Comparative Genomic Analysis of the Marine Cyanobacterium Acaryochloris marina MBIC10699 Reveals the Impact of Phycobiliprotein Reacquisition and the Diversity of Acaryochloris Plasmids

Acaryochloris is a marine cyanobacterium that synthesizes chlorophyll d, a unique chlorophyll that absorbs far-red lights. Acaryochloris is also characterized by the loss of phycobiliprotein (PBP), a photosynthetic antenna specific to cyanobacteria; however, only the type-strain A. marina MBIC11017 retains PBP, suggesting that PBP-related genes were reacquired through horizontal gene transfer (HGT). Acaryochloris is thought to have adapted to various environments through its huge genome size and the genes acquired through HGT; however, genomic information on Acaryochloris is limited. In this study, we report the complete genome sequence of A. marina MBIC10699, which was isolated from the same area of ocean as A. marina MBIC11017 as a PBP-less strain. The genome of A.marina MBIC10699 consists of a 6.4 Mb chromosome and four large plasmids totaling about 7.6 Mb, and the phylogenic analysis shows that A.marina MBIC10699 is the most closely related to A. marina MBIC11017 among the Acaryochloris species reported so far. Compared with A. marina MBIC11017, the chromosomal genes are highly conserved between them, while the genes encoded in the plasmids are significantly diverse. Comparing these genomes provides clues as to how the genes for PBPs were reacquired and what changes occurred in the genes for photosystems during evolution.

Effects of Light and Oxygen on Chlorophyll d Biosynthesis in a Marine Cyanobacterium Acaryochloris marina

A marine cyanobacterium Acaryochloris marina synthesizes chlorophyll (Chl) d as a major Chl. Chl d has a formyl group at its C3 position instead of a vinyl group in Chl a. This modification allows Chl d to absorb far-red light addition to visible light, yet the enzyme catalyzing the formation of the C3-formyl group has not been identified. In this study, we focused on light and oxygen, the most important external factors in Chl biosynthesis, to investigate their effects on Chl d biosynthesis in A. marina. The amount of Chl d in heterotrophic dark-grown cells was comparable to that in light-grown cells, indicating that A. marina has a light-independent pathway for Chl d biosynthesis. Under anoxic conditions, the amount of Chl d increased with growth in light conditions; however, no growth was observed in dark conditions, indicating that A. marina synthesizes Chl d normally even under such "micro-oxic" conditions caused by endogenous oxygen production. Although the oxygen requirement for Chl d biosynthesis could not be confirmed, interestingly, accumulation of pheophorbide d was observed in anoxic and dark conditions, suggesting that Chl d degradation is induced by anaerobicity and darkness.

Chlorophyll and Pheophytin Dephytylating Enzymes Required for Efficient Repair of PSII in Synechococcus elongatus PCC 7942.

The Chlorophyll Dephytylase1 (CLD1) and pheophytinase (PPH) proteins of Arabidopsis thaliana are homologous proteins characterized respectively as a dephytylase for chlorophylls (Chls) and pheophytin a (Phein a) and a Phein a-specific dephytylase. Three genes encoding CLD1/PPH homologs (dphA1, dphA2 and dphA3) were found in the genome of the cyanobacterium Synechococcus elongatus PCC 7942 and shown to be conserved in most cyanobacteria. His6-tagged DphA1, DphA2 and DphA3 proteins were expressed in Escherichia coli, purified to near homogeneity, and shown to exhibit significant levels of dephytylase activity for Chl a and Phein a. Each DphA protein showed similar dephytylase activities for Chl a and Phein a, but the three proteins were distinct in their kinetic properties, with DphA3 showing the highest and lowest Vmax and Km values, respectively, among the three. Transcription of dphA1 and dphA3 was enhanced under high-light conditions, whereas that of dphA2 was not affected by the light conditions. None of the dphA single mutants of S. elongatus showed profound growth defects under low (50 µmol photons m-2 s-1) or high (400 µmol photons m-2 s-1) light conditions. The triple dphA mutant did not show obvious growth defects under these conditions, either, but under illumination of 1,000 µmol photons m-2 s-1, the mutant showed more profound growth retardation compared with wild type (WT). The repair of photodamaged photosystem II (PSII) was much slower in the triple mutant than in WT. These results revealed that dephytylation of Chl a or Phein a or of both is required for efficient repair of photodamaged PSII.

