Colicinogenic Factor Research Articles

Degradation of DNA in a temperature-sensitive mutant of Escherichia coli defective in DNA synthesis, harboring the colicinogenic factor E 1

1. The temperature-sensitive DNA replication mutant Escherichia coli CR 34 43 (Col E 1) stops chromosomal DNA synthesis immediately after the temperature is shifted to 43°, while allowing Col E 1 DNA synthesis to occur at a reduced rate. Small double-stranded fragments of chromosomal DNA are observed at the nonpermissive temperature. 2. The DNA of E. coli CR 34 43 (Col E 1), labeled at the permissive temperature for two generations, is degraded in a slow reaction at the nonpermissive temperature. Both the chromosomal and the extrachromosomal DNA are affected by this nuclease reaction. 3. When the DNA of E. coli CR 34 43 (Col E 1) is pulse labeled for short periods of time at the permissive temperature, a rapid breakdown of the pulse in the chromosomal DNA is observed, the extent of which is dependent on the pulse time. The extrachromosomal Col E 1 DNA labeled during the pulse period is completely resistant to this nuclease action. 4. Based on sedimentation analyses of the acid-insoluble breakdown products of the pulse-labeled DNA, it appears likely that an endonuclease with nicking properties participates in the degradation of the pulse-labeled region of the DNA at the restrictive temperature.

DNA polymerase as a requirement for the maintenance of the bacterial plasmid colicinogenic factor E 1

The extrachromosomal elements Col V, Col I, F′14, R1 , and R64 can be transferred by conjugation into and maintained in Escherichia coli cells carrying the pol A1 mutation. The Col E 2 factor can be transferred into pol A1 cells but is poorly maintained. The Col E 2 cells show a segregation to Col − at a rate of about 50% per generation. The Col E 1 factor cannot be transferred into pol A1 cells and is lost when the pol A1 mutation is tranduced into Col E 1 containing cells. Evidence is provided that DNA polymerase is essential to the maintenance of the Col E 1 factor.

Existence of the colicinogenic factor-sex factor [formula omitted]-P9 as a supercoiled circular DNA-protein relaxation complex

The colicinogenic factor ColE 1 has previously been isolated as a supercoiled circular DNA-protein complex designated relaxation complex. When treated with proteases, strong ionic detergents, or alkali, the supercoiled DNA in the complex is converted to the relaxed, or open circular, form. In these studies the colicinogenic factor-sex factor ColI b -P9 has been purified from Escherichia coli as a circular DNA molecule with sedimentation values of 73S and 45S for the supercoiled and open circular forms, respectively, and as a supercoiled DNA-protein relaxation complex with properties similar to those described for the ColE 1 relaxation complex.

Chloramphenicol and puromycin as inducers of colicinogenic bacteria

Addition of chloramphenicol or puromycin to colicinogenic bacteria carrying the colicinogenic factor E2 resulted, after resumption of protein synthesis, in the induction of colicin. An isogenic lysogenic strain carrying prophage λ was not induced by chloramphenicol.

Evidence for the existence of the colicinogenic factors E 2 and E 3 as supercoiled circular dna-protein relaxation complexes

Colicinogenic factor E 1 previously has been isolated as a supercoiled circular DNA-protein complex that undergoes a conversion to the relaxed, or open circular, state when the complex is treated with certain proteases, strong ionic detergents, or alkali. In this study, data is presented demonstrating the existence of similar relaxation complexes for the bacterial colicinogenic factors E 2 and E 3 of Escherichia coli .

Studies on extrachromosomal DNA elements. Replication of the colicinogenic factor Col E1 in two temperature sensitive mutants of Escherichia coli defective in DNA replication.

The replication of the colicinogenic factor Col E1 has been studied in two mutants of Escherichia coli with different thermosensitive lesions in DNA replication. One of these mutants E. coli ts 120/6 (Col E1) presumably synthesizes a thermolabile initiator for DNA replication. This mutant is not capable of supporting the synthesis of the Col E1 DNA at the restrictive temperature. The other mutant E. coli ts CR 34/43 (Col E1 DNA at the restrictive temperature at a reduced rate. The Col E1 DNA forms synthesized under the non‐permissive conditions are abnormal. The banding pattern of the DNA in ethidium bromide‐cesium chloride gradients and the sedimentation profile in sucrose gradients suggest that multiple circular and possibly catenated forms of Col E1 DNA are generated under these non‐permissive conditions.Mutant E. coli ts CR 34/43 (Col E1) shows a high colicin production upon induction with mitomycin C at 43°, while mutant E. coli ts 120/6 (Col E1) fails to synthesize colicin at the restrictive temperature.

