Tatsuya Tomo

Niche adaptation and genome expansion in the chlorophyll d-producing cyanobacterium Acaryochloris marina

Acaryochloris marina is a unique cyanobacterium that is able to produce chlorophyll d as its primary photosynthetic pigment and thus efficiently use far-red light for photosynthesis. Acaryochloris species have been isolated from marine environments in association with other oxygenic phototrophs, which may have driven the niche-filling introduction of chlorophyll d. To investigate these unique adaptations, we have sequenced the complete genome of A. marina. The DNA content of A. marina is composed of 8.3 million base pairs, which is among the largest bacterial genomes sequenced thus far. This large array of genomic data is distributed into nine single-copy plasmids that code for >25% of the putative ORFs. Heavy duplication of genes related to DNA repair and recombination (primarily recA) and transposable elements could account for genetic mobility and genome expansion. We discuss points of interest for the biosynthesis of the unusual pigments chlorophyll d and α-carotene and genes responsible for previously studied phycobilin aggregates. Our analysis also reveals that A. marina carries a unique complement of genes for these phycobiliproteins in relation to those coding for antenna proteins related to those in Prochlorococcus species. The global replacement of major photosynthetic pigments appears to have incurred only minimal specializations in reaction center proteins to accommodate these alternate pigments. These features clearly show that the genus Acaryochloris is a fitting candidate for understanding genome expansion, gene acquisition, ecological adaptation, and photosystem modification in the cyanobacteria.

Identification of the special pair of photosystem II in a chlorophyll d-dominated cyanobacterium

The composition of photosystem II (PSII) in the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina MBIC 11017 was investigated to enhance the general understanding of the energetics of the PSII reaction center. We first purified photochemically active complexes consisting of a 47-kDa Chl protein (CP47), CP43′ (PcbC), D1, D2, cytochrome b559, PsbI, and a small polypeptide. The pigment composition per two pheophytin (Phe) a molecules was 55 ± 7 Chl d, 3.0 ± 0.4 Chl a, 17 ± 3 α-carotene, and 1.4 ± 0.2 plastoquinone-9. The special pair was detected by a reversible absorption change at 713 nm (P713) together with a cation radical band at 842 nm. FTIR difference spectra of the specific bands of a 3-formyl group allowed assignment of the special pair. The combined results indicate that the special pair comprises a Chl d homodimer. The primary electron acceptor was shown by photoaccumulation to be Phe a, and its potential was shifted to a higher value than that in the Chl a/Phe a system. The overall energetics of PSII in the Chl d system are adjusted to changes in the redox potentials, with P713 as the special pair using a lower light energy at 713 nm. Taking into account the reported downward shift in the potential of the special pair of photosystem I (P740) in A. marina, our findings lend support to the idea that changes in photosynthetic pigments combine with a modification of the redox potentials of electron transfer components to give rise to an energetic adjustment of the total reaction system.

Characterization of Highly Purified Photosystem I Complexes from the Chlorophyll d-dominated Cyanobacterium Acaryochloris marina MBIC 11017

Photochemically active photosystem (PS) I complexes were purified from the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina MBIC 11017, and several of their properties were characterized. PS I complexes consist of 11 subunits, including PsaK1 and PsaK2; a new small subunit was identified and named Psa27. The new subunit might replace the function of PsaI that is absent in A. marina. The amounts of pigments per one molecule of Chl d′ were 97.0 ± 11.0 Chl d, 1.9 ± 0.5 Chl a, 25.2 ± 2.4 α-carotene, and two phylloquinone molecules. The light-induced Fourier transform infrared difference spectroscopy and light-induced difference absorption spectra reconfirmed that the primary electron donor of PS I (P740) was the Chl d dimer. In addition to P740, the difference spectrum contained an additional band at 728 nm. The redox potentials of P740 were estimated to be 439 mV by spectroelectrochemistry; this value was comparable with the potential of P700 in other cyanobacteria and higher plants. This suggests that the overall energetics of the PS I reaction were adjusted to the electron acceptor side to utilize the lower light energy gained by P740. The distribution of charge in P740 was estimated by a density functional theory calculation, and a partial localization of charge was predicted to P1 Chl (special pair Chl on PsaA). Based on differences in the protein matrix and optical properties of P740, construction of the PS I core in A. marina was discussed.

