Antisense RNA regulates glutamine synthetase in a heterocyst-forming cyanobacterium.

Glutamine synthetase (GS) is a key enzyme involved in nitrogen assimilation and the maintenance of C/N balance, and it is strictly regulated in all bacteria. In cyanobacteria, GS expression is controlled by nitrogen control A (NtcA) transcription factor, which operates global nitrogen regulation in these photosynthetic organisms. Furthermore, posttranslational regulation of GS is operated by protein-protein interaction with GS inactivating factors (IFs). In this study, we describe an additional regulatory mechanism involving an antisense RNA. In Nostoc sp. PCC 7120, the gifA gene (encoding GS inactivating factor IF7) is transcribed downstream of the GS (glnA) gene, from the opposite strand, and the gifA mRNA extends into the glnA coding sequence in antisense orientation. Therefore, the dual RNA transcript that encodes gifA constitutes two functional regions: a 5' protein-coding region, encoding IF7, and a 3' untranslated region that acts as an antisense to glnA. By increasing the levels of such antisense RNA either in cis or in trans, we demonstrate that the amount of GS activity can be modulated by the presence of the antisense RNA. The tail-to-tail disposition of the glnA and gifA genes observed in many cyanobacterial strains from the Nostocales clade suggests the prevalence of such antisense RNA-mediated regulation of GS in this group of cyanobacteria.

A nitrogen stress-inducible small RNA regulates CO2 fixation in Nostoc.

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.

NsiR1, a small RNA with multiple copies, modulates heterocyst differentiation in the cyanobacterium Nostoc sp. PCC 7120.

Upon nitrogen starvation, filamentous cyanobacteria develop heterocysts, specialized cells devoted to the fixation of atmospheric nitrogen. Differentiation of heterocyst at semi-regular intervals along the filaments requires complex structural and functional changes that are under the control of the master transcriptional regulator HetR. NsiR1 (nitrogen stress-induced RNA 1) is a HetR-dependent non-coding RNA that is expressed specifically in heterocysts from a very early stage of differentiation. In the genome of Nostoc sp. PCC 7120 there are 12 tandem copies of nsiR1 (nsiR1.1 to nsiR1.12), seven of them with identical sequence (nsiR1.3 to nsiR1.9) and the others slightly divergent. nsiR1.1 is transcribed antisense to the 5' UTR of hetF, a gene required for heterocyst development. Here, we show that binding of NsiR1.1 inhibits translation of the hetF mRNA by inducing structural changes in its 5' UTR. Altered levels of NsiR1 result in different phenotypic alterations including enlarged cell size and delayed heterocyst development that could be related to a reduced amount of HetF.

A Heterocyst-Specific Antisense RNA Contributes to Metabolic Reprogramming in Nostoc sp. PCC 7120.

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.

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

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.

Dynamics of transcriptional start site selection during nitrogen stress-induced cell differentiation in Anabaena sp. PCC7120.

The fixation of atmospheric N(2) by cyanobacteria is a major source of nitrogen in the biosphere. In Nostocales, such as Anabaena, this process is spatially separated from oxygenic photosynthesis and occurs in heterocysts. Upon nitrogen step-down, these specialized cells differentiate from vegetative cells in a process controlled by two major regulators: NtcA and HetR. However, the regulon controlled by these two factors is only partially defined, and several aspects of the differentiation process have remained enigmatic. Using differential RNA-seq, we experimentally define a genome-wide map of >10,000 transcriptional start sites (TSS) of Anabaena sp. PCC7120, a model organism for the study of prokaryotic cell differentiation and N(2) fixation. By analyzing the adaptation to nitrogen stress, our global TSS map provides insight into the dynamic changes that modify the transcriptional organization at a critical step of the differentiation process. We identify >900 TSS with minimum fold change in response to nitrogen deficiency of eight. From these TSS, at least 209 were under control of HetR, whereas at least 158 other TSS were potentially directly controlled by NtcA. Our analysis of the promoters activated during the switch to N(2) fixation adds hundreds of protein-coding genes and noncoding transcripts to the list of potentially involved factors. These data experimentally define the NtcA regulon and the DIF(+) motif, a palindrome at or close to position -35 that seems essential for heterocyst-specific expression of certain genes.

