Isolation of Deoxyribonucleic Acid Methylase Mutants of Escherichia coli K-12

Fourteen deoxyribonucleic acid (DNA) and 10 ribonucleic acid (RNA) methylation mutants were isolated from Escherichia coli K-12 by examining the ability of nucleic acids prepared from clones of unselected mutagenized cells to accept methyl groups from wild-type crude extract. Eleven of the DNA methylation mutants were deficient in 5-methylcytosine (5-MeC) and were designated Dcm. Three DNA methylation mutants were deficient in N(6)-methyladenine (N(6)-MeA) and were designated Dam. Extracts of the mutants were tested for DNA-cytosine:S-adenosylmethionine and DNA-adenine:S-adenosylmethionine methyltransferase activities. With one exception, all of the mutants had reduced or absent activity. A representative Dcm mutation was located at 36 to 37 min and a representative Dam mutation was located in the 60-to 66-min region on the genetic map. The Dcm mutants had no obvious associated phenotypic abnormality. The Dam mutants were defective in their ability to restrict lambda. None of the mutations had the effect of being lethal.     

Effect of Mutations in Deoxyribonucleic Acid Repair Pathways on the Sensitivity of Escherichia coli K-12 Strains to Nitrofurantoin

Escherichia coli K-12 strains that carry mutations in one or more genes coding for proteins involved in repair of deoxyribonucleic acid lesions are more sensitive to the antibiotic nitrofurantoin than are the nonmutant parent strains.     

Deoxyribonucleic Acid Transferred from Ultraviolet-Irradiated Excision-Defective Hfr Cells of Escherichia coli K-12

Deoxyribonucleic acid (DNA) transfer from (3)H-thymine-labeled Hfr cells has been measured by determining the amount of radioactivity remaining after selective lysis of the donor cells in the mating mixture. DNA transfer was less effectively reduced by ultraviolet irradiation of excision-defective Hfr cells than was the yield of recombinants. The buoyant density of DNA transferred from unirradiated and irradiated Hfr cells was equivalent to that of double-stranded DNA. Mating-dependent DNA synthesis in the recipient has been measured by mating Hfr cells deficient in thymidine kinase with irradiated thymine-requiring F(-) cells in the presence of (3)H-thymine. The extent of such DNA synthesis approximated the amount of DNA transferred from unirradiated donors. Neither DNA transfer nor mating-dependent DNA synthesis could be reliably measured when both parents were irradiated. It is proposed that transferred Hfr DNA is replicated in the recipient and that this replication still occurs when the Hfr DNA contains dimers.     

Effect of Thymine Starvation on Deoxyribonucleic Acid Repair Systems of Escherichia coli K-12

Thymine starvation of Escherichia coli K-12 results in greatly increased sensitivity to ultraviolet light (UV). Our studies, using isogenic strains carrying rec and uvr mutations, have shown the following. (i) Common to all strains tested is a change from multihit to single-hit kinetics of survival to UV after 60 min of thymine starvation. However, the limiting slope of UV survival curves decreases in the rec(+)uvr(+) strain and changes very little in several rec mutant strains and one uvrB mutant strain. Thus, when either the rec or uvr system is functioning alone, the limiting slopes of the UV survival curves are relatively unaffected by thymine starvation. (ii) Thymine starvation does not significantly inhibit repair processes carried out by either repair system alone; i.e., host cell reactivation of irradiated phage (carried out by the uvr system), excision of thymine dimers (uvr), or X-ray repair (rec). (iii) In a rec(+)uvr(+) strain, repair appears to be a synergistic rather than additive function of the two systems. However, after thymine starvation, repair capacity is reduced to about the sum of the repair capacities of the independent systems. (iv) The kinetics of thymineless death are not changed by rec and uvr mutations. This indicates that the lesions responsible for thymineless death are not repaired by rec or uvr systems. (v) Withholding thymine from thy rec(+)uvr(+) bacteria not undergoing thymineless death has no effect on UV sensitivity. Under these conditions one sees higher than normal UV resistance in the presence or absence of thymine. This is due to increased repair carried out by the uvr system. To explain these results we postulate that thymine starvation does not inhibit either the rec or uvr repair pathway directly. Rather it appears that thymine starvation results in increased UV sensitivity in part by inhibiting a function which normally carries out efficient coordination of rec and uvr pathways.     

