The content of 4-amino-4-deoxy-L-arabinopyranose (L-Arap4N) and the phosphate substitution pattern of the LPS of various strains from Salmonella minnesota, Yersinia enterocolitica and Proteus mirabilis was determined by GC/MS, HPLC and 31P-NMR. These data allowed us to examine the possible role of these components for the polymyxin B-binding capacity of LPS and for the minimal inhibiting concentration (MIC) and the minimal bactericidal concentration (MBC) of polymyxins B and E towards the respective R-mutants. Contrary to other investigated Re-, Rd- and Rc-mutants of S. minnesota, strain R595 (Re-mutant) showed about a 90% substitution of the ester-linked phosphate-group with L-Arap4N, whereas the L-Arap4N content of the other S. minnesota strains amounted to 17-25%. Neither the binding capacity of LPS to polymyxin B, determined by a bioassay, nor the MIC- and MBC-values of the R-mutants were significantly affected by this alteration. Similar results were obtained after using the temperature-dependent changes in the L-Arap4N-content and phosphate substitution pattern of Y. enterocolitica 75R. In order to explore the relevant polymyxin B binding site, lipid A samples with or without substitution of their ester-linked phosphate group were prepared and subjected to the polymyxin-binding assay. The results obtained so far indicated that the inner core bound L-Arap4N, detected in all resistant strains investigated, may play a decisive role in the decreased binding of polymyxin B, responsible for the bacterial resistance towards polymyxin(s).

The inner-core region of the lipopolysaccharide of an UDPGalNAc-4-epimerase-deficient mutant of Yersinia enterocolitica 0 : 3, designated as Ye75R, was investigated using methylation analysis, 1 D-"C-NMR and 2D-"C-NMR and 'H-NMR, as well as 31P-NMR, fast-atom-bombardment mass spectrometry (FAB MS) and FAB M S N S in positive and negative modes. The isolated core heptasaccharide (0s) was composed of 2 units D-glucose, 3 units LD-heptose and 1 unit each of DD-heptose and 3-deoxy-~-manno-octulosonic acid. Methylation analysis indicated that 0s was highly branched with terminal location of the two glucoses and the DD-heptose unit, which was partially (to about 40%) phosphorylated at C7. These combined studies allowed us to formulate the structure of the inner core region as shown in Scheme 1.The substitution of the 7-position of the terminally linked DD-heptose unit by phosphate could be recognized by MS characterization of permethylated ~~-heptose-7-phosphate (alditol acetate) and the extent of the substitution by the ratio of the two well separated 'H signals of DD-heptose in 500-MHz 'H-NMR. Negative FAB MS of 0s also indicated the presence of smaller amounts of two hexasaccharides, differing from 0s in lacking either one terminal unit of D-glucose or of the tenninal DD-heptose, and additionally of a pentasaccharide lacking two heptosyl units, namely the terminal DD-heptose and and the subterminal LD-heptose. The presence of the smaller oligosaccharides in the 0s fraction was also recognized by the methylation analysis.Structural studies on the R-core region of a number of enterobacterial lipopolysaccharides (LPS) and of LPS of some phylogenetically rather remote bacterial species have been carried out in the past [l, 21. The core region, together with the lipid A component, are the two structural regions which are present in almost all Gram-negative bacteria and, as such, are of interest especially with regard to their immunoreactive properties [l]

The double bond of dodecenoic acid in the endotoxin-antagonistically acting lipid A of Rhodobacter capsulatus, strains 37b4 and St Louis, was found to have cis-configuration. The position of the double bond was ω7. The mono-unsaturated fatty acids of lipid A from a number of additionally investigated strains of various species of the α-, β-, and γ-subgroups of Proteobacteria (Agrobacterium spp., Azospirillum spp., Rhizobium meliloti, Rhodobacter sphaeroides, Rhodomicrobium vannielii, Rhodopseudomonas blastica, Rhodospirillum salinarum, Sphaerotilus natans, Thiobacillus spp. and Yersinia enterocolitica) have also cis -configuration with the double bond in the ω7-position (one exception), suggesting the anaerobic pathway of biosynthesis to be common for most of them.