Characterization of the ColE1 mobilization region and its protein products

Summary A third of the 6.6 kb genome of ColE1 is devoted to mobilization (mob) genes necessary to promote its specific transfer in the presence of conjugative plasmids. Themob region is genetically complex: twomob genes are entirely overlapped by a third. Oligonucleotide-directed mutagenesis was used to insert an amber codon into one of the overlapped genes and make possible a full complementation analysis ofmob. Fourmob genes essential for mobilization by R64drd11 were thus identified. Fragments ofmob were subcloned under control of the P_tac promoter in a suitable vector, overexpressed in minicells and the mobilization proteins visualized. A comprehensive alignment of themob region of ColE1 with those of its close relatives ColK and ColA demonstrating that the four essentialmob genes are conserved is also presented.     

Polar mobilization of the Escherichia coli chromosome by the ColE1 transfer origin

Summary Mobilization of the plasmid ColE1 from cells containing a conjugative plasmid (such as F) requires the synthesis of ColE1 mob proteins, and the presence, in cis, of bom (basis of mobility), a region of ColE1 containing the origin of transfer (oriT). The process of ColE1 transfer is thought to resemble that of the conjugative plasmid F, although the plasmids share little sequence homology. In F, conjugation is preceded by a strand-specific nicking event at oriT. The nicked strand is then conducted to the recipient with the 5 end leading. This is believed also to occur with ColE1, but direct biochemical confirmation has been precluded by its small size (6.65 kb). To test this hypothesis genetically, a novel method, using a dv-based vector, has been devised to site-specifically integrate bom (or any other cloned sequence) into the chromosome of Escherichia coli. When provided with suitable mobilizing plasmids, such strains were found to transfer the chromosome in a polar way. From these data, the orientation of transfer of ColE1 was deduced and shown to be analogous to F.     

Conjugal transfer of cloning vectors derived from ColE1.

The transfer properties of five cloning vectors derived from ColE1 were studied. Two of the vectors (pSF2124 and pGM706) behaved like wild type ColE1 in that they could be transferred efficiently in the presence of the conjugative plasmid F. The mobilization of the remaining three vectors (pMB9, PBR313 and pBR322) by F was barely detectable. The transfer defect in pBR313 and pBR322 could be complemented by ColK when R64drd11, but not F, was used as the conjugative plasmid. The transferred plasmids could be recovered unchanged from recipients. Conjugal transfer is a potentially useful technique for screening hybrid plasmids in low-risk cloning experiments involving poorly transformable strains.     

Site-specific recombination between ColE1 tcer and NTP16 nmr sites in vivo

The site-specific recombination system used by multicopy plasmids of the ColE1 family uses two identical plasmid-encoded recombination sites and four bacterial proteins to catalyze the recombination reaction. In the case of the Escherichia coli plasmid ColE1, the recombination site, cer, is a 280 by DNA sequence which is acted on by the products of the argR, pepA, xerC and xerD genes. We have constructed a model system to study this recombination system, using tandemly repeated recombination sites from the plasmids ColE1 and NTP16. These plasmids have allowed us precisely to define the region of strand exchange during site-specific recombination, and to derive a model for cer intramolecular site-specific recombination.     

Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid.

Construction and characterization of a class of multicopy plasmid cloning vehicles containing the replication system of miniplasmid P15A are described. The constructed plasmids have cleavage sites within antibiotic resistance genes for a variety of commonly employed site-specific endonucleases, permitting convenient use of the insertional inactivation procedure for the selection of clones that contain hybrid DNA molecules. Although the constructed plasmids showed DNA sequence homology with the ColE1 plasmid within the replication region, were amplifiable by chloramphenicol or spectinomycin, required DNA polymerase I for replication, and shared other replication properties with ColE1, they were nevertheless compatible with ColE1. P15A-derived plasmids were not self-transmissible and were mobilized poorly by Hfr strains; however, mobilization was complemented by the presence of a ColE1 plasmid within the same cell.     