The nature of colicin K from Proteus mirabilis.

The colicinogenic factor K has been transferred from E. coli K 235 to Proteus mirabilis. The DNA of the colicinogenic Proteus has been shown to contain a small amount of a satellite DNA which presumably harbors the Col K factor. In the presence of mitomycin C the colicinogenic Proteus secretes colicin K into the growth medium. The bacteriocin has been purified by chromatography and obtained as an immunologically homogeneous substance unconjugated with other antigens of the Proteus bacillus. Proteus colicin K is a protein of relatively low molecular weight. It contains all of the usual amino acids except cysteine and is free of lipids and polysaccharides. The bacteriocin can be separated by electrofocusing into two major components. The latter have the same biological properties but differ in their specific electrical charges.

Characterization of DNA of colicinogenic factor E1 in a providence strain.

Characterization of DNA of colicinogenic factor E1 in a providence strain.

Evolution of a Site of Specific Genetic Homology on the Chromosome of Escherichia coli

Integration of the factors F(v) and F into the chromosome of a substrain of Escherichia coli K-12 has been studied. The F(v) factor is a fertility factor derived from Col V, lacking the ability to govern the production of colicin V. The derivatives of an Hfr(v) (Hfr isolated from a V colicinogenic parent) strain, PK2 (initially isolated from C600 V(+)), were shown to retain a unique bidirectional sex factor affinity locus between recA and pheA. This site shows no affinity for the E. coli K-12 F factor as shown by inability to isolate Hfr strains with origins in this region from a parental strain containing a cytoplasmic F factor. However this area exhibits two regions of homology to the V colicinogenic factor. One gives rise to Hfr(v) strains identical to the original Hfr(v) strain, PK2, with an origin and polarity of transfer designated pheA-CC injecting markers in the order pheA-his-trp-pro. The second gives rise to strains apparently originating at the same site but with reverse polarity designated recA-C, transferring markers in the order recA-thyA-str-xyl. For strains possessing the F(v) factor only the second homology is apparent. A model for the evolution of these strains is presented.

Characterization of multiple circular DNA forms of colicinogenic factor E-1 from Proteus mirabilis.

Characterization of multiple circular DNA forms of colicinogenic factor E-1 from Proteus mirabilis.

Circular DNA forms of colicinogenic factors E1, E2 and E3 from Escherichia coli.

Abstract Colicinogenic factor E1 (Col E1) DNA isolated from Escherichia coli by phenol extraction followed by dye-buoyant centrifugation was found to consist almost exclusively of the monomer 23 s supercoiled circular DNA form. Little if any of the larger multiple forms which are characteristic of Col E1 DNA isolated from Proteus mirabilis were observed. The Col E1 DNA isolated from E. coli was shown to be similar, if not identical, to the monomer form of P. mirabilis by co-purification through alkali denaturation, neutralization and nitrocellulose filtration, and by co-sedimentation in sucrose gradients. The Col E1 DNA of E. coli also gives rise to a 17 s form with first-order kinetics during degradation with pancreatic deoxyribonuclease, as does the 23 s form isolated from P. mirabilis . A rapid technique of isolation of closed circular DNA molecules from E. coli , consisting of direct dye—buoyant centrifugation of a sheared sarkosyl lysate was developed. Using this technique, Col E1 from E. coli was again isolated as the 23 s form exclusively, while colicinogenic factors E2 and E3 were both found to be predominantly 25 s DNA forms. The sedimentation properties of the Col E2 and Col E3 DNA indicate a molecular weight of 5 × 10 6 for these factors.

Isolation of high-frequency recombining strains from Escherichia coli containing the V colicinogenic factor.

The isolation and characterization of high-frequency recombining strains from different Escherichia coli host cells containing either the F factor or the Col V factor are described. The strains (with one exception) formed from three of the V(+) parents showed the same origin and polarity of transfer (xyl-arg-pro-trp-his-mal). The Hfr strains formed from the one remaining V(+) and the F(+) host cells showed a greater variety in their points of origin. In addition, several Hfr strains isolated from V(+) parents lost the ability to produce colicin V. F(v) (+) segregants of these were isolated, and the F(v) factors appeared to retain their preferential site for Hfr formation, but they lacked other propertes controlled by the Col V factor. Chromosomal integration of episomes and its relation to the fertility of F(+) and V(+) strains are discussed. Production of colicin V appeared to be uninfluenced by the state of the Col V factor within the cell.

A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells.

