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Tunable protein degradation in bacteria.

Cameron DE, Collins JJ - Nat. Biotechnol. (2014)

Bottom Line: Here we use components of the Mesoplasma florum transfer-messenger RNA system to create a synthetic degradation system that provides both independent control of steady-state protein level and inducible degradation of targeted proteins in Escherichia coli.We demonstrate application of this system in synthetic circuit development and control of core bacterial processes and antibacterial targets, and we transfer the system to Lactococcus lactis to establish its broad functionality in bacteria.We create a 238-member library of tagged essential proteins in E. coli that can serve as both a research tool to study essential gene function and an applied system for antibiotic discovery.

View Article: PubMed Central - PubMed

Affiliation: 1] Howard Hughes Medical Institute, Boston University, Boston, Massachusetts, USA. [2] Center of Synthetic Biology, Boston University, Boston, Massachusetts, USA. [3] Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA.

ABSTRACT
Tunable control of protein degradation in bacteria would provide a powerful research tool. Here we use components of the Mesoplasma florum transfer-messenger RNA system to create a synthetic degradation system that provides both independent control of steady-state protein level and inducible degradation of targeted proteins in Escherichia coli. We demonstrate application of this system in synthetic circuit development and control of core bacterial processes and antibacterial targets, and we transfer the system to Lactococcus lactis to establish its broad functionality in bacteria. We create a 238-member library of tagged essential proteins in E. coli that can serve as both a research tool to study essential gene function and an applied system for antibiotic discovery. Our synthetic protein degradation system is modular, does not require disruption of host systems and can be transferred to diverse bacteria with minimal modification.

