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PubMed1981

Construction of hybrid plasmids containing the lysA gene of Escherichia coli: studies of expression in Escherichia coli and Saccharomyces cerevisiae.

The lysA gene of Escherichia coli has been cloned from a lambda transducing phage on various plasmids, present in different copy numbers in bacterial cells. Synthesis of the product of this gene, diaminopimelate (DAP)-decarboxylase, and its regulation have been studied. Expression does not follow a simple gene dosage effect, maximal expression already being obtained with a six-copy plasmid. This result suggests that either a positive or an autogenous regulatory mechanism is involved. We also used one of the hybrid plasmids to look for expression of the bacterial lysA gene in Saccharomyces cerevisiae. The results indicate that the product of the E. coli gene is not actively translated in yeast.

Bacterial Proteins↗
PubMed1986

Nucleotide sequence of the Escherichia coli hisD gene and of the Escherichia coli and Salmonella typhimurium hisIE region.

In this paper we report the nucleotide sequence of the hisD gene of Escherichia coli and of the his IE region of both E. coli and Salmonella typhimurium. The hisD gene codes for a bifunctional enzyme, L-histidinol:NAD+ oxidoreductase, of 434 amino acids with a molecular mass of 46,199 daltons. We established that the hisIE region of both S. typhimurium and E. coli is composed of a single gene and not, as previously believed, of two separate genes. The derived amino acid sequence indicates that the hisIE gene codes for a bifunctional protein of 203 amino acids with an approximate molecular mass of 22,700 daltons. We also determined the nucleotide sequence of a deletion mutant in S. typhimurium which abolishes the hisF and hisI functions but retains the hisE function. We deduced that the mutant produces a chimeric protein fusing the aminoterminal region of the upstream hisF gene to the carboxyl-terminal domain of the hisIE gene which encodes for the hisE function. In view of these results the structural and functional organization of the histidine operon in enteric bacteria needs to be revised. The operon is composed of only 8 genes and the pathway leading to the biosynthesis of the amino acid requires 11 enzymatic steps.

Amino Acid Sequence↗
PubMed1990

Mobilization of Escherichia coli R1 silver-resistance plasmid pJT1 by Tn5-Mob into Escherichia coli C600.

Escherichia coli R1 is an Ag(+)-resistant strain that, as we have shown recently, harbours at least two large plasmids, pJT1 (83 kb) and pJT2 (77 kb). Tn5-Mob was introduced into the E. coli R1 host replicon via conjugation on membrane filters. The transfer functions of plasmid RP4-4 were provided in this process and Tn5-Mob clones mated with E. coli C600 yielded Ag(+)-resistant transconjugants. This mobilization procedure allowed transfer and expression of pJT1 Ag+ resistance in E. coli C600. Prior to use of Tn5-Mob mobilization, it was not possible to transfer Ag(+)-resistant determinant(s) into E. coli by conjugation or transformation including high-voltage electroporation. E. coli C600 containing PJT1 and PJT2 displayed decreased accumulation of Ag+ similar to E. coli R1.E. coli C600 could not tolerate 0.1 and 0.5 mM Ag+, rapidly accumulated Ag+ and became non-viable. Tn5-Mob mobilization may be useful in the study of metal resistance in bacteria, especially in strains not studied for resistance mechanisms.

Conjugation, Genetic↗
PubMed1993

Polypyrimidine/polypurine sequence in plasmid DNA enhances formation of dimer molecules in Escherichia coli. Dimerization of plasmid DNA in Escherichia coli.

Formation of dimer molecules of a recombinant plasmid, pTIR10, which carries a pyrimidine/purine-biased stretch occurs about 6-fold more efficiently than for the control plasmid pUC19 in Escherichia coli strain JM107. Since pyrimidine/purine-biased sequences have a potential to form unusual DNA structures, this observation suggests that the inserted sequence affects the replication process of plasmid DNA, probably by forming a triple helix under physiological conditions.

