Jürgen Weckesser

The structure of porin from Rhodobacter capsulatus at 1.8 Å resolution

The structure of the porin from Rhodobacter capsulanus was determined at a resolution of 1.8 Å. The analysis started from a closely related crystal structure that had been solved at a medium resolution of 3 Å using multiple isomorphous replacement and solvent flattening. The new structure contains the complete sequence of 301 amino acid residues. Refinement of the model is under way: the present R‐factor is 22% with good geometry. Except for the lengths of several loops, the resulting chain fold corresponds to the medium resolution model. The membrane channel is lined by a large number of ionogenic side chains with characteristic segregation of differently charged groups.

The three-dimensional structure of porin from Rhodobacter capsulatus at 3 Å resolution

The crystal structure of porin from Rhodobacter capsulatus strain 37b4 has been solved at 3.0 Å (1 Å = 0.1 nm) resolution by multiple isomorphous replacement and solvent‐flattening. The three pores of the trimer are well denned in the electron density map. Each pore consists of a 16‐stranded β‐barrel which traverses the membrane as a tube. Near its center the tube is narrowed by chain segments protruding from the inner wall of the barrel that form an eye‐let with an irregular cross‐section of about 6 Å by 10 Å. The eye‐let has an axial length of about 10 Å; it defines the exclusion limit for diffusing particles.

Microcystins (hepatotoxic heptapeptides) in german fresh water bodies

In 1995 and 1996 a total of 55 German water bodies with different trophic states were investigated for the presence of potentially microcystin‐producing cyanobacteria. The seston biomass of over 500 samples was analyzed by HPLC to assess microcystin content. The highest microcystin content per dry weight was found when Planktothrix rubescens was dominant, followed by Planktothrix agardhii and Microcystis spp. The microcystin to chlorophyll‐a ratio mostly varied between 0.1 and 0.5, with maxima of 1–2. In over 70% of the samples from pelagic water, particulate microcystin concentrations were below 10 μg L−1. By contrast, spatial and temporal concentrations varied by 4 orders of magnitude (2–25,000 μg L−1) at bathing sites in 1997 during a mass development of Microcystis spp. The seasonal peak of development of Microcystis spp. occurred from June to September and of Planktothrix agardhii from September to November. Planktothrix rubescens, however, was almost perennial. The seasonal levels of these cyanobacteria were clearly reflected by microcystin concentrations in the water bodies. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 13–22, 1999

6 Analysis of Lipopolysaccharides of Gram-Negative Bacteria

Publisher Summary This chapter discusses the analysis of lipopolysaccharides (LPS) of gram-negative bacteria. LPS are characteristic components of the cell wall of all Gram-negative bacteria and of at least some cyanobacteria. They are localized in the outer layer of the outer membrane, and are, in noncapsulated strains, exposed on the cell surface. They contribute to the integrity of the outer membrane and, as has been shown for enteric bacteria, protect the cell against the action of bile salts and (lipophilic) antibiotics. Isolated lipopolysaccharide, upon injection into higher organisms, exhibits a variety of biological (“endotoxic”) activities, such as the induction of fever, changes in the white blood cell count, diarrhoea, or even shock and death. Since the toxic activities are because of substances released from disintegrating bacteria, and not to an excreted exotoxin, lipopolysaccharide has been termed an endotoxin. Thus, the terms lipopolysaccharide, O-antigen, and endotoxin are used synonymously and describe either the chemical structure or discrete biological functions of the same substance.

Identification of a 2,3-diamino-2,3-dideoxyhexose in the lipid a component of lipopolysaccharides of Rhodopseudomonas viridis and Rhodopseudomonas palustris

A hitherto unknown amino sugar (Compound A), detected in acid hydrolyzates of lipopolysaccharides of Rhodopseudomonas viridis and Rhodopseudomonas palustris, is present in the Lipid A component but not in the O-specific part of the lipopolysaccharides. 2-Amino-2-deoxy-D-glucose is lacking in the purified Lipid A of both strains. Compound A, characterized by a very high migration in paper electrophoresis was obtained in a pure state by ion-exchange chromatography and shown by m.s. of the alditol acetate to be a 2, 3-diamino-2, 3-dideoxyhexose. G.l.c. and periodate oxidation excluded all possible stereoisomers with the exception of 2, 3-diamino-2, 3-dideoxyglucose and 2, 3-diamino-2, 3-dideoxydose. G.l.c. of the alditol acetates of Compound A and of the glucose derivative suggests that Compound A is 2, 3-diamino-2, 3-dideoxyglucose. The significance of the occurrence of this new aminodeoxy sugar in the Lipid A component of Rhodopseudomonas viridis and Rhodopseudomonas palustris O-antigens for the biological properties of the respective lipopolysaccharides and for the taxonomy of the Rhodospirillaceae family is discussed.

