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.     

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.     

Effect of ColV plasmids on the hydrophobicity of Escherichia coli

Abstract The hydrophobicity of E. coli strains carrying or lacking the colicin V ( ColV ) plasmids, ColV , I-K94 or ColV -K30 was assayed. ColV ⁺ derivatives of strain 1829, produced by conjugation or transformation, were more hydrophobic than either the original 1829 parental strain or a Col ^- derivative formed by curing 1829 ColV -K30 of its plasmid by an SDS/high temperature growth technique. Transfer of ColV into other E. coli strains also led to increased hydrophobicity. This effect of ColV plasmids was observed for organisms grown at 37°C; ColV ⁺ and ColV^- strains did not differ in hydrophobicity of grown at 21°C. This finding and other studies suggest that sex pili may be involved in the increased hydrophobicity.     

Isolation of Hfr Strains from R+ and ColV2+ Strains of Escherichia coli and Derivation of an R′lac Factor by Transduction

Temperature-resistant revertants were isolated from Escherichia coli strains carrying a temperature-sensitive dnaA mutation (initiation of chromosome replication) and either a repressed or a derepressed F-like R factor or a ColV2 factor. Many of the revertants had all the properties of Hfr strains, with a variety of directions and origins of transfer. From one such revertant, episomes carrying the R factor and part of the lac region (R'lac) could be isolated by transduction. This system offers a good selection for Hfr strains produced by integration of various episomes and for the isolation of R' factors.     

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.     

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.     

Sequence rearrangements in the plasmid ColV,I-K94

ColV,I-K94 is a conjugative (F-like) plasmid specifying colicins V and I. Numerous rearrangements were observed in ColV,I-K94 plasmids from three culture collections and in derivatives which specified larger inhibition zones and increased titers of colicin V. Several of the rearrangements in ColV,I-K94 derivatives specifying higher titers of colicin V involved a 1.3 ± 0.08-kb inverted repeat sequence which was separated by a 3.3 ± 0.2-kb sequence. This was rearranged in one derivative, pKH46, such that the 3.3-kb sequence became flanked by a 2.5 ± 0.15-kb inverted repeat which was subsequently altered to form a 2.1 ± 0.1-kb inverted repeat. In two other derivatives, the 3.3-kb sequence was deleted together with all of the flanking inverted repeat (in pKH39-2) or part of each repeat (in pKH39-1). A 0.5 ± 0.1-kb sequence adjacent to one of the 1.3-kb repeats was inverted or substituted in both of these plasmids. A novel 12.9 ± 0.7-kb inverted duplication separated by a 7.3 ± 0.4-kb sequence was present in the ColV,I-K94 derivative, pKH41, and insertion of the γδ element was detected in another derivative, pKH46-1, after this nonconjugative plasmid had been mobilized by the F plasmid. The genes for colicin V and for immunity to colicin V were mapped to a 3.6-kb sequence. The copy numbers of both pKH39-2 and pKH46-1::γδ were greater than those of pKH38 from which they were derived.     

Nitrosoguanidine Assay of Episomal Integration in Escherichia coli

Mutations affecting utilization of lactose and resistance to the male-specific phages f1, f2, and Qbeta tend to occur simultaneously more often than expected by chance in Hfr strains whose origin of transfer is close to the genes for lactose utilization, but not in F(+) strains. Strains derived from the Hfr, but exhibiting poor ability to transfer early chromosomal genes, may or may not show this comutation phenomenon. These results support the concept that the F factor is integrated into the Hfr chromosome during vegetative growth, but is autonomous in the F(+) strains and could serve as an assay for episomal localization.     

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.     

The ColV, I-K94 plasmid and heat sensitivity of Escherichia coli

The ColV, I-K94 plasmid markedly increased the heat sensitivity of Escherichia coli K12 but had no sensitizing effect on a wild E. coli isolate. The plasmid effect on strain K12 appeared to result from ColV-encoded transfer and colicin components possibly due to their effects on membrane properties.     

