Role of Sigma Factors in Controlling Global Gene Expression in Light/Dark Transitions in the Cyanobacterium Synechocystis sp. Strain PCC 6803

We report on differential gene expression in the cyanobacteriumSynechocystis sp. strain PCC 6803 after light-dark transitionsin wild-type, ${Delta}$ sigB, and ${Delta}$ sigD strains. We also studied the effectof day length in the presence of glucose on a ${Delta}$ sigB ${Delta}$ sigE mutant.Our results indicated that the absence of SigB or SigD predominatelyaltered gene expression in the dark or in the light, respectively.In the light, approximately 350 genes displayed transcript levelsin the ${Delta}$ sigD strain that were different from those of the wildtype, with over 200 of these up-regulated in the mutant. Inthe dark, removal of SigB altered more than 150 genes, and thelevels of 136 of these were increased in the mutant comparedto those in the wild type. The removal of both SigB and SigEhad a major impact on gene expression under mixotrophic growthconditions and resulted in the inability of cells to grow inthe presence of glucose with 8-h light and 16-h dark cycles.Our results indicated the importance of group II ${sigma}$ factors inthe global regulation of transcription in this organism andare best explained by using the ${sigma}$ cycle paradigm with the stochasticrelease model described previously (R. A. Mooney, S. A. Darst,and R. Landick, Mol. Cell 20:335-345, 2005). We combined ourresults with the total protein levels of the ${sigma}$ factors in thelight and dark as calculated previously (S. Imamura, S. Yoshihara,S. Nakano, N. Shiozaki, A. Yamada, K. Tanaka, H. Takahashi,M. Asayama, and M. Shirai, J. Mol. Biol. 325:857-872, 2003;S. Imamura, M. Asayama, H. Takahashi, K. Tanaka, H. Takahashi,and M. Shirai, FEBS Lett. 554:357-362, 2003). Thus, we concludedthat the control of global transcription is based on the amountof the various ${sigma}$ factors present and able to bind RNA polymerase.

INTRODUCTION

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INTRODUCTION

Cyanobacteria represent a diverse group of organisms that occupyhabitats from oceans to lakes to soil. They are forced to dealwith many environmental changes, and the impact of many suchstresses has been investigated for various strains. One environmentalfactor that affects cyanobacteria each day in every habitatis the changing light conditions. The growth characteristicsof cyanobacteria and the response of the transcriptional machineryto different light conditions have been studied in some detail(1-4, 10, 14, 24).

The effort to understand how light affects the transcriptionof cyanobacterial genes, especially those encoding photosynthesisproteins, includes a number of microarray studies that describeddifferential transcription based on acclimation to high levelsof light (14, 15), response to UV-B and white light (16), andlight-to-dark transitions (10). More recently, Muramatsu andHihara (33, 34) have studied the high-light-responsive promoterof the psaAB operon as a follow up to a high-light microarrayexperiment (14). They concluded that the coordinated high-lightresponse of the photosystem I (PSI) genes is achieved by AT-richupstream sequences in Synechocystis sp. strain PCC 6803 (34).The experiments performed by Gill et al. (10) were closest tothose to be discussed in this paper, since they also put wild-typecultures through light-to-dark and dark-to-light transitions.However, that study did not include mutants in any of the keytranscriptional factors, as will be described herein.

The presence of multiple sigma factors represents one mechanismof regulating gene expression. Similar to Escherichia coli,cyanobacteria such as Synechocystis sp. strain PCC 6803 havean RNA polymerase (RNAP) that is able to bind different ${sigma}$ factors;these subunits specify transcriptional initiation at appropriatepromoters. Synechocystis sp. strain PCC 6803 has an essentialgroup 1 principal ${sigma}$ factor and four group 2 ${sigma}$ factors (see theCyanobase at http://www.kazusa.or.jp/cyano/cyano.html) (21).The group 2 ${sigma}$ factors (SigB, SigC, SigD, and SigE) are similarto the principal SigA in sequence and structure but are nonessentialfor any known condition (12, 32). Different group 2 ${sigma}$ factorshave been shown to modulate gene expression under differentconditions. For example, the ${sigma}$ factor, SigB, mediates transcriptionalresponses after exposure to heat shock and upon entering stationaryphase (9, 19, 44). Both SigB and SigC are up-regulated in stationaryphase and exhibit regulation of gene expression under low-nitrogenconditions (3, 18). SigE is involved in the response to nitrogendepletion as well as being a positive regulator of sugar catabolism(35). The group 2 ${sigma}$ factors also respond to light/dark stimuli;e.g., SigB and SigD have demonstrated antagonistic dark/light-inducedexpression via changes in redox potential and SigE was shownto accumulate in the light but did so more slowly than SigD(17).

Studies have indicated extensive regulation between the ${sigma}$ factorsthat are specific to particular growth conditions. For example,SigB and SigC were able to regulate each other's transcriptionallevels but with somewhat different effects in exponential- orstationary-phase conditions (18). Additionally, Imamura et al.(17) showed that SigC down-regulated SigB on transition fromdark to light. Recently, Yoshimura et al. (50) indicated thatSigD and SigE also negatively regulated SigB in the light. Cross-talkamong group 2 ${sigma}$ factors has been shown at the transcript level.For example, the work of Lemeille et al. (25) included nitrogendeprivation and long-term growth as experimental conditions,and they suggested a network of transcriptional interactionsbetween the group 2 ${sigma}$ factors. The recent work of Matsui et al.(27) indicated that the group 3 sigma factors may play a rolein these interactions. Importantly, Imamura et al. (19) purifiedall group 1 and group 2 ${sigma}$ factors and determined their intracellularlevels under steady-state growth conditions, as well as theirgrowth-phase-dependent changes. We will relate the levels ofthe ${sigma}$ factor proteins to the changes we find in global gene expressionwhen individual ${sigma}$ factors are no longer present in the cell.

