Characterization of genetic elements required for site-specific integration of Lactobacillus delbrueckii subsp. bulgaricus bacteriophage mv4 and construction of an integration-proficient vector for Lactobacillus plantarum.

Temperate phage mv4 integrates its DNA into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus strains via site-specific recombination. Nucleotide sequencing of a 2.2-kb attP-containing phage fragment revealed the presence of four open reading frames. The larger open reading frame, close to the attP site, encoded a 427-amino-acid polypeptide with similarity in its C-terminal domain to site-specific recombinases of the integrase family. Comparison of the sequences of attP, bacterial attachment site attB, and host-phage junctions attL and attR identified a 17-bp common core sequence, where strand exchange occurs during recombination. Analysis of the attB sequence indicated that the core region overlaps the 3' end of a tRNA(Ser) gene. Phage mv4 DNA integration into the tRNA(Ser) gene preserved an intact tRNA(Ser) gene at the attL site. An integration vector based on the mv4 attP site and int gene was constructed. This vector transforms a heterologous host, L. plantarum, through site-specific integration into the tRNA(Ser) gene of the genome and will be useful for development of an efficient integration system for a number of additional bacterial species in which an identical tRNA gene is present.     

Bacteriophage T12 of Streptococcus pyogenes integrates into the gene encoding a serine tRNA

The region of temperate bacteriophage T12 responsible for integration into the chromosome of Streptococcus pyogenes has been identified. The integrase gene ( int ) and the phage attachment site ( attP ) are found immediately upstream of the gene for speA , the latter of which is known to be responsible for the production of erythrogenic toxin A (also known as pyrogenic exotoxin A). The integrase gene has a coding capacity for a protein of 41 457 Da, and the C-terminus of the deduced protein is similar to other conserved C-terminal regions typical of phage integrases. Upstream of int is a second open reading frame, which is capable of encoding an acidic protein of 72 amino acids (8744 Da); the position of this region in relation to int suggests it to be the phage excisionase gene ( xis ). The arms flanking the integrated prophage ( attL and attR ) were identified, allowing determination of the sequences of the phage ( attP ) and bacterial ( attB ) attachment sites. A fragment containing the integrase gene and attP was cloned into a streptococcal suicide vector; when introduced into S. pyogenes by electrotransformation, this plasmid stably integrated into the bacterial chromosome at attB . The insertion site for the phage into the S. pyogenes chromosome was found to be in the anticodon loop of a putative type II gene for a serine tRNA. attP and attB share a region of identity that is 96 bp in length; this region of identity corresponds to the 3' end of the tRNA gene such that the coding sequence remains intact after integration of the prophage. The symmetry of the core region of att may set this region apart from previously described phage attachment sites (Campbell, 1992), and may play a role in the biology of this medically important bacteriophage.     

Characterization of the genetic elements required for site-specific integration of plasmid pSE211 in Saccharopolyspora erythraea.

The 18.1-kilobase plasmid pSE211 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site. Restriction analysis of the integrated plasmid, pSE211int, and adjacent chromosomal sequences allowed identification of attP, the plasmid attachment site. Nucleotide sequencing of attP, attB, attL, and attR revealed a 57-base-pair sequence common to all sites with no duplications of adjacent plasmid or chromosomal sequences in the integrated state, indicating that integration takes place through conservative, reciprocal strand exchange. An analysis of the sequences indicated the presence of a putative gene for Phe-tRNA at attB which is preserved at attL after integration has occurred. A comparison of the attB site for a number of actinomycete plasmids is presented. Integration at attB was also observed when a 2.4-kilobase segment of pSE211 containing attP and the adjacent plasmid sequence was used to transform a pSE211- host. Nucleotide sequencing of this segment revealed the presence of two complete open reading frames (ORFs) and a segment of a third ORF. The ORF adjacent to attP encodes a putative polypeptide 437 amino acids in length that shows similarity, at its C-terminal domain, to sequences of site-specific recombinases of the integrase family. The adjacent ORF encodes a putative 98-amino-acid basic polypeptide that contains a helix-turn-helix motif at its N terminus which corresponds to domains in the Xis proteins of a number of bacteriophages. A proposal for the function of this polypeptide is presented. The deduced amino acid sequence of the third ORF did not reveal similarities to polypeptide sequences in the current data banks.     

The actinophage RP3 DNA integrates site-specifically into the putative tRNA(Arg)(AGG) gene of Streptomyces rimosus.

