C. Fred Fox

The effect of membrane lipid unsaturation on glycoside transport

Abstract The effect of temperature upon the rates of β-galactoside and β-glucoside transport has been studied in an unsaturated fatty acid auxotroph of E. coli K12 grown on oleic acid or linoleic acid supplemented media. Arrhenius plots of the data for both transport systems are biphasic with changes in slope extrapolating to approximately 13° and 7°C for cells grown with oleate and linoleate supplementation respectively. The slopes of the curves are, however, similar for both fatty acids above and below the transition points. These data suggest that the membrane may exist in two physical states, one above and the other below a transition point determined by the composition of the lipid phase.

Biogenesis of microbial transport systems: Evidence for coupled incorporation of newly synthesized lipids and proteins into membrane

Abstract The biogenesis of the independent β-galactoside and β-glueoside transport systems of Escherichia coli K12 has been studied using an unsaturated fatty acid auxotroph. The response of transport rate to temperature was determined for cells grown with different fatty acid supplements. A change in the slope of an Arrhenius plot for transport rate was obtained at transition temperatures unique for each of the five fatty acid supplements tested. Both of the transport systems studied here had identical transition temperatures when the cells were grown with the same fatty acid supplement, indicating that the transport temperature characteristics are determined primarily by the properties of the lipid phase of the membrane. The specification of the transport temperature profile by membrane lipid composition was exploited in the study of transport system biogenesis. When the auxotroph was grown, for example, in a medium supplemented with oleic acid and then shifted to growth in a medium supplemented with linoleic acid for a brief period of induction of the transport system, the effect of temperature on transport was characteristic of cells grown entirely in medium supplemented with linoleic acid. In contrast, when cells induced for transport in medium supplemented with oleic acid were subsequently shifted to medium supplemented with linoleic acid for up to one generation of growth, the transport temperature profile remained characteristic of cells grown entirely with the first fatty acid supplement. These and the accompanying data clearly indicate a preferential association of newly synthesized transport proteins with newly synthesized lipids.

Evidence for stable attachment of DNA to membrane at the replication origin of Escherichia coli.

The formation of pulse labelled DNA which remains firmly attached to membrane after further growth in unlabelled medium has been studied at different periods of the cell cycle using synchronized cultures of Escherichia coli. The maximal labelling of this species of membrane bound DNA occurs near the time of cell division. This provides evidence for association of DNA at the replication origin with the cell membrane.

Synthesis and properties of a complex of polyriboguanylic acid and polyribocytidylic acid

RNA polymerase from Micrococcus lysodeikticus has been used to synthesize rG : rC† from rC and GTP. The rG: rC product appears to consist of one continuous rC strand and many short pieces of rG comprising the other strand. rG:rC is completely resistant to pancreatic RNase at 37°C. Attempts at thermal dissociation of the complex were unsuccessful. Dissociation can be achieved in alkali, for which the p K a is about 12·5 at 25°C. Alkaline dissociation is essentially reversible, but the complex formed by neutralization is distinguishable from the rG:rC synthesized initially. The density of rG:rC in cesium sulfate is 1·685.

The β-glucoside system of Escherichia coli II. Kinetic evidence for a phosphoryl-enzyme II intermediate

Abstract The β-glucoside specific enzyme II (enzyme II bgl ) of the phosphoenolpyruvate dependent phosphotransferase system of Escherichia coli exhibits properties indicative of a “Ping-Pong” kinetic mechanism.

Membrane assembly in Escherichia coli II. Segregation of preformed and newly formed membrane proteins into cells and minicells

Abstract Cells of a minicell producing strain of Escherichia coli were freed of minicells and innoculated into fresh medium for further growth. The protein composition of the minicell membranes formed during periods of further growth reflected that of the total cellular membrane proteins, rather than being representative of the membrane proteins formed during the period of production of the minicells. These data, when viewed together with published observations on minicell morphogenesis, do not support models of membrane assembly where the membrane protein matrix grows preferentially either at the poles or at the equitorial perimeter of this organism with conservation of structure of preformed and newly formed membrane.

Membrane assembly in Escherichia coli I. Segregation of preformed and newly formed membrane into daughter cells

Abstract Density labeling of the lipid phase of membranes has been used to test the possibilities that the cytoplasmic membrane of Escherichia coli is assembled at one or two fixed foci, either at the equitorial perimeter or at the poles of the organism. Synchronized cultures of E. coli were switched from light to heavy medium, and samples were removed after either one or two cell division cycles of growth in heavy medium and processed by equilibrium density gradient centrifugation. No separation of definitively light or heavy membrane bands was observed indicating that preformed and newly formed membranes are not assembled at one or two fixed foci and then segregated with conservation of structure.