Light-activated Heterotrophic Growth Conditions Research Articles

The NAD Kinase Slr0400 Functions as a Growth Repressor in Synechocystis sp. PCC 6803.

NADP+, the phosphorylated form of nicotinamide adenine dinucleotide (NAD), plays an essential role in many cellular processes. NAD kinase (NADK), which is conserved in all living organisms, catalyzes the phosphorylation of NAD+ to NADP+. However, the physiological role of phosphorylation of NAD+ to NADP+ in the cyanobacterium Synechocystis remains unclear. In this study, we report that slr0400, an NADK-encoding gene in Synechocystis, functions as a growth repressor under light-activated heterotrophic growth conditions and light and dark cycle conditions in the presence of glucose. We show, via characterization of NAD(P)(H) content and enzyme activity, that NAD+ accumulation in slr0400-deficient mutant results in the unsuppressed activity of glycolysis and tricarboxylic acid (TCA) cycle enzymes. In determining whether Slr0400 functions as a typical NADK, we found that constitutive expression of slr0400 in an Arabidopsis nadk2-mutant background complements the pale-green phenotype. Moreover, to determine the physiological background behind the growth advantage of mutants lacking slr04000, we investigated the photobleaching phenotype of slr0400-deficient mutant under high-light conditions. Photosynthetic analysis found in the slr0400-deficient mutant resulted from malfunctions in the Photosystem II (PSII) photosynthetic machinery. Overall, our results suggest that NADP(H)/NAD(H) maintenance by slr0400 plays a significant role in modulating glycolysis and the TCA cycle to repress the growth rate and maintain the photosynthetic capacity.

Thylakoid Localized Type 2 NAD(P)H Dehydrogenase NdbA Optimizes Light-Activated Heterotrophic Growth of Synechocystis sp. PCC 6803.

NdbA, one of the three type 2 NAD(P)H dehydrogenases (NDH-2) in Synechocystis sp. PCC 6803 (hereafter Synechocystis) was here localized to the thylakoid membrane (TM), unique for the three NDH-2s, and investigated with respect to photosynthetic and cellular redox metabolism. For this purpose, a deletion mutant (ΔndbA) and a complementation strain overexpressing NdbA (ΔndbA::ndbA) were constructed. It is demonstrated that NdbA is expressed at very low level in the wild-type (WT) Synechocystis under photoautotrophic (PA) growth whilst substantially higher expression occurs under light-activated heterotrophic growth (LAHG). The absence of NdbA resulted in non-optimal growth of Synechocystis under LAHG and concomitantly enhanced the expression of photoprotection-related flavodiiron proteins and carbon acquisition-related proteins as well as various transporters, but downregulated a few iron homeostasis-related proteins. NdbA overexpression, on the other hand, promoted photosynthetic pigmentation and functionality of photosystem I under LAHG conditions while distinct photoprotective and carbon concentrating proteins were downregulated. NdbA overexpression also exerted an effect on the expression of many signaling and gene regulation proteins. It is concluded that the amount and function of NdbA in the TM has a capacity to modulate the redox signaling of gene expression, but apparently has a major physiological role in maintaining iron homeostasis under LAHG conditions. LC-MS/MS data are available via ProteomeXchange with identifier PXD011671.

Role of Type 2 NAD(P)H Dehydrogenase NdbC in Redox Regulation of Carbon Allocation in Synechocystis.

NAD(P)H dehydrogenases comprise type 1 (NDH-1) and type 2 (NDH-2s) enzymes. Even though the NDH-1 complex is a well-characterized protein complex in the thylakoid membrane of Synechocystis sp. PCC 6803 (hereafter Synechocystis), the exact roles of different NDH-2s remain poorly understood. To elucidate this question, we studied the function of NdbC, one of the three NDH-2s in Synechocystis, by constructing a deletion mutant (ΔndbC) for a corresponding protein and submitting the mutant to physiological and biochemical characterization as well as to comprehensive proteomics analysis. We demonstrate that the deletion of NdbC, localized to the plasma membrane, affects several metabolic pathways in Synechocystis in autotrophic growth conditions without prominent effects on photosynthesis. Foremost, the deletion of NdbC leads, directly or indirectly, to compromised sugar catabolism, to glycogen accumulation, and to distorted cell division. Deficiencies in several sugar catabolic routes were supported by severe retardation of growth of the ΔndbC mutant under light-activated heterotrophic growth conditions but not under mixotrophy. Thus, NdbC has a significant function in regulating carbon allocation between storage and the biosynthesis pathways. In addition, the deletion of NdbC increases the amount of cyclic electron transfer, possibly via the NDH-12 complex, and decreases the expression of several transporters in ambient CO2 growth conditions.

