New method for generating deletions and gene replacements in Escherichia coli.

We describe a method for generating gene replacements and deletions in Escherichia coli. The technique is simple and rapid and can be applied to most genes, even those that are essential. What makes this method unique and particularly effective is the use of a temperature-sensitive pSC101 replicon to facilitate the gene replacement. The method proceeds by homologous recombination between a gene on the chromosome and homologous sequences carried on a plasmid temperature sensitive for DNA replication. Thus, after transformation of the plasmid into an appropriate host, it is possible to select for integration of the plasmid into the chromosome at 44 degrees C. Subsequent growth of these cointegrates at 30 degrees C leads to a second recombination event, resulting in their resolution. Depending on where the second recombination event takes place, the chromosome will either have undergone a gene replacement or retain the original copy of the gene. The procedure can also be used to effect the transfer of an allele from a plasmid to the chromosome or to rescue a chromosomal allele onto a plasmid. Since the resolved plasmid can be maintained by selection, this technique can be used to generate deletions of essential genes.     

Alielic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker

An improved method for allele replacement in Pseudomonas aeruginosa was developed. The two main ingredients of the method are: (i) novel ColE1-type cloning vectors derived from pBR322 and pUC19; and (ii) a family of cassettes containing a portable oriT, the sacB gene from Bacillus subtilis as a counter-selectable marker, and a chloramphenicol-resistance gene allowing positive selection of both oriT and sacB. Introduction of plasmid-borne DNA into the chromosome was achieved in several steps. The DNA to be exchanged was first cloned into the new ColE1-type vectors. After insertion of the oriT and sacB sequences, these plasmid were conjugally transferred into P. aeruginosa and plasmid integrants were selected. Plating on sucrose-containing medium allowed positive selection for both plasmid excision and curing since Pseudomonas aeruginosa strains containing the sacB gene in single- or multiple copy were highly sensitive to 5% sucrose in rich medium. This procedure was successfully used to introduce an agmR mutation into P. aeruginosa wild-type strain PAO1 and should allow the exchange of any DNA segment into any non-essential regions of the P. aeruginosa chromosome.     

A simple method for multiple modification of the Escherichia coli K-12 chromosome

We developed a simple method of generating markerless deletions in the Escherichia coli chromosome. The method consists of two recombination events stimulated by lambda Red recombinase. The first recombination replaced a target region with a marker cassette and the second then eliminated the marker cassette. The marker cassette included an antibiotic resistant gene and a negative selection marker (Bacillus subtilis sacB). Since sacB makes E. coli sensitive to sucrose, a markerless deletion strain was successfully selected using its sucrose-resistant phenotype. To stimulate these recombination events, 1-kbp homologous sequences adjacent to the target region were connected to both ends of the marker cassette or connected to each other by PCR. The average efficiency of the recombinations was 24% and 93% respectively. Eliminating the marker cassette with a fragment including an additional sequence, insertion was also possible. This markerless deletion method should be useful in creating a highly modified E. coli chromosome.     

Markerless gene replacement in Escherichia coli stimulated by a double-strand break in the chromosome.

A simple and efficient gene replacement method, based on the recombination and repair activities of the cell, was developed. The method permits the targeted construction of markerless deletions, insertions and point mutations in the Escherichia coli chromosome. A suicide plasmid, carrying the mutant allele and the recognition site of meganuclease I- Sce I, is inserted into the genome by homologous recombination between the mutant and the wild-type (wt) alleles. Resolution of this cointegrate by intramolecular recombination of the allele pair results in either a mutant or a wt chromosome which can be distinguished by allele-specific PCR screening. The resolution process is stimulated by introducing a unique double-strand break (DSB) into the chromosome at the I- Sce I site. Cleavage by the nuclease not only enhances the frequency of resolution by two to three orders of magnitude, but also selects for the resolved products. The DSB-stimulated gene replacement method can be used in recombination-proficient E.coli cells, does not require specific growth conditions, and is potentially applicable in other microorganisms. Use of the method was demonstrated by constructing a 17-bp and a 62-kb deletion in the MG1655 chromosome. Cleavage of the chromosome induces the SOS response but does not lead to an increased mutation rate.     

