2,3-Diamino-2,3-dideoxyuronic and 5,7-diamino-3,5,7,9-tetradeoxynonulosonic acids: new components of bacterial polysaccharides

Abstract The discovery of the derivatives of 2,3-diamino-2,3-dideoxyuronic acids and 5,7-diamino-3,5,7,9-tetradeoxynonulosonic acids in bacterial polysaccharides enlarges the list of natural monosaccharides. Many of the new sugars carry unusual N -substituents, such as formyl, ( R )-3-hydroxybutyryl, and acetimidoyl groups. They are most characteristics of O-chains of Pseudomonas aeruginosa lipopolysaccharides, composed almost exclusively of amino sugars or amphoteric amino sugars; the latter seem to play an important role as serological determinants. The identification of these sugars and the structural determination of the O-specific polysaccharides provide the chemical basis for the classification of P. aeruginosa strains. Some of the new monosaccharides enter also the polysaccharides from some other bacteria.     

The O-acetylation patterns in the O-antigens of Hafnia alvei strains PCM 1200 and 1203, serologically closely related to PCM 1205

Serological tests revealed immunochemical similarities between the lipopolysaccharides of Hafnia alvei strains PCM 1200, 1203 and 1205. Immunoblotting and ELISA showed cross-reactions between the strains. NMR spectroscopy showed that the O-deacetylated O-specific polysaccharides isolated from lipopolysaccharides of H. alvei strains PCM 1200 and 1203 possessed the same composition and sequence as the O-deacetylated O-specific polysaccharide of H. alvei strain PCM 1205, that is a glycerol teichoic-acid-like polymer with a repeating unit of the following structure: [carbohydrate structure: see text] NMR spectroscopic studies of the polysaccharides concluded that O-3 of the side chain beta-D-GlcpNAc is partially O-acetylated (50-80%) in both investigated strains. In strain PCM 1203 an additional O-acetyl group (50-80%) is linked to O-6 of the chain -->3)-alpha-D-GlcpNAc-(1--> residue. The structural features of the isolated O-specific polysaccharides were also the same as those of the O-specific polysaccharides on the bacterial cells directly observed by the HR-MAS NMR technique.     

Lipopolysaccharides of Salmonella T mutants

The composition of lipopolysaccharides derived from various Salmonella T forms was studied. All T1-form lipopolysaccharides examined contained 14 to 22% each of both d-galactose and pentose in addition to 4 to 9% each of ketodeoxyoctonic acid, heptose, d-glucosamine, and d-glucose. The pentose was identified as d-ribose. The T2-form lipopolysaccharide examined did not contain a significant amount of pentose, nor more than the usual amounts of d-galactose. Periodate oxidation of T1 (lipo) polysaccharides followed by NaBH(4) reduction revealed that ribose was almost quantitatively protected, galactose was destroyed, and threitol and mannose were newly formed. The latter two products probably originated from 4-linked galactose and heptose, respectively. Ribose and galactose were found in specific precipitates of T1 lipopolysaccharide with anti-T1 antiserum but were not found in specific precipitates of alkali-treated T1 lipopolysaccharide and of Freeman degraded polysaccharide with anti-T1 serum Ribose and galactose are present in these degraded preparations in the form of nondialyzable polymers. The T1-form mutant lipopolysaccharides lacked the O-specific sugars constituting the side-chains in the wild-type antigens. They did not produce the soluble O-specific haptenic polysaccharide known to be accumulated in RI strains. With these properties, T1 lipopolysaccharides resemble RII lipopolysaccharides. Like RII degraded polysaccharides, T1-degraded polysaccharides also contained glucosamine. Furthermore, strong cross-reactions were found to exist between T1 and RII lipopolysaccharides in both hemagglutination inhibition assays and in precipitation tests. It is proposed that T1 lipopolysaccharides represent RII lipopolysaccharides to which polymers consisting of ribose and galactose are attached.     

