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| Status |
Public on Feb 06, 2023 |
| Title |
probe based bacterial single-cell RNA sequencing predicts toxin regulation |
| Organisms |
Escherichia coli; Bacillus subtilis; Clostridium perfringens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Clonal bacterial populations rely on transcriptional variation across individual cells to commit to specialized states that increase the population’s fitness. Such heterogeneous gene expression is implicated in fundamental microbial processes including sporulation, cell communication, detoxification, substrate utilization, competence, biofilm formation, and motility1. To identify specialized cell states and determine the processes by which they develop, isogenic bacterial populations need to be studied at the single cell level2,3. Here, we developed ProBac-seq a method that uses libraries of DNA probes and leverages an existing commercial microfluidic platform to conduct bacterial single cell RNA sequencing. We sequenced the transcriptome of thousands of individual bacterial cells per experiment, detecting several hundred transcripts per cell on average. When applying this method to the model organisms Bacillus subtilis and Escherichia coli, we correctly identify known cell states and uncover previously unreported transcriptional heterogeneity. In the context of bacterial pathogenesis, single cell RNA-seq of the pathogen Clostridium perfringens reveals that toxin is differentially expressed by a subpopulation of cells with a distinct transcriptional profile. We further show that the size of the toxin producing subpopulation and the secreted toxin levels can be downregulated by providing acetate, a short chain fatty acid highly prevalent in the gut. Overall, we demonstrate that our high throughput, highly resolved single cell transcriptomic platform can be broadly used to uncover heterogeneity in isogenic microbial populations and identify perturbations that can impact pathogenicity.
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| Overall design |
Before microfluidic encapsulation, bacteria were fixed in 1% paraformaldehyde to preserve transcripts and permeabilized by mild lysozyme treatment. Permeabilized bacteria were then incubated with the corresponding DNA probe library. Non-hybridized probes were removed with repeated washes . Next, the bacteria were run through a 10X Chromium Controller, where the DNA probes were captured and barcoded in a manner analogous to the barcoding of the transcriptome of eukaryotic cells
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| Contributor(s) |
McNulty R, Sritharan D, Pahng SH, Meisch JP, Liu S, Brennan MA, Saxer G, Hormoz S, Rosenthal AZ |
| Citation(s) |
37012420 |
| Submission date |
Jan 26, 2023 |
| Last update date |
May 08, 2023 |
| Contact name |
Adam Rosenthal |
| E-mail(s) |
adam.z.rosenthal@gmail.com
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| Phone |
6263939102
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| Organization name |
UNC
|
| Department |
Microbiology and Immunology
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| Lab |
Adam Rosenthal
|
| Street address |
125 Mason Farm Marsico Hall rm6201
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| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599 |
| Country |
USA |
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| Platforms (4)
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| GPL32081 |
NextSeq 2000 (Escherichia coli) |
| GPL32426 |
NextSeq 2000 (Bacillus subtilis) |
| GPL33060 |
NextSeq 2000 (Bacillus subtilis; Escherichia coli) |
| GPL33061 |
NextSeq 2000 (Clostridium perfringens) |
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| Samples (8)
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| Relations |
| BioProject |
PRJNA928315 |
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