The effect of rifampicin on the developmental phases of germinating spores of Clostridum sp., MSp+.

The effect of rifampicin on the developmental phases of germinating spores of Clostridium botulinum, MSp+, has been studied. At sublethal concentrations of rifampicin (0.05 ng/ml) the time periods required for outgrowth and vegetative growth was significantly prolonged because of the inhibition of RNA and protein synthesis. However, rifampicin had essentially no effect on DNA synthesis or on subsequent spore formation. Chemical analyses showed that the amount of protein present in vegetative cells of the rifampicin-treated cultures was twice as great as in the untreated cultures but the total protein content of endospores was the same in both cases. It was revealed in ultrastructural studies of rifampicin (0.1 ng/ml) treated cultures, examined after 22 h, that septum formation and normal cell division of the emerging cell was blocked and a few cells showed constriction which produced one normal and one protoplast-like daughter cell.     

Sensitivity of DNA-repair-deficient strains of Escherichia coli to rifampicin killing

We have analyzed the role of RNA polymerase in DNA repair using the antibiotic rifampicin which binds specifically to the beta subunit of the enzyme. Several DNA-repair-deficient strains such as recA, uvr, and polA, and their isogenic parents were used for this study. All repair-deficient strains were found to be hypersensitive to rifampicin killing. Compared to the isogenic parent strains, recA strains are about 50 times more sensitive and the polA strain is about 100 times more sensitive to rifampicin killing. UvrA and uvrB strains are slightly more sensitive to rifampicin than the wild-type strains. The hypersensitivity of repair-deficient strains to rifampicin killing is totally abolished by the introduction of rifampicin-resistant mutations into these strains. We have examined the effect of rifampicin on RNA and protein synthesis in repair-deficient and -proficient strains. RNA and protein synthesis were found to be inhibited by rifampicin to the same extent among all the strains tested. The results also show that the resumption of DNA synthesis was significantly disrupted in DNA-repair-deficient strains following drug removal. Taken together these results suggest that RNA polymerase plays an essential role in DNA metabolism and such function may be replaced by polA and recA gene products and to a lesser extend by uvrA and uvrB gene products.     

Rifampicin Inhibition of Ribonucleic Acid and Protein Synthesis in Normal and Ethylenediaminetetraacetic Acid-Treated Escherichia coli

The kinetics of ribonucleic acid (RNA) and protein synthesis in rifampicin-inhibited normal and ethylenediaminetetraacetic acid (EDTA)-treated Escherichia coli was measured. Approximately 200-fold higher external concentrations of rifampicin were needed to produce a level of inhibition in normal cells comparable to that observed in EDTA-treated cells. The rates of RNA and protein synthesis in both kinds of cells decreased exponentially, after an initial lag phase, at all rifampicin concentrations tested. The lag phase was longer and the final exponential slope less for protein synthesis than for RNA synthesis at a given rifampicin concentration. Below certain rifampicin concentrations, both the lag phase and the subsequent exponential decrease in the rates of RNA and protein synthesis were found to be rifampicin concentration dependent. At greater concentrations only the time of the lag phase was decreased by higher rifampicin concentrations, whereas the slope of the exponential decrease in the rates of RNA and protein synthesis was unaffected. In all cases, the exponential decrease continued to at least a 99.8% inhibition of the original rate of synthesis. These in vivo results are consistent with the mode of rifampicin action determined from in vitro studies; rifampicin prevents initiations of RNA polymerase on deoxyribonucleic acid, but not its propagation, by binding the enzyme essentially irreversibly. The results also indicate the size distribution of messenger RNA molecules in E. coli under our conditions.     

Inhibition of lipid synthesis in Bacillus amyloliquefaciens by inhibitors of protein synthesis

Inhibitors of protein synthesis reduce the level of lipid production in Bacillus amyloliquefaciens, both in growing cells and in non-dividing washed-cell suspensions. This effect is primarily on phospholipids rather than acetone-soluble lipids. Although the cells contain five major phospholipids the washed-cell supernatants contain only one phospholipid species whose accumulation is sensitive to chloramphenicol but insensitive to rifampicin.     

