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.     

Molecular genetics of the chloramphenicol-resistance transposon Tn4451 from Clostridium perfringens: the TnpX site-specific recombinase excises a circular transposon molecule

The chloramphenicol-resistance transposon Tn4451 undergoes precise conjugative deletion from its parent plasmid piP401 in Clostridium perfringens and precise spontaneous excision from multicopy plasmids in Escherichia coli. The complete nucleotide sequence of the 6338 bp transposon was determined and it was found to encode six genes. Genetic analysis demonstrated that the largest Tn4451-encoded gene, tnpX, was required for the spontaneous excision of the transposon in both E. coli and C. perfringens, since a Tn4451 derivative that lacked a functional tnpX gene was completely stable in both organisms. Because the ability of this derivative to excise was restored by providing the tnpX gene on a compatible plasmid, it was concluded that this gene encoded a trans-acting site-specific recombinase. Allelic exchange was used to introduce the tnpXΔ allele onto plP401 and it was shown that TnpX was also required for the conjugative excision of Tn4451 in C. perfringens. It was also shown by hybridization and polymerase chain reaction (PCR) studies that TnpX-mediated transposon excision resulted in the formation of a circular form of the transposon. The TnpX recombinase was unique because it potentially contained the motifs of two independent site-specific recombinase families, namely the resolvase/invertase and integrase families. Sequence analysis indicated that the resolvase/invertase domain of TnpX was likely to be involved in the excision process by catalysing the formation of a 2bp staggered nick on either side of the GA dinucleotide located at the ends of the transposon and at the junction of the circular form. The other Tn4451-encoded genes include tnpZ, which appears to encode a second potential site-specific recombinase. This protein has similarity to plasmid-encoded Mob/Pre proteins, which are involved in plasmid mobilization and multimer formation. Located upstream of the tnpZ gene was a region with similarity to the site of interaction of these mobilization proteins.     

Characterization and transferability of Clostridium perfringens plasmids.

Two strains of Clostridium perfringens resistant to clindamycin (Cl), chloramphenicol (Cm), erythromycin (Em), and tetracycline (Tc) were isolated in France in 1974 and 1975. For one of these strains, curing experiments and molecular characterization of the extrachromosomal DNA clearly demonstrate the existence of two plasmids, plP401 (54 kilobases) and plP402 (63 kilobases), which, respectively, code for Tc Cm and Em Cl resistance. With mixed cultures, the Tc Cm plasmid is transferable to sensitive strains of C. perfringens; a segregation of these markers is frequently observed during mating experiments. In contrast, the transfer of the naturally occurring plasmid Em Cl does not occur at a significant rate. In performing transfer experiments in axenic mice, we obtained a Cl^r Em^r Tc^r transcipient whose chromosomal properties are those of a hybrid. When used in mating as a parental strain, this strain promotes chromosomal gene exchange. The role of the plasmid in this phenomenon is discussed, these transcipients being generally Cl^r Em^r Tc^r. The plasmid transfer is not limited to antibiotic resistance plasmids, the transferability of a bacteriocinogenic plasmid, plP404, harbored by C. perfringens BP6K-N5 being shown also. The transfer mechanism remains to be proved; it might be a conjugation process, a cell-to-cell contact being necessary for the transfer.     

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.     

pBECKS2000: a novel plasmid series for the facile creation of complex binary vectors, which incorporates "clean-gene" facilities

A new plasmid series has been created for Agrobacterium-mediated plant transformation. The pBECKS2000 series of binary vectors exploits the Cre/loxP site-specific recombinase system to facilitate the construction of complex T-DNA vectors. The new plasmids enable the rapid generation of T-DNA vectors in which multiple genes are linked, without relying on the availability of purpose-built cassette systems or demanding complex, and therefore inefficient, ligation reactions. The vectors incorporate facilities for the removal of transformation markers from transgenic plants, while still permitting simple in vitro manipulations of the T-DNA vectors. A `shuttle' or intermediate plasmid approach has been employed. This permits independent ligation strategies to be used for two gene sets. The intermediate plasmid sequence is incorporated into the binary vector through a plasmid co-integration reaction which is mediated by the Cre/loxP site-specific recombinase system. This reaction is carried out within Agrobacterium cells. Recombinant clones, carrying the co-integrative binary plasmid form, are selected directly using the antibiotic resistance marker carried on the intermediate plasmid. This strategy facilitates production of co-integrative T-DNA binary vector forms which are appropriate for either (1) transfer to and integration within the plant genome of target and marker genes as a single T-DNA unit; (2) transfer and integration of target and marker genes as a single T-DNA unit but with a Cre/loxP facility for site-specific excision of marker genes from the plant genome; or (3) co-transfer of target and marker genes as two independent T-DNAs within a single-strain Agrobacterium system, providing the potential for segregational loss of marker genes. Received: 30 July 1998 / Accepted: 2 November 1998     