Saccharides of seven Pseudomonas aeruginosa immunotypes

Abstract The structures of O-specific polysaccharides obtained by mild acid degredation of lipopolysaccharides (LPS) from seven Pseudomonas aeruginosa Fisher's immunotypes have been studied. The polysaccharides consist mainly of monoamino and diamino sugars, frequently also carrying acidic functions. Some of the sugars were detected in nature for the O-specific polysaccharides of the immunotypes 2, 3, 4, 5 and 6 are identical to those of the polysaccharides of the 011; 0(2a)2c; 01; 010a, 10b and 07a, 7d Lányi-Bergan serological subgroups respectively, whereas no analogues have been found for the immunotypes 1 and 7. Some cross-reactions between the LPS of different immunotypes were observed in passive haemagglutination tests; the results of inhibition of passive haemagglutination and agar gel immunoprecipitation point, however, to a specificity of the LPS. Many of the LPS of the seven Pseudomonas aeruginosa immunotypes manifest rather a high cross-protective activity in active immunization tests in mice. The nature of the cross-protective activity of the LPS is discussed.     

Antigenic polysaccharides of bacteria. 18. Structure of O-specific polysaccharide chains of Pseudomonas aeruginosa 05 (Lányi) lipopolysaccharide

Polysaccharide chains of P. aeruginosa O5a, b, c, O5a, b, d and O5a, d (Lányi classification) lipopolysaccharides contain D-xylose, N-acetyl-D-fucosamine (FucNAc) and a derivative of 5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid, PseN2) carrying acetyl or (R)-3-hydroxybutyryl (Hb) and formyl (Fm) groups as N-acyl substituents. Degradation of the lipopolysaccharides with dilute acetic acid caused depolymerisation of the polysaccharide chains as a result of cleavage of glycosidic linkage of pseudaminic acid to give trisaccharides representing chemical repeating units of the polysaccharides. Basing on analysis of the trisaccharides using 1H and 13C NMR spectroscopy and mass-spectrometry, the following structures of the polysaccharide chains were established: (Formula: see text). O5a, d polysaccharide is identical to P. aeruginosa immunotype 6 O-specific polysaccharide.     

Saccharides of seven Pseudomonas aeruginosa immunotypes

The structures of O-specific polysaccharides obtained by mild acid degradation of lipopolysaccharides (LPS) from seven Pseudomonas aeruginosa Fisher's immunotypes have been studied. The polysaccharides consist mainly of monoamino and diamino sugars, frequently also carrying acidic functions. Some of the sugars were detected in nature for the first time in these organisms. The structures of the O-specific polysaccharides of the immunotypes 2, 3, 4, 5 and 6 are identical to those of the polysaccharides of the 011; 0(2a)2c; 01; 010a, 10b and 07a, 7d Lányi-Bergan serological subgroups respectively, whereas no analogues have been found for the immunotypes 1 and 7. Some cross-reactions between the LPS of different immunotypes were observed in passive haemagglutination tests; the results of inhibition of passive haemagglutination and agar gel immunoprecipitation point, however, to a specificity of the LPS. Many of the LPS of the seven Pseudomonas aeruginosa immunotypes manifest rather a high cross-protective activity in active immunization tests in mice. The nature of the cross-protective activity of the LPS is discussed.     

Structural studies on lipopolysaccharides of serologically non-typable strains of Helicobacter pylori, AF1 and 007, expressing Lewis antigenic determinants.

