Versatile Use oforiCPlasmids for Functional Genomics ofMycoplasma capricolumsubsp.capricolum

Abstract

Replicative oriC plasmids were recently developed for several mollicutes, including three Mycoplasma species belonging to the mycoides cluster that are responsible for bovine and caprine diseases: Mycoplasma mycoides subsp. mycoides small-colony type, Mycoplasma mycoides subsp. mycoides large-colony type, and Mycoplasma capricolum subsp. capricolum. In this study, oriC plasmids were evaluated in M. capricolum subsp. capricolum as genetic tools for (i) expression of heterologous proteins and (ii) gene inactivation by homologous recombination. The reporter gene lacZ, encoding ␤-galactosidase, and the gene encoding spiralin, an abundant surface lipoprotein of the related mollicute Spiroplasma citri, were successfully expressed. Functional Escherichia coli ␤-galactosidase was detected in transformed Mycoplasma capricolum subsp. capricolum cells despite noticeable codon usage differences. The expression of spiralin in M. capricolum subsp. capricolum 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 lppA encoding lipoprotein A was achieved in M. capricolum subsp. capricolum with plasmids harboring a replication origin derived from S. citri. Our results suggest that the selection of the infrequent events of homologous recombination could be enhanced by the use of oriC 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.Mycoplasmas are small bacteria from the class Mollicutes that lack a cell wall and are characterized by a genome with a low percent GϩC (for a review, see reference 27). In contrast to the large wealth of data extracted from the analysis of their genome sequences (2), there is still a general lack of efficient genetic tools for the functional genomics of these bacteria. Transposon-based strategies have been used to generate random insertion mutants in a few mycoplasma species, but the attempts to develop cloning vectors from endogenous plasmids and viruses have encountered limited success (for a review, see reference 28). Recently, oriC-based replicative plasmids were developed for three mycoplasmas that cause economically important diseases in ruminants and belong to the mycoides cluster: Mycoplasma mycoides subsp. mycoides large-colony type, Mycoplasma mycoides subsp. mycoides small-colony type, and Mycoplasma capricolum subsp. capricolum (20). As previously shown for Mycoplasma pulmonis (6) and for another mollicute, Spiroplasma citri (38), the oriC plasmids that harbor the chromosomal dnaA gene and the adjacent DnaA box sequences were efficiently replicated in their respective hosts. Moreover, by h...

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

“…This is the first report, to our knowledge, of oriC plasmids for members of the Pneumoniae phylogenetic group. In other mollicutes, oriC plasmids have been used to inactivate genes or to express exogenous genes (Cordova et al, 2002;Duret et al, 1999;Janis et al, 2005;Lartigue et al, 2002), so these oriC plasmids are likely to be useful tools for genetic research in M. gallisepticum and M. imitans.…”

“…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.…”

Detection and quantification of intergenic transcription in Mycoplasma hyopneumoniae

“…The heat-shock response has been studied in a limited number of mycoplasma species (Madsen et al, 2006b;Musatovova et al, 2006;Weiner et al, 2003), and it is clear that a transcriptional activator, HrcA, is involved in regulating transcription of two heat-shock genes (Chang et al, 2008). In addition, transcriptional start sites have been mapped for a limited number of genes (Hyman et al, 1988;Taschke & Herrmann, 1988;Taschke et al, 1986;Weiner et al, 2000), and promoter-probe vectors have been used in several different species (Janis et al, 2005;Knudtson & Minion, 1993, 1994Lluch-Senar et al, 2007). These studies show that the 210 box of the Pribnow sequence is relatively conserved, but not the 235 sequence.…”

Xer1-Mediated Site-Specific DNA Inversions and Excisions in Mycoplasma agalactiae

“…This raised the intriguing possibility that such Xer1-mediated excisions also occur in the native M. agalactiae system. For further analysis of such excision events in the native system, we tested the feasibility of using the lacZ reporter tool in M. agalactiae, as lacZ is known to be expressed successfully in few other mycoplasma species, to study gene expression by use of promoter probe vectors (15,19,22,23). We developed an excision assay based on blue-white phenotype selection to study Xer1-mediated excisions in M. agalactiae, thus displaying a novel application of the lacZ reporter gene in mycoplasmas.…”