Recombinant F′ Factors from Escherichia coli K-12 Strains Carrying recB or recC

The frequency of genetic exchanges between F' factors and the bacterial chromosome was studied in recombination-deficient Escherichia coli mutants under conditions in which the recombinant F' factors were immediately transferred to new hosts. In a series of double matings, F101-1 thr(+)leu(-) episomes were first transferred into each of four intermediate F(-)thr(-)leu(+) strains carrying various rec alleles. After the original F' donors were killed with phage T6, the F101-1 episomes were then transferred from the intermediate cells to F(-)thr(-)leu(-)Str(R)recA(-) females. Recipients of nonrecombinant episomes formed Thr(+) (Str(R)) colonies, and recipients of recombinant episomes formed Leu(+)(Str(R)) colonies. A comparison of the numbers of Leu(+)(Str(R)) and Thr(+)(Str(R)) colonies shows that recB(-) males formed 18 to 21% and recC(-) formed 47 to 60% of the wild-type level of recombinant episomes that could be detected after transfer. No recombinant episomes were detected using a recA(-) intermediate strain. If the intermediate strains harboring the F101 episomes were purified, allowed to grow for 50 generations, and then mated with the recA(-) recipient, recombinant episomes were transferred at 8% of the wild-type level for recB(-) and 13% for recC(-). In contrast, only 0.4 and 0.6% of the normal number of recombinants were obtained from crosses between Hfr Cavalli donors and the same recB(-) and recC(-) strains. Recombinant episomes were detected with greater frequency among newly formed rec(+), recB(-), and recC(-) partial diploids than in those which were 50 generations old.     

Suppression of the Formation of Polygenotypic Recombinant Colonies by a maf Mutation in Mating with Hfrh

W3011, a Cavalli-type Hfr (HfrC), was mated with F-KY9474, maf-1, which cannot maintain F or F-like plasmids, and with F-OU9474, Maf+, a spontaneous revertant of KY9474. The recombinant colonies obtained were 100% monogenotypic from KY9474 and 90% monogenotypic from OU9474. On the other hand, in matings with OU11, a Hayes-type Hfr (HfrH), and these two F- strains, recombinant colonies derived from KY9474 showed only 22% polygenotypic recombinant colonies; whereas, those derived from OU9474 showed a high production rate (57%) of polygenotypic recombinant colonies. Among the polygenotypic recombinant colonies derived from KY9474 maf-1, 50% contained three or more recombinant types. These were probably derived from a small fraction of Maf+ revertants in the KY9474 population, as suggested by the results of mating this strain with M80, an F' strain that contains an amber mutation in traH. These results support the hypothesis that the donor DNA fragments derived from an HfrH can undergo a limited replication in the recipient to produce polygenotypic recombinant colonies, whereas those derived from HfrC cannot.     

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.     

High frequency FP2 donor of Pseudomonas aeruginosa PAO

Summary After NG mutagenesis an FP2 donor was isolated which exhibited an enhanced conjugational capacity for chromosomal genes. The recombination frequency was increased by two orders of magnitude as compared to the parenal strain. In plate matings recombinants arose at a frequency up to 5×10^-1 per donor cell. Late markers also recombined efficiently.An Hfr state of the donor strain was supported by (i) the high recombination frequency, (ii) the incompatibility reaction with plasmid pRO271 (=FP2::Tn401) and (iii) the clearcut transfer kinetics in interrupted matings, even for a late marker.     

Inhibition of Replication of an F′lac Episome in Hfr Cells of Escherichia coli

Hfr strains of Escherichia coli K-12 were found capable of accepting a F'lac episome during mating, with a frequency approximating that of F(-) strains. However, the F'lac episome was unable to replicate in the Hfr cells, and was diluted out during the growth of the culture. The lac(+) gene of the episome can be "rescued" by recombination into the host chromosome, as shown by the appearance of variegated recombinant colonies on a lactose-fermentation indicator medium. In recA Hfr strains, however, no lac(+) offspring were obtained in crosses with F'lac donors. The induced synthesis of beta-galactosidase in F'lac(+) x Hfr zygotes was studied. Rates of enzyme synthesis were approximately constant with respect to time as expected from unilinear inheritance of the F'lac episome. However, the rate of synthesis eventually increased, presumably due to integration of the lac(+) gene in some of the zygotes. In F'lac(+) x recA Hfr zygotes the rate of beta-galactosidase synthesis remained constant with respect to time, as expected.     

