Conjugal Deoxyribonucleic Acid Replication by Escherichia coli K-12: Stimulation in dnaB(ts) Donors by Minicells

R64-11(+) donor cells that are thermosensitive for vegetative DNA replication will synthesize DNA at the restrictive temperature when recipient minicells are present. This is conjugal DNA replication because it is R64-11 DNA that is being synthesized and there is no DNA synthesis if minicells that cannot be recipients of R64-11 DNA are used. The plasmid DNA present in the donor cells before mating is transferred to recipient minicells within the first 20 min of mating, but additional copies of plasmid DNA synthesized during the mating continue to be transferred for at least 90 min. However, the transfer of R64-11 DNA to minicells is not continuous because the plasmid DNA in minicells is the size of one R64-11 molecule or smaller, and there are delays between the rounds of plasmid transfer. DNA is synthesized in minicells during conjugation, but this DNA has a molecular weight much smaller than that of R64-11. Thus, recipient minicells are defective and are not able to complete the synthesis of a DNA strand complementary to the single-stranded R64-11 DNA received from the donor cell.     

Comparison of Proteins Involved in Pilus Synthesis and Mating Pair Stabilization from the Related Plasmids F and R100-1: Insights into the Mechanism of Conjugation

F and R100-1 are closely related, derepressed, conjugative plasmids from the IncFI and IncFII incompatibility groups, respectively. Heteroduplex mapping and genetic analyses have revealed that the transfer regions are extremely similar between the two plasmids. Plasmid specificity can occur at the level of relaxosome formation, regulation, and surface exclusion between the two transfer systems. There are also differences in pilus serology, pilus-specific phage sensitivity, and requirements for OmpA and lipopolysaccharide components in the recipient cell. These phenotypic differences were exploited in this study to yield new information about the mechanism of pilus synthesis, mating pair stabilization, and surface and/or entry exclusion, which are collectively involved in mating pair formation (Mpf). The sequence of the remainder of the transfer region of R100-1 (trbA to traS) has been completed, and the complete sequence is compared to that of F. The differences between the two transfer regions include insertions and deletions, gene duplications, and mosaicism within genes, although the genes essential for Mpf are conserved in both plasmids. F+ cells carrying defined mutations in each of the Mpf genes were complemented with the homologous genes from R100-1. Our results indicate that the specificity in recipient cell recognition and entry exclusion are mediated by TraN and TraG, respectively, and not by the pilus.     

Recombinant F′ Factors from Escherichia coli K-12 Strains Carrying recB or recC

The frequency of genetic exchanges between F' factors and the bacterial chromosome was studied in recombination-deficient Escherichia coli mutants under conditions in which the recombinant F' factors were immediately transferred to new hosts. In a series of double matings, F101-1 thr(+)leu(-) episomes were first transferred into each of four intermediate F(-)thr(-)leu(+) strains carrying various rec alleles. After the original F' donors were killed with phage T6, the F101-1 episomes were then transferred from the intermediate cells to F(-)thr(-)leu(-)Str(R)recA(-) females. Recipients of nonrecombinant episomes formed Thr(+) (Str(R)) colonies, and recipients of recombinant episomes formed Leu(+)(Str(R)) colonies. A comparison of the numbers of Leu(+)(Str(R)) and Thr(+)(Str(R)) colonies shows that recB(-) males formed 18 to 21% and recC(-) formed 47 to 60% of the wild-type level of recombinant episomes that could be detected after transfer. No recombinant episomes were detected using a recA(-) intermediate strain. If the intermediate strains harboring the F101 episomes were purified, allowed to grow for 50 generations, and then mated with the recA(-) recipient, recombinant episomes were transferred at 8% of the wild-type level for recB(-) and 13% for recC(-). In contrast, only 0.4 and 0.6% of the normal number of recombinants were obtained from crosses between Hfr Cavalli donors and the same recB(-) and recC(-) strains. Recombinant episomes were detected with greater frequency among newly formed rec(+), recB(-), and recC(-) partial diploids than in those which were 50 generations old.     

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.     

Mapping Loci for Surface Exclusion and Incompatibility on the F Factor of Escherichia coli K-12

A series of Hfr deletion strains carrying deletions extending different distances into the integrated F factor have been used to map loci for surface exclusion (traS) and for incompatibility (inc) on the Escherichia coli K-12 sex factor F. traS mapped between traG and traD. It forms a part of the large operon, including all the known transfer genes except traJ, and is co-controlled with these. The product of traS is not required for formation of the F pilus. inc mapped between the phi(R) (11) locus and the origin of transfer; it is therefore one of the earliest loci transferred during conjugation.     

