Carbon isotope fractionation by anoxygenic phototrophic bacteria in euxinic Lake Cadagno.

Anoxygenic phototrophic bacteria utilize ancient metabolic pathways to link sulfur and iron metabolism to the reduction of CO2 . In meromictic Lake Cadagno, Switzerland, both purple sulfur (PSB) and green sulfur anoxygenic phototrophic bacteria (GSB) dominate the chemocline community and drive the sulfur cycle. PSB and GSB fix carbon utilizing different enzymatic pathways and these fractionate C-isotopes to different extents. Here, these differences in C-isotope fractionation are used to constrain the relative input of various anoxygenic phototrophs to the bulk community C-isotope signal in the chemocline. We sought to determine whether a distinct isotopic signature of GSB and PSB in the chemocline persists in the settling fraction and in the sediment. To answer these questions, we also sought investigated C-isotope fractionation in the water column, settling material, and sediment of Lake Cadagno, compared these values to C-isotope fractionation of isolated anoxygenic phototroph cultures, and took a mass balance approach to investigate relative contributions to the bulk fractionation signature. We found a large C-isotope fractionation between dissolved inorganic carbon (DIC) and particulate organic carbon (POC) in the Lake Cadagno chemocline. This large fractionation between the DIC and POC was also found in culture experiments carried out with anoxygenic phototrophic bacteria isolated from the lake. In the Lake Cadagno chemocline, anoxygenic phototrophic bacteria controlled the bulk C-isotope fractionation, but the influence of GSB and PSB differed with season. Furthermore, the contribution of PSB and GSB to bulk C-isotope fractionation in the chemocline could be traced in the settling fraction and in the sediment. Taken together with other studies, such as lipid biomarker analyzes and investigations of other stratified lakes, these results offer a firmer understanding of diagenetic influences on bacterial biomass.

Light-driven oxidation of polysaccharides by photosynthetic pigments and a metalloenzyme.

Oxidative processes are essential for the degradation of plant biomass. A class of powerful and widely distributed oxidative enzymes, the lytic polysaccharide monooxygenases (LPMOs), oxidize the most recalcitrant polysaccharides and require extracellular electron donors. Here we investigated the effect of using excited photosynthetic pigments as electron donors. LPMOs combined with pigments and reducing agents were exposed to light, which resulted in a never before seen 100-fold increase in catalytic activity. In addition, LPMO substrate specificity was broadened to include both cellulose and hemicellulose. LPMO enzymes and pigment derivatives common in the environment of plant-degrading organisms thus form a highly reactive and stable light-driven system increasing the turnover rate and versatility of LPMOs. This light-driven system may find applications in biotechnology and chemical processing.

X-ray scattering and electron cryomicroscopy study on the effect of carotenoid biosynthesis to the structure of Chlorobium tepidum chlorosomes.

Chlorosomes, the main antenna complexes of green photosynthetic bacteria, were isolated from null mutants of Chlorobium tepidum, each of which lacked one enzyme involved in the biosynthesis of carotenoids. The effects of the altered carotenoid composition on the structure of the chlorosomes were studied by means of x-ray scattering and electron cryomicroscopy. The chlorosomes from each mutant strain exhibited a lamellar arrangement of the bacteriochlorophyll c aggregates, which are the major constituents of the chlorosome interior. However, the carotenoid content and composition had a pronounced effect on chlorosome biogenesis and structure. The results indicate that carotenoids with a sufficiently long conjugated system are important for the biogenesis of the chlorosome baseplate. Defects in the baseplate structure affected the shape of the chlorosomes and were correlated with differences in the arrangement of lamellae and spacing between the lamellar planes of bacteriochlorophyll aggregates. In addition, comparisons among the various mutants enabled refinement of the assignments of the x-ray scattering peaks. While the main scattering peaks come from the lamellar structure of bacteriochlorophyll c aggregates, some minor peaks may originate from the paracrystalline arrangement of CsmA in the baseplate.

Chromosomal gene inactivation in the green sulfur bacterium Chlorobium tepidum by natural transformation.

