Studies on superinfection immunity among transmissible plasmids in Escherichia coli

Superinfection immunity was studied by a method which permits the specific labeling of plasmid DNA following its entry into a recipient cell during conjugation. By measuring the incorporation of [³H]thymine during matings between a donor strain of Escherichia coli K12 carrying the R factor, Rl, and various recipients, we found that the presence in the recipient of a plasmid closely related to R1 (F or R factor 222), or isogenic to it (resistance transfer factor, from Rl), resulted in a reduction of 80 to 90% in the rate of [³H]thymine incorporation, relative to a mating with a plasmid-negative (F ^?) recipient. The DNA present in these recipients after 60 minutes of mating was further examined by neutral sucrose gradient eentrifugation. The DNA in the F⁺ and F ^? recipients sedimented similarly, in two major peaks at 50 S (relaxed circles) and 75 S (supercoiled circles). However, the DNA in the RTF recipient sedimented at rates intermediate between 50 S and 75 S. Pulse-chase experiments revealed that the DNA species seen after 60 minutes of an R1 × RTF mating are normal replicative intermediates which have disappeared by 60 minutes in the R1 × F^? or R1 × F⁺ matings.These data support genetic evidence suggesting that superinfection immunity is due to two distinct effects—entry exclusion and plasmid incompatibility. Thus, F (related to R1 but genetically compatible with it), as well as the incompatible plasmids, 222 and the RTF of R1 itself, when present in the recipient, greatly reduce the total synthesis of newly introduced R1 DNA in the recipient. We interpret this effect as entry exclusion. Incompatibility, manifested by RTF, but not F, further reduces the efficiency of conjugation by slowing the rate at which a newly acquired plasmid is replicated.     

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.     

The requirements for conjugal DNA synthesis in the donor strain during flac transfer.

Although neither rifampicin nor spectinomycin had any effect on the frequency of Flac transfer by a sensitive donor, rifampicin but not spectinomycin prevented donor conjugal DNA synthesis as measured in matings between a dnaB donor and a tdk recipient. An untranslated RNA species is therefore probably required for this synthesis, although transfer took place even in its absence. Donor conjugal DNA synthesis was abolished in a dnaE donor, showing that DNA polymerase III is responsible for this process; again, plasmid DNA transfer was not affected.Flac mutants lacking the F pilus gave neither donor conjugal DNA synthesis nor plasmid DNA transfer, probably because they could not receive a “mating signal” to activate the transfer process. The products of traI and traM were also required both for donor conjugal DNA synthesis and for physical transfer of plasmid DNA, probably being involved in the conversion of covalently closed circular plasmid DNA into the open circular form that is the substrate for the independent although normally simultaneous synthesis and transfer steps. In contrast, donor conjugal DNA synthesis took place at a normal rate in both piliated traG and traN mutants, and at a reduced rate in traD mutants, although in no case was there physical transfer of plasmid DNA. These gene products are therefore required for DNA transfer to the recipient, and in addition, the absence of the traD product may hinder DNA synthesis.Based upon these results, a scheme for the processing of DNA during conjugation is presented.     

The Virulence Plasmid of Salmonella typhimurium Is Self-Transmissible

Most isolates of Salmonella enterica serovar Typhimurium contain a 90-kb virulence plasmid. This plasmid is reported to be mobilizable but nonconjugative. However, we have determined that the virulence plasmid of strains LT2, 14028, and SR-11 is indeed self-transmissible. The plasmid of strain SL1344 is not. Optimal conjugation frequency requires filter matings on M9 minimal glucose plates with a recipient strain lacking the virulence plasmid. These conditions result in a frequency of 2.9 × 10⁻⁴ transconjugants/donor. Matings on Luria-Bertani plates, liquid matings, or matings with a recipient strain carrying the virulence plasmid reduce the efficiency by up to 400-fold. Homologs of the F plasmid conjugation genes are physically located on the virulence plasmid and are required for the conjugative phenotype.     

On the ability of Salmonella typhimurium cells to form deoxycytidine nucleotides.

