A bacterium from a mountain lake harvests light using both proton-pumping xanthorhodopsins and bacteriochlorophyll-based photosystems.

Photoheterotrophic bacteria harvest light energy using either proton-pumping rhodopsins or bacteriochlorophyll (BChl)-based photosystems. The bacterium Sphingomonas glacialis AAP5 isolated from the alpine lake Gossenköllesee contains genes for both systems. Here, we show that BChl is expressed between 4°C and 22°C in the dark, whereas xanthorhodopsin is expressed only at temperatures below 16°C and in the presence of light. Thus, cells grown at low temperatures under a natural light-dark cycle contain both BChl-based photosystems and xanthorhodopsins with a nostoxanthin antenna. Flash photolysis measurements proved that both systems are photochemically active. The captured light energy is used for ATP synthesis and stimulates growth. Thus, S. glacialis AAP5 represents a chlorophototrophic and a retinalophototrophic organism. Our analyses suggest that simple xanthorhodopsin may be preferred by the cells under higher light and low temperatures, whereas larger BChl-based photosystems may perform better at lower light intensities. This indicates that the use of two systems for light harvesting may represent an evolutionary adaptation to the specific environmental conditions found in alpine lakes and other analogous ecosystems, allowing bacteria to alternate their light-harvesting machinery in response to large seasonal changes of irradiance and temperature.

Psb35 Protein Stabilizes the CP47 Assembly Module and Associated High-Light Inducible Proteins during the Biogenesis of Photosystem II in the Cyanobacterium Synechocystis sp. PCC6803.

Photosystem II (PSII) is a large membrane protein complex performing primary charge separation in oxygenic photosynthesis. The biogenesis of PSII is a complicated process that involves a coordinated linking of assembly modules in a precise order. Each such module consists of one large chlorophyll (Chl)-binding protein, number of small membrane polypeptides, pigments and other cofactors. We isolated the CP47 antenna module from the cyanobacterium Synechocystis sp. PCC 6803 and found that it contains a 11-kDa protein encoded by the ssl2148 gene. This protein was named Psb35 and its presence in the CP47 module was confirmed by the isolation of FLAG-tagged version of Psb35. Using this pulldown assay, we showed that the Psb35 remains attached to CP47 after the integration of CP47 into PSII complexes. However, the isolated Psb35-PSIIs were enriched with auxiliary PSII assembly factors like Psb27, Psb28-1, Psb28-2 and RubA while they lacked the lumenal proteins stabilizing the PSII oxygen-evolving complex. In addition, the Psb35 co-purified with a large unique complex of CP47 and photosystem I trimer. The absence of Psb35 led to a lower accumulation and decreased stability of the CP47 antenna module and associated high-light-inducible proteins but did not change the growth rate of the cyanobacterium under the variety of light regimes. Nevertheless, in comparison with WT, the Psb35-less mutant showed an accelerated pigment bleaching during prolonged dark incubation. The results suggest an involvement of Psb35 in the life cycle of cyanobacterial Chl-binding proteins, especially CP47.

Unique double concentric ring organization of light harvesting complexes in Gemmatimonas phototrophica.

The majority of life on Earth depends directly or indirectly on the sun as a source of energy. The initial step of photosynthesis is facilitated by light-harvesting complexes, which capture and transfer light energy into the reaction centers (RCs). Here, we analyzed the organization of photosynthetic (PS) complexes in the bacterium G. phototrophica, which so far is the only phototrophic representative of the bacterial phylum Gemmatimonadetes. The isolated complex has a molecular weight of about 800 ± 100 kDa, which is approximately 2 times larger than the core complex of Rhodospirillum rubrum. The complex contains 62.4 ± 4.7 bacteriochlorophyll (BChl) a molecules absorbing in 2 distinct infrared absorption bands with maxima at 816 and 868 nm. Using femtosecond transient absorption spectroscopy, we determined the energy transfer time between these spectral bands as 2 ps. Single particle analyses of the purified complexes showed that they were circular structures with an outer diameter of approximately 18 nm and a thickness of 7 nm. Based on the obtained, we propose that the light-harvesting complexes in G. phototrophica form 2 concentric rings surrounding the type 2 RC. The inner ring (corresponding to the B868 absorption band) is composed of 15 subunits and is analogous to the inner light-harvesting complex 1 (LH1) in purple bacteria. The outer ring is composed of 15 more distant BChl dimers with no or slow energy transfer between them, resulting in the B816 absorption band. This completely unique and elegant organization offers good structural stability, as well as high efficiency of light harvesting. Our results reveal that while the PS apparatus of Gemmatimonadetes was acquired via horizontal gene transfer from purple bacteria, it later evolved along its own pathway, devising a new arrangement of its light harvesting complexes.

