Regulation of pSYSA defense plasmid copy number in Synechocystis through RNase E and a highly transcribed asRNA

In the model cyanobacterium Synechocystis sp. PCC 6803, four large and three smaller plasmids exist. The ~100 kb plasmid pSYSA was previously characterized as specialized on defense functions by encoding all three CRISPR-Cas systems and several toxin-antitoxin systems. The expression of genes located on pSYSA depends on the plasmid copy numbers in the cell. We found a positive correlation between pSYSA copy numbers and the level of the endoribonuclease E. As molecular basis for this correlation we identified the RNase E-mediated cleavage within the ssr7036 transcript. Together with a cis-located abundant antisense RNA (asRNA1), this mechanism resembles strikingly the control of ColE1-type plasmids by two overlapping RNAs, RNA I and II, in enterobacteria. In the ColE1 mechanism, two non-coding RNAs interact, supported by the small protein Rop, which is encoded separately. In contrast, in pSYSA the similar-sized protein Ssr7036 is encoded within one of the interacting RNAs and it is this mRNA that likely primes pSYSA replication. Essential for plasmid replication is furthermore the downstream encoded protein Slr7037 featuring primase and helicase domains because deletion of slr7037 led to the integration of pSYSA into the chromosome or other large plasmid pSYSX. These findings open new perspectives on the development of shuttle vectors for genetic engineering and of modulating the activity of the entire CRISPR-Cas apparatus in this cyanobacterium.