Genetic analysis of transfer and incompatibility functions within the IncHI plasmid R27

This study was undertaken to establish a transfer complementation system for IncH plasmids and to locate regions of incompatibility within the HI1 plasmid, R27. Two regions of R27 were found to contribute to incompatibility as determined by incompatibility testing with fragments of R27 cloned in cosmid vectors. One of these regions hybridized with the IncHI1 rep probe (Couturier et al., Microbiol. Rev. 52, 375-395, 1988). Complementation analysis was carried out using transfer-deficient mutants of R27 in combination with pHH1508a. Cosmid vectors, which contained cloned restriction fragments of R27, were able to complement selected R27 Tra- mutants, enabling the transfer-deficient plasmid to transfer at near-normal frequencies. Complementation of R27 Tra- plasmids by pHH1508a at both 26 and 37 degrees C was shown to occur, but was host-dependent in its degree. These results suggest that the transfer mechanisms of IncHI and IncHII plasmids are related.     

Cloning and characterization of a replicon region of the IncHII plasmid pHH1457

A replicative region of the large conjugative plasmid pHH1457 (incompatibility group HII (IncHII)) was cloned. A 1.4-kbp region, in a stable pSBII14 clone, containing a PolI-independent replicon and determinants for the HII incompatibility phenotype, was selected and characterized. High incompatibility with IncHII plasmids was corroborated. Independent replication of the insert was demonstrated by ligation to an antibiotic resistance cassette. pSBII14 was used as a probe to identify IncHII plasmids from other members of the H complex: IncHI (IncHI1, IncHI2 and IncHI3 subgroups). Hybridization experiments revealed a high homology with the replication region of IncHII plasmids, but not with IncHI1 or IncHI3 plasmid prototypes. Homology with IncHI2 plasmids was observed, suggesting the presence of IncHII-like replicons among this subgroup of plasmids. This is the first report of the characterization of an IncHII plasmid maintenance region.     

F plasmid incompatibility and copy number genes: Their map locations and interactions

The genes coding for vegetative F plasmid replication, replication control, and incompatibility are known to map between the kilobase coordinates 40.3 and 49.3 (abbreviated 40.3–49.3F). We have subdivided this region of the F genome by a combination of in vivo and in vitro genetic techniques and have constructed F:pSC101 hybrid plasmids which contain the F DNA sequences having the approximate coordinates 41–43, 43–46, and 46–49F. We find that hybrids with regions 43–46 and 46–49F are incompatible with an F′lac⁺ plasmid while the hybrid with the region 41–43F is compatible. We have also constructed similar F:pSC101 hybrid plasmids with the regions 43–46 and 46–49F derived from mini-F plasmid copy number mutants. We find that hybrids made from three independent F copy number mutants show a loss of the incompatibility function associated with the 43–46F region and retention of the incompatibility function associated with 46–49F region. Moreover, spontaneous revertants, selected for regain of the 43–46F incompatibility function, have also regained normal control over their copy numbers. We also find that copy number mutations map in the 43–46F region. From our results we conclude (i) that F contains at least two inc⁺ loci, designated incA⁺ (46–49F) and incB⁺ (43–46F), and (ii) that gene(s) regulating F copy number may be related to the incB⁺ gene(s).     

Construction and BamHL analysis of chimeric plasmids containing EcoRL DNA fragments of the F sex factor.

The construction of seven chimeric plasmids (pRS series) carrying EcoRI endonuclease-generated segments of the F sex factor cloned onto the vector pSC101 is described. BamHI endonuclease analysis of these seven plasmids, the six previously described pRS plasmids (Skurray, R. A., Nagaishi, H., and Clark, A. J. (1976) Proc. Nat. Acad. Sci. USA73, 64–68) and F plasmid DNA has enabled a partial BamHI map of F to be constructed; the orientation of insertion of F DNA segments into the pSC101 vector was also established for nine of the pRS plasmids. Results indicate that in the absence of their normal promoter, F cistrons cloned into the EcoRI site of pSC101 are expressed regardless of orientation of insertion although there is a preferred orientation for high levels of expression.     

