Roles of Close Homologues SigB and SigD in Heat and High Light Acclimation of the Cyanobacterium Synechocystis sp. PCC 6803

Acclimation of cyanobacterium Synechocystis sp. PCC6803 to suboptimal conditions is largely dependent on adjustments of gene expression, which is highly controlled by the σ factor subunits of RNA polymerase (RNAP). The SigB and SigD σ factors are close homologues. Here we show that the sigB and sigD genes are both induced in high light and heat stresses. Comparison of transcriptomes of the control strain (CS), ΔsigB, ΔsigD, ΔsigBCE (containing SigD as the only functional group 2 σ factor), and ΔsigCDE (SigB as the only functional group 2 σ factor) strains in standard, high light, and high temperature conditions revealed that the SigB and SigD factors regulate different sets of genes and SigB and SigD regulons are highly dependent on stress conditions. The SigB regulon is bigger than the SigD regulon at high temperature, whereas, in high light, the SigD regulon is bigger than the SigB regulon. Furthermore, our results show that favoring the SigB or SigD factor by deleting other group 2 σ factors does not lead to superior acclimation to high light or high temperature, indicating that all group 2 σ factors play roles in the acclimation processes.


Introduction
Efficient recycling and sustainable production of valuable compounds are major challenges of the future. Development of carbon neutral production systems and improvement of recycling could help to prevent further global warming and reduce pollution. Municipal, piggery, and dairy wastewaters, as well as anaerobic digestion reject waters, are typically nutrient rich, containing plenty of ammonium, nitrate, and phosphate [1,2]. Cyanobacteria are efficient in collecting nutrients [3] and many valuable products, including ethanol, isopropanol, butanol, biohydrogen and alkanes, have already been produced in cyanobacteria [4]. In the most tempting scenario, cyanobacteria are engineered to produce high value compounds, not only biomass, and simultaneously remediate wastewaters. For biotechnical applications, robust strains with high acclimation capacity are beneficial. Keeping up constant optimal environmental conditions, such as optimal temperature and constant light, is expensive, and using wastewater as a growth medium might cause additional problems; wastewaters typically contain harmful chemicals, and their nutrient balance is variable and not optimal.
Acclimation of cyanobacteria to suboptimal conditions is strongly dependent on changes in gene expression. In cyanobacteria, regulatory σ subunits of the RNA polymerase (RNAP) play major roles in acclimation responses. In optimal growth conditions, the RNAP core mainly recruits the primary σ factor that guides RNAP to transcribe housekeeping genes [5]. In suboptimal conditions, RNAP holoenzymes bearing stress-condition-specific alternative σ factor(s) become abundant and change the transcriptome to allow acclimation The glucose tolerant strain of Synechocystis sp. PCC 6803 (CS) was used as a control and host strain [8]. The construction of ∆sigB, ∆sigD [8], ∆sigBCE, and ∆sigCDE [17] strains has been described earlier. Single and triple inactivation strains were maintained on BG-11 plates in standard growth conditions in the presence of appropriate antibiotics, as described earlier [8,17], but antibiotics were omitted from liquid cultures.
To study the sigB and sigD promoters, P sigB -and P sigD -lux strains were constructed. First, a nourseotricine resistance cassette (NAT) with the rrnBT1 transcription terminator sequence of E. coli was released as a HindII fragment from plasmid pGEM-NATter (generous gift from L. Lopez-Maury), and the single stranded ends of the DNA fragment were filled with Klenow enzyme. This NAT fragment was then cloned into the SmaI site after the luxCDABE operon into the pTETlux plasmid (generous gift from Marko Virta) to obtain the pTETluxNAT plasmid. Either the 402 bp long fragments of upstream from the coding region of the sigB gene ( Figure 1A) or 284 bp long upstream fragment of the sigD gene ( Figure 1B) was used to control the expression of the lux operon. For insertion to the Synechocystis chromosome, the 200 bp long upstream and downstream regions of the psbA1 gene were inserted to the both ends of the construct containing the lux operon under the sigB or sigD promoter and NAT cassette ( Figure 1A,B). The psbA1 gene was selected, as it is practically silent in all conditions used in this study and only expressed in low oxygen conditions [18,19]. Synthetic DNA fragments were purchased from GenScript Biotech (New Jersey, NJ, USA). Then plasmids pP sigB lux and pP sigD lux were transformed to the glucose tolerant control strain of Synechocystis sp. PCC6803. Colonies appeared after one week on BG-11 plates supplemented with nourseothricin (10 µg/mL), and they were streaked on new selective plates once a week for eight weeks. Thereafter, the segregation of PsigB-and PsigD-lux strains was verified with PCR, as described in [10], using the primers 5 -CCGCTACCACCTGTTTTATTA-3 and 5 -TCGGCCCAGGTGCTCACG-3 . Finally, the Life 2022, 12, 162 3 of 17 modified areas of the genome in PsigB-and PsigD-lux strains were sequenced to confirm that they were as planned.
Life 2022, 12,162 3 of 17 appeared after one week on BG-11 plates supplemented with nourseothricin (10 μg/mL), and they were streaked on new selective plates once a week for eight weeks. Thereafter, the segregation of PsigB-and PsigD-lux strains was verified with PCR, as described in [10], using  the  primers  5′-CCGCTACCACCTGTTTTATTA-3′  and  5′-TCGGCCCAGGTGCTCACG-3′. Finally, the modified areas of the genome in PsigB-and PsigD-lux strains were sequenced to confirm that they were as planned. was inserted upstream of the promoter-less lux operon, and nourseotricine antibiotic cassette (NAT) was added after the lux operon for antibiotic selection. The promoter-lux-NAT construct was inserted in the middle of the non-essential psbA1 gene, and the CS strain of Synechocystis was transformed with linearized plasmids to produce PsigB-and PsigD-lux strains. HR1 and HR2 are proximal areas of the psbA1 gene used for homologous recombination. (C) PCR verification of PsigBand PsigD-lux strains. The amplicon in the CS strain is 829, 8043 in PsigB-lux, and 7925 bp in PsigDlux. The primers are marked as P1 and P2 in (A) and (B). (D) Luciferase activity was measured from control (CS), PsigB-, and PsigD-lux strains (3-day old cultures) at standard growth conditions or after 15 min of high light or temperature treatments. The luciferase substrate, 0.1 mM decanal, was added to the samples 15 min prior to measurements.

