Retrotransfer in Escherichia coli conjugation: bidirectional exchange or de novo mating?

DNA can be transferred among eubacteria and to plants and fungi by related, plasmid-mediated processes collectively referred to as bacterial conjugation. Conjugation occurs between cells in contact with one another and results in the unidirectional delivery of DNA from a bacterial donor to a recipient. Recent experiments that have reexamined the directionality of DNA flow during conjugation have come to different conclusions, some suggesting that genetic material also flows from recipient cells into the donor and that this process, termed retrotransfer, is likewise directed by donor-encoded functions. Given that bacteria are perhaps united with all living creatures by conjugation, the possibility of gene flow into donor bacteria during conjugation raises interesting evolutionary and biocontainment issues. Here we report that plasmid transmission from bacterial recipients to donors is not a donor-mediated event. Movement of genetic material from recipients to donors was inhibited by streptomycin, which does not inhibit the conjugative donor, indicating that retrotransfer requires gene expression in recipients. Furthermore, retrotransfer was reduced in matings mediated by plasmids that encode strong entry exclusion, to a similar degree as matings between two donors. Therefore we suggest that retrotransfer is in fact newly initiated conjugation between transconjugants and donors.     

Surrogate genetics: the use of bacterial hybrids as a genetic tool

Experimental dissection of bacterial genomes requires a well-developed set of genetic tools, but many bacteria lack the essential tools required for genetic analysis. Recombination of a region of chromosomal DNA from poorly characterized donor bacteria with the chromosome of a suitable surrogate host creates a genetically malleable hybrid, providing a short-cut for the detailed genetic analysis of the substituted genes. However, recombination between closely related but nonidentical DNA sequences ("homeologous recombination") is strongly inhibited, posing a powerful barrier to gene exchange between bacteria and a major impediment to the construction of genetic hybrids. By taking advantage of mutS and recD mutant recipients, it is possible to effectively overcome the recombination barrier, allowing construction of genetic hybrids in a related surrogate host. Once stably recombined into the recipient chromosome, the donor DNA can be studied with all the genetic tools available in the surrogate host. In addition to facilitating standard genetic analysis, use of a surrogate host can provide novel approaches to study the physiological roles of unique genes from poorly characterized bacteria.     

Identification of host genes that affect acquisition of an integrative and conjugative element in Bacillus subtilis

Conjugation, a major type of horizontal gene transfer in bacteria, involves transfer of DNA from a donor to a recipient using donor‐encoded conjugation machinery. Using a high‐throughput screen (Tn‐seq), we identified genes in recipients that contribute to acquisition of the integrative and conjugative element ICEBs1 by Bacillus subtilis. We found that null mutations in some genes caused an increase, and others a decrease in conjugation efficiency. Some mutations affected conjugation only when present in recipients. Other mutations affected conjugation when present in donors or recipients. Most of the genes identified are known or predicted to affect the cell envelope. Several encode enzymes involved in phospholipid biosynthesis and one encodes a homologue of penicillin‐binding proteins. Two of the genes identified also affected conjugation of Tn916, indicating that their roles in conjugation may be general. We did not identify any genes in recipients that were essential for ICEBs1 conjugation, indicating that if there are such genes, then these are either essential for cell growth or redundant. Our results indicate that acquisition of ICEBs1, and perhaps other conjugative elements, is robust and not easily avoided by mutation and that several membrane‐related functions affect the efficiency of conjugation.     

Validity of mechanism of gene transfer in the process called conjugation in bacteria

We have attempted a new evaluation of the process of conjugation in bacteria, because of some basic dissimilarities observed between this and that of eukaryotes, or plants and animals. Reference donor and recipient strains, widely used to prove conjugation in bacteria, were chosen; addition of DNase during the conjugation process, led to an unexpected but highly reproducible increase in the transconjugant colony counts (TCC; ca. > or = 1 log), when compared with that of the controls without DNase. Transconjugants were also obtained when the same live donors were substituted with the UV-killed ones although the TCC was very low initially. Contrarily, donors treated with DNA-intercalating agents, e.g. acridine orange or ethidium bromide, resulted in a complete failure to produce transconjugants. There was a quantitative relationship between the DNase used on donors and levels of DNA sugars/nucleotides/DNA, which possibly resulted from interaction between the DNase and DNA being present/produced on the donor surface. This may be indicative of what may actually happen in the donor-recipient mixtures in the conjugation test proper, where the recipient DNase may activate a donor DNA production cycle. The evidences presented did not suggest that the donor DNA in the conjugation process is actually vestibuled through any intercellular conjugation passages, and is susceptible to the action of DNase or the intercalating dyes.     