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Tunable control of endogenous bacterial systems and antibacterial targets(a) Schematic of our recombineering method for genomic insertion of pdt variants, adapted from Datsenko and Wanner30. Red recombinase-assisted insertion of the desired pdt variant is followed by Flp recombinase-mediated excision of the accompanying kanR cassette. The resulting insertion contains the C-terminal pdt variant fusion and a 106 bp scar including the remaining FRT site. (b) Growth of strains following protease-driven depletion of MurA. Protease induction during early exponential phase growth (arrow) causes cells containing murA-pdt#1 to lyse within 1 hour, as measured by optical density (OD600). Cells containing the weakened pdt letter variants show a delayed response. Error bars show the standard deviation from the mean of six biological replicates. See Supplementary Figure 7 for data showing wild-type growth of non-induced cells. (c) DIC-fluorescence overlay images of cells after ATc induction for 3 hours. Cells containing ftsZ-pdt#5 form filaments while untagged wild-type bacteria maintain normal length. The fluorescence micrograph overlay showing constitutive GFP expression serves as a visual aid. Scale bars are 10 μm. (d) Disk diffusion assay on a chemotactic motility plate shows loss of chemotactic motility due to pdt-dependent CheZ degradation. Cells were stabbed into the chemotaxis plate following addition of 250 ng ATc to the center disk. Scale bar is 6 mm. (e) Cells containing murA-pdt#1D show increased sensitivity to fosfomycin upon simultaneous induction with 4 ng/ml ATc (induced). OD600 measurements were taken 4 hours after ATc and fosfomycin treatment and are presented as a percent of the OD600 of cells not exposed to fosfomycin (untreated). In the murA-pdt#1D strain, P < 0.001 when comparing uninduced and induced cells for fosfomycin concentrations between 0.05 and 2 μg/ml. See Supplementary Figure 8 for additional data. (f) Pdt-dependent degradation of RecA causes hypersensitivity to the quinolone norfloxacin that matches the known hypersensitivity of a recA deletion strain (ΔrecA). Where indicated, cells were induced with 50 ng/ml ATc for 2 hours before treatment with norfloxacin (25 ng/ml) for 2 hours. Survival was measured by colony forming units (CFU) and is presented as a percent of CFUs measured immediately before norfloxacin treatment. P < 0.001 for ATc induction of the recA-pdt#3 strain. (g) Scatter plot displaying the relative growth and CFU count of EPD library members after targeted mf-Lon degradation. Growth and CFU measurements are displayed as a ratio of induced/uninduced cells at 4 hours after ATc induction (50 ng/ml). CFU data points were placed at 1.0 × 10−6, the limit of detection, when colonies were not recovered from the induced well. Error bars show the standard deviation from the mean of three biological replicates. CFU ratios represent a single experiment. See Supplementary Table 2 for details. (h) Histogram of propidium iodide (PI) staining for EPD member plsB-pdt#1. PI was measured by flow cytometry 2 hours after induction with 50 ng/ml ATc. The percentage of cells that are PI+ is displayed. The data are normalized to mode and are representative of three biological replicates.
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Figure 4: Tunable control of endogenous bacterial systems and antibacterial targets(a) Schematic of our recombineering method for genomic insertion of pdt variants, adapted from Datsenko and Wanner30. Red recombinase-assisted insertion of the desired pdt variant is followed by Flp recombinase-mediated excision of the accompanying kanR cassette. The resulting insertion contains the C-terminal pdt variant fusion and a 106 bp scar including the remaining FRT site. (b) Growth of strains following protease-driven depletion of MurA. Protease induction during early exponential phase growth (arrow) causes cells containing murA-pdt#1 to lyse within 1 hour, as measured by optical density (OD600). Cells containing the weakened pdt letter variants show a delayed response. Error bars show the standard deviation from the mean of six biological replicates. See Supplementary Figure 7 for data showing wild-type growth of non-induced cells. (c) DIC-fluorescence overlay images of cells after ATc induction for 3 hours. Cells containing ftsZ-pdt#5 form filaments while untagged wild-type bacteria maintain normal length. The fluorescence micrograph overlay showing constitutive GFP expression serves as a visual aid. Scale bars are 10 μm. (d) Disk diffusion assay on a chemotactic motility plate shows loss of chemotactic motility due to pdt-dependent CheZ degradation. Cells were stabbed into the chemotaxis plate following addition of 250 ng ATc to the center disk. Scale bar is 6 mm. (e) Cells containing murA-pdt#1D show increased sensitivity to fosfomycin upon simultaneous induction with 4 ng/ml ATc (induced). OD600 measurements were taken 4 hours after ATc and fosfomycin treatment and are presented as a percent of the OD600 of cells not exposed to fosfomycin (untreated). In the murA-pdt#1D strain, P < 0.001 when comparing uninduced and induced cells for fosfomycin concentrations between 0.05 and 2 μg/ml. See Supplementary Figure 8 for additional data. (f) Pdt-dependent degradation of RecA causes hypersensitivity to the quinolone norfloxacin that matches the known hypersensitivity of a recA deletion strain (ΔrecA). Where indicated, cells were induced with 50 ng/ml ATc for 2 hours before treatment with norfloxacin (25 ng/ml) for 2 hours. Survival was measured by colony forming units (CFU) and is presented as a percent of CFUs measured immediately before norfloxacin treatment. P < 0.001 for ATc induction of the recA-pdt#3 strain. (g) Scatter plot displaying the relative growth and CFU count of EPD library members after targeted mf-Lon degradation. Growth and CFU measurements are displayed as a ratio of induced/uninduced cells at 4 hours after ATc induction (50 ng/ml). CFU data points were placed at 1.0 × 10−6, the limit of detection, when colonies were not recovered from the induced well. Error bars show the standard deviation from the mean of three biological replicates. CFU ratios represent a single experiment. See Supplementary Table 2 for details. (h) Histogram of propidium iodide (PI) staining for EPD member plsB-pdt#1. PI was measured by flow cytometry 2 hours after induction with 50 ng/ml ATc. The percentage of cells that are PI+ is displayed. The data are normalized to mode and are representative of three biological replicates.

Mentions: A major goal in microbial biotechnology is to develop tools to control and manipulate endogenous bacterial systems, so next we sought to target native E. coli pathways for control by our system. We developed a modified recombineering method to insert pdt tags into the E. coli genome (Fig. 4a) and began by targeting MurA, an essential enzyme involved in peptidoglycan biosynthesis14 whose depletion causes cell lysis measurable by a drop in optical density. The murA-pdt#1 genomic fusion caused observable cell lysis 45 minutes after mf-Lon induction (Fig. 4b), and the delayed phenotypic response of the hybrid variants pdt#1A and pdt#1B correlates well with the temporal delay seen for letter variants in the toggle switch and GFP degradation assays (see Fig. 2d and Fig. 3c). Importantly, cells containing murA-pdt fusions display the same growth rate as wild-type cells in the absence of mf-Lon induction, demonstrating that the pdt variants do not interfere with MurA function or regulation (Supplementary Fig. 7a). Sequence analysis of cells with murA-pdt#1 that escape ATc-induced cell lysis showed mutations in mf-lon or its promoter that block its expression.


Tunable protein degradation in bacteria.

