Jochen R. Golecki

Visualization of the supramolecular architecture of chlorosomes (chlorobium type vesicles) in freeze-fractured cells of Chloroflexus aurantiacus

Freeze-fracture electron microscopy has been used to investigate the size, form, distribution and supramolecular organization of chlorosomes (chlorobium type vesicles) in Chloroflexus aurantiacus J-10fl, a phototrophic, filamentous gliding bacterium. The chlorosomes, that appear tightly attached to the cytoplasmic membrane, have the form of flat, elongated sacs with rounded ends, and measure 106±24×32±10×12±2nm. They are randomly distributed, and in most instances their longitudinal axis makes an angle of 30–60° to the filament axis. Each chlorosome consists of a core and an approx. 2 nm thick envelope. The core is filled with rod-shaped elements (approx. 5.2 nm in diameter) made up of globular subunits with a periodicity of approx. 6 nm. The rod elements extend the full length of the chlorosome. The membrane-associated envelope layer is marked by extremely fine striations with a repeating distance of 2.5–3nm, while the envelope layer adjacent to the cytoplasm exhibits no discernable substructure. The margins of the vesicles are delineated by regularly spaced 7 nm particles.No information is yet available on the organization of the cytoplasmic membrane areas to which the vesicles are attached since the fracture plane always passes into the adjacent vesicles in such region rather than continuing through the membrane. Upon cooling of the cells large particle-free areas develop in the cytoplasmic membrane. Simultaneously the chlorosomes become crowded into the remaining particle-rich areas, where some seem to fuse with each other to form

Supramolecular organization of chlorosomes (chlorobium vesicles) and of their membrane attachment sites in Chlorobium Limicola

The photosynthetic green bacterium Chlorobium limicola 6230 has been examined by freeze-fracture electron microscopy to investigate the size, form, distribution and supramolecular architecture of its chlorosomes (chlorobium vesicles) as well as the chlorosome attachment sites on the cytoplasmic membrane. The oblong chlorosomes that underlie the cytoplasmic membrane show a considerable variation in size from about 40 X 70 nm to 100 X 260 nm and exhibit no particular orientation. The chlorosome core, which appears to be hydrophobic in nature, contains between 10 and 30 rod-shaped elements (approx. 10 nm in diameter) surrounded by an unetchable matrix. The rod elements are closely packed and extend the full length of the chlorosome. Separating the chlorosome core from the cytoplasm is a approx. 3 nm thick lipid-like envelope layer, which exhibits no substructure. A 5-6 nm thick, crystalline baseplate connects the chlorosome to the cytoplasmic membrane. The ridges of the baseplate lattice make an angle of between 40 degrees and 60 degrees with the longitudinal axis of the chlorosome and have a repeating distance of approx. 6 nm. In addition, each ridge exhibits a granular substructure with a periodicity of approx. 3.3 nm. The cytoplasmic membrane regions adjacent to the baseplates are enriched in large (greater than 9 nm) intramembrane particles, most of which belong to approx. 10 nm and approx. 12.5 nm particle size categories. Each chlorosome attachment site contains between 20 and 30 very large (greater than 12.0 nm diameter) intramembrane particles. The following interpretive model of a chlorosome is discussed in terms of biophysical, biochemical and structural information reported by others: it is proposed that the bacteriochlorophyll c (BChl c; chlorobium chlorophyll) is located in the rod elements of the core and that it is complexed with specific proteins. The cytoplasm-associated envelope layer is depicted as consisting of a monolayer of galactosyl diacylglycerol molecules. BChl alpha-protein complexes in a planar lattice configuration most likely make up the crystalline baseplate. The greater than 12-nm particles in the chlorosome attachment sites of the cytoplasmic membrane, finally, may correspond to complexes containing a reaction center and non-crystalline light-harvesting BChl alpha. The crystalline nature of the baseplate is consistent with the notion that it serves two functions: besides transferring excitation energy to the reaction centers it could also function as a distributor of this energy amongst the reaction centers.

