Differential accumulation of nif structural gene mrna in Azotobacter vinelandii. Research Center, Montana State University, Bozeman, Montana 59717

Transcription

1 JB Accepts, published online ahead of print on 1 July 2011 J. Bacteriol. doi: /jb Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved Differential accumulation of nif structural gene mrna in Azotobacter vinelandii Trinity L. Hamilton 1, Marty Jacobson 3, Marcus Ludwig 2, Eric S. Boyd 1, Donald A. Bryant 1,2, Dennis R. Dean 3*, and John W. Peters 1* Department of Chemistry and Biochemistry and the Astrobiology Biogeocatalysis Research Center, Montana State University, Bozeman, Montana Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 3 Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia *Corresponding Authors: Dennis R. Dean Department of Biochemistry 110 Fralin Hall Virginia Tech Blacksburg, Virginia Tel: Fax: deandr@vt.edu John W. Peters Department of Chemistry and Biochemistry 103 Chemistry and Biochemistry Building Montana State University Bozeman, Montana Tel: Fax: john.peters@chemistry.montana.edu 34 1

2 35 Abstract Northern analysis was employed to investigate mrna produced by mutant strains of Azotobacter vinelandii with defined deletions in the nif structural genes and in the intergenic noncoding regions. The results indicate that intergenic RNA secondary structures effect the differential accumulation of transcripts supporting the high Fe protein to MoFe protein ratio required for optimal diazotrophic growth. 2

3 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). nif-encoded genes are located in two clusters in the model diazotroph, A. vinelandii. The major nif cluster encodes the structural genes and most of the biosynthetic machinery which are organized in several contiguous operons (6), and a minor nif cluster contains the nif regulatory elements and the remaining, necessary biosynthetic genes (8). The major nif cluster in A. vinelandii was sequenced over three 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 nif operon and are cotranscribed from a single promoter, the nifh promoter, along with nift, nify, orf1, and lrv (6). The nifh promoter is efficient and 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 vitro characterization of nitrogenase from a variety of microbial sources indicates that 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 nif structural proteins (7), and immunoblotting has 3

4 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 nifh mrna accumulates at ~ 3-fold higher levels than nifd and nifk messages in A. vinelandii grown diazotrophically (4). Because the genes encoding the nitrogenase Fe protein and MoFe protein are co-transcribed in A. vinelandii, a mechanism must exist to control differential nif structural gene transcript abundance. In order to assess possible mechanisms resulting in the differential accumulation of nif structural gene mrna and nitrogenase component proteins, northern blot hybridization analysis was performed probing mrnas encoding nifh. Wild-type A. vinelandii and deletion strains were grown in fixed-n replete, modified Burk medium (17), and cells were de-repressed to induce expression of nitrogenase as previously described (16). Deletion strains were constructed as previously described (1,6). Samples for northern blot hybridization were collected at 10-min intervals for 120 min. RNA extraction, glyoxylation and electrophoresis were performed as described (10, 11). Transfer of RNA onto GeneScreen TM 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, 32 P-labelled probe pmjh5 purified from E. coli as previously described (7). The results revealed differential accumulation of major transcripts that could correspond to nifh, nifhd, nifhdk, and 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 4

5 analysis, only the bands corresponding to putative nifhd and nifhdk transcripts 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 nifh probe (Fig. 1B). These results showed that a deletion within nifd had no effect on the size of the accumulated nifh transcripts but resulted in accumulation in smaller transcripts assigned to nifhd and nifhdk (DJ100, Fig. 1B). Similarly, a deletion in nifk had no effect on the size of the transcripts corresponding to nifh or nifhd but resulted in the accumulation of a smaller transcript assigned to nifhdk (DJ13, Fig. 1B). Strains that had deletions that spanned portions of nifh and nifd (DJ46, Fig. 1B) or nifd and 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 nifh that encompassed the region corresponding to the nifh-specific probe showed no nifh hybridizing transcripts (DJ54, Fig. 1B). These results are consistent with the aforementioned results showing that the major nif operon produces three major transcripts that correspond to nifh, nifhd, and nifhdk. It is interesting that during the transition to nitrogen fixing conditions, the relative abundance 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 encoding solely nifh increased in abundance relative to the longer transcripts. The elevated transcript levels for nifh, compared to 5

6 nifdk expression 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 nifd relative to 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 nif structural gene operon revealed potential secondary structures between nifh and nifd, between nifd and nifk, and downstream from nifk (5, 19; Fig. 2A). Free energy (ΔG) values of and kcal/mol were calculated for the structures between nifh and nifd and nifd and nifk, respectively (Fig. 2A). Additional structures predicted just downstream of nifk had free energy values of and kcal/mol. Transcript profiling of A. vinelandii under 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 nifd and nifk and 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 nifh clearly indicates that transcript levels declined upstream of the first structure observed in the operon located between nifh and 6

7 nifd (Fig 2A). An inability to produce any detectable cdna spanning the proposed secondary structure located within the nifh nifd intergenic 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 nifk integenic region and downstream of nifk (data not shown). To examine the role of the intergenic regions in the differential accumulation of nif structural gene operon directly, a strain was constructed (DJ81) that had an 87 basepair deletion spanning the proposed secondary structure located in the nifh-nifd intergenic region. Strain DJ81 was capable of diazotrophic growth at rates comparable to 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 nifh transcripts corresponding the nifhd and nifhdk and a very minor transcript that could correspond to nifh, nifd, nifk, nift, nify, orf1, and lrv (Fig. 2B). In this strain, no nifhonly transcripts were detected. Similar to wild-type (Fig. 1A), the first major accumulating mrna during nif-derepression of DJ81 corresponds to nifhdk, with the shorter nifhd transcript 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 nifh promoter 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 7

