Genetic Mapping of the Locus for Detergent-Resistant Phospholipase A (pldA) in Escherichia coli K-12

An Escherichia coli K-12 mutant deficient for detergent-resistant (DR) phospholipase A, a principal enzyme catalyzing the first step in phospholipid degradation, was characterized genetically. The mutation was found to affect the locus pldA (phospholipid degradation), which is cotransducible both with ilv and metE at a frequencies of 13 and 78%, respectively, and shown to lie between the ilv and metE loci on the E. coli chromosome. DR phospholipase A(1) and A(2) activities were simultaneously transduced with pldA(+) by phage P1; therefore it is proposed that DR phospholipase A has both activities.     

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.     

Mutant of Escherichia coli K-12 defective in D-glucosamine biosynthesis

A mutant was isolated from a derivative of Escherichia coli K-12 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. This mutant contained normal levels of 2-amino-2-deoxy-d-glucose-6-phosphate ketol-isomerase (deaminating) (EC 5.3.1.10), but no detectable activity of l-glutamine:d-fructose-6-phosphate amino-transferase (EC 2.6.1.16). It required either N-acetyl-d-glucosamine or d-glucosamine for growth, and went into rapid lysis when the supply of these compounds was exhausted. In medium containing 11% sucrose, the cells were converted into spheroplasts in the absence of d-glucosamine.     

Mutant of Escherichia coli K-12 Missing Acetolactate Synthase Activity

A mutant requiring isoleucine and valine for growth, because of the absence of acetolactate synthase activity, has been isolated. At least one of three different genes (ilvG, ilvB, ilvI) is required for the expression of acetolactate synthase activity, thus suggesting the presence of three different acetolactate synthase isoenzymes.     

Endonuclease V (nfi) Mutant of Escherichia coli K-12

Endonuclease V (deoxyinosine 3′ endonuclease), the product of the nfi gene, has a specificity that encompasses DNAs containing dIMP, abasic sites, base mismatches, uracil, and even untreated single-stranded DNA. To determine its importance in DNA repair pathways, nfi insertion mutants and overproducers (strains bearing nfi plasmids) were constructed. The mutants displayed a twofold increase in spontaneous mutations for several markers and an increased sensitivity to killing by bleomycin and nitrofurantoin. An nfi mutation increased both cellular resistance to and mutability by nitrous acid. This agent should generate potential cleavage sites for the enzyme by deaminating dAMP and dCMP in DNA to dIMP and dUMP, respectively. Relative to that of a wild-type strain, an nfi mutant displayed a 12- to 1,000-fold increase in the frequency of nitrite-induced mutations to streptomycin resistance, which are known to occur in A · T base pairs. An nfi mutation also enhanced the lethality caused by a combined deficiency of exonuclease III and dUTPase, which has been attributed to unrepaired abasic sites. However, neither the deficiency nor the overproduction of endonuclease V affected the growth of the single-stranded DNA phages M13 or X174 nor of Uracil-containing bacteriophage λ. These results suggest that endonuclease V has a significant role in the repair of deaminated deoxyadenosine (deoxyinosine) and abasic sites in DNA, but there was no evidence for its cleavage in vivo of single-stranded or uracil-containing DNA.     

Mutant Strains of Escherichia coli K-12 Unable to Form Ubiquinone

A strain of Escherichia coli was isolated which was unable to form ubiquinone. This mutant was obtained by selecting strains unable to grow on malate as sole source of carbon. Such strains were further screened by examination of the quinone content of cells grown on a glucose medium. A mutant unable to form vitamin K was also isolated by this procedure. A genetic analysis of the ubiquinoneless strain showed that it possessed two mutations affecting ubiquinone biosynthesis.     

Isolation and Characterization of a Phosphonomycin-Resistant Mutant of Escherichia coli K-12

A mutant was isolated from Escherichia coli K-12 which showed increased resistance towards phosphonomycin, a new bactericidal antibiotic recently isolated from strains of Streptomyces. Evidence is presented which suggests that this mutant is resistant to lysis by phosphonomycin because of a lower affinity of phosphoenolpyruvate: uridine diphospho-N-acetylglucosamine enolpyruvyl transferase for this antibiotic. This mutant was also found to be temperature-sensitive in growth. At 42 C mutant cells grew poorly, and the rate of incorporation of (3)H-diaminopimelic acid into trichloroacetic acid-insoluble material was also greatly reduced. Genetic studies indicate that the increased resistance toward phosphonomycin and temperature sensitivity in growth of this mutant are probably the consequences of a single mutation.     

