A glucose-tolerant non-motile strain (GT strain) of Synechocystis sp. PCC 6803 was grown at 32°C in BG-11 medium containing 20 mM HEPES-NaOH, pH 7.0, under continuous illumination at 20 μmol photons m⁻² s⁻¹ with bubbling of air. The lexA (sll1626)-disrupted mutant (ΔlexA) was grown under the same conditions, except that 20 μg mL⁻¹ kanamycin (Km) was added to the medium. Cell density was estimated by measuring OD₇₃₀ using a spectrophotometer (model UV-160A, Shimadzu).

The coding region of lexA (612 bp, from nucleotide 1319330 to 1318719 according to numbering in CyanoBase) was disrupted by insertion of a kanamycin resistance (Km^r) cassette. The upstream and downstream fragments including the lexA coding sequence were amplified by PCR from the genomic DNA of the WT strain using the primer sets lexA-F and Km-lexA-R (for amplification of 404 bp upstream fragment, from nucleotide 1319525 to 1319122) and Km-lexA-F and lexA-R (for amplification of 394 bp downstream fragment, from nucleotide 1318996 to 1318603; Table S1). Km^r cassette was PCR amplified from the pRL161 plasmid using the primer set Km-F and Km-R (Table S1). The amplified lexA fragments and Km^r cassette were fused together by the fusion PCR method (Wang et al., 2002) using the primer set lexA-F and lexA-R. The WT strain was transformed with the fusion PCR product and transformants (ΔlexA mutant) were selected in the presence of Km.

Isolation of total RNA by the hot phenol method and RNA gel blot analyses, using DIG RNA Labeling and Detection Kit (Roche), were performed as described previously (Muramatsu and Hihara, 2003). Template DNA fragments for in vitro transcription to generate RNA probes were prepared by PCR using the primers shown in Table S1.

Total proteins were extracted from Synechocystis cells as described previously (Ishii and Hihara, 2008) and separated by 15% (w/v) SDS-PAGE, followed by electroblotting onto PVDF membranes (Immobilon-P; Millipore). Immunodetection was done using a rabbit polyclonal antibody raised against His-LexA recombinant protein. Goat anti-rabbit IgG conjugated to alkaline phosphatase was used as a secondary antibody.

In vivo absorption spectra of whole cells suspended in BG-11 medium were measured at room temperature using a spectrophotometer (V-650 Spectrometer, JASCO) with ISV-722 integrating sphare. Chlorophyll and phycocyanin contents were calculated from the peak heights of absorption spectra using the equations described in Arnon et al. (1974).

RNA-seq analysis was carried out using cultures at OD₇₃₀ = 0.5 and OD₇₃₀ = 1.0 with three biological replicates. WT and ΔlexA were inoculated into new media at OD₇₃₀ = 0.1 and incubated for 50 and 80 h, respectively, to be harvested at OD₇₃₀ = 0.5. Similarly, WT and ΔlexA were inoculated at OD₇₃₀ = 0.1 and incubated for 70 and 120 h, respectively, to be harvested at OD₇₃₀ = 1.0. Isolation of total RNA by the hot phenol method was performed as described previously (Muramatsu and Hihara, 2003). To eliminate genomic DNA from total RNA samples, each sample was added with DNase I (TaKaRa) and incubated at 37°C for 3 h. Total RNA concentration was measured with Nanodrop 2000 (Thermo Fisher Scientific). The Ribo-Zero Magnetic Kit for Bacteria (Epicentre) was used to remove ribosomal RNA from each sample. Concentration and quality of mRNA samples were examined using an Agilent 2100 Bioanalyzer. TruSeq RNA Sample Prep Kit v2 (Illumina) was used for cDNA library construction, and the libraries were sequenced using the Illumina MiSeq system. 12 samples in total were analyzed using two cartridge of MiSeq Reagent Kit v3 (Illumina).

A total of 64 million reads data was obtained from 12 samples. To quantify expression level of each gene, nucleotide sequences of obtained reads were mapped to the genomic sequence of GT-I strain of S.6803 (Kanesaki et al., 2012) (NC_017038.1; http://www.ncbi.nlm.nih.gov/nuccore/NC_017038) using CLC Genomics Workbench 7.5.1 software (Qiagen). Raw read counts were divided by length of the transcripts and total number of million mapped reads in each sample to obtain reads per kilobase per million (RPKM) values (Mortazavi et al., 2008). TCC package of R software (Sun et al., 2013) was used to detect the differentially expressed genes between WT and ΔlexA. A false discovery rate of <0.01 was considered to be significant.

