Bacteriophage Mu Transposition Research Articles

High-Throughput Mapping of Chromosomal Conformations in E. coli Under Physiological Conditions Using Massively Multiplexed Mu Transposition.

Many approaches for measuring three-dimensional chromosomal conformations rely upon formaldehyde crosslinking followed by subsequent proximity ligation, a family of methods exemplified by 3C, Hi-C, etc. Here we provide an alternative crosslinking-free procedure for high-throughput identification of long-range contacts in the chromosomes of enterobacteria, making use of contact-dependent transposition of phage Mu to identify distant loci in close contact. The procedure described here will suffice to provide a comprehensive map of transposition frequencies between tens of thousands of loci in a bacterial genome, with the resolution limited by the diversity of the insertion site library used and the sequencing depth applied.

Deep sequencing reveals new roles for MuB in transposition immunity and target-capture, and redefines the insular Ter region of E. coli

BackgroundThe target capture protein MuB is responsible for the high efficiency of phage Mu transposition within the E. coli genome. However, some targets are off-limits, such as regions immediately outside the Mu ends (cis-immunity) as well as the entire ~ 37 kb genome of Mu (Mu genome immunity). Paradoxically, MuB is responsible for cis-immunity and is also implicated in Mu genome immunity, but via different mechanisms. This study was undertaken to dissect the role of MuB in target choice in vivo.ResultsWe tracked Mu transposition from six different starting locations on the E. coli genome, in the presence and absence of MuB. The data reveal that Mu’s ability to sample the entire genome during a single hop in a clonal population is independent of MuB, and that MuB is responsible for cis-immunity, plays a minor role in Mu genome immunity, and facilitates insertions into transcriptionally active regions. Unexpectedly, transposition patterns in the absence of MuB have helped extend the boundaries of the insular Ter segment of the E. coli genome.ConclusionsThe results in this study demonstrate unambiguously the operation of two distinct mechanisms of Mu target immunity, only one of which is wholly dependent on MuB. The study also reveals several interesting and hitherto unknown aspects of Mu target choice in vivo, particularly the role of MuB in facilitating the capture of promoter and translation start site targets, likely by displacing macromolecular complexes engaged in gene expression. So also, MuB facilitates transposition into the restricted Ter region of the genome.

A Well-Mixed E. coli Genome: Widespread Contacts Revealed by Tracking Mu Transposition

The three-dimensional structures of chromosomes are increasingly being recognized as playing a major role in cellular regulatory states. The efficiency and promiscuity of phage Mu transposition was exploited to directly measure invivo interactions between genomic loci in E.coli. Two global organizing principles have emerged: first, the chromosome is well-mixed and uncompartmentalized, with transpositions occurring freely between all measured loci; second, several gene families/regions show "clustering": strong three-dimensional co-localization regardless of linear genomic distance. The activities of the SMC/condensin protein MukB and nucleoid-compacting protein subunit HU-α are essential for the well-mixed state; HU-α is also needed for clustering of 6/7 ribosomal RNA-encoding loci. The data are explained by a model in which the chromosomal structure is driven by dynamic competition between DNA replication and chromosomal relaxation, providing a foundation for determining how region-specific properties contribute to both chromosomal structure and gene regulation.

A Dynamic <i>E. coli</i> Genome: Widespread DNA Contacts Revealed by Monitoring Mu Transposition

The problem of compacting genomes while still performing cellular processes is common to all life forms. The current view for E. coli is a compacted genome with well-organized domains where replication initiates and terminates. Some studies have suggested that these and other regions are sequestered or compartmentalized such that there is little to no interaction between them. In this work, we have exploited the high efficiency and promiscuity of phage Mu transposition to directly measure the in vivo rates of interactions between genomic loci, and have developed new tools for analyzing the proximity of loci across the genome. We observe widespread contacts between all regions of the E. coli chromosome, revealing a dynamic, effectively un-compartmentalized genome. We detect long-range interactions between several genes in different gene families such as dna and rrna, implicating spatial proximity of many distantly co-regulated genes for the first time in a prokaryote. We also see a higher interaction between the two halves of the chromosome during replication, consistent with the deduced proximity and higher mobility of the chromosomal arms as they segregate during replication. Our work advances a new view of genome organization in E. coli.

The N-terminal domain of MuB protein has striking structural similarity to DNA-binding domains and mediates MuB filament-filament interactions.