Conditions for inactivating chromosomal genes of Chlorobium tepidum by natural transformation and homologous recombination were established. As a model, mutants unable to perform nitrogen fixation were constructed by interrupting nifD with various antibiotic resistance markers. Growth of wild-type C. tepidum at 40 degrees C on agar plates could be completely inhibited by 100 microg of gentamicin ml(-1), 2 microg of erythromycin ml(-1), 30 microg of chloramphenicol ml(-1), or 1 microg of tetracycline ml(-1) or a combination of 300 microg of streptomycin ml(-1) and 150 microg of spectinomycin ml(-1). Transformation was performed by spotting cells and DNA on an agar plate for 10 to 20 h. Transformation frequencies on the order of 10(-7) were observed with gentamicin and erythromycin markers, and transformation frequencies on the order of 10(-3) were observed with a streptomycin-spectinomycin marker. The frequency of spontaneous mutants resistant to gentamicin, erythromycin, or spectinomycin-streptomycin was undetectable or significantly lower than the transformation frequency. Transformation with the gentamicin marker was observed when the transforming DNA contained 1 or 3 kb of total homologous flanking sequence but not when the transforming DNA contained only 0.3 kb of homologous sequence. Linearized plasmids transformed at least an order of magnitude better than circular plasmids. This work forms a foundation for the systematic targeted inactivation of genes in C. tepidum, whose 2.15-Mb genome has recently been completely sequenced.

Quenching of bacteriochlorophyll fluorescence in chlorosomes from Chloroflexus aurantiacus by exogenous quinones.

The quenching of bacteriochlorophyll (BChl) c fluorescence in chlorosomes isolated from Chloroflexus aurantiacus was examined by the addition of various benzoquinones, naphthoquinones (NQ), and anthraquinones (AQ). Many quinones showed strong quenching in the micromolar or submicromolar range. The number of quinone molecules bound to the chlorosomes was estimated to be as small as one quinone molecule per 50 BChl c molecules. Quinones which exhibit a high quenching effect have sufficient hydrophobicity and one or more hydroxyl groups in the alpha positions of NQ and AQ. Chlorobiumquinone has been suggested to be essential for the endogenous quenching of chlorosome fluorescence in Chlorobium tepidum under oxic conditions. We suggest that the quenching effect of chlorobiumquinone in chlorosomes from Chl. tepidum is related to the 1'-oxo group neighboring the dicarbonyl group.

Electronic energy transfer involving carotenoid pigments in chlorosomes of two green bacteria: Chlorobium tepidum and Cholroflexus aurantiacus.

Electronic energy transfer processes in chlorosomes isolated from the green sulphur bacterium Chlorobium tepidum and from the green filamentous bacterium Chloroflexus aurantiacus have been investigated. Steady-state fluorescence excitation spectra and time-resolved triplet-minus-singlet (TmS) spectra, recorded at ambient temperature and under non-reducing or reducing conditions, are reported. The carotenoid (Car) pigments in both species transfer their singlet excitation to bacteriochlorophyll c (BChlc) with an efficiency which is high (between 0.5 and 0.8) but smaller than unity; BChlc and bacteriochlorophyll a (BChla) transfer their triplet excitation to the Car's with nearly 100% efficiency. The lifetime of the Car triplet states is approximately 3 micros, appreciably shorter than that of the Car triplets in the light-harvesting complex II (LHCII) in green plants and in other antenna systems. In both types of chlorosomes the yield of BChlc triplets (as judged from the yield of the Car triplets) remains insensitive to the redox conditions. In notable contrast the yield of BChlc singlet emission falls, upon a change from reducing to non-reducing conditions, by factors of 4 and 35 in Cfx. aurantiacus and Cb. tepidum, respectively. It is possible to account for these observations if one postulates that the bulk of the BChlc triplets originate either from a large BChlc pool which is essentially non-fluorescent and non-responsive to changes in the redox conditions, or as a result of a process which quenches BChlc singlet excitation and becomes more efficient under non-reducing conditions. In chlorosomes from Cfx. aurantiacus whose Car content is lowered, by hexane extraction, to 10% of the original value, nearly one-third of the photogenerated BChlc triplets still end up on the residual Car pigments, which is taken as evidence of BChlc-to-BChlc migration of triplet excitation; the BChlc triplets which escape rapid static quenching contribute a depletion signal at the long-wavelength edge of the Qy absorption band, indicating the existence of at least two pools of BChlc.

