Azospirillum brasilense: Mode of Action

Biological Nitrogen Fixation Mechanism

Azospirillum brasilense converts atmospheric nitrogen (N₂) into plant-available ammonium (NH₄⁺) through the nitrogenase enzyme complex under microaerobic conditions. The enzyme consists of two essential components: the dinitrogenase protein (MoFe protein, encoded by nifDK) containing a molybdenum-iron cofactor where N₂ reduction occurs, and the dinitrogenase reductase protein (Fe protein, encoded by nifH) that transfers electrons to the nitrogenase protein. (academic.oup+1)

Regulatory Control Systems

Transcriptional Regulation

The nitrogen fixation genes are organized in a major 30 kb nif gene cluster containing the nifHDK operon, with separately transcribed nifA and nifB genes. Expression is controlled by the NtrBC two-component regulatory system and the alternative sigma factor σ⁵⁴ (RpoN). Unlike Klebsiella pneumoniae, transcription of nifA in A. brasilense does not require NtrBC, and nifHDK expression is primarily controlled through posttranslational regulation of NifA activity. (pubmed.ncbi.nlm.nih+1)

Post-translational Regulation

A. brasilense employs a sophisticated dual regulatory mechanism for rapid nitrogenase inactivation. The primary system involves reversible ADP-ribosylation of the nitrogenase Fe protein mediated by DraT (dinitrogenase reductase ADP-ribosyltransferase) and DraG (dinitrogenase reductase activating glycohydrolase) enzymes. A second independent mechanism exists that can partially inhibit nitrogenase activity in response to ammonium, even when ADP-ribosylation is eliminated.(pmc.ncbi.nlm.nih+2)

Phytohormone Production and Root Morphology Alteration

Indole-3-Acetic Acid (IAA) Biosynthesis

A. brasilense produces significant amounts of IAA through the indole-3-pyruvate (IPyA) pathway. The key enzyme indole-3-pyruvate decarboxylase (IpdC) converts indole-3-pyruvic acid to IAA, with the ipdC gene being essential for bacterial IAA production. IAA production reaches 10.8 μg/ml in strain Cd and varies significantly between strains. (springer+4)

IAA serves a dual function - it promotes plant growth while also protecting the bacterium from toxic effects of indole intermediates by maintaining membrane potential homeostasis and regulating bacterial translation. ipdC mutants show reduced growth rates, altered physiology, and more depolarized membrane potential compared to wild-type strains. (pubmed.ncbi.nlm.nih+1)

Additional Phytohormones

A. brasilense produces multiple plant hormones including gibberellic acid (GA₃) at concentrations up to 0.66 μg/ml, zeatin (cytokinin) up to 2.37 μg/ml, abscisic acid (ABA) up to 0.077 μg/ml, and ethylene. The bacterium can hydrolyze GA₂₀-glucosyl conjugates and perform 3β-hydroxylation to convert GA₂₀ to bioactive GA₁.pubmed.ncbi.nlm.nih+2

Root Architecture Modification

IAA produced by A. brasilense causes dramatic changes in root morphology including decreased primary root length and increased root hair formation. These effects are completely abolished in ipdC mutants and can be mimicked by exogenous IAA application. The altered root architecture enables plants to explore larger soil volumes for nutrient and water acquisition.academic.oup+1

Root Colonization and Chemotaxis Mechanisms

Motility-Dependent Colonization

A. brasilense employs active motility and chemotaxis as essential mechanisms for root surface colonization. Motile strains can travel from inoculated roots to non-inoculated roots, forming characteristic band-type colonization patterns composed of bacterial aggregates encircling limited root regions. Non-motile mutants remain at inoculation sites and show severely impaired colonization ability.pmc.ncbi.nlm.nih+2