MuB is an ATP-dependent DNA-binding protein that regulates the activity of MuA transposase and delivers the target DNA for transposition of phage Mu. Mechanistic insight into MuB function is limited to its AAA+ ATPase module, which upon ATP binding assembles into helical filaments around the DNA. However, the structure and function of the flexible N-terminal domain (NTD) appended to the AAA+ module remains uncharacterized. Here we report the solution structure of MuB NTD determined by NMR spectroscopy. The structure reveals a compact domain formed by four α-helices connected by short loops, and confirms the presence of a helix-turn-helix motif. High structural similarity and sequence homology with λ repressor-like DNA-binding domains suggest a possible role of MuB NTD in DNA binding. We also demonstrate that the NTD directly mediates the ability of MuB to establish filament-filament interactions. These findings lead us to a model in which the NTD interacts with the AAA+ spirals and perhaps also with the DNA bound within the filament, favoring MuB polymerization and filament clustering. We propose that the MuB NTD-dependent filament interactions might be an effective mechanism to bridge distant DNA regions during Mu transposition.

An assay to monitor the activity of DNA transposition complexes yields a general quality control measure for transpositional recombination reactions.

Transposon-based technologies have many applications in molecular biology and can be used for gene delivery into prokaryotic and eukaryotic cells. Common transpositional activity measurement assays suitable for many types of transposons would be beneficial, as diverse transposon systems could be compared for their performance attributes. Therefore, we developed a general-purpose assay to enable and standardize the activity measurement for DNA transposition complexes (transpososomes), using phage Mu transposition as a test platform. This assay quantifies transpositional recombination efficiency and is based on an in vitro transposition reaction with a target plasmid carrying a lethal ccdB gene. If transposition targets ccdB, this gene becomes inactivated, enabling plasmid-receiving Escherichia coli cells to survive and to be scored as colonies on selection plates. The assay was validated with 3 mini-Mu transposons varying in size and differing in their marker gene constitution. Tests with different amounts of transposon DNA provided a linear response and yielded a 10-fold operational range for the assay. The colony formation capacity was linearly correlated with the competence status of the E.coli cells, enabling normalization of experimental data obtained with different batches of recipient cells. The developed assay can now be used to directly compare transpososome activities with all types of mini-Mu transposons, regardless of their aimed use. Furthermore, the assay should be directly applicable to other transposition-based systems with a functional in vitro reaction, and it provides a dependable quality control measure that previously has been lacking but is highly important for the evaluation of current and emerging transposon-based applications.

MuB gives a new twist to target DNA selection

Transposition target immunity is a phenomenon observed in some DNA transposons that are able to distinguish the host chromosome from their own DNA sequence, thus avoiding self-destructive insertions. The first molecular insight into target selection and immunity mechanisms came from the study of phage Mu transposition, which uses the protein MuB as a barrier to self-insertion. MuB is an ATP-dependent non-specific DNA binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. However, a detailed mechanistic understanding of MuB functioning was hindered by the poor solubility of the MuB-ATP complexes. Here we comment on the recent discovery that MuB is an AAA+ ATPase that upon ATP binding assembles into helical filaments that coat the DNA. Remarkably, the helical parameters of the MuB filament do not match those of the bound DNA. This intriguing mismatch symmetry led us to propose a model on how MuB targets DNA for transposition, favoring DNA bending and recognition by the transposase at the filament edge. We also speculate on a different protective role of MuB during immunity, where filament stickiness could favor the condensation of the DNA into a compact state that occludes it from the transposase.

The μ transpososome structure sheds light on DDE recombinase evolution.

Studies of bacteriophage Mu transposition paved the way for understanding retroviral integration and V(D)J recombination as well as many other DNA transposition reactions. Here we report the structure of Mu transposase (MuA) in complex with bacteriophage DNA ends and target DNA, determined from data that extend anisotropically to 5.2/5.2/3.7Å resolution, in conjunction with previously-determined structures of individual domains. The highly intertwined structure illustrates why chemical activity depends on formation of the synaptic complex, and reveals that individual domains play different roles when bound to different sites. It also suggests explanations for the increased stability of the final product complex and for its preferential recognition by the ATP-dependent unfoldase ClpX. Although MuA and many other recombinases share a structurally conserved “DDE” catalytic domain, comparisons among the limited set of available complex structures suggest that some conserved features, such as catalysis in trans and target DNA bending, arose through convergent evolution because they are important for function.

