we designated hupA, hupB, hupY and hupC; these genes potentially encode polypeptides with predicted masses of 12.6, 33.3, 80.4 and 9.0 kDa, respectively. This cluster of genes was shown to be essential for hydrogenase activity by insertion mutagenesis using antibiotic-resistance gene cassettes and a Tn5 derivative carrying a promoterless lacZ gene. A 10.5-kb fragment of DNA beginning 3.4 kb downstream from hupL, and including the sequenced region, was able to complement hupA and hupY mutants, supporting earlier evidence for a promoter downstream from hupSL. The deduced amino acid sequences of hupA, hupB and hupC are homologous to the Escherichia coli hypA, hypB and hypC gene products, respectively. Of particular interest is the fact that there is no homologue of the hupY gene product in the E. coli hyp operon. Mutations in hupY or hupB had little effect on beta-galactosidase activity in a strain also carrying a hupL::lacZ fusion, showing that hupY and hupB are not major factors in regulating the transcription of the hydrogenase structural genes.

During the reduction of N2 by V-nitrogenase at 30 degrees C, some hydrazine (N2H4) is formed as a product in addition to NH3 [Dilworth and Eady (1991) Biochem. J. 277, 465-468]. We show here the following. (1) That over the temperature range 30-45 degrees C the apparent Km for the reduction of N2 to yield these products is the same, but increases from 30 to 58 kPa of N2. On increasing the temperature from 45 degrees C to 50 degrees C, little change occurred in the rate of reduction of protons to H2; the rate of N2H4 production increased, but the rate of NH3 formation decreased 7-fold. (2) Temperature-shift experiments from 42 to 50 degrees C or from 50 to 42 degrees C showed that this selective loss of the ability to reduce N2 to NH3 was reversible. The effects we observe are consistent with the existence of different conformers of the VFe-protein at the two temperatures, that predominating at 50 degrees C being largely unable to reduce N2 to ammonia. (3) Measurement of the ratio between H2 evolution and N2 reduced to NH3 at N2 pressures up to 339 kPa for both Mo- and V-nitrogenases gave limiting H2/N2 values of 1.13 +/- 0.13 for Mo-nitrogenase and 3.50 +/- 0.03 for V-nitrogenase. Since for Mo-nitrogenase our measured value for the ratio at 339 kPa is the same as that derived by Simpson and Burris [(1984) Science 224, 1095-1097] at 5650 kPa, there appears to be little or no divergence from the predictions based on the apparent Km for N2. These data then suggest that there may be a fundamentally different mechanism for N2 binding to V-nitrogenase compared with Mo-nitrogenase. (4) We did not detect any N2H4 as a product of N2 reduction by Mo-nitrogenase over the temperature range investigated; however, at 50 degrees C this system reduced acetylene (C2H2) to yield some ethane (C2H6), in addition to ethylene (C2H4), a reaction normally associated with Mo-independent nitrogenases.

The nucleotide sequence spanning the nifH gene and part of the nifD gene encoding the molybdenum nitrogenase from Azotobacter chroococcum was determined. The transcription start point of the nifH promoter was mapped, and a potential transcriptional attenuator was located between the nifH and nifD genes.