Detergent-resistant phospholipase A of Escherichia coli K-12. Purification and properties.

Detergent-resistant phospholipase A, which is tightly bound to the outer membranes of Escherichia coli K-12 cells, was purified approximately 2000-fold to near homogeneity by solubilization with sodium dodecylsulfate and butan-1-ol, acid precipitation, acetone fractionation and column chromatographies on Sephadex G-100 in the presence of sodium dodecylsulfate and on DEAE-cellulose in the presence of Triton X-100. The final preparation showed a single band in the sodium dodecylsulfate gel system. The enzyme hydrolyzes both the 1-acyl and 2-acyl chains of phosphatidylethanolamine or phosphatidylcholine. It also attacks 1-acyl and 2-acylglycerylphosphorylethanolamine. Thus, this enzyme shows not only phospholipase A1 and lysophospholipase L1 activities but also phospholipase A2 and lysophospholipase L2 activities. The enzyme lost its activity completely on incubation at 80 degrees C for 5 min at either pH 6.4 or pH 8.0. It was stable in 0.5% sodium dodecylsulfate at below 40 degrees C. The enzyme was inactivated on incubation for 5 min at 90 degrees C in 1% sodium dodecylsulfate/1% 2-mercaptoethanol/4 M urea. The native and inactivated enzymes showed different protein bands with RF values corresponding to Mr 21 000 and Mr 28 000 respectively, in a sodium dodecylsulfate gel system. Triton X-100 seemed to protect the enzyme from inactivation. The purified enzyme was fully active on phosphatidylethanolamine in the presence of 0.0002% or 0.05% Triton X-100. The enzyme requires Ca2+. From its properties this enzyme seems to be identical with the enzyme purified from crude extracts of Escherichia coli B by Scandella and Kornberg. However, it differs from the latter in its positional specificity and susceptibility to sodium dodecylsulfate. Possible explanation of the difference of positional specificity of the two preparations is also described.     

Synthesis of acyl phosphatidylglycerol from phosphatidylglycerol in Escherichia coli K-12. Evidence for the participation of detergent-resistant phospholipase A and heat-labile membrane-bound factor(s).

The conversion of phosphatidylglycerol to acyl phosphatidylglycerol by extracts of Escherichia coli K-12 strains was examined under various conditions. The maximum rate of conversion was observed at pH 7.2 in the presence of 50% (v/v) diethyl ether and 10 mM CaCl2. This conversion was found to involve two sequential reactions: (1) The formation of 2-acyl glycerophosphoglycerol and 2-acyl glycerophosphoethanolamine from phosphatidylglycerol and phosphatidylethanolamine, respectively, by detergent-resistant phospholipase A in the presence of Ca2+ and (2) transfer of the acyl group of 2-acyl lysophospholipid to phosphatidylglycerol by a heat-labile factor(s) in the presence of diethyl ether. Neither fatty, acid acyl-CoA nor 1-acyl lysophospholipid could act as an acyl donor for phosphatidylglycerol. The heat-labile factor(s) was found in both the inner membrane and supernatant fractions.     

Properties of Mitomycin C-sensitive Mutants of Escherichia coli K-12

Strains hypersensitive to mitomycin C (MC) were isolated from Escherichia coli K-12 after treatment with nitrosoguanidine. Of 43 MC-sensitive strains tested for their ultraviolet light (UV) sensitivity and for their ability to reactivate UV-inactivated λ phage, 38 were found to be insensitive to UV irradiation and to be able to reactivate UV-irradiated bacteriophage λ. Some properties of the MC-sensitive, uvr⁺ mutants were analyzed. Synthesis of deoxyribonucleic acid (DNA) in MC-sensitive, uvr⁺ mutants was inhibited at a lower concentration of MC than in the wild-type strain. Mutant cells, labeled with ³H-thymidine and then exposed to MC, released radioactivity as low molecular weight compounds. The amount of radioactivity released was the same as that from the wild-type strain. MC-sensitive, uvr⁺ mutants, as well as the corresponding wild-type strain, were equally susceptible to induction of prophage 80 by UV irradiation. However, MC induction of prophage was achieved in MC-sensitive, uvr⁺ mutants at a lower concentration of the antibiotic than in the wild-type strain. Genetic experiments indicated that a gene controlling MC sensitivity is located close to that determining lactose fermentation of E. coli. It is situated on episome F′13, and the wild type is dominant to the MC-sensitive allele.     

