Analysis of the multimer resolution system encoded by the parCBA operon of broad-host-range plasmid RP4

The broad-host-range plasmid RP4 encodes a highly efficient partitioning function, termed par, that is capable of stabilizing plasmids in a variety of Gram-negative bacteria independently of the nature of the replicon. The mechanism responsible for plasmid stabilization by this locus appears to be a complex system which includes a site-specific recombination system mediating resolution of plasmid multimers. In this report we present a detailed study on this multimer resolution system (mrs). The parA gene encodes two forms of a resolvase capable of catalysing site-specific recombination between specific sites situated in the promoter region of the parCBA operon. The two ParA proteins that are produced as a result of independent translation initiation at two different start codons within the same open reading frame were overexpressed in Escherichia coli and partially purified. Both forms of the enzyme are able to recombine a supercoiled cointegrate substrate containing two cis-acting elements with the same orientation in an in vitro resolution assay. ParA-mediated, site-specific recombination was found to be independent of any other gene product encoded by the RP4 par locus in vitro and in vivo. The DNA-binding sites for the ParA resolvase were determined using DNase I protection experiments. The results identified three binding sites within the mrs cis-acting region. Both the biochemical properties of the ParA protein and the organization of the cis-acting recombination site revealed a high degree of similarity to the site-specific recombination systems of Tn3-llke transposable elements suggesting an evolutionary retationship.     

Molecular characterization of the resolvase gene, res, carried by a multicopy plasmid from Clostridium perfringens: common evolutionary origin for prokaryotic site-specific recombinases

Clostridium perfringens strain CPN50 harbours a 10.2 kb plasmid known as pIP404 which, in addition to a set of UV-inducible genes involved in bacteriocin production, carries res, a gene probably encoding a site-specific recombinase. The RES protein is highly homologous to the resolvases of transposons from both Gram-negative and Gram-positive bacteria as well as enzymes involved in site-specific DNA inversion. A likely role for the RES protein would be to stabilize pIP404 by reducing the number of plasmid multimers resulting from homologous recombination. A putative resolution site for RES action was found overlapping the res promoter. Phylogenetic analysis of the primary structures of ten site-specific recombinases suggested a common descent and showed the RES protein to be closest to the resolvase encoded by Tn917 from Streptococcus faecalis.     

Tn5053 family transposons are res site hunters sensing plasmidal res sites occupied by cognate resolvases

DNA sequence database search revealed that most of Tn5053/Tn402 family transposons inserted into natural plasmids were located in putative res regions upstream of genes encoding various resolvase-like proteins. Some of these resolvase genes belonged to Tn3 family transposons and were closely related to the tnpR genes of Tn1721 and a recently detected Tn5044. Using recombinant plasmids containing fragments of Tn1721 or Tn5044 as targets in transposition experiments, we have demonstrated that Tn5053 displays striking insertional preference for the res regions of these transposons: more than 70% of Tn5053 insertion events occur in clusters inside the target res regions, while most remaining insertion events occur no further than 200 base pairs away from both sides of the res regions. We demonstrate that Tn5053 insertions (both into and outside a res region of the target plasmid) require the presence of a functional cognate resolvase gene either in cis or in trans. To our knowledge, this is the first case when a site-specific recombination system outside a transposon has been shown to be involved in transposition.     

Use of a transposon-encoded site-specific resolution system for construction of large and defined deletion mutations in bacterial chromosome

A class II transposon, Tn1722, encodes a site-specific resolution system, in which the resolvase (TnpR) efficiently catalyzes intramolecular recombination between the two directly oriented copies of the resolution site (res), leading to precise excision of the intervening DNA region. This property was exploited to develop the general strategies to introduce the large and defined deletion mutations into the bacterial chromosome. The Tn1722 res site was inserted into the plasmid carrying a cloned chromosomal fragment, and the resulting plasmid was integrated into a Tn1722-containing target chromosome by single crossover-mediated homologous recombination. The plasmid integrant carrying the two copies of the res site in the same orientation could efficiently excise the chromosomal region locating between the two res sites by means of the site-specific resolution system. Such site-specific deletion could be also detected by appropriate integration of the res–tnpR-containing plasmid into the chromosome in which another copy of the res site had been inserted through allelic exchange. This latter strategy was further modified to isolate the deletion mutations that were free of the resistance markers used for introduction of the res site and the res–tnpR block into the target chromosome. The deletion systems were applied to analyze the 103-kb pvd region of Pseudomonas aeruginosa PAO carrying most of the pyoverdin biosynthetic genes. Successful isolation of the mutation lacking more than a 100-kb fragment in the pvd region indicated that this region did not carry any essential genes.     