Mutant Strains of Escherichia coli K-12 Exhibiting Enhanced Sensitivity to 5-Methyltryptophan

Eighteen mutants (designated MT(s)), isolated in Escherichia coli K-12, showed increased sensitivity to inhibition of growth by 5-methyltryptophan. All mutants were also much more sensitive to 4-methyltryptophan and 7-azatryptophan but exhibited near normal sensitivity to 5-fluorotryptophan and 6-fluorotryptophan. All of the mutations were linked to the trp operon. Their locations within the trp operon were established by deletion mapping. There was good agreement between the map position of an MT(s) mutation and a lowered activity of one of the tryptophan pathway enzymes. Three mutants, one of which contained a mutation that mapped within the trpE gene, were deficient in their ability to use glutamine as an amino donor in the formation of anthranilic acid. Another trpE mutation led to the production of an anthranilate synthetase with an increased sensitivity to feedback inhibition by tryptophan.     

NAD-dependent Recovery of Cell Division in UV-irradiated Escherichia coli B and Lon- Mutant of Escherichia coli K-12

We found that both benzyl isothiocyanate (ITC) and phenyl ITC inhibited respiration in the mitochondria in an electrophilic reaction-dependent manner. ITC-induced mitochondrial swelling and cytochrome c release were prevented by cyclosporin A, indicating that they are mediated through the ITC moiety-dependent reaction to critical thiol groups for the opening of membrane permeability transition-dependent pores.     

Mutant of Escherichia coli K-12 Defective in the Transport of Basic Amino Acids

Escherichia coli K-12 possesses two active transport systems for arginine, two for ornithine, and two for lysine. In each case there is a low- and a high-affinity transport system. They have been characterized kinetically and by response to competitive inhibition by arginine, lysine, ornithine and other structurally related amino acids. Competitors inhibit the high-affinity systems of the three amino acids, whereas the low-affinity systems are not inhibited. On the basis of kinetic evidence and competition studies, it is concluded that there is a common high-affinity transport system for arginine, ornithine, and lysine, and three low-affinity specific ones. Repression studies have shown that arginine and ornithine repress each other's specific transport systems in addition to the repression of their own specific systems, whereas lysine represses its own specific transport system. The common transport system was found to be repressible only by lysine. A mutant was studied in which the uptake of arginine, ornithine, and lysine is reduced. The mutation was found to affect both the common and the specific transport systems.     

Ozone-induced damage of Escherichia coli K-12

Escherichia coli K-12 transformed with pACYC184 plasmid DNA was exposed to ozone (O₃) in aqueous solution. The damage to the membrane, protein, plasmid DNA, and cell survival were investigated. Cell viability was unaffected by short-term O₃ exposure (1–5 min) but membrane permeability was compromised as indicated by protein and nucleic acid leakage and lipid oxidation. The intracellular components, protein and DNA, remained intact. With longer durations of O₃ exposure (up to 30 min) cell viability decreased with a more significant increase in lipid oxidation and protein and nucleic acid leakage. The proteins leaking out were further oxidized by O₃. The total intracellular proteins run on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and plasmid DNA run on agarose gel, showed progressive degradation corresponding to the decrease in cell viability. The data indicate that membrane components are the primary targets of O₃ damage with subsequent reactions involving the intracellular components, protein and DNA. Received: 18 Apirl 1996 / Received revision: 26 July 1996 / Accepted: 5 August 1996     

Potassium-dependant mutants of Escherichia coli K-12

Mutants of Escherichia coli K-12 that grow more slowly in media containing low concentrations of K have been isolated. All independent mutants of this type which have been studied carry a mutation in a small region of the bacterial chromosome between the supE and gal loci. The growth rate of the mutants is the same as that of the parental strains in medium containing more than 1 mm K, but is only 50% that of the parent when the K concentration is reduced to 0.1 mm. The mutants do not appear to have a primary alteration in K transport, and are therefore referred to as K-dependent. The abbreviation kdp is proposed for this class of mutant.     

Flavodoxin mutants of Escherichia coli K-12

The flavodoxins are flavin mononucleotide-containing electron transferases. Flavodoxin I has been presumed to be the only flavodoxin of Escherichia coli, and its gene, fldA, is known to belong to the soxRS (superoxide response) oxidative stress regulon. An insertion mutation of fldA was constructed and was lethal under both aerobic and anaerobic conditions; only cells that also had an intact (fldA(+)) allele could carry it. A second flavodoxin, flavodoxin II, was postulated, based on the sequence of its gene, fldB. Unlike the fldA mutant, an fldB insertion mutant is a viable prototroph in the presence or absence of oxygen. A high-copy-number fldB(+) plasmid did not complement the fldA mutation. Therefore, there must be a vital function for which FldB cannot substitute for flavodoxin I. An fldB-lacZ fusion was not induced by H(2)O(2) and is therefore not a member of the oxyR regulon. However, it displayed a soxS-dependent induction by paraquat (methyl viologen), and the fldB gene is preceded by two overlapping regions that resemble known soxS binding sites. The fldB insertion mutant did not have an increased sensitivity to the effects of paraquat on either cellular viability or the expression of a soxS-lacZ fusion. Therefore, fldB is a new member of the soxRS (superoxide response) regulon, a group of genes that is induced primarily by univalent oxidants and redox cycling compounds. However, the reactions in which flavodoxin II participates and its role during oxidative stress are unknown.     

