Studies on mode of action of a bacteriocin from Clostridium septicum.

A bacteriocin was found in the supernatant fluid of Clostridium septicum strain Ovinus. Sensitivity to the bacteriocin was confined to other strains of C. septicum and to strains of C. chauvoei; the other Gram-positive and Gram-negative bacteria tested for sensitivity were unaffected by the bacteriocin. The bacteriocin killed sensitive cells rapidly but cell lysis did not appear to be involved. The bacteriocin inhibited protein and RNA synthesis immediately after its addition to sensitive cells; DNA synthesis was inhibited 10 min later.     

Action of heliomycin on protein and mucleic acid synthesis in Staphylococcus aureus]

Heliomycin inhibited synthesis of RNA in Staph. aureua which was clearly shown in the study of the antibiotic effect on RNA synthesis in the lag phase of the culture development: heliomycin markedly lowered the maximum RNA level in the biomass observed in the culture at the beginning of the exponential growth. On further growth of the culture heliomycin induced a significant retardness of the process of the natural decrease in the RNA biomass level resulting in increased content of RNA in the cells growing in the presence of heliomycin as compared to the control culture. Retarded natural decrease in the RNA biomass level in the presence of heliomycin was observed also on the antibiotic addition just at the beginning of the exponential growth, during the period of maximum RNA accumulation in the cells. Heliomycin had no effect on synthesis and biomass levels of DNA. Heliomycin inhibited the protein synthesis and was close to chloramphenicol by the level of inhibition of the summation protein synthesis in the biomass. However, comparison of the effect of the above antibiotics on synthesis of beta-galactosidase, an individual enzyme protein showed that heliomycin was much less active as an inhibitor of protein synthesis in comparison to chloramphenicol.     

Stringent control of ribonucleic acid synthesis in Bacillus subtilis treated with granaticin.

The antibiotic granaticin interferes in Bacillus subtilis with the charging process of tRNALeu causing both the arrest of protein synthesis and bacteriostasis [A. Ogilvie, K. Wiebauer & W. Kersten (1975) Biochem. J. 152, 511-515]. A concomitant inhibition of RNA synthesis is observed. This inhibition was studied with mutant strains of B. subtilis. 2. Granaticin inhibits protein and RNA synthesis in stringently controlled B. subtilis (rel+) to about the same extent. In a relaxed mutant strain (rel-) of B. subtilis, protein synthesis is also inhibited, but the accumulation of RNA continues after the addition of the drug. 3. Chloramphenicol, which is known to abolish the stringent control mechanism, added simultaneously with granaticin, allows the synthesis of RNA to proceed in the stringent strain. 4. Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) accumulate in granaticin-treated stringently controlled B. subtilis but not in the rel- mutant. 5. It is concluded that the inhibition of RNA synthesis granaticin can adequately be explained as a stringent response caused by the interference by the drug with leucyl-tRNA synthetase.     

Mode of Action of Myxin on Escherichia coli

The effect of the new antibiotic, myxin, on the syntheses of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein in Escherichia coli (strains B and 15T(-)) was examined. Within 7 min of the addition of myxin at 5 mug/ml, the synthesis of new bacterial DNA was almost completely inhibited. This was followed by an extensive degradation of the pre-existing DNA to an acid-soluble form. All of the evidence indicated that the primary effect of the antibiotic was on cellular DNA. The synthesis of RNA was completely inhibited after 15 min of exposure to myxin (5 mug/ml), and the synthesis of protein was markedly reduced after 30 min. There was no measurable breakdown of either RNA or protein in the myxin-treated cells. A marked stimulation of (14)C-uracil incorporation was found in the presence of myxin in 15T(-) cells only. This did not result from an increased rate of RNA synthesis but was due to an increase in the proportion of exogenous uracil, relative to endogenous uracil, incorporated into cellular RNA. This probably reflected a partial inhibition of the biosynthesis of uridine monophosphate from orotate. At 4.5 mug of myxin per ml and with 0.8 x 10(8) cells per ml, 50% of the antibiotic was reduced in 15 min from the biologically active oxidized form to the biologically inactive state. Under these conditions, a maximum of 0.6% (27 mumug/ml) of the myxin was retained in the cells.     

