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.
INTRODUCTION
Nitrogen is an essential component of all biological building blocks, including DNA, RNA, and proteins. All forms of life on earth are dependent on a suitable supply of this important element. Through the action of biological nitrogen fixation, nature has evolved very complicated methods to harness the large reservoir of nitrogen available in the atmosphere. Our principal understanding of this process is based upon studies of the Mo-dependent nitrogenase system, which is the best characterized of the various nitrogenase classes (1, 2). In the fundamental sense, biological nitrogen fixation through the Mo-dependent nitrogenase relies on the action of the two-component enzyme system composed of the Fe protein (NifH) and the MoFe protein (NifDK). However, in actual application, biological nitrogen fixation requires a large repertoire of enzymes that provide reducing equivalents, chemical energy in the form of ATP, biosynthetic pathways to assemble complex metal cofactors, and an environment that is suitable for the function of an oxygen-sensitive enzyme. Only a small percentage of organisms, termed diazotrophs, can fix nitrogen, and of these organisms, the majority do so under anaerobic conditions. Among the small fraction of organisms that accomplish biological nitrogen fixation during aerobic growth, Azotobacter vinelandii serves as a model organism (3).
Our understanding of nitrogenase comes from a combination of in vitro studies with the isolated enzymes and in vivo studies of cause and effect based on the manipulation of specific genes within nitrogen-fixing organisms. Additionally, biological nitrogen fixation is highly regulated depending on the needs of the cell and the environmental conditions where it is found (4–7). For this reason, within a large culture containing many cells, it would be expected that only a fraction of cells are actively fixing nitrogen at any given time, as they regulate this system based on the need for intracellular nitrogen, even when grown under nitrogen-fixing conditions (8). The isolation of a strain that is deregulated for nitrogen fixation, and, thus, produces elevated levels of ammonium that go well beyond the needs of the cell, is a powerful tool to distinguish the genes required to maintain a nitrogen-fixing phenotype from those that are cycling between expression and repression based on the need for internal nitrogen (7, 9). The Kennedy laboratory isolated such a strain of A. vinelandii in 1992 (10), which produced elevated levels of ammonium that went well beyond the needs of the cell. This strain was based on a disruption of the antiactivator nifL gene while the nifA gene and ribosome binding sites (RBSs) remained intact. Strains producing elevated levels of ammonium are of interest in many agricultural settings (6, 11–16). We have reconstructed a similar strain by using approaches taken by Bali and coworkers (Kennedy laboratory) as a guide for producing a strain with a similar phenotype, A. vinelandii strain AZBB163 (10, 12). This strain produces elevated levels of ammonium throughout all stages of growth, achieving levels of ammonium in spent culture that surpass 30 mM at 30°C using Burk's medium (B medium), which contains approximately 60 mM sucrose as the sole carbon source, and matches the phenotype described in previous reports by the Kennedy and Drummond laboratories (4, 10).
In this study, we evaluated the global transcriptional profile of the high-ammonium-excreting A. vinelandii strain AZBB163 during ammonium accumulation in the stationary phase of growth and compared this to the patterns of gene expression observed for the wild-type A. vinelandii strain from which it was constructed. In addition to an evaluation of the genes for the Mo-dependent nitrogenase system and related cofactor biosynthesis genes, we also evaluated genes thought to be important for recycling the hydrogen by-product of biological nitrogen reduction and providing the required chemical energy needed in the form of ATP. These results reveal changes in energy metabolism and metal acquisition and utilization that can be rationalized in the context of the increased demands of nitrogenase biosynthesis, which supports excess ammonium production based on elevated levels of nitrogenase.
RESULTS
Ammonium production and sample selection for transcriptome sequencing (RNA-Seq). A. vinelandii strain AZBB163, hereafter referred to as AZBB163, produces copious quantities of ammonium when grown on standard B medium lacking exogenously provided ammonium, which differentiates this strain from the A. vinelandii wild type. Transcriptional studies of A. vinelandii grown diazotrophically (in the absence of supplemented ammonium), hereafter referred to as AvWT(−NH4+), versus A. vinelandii grown in the presence of ammonium, hereafter referred to as AvWT(+NH4+), highlighted many of the key genes involved in nitrogen fixation in this model strain (6). However, A. vinelandii tightly regulates nitrogen fixation based on the internal nitrogen requirements of the cell and nutrient availability (4–7), while AZBB163 has been altered to deregulate nitrogen fixation, which results in the hyperaccumulation of ammonium in the extracellular space (4, 10, 12). For this reason, AZBB163 offers a unique opportunity to evaluate which genes are essential for maintaining this high-ammonium-accumulating phenotype and compare these data to what is found for AvWT(+NH4+) and AvWT(−NH4+).
To provide insight into the differences between AZBB163 and the A. vinelandii wild-type strain, a careful selection of samples and controls was done so that proper comparisons of the conditions under which AZBB163 accumulates ammonium could be made. As shown in Fig. 1, AZBB163 experienced an initial rate of growth that was comparable to what was seen for AvWT(−NH4+), although the final optical density (OD) obtained for AZBB163 was lower than that found for the A. vinelandii wild type [the AZBB163 cell mass was less than one-quarter of what could be obtained for AvWT(+NH4+) or AvWT(−NH4+)]. AvWT(+NH4+) transitioned from exponential phase to early stationary phase after approximately 12 h. An analysis of the ammonium levels remaining in the medium for this baseline sample revealed that large quantities of ammonium were still present during the stationary phase (Fig. 1, top), indicating that nitrogen limitation was not responsible for this transition to the stationary phase. Part of this limitation was attributed to a gradual drop in the pH of the culture grown under these conditions (see Fig. S1 in the supplemental material). AvWT(+NH4+) was intended to represent a physiological state where nitrogen fixation is minimized due to the presence of sufficient amounts of fixed nitrogen still available to the cells within the medium (which is supported by the fragments per kilobase of transcript per million mapped reads [FPKM] values obtained for this sample set, as described below). AvWT(−NH4+) (grown diazotrophically) transitioned into stationary phase at approximately 24 h, while AZBB163 (also grown diazotrophically) reached stationary phase just after 24 h. While wild-type A. vinelandii does not release significant quantities of ammonium into the medium, AZBB163 released ammonium throughout growth, although the maximum rate of ammonium accumulation was found only once the cell density was maximized following the transition from exponential-phase growth to early-stationary-phase growth (between day 2 and day 4).
