Structural elucidation of the outer core tetrasaccharide isolated from the LPS of Rhizobium leguminosarum bv. trifolii strain 24.

The outer core oligosaccharide (OS) was isolated from the lipopolysaccharide (LPS) of Rhizobium leguminosarum bv. trifolii strain 24 after Smith degradation and then studied by sugar and methylation analyses along with NMR and mass spectrometry methods. Negative-ion electrospray (ESI-MS) mass spectrum showed two molecular ions at m/z 686.3 and 728.3, which corresponded to the core OS having the composition Rha2QuiNAcKdh. The mass difference between both ions indicated that the higher molecule mass represented the mono O-acetylated variant of the OS. The sequence of the oligosaccharide was reflected in CID MS/MS spectra. In turn, NMR spectroscopy confirmed the composition and glycosylation pattern of the core OS and provided additional evidence on its structure. 2D NMR experiments revealed that the terminal Rhap is acetylated at position O-2. Moreover, 3-deoxyheptulosonic acid (Kdh), which was detected at the reducing terminus of the OS, was evidently derived from the Kdo as a result of Smith degradation. In addition, the higher intensity of signals for a six-membered pyranose ring of Kdhp over 2,7-anh-Kdhf seemed to indicate prevalence of this form of the sugar in the OS-derived species. Based on the data obtained, the following structure of the outer core tetrasaccharide, which probably links the O-chain polysaccharide to the inner core in the LPS of R. leguminosarum bv. trifolii strain 24, was established: α-L-Rhap-2-OAc*-(1-->3)-α-L-Rhap-(1-->3)-ß-D-QuipNAc-(1-->4)-Kdo * ~ 50%. .

Identification of unusual phospholipid fatty acyl compositions of Acanthamoeba castellanii.

Acanthamoeba are opportunistic protozoan pathogens that may lead to sight-threatening keratitis and fatal granulomatous encephalitis. The successful prognosis requires early diagnosis and differentiation of pathogenic Acanthamoeba followed by aggressive treatment regimen. The plasma membrane of Acanthamoeba consists of 25% phospholipids (PL). The presence of C20 and, recently reported, 28- and 30-carbon fatty acyl residues is characteristic of amoeba PL. A detailed knowledge about this unusual PL composition could help to differentiate Acanthamoeba from other parasites, e.g. bacteria and develop more efficient treatment strategies. Therefore, the detailed PL composition of Acanthamoeba castellanii was investigated by 31P nuclear magnetic resonance spectroscopy, thin-layer chromatography, gas chromatography, high performance liquid chromatography and liquid chromatography-mass spectrometry. Normal and reversed phase liquid chromatography coupled with mass spectrometric detection was used for detailed characterization of the fatty acyl composition of each detected PL. The most abundant fatty acyl residues in each PL class were octadecanoyl (18∶0), octadecenoyl (18∶1 Δ9) and hexadecanoyl (16∶0). However, some selected PLs contained also very long fatty acyl chains: the presence of 28- and 30-carbon fatty acyl residues was confirmed in phosphatidylethanolamine (PE), phosphatidylserine, phosphatidic acid and cardiolipin. The majority of these fatty acyl residues were also identified in PE that resulted in the following composition: 28∶1/20∶2, 30∶2/18∶1, 28∶0/20∶2, 30∶2/20∶4 and 30∶3/20∶3. The PL of amoebae are significantly different in comparison to other cells: we describe here for the first time unusual, very long chain fatty acids with Δ5-unsaturation (30∶35,21,24) and 30∶221,24 localized exclusively in specific phospholipid classes of A. castellanii protozoa that could serve as specific biomarkers for the presence of these microorganisms.

Ether-type moieties in the lipid part of glycoinositolphospholipids of Acanthamoeba rhysodes.