Hydroxamate-mediated transport of iron controlled by ColV plasmids.

A new high-affinity system for iron transport, associated with the presence of ColV plasmids, has been detected in Escherichia coli and partially characterized. The presence of such "iron-transport plasmids" in E. coli cells that are defective in enterochelin-mediated transport of iron enabled them to grow in media to which 2,2'-dipyridyl had been added to reduce availability of iron. In addition, the presence of plasmid deoxyribonucleic acid in a mutant defective in enterochelin biosynthesis was associated with a marked increase in the rate of radioactive-iron uptake. Plasmid-determined uptake of iron was distinct from previously recognized systems for iron transport in E. coli K-12, and the colicin V molecule appeared not to be directly involved. Hydroxylamine-nitrogen could be detected in cell pellets of ColV+ cultures, and similar material was detected in supernatant fluids of late log- or stationary-phase cultures. The hydroxamate material was not detected in cell pellets or culture supernatants of strains from which plasmids had been eliminated, and a 95% decrease in hydroxamate synthesis was observed when cells were grown in minimal medium containing 2 microM iron.     

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.     

Characterization of Escherichia coli serogroups causing meningitis, sepsis and enteritis. II. Classification of Escherichia coli O78 strains by phage sensitivity, colicin type and antibiotic resistance.

Escherichia coli O78: K80 strains isolated from an outbreak of meningitis, sepsis and enteritis in infants, were compared with O78: K80 strains from sporadic cases of enteritis, healthy carriers and animals. The strains were uniform in antigenic structure and phage pattern but differed in colicinogenicity. The epidemic strains and calf-pathogenic cultures produced colicin V, the remaining isolates were characterized by other types of colicin or were not colicinogenic. Col V+ strains multiplied in the mouse peritoneal cavity more readily and killed the animals at significantly lower doses than did col V- strains. One half of antibiotic resistant O78: K80 strains carried R factor. The spread of R factor could be followed by phage restriction experiments.     

Suppression by the ColV,I-K94 plasmid of inhibitor sensitivities in ompA mutants of Escherichia coli

A series of ompA mutants derived from Escherichia coli K12 strains showed increased sensitivity (compared with the ompA+ parents) to aminoglycoside antibiotics and to other cationic agents including polymyxin B. One tested mutant also showed increased sensitivity to nafcillin and fusidic acid, but not to the hydrophilic ampicillin. All these inhibitor sensitivities in the ompA mutants were suppressed by ColV, I-K94 and by certain other ColV plasmids, but not by any of the other tested large plasmids. Suppression correlated with the production of the VmpA protein, but transfer and colicin components were not needed for suppression. Further comparison of the ompA and vmpA genes and their products was made and it indicated that there is little if any homology between the genes, that the synthesis of their products is regulated by quite different mechanisms, and that regions of these gene products exposed at the cell surface show different susceptibility to protease attack after denaturation.     

Specific Action of Sodium Dodecyl Sulfate on the Sex Factor of Escherichia coli K-12 Hfr Strains

A specific action of sodium dodecyl sulfate (SDS) on the sex (F) factor in the integrated state of Escherichia coli K-12 Hfr H strain is reported. Growth of Hfr cells in Penassay Broth containing SDS results in the elimination of part or all of the F factor, yielding low and nonfertile variants of defective Hfr type and F⁺ cells and also F⁻ derivatives. Appearance of such variants was generally observed after the culture reached stationary phase. The frequencies of F⁻ cells then increased. F⁻ cells were usually isolated as the major population among survivors. Some defective variants of Hfr cells with an intermediate fertility between standard Hfr and F⁺ cells had lost sensitivity toward the male-specific ribonucleic acid phage M12. Other defective Hfr variants with as much or less fertility than standard F⁺ cells had also all lost sensitivity to phage M12. On single-colony isolation, they segregated nonfertile female H cells which, when infected with F, could restore high fertility with oriented transfer of the chromosome the same as that of the original Hfr H. Also, sensitivity to phage M12 was regained. Female H cells were characterized as those lacking fertility but still retaining a small segment of F or sfa locus at the original part of the chromosome, where newly infected F could attach. Similar results were obtained with two other Hfr strains. A possible mechanism of the specific action of SDS is discussed.     

