Cell-type specificity of the Anabaena fdxN-element rearrangement requires xisH and xisl

The fdxN element, along with two other DNA elements, is excised from the chromosome during heterocyst differentiation in Anabaena sp. strain PCC 7120. Previous work showed that rearrangement of the fdxN element requires the xisF gene, which encodes a site-specific recombinase, and suggested that at least one other heterocyst-specific factor is involved. Here we report that the xisH and xisl genes are necessary for the heterocyst-specific excision of the fdxN element. Deletion of a 3.2 kb region downstream of the xisF gene blocked the fdxN-element rearrangement in hetero-cysts. The 3.2 kb deletion was complemented by the two overlapping genes xisH and xisl. Interestingly, extra copies of xlsHI on a replicating plasmid resulted in the xisF-dependent excision of the fdxN element in vegetative cells. Therefore, xisHI are involved in the control of cell-type specificity of the fdxN rearrangement. The xisHI genes had no effect on the two other DNA rearrangements. The xisHl-induced excision of the fdxN element produced strains lacking the element and demonstrates that the 55 kb element contains no essential genes. xisH and xisl do not show similarity to any known genes.     

Expression of the Anabaena sp. strain PCC 7120 xisA gene from a heterologous promoter results in excision of the nifD element.

An 11-kilobase-pair element interrupts the nifD gene in vegetative cells of Anabaena sp. strain PCC 7120. The nifD element normally excises only from the chromosomes of cells that differentiate into nitrogen-fixing heterocysts. The xisA gene contained within the element is required for the excision. Shuttle vectors containing the Escherichia coli tac consensus promoter fused to various 5' deletions of the xisA gene were constructed and conjugated into Anabaena sp. strain PCC 7120 cells. Some of the expression plasmids resulted in excision of the nifD element in a high proportion of vegetative cells. Excision of the element required deletion of an xisA 5' regulatory region which presumably blocks expression in Anabaena sp. strain PCC 7120 vegetative cells but not in E. coli. Strains lacking the nifD element grew normally in medium containing a source of combined nitrogen and showed normal growth and heterocyst development in medium lacking combined nitrogen. The xisA gene was shown to be the only Anabaena gene required for the proper rearrangement in E. coli of a plasmid containing the borders of the nifD element.     

Identification and sequence of a gene required for a developmentally regulated DNA excision in Anabaena

Vegetative cells of the cyanobacterium Anabaena contain an 11 kb DNA element within the coding region of the nifD gene. This element is excised by site-specific recombination between directly repeated 11 bp sequences at each of its ends during differentiation of nitrogen-fixing cells called heterocysts. Site-specific recombination, leading to the same rejoined nifD gene, was observed during propagation in E. coli of a fragment containing the 11 kb element and flanking sequences. An assay for excision of the element in E. coli was developed, based on mini-Mu-lac transposition into the element. Since the 11 kb element lacks an origin of replication, its excision results in loss of lac and conversion of blue colony-forming cells to white on X-gal plates. Insertion and deletion mutagenesis identified a region of the element needed for excision. Mutations in this region could be complemented by a 6 kb fragment containing an open reading frame that runs counter to those of the nif genes, beginning 240 bp from the recombination site.     

