DnaA modulates gene expression and morphology of the Lyme Disease spirochete

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 spirochetes 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. IMPORTANCELyme disease is the most prevalent tick-borne infection in the Northern Hemisphere. Borrelia burgdorferi, the causative spirochete bacteria, has been maintained in nature for millennia in a consistent enzootic cycle between Ixodes ticks and various small vertebrate hosts. During the ticks blood meal, B. burgdorferi substantially increases its replication rate, alters its repertoire of outer surface proteins, and disseminates into the new vertebrate host. Across eubacteria, DnaA is the master regulatory protein that initiates chromosomal replication and acts as a transcription factor to regulate specific pathways. Here, we describe the roles that B. burgdorferi DnaA has on the physiology and gene expression of this medically important pathogen.