Some effects of antibiotics on bacterial polyribosomes as studied by gel electrophoresis

Bacterial polyribosomes possess characteristic electrophoretic mobilities in agarose-acrylamide composite gels. In cells whose normal protein synthesis is inhibited by certain antibiotics, the resolution of the gel electrophoresis technique has permitted the detection of specific increases in the mobility of the polyribosomes. Antibiotics producing these changes in polyribosome mobility include inhibitors of the 30 S as well as the 50 S subunit.The in vivo action of streptomycin has been studied in some detail. Streptomycin alters the polyribosomes of sensitive strains, haploid as well as heterodiploid, but does not alter polyribosomes of strains resistant to or dependent upon streptomycin. Streptomycin-altered polyribosomes are stable in vivo for more than one hour and exhibit a considerably prolonged run-off time following rifampicin treatment. They are also significantly more resistant to the in vitro RNase degradation than control ribosomes. The subunit composition ($\text{50}\text{S}\text{30}\text{S}$) of the altered polyribosomes remains unchanged from the control (1:1).Since the electrophoretic mobility of monosomes remains unchanged during the antibiotic treatment, the evidence presented suggests that the alteration of polyribosome mobility involves a stacking of the ribosomes on mRNA.     

Magnesium starvation of Aerobacter aerogenes. I. Changes in nucleic acid composition

Aerobacter aerogenes incubated in a medium containing all factors necessary for exponential growth except Mg⁺⁺ continued to synthesize nucleic acids and proteins for more than 70 hr, provided the major carbon source was in excess at all times. After 24 hr of Mg⁺⁺ starvation, deoxyribonucleic acid content in the culture had increased 10-fold. In contrast, the viable-cell count increased only about threefold during the first few hours and then remained approximately constant for the subsequent 70 hr. After specified intervals of Mg⁺⁺ starvation, extracts of the bacteria, or ribonucleic acid (RNA) purified from them, was centrifuged through gradients of sucrose to separate transfer RNA from ribosomal components. After correcting for losses, we obtained the following results. (i) There was a progressive rise in the content of transfer RNA competent to accept amino acids and during starvation it remained completely stable. (ii) In contrast, the contents of normally sedimenting ribosomal RNA and ribosomal subunits (30 and 50S) remained approximately constant for more than 24 hr. This did not result from stability of ribosomes made prior to starvation together with an inhibition of synthesis of new particles. Rather, ribosomes were continually breaking down and being replaced by an equivalent number of new ones. (iii) The breakdown of ribosomes appeared to be sequentially ordered; polysomes yielded 70S monomers, which then gave 30 and 50S particles, and these disintegrated to smaller units and finally to acid-soluble products. (iv) Furthermore, the particles derived from breakdown do not appear to exchange with subparticles on the path of assembly. Thus, ribosome decay was age-dependent and ribosomal RNA molecules had a minimal life expectancy of 90 min; however, some survived much longer.     

ISOLATION OF CYTOPLASMIC AND CHLOROPLAST RIBOSOMES AND THEIR DISSOCIATION INTO ACTIVE SUBUNITS FROM CHLAMYDOMONAS REINHARDTII

A mixture of cytoplasmic (80S) and chloroplast (70S) ribosomes from Chlamydomonas reinhardtii was freed of contaminating membranes by sedimentation of the postmitochondrial supernatant through a layer of 1.87 M sucrose. The purified ribosomes were separated into 80S and 70S fractions by centrifugation at a relatively low speed on a 10–40% sucrose gradient containing 25 mM KCl and 5 mM MgCl₂. Both the 80S and 70S ribosomes were dissociated into compact subunits by centrifugations in 5–20% high-salt sucrose gradients. The dissociations of both ribosomal species under these conditions were not affected by the addition of puromycin, indicating that the ribosomes as isolated were devoid of nascent chains. Subunits derived from the 80S ribosomes had apparent sedimentation coefficients of 57S and 37S whereas those from the 70S ribosomes had apparent sedimentation coefficients of 50S and 33S. In the presence of polyuridylic acid and cofactors, the 80S and 70S ribosomes incorporated [¹⁴C]phenylalanine into material insoluble in hot TCA. The requirements for incorporation were found to be similar to those described for eukaryotic and prokaryotic ribosomes. Experiments with antibiotics showed that the activity of the 80S ribosomes was sensitive to cycloheximide, whereas that of the 70S ribosomes was inhibited by streptomycin. The isolated subunits, when mixed together in an incorporation medium, were also active in the polymerization of phenylalanine in vitro.     

