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Epitope tagging of chromosomal genes in Salmonella

Uzzau, Sergio and Figueroa-Bossi, Nara and Rubino, Salvatore and Bossi, Lionello (2001) Epitope tagging of chromosomal genes in Salmonella. Proceedings of The National Academy of Sciences, Vol. 98 (26), p. 15264-15269. eISSN 1091-6490. Article.

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DOI: 10.1073/pnas.261348198

Abstract

We have developed a simple and efficient procedure for adding an epitope-encoding tail to one or more genes of interest in the bacterial chromosome. The procedure is a modification of the gene replacement method of Datsenko and Wanner [Datsenko, K. A. & Wanner, B. L. (2000) Proc. Natl. Acad. Sci. USA 97, 6640–6645]. A DNA module that begins with the epitope-encoding sequence and includes a selectable marker is amplified by PCR with primers that carry extensions (as short as 36 nt) homologous to the last portion of the targeted gene and to a region downstream from it. Transformation of a strain expressing bacteriophage ʎ red functions yields recombinants carrying the targeted gene fused to the epitope-encoding sequence. The resulting C-terminal-tagged protein can be identified by standard immuno-detection techniques. In an initial application of the method, we have added the sequences encoding the FLAG and 3xFLAG and influenza virus hemagglutinin epitopes to various genes of Salmonella enterica serovar Typhimurium, including putative and established pathogenic determinants present in prophage genomes. Epitope fusion proteins were detected in bacteria growing in vitro, tissue culture cells, and infected mouse tissues. This work identified a prophage locus specifically expressed in bacteria growing intracellularly. The procedure described here should be applicable to a wide variety of Gram-negative bacteria and is particularly suited for the study of intracellular pathogens.

Item Type:Article
ID Code:1148
Status:Published
Refereed:Yes
Uncontrolled Keywords:Salmonella, bacterial chromosome, epitope tagging
Subjects:Area 06 - Scienze mediche > MED/07 Microbiologia e microbiologia clinica
Divisions:001 Università di Sassari > 01 Dipartimenti > Scienze biomediche
Publisher:The National Academy of Sciences of the USA
eISSN:1091-6490
Publisher Policy:Depositato per gentile concessione dell'Editore. È vietato duplicare e postare il PDF. E' consentito l'uso a solo scopo di studio e di ricerca.
Deposited On:18 Aug 2009 10:04

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