A determination of mutagen specificity in bacteria using nonsense mutants of bacteriophage T4

Osborn, Mary; Person, Stanley; Phillips, Stephen L.; Funk, Fred

doi:10.1016/0022-2836(67)90314-2

Cited by 152 publications

(44 citation statements)

References 18 publications

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“…The mutation is most likely a single-point mutation because reversion to usual MICs for both drugs occurred in about 1 in 105 clones. Also, methanesulfonic ethyl ester mutagenesis has a high probability of producing point transitions (16 Fig. 1.…”

Section: Resultsmentioning

confidence: 99%

Effect of Enzymatic Adenylylation on Dihydrostreptomycin Accumulation in Escherichia coli Carrying an R-Factor: Model Explaining Aminoglycoside Resistance by Inactivating Mechanisms

Dickie

Bryan

Pickard

1978

Antimicrob Agents Chemother

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Strains of Escherichia coli carrying R-factor R71(a), which codes for a streptomycin-spectinomycin adenylyltransferase, have elevated levels of resistance to dihydrostreptomycin (DHS) compared with isogenic R-bacteria. DHS accumulated by whole cells and spheroplasts of R+ bacteria is lower than that observed for R-strains, a result of the absence of the second and more rapid of the two energy-dependent phases of DHS uptake seen in susceptible E. coli. A mutant of R+ E. coli with reduced DHS resistance has been shown to have reduced levels of streptomycin-spectinomycin adenylyltransferase activity as well as enhanced drug accumulation. Actively accumulated DHS was recovered from R+ cells as the adenylylated derivative. Neither was inactivated antibiotic detected in culture filtrates, nor was actively accumulated drug lost from R+ cells under normal conditions. The cellular distribution of actively accumulated DHS in R+ and Rcells was found to be the same. Membranes isolated from these cells retained only a small fraction (=1%) of the total cell-associated drug. The R+ derivative of a mutant with defective energy transduction (E. coli NR-70) and reduced ability to transport aminoglycosides has a significantly higher minimal inhibitory concentration of DHS than its R+ parent (strain 7). Streptomycin-spectinomycin adenylyltransferase activity, from comparisons of Km values and total activities of enzyme, was the same in both strains. The enzyme has been localized to the exterior surface of the bacterial inner membrane, although isolated membranes lacked detectable enzyme activity. The preceding observations are consistent with the proposal that the level of R71(a)-mediated DHS resistance is the outcome of competition between the rate of adenylylation and the rate of the first energy-dependent phase of DHS transport. When the rate of adenylylation exceeds the first energy-dependent phase, adenylylated DHS is accumulated, apparently in a manner identical to the accumulation of DHS. Unlike DHS, adenylylated DHS does not interact with ribosomes, and, consequently, there is a failure to initiate ribosomally dependent sequelae such as the second energydependent phase of accumulation, inhibition of protein synthesis, and/or misreading of mRNA. R-factor-specified enzymatic modification of aminoglycoside antibiotics has been shown repeatedly to be a clinically important mechanism of resistance to aminoglycosides in bacteria. However, the precise mechanism by which enzymatic modification causes resistance is unknown. It has been shown in certain instances that modified aminoglycosides are no longer as effective at inhibiting protein synthesis or producing misreading of mRNA (23). These observations fail to explain aminoglycoside resistance because it has been demonstrated that only a very small fraction (less than 1%) of the total available, active aminoglycoside in the bacterial growth medium is detoxified. Thus, bacteria possessing aminoglycoside-inactivating enzymes are able to multiply in a milieu of almost entirely activ...

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Section: Resultsmentioning

confidence: 99%

Effect of Enzymatic Adenylylation on Dihydrostreptomycin Accumulation in Escherichia coli Carrying an R-Factor: Model Explaining Aminoglycoside Resistance by Inactivating Mechanisms

Dickie

Bryan

Pickard

1978

Antimicrob Agents Chemother

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“…In contrast, earlier studies in other systems generally relied upon the analysis ofreversion at a rather limited number of sites (4)(5)(6). Often the mutants analyzed in such reversion studies were originally induced by the mutagen (7); therefore, the possibility existed that preferentially mutable sites or hotspots had been selected and that these might behave atypically.…”

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confidence: 99%

“…Alternatively, the original mutation might have removed a critical DNA target sequence rendering the site practically immutable. Moreover, in studies of the reversion of nonsense mutations, the majority of "revertants" often occur not in the structural gene but at suppressor loci, which behave differently in their responses to UV light (4,8,9). The lacl system allows an examination of forward mutagenesis at 65 individual sites where nonsense mutations can arise by a single base substitution.…”

mentioning

confidence: 99%

Mutational specificity of UV light in Escherichia coli: indications for a role of DNA secondary structure.

Todd¹,

Glickman²

1982

Proc. Natl. Acad. Sci. U.S.A.