The process of transcriptional initiation and elongation wasdescribed as a ${sigma}$ cycle in which ${sigma}$ associates with the RNAP toinitiate transcription and then dissociates after the formationof a stable elongation complex (7, 45). The polymerase can bindanother ${sigma}$ factor and may be reprogrammed by different ${sigma}$ factorsin each round of transcription. There is a great deal of evidenceto support this ${sigma}$ cycle paradigm, but the initial model did nottake into account interactions with multiple ${sigma}$ factors involvedwith global regulation. Thus, a number of versions of the ${sigma}$ cyclemodel have been proposed that try to account for the binding-dissociation-rebindingof ${sigma}$ factors to RNAP (6, 11, 31, 37, 40). Six different modelswere reviewed by Mooney et al. (30), and they compared the modelsusing recent experimental evidence. They concluded that thestochastic release model best conforms to both the in vitroand in vivo data. In this model, the affinity of the ${sigma}$ factorfor the RNAP decreases significantly after promoter release,rather than ${sigma}$ release at a specific stage of elongation, as suggestedby the obligate release model. Furthermore, in the stochasticrelease model, the dissociation of the ${sigma}$ factor from the RNAPmay be altered by factors including concentration of the RNAPcore and other ${sigma}$ factors. The authors suggest that the main postulateof the ${sigma}$ cycle, wherein ${sigma}$ factors compete for binding to the polymeraseafter each round of mRNA synthesis, is still the operative modelfor transcriptional initiation in bacteria. This is consistentwith competition between the ${sigma}$ factors, such that elevated levelsof alternative ${sigma}$ factors are able to direct transcription tospecific promoters and maintain the cell's ability to respondrapidly to environmental fluctuations.

In this report, we describe the differential transcription ofSynechocystis sp. strain PCC 6803 as cells transition from darkto light in the morning and from light to dark in the evening.We also performed similar experiments using mutations in sigBand sigD to investigate the roles of these ${sigma}$ factors at light/darktransitions. In addition, we examined autotrophic and mixotrophicgrowth of strains lacking combinations of SigB, SigD, and SigEto determine the physiological importance of these subunits.Finally, we analyzed the impact of light/dark transitions whencells are growing mixotrophically in the presence of glucoseusing the wild-type strain and a strain lacking SigB and SigE.We present a hypothesis that combines our data on transcriptionalregulation with the relative amounts of ${sigma}$ factors as determinedby Imamura et al. (17, 19) and that provides a simple, yet comprehensive,model of gene regulation as cells progress through their diurnalcycle.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Bacterial strains and growth conditions. The glucose-tolerant strain Synechocystis sp. strain PCC 6803was used throughout this study (48). Cultures were grown at30 ± 2°C using cool white fluorescent light at anintensity of $~$ 30 µE·m^–2·s^–1,with shaking at 125 rpm in BG-11 medium. The cell density ofthe cultures was determined by the optical density at 750 nmas previously described (8, 28). The antibiotic concentrationsused for the mutant strains were the following: 25 µg/mlkanamycin, 25 µg/ml spectinomycin, and 15 µg/mlchloramphenicol.

Construction of mutants. Mutations of sigB (sll0306), sigD (sll2012), and sigE (sll1689)were constructed by interruption or deletion of part of eachgene and insertion of an antibiotic resistance cassette, asdescribed by Singh et al. (44). A spectinomycin resistance cassettewas used to create ${Delta}$ sigB and ${Delta}$ sigE mutants, and a kanamycin resistancecassette was used to produce the ${Delta}$ sigD strain. Double mutantslacking SigB and SigD as well as SigD and SigE were producedusing the same constructs as those for the single mutants. Inthe case of the ${Delta}$ sigB ${Delta}$ sigE and ${Delta}$ sigB ${Delta}$ sigD ${Delta}$ sigE strains, the sigEgene contained a kanamycin resistance cassette and a chloramphenicolresistance cassette, respectively. Complete segregation of themutants was obtained as confirmed by PCR and Southern blotting(data not shown).

RNA isolation. Total RNA was extracted and purified using phenol-chloroformextraction and CsCl gradient purification as previously described(38, 42).

Microarray design. The microarray platform and construction were as described byPostier et al. (36). The cDNA labeling, glass treatment, prehybridization,and hybridization protocols were described in detail previously(41). Microarray experiments involved a loop design that allowedcomparison of all conditions by using an analysis of variance(ANOVA) model (26, 41). This work is comprised of four separateloop experiments. In the first two experiments, we analyzedthe effect of light or dark treatment on the wild type, on eachof two mutant strains, and on the relationship between the wildtype and the mutants. The third experiment examined the impactof the length of dark incubation on mixotrophically grown wild-typecells, and the fourth experiment compared a mutant strain tothe wild-type strain as described for the first two experiments.

The first loop compared the effects of light or dark treatmentof the wild-type strain to that of the ${Delta}$ sigB strain, and thesecond compared the light or dark treatment of the wild-typestrain to that of the ${Delta}$ sigD strain. In both experiments, cellswere grown under photoautotrophic conditions with constant lightfor 72 h and then were transferred to a 12-h dark and 12-h lightregimen. Cells were harvested at 1 h following transition tolight (L1) and 1 h following transition to dark (D1), as shownin Fig. 1A. The third loop experiment allowed examination ofthe wild type at different time points following a transitionto darkness. Cells were grown photoautotrophically in constantlight for 48 h before being transferred to a 16-h dark and 8-hlight regimen; 5 mM glucose was added at 40 h of this regimen.Samples were collected at 7 h light (GL7), 1 h dark (GD1), 8h dark (GD8), and 16 h dark (GD16), as shown in Fig. 1D. Thefourth loop compared the light or dark treatment of the wild-typestrain to that of the ${Delta}$ sigB ${Delta}$ sigE strain grown under the samemixotrophic conditions as those used for the third loop. Sampleswere collected at 1 h following transition to dark (GD1) andat 1 h following transition to light (GL1) (Fig. 1D). Biologicalvariation was sampled by pooling RNA extracted from three experimentsprior to labeling and hybridization.

