Bioaccumulation and molecular effects of sediment-bound metals in zebrafish embryos.

Predicting the bioavailability and effects of metals in sediments is of major concern in context with sediment risk assessment. This study aimed to investigate the bioavailability and molecular effects of metals spiked into riverine sediments to zebrafish (Danio rerio) embryos. Embryos were exposed to a natural and an artificial sediment spiked with cadmium (Cd), copper (Cu), nickel (Ni) and zinc (Zn) individually or as a mixture at concentrations ranging from 150 to 3000 mg/kg dry weight (dw) over 48 h, and uptake of metals was determined. Furthermore, transcript abundances of the metallothioneins MT1 and MT2, the metal-responsive element-binding transcription factor (MTF) and the genes sod1, hsp70 and hsp90α1 were measured as indicators of metal-induced or general cellular stress. D. rerio embryos accumulated metals from sediments at concentrations up to 100 times greater than those spiked to the sediment with the greatest bioaccumulation factor (BAF) for Cu from artificial sediment (275.4 ± 41.9 (SD)). Embryos accumulated greater concentrations of all metals from artificial than from natural sediment, and accumulation was greater when embryos were exposed to individual metals than when they were exposed to the mixture. Exposure of embryos to Zn or the mixture exhibited up to 30-fold greater transcript abundances of MT1, MT2 and hsp70 compared to controls which is related to significant uptake of Zn from the sediment. Further changes in transcript abundances could not be related to a significant uptake of metals from sediments. These studies reveal that metals from spiked sediments are bioavailable to D. rerio embryos directly exposed to sediments and that the induction of specific genes can be used as biomarkers for the exposure of early life stages of zebrafish to metal-contaminated sediments.

Variability of sediment-contact tests in freshwater sediments with low-level anthropogenic contamination--determination of toxicity thresholds.

Freshwater sediments with low levels of anthropogenic contamination and a broad range of geochemical properties were investigated using various sediment-contact tests in order to study the natural variability and to define toxicity thresholds for the various toxicity endpoints. Tests were performed with bacteria (Arthrobacter globiformis), yeast (Saccharomyces cerevisiae), nematodes (Caenorhabditis elegans), oligochaetes (Lumbriculus variegatus), higher plants (Myriophyllum aquaticum), and the eggs of zebrafish (Danio rerio). The variability in the response of some of the contact tests could be explained by particle size distribution and organic content. Only for two native sediments could a pollution effect not be excluded. Based on the minimal detectable difference (MDD) and the maximal tolerable inhibition (MTI), toxicity thresholds (% inhibition compared to the control) were derived for each toxicity parameter: >20% for plant growth and fish-egg survival, >25% for nematode growth and oligochaete reproduction, >50% for nematode reproduction and >60% for bacterial enzyme activity.

Membrane-associated c-type cytochromes from the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum: purification and characterization of cytochrome c553.

Tetraheme cytochromes involved in photosynthetic electron transport have previously been described associated with the reaction centers of purple photosynthetic bacteria; however, similar heme proteins have not until now been characterized in the phylogenetically distinct green sulfur bacteria. In this paper we describe the first isolation and characterization of a multitheme, membrane-associated cytochrome from a green sulfur bacterium, Chlorobium limicola forma thiosulfatophilum. We show that this cytochrome contains a single polypeptide of 32 kDa apparent molecular mass on SDS-PAGE and has a characteristic broad alpha-band absorption at 553 nm. By both low-temperature absorption and electron paramagnetic resonance spectroscopy, we demonstrate that there are at least four distinct heme groups.

Symmetric structural features and binding site of the primary electron donor in the reaction center of Chlorobium.

