Project description:Green photosynthetic bacteria harvest light and perform photosynthesis in low-light environments, and contain specialized antenna complexes to adapt to this condition. We performed small-angle neutron scattering (SANS) studies to obtain structural information about the photosynthetic apparatus, including the peripheral light-harvesting chlorosome complex, the integral membrane light-harvesting B808-866 complex, and the reaction center (RC) in the thermophilic green phototrophic bacterium Chloroflexus aurantiacus. Using contrast variation in SANS measurements, we found that the B808-866 complex is wrapped around the RC in Cfx. aurantiacus, and the overall size and conformation of the B808-866 complex of Cfx. aurantiacus is roughly comparable to the LH1 antenna complex of the purple bacteria. A similar size of the isolated B808-866 complex was suggested by dynamic light scattering measurements, and a smaller size of the RC of Cfx. aurantiacus compared to the RC of the purple bacteria was observed. Further, our SANS measurements indicate that the chlorosome is a lipid body with a rod-like shape, and that the self-assembly of bacteriochlorophylls, the major component of the chlorosome, is lipid-like. Finally, two populations of chlorosome particles are suggested in our SANS measurements.
Project description:The green phototrophic bacteria contain a unique complement of chlorophyll pigments, which self-assemble efficiently into antenna structures known as chlorosomes with little involvement of protein. The few proteins found in chlorosomes have previously been thought to have a primarily structural function. The biosynthetic pathway of the chlorosome pigments, bacteriochlorophylls c, d, and e, is not well understood. In this report, we used spectroscopic, proteomic, and gene expression approaches to investigate the chlorosome proteins of the green filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus. Surprisingly, Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase, AcsF, was identified under anaerobic growth conditions. The AcsF protein was found in the isolated chlorosome fractions, and the proteomics analysis suggested that significant portions of the AcsF proteins are not accessible to protease digestion. Additionally, quantitative real-time PCR studies showed that the transcript level of the acsF gene is not lower in anaerobic growth than in semiaerobic growth. Since the proposed enzymatic activity of AcsF requires molecular oxygen, our studies suggest that the roles of AcsF in C. aurantiacus need to be investigated further.
Project description:The green filamentous bacterium Chloroflexus aurantiacus employs chlorosomes as photosynthetic antennae. Chlorosomes contain bacteriochlorophyll aggregates and are attached to the inner side of a plasma membrane via a protein baseplate. The structure of chlorosomes from C. aurantiacus was investigated by using a combination of cryo-electron microscopy and X-ray diffraction and compared with that of Chlorobi species. Cryo-electron tomography revealed thin chlorosomes for which a distinct crystalline baseplate lattice was visualized in high-resolution projections. The baseplate is present only on one side of the chlorosome, and the lattice dimensions suggest that a dimer of the CsmA protein is the building block. The bacteriochlorophyll aggregates inside the chlorosome are arranged in lamellae, but the spacing is much greater than that in Chlorobi species. A comparison of chlorosomes from different species suggested that the lamellar spacing is proportional to the chain length of the esterifying alcohols. C. aurantiacus chlorosomes accumulate larger quantities of carotenoids under high-light conditions, presumably to provide photoprotection. The wider lamellae allow accommodation of the additional carotenoids and lead to increased disorder within the lamellae.