Growth at Standard, High Temperature and High Light Conditions
The cells were grown as 30-mL batch cultures in 100-mL Erlenmyer flasks in BG-11 medium that was buffered with 20 mM Hepes, pH 7.5. For standard growth conditions, cells were grown at 32 °C under continuous illumination at the photosynthetic photon flux density (PPFD) of 40 μmol m −2 s −1 in ambient air. The light source was a mixture of fluorescent tubes, light colors 865 and 840 (Osram (Munich, Germany)/Philips (Amsterdam, The Netherlands)). The flasks were shaken at 90 rpm.
To induce long-term, high temperature stress, the OD730 of the cell culture was set to 0.06, and 30-mL cell cultures in 100-mL Erlenmyer flasks were grown in Hepes-buffered Figure 1. Activities of sigB and sigD promoters in high light and temperature. The 402 bp fragment upstream of the sigB coding region (A) or 284 bp fragment upstream of the sigD coding region (B) was inserted upstream of the promoter-less lux operon, and nourseotricine antibiotic cassette (NAT) was added after the lux operon for antibiotic selection. The promoter-lux-NAT construct was inserted in the middle of the non-essential psbA1 gene, and the CS strain of Synechocystis was transformed with linearized plasmids to produce PsigB-and PsigD-lux strains. HR1 and HR2 are proximal areas of the psbA1 gene used for homologous recombination. (C) PCR verification of PsigB-and PsigD-lux strains. The amplicon in the CS strain is 829, 8043 in PsigB-lux, and 7925 bp in PsigD-lux. The primers are marked as P1 and P2 in (A,B). (D) Luciferase activity was measured from control (CS), PsigB-, and PsigD-lux strains (3-day old cultures) at standard growth conditions or after 15 min of high light or temperature treatments. The luciferase substrate, 0.1 mM decanal, was added to the samples 15 min prior to measurements.

Growth at Standard, High Temperature and High Light Conditions
The cells were grown as 30-mL batch cultures in 100-mL Erlenmyer flasks in BG-11 medium that was buffered with 20 mM Hepes, pH 7.5. For standard growth conditions, cells were grown at 32 • C under continuous illumination at the photosynthetic photon flux density (PPFD) of 40 µmol m −2 s −1 in ambient air. The light source was a mixture of fluorescent tubes, light colors 865 and 840 (Osram (Munich, Germany)/Philips (Amsterdam, The Netherlands)). The flasks were shaken at 90 rpm.
To induce long-term, high temperature stress, the OD 730 of the cell culture was set to 0.06, and 30-mL cell cultures in 100-mL Erlenmyer flasks were grown in Hepes-buffered (pH 7.5) BG-11 medium at 42 • C or at 40 • C, as indicated, PPFD 40 µmol m −2 s −1 , ambient air. Cells were grown in Climatic Chamber KK1200 (Pol-Eko Aparatura ® , WodzisławŚląski, Poland), the same light source as in standard conditions was used, and the temperature was monitored during experiments. Three independent biological replicates were tested.
Growth was monitored by measuring OD 730 once a day. Dense cultures were diluted, so that the measured OD 730 did not exceed 0.4, and the dilutions were taken into account when the results were calculated. In vivo absorption spectra were measured from 2-day old cells, as described in [16].

