New Structures of the O-Specific Polysaccharides of Bacteria of the Genus Proteus. 1. Phosphate-Containing Polysaccharides

The O-specific polysaccharide chains (O-antigens) of the lipopolysaccharides of five Proteus strains, P. vulgaris O17, P. mirabilis O16 and O33, and P. penneri 31 and 103, were found to contain phosphate groups that link the non sugar components, e.g., ethanolamine and ribitol. The polysaccharides of P. mirabilis O16 and P. penneri 103 include ribitol phosphate in the main chain and thus resemble ribitol teichoic acids of Gram-positive bacteria. The structures of the polysaccharides were elucidated using NMR spectroscopy, including two-dimensional ¹H, ¹H correlation spectroscopy (COSY and TOCSY), nuclear Overhauser effect spectroscopy (NOESY or ROESY), and H-detected ¹H, ¹³C and ¹H, ³¹P heteronuclear multiple-quantum coherence spectroscopy (HMQC), along with chemical methods. The structures determined are unique among the bacterial polysaccharides and, together with the data obtained earlier, represent the chemical basic for classification of Proteus strains. Based on structural similarities of the O-specific polysaccharides and serological relationships between the O-antigens, we propose to extend Proteus serogroups O17 and O19 by including P. penneri strains 16 and 31, respectively.     

The structure of the carbohydrate backbone of the core–lipid A region of the lipopolysaccharide from Proteus penneri strain 40: new Proteus strains containing open-chain acetal-linked N-acetylgalactosamine in the core part of the LPS

Analysis of the core part of the LPS from several strains of Proteus revealed that P. penneri strains 2, 11, 19, 107, and P. vulgaris serotypes O4 and O8 have the same structure with a new type of linkage between monosaccharides–an open-chain acetal — that was previously determined for P. vulgaris OX2 and P. penneri 17. The LPS from P. penneri strain 40 contains the same structure substituted with one additional monosaccharide:

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where (1S)-GalaNAc¹ is a residue of N-acetyl- -galactosamine in the open-chain form. It is connected as a cyclic acetal to positions 4 and 6 of the galactosamine residue having a free amino group. All other sugars are in the pyranose form.     

NMR natural abundance estimation of13C-metabolic solutes in normal and habituated sugarbeet cell lines

Summary NMR (nuclear magnetic resonance) spectroscopy was used to identify metabolic solutes in one normal and two habituated sugarbeet cell lines (Beta vulgaris L.altissima) obtained from the same mother strain. This technique was applied to investigate the intracellular naturally occurring¹³C isotopes (1.1% of total natural carbon) in living sugarbeet suspension cells and perchloric cell extracts. A combination of¹H,¹³C, double-quantum filter correlation spectroscopy, heteronuclear multiple-bond correlation, and heteronuclear multiple-quantum coherence spectra from perchloric cell extracts enabled us to identify the main compounds in the different extract solutions. This was verified by spiking the solutions with small amounts of reference compounds to exclude the influence exerted by pH on the chemical shifts of the different compounds in the¹H and¹³C spectra. The comparison of the three sugarbeet cell lines' NMR spectra showed the presence of sucrose, glucose, and fructose in the three strains. On the other hand, it revealed a strong discrepancy between metabolic solutes. Spectra from the habituated lines showed the presence of glutamine. Some amino acids such as alanine or valine, and unidentified signals corresponding to aromatic rings were only characterized in the habituated nonorganogenic cells. On the basis of these¹³C NMR data we assumed that the discrepancy between the different sugarbeet cell lines could be due to an increase in the metabolic activity of the habituated cell lines in relation to their autonomous growth.Abbreviations DQF-COSY double-quantum filter correlation spectroscopy - HO habituated organogenous - HNO habituated nonorganogenous - HMBC heteronuclear multiple-bond correlation - HMQC heteronuclear multiple-quantum coherence - N normal - NMR nuclear magnetic resonance - TSP sodium tetradeutero-3-(trimethylsilyl)-propionate     