The Proteome and Lipidome of Synechocystis sp. PCC 6803 Cells Grown under Light-Activated Heterotrophic Conditions*[S

Cyanobacteria are photoautotrophic prokaryotes with a plant-like photosynthetic machinery. Because of their short generation times, the ease of their genetic manipulation, and the limited size of their genome and proteome, cyanobacteria are popular model organisms for photosynthetic research. Although the principal mechanisms of photosynthesis are well-known, much less is known about the biogenesis of the thylakoid membrane, hosting the components of the photosynthetic, and respiratory electron transport chain in cyanobacteria. Here we present a detailed proteome analysis of the important model and host organism Synechocystis sp. PCC 6803 under light-activated heterotrophic growth conditions. Because of the mechanistic importance and severe changes in thylakoid membrane morphology under light-activated heterotrophic growth conditions, a focus was put on the analysis of the membrane proteome, which was supported by a targeted lipidome analysis. In total, 1528 proteins (24.5% membrane integral) were identified in our analysis. For 641 of these proteins quantitative information was obtained by spectral counting. Prominent changes were observed for proteins associated with oxidative stress response and protein folding. Because of the heterotrophic growth conditions, also proteins involved in carbon metabolism and C/N-balance were severely affected. Although intracellular thylakoid membranes were significantly reduced, only minor changes were observed in their protein composition. The increased proportion of the membrane-stabilizing sulfoqinovosyl diacyl lipids found in the lipidome analysis, as well as the increased content of lipids with more saturated acyl chains, are clear indications for a coordinated synthesis of proteins and lipids, resulting in stabilization of intracellular thylakoid membranes under stress conditions.

Inhibition of chlorophyll biosynthesis at the protochlorophyllide reduction step results in the parallel depletion of Photosystem I and Photosystem II in the cyanobacterium Synechocystis PCC 6803

In most oxygenic phototrophs, including cyanobacteria, two independent enzymes catalyze the reduction of protochlorophyllide to chlorophyllide, which is the penultimate step in chlorophyll (Chl) biosynthesis. One is light-dependent NADPH:protochlorophyllide oxidoreductase (LPOR) and the second type is dark-operative protochlorophyllide oxidoreductase (DPOR). To clarify the roles of both enzymes, we assessed synthesis and accumulation of Chl-binding proteins in mutants of cyanobacterium Synechocystis PCC 6803 that either completely lack LPOR or possess low levels of the active enzyme due to its ectopic regulatable expression. The LPOR-less mutant grew photoautotrophically in moderate light and contained a maximum of 20% of the wild-type (WT) Chl level. Both Photosystem II (PSII) and Photosystem I (PSI) were reduced to the same degree. Accumulation of PSII was mostly limited by the synthesis of antennae CP43 and especially CP47 as indicated by the accumulation of reaction center assembly complexes. The phenotype of the LPOR-less mutant was comparable to the strain lacking DPOR that also contained <25% of the wild-type level of PSII and PSI when cultivated under light-activated heterotrophic growth conditions. However, in the latter case, we detected no reaction center assembly complexes, indicating that synthesis was almost completely inhibited for all Chl-proteins, including the D1 and D2 proteins.

Involvement of Carotenoids in the Synthesis and Assembly of Protein Subunits of Photosynthetic Reaction Centers of Synechocystis sp. PCC 6803

The crtB gene of Synechocystis sp. PCC 6803, encoding phytoene synthase, was inactivated in the Delta crtH mutant to generate a carotenoidless Delta crtH/B double mutant. Delta crtH mutant cells were used because they had better transformability than wild-type cells, most probably due to their adaptation to partial carotenoid deficiency. Cells of the Delta crtH/B mutant were light sensitive and could grow only under light-activated heterotrophic growth conditions in the presence of glucose. Carotenoid deficiency did not significantly affect the cellular content of phycobiliproteins while the chlorophyll content of the mutant cells decreased. The mutant cells exhibited no oxygen-evolving activity, suggesting the absence of photochemically active PSII complexes. This was confirmed by 2D electrophoresis of photosynthetic membrane complexes. Analyses identified only a small amount of a non-functional PSII core complex lacking CP43, while the monomeric and dimeric PSII core complexes were absent. On the other hand, carotenoid deficiency did not prevent formation of the cytochrome b(6)f complex and PSI, which predominantly accumulated in the monomeric form. Radioactive labeling revealed very limited synthesis of inner PSII antennae, CP47 and especially CP43. Thus, carotenoids are indispensable constituents of the photosynthetic apparatus, being essential not only for antioxidative protection but also for the efficient synthesis and accumulation of photosynthetic proteins and especially that of PSII antenna subunits.