Imaging Cell Cultures
Images of cultures were taken with a Canon EOS 250D camera (Canon, Tokio, Japan) equipped with a Canon Compact-Macro EF 50 mm lens (Canon, Tokio, Japan), after 2 and 3 days of growth. For single cell imaging, cells were diluted to OD 730 of 0.0175, 100 µL of each culture was transferred to a 96-well plate, and images were taken with a Nikon Eclipse Ti2-E-microscope (Nikon, Tokio, Japan) and Nikon DS-Fi3-camera (Nikon, Tokio, Japan).

Activities of sigB and sigD Promoters
The lux reporter gene operon was expressed under the sigB or sigD promoters. The construction of these strains is described above. Bacterial strains containing the whole lux operon are expected to produce light without the addition of a substrate. However, in Synechocystis, we found that the addition of the substrate decanal greatly enhanced the light signal. The CS, PsigB-lux, and PsigD-lux cultures were grown in standard growth conditions for three days. Prior to measurements, 200 µL of cell cultures were supplemented with 0.1 mM decanal in microplate wells (black, flat, clear bottom polystyrol, Corning), after which cells were incubated in standard, high temperature (40 • C) or high light (PPFD 380 µmol m −2 s −1 ) conditions for 15 min, and then luminescence and OD 730 were measured with a plate reader (Infinite Pro200, Tecan, Männedorf Switzerland). Luminescence values were calculated relative to OD 730 .

RNAseq Analysis
Thirty-ml cell cultures were grown for three days in standard conditions (to OD 730 0.6). Cells were then collected from the standard conditions or treated for 1 h at the PPFD of 750 µmol photons m −2 s −1 or 1 h at 42 • C, as indicated, maintaining the other conditions same as in the standard conditions. Three biological replicates were prepared for each treatment. A total of 15 mL of cell suspension was poured to a pre-frozen centrifugation tube, and a cell pellet was collected by centrifugation at 7000× g for 5 min at 4 • C. Cell pellets were frozen in liquid nitrogen. Isolation of total RNA was done with the hot phenol method [20]. Sequencing libraries for total RNA samples were prepared using Illumina TruSeq Stranded mRNA kit protocol. The quality of the library was confirmed using the Advanced Analytical Fragment Analyzer (Advanced Analytical Technologies, Heidelberg, Germany), and the concentrations of the libraries were quantified with Qubit ® Fluorometric Quantitation (Life Technologies, ThermoFisher, Waltham, MA, USA). Sequencing was done with a HiSeq2500 Next Generation Sequencing platform at the Finnish Functional Genomics Centre. The quality control of raw sequencing reads was performed with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ accesed on 20 November 2020). Reads were aligned to the Synechocystis sp. PCC 6803 (Moscow strain) reference genome (Ensemble release 35) using HISAT2 [21]. HTseq-count [22] was used to calculate summarized read counts for each gene, except that the two ribosomal RNA operons were excluded from the analysis. DESeq2 [23] was used to identify differentially expressed genes. Wald test statistic is used for hypothesis testing in DESeq2. Genes with a false discovery rate, <0.05, were considered to show significantly changed expression. Data analysis was done with the CSC's Chipster v.4 platform [24]. We considered the gene to be up-or down-regulated in a mutant strain if its expression was at least two-fold up-regulated (log 2 fold change ≥ 1) or down-regulated to one half or less (log 2 fold change ≤ −1), compared to the CS strain in the same conditions, respectively.
For regulon analysis, we chose genes showing a log 2 fold change ≥ 1 or ≤ −1 in at least one of the mutant strains, compared to the control strain in the same conditions. Genes were assigned to the SigB regulon if they were significantly down-regulated both in ∆sigB and ∆sigBCE strains, but not in the other strains (up-regulation in ∆sigCDE was allowed), or significantly up-regulated both in ∆sigB and ∆sigBCE strains, but not in the other mutant strains (down-regulation in ∆sigCDE was allowed). Genes were assigned to the SigD regulon if they significantly down-regulated both in ∆sigD and ∆sigCDE strains, but not in the other mutant strains (up-regulation in ∆sigBCE was allowed), or significantly up-regulated both in ∆sigD and ∆sigCDE strains, but not in the other mutant strains (down-regulation in ∆sigBCE was allowed). Genes simultaneously significantly down-or up-regulated in both triple mutant strains, but not in the single mutants, were considered to belong the SigC and/or SigE regulons. Since the ∆sigC and ∆sigE strains were not included in the study, we cannot tell whether a gene belongs to the SigC or the SigE regulon. To visualize the results, heat maps were created using a web tool [25]. RNAseq data are available in GEO (accession GSE192357).

