Bacteria of the genus Mycoplasma have recently attracted considerable interest as model organisms in synthetic and systems biology. In particular, Mycoplasma pneumoniae is one of the most intensively studied organisms in the field of systems biology. However, the genetic manipulation of these bacteria is often difficult due to the lack of efficient genetic systems and some intrinsic peculiarities such as an aberrant genetic code. One major disadvantage in working with M. pneumoniae is the lack of replicating plasmids that can be used for the complementation of mutants and the expression of proteins. In this study, we have analysed the genomic region around the gene encoding the replication initiation protein, DnaA, and detected putative binding sites for DnaA (DnaA boxes) that are, however, less conserved than in other bacteria. The construction of several plasmids encompassing this region allowed the selection of plasmid pGP2756 that is stably inherited and that can be used for genetic experiments, as shown by the complementation assays with the glpQ gene encoding the glycerophosphoryl diester phosphodiesterase. Plasmid-borne complementation of the glpQ mutant restored the formation of hydrogen peroxide when bacteria were cultivated in the presence of glycerol phosphocholine. Interestingly, the replicating plasmid can also be used in the close relative, Mycoplasma genitalium but not in more distantly related members of the genus Mycoplasma. Thus, plasmid pGP2756 is a valuable tool for the genetic analysis of M. pneumoniae and M. genitalium.Read less <