Conjugal Deoxyribonucleic Acid Replication by Escherichia coli K-12: Effect of Chloramphenicol and Rifampin

Conjugal replication of R64-11 deoxyribonucleic acid (DNA) and the concomitant transfer of R64-11 DNA to DNA-deficient minicells are dependent upon processes that are inhibited by rifampin and chloramphenicol. The rifampin-sensitive product is not present in vegetatively growing cells and is needed to initiate both conjugal DNA replication in donor cells and DNA transfer to recipient minicells. If the rifampin-sensitive product is a ribonucleic acid (RNA) molecule (rather than RNA polymerase itself), our data indicate that this RNA species required for initiation of conjugal activity does not need to be translated into a protein product. The chloramphenicol-sensitive product(s) is present in vegetatively growing cells in sufficient quantity to permit most donor cells to carry out one round of plasmid conjugal replication and transfer. The initiation of second and subsequent rounds of conjugal replication and transfer are dependent on the synthesis of both the rifampin-sensitive and chloramphenicol-sensitive products. Our results demonstrate a correspondence between the amount of conjugal DNA replication in the donor and the amount of DNA transferred to recipient minicells under all conditions, and therefore suggest but do not prove that plasmid transfer is dependent on conjugal DNA replication. The results also add additional proof that R64-11 transfer to minicells is discontinuous. All of these results are discussed in regard to further refinements of old models for the mechanism of conjugal transfer as well as a more radical departure from current dogma.     

Effect of Iododeoxyuridine upon Conjugation and the Fate of Transferred Deoxyribonucleic Acid in Escherichia coli K-12

The incorporation of 5-iododeoxyuridine (IUdR) into Escherichia coli K-12 deoxyribonucleic acid (DNA) has been found to decrease significantly the viability of female strains A288 and JC411(r) but to have only minor effect upon their ability to act as conjugational recipients and to perform recombination after conjugation. In contrast, IUdR incorporation into male strain HfrC appears to interfere with both chromosome transfer and genetic recombination. By using IUdR to densitylabel female DNA, and carrying out large-scale matings with (3)H-thymidine-labeled male cells, we examined the fate of transferred DNA. After a 30-min mating, the T6-sensitive male cells were lysed, and the DNA of the merozygotes and remaining female cells was isolated. Initial centrifugation of this DNA in a CsCl gradient showed that the male and female DNA species were associated. The nature of this association of the parental DNA species was determined by formaldehyde denaturation followed by CsCl centrifugation. Denaturation of DNA isolated immediately after T6 lysis gave a peak of radioactivity banding at the density of light single-stranded DNA. However, denaturation of DNA isolated after T6 lysis and dilution of the cells into fresh medium, exhibited peaks of radioactivity banding at positions corresponding to single-stranded, density-labeled DNA. The results indicate that recombination after conjugation in E. coli takes place by a breakage-and-reunion mechanism. The process of recombination can be separated into two stages. In the first stage, the donor and recipient DNA molecules become associated. The second stage consists of the formation of phosphodiester bonds between the donor and recipient segments comprising the recombinant molecule.     

Glycolate Uptake by Mutant Strains of Escherichia coli K-12

Mutant strains of Escherichia coli K-12 were shown to be impaired in their ability to assimilate glycolate-2-(14)C. One strain (Glc-103) has lost the ability to oxidize glycolate; another strain (Glc-102) was relatively impermeable to the compound. A third strain (Glc-104) had undergone a similar loss in permeability, and, in addition, was deranged in the synthesis of either glyoxylate reductase or malate synthase G.     

