The temperature-regulated DEAD-box RNA helicase CrhR interactome: Autoregulation and photosynthesis-related transcripts.

RNA helicases play crucial functions in RNA biology. In plants, RNA helicases are encoded by large gene families, performing roles in abiotic stress responses, development, the post-transcriptional regulation of gene expression as well as house-keeping functions. Several of these RNA helicases are targeted to the organelles, mitochondria and chloroplasts. Cyanobacteria are the direct evolutionary ancestors of plant chloroplasts. The cyanobacterium Synechocystis 6803 encodes a single DEAD-box RNA helicase, CrhR, that is induced by a range of abiotic stresses, including low temperature. Though the ΔcrhR mutant exhibits a severe cold-sensitive phenotype, the physiological function(s) performed by CrhR have not been described. To identify transcripts interacting with CrhR, we performed RNA co-immunoprecipitation with extracts from a Synechocystis crhR deletion mutant expressing the FLAG-tagged native CrhR or a K57A mutated version with an anticipated enhanced RNA binding. The composition of the interactome was strikingly biased towards photosynthesis-associated and redox-controlled transcripts. A transcript highly enriched in all experiments was the crhR mRNA, suggesting an auto-regulatory molecular mechanism. The identified interactome explains the described physiological role of CrhR in response to the redox poise of the photosynthetic electron transport chain and characterizes CrhR as an enzyme with a diverse range of transcripts as molecular targets.

The Ssl2245-Sll1130 Toxin-Antitoxin System Mediates Heat-induced Programmed Cell Death in Synechocystis sp. PCC6803.

Two putative heat-responsive genes, ssl2245 and sll1130, constitute an operon that also has characteristics of a toxin-antitoxin system, thus joining several enigmatic features. Closely related orthologs of Ssl2245 and Sll1130 exist in widely different bacteria, which thrive under environments with large fluctuations in temperature and salinity, among which some are thermo-epilithic biofilm-forming cyanobacteria. Transcriptome analyses revealed that the clustered regularly interspaced short palindromic repeats (CRISPR) genes as well as several hypothetical genes were commonly up-regulated in Δssl2245 and Δsll1130 mutants. Genes coding for heat shock proteins and pilins were also induced in Δsll1130 We observed that the majority of cells in a Δsll1130 mutant strain remained unicellular and viable after prolonged incubation at high temperature (50 °C). In contrast, the wild type formed large cell clumps of dead and live cells, indicating the attempt to form biofilms under harsh conditions. Furthermore, we observed that Sll1130 is a heat-stable ribonuclease whose activity was inhibited by Ssl2245 at optimal temperatures but not at high temperatures. In addition, we demonstrated that Ssl2245 is physically associated with Sll1130 by electrostatic interactions, thereby inhibiting its activity at optimal growth temperature. This association is lost upon exposure to heat, leaving Sll1130 to exhibit its ribonuclease activity. Thus, the activation of Sll1130 leads to the degradation of cellular RNA and thereby heat-induced programmed cell death that in turn supports the formation of a more resistant biofilm for the surviving cells. We suggest to designate Ssl2245 and Sll1130 as MazE and MazF, respectively.

A membrane-bound cAMP receptor protein, SyCRP1 mediates inorganic carbon response in Synechocystis sp. PCC 6803.

The availability of inorganic carbon (Ci) as the source for photosynthesis is fluctuating in aquatic environments. Despite the involvement of transcriptional regulators CmpR and NdhR in regulating genes encoding Ci transporters at limiting CO2, the Ci-sensing mechanism is largely unknown among cyanobacteria. Here we report that a cAMP-dependent transcription factor SyCRP1 mediates Ci response in Synechocystis. The mutant ∆sycrp1 exhibited a slow-growth phenotype and reduced maximum rate of bicarbonate-dependent photosynthetic electron transport (Vmax) compared to wild-type at the scarcity of CO2. The number of carboxysomes was decreased significantly in the ∆sycrp1 at low CO2 consistent with its reduced Vmax. The DNA microarray analysis revealed the upregulation of genes encoding Ci transporters in ∆sycrp1. The membrane-localized SyCRP1 was released into the cytosol in wild-type cells shifted from low to high CO2 or upon cAMP treatment. Soluble His-tagged SyCRP1 was shown to target DNA-binding sites upstream of the Ci-regulated genes sbtA and ccmK3. In addition, cAMP enhanced the binding of SyCRP1 to its target sites. Our data collectively suggest that the Ci is sensed through the second messenger cAMP releasing membrane-bound SyCRP1 into cytoplasm under sufficient CO2 conditions. Hence, SyCRP1 is a possible regulator of carbon concentrating mechanism, and such a regulation might be mediated via sensing Ci levels through cAMP in Synechocystis.