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.     

Mode of insertion of the broad-host-range plasmid RP4 and its derivatives into the chromosome of Myxococcus xanthus

The mode of insertion of the broad-host-range plasmid RP4 into the chromosome of Myxococcus xanthus strain DZ1 has been analyzed. The plasmid integrated in numerous sites of the chromosome and generated insertional mutations. There is a hot spot of integration located between 31.5 and 34.5 kb clockwise from the EcoRI site of the plasmid. In the absence of this segment the insertion can, however, take place, but much less efficiently. The presence of transposable elements on the plasmid decreases severely the insertion frequency. Once integrated, RP4 could be transferred back to Escherichia coli, either by precise excision or with a segment of the Myxococcus chromosome. The role of site-specific recombination in RP4 integration is discussed.     

Genetic system for reversible integration of DNA constructs and lacZ gene fusions into the Escherichia coli chromosome

A plasmid system for site-specific integration into and excision and recovery of gene constructs and lacZ gene fusions from the Escherichia coli chromosome was developed. Plasmid suicide vectors utilizing the origin of replication of R6K plasmids and containing the attP sequence of bacteriophage lambda, multiple cloning site, and antibiotic resistance markers facilitate reversible integration into the E. coli chromosome by site-specific recombination. Additional vectors permit construction of lacZ gene fusions in three possible reading frames for recombination with the bacterial chromosome. These suicide vectors can be propagated in newly constructed E. coli strains that harbor different pir alleles. Two helper plasmids that encode the necessary gene products for integration (Int) and excision (Int and Xis) were also constructed. This plasmid system was shown to be a reliable and efficient means to integrate and subsequently recover plasmids from the E. coli attB site.     

Marker-free site-specific gene integration in rice based on the use of two recombination systems

Transgene integration mediated by heterologous site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. This approach of plant transformation generates a precise site-specific integration (SSI) structure consisting of a single copy of the transgene construct. As a result, stable transgene expression correlated with promoter strength and gene copy number is observed among independent transgenic lines and faithfully transmitted through subsequent generations. Site-specific integration approaches use selectable marker genes, removal of which is necessary for the implementation of this approach as a biotechnology application. As SSR systems are also excellent tools for excising marker genes from transgene locus, a molecular strategy involving gene integration followed by marker excision, each mediated by a distinct recombination system, was earlier proposed. Experimental validation of this approach is the focus of this work. Using FLPe-FRT system for site-specific gene integration and heat-inducible Cre-lox for marker gene excision, marker-free SSI lines were developed in the first generation itself. More importantly, progeny derived from these lines inherited the marker-free locus, indicating efficient germinal transmission. Finally, as the transgene expression from SSI locus was not altered upon marker excision, this method is suitable for streamlining the production of marker-free SSI lines.     

Structural analysis of plasmid and chromosomal loci involved in site-specific excision and integration of the SLP1 element of Streptomyces coelicolor.

SLP1int (integrated [int] form of Streptomyces lividans plasmid 1 [SLP1]) is a Streptomyces coelicolor A3(2) transmissible sequence capable of autonomous replication as well as site-specific integration into and excision from the S. coelicolor chromosome. We report here that the plasmid and chromosomal loci involved in the integration of SLP1 and the two loci at which the recombination occurs during excision all share at least 111 base pairs of a 112-base-pair DNA sequence. Recombinational cross-over during integration or excision occurred nonrandomly within the common att sequence at or near a 25-base-pair inverted repeat. We suggest that chromosomally integrated plasmidogenic segments such as SLP1int may be involved in the acquisition and structural organization of genes encoding the diverse metabolic capabilities observed in different streptomycetes.     

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.     

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     

Site-specific recombination in human cells catalyzed by the wild-type integrase protein of coliphage HK022

The activity of the Integrase (Int) protein encoded by coliphage HK022 was tested in a human cell culture. Plasmids were constructed as substrates that carry the sites of the integration reaction (attP and attB) or the sites of excision (attL and attR). The site-specific recombination reactions were monitored in cis and in trans configurations by the expression of the green fluorescent protein (GFP) as a reporter. Cells cotransfected with the substrate plasmid(s) and with a plasmid that expresses the wild-type Int show efficient integration as well as excision in both configurations. The wild-type Int was active in the human cells without the need to supply the accessory proteins integration host factor (IHF) and excisionase (Xis) that are indispensable for the reaction in the bacterial host.     

