RNA Processing of Nitrogenase Transcripts in the CyanobacteriumAnabaena variabilis

Abstract

Little is known about the regulation of nitrogenase genes in cyanobacteria. Transcription of the nifH1 and vnfH genes, encoding dinitrogenase reductases for the heterocyst-specific Mo-nitrogenase and the alternative V-nitrogenase, respectively, was studied by using a lacZ reporter. Despite evidence for a transcription start site just upstream of nifH1 and vnfH, promoter fragments that included these start sites did not drive the transcription of lacZ and, for nifH1, did not drive the expression of nifHDK1. Further analysis using larger regions upstream of nifH1 indicated that a promoter within nifU1 and a promoter upstream of nifB1 both contributed to expression of nifHDK1, with the nifB1 promoter contributing to most of the expression. Similarly, while the region upstream of vnfH, containing the putative transcription start site, did not drive expression of lacZ, the region that included the promoter for the upstream gene, ava4055, did. Characterization of the previously reported nifH1 and vnfH transcriptional start sites by 5RACE (5 rapid amplification of cDNA ends) revealed that these 5 ends resulted from processing of larger transcripts rather than by de novo transcription initiation. The 5 positions of both the vnfH and nifH1 transcripts lie at the base of a stem-loop structure that may serve to stabilize the nifHDK1 and vnfH specific transcripts compared to the transcripts for other genes in the operons providing the proper stoichiometry for the Nif proteins for nitrogenase synthesis.Anabaena variabilis ATCC 29413 is a filamentous cyanobacterium that fixes atmospheric nitrogen under oxic growth conditions. After removal of fixed nitrogen from the growth medium, ca. 5 to 10% of the vegetative cells differentiate into specialized cells called heterocysts, in which nitrogen fixation occurs (60, 62). Heterocysts protect the oxygen-labile nitrogenase from external oxygen by synthesizing a glycolipid layer that limits oxygen diffusion into the cell (30,57,58). Internal oxygen is low in heterocysts because they lack oxygen-evolving photosystem II activity and they have increased respiration (29,53). A. variabilis has three nitrogenases, but each functions under different environmental conditions (reviewed in reference 46). The primary nitrogenase is the heterocyst-specific Mo-nitrogenase encoded by the nif1 genes (44, 45). A. variabilis also has an alternative heterocyst-specific V-nitrogenase, encoded by the vnf genes, that is only expressed when Mo is limiting (32, 44). A second Mo-nitrogenase, encoded by the nif2 genes (47-49), functions only under anoxic conditions in vegetative cells and heterocysts. Synthesis of all three nitrogenases is repressed in cells grown with a source of fixed nitrogen.Synthesis of a functional nitrogenase requires the products of many genes, several of which are involved in production and insertion of the FeMo-cofactor that is found in most nitrogenases (reviewed in reference 37). These genes include nifB, nifS, nifU, nifH, nifD, nifK, nifE, nifN, nifX, and nifW. The eight genes of...

Regulation of Nitrogenase Gene Expression by Transcript Stability in the Cyanobacterium Anabaena variabilis

“…A 6-kb SacI fragment of pRL2948a was arrow. The solid rightward arrows indicate transcription start sites (tss) upstream of nifB1, in nifU1, in nifE1, and upstream of hesA1, while the dashed arrows indicate transcription processing sites upstream of nifH1, in ava3935, and upstream of fdxH, as determined previously (33) and in this study. (B) ␤-Galactosidase activities for strains containing fusions of four different regions upstream of nifE1 to a promoterless lacZ.…”

“…Apparent transcription start sites have been identified upstream of nifB (25,26,(33)(34)(35), nifH (26,36), hesA (30,35), and fdxH (31). Although the putative nifH transcription start site has been identified several times, we were unable to drive transcription of lacZ using a DNA fragment that included the entire niU1-nifH1 intergenic region of A. variabilis, and we showed that the 5= end of the nifH1 transcript in A. variabilis is a processed transcript rather than a transcription initiation site; thus, there is no promoter upstream of nifH1 (33). In contrast, nifB1 has a promoter that initiates transcription, and there is a promoter within the coding region of nifU1 (33), which has been verified by transcriptome sequencing (RNA-seq) (35).…”

The sigE Gene Is Required for Normal Expression of Heterocyst-Specific Genes in Anabaena sp. Strain PCC 7120

“…Demonstrating direct regulation of nifH expression by SigE will require in vitro transcription assays using purified components. However, these experiments are especially challenging because the cis-acting sequences that constitute the nifH promoter have not been identified in Anabaena PCC 7120, and recent data with Anabaena variabilis suggest that multiple distant promoters and RNA-processing events may be involved in expression of the nifHDK operon (50).…”

“…The regulation of the Anabaena PCC 7120 nifHDK operon has been studied for over 25 years (14,18,20,43,44,50,53); however, little is known about the transcription components that regulate this operon. Our results suggest that sigE is in -FIG.…”

Transcriptional regulators ChlR and CnfR are essential for diazotrophic growth in nonheterocystous cyanobacteria

“…Ungerer et al (36) reported that the high expression of nifH1 is supported by two promoters: a strong one is located upstream of nifB1, and the other weak one is located in the coding region of nifU1 in A. variabilis. In addition, they pointed out that a secondary structure of 5′ UTR just upstream of nifH1 might contribute to stabilize the processed nifH1 transcript.…”