Integrase Family Of Site-specific Recombinases Research Articles

Cre- lox -Based System for Multiple Gene Deletions and Selectable-Marker Removal in Lactobacillus plantarum

The classic strategy to achieve gene deletion variants is based on double-crossover integration of nonreplicating vectors into the genome. In addition, recombination systems such as Cre-lox have been used extensively, mainly for eukaryotic organisms. This study presents the construction of a Cre-lox-based system for multiple gene deletions in Lactobacillus plantarum that could be adapted for use on gram-positive bacteria. First, an effective mutagenesis vector (pNZ5319) was constructed that allows direct cloning of blunt-end PCR products representing homologous recombination target regions. Using this mutagenesis vector, double-crossover gene replacement mutants could be readily selected based on their antibiotic resistance phenotype. In the resulting mutants, the target gene is replaced by a lox66-P(32)-cat-lox71 cassette, where lox66 and lox71 are mutant variants of loxP and P(32)-cat is a chloramphenicol resistance cassette. The lox sites serve as recognition sites for the Cre enzyme, a protein that belongs to the integrase family of site-specific recombinases. Thus, transient Cre recombinase expression in double-crossover mutants leads to recombination of the lox66-P(32)-cat-lox71 cassette into a double-mutant loxP site, called lox72, which displays strongly reduced recognition by Cre. The effectiveness of the Cre-lox-based strategy for multiple gene deletions was demonstrated by construction of both single and double gene deletions at the melA and bsh1 loci on the chromosome of the gram-positive model organism Lactobacillus plantarum WCFS1. Furthermore, the efficiency of the Cre-lox-based system in multiple gene replacements was determined by successive mutagenesis of the genetically closely linked loci melA and lacS2 in L. plantarum WCFS1. The fact that 99.4% of the clones that were analyzed had undergone correct Cre-lox resolution emphasizes the suitability of the system described here for multiple gene replacement and deletion strategies in a single genetic background.

Inversion within the haloalkaliphilic virus phi Ch1 DNA results in differential expression of structural proteins.

The sequence of phi Ch1 contains an open reading frame (int1) in the central part of its genome that belongs to the lambda integrase family of site-specific recombinases. Sequence similarities to known integrases include the highly conserved tetrad R-H-R-Y. The flanking sequences of int1 contain several direct repeats of 30 bp in length (IR-L and IR-R), which are orientated in an inverted direction. Here, we show that a recombination active region exists in the genome of phi Ch1: the number of those repeats, non-homologous regions within the repeat clusters IR-L and IR-R and the orientation of the int1 gene vary in a given virus population. Within this study, we identified circular intermediates, composed of the int1 gene and the inwards orientated repeat regions IR-L and IR-R, which could be involved in the recombination process itself. IR-L and IR-R are embedded within ORF34 and ORF36 respectively. As a consequence of the inversion within this region of phi Ch1, the C-terminal parts of the proteins encoded by ORF34 and 36 are exchanged. Both proteins, expressed in Escherichia coli, interact with specific antisera against whole virus particles, indicating that they could be parts of phi Ch1 virions. Expression of the protein(s) in Natrialba magadii could be detected 98 h after inoculation, which is similar to other structural proteins of phi Ch1. Taken together, the data show that the genome of phi Ch1 contains an invertible region that codes for a recombinase and structural proteins. Inversion of this segment results in a variation of these structural proteins.

Streptomyces coelicolor A3(2) plasmid SCP2*: deductions from the complete sequence.