Determinants that target the integrase of phage HK022 into the mammalian nucleus

The integrase (Int) protein of coliphage HK022 catalyzes the site-specific integration and excision of the phage into and from its Escherichia coli host chromosome. Int expressed from a plasmid in COS1 monkey cells is localized in the nucleus, as is a fusion protein between Int and the green fluorescent protein (GFP). Mutation analysis of the GFP-Int fusion has revealed in Int two regions of positively charged amino acid residues that cooperate in the nuclear localization. One region harbors residues Arg90 and Arg93. The other, which spans residues 307-340 belongs to the catalytic domain of Int, is rich in basic residues and is strongly conserved within the Int protein family. Being localized in the nucleus renders Int of HK022 as a potential recombinase for site-specific gene manipulations in mammals.     

A method to maintain introduced DNA sequences stably and safely on the bacterial chromosome: application of prophage integration and subsequent designed excision

By application of prophage integration and subsequent intended excision, a method to maintain an introduced DNA sequence stably onto a bacterial chromosome has been proposed. Recently-constructed integration plasmids using Campbell-type prophage integration system in Lactobacillus casei strain Shirota and its temperate phage phi FSW was modified for this purpose and a chloramphenicol (Cm)-resistance gene was used as a model passenger DNA. On the integration plasmid having an erythromycin (Em)-resistance gene as a selection marker, N- and C-terminally-truncated Cm-resistance genes were inserted into both sides of the attP of phi FSW, within which the site-specific recombination took place with the attB of phi FSW on the recipient chromosome through the phi FSW integrase. Primary integrants of the modified plasmid (integration-excision vector) exhibiting Em-resistant and Cm-sensitive phenotype generated Em-sensitive and Cm-resistant derivatives under the nonselective conditions. Sequence analyses showed that one copy of the complete Cm-resistance gene resided at the attachment site on the host chromosome and the other vector-derived sequences were excised probably by endogenous homologous recombination in the host cells to derive final integrants. The Cm-resistant phenotype of the final integrants was stable for more than 50 generations under non-selective conditions. Frequency of the homologous recombination suggests that negative selection is also adoptable. Thus, this method using the integration-excision vector gives a stable and safe derivatives of the strain and is likely to be applicable to various bacteria, since Campbell-type prophage integration system and homologous recombination are prevalent among bacteria.     

Attachment site of the genetic element e14.

The Escherichia coli K-12 genetic element, e14, contains a 216-base-pair region that is homologous to a portion of the host chromosome. This region serves as the integration site for the element. The 216-base-pair homology is interrupted by 28 mismatches distributed through the sequence. The actual integrative crossover occurs within the first 11 base pairs from one end of the region. To test factors which affect e14 site-specific recombination, we cloned the attachment sites of free e14 and the host chromosome into the same plasmid. The cloned attachment sites recombined intramolecularly in a process that required the presence of a chromosomal copy of e14 in the host cell as well as the induction of SOS. Recombination events that mimicked both integration and excision occurred under the same conditions and to roughly the same extent.     

Localization and nucleotide sequences of genes mediating site-specific recombination of the SLP1 element in Streptomyces lividans.

