M. J. Osborn

Inhibition of dihydrofolic reductase by aminopterin and amethopterin.

Summary The enzyme, dihydrofolic reductase, which catalyzes the TPNH-linked reduction of dihydrofolic acid to tetrahydrofolic acid, is inhibited non-competitively by the folic acid antagonists, Aminopterin and Amethopterin. Values of 1 × 10-9 M and 2.3 × 10-9 M were obtained for the inhibitor constants (KI) of Aminopterin and Amethopterin, respectively.

Lipopolysaccharide of the Gram-Negative Cell Wall Biosynthesis of a complex heteropolysaccharide occurs by successive addition of specific sugar residues

The use of mutants of Salmonella typhimurium in which biosynthesis of specific lipopolysaccharide precursors is blocked has made possible both biosynthetic studies and structural analyses which provide the basis for the structure of the core polysaccharide shown in Fig. 6. The simplest mutant, which is unable to synthesize UDP-glucose, forms only the backbone structure, containing heptose, phosphate, and keto-deoxyoctonate. To this backbone are attached side chains containing glucose, galactose, and N-acetylglucosamine. The resulting core structure is found in the lipopolysaccharide of the rough strain, as well as in that of the GDP-mannose- deficient mutant. In the wild type organism, long O-antigenic chains composed of repeating units containing galactose, mannose, rhamnose, and abequose are linked to the core, perhaps to the N-acetylglucosamine residue, as indicated in Fig. 6. The rough phenotype could presumably arise from mutation either at the level of nucleotide sugar synthesis or at some stage in assembly or attachment of the O-antigenic side chains. The pathways of nucleotide sugar synthesis appear to be normal in most rough strains of S. typhimurium (42), a finding which suggests loss of a lipopolysaccharide transferase reaction in these mutants. The site of the enzymatic defect has not yet been established in these cases, but two distinct genetic types of rough mutants have been detected (18). It is interesting to speculate about the function of the lipopolysaccharide. The lipopolysaccharide can account for as much as 5 percent of the dry weight of the cell, and its synthesis clearly involves major expenditure both of energy and of material. Yet loss of the antigenic side chains, or even of a major part of the core structure, appears to have little or no effect on the ability of the organism to survive under laboratory conditions, since the rough and mutant strains grow as well as the wild type does. However, only the wild types, possessing the complete antigenic side chains, are pathogenic. It is possible that the lipopolysaccharide is an important factor in aiding the bacterium to evade host defense mechanisms, such as phagocytosis. Such a role is well established for the capsular polysaccharides of the pneumococci. No mutants have thus far been detected which lack the backbone or lipid portions of the lipopolysaccharide. It may be that these parts of the lipopolysaccharide play an essential role in the physiology of the organism

Enzymes of phospholipid metabolism: Localization in the cytoplasmic and outer membrane of the cell envelope of Escherichia coli and Salmonella typhimurium

Abstract The phospholipid biosynthetic enzymes of the cell envelope of Escherichia coli and Salmonella typhimurium had highest specific activities in the cytoplasmic membrane fraction and lowest specific activities in the outer membrane fraction obtained by isopycnic sucrose density gradient centrifugation of the total membrane fraction of lysozyme-EDTA spheroplasts. This finding suggests that the site of phospholipid synthesis is the cytoplasmic membrane. Several degradative enzymes of phospholipid metabolism had highest specific activities in the outer membrane fraction and lowest specific activities in the cytoplasmic membrane. These data suggest an intracellular separation of the degradative and biosynthetic enzymes of phospholipid metabolism.

A polymerization-depolymerization model that accurately generates the self-sustained oscillatory system involved in bacterial division site placement

Determination of the proper site for division in Escherichia coli and other bacteria involves a unique spatial oscillatory system in which membrane-associated structures composed of the MinC, MinD and MinE proteins oscillate rapidly between the two cell poles. In vitro evidence indicates that this involves ordered cycles of assembly and disassembly of MinD polymers. We propose a mathematical model to explain this behavior. Unlike previous attempts, the present approach is based on the expected behavior of polymerization-depolymerization systems and incorporates current knowledge of the biochemical properties of MinD and MinE. Simulations based on the model reproduce all of the known topological and temporal characteristics of the in vivo oscillatory system.

BIOSYNTHESIS OF O‐ANTIGEN in SALMONELLA TYPHIMURIUM

Studies of the mechanism of biosynthesis of the lipopolysaccharides of the Enterobacteriaceae have, until recently, been limited to the “core” region of the molecule (FIGURE 1). (For summary see references 1 and 2.) Within the past year, however, several laboratories have reported the biosynthesis of the antigenic side chain in cell-free preparations from species of Salmonella. Zeleznick et ~ 1 . ~ used the cell envelope fraction prepared from a mutant strain of S. typhimurium deficient in the synthesis of GDP-mannose. These workers described the synthesis of a macromolecular product containing galactose, rhamnose, and mannose in the same ratio and sequence as reported for the repeating unit of the authentic 0-antigen of this Partial acid hydrolysates of the enzymic product contained the trisaccharide a-galactosylmannosyl-rhamnose. Abequose, the. remaining component of authentic 0-antigen side chains, occurs as nofireducing monosaccharide branches on the trisaccharide repeating units, and it was concluded that the absence of its precursor (CDP-abequose) from the reaction mixtures did not preclude the formation of the macromolecule. Comparable results were reported by Nikaido and Nikaido,‘ who employed a mutant strain of s. typhimurium deficient in the synthesis of TDPrhamnose. With cell-free preparations of S . anatum, Robbins et ~ 1 . ~ observed the synthesis of a similar macromolecule from the appropriate sugar nucleotides. The 0-antigenic side chain of lipopolysaccharide from the latter organism contains the sugars galactose, mannose, and rhamnose in the same sequence as is found in S. typhimurium but is lacking in abequose. The structure of the core region of the molecule has been established by chemical analysis of the lipopolysaccharide isolated from a variety of rough mutants and by biosynthetic studies utilizing mutants deficient in the biosynthesis of UDP-galactose and/or UDP-glucose. This portion of the lipopolysaccharide molecule does not contain repeating sequences of sugars (FIGURE 1) and the biosynthetic work indicates that it is formed by the stepwise addition of sugars according to the following general equation:

Incorporation of lipid vesicles by Salmonella.

The interest of this laboratory in the interaction of phospholipid vesicles with bacterial cells developed out of studies on the mechanism of assembly of the outer membrane of Salmonella, specifically the mechanism of interrnembrane translocation of lipids in these organisms. During the course of these studies it became desirable to incorporate exogenous lipids into the outer membrane of intact cells. Although incorporatiori of vesicle-derived lipids into the cell membrane of rnycoplasma and eucaryotic cells 2-1 was well established, no information was available as to the potential use of this technique with intact bacteria. Gram-negative bacteria, such as Salmonella, are bounded by a membranous structure, the so-called outer membrane, which overlies the cytoplasmic membrane and the peptidoglycan layer of the wall and is exposed at the surface of the ceK5 The outer membrane should therefore be accessible to interaction with added phospholipid vesicles. The results summarized here show that vesicle-derived lipids are indeed transferred to intact cells by a process which results in bulk incorporation of all components of the vesicle bilayer into the outer membrane. The procedure has been employed to investigate intermembrane translocation of lipids in Salmonella, and may provide a generally useful technique for introduction of exogenous molecules of interest into the membranes of viable cells.