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Background: Large serine integrases (LSIs, derived from temperate phages) have been adapted for use in a multipart DNA assembly process in vitro, called serine integrase recombinational assembly (SIRA). The versatility, efficiency, and fidelity of SIRA is limited by lack of a sufficient number of LSIs whose activities have been characterized in vitro., Methods and Major Results: In this report, we compared the activities in vitro of 10 orthogonal LSIs to explore their suitability for multiplex SIRA reactions. We found that Bxb1, ϕR4, and TG1 integrases were the most active among the set we studied, but several others were also usable. As proof of principle, we demonstrated high-efficiency one-pot assembly of six DNA fragments (made by PCR) into a 7.5 kb plasmid that expresses the enzymes of the β-carotenoid pathway in Escherichia coli, using six different LSIs. We further showed that a combined approach using a few highly active LSIs, each acting on multiple pairs of att sites with distinct central dinucleotides, can be used to scale up "poly-part" gene assembly and editing., Conclusions and Implications: We conclude that use of multiple orthogonal integrases may be the most predictable, efficient, and programmable approach for SIRA and other in vitro applications., (© 2022 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

The structure and diversity of all open microbial communities are shaped by individual births, deaths, speciation and immigration events; the precise timings of these events are unknowable and unpredictable. This randomness is manifest as ecological drift in the population dynamics, the importance of which has been a source of debate for decades. There are theoretical reasons to suppose that drift would be imperceptible in large microbial communities, but this is at odds with circumstantial evidence that effects can be seen even in huge, complex communities. To resolve this dichotomy we need to observe dynamics in simple systems where key parameters, like migration, birth and death rates can be directly measured. We monitored the dynamics in the abundance of two genetically modified strains of Escherichia coli, with tuneable growth characteristics, that were mixed and continually fed into 10 identical chemostats. We demonstrated that the effects of demographic (non-environmental) stochasticity are very apparent in the dynamics. However, they do not conform to the most parsimonious and commonly applied mathematical models, where each stochastic event is independent. For these simple models to reproduce the observed dynamics we need to invoke an 'effective community size', which is smaller than the census community size., (© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Decatenation is a crucial in vivo reaction of DNA topoisomerases in DNA replication and is frequently used in in vitro drug screening. Usually this reaction is monitored using kinetoplast DNA as a substrate, although this assay has several limitations. Here we have engineered a substrate for Tn 3 resolvase that generates a singly-linked catenane that can readily be purified from the DNA substrate after restriction enzyme digestion and centrifugation. We show that this catenated substrate can be used with high sensitivity in topoisomerase assays and drug-inhibition assays.

A device that counts and records the number of events experienced by an individual cell could have many uses in experimental biology and biotechnology. Here, we report a DNA-based 'latch' that switches between two states upon each exposure to a repeated stimulus. The key component of the latch is a DNA segment whose orientation is inverted by the actions of ϕC31 integrase and its recombination directionality factor (RDF). Integrase expression is regulated by an external input, while RDF expression is controlled by the state of the latch, such that the orientation of the invertible segment switches efficiently each time the device receives an input pulse. Recombination occurs over a time scale of minutes after initiation of integrase expression. The latch requires a delay circuit, implemented with a transcriptional repressor expressed in only one state, to ensure that each input pulse results in only one inversion of the DNA segment. Development and optimization of the latch in living cells was driven by mathematical modelling of the recombination reactions and gene expression regulated by the switch. We discuss how N latches built with orthogonal site-specific recombination systems could be chained together to form a binary ripple counter that could count to 2N - 1., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Transposons are invaluable biological tools for the genetic manipulation of microorganisms. ISY100 from Synechocystis sp. PCC6803 is a member of the Tc1/ mariner /IS 630 superfamily, and is characterized by high transposition efficiency and a strong preference for TA target sequences. In this paper, we describe the design and application of a mini-ISY100 suicide vector for the in vivo creation of stable random transposon insertion libraries. The system was successfully applied in seven species belonging to four different orders of γ proteobacteria. In all cases, delivery using conjugation consistently showed the highest transposition efficiency compared to chemical transformation or electroporation. We determined the frequency of transposon insertions in all the species and proved the utility of the system by identifying genes involved in colony coloration in Shewanella oneidensis . The ease and the efficiency of the protocol developed here allow the creation of complete knock-out libraries in an extensive range of host microorganisms in less than a week with no requirement for preparatory modification.