In contrast to other Helicobacter pylori strains, which have serologically detectable Lewis(x)+ (Le(x)) and Lewis(y)++ (++Le(y)) antigenic determinants in the O-specific polysaccharide chains of the lipopolysaccharides, H. pylori AF1 and 007 were non-typable with anti-Le(x) and anti-Le(y) antibodies. The carbohydrate portions of the lipopolysaccharides were liberated by mild acid hydrolysis and subsequently studied by sugar and methylation analyses, 1H-NMR spectroscopy and electrospray ionization-mass spectrometry. Compared with each other, and with lipopolysaccharides of strains studied previously, the lipopolysaccharides of both AF1 and 007 showed similarities, but also differences, in the structures of the core region and O-specific polysaccharide chains. The O-specific polysaccharide chains of both strains consisted of a short or long polyfucosylated poly-N-acetyl-beta-lactosamine chains, which were distinguished from those of other strains by a high degree of fucosylation producing a polymeric Le(x)chain terminating with Le(x) or Le(y) units:[sequence: see text] where n = 0 or 1 in strain AF1 and 0 in strain 007, m = 0-2, 6-7 in strain AF1 and m = 0-2, 6-7 or approximately 40 in strain 007, the medium-size species being predominant. Therefore, compared with other strains, the lack of reactivity of lipopolysaccharide of H. pylori AF1 and 007 with anti-Le(x) and anti-Le(y) may reflect the presence of a polymeric Le(x) chain and has important implications for serological and pathogenesis studies. As the substitution pattern of a D-glycero-D-manno-heptose residue in the outer core varied in the two strains, and an extended DD-heptan chain was present in some lipopolysaccharide species but not in others, this region was less conservative than the inner core region. The inner core L-glycero-D-manno-heptose region of both strains carried a 2-aminoethyl phosphate group, rather than a phosphate group, as reported previously for other H. pylori strains.     

Antigenic polysaccharides of bacteria. 21. The structure of O-specific polysaccharide chains and serological specificity of lipopolysaccharides from 7 Pseudomonas aeruginosa immunotypes

On mild acid degradation on lipopolysaccharides of seven Pseudomonas aeruginosa immunotypes, O-specific polysaccharides were obtained and their structures established. A peculiar feature of the polysaccharides is the presence of various, mostly acidic, mono- and diaminosugars, many of which have not previously been found in nature. The absence of serological cross-reactions (inhibition of passive haemagglutination) between lipopolysaccharides of seven immunotypes correlates with the absence of any common oligosaccharide fragments in their O-specific chains. The data obtained revealed structural and serological interrelations between O-antigens of seven immunotypes and P. aeruginosa O-serotypes, and showed that immunotypes 1 and 7 should be included into the serological classification scheme as individual O-serotypes.     

Antigenic polysaccharides of bacteria. 26. Structure of O-specific polysaccharides from Pseudomonas cerasi 467 and Pseudomonas syringae pv. syringae strains 218 and P-55 belonging to serogroups II and III

Serologically active O-specific polysaccharides were obtained on mild acid hydrolysis of lipopolysaccharides from Pseudomonas cerasi 467 and Pseudomonas syringae pv. syringae strains 218 and P-55. On the basis of 1H- and 13C-NMR analysis, it was concluded that the P. cerasi polysaccharide has the following structure: ----3)-alpha-D-Rhap-(1----3)-alpha-D-Rhap-(1----2)-alpha-D-+ ++Rhap-(1---- which is identical to that of O-specific polysaccharide from P. syringae pv. morsprunorum C28 (Smith A. R. W. et al. Eur. J. Biochem., 1985, V. 149, No 1, p. 73-78). The polysaccharides from P. syringae pv. syringae strains possess the same backbone but differ by the presence of D-fucose as monosaccharide branches. Methylation and 1H- and 13C-NMR analysis revealed the following structure of these polysaccharides: (Formula: see text). The degree of substitution of the backbone trisaccharide units by the fucofuranose residues is about 35% for the strain 218 and about 85% for the strain P-55.     

Structure of the glycerol phosphate-containing O-specific polysaccharide and serological studies on the lipopolysaccharides of Citrobacter werkmanii PCM 1548 and PCM 1549 (serogroup O14)

The O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide of Citrobacter werkmanii PCM 1548 and PCM 1549 (serogroup O14) and found to contain D-glucose, D-glucosamine and glycerol-1-phosphate in molar ratios 2 : 2 : 1. Based on methylation analysis and 1H and 13C nuclear magnetic resonance spectroscopy data, it was established that the O-specific polysaccharides from both strains have the identical branched tetrasaccharide repeating unit with 3,6-disubstituted GlcNAc, followed by 2,4-disubstituted Glc residues carrying at the branching points lateral residues of Glc and GlcNAc at positions 6 and 2, respectively. Glycerol-1-phosphate is linked to position 6 of the chain Glc. All sugars have a beta configuration, except for the side-chain Glc, which is alpha. Serological studies revealed a close relatedness of the lipopolysaccharides of C. werkmanii PCM 1548 and PCM 1549, both belonging to serogroup O14. In immunoblotting, anti-C. werkmanii PCM 1548 serum showed no cross-reactivity with the O-polysaccharide bands of the lipopolysaccharides of Citrobacter youngae PCM 1550 (serogroup O16) and Hafnia alvei PCM 1207, also containing a lateral glycerol phosphate residue.     