Ether lipids were identified among components liberated with HF and nitrous acid deamination from Acanthamoeba rhysodes whole cells and its membrane glycoinositolphospholipids (GIPL). Liberated ether glycerols were converted to various derivatives that served characterization thereof. These included TMS and isopropylidene derivatives, oxidation with sodium periodate to aldehyde followed by reduction with NaBH4 to alcohol, and reaction of the alcohol with acetic anhydrite to form acetate derivatives. Periodate sensitivity demonstrated that the alkyl side chains were linked to the sn-1 position of glycerol. Combined information from TLC, GC-MS analysis, MALDI-TOF spectrometry, and chemical degradation experiments indicated the presence of ether-linked saturated normal and branched hydrocarbons with a length of C20-23 in the phospholipid fraction, C20-24 in free GPI, and C21-23 in the LPG polymer. The distribution of particular classes of alkylglycerols was similar for phospholipid and GPI fractions, and amounted to 2.62% (±0.04-0.28) 1-O-eicosanyl-sn-glycerol, 16.66% (±0.32-1.1) 1-O-uncosanyl-sn-glycerol, 9.18% (±0.33-1.37) anteiso-1-O-docosanyl-sn-glycerol, 47.56% (±0.32-2.14) 1-O-docosanyl-sn-glycerol, 20.56% (±0.58-1.67) anteiso-1-O-tricosanyl-sn-glycerol, and 2.34% (±0.12-0.63) 1-O-tricosanyl-sn-glycerol. For LPG preparation, the most abundant were anteiso-1-O-tricosanyl-sn-glycerol (57.26%) and 1-O-docosanyl-sn-glycerol (30.12%). The data from TLC and GC-MS analysis showed that ether lipids from phospholipids probably represent the lyso-alkylglycerol type, while those derived from GIPL are alkylacylglycerol moieties.

New long chain bases in lipophosphonoglycan of Acanthamoeba castellanii.

The polymer called lipophosphonoglycan (LPG) was isolated from Acanthamoeba castellanii membranes after exhaustive delipidation and butanol extraction. A novel extremely long phytosphingosine was revealed in glycoinositolphosphosphingolipid (GIPSL). All data obtained by gas-liquid chromatography coupled with MS analyses of products liberated during acid methanolysis and products of sodium metaperiodate and permanganate-periodate oxidations showed an unusual pattern of long chain bases (LCB) with branched bases (anteiso-C24, anteiso-C25, anteiso-C26, iso-C26, anteiso-C27, and anteiso-C28) and normal ones (C24, C25, C26, C27). The phytosphingosines with hexa-, hepta-, and octacosanoic chains have not been detected in Acanthamoeba cells up to now. Also, the isomer configuration of long chain bases in LPG of A. castellanii was not defined in earlier reports. In the GC-MS chromatograms, the component forming a peak corresponding to anteiso-C25 phytosphingosine was the most abundant and constituted more than 50 % of all LCB.

Structural analysis of the O-specific polysaccharide from the lipopolysaccharide of Aeromonas veronii bv. sobria strain K49.

The O-specific polysaccharide obtained by mild-acid degradation of the lipopolysaccharide from Aeromonas veronii bv. sobria strain K49 was studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy. The sequence of the sugar residues was determined using (1)H,(1)H NOESY and (1)H,(13)C HMBC experiments. The O-specific polysaccharide was found to be a high molecular mass polysaccharide composed of repeating units of the structure: →2)-ß-D-Quip3NAc-(1→3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-D-FucpNAc-(1→ ESI MS confirmed the pentasaccharide structure of the repeating unit, as the molecular mass peaks seen in the spectrum differed by 812.34 u, a value corresponding to the calculated molecular mass of the O-unit.

Structural studies of the O-specific polysaccharide from the lipopolysaccharide of Mesorhizobium huakuii strain S-52, the symbiotic partner of Astragalus sinicus.

The O-specific polysaccharide (OPS) obtained by mild-acid degradation of the lipopolysaccharide isolated from Mesorhizobium huakuii strain S-52 was studied by sugar and ethylation analyses along with (1)H and (13)C NMR spectroscopy. It was concluded that the OPS was composed of trisaccharide repeating units containing two residues of 6-deoxy-l-talose (6dTal) and one l-rhamnose (Rha), whose sequence in the OPS was determined by NOESY and HMBC experiments. The minor 3-O-acetylation (about 10%) of 6-deoxytalose glycosidically substituted at position-2 was judged by relative signal intensities of corresponding O-acetylated and non-acetylated 6dTal residues. Moreover, it was found that the non-reducing end of the OPS repeating unit was occupied by 3-O-methyl-d-fucose, which terminated the O-chain as a cap-residue. These data defined the structure of the OPS as: α-3-OMe-d-Fucp-(1→[2)-α-l-6dTalp-(1→3)-α-l-6dTalp-(1→2)-α-l-Rhap-(1→](n).

