RNA-seq and especially differential RNA-seq-type transcriptomic analyses (dRNA-seq) are powerful analytical tools, as they not only provide insights into gene expression changes but also provide detailed information about all promoters active at a given moment, effectively giving a deep insight into the transcriptional landscape. Synechocystis sp. PCC 6803 (Synechocystis 6803) is a unicellular model cyanobacterium that is widely used in research fields from ecology, photophysiology to systems biology, modelling and biotechnology. Here, we analysed the response of the Synechocystis 6803 primary transcriptome to different, environmentally relevant stimuli. We established genome-wide maps of the transcriptional start sites active under 10 different conditions relevant for photosynthetic growth and identified 4,091 transcriptional units, which provide information about operons, 5′ and 3′ untranslated regions (UTRs). Based on a unique expression factor, we describe regulons and relevant promoter sequences at single-nucleotide resolution. Finally, we report several sRNAs with an intriguing expression pattern and therefore likely function, specific for carbon depletion (CsiR1), nitrogen depletion (NsiR4), phosphate depletion (PsiR1), iron stress (IsaR1) or photosynthesis (PsrR1). This dataset is accompanied by comprehensive information providing extensive visualization and data access to allow an easy-to-use approach for the design of experiments, the incorporation into modelling studies of the regulatory system and for comparative analyses.

Cyanobacteria are prokaryotes that can assimilate inorganic carbon via oxygenic photosynthesis, which results in the formation of organic compounds essentially from CO₂, water, and light. Increasing concerns regarding the increase in atmospheric CO₂ due to fossil energy usage fueled the idea of a photosynthesis-driven and CO₂-neutral, i.e., cyanobacteria-based biotechnology. The ability of various cyanobacteria to tolerate high and/or fluctuating salinities attenuates the requirement of freshwater for their cultivation, which makes these organisms even more interesting regarding a sustainable utilization of natural resources. However, those applications require a detailed knowledge of the processes involved in salt acclimation. Here, we review the current state of our knowledge on the regulation of compatible solute accumulation in cyanobacteria. The model organism Synechocystis sp. PCC 6803 responds to increasing salinities mainly by the accumulation of glucosylglycerol (GG) and sucrose. After exposure toward increased salt concentrations, the accumulation of the main compatible solute GG is achieved by de novo synthesis. The key target of regulation is the enzyme GG-phosphate synthase (GgpS) and involves transcriptional, posttranscriptional, and biochemical mechanisms. Recently, the GG-degrading enzyme GG hydrolase A (GghA) was identified, which is particularly important for GG degradation during exposure to decreasing salinities. The inversely ion-regulated activities of GgpS and GghA could represent the main model for effectively tuning GG steady state levels according to external salinities. Similar to GG, the intracellular amount of sucrose is also salt-regulated and seems to be determined by the balance of sucrose synthesis via sucrose-phosphate synthase (Sps) and its degradation via invertase (Inv). In addition to their role as stress protectants, both compatible solutes also represent promising targets for biotechnology. Hence, the increasing knowledge on the regulation of compatible solute accumulation not only improves our understanding of the stress physiology of cyanobacteria but will also support their future biotechnological applications.

Glutamine synthetase (GS), a key enzyme in biological nitrogen assimilation, is regulated in multiple ways in response to varying nitrogen sources and levels. Here we show a small regulatory RNA, NsiR4 (nitrogen stress-induced RNA 4), which plays an important role in the regulation of GS in cyanobacteria. NsiR4 expression in the unicellular Synechocystis sp. PCC 6803 and in the filamentous, nitrogen-fixing Anabaena sp. PCC 7120 is stimulated through nitrogen limitation via NtcA, the global transcriptional regulator of genes involved in nitrogen metabolism. NsiR4 is widely conserved throughout the cyanobacterial phylum, suggesting a conserved function. In silico target prediction, transcriptome profiling on pulse overexpression, and site-directed mutagenesis experiments using a heterologous reporter system showed that NsiR4 interacts with the 5′UTR of gifA mRNA, which encodes glutamine synthetase inactivating factor (IF)7. In Synechocystis, we observed an inverse relationship between the levels of NsiR4 and the accumulation of IF7 in vivo. This NsiR4-dependent modulation of gifA (IF7) mRNA accumulation influenced the glutamine pool and thus <mml:math><mml:mrow><mml:mi mathvariant="normal">N</mml:mi><mml:msubsup><mml:mi mathvariant="normal">H</mml:mi><mml:mi mathvariant="normal">4</mml:mi><mml:mi mathvariant="normal">+</mml:mi></mml:msubsup></mml:mrow></mml:math>NH4+ assimilation via GS. As a second target, we identified ssr1528, a hitherto uncharacterized nitrogen-regulated gene. Competition experiments between WT and an ΔnsiR4 KO mutant showed that the lack of NsiR4 led to decreased acclimation capabilities of Synechocystis toward oscillating nitrogen levels. These results suggest a role for NsiR4 in the regulation of nitrogen metabolism in cyanobacteria, especially for the adaptation to rapid changes in available nitrogen sources and concentrations. NsiR4 is, to our knowledge, the first identified bacterial sRNA regulating the primary assimilation of a macronutrient.

Little is known so far about RNA regulators of photosynthesis in plants, algae, or cyanobacteria. The small RNA PsrR1 (formerly SyR1) has been discovered in Synechocystis sp PCC 6803 and appears to be widely conserved within the cyanobacterial phylum. Expression of PsrR1 is induced shortly after a shift from moderate to high-light conditions. Artificial overexpression of PsrR1 led to a bleaching phenotype under moderate light growth conditions. Advanced computational target prediction suggested that several photosynthesis-related mRNAs could be controlled by PsrR1, a finding supported by the results of transcriptome profiling experiments upon pulsed overexpression of this small RNA in Synechocystis sp PCC 6803. We confirmed the interaction between PsrR1 and the ribosome binding regions of the psaL, psaJ, chlN, and cpcA mRNAs by mutational analysis in a heterologous reporter system. Focusing on psaL as a specific target, we show that the psaL mRNA is processed by RNase E only in the presence of PsrR1. Furthermore, we provide evidence for a posttranscriptional regulation of psaL by PsrR1 in the wild type at various environmental conditions and analyzed the consequences of PsrR1-based regulation on photosystem I. In summary, computational and experimental data consistently establish the small RNA PsrR1 as a regulatory factor controlling photosynthetic functions.