Covalently closed circular duplex DNA’s are now known to be widespread among living organisms. This DNA structure, originally identified in polyoma viral DNA,1’2 has been assigned to the mitochondrial DNA’s in ox3 and sheep heart,4 in mouse and chicken liver,3 and in unfertilized sea urchin egg.5 The animal viral DNA’s—polyoma, SV40,6 rabbit7 and human8 papilloma—the intracellular forms of the bacterial viral DNA’s—φX174,9, 10 lambda,11, 12 M13,13 and P2214—and a bacterial plasmid DNA, the colicinogenic factor E2,15 have all been shown to exist as closed circular duplexes. Other mitochondrial DNA’s16, 17 and a portion of the DNA from boar sperm18 have been reported to be circular, but as yet have not been shown to be covalently closed.

Influence of the State of Fimbriation on Transmission of the Colicinogenic Factor colI between Strains of Shigella flexneri

SUMMARY: The colicinogenic factor colI derived from Shigella sonnei colicine type 2 (Abbott & Shannon, 1958) was transmitted between different strains of Shigella flexneri in serotypes 1a, 1b, 2a, 2b, 3a, 3c, 4a, 4b, 4c, 5a, X and Y when donor and acceptor strains were grown together in mixed culture in aerobic static broth for a period of 20 hr or 48 hr at 37·. The rate of transfer was influenced by the state of fimbriation of the acceptor bacteria, but not by that of the donor bacteria. The proportion of acceptor organisms acquiring colI was very high, e.g. 15-40 % in 20 hr and 50-95 % in 48 hr, when the acceptor was genotypically fimbriate and in the fimbriate phase (Fim+F); but it was much lower, e.g. 0-5 % in 20 hr and 1-15 % in 48 hr, when the acceptor was either genotypically non-fimbriate (Fim−) or genotypically fimbriate but in the non-fimbriate phase (Fim+N). Although it facilitated the transmission of colI, the presence of fimbriae was not essential for transmission, since transmission at a low rate was obtained in most crosses of Fim− donors with Fim− acceptors. Only 1 out of 17 strains of S. flexneri tested as acceptors failed to acquire colI in any test. Transmission did not take place within the first 2 hr of mixed culture; it occurred mainly during the period of slow growth after the end of the exponential phase (i.e. between 8 and 48 hr) when, with fimbriate organisms, a pellicle of bacteria had developed on the surface of the broth. The rate of transmission was very much decreased when pellicle formation was prevented by intermittent or continuous agitation of the culture. Experiments in which the donor bacteria were killed by adding streptomycin at 8 hr showed that an extensive ‘epidemic’ spread of colI took place within the acceptor population during the later stages of culture. When stationary-phase (1-10 days) broth cultures of donor and acceptor bacteria were mixed together and incubated without the addition of fresh broth, the rates of transfer at 20 and 48 hr were as high as in the mixed cultures grown from small inocula. Transmission also occurred with high frequency in a defined medium containing glucose, NH4 + and nicotinic acid.

Behavior of Some Episomal Elements in a Recombination‐Deficient Mutant of Escherichia coli

ABSTRACTConjugal transfer and autonomous replication of some episomes occurred normally in a recombination‐deficient (Rec–) mutant of Escherichia coli K‐12. Transduction with phage Plbt of an R factor also occurred normally in this Rec– mutant, but complete or abortive transduction with Plbt of chromosomal genes did not occur. In contrast, transduction of galactose genes by phage λdg occurred in the Rec– bacteria as frequently as in the Rec+ strain. It was shown that phage Plbt does not grow at all on the Rec–bacteria. Recombination between two different R factors, two mutants of phage λ and two mutants of phage T4 occurred normally in the Rec– bacteria, but did not give a Rec+ phenotype to the host bacteria. Colicinogenic factor I made the Rec– host bacteria more resistant to ultraviolet light but the colicinogenic strain was still infertile in the crosses with the Hfr srains of E. coli K‐12.

EFFECT OF THYMINE STARVATION ON CHROMOSOME TRANSFER DURING CONJUGATION IN <i>ESCHERICHIA COLI</i>

Two Hfr strains which had recieved the thymine requiring mutation of strain 15T-, but the colicinogenic factor were isolated. Mating experiments with these Hfr's revealed that thymine starvation of Hfr cells inhibits chromosome transfer. Uracil and histidine double starvation in Hfr cells, rather than being inhibitory, protects them from the spontaneous interruption of the process. The inhibitory effect of thymine starvation is differentially active, the more distant from the origin of an Hfr chromosome a given genetic marker is located, the more strongly transfer of that marker is inhibited. This inhibitory effect is (at least, partially) reversible.

