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.     

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.     

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.     

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.     

Lethal zygosis in recombination-deficient mutants of Escherichia coli.

Use of nonselective medium for plating cells following mating has revealed that Rec recipient strains of E. coli may be killed as a result of conjugation. Sensitivity of RecA-, RecB-, and RecC- recipients increases with ratio of donor: recipient cells in mating mixtures and with time of mating. A Rec+ recipient shows no lethal zygosis in these experiments performed without aeration. Cell contact does not seem to be responsible for the sensitivity of Rec- strains, since lethality is prevented when cell contact is permitted but DNA transfer is not. Thus, an event(s) occuring subsequent to entry of donor DNA appears to cause lethality in Rec- recipients.     

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.     

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.     

Derepression of F factor function in Salmonella typhimurium

In Salmonella typhimurium LT2 the F factor of Escherichia coli K-12 replicates normally but is repressed; Flac+ cells give no visible lysis on solid media with male-specific phages, low frequency transfer of Flac+ (0.001-0.007 per donor cell), few f2 infective centers (0.002-0.006 per cell), and they propagate male-specific phages to low titers. Thus they display a Fin+ (fertility inhibition) phenotype. This repression, owing to pSLT, a 60 Mdal plasmid normally resident in S. typhimurium, was circumvented by the following materials: (i) Flac+ plasmids from E. coli with mutations in finP or traO; (ii) a S. typhimurium line which had been cured of pSLT; (iii) pKZl, a KmR plasmid in the same Inc group as pSLT, which caused expulsion of pSLT and made Fin- lines; (iv) F-Fin- mutants which originated spontaneously and which are present in most Hfr strains of S. typhimurium. Strains which are derepressed for F function by the above methods give visible lysis on solid media with male-specific phages, ca. 1.0 Lac+ recombinants per donor cell in conjugal transfer, ca. 0.82 f2 infective centers per cell, over 80% of cells with visible F pili, and propagation of male-specific phages to high titer. These data confirm earlier observations that pSLT represses F by the FinOP system. In addition, it shows that there is no other mechanism which represses F function in S. typhimurium. If donor function is derepressed by one of the above methods, and if rough recipient strains are used, F-mediated conjugation in S. typhimurium LT2 is as efficient as in E. coli K-12.     

Escherichia coli K-12 F? mutants that form mating aggregates but form transconjugants with low frequencies

Summary We have isolated Escherichia coli F^– mutants which, when mated with either Hfr or F, can form stable mating aggregates well but produce transconjugants with reduced frequencies. Selection procedure and other tests rule out the possibility that they are Rec^– strains. These mutants can be classified into two types: type I mutants can induce conjugal DNA replication in the donor, yet form transconjugants poorly; whereas, type II mutants induce conjugal DNA replication with poor efficiencies in the donor. Further tests indicate that type I mutants are very sensitive to lethal zygosis and their membranes, both inner and outer, show alterations in protein composition, whereas type II mutants are insensitive to lethal zygosis, and have an obvious alteration in the protein composition of their outer membrane. These results suggest that type I is defective in transconjugant formation primarily due to a change in the inner membrane, whereas type II is defective in generating a mating signal, which induces donor conjugal DNA replication, due to an alteration in the outer membrane.     

Isolation and characterization of Hfr strains of Erwinia amylovora

Hfr strains (Hfr 159 and its derivatives, Hfr 160 and Hfr 161) were constructed from Erwinia amylovora ICPB EA178 by introducing an Escherichia coli F'his+ plasmid and then selecting for integration of F'his+ after treatment with acridine orange. The Hfr strains were relatively stable upon repeated transfers on nonselective media. Interrupted mating experiments and analyses of inheritance of unselected markers showed that his+ is transferred by Hfr 159 as the proximal marker at a relatively high frequency (about 5 x 10(-4) recombinants per input donor cell), followed by ilv+, orn+, arg+, pro+, rbs+, met+, trp+, leu+, ser+, and thr+ (not necessarily in that precise order). The donor strains, previously constructed in E. amylovora by integration of F'lac+ from E. coli transfer cys+ as the proximal marker followed by ser+. Further analysis of one of those earlier donor strains, Hfr99, showed that ser+ is followed by arg+, orn+, met+, pro+, leu+, ilv+, rbs+, his+, trp+, and thr+ (not necessarily in that precise order). Thus, the Hfr strains constructed by integration of F'his+ are different, in terms of origin and direction of transfer, from those derived from integration of F'lac+. The applicability of these Hfr strains to mapping the genes on the E. amylovora chromosome is indicated.     

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.     

Rsf mutants of Escherichia coli HfrC defective in the production of the factor stimulating recombination in conjugation

Summary Male strains of Escherichia coli K12 excrete a protein which stimulates recombination in conjugation. The properties of four Rsf^- mutants unable to produce this recombination-stimulating factor (RSF) have been studied. Two of the mutants have a pleiotropic phenotype which includes hypersensitivity to the lethal effects of ultraviolet (UV) and monofunctional alkylating agents (MAA) and markedly decreased growth rates at elevated temperatures. The latter property is associated with a diminished rate of DNA synthesis. Many more single-strand breaks are detected in the DNA of the pleiotropic mutants after MAA treatment than in the wild type, which are, however, repaired during incubation of the Rsf^- cells after the treatment. No changes in UV-induced DNA breakdown or in host-cell reactivation of bacteriophage T1 have been detected in the mutant. The complete restoration of the wild type characters in Rsf⁺ revertants of these mutants proves that their complex phenotype is due to a single pleiotropic mutation. The integration of the wild type F factor into the chromosome of a derivative of a pleiotropic mutant retaining a portion of a previously integrated sex factor results in the complete restoration of the wild phenotype, which implies that the Rsf^- mutation is located in the episomal DNA. These results show that some products specified by the F factor are necessary for the maintaince of the wild phenotype of Hfr cells. Possible mechanisms of this phenomenon are discussed.     

