On the mechanism of conjugation in Escherichia coli K 12

Summary The phenomenon of conjugation consists of many stages. The most important are: the formation of contacts between mating cells, the transfer of DNA from the donor to the recipient, and the integration of the transfered DNA fragments into the chromosome of the recipient. Only after completion of all these stages are recombinants formed. With the aid of specific inhibitiors (nalidixic acid, FUDR), thymine starvation, and use of special thermosensitive mutants it is possible to study the role of DNA synthesis during every stage of conjugation. It was demonstrated that the genetic transfer is due to semiconservative DNA-replication in the donor cell. The fragments of DNA transfered are synthesized in the period of mating by a special replication system (F-replicon). In case of T _DNA ^S mutants unable to grow at 41°, the ability to synthesize DNA during conjugation is preserved.The inhibition of the DNA synthesis in the donor cell by poisons leads to complete inhibition of genetic transfer. The third stage — formation of recombinants requires DNA synthesis in the recipient cell and is inhibited by poisoning, thymine starvation or T _DNA ^S mutations in the recipient. In cases where recombination is not involved (i.e. sexduction) the inhibition of DNA synthesis in the recipient has no significant effect.     

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.     

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.     

Identification of individual sex-factor DNA strands and their replication during conjugation in thermosensitive DNA mutants of Escherichia coli

Covalent circular sex-factor DNA has been isolated from donor and recipient cells during the conjugation of normal and temperature-sensitive DNA mutants of Escherichia coli. Single strands of sex-factor DNA were centrifuged in cesium chloride-poly(U,G) gradients to give two components that have been identified by annealing experiments as the separated complementary strands. When matings are performed with either DNA temperature-sensitive donor or recipient cells, the inhibition of vegetative DNA synthesis at the restrictive temperature does not interfere with transfer and circularization of the sex-factor DNA. If DNA is isolated from temperature-sensitive donor cells mated at the restrictive temperature, a specific stimulation of sex-factor DNA synthesis can be demonstrated. By separating the complementary strands of the sex-factor in a cesium chloride-poly (U,G) gradient, this DNA synthesis has been found to be asymmetric. The sex-factor DNA strand which is synthesized in the donor has the same polarity as the strand which is transferred to the recipient.     

Effect of mismatched base pairs on the fate of donor DNA in transformation of Streptococcus pneumoniae

Summary Investigation of the mechanism that discriminates against mismatched base pairs in transformation of Streptococcus pneumoniae of genotype hex ⁺ was based on the use of a radioactively labeled cloned fragment of pneumococcal DNA as donor in transformation. The fate of the donor label was followed by lysis of the transformed cells and separation by agarose gel electrophoresis of DNA fragments generated by restriction endonucleases. As a result of Hex action, most of the donor DNA fragment, which was a few kilobases in length, was lost when a mismatched base pair occurred between donor and recipient DNA. This was not observed in hex ^- recipient cells. Kinetic studies of mismatch-induced donor DNA loss showed that the process is faster in strain 800, an R6 derivative, than in DP 1601, a strain of different origin. In the latter strain, the amount of donor label that becomes double stranded rises substantially, indicating extensive formation of donorrecipient heteroduplex structures, before falling to the expected level. At 30°C the process is essentially completed 15 min after entry.     

Physiological and genetical studies on a mutant of Salmonella typhimurium which is temperature-sensitive for DNA synthesis

Summary The following evidence supports the view that a temperature-sensitive mutant of Salmonella typhimurium (11 G) is defective in DNA synthesis initiation: a) the increment in DNA synthesis at 38° is abolished by prior completion of rounds of replication at 25°. b) The extent of the increment at 38° is greatly increased by prior growth in the presence of a DNA synthesis inhibitor. c) Density gradient centrifugation demonstrates that the terminal region of the chromosomes is preferentially replicated at 38°. d) Preferential replication of the chromosome origins occurs at 25° after a period at 38°. The parental strain in the presence of a DNA synthesis inhibitor or the mutant at 38° (without inhibitor) show increased sensitivity to the detergent sodium deoxycholate, possibly due to a secondary effect of DNA synthesis inhibition on membrane composition. Strains of 11 G carrying episomes transfer the episomes very poorly at 38° suggesting a rôle for the chromosomal initiation apparatus in episome transfer. Continued cell division of 11 G with the production of DNA-less cells at 38° is not due to the presence of a rec mutation and no secondary mutation responsible for such division has been found. The lesion maps close to leu on the Salmonella chromosome.     

