An orphan response regulator Sll0649 involved in cadmium tolerance and metal homeostasis in photosynthetic Synechocystis sp PCC 6803

Photosynthetic cyanobacteria are sensitive to toxicity of metal cadmium (Cd2+. Although metabolic responses against Cd2+ exposure have been described, the related regulatory mechanism is still unclear in cyanobacteria. In this study, we identified in Synechocystis sp. PCC 6803 a response regulator (RR)-encoding gene s110649, whose mutant was more sensitive to Cd2+ stress. Further phenotypic analysis revealed that Delta 110649 becomes more sensitive to Cu2+, Fe2+, Mn2+ and Zn2+ stress as well. Using a quantitative iTRAQ-LC-MS/MS proteomics approach, we showed that a total of 156 and 151 unique proteins were downand up-regulated for at least 2-fold in the M110649 mutant grown under Cd2+ stress, respectively. In addition, electrophoretic mobility shift assays showed that S110649 was able to bind directly to the upstream regions of s111598 and s1r0798, which encode an Mn2+ transporter MntC and a Zn2+ transporting P-type ATPases ZiaA, respectively, suggesting that 5110649 was involved in Cd2+ tolerance by regulating and maintaining intracellular metal homeostasis. The involvement of s111598 and s1r0798 genes in Cd2+ tolerance was also verified by comparative mutant analyses. The study provided a proteomic description of the Cd2+ response network mediated by the response regulator S110649, and revealed novel insights on the metal-tolerance mechanism in Synechocystis. Biological significance As a major pollutant on earth, Cd2+ is toxic to both prokaryotic and eukaryotic organisms. It is thus important to obtain a better understanding of cellular response to Cd2+ and the related regulatory mechanism. In this study, by screening 44 gene knockout mutants of putative RR-encoding genes in Synechocystis for their sensitivity change to Cd2+ stress, we identified the orphan RR, S1r0649, involved in Cd2+ tolerance in Synechocystis. The Delta sll0649 mutant was also found to be more sensitive to high-concentration Cu2+, Fe2+, Mn2+ and Zn2+, when compared with the wild type. Using an iTRAQ-LC-MSMS based quantitative proteomic analysis coupled with EMSAs, we found that, in addition to its positive regulation on genes directly related to Cd2+ utilization, S110649 can also functions as a key positive regulator either directly or indirectly on expression of multiple genes related to transporting and utilization of several other metal ions. The study provided a proteomic description of the Cd2+ response network mediated by the response regulator Sll0649, and revealed novel insights on the metal-tolerance mechanism in Synechocystis. (C) 2014 Elsevier B.V. All rights reserved.