The Path to Thioredoxin and Redox Regulation in Chloroplasts.

After a brief discussion of my graduate work at Duke University, I describe a series of investigations on redox proteins at the University of California, Berkeley. Starting with ferredoxin from fermentative bacteria, the Berkeley research fostered experiments that uncovered a pathway for fixing CO2 in bacterial photosynthesis. The carbon work, in turn, opened new vistas, including the discovery that thioredoxin functions universally in regulating the Calvin-Benson cycle in oxygenic photosynthesis. These experiments, which took place over a 50-year period, led to the formulation of a set of biological principles and set the stage for research demonstrating a role for redox in the regulation of previously unrecognized processes extending far beyond photosynthesis.

Coupling dark metabolism to electricity generation using photosynthetic cocultures.

We investigated the role of green sulfur bacteria inlight-responsive electricity generation in microbial electrochemical cells (MXCs). We operated MXCs containing either monocultures or defined cocultures of previously enriched phototrophic Chlorobium and anode-respiring Geobacter under anaerobic conditions in the absence of electron donor. Monoculture control MXCs containing Geobacter or Chlorobium neither responded to light nor produced current, respectively. Instead, light-responsive current generation occurred only in coculture MXCs. Current increased above background levels only in the dark and declined slowly over 96 h. This pattern suggested that Chlorobium exhausted intracellular glycogen reserves via dark fermentation to supply an electron donor, presumably acetate, to Geobacter. With medium containing sulfide as the sole photosynthetic electron donor, current generation had a similar and reproducible negative light response. To investigate whether this metabolic interaction also occurred without an electrode, we performed coculture experiments in batch serum bottles. In this setup, sulfide served as the sole electron donor, whose oxidation by Chlorobium was required to provide S(0) as the electron acceptor to Geobacter. Copies of Geobacter 16S rDNA increased approximately 14-fold in batch bottle cocultures containing sulfide compared to those lacking sulfide, and did not decline after termination of sulfide feeding. These results suggest that products of both photosynthesis and dark fermentation by Chlorobium were sufficient both to yield an electrochemical response by Geobacter biofilms, and to promote Geobacter growthin batch cocultures. Our work expands upon the fusion of MXCs with coculture techniques and reinforces the utility of microbial electrochemistry for sensitive, real-time monitoring of microbial interactions in which a metabolic intermediate can be converted to electrical current.

Metabolic analysis of Chlorobium chlorochromatii CaD3 reveals clues of the symbiosis in 'Chlorochromatium aggregatum'.

A symbiotic association occurs in 'Chlorochromatium aggregatum', a phototrophic consortium integrated by two species of phylogenetically distant bacteria composed by the green-sulfur Chlorobium chlorochromatii CaD3 epibiont that surrounds a central ß-proteobacterium. The non-motile chlorobia can perform nitrogen and carbon fixation, using sulfide as electron donors for anoxygenic photosynthesis. The consortium can move due to the flagella present in the central ß-protobacterium. Although Chl. chlorochromatii CaD3 is never found as free-living bacteria in nature, previous transcriptomic and proteomic studies have revealed that there are differential transcription patterns between the symbiotic and free-living status of Chl. chlorocromatii CaD3 when grown in laboratory conditions. The differences occur mainly in genes encoding the enzymatic reactions involved in nitrogen and amino acid metabolism. We performed a metabolic reconstruction of Chl. chlorochromatii CaD3 and an in silico analysis of its amino acid metabolism using an elementary flux modes approach (EFM). Our study suggests that in symbiosis, Chl. chlorochromatii CaD3 is under limited nitrogen conditions where the GS/GOGAT (glutamine synthetase/glutamate synthetase) pathway is actively assimilating ammonia obtained via N2 fixation. In contrast, when free-living, Chl. chlorochromatii CaD3 is in a condition of nitrogen excess and ammonia is assimilated by the alanine dehydrogenase (AlaDH) pathway. We postulate that 'Chlorochromatium aggregatum' originated from a parasitic interaction where the N2 fixation capacity of the chlorobia would be enhanced by injection of 2-oxoglutarate from the ß-proteobacterium via the periplasm. This consortium would have the advantage of motility, which is fundamental to a phototrophic bacterium, and the syntrophy of nitrogen and carbon sources.

Comparison of the physical characteristics of chlorosomes from three different phyla of green phototrophic bacteria.