Plasmid SCP2* is a 31 kb, circular, low-copy-number plasmid originally identified in Streptomyces coelicolor A3(2) as a fertility factor. The plasmid was completely sequenced. The analysis of the 31 317 bp sequence revealed 34 ORFs encoding putative proteins from 31 to 710 aa long, most of them lacking similarity to known proteins. Three functional regions had been identified previously: the replication region, the transfer and spreading region, and the stability region. Three genes were identified in the stability region which contribute to the stability of SCP2 as shown by plasmid stability testing. The first gene, mrpA, encodes a new member of the lambda integrase family of site-specific recombinases. The two genes downstream of mrpA were called parA and parB. The gene product, ParA, shows similarity to a family of ATPases involved in plasmid partition. An increase of plasmid stability could be seen only when both genes were present. By deletion analysis, the replication region could be narrowed down to a 1.6 kb region, consisting of a 650 bp non-coding region and two genes, repI and repII, encoding proteins of 161 and 131 aa. Only RepI exhibits similarities to DNA binding elements and contains a putative helix-turn-helix motif. The traA gene that is essential for DNA transfer and pock formation was identified previously. Upstream of traA, 10 ORFs were found in the same orientation as traA which might be involved in conjugation and DNA spreading, together with one gene in the opposite orientation with similarities to transcriptional regulators of DNA transfer. Two transposable elements were found on SCP2*. IS1648 belongs to the IS3 family of insertion sequences. The second element, Tn5417, shows the highest similarity to the Tn4811 element located in the terminal inverted repeats of the Streptomyces lividans chromosome.

Site-specific recombination system encoded by toluene catabolic transposon Tn4651.

The 56-kb class II toluene catabolic transposon Tn4651 from Pseudomonas putida plasmid pWW0 is unique in that (i) its efficient resolution requires, in addition to the 0.2-kb resolution (res) site, the two gene products TnpS and TnpT and (ii) the 2.4-kb tnpT-res-tnpS region is 48 kb apart from the tnpA gene (M. Tsuda, K.-I. Minegishi, and T. Iino, J. Bacteriol. 171:1386-1393, 1989). Detailed analysis of the 2.4-kb region revealed that the tnpS and tnpT genes encoding the putative 323- and 332-amino-acid proteins, respectively, were transcribed divergently with an overlapping 59-bp sequence in the 203-bp res site. The motifs (the R-H-R-Y tetrad in domains I and II with proper spacing) commonly conserved in the integrase family of site-specific recombinases were found in TnpS. In contrast, TnpT did not show any significant amino acid sequence homology to the other proteins that are directly or indirectly involved in recombination. Analysis of site-specific recombination under the Escherichia coli recA cells indicated that (i) the site-specific resolution between the two copies of the res site on a single molecule was catalyzed by TnpS, (ii) the functional res site was located within a 95-bp segment, and (iii) TnpT appeared to have the role of enhancing the site-specific resolution. It was also found that TnpS catalyzed the site-specific recombination between the res sites located at two different molecules to form a cointegrate molecule. Site-specific mutagenesis of the conserved tyrosine residue in TnpS led to the loss of both the resolution and the integration activities, indicating that such a residue took part in both types of recombination.

The integrase family of recombinase: organization and function of the active site.

The integrase family of site-specific recombinases (also called the tyrosine recombinases) mediate a wide range of biological outcomes by the sequential exchange of two pairs of DNA strands at defined phosphodiester positions. The reaction produces a recombinant arrangement of the DNA sequences flanking the cross-over region. The crystal structures of four integrase family members have revealed very similar three-dimensional protein folds that belie the large diversity in amino acid sequences among them. The active sites are similar in organization to those seen in structures of eukaryotic type IB topoisomerases, and conservation of catalytic mechanism is expected. The crystal structures, combined with previous biochemical knowledge, allow the refinement of models for recombination and the assignment of catalytic function to the active site residues. However, each system has its own peculiarities, and the exact sequence of events that allows the reaction to proceed from the first exchange reaction to the second is still unclear for at least some family members.