The temperate actinophage RP3 integrates site-specifically into the chromosome of Streptomyces rimosus R6-554. The phage attachment site attP and the hybrid attachment sites of the integrated prophage--attL and attR--were cloned and sequenced. The 54nt core sequence, common to all RP3 related attachment sites, comprises the 3' terminal end of a putative tRNA(Arg)(AGG) gene. AttB bears the complete tRNA gene which is restored in attL after integration. A 7.5kb HindIII fragment, bearing attP, was used to construct an integrative plasmid to simulate the integration process in vivo and to localize the phage genes necessary for site specific integration. The int and xis genes were sequenced and compared to other recombination genes.     

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.     

Site-Specific Recombination of Temperate Myxococcus xanthus Phage Mx8: Regulation of Integrase Activity by Reversible, Covalent Modification

Temperate Myxococcus xanthus phage Mx8 integrates into the attB locus of the M. xanthus genome. The phage attachment site, attP, is required in cis for integration and lies within the int (integrase) coding sequence. Site-specific integration of Mx8 alters the 3' end of int to generate the modified intX gene, which encodes a less active form of integrase with a different C terminus. The phage-encoded (Int) form of integrase promotes attP x attB recombination more efficiently than attR x attB, attL x attB, or attB x attB recombination. The attP and attB sites share a common core. Sequences flanking both sides of the attP core within the int gene are necessary for attP function. This information shows that the directionality of the integration reaction depends on arm sequences flanking both sides of the attP core. Expression of the uoi gene immediately upstream of int inhibits integrative (attP x attB) recombination, supporting the idea that uoi encodes the Mx8 excisionase. Integrase catalyzes a reaction that alters the primary sequence of its gene; the change in the primary amino acid sequence of Mx8 integrase resulting from the reaction that it catalyzes is a novel mechanism by which the reversible, covalent modification of an enzyme is used to regulate its specific activity. The lower specific activity of the prophage-encoded IntX integrase acts to limit excisive site-specific recombination in lysogens carrying a single Mx8 prophage, which are less immune to superinfection than lysogens carrying multiple, tandem prophages. Thus, this mechanism serves to regulate Mx8 site-specific recombination and superinfection immunity coordinately and thereby to preserve the integrity of the lysogenic state.     

Site-specific integration in Saccharopolyspora erythraea and multisite integration in Streptomyces lividans of actinomycete plasmid pSE101.

An 11.3-kilobase-pair plasmid, designated pSE101, exists in Saccharopolyspora erythraea NRRL 2338 as an integrated sequence (pSE101int) at a unique chromosomal location and in the free form in less than an average of 1 copy per 10 chromosomes. The plasmid sequence is missing from S. erythraea NRRL 2359. Restriction maps of the free and integrated forms of pSE101 showed point-to-point correspondence. Plasmid pECT2 was constructed by ligation of pSE101, pBR322, and the gene for thiostrepton resistance (tsr). When introduced by polyethylene glycol-mediated transformation into protoplasts of S. erythraea NRRL 2359, all thiostrepton-resistant regenerants examined were found to carry a single copy of pECT2 in the integrated state at a single chromosomal site. The chromosomal site of pECT2 integration in strain NRRL 2359 (attB) corresponded to the chromosomal location of pSE101int in strain NRRL 2338. The plasmid crossover site (attP) was mapped to the plasmid site that corresponded to the site of interruption of the plasmid sequence in the host carrying pSE101int, indicating that site-specific integrative recombination had occurred. An additional 2.8-kilobase-pair chromosomal sequence homologous to a segment of pSE101 was also observed in strains NRRL 2338 and NRRL 2359. After introduction of pECT2 into Streptomyces lividans, approximately half of the transformants examined were found to carry the plasmid as a stable, autonomously replicating element. The other half carried a single copy of pECT2 as an integrated sequence, but the location of pECT2int in Streptomyces lividans varied from one transformant to another. In each case, integrative crossover used the attP site. A model is proposed to account for the determination of the particular state of pSE101 in Streptomyces lividans.     

