Structural polymorphisms of rough mutant lipopolysaccharides Rd to Ra from Salmonella minnesota

The structural polymorphisms of rough mutant lipopolysaccharides (LPS) Rd, Rc, Rb, and Ra from Salmonella minnesota (strains R4, R7, Rz, R5, R345, and R60, respectively) were investigated as a function of temperature, water content, and Mg2+ concentration. The gel to liquid crystalline (beta<==>alpha) phase transition temperature (Tc) and the state of order within each phase were measured by Fourier transform infrared spectroscopy. The amount of bound water was determined by differential scanning calorimetry and the three-dimensional structures in each phase state were characterized by synchrotron radiation X-ray diffraction. The results indicate an extremely complex dependence of the structural behavior of LPS on ambient conditions. The beta<==>alpha acyl chain melting temperatures at high water contents (95-97%), Tc = 31 to 32 degrees C for LPS Rd, 33 to 35 degrees C for LPS Rc to Rb, and 36 degrees C for LPS Ra, increase with decreasing water content and in the presence of Mg2+ cations with a concomitant broadening of the transition range. Below 30 to 50% water content, no distinct phase transitions can be observed. These effects are most pronounced for LPS with the shortest sugar chains. Below 50% water content, only lamellar structures can be observed in the temperature range 5 to 80 degrees C, independent of the Mg2+ concentration. Above 50% water concentration, for large [LPS]:[Mg2+] molar ratios the predominant structure above Tc is nonlamellar; for smaller [LPS]:[Mg2+] molar ratios a superposition of lamellar and nonlamellar structures is found. For all LPS Rd to Rb at low Mg2+ concentrations, the phase transition is connected with a change in the three-dimensional structure from lamellar or mixed lamellar/nonlamellar to a pure nonlamellar, probably cubic structure. The tendency to form non-lamellar structures decreases with the length of the core oligosaccharide. At an equimolar ratio of [LPS] and [Mg2+] a multibilayered organization is observed. Some of the nonlamellar structures are cubic phases with periodicities between 12 and 16 nm. The molecular dimensions of the single endotoxin molecules in the absence and the presence of external water are estimated from the different lamellar periodicities, including those of free lipid A and deep rough mutant LPS Re. These observations are discussed with respect to the biological importance of LPS as a potent inducer of biological effects in mammals.