PhoA gene fusions in Legionella pneumophila generated in vivo using a new transposon, MudphoA

To enable effective use of phoA gene fusions in Legionella pneumophila, we constructed MudphoA, a derivative of the mini-Mu phage Mu dII4041, which is capable of generating gene fusions to the Escherichia coli alkaline phosphatase gene (EC 3.1.3.1). Although an existing fusion-generating transposon, TnphoA, has been a useful tool for studying secreted proteins in other bacteria, this transposon and other Tn5 derivatives transpose inefficiently in Legionella pneumophila, necessitating the construction of a more effective vector for use in this pathogen. Using MudphoA we generated fusions to an E. coli gene encoding a periplasmic protein and to an L. pneumophila gene encoding an outer membrane protein; both sets of fusions resulted in alkaline phosphatase activity. We have begun to use MudphoA to mutate secreted proteins of L. pneumophila specifically, since this subset of bacterial proteins is most likely to be involved in host-bacterial interactions. This modified transposon may be useful for studies of other bacteria that support transposition of Mu, but not Tn5, derivatives.     

Efficient insertion mutagenesis strategy for bacterial genomes involving electroporation of in vitro-assembled DNA transposition complexes of bacteriophage mu

An efficient insertion mutagenesis strategy for bacterial genomes based on the phage Mu DNA transposition reaction was developed. Incubation of MuA transposase protein with artificial mini-Mu transposon DNA in the absence of divalent cations in vitro resulted in stable but inactive Mu DNA transposition complexes, or transpososomes. Following delivery into bacterial cells by electroporation, the complexes were activated for DNA transposition chemistry after encountering divalent metal ions within the cells. Mini-Mu transposons were integrated into bacterial chromosomes with efficiencies ranging from 10(4) to 10(6) CFU/microg of input transposon DNA in the four species tested, i.e., Escherichia coli, Salmonella enterica serovar Typhimurium, Erwinia carotovora, and Yersinia enterocolitica. Efficiency of integration was influenced mostly by the competence status of a given strain or batch of bacteria. An accurate 5-bp target site duplication flanking the transposon, a hallmark of Mu transposition, was generated upon mini-Mu integration into the genome, indicating that a genuine DNA transposition reaction was reproduced within the cells of the bacteria studied. This insertion mutagenesis strategy for microbial genomes may be applicable to a variety of organisms provided that a means to introduce DNA into their cells is available.     

Efficient Insertion Mutagenesis Strategy for Bacterial Genomes Involving Electroporation of In Vitro-Assembled DNA Transposition Complexes of Bacteriophage Mu

An efficient insertion mutagenesis strategy for bacterial genomes based on the phage Mu DNA transposition reaction was developed. Incubation of MuA transposase protein with artificial mini-Mu transposon DNA in the absence of divalent cations in vitro resulted in stable but inactive Mu DNA transposition complexes, or transpososomes. Following delivery into bacterial cells by electroporation, the complexes were activated for DNA transposition chemistry after encountering divalent metal ions within the cells. Mini-Mu transposons were integrated into bacterial chromosomes with efficiencies ranging from 10⁴ to 10⁶ CFU/μg of input transposon DNA in the four species tested, i.e., Escherichia coli, Salmonella enterica serovar Typhimurium, Erwinia carotovora, and Yersinia enterocolitica. Efficiency of integration was influenced mostly by the competence status of a given strain or batch of bacteria. An accurate 5-bp target site duplication flanking the transposon, a hallmark of Mu transposition, was generated upon mini-Mu integration into the genome, indicating that a genuine DNA transposition reaction was reproduced within the cells of the bacteria studied. This insertion mutagenesis strategy for microbial genomes may be applicable to a variety of organisms provided that a means to introduce DNA into their cells is available.     

