Hfr-mediated conjugative transfer of pBR322 vector carrying the chromosomal DNA of Escherichia coli

Hybrid plasmids consisting of a non-mobilized plasmid, pBR322, and a segment of chromosomal DNA of Escherichia coli could be transferred from an Hfr donor to recipient cells by a bacterial mating. When the chromosomal DNA in the plasmid corresponded to the early transfer region of the Hfr, the frequency of the transfer was high. The recA function of both donor and recipient cells was required in the transfer. The physical association of the hybrid plasmid with the transferring Hfr chromosome through the homologous sequences may mediate the transfer of the non-mobilized plasmid. This phenomenon is useful for the determination of the chromosomal location of an unidentified fragment cloned in a non-mobilized plasmid.     

Plasmid DNA transfer in Mycobacterium smegmatis involves novel DNA rearrangements in the recipient, which can be exploited for molecular genetic studies

The establishment of molecular genetic techniques is essential for development of new treatments for mycobacterial infections. To this end, we recently described a novel DNA transfer process that occurs in the model mycobacterial organism Mycobacterium smegmatis. This transfer system is most like conjugal DNA transfer in that it requires two viable parents, is DNAse resistant and occurs between distinct donor and recipient strains. Cis-acting sequences called bom, which confer transferability, are distinct from the prototypical oriT sites of conjugative plasmids, as they occur at multiple locations in the chromosome and require RecA in the recipient to mediate plasmid recircularization. Here, we show that a plasmid containing two of these bom regions can undergo several fates in the recipient cell, each of which require recipient recombination functions. The products of plasmid transfer that we observed provide further insights toward a model for DNA transfer. Furthermore, we have taken advantage of the recombination events that occur in the recipient to develop simple procedures for capturing, or replacing specific segments of the recipient chromosome. To demonstrate the potential of the system, we describe the capture and deletion of 25 kb of the M. smegmatis chromosome, and targeted-allele exchange of the recipient recB and recD genes. Using these transfer-mediated rearrangements, we demonstrate that homology with the recipient chromosome and RecB, but not RecD, are essential for DNA transfer.     

Conjugal transfer of chromosomal DNA in Mycobacterium smegmatis

The genus Mycobacterium includes the major human pathogens Mycobacterium tuberculosis and Mycobacterium leprae . The development of rational drug treatments for the diseases caused by these and other mycobacteria requires the establishment of basic molecular techniques to determine the genetic basis of pathogenesis and drug resistance. To date, the ability to manipulate and move DNA between mycobacterial strains has relied on the processes of transformation and transduction. Here, we describe a naturally occurring conjugation system present in Mycobacterium smegmatis , which we anticipate will further facilitate the ability to manipulate the mycobacterial genome. Our data rule out transduction and transformation as possible mechanisms of gene transfer in this system and are most consistent with conjugal transfer. We show that recombinants are not the result of cell fusion and that transfer occurs from a distinct donor to a recipient. One of the donor strains is mc²155, a highly transformable derivative that is considered the prototype laboratory strain for mycobacterial genetics; the demonstration that it is conjugative should increase its genetic manipulability dramatically. During conjugation, extensive regions of chromosomal DNA are transferred into the recipient and then integrated into the recipient chromosome by multiple recombination events. We propose that DNA transfer is occurring by a mechanism similar to Hfr conjugation in Escherichia coli .     

Translocation of ampicillin transposon Tn1 in Escherichia coli cells during sexual reproduction

The efficiency of Tn1 transposition has been shown to increase considerably in course of bacterial conjugation. Usually, the frequency of Tn1 transposition from plasmid pSA2001, a derivative of RP4, into the chromosome never exceeded 0.1% per cell. Percentage of His+ transconjugants, marked by transposon Tn1 during conjugation between Hfr donor, carrying plasmid pSA2001, and auxotrophic recipient, was about 30%. Transposon Tn1 transfer into the recipient cells does not depend on the recA+ gene function in donor cells or on conjugative transfer of plasmid pSA2001. The transfer requires the recA+ gene function in recipients as well as the Hfr function in donor cells. Southern's blot-hybridization revealed the insertion of transposon Tn1 into the different sites of the chromosome of His+ transconjugants. The transposon inserted during conjugation retains the ability to potential further translocation into new sites on the chromosomal DNA.     

