Michael Artman

Effect of magnesium and spermine on the aggregation of bacterial and mammalian ribosomes.

Abstract A strikingly different behaviour of Escherichia coli and mouse-liver ribonucleoprotein particles towards Mg 2+ and spermine was observed. Electron micrographs of E. coli ribosomes in 10 mM Mg 2+ showed the characteristic features of a mixed ribosomal population consisting of monomers and small clusters of monomers, “polysomes”. Suspensions of mouse-liver ribosomes in media containing 10 mM Mg 2+ , on the other hand, gave rise to the appearance of extremely large ribosomal aggregates consisting of a minimum of 100 ribosomal particles. These ribosomal aggregates were shown to be held by Mg 2+ alone, the presence of messenger RNA being superfluous. The lowest Mg 2+ concentration at which mouse-liver ribosomes formed aggregates was 5 mM. 30-S and 50-S ribosomal sub-units of E. coli associated into the 70-S form at concentration of spermine of 0.4 mM. Increasing the concentration of spermine to 0.8 mM gave rise to the appearance of 80-S and 100-S ribosomal particles. At 1 mM spermine E. coli ribosomes were reversibly and quantitatively precipitated. Precipitation of mouse-liver ribosomes commenced at 0.1 mM spermine. Quantitative precipitation of mouse-liver ribosomes occurred at a concentration of spermine which was optimal for the preservation of bacterial ribonucleoprotein in the 70-S form (0.4 mM spermine).

Studies on streptomycin-dependent bacteria: Uptake of 14C streptomycin by a streptomycin-dependent mutant of Escherichia coli

Uptake of [24C]streptomycin by growing cells and non-proliferating suspensions of a streptomycin-dependent mutant, Sd-4, of Escherichia coli has been studied. The uptake of streptomycin has been shown to be an instantaneous and temperature-independent process. The magnitude of streptomycin uptake has been found to be a function of ionic strength of the medium. Maximal uptake has been observed from salt-free solutions. Increase in ionic strength of the medium brought about a decrease in streptomycin uptake as well as a release of cell-bound streptomycin. Minimum of streptomycin that had to be added to the growth of medium in order to nesure optimal growth of the organism has been found to constitute an approx. 30-fold excess over that actually taken up by the cells. The necessity of adding streptomycin to the growth medium in such a large excess as well as the low streptomycin uptake and release of the cell-bound streptomycin in salt solutions have been explained by a dynamic equilibrium that exists between cell-bound streptomycin and inorganic ions, in all likelihood cations, of the medium i.e. a competition between inorganic ions and streptomycin for common binding sites of the cell. The data indicate that streptomycin is held by the cell of the streptomycin dependent of Escherichia coli through electrostatic interaction of opposite charges, i.e. the positively-charged groups of streptomycin and the negatively-charged groups of cell receptors.

Effect of glucose on the utilization of succinate and the activity of tricarboxylic acid-cycle enzymes in Escherichia coli

Abstract When cultures of Escherichia coli growing exponentially in a glucose-saltys medium were transferred to a similar medium containing succinate as the sole source of carbon, a lag in growth of about 4 h was observed. The rate of O2 uptake by non-proliferating suspensions of glucose-grown cells in the presence of succinate was about 9% of that observed with succinate-grown cells. The rate of O2 uptake by cell-free extracts of glucose-grown cells in the presence of succinate (and other tricaboxylic acid cycle intermediates) and the activity of tricaboxylic acid-cylce enzymes in these extracts were 2–4-fold lower than the rate of O2 uptake and the coresponding enzymatic activities in extracts of succinate-grown cells. The intracellular concentration of “succinate” following incubation with [14C]succinate was 3–5 fold higher in succinate- grown than in glucose-grown cells. It is concluded that the lag in growth observed upon transfer of glucose-grown E. coli to a succinate medium is a combined effect of the repression by glucose of tricarboxylic acid-cycle enzymes and the active transport of succinate into the cell.

Studies on streptomycin-dependent bacteria: Effect of streptomycin on protein synthesis by streptomycin-sensitive, streptomycin-resistant and streptomycin-dependent, mutants of Escherichia coli

Abstract The effect of streptomycin on the synthesis of protein by streptomycin-sensitive, resistant-, and dependent-, mutants of Escherichia coli has been studied. The effect of streptomycin on sensitive cells was examined under conditions which did not affect the viability of the cell. It has been shown that streptomycin preferentially inhibits the induced synthesis of β-galactosidase (β- d -galactoside galactohydrolase,EC 3.2.1.23 ) by sensitive cells in comparison with the synthesis of total protein. Evidence has been presented to support the assumption that the binding of streptomycin to ribosomes of sensitive cells precludes the attachment of messenger RNA. The preferential inhibition of the induced synthesis of β-galactosidase when compared with the synthesis of total protein has been interpreted to mean that, in Escherichia coli , in addition to proteins synthesized through the mediation of labile messenger RNA, there are proteins synthesized through the mediation of relatively stable messenger RNA. On the basis of the results of our experiments on the action of streptomycin on the synthesis of protein by non-proliferating suspensions of sensitive cells which closely parallel those obtained by numerous workers with cell-free systems, it has been concluded that intact cells of Escherichia coli are permeable to streptomycin. Evidence has been presented to show that the magnitude of the uptake of streptomycin by sensitive, and resistant, cells is irrelevant to studies on the mode of action of this drug. Protein synthesis by resistant cells is unaffected by streptomycin. The growth of streptomycin-dependent cells without streptomycin results in a gradual decrease in the rate of protein synthesis per unit of bacterial mass as the latter is diluted by growth. The addition of streptomycin to dependent cells, starved of streptomycin, failed to restore the normal rate of protein synthesis. It has been concluded that streptomycin is required for the synthesis of some component of the protein-synthesizing system of streptomycin-dependent mutants of Escherichia coli and not merely for the proper functioning of the component in question. In the light of these findings the unitary hypothesis of Spotts and Stanier has been discussed.

Studies on streptomycin dependent bacteria: Effect of growth in limiting amounts of streptomycin on respiration and fermentation of a streptomycin dependent mutant of Escherichia coli

Respiration and glycolytic of a streptomycin dependent mutant of Escherichia coli Sd-4, grown with optimal and suboptimal amounts of streptomycin were studied. n nThe glycolytic activity of cells grown in limiting amounts of streptomycin was considerably higher than that of cells grown at optimal concentration of streptomycin. n nOn the other hand, the respiration of cells grown with limiting amount of streptomycin was two to five-fold lower than that of cells grown with optimal amounts of sterptomycin. n nIt was found that the deficient cells lacked the carbon monoxide-sensitive pathway of respiration.

Studies on the fate of parental bacteriophage RNA in the host

Abstract MS2 phage particles incapable of forming plaques are capable of RNA injection. The RNA injected by non-viable phage comprises about 90% of the total phage RNA which penetrates the host cells. This RNA is soon after injection converted into all nucleic acid components of the cell. At the same time about 10% of the parental phage RNA was found in the double stranded form, a value which corresponds to the total amount of RNA injected by viable phage, indicating quantitative conversion of this RNA into replicative form, soon after injection.

Messenger RNA from normal and T4 phage-infected Escherichia coli: Isolation and size distribution

Abstract An apparently non-fragmented messenger RNA free of ribosomal RNA was isolated from normal and phage-infected cells of Escherichia coli. Messenger RNA was purified by the use of our recently developed techniques for the extraction of non-fragmented rapidly labeled RNA and for hybridization and subsequent elution of non-fragmented RNA from DNA · RNA hybrids.