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We used the lad forward mutagenesis system to determine the mutational specificity of UV-induced mutation in a repair-proficient (Uvr') and a repair-deficient (AUvrB) strain ofEscherichia coli. The spectra recovered at similar levels of mutagenesis were similar, the exception being a mutational hotspot at site A24 specific to the AUvrB strain. Mutations induced at this hotspot, as well as those induced at other mutational hotspots that were found to be common to both the Uvr' and Uvr-strains, involve G-C -* APT transitions. All of the hotspots are at sites of potential dipyrimidine photoproducts, such as thymine-cytosine and cytosine-cytosine dimers, or of pyrimidine-cytosine photoproduct Py-C* lesions. Each of these hotspots occurs at a site in the potential hairpin loop of quasi-palindromic sequences. These observations suggest an important role for DNA structure in determining the fate of UV-induced premutational lesions.The lacI system of E. coli provides a method for determining UV-induced mutational specificity at a large number of sites (1-3). In contrast, earlier studies in other systems generally relied upon the analysis ofreversion at a rather limited number of sites (4-6). Often the mutants analyzed in such reversion studies were originally induced by the mutagen (7); therefore, the possibility existed that preferentially mutable sites or hotspots had been selected and that these might behave atypically. Alternatively, the original mutation might have removed a critical DNA target sequence rendering the site practically immutable. Moreover, in studies of the reversion of nonsense mutations, the majority of "revertants" often occur not in the structural gene but at suppressor loci, which behave differently in their responses to UV light (4, 8, 9). The lacl system allows an examination of forward mutagenesis at 65 individual sites where nonsense mutations can arise by a single base substitution. Because both the DNA sequence and the location of the nonsense mutations have been established (10), each mutation can be attributed to a specific transition or transversion event.Detailed knowledge ofthe sites at which mutagenesis occurs and of the specific base changes produced may yield important clues about the premutagenic lesions and how they are processed. In the case ofUV irradiation, the mutational mechanism is poorly understood. Photoreactivation experiments suggest that mutagenesis in both excision-proficient and excision-deficient strains requires pyrimidine dimers (11,12). Nevertheless, considerable evidence exists that not all UV-induced mutagenesis occurs at the sites of induced lesions (13). For example, undamaged bacteriophage A plated on UV-irradiated hosts show high levels of recA+-dependent mutagenesis ("indirect mutagenesis") (14). In a current model, UV-induced mutagenesis in E. coli proceeds through a recA+-dependent, inducible error-prone repair pathway called "SOS" repair [reviewed by Witkin (13)]. It is postulated that, where alternative repair pathways are unable to function...

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“…In order to determine the type of mutations induced by SspC, we analyzed the ability of a number of bacteriophage T4 mutants to grow on the E. coli mutants (3,10,14). The E. coli strain used for this analysis was WU3610, a derivative of strain B/r containing an ochre mutation rendering the strain tyrosine dependent and an amber mutation causing leucine dependence.…”

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confidence: 99%

Mutation and killing of Escherichia coli expressing a cloned Bacillus subtilis gene whose product alters DNA conformation

et al. 1992

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Expression of the BaciUus subtilis gene coding for SspC, a small, acid-soluble protein, caused both killing and mutation in a number of Escherichia coli B and K-12 Table 2 was with ampicillin (50 ,ug/ml); the other two plasmids were selected for with chloramphenicol (10 ,ug/ml). To construct plasmid pET-3SspC, the 0.6-kb HindIII fragment encompassing the sspC structural gene was isolated from pPS708wt. The ends of the latter fragment were filled in with the large fragment of E. coli DNA polymerase, and it was then ligated into the BamHI site of plasmid pET-3 after the BamHI ends had also been filled in. The ligation mixture was then used to transform strain HMS174 to ampicillin resistance, and colonies carrying the sspC gene were detected by colony hybridization. A strain with a plasmid in which the sspC coding sequence was just downstream of and in the correct orientation relative to the plasmid's T7 RNA polymerase promoter was then identified by digestion with EcoRV (13). The resulting plasmid, pET3SspC, was then introduced into E. coli BL21 and BL21(DE3), with or without the last two plasmids listed in Table 2.Mutation assays. Cells were grown at 37°C (unless other-

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A determination of mutagen specificity in bacteria using nonsense mutants of bacteriophage T4

Cited by 152 publications

References 18 publications

Effect of Enzymatic Adenylylation on Dihydrostreptomycin Accumulation in Escherichia coli Carrying an R-Factor: Model Explaining Aminoglycoside Resistance by Inactivating Mechanisms

Effect of Enzymatic Adenylylation on Dihydrostreptomycin Accumulation in Escherichia coli Carrying an R-Factor: Model Explaining Aminoglycoside Resistance by Inactivating Mechanisms

Mutational specificity of UV light in Escherichia coli: indications for a role of DNA secondary structure.

Mutation and killing of Escherichia coli expressing a cloned Bacillus subtilis gene whose product alters DNA conformation

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