AUTHOR=Wong Jessica K. , Crowley Michael A. , Bankhead Troy 

TITLE=Deletion of a Genetic Region of lp17 Affects Plasmid Copy Number in Borrelia burgdorferi

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=12

YEAR=2022

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2022.884171

DOI=10.3389/fcimb.2022.884171

ISSN=2235-2988

ABSTRACT=<p><italic>Borrelia burgdorferi</italic>, the Lyme disease pathogen, is maintained in its enzootic life cycle through complex gene regulatory pathways encoded on its uniquely fragmented genome. This genome consists of over 20 plasmids, and the regulatory mechanisms of plasmid maintenance and replication are largely unknown. The <italic>bbd21</italic> gene, encoded on lp17 and a member of the paralogous family 32 proteins, was originally proposed to be a putative <italic>parA</italic> orthologue involved with plasmid partitioning; however, this function has not been confirmed to date. To determine the role of <italic>bbd21</italic> in <italic>B. burgdorferi</italic>, we utilized targeted gene deletion and discovered <italic>bbd21</italic> and <italic>bbd22</italic> are co-transcribed. The effects of <italic>bbd21</italic> and <italic>bbd22</italic> deletion on plasmid copy number and mammalian infectivity were assessed. By qPCR, lp17 copy number did not differ amongst strains during mid-exponential and stationary growth phases. However, after <italic>in vitro</italic> passaging, the mutant strain demonstrated an 8-fold increase in lp17 copies, suggesting a cumulative defect in plasmid copy number regulation. Additionally, we compared lp17 copy number between <italic>in vitro</italic> and mammalian host-adapted conditions. Our findings showed 1) lp17 copy number was significantly different between these growth conditions for both the wild type and <italic>bbd21-bbd22</italic> deletion mutant and 2) under mammalian host-adapted cultivation, the absence of <italic>bbd21-bbd22</italic> resulted in significantly decreased copies of lp17. Murine infection studies using culture and qPCR demonstrated <italic>bbd21</italic>-<italic>bbd22</italic> deletion resulted in a tissue colonization defect, particularly in the heart. Lastly, we showed <italic>bbd21</italic> transcription appears to be independent of direct <italic>rpoS</italic> regulation based on similar expression levels in wild type and Δ<italic>rpoS</italic>. Altogether, our findings indicate the <italic>bbd21</italic>-<italic>bbd22</italic> genetic region is involved with regulation of lp17 plasmid copy number. Furthermore, we propose the possibility that lp17 plasmid copy number is important for microbial pathogenesis by the Lyme disease spirochete.</p>