Reversion of Frameshift Mutations by Mutator Genes in Escherichia coli

The Escherichia coli mutator genes mutU4, mutS3, and mut-25 (a possible allele of mutL), previously known to induce transitional base changes, increased significantly the frequencies of reversion of lacZ frameshift mutations. mutT1, previously shown to induce only the transversion of adenine-thymine to cytosine-guanine, had no effect on the reversion of lacZ frameshift mutations. With mutator genes other than mutT1, small increases were found in the frequencies of reversion of trpA frameshift mutations.     

Adaptive Reversion of a Frameshift Mutation in Escherichia Coli

Mutation rates are generally thought not to be influenced by selective forces. This doctrine rests on the results of certain classical studies of the mutations that make bacteria resistant to phages and antibiotics. We have studied a strain of Escherichia coli which constitutively expresses a lacI-lacZ fusion containing a frameshift mutation that renders it Lac-. Reversion to Lac+ is a rare event during exponential growth but occurs in stationary cultures when lactose is the only source of energy. No revertants accumulate in the absence of lactose, or in the presence of lactose if there is another, unfulfilled requirement for growth. The mechanism for such mutation in stationary phase is not known, but it requires some function of RecA which is apparently not required for mutation during exponential growth.     

Growth Phase-Dependent Regulation of Vsr Endonuclease May Contribute to 5-Methylcytosine Mutational Hot Spots in Escherichia coli

Using rabbit polyclonal antibodies, we have shown that the Dcm cytosine methylase of Escherichia coli is maintained at a constant level during cell growth, while Vsr endonuclease levels are growth phase dependent. Decreased production of Vsr relative to Dcm during the log phase may contribute substantially to the mutability of 5-methylcytosine.     

Thymineless Death in Escherichia coli 15T− and Recombinants of 15T− and Escherichia coli K-12

Thymineless death was examined in Escherichia coli 15T(-) and recombinants of 15T(-) and E. coli K-12. Those strains that were very sensitive to thymine deprivation were also very sensitive to a variety of inducing agents (mitomycin C, ultraviolet light, hydroxyurea, and nalidixic acid). Those strains that were relatively resistant to thymineless death were also relatively resistant to the inducing agents. After exposure to thymineless death and the inducing agents, sensitive strains lysed, produced colicin, and had phage particles in their lysates. These strains also showed an increase in the 6-methyladenine content of their deoxyribonucleic acid (DNA) and an increase in the DNA methylase activity of their crude extracts under these conditions. None of these effects was noted in the strains relatively resistant to thymineless death and the inducing agents. These data indicate that there are two types of thymineless death. One is represented by the strains that are very sensitive to thymine deprivation and other inducing agents and is secondary to the induction of phage psi. The strains more resistant to thymine deprivation and the other inducing agents undergo a non-phage-mediated thymineless death. The mechanism of this latter process is currently under study.     

Role of the Escherichia coli SurA Protein in Stationary-Phase Survival

SurA is a periplasmic peptidyl-prolyl isomerase required for the efficient folding of extracytoplasmic proteins. Although the surA gene had been identified in a screen for mutants that failed to survive in stationary phase, the role played by SurA in stationary-phase survival remained unknown. The results presented here demonstrate that the survival defect of surA mutants is due to their inability to grow at elevated pH in the absence of ς^S. When cultures of Escherichia coli were grown in peptide-rich Luria-Bertani medium, the majority of the cells lost viability during the first two to three days of incubation in stationary phase as the pH rose to pH 9. At this time the surviving cells resumed growth. In cultures of surA rpoS double mutants the survivors lysed as they attempted to resume growth at the elevated pH. Cells lacking penicillin binding protein 3 and ς^S had a survival defect similar to that of surA rpoS double mutants, suggesting that SurA foldase activity is important for the proper assembly of the cell wall-synthesizing apparatus.     