Intracellular Population Genetics: Evidence for Random Drift of Mitochondrial Allele Frequencies in SACCHAROMYCES CEREVISIAE and SCHIZOSACCHAROMYCES POMBE

We report evidence for random drift of mitochondrial allele frequencies in zygote clones of Saccharomyces cerevisiae and Schizosaccharomyces pombe. Monofactorial and bifactorial crosses were done, using strains resistant or sensitive to erythromycin (alleles E^R, E^S), oligomycin (O^R, O^S), or diuron (D^R, D^S). The frequencies of resistant and sensitive cells (and thus the frequencies of the resistant and sensitive alleles) were determined for each of a number of clones of diploid cells arising from individual zygotes. Allele frequencies were extremely variable among these zygote clones; some clones were "uniparental," with mitochondrial alleles from only one parent present. These observations suggest random drift of the allele frequencies in the population of mitochondrial genes within an individual zygote and its diploid progeny. Drift would cease when all the cells in a clone become homoplasmic, due to segregation of the mitochondrial genomes during vegetative cell divisions. To test this, we delayed cell division (and hence segregation) for varying times by starving zygotes in order to give drift more time to operate. As predicted, delaying cell division resulted in an increase in the variance of allele frequencies among the zygote clones and an increase in the proportion of uniparental zygote clones. The changes in form of the allele frequency distributions resembled those seen during random drift in finite Mendelian populations. In bifactorial crosses, genotypes as well as individual alleles were fixed or lost in some zygote clones. However, the mean recombination frequency for a large number of clones did not increase when cell division was delayed. Several possible molecular mechanisms for intracellular random drift are discussed.     

Two Genetic Loci for Resistance to Kasugamycin in Escherichia coli

There are two loci for resistance to the antibiotic kasugamycin (Ksg) in Escherichia coli. Mutations at ksgA resulted in 30S ribosomal subunit resistance to Ksg. The map location of ksgA was near minute 0.5: ksgA was 95% cotransducible with pdxA, and the apparent gene order was thr... ksgA... pdxA. Studies in stable ksgA/ksgA⁺ merodiploids showed that sensitivity was dominant over resistance. Mutations at a second gene (ksgB), located between minutes 25 and 39, resulted in phenotypic Ksg^R indistinguishable from ksgA mutations, but ribosomes from ksgB strains were sensitive to the drug in vitro. Spontaneous and induced mutations to Ksg^R were usually of the ksgA (ribosomal) type.     

Effect of streptomycin on the response of Escherichia coli ribosomes to the dissociation factor

Ribosomes recovered from cells of Escherichia coli treated with streptomycin are impaired in their response to the dissociation factor. This effect of Str evidently depends on a direct action on the ribosome and not on stabilization of a complex with normal ligands. Thus, such Str-ribosomes lack firmly bound transfer RNA; treatment with puromycin does not remove the resistance to dissociation; and similar resistance is produced when free ribosomes (in the absence of normal ligands) are exposed to Str in buffer and then washed.The impairment of dissociation of Str-ribosomes in cells is evidently incomplete, for these ribosomes maintain a reduced polysome level by engaging in cyclic abortive reinitiation (see preceding paper, Wallace &; Davis, 1973) and this process requires formylation (and hence presumably dissociation). Str-inhibited dissociation may be the limiting step in this reinitiation, for the polysome level is much lower in Str-treated cells of strain W than in those of K12, and Str impairs dissociation much more with ribosomes of the former strain.     