Origin and fate of the 3' ends of single-stranded DNA generated by conjugal transfer of plasmid r1162

During conjugation, a single strand of DNA is cleaved at the origin of transfer (oriT) by the plasmid-encoded relaxase. This strand is then unwound from its complement and transferred in the 5'-to-3' direction, with the 3' end likely extended by rolling-circle replication. The resulting, newly synthesized oriT must then be cleaved as well, prior to recircularization of the strand in the recipient. Evidence is presented here that the R1162 relaxase contains only a single nucleophile capable of cleaving at oriT, with another molecule therefore required to cleave at a second site. An assay functionally isolating this second cleavage shows that this reaction can take place in the donor cell. As a result, there is a flux of strands with free 3' ends into the recipient. These ends are susceptible to degradation by exonuclease I. The degree of susceptibility is affected by the presence of an uncleaved oriT within the strand. A model is presented where these internal oriTs bind and trap the relaxase molecule covalently bound to the 5' end of the incoming strand. Such a mechanism would result in the preferential degradation of transferred DNA that had not been properly cleaved in the donor.     

Identification of the origin of transfer (oriT) and DNA relaxase required for conjugation of the integrative and conjugative element ICEBs1 of Bacillus subtilis

Integrative and conjugative elements (ICEs), also known as conjugative transposons, are mobile genetic elements that can transfer from one bacterial cell to another by conjugation. ICEBs1 is integrated into the trnS-leu2 gene of Bacillus subtilis and is regulated by the SOS response and the RapI-PhrI cell-cell peptide signaling system. When B. subtilis senses DNA damage or high concentrations of potential mating partners that lack the element, ICEBs1 excises from the chromosome and can transfer to recipients. Bacterial conjugation usually requires a DNA relaxase that nicks an origin of transfer (oriT) on the conjugative element and initiates the 5'-to-3' transfer of one strand of the element into recipient cells. The ICEBs1 ydcR (nicK) gene product is homologous to the pT181 family of plasmid DNA relaxases. We found that transfer of ICEBs1 requires nicK and identified a cis-acting oriT that is also required for transfer. Expression of nicK leads to nicking of ICEBs1 between a GC-rich inverted repeat in oriT, and NicK was the only ICEBs1 gene product needed for nicking. NicK likely mediates conjugation of ICEBs1 by nicking at oriT and facilitating the translocation of a single strand of ICEBs1 DNA through a transmembrane conjugation pore.     

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.     

Blending genomes: distributive conjugal transfer in mycobacteria,a sexier form of HGT

This review discusses a novel form of horizontal gene transfer (HGT) found in mycobacteria called Distributive Conjugal Transfer (DCT). While satisfying the criteria for conjugation, DCT occurs by a mechanism so distinct from oriT‐mediated conjugation that it could be considered a fourth category of HGT. DCT involves the transfer of chromosomal DNA between mycobacteria and, most significantly, generates transconjugants with mosaic genomes of the parental strains. Multiple segments of donor chromosomal DNA can be co‐transferred regardless of their location or the genetic selection and, as a result, the transconjugant genome contains many donor‐derived segments; hence the name DCT. This distinguishing feature of DCT separates it from the other known mechanisms of HGT, which generally result in the introduction of a single, defined segment of DNA into the recipient chromosome (Fig. 1 ). Moreover, these mosaic progeny are generated from a single conjugal event, which provides enormous capacity for rapid adaptation and evolution, again distinguishing it from the three classical modes of HGT. Unsurprisingly, the unusual mosaic products of DCT are generated by a conjugal mechanism that is also unusual. Here, we will describe the unique features of DCT and contrast those to other mechanisms of HGT, both from a mechanistic and an evolutionary perspective. Our focus will be on transfer of chromosomal DNA, as opposed to plasmid mobilization, because DCT mediates transfer of chromosomal DNA and is a chromosomally encoded process.     

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.     

Formation of delta tra F' plasmids: specific recombination at oriT

Delta tra F' plasmids can be isolated from matings between Hfr donors and recA- recipients, with selection for transfer of proximal chromosomal genes. Previous experiments indicate that F DNA from the neighborhood of the transfer origin up to the proximal junction with the chromosomal DNA is present on these plasmids, together with chromosomal segments, some of which belong to distinct size classes. We have sequenced across the novel joints contained in five delta tra FproA+ plasmids and in five delta tra FpurE+ plasmids, and we have compared these with the F sequence near oriT and with a chromosomal site near purE. The previously reported specificity in formation of some of these classes is confirmed at the nucleotide sequence level. The F DNA in nine of these novel joints extended beyond the nicking sites identified by others in lambda oriT+ bacteriophages up to a position between two sequenced oriT- mutations. Small plasmids containing these novel joints are mobilized in trans by pOX38 at frequencies less than 5 X 10(-7) times the mobilization frequencies for similar plasmids that contain oriT. The relations of these findings to the location of the nicking site at oriT are discussed.     