Super-activator variants of the cyanobacterial transcriptional regulator ChlR essential for tetrapyrrole biosynthesis under low oxygen conditions.

ChlR is a MarR-type transcriptional regulator that activates the transcription of the chlAII-ho2-hemN operon in response to low oxygen conditions in the cyanobacterium Synechocystis sp. PCC 6803. Upon exposure to low oxygen conditions, ChlR activates transcription of the operon that encodes enzymes critical to tetrapyrrole biosynthesis under low oxygen conditions. We previously identified a super-activator variant, D35H, of ChlR that constitutively activates transcription of the operon. To gain insight into the low-oxygen induced activation of ChlR, we obtained eight additional super-activator variants of ChlR including D35H from pseudorevertants of a chlAI-disrupted mutant. Most substitutions were located in the N-terminal region of ChlR. Mapping of the substituted amino acid residues provided valuable structural insights that uncovered the activation mechanism of ChlR.

Accessory Proteins of the Nitrogenase Assembly, NifW, NifX/NafY, and NifZ, Are Essential for Diazotrophic Growth in the Nonheterocystous Cyanobacterium Leptolyngbya boryana.

Since nitrogenase is extremely vulnerable to oxygen, aerobic or micro-aerobic nitrogen-fixing organisms need to create anaerobic microenvironments in the cells for diazotrophic growth, which would be one of the major barriers to express active nitrogenase in plants in efforts to create nitrogen-fixing plants. Numerous cyanobacteria are able to fix nitrogen with nitrogenase by coping with the endogenous oxygen production by photosynthesis. Understanding of the molecular mechanisms enabling to the coexistence of nitrogen fixation and photosynthesis in nonheterocystous cyanobacteria could offer valuable insights for the transfer of nitrogen fixation capacity into plants. We previously identified the cnfR gene encoding the master regulator for the nitrogen fixation (nif) gene cluster in the genome of a nonheterocystous cyanobacterium Leptolyngbya boryana, in addition to initial characterization of the nif gene cluster. Here we isolated nine mutants, in which the nif and nif-related genes were individually knocked out in L. boryana to investigate the individual functions of (1) accessory proteins (NifW, NifX/NafY, and NifZ) in the biosynthesis of nitrogenase metallocenters, (2) serine acetyltransferase (NifP) in cysteine supply for iron-sulfur clusters, (3) pyruvate formate lyase in anaerobic metabolism, and (4) NifT and HesAB proteins. ΔnifW, ΔnifXnafY, and ΔnifZ exhibited the most severe phenotype characterized by low nitrogenase activity (<10%) and loss of diazotrophic growth ability. The phenotypes of ΔnifX, ΔnafY, and ΔnifXnafY suggested that the functions of the homologous proteins NifX and NafY partially overlap. ΔnifP exhibited significantly slower diazotrophic growth than the wild type, with lower nitrogenase activity (22%). The other four mutants (ΔpflB, ΔnifT, ΔhesA, and ΔhesB) grew diazotrophically similar to the wild type. Western blot analysis revealed a high correlation between nitrogenase activity and NifD contents, suggesting that NifD is more susceptible to proteolytic degradation than NifK in L. boryana. The phenotype of the mutants lacking the accessory proteins was more severe than that observed in heterotrophic bacteria such as Azotobacter vinelandii, which suggests that the functions of NifW, NifX/NafY, and NifZ are critical for diazotrophic growth of oxygenic photosynthetic cells. L. boryana provides a promising model for studying the molecular mechanisms that produce active nitrogenase, to facilitate the creation of nitrogen-fixing plants.

PAS-histidine kinases PHK1 and PHK2 exert oxygen-dependent dual and opposite effects on gametophore formation in the moss Physcomitrella patens.