Based on these results, samples from AZBB163 were selected to survey gene transcription during this extended period of ammonium accumulation between days 2 and 4. Specific time points at which the cultures were sampled are indicated by asterisks in Fig. 1 for each of the three strains or conditions selected to make these comparisons, and multiple asterisks indicate multiple samples. The selection of samples across this range of time points was purposely done to minimize transitory changes in transcription levels of genes not associated with this nitrogen-fixing phenotype while highlighting those genes that were consistently transcribed at elevated levels during extracellular ammonium accumulation in AZBB163. The elevated OD values for AvWT(−NH4+) are partially attributed to substantial polyhydroxybutyrate accumulation under these conditions, which is diminished when the alternative ΔphbBAC strain is grown under the same conditions (Fig. S2). These experiments also revealed that the accumulation of ammonium in the medium resulted in an increase in the pH of the AZBB163 culture over time (Fig. S1), which was reported previously (4, 10).
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).
Transcriptional levels of nitrogen fixation genes.The AvWT(+NH4+) culture was used as a benchmark to which transcriptional levels in both AvWT(−NH4+) and AZBB163 were compared. Our initial comparison of genes included the two nitrogen fixation (nif) clusters that were highlighted previously as a primary reference point versus other genes (6). Figure 2 provides a graphical representation of the FPKM values obtained from the RNA-Seq analysis. This method of presentation was selected not only to provide a view of the transcriptional changes between different strains or conditions but also to show the relative quantities of transcripts found for individual genes. The results shown in Fig. 2 represent the averages and standard deviations of the FPKM values for all time points of sampling. Results for individual samples can be downloaded at the NCBI Gene Expression Omnibus (GEO).
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.
As expected, the AZBB163 nifH gene encoding the iron protein (Fe protein) (NifH) of the Mo-based nitrogenase system showed the highest fold increase in transcription, with a >500-fold increase in transcriptional levels versus those for AvWT(+NH4+) based on RNA-Seq analysis. The nifD and nifK genes, which code for the two subunits of the MoFe protein (NifDK), also showed very high levels of transcription in AZBB163. While the transcriptional levels of most of the genes associated with nitrogen fixation in AvWT(−NH4+) were also elevated, the differences were much smaller than what was found for AZBB163, as was found for nifH, which showed an increase of only ∼20-fold. Many of the accessory proteins and biosynthetic proteins involved in the cluster biosynthesis of the metalloclusters also showed elevated levels of transcription of their respective genes, with general trends that were very similar to what was reported in a previous transcriptional analysis of nitrogen fixation genes in the wild-type strain alone during exponential growth versus growth with ammonium repression (6, 16). In general, the levels of transcription found for many of the nif genes in our study were significantly higher for AZBB163 than for AvWT(−NH4+). In addition to the genes highlighted in the major nif cluster, we also found elevated levels of the genes of the minor nif cluster, including the nifB gene downstream of the nifA gene. Since the modification resulting in elevated ammonium production in AZBB163 is based on a disruption of the nifL gene and is dependent on the directional insertion of a kanamycin cassette and the specific sequence of this insert to achieve the high-ammonium-production phenotype (4, 10, 12), we were particularly interested in whether this modification resulted in elevated transcription levels of nifA. Levels of nifA transcription were similar in AZBB163, AvWT(+NH4+), and AvWT(−NH4+) (Fig. 2), indicating that this phenotype is not related to a dramatic increase in nifA levels. One prominent feature of the transcriptional levels of the genes in this region was found for the genes upstream of nifL, which compose one of the two rnf gene clusters. All seven genes of this rnf cluster (Avin_50920 to Avin_50980) showed significant increases in transcriptional levels (8- to 22-fold), while the other set of rnf genes (Avin_19220 to Avin_19270) showed no significant increases (<2-fold) in transcription levels (see Fig. S3 in the supplemental material). These results illustrate the important coupling of these rnf genes to the nif genes in A. vinelandii and agree with findings from a previous study related to the two rnf clusters in A. vinelandii (9). The levels of genes of the fix cluster, which has also been implicated as a potential source of electrons for nitrogen fixation (6, 17), were elevated in AZBB163 versus AvWT(+NH4+), but the increases in transcription levels that were found were similar to those found for AvWT(−NH4+) (Fig. S3).
Nitrogenase activity.To confirm that the elevated transcript levels found for nifH and nifDK correlated directly with intracellular nitrogenase activity, we measured the full-cell activity of nitrogenase using a standard acetylene reduction assay (Fig. 3). The nitrogenase activity of AZBB163 was markedly higher than that of AvWT(−NH4+) at each daily time point of measurement during the experiment. The highest level of nitrogenase activity was found on day 1 for AZBB163, which then dropped to about one-half of the maximal activity for the next 3 days but remained relatively constant during days 2 through 4. In contrast, the level of nitrogenase activity of AvWT(−NH4+) was highest at day 2 but then dropped to very low levels on days 3 and 4. These differences correspond to 3- to 30-fold differences in nitrogenase activity between these two strains on days 1 to 3, which correlate with the increases in transcriptional levels seen for nifH from the RNA-Seq samples over the same time frame (Fig. 1 to 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).
Transcriptional levels of hydrogenase genes.The production of ammonia by the nitrogenase enzyme system produces hydrogen gas as a by-product of biological nitrogen fixation (1). Nitrogen-fixing organisms generally contain one or more hydrogenase enzyme systems to recycle the electrons from this hydrogen gas to drive other cellular processes and conserve energy. This process is generally assumed to be tightly regulated in these organisms, and A. vinelandii contains two distinct hydrogenase enzymes and associated genes involved in cofactor biosynthesis (3).