Significance of filter mating in integrative incompatibility test for plasmid classification

For classification of plasmids in epidemiological studies, an integrative incompatibility test using liquid mating was developed by Sasakawa et al (Plasmid 3: 116-127, 1980). This test was designed to compare the relative mating frequency of a donor carrying a test plasmid with that of recA recipients carrying various integrated plasmids. To improve the accuracy of this method by increasing transfer frequency of a test plasmid, filter mating was introduced. A transfer frequency 10 to 30,000 times higher than that achieved by liquid mating was attained by filter mating. The degree of increase varied among the incompatibility groups and the majority of members belonging to the same incompatibility group exhibited a similar degree of increase. Standard plasmids were classified correctly with the integrative incompatibility test using filter mating. Out of 26 naturally occurring plasmids of poor transferability in liquid mating, all of domestic animal origin, 25 were correctly classified as IncH with the integrative incompatibility test using filter mating. Moreover, the method is capable of subdividing IncH plasmids directly into IncH1 and IncH2 , because IncH2 , but not IncH1 , plasmids showed incompatibility with the integrated plasmid, R478 , of the IncH2 group.     

Study of the incompatibility and replication of the 70-kb virulence plasmid of Yersinia

The 70-kb virulence plasmid, vir, from four Yersinia enterocolitica and one Y. pseudotuberculosis strains are incompatible with IncFI plasmids FLac and R386 while they are compatible with plasmids representing nine other incompatibility groups. Hybridization experiments carried out on one of these virulence plasmids showed that it contains the F incompatibility determinant D, incD. This determinant was cloned onto pACYC184 and the recombinant clone expressed incompatibility with FLac. We conclude that the incompatibility observed between F or R386 and the 70-kb virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis is mediated by incD. Replication genes (rep) from the same plasmid were cloned independently in Escherichia coli. Rep and incD map on two different BamHI fragments. Surprisingly, the replicon isolated is not sensitive to inc D incompatibility. Apart from incD, vir and F share extremely little homology. In particular, there is no evidence for the presence of an F-like transfer operon on vir.     

Genetic studies of F plasmid maintenance genes

We have used the mutagenic potential of the ampicillin resistance transposon Tn3 and in vitro deletion techniques to study essential regions for maintenance of mini-F plasmids. Our parental mini-F plasmid contains the 40.3 to 40.8F and 43.1 to 49.3F sequences, a total of 6.7 kilobases (kb). From a spectrum of insertion and deletion mutants, we find only two insertion regions; they map near the 45.8- and 46.4-kb coordinates. In each region the orientation of Tn3 insertion is unique and different from that of the other region. Spontaneous deletions extend from either region in a common direction which is toward the 49.3-kb coordinate. One deletion plasmid, pBK138-2, which arose from a combination of in vitro and spontaneous deletion events, contains just the 44- to 45.8-kb sequences and the ampicillin resistance gene of Tn3. As shown by J. Wechsler and B. C. Kline (1980, Plasmid 4, 276–280), the 45.8- to 46.3-kb sequences specify F sensitivity to the plasmid curing agent, acridine orange. Since sensitivity to acridine orange is a property of normal F maintenance, the 45.8- to 46.35-kb sequences also likely are required for normal plasmid maintenance.     

Molecular homology and incompatibility relationships between F and IncH1 plasmids

IncH1 plasmids and the F plasmid of Escherichia coli display one-way compatibility. An entering IncH1 plasmid is incompatible with a resident F plasmid, but is compatible when it is the resident plasmid. There is little molecular homology between IncH1 plasmids and the F plasmid. A single 5 MDal EcoRI restriction enzyme fragment from digests of several IncH1 plasmids hybridizes with probes constructed from the primary replication region of F. Homology can be demonstrated only with the gene for the essential replication protein of F (gene E), but the expression of incompatibility behaviour appears to be associated with the presence of the secondary replicon of the F plasmid. Thus R27 and F are compatible under growth conditions allowing replication and maintenance of F by the secondary replicon. However, a mutant F plasmid which lacks the secondary replicon of F is incompatible with R27 in both directions, irrespective of the growth conditions used.     

IncD, a genetic locus in F responsible for incompatibility with several plasmids of the IncFI group

Summary Cloning of mini-F DNA segments has led to the identification and mapping of a locus, incD, involved in incompatibility reactions with many IncFI plasmids. The cloned incD locus expressed incompatibility with F, R386, and six other IncFI plasmids but not with ColV3-K30 or pHH507 which lack sequence homology with the incD region. A sequence of 360 bp (48.66–49.02 FKB) was found to be sufficient for expression of incD incompatibility. Multicopy vectors containing incD are compatible with each other, but can be displaced by mini-F plasmids deleted for incD. These results indicate that incD-mediated incompatibility reactions require the presence of replication genes to which incD is normally linked. The degree of incompatibility exercised by incD is moderate compared with that of other inc loci in F, suggesting that incD is involved in an aspect of plasmid maintennce, such as partition, different from the functions of the other inc loci.     