Gene transfer between Salmonella enterica serovar Typhimurium inside epithelial cells

Virulence and antibiotic resistance genes transfer between bacteria by bacterial conjugation. Conjugation also mediates gene transfer from bacteria to eukaryotic organisms, including yeast and human cells. Predicting when and where genes transfer by conjugation could enhance our understanding of the risks involved in the release of genetically modified organisms, including those being developed for use as vaccines. We report here that Salmonella enterica serovar Typhimurium conjugated inside cultured human cells. The DNA transfer from donor to recipient bacteria was proportional to the probability that the two types of bacteria occupied the same cell, which was dependent on viable and invasive bacteria and on plasmid tra genes. Based on the high frequencies of gene transfer between bacteria inside human cells, we suggest that such gene transfers occur in situ. The implications of gene transfer between bacteria inside human cells, particularly in the context of antibiotic resistance, are discussed.     

Determination of the mechanism of retrotransfer by mechanistic mathematical modeling.

Two mathematical models to elucidate the mechanism of retromobilization (or retrotransfer), that is, the ability of conjugative plasmids to mobilize genes into the cell containing the conjugative plasmid, were developed. This study deals with retromobilization of nonconjugative plasmids (Tra-Mob+). Plasmid transfer was modeled by two mass action models. The first is based on the hypothesis that retromobilization of the Tra-Mob+ vector occurs in one step, by means of the pilus formed by the Tra+ plasmid in the original host. In the second model, retromobilization is considered to be a two-step process involving two transfer events. The first step involves the transfer of the Tra+ plasmid from the recipient cell to the donor of the nonconjugative vector, and during the second encounter the nonconjugative vector is mobilized toward the recipient. Since the relationships between the number of transconjugants and the number of recipients for the two models are different, filter matings were performed for short time periods with different initial densities of the recipient population. Comparison of the numbers of transconjugants with the results of the mathematical equations confirmed the hypothesis that retromobilization is a one-step conjugation process.     

Flow cytometric analysis of Enterococcus faecalis aph2"(+) response to aph2" (-) strains with high and low gene transfer rate

Resistance genes, as aph2" are usually encoded on conjugate plasmids and spread with high rate 2 among Gram-positive cocci. The conjugation is inducted by recipient strains by secreting specific pheromone involved in formation of mating aggregates with donor cells. The project aimed to check if strain with lower rate of gene transfer differ also from strains with high gene transfer in ability to aggregate to donor strains. In our study we used two aph2"(+), three aph2"(-) with low transfer e and three aph2"(-) with high transfer strains. Each time one aph2"(+) and one aph2" strains were cultivated for 18h in BHI. The bacteria was washed, stained with carboksyfluorescein, and analyzed by flow cytometry in FACS BD cytometr. Relative fluorescence and size of aggregation was used to compare influence on particular stains. In result of induction of aph2"(+) strains with aph2" recipients with high transfer rate we observed increase of size and number aggregates. Surprisingly, induction with aph2" recipients with low transfer rate result in two different reaction of aph2"(+) donors. In case of one of the , according to expectation we do not observe increase of aggregation. Second of the donors aggregate with induction with aph2" recipients with low transfer rate, but in contrast to reaction to presence of other recipients, fluorescence of such aggregates increased. The results show that strain with lower rate of gene transfer in fact differ from strains with high gene transfer in ability to aggregate to donor strains ant that analysis of aggregation alone is insufficient to distinguish between recipients of high and low transfer rate.     