Chlorosomes, the major antenna complexes in green sulphur bacteria, filamentous anoxygenic phototrophs, and phototrophic acidobacteria, are attached to the cytoplasmic side of the inner cell membrane and contain thousands of bacteriochlorophyll (BChl) molecules that harvest light and channel the energy to membrane-bound reaction centres. Chlorosomes from phototrophs representing three different phyla, Chloroflexus (Cfx.) aurantiacus, Chlorobaculum (Cba.) tepidum and the newly discovered "Candidatus (Ca.) Chloracidobacterium (Cab.) thermophilum" were analysed using PeakForce Tapping atomic force microscopy (PFT-AFM). Gentle PFT-AFM imaging in buffered solutions that maintained the chlorosomes in a near-native state revealed ellipsoids of variable size, with surface bumps and undulations that differ between individual chlorosomes. Cba. tepidum chlorosomes were the largest (133×57×36nm; 141,000nm(3) volume), compared with chlorosomes from Cfx. aurantiacus (120×44×30nm; 84,000nm(3)) and Ca. Cab. thermophilum (99×40×31nm; 65,000nm(3)). Reflecting the contributions of thousands of pigment-pigment stacking interactions to the stability of these supramolecular assemblies, analysis by nanomechanical mapping shows that chlorosomes are highly stable and that their integrity is disrupted only by very strong forces of 1000-2000pN. AFM topographs of Ca. Cab. thermophilum chlorosomes that had retained their attachment to the cytoplasmic membrane showed that this membrane dynamically changes shape and is composed of protrusions of up to 30nm wide and 6nm above the mica support, possibly representing different protein domains. Spectral imaging revealed significant heterogeneity in the fluorescence emission of individual chlorosomes, likely reflecting the variations in BChl c homolog composition and internal arrangements of the stacked BChls within each chlorosome.

A variety of glycolipids in green photosynthetic bacteria.

The compositions of glycolipids in the following seven strains of green photosynthetic bacteria were investigated at the molecular level using LC-MS coupled with an evaporative light scattering detector: Chlorobium (Chl.) limicola strains Larsen (30 °C as the optimal cultivation temperature) and DSM245 (30 °C), Chlorobaculum (Cba.) tepidum strain ATCC49652 (45 °C), Cba. parvum strain NCIB8327 (30 °C), Cba. limnaeum strain 1549 (30 °C), Chl. phaeovibrioides DSM269 (30 °C), and Chloroflexus (Cfl.) aurantiacus strain J-10-fl (55 °C). Dependence of the molecular structures of glycolipids including the chain-length of their acyl groups upon bacterial cultivation temperatures was clearly observed. The organisms with their optimal temperatures of 30, 45, and 55 °C dominantly accumulated glycolipids possessing the acyl chains in the range of C(15)-C(16), C(16)-C(17), and C(18)-C(20), respectively. Cba. tepidum with an optimal temperature of 45 °C preferred the insertion of a methylene group to produce finally a C(17)-cyclopropane chain. Cfl. aurantiacus cultured optimally at 55 °C caused a drastic increase in the chain-length. Notably, the length of such acyl groups corresponded to that of the esterifying chain in the 17-propionate residues of self-aggregative bacteriochlorophylls-c/d/e, indicating stabilization of their supramolecular structures through hydrophobic interactions among those hydrocarbon chains. Based on the detailed compositions of glycolipids, a survival strategy of green photosynthetic bacteria grown in the wide range of temperatures is discussed.

[On structure and function of "chlorosoma" of green bacteria].

An assertion is substantiated that what is widely termed as chlorosoma of green bacteria--is not a bioparticle, but simply microscopic bacteriochlorophyll-c crystals. Apparently the creation of "chlorosoma" represents the first mostly unsuccessful evolutionary attempt to produce the regulatory mechanism in photosynthesis, which should react to the variations in the intensity of solar light reaching earth surface. It could not be successful without bacteriochlorophyll cooperation with proteins.

Two-dimensional spectroscopy can distinguish between decoherence and dephasing of zero-quantum coherences.

Recent experiments on a variety of photosynthetic antenna systems have revealed that coherences among electronic states persist longer than previously anticipated. In an ensemble measurement, the observed dephasing of a coherent state can occur because of either disorder across the ensemble or decoherence from interactions with the bath. Distinguishing how much such disorder affects the experimentally observed dephasing rate is paramount for understanding the role that quantum coherence may play in energy transfer through these complexes. Here, we show that two-dimensional electronic spectra can distinguish between the limiting cases of homogeneous dephasing (decoherence) and inhomogeneous dephasing by examining how the quantum beat frequency changes within a cross peak. For the antenna complex LH2 isolated from Rhodobacter sphaeroides , we find that dephasing of the coherence between the B850 and B800 rings arises predominantly from inhomogeneity. In contrast, within the Fenna-Matthews-Olson (FMO) complex from Chlorobium tepidum , dephasing of the coherence between the first two excitons appears quite homogeneous. Thus, the observed dephasing rate sets an upper bound on decoherence for the LH2 complex while establishing both an upper and lower bound for the FMO complex.

Temperature- and time-dependent changes in the structure and composition of glycolipids during the growth of the green sulfur photosynthetic bacterium Chlorobaculum tepidum.