Conditional change of DNA replication control in an RNA polymerase mutant of Escherichia coli

The infectivity of the soybean symbiont Rhizobium japonicum changed two- to fivefold with culture age for strains 110 ARS, 138 Str Spc, and 123 Spc, whereas culture age had relatively little effect on the infectivity of strains 83 Str and 61A76 Str. Infectivity was measured by determining the number of nodules which developed on soybean primary roots in the zone which contained developing and preemergent root hairs at the time of inoculation. Root cells in this region of the host root are susceptible to Rhizobium infection, but this susceptibility is lost during acropetal development and maturation of the root cells within a period of 4 to 6 h (T. V. Bhuvaneswari, B. G. Turgeon, and W. D. Bauer, Plant Physiol. 66:1027-1031, 1980). Profiles of nodulation frequency at different locations on the root were not affected by the age of the R. japonicum cultures, indicating that culture age affected the efficiency of Rhizobium infection rather than how soon infections were initiated after inoculation. Inoculum dose-response experiments also indicated that culture age affected the efficiency of infection. Two strains, 61A76 Str and 83 Str, were relatively inefficient at all culture ages, particularly at low inoculum doses. Changes in infectivity with culture age were reasonably well correlated with changes in the proportion of cells in a culture capable of binding soybean lectin. Suspensions of R. japonicum in water were found to retain their viability and infectivity.     

Synthesis,antimycobacterial and cytotoxic activity of α,β-unsaturated amides and 2,4-disubstituted oxazoline derivatives

The synthesis of six α,β,-unsaturated amides and six 2,4-disubstituted oxazolines derivatives and their evaluation against two Mycobacterium tuberculosis strains (sensitive H37Rv and a resistant clinical isolate) is reported. 2,4-Disubstituted oxazolines (S)-3b,d,e were the most active in the sensitive strain with a MIC of 14.2, 13.6 and 10.8 μM, respectively, and the compounds (S)-3d,f were the most active against resistant strain with a MIC of 6.8 and 7.4 μM. The ex-vivo evaluation of hepatotoxicity on precision-cut rat liver slices was also tested for the α,β-unsaturated amides (S)-2b and (S)-2d,f and for the oxazolines (S)-3b and (S)-3d,f at different concentrations (5, 15 and 30 μg/mL). The results indicate that these compounds possess promising antimycobacterial activity and at the same time are not hepatotoxic. These findings open the possibility for development of new drugs against tuberculosis.     

Phenotypic Suppression in Escherichia coli by Chloramphenicol and Other Reversible Inhibitors of the Ribosome

Antibiotics that interfere reversibly with various aspects of ribosomal function (chloramphenicol, tetracycline, erythromycin, and spectinomycin) are shown to antagonize, at barely inhibitory concentrations, the inhibitory effect of low concentrations of streptomycin (SM) on the growth of Escherichia coli. Paradoxically, these compounds can also replace SM in supporting the growth of conditionally SM-dependent mutants. Chloramphenicol produced about as much phenotypic suppression as SM in SM-sensitive strains, but less than that attainable with high concentrations of SM in resistant strains. The antagonism to SM inhibition and the phenotypic suppression appear to be specific for those growth inhibitors that act on the ribosome. Since inhibitors of the 50S subunit of the ribosome (chloramphenicol, erythromycin) are as active as inhibitors of the 30S subunit, it is suggested that phenotypic suppression by borderline concentrations of ribosome inhibitors does not necessarily depend on an alteration of the recognition region of the ribosome. Alternatively, partial inhibition of the ribosomes might change the environment in a way that would influence the frequency of misreading. Phenotypic suppression by a low concentration of SM as well as by chloramphenicol was found to depend on the presence of a trace of the required growth factor.     

Ribosomal RNA and protein mutants resistant to spectinomycin.

We have compared the influence of spectinomycin (Spc) on individual partial reactions during the elongation phase of translation in vitro by wild-type and mutant ribosomes. The data show that the antibiotic specifically inhibits the elongation factor G (EF-G) cycle supported by wild-type ribosomes. In addition, we have reproduced the in vivo Spc resistant phenotype of relevant ribosome mutants in our in vitro translation system. In particular, three mutants with alterations at position 1192 in 16S rRNA as well as an rpsE mutant with an alteration of protein S5 were analysed. All of these ribosomal mutants confer a degree of Spc resistance for the EF-G cycle in vitro that is correlated with the degree of growth rate resistance to the antibiotic in culture.     

