| Abstract |
Chlorobaculum tepidum is a Gram-negative bacterium which is characterized by its ability to perform anoxygenic photosynthesis in which oxidation of inorganic sulfur compounds (sulfide, polysulfide or thiosulfate) or H2 is coupled to the production of strongly reducing, soluble ferredoxins by the way of a type I (iron–sulfur-type) reaction center. Carbon dioxide is assimilated by the reverse tricarboxylic acid cycle. In cultures of Cba.tepidum that contain both sulfide and thiosulfate, sulfide is oxidized preferentially while sulfur globules are formed. Following sulfide depletion, thiosulfate and sulfur globules are oxidized to sulfate. The molecular mechanism of this phenomenon is poorly understood.
To gain insight into the sulfur metabolism and the sulfur transport though the membrane, the membrane proteome of Cba. tepidum grown under different conditions was analysed. Cba. tepidum cells were grown under anaerobic conditions for 24–30 h at 480C in the presence of both 7,7mM sulfide and 4mM of thiosulfate (source A), 4mM of thiosulfate (source B) and 7,7mM of sulfide (source C). In order to study the effect that thiosulfate had on the formation of elementar sulfur, the bacterium was grown in the two different sources A and C. The growth rate of the bacterial cells as indicated by the Bchl c concentration, the oxidation rate of sulfide as indicated by elementar sulfur’s concentration and the transformation of the sulfur compounds to sulfate were measured and the results showed that Cba. tepidum reaches its optimal growth between 26 to 28 hours and it is favored when it is cultured in the A source. Furthermore, the bacterium was cultured in the addition of increasing concentration of sulfide, which is the major electron donor in photosynthesis. The results showed that this addition was proportional to the concentration of Bchl c and therefore to the cell yield of the bacterium.
For the further understanding of the mechanism of sulfur metabolism, the whole membrane proteome of the bacterium, in its different growth conditions, was analyzed. The whole proteome was subfractionated in the membrane and the water-soluble proteome. Analysis of the membrane proteome from Cba tepidum was achieved by using two-dimensional SDS-Tricine electrophoresis. Protein spots are separated after the coupling of two SDS-gels of different total acrylamide concentration. From the 59 protein spots that were excised from the gel and treated with trypsin, only the 56 were identified by the mass spectrometer MALDI-TOF MS/MS. The results showed that most of these proteins are being critical in photosynthesis, electron transport and translation. The location of the proteins inside the cell, showed the presence of ribosomal proteins in a rate of 32%, in the proteome of Cba tepidum. Therefore, the removal of these proteins from the membranes using EDTA, was tested. The enriched membrane proteome was then collected and analyzed using two-dimensional SDS-PAGE. The results, as determined by MALDI-TOF MS/MS, showed a significant percentage, 52%, of proteins were ribosomal. For this reason the idea of the enrichment of the membrane proteome was rejected. Thus, the total membrane proteomes of the two other sources of the bacterium were analyzed by two-dimensional SDS-PAGE and the protein spots that obtained did not exceed in number those of the gel of source A. The identifications of the protein spots of the gels of sources B and C, their location within the cell and the predicted from the genome function, showed no significant differences. The only difference perhaps is the presence of less ribosomal proteins during growth of the bacterium in source A.
Cba tepidum is a bacterium that is distinguished because of the large light-harvesting complexes-antennas that contains. These complexes are called chlorosomes, are attached to the cytoplasmic membrane through the FMO protein and are able to capture the light. Most components of the sulfur metabolism have been known in biophysical level, however, the structural configuration of these light-harvesting organs within the cell is not fully understood. For the first time, in this study, a three dimensional imaging of the architecture of the cell of Cba. tepidum, using cryo-electron microscopy, is presented. The combination of cryo-preparation, keeping samples in their natural hydrated environment, and three-dimensional (3D) electron tomographic imaging of intact cells allowed insights into the in situ organization of cells at macromolecular resolution.
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Μελέτη του μεταβολισμού του θείου στο φωτοσυνθετικό θειοβακτήριο Chlorobaculum tepidum
