Quinolone-resistant mutations of the gyrA gene of Escherichia coli

Summary DNA fragments of 8.5 kb containing the gyrA gene were cloned from Escherichia coli KL-16 and from four spontaneous gyrA mutants which showed various levels of resistance to quinolones. The gyrA gene was situated at about 4 kb in front of the nrdA gene and transcribed counterclockwise on the E. coli chromosome. It encoded a polypeptide of 875 amino acids with a molecular weight of about 97000. The four gyrA mutations were located strikingly close to one another within a small region near the N-terminus of the gyrA polypeptide, i.e., nucleotide changes from C to T, from C to G, from G to T and from G to T at nucleotides 248, 248, 318 and 199, respectively, resulting in amino acid changes from Ser to Leu, from Ser to Trp, from Gln to His and from Ala to Ser at amino acids 83, 83, 106 and 67, respectively. These mutations were situated in the relatively hydrophilic regions of the GyrA polypeptide and close to Tyr at amino acid 122 which has been shown to be the site covalently bound to DNA.     

An Escherichia coli plasmid-based, mutational system in which supF mutants are selectable: insertion elements dominate the spontaneous spectra.

A new system is described to determine the mutational spectra of mutagens and carcinogens in Escherichia coli; data on a limited number (142) of spontaneous mutants is presented. The mutational assay employs a method to select (rather than screen) for mutations in a supF target gene carried on a plasmid. The E. coli host cells (ES87) are lacI⁻ (am26), and carry the lacZΔM15 marker for -complementation in β-galactosidase. When these cells also carry a plasmid, such as pUB3, which contains a wild-type copy of supF and lacZ-, the lactose operon is repressed (off). Furthermore, supF suppression of lacl^um26 results in a lactose repressor that has an uninducible, lacl^S genotype, which makes the cells unable to grow on lactose minimal plates. In contrast, spontaneous or mutagen-induced supF⁻ mutations in pUB3 prevent suppresion of lacl^am26 and result in constitutive expression of the lactose operon, which permits growth on lactose minimal plates. The spontaneous mutation frequency in the supF gene is 0.7 and 1.0 × 10⁻⁶ without and with SOS induction, respectively. Spontaneous mutations are dominated by large insertions (67% in SOS-uninduced and 56% in SOS-induced cells), and their frequency of appearance is largely unaffected by SOS induction. These are identified by DNA sequencing to be Insertion Element: IS1 dominates, but IS4, IS5, gamma-delta and IS10 are also obtained. Large deletions also contribute significantly (19% and 15% for - SOS and +SOS, respectively), where a specific deletion between a 10 base pair direct repeat dominates; the frequency of appearance of these mutations also appears to be unaffected by SOS induction. In contrast, SOS induction increases base pairing mutations (13% and 27% for -SOS and +SOS, respectively), The ES87/pUB3 system has many advantages for determining mutational spectra, including the fact that mutant isolation is fast and simple, and the determination of mutational changes is rapid because of the small size of supF.     

Role of the RuvAB protein in avoiding spontaneous formation of deletion mutations in the Escherichia coli K-12 endogenous tonB gene

The endogenous tonB gene of Escherichia coli was used as a target for spontaneous deletion mutations which were isolated from ruvAB-, recG-, and ruvC- cells. The rates of tonB mutation were essentially the same in ruv+, ruvAB-, recG-, and ruvC- cells. We analyzed tonB mutants by sequencing. In the ruv+, recG-, and ruvC- strains, the spectra were different from those obtained from the ruvAB- cells, where deletions dominated followed by IS insertions, base substitutions, and frameshifts, in that order. We then analyzed the tonB-trp large deletion, due to simultaneous mutations of the trp operon, and found that the frequency in ruvAB- was higher than those in ruv+, recG-, and ruvC- cells. To characterize deletion formation further, we analyzed all the tonB mutants from one colicin plate. Seven deletions were identified at five sites from the 45 tonB mutants of ruv+ cells and 24 deletions at 11 sites from the 43 tonB mutants of ruvAB- cells. Thus, the ruvAB- strain is a deletion mutator. We discuss the role of RuvAB in avoiding deletions.     

Probing the antigenicity of E. coli l-asparaginase by mutational analysis

A strategy, termed alanine-scanning mutagenesis, was used to identify the amino acid residues which are critical to the antigenicity of Escherichia coli l-asparaginase (l-ASP). Three continuous alkaline residues, 195RKH197, were mutated to Ala selectively. Four mutant recombinant l-ASPs were constructed and expressed in E. coli, and then purified. The purified mutants showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were more than 95% pure by reverse high-perfomance liquid chromatography. The activities of wild-type and m l-ASPs in the fermentative medium were all about 130 U/mL. The change from 195RKH 197 to 195AAA 197 reduced the antigenicity ofhe enzyme greatly as shown in competition enzyme-linked immunosorbent assay using polyclonal antibodies raised against the wild-type l-ASP from rabbits. The results show that residues 195RKH197 of E. coli l-ASP are critical to its antigenicity. These authors contributed equally to this work.     

