Escherichia coli BL21 (DE3) and E. coli DH5α were grown in the standard liquid LB medium or on agar plate with appropriate antibiotic (i.e., 10 μg/mL kanamycin) at 37°C using a shaking incubator at 130 rpm or incubator (Honour, Tianjin, China). Wild type Synechocystis (WT), mutants and the constructed strains were grown on agar plate or in BG11 medium at pH 7.5 using an illuming incubator or shaking incubator at a light intensity of approximately 50 μmol photons m^-2s^-1 and 130 rpm at 30°C (Honour, Tianjin, China). Medium for mutants and constructed strains was supplemented with appropriate antibiotic(s) (i.e., 10 μg/mL chloramphenicol and/or 10 μg/mL kanamycin). All strains and plasmids used in this study were listed in Table 1.

All primers used in this study were listed in Supplementary Table S1.

For gene deletion, the homologous recombination method was employed for the construction of gene knockout fragments for slr0946 (Chen et al., 2014b). Briefly, the chloramphenicol resistance cassette (amplified from pACYC184), two flanking homologous arms (about 1 kb) were employed for overlapping PCR and replacing the target gene of Synechocystis by natural transformation. The successful knockout mutant was confirmed by PCR and purified via successive passages.

For gene complementation and overexpression, a replicative vector pJA2 kindly provided by Prof. Paul Hudson (KTH Royal Institute of Technology of Sweden) was employed to overexpress sll0649, sll1598, slr0798, and slr0946, respectively (Huang et al., 2010; Kaczmarzyk et al., 2014). The resulting plasmid pJA2-sll0649 and pJA2-slr0946 was, respectively, back introduced into Δsll0649 and Δslr0946, leading to complementation strains Δsll0649-pJA0649 and Δslr0946-pJA0946. In addition, the resulting plasmid pJA2-slr0946, pJA2-sll0649, pJA2-sll1598, and pJA2-slr0798 were, respectively, introduced into the WT, leading to the overexpression strains WT-pJA0946, WT-pJA0649, WT-pJA1598, and WT-pJA0798. The transformation was performed using GenePulser Xcell (Bio-Rad, Hercules, CA, United States) (Sun et al., 2017). The positive colonies were validated by PCR.

For growth patterns, 5 mL fresh cells at OD_{630 nm} = 0.2 were collected by centrifugation (4°C, 3000 × g for 15 min) and then were inoculated into 25 mL BG11 liquid medium in a 100 mL flask with or without CdSO₄, each with three replicates (the concentration of CdSO₄ was 4.6 μM for WT and deletion mutants but 5.0 μM for WT and overexpression strains). Cell density was measured on an ELx808 Absorbance Microplate Reader (BioTek, Winooski, VT, United States) at OD₆₃₀ (Sun et al., 2017). Growth experiments were repeated at least three times to confirm the phenotype.

Overexpression and purification of His₆-Sll0649 protein were carried out as described previously (Chen et al., 2014b). Briefly, the sll0649 gene was amplified and then cloned to pET46 Ek/LIC vector, resulting in the plasmid pET46-sll0649. The pET46-sll0649 plasmid was then transformed into E. coli BL21 (DE3). The expression of His₆-Sll0649 was induced by 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and followed by incubation at 22°C overnight. His₆-Sll0649 was purified by the Ni-NTA agarose chromatography (GE healthcare, Uppsala, Sweden).

