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Chapter 82 : Novel Use of Nitroreductase as a Counterselectable Marker in Allelic Vector Exchange to Create Philadelphia-1 Mutants

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Novel Use of Nitroreductase as a Counterselectable Marker in Allelic Vector Exchange to Create Philadelphia-1 Mutants, Page 1 of 2

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Abstract:

This chapter tests the feasibility of replacing the gene with another suicide gene, , from , which encodes an NADPH oxygen insensitive nitroreductase. The authors show that the improved suicide vector pKBOXR () and use of metronidazole in counterselection is superior to the in . encodes an oxygen insensitive NADPH nitroreductase which exhibits high substrate specificity for the prodrug metronidazole, which is rapidly reduced to DNA damaging adducts of hydroxylamine, a bactericidal agent, thereby acting as a potential and novel counterselectable marker To create a gene knockout construct, flanking regions of ~500 bp of the targeted gene were individually PCR amplified and ligated into the multiple cloning site of an appropriate high-copy cloning vector (i.e., pBluescript, pUC19), after which an antibiotic cassette (kanamycin or gentamicin) was inserted between the two flanking regions. To date, utilization of the novel allelic exchange vector pKBOXR in our laboratory has generated several successful chromosomal gene knockouts of , , , and in Lp02. The pKBOXR vector appears to be more efficient than pBOC20 in generating knockout gene mutant strains, with the additional benefit that is not subjected to osmotic stress during the counterselectable process.

Citation: C. Brassinga A, A. Croxen M, J. Shoemaker C, G. Morash M, J. LeBlanc J, S. Hoffman P. 2006. Novel Use of Nitroreductase as a Counterselectable Marker in Allelic Vector Exchange to Create Philadelphia-1 Mutants, p 339-342. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch82

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Reverse Transcriptase PCR
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Figures

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FIGURE 1

Vector map of pKBOXR with unique multiple cloning restriction sites.

Citation: C. Brassinga A, A. Croxen M, J. Shoemaker C, G. Morash M, J. LeBlanc J, S. Hoffman P. 2006. Novel Use of Nitroreductase as a Counterselectable Marker in Allelic Vector Exchange to Create Philadelphia-1 Mutants, p 339-342. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch82
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Image of FIGURE 2
FIGURE 2

Target gene knockout strategy. Drawings are not to scale, and restriction sites are used as examples.

Citation: C. Brassinga A, A. Croxen M, J. Shoemaker C, G. Morash M, J. LeBlanc J, S. Hoffman P. 2006. Novel Use of Nitroreductase as a Counterselectable Marker in Allelic Vector Exchange to Create Philadelphia-1 Mutants, p 339-342. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch82
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References

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1. Brassinga, A. K.,, M. F. Hiltz,, G. R. Sisson,, M. G. Morash,, N. Hill,, E. Garduno,, P. H. Edelstein,, R. A. Garduno, and, P. S. Hoffman. 2003. A 65-kilobase pathogenicity island is unique to Philadelphia-1 strains of Legionella pneumophila. J. Bacteriol. 185:46304637.
2. Cazalet, C.,, C. Rusniok,, H. Bruggemann,, N. Zidane,, A. Magnier,, L. Ma,, M. Tichit,, S. Jar-raud,, C. Bouchier,, F. Vandenesch,, F. Kunst,, J. Etienne,, P. Glaser, and, C. Buchrieser. 2004. Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity. Nat. Genet. 36:11651173.
3. Chien, M.,, I. Morozova,, S. Shi,, H. Sheng,, J. Chen,, S. M. Gomez,, G. Asamani,, K. Hill,, J. Nuara,, M. Feder,, J. Rineer,, J. J. Greenberg,, V. Steshenko,, S. H. Park,, B. Zhao,, E. Teplits-kaya,, J. R. Edwards,, S. Pampou,, A. Georg-hiou,, I. C. Chou,, W. Iannuccilli,, M. E. Ulz,, D. H. Kim,, A. Geringer-Sameth,, C. Golds-berry,, P. Morozov,, S. G. Fischer,, G. Segal,, X. Qu,, A. Rzhetsky,, P. Zhang,, E. Cayanis,, P. J. De Jong,, J. Ju,, S. Kalachikov,, H. A. Shuman, and, J. J. Russo. 2004. The genomic sequence of the accidental pathogen Legionella pneumophila. Science 305:19661968.
4. Fields, B. S.,, R. F. Benson, and, R. E. Besser. 2002. Legionella and Legionnaires’ disease: 25 years of investigation. Clin. Microbiol. Rev. 15:506526.
5. O’Connell, W. A.,, J. M. Bangsborg, and, N. P. Cianciotto. 1995. Characterization of a Legionella micdadei mip mutant. Infect. Immun. 63:28402845.
6. O’Connell, W. A.,, E. K. Hickey, and, N. P. Cianciotto. 1996. A Legionella pneumophila gene that promotes hemin binding. Infect. Immun. 64:842848.
7. Sisson, G.,, J. Y. Jeong,, A. Goodwin,, L. Bryden,, N. Rossler,, S. Lim-Morrison,, A. Raudoni-kiene,, D. E. Berg, and, P. S. Hoffman. 2000 Metronidazole activation is mutagenic and causes DNA fragmentation in Helicobacter pylori and in Escherichia coli containing a cloned H. pylori RdxA(+) (Nitroreductase) gene. J. Baderiol. 182:50915096.

Tables

Generic image for table
TABLE 1

Efficiency of plating of Lp02 strains with and without metronizadole (20 μg/ml)

Citation: C. Brassinga A, A. Croxen M, J. Shoemaker C, G. Morash M, J. LeBlanc J, S. Hoffman P. 2006. Novel Use of Nitroreductase as a Counterselectable Marker in Allelic Vector Exchange to Create Philadelphia-1 Mutants, p 339-342. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch82

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