Red-shifted light-harvesting system of freshwater eukaryotic alga Trachydiscus minutus (Eustigmatophyta, Stramenopila).

Survival of phototrophic organisms depends on their ability to collect and convert enough light energy to support their metabolism. Phototrophs can extend their absorption cross section by using diverse pigments and by tuning the properties of these pigments via pigment-pigment and pigment-protein interaction. It is well known that some cyanobacteria can grow in heavily shaded habitats by utilizing far-red light harvested with far-red-absorbing chlorophylls d and f. We describe a red-shifted light-harvesting system based on chlorophyll a from a freshwater eustigmatophyte alga Trachydiscus minutus (Eustigmatophyceae, Goniochloridales). A comprehensive characterization of the photosynthetic apparatus of T. minutus is presented. We show that thylakoid membranes of T. minutus contain light-harvesting complexes of several sizes differing in the relative amount of far-red chlorophyll a forms absorbing around 700 nm. The pigment arrangement of the major red-shifted light-harvesting complex is similar to that of the red-shifted antenna of a marine alveolate alga Chromera velia. Evolutionary aspects of the algal far-red light-harvesting complexes are discussed. The presence of these antennas in eustigmatophyte algae opens up new ways to modify organisms of this promising group for effective use of far-red light in mass cultures.

Quenching of chlorophyll triplet states by carotenoids in algal light-harvesting complexes related to fucoxanthin-chlorophyll protein.

We have used time-resolved absorption and fluorescence spectroscopy with nanosecond resolution to study triplet energy transfer from chlorophylls to carotenoids in a protective process that prevents the formation of reactive singlet oxygen. The light-harvesting complexes studied were isolated from Chromera velia, belonging to a group Alveolata, and Xanthonema debile and Nannochloropsis oceanica, both from Stramenopiles. All three light-harvesting complexes are related to fucoxanthin-chlorophyll protein, but contain only chlorophyll a and no chlorophyll c. In addition, they differ in the carotenoid content. This composition of the complexes allowed us to study the quenching of chlorophyll a triplet states by different carotenoids in a comparable environment. The triplet states of chlorophylls bound to the light-harvesting complexes were quenched by carotenoids with an efficiency close to 100%. Carotenoid triplet states were observed to rise with a ~5 ns lifetime and were spectrally and kinetically homogeneous. The triplet states were formed predominantly on the red-most chlorophylls and were quenched by carotenoids which were further identified or at least spectrally characterized.

Pigment configuration in the light-harvesting protein of the xanthophyte alga Xanthonema debile.

The soil chromophyte alga Xanthonema (X.) debile contains only non-carbonyl carotenoids and Chl-a. X. debile has an antenna system denoted Xanthophyte light-harvesting complex (XLH) that contains the carotenoids diadinoxanthin, heteroxanthin, and vaucheriaxanthin. The XLH pigment stoichiometry was calculated by chromatographic techniques and the pigment-binding structure studied by resonance Raman spectroscopy. The pigment ratio obtained by HPLC was found to be close to 8:1:2:1 Chl-a:heteroxanthin:diadinoxanthin:vaucheriaxanthin. The resonance Raman spectra suggest the presence of 8-10 Chl-a, all of which are 5-coordinated to the central Mg, with 1-3 Chl-a possessing a macrocycle distorted from the relaxed conformation. The three populations of carotenoids are in the all-trans configuration. Vaucheriaxanthin absorbs around 500-530 nm, diadinoxanthin at 494 nm and heteroxanthin at 487 nm at 4.5 K. The effective conjugation length of heteroxanthin and diadinoxanthin has been determined as 9.4 in both cases; the environment polarizability of the heteroxanthin and diadinoxanthin binding pockets is 0.270 and 0.305, respectively.