I-region linked complementing loci in resistance to viral leukemogenesis in the mouse

A-RadLV, a variant of the radiation leukemia virus, inoculated intrathymically into adult mice, causes a high frequency of leukemia in haplotypesb, f, k, d, p andj on the B10 background, whereas H-2^S mice are resistant. Resistance is dominant and segregates withH-2 ^S in the offspring of (b×s)b and (b×t ₂)b backcrosses. Analysis of recombinant strains revealed that resistance is associated withI-A andI-B. B10.A(5R), a recombinant of two sensitive haplotypes, was found to be resistant, suggesting intra-H-2-gene complementation. The resistance of such complementing loci was demonstrated also in thetrans position by testing F₁ mice bred from sensitive parents. These data are taken to suggest thatI-region linked complementing loci, similar to classicalIr genes, may be involved in resistance to murine leukemia.     

Postadaptational Resistance to Benzalkonium Chloride and Subsequent Physicochemical Modifications of Listeria monocytogenes

Many studies have demonstrated that bacteria, including Listeria monocytogenes, are capable of adapting to disinfectants used in industrial settings after prolonged exposure to sublethal concentrations. However, the consequent alterations of the cell surface due to sanitizer adaptation of this pathogen are not fully understood. Two resistant and four sensitive L. monocytogenes strains from different sources were progressively subcultured with increasing sublethal concentrations of a surfactant, benzalkonium chloride (BC). To evaluate the effects of acquired tolerance to BC, parent and adapted strains were compared by using several morphological and physiological tests. Sensitive strains showed at least a fivefold increase in the MIC, while the MIC doubled for resistant strains after the adaptation period. The hydrophobicities of cells of parent and adapted strains were similar. Serological testing indicated that antigen types 1 and 4 were both present on the cell surface of adapted cells. The data suggest that efflux pumps are the major mechanism of adaptation in sensitive strains and are less important in originally resistant isolates. A different, unknown mechanism was responsible for the original tolerance of resistant isolates. In an originally resistant strain, there was a slight shift in the fatty acid profile after adaptation, whereas sensitive strains had similar profiles. Electron micrographs revealed morphological differences after adaptation. The changes in cell surface antigens, efflux pump utilization, and fatty acid profiles suggest that different mechanisms are used by resistant and sensitive strains for adaptation to BC.     

Invasive Salmonella Typhimurium ST313 with Naturally Attenuated Flagellin Elicits Reduced Inflammation and Replicates within Macrophages

Invasive non-typhoidal Salmonella (iNTS) are an important cause of septicemia in children under the age of five years in sub-Saharan Africa. A novel genotype of Salmonella enterica subsp. enterica serovar Typhimurium (multi-locus sequence type [ST] 313) circulating in this geographic region is genetically different to from S. Typhimurium ST19 strains that are common throughout the rest of the world. S. Typhimurium ST313 strains have acquired pseudogenes and genetic deletions and appear to be evolving to become more like the typhoidal serovars S. Typhi and S. Paratyphi A. Epidemiological and clinical data show that S. Typhimurium ST313 strains are clinically associated with invasive systemic disease (bacteremia, septicemia, meningitis) rather than with gastroenteritis. The current work summarizes investigations of the broad hypothesis that S. Typhimurium ST313 isolates from Mali, West Africa, will behave differently from ST19 isolates in various in vitro assays. Here, we show that strains of the ST313 genotype are phagocytosed more efficiently and are highly resistant to killing by macrophage cell lines and primary mouse and human macrophages compared to ST19 strains. S. Typhimurium ST313 strains survived and replicated within different macrophages. Infection of macrophages with S. Typhimurium ST19 strains resulted in increased apoptosis and higher production of proinflammatory cytokines, as measured by gene expression and protein production, compared to S. Typhimurium ST313 strains. This difference in proinflammatory cytokine production and cell death between S. Typhimurium ST19 and ST313 strains could be explained, in part, by an increased production of flagellin by ST19 strains. These observations provide further evidence that S. Typhimurium ST313 strains are phenotypically different to ST19 strains and instead share similar pathogenic characteristics with typhoidal Salmonella serovars.     

