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.     

Analysis of insertions of the determinant of drug resistance to kanamycin and chloramphenicol into bacteriophage lambda att80

The insertion sites of elements Tn9 and Tn601 which determine chloramphenicol and kanamycin resistance have been detected restriction analysis. The functioning of transposons i.e. their stability or instability, has been found to influence the specificity of their insertions into the genome of lambda att80 bacteriophage. During transposition from stable integration sites both transposons are inserted into the regions of the lambda att80 bacteriophage genome, definite for each transposon. However, during transposition from the site of unstable integration both determinants of drug resistance are inserted into different regions of the phage genome.     

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.     

A system of transposon mutagenesis for bacteriophage T4

We have developed a system of transposon mutagenesis for bacteriophage T4. The transposon is a plasmid derivative of Tn5 which contains the essential T4 gene 24, permitting a direct selection for transposition events into a gene 24-deleted phage. The transposition occurred at a frequency of only 10(-7) per progeny phage, even though a dam- host was used to increase transposition frequency. Phage strains with a transposon insert were distinguished from most pseudorevertants of the gene 24 deletion by plaque hybridization using a transposon-specific probe. Mapping analysis showed that the transposon inserts into a large number of sites in the T4 genome, probably with a preference for certain regions. The transposon insertions in four strains were analysed by DNA sequencing using primers that hybridize to each end of the transposon and read out into the T4 genome. In each case, a 9 bp T4 target sequence had been duplicated and the insertions had occurred exactly at the IS50 ends of the transposon, demonstrating that bona fide transposition had occurred. Finally, the transposon insert strains were screened on the TabG Escherichia coli strain, which inhibits the growth of T4 motA mutants, and a motA transposon insert strain was found.     

Minimization of the Escherichia coli genome using a Tn5-targeted Cre/loxP excision system

An increasing number of microbial genomes have been completely sequenced, and functional analyses of these genomic sequences are under way. To facilitate these analyses, we have developed a genome-engineering tool for determining essential genes and minimizing bacterial genomes. We made two large pools of independent transposon mutants in Escherichia coli using modified Tn5 transposons with two different selection markers and precisely mapped the chromosomal location of 800 of these transposons. By combining a mapped transposon mutation from each of the mutant pools into the same chromosome using phage P1 transduction and then excising the flanked genomic segment by Cre-mediated loxP recombination, we obtained E. coli strains in which large genomic fragments (59-117 kilobases) were deleted. Some of these individual deletions were then combined into a single "cumulative deletion strain" that lacked 287 open reading frames (313.1 kilobases) but that nevertheless exhibited normal growth under standard laboratory conditions.     

Enterohemolysin production is associated with a temperate bacteriophage in Escherichia coli serogroup O26 strains.

A temperate bacteriophage that determines the expression of enterohemolysin was isolated from Escherichia coli O26 strain C3888. The genetic determinant associated with enterohemolysin production (E-Hly determinant) was cloned from EcoRI-digested bacteriophage DNA in vector plasmid pUC8. pUC8 recombinant plasmid pEO19 carries a 3.7-kb EcoRI insert of phage DNA, and enterohemolysin was expressed in E. coli K-12 after transformation. Hemolysin-negative derivatives of pEO19 were generated by transposon mutagenesis with Tn1725. By subcloning, the phage E-Hly determinant was assigned to a 2,150-bp piece of DNA which is flanked by EcoRI and AccI restriction sites. The enterohemolysin-producing recombinant strains and wild-type strain C3888 express a 60-kDa protein which was detected in the bacterial outer membrane by Western immunoblotting. Biologically active enterohemolysin was detected only in bacteria grown to the stationary phase, and the hemolysin was not released into the culture medium. Lysis of erythrocytes was inhibited by 30 mM dextran 4, which functions as an osmotic protectant without destroying the enterohemolysin itself.     

Global mapping of transposon location

Transposable genetic elements are ubiquitous, yet their presence or absence at any given position within a genome can vary between individual cells, tissues, or strains. Transposable elements have profound impacts on host genomes by altering gene expression, assisting in genomic rearrangements, causing insertional mutations, and serving as sources of phenotypic variation. Characterizing a genome's full complement of transposons requires whole genome sequencing, precluding simple studies of the impact of transposition on interindividual variation. Here, we describe a global mapping approach for identifying transposon locations in any genome, using a combination of transposon-specific DNA extraction and microarray-based comparative hybridization analysis. We use this approach to map the repertoire of endogenous transposons in different laboratory strains of Saccharomyces cerevisiae and demonstrate that transposons are a source of extensive genomic variation. We also apply this method to mapping bacterial transposon insertion sites in a yeast genomic library. This unique whole genome view of transposon location will facilitate our exploration of transposon dynamics, as well as defining bases for individual differences and adaptive potential.     

