Identification of a New Target slr0946 of the Response Regulator Sll0649 Involving Cadmium Tolerance in Synechocystis sp. PCC 6803

Abstract

Survival of photosynthetic cyanobacteria is challenged by environmental contaminations like heavy metals. Among them, deciphering the regulatory mechanisms for cadmium (Cd) in cyanobacteria would facilitate the construction of Cd-resistant strains. In this study, the DNA-Affinity-Purified-chromatin immunoprecipitation assay was employed to identify the direct targets of Sll0649, which was a Cd2+-related response regulator identified in our previous work in model cyanobacteria Synechocystis sp. PCC 6803. As a result, the promoter region of slr0946 encoding the arsenate reductase was enriched fourfolds by quantitative real time PCR analysis. Further, deletion of slr0946 led to a sensitive phenotype to Cd2+ stress compared with the wild type (WT) and the sensitive phenotype of Δslr0946 could be rescued by complementation assay via introducing slr0946 back into Δslr0946. Finally, individually overexpression of slr0946 as well as two Cd2+-related genes identified priviously (i.e., sll1598 and slr0798) in WT could significantly improve the tolerance of Synechocystis sp. PCC 6803 to Cd2+. This study provided a better understanding of the tolerance mechanism to Cd2+ in cyanobacteria and also feasible strategies for tolerance modifications to heavy metals in the future.

Adaptive laboratory evolution of cadmium tolerance in Synechocystis sp. PCC 6803

“…Finally, Slr0798 is an SmtB-like repressor concerning zinc-transporting P-type ATPase involved in zinc tolerance [ 44 ]. Consistent with the previous study in Synechocystis , overexpression of slr0798 gene with a replicative vector pJA2 could significantly enhance Cd 2+ tolerance [ 23 ]. Unlike SmtB, Slr0798 triggered excess Zn 2+ expulsion by via Slr0798-mediated efflux into the periplasm, which we supposed was the same mode for Cd 2+ .…”

“…Wild-type (WT) Synechocystis strain was evolved by serial passaging for 128 passages (802 days) in BG11 medium supplemented with CdSO 4 , as a selective pressure to enrich population with Cd 2+ tolerance. The starting Cd 2+ concentration for WT was set as 4.6 µM as our previous study showed that WT showed a slight growth deficiency at this Cd 2+ concentration level [ 23 ]. Under normal BG11 medium without CdSO 4 , Synechocystis could achieve late exponential phase (OD 750 nm = 1.5) from an initial inoculum (OD 750 nm = 0.1) within 96 h. When CdSO 4 was added, cell growth rate decreased obviously.…”

Tools to map target genes of bacterial two‐component system response regulators

“…Similar to the transcriptomics approach, targets for RRs may be identified by measuring protein abundances between WT and mutant strains. Proteomics studies are often accompanied by transcriptomics, metabolomics or ChIP‐seq analyses (Wang et al ., , ; Sepulveda and Lupas, ; Sun et al ., ; Reed et al ., ).…”

A simple, switchable pili‐labelling method by plasmid‐based replacement of pilin

“…For the construction of pJA‐tho‐pilA, the mutated pilA was obtained in the same way. Then the backbone of pJA2‐ Sll0649 (Sun et al ., 2017), the Ptrc‐tho promoter, the T rbcl terminator and the mutated pilA was obtained by primers GG‐PILA‐F and GG‐PILA‐R, GG‐THO‐F and GG‐THO‐R, GG‐TRBCL‐F and GG‐TRBCL‐R, GG‐PJA‐F and‐ GG‐PJA‐R, respectively, and then assembled by golden gate assembly.…”