Recombinogenic properties of Pyrococcus furiosus strain COM1 enable rapid selection of targeted mutants

We recently reported the isolation of a mutant of Pyrococcus furiosus, COM1, that is naturally and efficiently competent for DNA uptake. While we do not know the exact nature of this mutation, the combined transformation and recombination frequencies of this strain allow marker replacement by direct selection using linear DNA. In testing the limits of its recombination efficiency, we discovered that marker replacement was possible with as few as 40 nucleotides of flanking homology to the target region. We utilized this ability to design a strategy for selection of constructed deletions using PCR products with subsequent excision, or "pop-out," of the selected marker. We used this method to construct a "markerless" deletion of the trpAB locus in the GLW101 (COM1 ΔpyrF) background to generate a strain (JFW02) that is a tight tryptophan auxotroph, providing a genetic background with two auxotrophic markers for further strain construction. The utility of trpAB as a selectable marker was demonstrated using prototrophic selection of plasmids and genomic DNA containing the wild-type trpAB alleles. A deletion of radB was also constructed that, surprisingly, had no obvious effect on either recombination or transformation, suggesting that its gene product is not involved in the COM1 phenotype. Attempts to construct a radA deletion mutation were unsuccessful, suggesting that this may be an essential gene. The ease and speed of this procedure will facilitate the construction of strains with multiple genetic changes and allow the construction of mutants with deletions of virtually any nonessential gene.     

A Simple Method for Multiple Modification of the Escherichia coli K-12 Chromosome

We developed a simple method of generating markerless deletions in the Escherichia coli chromosome. The method consists of two recombination events stimulated by λ Red recombinase. The first recombination replaced a target region with a marker cassette and the second then eliminated the marker cassette. The marker cassette included an antibiotic resistant gene and a negative selection marker (Bacillus subtilis sacB). Since sacB makes E. coli sensitive to sucrose, a markerless deletion strain was successfully selected using its sucrose-resistant phenotype. To stimulate these recombination events, 1-kbp homologous sequences adjacent to the target region were connected to both ends of the marker cassette or connected to each other by PCR. The average efficiency of the recombinations was 24% and 93% respectively. Eliminating the marker cassette with a fragment including an additional sequence, insertion was also possible. This markerless deletion method should be useful in creating a highly modified E. coli chromosome.     

Excision and episomal replication of cauliflower mosaic virus integrated into a plant genome

Transgenic Arabidopsis (Arabidopsis thaliana) plants containing a monomeric copy of the cauliflower mosaic virus (CaMV) genome exhibited the generation of infectious, episomally replicating virus. The circular viral genome had been split within the nonessential gene II for integration into the Arabidopsis genome by Agrobacterium tumefaciens-mediated transformation. Transgenic plants were assessed for episomal infections at flowering, seed set, and/or senescence. The infections were confirmed by western blot for the CaMV P6 and P4 proteins, electron microscopy for the presence of icosahedral virions, and through polymerase chain reaction across the recombination junction. By the end of the test period, a majority of the transgenic Arabidopsis plants had developed episomal infections. The episomal form of the virus was infectious to nontransgenic plants, indicating that no essential functions were lost after release from the Arabidopsis chromosome. An analysis of the viral genomes recovered from either transgenic Arabidopsis or nontransgenic turnip (Brassica rapa var rapa) revealed that the viruses contained deletions within gene II, and in some cases, the deletions extended to the beginning of gene III. In addition, many of the progeny viruses contained small regions of nonviral sequence derived from the flanking transformation vector. The nature of the nucleotide sequences at the recombination junctions in the circular progeny virus indicated that most were generated by nonhomologous recombination during the excision event. The release of the CaMV viral genomes from an integrated copy was not dependent upon the application of environmental stresses but occurred with greater frequency with either age or the late stages of plant maturation.     

High-efficiency gene inactivation and replacement system for gram-positive bacteria.

A system for high-efficiency single- and double-crossover homologous integration in gram-positive bacteria has been developed, with Lactococcus lactis as a model system. The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG+host5, which contains a pBR322 replicon for propagation in Escherichia coli at 37 degrees C. A nested set of L. lactis chromosomal fragments cloned onto pG+host5 were used to show that the single-crossover integration frequency was logarithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb. Using random chromosomal 1-kb fragments, we showed that homologous integration can occur along the entire chromosome. We made use of the reported stimulatory effect of rolling-circle replication on intramolecular recombination to develop a protocol for gene replacement. Cultures were first maintained at 37 degrees C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28 degrees C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy. More than 50% of cells underwent replacement recombination when selection was applied for the replacing gene. Between 1 and 40% of cells underwent replacement recombination when no selection was applied. Chromosomal insertions and deletions were obtained in this way. These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector. This procedure is applicable to numerous gram-positive bacteria.     