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FIG. 1. (A) Photoautotrophic growth of cultures from the ${Delta}$ sigB and ${Delta}$ sigD experiments. The bar (top) represents the light-dark regimen used in these experiments. Cells were grown for 72 h in continuous light before beginning the 12-h light/12-h dark cycles. The growth curves for the wild-type (square), ${Delta}$ sigB (triangle), and ${Delta}$ sigD (diamond) strains, as measured by the optical density at 750 nm (OD₇₅₀), are shown to scale beneath the bar. The data are the averages ± standard errors of three independent measurements. The arrows represent the time points L1 and D1, at which cells were harvested and RNA was extracted and used for microarray analysis. (B and C) Histogram of genes differentially expressed 1.5-fold or more (FDR = 0.05) at L1 and D1. The numbers of differentially expressed genes for different functional categories are plotted on the x axis, with genes up-regulated in the mutant shown above the zero line on the y axis and genes down-regulated shown beneath zero line. (B) Comparison of the ${Delta}$ sigB strain to the wild type. (C) Comparison of the ${Delta}$ sigD strain to the wild type. Genes are presented as Cyanobase functional categories, as defined in the key beneath the graphs. (D) The light-dark regimen for mixotrophic growth of wild-type and ${Delta}$ sigB ${Delta}$ sigE strains (top). The cells were grown for 72 h in the light before transfer to a 16-h dark/8-h light cycle. The addition of glucose (Glu) to a final concentration of 5 mM is indicated. The arrows on the top bar represent time points L7, D1, D8, and D16, the points at which wild-type cells were harvested and RNA was extracted and used for microarray analysis. The arrows on the second bar represent time points D1 and L1, the points at which wild-type and ${Delta}$ sigB ${Delta}$ sigE cells were harvested and RNA was extracted and used for microarray analysis. The growth curves for the wild type (square) and ${Delta}$ sigB ${Delta}$ sigE (diamond), as measured by the OD₇₅₀, are shown to scale beneath the bar. The data are the averages ± standard errors of three independent measurements. (E and F) Histogram of genes differentially expressed 1.5-fold or more (FDR = 0.05). (E) Genes differentially regulated in the wild type at L7 compared to D1 and D16. (F) Genes differentially regulated in the ${Delta}$ sigB ${Delta}$ sigE strain compared to the wild type at L1 and D1. The genes were divided into functional categories as described for panels B and C.

Data analysis. Spot intensities of the images were quantified by using Quantarray3.0 (Packard BioChip Technologies, Boston, MA). Data for theslides used in each experiment then were collated into fourdata sets (one for each experiment) by using SAS (version 8.02;SAS Institute, Cary, NC). The local background was subtractedfrom each spot. For each replicate block on a slide, there were480 empty spots, and there were three replicates per slide.We examined the intensities for these empty spots and declareddata from a nonempty spot to be detected if the background-correctedintensity of the spot was greater than that for 95% of the emptyspots. If all the spots for a given gene were not detected onall the slides in an experiment, then the gene was consideredto be off and was not analyzed further (282 genes in the ${Delta}$ sigBexperiment, 652 genes in the ${Delta}$ sigD experiment, 326 in the wild-typemixotrophic growth experiment, and 414 genes in the ${Delta}$ sigB ${Delta}$ sigEexperiment). We then calculated the log of the background-correctedsignals that were normalized to the slide median (the medianfor all non-control spots detected). Three of the experimentscontained two genotypes (mutant and wild type) and two stimuli(mutant and wild type for light and dark transitions) for atotal of four treatment combinations. The effects of the mutantand the light/dark stimuli were examined in an ANOVA essentiallyas described previously (22, 23, 26, 41). Our normalizationand overall statistical approach enabled us to make comparisonsamong our experiments without the need for renormalization (26,41).

The above analysis included the use of the very stringent Bonferronicorrection, which emphasizes very strong effects when many testsare performed (47). Many such analyses now use the false discoveryrate (FDR), which controls the proportion of significant resultsthat are type I errors (false rejection of the null hypothesis)(5, 47). Once our initial analysis was completed, we used theFDR of 5% to control the number of false positives in gene lists.Genes with such FDRs (corresponding to 5% expected false positives)and that exhibited a change of at least 1.5-fold were consideredinteresting and were retained for further analysis. The entiredata set for the ${Delta}$ sigB, ${Delta}$ sigD experiments, the wild-type timecourse, and the ${Delta}$ sigB ${Delta}$ sigE experiment are shown in Table S1 inthe supplemental material. These data include the fold changesand the various P values from the ANOVA analysis.

qPCR. RNA was treated with DNase I (Invitrogen, Carlsbad, CA) for1 h at 37°C, and successful DNase treatment was confirmedby quantitative PCR (qPCR) on each DNase-treated RNA sample.Reverse transcription was performed using Superscript II (Invitrogen)and random primers by following the manufacturer's instructions.Primer Express software (Applied Biosystems, Foster City, CA)was used to design primers for the following genes: slr2072(forward, 5'-GTTGGGTACCAGGGCGATTA-3'; reverse, 5'-CTTTGACCGCCTCCACTTTC-3'),sll0329 (forward, 5'-CGGCTTGAGCAACGAACA-3'; reverse, 5'-TCATCGGTTTGATTCCATTGG-3'),slr0474 (forward, 5'-CCTTGTCCAGGAGGTGTTGAA-3'; reverse, 5'-CCCGGAGAATGATCAGTTCGT-3'),slr1311 (forward, 5'-GGTTGGTTCGGTACCTTGATGA-3'; reverse, 5'-CGGCGATGAAGGCAATG-3'),and slr1667 (forward, 5'-GATTGTTCTGATGCTACTGGTTGTG-3'; reverse,5'-AACTTGCTCTTCTGCGATTGC-3'). qPCR was performed on the ABI7300 real-time PCR system (Applied Biosystems) using a SYBRgreen master mix and 50 nM primers (Applied Biosystems) at 95°Cfor 10 min and then 40 cycles of 95°C for 15 s and 60°Cfor 1 min. The slr2072 transcript abundance was used as an internalcontrol for normalization, as microarray data from this studyand previous studies (9, 41, 44) indicated that the transcriptlevel for slr2072 was unchanged under a number of growth conditions.Standard curves used for each primer set indicated similar efficienciesfor the slr2072 primers and all other primer pairs in the rangeof 1 ng to 10 pg cDNA. The results represent the means of triplicatetechnical replicates for duplicate biological samples.

