The structure of the glycerophosphate-containing O-specific polysaccharide from <Emphasis Type="Italic">Escherichia coli</Emphasis> O130

A phosphorylated O-specific polysaccharide was obtained by mild acidic degradation of the lipopolysaccharide from the enteric bacterium Escherichia coli O130 and characterized by the methods of chemical analysis, including dephosphorylation and ¹H and ¹³C NMR spectroscopy. The polysaccharide was shown to be composed of branched tetrasaccharide repeating units containing two N-acetyl-D-galactosamine residues, D-galactose, D-glucose, and glycerophosphate residues (one of each). The polysaccharide has the following structure, which is unique among the known bacterial polysaccharides:

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Bacterial antigen polysaccharides. 43. Structure for the O-specific polysaccharide of <Emphasis Type="Italic">Providencia alcalifaciens</Emphasis> O46

Acidic O-specific polysaccharide containing D-glucose, D-glucuronic acid, L-fucose, and 2-acetamido-2-deoxy-D-glucose was obtained by mild acid degradation of lipopolysaccharide from Providencia alcalifaciens O46. The following structure of the hexasaccharide repeating unit of the O-specific polysaccharide was established using methylation analysis along with ¹H and ¹³C NMR spectroscopy, including 2D ¹H, ¹H-COSY, TOCSY, ROESY, ¹H, ¹³C-HSQC, and HMQC-TOCSY experiments:

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Antigenic polysaccharides of bacteria: 40. The structures of O-specific polysaccharides from <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> types 3 and 9 and <Emphasis Type="Italic">S. boydii</Emphasis> type 4 revised by NMR spectroscopy

The reported structures of O-specific polysaccharides from three type strains of Shigella bacteria were corrected by modern NMR techniques. The revisions concerned the configuration of the O-glycoside linkage (S. dysenteriae type 3, structure 1), the positions of monosaccharide residue glycosylation and acetalation by pyruvic acid (S. dysenteriae type 9, structure 2), and the attachment position of the side monosaccharide chain (S. boydii type 4, structure 3).

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A pseudoaminic acid-containing O-specific polysaccharide from a marine bacterium Cellulophaga fucicola

The O-polysaccharide was isolated from the lipopolysaccharide of Cellulophaga fucicola and studied by chemical analyses along with (1)H and (13)C NMR spectroscopy. The following new structure of the O-polysaccharide of C. fucicola containing 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid residue (pseudoaminic acid, Psep) was elucidated as the following:     

Antigenic polysaccharides of bacteria: 41. Structures of the O-specific polysaccharides of <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> types 4 and 5 revised by NMR spectroscopy

The earlier established structures of the acidic O-specific polysaccharides from two typical strains of the Shigella dysenteriae bacterium were revised using modern NMR spectroscopy techniques. In particular, the configurations of the glycosidic linkages of GlcNAc (S. dysenteriae type 4) and mannose (S. dysenteriae type 5) residues were corrected. In addition, the location of the sites of non-stoichiometric O-acetylation in S. dysenteriae type 4 was determined: the lateral fucose residue was shown to be occasionally O-acetylated; also, theposition of the O-acetyl group present at the stoichiometric quantity in S. dysenteriae type 5 was corrected. The revised structures of the polysaccharides studied are shown below. The known identity of the O-specific polysaccharide structures of S. dysenteriae type 5 and Escherichia coli O58 was confirmed by ¹³C NMR spectroscopy and, hence, the structure of the E. coli O58 polysaccharide should be revised in the same manner.

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where L-Rhap3Rlac2Ac is 2-O-acetyl-3-O-[(R-1-carboxyethyl]-L-rhamnose

     

Characterization of the cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex from marine green alga, <Emphasis Type="Italic">Bryopsis corticulans</Emphasis>