The green sulfur photosynthetic bacterium Chlorobaculum (Cba.) tepidum (previously known as Chlorobium tepidum), which grows at an optimal temperature of around 45 °C, biosynthesized unique disaccharide rhamnosylgalactosyldiacylglyceride (RGDG) having a methylene-bridged palmitoleyl (17:Cyc) and a palmitoyl group (16:0) as the two acyl chains in a molecule [RGDG(17:Cyc,16:0)], together with the corresponding monosaccharide monogalactosyldiacylglyceride (MGDG). Here, we report changes in the structure and composition of the glycolipids that are dependent upon the temperature and period of cultivation. With a decrease in temperature to 25 °C, the two major glycolipids were almost completely eliminated, and MGDG with a palmitoleyl (16:1) and a (16:0) group concomitantly became the major glycolipid. MGDG(16:1,16:0) corresponded to the removal of an α-rhamnosyl and a cyclopropyl methylene group from RGDG(17:Cyc,16:0) and the lack of the CH(2) group in MGDG(17:Cyc,16:0). The structural conversion was almost reversible when the Cba. tepidum adapted to low and high temperatures was cultured again at 45 and 25 °C, respectively. Moreover, during this cultivation, the structure and composition of glycolipids were sequentially changed: MGDG(16:1,16:0), MGDG(17:Cyc,16:0), and RGDG(17:Cyc,16:0) predominated in the exponential, stationary and late phases of the cultivation, respectively. On the basis of these time-dependent changes, the unique disaccharide RGDG(17:Cyc,16:0) was thought to be created by the site-specific transfer of an α-rhamnosyl group to MGDG(17:Cyc,16:0) after insertion of a methylene group into the precursor MGDG(16:1,16:0). These culturing temperature- and time-dependent changes in glycolipids at the molecular level allow us to discuss their biosynthesis as well as physiological function in green photosynthetic bacteria.

Robustness of electronic coherence in the Fenna-Matthews-Olson complex to vibronic and structural modifications.

We present the first two-dimensional electronic spectra of photosynthetic antenna complexes bearing modifications to the protein and the chromophores. The vibronic structure of the Fenna-Matthews-Olson complex was altered by near-complete substitution of 13C for naturally abundant carbon and separately by randomly distributed partial deuteration. The structure and arrangement of the bacteriochlorophyll a chromophores were modified by deletion of the gene encoding the enzyme responsible for reducing the isoprenoid tail of the bacteriochlorophylls. Analysis of the time-dependent amplitude of the crosspeak corresponding to excitons 1 and 2 indicates that these modifications do not affect the frequency or dephasing of the beating observed in this particular peak. This result leads us to conclude that this beating indeed arises from electronic coherence and not vibrational wavepacket motion. We further conclude that the protection of zero-quantum coherences afforded by the protein matrix of this photosynthetic complex is not the result of a finely-tuned series of system-bath interactions perfected by billions of years of evolution but rather a simple downstream property of a close arrangement of chromophores within a phonon bath. We conclude with a brief discussion of the outstanding questions and possible applications of this phenomenon.

Direct counting of submicrometer-sized photosynthetic apparatus dispersed in medium at cryogenic temperature by confocal laser fluorescence microscopy: estimation of the number of bacteriochlorophyll c in single light-harvesting antenna complexes chlorosomes of green photosynthetic bacteria.

The number of pigments in single light-harvesting complexes (chlorosomes) were calculated by imaging single chlorosomes in a frozen buffer at cryogenic temperature with a confocal laser fluorescence microscope and pigment extraction. Chlorosomes were isolated from two types of green photosynthetic bacteria Chlorobium (Chl.) tepidum and Chloroflexus (Cfl.) aurantiacus and were individually imaged in the frozen medium. Each fluorescence spot observed mainly came from a single chlorosome and was ascribable to self-aggregates of bacteriochlorophyll (BChl) c molecules as core parts of chlorosomes. A three-dimensional distribution of fluorescence of single chlorosomes was analyzed, and the number of chlorosomes in a volume of 54,000 microm(3) was counted directly. On the basis of the results, averaged numbers of the BChl c molecules contained in a single chlorosome of Chl. tepidum and Cfl. aurantiacus were determined to be 1.4 x 10(5) and 9.6 x 10(4), respectively. The present numbers are almost comparable to those estimated by other methods (Martinez-Planells et al., Photosynth. Res. 2002, 71, 83 and Montaño et al., Biophys. J. 2003, 85, 2560).

Chlorobium chlorochromatii sp. nov., a symbiotic green sulfur bacterium isolated from the phototrophic consortium "Chlorochromatium aggregatum".

