Differential Accumulation of nifStructural Gene mRNA in Azotobacter vinelandii

TEXT

Biological nitrogen fixation occurs by the activity of nitrogenase, which exists as a complex metalloenzyme composed of two easily separable components. The Fe protein component (encoded by nifH) serves as the obligate electron donor to the MoFe protein component (encoded by nifDK), which contains the active site for dinitrogen reduction (12). In addition to the structural proteins, the nitrogenase enzyme requires extensive biosynthetic machinery consisting of enzymes, scaffolds, and carrier proteins to assemble the metalloclusters required for catalysis (3, 15). nifgenes are located in two clusters in the model diazotroph, Azotobacter vinelandii. The major nifcluster contains the structural genes and those for most of the biosynthetic machinery, which are organized in several contiguous operons (6), and a minor nifcluster contains the nifregulatory elements and the remaining, necessary biosynthetic genes (8). The major nifcluster in A. vinelandiiwas sequenced more than 3 decades ago and has been the subject of a number of gene deletion and mutation studies, which laid the groundwork for our current understanding of the operon structure and regulation of nif(6). The genes encoding the nitrogenase structural proteins, nifH, nifD, and nifK, are located in the major nifoperon and are cotranscribed from a single promoter, the nifHpromoter, along with nifT, nifY, orf1, and lrv(6). The nifHpromoter is efficient and is effectively regulated, driving the rapid and abundant expression of nitrogenase in the absence of fixed N and conversely an abrupt decline in nitrogenase component transcription in the presence of fixed N (16).

In vitrocharacterization of nitrogenase from a variety of microbial sources indicates that the highest nitrogenase activities are observed at high Fe protein-to-MoFe protein ratios. In line with these observations, Northern blot hybridization analyses have indicated differential expression of the nifstructural proteins (7), and immunoblotting has shown that the Fe protein occurs in significant excess compared to MoFe protein levels in cultures grown under diazotrophic conditions (2, 7). Moreover, global transcriptional analyses indicate that nifHmRNA accumulates at ∼3-fold-higher levels than nifDand nifKmessages in A. vinelandiigrown diazotrophically (4). Because the genes encoding the nitrogenase Fe protein and MoFe protein are cotranscribed in A. vinelandii, a mechanism must exist to control differential nifstructural gene transcript abundance.

In order to assess possible mechanisms resulting in the differential accumulation of nifstructural gene mRNA and nitrogenase component proteins, Northern blot hybridization analysis was performed, probing mRNAs for nifH. Wild-type A. vinelandiiand deletion strains were grown in fixed-N replete, modified Burk medium (17), and cells were derepressed to induce expression of nitrogenase as previously described (16). Deletion strains were constructed as previously described (1, 6, 13, 14). Samples for Northern blot hybridization were collected at 10-min intervals for 120 min. RNA extraction, glyoxylation, and electrophoresis were performed as described previously (10, 11). Transfer of RNA onto GeneScreen hybridization membranes and subsequent hybridization were performed according to the manufacturers' instructions (Dupont). Approximately 10 μg of RNA from each sample was analyzed by Northern blot hybridization using a nifH-specific, ³²P-labeled probe, pMJH5, purified from Escherichia colias previously described (7). The results revealed differential accumulation of major transcripts that could correspond to nifH, nifHD, and nifHDKand a very minor transcript corresponding to the length of the entire operon, nifH, nifD, nifK, nifT, nifY, orf1, and lrv(Fig. 1A). The identity of the three most abundant transcripts was established in two ways. First, when a nifD-specific hybridization probe was used for Northern blot analysis, only the bands corresponding to putative nifHDand nifHDKtranscripts were detected (data not shown). In a second series of experiments, strains having defined deletions within the structural gene region were analyzed by Northern blot analysis using a nifHprobe (Fig. 1B). These results showed that a deletion within nifDhad no effect on the size of the accumulated nifHtranscripts but resulted in accumulation of smaller transcripts assigned to nifHDand nifHDK(DJ100) (Fig. 1B). Similarly, a deletion in nifKhad no effect on the size of the transcripts corresponding to nifHor nifHDbut resulted in the accumulation of a smaller transcript assigned to nifHDK(DJ13) (Fig. 1B). Strains that had deletions that spanned portions of nifHand nifD(DJ46) (Fig. 1B) or nifDand nifK(DJ33) (Fig. 1B) resulted in the accumulation of only two major transcripts, which suggested that elements leading to differential accumulation are likely to be located within the intergenic regions. A strain having a deletion of nifHthat encompassed the region corresponding to the nifH-specific probe showed no nifHhybridizing transcripts (DJ54) (Fig. 1B). These results are consistent with the aforementioned results showing that the major nifoperon produces three major transcripts that correspond to nifH, nifHD, and nifHDK.

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Fig. 1.

(A) Northern blot hybridization analysis of total RNA isolated from wild-type A. vinelandiiusing pMJH5, a nifH-specific probe. Cells were derepressed in N-free medium, and total RNA was extracted at 10-min increments from 0 to 120 min. The black arrow indicates the minor band resulting from the entire nifstructural gene operon. (B) Northern blot hybridization analysis of total RNA isolated from A. vinelandiistrains with deletions spanning regions of the structural genes indicated, performed by using pMJH5, a nifH-specific probe. Cells were derepressed in N-free medium, and total RNA was extracted 60 min after derepression. Nif⁺strains were capable of diazotrophic growth; Nif⁻strains were not.