Animals↗
PubMed1975

Ineffectiveness of rifampicin in inhibiting RNA synthesis in Escherichia coli and T(4)-infected Escherichia coli cells after exposure to ultraviolet radiation.

Resdiual RNA synthesis in Escherichia coli cells and T(4)-infected E. coli cells exposed to ultraviolet radiation is insensitive to rifampicin. On the other hand, residual RNA synthesis in these cells after gamma irradiation is further inhibited by rifampicin. The specific effect exerted by ultraviolet irradiation on transcription seems to arise from alterations in the DNA structure rather than in bacterial RNA polymerase since RNA synthesis in ultraviolet-irradiated E. coli cells infected with unirradiated T(4) is sensitive to rifampicin.

Coliphages↗
PubMed1989

Lethality of hydrogen peroxide in wild type and superoxide dismutase mutants of Escherichia coli. (A hypothesis on the mechanism of H2O2-induced inactivation of Escherichia coli).

The toxicity of H2O2 in Escherichia coli wild type and superoxide dismutase mutants was investigated under different experimental conditions. Cells were either grown aerobically, and then treated in M9 salts or K medium, or grown anoxically, and then treated in K medium. Results have demonstrated that the wild type and superoxide dismutase mutants display a markedly different sensitivity to both modes of lethality produced by H2O2 (i.e. mode one killing, which is produced by concentrations of H2O2 lower than 5 mM, and mode two killing which results from the insult generated by concentrations of H2O2 higher than 10 mM). Although the data obtained do not clarify the molecular basis of H2O2 toxicity and/or do not explain the specific function of superoxide ions in H2O2-induced bacterial inactivation, they certainly demonstrate that the latter species plays a key role in both modes of H2O2 lethality. A mechanism of H2O2 toxicity in E. coli is proposed, involving the action of a hypothetical enzyme which should work as an O2-. generating system. This enzyme should be active at low concentrations of H2O2 (less than 5 mM) and high concentrations of the oxidant (greater than 5 mM) should inactivate the same enzyme. Superoxide ions would then be produced and result in mode one lethality. The resistance at intermediate H2O2 concentrations may be dependent on the inactivation of such enzyme with no superoxide ions being produced at levels of H2O2 in the range 5-10 mM. Mode two killing could be produced by the hydroxyl radical in concert with superoxide ions, chemically produced via the reaction of high concentrations of H2O2 (greater than 10 mM) with hydroxyl radicals. The rate of hydroxyl radical production may be increased by the higher availability of Fe2+ since superoxide ions may also reduce trivalent iron to the divalent form.

Aerobiosis↗
PubMed1987

Effects of the Escherichia coli SSB protein on the binding of Escherichia coli RecA protein to single-stranded DNA. Demonstration of competitive binding and the lack of a specific protein-protein interaction.

The effect of the Escherichia coli single-stranded DNA binding (SSB) protein on the stability of complexes of E. coli RecA protein with single-stranded DNA has been investigated through direct DNA binding experiments. The effect of each protein on the binding of the other to single-stranded DNA, and the effect of SSB protein on the transfer rate of RecA protein from one single-stranded DNA molecule to another, were studied. The binding of SSB protein and RecA protein to single-stranded phage M13 DNA is found to be competitive and, therefore, mutually exclusive. In the absence of a nucleotide cofactor, SSB protein binds more tightly to single-stranded DNA than does RecA protein, whereas in the presence of ATP-gamma-S, RecA protein binds more tightly than SSB protein. In the presence of ATP, an intermediate result is obtained that depends on the type of DNA used, the temperature, and the magnesium ion concentration. When complexes of RecA protein, SSB protein and single-stranded M13 DNA are formed under conditions of slight molar excess of single-stranded DNA, no effect of RecA protein on the equilibrium stability of the SSB protein-single-stranded DNA complex is observed. Under similar conditions, SSB protein has no observed effect on the stability of the RecA protein-etheno M13 DNA complex. Finally, measurements of the rate of RecA protein transfer from RecA protein-single-stranded DNA complexes to competing single-stranded DNA show that there is no kinetic stabilization of the RecA protein-etheno M13 DNA complex by SSB protein, but that a tenfold stabilization is observed when single-stranded M13 DNA is used to form the complex. However, this apparent stabilizing effect of SSB protein can be mimicked by pre-incubation of the RecA protein-single-stranded M13 DNA complex in low magnesium ion concentration, suggesting that this effect of SSB protein is indirect and is mediated through changes in the secondary structure of the DNA. Since no direct effect of SSB protein is observed on either the equilibrium or dissociation properties of the RecA protein-single-stranded DNA complex, it is concluded that the likely effect of SSB protein in the strand assimilation reaction is on a slow step in the association of RecA protein with single-stranded DNA. Direct evidence for this conclusion is presented in the accompanying paper.

