Growth and indole-3-acetic acid biosynthesis of Azospirillum brasilense Sp245 is environmentally controlled

Batch and fed batch cultures of Azospirillum brasilense Sp245 were conducted in a bioreactor. Growth response, IAA biosynthesis and the expression of the ipdC gene were monitored in relation to the environmental conditions (temperature, availability of a carbon source and aeration). A. brasilense can grow and produce IAA in batch cultures between 20 and 38 degrees C in a standard minimal medium (MMAB) containing 2.5 gl(-1)l-malate and 50 microgml(-1) tryptophan. IAA synthesis requires depletion of the carbon source from the growth medium in batch culture, causing growth arrest. No significant amount of IAA can be detected in a fed batch culture. Varying the concentration of tryptophan in batch experiments has an effect on both growth and IAA synthesis. Finally we confirmed that aerobic growth inhibits IAA synthesis. The obtained profile for IAA synthesis coincides with the expression of the indole-3-pyruvate decarboxylase gene (ipdC), encoding a key enzyme in the IAA biosynthesis of A. brasilense.     

Identification and sequencing of pyrG, the CTP synthetase gene of Azospirillum brasilense Sp7

Abstract An 18.5-kb DNA fragment carrying the trpGDC cluster of Azospirillum brasilense Sp7 was previously cloned, yielding cosmid pAB1005. Attempts to identify trpA in the vicinity of trpGDC failed but led to the detection of a locus strongly homologous to pyrG , the structural gene for the CTP synthetase. The function of the A. brasilense pyrG gene was verified by complementation of the cytidine-requiring PyrG-deficient mutant JF646 of Escherichia coli . A second open reading frame was identified downstream of pyrG . The deduced amino acid sequence showed homology to dienelactone hydrolases of Pseudomonas and Alcaligenes , enzymes involved in utilization of halogenated aromatic compounds.     

A universal stress protein of Porphyromonas gingivalis is involved in stress responses and biofilm formation

Porphyromonas gingivalis is recognized as one of the major periodontal pathogens in subgingival plaque, which is implicated in the progression of chronic periodontal disease. We analyzed the role of upsA in P. gingivalis 381 and its uspA-deficient mutant CW301 under various stress conditions. In general, the uspA mutant was less tolerant to a variety of environmental stresses relative to the parental strain. In addition, gene expression of uspA is upregulated during biofilm formation. Biofilm formation of the uspA mutant was also less than that of strain 381. In conclusion, the uspA gene affecting the stress responses of P. gingivalis is required for optimal biofilm formation.     

Regulation of nitrogen fixation in Azospirillum brasilense Sp7: Involvement of nifA, glnA and glnB gene products

The expression of nifA-, niH- and nifB-lacZ fusions was examined in different mutants of Azospirillum brasilense. Mutations in nifA, glnA and glnB severely impaired the expression of nifH- and nifB-lacZ fusions. By contrast, a nifA-lacZ fusion was not affected in a nifA or a glnB background and was only partially impaired in glnA mutants. It is proposed that in A. brasilense, the PII protein and glutamine synthetase are involved in a post-translational modification of NifA.     

A CheR/CheB fusion protein is involved in cyst cell development and chemotaxis in Azospirillum brasilense Sp7

We here report the sequence and functional analysis of cstB of Azospirillum brasilense Sp7. The predicted cstB contains C-terminal two PAS domains and N-terminal part which has similarity with CheB-CheR fusion protein. cstB mutants had reduced swarming ability compared to that of A. brasilense wild-type strain, implying that cstB was involved in chemotaxis in A. brasilense. A microscopic analysis revealed that cstB mutants developed mature cyst cells more quickly than wild type, indicating that cstB is involved in cyst formation. cstB mutants were affected in colony morphology and the production of exopolysaccharides (EPS) which are essential for A. brasilense cells to differentiate into cyst-like forms. These observations suggested that cstB was a multi-effector involved in cyst development and chemotaxis in A. brasilense.     

