Replication defective RP4 plasmids recovered after chromosomal integration

pHH6000 is a composite replicon made by the in vitro ligation of the IncP plasmid RP4 to a fragment of bacteriophage lambda capable of autonomous replication. Derivatives were selected in which it had integrated into the Escherichia coli chromosome by homologous recombination with the resident lambda prophage, and plasmids were subsequently regenerated from the integrated molecules. Although of the same molecular size as pHH6000, all had altered properties: those recovered from the chromosome of cells simultaneously carrying a distinguishable autonomous IncP plasmid showed a 100- to 1000-fold reduction in their ability to become established in a lambda lysogen; those regenerated from cells with no autonomous IncP plasmid were no longer RP4 replicons, now being dependent on replication functions encoded by the lambda DNA they carry and therefore unable to form a plasmid in a lambda lysogen. This second class of plasmids still exhibited normal RP4 incompatibility and stability even though neither property is encoded by the lambda replicator DNA. It was concluded that expression of RP4 incompatibility and partitioning control do not require an intact RP4 replicon. The data also suggest that the presence in the chromosome of a normal RP4 molecule may be deleterious to the host, although the manner in which the integrated molecules were obtained allows other explanations. The composite plasmids replicating from cloned lambda genes should be useful in analysis of the regulated distribution of RP4 molecules at cell division.     

Cloning and localization of the replication and mobilization regions in the D-plasmid of Pseudomonas putida pBS286 (IncP-9) with a broad host range

Rep-mob loci of naphthalene degradative plasmid pBS286 (IncP-9) have been cloned on the Escherichia coli vectors pUC19 and pUBR322. These loci confer to recombinant plasmids pBS952 and pBS953 the ability for effective mobilization by RP4 (IncP-1) and F plasmid, as well as constant maintenance in various gram-negative bacteria. Localization of cloned sequences in the restriction fragments of conservative part of the pBS286 genome was established. The data obtained correlate with the analysis of plasmids pBS950 and pBS951 which are spontaneous mini-derivatives of pBS286 and pBS292 (delta NPL1::Tn1/Tra+ Nah-) plasmids formed during transformation of E. coli HB101 cells. Plasmids pBS952 and pBS953 retain the incompatibility properties of parental IncP-9 replicon. These recombinant derivatives can be used for construction of bhr vectors with required properties and compatible with bhr vectors constructed on the basis of plasmids from the IncP-1 and IncP-4 groups.     

Second replicon in ColV2-K94 mediates the stable coexistence of two incompatible plasmids.

The colicin-producing plasmid pWS12, a Tn903 derivative of ColV2-K94, was found to be incompatible with the IncFI plasmids KLF1 and R386. It was compatible with the IncFII plasmids R538 and R100. Three overlapping mini-ColV derivatives, pWS15, pWS16 and pWS17, were obtained by restriction digestion of pWS12. Unlike pWS12, pWS16 exhibited incompatibility with both IncFI and IncFII plasmids, whereas the pWS15 and pWS17 plasmids expressed IncFII incompatibility but not the IncFI incompatibility of their parental ColV plasmid. We show that, although pWS12 has an IncFII replicon, Rep1, it does not normally express IncFII incompatibility because a second replicon, Rep2 (homologous to the secondary replicon of F), functions during the stable coexistence of the plasmid with IncFII plasmids. When Rep2 is deleted (as in the mini-ColV plasmids) or made nonfunctional (as in a PolA mutant strain), ColV then behaves as an IncFII plasmid.     

Nature of the compatible inheritance of hybrid plasmid pAS8 and its deletion mutants with replicon ColE1]

Hybrid plasmid pAS8, that consists of RP4 and ColE1 replicons, is incompatible with RP4 but co-exists with ColE1 replicon. The deletion mutants of pAS8, that replicates under the control of ColE1 replicon only, are incompatible with ColE1 derivatives, although the copy number of pAS8 and its deletion mutants in the cell is the same (1-3 per the chromosome). Incompatibility effect of plasmids is expressed in a sharp decrease in transformant's yield during selection of incoming and resident plasmids markers. In this case incompatibility is a very fast process, that leads to the elimination of resident or incoming plasmid just before plating on the selective medium. On the base of negative control theory, the repressors yield, synthesized in the presence of 1-3 copies of the deletion mutant is enough for the expression of ColE1-specific incompatibility. This ColE1 incompatibility is probably connected with the functional activity of ColE1 replicon. When ColE1 factor replicates under the control of RP4 replicon the expression of ColE1-specific incompatibility does not occur. Possibly, the incompatibility effect takes place when pAS8 deletion mutants cause the synthesis of ColE1-specific repressor. Also, the replicons of ColE1 may competein the membrane attachment site.     