Differential effects of cycloheximide on protein and RNA synthesis as a function of dose

The $\text{in}$$\text{vivo}$ dose response of rat liver protein and DNA synthesis to cycloheximide have been determined. Protein synthesis was quite sensitive to relatively low doses of cycloheximide being inhibited by more than 90% with 1.5 mg/kg. Maximal inhibition of 98% was achieved with 5 mg/kg. There was no inhibition of RNA synthesis with this dose of cycloheximide. Larger doses of cycloheximide did lead to quite marked inhibition of RNA synthesis without any change in the already maximally inhibited rate of protein synthesis. This differential effect of cycloheximide on protein and RNA synthesis as a function of dose indicates that the inhibition of RNA synthesis caused by the antibiotic is not a consequence of the inhibition of protein synthesis but related otherwise to the effects of large doses of cycloheximide.     

Inhibition of protein synthesis by polypeptide antibiotics.. II. In vitro protein synthesis

Ennis, Herbert L. (St. Jude Children's Research Hospital, Memphis, Tenn.). Inhibition of protein synthesis by polypeptide antibiotics. II. In vitro protein synthesis. J. Bacteriol. 90:1109-1119. 1965.-This investigation has shown that the polypeptide antibiotics of the PA 114, vernamycin, and streptogramin complexes are potent inhibitors of the synthetic polynucleotide-stimulated incorporation of amino acids into hot trichloroacetic acid-insoluble peptide. The antibiotics inhibited the transfer of amino acid from aminoacyl-soluble ribonucleic acid (s-RNA) to peptide. The A component of the antibiotic complex was active alone in inhibiting in vitro protein synthesis, whereas the B fraction was totally inactive. However, the A component, when in combination with the B component, gave a greater degree of inhibition than that observed with the A fraction alone. On the other hand, the endogenous incorporation of amino acid was much less susceptible to inhibition than the incorporation of the corresponding amino acid in a system stimulated by synthetic polynucleotide. In addition, synthesis of polyphenylalanine stimulated by polyuridylic acid was inhibited to a greater extent when the antibiotics were added before the addition of polyuridylic acid to the reaction mixture than when the antibiotics were added after the polynucleotide had a chance to attach to the ribosomes. However, the antibiotics apparently did not inhibit the binding of C(14)-polyuridylic acid or C(14)-phenylalanyl-s-RNA to ribosomes. The antibiotics did not affect the normal release of nascent protein from ribosomes and did not disturb protein synthesis by causing misreading of the genetic code. The antibiotics bind irreversibly to the ribosome, or destroy the functional identity of the ribosome. The antibiotic action is apparently a result of the competition between antibiotic and messenger RNA for a functional site(s) on the ribosome.     

RELATIONSHIP BETWEEN RNA SYNTHESIS, CELL DIVISION, AND MORPHOLOGY OF MAMMALIAN CELLS : I. Puromycin Aminonucleoside As An Inhibitor of RNA Synthesis and Division in HeLa Cells

Logarithmically growing HeLa cell monolayers were treated with a range of concentrations of puromycin aminonucleoside (AMS). The effects of AMS were studied by the following means: microscope examination of treated cells; enumeration of the cell number using an electronic particle counter; analyses for DNA, RNA, and protein content; incorporation of P³² and H³-thymidine into nucleic acids; and fractionation of nucleic acids by column chromatography. Taking the rate of incorporation of the isotopic precursor as a measure of nucleic acid synthesis, it was found that concentrations of the inhibitor which had a rapid effect on the rate of cell division inhibited the synthesis of all types of nucleic acids and of protein, but depressed ribosomal RNA synthesis most markedly. Lower concentrations of AMS selectively inhibited ribosomal RNA and, to a lesser extent, transfer RNA synthesis. Partial inhibition of ribosomal RNA synthesis with low doses had no effect on the rate of cell division within the period studied (3 generation times). The cell content of RNA returned to normal when the inhibitor was removed.     