Structure determination of the O-specific polysaccharides from Citrobacter O4- and O27-lipopolysaccharides by methylation analysis and one- and two-dimensional 1H-NMR spectroscopy

Using sugar and methylation analyses, and one- and two-dimensional 1H-NMR spectroscopy at 500 MHz it was established that poly-beta-1,2-4-deoxy-D-arabinohexopyranose occurs as O-specific chains of lipopolysaccharides in Citrobacter serotypes O4, O27 and O36. Strong serological cross-reactivity between these serotypes is in full agreement with the chemical identity of their O-specific polysaccharides.     

Sudies of O-specific polysaccharide chains of Pseudomonas solanacearum lipopolysaccharides consisting of structually different repeating units.

The structure of the O-antigenic ploysaccharide chains of lipopolysaccharides of a number of Pseudomonas solanacearum strains were elucidated mainly with the help of methylation analysis and ¹³CNMR spectroscopy, including a computer-assisted ¹³CNMR-based analysis. Six structually distinct but related polysaccharides were identified. They have a backbone which is built up of three -rhammoyranosyl resides and one 2-acetamido-2-deoxy- -glucopyranosyl residue, and is unsubtituted or substituted with a residue of -xylopyranose or -rhamnopyranose as a monosaccharide side chain. The lipopolysaccharides of most of the strains contain polysaccharide chains consisting of at least two structually different types of repeating units. Three of the polysaccharides are common to more than one strain.     

Somatic antigens of Pseudomonas aeruginosa. The structure of the O-specific polysaccharide chains of lipopolysaccharides of P. aeruginosa serogroup O4 (Lányi) and related serotype O6 (Habs) and immunotype 1 (Fisher)

Acidic O-specific polysaccharides were isolated on mild acidic degradation of lipopolysaccharides of Pseudomonas aeruginosa serotypes O4a,b, O4a,c, O4a,d (Lányi classification) and serologically related to them serotype O6 (Habs classification) and immunotype 1 (Fisher classification). The polysaccharides had identical monosaccharide composition and were built up of L-rhamnose, 2-acetamido-2,6-dideoxy-D-glucose,2-formamido-2-deoxy-D-galacturonic acid and 2-acetamido-2-deoxy-D-galactouronamide residues. The latter two derivatives of D-galactosaminuronic acid were found in nature for the first time. All the polysaccharides, but Lányi serotype O4a,c, contained O-acetyl groups. The polysaccharides were readily de-O-acetylated with aqueous triethylamine and de-N-formylated with dilute hydrochloric acid. De-N-formylated polysaccharide of serotype O4a,c was selectively cleaved with nitrous acid upon 2-amino-2-deoxygalacturonic acid residues to form a tetrasaccharide with a 2,5-anhydrotaluronic acid residue on the reducing end. The tetrasaccharide represented a modified repeating unit of the polysaccharide. Solvolysis of all intact polysaccharides with hydrogen fluoride selectively split the glycosidic linkages of 6-deoxy sugars to give the same trisaccharide, including both derivatives of galactosaminuronic acid and having 2-acetamido-2,6-dideoxyglucose on the reducing end. Structural investigation of the oligosaccharides obtained together with methylation analysis and 13C nuclear magnetic resonance data revealed the following structures of the O-specific polysaccharides: (Formula: see text) An independent confirmation of the structures of the repeating units was obtained as the result of full interpretation of the 13C nuclear magnetic resonance spectra of the intact and modified polymers. Spectral data analysis revealed a number of regularities in the effects of glycosidation connecting their values with the anomeric and absolute configuration of pyranose residues. The data on the structures of the O-specific polysaccharides indicated that each of the five P. aeruginosa strains under study should be considered as an individual O-serotype within one O-serogroup.     