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.     

In vitro DNA and RNA synthesis by human platelets.

In vitro incorporation of [Me-3H] thymidine and [5-3H] uridine into human platelets was demonstrated. Thymidine incorporation was inhibited by three specific inhibitors of DNA synthesis: hydroxyurea, cytosine arabinoside and daunomycin. The effect was dose-dependent. Uridine uptake by platelets was found to be inhibited by specific inhibitors of RNA synthesis such as actinomycin D, rifampicin and vincristine, the effect of actinomycin D being dose dependent. The drug also led to a time-dependent inhibition of protein synthesis when preincubated with platelets. The platelet RNA profile on polyacrylamide gel was demonstrated to be similar to that of embryonic mouse erythroblast RNA. Synthesis of all three fractions, 28 S, 18 S and 4 S, was inhibited by actinomycin D. These findings show that human platelets are capable of DNA and RNA synthesis, and that these activities play a role in controlling protein synthesis in these cells. Detectable amounts of DNA have been found in whole human platelets, and in isolated mitochondria derived from these cells. Isolated platelet mitochondria incorporated [3H] thymidine and [3H] uridine into their macromolecules. These activities were inhibited by daunomycin and by both rifampicin and actinomycin D, respectively. These results support the assumption that DNA and RNA synthesis found in intact cell preparations takes place most probably in platelet mitochondria.     

DNA synthesis in Lactobacillus acidophilus in the absence of RNA and protein synthesis: a study with rifampicin

Summary Synthesis of DNA in Lactobacillus acidophilus R-26 was investigated in the presence of rifampicin which inhibits RNA and protein synthesis. Increments in DNA of between 140 and 200% were found under these conditions. Indirect evidence is presented that these large increments are not due to the presence of several replication points in the bacterial chromosome at the time of addition of the drug. Incubation with rifampicin was found to result in a progressive decrease in the capacity for DNA synthesis. This decrease was independent of the amount of DNA synthesized during incubation with rifampicin but was dependent on the time of incubation in the presence of the drug. It is proposed that inhibition of protein and RNA synthesis in L. acidophilus does not inhibit initiation of new cycles of chromosome replication but results in a progressive loss of the capacity to replicate DNA.     

Inhibition of Bacteriophage ϕX174 Replicaiive-form DNA Replication by Rifampicin

?X174 DNA synthesis as well as phage production was inhibited by rifampicin when added in early phase of infection. Rifampicin did not inhibit the formation of parental duplex replicative-form, RF, and it inhibited the synthesis of progeny RF under conditions where protein synthesis was not necessary to be synthesized continuously. In addition, replication of parental RF into progeny RF was inhibited by rifampicin under conditions where a high concentration of chloramphenicol did not affect the replication. Consequently, it could be concluded that RNA synthesis other than that required for protein synthesis was necessary for both the initiation and continuation of RF replication.     

The effect of translation and transcription inhibitors on the synthesis of periplasmic phosphatases in E. coli.

Previous studies by others have indicated that the synthesis of secreted enzymes is unusually sensitive to many translation inhibitors and resistant, for about 30 min, to rifampicin. We have studied the sensitivity of secreted (periplasmic) phosphatases to such inhibitors. Alkaline phosphatase synthesis is more sensitive than total protein synthesis to tetracyclin and spectinomycin, but not to sparsomycin, streptomycin, chloramphenicol, kasugamycin, blasticidin S or thiostrepton; it is slightly more resistant than total protein synthesis to the latter two antibiotics. Acid hexose-phosphatase was also preferentially sensitive to tetracyclin and spectinomycin and also to kasugamycin. beta-galactosidase was also included in the study, as an intracellular enzyme, and was found to be preferentially inhibited ("repressed"), sometimes transiently, by all eight translation inhibitors. This effect did not seem to be mediated through cyclic AMP or guanosine tetraphosphate; the "repression" was still evident in mutants with altered rho factor indicating that it may also not be related to artificial polarity. Synthesis of both periplasmic phosphatases was immediately inhibited by rifampicin. These results differ from those found in previous studies with other organisms and suggest a reappraisal of the usual interpretation of these phenomena.     