Induction of autolysis in Bacillus subtilis by ochratoxin A.

Ochratoxin A (OTA) added during the exponential growth phase at a concentration higher than 12 microgram/ml caused autolysis of Bacillus subtilis. Optical density of cultures decreased, and at higher concentrations the cultures became sterile. Optimum OTA-induced lysis was about pH 5. At concentrations below 10 microgram/ml, protein synthesis was inhibited more strongly than RNA synthesis. Cell wall synthesis was also strongly inhibited. A fraction extracted from the lysates had the property of a lysis inhibitor. The relevance of this fraction in respect to autolysis is discussed.     

The effect of trimethoprim on macromolecular synthesis in Escherichia coli. General effects on ribonucleic acid and protein synthesis

In trimethoprim-inhibited RC(str) strains of Escherichia coli, the expression of the RC control of stable RNA synthesis arose primarily from a decrease in the intracellular concentrations of glycine and methionine, and not from inhibition of the initiation of new protein chains. In non-supplemented cultures, experiments with rifampicin showed that the immediate response to the addition of trimethoprim was a rapid decrease in the rate of initiation of RNA chains. This was followed after a few minutes by a sufficiently large fall in the rate of endogenous synthesis of nucleotide bases to cause a decrease in the rate of RNA chain polymerization. Inhibition of RNA chain initiation was thus overridden by an accumulation of DNA-dependent RNA polymerases upon the cistrons. RC(rel) strains also accumulated polymerases upon the DNA in similar circumstances, but did not suffer the initial effects on chain initiation. If purines were supplied before adding trimethoprim, RC(str) strains polymerized RNA chains at normal rates, but initiation rates were permanently decreased. In either situation, an increased% of the RNA formed was mRNA. However, in RC(rel) strains supplemented with bases, trimethoprim did not affect either the rate of initiation of new chains or their rates of polymerization or the relative rates of synthesis of stable RNA and mRNA. Protein synthesis was also severely inhibited by trimethoprim. Though the addition of glycine and methionine to base-supplemented, trimethoprim-inhibited RC(str) strains did not apparently affect the decreased rate of protein synthesis, RNA accumulation resumed at its normal rate. Thus, the inhibition of protein chain initiation had no effect on the rate of RNA accumulation in either RC(str) or RC(rel) bacteria. The RC control does not express itself through inhibitions of protein synthesis at this level.     

Inhibition of bacterial growth by metal salts. A survey of effects on the synthesis of ribonucleic acid and protein

Inhibition of the growth of Escherichia coli M.R.E. 600 by six different metal salts was accompanied by a greater decrease in the synthesis of RNA than in that of protein. The action of cobalt chloride was exceptional; inhibited cells made an excess of RNA to an extent depending on the concentration of Co(2+), the time of incubation and the concentration of Mg(2+) in the medium. Preferential synthesis of RNA in the presence of cobalt chloride was not confined to E. coli but occurred to various extents in some, but not all, of the other micro-organisms that were tested. Possible reasons for the special effect of Co(2+) are discussed.     

Baciphelacin: a new eukaryotic translation inhibitor

Baciphelacin an antibiotic produced by Bacillus thiaminolyticus was a potent inhibitor of protein synthesis in HeLa cells and other mammalian cell lines. It had no effect on DNA or RNA synthesis. Concentrations of baciphelacin around 10⁻⁷ M inhibited protein synthesis by 50% in intact cells. The antibiotic had no effect on protein synthesis in Saccharomyces cerevisiae or Escherichia coli, but inhibited the protozoan Trypanosoma brucei. In vitro protein synthesis in a rabbit reticulocyte cell-free system was blocked by baciphelacin. However, translation of globin mRNA in a wheat cell-free system was not affected by this antibiotic. Baciphelacin had no activity against a number of cell-free systems used to measure different steps of translation, including binding of substrates to the ribosome, peptide bond formation and polyphenylalanine synthesis. Therefore, it is assumed that it affects the initiation of translation or the charging of tRNA. Finally, the inhibition of protein synthesis by compounds structurally related to baciphelacin was tested and their effects compared to baciphelacin.     