Structure of the glycerol phosphate-containing O-polysaccharides and serological studies of the lipopolysaccharides of Proteus mirabilis CCUG 10704 (OE) and Proteus vulgaris TG 103 classified into a new Proteus serogroup, O54

O-Polysaccharides were obtained from the lipopolysaccharides of Proteus mirabilis CCUG 10704 (OE) and Proteus vulgaris TG 103 and studied by chemical analyses and one- and two-dimensional (1)H and (13)C nuclear magnetic resonance spectroscopy, including rotating-frame nuclear Overhauser effect spectroscopy, H-detected (1)H,(13)C heteronuclear single-quantum spectroscopy and (1)H,(31)P heteronuclear multiple-quantum spectroscopy experiments. The Proteus mirabilis OE polysaccharide was found to have a trisaccharide repeating unit with a lateral glycerol phosphate group. The Proteus vulgaris TG 103 produces a similar O-polysaccharide, which differs in incomplete substitution with glycerol phosphate (c. 50% of the stoichiometric amount) and the presence of an O-acetyl group at position 6 of the 2-acetamido-2-deoxygalactose (GalNAc) residue. These structures are unique among the known bacterial polysaccharide structures. Based on the structural and serological data of the lipopolysaccharides, it is proposed to classify both strains studied into a new Proteus serogroup, O54, as two subgroups, O54a,54b and O54a,54c. The serological relatedness of the Proteus O54 and some other Proteus lipopolysaccharides is discussed.     

Structure of the O-specific polysaccharide of the bacterium Proteus vulgaris O23

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of the bacterium Proteus vulgaris O23 (strain PrK 44/57) and found to contain 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, and D-galacturonic acid. Based on 1H- and 13C-NMR spectroscopic studies, including two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments, the following structure of the branched tetrasaccharide repeating unit of the polysaccharide was established: [figure], where the degree of O-acetylation of the terminal GalA residue at position 4 is about 80%. A structural similarity of the O-specific polysaccharides of P. vulgaris O23 and P. mirabilis O23 is discussed.     

1H, 13C and 15N NMR assignments and secondary structure of the paramagnetic form of rat cytochrome b 5

Summary Modern multidimensional double- and triple-resonance NMR methods have been applied to assign the backbone and side-chain ¹³C resonances for both equilibrium conformers of the paramagnetic form of rat liver microsomal cytochrome b ₅. The assignment of backbone ¹³C resonances was used to confirm previous ¹H and ¹⁵N resonance assignments [Guiles, R.D. et al. (1993) Biochemistry, 32, 8329–8340]. On the basis of short- and medium-range NOEs and backbone ¹³C chemical shifts, the solution secondary structure of rat cytochrome b ₅ has been determined. The striking similarity of backbone ¹³C resonances for both equilibrium forms strongly suggests that the secondary structures of the two isomers are virtually identical. It has been found that the ¹³C chemical shifts of both backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts can be observed, is evaluated through calculations of the magnitude of such shifts.Abbreviations DANTE delays alternating with nutation for tailored excitation - DEAE diethylaminoethyl - DQF-COSY 2D double-quantum-filtered correlation spectroscopy - EDTA ethylenediaminetetraacetic acid - HCCH-TOCSY 3D proton-correlated carbon TOCSY experiment - HMQC 2D heteronuclear multiple-quantum correlation spectroscopy - HNCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons - HNCO 3D triple-resonance experiment correlating amide protons, amide nitrogens and carbonyl carbons - HNCOCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons via carbonyl carbons - HOHAHA 2D homonuclear Hartmann-Hahn spectroscopy - HOHAHA-HMQC 3D HOHAHA relayed HMQC - HSQC 2D heteronuclear single-quantum correlation spectroscopy - IPTG isopropyl thiogalactoside - NOESY 2D nuclear Overhauser enhancement spectroscopy - NOESY-HSQC 3D NOESY relayed HSQC - TOCSY 2D total correlation spectroscopy - TPPI time-proportional phase incrementation - TSP trimethyl silyl propionate     