Dual-state genetic switches that can change their state in response to input signals can be used in synthetic biology to encode memory and control gene expression. A transcriptional toggle switch (TTS), with two mutually repressing transcription regulators, was previously used for switching between two expression states. In other studies, serine integrases have been used to control DNA inversion switches that can alternate between two different states. Both of these switches use two different inputs to switch ON or OFF. Here, we use mathematical modelling to design a robust one-input binary switch, which combines a TTS with a DNA inversion switch. This combined circuit switches between the two states every time it receives a pulse of a single-input signal. The robustness of the switch is based on the bistability of its TTS, while integrase recombination allows single-input control. Unidirectional integrase-RDF-mediated recombination is provided by a recently developed integrase-RDF fusion protein. We show that the switch is stable against parameter variations and molecular noise, making it a promising candidate for further use as a basic element of binary counting devices., (© 2018 The Authors.)

Bacteriophage serine integrases are extensively used in biotechnology and synthetic biology for assembly and rearrangement of DNA sequences. Serine integrases promote recombination between two different DNA sites, attP and attB, to form recombinant attL and attR sites. The 'reverse' reaction requires another phage-encoded protein called the recombination directionality factor (RDF) in addition to integrase; RDF activates attL × attR recombination and inhibits attP × attB recombination. We show here that serine integrases can be fused to their cognate RDFs to create single proteins that catalyse efficient attL × attR recombination in vivo and in vitro, whereas attP × attB recombination efficiency is reduced. We provide evidence that activation of attL × attR recombination involves intra-subunit contacts between the integrase and RDF moieties of the fusion protein. Minor changes in the length and sequence of the integrase-RDF linker peptide did not affect fusion protein recombination activity. The efficiency and single-protein convenience of integrase-RDF fusion proteins make them potentially very advantageous for biotechnology/synthetic biology applications. Here, we demonstrate efficient gene cassette replacement in a synthetic metabolic pathway gene array as a proof of principle., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Assembling multiple DNA fragments into functional plasmids is an important and often rate-limiting step in engineering new functions in living systems. Bacteriophage integrases are enzymes that carry out efficient recombination reactions between short, defined DNA sequences known as att sites. These DNA splicing reactions can be used to assemble large numbers of DNA fragments into a functional circular plasmid in a method termed serine integrase recombinational assembly (SIRA). The resulting DNA assemblies can easily be modified by further recombination reactions catalyzed by the same integrase in the presence of its recombination directionality factor (RDF). Here we present a set of protocols for the overexpression and purification of bacteriophage ϕC31 and Bxb1 integrase and RDF proteins, their use in DNA assembly reactions, and subsequent modification of the resulting DNA assemblies.

Serine integrases catalyse site-specific recombination to integrate and excise bacteriophage genomes into and out of their host's genome. These enzymes exhibit remarkable directionality; in the presence of the integrase alone, recombination between attP and attB DNA sites is efficient and irreversible, giving attL and attR products which do not recombine further. However, in the presence of the bacteriophage-encoded recombination directionality factor (RDF), integrase efficiently promotes recombination between attL and attR to re-form attP and attB The DNA substrates and products of both reactions are approximately isoenergetic, and no cofactors (such as adenosine triphosphate) are required for recombination. The thermodynamic driving force for directionality of these reactions is thus enigmatic. Here, we present a minimal mathematical model which can explain the directionality and regulation of both 'forward' and 'reverse' reactions. In this model, the substrates of the 'forbidden' reactions (between attL and attR in the absence of RDF, attP and attB in the presence of RDF) are trapped as inactive protein-DNA complexes, ensuring that these 'forbidden' reactions are extremely slow. The model is in good agreement with the observed in vitro kinetics of recombination by ϕC31 integrase, and defines core features of the system necessary and sufficient for directionality., (© 2017 The Authors.)

