Uwe J. Jürgens

Structures of two bacteriohopanoids with acyclic pentol side-chains from the cyanobacterium Nostoc PCC 6720

Two bacteriohopanepentols 2 and 3 have been isolated from the cyanobacterium Nostoc PCC 6720. Their structures were elucidated by 1H, 13C-NMR and mass spectrometry. The absolute configurations of the acyclic 1,2- and 1,2-/1,3-mixed pentol side-chains were determined by bichromophoric exciton coupled dichroism on microgram scale. An improved first step anthroylation in the two-step derivatization is also described.

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.

Orinithine as a constituent of the peptidoglycan of Chloroflexus aurantiacus, diaminopimelic acid in that of Chlorobium vibrioforme f. thiosulfatophilum

L-Ornithine is the only diamino acid of the peptidoglycan of the gliding phototrophic Chloroflexus aurantiacus. The other constituents are L- and D-alanine, D-glutamic acid, N-acetyl-glucosamine and N-acetyl-muramic acid (in part as muramic acid-6-phosphate), all in approximate equimolar ratios to L-ornithine, aside from small amounts of glycine and histidine. Furthermore unlike typical Gram-negative bacteria, protein is not bound to this peptidoglycan. Instead, the rigid layer (sodium dodecyl sulfate insoluble cell wall fraction) contained large amounts of a complex polysaccharide consisting of sugar O-methyl ethers, hexoses and pentoses. Its binding site is presumably muramic acid-6-phosphate of the peptidoglycan.In contrast, in Chlorobium vibrioforme f. thiosulfatophilium, meso-diaminopimelic acid was found as the only diamino acid of this peptidoglycan. As with other Gramnegative bacteria, L- and D-alanine, D-glutamic acid, N-acetyl-glucosamine and N-acetyl-muramic acid (no muramic acid-6-phosphate) were observed in approximate equimolar ratios to meso-diaminopimelic acid, except a lower D-alanine content. The rigid layer of Chlorobium vibrioforme f. thiosulfatophilum contained protein, and there were no indications for a complex polysaccharide comparable to that of Chloroflexus aurantiacus.

Isolation and Chemical Analysis of the Sheaths of the Filamentous Cyanobacteria Calothrix parietina and C. scopulorum

SUMMARY: The sheaths of two species of cyanobacteria, Calothrix parietina (two strains) and C. scopulorum (one strain), were studied. Their fine structure showed osmiophilic fibres running parallel to the cell surface. Enriched sheath fractions were obtained from both species in high yields in the phenol/water interphase after hot phenol/water extraction of cell homogenates. The purified sheaths of the two species had similar chemical compositions, with about 50% (of sheath dry weight) neutral sugars (galactose, glucose, mannose, xylose, arabinose, rhamnose, fucose, a 2-O-methylhexose and an unidentified 2-O-methyl sugar), 5% amino acids and small amounts of glucosamine and galacturonic acid. The sheath composition was independent of changes in the iron or phosphate content of the culture medium. The sheath was insoluble in water, and its chemical composition remained essentially unchanged on application of drastic extraction methods, including boiling in 2% (w/v) SDS. The sheath bound heavy metals (up to at least 0.7% of sheath dry weight) with the effectiveness (measured as absolute quantities) Fe > Zn > Cu > Ni > Mn > Mo > Co. Ni, Cu, Zn and Fe were highly enriched relative to their concentration in the culture medium. The concentration factors for Mo. Mn, Ca, Na and K were low.

Orientation of carotenoids in the outer membrane of Synechocystis PCC 6714 (cyanobacteria).

The orientation of outer membrane carotenoids from Synechocystis PCC 6714 and Synechococcus PCC 6307 was studied by linear dichroism spectrophotometry. Uniaxially oriented, tilted outer membrane films revealed a significant linear dichroism after rotating the polarization vector of the incident light beam, indicating a predominant orientation of the carotenoid transition moments perpendicular to the outer membrane plane. Values for the reduced dichroism at the absorbance maxima presented a linear correlation to a function of the tilt angle (sin2 alpha).

Isolation and characterization of cell wall components of the unicellular cyanobacterium Synechococcus sp. PCC 6307

SUMMARY: Cell walls and outer membranes, free of thylakoids and cytoplasmic membranes, were isolated from the unicellular cyanobacterium Synechococcus sp. PCC 6307. Electron microscopy revealed C-shaped cell wall fragments, which were partially converted to outer membrane vesicles after removal of the peptidoglycan by lysozyme digestion. The major constituents of the outer membrane were proteins and lipopolysaccharide, while lipids and carotenoids were minor components. The polypeptide patterns of the outer membranes were dominated by two major proteins (M r 52000 and 54000). Five strongly polar lipids (unidentified), free fatty acids and small amounts of sulpholipid were detected in extracts of partially purified cell wall fractions, but monogalactosyldiglyceride and digalactosyldiglyceride were not found. The peptidoglycan layer (10 nm thick) was also isolated. Its chemical composition indicated an Alγ-type structure. The degree of cross-linkage was 57%. A polysaccharide, consisting of fucose, mannose, galactose and glucose, was bound to the peptidoglycan, most likely via muramic acid 6-phosphate.