The levels of transcription of the two hydrogenase gene clusters in A. vinelandii (Fig. 4) did not correlate with what was found for the nitrogen fixation genes in AZBB163. While genes associated with nitrogen fixation were highly transcribed (Fig. 2), those encoding the two hydrogenases showed only minimal (∼2-fold) increases versus the wild type for the membrane-bound hydrogenase (Avin_50440 to Avin_50590) and showed decreases in the transcription of the genes encoding the soluble hydrogenase (Avin_04350 to Avin_04410). This was of interest because it was anticipated that the elevated levels of ammonium accumulating in the growth medium in AZBB163 would be accompanied by elevated intracellular hydrogen production and would induce the hydrogenase genes to be upregulated for transcription as well, although this feature has not been reported for Azotobacter to the best of our knowledge.
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.
Hydrogen uptake.Various methods are available to measure intracellular hydrogenase activity. As part of this work, we developed a method that monitors the potential of cells to consume externally spiked hydrogen by direct measurement. In these experiments, a known quantity of hydrogen (250 μl) was added to a sealed serum vial (20 ml) containing 3 ml of culture, and the amount of hydrogen was quantified after a fixed point of time, when approximately one-third of the hydrogen had been consumed for the wild-type strain. AvWT(−NH4+) and AZBB163 yielded similar levels of hydrogen uptake activity on days 1 and 2 (Fig. 5). On day 3, levels of hydrogen consumption were slightly increased for AZBB163 but remained similar to days 1 and 2 for AvWT(−NH4+). AvWT(+NH4+) consumed a smaller quantity of hydrogen per milligram of protein on all 3 days. The AvWT(+NH4+) culture required supplementation with 20 mM morpholinepropanesulfonic acid (MOPS) buffer and daily adjustment with NaOH to maintain a suitable pH for performing measurements throughout the entire experiment. None of the three primary cultures [AZBB163, AvWT(−NH4+), and AvWT(+NH4+)] released measurable amounts of hydrogen in the gas phase when similar assays were performed without any added external hydrogen, indicating that hydrogenase levels within the cell are sufficient to ensure that no excess hydrogen produced by the elevated levels of nitrogenase found in AZBB163 is being lost to the atmosphere in this strain. These results in combination with the results obtained from RNA-Seq (Fig. 4) indicated a more likely role and correlation for the membrane-associated hydrogenase (Avin_50440 to Avin_50590) in being responsible for this measured activity than for the soluble hydrogenase (Avin_04350 to Avin_04410). To further probe the roles of each hydrogenase cluster, two strains were constructed from AZBB163 by disrupting each of the hydrogenase clusters independent of one another, yielding strains AZBB326 and AZBB328 (Table 1). Strain AZBB326, which contained a disruption in the soluble hydrogenase, resulted in no discernible change in hydrogen uptake activity versus strain AZBB163, while AZBB328, which contained a disruption in the membrane-associated hydrogenase, lost the ability to consume hydrogen and resulted in a slight increase in hydrogen levels at days 2 and 3 in this experiment (for both samples containing no added hydrogen and for the samples spiked with 250 μl of hydrogen [Fig. 5]), indicating that some hydrogen was being released by this strain. The similar levels of hydrogen uptake activity for AZBB163 and AvWT(−NH4+) (Fig. 5), while nitrogenase activity was dramatically increased (Fig. 3), indicate that nitrogenase activity and hydrogen uptake activity are not necessarily as tightly coupled as one might expect based on our current model for the nitrogenase-hydrogenase relationship (18, 19).
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).
Mutant strains constructed and/or used in this study
Transcriptional levels of ATP synthase genes.The production of ammonia by the nitrogenase enzyme is dependent on ATP hydrolysis by the Fe protein to provide the chemical energy necessary to drive the energy-intensive dinitrogen reduction reaction (1). A. vinelandii contains two clusters of the ATP synthase genes (3). Since the ATP synthase is an integral membrane-spanning protein, it was unclear whether transcription would correlate with increased nitrogenase activity. The transcriptional levels of the first ATP synthase gene cluster (Avin_19670 to Avin_19750) decreased between 2- and 3-fold for AZBB163 versus what was found for AvWT(+NH4+) (Fig. 6). In contrast, the transcription level of the second ATP synthase gene cluster (Avin_52150 to Avin_52220) increased between 2- and 3-fold for AZBB163 (Fig. 6). Additionally, the FPKM values showed that the second ATP synthase gene cluster was found to have an elevated basal level of transcription versus those of the first ATP synthase gene cluster. These results indicate that A. vinelandii might prefer the second ATP synthase gene cluster over the first for providing the necessary ATP to drive nitrogen fixation.
Transcriptional levels of siderophore biosynthetic genes.Following the genes involved in nitrogen fixation, the cluster of genes with the largest increase in transcription in AZBB163 included genes associated with siderophore biosynthesis. Azotobacter is known to produce two primary classes of siderophores. The first class of siderophores includes catechol-derived compounds, and Azotobacter is known to produce at least four different siderophores that fit within this class (20–22). The second class contains a pyoverdine chromophore attached to a peptide backbone and is produced by the action of nonribosomal peptide synthases. The best-characterized pyoverdine-based siderophore of Azotobacter is called azotobactin (21, 23, 24). Previous studies that disrupted genes from a specific cluster of genes (Avin_25560, Avin_25570, Avin_25580, Avin_25600, Avin_25640, and Avin_25650) resulted in stark decreases in azotobactin fluorescence, indicating that these genes are all integrally involved in the biosynthesis of azotobactin (22).
Elevated levels of transcription were found for the entire cluster of genes associated with azotobactin biosynthesis (Avin_25550 through Avin_25660), especially for the small mbtH-like gene (Avin_25630) (see Fig. 7). In addition to this cluster of genes, an additional cluster of genes upstream of the azotobactin-associated cluster (Avin_25410 to Avin_25470) was also highly transcribed (see Fig. 7). It is interesting to note that these two clusters of genes are separated from one another by a transposon, which may indicate that they were once part of a single cluster. While the genes associated with azotobactin biosynthesis were transcribed at much higher levels in AZBB163, those associated with catechol biosynthesis (Avin_21180 to Avin_21230) showed very little difference compared to what was seen for AvWT(+NH4+). The catechol-based siderophore system is generally associated with iron acquisition, while azotobactin has been linked to the acquisition of both iron and molybdenum (21, 22). This difference in the transcription of the siderophore-associated genes could be either related to a coupling of these genes to those of nitrogen fixation or the result of extensive metal depletion arising from elevated levels of nitrogenase in the AZBB163 strain, resulting in a metal deficiency within the cell, which then induces the transcription of these genes.
