What makes the bacteriophage lambda red system

Conjugational and transductional recombination normally proceeds through the RecBCD pathway, in which the functions of recB and recC are essential the recD -encoded subunit, discovered much later, is not essential. If the RecBCD pathway was blocked by mutation of either gene, further mutation of the bacterium could induce either the RecE or the RecF pathway, restoring recombination proficiency. Red recombination was thus considered to be a separate pathway. If the only partner available for recombination with the dsDNA end is an unbroken circular homologous duplex, then recombination proceeds via strand invasion, and is dependent upon the bacterial RecA protein.

Pathways of Red-mediated double-strand break repair. Invasion left is efficient only in the presence of RecA, whereas annealing is efficient in the absence of RecA [ 12 ]. The genes gam, bet , and exo , expressed at moderate levels under control of the lac promoter from a multicopy plasmid, were found to be non-toxic to E. The plasmid-encoded Red system could restore recombination proficiency in conjugational crosses to a recB recC E.

The RecF dependence of Red-mediated conjugational recombination was consistent with a well-known pattern of similarities between the Red and RecE pathways. The genes of the RecE pathway, recE and recT , are encoded by a cryptic prophage, called Rac, which is found in certain strains of E. Like exo, recE encodes an exonuclease; like bet, recT encodes a ssDNA-binding strand-exchange protein [ 17 ]. Another technical advance in the study of Red-mediated bacterial recombination came from replacing the E.

In the resulting strain, unlike a cell bearing a multicopy plasmid with red genes, it was possible to knock out most of the known recombination genes of E. The contributions of the encoded recombination proteins to Red-mediated recombination could then be readily assessed. A surprising finding from this line of experiment was that knocking out the RecE and RecF pathway genes recJ or recG increased the efficiency of Red-mediated recombination.

The degree of dependence of Red-mediated recombination on other bacterial genes was found to vary with the nature of the recombining DNA species, as well as with the presence or absence of RecG [ 20—22 ]. These observations, and others described below, indicated clearly that the two Red pathways invoked by Stahl and coworkers — invasion and annealing — are themselves multiply branched downstream.

The Red-mediated break-join recombination event for which we have, at present, the clearest description, occurs between a linear dsDNA species and the bacterial chromosome, in a strain in which the recC-ptr-recB-recD gene cluster has been replaced with the genes of the Red system, and recG has been deleted. The phage's chromosome, which is unable to transcribe its lytic genes or replicate, is cut by a cellular restriction endonuclease, releasing a linear dsDNA.

The linear molecule, bearing the gene for chloramphenicol acetyltransferase cat flanked on either side by 1. Chromosomal gene replacement by a linear dsDNA fragment, either released from an infecting phage by the action of an endogenous restriction endonuclease, or introduced directly into the cell by electroporation.

The production of recombinants in these crosses between a phage-delivered linear dsDNA and the bacterial chromosome depends on recA, recF, recO, recR, recQ, ruvAB , and ruvC ; it is independent of recN [ 22 ]; unpublished results.

This mechanism accounts fairly well for events in the absence of RecG; in the presence of RecG, this particular pathway is partly impeded, but, evidently, others become available [ 21 ]. Red-mediated replacement of lac with cat in the chromosome of E. The depicted molecular events would have to take place on both sides of the cat gene to generate a recombinant; for clarity, only one side is shown. The double-stranded end initiates recombination.

Murphy [ 20 ] found that the Red system, plasmid- or chromosome-encoded, would promote recombination between the bacterial chromosome and linear dsDNA molecules introduced into the cell via electroporation. The Red system was far more efficient than systems used previously for making gene replacements in E. The Red system works in other bacteria as well. It has been used to make gene replacements in Salmonella E. Kofoid and J. Roth, personal communication , as well as enteropathogenic E.

Murphy, personal communication. All of the research described above involved recombination between DNA segments with identical sequences of several hundred bases or more. For purposes of genetic engineering, recombination reactions based on short DNA sequences, of a size readily synthesized, is far more useful. Stewart and coworkers [ 4 ] showed that the RecET system, as well as the Red system, would promote such events. Their experimental approach involved co-electroporation of an E.

The linear species was PCR-generated, and encoded a selectable drug resistance marker. The PCR primers included up to 60 bases of plasmid sequences, which, as homologous flanks in the PCR products, promoted recombination with the plasmid, generating a new plasmid in which the drug resistance marker had replaced specified plasmid sequences. Two groups of researchers developed efficient methods, based on the use of the Red system, for replacing genes in the E. The Wanner group obtained high-efficiency recombination between the E.

The Red system also promotes high-frequency recombination between the E. Ellis and D. Court, personal communication; F. Stewart, personal communication. Mismatch repair, or actual incorporation of the base-paired oligonucleotide into one of the replication products, could then lead to gene conversion.

A new mechanistic question emerges from studies of the Red system in genetic engineering: How do short-homology e. Perhaps it attacks a structure which is formed in the course of short-homology recombination, but not in long-homology recombination. Methods Citations. Results Citations. Topics from this paper. Genetic Engineering Hyperactive behavior Homologous Recombination. Paper Mentions.

Blog Post. Small Things Considered. Citation Type. Hernalsteens, unpublished results. The modification of the Red Recombinase system reported in the present study offer some solutions for constructing recombinant bacteriophages. This study focused on the construction of recombinant stx -phages carrying antibiotic resistance genes within the stx operon.

