Some O-polysaccharides (O-antigens) of the lipopolysaccharides of bacteria of the genus Cronobacter contain 3-deoxy- d -manno-oct-2-ulosonic acid. O-Polysaccharides of Cronobacter turicensis G3874 and Cronobacter muytjensii G3886 containing this unit were found to be structurally related to each other and to those of Cronobacter dublinensis HPB 3169 and Cronobacter sakazakii G2706 (serotype O5) studied earlier. The O-polysaccharides differ in the N -acyl group on a 3-amino-3,6-dideoxy- d -glucose residue [( R )-3-hydroxybutanoyl vs. acetyl] or in the presence or absence of the O-acetyl group on an l -rhamnose residue only. Structures of the new O-antigens were established by chemical methods and by 1D and 2D 1 H and 13 C NMR spectroscopy applied to polysaccharides and pentasaccharides obtained by mild alkaline O-deacylation and mild acid hydrolysis of the isolated lipopolysaccharides, respectively.

The K5 capsular polysaccharide (CPS) was isolated from the bacterium Acinetobacter baumannii (A. baumannii) SDF and studied by 1D and 2D 1H and 13C NMR spectroscopy before and after O-deacetylation and partial acid hydrolysis. The CPS was found to be composed of branched tetrasaccharide repeats (K units) containing 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic acid (Leg5Ac7Ac). The KL5 capsule biosynthesis gene cluster at the K locus is consistent with the composition and structure of the CPS. KL5 is almost identical to the KL7 gene ulosonic acid (Leg5Ac7Ac A. baumannii LUH5533 that has been characterized earlier, and differs only in the gene for Wzy polymerase that is responsible for the formation of the linkage between the K units. The K5 CPS from strain SDF and the K7 CPS from strain LUH5533 have the same K-unit structure but a different linkage between the K units, which is formed by distinct Wzy polymerases. As opposite to Leg5Ac7Ac in the K7 CPS, this monosaccharide is O-acetylated at position 4 in the K5 CPS. No acetyltransferase for this modification of the K5 CPS is present in the KL5 gene cluster, and hence it is encoded by a gene located elsewhere in the genome.

The structure of О-polysaccharide (О-antigen) of Escherichia coli O95 was established using the analysis of deacylated lipopolysaccharide of E. coli O95 by 2D 1H and 13С NMR spectroscopy. It was found to consist of disaccharide repeating units containing one of each 3-substituted residues of d-fucose and d-threo-pent-2-ulose (xylulose).

O-Polysaccharides (O-antigens) of a number of genetically related Escherichia coli O-serogroups (O17, O44, O73, O77, and O106) and Salmonella enterica O:6,14 possess an identical main chain composed of d-GlcNAc and d-Man residues and differ from each other by the absence or presence of glucose side chains at various positions. Using two-dimensional NMR spectroscopy, we established the structure of the O-polysaccharide of E. coli O106 having two glucose side chains in a hexasaccharide repeating unit.

Carbohydrate Structure Database (CSDB) is a regularly updated database containing structural, taxonomic, bibliographic, NMR spectroscopic, and other information on carbohydrates and their derivatives obtained from prokaryotes, plants, and fungi. CSDB claims for full coverage and high data quality. It serves as a platform for various search strategies, tools for NMR spectrum and structure prediction, and instruments for statistical analysis. CSDB is freely available via the Internet at http://csdb.glycoscience.ru.

Undecyl phosphate derivatives with new fluorescent labels, 11-[(2-pyridyl)amino]undecyl phosphate and 11-[(9-anthracenylcarbonyl)amino]undecyl phosphate, were synthesized. These compounds were shown to be acceptor substrates of the galactosyl phosphate residue in the enzymatic reaction catalyzed by galactosyl phosphotransferase from Salmonella anatum or Salmonella newport membrane preparations.

11-[(2-Pyridyl)amino]undecyl phosphate and 11-[(9-anthracenylcarbonyl)amino]undecyl phosphate were chemically synthesized. The abiliy of these new fluorescent derivatives of undecyl phosphate to serve as acceptor substrate of galactosyl phosphate residue in the enzymic reaction catalyzed by galactosylphosphotransferase from Salmonella anatum or Salmonella newport membrane preparation was demonstrated.

Quaternization of triethylamine, 4-dimethylaminopyridine, and N -methylimidazole with moraprenyl bromide gives the corresponding quaternary ammonium salts.

