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.     

A Comparative Study of Two F-like Colicin Factors, ColV2 and ColV3, in Escherichia coli K-12

Of three colicin factors, each determining the synthesis of a colicin V in three wild-type Escherichia coli strains studied, two were shown to have sex-factor activities. In E. coli K-12, these activities resembled those of the F sex factor (including rapid and efficient self-transmission in exponentially growing cultures, adsorption of "male-specific" ribonucleic acid phage, production of "female phenocopies," elimination by acridine orange, and chromosomal transfer dependent upon recombination with host bacterium) and differed in this way from those of the colicin I sex factor (ColI). The two V factors, ColV2 and ColV3, differed in their efficiency of plating male-specific phage and in the pattern of transfer of chromosomal markers. Furthermore, although neither factor could stably coexist with F within the same cell, they showed markedly different exclusion effects. In general, ColV2 excluded F and ColV3 was excluded by F, irrespective of which sex factor was preestablished in the cell. An exception to this was the ability of ColV2 to stabilize in any one of a series of Hfr strains, giving rise to strains which in the majority of cases showed normal Hfr and colicinogenic properties.     

Incompatibility of Integrated Sex Factors in Double Male Strains of ESCHERICHIA COLI

Several strains of Escherichia coli K-12 harboring two F factors were isolated from Hfr x Hfr crosses. These strains were transiently capable of initiating chromosome transfer from two separate points of origin, and of transferring two different sex factors as integrated chromosomal markers. Each strain tested invariably reverted to a simple Hfr by loss of one of the inherited F factors. The F factor persisting in the revertant was, in nearly every case, that which had been inherited from the recipient Hfr parent.     

Genetic exchange between Escherichia coli strains in the mouse intestine

Germ-free mice contaminated with selected Escherichia coli strains were used for experiments designed to demonstrate gene transfer and recombinant formation in vivo. The well-characterized conjugation system of E. coli K-12 was examined in these experiments. Contamination of germ-free mice with a polyauxotrophic F(-) strain followed by the addition of isogenic Hfr, F', or F(+) strains resulted in the appearance of all recombinant classes at frequencies that would be expected from an in vitro mating experiment. Inheritance of unselected donor markers occurred at frequencies that were dependent on linkage relationships established in experiments in vitro. The presence of Lactobacillus had no influence on gene transfer and recombinant formation in an F' x F(-) in vivo mating. The R factor ROR-1 was transferred from E. coli strain M7-18 to an E. coli F(-) strain in the mouse intestine.     

Characterization of V-Prime Factors in Escherichia coli K-12

An episome derived from an Hfr_v (Hfr isolated from a V colicinogenic parent) strain of Escherichia coli K-12 was isolated and characterized. The direction of gene transfer was inverted from that in the original parental strain.     

Reinitiation of chromosome replication in the presence of chloramphenicol under an integratively suppressed state by R6K.

The autonomous replication of an R plasmid, R6K (amp, str) was shown not to be affected by chloramphenicol. It provoked integrative suppression and gave rise to Hfr strains when integrated into the chromosome of a strain of Escherichia coli K-12 with a temperature-sensitive mutation in the gene, dnaA. An Hfr strain designated as Hfr(R6K) no. 1 was thus obtained and characterized. It was not completely stable as shown by a plating efficiency of 0.6 at 42 C relative to that at 30 C. The density labeling and the ultracentrifugation analysis suggested that the deoxyribonucleic acid replication in this Hfr strain did not stop immediately after completion of the round already started before temperature shift-up and the addition of chloramphenicol. These observations are discussed in relation to a possibility that the chromosome replication of this Hfr strain is under the control of the integrated plasmid at a nonpermissive temperature.     

Isolation and Characterization of a Recombination-Deficient Hfr Strain

The isolation of a rec(-) Hfr strain of Escherichia coli K-12 is described. The method used consisted of mating AB2463 F(-) Rec(-) His(-) Lac(-) with P4X6 Hfr Rec(+) His(+) Lac(+), selecting Rec(-) His(-) Lac(+) recombinants, and searching for Hfr strains. One Hfr rec(-) strain, no. 12, was used as donor in crosses with Rec(+) and Rec(-) recipients. Crosses with Rec(+) recipients are fertile, and those with Rec(-) recipients are almost infertile, the frequency of recombinants being 10(-2) to 10(-3) that found with Rec(+) recipients. The Rec(-) mutant marker is transfered to and integrated into Rec(+) recipients. Zygotic induction of prophage lambda is observed in crosses between two Rec(-) strains. In crosses of F(-) Rec(-) with Hfr Rec(-), the gradient of integration frequencies for markers progressively more distant from the origin is steeper than in the Rec(+) x Rec(-) or the Rec(-) x Rec(+) crosses.     