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.     

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 .     

Replication of the deoxyribonucleic acid of multiple-drug-resistance factor in Escherichia coli.

1. It was shown that a system previously described for labelling R-factor DNA during transfer to an irradiated recipient strain of Escherichia coli did not allow high selectivity in the incorporation of thymine into R-factor DNA. 2. Lack of selectivity was shown to be due to cross-feeding from recipient to donor strain. 3. An improved system using a nalidixic acid-resistant recipient strain is described in which incorporation of thymine into the DNA of donor cells is minimized by addition of nalidixic acid after completion of transfer of the plasmid during conjugation.     

Temperature sensitive initiation of DNA synthesis in a mutant of Salmonella typhimurium

Summary A mutant (dna-1) of Salmonella typhimurium defective in DNA synthesis is described. DNA synthesis is stopped in this mutant at 42° after a residual synthesis amounting to about 50 to 60% of the total cellular DNA in minimal medium and about 120 to 200% in a medium enriched with amino acids. Reshift back to permissive temperature after the inhibition of DNA synthesis at 42° allows for recovery of DNA synthesis after a lag of about 30 min. Protein synthesis is required during that lag for the recovery of DNA synthesis at permissive temperature. The density transfer experiments indicate that in the mutant dna-1 chromosome termini are replicated normally at 42° while the initiation of new rounds of replication is inhibited although the mutation is probably leaky at this temperature. The mutant is hypersensitive to sodium deoxycholate at 42° which suggests alteration of the membrane structure.     

Constitutive expression of SOS functions and modulation of mutagenesis resulting from resolution of genetic instability at or near the recA locus of Escherichia coli

Summary Cellular activities normally inducible by DNA damage (SOS functions) are expressed, without DNA damage, in recA441 (formerly tif-1) mutants of Escherichia coli at 42° C but not at 30° C. We describe a strain (SC30) that expresses SOS functions (including mutator activity, prophage induction and copious synthesis of recA protein) constitutively at both temperatures. SC30 is one of four stable subclones (SC strains) derived from an unstable recombinant obtained in a conjugation between a recA441 K12 donor and a recA ⁺ B/r-derived recipient. SC30 does not owe its SOS-constitutive phenotype to a mutation in the lexA gene (which codes the repressor of recA and other DNA damage-inducible genes), since it is lexA ⁺. Each of the SC strains expresses SOS functions in a distinctively anomalous way. We show that the genetic basis for the differences in SOS expression among the SC strains is located at or very near the recA locus. We propose that resolution of genetic instability in this region, in the original recombinant, has altered the pattern of expression of SOS functions in the SC strains.     

Temperature-sensitive initiation of DNA replication in a mutant of Escherichia coli K12

Summary A mutant of E. coli K12 appears to be temperature-sensitive in the process of initiation of DNA replication. After a temperature shift from 33 to 42°C, the amount of residual DNA synthesis (Fig. 1) and the number of residual cell divisions (Figs. 2,4) indicate that rounds of DNA replication in process are completed, but new rounds cannot be initiated. Following the alignment of chromosomal DNA by amino acid starvation at 33° C no residual DNA synthesis at 42°C takes place (Fig. 5). When the temperature is lowered to 33°C after a period of inhibition at 42°C, the following observations are made: 1. DNA replication resumes and proceeds synchroneously, (Figs. 7, 8a), 2. cells start to divide again only after a lag period of about 1 hour 3. a temporary increase in cell volume is correlated with the frequency of initiation of DNA synthesis (Fig. 8a, b). In a lysogenic mutant strain prophage is inducible; with all bacteriophages tested, replication of phage DNA is not inhibited at 42°C.     