A New Role for Translation Initiation Factor 2 in Maintaining Genome Integrity

Escherichia coli translation initiation factor 2 (IF2) performs the unexpected function of promoting transition from recombination to replication during bacteriophage Mu transposition in vitro, leading to initiation by replication restart proteins. This function has suggested a role of IF2 in engaging cellular restart mechanisms and regulating the maintenance of genome integrity. To examine the potential effect of IF2 on restart mechanisms, we characterized its influence on cellular recovery following DNA damage by methyl methanesulfonate (MMS) and UV damage. Mutations that prevent expression of full-length IF2-1 or truncated IF2-2 and IF2-3 isoforms affected cellular growth or recovery following DNA damage differently, influencing different restart mechanisms. A deletion mutant (del1) expressing only IF2-2/3 was severely sensitive to growth in the presence of DNA-damaging agent MMS. Proficient as wild type in repairing DNA lesions and promoting replication restart upon removal of MMS, this mutant was nevertheless unable to sustain cell growth in the presence of MMS; however, growth in MMS could be partly restored by disruption of sulA, which encodes a cell division inhibitor induced during replication fork arrest. Moreover, such characteristics of del1 MMS sensitivity were shared by restart mutant priA300, which encodes a helicase-deficient restart protein. Epistasis analysis indicated that del1 in combination with priA300 had no further effects on cellular recovery from MMS and UV treatment; however, the del2/3 mutation, which allows expression of only IF2-1, synergistically increased UV sensitivity in combination with priA300. The results indicate that full-length IF2, in a function distinct from truncated forms, influences the engagement or activity of restart functions dependent on PriA helicase, allowing cellular growth when a DNA–damaging agent is present.

A method to sequence and quantify DNA integration for monitoring outcome in gene therapy

Human genetic diseases have been successfully corrected by integration of functional copies of the defective genes into human cells, but in some cases integration of therapeutic vectors has activated proto-oncogenes and contributed to leukemia. For this reason, extensive efforts have focused on analyzing integration site populations from patient samples, but the most commonly used methods for recovering newly integrated DNA suffer from severe recovery biases. Here, we show that a new method based on phage Mu transposition in vitro allows convenient and consistent recovery of integration site sequences in a form that can be analyzed directly using DNA barcoding and pyrosequencing. The method also allows simple estimation of the relative abundance of gene-modified cells from human gene therapy subjects, which has previously been lacking but is crucial for detecting expansion of cell clones that may be a prelude to adverse events.

Universal platform for quantitative analysis of DNA transposition

BackgroundCompleted genome projects have revealed an astonishing diversity of transposable genetic elements, implying the existence of novel element families yet to be discovered from diverse life forms. Concurrently, several better understood transposon systems have been exploited as efficient tools in molecular biology and genomics applications. Characterization of new mobile elements and improvement of the existing transposition technology platforms warrant easy-to-use assays for the quantitative analysis of DNA transposition.ResultsHere we developed a universal in vivo platform for the analysis of transposition frequency with class II mobile elements, i.e., DNA transposons. For each particular transposon system, cloning of the transposon ends and the cognate transposase gene, in three consecutive steps, generates a multifunctional plasmid, which drives inducible expression of the transposase gene and includes a mobilisable lacZ-containing reporter transposon. The assay scores transposition events as blue microcolonies, papillae, growing within otherwise whitish Escherichia coli colonies on indicator plates. We developed the assay using phage Mu transposition as a test model and validated the platform using various MuA transposase mutants. For further validation and to illustrate universality, we introduced IS903 transposition system components into the assay. The developed assay is adjustable to a desired level of initial transposition via the control of a plasmid-borne E. coli arabinose promoter. In practice, the transposition frequency is modulated by varying the concentration of arabinose or glucose in the growth medium. We show that variable levels of transpositional activity can be analysed, thus enabling straightforward screens for hyper- or hypoactive transposase mutants, regardless of the original wild-type activity level.ConclusionsThe established universal papillation assay platform should be widely applicable to a variety of mobile elements. It can be used for mechanistic studies to dissect transposition and provides a means to screen or scrutinise transposase mutants and genes encoding host factors. In succession, improved versions of transposition systems should yield better tools for molecular biology and offer versatile genome modification vehicles for many types of studies, including gene therapy and stem cell research.

