Vibronic coupling explains the ultrafast carotenoid-to-bacteriochlorophyll energy transfer in natural and artificial light harvesters.

The initial energy transfer steps in photosynthesis occur on ultrafast timescales. We analyze the carotenoid to bacteriochlorophyll energy transfer in LH2 Marichromatium purpuratum as well as in an artificial light-harvesting dyad system by using transient grating and two-dimensional electronic spectroscopy with 10 fs time resolution. We find that Förster-type models reproduce the experimentally observed 60 fs transfer times, but overestimate coupling constants, which lead to a disagreement with both linear absorption and electronic 2D-spectra. We show that a vibronic model, which treats carotenoid vibrations on both electronic ground and excited states as part of the system's Hamiltonian, reproduces all measured quantities. Importantly, the vibronic model presented here can explain the fast energy transfer rates with only moderate coupling constants, which are in agreement with structure based calculations. Counterintuitively, the vibrational levels on the carotenoid electronic ground state play the central role in the excited state population transfer to bacteriochlorophyll; resonance between the donor-acceptor energy gap and the vibrational ground state energies is the physical basis of the ultrafast energy transfer rates in these systems.

Identifying residues that cause pH-dependent reduction potentials.

The pH dependence of the reduction potential E° for a metalloprotein indicates that the protonation state of at least one residue near the redox site changes and may be important for its activity. The responsible residue is usually identified by site-specific mutagenesis, which may be time-consuming. Here, the titration of E° for Chromatium vinosum high-potential iron-sulfur protein is predicted to be in good agreement with experiment using density functional theory and Poisson-Boltzmann calculations if only the sole histidine undergoes changes in protonation. The implementation of this approach into CHARMMing, a user-friendly web-based portal, allows users to identify residues in other proteins causing similar pH dependence.

Investigation of the redox interaction between Mn-bicarbonate complexes and reaction centers from Rhodobacter sphaeroides R-26, Chromatium minutissimum, and Chloroflexus aurantiacus.

The change in the dark reduction rate of photooxidized reaction centers (RC) of type II from three anoxygenic bacteria (Rhodobacter sphaeroides R-26, Chromatium minutissimum, and Chloroflexus aurantiacus) having different redox potentials of the P(+)/P pair and availability of RC for exogenous electron donors was investigated upon the addition of Mn(2+) and HCO(3)(-). It was found that the dark reduction of P(870)(+) from Rb. sphaeroides R-26 is considerably accelerated upon the combined addition of 0.5 mM MnCl(2) and 30-75 mM NaHCO(3) (as a result of formation of "low-potential" complexes [Mn(HCO(3))(2)]), while MnCl(2) and NaHCO(3) added separately had no such effect. The effect is not observed either in RC from Cf. aurantiacus (probably due to the low oxidation potential of the primary electron donor, P(865), which results in thermodynamic difficulties of the redox interaction between P(865)(+) and Mn(2+)) or in RC from Ch. minutissimum (apparently due to the presence of the RC-bound cytochrome preventing the direct interaction between P(870)(+) and Mn(2+)). The absence of acceleration of the dark reduction of P(870)(+) in the RC of Rb. sphaeroides R-26 when Mn(2+) and HCO(3)(-) were replaced by Mg(2+) or Ca(2+) and by formate, oxalate, or acetate, respectively, reveals the specificity of the Mn2+-bicarbonate complexes for the redox interaction with P(+). The results of this work might be considered as experimental evidence for the hypothesis of the participation of Mn(2+) complexes in the evolutionary origin of the inorganic core of the water oxidizing complex of photosystem II.

Anoxygenic photo- and chemo-synthesis of phototrophic sulfur bacteria from an alpine meromictic lake.