SLP1 is a 17.2-kbp genetic element indigenous to the Streptomyces coelicolor chromosome. During conjugation, SLP1 can undergo excision and subsequent site-specific integration into the chromosomes of recipient cells. We report here the localization, nucleotide sequences, and initial characterization of the genes mediating these recombination events. A region of SLP1 adjacent to the previously identified site of integration, attP, was found to be sufficient to promote site-specific integration of an unrelated Streptomyces plasmid. Nucleotide sequence analysis of a 2.2-kb segment of this region reveals two open reading frames that are adjacent to and transcribed toward the attP site. One of these, the 1,365-bp int gene of SLP1, encodes a predicted 50.6-kDa basic protein having substantial amino acid sequence similarity to a family of site-specific recombinases that includes the Escherichia coli bacteriophage lambda integrase. A linker insertion in the 5' end of the cloned int gene prevents integration, indicating that Int is essential for promoting integration. An open reading frame (orf61) lying immediately 5' to int encodes a predicted 7.1-kDa basic peptide showing limited sequence similarity to the excisionase (xis) genes of other site-specific recombination systems.     

High efficiency, site-specific excision of a marker gene by the phage P1 cre-loxP system in the yellow fever mosquito, Aedes aegypti

The excision of specific DNA sequences from integrated transgenes in insects permits the dissection in situ of structural elements that may be important in controlling gene expression. Furthermore, manipulation of potential control elements in the context of a single integration site mitigates against insertion site influences of the surrounding genome. The cre-loxP site-specific recombination system has been used successfully to remove a marker gene from transgenic yellow fever mosquitoes, Aedes aegypti. A total of 33.3% of all fertile families resulting from excision protocols showed evidence of cre-loxP-mediated site-specific excision. Excision frequencies were as high as 99.4% within individual families. The cre recombinase was shown to precisely recognize loxP sites in the mosquito genome and catalyze excision. Similar experiments with the FLP/FRT site-specific recombination system failed to demonstrate excision of the marker gene from the mosquito chromosomes.     

Integration and excision of pMC7105 in Pseudomonas syringae pv. phaseolicola: involvement of repetitive sequences.

The site for integration of pMC7105 into the chromosome of Pseudomonas syringae pv. phaseolicola has been mapped to a 2.6-kilobase-pair (kb) Bg/II-EcoRI fragment on this 150-kb indigenous plasmid. Selected excision plasmids resulting from imprecise excision of pMC7105 were used to identify one of the plasmid-chromosome juncture fragments and to characterize the mechanism of recombination from the chromosome. A 14.2-kb BamHI plasmid-chromosome juncture fragment has been identified in pEX8060 (234 kb), an excision plasmid which carries approximately 90 kb of chromosomal sequences to the left of the site of integration. This fragment contains a portion of the 2.6-kb Bg/II-EcoRI fragment as well as chromosomal sequences. Blot hybridization with a probe made from selected fragments of pMC7105 revealed three distinct repetitive sequences, RS-I, RS-II, and RS-III, on this plasmid. The 2.6-kb fragment, to which the site of integration maps, also contains RS-II. Five copies of RS-II are present in pMC7105, and more than 20 copies are present in the chromosome. Eight small excision plasmids were shown to result from recombination among fragments of pMC7105 that contain common repetitive sequences. The results indicate that integration and excision of pMC7105 occur through general recombination at homologous repetitive sequences.     

The chromosomal integration site for the Streptomyces plasmid SLP1 is a functional tRNATyr gene essential for cell viability

The genetic element SLP1 exists in nature as a single DNA segment integrated into the genome of Streptomyces coelicolor. Upon mating with Streptomyces lividans, a closely related species, SLP1 undergoes precise excision from its chromosomal site and is transferred into the recipient where it integrates chromosomally. Previous work has shown that integration and excision involve site-specific recombination between a chromosomal site, attB, and a virtually identical sequence, attP, on SLP1. We demonstrate here by means of gene replacement that a tRNA(Tyr) sequence that overlaps part of the attB site of S. lividans is both biologically functional and essential for cell viability. The requirement for this tRNA gene has been used to stabilize the inheritance of a segrationally unstable plasmid in cells lacking a chromosomal attB site. The evolution of an essential DNA locus as an attachment site for a chromosomally integrating genetic element represents a novel mechanism of biological adaptation.     

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.     

The sre gene (ORF469) encodes a site-specific recombinase responsible for integration of the R4 phage genome.