Deoxyribonucleic Acid Transferred from Ultraviolet-Irradiated Excision-Defective Hfr Cells of Escherichia coli K-12

Deoxyribonucleic acid (DNA) transfer from (3)H-thymine-labeled Hfr cells has been measured by determining the amount of radioactivity remaining after selective lysis of the donor cells in the mating mixture. DNA transfer was less effectively reduced by ultraviolet irradiation of excision-defective Hfr cells than was the yield of recombinants. The buoyant density of DNA transferred from unirradiated and irradiated Hfr cells was equivalent to that of double-stranded DNA. Mating-dependent DNA synthesis in the recipient has been measured by mating Hfr cells deficient in thymidine kinase with irradiated thymine-requiring F(-) cells in the presence of (3)H-thymine. The extent of such DNA synthesis approximated the amount of DNA transferred from unirradiated donors. Neither DNA transfer nor mating-dependent DNA synthesis could be reliably measured when both parents were irradiated. It is proposed that transferred Hfr DNA is replicated in the recipient and that this replication still occurs when the Hfr DNA contains dimers.     

Heteroduplex analysis of tra delta f'' plasmids and the mechanism of their formation.

Four tra delta FargG+ plasmids, derived from matings between Hfr AB312 and a recA recipient, have been shown to have deletions of at least 50% of the F genome, including the region in which the tra genes map. The mutant plasmids do contain the F genes required for plasmid maintenance. Correlations can be made between, on the one hand, the F genes present on the tradelta F' plasmids and the F genes transferred early by an Hfr donor, and, on the other hand, the F genes deleted from the tradelta F' plasmids and the F genes transferred late by an Hfr donor. A biased representation of proximally and distally transferred chromosomal markers among the tradelta F' elements was also demonstrated. Taken Taken together, the asymmetrical representation of Hfr genes and the cis dominance of the Tra phenotype of these mutants can best be explained by the hypothesis that the tradelta F' plasmids are formed by repliconation of the transferred exogenote in a recA recipient.     

Chromosome Transfer and Recombinant Formation with Deoxyribonucleic Acid Temperature-Sensitive Strains of Escherichia coli

Haploid recombinant yield is reduced in matings conducted at 42.5 C between deoxyribonucleic acid (DNA) temperature-sensitive [dna⁻(TS)] recipients unable to synthesize DNA at 42.5 C and any of the three major donor types (Hfr, F⁺, F′) of Escherichia coli. No such reduction is observed in matings conducted at 42.5 C when the dna⁻(TS) mutation is in the donor parent. Evidence is presented which indicates that chromosome transfer from donors to recipients unable to replicate DNA at 42.5 C during vegetative growth occurs at normal frequencies when the mating is conducted at 42.5 C. It is concluded that some stage in haploid recombinant formation is adversely affected in dna⁻(TS) recipients mated at the temperature restrictive for DNA synthesis.     

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.     

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.     

Linkage distortion following conjugational transfer of sbcC+ to recBC sbcBC strains of Escherichia coli.

Conjugational recombination in Escherichia coli depends normally on RecBCD enzyme, a multifunctional nuclease and DNA helicase produced by the recB, recC, and recD genes. However, recombination can proceed efficiently without RecBCD in recB or recC strains carrying additional mutations in both the sbcB and sbcC genes. Recombination in these strains, sometimes referred to as the RecF pathway, requires gene products that are not essential in the RecBCD-dependent process predominating in the wild type. It has also been reported to produce a different spectrum of recombinant genotypes in crosses with Hfr donors. However, the sbcC+ gene was unknowingly transferred to the recipient strain in some of these crosses, and this may have affected the outcome. This possibility was examined by conducting parallel crosses with Hfr donors that were either wild type or mutant for sbcC. Transfer of sbcC+ from an Hfr donor is shown to alter the frequency of recombinant genotypes recovered. There is a severe reduction in progeny that inherit donor markers linked to the sbcC+ allele and an increase in the incidence of multiple exchanges. Colonies of mixed genotype for one or more of the unselected proximal markers are also much more prevalent. Since the yield of recombinants is lower than normal, these changes are attributed to the reduced viability of recombinants that inherit sbcC+ from the Hfr donor. When the Hfr donor used is also mutant for sbcC, the yield of recombinants is greater and the frequencies of the different genotypes recovered are similar to those obtained in crosses with a rec+ sbc+ recipient, in which transfer of sbcC+ has no apparent effect. Earlier studies are re-examined in light of these findings. It is concluded that, while recombination in recBC sbcBC strains involves different enzymes, the underlying molecular mechanism is essentially the same as that in the wild type.     