Mechanism of retrotransfer in conjugation: prior transfer of the conjugative plasmid is required.

Bacterial conjugation normally involves the unidirectional transfer of DNA from donor to recipient. Occasionally, conjugation results in the transfer of DNA from recipient to donor, a phenomenon known as retrotransfer. Two distinct models have been generally considered for the mechanism of retrotransfer. In the two-way conduction model, no transfer of the conjugative plasmid is required. The establishment of a single conjugation bridge between donor and recipient is sufficient for the transfer of DNA in both directions. In the one-way conduction model, transfer of the conjugative plasmid to the recipient is required to allow the synthesis of a new conjugation bridge for the transfer of DNA from recipient to donor. We have tested these models by the construction of a mutant of the self-transmissible, IncP plasmid RK2lac that allows the establishement of the conjugation bridge but is incapable of self-transfer. Four nucleotides of the nic region of the origin of transfer (oriT) were changed directly in the 67-kb plasmid RK2lac by a simple adaptation of the vector-mediated excision (VEX) strategy for precision mutagenesis of large plasmids (E. K.Ayres, V. J. Thomson, G. Merino, D. Balderes, and D. H. Figurski, J. Mol. Biol. 230:174-185, 1993). The resulting RK2lac oriT1 mutant plasmid mobilizes IncQ or IncP oriT+ plasmids efficiently but transfers itself at a frequency which is 10(4)-fold less than that of the wild type. Whereas the wild-type RK2lac oriT+ plasmid promotes the retrotransfer of an IncQ plasmid from Escherichia coli or Pseudomonas aeruginosa recipients, the RK2lac oriT1 mutant is severely defective in retrotransfer. Therefore, retrotransfer requires prior transfer of the conjugative plasmid to the recipient. The results prove that retrotransfer occurs by two sequential DNA transfer events.     

Role and specificity of plasmid RP4-encoded DNA primase in bacterial conjugation.

The role of the DNA primase of IncP plasmids was examined with a derivative of RP4 containing Tn7 in the primase gene (pri). The mutant was defective in mediating bacterial conjugation, with the deficiency varying according to the bacterial strains used as donors and recipients. Complementation tests involving recombinant plasmids carrying cloned fragments of RP4 indicated that the primase acts to promote some event in the recipient cell after DNA transfer and that this requirement can be satisfied by plasmid primase made in the donor cell. It is proposed that the enzyme or its products or both are transmitted to the recipient cell during conjugation, and the role of the enzyme in the conjugative processing of RP4 is discussed. Specificity of plasmid primases was assessed with derivatives of RP4 and the IncI1 plasmid ColIb-P9, which is known to encode a DNA primase active in conjugation. When supplied in the donor cell, neither of the primases encoded by these plasmids substituted effectively in the nonhomologous conjugation system. Since ColIb primase provided in the recipient cell acted weakly on transferred RP4 DNA, it is suggested that the specificity of these enzymes reflects their inability to be transmitted via the conjugation apparatus of the nonhomologous plasmid.     

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.     

Single-stranded conjugation in Escherichia coli K-12

Summary The mutation BT43 in the gene dnaB leads to the inhibition of vegetative and conjugational DNA synthesis at 42°. The consequences in case of conjugation are very unusual. The fragment of donor DNA tramsmitted to the recipient cell remains single-stranded and is integrated as such into the recipient chromosome similar to the main events during transformation. We call this process single-stranded (SS) conjugation.The evidence for this statement comes from the measurement of the time of expression of the gene tsx, containing the genetic information for the receptor of phage T6. The gene tsx is introduced into a dnaBT43 recipient cell alternatively by two different donors Hfr H and Hfr C, which are characterized by opposite directions of transfer. Therefore both donors introduce into the recipient cell alternatively the informational or noninformational DNA strand. If conjugation is performed at a nonpermissive temperature, the transferred DNA piece remains single-stranded and is integrated as such into the recipient chromosome. If it is the informational strand (case of Hfr H), it is transcribed very fast and yields the protein in question in about 20 min. If the noninformational strand is integrated (Hfr C) about 40 min additional time is required to effect cell division.SS-conjugation is very sensitive to the action of exonucleases Exo I and Exo V and is much enhanced in the absence of both nucleases in the recipient.The exogenous DNA pieces are integrated as short insertions, this leads to the disjoining of linked markers and to a very short scale of the genetic map. Because the donor DNA undergoes recombination in the single-stranded state heteroduplex regions originate which are subsequently corrected by the enzymes of the recipient cell. The situation leads to a very special but predictable heterogeneity of the progeny of transconjugants.The fact of the existence of this special process, SS-conjugation, drastically different from common conjugation in many respects, suggests that common conjugation leads to the integration of double-stranded DNA pieces into the recipient chromosome.     