An Association of Genotypes and Antimicrobial Resistance Patterns among Salmonella Isolates from Pigs and Humans in Taiwan

We collected 110 Salmonella enterica isolates from sick pigs and determined their serotypes, genotypes using pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility to 12 antimicrobials and compared the data with a collection of 18,280 isolates obtained from humans. The pig isolates fell into 12 common serovars for human salmonellosis in Taiwan; S. Typhimurium, S. Choleraesuis, S. Derby, S. Livingstone, and S. Schwarzengrund were the 5 most common serovars and accounted for a total of 84% of the collection. Of the 110 isolates, 106 (96%) were multidrug resistant (MDR) and 48 (44%) had PFGE patterns found in human isolates. S. Typhimurium, S. Choleraesuis, and S. Schwarzengrund were among the most highly resistant serovars. The majority of the 3 serovars were resistant to 8–11 of the tested antimicrobials. The isolates from pigs and humans sharing a common PFGE pattern displayed identical or very similar resistance patterns and Salmonella strains that caused severe infection in pigs were also capable of causing infections in humans. The results indicate that pigs are one of the major reservoirs to human salmonellosis in Taiwan. Almost all of the pig isolates were MDR, which highlights the necessity of strictly regulating the use of antimicrobials in the agriculture sector in Taiwan.     

Extrachromosomal Element Delta in DROSOPHILA MELANOGASTER. VIII. Inseparable Association with Sensitive Second Chromosome

The presence of delta is always accompanied by a sensitive second chromosome in Drosophila melanogaster or vice versa. The separability of delta and the chromosome line was investigated in experiments designed to eliminate delta or to suppress its multiplication for a number of generations. Cy/S^b-5 males which transmit a minute amount of delta b (retained by chromosome S^b, kills S^b/S^b and S^b/S^r zygotes) to their progeny were backcrossed for 61 generations to Cy/Pm females which carried no delta. After backcrossing 465 Cy/S^b-5 males were individually examined for retention of delta, and all were found to retain delta b in its latent state. A homozygous strain for the S^r-20B chromosome which retained a minute amount of delta r (retained by chromosome S^r, kills S^r/S^r zygotes) was obtained. 1,252 Cy/S^r-20B females derived from the homozygous strains at the 21st generation were tested individually for delta retention. All females tested were found to retain delta r in its latent state. The delta retention in S^b-5 chromosome lines isolated from S^r-Cy/S^b-5 heterozygous strains which carried delta r but not delta b was examined. The descendant Cy/S^b-5 lines from the heterozygous strains had accumulated their specific delta, delta b. These findings are consistent with those obtained in an earlier study, and lead to the conclusion that delta is associated inseparably with each specific sensitive chromosome.     

Antimalarial sesquiterpene lactones from Distephanus angulifolius

Combined use of bioassay-guided fractionation based on in vitro antiplasmodial assay and dereplication based on HPLC-PDA-MS-SPE-NMR led to isolation of (6S,7R,8S)-14-acetoxy-8-[2-hydroxymethylacrylat]-15-helianga-1(10),4,11(13)-trien-15-al-6,12-olid and (5R,6R,7R,8S,10S)-14-acetoxy-8-[2-hydroxymethylacrylat]-elema-1,3,11(13)-trien-15-al-6,12-olid, along with vernodalol, vernodalin, and 11,13β-dihydroxyvernodalin from extract of Distephanus angulifolius. All compounds were identified by spectroscopic methods, including 1D and 2D homo- and heteronuclear NMR experiments. The isolated compounds showed IC₅₀ values in the range 1.6-3.8 μM and 2.1-4.9 μM against chloroquine sensitive D10 and chloroquine resistant W2 Plasmodium falciparum strains, respectively.     

Letter: Unequal contribution to ribosomal assembly of both str alleles in Escherichia coli merodiploids and its relationship to the dominance phenomenon

Two Escherichia coli strains were constructed which are reciprocal diploids for the str locus and isogenic for this region of the chromosome (str^s/′F str^r and str^r/′Fstr³). During exponential growth the steady-state ribosomal pools of both merodiploids are comprised of about 90%, or more, of streptomycin-sensitive ribosomes. Little effect of allele position was found. A similar preponderance of sensitive 30 S subunits over those that are resistant has been found during limited subunit reconstitution when an equimolar mixture of ribosomal proteins from both phenotypes was initially present. The results indicate that the rates of 30 S subunit assembly of both phenotypes are different, and that the sensitive sub-units predominate over the resistant subunits, suggesting that the difference in biosynthetic rate may be the basis for the dominance of this phenotype in vivo. An explanation for some aspects of the physiology of str diploids have been suggested in terms of these findings.     

