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Applied and Environmental Microbiology, June 2005, p. 2888-2893, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.2888-2893.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Versatile Use of oriC Plasmids for Functional Genomics of Mycoplasma capricolum subsp. capricolum{dagger}

Carole Janis,1,{ddagger} Carole Lartigue,1,{ddagger} Joachim Frey,2 Henri Wróblewski,3 François Thiaucourt,4 Alain Blanchard,1 and Pascal Sirand-Pugnet1*

UMR Génomique Développement Pouvoir Pathogène, INRA, Université Victor Segalen Bordeaux 2, BP 81, 33883 Villenave d'Ornon Cedex, France,1 Institute of Veterinary Bacteriology, University of Berne, Laenggasstrasse 122, CH-3012 Berne, Switzerland,2 UMR CNRS 6026, Université de Rennes I, Campus de Beaulieu, 35042 Rennes Cedex, France,3 CIRAD-EMVT, Santé Animale, TA30/G, Campus International de Baillarguet, 34398 Montpellier Cedex 5, France4

Received 27 September 2004/ Accepted 21 December 2004

Replicative oriC plasmids were recently developed for several mollicutes, including three Mycoplasma species belonging to the mycoides cluster that are responsible for bovine and caprine diseases: Mycoplasma mycoides subsp. mycoides small-colony type, Mycoplasma mycoides subsp. mycoides large-colony type, and Mycoplasma capricolum subsp. capricolum. In this study, oriC plasmids were evaluated in M. capricolum subsp. capricolum as genetic tools for (i) expression of heterologous proteins and (ii) gene inactivation by homologous recombination. The reporter gene lacZ, encoding ß-galactosidase, and the gene encoding spiralin, an abundant surface lipoprotein of the related mollicute Spiroplasma citri, were successfully expressed. Functional Escherichia coli ß-galactosidase was detected in transformed Mycoplasma capricolum subsp. capricolum cells despite noticeable codon usage differences. The expression of spiralin in M. capricolum subsp. capricolum was assessed by colony and Western blotting. Accessibility of this protein at the cell surface and its partition into the Triton X-114 detergent phase suggest a correct maturation of the spiralin precursor. The expression of a heterologous lipoprotein in a mycoplasma raises potentially interesting applications, e.g., the use of these bacteria as live vaccines. Targeted inactivation of gene lppA encoding lipoprotein A was achieved in M. capricolum subsp. capricolum with plasmids harboring a replication origin derived from S. citri. Our results suggest that the selection of the infrequent events of homologous recombination could be enhanced by the use of oriC plasmids derived from related mollicute species. Mycoplasma gene inactivation opens the way to functional genomics in a group of bacteria for which a large wealth of genome data are already available and steadily growing.

* Corresponding author. Mailing address: INRA, Centre de Recherche de Bordeaux, UMR Génomique Développement Pouvoir Pathogène, 71 avenue Edouard Bourlaux, BP 81, 33883 Villenave d'Ornon Cedex, France. Phone: (33) 5 57 12 23 59. Fax: (33) 5 57 12 23 69. E-mail: sirand{at}bordeaux.inra.fr.

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

{ddagger} C.J. and C.L. contributed equally to the work.

Applied and Environmental Microbiology, June 2005, p. 2888-2893, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.2888-2893.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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