Light‐induced gene expression of fructose 1,6‐bisphosphate aldolase during heterotrophic growth in a cyanobacterium, Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803 exhibits light-activated heterotrophic growth (LAHG) under dark conditions with glucose as a carbon source. The light activation is remarkable at a late period of photoautotrophic preculture, such as the late-linear and stationary growth phases. To understand the physiological effects of light irradiation and glucose under LAHG conditions, their effects on the expression of soluble proteins were analyzed by means of 2D-PAGE. Various soluble proteins, which were minimal under photoautotrophic preculture conditions, were observed clearly under LAHG conditions, suggesting that proteins were synthesized actively under these conditions. Fructose 1,6-bisphosphate aldolase, one of the glycolytic enzymes, was found to be induced under LAHG conditions on 2D-PAGE. The activity of fructose 1,6-bisphosphate aldolase, which had decreased during photoautotrophic preculture, also increased under LAHG conditions, similar to the mRNA level of the encoding gene, fbaA. In addition, we found that a deletion mutant of sll1330, a putative gene containing a helix-turn-helix DNA-binding motif, could not grow under LAHG conditions, whereas it could grow photoautotrophically. The increases in the protein level of FbaA and fbaA gene expression observed in wild-type cells under LAHG conditions were greatly inhibited in the deletion mutant. These results suggest that the regulation of fbaA gene expression by way of sll1330 is one of the important processes in Synechocystis sp. PCC 6803 under light pulse LAHG conditions.

Sll1330 controls the expression of glycolytic genes in Synechocystis sp. PCC 6803

In the complete annotated genome sequences of cyanobacterium Synechocystis sp. PCC 6803, one can find many putative genes for two-component response regulators that include a helix-turn-helix DNA-binding domain. The mRNA level of one of the putative genes, sll1330, was increased by glucose, especially in the presence of light. We successfully disrupted the sll1330 gene by targeted mutagenesis with a spectinomycin resistance cassette. Δ sll1330 could not grow well under light-activated heterotrophic growth conditions. Analyses of the expression of glycolytic genes revealed that the mRNA levels of five glycolytic genes, that is, glk ( sll0593), pfkA ( sll1196), fbaA ( sll0018), gpmB ( slr1124), and pk ( sll0587), were decreased, and were regulated by Sll1330 under light and glucose-supplemented conditions. The Synechocystis sp. PCC 6803 genome each encodes two isozymes for these five glycolytic genes, suggesting that each of the two isozymes is regulated by Sll1330 at the mRNA level.

Positive Regulation of Sugar Catabolic Pathways in the Cyanobacterium Synechocystis sp. PCC 6803 by the Group 2 σ Factor SigE

The sigE gene of Synechocystis sp. PCC 6803 encodes a group 2 sigma factor for RNA polymerase and has been proposed to function in transcriptional regulation of nitrogen metabolism. By using microarray and Northern analyses, we demonstrated that the abundance of transcripts derived from genes important for glycolysis, the oxidative pentose phosphate pathway, and glycogen catabolism is reduced in a sigE mutant of Synechocystis maintained under the normal growth condition. Furthermore, the activities of the two key enzymes of the oxidative pentose phosphate pathway, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, encoded by the zwf and gnd genes were also reduced in the sigE mutant. The dark enhancements in both enzyme activity and transcript abundance apparent in the wild type were eliminated by the mutation. In addition, the sigE mutant showed a reduced rate of glucose uptake and an increased intracellular level of glycogen. Moreover, it was unable to proliferate under the light-activated heterotrophic growth conditions. These results indicate that SigE functions in the transcriptional activation of sugar catabolic pathways in Synechocystis sp. PCC 6803.

Effects of glycerol and high temperatures on structure and function of phycobilisomes in Synechocystis sp. PCC 6803

The effects of glycerol and high temperatures on structure and function of phycobilisomes (PBSs) in vivo were investigated in a chlL deletion mutant of the cyanobacterium Synechocystis sp. PCC 6803. When the mutant was grown under light-activated heterotrophic growth conditions, it contained intact and functional PBSs, but essentially no chlorophyll and photosystems. So the structural and functional changes of the mutant PBSs in vivo can be handily detected by measurement of low temperature (77 K) fluorescence emission spectra. High concentration glycerol induced an obvious disassembly of PBSs and the dissociation of phycocyanins in the rod substructures into their oligomers and monomers. PBSs also disassembled at high temperatures and allophycocyanins were more sensitive to heat stress than phycocyanins. Our results demonstrate that the chlL − mutant strain is an advantageous model for studying the mechanisms of assembly and disassembly of protein complexes in vivo.