Heat Stress Activates Fission Yeast Spc1/StyI MAPK by a MEKK-Independent Mechanism

Fission yeast Spc1/StyI MAPK is activated by many environmental insults including high osmolarity, oxidative stress, and heat shock. Spc1/StyI is activated by Wis1, a MAPK kinase (MEK), which is itself activated by Wik1/Wak1/Wis4, a MEK kinase (MEKK). Spc1/StyI is inactivated by the tyrosine phosphatases Pyp1 and Pyp2. Inhibition of Pyp1 was recently reported to play a crucial role in the oxidative stress and heat shock responses. These conclusions were based on three findings: 1) osmotic, oxidative, and heat stresses activate Spc1/StyI in wis4 cells; 2) oxidative stress and heat shock activate Spc1/StyI in cells that express Wis1AA, in which MEKK consensus phosphorylation sites were replaced with alanine; and 3) Spc1/StyI is maximally activated in Δpyp1 cells. Contrary to these findings, we report: 1) Spc1/StyI activation by osmotic stress is greatly reduced in wis4 cells; 2) wis1-AA and Δwis1 cells have identical phenotypes; and 3) all forms of stress activate Spc1/StyI in Δpyp1 cells. We also report that heat shock, but not osmotic or oxidative stress, activate Spc1 in wis1-DD cells, which express Wis1 protein that has the MEKK consensus phosphorylation sites replaced with aspartic acid. Thus osmotic and oxidative stress activate Spc1/StyI by a MEKK-dependent process, whereas heat shock activates Spc1/StyI by a novel mechanism that does not require MEKK activation or Pyp1 inhibition.     

Misreading of the ribosomal suppressor SUP46 due to an altered 40 S subunit in yeast

The dominant suppressor, SUP46, in the yeast Saccharomyces cerevisiae acts on a wide range of different types of mutations. The incorporation of phenylalanine and the misincorporation of leucine in a cell-free system programmed with poly(U) indicated that the ribosomes from a SUP46 strain produce abnormally high rates of translation errors. Furthermore, the cell-free translation system was used to demonstrate that the SUP46 defect resides in the 40 S ribosomal subunit. The growth of SUP46 strains was shown to be unusually sensitive to paromomycin, an aminoglycoside antibiotic that is known to induce translation errors. In addition, paromomycin stimulated mistranslation with SUP46 ribosomes to a greater extent than with normal ribosomes. These results indicate that SUP46 suppression is caused by increased translation errors as a result of the mutationally altered 40 S ribosomal subunit. Paromomycin appears to produce translation errors in SUP46 strains at rates that are too high for cellular growth.     

Antimicrobial Susceptibility of Clostridium difficile from Different Sources

A total of 79 Clostridium difficile strains from healthy young and elderly adults, elderly patients without gastrointestinal disease, elderly patients receiving antibiotics without gastrointestinal complications, and elderly patients with antibiotic-associated diarrhea or pseudomembranous colitis were tested for their susceptibilities to 24 antimicrobial agents. All of the 79 strains were inhibited by low concentrations of rifampicin, metronidazole, fusidic acid, vancomycin, ampicillin, and penicillin G. The strains were highly resistant to aminoglycosides, trimethoprim, sulfamethoxazole, nalidixic acid, and cycloserine and often resistant to neomycin, cefoxitin, and cefalexin. Wide variations in the susceptibility of C. difficile strains to erythromycin, clindamycin, lincomycin, chloramphenicol, and tetracycline were found. Strains resistant to erythromycin, clindamycin, and lincomycin were more frequently found among strains isolated from elderly adults than those isolated from young adults, with particularly high frequency among strains isolated from elderly patients receiving antibiotics. None of the 23 strains isolated from healthy young adults was resistant to chloramphenicol. All of the 14 strains resistant to erythromycin, clindamycin, lincomycin, and chloramphenicol were sensitive to tetracycline and all of the 15 strains resistant to erythromycin, clindamycin, lincomycin, and tetracycline were sensitive to chloramphenicol. Only one out of 19 tetracycline-resistant strains was highly toxigenic, whereas 42 (70%) of 60 sensitive strains were highly toxigenic.