Two-component systems, versatile signaling mechanisms based on phosphate transfer between component proteins, must have played important roles in adaptation and diversification processes in land plant evolution. We previously demonstrated that two Per-Arnt-Sim (PAS)-histidine kinases, PHK1 and PHK2, repress gametophore formation in the moss Physcomitrella patens under aerobic conditions, and that, in eukaryotes, the presence of their homologs is restricted to early-diverging streptophyte linages. We assessed here whether or not PHKs play a role in oxygen signaling. When submerged under water, the double disruption line for PHK1 and PHK2 formed fewer gametophores than the wild-type line (WT) both under light-dark cycles or continuous light, indicating that PHKs promote gametophore formation under an aquatic environment, in contrast to aerobic conditions. Similarly, in an artificial low-oxygen condition, the double disruption line formed fewer gametophores than WT. These results indicate that PHKs exert dual and opposite effects on gametophore formation depending on oxygen status. This study adds important insight into functional versatility and evolutionary significance of two-component systems in land plants.

In vivo transposon tagging in the nonheterocystous nitrogen-fixing cyanobacterium Leptolyngbya boryana.

Nitrogenase is an oxygen-vulnerable metalloenzyme that catalyzes nitrogen fixation. It largely remains unknown how nitrogenase coexists with oxygenic photosynthesis in nonheterocystous cyanobacteria, since there have been no appropriate model cyanobacteria so far. Here, we demonstrate in vivo transposon tagging in the nonheterocystous cyanobacterium Leptolyngbya boryana as a forward genetics approach. By conjugative transfer, a mini-Tn5-derived vector, pKUT-Tn5-Sm/Sp, was transferred from Escherichia coli to L. boryana cells. Of 1839 streptomycin-resistant colonies, we isolated three mutants showing aberrant diazotrophic growth. Genome resequencing identified the insertion sites of the transposon in the mutants. This in vivo transposon tagging mutagenesis of L. boryana provides a promising system to investigate molecular mechanisms to resolve the Oxygen Paradox between nitrogen fixation and oxygenic photosynthesis in cyanobacteria.

Functional expression of an oxygen-labile nitrogenase in an oxygenic photosynthetic organism.

Transfer of nitrogen fixation ability to plants, especially crops, is a promising approach to mitigate dependence on chemical nitrogen fertilizer and alleviate environmental pollution caused by nitrogen fertilizer run-off. However, the need to transfer a large number of nitrogen fixation (nif) genes and the extreme vulnerability of nitrogenase to oxygen constitute major obstacles for transfer of nitrogen-fixing ability to plants. Here we demonstrate functional expression of a cyanobacterial nitrogenase in the non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803). A 20.8-kb chromosomal fragment containing 25 nif and nif-related genes of the diazotrophic cyanobacterium Leptolyngbya boryana was integrated into a neutral genome site of Synechocystis 6803 by five-step homologous recombination together with the cnfR gene encoding the transcriptional activator of the nif genes to isolate CN1. In addition, two other transformants CN2 and CN3 carrying additional one and four genes, respectively, were isolated from CN1. Low but significant nitrogenase activity was detected in all transformants. This is the first example of nitrogenase activity detected in non-diazotrophic photosynthetic organisms. These strains provide valuable platforms to investigate unknown factors that enable nitrogen-fixing growth of non-diazotrophic photosynthetic organisms, including plants.

Mechanisms of drought-induced dissipation of excitation energy in sun- and shade-adapted drought-tolerant mosses studied by fluorescence yield change and global and target analysis of fluorescence decay kinetics.

Some mosses stay green and survive long even under desiccation. Dissipation mechanisms of excess excitation energy were studied in two drought-tolerant moss species adapted to contrasting niches: shade-adapted Rhytidiadelphus squarrosus and sun-adapted Rhytidium rugosum in the same family. (1) Under wet conditions, a light-induced nonphotochemical quenching (NPQ) mechanism decreased the yield of photosystem II (PSII) fluorescence in both species. The NPQ extent saturated at a lower illumination intensity in R. squarrosus, suggesting a larger PSII antenna size. (2) Desiccation reduced the fluorescence intensities giving significantly lower F 0 levels and shortened the overall fluorescence lifetimes in both R. squarrosus and R. rugosum, at room temperature. (3) At 77 K, desiccation strongly reduced the PSII fluorescence intensity. This reduction was smaller in R. squarrosus than in R. rugosum. (4) Global and target analysis indicated two different mechanisms of energy dissipation in PSII under desiccation: the energy dissipation to a desiccation-formed strong fluorescence quencher in the PSII core in sun-adapted R. rugosum (type-A quenching) and (5) the moderate energy dissipation in the light-harvesting complex/PSII in shade-adapted R. squarrosus (type-B quenching). The two mechanisms are consistent with the different ecological niches of the two mosses.