Excision Repair Characteristics of recB−res− and uvrC− Strains of Escherichia coli

An Escherichia coli strain carrying the recB21 and res-1 mutations showed an abnormally low level of colony-forming ability although it grew essentially normally in liquid medium. The recB21 res-1 strain showed little, if any, of the ultraviolet (UV)-induced deoxyribonucleic acid (DNA) breakdown characteristic of the res-1 mutant. Nevertheless, the double mutant was far more sensitive to UV than either the res-1 or the recB21 strain. When compared with a wild-type strain, the rate of release of dimers from UV-irradiated DNA was very slow in the recB21 res-1, but normal in the res-1 recB(+) or recB21 res(+) mutants. However, the ratio of dimer-to-thymine released into the acid-soluble fraction was three times higher than the wild type in recB21 res(+) and recB21 res-1 and only one-tenth as high as the wild type in res-1 rec(+). Alkaline sucrose gradient centrifugation revealed occurrence of single-strand incision of UV-irradiated DNA and the restitution of nicked DNA at a similar rate in the recB21 res-1 and recB21 res(+) strains. Mutants uvrC(-) showed increased amounts of nicks in their DNA with increasing incubation time after UV irradiation, although no detectable amounts of dimers were excised from UV-irradiated DNA. From these results, it is concluded that the increased sensitivity of the res-1 strain to UV light is due to a reduced ability to excise dimers from UV-irradiated DNA and that the high rate of UV-induced breakdown of DNA is not the primary cause. A possible role of uvrC gene in the excision repair is discussed.     

Reversion of a streptomycin-dependent strain of Escherichia coli

Summary A streptomycin dependent, spectinomycin resistant mutant ofEscherichia coli was used to select spontaneous phenotypic revertants to non-dependence on streptomycin. The ribosomes from one such revertant, which is inhibited by both streptomycin and spectinomycin, were analyzedin vitro. The altered protein responsible for the suppression of the streptomycin dependent phenotype was identified; this protein is 30S-10. The genetic locus for this mutation is a newly identified locus and it has been positioned close to thestr locus. The identification of the altered component responsible for the suppression of the spectinomycin resistant phenotype may be the same as that for the streptomycin dependent phenotype, but this is unproven.     

Functional interactions between the MutL and Vsr proteins of Escherichia coli are dependent on the N-terminus of Vsr

The crystal structure of the Escherichia coli Vsr endonuclease bound to a C(T/G)AGG substrate revealed that the DNA is held by a pincer composed of a trio of aromatic residues which intercalate into the major groove, and an N-terminus alpha helix which lies across the minor groove. We have constructed an N-terminus truncation (Delta14) which removes most of the alpha helix. The mutant is still fairly proficient in mediating very short patch repair. However, its endonuclease activity is considerably reduced and, in contrast to that of the wild type protein, cannot be stimulated by MutL. We had shown previously that excess Vsr in vivo causes mutagenesis, probably by inhibiting the participation of MutL in mismatch repair. The Delta14 mutant has diminished mutagenicity. In contrast, four enzymatically inactive mutants, with intact N-termini, are as mutagenic as the wild type protein. On the basis of these results we suggest that MutL causes a conformational change in the N-terminus of Vsr which enhances Vsr activity, and that this functional interaction between Vsr and MutL decreases the ability of MutL to carry out mismatch repair.     

Reversion Instability in the Galactose operon of Escherichia coli

The gal-3 mutation, which had been shown previously to produce unstable revertants, was combined in three instances with recA, a mutation which suppresses recombination. Unstable revertants were produced in the gal-3 recA recombinants qualitatively as frequently as in the absence of the recA gene, and it is concluded that a recombination mechanism is not the basis of the instability observed.     

Reversion of Ribosomal Helix Formation in Escherichia coli

After transfer into fresh medium, Escherichia coli cells containing ribosomal helices resume growth without a lag period. The helices disappear within 15 min after transfer, the number of 70S ribosomes decreases, and a steady-state ribosomal profile appears within one cell generation time. Subunits isolated from the helices support in vitro protein synthesis, but efficiency is optimal only when supplemented with an undetermined factor that is contained in the S-100 fraction of log-phase cells. The data suggest a possible role of helices as ribosomal reserve units.     