A newly identified, essential catalytic residue in a critical secondary structure element in the integrase family of site-specific recombinases is conserved in a similar element in eucaryotic type IB topoisomerases

The integrase family of site-specific recombinases catalyzes conservative rearrangements between defined segments of DNA. A highly conserved tetrad (RHRY) of catalytic residues is essential for this process. This tetrad is dispersed in two motifs in the linear sequence, but is configured appropriately in the catalytic pocket to execute the strand cleavage and rejoining reactions. A third conserved motif has been identified in the Xer subgroup of the integrase family. Mutational analysis of 12 conserved residues in this motif in the XerD protein from Salmonella typhimurium led to the identification of an essential fifth catalytic residue (lysine 172) which is implicated in strand cleavage or exchange. This lysine residue occupies part of the turn of an antiparallel β-hairpin which forms one side of the catalytic cleft in XerD, and is found at similar positions among evolutionarily diverse integrase family members. Related antiparallel β-hairpins are present in eucaryotic type IB topoisomerase enzymes which also contain a critical lysine residue in the turn of the hairpin. In both the integrase family and eucaryotic type IB topoisomerases, the catalytic lysine residues are in close contact with the substrates and may play similar roles in influencing the reactivity of the phosphotyrosine intermediates formed during reactions catalyzed by both enzymes.

Structure and mechanism in site-specific recombination

Three-dimensional structural information on the integrase family of site-specific recombinases has only recently become available, with the crystal structures of catalytic domains, full-length proteins and protein–DNA complexes of this family reported over the past two years. These results have led to a model for the overall architecture and active site stereochemistry of the recombination pathway that addresses a number of interesting mechanistic issues.

Site-specific DNA binding using a variation of the double stranded RNA binding motif.

The integrase family of site-specific recombinases catalyze a diverse array of DNA rearrangements in archaebacteria, eubacteria and yeast. The solution structure of the DNA binding domain of the integrase protein from the conjugative transposon Tn916 has been determined using NMR spectroscopy. The structure provides the first insights into distal site DNA binding by a site-specific integrase and reveals that the N-terminal domain is structurally similar to the double stranded RNA binding domain (dsRBD). The results of chemical shift mapping experiments suggest that the integrase protein interacts with DNA using residues located on the face of its three stranded beta-sheet. This surface differs from the proposed RNA binding surface in dsRBDs, suggesting that different surfaces on the same protein fold can be used to bind DNA and RNA.

A site-specific recombinase is required for competitive root colonization by Pseudomonas fluorescens WCS365.

A colonization mutant of the efficient root-colonizing biocontrol strain Pseudomonas fluorescens WCS365 is described that is impaired in competitive root-tip colonization of gnotobiotically grown potato, radish, wheat, and tomato, indicating a broad host range mutation. The colonization of the mutant is also impaired when studied in potting soil, suggesting that the defective gene also plays a role under more natural conditions. A DNA fragment that is able to complement the mutation for colonization revealed a multicistronic transcription unit composed of at least six ORFs with similarity to lppL, lysA, dapF, orf235/233, xerC/sss, and the largely incomplete orf238. The transposon insertion in PCL1233 appeared to be present in the orf235/233 homologue, designated orf240. Introduction of a mutation in the xerC/sss homologue revealed that the xerC/sss gene homologue rather than orf240 is crucial for colonization. xerC in Escherichia coli and sss in Pseudomonas aeruginosa encode proteins that belong to the lambda integrase family of site-specific recombinases, which play a role in phase variation caused by DNA rearrangements. The function of the xerC/sss homologue in colonization is discussed in terms of genetic rearrangements involved in the generation of different phenotypes, thereby allowing a bacterial population to occupy various habitats. Mutant PCL1233 is assumed to be locked in a phenotype that is not well suited to compete for colonization in the rhizosphere. Thus we show the importance of phase variation in microbe-plant interactions.

Selection of novel, specific single-stranded DNA sequences by Flp, a duplex-specific DNA binding protein.