RESULTS

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RESULTS

Differentially regulated genes in the wild type at L1 compared to D1. The design of the experiment is presented in Fig. 1A. The cultureswere grown in continuous light for 3 days and then in 12-h darkand 12-h light periods. Samples were taken after 1 h in thelight (L1) and after 1 h in the dark (D1). As indicated in Materialsand Methods, we considered genes to be differentially regulatedif they showed a fold change of $≥$ 1.5 with an FDR of 0.05. Forthe wild type, the expression of 19% of the chromosomal genesmet these criteria (605/3,165 genes) at L1 compared to D1, andabout two-thirds of these transcripts (399/605 genes) were up-regulatedat L1 (Table 1). This is similar to the number of genes (387)positively regulated following 30 to 90 min of light in theexperiment of Gill et al. (10). It should be noted that Gillet al. (10) compared light-exposed samples to 24-h dark-incubatedsamples, not 1-h-dark samples, as in our experiment.

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TABLE 1. Differentially regulated genes organized by functional category^a

The differentially expressed genes were divided into functionalcategories according to the Cyanobase designation, and the numberof genes in each category is shown in Table 1. The functionalcategories (excluding hypothetical genes) with the largest percentageof differentially regulated genes at L1 relative to D1 werephotosynthesis and respiration (67/141 genes) and translation(67/168), and these were mostly up-regulated. Other categoriescontaining elevated levels of transcripts after 1 h of growthin the light included biosynthesis of cofactors, prostheticgroups, and carriers (15 genes); cell envelope (13 genes); energymetabolism (15 genes); regulatory functions (10 genes); andtransport and binding proteins (19 genes). Many up-regulatedgenes belonged to the hypothetical (82) or unknown (41) functionalcategories, and more than 50% (111/206 genes) of the transcriptsdown-regulated also belonged to these categories (Table 1).Other down-regulated genes encoded products that are involvedin energy metabolism (15), regulatory functions (12), transportand binding proteins (13), and biosynthesis of cofactors, prostheticgroups, and carriers (12). These data indicated that genes inmany functional categories were affected when cells sensed light.

The absence of SigB predominately alters gene expression in the dark. One objective of this study was to determine the impact on globaltranscription of removing various ${sigma}$ factors. The growth curvesfor the wild type, ${Delta}$ sigB, and ${Delta}$ sigD under photoautotrophic growthconditions are presented in Fig. 1A, and it can be seen thatgrowth was virtually identical among the three cultures. Removalof SigB led to very little difference in transcript levels fromthose of the wild type at L1, and only 48 genes (31 hypotheticalor unknown) were differentially regulated in the mutant comparedto the wild type (Table 1 and Fig. 1B). Selected genes thatdemonstrated significant transcriptional changes at L1 in ${Delta}$ sigBrelative to the wild type are shown in Table S2 in the supplementalmaterial. In contrast, 160 genes were differentially regulatedat D1 in ${Delta}$ sigB relative to the wild type (Table 1), and the majorityof these (136 genes) were increased in the mutant. Genes up-regulatedin the mutant included 28 that encoded proteins involved intranslation: 10 with regulatory functions, 9 encoding photosynthesisand respiration proteins, and 6 others involving energy metabolism(Table 1; also see Table S2 in the supplemental material).

Absence of SigD alters expression of more genes in the light than in the dark. The removal of SigD resulted in considerably more changes inthe global transcription pattern (Table 1 and Fig. 1C). In thelight, some 345 genes displayed transcript levels in ${Delta}$ sigD differentfrom those of the wild type, with 214 of these genes up-regulatedin the mutant. The genes with elevated transcript levels includedgenes encoding proteins involved in photosynthesis and respiration(22 genes), amino acid biosynthesis (15 genes), energy metabolism(12 genes), regulation (12 genes), and translation (12 genes).In the dark, fewer genes showed differential expression, although149 genes in the ${Delta}$ sigD strain were regulated differently thanthose of the wild type. It was evident that the removal of SigDhad a more profound effect on transcription in the light thanremoval of SigB. On the other hand, although they both affecteda similar number of genes in the dark, the removal of SigB resultedin the up-regulation of the genes affected, whereas SigD affectedtranscription in both directions (Table 1; Fig. 1B and C).

Differential expression of selected genes. Transcription of genes encoding photosynthesis and respirationproteins demonstrated light-responsive regulation during dark-lighttransitions, with the photosynthesis genes mostly up-regulatedin the light. This pattern can be seen clearly in Table 2, wherewe list select groups of differentially regulated genes involvedin photosynthesis or with central metabolism. Similar to theresults of Gill et al. (10), there was up-regulation of genesthat encode proteins involved with the structure and assemblyof the photosynthetic membrane. This included phycobilisomegenes encoding components of both the peripheral rods (phycocyanin)and core (allophycocyanin), six PSI genes, genes encoding anumber of core components of PSII (also observed in reference13), and both ATP synthase operons. Table 2 displays the transcriptionalchanges in the light compared to the dark for the wild typefor both the light-dark experiments as well as for the respectivemutant in each experiment. The comparison of the changes inthe wild type is informative and provides an idea of the closecorrespondence of the results between such repetitions.

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TABLE 2. Relative transcript abundance of selected differentially regulated genes^a

In our experiments, the photosynthesis genes were always representedamong the genes with the highest transcript levels. Thus, photosynthesisgenes represented 20 to 25% of the top 200 transcript levelsin both the light and the dark (based on the data prior to normalization).Light-responsive expression in many photosynthesis genes thathas been reported previously (10) included the up-regulationof psbA transcripts encoding the PSII protein D1 (39). The foldchanges we observed were attenuated in two ways. First, thetwo psbA transcripts, psbA2 (slr1311) and psbA3 (sll1867), couldnot be distinguished by the microarray, as the coding regionsdiffer by only 4 nucleotides. In addition, in comparisons ofL1 to D1, the fold changes in the psbA2 and psbA3 transcriptsoften were less than 1.5-fold due to the high transcript levelsunder both light and dark conditions. This was not unexpected,as the psbA transcript levels have been shown to remain elevatedfollowing at least 1 h of dark incubation (29), and our darkincubation time was 1 h. In addition, the psbA transcript levelswere so abundant in the dark that further elevation of expressionin the light may be outside the dynamic range of our detectionsystem. This also may explain why we do not observe the $~$ 1.5-foldreduction of both psbA2 and psbA3 transcripts in the absenceof SigD compared to the wild type, which were reported by Imamuraet al. (17).