Determination of restriction sites and the nucleotide sequence surrounding the relaxation site of ColE1

The relaxation site of ColE1 has been located within the restriction fragment HpaII L, which is 148 base-pairs in length. Restriction mapping data indicate that the relaxation nick (the presumptive origin of transfer) of ColE1 is located at a distance of 250 to 300 nucleotides away from the replication origin, downstream in the direction of replication. This result is consistent with the observation made by Inselburg (1977), that the relaxation phenomenon probably does not play a direct role in vegetative replication of ColE1. The sequence of 185 nucleotides surrounding the relaxation site has been determined and this contains a translational symmetry and several 2-fold rotational symmetries. These symmetric elements may be recognition sites for proteins involved in the conjugal transfer of ColE1. The sequence further demonstrates that the relaxation site, unlike the cis A nicking site of φX174, is located in an intercistronic region. The site of the relaxation break has a 2-fold rotational symmetry.     

Regions of homology in small colicinogenic plasmids

Restriction maps have been constructed for the colicinogenic plasmids (ColA, ColD, and ColK. Their regions of homology with the ColE1 plasmid and its deletion derivative pAO3 carrying the region responsible for autonomous replication of ColE1 plasmid were determined by means of blotting hybridization and heteroduplex analysis. The plasmids ColA, ColD, and ColK were shown to contain DNA fragments homologous to the region of ColE1 involved in the regulation of replication.     

Site-specific recombination between ColE1 tcer and NTP16 nmr sites in vivo

The site-specific recombination system used by multicopy plasmids of the ColE1 family uses two identical plasmid-encoded recombination sites and four bacterial proteins to catalyze the recombination reaction. In the case of the Escherichia coli plasmid ColE1, the recombination site, cer, is a 280 by DNA sequence which is acted on by the products of the argR, pepA, xerC and xerD genes. We have constructed a model system to study this recombination system, using tandemly repeated recombination sites from the plasmids ColE1 and NTP16. These plasmids have allowed us precisely to define the region of strand exchange during site-specific recombination, and to derive a model for cer intramolecular site-specific recombination.     

Characteristics of replication of small colicinogenic plasmids

Specificity of small multicopy colicinogenic plasmids ColA, ColD, ColE2 and ColK replication has been compared with the one of ColE1 plasmid. Copy number for these plasmids per host cell has been estimated under the normal conditions of cellular growth and under the conditions of chloramphenicol-inhibited growth. DNA polymerase I and dnaB protein, an obligatory component for elongation step in replication, have been shown to be necessary for the plasmids replication. Initiation of plasmids replication has been demonstrated to be independent of dnaA and dnaC proteins. Replication of plasmid ColE2, being similar in its main features to replication of other plasmids from this group, has an important distinction. It requires de novo protein synthesis implying that ColE2 replicon may be different from ColA, ColD, ColK, ColE1 replicons. Thus study of the inducible A, D, K, El colicin synthesis coded by the corresponding plasmids has revealed the similarity regulation of genes, determining the synthesis of each of the mentioned colicins.     

Analysis of mobilization elements in plasmids from Shigella flexneri.

The mobilization properties of three plasmids were examined after cotransfer from Shigella flexneri to Escherichia coli. The largest plasmid, pCN1, was shown to be a conjugative R factor that could promote its own transfer and allow cotransfer of a 4.1-kilobase plasmid, pCN3; mobilization of the third plasmid, pCN2 (6.3 kilobases), required the presence of both pCN1 and pCN3. Sequences from pCN2 and pCN3 homologous to the bom (basis of mobilization) sites of ColE1 and pBR322 were localized by analysis of site-specific deletion derivatives generated in vivo during the transfer of composite plasmids and were characterized by DNA sequencing.     