Serine integrases, DNA site-specific recombinases used by bacteriophages for integration and excision of their DNA to and from their host genomes, are increasingly being used as tools for programmed rearrangements of DNA molecules for biotechnology and synthetic biology. A useful feature of serine integrases is the simple regulation and unidirectionality of their reactions. Recombination between the phage attP and host attB sites is promoted by the serine integrase alone, giving recombinant attL and attR sites, whereas the 'reverse' reaction (between attL and attR) requires an additional protein, the recombination directionality factor (RDF). Here, we present new experimental data on the kinetics and regulation of recombination reactions mediated by ϕC31 integrase and its RDF, and use these data as the basis for a mathematical model of the reactions. The model accounts for the unidirectionality of the attP × attB and attL × attR reactions by hypothesizing the formation of structurally distinct, kinetically stable integrase-DNA product complexes, dependent on the presence or absence of RDF. The model accounts for all the available experimental data, and predicts how mutations of the proteins or alterations of reaction conditions might increase the conversion efficiency of recombination., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Metabolic pathway engineering in microbial hosts for heterologous biosynthesis of commodity compounds and fine chemicals offers a cheaper, greener, and more reliable method of production than does chemical synthesis. However, engineering metabolic pathways within a microbe is a complicated process: levels of gene expression, protein stability, enzyme activity, and metabolic flux must be balanced for high productivity without compromising host cell viability. A major rate-limiting step in engineering microbes for optimum biosynthesis of a target compound is DNA assembly, as current methods can be cumbersome and costly. Serine integrase recombinational assembly (SIRA) is a rapid DNA assembly method that utilizes serine integrases, and is particularly applicable to rapid optimization of engineered metabolic pathways. Using six pairs of orthogonal attP and attB sites with different central dinucleotide sequences that follow SIRA design principles, we have demonstrated that ΦC31 integrase can be used to (1) insert a single piece of DNA into a substrate plasmid; (2) assemble three, four, and five DNA parts encoding the enzymes for functional metabolic pathways in a one-pot reaction; (3) generate combinatorial libraries of metabolic pathway constructs with varied ribosome binding site strengths or gene orders in a one-pot reaction; and (4) replace and add DNA parts within a construct through targeted postassembly modification. We explain the mechanism of SIRA and the principles behind designing a SIRA reaction. We also provide protocols for making SIRA reaction components and practical methods for applying SIRA to rapid optimization of metabolic pathways., (© 2016 Elsevier Inc. All rights reserved.)

Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by C31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. C31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition.

Xer site-specific recombination at cer and psi converts bacterial plasmid multimers into monomers so that they can be efficiently segregated to both daughter cells at cell division. Recombination is catalysed by the XerC and XerD recombinases acting at ~30 bp core sites, and is regulated by the action of accessory proteins bound to accessory DNA sequences adjacent to the core sites. Recombination normally occurs only between sites in direct repeat in a negatively supercoiled circular DNA molecule, and yields two circular products linked together in a right-handed four-node catenane with antiparallel sites. These and other topological results are explained by a model in which the accessory DNA sequences are interwrapped around the accessory proteins, trapping three negative supercoils so that strand exchange by the XerC and XerD yields the observed four-node catenane.

Integrases, such as that of the Streptomyces temperate bacteriophage ϕC31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. ϕC31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a "subunit rotation" mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining att sites, and that many rounds of subunit rotation can occur before the strands are religated. We have analyzed the mechanism of ϕC31 integrase-mediated recombination in a topologically constrained experimental system using hybrid "phes" recombination sites, each of which comprises a ϕC31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180° rotation. However, multiple processive "360° rotation" rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory "gating" mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round.

Transposons of the Tc1/mariner family have been used to integrate foreign DNA stably into the genome of a large variety of different cell types and organisms. Integration is at TA dinucleotides located essentially at random throughout the genome, potentially leading to insertional mutagenesis, inappropriate activation of nearby genes, or poor expression of the transgene. Here, we show that fusion of the zinc-finger DNA-binding domain of Zif268 to the C-terminus of ISY100 transposase leads to highly specific integration into TA dinucleotides positioned 6-17 bp to one side of a Zif268 binding site. We show that the specificity of targeting can be changed using Zif268 variants that bind to sequences from the HIV-1 promoter, and demonstrate a bacterial genetic screen that can be used to select for increased levels of targeted transposition. A TA dinucleotide flanked by two Zif268 binding sites was efficiently targeted by our transposase-Zif268 fusion, suggesting the possibility of designer 'Z-transposases' that could deliver transgenic cargoes to chosen genomic locations.

