The anaerobic oxidation of dihydroorotate by Escherichia coli K-12

The oxidation of dihydroorotate under anaerobic conditions has been examined using various mutant strains of Escherichia coli K-12. This oxidation in cells grown anaerobically in a glucose minimal medium is linked via menaquinone to the fumarate reductase enzyme coded for by the frd gene and is independent of the cytochromes. The same dihydroorotate dehydrogenase protein functions in both the anaerobic and aerobic oxidation of dihydroorotate. Ferricyanide can act as an artificial electron acceptor for dihydroorotate dehydrogenase and the dihydroorotate-menaquinone-ferricyanide reductase activity can be solubilised by 2 M guanidine · HCl with little loss of activity.     

A permeabilized-cell assay for transaminase B activity in Escherichia coli K-12

The assay for transaminase B (EC 2.6.1.6) activity, developed by D. E. Duggan and J. A. Wechsler (1973, Anal. Biochem.51, 67–79) has been modified to allow for the measurement of activity in Escherichia coli cells made permeable by cetyltrimethylammonium bromide (CETAB). A concentration of 10 mg% CETAB was found to be most effective in treating the cells without having a significant effect on transaminase B activity. Extraction of the dinitrophenylhydrazone of 2-oxoisovalerate by toluene was not affected by the CETAB treatment. We further report that the Na₂CO₃ extraction step is not required to measure color formed by the dinitrophenylhydrazone of 2-oxoisovalerate. This CETAB-treated cell assay is accurate to study transaminase B activity through most of the logarithmic phase of growth of Escherichia coli.     

Cloning of the Escherichia coli K-12 dihydrofolate reductase gene following Mu-mediated transposition

The dihydrofolate reductase structural gene, folA, has been cloned into the multicopy vector pBR322 following the gene's enrichment by bacteriophage Mu-mediated transposition. Strains carrying the resultant plasmid, pJFMS, produce 25 to 30 times more dihydrofolate reductase than control strains. Consequently they are resistant to trimethoprim, an inhibitor of this enzyme. This elevation in enzyme production is due to an increase in the number of folA gene copies per cell. The higher yield of dihydrofolate reductase obtained will be extremely useful for purifying and characterising this trimethoprim-sensitive chromosomally derived enzyme. The plasmid will also be invaluable for studying the structure, function and regulation of dihydrofolate reductase.     

A comparative study of the acetohydroxy acid synthase isoenzymes of Escherichia coli K-12

We studied the properties of the two acetohydroxy acid synthase isoenzymes expressed in wild type Escherichia coli K-12 in two isogenic strains, PS1035 (containing only acetohydroxy acid synthase III) and PS1036 (containing only acetohydroxy acid synthase I). The pH dependence is different for the two enzymes: acetohydroxy acid synthase I shows optimum activity at neutral pH, while acetohydroxy acid synthase III is most active at alkaline pH. Both activities require Mg²⁺ and thiamine pyrophosphate, but acetohydroxy acid synthase I, as compared to acetohydroxy acid synthase III, has a specific requirement for flavin adenine dinucleotide. Acetohydroxy acid synthase I is also more resistant to valine inhibition but more sensitive to inactivating conditions such as dialysis and temperature. The catalytic role of acetohydroxy acid synthase I in the synthesis of α-acetolactate is characterized by a higher affinity for pyruvate and a lower sensitivity to inhibition by α-ketobutyrate.     

Effect of changes in fatty acid composition of phospholipid species on the β-galactoside transport system of Escherichia coli K-12

On lowering the growth temperature of Escherichia coli K-12 from 37 to 17 °C, the cells resumed growth after a lag period of 40 min. During the lag period, the transition points in Arrhenius plots of the preinduced β-galactoside transport system were not changed while the saturated/unsaturated fatty acids ratio decreased gradually in phosphatidylethanolamine, rapidly in phosphatidylglycerol and little in cardiolipin.     

Cell division after inhibition of chromosome replication in Escherichia coli

The residual cell divisions after thymine starvation of exponential cultures of TJK16, a thymine-requiring derivative of Escherichia coli B/r, were evaluated. The results indicate that under the conditions used (glucose minimal medium 37 °), (1) only cells that had terminated a round of replication divided; (2) once termination had occurred, thymine starvation and replication no longer affected the time of cell division; (3) synchronously terminating subpopulations of cells began to divide about 17 min after termination; after that time, the rate of division decreased exponentially. The results confirm the previously inferred asymmetric distribution of D-periods in an exponential population of E. coli bacteria and suggest that an event associated with termination of replication is required for cell division. The method of data evaluation presented can be used to determine the duration of the D-period and to find the parameter values (halflife and onset) of the stochastic phase of the D-period in exponential cultures, eliminating the need for synchronization procedures.     

Unidirectional replication in Escherichia coli of three small plasmids derived from R factor R12

Replicating molecules of three small plasmids, pSM1, pSM2, and pSM3, were isolated from a CsCl density gradient containing ethidium bromide. These plasmids are all derived from R12, a mutant of NR1 (same as R100). By means of pulse-labeling experiments, the replicating forms were located at buoyant densities intermediate between those of the closed circular and open circular DNA bands. These molecules were analyzed by electron microscopy following digestion with restriction endonucleases. Digestion of pSM2 with EcoR1 and with HindIII revealed the presence of a single origin of replication located 1.72 kilobases (kb) from the EcoR1 cutting site (2.04 kb from the HindIII cutting site). These experiments also demonstrated that replication occurs in a unidirectional mode from the origin. Analysis of EcoR1-cleaved replicating molecules of pSM1 and pSM3, which carry common sequences completely or partly homologous to pSM2, provides further evidence for the unidirectional replication of these plasmids from a common origin. The site of the origin of replication was fixed at 85.5 on the kilobase map of R100. This origin, which is located in the RTF region, probably corresponds to one of the replication origins of R100.     