Properties and incompatibility behavior of miniplasmids derived from the bireplicon plasmid pCG86

Summary Many plasmids belonging to the F incompatibility groups contain more than one basic replicon. The chimeric plasmid pCG86 is an example of such a multireplicon plasmid. The two basic replicons of pCG86, RepFIIA/FIC and RepFIB have been cloned and re-ligated, the copy numbers of the clones have been determined, and the incompatibility behavior of plasmids containing the ligated replicons and the individual replicons has been studied. The bireplicon plasmids are not expected to be incompatible as recipients with monoreplicon RepFIB or RepFIIA/RepFIC plasmids, since when one replicon is challenged by an incoming replicon, the other should be able to handle the plasmid's replication. In our studies, we found that challenge with either monoreplicon plasmid resulted in incompatibility. This incompatibility was increased in bireplicon plasmids in which RepFIB was duplicated. We conclude that in the bireplicon plasmids, challenging the replication control of one replicon by an incompatible plasmid can interfere with the replication originating from the second replicon.     

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.     

Homology of plasmids with a wide range of hosts

According to blotting hybridization and heteroduplex analysis, plasmids R751, R906 and RP4 of Inc Pi group have continuous regions of homology. These homologous regions were mapped on the R751 and RP4-derived pRP401 deletion mutant DNAs. The plasmid pRP401 (m.w. 21.9 kg) retains the broad host range property and has two regions of intensive homology with other Inc P-1 plasmid DNAs. These regions are localized at 8.2-12.0 kb and 13.9-21.9 kb of the physical map of pRP401 plasmid. Homologous regions of pRP401 DNA include at least the replication genes (oriV, trfA, trfB) as well as genes kilB, korA, korB and probably kilC. The data strongly point out that the broad host range plasmids have the same principle of structural and functional organization.     

A new IncQ plasmid R89S: Properties and genetic organization

The new small (8.18 kb) streptomycin-resistant multicopy plasmid R89S of the Q group incompatibility is described. In contrast to other IncQ plasmids, replication of R89S is dependent on DNA polymerase 1 and proceeds in the absence of de novo protein synthesis. According to our data up to now, the host spectrum of the plasmid R89S is limited to Enterobacteriaceae. A genetic map of the plasmid R89S has been prepared through the construction of deletion and insertion derivatives. Phenotypic analysis of these derivatives has identified the location of genes encoding resistance to streptomycin, and the region essential for mobilization of R89S. The origin of vegetative replication has been located within a 0.7-kb fragment. Another region highly homologous to oriV of the plasmid RSF1010, but not functioning as an origin of replication, was localized. Two regions involved in the expression of incompatibility have also been identified. The data from the restriction analyses, DNA-DNA hybridization, and genetic experiments enable us to assume that the plasmid R89S is a naturally occurring recombinant between part of an IncQ plasmid and another narrow host range replicon of unknown incompatibility group.     

Isolation and evolutionary analysis of a RepFVIB replicon of the plasmid pSU212

We have isolated at least two different replication regions from pSU401, a Tn802 insertion derivative of the IncFVI plasmid pSU212. One of the replication regions (RepFVIB) is highly homologous to the RepFIIA replicon of IncFII plasmids, and thus belongs to the RepFIIA family. We have also cloned the incompatibility determinant (incFVI) and the copy number control gene (cop) from RepFVIB and determined their nucleotide sequences. The analysis of the sequences supports the idea of a modular evolution of RepFIIA plasmids.     

Molecular homology and incompatibility in the IncFI plasmid Group

The usual grounds for the inclusion of a plasmid in a particular incompatibility group are its mutual incompatibility with a type plasmid of that group, and, in some cases, the demonstration of shared regions of specific homology, presumed to be related to DNA replication. We have found that some plasmids classified as IncFI on genetical grounds share no homology with the previously described incompatibility regions of F on the basis of hybridization of specific radioactive probes to restriction enzyme digests of DNA from these plasmids. Others show homology with some or all of the regions of the F plasmid that can express incompatibility. The incompatibility behaviour of these plasmids has been examined to determine the relationship between the possession of regions of homology and the expression of incompatibility. Three plasmids, ColV3-K30, pHH507 and Entp307, show homology only with the secondary replicon of F and appear to use sequences homologous with the secondary F replicon in their replication. The results are consistent with the propositions that some contemporary IncFI plasmids arose by the integration of several replicons, and, in general, the replicon not being used for replicon expresses its incompatibility, as does the replicon being used for replication. We conclude that incompatibility of two plasmids with F does not necessarily demonstrate relatedness of the plasmids to each other, and that inclusion within the IncFI group can result from the possession of any of several combinations of inc sequences.     

