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E Cundliffe

Publications and source records attributed to E Cundliffe.

At least 55 records · Page 3Linked to original sources
PubMed1989

Use of a fractionated coupled transcription-translation system in the study of ribosomal resistance mechanisms in antibiotic-producing Streptomyces.

The coupled transcription-translation system, formerly involving extracts of Streptomyces lividans, has been developed such that it functions with ribosomes (or their subunits) from at least 20 different Streptomyces species. This fractionated system has been used to investigate the antibiotic responses of ribosomes from various Streptomyces which synthesize inhibitors of protein synthesis. Of the 11 organisms included in this study, two strains possessed ribosomes that were specifically resistant to the autogenous antibiotic. These were Streptomyces pactum and Streptomyces karnatakensis, both of which produce pactamycin. Ribosomal subunit exchange analysis further demonstrated that resistance to pactamycin in those strains is due to some property of the 30S ribosomal subunits.

Anti-Bacterial Agents↗
PubMed1989

Methylation of 23S rRNA caused by tlrA (ermSF), a tylosin resistance determinant from Streptomyces fradiae.

Ribosomes from Streptomyces griseofuscus expressing tlrA, a resistance gene isolated from the tylosin producer Streptomyces fradiae, are resistant to macrolide and lincosamide antibiotics in vitro. The tlrA product was found to be a methylase that introduces two methyl groups into a single base within 23S rRNA, generating N6,N6-dimethyladenine at position 2058. This activity is therefore similar to the ermE resistance mechanism in Saccharopolyspora erythraea (formerly Streptomyces erythraeus).

Drug Resistance, Microbial↗
PubMed1988

Site-directed mutagenesis of Escherichia coli 23 S ribosomal RNA at position 1067 within the GTP hydrolysis centre.

Site-directed mutagenesis has been used to change, specifically, residue 1067 within 23 S ribosomal RNA of Escherichia coli. This nucleoside (adenosine in the wild-type sequence) lies within the GTPase centre of the larger ribosomal subunit and is normally the target for the methylase enzyme responsible for resistance to the antibiotic thiostrepton. The performance of the altered ribosomes was not impaired in cell-free protein synthesis nor in GTP hydrolysis assays (although the 3 mutant strains grew somewhat more slowly than wild-type) but their responses to thiostrepton did vary. Thus, ribosomes containing the A to C or A to U substitution at residue 1067 of 23 S rRNA were highly resistant to the drug, whereas the A to G substitution resulted in much lesser impairment of thiostrepton binding and the ribosomes remained substantially sensitive to the antibiotic. These data reinforce the hypothesis that thiostrepton binds to 23 S rRNA at a site that includes residue A1067. They also exclude any possibility that the insensitivity of eukaryotic ribosomes to the drug might be due solely to the substitution of G at the equivalent position within eukaryotic rRNA.

Binding Sites↗
PubMed1988

Cloning and characterization of a DNA gyrase B gene from Streptomyces sphaeroides that confers resistance to novobiocin.

A gyrB gene from Streptomyces sphaeroides, a producer of novobiocin, has been cloned in Streptomyces lividans, where it conferred resistance to novobiocin. The Streptomyces gyrB gene was sufficiently similar to a Bacillus subtilis gyrB probe to be specifically recognized during Southern analysis. Partial purification of DNA gyrase by affinity chromatography revealed the presence of two such activities (differing in their responses to novobiocin) in the clone. The product of the cloned gene, a novobiocin-resistant DNA gyrase B subunit, was identified in vitro by coupled transcription--translation as a 79-kd protein.

Cloning, Molecular↗
PubMed1988

The binding site for ribosomal protein L2 within 23S ribosomal RNA of Escherichia coli.

Ribosomal protein L2 from Escherichia coli binds to and protects from nuclease digestion a substantial portion of 'domain IV' of 23S rRNA. In particular, oligonucleotides derived from the sequence 1757-1935 were isolated and shown to rebind specifically to protein L2 in vitro. Other L2-protected oligonucleotides, also derived from domain IV (i.e. from residues 1955-2010) did not rebind to protein L2 in vitro nor did others derived from domain I. Given that protein L2 is widely believed to be located in the peptidyl transferase centre of the 50S ribosomal subunit, these data suggest that domain IV of 23S rRNA is also present in that active site of the ribosomal enzyme.

Base Sequence↗
PubMed1987

Sites of action of two ribosomal RNA methylases responsible for resistance to aminoglycosides.

Methylation of either of two residues (G-1405 or A-1408) within bacterial 16 S ribosomal RNA results in high level resistance to specific combinations of aminoglycoside antibiotics. The product of a gene that originated in Micromonospora purpurea (an actinomycete that produces gentamicin) gives resistance to kanamycin plus gentamicin by converting residue G-1405 to 7-methylguanosine. Resistance to kanamycin plus apramycin results from conversion of residue A-1408 to 1-methyladenosine catalysed by the product of a gene from Streptomyces tenjimariensis.

Aminoglycosides↗
PubMed1987

On the nature of antibiotic binding sites in ribosomes.

The ribosome is an enzyme and enzymes have active sites. Antibiotics that affect ribosomal function can be considered as enzyme inhibitors (or regulators) and it is therefore pertinent to identify their molecular targets as a means of studying the active sites of the particle. The methods available for doing this are considered and, in general terms, the data are evaluated. The conclusion is reached that there exists virtually no compelling evidence that antibiotics bind primarily to ribosomal proteins. Rather, studies of antibiotic resistance in various systems strongly suggest that ribosomal RNA is the primary target for a number of drugs. Moreover, in at least one case (relating to the antibiotic thiostrepton), such an effect can be demonstrated directly. These conclusions imply a fundamental role for RNA in ribosomal function.

