In vitro uridylylation of the Azospirillum brasilense N-signal transducing GlnZ protein

Azospirillum brasilense is a diazotroph which associates with important agricultural crops. The nitrogen fixation process in this organism is highly regulated by ammonium and oxygen, and involves several proteins including the two PII-like proteins, GlnB and GlnZ. Although these proteins are structurally very similar, they play different roles in the control of nitrogen fixation. In this work, we describe the expression, purification, and uridylylation of the GlnZ protein of A. brasilense strain FP2. The amplified glnZ gene was sub-cloned and expressed as a His-tagged fusion protein. After purification, we obtained 30-40 mg of purified GlnZ per liter of culture. This protein was purified to 99% purity and assayed for in vitro uridylylation using a partially purified Escherichia coli GlnD as a source of uridylylyl-transferase activity. Analyses of the uridylylation reactions in non-denaturing and denaturing polyacrylamide gel electrophoresis showed that up to 74% of GlnZ monomers were modified after 30 min reaction. This covalent modification is strictly dependent on ATP and 2-ketoglutarate, while glutamine acts as an inhibitor and promotes deuridylylation.     

Nitrogenase switch-off by ammonium ions in Azospirillum brasilense requires the GlnB nitrogen signal-transducing protein

Nitrogenase activity in several diazotrophs is switched off by ammonium and reactivated after consumption. The signaling pathway to this system in Azospirillum brasilense is not understood. We show that ammonium-dependent switch-off through ADP-ribosylation of Fe protein was partial in a glnB mutant of A. brasilense but absent in a glnB glnZ double mutant. Triggering of inactivation by anaerobic conditions was not affected in either mutant. The results suggest that glnB is necessary for full ammonium-dependent nitrogenase switch-off in A. brasilense.     

Repressor Mutant Forms of the Azospirillum brasilense NtrC Protein

The Azospirillum brasilense mutant strains FP8 and FP9, after treatment with nitrosoguanidine, showed a null Nif phenotype and were unable to use nitrate as their sole nitrogen source. Sequencing of the ntrC genes revealed single nucleotide mutations in the NtrC nucleotide-binding site. The phenotypes of these strains are discussed in relation to their genotypes.     

Inhibition of Biosynthesis and Activity of Nitrogenase in Azospirillum brasilense Sp7 Under Salinity Stress

Azospirillum brasilense is a microaerophilic, plant growth-promoting bacterium, whose nitrogenase activity has been shown to be sensitive to salinity stress. Growth of A. brasilense in semi-solid medium showed that diazotrophic growth in N-free medium was relatively less sensitive to high NaCl concentrations (200–400 mM) than that in presence of NH₄ ⁺. Increase in salinity stress to diazotrophic A. brasilense in the semi-solid medium led to the migration of the pellicle to deeper anaerobic zones. Assays of acetylene reduction and nifH-lacZ and nifA-lacZ fusions indicated that salinity stress inhibited nitrogenase biosynthesis more strongly than nitrogenase activity. Under salt stress, the amount of dinitrogenase reductase inactivated by ADP-ribosylation was strongly reduced, indicating that the dinitrogenase reductase ADP ribosyl transferase (DRAT) activity was also inhibited by increased NaCl concentrations. Movement of the pellicle to the anaerobic zone and inhibition of DRAT might be adaptive responses of A. brasilense to salinity stress under diazotrophic conditions. Supplementation of glycine betaine, which alleviates salt stress, partially reversed both responses. Received: 2 August 2001 / Accepted: 28 August 2001     

Denitrification by Azospirillum brasilense Sp 7

Nitrous oxide reduction can consistently be demonstrated with high activities in cells of Azospirillum brasilense Sp 7 which are grown anaerobically in the presence of low amounts of nitrite. Azospirillum can even grow anaerobically with nitrous oxide in the absence of any other respiratory electron acceptor. Nitrous oxide reduction by Azospirillum is inhibited by acetylene, amytal and weakly by carbon monoxide. Azospirillum converts nitrous oxide to molecular nitrogen without the formation of ammonia. The cells must, therefore, be supplied with ammonia from nitrogen fixation during anaerobic growth with nitrous oxide. When no other nitrogen compound besides nitrous oxide is available in the medium, the bacteria synthesize nitrogenase from protein reserves in about 2 h. Nitrogenase synthesis is blocked by chloramphenicol under these conditions. In contrast, the addition of nitrate or nitrite to the medium represses the synthesis of nitrogenase. Nitrous oxide reduction by Azospirillum and other microorganisms is possibly of ecological significance, because the reaction performed by the bacteria may remove nitrous oxide from soils.     

Plasmids of Azospirillum brasilense

The cells from natural isolates of A. Brasilense were found to harbour 1 to 4 plasmids with the molecular masses within the 27-300 Md range. 100 Md plasmids are specific for this bacterial species. Strains isolated from the roots of cereals (wheat, maize, barley) have more heterogeneous plasmid composition as compared to the strains isolated from soil.     