Effect of the heat shock reaction on the phenotypic manifestation of mutations in rifampicin resistance affecting the gene of the RNA polymerase beta-subunit under the control of the lactose promotor

Plasmids bearing the rifampicin-resistant RNA polymerase beta-subunit gene under control of the lac promoter failed to provide Escherichia coli cells with resistance to rifampicin, despite the accumulation of large quantities of the resistant subunit. The effect proved to be connected with the aggregation of the plasmid-borne subunit. Conditions that induce synthesis of heat-shock proteins make the plasmid-containing cells resistant to rifampicin. This finding suggests that heat-shock proteins prevent the formation of protein aggregates.     

Rifampicin and chloramphenicol effects on DNA replication in thymine-prestarved Escherichia coli B/r WP2 thy trp.

When cultures of Escherichia coli B/r WP2 thy trp were prestarved for thymine for 30 min, DNA replication after readdition of thymine was limited to an increase of about 100% in the presence of rifampicin, an antibiotic which inhibits DNA-dependent RNA polymerase. However, chloramphenicol, an antibiotic which blocks protein but not RNA synthesis, did not limit replication. After prolonged thymine prestarvation (55 min) DNA increased only about 50% in the presence of rifampicin, but no such limitation occurred in the presence of chloramphenicol. The ability of a high concentration of rifampicin to limit DNA replication was eliminated by addition of either high or low concentrations of chloramphenicol, indicating that stoichiometric interaction of the antibiotics is not responsible for this effect.     

T4 phage and T4 ghosts inhibit f2 phage replication by different mechanisms

Both T4 phage and DNA-free ghosts inhibit replication of RNA phage f2. Most but not all of the effects by T4 upon f2 growth can be blocked by the addition of rifampicin prior to T4 superinfection; by contrast, the inhibition of f2 synthesis by T4 ghosts cannot be blocked by rifampicin. This indicates that inhibition by intact T4 requires gene function, while inhibition by ghosts does not. There is a small, multiplicity-dependent inhibition by viable T4 on f2 growth in the presence of rifampicin which may be similar to the gene function-independent inhibition by T4 ghosts. With one viable T4 per cell, there appears to be no effect by viable T4 upon f2 growth which does not require T4 gene action. Moreover, increasing multiplicities of viable T4 appear to inhibit T4 replication as well.In the absence of rifampicin, pre-existing f2 single and double-stranded RNA are degraded after superinfection by viable T4, but remain stable after superinfection by ghosts. However, no new f2 RNA is synthesized after superinfection with either. In the presence of rifampicin, f2-specific protein synthesis is largely unaffected by viable T4, but is completely inhibited by ghosts. Both Escherichia coli, as well as f2-speciflc polysomes disappear in the presence of ghosts.We conclude that, at low multiplicities, T4 phage and T4 ghosts inhibit replication of f2 phage, and presumably host syntheses, by different mechanisms.     

Induction of prophage lambda does not require full induction of RecA protein synthesis

In mitomycin C-treated lambda lysogens, even though the rate of synthesis of RecA protein was greatly reduced by a low concentration of rifampicin (4 microgram/ml), induction of prophage lambda occurred readily as assessed by (i) cell lysis of the lysogens, (ii) production of progeny phage, and (iii) extensive cleavage of lambda repressor. The extent and the rate of cleavage of lambda repressor were not significantly affected by the low rate of synthesis of RecA protein resulting from rifampicin action. However, the yield of phage progeny was reduced and lysis of the cells was slightly delayed. We conclude that in RecA+ bacteria, induction of prophage lambda does not require full induction of RecA protein synthesis.     

