During DNA replication in Borrelia burgdorferi, replication of linear elements results in an inverted dimer repeat of the telomere which is subsequently resolved by the telomere resolvase, ResT. ResT has been demonstrated as essential and a knock-out strain has never been produced. Using a conditional expression system a resT knock-out strain has been produced and used to investigate the effects of ResT depletion of spirochete viability as well as genome stability. ResT depleted spirochetes ceased to divide after ResT depletion, however, remained viable up to 16 days suggesting an arrested state of the spirochetes. Southern blots of the DNA revealed a shift of linear plasmid DNA to a higher molecular weight representing the circular intermediate. DNA replication did not continue in spirochetes after ResT depletion suggesting ResT may play a larger role in replication than previously thought.