Deoxyribonucleic Acid Synthesis and Cell Division in a lon− Mutant of Escherichia coli

The lon(-) mutants of Escherichia coli form long filamentous cells after temporary inhibition of deoxyribonucleic acid (DNA) synthesis by ultraviolet irradiation, treatment with nalidixic acid, or thymine starvation. The kinetics of DNA synthesis and cell division after a period of thymine starvation have been compared in lon(+) and lon(-) cells. After this treatment, both kinds of cells recover their normal DNA to mass ratio with the same kinetics. In contrast to previous reports, cell division is found to recommence in both lon(+) and in lon(-) cells after such a temporary period of inhibition of DNA synthesis. However, the delay separating the recommencement of DNA synthesis and of cell division is approximately three times as long in lon(-) as in lon(+) cells. Low concentrations of penicillin inhibit cell division in both lon(+) and lon(-) cells. In this case, cell division recommences with the same kinetics in both strains after the removal of penicillin. This suggests that different steps in the cell division process are blocked by inhibition of DNA synthesis and by penicillin treatment. The lon(-) mutation appears to affect the former of these steps.     

Thymineless Death in polA+ and polA− Strains of Escherichia coli

The extreme sensitivity of polA(-) cells to thymineless death is due, primarily, to the absence of an extended lag prior to the commencement of death. Once thymineless death has commenced, the rate in polA(-) cells is only slightly faster than in polA(+) cells.     

Antimicrobial Drug Resistance Genes Do Not Convey a Secondary Fitness Advantage to Calf-Adapted Escherichia coli

Maintenance of antimicrobial drug resistance in bacteria can be influenced by factors unrelated to direct selection pressure such as close linkage to other selectively advantageous genes and secondary advantage conveyed by antimicrobial resistance genes in the absence of drug selection. Our previous trials at a dairy showed that the maintenance of the antimicrobial resistance genes is not influenced by specific antimicrobial selection and that the most prevalent antimicrobial resistance phenotype of Escherichia coli is specifically selected for in young calves. In this paper we examine the role of secondary advantages conveyed by antimicrobial resistance genes. We tested antimicrobial-susceptible null mutant strains for their ability to compete with their progenitor strains in vitro and in vivo. The null mutant strains were generated by selection for spontaneous loss of resistance genes in broth supplemented with fusaric acid or nickel chloride. On average, the null mutant strains were as competitive as the progenitor strains in vitro and in newborn calves (in vivo). Inoculation of newborn calves at the dairy with antimicrobial-susceptible strains of E. coli did not impact the prevalence of antimicrobial-resistant E. coli. Our results demonstrate that the antimicrobial resistance genes are not responsible for the greater fitness advantage of antimicrobial-resistant E. coli in calves, but the farm environment and the diet clearly exert critical selective pressures responsible for the maintenance of antimicrobial resistance genes. Our current hypothesis is that the antimicrobial resistance genes are linked to other genes responsible for differential fitness in dairy calves.     

Determining the Optimal Thymidine Concentration for Growing Thy− Escherichia coli Strains

Changes of thymidine concentration in the growth medium affect the chromosome replication time of Thy⁻ strains without at the same time causing a detectable difference in the growth rate (R. H. Pritchard and A. Zaritsky, Nature 226:126–131, 1970). Consequently, the optimal thymidine concentration cannot be determined by ascertaining which concentration produces the highest growth rate. Here we present a method for determining the optimal thymidine concentration of any Thy⁻ Escherichia coli strain. Using this method, we found that the E. coli “wild-type” strain MG1655 has a partial Thy⁻ phenotype.     

Defective Excision Repair of Pyrimidine Dimers in the Ultraviolet-Sensitive Escherichia coli ras− Mutant

The ras(-) mutant of Escherichia coli K-12 is sensitive to ultraviolet (UV) light but only slightly sensitive to X-irradiation (1.5-fold increase). Other phenotypic properties include normal recombination ability and normal host cell reactivation ability but an abnormally high frequency of UV-induced mutation. The response of the ras(-) mutant to UV has been studied biochemically. After low doses of UV, the ras(-) mutant degraded excessive amounts of deoxyribonucleic acid, and long delays in resumption of deoxyribonucleic acid synthesis occurred. Pyrimidine dimers were excised at the normal rate. Although the mutant had the capability of initiating repair replication, the process was not completed after the high UV dose required to allow detection of repair replication. The ras(-) mutant, after low UV doses, left three to four times as many single-strand breaks not rejoined as did the wild-type strain.     