Plasmid location,cloning, and sequence analysis of the gene encoding a 27.3-kilodalton cytolytic protein fromBacillus thuringiensis subsp.morrisoni (PG-14)

A gene encoding the 27.3 kilodalton cytolytic protein toxin in the mosquitocidal isolate (PG-14) ofBacillus thuringiensis subsp.morrisoni (BTM) was cloned and sequenced, and compared with the homologous gene inB. thuringiensis subsp.israelensis (BTI). The BTM gene was determined to be located on a 140 kb plasmid by use of a synthetic 20-base nucleotide probe derived from the sequence of the BTI gene. A 9.4-kbHind III plasmid fragment containing the BTM cytolytic protein gene was cloned into the plasmid vector pUC12, and subsequently subcloned and sequenced. Comparison of the nucleotide sequence of the BTM gene with that of the homologous BTI gene revealed only a single base difference; the base at position 310 in BTM is guanine, whereas in BTI it is cytosine. This single base change results in the occurrence of alanine rather than proline at amino acid residue 82 in BTM. Analysis of the secondary structure and hydropathic profile of the BTM protein indicates that alanine at this position increases both the propensity to form an -helical structure and the hydrophobicity in the vicinity of this residue. Thus, the BTM toxin is potentially more cytolytic than the homologous protein of BTI.     

Characterization of a region of the Enterococcus faecalis plasmid pAMβ1 which enhances the segregational stability of pAMβ1-derived cloning vectors in Bacillus subtilis

The nucleotide sequence of a 2.13-kb EcoRI-HindIII, pAMβ1-derived fragment, isolated from the gram-positive cloning vector pHV1431, has been determined and shown to encode two ORFs. ORF H encodes for a protein of 23,930 Da which exhibits substantial homology to bacterial site-specific recombinases, particularly the resolvases of the gram-positive transposons Tn917 (30.3% identity) and Tn552 (31.6% identity) and the clostridial plasmid pIP404 (27.1% identity). The second ORF (I) is incomplete and encodes a polypeptide which has significant homology with Escherichia coli topoisomerase I (26.0% identity). Insertion of either the entire 2.13-kb EcoRI-HindIII fragment or a 0.73-kb EcoRI-DraI subfragment encoding only the resolvase into the pAMβ1-based cloning vector pMTL500E causes a significant enhancement of segregational stability (from 6.5 × 10⁻² to 3.0–4.0 × 10⁻³ plasmid loss per cell per generation). Improved segregational stability is mirrored by a reduction in plasmid polymerization. The introduction of a stop codon into the resolvase coding region negates its ability to promote segregational stability. It is proposed that the identified determinant stabilizes pAMβ1-based vectors in Bacillus subtilis by maintaining the plasmid population in the monomeric state, thereby reducing the chances of producing plasmid-free segregants.     

Maintenance of the bacteriocinogenic plasmid Clo DF13 in Escherichia coli cells. II. Specific recombination functions involved in plasmid maintenance

Summary Clo DF13 plasmids that are present at high copy-number in bacterial cells, such as Clo DF13 cop1 Ts, cop2 and cop3 are not stably inherited in the progeny, when certain plasmid DNA regions have been deleted. We have localized two Clo DF13 DNA regions involved in stable maintenance through accurate partitioning (par) namely parA, located between 71% and 72% and parB, located between 45% and 50% on the Clo DF13 genome. The instability of these cop plasmids which is accompanied by the formation of high amounts of multimeric DNA molecules, could be abolished by the insertion of transposon Tn901 into the plasmid genome. In particular that part of Tn901, that encodes for the site-specific recombination/ resolution system, appeared to be essential for stabilizing plasmid molecules. Wild-type parA^- and/or parB^- Clo DF13 plasmids, in contrast to cop mutants lacking these regions, are stably maintained during subsequent cell division, indicating that other (host specified) functions contribute to plasmid stability. Analysis of the role of host recombination systems in plasmid partitioning revealed that the recA function has no influence and recBC contributes only weakly to plasmid stability. With respect to the recE pathway, however, we found that in a recE proficient host all plasmids, even those lacking parA and/or parB, are stably maintained, indicating that the function of parA and parB can be replaced not only by the site-specific resolution functions of transposon Tn901, but also by the recE system. The possible role of plasmid specified and host specified functions in plasmid partitioning will be discussed.     

