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.     

Multicopy plasmid stability in Escherichia coli requires host-encoded functions that lead to plasmid site-specific recombination

Summary The heritable stability of the multicopy plasmid ColE1 and its natural relatives, requires the presence in the plasmid of a site (cer in ColE1) that acts as a substrate for site-specific recombination, thereby maintaining plasmids in the monomeric state. Multimerization, promoted by homologous recombination, leads to plasmid loss. Here we show that the Escherichia coli chromosome encodes at least two unlinked functions that act on cer and its analogous sites, to promote stabilizing site-specific recombination. One of these functions is encoded by a gene residing on a cosmid that also contains the argI and pyrB genes, mapping it to the 96–97 min region of the E. coli map.     

RecA independent,site-specific recombination between ColE1 or ColK and a miniplasmid they complement for mobilization and relaxation: Implications for the mechanism of DNA transfer during mobilization

We report a novel type of recA independent recombination between plasmids ColE1 or ColK and a naturally occurring miniplasmid (pLG500). This miniplasmid can be complemented for mobilization and relaxation in the presence of ColE1 or ColK. Recombination between ColE1 and pLG500, or ColK and pLG500, was site-specific, and was only detected following the mobilization of these plasmids. The composite plasmids thus formed were stable, but recombination (resulting in dissociation of their component replicons) was again detected following mobilization. For ColE1, the site at which cointegration with pLG500 occurred was mapped to within 47 base pairs of the relaxation nicking site; for ColK, the recombination site was localized to the same region as its genetically defined transfer origin. The generation of these cointegrate plasmids is consistent with the hypothesis that mobilization entails relaxation nicking, transfer of the nicked single strand of DNA, and recircularization of the transferred single strand by ligation of 3′ and 5′ termini by the relaxation protein bound to the 5′ nick terminus. Since both plasmids are mobilized by the same proteins, their cointegration can be explained as a consequence of the ligation of the 5′ end of one plasmid to the 3′ end of the other, and vice versa.     

The complete nucleotide sequence of the colicinogenic plasmid ColA. High extent of homology with ColE1

Summary The complete nucleotide sequence of the colicinogenic plasmid ColA has been determined. The plasmid DNA consists of 6720 bp (molecular weight 4.48×10⁶). Fifteen putative biological functions have been identified using the functional map previously determined. These include 11 genes and 3 DNA sites. Nine genes encode proteins of which 3 have been fully characterized. The replication region of ColA coding for RNAI and RNAII is highly homologous to that of ColE1 andClo DF13. The same holds true for the site-specific recombination region containing palindromic symmetry and involved in stable maintenance of the plasmids. A high percentage of homology has been detected for putative mobility proteins encoded by ColA and ColE1. The exclusion proteins are also highly homologous.     

Suppression of Co1E1 replication properties by the Inc P-1 plasmid RK2 in hybrid plasmids constructed in vitro.

Hybrid plasmids were constructed in vitro by linking the Inc P-1 broad host range plasmid RK2 to the colicinogenic plasmid ColE1 at their EcoRI endonuclease cleavage sites. These plasmids were found to be immune to colicin E1, non-colicin-producing, and to exhibit all the characteristics of RK2 including self-transmissibility. These joint replicons have a copy number of 5 to 7 per chromosome which is typical of RK2, but not ColE1. Unlike ColE1, the plasmids will not replicate in the presence of chloramphenicol and are maintained in DNA polymerase I mutants of Escherichia coli. In addition, only RK2 incompatibility is expressed, although functional ColE1 can be rescued from the hybrids by EcoRI cleavage. This suppression of ColE1 copy number and incompatibility was found to be a unique effect of plasmid size on ColE1 properties. However, the inhibition of ColE1 or ColE1-like plasmid replication in chloramphenicol-treated cells is a specific effect of RK2 or segments of RK2 (Cri⁺ phenotype). This phenomenon is not a function of plasmid size and requires covalent linkage of RK2 DNA to ColE1. A specific region of RK2 (50.4 to 56.4 × 10³ base-pairs) cloned in the ColE1-like plasmid pBR313 was shown to carry the genetic determinant(s) for expression of the Cri⁺ phenotype.     

