Biology subjects

Stevenson, B.

Publications and source records attributed to Stevenson, B..

8 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↗

bioRxiv2022

Inducible CRISPRi-based operon silencing and selective in trans gene complementation in Borrelia burgdorferi

To accelerate genetic studies on the Lyme disease pathogen Borrelia burgdorferi, we developed an enhanced CRISPR interference (CRISPRi) approach for IPTG-inducible repression of specific B. burgdorferi genes. The entire system is encoded on a compact 11-kb shuttle vector plasmid that allows for inducible expression of both the sgRNA module and a non-toxic codon-optimized dCas9 protein. We validated this CRISPRi system by targeting the genes encoding for OspA and OspB, abundant surface lipoproteins co-expressed by a single operon, and FlaB, the major subunit forming the periplasmic flagella. As in other systems, sgRNAs complementary to the non-template strand were consistently effective in gene repression, with 4- to 994-fold reductions in targeted transcript levels and concomitant reductions of in proteins levels. Furthermore, we showed that ospAB knockdowns could be selectively complemented in trans for OspA expression via the insertion of synonymous or non-synonymous CRISPRi-resistant PAM mutant (PAM*) ospA alleles into a unique site within the plasmid. Together, this establishes CRISPRi PAM* as a robust new genetic tool to simplify the study of B. burgdorferi genes, bypassing the need for gene disruptions by allelic exchange and avoiding rare-codon toxicity from heterologous expression of dCas9. SIGNIFICANCEBorrelia burgdorferi, the causative agent of Lyme disease, is a tick-borne pathogen of global importance. Here, we expand the genetic toolbox for studying B. burgdorferi physiology and pathogenesis by establishing a single-plasmid-based CRISPRi system with optional in trans complementation for the functional study of essential and non-essential proteins.

microbiology↗

bioRxiv2022

Erp and Rev adhesins of the Lyme disease spirochete's ubiquitous cp32 prophages assist the bacterium during vertebrate infection

Almost all spirochetes in the genus Borrelia (sensu lato) naturally contain multiple variants of closely related prophages. In the Lyme disease borreliae, these prophages are maintained as circular episomes that are called cp32s (circular plasmid 32kb). The cp32s of Lyme agents are particularly unique in that they encode two distinct families of lipoproteins, Erp and Rev, that are expressed on the bacterias outer surface during infection of vertebrate hosts. All identified functions of those outer surface proteins involve interactions between the spirochetes and host molecules: Erp proteins bind plasmin(ogen), laminin, glycosaminoglycans, and/or components of complement, and Rev proteins bind fibronectin. Thus, cp32 prophages provide their bacterial hosts with surface proteins that can enhance infection processes, thereby facilitating their own survival. Horizontal transfer via bacteriophage particles increases spread of beneficial alleles and creates diversity among Erp and Rev proteins.

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↗

bioRxiv2018

DNA Methylation by Restriction Modification Systems Affects the Global Transcriptome Profile in Borrelia burgdorferi

Prokaryote restriction modification (RM) systems serve to protect bacteria from potentially detrimental foreign DNA. Recent evidence suggests that DNA methylation by the methyltransferase (MTase) components of RM systems can also have effects on transcriptome profiles. The causative agent of Lyme disease, Borrelia burgdorferi, encodes two RM systems with N6-Methyladenosine (m6A) MTase activity. The specific recognition and/or methylation sequences have not been identified for either B. burgdorferi MTase, and it is not currently known whether these RM systems influence transcriptome profiles. In the current study, Single Molecule Real Time sequencing was utilized to map genome-wide m6A sites, and to identify consensus modified motifs in wild-type B. burgdorferi as well as isogenic MTase mutants. Four conserved m6A motifs were identified, and were fully attributable to the presence of specific MTases. Whole-genome transcriptome changes were observed in conjunction with the loss of MTase enzymes, indicating that DNA methylation by RM systems has effects on gene expression in B. burgdorferi. The results of this study provide a comprehensive view of the DNA methylation pattern in B. burgdorferi, and the accompanying gene expression profiles add to the emerging body of research on RM systems and gene regulation in bacteria.\n\nIMPORTANCELyme disease is the most prevalent vector-borne disease in North America, and is classified by the Centers for Disease Control and Prevention (CDC) as an emerging infectious disease with an expanding geographical area of occurrence. Previous studies have shown that the causative bacterium, Borrelia burgdorferi, methylates its genome using restriction modification systems that allow for the distinction of self from foreign DNA. Although much research has focused on the regulation of gene expression in B. burgdorferi, the effects of DNA methylation on gene regulation has not been evaluated. The current study characterizes the patterns of DNA methylation by restriction modification systems in B. burgdorferi, and evaluates the resulting effects on gene regulation in this important pathogen.

microbiology↗