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

Abstract

Photosynthetic cyanobacteria are sensitive to toxicity of metal cadmium (Cd(2+)). Although metabolic responses against Cd(2+) 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 sll0649, whose mutant was more sensitive to Cd(2+) stress. Further phenotypic analysis revealed that ∆sll0649 becomes more sensitive to Cu(2+), Fe(2+), Mn(2+) and Zn(2+) stress as well. Using a quantitative iTRAQ-LC-MS/MS proteomics approach, we showed that a total of 156 and 151 unique proteins were down- and up-regulated for at least 2-fold in the ∆sll0649 mutant grown under Cd(2+) stress, respectively. In addition, electrophoretic mobility shift assays showed that Sll0649 was able to bind directly to the upstream regions of sll1598 and slr0798, which encode an Mn(2+) transporter MntC and a Zn(2+) transporting P-type ATPases ZiaA, respectively, suggesting that Sll0649 was involved in Cd(2+) tolerance by regulating and maintaining intracellular metal homeostasis. The involvement of sll1598 and slr0798 genes in Cd(2+) tolerance was also verified by comparative mutant analyses. The study provided a proteomic description of the Cd(2+) response network mediated by the response regulator Sll0649, and revealed novel insights on the metal-tolerance mechanism in Synechocystis.

Biological significance: As a major pollutant on earth, Cd(2+) is toxic to both prokaryotic and eukaryotic organisms. It is thus important to obtain a better understanding of cellular response to Cd(2+) 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 Cd(2+) stress, we identified the orphan RR, Slr0649, involved in Cd(2+) tolerance in Synechocystis. The ∆sll0649 mutant was also found to be more sensitive to high-concentration Cu(2+), Fe(2+), Mn(2+) and Zn(2+), 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 Cd(2+) utilization, Sll0649 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 Cd(2+) response network mediated by the response regulator Sll0649, and revealed novel insights on the metal-tolerance mechanism in Synechocystis.

Keywords: Cadmium; Proteomics; Response regulator; Synechocystis; Tolerance.