Deletion of a 55-kilobase-pair DNA element from the chromosome during heterocyst differentiation of Anabaena sp. strain PCC 7120.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 produces terminally differentiated heterocysts in response to a lack of combined nitrogen. Heterocysts are found approximately every 10th cell along the filament and are morphologically and biochemically specialized for nitrogen fixation. At least two DNA rearrangements occur during heterocyst differentiation in Anabaena sp. strain PCC 7120, both the result of developmentally regulated site-specific recombination. The first is an 11-kilobase-pair (kb) deletion from within the 3' end of the nifD gene. The second rearrangement occurs near the nifS gene but has not been completely characterized. The DNA sequences found at the recombination sites for each of the two rearrangements show no similarity to each other. To determine the topology of the rearrangement near the nifS gene, cosmid libraries of vegetative-cell genomic DNA were constructed and used to clone the region of the chromosome involved in the rearrangement. Cosmid clones which spanned the DNA separating the two recombination sites that define the ends of the element were obtained. The restriction map of this region of the chromosome showed that the rearrangement was the deletion of a 55-kb DNA element from the heterocyst chromosome. The excised DNA was neither degraded nor amplified, and its function, if any, is unknown. The 55-kb element was not detectably transcribed in either vegetative cells or heterocysts. The deletion resulted in placement of the rbcLS operon about 10 kb from the nifS gene on the chromosome. Although the nifD 11-kb and nifS 55-kb rearrangements both occurred under normal aerobic heterocyst-inducing conditions, only the 55-kb excision occurred in argon-bubbled cultures, indicating that the two DNA rearrangements can be regulated differently.     

Excision of an 11-kilobase-pair DNA element from within the nifD gene in anabaena variabilis heterocysts.

The 3' region of the Anabaena variabilis nifD gene contains an 11-kilobase-pair element which is excised from the chromosome during heterocyst differentiation. We have sequenced the recombination sites which border the element in vegetative cells and the rearranged heterocyst sequences. In vegetative cells, the element was flanked by 11-base-pair direct repeats which were identical to the repeats present at the ends of the nifD element in Anabaena sp. strain PCC 7120 (Anabaena strain 7120). Although Anabaena strain 7120 and A. variabilis are quite distinct in many ways, the overall sequence similarity between the two strains for the regions sequenced was 96%. Like the Anabaena strain 7120 element, the A. variabilis element was excised in heterocysts to produce a functional nifD gene and a free circularized element which was neither amplified nor degraded. The Anabaena strain 7120 xisA gene is located at the nifK-proximal end of the nifD element and is required for excision of the element in heterocysts. The A. variabilis element also contained an xisA gene which could complement a defective Anabaena strain 7120 xisA gene. A. variabilis did not contain the equivalent of the Anabaena strain 7120 fdxN 55-kilobase-pair element.     

The integrated conjugative plasmid pSAM2 of Streptomyces ambofaciens is related to temperate bacteriophages.

Streptomyces ambofaciens ATCC23877 and derivatives contain the 11-kb element pSAM2 present in an integrated state or as a free and integrated plasmid. This element, able to integrate site-specifically in the genome of different Streptomyces species, is conjugative and mobilizes chromosomal markers. Besides these plasmid functions, we have shown that the site-specific recombination system of pSAM2 presents strong similarities with that of several temperate phages. The integration event is promoted by a site-specific recombinase of the integrase family. The int gene encoding this integrase is closely linked to the plasmid attachment site (attP). A small open reading frame (ORF) overlaps the int gene and the predicted protein exhibits similarities with Xis proteins involved in phages excision. The integrated copy of pSAM2 in strain ATCC23877 is flanked by att sequences (attL and attR). Another att sequence (attX) is present in this strain and attX and attL are the boundaries of a 42-kb fragment (xSAM1) absent, as well as pSAM2, from S.ambofaciens DSM40697. Sequences partially similar to pSAM2 int gene are found near the chromosomal integration zone in both S.ambofaciens strains. The possible origin of pSAM2, an element carrying plasmid as well as phage features, is discussed.     

Developmental rearrangement of cyanobacterial nif genes: nucleotide sequence, open reading frames, and cytochrome P-450 homology of the Anabaena sp. strain PCC 7120 nifD element.

An 11-kbp DNA element of unknown function interrupts the nifD gene in vegetative cells of Anabaena sp. strain PCC 7120. In developing heterocysts the nifD element excises from the chromosome via site-specific recombination between short repeat sequences that flank the element. The nucleotide sequence of the nifH-proximal half of the element was determined to elucidate the genetic potential of the element. Four open reading frames with the same relative orientation as the nifD element-encoded xisA gene were identified in the sequenced region. Each of the open reading frames was preceded by a reasonable ribosome-binding site and had biased codon utilization preferences consistent with low levels of expression. Open reading frame 3 was highly homologous with three cytochrome P-450 omega-hydroxylase proteins and showed regional homology to functionally significant domains common to the cytochrome P-450 superfamily. The sequence encoding open reading frame 2 was the most highly conserved portion of the sequenced region based on heterologous hybridization experiments with three genera of heterocystous cyanobacteria.     

