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Journal of Bacteriology, October 2002, p. 5426-5435, Vol. 184, No. 19
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.19.5426-5435.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification of the Origin of Replication of the Mycoplasma pulmonis Chromosome and Its Use in oriC Replicative Plasmids

Caio M. M. Cordova,1 Carole Lartigue,2 Pascal Sirand-Pugnet,2 Joël Renaudin,3 Regina A. F. Cunha,1 and A. Blanchard2*

University of Sao Paulo, Analises Clinica & Toxicologicas, Faculdade de Ciencias Farmaceuticas, Sao Paulo 05508-900, Brazil,1 Laboratoire des Interactions Plantes-Pathogènes,2 Laboratoire de Biologie Cellulaire et Moléculaire, INRA-Université Victor Segalen Bordeaux 2, 33883 Villenave d'Ornon Cedex, France3

Received 12 March 2002/ Accepted 10 July 2002

Mycoplasma pulmonis is a natural rodent pathogen, considered a privileged model for studying respiratory mycoplasmosis. The complete genome of this bacterium, which belongs to the class Mollicutes, has recently been sequenced, but studying the role of specific genes requires improved genetic tools. In silico comparative analysis of sequenced mollicute genomes indicated the lack of conservation of gene order in the region containing the predicted origin of replication (oriC) and the existence, in most of the mollicute genomes examined, of putative DnaA boxes lying upstream and downstream from the dnaA gene. The predicted M. pulmonis oriC region was shown to be functional after cloning it into an artificial plasmid and after transformation of the mycoplasma, which was obtained with a frequency of 3 x 10-6 transformants/CFU/µg of plasmid DNA. However, after a few in vitro passages, this plasmid integrated into the chromosomal oriC region. Reduction of this oriC region by subcloning experiments to the region either upstream or downstream from dnaA resulted in plasmids that failed to replicate in M. pulmonis, except when these two intergenic regions were cloned with the tetM determinant as a spacer in between them. An internal fragment of the M. pulmonis hemolysin A gene (hlyA) was cloned into this oriC plasmid, and the resulting construct was used to transform M. pulmonis. Targeted integration of this genetic element into the chromosomal hlyA by a single crossing over, which results in the disruption of the gene, could be documented. These mycoplasmal oriC plasmids may therefore become valuable tools for investigating the roles of specific genes, including those potentially implicated in pathogenesis.

* Corresponding author. Mailing address: INRA, Centre de Recherche de Bordeaux, Institut de Biologie Végétale Moléculaire, 71, avenue Edouard Bourlaux, BP 81, 33883 Villenave D'Ornon Cedex, France. Phone: (33) 5 57 12 23 93. Fax: (33) 5 57 12 23 69. E-mail: ablancha{at}bordeaux.inra.fr.

Journal of Bacteriology, October 2002, p. 5426-5435, Vol. 184, No. 19
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.19.5426-5435.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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