The complete data set for all genes in all four experimentsis provided in Table S1 in the supplemental material. This tableincludes the P values obtained from the ANOVA and indicateshow individual genes were affected after cells went betweendark and light growth conditions, either in a wild-type backgroundor in the absence of SigB, SigD, or SigB and SigE. A few genesfrom important functional categories are shown in Table 2 andin Table S2 in the supplemental material. Certain genes thatclearly were light regulated were altered by the absence ofSigB or SigD; these are shown in Table 2. This included photosynthesisgenes, such as ATPase genes, that were enhanced by the lightin the wild type and to a similar level in ${Delta}$ sigB, but most werefurther up-regulated in ${Delta}$ sigD. Similarly, genes encoding manyphycobilisome components were up-regulated by the light in thewild-type and ${Delta}$ sigB strains. Many of these genes were furtherincreased in the ${Delta}$ sigD strain in the light, and some also wereelevated in the dark in the ${Delta}$ sigD strain compared to wild-typelevels (e.g., cpcC2, cpcC1, and cpcD). Similarly, the removalof SigD had a greater impact than the removal of SigB on PSIand PSII genes (Table 2). Key chaperone genes had previouslybeen shown to be up-regulated in the light (10), and these wereup-regulated in this study in the wild type and in the ${Delta}$ sigBand ${Delta}$ sigD strains. The enhancement in transcript level was virtuallyidentical between the wild type and ${Delta}$ sigB, whereas the genesgroES and groEL2 were further up-regulated in ${Delta}$ sigD (Table 2).Importantly, most of the genes encoding ribosomal proteins had $~$ 2-fold more abundant transcripts in the light than in the darkfor the wild-type, ${Delta}$ sigD, and, to a lesser extent, ${Delta}$ sigB strain(see Table S1 in the supplemental material).

The transition from light to dark resulted in a shift from photosynthesisand the accumulation of carbohydrates to sugar catabolism throughglycolysis and the oxidative pentose phosphate pathway (OPPpathway). A number of genes that are involved in sugar catabolismand that are regulated by a histidine kinase (Hik8) or by SigE(35, 43) exhibited light-sensitive transcription. In the wildtype, four genes involved in glycolysis were differentiallyregulated at L1 compared to their regulation at D1 (see TableS2 in the supplemental material). This included fbaA and pgk,which were up-regulated in the light, whereas the levels oftranscripts of both phosphofructokinase genes (pfkB1 [sll1196]and pfkB2 [sll0745]) were elevated in the dark (Table 2) (43).The strain ${Delta}$ sigD had decreased levels of the fbaA transcriptin the dark and elevated pgk transcript in the light, and thesefindings are shown in Table 2 as increased fold changes betweenlight and dark gene expression. Other glycolysis genes thatwere unchanged in the wild type but were up-regulated in ${Delta}$ sigDin the light included eno, fbaI, pgi, and pgm. In addition,the transcript encoding gap1 was unchanged in the wild typeat L1 compared to its status at D1, as previously reported (43),but was down-regulated in the ${Delta}$ sigD mutant in the light. In general,the transcription of these genes was affected more by the lackof SigD than the lack of SigB (Table 2).

The OPP pathway plays an important role in glucose breakdownand generation of reducing power (43). In the wild type, down-regulatedtranscripts in the light included the genes encoding two keyenzymes in the OPP pathway, gnd and zwf, as well as the glucose6-phosphate dehydrogenase assembly protein (opcA [slr1734])and transaldolase (tal [slr1793]). Changes in transcript abundanceagreed with previous reports of dark-responsive regulation ofthese four genes (35, 43). Two genes up-regulated by light inthe wild type were rpe and tktA (Table 2). Overall, more ofthe genes encoding energy metabolism enzymes were differentiallyregulated in the ${Delta}$ sigD strain compared to the wild type thanin the ${Delta}$ sigB strain compared to the wild type. The numbers ofdifferentially regulated genes are shown in Table 1.

Photoautotrophic, mixotrophic, and heterotrophic growth of ${sigma}$ factor mutants. We next studied the effect of removing SigB, SigD, and SigEon growth under photoautotrophic, mixotrophic, and heterotrophicconditions. The ${Delta}$ sigB, ${Delta}$ sigD, and ${Delta}$ sigB ${Delta}$ sigD strains had doublingtimes comparable to those of the wild type under photoautotrophicgrowth conditions (Fig. 1A). The absence of either SigB or SigDaltered some of the transcripts encoding components of sugarmetabolism following transition from light to dark (Table 2;also see Table S1 in the supplemental material). Nonetheless,the ${Delta}$ sigB, ${Delta}$ sigD, and ${Delta}$ sigB ${Delta}$ sigD strains had doubling times similarto those of the wild type in mixotrophic conditions, includingunder different light regimens (Table 3). A number of genesinvolved in sugar catabolism exhibited light-sensitive transcriptionand were shown to be regulated by SigE (35), and we observedthat ${Delta}$ sigE exhibited decreased mixotrophic growth with 12-h/12-hor 8-h/16-h light/dark regimens (Table 3). Under these conditions,the removal of SigB or SigD from the ${Delta}$ sigE strain reduced growth,such that the ${Delta}$ sigB ${Delta}$ sigE strain grew less than the ${Delta}$ sigD ${Delta}$ sigEstrain, and the ${Delta}$ sigB ${Delta}$ sigD ${Delta}$ sigE strain was unable to grow in12-h/12-h light/dark or 8-h/16-h light/dark regimens (Table3). Similarly, the transfer of cultures grown on BG-11 plates(supplemented with 5 mM glucose) from continuous light to continuousdark resulted in decreased growth of the ${Delta}$ sigE strain comparedto that of the wild type and a further decrease in growth ofthe ${Delta}$ sigB ${Delta}$ sigE, ${Delta}$ sigD ${Delta}$ sigE, and ${Delta}$ sigB ${Delta}$ sigD ${Delta}$ sigE strains (Fig. 2).However, under autotrophic conditions with different light regimensand mixotrophic conditions with 24 h of light, growth of thesestrains was similar to that of the wild type (Fig. 2). Theseresults indicated that whereas SigE is the principal group 2 ${sigma}$ factor involved in sugar catabolism, SigB and SigD do playat least a secondary role in regulating gene expression at light/darktransitions and, thus, affect cell viability when SigE is absent.