Adenosine Triphosphate↗
PubMed1987

Effects of Escherichia coli SSB protein on the single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein. Evidence that SSB protein facilitates the binding of RecA protein to regions of secondary structure within single-stranded DNA.

The effect that Escherichia coli single-stranded DNA binding (SSB) protein has on the single-stranded DNA-dependent ATPase activity of RecA protein is shown to depend upon a number of variables such as order of addition, magnesium concentration, temperature and the type of single-stranded DNA substrate used. When SSB protein is added to the DNA solution prior to the addition of RecA protein, a significant inhibition of ATPase activity is observed. Also, when SSB protein is added after the formation of a RecA protein-single-stranded DNA complex using either etheno M13 DNA, poly(dA) or poly(dT), or using single-stranded phage M13 DNA at lower temperature (25 degrees C) and magnesium chloride concentrations of 1 mM or 4 mM, a time-dependent inhibition of activity is observed. These results are consistent with the conclusion that SSB protein displaces the RecA protein from these DNA substrates, as described in the accompanying paper. However, if SSB protein is added last to complexes of RecA protein and single-stranded M13 DNA at elevated temperature (37 degrees C) and magnesium chloride concentrations of 4 mM or 10 mM, or to poly(dA) and poly(dT) that was renatured in the presence of RecA protein, no inhibition of ATPase activity is observed; in fact, a marked stimulation is observed for single-stranded M13 DNA. A similar effect is observed if the bacteriophage T4-coded gene 32 protein is substituted for SSB protein. The apparent stoichiometry of DNA (nucleotides) to RecA protein at the optimal ATPase activity for etheno M13 DNA, poly(dA) and poly(dT) is 6(+/- 1) nucleotides per RecA protein monomer at 4 mM-MgCl2 and 37 degrees C. Under the same conditions, the apparent stoichiometry obtained using single-stranded M13 DNA is 12 nucleotides per RecA protein monomer; however, the stoichiometry changes to 4.5 nucleotides per RecA protein monomer when SSB protein is added last. In addition, a stoichiometry of four nucleotides per RecA protein can be obtained with single-stranded M13 DNA in the absence of SSB protein if the reactions are carried out in 1 mM-MgCl2. These data are consistent with the interpretation that secondary structure within the natural DNA substrate limits the accessibility of RecA protein to these regions. The role of SSB protein is to eliminate this secondary structure and allow RecA protein to bind to these previously inaccessible regions of the DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗
PubMed1991

Factor-independent activation of Escherichia coli rRNA transcription. II. characterization of complexes of rrnB P1 promoters containing or lacking the upstream activator region with Escherichia coli RNA polymerase.