Response of Promoters of sigB and sigD Genes to Heat and High Light
To study the specific roles of SigB and SigD factors, we first measured the response of sigB and sigD promoters to heat and high light. To that end, the predicted promoter regions of sigB and sigD genes were cloned in front of the luxCDABE operon from Photorhabdus luminescens, and the non-essential psbA1 gene was replaced from Synechocystis genome with these constructs to produce PsigB-and PsigD-lux strains ( Figure 1A-C). The whole luxCD-ABE operon, as such, is able to induce production of luminescence in E. coli. However, in Synechocystis, measured luminescence levels were low, without the addition of the substrate decanal; the decanal was, therefore, added to cell samples 15 min prior to measurements.
The background luminescence signal from the control strain without the lux construct was negligibly low in all studied conditions ( Figure 1D). Luminescence levels remained low in PsigB-and PsigD-lux strains in standard growth conditions ( Figure 1D). In high light, luminescence of the PsigD-lux strain increased almost five-fold, whereas that of the PsigB-lux strain was tripled ( Figure 1D). At high temperature, the luminescence levels of PsigB-and PsigD-lux strains were 2.2-and 1.4-fold higher than in the standard growth conditions, respectively. These results indicate that both sigB and sigD genes are activated by high light and temperature, suggesting that SigB and SigD play roles in acclimation to these stress conditions.

Comparison of Transcript Profiles of sigB and sigD Deletion Strains in Standard Growth Conditions
To better understand the roles of SigB and SigD σ factors, we compared transcriptomes of ∆sigB, ∆sigD, ∆sigBCE (with SigD as the only functional group 2 σ factor), and ∆sigCDE (SigB as the only functional group 2 σ factor) strains. Total RNA was analyzed from three biological replicates with RNAseq. In addition to our standard growth conditions, we also analyzed samples after 1 h treatments at high temperature (42 • C) or light (PPFD 750 µmol m −2 s −1 ). We selected 1 h treatment to allow the accumulation of SigB and SigD proteins after the stress-induced induction of sigB and sigD genes [5].
All genes showing statistically significant difference in at least one of the mutant strains, compared to the CS strain in standard growth conditions, are listed in Supplemen-  Table S1. For further analysis, we selected only genes whose expression was at least twice of that measured in CS or down-regulated to only half of that measured in CS in at least one of the mutant strains. Using these criteria, 43 genes showed differential expression in at least one of the mutant strains, compared to the control strain (Supplemental Table S1; Figure 2A); the inactivated group 2 σ factors were not included in these analyses. In the triple mutants, more genes (18 in ∆sigBCE and 22 in ∆sigCDE) showed at least 2-fold differential expression than in the single mutants (12 in ∆sigB and 5 in ∆sigD). The majority of differentially expressed genes, 24, belongs to Cyanobase categories unknown or hypothetical.
ΔsigCDE (SigB as the only functional group 2 σ factor) strains. Total RNA was analyzed from three biological replicates with RNAseq. In addition to our standard growth conditions, we also analyzed samples after 1 h treatments at high temperature (42 °C) or light (PPFD 750 μmol m −2 s −1 ). We selected 1 h treatment to allow the accumulation of SigB and SigD proteins after the stress-induced induction of sigB and sigD genes [5].
All genes showing statistically significant difference in at least one of the mutant strains, compared to the CS strain in standard growth conditions, are listed in Supplemental Table S1. For further analysis, we selected only genes whose expression was at least twice of that measured in CS or down-regulated to only half of that measured in CS in at least one of the mutant strains. Using these criteria, 43 genes showed differential expression in at least one of the mutant strains, compared to the control strain (Supplemental Table S1; Figure 2A); the inactivated group 2 σ factors were not included in these analyses. In the triple mutants, more genes (18 in ΔsigBCE and 22 in ΔsigCDE) showed at least 2-fold differential expression than in the single mutants (12 in ΔsigB and 5 in ΔsigD). The majority of differentially expressed genes, 24, belongs to Cyanobase categories unknown or hypothetical.  We selected genes showing either two-fold up-or down-regulation in at least one of the mutant strains for regulon analysis. The criteria placing genes to different regulons are described in Section 2.5. Note that when counting differently expressed genes in a mutant strain, we only count those ones that show at least two-fold expression differences, whereas in regulon analysis genes whose expression difference is less than two-fold, but significant, were counted. In standard conditions, the SigB regulon comprises of the norB, slr0284, slr1236, and slr1207 genes, whose expression was down-regulated in ∆sigB and ∆sigBCE (Figure 2A). SigD-dependent transcripts were the slr1612, ssl1263, fed7, and ycf35 genes (Figure 2A). Nine genes (tal, ssl1533, gnd, sll0098, sll0543, slr0978, hik35, opcA, and slr0869) were down-regulated and one (sll1219) was up-regulated in both triple inactivation strains, but not in the single mutants, suggesting that these genes are controlled by SigC or SigE factors rather than by SigB or SigD factors (Figure 2A). Two of the genes, sll0407 and ssr2153, were significantly down-regulated in all mutant strains. Overall, transcriptional differences between the strains were small; accordingly, similar growth rates were measured for all strains in standard growth conditions ( Figure 2B).

