The endotoxically active cell wall glycolipid of a highly deficient heptose-less R mutant of Salmonella minnesota, consisting of 2-keto-3-deoxyoctonic acid (KDO) and lipid A, was subjected to hydrazinolysis. Besides fatty acid hydrazides, a water-soluble main fraction was obtained, containing KDO, glucosamine, and ester phosphate and free phosphorylethanolamine. The application of two procedures of partial hydrolysis, followed by paper electrophoretic separation, led to seven oligosaccharides.Their structures were evaluated with the aid of the following analytical methods: further acid degradation, borohydride reduction, ninhydrin degradation, the Morgan-Elson reaction, treatment with phosphomonoesterase and N-acetylglucosaminidases. The results show that the backbone of lipid A contains phosphorylated glucosaminyl-tglucosamine disaccacharide units, probably linked /3-1,6, to which a trisaccharide composed of three KDO rehidues is linked ketosidically to form a pentasaccharide which appears to be a common structure in many Enterobacteriaceae.

Composition of cell wall peptidoglycan was studied in the gram‐negative bacterium Proteus mirabilis and in different wall‐defective growth forms of this organism which survive and multiply in the presence of penicillins. The results show that action of the classical benzylpenicillin does not proceed by a uniform mechanism of inhibition of peptide crosslinkage in peptidoglycan. Peptidoglycan of the unstable spheroplast L‐form of P. mirabilis grown in medium with 120 μg/ml benzylpenicillin contained monomers, and peptide‐crosslinked dimers and trimers of disaccharide‐peptide building blocks in a similar ratio as peptidoglycan of the normal bacterial form. Moreover, peptide side‐chains in peptidoglycan from penicillin‐grown L‐form spheroplasts were predominantly tetrapeptides l‐alanyl‐d‐γ‐glutamyl‐meso‐diaminopimelyl‐d‐alanine. Thus, specific penicillin‐sensitive enzymes of peptidoglycan synthesis, peptidoglycan transpeptidase and dd‐carboxypeptidase, must have remained uninhibited by the antibiotic in the L‐form. L‐form peptidoglycan, however, differed characteristically from peptidoglycan of normal Proteus bacteria by having a much lower content of O‐acetyl groups linked to muramic acid residues of the polymer. P. mirabilis grew actively in the form of osmotically stable spheres in medium with up to 100 μg/ml of the amidino penicillin mecillinam. Neither the peptide crosslinkage nor the O‐acetyl content of peptidoglycan were influenced to any great extent by mecillinam. Only the combined action of benzylpenicillin and mecillinam caused complete inhibition of growth in P. mirabilis, concomitant with deformation of cells to very large spheres. It is hypothesized that benzylpenicillin and mecillinam each inhibit different parts of a multiple, isofunctional enzyme system whose total inhibition requires the presence of both antibiotics.

Outer membrane proteins extracted from isolated cell walls of Proteus mirabilis were able to combine with cell wall phospholipids in a model membrane system. The presence of outer membrane proteins in vesicular model membranes mediated the release of previously entrapped [14C]sucrose while [3H]inulin was retained. Incorporation of lipopolysaccharide from the same cell walls was not required for the formation of such selectively permeable membranes.

Four distinct Proteus mirabilis strains were extracted by the phenol/water procedure. After ultracentrifugation of the dialyzed water phase, the pelleted lipopolysaccharide was purified and analyzed. The sugar composition of this lipopolysaccharide fraction I was similar for all four strains, containing only small amounts of strain-specific constituents. A second lipopolysaccharide fraction was isolated from the supernatant above (termed L1 fraction) after removal of nucleic acids. DEAE-cellulose chromatography indicated that this material is not a polysaccharide but rather a water-soluble lipopolysaccharide containing strain-specific constituents such as uronic acids, amino acids, amino sugars, neutral sugars, ethanolamine and phosphate, depending on the strain from which lipopolysaccharide I1 was isolated.Lipopolysaccharides are main constituents of the outer membrane of gramnegative bacteria. A large body of knowledge has been accumulated during the last decade concerning the chemical structure, biosynthesis and biological function of lipopolysaccharide from the Escherichia coli-Salmonella group (for review see [ 1,2]).The lipopolysaccharide from Proteus mirabilis has not been characterized very well. Kotelko and coworkers [3] reported the isolation of two different polysaccharides by acidic extraction procedures. They also showed that in a number of Proteus mirabilis strains uronic acids are constituents of the lipopolysaccharide rather than of a capsular or K-like substance [4,5]. Dmitriev et al.[6] extracted the polysaccharides from various Proteus strains with hot acetic acid and classified these strains as smooth or rough strains according to the elution pattern of the extracted polysaccharides on a Sephadex G50 column.In this paper a convenient procedure is reported for the isolation and separation of two different lipopolysaccharide fractions from each of four distinct Proteus mirabilis strains. Whereas one of these fractions resembles the lipopolysaccharide extracted from smooth strains of Salmonella, the other resembles the lipopolysaccharide extracted from rough strains of Salmonella. The smooth type lipopolysaccharide from Proteus mirabilis contains either uronic acids or phos- phate esters as 0 side chain constituents, in addition to neutral sugars and amino sugars or amino acids. MATERIALS AND METHODS Organisms and Growth ConditionsStrains D52, VI and 19 were obtained from Dr H. H. Martin (this laboratory); strain 19 Q was obtained from Dr G. Schmidt (Max-Planck-Institut fur Immunbiologie, Freiburg i. Br., Germany) and had originally been received from Dr F. Qrskov (Statens Serum Institute, Kobenhavn, Denmark). Bacteria were cultivated in complex medium as described previously [7]. Some batches were kindly grown by Dr S. Schlecht (Max-Planck-Institut fur Immunbiologie, Freiburg i. Br., Germany) [8]. Isolation of L@opolysaccharideWhole cells (20-30 g dry weight) were extracted by the hot phenollwater procedure of Westphal and Jann [9]. The water phase was dialyzed extensively against water, concen...

The immunoglobulin M (IgM) and the IgG1, IgG2ab, and IgG3 subclasses of plaque-forming cells (PFC) specific for lipopolysaccharide (LPS) were measured after immunization of mice with LPS alone and compared with the responses to LPS in combination with nonbacterial proteins and with bacterial membrane phospholipid vesicles or two major outer membrane proteins from Proteus mirabilis. The relative numbers of IgG PFC belonging to the IgG1, IgG2, or IgG3 subclasses induced by immunization with LPS alone depended upon the type of LPS administered. Phospholipids and the proteins effected characteristic alterations in not only the strength but also the subclass of the IgG responses to LPS. The results suggest that the hydrophobic-hydrophilic nature or state of aggregation of the preparations plays a role in the induction of IgG1 and IgG2 subclasses of PFC specific for LPS. Complex formation with LPS and adjuvant was apparently necessary to obtain these effects.