Interaction between colicinogenic factor V and the integrated F factor in an Hfr strain of Escherichia coli.

Kahn, Phyllis L. (Princeton University, Princeton, N.J.), and Donald R. Helinski. Interaction between colicinogenic factor V and the integrated F factor in an Hfr strain of Escherichia coli. J. Bacteriol. 90:1276-1282. 1965.-The production of colicin V by strains of Escherichia coli is determined by a colicinogenic factor, colV. The colV factor possesses a genetic determinant of fertility, F(v). V(+)F(v) (+) cells are characteristically susceptible to a male-specific phage, mu, and able to transfer the colV factor and chromosomal markers to recipient cells. The present work describes an interaction of the colV factor with the chromosome of the Hfr strain, HfrH. A colV-containing HfrH strain, designated HfrHV(+)F(v) (+)(l), was isolated and shown to be insensitive to phage mu and impaired in its fertility properties. Loss of the colV factor by this strain, either spontaneous or induced by acridine orange, resulted in a further 10(3)- or 10(4)-fold loss in fertility. This additional loss of fertility was restored by reinfection of these strains with the colV factor. The colV interaction with the HfrH chromosome also can result in defects in the fertility properties of the colV factor. Altered colV factors were found in recombinants isolated from a cross between the HfrHV(+)F(v) (+)(l) strain and F(-) recipients. It is postulated that in the HfrHV(+)F(v) (+)(l) strain an interaction of the colV episome with the integrated F region of the chromosome occurs, with a resulting modification of the fertility properties of the HfrH strain. This interaction can also result in a defect in certain properties of the colV factor.

Characterization of colicinogenic factor E 1 from a non-induced and a mitomycin C-induced Proteus strain

Colicinogenic factor E 1 ( colE 1 ) was transferred by conjugation from Escherichia coli K30 to a non-colicinogenic Proteus mirabilis strain. Colicin E 1 produced by the Proteus E 1 + strain has a host range specificity identical to that of the colicin E 1 synthesized by E. coli E 1 + strains. DNA was extracted from the Proteus E 1 + strain and examined by equilibrium centrifugation in a CsCl density gradient. Band profiles of this unfractionated DNA indicated a single DNA species with a buoyant density of 1·700 ± 0·001 g/cm 3 . Fractionation of this DNA on methylated albumin columns, however, revealed in the early fractions an additional DNA with a buoyant density of 1·710·0·002 g/cm 3 . This satellite material amounted to about 0·3% of the total cellular DNA. The minor DNA component was not observed in the DNA of the parental non-colicinogenic Proteus strain or in a non-colicinogenic variant of Proteus E 1 + . The conclusion that this new DNA detected in the Proteus E 1 + strain represents colicinogenic factor E 1 is also supported by the apparent single labeling of this factor with tritium when the colicinogenic and non-colicinogenic Proteus strains were labeled with [ 3 H]thymine and [ 14 C]thymine, respectively. Colicin E 1 production was induced by treatment of the colicinogenic Proteus strain with mitomycin C. Under these conditions, the buoyant density of the satellite DNA is not altered, but there is a considerable increase in the amount of this minor DNA component. Furthermore, under conditions which lead to different levels of colicin production, the increase in the colE 1 DNA is approximately proportional to the magnitude of increase in colicin production.

THE TRANSFER OF TWO EPISOMES, COLICINOGENIC FACTOR I AND RESISTANCE TRANSFER FACTOR, IN SHIGELLA FLEXNERI STRAINS BY CROSSES BETWEEN STRAINS EACH POSSESSING A SINGLE EPISOME.

It was shown that bilateral transfer of two episomes col I and RTF, carried singly in different strains of Shigella flexneri, could occur at the same time. The transfer of col I occurred at a considerably higher frequeney than did the transfer of RTF. Whilst the presence of col I in the acceptor organism appeared not to affect the frequency of transfer of RTF it was observed that, in some cases at least, the presence of RTF in the acceptor organism lowered the frequency of col I transfer.

Interactions between colicinogenic factors and fertility factors

Colicinogenic factors E 2, E 1, I, V and B are known to be transferred in conjugation among strains of Escherichia coli K12 as extrachromosomal particles ( Nagel de Zwaig, Antón and Puig, 1962; Clowes, 1963; Nagel de Zwaig, 1963; Puig, 1963). The present report is based on further observations on the behaviour of E. coli K12 strains colicinogenic for colicin V which suggest the existence of a relationship between colicinogenic factor V, colicinogenic factor I and the F factor.