High-frequency transfer of a naturally occurring chromosomal tetracycline resistance element in the ruminal anaerobe Butyrivibrio fibrisolvens.

Butyrivibrio fibrisolvens strains resistant to tetracycline were isolated from the bovine rumen. Two of three Tcr B. fibrisolvens tested were able to donate tetracycline resistance at frequencies ranging from 10(-7) to 10(-1) per donor cell in anaerobic filter matings to a rifampin-resistant mutant of the type strain of B.fibrisolvens, 2221R. The recipient strain 2221R exhibited rapid autoaggregation, which might be a factor in the high transfer rates observed. Tcr transconjugants of B. fibrisolvens 2221R were also capable of further transferring tetracycline resistance to a fusidic acid-resistant mutant, 2221F. Comparison of genomic DNAs by pulsed-field gel electrophoresis demonstrated altered band profiles in transconjugants, consistent with the acquisition of a large mobile chromosomal element. The transferable elements from the two B. fibrisolvens donors 1.23 and 1.230 (TnB123 and TnB1230, respectively) showed the same preferred insertion site in the B. fibrisolvens 2221R chromosome and are likely to be similar, or identical, elements. Hybridization experiments showed no close relationship between TnB1230 and int-xis regions from Tn916 or Tn5253. Although DNA from the B. fibrisolvens donor strains hybridized with probes carrying tet(M) or tet(O) sequences, transconjugants were found to have acquired a distinct band that hybridized only weakly with these probes, suggesting that a second, distantly related Tcr determinant had been transferred.     

Role of sog polypeptides specified by plasmid ColIb-P9 and their transfer between conjugating bacteria.

The sog gene of the conjugative plasmid ColIb-P9 specifies two sequence-related polypeptides with the N-terminal third of the larger product having DNA primase activity. To resolve the function of the C-terminal portion of the polypeptides, we constructed a ColIb mutant containing a Tn5 insertion in the 3' region of sog. The mutation truncated sog gene products without inactivating DNA primase and rendered the plasmid defective in conjugation. Tests for the presence of conjugative pili, for complementation by a sog+ recombinant, and for mobilization of small origin of transfer (oriT) recombinant plasmids indicated that the mutant ColIb allows conjugative aggregation of cells but it is defective in DNA transfer at some stage subsequent to its initiation at oriT. Physical evidence is given that normal sog polypeptides are among a group of proteins transferred selectively from the donor to the recipient cell by a conjugation-specific process. No transfer of the mutant sog proteins was detected. It is proposed that the C-terminal region of sog polypeptides facilitates transfer of single-stranded ColIb DNA between conjugating cells following initiation of transfer at the oriT site, and that in this role the proteins are transmitted to the recipient cell.     

Conjugal transfer in Lactococcus lactis of a 68-kilobase-pair chromosomal fragment containing the structural gene for the peptide bacteriocin nisin.

Nisin-producing transconjugants were generated by mating nisin-producing strains of Lactococcus lactis subsp. lactis with derivatives of L. lactis subsp. lactis LM0230. The sucrose-utilizing ability and reduced bacteriophage sensitivity were also transferred with the nisin-producing character. Pulsed-field gel electrophoretic analysis of genomic DNA from donor, recipient, and nisin-producing transconjugants indicated that 68 kbp of DNA was transferred from the chromosome of the donor into the chromosome of the recipient in the conjugation process. The location of the transferred nisin structural gene spaN in the transconjugant HID500 was not stable, and cultures of strain HID500 were a mixture of different genotypes in which spaN was located at different positions in the chromosome on different SmaI fragments. ApaI, BglI, BssHII, NciI, SalI, and SmaI digests of genomic DNA were used to map the location of spaN in a donor (DL11) and a nisin-producing transconjugant (HID504).     