Meromictic lakes are interesting ecosystems to study anaerobic microorganisms due their permanent stratification allowing the formation of a stable anoxic environment. The crenogenic meromictic Lake Cadagno harbors an important community of anoxygenic phototrophic sulfur bacteria responsible for almost half of its total productivity. Besides their ability to fix CO2 through photosynthesis, these microorganisms also showed high rates of dark carbon fixation via chemosyntesis. Here, we grew in pure cultures three populations of anoxygenic phototrophic sulfur bacteria previously isolated from the lake, accounting for 72.8% of the total microbial community and exibiting different phenotypes: (1) the motile, large-celled purple sulfur bacterium (PSB) Chromatium okenii, (2) the small-celled PSB Thiodictyon syntrophicum and (3) the green sulfur bacterium (GSB) Chlorobium phaeobacteroides. We measured their ability to fix CO2 through photo- and chemo-synthesis, both in situ in the lake and in laboratory under different incubation conditions. We also evaluated the efficiency and velocity of H2S photo-oxidation, an important reaction in the anoxygenic photosynthesis process. Our results confirm that phototrophic sulfur bacteria strongly fix CO2 in the presence of light and that oxygen increases chemosynthesis at night, in laboratory conditions. Moreover, substancial differences were displayed between the three selected populations in terms of activity and abundance.

Heterotrophic euglenid Rhabdomonas costata resembles its phototrophic relatives in many aspects of molecular and cell biology.

Euglenids represent a group of protists with diverse modes of feeding. To date, only a partial genomic sequence of Euglena gracilis and transcriptomes of several phototrophic and secondarily osmotrophic species are available, while primarily heterotrophic euglenids are seriously undersampled. In this work, we begin to fill this gap by presenting genomic and transcriptomic drafts of a primary osmotroph, Rhabdomonas costata. The current genomic assembly length of 100 Mbp is 14× smaller than that of E. gracilis. Despite being too fragmented for comprehensive gene prediction it provided fragments of the mitochondrial genome and comparison of the transcriptomic and genomic data revealed features of its introns, including several candidates for nonconventional types. A set of 39,456 putative R. costata proteins was predicted from the transcriptome. Annotation of the mitochondrial core metabolism provides the first data on the facultatively anaerobic mitochondrion of R. costata, which in most respects resembles the mitochondrion of E. gracilis with a certain level of streamlining. R. costata can synthetise thiamine by enzymes of heterogenous provenances and haem by a mitochondrial-cytoplasmic C4 pathway with enzymes orthologous to those found in E. gracilis. The low percentage of green algae-affiliated genes supports the ancestrally osmotrophic status of this species.

Co-occurrence of denitrification and nitrogen fixation in a meromictic lake, Lake Cadagno (Switzerland).

The nitrogen cycling of Lake Cadagno was investigated by using a combination of biogeochemical and molecular ecological techniques. In the upper oxic freshwater zone inorganic nitrogen concentrations were low (up to approximately 3.4 microM nitrate at the base of the oxic zone), while in the lower anoxic zone there were high concentrations of ammonium (up to 40 microM). Between these zones, a narrow zone was characterized by no measurable inorganic nitrogen, but high microbial biomass (up to 4 x 10(7) cells ml(-1)). Incubation experiments with (15)N-nitrite revealed nitrogen loss occurring in the chemocline through denitrification (approximately 3 nM N h(-1)). At the same depth, incubations experiments with (15)N(2)- and (13)C(DIC)-labelled bicarbonate, indicated substantial N(2) fixation (31.7-42.1 pM h(-1)) and inorganic carbon assimilation (40-85 nM h(-1)). Catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and sequencing of 16S rRNA genes showed that the microbial community at the chemocline was dominated by the phototrophic green sulfur bacterium Chlorobium clathratiforme. Phylogenetic analyses of the nifH genes expressed as mRNA revealed a high diversity of N(2) fixers, with the highest expression levels right at the chemocline. The majority of N(2) fixers were related to Chlorobium tepidum/C. phaeobacteroides. By using Halogen In Situ Hybridization-Secondary Ion Mass Spectroscopy (HISH-SIMS), we could for the first time directly link Chlorobium to N(2) fixation in the environment. Moreover, our results show that N(2) fixation could partly compensate for the N loss and that both processes occur at the same locale at the same time as suggested for the ancient Ocean.

