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Applied and Environmental Microbiology, August 2008, p. 4772-4775, Vol. 74, No. 15
0099-2240/08/$08.00+0     doi:10.1128/AEM.00134-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Plasmid pCS1966, a New Selection/Counterselection Tool for Lactic Acid Bacterium Strain Construction Based on the oroP Gene, Encoding an Orotate Transporter from Lactococcus lactis{triangledown}

Christian Solem, Els Defoor, Peter Ruhdal Jensen, and Jan Martinussen*

Center for Systems Microbiology, Department for Systems Biology, Building 301, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark

Received 15 January 2008/ Accepted 18 March 2008

In this paper we describe the new selection/counterselection vector pCS1966, which is suitable for both sequence-specific integration based on homologous recombination and integration in a bacteriophage attachment site. This plasmid harbors oroP, which encodes a dedicated orotate transporter, and can replicate only in Escherichia coli. Selection for integration is performed primarily by resistance to erythromycin; alternatively, the ability to utilize orotate as a pyrimidine source in a pyrimidine auxotrophic mutant could be utilized. Besides allowing the cell to utilize orotate, the transporter renders the cell sensitive to 5-fluoroorotate. This sensitivity is used to select for loss of the plasmid. When expressed from its own promoter, oroP was toxic to E. coli, whereas in Lactococcus lactis the level of expression of oroP from a chromosomal copy was too low to confer 5-fluoroorotate sensitivity. In order to obtain a plasmid that confers 5-fluoroorotate sensitivity when it is integrated into the chromosome of L. lactis and at the same time can be stably maintained in E. coli, the expression of the oroP gene was controlled from a synthetic promoter conferring these traits. To demonstrate its use, a number of L. lactis strains expressing triosephosphate isomerase (tpiA) at different levels were constructed.

* Corresponding author. Mailing address: Center for Systems Microbiology, Department of Systems Biology, Building 301, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark. Phone: 45 45 25 24 98. Fax: 45 45 88 26 60. E-mail: jma{at}biocentrum.dtu.dk

{triangledown} Published ahead of print on 6 June 2008.

Applied and Environmental Microbiology, August 2008, p. 4772-4775, Vol. 74, No. 15
0099-2240/08/$08.00+0     doi:10.1128/AEM.00134-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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