Elvira Olmedo-Verd

In the absence of fixed nitrogen, some filamentous cyanobacteria differentiate heterocysts, specialized cells devoted to fixing atmospheric nitrogen (N₂). This differentiation process is controlled by the global nitrogen regulator NtcA and involves extensive metabolic reprogramming, including shutdown of photosynthetic CO₂ fixation in heterocysts, to provide a microaerobic environment suitable for N₂ fixation. Small regulatory RNAs (sRNAs) are major post-transcriptional regulators of gene expression in bacteria. In cyanobacteria, responding to nitrogen deficiency involves transcribing several nitrogen-regulated sRNAs. Here, we describe the participation of nitrogen stress-inducible RNA 4 (NsiR4) in post-transcriptionally regulating the expression of two genes involved in CO₂ fixation via the Calvin cycle: glpX, which encodes bifunctional sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (SBPase), and pgk, which encodes phosphoglycerate kinase (PGK). Using a heterologous reporter assay in Escherichia coli, we show that NsiR4 interacts with the 5′-untranslated region (5′-UTR) of glpX and pgk mRNAs. Overexpressing NsiR4 in Nostoc sp. PCC 7120 resulted in a reduced amount of SBPase protein and reduced PGK activity, as well as reduced levels of both glpX and pgk mRNAs, further supporting that NsiR4 negatively regulates these two enzymes. In addition, using a gfp fusion to the nsiR4 promoter, we show stronger expression of NsiR4 in heterocysts than in vegetative cells, which could contribute to the heterocyst-specific shutdown of Calvin cycle flux. Post-transcriptional regulation of two Calvin cycle enzymes by NsiR4, a nitrogen-regulated sRNA, represents an additional link between nitrogen control and CO₂ assimilation.

NsiR3 (nitrogen‐stress induced RNA 3) is a small non‐coding RNA strongly conserved in heterocyst‐forming cyanobacteria. In Nostoc sp. PCC 7120, transcription of NsiR3 is induced by nitrogen starvation and depends on the global nitrogen regulator NtcA. A conserved NtcA binding site is centered around position ‐42.5 with respect to the transcription start site of NsiR3 homologs, and NtcA binds in vitro to a DNA fragment containing this sequence. In the absence of combined nitrogen NsiR3 expression is induced in all cells along the Nostoc filament but much more strongly in heterocysts, differentiated cells devoted to nitrogen fixation. Co‐expression analysis of transcriptomic data obtained from microarrays hybridized with RNA obtained from Nostoc wild type or mutant strains grown in the presence of ammonium or in the absence of combined nitrogen revealed that the expression profile of gene putA (proline oxidase) correlates negatively with that of NsiR3. Using a heterologous system in Escherichia coli, we show that NsiR3 binds to the 5’‐UTR of putA mRNA, resulting in reduced expression of a reporter gene. Overexpression of NsiR3 in Nostoc resulted in strong reduction of putA mRNA accumulation, further supporting the negative regulation of putA by NsiR3. The higher expression of NsiR3 in heterocysts versus vegetative cells of the N₂‐fixing filament could contribute to the previously described absence of putA mRNA and of the catabolic pathway to produce glutamate from arginine via proline specifically in heterocysts. Post‐transcriptional regulation by NsiR3 represents an indirect NtcA‐operated regulatory mechanism of putA expression.

Zur regulators control zinc homeostasis by repressing target genes under zinc-sufficient conditions in a wide variety of bacteria. This paper describes how part of a survey of duplicated genes led to the identification of the open reading frame all2473 as the gene encoding the Zur regulator of the cyanobacterium Anabaena sp. strain PCC 7120. All2473 binds to DNA in a zinc-dependent manner, and its DNA-binding sequence was characterized, which allowed us to determine the relative contribution of particular nucleotides to Zur binding. A zur mutant was found to be impaired in the regulation of zinc homeostasis, showing sensitivity to elevated concentrations of zinc but not other metals. In an effort to characterize the Zur regulon in Anabaena, 23 genes containing upstream putative Zur-binding sequences were identified and found to be regulated by Zur. These genes are organized in six single transcriptional units and six operons, some of them containing multiple Zur-regulated promoters. The identities of genes of the Zur regulon indicate that Anabaena adapts to conditions of zinc deficiency by replacing zinc metalloproteins with paralogues that fulfill the same function but presumably with a lower zinc demand, and with inducing putative metallochaperones and membrane transport systems likely being involved in the scavenging of extracellular zinc, including plasma membrane ABC transport systems and outer membrane TonB-dependent receptors. Among the Zur-regulated genes, the ones showing the highest induction level encode proteins of the outer membrane, suggesting a primary role for components of this cell compartment in the capture of zinc cations from the extracellular medium.

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that fixes N ₂ in specialized cells called heterocysts, which differentiate from vegetative cells in a process that requires the nitrogen control transcription factor NtcA. 2-Oxoglutarate-stimulated binding of purified NtcA to wild-type and modified versions of the ntcA gene promoter from Anabaena sp. was analyzed by mobility shift and DNase I footprinting assays, and the role of NtcA-binding sites in the expression of the ntcA gene during heterocyst differentiation was studied in vivo by using an ntcA-gfp translational fusion and primer extension analysis. Mutation of neither of the two identified NtcA-binding sites eliminated localized expression of ntcA in proheterocysts, but mutation of both sites led to very low, nonlocalized expression.

The ntcA gene encodes an N-control transcriptional regulator in cyanobacteria. In the N(2)-fixing, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, ntcA is an autoregulatory gene that is transcribed from a complex promoter region that includes a constitutive promoter (P(2)) and promoters that are induced upon N step-down (P(1) and P(3)). Expression of ntcA was investigated with the use of an ntcA-gfp translational fusion, which was introduced both in the natural ntcA locus and in a heterologous genomic place. Induction of ntcA-gfp took place after N step-down in all the cells of the filament, but at especially high levels in developing heterocysts. Localized induction could be driven independently by P(3) and P(1).

All4312, encoded by open reading frame all4312 in the genome of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, exhibits a CheY-l

Heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120 depends on both the global nitrogen control transcription factor NtcA and the cell differentiation regulatory protein HetR, with expression of nt cA and hetR being dependent on each other. In this study we constructed strains that constitutively express the nt cA gene leading to high levels of NtcA protein irrespective of the nitrogen source, and we analyzed the effects of such NtcA levels on heterocyst differentiation. In the NtcA-overproducing strain, heterocyst differentiation, induction of NtcA-dependent heterocyst development genes or operons such as devBCA or the cox2 operon, and NtcA-dependent excision of the 11-kb nifD -intervening element only took place under nitrogen deficiency. Although functional heterocysts were produced in response to nitrogen step-down, the NtcA overproducing strain could not grow diazotrophically. Overexpression of ntcA in a hetR background promoted expression of devBCA in response to ammonium withdrawal and excision of the 11-kb element even in the presence of combined nitrogen. Our results show that some NtcA-dependent heterocyst-related genes can be expressed independently of HetR.