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TABLE 3. Mixotrophic growth characteristics of Synechocystis sp. strain PCC 6803 wild type and sigma factor mutants under different light regimens

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FIG. 2. Effect of sigma mutations on heterotrophic growth. Cultures of the wild type (WT) and sigma mutants were grown photoautotrophically and were spotted (at the optical density at 750 nm indicated) onto BG-11 plates containing 5 mM glucose. Plates were incubated in the light for 96 h (left gels) and then were transferred to the dark for 96 h (right gels).

Previous microarray analyses indicated that during long-termgrowth in continuous light, the presence or absence of glucosehad little impact on gene expression in Synechocystis sp. strainPCC 6803 (20, 46). Furthermore, Yang et al. (49) reported thatcultures grown with glucose in the light and dark showed alteredexpression of only 2 out of 13 transcripts examined, but theexpression level of many proteins was altered. The phenotypeof the SigE mutants was seen following periods of heterotrophicgrowth, indicating the importance of ${sigma}$ -factor-mediated transcriptionalregulation under these conditions. Therefore, we examined changesin gene expression in the presence of glucose on transfer fromlight to dark under conditions that resulted in impaired growthof the strains that lacked SigE in combination with SigB and/orSigD.

Differential gene expression of the wild type during mixotrophic growth. In wild-type cells grown under mixotrophic conditions (8 h oflight/16 h of dark), only $~$ 3 to 5% of genes were differentiallyexpressed in the light (7 h) compared to expression in the dark(1 or 16 h) (Fig. 1D). A large number of transcripts encodingthe ribosomal protein genes were down-regulated (1.5- to 4.4-fold)on transition from light to dark, and the levels of many ofthese transcripts also were decreased following 16 h in thedark compared to their levels after 7 h in light (Fig. 1E; alsosee Table S1 in the supplemental material). A small number ofgenes from several functional categories, including photosynthesis,energy metabolism, and regulatory proteins, were differentiallyregulated following transition from light to dark (see TableS1 in the supplemental material).

The fourth microarray experiment compared gene expression ofthe wild type to that of the ${Delta}$ sigB ${Delta}$ sigE mutant, as representedby the lower bar in Fig. 1D. This experiment included comparisonof gene expression in wild-type cells incubated for 1 h in thedark to expression in wild-type cells incubated for 1 h in thelight (Fig. 1D), which showed that $~$ 7% of transcripts were differentiallyregulated, of which about 61% were up-regulated (Table 1). Morethan half of these genes belonged to the unknown, hypothetical,or other functional categories. Genes encoding many of the ribosomalproteins were up-regulated in the light compared to the dark(1.7- to 6.7-fold), consistent with results of the previousmicroarray experiments in which there was down-regulation ofribosomal protein genes following transfer from light to dark.Genes up-regulated in the light included photosynthesis andrespiration genes (e.g., ATP synthase and CO₂-concentratingmechanism genes) and genes encoding regulatory proteins. Differentiallyregulated genes involved in energy metabolism included fivein glycolysis and five in the OPP pathway (see Table S3 in thesupplemental material).

The absence of SigB and SigE has a major impact on gene expression under mixotrophic growth conditions. Levels of gene expression of the wild type and the ${Delta}$ sigB ${Delta}$ sigEmutant were compared for cells grown in the dark (1 h) and thelight (1 h) under mixotrophic conditions with 8 h of light and16 h of dark. Figure 1D shows the experimental design as wellas the finding that the ${Delta}$ sigB ${Delta}$ sigE mutant grows more slowly thanthe wild type after the addition of glucose under these short-dayconditions. At D1, 883 genes were regulated differently thanthey were at L1 in the ${Delta}$ sigB ${Delta}$ sigE strain, almost double the numberof genes that were differentially expressed in the wild type.In the dark, 267 genes were differentially regulated in themutant compared to the wild type, of which almost half encodedhypothetical, unknown, or other proteins. As shown in Fig. 1F,many of the genes (45) encoding ribosomal proteins were up-regulatedin the ${Delta}$ sigB ${Delta}$ sigE mutant in the dark (1.5- to 3.5-fold), consistentwith the up-regulation of genes encoding ribosomal proteinsthat was observed with the ${Delta}$ sigB mutant in the dark under photoautotrophicconditions. Glycolysis and OPP pathway genes were down-regulatedin the dark in the ${Delta}$ sigB ${Delta}$ sigE strain compared to their expressionin the wild type (see Table S3 in the supplemental material).A number of these genes were reported to be down-regulated inthe absence of SigE in the dark (35).

In the light, more than 30% of the genes (998) were differentiallyregulated in the ${Delta}$ sigB ${Delta}$ sigE strain compared to the wild type,with 618 of these genes down-regulated in the mutant. Functionalcategories containing down-regulated genes included photosynthesisand respiration (78 genes), translation (63 genes, mostly ribosomalprotein genes), regulatory function (40 genes), and energy metabolism(29 genes). This large number of differentially expressed genesreflects the significant change in growth status brought aboutby the lack of both SigB and SigE under these growth conditions.