Pigments and proteins in green bacterial chlorosomes studied by matrix-assisted laser desorption ionization mass spectrometry.

We have used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for mass determination of pigments and proteins in chlorosomes, the light-harvesting organelles from the photosynthetic green sulfur bacterium Chlorobium tepidum. By applying a small volume (1 microL) of a concentrated suspension of isolated chlorosomes directly to the target of the mass spectrometer we have been able to detect bacteriochlorophyll a and all the major homologs of bacteriochlorophyll c. The peak heights of the different bacteriochlorophyll c homologs in the MALDI spectra were proportional to peak areas obtained from HPLC analysis of the same sample. The same result was also obtained when whole cells of Chl. tepidum were applied to the target, indicating that MALDI-MS can provide a rapid method for obtaining a semiquantitative determination or finger-print of the bacteriochlorophyll homologs in a small amount of green bacterial cells. In addition to information on pigments, the MALDI spectra also contained peaks from chlorosome proteins. Thus we have been able with high precision to confirm the molecular masses of the chlorosome proteins CsmA and CsmE which have been previously determined by conventional biochemical and genetic methods, and demonstrate the presence of truncated versions of CsmA and CsmB.

Pheophytinization of bacteriochlorophyll c and energy transfer in cells of Chlorobium tepidum.

Bacteriochlorophyll (BChl) c in whole cells of Chlorobium tepidum grown at 46 degrees C changed into bacteriopheophytin (BPhe) c within 10 days after reaching full growth. When a small amount of C. tepidum cells in which BChl c had been completely pheophytinized were transferred to a new culture medium, normal growth was observed after a short lag phase, and the absorption spectrum of the growing cells showed the presence of a normal amount of BChl c. During the growth of C. tepidum in the new culture, the BChl c concentration was nearly proportional to the cell density measured by turbidity (OD640). These results indicate that C. tepidum can survive even when BChl c has been completely pheophytinized and that BChl c is newly synthesized in such cells when transferred to a new culture medium. In partly pheophytinized cells, upon excitation of BPhe c at 550 nm the fluorescence emission spectrum showed maxima at 775 and 810 nm, which correspond to emissions from BChl c and BChl a, respectively. This indicates energy transfer from BPhe c to BChl c and BChl a. In cells in which BChl c was completely pheophytinized, fluorescence measurements were indicative of direct energy transfer from BPhe c to baseplate BChl a. These findings suggest that when BChl c in C. tepidum cells is pheophytinized, the product (BPhe c) remains in the chlorosomes and continues to work as a light-harvesting pigment.

Menaquinone-7 in the reaction center complex of the green sulfur bacterium Chlorobium vibrioforme functions as the electron acceptor A1.

Photosynthetically active reaction center complexes were prepared from the green sulfur bacterium Chlorobium vibrioforme NCIMB 8327, and the content of quinones was determined by extraction and high-performance liquid chromatography. The analysis showed a stoichiometry of 1.7 molecules of menaquinone-7/reaction center. No other quinones were detected in the isolated reaction centers, whereas membrane preparations also contained chlorobiumquinone. The possible involvement of quinones in electron transport was investigated by electron paramagnetic resonance (EPR) spectroscopy. A highly anisotropic radical was detected by Q-band EPR spectroscopy in both membranes and isolated reaction centers following dark reduction with sodium dithionite and photoaccumulation at 205 K. At 34 GHz, the EPR spectrum is characterized by a g tensor with gxx = 2.0063, gyy = 2.0052, gzz = 2.0020 and delta B of 0.7 mT, consistent with its identification as a quinone. This spectrum is highly similar in terms of g values and line widths to photoaccumulated A1- in photosystem I of Synechococcus sp. PCC 7002. The results indicate that menaquinone-7 in the green sulfur bacterial reaction center is analogous to phylloquinone in photosystem I.