SigB Regulon Is Bigger Than SigD Regulon at High Temperature
In a high temperature treatment, the number of differently expressed genes in the mutant strains was higher than in standard growth conditions (Supplemental Table S2). In total, 221 genes were at least two-fold up-or down-regulated in at least one of the mutant strains, compared to the control strain after 1 h treatment at 42 • C (Figure 3). In the ∆sigB strain 25 genes and in ∆sigD strain 7 genes were differently regulated. In ∆sigCDE and ∆sigBCE, 51 and 172 genes were differently expressed, in comparison to the CS strain, respectively. Of these genes, 27 genes were up-regulated and 31 down-regulated in both triple inactivation strains but showed normal expression in both single mutants (Figure 3), indicating that the expression of 58 (27 + 31) genes was dependent on SigC or SigE factors, not SigB or SigD factors.
Only a few genes can be considered to belong to the SigD regulon at high temperature. The SigD regulon includes the down-regulated genes ycf35, rre12, ssl1263, sll1862, and slr0888, as well as the cmp operon ( Figure 4). The cmp genes encode the bicarbonate transporter BCT1, and Rre12 has been connected to chemotaxis in unfavorable conditions, whereas the biological functions of other genes in the regulon remains to be solved.
The SigB high-temperature regulon comprised of 36 genes. The up-regulated genes include pilA1 (a major structural protein of the pilus), glcD (a subunit of glycolate oxidase), rps12 (a protein of the small ribosomal subunit), hemA (transfer RNA-Gln reductase), and the slr0442 and slr1686 genes. The down-regulated genes include exbD (a putative biopolymer export protein), ftsH (a PSII repair machinery metalloprotease), gdhB (a putative glucose dehydrogenase), psb28-2 (Photosystem II subunit), clpC (ATPase subunit of Clp protease), pacS (copper-transporting P-type ATPase), trxA (thioredoxin), sufA (FeS cluster assembly accessory/regulatory protein), and ctpB (periplasmic carboxyterminal protease) genes, as well as 23 open reading frames encoding proteins with unknown functions ( Figure 3). As so many genes of this regulon belong to categories hypothetical/unknown, it remains to be elucidated how the SigB regulon actually helps cells to acclimate to high temperature. Gene expression results suggest that the SigB factor might play a more important role at high temperature than the SigD factor, as the SigB regulon is six times bigger than the SigD regulon. However, it should be noted that more genes actually belong to the SigC and SigE regulons (58 genes) than the SigB and SigD regulons (42 genes) at high temperature.  temperature treatment; after the third day, the cultures started to decline and died in less than one week. These results indicate that 42 °C is a too high temperature for maintaining Synechocystis cells. At 40 °C, CS cells continued to grow for a whole one-week experiment, indicating that 40 °C is a permissive condition ( Figure 4B). At 40 °C, growth of the triple inactivation strains did not differ from that measured for CS. Single mutants grew like the CS for the first three days; after that, the growth of the ΔsigD strain decelerated more than that of the other strains.  The importance of SigB and SigD during long-term exposure to high temperature was tested by growing cells at 42 • C. In comparison to the standard growth conditions, growth of all strains slowed down at 42 • C. After the first day at 42 • C, the OD 730 was only half of that measured in the standard growth conditions ( Figure 4A). Both triple inactivation strains grew more slowly than the CS strain at 42 • C, whereas ∆sigD and ∆sigB strains grew like the CS strain. The growth rates of all strains decreased upon prolonged high temperature treatment; after the third day, the cultures started to decline and died in less than one week. These results indicate that 42 • C is a too high temperature for maintaining Synechocystis cells. At 40 • C, CS cells continued to grow for a whole one-week experiment, indicating that 40 • C is a permissive condition ( Figure 4B). At 40 • C, growth of the triple inactivation strains did not differ from that measured for CS. Single mutants grew like the CS for the first three days; after that, the growth of the ∆sigD strain decelerated more than that of the other strains.

