Jobst Gmeiner

Interaction of lipopolysaccharide with detergents and its possible role in the detergent resistance of the outer membrane of gram-negative bacterua

In the presence of MgCl2, amounts of detergents which disrupted phospholipid vesicles caused lipopolysaccharide I from Proteus mirabilis to aggregate and form vesicular, membrane-like structures. Vesicle formation with P. mirabilis lipopolysaccharide II containing longer O-polysaccharide chains was extremely poor. Lipopolysaccharides of Salmonella minnesota R mutants (chemotypes Ra, Rc and Re) displayed a growing tendency for vesicle formation with increasing deficiency of the R core polysaccharide. Lipopolysaccharides of chemotypes Rc and Re produced vesicles even in the absence of MgCl2 and detergent. Spherical aggregates consisting of P. mirabilis lipopolysaccharide I MgCl2 and detergent were unable to either entrap or retain [14C]-sucrose, [3H=inulin or [3H]dextran. On the other hand, S. minnesota R mutant lipopolysaccharides of chemotypes Rc and Re could entrap all three saccharides and retain them for at least short periods of time. Leakage of [3H]-inulin out of re-lipopolysaccharide vesicles was greatly retarded by addition of MgCl2 to the vesicle system. Incorporation of P. mirabilis lipopolysaccharide I or S. minnesota Rc lipopolysaccharide into phospholipid vesicles protected these model membranes from disruption by detergent. This suggested a similar protective function of lipopolysaccharide in the outer membrane of enteric bacteria against the action of surfactants occurring in their normal intestinal habitat.

Characterization of minor fragments after digestion of Escherichia coli murein with endo-N,O-diacetylmuramidase from Chalaropsis, and determination of glycan chain length.

The murein (‘peptidoglycan) of Escherichia coli consists of glycan chains composed of alternating N-acetylated glucosamine and muramic acid with trior tetrapeptide side chains attached to the O-lactyl groups of the muramic acid residues. Using hen egg white lysozyme for murein degradation predominantly disaccharide peptide monomers, called C5 and C6, and peptidecrosslinked bis-disaccharide peptide dimers, called C3 and C4, were obtained [ 1,2]. However, small amounts of monomer species exhibiting higher RF-values in chromatographic systems were also observed. In addition, C4 was identified as a ringshaped dimer formed by a transglycosylase reaction during murein degradation with lysozyme [ 1,2]. lated. Digestion of murein with endo-N,@diacetylmuramidase from Chalaropsis, separation of monomers and dimers and the analytical procedures employed were detailed in [3-S].

Covalent linkage of lipoprotein to peptidoglycan is not essential for outer membrane stability inProteus mirabilis

Isolated rigid layers fromProteus mirabilis harvested at different growth phases were degraded by endo-N-acetylmuramidase fromChalaropsis B, and the degradation products were investigated. The results show the complete absence of covalently linked lipoprotein in exponential-phase cultures. Stationary cells, however, possess covalently linked lipoprotein in amounts similar to those found inEscherichia coli orSalmonella typhimurium during all growth phases. The overall peptidoglycan structure did not change during transition from logarithmic to stationary growth. Implications of these findings for the organization of the outer membrane are discussed.

N‐acetylglucosaminyl N‐acetylmuramyl‐dipeptide, a novel murein building block formed during the cell division cycle of Proteus mirabilis

The murein (peptidoglycan) of Gram-negative bacteria consists of glycan chains containing alternatingN-acetylated residues of glucosamine and muramic acid. The muramic acid residues are substituted by tetrapeptide units consisting of Lalanyl-y-D-glutamyl(L)-meso-diaminopimelyl-(L)-D-alanine, part of which form crosslinkages between neighbouring glycan chains [ 11. Proteus mirubilis murein is unique in regard to the O-acetylation of a portion of its N-acetylmuramic acid residues [ 2,3 J . Working with synchronized cells of this organism and labelling the murein with radioactive N-acetyl-D[ 1 -14C]glucosamine in vivo, we detected 2 new uncrosslinked building blocks after enzymatic digestion of the isolated murein with endo-N,O-diacetylmuramidase from C’halaropsis. Here, we present analytical data which identify these new compounds as 0-acetylated and non-O-acetylated N-acetylglucosaminyl-N-acetylmuramyl-dipeptides.