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.     

Integration and excision by the large serine recombinase phiRv1 integrase

The Mycobacterium tuberculosis prophage-like element phiRv1 encodes a site-specific recombination system utilizing an integrase of the serine recombinase family. Recombination occurs between a putative attP site and the host chromosome, but is unusual in that the attB site lies within a redundant repetitive element (REP13E12) of which there are seven copies in the M. tuberculosis genome; four of these elements contain attB sites suitable for phiRv1 integration in vivo. Although the mechanism of directional control of large serine integrases is poorly understood, a recombination directionality factor (RDF) has been identified that is required for phiRv1 integrase-mediated excisive recombination in vivo. Here we describe defined in vitro recombination reactions for both phiRv1 integrase-mediated integration and excision and show that the phiRv1 RDF is not only required for excision but inhibits integrative recombination; neither reaction requires DNA supercoiling, host factors, or high-energy cofactors. Integration, excision and excise-mediated inhibition of integration require simple substrates sites, indicating that the control of directionality does not involve the manipulation of higher-order protein-DNA architectures as described for the tyrosine integrases.     

Insertion of bacteriophage phiFSW into the chromosome of Lactobacillus casei strain Shirota (S-1): characterization of the attachment sites and the integrase gene

The integrase gene (int) on the genome of φFSW, which is a temperate bacteriophage of Lactobacillus casei strain Shirota (formerly denoted as S-1), and the four attachment sites on the genomes of the phage and its host were characterized by sequencing. The φFSW integrase was found to belong to the integrase family of site-specific tyrosine recombinase. The attachment sites shared a 40bp common core within which an integrative site-specific recombination occurs. The common core was flanked on one side by an additional segment of high sequence similarity. An integration plasmid, consisting of int, the phage attachment site (attP), and a selectable marker, inserted stably into the bacterial attachment site (attB) within the common core, as did the complete prophage genome at a frequency of more than 10(3)/microg of plasmid DNA. This plasmid was used as a test system for a preliminary mutational analysis of int and attP. The attB common core was located within and near the end of an open reading frame that appears to encode a homolog to glucose 6-phosphate isomerase, an enzyme of the glycolytic pathway. It is unlikely that the prophage integration inactivates this protein, since a change of only the C-terminal amino acid is predicted because of the sequence similarity between attP and attB.     

Site-specific cassette exchange and germline transmission with mouse ES cells expressing phiC31 integrase

Currently two site-specific recombinases are available for engineering the mouse genome: Cre from P1 phage and Flp from yeast. Both enzymes catalyze recombination between two 34-base pair recognition sites, lox and FRT, respectively, resulting in excision, inversion, or translocation of DNA sequences depending upon the location and the orientation of the recognition sites. Furthermore, strategies have been designed to achieve site-specific insertion or cassette exchange. The problem with both recombinase systems is that when they insert a circular DNA into the genome (trans event), two cis-positioned recognition sites are created, which are immediate substrates for excision. To stabilize the trans event, functional mutant recognition sites had to be identified. None of the systems, however, allowed efficient selection-free identification of insertion or cassette exchange. Recently, an integrase from Streptomyces phage phiC31 has been shown to function in Schizosaccharomyces pombe and mammalian cells. This enzyme recombines between two heterotypic sites: attB and attP. The product sites of the recombination event (attL and attR) are not substrates for the integrase. Therefore, the phiC31 integrase is ideal to facilitate site-specific insertions into the mammalian genome.     