The Bacteroides mobilizable transposon Tn4555 integrates by a site-specific recombination mechanism similar to that of the gram-positive bacterial element Tn916.

The Bacteroides mobilizable transposon Tn4555 is a 12.2-kb molecule that encodes resistance to cefoxitin. Conjugal transposition is hypothesized to occur via a circular intermediate and is stimulated by coresident tetracycline resistance elements and low levels of tetracycline. In this work, the ends of the transposon were identified and found to consist of 12-bp imperfect inverted repeats, with an extra base at one end. In the circular form, the ends were separated by a 6-bp "coupling sequence" which was associated with either the left or the right transposon terminus when the transposon was inserted into the chromosome. Tn4555 does not duplicate its target site upon insertion. Using a conjugation-based transposition assay, we showed that the coupling sequence originated from 6 bases of genomic DNA flanking either side of the transposon prior to excision. Tn4555 preferentially transposed into a 589-bp genomic locus containing a 207-bp direct repeat. Integration occurred before or after the repeated sequence, with one integration site between the two repeats. These observations are consistent with a transposition model based on site-specific recombination. In the bacteriophage lambda model for site-specific recombination, the bacteriophage recombines with the Escherichia coli chromosome via a 7-bp "crossover" region. We propose that the coupling sequence of Tn4555 is analogous in function to the crossover region of lambda but that unlike the situation in lambda, recombination occurs between regions of nonhomologous DNA. This ability to recombine into divergent target sites is also a feature of the gram-positive bacterial transposon Tn916.     

The construction and preliminary analysis of a Tn5 transposon based random mutant library of baculovirus

A transposon-based random mutation library of AcMNPV,the type species of baculovirus,was constructed using a Tn5 transposon.The green fluorescence protein gene under the control of the Drosophila hsp70 promoter was inserted into the transposon for easy tracking in insect cells.In vitro transposition was carried out using the transposon and AcMNPV genomic DNA to allow the random insertion of the transposon into the virus genome.The transposed genome was then used to transfect Sf21 insect cells,and a library of mutant viruses capable of expressing green fluorescence protein was obtained.Two mutant viruses,B9F and Li6A were isolated,and the sites of transposon insertion were determined to be within the coding regions of the 94k and p10 genes,respectively.Both genes were determined to be nonessential in viral replication and infection.This technique will be very useful in the functional study of baculovirus genes.     

Developing live Shigella vaccines using lambda Red recombineering

Live attenuated Shigella vaccines have shown promise in inducing protective immune responses in human clinical trials and as carriers of heterologous antigens from other mucosal pathogens. In the past, construction of Shigella vaccine strains relied on classical allelic exchange systems to genetically engineer the bacterial genome. These systems require extensive in vitro engineering of long homologous sequences to create recombinant replication-defective plasmids or phage. Alternatively, the lambda red recombination system from bacteriophage facilitates recombination with as little as 40 bp of homologous DNA. The process, referred to as recombineering, typically uses an inducible lambda red operon on a temperature-sensitive plasmid and optimal transformation conditions to integrate linear antibiotic resistance cassettes flanked by homologous sequences into a bacterial genome. Recent advances in recombineering have enabled modification of genomic DNA from bacterial pathogens including Salmonella, Yersinia, enteropathogenic Escherichia coli, or enterohemorrhagic E. coli and Shigella. These advances in recombineering have been used to systematically delete virulence-associated genes from Shigella, creating a number of isogenic strains from multiple Shigella serotypes. These strains have been characterized for attenuation using both in vivo and in vitro assays. Based on this data, prototypic Shigella vaccine strains containing multiple deletions in virulence-associated genes have been generated.     

Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis.

We have developed efficient methods for creating artificial transposons and inserting these transposons into plasmid targets in vitro, primarily for the purpose of DNA mapping and sequencing. A novel plasmid has been engineered to convert virtually any DNA sequence, or combination of sequences, into an artificial transposon; hence, custom transposons containing any desired feature can be easily designed and constructed. Such transposons are then efficiently inserted into plasmid targets, in vitro, using the integrase activity present in yeast Ty1 virus-like particles. A single in vitro integration reaction, which resembles a simple restriction digestion in the complexity of the reaction, gives rise to thousands of recoverable insertion events within DNA target molecules; this frequency approaches one insertion per phosphodiester bond in typical plasmids. Importantly, transposon insertions are recovered from all regions of DNA inserts carried on plasmid targets, indicating that integration is a random or nearly-random process. Because of its versatility, this technology offers a generalized method of generating recombinant DNA molecules of a desired structure. We have adapted this system for DNA sequencing by developing a customized artificial transposon to insert new primer binding sites into internal regions of DNA inserts carried on cloning vectors. Transposon insertions have been generated throughout several different yeast and human DNA inserts carried on plasmids, allowing the efficient recovery of sequence information from these inserts. Our results demonstrate the overall utility of this method for both small and large-scale DNA sequencing, as well as general DNA restructuring, and indicate that it could be adapted for use with a number of additional applications including functional genetic analysis.     