Somatic antigens of Pseudomonas aeruginosa. The structure of O-specific polysaccharide chains of the lipopolysaccharides from P. aeruginosa O5 (Lányi) and immunotype 6 (Fisher)

Lipopolysaccharides were isolated from dry bacterial cells of Pseudomonas aeruginosa O5a,b,c, O5a,b,d, O5a,d (Lányi classification) and immunotype 6 (Fisher classification) by the Westphal procedure. Their polysaccharide chains were built up of trisaccharide repeating units containing D-xylose, 2-acetamido-2,6-dideoxy-D-galactose and a new sialic acid-like sugar, the di-N-acyl derivative of 5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic (pseudaminic) acid. Formyl, acetyl and (R)-3-hydroxybutyryl groups were identified as the N-acyl substituents of the last monosaccharide; O5a,b,c and O5a,b,d lipopolysaccharides also contained O-acetyl groups. The glycosidic linkage of pseudaminic acid was extremely labile towards acids, and mild acid degradation of the lipopolysaccharides produced, instead of the O-specific polysaccharides, their trisaccharide fragments with pseudaminic acid at the reducing terminus. Similar degradation of immunotype 6 lipopolysaccharides, followed by oxidation with sodium metaperiodate, resulted in a disaccharide fragment due to destruction of xylose. In contrast the glycosidic linkage of pseudaminic acid proved to be more stable towards treatment with hydrogen fluoride than those of xylose and N-acetylfucosamine. As a result, solvolysis of immunotype 6 lipopolysaccharide with hydrogen fluoride in methanol gave methyl glycosides of a disaccharide and a trisaccharide with pseudaminic acid at the non-reducing terminus. Mild acid hydrolysis of these oligosides afforded free 5-N-acetyl-7-N-formylpseudaminic acid, which was identified by the 1H ande 13C nuclear magnetic resonance data, as well as by the mass spectrum of the corresponding fully methylated aldonic acid. As a result of the identification of all oligosaccharides obtained and comparative analysis of the 13C nuclear magnetic resonance spectra of the oligosaccharides and lipopolysaccharides the following structures were established for the repeating units of the polysaccharide chains of the lipopolysaccharides: (Formula: see text) where D-Xyl = D-xylose, D-FucNAc = 2-acetamido-2,6-dideoxy-D-galactose, Pse5N7NFm = 5-amino-3,5,7,9-tetradeoxy-7-formamido-L-glycero-L-manno-nonulosonic+ ++ acid (7-N-formylpseudaminic acid). All the polysaccharides have an identical carbohydrate skeleton and differ from each other by the acyl substituent at N-5 of pseudaminic acid [acetyl or (R)-3-hydroxybutyryl group] or by the presence or absence of the O-acetyl group at position 4 of N-acetylfucosamine. The data obtained account properly for the O specificity of the studied P. aeruginosa strains.     

Atypical R-S dissociation in Azospirillum brasilense

It was found that atypical R-S dissociation in the type strain A. brasilense Sp7 is not accompanied by drastic changes in the carbohydrate moieties of bacterial lipopolysaccharides but is rather due to different contributions of two O-specific polysaccharides (found in both R and S dissociants) to the age-dependent architectonics of the cell surface.     

Somatic antigens of Pseudomonas aeruginosa. The structure of O-specific polysaccharide chains of the lipopolysaccharides from P. aeruginosa II (Sandvik) and V (IM-1, Verder-Evans)

