Mutation induction by thymine deprivation in Escherichia coli B/R. II. Mutation in the repressed and derepressed tryptophan operon.

True Trp⁺ reversions are induced by thymine deprivation in cells with repressed trp operons as efficiently as in derepressed cells. At least part of the mutations are fixed during thymine starvation, i.e. in the absence of net DNA synthesis. The hypothesis is put forward that thymineless mutagenesis is due to repair-replication under limited concentrations of 5′-dTTP, performed by an inducible error-prone “DNA-polymerizing activity” on single-strand gaps.     

Escherichia coli B/r leuK mutant lacking pseudouridine synthase I activity.

Escherichia coli B/r strain EB146 containing mutation leuK16 has elevated levels of enzymes involved in the synthesis of leucine, valine, isoleucine, histidine, and tryptophan (Brown et al., J. Bacteriol. 135:542-550, 1978). We show here that strain EB146 (leuK16) has properties that are similar to those of E. coli and Salmonella typhimurium hisT strains. In tRNA1Leu from both hisT and leuK strains, positions 39 and 41 are uridine residues rather than pseudouridine residues. Furthermore, in tRNA3Leu and tRNA4Leu from a leuK strain, uridine residues at positions 39 and 40, respectively, are unmodified. Pseudouridine synthase I activity is missing in extracts of strain EB146 (leuK16), and extracts of strain EB146 (leuK16) and of a hisT strain do not complement one another in vitro. Four phenotypes of strain EB146 (leuK16), leucine excretion, wrinkled colony morphology, and elevated levels of leu and his enzymes, are complemented by a plasmid having a 1.65-kilobase DNA fragment containing the E. coli K-12 hisT locus. These results indicate that either leuK codes for pseudouridine synthase I (and is thus a hisT locus in reality) or, less likely, it codes for a product that affects the synthesis or activity of pseudouridine synthase I.     

Mutation frequency decline in Escherichia coli B/r after mutagenesis with ethyl methanesulfonate

Nonsense-defective auxotrophic strains of Escherichia coli B/r were used to study mutation frequency decline (MFD) after mutagenesis with ethyl methanesulfonate (EMS). The mutation frequencies for prototrophic revertants that were either converted or de novo glutamine tRNA suppressor mutations declined as treated auxotrophic parental cells were incubated with glucose but without required amino acids (a condition typically producing MFD). The decline for converted suppressor mutations was more rapid than the decline for de novo suppressor mutations after low or moderate EMS treatment, but both suppressor mutation types showed the same slow decline after extensive treatment. The declines for both types of suppressor mutation were eliminated in uvrA-defective cells, and the rapid decline seen for converted suppressor mutations appeared as a slow decline in mfd-defective cells. The results are interpreted that true MFD (the rapid process) affects only the EMS-induced converted glutamine tRNA suppressor mutations. This would account for the rapid decline that is blocked in cells with an mfd defect and in cells with deficient excision repair activity (uvrA or excessive DNA damage). In addition, a second non-specific antimutation mechanism is proposed that is dependent on excision repair only and accounts for the slow decline seen with converted suppressor mutations in some instances and with de novo suppressor mutations at all times. The true MFD mechanism may consist of a physiologically dependent facilitated excision repair specifically for premutational residues located in the transcribed strand of the target DNA sequence (for O6-ethylguanine in cells treated with ethyl methanesulfonate or pyrimidine-pyrimidine photoproducts after UV irradiation).     

Metabolic Regulation by Homoserine in Escherichia coli B/r

A mathematical analysis of branched pathway regulation has led to the prediction of a novel homoserine control in Escherichia coli B. Experimental support for such control is presented in this paper. Homoserine, the precursor of both threonine and methionine, inhibits nicotinamide adenine dinucleotide phosphate (NADP(+))-specific glutamate dehydrogenase (EC 1.4.1.4), the enzyme catalyzing the first reaction in ammonia assimilation. Physiological and biochemical evidence for this effect are offered. Homoserine depresses the growth rate of the organism, and glutamate, the product of the inhibited reaction, reverses this effect. The NADP(+)-specific glutamate dehydrogenase activity in cell-free extracts is inhibited by homoserine, and this inhibition parallels the restriction of growth rate. These effects are found in other enteric bacteria which share a similar overall pattern of control for the amino acids derived from aspartate. On the other hand, a sampling of more distantly related species which have different pathways and/or regulatory patterns provides no evidence for homoserine inhibition of the glutamate dehydrogenase reaction.     