The structure of flavocytochrome c sulfide dehydrogenase from a purple phototrophic bacterium.

The structure of the heterodimeric flavocytochrome c sulfide dehydrogenase from Chromatium vinosum was determined at a resolution of 2.53 angstroms. It contains a glutathione reductase-like flavin-binding subunit and a diheme cytochrome subunit. The diheme cytochrome folds as two domains, each resembling mitochondrial cytochrome c, and has an unusual interpropionic acid linkage joining the two heme groups in the interior of the subunit. The active site of the flavoprotein subunit contains a catalytically important disulfide bridge located above the pyrimidine portion of the flavin ring. A tryptophan, threonine, or tyrosine side chain may provide a partial conduit for electron transfer to one of the heme groups located 10 angstroms from the flavin.

Molecular diversity of the ribulose-1,5-diphosphate carboxylase from photosynthetic microorganisms.

The ribulose-1,5-diphosphate carboxylases from green and blue-green algae and the purple sulfur photosynthetic bacterium Chromatium are proteins with high molecular weights and with sedimentation coefficients of 18 to 21 Svedberg units. The carboxylases of the Athiorhodaceae are smaller, that of Rhodospirillum rubrum being a 6.2S molecule, and those of the two species of Rhodopseudomonas are 12S and 14.5S.

Chromatium species: an emerging bioindicator of crude oil pollution of tidal mud flats in the Niger Delta mangrove ecosystem, Nigeria.

Establishing microbiological indices for the monitoring of environmental decay by crude oil pollution in the Niger Delta region has been a major concern of our current researches. Chromatium species, a purple, Gram positive pleomorphic, motile, microaerophlic sulfur bacterium offers a good potential for use in the assessment of the short term effects of oil pollution of tidal mud flats in the Niger Delta mangrove ecosystem. Its response to the November 22, 2003 spillage at the Qua Iboe Estuary and the adjoining Cross River Estuary was investigated. Our results have revealed that the sulphur bacterium is easily identified and widely distributed in the epipellic sediment of the mangrove ecosystem but very sensitive to hydrocarbon pollution. The bacterium was readily detected in the tidal mud flats containing as much as 2.0 mg kg(-1) but not detected in sediment with THC level of 3.65 mg kg(-1) and above. It is thus, suggestive that the threshold and lethal limits of effect of hydrocarbons against the sulfur bacterium lies between 2.04 and 3.65 mg kg(-1). These imply that in any case of crude oil pollution that Chromatium is not detected during monitoring the THC levels of the sediment may have been raised to a level close to or above 3.65 mg kg(-1). Statistical analysis of the relationship between THC level and density of Chromatium in sediment revealed a significant (p < 0.05) negative relationship (r = -0.85) in Qua Iboe mangrove ecosystem as against an insignificant (p > 0.05) relationship (r = -0.41) recorded for the Cross River mangrove ecosystem which served as the control. The result indicates that oil pollution affect the homeostatic status of Chromatium in tidal mud flats despite its even distribution (R2 = 71.4%). The finding though not definitive may contribute to the hierarchical process of oil pollution assessment in the Niger Delta region of Nigeria. However, its effective utilization will require not only the development of a selective medium for enumeration and isolation of the bacterium but also the establishment of a defined dose-response relationship under controlled conditions which requires further research.

[Physilogical and biochemical properties of bacteria of Chromatium genus, isolated from water bodies enriched with hydrogen sulfide].

Pure cultures of purple sulfur bacteria, which were attributed to genus Chromatium, were isolated from water bodies of the Yavoriv sulfur deposit. Both cultures perform anoxygenic photosynthesis and contain bacteriochlorophyll a and carotenoids of spirilloxanthin group. Isolated bacteria grow photolithoauthotrophically, photolithoheterotrophically and photoorganoheterotrophically. Hydrogen sulphide, sulfur and thiosulfate were used as inorganic electron donors. Bacteria were resistant to high hydrogen sulphide concentrations and assimilated it effectively in the process of anoxygenic photosynthesis. Isolated bacteria are considered as promising models for creation of biotechnologic ecosystems, which will be used for treatment of media polluted with sulfur compounds.

