Lipopolysaccharide Core Structures in \u3cem\u3eRhizobium etli\u3c/em\u3e and Mutants Deficient in \u3cem\u3eO\u3c/em\u3e-Antigen

Lipopolysaccharide (LPS) is a major component of the bacterial outer membrane, and for Rhizobium spp. has been shown to play a critical role in the establishment of an effective nitrogen-fixing symbiosis with a legume host. Many genes required for O-chain polysaccharide synthesis are in the lps α region of the CE3 genome; this region may also carry lps genes required for core oligosaccharide synthesis. The LPSs from several strains mutated in the α region were isolated, and their mild acid released oligosaccharides, purified by high performance anion-exchange chromatography, were characterized by electrospray- and fast atom bombardment-mass spectrometry, NMR, and methylation analysis. The LPSs from several mutants contained truncated O-chains, and the core region consisted of a (3-deoxy-D-manno-2-octulosomic acid) (Kdo)-(26)-α-Galp-(16)-[α-GalpA-(14)]-α-Manp-(15)-Kdop (3-deoxy-D-manno-2-octulosomic acid) (Kdo)pentasaccharide and a α-GalpA-(14)-[α-GalpA-(15)]-Kdop trisaccharide. The pentasaccharide was altered in two mutants in that it was missing either the terminal Kdo or the GalA residue. These results indicate that the lps α region, in addition to having the genes for O-chain synthesis, contains genes required for the transfer of these 2 residues to the core region. Also, the results show that an LPS with a complete core but lacking an O-chain polysaccharide is not sufficient for an effective symbiosis

Influence of the supramolecular structure of free lipid A on its biological activity

The three-dimensional supramolecular structures and the states of order of the acyl chains of lipid A from different Gram-negative species were investigated at 40°C, high water content (80–90%), and different [lipid A]/[Mg2+] molar ratios using synchrotron radiation X-ray diffraction and Fourier-transform infrared spectroscopy. Measurements were made on free lipid A from Salmonella minnesota R595, mono- and bi-phosphoryl, as well as those from the non-enterobacterial strains Rhodobacter capsulatus 37 b4, Rhodopseudomonas viridis F, and Rhodocyclus gelatinosus 29/1. Parallel to differences in their chemical primary structure, the structural polymorphisms and states of order at 37°C of the non-enterobacterial lipid A were found to be different from those of enterobacterial lipid A. A clear correlation between the supramolecular structure and previously determined biological activities was found. Lipid A with a strong preference for lamellar structures (Rb. capsulatus and Rp. viridis) are endotoxically inactive and lack cytokine-inducing capacity; the compounds assuming a mixed lamellar/nonlamellar structure (monophosphoryl lipid A from S. minnesota) are of lower toxicity in vivo, but may induce cytokines in vitro; those lipid A with a strong tendency to form non-lamellar inverted structures (lipid A from S. minnesota and Rc. gelatinosus) exhibit full endotoxicity in vitro and in vivo. In contrast, anti-complementary activity is most pronounced for compounds with lamellar and least expressed for those with inverted structures. The states of order at 37°C vary non-systematically, exhibiting the highest values for lipid A of S. minnesota and the lowest for that of Rc. gelatinosus.We propose to extend the term ‘endotoxic conformation’, which is used to describe the conformation of a single lipid A molecule required for optimal triggering of biological effects, to ‘endotoxic supramolecular conformation’ which denotes the particular organization of lipid A aggregates in physiological fluids causing biological activity