Spectrum of spontaneous mutations in a cDNA of the human hprt gene integrated in chromosomal DNA

Summary Altered sequences were determined of 52 independent spontaneous mutations occuring in a cDNA of the human hypoxanthine phosphoribosyltransferase (hprt) gene, which was integrated into chromosomal DNA of the mouse cell as a part of the retroviral shuttle vector. Spontaneous mutations comprised a variety of events: base substitutions, frameshifts, deletions, duplications, and complex mutational events, and were distributed randomly over the coding region of the gene. Frameshifts were the most frequent mutational event (38%), and base substitutions were the next most frequent (25%), followed by deletions (19%). Frameshift and deletion mutations commonly occurred preferentially at sites flanked by short direct repeats. Short inverted repeats were frequently found to be associated with duplication and complex mutational events. Analysis of the sequence alterations in the mutant genes suggests that misalignment mutagenesis represents an important molecular mechanism for the generation of spontaneous mutations in eukaryotic cells.     

A.T----C.G transversions and their prevention by the Escherichia coli mutT and mutHLS pathways

Summary Escherichia coli mutT strains are strong mutators yielding only A · T C · G transversion mutations. These are thought to result from uncorrected (or unprevented) A · G mispairings during DNA replication. We have investigated the interaction of mutT-induced replication errors with the mutHLS-encoded postreplicative mismatch repair system. By measuring mutation frequencies in both forward and reversion systems, we have demonstrated that mutTmutL and mutTmutS double mutators produce no more mutants than expected from simple additivity of the frequencies in the single mutators. We conclude that mutT-induced A · G replication errors are not recognized by the MutHLS system. On the other hand, direct measurements of mismatch repair by transfection of bacteriophage M13mp2 heteroduplex DNA as well as mutational data from strains other than mutT indicate that the MutHLS system can repair at least certain A · G mispairs. We suggest that A · G mispairs may exist in several different conformations, some of which are recognized by the MutHLS system. However, the A · G mispairs normally prevented by the mutT function are refractory to mismatch repair, indicating that they may represent a structurally distinct class.     

RexAB proteins of bacteriophage lambda enhance the effect of photolyase-dimer complexes on lacZ gene expression in Escherichia coli.

Summary Expression of the lacZ gene in Escherichia coli is inactivated by exposure to ultraviolet light (UV). Inactivation is exceptionally effective when cells contain amplified levels of DNA photolyase (which forms complexes with pyrimidine dimers in the absence of light for actual photoreversal) and a prophage. Without amplified photolyase, the prophage or both, inactivation rates are similar and much lower. UV-inactivation of lacZ gene expression in the presence of both amplified photolyase and is even more effective if cI857 is used in place of the wildtype prophage but is wholly unexceptional if the prophage carries defects in the genes rexA or rexB. When Rex AB proteins are provided by expression from a plasmid and the cell also contains amplified photolyase, exceptional inactivation rates again obtain; in fact inactivation is most effective under these conditions. The data are considered to reveal a role for Rex AB proteins, which mediate superinfection exclusion, in the exceptional inactivation of gene expression by photolyase bound to pyrimidine dimers in DNA. Photolyase-dimer complexes may mimic the structure of certain complexes that arise during phage development and thus influence Rex A and/or B proteins, thereby shutting down cell metabolism.     

DNA sequence analysis of spontaneous and γ-radiation (anoxic)-induced lacI mutations in Escherichia coliumuC122::Tn5: differential requirement for umuC at G · C vs. A · T sites and for the production of transversions vs. transitions

Escherihica coliumC122::Tn5 cells were γ-radiated (¹³⁷Cs, 750 Gy, under N₂), and lac-constitutive mutants were produced at 36% of the wild-type level (the umC strain was not deficient in spontaneous mutagenesis, and the mutational spectrum determined by sequencing 263 spontaneous lacI^d mutations was very similar to that for the wild-type strain). The specific nature of the umC strain's partial radiation was determined by sequencing 325 radiation-induced lacI^d mutations. The yields of radiation-induced mutation classes in the umC strain (as a percentage of the wild-type yield) were: 80% for A · T → G · C transitions, 70% for multi-base additions, 60% for single-base deletions, 53% for A · T → C · G transversions, 36% for G · C → A · T transitions, 25% for multi-base deletions, 21% for A · T → T · A transversions, 11% for G · C → C · G transversions, 9% for G · C → T · A transversions and 0% for multiple mutations. Based on these deficiencies and other factors, it is concluded that the umC strain is near-normal for A · T → G · C transitions, single-base deletions and possibly A · T → C · G transversions; is generally deficient for mutagenesis at G · C sites fro transversions, and is grossly deficient in multiple mutations. Damage at G · C sites seems more difficult for translesion DNA synthesis to bypass than damage at A · T sites, and especially when trying to produced a transversion. The yield of G · C → A · T transitions in the umC strain *36% of the wild-type level) argues that a basic sites are involved in no more than 64% of γ-radiation-induced base substitutions in the wild-type strain. Altogether, these data suggest that the UmuC and UmuD′ proteins facilitate, rather than being absolutely required for, translesion DNA synthesis; with the degree of facilitation being dependent both on the nature of the noncoding DNA damage, i.e., at G · C vs A · T sites, and on the nature of the misincorporated base, i.e., whether it induces transversions or transitions.     

