The "missing" typical Rhizobium leguminosarum O antigen is attached to a fatty acylated glycerol in R. leguminosarum bv. trifolii 4S, a strain that also lacks the usual tetrasaccharide "core" component.

Rhizobium leguminosarum bv. trifolii 4S has a lipopolysaccharide O antigen that lacks galactose and many of the typical glycosyl components found in related strains. Here, we show that it also lacks the typical core tetrasaccharide but synthesizes an alternative glycolipid that contains galactose and the typical O-antigen glycosyl components, suggesting that in this strain, the O antigen is transferred to an alternative lipid acceptor.

Common links in the structure and cellular localization of Rhizobium chitolipooligosaccharides and general Rhizobium membrane phospholipid and glycolipid components.

Several common links between the structural chemistry of the chitolipooligosaccharides of Rhizobium and the general rhizobial membrane lipid and lipopolysaccharide chemistry of these bacteria have been uncovered. Aspects of common chemistry include sulfation, methylation, and the position and extent of fatty acyl chain unsaturation. We find that bacteria which are known to synthesize sulfated chitolipooligosaccharides (such as Rhizobium meliloti strains and the broad-host-range Rhizobium species strain NGR234) also have sulfated lipopolysaccharides. Their common origins of sulfation have been demonstrated by using mutants which are known to be impaired in sulfating their chitolipooligosaccharides. In such cases, there is a corresponding diminution or complete lack of sulfation of the lipopolysaccharides. The structural diversity of the fatty acids observed in the chitolipooligosaccharides is also observed in the other membrane lipids. For instance, the doubly unsaturated fatty acids which are known to be predominant components of R. meliloti chitolipooligosaccharides were also found in the usual phospholipids and glycolipids. Also, the known functionalization of the chitolipooligosaccharides of R. sp. NGR234 by O- and N-methylation was also reflected in the lipopolysaccharide of this organism. The common structural features of chitolipooligosaccharides and membrane components are consistent with a substantial degree of biosynthetic overlap and a large degree of cellular, spatial overlap between these molecules. The latter aspect is clearly demonstrated here since we show that the chitolipooligosaccharides are, in fact, normal membrane components of Rhizobium. This increases the importance of understanding the role of the bacterial cell surface chemistry in the Rhizobium/legume symbiosis and developing a comprehensive understanding of the highly integrated membrane lipid and glycolipid chemistry of Rhizobium.

Occurrence of sulfoquinovosyl diacylglycerol in some members of the family Rhizobiaceae.

A radiolabeled component of a membrane extract of Rhizobium meliloti 2011 cells grown in the presence of 35S-labeled sulfate was isolated by silica flash chromatography and purified by high performance liquid chromatography (HPLC). Based on 1- and 2-dimensional nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric analyses, the structure of the compound was determined to be sulfoquinovosyl diacylglycerol (SQDG). NMR analyses indicated substantial heterogeneity in the fatty acid composition and that an important group was the cyclopropyl fatty acids. This first report of the occurrence of SQDG outside of the plant kingdom, photosynthetic bacteria or diatoms deserves special attention as, in this case, the bacterium is one that can fix nitrogen in symbiosis with plants. The origins of the bacterium's ability to synthesize this class of membrane lipids is an important question. Membrane extracts of other strains of the family Rhizobiaceae were screened for the presence of SQDG. The occurrence of SQDG in the symbiotic organisms was confirmed, while no SQDG was detected in either the Agrobacterium tumefaciens or the Escherichia coli strains tested. The current function of these lipids in symbiosis and the commonality of the ability of bacteria that function as plant symbionts to synthesize such molecules are all germane to studies of the Rhizobium/legume symbiosis.

N,N-(2,4-dinitrophenyl)octylamine derivatives for the isolation, purification, and mass spectrometric characterization of oligosaccharides.

A simple, sensitive method for the structural characterization of oligosaccharides by fast atom bombardment-mass spectrometry (FAB-MS) has been designed. Oligosaccharides are labeled with a uv chromophore (which also serves as a charge stabilizing group) and with a hydrophobic alkyl tail. The chromophore, a 2,4-dinitrophenyl group, aids uv detection during HPLC and stabilizes negative ion species formed during analysis by FAB-MS. The hydrophobic tail, provided by an octyl group, enhances the surface activity of the analytes and makes them amenable to separation by reverse-phase chromatography using a C18 bonded phase. This method was applied to the structural analysis of the components of a mixture of starch maltodextrins with a degree of polymerization 1-16, to the analysis of the structure of pure maltohexaose, and to a previously characterized oligosaccharide from a Rhizobium capsular polysaccharide. The method gave a good yield of [M-H]- anions for the derivatized compounds, which in most cases were detectable at a level of about 1 pmol. In the case of maltohexaose, four series of sequence anions corresponding to sequential loss of glycosyl residues from the reducing and nonreducing end by different mechanisms were observed. The mixture of derivatized malto-oligosaccharides could easily be separated by HPLC. Based on the relative proportions of the individual oligomers in the mixture calculated from HPLC analysis, even though the higher oligomers were present in amounts of about 0.1%, they could still be easily detected in mass spectra of the entire mixture.(ABSTRACT TRUNCATED AT 250 WORDS)