Multimer resolution systems of ColE1 and ColK: localisation of the crossover site

Summary We have identified and characterised a stability function encoded by the high copy plasmid ColK. The function is analogous to ColE1 cer and maximises stability by maintaining plasmids in the monomeric state. In vivo recombination between cer and ckr (which share more than 90% homology at the DNA sequence level) produced a functional hybrid. Sequence analysis of hybrids indicates that recombination involving cer and ckr is site-specific and occurs within a 35 bp region of DNA which contains palindromic symmetry.     

A complementation analysis of mobilization deficient mutants of the plasmid colE1

Summary Hydroxylamine was used to induce mutants of the ColE1 derived plamid pML2 that are inefficiently mobilized (Mob^-) during conjugation by an Hfr donor. The ability of those mutants to be complemented by deletion mutants and Tn3 insertion mutants of ColE1 was examined. Three complementation groups were identified and localized on the ColE1 genetic map (Mob1, Mob2, and Mob3). One hydroxylamine mutant was not complemented by any mobilization deficient mutant but was complemented by mobilizable ColE1 mutants. Two hydroxylamine mutants were not complemented by any ColE1 derivatives. A mutant that had its relaxation nick site deleted had a markedly reduced mobilizability. The relationship between DNA relaxation, replication and mobilization is considered.     

The relaxosome protein MobC promotes conjugal plasmid mobilization by extending DNA strand separation to the nick site at the origin of transfer

The frequency of conjugal mobilization of plasmid R1162 is decreased approximately 50-fold if donor cells lack MobC, one of the plasmid-encoded proteins making up the relaxosome at the origin of transfer ( oriT  ). The absence of MobC has several different effects on oriT DNA. Site- and strand-specific nicking by MobA protein is severely reduced, accounting for the lower frequency of mobilization. The localized DNA strand separation required for this nicking is less affected, but becomes more sensitive to the level of active DNA gyrase in the cell. In addition, strand separation is not efficiently extended through the region containing the nick site. These effects suggest a model in which MobC acts as a molecular wedge for the relaxosome-induced melting of oriT DNA. The effect of MobC on strand separation may be partially complemented by the helical distortion induced by supercoiling. However, MobC extends the melted region through the nick site, thus providing the single-stranded substrate required for cleavage by MobA.     

Rifampicin-resistant initiation of DNA synthesis on the isolated strands of ColE plasmid DNA.

The opposite strands of the ColE1 and ColE3 plasmids were isolated as circular single-stranded DNA molecules. These molecules were compared with M13 and phi X174 viral DNA with respect to their capacity to function as templates for in vitro DNA synthesis by a replication enzyme fraction from Escherichia coli. It was found for both ColE plasmids that the conversion of H as well as L strands to duplex DNA molecules closely resembles phi X174 complementary strand synthesis and occurs by a rifampicin-resistant priming mechanism involving the dnaB, dnaC, and dnaG gene products. Restriction analysis of partially double-stranded intermediates indicates that preferred start sites for DNA synthesis are present on both strands of the ColE1 HaeII-C fragment. Inspection of the nucleotide sequence of this region reveals structural similarities with the origin of phi X174 complementary strand synthesis. We propose that the rifampicin-resistant initiation site (rri) in the ColE1 L strand is required for the priming of discontinuous lagging strand synthesis during vegetative replication and that the rri site in the H strand is involved in the initiation of L strand synthesis during conjugative transfer.     

Mechanism of plasmid ColEl and pMB-9 mobilization by plasmid F'lac+ in Escherichia coli K-12]

The genetic control and mechanism of mobilization of the non-conjugative plasmids ColE1 and pMB-9 by the conjugative plasmids was orived to be recA-independent process in contrast to the mobilization of the chromosomal marker pro. Acridine orange and ethidium bromide curing data together with the results of electrophoretic analysis of plasmid DNA suggest that the plasmids F' lac+ and pMB-9 as well as F' lac+ and ColE1 remain autonomous after their contransfer to recipient cells. These data argue in favour of non-recombinational nature of the plasmid mobilization process. The possibility of transmission of a non-conjugative plasmid without transmission of a conjugative one from the donor strain carrying both plasmids was established. The results obtained are discussed with respect to the hypothesis on the effect of diffusible products encoded by the conjugative plasmid and required of the mobilization of the non-conjugative plasmid.     