A key step in cut-and-paste DNA transposition is the pairing of transposon ends before the element is excised and inserted at a new site in its host genome. Crystallographic analyses of the paired-end complex (PEC) formed from precleaved transposon ends and the transposase of the eukaryotic element Mos1 reveals two parallel ends bound to a dimeric enzyme. The complex has a trans arrangement, with each transposon end recognized by the DNA binding region of one transposase monomer and by the active site of the other monomer. Two additional DNA duplexes in the crystal indicate likely binding sites for flanking DNA. Biochemical data provide support for a model of the target capture complex and identify Arg186 to be critical for target binding. Mixing experiments indicate that a transposase dimer initiates first-strand cleavage and suggest a pathway for PEC formation.

The multiresistance plasmid pJHCMW1, first identified in a Klebsiella pneumoniae strain isolated from a neonate with meningitis, includes a Xer recombination site, mwr, with unique characteristics. Efficiency of resolution of mwr-containing plasmid dimers is strongly dependent on the osmotic pressure of the growth medium. An increase in supercoiling density of plasmid DNA was observed as the osmotic pressure of the growth culture decreased. Reporter plasmids containing directly repeated mwr, or the related cer sites were used to test if DNA topological changes were correlated with significant changes in efficiency of Xer recombination. Quantification of Holliday junctions showed that while recombination at cer was efficient at all levels of negative supercoiling, recombination at mwr became markedly less efficient as the level of supercoiling was reduced. These results support a model in which modifications at the level of supercoiling density caused by changes in the osmotic pressure of the culture medium affects resolution of mwr-containing plasmid dimers, a property that separates mwr from other Xer recombination target sites.

In the lysogenic state, bacteriophage P1 is maintained as a low copy-number circular plasmid. Site-specific recombination at loxP by the phage-encoded Cre protein keeps P1 monomeric, thus helping to ensure stable plasmid inheritance. Two Escherichia coli DNA-binding proteins, PepA and ArgR, were recently reported to be necessary for maintenance or establishment of P1 lysogeny. PepA and ArgR bind to regulatory DNA sequences upstream of the ColE1 cer recombination site to regulate site-specific recombination by the XerCD recombinases. This recombination keeps ColE1 in a monomeric state and helps to ensure stable plasmid maintenance. It has been suggested that ArgR and PepA play a similar role in P1 maintenance, regulating Cre recombination by binding to DNA sequences upstream of loxP. Here, we show that ArgR does not bind to its proposed binding site upstream of loxP, and that Cre recombination at loxP in its natural P1 context is not affected by PepA and ArgR in vitro. When sequences upstream of loxP were mutated to allow ArgR binding, PepA and ArgR still had no effect on Cre recombination. Our results demonstrate that PepA requires specific DNA sequences for binding, and that PepA and ArgR have no direct role in Cre recombination at P1 loxP.

The Synechocystis sp. PCC6803 insertion sequence ISY100 (ISTcSa) belongs to the Tc1/mariner/IS630 family of transposable elements. ISY100 transposase was purified and shown to promote transposition in vitro. Transposase binds specifically to ISY100 terminal inverted repeat sequences via an N-terminal DNA-binding domain containing two helix-turn-helix motifs. Transposase is the only protein required for excision and integration of ISY100. Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3' ends and two nucleotides inside the 5' ends. Cleavage of short linear substrates containing a single transposon end was less precise. Transposase also catalysed strand transfer, covalently joining the transposon 3' end to the target DNA. When a donor plasmid carrying a mini-ISY100 was incubated with a target plasmid and transposase, the most common products were insertions of one transposon end into the target DNA, but insertions of both ends at a single target site could be recovered after transformation into Escherichia coli. Insertions were almost exclusively into TA dinucleotides, and the target TA was duplicated on insertion. Our results demonstrate that there are no fundamental differences between the transposition mechanisms of IS630 family elements in bacteria and Tc1/mariner elements in higher eukaryotes.