Summary The fate of the donor DNA after conjugation in Escherichia coli was studied through crosses with a Hfr lacZ5 donor and several F^- lacZ22 recipients. The fate of the donor allele was studied by assaying the -galactosidase activity formed by complementation between the lacZ5 allele and the lacZ22 allele. We used continuous cultures of the recipient in order to be able to study the fate of the donor DNA during many generations under constant physiological conditions. We could show that the donor DNA allele is inactivated in Rec⁺, recA171 and recB21 recipient cells. The inactivation rate depends on the nature of the recipient, Rec⁺ or recombination deficient, and especially in the case of the recombination deficient mutants on the growth rate of the recipient.     

The maintenance affinities of KLF-1 proximal F merogenotes inEscherichia coli

Summary In conjugation with donor strains carrying proximal F merogenotes of KLF-1 type about 100-fold lower frequency ofLeu ⁺ orLac ⁺ recombinants was found. The determination of the level of β-galactosidase synthesis during the initial period of mating indicated that the transfer process of plasmid DNA was not impaired. Among the recombinants selected a large fraction have not expressed the plasmic fertility functions. This phenomenon was found to be replicon specific and was observed only with proximal F merogenotes but not with classical F'lac and F'ORF-1 elements or RI-19 plasmid. The expression of KLF-1 plasmid functions in the cell seems to be affected by a chromosomal gene of the proximal F merogenote closely linked toleu marker.     

Chromosome Transfer by Autonomous Transmissible Plasmids: the Role of the Bacterial Recombination (rec) System

The ability of autonomous transmissible plasmids or sex factors to transfer chromosomal genes to F⁻ recipient bacteria has been investigated by using a series of rec⁺ and recA⁻ donor strains. It is concluded that chromosome transfer by most sex factors is virtually dependent upon the functional integrity of the bacterial recombination system. However, evidence is presented that suggests the existence of plasmid-specified mechanisms of interaction with the chromosome which are independent of the bacterial recombination system.     

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.     

A competence specific inducible protein promotes in vivo recombination in Streptococcus sanguis

Summary We describe the first example of a recombination-specific protein induced during the development of competence for transformation in Streptococcus sanguis. Elaborated in response to stimulation by competence-protein, the 51,000 Molecular Weight (MW) polypeptide is one of at least 10 new polypeptides transiently induced during the competence phase. Biochemical and genetic analyses of the parental, cipA⁺ (competence specific inducible polypeptide A), and mutant, cipA, strains have shown that the 51,000 MW polypeptide has two roles: its low level constitutive synthesis is required for repair of damage to DNA due to UV light and methylmethane sulfonate; its induced synthesis (3–6x10⁴ copies/cell) during the competence phase is essential for promoting recombination between donor single-stranded DNA and the recipient chromosome. Also, ccc plasmid donor DNA transformation, which occurs as a decreasing probability of the increasing donor plasmid MW, requires the inducible function specified by the 51,000 MW polypeptide. The MW independent low level transformation with ccc plasmids, the inheritance of plasmids by conjugation, and the stable maintenance of plasmids introduced by transformation and conjugation, respectively, are independent of the function specified by the 51,000 MW polypeptide.     

High Frequency Conjugative Mobilization Accomplished by a Natural Bacillus subtilisStrain Carrying a Large Plasmid

Conjugative properties of the strain Bacillus subtiliscarrying a large plasmid approximately 95 kb in size and isolated in Belarus from forest soil were described. The staphylococcal plasmid pUB110 that had previously been introduced into this strain was transferred to recipient cells of the Bacillus subtilis168 strain with a frequency of approximately 10^–2. The transfer occurred with approximately the same frequency both upon donor and recipient cell contact on the surface of membranes and in a liquid medium. The latter fact makes this system suitable as a model for studying conjugative mobilization in bacilli. A large plasmid cannot be transferred to recipients. An optimal temperature for conjugation of donor and recipient cells was 37°C, but conjugation also proceeded at lower temperatures, up to 21°C.     

