Date of Award
Doctor of Philosophy
Veterinary Microbiology and Preventive Medicine
Ricardo F. Rosenbusch
In attempts to better understand the basic physiology and pathogenicity of the bovine-pathogenic Mycoplasma bovisstrain M23, we have developed genetic tools useful for M. bovis. Using the tools, a large scale transposon insertion mutant library was generated. Genetic analysis of the mutant library revealed that 324 out of 663 genes annotated in M. bovis were disrupted by 1,142 transposition events. By comparative genomic analysis, we defined 21 non-essential genes in M. bovis from among genes categorized as essential in other mycoplasma species, thereby, further reducing the size of the minimal genome complement of an independently living organism. From the mutant library, mutants defective in the ability to uptake thymidine were isolated. These were mutants with disruptions in the genes encoding the components of an ABC transport system. Substrate uptake studies proved that all components of the transporter are absolutely required for biological function of this thymidine high-affinity ABC transporter. 3H-thymidine uptake was sensitive to the presence of several nucleosides but insensitive to both nucleobases and pentoses, indicating that the transporter possesses a substrate binding protein with specificity to a portion of the broad chemical configuration of nucleosides, but not nucleobases or the ribose moiety. To define pathogenicity factors of M. bovis, a signature-tagged mutagenesis assay was developed using the mutant library and applied to determine the genes required for establishment of systemic infection and colonization on the respiratory tract of cattle. The genes identified as defective in non-invasive mutants were a putrescine/spermidine transporter permease gene (potC), uncharacterized ABC transport system permease genes, and an oligopeptide ABC transport system ATP-binding domain gene (oppF). Mutants of the last two genes were also unable to persist on the tracheal mucosa of cattle.
In summary, significant advances have been made in the characterization of the genome of M. bovis, and in the genetic manipulation of the genome. A high affinity thymidine transporter was characterized genetically and functionally, and several genes involved in pathogenicity were identified. These novel accomplishments have significantly advanced molecular knowledge about the biology and pathogenicity of this important pathogen of cattle.
Lee, Nakhyung, "Characterization of an ATP-binding cassette (ABC) transport system involved in nucleoside uptake in Mycoplasma bovis strain M23, and discovery of its pathogenicity genes" (2009). Graduate Theses and Dissertations. 10807.