A method for isolating nonsense suppressors in enterobacteriaceae using an amber mutant of the drug resistance factor R1

Abstract

We describe here the isolation of a mutant derivative of the drug resistance factor R1 (Meynell and Datta, 1966) that carries a nonsense mutation in a gene determining resistance to penicillins. We have used this mutant R1 to isolate derivatives of Escherichia coli and Klebsiella pneumoniae that contain nonsense suppressors (Sup- strains) by screening penicillin-resistant revertants of strains containing the mutant R factor for the presence of such suppressors. This obviates the need to have known nonsense mutations in chromosomal genes. Theoretically, suppressor-containing derivatives of any bacterial species that can maintain and express R1 can be constructed.

Allele Specific and Locus Non-specific Suppressors in Aspergillus nidulans

“…In bacteria and viruses, several different approaches for the direct identification of nonsense suppressors are available (Benzer & Champe, 1962;Kennedy & Crowlesmith, 1975;Mindich et al, 1976). In Eukaryota, no such approach exists.…”

Characterization of beta-lactamase-deficient (bla) mutants of the R plasmid R1 in Escherichia coli K-12 and comparison with similar mutants of RP1

“…Bacterial strains were all Escherichia coli K-12 derivatives. UB1301 and UB1325, our laboratory designations for W3110 Rifr and J6-2 Nalr, respectively (20), were the standard host strains. JC7620, a recB recC sbcB strain (21), was used as a recipient for transformation.…”

“…Both plasmids carry the bla gene encoding the type IIIa (TEM) ,B-lactamase. bla mutants were isolated by nitrosoguanidine mutagenesis, as described previously (20 (7,8). RP1 bla-6 (this paper) was isolated in the same way as were the Rl bla mutants, except that the host strain used was UB1139 (10).…”

“…RP1 bla-6 (this paper) was isolated in the same way as were the Rl bla mutants, except that the host strain used was UB1139 (10). Only one mutant was isolated from 100,000 colonies screened, a frequency only 1% of that obtained with Ri (20) 7 h to permit conjugation and recombination. Portions of0.5 ml were then inoculated into 100 ml of nutrient broth and grown overnight to permit the expression of carbenicillin resistance in cells carrying recombinant bla' plasmids.…”

“…Conjugation was carried out by using standard procedures (20). Transformation (5) was performed as described by Bennett et al (1).…”