A motif in the C-terminal domain of phiC31 integrase controls the directionality of recombination

Bacteriophage C31 encodes an integrase, which acts on the phage and host attachment sites, attP and attB, to form an integrated prophage flanked by attL and attR. In the absence of accessory factors, C31 integrase cannot catalyse attL x attR recombination to excise the prophage. To understand the mechanism of directionality, mutant integrases were characterized that were active in excision. A hyperactive integrase, Int E449K, gained the ability to catalyse attL x attR, attL x attL and attR x attR recombination whilst retaining the ability to recombine attP x attB. A catalytically defective derivative of this mutant, Int S12A, E449K, could form stable complexes with attP/attB, attL/attR, attL/attL and attR/attR under conditions where Int S12A only complexed with attP/attB. Further analysis of the Int E449K-attL/attR synaptic events revealed a preference for one of the two predicted synapse structures with different orientations of the attL/attR sites. Several amino acid substitutions conferring hyperactivity, including E449K, were localized to one face of a predicted coiled-coil motif in the C-terminal domain. This work shows that a motif in the C-terminal domain of C31 integrase controls the formation of the synaptic interface in both integration and excision, possibly through a direct role in protein-protein interactions.     

Site-specific integration of the temperate bacteriophage phi adh into the Lactobacillus gasseri chromosome and molecular characterization of the phage (attP) and bacterial (attB) attachment sites.

The temperate bacteriophage phi adh integrates its genome into the chromosomal DNA of Lactobacillus gasseri ADH by a site-specific recombination process. Southern hybridization analysis of BclI-digested genomic DNA from six relysogenized derivatives of the prophage-cured strain NCK102 displayed phage-chromosomal junction fragments identical to those of the lysogenic parent. The phi adh attachment site sequence, attP, was located within a 365-bp EcoRI-HindIII fragment of phage phi adh. This fragment was cloned and sequenced. DNA sequence analysis revealed striking features common to the attachment sites of other site-specific recombination systems: five direct repeats of the sequence TGTCCCTTTT(C/T) and a 14-bp inverted repeat. Oligonucleotides derived from the sequence of the attP-containing fragment enabled us to amplify predicted junction fragment sequences and thus to identify attL, attR, and attB. The core region was defined as the 16-bp sequence TACACTTCTTAGGAGG. Phage-encoded functions essential for site-specific insertion of phage phi adh were located in a 4.5-kb BclI fragment. This fragment was cloned in plasmid pSA34 to generate the insertional vector pTRK182. Plasmid pTRK182 was introduced into L. gasseri NCK102 by electroporation. Hybridization analysis showed that a single copy of pTRK182 had integrated at the attB site of the NCK102 erythromycin-resistant transformants. This is the first site-specific recombination system described in lactobacilli, as well as the first attP-based site-specific integration vector constructed for L. gasseri ADH.     

Identification of int and attP on the genome of lactococcal bacteriophage Tuc2009 and their use for site-specific plasmid integration in the chromosome of Tuc2009-resistant Lactococcus lactis MG1363.

The DNA sequence of the int-attP region of the small-isometric-headed lactococcal bacteriophage Tuc2009 is presented. In this region, an open reading frame, int, which potentially encodes a protein of 374 amino acids, representing the Tuc2009 integrase, was identified. The nucleotide sequence of the bacteriophage attachment site, attP, and the sequences of attB, attL, and attR in the lysogenic host Lactococcus lactis subsp. cremoris UC509 were determined. A sequence almost identical to the UC509 attB sequence was found to be present in the plasmid-free Tuc2009-resistant L. lactis subsp. cremoris MG1363. This site could be used for the site-specific integration of a plasmid carrying the Tuc2009 int-attP region in the chromosome of MG1363, thereby demonstrating that the application of chromosomal insertion vectors based on bacteriophage integration functions is not limited to the prophage-cured original host strain of the phage.     

Mutational analysis of integrase arm-type binding sites of bacteriophage lambda. Integration and excision involve distinct interactions of integrase with arm-type sites

Integrative recombination between specific attachment (att) regions of the bacteriophage lambda genome (attP) and the Escherichia coli genome (attB) results in a prophage flanked by the hybrid recombinant sites attL and attR. Each att site contains sequences to which proteins involved in recombination bind. Using site-directed mutagenesis, we have constructed a related set of point mutations within each of the five Int "arm-type" binding sites located within attP, attL and attR. Footprint analyses of binding demonstrate that mutating the arm-type sites significantly disrupts the binding of Int. Recombination analyses of mutant att sites in vivo and in vitro demonstrate that only three wild-type arm-type sites within attP are required for efficient integrative recombination. Similar analyses demonstrate that efficient excision can occur with two other different sets of wild-type arm-type sites in attL and attR. These results demonstrate that integrative and excisive recombination may involve interactions of Int with distinct and different subsets of arm-type sites.     

Integration of bacteriophage Mx8 into the Myxococcus xanthus chromosome causes a structural alteration at the C-terminal region of the IntP protein.