Description of an incompatibility mutant of Escherichia coli

A mutant Hfr strain of Escherichia coli which has an impaired incompatibility function but is normal for other F factor functions has been isolated. This Inc(-) Hfr permits the maintenance and transfer of both the integrated F factor and an F' factor. F' factors have been isolated from the integrated F factor of the Inc(-) Hfr strain. When these episomes were tested in matings with Hfr or F' strains, they did not differ in any observed way from wild-type F' factors.     

Fertility of Salmonella typhimurium Crosses with Escherichia coli

At least one factor that causes low fertility of Salmonella typhimurium LT2 strains in crosses with Escherichia coli K-12 Hfr's can be inhibited by growing the female strains in supplemented minimal salts medium rather than in nutrient broth and by incubating the female strains at 50 C immediately before mating with the Hfr. These pretreatments can enhance the recovery of prototrophic recombinants for markers injected early by the Hfr by a factor of as much as 10(4). The heat treatment is effective only on the female in intergeneric crosses and gradually loses (within 50 min) its effectiveness after return of heat-treated cells to 37 C. It is concluded that the restriction system of the female is heat-sensitive. Since markers injected late by the male enter females in which the heat-impaired restriction system has recovered, few recombinants for late markers are found. The presence of the leading end of an E. coli Hfr in an S. typhimurium-E. coli hybrid enhances by up to sevenfold the frequency of lac(+) recombinants in subsequent crosses with an E. coli Hfr if the E. coli segment is integrated into the chromosome of the hybrid; the effect is less marked if the E. coli segment is not integrated.     

Inversion of Transfer Modes and Sex Factor-Chromosome Interactions in Conjugation in Escherichia coli

A study was made of the mating properties of an unusual system of interconvertible donor strains of Escherichia coli K-12: Ra-1, Ra-2, and RaF⁺. The Ra-1 and Ra-2 strains are Hfr strains whose origins are widely separated on the chromosome and whose transfer modes proceed in the opposite direction from one another. When Ra-1 cells were mated with females, a small fraction of the donors transferred markers via the Ra-2 mode. This effect was enhanced by preconjugal ultraviolet (UV) treatment of the Ra-1 cells. Among the survivors of UV-treated Ra-1 cells, a few stable Ra-2 cells were found. When Ra-2 cells were used as the donors, some of them were found to mate via the Ra-1 mode, in analogy with the Ra-1 to Ra-2 alteration with inversion of F mentioned above. Related experiments suggested that the inversion occurs by detachment of the F factor from one Hfr origin locus, followed by reassociation of the F factor with the other Hfr origin locus. Both the Ra-1 and Ra-2 strains reverted spontaneously to an F⁺ strain, called RaF⁺. Cultures of RaF⁺ cells were found to mate primarily according to the Ra-1 and Ra-2 transfer modes, with smaller contributions also coming from transfer modes with origins elsewhere on the chromosome in a way which is similar to the transfer of markers from a normal F⁺ strain. The RaF⁺ sex factor was found to be wild type, whereas the chromosome was found to carry irregularities (sex factor affinity loci) at the locations of the Ra-1 and Ra-2 origins. Only about 10% of the donor capacity of the RaF⁺ strain was due to stable spontaneous Hfr cells in cultures of RaF⁺ cells.     

Convenient construction of recA deletion derivatives of Escherichia coli.

Two Escherichia coli K-12 Hfr strains have been constructed which transfer a recA deletion, which is highly linked to a Tn10 insertion conferring tetracycline resistance, early during conjugation. These strains transfer the recA deletion in opposite directions with different origins of transfer, allowing for preservation of desirable recipient strain markers either clockwise or counterclockwise of recA.     

