Genetic Determinants of Ammonium Excretion in nifL Mutants of Azotobacter vinelandii

Abstract

The ubiquitous diazotrophic soil bacterium Azotobacter vinelandii has been extensively studied as a model organism for biological nitrogen fixation (BNF). In A. vinelandii, BNF is regulated by the NifL-NifA two-component system, where NifL acts as an antiactivator that tightly controls the activity of the nitrogen fixation-specific transcriptional activator NifA in response to redox, nitrogen, and carbon status. While several studies reported that mutations in A. vinelandii nifL resulted in the deregulation of nitrogenase expression and the release of large quantities of ammonium, knowledge about the specific determinants for this ammonium-excreting phenotype is lacking. In this work, we report that only specific disruptions of nifL lead to large quantities of ammonium accumulated in liquid culture (∼12 mM). The ammonium excretion phenotype is associated solely with deletions of NifL domains combined with the insertion of a promoter sequence in the orientation opposite that of nifLA transcription. We further demonstrated that the strength of the inserted promoter could influence the amounts of ammonium excreted by affecting rnf1 gene expression as an additional requirement for ammonium excretion. These ammonium-excreting nifL mutants significantly stimulate the transfer of fixed nitrogen to rice. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops. IMPORTANCE There is considerable interest in the engineering of ammonium-excreting bacteria for use in agriculture to promote the growth of plants under fixed-nitrogen-limiting conditions. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops.

Keywords: A. vinelandii; ammonium excretion; biofertilizer; nifL; nitrogen fixation; regulation; rice; transfer of fixed nitrogen.

FIG 1

Map of the nifLA region of A. vinelandii showing the restriction sites used for manipulations and the positions of KIXX and promoter inserts. (a) Domain structure of A. vinelandii NifL. The numbers refer to the primary amino acid (aa) sequence of the A. vinelandii NifL protein and mark the approximate boundaries of its N-terminal and C-terminal domains. The locations of the PAS domains (PAS1 and PAS2), the Q-linker/DHp domain, and the apparent ATP-binding-site GHKL domain are indicated. (b) Map of the nifL region of A. vinelandii showing the restriction sites used for manipulations and the positions of KIXX and promoter inserts. The arrows mark the directions of transcription of the aph, cydAB, and cycB promoters in the respective strains. AvFM1*, ΔnifL (PAS domains), with p_aph_KIXX in an orientation opposite that of nifLA; AvFM2*, ΔnifL (GHKL domain), with p_aph_KIXX in an orientation opposite that of nifLA; AvFM3*, ΔnifL (PAS, Q-linker/DHp, and GHKL domains), with p_aph_KIXX in an orientation opposite that of nifLA; AvFM4, ΔnifL (PAS domains), with p_aph_KIXX in the same orientation as that of nifLA; AvFM5, ΔnifL (GHKL domain), with p_aph_KIXX in the same orientation as that of nifLA; AvFM10, ΔnifL (PAS domains), with KIXX in an orientation opposite that of nifLA; AvFM11, ΔnifL (GHKL domain), with KIXX in an orientation opposite that of nifLA; AvFM12*, ΔnifL (PAS domains), with p_aph in an orientation opposite that of nifLA; AvFM13*, ΔnifL (PAS domains), with p_cydAB in an orientation opposite that of nifLA; AvFM14*, ΔnifL (PAS domains), with p_cycB in an orientation opposite that of nifLA; AvFM15*, ΔnifL (GHKL domain), with p_aph in an orientation opposite that of nifLA; AvFM16*, ΔnifL (GHKL domain), with p_cydAB in an orientation opposite that of nifLA; AvFM17*, ΔnifL (GHKL domain), with p_cycB in an orientation opposite that of nifLA. Ammonium-excreting strains are indicated with an asterisk.