Gene Transmission Among Strains of Erwinia amylovora

Stable donor strains of Erwinia amylovora were obtained from strain EA178R(1) (harboring an Escherichia coli F'lac) by selection for clones resistant to curing by acridine orange. These donor strains (EA178R(1)-99 and EA178R(1)-111) transfer chromosomal markers (arg, cys, gua, ilv, met, pro, ser, trp); the frequency of the appearance of recombinants prototrophic for Cys, Gua, Met, Ser, and Trp is highest (> 10(-5)), followed by recombinants prototrophic for Arg, Ilv, and Pro (10(-7) to 10(-5)). The results of interrupted matings, as well as the frequency of transmission of various markers, suggest that cys is transferred as an early marker by both donor strains. The Hfr state of these donor strains is rather likely on the basis of the following observations. The donor strains exhibit a relatively efficient and possibly oriented chromosome transfer; the Lac(+) character is not cured by acridine orange in these donor strains; and these donor strains do not transfer F.     

Chromosomal Integration of F′ Factors in Recombination-Deficient Hfr Strains of Escherichia coli

In matings between F′ donors and recombination-deficient Hfr recipients, we isolated progeny which transferred both episomal markers and Hfr markers early and with high frequency. A number of these progeny had two integrated sex factors. Investigation of these double Hfr strains showed that the F′ nearly always integrated in a homologous region of the chromosome. In any particular mating system integration was specific as to location and direction of chromosome transfer.     

Conjugational Recombination in Escherichia Coli: Genetic Analysis of Recombinant Formation in Hfr X F(-) Crosses

The formation of recombinants during conjugation between Hfr and F(-) strains of Escherichia coli was investigated using unselected markers to monitor integration of Hfr DNA into the circular recipient chromosome. In crosses selecting a marker located ~500 kb from the Hfr origin, 60-70% of the recombinants appeared to inherit the Hfr DNA in a single segment, with the proximal exchange located >300 kb from the selected marker. The proportion of recombinants showing multiple exchanges increased in matings selecting more distal markers located 700-2200 kb from the origin, but they were always in the minority. This effect was associated with decreased linkage of unselected proximal markers. Mutation of recB, or recD plus recJ, in the recipient reduced the efficiency of recombination and shifted the location of the proximal exchange (s) closer to the selected marker. Mutation of recF, recO or recQ produced recombinants in which this exchange tended to be closer to the origin, though the effect observed was rather small. Up to 25% of recombinant colonies in rec(+) crosses showed segregation of both donor and recipient alleles at a proximal unselected locus. Their frequency varied with the distance between the selected and unselected markers and was also related directly to the efficiency of recombination. Mutation of recD increased their number by twofold in certain crosses to a value of 19%, a feature associated with an increase in the survival of linear DNA in the absence of RecBCD exonuclease. Mutation of recN reduced sectored recombinants in these crosses to ~1% in all the strains examined, including recD. A model for conjugational recombination is proposed in which recombinant chromosomes are formed initially by two exchanges that integrate a single piece of duplex Hfr DNA into the recipient chromosome. Additional pairs of exchanges involving the excised recipient DNA, RecBCD enzyme and RecN protein, can subsequently modify the initial product to generate the spectrum of recombinants normally observed.     

The effect of UV irradiation on the capacity of an Hfr recA strain of Escherichia coli to act as donor

Summary The ability of a recA Hfr strain of Escherichia coli to form colonies is extremely sensitive to inhibition by ultraviolet light (Fig. 2). Furthermore, in this strain the synthesis of DNA is stopped completely by a dose of 385 ergs/mm² of UV (Fig. 3). Nevertheless, the ability of this recA Hfr strain to act as a donor in sexual recombination was no more sensitive to UV than that of a wild type donor (Fig. 1). Furthermore, when irradiated and mated with a recA female, in which DNA synthesis was also inhibited by UV (Fig. 3), there was a net synthesis of DNA as measured by the incorporation of C¹⁴ thymidine (Fig. 4). By using nalidixic acid resistant recA donors and recipients in all combinations, irradiating with UV and treating with nalidixic acid during mating, it is shown that DNA was synthesized by the donor (Fig. 5). It is concluded that synthesis of DNA directed by the sex factor during mating in a recA donor is not as sensitive to inhibition by UV as normal DNA synthesis in a recA donor.     