卵母细胞特异表达fC31整合酶载体pZP3-INT的构建及其在小鼠卵母细胞中的表达

链霉菌噬菌体fC31整合酶是一种位点特异性重组酶(Site-specific recombinase, SSR), 可介导链霉菌噬菌体attP位点(Phage attachment site)和链霉菌基因组attB位点(Bacterial attachment site)间的单向重组。为探讨它能否应用于卵母细胞特定基因的重组, 文章采用卵巢针刺取卵法采集生发泡(GV)期小鼠卵母细胞, 将卵透明带糖蛋白3(ZP3)启动子驱动的fC31整合酶表达载体pZP3-INT和检测fC31整合酶位点特异性重组功能的重组质粒载体pBCPB⁺, 通过显微注射导入到小鼠卵母细胞中。培养48 h后, RT-PCR检测fC31整合酶mRNA表达以及PCR检测pBCPB⁺载体发生重组的情况。结果表明: 载体pZP3-INT在卵母细胞中表达fC31 整合酶mRNA; 并且pBCPB⁺载体发生了位点特异性重组, 提示fC31整合酶在卵母细胞中可以介导位点特异性重组反应。     

Mycobacteriophage Bxb1 integrates into the Mycobacterium smegmatis groEL1 gene

Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis and forms stable lysogens in which the Bxb1 genome is integrated into the host chromosome. Bxb1 encodes an integrase of the large serine recombinase family that catalyses integration and excision of the Bxb1 genome. We show here that Bxb1 integrates into a chromosomal attB site located within the 3' end of the groEL1 gene such that integration results in alteration of the C-terminal 21 amino acid residues. An integration-proficient plasmid vector containing the Bxb1 integrase gene and flanking DNA sequences efficiently transforms M. smegmatis via integration at attB. Bxb1-integrated recombinants are stable and fully compatible with L5 integration vectors. Strand exchange occurs within an 8 bp common core sequence present in attB and within an attP site situated immediately upstream of the phage integrase gene. Establishment of a defined in vitro system for Bxb1 integration shows that recombination occurs efficiently without requirement for high-energy cofactors, divalent metals, DNA supercoiling or additional proteins.     

Genetic surgery in fungi: employing site-specific recombinases for genome manipulation

Site-specific recombination mediates the rearrangement of nucleic acids by the virtue of an recombinase acting on specific recognition sequences. Recombining activities belong either to the tyrosine- or serine-type group, based on the presence of specific residues in the catalytic centre, which can be further subdivided into families due to additional criteria. The most prominent systems are the λ phage integrase acting on att sites; the Cre recombinase from bacteriophage P1 with its loxP attachment sites; the FLP/FTR system of fungal origin, where it is required for 2-μm plasmid replication/amplification in yeast; and the prokaryotic β-recombinase that recombines six sites specifically in cis. Each of these has been exploited in fungal hosts of biotechnological, medical or general relevance, mainly for cloning projects, approaches of gene targeting, genome modification or screening purposes. With their precise and defined mode of action are site-specific recombination systems eminently suited for genetic tasks in fungi, like they are executed in functional studies at high throughput or modern approaches of synthetic biology.     

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.     

Rearrangements of nitrogen fixation (nif) genes in the heterocystous cyanobacteria

In the vegetative cells of heterocystous cyanobacteria, such asAnabaena, two Operons harbouring the nitrogen fixaton (nif) genes contain two separate intervening DNA elements resulting in the dispersion of genes and impaired gene expression. A 11 kb element disrupts thenifD gene in thenifH, D-K operon. It contains a 11 bp sequence (GGATTACTCCG) directly repeated at its ends and harbours a gene,xisA, which encodes a site-specific recombinase. A large 55 kb element interrupts thefdxN gene in thenifB fdxN-nifS-nifU operon. It contains two 5 bp direct repeats (TATTC) at its ends and accommodates at least one gene,xisF, which encodes another site-specific recombinase. During heterocyst differentiation both the discontinuities are precisely excised by two distinct site-specific recombination events. One of them is brought about by the XisA protein between the 11 bp direct repeats. The second one is caused by the XisF protein and occurs between the 5 bp direct repeats. As a consequence the 11kb and 55 kb elements are removed from the chromosome as circles and functionalnif Operons are created. Nitrogenase proteins are then expressed from the rearranged genes in heterocysts and aerobic nitrogen fixation ensues. How these elements intruded thenif genes and how and why are they maintained in heterocystous cyanobacteria are exciting puzzles engaging considerable research effort currently. The unique developmental regulation of these gene rearrangements in heterocystous cyanobacteria is discussed.     