Rearrangements in the staphylococcal β-lactamase-encoding plasmid, plP1066, including a DNA inversion that generates two alternative transposons

The plasmid plP1066, harboured by a methicillin-resistant Staphylococcus aureus strain isolated in France, carries genes specifying β-lactamase. This plasmid undergoes numerous rearrangements. One of these was an insertion, between the genes binR and sin encoding resolvases, of a 16 kb element which displayed the characteristic features of a transposon. This putative transposon, named Tn 5404 , carried genes encoding proteins involved in its transposition, as well as a resolution system, which were indistinguishable from those of the S. aureus transposon Tn 552 . These were: p480 encoding a probable transposase, p271 encoding a putative ATP-binding protein, binL encoding a resolvase, and a resolution site, resL . In addition, Tn 5404 carried aminoglycoside-resistance genes ( aphA, str ) and the insertion sequence IS 1181 . Tn 5404 contained at its termini 116 bp imperfect inverted repeats, similar to those of Tn 552 , and was flanked by 6 bp direct repeats. Insertion of Tn 5404 close to resR and to the structural and regulatory β-lactamase genes ( blaZ, blal, blaR1 ) of plP1066, generated a 3.5 kb invertible segment flanked by inversely repeated resolution sites ( resR, resL ). This invertible segment, which carried p480 , p271 and binL , generated Tn 552 or Tn 5404 , depending on its orientation. Thus, these two transposons share their transposition and resolution systems.     

Purification and DNA binding of the D protein, a putative resolvase of the F-factor of Escherichia coli

Summary The D protein encoded by plasmid mini-F promotes resolution of plasmid cointegrates or dimers of the F-factor or mini-F. In addition, two rfsF sequences are essential for this site-specific, recA-independent recombination event. The D gene was cloned into an expression vector and the gene product was overproduced in Escherichia coli and purified to homogeneity. The sequence of the N-terminus of the D protein was determined, thus permitting identification of the correct translational start codon in the nucleotide sequence that results in a 29.6 kDa protein. The binding site for the purified D protein is located within the mini-F NcoIHpaI DNA fragment (192 bp). Binding seems to be affected by DNA methylation, since the protein did not bind to DNA isolated from a dam mutant of E. coli. The binding site, which is a region of approximately 28 bp and is located 160 by downstream of the rfsF site, was identified by DNase I footprinting using fluorescence labelled DNA.     

Involvement of the inverted repeat of the yeast 2-micron plasmid in Flp site-specific and RAD52-dependent homologous recombination

Site-specific recombination within the Saccharomyces cerevisiae 2-micron DNA plasmid is catalyzed by the Flp recombinase at specific Flp Recognition Target (FRT) sites, which lie near the center of two precise 599-bp Inverted Repeats (IRs). However, the role of IR DNA sequences other than the FRT itself for the function of the Flp reaction in vivo is not known. In the present work we report that recombination efficiency differs depending on whether the FRT or the entire IR serves as the substrate for Flp. We also provide evidence for the involvement of the IR in RAD52-dependent homologous recombination. In contrast, the catalysis of site-specific recombination between two FRTs does not require the function of RAD52. The efficiency of Flp site-specific recombination between two IRs cloned in the same orientation is about one hundred times higher than that obtained when only the two FRTs are present. Moreover, we demonstrate that a single IR can activate RAD52-dependent homologous recombination between two flanking DNA regions, providing new insights into the role of the IR as a substrate for recombination and a new experimental tool with which to study the molecular mechanism of homologous recombination. Received: 14 June 1999 / Accepted: 3 November 1999     

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.     

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.     

The region essential for efficient autonomous replication of pSa in Escherichia coli

Summary Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication. By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined. A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced. A 36 kDa RepA protein was encoded in the region essential for replication. Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences. Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene. Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative. Another open reading frame (ORF) was found in the region required for the stability of the plasmid. The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins. A possible role of this putative protein in stable maintenance of the plasmid is discussed.     

