Dataset Information

Role of RNA secondary structure and processing in stability of the nifH1 transcript in the cyanobacterium Anabaena variabilis.

ABSTRACT:

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In the cyanobacterium Anabaena variabilis ATCC 29413, aerobic nitrogen fixation occurs in micro-oxic cells called heterocysts. Synthesis of nitrogenase in heterocysts requires expression of the large nif1 gene cluster, which is primarily under the control of the promoter for the first gene, nifB1. Strong expression of nifH1 requires the nifB1 promoter but is also controlled by RNA processing, which leads to increased nifH1 transcript stability. The processing of the primary nifH1 transcript occurs at the base of a predicted stem-loop structure that is conserved in many heterocystous cyanobacteria. Mutations that changed the predicted secondary structure or changed the sequence of the stem-loop had detrimental effects on the amount of nifH1 transcript, with mutations that altered or destabilized the structure having the strongest effect. Just upstream from the transcriptional processing site for nifH1 was the promoter for a small antisense RNA, sava4870.1. This RNA was more strongly expressed in cells grown in the presence of fixed nitrogen and was downregulated in cells 24 h after nitrogen step down. A mutant strain lacking the promoter for sava4870.1 showed delayed nitrogen fixation; however, that phenotype might have resulted from an effect of the mutation on the processing of the nifH1 transcript. The nifH1 transcript was the most abundant and most stable nif1 transcript, while nifD1 and nifK1, just downstream of nifH1, were present in much smaller amounts and were less stable. The nifD1 and nifK1 transcripts were also processed at sites just upstream of nifD1 and nifK1.

Importance

In the filamentous cyanobacterium Anabaena variabilis, the nif1 cluster, encoding the primary Mo nitrogenase, functions under aerobic growth conditions in specialized cells called heterocysts that develop in response to starvation for fixed nitrogen. The large cluster comprising more than a dozen nif1 genes is transcribed primarily from the promoter for the first gene, nifB1; however, this does not explain the large amount of transcript for the structural genes nifH1, nifD1, and nifK1, which are also under the control of the distant nifB1 promoter. Here, we demonstrate the importance of a predicted stem-loop structure upstream of nifH1 that controls the abundance of nifH1 transcript through transcript processing and stabilization and show that nifD1 and nifK1 transcripts are also controlled by transcript processing.

SUBMITTER: Pratte BS

PROVIDER: S-EPMC4372750 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Publications

Role of RNA secondary structure and processing in stability of the nifH1 transcript in the cyanobacterium Anabaena variabilis.

Pratte Brenda S BS Ungerer Justin J Thiel Teresa T

Journal of bacteriology 20150209 8

<h4>Unlabelled</h4>In the cyanobacterium Anabaena variabilis ATCC 29413, aerobic nitrogen fixation occurs in micro-oxic cells called heterocysts. Synthesis of nitrogenase in heterocysts requires expression of the large nif1 gene cluster, which is primarily under the control of the promoter for the first gene, nifB1. Strong expression of nifH1 requires the nifB1 promoter but is also controlled by RNA processing, which leads to increased nifH1 transcript stability. The processing of the primary ni ...[more]

PMID: 25666132

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Project description:Anabaena variabilis is a filamentous cyanobacterium that is capable to differentiate specialized cells, the heterocysts and akinetes, to survive under different stress conditions. Under nitrogen limited condition, heterocysts provide the filament with nitrogen by fixing N2. Akinetes are spore-like dormant cells that allow survival during adverse environmental conditions. Both cell types are characterized by the presence of a thick multilayered envelope, including a glycolipid layer. While in the heterocyst this glycolipid layer is required for the maintenance of a microoxic environment and nitrogen fixation, its function in akinetes is completely unknown. Therefore, we constructed a mutant deficient in glycolipid synthesis and investigated the performance of heterocysts and akinetes in that mutant strain. We chose to delete the gene Ava_2595, which is homolog to the known hglB gene, encoding a putative polyketide synthase previously shown to be involved in heterocyst glycolipid synthesis in Anabaena sp. PCC 7120, a species which does not form akinetes. Under the respective conditions, the Ava_2595 null mutant strain formed aberrant heterocysts and akinete-like cells, in which the specific glycolipid layers were absent. This confirmed firstly that both cell types use a glycolipid of identical chemical composition in their special envelopes and, secondly, that HglB is essential for glycolipid synthesis in both types of differentiated cells. As a consequence, the mutant was not able to fix N2 and to grow under diazotrophic conditions. Furthermore, the akinetes lacking the glycolipids showed a severely reduced tolerance to stress conditions, but could germinate normally under standard conditions. This demonstrates the importance of the glycolipid layer for the ability of akinetes as spore-like dormant cells to withstand freezing, desiccation, oxidative stress and attack by lytic enzymes. Our study established the dual role of the glycolipid layer in fulfilling different functions in the evolutionary-related specialized cells of cyanobacteria. It also indicates the existence of a common pathway involving HglB for the synthesis of glycolipids in heterocysts and akinetes.

Project description:Anabaena variabilis ATCC 29413 has one Mo nitrogenase that is made under oxic growth conditions in specialized cells called heterocysts and a second Mo nitrogenase that is made only under anoxic conditions in vegetative cells. The two large nif gene clusters responsible for these two nitrogenases are under the control of the promoter of the first gene in the operon, nifB1 or nifB2 Despite differences in the expression patterns of nifB1 and nifB2, related to oxygen and cell type, the regions upstream of their transcription start sites (tss) show striking homology, including three highly conserved sequences (CS). CS1, CS2, and the region just upstream from the tss were required for optimal expression from the nifB1 promoter, but CS3 and the 5' untranslated region (UTR) were not. Hybrid fusions of the nifB1 and nifB2 upstream regions revealed that the region including CS1, CS2, and CS3 of nifB2 could substitute for the similar region of nifB1; however, the converse was not true. Expression from the nifB2 promoter region required the CS1, CS2, and CS3 regions of nifB2 and also required the nifB2 5' UTR. A hybrid promoter that was mostly nifB2 but that had the region from about position -40 to the tss of nifB1 was expressed in heterocysts and in anoxic vegetative cells. Thus, addition of the nifB1 promoter region (from about position -40 to the tss of nifB1) in the nifB hybrid promoter supported expression in heterocysts but did not prevent the mostly nifB2 promoter from also functioning in anoxic vegetative cells. In the filamentous cyanobacterium Anabaena variabilis, two Mo nitrogenase gene clusters, nif1 and nif2, function under different environmental conditions in different cell types. Little is known about the regulation of transcription from the promoter upstream of the first gene of the cluster, which drives transcription of each of these two large operons. The similarity in the sequences upstream of the primary promoters for the two nif gene clusters belies the differences in their expression patterns. Analysis of these nif promoters in strains with mutations in the conserved sequences and in strains with hybrid promoters, comprising parts from nif1 and nif2, provides strong evidence that each promoter has key elements required for cell-type-specific expression of the nif1 and nif2 gene clusters.

Dataset Information

Role of RNA secondary structure and processing in stability of the nifH1 transcript in the cyanobacterium Anabaena variabilis.

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Importance

Publications

Role of RNA secondary structure and processing in stability of the nifH1 transcript in the cyanobacterium Anabaena variabilis.

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