A pure, active cytochrome b ₆ f was isolated from the chloroplasts of the marine green alga, Bryopsis corticulans. To investigate and characterize this cytochrome b ₆ f complex, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), absorption spectra measurement and HPLC were employed. It was shown that this purified complex contained four large subunits with apparent molecular masses of 34.8, 24, 18.7 and 16.7 kD. The ratio of Cyt b ₆ to Cytf was 2.01 : 1. The cytochromeb ₆ f was shown to catalyze the transfer of 73 electrons from decylplastoquinol to plastocyanin–ferricyanide per Cyt f per second. α-Carotene, one kind of carotenoid that has not been found to present in cytochrome b ₆ f complex, was discovered in this preparation by reversed phase HPLC. It was different from β-carotene usually found in cytochrome b ₆ f complex. The configuration of the major α-carotene component was assigned to be 9-cis by resonance Raman spectroscopy. Different from the previous reports, the configuration of this α-carotene in dissociated state was determined to be all-trans. Besides this carotene, chlorophyll a was also found in this complex. It was shown that the molecular ratios of chlorophylla, cis and all-trans-α-carotene to Cyt f in this complex were 1.2, 0.7 and 0.2, respectively.     

Exopolysaccharide from surface-liquid culture of <Emphasis Type="Italic">Clonostachys</Emphasis><Emphasis Type="Italic">rosea</Emphasis> originates from autolysis of the biomass

We describe the purification and chemical characterization of galactomannans that appear both in the biomass and the culture broth during surface-liquid culture of the fungus Clonostachys rosea, a common facultative saprophyte that has potential to be used as a biological control agent against several plant pathogenic fungi, insects and nematodes. The galactomannans from both sources had comparable ratios of Man, Gal and Glc and the similarity were confirmed by ¹H, ¹³C NMR, HMQC, and COSY spectra. We propose that the galactomannan in the culture broth originates from autolysis of the biomass, based not only on the similarity that it has with the galactomannan extracted from the biomass but also on the fact that its concentration increased rapidly after glucose depletion from the medium, when biomass concentration was falling. Polysaccharides from C. rosea have not previously been characterized; we show that the characteristics of the galactomannans are consistent with those that have been reported for other members of the Bionectriaceae, the family to which C. rosea belongs.     

A completed structure of the O-polysaccharide from <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> type 10

The structure of the O-specific polysaccharide from Shigella dysenteriae type 10, which has been reported previously in Bioorganic chemistry (1977, vol.3, pp. 1219–1225), is refined: →2)-β-D-Manp-(1→3)-α-D-ManpNAc-(1→3)-β-L-Rhap-(1→4)-α-D-GlcpNAc-(1→.     

Removal of <Emphasis Type="Italic">p</Emphasis>-chlorophenol by the marine microalga <Emphasis Type="Italic">Tetraselmis marina</Emphasis>

The ability of Tetraselmis marina, a green coastal microalga, to remove chlorophenols under photoautotrophic conditions was investigated. T.marina was able to grow in the presence of 20 mg L⁻¹ of the phenolic compounds tested. The EC₅₀ (growth rate) value of p-chlorophenol (p-CP) to T.marina was found to be 25.5 mg L⁻¹. The microalga was able to remove chlorophenols, showing higher efficiency for p-CP. The effect of photoregime and NaHCO₃ concentration on p-CP removal was investigated. Under continuous illumination with 1 g L⁻¹ NaHCO₃ initial concentration T.marina removed 65% of 20 mg L⁻¹ in a 10-day cultivation period.     

Structural study of bergenan,a pectin from <Emphasis Type="Italic">Bergenia crassifolia</Emphasis>

A pectin polysaccharide named bergenan was isolated from the freshly collected leaves of the leather bergenia Bergenia crassifolia by extraction with an aqueous solution of ammonium oxalate. The main component of its carbohydrate chain was shown to be the residues of D-galacturonic acid (about 80%). In addition, the polysaccharide contains the residues of galactose, arabinose, and rhamnose; their total content is less than 15%. It was shown that the bergenan samples from bergenia leaves collected at different vegetation periods (from July to September) do not substantially differ either in monosaccharide composition or in the viscosity of their aqueous solutions. The results of enzymatic hydrolysis by α-1,4-galacturonase (pectinase), partial acidic hydrolysis, NMR spectroscopy, and methylation with subsequent analysis of the results by GC-MS indicate that the bergenan macromolecule contains the regions of a linear α-1,4-D-galactopyranosyluronan and rhamnogalacturonan-I (RG-I). Galacturonan responds for a greater part of the macromolecule. A considerable amount of its constitutent galacturonic acid residues are present as methyl esters. The side chains in RG-I are attached to the rhamnopyranose residues of the backbone by a 1,4-linkages and are composed of the residues of terminal arabinofuranose and galactopyranose, 1,5-linked α-arabinofuranose, and 1,4- and 1,6-linked β-galactopyranose. The branching points of the side chains of the RG-I molecule are 3,4- and 3,6-di-O-substituted galactose residues.     