Superinfection with R Factors by Transduction in Escherichia coli and Salmonella typhimurium

Superinfection immunity is found in the conjugal transfer of R factors between two fi(+) R factors and between two fi(-) R factors (fi = fertility inhibition), as we reported previously. In contrast, no reduction in the frequencies of transduction of an fi(+) R factor 222 was caused by the presence of fi(+) R factors in the recipients in transduction systems with phage P1kc in Escherichia coli K-12 and with phage P22 in Salmonella typhimurium LT-2. The absence of superinfection immunity in transduction may be due to the difference in the route of entry of the R factor. The frequencies of transduction of an fi(+) R factor were reduced, although slightly, by the presence of fi(-) R factors in the recipients. This reduction is probably due to host-controlled restriction of the entering fi(+) R factor by the fi(-) R factors in the recipients, since transduction of an fi(+) R factor by the transducing phage propagated on the strain carrying both fi(+) and fi(-) R factors was not reduced by the presence of homologous fi(-) R factors in the recipients. The fi(+) R factor 222, when transduced to the recipient strains carrying other R factors, recombined genetically at high frequencies with these resident R factors, regardless of their fi type.     

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.     

Conjugal Deoxyribonucleic Acid Replication by Escherichia coli K-12: Effect of Chloramphenicol and Rifampin

Conjugal replication of R64-11 deoxyribonucleic acid (DNA) and the concomitant transfer of R64-11 DNA to DNA-deficient minicells are dependent upon processes that are inhibited by rifampin and chloramphenicol. The rifampin-sensitive product is not present in vegetatively growing cells and is needed to initiate both conjugal DNA replication in donor cells and DNA transfer to recipient minicells. If the rifampin-sensitive product is a ribonucleic acid (RNA) molecule (rather than RNA polymerase itself), our data indicate that this RNA species required for initiation of conjugal activity does not need to be translated into a protein product. The chloramphenicol-sensitive product(s) is present in vegetatively growing cells in sufficient quantity to permit most donor cells to carry out one round of plasmid conjugal replication and transfer. The initiation of second and subsequent rounds of conjugal replication and transfer are dependent on the synthesis of both the rifampin-sensitive and chloramphenicol-sensitive products. Our results demonstrate a correspondence between the amount of conjugal DNA replication in the donor and the amount of DNA transferred to recipient minicells under all conditions, and therefore suggest but do not prove that plasmid transfer is dependent on conjugal DNA replication. The results also add additional proof that R64-11 transfer to minicells is discontinuous. All of these results are discussed in regard to further refinements of old models for the mechanism of conjugal transfer as well as a more radical departure from current dogma.     

Effect of Bacillus subtilis BsuM restriction-modification on plasmid transfer by polyethylene glycol-induced protoplast fusion

Polyethylene glycol (PEG)-induced cell fusion is a promising method to transfer larger DNA from one cell to another than conventional genetic DNA transfer systems. The laboratory strain Bacillus subtilis 168 contains a restriction (R) and modification (M) system, BsuM, which recognizes the sequence 5'-CTCGAG-3'. To study whether the BsuM system affects DNA transfer by the PEG-induced cell fusion between R(+)M(+) and R(-)M(-) strains, we examined transfer of plasmids pHV33 and pLS32neo carrying no and eight BsuM sites, respectively. It was shown that although the transfer of pLS32neo but not pHV33 from the R(-)M(-) to R(+)M(+) cells was severely restricted, significant levels of transfer of both plasmids from the R(+)M(+) to R(-)M(-) cells were observed. The latter result shows that the chromosomal DNA in the R(-)M(-) cell used as the recipient partially survived restriction from the donor R(+)M(+) cell, indicating that the BsuM R(-)M(-) strain is useful as a host for accepting DNA from cells carrying a restriction system(s). Two such examples were manifested for plasmid transfer from Bacillus circulans and Bacillus stearothermophilus strains to a BsuM-deficient mutant, B. subtilis RM125.     

Synthesis of Ribonucleic Acid and Protein in Plasmid-Containing Minicells of Escherichia coli K-12

Unlike the deoxyribonucleic acid (DNA)-deficient minicells produced by F(-) parents, minicells produced by plasmid-containing strains contain significant amounts of plasmid DNA. We examined the ability of plasmid-containing minicells to synthesize ribonucleic acid (RNA) and protein. In vivo, minicells produced by F(-) parents are unable to incorporate radioactive precursors into acid-insoluble RNA or protein, whereas minicells produced by F', R(+), or Col(+) parents are capable of such synthesis. Using a variety of approaches, including polyacrylamide gel analysis of the RNA species produced and electron microscope autoradiography, we demonstrated that the synthesis observed in minicell preparations is a property of the plasmid-containing minicells and not a result of the few cells (approximately 1 per 10(6) minicells) contaminating the preparations. That the observed synthesis is of biological importance is suggested by the ability of plasmid-containing minicells to yield viable phage upon infection with T4.     

