Biology subjects

Jusufovic, N.

Publications and source records attributed to Jusufovic, N..

3 recordsLinked to original sources

bioRxiv2024

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.

microbiology↗

bioRxiv2023

Quantitative analyses of interactions between SpoVG and RNA/DNA

1.The Borrelia burgdorferi SpoVG protein has previously been found to be a DNA- and RNA-binding protein. To aid in the elucidation of ligand motifs, affinities for numerous RNAs, ssDNAs, and dsDNAs were measured and compared. The loci used in the study were spoVG, glpFKD, erpAB, bb0242, flaB, and ospAB, with particular focus on the untranslated 5 portion of the mRNAs. Performing binding and competition assays yielded that the 5 end of spoVG mRNA had the highest affinity while the lowest observed affinity was to the 5 end of flaB mRNA. Mutagenesis studies of spoVG RNA and ssDNA sequences suggested that the formation of SpoVG-nucleic acid complexes are not entirely dependent on either sequence or structure. Additionally, exchanging uracil for thymine in ssDNAs did not affect protein-nucleic acid complex formation.

microbiology↗

bioRxiv2023

Borrelia burgdorferi PlzA is a c-di-GMP dependent DNA and RNA binding protein

The PilZ domain-containing protein, PlzA, is the only known cyclic di-GMP binding protein encoded by all Lyme disease spirochetes. PlzA has been implicated in the regulation of many borrelial processes, but the effector mechanism of PlzA was not previously known. Here we report that PlzA can bind DNA and RNA and that nucleic acid binding requires c-di-GMP, with the affinity of PlzA for nucleic acids increasing as concentrations of c-di-GMP were increased. A mutant PlzA that is incapable of binding c-di-GMP did not bind to any tested nucleic acids. We also determined that PlzA interacts predominantly with the major groove of DNA and that sequence length plays a role in DNA binding affinity. PlzA is a dual-domain protein with a PilZ-like N-terminal domain linked to a canonical C-terminal PilZ domain. Dissection of the domains demonstrated that the separated N-terminal domain bound nucleic acids independently of c-di-GMP. The C-terminal domain, which includes the c-di-GMP binding motifs, did not bind nucleic acids under any tested conditions. Our data are supported by computational docking, which predicts that c-di-GMP binding at the C-terminal domain stabilizes the overall protein structure and facilitates PlzA-DNA interactions via residues in the N-terminal domain. Based on our data, we propose that levels of c-di-GMP during the various stages of the enzootic life cycle direct PlzA binding to regulatory targets.

microbiology↗