Structural characterization of the O-specific polysaccharide from the lipopolysaccharide of the fish pathogen Aeromonas bestiarum strain P1S.

The O-specific polysaccharide obtained by mild-acid degradation of lipopolysaccharide of Aeromonas bestiarum P1S was studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy. The sequence of the sugar residues was determined using (1)H,(1)H NOESY and (1)H,(13)C HMBC experiments. The O-specific polysaccharide was found to be a high-molecular-mass polysaccharide composed of tetrasaccharide repeating units of the structure [formula in text]. Since small amounts of a terminal Quip3N residue were identified in methylation analysis, it was assumed that the elucidated structure also represented the biological repeating unit of the O-specific polysaccharide.

Legionella bozemanae synthesizes phosphatidylcholine from exogenous choline.

The phospholipid class and fatty acid composition of Legionella bozemanae were determined using thin-layer chromatography, gas-liquid chromatography, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were the predominant phospholipids, while phosphatidyl-N-monomethylethanolamine, phosphatidylglycerol, and phosphatidyl-N,N-dimethylethanolamine were present at low concentrations. With the use of the LC/MS technique, PC16:0/15:0, PC17:/15:0, and PE16:1/15:0 were shown to be the dominant phospholipid constituents, which may be taxonomically significant. Two independent phosphatidylcholine synthesis pathways (the three-step methylation and the one-step CDP-choline pathway) were present and functional in L. bozemanae. In the genome of L. bozemanae, genes encoding two potential phosphatidylcholine forming enzymes, phospholipid N-methyl transferase (PmtA) and phosphatidylcholine synthase (Pcs), homologous to L. longbeachae, L. drancourtii, and L. pneumophila pmtA and pcs genes were identified. Genes pmtA and pcs from L. bozemanae were sequenced and analyzed on nucleotide and amino acid levels. Bacteria grown on an artificial medium with labelled choline synthesized phosphatidylcholine predominantly via the phosphatidylcholine synthase pathway, which indicates that L. bozemanae phosphatidylcholine, similarly as in other bacteria associated with eukaryotes, is an important determinant of host-microbe interactions.

Morphology and general characteristics of bacteriophages infectious to Robinia pseudoacacia mesorhizobia.

Four phages infectious to Mesorhizobium strains were identified in soil samples taken from local Robinia pseudoacacia stands. Based on their polyhedral heads and short noncontractile tails, three of the phages, Mlo30, Mam12, and Mam20, were assigned to group C of Bradley's classification, the Podoviridae family, while phage Mlo1, with its elongated hexagonal head and a long flexible tail represented subgroup B2 bacteriophages, the Siphoviridae family. The phages were homogeneous in respect of their virulence, as they only lysed Mesorhizobium strains, but did not affect strains of Rhizobium or Bradyrhizobium. On the basis of one-step growth experiments, the average virus yield was calculated as approximately 10-25 phage particles for phages Mlo30, Mam12 and Mam20, and as many as 100-120 for phage Mlo1. The rate of phage adsorption to heat-treated cells showed differences in the nature of their receptors, which seemed to be thermal sensitive, thermal resistant, or a combination of the two. Only the receptor for phage Mlo30 was likely to be an LPS molecule, which was supported by a neutralization test. The smooth LPS with O-antigenic chains of the phage-sensitive M. loti strain completely reduced the bactericidal activity of virions at a concentration of 1 µg/ml. The molecular weights of phage DNAs estimated from restriction endonuclease cleavage patterns were in the range from approximately 39 kb for group C phages to approximately 80 kb for B2.

The structure of the O-specific polysaccharide from the lipopolysaccharide of Aeromonas bestiarum strain 207.

