Abstract
All bacteria encode a multifunctional DNA-binding protein, DnaA, which initiates chromosomal replication. Despite having the most complex, segmented bacterial genome, little is known about Borrelia burgdorferi DnaA and its role in maintaining the spirochete's physiology. In this work we utilized inducible CRISPR-interference and overexpression to modulate cellular levels of DnaA to better understand this essential protein. Dysregulation of DnaA, either up or down, increased or decreased cell lengths, respectively, while also significantly slowing replication rates. Using fluorescent microscopy, we found the DnaA CRISPRi mutants had increased numbers of chromosomes with irregular spacing patterns. DnaA-depleted spirochetes also exhibited a significant defect in helical morphology. RNA-seq of the conditional mutants showed significant changes in the levels of transcripts involved with flagellar synthesis, elongation, cell division, virulence, and other functions. These findings demonstrate that the DnaA plays a commanding role in maintaining borrelial growth dynamics and protein expression, which are essential for the survival of the Lyme disease spirochete.
Published Version
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