The SigD Regulon Is Bigger in High Light Than the SigB Regulon
After the high light treatment (1h, PPFD 750 µmol m −2 s −1 ), 8 genes were at least two-fold down-regulated and 5 genes were two-fold up-regulated in ∆sigB, compared to the CS strains, whereas, in ∆sigD, 29 genes were down-regulated and 9 were up-regulated (Supplemental Table S3, Figure 5). In ∆sigBCE and ∆sigCDE strains, 38 and 80 genes were down-regulated, respectively, whereas only 9 and 34 genes were up-regulated in those strains, respectively. The SigC or SigE regulons (regulated in both triple inactivation strains, but not in single mutants) comprise of 23 down-regulated and 7 up-regulated genes ( Figure 5), 1 and 6 of which were also, respectively, down-or up-regulated at high temperature.  The SigB high-light regulon comprised of an up-regulated glm gene (glucose-1-phosphate adenylyltransferase) and down-regulated ndhD2, ssl2501, slr1236, sll0847, and slr0284 genes ( Figure 5). For these, only ssl2501 belongs to the SigB high-temperature regulon.
The SigD high light regulon included 51 down-regulated and 11 up-regulated genes ( Figure 5). Up-regulated SigD regulon genes comprised of the pilA1, atpC, atpI, atpH, menA (plastoquinone synthesis), murI (peptidoglycan synthesis), pgk (phosphoglycerate kinase), fbpI (Lys propinyilation), slr0358, slr0013, and slr1177 genes. Interestingly, the pilA1 gene was found among the SigB regulon genes at high temperature. In addition, many ATP synthase genes were up-regulated in high light in the ∆sigD and ∆sigCDE strains missing the SigD factor, whereas, at high temperature, ATP synthase genes were up-regulated in the ∆sigBCE strains that contain SigD as only the functional group 2 σ factor. These results indicate that the expression of a particular gene might be dependent on different σ factors, depending on prevailing environmental conditions.
We next tested the growth of mutant strains at the same high light intensity, where RNAseq analysis was carried out. Growth of cells at the PPFD of 750 µmol photons m −2 s −1 ( Figure 6A) induced the formation of cell aggregates in all strains and, especially, strains ∆sigB and ∆sigBCE produced numerous aggregates ( Figure 6B). However, mixing cell cultures for a couple of minutes by pipetting broke up the aggregates ( Figure 6C), allowing for OD 730 measurements.
In standard conditions, Synechocystis cultures have an intense green color ( Figure 6D), but in high light colors of cultures turn to different tones of olive and yellowish-brown, depending on the mutant strain ( Figure 6B). Although cell cultures had different appearances, the growth of the different strains did not differ much, as only ∆sigBCE grew slightly faster than the other strains on the first day ( Figure 6A). As a high light treatment at PPFD 750 µmol photons m −2 s −1 did not improve growth, compared to the standard conditions, we next tested if a reduction of irradiation to PPFD 500 µmol photons m −2 s −1 would improve growth.
According to the OD 730 measurements, the growth rate at PPFD 500 µmol photons m −2 s −1 remained similar as at 750 µmol photons m −2 s −1 , and a great variability in culture colors between the strains was observed ( Figure 6E,F). Obviously, high light-induced pigment changes differ between the mutant strains. In vivo absorption spectra measurements showed that contents of phycobilin and chlorophyll a were similar in all strains in standard growth conditions, but carotenoid content was higher in ∆sigCDE and ∆sigD strains than in the other strains ( Figure 6G). The olive color of the CS strain in high light was due to simultaneous increase of carotenoid content and reduction of chlorophyll a and phycobilin contents, compared to standard growth conditions ( Figure 6G). The intense yellowishbrown color of the ∆sigCDE strain in high light is due to high accumulation of carotenoids and less pronounced reduction of chlorophyll a and phycobilin than in the CS strain ( Figure 6G). The ∆sigD strain shows a yellowish appearance in high light ( Figure 6B,F). The absorption spectra show that carotenoids are highly induced in the ∆sigD strain in high light, whereas chlorophyll a and phycobilin are more drastically reduced than in the other strains; together, these pigment changes explain the yellowish color of the ∆sigD culture in high light. Interestingly, the behavior of the ∆sigBCE strain that contains SigD as the only functional group 2 σ factor was opposite to that of ∆sigD strain. The ∆sigBCE strain remained greener in high light than the other strains ( Figure 6B,F), because it retained more chlorophyll a and phycobilin that the control strain ( Figure 6G). These results suggest that SigD plays an important role in the adjustment of photosynthetic pigments, according to light intensity. but ΔsigCDE and ΔsigD strains remained more yellowish than the other strains ( Figure  6H).  Finally, we reduced PPFD to 120 µmol m −2 s −1 . At PPFD 120 µmol m −2 s −1 , the CS strain grew 15% faster than in the standard growth conditions (Figures 6H and 2B). The ∆sigBCE strain grew slightly better than the other strains, but otherwise all mutants grew like the CS. Color differences between the strains were not as notable as in higher light conditions, but ∆sigCDE and ∆sigD strains remained more yellowish than the other strains ( Figure 6H).