Construction and characterization of multicopy plasmids containing the entire F transfer region

The restriction endonucleases HindIII and/or BamI have been used to clone the entire F transfer region into pBR322 to create a series of transfer-proficient multicopy plasmids. Despite the insertion of 40.7- to 55.9-kilobase F fragments, the plasmid copy numbers remained high, at 25–40 copies per cell. One of the chimeric plasmids contained the F replication and incompatibility region, and its high copy number confirmed that replication of the cointegrate was governed by pBR322. Despite the 30- to 40-fold increase in tra gene copy number compared to Flac, the transfer frequencies, number of pili per cell, and syntheses of the individual traT and traI proteins were increased only by about 5-fold. The level of tra mRNA in cells carrying the multicopy transfer-proficient plasmids was also increased only by about 5-fold, suggesting that its relative synthesis or stability was reduced in this situation. Nonetheless, the increased production of tra DNA, mRNA, and protein makes cells carrying the multicopy conjugative plasmids excellent sources of these products.     

A novel plasmid-mediated DNA restriction-modification system in E. coli

R plasmids from 101 clinical isolates were transferred to E. coli J62 by conjugation and tested for the presence of R plasmid-mediated restriction-modification DNA systems. Thirty R plasmids were found to inhibit phage λ. vir development. Ten plasmids determined restriction modification system; nine of them proved identical with R.M. EcoRII. One transconjugant, E. coli J62 pLG74, was shown to have a restriction-modification system different from all the known R plasmid-mediated systems. Site-specific endonuclease has been isolated from E. coli J62 pLG74 which differed from all the known restriction endonucleases in the number of cleavage sites on phages λ, φX 174, virus SV40, plasmid pBR322 DNA molecules.     

Incompatibility between Flac, R386, and F:pSC101 recombinant plasmids: the specificity of F incompatibility genes.

The specificity of F incompatibility genes (inc⁺) has been studied with the Flac and R386 plasmids, members of the IncFI incompatibility group. Recently, two inc⁺ regions, incA (46.4–49.3F) and incB (43.1–46.4F) were identified by cloning these F sequences onto pSC101 and subsequently demonstrating incompatibility of the recombinants with Flac. It is shown here that the FincA⁺ recombinant is incompatible with both Flac and R386 while the FincB⁺ recombinant is incompatible only with Flac. Also, a plasmid mutant is described that has reduced incompatibility against Flac and R386. The mutation is located on the BamHI restriction fragment that contains the FincA region. These genetic findings are consistent with the deduction of Palchaudhuri and Maas, based on heteroduplex analysis of IncFI plasmids, that placed the IncFI determinant in the 46.4–48.6F region. The findings also indicate that the FincB⁺ gene product, which has been implicated in negative control of F copy number, is specific for the F replicon.     

Variable spatial selection with two stages of migrations and comparisons between different timings

The conditions for a protected polymorphism for a general multideme population model subject to selection-migration interaction involving two stages of migration are determined. Migration occurs for adults from demes to mating areas and there exists a distinct distribution governing dispersal of offspring from mating areas to deme sites. A number of characterizations of the stable equilibrium configurations are presented. Some implications accruing due to the existence of multiple mating areas separated from the deme sites are discussed. In this framework the special nature of the Levene (Amer. Naturalist87, 331–333, 1953) and Strobeck (Amer. Naturalist108, 152–156, 1974) population subdivision models are elucidated.     

Specification of surface mating systems among conjugative drug resistance plasmids in Escherichia coli K-12

Representative plasmids for most incompatibility groups in Escherichia coli K-12 were transferred to a "bald" strain to compare transfer frequencies for liquid and solid media. Standard broth matings were used for a liquid environment, but for solid surface mating, conjugation was allowed to take place on nutrient plates before washing off the cells for transconjugant selection on plates containing appropriate drugs. Plasmids that determine rigid pili transferred at least 2,000x better on plates than in broth. Some plasmids that determine thick flexible pili transferred 45 to 470x better, whereas others transferred equally well in both environments, as did plasmids of the I complex, which determine thin flexible pili. These results clearly distinguished a number of surface mating systems where most plasmids were derepressed for transfer and determined conjugative pili constitutively. The temperature-independent IncH2 plasmid R831b transferred best on plates, but other IncH plasmids transferred equally well in broth. This inconsistency led to the reclassification of R831b as IncM.     