Characterization of the lipopolysaccharides from eight strains of the cyanobacterium Synechococcus

Lipopolysaccharides have been isolated from eight strains of the unicellular cyanobacterium Synechococcus. Fucose, mannose, galactose, glucose and glucosamine were found in all of the lipopolysaccharides investigated. Additionally, strain-specific sugars are present and permit the chemotyping of lipopolysaccharide. Chemotype I, comprising three strains with a high G+C content of DNA (71-66 mol%), is characterized by a high rhamnose portion and by 3,6-dideoxy-d-arabino-hexose (tyvelose). Chemotype III, represented by three strains with a low G+C content of DNA (55-48 mol%), contains a mannose-polymer with small amounts of 3-O-methyl-mannose, 4-O-methyl-mannose, 2-keto-3-deoxyoctonate and mannosamine. Lipopolysaccharides of the two strains of chemotype II contain 2,3,4-tri-O-methyl-arabinose.Lipid A is difficult to split off from the polysaccharide moiety, but is present in all lipopolysaccharides from the Synechococcus strains. The presence of Lipid A is supported by the finding of β-hydroxy fatty acids, predominantly β-hydroxypalmitic acid. The distribution of branched β-hydroxy fatty acids, detected in small amounts, parallels chemotyping of lipopolysaccharide based on the sugar composition. The phosphorus content of the lipopolysaccharides is low.The pyrogenicity of lipopolysaccharides from two strains is low. Synechococcus lipopolysaccharides have little reactivity in antisera raised in rabbits against homologous cells. As far as tested they do not migrate in immunoelectrophoresis. This confirms the neutral character or low negative charge of Synechococcus lipopolysaccharides.

Prediction of the general structure of OmpF and PhoE from the sequence and structure of porin from Rhodobacter capsulatus. Orientation of porin in the membrane

By comparing the hydrophilicity profiles and sequences of porin from Rhodobacter capsulatus with those of OmpF and PhoE from Escherichia coli, a set of insertions and deletions for alignment of the sequences has been deduced. With this alignment a similar folding of OmpF and PhoE has been predicted as found by X-ray structure analysis of porin from Rhodobacter capsulates. Furthermore, the orientation of the porin trimer in the outer membrane was inferred from topological data on PhoE. According to this result a single channel of approx. 30 A diameter starts at the outer surface. Near the middle of the outer membrane bilayer this channel branches out into three separate channels, each running within a single porin monomer to the periplasmic surface.

Lipopolysaccharides in four strains of the unicellular cyanobacterium Synechocystis

Lipopolysaccharides were found in four strains of Synechocystis. Depending on the strain, they were extracted into either the water or into the phenol phase of phenol-water extracts.The lipopolysaccharides of all four strains contain fucose, mannose, galactose, glucose and glucosamine. l-Glycero-d-mannoheptose and 2-keto-3-deoxyoctonate are lacking. The strain-specific sugars are dominated by O-methyl sugars in three of the four strains: Synechocystis PCC 6803 contains 2,3-di-O-methyl-fucose, 2-O-methyl-fucose and 2-O-methylxylose. In Synechocystis PCC 6807 a 6-O-methylheptose and 2-O-methyl-mannose, in Synechocystis PCC 6308 2-O-methyl-mannose was identified.Lipid A, although difficult to be split off from the polysaccharide moiety, is indicated in all four strains by the presence of β-hydroxypalmitic and β-hydroxymyristic acids and of glucosamine. In addition, a branched β-hydroxypentadecanoic acid (anteiso) was found. The phosphorus content of the four lipopolysaccharides amounts to less than 0.3% of dry weight.The lipopolysaccharides from Synechocystis show O-specific activity. Their reactivity in homologous O-antisera, however, is low when tested by passive hemagglutination. In immunoelectrophoresis, no migration of lipopolysaccharide was observed.

Lipopolysaccharides and polysaccharides of the cell envelope of toxic Microcystis aeruginosa strains

Lipopolysaccharides were obtained from three strains (PCC 7806, PCC 7820 and UV-017) of the waterbloom-forming cyanobacterium Microcystis aeruginosa. 3-Hydroxy fatty acids (3-OH-14:0, 3-OH-16:0, 3-OH-18:0) in addition to other fatty acids were identified in all three lipopolysaccharides. Glucosamine, the only amino sugar found, presumably represents the backbone amino sugar of the phosphate-free lipid A moiety. Heptoses were absent and 2-keto-3-deoxyoctonate was not detected in all three lipopolysaccharides. Strain-specificity was revealed in the complex composition of the polysaccharide moieties. Strains PCC 7806 and UV-017 were of the same chemotype, it differs from that of strain PCC 7820. Polysaccharides with strain-specific chemical compositions different from those of the respective lipopolysaccharides were obtained from each strain. The polysaccharides are likely to be ascribed to external cell envelope layers, their sugar specificity was in parallel with the O-chain chemotypes of the lipopolysaccharides of the three strains.

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.