FIG 2

Extracellular ammonium concentrations in cultures of nifL mutant strains generated with the KIXX cassette containing the aph promoter and the nifL mutant strains generated with the aph promoter under diazotrophic conditions. (a) Bar graph showing the quantification of ammonium present in the medium at the indicated time points for the AvFM1, AvFM2, and AvFM3 strains. (b) Bar graph showing the quantification of ammonium present in the medium at the indicated time points for the AvFM12 and AvFM15 strains. The changes in extracellular ammonium levels are presented as moles of ammonium excreted per kilogram of protein. The results show the means and standard deviations (error bars) for data from triplicate experiments. ns indicates not statistically significant (P > 0.05) according to an unpaired t test. Results of the unpaired t test for extracellular ammonium concentrations between the AvFM1 and AvFM2 strains are a P value of 0.14 at 24 h and a P value of 0.05 at 48 h, respectively; results between the AvFM1 and AvFM3 strains are a P value of 0.09 at 24 h and a P value of 0.36 at 48 h, respectively; and results between the AvFM2 and AvFM3 strains are a P value of 0.09 at 24 h and a P value of 0.05 at 48 h, respectively. Results of the unpaired t test for extracellular ammonium concentrations between AvFM12 and AvFM15 are a P value of 0.79 at 24 h and a P value of 0.89 at 48 h, respectively.

FIG 3

Changes in nifA, nifH, rnfA1, and rnfD1 transcript levels. (a) Bar graph showing the n-fold changes in nifA transcripts under diazotrophic growth conditions (B) relative to nondiazotrophic growth conditions (BN) in the AvFM2, AvFM9, AvFM11, and AvFM15 strains compared to the wild-type (WT) strain (control). (b and c) Bar graphs showing the n-fold changes in nifH transcripts under nondiazotrophic (b) and diazotrophic (c) growth conditions in the AvFM2, AvFM9, AvFM11, and AvFM15 strains compared to the WT strain (control). (d and e) Bar graphs showing the n-fold changes in the rnfA1 (d) and rnfD1 (e) transcripts under diazotrophic growth conditions relative to nondiazotrophic growth conditions of the AvFM2, AvFM9, AvFM15, AvFM16, and AvFM17 strains compared to the WT strain (control). The results were normalized to gyrB transcript levels, which remained constant under the different experimental conditions tested. The results show the means and standard deviations (error bars) for data from biological triplicates. An asterisk indicates a significant difference (P < 0.05), and ns indicates not statistically significant (P > 0.05), according to an unpaired t test. For panel a, results of the unpaired t test for the n-fold changes in nifA transcripts between the WT strain (control) and the AvFM2, AvFM9, AvFM11, or AvFM15 strain under diazotrophic growth conditions relative to nondiazotrophic growth conditions are a P value of 0.5 (AvFM2), a P value of 0.09 (AvFM9), a P value of 0.66 (AvFM11), and a P value of 0.82 (AvFM15), respectively. For panel b, results of the unpaired t test for the n-fold changes in nifH transcripts between the WT strain and the AvFM2, AvFM9, AvFM11, or AvFM15 strain under nondiazotrophic growth conditions are a P value of 4.62E−07 (AvFM2), a P value of 8.31E−07 (AvFM9), a P value of 7.97E−07 (AvFM11), and a P value of 4.68E−07 (AvFM15), respectively. For panel c, results of the unpaired t test for the n-fold changes in nifH transcripts between the WT strain and the AvFM2, AvFM9, AvFM11, or AvFM15 strain under diazotrophic growth conditions are a P value of 0.31 (AvFM2), a P value of 0.22 (AvFM9), a P value of 0.13 (AvFM11), and a P value of 0.21 (AvFM15), respectively. For panel d, results of the unpaired t test for the n-fold changes in rnfA1 transcripts between the WT strain and the AvFM2, AvFM9, AvFM15, AvFM16, or AvFM17 strain under diazotrophic growth conditions relative to nondiazotrophic growth conditions are a P value of 3.25E−03 (AvFM2), a P value of 0.25 (AvFM9), a P value of 2.77E−03 (AvFM15), a P value of 2.61E−03 (AvFM16), and a P value of 5.39E−04 (AvFM17), respectively. For panel e, results of the unpaired t test for the n-fold changes in rnfD1 transcripts between the WT strain and the AvFM2, AvFM9, AvFM15, AvFM16, or AvFM17 strain under diazotrophic growth conditions relative to nondiazotrophic growth conditions are a P value of 2.18E−02 (AvFM2), a P value of 0.17 (AvFM9), a P value of 5.04E−02 (AvFM15), a P value of 2.42E−02 (AvFM16), and a P value of 8.72E−04 (AvFM17), respectively. a.u, arbitrary units.