Modular antenna of photosystem I in secondary plastids of red algal origin: a Nannochloropsis oceanica case study.

Photosystem I (PSI) is a multi-subunit integral pigment-protein complex that performs light-driven electron transfer from plastocyanin to ferredoxin in the thylakoid membrane of oxygenic photoautotrophs. In order to achieve the optimal photosynthetic performance under ambient irradiance, the absorption cross section of PSI is extended by means of peripheral antenna complexes. In eukaryotes, this role is played mostly by the pigment-protein complexes of the LHC family. The structure of the PSI-antenna supercomplexes has been relatively well understood in organisms harboring the primary plastid: red algae, green algae and plants. The secondary endosymbiotic algae, despite their major ecological importance, have so far received less attention. Here we report a detailed structural analysis of the antenna-PSI association in the stramenopile alga Nannochloropsis oceanica (Eustigmatophyceae). Several types of PSI-antenna assemblies are identified allowing for identification of antenna docking sites on the PSI core. Instances of departure of the stramenopile system from the red algal model of PSI-Lhcr structure are recorded, and evolutionary implications of these observations are discussed.

Molecular basis of chromatic adaptation in pennate diatom Phaeodactylum tricornutum.

The remarkable adaptability of diatoms living in a highly variable environment assures their prominence among marine primary producers. The present study integrates biochemical, biophysical and genomic data to bring new insights into the molecular mechanism of chromatic adaptation of pennate diatoms in model species Phaeodactylum tricornutum, a marine eukaryote alga possessing the capability to shift its absorption up to ~700 nm as a consequence of incident light enhanced in the red component. Presence of these low energy spectral forms of Chl a is manifested by room temperature fluorescence emission maximum at 710 nm (F710). Here we report a successful isolation of the supramolecular protein complex emitting F710 and identify a member of the Fucoxanthin Chlorophyll a/c binding Protein family, Lhcf15, as its key building block. This red-shifted antenna complex of P. tricornutum appears to be functionally connected to photosystem II. Phylogenetic analyses do not support relation of Lhcf15 of P. tricornutum to other known red-shifted antenna proteins thus indicating a case of convergent evolutionary adaptation towards survival in shaded environments.

Native FMO-reaction center supercomplex in green sulfur bacteria: an electron microscopy study.

Chlorobaculum tepidum is a representative of green sulfur bacteria, a group of anoxygenic photoautotrophs that employ chlorosomes as the main light-harvesting structures. Chlorosomes are coupled to a ferredoxin-reducing reaction center by means of the Fenna-Matthews-Olson (FMO) protein. While the biochemical properties and physical functioning of all the individual components of this photosynthetic machinery are quite well understood, the native architecture of the photosynthetic supercomplexes is not. Here we report observations of membrane-bound FMO and the analysis of the respective FMO-reaction center complex. We propose the existence of a supercomplex formed by two reaction centers and four FMO trimers based on the single-particle analysis of the complexes attached to native membrane. Moreover, the structure of the photosynthetic unit comprising the chlorosome with the associated pool of RC-FMO supercomplexes is proposed.

Architecture of the light-harvesting apparatus of the eustigmatophyte alga Nannochloropsis oceanica.