Sequence Analysis of a Small Cryptic Plasmid Isolated from Streptococcus suis Serotype 2

A small cryptic plasmid designated pSSU1 was isolated from Streptococcus suis serotype 2 strain DAT1. The complete sequence of pSSU1 was 4975 bp and contained six major open reading frames (ORFs). ORF1 and ORF2 encode for proteins highly homologous to CopG and RepB of the pMV158 family, respectively. ORF5 encodes for a protein highly homologous to Mob of pMV158. ORF4 encodes for a protein highly homologous to orf3 of pVA380-1 of S. ferus, but its function is unknown. There was no similarity between ORF3 and ORF6 and other protein sequences. In this plasmid, the ORF1 (CopG protein) was preceded by two multiples of direct repeat and the conserved nucleotides that could be the double-strand origin (DSO) of rolling circle replication (RCR) mechanism. The ORF5 (Mob protein) was followed by a potential hairpin loop that could be the single-strand origin (SSO) of RCR mechanism. The sequence, which was complementary to the leader region of Rep mRNA, was homologous to the countertranscribed RNA (ctRNA) of pLS1. Moreover, a 5-amino acid conserved sequence was found in C terminal of Rep and putative Rep proteins of several pMV158 family plasmids. These observations suggest that this plasmid replicates by use of the rolling circle mechanism. Received: 26 June 1999 / Accepted: 10 August 1999     

Homologous Recombination in Zebrafish ES Cells

Targeted insertion of a plasmid by homologous recombination was demonstrated in zebrafish ES cell cultures. Two selection strategies were used to isolate ES cell colonies that contained targeted plasmid insertions in either the no tail or myostatin I gene. One selection strategy involved the manual isolation of targeted cell colonies that were identified by the loss of fluorescent protein gene expression. A second strategy used the diphtheria toxin A-chain gene in a positive-negative selection approach. Homologous recombination was confirmed by PCR, sequence and Southern blot analysis and colonies isolated using both selection methods were expanded and maintained for multiple passages. The results demonstrate that zebrafish ES cells have potential for use in a cell-mediated gene targeting approach.     

TKO'ed: Lox,stock and barrel

The generation of panels of mutant mice by homologous recombination has greatly increased the ability to assess the function of particular gene products in vivo. The ability to control the developmental stage, the tissue and the nature of the mutation would be an important innovation. A recent report⁽¹⁾ demonstrates that the conservative site-specific recombination of bacteriophage P1, namely Cre-lox, can be used successfuly in combination with homologous recombination to generate temporal-and cell-restricted mutations in vivo. The technical advance allows a greater flexibility in gene targeting and will have a significant impact on how complex gene functions are studied in vivo.     

Characterization of the Novel Fusobacterium nucleatum Plasmid pKH9 and Evidence of an Addiction System

Fusobacterium nucleatum is an important oral anaerobic pathogen involved in periodontal and systemic infections. Studies of the molecular mechanisms involved in fusobacterial virulence and adhesion have been limited by lack of systems for efficient genetic manipulation. Plasmids were isolated from eight strains of F. nucleatum. The smallest plasmid, pKH9 (4,975 bp), was characterized and used to create new vectors for fusobacterial genetic manipulation. DNA sequence analysis of pKH9 revealed an open reading frame (ORF) encoding a putative autonomous rolling circle replication protein (Rep), an ORF predicted to encode a protein homologous to members of the FtsK/SpoIIIE cell division-DNA segregation protein family, and an operon encoding a putative toxin-antitoxin plasmid addiction system (txf-axf). Deletion analysis localized the pKH9 replication region in a 0.96-kbp fragment. The pKH9 rep gene is not present in this fragment, suggesting that pKH9 can replicate in fusobacteria independently of the Rep protein. A pKH9-based, compact Escherichia coli-F. nucleatum shuttle plasmid was constructed and found to be compatible with a previously described pFN1-based fusobacterial shuttle plasmid. Deletion of the pKH9 putative addiction system (txf-axf) reduced plasmid stability in fusobacteria, indicating its addiction properties and suggesting it to be the first plasmid addiction system described for fusobacteria. pKH9, its genetic elements, and its shuttle plasmid derivatives can serve as useful tools for investigating fusobacterial properties important in biofilm ecology and pathogenesis.     

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.