Detection of transcribable recombination products following conjugation in rec+, reCB- and recC-strains of Escherichia coli K12

By crossing Hfr and F^? strains of Escherichia coli which carry non-identical (but non-complementing) lacZ^? mutations, the detection of β-galactosidase produced from LacZ⁺ recombination products is possible, beginning 60 minutes after transfer of the Hfr lac^? allele. This system was used to show that when the F^? cells carry recB^?, almost normal amounts of LacZ⁺ enzyme are formed even though the number of viable recombinants is less than 1% of the Rec⁺ level. A similar result is found when the F^? cells carry recC^?. In contrast, LacZ⁺ enzyme activity is not detected either when RecA^? F^? cells are used or in a stable RecA^? merodiploid carrying the two lacZ^? alleles.     

Integration of donor DNA in bacterial conjugation

Conjugation between ¹³C¹⁵N- and ³H-labelled hybrid donors and ¹³C¹⁵N-labelled hybrid recipients of Escherichia coli gives rise to recombinant radioactive DNA of density greater than labelled hybrid. The donor radioactivity is present, in these molecules, in discrete heavy segments covalently attached to the light strand. When light radioactive Hfr cells are mated to heavy F^? cells in light medium, the donor label appears, in DNA extracted from the F^? cells, in labelled hybrid molecules. The radioactivity in these molecules is exclusively in the light strand. The insertion of donor material is thus restricted to a single newly formed strand of the recipient DNA and double-strand integrations do not occur. A temperature-sensitive recipient containing the dna B mutation ts₄₃ accumulates single-stranded Hfr DNA if mating is carried out at the nonpermissive temperature. The formation of a complementary strand in the recipient does not, therefore, appear to be necessary for continued transfer of Hfr DNA.     

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.     

Participation of RNase I in MS2 Phage Growth

Participation of RNase I in the growth of phage on infection with bacteriophage MS2 was studied.

Some strains of uracil-requiring E. coli were isolated, grown in MS broth, and transferred to a minimal medium to exhaust the pool of nucleotides. The phage was then added to the cells grown in uracil-deficient medium. The growth of phage was observed to occur at the burst size of two hundreds in strains of E. coli K12S (F⁺) U^? and C600 (F⁺) U^?, which possessed RNase I, but not in strains, A19 (Hfr) U^? and Q13 (Hfr) U^?, which lacked RNase I.

A marked increase in acid-soluble fraction was observed with E. coli K12S (F⁺) U^? and C600 (F⁺) U^?, whereas the increase was little with E. coli A10 (Hfr) U^? and Q13 (Hfr) U^? Conditions for the growth of phage in uracil-deficient medium were investigated and the effect of antibiotics were also investigated.     

DNA synthesis during bacterial conjugation. I. Effect of mating on DNA replication in Escherichia coli Hfr

Conjugation in Escherichia coli involves an oriented transfer of DNA from the Hfr to the F^?. We have examined the course of DNA replication in a donor cell while it is transferring its DNA. Using isotopic density shift for estimating replication, we have shown that mating is accompanied by initiation of a new round of DNA replication in Hfr cells. With the onset of F-mediated transfer replication, the normal vegetative replication in the Hfr appears to be suppressed. Experiments with F′ donors indicate that the transfer of the chromosome is necessary for switching off vegetative replication.     

Chromosome transfer and Hfr formation by F in rec+ and recA strains of Escherichia coli K12.

We attempted to assess the role of Hfr clones in chromosome transfer by F⁺ populations. We thought that any Hfr-independent component of fertility might be affected to a different extent by the recA mutation than was the Hfr component. However, the rate of Hfr formation and the efficiency of chromosome transfer were reduced to an equal extent (× 100-fold) by the recA mutation. Such experiments therefore provide no evidence for an Hfr-independent component. It appeared that Type II strains, which were thought to suffer a defect in Hfr formation, actually produced fertile clones but had a secondary defect which affected the persistence of these clones. Thus, evidence from Type II strains is also not useful for examining the quantitative contribution of Hfr cells to F⁺ transfer.     

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.     

Physiology of Escherichia coli K-12 During Conjugation: Altered Recipient Cell Functions Associated with Lethal Zygosis

The number of viable F⁻ cells decreases when Escherichia coli recipient cells are mixed with an excess of Hfr cells. Evidence is presented showing that lethal zygosis was accompanied by changes in the physiology of the recipient cells, including (i) inhibition of deoxyribonucleic acid synthesis, (ii) inhibition of β-galactosidase induction, (iii) altered transport and accumulation of galactosides, and (iv) leakage of β-galactosidase into the supernatant fluid. The results are discussed in terms of possible conjugation-associated changes that, at high Hfr to F⁻ ratios, lead to lethal zygosis.     