A symbiotic green sulfur bacterium, strain CaD, was isolated from an enrichment culture of the phototrophic consortium "Chlorochromatium aggregatum". The capability of the epibiont to grow in pure culture indicates that it is not obligately symbiotic. Cells are Gram-negative, nonmotile, rod-shaped and contain chlorosomes. Strain CaD is obligately anaerobic and photolithoautotrophic, using sulfide as electron donor. Acetate and peptone are photoassimilated in the presence of sulfide and hydrogencarbonate. Photosynthetic pigments contain bacteriochlorophylls a and c, and gamma-carotene and OH-gamma-carotene glucoside laurate as the dominant carotenoids. In cells from pure cultures, chlorosomes are equally distributed along the inner face of the cytoplasmic membrane. In contrast, the distribution of the chlorosomes in symbiotic epibiont cells is uneven, with chlorosomes being entirely absent at the site of attachment to the central bacterium. The symbiotic epibiont cells display a conspicuous additional layered structure at the attachment site. The G + C content of genomic DNA of strain CaD is 46.7 mol%. On the basis of 16S rRNA sequence comparison, the strain is distantly related to Chlorobium species within the green sulfur bacteria phylum (

[Ecophysiological properties of photosynthesizing bacteria from the Black Sea chemocline zone].

In May 1998, during the fifty-first voyage on board the research vessel Professor Vodyanitskii, a comparative study was conducted of the species diversity of green and purple sulfur bacteria in the water column of the chemocline zone at deep-sea stations and on the bottom surface of the Black Sea shallow regions. At three deep-sea stations, the accumulation of photosynthesizing bacteria in the chemocline zone at a depth of 85-115 m was revealed on the basis of the distribution of potential values of carbon dioxide light fixation. The location of the site of potential carbon dioxide light fixation suggests that the photosynthesis may be determined by the activity of the brown Chlorobium sp., revealed earlier at these depths. Enrichment cultures of brown sulfur bacteria were obtained from samples taken at the deep-sea stations. By morphology, these bacteria, assigned to Chlorobium sp., appear as nonmotile straight or slightly curved rods 0.3-0.5 x 0.7-1.2 microm in size; sometimes, they form short chains. Ultrathin sections show photosynthesizing antenna-like structures, chlorosomes, typical of Chlorobiaceae. The cultures depended on the presence of NaCl (20 g/l) for growth, which corresponds to the mineralization of Black Sea water. The bacteria could grow photoautotrophically, utilizing sulfide, but the Black Sea strains grew much more slowly than the known species of brown sulfur bacteria isolated from saline or freshwater meromictic lakes. The best growth of the strains studied in this work occurred in media containing ethanol (0.5 g) or sodium acetate (1 g/l) and low amounts of sulfide (0.4 mM), which is consistent with the conditions of syntrophic growth with sulfidogens. The data obtained allow us to conclude that the cultures of brown sulfur bacteria are especially adapted to developing at large depths under conditions of electron donor deficiency owing to syntrophic development with sulfate reducers. The species composition of the photosynthetic bacteria developing in the bottom sediments of shallow stations differed substantially from that observed at deep-sea stations. Pure cultures of the green Chlorobium sp. BS 1C and BS 2C (chlorobactin as the carotenoid), purple sulfur bacteria Chromatium sp. BS 1Ch (containing spirilloxanthine series pigments), and Thiocapsa marina BS 2Tc (containing the carotenoid okenone) were obtained from samples of sediments at shallow-water stations. Brown sulfur bacteria were absent in the sediment samples obtained from the Black Sea shallow-water stations 1 and 2.

The role of carotenoids in the photoadaptation of the brown-colored sulfur bacterium Chlorobium phaeobacteroides.

The brown-colored sulfur bacterium Chlorobium (Cb.) phaeobacteroides 1549 (new name, Chlorobaculum limnaeum 1549) contains many kinds of carotenoids as well as bacteriochlorophyll (BChl) e. These carotenoids were identified with C18-high-performance liquid chromatography, absorption, mass and proton nuclear magnetic resonance spectroscopies and were divided into two groups: the first is carotenoid with one or two phi-end groups such as isorenieratene and beta-isorenieratene and the second is carotenoid with one or two beta-end groups such as p-zeacarotene, beta-carotene and 7,8-dihydro-beta-carotene. The latter 7,8-dihydro-beta-carotene was found to be a novel carotenoid in nature. OH-gamma-Carotene glucoside laurate and OH-chlorobactene glucoside laurate were also found as minor components. The distribution of BChl e homologs in Cb. phaeobacteroides cultivated under various light intensities did not change, but the carotenoid to BChl e ratio changed markedly: carotenoid with the phi-end group maintained the same ratio to BChl e, whereas that with the beta-end group increased with increasing light intensity. The cells cultured under low-light intensity contained more phi-end carotenoids than beta-end. In Cb. phaeobacteroides the wavelength of the Qy band of BChl e aggregates did not change. We suggested that Cb. phaeobacteroides photoadapts to light intensity by changing the carotenoid composition.