Molecular characterization of the resolvase gene, res, carried by a multicopy plasmid from Clostridium perfringens: common evolutionary origin for prokaryotic site-specific recombinases

Clostridium perfringens strain CPN50 harbours a 10.2 kb plasmid known as plP404 which, in addition to a set of UV-inducible genes involved in bacteriocin production, carries res, a gene probably encoding a site-specific recombinase. The RES protein is highly homologous to the resolvases of transposons from both Gram-negative and Gram-positive bacteria as well as enzymes involved in site-specific DNA inversion. A likely role for the RES protein would be to stabilize plP404 by reducing the number of plasmid multimers resulting from homologous recombination. A putative resolution site for RES action was found overlapping the res promoter. Phylogenetic analysis of the primary structures of ten site-specific recombinases suggested a common descent and showed the RES protein to be closest to the resolvase encoded by Tn917 from Strepfococcus faecalis.     

Genetic properties of chromosomally integrated 2 mu plasmid DNA in yeast

We obtained strains of yeast with large segments of 2 mu plasmid DNA integrated at several chromosomal locations by selecting genetically for recombination between a chromosomal sequence carried on a 2 mu-circle-containing hybrid plasmid and a homologous sequence on the chromosome. In all diploids examined, the presence of 2 mu circle sequences causes a marked instability of the chromosome into which the 2 mu DNA is inserted. Although in some cases the loss of genetic markers is due to physical loss of the entire chromosome, in most cases the loss of markers appears to be due to a mitotic homozygotization of markers: the allelic information from the homologous chromosome replaces the information distal to the integrated 2 mu DNA. The instability caused by integrated 2 mu DNA sequences requires the activity of the specialized site-specific recombination system encoded by the 2 mu plasmid. We propose that the presence of integrated 2 mu DNA allows efficient integration of additional copies of the intact 2 mu plasmid by the action of the plasmid-coded special recombination system. Unequal sister-strand exchanges within the inverted repetition would result in the formation of dicentric chromosomes whose breakage during mitosis might begin a cycle analogous to the breakage-fusion-bridge cycle described many years ago in maize.     

Mechanism of integration of the broad-host-range plasmid RP4 into the chromosome of Myxococcus xanthus

The site-specific recombination mechanism through which the plasmid RP4 has been previously shown to integrate into the chromosome of Myxococcus xanthus has been investigated further. Once integrated in one of the numerous chromosomal sites from two different strains, through a precise site on the plasmid, the latter can be excised either precisely or after a definite 14.5-kb deletion. In some cases, the integration is followed by different DNA rearrangements that yield a higher rate of excision and integration. A model for the site-specific integration and excision of the plasmid is proposed.     

Specificity of DNA recognition in the nucleoprotein complex for site-specific recombination by Tn21 resolvase.

Resolvases from Tn3-like transposons catalyse site-specific recombination at res sites. Each res site has 3 binding sites for resolvase, I, II, and III. The res sites in Tn3 and Tn21 have similar structures at I and II but they differ at III. Mutagenesis of the Tn21 res site showed that sub-site III is essential for recombination though the sequences in III that are recognized by Tn21 resolvase are positioned differently from the equivalent sequences in the Tn3 site. The deletion of III caused a 1,000-fold drop in the rate of recombination. But other mutations at III, changing 3 or 4 consecutive base pairs, caused only 1.5- to 4-fold decreases in rate, even when the mutations were in target sequences for this helix-turn-helix protein. The reason why Tn21 resolvase has similar activities at a number of different DNA sequences may be due to the multiplicity of protein-protein and protein-DNA interactions in its recombinogenic complex. This lack of precision may be a general feature of nucleoprotein complexes.     