Flp is a member of the integrase family of site-specific recombinases. Flp is known to be a double-stranded (ds)DNA binding protein that binds sequence specifically to the 13 bp binding elements in the FRT site (Flprecognitiontarget). We subjected a random pool of oligonucleotides to the in vitro binding site selection method and have unexpectedly recovered a series of single-stranded oligonucleotides to which Flp binds with high affinity. These single-stranded oligonucleotides differ in sequence from the duplex FRT site. The minimal length of the oligonucleotides which is active is 29 nt. This single strand-specific DNA binding activity is located in the same C-terminal 32 kDa domain of Flp in which the site-specific dsDNA binding activity resides. Competition studies suggest that the apparent affinity of Flp for single-stranded oligonucleotide is somewhat less than for a complete duplex FRT site but greater than for a single duplex 13 bp binding element. We have also shown that Cre, another member of the integrase family of site-specific recombinases, also exhibits single-stranded DNA binding similar to that of Flp.

Asymmetry in Flp-mediated cleavage.

Flp is a member of the integrase family of site-specific recombinases. Members of the integrase family mediate DNA strand cleavage via a transesterification reaction involving an active site tyrosine residue. The first step of the reaction results in covalent linkage of the protein to the 3'-phosphoryl DNA terminus, leaving a 5'-hydroxyl group at the site of the nick. We have used Flp recognition target (FRT) sites containing a 5'-bridging phosphorothioate linkage at the site of Flp cleavage to accumulate intermediates in which Flp is covalently bound at a cleavage site. We have probed these intermediates with dimethylsulfate using methylation protection and find that Flp-mediated cleavage is associated with protection of two adenine residues that are opposite the sites of cleavage and covalent attachment by Flp. Methylation interference studies showed that cleavage and covalent attachment are also accompanied by differences in the contacts of Flp with each of the two cleavage sites and with the surrounding symmetry elements. Therefore, we provide evidence that Flp-mediated cleavage and covalent attachment result in changes to the conformation of the Flp-FRT complex. These changes may be required for Flp-mediated strand exchange activity.

Site-specific recombination: Synapsis and strand exchange revealed

Recently determined crystal structures of several members of the λ integrase family of site-specific recombinases have provided insights into the cis versus trans action of active site constituents, and the processes of synapsis and strand exchange.

Specific DNA cleavage mediated by the integrase of conjugative transposon Tn916.

The conjugative transposon Tn916 encodes a protein called INT(Tn916) which, based on DNA sequence comparisons, is a member of the integrase family of site-specific recombinases. Integrase proteins such as INT(lambda), FLP, and XERC/D that promote site-specific recombination use characteristic, conserved amino acid residues to catalyze the cleavage and ligation of DNA substrates during recombination. The reaction proceeds by a two-step transesterification reaction requiring the formation of a covalent protein-DNA intermediate. Different requirements for homology between recombining DNA sites during integrase-mediated site-specific recombination and Tn916 transposition suggest that INT(Tn916) may use a reaction mechanism different from that used by other integrase recombinases. We show that purified INT(Tn916) mediates specific cleavage of duplex DNA substrates containing the Tn916 transposon ends and adjacent bacterial sequences. Staggered cleavages occur at both ends of the transposon, resulting in 5' hydroxyl protruding ends containing coupling sequences. These are sequences that are transferred with the transposon from donor to recipient during conjugative transposition. The nature of the cleavage products suggests that a covalent protein-DNA linkage occurs via a residue of INT(Tn916) and the 3'-phosphate group of the DNA. INT(Tn916) alone is capable of executing the strand cleavage step required for recombination during Tn916 transposition, and this reaction probably occurs by a mechanism similar to that of other integrase family site-specific recombinases.

Molecular characterization of a deletion-prone region of plasmid pAE1 of Alcaligenes eutrophus H1

A 93-kb region (D region) of plasmid pAE1 of Alcaligenes eutrophus H1 has been found to have a high rate of spontaneous deletion. In this study, we constructed a restriction endonuclease map and carried out limited sequencing of an approximately 100-kb region from pAE1 which includes the D region (the deleted region) in order to detect and characterize repetitive sequences. Two types of repetitive sequences, the R1 and R2 sequences, were observed to flank the D region; within the D region are three copies of insertion element ISAE1. The R1 and R2 sequences are arranged in direct and inverted orientations, respectively. Molecular analysis of the end product of the deletion is consistent with the hypothesis that the loss of the D-region DNA is the result of recombination between two copies of the R1 sequence. The R1 sequence encodes a 415-amino-acid protein which exhibits substantial sequence similarity to the lambda integrase family of site-specific recombinases. Its genetic function remains to be determined.