A region upstream from the Escherichia coli rrnB P1 promoter, the upstream activator region (UAR), increases the activity of the promoter in vivo and the rate of association with RNA polymerase (E sigma 70) in vitro in the presence of the two initiating nucleotides. We have used four types of chemical and enzymatic footprinting probes to determine whether rrnB P1-E sigma 70 complexes formed in the presence of the initiating nucleotides (RPinit) differ from typical open complexes (RPo) formed in the absence of the initiating nucleotides and to examine the structural differences between rrnB P1 complexes containing the UAR and those lacking the UAR. We find that the rrnB P1-RPinit complex closely resembles open complexes formed at other E sigma 70 promoters, indicating that the formation of the first phosphodiester bond does not result in a major rearrangement of the promoter-RNA polymerase complex. An unusual potassium permanganate modification at position -18 in both RPo and RPinit indicates the possible presence of a subtle difference in the -10, -35 spacer structure compared to some other E. coli promoters. We show that the E sigma 70-rrnB P1 complex formed with the promoter containing the UAR has DNase I and hydroxyl radical cleavage patterns in the -50 region different from those observed with the same promoter lacking the UAR. These results are interpreted to indicate that E sigma 70 may interact with a region further upstream from that contacted by RNA polymerase bound at most other promoters and/or that unusual structural properties of this region are induced by bound E sigma 70.

Base Sequence↗
PubMed1983

Molecular cloning of the glyceraldehyde-3-phosphate dehydrogenase genes of Bacillus stearothermophilus and Escherichia coli, and their expression in Escherichia coli.

Recombinant plasmids derived from pBR322, which carry gap genes coding for the D-glyceraldehyde-3-phosphate dehydrogenases (GAPDH) of Bacillus stearothermophilus and Escherichia coli, have been isolated. The selection was carried out by complementation of an E. coli gapam mutant. Two plasmids containing B. stearothermophilus and E. coli DNA inserts of 4.3 kb and 1.4 kb, respectively, were characterized. Transformation of the E. coli mutant with either of the recombinant plasmids lead to a very high expression of the GAPDH activity. GAPDH produced by the strain containing the B. stearothermophilus gap gene was characterized by immunological cross-reactivity with antiserum raised against pure B. stearothermophilus GAPDH.

Base Sequence↗
PubMed2005

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 522/C1 and the international type strain from Escherichia coli O 178.

The structure of the O-antigenic polysaccharide (PS) from the enteroaggregative Escherichia coli strain 522/C1 has been determined. Component analysis and (1)H and (13)C NMR spectroscopy techniques were used to elucidate the structure. Inter-residue correlations were determined by (1)H,(1)H-NOESY and (1)H,(13)C-heteronuclear multiple-bond correlation experiments. The PS is composed of pentasaccharide repeating units with the following structure: [ structure: see text]. Analysis of NMR data reveals that on average the PS consists of four repeating units and indicates that the biological repeating unit contains an N-acetylgalactosamine residue at its reducing end. Serotyping of the E. coli strain 522/C1 showed it to be E. coli O 178:H7. Determination of the structure of the O-antigen PS of the international type strain from E. coli O 178:H7 showed that the two polysaccharides have identical repeating units. In addition, this pentasaccharide repeating unit is identical to that of the capsular polysaccharide from E. coli O9:K 38, which also contains O-acetyl groups.

Carbohydrate Sequence↗
PubMed1994

Explanation for different types of regulation of arginine biosynthesis in Escherichia coli B and Escherichia coli K12 caused by a difference between their arginine repressors.