Cameron DE, Collins JJ - Nat. Biotechnol. (2014)

Tunable control of endogenous bacterial systems and antibacterial targets(a) Schematic of our recombineering method for genomic insertion of pdt variants, adapted from Datsenko and Wanner30. Red recombinase-assisted insertion of the desired pdt variant is followed by Flp recombinase-mediated excision of the accompanying kanR cassette. The resulting insertion contains the C-terminal pdt variant fusion and a 106 bp scar including the remaining FRT site. (b) Growth of strains following protease-driven depletion of MurA. Protease induction during early exponential phase growth (arrow) causes cells containing murA-pdt#1 to lyse within 1 hour, as measured by optical density (OD600). Cells containing the weakened pdt letter variants show a delayed response. Error bars show the standard deviation from the mean of six biological replicates. See Supplementary Figure 7 for data showing wild-type growth of non-induced cells. (c) DIC-fluorescence overlay images of cells after ATc induction for 3 hours. Cells containing ftsZ-pdt#5 form filaments while untagged wild-type bacteria maintain normal length. The fluorescence micrograph overlay showing constitutive GFP expression serves as a visual aid. Scale bars are 10 μm. (d) Disk diffusion assay on a chemotactic motility plate shows loss of chemotactic motility due to pdt-dependent CheZ degradation. Cells were stabbed into the chemotaxis plate following addition of 250 ng ATc to the center disk. Scale bar is 6 mm. (e) Cells containing murA-pdt#1D show increased sensitivity to fosfomycin upon simultaneous induction with 4 ng/ml ATc (induced). OD600 measurements were taken 4 hours after ATc and fosfomycin treatment and are presented as a percent of the OD600 of cells not exposed to fosfomycin (untreated). In the murA-pdt#1D strain, P < 0.001 when comparing uninduced and induced cells for fosfomycin concentrations between 0.05 and 2 μg/ml. See Supplementary Figure 8 for additional data. (f) Pdt-dependent degradation of RecA causes hypersensitivity to the quinolone norfloxacin that matches the known hypersensitivity of a recA deletion strain (ΔrecA). Where indicated, cells were induced with 50 ng/ml ATc for 2 hours before treatment with norfloxacin (25 ng/ml) for 2 hours. Survival was measured by colony forming units (CFU) and is presented as a percent of CFUs measured immediately before norfloxacin treatment. P < 0.001 for ATc induction of the recA-pdt#3 strain. (g) Scatter plot displaying the relative growth and CFU count of EPD library members after targeted mf-Lon degradation. Growth and CFU measurements are displayed as a ratio of induced/uninduced cells at 4 hours after ATc induction (50 ng/ml). CFU data points were placed at 1.0 × 10−6, the limit of detection, when colonies were not recovered from the induced well. Error bars show the standard deviation from the mean of three biological replicates. CFU ratios represent a single experiment. See Supplementary Table 2 for details. (h) Histogram of propidium iodide (PI) staining for EPD member plsB-pdt#1. PI was measured by flow cytometry 2 hours after induction with 50 ng/ml ATc. The percentage of cells that are PI+ is displayed. The data are normalized to mode and are representative of three biological replicates.
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Related In: Results  -  Collection