RNase P RNA from the recently evolved plastid of Paulinella and from algae.

The RNase P RNA catalytic subunit (RPR) encoded in some plastids has been found to be functionally defective. The amoeba Paulinella chromatophora contains an organelle (chromatophore) that is derived from the recent endosymbiotic acquisition of a cyanobacterium, and therefore represents a model of the early steps in the acquisition of plastids. In contrast with plastid RPRs the chromatophore RPR retains functionality similar to the cyanobacterial enzyme. The chromatophore RPR sequence deviates from consensus at some positions but those changes allow optimal activity compared with mutated chromatophore RPR with the consensus sequence. We have analyzed additional RPR sequences identifiable in plastids and have found that it is present in all red algae and in several prasinophyte green algae. We have assayed in vitro a subset of the plastid RPRs not previously analyzed and confirm that these organelle RPRs lack RNase P activity in vitro.

Nitrogen-regulated antisense transcription in the adaptation to nitrogen deficiency in Nostoc sp. PCC 7120.

Transcriptomic analyses using high-throughput methods have revealed abundant antisense transcription in bacteria. Antisense transcription is often due to the overlap of mRNAs with long 5' or 3' regions that extend beyond the coding sequence. In addition, antisense RNAs that do not contain any coding sequence are also observed. Nostoc sp. PCC 7120 is a filamentous cyanobacterium that, under nitrogen limitation, behaves as a multicellular organism with division of labor among two different cell types that depend on each other, the vegetative CO2-fixing cells and the nitrogen-fixing heterocysts. The differentiation of heterocysts depends on the global nitrogen regulator NtcA and requires the specific regulator HetR. To identify antisense RNAs potentially involved in heterocyst differentiation, we assembled the Nostoc transcriptome using RNA-seq analysis of cells subjected to nitrogen limitation (9 or 24 h after nitrogen removal) in combination with a genome-wide set of transcriptional start sites and a prediction of transcriptional terminators. Our analysis resulted in the definition of a transcriptional map that includes >4,000 transcripts, 65% of which contain regions in antisense orientation to other transcripts. In addition to overlapping mRNAs, we identified nitrogen-regulated noncoding antisense RNAs transcribed from NtcA- or HetR-dependent promoters. As an example of this last category, we further analyzed an antisense (as_gltA) of the gene-encoding citrate synthase and showed that transcription of as_gltA takes place specifically in heterocysts. Since the overexpression of as_gltA reduces citrate synthase activity, this antisense RNA could eventually contribute to the metabolic remodeling that occurs during the differentiation of vegetative cells into heterocysts.

The Heterocyst-Specific Small RNA NsiR1 Regulates the Commitment to Differentiation in Nostoc.

Heterocysts are specialized cells that filamentous cyanobacteria differentiate for the fixation of atmospheric nitrogen when other nitrogen sources are not available. Heterocyst differentiation at semiregular intervals along the filaments requires complex structural and metabolic changes that are under the control of the master transcriptional regulator HetR. NsiR1 (nitrogen stress-induced RNA 1) is a HetR-dependent noncoding RNA that is expressed from multiple chromosomal copies, some identical, some slightly divergent in sequence, specifically in heterocysts from very early stages of differentiation. We have previously shown that NsiR1 inhibits translation of the overlapping hetF mRNA by an antisense mechanism. Here, we identify alr3234, a hetP-like gene involved in the regulation of commitment (point of no return) to heterocyst differentiation, as a target of NsiR1. A strain overexpressing one of the identical copies of NsiR1 commits to heterocyst development earlier than the wild type. The posttranscriptional regulation exerted by NsiR1 on the expression of two genes involved in heterocyst differentiation and commitment, hetF and alr3234, adds a new level of complexity to the network of transcriptional regulation and protein-protein interactions that participate in heterocyst differentiation. IMPORTANCE Heterocysts are nitrogen-fixing specialized cells that appear at semiregular intervals along cyanobacterial filaments upon nitrogen starvation. The differentiation and patterning of heterocysts is a model for the study of cell differentiation in multicellular prokaryotes. The regulation of differentiation, which is only partially understood, includes transcriptional changes, factor diffusion between cells, and protein-protein interactions. This work describes the identification of a novel target for NsiR1, a small RNA (sRNA) encoded in multiple slightly divergent copies, and shows how different copies of "sibling" sRNAs regulate the expression of different targets involved in one of the few examples of a differentiation process in prokaryotes.