Phage Mu Transposition Immunity: Protein Pattern Formation along DNA by a Diffusion-Ratchet Mechanism

DNA transposons integrate into host chromosomes with limited target sequence specificity. Without mechanisms to avoid insertion into themselves, transposons risk self-destruction. Phage Mu avoids this problem by transposition immunity, involving MuA-transposase and MuB ATP-dependent DNA-binding protein. MuB-bound DNA acts as an efficient transposition target, but MuA clusters bound to Mu DNA ends activate the MuB-ATPase and dissociate MuB from their neighborhood before target site commitment, making the regions near Mu ends a poor target. This MuA-cluster-MuB interaction requires formation of DNA loops between the MuA- and the MuB-bound DNA sites. At early times, MuB clusters are disassembled via loops with smaller average size, and at later times, MuA clusters find distantly located MuB clusters by forming loops with larger average sizes. We demonstrate that iterative loop formation/disruption cycles with intervening diffusional steps result in larger DNA loops, leading to preferential insertion of the transposon at sites distant from the transposon ends.

LettingEscherichia coliTeach Me About Genome Engineering

A career of following unplanned observations has serendipitously led to a deep appreciation of the capacity that bacterial cells have for restructuring their genomes in a biologically responsive manner. Routine characterization of spontaneous mutations in the gal operon guided the discovery that bacteria transpose DNA segments into new genome sites. A failed project to fuse lambda sequences to a lacZ reporter ultimately made it possible to demonstrate how readily Escherichia coli generated rearrangements necessary for in vivo cloning of chromosomal fragments into phage genomes. Thinking about the molecular mechanism of IS1 and phage Mu transposition unexpectedly clarified how transposable elements mediate large-scale rearrangements of the bacterial genome. Following up on lab lore about long delays needed to obtain Mu-mediated lacZ protein fusions revealed a striking connection between physiological stress and activation of DNA rearrangement functions. Examining the fate of Mudlac DNA in sectored colonies showed that these same functions are subject to developmental control, like controlling elements in maize. All these experiences confirmed Barbara McClintock's view that cells frequently respond to stimuli by restructuring their genomes and provided novel insights into the natural genetic engineering processes involved in evolution.

Generation of Single-Copy Transposon Insertions in Clostridium perfringens by Electroporation of Phage Mu DNA Transposition Complexes

Transposon mutagenesis is a tool that is widely used for the identification of genes involved in the virulence of bacteria. Until now, transposon mutagenesis in Clostridium perfringens has been restricted to the use of Tn916-based methods with laboratory reference strains. This system yields primarily multiple transposon insertions in a single genome, thus compromising its use for the identification of virulence genes. The current study describes a new protocol for transposon mutagenesis in C. perfringens, which is based on the bacteriophage Mu transposition system. The protocol was successfully used to generate a single-insertion mutant library both for a laboratory strain and for a field isolate. Thus, it can be used as a tool in large-scale screening to identify virulence genes of C. perfringens.

3P-090 バクテリオファージMuの標的免疫反応の光学顕微鏡下での直接観察(核酸結合蛋白質,第47回日本生物物理学会年会)

3P-090 バクテリオファージMuの標的免疫反応の光学顕微鏡下での直接観察(核酸結合蛋白質,第47回日本生物物理学会年会)

DNA transposition target immunity and the determinants of the MuB distribution patterns on DNA

MuB, an ATP-dependent DNA-binding protein, is critical for the selection of target sites on the host chromosome during the phage Mu transposition. We developed a multichannel fluidic system to study the MuB-DNA interaction dynamics at the single DNA molecule level by total internal reflection fluorescence microscopy. We analyzed the distribution of MuB along DNA during the assembly and disassembly of MuB polymers on immobilized DNA molecules. The results reveal the absence of a significant correlation of MuB polymer distribution between the assembly and disassembly phases. These observations argue against a model in which MuB polymers on DNA represent a mixture of higher and lower affinity forms, with higher affinity forms being the first to appear and the last to disappear. Instead, assembly and disassembly of MuB polymers involve independent stochastic events. Additionally, we demonstrate that MuB disassembles from the polymer ends at a higher rate than from internal regions of the polymer and MuA stimulates MuB disassembly both at the polymer ends and internally.

Interactions of Phage Mu Enhancer and Termini that Specify the Assembly of a Topologically Unique Interwrapped Transpososome