The Effect of Two Amino acid Residue Substitutions via RNA Editing on Dark-operative Protochlorophyllide Oxidoreductase in the Black Pine Chloroplasts.

Dark-operative protochlorophyllide oxidoreductase (DPOR) is a key enzyme to produce chlorophyll in the dark. Among photosynthetic eukaryotes, all three subunits chlL, chlN, and chlB are encoded by plastid genomes. In some gymnosperms, two codons of chlB mRNA are changed by RNA editing to codons encoding evolutionarily conserved amino acid residues. However, the effect of these substitutions on DPOR activity remains unknown. We first prepared cyanobacterial ChlB variants with amino acid substitution(s) to mimic ChlB translated from pre-edited mRNA. Their activities were evaluated by measuring chlorophyll content of dark-grown transformants of a chlB-lacking mutant of the cyanobacterium Leptolyngbya boryana that was complemented with pre-edited mimic chlB variants. The chlorophyll content of the transformant cells expressing the ChlB variant from the fully pre-edited mRNA was only one-fourth of the control cells. Co-purification experiments of ChlB with Strep-ChlN suggested that a stable complex with ChlN is greatly impaired in the substituted ChlB variant. We then confirmed that RNA editing efficiency was markedly greater in the dark than in the light in cotyledons of the black pine Pinus thunbergii. These results indicate that RNA editing on chlB mRNA is important to maintain appropriate DPOR activity in black pine chloroplasts.

Harvesting Far-Red Light by Chlorophyll f in Photosystems I and II of Unicellular Cyanobacterium strain KC1.

Cells of a unicellular cyanobacterium strain KC1, which were collected from Japanese fresh water Lake Biwa, formed chlorophyll (Chl) f at 6.7%, Chl a' at 2.0% and pheophytin a at 0.96% with respect to Chl a after growth under 740 nm light. The far-red-acclimated cells (Fr cells) formed extra absorption bands of Chl f at 715 nm in addition to the major Chl a band. Fluorescence lifetimes were measured. The 405-nm laser flash, which excites mainly Chl a in photosystem I (PSI), induced a fast energy transfer to multiple fluorescence bands at 720-760 and 805 nm of Chl f at 77 K in Fr cells with almost no PSI-red-Chl a band. The 630-nm laser flash, which mainly excited photosystem II (PSII) through phycocyanin, revealed fast energy transfer to another set of Chl f bands at 720-770 and 810 nm as well as to the 694-nm Chl a fluorescence band. The 694-nm band did not transfer excitation energy to Chl f. Therefore, Chl a in PSI, and phycocyanin in PSII of Fr cells transferred excitation energy to different sets of Chl f molecules. Multiple Chl f forms, thus, seem to work as the far-red antenna both in PSI and PSII. A variety of cyanobacterial species, phylogenically distant from each other, seems to use a Chl f antenna in far-red environments, such as under dense biomats, in colonies, or under far-red LED light.

A novel "oxygen-induced" greening process in a cyanobacterial mutant lacking the transcriptional activator ChlR involved in low-oxygen adaptation of tetrapyrrole biosynthesis.

ChlR activates the transcription of the chlAII-ho2-hemN operon in response to low-oxygen conditions in the cyanobacterium Synechocystis sp. PCC 6803. Three genes in the operon encode low-oxygen-type enzymes to bypass three oxygen-dependent reactions in tetrapyrrole biosynthesis. A chlR-lacking mutant, ΔchlR, shows poor photoautotrophic growth due to low chlorophyll (Chl) content under low-oxygen conditions, which is caused by no induction of the operon. Here, we characterized the processes of etiolation of ΔchlR cells in low-oxygen conditions and the subsequent regreening of the etiolated cells upon exposure to oxygen, by HPLC, Western blotting, and low-temperature fluorescence spectra. The Chl content of the etiolated ΔchlR cells incubated under low-oxygen conditions for 7 days was only 10% of that of the wild-type with accumulation of almost all intermediates of the magnesium branch of Chl biosynthesis. Both photosystem I (PSI) and photosystem II (PSII) were significantly decreased, accompanied by a preferential decrease of antenna Chl in PSI. Upon exposure to oxygen, the etiolated ΔchlR cells resumed to produce Chl after a short lag (∼2 h), and the level at 72 h was 80% of that of the wild-type. During this novel "oxygen-induced" greening process, the PSI and PSII contents were largely increased in parallel with the increase in Chl contents. After 72 h, the PSI content reached ∼50% of the wild-type level in contrast to the full recovery of PSII. ΔchlR provides a promising alternative system to investigate the biogenesis of PSI and PSII.