The O-specific polysaccharide obtained by mild-acid degradation of Aeromonas bestiarum 207 lipopolysaccharide was studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy. The sequence of the sugar residues was determined by ROESY and HMBC experiments. It is concluded that the O-polysaccharide is composed of branched pentasaccharide repeating units of the following structure: [structure: see the text]

Structure of the O-polysaccharides of the lipopolysaccharides of Mesorhizobium loti HAMBI 1148 and Mesorhizobium amorphae ATCC 19655 containing two O-methylated monosaccharides.

The O-polysaccharide of Mesorhizobium loti HAMBI 1148 was obtained by mild acid degradation of the lipopolysaccharide and studied by sugar and methylation analyses, Smith degradation, and (1)H and (13)C NMR spectroscopies, including 2D (1)H/(1)H COSY, TOCSY, ROESY, and H-detected (1)H/(13)C HSQC experiments. The O-polysaccharide was found to have a branched hexasaccharide-repeating unit of the following structure: [Formula: see text] where 2-acetamido-2-deoxy-4-O-methyl-D-glucose (D-GlcNAc4Me) and methyl group on 2-substituted D-rhamnose (Me) shown in italics are present in approximately 80% and approximately 40% repeating units, respectively. Similar studies of the O-polysaccharide from Mesorhizobium amorphae ATCC 19655 by sugar analysis and NMR spectroscopy revealed essentially the same structure but a higher content of 3-O-methyl-D-rhamnose ( approximately 70%).

Chemical structure and biological significance of lipopolysaccharide from Legionella.

Legionella are aerobic, gram-negative, motile, rod-shaped bacteria, which form a distinct taxonomic unit within the gamma - 2 subdivision of the Proteobacteria. The reservoirs of Legionella are natural or man-made water systems where the bacteria survive and disseminate as obligate intracellular parasites of free living protozoa. In the human lung, the bacteria invade alveolar macrophages inducing the potentially lethal pneumonia commonly known as Legionnaires' disease. Although all Legionella species are considered potentially pathogenic for humans, Legionella pneumophila is the aetiological agent responsible for most reported cases of community- and nosocomially-acquired legionellosis. The O-polysaccharide in the lipopolysaccharide of L. pneumophila is composed of a repeating homopolymer of alpha-(2-->4)-linked 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (legionaminic acid). The outer region of the core enriched with 6-deoxy sugars and N- and O- acetylated sugars as well as the highly N- and O-acylated O-chain contribute to a high hydrophobicity of the bacterial surface, which enables these bacteria to spread. Lipids A from Legionella contain a backbone with 2,3-diamino-2,3-dideoxy-D-glucose and unusual fatty acids. The present article indicates some patents useful in the diagnostics of Legionnaires' disease.

Localization of the attachment site of oligoglucans to Mesorhizobium loti HAMBI 1148 murein.

The location and nature of the linkage between peptidoglycan and oligoglucans in the cell wall of Mesorhizobium loti HAMBI 1148 have been defined by the analysis of nitrous acid deamination of peptidoglycan glucosaminyl residues. The MurNH(2)-Glc(n) fraction was obtained after converting deaminoacylated and N-deacetylated muramyl residues in the cell wall preparation to lactam forms which were stable during subsequent deamination, followed by reduction and opening of the lactams. GC/MS analysis of this material, subjected to partial hydrolysis and reduction or to methanolysis followed by peracetylation, confirmed the presence of glucosyl residues glycosidically attached to muramic acid. The MALDI-TOF spectroscopic analysis of the deaminated material also revealed the presence of [M-H](-) or [M+Na-2H](-) ions representing fragments containing muramic acid with one to three linked glucose residues. The analysis of fully methylated neutral oligosaccharides released from the peptidoglycan with lysozyme followed by borohydride reduction showed the presence of di- and trisaccharides lacking the reducing end.

The incomplete substitution of lipopolysaccharide with O-chain prevents the establishment of effective symbiosis between Mesorhizobium loti NZP2213.1 and Lotus corniculatus.

