A B S T R A C T Clinical isolates of enterococci (Streptococcus faecalis) with high-level resistance to both streptomycin and kanamycin (minimal inhibitory concentration >2,000 ,ug/ml), and resistant to synergism with penicillin and streptomycin or kanamycin were examined for aminoglycoside-inactivating enzymes. All of the 10 strains studied had streptomycin adenylyltransferase and neomycin phosphotransferase activities; the latter enzyme phosphorylated amikacin as well as its normal substrates, such as kanamycin. Substrate profiles of the neomycin phosphotransferase activity suggested that phosphorylation occurred at the 3'-hydroxyl position, i.e., aminoglycoside 3'-phosphotransferase. A transconjugant strain, which acquired high-level aminoglycoside resistance and resistance to antibiotic synergism after mating with a resistant clinical isolate, also acquired both enzyme activities. Quantitative phosphorylation of amikacin in vitro by a sonicate of the transconjugant strain inactivated the antibiotic, as measured by bioassay, and the phosphorylated drug failed to produce synergism when combined with penicillin against a strain sensitive to penicillin-amikacin synergism.No differences were found in the sensitivity of ribosomes from a sensitive and resistant strain when exam-
1. The novel aminoglycoside antibiotic apramycin is shown to be a potent inhibitor of protein synthesis in bacteria both invivo and invitro. 2. In cell‐free systems from Escherichia coli programmed with poly(U), apramycin induces translation errors, as assayed by incorporation of leucine, isoleucine and serine, although this effect occurs only to a limited extent. 3. Apramycin inhibits the translocation step of protein synthesis both invivo, in protoplasts of Bacillus megaterium, and invitro, in cell‐free systems from E. coli. It is proposed that this is the primary inhibitory effect of the drug.
It has been shown (Matthaei, Jones, Martin & Nirenberg, 1962; Bretscher & Grunberg-Manago, 1962) that poly Ut can stimulate the incorporation not only of phenylalanine but also of leucine in the Eacherichia coli cell-free system. This phenomenon has also been observed with the 30000g supernatant from BaciUsstearothertmophilus (Friedman & Weinstein, 1966). Several factors such as Mg2+ and temperature (Szer & Ochoa, 1964), antibiotics (Davies, Gilbert & Gorini, 1964), polyamines (Friedman & Weinstein, 1964 and pH (Grunberg-Manago & Dondon, 1965) are known to influence the fidelity of translation of synthetic polynucleotides in different systems. Since the E. coli cell-free system translates poly tJ with much higher fidelity than that from B. stearothermophilus, experiments were performed to study the relative contributions of ribosomes and supernatant in determining the accuracy of translation in mixed systems.Materials. B. stearothermophilus (obtained from the Department of Microbiology, UJniversity of Warsaw) was grown at 600 in vigorously agitated medium containing 5g. of yeast extract, lOg. of Proteobak, 5g. of glucose and 5g. of NaCl in 11.; the pH of medium was 7-2. Cells were harvested in the exponential phase (E660 0.5), washed twice with standard buffer [lOmM-tris (pH 7.8)-14mMmagnesium acetate-60mm-KCl-6mM--mercaptoethanol] and stored at -40°.E. coli B was grown and harvested as described by Perzynski & Szafranski (1967).Cells were disrupted in the Eaton (1962) press, digested with deoxyribonuclease and centrifuged at 30000g for 30min. (S-30 fraction). The S-30 fraction from B. stearothermophilus was preincubated for 10min. at 60°; the corresponding fraction from E. coli for 40min. at 370 as described by Nirenberg & Matthaei (1961). The S-30 supernatants were centrifuged at 122000g for 2-5hr., ribosomes were collected and the upper two-thirds
0-Methylhydroxylamine (methoxyamine) was used for selective modification of cytosine residues in Escherichia coli 16-S rRNA. It was shown that cytosines accessible for methoxyamination are randomly distributed along the 16-S rRNA chain.Preparations of methoxyaminated 16-S rRNA, containing 2 -130 modified cytosines/chain, still retained the ability to bind 30-S proteins, but the physical assembly of reconstituted particles was incorrect. The protein compositions of the reconstituted and native particles did not differ qualitatively from each other. However, the amount of protein in reconstituted particles decreased with an increasing number of methoxyaminated cytosines in 16-S rRNA.The particles obtained sedimented slower than native 30-S subunits, lost their ability to associate with 50-S ribosomes and to bind native phage f2 RNA. In contrast, modification of 16-S rRNA did not affect binding of poly(U) by reconstituted particles.Chemical modification of RNA is one of the ways to study the relationship between its structure and function. In the previous experiments with ribosomal RNA, however, unspecific reagents, which alter different bases were used. Modification of 16-S rRNA by nitrous acid led to deamination of adenine, guanine and cytosine and blocked the incorporation of phenylalanine in the presence of reconstituted particles containing this RNA [l]. Similar results were obtained by using monoperphthalic acid, which alters adenine and cytosine [2].In our experiments 0-methylhydroxylamine (methoxyamine) was employed as a reagent for selective modification of cytosines in the single-stranded regions of E. coli 16-S rRNA. The effects of this alteration on the reconstitution of 30-S particles, the reassociation of the resulting 30-S with 50-S subunits and binding of poly(U) and f2 RNA to modified ribosomes are described. Attempts were also undertaken to localize methoxyaminated cytosines in the 16-S rRNA molecule.Recent ideas about the participation of cytosines in 16-S rRNA in binding of mRNA to 30-S subunits [3,4] and the involvement of ribosomal RNA-s in the association of subunits [5] made these experiments additionally attractive.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.