Identification of a Proteus penneri isolate as the causal agent of red body disease of the cultured white shrimp Penaeus vannamei and its control with Bdellovibrio bacteriovorus

Bacteriosis has become a major economic problem in the farming of the Pacific white shrimp Penaeus vannamei. However, no definitive data are available about Proteus penneri infection in cultured P. vannamei and its control. In this study, a virulent strain NC was isolated from diseased P. vannamei suffering from red body disease and identified as a P. penneri isolate through phylogenetic analysis and ATB 32GN system. A phylogenetic constructed tree using the neighbour-joining method identified the NC isolate as a P. penneri strain. In addition, Bdellovibrio bacteriovorus conferred significant protection against P. penneri: it exhibited significant bacteriolytic effects on the pathogenic P. penneri, had a wide prey range towards Proteus pathogens, and displayed a good protective efficacy on experimental P. penneri infection in P. vannamei. To the best of our knowledge, this is the first report of farmed P. vannamei infected with P. penneri and its control with B. bacteriovorus.     

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O60

An O-polysaccharide (O-antigen) was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O60 and studied by sugar and methylation analyses as well as ¹H and ¹³C NMR spectroscopy, including 2D ROESY and ¹H,¹³C HMBC experiments in D₂O and a ROESY experiment in a 9:1 H₂O–D₂O mixture to reveal correlations for NH protons. It was found that the polysaccharide is built up of linear pentasaccharide repeating units containing an amide of d-glucuronic acid with l-serine and has the following structure:The O-antigen studied is structurally and serologically closely related to the O-antigen of Proteus vulgaris O44.     

Enzymatic synthesis and characterization of arbutin glucosides using glucansucrase from Leuconostoc mesenteroides B-1299CB

Two arbutin glucosides were synthesized via the acceptor reaction of a glucansucrase from Leuconostoc mesenteroides B-1299CB with arbutin and sucrose. The glucosides were purified by Bio-gel P-2 column chromatography and high-performance liquid chromatography, and the structures were elucidated as 4-hydroxyphenyl β-isomaltoside (arbutin-G1), 4-hydroxyphenyl β-isomaltotrioside (arbutin-G2), according to the results of ¹H, ¹³C, heteronuclear single-quantum coherence, ¹H-¹H COSY, and heteronuclear multiple-bond correlation analyses. Arbutin glucoside (4-hydroxyphenyl β-isomaltoside) exhibited slower effects on 1,1-diphenyl-2-picrylhydrazyl radical scavenging and similar effects on tyrosinase inhibition, and increased inhibitory effect on matrix metalloproteinase-1 production induced by UVB than arbutin. Young Hwan Moon and Seung Hee Nam contributed equally to this work.     

The primary electron acceptor of green sulfur bacteria,bacteriochlorophyll 663, is chlorophyll a esterified with Δ 2,6-phytadienol

The primary electron acceptor of green sulfur bacteria, bacteriochlorophyll (BChl) 663, was isolated at high purity by an improved purification procedure from a crude reaction center complex, and the molecular structure was determined by fast atom bombardment mass spectroscopy (FAB-mass), ¹H- and ¹³C-NMR spectrometry, double quantum filtered correlation spectroscopy (DQF-COSY), heteronuclear multiple-quantum coherence (HMQC) and heteronuclear multiple-bond correlation (HMBC) spectral measurements. BChl 663 was 2.0 mass units smaller than plant Chl a. The NMR spectra showed that the macrocycle was identical to that of Chl a. In the esterifying alcohol, a singlet P7¹ signal was observed at the high-field side of the singlet P3¹ signal in BChl 663, while a doublet peak of P7¹ overlapped that of P11¹ in Chl a. A signal of P7-proton, seen in Chl a, was lacking, and the P6-proton appeared as a triplet signal near the triplet P2-proton signal in BChl 663. These results indicate the presence in BChl 663 of a C=C double bond between P6 and P7 in addition to that between P2 and P3. The structure of BChl 663 was hence concluded to be Chl a esterified with 2,6-phytadienol instead of phytol. In addition to BChl 663, two molecules of the 13²-epimer of BChl a, BChl a′, were found to be present per reaction center, which may constitute the primary electron donor. This revised version was published online in June 2006 with corrections to the Cover Date.     