Energy Taxis and Chemical Sensing

Root colonization is mediated by energy taxis through the Tlp1 transducer protein. A. brasilense navigates toward metabolizable compounds in root exudates that affect intracellular energy levels. The bacterium responds to specific chemicals including organic acids (malate, succinate), sugars, and amino acids found in root exudates. Metabolism-dependent chemotaxis contributes to the broad host range observed in Azospirillum-plant associations.journals.asm+2

Two-Phase Attachment Process

Colonization involves a two-step process: initial adsorption mediated by the polar flagellum whose flagellin protein facilitates motility-dependent attachment, followed by anchoring through surface polysaccharides that enable stable root surface colonization. (academic.oup+1)

Stress Tolerance and ACC Deaminase Activity

Ethylene Regulation

A. brasilense produces ACC deaminase enzyme which cleaves the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) into ammonia and α-ketobutyrate. This reduces plant ethylene levels during stress conditions, preventing growth-inhibitory effects of stress ethylene. ACC deaminase activity is constitutively expressed but can be enhanced under stress conditions. (pmc.ncbi.nlm.nih+1)

Plants treated with ACC deaminase-producing A. brasilense show enhanced stress tolerance to flooding, drought, salinity, pathogen attack, and metal toxicity. The bacterium itself contains a functional ethylene receptor (AzoEtr1) that responds to plant ethylene signals.(nature+2)

Multiple Stress Protection Mechanisms

A. brasilense confers stress tolerance through various mechanisms including osmotic adjustment, antioxidant enzyme activation, and synthesis of stress-protective compounds like trehalose. The bacterium modifies plant ion selectivity during salt stress, restricting sodium uptake while promoting potassium absorption. (frontiersin+1)

Biofilm Formation and Surface Colonization

Cyclic-di-GMP Regulation

Biofilm formation is regulated by the c-di-GMP signaling system involving diguanylate cyclases like CdgA. The cdgA gene is essential for biofilm formation and exopolysaccharide (EPS) production. Biofilms consist of bacterial aggregates embedded in a matrix of EPS, extracellular DNA, and fibrillar material.pubmed.ncbi.nlm.nih+2

Ethylene-Mediated Biofilm Modulation

Plant ethylene reduces biofilm formation in A. brasilense through the AzoEtr1 ethylene receptor. Ethylene treatment decreases EPS production and cell aggregation, preventing surface attachment. This represents a novel cross-kingdom signaling mechanism where plant hormones directly influence bacterial colonization behavior.(pmc.ncbi.nlm.nih)

Mineral Nutrition Enhancement

Phosphate Availability

While A. brasilense strains Cd and Az39 show limited phosphate solubilization ability in standard assays , some strains can solubilize phosphate through organic acid production that reduces medium pH. Co-inoculation with specialized phosphate-solubilizing bacteria enhances phosphate availability. ( citeseerx.ist.psu+3)

Iron Acquisition and Siderophore Production

A. brasilense strains show variable siderophore production depending on strain and culture conditions. While strains Cd and Az39 tested negative for siderophore production in standard assays, other studies suggest potential iron chelation mechanisms exist. (pubmed.ncbi.nlm.nih+1)

Polyamine Production

A. brasilense produces significant quantities of polyamines including spermidine (up to 155 nmol/ml), putrescine, spermine, and cadaverine. Polyamines function as growth regulators and stress protectants, with production patterns influenced by culture medium composition.

(citeseerx.ist.psu+1)

Agricultural Field Performance

Yield Enhancement Mechanisms

Field studies demonstrate that A. brasilense inoculation can substitute for 25-50% of nitrogen fertilizer applications without yield reduction. Meta-analyses of Brazilian field trials show consistent positive responses in maize and wheat yields. The bacterium's effectiveness results from the synergistic combination of nitrogen fixation, phytohormone production, stress tolerance enhancement, and improved nutrient uptake.(pmc.ncbi.nlm.nih+3)

Survival and Persistence

A. brasilense survives on root surfaces for several weeks under field conditions, maintaining populations sufficient for continued plant growth promotion. The bacterium forms protective biofilms that enhance survival under environmental stress.(nature+2)