Quantitative relationship between bacteriochlorophyll content, cytoplasmic membrane structure and chlorosome size in Chloroflexus aurantiacus

Continuous cultures of Chloroflexus aurantiacus were cultivated in a chemostat in the light with varying bacteriochlorophyll (BChl) a/c ratios by changing the growth rate. Under these culture conditions all cells were homogeneously and reproducibly equipped with chlorosomes. In order to determine the number and size of chlorosomes in relation to different BChl contents morphometric measurements were performed on electron micrographs. The linear increase of BChl a contents coincided with an increasing number of chlorosomes per membrane area and per bacterium rather than with an enlargement of the average size of chlorosomes. The numbers of chlorosomes and therefore the percentage of chlorosome-covered cytoplasmic membrane increased linearly with increasing BChl a contents. The average size of the baseplates was largely constant in all cultures (mean 3,222±836 nm2). However, within individual cells the size of baseplates varied by a factor of 3.0, especially by the variation of the length. The exponential increase in BChl c contents coincided with an increasing number of chlorosomes (up to a factor of 2.3) and an enlargement of the average chlorosome volume (up to a factor of 1.9). The number of BChl a molecules per chlorosome was about 1,484±165, thus the number of reaction centers per chlorosome was 58±12. The data suggest, firstly, that BChl a is confined to areas (cytoplasmic membrane plus baseplate) as represented by the chlorosome attachment sites; secondly, that the degree of packing of BChl c molecules within chlorosomes increases with increasing BChl c contents.

Membranes and Chlorosomes of Green Bacteria: Structure, Composition and Development

Although belonging to evolutionary distantly related groups, cells of members of the Chlorobiaceae and the Chloroflexaceae exhibit structurally and functionally comparable substructures. While the photochemical reaction center complex plus a light-harvesting unit are housed in the peripheral cytoplasmic membrane (CM) system, an accessory light-harvesting unit is localized in specialized structures, the chlorosomes, underlying the CM. In this chapter, the present knowledge on the fine structure of chlorosomes and the CM is reviewed. After a description of methods commonly employed to isolate chlorosomes and CM, data of chemical analyses of both subcellular fractions are detailed. In spite of considerable similarities in the overall ultrastructural and functional properties, the chemical composition reveals significant differences between chlorosomes and CM, when isolated from Chlorobium and Chloroflexus, respectively. The same holds true with respect to the supramolecular organization, particularly of chlorosomes and elements involved in their connection to the CM. Since the photosynthetic apparatus of green bacteria is composed of two different moieties, i.e. the chlorosomes and the CM-bound unit, the important questions arise how these different units are synthesized, how the synthesis of individual constituents is controlled and how the syntheses of chlorosomes and the CM-bound units are coordinated. In the present contribution these problems are approached on the basis of the pigments characteristic of both units. In addition, the knowledge on polypeptide formation is presented. These data are combined with the respective changes in number and size of chlorosomes. In spite of a considerable amount of detailed information available as yet, it is finally concluded that considerable research efforts are still required in order to understand the development of the biologically unique type of photosynthetic apparatus characteristic of the green bacteria.

Studies on ultrastructure and composition of cell walls of the cyanobacterium Anacystis nidulans

The multilayered cell wall of the cyanobacterium Anacystis nidulans was studied by the freezeetching technique. A characteristic fracture face in the outer cell wall was demonstrated which is densely packed with particles of a diameter of 60–75 Å. This particle layer is comparable with layers which have been described in many cell walls of Gram-negative prokaryotes.The outer membrane of the cell wall was solubilised by extraction with phenol/water or sodium dodecyl sulfate (SDS). In the SDS-extract 31 bands were separated by polyacrylamide gel electrophoresis, among them 3–5 major proteins with molecular weights of approximately 60, 40, and 10 kdaltons, respectively. Several polypeptides of the Anacystis cell wall were comparable in their mobility with polypeptides extracted from cell walls of different Gramnegative bacteria. The analysis of the SDS-unsoluble electron dense layer (sacculi) revealed the typical components of peptidoglycan diaminopimelic acid, muramic acid, glutamic acid, glucosamine and alamine in the molar ratio of 1.0:0.9:1.1:1.5:1.9. In addition, other amino acids (molar ratio from 0.05–0.36), mannosamine (molar ratio 0.54), and lipopolysaccharide components were detected in low concentration.

