Colicinogenic Factor Research Articles

Modeling of the acute pyelonephritis in animals of various kind

Experiments have been carried out with two kinds of laboratory animals: 27 rabbits of chinchilla breed with the age of 3—4 months and the weight of the body from 1740 to 2 290 grams (1 993,1 ± 243,2) and 49 white alley male rats with the weight of the body of 180—220 g (195,4 ± 27,3). As an infectious organism, the daily dredge hemolysinogen E. coli with colicinogenic factor Ca 38 was used. Roots of introduction of dredge E. coli were intravenous, transurethral, transcapsular in parenchyma kidneys, enteral and endotracheal (in rats). As the animals were infected through various entrance gates, three variants of acute infections of the uric system were obtained: purulent-destructive defeats, tubular-interstitial infringements and asymptomatic bacteriuria. High virulence of the urostrain E. coli , used in the experiment, is demonstrated. The hypothesis about the universal mechanism of infection of the uric system "from top to down", i.e. hematogenic, has been formulated.

Synthesis and repair of RNA-containing supercoiled plasmid ColE1 DNA in Escherichia coli

Supercoiled plasmid molecules sensitive to nicking by RNase or alkali have been shown to accumulate during replication of colicinogenic factor E1 (ColE1) in Escherichia coli in the presence of chloramphenicol. The possibility that this sensitivity is due to the covalent integration of RNA molecules during the synthesis of plasmid DNA is supported by the demonstration that (a) strands of supercoiled ColE1 newly replicated in the presence of chloramphenicol exhibit sensitivity to RNase and alkali treatment, while (b) RNase- and alkali-resistant circular strands of plasmid DNA synthesized either before or after the addition of chloramphenicol remain resistant during subsequent replication of the plasmid in the presence of chloramphenicol. Furthermore, newly made plasmid DNA strands cannot act as templates for further rounds of replication if they possess an RNA segment. The existence of a repair mechanism for the removal of the RNA segment from supercoiled ColE1 DNA molecules was demonstrated by pulse-chase experiments. It was observed that the proportion of RNase-sensitive molecules is considerably higher in pulse-labeled as compared to continuously labeled ColE1 DNA synthesized in the presence of chloramphenicol, and the proportion of pulse-labeled ColE1 DNA that is RNase sensitive is greatly reduced during a chase period. Removal of the RNA segment is also carried out effectively at the restrictive temperature in temperature-sensitive DNA polymerase I mutants. In a survey of other bacterial mutants defective in the repair of damaged DNA, a substantial increase in the rate of accumulation of RNase-and alkali-sensitive supercoiled ColE1 DNA in the presence of chloramphenicol was observed in recBC and uvrA mutants in comparison with the wild-type strains.

Re-examination of the ColE1 DNA regions essential for autonomous replication and their functions.

Starting from pAO3, a plasmid consisting of a quarter of colicinogenic factor E1 (ColE1) DNA, various small ColE1 derivatives were constructed by in vitro recombination and their ability to achieve autonomous replication was examined. The 436 base pair HaeIII-C fragment of pAO3 contained information for replication when it was recombined with the non-replicating Amp fragment. However, when it was connected to other DNA fragments, the resulting hybrid molecules were not isolated as plasmids. The present results indicate that the additional region of about 240 base pairs next to the HaeIII-C fragment of ColE1 is also essential for the maintenance of a plasmid state. Moreover, using various small ColE1 derivatives, the DNA region responsible for the interference and incompatibility functions of ColE1 DNAs was located. The results indicate that the interference and incompatibility functions are coded by the same ColE1 DNA segment and are not essential for the maintenance of a plasmid state.

The relaxation complex of colicinogenic factor E1: Electrophoretic properties and a novel assay system for sodium dodecyl sulfate-mediated induction

The relaxation complex of plasmid colicinogenic factor E1 is demonstrated to be stable during agarose gel electrophoresis in low ionic strength buffer and to migrate as a distinct species having a 3% reduction in electrophoretic mobility relative to uncomplexed native supercoiled DNA. Treatment of complex with sodium dodecyl sulfate results in the apparent activation of a repressed endonucleolytic activity (induction) resulting in conversion to a species having a mobility coincident with nicked circular DNA. We have further demonstrated that if sodium dodecyl sulfate is applied to an agarose gel at times subsequent to the application of relaxation complex, sodium dodecyl sulfate will overtake the DNA, cause induction, and result in complete separation of induced complex from all other DNA forms. This technique constitutes a rapid, sensitive, and quantitative assay for induction as well as a single-step purification procedure. Induced complex can be readily isolated from preparative gels by electroelution and demonstrated to be a substrate for DNA polymerase I in a nick translation reaction. Incorporation of radioactive nucleotides occurs specifically in a 460 base pair restriction fragment known to contain the relaxation cleavage site, the likely origin of transfer of the heavy strand during F-factor-mediated plasmid mobilization.

