Transcriptional and Proteolytic Regulation of the Toxin-Antitoxin Locus vapBC10 (ssr2962/slr1767) on the Chromosome of Synechocystis sp PCC 6803

VapBC toxin-antitoxin (TA) systems are defined by the association of a PIN-domain toxin with a DNA-binding antitoxin, and are thought to play important physiological roles in bacteria and archaea. Recently, the PIN-associated gene pair PIN-COG2442 was proposed to encode VapBC-family TA system and found to be abundant in cyanobacteria. However, the features of these predicted TA loci remain under investigation. We here report characterization of the PIN-COG2442 locus vapBC10 (ssr2962/slr1767) on the chromosome of Synechocystis sp. PCC 6803. RT-PCR analysis revealed that the vapBC10 genes were co-transcribed under normal growth conditions. Ectopic expression of the PIN-domain protein VapC10 caused growth arrest of Escherichia coli that does not possess vapBC TA locus. Coincidentally, this growth-inhibition effect could be neutralized by either simultaneous or subsequent production of the COG2442-domain protein VapB10 through formation of the TA complex VapBC10 in vivo. In contrast to the transcription repression activity of the well-studied antitoxins, VapB10 positively auto-regulated the transcription of its own operon via specific binding to the promoter region. Furthermore, in vivo experiments in E. coli demonstrated that the Synechocystis protease ClpXP2s, rather than Lons, could cleave VapB10 and proteolytically activate the VapC10 toxicity. Our results show that the PIN-COG2442 locus vapBC10 encodes a functional VapBC TA system with an alternative mechanism for the transcriptional auto-regulation of its own operon.