One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.
Journal: 2000/July - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
Abstract:
We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.
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Proc Natl Acad Sci U S A 97(12): 6640-6645

One-step inactivation of chromosomal genes in <em>Escherichia coli</em> K-12 using PCR products

Department of Biological Sciences, Purdue University, West Lafayette, IN 47907
To whom reprint requests should be addressed. E-mail: ude.eudrup.oib.oblib@WLB.
Communicated by Jonathan Beckwith, Harvard Medical School, Boston, MA
Communicated by Jonathan Beckwith, Harvard Medical School, Boston, MA
Received 2000 Feb 13; Accepted 2000 Apr 11.

Abstract

We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage λ Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.

Keywords: bacterial genomics, FLP recombinase, FRT sites, Red recombinase
Abstract

The availability of complete bacterial genome sequences has provided a wealth of information on the molecular structure and organization of a myriad of genes and ORFs whose functions are poorly understood. A systematic mutational analysis of genes in their normal location can provide significant insight into their function. Although a number of general allele replacement methods (17) can be used to inactivate bacterial chromosomal genes, these all require creating the gene disruption on a suitable plasmid before recombining it onto the chromosome. In contrast, genes can be directly disrupted in Saccharomyces cerevisiae by transformation with PCR fragments encoding a selectable marker and having only 35 nt of flanking DNA homologous to the chromosome (8). This PCR-mediated gene replacement method has greatly facilitated the generation of specific mutants in the functional analysis of the yeast genome; it relies on the high efficiency of mitotic recombination in yeast (9). Directed disruption of chromosomal genes can also be done in Candida albicans by using similar PCR fragments with 50- to 60-nt homology extensions (10).

In contrast to yeast and a few naturally competent bacteria, most bacteria are not readily transformable with linear DNA. One reason Escherichia coli is not so transformable is because of the presence of intracellular exonucleases that degrade linear DNA (11). However, recombination-proficient mutants lacking exonuclease V of the RecBCD recombination complex are transformable with linear DNA (12). Recombination can occur in recB or recC mutants carrying a suppressor (sbcA or sbcB) mutation that activates an alternative recombination pathway; sbcA activates the RecET recombinase of the Rac prophage, whereas sbcB enhances recombination by the RecF pathway (13). Such recBC sbcB mutants have been especially useful for recombining in vitro constructed mutations onto the E. coli chromosome by using linear DNA (14). The discovery that recD mutants are recombinase proficient but lack exonuclease V (15, 16) has led to using singly mutated recD derivatives of E. coli (1) in similar gene disruption experiments.

It has been known for a long time that many bacteriophages encode their own homologous recombination systems (17). It has also recently been shown that the λ Red (γ, β, exo) function promotes a greatly enhanced rate of recombination over that exhibited by recBC sbcB or recD mutants when using linear DNA (18). Yet this system has produced no chromosomal gene disruptions when using PCR fragments with short homology extensions (unpublished data). A system has been developed that uses the RecET recombinase to disrupt plasmid-borne genes with such fragments (19); it has also been used to make a single chromosomal deletion, but in that instance very long (138-nt) primers were used.

Here we describe a procedure based on the Red system that has allowed us to make more than 40 different disruptions on the E. coli chromosome without a single failure. The basic strategy is to replace a chromosomal sequence (e.g., gene B in Fig. Fig.1)1) with a selectable antibiotic resistance gene that is generated by PCR by using primers with 36-nt homology extensions (H1 and H2). This is accomplished by Red-mediated recombination in these flanking homologies. After selection, the resistance gene can also be eliminated by using a helper plasmid expressing the FLP recombinase, which acts on the directly repeated FRT (FLP recognition target) sites flanking the resistance gene. The Red and FLP helper plasmids can be simply cured by growth at 37°C because they are temperature-sensitive replicons.

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A simple gene disruption strategy. H1 and H2 refer to the homology extensions or regions. P1 and P2 refer to priming sites.

Acknowledgments

This manuscript is dedicated to the memory of H. E. Umbarger who died on November 15, 1999. We thank individuals cited in the text for samples; Don Court, Jean-Marc Ghigo, and Kenan Murphy for communicating unpublished results; Jill Hutchcroft and Irwin Tessman for critically reading the manuscript; and lab members for helpful discussions. This research was supported by Award MCB-9730034 from the National Science Foundation.

Acknowledgments

Abbreviations

Bapbacterial alkaline phosphatase
CmRchloramphenicol-resistant
FRTFLP recognition target
KmRkanamycin-resistant
kankanamycin resistance gene
catchloramphenicol resistance gene
Abbreviations

Footnotes

Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.120163297.

Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.120163297

Footnotes

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