Protein synthesis induced by infection with packaged λdv plasmid

Plasmid λdv or imm²¹dv DNA was joined to a λ arm having a cos site. This recombinant plasmid can be packaged in a λ head, and used to infect Escherichia coli K12 cells. The injected DNA molecules become plasmids in cells. By adding these particles to uv-irradiated uvrA cells, the packageable λdv or imm²¹dv plasmids can be induced to synthesize proteins coded by genes on the plasmid genome. The packageable plasmid system is thus suitable for studying on synthesis and regulation of plasmid-coded biopolymers. Analyses of the dv-coded proteins in gel electrophoreses revealed that among several genes carried on the dv plasmid genome, only those genes that are members of the pRoR-tof-cII-O-P operon can be expressed. Evidence has been presented to show that expression of this operon, which is directly correlated with replication of the genome, is only partially allowed in cells perpetuating the dv plasmid. These observations are discussed in connection with the autorepressor model (D. E. Berg, 1974, Virology 62, 224–233; K. Matsubara, 1976, J. Mol. Biol. 102, 427–439) that genetically accounts for the control mechanism of plasmid replication.     

Molecular studies of FIme resistance plasmids, particularly in epidemic Salmonella typhimurium.

Summary The molecular sizes of F₁ me resistance plasmids from strains of Salmonella typhimurium, S. wien and S. typhi were within the range 87.9–102.6×10⁶ daltons. DNA reassociation studies indicated that the plasmids from these hosts had at least 80% of their nucleotide sequences in common. A high proportion of F₁ me plasmids cannot mediate their own transfer. The non-autotransferring property of such plasmids is the result of DNA deletion; a non-autotransferring F₁ me plasmid was about 10×10⁶ daltons shorter than autotransferring representatives of the group, and its DNA showed 100% homology with them. Plasmids of the F₁ me group are incompatible with the F factor and with F₁R factors. F₁ me plasmids are incompatible with the fi ⁺ MP10 plasmid of S. typhimurium, whereas F and F₁ factors are compatible with MP10 (Anderson et al., 1977). There was no significant DNA homology between members of the F₁ me group and MP10, and these plasmids may share only a small region of DNA responsible for their incompatibility. The F₁ me R factors examined had 29–37% DNA homology with the F factor, and 50–58% homology with the F₁ resistance plasmid, R162. Molecular examination therefore supports the genetic differentiation of members of the F₁ me group from other F-like plasmids. Both types of investigation can thus be used in epidemiological studies of bacterial strains carrying resistance or other plasmids.     

IncH plasmids in Escherichia coli strains isolated from broiler chicken carcasses.

Plasmids conferring tellurite resistance were transferred at low temperature (27 degrees C) from Escherichia coli strains isolated from chicken carcasses at the time of slaughter and after storage. They belonged to group IncH, as evidenced by their large molecular weight and incompatibility with plasmid pIP233. E. coli strains contaminating chickens meat can thus represent a source of IncH plasmids in the food chain of humans.     

Expression of incompatibility by derivatives of the broad host-range inc P-1 plasmid RK2.

Summary A segment of DNA encoding incompatibility on the inc P-1 plasmid pRK248 was identified by the analysis of deletions generated in vitro, and then cloned into several unrelated and mutually compatible plasmids. These derivatives were tested for expression of P-1 incompatibility. It was demonstrated by transformation experiments that P-1 plasmids were efficiently eliminated from an E. coli host following introduction of any one of the derivatives. However, all the derivatives were compatible with each other. The cloned segment of pRK248 DNA is itself capable of autonomous replication, without being cloned into any plasmid, if plasmid-specified gene products are provided in trans. This satellite plasmid is eliminated from the cell by the inc P-1 plasmid pRK286. The results argue against a partitioning mechanism as the basis for P-1 incompatibility but are consistent with incompatibility being the consequence of negative regulation of copy number. For the inc P-1 system, susceptibility of the plasmid to elimination, but not its ability to eliminate, requires that the P-1 replication system is active.