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Cyanobacteria are photoautotrophic prokaryotes with a plant-like photosynthetic machinery. Because of their short generation times, the ease of their genetic manipulation, and the limited size of their genome and proteome, cyanobacteria are popular model organisms for photosynthetic research. Although the principal mechanisms of photosynthesis are well-known, much less is known about the biogenesis of the thylakoid membrane, hosting the components of the photosynthetic, and respiratory electron transport chain in cyanobacteria. Here we present a detailed proteome analysis of the important model and host organism Synechocystis sp. PCC 6803 under light-activated heterotrophic growth conditions. Because of the mechanistic importance and severe changes in thylakoid membrane morphology under light-activated heterotrophic growth conditions, a focus was put on the analysis of the membrane proteome, which was supported by a targeted lipidome analysis. In total, 1528 proteins (24.5% membrane integral) were identified in our analysis. For 641 of these proteins quantitative information was obtained by spectral counting. Prominent changes were observed for proteins associated with oxidative stress response and protein folding. Because of the heterotrophic growth conditions, also proteins involved in carbon metabolism and C/N-balance were severely affected. Although intracellular thylakoid membranes were significantly reduced, only minor changes were observed in their protein composition. The increased proportion of the membrane-stabilizing sulfoqinovosyl diacyl lipids found in the lipidome analysis, as well as the increased content of lipids with more saturated acyl chains, are clear indications for a coordinated synthesis of proteins and lipids, resulting in stabilization of intracellular thylakoid membranes under stress conditions.

Partial recovery of light-independent chlorophyll biosynthesis in the chlL-deletion mutant of Synechocystis sp. PCC 6803.

A chlL-deletion mutant of Synechocystis sp. PCC 6803 designated as chlL- was unable to make significant amounts of chlorophyll in darkness. However, an apparent pseudorevertant has been generated spontaneously that can synthesize an increased amount of chlorophyll under light-activated heterotrophic growth conditions. Under these conditions, the chlorophyll content in this pseudorevertant was about 20% of that in the wild-type strain and about 4 times more than that in the original and in the recently recreated chlL-deletion mutant. This is paralleled by increased performance of dark-grown cells in terms of chlorophyll fluorescence induction and oxygen evolution rates in the pseudorevertant versus in the original mutant. PCR analysis confirmed that the chlL- -pseudorevertant mutant still lacked the chlL gene. These results imply that the light-independent chlorophyll biosynthesis pathway was partly recovered.

Chlorophyll a availability affects psbA translation and D1 precursor processing in vivo in Synechocystis sp. PCC 6803.

Transcript accumulation and translation of psbA as well as processing of the D1 precursor protein were investigated in relation to chlorophyll availability in vivo in cyanobacterial strains lacking photosystem I (PS I). The psbA transcript level was almost independent of chlorophyll availability and was approximately 3-fold lower in darkness than in continuous light (5 microE m-2 s-1). Upon illumination, it reached a steady-state level within several hours. Upon growth under light-activated heterotrophic growth conditions (LAHG) in the PS I-less strain, D1 synthesis occurred immediately upon illumination. However, in PS I-less/chlL- cells, which lacked the light-independent chlorophyll biosynthesis pathway and had very low chlorophyll levels after LAHG growth, very little D1 synthesis occurred upon illumination, and the synthesis rate increased with time. This result suggests a translational control of D1 biosynthesis related to chlorophyll availability. Upon illumination, initially a high level of the nonprocessed D1 precursor was observed by pulse labeling and immunodetection in LAHG-grown PS I-less/chlL- cells but not in PS I-less cells. A significant amount of the D1 precursor eventually was processed to mature D1, and the half-life of the D1 precursor decreased as the chlorophyll content of the cells increased. The D1 processing enzyme CtpA was found to be present at similar levels regardless illumination or chlorophyll levels. We conclude that, directly or indirectly, chlorophyll availability is needed for D1 translation as well as for efficient processing of the D1 precursor.

Light-induced expression of fatty acid desaturase genes.