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.     

A Fast and Practical Yeast Transformation Method Mediated by Escherichia coli Based on a Trans-Kingdom Conjugal Transfer System: Just Mix Two Cultures and Wait One Hour

Trans-kingdom conjugation is a phenomenon by which DNA is transferred into a eukaryotic cell by a bacterial conjugal transfer system. Improvement in this method to facilitate the rapid co-cultivation of donor bacterial and recipient eukaryotic cell cultures could make it the simplest transformation method, requiring neither isolation of vector DNA nor preparation of competent recipient cells. To evaluate this potential advantage of trans-kingdom conjugation, we examined this simple transformation method using vector combinations, helper plasmids, and recipient Saccharomyces cerevisiae strains. Mixing donor Escherichia coli and recipient S. cerevisiae overnight cultures (50 μL each) consistently yielded on the order of 10¹ transformants using the popular experimental strain BY4742 derived from S288c and a shuttle vector for trans-kingdom conjugation. Transformation efficiency increased to the order of 10² using a high receptivity trans-kingdom conjugation strain. In addition, either increasing the amount of donor cells or pretreating the recipient cells with thiols such as dithiothreitol improved the transformation efficiency by one order of magnitude. This simple trans-kingdom conjugation-mediated transformation method could be used as a practical yeast transformation method upon enrichment of available vectors and donor E. coli strains.     

Molecular fate of heterologous bacterial DNA in competent Bacillus subtilis: further characterization of unstable association between donor and recipient DNA and the involvement of the cellular membrane

Summary Although heterospecific transformation is extremely inefficient and very little heterologous donor DNA integrates into the recipient chromosome in a stable way, we have previously shown that B. pumilus DNA entering competent B. subtilis efficiently associates with the recipient chromosome in an unstable way. This association can be stabilized by photocrosslinking in the presence of 4,5,8-trimethylpsoralen; it depends on the recombination proficiency of the recipient strain and on strand-separation of the recipient chromosome (te Riele and Venema 1982b). The present study provides further evidence that the heterologous donor DNA and the recipient DNA are associated by regions of base-pairing. Based on the high sensitivity of the donor moiety in the complex to nuclease S1 (90%) and the high sensitivity of the complex to moderate denaturing conditions (Tm=48°C), we presume that donor and recipient DNA are associated either by several short sequences of 15–25 fairly well matched base pairs or by a region of base-pairing of about 200 bases, which contains 25% of mismatches. During incubation, the unstable complex disappears, probably due to nucleolytic degradation.The unstable heterologous donor-recipient complex (DRC) was found to be membrane-bound. However, in contrast to homologous DRC, the unstable heterologous DRC remains membrane bound during incubation. Apparently, the predominantly single-stranded character of the heterologous DRC prevents release of the complex from the membrane.Abbreviations DRC donor-recipient complex - TMP 4,5,8-trimethyl-psoralen - DNAase I deoxyribonuclease 1 - TCA trichloroacetic acid     

DNA synthesis in P1 infected E. coli mutants temperature-sensitive in DNA replication

Summary P1 DNA is synthesized in the E. coli ts dna mutants 165/70 (elongation defect) and 252 (initiation defect) at elevated temperatures. In strain 165/70, P1 infection at 41°C leads to phage production accompanied by a transient recovery of bacterial DNA synthesis. No phages are produced byt P1 DNA is still synthesized in strain 252 if infected after host DNA replication has come to a halt at 42°C.     

