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Applied and Environmental Microbiology, May 2009, p. 3093-3105, Vol. 75, No. 10
0099-2240/09/$08.00+0     doi:10.1128/AEM.02502-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Development and Application of a upp-Based Counterselective Gene Replacement System for the Study of the S-Layer Protein SlpX of Lactobacillus acidophilus NCFM ^,

Yong Jun Goh,¹ M. Andrea Azcárate-Peril,¹ Sarah O'Flaherty,¹ Evelyn Durmaz,¹ Florence Valence,² Julien Jardin,² Sylvie Lortal,² and Todd R. Klaenhammer^1*

Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, North Carolina 27695,¹ INRA—Technologie Laitière, 65 Rue de St. Brieuc, 35042 Rennes Cédex, France²

Received 23 October 2008/ Accepted 16 February 2009

In silico genome analysis of Lactobacillus acidophilus NCFM coupled with gene expression studies have identified putative genes and regulatory networks that are potentially important to this organism's survival, persistence, and activities in the gastrointestinal tract. Correlation of key genotypes to phenotypes requires an efficient gene replacement system. In this study, use of the upp-encoded uracil phosphoribosyltransferase (UPRTase) of L. acidophilus NCFM was explored as a counterselection marker to positively select for recombinants that have resolved from chromosomal integration of pORI-based plasmids. An isogenic mutant carrying a upp gene deletion was constructed and was resistant to 5-fluorouracil (5-FU), a toxic uracil analog that is also a substrate for UPRTase. A 3.0-kb pORI-based counterselectable integration vector bearing a upp expression cassette, pTRK935, was constructed and introduced into the upp host harboring the pTRK669 helper plasmid. Extrachromosomal replication of pTRK935 complemented the mutated chromosomal upp allele and restored sensitivity to 5-FU. This host background provides a platform for a two-step plasmid integration and excision strategy that can select for plasmid-free recombinants with either the wild-type or mutated allele of the targeted gene in the presence of 5-FU. The efficacy of the system was demonstrated by in-frame deletion of the slpX gene (LBA0512) encoding a novel 51-kDa secreted protein associated with the S-layer complex of L. acidophilus. The resulting slpX mutant exhibited lower growth rates, increased sensitivity to sodium dodecyl sulfate, and greater resistance to bile. Overall, this improved gene replacement system represents a valuable tool for investigating the mechanisms underlying the probiotic functionality of L. acidophilus.

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* Corresponding author. Mailing address: Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Box 7624, Raleigh, NC 27695. Phone: (919) 515-2972. Fax: (919) 513-0014. E-mail: klaenhammer{at}ncsu.edu

Published ahead of print on 20 March 2009.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, May 2009, p. 3093-3105, Vol. 75, No. 10
0099-2240/09/$08.00+0     doi:10.1128/AEM.02502-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.