Biosynthesis of gold nanoparticles by Azospirillum brasilense

Plant-associated nitrogen-fixing soil bacteria Azospirillum brasilense were shown to reduce the gold of chloroauric acid to elemental gold, resulting in formation of gold nanoparticles. Extracellular phenoloxidizing enzymes (laccases and Mn peroxidases) were shown to participate in reduction of Au⁺³ (HAuCl₄) to Au⁰. Transmission electron microscopy revealed accumulation of colloidal gold nanoparticles of diverse shape in the culture liquid of A. brasilense strains Sp245 and Sp7. The size of the electron-dense nanospheres was 5 to 50 nm, and the size of nanoprisms varied from 5 to 300 nm. The tentative mechanism responsible for formation of gold nanoparticles is discussed.     

Regulation of nitrogenase activity by oxygen in Azospirillum brasilense and Azospirillum lipoferum.

The nitrogenase activity of the microaerophilic bacteria Azospirillum brasilense and A. lipoferum was completely inhibited by 2.0 kPa of oxygen (approximately 0.02 atm of O2) in equilibrium with the solution. The activity could be partially recovered at optimal oxygen concentrations of 0.2 kPa. In contrast to the NH4+ switch off, no covalent modification of the nitrogenase reductase (Fe protein) was involved, as demonstrated by Western-blotting and 32P-labeling experiments. However, the inhibition of the nitrogenase activity under anaerobic conditions was correlated with covalent modification of the Fe protein. In contrast to the NH4+ switch off, no increase in the cellular glutamine pool and no modification of the glutamine synthetase occurred under anaerobic switch-off conditions. Therefore, a redox signal, independent of the nitrogen control of the cell, may trigger the covalent modification of the nitrogenase reductase of A. brasilense and A. lipoferum.     

Production of vitamins by Azospirillum brasilense in chemically-defined media

The production of vitamins by Azospirillum brasilense was studied in chemically-defined media amended with malate, gluconate and fructose. The liberation of vitamins was significantly affected by the presence of different carbon sources and the age of the culture. Thiamine, niacin and pantothenic acid were produced in large amounts. Thiamine and riboflavin were produced only in culture containing malate or fructose. Biotin was not detected in the supernatants of the culture media.     

Fructose catabolism in Azospirillum brasilense and Azospirillum lipoferum.

The pathways for catabolism of fructose were investigated in the type strains of Azospirillum lipoferum and Azospirillum brasilense grown aerobically with (NH4)2SO4 as the nitrogen source. When grown on fructose, the former species possessed a complete Entner-Doudoroff pathway, whereas the latter species lacked activity for glucose-6-phosphate dehydrogenase. Both species possessed a complete catabolic Embden-Meyerhof-Parnas pathway. Neither species possessed the key enzyme of the hexose monophosphate pathway, 6-phosphogluconate dehydrogenase. Both species could phosphorylate fructose to fructose-1-phosphate by means of a phosphoenolpyruvate-phosphotransferase system, and high activities of 1-phosphofructokinase occurred. Both species possessed glucokinase activity, but only A. lipoferum had hexokinase activity; moreover, the cells of A. brasilense were nearly impermeable to glucose, accounting for the inability of this species to grow on glucose. Both species possessed pyruvate dehydrogenase, a complete tricarboxylic acid cycle, a glyoxylate shunt, and malic enzyme. Analysis of the acidic end products for both species indicated the formation of only small amounts of various organic acids, and most of the titratable acidity was due to utilization of the ammonium ions of the medium. Gluconic acid was not formed during growth of either species on fructose but was detected during growth of A. lipoferum on glucose; this species also possessed an NADP-linked glucose dehydrogenase and gluconokinase.     

Detection of chemotaxis in Azospirillum brasilense

Azospirillum brasilense strain Cd responded chemotactically to amino acids, sugars and organic acids. Chemotactic rings were observed in semisolid agar plates containing oxidizable substrates. Increasing sodium succinate concentration decreased the velocity of ring expansion. Chemotactic activity of Azospirillum was also examined by a newly developed technique using a channelled chamber. Varying the concentrations of aspartic and glutamic acids affected the chemotactic response of the bacteria. In both assays chemotaxis was obtained under conditions that prevented aerotaxis.     

Cell Ultrastructure in Azospirillum brasilense Biofilms

Microbiology - Due to the primary localization of both epiphytic and endophytic plant growth-promoting rhizobacteria on the surface of the plant root system, biofilm formation is an adaptive trait...     

Atypical R-S dissociation in Azospirillum brasilense

It was found that atypical R-S dissociation in the type strain A. brasilense Sp7 is not accompanied by drastic changes in the carbohydrate moieties of bacterial lipopolysaccharides but is rather due to different contributions of two O-specific polysaccharides (found in both R and S dissociants) to the age-dependent architectonics of the cell surface.     