By placing loxP adjacent to the accessory sequences from the Xer/psi multimer resolution system, we have imposed topological selectivity and specificity on Cre/loxP recombination. In this hybrid recombination system, the Xer accessory protein PepA binds to psi accessory sequences, interwraps them, and brings the loxP sites together such that the product of recombination is a four-node catenane. Here, we investigate communication between PepA and Cre by varying the distance between loxP and the accessory sequences, and by altering the orientation of loxP. The yield of four-node catenane and the efficiency of recombination in the presence of PepA varied with the helical phase of loxP with respect to the accessory sequences. When the orientation of loxP was reversed, or when half a helical turn was added between the accessory sequences and loxP, PepA reversed the preferred order of strand exchange by Cre at loxP. The results imply that PepA and the accessory sequences define precisely the geometry of the synapse formed by the loxP sites, and that this overcomes the innate preference of Cre to initiate recombination on the bottom strand of loxP. Further analysis of our results demonstrates that PepA can stimulate strand exchange by Cre in two distinct synaptic complexes, with the C-terminal domains of Cre facing either towards or away from PepA. Thus, no specific PepA-recombinase interaction is required, and correct juxtaposition of the loxP sites is sufficient to activate Cre in this system.

PepA is an aminopeptidase and also functions as a DNA-binding protein in two unrelated systems in Escherichia coli: Xer site-specific recombination and transcriptional regulation of carAB. In these systems, PepA binds to and brings together distant segments of DNA to form interwrapped, nucleosome-like structures. Here we report the selection of PepA mutants that were unable to support efficient Xer recombination. These mutants were defective in DNA-binding and in transcriptional regulation of carAB, but had normal peptidase activity. The mutations define extended patches of basic residues on the surface of the N-terminal domain of PepA that flank a previously proposed DNA-binding groove in the C-terminal domain of PepA. Our results suggest that DNA passes through this C-terminal groove in the PepA hexamer, and is bound by N-terminal DNA-binding determinants at each end of the groove. Based on our data, we propose a new model for the Xer synaptic complex, in which two recombination sites are wrapped around a single hexamer of PepA, bringing the cross-over sites together for strand exchange by the Xer recombinases. In this model, PepA stabilizes negative plectonemic interwrapping between two segments of DNA by passing one segment through the C-terminal groove while the other is held in place in a loop over the groove.

The product of Xer recombination at directly repeated psi sites on a circular unknotted DNA molecule is a right-hand four-noded catenane. Here, we use tangle equations to analyze the topological changes associated with Xer recombination at psi. This mathematical method allows computation of all possible topological pathways consistent with the experimental data. We give a rigorous mathematical proof that, under reasonable biological assumptions, there are only three solutions to the tangle equations. One of the solutions corresponds to a synaptic complex with antiparallel alignment of recombination core sites, the other two correspond to parallel alignment of cores. We show that all three solutions can be unified into a single three-dimensional model for Xer recombination. Thus the three distinct mathematical solutions do not necessarily represent distinct three-dimensional pathways, and in this case the three distinct tangle solutions are different planar projections of the same three-dimensional configuration.

Site-specific recombination by the Cre recombinase takes place at a simple DNA site (loxP), requires no additional proteins and gives topologically simple recombination products. In contrast, cer and psi sites for Xer recombination contain approximately 150 bp of accessory sequences, require accessory proteins PepA, ArgR and ArcA, and the products are specifically linked to form a four-noded catenane. Here, we use hybrid sites consisting of accessory sequences of cer or psi fused to loxP to probe the function of accessory proteins in site-specific recombination. We show that PepA instructs Cre to produce four-noded catenane, but is not required for recombination at these hybrid sites. Mutants of Cre that require PepA and accessory sequences for efficient recombination were selected. PepA-dependent Cre gave products with a specific topology and displayed resolution selectivity. Our results reveal that PepA acts autonomously in the synapsis of psi and cer accessory sequences and is the main architectural element responsible for intertwining accessory site DNA. We suggest that accessory proteins can activate recombinases simply by synapsing the regulatory DNA sequences, thus bringing the recombination sites together with a specific geometry. This may occur without the need for protein-protein interactions between accessory proteins and the recombinases.

Xer site-specific recombination in Escherichia coli converts plasmid multimers to monomers, thereby ensuring their correct segregation at cell division. Xer recombination at the psi site of plasmid pSC101 is preferentially intramolecular, giving products of a single topology. This intramolecular selectivity is imposed by accessory proteins, which bind at psi accessory sequences and activate Xer recombination at the psi core. Strand exchange proceeds sequentially within the psi core; XerC first exchanges top strands to produce Holliday junctions, then XerD exchanges bottom strands to give final products. In this study, recombination was analysed at sites in which the psi core was inverted with respect to the accessory sequences. A plasmid containing two inverted-core psi sites recombined with a reversed order of strand exchange, but with unchanged product topology. Thus the architecture of the synapse, formed by accessory proteins binding to accessory sequences, determines the order of strand exchange at psi. This finding has important implications for the way in which accessory proteins interact with the recombinases.