Cell wall and sheath constituents of the cyanobacterium Gloeobacter violaceus

Sheaths isolated from Gloeobacter violaceus were found to be composed of a major polysaccharide moiety (glucose, galactose, rhamnose, mannose, arabinose), a protein moiety, and negatively charged components (glucuronic acids, phosphate, sulfate). Outer membrane polypeptide patterns were dominated by two major peptidoglycan-associated proteins (Mr 62,000 and 53,000). Lipopolysaccharide constituents were glucosamine, 3-hydroxy fatty acids (3-OH-14:0, anteiso-3-OH-15:0, 3-OH-16:0, 3-OH-18:0), carbohydrates, and phosphate. A1γ-type peptidoglycan and non-peptidoglycan components (mannosamine, glucose, mannose, and glucosamine) indicated the presence of a peptidoglycan-polysaccharide complex in the cell walls of Gloeobacter violaceus.

Chemical analyses on cell envelope polymers of the halophilic, phototrophic Rhodospirillum salexigens

Whole cells of Rhodospirillum salexigens, an obligatory halophilic bacterium, have a very low peptidoglycan content (0.17 μmol muramic acid/mg cell dry weight) which is not sufficient to form a sacculus structure. The isolated peptidoglycan contains glucosamine: muramic acid: diaminopimelic acid: alanine: glutamic acid in molar ratios of 1:1:1:2:3. The degree of cross linking is 30%. A polysaccharide consisting of glucosamine, an unknown compound X and a 2-amino-2-deoxy-pentose (relative molar ratios; 1:2:1) was extracted into the water phase of phenol water extracts of whole cells. The polysaccharide co-sedimented with peptidoglycan when cell homogenates were centrifuged in the presence of ≥4% NaCl (100,000xg, 4 h) or on a sucrose gradient (20–60% sucrose, 28,000xg, 16 h) in the presence or absence of NaCl and/or EDTA.Lack of β-hydroxy fatty acids and of 2-keto-3-deoxyoctonate in all phenol-water extract fractions as well as in the whole cell hydrolysate indicates the absence of common outer membrane lipopolysaccharide in R. salexigens. Removal of the cell surface layer exposed six proteins to labeling with radioactive iodine catalyzed by lactoperoxidase. These proteins are suggested to be constituents of the “outer membrane” of R. salexigens.

Isolation and Chemical Characterization of the Sheath from the Cyanobacterium Chroococcus minutus SAG B.41.79

SUMMARY: The sheath of the unicellular cyanobacterium Chroococcus minutus SAG B.41.79 was isolated from a crude cell envelope fraction by discontinuous sucrose gradient centrifugation, and was further purified by treatment with lysozyme followed by Triton X-100 or sodium dodecyl sulphate (SDS) extraction. The absence of muramic and diaminopimelic acids and of β-hydroxy fatty acid showed the fraction to be free from cell wall components. The sheath had a fibrillar fine structure with the fibres parallel to the cell surface. The total neutral sugar content was 45.9% (w/w). The main sugars were glucose and 2-O-methyl-6-deoxyhexose. Additional O-methyl sugars, 2-O-methylhexose, 3-O-methylhexose and a 2-O-methyl sugar (not further identified), were present. Protein could not be completely removed from the sheath fraction by treatment with boiling SDS. The contents of fatty acids, phosphorus, uronic acids and glucosamine in the fraction were all less than 0.5% (w/w).

Ultrastructural studies on the membrane systems and cell inclusions of the filamentous prochlorophyte Prochlorothrix hollandica

The outer membrane of Prochlorothrix hollandica is covered with a network of fine fibrils on its surface and separated from the cytoplasmic membrane by an electrondense peptidoglycan layer (8 to 20 nm thick). The thylakoid membranes are arranged in stacked and unstacked regions which present four characteristic fracture faces with different numbers and sizes of intramembrane particles. Cell inclusions such as polyhedral bodies (carboxysomes), ribosomes, and polyphosphate granules were found in Prochlorothrix hollandica. Another type of cell inclusions was identified by its characteristic shape (a cylindre with conical caps) and a regular striation as gas vesicles. It is concluded that the organism is in its morphological structure similar to the cyanobacteria.