Isolation of the replication and partitioning regions of the Salmonella typhimurium virulence plasmid and stabilization of heterologous replicons.

Although the virulence plasmid of Salmonella typhimurium has a copy number of one to two per chromosome, plasmid-free segregants are produced at a rate less than 10(-7) per cell per generation. Three regions appear to be involved in the maintenance of this virulence plasmid. The first two, repB and repC, are functional replicons hybridizing with IncFII and IncFI plasmids, respectively, neither exhibiting the segregational stability of the parent virulence plasmid. The third region, par, cloned as a 3.9-kilobase Sau3A fragment, is not a functional replicon but exhibits incompatibility with the virulence plasmid. Subsequent tests revealed the ability of this 3.9-kilobase par insert to increase the stability of pACYC184 in S. typhimurium from less than 34% to 99% plasmid-containing cells after 50 generations. In addition, the par region increased the stability of oriC, R388, and repC replicons in both S. typhimurium and Escherichia coli hosts. The par region encodes 44,000- and 40,000-molecular-weight proteins essential for the Par+ phenotype but not for the Inc+ phenotype. Although actual sequestering of plasmids within the cell was not demonstrated, all results indicate that the par region described is an actual partitioning locus, similar in organization to those described for plasmids F, P1, and NR1.     

RP1 properties and fertility inhibition among P, N, W, and X incompatibility group plasmids.

Incompatibility group P plasmids demonstrate strong entry exclusion properties. Stringent incompatibility is also observed in the absence of entry exclusion. These observations have been facilitated by the study of a nontransmissible plasmid, RP1-S2, derived from RP1 by transductional shortening. RP1-S2 retains carbenicillin and tetracycline resistances as well as loci that cause either the loss of P plasmids (incp) or a locus specifying susceptibility to curing (sinp) in the presence of a P plasmid. RP1-S2 can be mobilized by an incompatibility group W plasmid, R388, and also freely forms recombinants with R388. P, N, and W incompatibility group plasmids all encode information for the receptor of the cell wall-adsorbing phage PRD1. Based on the premise that the location of this receptor is analogous to entry exclusion factors for F-like plasmids and hence a regulated transfer region determinant, we tested fertility inhibition relationships among these plasmid groups. We detected both reciprocal and nonreciprocal fertility inhibition relationships for bacteria containing various combinations of W, N, and P group plasmids. The nonreciprocal nature of some combinations, we believe, reflects the identity of the point mutation reading to derepression of the plasmid in question. Reciprocal fertility inhibition, on the other hand, may reflect the reconstruction of a fertility inhibition system through complementation. An X incompatibility group plasmid, known to affect the fertility of an N group plasmid, was also shown to inhibit P plasmid fertility. These observations may indicate a possible evolutionary relationship(s) of plasmids unrelated by the criteria of incompatibility, pilus phage specificity, or plasmid host range.     

Isolation and location on the R27 map of two replicons and an incompatibility determinant specific for IncHI1 plasmids.

Two replicons were isolated independently from different IncHI1 plasmids. One was isolated from R27, and a second was isolated from pIP522. We demonstrate, by DNA-DNA hybridization experiments, that these maintenance regions are different and that they are specific to, and carried by, all IncHI1 plasmids tested. In view of this specificity we decided to designate the replicon isolated from R27 as RepHI1A and the replicon isolated from pIP522 as RepHI1B. These two autoreplicative regions are not related to a third replicon present in all IncHI1 plasmids that bears homology with RepFIA and that expresses the characteristic incompatibility of IncHI1 subgroup plasmids toward F factor (D. Saul, D. Lane, and P. L. Bergquist, Mol. Microbiol. 2:219-225, 1988; D. E. Taylor, R. W. Hedges, and P. L. Bergquist, J. Gen. Microbiol. 131:1523-1530, 1985). These results demonstrate that all IncHI1 plasmids tested contain at least three replicons. An incompatibility (Inc) region that hybridizes specifically to all the IncHI1 plasmids was previously isolated (M. Couturier, F. Bex, P. L. Bergquist, and W. K. Maas, Microbiol. Rev. 52:375-395, 1988). Although this Inc locus is not located in an autoreplicative region of IncHI1 plasmids, we observed that this locus stabilizes a low-copy-number replicon. This Inc locus is probably a component of an active partition locus involved in the maintenance of IncHI1 plasmids. The nucleotide sequence of the Inc region contains direct repeats of 31 bp. In addition, this incompatibility determinant hybridizes specifically with IncHI1 plasmids but expresses incompatibility toward plasmids of both IncHI subgroups (IncHI1 and IncHI2). In this communication, we present the mapping of these maintenance elements on the R27 genome.     