Site-specific integrative elements of rhizobiophage 16-3 can integrate into proline tRNA (CGG) genes in different bacterial genera.

The integrase protein of the Rhizobium meliloti 41 phage 16-3 has been classified as a member of the Int family of tyrosine recombinases. The site-specific recombination system of the phage belongs to the group in which the target site of integration (attB) is within a tRNA gene. Since tRNA genes are conserved, we expected that the target sequence of the site-specific recombination system of the 16-3 phage could occur in other species and integration could take place if the required putative host factors were also provided by the targeted cells. Here we report that a plasmid (pSEM167) carrying the attP element and the integrase gene (int) of the phage can integrate into the chromosomes of R. meliloti 1021 and eight other species. In all cases integration occurred at so-far-unidentified, putative proline tRNA (CGG) genes, indicating the possibility of their common origin. Multiple alignment of the sequences suggested that the location of the att core was different from that expected previously. The minimal attB was identified as a 23-bp sequence corresponding to the anticodon arm of the tRNA.     

Excision of the high-pathogenicity island of Yersinia pseudotuberculosis requires the combined actions of its cognate integrase and Hef, a new recombination directionality factor

The Yersinia high-pathogenicity island (HPI) encodes the siderophore yersiniabactin-mediated iron uptake system. The HPI of Yersinia pseudotuberculosis I has previously been shown to be able to excise precisely from the bacterial chromosome by recombination between the attB-R and attB-L sites flanking the island. However, the nature of the Y. pseudotuberculosis HPI excision machinery remained unknown. We show here that, upon excision, the HPI forms an episomal circular molecule. The island thus has the ability to excise from the chromosome, circularize and reintegrate itself, either in the same location or in another asn tRNA copy. We also demonstrate that the HPI-encoded bacteriophage P4-like integrase (Int) plays a critical role in HPI excision and that, like phage integrases, it acts as a site-specific recombinase that catalyses both excision and integration reactions. However, Int alone cannot efficiently promote recombination between the attB-R and attB-L sites, and we demonstrate that a newly identified HPI-borne factor, designated Hef (for HPI excision factor) is also required for this activity. Hef belongs to a family of recombination directionality factors. Like the other members of this family, Hef probably plays an architectural rather than a catalytic role and promotes HPI excision from the chromosome by driving the function of Int towards an excisionase activity. The fact that the HPI, and probably several other pathogenicity islands, carry a machinery of integration/excision highly similar to those of bacteriophages argues for a phage-mediated acquisition and transfer of these elements.     

Phage R4 integrase mediates site-specific integration in human cells.

The R4 integrase is a site-specific, unidirectional recombinase derived from the genome of phage R4 of Streptomyces parvulus. Here we define compact attB and attP recognition sites for the R4 integrase and express the enzyme in mammalian cells. We demonstrate that R4 integrase functions in human cells, performing efficient and precise recombination between R4 attB and attP sites cloned on an extrachromosomal vector. We also provide evidence that the enzyme can mediate integration of an incoming plasmid bearing an attB or attP site into endogenous sequences in the human genome. Furthermore, when R4 attB and attP sites are placed into the human genome, either by random integration or at a specific sequence by using the phi C31 integrase, they act as targets for integration of incoming plasmids bearing R4 att sites. The R4 integrase has immediate utility as a site-specific integration tool for genome engineering, as well as potential for further development.