Mx8 is a generalized transducing phage that infects Myxococcus xanthus cells. This phage is lysogenized in M. xanthus cells by the integration of its DNA into the host chromosome through site-specific recombination. Here, we characterize the mechanism of Mx8 integration into the M. xanthus chromosome. The Mx8 attachment site, attP, the M. xanthus chromosome attachment site, attB, and two phage-host junctions, attL and attR, were cloned and sequenced. Sequence alignments of attP, attB, attL, and attR sites revealed a 29-bp segment that is absolutely conserved in all four sequences. The intP gene of Mx8 was found to encode a basic protein that has 533 amino acids and that carries two domains conserved in site-specific recombinases of the integrase family. Surprisingly, the attP site was located within the coding sequence of the intP gene. Hence, the integration of Mx8 into the M. xanthus chromosome results in the conversion of the intP gene to a new gene designated intR. As a result of this conversion, the 112-residue C-terminal sequence of the intP protein is replaced with a 13-residue sequence. A 3-base deletion within the C-terminal region had no effect on Mx8 integration into the chromosome, while a frameshift mutation with the addition of 1 base at the same site blocked integration activity. This result indicates that the C-terminal region is required for the enzymatic function of the intP product.     

Integration of the temperate phage phiU into the putative tRNA gene on the chromosome of its host Rhizobium leguminosarum biovar trifolii

The plasmid pCI6, carrying the attP site of the temperate phage phiU, integrates into the attB site on the chromosome of Rhizobium leguminosarum biovar trifolii strain 4S. The 4 kb EcoRI-HindIII region of pCI6 involved in site-specific integration was subcloned as the attP fragment of phage phiU and sequenced. The attL fragment, one of the new DNA junctions generated from the insertion of pCI6 into the chromosome of the host Rhizobium, was used as a hybridization probe for isolation of the attB fragment of strain 4S. The nucleotide sequence of the 2 kb PstI fragment of strain 4S, which hybridized with the attL fragment, was decided and compared with that of the attP fragment. A 53 bp common sequence was expected to be the core sequence of site-specific integration between phage phiU and strain 4S. One of the ORFs on the attP fragment, which was located adjacent to the core sequence, had structural homology to the integrase family. However, the attB fragment showed high homology with the tRNA genes of Agrobacterium tumefaciens and E. coli. A 47 bp sequence of the 53 bp core sequence overlapped with this tRNA-like sequence. This indicates that the target site of phage phiU integration is the putative tRNA gene on the chromosome of the Rhizobium host.     

Characterization of genetic elements required for site-specific integration of the temperate lactococcal bacteriophage phi LC3 and construction of integration-negative phi LC3 mutants.

The genetic elements required for the integration of the temperate lactococcal bacteriophage phi LC3 into the chromosome of its bacterial host, Lactococcus lactis subsp. cremoris, were identified and characterized. The phi LC3 phage attachment site, attP, was mapped and sequenced. DNA sequence analysis of attP and of the bacterial attachment site, attB, as well as the two phage-host junctions, attR and attL, in the chromosome of a phi LC3 lysogen, identified a 9-bp common core region, 5'-TTCTTCATG'-3, within which the strand exchange reaction takes place during integration. The attB core sequence is located within the C-terminal part of an open reading frame of unknown function. The phi LC3 integrase gene (int), encoding the phi LC3 site-specific recombinase, was identified and is located adjacent to attP. The phi LC3 Int protein, as deduced from the nucleotide sequence, is a basic protein of 374 amino acids that shares significant sequence similarity with other site-specific recombinases of the integrase family. Phage phi LC3 int- and int-attP-defective mutants, conferring an abortive lysogenic phenotype, were constructed.     

Control of directionality in the site-specific recombination system of the Streptomyces phage phiC31

The genome of the Streptomyces temperate phage phiC31 integrates into the host chromosome via a recombinase belonging to a novel group of phage integrases related to the resolvase/invertase enzymes. Previously, it was demonstrated that, in an in vitro recombination assay, phiC31 integrase catalyses integration (attP/attB recombination) but not excision (attL/attR). The mechanism responsible for this recombination site selectivity was therefore investigated. Purified integrase was shown to bind with similar apparent binding affinities to between 46 bp and 54 bp of DNA at each of the attachment sites, attP, attB, attL and attR. Assays using recombination sites of 50 bp and 51 bp for attP and attB, respectively, showed that these fragments were functional in attP/attB recombination and maintained strict site selectivity, i.e. no recombination between non-permissive sites, such as attP/attP, attB/attL, etc., was observed. Using bandshifts and supershift assays in which permissive and non-permissive combinations of att sites were used in the presence of integrase, only the attP/attB combination could generate supershifts. Recombination products were isolated from the supershifted complexes. It was concluded that these supershifted complexes contained the recombination synapse and that site specificity, and therefore directionality, is determined at the level of stable synapse formation.     