Identification and mapping of the replication genes of an R factor, R100-1, integrated into the chromosome of Escherichia coli K-12

A stable Hfr strain of Escherichia coli K-12 was obtained by integrative suppression by an R factor, R100-1. The R factor was integrated into the right of 81 min, and chromosome transfer occurred counterclockwise. Mating experiments revealed two linkage groups of genes on the R factor. Drug-resistant transductants of a dnaA-ts recipient from an R-factor Hfr and from an R(+) strain differ in their drug resistance patterns, temperature sensitivity, and transferability of drug resistance as well as chromosome markers. Transductants that transferred chromosome markers were further classified as to the origin and direction of chromosome transfer. For temperature-sensitive transductants, the reversion frequency to temperature resistance was determined, and these revertants were scored for transfer of drug resistance as well as chromosome markers. Two genes responsible for integrative suppression (designated as repA) and the other for autonomous replication (designated as repB) were identified and mapped. The arrangement of genes on the R factor is... (sul, str, cml)... repA... tra... (tet, repB).... The map of the autonomously replicating R factor is probably a circle connecting both sides of this linear map. Thus, a method has been established to map a plasmid that could not finely be analyzed under autonomous state by transduction. It also permits genetic analysis of genes responsible for replication of the plasmid without making use of a conditional mutant of itself but with that of the host, dnaA.     

ColB2-K77, a Fertility-Repressed F-Like Sex Factor

The colicinogenic B factor, transferred from Escherichia coli strain K77 (and termed ColB2-K77 or ColB2) to an E. coli K12 F(-) strain, is capable of promoting its own transfer to other K12 F(-) strains at a low rate (from LFC cultures) which can be increased under special conditions (HFC cultures). LFC cultures of K12 (ColB2)(+) F(-) strains show a low level of adsorption of F-specific phage particles which also increases under HFC conditions. The ColB2 factor is thus inferred to be an F-like sex factor which is repressed in its fertility. This repression is concluded to be due to a cytoplasmic repressor since, when ColB2 is present in cells containing an F factor (either autonomous or integrated), F fertility is also repressed as shown by the inability of such (ColB2)(+)F(+) [or (ColB2)(+)Hfr] strains to plaque F-specific phages, and by a reduction in the level of chromosomal transfer from such strains, compared to the corresponding F(+) (or Hfr) control strains. Mutants of the ColB2 factor in which fertility is no longer repressed (fertility derepressed or Fdr mutants) have been isolated. The ColB2Fdr mutant strains do not appear to be able to mobilize chromosomal transfer, although they have acquired F-specific phage sensitivity demonstrable by plaque formation and they transfer their colicin factor at high frequency and are well piliated. The Fdr mutation is presumed to result in the inability to synthesize the cytoplasmic fertility repressor since the ColB2Fdr factor does not repress the fertility of an F factor when present in the same host strain. A fertility-repressed drug resistance factor of the R(f) type is not stable in the presence of a ColB2 factor in the same cell and is eliminated in about 10% of the cells per generation. In contrast, another factor characteristic of the R(i) type is fully compatible with ColB2. Under conditions artificially stabilizing (ColB2Fdr)(+) (Rf)(+) strains, the enhanced fertility of ColB2Fdr is not repressed by the presence of the R factor, nor does the presence of R(f) in the intermediate strain of an HFC (for ColB2) system inhibit the normal increase in ColB2 transmissibility. It is concluded that the repressors of R(f) and ColB2, although both active on F fertility, are different; this may indicate that at least two independently repressible cistrons are involved in the expression of fertility characteristics.     

Effect of Rec− Mutations on the Activity of Colicinogenic Factors

The effect of two Rec(-) mutations (AB2463 and JC1553) on the ability of a cell to accept, maintain, and express the colicinogenic factors ColE(1), ColE(2), and ColV was examined in Escherichia coli. These mutations had no observable effect on the colicinogenic properties of the ColV factor, but prevented the spontaneous and induced production of colicins E(1) and E(2) which are determined by the ColE(1) and ColE(2) factors, respectively. The two Rec(-) mutations had no apparent effect on the ability of the cells to acquire, maintain, or transfer the ColE(1) and ColE(2) factors. These mutations did not affect the expression of immunity by any of the three Col factors. ColE(1) and ColE(2) were also shown to be indirectly induced by mating F(-) cells carrying these Col factors with ultraviolet-irradiated, non-colicinogenic, Hfr and F(+) cells. Indirect induction of colicin production occurred when either an irradiated F(+) Rec(+) or F(+) Rec(-) strain was employed as the donor strain.     

Replication of Fpoh+ plasmid in mafA mutants of Escherichia coli defective in plasmid maintenance.