The aggregation-mediated conjugation system of Bacillus thuringiensis subsp. israelensis: Host range and kinetics of transfer

The aggregation-mediated conjugation system in Bacillus thuringiensis subsp. israelensis encoded on the plasmid pXO16 is characterized by the formation of aggregates when Agr⁺ and Agr⁻ cells are socialized in exponential growth. Using the aggregation phenotypes, we have identified potential recipients of the aggregation-plasmid pXO16 among Bacillus cereus, Bacillus subtilis, Bacillus megaterium, Bacillus sphaericus, and 24 subspecies of B. thuringiensis. We found 14 Agr⁻ strains, i.e., potential recipients of the aggregation system encoded by plasmid pXO16. Five strains contained a conjugative apparatus of their own and were excluded from further examinations. To monitor the transfer of plasmid pXO16, we constructed a transposon insertion of the plasmid with Tn5401. The study of the plasmid transfer of pXO16::Tn5401 indicated the secretion of bacteriocins from both donor strain and recipient strains. Only one out of the nine strains examined was unable to receive the aggregation-plasmid pXO16 and express the aggregation phenotype and the conjugative abilities. It was found that the transfer of plasmid pXO16 to Bacillus thuringiensis subsp. israelensis Agr⁻ strains was 100%. All recipients had acquired the aggregation-plasmid pXO16 and converted to the Agr⁺ phenotype. Received: 29 February 1996 / Accepted: 26 March 1996     

Genetic Drift Suppresses Bacterial Conjugation in Spatially Structured Populations

Conjugation is the primary mechanism of horizontal gene transfer that spreads antibiotic resistance among bacteria. Although conjugation normally occurs in surface-associated growth (e.g., biofilms), it has been traditionally studied in well-mixed liquid cultures lacking spatial structure, which is known to affect many evolutionary and ecological processes. Here we visualize spatial patterns of gene transfer mediated by F plasmid conjugation in a colony of Escherichia coli growing on solid agar, and we develop a quantitative understanding by spatial extension of traditional mass-action models. We found that spatial structure suppresses conjugation in surface-associated growth because strong genetic drift leads to spatial isolation of donor and recipient cells, restricting conjugation to rare boundaries between donor and recipient strains. These results suggest that ecological strategies, such as enforcement of spatial structure and enhancement of genetic drift, could complement molecular strategies in slowing the spread of antibiotic resistance genes.     

Genetic Drift Suppresses Bacterial Conjugation in Spatially Structured Populations

Conjugation is the primary mechanism of horizontal gene transfer that spreads antibiotic resistance among bacteria. Although conjugation normally occurs in surface-associated growth (e.g., biofilms), it has been traditionally studied in well-mixed liquid cultures lacking spatial structure, which is known to affect many evolutionary and ecological processes. Here we visualize spatial patterns of gene transfer mediated by F plasmid conjugation in a colony of Escherichia coli growing on solid agar, and we develop a quantitative understanding by spatial extension of traditional mass-action models. We found that spatial structure suppresses conjugation in surface-associated growth because strong genetic drift leads to spatial isolation of donor and recipient cells, restricting conjugation to rare boundaries between donor and recipient strains. These results suggest that ecological strategies, such as enforcement of spatial structure and enhancement of genetic drift, could complement molecular strategies in slowing the spread of antibiotic resistance genes.     

R-Plasmid Transfer to and from Escherichia coli Strains Isolated from Human Fecal Samples