Transforming DNA Uptake Gene Orthologs Do Not Mediate Spontaneous Plasmid Transformation in Escherichia coli

Spontaneous plasmid transformation of Escherichia coli occurs on nutrient-containing agar plates. E. coli has also been reported to use double-stranded DNA (dsDNA) as a carbon source. The mechanism(s) of entry of exogenous dsDNA that allows plasmid establishment or the use of DNA as a nutrient remain(s) unknown. To further characterize plasmid transformation, we first documented the stimulation of transformation by agar and agarose. We provide evidence that stimulation is not due to agar contributing a supplement of Ca²⁺, Fe²⁺, Mg²⁺, Mn²⁺, or Zn²⁺. Second, we undertook to inactivate the E. coli orthologues of Haemophilus influenzae components of the transformation machine that allows the uptake of single-stranded DNA (ssDNA) from exogenous dsDNA. The putative outer membrane channel protein (HofQ), transformation pseudopilus component (PpdD), and transmembrane pore (YcaI) are not required for plasmid transformation. We conclude that plasmid DNA does not enter E. coli cells as ssDNA. The finding that purified plasmid monomers transform E. coli with single-hit kinetics supports this conclusion; it establishes that a unique monomer molecule is sufficient to give rise to a transformant, which is not consistent with the reconstitution of an intact replicon through annealing of partially overlapping complementary ssDNA, taken up from two independent monomers. We therefore propose that plasmid transformation involves internalization of intact dsDNA molecules. Our data together, with previous reports that HofQ is required for the use of dsDNA as a carbon source, suggest the existence of two routes for DNA entry, at least across the outer membrane of E. coli.The spontaneous transformation of Escherichia coli with plasmid DNA on nutrient-containing agar plates was described in at least three independent articles (14, 23, 24). However, no attempt to characterize the mechanism of plasmid DNA uptake has been reported. Genomic analysis revealed the presence in E. coli of a set of genes homologous to those required for DNA uptake in naturally transformable species, including the gram-positive Bacillus subtilis and Streptococcus pneumoniae and the gram-negative Haemophilus influenzae and Neisseria gonorrhoeae (9). The machine they potentially encode would allow the uptake of single-stranded DNA (ssDNA) from an exogenous double-stranded DNA (dsDNA) substrate in E. coli (Fig. ​(Fig.1).1). HofQ (called ComE in reference 7) is the ortholog of the PilQ secretin of N. gonorrhoeae, which constitutes a transmembrane channel required for exogenous dsDNA to traverse the outer membrane (OM) and reach the so-called transformation pseudopilus (8). According to the Bacillus subtilis paradigm (8), assembly of the pseudopilus requires a prepilin peptidase (PppA; called PilD in reference 7), a traffic NTPase (HofB; called PilB in reference 7), and a polytopic membrane protein (HofC; called PilC in reference 7). The pseudopilus, which would include PpdD (called PilA in reference 7), provides access for dsDNA to its receptor, YbaV (called ComE1 in reference 7), through the peptidoglycan. Degradation of one strand by an unidentified nuclease (N) would allow uptake of ssDNA through YcaI (called Rec2 in reference 7), a channel in the inner membrane. Finally, DprA (also named Smf) would be required to protect internalized ssDNA from endogenous nucleases, as shown in S. pneumoniae (4), and to assist the processing of ssDNA into transformants (16).Open in a separate windowFIG. 1.Diagrammatic representation of the putative E. coli DNA uptake machine. The E. coli orthologues of proteins required involved in the uptake of transforming DNA in naturally transformable species, including B. subtilis, S. pneumoniae, H. influenzae, and N. gonorrhoeae, were identified by genomic analysis (9). GspD is a PilQ paralogue (25% identity over 278 residues), which was considered in the present study as a possible alternative route for dsDNA across the OM. A prepilin peptidase (PppA; called PilD in reference 7) required for maturation and export of proteins constituting the transformation pseudopilus (see Table S1 in the supplemental material) is not drawn on this diagram. (Additional information regarding the relationship between E. coli and H. influenzae transformation genes, and a table listing the various alternative names used in the literature are available in the supplemental material.). Red crosses indicate components of the putative DNA uptake machine inactivated during this work. IM, inner membrane.In H. influenzae, transformation genes are preceded by unusual CRP (for cyclic AMP receptor protein) binding sites, now called CRP-S (7), that absolutely require a second protein, Sxy (also called TfoX), in addition to CRP for induction (19). Interestingly, bioinformatics analysis revealed the conservation of CRP-S sites in front of the corresponding E. coli genes (7), including all of the genes encoding the proteins shown in Fig. ​Fig.11 (except GspD). Furthermore, some of these genes were experimentally demonstrated to require CRP, cAMP (CRP''s allosteric effector), and Sxy for induction in E. coli, providing support to the view that CRP-S sites control a bona fide transformation regulon in this bacterium (7). However, the involvement of E. coli transformation genes in DNA uptake has not been documented, except for hofQ, which was reported to be required for the use of dsDNA as a nutrient (11, 18). Although the functionality of the E. coli transformation genes has not been confirmed experimentally, it is of note that the bioinformatics identification of a complete set of transformation genes in two other species not previously known to be naturally transformable, Streptococcus thermophilus and Bacillus cereus, opened the way to the demonstration of genetic transformation in these species (6, 15a).To characterize further spontaneous plasmid transformation in E. coli, we first identified parameters affecting plasmid transformation frequencies on plates. We then undertook to inactivate genes encoding the putative transformation-related DNA uptake machinery of E. coli (Fig. ​(Fig.1)1) and to compare the rate of spontaneous plasmid transformation in the corresponding mutants and in their wild-type parent. In addition, to get an insight into the process of plasmid DNA entry, we characterized the kinetics of plasmid monomer transformation because it was shown in S. pneumoniae that regeneration of an intact plasmid replicon requires the independent uptake (via the transformation machine) of complementary ssDNA from two monomers (21). Finally, we discuss the possible significance of our data regarding the entry of exogenous dsDNA in E. coli in the light of previous findings on the use of dsDNA as a carbon source in this species (11, 18).     