Transfer inhibition of RP4 by F factor

When RP4 and F factors were brought together into one E. coli cell, the F factor reduced 500-1000-fold the frequency of transfer of RP4. However, F had almost no effect on the surface exclusion and pilus formation by RP4. In contrast, RP4 did not affect the transfer of F. Using in vitro recombinant DNA techniques, a gene of F responsible for the above-mentioned transfer inhibition of RP4 was located within the BamHI fragment (40.4-42.8 kb) of the mini-F sequence on F. From the result of product analysis using minicells, the responsible gene in the BamHI fragment was inferred to encode the 33 K protein.     

Genetic Analysis of the Role of the Transfer Gene, traN, of the F and R100-1 Plasmids in Mating Pair Stabilization during Conjugation

Mating pair stabilization occurs during conjugative DNA transfer whereby the donor and recipient cells form a tight junction which requires pili as well as TraN and TraG in the donor cell. The role of the outer membrane protein, TraN, during conjugative transfer was examined by introduction of a chloramphenicol resistance cassette into the traN gene on an F plasmid derivative, pOX38, to produce pOX38N1::CAT. pOX38N1::CAT was greatly reduced in its ability to transfer DNA, indicating that TraN plays a greater role in conjugation than previously thought. F and R100-1 traN were capable of complementing pOX38N1::CAT transfer equally well when wild-type recipients were used. F traN, but not R100-1 traN, supported a much lower level of transfer when there was an ompA mutation or lipopolysaccharide (LPS) deficiency in the recipient cell, suggesting receptor specificity. The R100-1 traN gene was sequenced, and the gene product was found to exhibit 82.3% overall similarity with F TraN. The differences were mainly located within a central region of the proteins (amino acids 162 to 333 of F and 162 to 348 of R100-1). Deletion analysis of F traN suggested that this central portion might be responsible for the receptor specificity displayed by TraN. TraN was not responsible for TraT-dependent surface exclusion. Thus, TraN, and not the F pilus, appears to interact with OmpA and LPS moieties during conjugation, resulting in mating pair stabilization, the first step in efficient mobilization of DNA.     

Conjugal Deoxyribonucleic Acid Replication by Escherichia coli K-12: Effect of Nalidixic Acid

During the conjugal transfer of the R64-11 plasmid at 42 C from donor cells thermosensitive for vegetative deoxyribonucleic acid (DNA) synthesis to recipient minicells, the plasmids are conjugally replicated in the donor cells. This conjugal replication is inhibited by nalidixic acid, and the degree of inhibition is comparable to the reduction in the amount of plasmid DNA transferred to the recipient minicells in the presence of the drug. In addition, the size of DNA transferred to the minicells and the fraction of conjugally replicated DNA in the donor cells that can be isolated as closed-circular plasmid DNA under alkaline conditions are both reduced by nalidixic acid. When the drug is added to a mating that is underway, the rate of conjugal replication is immediately reduced. This change is accompanied by a reduction in the amount of conjugally replicated DNA in the donor cells that can be isolated as closed-circular plasmid DNA. Furthermore, conjugally replicated plasmid DNA that is not associated with the donor cell membrane becomes membrane bound after the addition of nalidixic acid.     

Role of Agrobacterium VirB11 ATPase in T-pilus assembly and substrate selection

The VirB11 ATPase is a subunit of the Agrobacterium tumefaciens transfer DNA (T-DNA) transfer system, a type IV secretion pathway required for delivery of T-DNA and effector proteins to plant cells during infection. In this study, we examined the effects of virB11 mutations on VirB protein accumulation, T-pilus production, and substrate translocation. Strains synthesizing VirB11 derivatives with mutations in the nucleoside triphosphate binding site (Walker A motif) accumulated wild-type levels of VirB proteins but failed to produce the T-pilus or export substrates at detectable levels, establishing the importance of nucleoside triphosphate binding or hydrolysis for T-pilus biogenesis. Similar findings were obtained for VirB4, a second ATPase of this transfer system. Analyses of strains expressing virB11 dominant alleles in general showed that T-pilus production is correlated with substrate translocation. Notably, strains expressing dominant alleles previously designated class II (dominant and nonfunctional) neither transferred T-DNA nor elaborated detectable levels of the T-pilus. By contrast, strains expressing most dominant alleles designated class III (dominant and functional) efficiently translocated T-DNA and synthesized abundant levels of T pilus. We did, however, identify four types of virB11 mutations or strain genotypes that selectively disrupted substrate translocation or T-pilus production: (i) virB11/virB11* merodiploid strains expressing all class II and III dominant alleles were strongly suppressed for T-DNA translocation but efficiently mobilized an IncQ plasmid to agrobacterial recipients and also elaborated abundant levels of T pilus; (ii) strains synthesizing two class III mutant proteins, VirB11, V258G and VirB11.I265T, efficiently transferred both DNA substrates but produced low and undetectable levels of T pilus, respectively; (iii) a strain synthesizing the class II mutant protein VirB11.I103T/M301L efficiently exported VirE2 but produced undetectable levels of T pilus; (iv) strains synthesizing three VirB11 derivatives with a four-residue (HMVD) insertion (L75.i4, C168.i4, and L302.i4) neither transferred T-DNA nor produced detectable levels of T pilus but efficiently transferred VirE2 to plants and the IncQ plasmid to agrobacterial recipient cells. Together, our findings support a model in which the VirB11 ATPase contributes at two levels to type IV secretion, T-pilus morphogenesis, and substrate selection. Furthermore, the contributions of VirB11 to machine assembly and substrate transfer can be uncoupled by mutagenesis.     