Mesorhizobium loti NZP2213.1 mutant obtained after random Tn5 mutagenesis of M. loti NZP2213 was inefficient in nitrogen fixation on Lotus corniculatus. The transposon insertion was located within an ORF with a sequence similarity to a putative glycosyl transferase from Caulobacter crescentus. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the mutant produced LPS of the same O-chain length but only half of the entire smooth LPS, compared to that of the parental strain. A greater diversity of the anomeric region as determined by NMR spectroscopy, reflected structural differences in the mutant repeating units represented by 6-deoxytalose, 2-OAc-6-deoxytalose, and 2-OMe-6-deoxytalose. In contrast to the completely O-acetylated 6-deoxytalose in wild-type OPS only partial O-acetylation was found in the mutant. The decrease of the LPS species with O-chains seems to be correlated with 6-deoxytalose deficiency. Microscopic examination of the nodules induced by the mutant revealed disturbances in infection thread development and premature senescence of symbiosomes. The impairment of mutant-induced symbiosomes to sustain latter stages of symbiosis could be a consequence of the decreased ratio of the hydrophobic to the hydrophilic LPSs.

Cellular envelope phospholipids from Legionella lytica.

The composition of phospholipids from the cellular envelope of Legionella lytica grown on artificial medium was determined by two-dimensional thin-layer chromatography. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidyl-N-monomethylethanolamine were the predominant phospholipids, while diphosphatidylglycerol, phosphatidylglycerol, and phosphatidyl-N,N-dimethylethanolamine were present at low concentrations. A trace amount of lipids carrying glycosyl residues was also observed. The fatty acids and their distribution in individual phospholipids were characterized using liquid chromatography/mass spectrometry (LC/MS), matrix-assisted laser desorption ionization-time of flight, and gas chromatography/MS methods. The characteristic feature of L. lytica phospholipids was the presence of an unbranched chain (which differentiates this bacterium from Legionella pneumophila) and branched iso and anteiso fatty acids as well as cis-9,10-methylenehexadecanoic acid. According to spectroscopic LC/MS data, the localization of saturated and unsaturated fatty acid residues on phosphorylglycerol was determined. Some aspects of the significance of phosphatidylcholine, one of the main phospholipids in L. lytica, are addressed and taxonomic implications of the data are discussed.

Alteration of O-specific polysaccharide structure of symbiotically defective Mesorhizobium loti mutant 2213.1 derived from strain NZP2213.

Mesorhizobium loti mutant 2213.1 derived from the wild-type strain NZP2213 by Tn5 mutagenesis showed impaired effectiveness of symbiosis with the host plant Lotus corniculatus (Turska-Szewczuk et al., 2007 Microbiol Res, in press). The inability of lipopolysaccharide (LPS) isolated from the mutant 2213.1 strain or de-O-acetylated LPS of the parental cells to inactivate phage A1 particles implicated alterations in the LPS structure. The O-specific polysaccharide of the mutant was studied by chemical analyses along with (1)H and (13)C NMR spectroscopy, which clearly confirmed alterations in the O-chain structure. 2D NMR data showed that the mutant O-polysaccharide consists of a tetrasaccharide repeating unit containing non-substituted as well as O-acetylated or O-methylated 6-deoxytalopyranose residues. Additionally, an immunogold assay revealed a reduced number of gold particles on the mutant bacteroid cell surface, which could result from both a diminished amount of an O-antigenic determinant in mutant LPS and modifications of structural epitopes caused by alterations in O-acetylation or O-methylation of sugar residues. Western immunoblot assay of alkaline de-O-acetylated lipophilic M. loti NZP2213 LPS showed no reactivity with homologous serum indicating a role of O-acetyl groups in its O-specificity.

Structural studies of the O-polysaccharide chain from the lipopolysaccharide of symbiotically enhanced mutant Mlo-13 of Mesorhizobium loti NZP2213.

The O-specific polysaccharide (OPS) of Mesorhizobium loti mutant Mlo-13 was obtained by mild-acid degradation of the lipopolysaccharide and studied by sugar and ethylation analyses, Smith degradation, as well as (1)H and (13)C NMR spectroscopy. The O-polysaccharide has a trisaccharide repeating unit composed of 6-deoxy-L-talose (L-6dTal), 4-O-acetyl-6-deoxy-L-talose (L-6dTal-4Ac) and L-rhamnose residues. An analysis of NMR spectra revealed that the rhamnose residue was substituted nonstoichiometrically with O-methyl group at C-3. The following structure of the repeating unit of the Mlo-13 OPS was established: [structure: see text].