The sss gene product, which affects pyoverdin production in Pseudomonas aeruginosa 7NSK2, is a site‐specific recombinease

Pyoverdin production by Pseudomonas aeruginosa strain 7NSK2 was induced by Zn(II) in the presence of iron. A mutant was isolated in which Zn(II) no longer induced pyoverdin production. The sss gene which was inactivated in this mutant was cloned and sequenced. Its protein sequence showed 50% identity to the XerC protein of Escherichia coli, which is a member of the lambda integrase family of site-specific recombinases. An open reading frame was found upstream of sss whose protein sequence showed strong identity to DapF, the diaminopimelate epimerase. In E. coli, xerC is part of a multicistronic unit that also contains dapF. The sss gene of P. aeruginosa could restore site-specific recombination at cer in an E. coli xerC mutant and the E. coli xerC gene could complement a genomic sss mutation in P. aeruginosa.

Tn5401, a new class II transposable element from Bacillus thuringiensis.

A new class II (Tn3-like) transposable element, designated Tn5401, was recovered from a sporulation-deficient variant of Bacillus thuringiensis subsp. morrisoni EG2158 following its insertion into a recombinant plasmid. Sequence analysis of the insert revealed a 4,837-bp transposon with two large open reading frames, in the same orientation, encoding proteins of 36 kDa (306 residues) and 116 kDa (1,005 residues) and 53-bp terminal inverted repeats. The deduced amino acid sequence for the 36-kDa protein shows 24% sequence identity with the TnpI recombinase of the B. thuringiensis transposon Tn4430, a member of the phage integrase family of site-specific recombinases. The deduced amino acid sequence for the 116-kDa protein shows 42% sequence identity with the transposase of Tn3 but only 28% identity with the TnpA transposase of Tn4430. Two small open reading frames of unknown function, designated orf1 (85 residues) and orf2 (74 residues), were also identified. Southern blot analysis indicated that Tn5401, in contrast to Tn4430, is not commonly found among different subspecies of B. thuringiensis and is not typically associated with known insecticidal crystal protein genes. Transposition was studied with B. thuringiensis by using plasmid pEG922, a temperature-sensitive shuttle vector containing Tn5401. Tn5401 transposed to both chromosomal and plasmid target sites but displayed an apparent preference for plasmid sites. Transposition was replicative and resulted in the generation of a 5-bp duplication at the target site. Transcriptional start sites within Tn5401 were mapped by primer extension analysis. Two promoters, designated PL and PR, direct the transcription of orf1-orf2 and tnpI-tnpA, respectively, and are negatively regulated by TnpI. Sequence comparison of the promoter regions of Tn5401 and Tn4430 suggests that the conserved sequence element ATGTCCRCTAAY mediates TnpI binding and cointegrate resolution. The same element is contained within the 53-bp terminal inverted repeats, thus accounting for their unusual lengths and suggesting an additional role for TnpI in regulating Tn5401 transposition.

Mechanism of cleavage and ligation by FLP recombinase: classification of mutations in FLP protein by in vitro complementation analysis.