Effect of root exudates on the exopolysaccharide composition and the lipopolysaccharide profile of Azospirillum brasilense Cd under saline stress

The effect of wheat root exudates on the exopolysaccharide (EPS) composition and the lipopolysaccharide (LPS) profile of Azospirillum brasilense Cd under saline stress was studied. EPS of A. brasilense Cd was composed of glucose (47%), mannose (3%), xylose (4%), fucose (28%), rhamnose (6%), arabinose (1%) and galactose (11%). Under saline stress, A. brasilense produced a totally different EPS, composed mainly of galactose. Root exudates induced changes in A. brasilense EPS composition only under normal conditions, consisting of higher amounts of arabinose and xylose compared with EPS of bacteria grown without root exudates. No changes were induced by root exudates when A. brasilense was grown under saline stress. Additionally, root exudates induced changes in the LPS profile, both under normal and stress conditions.     

Wheat inoculation with Azospirillum brasilense Sp6 and some mutants altered in nitrogen fixation and indole-3-acetic acid production

Abstract Inoculation of wheat seedlings with Azospirillum brasilense Sp6 produced an increase in the number and length of the lateral roots as a plant response. Inoculation with a Nif⁻ mutant, A. brasilense SpF103, which is producer of indole-3-acetic acid (IAA), yielded a very similar plant response. However, inoculation with a Nif⁻ mutant, A. brasilense SpF57, which is a low producer of IAA, did not elitic any response from the plant. The data suggest that the root system response of wheat seedlings to bacterial inoculation is due mainly to production of auxin-type substances by the microorganism.     

Glycogen phosphorylase <Emphasis Type="Italic">b</Emphasis> and phosphorylase kinase binding to glycogen under molecular crowding conditions. Inhibitory effect of FAD

Dynamic light scattering was used to study the interaction of phosphorylase kinase (PhK) and glycogen phos-phorylase b (Phb) from rabbit skeletal muscle with glycogen under molecular crowding conditions arising from the presence of 1 M trimethylamine N-oxide and at physiological ionic strength. The mean value of hydrodynamic radius of the initial glycogen particles was 52 nm. Crowding stimulated Phb and PhK combined binding on glycogen particles. Two-stage character of PhK binding to glycogen particles containing adsorbed Phb was found in the presence of the crowding agent. At the initial stage, limited size particles with hydrodynamic radius of ～220 nm are formed, whereas the second stage is accompanied by linear growth of hydrodynamic radius. Flavin adenine dinucleotide (FAD) selectively inhibited PhK binding at the second stage. The data indicate that in the first stage Phb is involved in PhK binding by glycogen particles containing adsorbed Phb, whereas PhK binding in the second stage does not involve Phb.     

Azospirillum brasilense Sp245 triggers cytokinin signaling in root tips and improves biomass accumulation in Arabidopsis through canonical cytokinin receptors

The plant growth promoting rhizobacterium Azospirillum brasilense Sp245 enhances biomass production in cereals and horticultural species and is an interesting model to study the physiology of the phytostimulation program. Although auxin production by Azospirillum appears to be critical for root architectural readjustments, the role of cytokinins in the growth promoting effects of Azospirillum remains unclear. Here, Arabidopsis thaliana seedlings were co-cultivated in vitro with A. brasilense Sp245 to assess whether direct contact of roots with bacterial colonies or exposure to the bacterial volatiles using divided Petri plates would affect biomass production and root organogenesis. Both interaction types increased root and shoot fresh weight but had contrasting effects on primary root length, lateral root formation and root hair development. Cell proliferation in root meristems analyzed with the CYCB1;1::GUS reporter decreased over time with direct contact, but was augmented by plant exposure to volatiles. Noteworthy, the expression of the cytokinin-inducible reporters TCS::GFP and ARR5::GUS increased in root tips in response to bacterial contact, without being affected by the volatiles. In A. thaliana having single (cre1-12, ahk2-2, ahk3-3), double (cre1-12/ahk2-2, cre1-12/ahk3-3, ahk2-2/ahk3-3) or triple (cre1-12/ahk2-2/ahk3-3) mutations in canonical cytokinin receptors, only the triple mutant had a marked effect on plant growth in response to A. brasilense. These results show that different mechanisms are elicited by A. brasilense, which influence the cytokinin-signaling pathway.     