Anti-inflammatory activity of polysaccharide from <Emphasis Type="Italic">Pholiota nameko</Emphasis>

Pholiota nameko polysaccharide (PNPS-1) has been isolated and purified by enzymatic hydrolysis, hot water extraction, ethanol precipitation, and ion-exchange and gel-filtration chromatography. The anti-inflammatory activity of PNPS-1 was evaluated in rodents using xylene-induced ear edema, egg albumin-, carrageenin-, and formaldehyde-induced paw edema, cotton pellet granuloma test, adhesion of peritoneal leukocytes in vitro, and ulcerogenic activity. The results showed that PNPS-1 (5 mg/ear) inhibited topical edema in the mouse ear and at 100, 200, and 400 mg/kg (intraperitoneally) it significantly suppressed the development of egg albumin-, carrageenin-, and formaldehyde-induced paw edema in the animals. PNPS-1 (100, 200, and 400 mg/kg, per oral) significantly inhibited the growth of granuloma tissues induced by subcutaneously implanted cotton pellets in rats by 10.96, 18.07, and 43.75%, respectively. PNPS-1 also inhibited spontaneous and phorbol-12-myristate-13-acetate-activated adhesion of peritoneal leukocytes in vitro. Further, both acute as well as chronic administration of PNPS-1 (100, 200, and 400 mg/kg, per oral) did not produce any gastric lesion in rats. In conclusion, these data indicated that PNPS-1 possesses significant anti-inflammatory activity suggesting its potential as an anti-inflammatory agent for use in the treatment of various inflammatory-related diseases.     

Composition,structure, and biological properties of lipopolysaccharides from different strains of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">atrofaciens</Emphasis>

The composition, structure, and certain biological properties of lipopolysaccharides (LPS) isolated from six strains of bacteria Pseudomonas syringae pv. atrofaciens pathogenic for grain-crops (wheat, rye) are presented. The LPS-protein complexes were isolated by a sparing procedure (extraction from microbial cells with a weak salt solution). They reacted with the homologous O sera and contained one to three antigenic determinants. Against the cells of warm-blooded animals (mice, humans) they exhibited the biological activity typical of endotoxins (stimulation of cytokine production, mitogenetic activity, etc.). The LCD of the biovar type strain was highly toxic to mice sensitized with D-galactosamine. The structural components of LPS macromolecules obtained by mild acidic degradation were characterized: lipid A, core oligosaccharide, and O-specific polysaccharide (OPS). Fatty acids 3-HO-C_10:0, C_12:0, 2-HO-C_12:0, 3-HO-C_12:0, C_16:0, C_16:1, C_18:0, and C_18:1 were identified in lipid A of all the strains, as well as the components of the hydrophilic part: glucosamine (GlcN), ethanolamine (EtN), phosphate, and phosphoethanolamine (EtN-P). In the core LPS, glucose (Glc), rhamnose (Rha), L-glycero-D-manno-heptose (Hep), GlcN, galactosamine (GalN), 2-keto-3-deoxy-D-mannooctonoic acid (KDO), alanine (Ala), and phosphate were present. The O chain of all the strains consisted of repeated elements containing a linear chain of three to four L-(two strains) or D-Rha (four strains) residues supplemented with a single residue of 3-acetamido-3,6-dideoxy-D-galactose (D-Fucp3Nac), N-acetyl-D-glucosamine (D-GlcpNAc), D-fucose (D-Fucf), or D-Rhap (strain-dependent) as a side substituent. In different strains the substitution position for Rha residues in the repeated components of the major rhamnan chain was also different. One strain exhibited a unique type of O-chain heterogeneity. Immunochemical investigation of the LPS antigenic properties revealed the absence of close serological relations between the strains of one pathovar; this finding correlates with the differences in their OPS structure. Resemblance between the investigated strains and other P. syringae strains with similar LPS structures was revealed. The results of LPS analysis indicate the absence of correlation between the OPS structure and the pathovar affiliation of the strains.     