In bacteria with circular chromosomes, homologous recombination can generate chromosome dimers that cannot be segregated to daughter cells at cell division. Xer site-specific recombination at dif, a 28-bp site located in the replication terminus region of the chromosome, converts dimers to monomers through the sequential action of the XerC and XerD recombinases. Chromosome dimer resolution requires that dif is positioned correctly in the chromosome, and the activity of FtsK, a septum-located protein that coordinates cell division with chromosome segregation. Here, we show that cycles of XerC-mediated strand exchanges form and resolve Holliday junction intermediates back to substrate irrespective of whether conditions support a complete recombination reaction. The C-terminal domain of FtsK is sufficient to activate the exchange of the second pair of strands by XerD, allowing both intra- and intermolecular recombination reactions to go to completion. Proper positioning of dif in the chromosome and of FtsK at the septum is required to sense the multimeric state of newly replicated chromosomes and restrict complete Xer reactions to dimeric chromosomes.

The structure of aminopeptidase A (PepA), which functions as a DNA-binding protein in Xer site-specific recombination and in transcriptional control of the carAB operon in Escherichia coli, has been determined at 2.5 A resolution. In Xer recombination at cer, PepA and the arginine repressor (ArgR) serve as accessory proteins, ensuring that recombination is exclusively intramolecular. In contrast, PepA homologues from other species have no known DNA-binding activity and are not implicated in transcriptional regulation or control of site-specific recombination. PepA comprises two domains, which have similar folds to the two domains of bovine lens leucine aminopeptidase (LAP). However, the N-terminal domain of PepA, which probably plays a significant role in DNA binding, is rotated by 19 degrees compared with its position in LAP. PepA is a homohexamer of 32 symmetry. A groove that runs from one trimer face across the 2-fold molecular axis to the other trimer face is proposed to be the DNA-binding site. Molecular modelling supports a structure of the Xer complex in which PepA, ArgR and a second PepA molecule are sandwiched along their 3-fold molecular axes, and the accessory sequences of the two recombination sites wrap around the accessory proteins as a right-handed superhelix such that three negative supercoils are trapped.

Xer site-specific recombination at the psi site from plasmid pSC101 displays topological selectivity, such that recombination normally occurs only between directly repeated sites on the same circular DNA molecule. This intramolecular selectivity is important for the biological role of psi, and is imposed by accessory proteins PepA and ArcA acting at accessory DNA sequences adjacent to the core recombination site. Here we show that the selectivity for intramolecular recombination at psi can be bypassed in multiply interlinked catenanes. Xer site-specific recombination occurred relatively efficiently between antiparallel psi sites located on separate rings of right-handed torus catenanes containing six or more nodes. This recombination introduced one additional node into the catenanes. Antiparallel sites on four-noded right-handed catenanes, the normal product of Xer recombination at psi, were not recombined efficiently. Furthermore, parallel psi sites on right-handed torus catenanes were not substrates for Xer recombination. These findings support a model in which psi sites are plectonemically interwrapped, trapping a precise number of supercoils that are converted to four catenation nodes by Xer strand exchange., (Copyright 1999 Academic Press.)

Two recombinases, XerC and XerD, act at the recombination sites psi and cer in plasmids pSC101 and Co1E1 respectively. Recombination at these sites maintains the plasmids in a monomeric state and helps to promote stable plasmid inheritance. The accessory protein PepA acts at both psi and cer to ensure that only intramolecular recombination takes place. An additional accessory protein, ArgR, is required for recombination at cer but not at psi. Here, we demonstrate that the ArcA/ArcB two-component regulatory system of Escherichia coli, which mediates adaptation to anaerobic growth conditions, is required for efficient recombination in vivo at psi. Phosphorylated ArcA binds to psi in vitro and increases the efficiency of recombination at this site. Binding of ArcA to psi may contribute to the formation of a higher order synaptic complex between a pair of psi sites, thus helping to ensure that recombination is intramolecular.