Metabolic function of glycogen phosphorylase and trehalose phosphorylase in fruit-body formation ofFlammulina velutipes

Glycogen phosphorylase in the vegetative mycelium ofFlammulina velutipes converts glycogen to α-glucose 1-phosphate (G1P) in the colony during fruit-body development. Glycogen may contribute to the synthesis of trehalose as the starting material in the vegetative mycelium during the fruiting process of the colony, and the trehalose produced is translocated into the fruit-bodies as the main carbohydrate substrate for their development. Trehalose phosphorylase activity in the vegetative mycelium was at a relatively high level until fruit-body initiation, suggesting the turnover of this disaccharide during the vegetative stage of the colony development. Trehalose phosphorylase activity in the stipes showed a peak level at the early phase of fruit-body development, suggesting the continuing phosphorolysis of trehalose by this enzyme. The stipes also showed a high specific activity of phosphoglucomutase at a sufficient level to facilitate the conversion of G1P to α-glucose 6-phosphate (G6P). In the pilei a large amount of G1P remained until the growth of the fruit-bodies ceased. Trehalase activities in the stipes and pilei were at a very low level, and this enzyme may not contribute to the catabolism of trehalose in the fruit-body development.     

Glycogen phosphorylase isoenzyme BB in diagnosis of myocardial ischaemic injury and infarction

This review deals with glycogen phosphorylase (GP) and its isoenzyme BB in the diagnosis of ischaemic myocardial injury. Early identification and confirmation of acute myocardial infarction is essential for correct patient care and disposition decision in the emergency department. In this respect, glycogen phosphorylase isoenzyme BB (GPBB) based on its metabolic function is an enzyme for early laboratory detection of ischaemia. In the aerobic heart muscle GPBB together with glycogen is tightly associated with the vesicles of the sarcoplasmic reticulum. Release of GPBB, the main isoform in the human myocardium, essentially depends on the degradation of glycogen, which is catalyzed by GP. Ischaemia is known to favour the conversion of bound GP in the b form into GP a, thereby accelerating glycogen breakdown, which is the ultimate prerequisite for getting GP into a soluble form being able to move freely in the cytosol. The efflux of GPBB into the extracellular fluid follows if ischaemia-induced structural alterations in the cell membrane become manifest. The clinical application of GPBB as a marker of ischaemic myocardial injury is a very promising tool for extending our knowledge of the severity of myocardial ischaemic events in the various coronary syndromes. The rational roots of this development were originated from Albert Wollenberger's research work on the biochemistry of cardiac ischaemia and the transient acceleration of glycogenolysis mainly brought about by GP activation.     

Localization and survival of Azospirillum brasilense Az39 in soybean leaves

In recent years, foliar inoculation has gained acceptance among the available methods to deliver plant beneficial micro-organisms to crops under field conditions. Colonization efficiency by such micro-organisms largely depends on their ability to survive when applied on the leaves. In this work, we evaluated the survival and localization of Azospirillum brasilense Az39 (Az39) in excised soybean leaves. Scanning electron microscopy and confocal laser scanning microscopy of a red fluorescent-transformed variant of Az39 were used to determine bacterial localization, while the most probable number and plate count methods were applied for bacterial quantification. Microscopic observations indicated a decrease in the number of Az39 cells on the leaf surface at 24 h after treatment, whereas midribs and cell–cell junctions of the inner leaf epidermis became highly populated zones. The presence of Az39 inside xylem vessels was corroborated at 6 h after bacterization. Az39 population did not significantly decrease throughout 24 h. We could visualize Az39 cells on the surface and in internal tissues of soybean leaves and recover them through culture methodologies. These results evidence the survival capacity of Az39 on and inside leaves and suggest a previously unnoticed endophytic potential for this well-known plant growth-promoting rhizobacteria strain.     