Exocellular Cyclic Dipeptides from a <Emphasis Type="Italic">Ruegeria</Emphasis> Strain Associated with Cell Cultures of <Emphasis Type="Italic">Suberites domuncula</Emphasis>

From cell cultures of Suberites domuncula was isolated a bacterial strain, SDC-1, which was identified by 16S ribosomal RNA sequence analysis as an -Proteobacterium of the genus Ruegeria. The occurrence of the strain in sponge cell culture could be explained by its resistance to the antibiotics used in the isolation of sponge cell cultures or by the preservation of SDC-1 by host sponge cells. The fatty acid composition of SDC-1 is characterized by branched C-12 methyl fatty acids. Two new and 8 known cyclic dipeptides were isolated and characterized from the fermentation broth of SDC-1. Cyclodipeptides are one of the families of cell-cell signaling compounds and may have some role to play in sponge-bacteria interactions.     

Structure of an acidic O-specific polysaccharide of the marine bacterium <Emphasis Type="Italic">Pseudoalteromonas agarivorans</Emphasis> KMM 232 (R-form)

An acidic O-specific polysaccharide containing L-rhamnose, 2-acetamido-2-deoxy-D-galactose, 2,6-dideoxy-2-(N-acetyl-L-threonine)amino-D-galactose, and 2-acetamido-2-deoxy-D-mannuronic acid was obtained by mild acid degradation of the lipopolysaccharide of the marine bacterium Pseudoalteromonas agarivorans KMM 232 (R-form) followed by gel-permeation chromatography. The polysaccharide was subjected to Smith degradation to give a modified polysaccharide with trisaccharide repeating unit containing L-threonine. The initial and modified polysaccharides were studied by sugar analysis and ¹H- and ¹³C-NMR spectroscopy, including COSY, TOCSY, ROESY, and HSQC experiments, and the structure of the branched tetrasaccharide repeating unit of the polysaccharide was established.     

The Structure of Uncommon Lipid A from the Marine Bacterium <Emphasis Type="Italic">Marinomonas communis</Emphasis> ATCC 27118<Superscript>T</Superscript>

Lipid A was obtained in a high yield (27%) by the hydrolysis of lipopolysaccharide from the marine gamma proteobacterium Marinomonas communis ATCC 27118^T with 1% AcOH. Using chemical analysis and 1D and 2D NMR spectroscopic and fast atom bombardment mass spectrometric methods, it was shown to be β-1′,6-linked D-glucosaminobiose 1-phosphate acylated with (R)-3-dodecanoyl- or (R)-3-decanoyloxydecanoic acid, (R)-3-{(R)-3-hydroxydecanoyloxy)]decanoic acid and (R)-3-hydroxydecanoic acid at the C2, C2′ and C3 positions, respectively. Uncommon structural peculiarities (a low acylation and phosphorylation degree) of the M .communis lipid A in comparison with those of terrestrial bacteria may be of pharmacological interest. The potential physiological meaning of this lipid A and compounds of similar structure are discussed.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 404–413.Original Russian Text Copyright © 2005 by Vorob’eva, A. Dmitrenok, P. Dmitrenok, Isakov, Krasikova, Solov’eva.The article was translated by the authors.     

Ammonium and attachment of <Emphasis Type="Italic">Rhodopirellula baltica</Emphasis>

A dimorphic life cycle has been described for the planctomycete Rhodopirellula baltica SH1^T, with juvenile motile, free-swimming cells and adult sessile, attached-living cells. However, attachment as a response to environmental factors was not investigated. We studied the response of R. baltica to nitrogen limitation. In batch cultures, ammonium limitation coincided with a dominance of free-swimming cells and a low number of aggregates. Flow cytometry revealed a quantitative shift with increasing ammonium availability, from single cells towards attached cells in large aggregates. During growth of R. baltica on glucose and ammonium in chemostats, an ammonium addition caused a macroscopic change of the growth behaviour, from homogeneous growth in the liquid phase to a biofilm on the borosilicate glass wall of the chemostat vessel. Thus, an ammonium limitation—a carbon to nitrogen supply ratio of 30:1—sustained free-living growth without aggregate formation. A sudden increase in ammonium supply induced sessile growth of R. baltica. These observations reveal a response of Rhodopirellula baltica cells to ammonium: they abandon the free-swimming life, attach to particles and form biofilms.     

