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

Abstract

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 ؋ 10 ؊6 transformants/CFU/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.The mollicutes are the smallest free-living microorganisms capable of autoreplication (33). They are related to grampositive eubacteria from which they evolved by a drastic reduction of genome size, being thus considered the best representatives of the concept of a minimal cell. (964 kbp [5]). Postgenomic approaches aiming at deciphering the role of specific genes discovered by in silico analysis now require efficient strategies to genetically manipulate these organisms. The transposon Tn916 or Tn4001 can transpose in all the mollicute genomes, at least for the species amenable to transformation, such as M. genitalium, M. pneumoniae, and M. pulmonis. In M. genitalium and M. pneumoniae, a global approach of gene inactivation with the transposon Tn4001 was used to define the genes that are essential for the life of these bacteria (22). Derivatives of this transposon, obtained by insertion of a chloramphenicol acetyltransferase gene and the tetM tetracycline resistance determinant, were also shown to transpose in M. pulmonis (12). The use of these genetic elements for gene inactivation, which relies on the random insertion of the transposon in the genome of the organism, does not allow specific targeting of a gene of interest. In addition, the complementation of mutants would require the cloning and expression of genes in...

Development of a replicable oriC plasmid for Mycoplasma gallisepticum and Mycoplasma imitans, and gene disruption through homologous recombination in M. gallisepticum

“…8), which contains the dnaA gene. The functional oriC plasmids for these species require regions upstream and downstream from the dnaA gene, with the exception of the smallest oriC plasmid for S. citri, which contains only a 163 bp region downstream from the dnaA gene (Cordova et al, 2002;Lartigue et al, 2002Lartigue et al, , 2003. The gene order in the oriC regions of M. gallisepticum and M. imitans is similar to those of M. pneumoniae and Mycoplasma genitalium (Fig.…”

“…Previous studies have reported that oriC plasmids containing larger oriC regions can easily integrate into the oriC region of genomic DNA through homologous recombination after in vitro passage (Cordova et al, 2002;Renaudin et al, 1995). In order to reduce the likelihood of this, a number of plasmids containing different lengths of the oriC region were tested for replication in M. gallisepticum.…”

“…8). These mycoplasmas belong to the Pneumoniae phylogenetic group and the putative oriC regions surround the soj gene but lie upstream of the dnaA gene (Cordova et al, 2002;Papazisi et al, 2003). We produced four oriC plasmid constructs that contained the soj gene and regions upstream and downstream of the soj gene in M. gallisepticum.…”

“…There has been some success in development of vectors for mollicutes using homologous origins of replication (oriC) and selectable antibiotic resistance markers. These plasmids are able to replicate extrachromosomally and have been used to inactivate target genes by homologous recombination (Cordova et al, 2002;Duret et al, 1999;Janis et al, 2005;Lartigue et al, 2002). In many cases oriC plasmids containing larger oriC regions have been found to integrate into the oriC region in the genomic DNA by homologous recombination following in vitro passage.…”

“…In many cases oriC plasmids containing larger oriC regions have been found to integrate into the oriC region in the genomic DNA by homologous recombination following in vitro passage. Plasmids containing a shorter oriC region are more stable and have been used to generate targeted homologous recombination (Cordova et al, 2002;Lartigue et al, 2002;Renaudin et al, 1995). Most of the oriC plasmids of mollicutes show host specificity, but plasmids containing the oriC of Mycoplasma mycoides subsp.…”