We present proteomic, spectroscopic, and phylogenetic analysis of light-harvesting protein (Lhc) function in oleaginous Nannochloropsis oceanica (Eustigmatophyta, Stramenopila). N. oceanica utilizes Lhcs of multiple classes: Lhcr-type proteins (related to red algae LHCI), Lhcv (VCP) proteins (violaxanthin-containing Lhcs related to Lhcf/FCP proteins of diatoms), Lhcx proteins (related to Lhcx/LhcSR of diatoms and green algae), and Lhc proteins related to Red-CLH of Chromera velia. Altogether, 17 Lhc-type proteins of the 21 known from genomic data were found in our proteomic analyses. Besides Lhcr-type antennas, a RedCAP protein and a member of the Lhcx protein subfamily were found in association with Photosystem I. The free antenna fraction is formed by trimers of a mixture of Lhcs of varied origins (Lhcv, Lhcr, Lhcx, and relatives of Red-CLH). Despite possessing several proteins of the Red-CLH-type Lhc clade, N. oceanica is not capable of chromatic adaptation under the same conditions as the diatom Phaeodactylum tricornutum or C. velia. In addition, a naming scheme of Nannochloropsis Lhcs is proposed to facilitate further work.

Novel type of red-shifted chlorophyll a antenna complex from Chromera velia: II. Biochemistry and spectroscopy.

A novel chlorophyll a containing pigment-protein complex expressed by cells of Chromera velia adapted to growth under red/far-red illumination [1]. Purification of the complex was achieved by means of anion-exchange chromatography and gel-filtration. The antenna is shown to be an aggregate of ~20kDa proteins of the light-harvesting complex (LHC) family, unstable in the isolated form. The complex possesses an absorption maximum at 705nm at room temperature in addition to the main chlorophyll a maximum at 677nm producing the major emission band at 714nm at room temperature. The far-red absorption is shown to be the property of the isolated aggregate in the intact form and lost upon dissociation. The purified complex was further characterized by circular dichroism spectroscopy and fluorescence spectroscopy. This work thus identified the third different class of antenna complex in C. velia after the recently described FCP-like and LHCr-like antennas. Possible candidates for red antennas are identified in other taxonomic groups, such as eustigmatophytes and the relevance of the present results to other known examples of red-shifted antenna from other organisms is discussed. This work appears to be the first successful isolation of a chlorophyll a-based far-red antenna complex absorbing above 700nm unrelated to LHCI.

Light harvesting complexes of Chromera velia, photosynthetic relative of apicomplexan parasites.

The structure and composition of the light harvesting complexes from the unicellular alga Chromera velia were studied by means of optical spectroscopy, biochemical and electron microscopy methods. Two different types of antennae systems were identified. One exhibited a molecular weight (18-19kDa) similar to FCP (fucoxanthin chlorophyll protein) complexes from diatoms, however, single particle analysis and circular dichroism spectroscopy indicated similarity of this structure to the recently characterized XLH antenna of xanthophytes. In light of these data we denote this antenna complex CLH, for "Chromera Light Harvesting" complex. The other system was identified as the photosystem I with bound Light Harvesting Complexes (PSI-LHCr) related to the red algae LHCI antennae. The result of this study is the finding that C. velia, when grown in natural light conditions, possesses light harvesting antennae typically found in two different, evolutionary distant, groups of photosynthetic organisms.

Supramolecular organization of fucoxanthin-chlorophyll proteins in centric and pennate diatoms.

Fucoxanthin-chlorophyll proteins (FCP) are the major light-harvesting proteins of diatom algae, a major contributor to marine carbon fixation. FCP complexes from representatives of centric (Cyclotella meneghiniana) and pennate (Phaeodactylum tricornutum) diatoms were prepared by sucrose gradient centrifugation and studied by means of electron microscopy followed by single particle analysis. The oligomeric FCP from a centric diatom were observed to take the form of unusual chain-like or circular shapes, a very unique supramolecular assembly for such antennas. The existence of the often disputed oligomeric form of FCP in pennate diatoms has been confirmed. Contrary to the centric diatom FCP, pennate diatom FCP oligomers are very similar to oligomeric antennas from related heterokont (Stramenopila) algae. Evolutionary aspects of the presence of novel light-harvesting protein arrangement in centric diatoms are discussed.