Discussion
The construction of robust cyanobacteria strains is one of the current challenges for biotechnology applications. Inactivation of the three other group 2 σ factors leads to twofold overproduction of the remaining SigB or SigD factor in the ∆sigCDE and ∆sigBCE strains, respectively [16]. As a previous study [16] has shown that ∆sigCDE and ∆sigBCE strains tolerate chemically-induced oxidative stress better than the CS strain, we considered the possibility that extra SigB or SigD factors in the triple inactivation strain would protect cells in high light or temperature conditions. However, that was not the case; actually, our results point to the involvement of all group 2 σ factors in different stress conditions. To test if the fitness of the cells in some stress conditions could be improved by overexpressing a particular σ factor should, therefore, be done with a wild-type strains as the host strain.
The comparison of transcriptomes of ∆sigB, ∆sigD, ∆sigCDE, and ∆sigBCE strains, in standard conditions and after 1 h of treatment in high light (750 µmol photons m −2 s −1 ) or at temperature (42 • C), revealed new features and confirmed old findings of gene regulation in Synechocystis. Group 2 σ factors indeed play only marginal roles in standard growth conditions but are important for acclimation to suboptimal conditions [6]. Our results revealed that although SigB and SigD factors are close homologs [8], their regulons do not overlap (Figures 3, 5 and 7). Furthermore, the comparison of up-and down-regulated genes after high light and temperature treatments showed that both SigB and SigD regulate different sets of genes in high light and temperature (Figure 7). Although our study did not focus on SigC or SigE regulons, transcriptomes of triple mutants contained numerous sugar catabolism and other genes belonging to SigE regulon [26,27], and our results indicate that the SigE regulon does not show similar high variation between the studied environmental condition as SigB and SigD regulons. As both triple inactivation strains grew slowly at high temperature, our results suggest that the SigE-dependent regulation of sugar catabolic genes and energy production by respiration might play an important role in high temperature stress, when temperature-sensitive photosynthesis [28] might not be fully functional. A detailed analysis of the photosynthetic and respiration reactions in heat stressed σ factor mutant strains would be interesting.