Production of bacteriocins and siderophore-like activity by Azospirillum brasilense

Sixty Azospirillum strains were tested for their bacteriocin production ability; twenty-seven (45%) were able to produce bacteriocins and inhibited the growth of one or more indicator strains in solid medium. Mitomycin C treatment enhanced the proportion to 80%. Sometimes large growth inhibition zones were formed, but not when FeCl3 was added in the medium. These inhibition zones probably result from the activity of siderophores. Partially purified bacteriocins produced by four strains were inactivated at pH 4, but were very stable between pH 5 to 10; bacteriocins produced by three strains lost their activity between 55 and 80 degrees C. Loss or decrease in the bacteriocin activity was observed with pronase E treatment; trypsin, lysozyme and alpha-amylase did not have an effect on bacteriocin activity. These findings show that the antagonism among azospirilla was due principally to the bacteriocins and sometimes probably due to siderophores, but not to bacteriophages or other substances.     

Multiple CheY Homologs Control Swimming Reversals and Transient Pauses in Azospirillum brasilense

Chemotaxis, together with motility, helps bacteria foraging in their habitat. Motile bacteria exhibit a variety of motility patterns, often controlled by chemotaxis, to promote dispersal. Motility in many bacteria is powered by a bidirectional flagellar motor. The flagellar motor has been known to briefly pause during rotation because of incomplete reversals or stator detachment. Transient pauses were previously observed in bacterial strains lacking CheY, and these events could not be explained by incomplete motor reversals or stator detachment. Here, we systematically analyzed swimming trajectories of various chemotaxis mutants of the monotrichous soil bacterium, Azospirillum brasilense. Like other polar flagellated bacterium, the main swimming pattern in A. brasilense is run and reverse. A. brasilense also uses run-pauses and putative run-reverse-flick-like swimming patterns, although these are rare events. A. brasilense mutant derivatives lacking the chemotaxis master histidine kinase, CheA4, or the central response regulator, CheY7, also showed transient pauses. Strikingly, the frequency of transient pauses increased dramatically in the absence of CheY4. Our findings collectively suggest that reversals and pauses are controlled through signaling by distinct CheY homologs, and thus are likely to be functionally important in the lifestyle of this soil organism.     

The random walk of Azospirillum brasilense

The bacterium Azospirillum brasilense has been frequently studied in laboratory experiments. It performs movements in space where long forward and backward runs on a straight line occur simultaneously with slow changes of direction of the line. A model is presented in which a correlated random walk on a line is joined to diffusion on a sphere of directions. For this transport system, a hierarchy of moment approximations is derived, ranging from a hyperbolic system with four dependent variables to a scalar damped wave equation (telegraph equation) and then to a single diffusion equation for particle density. The original parameters are compounded in the diffusion quotient. The effects of these parameters, such as particle speed or turning rate, on the diffusion coefficient are discussed in detail.     

Aerotactic response of Azospirillum brasilense.

Five strains of Azospirillum brasilense and two of Azospirillum spp., from Israel, responded to self-created and preformed oxygen gradients by forming aerotactic bands in capillary tubes and actively moving toward a specific zone with low dissolved oxygen. Increasing the oxygen concentration in capillaries containing phosphate buffer increased the number of attracted bacteria and decreased band velocity. High O2 concentrations and H2O2 temporarily repulsed the bacteria, causing the formation of a bacterial arc around the capillary mouth. There was no band formation under anaerobic conditions, although the bacteria remained highly motile. Exogenous energy sources were unnecessary for aerotaxis in Azospirillum spp. The addition of oxidizable substrates to the capillary slightly enhanced aerotaxis, possibly by accelerating O2 consumption. Aerotactic band formation was affected by pH, bacterial concentration and age, incubation time, and respiratory inhibitors, but not by the lack of combined nitrogen in the growth medium. It is proposed that aerotaxis plays a role in the capacity of Azospirillum spp. to reach an environment suitable for N2 fixation.     

Root-to-Root Travel of the Beneficial Bacterium Azospirillum brasilense

The root-to-root travel of the beneficial bacterium Azospirillum brasilense on wheat and soybean roots in agar, sand, and light-textured soil was monitored. We used a motile wild-type (Mot⁺) strain and a motility-deficient (Mot^-) strain which was derived from the wild-type strain. The colonization levels of inoculated roots were similar for the two strains. Mot⁺ cells moved from inoculated roots (either natural or artificial roots in agar, sand, or light-textured soil) to noninoculated roots, where they formed a band-type colonization composed of bacterial aggregates encircling a limited part of the root, regardless of the plant species. The Mot^- strain did not move toward noninoculated roots of either plant species and usually stayed at the inoculation site and root tips. The effect of attractants and repellents was the primary factor governing the motility of Mot⁺ cells in the presence of adequate water. We propose that interroot travel of A. brasilense is an essential preliminary step in the root-bacterium recognition mechanism. Bacterial motility might have a general role in getting Azospirillum cells to the site where firmer attachment favors colonization of the root system. Azospirillum travel toward plants is a nonspecific active process which is not directly dependent on nutrient deficiency but is a consequence of a nonspecific bacterial chemotaxis, influenced by the balance between attractants and possibly repellents leaked by the root.