Hfq is required for optimal nitrate assimilation in the Cyanobacterium Anabaena sp. strain PCC 7120.

Hfq is an RNA binding protein involved in posttranscriptional regulation of gene expression in bacteria. It acts by binding to regulatory small RNAs (sRNAs), which confer specificity for the regulation. Recently, orthologues of the Hfq protein were annotated in cyanobacterial genomes, although its capacity to regulate gene expression by interacting with sRNAs has not been yet demonstrated. Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that, in the absence of combined nitrogen, is able to fix atmospheric nitrogen by differentiating specialized cells called heterocysts. We have generated an hfq knockout mutant of Anabaena sp. PCC 7120. Deletion of this gene results in differentiation of heterocysts in the presence of nitrate, suggesting a defect in nitrate assimilation. We show that hfq mutant cells are affected in transport and use of nitrate and nitrite. An analysis of the expression of several genes in the nir operon, encoding different elements of the nitrate assimilation pathway, demonstrates a downregulation of their transcription in mutant cells. We also observed that genes ntcB and cnaT, involved in the regulation of the nir operon, show a lower expression in cells lacking Hfq. Finally, when hfq was reintroduced in the mutant, heterocyst differentiation was no longer observed in the presence of nitrate. Therefore, our results indicate that the RNA chaperone Hfq is involved in the regulation of the nir operon, although the mechanism for this regulation is still unknown.

A functional RNase P protein subunit of bacterial origin in some eukaryotes.

RNase P catalyzes 5'-maturation of tRNAs. While bacterial RNase P comprises an RNA catalyst and a protein cofactor, the eukaryotic (nuclear) variant contains an RNA and up to ten proteins, all unrelated to the bacterial protein. Unexpectedly, a nuclear-encoded bacterial RNase P protein (RPP) homolog is found in several prasinophyte algae including Ostreococcus tauri. We demonstrate that recombinant O. tauri RPP can functionally reconstitute with bacterial RNase P RNAs (RPRs) but not with O. tauri organellar RPRs, despite the latter's presumed bacterial origins. We also show that O. tauri PRORP, a homolog of Arabidopsis PRORP-1, displays tRNA 5'-processing activity in vitro. We discuss the implications of the striking diversity of RNase P in O. tauri, the smallest known free-living eukaryote.

NsiR3, a nitrogen stress-inducible small RNA, regulates proline oxidase expression in the cyanobacterium Nostoc sp. PCC 7120.

NsiR3 (nitrogen stress-inducible RNA 3) is a small noncoding 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. DATABASE: Microarray data are available in GEO database under accession numbers GSE120377 and GSE150191.

The Integrity of the Cell Wall and Its Remodeling during Heterocyst Differentiation Are Regulated by Phylogenetically Conserved Small RNA Yfr1 in Nostoc sp. Strain PCC 7120.