In Escherichia coli K12, formation of the enzymes of arginine biosynthesis are controlled by arginine, with complete repression during growth with added arginine, severe repression (about 95%) during growth without added arginine and complete derepression during arginine-limited growth. In E. coli B, the degree of repression is not correlated with arginine concentrations. Under all conditions of growth enzyme formation is repressed, with repression being somewhat less in a medium with arginine than in a medium without arginine. These differences in repressibility between the two strains have been shown previously to be due to the presence of different alleles of argR, the gene for the arginine repressor. Here we have compared the binding of the two repressors to the operator sites of argF (ARG boxes). In DNase I footprinting and gel retardation experiments with argF ARG boxes we have shown that the arginine repressor of E. coli K12 bound to arginine (ArgRK-arg) has a greater affinity than the arginine repressor of E. coli B bound to arginine (ArgRB-arg), whereas free ArgRB (ArgRBf) has a much stronger affinity than free ArgRK (ArgRKf). The stronger binding of ArgRBf can explain the repression seen in E. coli B during arginine-limited growth and indicates that ArgRBf, but not ArgRKf, is able to repress enzyme synthesis under physiological conditions. The weaker repression of E. coli B than of E. coli K12 seen in the presence of arginine can be explained by the lower affinity of ArgRB-arg for operator sites as compared to ArgRK-arg. Another contributing cause for the weaker repression is the reduction of ArgRBf concentration due to autoregulation of the gene for the repressor. Thus the combined effects of repression by ArgRBf, but not ArgRKf, with the weaker repression by ArgRB-arg as compared to ArgRK-arg, convert the arginine dependent regulation in E. coli K12 to arginine independent regulation in E. coli B.

Arginine↗
PubMed2000

Capacity of mercury volatilization by mer (from Escherichia coli) and glutathione S-transferase (from Schistosoma mansoni) genes cloned in Escherichia coli.

A study was carried out to evaluate the capacity for mercury volatilization by genetically engineered strains that express the mer and glutathione S-transferase genes from Escherichia coli and Schistosoma mansoni, respectively. This method enabled strains containing simultaneously mer and glutathione S-transferase genes to grow in high concentrations of mercuric chloride (30 microg/ml) and to volatilize part of the mercury (248 microg/g cell dry wt.) present in the culture medium, while strains bearing only a single gene, did not have the same behavior. Up to 70% of the total mercury of bacterial volatilization occurred in the first 4 h. Although the findings were preliminary, the genetically engineered strain containing simultaneously the mer and glutathione S-transferase genes show a great potential for bioremediation. It may be used in a closed system to remove by volatilization, and recover mercury (Hg0) from contaminated effluents, such as industrial effluent, for instance.

Animals↗
PubMed2000

Combined suicide gene therapy for human colon cancer cells using adenovirus-mediated transfer of escherichia coli cytosine deaminase gene and Escherichia coli uracil phosphoribosyltransferase gene with 5-fluorocytosine.

The virus-directed enzyme/prodrug system using the Escherichia coli cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) suffers from a sensitivity limitation in many tumor cells. The E. coil uracil phosphoribosyltransferase (UPRT), which is a pyrimidine salvage enzyme, directly converts 5-fluorouracil (5-FU) to 5-fluorouridine monophosphate at the first step of its activating pathway. To improve the antitumoral effect of the CD/5-FC system, we investigated a combined suicide gene transduction therapy for human colon cancer cells using two separate adenovirus vectors expressing the E. coli CD and E. coli UPRT genes and systemic 5-FC administration (the CD, UPRT/5-FC system). The present study demonstrates that the CD, UPRT/5-FC system generates a co-operative effect of CD and UPRT, resulting in dramatic increases in both RNA- and DNA-directed active forms, including 5-fluorouridine triphosphate incorporated into RNA, 5-fluorodeoxyuridine monophosphate, and the thymidylate synthase inhibition rate, compared with the CD/5-FC system. Furthermore a significant increase in the 5-FC sensitivity of colon cancer cells was demonstrated in the CD, UPRT/5-FC system compared with the CD/5-FC system in vitro and in vivo. These results suggest that the CD, UPRT/5-FC system is a powerful approach in gene therapy for colorectal cancer.

Adenoviridae↗
PubMed1967

Resistance of Escherichia coli to tetracyclines. Changes in permeability to tetracyclines in Escherichia coli bearing transferable resistance factors.