Ion fluxes during T5 bacteriophage infection of Escherichia coli

When T5 bacteriophage infects Escherichia coli B, 42 K + is immediately released from cells that have been preloaded with this ion. The rate of ion release and the total amount released are dependent on the multiplicity of infection and are not diminished by the use of mutants which can only inject 8% of their DNA. Normally, the ion release stops at about 6 min postinfection. If the host cells contain the colicinogenic factor, Col Ib, so that the infection is abortive, K + release continues. Evidence is presented to show that this continued ion release cannot be explained by a “damage and repair” hypothesis. The results are, however, consistent with the interpretation of membrane depolarization due to ion pore formation as the cause of the abortive infection.

Amino acid and sugar transport in Escherichia coli (ColIb) during abortive infection by bacteriophage T5

T5 bacteriophage cannot replicate in Escherichia coli containing the colicinogenic factor ColIb. We show that active transport of proline and glutamine begins to decline at about 10 min after infection, the same time at which macromolecular synthesis stops during abortive infection. Uptake of alpha-methylglucoside is stimulated, however, and this change is evident even by 5 min after infection. These changes in membrane function do not occur during infections that are productive because of mutations on the plasmid or phage. The results suggest that the abortive infection is caused by membrane depolarization.

Isolation and characterization of a plaque-forming lambda bacteriophage carrying a ColE1 plasmid.

A plaque-forming lambdaimm434 bacteriophage carrying the entire genome of colicinogenic factor E1 has been isolated and characterized. This phage, lambdaimm434ColE1, can lysogenize as a stable plasmid within a recombination-deficient Escherichia coli cell that lacks the normal attachment site for lambda phage. Furthermore, it has been found that lambdaimm434ColE1 phage carrying amber mutations in the O and P genes of the lambda genome, i.e., lambdaimm434OamPamColE1, behaves as a plaque-forming phage, and this finding suggests that the ColE1 factor DNA permits replication of the DNA of the plaque-forming phage.

Restoration by T4 ligase of DNA sequences sensitive to “flush”-cleaving restriction enzyme

Fouteen "flush"-ended segments originate from the action of the restriction endonuclease Hae III of Haemophilus aegiptius on the DNA of the colicinogenic factor ColE 1 (A. Oka and M. Takanami, Nature, 264, 191, 1976). They are joined by the T4 polynucleotide ligase. The reaction can be monitored by gel electrophoresis, electron microscopy and resistance to phosphatase of the 5'-32P labelled ends. The joined products are a random recombination of the original segments, and can be cleaved by the same Hae III endonuclease to restore the exact electrophoretic pattern of the Hae III-cut ColE 1 DNA. In a properly diluted mixture of 5'-32P segments treated with T4 ligase, the level of phosphatase resistance is very close to the frequency of circle-formation as determined by electron microscopy: thus, the joining of the "flush"-ends involves the formation of circular structures covalently closed in both strands.

Alkali-labile colicinogenic factor E1 DNA molecules formed in the presence of N-methyl-N′-nitro-N-nitrosoguanidine

Plasmid ColE1 DNA of E. coli was used as a target DNA molecule to analyse the structural modification of DNA by N-methyl-N′-nitro-N-nitrosoguanidine. When the low concentration of this drug was used, both cell growth and overall DNA synthesis were neither stimulated nor inhibited, and plasmid DNA molecules were isolated as closed circles after replication. These molecules were stable for the ribonuclease treatment, but became susceptible to the alkaline hydrolysis. Such alkali-labile sites of ColE1 DNA were found in the parental strands and randomly distributed from the restriction endonuclease EcoR1 cleavage site.