The higher-order DNA–protein complex that carries out the chemical steps of phage Mu transposition is organized by bridging interactions among three DNA sites, the left (L) and right (R) ends of Mu, and an enhancer element (E), mediated by the transposase protein MuA. A subset of the six subunits of MuA associated with their cognate sub-sites at L and R communicate with the enhancer to trigger the stepwise assembly of the functional transpososome. The DNA follows a well-defined path within the transpososome, trapping five supercoil nodes comprising two E-R crossings, one E-L crossing and two L-R crossings. The enhancer is a critical DNA element in specifying the unique interwrapped topology of the three-site LER synapse. In this study, we used multiple strategies to characterize Mu end–enhancer interactions to extend, modify and refine those inferred from earlier analyses. Directed placement of transposase subunits at their cognate sub-sites at L and R, analysis of the protein composition of transpososomes thus obtained, and their characterization using topological methods define the following interactions. R1–E interaction is essential to promote transpososome assembly, R3–E interaction contributes to the native topology of the transpososome, and L1–E and R2–E interactions are not required for assembly. The data on L2–E and L3–E interactions are not unequivocal. If they do occur, either one is sufficient to support the assembly process. Our results are consistent with two R–E and perhaps one L–E, being responsible for the three DNA crossings between the enhancer and the left and right ends of Mu. A 3D representation of the interwrapped complex (IW) obtained by modeling is consistent with these results. The model reveals straightforward geometric and topological relationships between the IW complex and a more relaxed enhancer-independent V-form of the transpososome assembled under altered reaction conditions.

SNP discovery by mismatch-targeting of Mu transposition

Single nucleotide polymorphisms (SNPs) represent a valuable resource for the mapping of human disease genes and induced mutations in model organisms. SNPs may become the markers of choice also for population ecology and evolutionary studies, but their isolation for non-model organisms with unsequenced genomes is often difficult. Here, we describe a rapid and cost-effective strategy to isolate SNPs that exploits the property of the bacteriophage Mu transposition machinery to target mismatched DNA sites and thereby to effectively detect polymorphic loci. To demonstrate the methodology, we isolated 164 SNPs from the unsequenced genome of the Glanville fritillary butterfly (Melitaea cinxia), a much-studied species in population biology, and we validated 24 of them. The strategy involves standard molecular biology techniques as well as undemanding MuA transposase-catalyzed in vitro transposition reactions, and it is applicable to any organism.

Isolation and Characterization of Biofilm Formation-Defective Mutants of Staphylococcus aureus

Staphylococcus aureus produces biofilm and this mode of colonization facilitates infections that are often difficult to treat and engender high morbidity and mortality. We have exploited bacteriophage Mu transposition methods to create an insertional mutant library in a highly biofilm-forming S. aureus clinical isolate. Our screen identified 38 insertions in 23 distinct genes together with one intergenic region that significantly reduced biofilm formation. Nineteen insertions were mapped in loci not previously known to affect biofilm in this organism. These include insertions in codY, srrA, mgrA, and fmtA, a putative DEAD-box helicase, two members of the zinc-metallo-beta lactamase/beta-CASP family, and a hypothetical protein with a GGDEF motif. Fifteen insertions occurred in the icaADBC operon, which produces intercellular adhesion antigen (PIA) and is important for biofilm formation in many strains of S. aureus and Staphylococcus epidermidis. Obtaining a high proportion of independent Em-Mu disruptions in icaADBC demonstrated both the importance of PIA for biofilm formation in this clinical strain and the strong validation of the screening procedure that concomitantly uncovered additional mutants. All non-ica mutants were further analyzed by immunoblotting and biochemical fractionation for perturbation of PIA and wall teichoic acid. PIA levels were diminished in the majority of non-ica insertional mutants. Three mutant strains were chosen and were functionally complemented for restored biofilm formation by transformation with plasmids carrying the cloned wild-type gene under the control of a xylose-inducible promoter. This is a comprehensive collection of biofilm-defective mutants that underscores the multifactorial genetic program underlying the establishment of biofilm in this insidious pathogen.

Meox1Cre: A mouse line expressing Cre recombinase in somitic mesoderm

We developed a novel strategy based on in vitro DNA transposition of phage Mu to construct vectors for "knock-in" of the gene encoding Cre recombinase into endogenous loci in embryonic stem cells. This strategy was used to introduce Cre into the mouse Meox1 locus, which was expected to drive Cre expression in the presomitic and somitic mesoderm. In embryos heterozygous for both Meox1(Cre) and R26R or Z/AP reporter alleles, specific and efficient recombination of the reporter alleles was detected in the maturing somites and their derivatives, including developing vertebrae, skeletal muscle, back dermis, as well as endothelium of the blood vessels invading the spinal cord and developing limbs. In contrast to the somitic mesoderm, Cre activity was not observed in the cranial paraxial mesoderm. Thus, the Meox1(Cre) allele allows detailed fate-mapping of Meox1-expressing tissues, including derivatives of the somitic mesoderm. We used it to demonstrate dynamic changes in the composition of the mesenchyme surrounding the developing inner ear. Meox1(Cre) may also be used for tissue-specific mutagenesis in the somitic mesoderm and its derivatives.