Mechanisms of strand replacement synthesis for plasmid DNA transferred by conjugation

A single strand of plasmid DNA is transferred during conjugation. We examined the mechanism of complementary strand synthesis in recipient cells following conjugative mobilization of derivatives of the IncQ plasmid R1162. A system for electroporation of donor cells, followed by immediate mating, was used to eliminate plasmid-specific replicative functions. Under these conditions, Escherichia coli recipients provided a robust mechanism for initiation of complementary strand synthesis on transferred DNA. In contrast, plasmid functions were important for efficient strand replacement in recipient cells of Salmonella enterica serovar Typhimurium. The mobilizing vector for R1162 transfer, the IncP1 plasmid R751, encodes a DNA primase with low specificity for initiation. This protein increased the frequency of transfer of R751 into Salmonella, but despite its low specificity, it was inactive on the R1162 derivatives. The R751 primase was slightly inhibitory for the transfer of both R751 and R1162 into E. coli. The results show that there is a chromosomally encoded mechanism for complementary strand synthesis of incoming transferred DNA in E. coli, while plasmid-specific mechanisms for this synthesis are important in Salmonella.     

Evaluation of a method to measure conjugal transfer of recombinant DNA in soil slurries

Release of recombinant microbes into the environment necessitates an evaluation of their ability to transfer genetic material. The present report evaluates a method to detect conjugal DNA plasmid transfer in soil slurries under various environmental conditions. DonorPseudomonas cepacia containing pR388::Tn1721 andP. cepacia recipient cultures were coincubated in soil slurries containing autoclaved or natural soil and treated with one or more of 14 experimental conditions. Conjugal mating frequency (transconjugants per initial donor) ranged from 4.8×10^–1 to 1.9×10^–7. Highest numbers of transconjugants, 1.5×10⁷ colony forming units/ml soil slurry, were observed following incubation at 35°C with an enriched nutrient supplement added to the soil. Low numbers of transconjugants, 10³ colony forming units/ml soil slurry, were observed when mating pairs were subjected to low nutrient or pH stress even though initial donor and recipient populations were maintained at high levels. This test system provides a simple way to estimate effects of changing environmental factors on plasmid transfer rates and on the survival of recombinant microorganisms. By use of soil collected from sites proposed to receive genetically engineered microorganisms, preliminary risk assessments can be obtained regarding the potential for gene transfer and microorganism survival with this soil slurry test system.     

Temperature-Sensitive Mutants for the Replication of Plasmids in ESCHERICHIA COLI II. Properties of Host and Plasmid Mutations

Host mutations in Escherichia coli K12 selected for the temperature-sensitive replication of the bacterial plasmid colicinogenic factor E(1) (ColE(1)) exhibit a pleiotropic effect with respect to the effect of the mutation on other extra-chromosomal elements. The mutations also vary with respect to the time of incubation of the cells at 43 degrees C required for complete cessation of ColE(1) DNA synthesis. While the synthesis of the bacterial chromosome appears unaffected, supercoiled ColE(1) DNA replication stops immediately in some mutants and gradually decreases during several generations of cell growth before stopping in others. Mutations isolated in the ColE(1) plasmid resulted in only a gradual cessation of ColE(1) DNA synthesis over several generations of cell growth at 43 degrees C. Conjugal transfer of the ColE(1) and ColV factors occurs normally in the host mutants when the transfer is carried out at the permissive temperature; however, the presence of a group I mutation in the donor cell prohibited conjugal transfer of either plasmid DNA at 43 degrees C to a normal recipient cell. Similarly, the presence of this mutation in the recipient prevented the establishment of ColE(1) or ColV in the mutant recipient cell upon conjugation with a normal donor at 43 degrees C. Various host ColE(1) replication mutants carrying either ColE(1) or ColE(2) were also defective in the mitomycin C-induced production of colicin E(1) or colicin E(2) at 43 degrees C. The majority of the host mutations examined exhibited a temperature sensitivity to growth in deoxycholate in addition to the inhibition of plasmid DNA replication, suggesting a membrane alteration in these mutants when grown at the restrictive temperature.