Genetic Evidence of Protein Transfer during Bacterial Conjugation

DNA can be transferred from eubacteria to at least plants, fungi, and all other eubacteria by related, plasmid-mediated conjugation. Little is known about the biochemistry of intraspecies or interspecies DNA transfer. Even less is known about what other molecules may accompany the DNA, or the direct or inheritable effects on recipients of these escort molecules. This report describes a genetic assay for detecting protein transfer during conjugation. The assay monitored phage lambda released from lysogenic recipients as a result of the concomitant delivery of the Escherichia coli RecA protein and plasmid DNA. The heretofore unexpected transfer of a donor chromosome-encoded protein initiates a heritable change in the recipient without altering its genetic make-up. The mechanism of transfer could be independent of transferred DNA.     

Methods for Detection of Conjugative Plasmid Transfer in Aquatic Environments

Donor and recipient counter selection was evaluated by selecting bacteria that received plasmid RP4 by conjugation on filters and in lake water microcosms. Three counter selection systems were compared; (i) Use of antibiotic-resistant recipients, (ii) use of an auxotrophic donor, and (iii) use of a donor with chromosomal suicide genes. Transfer efficiencies of transconjugants per recipient obtained with the three different counter selection systems in filter-matings were not significantly different. Some nalidixic acid-resistant recipients became partly sensitive to nalidixic acid after receiving the plasmid. Use of an auxotrophic donor was a feasible and easy way to recover indigenous transconjugants. A strain with two copies of the suicide gene gef was successfully eliminated in filter-matings, but elimination of the donor in microcosms by induction of the suicide genes did not succeed. Thus, this counter selection system was not usable in microcosm experiments. Received: 3 March 1998 / Accepted: 15 May 1998     

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.     

Recipient Range of IncP-7 Conjugative Plasmid pCAR2 from Pseudomonas putida HS01 is Broader than from Other Pseudomonas Strains

The carbazole-degradative plasmid pCAR2 was isolated from Pseudomonas putida and had a genetic structure similar to that of pCAR1, the IncP-7 archetype plasmid. Mating analyses of pCAR2 with various recipient strains showed that it could transfer from HS01 to Pseudomonas recipients: P. chlororaphis, P. fluorescens, P. putida, P. resinovorans and P. stutzeri. The range of recipients changed when different hosts were used as a donor of pCAR2. The range of the plasmid from strain HS01 was broader than that using P. resinovorans CA10dm4 or P. putida KT2440. When pCAR1 or pCAR2 was transferred from the same cell background, the range and frequency of conjugation were now similar. Quantitative RT-PCR analyses indicated that tra/trh genes on both plasmids were similarly transcribed in each donor strain suggesting that the conjugative machinery of both plasmids may function similarly, and that other host factors are affecting the recipient range and frequency of conjugation.     

