Janis, Carole Lartigue, Carole Frey, Joachim Wróblewski, Henri Thiaucourt, François Blanchard, Alain Sirand-Pugnet, Pascal <jats:title>ABSTRACT</jats:title> <jats:p>Replicative <jats:italic>oriC</jats:italic> plasmids were recently developed for several mollicutes, including three <jats:italic>Mycoplasma</jats:italic> species belonging to the mycoides cluster that are responsible for bovine and caprine diseases: <jats:italic>Mycoplasma mycoides</jats:italic> subsp. <jats:italic>mycoides</jats:italic> small-colony type, <jats:italic>Mycoplasma mycoides</jats:italic> subsp. <jats:italic>mycoides</jats:italic> large-colony type, and <jats:italic>Mycoplasma capricolum</jats:italic> subsp. <jats:italic>capricolum</jats:italic>. In this study, <jats:italic>oriC</jats:italic> plasmids were evaluated in <jats:italic>M. capricolum</jats:italic> subsp. <jats:italic>capricolum</jats:italic> as genetic tools for (i) expression of heterologous proteins and (ii) gene inactivation by homologous recombination. The reporter gene <jats:italic>lacZ</jats:italic>, encoding β-galactosidase, and the gene encoding spiralin, an abundant surface lipoprotein of the related mollicute <jats:italic>Spiroplasma citri</jats:italic>, were successfully expressed. Functional <jats:italic>Escherichia coli</jats:italic> β-galactosidase was detected in transformed <jats:italic>Mycoplasma capricolum</jats:italic> subsp. <jats:italic>capricolum</jats:italic> cells despite noticeable codon usage differences. The expression of spiralin in <jats:italic>M. capricolum</jats:italic> subsp. <jats:italic>capricolum</jats:italic> 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 <jats:italic>lppA</jats:italic> encoding lipoprotein A was achieved in <jats:italic>M. capricolum</jats:italic> subsp. <jats:italic>capricolum</jats:italic> with plasmids harboring a replication origin derived from <jats:italic>S. citri</jats:italic>. Our results suggest that the selection of the infrequent events of homologous recombination could be enhanced by the use of <jats:italic>oriC</jats:italic> 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.</jats:p> application/pdf https://sonar.ch/global/documents/244529 eng info:eu-repo/semantics/altIdentifier/doi/10.1128/aem.71.6.2888-2893.2005 info:eu-repo/semantics/altIdentifier/issn/0099-2240 info:eu-repo/semantics/openAccess Applied and Environmental Microbiology. - American Society for Microbiology. - 2005, vol. 71, no. 6, p. 2888-2893 Biotechnology Food Science Ecology Applied Microbiology and Biotechnology Versatile Use of oriC Plasmids for Functional Genomics of Mycoplasma capricolum subsp. capricolum http://purl.org/coar/resource_type/c_6501