Studies Using Mutant Strains of Azospirillum brasilense Reveal That Atmospheric Nitrogen Fixation and Auxin Production Are Light Dependent Processes

Abstract

As the use of microbial inoculants in agriculture rises, it becomes important to understand how the environment may influence microbial ability to promote plant growth. This work examines whether there are light dependencies in the biological functions of Azospirillum brasilense, a commercialized prolific grass-root colonizer. Though classically defined as non-phototrophic, A. brasilense possesses photoreceptors that could perceive light conducted through its host's roots. Here, we examined the light dependency of atmospheric biological nitrogen fixation (BNF) and auxin biosynthesis along with supporting processes including ATP biosynthesis, and iron and manganese uptake. Functional mutants of A. brasilense were studied in light and dark environments: HM053 (high BNF and auxin production), ipdC (capable of BNF, deficient in auxin production), and FP10 (capable of auxin production, deficient in BNF). HM053 exhibited the highest rate of nitrogenase activity with the greatest light dependency comparing iterations in light and dark environments. The ipdC mutant showed similar behavior with relatively lower nitrogenase activity observed, while FP10 did not show a light dependency. Auxin biosynthesis showed strong light dependencies in HM053 and FP10 strains, but not for ipdC. Ferrous iron is involved in BNF, and a light dependency was observed for microbial ⁵⁹Fe²⁺ uptake in HM053 and ipdC, but not FP10. Surprisingly, a light dependency for ⁵²Mn²⁺ uptake was only observed in ipdC. Finally, ATP biosynthesis was sensitive to light across all three mutants favoring blue light over red light compared to darkness with observed ATP levels in descending order for HM053 > ipdC > FP10.

Keywords: ATP biosynthesis; Azospirillum brasilense; biological nitrogen fixation; light stimulation; microbial auxin biosynthesis; micronutrient uptake.

Figure 1

Light dependencies for HM053, ipdC, and FP10 strains of A. brasilense. Panels (A–C) represents full spectrum white light (200 µmol m⁻² s⁻¹) versus darkness data collected from ARAs performed on N = 3 plants (labeled P1–P3) that were inoculated with each of the bacteria strains. Panels (D,E) represent red light versus blue light data collected from ARAs performed on N = 3 plants that were inoculated with either HM053 or ipdC bacteria. Light intensities for red and blue light studies were 800 µmol m⁻² s⁻¹, measuring 4 times the intensity of the full spectrum white light.

Figure 2

Comparative measurements of cellular ATP concentrations are presented as pmoles of ATP per mg of dry weight (mg⁻¹ DW) of extracted bacteria cells for HM053, ipdC, and FP10 mutant strains of A. brasilense bacteria. Bacteria were grown in liquid cultures at 30 °C for 48 h under red light, blue light, and in darkness. Light intensities were 200 µmol m⁻¹ s⁻¹. Data bars reflect average values ± SE for N = 6 replicates. Levels of significance are shown by the p-values where p < 0.05 was statistically significant.

Figure 3

Light dependencies for ⁵⁹Fe uptake as ⁵⁹Fe³⁺ in HM053, ipdC, and FP10 bacteria (Panel (A–C)), and as ⁵⁹Fe²⁺ HM053, ipdC, and FP10 bacteria (Panel (D–F)). Studies were conducted in triplicate for each time point measured extending out to 5 h incubation with radiotracer. Panels (G,H) also depict how the original oxidation state of the ⁵⁹Fe tracer was metabolically transformed to its other oxidation state over time after being taken up by the bacteria. Data in Panel G represent the percent metabolic transformation of ⁵⁹Fe³⁺-to-⁵⁹Fe²⁺.after administration of ⁵⁹Fe³⁺. Data in Panel H represent the percent metabolic transformation of ⁵⁹Fe²⁺-to-⁵⁹Fe³⁺ after administration of ⁵⁹Fe²⁺.

Figure 4

Panel (A): total microbial auxin content presented as nmole auxin per 10⁸ colony forming units (CFUs). Treatments included light (equal intensities of red and blue light) versus darkness and examined across the functional mutants of A. brasilense including HM053, ipdC, and FP10. Data bars reflect average values ± SE for N = 6 replicates. Panel (B): relative percent of auxin excreted by the bacteria cells after 48 h of growth under light treatment.

Figure 5

The shikimate pathway common to plants and microorganisms involves seven enzymatic steps that starts with the Aldol condensation of phosphoenol pyruvate (PEP) and erythrose-4-phosphate. At least three of the seven enzymatic steps in this pathway (highlighted in blue) are known to involve divalent manganese.

Figure 6

Light dependencies for ⁵²Mn²⁺ uptake in HM053, ipdC, and FP10 bacteria (Panel (A–C)). Studies were conducted in triplicate for each time point measured extending out to 5 h incubation with radiotracer.

Figure 7

Principle Component Analyses comparing uptake of ⁵⁹Fe³⁺ and ⁵⁹Fe²⁺ (left panel), and ⁵⁹Fe²⁺ and ⁵²Mn²⁺ (right panel) as a function of treatment type (light vs. darkness) and microbial type.