Background: Cyanobacteria harbor a vast array of toxin-antitoxin modules, but their roles remain largely unknown. Results: The sll7003/ssl7004 system involves a VapC-type RNA endonuclease targeting single-stranded regions with little specificity. Conclusion: Seven different TA systems plus three free-standing components are located on plasmid pSYSA. Significance: Some bacterial plasmids are safeguarded against post-segregational loss by multiple TA systems and an aggressive type of RNA-targeting toxicity.

Bacterial toxin–antitoxin (TA) systems are genetic elements, which are encoded by plasmid as well as chromosomal loci. They mediate plasmid and genomic island maintenance through post-segregational killing mechanisms but may also have milder effects, acting as mobile stress response systems that help certain cells of a population in persisting adverse growth conditions. Very few cyanobacterial TA system have been characterized thus far. In this work, we focus on the cyanobacterium Synechocystis 6803, a widely used model organism. We expand the number of putative Type II TA systems from 36 to 69 plus seven stand-alone components. Forty-seven TA pairs are located on the chromosome and 22 are plasmid-located. Different types of toxins are associated with various antitoxins in a mix and match principle. According to protein domains and experimental data, 81% of all toxins in Synechocystis 6803 likely exhibit RNase activity, suggesting extensive potential for toxicity-related RNA degradation and toxin-mediated transcriptome remodeling. Of particular interest is the Ssr8013–Slr8014 system encoded on plasmid pSYSG, which is part of a larger defense island or the pSYSX system Slr6056–Slr6057, which is linked to a bacterial ubiquitin-like system. Consequently, Synechocystis 6803 is one of the most prolific sources of new information about these genetic elements.

Two putative heat-responsive genes, and, 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 Δ and Δ mutants. Genes coding for heat shock proteins and pilins were also induced in Δ We observed that the majority of cells in a Δ 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.