Overproduction and high-yield purification of phospholipase A from the outer membrane of Escherichia coli K-12

The cloned gene for the outer-membrane-bound phospholipase A from Escherichia coli was placed under control of the strong PL promoter of phage lambda. Induction of PL resulted in a 250-fold overexpression up to about 2% total cellular protein. This overproduced enzyme was indistinguishable from the wild-type enzyme. A homogeneous phospholiphase A preparation was obtained in high yield from overproducing bacteria, using the zwitterionic detergent C12-Sulfobetaine and anion-exchange chromatography. Detergent gradients were found to exert great influence on the elution characteristics. Considerations for the choice of optimal detergent gradients are discussed. The purified enzyme migrated as a single 29-kDa band in SDS/polyacrylamide gels, and required Ca(II) for activity. Maximum activity was displayed by enzyme samples taken from solutions with detergent concentrations near the critical micelle concentration. However, upon switching from high to optimal detergent concentration, maximum activity was restored in several hours, probably reflecting a slow conformational transition of the protein. Because the final pure protein was found to hydrolyze phospholipids in the intact erythrocyte membrane, a densely packed bilayer, we assume that this protein is in its biological native state.     

Uroporphyrin-accumulating mutant of Escherichia coli K-12.

An uroporphyrin III-accumulating mutant of Escherichia coli K-12 was isolated by neomycin. The mutant, designated SASQ85, was catalase deficient and formed dwarf colonies on usual media. Comparative extraction by cyclohexanone and ethyl acetate showed the superiority of the former for the extraction of the uroporphyrin accumulated by the mutant. Cell-free extracts of SASQ85 were able to convert 5-aminolevulinic acid and porphobilinogen to uroporphyrinogen, but not to copro- or protoporphyrinogen. Under the same conditions cell-free extracts of the parent strain converted 5-aminolevulinic to uroporphyringen, coproporphyrinogen, and protoporphyrinogen. The conversion of porphobilinogen to uroporphyrinogen by cell-free extracts of the mutant was inhibited 98 and 95%, respectively, by p-chloromercuribenzoate and p-chloromercuriphenyl-sulfonate, indicating the presence of uroporphyrinogen synthetase activity in the extracts. Spontaneous transformation of porphobilinogen to uroporphyrin was not detectable under the experimental conditions used [4 h at 37 C in tris(hydroxymethyl)aminomethane-potassium phosphate buffer, pH 8.2]. The results indicate a deficient uroporphyrinogen decarboxylase activity of SASQ85 which is thus the first uroporphyrinogen decarboxylase-deficient mutant isolated in E. coli K-12. Mapping of the corresponding locus by P1-mediated transduction revealed the frequent joint transduction of hemE and thiA markers (frequency of co-transduction, 41 to 44%). The results of the genetic analysis suggest the gene order rif, hemE, thiA, metA; however, they do not totally exclude the gene order rif, thiA, hemE, metA.     

Cadmium uptake in Escherichia coli K-12.

109Cd2+ uptake by Escherichia coli occurred by means of an active transport system which has a Km of 2.1 microM Cd2+ and a Vmax of 0.83 mumol/min X g (dry weight) in uptake buffer. 109Cd2+ accumulation was both energy dependent and temperature sensitive. The addition of 20 microM Cd2+ or Zn2+ (but not Mn2+) to the cell suspensions preloaded with 109Cd2+ caused the exchange of Cd2+. 109Cd2+ (0.1 microM) uptake by cells was inhibited by the addition of 20 microM Zn2+ but not Mn2+. Zn2+ was a competitive inhibitor of 109Cd2+ uptake with an apparent Ki of 4.6 microM Zn2+. Although Mn2+ did not inhibit 109Cd2+ uptake, the addition of either 20 microM Cd2+ or Zn2+ prevented the uptake of 0.1 microM 54Mn2+, which apparently occurs by a separate transport system. The inhibition of 54Mn2+ accumulation by Cd2+ or Zn2+ did not follow Michaelis-Menten kinetics and had no defined Ki values. Co2+ was a competitive inhibitor of Mn2+ uptake with an apparent Ki of 34 microM Co2+. We were unable to demonstrate an active transport system for 65Zn2+ in E. coli.     

Some Properties of Excision-defective Recombination-deficient Mutants of Escherichia coli K-12

Strains of Escherichia coli that carry the mutation uvrA6 show no measurable excision of pyrimidine dimers and are easily killed by ultraviolet (UV) light, whereas strains that carry recA13 are defective in genetic recombination and are also UV-sensitive. An Hfr strain carrying uvrA6 was crossed with an F⁻ strain carrying recA13. Among the recombinants identified, one carrying uvrA recA proved to be of exceptional sensitivity to UV light. It is estimated from the UV dose (0.2 erg/mm² at 253.7 nm) required to reduce the number of colony-forming cells by one natural logarithm that about 1.3 pyrimidine dimers were formed in a genome of 5 × 10⁶ base pairs for each lethal event. This double mutant is 40 times more UV-sensitive than the excision-defective strain carrying uvrA6. The replication of one pyrimidine dimer is generally a lethal event in strains carrying recA13. Spontaneous breakdown and UV-induced breakdown of the deoxyribonucleic acid (DNA) of cells of the various genotypes were estimated by growing the cells in medium containing ³H-thymidine and measuring both acid-precipitable and acid-soluble radioactivity. The UV-induced degradation in strains with recA13 did not require the uvr⁺ genes and hence appears to depend upon a mechanism other than dimer excision. The greater level of survival after irradiation in Rec⁺ as compared to Rec⁻ bacteria may be due to a recovery mechanism involving the reconstruction of the bacterial chromosome through genetic exchanges which occur between the newly replicated sister duplexes and which effectively circumvent the damaged bases remaining in the DNA.     