A recent report highlighted the potential importance of vibrioferrin as a third class of siderophore in A. vinelandii. Vibrioferrin was found at the highest concentrations among all siderophores obtained from stationary-phase A. vinelandii cultures based on a liquid chromatography-mass spectrometry (LC-MS) approach (25). Our results found no significant changes in the transcription levels of many of the key genes associated with vibrioferrin production (Avin_09320 to Avin_09360) in AZBB163 versus what was found for AvWT(−NH4+).
Ammonium production in the presence of elevated concentrations of metals.Transcriptional analysis of AZBB163 indicated that the culture may be metal limited based on the elevated transcription levels of genes associated with siderophore production. As such, we were interested to see if the addition of specific metals would result in an improvement in ammonium accumulation by this strain. To test this hypothesis, we grew AZBB163 in standard B medium (26) and also in B medium containing elevated levels of iron (3-fold increase from 18 to 54 μM), molybdenum (5-fold increase from 1 to 5 μM), or both. The results from this experiment revealed an improvement in ammonium production by AZBB163 in the presence of elevated concentrations of molybdenum (see Fig. 8) throughout the experiment. Elevated levels of iron resulted in only a minor improvement, while the inclusion of elevated concentrations of molybdenum and iron gave a similar result to what was found for elevated concentrations of molybdenum alone. No change in the growth rate or cell yield was observed in this experiment based on elevated levels of these metals. We performed analysis of variance (ANOVA) on the ammonium levels at days 3 and 4 of culture with a linear model as a function of iron, molybdenum, and the possible improvement of ammonium production by increasing the concentrations of both metals. We found no significant improvement with iron alone, but molybdenum by itself or combined with iron led to a significant improvement (P < 0.05). These results demonstrate that the limiting metal for AZBB163 is likely molybdenum.
Additional highly transcribed genes.In addition to the regions of the genome described above (Fig. 2, 4, 6, and 7), several other genes were transcribed at elevated levels (Table 2). All genes that showed at least a 5-fold increase in transcription in AZBB163 versus AvWT(+NH4+) (P < 0.05 based on Welch's t test), that met a basic size requirement (>180 bases), and that were not included in Fig. 2, 4, 6, or 7 are shown in Table 2. The top two genes from this secondary analysis included a potential globin and an ATPase that cluster together in the genome (Avin_18050 and Avin_18070). Genes coding for outer membrane proteins such as OmpW and OmpA (Avin_06590, Avin_06610, and Avin_41140) were also transcribed at elevated levels. Several additional genes associated with metal scavenging that were not included as part of the other siderophore clusters described above (Fig. 7) were also highly transcribed, including those encoding a ferric uptake regulation-associated protein (Avin_27170), a small cluster of genes encoding several siderophore-related proteins (Avin_50350 to Avin_50380), and a gene encoding a molybdenum transporter (Avin_50670). Other genes transcribed at elevated levels included those encoding a diheme cytochrome c peroxidase (Avin_10230); a fumarate hydratase (Avin_27180), which was adjacent to the ferric uptake regulation-associated protein described above (Avin_27170); a subunit of the tryptophan synthase (Avin_02120); and several hypothetical proteins (Avin_10380, Avin_01250, Avin_02520, Avin_25150, and Avin_50410). These genes and others are shown in Table 2. An additional analysis of global gene transcriptional levels resulting from these experiments is presented in the form of various volcano plots (Fig. S4 through S6).
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.
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.
Highly transcribed genes in AZBB163 and AvWT(−NH4+)
Simple survey of full-cell protein expression of nitrogenase proteins.A complete proteomics survey of global protein expression is outside the scope of this study. However, based on the mRNA levels of the nitrogenase genes nifH, nifD, and nifK (Fig. 2) and the elevated levels of intracellular nitrogenase activity found (Fig. 3), it was expected that these gene products should be present as significant proportions of the whole-cell protein content. To test this, we ran full-cell sodium dodecyl sulfate (SDS)-extracted protein fractions on a large (15-cm by 18-cm) SDS-PAGE gel to compare AZBB163 protein expression levels to those of AvWT(−NH4+) and AvWT(+NH4+). This analysis also included a sample of A. vinelandii wild-type cells that were first grown with ammonium provided to the culture and then harvested and resuspended in medium devoid of ammonium, which is the general approach taken to induce nitrogenase for protein production strategies using this important enzyme (27). Three protein bands corresponding to the NifH and NifDK subunits of the Fe protein and MoFe protein were found to be expressed at significantly higher concentrations in AZBB163 than in AvWT(−NH4+) or AvWT(+NH4+) (see Fig. 9). These results correlate well with data from a previous report by Brewin et al. showing the expression levels of the same genes in their equivalent constructs (4).
DISCUSSION
Identification of key genes involved in nitrogen fixation. A. vinelandii strain AZBB163 accumulates extensive quantities of ammonium in spent medium during normal growth on sucrose as the carbon source and has been essentially deregulated for nitrogen fixation. This strain was constructed to replicate a phenotype reported originally by Bali and coworkers and again by Brewin and coworkers in 1992 and 1999, respectively (4, 10). At the time of those original reports, the tools required to perform a global transcriptional analysis of these strains were not available. To date, such studies of these deregulated strains have not been pursued, even though this phenotype could offer important insights for our understanding of the requirements to sustain continued high-level ammonium accumulation in an aerobic nitrogen-fixing bacterium. By collecting samples at multiple time points during the stationary phase of growth, when AZBB163 is accumulating ammonium, the transcription of additional genes that might fluctuate due to the growth state of the culture should be minimized. Additionally, since nitrogenase is so tightly regulated within the cell (in the wild-type strain), nitrogen production likely fluctuates in the A. vinelandii wild-type strain within individual cells based on the stage of growth of the culture and intracellular nitrogen availability, similar to what has been found for Klebsiella oxytoca (8). For these reasons, AZBB163 may serve as an invaluable tool to study the potential for ammonium production above and beyond the needs of each individual cell. To ensure that the cultures had not succumbed to potential cheaters that might have evolved within the culture to take advantage of the excess available ammonium (12), cultures were monitored throughout growth for continued ammonium production during the collection of samples for RNA isolation.