Mechanism of integration of the broad-host-range plasmid RP4 into the chromosome of Myxococcus xanthus

The site-specific recombination mechanism through which the plasmid RP4 has been previously shown to integrate into the chromosome of Myxococcus xanthus has been investigated further. Once integrated in one of the numerous chromosomal sites from two different strains, through a precise site on the plasmid, the latter can be excised either precisely or after a definite 14.5-kb deletion. In some cases, the integration is followed by different DNA rearrangements that yield a higher rate of excision and integration. A model for the site-specific integration and excision of the plasmid is proposed.     

Definition of three resolvase binding sites at the res loci of Tn21 and Tn1721.

The dual functions of resolvase, site-specific recombination and the regulation of its own expression from tnpR, both require the interaction of this protein with the DNA sequence at res, but the specificity of this interaction differs between groups of Tn3-like elements. In this study, DNA fragments that contained res from Tn21 or Tn1721 were subjected to either cleavage by DNase I or methylation by dimethyl sulphate in the presence of the purified resolvase from Tn21 or Tn1721. These experiments showed that each resolvase bound to the same three sites (I, II and III) within res from Tn1721 and to an equivalent series of three sites on Tn21: the differences in the amino acid sequences of the two proteins did not affect their interaction with either DNA. The DNA sequences at each site had some similarities and, in conjunction with data from the related transposon Tn501, a consensus was established. However, the three sites are functionally distinct: site I (tnpR-distal) spans the recombination cross-over point and sites II and III (tnpR-proximal) overlap the promoter of tnpR. The binding sites on these transposons were compared with those in the gamma delta/Tn3 system: the similarities between the two groups of transposons revealed some general features of resolvase-DNA interactions while the differences in fine structure elucidated the specificity of each resolvase.     

Insertional mutagenesis in Bacillus subtilis: mechanism and use in gene cloning

The plasmid pHV32, which replicates in Escherichia coli but not in Bacillus subtilis, transformed B. subtilis-competent cells efficiently when linked in vitro to EcoRI B. subtilis DNA segments. The transformed clones carried pHV32 inserted in their chromosomes, and often displayed a mutant phenotype. One of the transformed clones carried pHV32 inserted close to the thyB gene. We cleaved the DNA extracted from this clone with BglII restriction endonuclease, for which no sites exist on pHV32, ligated the released segments and used them to transform E. coli selecting for pHV32-carried genetic markers. The transformants harbored a hybrid plasmid which carried the B. subtilis thyB gene. Circular molecules composed of pHV32 joined to B. subtilis DNA inserted into the chromosome by a Campbell-like recombination event. Linear molecules, in which pHV32 was flanked by two non-adjacent DNA segments, underwent a double cross-over recombination with the chromosome. In this case the chromosomal sequences between the non-adjacent segments were deleted, and replaced by pHV32 sequences.     

A bacterial model system for chromosomal targeting.

A system that permits efficient site-specific chromosomal targeting of foreign DNA on the Escherichia coli chromosome has been developed, using the FLP site-specific recombination system derived from the yeast 2 mu plasmid. The system demonstrates the feasibility of using site-specific recombination for this purpose, and provides a means to gather information on parameters that may affect chromosomal targeting to guide efforts to establish similar systems in higher eukaryotes. In this model system, the efficiency of integration of foreign DNA is affected by the location of the target site in the chromosome, and the structure of the recombination sites.     

Microcin H47 system: an Escherichia coli small genomic island with novel features