R-plasmid-mediated chromosome mobilization in Bordetella pertussis

Antibiotic-resistant and auxotrophic mutants of Bordetella pertussis were isolated. These were used as recipients for the uptake from Escherichia coli of broad-host-range R plasmids R68.45, RP1, and RP1 and RP4 carrying transposons Tn501 and Tn7 respectively. B. pertussis transconjugants from these crosses were used as donors to mobilize StrR, NalR, thr+ and gly+ chromosomal markers to B. pertussis or to B. parapertussis recipient strains. The frequency of plasmid transfer varied and depended on the donor and recipient strains used. Differences in chromosome mobilization frequencies of individual markers were observed and appeared to depend on the presence or absence of transposons Tn501 and Tn7 on the plasmid. Linkage was detected between the gly+ and NalR markers.     

Transfer of plasmids pBR322 and pBR325 in wastewater from laboratory strains of Escherichia coli to bacteria indigenous to the waste disposal system

Laboratory strains of Escherichia coli containing plasmid pBR325 (or pBR322) were coincubated with a mobilizer strain of E. coli (containing the conjugative plasmid R100-1) and a recipient strain of bacteria. Bacterial strains isolated from raw wastewater or a plasmid-free E. coli laboratory strain served as recipients. Transfer of the pBR plasmid into the recipient strain occurred during a 25-h coincubation in either L broth or sterilized wastewater; transfer frequencies were several orders of magnitude lower in wastewater. After the coincubation, recipients exhibited both plasmid-encoded phenotypic characteristics and an altered plasmid profile, as shown by agarose gel electrophoresis of purified plasmid DNA.     

Mating variation by DNA inversions of shufflon in plasmid R64

Gene organization of the 54-kb transfer region of IncI1 plasmid R64 was deduced from the DNA sequence. Forty-eight ORFs were found in this region. A unique DNA rearrangement designated shufflon is located at the downstream region of an operon responsible for synthesis of thin pilus. The shufflon of R64 consists of four DNA segments, designated as A, B, C, and D, which are flanked and separated by seven 19-bp repeat sequences. Site-specific recombination mediated by the product of the rci gene between any two inverted repeats results in a complex DNA rearrangement. An analysis of open reading frames revealed that the shufflon is a biological switch to select one of seven C-terminal segments of the pilV genes. The products of pilV genes were shown to be components of thin pilus which was required for liquid mating.Seven R64 derivatives where the pilV genes were fixed in the seven C-terminal segments were constructed and their transfer frequencies in liquid mating were measured using various bacterial strains as recipients. Transfer frequencies of R64 in liquid mating strongly depended on the combination of C-terminal segments of the pilV genes in donor cells and bacterial strains of recipient cells, suggesting that the shufflon determines the recipient specificity in liquid mating of plasmid R64.     

Transfer of plasmids pBR322 and pBR325 in wastewater from laboratory strains of Escherichia coli to bacteria indigenous to the waste disposal system.

Laboratory strains of Escherichia coli containing plasmid pBR325 (or pBR322) were coincubated with a mobilizer strain of E. coli (containing the conjugative plasmid R100-1) and a recipient strain of bacteria. Bacterial strains isolated from raw wastewater or a plasmid-free E. coli laboratory strain served as recipients. Transfer of the pBR plasmid into the recipient strain occurred during a 25-h coincubation in either L broth or sterilized wastewater; transfer frequencies were several orders of magnitude lower in wastewater. After the coincubation, recipients exhibited both plasmid-encoded phenotypic characteristics and an altered plasmid profile, as shown by agarose gel electrophoresis of purified plasmid DNA.     

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.