Xer site-specific recombination at ColE1 cer converts plasmid multimers into monomers, thus ensuring the heritable stability of ColE1. Two related recombinase proteins, XerC and XerD, catalyse the strand exchange reaction at a 30 bp recombination core site. In addition, two accessory proteins, PepA and ArgR, are required for recombination at cer. These two accessory proteins are thought to act at 180 bp of accessory sequences adjacent to the cer recombination core to ensure that recombination only occurs between directly repeated sites on the same molecule. Here, we demonstrate that PepA and ArgR interact directly with cer, forming a complex in which the accessory sequences of two cer sites are interwrapped approximately three times in a right-handed fashion. We present a model for this synaptic complex, and propose that strand exchange can only occur after the formation of this complex.

Holliday junctions are key intermediates in homologous genetic recombination. Their resolution requires specialised nucleases that nick pairs of strands at the junction point, leading to the separation of mature recombinants. Resolution occurs in either of two orientations, according to which DNA strands are cut. We show that DNA topology can determine the efficiency and outcome of a recombination reaction. Using two Holliday junction resolvases, Escherichia coli RuvC protein and T7 endonuclease I, we observed that supercoiled figure-8 DNA molecules containing Holliday junctions were resolved with a specific orientation bias, and that this bias was reversed by the presence of a topological tether (catenation). In contrast, when all topological constraints were removed by restriction digestion, the recombination intermediates were resolved equally in the two orientations. These results show that topological constraints affecting Holliday junction structure influence the orientation of resolution by cellular resolvases.

The product topology of Xer-mediated site-specific recombination at plasmid sites has been determined. The product of deletion at pSC101 psi is a right-handed antiparallel 4-noded catenane. The ColE1 cer deletion product has an identical topology, except that only one pair of strands is exchanged. These specific product topologies imply that the productive synaptic complex and the strand exchange mechanism have fixed topologies. Further analysis suggests that synapsis traps exactly three negative supercoils between recombining sites, and that strand exchange introduces a further negative topological node in the deletion reaction. We present a model in which the requirement for a specific synaptic stucture, with two recombination sites interwrapped around the accessory proteins ArgR and PepA, ensures that recombination only occurs efficiently between directly repeated sites on the same DNA molecule.

The Xer site-specific recombination system acts at ColE1 cer and pSC101 psi sites to ensure that these plasmids are in a monomeric state prior to cell division. We show that four proteins, ArgR, PepA, XerC and XerD are necessary and sufficient for recombination between directly repeated cer sites on a supercoiled plasmid in vitro. Only PepA, XerC and XerD are required for recombination at psi in vitro. Recombination at cer and psi in vitro requires negative supercoiling and is exclusively intramolecular. Strand exchange at cer produces Holliday junction-containing products in which only the top strands have been exchanged. This reaction requires the catalytic tyrosine residue of Xer C but not that of XerD. Recombination at psi gives catenated circular resolution products. Strand exchange at psi is sequential. XerC catalyses the first (top) strand exchange to make a Holiday junction intermediate and XerD catalyses the second (bottom) strand exchange.

A typing method for bacteria was developed and applied to several species, including Escherichia coli and Actinobacillus actinomycetemcomitans. Total genomic DNA was digested with a restriction endonuclease, and fragments were enabled with [alpha-32P]dATP by using the Klenow fragment of DNA polymerase and separated by electrophoresis in 6% polyacrylamide/8 M urea (sequencing gel). Depending on the restriction endonuclease and the bacterium, the method produced approximately 30-50 well-separated fragments in the size range of 100-400 nucleotides. For A. actinomycetemcomitans, all strains had bands in common. Nevertheless, many polymorphisms could be observed, and the 31 strains tested could be classified into 29 distinct types. Furthermore, serotype-specific fragments could be assigned for the three serotypes investigated. The method described is very sensitive, allowing more distinct types to be distinguished than other commonly used typing methods. When the method was applied to 10 other clinically relevant bacterial species, both species-specific bands and strain-specific bands were found. Isolates from different locations of one patient showed indistinguishable patterns. Computer-assisted analysis of the DNA fingerprints allowed the determination of similarity coefficients. It is concluded that genomic fingerprinting by restriction fragment end labeling (RFEL) is a powerful and generally applicable technique to type bacterial species.