Supramolecular organization of photosynthetic membrane proteins in the chlorosome-containing bacterium Chloroflexus aurantiacus.

The arrangement of core antenna complexes (B808-866-RC) in the cytoplasmic membrane of filamentous phototrophic bacterium Chloroflexus aurantiacus was studied by electron microscopy in cultures from different light conditions. A typical nearest-neighbor center-to-center distance of ~18 nm was found, implying less protein crowding compared to membranes of purple bacteria. A mean RC:chlorosome ratio of 11 was estimated for the occupancy of the membrane directly underneath each chlorosome, based on analysis of chlorosome dimensions and core complex distribution. Also presented are results of single-particle analysis of core complexes embedded in the native membrane.

A photoheterotrophic bacterium from Iceland has adapted its photosynthetic machinery to the long days of polar summer.

During their long evolution, anoxygenic phototrophic bacteria have inhabited a wide variety of natural habitats and developed specific strategies to cope with the challenges of any particular environment. Expression, assembly, and safe operation of the photosynthetic apparatus must be regulated to prevent reactive oxygen species generation under illumination in the presence of oxygen. Here, we report on the photoheterotrophic Sediminicoccus sp. strain KRV36, which was isolated from a cold stream in north-western Iceland, 30 km south of the Arctic Circle. In contrast to most aerobic anoxygenic phototrophs, which stop pigment synthesis when illuminated, strain KRV36 maintained its bacteriochlorophyll synthesis even under continuous light. Its cells also contained between 100 and 180 chromatophores, each accommodating photosynthetic complexes that exhibit an unusually large carotenoid absorption spectrum. The expression of photosynthesis genes in dark-adapted cells was transiently downregulated in the first 2 hours exposed to light but recovered to the initial level within 24 hours. An excess of membrane-bound carotenoids as well as high, constitutive expression of oxidative stress response genes provided the required potential for scavenging reactive oxygen species, safeguarding bacteriochlorophyll synthesis and photosystem assembly. The unique cellular architecture and an unusual gene expression pattern represent a specific adaptation that allows the maintenance of anoxygenic phototrophy under arctic conditions characterized by long summer days with relatively low irradiance.IMPORTANCEThe photoheterotrophic bacterium Sediminicoccus sp. KRV36 was isolated from a cold stream in Iceland. It expresses its photosynthesis genes, synthesizes bacteriochlorophyll, and assembles functional photosynthetic complexes under continuous light in the presence of oxygen. Unraveling the molecular basis of this ability, which is exceptional among aerobic anoxygenic phototrophic species, will help to understand the evolution of bacterial photosynthesis in response to changing environmental conditions. It might also open new possibilities for genetic engineering of biotechnologically relevant phototrophs, with the aim of increasing photosynthetic activity and their tolerance to reactive oxygen species.

Secretion of extracellular vesicles during ontogeny of the tapeworm Schistocephalus solidus.

We provide the first ultrastructural evidence of the secretion of extracellular vesicles (EVs) across all parasitic stages of the tapeworm Schistocephalus solidus (Müller, 1776) (Cestoda: Diphyllobothriidea) using a laboratory life cycle model. We confirmed the presence of EV-like bodies in all stages examined, including the hexacanth, procercoids in the copepod, Macrocyclops albidus (Jurine, 1820), plerocercoids from the body cavity of the three-spined stickleback, Gasterosteus aculeatus Linnaeus, and adults cultivated in artificial medium. In addition, we provide description of novel tegumental structures potentially involved in EV biogenesis and the presence of unique elongated EVs similar to those previously described only in Fasciola hepatica Linnaeus, 1758 (Trematoda), Hymenolepis diminuta (Rudolphi, 1819) (Cestoda), and Trypanosoma brucei Plimmer et Bradford, 1899 (Kinetoplastida).

Characterisation of the photosynthetic complexes from the marine gammaproteobacterium Congregibacter litoralis KT71.