Differentiation of the membrane system in cells of Rhodopseudomonas capsulata after transition from chemotrophic to phototrophic growth conditions

Aerobically in the dark grown cultures of Rhodopseudomonas capsulata were shifted to low oxygen partial pressure for 30 min and afterwards to phototrophic conditions (anaerobic, light). During 210 min of adaptation to a phototrophic mode of life the bacteriochlorophyll (BChl) concentration increased 53-fold (doubling time 40 min) and the carotenoid content six fold. Growth was delayed. The light membrane fraction from chemotrophic and induced phototrophic cells contained low concentrations of small photosynthetic units (reaction center+light harvesting BChl B870), and low respiratory activities, especially of succinatecytochrome c oxidase. The heavy membrane fraction, i.e. the intracytoplasmic chromatophore fraction, increased during adaptation approximately 9-fold in surface area per cell, 42-fold in BChl content, 7-fold in reaction center content and 6-fold in the size of the photosynthetic unit.Phospholipid and fatty acid content and patterns changed slightly during adaptation.

Putative function of cytochrome b559 as a plastoquinol oxidase

The function of cytochrome b559 (cyt b559) in photosystem II (PSII) was studied in a tobacco mutant in which the conserved phenylalanine at position 26 in the beta-subunit was changed to serine. Young leaves of the mutant showed no significant difference in chloroplast ultra structure or in the amount and activity of PSII, while in mature leaves the size of the grana stacks and the amount of PSII were significantly reduced. Mature leaves of the mutant showed a higher susceptibility to photoinhibition and a higher production of singlet oxygen, as shown by spin trapping electron paramagnetic resonance (EPR) spectroscopy. Oxygen consumption and superoxide production were studied in thylakoid membranes in which the Mn cluster was removed to ensure that all the cyt b559 was present in its low potential form. In thylakoid membranes, from wild-type plants, the larger fraction of superoxide production was 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive. This type of superoxide formation was absent in thylakoid membranes from the mutant. The physiological importance of the plastoquinol oxidation by cyt b559 for photosynthesis is discussed.

Characterization of the cell wall of the unicellular cyanobacterium Synechocystis PCC 6714

Cell walls free of cytoplasmic- and thylakoid membranes were isolated from Synechocystis PCC 6714 by sucrose density gradient centrifugation and extraction with Triton X-100. The Triton-insoluble cell wall fraction retained the multilayered fine structure. Peptidoglycan, proteins, polysaccharides, lipopolysaccharides, lipids and carotenoids were found as constituents of the cell wall. Polypeptide and lipid patterns of cell walls were completely different from that of the cytoplasmic/thylakoid membrane fraction. The purified cell walls contained about twelve outer membrane proteins. The two major polypeptides (Mr 67,000 and 61,000) were found to be associated with the peptidoglycan by ionic interactions.Myxoxanthophyll (major carotenoid), related carotenoid-glycosides and zeaxanthin were the predominating carotenoids of the cell wall of Synechocystis PCC 6714 over echinenone and β-carotene. A polar unknown carotenoid was observed, the absorption spectrum of which resembled that of myxoxanthophyll. It was exclusively found in cell walls, but not in the cytoplasmic/thylakoid membrane fraction.

Race cultivar-specific differences in callose deposition in soybean roots following infection with Phytophthora megasperma f.sp. glycinea.

Primary roots of soybean (Glycine max (L.), Merrill, cv. Harosoy 63) seedlings were inoculated with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea and total callose was determined at various times after inoculation. From 4 h onward, total callose was significantly higher in roots showing the resistant rather than the susceptible response. Local callose deposition in relation to location of fungal hyphae was determined in microtome sections by its specific fluorescence with sirofluor and was quantified on paper prints with an image-analysis system. Callose deposition, which occurs adjacent to hyphae, was found soon after inoculation (2, 3 and 4 h post inoculation) only in roots displaying the resistant response, and was also higher at 5 and 6 h after inoculation in these resistant roots than in susceptible roots. Early callose deposition in the incompatible root-fungus reaction could be a factor in resistance of soybean against P. megasperma.

Spectroscopical studies on the light-harvesting pigment-protein complex II from dark-aerobic and light-anaerobic grown cells of Rhodobacter sulfidophilus

The photosynthetic bacterium Rhodobacter sulfidophilus can grow and synthesize photosynthetic pigments under dark-aerobic as well as light-anaerobic growth conditions. Under both growth conditions intracytoplasmic membrane vesicles (diameter about 35 nm) are formed. The light-harvesting (LH) pigment-protein complex II, isolated from dark-aerobic and light-anaerobic grown cells, consists of two small polypeptides (