DnaA modulates gene expression and morphologyof 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 spirochete's physiology. 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, significantly slowed replication and increased or decreased cell lengths. Using fluorescent microscopy, we found the DnaA CRISPRi mutants had increased numbers of chromosomes with irregular spacing patterns. The 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.

Borrelia burgdorferi PlzA is a cyclic-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 and G-C content play 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.

Borrelia burgdorferi PlzA is a cyclic-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.

Quantitative analyses of interactions between SpoVG and RNA/DNA.

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.

Gac Is a Transcriptional Repressor of the Lyme Disease Spirochete's OspC Virulence-Associated Surface Protein.

The OspC outer-surface lipoprotein is essential for the Lyme disease spirochete's initial phase of vertebrate infection. Bacteria within the midguts of unfed ticks do not express OspC but produce high levels when ticks begin to ingest blood. Lyme disease spirochetes cease production of OspC within 1 to 2 weeks of vertebrate infection, and bacteria that fail to downregulate OspC are cleared by host antibodies. Thus, tight regulation of OspC levels is critical for survival of Lyme borreliae and, therefore, an attractive target for development of novel treatment strategies. Previous studies determined that a DNA region 5' of the ospC promoter, the ospC operator, is required for control of OspC production. Hypothesizing that the ospC operator may bind a regulatory factor, DNA affinity pulldown was performed and identified binding by the Gac protein. Gac is encoded by the C-terminal domain of the gyrA open reading frame from an internal promoter, ribosome-binding site, and initiation codon. Our analyses determined that Gac exhibits a greater affinity for ospC operator and promoter DNAs than for other tested borrelial sequences. In vitro and in vivo analyses demonstrated that Gac is a transcriptional repressor of ospC. These results constitute a substantial advance to our understanding of the mechanisms by which the Lyme disease spirochete controls production of OspC. IMPORTANCE Borrelia burgdorferi sensu lato requires its surface-exposed OspC protein in order to establish infection in humans and other vertebrate hosts. Bacteria that either do not produce OspC during transmission or fail to repress OspC after infection is established are rapidly cleared by the host. Herein, we identified a borrelial protein, Gac, that exhibits preferential affinity to the ospC promoter and 5' adjacent DNA. A combination of biochemical analyses and investigations of genetically manipulated bacteria demonstrated that Gac is a transcriptional repressor of ospC. This is a substantial advance toward understanding how the Lyme disease spirochete controls production of the essential OspC virulence factor and identifies a novel target for preventative and curative therapies.

Quantitative analyses of interactions between SpoVG and RNA/DNA.

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.