As expected, the genes coding for the Fe protein (nifH) and the MoFe protein (nifDK) were very highly transcribed in AZBB163 (Fig. 2), and both NifH and NifDK were found at noticeably higher levels by SDS-PAGE analysis of full-cell protein fractions from this strain (see Fig. 9), which agrees with what was reported previously for the original construction of A. vinelandii MV376 (4, 10). These results also correlate with the higher levels of intracellular nitrogenase activity (Fig. 3) and the dramatic increases in extracellular ammonium accumulation in this strain (Fig. 1). The marked increases in both the transcription and translation of these key nitrogenase-associated genes and their enzyme products during stationary phase demonstrate not only the importance of AZBB163 for ammonium production and transcriptomics analysis but also the potential benefit that the strain might have for laboratories isolating nitrogenase for in vitro assays. AZBB163 could serve as a beneficial strain for the expression of elevated levels of nitrogenase components without the need for an external stimulus to induce the expression of these genes, as is often done for biochemical studies (27).
Preference for supplying necessary ATP.Biological nitrogen fixation requires high levels of reducing equivalents and chemical energy in the form of ATP to drive this energy-intensive process. The transcriptional results from AZBB163 (Fig. 6) indicate that nitrogen fixation in A. vinelandii may prefer a specific ATP synthase gene cluster (Avin_52150 to Avin_52230) over the alternative ATP synthase gene cluster (Avin_19670 to Avin_19750). The preferred ATP synthase also yielded 3-fold-higher average FPKM background levels in AvWT(+NH4+) than did the alternative ATP synthase, potentially indicating that this is the more prominent of the two ATP synthases present in A. vinelandii. The changes in the transcriptional levels of both of these two ATP synthase clusters were minor (Fig. 6), indicating that the available ATP synthase levels in the cell were sufficient to drive this process without the need for extensive increases in the transcription of ATP synthase genes to match the changes in transcription that were seen for the nitrogenase enzymes and nitrogenase cofactor biosynthesis-associated genes.
Correlations with enzymes thought to provide reducing equivalents.Three gene clusters have been implicated in potentially providing the necessary reducing equivalents for nitrogen fixation (6, 28–31). The genes of the rnf1 cluster (Avin_50920 to Avin_50980) showed the largest increase in the transcription level [∼16-fold on average for AZBB163/AvWT(+NH4+)], which was significantly higher than the ∼2-fold average increase in the transcription of these genes found for AvWT(−NH4+)/AvWT(+NH4+) (Fig. 2; see also Fig. S3 in the supplemental material). The genes associated with the fix system (Avin_10510 to Avin_10550) showed an elevated level of transcription in AZBB163 [∼5-fold higher on average for AZBB163/AvWT(+NH4+)] (Fig. S3), although the significance as determined by a t test was not as robust for AZBB163 versus AvWT(+NH4+) (P > 0.05), even though statistical significance (P < 0.05) was found for AvWT(−NH4+) versus AvWT(+NH4+). Importantly, while the rnf1 cluster showed an elevated level of transcription in AZBB163/AvWT(+NH4+) that was far higher than that of the rnf1 cluster in AvWT(−NH4+)/AvWT(+NH4+), the changes in the levels of transcription of the fix cluster were very similar in AZBB163 and AvWT(−NH4+), indicating that the fix gene cluster is not as tightly coupled to the elevated-ammonium phenotype as the rnf1 cluster. The levels of transcription of the second rnf cluster (Avin_19220 to Avin_19270) showed very little change among the three sets of samples (Fig. S3), which is in agreement with data from a previous report by Curatti et al. (9), who found that the second rnf cluster was constitutively expressed independent of nitrogen availability.
The rnf1 gene cluster is immediately upstream of nifL in the minor nif gene cluster (Fig. 2) and showed levels of transcription that were similar to those of many of the genes of the minor nif gene cluster. While these results represent only indirect evidence, they indicate that the rnf1 cluster plays the greatest potential role of the three electron transport systems in providing the electrons necessary to drive increased biological nitrogen fixation in AZBB163. One aspect of these findings that is still uncertain is whether the elevated levels of transcription found for the rnf1 cluster are solely the result of regulation by NifA or whether they might be enhanced by elevated directional transcription from the strong aph promoter present in the kanamycin cassette that was inserted into AZBB163 (Fig. 2). The potential that the aph promoter might play a role in enhancing the transcription of the rnf1 cluster was originally pointed out by Brewin et al. (4) in their evaluation of the MV376 strain. The determination of whether aph plays a role in elevated transcription levels will require the construction, transcriptional analysis, and ammonium accumulation determinations of additional strains. Brewin et al. constructed a NifA overexpression strain based on the ptac promoter, which was integrated into the same region of the genome as for their strain MV376 (4). In that construct, the aph promoter and kanamycin cassette were inserted in the direction opposite from that in MV376 and the strain that we constructed here (AZBB163), and those authors reported that they could also achieve the same elevated levels of ammonium when they overexpressed NifA by adding isopropyl-β-d-thiogalactopyranoside (IPTG). This result indicates that the elevated transcription level of the rnf1 cluster is not related to an increase in transcription by the aph promoter and is instead solely the result of nifA. The rnf1 complex of A. vinelandii has not been studied to the same degree as those of Rhodobacter capsulatus, Acetobacterium woodii, and Vibrio cholerae (28, 29, 31), but studies that deleted each rnf gene cluster alone and in combination (9) found that the loss of these clusters resulted in inactive and iron-deficient forms of the Fe protein (NifH).