The specialized secretory apparatus ESX-1 is essential for DNA transfer in Mycobacterium smegmatis

Conjugal DNA transfer in Mycobacterium smegmatis occurs by a mechanism distinct from plasmid-mediated DNA transfer. Previously, we had shown that the secretory apparatus, ESX-1, negatively regulated DNA transfer from the donor strain; ESX-1 donor mutants are hyper-conjugative. Here, we describe a genome-wide transposon mutagenesis screen to isolate recipient mutants. Surprisingly, we find that a majority of insertions map within the esx-1 locus, which encodes the secretory apparatus. Thus, in contrast to its role in donor function, ESX-1 is essential for recipient function; recipient ESX-1 mutants are hypo-conjugative. In addition to esx-1 genes, our screen identifies novel non- esx-1 loci in the M. smegmatis genome that are required for both DNA transfer and ESX-1 activity. DNA transfer therefore provides a simple molecular genetic assay to characterize ESX-1, which, in Mycobacterium tuberculosis , is necessary for full virulence. These findings reinforce the functional intertwining of DNA transfer and ESX-1 secretion, first described in the M. smegmatis donor. Moreover, our observation that ESX-1 has such diametrically opposed effects on transfer in the donor and recipient, forces us to consider how proteins secreted by the ESX-1 apparatus can function so as to modulate two seemingly disparate processes, M. smegmatis DNA transfer and M. tuberculosis virulence.     

Conjugal transfer in Lactococcus lactis of a 68-kilobase-pair chromosomal fragment containing the structural gene for the peptide bacteriocin nisin.

Nisin-producing transconjugants were generated by mating nisin-producing strains of Lactococcus lactis subsp. lactis with derivatives of L. lactis subsp. lactis LM0230. The sucrose-utilizing ability and reduced bacteriophage sensitivity were also transferred with the nisin-producing character. Pulsed-field gel electrophoretic analysis of genomic DNA from donor, recipient, and nisin-producing transconjugants indicated that 68 kbp of DNA was transferred from the chromosome of the donor into the chromosome of the recipient in the conjugation process. The location of the transferred nisin structural gene spaN in the transconjugant HID500 was not stable, and cultures of strain HID500 were a mixture of different genotypes in which spaN was located at different positions in the chromosome on different SmaI fragments. ApaI, BglI, BssHII, NciI, SalI, and SmaI digests of genomic DNA were used to map the location of spaN in a donor (DL11) and a nisin-producing transconjugant (HID504).     

DNA recognition by F factor TraI36: highly sequence-specific binding of single-stranded DNA

The TraI protein has two essential roles in transfer of conjugative plasmid F Factor. As part of a complex of DNA-binding proteins, TraI introduces a site- and strand-specific nick at the plasmid origin of transfer (oriT), cutting the DNA strand that is transferred to the recipient cell. TraI also acts as a helicase, presumably unwinding the plasmid strands prior to transfer. As an essential feature of its nicking activity, TraI is capable of binding and cleaving single-stranded DNA oligonucleotides containing an oriT sequence. The specificity of TraI DNA recognition was examined by measuring the binding of oriT oligonucleotide variants to TraI36, a 36-kD amino-terminal domain of TraI that retains the sequence-specific nucleolytic activity. TraI36 recognition is highly sequence-specific for an 11-base region of oriT, with single base changes reducing affinity by as much as 8000-fold. The binding data correlate with plasmid mobilization efficiencies: plasmids containing sequences bound with lower affinities by TraI36 are transferred between cells at reduced frequencies. In addition to the requirement for high affinity binding to oriT, efficient in vitro nicking and in vivo plasmid mobilization requires a pyrimidine immediately 5' of the nick site. The high sequence specificity of TraI single-stranded DNA recognition suggests that despite its recognition of single-stranded DNA, TraI is capable of playing a major regulatory role in initiation and/or termination of plasmid transfer.     

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.     

Integration of donor DNA in bacterial conjugation

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

Chromosomal gene capture mediated by the Pseudomonas putida TOL catabolic plasmid.

The Pseudomonas putida TOL plasmid pWW0 is able to mediate chromosomal mobilization in the canonical unidirectional way, i.e., from donor to recipient cells, and bidirectionally, i.e., donor-->recipient-->donor (retrotransfer). Transconjugants are recipient cells that have received DNA from donor cells, whereas retrotransconjugants are donor bacteria that have received DNA from a recipient. The TOL plasmid pWW0 is able to directly mobilize and retromobilize a kanamycin resistance marker integrated into the chromosome of other P. putida strains, a process that appears to involve a single conjugational event. The rate of retrotransfer (as well as of direct transfer) of the chromosomal marker is influenced by the location of the kanamycin marker on the chromosome and ranges from 10(-3) to less than 10(-8) retrotransconjugants per donor (transconjugants per recipient). The mobilized DNA is incorporated into the chromosome of the retrotransconjugants (transconjugants) in a process that seems to occur through recombination of highly homologous flanking regions. No interspecific mobilization of the chromosomal marker in matings involving P. putida and the closely related Pseudomonas fluorescens, which belongs to rRNA group I, was observed.     

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

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