19013-36-6Relevant academic research and scientific papers
Structure of the unusual Sinorhizobium fredii HH103 lipopolysaccharide and its role in symbiosis
Di Lorenzo, Flaviana,Speciale, Immacolata,Silipo, Alba,Alías-Villegas, Cynthia,Acosta-Jurado, Sebastián,Rodríguez-Carvajal, Miguel-ángel,Dardanelli, Marta S.,Palmigiano, Angelo,Garozzo, Domenico,Ruiz-Sainz, José-Enrique,Molinaro, Antonio,Vinardell, José-María
, p. 10969 - 10987 (2021/01/07)
Rhizobia are soil bacteria that form important symbiotic associations with legumes, and rhizobial surface polysaccharides, such as K-antigen polysaccharide (KPS) and lipopolysaccharide (LPS), might be important for symbiosis. Previously, we obtained a mutant of Sinorhizobium fredii HH103, rkpA, that does not produce KPS, a homopolysaccharide of a pseudaminic acid derivative, but whose LPS electrophoretic profile was indistinguishable from that of the WT strain. We also previously demonstrated that the HH103 rkpLMNOPQ operon is responsible for 5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybutyramido)-L-glyc-ero-L-manno-nonulosonic acid [Pse5NAc7(3OHBu)] production and is involved in HH103 KPS and LPS biosynthesis and that an HH103 rkpM mutant cannot produce KPS and displays an altered LPS structure. Here, we analyzed the LPS structure of HH103 rkpA, focusing on the carbohydrate portion, and found that it contains a highly heterogeneous lipid A and a peculiar core oligosaccharide composed of an unusually high number of hexuronic acids containing b-configured Pse5NAc7(3OHBu). This pseudaminic acid derivative, in its a-configuration, was the only structural component of the S. fredii HH103 KPS and, to the best of our knowledge, has never been reported from any other rhizobial LPS. We also show that Pse5NAc7(3OHBu) is the complete or partial epitope for a mAb, NB6-228.22, that can recognize the HH103 LPS, but not those of most of the S. fredii strains tested here. We also show that the LPS from HH103 rkpM is identical to that of HH103 rkpA but devoid of any Pse5NAc7(3OHBu) residues. Notably, this rkpM mutant was severely impaired in symbiosis with its host, Macroptilium atropurpureum.
Gas chromatography/electron-capture negative ion mass spectrometry for the quantitative determination of 2- and 3-hydroxy fatty acids in bovine milk fat
Jenske, Ramona,Vetter, Walter
experimental part, p. 5500 - 5505 (2010/03/25)
2- and 3-hydroxy fatty acids (2- and 3-OH-FAs) are bioactive substances reported in sphingolipids and bacteria. Little is known of their occurrence in food. For this reason, a method suitable for the determination of OH-FAs at trace levels in bovine milk fat was developed. OH-FAs (and conventional fatty acids in samples) were converted into methyl esters and the hydroxyl group was derivatized with pentafluorobenzoyl (PFBO) chloride to give PFBO-O-FA methyl esters. These derivatives with strong electron affinity were determined by gas chromatography interfaced to mass spectrometry using electron-capture negative ion in the selected ion monitoring mode (GC/ECNI-MS-SIM). This method proved to be highly sensitive and selective for PFBO-O-FA methyl esters. For the analysis of samples, two internal standards were used. For this purpose, 9,10-dideutero-2-OH-18:0 methyl ester (ISTD-1) from 2-OH-18:1(9c) methyl ester as well as the ethyl ester of 3-PFBO-O-12:0 (ISTD-2) was synthesized. ISTD-1 served as a recovery standard whereas ISTD-2 was used for GC/MS measurements. The whole-sample cleanup consisted of accelerated solvent extraction of dry bovine milk, addition of ISTD 1, saponification, conversion of fatty acids into methyl esters by use of boron trifluoride, separation of the methyl esters of OH-FAs from nonsubstituted FAs on activated silica, conversion of OH-FAs methyl esters into PFBO-O-FA methyl esters, addition of ISTD-2, and measurement by GC/ECNI-MS-SIM. By this method, ten OH-FAs were quantified in bovine milk fat with high precision in the range from 0.02 ± 0.00 to 4.49 ± 0.29 mg/100 g of milk fat.
