Biology subjects

Saylor, T. C.

Publications and source records attributed to Saylor, T. C..

4 recordsLinked to original sources

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↗

bioRxiv2022

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 spirochetes 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 1-2 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 the mechanisms by which the Lyme disease spirochete controls production of OspC. ImportanceBorrelia burgdorferi (sensu lato) requires its surface-exposed OspC protein in order to establish infection of 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.

microbiology↗

bioRxiv2022

Borrelia burgdorferi DnaA and the nucleoid-associated protein EbfC coordinate expression of the dnaX-ebfC operon

Borrelia burgdorferi, the spirochete agent of Lyme disease, has evolved within a consistent infectious cycle between tick and vertebrate hosts. The transmission of the pathogen from tick to vertebrate is characterized by rapid replication and a change in the outer surface protein profile. EbfC, a highly conserved nucleoid-associated protein, binds throughout the borrelial genome affecting expression of many genes, including the Erp outer surface proteins. In B. burgdorferi, like many other bacterial species, ebfC is co-transcribed with dnaX, an essential component of the DNA polymerase III holoenzyme, which facilitates chromosomal replication. The expression of the dnaX-ebfC operon is tied to the spirochetes replication rate, but the underlying mechanism for this connection was unknown. In this work, we provide evidence that the expression of dnaX-ebfC is controlled by direct interactions of DnaA, the chromosomal replication initiator, and EbfC at the unusually long dnaX-ebfC 5 UTR region. Both proteins bind to the 5 UTR DNA, with EbfC also binding to the RNA. The DNA binding of DnaA to this region was similarly impacted by ATP/ADP. In vitro studies characterized DnaA as an activator of dnaX-ebfC and EbfC as an anti-activator. We further found evidence that DnaA may regulate other genes essential for replication. IMPORTANCEThe dual-life cycle of Borrelia burgdorferi, the causative agent of Lyme disease, is characterized by periods of rapid and slowed replication. The expression patterns of many of the spirochetes virulence factors are impacted by these changes in replication rates. The connection between replication and virulence can be understood at the dnaX-ebfC operon. DnaX is a component of the DNA polymerase III holoenzyme that facilitates replication. EbfC is a nucleoid-associated protein that regulates the infection-associated outer surface Erp proteins, as well as other transcripts. The expression of dnaX-ebfC is tied to replication rate, which we demonstrate is mediated by DnaA, the master chromosomal initiator protein and transcription factor, and EbfC.

microbiology↗