Integration of phage Mu into the RP4::D3112A-plasmid in Escherichia coli cells

Hybrid plasmids obtained as a result of Mu phage insertions into the RP4::D3112 plasmid in Escherichia coli cells were studied. Stable maintenance of RP4::D3112 plasmid in E. coli cells was provided by using the D3112 phage genome with a point polar mutation in the A gene which prevented early genes' expression. The presence of D3112A- in the RP4 plasmid has been shown to have no effect on efficiency of phage Mu transposition into this plasmid. Moreover, RP4 and D3112 genomes were equivalent targets for Mu integration. The integration of transposable phage into genome of nonrelated phage can be used as one of the approaches to construct recombinant phage genomes in vivo in the absence of DNA homology.     

THE EVOLUTION OF TRANSPOSABLE ELEMENTS: CONDITIONS FOR ESTABLISHMENT IN BACTERIAL POPULATIONS

Previous theoretical studies have shown that bacterial transposons can become established in populations by infectious transfer, even if they reduce the fitness of their host cells. Conditions for the persistence of “parasitic” transposons are, however, restrictive: i) transposition must be replicative, rather than conservative; ii) the rate of transposition must be greater than the loss in host fitness caused by the transposon; and iii) cells must exchange plasmids at rates greater than the fitness cost of the transposon. I sought to test the validity of the model underlying this theory by performing experiments with laboratory populations of the bacterium Escherichia coli, the conjugative plasmid R100, and the transposons Tn3 and Tn5. A plasmid-borne transposon was introduced at low frequency into a population of bacteria carrying the same plasmid without the transposon in a habitat where the transposon offered no benefit to its host. The fate of the invading transposon was followed by tracking the various bacterial populations appearing in the cultures. Using independent estimates of the parameters of the model, predicted population changes were generated with numerical solutions of the model, and these were compared to experimental results. Plasmids transferred into new hosts as predicted by the model, and the resulting transconjugant populations either maintained a steady low density or rose slowly in abundance. Transposition appeared to play no role in population changes. Abundance of all cell types fit theoretical predictions of a system with no transposition, despite evidence that transposition was taking place. This is exactly what the model predicted. It thus appears unlikely that deleterious or neutral transposons have much impact on the genetics of bacterial populations. This is consistent with the hypothesis that most bacterial transposons are not parasitic DNA, but rather invade and persist in populations by providing a fitness advantage to cells carrying them.     

Binding of an Escherichia coli double-stranded DNA virus PRD1 to a receptor coded by an IncP-type plasmid.

IncP plasmid RP1 Tra regions are needed to assemble the receptor for lipid-containing double-stranded DNA bacteriophage PRD1 on the cell surface. Using radioactively labeled phage and electron microscopic techniques, we showed that the surfaces of Salmonella typhimurium(RP1) and Escherichia coli(RP1) cells contained approximately 50 and 20 PRD1 binding sites, respectively. Expression of the receptor was growth phase dependent and was highest at late logarithmic or early stationary phase. The PRD1-resistant RP1 transposon mutants isolated were all Tra-, and the transposons were located in both the Tra1 and Tra2 regions.     

Rapid construction of adenoviral vectors by lambda phage genetics

Continued improvements of adenoviral vectors require the investigation of novel genome configurations. Since adenovirus can be generated directly by transfecting packaging cell lines with viral genomes isolated from plasmid DNA, it is possible to separate genome construction from virus production. In this way failure to generate a virus is not associated with an inability to generate the desired genome. We have developed a novel lambda-based system that allows rapid modification of the viral genome by double homologous recombination in Escherichia coli. The recombination reaction and newly generated genome may reside in a recombination-deficient bacterial host for enhanced plasmid stability. Furthermore, the process is independent of any restriction endonucleases. The strategy relies on four main steps: (i) homologous recombination between an adenovirus cosmid and a donor plasmid (the donor plasmid carries the desired modification[s] and flanking regions of homology to direct its recombination into the viral genome); (ii) in vivo packaging of the recombinant adenoviral cosmids during a productive lambda infection; (iii) transducing a recombination-deficient E. coli lambda lysogen with the generated lysate (the lysogen inhibits the helper phage used to package the recombinant andenoviral cosmid from productively infecting and destroying the host bacteria); (iv) effectively selecting for the desired double-recombinant cosmid. Approximately 10,000 double-recombinant cosmids are recovered per reaction with essentially all of them being the correct double-recombinant molecule. This system was used to generate quickly and efficiently adenoviral genomes deficient in the E1/E3 and E1/E3/E4 regions. The basis of this technology allows any region of the viral genome to be readily modified for investigation of novel configurations.     