The plasmid of Salmonella typhimurium LT2

Summary Methods of clonal analysis were applied to the study of heterogeneity of the progeny after crosses of 4 donor strains (Hfr H, Hfr C, KL 16 and KL 99) with 3 recipient strains (PC 0212, AB 712 and ECK 022). Three markers were used in each cross. The distal one was the selective marker. The inheritance of two additional proximal markers characterized the heterogeneity of clones originating from particular zygotes. In most crosses the percentage of heterogeneity exceeded 30. One of the recipient strains, obtained by conjugation of the conventional strain PC 0212 with the donor Hfr H revealed unusual properties in respect to heterogeneity. Exconjugants derived from this recipient (ECK 022) and donor Hfr H and Hfr C had a heterogeneity index of about 5%. It is shown that this unusual behavior reflects a very fast process of segregation of recombinants.In crosses with the donors KL 16 and KL 99 the same recipient revealed normal indices of heterogeneity. All these data are explained assuming that there exists a specific genetic marker which determines the process of decay of merozygotes. Tentatively it is called het. Its approximate localization was deduced from specifically designed experiments, in which the heterogeneity of the progeny was found very different, when the donor KL 16 transmitted different parts of its chromosome to the recipient ECK 022.     

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.     

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.     

Isolation of a hybrid F' factor-carrying Escherichia coli lactose region and Salmonella typhimurium histidine region, F42-400 (F' ts114 lac+, his+): its partial characterization and behavior in Salmonella typhimurium.

Episome F' ts114 lac+ (F42-114) was transferred into Salmonella typhimurium carrying an F'his+ (FS400) episome, and fused episome F' ts114 lac+, his+ (F42-400) was obtained. Episome F42-400 could be transferred to S. typhimurium, Escherichia coli and Klebsiella pneumoniae. Identification of the episome was based on: (i) temperature sensitivity of the Lac+ and His+ phenotypes; (ii) the fact that F- segregants, obtained after temperature curing or acridine orange curing, were simultaneously Lac- and His-; and (iii) linkage of lac+ with his+ in episomal transfers to E. coli and S. typhimurium. The frequency of episome transfer was influenced by the genotype of the donor. Plasmid LT2, prevalent in S. typhimurium LT2 strains, was suggested to be responsible for the low fertility of S. typhimurium donors. Episome F42-400 was capable of chromosome mobilization, and the extent of chromosome mobilization was not influenced by the presence or absence of the histidine region on the donor chromosome. Growth in a defined medium with acridine orange was able to cure F42-400. The frequency of curing was increased (the frequency of His+ cells was 0.0001%) if the cells were grown at 40 C in the presence of acridine orange. Selection for temperature-resistant Lac+, His+ derivatives in a strain without histidine deletion yielded Hfr strains. However, similar and stronger selections in strains without the chromosomal histidine region failed to yield Hfr strains. Our inability to obtain Hfr's in strains without the chromosomal histidine region was explained by assuming that the episome F42-400 has lost the F sites involved in integration into the S. typhimurium chromosome.     

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.     

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.     

Transfer and incorporation of genes controlling beta-D-galactosidase synthesis from Hfr and F' donors of Escherichia coli

Ganesan, Ann K. (Syntex Institute of Molecular Biology, Palo Alto, Calif.), and Boris Rotman. Transfer and incorporation of genes controlling beta-d-galactosidase synthesis from Hfr and F' donors of Escherichia coli. J. Bacteriol. 92:1378-1382. 1966.-Comparisons were made between Hfr(1) and F(13) donors with respect to the frequency of transfer and incorporation of genes controlling beta-d-galactosidase synthesis. The Hfr(1) donor transfers these genes as part of the chromosome, and the F(13) donor transfers them by F-duction. The criterion used for gene transfer was the acquisition by recipient cells of the ability to synthesize the enzyme, beta-d-galactosidase, measured by fluorogenic assays at the single-cell level. The criterion for incorporation was the formation of lac(+) recombinant colonies. It was found that the two types of donor showed the same frequency of gene transfer, but the probability of incorporation was 10-fold higher in F(13) matings than in Hfr(1) matings. In the former, between 46 and 97% of the merozygotes produced recombinant colonies; in the latter, 2 to 6% did so.     

Expression of a mutation affecting F incompatibility in the integrated but not the autonomous state of F.

Previously we have described a mutant Hfr strain in which incompatibility between the integrated F factor and an autonomous F-prime (F') factor was abolished. The mutation (inc) was located in the integrated F factor. F-prime factors isolated from the mutant Hfr strain have the same incompatibility behavior as those isolated from normal Hfr strains. Reintegration of these F' factors into the chromosome restores the Inc- phenotype characteristic of the mutant Hfr. The inc mutation thus affects incompatibility between integrated F and autonomous F(Fi-Fa incompatibility) but not incompatibility between two autonomous F factors (Fa-Fa incompatibility). The implications of this finding for the mechanism of plasmid incompatibility are discussed.