1. When strains of Escherichia coli, bearing transferable factors for resistance to the tetracyclines (R-factors), and previously cultured in the absence of the tetracyclines, are grown for 15-30min. in a low, subinhibitory, concentration (10mug./ml.) of oxytetracycline or tetracycline, there is a rapid and striking increase in resistance to oxytetracycline or tetracycline, this being associated with a marked fall in the absorption of the drug by the cells. 2. Very short preincubation (1min.) with oxytetracycline, followed by growth for 15-30min. in drug-free medium, produces a marked fall in the absorption of the drug by the resistant cells. Preincubation for 30min. with very low concentrations (0.05mug./ml.) of oxytetracycline produces a similar effect. 3. beta-Apo-oxytetracycline, which has very little antibacterial activity, also induces a decreased absorption of oxytetracycline. 4. The ability to exclude oxytetracycline is retained by preincubated resistant cells after growth for 2hr. in drug-free medium. However, after growth for 16hr. in drug-free medium, the cells absorb oxytetracycline freely. 5. Chloramphenicol and proflavine inhibit the adaptive decrease in tetracycline absorption. 5-Fluorouracil has only a slight effect. 6. Spheroplasts prepared from resistant cells show an impaired response to preincubation with tetracycline, compared with intact cells. 7. The relevance of these results to the probable mechanism of tetracycline resistance in R-factor-bearing E. coli is discussed.

Acridines↗
PubMed1998

Applicability of a model for non-pathogenic Escherichia coli for predicting the growth of pathogenic Escherichia coli.

A model was developed for the temperature dependence of growth rate of a non-pathogenic Escherichia coli strain. The suitability of that model for predicting the growth rate of pathogenic E. coli strains was assessed. Growth rates of pathogenic strains were found to be adequately described by the model. Model predictions were also found to describe sufficiently well-published growth rate data for non-pathogenic E. coli on mutton carcase surfaces and E. coli O157:H7 in ground roasted beef, milk, and on cantaloupes and water melons. In addition, E. coli O157:H7 was found to grow in the region of 44-45 degrees celsius.

Escherichia coli↗
PubMed1970

On the process of cellular division in Escherichia coli. V. Incorporation of deoxynucleoside triphosphates by DNA thermosensitive mutants of Escherichia coli also lacking DNA polymerase activity.

Double mutants of Escherichia coli were constructed by combining DNA thermosensitive mutations affecting either DNA initiation (DnaA) or elongation (DnaB) with the Pol A1 mutation, which abolishes DNA polymerase activity. Incorporation of labeled deoxynucleoside triphosphates was studied in these mutants using toluene-treated cells. This incorporation was found to be exactly correlated with the capacity of the mutants to synthesize DNA in vivo.

DNA Nucleotidyltransferases↗
PubMed1993

Heterogeneity of the principal sigma factor in Escherichia coli: the rpoS gene product, sigma 38, is a second principal sigma factor of RNA polymerase in stationary-phase Escherichia coli.

The rpoS gene of Escherichia coli encodes a putative RNA polymerase sigma factor that is considered to be the central regulator of gene expression in stationary phase. The gene product (sigma 38) was overproduced using the cloned rpoS gene and purified to homogeneity. Reconstituted RNA polymerase holoenzyme (E sigma 38) was found to recognize in vitro a number of typical sigma 70-type promoters, including the lacUV5 and trp promoters. Some, however, were recognized exclusively or preferentially by E sigma 70, whereas at least one, fic, was favored by E sigma 38. Thus E. coli promoters can be classified into three groups: the first group is recognized by E sigma 70 and E sigma 38, but the second and third groups are recognized substantially by either E sigma 70 or E sigma 38 alone. In contrast to other minor sigma factors, sigma 38 shares a set of amino acid sequences common among the principal sigma factors of eubacteria and is therefore a member of the RpoD-related protein family. The intracellular level of sigma 38 was demonstrated to increase in vivo upon entry into stationary phase. These results together indicate that sigma 38 is a second principal sigma factor in stationary-phase E. coli.

Bacterial Proteins↗