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/ micro g 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.