Yfr1 is a strictly conserved small RNA in cyanobacteria. A bioinformatic prediction to identify possible interactions of Yfr1 with mRNAs was carried out by using the sequences of Yfr1 from several heterocyst-forming strains, including Nostoc sp. strain PCC 7120. The results of the prediction were enriched in genes encoding outer membrane proteins and enzymes related to peptidoglycan biosynthesis and turnover. Heterologous expression assays with Escherichia coli demonstrated direct interactions of Yfr1 with mRNAs of 11 of the candidate genes. The expression of 10 of them (alr2458, alr4550, murC, all4829, all2158, mraY, alr2269, alr0834, conR, patN) was repressed by interaction with Yfr1, whereas the expression of amiC2, encoding an amidase, was increased. The interactions between Yfr1 and the 11 mRNAs were confirmed by site-directed mutagenesis of Yfr1. Furthermore, a Nostoc strain with reduced levels of Yfr1 had larger amounts of mraY and murC mRNAs, supporting a role for Yfr1 in the regulation of those genes. Nostoc strains with either reduced or increased expression of Yfr1 showed anomalies in cell wall completion and were more sensitive to vancomycin than the wild-type strain. Furthermore, growth in the absence of combined nitrogen, which involves the differentiation of heterocysts, was compromised in the strain overexpressing Yfr1, and filaments were broken at the connections between vegetative cells and heterocysts. These results indicate that Yfr1 is an important regulator of cell wall homeostasis and correct cell wall remodeling during heterocyst differentiation.IMPORTANCE Bacterial small RNAs (sRNAs) are important players affecting the regulation of essentially every aspect of bacterial physiology. The cell wall is a highly dynamic structure that protects bacteria from their fluctuating environment. Cell envelope remodeling is particularly critical for bacteria that undergo differentiation processes, such as spore formation or differentiation of heterocysts. Heterocyst development involves the deposition of additional layers of glycolipids and polysaccharides outside the outer membrane. Here, we show that a cyanobacterial phylogenetically conserved small regulatory RNA, Yfr1, coordinates the expression of proteins involved in cell wall-related processes, including peptidoglycan metabolism and transport of different molecules, as well as expression of several proteins involved in heterocyst differentiation.

The Nitrogen Stress-Repressed sRNA NsrR1 Regulates Expression of all1871, a Gene Required for Diazotrophic Growth in Nostoc sp. PCC 7120.

Small regulatory RNAs (sRNAs) are post-transcriptional regulators of bacterial gene expression. In cyanobacteria, the responses to nitrogen availability, that are mostly controlled at the transcriptional level by NtcA, involve also at least two small RNAs, namely NsiR4 (nitrogen stress-induced RNA 4) and NsrR1 (nitrogen stress-repressed RNA 1). Prediction of possible mRNA targets regulated by NsrR1 in Nostoc sp. PCC 7120 allowed, in addition to previously described nblA, the identification of all1871, a nitrogen-regulated gene encoding a protein of unknown function that we describe here as required for growth at the expense of atmospheric nitrogen (N2). We show that transcription of all1871 is induced upon nitrogen step-down independently of NtcA. All1871 accumulation is repressed by NsrR1 and its expression is stronger in heterocysts, specialized cells devoted to N2 fixation. We demonstrate specific interaction between NsrR1 and the 5' untranslated region (UTR) of the all1871 mRNA, that leads to decreased expression of all1871. Because transcription of NsrR1 is partially repressed by NtcA, post-transcriptional regulation by NsrR1 would constitute an indirect way of NtcA-mediated regulation of all1871.

NsrR1, a Nitrogen Stress-Repressed sRNA, Contributes to the Regulation of nblA in Nostoc sp. PCC 7120.

Small regulatory RNAs (sRNAs) are currently considered as major post-transcriptional regulators of gene expression in bacteria. The interplay between sRNAs and transcription factors leads to complex regulatory networks in which both transcription factors and sRNAs may appear as nodes. In cyanobacteria, the responses to nitrogen availability are controlled at the transcriptional level by NtcA, a CRP/FNR family regulator. In this study, we describe an NtcA-regulated sRNA in the cyanobacterium Nostoc sp. PCC 7120, that we have named NsrR1 (nitrogen stress repressed RNA1). We show sequence specific binding of NtcA to the promoter of NsrR1. Prediction of possible mRNA targets regulated by NsrR1 allowed the identification of nblA, encoding a protein adaptor for phycobilisome degradation under several stress conditions, including nitrogen deficiency. We demonstrate specific interaction between NsrR1 and the 5'-UTR of the nblA mRNA, that leads to decreased expression of nblA. Because both NsrR1 and NblA are under transcriptional control of NtcA, this regulatory circuit constitutes a coherent feed-forward loop, involving a transcription factor and an sRNA.

tRNase Z.

