bZur 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.

The ntcA gene encodes an N-control transcriptional regulator in cyanobacteria. In the N 2 -fixing, heterocystforming 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 .Some filamentous cyanobacteria, such as those of the genera Anabaena and Nostoc, develop heterocysts, which are cells specialized in N 2 fixation (9,11,17,31). The initiation of heterocyst differentiation is dependent on the NtcA transcription factor, which is a global regulator of nitrogen assimilation (8,10,11,30), as well as on other early acting developmental regulatory genes, such as hetR (3, 4). The hetR gene is induced after N step-down in an NtcA-dependent manner (8,21). The ntcA gene is autoregulatory, and induction of ntcA in Anabaena sp. strain PCC 7120 takes place after N step-down (21, 24). NtcA binds to DNA sites bearing the sequence signature GTAN 8 TAC (10, 13), and the activity of NtcA as a transcriptional regulator is positively modulated by 2-oxoglutarate, a metabolite that lies at the link of C and N metabolisms in cyanobacteria (12,14,22,26,28,29). Induction of ntcA also requires the action of HetR (21). Although HetR dependency is suggestive of induction in heterocysts or developing heterocysts (proheterocysts), localized induction has not been demonstrated for ntcA.The ntcA gene is transcribed from a complex promoter region that gives rise to three transcription start points (TSPs) (21, 24). Promoter P 1 produces a TSP at Ϫ49 (that is, 49 nucleotides upstream from the translational start of ntcA), P 2 produces a TSP at Ϫ136, and P 3 produces a TSP at Ϫ180. P 2 functions constitutively, i.e., independent of the N source. However, an NtcA binding site of sequence GTAN 8 AAC, which is centered at Ϫ143.5 and has been footprinted with DNase I (24), overlaps the Ϫ10 hexamer of this promoter (21). P 1 also functions in filaments grown with different N sources, but its utilization increases after 6 to 12 h of N deprivation in an NtcA-and HetR-dependent manner, and it is very strong in heterocysts. P 3 functions only in the absence of combined N, being transiently induced after 6 to 12 h of N deprivation in an NtcA-and HetR-dependent manner (21). A second putative NtcA binding site, which exhibits a perfect NtcA sequence signature, GTAN 8 TAC, is found in the ntcA promoter region centered at Ϫ103.5. Although this NtcA binding site did not show up in DNase I footprinting analysis (24), two retarded bands have been observed in band shift assays with NtcA and a DNA fragment of th...

Summary Nitrogen is frequently limiting microbial growth in the environment. As a response, many filamentous cyanobacteria differentiate heterocysts, cells devoted to N2 fixation. Heterocyst differentiation is under the control of the master regulator HetR. Through the characterization of the HetR‐dependent transcriptome in Nostoc sp. PCC 7120, we identified the new candidate genes likely involved in heterocyst differentiation. According to their maximum induction, we defined E‐DIF (early in differentiation) and L‐DIF (late in differentiation) genes. Most of the genes known to be involved in the critical aspects of heterocyst differentiation or function were also classified into these groups, showing the validity of the approach. Using fusions to gfp, we verified the heterocyst‐specific transcription of several of the found genes, antisense transcripts and potentially trans‐acting sRNAs. Through comparative sequence analysis of promoter regions, we noticed the prevalence of the previously described DIF1 motif and identified a second motif, called DIF2, in other promoters of the E‐DIF cluster. Both motifs are widely conserved in heterocystous cyanobacteria. We assigned alr2522 as a third member, besides nifB and nifP, to the CnfR regulon. The elements identified here are of interest for understanding cell differentiation, engineering of biological nitrogen fixation or production of O2‐sensitive molecules in cyanobacteria.