The regulatory relationship of SigB, SigD, and SigE to histidine kinases. There is reciprocal regulation between the group 2 ${sigma}$ factorsand specific histidine kinases (35, 43). As shown in Table 4,the removal of SigB and, to a lesser extent, the removal ofSigD, but especially the removal of both SigB and SigE, hada dramatic effect on the transcription levels of a number ofhistidine kinases. In the case of hik31 (and its possible responseregulator, rre34), the absence of SigB increased the transcriptlevel in the dark. However, the absence of both SigB and SigEdecreased the hik31 transcript level in ${Delta}$ sigB ${Delta}$ sigE in the lightand, to a lesser extent, in the dark, and the removal of SigDdecreased transcript levels in the light. The hik8 gene wasaffected only when both SigB and SigE were missing, and thetranscript level then was decreased in the light and was decreasedsignificantly in the dark (Table 4). The hik12 gene was affectedin a similar fashion. On the other hand, SigD regulated hik16,as seen by the increased hik16 transcript level in the ${Delta}$ sigDstrain in both the light and the dark.

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TABLE 4. Fold change of transcript abundance for selected differentially regulated genes under different growth conditions at L1 and D1^a

Validation of microarray results by qPCR. The microarray data were validated by qPCR using two biologicalreplicates (Table 5). The comparisons demonstrated excellentcorrespondence for genes that were up-regulated in the light(slr1667) and up-regulated in the dark (sll0329 and slr0474)and for psbA2, which showed relatively small fold changes duringthe 1-h transition. From a quantitative perspective, the ratiosdetermined by microarray analysis appeared to be underestimatedcompared to those from qPCR. The qPCR data probably are moreaccurate for genes with particularly high or low transcriptlevels; this includes the psbA transcript, among the most abundanttranscripts, and slr1667, which has low transcript levels. However,in the case of the ${Delta}$ sigD strain, the psbA transcript levels fromthe microarray experiment appeared to be consistent with dataof Yoshimura et al. (50), who reported little change in transcriptlevels on transfer from dark to light. They also are in agreementwith our Northern blot analysis with an aliquot of the sametotal RNA samples as those used for the microarray experiments(data not shown). For all other samples and genes, there wasquantitative agreement between the qPCR and microarray data.

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TABLE 5. Comparison of transcript abundance at L1 to that at D1 using qPCR and microarray data for wild-type, ${Delta}$ sigB, and ${Delta}$ sigD Synechocystis sp. strain PCC 6803 strains^a

DISCUSSION

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DISCUSSION

Our results indicate the importance of the group 2 ${sigma}$ factorsSigB, SigD, and SigE in the global regulation of transcriptionin Synechocystis sp. strain PCC 6803. We then needed to determinewhy so many genes in so many functional categories were affectedby the deletion of a specific sigma factor. The results canbest be explained by using the ${sigma}$ cycle paradigm (45) and particularlythe stochastic release model described previously (30). Mooneyet al. (30) have summarized the evidence for and against a halfdozen different possible models of ${sigma}$ action and concluded thatdata support stochastic release over the obligate and nonreleasemodels. In addition, the authors discuss more nuanced modesof ${sigma}$ action, including models referred to as promoter-proximal ${sigma}$ pause, rebinding ${sigma}$ pause, and the hypothetical role of ${sigma}$ as anantitermination factor. Each model is consistent with resultsfrom specific promoter systems, and there is currently no wayto compare such details in our system. Thus, we will refer onlyto the basic stochastic release model. In this model, a poolof ${sigma}$ factors competes for binding to the core RNAP to form anopen complex. The affinity of ${sigma}$ for the RNAP decreases in theelongation complex, but release of the ${sigma}$ factor occurs stochasticallyafter the RNAP has initiated transcription. The release is affectedby factors such as RNAP and ${sigma}$ factor concentration and occursduring each transcription cycle, allowing ${sigma}$ competition for rebindingof the RNAP. Therefore, control of global transcription willbe based on the amount of the various sigma factors presentand able to bind to the RNAP.

The model depicted in Fig. 3 is based on our transcription dataas well as the levels of the ${sigma}$ factors in the light and darkas calculated by Imamura et al. (19). In general, the group3 ${sigma}$ factors were below the detectable level. In the wild type,the transition from growth in the light to growth in the darkgenerated an increase in SigB and a decrease in SigE. In thewild type, the major impact of this transition on global regulationwas a decrease of the transcript levels of genes encoding ribosomalproteins and photosynthesis proteins. At the same time, genesencoding enzymes involved in energy metabolism, biosynthesisof various cofactors and prosthetic groups, regulatory functions,and transport and binding proteins tended to increase. In addition,many genes annotated as hypothetical or unknown also increasedas cells went into the dark phase. This pattern was reversedas the cells were exposed to the light, where SigB decreasedtwofold and SigD and SigE increased twofold.

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FIG. 3. Relationship between sigma factor abundance and the impact of SigB or SigD removal on global regulation of gene expression in light-to-dark and dark-to-light transitions. (A) Wild-type Synechocystis sp. strain PCC 6803. The relative amounts of the sigma factor proteins are represented by the numbers above the sigma factors, based on the calculations of Imamura et al. (19). A twofold increase in SigB and a twofold decrease in SigE were observed following transfer to the dark for 1 h. Functional groups containing differentially regulated genes at 1 h of dark and 1 h of light are shown in boxes; the up arrow indicates that transcripts were up-regulated in the light compared to their status in the dark, and the two-headed arrow indicates that there were both up- and down-regulated genes in these groups. The rpl and rps genes encode ribosome protein large subunits and small subunits, respectively. (B) Differentially regulated genes in ${Delta}$ sigB compared to regulation in the wild type at 1 h of dark and 1 h of light. The up arrow indicates functional categories that contained up-regulated transcripts in the mutant compared to the wild type. (C) Differentially regulated genes in the ${Delta}$ sigD strain compared to the wild type at 1 h of dark and 1 h of light. The up arrow indicates functional categories that contain up-regulated transcripts in the mutant, and the two-headed arrow indicates both increased and decreased transcript abundance in genes belonging to these functional categories.