Two unique cyanobacteria lead to a traceable approach of the first appearance of oxygenic photosynthesis

The evolutionary route from anoxygenic photosynthetic bacteria to oxygenic cyanobacteria is discontinuous in terms of photochemical/photophysical reaction systems. It is difficult to describe this transition process simply because there are no recognized intermediary organisms between the two bacterial groups. Gloeobacter violaceus PCC 7421 might be a model organism that is suitable for analysis because it still possesses primordial characteristics such as the absence of thylakoid membranes. Whole genome analysis and biochemical and biophysical surveys of G. violaceus have favored the hypothesis that it is an intermediary organism. On the other hand, species differentiation is an evolutionary process that could be driven by changes in a small number of genes, and this process might give fair information more in details by monitoring of those genes. Comparative studies of genes, including those in Acaryochloris marina MBIC 11017, have provided information relevant to species differentiation; in particular, the acquisition of a new pigment, chlorophyll d, and changes in amino acid sequences have been informative. Here, based on experimental evidence from these two species, we discuss some of the evolutionary pathways for the appearance and differentiation of cyanobacteria.

Replacement of chlorophyll with di-vinyl chlorophyll in the antenna and reaction center complexes of the cyanobacterium Synechocystis sp. PCC 6803: characterization of spectral and photochemical properties.

Chlorophyll (Chl) a in a cyanobacterium Synechocystis sp. PCC 6803 was replaced with di-vinyl (DV)-Chl a by knock-out of the specific gene (slr1923), responsible for the reduction of a 8-vinyl group, and optical and photochemical properties of purified photosystem (PS) II complexes (DV-PS II) were investigated. We observed differences in the peak wavelengths of absorption and fluorescence spectra; however, replacement of Chl a with DV-Chl a had limited effects. On the contrary, photochemical reactions were highly sensitive to high-light treatments in the mutant. Specifically, DV-Chl a was rapidly bleached under high-light conditions, and we detected significant dissociation of complexes and degradation of D1 proteins (PsbA). By comparing the SDS-PAGE patterns observed in this study to those observed in spinach chloroplasts, this degradation is assigned to the acceptor-side photoinhibition. The delayed fluorescence in the nanosecond time region at 77 K was suppressed in DV-PS II, possibly increasing triplet formation of Chl molecules. Our findings provide insight into the evolutionary processes of cyanobacteria. The effects of pigment replacement on the optimization of reactions are discussed.

Isolation and spectral characterization of Photosystem II reaction center from Synechocystis sp. PCC 6803.

We isolated highly-purified photochemically active photosystem (PS) II reaction center (RC) complexes from the cyanobacterium Synechocystis sp. PCC 6803 using a histidine-tag introduced to the 47xa0kDa chlorophyll protein, and characterized their spectroscopic properties. Purification was carried out in a one-step procedure after isolation of PS II core complex. The RC complexes consist of five polypeptides, the same as in spinach. The pigment contents per two molecules of pheophytin a were 5.8xa0±xa00.3 chlorophyll (Chl) a and 1.8xa0±xa00.1 β-carotene; one cytochrome b559 was found per 6.0 Chl a molecules. Overall absorption and fluorescence properties were very similar to those of spinach PS II RCs; our preparation retains the best properties so far isolated from cyanobacteria. However, a clear band-shift of pheophytin a and β-carotene was observed. Reasons for these differences, and RC composition, are discussed on the basis of the three-dimensional structure of complexes.

Spectral properties of the CP43-deletion mutant of Synechocystis sp. PCC 6803

Spectral properties, particularly fluorescence spectra and their time-dependent behavior, were investigated for a mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking the 43xa0kDa chlorophyll-protein (CP43, PsbC). Lack of CP43 was confirmed by a size shift of the corresponding gene and by Western blotting. The CP43-deletion mutant grown under heterotrophic conditions accumulated a small amount of photosystem (PS) II, but virtually no PS II fluorescence was observed. A 686-nm fluorescence band was clearly observed by phycocyanin excitation, coming from the terminal pigments of phycobilisomes. In contrast, no PS I fluorescence was detected by phycocyanin excitation when accumulation of PS II components was not proved by a fluorescence excitation spectrum, indicating that energy transfer to PS I chlorophyll a was mediated by PS II chlorophyll a. Direct connection of phycobilisomes with PS I was not suggested. Based on these fluorescence properties, the energy flow in the CP43-deletion mutant cells is discussed.

