Role of the nifB1 and nifB2 Promoters in Cell-Type-Specific Expression of Two Mo Nitrogenases in the Cyanobacterium Anabaena variabilis ATCC 29413

Abstract

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 Ϫ40 to the tss of nifB1 was expressed in heterocysts and in anoxic vegetative cells. Thus, addition of the nifB1 promoter region (from about position Ϫ40 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. IMPORTANCE 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.KEYWORDS Anabaena, cyanobacteria, heterocysts, nitrogenase, regulation, transcriptional regulation F ilamentous heterocystous cyanobacteria fix atmospheric nitrogen under oxic growth conditions in specialized cells called heterocysts that differentiate in a semiregular pattern in a filament. They comprise about 5 to 10% of the cells in filaments in cultures grown in an environment that is deficient in fixed nitrogen (1-5). Heterocysts are microoxic; they have a glycolipid layer that restricts oxygen diffusion into the cell, lack components of oxygen-evolving photosystem II, and have high levels of respiration, which together protect the oxygen-labile nitrogenase from oxygen (6-10).

Elements of the heterocyst‐specific transcriptome unravelled by co‐expression analysis in Nostoc sp. PCC 7120

“…In contrast to these findings for the E‐DIF cluster, the comparison of promoters in the L‐DIF cluster only led to the identification of conserved motifs that corresponded to the sequences associated to the CnfR (a.k.a. PatB, alr2512 , also in the L‐DIF cluster)‐regulated expression of the nifB promoter of Anabaena variabilis ATCC 29413 (Vernon et al ., ) and the nifB and nifP promoters of the non‐heterocystous cyanobacterium Leptolyngbya boryana (Tsujimoto et al ., ) (Supporting Information Fig. S9A).…”

“…The slightly different temporal profiles observed for induction of L‐DIF elements, likely due to the participation of several different regulators, is consistent with a limited identification of conserved promoter elements in this group. Nevertheless, the array of conserved sequences defined as CnfR‐regulated promoters for nifB (Vernon et al ., ) and nifP (Tsujimoto et al ., ) could also be identified in the promoter driving transcription of another gene cluster with late DIF profile ( alr2522‐alr2524 ), suggesting these genes might also be under the control of CnfR in Nostoc sp. PCC 7120.…”

“…Mutational analysis clearly demonstrated the connection between this DIF2 motif and heterocyst‐specific expression. Finally, a previously defined motif mediating the CnfR‐regulated expression of a promoter upstream of nifB (Vernon et al ., ) or nifP (Tsujimoto et al ., ) was also identified in a third transcriptional unit in the L‐DIF group. Our results show that substantially more genes, elements and factors might be involved in the genetic program that leads to the differentiation of cyanobacterial heterocysts than anticipated so far.…”

Developmentally Regulated Genome Editing in Terminally Differentiated N2-Fixing Heterocysts ofAnabaena cylindricaATCC 29414

“…Much research has demonstrated that nitrogen fixation in diazotrophic bacteria is tightly regulated at the transcriptional level 30, 31, 32, 33, 34, 35, 36 . Here, we along with previous studies 21, 22, 23, 37 demonstrate that nitrogen fixation in heterocyst-forming cyanobacteria is also developmentally regulated at the genomic level.…”

Regulatory systems for gene expression control in cyanobacteria

“…Another vegetative cell-specific promoter is PnifB2 from Av_29413. It controls the expression of a second nitrogenase in Av_29413, which, in contrast to the above-described enzyme controlled by PnifB1, is exclusively produced in vegetative cells during anaerobic conditions (Thiel et al 1995;Vernon et al 2017). Moreover, an expression system specific for prospective and immature heterocysts was reported in A_7120 (Muro-Pastor 2014).…”