Project description:BACKGROUND: Chloroflexus aurantiacus is a thermophilic filamentous anoxygenic phototrophic (FAP) bacterium, and can grow phototrophically under anaerobic conditions or chemotrophically under aerobic and dark conditions. According to 16S rRNA analysis, Chloroflexi species are the earliest branching bacteria capable of photosynthesis, and Cfl. aurantiacus has been long regarded as a key organism to resolve the obscurity of the origin and early evolution of photosynthesis. Cfl. aurantiacus contains a chimeric photosystem that comprises some characters of green sulfur bacteria and purple photosynthetic bacteria, and also has some unique electron transport proteins compared to other photosynthetic bacteria. METHODS: The complete genomic sequence of Cfl. aurantiacus has been determined, analyzed and compared to the genomes of other photosynthetic bacteria. RESULTS: Abundant genomic evidence suggests that there have been numerous gene adaptations/replacements in Cfl. aurantiacus to facilitate life under both anaerobic and aerobic conditions, including duplicate genes and gene clusters for the alternative complex III (ACIII), auracyanin and NADH:quinone oxidoreductase; and several aerobic/anaerobic enzyme pairs in central carbon metabolism and tetrapyrroles and nucleic acids biosynthesis. Overall, genomic information is consistent with a high tolerance for oxygen that has been reported in the growth of Cfl. aurantiacus. Genes for the chimeric photosystem, photosynthetic electron transport chain, the 3-hydroxypropionate autotrophic carbon fixation cycle, CO2-anaplerotic pathways, glyoxylate cycle, and sulfur reduction pathway are present. The central carbon metabolism and sulfur assimilation pathways in Cfl. aurantiacus are discussed. Some features of the Cfl. aurantiacus genome are compared with those of the Roseiflexus castenholzii genome. Roseiflexus castenholzii is a recently characterized FAP bacterium and phylogenetically closely related to Cfl. aurantiacus. According to previous reports and the genomic information, perspectives of Cfl. aurantiacus in the evolution of photosynthesis are also discussed. CONCLUSIONS: The genomic analyses presented in this report, along with previous physiological, ecological and biochemical studies, indicate that the anoxygenic phototroph Cfl. aurantiacus has many interesting and certain unique features in its metabolic pathways. The complete genome may also shed light on possible evolutionary connections of photosynthesis.
Project description:Superoxide dismutase from the thermophilic anoxygenic photosynthetic bacterium Chloroflexus aurantiacus was cloned, purified, and characterized. This protein is in the manganese- and iron-containing family of superoxide dismutases and is able to use both manganese and iron catalytically. This appears to be the only soluble superoxide dismutase in C. aurantiacus. Iron and manganese cofactors were identified by using electron paramagnetic resonance spectroscopy and were quantified by atomic absorption spectroscopy. By metal enrichment of growth media and by performing metal fidelity studies, the enzyme was found to be most efficient with manganese incorporated, yet up to 30% of the activity was retained with iron. Assimilation of iron or manganese ions into superoxide dismutase was also found to be affected by the growth conditions. This enzyme was also found to be remarkably thermostable and was resistant to H2O2 at concentrations up to 80 mM. Reactive oxygen defense mechanisms have not been previously characterized in the organisms belonging to the phylum Chloroflexi. These systems are of interest in C. aurantiacus since this bacterium lives in a hyperoxic environment and is subject to high UV radiation fluxes.
Project description:Early-time dynamics of absorbance changes (light minus dark) in the long-wavelength Qy absorption band of bacteriochlorophyll dimer P of isolated reaction centers (RCs) from thermophilic green bacterium Chloroflexus (Cfx.) aurantiacus was studied by difference pump-probe spectroscopy with 18-fs resolution at cryogenic temperature. It was found that the stimulated emission spectrum gradually moves to the red on the ~100-fs time scale and subsequently oscillates with a major frequency of ~140?cm-1. By applying the non-secular Redfield theory and linear susceptibility theory, the coherent dynamics of the stimulated emission from the excited state of the primary electron donor, bacteriochlorophyll dimer P*, was modeled. The model showed the possibility of an extremely fast transition from the locally excited state P1* to the spectrally different excited state P2*. This transition is clearly seen in the kinetics of the stimulated emission at 880 and 945?nm, where mostly P1* and P2* states emit, respectively. These findings are similar to those obtained previously in RCs of the purple bacterium Rhodobacter (Rba.) sphaeroides. The assumption about the existence of the second excited state P2* helps to explain the complicated temporal behavior of the ?A spectrum measured by pump-probe spectroscopy. It is interesting that, in spite of the strong coupling between the P1* and P2* states assumed in our model, the form of the coherent oscillations is mainly defined by pure vibrational coherence in the excited states. A possible nature of the P2* state is discussed.
