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.

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.

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.

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.

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.

Summary 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.

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 (SWATH-MS) pipeline to analyse 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, defence-related proteins, and cell wall modification. In Nostoc punctiforme we found increased expression in 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 signalling pathway (CSSP) show a 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.