The Proteolytic Activation of the relNEs (ssr1114/slr0664) Toxin–Antitoxin System by Both Proteases Lons and ClpP2s/Xs of Synechocystis sp. PCC 6803

Abstract

The proteolytic regulation of toxin-antitoxin (TA) systems has been well studied in Escherichia coli but remains unclear in other bacteria. A chromosomal gene pair ssr1114/slr0664, named relNEs, of Synechocystis sp. PCC 6803 forms a TA system belonging to rel family. Here, we used E. coli strain BL21 (DE3) as a host to characterize the proteolytic regulation of relNEs. The proteases of this strain could not degrade the antitoxin RelN, and the ectopic production of the ATP-dependant protease Lons or ClpP2s/Xs of Synechocystis sp. PCC6803 did not affect E. coli growth. Either Lons or ClpP2s/Xs was able to degrade RelN resulting in growth arrest of E. coli due to the activation of RelEs's toxicity, and the presence of RelEs could protect RelN to a certain extent against Lons and ClpP2s/Xs. Our observations suggest that both Lons and ClpP2s/Xs are responsible for RelN proteolysis in the native host under certain conditions. RelN is the first protein substrate identified for cyanobacterial ATP-dependent proteases.

Characterization of a Chromosomal Type II Toxin–Antitoxin System mazEaFa in the Cyanobacterium Anabaena sp. PCC 7120

“…The unicellular cyanobacterium, Synechocystis sp. PCC 6803, contains as many as 34 putative TA systems [15]–[17], including our previously characterized reNlEs operon [31], [52]. Characterization of this TA system carried out in E. coli demonstrated that the relNEs operon constitutes a “hybrid” TA systems where the toxin gene relEs belonging to the relE family is paired with the antitoxin gene relN with no sequence homology to characterized antitoxin genes [52].…”

“…Furthermore, the antitoxin protein RelN in E. coli could be degraded by both Lons and ClpP2s proteases from Synechocystis sp. PCC 6803, thus resulting in the activation of the toxin RelEs from the RelN-RelEs complex [31]. Anabaena encodes at least 38 putative type II TA systems [15]–[17].…”

“…To construct the plasmids for selective expression of MazEa and MazFa, the plasmid pJS298 was used, which contains P BAD and P T7 as well as the genes araC and lacI encoding the regulator proteins of the respective promoters [31]. mazFa was amplified using the primers mazFa-N and mazFa-K, digested with Nde I/ Kpn I, and cloned behind P T7 of pJS298 generating pJS301.…”

Toxin-Antitoxin Systems on the Large Defense Plasmid pSYSA of Synechocystis sp. PCC 6803

“…However, the only cyanobacterial TA system that has been experimentally characterized thus far is the chromosomally located relNE (ssr1114/slr0664) TA system of Synechocystis and its proteolytic regulation by ATP-dependent proteases (16).…”

Conditional, Temperature-Induced Proteolytic Regulation of Cyanobacterial RNA Helicase Expression

“…strain PCC 6803 encodes 62 peptidases (17). Proteolysis in cyanobacteria has been associated with the degradation of a few specific proteins (18)(19)(20)(21)(22)(23)(24) or with stress response pathways (25)(26)(27).…”