Isolation and structural determination of a unique polysaccharide containing mannofuranose from the cell wall of the fungus <Emphasis Type="Italic">Acrospermum compressum</Emphasis>

The alkali-extractable and water-soluble fungal polysaccharide F1SS isolated from the cell wall of Acrospermum compressum has been studied by methylation analyses, reductive cleavage and 1D- and 2D-NMR spectroscopy. The polysaccharide consists of a regular disaccharide repeating unit with the structure: The mannan core was obtained by mild hydrolysis of the polysaccharide F1SS and its structure was deduced to be composed of a skeleton of α-(1→6)-mannopyranan, with around 1 out of 11 residues substituted at position 2 by short chains (one to six units) of 2-substituted mannopyranoses. DOSY experiments provided molecular sizes of 60 kDa and 2.5 kDa for the polysaccharide F1SS and the mannan core, respectively. This is the first report of a fungal mannofuranose-containing cell wall polysaccharide.     

Antimicrobial screening and active compound isolation from marine bacterium NJ6-3-1 associated with the sponge <Emphasis Type="Italic">Hymeniacidon perleve</Emphasis>

Twenty-nine marine bacterial strains were isolated from the sponge Hymeniacidon perleve at Nanji island, and antimicrobial screening showed that eight strains inhibited the growth of terrestrial microorganisms. The strain NJ6-3-1 with wide antimicrobial spectrum was identified as Pseudoalteromonas piscicida based on its 16S rRNA sequence analysis. The major antimicrobial metabolite, isolated through bioassay-guide fractionation of TLC bioautography overlay assay, was identified as norharman (a beta-carboline alkaloid) by EI-MS and NMR.     

Transfer of the chromosomally encoded tetracycline resistance gene <Emphasis Type="Italic">tet</Emphasis>(M) from marine bacteria to <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Enterococcus faecalis</Emphasis>

The transferability of the tetracycline (TC) resistance gene tet(M) from marine bacteria to human enteric bacteria was examined by a filter-mating method. Vibrio spp., Lactococcus garvieae, Bacillus spp., Lactobacillus sp., and Paenibacillus sp. were used as donors, and Escherichia coli JM109 and Enterococcus faecalis JH2-2 were used as recipients. The combination of Vibrio spp. and E. coli resulted in 5/68 positive transconjugants with a transfer rate of 10⁻⁷ to 10⁻³; however, no transfer was observed with E. faecalis. In case of L. garvieae and E. faecalis, 6/6 positive transconjugants were obtained with a transfer rate of 10⁻⁶ to 10⁻⁵; however, no transfer was observed with E. coli. The tet(M) gene of Bacillus, Lactobacillus, and Paenibacillus were not transferred to either E. coli or E. faecalis. tet(M) transfer was confirmed in positive E. coli and E. faecalis transconjugants by polymerase chain reaction (PCR) and Southern hybridization. All the donor strains did not harbor plasmids, while they all harbored transposon Tn916. In the transconjugants, the transposon was not detected by PCR, suggesting the possible transfer of tet(M) from the marine bacterial chromosome to the recipient chromosome. This is the first report to show that tet(M) can be transferred from marine bacteria to human enteric bacteria in a species-specific manner.     

Synthesis of (<Emphasis Type="Italic">2S</Emphasis>,<Emphasis Type="Italic">4S</Emphasis>)-4-phenylamino-5-oxoproline derivatives

Summary. The paper describes the synthesis of (2S,4S)-4-(N-Ts)- and (2S,4S)-4-(N-Boc)-phenylamino-5-oxoprolines (pyroglutamic acid). These derivatives have been shown to be useful for synthesis of their amides and peptides in spite of steric hindrances caused by bulky groups adjacent to the reaction centre. Under the conditions applied no lactam ring opening and no loss of stereochemical integrity of any of the chiral centres were observed, which has been confirmed by NMR techniques. Received December 29, 2000 Accepted June 26, 2001