Molecular Studies on Entry Exclusion in Escherichia coli Minicells

Minicells produced by abnormal cell division in a strain of Escherichia coli (K-12) have been employed here to investigate the phenomenon of "entry exclusion." When purified minicells from strains containing F' or R factors, or both, are mated with radioactive thymidine-labeled Hfr or R(+) donors, the recipient minicells can be conveniently separated from normal-sized donors following mating, and the products of conjugation can be analyzed in the absence of donors and of further growth of the recipients. Transmissible plasmids or episomes are transferred less efficiently to purified minicells derived from strains carrying similar or related elements than to strains without them. Measurement of deoxyribonucleic acid (DNA) degradation and determination of weight-average molecular weights following transfer indicate that degradation of transferred DNA or transfer of smaller pieces cannot account for the comparative reduction in transfer to entry-excluding recipients. Therefore, we conclude that entry exclusion operates to prevent the physical entry of DNA into recipients expressing the exclusion phenotype. The R-produced repressor (product of the drd(+) gene), which represses fertility (i.e., ability to act as donor), reduces exclusion mediated by R or F factor, or both, in matings between strains carrying homologous elements. Furthermore, the data suggest that the presence of the F pilus or F-like R pilus on recipient cells ensures maximum expression of the exclusion phenotype but is not essential for its expression. In contrast to previous suggestions, we found no evidence for a reduction of entry exclusion attributable to the DNA temperature-sensitive chromosomal mutation dnaB(TS).     

Effect of Genomic Location on Horizontal Transfer of a Recombinant Gene Cassette Between Pseudomonas Strains in the Rhizosphere and Spermosphere of Barley Seedlings

The use of genetically engineered bacteria in natural environments constitutes a risk of transfer of recombinant DNA to the indigenous bacteria. However, chromosomal genes are believed to be less likely to transfer than genes on mobilizable and conjugative plasmids. To study this assumption, horizontal transfer of a recombinant gene cassette inserted into the chromosome of a Pseudomonas stutzeri strain, into a mobilizable plasmid (pAGM42), and into a conjugative plasmid (pKJK5) isolated from barley rhizosphere was investigated. Horizontal transfer efficiencies of the gene cassette inserted into a conjugative plasmid was 8.20 × 10⁻³ transconjugants/(donors × recipients)^1/2 in the rhizosphere and 4.57 × 10⁻² transconjugants/(donors × recipients)^1/2 in the spermosphere. Mobilization of the plasmid pAGM42 by the plasmids RP4 and pKJK5 was also detected at high levels in the microcosms, transfer efficiencies were up to 4.36 × 10⁻³ transconjugants/(donors × recipients)^1/2. Transfer of chromosomal encoded genes could not be detected in the microcosms by conjugation or transformation. However, transformation did occur by using the same bacterial strains under laboratory conditions. The rhizosphere and especially the spermosphere thus proved to be hot spot environments providing favorable conditions for gene transfer by mobilization and conjugation, but these environments did not support transformation at a detectable level. Received: 21 July 2000 / Accepted: 21 August 2000     

Analysis of the sequence and gene products of the transfer region of the F sex factor.

Bacterial conjugation results in the transfer of DNA of either plasmid or chromosomal origin between microorganisms. Transfer begins at a defined point in the DNA sequence, usually called the origin of transfer (oriT). The capacity of conjugative DNA transfer is a property of self-transmissible plasmids and conjugative transposons, which will mobilize other plasmids and DNA sequences that include a compatible oriT locus. This review will concentrate on the genes required for bacterial conjugation that are encoded within the transfer region (or regions) of conjugative plasmids. One of the best-defined conjugation systems is that of the F plasmid, which has been the paradigm for conjugation systems since it was discovered nearly 50 years ago. The F transfer region (over 33 kb) contains about 40 genes, arranged contiguously. These are involved in the synthesis of pili, extracellular filaments which establish contact between donor and recipient cells; mating-pair stabilization; prevention of mating between similar donor cells in a process termed surface exclusions; DNA nicking and transfer during conjugation; and the regulation of expression of these functions. This review is a compendium of the products and other features found in the F transfer region as well as a discussion of their role in conjugation. While the genetics of F transfer have been described extensively, the mechanism of conjugation has proved elusive, in large part because of the low levels of expression of the pilus and the numerous envelope components essential for F plasmid transfer. The advent of molecular genetic techniques has, however, resulted in considerable recent progress. This summary of the known properties of the F transfer region is provided in the hope that it will form a useful basis for future comparison with other conjugation systems.