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.     

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.     

The replication of Escherichia coli chromosome can be initiated by integrated ColE1-type plasmids

Summary When integrated in the chromosome of E. coli by site-specific recombination of , the ColE1 type replicator provokes the phenotypic suppression of CRT46 dnaA strain. The level of this suppression depends on the orientation of ColE1 replicator in the chromosome of bacteria. Integrative plasmids (intmids) coexist in both autonomous and extrachromosomal states in the same cell.     

Peptidase activity of Escherichia coli aminopeptidase A is not required for its role in Xer site-specific recombination

Xer site-specific recombination is required for the stable inheritance of multicopy plasmids and the normal segregation of the bacterial chromosome in Escherichia coli.Two related recombinases and two accessory proteins are essential for Xer-mediated recombination at cer, a recombination site in the plasmid ColE1 The accessory proteins, ArgR and PepA, function in ensuring that the Xer recombination reaction acts exclusively intramolecularly, converting plasmid dimers into monomers and not vice versa. PepA is an amino-exopeptidase, but its molecular role in the Xer recombination mechanism is unclear. Here we show that a mutation directed at the presumptive active site of PepA creates a protein with no detectable peptidase activity in vitro or in vivo, but which still functions normality in Xer site-specific recombination at cer.     

recB recJ mutants ofSalmonella typhimurium are deficient in transductional recombination,DNA repair and plasmid maintenance

recB recJ mutants ofSalmonella typhimurium are deficient in transduction of chromosomal markers and ColE1-derived plasmids, and also in the maintenance of ColE1 and F plasmids. Plasmid instability is less severe inrecD recJ strains; ColE1 plasmid DNA preparations from these strains show an increased yield of high molecular weight (HMW) linear multimers and a concomitant reduction in plasmid monomers compared to the wild type. Plasmids remain unstable inrecA recD recJ mutants; since these do not produce HMW linear concatemers, we propose that a decrease in monomer production leads to plasmid instability.recB recJ strains also display decreased viability, a component of which may be related to their deficiency in DNA repair. In contrast to their severe defects in recombination, DNA repair and plasmid maintenance,recB recJ mutants ofS. typhimurium behave similarly to the wild type in the segregation of chromosome duplications. The latter observation suggests that neither RecBCD nor RecJ functions are required for chromosomal recombination events that do not involve the use of free ends as recombination substrates.     

The arginine repressor is essential for plasmid-stabilizing site-specific recombination at the ColE1 cer locus.

The heritable stability in Escherichia coli of the multicopy plasmid ColE1 and its natural relatives requires that the plasmids be maintained in the monomeric state. Plasmid multimers, that arise through recA-dependent homologous recombination, are normally converted to monomers by a site-specific recombination system that acts at a specific plasmid site (cer in ColE1). No plasmid functions that act at this site have been identified. In contrast, two unlinked E.coli genes that encode functions required for cer-mediated site-specific recombination have been identified. Here we describe the isolation and characterization of one such gene (xerA) and show it to be identical to the gene encoding the repressor of the arginine biosynthetic genes (argR). The argR protein binds to cer DNA both in vivo and in vitro in the presence of arginine. We believe this binding is required to generate a higher order protein-DNA complex within the recombinational synapse. The argR gene of Bacillus subtilis complements an E.coli argR deficiency for cer-mediated recombination despite the two proteins having only 27% amino acid identity.     

Characterization of the ColE1-Km plasmids pCR1 and pCR11 by electron microscope and restriction endonuclease mapping.