Involvement of the recB-recC Nuclease (Exonuclease V) in the Process of X-Ray-Induced Deoxyribonucleic Acid Degradation in Radiosensitive Strains of Escherichia coli K-12

The ras, polA, exrA, recA, and uvrD3 strains of Escherichia coli K-12 degrade their deoxyribonucleic acid more extensively than wild-type strains after X irradiation. The relationship of the recB-recC nuclease (exonuclease V) to the degradation process in these strains was determined by comparing the degradation response of the original strains with that of strains containing an additional recB21 or recC22 mutation. The initial rate of degradation in ras, polA12, exrA, and recA13 strains after an exposure of 20 to 30 kR was reduced more than 10-fold by the presence of an additional recB21 or recC22 mutation. The extent of degradation in these irradiated strains after 90 to 120 min of incubation was reduced two- to fivefold. In the uvrD3 strain, a recC22 mutation caused a fourfold decrease in initial degradation rate and reduced the extent of degradation after 90 min of incubation by a factor of 1.6. The results are consistent with the statement that the degradation process is normally dependent on exonuclease V activity. However, the observation that 10 to 30% degradation always occurred even in recB or recC strains, which lack this enzyme, suggests that alternative degradation mechanisms exist.     

Mapping of a Mutation, polB100, Affecting Deoxyribonucleic Acid Polymerase II in Escherichia coli K-12

Direct assay for deoxyribonucleic acid polymerase II in extracts has been used to screen recombinants for the polB allele in Hfr × F⁻ crosses, F-ductants in episome transfer, and transductants in generalized transduction by phage P1. The polB gene is located at 2 min on the Escherichia coli linkage map; it is 39 to 64% co-transducible with leu. A mutant, E. coli polA1 polB100 polC (ts), deficient in deoxyribonucleic acid polymerases I and II and having a thermolabile deoxyribonucleic acid polymerase III, has been prepared by the P1-mediated cross: P1 (HMS85 polB100) × E. coli BT1026 polA1 polC (ts) leu⁻.     

Acid response of exponentially growing Escherichia coli K-12

Induction of acid tolerance response (ATR) of exponential-phase Escherichia coli K-12 cells grown and adapted at different conditions was examined. The highest level of protection against pH 2.5 challenges was obtained after adaptation at pH 4.5-4.9 for 60 min. To study the genetic systems, which could be involved in the development of log-phase ATR, we investigated the acid response of E. coli acid resistance (AR) mutants. The activity of the glutamate-dependent system was observed in exponential cells grown at pH 7.0 and acid adapted at pH 4.5 in minimal medium. Importantly, log-phase cells exhibited significant AR when grown in minimal medium pH 7.0 and challenged at pH 2.5 for 2 h without adaptation. This AR required the glutamate-dependent AR system. Acid protection was largely dependent on RpoS in unadapted and adapted cells grown in minimal medium. RpoS-dependent oxidative, glutamate and arginine-dependent decarboxylase AR systems were not involved in triggering log-phase ATR in cells grown in rich medium. Cells adapted at pH 4.5 in rich medium showed a higher proton accumulation rate than unadapted cells as determined by proton flux assay. It is clear from our study that highly efficient mechanisms of protection are induced, operate and play the main role during log-phase ATR.     