In vitro cleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins.

We have used purified RSF1010 mobilization proteins to reproduce in vitro a strand-specific nicking at the plasmid origin of transfer, oriT. In the presence of Mg2+, the proteins MobA (78-kDa form of RSF1010 DNA primase), MobB, and MobC and supercoiled or linear duplex oriT DNA form large amounts of a cleavage complex, which is characterized by its sensitivity to protein-denaturant treatment. Upon addition of SDS to such a complex, a single strand break is generated in the DNA, and MobA is found linked to the 5' nick terminus, presumably covalently. The double-strand nicking activity of MobA requires, in addition to Mg2+, the presence of MobC and is stimulated by the presence of MobB. The nick site has been shown by DNA sequencing to lie at the position cleaved in vivo during transfer, between nucleotides 3138/3139 in the r strand of RSF1010. We have found that MobA will also cleave DNA at sites other than oriT if the DNA is present in single-stranded form. Breakage in this case occurs in the absence of denaturing conditions, and after prolonged incubation, reclosure can be demonstrated.     

Complementation of transfer deficient ColE1 mutants

Summary The transfer defect of some ColE1 mutants is complemented by ColE1 or ColK, but not by ColE2. This implies that at least one ColE1-specified protein or RNA is normally needed for ColE1 conjugal transfer. The gene(s) postulated for this function lies within a region whose length is at most 50% of the genome.     

Evolution of ColE1-like plasmids across γ-Proteobacteria: From bacteriocin production to antimicrobial resistance

Antimicrobial resistance is one of the major threats to Public Health worldwide. Understanding the transfer and maintenance of antimicrobial resistance genes mediated by mobile genetic elements is thus urgent. In this work, we focus on the ColE1-like plasmid family, whose distinctive replication and multicopy nature has given rise to key discoveries and tools in molecular biology. Despite being massively used, the hosts, functions, and evolutionary history of these plasmids remain poorly known. Here, we built specific Hidden Markov Model (HMM) profiles to search ColE1 replicons within genomes. We identified 1,035 ColE1 plasmids in five Orders of γ-Proteobacteria, several of which are described here for the first time. The phylogenetic analysis of these replicons and their characteristic MOB_P5/HEN relaxases suggest that ColE1 plasmids have diverged apart, with little transfer across orders, but frequent transfer across families. Additionally, ColE1 plasmids show a functional shift over the last decades, losing their characteristic bacteriocin production while gaining several antimicrobial resistance genes, mainly enzymatic determinants and including several extended-spectrum betalactamases and carbapenemases. Furthermore, ColE1 plasmids facilitate the intragenomic mobilization of these determinants, as various replicons were identified co-integrated with large non-ColE1 plasmids, mostly via transposases. These results illustrate how families of plasmids evolve and adapt their gene repertoires to bacterial adaptive requirements.     

Role of the origin of transfer in termination of strand transfer during bacterial conjugation.

Conjugal transfer of the broad-host-range plasmid R1162 is initiated and terminated at the nic site within the 38-bp origin of transfer (oriT). Termination involves ligation of the transferred single strand by the plasmid-encoded MobA protein. Several different assays were used to identify the oriT DNA required for termination. For plasmids containing two oriTs, with transfer initiated at one and terminated at the other, the inverted repeat within oriT is important for termination. Deletion of the outer arm reduces the termination frequency; those terminations that do occur probably depend upon nicking at this oriT prior to transfer. The locations of second-site suppressor mutations indicate that base pairing between the arms of the inverted repeat is important for termination. In vitro, the inverted repeat is not required for specific cleavage of single-stranded DNA at nic, but competition experiments indicate that oriTs with the inverted repeat are preferentially cleaved. We propose that the function of the oriT inverted repeat is to trap the plasmid-encoded MobA protein at the end of a round of strand transfer, thus ensuring that the protein is available for the ligation step.