Possibly the most abundant group of anoxygenic phototrophs are marine photoheterotrophic Gammaproteobacteria belonging to the NOR5/OM60 clade. As little is known about their photosynthetic apparatus, the photosynthetic complexes from the marine phototrophic bacterium Congregibacter litoralis KT71 were purified and spectroscopically characterised. The intra-cytoplasmic membranes contain a smaller amount of photosynthetic complexes when compared with anaerobic purple bacteria. Moreover, the intra-cytoplasmic membranes contain only a minimum amount of peripheral LH2 complexes. The complexes are populated by bacteriochlorophyll a, spirilloxanthin and two novel ketocarotenoids, with biophysical and biochemical properties similar to previously characterised complexes from purple bacteria. The organization of the RC-LH1 complex has been further characterised using cryo-electron microscopy. The overall organisation is similar to the complex from the gammaproteobacterium Thermochromatium tepidum, with the type-II reaction centre surrounded by a slightly elliptical LH1 antenna ring composed of 16 αß-subunits with no discernible gap or pore. The RC-LH1 and LH2 apoproteins are phylogenetically related to other halophilic species but LH2 also to some alphaproteobacterial species. It seems that the reduction of light-harvesting apparatus and acquisition of novel ketocarotenoids in Congregibacter litoralis KT71 represent specific adaptations for operating the anoxygenic photosynthesis under aerobic conditions at sea.

Localization of Pcb antenna complexes in the photosynthetic prokaryote Prochlorothrix hollandica.

The freshwater filamentous green oxyphotobacterium Prochlorothrix hollandica is an unusual oxygenic photoautotrophic cyanobacterium differing from most of the others by the presence of light-harvesting Pcb antenna binding both chlorophylls a and b and by the absence of phycobilins. The pigment-protein complexes of P. hollandica SAG 10.89 (CCAP 1490/1) were isolated from dodecylmaltoside solubilized thylakoid membranes on sucrose density gradient and characterized by biochemical, spectroscopic and immunoblotting methods. The Pcb antennae production is suppressed by high light conditions (>200 mumol photons m(-2) s(-1)) in P. hollandica. PcbC protein was found either in higher oligomeric states or coupled to PS I (forming antenna rings around PS I). PcbA and PcbB are most probably only very loosely bound to photosystems; we assume that these pigment-protein complexes function as low light-induced mobile antennae. Further, we have detected alpha-carotene in substantial quantities in P. hollandica thylakoid membranes, indicating the presence of chloroplast-like carotenoid synthetic pathway which is not present in common cyanobacteria.

Structure of PSI, PSII and antennae complexes from yellow-green alga Xanthonema debile.

Photosynthetic carbon fixation by Chromophytes is one of the significant components of a carbon cycle on the Earth. Their photosynthetic apparatus is different in pigment composition from that of green plants and algae. In this work we report structural maps of photosystem I, photosystem II and light harvesting antenna complexes isolated from a soil chromophytic alga Xanthonema debile (class Xanthophyceae). Electron microscopy of negatively stained preparations followed by single particle analysis revealed that the overall structure of Xanthophytes' PSI and PSII complexes is similar to that known from higher plants or algae. Averaged top-view projections of Xanthophytes' light harvesting antenna complexes (XLH) showed two groups of particles. Smaller ones that correspond to a trimeric form of XLH, bigger particles resemble higher oligomeric form of XLH.

Extracellular vesicles secreted by model tapeworm Hymenolepis diminuta: biogenesis, ultrastructure and protein composition.

We provided the first known evidence of the presence and release of extracellular vesicles in adults of important model tapeworm Hymenolepis diminuta. Two different subtypes have been observed on the surface of the worm and among the secretory products confirmed by several microscopical methods. Proteomic analysis revealed the presence of parasite-specific proteins as well as those of the host in purified extracellular vesicles. Among the protein cargo, we identified potential drug targets, vaccine candidates and H. diminuta antigens. Finally, the protein composition further revealed proteins participating in the endosomal complex required for transport-dependent biogenesis pathway.