The restriction endonucleases in Bacillus amyloliquefaciens N strain. Substrate specificities

Two species of restriction endonuclease were isolated by gel filtration and DEAE-cellulose chromatography from a cell-free extract of Bacillus amyloliquefaciens (B. subtilis) N strain; a lower molecular weight endonuclease (endo-nuclease R BamNI) and a higher molecular-weight one (endonuclease R. Bam-Nx). Both of them required only Mg 2+ for their activities. Endonuclease R. Bam-Nx introduced a larger number of site-specific scissions in Escherichia coli phage γ DNA than endonuclease R. BamNI did. Endonuclease R. BamNx cleaved Bacillus phage ø105C DNA at the specific sites which are classified into two groups: one type of sites is modified by B. amyloliquefaciens H strain in vivo while the other is not affected. It was also active on DNAs of E. coli phage T7, λdvl, Simian virus 40 (SV40) and colicinogenic factor ColEl and was inactive on DNAs of Bacillus phages ø29 and M2. Endonuclease R. BamNI cleaved DNA in the same nucleotide sequences as endonuclease R. BamHI isolated from H strain by Wilson and Young. This endonuclease was active on DNAs of phage λ, λdvl and SV40, and was inactive on DNAs of phages ø105C, ø29, M2 and T7, and ColEI DNA.

Phenotypic difference between uvr A and uvr B mutants of E. coli.

THE synthesis of colicin E1, an antibiotic protein, by cells of Escherichia coli is coded for by a bacterial plasmid, colicinogenic factor E1 (ColE1). The synthesis is induced by agents such as ultraviolet light1 or mitomycin C (mit C)2. Ultraviolet and mit C are known to modify the base portions of DNA to form pyrimidine dimers3 and cross linkages between complementary strands4, respectively. Damage to DNA caused by ultraviolet or mit C is repairable by a similar molecular mechanism5,6, but the relationship between these repair systems and the induction process of ColE1 synthesis has not been established unequivocally. We have analysed the induction of the colicin by ultraviolet and mit C in various ultraviolet-sensitive mutants to which the colicinogenic factor was transferred. Among the mutants tested, the recA mutant did not synthesise the colicin in response to ultraviolet or mit C as reported previously7. In addition, however, we have now found that the induction of the colicin synthesis is greatly delayed in the uvrB mutant, but is normal in the uvrA mutant.

Biochemical construction and selection of hybrid plasmids containing specific segments of the Escherichia coli genome.

Using a poly(dA-dT) "connector" method, a population of annealed hybrid circular DNAs was constructed in vitro; each hybrid DNA circle containing one full-length molecule of poly(dT)-tailed DNA from E1 colicinogenic factor (Col E1) fragmented by EcoRI endonuclease annealed to any one of a collection of poly(dA)-tailed linear DNA fragments of the entire E. coli genome. This annealed, but unligated, hybrid DNA was used to transform several different auxotrophic mutants of E. coli, and by direct selection, bacterial clones were isolated which contained specific hybrid plasmids. In this manner, bacterial strains containing Col E1 hybrid plasmids carrying the entire tryptophan operon or the arabinsoe and leucine operons were isolated. The methods described should allow the molecular cloning of any portion of the E. coli genome by selection from a pool of DNA molecules containing at least several hundred different hybrids representing the entire bacterial genome.

Replication of Colicinogenic Factor E1 DNA in Plasmolysed Escherichia coli Cells

Plasmolysed chloramphenicol-treated Escherichia coli cells carrying the colicinogenic factor E1 utilize deoxynucleoside triphosphates for the semi-conservative synthesis of Col E1 DNA. Col E1 DNA replication in plasmolysed cells can be dissociated into two temporally separated processes: (a) a rifampicin-sensitive RNA synthesis, which is stimulated by adenosine 3':5'-monophosphate (cyclic AMP) and requires all four ribonucleoside triphosphates and (b) an ATP-dependent DNA synthesis, which is inhibited by arabinosylnucleoside triphosphates and sulfhydryl-blocking reagents. Thes two processes exhibit different sensitivities to inhibition by polyamines and actinomycin D.

The fate of a bacterial plasmid in mammalian cells.