Potential roles for the glnB and ntrYX genes in Azospirillum brasilense

Three Azospirillum brasilense mutants constitutive for nitrogen fixation (Nif(C)) in the presence of NH4(+) and deficient in nitrate-dependent growth were used as tools to define the roles of the glnB and ntrYX genes in this organism. Mutant HM14 was complemented for nitrate-dependent growth and NH4(+) regulation of nitrogenase by plasmid pL46 which contains the ntrYX genes of A. brasilense. Mutant HM26 was restored for NH4(+) regulation and nitrate-dependent growth by plasmid pJC1, carrying the A. brasilense glnB gene expressed from a constitutive promoter. Mutant HM053, on the other hand, was not complemented for NH4(+) regulation of nitrogenase and nitrate-dependent growth by both plasmids pJCI and pL46. The levels and control of glutamine synthetase activity of all mutants were not affected by both plasmids pL46 (ntrYX) and pJC1 (glnB). These results support the characterization of strains HM14 as an ntrYX mutant and strain HM26 as a glnB mutant and the involvement of ntrYX and glnB in the regulation of the general nitrogen metabolism in A. brasilense.     

Strain-specific salt tolerance and osmoregulatory mechanisms in Azospirillum brasilense

Salinity stress inhibits the growth and nitrogen fixation ability of the plant growth-promoting rhizobacterium Azospirillum brasilense. Five strains of A. brasilense were isolated from the rhizosphere of Indian cereals and grasses and identified on the basis of their phenotypic features and 16S rRNA gene sequence. The five Indian isolates and two standard strains of A. brasilense, Sp7 and Cd, showed notable differences in growth, acetylene-reducing activity under salt stress, and ability to take up and use glycine betaine for the restoration of growth and acetylene-reducing activity under salt stress. Salt stress also enhanced the production of exopolysaccharides and cell aggregates, the extent of which varied in different strains of A. brasilense at different carbon to nitrogen ratios in the culture medium. It can be concluded that the production of exopolysaccharides and cell aggregates is a more consistent physiological response of A. brasilense to salt stress than is the uptake and osmoprotection by glycine betaine.     

Plastidial phosphorylase is required for normal starch synthesis in Chlamydomonas reinhardtii

Among the three distinct starch phosphorylase activities detected in Chlamydomonas reinhardtii, two distinct plastidial enzymes (PhoA and PhoB) are documented while a single extraplastidial form (PhoC) displays a higher affinity for glycogen as in vascular plants. The two plastidial phosphorylases are shown to function as homodimers containing two 91-kDa (PhoA) subunits and two 110-kDa (PhoB) subunits. Both lack the typical 80-amino-acid insertion found in the higher plant plastidial forms. PhoB is exquisitely sensitive to inhibition by ADP-glucose and has a low affinity for malto-oligosaccharides. PhoA is more similar to the higher plant plastidial phosphorylases: it is moderately sensitive to ADP-glucose inhibition and has a high affinity for unbranched malto-oligosaccharides. Molecular analysis establishes that STA4 encodes PhoB. Chlamydomonas reinhardtii strains carrying mutations at the STA4 locus display a significant decrease in amounts of starch during storage that correlates with the accumulation of abnormally shaped granules containing a modified amylopectin structure and a high amylose content. The wild-type phenotype could be rescued by reintroduction of the cloned wild-type genomic DNA, thereby demonstrating the involvement of phosphorylase in storage starch synthesis.     

Disruption of dTDP-rhamnose biosynthesis modifies lipopolysaccharide core, exopolysaccharide production, and root colonization in Azospirillum brasilense

The interaction between Azospirillum brasilense and plants is not fully understood, although several bacterial surface components like exopolysaccharides (EPS), flagella, and capsular polysaccharides are required for attachment and colonization. While in other plant-bacteria associations (Rhizobium-legume, Pseudomonas-potato), lipopolysaccharides (LPS) play a key role in the establishment of an effective association, their role in the root colonization by Azospirillum had not been determined. In this study, we isolated a Tn5 mutant of A. brasilense Cd (EJ1) with an apparently modified LPS core structure, non-mucoid colony morphology, increased EPS production, and affected in maize root colonization. A 3790-bp region revealed the presence of three complete open reading frames designated rmlC, rmlB and rmlD. The beginning of a fourth open reading frame was found and designated rmlA. These genes are organized in a cluster which shows homology to the cluster involved in the synthesis of dTDP-rhamnose in other bacteria. Additionally, the analysis of the monosaccharide composition of LPSs showed a diminution of rhamnose compared to the wild-type strain.