A class of F' plasmids, designated Fpoh+, was previously shown to be able to replicate extra-chromosomally on Hfr strains by virtue of carrying the specific site or region poh+ (permissive on Hfr) of the E. coli chromosome (Hiraga, 1975, 1976a). These plasmids were now found to replicate on E. coli mafA mutants (mafA1 and mafA23) that cannot support vegetative replication of F and some other F-like plasmids. The derivatives of Fpoh+ that have lost the poh+ site, on the other hand, failed to replicate on mafA mutants. These mutants harboring Fpoh+ (but not Poh- derivatives thereof) exhibit abnormal cell division and form elongated cells, presumably due to competition between Fpoh+ and the host chromosome for some factor(s) essential for the initiation of DNA replication of the both replicons. It is tentatively concluded that the poh+ site is required for F' plasmids to replicate on mafA mutants as well as on Hfr strains. In view of the fact that the mechanism of inhibition of autonomous F DNA replication in mafA mutants and in Hfr strains are clearly different, the present data seem to provide strong support to the notion that the poh+ region contains the replication origin of the E. coli chromosome.     

Conservation and transfer of Escherichia coli genetic segments by partial diploid Hfr strains of Salmonella typhosa

Heterozygous, partial diploid hybrids were obtained in a Salmonella typhosa Hfr strain by using it as the recipient in a mating with the Escherichia coli Hfr donor WR2004 (O...proA...leu). Three of these S. typhosa Hfr hybrids were observed to mobilize and transfer the diploid E. coli genes, at high frequencies, to an E. coli recipient. The gradient of transfer frequencies of E. coli markers from these S. typhosa Hfr hybrids was similar to that observed with E. coli Hfr WR2004, from which they were derived. Interrupted matings with one of these S. typhosa Hfr hybrids, designated WR4272, showed the entry times for the proA, thr(-)leu, and argB E. coli diploid markers to be identical to the times obtained for these markers with E. coli Hfr WR2004. Also, the pattern of unselected inheritance of the diploid E. coli markers of S. typhosa Hfr hybrid WR4272 was similar to that observed with the chromosomal markers of E. coli Hfr WR2004. It was concluded that S. typhosa Hfr hybrid WR4272 contains, in addition to its Salmonella genome, a physically continuous E. coli chromosomal segment which is genetically complete from proA to at least the strA locus. The two other S. typhosa Hfr hybrids, on the basis of transmission frequency gradients, appeared to contain a continuous E. coli diploid segment complete from proA through the fuc locus. Other classes of S. typhosa Hfr hybrids, derived from mating with E. coli Hfr WR2010 (O...tna...xyl), were also observed to transfer E. coli genes at high frequency.     

Mapping Loci for Surface Exclusion and Incompatibility on the F Factor of Escherichia coli K-12

A series of Hfr deletion strains carrying deletions extending different distances into the integrated F factor have been used to map loci for surface exclusion (traS) and for incompatibility (inc) on the Escherichia coli K-12 sex factor F. traS mapped between traG and traD. It forms a part of the large operon, including all the known transfer genes except traJ, and is co-controlled with these. The product of traS is not required for formation of the F pilus. inc mapped between the phi(R) (11) locus and the origin of transfer; it is therefore one of the earliest loci transferred during conjugation.     

Localization of Rsf- mutation in Escherichia coli HfrC7]

The contransduction frequency of MAAs, UVs phenotype of Escherichia coli HfrC7 and its 7-51F- derivative with purE markers is found to be 1-2% which indicates that the mutation N 7 is located close to the F integration site in HfrC strain. E. coli strains K-12 7-51F+ and 7-51ColV2+ transfer chromosome markers in the same direction as does HfrC strain. The results suggest the presence of an integrated F fragment (sfa locus) into K-12 7-51F- chromosome.     

Spontaneous emergence of an Hfr strain with a cit plasmid from natural isolates of citrate-positive Escherichia coli bovine origin.

From citrate-utilizing (Cit+) Escherichia coli strain C53 of bovine origin, strains C53A and C53B were obtained. Upon mating with recA+ but not with recA mutant recipients of K-12, C53A produced chromosomal recombinants at quite high frequencies, leading to the following conclusions: (i) C53A is an Hfr strain; (ii) the site of integration of the Cit plasmid (IncH1) is between metA (89 min) and ara (1 min); (iii) the direction of chromosome transfer is clockwise; and (iv) the plasmid-associated determinants are transferred as the terminal markers. A transductant of a dnaA(Ts) strain, CRT46, which acquired Cit determinants from a recombinant, SG13, was also an Hfr strain similar to SG13, and thermoresistant due to suppressive integration. On the other hand, unstable C53B did not produce recombinants, but the frequency of RecA-independent transfer of the Cit plasmid was high, indicating that the Cit plasmid (IncH1) exists autonomously in C53B. Attempts to isolate an Hfr strain from C53B failed.     

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.