Genomic islands are DNA regions containing variable genetic information related to secondary metabolism. Frequently, they have the ability to excise from and integrate into replicons through site-specific recombination. Thus, they are usually flanked by short direct repeats that act as attachment sites, and contain genes for an integrase and an excisionase which carry out the genetic exchange. These mobility events would be at the basis of the horizontal transfer of genomic islands among bacteria.Microcin H47 is a ribosomally-synthesized antibacterial peptide that belongs to the group of chromosome-encoded microcins. The 13 kb-genetic system responsible for its production resides in the chromosome of the Escherichia coli H47 strain and is flanked by extensive and imperfect direct repeats. In this work, both excision and integration of the microcin H47 system were experimentally detected. The analyses were mainly performed in E. coli K12 cells carrying the microcin system cloned in a multicopy plasmid. As expected of a site-specific recombination event, the genetic exchange also occurred in a context deficient for homologous recombination. The DNA sequence of the attachment sites resulting from excision were hybrid between the sequences of the direct repeats. Unexpectedly, different hybrid attachment sites appeared which resulted from recombination in four segments of identity between the direct repeats. Genes encoding the trans-acting proteins responsible for the site-specific recombination were shown to be absent in the microcin H47 system. Therefore, they should be provided by the remaining genetic context, not only in the H47 strain but also in E. coli K12 cells, where both excision and integration occurred. Moreover, a survey of the attachment sites in data banks revealed that they are widely spread among E. coli strains. It is concluded that the microcin system is a small island -H47 genomic island- that would employ a parasitic strategy for its mobility.     

Prophage lambda induces terminal recombination in Escherichia coli by inhibiting chromosome dimer resolution. An orientation-dependent cis-effect lending support to bipolarization of the terminus

A prophage lambda inserted by homologous recombination near dif, the chromosome dimer resolution site of Escherichia coli, is excised at a frequency that depends on its orientation with respect to dif. In wild-type cells, terminal hyper- (TH) recombination is prophage specific and undetectable by a test involving deletion of chromosomal segments between repeats identical to those used for prophage insertion. TH recombination is, however, detected in both excision and deletion assays when Deltadif, xerC, or ftsK mutations inhibit dimer resolution: lack of specialized resolution apparently results in recombinogenic lesions near dif. We also observed that the presence near dif of the prophage, in the orientation causing TH recombination, inhibits dif resolution activity. By its recombinogenic effect, this inhibition explains the enhanced prophage excision in wild-type cells. The primary effect of the prophage is probably an alteration of the dimer resolution regional control, which requires that dif is flanked by suitably oriented (polarized) stretches of DNA. Our model postulates that the prophage inserted near dif in the deleterious orientation disturbs chromosome polarization on the side of the site where it is integrated, because lambda DNA, like the chromosome, is polarized by sequence elements. Candidate sequences are oligomers that display skewed distributions on each oriC-dif chromosome arm and on lambda DNA.     

Chromosome engineering in Saccharomyces cerevisiae by using a site-specific recombination system of a yeast plasmid.

We have developed an effective method to delete or invert a chromosomal segment and to create reciprocal recombination between two nonhomologous chromosomes in Saccharomyces cerevisiae, using the site-specific recombination system of pSR1, a circular cryptic DNA plasmid resembling 2 microns DNA of S. cerevisiae but originating from another yeast, Zygosaccharomyces rouxii. A 2.1-kilobase-pair DNA fragment bearing the specific recombination site on the inverted repeats of pSR1 was inserted at target sites on a single or two different chromosomes of S. cerevisiae by using integrative vectors. The cells were then transformed with a plasmid bearing the R gene of pSR1, which encodes the site-specific recombination enzyme and is placed downstream of the GAL1 promoter. When the transformants were cultivated in galactose medium, the recombination enzyme produced by expression of the R gene created the modified chromosome(s) by recombination between two specific recombination sites inserted on the chromosome(s).     

Identification and characterization of Tn4656, a novel class II transposon carrying a set of toluene-degrading genes from TOL plasmid pWW53

It has been reported that the toluene-degrading (xyl) genes from Pseudomonas putida plasmid pWW53 are able to translocate to broad-host-range drug resistance plasmid RP4, and pWW53-4 is one of the smallest RP4 derivatives (H. Keil, S. Keil, R. W. Pickup, and P. A. Williams, J. Bacteriol. 164:887-895, 1985). Our investigation of pWW53-4 in this study demonstrated that such a translocated region that is 39 kb long is a transposon. This mobile element, Tn4656, was classified as a class II transposon since its transposition occurred by a two-step process: transposase (TnpA)-mediated formation of the cointegrate and resolvase (TnpR)-mediated site-specific resolution of the cointegrate at the two copies of the res site. The Tn4656 TnpA and TnpR functions encoded in the rightmost 4-kb region were found to be exchangeable with those specified by other Tn1721-related class II transposons, including another toluene transposon, Tn4653. Sequence analysis of the transposition-related genes and sites of Tn4656 also supported the hypothesis that this transposon is closely related to the Tn1721-related transposons. The lower transposition frequency of Tn4656 has been suggested to be due to the unique nucleotide sequence of one of the terminal 39-bp inverted repeats.     