Structural-functional organization of the incompatibility group P plasmid pBS221

Plasmid pBS221 was physically mapped for restriction endonucleases EcoRI, BamHI, BglII, HindIII. The regions essential for the plasmid existence and participating in replication (oriV trfA*) and mobilization (mob) were cloned. The tet determinant and oriV trfA* regions were localized on the physical map of the plasmid. A DNA sequence homologous to genes of Tn501 mer operon was detected in this plasmid. The studies on homology of plasmids RP4 (IncP alpha), R751 (IncP beta) and pBS221 plasmid suggest that the latter belongs to the IncP beta subgroup.     

Elimination of broad-host range plasmid vectors in Escherichia coli by curring agents

A comparative study was made of the susceptibility of broad-host range vector plasmids belonging to Inc P1 and Q groups in Escherichia coli to various curing agents. Plumbagin and SDS eliminated RP4 (Inc P1 group) plasmid whereas pKT231 (Inc Q) and pRK2013 (having ColE1 replicon) were eliminated by hexamine ruthenium (III) chloride, alpha-santonin, coumermycin A1 and cis-dichloro diamine platinum (II). The curing activity of these agents was specific.     

Isolation of transmissible cointegrates of Yersinia pestis plasmids pYV and pYT with the plasmid RP4::Mu cts 62, IncP1

The transmissible cointegrates of the Yersinia pestis plasmids pYV and pYT with the broad host range plasmid RP4::Mu cts62 of the incompatibility group IncP have been constructed by the in vivo recombination. The cointegrative plasmid pKR14 (pYV76 omega RP4::Mu cts62) conferred on the transconjugants the properties of Ca2(+)-dependence at 37 degrees C, V-antigen synthesis, RP4 plasmid markers (ApR, KmR, TcR), immunity to the lysis by the bacteriophage Mu cts62 and incompatibility with the homologous replicon pYV76. Cointegrates pKR103 and pKR106 (pYT omega RP4::Mu cts62) conferred on the transconjugant clones the ability to synthesize the "mouse" toxin and fraction I. The capability of Escherichia coli cells to synthesize the latter products has been demonstrated together with the deficiency of these cells to transport the synthesized fraction I to the cell surface.     

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.     

Comparative analysis of the P-1-group plasmids of incompatibility

Wide host range plasmids (IncP-1) R906, R751 and R702 have several cleavage sites for BamHI, HindIII and EcoRI enzymes, in contrast to RP4 plasmid. Using these enzymes, deletion mutants of R906 plasmid have been obtained in vitro which only lost short DNA fragments (1 to 14 kb). A narrow host range pAV1 plasmid of the same incompatibility group has been transformed into the cells of Escherichia coli. pAV1 is stably maintained in the new host and retains its narrow host range in the course of conjugation. Different restriction fragments of R702, R751, R906 and R906-derived deletion mutants hybridize with the nick-translated probe of RP4 DNA. It is suggested that the wide host range plasmids have a similarity in structural and functional organization.     

Dispersion of a gene that codifies fosfomycin resistance among plasmids from enterobacteriaceae isolated from sewage

Abstract The presence of plasmid encoded resistance to fostomycin among enterobacteriaceae isolated from sewage was studied. These plasmids found were classified into 7 varieties according to their original host, size, resistance determinants, restriction pattern and incompatibility group. All of them were related to the Inc M group, indicating a probable common origin from an ancestral replicon. The bacteria carrying these plasmids modified fosfomycin as did those carrying fosfomycin resistant (Fo^r) hospital plasmids. The plasmids showed positive hybridization with a 1-kb DNA fragment which carried a Fo^r-determinant from a hospital plasmid.     

Characteristics of RP4 tellurite-resistance transposon Tn521

A restriction map of the tellurite-resistance (Ter) transposon Tn521 (parent plasmid RP4Ter) was prepared. Five sites from RP4Ter, including the EcoRI origin, were found in pIN25::Tn521. Tn521 was inserted into a transferable 27.5 kb vector (pCU109) to make three different insertion mutants, in which the size of Tn521 was measured accurately at 4.5 kb. Unlike the Ter of IncHI2 plasmids, that of Tn521 in RP4Ter was non-inducible. Ter was expressed in five widely differing bacterial species to which RP4Ter was transferred from Escherichia coli. Electron micrographs of bacteria expressing the Ter of RP4Ter, H complex plasmids, and chromosomal mutants, all revealed similar tellurium metal crystallites when the bacteria were grown in potassium tellurite medium. No other Ter determinants were found amongst 54 plasmids representing most incompatibility groups (excluding the H complex).