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.
Highlights
The spirochete Borrelia burgdorferi causes Lyme disease, an increasingly prevalent infection
Using a transcriptional fusion to mCherry, we showed that Psyn is active in B. burgdorferi (Fig. S1C and D)
We introduced the sgRNA-expressing shuttle vectors into appropriate B. burgdorferi host strains, generating clustered regularly interspaced palindromic repeats interference (CRISPRi) strains for depletion of FlaB, FtsI, MreB, and RodA (Table 2)
Summary
The spirochete Borrelia burgdorferi causes Lyme disease, an increasingly prevalent infection. To help speed up the discovery process, we adapted a clustered regularly interspaced palindromic repeats interference (CRISPRi) platform for use in B. burgdorferi. 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 2 orders of magnitude. 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 implicate MreB and RodA in the particular mode of cell wall elongation of these bacteria. 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
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