The ColE1-Km plasmids pCR1 and pCR11 have been characterized by electron microscope techniques. Their sizes have been determined to be 13.1 and 9.2 kb, respectively, and heteroduplex studies show that the plasmids differ in the presence of a 3.9 kb deletion in the ColE1 region of pCR11. Both contain a nontandem inverted duplication of a 1.06 kb sequence. The single HindIII site, 3.5 kb from the EcoR1 site, lies in the 0.97 kb of DNA between the inverted repeat sequences. Since DNA insertions at the HindIII site destroy kanamycin resistance, it can be concluded that the kanamycin phosphotransferase gene is contained in this region. Electron microscopy of self-annealed plasmids treated with restriction endonucleases shows that each inverted duplication sequence contains one HindII site and at least two SmaI sites.     

Xer-mediated site-specific recombination at cer generates Holliday junctions in vivo.

Normal segregation of the Escherichia coli chromosome and stable inheritance of multicopy plasmids such as ColE1 requires the Xer site-specific recombination system. Two putative lambda integrase family recombinases, XerC and XerD, participate in the recombination reactions. We have constructed an E. coli strain in which the expression of xerC can be tightly regulated, thereby allowing the analysis of controlled recombination reactions in vivo. Xer-mediated recombination in this strain generates Holliday junction-containing DNA molecules in which a specific pair of strands has been exchanged in addition to complete recombinant products. This suggests that Xer site-specific recombination utilizes a strand exchange mechanism similar or identical to that of other members of the lambda integrase family of recombination systems. The controlled in vivo recombination reaction at cer requires recombinase and two accessory proteins, ArgR and PepA. Generation of Holliday junctions and recombinant products is equally efficient in RuvC- and RuvC+ cells, and in cells containing a multicopy RuvC+ plasmid. Controlled XerC expression is also used to analyse the efficiency of recombination between variant cer sites containing sequence alterations and heterologies within their central regions.     

Site-specific deletions in the tomato genome by the CinH-<Emphasis Type="Italic">RS2</Emphasis> and ParA-<Emphasis Type="Italic">MRS</Emphasis> recombination systems

We have tested the CinH-RS2 and ParA-MRS site-specific deletion systems in tomato (Solanum lycopersicum L.). The ParA-MRS system is derived from the broad-host-range plasmid RK2, where the 222 aa ParA recombinase recognizes a 133 bp multimer resolution site (MRS). The CinH-RS2 system is derived from Acinetobacter plasmids pKLH2 and pKLH204, where the 188 amino acid CinH recombinase recognizes a 113-bp recombination site known as RS2. In this study, target lines containing a DNA segment flanked by recombination sites were crossed to recombinase-expressing lines producing CinH or ParA recombinase. CinH-mediated recombination of RS2 substrates was detected in 2 of 3 F₁ plants that harbor both the target and recombinase loci. On the other hand, recombination mediated by ParA was not detected among F₁ plants, but was found among 13 of 47 F₂ plants. These data show that both systems can mediate site-specific DNA deletion in the tomato genome, and, upon further refinement, can provide additional molecular tools for tomato improvement through precise genome manipulation. As the target construct also contains additional recombination sites for site-specific integration by other recombination systems, these tomato lines could be used for future testing of gene stacking through site-specific integration.     

Characterization of pECL18 and pKPN2: a proposed pathway for the evolution of two plasmids that carry identical genes for a Type II restriction-modification system

The primary structures of the plasmids pECL18 (5571 bp) and pKPN2 (4196 bp) from Escherichia coli and Klebsiella pneumoniae, respectively, which carry genes for a Type II restriction-modification system (RMS2) with the specificity 5'-CCNGG-3', were determined in order to elucidate the structural relationship between them. The data suggest a possible role for recombination events at bom (basis of mobility) regions and the sites of resolution of multimer plasmid forms (so-called cer sequences) in the structural evolution of multicopy plasmids. Analysis of the sequences of pECL18 and pKPN2 showed that the genes for RM* Ecl18kI and RM* Kpn2kI, and the sequences of the rep (replication) regions in the two plasmids, are almost identical. In both plasmids, these regions are localized between the bom regions and the cer sites. The rest of the pECL18 sequence is almost identical to that of the mob (mobilization) region of ColE1, and the corresponding segment of pKPN2 is almost identical to part of pHS-2 from Shigella flexneri. The difference in primary structures results in different mobilization properties of pECL18 and pKPN2. The complete sequences of pECL18, pKPN2 and the pairwise comparison of the sequences of pECL18, pKPN2, ColE1 and pHS-2 suggest that plasmids may exchange DNA units via site-specific recombination events at bom and cer sites. In the course of BLASTN database searches using the cer sites of pECL18 and pKPN2 as queries, we found twenty cer sites of natural plasmids. Alignment of these sequences reveals that they fall into two classes. The plasmids in each group possess related segments between their cer and bom sites.     