The sre gene (ORF469) of the R4 phage encodes a protein similar to the resolvase-DNA invertase family proteins. Insertional gene disruption of sre prevented a lysogen from entering the lytic cycle, implying that Sre protein is a site-specific recombinase needed for excision of the R4 prophage genome (M. Matsuura, T. Noguchi, T. Aida, M. Asayama, H. Takahashi, and M. Shirai, J. Gen. Appl. Microbiol. 41:53-61, 1995). To determine whether this sre gene is also necessary for the integration reaction, we studied its function by integration plasmid analysis. When deletions, frameshifts, and site-directed mutations that caused an amino acid substitution of Ser-17 for Ala were introduced into the sre structural gene, transformation efficiency of Streptomyces parvulus 2297 with these plasmid DNAs was severely reduced. However, an adenine insertion just before the possible initiation codon of the sre gene did not significantly decrease the efficiency. These data suggest that the Sre protein is a site-specific recombinase responsible for integration of the R4 phage genome.     

Site-Specific Recombination of Bacteriophage P22 Does Not Require Integration Host Factor

Site-specific recombination by phages lambda and P22 is carried out by multiprotein-DNA complexes. Integration host factor (IHF) facilitates lambda site-specific recombination by inducing DNA bends necessary to form an active recombinogenic complex. Mutants lacking IHF are over 1,000-fold less proficient in supporting lambda site-specific recombination than wild-type cells. Although the attP region of P22 contains strong IHF binding sites, in vivo measurements of integration and excision frequencies showed that infecting P22 phages can perform site-specific recombination to its maximum efficiency in the absence of IHF. In addition, a plasmid integration assay showed that integrative recombination occurs equally well in wild-type and ihfA mutant cells. P22 integrative recombination is also efficient in Escherichia coli in the absence of functional IHF. These results suggest that nucleoprotein structures proficient for recombination can form in the absence of IHF or that another factor(s) can substitute for IHF in the formation of complexes.     

Site-specific deletions of chromosomally located DNA segments with the multimer resolution system of broad-host-range plasmid RP4.

The multimer resolution system (mrs) of the broad-host-range plasmid RP4 has been exploited to develop a general method that permits the precise excision of chromosomal segments in a variety of gram-negative bacteria. The procedure is based on the site-specific recombination between two directly repeated 140-bp resolution (res) sequences of RP4 effected by the plasmid-borne resolvase encoded by the parA gene. The efficiency and accuracy of the mrs system to delete portions of chromosomal DNA flanked by res sites was monitored with hybrid mini-Tn5 transposons in which various colored (beta-galactosidase and catechol 2,3 dioxygenase) or luminescent (Vibrio harveyi luciferase) phenotypic markers associated to res sequences were inserted in the chromosome of the target bacteria and exposed in vivo to the product of the parA gene. The high frequencies of marker excision obtained with different configurations of the parA expression system suggested that just a few molecules of the resolvase are required to achieve the site-specific recombination event. Transient expression of parA from a plasmid unable to replicate in the target bacterium was instrumental to effect differential deletions within complex hybrid transposons inserted in the chromosome of Pseudomonas putida. This strategy permits the stable inheritance of heterologous DNA segments virtually devoid of the sequences used initially to select their insertion.     

Cre/lox位点特异重组系统在转基因植物中的应用

位点特异重组系统由重组酶和相应的重组酶识别位点组成,通过两者间的相互作用,实现外源基因精确整合与切除等一系列遗传操作.主要可分为Cre/lox系统、FLP/frt系统、R/RS系统和Gin/gix系统.目前,研究最充分应用最广泛的位点特异重组系统为Cre/lox系统.此系统为位点特异重组系统家族中的一员,由38.5kDCre重组酶和34bplox位点组成,最早被应用于动物转基因研究,包括基因敲除、基因激活、基因易位等.近年来,随着研究的深入,Cre/lox系统被逐步应用到植物研究中,并在诸多领域取得重大进展.本文总结归纳了Cre/lox系统在定点整合、定点切除以及叶绿体转化等方面的最新研究成果,旨在为利用Cre/lox系统构建环境安全和高效表达的植物遗传转化体系提供参考.