Draft Genome Sequence of Chromatium okenii Isolated from the Stratified Alpine Lake Cadagno.

Blooms of purple sulfur bacteria (PSB) are important drivers of the global sulfur cycling oxidizing reduced sulfur in intertidal flats and stagnant water bodies. Since the discovery of PSB Chromatium okenii in 1838, it has been found that this species is characteristic of for stratified, sulfidic environments worldwide and its autotrophic metabolism has been studied in depth since. We describe here the first high-quality draft genome of a large-celled, phototrophic, γ-proteobacteria of the genus Chromatium isolated from the stratified alpine Lake Cadagno, C. okenii strain LaCa. Long read technology was used to assemble the 3.78 Mb genome that encodes 3,016 protein-coding genes and 67 RNA genes. Our findings are discussed from an ecological perspective related to Lake Cadagno. Moreover, findings of previous studies on the phototrophic and the proposed chemoautotrophic metabolism of C. okenii were confirmed on a genomic level. We additionally compared the C. okenii genome with other genomes of sequenced, phototrophic sulfur bacteria from the same environment. We found that biological functions involved in chemotaxis, movement and S-layer-proteins were enriched in strain LaCa. We describe these features as possible adaptions of strain LaCa to rapidly changing environmental conditions within the chemocline and the protection against phage infection during blooms. The high quality draft genome of C. okenii strain LaCa thereby provides a basis for future functional research on bioconvection and phage infection dynamics of blooming PSB.

Epigenetics in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a disease characterized by uncontrolled proliferation of clonal neoplastic hematopoietic precursor cells. This leads to the disruption of normal hematopoiesis and bone marrow failure. Major breakthroughs in the past have contributed to our understanding of the genetic failures and the changed biology in AML cells that underlie the initiation and progression of the disease. It is now recognized that not only genetic but also epigenetic alterations are similarly important in this process. Since these alterations do not change the DNA sequences and are pharmacologically reversible, they have been regarded as optimal targets for what is now known as epigenetic therapy. In this review, we will discuss our current understanding of normal epigenetic processes, outline our knowledge of epigenetic alterations in AML, and discuss how this information is being used to improve current therapy of this disease.

Mechanical Unfolding Pathway of the High-Potential Iron-Sulfur Protein Revealed by Single-Molecule Atomic Force Microscopy: Toward a General Unfolding Mechanism for Iron-sulfur Proteins.

High-potential iron-sulfur proteins (HiPIPs) are an important class of metalloproteins with a [4Fe-4S] cluster coordinated by four cysteine residues. Distinct from other iron-sulfur proteins, the cluster in HiPIP has a high reduction potential, making it an essential electron carrier in bacterial photosynthesis. Here, we combined single-molecule atomic force microscopy and protein engineering techniques to investigate the mechanical unfolding mechanism of HiPIP from Chromatium tepidum (cHiPIP). We found that cHiPIP unfolds in a two-step fashion with the protein sequence sequestered by the iron-sulfur center as a stable unfolding intermediate state. The rupture of the iron-sulfur center of cHiPIP proceeds in two distinct parallel pathways; one pathway involves the concurrent rupture of multiple iron-thiolate bonds, and the other one involves the sequential rupture of the iron-thiolate bonds. This mechanistic information was further confirmed by mutational studies. We found that the rupture of the iron-thiolate bonds in reduced and oxidized cHiPIP occurred in the range of 150-180 pN at a pulling speed of 400 nm/s, similar to that measured for iron-thiolate bonds in rubredoxin and ferredoxin. Our results may have important implications for understanding the general unfolding mechanism governing iron-sulfur proteins, as well as the mechanism governing the mechanical rupture of the iron-sulfur center.