In addition, this methodology could be useful for prophages infecting other enterobacteria, since the method has been applied to Salmonella [ 21 ]; Shigella [ 39 ]; Serratia [ 40 ]; and Yersinia [ 41 ], which also carry prophages encoding virulence genes [ 42 — 44 ]. Bacteriophage W [ 26 ] was induced from E. SOB and SOC media [ 45 ] were used to prepare electrocompetent cells and for recovery after transformation.

Plasmid pKD3 Genbank AY [ 20 ] was used to obtain the chloramphenicol acetyl transferase gene cat , which confers resistance to chloramphenicol. Amsterdam, the Netherlands was used as a control for the transformation. The oligonucleotides used in this study are described in Table 3. Construction of the amplimers containing tet and cat genes, inserted in the stx 2 gene, was performed as follows. Primer pairs amplified from the stxA 2 subunit initial codon to the 5' region of each resistance cassette 5' fragment and from the 3' region of each resistance cassette to the final codon of the stxB 2 subunit 3' fragment Fig 1.

Amplimers of the 5' fragment, the respective resistance gene and the 3' fragment for each resistance gene were annealed at their overlapping region underlined letters in Table 3. The final product was used for the transformation in the lysogens.

The probes were labeled by incorporating digoxigenindeoxy-uridine-triphosphate Roche Diagnostics, Barcelona, Spain during PCR, as described in Muniesa et al. Colony hybridization was performed as previously described [ 37 ].

They were then washed in 0. The DIG-labeled probes were prepared as described above. They were then washed in 2 ml of ice-cold double distilled water. The cells were mixed with the corresponding amount of DNA plasmid or PCR-amplified, see results in an ice-cold microcentrifuge tube and transferred to a 0. The cells were electroporated at 2. Immediately after electroporation, 1 ml of SOC medium [ 47 ] was added to the cuvette.

Cells were concentrated ten-fold from a 1 ml culture before plating on selective media. A protocol modified from the one-step inactivation method using the Red recombinase system, as proposed by Datsenko and Wanner [ 20 ], was performed to obtain recombinant phages. Antibiotic resistance cassettes were inserted inside the truncated stx 2 gene of each phage to obtain recombinant phages carrying the resistance markers.

For this purpose, plasmid pKD46 encoding the Red recombinase was transformed by electroporation, as described above. After recovery in SOC medium and incubation for 4 hours, recombinant clones were selected on medium containing the appropriate antibiotic. Presumptive colonies were confirmed by PCR, using the rho primer and the respective primer for each antibiotic cassette.

Cycling times and temperatures were according to the properties of the primer pairs. Positive clones were also further confirmed by sequencing. PCR amplimers of the stx gene containing each antibiotic resistance gene and PCR amplimers obtained to characterise recombinant phages were sequenced.

The oligonucleotides used for sequencing are described in Table 3. All sequencing was performed in duplicate. BLAST analyses were carried out with the tools available on the web [ 48 ]. Growth was measured with a spectrophotometer Spectronic , Milton Roy. Cultures were then further incubated overnight.

To evaluate the transduction capacity of the recombinant phages, they were used to convert E. Antibiotic resistant colonies were tested by colony blot and confirmed by PCR amplification of the truncated stx 2 gene containing the antibiotic marker.

Curr Opin Microbiol. Casjens S: Prophages and bacterial genomics: what have we learned so far?. Mol Microbiol. Smith GR: Homologous recombination in procaryotes.

Microbiol Rev. Lederberg E: Lysogenicity in E. Google Scholar. Trends Microbiol. Infect Immun. Appl Environ Microbiol. J Bacteriol. Slater S, Maurer R: Simple phage-based system for generating allele replacements in Escherichia coli. Link AJ, Phillips D, Church GM: Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli : application to open reading frame characterization. Poteete AR: What makes the bacteriophage lambda Red system useful for genetic engineering: Molecular mechanism and biological function.

Emerg Infect Dis. Article PubMed Google Scholar. Article Google Scholar. Int J Med Microbiol. Lambda red-mediated gene manipulation in gram-negative bacteria. J Mol Biol. BMC Mol Biol. Mol Genet Genomics. Epub Hanahan D: Studies on transformation of Escherichia coli with plasmids. Nucleic Acids Res. National Center for Biotechnology Information. Download references. Muniesa is a researcher of the "Ramon y Cajal" program of the Spanish government. You can also search for this author in PubMed Google Scholar.

It also has the flexibility to modify the E. These enzymes then catalyze the homologous recombination of the substrate with the target DNA sequence. This means cloning occurs in vivo , as compared to restriction enzyme cloning where the genetic changes occur in a test tube. The lambda red recombineering system has three components Figure 1 : 1 Exo, 2 Beta, and 3 Gam. All three are required for recombineering with a dsDNA substrate; however, only Beta is required when generating a modification with an ssDNA substrate.

Only Beta expression is required for recombineering with an ssDNA oligo substrate. Figure 1: Components of the Lambda Red Recombineering system. See text for details. Below is a brief outline of a generic lambda red recombineering experiment Figure 2. In the following sections, key steps that differ from traditional restriction enzyme cloning will be explained in greater detail. Figure 2: Overview of using Lambda Red recombineering system to replace a gene of interest with an antibiotic resistance cassette.

The best applications for dsDNA inserts include: large insertions or deletions, including selectable DNA fragments, such as antibiotic resistance genes , as well as non-selectable DNA fragments, such as gene replacements and tags. The typical frequency of recombinants is 1 positive clone out of 10 4 to 10 5 colonies. This is an easy fix, but can only be used in limited circumstances.

An alternative approach is to flank the desired change with silent changes in the wobble codons, i.