The transposon Tn1 as a probe for studying ColE1 structure and function.

Summary Insertion of the transposable genetic element Tn1 into different sites of plasmid ColE1 results in a number of mutant phenotypes. Whereas all plasmids examined were present in normal amount, all showed reduced immunity to killing by colicin E1. Of six insertions isolated after conjugation, five fail to produce colicin, are conjugally proficient (transmissible), and map within a 500 nucleotide region of the genome. The other is conjugally deficient, produces colicin normally and maps close to two others with a similar phenotype isolated after transformation. Of four others isolated after transformation, two have similar properties to the original five transmissible plasmids. The other two are nontransmissible and produce colicin. Non-transmissibility is correlated with reduced relaxation complex. Patterns of protein synthesis in minicels by ColE1 and ColE1:: Tn1 plasmids have been examined: all ColE1 plasmids containing Tn1 show an altered pattern of ColE1 protein synthesis in addition to three presumptive Tn1-specified proteins, one of which is shown to be -lactamase. ColE1:: Tn1 plasmids can be inserted into the conjugative plasmid R64drd11 to form a cointegrate in which ColE1 and Tn1 function can be expressed.     

Xer-mediated site-specific recombination in vitro.

The Xer site-specific recombination system acts at ColE1 cer and pSC101 psi sites to ensure that these plasmids are in a monomeric state prior to cell division. We show that four proteins, ArgR, PepA, XerC and XerD are necessary and sufficient for recombination between directly repeated cer sites on a supercoiled plasmid in vitro. Only PepA, XerC and XerD are required for recombination at psi in vitro. Recombination at cer and psi in vitro requires negative supercoiling and is exclusively intramolecular. Strand exchange at cer produces Holliday junction-containing products in which only the top strands have been exchanged. This reaction requires the catalytic tyrosine residue of Xer C but not that of XerD. Recombination at psi gives catenated circular resolution products. Strand exchange at psi is sequential. XerC catalyses the first (top) strand exchange to make a Holiday junction intermediate and XerD catalyses the second (bottom) strand exchange.     

Escherichia coli XerC recombinase is required for chromosomal segregation at cell division

XerC is a site-specific recombinase of the bacteriophage lambda integrase family that is encoded by xerC at 3700 kbp on the genetic map of Escherichia coli. The protein was originally identified through its role in converting multimers of plasmid ColE1 to monomers; only monomers are stably inherited. Here we demonstrate that XerC also has a role in the segregation of replicated chromosomes at cell division. xerC mutants form filaments with aberrant nucleotides that appear unable to partition correctly. A DNA segment (dif) from the replication terminus region of the E. coli chromosome binds XerC and acts as a substrate for XerC-mediated site-specific recombination when inserted into multicopy plasmids. This dif segment contains a region of 28 bp with sequence similarity to the crossover region of ColE1 cer. The cell division phenotype of xerC mutants is suppressed in strains deficient in homologous recombination, suggesting that the role of XerC/dif in chromosomal metabolism is to convert any chromosomal multimers (arising through homologous recombination) to monomers.     

Use of gene replacement to construct Escherichia coli strains carrying mutations in two genes required for stability of multicopy plasmids.

Escherichia coli mutants completely defective in ColE1 cer-mediated site-specific recombination have been mapped to two genes, xerA and xerB. In this study, xerA xerB double mutants were constructed by gene replacement with a lambda dv plasmid and were shown to be both viable and defective in ColE1 site-specific recombination.