RESUMEN
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.
Asunto(s)
Antracenos/química , Colorantes Fluorescentes/química , Organofosfatos/química , Piridinas/química , Transferasas/metabolismo , Antracenos/síntesis química , Antracenos/metabolismo , Membrana Celular/enzimología , Colorantes Fluorescentes/síntesis química , Organofosfatos/síntesis química , Organofosfatos/metabolismo , Piridinas/síntesis química , Piridinas/metabolismo , Salmonella/enzimología , Transferasas/químicaRESUMEN
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.
Asunto(s)
Aciltransferasas/genética , Pisum sativum/microbiología , Rhizobium leguminosarum/fisiología , Simbiosis , Secuencia de Carbohidratos , Genes Bacterianos , Datos de Secuencia Molecular , Mutagénesis Insercional , Fijación del Nitrógeno/genética , Pisum sativum/ultraestructura , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/metabolismo , Rhizobium leguminosarum/enzimología , Rhizobium leguminosarum/genética , Simbiosis/genéticaRESUMEN
Symbiotic nitrogen-fixing bacteria Rhizobium leguminosarum by. 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.
Asunto(s)
Proteínas Bacterianas/genética , Glicosiltransferasas/genética , Proteínas de la Membrana/genética , Polisacáridos Bacterianos/biosíntesis , Rhizobium leguminosarum/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/análisis , Western Blotting , Clonación Molecular , Escherichia coli/genética , Glicosiltransferasas/análisis , Proteínas de la Membrana/análisis , Datos de Secuencia Molecular , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/genética , Rhizobium leguminosarum/genética , Alineación de SecuenciaRESUMEN
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 2-acetamido-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.
Asunto(s)
Salmonella arizonae/metabolismo , Uridina Difosfato N-Acetilglucosamina/metabolismo , Azúcares de Uridina Difosfato/biosíntesis , Animales , Deshidrogenasas de Carbohidratos/metabolismo , Cromatografía Líquida de Alta Presión , Cromatografía en Papel , Citoplasma/enzimología , Electroforesis en Papel , Salmonella arizonae/enzimología , PorcinosRESUMEN
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.
Asunto(s)
Antígenos Bacterianos/biosíntesis , Lipopolisacáridos/biosíntesis , Salmonella/metabolismo , Terpenos/química , Antígenos O , Fosforilación , Salmonella/enzimología , Estereoisomerismo , Especificidad por SustratoRESUMEN
A series of polyprenyl phosphates with modified structure of polyprenyl residue was prepared through phosphorylation of polyprenyl trichloroacetimidates with phosphoric acid. Interaction of polyprenols with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile was found to represent a very efficient, simple and general method for the synthesis of polyprenyl phosphates. A procedure was developed for smooth conversion of polyprenyl pyrophosphates into the monophosphates through hydrolysis in the presence of 4-dimethylaminopyridine. The polyprenyl phosphates prepared were studied as substrates for the enzymes of Salmonella anatum O-specific polysaccharide biosynthesis. Correct stereochemistry of alpha- and beta-isoprenic units was found to be essential for substrate efficiency. At the more remote positions of the hydrocarbon chain just the presence of isoprenic units of any configuration seems necessary. Some changes in position of the phosphate group may be permissible without significant loss of substrate properties.
Asunto(s)
Antígenos O , Azúcares de Poliisoprenil Fosfato/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Salmonella/metabolismo , Fosfatos de Poliisoprenilo/síntesis química , Estereoisomerismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Antígenos Bacterianos , Polisacáridos Bacterianos/síntesis química , Salmonella/inmunología , Inmunoquímica , Antígenos O , Polímeros , Polisacáridos Bacterianos/inmunologíaRESUMEN
Polyprenyl phosphates of different structure were prepared and their ability to serve as sugar acceptors in the biosynthesis of O-specific polysaccharides of Salmonella anatum was investigated. It was demonstrated that C30-C80-polyprenyl phosphates with unsaturated alpha-isoprene unit were as active as natural acceptor (undecaprenyl phosphate) in this enzymic system. C15- and C100-polyprenyl phosphates of this series were less effective in O-antigen repeating unit formation. Citronellyl and dolichyl phosphates (derivatives of C10- and C105-polyprenols, respectively, with saturated alpha-isoprene unit) were poor substrates. For polymerization of repeating units, the polyprenol chain-length is of utmost importance: its shortening results in a marked drop in the efficiency of respective compounds as substrates.
Asunto(s)
Antígenos Bacterianos , Galactosiltransferasas , Azúcares de Poliisoprenil Fosfato/síntesis química , Polisacáridos Bacterianos/biosíntesis , Salmonella/inmunología , Antígenos O , Fosfatos de Poliisoprenilo/síntesis química , Polímeros , Especificidad por SustratoRESUMEN
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.