Correlation between umuC induction and Salmonella mutagenicity assay for quinolone antimicrobial agents

Abstract A novel genetic procedure is described to identify stress genes in Bacillus subtilis by insertion mutagenesis. In addition, this method allows the rapid mapping of the mutation and the establishment of the DNA sequence of the gene impaired by the mutation. Small restriction fragments of chromosomal DNA of B. subtilis are inserted into a pBR322-based vector, the recombinant plasmids are transformed into B. subtilis , and integrants are selected which arise by recombination between the insert and its homologous region within the bacterial chromosome. About two dozen heat-, cold- and salt-sensitive mutants were isolated. Four mutations were mapped using PBS1 transduction, and the physiology of one salt-sensitive mutant was analysed.     

Study of chromosome rearrangements associated with the trpE26 mutation of Bacillus subtilis

Chromosome rearrangements involved in the formation of merodiploid strains in the Bacillus subtilis 168-166 system were explained by postulating the existence of intrachromosomal homology regions. This working hypothesis was tested by analysing sequences and restriction patterns of the, as yet uncharacterized, junctions between chromosome segments undergoing rearrangements in parent, 168 trpC2 and 166 trpE26, as well as in derived merodiploid strains. Identification, at the Ia/Ib chromosome junction of both parent strains, of a 1.3 kb segment nearly identical to a segment of prophage SPbeta established the existence of one of the postulated homology sequences. Inspection of relevant junctions revealed that a set of different homology regions, derived from prophage SPbeta, plays a key role in the formation of so-called trpE30, trpE30+, as well as of new class I merodiploids. Analysis of junctions involved in the transfer of the trpE26 mutation, i.e. simultaneous translocation of chromosome segment C and rotation of the terminal relative to the origin moiety of the chromosome, did not confirm the presence of any sequence suitable for homologous recombination. We propose a model involving simultaneous introduction of four donor DNA molecules, each comprising a different relevant junction, and their pairing with the junction regions of the recipient chromosome. The resolution of this structure, resting on homologous recombination, would confer the donor chromosome structure to the recipient, achieving some kind of 'transstamping'. In addition, a rather regular pattern of inverse and direct short sequence repeats in regions flanking the breaking points could be correlated with the initial, X-ray-induced, rearrangement.     

Phosphoribosylpyrophosphate synthetase of Bacillus subtilis. Cloning, characterization and chromosomal mapping of the prs gene

The gene (prs) encoding phosphoribosylpyrophosphate (PRPP) synthetase has been cloned from a library of Bacillus subtilis DNA by complementation of an Escherichia coli prs mutation. Flanking DNA sequences were pruned away by restriction endonuclease and exonuclease BAL 31 digestions, resulting in a DNA fragment of approx. 1.8 kb complementing the E. coli prs mutation. Minicell experiments revealed that this DNA fragment coded for a polypeptide, shown to be the PRPP synthetase subunit, with an Mr of approx. 40,000. B. subtilis strains harbouring the prs gene in a multicopy plasmid contained up to nine-fold increased PRPP synthetase activity. The prs gene was cloned in an integration vector and the resulting hybrid plasmid inserted into the B. subtilis chromosome by homologous recombination. The integration site was mapped by transduction and the gene order established as purA-guaA-prs-cysA.     

Integration of linear, heterologous DNA molecules into the Bacillus subtilis chromosome: mechanism and use in induction of predictable rearrangements.

Linear DNA molecules composed of a central region nonhomologous with the Bacillus subtilis chromosome and two flanking regions homologous with the chromosome can integrate into the chromosome, provided that the homologous regions have the same relative orientation. The resulting chromosome can be maintained in a haploid or in a merodiploid cell together with a parental chromosome. This can most easily be explained by supposing that the integration occurs by crossing over at each homologous region and that a part of the chromosome between these regions is deleted and replaced by the central nonhomologous region of the integrating molecule. If no essential genes were replaced during that process a haploid cell would be obtained; if essential genes were replaced a merodiploid cell would be obtained. The use of appropriate linear molecules therefore should allow the induction of deletions, extending from a given chromosomal site in a predetermined direction, and defined duplications in the B. subtilis chromosome.     

Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli

Red recombination using PCR-amplified selectable markers is a well-established technique for mutagenesis of large DNA molecules in Escherichia coli. The system has limited efficacy and versatility, however, for markerless modifications including point mutations, deletions, and particularly insertions of longer sequences. Here we describe a procedure that combines Red recombination and cleavage with the homing endonuclease I-SceI to allow highly efficient, PCR-based DNA engineering without retention of unwanted foreign sequences. We applied the method to modification of bacterial artificial chromosome (BAC) constructs harboring an infectious herpesvirus clone to demonstrate the potential of the mutagenesis technique, which was used for the insertion of long sequences such as coding regions or promoters, introduction of point mutations, scarless deletions, and insertion of short sequences such as an epitope tag. The system proved to be highly reliable and efficient and can be adapted for a variety of different modifications of BAC clones, which are fundamental tools for applications as diverse as the generation of transgenic animals and the construction of gene therapy or vaccine vectors.     

Cre/lox system and PCR-based genome engineering in Bacillus subtilis

We have developed a fast and accurate method to engineer the Bacillus subtilis genome that involves fusing by PCR two flanking homology regions with an antibiotic resistance gene cassette bordered by two mutant lox sites (lox71 and lox66). The resulting PCR products were used directly to transform B. subtilis, and then transient Cre recombinase expression in the transformants was used to recombine lox71 and lox66 into a double-mutant lox72 site, thereby excising the marker gene. The mutation process could also be accomplished in 2 days by using a strain containing a cre isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible expression cassette in the chromosome as the recipient or using the lox site-flanked cassette containing both the cre IPTG-inducible expression cassette and resistance marker. The in vivo recombination efficiencies of different lox pairs were compared; the lox72 site that remains in the chromosome after Cre recombination had a low affinity for Cre and did not interfere with subsequent rounds of Cre/lox mutagenesis. We used this method to inactivate a specific gene, to delete a long fragment, to realize the in-frame deletion of a target gene, to introduce a gene of interest, and to carry out multiple manipulations in the same background. Furthermore, it should also be applicable to large genome rearrangement.     

Somatic DNA recombination yielding circular DNA and deletion of a genomic region in embryonic brain

In this study, a mouse genomic region is identified that undergoes DNA rearrangement and yields circular DNA in brain during embryogenesis. External region-directed inverse polymerase chain reaction on circular DNA extracted from late embryonic brain tissue repeatedly detected DNA of this region containing recombination joints. Wide-range genomic PCR and digestion-circularization PCR analysis showed this region underwent recombination accompanied with deletion of intervening sequences, including the circularized regions. This region was mapped by fluorescence in situ hybridization to C1 on mouse chromosome 16, where no gene and no physiological DNA rearrangement had been identified. DNA sequence in the region has segmental homology to an orthologous region on human chromosome 3q.13. These observations demonstrated somatic DNA recombination yielding genomic deletions in brain during embryogenesis.     

Two Types of Sites Required for Meiotic Chromosome Pairing in Caenorhabditis Elegans

Previous studies have shown that isolated portions of Caenorhabditis elegans chromosomes are not equally capable of meiotic exchange. These results led to the proposal that a homolog recognition region (HRR), defined as the region containing those sequences enabling homologous chromosomes to pair and recombine, is localized near one end of each chromosome. Using translocations and duplications we have localized the chromosome I HRR to the right end. Whereas the other half of chromosome I did not confer any ability for homologs to pair and recombine, deficiencies in this region dominantly suppressed recombination to the middle of the chromosome. These deletions may have disrupted pairing mechanisms that are secondary to and require an HRR. Thus, the processes of pairing and recombination appear to utilize at least two chromosomal elements, the HRR and other pairing sites. For example, terminal sequences from other chromosomes increase the ability of free duplications to recombine with their normal homologs, suggesting that telomere-associated sequences, homologous or nonhomologous, play a role in facilitating meiotic exchange. Recombination can also initiate at internal sites separated from the HRR by chromosome rearrangement, such as deletions of the unc-54 region of chromosome I. When crossing over was suppressed in a region of chromosome I, compensatory increases were observed in other regions. Thus, the presence of the HRR enabled recombination to occur but did not determine the distribution of the crossover events. It seems most likely that there are multiple initiation sites for recombination once homolog recognition has been achieved.     

Genetic analysis of sacB, the structural gene of a secreted enzyme, levansucrase of Bacillus subtilis Marburg

The structural gene sacB encoding B. subtilis levansucrase, a secreted enzyme, expresses in E. coli. E. coli hosts of the sacB gene are poisoned by sucrose. This property allowed a powerful selection of mutants affected in the cloned gene. The plasmidic mutations were readily introduced in the B. subtilis chromosome. Using a collection of plasmids bearing various deletions extending in sacB we developed a technique of deletion mapping based on plasmid integration in the chromosome of B. subtilis. A generalization of this technique is discussed.