When hamster cells are infected with the bacterial plasmid colicinogenic factor E1 (ColE1), as much as 5-8% of the input plasmid radioactivity is found in the recipient cell, mainly in the nuclear fraction. Density shift experiments with bromodeoxyuridine labeled ColE1 DNA indicate that part of the input DNA may be replicated in the nucleus. ColE1 specific RNA but no colicin E1, can be detected during the first two generations after the uptake of ColE1 DNA. However, extrachromosomal ColE1 DNA is unstable in the mammalian cells and is degraded to acid soluble fragments after a few generations.

Breaks induced in supercoiled colicinogenic factor E1 DNA as a result of the decay of incorporated tritium

The supercoiled DNA form of the colicinogenic factor E1, labeled with tritiated thymidine, was stored at −25°C and aliquots examined periodically for breaks in the DNA. The majority of the breaks were single-stranded and took place in the labeled strand of the DNA. Double-stranded breaks due to the tritium disintegration occurred at about one-sixth the efficiency for single-stranded breaks. The formation of alkaline-sensitive bonds as a result of the tritium decay was not observed.

Abortive infection by bacteriophage BF23 due to the colicin Ib factor: II. Involvement of pre-early proteins

The mechanism by which the growth of phage BF23 is arrested in cells carrying colicinogenic factor Ib involves certain phage-specific pre-early proteins. BF23 induces the extensive formation of proteins lc and ld, but very little formation of protein le, whereas BF23h − (a mutant that is not arrested in cells carrying colicinogenic factor Ib) induces very small amounts of proteins lc and ld, but extensive amounts of protein le. Proteins lc and ld may be oligomers of protein le, and it is the presence of these putative oligomers that is necessary for the arrest of phage growth.

Replication of colicinogenic factor E 1 DNA: evidence for a discontinuous replication mechanism.

The mechanism of Col E 1 DNA replication was investigated in a plasmolysed cell system prepared from chloramphenicoltreated E. coli JC 411 (Col E 1). After pulse-labelling with (3)H-dTTP a considerable fraction of the newly synthesized DNA was recovered as single-stranded fragments. Upon alkali denaturation the pulse label was found in DNA chains sedimenting slower than unit length Col E 1 strands with a prominent peak at 5 S. During a chase with unlabeled precursors the label is transferred nearly completely into supercoiled Col E 1 DNA. DNA ligase appears to be required for the joining of the 5 S pieces since in the absence of NAD an accumulation of short fragments is observed.

Determination of the length of the V colicinogenic factor and a derivative fertility factor.

Plasmid deoxyribonucleic acid from Escherichia coli harboring either the V colicinogenic factor (Col V) or the derivative fertility factor (Fv), (a non-colicin-producing fertility factor derived from Col V) were separated by cesium chloride-ethidium bromide centrifugation and examined by electron microscopy. Results indicate a deletion of 11 mum of deoxyribonucleic acid in the transition from Col V to Fv.

Isolation of circular DNA molecules from whole cellular DNA by use of ATP-dependent deoxyribonuclease.

A technique is described for isolation of plasmid DNA in closed and open circular double-stranded forms from bacterial cells, by use of ATP-dependent deoxyribonuclease purified from Micrococcus luteus. This DNase, acting only upon linear DNA molecules, degrades all bacterial chromosomal DNA extracted in the linear form. Circular plasmid DNAs are left intact, and are then separated by sedimentation through a sucrose gradient. Unlike previous techniques for analysis of plasmid DNA, this technique can be used to isolate not only closed circular DNA but also open circular DNA. Several plasmids, such as those from phage (lambdadv1 and lambdadv21), a colicinogenic factor (Col E2), a sex factor (F(8)' gal), and "minicircles" in Escherichia coli 15, in both the open and closed circular forms, were well separated from chromosomal DNA by this technique.

Replication of colicinogenic factor E1 in Escherichia coli. Properties of newly replicated supercoils.

Replication of the colicinogenic factor E1 (Col E1) has been studied in exponentially growing cells and in cells treated with chloramphenicol. Whereas about 80% of non‐replicating Co1E1 DNA is isolated from the cytoplasm, the membrane fraction is enriched for newly replicated DNA. Equal amounts of pulse label are incorporated into each of the strands of Col E1 supercoils isolated after a short pulse, indicating a symmetrical replication mechanism. The replication time of the Co1E1 plasmid is about 2 min at 33°C. The synthesis of Co1E1 supercoils was reduced to less than 10% of the control level 4 min after shift to 42 °C in a temperature‐sensitive mutant for DNA polymerase I.