Transcriptional Analysis of an Ammonium-Excreting Strain of Azotobacter vinelandii Deregulated for Nitrogen Fixation

Abstract

Biological nitrogen fixation is accomplished by a diverse group of organisms known as diazotrophs and requires the function of the complex metalloenzyme nitrogenase. Nitrogenase and many of the accessory proteins required for proper cofactor biosynthesis and incorporation into the enzyme have been characterized, but a complete picture of the reaction mechanism and key cellular changes that accompany biological nitrogen fixation remain to be fully elucidated. Studies have revealed that specific disruptions of the antiactivator-encoding gene nifL result in the deregulation of the nif transcriptional activator NifA in the nitrogen-fixing bacterium Azotobacter vinelandii, triggering the production of extracellular ammonium levels approaching 30 mM during the stationary phase of growth. In this work, we have characterized the global patterns of gene expression of this high-ammonium-releasing phenotype. The findings reported here indicated that cultures of this high-ammonium-accumulating strain may experience metal limitation when grown using standard Burk's medium, which could be amended by increasing the molybdenum levels to further increase the ammonium yield. In addition, elevated levels of nitrogenase gene transcription are not accompanied by a corresponding dramatic increase in hydrogenase gene transcription levels or hydrogen uptake rates. Of the three potential electron donor systems for nitrogenase, only the rnf1 gene cluster showed a transcriptional correlation to the increased yield of ammonium. Our results also highlight several additional genes that may play a role in supporting elevated ammonium production in this aerobic nitrogen-fixing model bacterium.IMPORTANCE The transcriptional differences found during stationary-phase ammonium accumulation show a strong contrast between the deregulated (nifL-disrupted) and wild-type strains and what was previously reported for the wild-type strain under exponential-phase growth conditions. These results demonstrate that further improvement of the ammonium yield in this nitrogenase-deregulated strain can be obtained by increasing the amount of available molybdenum in the medium. These results also indicate a potential preference for one of two ATP synthases present in A. vinelandii as well as a prominent role for the membrane-bound hydrogenase over the soluble hydrogenase in hydrogen gas recycling. These results should inform future studies aimed at elucidating the important features of this phenotype and at maximizing ammonium production by this strain.

Keywords: Rnf complex; ammonium; hydrogenase; nifL; nitrogenase.

FIG 1

Ammonium levels and growth rates of A. vinelandii strains cultured under different conditions and used to collect samples for RNA sequencing studies. (Top) Ammonium levels quantified from the supernatants of A. vinelandii strain AZBB163 grown in the absence of exogenous ammonium and wild-type A. vinelandii grown in the presence of exogenous ammonium (targeted ∼20 mM initial concentration). Wild-type A. vinelandii grown without the addition of exogenous ammonium was also analyzed for ammonium levels, but the results obtained were below 50 μM for all samples and are not included on the graph for clarity. (Bottom) OD at 600 nm (OD₆₀₀) measurements for A. vinelandii strain AZBB163 grown in standard B medium (no exogenous ammonium provided) and for wild-type A. vinelandii grown with and without exogenously added ammonium. Results for ODs are plotted on a log₂-based scale (y axis). Asterisks shown on both graphs indicate time points and numbers of samples that were withdrawn for RNA-Seq analysis. All samples were cultured at 30°C. Results shown in each graph represent results from at least triplicate samples (n ≥ 3).

FIG 2

Transcriptional comparisons of primary nitrogen fixation genes. Shown are the transcriptional levels found in A. vinelandii strain AZBB163 grown in the absence of exogenously provided ammonium in the growth medium versus wild-type A. vinelandii (strain DJ) grown with excess ammonium (≥10 mM throughout the experiment) in the medium. Wild-type A. vinelandii without ammonium is also shown as a control. All FPKM results are plotted on a log₂-based scale for global comparisons of transcriptional levels as well as differential transcription between samples. A scale of fold changes is included beside the nifH and rnfD1 genes for reference. The transcriptional levels of the major nitrogen fixation (nif) cluster are shown on the left. A graphical representation of each of the nif gene regions is included for reference at the top, along with a color-based key illustrating the fold changes in transcription for A. vinelandii strain AZBB163 without exogenously added ammonium versus wild-type A. vinelandii with exogenously added ammonium. The transcriptional levels of the minor nif cluster are shown on the right. This region includes the nifL gene, which is the insertion point of the kanamycin cassette (depicted graphically in red) that results in the elevated-ammonium phenotype of A. vinelandii strain AZBB163 (shown below the graphical representation of that region) (4, 10, 12). Results presented represent the averages and standard deviations for all of the samples drawn from that data set (n ≥ 4), as indicated for each sample set in Fig. 1. See Table S1 in the supplemental material for a comparison of the statistical significances between each sample set shown for individual genes. cons., conserved; hyp., hypothetical; rel., related; XRE fam., xenobiotic response element family.

FIG 3

Full-cell nitrogenase activities of the wild-type A. vinelandii strain and strain AZBB163 under diazotrophic growth conditions. Strains were cultured aerobically at 30°C. Results presented represent the averages and standard deviations (n = 3).