Effect of the physiological activity of Clostridium cells on their sensitivity to antibiotics]

Actively multiplicating cells of C1. perfringens proved to be more sensitive to 7-chlor-7-desoxylincomycin and rifampicin than the cells in the phase of the population dying. The bactericidal effect of the antibiotics on Clostridia vegetating at a temperature range within 37--4degrees was studied. Determination of the content of higher fatty acids in the cultivation medium with the method of gas chromatography showed that the metabolic processes in the bacterial cells went on at a temperature of 4degrees. Sensitivity of Clostridia to antibiotics at 20 and 4degrees lowered. However, all antibiotics inhibited the cell viability under such conditions. The inhibitors of the intracellular protein synthesis, i. e. rifampicin, 7-chlor-7-desoxylincomycin and morphocycline proved to be most active. The effect of beta-lactame antibiotics, i. e. cephaloridine and benzylpenicillin was reliable though lower.     

The effect of growth conditions on inducible, recA-dependent resistance to X rays in Escherichia coli

Escherichia coli cells grown to logarithmic phase in, and plated on, rich medium (yeast extract-nutrient broth) were more resistant to X rays, ultraviolet (uv) radiation, and methyl methanesulfonate (MMS) than cells grown in, and plated on, minimal medium. We have called this enhanced survival capability medium-dependent resistance (MDR). The magnitude of MDR observed after oxic X irradiation was greater than that observed after anoxic X irradiation, uv irradiation, or MMS treatment. MDR was not observed in stationary-phase cells with X or uv radiation. MDR was associated with an increased ability to repair X-ray-induced DNA single-strand breaks, and with reduced X-ray-induced DNA degradation and protein synthesis retardation. Postirradiation protein synthesis was concluded to be critical in allowing the high X-ray survival associated with MDR, because of the large radiosensitization caused by a postirradiation growth medium shift down or treatment with rifampicin (RIF), recA protein must be at least one of the proteins whose synthesis is critical to MDR, as judged by the absence of MDR or a RIF effect in X-irradiated recA and lexA mutants. The results with X-irradiated temperature-conditional recA cells suggest that it is only after cells have been damaged that the recA gene plays a role in MDR.     

Comparative study of the effect of different rifamycins on bacterial cell metabolism and on the RNA-polymerase reaction in a cell-free system]

The results of the study on the inhibitory effect of a number of rifamycin derivatives, such as rifamycin B, rifamycin O, rifamycin, rifamycin A, 25-desacetylrifampicin and rifampicin are presented. It was shown that rifampicin had the highest inhibitory effect on the synthesis of RNA in the cells of E. coli and Staph. aureus. It inhibited the above process by 93.0 and 98.8 per cent respectively. The data on the cells of Staph. aureus, as well as the data on comparison of the inhibitory effect of rifampicin derivatives with respect to the RNA-polymerase reaction in acellular systems are presented.     

An examination of the inhibitory effects of three antibiotics in combination on ribosome biosynthesis in Staphylococcus aureus

Although a number of different antibiotics are used to combat staphylococcal infections, resistance has continued to develop. The use of rifampicin and ciprofloxacin in combination with azithromycin, known for its inhibitory effects on the bacterial ribosome, can create potential synergistic effects on ribosomal subunit synthesis rates. In this work, combination antibiotic treatments gave a significant decrease in cell numbers following growth in the presence of ciprofloxacin or rifampicin with azithromycin compared to those grown with azithromycin or rifampicin alone. DNA, RNA and protein synthesis rates were reduced with single antibiotic treatments and showed further decreases when drug combinations were used. 70S ribosome levels were reduced with every antibiotic treatment. DNA gyrase subunits A and B showed significant decreases for double and triple antibiotic-treated samples. Ribosomal subunit synthesis rates were diminished for each different antibiotic combination. Turnover of 16S and 23S rRNA was also observed in each case and was stimulated by antibiotic combinations. The frequency of spontaneous resistance was reduced in all double selections, and no triply resistant mutants were found.