Endogenous Superoxide Dismutase Levels Regulate Iron-Dependent Hydroxyl Radical Formation in Escherichia coli Exposed to Hydrogen Peroxide

Aerobic organisms contain antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, to protect them from both direct and indirect effects of reactive oxygen species, such as O₂^·− and H₂O₂. Previous work by others has shown that Escherichia coli mutants lacking SOD not only are more susceptible to DNA damage and killing by H₂O₂ but also contain larger pools of intracellular free iron. The present study investigated if SOD-deficient E. coli cells are exposed to increased levels of hydroxyl radical (^·OH) as a consequence of the reaction of H₂O₂ with this increased iron pool. When the parental E. coli strain AB1157 was exposed to H₂O₂ in the presence of an α-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone (4-POBN)–ethanol spin-trapping system, the 4-POBN–^·CH(CH₃)OH spin adduct was detectable by electron paramagnetic resonance (EPR) spectroscopy, indicating ^·OH production. When the isogenic E. coli mutant JI132, lacking both Fe- and Mn-containing SODs, was exposed to H₂O₂ in a similar manner, the magnitude of ^·OH spin trapped was significantly greater than with the control strain. Preincubation of the bacteria with the iron chelator deferoxamine markedly inhibited the magnitude of ^·OH spin trapped. Exogenous SOD failed to inhibit ^·OH formation, indicating the need for intracellular SOD. Redox-active iron, defined as EPR-detectable ascorbyl radical, was greater in the SOD-deficient strain than in the control strain. These studies (i) extend recent data from others demonstrating increased levels of iron in E. coli SOD mutants and (ii) support the hypothesis that a resulting increase in ^·OH formation generated by Fenton chemistry is responsible for the observed enhancement of DNA damage and the increased susceptibility to H₂O₂-mediated killing seen in these mutants lacking SOD.     

Modeling of Escherichia coli Endonuclease V structure in complex with DNA

Endonuclease V (EndoV) is a metal-dependent DNA repair enzyme involved in removal of deaminated bases (e.g., deoxyuridine, deoxyinosine, and deoxyxanthosine), with pairing specificities different from the original bases. Homologs of EndoV are present in all major phyla from bacteria to humans and their function is quite well analyzed. EndoV has been combined with DNA ligase to develop an enzymatic method for mutation scanning and has been engineered to obtain variants with different substrate specificities that serve as improved tools in mutation recognition and cancer mutation scanning. However, little is known about the structure and mechanism of substrate DNA binding by EndoV. Here, we present the results of a bioinformatic analysis and a structural model of EndoV from Escherichia coli in complex with DNA. The structure was obtained by a combination of fold-recognition, comparative modeling, de novo modeling and docking methods. The modeled structure provides a convenient tool to study protein sequence-structure-function relationships in EndoV and to engineer its further variants.