The iron-stress activated RNA 1 (IsaR1) coordinates osmotic acclimation and iron starvation responses in the cyanobacterium Synechocystis sp. PCC 6803.

In nature, microorganisms are exposed to multiple stress factors in parallel. Here, we investigated the response of the model cyanobacterium Synechocystis sp. PCC 6803 to simultaneous iron limitation and osmotic stresses. Iron is a major limiting factor for bacterial and phytoplankton growth in most environments. Thus, bacterial iron homeostasis is tightly regulated. In Synechocystis, it is mediated mainly by the transcriptional regulator FurA and the iron-stress activated RNA 1 (IsaR1). IsaR1 is an important riboregulator that affects the acclimation of the photosynthetic apparatus to iron starvation in multiple ways. Upon increases in salinity, Synechocystis responds by accumulating the compatible solute glucosylglycerol (GG). We show that IsaR1 overexpression causes a reduction in the de novo GG synthesis rate upon salt shock. We verified the direct interaction between IsaR1 and the 5'UTR of the ggpS mRNA, which in turn drastically reduced the de novo synthesis of the key enzyme for GG synthesis, glucosylglycerol phosphate synthase (GgpS). Thus, IsaR1 specifically interferes with the salt acclimation process in Synechocystis, in addition to its primary regulatory function. Moreover, the salt-stimulated GgpS production became reduced under parallel iron limitation in WT - an effect which is, however, attenuated in an isaR1 deletion strain. Hence, IsaR1 is involved in the integration of the responses to different environmental perturbations and slows the osmotic adaptation process in cells suffering from parallel iron starvation.

Inactivation of invertase enhances sucrose production in the cyanobacterium Synechocystis sp. PCC 6803.

Sucrose is naturally synthesized by many cyanobacteria under high salt conditions, which can be applied to produce this widely used feedstock. To improve sucrose production with the moderate halo-tolerant cyanobacterium Synechocystis sp. PCC 6803, we identified and biochemically characterized the sucrose-degrading invertase. Inactivating the invertase encoding gene sll0626 (inv) significantly increased cellular sucrose levels; interestingly sucrose over-accumulation was also observed under NaCl-free conditions. The subsequent inactivation of inv in the mutant ΔggpS, which cannot synthesize the major compatible solute glucosylglycerol, resulted in further enhanced sucrose accumulation in the presence of 1.5 % NaCl. Then, inv mutation was introduced into the previously obtained sucrose-producing strain WD25 (Du W, Liang F, Duan Y, Tan X, Lu X. Metab Eng 2013;19:17-25), which resulted in almost 40 % higher sucrose accumulation. These findings show that invertase is an interesting target in obtaining efficient sucrose production in cyanobacterial host cells.

The glucosylglycerol-degrading enzyme GghA is involved in acclimation to fluctuating salinities by the cyanobacterium Synechocystis sp. strain PCC 6803.

The ggpS gene, which encodes the key enzyme for the synthesis of the compatible solute glucosylglycerol (GG), has a promoter region that overlaps with the upstream-located gene slr1670 in the cyanobacterium Synechocystissp. PCC 6803. Like ggpS, the slr1670 gene is salt-induced and encodes a putative glucosylhydrolase. A mutant strain with a slr1670 deletion was generated and found to be unable to adapt the internal GG concentrations in response to changes in external salinities. Whereas cells of the wild-type reduced the internal pool of GG when exposed to gradual and abrupt hypo-osmotic treatments, or when the compatible solute trehalose was added to the growth medium, the internal GG pool of ∆slr1670 mutant cells remained unchanged. These findings indicated that the protein Slr1670 is involved in GG breakdown. The biochemical activity of this GG-hydrolase enzyme was verified using recombinant Slr1670 protein, which split GG into glucose and glycerol. These results validate that Slr1670, which was named GghA, acts as a GG hydrolase. GghA is involved in GG turnover in fluctuating salinities, and similar proteins are found in the genomes of other GG-synthesizing cyanobacteria.

Salt-Regulated Accumulation of the Compatible Solutes Sucrose and Glucosylglycerol in Cyanobacteria and Its Biotechnological Potential.

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.