Use of Mu phages to isolate transposon insertions juxtaposed to given genes ofEscherichia coli

The small sizes of the DNA fragments transduced by lysates of phage Mu and of mixed lysates of Mu and mini-Mu18A-1 (an internally deleted Mu phage) provide a method for the selection of insertions of transposon Tn10 located very close to givenEscherichia coli genes. Generalized transduction with Mu lysates selected for those insertions located within 38 kilobase pairs of the gene of interest whereas insertions located within about half that distance are directly selected by use of mini-Mu phages. Use of these transduction systems avoids screening of individual colonies by phage P1 transduction for those transposon insertions closely linked to a given gene. Such insertions are most useful for localized mutagenesis and for in vitro molecular cloning.     

Physical characterization of mini-mu and mini-D108

Several derivatives of phages Mu and D108 have been isolated that carry an internal deletion generated by one of the IS1 components of a Tn9 transposon located in the A, B, or S gene of the prenatal phage. The deletions remove most of the lytic functions of the phage but leave intact either genes A and B or gene A and the left and the right end of the phages. These deleted derivatives, called mini-Mu and mini-D108, were physically characterized by electron microscopy and digestion with restriction enzymes. Mini-Mu and mini-D108, which carry an antibiotic resistance marker, are described and some of their genetic properties are summarized in the paper by Toussaint et al. (1981).     

Construction and characteristics of mini-Mu phages

The paper reports on the principles of construction, physical characterization and results of preliminary genetic investigation of hybrid plasmids containing Mu DNA sequences or deletion derivatives of phage Mu, the so-called mini-Mu phages. The mini-Mu were obtained by joining both phage ends within one plasmid in a regular orientation. A collection obtained by in vitro manipulations included 14 recombinant plasmids containing different DNA fragments of the Mu genome. Seven plasmids have both ends of phage Mu, three plasmids containing regularly oriented ends, i.e. mini-phages of different size: the mini-Mu5 (11 kb) within pRM8 plasmid, the mini-Mu4 Ap (18 kb) within pRM6 and the mini-mini-Mu (4.4 kb) within pRM5. The collection comprises mini-Mu phages with the gene kil inactivated after treatment with hydroxylamine. Biological properties of the hybrid plasmids have been preliminary studied.     

Mini-Mu mediates deletion-inversions in vivo by intra-transposon transposition

We have shown that a mini-Mu can transpose into itself in vivo to generate a circle containing only transposon sequences. This deletion-inversion product, which has previously been observed in vitro, is formed by non-replicative transposition and has directly repeated Mu ends. It therefore cannot undergo further rounds of transposition and retains the two copies of the target sequence duplicated in the event. Thus we have been able to confirm that a mini-Mu can undergo non-replicative reactions in vivo and that these generate a 5 bp target site duplication, as has been shown to occur following replicative transposition and lysogenization with Mu.     

Electron microscopic analysis of in vitro transposition intermediates of bacteriophage Mu DNA

Bacteriophage Mu is a highly efficient transposon and the only moveable element for which an in vitro transposition system has been reported. Recently, this system has been used by Craigie and Mizuuchi [Cell 41 (1985) 867-876] to identify and biochemically characterize intermediates in the transposition process. We have utilized the in vitro transposition system to generate intermediates in the transposition process and have analyzed these intermediates by electron-microscopic methods. Partial denaturation mapping has shown the intermediates to be theta-shaped structures in which the phi X174 target DNA is joined to the mini-Mu plasmid at the ends of the Mu genome. Our results are in agreement with the previous biochemical studies and the type of intermediate we observe is exactly what is predicted by the Shapiro model of transposition [Proc. Natl. Acad. Sci. USA 76 (1979) 1933-1937].     