Reducing red color intensity of seafood wastewater in facultative pond.

Studies were carried out on the growth of Chromatium sp. on seafood wastewater (SFWW), which under facultative conditions and light exposure produced red pigment. The strain grew and utilized organic matter in both dark and light exposure conditions, but it produced red pigment when exposed to light. The growth was repressed by aerobic condition. The red color intensity was reduced by about 32.5+/-1.5 and 70.8+/-2.8% when kept under dark and static conditions, or aerobic and light exposure conditions, respectively. The COD of SFWW and the number of cells of Chromatium sp. were also rapidly reduced by about 78.6+/-2.7 and 92.0+/-1.0%, respectively, under aerobic and light exposure condition. KNO3 and FeCl3 also reduced red color intensity and maximum removal of organic matter and red color were 30 and 4 mg/l, respectively. Aerobic conditions increased the color removal efficiency with 30 mg/l KNO3 and 4 mg/l FeCl3 treatments up to 96.5+/-1 and 98.9+/-1%, respectively.

Characterization of glutathione amide reductase from Chromatium gracile. Identification of a novel thiol peroxidase (Prx/Grx) fueled by glutathione amide redox cycling.

Among the Chromatiaceae, the glutathione derivative gamma-l-glutamyl-l-cysteinylglycine amide, or glutathione amide, was reported to be present in facultative aerobic as well as in strictly anaerobic species. The gene (garB) encoding the central enzyme in glutathione amide cycling, glutathione amide reductase (GAR), has been isolated from Chromatium gracile, and its genomic organization has been examined. The garB gene is immediately preceded by an open reading frame encoding a novel 27.5-kDa chimeric enzyme composed of one N-terminal peroxiredoxin-like domain followed by a glutaredoxin-like C terminus. The 27.5-kDa enzyme was established in vitro to be a glutathione amide-dependent peroxidase, being the first example of a prokaryotic low molecular mass thiol-dependent peroxidase. Amino acid sequence alignment of GAR with the functionally homologous glutathione and trypanothione reductases emphasizes the conservation of the catalytically important redox-active disulfide and of regions involved in binding the FAD prosthetic group and the substrates glutathione amide disulfide and NADH. By establishing Michaelis constants of 97 and 13.2 microm for glutathione amide disulfide and NADH, respectively (in contrast to K(m) values of 6.9 mm for glutathione disulfide and 1.98 mm for NADPH), the exclusive substrate specificities of GAR have been documented. Specificity for the amidated disulfide cofactor partly can be explained by the substitution of Arg-37, shown by x-ray crystallographic data of the human glutathione reductase to hydrogen-bond one of the glutathione glycyl carboxylates, by the negatively charged Glu-21. On the other hand, the preference for the unusual electron donor, to some extent, has to rely on the substitution of the basic residues Arg-218, His-219, and Arg-224, which have been shown to interact in the human enzyme with the NADPH 2'-phosphate group, by Leu-197, Glu-198, and Phe-203. We suggest GAR to be the newest member of the class I flavoprotein disulfide reductase family of oxidoreductases.

The use of a water-soluble carbodiimide to study the interaction between Chromatium vinosum flavocytochrome c-552 and cytochrome c.

The interaction between horse heart cytochrome c and Chromatium vinosum flavocytochrome c-552 was studied using the water-soluble reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Treatment of flavocytochrome c-552 with EDC was found to inhibit the sulfide: cytochrome c reductase activity of the enzyme. SDS gel electrophoresis studies revealed that EDC treatment led to modification of carboxyl groups in both the Mr 21 000 heme peptide and the Mr 46 000 flavin peptide, and also to the formation of a cross-linked heme peptide dimer with an Mr value of 42 000. Both the inhibition of sulfide: cytochrome c reductase activity and the formation of the heme peptide dimer were decreased when the EDC modification was carried out in the presence of cytochrome c. In addition, two new cross-linked species with Mr values of 34 000 and 59 000 were formed. These were identified as cross-linked cytochrome c-heme peptide and cytochrome c-flavin peptide species, respectively. Neither of these species were formed in the presence of a cytochrome c derivative in which all of the lysine amino groups had been dimethylated, demonstrating that EDC had cross-linked lysine amino groups on native cytochrome c to carboxyl groups on the heme and flavin peptides. A complex between cytochrome c and flavocytochrome c-552 was required for cross-linking to occur, since ionic strengths above 100 mM inhibited cross-linking.