Structure of a new acidic O-antigen of Proteus vulgaris O22 containing O-acetylated 3-acetamido-3,6-dideoxy-D-glucose.

The acidic O-specific polysaccharide of Proteus vulgaris O22 was studied using 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC) experiments, and the following structure for the branched pentasaccharide repeating unit was established: [sequence: see text] where Qui3NAc is 3-acetamido-3,6-dideoxyglucose, O-acetylation of QuiNAc at position 4 is stoichiometric and at position 2 nonstoichiometric. Serological relationships of P. vulgaris O22 with some other Proteus strains were substantiated on the level of the O-antigen structures.     

Quantitative evaluation of NMR and MRI methods to measure sucrose concentrations in plants

Summary Developing pea (Pisum sativum L.) seeds were chosen to evaluate the performance of various nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) methods of detecting sucrose in plants. The methods included chemical shift selective imaging (CHESS), heteronuclear correlation via¹³C-¹H coupling (HMQC), and homonuclear correlation via¹H-¹H coupling (DQF). The same experiments were also performed on sucrose phantom samples to evaluate the methods in the absence of the line broadening observed in plant systems. Using the spin echo technique for multi-slice imaging, we could discern the detailed internal structure of the intact seed with a resolution of tens of microns. The proton spin-lattice relaxation time and linewidth as a function of the age of the seed were measured to optimize the efficiency of the NMR and MR experiments. The age-dependent changes in these NMR parameters are consistent with the accumulation of insoluble starch as age increases. Both the NMR and MRI results are in accord with the results of chemical analysis, which reveal that the sucrose concentration is higher in the embryo than in the seed coat, and glucose is at low concentration throughout the seed. Of the three methods for proton observation, the enhanced version of the CHESS approach (CD-CHESS) provides the best combination of sucrose detection and water suppression. Direct observation of¹³C is preferable to indirect detection using HMQC because of water signal bleed-through in samples with large (>200 Hz) linewidths.Abbreviations CD-CHESS continuous wave decoupling chemical shift selective imaging - CHESS chemical shift selective imaging - CSI chemical shift imaging - CW continuous wave - DQF homonuclear double quantum filtering - FOV field of view - FW fresh weight - GHMQC gradient version of the heteronuclear multiple quantum coherence     

Structure of a 2-aminoethyl phosphate-containing O-specific polysaccharide of Proteus penneri 63 from a new serogroup O68.

Lipopolysaccharide of Proteus penneri strain 63 was degraded by mild acid to give a high molecular mass O-specific polysaccharide that was isolated by gel-permeation chromatography. Sugar and methylation analyses and NMR spectroscopic studies, including two-dimensional 1H, 1H COSY, TOCSY rotating-frame NOE spectroscopy, H-detected 1H,13C and 1H,31P heteronuclear multiple-quantum coherence (HMQC), and 1H, 13C HMQC-TOCSY experiments, demonstrated the following structure of the polysaccharide: where FucNAc is 2-acetamido-2,6-dideoxygalactose and PEtn is 2-aminoethyl phosphate. The polysaccharide studied shares some structural features, such as the presence of D-GlcNAc6PEtn and an alpha-L-FucNAc-(1-->3)-D-GlcNAc disaccharide, with other Proteus O-specific polysaccharides. A marked cross-reactivity of P. penneri 63 O-antiserum with P. vulgaris O12 was observed and substantiated by a structural similarity of the O-specific polysaccharides of the two strains. In spite of this, the polysaccharide of P. penneri 63 has the unique structure among Proteus O-antigens, and therefore a new, separate serogroup, O68, is proposed for this strain.     