The coding region of the lexA gene was amplified by PCR using the primers lexA-NdeI-F and lexA-XhoI-R (Table S1), containing NdeI and XhoI sites at their 5′ end, respectively. The amplified lexA coding fragment was cloned into the pT7Blue T-vector (Novagen), digested with NdeI and XhoI and subcloned into the same restriction sites in pET28a vector (Novagen) to express the LexA protein with an N-terminal 6 × His-tag.

E. coli BL21(DE3) harboring the His-LexA expression construct was grown to an OD₆₀₀ = 0.6 in 250 mL of 2 × yeast extract-tryptone (YT) medium containing 20 μg mL⁻¹ Km at 37°C and induced with 0.013% of isopropyl β-D-thiogalactoside for 3 h. The cells were pelleted by centrifugation at 5800 g for 2 min, resuspended in 50 mM sodium phosphate buffer, pH 7.4, containing 0.5 M NaCl and 60 mM imidazole, and disrupted by three rounds of sonication with Sonifier 450 (Branson) for 2 min with interval of 1 min on ice. After the removal of whole cells and insoluble material by centrifugation, the soluble protein fraction was filtered through a 0.2 μm filter (DISMIC-25CS; ADVANTEC). His-LexA was purified by nickel-affinity column chromatography using a HisTrap FF crude (GE Healthcare). The soluble protein fraction was applied to the column equilibrated with 20 mM phosphate buffer, pH 7.4, containing 0.5 M NaCl and 60 mM imidazole, washed with 20 mM phosphate buffer, pH 7.4, containing 0.5 M NaCl and 80 mM imidazole, and eluted with 20 mM phosphate buffer, pH 7.4, containing 0.5 M NaCl and 300 mM imidazole. Purified His-LexA was desalted by a HiTrap Desalting column (GE Healthcare). Protein composition was examined by 15% (w/v) SDS-PAGE followed by staining with Coomassie Brilliant Blue R-250.

Probes for DNA gel mobility shift assays were obtained by PCR amplification with primers shown in Table S1 using genomic DNA as a template. The 3′ end of the DNA fragment for each probe was labeled with digoxigenin (DIG)-ddUTP by using the terminal transferase method according to the manufacturer's instructions (DIG gel shift kit 2nd generation; Roche). Gel mobility shift assays were performed by using a DIG gel shift kit 2nd generation (Roche) according to the manufacturer's instruction except that 1 mM DTT was added to the reaction mixture.

To reveal the function of LexA in GT strain of S.6803, we disrupted the lexA gene by inserting a Km^r cassette within the coding region (Figure 1A). Although a fully segregated mutant was not obtained (Figure 1B), RNA gel blot and immunoblot analyses revealed that both the lexA transcript (Figure 1C) and LexA protein (Figure 1D) levels were below the detection limit in the partially segregated mutant (ΔlexA) grown under normal growth conditions. Under the same conditions, ΔlexA displayed several abnormal phenotypes. The doubling time of ΔlexA was longer (31.4 h) than that of WT (19.5 h) at log phase, whereas the difference in growth rate between strains became smaller at stationary phase (Figure 1E). Amounts of chlorophyll and phycocyanin in ΔlexA calculated from the peak heights of cellular absorption spectra were 93 and 80% of WT levels, respectively (Figure 1F). Microscopic observation revealed that cell size of ΔlexA was heterogeneous and tended to be larger than that of WT (Figure 1G).

To investigate the difference in gene expression profile between WT and ΔlexA, total RNA was isolated from cultures incubated under normal growth conditions and RNA-seq analysis was performed. Figure 2 shows MA plots of the gene expression data obtained from cultures at OD₇₃₀ = 0.5 and OD₇₃₀ = 1.0. There were 1011 genes differentially expressed between strains at OD₇₃₀ = 0.5 as shown in magenta (Figure 2A). Among them, expression levels of 315 genes were more than two-fold higher and those of 28 genes were more than two-fold lower in ΔlexA than in WT (Table S2). In the case of WT and ΔlexA cells at OD₇₃₀ = 1.0, there were 447 genes differentially expressed between strains (Figure 2B). Among them, expression levels of 360 genes were more than two-fold higher and those of 21 genes were more than two-fold lower in ΔlexA than in WT (Table S3).