The protein binding interactions of the constituent bacteriochlorophyll a molecules of the primary electron donor, P840, in isolated reaction centers from Chlorobium limicola f thiosulphatophilum and the electronic symmetry of the radical cation P840+. were determined using near-infrared Fourier transform (FT) Raman spectroscopy excited at 1064 nm. The FT Raman vibrational spectrum of P840 indicates that it is constituted of a single population of BChl a molecules which are spectrally indistinguishable. The BChl a molecules of P840 are pentacoordinated with only one axial ligand on the central Mg atom, and the pi-conjugated C2 acetyl and C9 keto carbonyls are free of hydrogen-bonding interactions. The FT Raman spectrum of P840+. exhibits a 1707 cm-1 band attributable to a BChl a C9 keto carbonyl group vibrational frequency that has upshifted 16 cm-1 upon oxidation of P840; this upshift is exactly one-half of that expected for the one-electron oxidation of monomeric BChl a in vitro. The 16 cm-1 upshift, thus, indicates that the resulting +1 charge is equally shared between two BChl a molecules. This situation is markedly different from that of the oxidized primary donor of the purple bacterial reaction center of Rhodobacter sphaeroides, (i) which exhibits a 1717 cm-1 band that has upshifted 26 cm-1, indicating an asymmetric distribution of the resulting +1 charge over the two constituent BChl a molecules, and (ii) whose H-bonding pattern with respect to the pi-conjugated carbonyl groups is asymmetric.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and binding site of the primary electron acceptor in the reaction center of Chlorobium.

In isolated, chlorosome-free reaction centers from Chlorobium limicola f thiosulphatophilum, a chlorin pigment exhibits a Qy absorption band at 672 nm (Feiler, U., Nitschke, W., & Michel, H. (1992) Biochemistry 31, 2608-2614). To characterize the chemical nature of this chlorin pigment and its interactions within the reaction-center protein, selective enhancement of its Raman scattering was achieved by resonant excitation within its Soret band. This is the first time that structural studies of this pigment were performed on the native reaction-center protein. The obtained resonance Raman spectra were consistent with a single population of a chlorophyll a(-like) pigment, possessing a vinyl group on ring I, but not with bacteriochlorophyll c or bacteriophaeophytin c. The stretching frequencies of the C9-keto carbonyl of this pigment indicates that it is H-bonded to the reaction-center protein. The strength of this H-bond is very close to those of the keto carbonyls of the primary electron acceptors in purple bacterial reaction centers and D1/D2 particles. Since in membranes of Chlorobiaceae a transient bleaching at 670 nm is due to the primary acceptor in the reaction center (Nuijs, A. M., Vasmel, H., Joppe, H. L. P., Duysens, L. N. M., & Amesz, J. (1985a) Biochim. Biophys. Acta 907, 24-34), we thus conclude that the primary acceptor in Chlorobium reaction centers is the characterized chlorophyll a(-like) pigment.(ABSTRACT TRUNCATED AT 250 WORDS)

Pigment interactions in chlorosomes of various green bacteria.

Resonance Raman experiments were performed on different green bacteria. With blue excitation, i.e. under Soret resonance or preresonance conditions, resonance Raman contributions were essentially arising from the chlorosome pigments. By comparing these spectra and those of isolated chlorosomes, it is possible to evaluate how the latter retain their native structure during the isolation procedures. The structure of bacteriochlorophyll oligomers in chlorosomes was interspecifically compared, in bacteriochlorophyllc- and bacteriochlorophylle- synthesising bacteria. It appears that interactions assumed by the 9-keto carbonyl group are identical inChlorobium limicola, Chlorobium tepidum, andChlorobium phaeobacteroides. In the latter strain, the 3-formyl carbonyl group of bacteriochlorophylle is kept free from intermolecular interactions. By contrast, resonance Raman spectra unambiguously indicate that the structure of bacteriochlorophyll oligomers is slightly different in chlorosomes fromChloroflexus auranticus, either isolated or in the whole bacteria.

Characterization of an improved reaction center preparation from the photosynthetic green sulfur bacterium Chlorobium containing the FeS centers FA and FB and a bound cytochrome subunit.

A photosynthetic reaction center complex was prepared from the green sulfur bacterium Chlorobium by solubilization of chlorosome-depleted membranes with lauryl maltoside, followed by anion-exchange chromatography and molecular sieve chromatography. The purified complex was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, optical spectroscopy, and EPR spectroscopy. The major bands migrated at apparent molecular masses of 50, 42, and 32 kDa (heme-staining) and additional weaker bands at 22, 15, and 12 kDa. The isolated reaction center complex contained about 40 bacteriochlorophyll alpha molecules per primary electron donor, P840, assayed by photooxidation. It was competent in stable low-temperature photoreduction of the FeS centers FA and FB. The spectra of these acceptors and their low-temperature photochemistry in the purified complex were the same as found in intact Chlorobium membranes and similar to what had been described for photosystem I from plants. Membrane-bound cytochrome c553 copurified with the reaction center complex. A ratio of about four hemes per P840 was determined. This result indicates that cytochrome c553 that is closely associated with the reaction center is a tetraheme cytochrome, as described for some purple bacteria.