Tc3 is a member of the Tc1/mariner family of transposable elements. All these elements have terminal inverted repeats, encode related transposases and insert exclusively into TA dinucleotides. We have studied the DNA binding properties of Tc3 transposase and found that an N-terminal domain of 65 amino acids binds specifically to two regions within the 462 bp Tc3 inverted repeat; one region is located at the end of the inverted repeat, the other is located approximately 180 bp from the end. Methylation interference experiments indicate that this N-terminal DNA binding domain of the Tc3 transposase interacts with nucleotides on one face of the DNA helix over adjacent major and minor grooves.

The Tc3 transposon of C. elegans belongs to a family of inverted repeat DNA transposons, found in many different phyla. We studied the mechanism of Tc3 transposition by expression of Tc3 transposase from a heat-shock promoter in transgenic nematodes. Transposition is accompanied by the appearance of linear extrachromosomal Tc3 DNA. Analysis of the ends of this presumed transposition intermediate shows that the transposon is excised incompletely: the 5' ends of the transposon lack two nucleotides. The 3' ends coincide with the last nucleotide of the integrated element and carry 3' hydroxyls. The nucleotides that are not coexcised with the transposon remain at the donor site and result in a characteristic footprint. A model is derived for the mechanism of Tc3 jumping that probably applies to the entire family of Tc1/mariner transposable elements.

Normal segregation of the Escherichia coli chromosome and stable inheritance of multicopy plasmids such as ColE1 requires the Xer site-specific recombination system. Two putative lambda integrase family recombinases, XerC and XerD, participate in the recombination reactions. We have constructed an E. coli strain in which the expression of xerC can be tightly regulated, thereby allowing the analysis of controlled recombination reactions in vivo. Xer-mediated recombination in this strain generates Holliday junction-containing DNA molecules in which a specific pair of strands has been exchanged in addition to complete recombinant products. This suggests that Xer site-specific recombination utilizes a strand exchange mechanism similar or identical to that of other members of the lambda integrase family of recombination systems. The controlled in vivo recombination reaction at cer requires recombinase and two accessory proteins, ArgR and PepA. Generation of Holliday junctions and recombinant products is equally efficient in RuvC- and RuvC+ cells, and in cells containing a multicopy RuvC+ plasmid. Controlled XerC expression is also used to analyse the efficiency of recombination between variant cer sites containing sequence alterations and heterologies within their central regions.

The commonly studied Caenorhabditis elegans strain Bristol N2 contains approximately 15 copies per genome of the transposon Tc3. However, Tc3 is not active in Bristol N2. Tc3 contains one major open reading frame (Tc3A). We have fused this open reading frame to an inducible promoter and expressed it in a transgenic Bristol N2 line. Tc3A expression resulted in frequent excision and transposition of endogenous Tc3 elements. This shows that the Bristol N2 genome contains Tc3 transposons that are cis proficient for transposition, but are immobile because Tc3A is absent. We demonstrate that recombinant Tc3A binds specifically to the terminal nucleotides of the Tc3 inverted repeat, indicating that Tc3A is the Tc3 transposase. Activation of Tc3 transposition in vivo was accompanied by the appearance of extrachromosomal, linear copies of Tc3. These may be intermediates in Tc3 transposition.

Site-specific recombination at the plasmid ColE1 cer site requires the Escherichia coli chromosomal gene xerC. The xerC gene has been localized to the 85-min region of the E. coli chromosome, between cya and uvrD. The nucleotide sequences of the xerC gene and flanking regions have been determined. The xerC gene encodes a protein with a calculated molecular mass of 33.8 kDa. This protein has substantial sequence similarity to the lambda integrase family of site-specific recombinases and is probably the cer recombinase. The xerC gene is expressed as part of a multicistronic unit that includes the dapF gene and two other open reading frames.

The heritable stability of ColE1 is dependent on a site-specific recombination system which acts to resolve plasmid multimers into monomers. This plasmid stabilizing recombination system requires the presence in cis of the ColE1 cer region, plus at least two trans-acting factors encoded by the xerA and xerB genes of Escherichia coli. The xerB gene has been cloned and sequenced and found to encode a polypeptide with a calculated mol. wt of 55.3 kd. The predicted amino acid sequence of this protein exhibits striking similarity to that of bovine lens leucine aminopeptidase (53 kd). The biological significance of this similarity is corroborated by genetic and biochemical evidence which suggests that xerB is identical to the E.coli and S.typhimurium pepA genes that encode aminopeptidase A.