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.     

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.     

Mutant of Escherichia coli K-12 Deficient for Detergent-Resistant Phospholipase A

A mutant deficient for detergent-resistant (DR) phospholipase A was isolated from Escherichia coli K-12. Because the enzyme is membrane-bound and the substrate is a lipid, a special procedure was developed for isolating mutants deficient for the enzyme from agar plates. A sodium dodecyl sulfate (SDS)-sensitive mutant was used as a parental strain for the isolation of DR phospholipase A-deficient mutant. Soft agar containing an unsaturated fatty acid auxotroph and SDS was poured over colonies of the parental strain. The cells were easily solubilized with SDS, and phospholipids were efficiently digested by DR phospholipase A from the colonies on an agar plate. Fatty acids released supported the growth of the indicator bacteria. After the cells of the parent were mutagenized with nitrosoguanidine, colonies which could not support the growth of an unsaturated fatty acid auxotroph in the presence of SDS were selected. Four mutants were isolated after in vitro scre[UNK]ning of DR phospholipase A activity of 30 halo-less clones. Since an extract of the parent strain mixed with that of a mutant strain was still active, it was concluded that the inability to hydrolyze phospholipids was not due to the accumulation of inhibitory substance; the activity of DR phospholipase A in the mutant was less than 1% of the parental activity. Physiological studies indicated that DR phospholipase A is not essential for the growth of E. coli.     

Identification and Characterization of a New Gene of Escherichia Coli K-12 Involved in Outer Membrane Permeability

Using a genetic selection for mutations which allow large maltodextrins to cross the outer membrane of Escherichia coli in the absence of the LamB maltoporin, we have obtained and characterized two mutations that define a new locus of E. coli. We have designated this locus imp for increased membrane permeability. Mapping studies show that the imp gene resides at approximately 1.2 min on the E. coli chromosome. The mutations alter the permeability of the outer membrane resulting in increased sensitivity to detergents, antibiotics and dyes. The mutations are nonreverting and codominant. Genetic analysis of the mutations suggest that the imp gene is an essential gene. We describe a general cloning strategy that can be used to clone both dominant and recessive alleles. Using this technique, we have cloned the wild-type and mutant imp alleles onto a low copy number plasmid.     

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.     

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.     

Mapping by Transposons of the Inversion Termini in Escherichia Coli K-12 Strain 1485in

Strain 1485IN carries a chromosomal inversion which corresponds to 35% of the chromosome and includes proC, trp and his genes. The termini of the inversion lie between the lac and proC loci and between his and cdd of the normal strain. Using Tn10 and Tn5 in transduction crosses between the normal and inversion strains, the termini were mapped to sites located approximately 0.25 min and 1.6 min away from proC and his, respectively within a region of roughly 4 kb long. The crosses where the normal strains carrying Tn10 near the terminus are donors and the inversion strain is a recipient, yielded unusual Tetr His- recombinants, which arose from illegitimate recombination leading to the replacement of a chromosomal his+ region with a transducing fragment carrying proC. Another rearrangement was detected between the normal and inversion strains in a region outside the inverted segment near the cdd locus.     

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.     

The Transport Systems for Selenomethionine/Methionine and Selenocystine/Cystine in Escherichia Coli K-12 Appear to be Cooperative

Dopamine transporters of bovine and rat striata were identified by their specific [³H]cocaine binding and cocaine-sensitive [³H]dopamine ([³H]DA) uptake. Both binding and uptake functions of bovine striatal transporters were potentiated by lectins. Concanavalin A (Con A) increased the velocity but did not change the affinity of the transporter for DA; however, it increased its affinity for cocaine without changing the number of binding sites. This suggests that the DA transporter is a glycoprotein and that Con A action on it produces conformational changes

Inorganic and organic mercury reagents inhibited both [³H]DA uptake and [³H]cocaine binding, though they were all more potent inhibitors of the former, n- Ethylmaleimide inhibited [³H]DA uptake totally but [³H]cocaine binding only partially. Also, n-pyrene maleimide had differential effects on uptake and binding, inhibiting uptake and potentiating binding. [³H]DA uptake was not affected by mercaptoethanol up to 100 mM, whereas [³H]cocaine binding was inhibited by concentrations above 10 mM. On the other hand, both uptake and binding were fairly sensitive to dimercaprol (< 1 mM). The effects of all these sulfhydryl reagents suggest that the DA transporter has one or more thiol group(s) important for both binding and uptake activities. The Ellman reagent and dithiopyridine were effective inhibitors of uptake and binding only at fairly high concentration (>10 mM). Loss of activity after treatment with the dithio reagents may be a result of reduction of a disulfide bond, which may affect the transporter conformation     

Partial purification and characterization of four endodeoxyribonuclease activities from Escherichia coli K-12

Four hitherto undescribed endodeoxyribonucleases, temporarily designated A₁, A₂, A₃, and B, have been isolated from E. coli K-12. Each requires Mg⁺⁺ and is not stimulated by ATP or S-adenosylmethionine. A₃ is strongly inhibited by Fe⁺⁺⁺ and weakly inhibited by ATP, S-adenosylmethionine, and DPN, whereas B is inhibited by caffeine. Each can be purified free of exonuclease or DNA-3′-phosphatase. A₁ (molecular weight approximately 72,000) cleaves single-stranded, circular fd DNA to form 3′-hydroxyl termini and introduces nicks and breaks in the closed, double-stranded replicative form DNA of fd (fd RFI). A₂ (molecular weight approximately 46,000) cleaves fd DNA and introduces nicks and breaks in RFI, forming 3′-hydroxyl- and 5′-phosphoryl termini. A₃ (molecular weight approximately 38,000) cleaves fd DNA to form 3′-hydroxyl termini and introduces only nicks in fd RFI. Irradiation of the RFI with ultraviolet light markedly increases the rate of hydrolysis by A₃. B appears to form 3′-phosphoryl termini with fd DNA, but its characterization is highly preliminary due to its instability.     

Increased Resistance to Acriflavine by Amplification of the acrA Gene of Escherichia coli K-12

The wild-type acrA⁺ gene of Escherichia coli K-12, cloned intoplasmid pAF1, was expressed as resistance to acriflavine (AF)in AF-sensitive acrA mutant cells (N43). When acrA⁺ genes wereamplified by treatment of cultures with chloramphenicol (50µg/ml), cells expressed much higher resistance to AF thanthat of the wild-type strain (N90). (Received November 22, 1989; Accepted July 7, 1990)     

Addition of Escherichia coli K-12 Growth Observation and Gene Essentiality Data to the EcoCyc Database

The sets of compounds that can support growth of an organism are defined by the presence of transporters and metabolic pathways that convert nutrient sources into cellular components and energy for growth. A collection of known nutrient sources can therefore serve both as an impetus for investigating new metabolic pathways and transporters and as a reference for computational modeling of known metabolic pathways. To establish such a collection for Escherichia coli K-12, we have integrated data on the growth or nongrowth of E. coli K-12 obtained from published observations using a variety of individual media and from high-throughput phenotype microarrays into the EcoCyc database. The assembled collection revealed a substantial number of discrepancies between the high-throughput data sets, which we investigated where possible using low-throughput growth assays on soft agar and in liquid culture. We also integrated six data sets describing 16,119 observations of the growth of single-gene knockout mutants of E. coli K-12 into EcoCyc, which are relevant to antimicrobial drug design, provide clues regarding the roles of genes of unknown function, and are useful for validating metabolic models. To make this information easily accessible to EcoCyc users, we developed software for capturing, querying, and visualizing cellular growth assays and gene essentiality data.     

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.