Metal limitations during accumulation of high levels of ammonium.Mo-nitrogenase contains several important metal cofactors, including the FeMo cofactor. These metal clusters are among the most complicated clusters known in nature and require substantial levels of iron to fill this requirement (1, 2, 21). Our results indicated that metal availability was limiting potential ammonium accumulation in AZBB163, as many of the genes required for metal homeostasis were highly transcribed (Fig. 7). The same increases in transcription were not found for AvWT(−NH4+), indicating that this metal-deficient phenotype may be related to the elevated expression level of the nitrogenase protein (see Fig. 9). Providing AZBB163 with elevated levels of iron and molybdenum resulted in a small but statistically significant improvement in ammonium accumulation within the cell, particularly with molybdenum (Fig. 8). Standard Burk's medium (26) provides ∼18-fold-higher levels of iron (∼18 μM) than of molybdenum (∼1 μM), and the full complement of metals for a functional nitrogenase complex (NifHDK) requires 19 iron atoms for each molybdenum atom (1). Based on these requirements, it was interesting that elevated levels of iron had little effect on the ammonium yield without the addition of molybdenum (Fig. 8). Molybdenum was implicated previously as being important for supporting nitrogen fixation (32–35), and based on the levels of the Fe protein and MoFe protein that were expressed in full cells of AZBB163 (Fig. 9), it is not surprising that metal availability might limit ammonium accumulation. These results were not anticipated but highlight the potential benefits of these studies for related fields, including those laboratories interested in the roles of siderophores and metal-trafficking proteins. While many genes were shown to be integral for the production of Azotobacter siderophores in previous reports (22), other genes that might also play a role in azotobactin production and transport are reported here (Fig. 7 and Table 2) and could be targeted in future studies to better characterize the role that these genes play in metal homeostasis within this species.
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).
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).
Additional highly transcribed genes.Many of the genes generally associated with nitrogen fixation showed increased transcription, in agreement with data from previous studies (6). However, some of the most interesting findings from this work are related to genes that are not commonly considered regarding nitrogen fixation. Our results highlight genes, such as those encoding a specific ATPase and a globin-like protein, that were highly transcribed in AZBB163. The potential role that a globin might play in nitrogen fixation is intriguing. Additional genes such as those coding for several outer membrane proteins might indicate a possible role that these genes play in maintaining the proper environment within the cell to sustain nitrogen fixation at elevated levels. A. vinelandii is an ideal model organism for the study of nitrogen fixation not only because it is very efficient at performing this process but also because it accomplishes this task while growing under aerobic conditions, even though the nitrogenase enzyme system itself is highly susceptible to oxygen (5).
Levels of nifA transcription.The modification of the genome that results in deregulated nitrogen fixation in AZBB163 is due to an insertion of a kanamycin cassette into the nifL gene (4, 10, 12). The NifL protein is a regulatory protein that behaves as an antiactivator of the σ54-dependent activator protein NifA. The NifL and NifA proteins function together in response to cellular signals such as redox, nitrogen, and carbon status and are further mediated by the signal transduction protein GlnK (36). The nifL gene lies immediately upstream of nifA (5). Simply deleting the entire nifL gene is not sufficient to result in the high-ammonium-accumulating phenotype found for AZBB163, although it results in an organism that can grow under diazotrophic conditions (Nif+) (12). Additionally, the phenotypes of AZBB163 and predecessor strains are dependent on both the specific antibiotic cassette used and the direction in which the cassette is inserted (4, 10, 12). For these reasons, it was interesting to look at the levels of transcription of nifA in AZBB163 versus the AvWT(+NH4+) and AvWT(−NH4+) strains. The transcriptional levels of nifA were similar for all three strains or growth conditions (Fig. 2). Brewin and coworkers reported decreased levels of expression of NifA in their MV376 strain. They also reported that a similar high-ammonium phenotype could be obtained in strain MV496 when a ptac promoter was placed upstream of nifA and IPTG was added, although transcription based on the ptac promoter resulted in significantly elevated levels of NifA expression and the highest levels of ammonium that they were able to obtain in their studies (4). The modifications made to AZBB163 do not appear to result in elevated levels of nifA, as was expected due to the specifics of how the kanamycin cassette must be inserted into the genome and the mutation that was required to achieve the Nif+ phenotype (12).
The specific reasons why certain modifications to nifL result in the release of ammonium, while others do not, are still uncertain (4, 10, 12). Attempts to generate strains where the aph gene is inserted in the same direction as nifA were unsuccessful. One explanation for this directional dependence of the aph and nifA genes is that this results in elevated levels of nifA such that the levels of ammonium become toxic (10). We were therefore interested in determining if an elevated ammonium level was toxic to the cells. As shown in Fig. 1, ammonium levels increased in a linear fashion for AZBB163, while the optical density of the culture remained constant. Transcriptional levels of key genes such as nifH also remained relatively constant in all of the samples of AZBB163 analyzed (Fig. 2). These results indicate that cellular function is not hindered by accumulating ammonium in the strain due to the gradual increase over time to more than 30 mM ammonium, similar to what was reported previously by Brewin et al. (4) for their MV496 strain, which required IPTG to induce the ptac promoter and yielded much higher levels of NifA.
Genes associated with ammonium uptake and assimilation.The elevated levels of ammonium resulting from the deregulation of nitrogenase expression and biological nitrogen fixation resulted in substantial quantities of ammonium accumulating in the growth medium for AZBB163. However, genes coding for the enzymes and transporters associated with ammonium transport and ammonium assimilation did not register significant differences in transcription levels for any of the samples tested as part of this study (see Fig. S7 in the supplemental material). This included amtB, which is proposed to protect the strain from losses of ammonium to the medium, and glnA, which encodes the enzyme that catalyzes the condensation of ammonia with glutamate, the primary entry point of ammonia into amino acid biosynthesis.