Table 1 shows the list of genes whose expression was affected by disruption of lexA. The higher resolution and better dynamic range of RNA-seq analysis compared to DNA microarray analysis enabled listing of small ORFs such as ssl1577, ggpR (ssl3076), ssr1251, ssr1473 and ssr3589, and genes with low expression level (low RPKM value) that cannot be detected by previous DNA microarray analyses. Differentially expressed genes can be categorized into several groups according to related cellular functions as mentioned below.

DNA gel mobility shift assay was performed to examine whether LexA directly regulates expression of putative target genes listed by RNA-seq analysis (Figure 3). We observed induction of the pilA7-pilA8 operon and repression of the pilA9-pilA10-pilA11 operon in ΔlexA (Table 1). Binding of His-LexA to the promoter regions of both operons (for the pilA7 operon from nucleotide 2222102 to 2222304 and for the pilA9 operon from nucleotide 755577 to 755778, according to numbering in CyanoBase) was observed, indicating that LexA directly activates or represses expression of these pilA operons. We also examined whether His-LexA binds to the upstream region of the two divergently transcribed operons, ggpS-glpD and slr1670-glpK-spoU-slr1674-hypA1, both of which are highly induced in ΔlexA. His-LexA bound to the promoter fragment of each operon (for the ggpS operon from nucleotide 1949371 to 1949186 and for the slr1670 operon from nucleotide 1949332 to 1949534). It is notable that LexA-binding site for the the ggpS operon is within the coding region of ggpR (nucleotide 1949372 to 1949100). Our results suggest that LexA binds to at least two binding site located in the intergenic region of the ggpS and slr1670 operons to repress their expression. Next, we examined the binding of LexA to the upstream region of PSI genes by using light-responsive promoter fragments containing the HLR1 sequence recognized by the response regulator RpaB (Seino et al., 2009). Binding of His-LexA to the promoter region of PSI genes was not observed (Figure 3) or much weaker than that to the pilA7, pilA9, ggpS, and slr1670 promoters and not reproducible. This indicates that decrease in expression levels of PSI genes in ΔlexA may be a secondary effect.

In this study, we created the gene-disrupted mutant of lexA in GT strain of S.6803 to obtain the comprehensive view of LexA regulon by RNA-seq analysis. Although Kamei et al. (2001) successfully obtained the fully-segregated lexA mutant from the motile PCC strain, in most cases the ΔlexA mutant invariably retained the WT copy of the lexA gene (Domain et al., 2004; Gutekunst et al., 2005) and we also could not obtain fully-segregated mutant (Figure 1B). The heterogeneous appearance of the ΔlexA mutant cells (Figure 1G) may be caused by difference in the extent of segregation. However, despite the existence of the WT copy of lexA, immunoblot analysis revealed that LexA protein level was below the detection limit in our mutant (Figure 1D).

To date, LexA in S.6803 has been reported to be involved in transcriptional regulation of genes related to various cellular functions. Our RNA-seq data are consistent with some of these reports, e.g., positive regulation of the hox operon reported by Gutekunst et al. (2005) and positive regulation of the pilA genes reported by Kamei et al. (2001). However, we could not observe the large effect of LexA depletion on carbon metabolism-related genes reported by Domain et al. (2004). Domain et al. isolated RNA for DNA microarray analysis from concentrated cultures incubated on plates for 2 h. The growth condition must be largely different from our liquid culture, which may cause the difference in gene expression profile. in vitro transcription/translation assay performed by Patterson-Fortin et al. (2006) showed that CrhR protein accumulation decreased in response to increasing LexA concentration. However, in our data, expression level of crhR was not affected by disruption of lexA.

RNA-seq data in this study suggested involvement of LexA in regulation of (1) phototactic motility, (2) accumulation of GG, (3) bidirectional hydrogenase, and (4) photosystem I and phycobilisome complexes. We also observed increase in expression level of genes related to iron and manganese uptake in ΔlexA at OD₇₃₀ = 1.0. LexA may be involved in stage specific repression of these genes, but it is also possible that these genes were upregulated as a consequence of iron and manganese limitation in the mutant culture during prolonged incubation. We will discuss regulation of cellular processes (1)–(4) by LexA in the following sections.