Endonuclease tRNase Z catalyzes the generation of the mature 3' end of tRNA precursors through specific endonucleolytic cleavage. The enzyme has been characterized from organisms representative of all domains of life as well as from organelles, and the crystal structure of three bacterial enzymes has been determined. This review presents an overview of its properties and what is known about its structure, substrate recognition, cleavage site definition, and potential practical applications.

Transformation of cyanobacteria.

Cyanobacteria are a diverse and successful group of bacteria defined by their ability to carry out oxygenic photosynthesis. They occupy diverse ecological niches and are important primary producers in the oceans. Cyanobacteria are amenable to genetic manipulation. Some strains are naturally transformable. Many others have been transformed in the lab by conjugation or electroporation. The ability to transform cyanobacteria has been determinant in the development of the molecular biology of these organisms and has been the basis of many of their biotechnological applications. Cyanobacteria are the source of natural products and toxins of potential use and can be engineered to synthesize substances of biotechnological interest. Their high protein and vitamin content makes them useful as a dietary supplement. Because of their ability to occupy diverse ecological niches, they can be used to deliver to the medium substances of interest or as biosensors.

A combinatorial strategy of alternative promoter use during differentiation of a heterocystous cyanobacterium.

Heterocystous cyanobacteria such as Nostoc sp. are filamentous photosynthetic organisms that, in response to nitrogen deficiency, undergo a differentiation process transforming certain, semi-regularly spaced cells into heterocysts, devoted to nitrogen fixation. During transition to a nitrogen-fixing regime, growth of most vegetative cells in the filament is temporarily arrested due to nutritional deprivation, but developing heterocysts require intense transcriptional activity. Therefore, the coexistence of arrested vegetative cells and actively developing prospective heterocysts relies on the simultaneous operation of somewhat opposite transcriptional programs. We have identified genes with multiple nitrogen-responsive transcriptional starts appearing in seemingly paradoxical combinations. For instance, sigA, encoding the RNA polymerase housekeeping sigma factor, is transcribed from one major nitrogen stress-repressed promoter and from a second, nitrogen stress-induced promoter. Here, we show that both promoters are expressed with complementary temporal dynamics. Using a gfp reporter we also show that transcription from the inducible promoter takes place exclusively in differentiating heterocysts and is already detected before any morphological or fluorescence signature of differentiation is observed. Tandem promoters with opposite dynamics could operate a compensatory mechanism in which repression of transcription from the major promoter operative in vegetative cells is offset by transcription from a new promoter only in developing heterocyst.

Identification of Conserved and Potentially Regulatory Small RNAs in Heterocystous 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.

Functional in situ evaluation of photosynthesis-protecting carotenoids in mutants of the cyanobacterium Synechocystis PCC6803.

Cyanobacteria possess different carotenoids as scavengers of reactive oxygen species. In Synechocystis PCC6803, zeaxanthin, echinenone, beta-carotene and myxoxanthophyll are synthesized. By disruption of the ketolase and hydroxylase genes, it was possible to obtain mutants devoid of either zeaxanthin, echinenone, or a combination of both carotenoids. With these mutants, their function in protecting photosynthetic electron transport under high light stress as well as chlorophyll and carotenoid degradation after initiation of singlet oxygen or radical formation was analyzed. Wild type Synechocystis is very well protected against high light-mediated photooxidation. Absence of echinenone affects photosynthetic electron transport to only a small extent. However, complete depletion of zeaxanthin together with a modification of myxoxanthophyll resulted in strong photoinhibition of overall photosynthetic electron transport as well as the photosystem II reaction. In the double mutant lacking both carotenoids the effects were additive. The light saturation curves of photosynthetic electron transport of the high light-treated mutants exhibited not only a lower saturation value but also smaller slopes. Using methylviologen or methylene blue as a radical or singlet oxygen generators, respectively, massive degradation of chlorophyll and carotenoids, indicative of photooxidative destruction of the photosynthetic apparatus, was observed, especially in the mutants devoid of zeaxanthin.