DNA-affinity-purified chip assays were employed to identify the genes that directly regulated by Sll0649. Promoter regions of 10 selected genes were amplified by PCR and incubated with the purified recombinant His₆-Sll0649 to allow the possible enrichment after elution according to the protocols described in the literature (Rajeev et al., 2011). Briefly, the binding reactions (500 μL) were set up with 12 to 18 μg of sheared Synechocystis genomic DNA (with an average length of 500 bp) and purified protein in the incubation buffer [20 mM Tris-HCl, pH 7.5; 1 mM dithiothreitol (DTT); 5 mM MgCl₂; 0.04 mg/mL BSA and 25% glycerol (v/v)]. The reactions were incubated at 25°C in a thermal cycler for 30 min; 50 μL of the reaction was then cleaned up by Qiaquick PCR purification columns (Qiagen, Hilden, Germany) and saved as input DNA. The rest was loaded to the Ni-NTA agarose chromatography that had been washed in the binding/wash buffer [20 mM Tris-HCl, pH 7.5; 10 mM MgCl₂; 50 mM KCl; 25% glycerol (v/v)]. The enriched DNA was specifically eluted from the resin with 500 μL elution buffer [20 mM Tris-HCl, pH 7.5; 500 mM NaCl; 600 mM imidazole; 10% glycerol (v/v)]. The enriched DNA fractions were cleaned up and saved as output DNA. Input DNA and output DNA were quantified using the Nanodrop 2000 (Thermo, CA, United States).

The qRT-PCR analysis was used to examine the enrichment fold of promoter regions of different genes after incubation with His₆-Sll0649. Primers for qRT-PCR analysis were designed using Primer Express 2.0. To differentiate PCR products from primer dimers, primers were selected to generate amplicons with sizes around 100–200 bp. Experimental steps are based on the description previously (Sun et al., 2017). Three technical replicates were performed for each sample. Data analysis was carried out using the StepOnePlus analytical software (Applied Biosystems, Foster City, CA, United States) and the 2^-ΔΔC_T method (Livak and Schmittgen, 2001). The rnpB gene encoding RNase P subunit B was used as an internal control (Chen et al., 2014a). Then the enrichment fold of output DNA was relatively quantified compared to that of input DNA. All primers were provided in Supplementary Table S1.

The EMSAs were performed as described previously (Chen et al., 2014b). Briefly, the promoter regions of slr0946 and slr1204 were amplified using the genomic DNA of Synechocystis and labeled with Cy5-labeled primer (5′-AGCCAGTGGCGATAAG-3′). The labeled PCR products were purified by QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). In each EMSA reaction, ∼10 ng of Cy5-labeled DNA probes was incubated with varying amount of His₆-Sll0649 protein in incubation buffer [1 mg/mL poly(dI–dC) (Roche, Basel, Switzerland), 20 mM Tris-base (pH 7.9), 1 mM DTT, 10 mM MgCl₂, 0.2 mg/mL BSA and 5% glycerol (v/v)] for 20 min at 25°C. After incubation, protein-bound DNA and free DNA were separated by 6% Native-PAGE and viewed under Typhoon (GE healthcare, Uppsala, Sweden).

In our previous work, a RR encoding gene sll0649 was identified involved in Cd²⁺ stress response in Synechocystis and Δsll0649 exhibited more sensitive phenotype to Cd²⁺ than WT (Chen et al., 2014b). In this work, to further confirm the involvement of sll0649 in Cd²⁺ tolerance, the sll0649 gene was placed under the control of the P_psbA2 promoter using a shuttle vector pJA2 and introduced back into the Δsll0649 mutant. The growth patterns among WT, Δsll0649 and Δsll0649-pJA0649 strains were then tested under normal medium and medium with 4.6 μM Cd²⁺. As illustrated in Figure 1, no obvious growth differences were observed for all three strains under both normal BG11 medium. Under 4.6 μM Cd²⁺ stress condition, the complementation stain (i.e., Δsll0649-pJA0649) was able to rescue the sensitive phenotype of the Δsll0649 to Cd²⁺, further suggesting the participation of sll0649 in Cd²⁺ regulation (Figure 1).

In our previous work, sll1598 and slr0798 have been identified as target genes of Sll0649 via EMSAs (Chen et al., 2014b). To further identify the new binding target(s) of Sll0649, DAP-chip strategy was employed. The full-length Sll0649 protein was first expressed in E. coli BL21(DE3) with a His₆-tag at its N-termini. Extractive Synechocystis genomic DNA was sheared into 500–600 bp by sonification as input DNA. Purified His₆-tagged Sll0649 proteins were incubated with sheared genomic DNA, and protein-bound DNA was purified using Ni-NTA resin to obtain the output DNA.