Phosphoglucomutase Mutants of Escherichia coli K-12

Bacteria with strongly depressed phosphoglucomutase (EC 2.7.5.1) activity are found among the mutants of Escherichia coli which, when grown on maltose, accumulate sufficient amylose to be detectable by iodine staining. These pgm mutants grow poorly on galactose but also accumulate amylose on this carbon source. Growth on lactose does not produce high amylose but, instead, results in the induction of the enzymes of maltose metabolism, presumably by accumulation of maltose. These facts suggest that the catabolism of glucose-1-phosphate is strongly depressed in pgm mutants, although not completely abolished. Anabolism of glucose-1-phosphate is also strongly depressed, since amino acid- or glucose-grown pgm mutants are sensitive to phage C21, indicating a deficiency in the biosynthesis of uridine diphosphoglucose or uridine diphosphogalactose, or both. All pgm mutations isolated map at about 16 min on the genetic map, between purE and the gal operon.     

Isolation of Escherichia coli B Mutant Deficient in Glutathione Biosynthesis

Certain edible large jellyfishes belonging to the order Rhizostomeae are consumed in large quantities in China and Japan. The exumbrella part of the edible jellyfish Stomolophus nomurai was cut and soaked in dilute hydrochloric acid solution (pH 3.0) for 12 h, and heated at 121 °C for 20 min. The immunostimulation effects of the jellyfish extract were examined. The jellyfish extract enhanced IgM production of human hybridoma HB4C5 cells 34-fold. IgM and IgG production of human peripheral blood lymphocytes (PBL) were also accelerated, 2.8- and 1.4-fold respectively. Moreover, production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α by human PBL was stimulated 100- and 17-fold respectively. Collagenase treatment inactivated the immunostimulation activity of the jellyfish extract. In addition, purified collagen from bovine Achilles’ tendon accelerated IgM production of hybridoma cells. These facts mean that collagen has an immunostimulation effect, and that the active substance in jellyfish extract is collagen.     

L-Serine-sensitive mutants of Escherichia coli K-12

While attempting to isolate d-serine-sensitive mutants of Escherichia coli K-12, we found a class of mutants sensitive to low concentrations of l-serine (10 to 25 mug/ml).     

Characterization and Genetic Analysis of a Mutant of Escherichia coli K-12 with Rounded Morphology

A morphological mutant of Escherichia coli K-12 that grows as round cells at 30, 37, or 42 C in a variety of complex and synthetic media has been isolated and characterized. The gene concerned, designated rodA, has been shown to be on the chromosome between the purE and pyrC loci and to be located at about minute 15. The rodA gene has been found to be co-transducible with the lip gene at a frequency of 95%. The rodA mutant showed an increased resistance to ultraviolet irradiation and a changed sensitivity to drugs. The resistance to ultraviolet irradiation and mitomycin C appears to be co-transducible with the rodA gene.     

A network model for biofilm development in Escherichia coli K-12

Proteins in any solution with a pH value that differs from their isoelectric point exert both an electric Donnan effect (DE) and colloid osmotic pressure. While the former alters the distribution of ions, the latter forces water diffusion. In cells with highly Cl^--permeable membranes, the resting potential is more dependent on the cytoplasmic pH value, which alters the Donnan effect of cell proteins, than on the current action of Na/K pumps. Any weak (positive or negative) electric disturbances of their resting potential are quickly corrected by chloride shifts. In many excitable cells, the spreading of action potentials is mediated through fast, voltage-gated sodium channels. Tissue cells share similar concentrations of cytoplasmic proteins and almost the same exposure to the interstitial fluid (IF) chloride concentration. The consequence is that similar intra- and extra-cellular chloride concentrations make these cells share the same Nernst value for Cl^-. Further extrapolation indicates that cells with the same chloride Nernst value and high chloride permeability should have similar resting membrane potentials, more negative than -80 mV. Fast sodium channels require potassium levels >20 times higher inside the cell than around it, while the concentration of Cl^- ions needs to be >20 times higher outside the cell. When osmotic forces, electroneutrality and other ions are all taken into account, the overall osmolarity needs to be near 280 to 300 mosm/L to reach the required resting potential in excitable cells. High plasma protein concentrations keep the IF chloride concentration stable, which is important in keeping the resting membrane potential similar in all chloride-permeable cells. Probable consequences of this concept for neuron excitability, erythrocyte membrane permeability and several features of circulation design are briefly discussed.