Acid-Adapted Strains of Escherichia coli K-12 Obtained by Experimental Evolution

Enteric bacteria encounter a wide range of pHs throughout the human intestinal tract. We conducted experimental evolution of Escherichia coli K-12 to isolate clones with increased fitness during growth under acidic conditions (pH 4.5 to 4.8). Twenty-four independent populations of E. coli K-12 W3110 were evolved in LBK medium (10 g/liter tryptone, 5 g/liter yeast extract, 7.45 g/liter KCl) buffered with homopiperazine-N,N′-bis-2-(ethanosulfonic acid) and malate at pH 4.8. At generation 730, the pH was decreased to 4.6 with HCl. By 2,000 generations, all populations had achieved higher endpoint growth than the ancestor at pH 4.6 but not at pH 7.0. All evolving populations showed a progressive loss of activity of lysine decarboxylase (CadA), a major acid stress enzyme. This finding suggests a surprising association between acid adaptation and moderation of an acid stress response. At generation 2,000, eight clones were isolated from four populations, and their genomes were sequenced. Each clone showed between three and eight missense mutations, including one in a subunit of the RNA polymerase holoenzyme (rpoB, rpoC, or rpoD). Missense mutations were found in adiY, the activator of the acid-inducible arginine decarboxylase (adiA), and in gcvP (glycine decarboxylase), a possible acid stress component. For tests of fitness relative to that of the ancestor, lacZ::kan was transduced into each strain. All acid-evolved clones showed a high fitness advantage at pH 4.6. With the cytoplasmic pH depressed by benzoate (at external pH 6.5), acid-evolved clones showed decreased fitness; thus, there was no adaptation to cytoplasmic pH depression. At pH 9.0, acid-evolved clones showed no fitness advantage. Thus, our acid-evolved clones showed a fitness increase specific to low external pH.     

Fatty Acid Replacements in a Fatty Acid Auxotroph of Escherichia coli

Unsaturated fatty acids having structural features which are different from those of the monoenoic acids normally synthesized by Escherichia coli can serve as growth factors for an auxotroph requiring unsaturated fatty acids. These analogues were incorporated into the phospholipids, as shown by gas-liquid and thin-layer chromatographic analysis of the phospholipid fatty acid composition. Some of these fatty acids were cisDelta(5)- and cis-Delta(9)-tetradecenoic, cis-Delta(11)-eicosenoic, cis,cis-Delta(11,14)-eicosadienoic, cis,cis,cis-Delta(11,14,17)-eicosatrienoic, trans-Delta(9)- and trans-Delta(11)-octadecenoic acids. Although partial degradation of some of these analogues to shorter even-chain homologues occurred, chain elongation of the exogenous fatty acids was not detected. Trans-olefinic acids were utilized without stereochemical or positional isomerization. These studies provide a basis for exploring the properties of the fatty acids and phospholipids required for the formation, structure, and function of membranes.     

Conjugal Deoxyribonucleic Acid Replication by Escherichia coli K-12: Stimulation in dnaB(ts) Donors by Minicells

R64-11(+) donor cells that are thermosensitive for vegetative DNA replication will synthesize DNA at the restrictive temperature when recipient minicells are present. This is conjugal DNA replication because it is R64-11 DNA that is being synthesized and there is no DNA synthesis if minicells that cannot be recipients of R64-11 DNA are used. The plasmid DNA present in the donor cells before mating is transferred to recipient minicells within the first 20 min of mating, but additional copies of plasmid DNA synthesized during the mating continue to be transferred for at least 90 min. However, the transfer of R64-11 DNA to minicells is not continuous because the plasmid DNA in minicells is the size of one R64-11 molecule or smaller, and there are delays between the rounds of plasmid transfer. DNA is synthesized in minicells during conjugation, but this DNA has a molecular weight much smaller than that of R64-11. Thus, recipient minicells are defective and are not able to complete the synthesis of a DNA strand complementary to the single-stranded R64-11 DNA received from the donor cell.     

Chromosome Transfer and Recombinant Formation with Deoxyribonucleic Acid Temperature-Sensitive Strains of Escherichia coli

Haploid recombinant yield is reduced in matings conducted at 42.5 C between deoxyribonucleic acid (DNA) temperature-sensitive [dna⁻(TS)] recipients unable to synthesize DNA at 42.5 C and any of the three major donor types (Hfr, F⁺, F′) of Escherichia coli. No such reduction is observed in matings conducted at 42.5 C when the dna⁻(TS) mutation is in the donor parent. Evidence is presented which indicates that chromosome transfer from donors to recipients unable to replicate DNA at 42.5 C during vegetative growth occurs at normal frequencies when the mating is conducted at 42.5 C. It is concluded that some stage in haploid recombinant formation is adversely affected in dna⁻(TS) recipients mated at the temperature restrictive for DNA synthesis.     