AIMS:To study the question whether acidic exopolysaccharide (EPS) modification, e.g. pyruvylation, plays any role in the development of Rhizobium leguminosarum/Pisum sativum symbiosis.METHOD AND RESULTS:The amino acid sequence deduced from the pssM gene, localized within the pss (polysaccharide synthesis) gene locus, was shown to be homologous to several known and putative ketal pyruvate transferases, including ExoV from Sinorhizobium meliloti and GumL from Xanthomonas campestris. Rh. l. bv. viciae strain VF39 carrying a Km-cassette insertion into the pssM gene was obtained by the gene replacement technique. Knock-out of pssM led to the absence of the pyruvic acid ketal group at the subterminal glucose in the repeating unit of EPS as it was shown by (13)C and (1)H nuclear magnetic resonance (NMR) analysis. Complementation in trans restored the EPS modification in the pssM mutant. Disruption of the pssM gene resulted also in the formation of aberrant non-nitrogen-fixing nodules on peas. Ultrastructural studies of mutant nodules revealed normal nodule invasion and release of bacteria into the plant cell cytoplasm, but further differentiation of bacteroids was impaired, and the existing symbiosomes underwent lysis.CONCLUSION:PssM encodes ketal pyruvate transferase involved in the modification of the Rh. l. bv. viciae EPS. The absence of subterminal glucose pyruvylation in the EPS repeating units negatively influences (directly or indirectly) the formation of the nitrogen-fixing symbiosis with peas.SIGNIFICANCE AND IMPACT OF THE STUDY:Our finding that the absence of modification even at the single position of EPS is likely to be crucial for establishment of nitrogen-fixing symbiosis argues in favour of the idea concerning their specific signalling role in this process.

An efficient method for the synthesis of moraprenyl bromide by the reaction of moraprenol (WT3C6—8—OH), the polyprenol from the mulberry tree Morus alba leaves, with bromo-trimethylsilane has been elaborated.

Symbiotic nitrogen-fixing bacteria Rhizobium leguminosarum bv. viciae VF39 secrete an acidic heteropolysaccharide, the biosynthesis of which involves the stage of polyprenyl diphosphate octasaccharide formation with its carbohydrate fragment corresponding to the repeating polymer unit. The amino acid analysis of the product of the pssA gene, we have earlier identified, showed its homology to bacterial polyisoprenyl phosphate hexose 1-phosphate transferases catalyzing the formation of phosphodiester bonds between polyprenyl phosphates and hexose 1-phosphates, whose donors are nucleotide sugars. The immunoblotting demonstrated that Rhizobium cells synthesize a protein with a molecular mass of 25 kDa, which implies the translation of the open reading frame occurring from the second initiating codon followed by the protein processing. It was shown that PssA is an integral membrane-bound protein involved in glucose 1-phosphate transfer from UDP-glucose to polyprenyl phosphate to form polyprenyl diphosphate glucose. These results suggest that the pssA gene encodes UDP-glucose:polyprenyl phosphate-glucosyl phosphotransferase.

Симбиотические азотфиксирующие бактерии Rhizobium leguminosarum bv. viciae VF39 секретируют кислый гетерополисахарид, биосинтез которого включает стадию образования полипренилдифосфатоктасахарида, углеводный фрагмент которого соответствует повторяющемуся звену полимера. Анализ аминокислотной последовательности продукта идентифицированного нами ранее гена pssA показал его гомологию с последовательностями бактериальных полиизопренилфосфат-гексозо-1-фосфат-трансфераз, катализирующих образование фосфодиэфирной связи между полипренилфосфатом и гексозо-1-фосфатами, донорами которых являются нуклеотидсахара. Методом иммуноблотинга показано, что в клетках ризобий синтезируется белок с молекулярной массой 25 кДа, что предполагает трансляцию открытой рамки считывания гена pssA со второго инициирующего кодона с последующей модификацией белка. Показано, что PssA является интегральным мембранным белком, участвующим в переносе глюкозо-1-фосфата с UDP-глюкозы на полипренилфосфат с образованием полипренилдифосфатглюкозы. На основании этих данных сделан вывод о том, что ген pssA кодирует UDP-глюкоза : полипренилфосфат–глюкозилфосфотрансферазу.

The conversion of uridine diphosphate N-acetyl-D-glucosamine into uridine diphosphate N-acetyl-L-fucosamine was demonstrated with enzymes from cytoplasmic fraction of Salmonella arizonae O:59 cells in the presence of NAD+ (NADP+) and NADPH. The reaction product was identified by ion-pair, reverse-phase HPLC with the use of synthetic nucleoside diphosphate sugar standards under conditions specially developed for separation of uridine diphosphate 2acetamido-2,6-dideoxyhexoses. L-Fucose dehydrogenase from porcine liver was shown to be applicable for determination of N-acetyl-L-fucosamine, this enzyme being used to confirm L-configuration of the amino sugar residue in the sugar nucleotide formed.

Phosphates of all-Z- and 3-demethyl-tri-trans, di-cis-hexaprenols have been prepared and studied as substrates for enzymes of the Salmonella anatum O-specific polysaccharide biosynthesis. Methyl group in alpha-isoprenic unit proved to be essential for the enzyme-substrate interaction, whereas the presence of E-isoprenic units near the omega-end of the polyprenol is not significant.

A partial purified polymerase from S. anatum was used for the synthesis of polysaccharide [-6) [14C]Man(beta 1-4)Rha(alpha 1-3)Gal(alpha 1-]n and its analogues containing D-glucose residue instead of D-galactose or D-mannose. Structures of these polymers were confirmed by methylation analysis and radioimmunochemical tests.

Chemical conversion of trisaccharide d-Man beta 1----4-L-Rha alpha 1----3-D-[6-C-3H]Gal into its moraprenyl pyrophosphate derivative is described. Treatment of the latter with the cell envelope preparation from S. anatum results in the formation of polysaccharide with the alpha 1----6 linkage between the trisaccharide units.