Arabitol provided by lichenous fungi enhances ability to dissipate excess light energy in a symbiotic green alga under desiccation.

Lichens are drought-resistant symbiotic organisms of mycobiont fungi and photobiont green algae or cyanobacteria, and have an efficient mechanism to dissipate excess captured light energy into heat in a picosecond time range to avoid photoinhibition. This mechanism can be assessed as drought-induced non-photochemical quenching (d-NPQ) using time-resolved fluorescence spectroscopy. A green alga Trebouxia sp., which lives within a lichen Ramalina yasudae, is one of the most common green algal photobionts. This alga showed very efficient d-NPQ under desiccation within the lichen thallus, whereas it lost d-NPQ ability when isolated from R. yasudae, indicating the importance of the interaction with the mycobiont for d-NPQ ability. We analyzed the water extracts from lichen thalli that enhanced d-NPQ in Trebouxia. Of several sugar compounds identified in the water extracts by nuclear magnetic resonance (NMR), mass spectrometry (MS) and gas chromatography (GC) analyses, only d-arabitol recovered d-NPQ in isolated Trebouxia to a level similar to that detected for R. yasudae thallus. Other sugar compounds did not help the expression of d-NPQ at the same concentrations. Thus, arabitol is essential for the expression of d-NPQ to dissipate excess captured light energy into heat, protecting the photobiont from photoinhibition. The relationship between mycobionts and photobionts is, therefore, not commensalism, but mutualism with each other, as shown by d-NPQ expression.

Dissipation of excess excitation energy by drought-induced nonphotochemical quenching in two species of drought-tolerant moss: desiccation-induced acceleration of photosystem II fluorescence decay.

Drought-tolerant mosses survive with their green color intact even after long periods of dehydration that would kill ordinary plants. The mechanism of dissipation of excitation energy under drought stress was studied in two species of drought-tolerant moss, Rhytidium rugosum and Ceratodon purpureus. They showed severe quenching of photosystem II chlorophyll fluorescence (PSII) after being dehydrated in the dark. Quenching was induced by the acceleration of the fluorescence decay rate. This drought-induced nonphotochemical quenching (designated d-NPQ) was fully reversed by rehydration. Global analysis of fluorescence decay at 77 K indicated rapid 46 ps transfer of excitation energy from the 680-690 nm PSII bands to a 710 nm band, and to 740-760 nm bands. The latter bands decayed to the ground state with the same time constant showing the rapid dissipation of excitation energy into heat. The quenching by d-NPQ in dry moss was stronger than that by PSII charge separation or nonphotochemical quenching (NPQ), which operates under hydrating conditions. Drought-tolerant mosses, thus, dissipate excess excitation energy into heat. The d-NPQ mechanism in moss resembles that reported in lichens, suggesting their common origin.

Molecular assembly of zinc chlorophyll derivatives by using recombinant light-harvesting polypeptides with His-tag and immobilization on a gold electrode.

LH1-α and -ß polypeptides, which make up the light-harvesting 1 (LH1) complex of purple photosynthetic bacteria, along with bacteriochlorophylls, have unique binding properties even for various porphyrin analogs. Herein, we used the porphyrin analogs, Zn-Chlorin and the Zn-Chlorin dimer, and examined their binding behaviors to the LH1-α variant, which has a His-tag at the C-terminus (MBP-rubα-YH). Zn-Chlorin and the Zn-Chlorin dimer could bind to MBP-rubα-YH and form a subunit-type assembly, similar to that from the native LH1 complex. These complexes could be immobilized onto Ni-nitrilotriacetic acid-modified Au electrodes, and the cathodic photocurrent was successfully observed by photoirradiation. Since Zn-Chlorins in this complex are too far for direct electron transfer from the electrode, a contribution of polypeptide backbone for efficient electron transfer was implied. These findings not only show interesting properties of LH1-α polypeptides but also suggest a clue to construct artificial photosynthesis systems using these peptide materials.