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.     

Regulation of the ColV plasmid-determined iron (III)-aerobactin transport system in Escherichia coli.

Regulation by iron was studied in Escherichia coli strains whose iron supply was entirely dependent on the iron(III)-aerobactin system determined by the ColV plasmid. By the insertion of phage Mu (Ap lac) into the ColV plasmid, mutants were selected that could no longer grow in iron-limited media. The inserted Mu (Ap lac) strongly reduced the amount of aerobactin and he cloacin receptor protein formed by the cells. Their production was no longer subject to regulation by iron. The Mu (Ap lac) insertion apparently led to a polar effect on the expression of the presumably closely linked genes that control the synthesis of aerobactin and the cloacin receptor protein. The expression of the beta-galactosidase gene on the inserted phage genome came under the control of the iron state of the cells. Under iron-limited growth conditions, the amount of beta-galactosidase synthesized was, depending on the strain studied, 6 to 30 times higher than under iron-sufficient growth conditions. In fur mutants with an impaired iron regulation of ll iron supply systems studied so far, high amounts of beta-galactosidase were synthesized independent of the cells' iron supply. The results demonstrate an iron-controlled promoter on the ColV plasmid which is subject to regulation by the chromosomal fur gene.     

F factor-mediated immunity to lethal zygosis in Escherichia coli K-12

Recipient (F(-)) cells of Escherichia coli are sensitive to an excess of Hfr donor cells. This phenomenon of lethal zygosis is associated with conjugation and is observed as a continuous fall in F(-) viable cells during liquid mating, or as inhibition of F(-) growth on solid media. One class of survivors, which arose in the zones of inhibition on solid media, was no longer sensitive to lethal zygosis and exhibited the following properties: sensitivity to male-specific phage, donor ability, and surface exclusion. Since these characteristics were sensitive to acridine orange treatment, the strains carry an F factor extrachromosomally. They are, however, defective in some way since they retain sensitivity to female-specific phage. Temporary sensitivity to lethal zygosis in these and in standard F(+) strains can be induced by the formation of F(-) phenocopies. We have suggested that there is an immunity to lethal zygosis (Ilz(+)) associated with the F factor and discuss the results in terms of this hypothesis.     

Alkali sensitivity in plasmid-bearing strains of Escherichia coli

Introduction of the ColV,I-K94 plasmid into any of four strains of Escherichia coli gave derivatives which grew less well than the parent at alkaline pH. The alkali sensitivity of the 1829 derivative resulted from the presence of the plasmid rather than from its introduction into a less alkali tolerant variant. Of two other ColV plasmids tested, one (ColV-K30) conferred substantial alkali sensitivity whereas the other (ColV-41) had little effect. Of several other plasmids examined, R124-F2 (which confers derepressed transfer properties) resembled ColV,I-K94 in its effect on alkali sensitivity and ColV-K98 produced a marked effect. The other plasmids (F lac , R124 itself, R1 and R483ColIa) had only a small effect. For ColV plasmids, it appears to be the presence of transfer and colicin components together which leads to the reduced tolerance to alkaline pHs.     

Iron-regulated synthesis and uptake of colicin V

Abstract Virulent strains of Escherichia coli frequently contain ColV plasmids. It is shown that synthesis of the marker protein, colicin V, is regulated by iron via the iron repressor encoded by the fur gene. Mutants in the Cir outer membrane protein, in tonB , as well as in the exbB gene are resistant to colicin V suggesting all three functions to be required for colicin V uptake.