Animals↗
PubMed1987

Cloning of aminoglycoside-resistance determinants from Streptomyces tenebrarius and comparison with related genes from other actinomycetes.

At least two aminoglycoside-resistance determinants from Streptomyces tenebrarius have been cloned separately in Streptomyces lividans. In each case, resistance (to kanamycin plus apramycin or to kanamycin plus gentamicin) was expressed at the level of the ribosome and involved specific methylation of 16S ribosomal RNA. Hybridization and restriction analysis revealed that related genes were present in other aminoglycoside-producing actinomycetes.

Actinomyces↗
PubMed1987

Ribosomal modification and resistance in antibiotic-producing organisms.

Antibiotic producing organisms defend themselves against their products in a variety of ways including modification of the normal target sites for antibiotic action. In the ribosomal context, some organisms that produce inhibitors of protein synthesis render their own ribosomes refractory to the autogenous drugs via specific methylation of ribosomal RNA. Such hints that antibiotics might normally recognize RNA within the ribosome, complement other data in suggesting that RNA might be intimately associated with--and, in evolutionary terms, might originally have constituted--various functional sites within the ribosome.

Anti-Bacterial Agents↗
PubMed1985

Studies of the GTPase domain of archaebacterial ribosomes.

Ribosomes from the methanogens Methanococcus vannielii and Methanobacterium formicicum catalyse uncoupled hydrolysis of GTP in the presence of factor EF-2 from rat liver (but not factor EF-G from Escherichia coli). In this assay, and in poly(U)-dependent protein synthesis, they were sensitive to thiostrepton. In contrast, ribosomes from Sulfolobus solfataricus did not respond to factor EF-2 (or factor EF-G) but possessed endogenous GTPase activity, which was also sensitive to thiostrepton. Ribosomes from the methanogens did not support (p)ppGpp production, but did appear to possess the equivalent of protein L11, which in E. coli is normally required for guanosine polyphosphate synthesis. Protein L11 from E. coli bound well to 23S rRNA from all three archaebacteria (as did thiostrepton) and oligonucleotides protected by the protein were sequenced and compared with rRNA sequences from other sources.

Anti-Bacterial Agents↗
PubMed1985

Methylation of 16S ribosomal RNA and resistance to the aminoglycoside antibiotics gentamicin and kanamycin determined by DNA from the gentamicin-producer, Micromonospora purpurea.

When DNA fragments from Micromonospora purpurea (the producer of gentamicin) were cloned in Streptomyces lividans, a gentamicin-resistant strain was obtained in which the ribosomes were highly resistant both to gentamicin and to kanamycin. Reconstitution analysis revealed that such resistance resulted from some property of their 16S RNA. Extracts from the clone contained methylase activity which acted on 16S RNA within E. coli 30S ribosomal subunits and rendered them resistant to gentamicin and kanamycin.

DNA, Bacterial↗
PubMed1985

Characterization in Micromonospora inyoensis of aminoglycoside acetyltransferase activity not previously encountered among actinomycetes.

Extracts of Micromonospora inyoensis contain aminoglycoside acetyltransferase activity not previously encountered among actinomycetes. Neomycin and its derivatives (excluding butirosin) are good substrates as is the novel aminoglycoside apramycin, whereas the gentamicins and kanamycins are utilized poorly if at all. Sisomicin, which is produced by M. inyoensis, is not acetylated. When neomycin was modified by extracts of M. inyoensis the product, most probably 3-N-acetylneomycin, was inactive against cell-free protein synthesis.

Acetyltransferases↗
PubMed1984

The binding site for ribosomal protein complex L8 within 23 s ribosomal RNA of Escherichia coli.

The ribosomal protein complex L8 of Escherichia coli consists of two dimers of protein L7/L12 and one monomer of protein L10. This pentameric complex and ribosomal protein L11 bind in mutually cooperative fashion to 23 S rRNA and protect specific fragments of the latter from digestion with ribonuclease T1. Oligonucleotides protected either by the L8 complex alone or by the complex plus protein L11 were isolated from such digests and shown to rebind specifically to these proteins. They were also subjected to nucleotide sequence analysis. The longest oligonucleotide, protected by the L8 complex alone, consisted of residues 1028-1124 of 23 S rRNA and included all the other RNA fragments produced in this study. Previously, protein L11 had been shown to protect residues 1052-1112 of 23 S rRNA. It is concluded that the binding sites for the L8 protein complex and for protein L11 are immediately adjacent within 23 S rRNA of E. coli.

Base Sequence↗
PubMed1984

Involvement of 16S ribosomal RNA in resistance of the aminoglycoside-producers Streptomyces tenjimariensis, Streptomyces tenebrarius and Micromonospora purpurea.

Resistance to aminoglycoside antibiotics in Micromonospora purpurea (the producer of gentamicin C complex), Streptomyces tenebrarius (the nebramycin producer) and Streptomyces tenjimariensis (which makes istamycin) occurs at the level of the ribosome. Reconstitution analysis has revealed, in each case, that 16S rRNA plays a critical role in determining such resistance.

Aminoglycosides↗
PubMed1984

Coupled transcription--translation in extracts of Streptomyces lividans.

A coupled transcription-translation system has been developed using extracts of Streptomyces lividans. Analysis by polyacrylamide gel electrophoresis revealed that proteins of the expected sizes were produced in systems programmed by pBR 322 or the Streptomyces plasmid pIJ 350. Also, linear forms of these plasmids retained template activity with no apparent loss of fidelity.

Bacterial Proteins↗