Isolation, characterization and mapping of the N15 phasmid insertion mutations]

Prophage of N15 temperate bacteriophage is stably maintained in Escherichia coli lysogens as a 46.33 kb linear plasmid. Using different transposons we obtained 18 insertion mutants of the N15 plasmid prophage. They were analysed for plaque formation ability, stability of the plasmid state and lysogenic conversion. Restriction mapping of the insertions allowed us to localize on the map the regions necessary for lytic growth and to map the lysogenic conversion gene. A recombinant phage encoding two antibiotic resistance genes was obtained. The phage contains an additional 4.77 kb DNA fragment (over 10% of the N15 genome).     

Conservation and variation of nucleotide sequences within related bacterial genomes: Escherichia coli strains.

Changes in the patterns produced by annealing restriction endonuclease digests of bacterial genomes with probe deoxyribonucleic acids (DNAs) containing small portions of a bacterial genome provide sensitive indicator of the degree of nucleotide sequence relatedness that exists in localized regions of the genomes of closely related bacteria. We have used five probe DNAs to explore the relatedness of parts of the genomes of six laboratory Escherichi coli strains. A range in in the amount of variability in the positions of restriction enzyme cleavage sites in the selected portions of the genomes was found. Portions of the genome that are believed to be inacative were more variable than portions that contained functional genes: the sites in and near regions of homology to phage lambda DNA in the genome showed the greatest variability. These regions probably represent remnants of cryptic prophages. Variability was assessed pairwise among four of the E. coli strains and ranged from 5 to > 25% base pair substitutions in the lambda-related regions. In contrast, the endonuclease cleavage sites in the trp, tna, lac, thy regions, and one other as-yet-unidentified segment of the genome were more highly conserved. It seems likely that these sites lie in genetic locations that are subject to functional constraints.     

Electron microscopic observation of new transposable elements inserted into P22 phage genome from R plasmids

By using phage P22spl, a deletion mutant of phage P22, the structures of two new transposons on P22 genomes were studied by the electron microscopic heteroduplex method. One of these was the Cm (chloramphenicol) transposon derived from an R plasmid, NR1, and the other the Km (kanamycin) transposon frin obr502. the heteroduplex between P22 phage DNAs with and without the Cm transposon revealed that the Cm transposon was similar in structure to the Tn9 element, a well-known Cm transposon derived from the R plasmid pMS14. On the other hand, the Km transposon of pNR502 was quite different in structure from other Km transposons reported previously. This transposon consists of a 6.8 kilobase (kb) segment of DNA, in which a short inverted repeat is contained. The heteroduplex experiments showed that a 4.5 kb segment of DNA was deleted from the P22 genome in the P22spl genome. Because of a shorter unit length of the genome, phage P22spl is considered to be useful of assaying various kinds of transposable elements.     

Reverse Genetics System for the Avian Coronavirus Infectious Bronchitis Virus

Major advances in the study of the molecular biology of RNA viruses have resulted from the ability to generate and manipulate full-length genomic cDNAs of the viral genomes with the subsequent synthesis of infectious RNA for the generation of recombinant viruses. Coronaviruses have the largest RNA virus genomes and, together with genetic instability of some cDNA sequences in Escherichia coli, this has hampered the generation of a reverse-genetics system for this group of viruses. In this report, we describe the assembly of a full-length cDNA from the positive-sense genomic RNA of the avian coronavirus, infectious bronchitis virus (IBV), an important poultry pathogen. The IBV genomic cDNA was assembled immediately downstream of a T7 RNA polymerase promoter by in vitro ligation and cloned directly into the vaccinia virus genome. Infectious IBV RNA was generated in situ after the transfection of restricted recombinant vaccinia virus DNA into primary chick kidney cells previously infected with a recombinant fowlpox virus expressing T7 RNA polymerase. Recombinant IBV, containing two marker mutations, was recovered from the transfected cells. These results describe a reverse-genetics system for studying the molecular biology of IBV and establish a paradigm for generating genetically defined vaccines for IBV.