Sulfur-dependent inhibition of protein and RNA synthesis by iron-grown Thiobacillus ferrooxidans

The addition of sulfur to iron-grown Thiobacillus ferrooxidans resulted in a rapid inhibition in the rates of protein synthesis and RNA synthesis. The inhibition of both functions was measured within 15 to 30 min and was maximal between 70 and 90% compared to the iron-grown controls. DNA synthesis, carbon dioxide fixation, and short-term ferrous oxidation rates of the bacteria growing on ferrous ions were not effected by sulfur addition, indicating that the sulfur addition was not perturbing general cellular energy metabolism. The inhibition caused by sulfur mimicked the effect of the RNA synthesis inhibitor, rifampicin, which inhibited both RNA and protein synthesis, but did not correspond with the translational inhibitor, chloramphenicol, which inhibited only protein synthesis in the first hour. Since chloramphenicol pretreatment did not block the sulfur effect, the inhibition of RNA synthesis following sulfur addition was not mediated through protein synthesis.     

Adaptation to cycloheximide of macromolecular synthesis in Tetrahymena

Cycloheximide (CHI) at 10 ng/ml partially inhibited protein synthesis in exponential cultures of Tetrahymena Sp. At 20 ng/ml or greater, inhibition was complete. When protein synthesis was inhibited to any extent, cell division ceased immediately. In all instances where measured, synthesis of RNA and DNA also ceased. After a period of delay, cellular functions reinitiated in the order: (i) protein synthesis, (ii) DNA synthesis and, (iii) RNA synthesis and cell division. The delay in cell division was divided into three phases of: I, zero; II, low; and, III, fully recovered rates of exponential protein synthesis. The length of the three phases increased with increasing concentration of CHI Prior growth of cells for one generation in the presence of 7.5 ng/ml CHI (facilitation) eliminated phase I and slightly decreased phases II and III following subsequent challenge with an inhibitory concentration of CHI. Facilitation for six generations further decreased phases II and III. Protein synthesis and cell division were not inhibited during facilitation In the culture, succinate dehydrogenase activity did not increase during the delay but increased normally at the onset of division. In contrast, NADPH-cytochrome c reductase activity continued to increase for an hour after inhibition of protein synthesis, was constant for a period and did not increase again until an hour after reinitiatoin of cell division and RNA synthesis Inhibition of division of all cells was immediate and reinitiation of synthesis and cell division was non-synchronous.     

Biochemical changes in lysogenic Bacillus stearothermophilus after bacteriophage induction

Welker, N. E. (University of Illinois, Urbana), and L. Leon Campbell. Biochemical changes in lysogenic Bacillus stearothermophilus after bacteriophage induction. J. Bacteriol. 90:1129-1137. 1965.-Cultures of Bacillus stearothermophilus 1503-4R (TP-1) continued to grow at an unaltered rate after induction with mitomycin C (MC). MC-induced cultures exhibited a 2.5-fold increase in cell number before lysis occurred. Prior to lysis, cells were observed to elongate and to contain areas of lesser density. Protein synthesis was slightly inhibited in MC- or ultraviolet light (UV)-induced cultures for a period of 5 to 10 min, and then proceeded at a rate identical to that in the noninduced culture. Ribonucleic acid (RNA) synthesis was not affected by MC induction. UV induction caused RNA synthesis to occur in two stages: in the first stage, the rate of RNA synthesis was one-third that observed in the noninduced culture and lasted for a period of 15 min; the second stage of RNA synthesis then proceeded at a rate identical to that in the noninduced culture. The synthesis of deoxyribonucleic acid (DNA) in an MC- or UV-induced culture occurred in two stages. In the first stage, DNA synthesis in induced cultures occurred at a rate of one-half (MC) and one-third (UV) of that observed in the noninduced culture. The first stage of DNA synthesis in MC- or UV-induced cultures lasted for 25 to 30 min and 15 to 20 min, respectively. In the second stage, the rate of DNA synthesis in MC- or UV-induced cultures occurred at a rate three times that of the noninduced culture. UV induction appeared to have a greater inhibitory effect than MC induction on protein, RNA, and DNA synthesis as well as phage yield. The differential rate (K) of inducible and constitutive alpha-amylase synthesis was inhibited by 75 and 100%, respectively, for a period of 20 min after MC induction. After 20 min, the K values for alpha-amylase synthesis were identical to those obtained in the absence of MC induction. The synthesis of TP-1 phage DNA occurred rapidly and was complete 25 min after MC induction, whereas bacterial DNA was degraded or its rate of synthesis was decreased. During the second stage of DNA synthesis, only bacterial DNA was synthesized, but at a rate greater than that found in the noninduced culture.     

Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Echerichia coli by pseudomonic acid

The mode of action of the antibiotic pseudomonic acid has been studied in Escherichia coli. Pseudomonic acid strongly inhibits protein and RNA synthesis in vivo. The antibiotic had no effect on highly purified DNA-dependent RNA polymerase and showed only a weak inhibitory effect on a poly(U)-directed polyphenylalanine-forming ribosomal preparation. Chloramphenicol reversed inhibition of RNA synthesis in vivo. Pseudomonic acid had little effect on RNA synthesis in a regulatory mutant, E. coli B AS19 RC(rel), whereas protein synthesis was strongly inhibited. In pseudomonic acid-treated cells, increased concentrations of ppGpp, pppGpp and ATP were observed, but the GTP pool size decreased, suggesting that inhibition of RNA synthesis is a consequence of the stringent control mechanism imposed by pseudomonic acid-induced deprivation of an amino acid. Of the 20 common amino acids, only isoleucine reversed the inhibitory effect in vivo. The antibiotic was found to be a powerful inhibitor of isoleucyl-tRNA synthetase both in vivo and in vitro. Of seven other tRNA synthetases assayed, only a weak inhibitory effect on phenylalanyl-tRNA synthetase was observed; this presumably accounted for the weak effect on polyphenylalanine formation in a ribosomal preparation. Pseudomonic acid also significantly de-repressed threonine deaminase and transaminase B activity, but not dihydroxyacid dehydratase (isoleucine-biosynthetic enzymes) by decreasing the supply of aminoacylated tRNA(Ile). Pseudomonic acid is the second naturally occurring inhibitor of bacterial isoleucyl-tRNA synthetase to be discovered, furanomycin being the first.     

Active target cell processes, possibly involving receptor-mediated endocytosis, are critical for expression of cytotoxicity by natural killer cell-derived cytolytic factor

Inhibitors of energy metabolism, 2-deoxyglucose and cyanide were shown to inhibit NKCF-mediated lysis of L929 target cells at the same molar concentrations that effectively inhibited cellular ATP levels and the toxic effects of pseudomonas toxin A. In addition, inhibitors of receptor-mediated endocytosis, cytochalasin B, a microtubule disrupter, and trifluoperazine, an inhibitor of clathrin-coat formation, inhibited NKCF-mediated lysis and expression of pseudomonas toxin activity, but had little effect upon cellular ATP. Lysomotropic agents chloroquine, ammonium chloride, and dansylcadavarine also inhibited both NKCF-mediated lysis and pseudomonas toxin activity. These results are similar to those involving diphtheria toxin and the plant toxins abrin, modeccin, and ricin, whose mode of action involves inhibition of protein synthesis following receptor-mediated endocytosis. However, it was determined that NKCF did not cause a decrease in the rate of protein synthesis up to the time of cell death. These results suggest that active target cell processes (possibly involving receptor-mediated endocytosis of NKCF) must occur for target cell lysis to be completed.