Correlations between transcriptional levels of wild-type A. vinelandii from this study and those from a previous report.A previous study of global transcriptional levels in A. vinelandii compared gene levels with and without ammonium supplementation in the presence of molybdenum or vanadium or in the absence of both (iron only) to induce the three different nitrogenase systems found in A. vinelandii (6). The approach taken in this report focused on the differences in the transcription levels of genes during stationary phase, when AZBB163 was accumulating large quantities of ammonium (Fig. 1), while in the previous report, those researchers performed sampling only during early exponential phase. We attribute these differences between the time points and stages of growth that were sampled, the methods used to enrich mRNA from tRNA and rRNA, and the increased numbers of time points and biological replicates that were sampled to the differences found between our AvWT(+NH4+) and AvWT(−NH4+) samples and the corresponding samples from that previous report. Many of the general trends found for the major and minor nif clusters of genes from the previous study and our study are in good agreement with one another (Fig. 2), with genes such as nifH, nifD, nifK, iscA, and nifB showing prominence in these two regions highlighted in both studies, although our results for the AvWT(+NH4+) and AvWT(−NH4+) samples did not show as large of an increase in fold changes for the nif-associated genes. Similarly, genes encoding housekeeping [Fe-S] proteins, including iscR, iscU, iscS, iscA, hscB, hscA, fdx, and iscX (Avin_40340 to Avin_40410), showed slightly decreased mRNA levels in our studies. In contrast, genes associated with type IV pili (pilG, pilE, pilA, pilN, and pilM), which were highlighted in the previous study and suggested to possibly be associated with an O2-protective mechanism, showed only slight increases in transcription levels [between 1- and 3-fold for AZBB163 and between 2- and 4-fold for AvWT(−NH4+)], indicating that the elevated levels reported previously might be more related to the exponential phase of growth under nitrogen limitation. The genes encoding cytochrome bd oxidase I (Avin_19880 and Avin_19890), the type II NADH-dependent oxidoreductase Ndh (Avin_12000), and the cytochrome o ubiquinol oxidase (Avin_11170 and Avin_11180) all showed no noticeable increases in transcription levels and in some cases showed a slight drop in transcription levels. The genes encoding the cytochrome cbb3 oxidase (Avin_19940 to Avin_20010) showed only minimal increases (∼1- to 3-fold) in transcription levels. These differences highlight the benefit of AZBB163 in providing an alternative data set related to this process, which is accompanied by a high-ammonium-accumulating phenotype and indicates the complementary nature of this data set and the results of the previous report to provide a broader picture of changes occurring within A. vinelandii to support high-ammonium accumulation.
The results presented in this report include only a small sampling of the extensive information available within this large data set. As with all such data sets, a detailed discussion of the importance of every gene for these organisms is well outside the scope of a single report. The RNA-Seq data from this study have been deposited in the NCBI GEO so that the entire data set may be evaluated by other laboratories. Many genes, especially those that were downregulated at the transcriptional level, were not discussed in detail, in part because these decreases were often not found to be statistically significant (P > 0.05), which may be related to the broader sampling approach that was purposely done to minimize the contributions of genes that had a high degree of variance (see Fig. S4 in the supplemental material). This data set also revealed many genes that showed only minimal changes in transcription levels in every sample under all three conditions (for example, Avin_37710, Avin_08100, Avin_37060, Avin_05040, Avin_18850, Avin_17430, Avin_14780, and Avin_36640), which could be suitable for use in future studies as reference genes. If we exclude any genes that contained an FPKM value of 0 (which includes many smaller genes), 378 genes showed a ≥2-fold increase in transcription levels in AZBB163 versus AvWT(+NH4+), and 199 showed a ≥2-fold decrease in transcription levels (P < 0.05) (Fig. S5). In many cases, the genes that showed significant decreases in transcription levels are seemingly correlated with a much higher level of variance in the results obtained within the AvWT(+NH4+) sample set, which might be partially attributed to a drop in the pH of this sample at later time points (Fig. S1).
Conclusions.This study provides a unique view of the genes involved in maintaining a high-ammonium-accumulating phenotype of a deregulated nitrogen-fixing A. vinelandii strain. The results revealed a lack of upregulation of hydrogenase gene expression and associated hydrogenase activity, which was surprising given the increased energy demands and the potential to recapture reducing equivalents through hydrogen oxidation in AZBB163. Additional salient observations include the inferred preference for a specific ATP synthase that might be more closely associated with nitrogen fixation and likely metal limitation based on an increased requirement for metals in AZBB163, which has significantly higher levels of nitrogenase associated with this phenotype. This metal limitation could be partially overcome by the inclusion of elevated molybdenum levels. Other genes that could be targeted to determine their role in nitrogen fixation in future studies were also revealed, which could shed light on how this model nitrogen-fixing bacterium is able to achieve high levels of nitrogen fixation while growing under aerobic conditions.
MATERIALS AND METHODS
Bacterial culture. A. vinelandii strain DJ (a highly transformable strain with diminished alginate production [3]) was obtained from Dennis Dean (Virginia Tech) and grown on standard Burk's medium (B medium) (26, 37) with or without ammonium (as ammonium sulfate) supplementation at 30°C and 180 rpm.
Genetic constructs. Escherichia coli JM109 was obtained from New England BioLabs (Ipswich, MA) and used to construct the plasmids described here. The construction of A. vinelandii strain AZBB163, which is deregulated for nitrogen fixation and produces enhanced levels of ammonium in the growth medium, was previously described (4, 10, 12). The construction of a strain lacking the ability to synthesize polyhydroxybutyrate (AZBB131) was described previously (38). A. vinelandii strains AZBB326 and AZBB328 were constructed as detailed in Table 1, by transforming AZBB163 with the plasmids described in Table 3. Strain AZBB208 is a synthetic construct meant to reconstruct the AZBB163 phenotype without requiring a spontaneous mutation step (12). The primers used to clone genes or genome segments and to confirm the final strains are listed in Table 4. Methods for the manipulation of A. vinelandii were previously described (26, 39).
Key plasmids and relative derivatives of these plasmids used for the construction of manipulated A. vinelandii strains
Key primers used in this study
Ammonium quantitation.The amount of ammonium was quantified as previously described (12), using a colorimetric approach. Samples were analyzed on a Cary 50 Bio spectrophotometer (Varian, Inc., Palo Alto, CA) by measuring the absorbance at 410 nm. To test the limitations of metal availability on ammonium accumulation rates, Burk's medium was prepared as described above, except that the iron levels were increased from 18 μM to 54 μM (3-fold) and the molybdenum levels were increased from 1 μM to 5 μM (5-fold).
