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, glutamine synthetase expression is controlled by nitrogen control A (NtcA) transcription factor, which operates global nitrogen regulation in these photosynthetic organisms. Furthermore, post-translational regulation of GS is operated by protein-protein interaction with GS inactivating factors (IFs). Here, we describe an additional regulatory mechanism involving an antisense RNA. In Nostoc sp. PCC 7120, the GS inactivating factor A (gifA) gene (encoding GS inactivating factor IF7) is transcribed downstream of the glutamine synthetase (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.

Quantitative Proteomics at Early Stages of the Symbiotic Interaction Between Oryza sativa and Nostoc punctiforme Reveals Novel Proteins Involved in the Symbiotic Crosstalk.

Symbiosis between cyanobacteria and plants is considered pivotal for biological nitrogen deposition in terrestrial ecosystems. Despite extensive knowledge of the ecology of plant-cyanobacterium symbioses, little is known about the molecular mechanisms involved in recognition between partners. Here, we conducted a quantitative sequential window acquisition of all theoretical fragment ion spectra mass spectrometry pipeline to analyze protein changes in Oryza sativa and Nostoc punctiforme during early events of symbiosis. We found differentially expressed proteins in both organisms linked to several biological functions, including signal transduction, adhesion, defense-related proteins and cell wall modification. In N. punctiforme we found increased expression of 62 proteins that have been previously described in other Nostoc-plant symbioses, reinforcing the robustness of our study. Our findings reveal new proteins activated in the early stages of the Nostoc-Oryza symbiosis that might be important for the recognition between the plant and the host. Oryza mutants in genes in the common symbiosis signaling pathway (CSSP) show reduced colonization efficiency, providing first insights on the involvement of the CSSP for the accommodation of N. punctiforme inside the plant cells. This information may have long-term implications for a greater understanding of the symbiotic interaction between Nostoc and land plants.

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.

CRISPR-Cas systems in multicellular cyanobacteria.

Novel CRISPR-Cas systems possess substantial potential for genome editing and manipulation of gene expression. The types and numbers of CRISPR-Cas systems vary substantially between different organisms. Some filamentous cyanobacteria harbor > 40 different putative CRISPR repeat-spacer cassettes, while the number of cas gene instances is much lower. Here we addressed the types and diversity of CRISPR-Cas systems and of CRISPR-like repeat-spacer arrays in 171 publicly available genomes of multicellular cyanobacteria. The number of 1328 repeat-spacer arrays exceeded the total of 391 encoded Cas1 proteins suggesting a tendency for fragmentation or the involvement of alternative adaptation factors. The model cyanobacterium Anabaena sp. PCC 7120 contains only three cas1 genes but hosts three Class 1, possibly one Class 2 and five orphan repeat-spacer arrays, all of which exhibit crRNA-typical expression patterns suggesting active transcription, maturation and incorporation into CRISPR complexes. The CRISPR-Cas system within the element interrupting the Anabaena sp. PCC 7120 fdxN gene, as well as analogous arrangements in other strains, occupy the genetic elements that become excised during the differentiation-related programmed site-specific recombination. This fact indicates the propensity of these elements for the integration of CRISPR-cas systems and points to a previously not recognized connection. The gene all3613 resembling a possible Class 2 effector protein is linked to a short repeat-spacer array and a single tRNA gene, similar to its homologs in other cyanobacteria. The diversity and presence of numerous CRISPR-Cas systems in DNA elements that are programmed for homologous recombination make filamentous cyanobacteria a prolific resource for their study. Abbreviations: Cas: CRISPR associated sequences; CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; C2c: Class 2 candidate; SDR: small dispersed repeat; TSS: transcriptional start site; UTR: untranslated region.

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.

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.

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.