Discussion
The construction of robust cyanobacteria strains is one of the current challenges for biotechnology applications. Inactivation of the three other group 2 σ factors leads to twofold overproduction of the remaining SigB or SigD factor in the ΔsigCDE and ΔsigBCE strains, respectively [16]. As a previous study [16] has shown that ΔsigCDE and ΔsigBCE strains tolerate chemically-induced oxidative stress better than the CS strain, we considered the possibility that extra SigB or SigD factors in the triple inactivation strain would protect cells in high light or temperature conditions. However, that was not the case; actually, our results point to the involvement of all group 2 σ factors in different stress conditions. To test if the fitness of the cells in some stress conditions could be improved by overexpressing a particular σ factor should, therefore, be done with a wild-type strains as the host strain.
The comparison of transcriptomes of ΔsigB, ΔsigD, ΔsigCDE, and ΔsigBCE strains, in standard conditions and after 1 h of treatment in high light (750 μmol photons m −2 s −1 ) or at temperature (42 °C), revealed new features and confirmed old findings of gene regulation in Synechocystis. Group 2 σ factors indeed play only marginal roles in standard growth conditions but are important for acclimation to suboptimal conditions [6]. Our results revealed that although SigB and SigD factors are close homologs [8], their regulons do not overlap (Figures 3, 5 and 7). Furthermore, the comparison of up-and down-regulated genes after high light and temperature treatments showed that both SigB and SigD regulate different sets of genes in high light and temperature (Figure 7). Although our study did not focus on SigC or SigE regulons, transcriptomes of triple mutants contained numerous sugar catabolism and other genes belonging to SigE regulon [26,27], and our results indicate that the SigE regulon does not show similar high variation between the studied environmental condition as SigB and SigD regulons. As both triple inactivation strains grew slowly at high temperature, our results suggest that the SigE-dependent regulation of sugar catabolic genes and energy production by respiration might play an important role in high temperature stress, when temperature-sensitive photosynthesis [28] might not be fully functional. A detailed analysis of the photosynthetic and respiration reactions in heat stressed σ factor mutant strains would be interesting.  SigB and SigD regulons at high temperature (42 • C) and light (PPFD 500 µmol photons m −2 s −1 ). Genes shown in blue were down-regulated, and those in red upregulated. Genes with bold font were up-regulated when the transcriptome of the CS strain was compared in high light or temperature to that of the CS strain in the standard growth conditions, and italics indicate genes down-regulated in the CS strain in the test conditions. Both triple inactivation strains grew more slowly than the CS or single inactivation strains at 42 • C ( Figure 4A), but this disadvantage of triple mutants disappeared at 40 • C ( Figure 4B). In triple mutants, this growth behavior might mainly be due to the missing SigC factor, as the ∆sigC strain has been shown to have serious growth defects at 43 • C, but not at 38 • C [28]. SigC is a growth restricting σ factor [29][30][31], and the formation of RNAP-SigC holoenzymes at high temperature occurs more regularly than at optimal temperatures [5]. In accordance with enhanced formation of growth restricting RNAP-SigC holoenzymes, the growth rate of the CS strain is very slow at 42 • C, in comparison to the optimal growth temperature (Figures 2 and 4A). RNAP-SigC induced adjustments of gene expression are important for heat acclimation. At high temperatures, numerous household genes, including transcription, translation, pigment synthesis, and photosynthesis genes, are not normally down-regulated upon temperature upshift in triple inactivation strains, and cells show early growth cessation at high temperature ( Figure 4). Obviously, the SigC controlled growth restriction is a central process for high temperature acclimation and retaining a functional SigC is a prerequisite for engineering temperature robustness of Synechocystis strains.
The amount of the RNAP-SigB holoenzyme is highly up-regulated at high temperature, whereas the amount of RNAP-SigD remains constant [5]; accordingly, more genes belong to the SigB regulon than to the SigD regulon at high temperature (Figures 3 and 7). The ∆sigB strain shows a low survival rate after a short term lethal temperature treatment (48 • C) and defects in the acquisition of acquired thermotolerance [10], but growth differences were not detected in milder heat stress (Figure 4; [10,11]). The overproduction of SigB in wild-type backgrounds has shown to increase heat resistance of Synechocystis cells [32], but we do not see the same effect in the ∆sigCDE strain that contains an abnormally high amount of SigB [16], but is missing the other group 2 σ factors.
Our results point to high flexibility and variability of high light acclimation. The growth rate of Synechocystis cells, at standard growth conditions (PPFD 40 µmol photons m −2 s −1 ) and in all tested high light conditions (120, 500 and 750 µmol photons m −2 s −1 ), remained similar in all mutant strains. One consequence of high light treatment is an accelerated rate of PSII damage. However, all our strains contained either the SigB or SigD factor, and it has been previously shown that the presence of either SigB or SigD can guarantee the normal high light-specific up-regulation of psbA genes that is required for efficient repair of photodamaged photosystem II centers [15].
The sigD gene is induced in high light [9,14], and the RNAP-SigD holoenzyme is accumulated in high quantities in high light [5], which explains why the SigD regulon is big in high light ( Figures 5 and 7). However, cells without the SigD factor did not show clear growth rate reductions in high light ( Figure 6). The ∆sigBCE strain shows up-regulation of RNAP-SigD holoenzyme formation, compared to the CS strain (Hakkila et al., 2019), and ∆sigBCE cells grow faster in the beginning of high light treatment, but this growth advantage disappeared after long high light treatment ( Figure 6A). The ∆sigBCE strain remains greener in high light than the other strains because the phycobilins and chlorophyll a are not degraded as much as in the other strains and the carotenoid content remains low. Previous studies have shown that the ∆sigBCE strain is more resistant against chemicallyinduced singlet oxygen and H 2 O 2 stresses than the control strain [3,16]. We have previously shown that the amount of the SigD protein is two times as high in the ∆sigBCE strain as the CS strain [16], suggesting that overproduction of SigD might offer protection against some reactive oxygen species. Obviously, the extra protection in the ∆sigBCE strain is not due to carotenoids, as the carotenoid content is lower in ∆sigBCE strain than in the other strains in high light ( Figure 6G). A high tendency of ∆sigBCE strain to form cell aggregates might function as a strategy to escape too high light. Both strains without the SigD factor (∆sigD and ∆sigCDE) were characterized by high accumulation of carotenoids ( Figure 6G). Carotenoids are especially important protective pigments, and Synechocystis cells are not viable in light without carotenoids [33]. Hence, the accumulation of carotenoids could be seen as another strategy to survive in high light.
The sigB gene is induced in high light (Figure 1), although to a lesser extent than the sigD gene, and similar induction of the RNAP-SigB holoezyme is not seen in high light as is seen for RNAP-SigD [3,16]. Our previous results have shown that high amounts of carotenoids, as well as Flv2, Flv4, and Sll0218 proteins, in ∆sigCDE reduce the production of singlet oxygen and confer resistance of PSII against light-induce damage; simultaneously, reduced contents of glutathione and Flv1/3 proteins led to more pronounced oxidative damage in ∆sigCDE than in the control strain. Due to these opposing effects, the ∆sigCDE grew like a control strain in high light but showed superior growth in H 2 O 2 stress [16]. Our results show that, actually, triple inactivation strains containing only SigD or SigB managed quite well in high light because their acclimation to some features of high light stress was superior, compared to the CS strains, and compensated for problems to acclimate other features of high light stress.

Conclusions
Our results show that the SigB and SigD factors control unique regulons with negligible overlay. Furthermore, the SigB regulon was found to be seven times bigger in heat stress than in high light and mainly comprised of genes with unknown functions. SigD regulon was bigger in high light than in heat stress and contained a different set of genes in different stress conditions. Our results point to the flexibility of the high light acclimation mechanism of Synechocystis and indicate that the SigD factor plays a role in adjusting pigment content, according to prevailing light conditions. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/life12020162/s1, Table S1: A list of genes whose expression is different (p < 0.05) at least in one of the mutant strains compared to the control strain (CS) in standard growth conditions., Table S2: A list of genes whose expression in at least in one mutant shows differential expression compared to that in the control strain after 1 h treatment at 40 • C., Table S3: A list of genes whose expression is significantly different (p < 0.05) compared to the control strain in at least of the mutant strains after 1 h treatment at photosynthetic photon flux density of 750 mmol m −2 s −1 .

Conflicts of Interest:
The authors declare no conflict of interest.