Ionizing Radiation Damage to the Folded Chromosome of Escherichia coli K-12: Repair of Double-Strand Breaks in Deoxyribonucleic Acid

The extremely gentle lysis and unfolding procedures that have been developed for the isolation of nucleoid deoxyribonucleic acid (DNA; K. M. Ulmer et al., J. Bacteriol. 138:475-485, 1979) yield undamaged, replicating genomes, thus permitting direct measurement of the formation and repair of DNA double-strand breaks at biologically significant doses of ionizing radiation. Repair of ionizing radiation damage to folded chromosomes of Escherichia coli K-12 strain AB2497 was observed within 2 to 3 h of post-irradiation incubation in growth medium. Such behavior was not observed after post-irradiation incubation in growth medium of a recA13 strain (strain AB2487). A model based on recombinational repair is proposed to explain the formation of 2,200 to 2,300S material during early stages of incubation and to explain subsequent changes in the gradient profiles. Association of unrepaired DNA with the plasma membrane is proposed to explain the formation of a peak of rapidly sedimenting material (greater than 3,100S) during the later stages of repair. Direct evidence of repair of double-strand breaks during post-irradiation incubation in growth medium was obtained from gradient profiles of DNA from ribonuclease-digested chromosomes. The sedimentation coefficient of broken molecules was restored to the value of unirradiated DNA after 2 to 3 h of incubation, and the fraction of the DNA repaired in this fashion was equal to the fraction of cells that survived at the same dose. An average of 2.7 double-strand breaks per genome per lethal event was observed, suggesting that one to two double-strand breaks per genome are repairable in E. coli K-12 strain AB2497.     

Accumulation of toxic concentrations of methylglyoxal by wild-type Escherichia coli K-12

Wild-type strains of Escherichia coli K-12 accumulate toxic concentrations of methylglyoxal when grown in medium containing adenosine 3',5'-monophosphate and either d-xylose, l-arabinose, or d-glucose-6-phosphate, independent of the presence of other carbon sources. Mutations at a locus called cxm specifically block methylglyoxal formation from xylose in the presence of adenosine 3',5'-monophosphate. Accumulation in medium containing xylose, studied in some detail, is dependent on the ability to utilize xylose and is associated with an increased rate of xylose utilization without changes in levels of xylose isomerase. These results suggest that adenosine 3',5'-monophosphate results in induction of excessively high levels of an early rate-limiting step in xylose metabolism. This step may be the transport of xylose into the cell. The resulting excessive rates of xylose catabolism could stimulate methylglyoxal formation by overburdening late steps in glycolysis.     

Isolation and Characterization of Supercoiled Circular Deoxyribonucleic Acid from Beta-Hemolytic Strains of Escherichia coli

Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated by cesium chloride centrifugation in the presence of ethidium bromide from a naturally occurring beta-hemolytic Escherichia coli strain (SC52). The open circular forms have contour lengths of 2.25 ± 0.1 μm, 24.0 ± 0.3 μm, and 29.5 ± 0.5 μm. The beta-hemolytic character of E. coli SC52 can be transferred by conjugation to a nonhemolytic recipient strain. Analysis of the supercoiled DNA of the hemolytic recipient demonstrated that the two large supercoiled DNA molecules of E. coli SC52 are transferred during this event, too. A beta-hemolytic laboratory E. coli strain and several of its derivatives have been shown to contain at least one circular DNA molecule, slightly larger in size than those isolated from E. coli SC52 and its conjugant. The possible significance of these DNA molecules for hemolysin production and transfer is discussed.