Nitrogenase activity.To determine nitrogenase activity, acetylene reduction assays were conducted on freshly grown cultures by using a derivation of a previously described protocol (40). Cultured cells were transferred into a glass serum vial fitted with a rubber septum. Ten percent of the headspace gas was replaced with acetylene, and the culture was incubated for 60 min with shaking at 200 rpm at 30°C. An aliquot of the headspace was removed and analyzed for ethylene by using a GC Trace 1300 gas chromatograph with a flame ionization detector (Thermo Scientific). Gases were separated on a TG-Bond Q column (30 m by 0.32 mm by 10.0 μm). The injector, detector, and oven temperatures were 200°C, 230°C, and 30°C, respectively. Nitrogenase activity was quantified against a standard curve prepared with ethylene gas as a standard.
Hydrogen consumption assays.Hydrogen uptake was measured by transferring a specific amount of the culture into 3 ml of buffer in a 20-ml serum vial. Care was taken to use a quantity of the culture that resulted in approximately one-third of the added hydrogen being consumed. Levels of activity consuming more than one-half of the hydrogen were avoided, as these results were limited by substrate availability associated with the low solubility of hydrogen gas. The serum vials were capped with stoppers and sealed, 250 μl of hydrogen gas at atmospheric pressure was added, and an aliquot of the headspace was then removed and analyzed to determine the initial amount of hydrogen present. The culture was incubated for 2 h at 30°C and at 180 rpm, and a sample was again withdrawn from the headspace and analyzed. The headspace gas composition was measured by using a gas chromatograph equipped with a thermal conductivity detector (GC-TCD) instrument (Shimadzu) with argon as the carrier gas, as described previously (27). For more-concentrated cultures, the cells were diluted to an optical density of between 0.3 and 2.5, depending on the activities found in preliminary experiments, and the quantities of protein added were quantified by using the Bradford assay reagent (41). Hydrogen production was quantified by using the same method but without the addition of any external hydrogen gas.
RNA isolation and RNA-Seq analysis.Samples of approximately 6 to 10 ml (OD of 2.0 or higher) were concentrated by centrifugation, and the supernatant was removed prior to being flash frozen in liquid nitrogen and stored at −80°C until needed. RNA was isolated by resuspending frozen cells in 1 ml of TRIzol reagent (Invitrogen, Grand Island, NY), and samples were vortexed intermittently for several minutes until they were fully dissolved. Following this, 200 μl of chloroform was added, and the mixture was vortexed and then centrifuged at 12,000 × g for 2 min. The upper phase was removed and further purified by using the Direct-zol RNA miniprep kit (Zymo Research, Irvine, CA). RNA was eluted, treated to remove background DNA using the RNase-free DNase I set (Zymo Research, Irvine, CA) according to the manufacturer's directions in a total volume of 100 μl for 10 min at room temperature, suspended in 300 μl of TRIzol, and again isolated by using the Direct-zol miniprep kit. The quantity of isolated RNA was measured by using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA). rRNAs and tRNAs were removed from the total isolated RNA by using the Ribo-Zero kit for Gram-negative bacteria (Illumina, Inc., San Diego, CA). Final mRNA fractions were concentrated by using the RNA Clean & Concentrator-5 system (Zymo Research, Irvine, CA). Final mRNA samples were submitted to the University of Minnesota Genomics Center for quality control analysis of RNA sizing using a BioAnalyzer (Agilent Technologies, Inc., Santa Clara, CA) and the subsequent creation of an RNA-Seq library. Samples were then sequenced by using a HiSeq 2500 Rapid Mode instrument with 100-bp paired-end reads.
Raw data from the RNA sequencing experiments were run through the rnaseq pipeline (tophat/hisat) for the analysis of high-throughput sequence data sets (42) at the University of Minnesota Supercomputing Institute. The pipeline utilized FastQC (43), Trimmomatic (44), and Bowtie 2 (45). Cuffquant and Cuffnorm were used to calculate FPKM values for each gene (46). Data were exported to Excel (Microsoft, Redmond, WA) for a further comparison of differences in transcriptional levels. Results presented represent the averages and standard deviations for each data set. Statistical measurement of significance for data sets was done with Welch's t test by using the t test function in Excel with a two-tailed distribution under the heteroscedastic parameters for two samples with unequal variance.
Total cell protein SDS-PAGE gels.To compare the general protein expression profiles of A. vinelandii strain AZBB163 to those of the wild-type strain under various growth conditions, liquid cultures of the wild-type strain grown at 30°C in Burk's medium with sufficient ammonium (20 mM) and grown diazotrophically overnight (no added nitrogen source) were cultured along with a sample that was first grown with sufficient ammonium (20 mM) and then transferred to fresh medium containing no ammonium for 2 h to derepress the nitrogenase. A. vinelandii strain AZBB163 was also grown diazotrophically in Burk's medium for comparison. Similar quantities of cells (∼100 mg [wet weight]) were collected under each of the four sample conditions by centrifugation at >12,000 × g for 2 min. Cells were flash frozen in liquid nitrogen. Cell pellets were thawed, suspended in 200 μl of 1% SDS, boiled for 10 min, vented and cooled for 5 min, and then spun at >15,000 × g for 10 min. The supernatant was loaded onto an SDS-PAGE gel to provide a simple overview of the expression levels in each strain.
ACKNOWLEDGMENTS
This work is supported by a grant (RC-0007-12) from the Initiative for Renewable Energy & the Environment (Institute on the Environment), the MnDRIVE transdisciplinary research initiative through the University of Minnesota based on funding from the state of Minnesota, and grants from the National Institute of Food and Agriculture (project numbers MIN-12-070 and MIN-12-081) to B.M.B.; the National Science Foundation (grant number NSF-1331098) to J.W.P.; and the Biotechnology Institute at the University of Minnesota for fellowship funding to V.N.
We thank Jon Bertram and Hanna Hondzo for assistance with performing specific experiments and Ying Zhang and the University of Minnesota Supercomputing Institute for assistance with data analysis using the pipeline to generate FPKM values. We thank Yaniv Brandvain for suggestions related to statistical analysis of our data.
FOOTNOTES
- Received 13 July 2017.
- Accepted 7 August 2017.
- Accepted manuscript posted online 11 August 2017.
Supplemental material for this article may be found at https://doi.org/10.1128/AEM.01534-17 .
- Copyright © 2017 American Society for Microbiology.