Synthesis of a tetrasaccharide related to the repeating unit of the O-antigen from Escherichia coli K-12

Synthesis of a tetrasaccharide related to the repeating unit of the O-antigen from Escherichia coli K-12 is reported in the form of its octyl glycoside. Syntheses of the 1,2-cis glycosidic linkages have been accomplished by using NIS in conjunction with H₂SO₄-silica, and it was found to be stereoselective and productive. The synthesized tetrasaccharide will be utilized as the substrate for galactofuranosyltransferase, WbbI.     

Regulation of the intracellular potassium concentration in Escherichia coli B 525

The mutant Escherichia coli B 525 requires histidine, leucine and methionine and an elevated extracellular K⁺ concentration for growth, and is unable to retain K⁺ tightly inside the cells when incubated in media supplemented with glucose, arabinose, galactose or lactose as the sole energy and carbon source. The loss of K⁺ from the cells of B 525 can be prevented by adding histidine and leucine, which react specifically and only in combination. In media supplemented with glycerol as the substrate, with glucose and NH₄⁺, or with glucose under anaerobic conditions, a stationary level of K⁺ inside the cells can be obtained without the addition of histidine-leucine.On the addition of ribose to glycerol-adapted cells of B 525 preincubated in glycerol media, the intracellular K⁺ decreased immediately and markedly. This decrease can be overcome by the addition of histidine-leucine.     

Effect of procaine hydrochloride on DNA repair in Escherichia coli

Liquid-holding recovery and rejoining of γ-radiation-induced DNA singlestrand scissions in Escherichia coli could be effectively inhibited by procaine hydrochloride at the concentration of 20 m M. At this concentration, the drug also reversibly altered cellular permeability barrier as evidenced from the uptake of acriflavin by bacterial cells.     

Changes in cell permeability following a marked reduction of saturated fatty acid content of Escherichia coli K-12

The present study examines the extent to which the fatty acid composition of the membrane lipid can be altered by nutritional means in mutants of Escherichia coli defective in total fatty acid synthesis. These changes are compared to those observed in wild type cells subjected to the same conditions of fatty acid supplementation. Abnormalities in physiological behavior of whole cells and membranes are related to extremes in fatty acid composition that can be produced in the mutant but not the wild type cells. In particular, when the saturated fatty acid of the membrane lipid is reduced below approx. 15% the barrier properties of the membrane toward small molecules such as K⁺ and a lactose analog decreases abruptly. This change is also reflected in the diminished temperature dependence of passive permeability and of NADH oxidase activity associated with the cytoplasmic membrane. Detailed studies on the properties of specific membrane function in relation to the physical behavior of membrane lipids should be possible with this biological system possessing a relatively simple membrane lipid structure in which the mole percentage of specific lipid components can be systematically varied.     

Localization and characterization of cytochromes from membrane vesicles of Escherichia coli K-12 grown in anaerobiosis with nitrate

Cytochromes b of anaerobically nitrate-grown Escherichia coli cells are analysed. Ascorbate phenazine methosulfate distinguishes low and high potential cytochromes b. Reduction kinetics performed at 559 nm presents a very complex pattern which can be analysed assuming that at least four b-type cytochromes are present. The electron transport chain from formate to oxygen would contain a low potential cytochrome b-556, a cytochrome b-558 associated to the oxidase, and a cytochrome d as the principal oxidase. Cytochrome o is also present, but seems to be functional only at low oxygen concentrations. A cytochrome b-556 associated to nitrate reductase is shown to belong to a branch of the formate-oxidase chain.2-N-Heptyl-4-hydroxyquinoline-N-oxide affects the reduction kinetics in a very complex way. One inhibition site is in evidence between cytochrome b-558 and cytochrome d; another between the cytochrome associated to nitrate reductase and the nitrate reductase. A third inhibition site is located in the common part of the formate-nitrate and the formate-oxidase systems.Ascorbate phenazine methosulfate is shown to donate electrons near cytochrome b-558.     

Expression of ultraviolet-induced restriction alleviation in Escherichia coli K-12. Detection of a λ phage fraction with a retarded mode of DNA injection

Ultraviolet-induced restriction alleviation is an SOS function which partially relieves the K-12-specific DNA restriction in Escherichia coli. Restriction alleviation is determined by observing elevated survival of unmodified phage λ in cells irradiated with ultraviolet prior to infection. We demonstrate that restriction of λ is also relieved when log-phase cells are irradiated as late as 50 min after adsorption of λ. At this time more than 60% of the λ DNA is already released as acid-soluble material from the cells. Experiments involving reextraction of λ DNA from infected cells and a mild detergent treatment removing adsorbed phages from the cellular surface showed that only a small specific fraction of all λ infections is destined to escape restriction due to restriction alleviation. This fraction (10–20%) has a retarded mode of DNA injection (60 min or longer) after adsorption which allows the expression of the restriction alleviation function before the phage DNA is exposed to restriction endonucleases. This behaviour of a fraction of λ phages explains why the SOS function restriction alleviation could initially be discovered. We show that the retarded mode of DNA injection is not required for another SOS function acting on λ DNA, the increased repair of ultraviolet-irradiated DNA (Weigle reactivation).