Reaction center photochemistry of Heliobacterium chlorum.

Reaction center photochemistry in Heliobacterium chlorum has been investigated by using EPR and flash absorption spectroscopy at low temperatures. The following results were obtained. At 5 K, in the presence of ascorbate, continuous illumination resulted in the formation of P798+ and a reduced iron-sulfur center designated FB (gz = 2.07, gy = 1.93, gx = 1.89). This state was stable at low temperatures, but the yield for this reaction was low, and it was estimated that it occurred only in about 3% of the centers upon the first flash. After continuous illumination of a dilute sample for 10 min, still only half of the centers attained this state. In most centers, flash excitation at 5 K produced a state which recombined with time constants of 2.5 ms (congruent to 80%) and 850 microseconds (congruent to 20%). These two phases were differently influenced by the redox state of the reaction center, indicating that two different acceptors were involved in the recombination reactions. When continuous illumination was given at 200 K, a second center, designated FA, was additionally reduced (gz = 2.05, gy = 1.95, gx = 1.90). High concentrations of dithionite resulted in the chemical reduction of FB and of most of FA; illumination at 200 K resulted in the further reduction of FA. Two triplet states were identified by EPR and optical spectroscopy. The amplitude of the narrower triplet (magnitude of D = 226 x 10(-4) cm-1) varied with the redox state of the iron-sulfur centers and was influenced by a component thought to be a quinone undergoing double reduction. It correlated with a triplet state observed by flash absorption spectroscopy showing a bleaching at 798 nm and is attributed to a triplet state formed by charge recombination in the reaction center. Its narrowness is taken as an indication of its origin on a pair of bacteriochlorophylls, and its orientation indicates an orientation of the chlorophyll ring plane perpendicular to the membrane plane. The second triplet had a wider splitting (magnitude of D = 242 x 10(-4) cm-1), did not vary systematically with redox conditions, corresponds to an optical spectrum with a maximum at 812 nm, and is not ordered in the membrane. It was thus attributed to a triplet located on a BChl g monomer in the antenna. The reaction center photochemistry in H. chlorum is comparable in many respects to that of photosystem I and green sulfur bacteria. Earlier contrasting conclusions are discussed and rationalized in light of the present results.

Photosynthetic reaction center of green sulfur bacteria studied by EPR.

Membrane preparations of two species of the green sulfur bacteria Chlorobium have been studied by EPR. Three signals were detected which were attributed to iron-sulfur centers acting as electron acceptors in the photosynthetic reaction center. (1) A signal from a center designated FB, (gz = 2.07, gy = 1.91, gx = 1.86) was photoinduced at 4 K. (2) A similar signal, FA (gz = 2.05, gy = 1.94, gx = 1.88), was photoinduced in addition to the FB signal upon a short period of illumination at 200 K. (3) Further illumination at 200 K resulted in the appearance of a broad feature at g = 1.78. This is attributed to the gx component of an iron-sulfur center designated FX. The designations of these signals as FB, FA, and FX are based on their spectroscopic similarities to signals in photosystem I (PS I). The orientation dependence of these EPR signals in ordered Chlorobium membrane multilayers is remarkably similar to that of their PS I homologues. A magnetic interaction between the reduced forms of FB and FA occurs, which is also very similar to that seen in PS I. However, in contrast to the situation in PS I, FA and FB cannot be chemically reduced by sodium dithionite at pH 11. This indicates redox potentials for FA and FB which are lower by at least 150 mV than their PS I counterparts. The triplet state of P840, the primary electron donor, could be photoinduced at 4 K in samples which had been preincubated with sodium dithionite and methyl viologen and then preilluminated at 200 K.(ABSTRACT TRUNCATED AT 250 WORDS)