Three different mechanisms of energy dissipation of a desiccation-tolerant moss serve one common purpose: to protect reaction centres against photo-oxidation.

Three different types of non-photochemical de-excitation of absorbed light energy protect photosystem II of the sun- and desiccation-tolerant moss Rhytidium rugosum against photo-oxidation. The first mechanism, which is light-induced in hydrated thalli, is sensitive to inhibition by dithiothreitol. It is controlled by the protonation of a thylakoid protein. Other mechanisms are activated by desiccation. One of them permits exciton migration towards a far-red band in the antenna pigments where fast thermal deactivation takes place. This mechanism appears to be similar to a mechanism detected before in desiccated lichens. A third mechanism is based on the reversible photo-accumulation of a radical that acts as a quencher of excitation energy in reaction centres of photosystem II. On the basis of absorption changes around 800 nm, the quencher is suggested to be an oxidized chlorophyll. The data show that desiccated moss is better protected against photo-oxidative damage than hydrated moss. Slow drying of moss thalli in the light increases photo-protection more than slow drying in darkness.

Two functional sites of phosphatidylglycerol for regulation of reaction of plastoquinone Q(B) in photosystem II.

Functional roles of an anionic lipid phosphatidylglycerol (PG) were studied in pgsA-gene-inactivated and cdsA-gene-inactivated/phycobilisome-less mutant cells of a cyanobacterium Synechocystis sp. PCC 6803, which can grow only in PG-supplemented media. 1) A few days of PG depletion suppressed oxygen evolution of mutant cells supported by p-benzoquinone (BQ). The suppression was recovered slowly in a week after PG re-addition. Measurements of fluorescence yield indicated the enhanced sensitivity of Q(B) to the inactivation by BQ. It is assumed that the loss of low-affinity PG (PG(L)) enhances the affinity for BQ that inactivates Q(B). 2) Oxygen evolution without BQ, supported by the endogenous electron acceptors, was slowly suppressed due to the direct inactivation of Q(B) during 10 days of PG depletion, and was recovered rapidly within 10h upon the PG re-addition. It is concluded that the loss of high-affinity PG (PG(H)) displaces Q(B) directly. 3) Electron microscopy images of PG-depleted cells showed the specific suppression of division of mutant cells, which had developed thylakoid membranes attaching phycobilisomes (PBS). 4) Although the PG-depletion for 14 days decreased the chlorophyll/PBS ratio to about 1/4, flourescence spectra/lifetimes were not modified indicating the flexible energy transfer from PBS to different numbers of PSII. Longer PG-depletion enhanced allophycocyanin fluorescence at 683nm with a long 1.2ns lifetime indicating the suppression of energy transfer from PBS to PSII. 5) Action sites of PG(H), PG(L) and other PG molecules on PSII structure are discussed.

Roles of two ATPase-motif-containing domains in cyanobacterial circadian clock protein KaiC.

Cyanobacterial clock protein KaiC has a hexagonal, pot-shaped structure composed of six identical dumbbell-shaped subunits. Each subunit has duplicated domains, and each domain has a set of ATPase motifs. The two spherical regions of the dumbbell are likely to correspond to two domains. We examined the role of the two sets of ATPase motifs by analyzing the in vitro activity of ATPgammaS binding, AMPPNP-induced hexamerization, thermostability, and phosphorylation of KaiC and by in vivo rhythm assays both in wild type KaiC (KaiCWT) and KaiCs carrying mutations in either Walker motif A or deduced catalytic Glu residues. We demonstrated that 1) the KaiC subunit had two types of ATP-binding sites, a high affinity site in N-terminal ATPase motifs and a low affinity site in C-terminal ATPase motifs, 2) the N-terminal motifs were responsible for hexamerization, and 3) the C-terminal motifs were responsible for both stabilization and phosphorylation of the KaiC hexamer. We proposed the following reaction mechanism. ATP preferentially binds to the N-terminal high affinity site, inducing the hexamerization of KaiC. Additional ATP then binds to the C-terminal low affinity site, stabilizing and phosphorylating the hexamer. We discussed the effect of these KaiC mutations on circadian bioluminescence rhythm in cells of cyanobacteria.