The araIc mutation in Escherichia coli B/r.

The araIc allele is a cis-acting mutation which has been used to define the araBAD promoter in Escherichia coli B/r. Nineteen araIc mutants were originally isolated by Englesberg and co-workers as Ara+ "revertants" of an araC deletion mutant (Englesberg et al. J. Mol. Biol. 43:281-298, 1969). The mutants constitutively expressed araBAD gene products in the absence of functional araC activator protein. Eight of the araIc mutations have been cloned by in vivo recombination onto pBR322-ara hybrid plasmids. Restriction and DNA sequence analysis of these araIc mutations showed that they result from a single base-pair change located at -35 in the araBAD promoter.     

Kinetics of minichromosome replication in Escherichia coli B/r.

Replication control of the minichromosome pAL2 was found to differ from that of the chromosome in synchronously dividing populations of Escherichia coli B/r. Initiation of minichromosome replication took place at an increasing rate throughout synchronous growth. No coupling to initiation of chromosome replication was detected. Minichromosome replication was further examined in a dnaA5(Ts) temperature-sensitive initiation mutant. When cultures held at nonpermissive temperature (41 degrees C) for 60 min were shifted to permissive temperature (25 degrees C), initiation of both pAL2 and chromosome replication ensued in two waves spaced 25 to 35 min apart. Evidence is presented that minichromosomes terminate replication by passing slowly through a series of dimeric intermediate forms before reaching the closed circular monomeric form. The consequence of this slow passage as a rate-limiting step in the initiation reaction is discussed.     

Isolation of rel mutants of Escherichia coli B/r.

A method that relies on the biological effect of near-UV (340-nm) irradiation is described by which large numbers of independent rel mutants of Escherichia coli B/r may be rapidly isolated.     

The cell cycle in Escherichia coli B/r

Cell division and DNA synthesis were measured in synchronous cultures of E. coll B/r growing in glucose minimal medium at 37 °. The kinetic curves were analysed in order to find the variability of replication initiation, termination, and cell division events during the cell cycle. It is inferred that under the conditions used, cells begin to divide 17 min (D₀ = minimum D-period) after each termination of chromosome replication with a constant probability per unit of time (half-life = 4·5–6 min). This randomness produces an asymmetric frequency distribution of D-periods, similar but mirror-symmetric frequency distributions of initiation and termination periods, a symmetric, non-Gaussian distribution of interdivision intervals, and complex kinetic changes in the rate of DNA synthesis as a function of cell age. The results suggest that replication and division are precisely controlled with respect to mass accumulation, and the apparent variability of cell cycle events would only result from the use of the time of cell separation as a reference point for the definition of cell age rather than initiation or termination of replication.     

Length growth of two Escherichia coli B/r substrains.

Length growth of synchronized Escherichia coli B/r substrain A (ATCC 12407) and B/r substrain F26 (Thy his) was followed with an electron microscope. Cells were grown with doubling times (tau) of 60 min (B/rA) and of 82 and 165 min (B/rF26). Different length growth patterns were found for the two substrains. In B/rF, the length growth rate increased about midway in the cell cycle. For tau = 165 min, the rate increase was preceded by a short period of slow growth. For B/r A (r = 60 min), this period seemed to occur at the beginning of the cell cycle. The possibility is raised that the different length growth patterns are related to different deoxyribonucleic acid replication patterns of the respective strains.     

Regulation of ribosomal protein synthesis in Escherichia coli B/r.