Small RNAs (sRNAs) are a growing class of non-protein-coding transcripts that participate in the regulation of virtually every aspect of bacterial physiology. Heterocystous cyanobacteria are a group of photosynthetic organisms that exhibit multicellular behavior and developmental alternatives involving specific transcriptomes exclusive of a given physiological condition or even a cell type. In the context of our ongoing effort to understand developmental decisions in these organisms we have undertaken an approach to the global identification of sRNAs. Using differential RNA-Seq we have previously identified transcriptional start sites for the model heterocystous cyanobacterium Nostoc sp. PCC 7120. Here we combine this dataset with a prediction of Rho-independent transcriptional terminators and an analysis of phylogenetic conservation of potential sRNAs among 89 available cyanobacterial genomes. In contrast to predictive genome-wide approaches, the use of an experimental dataset comprising all active transcriptional start sites (differential RNA-Seq) facilitates the identification of bona fide sRNAs. The output of our approach is a dataset of predicted potential sRNAs in Nostoc sp. PCC 7120, with different degrees of phylogenetic conservation across the 89 cyanobacterial genomes analyzed. Previously described sRNAs appear among the predicted sRNAs, demonstrating the performance of the algorithm. In addition, new predicted sRNAs are now identified that can be involved in regulation of different aspects of cyanobacterial physiology, including adaptation to nitrogen stress, the condition that triggers differentiation of heterocysts (specialized nitrogen-fixing cells). Transcription of several predicted sRNAs that appear exclusively in the genomes of heterocystous cyanobacteria is experimentally verified by Northern blot. Cell-specific transcription of one of these sRNAs, NsiR8 (nitrogen stress-induced RNA 8), in developing heterocysts is also demonstrated.

All4312, encoded by open reading frame all4312 in the genome of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, exhibits a CheY-like receiver domain and an output domain similar to that of OmpR, characteristic of two-component response regulators. Expression of all4312 was directly regulated by NtcA, the global transcriptional regulator of nitrogen assimilation in cyanobacteria. Features characteristic of NtcA-activated promoters were also found upstream from genes encoding All4312 homologues in several other cyanobacterial genomes. Expression of all4312 was however unaffected in a mutant of hetR, which encodes a regulator triggering heterocyst development. The function of All4312 may be related to the cellular response to nitrogen deprivation.

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 ntcA and hetR being dependent on each other. In this study we constructed strains that constitutively express the ntcA 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.

Background: Aminoacyl-tRNA synthetases (aaRS) are enzymes that couple tRNAs to their cognate amino acids. Results: aaRSs from cyanobacteria containing the novel CAAD protein domain are localized in the thylakoid membrane. Conclusion: Confinement of aaRSs to the membrane is advantageous for cyanobacteria under specific conditions. Significance: These findings provide the first description of membrane-bound aaRSs.

Upon nitrogen deficiency, some filamentous cyanobacteria differentiate specialized cells, called heterocysts, devoted to N2 fixation. Heterocysts appear regularly spaced along the filaments and exhibit structural and metabolic adaptations, such as loss of photosynthetic CO2 fixation or increased respiration, to provide a proper microaerobic environment for its specialized function. Heterocyst development is under transcriptional control of the global nitrogen regulator NtcA and the specific regulator HetR. Transcription of a large number of genes is induced or repressed upon nitrogen deficiency specifically in cells undergoing differentiation. In recent years, the HetR regulon has been described to include heterocyst-specific trans-acting small RNAs and antisense RNAs (asRNAs), suggesting that there is an additional layer of post-transcriptional regulation involved in heterocyst development. Here, we characterize in the cyanobacterium Nostoc (Anabaena) sp. PCC 7120 an asRNA, that we call as_glpX, transcribed within the glpX gene encoding the Calvin cycle bifunctional enzyme sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (SBPase). Transcription of as_glpX is restricted to heterocysts and is induced very early during the process of differentiation. Expression of as_glpX RNA promotes the cleavage of the glpX mRNA by RNase III, resulting in a reduced amount of SBPase. Therefore, the early expression of this asRNA could contribute to the quick shut-down of CO2 fixation in those cells in the filament that are undergoing differentiation into heterocysts. In summary, as_glpX is the first naturally occurring asRNA shown to rapidly and dynamically regulate metabolic transformation in Nostoc heterocysts. The use of antisense transcripts to manipulate gene expression specifically in heterocysts could became a useful tool for metabolic engineering in cyanobacteria.