8 response to deletion of the intergenic regions clearly demonstrates the role of intergenic RNA secondary structures in differential accumulation of nif structural gene mrnas. The intergenic secondary structures could function as premature termination sites and/or 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 Fig. 2B) seems 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 nif structural genes or protein subunit stabilities. Downloaded from on April 6, 2018 by guest 8

9 Acknowledgements. This work was supported by the NASA Astrobiology Institute grant NNA08C-N85A to J.W.P. and NASA Astrobiology: Exobiology and Evolutionary Biology, Award NNX09AM87G (D.A.B.) and NSF MCB (D.R.D). T.L.H. was supported by an NSF-Integrated Graduate Educational Research and Training fellowship grant and E.S.B. was supported by a fellowship from the NASA Astrobiology Institute Postdoctoral Program. Downloaded from on April 6, 2018 by guest 9

10 References 1. Brigle, K. E., R. A. Setterquist, D. R. Dean, J. S. Cantwell, M. C. Weiss, and W. E. Newton Site-directed mutagenesis of the nitrogenase MoFe protein of Azotobacter vinelandii. Proc. Natl. Acad. Sci. USA. 84: Dingler, C., J. Kuhla, H. Wassink, and J. Oelze Levels and activities of nitrogenase proteins in Azotobacter vinelandii grown at different dissolved oxygen concentrations. J. Bacteriol. 170: Dos Santos, P. C., and D. R. Dean Coordination and fine-tuning of nitrogen fixation in Azotobacter vinelandii. Mol. Microbiol. 79: Hamilton, T. L., M. Ludwig, R. Dixon, E. S. Boyd, P. C. Dos Santos, J. C. Setubal, D. B. Bryant, D. R. Dean, and J. W. Peters Transcriptional profiling of nitrogen fixation in Azotobacter vinelandii. J. Bacteriol. Submitted. 5. Hofacker, I. L Vienna RNA secondary structure server. Nucl. Acids. Res. 31: Jacobson, M. R., K. E. Brigle, L. T. Bennett, R. A. Setterquist, M. S. Wilson, V. L. Cash, J. Beynon, W. E. Newton, and D. R. Dean Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii. J. Bacteriol. 171: Jacobson, M. R., R. Premakumar, and P. E. Bishop Transcriptional regulation of nitrogen fixation by molybdenum in Azotobacter vinelandii. J. Bacteriol. 167: Joerger, R. D., and P. E. Bishop Nucleotide sequence and genetic analysis of the nifb-nifq region from Azotobacter vinelandii. J Bacteriol 170: Kertesz, M., Y. Wan, E. Mazor, J. L. Rinn, R. C. Nutter, H. Y. Chang, and E. Segal. Genome-wide measurement of RNA secondary structure in yeast. Nature. 467: Krol, A. J., J. G. Hontelez, B. Roozendaal, and A. van Kammen On the operon structure of the nitrogenase genes of Rhizobium leguminosarum and Azotobacter vinelandii. Nucl. Acids. Res. 10: McMaster, G. K., and G. G. Carmichael Analysis of single- and doublestranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. USA. 74: Peters, J. W., and R. K. Szilagyi Exploring new frontiers of nitrogenase structure and mechanism. Curr. Opin. Chem. Biol. 10: Robinson, A. C., B. K. Burgess, and D. R. Dean Activity, reconstitution, and accumulation of nitrogenase components in Azotobacter vinelandii mutant strains containing defined deletions within the nitrogenase structural gene cluster. J. Bacteriol. 166: Robinson, A. C., D. R. Dean, and B. K. Burgess Iron-molybdenum cofactor biosynthesis in Azotobacter vinelandii requires the iron protein of nitrogenase. J. Biol. Chem. 262: Rubio, L. M., and P. W. Ludden Biosynthesis of the iron-molybdenum cofactor of nitrogenase. Annu. Rev. Microbiol. 62:

11 Shah, V. K., L. C. Davis, and W. J. Brill Nitrogenase. I. Repression and derepression of the iron-molybdenum and iron proteins of nitrogenase in Azotobacter vinelandii. Biochim. Biophys. Acta. 256: Strandberg, G. W., and P. W. Wilson Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii. Can. J. Microbiol. 14: Underwood, J. G., A. V. Uzilov, S. Katzman, C. S. Onodera, J. E. Mainzer, D. H. Mathews, T. M. Lowe, S. R. Salama, and D. Haussler. FragSeq: transcriptome-wide RNA structure probing using high-throughput sequencing. Nat. Methods. 7: Zuker, M Mfold web server for nucleic acid folding and hybridization prediction. Nucleic. Acids. Res. 31: Downloaded from on April 6, 2018 by guest 11

12 Figure Legends. FIG. 1. (A) Northern blot hybridization analysis of total RNA isolated from wild-type A. vinelandii. Cells were de-repressed in N-free media and total RNA was extracted at 10- min increments from 0 to 120 min. Black arrow indicates the minor band resulting from the entire structural gene operon. (B) Northern blot hyrbidization analysis of total RNA isolated from A. vinelandii strains with deletions spanning regions of the structural genes indicated. Cells were de-repressed in N-free media and total RNA was extracted 60 min. after derepression. Nif + strains were capable of diazotrophic growth, Nif - were not. FIG. 2. (A) Identification of RNA secondary structures between the nif structural genes. ΔG values are given in kcal/mol. Transcript abundance of the intergenic region between nifh and nifd plotted against the location of these genes in the genome. The box indicates the location of the RNA secondary structure. For detailed methods of the transcriptional profiling, see reference (4). (B) Northern blot hybridization analysis of total RNA isolated from mutant strain DJ81. Cells were de-repressed in N-free media and total RNA was extracted at 10-min increments from 0 to 120 min. 12

13

14