The lipopolysaccharides from two serologically related strains of Pseudomonas aeruginosa II (Sandvik classification) and V (IM-1, Verder-Evans classification) were isolated by the Westphal method and cleaved with 1% acetic acid. The O-specific polysaccharide from Sandvik II lipopolysaccharide involved L-rhamnose, N-acetyl-D-quinovosamine, and N-acetyl-D-galactosaminuronic acid in the ratio 2:1:1, as well as O-acetyl groups. The Verder-Evans V O-specific polysaccharide was found to contain just the same sugars and, in addition, D-glucose, the monosaccharide ratio being 2:1:1:1. On the basis of methylation analysis, Smith degradation, solvolysis with anhydrous hydrogen fluoride and 1H and 13C nuclear magnetic resonance data, it was concluded that the polysaccharides have identical main chains, and there were established the following structures of their repeating units: -4)-alpha-D-GalNAcA-(1-3)-beta-D-QuiNAc-(1-2)-alpha-L-Rha-(1 -3)-alpha-L-Rha-(1- increases 3 60% OAc Sandvik II -4)-alpha-D-GalNAcA-(1-3)-beta-D-QuiNAc-(1-2)-alpha-L-Rha-(1 -3)-alpha-L-Rha-(1- increases 3 alpha-D-Glc Verder-Evans V The data obtained allow us to substantiate the combining of the strains studied into one O-serogroup as two individual O-serotypes.     

Atypical R–S Dissociation in Azospirillum brasilense

It was found that atypical R–S dissociation in the type strain A. brasilense Sp7 is not accompanied by drastic changes in the polysaccharide moieties of bacterial lipopolysaccharides but is rather due to different contributions of two O-specific polysaccharides (found in both R and S dissociants) to the age-dependent architectonics of the cell surface.     

Structure of O-specific side chains of lipopolysaccharides from Yersinia pseudotuberculosis

Lipopolysaccharide prepared from cells of Yersinia (Pasteurella) pseudotuberculosis of serogroups I, II, III, IV, and V is known to contain the 3,6-dideoxyhexose (DDH) paratose, abequose, paratose, tyvelose, and ascarylose in its respective O-specific side chains. Lipopolysaccharides or lipid-free polysaccharides of all of the 10 known serogroups and subgroups were subjected to methylation analysis and determined as alditol acetates by gas-liquid chromatography and mass spectrometry. The results indicated that the O-specific side chains of nine serotypes are composed of oligosaccharide repeating units in the form of four alternative general structures in which a terminal DDH may vary. These structures are DDH [Formula: see text] 6-deoxy-d-manno-heptose [Formula: see text] d-galactose (serogroups IA, IIA, and IVB), DDH [Formula: see text] d-mannose [Formula: see text] l-fucose (serogroups IB and IIB), and two configurations similar to the latter except that the 4-position of l-fucose was either linked to the d-mannose residue (serogroups VA and VB) or to the DDH residue (serogroups III and IVA). In contrast, O-groups in lipopolysaccharide of the newly discovered serogroup VI contained the DDH colitose and 2-acetamido-2-deoxy-d-galactose. Accordingly, all five known types of DDH have now been detected in lipopolysaccharides of Y. pseudotuberculosis. The sugar 6-deoxy-d-manno-heptose, present in O-specific side chains of serogroups IA, IIA, and IVB, has not yet been reported to occur elsewhere in nature.     

The immunochemistry of Salmonella chemotype VI O-antigens. The structure of oligosaccharides from Salmonella group G (o 13,22) lipopolysaccharides

1. Lipopolysaccharides have been isolated from ;smooth' (S) strains of Salmonella friedenau and Salmonella poona by the phenol-water method and purified in the preparative ultracentrifuge. 2. These lipopolysaccharides are serologically indistinguishable and on partial acid hydrolysis the same series of oligosaccharides was obtained in each instance. 3. The results of quantitative micro-analysis, borohydride reduction, periodate oxidation, Morgan-Elson reactions and enzymic hydrolysis with beta-galactosidase on the isolated oligosaccharides indicate that the O-specific side chains of these lipopolysaccharides have a repeating pentasaccharide unit that is beta-d-galactosyl-(1-->3)-N-acetylgalactosaminyl-(1-->3)-N-acetylgalactosaminyl-(1-->4)-l-fucose with a d-glucose residue bound at an undetermined point on this structure. 4. Two oligosaccharides, a glucosyl-galactose and an N-acetylglucosaminylglucose, have also been isolated and these seem to be identical with oligosaccharides obtained from ;rough' (R) Salmonella lipopolysaccharides. These findings are in accordance with the view that Salmonella S-lipopolysaccharides have a core that consists of R-lipopolysaccharide.     

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.