Unique Optical Properties of LHC II Isolated from Codium fragile – Its Correlation to Protein Environment

A Marine Green Alga, Codium Fragile, Contains A Specific Keto-Carotenoid, Siphonaxanthin (Siph), That Shows A Characteristic In Vivo Absorption Band At 535 Nm In Lhc Ii. This Band Is Ecologically Advantageous Under The Green Light-Rich Underwater Conditions, However It Is Not Detected In Solutions. In Isolated Lhc Ii Complexes, An Intensity Of The 535-Nm Absorption Band Of Siph Was Clearly Enhanced, And The Efficiency Of Energy Transfer From Siph To Chl Was Observed To Be Very High. We Confirmed That This Band Originated From A New Electronic Excited State (SX) Between The S2 And S1 States Based On Fluorescence Anisotropy Decay Of Codium Chloroplasts (Akimoto Et Al. 2004) And Isolated Lhc Ii Complexes (Akimoto Et Al. 2007), And Proposed That This SX State Derives From Distortion Of Siph In The Protein Environment. To Estimate The Effect Of Amino Acid Residues Close To Siph Molecules, We Determined A Nucleotide Sequence By Isolation Of A Major Lhcb Gene With Rt-Pcr Using Degenerate Oligonucleotide Primers. Although The Deduced Amino Acid Sequence Of Codium Lhc Ii Showed High Similarity To Those Of Other Lhc Ii, Some Codium-Specific Amino Acid Substitutions Were Observed. We Considered The Local Effect Of The Substitutions To The Electronic State Of Siph. A Possible Model Of The Molecular Interaction Of Siph And Protein Moiety In Codium Lhc Ii Will Be Discussed.

Detection of the D0→D1 transition of β-carotene radical cation photoinduced in photosystem II

The D0→D1 absorption band of a β-carotene radical cation in the near-infrared (NIR) region was detected for the first time in photosystem II (PSII). PSII-enriched membranes and isolated reaction center (RC) complexes (D1/D2/Cytb559) from spinach were illuminated at 80 and 150 K, respectively, in the presence of electron acceptors. In both preparations, UV-Vis-NIR difference spectra upon illumination exhibited a medium-intensity band at ∼1450 nm along with a strong band at ∼990 nm. The latter band has been assigned to the D0→D2 transition of the radical cation of the β-carotene in the secondary electron transfer pathway in PSII. These NIR bands disappeared at 210 K in the PSII membranes, and diminished their intensities in the RC complexes partially depleted of carotenoid. The absence or diminish of the β-carotene cation with little change in the formation of chlorophyll cations under these conditions were also confirmed by detecting light-induced FTIR difference spectra in the mid-IR region. From these results, it was concluded that the NIR band observed at ∼1450 nm arose from the D0→D1 transition of the β-carotene radical cation. It was shown that the observed band in the RC complexes was a mixture of the band of one β-carotene cation (Car507+) at 1464 nm and that of the other cation (Car489+) at a wavelength shorter than 1434 nm, indicating that the D0→D1 transition is sensitive to the protein environment. It is proposed that the position and the relative intensity of the D0→D1 band together with the well-known D0→D2 band can be useful monitors to investigate the properties of the radical cation and the molecular interaction of β-carotene in the PSII proteins.

Identification of the Special Pair and ChlZ of Photosystem II in Acaryochloris marina

The special pair and ChlorophyllZ (ChlZ) of the photosystem II (PSII) in the Chl d-dominated cyanobacterium, Acaryochlroris marina were studied using FT-IR and electronic absorption difference spectroscopy. We purified photochemically active complexes consisting of CP47, CP43′, D1, D2, cytochrome b 559, PsbI, and a small polypeptide. The special pair was detected by a reversible absorption change at 713 nm (P713) together with a cation radical band at 842 nm. FT-IR difference spectra of the specific bands of a 3-formyl group allowed assignment of the special pair. The combined results indicate that the special pair comprises a Chl d homodimer. Two molecules of ChlZ were also identified as Chl d using FT-IR difference spectra and UV/Vis absorption difference spectra. The primary electron acceptor was identified to be pheophytin (Phe) a, and its potential was shifted to a higher value than that in the Chl a/Phe a system. The overall energetics of PSII in the Chl d system are adjusted to changes in the redox potentials, with P713 as the special pair using a lower light energy at 713 nm. Our findings support the idea that changes in photosynthetic pigments combine with a modification of the redox potentials of electron transfer components to give rise to an energetic adjustment of the total reaction system.