Plasmid stability during in vitro propagation of Borrelia burgdorferi assessed at a clonal level

Borrelia burgdorferi causes Lyme disease in humans. The genome of the sequenced type strain B31 MI consists of a linear chromosome, 12 linear plasmids, and 9 circular plasmids. Previous studies by other investigators indicated that some of these plasmids are essential for the survival of the spirochetes in vivo but not in vitro. We have studied plasmid stability during in vitro growth at 23 and 35 degrees C, conditions that approximate the temperatures of the tick vector and the mammalian host, respectively. Starting with two clones that have all 21 plasmids, we investigated plasmid maintenance within the population and on a clonal level. After three passages (27 generations), the cultures were no longer homogeneous and some derivative clones had already lost multiple plasmids. Despite this, one of six clones analyzed after 25 passages (225 generations) retained all but one plasmid (cp9) and was able to complete the mouse-tick-mouse infectious cycle. We analyzed protein composition and regulation of gene expression of clones differing in plasmid content after serial passages. All clones tested exhibited temperature-regulated expression of several proteins, including OspC. In addition, analysis of cultures inoculated from frozen stocks suggests that freezing and/or thawing contributes to heterogeneity in the outgrowth population with respect to plasmid content. Our investigations show that in vitro propagation of a clone leads to a heterogeneous population but that virulent clones can persist through extended passage. We therefore conclude that isogenicity of clones must be confirmed irrespective of their in vitro passage history.