Transcription factor (TF)–promoter pairs have been repurposed from native hosts to provide tools to measure intracellular biochemical production titer and dynamically control gene expression. Most often, native TF–promoter systems require rigorous screening to obtain desirable characteristics optimized for biotechnological applications. High-throughput techniques may provide a rational and less labor-intensive strategy to engineer user-defined TF–promoter pairs using fluorescence-activated cell sorting and deep sequencing methods (sort-seq). Based on the designed promoter library’s distribution characteristics, we elucidate sequence–function interactions between the TF and DNA. In this work, we use the sort-seq method to study the sequence–function relationship of a σ54-dependent, butanol-responsive TF–promoter pair, BmoR-PBMO derived from Thauera butanivorans, at the nucleotide level to improve biosensor characteristics, specifically an improved dynamic range. Activities of promoters from a mutagenized PBMO library were sorted based on gfp expression and subsequently deep sequenced to correlate site-specific sequences with changes in dynamic range. We identified site-specific mutations that increase the sensor output. Double mutant and a single mutant, CA(129,130)TC and G(205)A, in PBMO promoter increased dynamic ranges of 4-fold and 1.65-fold compared with the native system, respectively. In addition, sort-seq identified essential sites required for the proper function of the σ54-dependent promoter biosensor in the context of the host. This work can enable high-throughput screening methods for strain development.
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This content will become publicly available on July 24, 2024
The master regulator for entry into sporulation in Bacillus subtilis becomes a mother cell‐specific transcription factor for forespore engulfment
The Spo0A transcription factor is activated by phosphorylation in starving
- NSF-PAR ID:
- 10464506
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Molecular Microbiology
- Volume:
- 120
- Issue:
- 3
- ISSN:
- 0950-382X
- Page Range / eLocation ID:
- p. 439-461
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Introduction MrpC, a member of the CRP/Fnr transcription factor superfamily, is necessary to induce and control the multicellular developmental program of the bacterium,
Myxococcus xanthus . During development, certain cells in the population first swarm into haystack-shaped aggregates and then differentiate into environmentally resistant spores to form mature fruiting bodies (a specialized biofilm).mrpC transcriptional regulation is controlled by negative autoregulation (NAR).Methods Wild type and mutant
mrpC promoter regions were fused to a fluorescent reporter to examine effects onmrpC expression in the population and in single cellsin situ . Phenotypic consequences of the mutantmrpC promoter were assayed by deep convolution neural network analysis of developmental movies, sporulation efficiency assays, and anti-MrpC immunoblot. In situ analysis of single cell MrpC levels in distinct populations were assayed with an MrpC-mNeonGreen reporter.Results Disruption of MrpC binding sites within the
mrpC promoter region led to increased and broadened distribution ofmrpC expression levels between individual cells in the population. Expression ofmrpC from the mutant promoter led to a striking phenotype in which cells lose synchronized transition from aggregation to sporulation. Instead, some cells abruptly exit aggregation centers and remain locked in a cohesive swarming state we termed developmental swarms, while the remaining cells transition to spores inside residual fruiting bodies.In situ examination of a fluorescent reporter for MrpC levels in developmental subpopulations demonstrated cells locked in the developmental swarms contained MrpC levels that do not reach the levels observed in fruiting bodies.Discussion Increased cell-to-cell variation in
mrpC expression upon disruption of MrpC binding sites within its promoter is consistent with NAR motifs functioning to reducing noise. Noise reduction may be key to synchronized transition of cells in the aggregation state to the sporulation state. We hypothesize a novel subpopulation of cells trapped as developmental swarms arise from intermediate levels of MrpC that are sufficient to promote aggregation but insufficient to trigger sporulation. Failure to transition to higher levels of MrpC necessary to induce sporulation may indicate cells in developmental swarms lack an additional positive feedback signal required to boost MrpC levels. -
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Abstract ELT-2 is the major transcription factor (TF) required for Caenorhabditis elegans intestinal development. ELT-2 expression initiates in embryos to promote development and then persists after hatching through the larval and adult stages. Though the sites of ELT-2 binding are characterized and the transcriptional changes that result from ELT-2 depletion are known, an intestine-specific transcriptome profile spanning developmental time has been missing. We generated this dataset by performing Fluorescence Activated Cell Sorting on intestine cells at distinct developmental stages. We analyzed this dataset in conjunction with previously conducted ELT-2 studies to evaluate the role of ELT-2 in directing the intestinal gene regulatory network through development. We found that only 33% of intestine-enriched genes in the embryo were direct targets of ELT-2 but that number increased to 75% by the L3 stage. This suggests additional TFs promote intestinal transcription especially in the embryo. Furthermore, only half of ELT-2's direct target genes were dependent on ELT-2 for their proper expression levels, and an equal proportion of those responded to elt-2 depletion with over-expression as with under-expression. That is, ELT-2 can either activate or repress direct target genes. Additionally, we observed that ELT-2 repressed its own promoter, implicating new models for its autoregulation. Together, our results illustrate that ELT-2 impacts roughly 20–50% of intestine-specific genes, that ELT-2 both positively and negatively controls its direct targets, and that the current model of the intestinal regulatory network is incomplete as the factors responsible for directing the expression of many intestinal genes remain unknown.
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Abstract During plant infection, fungi secrete effector proteins in coordination with distinct infection stages. Thus, the success of plant infection is determined by precise control of effector gene expression. We analysed the
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Transcription factor (TF)–promoter pairs have been repurposed from native hosts to provide tools to measure intracellular biochemical production titer and dynamically control gene expression. Most often, native TF–promoter systems require rigorous screening to obtain desirable characteristics optimized for biotechnological applications. High-throughput techniques may provide a rational and less labor-intensive strategy to engineer user-defined TF–promoter pairs using fluorescence-activated cell sorting and deep sequencing methods (sort-seq). Based on the designed promoter library’s distribution characteristics, we elucidate sequence–function interactions between the TF and DNA. In this work, we use the sort-seq method to study the sequence–function relationship of a σ 54 -dependent, butanol-responsive TF–promoter pair, BmoR-P BMO derived from Thauera butanivorans , at the nucleotide level to improve biosensor characteristics, specifically an improved dynamic range. Activities of promoters from a mutagenized P BMO library were sorted based on gfp expression and subsequently deep sequenced to correlate site-specific sequences with changes in dynamic range. We identified site-specific mutations that increase the sensor output. Double mutant and a single mutant, CA(129,130)TC and G(205)A, in P BMO promoter increased dynamic ranges of 4-fold and 1.65-fold compared with the native system, respectively. In addition, sort-seq identified essential sites required for the proper function of the σ 54 -dependent promoter biosensor in the context of the host. This work can enable high-throughput screening methods for strain development.more » « less
