Abstract
Mycoplasma bovis is a cause of pneumonia, mastitis, arthritis and otitis media in cattle throughout the world. However, despite its clinical significance, there is a paucity of tools to genetically manipulate it, impeding our capacity to further explore the molecular basis of its virulence. To address this limitation, we developed a series of homologous and heterologous replicable plasmids from M. bovis and M. agalactiae. The shortest replicable oriC plasmid based on the region downstream of dnaA in M. bovis was 247 bp and contained two DnaA boxes, while oriC plasmids based on the region downstream of dnaA in M. agalactiae strains 5632 and PG2 were 219 bp and 217 bp in length, respectively, and contained only a single DnaA box. The efficiency of transformation in M. bovis and M. agalactiae was inversely correlated with the size of the oriC region in the construct, and, in general, homologous oriC plasmids had a higher transformation efficiency than heterologous oriC plasmids. The larger pWholeoriC45 and pMM21-7 plasmids integrated into the genomic oriC region of M. bovis, while the smaller oriC plasmids remained extrachromosomal for up to 20 serial passages in selective media. Although specific gene disruptions were not be achieved in M. bovis in this study, the oriC plasmids developed here could still be useful as tools in complementation studies and for expression of exogenous genes in both M. bovis and M. agalactiae.
Highlights
Mycoplasma bovis is a major cause of chronic caseonecrotic bronchopneumonia in calves [1,2]
M. bovis strain PG45 was transformed with the homologous oriC plasmid pWholeoriC45, containing the dnaA gene and its upstream and downstream intergenic regions along with the tetM determinant, which confers tetracycline resistance in Mollicutes
M. agalactiae strain PG2 was transformed with the homologous oriC plasmid pMM21-7 [28], containing the 1.3 kbp oriC region of M. agalactiae strain 5632
Summary
Mycoplasma bovis is a major cause of chronic caseonecrotic bronchopneumonia in calves [1,2]. Its contribution to bovine respiratory tract disease is commonly complicated by concurrent infection with a number of viral and bacterial pathogens, including bovine herpesvirus type 1, PLOS ONE | DOI:10.1371/journal.pone.0119000. The genome sequence of the M. bovis type strain PG45, as well as the Hubei-1 and HB0801 strains, have been completed and published recently [8,9,10], but the paucity of genetic tools for generating targeted gene knockouts and for complementation studies impedes our ability to examine gene function in M. bovis. Transposon mutagenesis using Tn916 and Tn4001 and their derivatives has been used to study the genetics of several human and animal mycoplasmas, but the random insertion of a transposon in the genome of an organism does not allow specific targeting of a gene of interest [11,12]
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