Site-specific recombination between cloned attP and attB sites from the Haemophilus influenzae bacteriophage HP1 propagated in recombination-deficient Escherichia coli.

Plasmids were constructed which contain both attP and attB DNA segments derived from the insertion sites of the lysogenic bacteriophage HP1 and its host, Haemophilus influenzae. Similar plasmids containing the two junction segments (attL and attR regions) between the phage genome and the lysogenic host chromosome were also prepared. The formation of recombinant dimer plasmids was observed when attP-attB plasmids were propagated in Escherichia coli HB101 (recA), while plasmids containing the junction segments did not form recombinant dimers. Deletion of the phage DNA segment adjacent to the attP site from the attP-attB constructions eliminated detectable recombination, suggesting that this sequence contains the gene encoding the HP1 integrase. No plasmid recombination was observed in strains of E. coli defective in integration host factor. This suggests that integration host factor is important in the expression or activity of the system which produces the site-specific recombination of sequences derived from HP1 and H. influenzae. Further, it suggests that a protein functionally analogous to E. coli integration host factor may be present in H. influenzae.     

Evolution of the family of pRN plasmids and their integrase-mediated insertion into the chromosome of the crenarchaeon Sulfolobus solfataricus

Plasmid pHEN7 from Sulfolobus islandicus was sequenced (7.83 kb) and shown to belong to the archaeal pRN family, which includes plasmids pRN1, pRN2, pSSVx and pDL10 that share a large conserved sequence region. pHEN7 is most closely related to pRN1 in this conserved region. It also shares a large variant region containing several homologous genes with pDL10, which is absent from the other plasmids. The variant region is flanked by the sequence motif TTAGAATGGGGATTC and similar duplicated motifs occur in plasmids pRN1 and pRN2, separated by a few bases. It is inferred that recombination at these sites produces the main genetic variability in the plasmid family. The conserved region of the plasmid, and duplicated copies of the motif, are also present in the genome of Sulfolobus solfataricus P2. Moreover, they are bordered by a partitioned integrase gene (int) and by a 45 bp perfect direct repeat corresponding to the downstream half of a tRNA(Val) gene. The integrase and the direct repeat are highly similar in sequence to the integrase and the chromosomal integration site (att), respectively, of the SSV1 virus, which integrates into the chromosome of Sulfolobus shibatae. Recombination at the att repeats in S. solfataricus would produce a novel plasmid, pXQ1, which carries both an intact integrase gene and a single integration site (att). This strongly suggests that the same mechanism of site-specific integration at a tRNA gene is used for both viruses and plasmids in Sulfolobus.     

Characterization of the Micromonospora rosaria pMR2 plasmid and development of a high G+C codon optimized integrase for site-specific integration

pMR2, an 11.1 kb plasmid was isolated from Micromonospora rosaria SCC2095, NRRL3718, and its complete nucleotide sequence determined. Analysis revealed 13 ORFs including homologs of a KorSA regulatory protein and TraB plasmid transfer protein found on other actinomycete plasmids. pMR2 contains att/int functions consisting of an integrase, an excisionase, and a putative plasmid attachment site (attP). The integrase gene contained a high frequency of codons rarely used in high G+C actinomycete coding regions. The gene was codon optimized for actinomycete codon usage to create the synthetic gene int-OPT. pSPRX740, containing an rpsL promoter and the att/int-OPT region, was introduced into Micromonospora halophytica var. nigra ATCC33088. Analysis of DNA flanking the pSPRX740 integration site confirmed site-specific integration into a tRNA(Phe) gene in the M. halopytica var. nigra chromosome. The pMR2 attP element and chromosomal attachment (attB) site contain a 63 bp region of sequence identity overlapping the 3' end of the tRNA(Phe) gene. Plasmids comprising the site-specific att/int-OPT functions of pMR2 can be used to integrate genes into the chromosome of actinomycetes with an appropriate tRNA gene. The development of an integrative system for Micromonospora will expand our ability to study antibiotic biosynthesis in this important actinomycete genus.