Simultaneous expression of a bacteriophage Mu transposase and repressor: a way of preventing killing due to mini-Mu replication

In vitro studies of bacteriophage Mu transposition have shown that the phage-encoded transposase and repressor bind the same sequences on the phage genome. We attempted to test that prediction in vivo and found that Mu repressor directly inhibits transposition. We also found that, in the absence of repressor, constitutive expression of Mu transposition functions pA and pB is lethal in Escherichia coli strains lysogenic for a mini-Mu and that this is the result of intensive replication of the mini-Mu. These findings have important consequences where such mini-Mus are used as genetic tools. We also tested whether in Erwinia chrysanthemi the effect of transposition functions on a resident mini-Mu was the same as in E. coli. We observed that expression of pA alone was lethal in E. chrysanthemi and that a large fraction of the survivors underwent precise excision of the mini-Mu.     

铜绿假单胞菌PA1550基因对泳动及蹭动的影响

[目的]:研究与铜绿假单胞菌运动能力相关的基因.[方法]:以一株临床分离的铜绿假单胞菌PA68做受体菌,应用人工Mu转座技术建立了库容为2000的突变子文库,从中筛选出泳动能力和蹭动能力丧失或减弱的突变子,通过基因克隆、测序,GenBankBLAST比对测序结果,互补基因表达确定与铜绿假单胞菌运动能力相关的基因.[结果]:突变子Y46在丧失了泳动运动能力的同时,蹭动能力也发生了减弱.在Y46突变子中,Mu转座子插入到功能完全未知的基因PA1550中.对极性效应及PA1550所在操纵子的分析表明,Mu转座子对插入点下游的基因的转录并不造成影响.[结论]:PA1550与铜绿假单胞菌的泳动及蹭动能力有关.     

Cloning of genes from members of the family Enterobacteriaceae with mini-Mu bacteriophage containing plasmid replicons.

An in vivo cloning system that uses derivatives of the Escherichia coli bacteriophage Mu with plasmid replicons has been extended to five different species of the family Enterobacteriaceae. Mu and these mini-Mu replicon elements were introduced into strains of E. coli, Shigella flexneri, Salmonella typhimurium, Citrobacter freundii, and Proteus mirabilis by infection, by transformation, or by conjugation with newly constructed broad-host-range plasmids containing insertions of these elements. Lysates from these cells, lysogenic for Mu and mini-Mu elements, were used to infect sensitive recipient strains of E. coli, S. typhimurium, and C. freundii. Drug-resistant transductants had mini-Mu replicon elements with inserts of different DNA sequences. All of the lysogens made could be induced to yield high phage titers, including those coming from strains that were resistant to Mu and Mu derivatives. Clones of 10 particular genes were isolated by their ability to complement specific mutations in the recipient strains, even in the presence of the E. coli K-12 restriction system. Some of the mini-Mu replicon elements used contained lac gene fusing segments and resulted in fusions of the lac operon to control regions in the cloned sequences.     

Mini-Mulac transposons with broad-host-range origins of conjugal transfer and replication designed for gene regulation studies in Rhizobiaceae

Novel mini-Mu derivatives were constructed, carrying a truncated lacZYA operon fused to the terminal 117 bp of the Mu S-end, for the isolation of translational lac fusions by mini-Mu-mediated insertion mutagenesis. Different selectable markers (chloramphenicol resistance; gentamycin resistance) were introduced to allow selection for mini-Mu insertions in different replicons and bacterial strains. A mini-Mulac derivative carrying the site for conjugal transfer of plasmid RP4 (oriT) and the origin of replication of the Agrobacterium rhizogenes Ri plasmid (oriRiHRI) was constructed to enable one-step lac-fusion mutagenesis of cloned (plasmid-borne) regions in Escherichia coli and efficient conjugal transfer of gene fusions to to a variety of Gram-negative bacteria. The conjugation frequency, stability and copy number of replicons carrying mini-Mulac derivatives with oriT and oriRiHRI in members of the Rhizobiaceae such as Rhizobium meliloti, Azorhizobium caulinodans ORS571 and Agrobacterium tumefaciens C58 was examined.     