When SigB was absent, there was very little change from thewild-type levels in the light, as shown in Fig. 1B and Table1. This is consistent with the low level of SigB in the light,when SigB represented less than 4% of the total sigma factors,as reported by Imamura et al. (17, 19) (Fig. 3). However, inthe dark, SigB expression increased twofold and many more genesshowed enhanced transcript levels relative to those of the wildtype, especially in genes encoding ribosomal proteins and manyhypothetical proteins. It is striking that the major effectof removing SigB was to up-regulate at least some genes in manydifferent categories. Thus, according to the stochastic releasemodel, the absence of SigB permitted increased binding of other ${sigma}$ factors to the RNAP and altered transcription of a select numberof genes, especially the major operon that encodes ribosomalproteins. As noted in Fig. 1A and Table 3, the absence of SigBalone has little impact on cell-doubling times under eitherphotoautotrophic or mixotrophic conditions. The role of SigBas a positive regulator of gene expression under heat shockconditions is consistent with this model. Following heat shock,SigB becomes the most abundant ${sigma}$ factor in the cell, increasingby 13-fold (19). The enhanced transcription of genes such asthose encoding heat shock proteins may be explained by increasedtranscription from SigB promoters due to increased amounts ofthe SigB/RNAP holoenzyme.

The absence of SigD has a more profound impact on global regulationof transcription. Compared to SigB, SigD represented a largerproportion of ${sigma}$ factors available in both the light and the dark;during growth in the light, SigD represented more than one-quarterof the total number of sigma factors. The results shown in Fig.1C clearly demonstrated a much greater influence of SigD removalin the light than in the dark. In the light, many genes exhibitedtranscription in ${Delta}$ sigD that was different compared to that inthe wild type, and many of them were up-regulated; this includednumerous changes in photosynthesis genes. In the absence ofSigD, approximately two-thirds of the genes differentially regulatedin the light were up-regulated, whereas only approximately halfof the genes differentially regulated in the dark were up-regulated.In accordance with the stochastic release model, we suggestthat, as well as abolishing SigD promoter specific transcription,the absence of SigD allowed increased transcription from othersigma factors binding to RNAP.

Taken together, the results for both mutants indicated thatSigB is much more important in the dark, whereas SigD has agreater influence on global transcription in the light. Thestochastic release model explains the gene expression changeswe observed but does not preclude other models of ${sigma}$ factor action.In fact, the stochastic release model may represent the basic ${sigma}$ -factor-regulatory mechanism that may be modified under specificconditions by more complex interactions.

The results with the ${Delta}$ sigB ${Delta}$ sigE double mutant can be interpretedin a similar fashion. The alteration in transcription in thedouble mutant in the presence of glucose is most pronouncedin the light, and in this case, many more genes were down-regulatedin the mutant than in the wild type. These changes probablyreflect different growth states of the wild type and mutant(Fig. 1D). In the dark, the single greatest impact was on thetranscript levels of genes encoding ribosomal proteins (Fig.1F). In ${Delta}$ sigB, the transcript level of the ribosomal proteinswas not decreased, as occurs in the wild type, such that therewas typically little difference between transcript levels inthe light and the dark (the transcript levels of some genesdecreased from 1.5- to 2.0-fold). Similarly, for ${Delta}$ sigB ${Delta}$ sigE,the transcript levels of the ribosomal protein genes were two-to fourfold higher in the dark than they were for the wild type.Indeed, the ribosomal protein genes represented 32 out of thetop 50 genes that showed increases in ${Delta}$ sigB ${Delta}$ sigE relative tothe levels for the wild type. Therefore, as cultures are grownover long periods with short days (8 h of light/16 h of dark),the cells retained high levels of ribosomal protein transcripts.This, combined with the down-regulation of genes involved inglycolysis and the OPP pathway in the dark, may adversely affectmany components of cell growth and metabolism and cause theimpaired mixotrophic growth under different light regimes. Inthe ${Delta}$ hik8 mutant, we showed down-regulation of ribosome proteingenes, indicating that hik8 is involved in positive regulationof these genes (43). This is consistent with the negative regulationof sigB by hik8 shown in Fig. 4, as the absence of SigB resultedin increased levels of ribosome protein gene transcripts (Fig.1B). Therefore, an increase in SigB in ${Delta}$ hik8 may be involvedin down-regulation of the ribosomal protein genes in this strain.Based on our data, we propose that the cell down-regulated thelevel of ribosomal proteins in the dark in order to conserveresources, and this may be a major reason why growth of cellsgrown in light-dark cycles is significantly slower than thatin continuous light.

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FIG. 4. Model of sigma factor and histidine kinase interactions in the light and the dark. Interactions are based on transcript abundance in the ${Delta}$ hik8, ${Delta}$ sigB, ${Delta}$ sigD, and ${Delta}$ sigE mutants compared to that of the wild type. The data are from Sherman and Singh (43), this study, and Osanai et al. (35) for the ${Delta}$ hik8 strain, ${Delta}$ sigB and ${Delta}$ sigD strains, and ${Delta}$ sigE strain, respectively. The light/dark conditions at which the results were obtained are shown in the key.

Our results indicated that ribosomal protein genes representone of the key regulated targets for the ${sigma}$ factors, as diagrammedin Fig. 4. In this figure, we summarize data from this studyand from recent papers (35, 43) and demonstrate the relationshipbetween the ${sigma}$ factors and select two-component histidine kinases.A recent paper developed a more complex network of sigma factorinteractions (50), but we feel that our hypothesis providesa more comprehensive understanding of this regulation. We suggestthat SigB is central to this regulatory network, especiallyin the dark. In the light, the absence of SigE affected SigBand cph1/rcp1, although the SigE level is relatively low andwas changed only twofold between the light and the dark (Fig.3). Removal of SigD altered transcription of a number of histidinekinases, including hik31. This histidine kinase, together withHik8 and SigE, is known to play a role in sugar metabolism.Thus, mutants lacking sigE, hik8, and hik31 demonstrated impairedgrowth under mixotrophic conditions and altered transcriptionof genes involved in glycolysis and/or the OPP pathway (20,35, 43) This model illustrates the complex balance between themany regulatory components involved in fine-tuning the metabolicresponse of the cyanobacterium to changes in its light environment.

ACKNOWLEDGMENTS

This research was funded by grant DE-FG02-99ER20342 from theDepartment of Energy.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biological Sciences, Purdue University, 201 S. University St., Hansen Hall, West Lafayette, IN 47907. Phone: (765) 494-8106. Fax: (765) 496-1496. E-mail: lsherman{at}purdue.edu

Published ahead of print on 24 August 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

REFERENCES

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