New Structures of the O-Specific Polysaccharides of Proteus. 4. Polysaccharides Containing Unusual Acidic N-Acyl Derivatives of 4-Amino-4,6-dideoxy-D-glucose

The structures of the O-polysaccharides of the lipopolysaccharides of Proteus mirabilis O7 and O49 were determined by chemical methods, mass spectrometry, including MS/MS, and NMR spectroscopy, including experiments run in an H2O/D2O mixture to reveal correlations for NH protons. The O-polysaccharides were found to contain N-carboxyacetyl (malonyl) and N-(3-carboxypropanoyl) (succinyl) derivatives of 4-amino-4,6-dideoxyglucose (4-amino-4-deoxyquinovose, Qui4N), respectively. The behavior of Qui4N derivatives with the dicarboxylic acids under conditions of acid hydrolysis and methanolysis was studied using GLC-MS.     

Unique backbone-water interaction detected in sphingomyelin bilayers with 1H/31P and 1H/13C HETCOR MAS NMR spectroscopy

Two-dimensional ¹H/³¹P dipolar heteronuclear correlation (HETCOR) magic-angle spinning nuclear magnetic resonance (NMR) is used to investigate the correlation of the lipid headgroup with various intra- and intermolecular proton environments. Cross-polarization NMR techniques involving ³¹P have not been previously pursued to a great extent in lipid bilayers due to the long ¹H-³¹P distances and high degree of headgroup mobility that averages the dipolar coupling in the liquid crystalline phase. The results presented herein show that this approach is very promising and yields information not readily available with other experimental methods. Of particular interest is the detection of a unique lipid backbone-water intermolecular interaction in egg sphingomyelin (SM) that is not observed in lipids with glycerol backbones like phosphatidylcholines. This backbone-water interaction in SM is probed when a mixing period allowing magnetization exchange between different ¹H environments via the nuclear Overhauser effect (NOE) is included in the NMR pulse sequence. The molecular information provided by these ¹H/³¹P dipolar HETCOR experiments with NOE mixing differ from those previously obtained by conventional NOE spectroscopy and heteronuclear NOE spectroscopy NMR experiments. In addition, two-dimensional ¹H/¹³C INEPT HETCOR experiments with NOE mixing support the ¹H/³¹P dipolar HETCOR results and confirm the presence of a H₂O environment that has nonvanishing dipolar interactions with the SM backbone.     

Structural elucidation of the O-antigenic polysaccharide from Escherichia coli O175

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O175 has been elucidated. Component analysis together with ¹H and ¹³C NMR spectroscopy experiments were used to determine the structure. Inter-residue correlations were determined by ¹H,¹H-NOESY, and ¹H,¹³C-heteronuclear multiple-bond correlation experiments. The PS is composed of pentasaccharide repeating units with the following structure:→2)-α-d-Glcp-(1→4)-α-d-GlcpA-(1→3)-α-d-Manp-(1→2)-α-d-Manp-(1→3)-β-d-GalpNAc-(1→Cross-peaks of low intensity from an α-linked glucopyranosyl residue were present in the ¹H,¹H-TOCSY NMR spectra. The α-d-Glcp residue is suggested to originate from the terminal part of the polysaccharide and consequently the biological repeating unit has a 3-substituted N-acetyl-d-galactosamine residue at its reducing end. The repeating unit of the E. coli O175 O-antigen is similar to those from E. coli O22 and O83, both of which carry an α-d-Glcp-(1→4)-d-GlcpA structural element, thereby explaining the reported cross-reactivities between the strains.     

Structural and serological studies on the O-antigen of Proteus mirabilis O14, a new polysaccharide containing 2-[(R)-1-carboxyethylamino]ethyl phosphate.

