| The Lyme spirochete Borrelia burgdorferi causes the most prevalent vector-borne infection in North America. In this study, the importance of DNA metabolizing genes for the infectivity, persistence and survival to DNA damage in B. burgdorferi was determined. During the infection of a vertebrate host, B. burgdorferi undergoes antigenic variation by DNA recombination at vlsE, which encodes for an immunogenic surface lipoprotein required for the persistence of the spirochete. In the present study, eight gene targets were disrupted and only the RuvAB Holiday junction branch migrase subunits affected the switching at vlsE and the persistence of B. burgdorferi in mice. The disruption of these eight genes was part of a wider study aiming to identify nucleic acid metabolizing genes involved in switching at vlsE. Although no other genes were found to strongly affect switching, the disruption of the DEAH-box RNA helicase HrpA abolished the infectivity of B. burgdorferi. Since the complementation of hrpA in trans could not be achieved, the restoration of the wild-type gene by allelic exchange was used as an alternate strategy for complementation. The restoration of the wild-type hrpA did restore infectivity, confirming the importance of hrpA. Point mutations were also introduced by allelic exchange in motifs required for either the RNA helicase or the ATPase activity of HrpA. To avoid an initial screen of a large number of clones by sequencing, a strategy was adapted to confirm by PCR the presence of the mutation in the gene. Infection of mice with these B. burgdorferi hrpA mutants confirmed that the RNA helicase activity, in addition of the ATPase activity, is required for the survival of B. burgdorferi in the mouse. Finally, a strategy was adapted to expediently compare the cell density of multiple cultures. This strategy was used to measure the importance of 25 nucleic acid metabolizing genes for survival of B. burgdorferi to DNA damage. Using this strategy, the nucleotide excision repair pathway was shown to be the sole repair pathway to be significantly involved in repair of UV-induced DNA damage in B. burgdorferi. |
