A CRISPR Interference Platform for Selective Downregulation of Gene Expression in Borrelia burgdorferi

Abstract

The spirochete Borrelia burgdorferi causes Lyme disease, an increasingly prevalent infection. While previous studies have provided important insight into B. burgdorferi biology, many aspects, including basic cellular processes, remain underexplored. To help speed up the discovery process, we adapted a CRISPR interference (CRISPRi) platform for use in B. burgdorferi. For efficiency and flexibility of use, we generated various CRISPRi template constructs that produce different basal and induced levels of dcas9 and carry different antibiotic resistance markers. We characterized the effectiveness of our CRISPRi platform by targeting the motility and cell morphogenesis genes flaB, mreB, rodA, and ftsI, whose native expression levels span two orders of magnitude. For all four genes, we obtained gene repression efficiencies of at least 95%. We showed by darkfield microscopy and cryo-electron tomography that flagellin (FlaB) depletion reduced the length and number of periplasmic flagella, which impaired cellular motility and resulted in cell straightening. Depletion of FtsI caused cell filamentation, implicating this protein in cell division in B. burgdorferi. Finally, localized cell bulging in MreB- and RodA-depleted cells matched the locations of new peptidoglycan insertion specific to spirochetes of the Borrelia genus. These results therefore implicate MreB and RodA in the particular mode of cell wall elongation of these bacteria. Collectively, our results demonstrate the efficiency and ease of use of our B. burgdorferi CRISPRi platform, which should facilitate future genetic studies of this important pathogen. IMPORTANCE Gene function studies are facilitated by the availability of rapid and easy-to-use genetic tools. Homologous recombination-based methods traditionally used to genetically investigate gene function remain cumbersome to perform in B. burgdorferi, as they often are relatively inefficient. In comparison, our CRISPRi platform offers an easy and fast method to implement as it only requires a single plasmid transformation step and IPTG addition to obtain potent (>95%) downregulation of gene expression. To facilitate studies of various genes in wild-type and genetically modified strains, we provide over 30 CRISPRi plasmids that produce distinct levels of dcas9 expression and carry different antibiotic resistance markers. Our CRISPRi platform represents a useful and efficient complement to traditional genetic and chemical methods to study gene function in B. burgdorferi.

Polyploidy, regular patterning of genome copies, and unusual control of DNA partitioning in the Lyme disease spirochete

“…Semisolid BSK-agarose plating medium 75 contained 2 parts of 1.7% agarose solution in water and 3 parts BSK-1.5 medium containing appropriate amounts of selective antibiotics, as needed, to yield in the final plating mix the concentrations listed above. Each plate was seeded with a maximum of 1 mL B. burgdorferi culture.…”

“…Exponentially growing B. burgdorferi cultures were diluted to 2×10 5 cells/mL in 30 mL BSK-II, grown in duplicate for 2 days, then RNA was extracted and quantified as previously described 75 . The cells were pelleted using a 10 min centrifugation at 4,300 x g in a swinging bucket rotor.…”

“…Exponentially growing B. burgdorferi cultures were diluted to 2x10 5 cells/mL in 30 mL BSK-II, grown in duplicate for 2 days, then RNA was extracted and quantified as previously described 75 .…”

“…Any larger volume vessels were kept loosely capped in the incubator. BSK-1.5 medium for plating was previously described 12,74,75 . Selection antibiotics were used at the following final concentrations in both liquid cultures and plates: kanamycin at 200 μg/mL 76 , gentamicin at 40 μg/mL 77 , streptomycin at 100 μg/mL 78 , hygromycin B at 300 μg/mL 75,79 and blasticidin S at 10 μg/mL 79 .…”

The Consistent Tick-Vertebrate Infectious Cycle of the Lyme Disease Spirochete Enables Borrelia burgdorferi To Control Protein Expression by Monitoring Its Physiological Status

“…However, a stepwise approach, from recombination of an antibiotic resistance-encoding mutation into the chromosome, followed by use of that locus to integrate circular DNA via single crossover, led to the development of numerous selectable markers, cloning vectors, fluorescent tags, and other genetic tools ( 42 – 60 ). CRISPR-interference (CRISPRi) methods were recently developed and have been exploited to knock down expression levels of B. burgdorferi proteins ( 61 ).…”