Membranes of the protoplast L-form of Proteus mirabilis

Isolated membranes of the cell wall-less stable protoplast L-form of Proteus mirabilis were characterized by density gradient centrifugation and by assay for their major chemical constituents, proteins, phospholipids and lipopolysaccharide, and for some specific marker enzymes of the cytoplasmic membrane. In most of the analyzed properties the L-form protoplast membrane resembled the bacterial cytoplasmic membrane, with some notable modifications. considerable amounts of lipopolysaccharide, normally an exclusive constituent of the outer membrane, were found. Furthermore, the L-form membranes contained the functions of the reduced nicotinamide adenine dinucleotide oxidase system, of d-lactate dehydrogenase (EC 1.1.1.28) and of succinate dehydrogenase (EC 1.3.99.1) at specific activities comparable to, or in some cases considerably higher than, those present in cytoplasmic membranes of the bacterial form. Of two peptidoglycan DD-carboxypetidase/transpeptidases (EC 3.4.17.8 and EC 2.3.2.10), which are normally present in the cytoplasmic membrane of the bacterial form of P. mirabilis, the membrane of the protoplast L-form contained only one. Electron microscopy of thin sectioned L-form protoplasts showed extensive heterogeneity of membraneous structures. In addition to the single membraneous integument, internal membrane-bounded vesicles and multiple stacks of membranes were present, as the result of unbalanced growth and membrane synthesis in the L-form state.

Murein and lipopolysaccharide biosynthesis in synchronized cells of Escherichia coli K 12 and the effect of penicillin G, mecillinam and nalidixic acid

The incorporation of radioactive N-acetylglucosamine into murein and lipopolysaccharide of synchronized cells of Escherichia coli K 12 was followed over 100 min in the presence of antibiotics. At 20 min intervals cell walls were prepared. Lipopolysaccharide and murein sacculi were isolated and the radioactivity was quantified in both polymers. Labelled, newly synthesized murein was characterized according to murein subunits linked to lipoprotein, and the degree of crosslinkage. Furthermore, murein subunits containing anhydromuramic acid were determined, permitting the calculation of the average glycan chain length. The results indicated that penicillin G at 30 μg/ml stimulated the incorporation of new murein subunits into sacculi followed by a sudden increase in lipopolysaccharide incorporation into the outer membrane. The degree of crosslinkage in murein synthesized in the presence of 30 μg/ml penicillin G was higher than in the control, and almost twice as high as in murein synthesized in the presence of 20 μg/ml nalidixic acid. Both antibiotics inhibited cell division at the concentrations indicated. Murein synthesized in the presence of 2 μg/ml mecillinam also showed higher crosslinkage. However, about twice as much anhydromuramic acid-containing subunits were observed as in the control. At the same time lipopolysaccharide incorporation into the outer membrane was stimulated two- to three-fold.

Molecular organization of the outer membrane of Salmonella typhimurium. Different release of lipopolysaccharide from wild type and lipopolysaccharide mutant cells by EDTA treatment.

Cells of Salmonella typhimurium wild type and of several well defined lipopolysaccharide mutants were treated with EDTA. The percentage release of lipopolysaccharide and phospholipid was determined. The results obtained show that the release of lipopolysaccharide by EDTA declines along with the gradually diminishing chain length of the lipopolysaccharide, althought the total amount of lipopolysaccharide was found to increase at the same time in the respective mutants. Implications of these findings for the organization of the outer membrane are discussed.

Characterization of a new murein-associated lipoprotein in the outer membrane of Proteus mirabilis.

A murein-associated outer membrane protein from Proteus mirabilis has been isolated. Since the protein carries ester- as well as amide-linked fatty acids it can be classified as a second outer membrane lipoprotein. An apparent molecular weight of 15,000 for this protein was determined from amino acid analysis and sodium dodecylsulfate/polyacrylamide gel electrophoresis. The amino acid composition, however, does not show similarities with the amino acid composition of the lipoprotein covalently linked to murein, which has a molecular weight of 7,300 as described previously in Proteus mirabilis.