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Figure 4: Tunable control of endogenous bacterial systems and antibacterial targets(a) Schematic of our recombineering method for genomic insertion of pdt variants, adapted from Datsenko and Wanner30. Red recombinase-assisted insertion of the desired pdt variant is followed by Flp recombinase-mediated excision of the accompanying kanR cassette. The resulting insertion contains the C-terminal pdt variant fusion and a 106 bp scar including the remaining FRT site. (b) Growth of strains following protease-driven depletion of MurA. Protease induction during early exponential phase growth (arrow) causes cells containing murA-pdt#1 to lyse within 1 hour, as measured by optical density (OD600). Cells containing the weakened pdt letter variants show a delayed response. Error bars show the standard deviation from the mean of six biological replicates. See Supplementary Figure 7 for data showing wild-type growth of non-induced cells. (c) DIC-fluorescence overlay images of cells after ATc induction for 3 hours. Cells containing ftsZ-pdt#5 form filaments while untagged wild-type bacteria maintain normal length. The fluorescence micrograph overlay showing constitutive GFP expression serves as a visual aid. Scale bars are 10 μm. (d) Disk diffusion assay on a chemotactic motility plate shows loss of chemotactic motility due to pdt-dependent CheZ degradation. Cells were stabbed into the chemotaxis plate following addition of 250 ng ATc to the center disk. Scale bar is 6 mm. (e) Cells containing murA-pdt#1D show increased sensitivity to fosfomycin upon simultaneous induction with 4 ng/ml ATc (induced). OD600 measurements were taken 4 hours after ATc and fosfomycin treatment and are presented as a percent of the OD600 of cells not exposed to fosfomycin (untreated). In the murA-pdt#1D strain, P < 0.001 when comparing uninduced and induced cells for fosfomycin concentrations between 0.05 and 2 μg/ml. See Supplementary Figure 8 for additional data. (f) Pdt-dependent degradation of RecA causes hypersensitivity to the quinolone norfloxacin that matches the known hypersensitivity of a recA deletion strain (ΔrecA). Where indicated, cells were induced with 50 ng/ml ATc for 2 hours before treatment with norfloxacin (25 ng/ml) for 2 hours. Survival was measured by colony forming units (CFU) and is presented as a percent of CFUs measured immediately before norfloxacin treatment. P < 0.001 for ATc induction of the recA-pdt#3 strain. (g) Scatter plot displaying the relative growth and CFU count of EPD library members after targeted mf-Lon degradation. Growth and CFU measurements are displayed as a ratio of induced/uninduced cells at 4 hours after ATc induction (50 ng/ml). CFU data points were placed at 1.0 × 10−6, the limit of detection, when colonies were not recovered from the induced well. Error bars show the standard deviation from the mean of three biological replicates. CFU ratios represent a single experiment. See Supplementary Table 2 for details. (h) Histogram of propidium iodide (PI) staining for EPD member plsB-pdt#1. PI was measured by flow cytometry 2 hours after induction with 50 ng/ml ATc. The percentage of cells that are PI+ is displayed. The data are normalized to mode and are representative of three biological replicates.
Mentions: A major goal in microbial biotechnology is to develop tools to control and manipulate endogenous bacterial systems, so next we sought to target native E. coli pathways for control by our system. We developed a modified recombineering method to insert pdt tags into the E. coli genome (Fig. 4a) and began by targeting MurA, an essential enzyme involved in peptidoglycan biosynthesis14 whose depletion causes cell lysis measurable by a drop in optical density. The murA-pdt#1 genomic fusion caused observable cell lysis 45 minutes after mf-Lon induction (Fig. 4b), and the delayed phenotypic response of the hybrid variants pdt#1A and pdt#1B correlates well with the temporal delay seen for letter variants in the toggle switch and GFP degradation assays (see Fig. 2d and Fig. 3c). Importantly, cells containing murA-pdt fusions display the same growth rate as wild-type cells in the absence of mf-Lon induction, demonstrating that the pdt variants do not interfere with MurA function or regulation (Supplementary Fig. 7a). Sequence analysis of cells with murA-pdt#1 that escape ATc-induced cell lysis showed mutations in mf-lon or its promoter that block its expression.

Bottom Line: Here we use components of the Mesoplasma florum transfer-messenger RNA system to create a synthetic degradation system that provides both independent control of steady-state protein level and inducible degradation of targeted proteins in Escherichia coli.We demonstrate application of this system in synthetic circuit development and control of core bacterial processes and antibacterial targets, and we transfer the system to Lactococcus lactis to establish its broad functionality in bacteria.We create a 238-member library of tagged essential proteins in E. coli that can serve as both a research tool to study essential gene function and an applied system for antibiotic discovery.

View Article: PubMed Central - PubMed

Affiliation: 1] Howard Hughes Medical Institute, Boston University, Boston, Massachusetts, USA. [2] Center of Synthetic Biology, Boston University, Boston, Massachusetts, USA. [3] Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA.

ABSTRACT
Tunable control of protein degradation in bacteria would provide a powerful research tool. Here we use components of the Mesoplasma florum transfer-messenger RNA system to create a synthetic degradation system that provides both independent control of steady-state protein level and inducible degradation of targeted proteins in Escherichia coli. We demonstrate application of this system in synthetic circuit development and control of core bacterial processes and antibacterial targets, and we transfer the system to Lactococcus lactis to establish its broad functionality in bacteria. We create a 238-member library of tagged essential proteins in E. coli that can serve as both a research tool to study essential gene function and an applied system for antibiotic discovery. Our synthetic protein degradation system is modular, does not require disruption of host systems and can be transferred to diverse bacteria with minimal modification.

Show MeSH
Related in: MedlinePlus