Our results of DNA gel mobility shift assay suggest that LexA binds to the upstream region of piA7, pilA9, ggpS and slr1670 to directly regulate their expression (Figure 3). To date, several nucleotide sequences for LexA binding site have been identified by DNA gel mobility shift assay, for example, 5′-TTTATTTGA ACTATTTTT-3′, 5′-TTTTTCGTTGTCTAA ATT-3′ (Oliveira and Lindblad, 2005), 5′-CTA-N₉(AT-rich)-CTA-3′ (Patterson-Fortin and Owttrim, 2008), and 5′-AGT AACTAGTTCG-3′ (Gutekunst et al., 2005) in S.6803 and 5′-TAG TACTAATGTTCTA-3′ in A.7120. (Mazón et al., 2004). However, these LexA binding sequences could not be found in the promoter fragments to which His-LexA bound. Instead, we found that a 5′-TTTTG(A/T)TNAC-3′ sequence commonly exists in these promoter fragments (Figure S1). The sequence is located around the putative transcription start site in the case of the negatively-regulated target genes, ggpS, piA7, and slr1670, whereas it is located further upstream region in the case of the positively-regulated pilA9 gene. It has been reported that a certain global transcriptional regulator, such as NtcA and RpaB in S.6803, can act as both repressor and activator dependent on the location of the binding site (García-Domínguez et al., 2000; Seino et al., 2009). Binding of the transcriptional regulator causes repression when its binding site overlaps the RNA polymerase-binding site, whereas activating effect is observed when the binding site is located further upstream. The location of 5′-TTTTG(A/T)TNAC-3′ sequence in four LexA-target promoters seems consistent with the scheme.

Results of RNA-seq analysis (Table 1) together with DNA gel mobility shift assay (Figure 3) suggest LexA in S.6803 can positively or negatively regulate various cellular processes such as phototactic motility, GG accumulation and hydogenase activity. Regulation of such a wide range of cellular processes by LexA was reported in other bacterial species. For example, the lexA mutant of Clostridium difficile showed pleiotrophic phenotypes such as filamentous structure due to inhibition of cell division, decreased sporulation, decrease in swimming motility and increased biofilm formation (Walter et al., 2015). In this case, LexA acts as a regulator of DNA damage in addition to the above mentioned biological functions. In contrast, DNA microarray data from different research groups (Kamei et al., 2001; Domain et al., 2004) and our RNA-seq data suggest LexA in S.6803 is not involved in regulation of SOS genes. In S.6803, expression of lexA and recA was not induced upon UV-irradiation (Domain et al., 2004; Patterson-Fortin et al., 2006). Similarly, in Anabaena sp. PCC 7120, expression of lexA was not induced upon UV-B exposure or treatment with a DNA damaging agent mitomycin C (Kumar et al., 2015). In these freshwater species, LexA-independent protection mechanism for DNA damage may have evolved and LexA may have become devoted to regulating other cellular processes. Then, what is the physiological meaning of the coordinated regulation of phototactic motility, GG accumulation, and hydogenase activity by LexA in S.6803? We searched for environmental conditions where LexA-target genes are coordinately regulated using CyanoEXpress gene expression database (http://cyanoexpress.sysbiolab.eu/) and found that salt stress causes induction of GG metabolism-related genes and repression of hox operon and pilA genes in WT (Shoumskaya et al., 2005; Dickson et al., 2012). The expression profile is similar to that observed by disruption of lexA (Table 1), indicating the possibility that transcriptional regulation by LexA is temporarily inactivated under salt stress conditions.

Recently, it has been suggested that the SOS response in the marine Synechococcus is regulated by LexA like E. coli (Blot et al., 2011; Tetu et al., 2013). Cyanobacterial LexA genes can be clustered into three groups, Clade A containing Gloeobacter violaceus PCC 7421, Clade C containing marine picocyanobacteria and Clade B containing most remaining species (Li et al., 2010). There may exist high degree of variation of LexA regulons among species belonging to these three clades. By examination of what kind of cellular processes LexA regulates, we will be able to know decision of each species about how to use the transcriptional regulator LexA for better adaptation to changing environment.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.