Pasteurella Bacteriophage Sex Specific in Escherichia coli

Phage H, thought to be specific for Pasteurella pestis, was shown to plate efficiently on F⁻ strains of Escherichia coli but not on F⁺, F′, or Hfr strains. The phage was adsorbed rapidly to F⁻ strains but was not adsorbed to strains carrying F. Comparison with seven other reported female-specific phages showed that, although phage H was similar to the other phages in some characteristics, the exceptionally low efficiency of plating (<10⁻⁹) on F-containing cells makes phage H a particularly useful female-specific phage.     

Specific Effects of a recB Mutation on the HfrH Strain of Escherichia coli

The recB268::Tn10 mutation was introduced into the HfrH strain of Escherichia coli. Compared with recB F⁻ and recB F⁺ cells, the viability of this mutant strain was much lower. Compared with wild-type HfrH, the recB derivative donated much shorter fragments of its chromosome to the recipient. It is suggested that the recB gene product (i.e., RecBCD enzyme) participates in Hfr transfer.     

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.     

Genetic Factors Affecting Recovery of Nonpoint Mutations in the Region of a Gene Coding for Ornithine Transcarbamylase: Involvement of Both the F Factor in Its Chromosomal State and the recA Gene

Mutants of E. coli K12 that overproduce ornithine transcarbamylase can be identified in Car^- strains because they permit utilization of citrulline as a carbamyl phosphate source, due to reversal of the normal OTCase reaction; they are called Cut mutants (citrulline utilizers). Hfr strains that carry the F factor adjacent to argF (one of two duplicate genes that code for ornithine transcarbamylase in E. coli K12) yield more Cut mutants than do F⁺ or F^- strains, or Hfr strains in which the F factor is not adjacent to argF. When Hfr strains in which the F factor is integrated adjacent to argF are made recA, they yield few Cut mutants. Many of the Cut mutants recovered from one of the Hfr strains used in the investigation (Hfr P4X) are unstable; the properties of these unstable mutations suggest that they carry aberrations in the region of the argF gene. Thus, the increased yields of Cut mutants probably result from aberrations that occur when the F factor is integrated adjacent to argF. The nature of these aberrations is not yet known. The unstable Cut mutants are to a large extent stabilized by recA; such stabilization is one of the properties of duplications. Other data indicate that the aberrations may be more complex than simple gene duplications; in particular properties of segregants and some recombinants derived from unstable Cut mutants are most easily interpreted by assuming that segregation from, and possibly formation of, the unstable mutants occurs in several stages.     

Some Effects of Nalidixic Acid on Conjugation in Escherichia coli K-12

The role of deoxyribonucleic acid (DNA) synthesis in the Escherichia coli conjugation system has been studied using nalidixic acid (NAL) to specifically inhibit DNA synthesis in matings between reciprocal combinations of male (Hfr) and female (F⁻) mutants resistant and sensitive to NAL; the physiological action of NAL on the strains utilized was also studied. Matings between combinations of mutants resistant (Nal^r) and sensitive (Nal^s) to NAL allow various parental functions to be established: pair formation studies show that the female cells are responsible for the slight decrease in pair formation when NAL is present in Hfr(Nal^s) × F⁻ (Nal^s) matings. Preformed mating pairs are stable in the presence of NAL. In matings between Hfr(Nal^s) and F⁻(Nal^r), the transfer gradient constant increases linearly with low NAL concentration (0.1 to 0.6 μg of NAL per ml). Higher concentrations of NAL (5 μg/ml) act on Nal^s males to rapidly stop chromosome transfer; under these conditions, however, DNA degradation is unmeasurable as determined from single-strand nicking in the male cells. This result is consistent with a model for chromosome transfer which requires DNA synthesis in the male cell. Inhibition of DNA synthesis (by 85%) in the female has no effect on conjugal chromosome transfer. High concentrations of NAL (>20 μg/ml) produce slight inhibition in chromosome transfer for the Hfr(Nal^r) × F⁻(Nal^r) mating tested; this effect is probably caused by action of NAL on the male. The inhibition of chromosomal transfer by NAL appears to be irreversible in the normal sense. A pulse of NAL, applied during transfer, immediately stops the transfer which is in progress. On removal of the NAL block, the temporal appearance of recombinants is consistent with the idea that a new round of transfer has commenced from the sex factor location on the male chromosome.     

Effect of tra mutations on F factor-specified immunity to lethal zygosis

Summary Hfr, F⁺, and F-prime cells are, unlike F^– cells, insensitive to an excess of Hfr donor cells, indicating that there is an F factor mediated immunity to lethal zygosis (Ilz). Results with Flac episomes carrying traJ, traS or various polar mutations in the tra region indicate that this immunity is independent of surface exclusion, of traJ control, and of all known genes within the tra operon. However, analysis of a series of strains with deletions in the F factor, extending from the right into the tra region, suggests that a gene for immunity to lethal zygosis is located within the tra region. We therefore conclude that Ilz is genetically complex, and present a hypothesis to account for these results.     