Strains of Escherichia coli recently isolated from human feces were examined for the frequency with which they accept an R factor (R1) from a derepressed fi⁺ strain of E. coli K-12 and transfer it to fecal and laboratory strains. Colicins produced by some of the isolates rapidly killed the other half of the mating pair; therefore, conjugation was conducted by a membrane filtration procedure whereby this effect was minimized. The majority of fecal E. coli isolates accepted the R factor at lower frequencies than K-12 F⁻, varying from 10⁻² per donor cell to undetectable levels. The frequencies with which certain fecal recipients received the R-plasmid were increased when its R⁺ transconjugant was either cured of the R1-plasmid and remated with the fi⁺ strain or backcrossed into the parental strain. The former suggests the loss of an incompatibility plasmid, and the latter suggests the modification of the R1-plasmid deoxyribonucleic acid (DNA). In general, the fecal R⁺E. coli transconjugants were less effective donors for K-12 F⁻ and heterologous fecal strains than was the fi⁺ K-12 strain, whereas the single strain of Citrobacter freundii examined was generally more competent. Passage of the R1-plasmid to strains of salmonellae reached mating frequencies of 10⁻¹ per donor cell when the recipient was a Salmonella typhi previously cured of its resident R-plasmid. However, two recently isolated strains of Salmonella accepted the R1-plasmid from E. coli K-12 R⁺ or the R⁺E. coli transconjugants at frequencies of 5 × 10⁻⁷ or less.     

Tetracycline-resistance inEscherichia coli; a genetic contribution

A non-transmissible tetracycline-resistance plasmid inE. coli was found to be transmissible by transduction and by conjugation with the aid of theE. coli K12 sex-factor. Transfer of the tetracycline-resistance plasmid (R-tet) by transduction or conjugation to anE. coli K12 Hfr strain revealed that the plasmid was incompatible with the integrated F-factor. Selection for tetracycline-resistance after conjugation or transduction yielded Hfr colonies which carried the tetracycline-resistance determinant as a chromosomal marker. The tetracycline-resistance determinant was integrated at the 1 min region of theE. coli chromosome map (Taylor and Trotter, 1967) between the markersara andleu. Apart from Hfr colonies with a chromosomal tetracycline-resistance determinant, F-gal⁺-mediated transfer of R-tet to strain Hfr R4 gave some colonies in which the tetracycline-resistance determinant was carried on a fused plasmid that, besides the resistance determinant, contained thegal ⁺ marker of the original F-gal ⁺. This fused plasmid is transmissible and confers to an F⁻ cell male-specific phage-sensitivity, like an F-factor does. It is suggested that this fused plasmid, which is compatible with the integrated F-factor in the Hfr R4 cells, arose by recombination between F-gal ⁺ and R-tet.     

The spread of plasmids in model populations of Escherichia coli K12.

Comparison of R100 with its derepressed derivative R100-1 showed that the capacity to repress tra function does not significantly affect the spread by retransfer of R100. F′lac was used to investigate the contributions of growth and transfer to spread of a plasmid through a recipient population. Ability to transfer F′lac was lost rapidly when donor cultures entered stationary phase, but aggregate-forming ability was lost much more slowly. Comparison of F′lactra⁺ with F′lactraH88, which is unable to retransfer from recipients, showed the importance of retransfer. We used a mathematical model to calculate the amount of retransfer needed to explain the rate of increase of F′lac progeny. This showed that the lag between a cell receiving F′lac and being able to retransfer it was a less important constraint on this rate of increase than the inherent rate of plasmid transfer by established donors.     

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.     

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.     

Host range of conjugation and replication functions of the Escherichia coli sex plasmid Flac. Comparison with the broad host-range plasmid RK2

The Escherichia coli conjugative plasmid Flac has a restricted host range, in that transfer to Pseudomonas aeruginosa is not detectable. The molecular basis for this host-range restriction was studied by a separate comparison of the replication and conjugation systems of Flac with those of the broad host-range plasmid RK2. The origin of transfer of Flac (oriT_F) was cloned onto a small RK2 replicon. The hybrid plasmid, pDG2906, could be transferred efficiently by both the Flac and RK2 conjugation systems to an E. coli recipient. The Flac conjugation system was able to transfer pDG2906 to P. aeruginosa, but only at a frequency of 10^?4 of that of the RK2 conjugation system. A second hybrid plasmid, containing the replication region of Flac with the transfer region of RK2, could not be established in P. aeruginosa. These results show that Flac is able to mediate low frequency transfer to P. aeruginosa, and that the lack of replication in Pseudomonas is ultimately responsible for the restricted host range.