The FLP recombinase of the 2 microns plasmid of Saccharomyces cerevisiae is a member of the integrase family of site-specific recombinases. Recombination catalyzed by members of this family proceeds via the ordered cleavage and religation of four strands of DNA. Although the amino acid sequences of integrase family members are quite different, each recombinase maintains an absolutely conserved tetrad of amino acids (R-191, H-305, R-308, Y-343; numbers are those of the FLP protein). This tetrad is presumed to reflect a common chemical mechanism for cleavage and ligation that has evolved among all family members. The tyrosine is the nucleophile that causes phosphodiester bond cleavage and covalently attaches to the 3'-PO4 terminus, whereas the other three residues have been implicated in ligation of strands. It has recently been shown that cleavage by FLP takes place in trans; that is, a FLP molecule binds adjacent to the site of cleavage but receives the nucleophilic tyrosine from a molecule of FLP that is bound to another FLP-binding element (J.-W. Chen, J. Lee, and M. Jayaram, Cell 69:647-658, 1992). These studies led us to examine whether the ligation step of the FLP reaction is performed by the FLP molecule bound adjacent to the cleavage site (ligation in cis). We have found that FLP promotes ligation in cis. Furthermore, using in vitro complementation analysis, we have classified several mutant FLP proteins into one of two groups: those proteins that are cleavage competent but ligation deficient (group I) and those that are ligation competent but cleavage defective (group II). This observation suggests that the active site of FLP is composed of several amino acid residues from each of two FLP molecules.

Mechanism of Cleavage and Ligation by FLP Recombinase: Classification of Mutations in FLP Protein by in Vitro Complementation Analysis

The FLP recombinase of the 2 microns plasmid of Saccharomyces cerevisiae is a member of the integrase family of site-specific recombinases. Recombination catalyzed by members of this family proceeds via the ordered cleavage and religation of four strands of DNA. Although the amino acid sequences of integrase family members are quite different, each recombinase maintains an absolutely conserved tetrad of amino acids (R-191, H-305, R-308, Y-343; numbers are those of the FLP protein). This tetrad is presumed to reflect a common chemical mechanism for cleavage and ligation that has evolved among all family members. The tyrosine is the nucleophile that causes phosphodiester bond cleavage and covalently attaches to the 3'-PO4 terminus, whereas the other three residues have been implicated in ligation of strands. It has recently been shown that cleavage by FLP takes place in trans; that is, a FLP molecule binds adjacent to the site of cleavage but receives the nucleophilic tyrosine from a molecule of FLP that is bound to another FLP-binding element (J.-W. Chen, J. Lee, and M. Jayaram, Cell 69:647-658, 1992). These studies led us to examine whether the ligation step of the FLP reaction is performed by the FLP molecule bound adjacent to the cleavage site (ligation in cis). We have found that FLP promotes ligation in cis. Furthermore, using in vitro complementation analysis, we have classified several mutant FLP proteins into one of two groups: those proteins that are cleavage competent but ligation deficient (group I) and those that are ligation competent but cleavage defective (group II). This observation suggests that the active site of FLP is composed of several amino acid residues from each of two FLP molecules.

Functional analysis of Box II mutations in yeast site-specific recombinases Flp and R: Significance of amino acid conservation within the Int family and the yeast sub-family

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that share approximately 30% amino acid matches. They exhibit a common reaction mechanism that appears to be conserved within the larger Integrase family of site-specific recombinases. Two regions of the proteins, designated as Box I and Box II, harbor, in addition to amino acid conservation, a significantly high degree of nucleotide sequence homology within their coding segments. Box II also contains two amino acids, a histidine and an arginine, that are invariant throughout the Int family. We have performed functional analysis of Flp and R variants carrying point mutations within the Box II segment. Several positions within Box II can tolerate substitutions with no effect, or only modest effects on recombination. Alterations of the Int family residues, His305 and Arg3O8, in the R protein lead to the arrest of recombination at the strand cleavage or the strand exchange step. This is very similar to previously observed “step-arrest” phenotypes in Flp variants altered at these positions and has strong implications for the catalytic mechanism of recombination. Flp and R variants at His305 and His309 can be complemented in half-site strand transfer by a corresponding Tyr343 to phenylalanine variant. In contrast to Arg308 Flp variants, which are efficiently complemented in half-site strand transfer by Flp(Y343F), no strong complementation has been observed between Arg308 variants of R and R (Y343F).

Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine.

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.