FIG 4

Extracellular ammonium concentrations in cultures of nifL mutant strains deficient for the Rnf1 complex under diazotrophic conditions. The bar graph shows the quantification of ammonium present in the medium at the indicated time points for the AvFM1, AvFM21, AvFM2, and AvFM22 strains. The changes in extracellular ammonium levels are presented as moles of ammonium excreted per kilogram of protein. The results show the means and standard deviations (error bars) for data from triplicate experiments. An asterisk indicates a significant difference (P < 0.05) according to an unpaired t test. Results of the unpaired t test for extracellular ammonium concentrations between the AvFM1 strain (control) and the AvFM21 strain are a P value of 3.37E−03 at 24 h and a P value of 5.72E−03 at 48 h, respectively. Results of the unpaired t test for extracellular ammonium concentrations between the AvFM2 strain (control) and the AvFM22 strain are a P value of 1.94E−05 at 24 h and a P value of 7.40E−06 at 48 h, respectively.

FIG 5

Extracellular ammonium concentrations in cultures of the nifL mutant strains generated with the aph, cydAB, and cycB promoters and activities of p_aph_lacZ, p_cydAB_lacZ, and p_cydA_lacZ reporter genes in A. vinelandii under diazotrophic growth conditions. (a) Bar graph showing the quantification of ammonium present in the medium at the indicated time points for the AvFM15, AvFM16, and AvF17 strains. The changes in extracellular ammonium levels are presented as moles of ammonium excreted per kilogram of protein. The results show the means and standard deviations (error bars) for data from triplicate experiments. (b) Bar graph showing the β-galactosidase activities of the AvFM18, AvFM19, and AvF20 strains carrying the p_aph_lacZ, p_cydAB_lacZ, and p_cydA_lacZ reporters grown under diazotrophic growth conditions. The results show the means and standard deviations (error bars) for data from triplicate experiments. An asterisk indicates a significant difference (P < 0.05), and ns indicates not statistically significant (P > 0.05), according to an unpaired t test. For panel a, results of the unpaired t test for extracellular ammonium concentrations between the AvFM15 strain (control) and the AvFM16 or AvFM17 strain are a P value of 0.87 (AvFM16) and a P value of 4.36E−02 (AvFM17) at 24 h and a P value of 0.66 (AvFM16) and a P value of 2.36E−03 (AvFM17) at 48 h, respectively. For panel b, results of the unpaired t test for β-galactosidase activities between the AvFM20 strain (control) and the AvFM18 or AvFM19 strain are a P value of 1.23E−04 (AvFM18) and a P value of 1.26E−04 (AvFM19), respectively.

FIG 6

¹⁵N incorporation experiments on rice plants (Oryza sativa) inoculated with A. vinelandii strains. Quantification of ¹⁵N incorporated into the rice plant tissues inoculated with the A. vinelandii wild-type (WT), DJ100 (ΔnifD), AvFM2, AvFM16, and AvFM17 strains was performed. The results show the means and standard deviations (error bars) for data from quintuplicate experiments. An asterisk indicates a significant difference (P < 0.05), and ns indicates not statistically significant (P > 0.05), according to an unpaired t test. Results of the unpaired t test for δ¹⁵N between the wild-type strain (control) and the DJ100, AvFM2, AvFM16, or AvFM17 strain are a P value of 0.24 (DJ100), a P value of 2.69E−02 (AvFM2), a P value of 4.21E−02 (AvFM16), and a P value of 1.85E−02 (AvFM17), respectively.