FIG 4

Transcriptional comparisons of hydrogenase-related genes. Shown are the transcriptional levels found in A. vinelandii strain AZBB163 grown in the absence of exogenously provided ammonium in the growth medium versus wild-type A. vinelandii (strain DJ) grown with excess ammonium (≥10 mM throughout the experiment) in the medium. Wild-type A. vinelandii grown without ammonium is also shown as a control. The soluble nickel-dependent hydrogenase cluster is shown at the top with several nonrelated flanking genes to illustrate the lower levels of transcription of these genes in A. vinelandii strain AZBB163 than of neighboring genes. The membrane-bound hydrogenase cluster is shown at the bottom, illustrating similar levels of transcription for both A. vinelandii strain AZBB163 and the A. vinelandii wild-type strain grown with and without exogenously provided ammonium. The results presented represent the averages and standard deviations for all of the samples drawn from that data set (n ≥ 4), as indicated for each sample set in Fig. 1. All FPKM values are plotted on a log₂-based scale. A scale of fold changes is included beside the hoxI gene for reference. See Table S1 in the supplemental material for a comparison of the statistical significances between each sample set shown for individual genes. cons., conserved; hyp., hypothetical; met., metallo; est., ester; hyd., hydrolase; sulf., sulfate; trans. glut., transglutaminase.

FIG 5

Hydrogen uptake activities measured for cells of wild-type A. vinelandii and strains AZBB163, AZBB326 (based on AZBB163), and AZBB328 (based on AZBB163). Strains were grown aerobically at 30°C. The results presented represent the averages and standard deviations for at least three samples (n ≥ 3).

FIG 6

Transcriptional differences in ATP biosynthesis genes. Shown are the transcriptional levels found in A. vinelandii strain AZBB163 grown in the absence of exogenously provided ammonium in the growth medium versus wild-type A. vinelandii (strain DJ) grown with excess ammonium (≥10 mM throughout the experiment) in the medium. Wild-type A. vinelandii grown without ammonium is also shown as a control. A. vinelandii contains two ATP biosynthesis gene clusters. These results illustrate that the first cluster (top graph) accumulated diminished levels of mRNAs for the genes involved in ATP biosynthesis in A. vinelandii strain AZBB163, while the second cluster (bottom graph) illustrates that mRNA levels are slightly elevated. Results presented represent the averages and standard deviations for all of the samples drawn from that data set (n ≥ 4), as indicated for each sample set in Fig. 1. All FPKM values are plotted on a log₂-based scale. See Table S1 in the supplemental material for a comparison of the statistical significances between each sample set shown for individual genes. syn., synthase; sub., subunit; perm., permease.

FIG 7

Transcriptional comparisons of siderophore production-related genes. Shown are the transcriptional levels found in A. vinelandii strain AZBB163 grown in the absence of exogenously provided ammonium in the growth medium versus wild-type A. vinelandii (strain DJ) grown with excess ammonium (≥10 mM throughout the experiment) in the medium. Wild-type A. vinelandii grown without exogenously provided ammonium is also shown as a control. A. vinelandii contains two separate classes of siderophores (22). The first class (top right) is based on the well-characterized catechol siderophores, while the second class (bottom) represents the pyoverdine-based siderophore azotobactin. Another small cluster of genes associated with siderophores in A. vinelandii (Avin_50330 to Avin_50380) (not shown) was also transcribed at elevated levels (Table 2). Results presented represent the averages and standard deviations for all of the samples drawn from that data set (n ≥ 4), as indicated for each sample set in Fig. 1. All FPKM values are plotted on a log₂-based scale. See Table S1 in the supplemental material for a comparison of the statistical significances between each sample set shown for individual genes. hyp., hypothetical; NRPS, nonribosomal peptide synthase; reg., regulatory; synth., synthase.

FIG 8

Ammonium yields of A. vinelandii strain AZBB163 grown in Burk's medium (B medium) versus growth in the same medium with elevated levels of metals. The graph at the top shows levels of ammonium found in standard B medium versus those found in samples supplemented with additional molybdenum (from 1 μM to 5 μM), iron (from 18 μM to 54 μM), or both. Results for day 3 and day 4 of growth are shown. All samples were grown from the same starting culture initiated at the same time and under the same conditions, and data represent results for triplicate samples (n = 3). Results were determined to be significantly different for additional molybdenum for both days based on an ANOVA (*, P < 0.05).

FIG 9

Full-cell protein expression profile of A. vinelandii strains. Shown are the full-cell SDS-extractable protein profiles obtained from various strains used in this study. Lane 1, A. vinelandii wild-type cells grown in B medium supplemented with 20 mM ammonium for 24 h, harvested, resuspended in B medium devoid of ammonium, and grown an additional 2 h before flash freezing to induce the expression of nitrogenase; lane 2, A. vinelandii wild-type cells grown in B medium devoid of ammonium for 24 h; lane 3, A. vinelandii wild-type cells grown B medium supplemented with 20 mM ammonium for 24 h; lane 4, A. vinelandii strain AZBB163 grown in B medium devoid of ammonium for 24 h; lane 5, protein ladder; lanes 7 through 12, A. vinelandii strain AZBB163 grown in B medium devoid of ammonium for 24 h, with successively diluted amounts of the protein extract added to each lane to better resolve the protein bands representing the two bands of the MoFe protein (NifDK) and the single band of the Fe protein (NifH).