This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Émond, E.
Right arrow Articles by LaPointe, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Émond, E.
Right arrow Articles by LaPointe, G.
Agricola
Right arrow Articles by Émond, E.
Right arrow Articles by LaPointe, G.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, April 2001, p. 1700-1709, Vol. 67, No. 4
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.4.1700-1709.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Molecular Characterization of a Theta Replication Plasmid and Its Use for Development of a Two-Component Food-Grade Cloning System for Lactococcus lactis

Éric Émond,1,* Richard Lavallée,1 Geneviève Drolet,1 Sylvain Moineau,2 and Gisèle LaPointe1

Centre de recherche STELA, Département des sciences des aliments et de nutrition,1 and Department of Biochemistry and Microbiology and Groupe de Recherche en Écologie Buccale (GREB),2 Université Laval, Québec, Canada G1K 7P4

Received 9 October 2000/Accepted 16 January 2001

pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putative cis-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the cis-acting ori region and the repB gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in Lactococcus lactis. A two-component food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of L. lactis DNA and has no selection marker. The companion pCOM1 is a repB-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in L. lactis if trans complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of L. lactis and two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.

* Corresponding author. Mailing address: Chr. Hansen, Inc. 9015 West Maple St., Milwaukee, WI 53214-4298 Phone: (414) 607-5739. Fax: (414) 607-5859. E-mail: eemond{at}chr-hansen-us.com.

Applied and Environmental Microbiology, April 2001, p. 1700-1709, Vol. 67, No. 4
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.4.1700-1709.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Breton, M., Duret, S., Arricau-Bouvery, N., Beven, L., Renaudin, J. (2008). Characterizing the replication and stability regions of Spiroplasma citri plasmids identifies a novel replication protein and expands the genetic toolbox for plant-pathogenic spiroplasmas. Microbiology 154: 3232-3244 [Abstract] [Full Text]  
  • Defoor, E., Kryger, M.-B., Martinussen, J. (2007). The orotate transporter encoded by oroP from Lactococcus lactis is required for orotate utilization and has utility as a food-grade selectable marker. Microbiology 153: 3645-3659 [Abstract] [Full Text]  
  • Sun, C., Zhou, M., Li, Y., Xiang, H. (2006). Molecular Characterization of the Minimal Replicon and the Unidirectional Theta Replication of pSCM201 in Extremely Halophilic Archaea. J. Bacteriol. 188: 8136-8144 [Abstract] [Full Text]  
  • Labrie, S., Bart, C., Vadeboncoeur, C., Moineau, S. (2005). Use of an {alpha}-Galactosidase Gene as a Food-Grade Selection Marker for Streptococcus thermophilus. J DAIRY SCI 88: 2341-2347 [Abstract] [Full Text]  
  • Sorvig, E., Skaugen, M., Naterstad, K., Eijsink, V. G. H., Axelsson, L. (2005). Plasmid p256 from Lactobacillus plantarum represents a new type of replicon in lactic acid bacteria, and contains a toxin-antitoxin-like plasmid maintenance system. Microbiology 151: 421-431 [Abstract] [Full Text]  
  • Vaillancourt, K., LeMay, J.-D., Lamoureux, M., Frenette, M., Moineau, S., Vadeboncoeur, C. (2004). Characterization of a Galactokinase-Positive Recombinant Strain of Streptococcus thermophilus. Appl. Environ. Microbiol. 70: 4596-4603 [Abstract] [Full Text]  
  • Alpert, C.-A., Crutz-Le Coq, A.-M., Malleret, C., Zagorec, M. (2003). Characterization of a Theta-Type Plasmid from Lactobacillus sakei: a Potential Basis for Low-Copy-Number Vectors in Lactobacilli. Appl. Environ. Microbiol. 69: 5574-5584 [Abstract] [Full Text]  
  • Boucher, I., Vadeboncoeur, C., Moineau, S. (2003). Characterization of Genes Involved in the Metabolism of {alpha}-Galactosides by Lactococcus raffinolactis. Appl. Environ. Microbiol. 69: 4049-4056 [Abstract] [Full Text]  
  • Cotter, P. D., Hill, C., Ross, R. P. (2003). A Food-Grade Approach for Functional Analysis and Modification of Native Plasmids in Lactococcus lactis. Appl. Environ. Microbiol. 69: 702-706 [Abstract] [Full Text]  
  • Boucher, I., Parrot, M., Gaudreau, H., Champagne, C. P., Vadeboncoeur, C., Moineau, S. (2002). Novel Food-Grade Plasmid Vector Based on Melibiose Fermentation for the Genetic Engineering of Lactococcus lactis. Appl. Environ. Microbiol. 68: 6152-6161 [Abstract] [Full Text]  
  • Bron, P. A., Benchimol, M. G., Lambert, J., Palumbo, E., Deghorain, M., Delcour, J., de Vos, W. M., Kleerebezem, M., Hols, P. (2002). Use of the alr Gene as a Food-Grade Selection Marker in Lactic Acid Bacteria. Appl. Environ. Microbiol. 68: 5663-5670 [Abstract] [Full Text]  
  • Vaillancourt, K., Moineau, S., Frenette, M., Lessard, C., Vadeboncoeur, C. (2002). Galactose and Lactose Genes from the Galactose-Positive Bacterium Streptococcus salivarius and the Phylogenetically Related Galactose-Negative Bacterium Streptococcus thermophilus: Organization, Sequence, Transcription, and Activity of the gal Gene Products. J. Bacteriol. 184: 785-793 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»