An O-specific polysaccharide was obtained by mild acid degradation of Proteus mirabilis O14 lipopolysaccharide (LPS) and found to contain D-galactose, 2-acetamido-2-deoxy-D-glalactose, phosphate, N-(2-hydroxyethyl)-D-alanine (D-AlaEtn), and O-acetyl groups. Studies of the initial and O-deacetylated polysaccharides using one- and two-dimensional 1H- and 13C-NMR spectroscopy, including COSY, TOCSY, NOESY, H-detected 1H,13C heteronuclear multiple-quantum coherence, and heteronuclear multiple-bond correlation experiments, demonstrated the following structure of the repeating unit: [equation: see text] This is the second bacterial polysaccharide reported to contain alpha-D-Galp6PAlaEtn, whereas the first one was the O-antigen of P. mirabilis EU313 taken erroneously as strain PrK 6/57 from the O3 serogroup [Vinogradov, E. V., Kaca, W., Shashkov, A.S., Krajewska-Pietrasik, D., Rozalski, A., Knirel, Y.A. & Kochetkov, N.K. (1990) Eur. J. Biochem., 188, 645-651]. Anti-(P. mirabilis O14) serum cross-reacted with LPS of P. mirabilis EU313 and vice versa in passive hemolysis and ELISA. Absorption of both O-antisera with the heterologous LPS decreased markedly but did not abolish the reaction with the homologous LPS. These and chemical data indicated that both strains have similar but not identical O-antigens. Therefore, we propose that P. mirabilis EU313 should belong to a new subgroup of the O14 serogroup.     

Structure of a glycerol teichoic acid-like O-specific polysaccharide of Proteus vulgaris O12.

A phosphorylated O-specific polysaccharide (O-antigen) was obtained by mild acid degradation of Proteus vulgaris O12 lipopolysaccharide and studied by sugar and methylation analyses, 1H-, 13C- and 31P-NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, H-detected 1H, 13C and 1H, 31P heteronuclear multiple-quantum coherence experiments. It was found that the polysaccharide consists of pentasaccharide repeating units connected via a glycerol phosphate group, and has the following structure: where FucNAc is 2-acetamido-2,6-dideoxygalactose and the degree of O-acetylation at position 4 of GalNAc is approximately 25%. Immunochemical studies with P. vulgaris O12 O-antiserum suggested that the lipopolysaccharide studied shares common epitopes with the lipopolysaccharide core of P. vulgaris O8 and with the O-antigens of P. penneri strains 8 and 63.     

Structure of the O-polysaccharide and serological studies of the lipopolysaccharide of Proteus penneri 60 classified into a new Proteus serogroup O70

An alkali-treated lipopolysaccharide of Proteus penneri strain 60 was studied by chemical analyses and 1H, 13C and 31P NMR spectroscopy, and the following structure of the linear pentasaccharide-phosphate repeating unit of the O-polysaccharide was established: 6)-alpha-D-Galp-(1-->3)-alpha-L-FucpNAc-(1-->3)-alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4NAc-(1-->6)-alpha-D-Glcp-1-P-(O--> Rabbit polyclonal O-antiserum against P. penneri 60 reacted with both core and O-polysaccharide moieties of the homologous LPS. Based on the unique O-polysaccharide structure and serological data, we propose to classify P. penneri 60 into a new, separate Proteus serogroup O70. A weak cross-reactivity of P. penneri 60 O-antiserum with the lipopolysaccharide of Proteus vulgaris O8, O15 and O19 was observed and discussed in view of the chemical structures of the O-polysaccharides.     

Structural studies of the O-antigenic polysaccharide from Escherichia coli O177

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O177 has been determined. Component analysis together with ¹H and ¹³C NMR spectroscopy experiments was used to determine the structure. Inter-residue correlations were determined by ¹H,¹³C-heteronuclear multiple-bond correlation and ¹H,¹H-NOESY experiments. PS is composed of tetrasaccharide repeating units with the following structure:→2)-α-l-Rhap-(1→3)-α-l-FucpNAc-(1→3)-α-l-FucpNAc-(1→3)-β-d-GlcpNAc-(1→An α-l-Rhap residue is suggested to be present at the terminal part of the polysaccharide, which on average is composed of ∼20 repeating units, since the ¹H and ¹³C chemical shifts of an α-linked rhamnopyranosyl group could be assigned by a combination of 2D NMR spectra. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. The repeating unit of the E. coli O177 O-antigen shares the →3)-α-l-FucpNAc-(1→3)-β-d-GlcpNAc-(1→ structural element with the O-antigen from E. coli O15 and this identity may then explain the reported cross-reactivity between the strains.