Silent mischief: bacteriophage Mu insertions contaminate products of Escherichia coli random mutagenesis performed using suicidal transposon delivery plasmids mobilized by broad-host-range RP4 conjugative machinery

Random transposon mutagenesis is the strategy of choice for associating a phenotype with its unknown genetic determinants. It is generally performed by mobilization of a conditionally replicating vector delivering transposons to recipient cells using broad-host-range RP4 conjugative machinery carried by the donor strain. In the present study, we demonstrate that bacteriophage Mu, which was deliberately introduced during the original construction of the widely used donor strains SM10 λpir and S17-1 λpir, is silently transferred to Escherichia coli recipient cells at high frequency, both by hfr and by release of Mu particles by the donor strain. Our findings suggest that bacteriophage Mu could have contaminated many random-mutagenesis experiments performed on Mu-sensitive species with these popular donor strains, leading to potential misinterpretation of the transposon mutant phenotype and therefore perturbing analysis of mutant screens. To circumvent this problem, we precisely mapped Mu insertions in SM10 λpir and S17-1 λpir and constructed a new Mu-free donor strain, MFDpir, harboring stable hfr-deficient RP4 conjugative functions and sustaining replication of Π-dependent suicide vectors. This strain can therefore be used with most of the available transposon-delivering plasmids and should enable more efficient and easy-to-analyze mutant hunts in E. coli and other Mu-sensitive RP4 host bacteria.     

Application of the bacteriophage Mu-driven system for the integration/amplification of target genes in the chromosomes of engineered Gram-negative bacteria—mini review

The advantages of phage Mu transposition-based systems for the chromosomal editing of plasmid-less strains are reviewed. The cis and trans requirements for Mu phage-mediated transposition, which include the L/R ends of the Mu DNA, the transposition factors MuA and MuB, and the cis/trans functioning of the E element as an enhancer, are presented. Mini-Mu(LR)/(LER) units are Mu derivatives that lack most of the Mu genes but contain the L/R ends or a properly arranged E element in cis to the L/R ends. The dual-component system, which consists of an integrative plasmid with a mini-Mu and an easily eliminated helper plasmid encoding inducible transposition factors, is described in detail as a tool for the integration/amplification of recombinant DNAs. This chromosomal editing method is based on replicative transposition through the formation of a cointegrate that can be resolved in a recombination-dependent manner. (E-plus)- or (E-minus)-helpers that differ in the presence of the trans-acting E element are used to achieve the proper mini-Mu transposition intensity. The systems that have been developed for the construction of stably maintained mini-Mu multi-integrant strains of Escherichia coli and Methylophilus methylotrophus are described. A novel integration/amplification/fixation strategy is proposed for consecutive independent replicative transpositions of different mini-Mu(LER) units with “excisable” E elements in methylotrophic cells.     

Mini-muduction: A new mode of gene transfer mediated by mini-Mu

Summary We compared the transducing properties of Mucts62 and Mucts62/mini-Mu lysates, using Mu immune and non immune Rec⁺ and recA recipient strains. The Mu/mini-Mu lysates transduced all bacterial markers tested 10 times more efficiently than the Mucts62 lysates in Rec⁺ recipients. Most of the transductants obtained after infection with the Mu/mini-Mu lysates result from the substitution of the mutated gene of the recipient by the wild type allele from the donor, most probably carried on the gigantic variable end linked to the mini-Mu genome.Moreover the Mu/mini-Mu lysates gave a new type of Rec-independent transduction that we called mini-muduction. Mini-muduction requires the activity of Mu gene A and provides transductants which carry the transduced marker surrounded by two mini-Mu genomes similarly oriented, and inserted at random location in the recipient chromosome. The mini-Mu/transduced DNA/mini-Mu structures are able to transpose spontaneously, for instance into a transmissible plasmid, in the presence of Mu gene A product.     