Self-Transfer and Genetic Recombination Mediated by P, the Sex Factor of Vibrio cholerae

Vibrio cholerae cells, infected with the sex factor P, produce discrete, plaque-like clearings when plated on lawns of P(-) cells. We investigated the nature of these clearings and conclude that they are probably sites of active mating. We developed a quantitative assay for P(+) cells and used it to study the kinetics of sex factor spread in broth cultures. Both established and newly infected donor populations were efficient sex factor donors, indicating that P is not self-repressed. We also investigated the kinetics of recombinant formation in broth matings. In 1-hr matings, we routinely found recombination frequencies of 10(-6) per donor cell. Kinetic studies of recombinant formation showed that the markers tested all appeared at early times. Thus P, the V. cholerae sex factor, seems to resemble F in its transfer properties.     

Isolation of High Frequency of Recombination Donors from Tn5 Chromosomal Mutants of Pseudomonas Putida Ppn and Recalibration of the Genetic Map

A Tn5 loaded derivative of the IncP-10 plasmid R91-5 (pMO75) was used as a suicide vector to generate random chromosomal insertion mutations in Pseudomonas putida PPN. Reintroduction of pMO75 into such mutants resulted in integration of the plasmid at the site of Tn5 insertion, giving rise to two classes of high frequency of donors recombination (Hfr) donors, transferring chromosome at high frequency (greater than 10(-1) per donor cell) in opposite directions. Consequently, Tn5 induced auxotrophic mutations could be equated with or distinguished from previously mapped mutations, and closely linked markers ordered, on the basis of marker recovery using the two classes of Hfr donor. The isolation of many new transfer origins allowed more accurate time-of-entry analysis than previously possible and resulted in the reduction of the genetic map from 103 min to 88 min.     

Naphthalene and Donor Cell Density Influence Field Conjugation of Naphthalene Catabolism Plasmids

We examined transfer of naphthalene-catabolic genes from donor microorganisms native to a contaminated site to site-derived, rifampin-resistant recipient bacteria unable to grow on naphthalene. Horizontal gene transfer (HGT) was demonstrated in filter matings using groundwater microorganisms as donors. Two distinct but similar plasmid types, closely related to pDTG1, were retrieved. In laboratory-incubated sediment matings, the addition of naphthalene stimulated HGT. However, recipient bacteria deployed in recoverable vessels in the field site (in situ) did not retrieve plasmids from native donors. Only when plasmid-containing donor cells and naphthalene were added to the in situ mating experiments did HGT occur.     

Naphthalene and donor cell density influence field conjugation of naphthalene catabolism plasmids

We examined transfer of naphthalene-catabolic genes from donor microorganisms native to a contaminated site to site-derived, rifampin-resistant recipient bacteria unable to grow on naphthalene. Horizontal gene transfer (HGT) was demonstrated in filter matings using groundwater microorganisms as donors. Two distinct but similar plasmid types, closely related to pDTG1, were retrieved. In laboratory-incubated sediment matings, the addition of naphthalene stimulated HGT. However, recipient bacteria deployed in recoverable vessels in the field site (in situ) did not retrieve plasmids from native donors. Only when plasmid-containing donor cells and naphthalene were added to the in situ mating experiments did HGT occur.     

Conjugation in Escherichia coli: Failure to Confirm the Transfer of Part of Sex Factor at the Leading End of the Donor Chromosome

Experiments were carried out attempting to determine whether part of sex factor is transferred at the leading end of the Hfr chromosome during conjugation. In the first experiment, an analysis was made of the donor properties of recombinant strains which had inherited the terminal but not the proximal marker from an Hfr. Secondly, recombinants integrating an extremely proximal marker from an Hfr were examined for the inheritance of a sex factor affinity locus adjacent to this marker. In the third experiment, proximal transfer of the wild-type allele of a temperature-sensitive sex factor mutation was looked for, using as recipient a temperature-sensitive Hfr strain, and as donor a wild-type Hfr isogenic with respect to the site of sex factor integration. In none of these experiments could the presence of sex factor material at the leading end be demonstrated. The results do not rule out the possibility that part of F is transferred proximally but only integrated at a very low frequency. They do, however, conflict with certain findings of other authors which, in the past, have been taken as evidence for the transfer of part of F at the leading end.