In cyanobacterial cells, fatty acid desaturation is one of the crucial steps in the acclimation processes to low-temperature conditions. The expression of all the four acyl lipid desaturase genes of Synechocystis PCC 6803 was studied as a function of temperature and separately as a function of light. We used cells grown at 25 degreesC in light-activated heterotrophic growth conditions. In these cells, the production of alpha-linolenic acid and 18:4 fatty acids was negligible and the synthesis of gamma-linolenic acid was remarkably suppressed compared with those of the cells grown photoautotrophically. The cells grown in the light in the presence of glucose showed no difference in fatty acid composition compared with cells grown photoautotrophically. The level of desC mRNA for delta9 desaturase was not affected by either the temperature or the light. It was constitutively expressed at 25 degreesC with and without illumination. The level of desB transcripts was negligible in the dark-grown cells and was enhanced about 10-fold by exposure of the cells to light. The maximum level of expression occurred within 15 min. The level of desA and desD mRNAs was higher in dark-grown cells than that of desB mRNA for omega3 desaturase. However, the induction of both desA and desD mRNAs for delta12 and delta6 desaturases, respectively, was enhanced by light about 10-fold. Rifampicin, chloramphenicol, and 3-(3, 4-dichlorophenyl)-1,1-dimethylurea completely blocked the induction of the expression of desA, desB, and desD. Consequently, we suggest the regulatory role of light via photosynthetic processes in the induction of the expression of acyl lipid desaturases.

Chlorophyll alpha synthesis upon interruption and deletion of por coding for the light-dependent NADPH: protochlorophyllide oxidoreductase in a photosystem-I-less/chlL- strain of Synechocystis sp. PCC 6803.

The gene coding for the light-dependent NADPH:protochlorophyllide oxidoreductase (POR) was interrupted or deleted in a Synechocystis sp. PCC 6803 strain lacking photosystem I (PS I) as well as ChlL, which takes part in light-independent catalysis of protochlorophyllide reduction. Interruption of por by a kanamycin-resistance cartridge between the codons for M263 and V264 (about 83% into the coding region) did not abolish POR activity, but resulted in a decrease in the protochlorophyllide-(PChlide)-binding capacity of POR. Deletion of por in the PS I-less/chlL- strain generated a mutant [PS I-less/chlL-/por (del)] which accumulated both monovinyl-PChlide and divinyl-PChlide and excreted PChlides into the medium. This mutant also synthesized small amounts of protochlorophyllide dihydrogeranylgeraniol ester (protochlorophyll) when it was grown under light-activated heterotrophic growth conditions. However, the mutant was still able to synthesize small amounts of normal chlorophyll a under weak continuous illumination, even though the quantum yield of chlorophyll a formation was reduced. Either protochlorophyll or PChlide reduction by an unspecific reductase or by a ChlB/ChlN complex could account for chlorophyll a synthesis in the PS I-less/chlL-/por (del) strain. Functional photosystem II (PS II) was assembled in this mutant, but the PS II/chlorophyll ratio was fourfold lower than in the PS I-less strain with normal chlorophyll synthesis. The PS I-less/chlL-/por (del) mutant had a 77-K fluorescence emission maximum at 685 nm but no peak or shoulder at 695 nm when the cells were excited at 435 nm. Much of the chlorophyll in the PS I-less/chlL-/por (del) mutant therefore seems to be associated with components other than PS II.

Light-dependent chlorophyll a biosynthesis upon chlL deletion in wild-type and photosystem I-less strains of the cyanobacterium Synechocystis sp. PCC 6803.

Part of the chlL gene encoding a component involved in light-independent protochlorophyllide reduction was deleted in wild type and in a photosystem I-less strain of Synechocystis sp. PCC 6803. In resulting mutants, chlorophyll biosynthesis was fully light-dependent. When these mutants were propagated under light-activated heterotrophic growth conditions (in darkness except for 15 min of weak light a day) for several weeks, essentially no chlorophyll was detectable but protochlorophyllide accumulated. Upon return of the chlL- mutant cultures to continuous light, within the first 6 h chlorophyll was synthesized at the expense of protochlorophyllide at a rate independent of the presence of photosystem I. Chlorophyll biosynthesized during this time gave rise to a 685 nm fluorescence emission peak at 77 K in intact cells. This peak most likely originates from a component different from those known to be directly associated with photosystems II and I. Development of 695 and 725 nm peaks (indicative of intact photosystem II and photosystem I, respectively) required longer exposures to light. After 6 h of greening, the rate of chlorophyll synthesis slowed as protochlorophyllide was depleted. In the chlL- strain, greening occurred at the same rate at two different light intensities (5 and 50 microE m-2 s-1), indicating that also at low light intensity the amount of light is not rate-limiting for protochlorophyllide reduction. Thus, in this system the rate of chlorophyll biosynthesis is limited neither by biosynthesis of photosystems nor by the light-dependent protochlorophyllide reduction. We suggest the presence of a chlorophyll-binding 'chelator' protein (with 77 K fluorescence emission at 685 nm) that binds newly synthesized chlorophyll and that provides chlorophyll for newly synthesized photosynthetic reaction centers and antennae.