A gene truncation strategy generating N- and C-terminal deletion variants of proteins for functional studies: mapping of the Sec1p binding domain in yeast Mso1p by a Mu in vitro transposition-based approach

Bacteriophage Mu in vitro transposition constitutes a versatile tool in molecular biology, with applications ranging from engineering of single genes or proteins to modification of genome segments or entire genomes. A new strategy was devised on the basis of Mu transposition that via a few manipulation steps simultaneously generates a nested set of gene constructions encoding deletion variants of proteins. C-terminal deletions are produced using a mini-Mu transposon that carries translation stop signals close to each transposon end. Similarly, N-terminal deletions are generated using a transposon with appropriate restriction sites, which allows deletion of the 5′-distal part of the gene. As a proof of principle, we produced a set of plasmid constructions encoding both C- and N-terminally truncated variants of yeast Mso1p and mapped its Sec1p-interacting region. The most important amino acids for the interaction in Mso1p are located between residues T46 and N78, with some weaker interactions possibly within the region E79–N105. This general-purpose gene truncation strategy is highly efficient and produces, in a single reaction series, a comprehensive repertoire of gene constructions encoding protein deletion variants, valuable in many types of functional studies. Importantly, the methodology is applicable to any protein-encoding gene cloned in an appropriate vector.     

Mini-Mu-duction as a test for genetic complementation in Escherichia coli.

In mini-Mu-duction, segments of host DNA bracketed between two copies of an internally deleted Mu phage (a mini-Mu) can be packaged within Mu phage particles. Upon infection of a second host strain, the DNA injected by these particles can insert into the chromosomal DNA in a reaction catalyzed by the phage A gene product (transposase), which is independent of homologous recombination. This results in a partially diploid host strain in which the duplicated host DNA is bracketed by two copies of the mini-Mu phage (Faelen et al., Mol. Gen. Genet. 176:191-197, 1979). The frequency of mini-Mu-duction reported previously was low (10(-8) to 10(-9) per recipient cell) thus limiting its use to rather stable mutational lesions. I have increased the frequency of mini-Mu-duction 10- to 100-fold by use of a helper phage lacking the kil gene and by UV irradiation of the phage stocks. I have also shown that mini-Mu-duction is a reliable complementation assay in rec+ as well as recA recipient strains. This genetic complementation test does not require prior gene localization and (due to the extended host range of phage Mu) should be applicable to many enterobacterial species.     

Anchor polymerase chain reaction display: a high-throughput method to resolve, score, and isolate dimorphic genetic markers based on interspersed repetitive DNA elements

Genes which confer a disease when mutated, or for which population variability contributes to a quantitative trait such as longevity or disease susceptibility, can be localized in the genetic map by use of an appropriately dense set of polymorphic DNA markers. Here we describe an anchor PCR method for high-throughput genotyping, which can be used to amplify the DNA segments flanking an interspersed repetitive sequence such as a transposon, and to limit the number of product bands per reaction to facilitate marker resolution. We used this method to amplify and display DNA fragments flanking the Tc1 transposable elements from different strains of the nematode Caenorhabditis elegans, varying widely in insert number, and to analyze marker segregation in recombinant inbred lines generated from an interstrain cross. Since essentially all eukaryotic genomes contain abundant interspersed repeat families, many of which are dimorphic (for presence or absence of specific elements) among populations, this method can be used for rapid genotyping and fine-scale chromosomal mapping in many species, including those for which extensive mapping and sequencing data do not yet exist.     

Transposition studies of mini-Mu plasmids constructed from the chemically synthesized ends of bacteriophage Mu

We describe below the chemical synthesis of the right and left ends of bacteriophage Mu and characterize the activity of these synthetic ends in mini-Mu transposition. Mini-Mu plasmids were constructed which carry the synthetic Mu ends together with the Mu A and B genes under control of the bacteriophage λ p_L promoter. Derepression of p_L leads to a high frequency of mini-Mu transposition (5.6 × 10⁻²) which is dependent on the presence of the Mu ends and the Mu A and B proteins. Five deletion mutants in the Mu ends were tested in the mini-Mu transposition system and their effects on transposition are described.