An Escherichia coli plasmid-based, mutational system in which supF mutants are selectable: Insertion elements dominate the spontaneous spectra

A new system is described to determine the mutational spectra of mutagens and carcinogens in Escherichia coli; data on a limited number (142) of spontaneous mutants is presented. The mutational assay employs a method to select (rather than screen) for mutations in a supF target gene carried on a plasmid. The E. coli host cells (ES87) are lacI⁻ (am26), and carry the lacZΔM15 marker for α-complementation in β-galactosidase. When these cells also carry a plasmid, such as pUB3, which contains a wild-type copy of supF and lacZ-α, the lactose operon is repressed (off). Furthermore, supF suppression of lacl^um26 results in a lactose repressor that has an uninducible, lacl^S genotype, which makes the cells unable to grow on lactose minimal plates. In contrast, spontaneous or mutagen-induced supF⁻ mutations in pUB3 prevent suppresion of lacl^am26 and result in constitutive expression of the lactose operon, which permits growth on lactose minimal plates. The spontaneous mutation frequency in the supF gene is 0.7 and 1.0 × 10⁻⁶ without and with SOS induction, respectively. Spontaneous mutations are dominated by large insertions (67% in SOS-uninduced and 56% in SOS-induced cells), and their frequency of appearance is largely unaffected by SOS induction. These are identified by DNA sequencing to be Insertion Element: IS1 dominates, but IS4, IS5, gamma-delta and IS10 are also obtained. Large deletions also contribute significantly (19% and 15% for - SOS and +SOS, respectively), where a specific deletion between a 10 base pair direct repeat dominates; the frequency of appearance of these mutations also appears to be unaffected by SOS induction. In contrast, SOS induction increases base pairing mutations (13% and 27% for -SOS and +SOS, respectively), The ES87/pUB3 system has many advantages for determining mutational spectra, including the fact that mutant isolation is fast and simple, and the determination of mutational changes is rapid because of the small size of supF.     

Spontaneous and 9-aminoacridine-induced frameshift mutagenesis: second-site frameshift mutation within the N-terminal region of the lacI gene of Escherichia coli

Summary A novel forward mutational system, based on the acquisition of an I^q-d dominant phenotype from an initial I^q− recessive state, was used to identify second-site frameshift mutation [±1(±3 _n ) events] within the N-terminal region of thelacI gene ofEscherichia coli. The DNA sequences are described of forty-six spontaneous and twenty 9-aminoacridine(9-AA)-induced second site mutations. Although −1 frameshift events dominate both spectra, the nature and site specificity of these events clearly distinguish two mutational distributions. The spontaneous distribution contains two −(A: T) frameshift hotspots; one within a monotonic A₅ run (9 occurrences), the other at a 5′-CACAACAAC-3′ sequence (12 occurrences). In contrast 17 of the 20 mutations recovered after 9-AA treatment involve the loss of a G: C pair, 14 of which occur at a single site (5′-CGGGC-3′). The striking specificity of the observed mutational hotspots is of interest since this open genetic target contains similar sequences which were infrequently recovered.     

Mutation probe of gene structure in E. coli: suppressor mutations in the seven-tRNA operon

Summary Cells defective in uracil-DNA glycosylase (ung:: Tn10) were used in two ways to reveal differences in select point mutations (GC to AT transitions) within the seven-tRNA operon of E. coli. The mutations were indicated as de novo or converted glutamine tRNA suppressor mutations in the genes glnU and/or glnV: (1) the kinetics of photoenzymatic monomerization of pyrimidine dimers quantitated by ung-dependent UV mutagenesis indicated more rapid repair of dimers at sites for converted suppressor mutation than of dimers at sites for de novo suppressor mutation, and (2) spontaneous deamination of cytosine was considerably more frequent at sites for converted suppressor mutation than at sites for de novo suppressor mutation. To explain these results we suggest the physical structure of the DNA in vivo is different at different sites in the seven-tRNA operon. The non-transcribed strand including specifically the anticodon region of the site for converted suppressor mutation may frequently be looped out in a single strand so that a T=C dimer is more accessible to DNA photolyase or a free cytosine residue of non-irradiated DNA is in an aqueous environment conducive to deamination. In addition, we analysed the spontaneous de novo suppressor mutation data to determine an estimate for the in vivo rate of cytosine deamination in double strand DNA of 3.2×10¹³/sec.