Project description:Enolase catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate during both glycolysis and gluconeogenesis, and is required by all three domains of life. Here, we report the purification and biochemical and structural characterization of enolase from Chloroflexus aurantiacus, a thermophilic anoxygenic phototroph affiliated with the green non-sulfur bacteria. The protein was purified as a homodimer with a subunit molecular weight of 46?kDa. The temperature optimum for enolase catalysis was 80°C, close to the measured thermal stability of the protein which was determined to be 75°C, while the pH optimum for enzyme activity was 6.5. The specific activities of purified enolase determined at 25 and 80°C were 147 and 300?U?mg(-1) of protein, respectively. K m values for the 2-phosphoglycerate/phosphoenolpyruvate reaction determined at 25 and 80°C were 0.16 and 0.03?mM, respectively. The K m values for Mg(2+) binding at these temperatures were 2.5 and 1.9?mM, respectively. When compared to enolase from mesophiles, the biochemical and structural properties of enolase from C. aurantiacus are consistent with this being thermally adapted. These data are consistent with the results of our phylogenetic analysis of enolase, which reveal that enolase has a thermophilic origin.
Project description:Using RecA as the phylogenetic marker, the relationships of the green sulfur bacterium Chlorobium tepidum and the green non-sulfur bacterium Chloroflexus aurantiacus to other eubacteria were investigated. The recA genes of the two organisms were cloned, and the resulting protein sequences aligned with 86 other eubacterial RecA sequences. Cb. tepidum was placed as the nearest relative to the Cytophaga/Flexibacter/Bacteriodes group, a relationship supported by results obtained with several phylogenetic markers. Cf. aurantiacus was placed near Chlamydia trachomatis and the high-GC Gram-positives; however, this branching pattern was not strongly supported statistically by bootstrap analyses. Possible reasons for this ambiguity are discussed.
Project description:Chloroflexus aurantiacus is an anoxygenic phototrophic bacterium. Its unique CO2 fixation pathway and primitive light-harvesting antenna complexes have attracted extensive research attentions. In this work, we investigated the photoheterotrophic growth of C. aurantiacus J-10-fl using acetate [at 55°C and without H2(g)]. The results indicate that glycine can promote anaerobic biomass production in a minimal medium by threefold to fivefold. Via (13)C-metabolite analysis, we observed that glycine was involved in serine synthesis. Instead of being used as a major carbon source, glycine was degraded to produce C1 units and NAD(P)H. Tracer experiments also suggest that photoheterotrophic cultures growing with a exogenous glycine source exhibited capabilities of assimilating CO2 via multiple routes (including the 3-hydroxypropionate pathway). Finally, glycylglycine, a commonly used culture buffer, also significantly enhanced photoheterotrophic growth of C. aurantiacus, probably due to its thermal or enzymatic breakdown to glycine.
Project description:Chloroflexus aurantiacus is a facultative autotrophic green nonsulfur bacterium that grows phototrophically in thermal springs and forms microbial mats with cyanobacteria. Cyanobacteria produce glycolate during the day (photorespiration) and excrete fermentation products at night. C. aurantiacus uses the 3-hydroxypropionate bi-cycle for autotrophic carbon fixation. This pathway was thought to be also suited for the coassimilation of various organic substrates such as glycolate, acetate, propionate, 3-hydroxypropionate, lactate, butyrate, or succinate. To test this possibility, we added these compounds at a 5 mM concentration to autotrophically pregrown cells. Although the provided amounts of H(2) and CO(2) allowed continuing photoautotrophic growth, cells immediately consumed most substrates at rates equaling the rate of autotrophic carbon fixation. Using [(14)C]acetate, half of the labeled organic carbon was incorporated into cell mass. Our data suggest that C. aurantiacus uses the 3-hydroxypropionate bi-cycle, together with the glyoxylate cycle, to channel organic substrates into the central carbon metabolism. Enzyme activities of the 3-hydroxypropionate bi-cycle were marginally affected when cells were grown heterotrophically with such organic substrates. The 3-hydroxypropionate bi-cycle in Chloroflexi is unique and was likely fostered in an environment in which traces of organic compounds can be coassimilated. Other bacteria living under oligotrophic conditions acquired genes of a rudimentary 3-hydroxypropionate bi-cycle, possibly for the same purpose. Examples are Chloroherpeton thalassium, Erythrobacter sp. strain NAP-1, Nitrococcus mobilis, and marine gammaproteobacteria of the OM60/NOR5 clade such as Congregibacter litoralis.