Then qRT-PCR was employed to determine the enrichment folds of different DNA regions. Ten candidate genes from upstream regions of sll0649 were selected according to the previous results of quantitative proteomics analysis (Chen et al., 2014b). Among these ten candidates, seven of them (i.e., sll0247, sll0248, slr0513, slr1204, slr0944, slr0945, and slr0946) were found down-regulated in Δsll0649 compared to WT under Cd²⁺ stress, and the other three (i.e., sll0041, sll0507, and sll0819) were randomly selected as negative controls. The rnpB gene was used as a control for normalization in this study. The results of qRT-PCR were listed in Table 2, in which slr0946 encoding the arsenate reductase was found enriched fourfolds among output Sll0649-bound DNAs compared to input DNA, suggesting it could be a new target of Sll0649.

In order to further verify the reliability of the new target slr0946, we performed EMSAs using purified His₆-Sll0649 and the promoter region of slr0946. Meanwhile, the slr1204 gene encoding degP was selected as the negative control. As shown in Figure 2, clear gel-shift pattern for the purified His₆-Sll0649 with Pslr0946 was investigated while no direct binding was observed for the His₆-Sll0649 with Pslr1204 under the testing condition, suggesting that Sll0649 was able to bind directly to the promoter region of slr0946.

To investigate the relationship of slr0946 with Cd²⁺ stress response, knockout mutant was generated by inserting the chloramphenicol resistance cassettes to the opening reading frame (ORF) of slr0946. The Δslr0946 mutant was viable and its growth rate in the normal BG11 medium was similar to that of the WT (Figure 3). However, under 4.6 μM Cd²⁺ stress condition, Δslr0946 was found more sensitive to Cd²⁺ than WT (Figure 3), indicating its involvement in Cd²⁺ stress response. We further constructed a complementary mutant named Δslr0946-pJA0946 by introducing the gene slr0946 back into Δslr0946 using a shuttle vector pJA2. As expected, the Δslr0946-pJA0946 strain was able to rescue the sensitive phenotype of Δslr0946 to Cd²⁺ in 4.6 μM Cd²⁺ stress (Figure 3), further confirming the participation of slr0946 in Cd²⁺ stress response.

Engineered Cd²⁺-resistant strains in Synechocystis could be promising and useful for further Cd²⁺ tolerance modifications in other cyanobacterial chassis. In this study, aiming to improve the Cd²⁺ resistance of Synechocystis, we respectively, overexpressed four genes related to Cd²⁺ resistance, i.e., sll0649, sll1598, slr0798, and slr0946 in WT. The constructed strains were named as WT-pJA0649, WT-pJA1598, WT-pJA0798, and WT-pJA0946, respectively.

Growth patterns showed no visible differences among all the four overexpression strains in the normal BG11 medium compared to WT (Figure 4). Excitingly, three of the four overexpression strains, i.e., WT-pJA1598, WT-pJA0798, and WT-pJA0946 had significant tolerance improvement compared to WT under 5.0 μM Cd²⁺ stress condition (Figures 4B–D). This indicated that overexpression of any of the three target genes of Sll0649 (i.e., sll1598, slr0798, and slr0946) could improve the tolerance of WT to Cd²⁺. However, we found that overexpression of sll0649 can’t improve the tolerance of WT to Cd²⁺ due to some unknown reason (Figure 4A). To address this issue, the expression level of sll0649 was measured by qRT-PCR in WT and WT-pJA0649. The result showed that the transcriptional level of sll0649 gene in WT-pJA0649 was over 10-folds than that in WT (data not shown), suggesting that overexpressing sll0649 gene can’t improve Cd²⁺ tolerance in Synechocystis.