Inactivation of the Escherichia coli K-12 twin-arginine translocation system promotes increased hydrogen production

The effect of deleting the genes encoding the twin-arginine translocation (Tat) system on H2 production by Escherichia coli strain MC4100 and its formate hydrogenlyase upregulated mutant (DeltahycA) was investigated. H2 evolution tests using two mutant strains defective in Tat transport (DeltatatC and DeltatatA-E) showed that the rate doubled from 0.88+/-0.28 mL H2 mg dry weight-1 L culture-1 in the parental strain, to 1.70+/-0.15 and 1.75+/-0.18 mL H2 mg dry weight-1 L culture-1, respectively, in the DeltatatC and DeltatatA-E strains. This increase was comparable to that of a previously characterized hydrogen over-producing E. coli strain carrying a DeltahycA allele. Construction of a tatC, DeltahycA double deletion strain did not increase hydrogen production further. Inactivation of the Tat system prevents correct assembly of the uptake hydrogenases and formate dehydrogenases in the cytoplasmic membrane and it is postulated that the subsequent loss of basal levels of respiratory-linked hydrogen and formate oxidation accounts for the observed increases in formate-dependent hydrogen evolution.     

Linkage map of Escherichia coli K-12, edition 8.

The linkage map of Escherichia coli K-12 depicts the arrangement of genes on the circular chromosome of this organism. The basic units of the map are minutes, determined by the time-of-entry of markers from Hfr into F- strains in interrupted-conjugation experiments. The time-of-entry distances have been refined over the years by determination of the frequency of cotransduction of loci in transduction experiments utilizing bacteriophage P1, which transduces segments of DNA approximately 2 min in length. In recent years, the relative positions of many genes have been determined even more precisely by physical techniques, including the mapping of restriction fragments and the sequencing of many small regions of the chromosome. On the whole, the agreement between results obtained by genetic and physical methods has been remarkably good considering the different levels of accuracy to be expected of the methods used. There are now few regions of the map whose length is still in some doubt. In some regions, genetic experiments utilizing different mutant strains give different map distances. In other regions, the genetic markers available have not been close enough to give accurate cotransduction data. The chromosome is now known to contain several inserted elements apparently derived from lambdoid phages and other sources. The nature of the region in which the termination of replication of the chromosome occurs is now known to be much more complex than the picture given in the previous map. The present map is based upon the published literature through June of 1988. There are now 1,403 loci placed on the linkage group, which may represent between one-third and one-half of the genes in this organism.     

Effect of triethyltin on Escherichia coli K-12.

Triethyltin (TET) stimulated the basal respiration of Escherichia coli K-12 membrane vesicles in chloride (Cl-) medium but it had little effect on respiration in sulphate (SO4(2-)) medium. Since this uncoupling activity was Cl- dependent it was attributed to the Cl-/hydroxide (OH-) exchange reaction known to be mediated by TET [1,2]. TET inhibited the oxidation of succinate by intact E. coli in both Cl- and SO4(2-) medium, but at the same concentration of TET, inhibition was always more extensive in Cl- than SO4(2-) medium. In Cl- medium uncoupling in membrane vesicles and inhibition of succinate oxidation in intact bacteria occurred over the same concentration range and it appeared that the same mechanism, i.e. Cl-/OH- exchange, was responsible for both effects. Inhibition of succinate oxidation in SO4(2-) medium was not substantial until the concentration of TET was greater than 10(-5) M. Although the nature of this inhibition could not be determined by experiments with membrane vesicles indirect evidence from growth experiments indicated that it was due to impairment of oxidative phosphorylation. The relationship between these biochemical findings and the bacteriocidal action of TET was examined by using various concentrations of anion and substrate in the growth medium. Growth was inhibited in media containing either Cl- or SO4(2-) as the main anion but at a particular concentration of TET, inhibition was greater in Cl- medium. Growth was also inhibited to a greater extent in succinate than glucose medium. Furthermore in either Cl- or SO4(2-) glucose medium, lactic acid production increased as the concentration of TET was increased. These findings imply that the bacteriocidal action of TET is related to its effect(s) on oxidative phosphorylation.