Conjugation and phage MS 2 infection with HfrEscherichia coli

Summary Hfr cells that are engaged in conjugation with F⁻ cells are killed by MS 2 phage with the same kinetics as are Hfr cells that are not conjugating. Preincubation with large numbers of MS 2 does interfere with the ability of Hfr cells to form recombinants. This interference does not have certain properties that might be expected if it were due to competition for nucleic acid transport, namely, enhancement in the absence of divalent cations and prevention by the presence of ribonuclease. A preliminary report of this work was presented at the Annual Meeting of the American Society for Microbiology, and appeared as abstract inBacteriological Proceedings (1969).     

Early transfer of genes determining transfer functions by some Hfr strains in Escherichia coli K12

Summary Sixty-eight Hfr strains were examined for their ability to transfer early in conjugation the transfer genes carried by the integrated sex factor. This was measured by mating these strains with F^- phenocopied recipient cultures of strains carrying transfer-deficient Flac ⁺ factors, and then measuring the ability of the recipient strains to transfer lac ⁺ to a further recipient strain. Most Hfr strains did not complement the missing transfer functions, though in some strains complementation was observed. It is concluded that on the sex factors of different Hfr strains either the site at which integration occurs or the origin of transfer must vary.     

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

Summary A class of F plasmids, designated Fpoh ⁺, was previously shown to be able to replicate extrachromosomally 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.     

Temperature-sensitive mutations affecting the replication of F-prime factors in Escherichia coli K 12

Summary More temperature-sensitive mutants affecting the replication of the F-gal⁺ episome of Escherichia coli K12 have been isolated. Eight of the mutations were located on F itself and three were located on the chromosome.The temperature sensitive F-gal⁺'s have been integrated into the chromosome to produce Hfr strains. These Hfr strains have transfer origins similar to Hfr Cavalli, and all show aberrant excision and transfer of elongated segments of the chromosome including the integrated F-gal to generate long merodiploids.The chromosomal mutations that govern the replication of F have been termed seg (for segregation). Wild-type F-gal⁺ can be integrated into seg cells at 42° C to give Hfrs, in a process analogous to integrative suppression in the formation of Hfrs from cells carrying mutations that are temperature-sensitive for chromosomal DNA replication (dnaA). A curious feature of an Hfr derived from a seg strain is that it also shows F-genote enlargement as well as normal transfer of chromosomal genetic marker. Preliminary transductional mapping data show that the mutation seg-2 is linked to the threonine locus (minute 0).     

Role of surface exclusion genes in lethal zygosis in Escherichia coli K12 mating

Summary We find that diaminopimelic acid in the recipient membrane is released into the medium during bacterial matings, indicating that membrane damage was inflicted on the recipient by the donor, probably for forming a channel for DNA transfer. When the damage is extensive, as in matings with an excess of Hfr bacteria, the F^- bacteria are killed (lethal zygosis). The transfer of a large amount of DNA in Hfr matings appears to enhance the killing. In analogous F⁺xF^- (Nal^r) matings, on the other hand, killing of F^- bacteria does not occur unless F plasmid transfer is inhibited by a substance like nalidixic acid. The F^- bacteria are killed, suggesting that F plasmids contain genes that express immunity to lethal zygosis in the recipient. For example, bacteria containing surface exclusion-deficient mutants of F plasmids, such as traS ^- and traT ^-, induce lethal zygosis in F^- bacteria and are susceptible to it. Various tra ^- polar mutants that abolish surface exclusion are also susceptible to lethal zygosis when mated with Hfr bacteria. Kinetic experiments indicate that in F⁺ (wild type) x F^- matings, immunity to lethal zygosis is expressed in the F^- recipient within 1/4 division time, whereas a complete expression of surface exclusion requires more than 1 division time. Thus, a complete change in all receptor sites seems to be required for the expression of surface exclusion.     

Early and late transfer of F genes by Hfr donors of E. coli K-12

Summary The results of short interrupted matings between an Hfr donor and a recipient strain carrying a temperature-sensitive replication mutant (frp ^–) of Flac demonstrate that the Hfr strain transfers this frp gene of F early in conjugation. This frp gene was also shown to function in the maintenance of mutant F plasmids which appear to be generated from the DNA transferred early in conjugation by Hfr donors. In the course of these experiments, it was further demonstrated that certain Hfr strains which had been described as transferring the tra genes early in fact transfer that region of F late in conjugation.