Analysis of Resuscitation Promoting Factor C of Mycobacterium avium subsp. paratuberculosis

Heirwegh, Meagan
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University of Guelph

Bacteria can deal with environmental stress in a number of ways. Some species halt processes necessary for cell growth and division, leaving those only absolutely necessary for survival until conditions improve. In Mycobacterium avium subspecies paratuberculosis (MAP) this strategy is known as entering the viable but non-culturable state (VBNC). Exit from this state is thought to be facilitated by resuscitation promoting factors (Rpfs) which were first identified in Micrococcus luteus as essential, growth promoting proteins. Micr. luteus culture supernatant was used to resuscitate VBNC MAP cells in a concentration dependent manner. It was hypothesized that genes encoding four different resuscitation promoting factors are present in the MAP genome. One of these genes, a homologue of the M. tuberculosis rpfC was identified and cloned from the MAP K10 genome. MAP RpfC was produced as a maltose binding protein fusion in Escherichia coli Rosetta (DE3). It was not possible to generate anti-RpfC antibodies in rabbits and rats. Antibodies were successfully generated in rabbits towards a synthetic peptide based on the consensus Rpf domain sequence. However, these antibodies could not detect recombinant RpfC. Potential markers for the VBNC state were identified through the use of reverse transcriptase PCR. This study begins to build a foundation for the study of Rpfs in MAP, an area that is currently poorly understood.

Mycobacterium avium, Paratuberculosis, Micrococcus luteus, Reverse transcriptase, Environmental stress