It is interesting that during the transition to nitrogen-fixing conditions, the relative abundances of the three nifH-specific transcripts changed noticeably. For example, inspection of the time course shown in Fig. 1A revealed that in the early stage of nifderepression, the major accumulating mRNA corresponds to nifHDK. In contrast, as the cells enter steady-state, nitrogen fixation conditions (∼90 min following derepression), the relative amounts of shorter transcripts solely for nifHincreased in abundance relative to the longer transcripts. The elevated transcript levels for nifH, compared to nifDKexpression under steady-state nitrogen-fixing conditions, is in line with establishing and maintaining the high Fe protein-to-MoFe protein ratios required for optimal nitrogenase catalytic activity. It is not clear why there is an apparent increased capacity for expression of nifDrelative to that of nifK, because these genes encode subunits of the MoFe protein, which are required in equimolar amounts. However, it could be a mechanism to counteract potential differences in translation efficiencies for these transcripts or in the stabilities of nitrogenase protein subunits.

A closer analysis of the nucleotide sequences in the regions that separate the coding regions of the nifstructural gene operon revealed potential secondary structures between nifHand nifD, between nifDand nifK, and downstream from nifK(5, 19) (Fig. 2A). Free energy (ΔG) values of −29.0 and −26.9 kcal/mol were calculated for the structures between nifHand nifDand nifDand nifK, respectively (Fig. 2A). Additional structures predicted just downstream of nifKhad free energy values of −29.0 and −44.0 kcal/mol. Transcript profiling of A. vinelandiiunder Mo-dependent, nitrogen-fixing conditions (4) indicates a specific decrease in transcript sequences corresponding to a very small region that spans the putative mRNA secondary structure (Fig. 2A). Similar decreases in transcript abundance were observed for the regions corresponding to the predicted structures between nifDand nifKand downstream of nifK(data not shown). Conventionally, RNA structures have been probed individually, but the advent of whole-transcriptome sequencing has precipitated the need for high-throughput RNA structure probing to complement transcriptional profiling (9, 18). However, mapping the end of the most abundant message corresponding to nifHclearly indicates that transcript levels declined upstream of the first structure observed in the operon located between nifHand nifD(Fig. 2A). An inability to produce any detectable cDNA spanning the proposed secondary structure located within the nifH-nifDintergenic region provides strong evidence for the formation of a secondary structure within this region. This is further supported by the failure to detect cDNA sequence reads within the regions that define the putative secondary structures in the nifD-nifKintergenic region and downstream of nifK(data not shown).

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Fig. 2.

(A) Identification of RNA secondary structures between the nifstructural genes. ΔGvalues are given in kcal/mol. The transcript abundance of the intergenic region between nifHand nifDis plotted against the location of these genes in the genome. The box indicates the location of the RNA secondary structure. For detailed methods of transcriptional profiling, see reference 4. (B) Northern blot hybridization analysis of total RNA isolated from A. vinelandiimutant strain DJ81 using pMJH5, a nifH-specific probe. Cells were derepressed in N-free medium, and total RNA was extracted at 10-min increments from 0 to 120 min.

To examine the role of the intergenic regions in the differential accumulation of the nifstructural gene operon directly, a strain was constructed (DJ81) that had an 87-bp deletion spanning the proposed secondary structure located in the nifH-nifDintergenic region. Strain DJ81 was capable of diazotrophic growth at rates comparable to those of the wild type under typical laboratory culture conditions (6) (data not shown). Northern blot hybridization analysis of this strain revealed the accumulation of only two major nifHtranscripts, corresponding to nifHDand nifHDK, and a very minor transcript, which could correspond to nifH, nifD, nifK, nifT, nifY, orf1, and lrv(Fig. 2B). In this strain, no nifH-only transcripts were detected. As with the wild type (Fig. 1A), the first major accumulating mRNA during nifderepression of DJ81 corresponds to nifHDK, with the shorter nifHDtranscript accumulating later. This result provides further evidence that the secondary structures act as processing and/or mRNA stabilizing sites that increase the lifetime of specific segments of the primary transcripts.

In summary, the Northern analysis of multiple deletion mutants indicates definitively that all nifH-specific transcripts observed originate from the nifHpromoter and correspond to nifH, nifHD, nifHDK, and a small proportion of a full-length transcript representing the entire operon. The elimination of specific transcript segments in response to deletion of the intergenic regions clearly demonstrates the role of intergenic RNA secondary structures in differential accumulation of nifstructural gene mRNAs. The intergenic secondary structures could function as premature termination sites and/or sites of processing and stabilization. The changes in the relative abundances of nifH-specific transcripts that are observed to occur over the course of depression (Fig. 1A and 2B) seem to favor the latter or imply the involvement of trans-acting elements in controlling the ratios of these transcripts for optimal diazotrophic growth. In this regard, the differential accumulation of mRNA represents one mechanism for the production of the appropriate relative abundances of the nitrogenase components for optimal catalytic function. This mechanism may also be important in compensating for differences in translation efficiencies of the nifstructural genes or protein subunit stabilities.