EPR studies of protein state transitions in Chromatium ferricytochrome c'.

Recent magnetic studies have shown that in the pH range 1 to 11 the bacterial heme protein ferricytochrome c' can undergo reversible transitions between various pure high-spin and quantum mechanically mixed (intermediate and high)-spin protein states. The EPR data presented here extend the recent work to high alkaline pH and show that above pH 11.6 reversible transitions occur between various pure high-spin and pure low-spin protein states. The new data and the previous magnetic studies of the protein are discussed in the context of the entatic nature of the iron-porphyrin complex in ferricytochrome c'. EPR data are also provided concerning an anomalous, but reversible, transition to a protein state with reduced heme iron, triggered by freezing and thawing at physiological pH.

Molecular cloning and sequencing of cytochrome c' from the phototrophic purple sulfur bacterium Chromatium vinosum.

The gene for cytochrome c' from Chromatium vinosum was cloned from a HindIII-SalI digest of genomic DNA. A 1.4 kbp fragment containing the gene was sequenced in both directions using the Sanger dideoxy method. The cytochrome c' gene codes for a 154-residue peptide, of which the last 131 amino acids match the previously determined sequence of the protein. The remaining 23 residues represent a signal sequence that is cleaved from the polypeptide upon translocation to the periplasmic space. An additional open reading frame on the other strand of the fragment codes for a peptide that contains four regions that are homologous to corresponding regions of the cytochrome b-type subunit of several Ni-Fe hydrogenases.

Visible absorption spectra of quantum mixed-spin ferric heme proteins.

Recently, it has been shown that the magnetic data for Chromatium ferricytochrome c' at pH 7 are consistent with quantum mechanically (as distinguished from thermally) mixed mid-spin (S = 3/2) and high-spin (S = 5/2) heme. Visible absorption spectra of the protein measured at 77 degrees K and 293 degrees K, pH 7, show peaks at 400, 490, and 632 nm. The observation of a 630 nm band in quantum mixed-spin heme spectra, and the spin state-dependence of the band intensity, are discussed in the context of the iron-ligand structure for quantum mixed-spin heme inferred from magnetic data.

Effects of surface potential and membrane potential on the midpoint potential of cytochrome c-555 bound to the chromatophore membrane of Chromatium vinosum.

The values of midpoint potential (Em) of cytochrome c-555 bound to the chromatophore membranes of a photosynthetic bacterium Chromatium vinosum was determined under various pH and salt conditions. After a long incubation at high ionic concentrations in the presence of carbonylcyanide m-chlorophenylhydrazone, which was added to abolish electrical potential difference between the inner and outer bulk phases of chromatophore, the Em value was almost constant at pH values between 4.0 and 8.4. With the decrease of salt concentration, the pH dependence of the Em value became more marked. Under low ionic conditions, Em became more positive with the decrease of pH. Addition of salt made the value more positive or negative at pH values higher or lower than 4.5, respectively. Divalent cation salts were more effective than monovalent cation salts in producing the positive shift of Em at pH 7.8. The Em value became more positive when the electrical potential of the inner side of the chromatophore was made more positive by the diffusion potential induced by the K+ concentration gradient in the presence of valinomycin. These results were explained by a change of redox potential at the inner surface of the chromatophore membrane, at which the cytochrome is assumed to be situated, due to the electrical potential difference with respect to the outer solution induced by the surface potential or membrane potential change. The values for the surface potential and the net surface charge density of the inner surface of the chromatophore membrane were estimated using the Gouy-Chapman diffuse double layer theory.