Thermosensitive vector derived from RP1 plasmid for detection of transposable elements

The involvement of the transposable DNA element of E. coli K12 chromosome in integrative recombination of RP1 plasmid was studied. Using temperature sensitive for replication plasmid RP1ts12--the derivative of RP1 which contains mutated transposon Tnl, it was shown that integration of RP1 into host chromosome and Hfr formation may occur according to a mechanism mediated by chromosome IS-elements. Plasmids that are desintegrated from the chromosome of these Hfrs contain discrete DNA segments (IS-elements) and possess elevated frequency of integration into chromosome of rec+ cells. The latter was used for selection of RP1ts12 recombinants carrying chromosome IS. For identification of IS involved in RP1 integration the number of independent RP1ts 12 recombinants was subjected to restriction and heteroduplex analysis. By analysing recombinants integrated into bacterial chromosome with frequency 5 X 10(-3), a new IS-element of E. coli K12 designated IS111 was discovered. IS111-element is about 1500bp of length, contains Smal, Pst1 and BamH1 restriction endonuclease sites and was found in the same position on the plasmid RP1 in two different orientations. IS-elements that have been revealed in a number of other RP1ts12 recombinants were preliminary identified as IS1-like elements. One recombinants plasmid was found to have an IS5-like elements. The activity of IS-elements inserted into RP1ts12 in recA-dependent integrative recombination was estimated. From the data of absolute and relative RP1ts12 integration frequencies mediated by IS111, IS1- and IS5-like elements a conclusion was made about the absence of E. coli K12 chromosome IS-elements in RP1 plasmid. The Hfr-formation and chromosomal gene transfer by recombinant plasmids RP1ts12: IS111 were studied. The possibility to use insertion RP1ts12 derivatives for the estimation of copies number, mapping and definition of orientation of IS-elements in bacterial chromosome and the possibilities for detection of transposable DNA elements using RP1ts12 in a wide range of gram-negative bacteria are discussed.     

Location of the site-specific recombination system of R46: a function necessary for plasmid maintenance

The R46 site-specific recombination system comprises a per (plasmid-encoded recombinase) gene and a site at which the gene product acts, the per site. The two functions have been cloned into pACYC184. They are encoded by sequences within a region of approximately 2 kb on the R46 genome. These R46 sequences are closely related to the site-specific recombination systems of the ampicillin resistance transposons collectively designated TnA. The R46 per function is interchangeable with the tnpR gene product of TnA. Both enzymes can mediate recombination between the related res and per sites in R46::TnA recombinant plasmids to generate site-specific deletions and inversions. Similar DNA rearrangements occur when TnA inserts into pACYC184 derivatives carrying the cloned R46 per functions. Carriage of this site-specific recombination system contributes to the stable maintenance of R46. By converting plasmid dimers to monomers the R46 per functions help to ensure equal partitioning at cell division.     

Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage phiC31 site-specific recombination system

The site-specific recombination system used by the Streptomyces bacteriophage phiC31 was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4+ marker, and a second plasmid expressing the phiC31 integrase gene. High-efficiency transformation to the Ura+ phenotype occurred when the integrase gene was expressed. Southern analysis revealed that the attB-ura4+ plasmid integrated into the chromosomal attP site. Sequence analysis showed that the attBxattP recombination was precise. In another approach, DNA with a ura4+ marker flanked by two attB sites in direct orientation was used to transform S. pombe cells bearing an attP duplication. The phiC31 integrase catalyzed two reciprocal cross-overs, resulting in a precise gene replacement. The site-specific insertions are stable, as no excision (the reverse reaction) was observed on maintenance of the integrase gene in the integrant lines. The irreversibility of the phiC31 site-specific recombination system sets it apart from other systems currently used in eukaryotic cells, which reverse readily. Deployment of the phiC31 recombination provides new opportunities for directing transgene and chromosome rearrangements in eukaryotic systems.