The differential synthesis rate of ribosomal protein (r-protein), alpha-r (synthesis rate of r-protein divided by synthesis rate of total protein), was measured during the cell division cycle. It was observed that alpha-r remained essentially constant and was not measurably affected by duplication of the r-protein gene cluster (i.e., str-spc region) during the process of chromosome replication. It was further observed that the rate of total protein synthesis and r-protein synthesis increased continuously and uniformly during the entire cell cycle. This gene dosage independence of the synthesis rate of r-protein was similar to that observed earlier for the synthesis of ribosomal ribonucleic acid (rRNA). These observations indicate that the synthesis rates of the protein and RNA components of the ribosome are coordinately balanced during the entire cell division cycle and are not significantly perturbed by duplication of the r-protein or rRNA genes. Furthermore, this balanced synthesis insures that neither free rRNA nor free r-protein accumulate in appreciable amounts during balanced growth.     

Mutation induction by thymine deprivation in Escherichia coli B/R : II. Mutation in the repressed and derpressed tryptophan operon

True Trp⁺ reversions are induced by thymine deprivation in cells with repressed trp operons as efficiently as in derepressed cells. At least part of the mutations are fixed during thymine starvation, i.e. in the absence of net DNA synthesis. The hypothesis is put forward that thymineless mutagenesis is due to repair-replication under limited concentrations of 5′-dTTP, performed by an inducible error-prone “DNA-polymerizing activity” on single-strand gaps.     

Plasmolysis of Escherichia coli B/r with Sucrose

Escherichia coli B/r cells were plasmolyzed in sucrose solutions and observed under phase contrast. The prevalence of plasmolysis under various conditions was noted, and the degree of plasmolysis was categorized as slight, extensive, or severe. The presence of ions reduced the prevalence of plasmolysis. Survival curves showed that extensive plasmolysis was not lethal to colony-forming ability.     

Radiation protection of Escherichia coli B/r by hydroxyl radical scavengers

We have used Escherichia coli B/r to test the proposal that hydroxyl radicals (.OH) are major contributors to lethal damage when bacteria in equilibrium with air or 100% nitrogen are exposed to ionizing radiation. In addition, we have tested the hypothesis that oxygen sensitizes bacterial cells to radiation by reacting at radical sites previously formed by reactions of .OH. Our results with B/r indicate that the involvement of OH radicals in damage may have been overestimated. We believe that simple .OH removal provides B/r with only a relatively small amount of protection in N2 and air. Although some .OH scavengers can have large protective effects in air, evidence supports the tentative conclusion that these effects are not based on simple .OH removal. If this conclusion is correct, then radiation sensitization by oxygen--at least of this bacterial strain--would be unrelated to reactions of .OH.     

Cell cycle analysis of F''lac replication in Escherichia coli B/r.

The timing and control of replication of an F'lac plasmid was investigated in two substrains of Escherichia coli B/r lac/F'lac growing at a variety of rates. The cellular content of covalently closed circular F'lac deoxyribonucleic acid and the cellular mass at the time of F'lac replication both increased as a function of growth rate. The timing of plasmid replication during the division cycle was determined by measuring the inducibility of beta-galactosidase in cells of different ages in exponentially growing cultures. At all growth rates, the rate of induced beta-galactosidase synthesis increased in a step-wise fashion during the division cycle, indicating that the F'lac plasmid replicated at a discrete time in the cycle. At growth rates greater than one doubling per h, the cell age at F'lac replication was indistinguishable from the cell age at chromosomal lac+ replication in an isogenic F- parent. The ratio of plasmids to chromosomal origins decreased from about 0.7 to 0.4 between growth rates of 1.0 to 2.5 doublings per h. These observations are all consistent with replication of F'lac at about the same time in the division cycle as replication of the homologous chromosomal region at these growth rates. This similarity in timing of replication of homologous deoxyribonucleic acid regions was not evident in slower-growing cells.     

Studies on the biosynthesis of acylphosphatidylglycerol in Escherichia coli B and B/r.

The present study has demonstrated that one molecule of acylphosphatidylglycerol was synthesized from two molecules of phosphatidylgycerol by the transacylation reaction in which phosphatidylglycerol acted both as an acyl donor and an acceptor. Phosphatidylethanolamine was identified as an another acyl donor, participating in acylphosphatidylglycerol formation. These results are discussed in terms of a new pathway for the turnover of phosphatidylglycerol in Escherichia coli.