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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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Protein degradation tag characterization(a) Schematic of the tunable protein degradation system where anhydrotetracycline (ATc) induced mf-Lon expression allows the protease to degrade constitutively expressed GFP in a pdt-dependent manner. Mutations in two pdt regions produced tag variants with altered recognition by mf-Lon (letters) or endogenous E. coli proteases (numbers). (b) Dot plot of pdt number variants that show altered steady-state levels. Fluorescence was measured by flow cytometry 6 hours after ATc induction of cells in exponential growth. As an experimental control, the pdt#3 variant was tested in a strain that did not contain the mf-Lon expression cassette (#3 con). Fluorescence units are arbitrary, with untagged GFP set to 100, and show the mean of six biological replicates. P < 0.001 for ATc induction of each pdt variant except #3 (con). (c–d) Flow cytometry measurements of GFP degradation following mf-Lon induction with 50 ng/ml ATc. Data show the geometric mean fluorescence of at least 5,000 cells as a percentage of the non-induced control for each pdt variant. (c) Pdt number variants maintain similar mf-Lon-mediated degradation dynamics. (d) Pdt letter variants display altered mf-Lon-mediated degradation rates. (e) Dot plot of hybrid pdt variants. Strains expressing the indicated GFP-pdt fusion were measured by flow cytometry 6 hours after ATc induction. Fluorescence units are arbitrary with untagged GFP set to 100, and show the mean of six biological replicates. P < 0.001 for ATc induction of each pdt variant. The data in all panels show the mean of at least three biological replicates, and the error bars show the standard deviation.
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Figure 1: Protein degradation tag characterization(a) Schematic of the tunable protein degradation system where anhydrotetracycline (ATc) induced mf-Lon expression allows the protease to degrade constitutively expressed GFP in a pdt-dependent manner. Mutations in two pdt regions produced tag variants with altered recognition by mf-Lon (letters) or endogenous E. coli proteases (numbers). (b) Dot plot of pdt number variants that show altered steady-state levels. Fluorescence was measured by flow cytometry 6 hours after ATc induction of cells in exponential growth. As an experimental control, the pdt#3 variant was tested in a strain that did not contain the mf-Lon expression cassette (#3 con). Fluorescence units are arbitrary, with untagged GFP set to 100, and show the mean of six biological replicates. P < 0.001 for ATc induction of each pdt variant except #3 (con). (c–d) Flow cytometry measurements of GFP degradation following mf-Lon induction with 50 ng/ml ATc. Data show the geometric mean fluorescence of at least 5,000 cells as a percentage of the non-induced control for each pdt variant. (c) Pdt number variants maintain similar mf-Lon-mediated degradation dynamics. (d) Pdt letter variants display altered mf-Lon-mediated degradation rates. (e) Dot plot of hybrid pdt variants. Strains expressing the indicated GFP-pdt fusion were measured by flow cytometry 6 hours after ATc induction. Fluorescence units are arbitrary with untagged GFP set to 100, and show the mean of six biological replicates. P < 0.001 for ATc induction of each pdt variant. The data in all panels show the mean of at least three biological replicates, and the error bars show the standard deviation.

Mentions: We renamed the mf-ssrA tag “pdt” (protein degradation tag) to minimize confusion with the E. coli ssrA tag, and incorporated it into a GFP-based test platform for inducible protein degradation in E. coli (Fig. 1a). To first engineer pdt variants that modify steady-state GFP levels in the absence of mf-Lon expression, we chose to target pdt residues 24–27 for mutagenesis due to the region’s partial homology with the ec-ssrA ClpA binding site5 and altered GFP-pdt stability in clpA, clpX, and clpP deletion strains (Supplementary Figs. 1 and 2). As seen in Figure 1b, we identified several pdt variants, denoted with numbers, that alter GFP steady-state levels and maintained near wild-type GFP degradation rates following mf-Lon expression. Importantly, untagged GFP remained largely unaffected by mf-Lon expression while the wild-type GFP-pdt fusion was reduced to 3% of its initial levels, confirming the specificity of pdt-mediated mf-Lon degradation seen by Gur and Sauer for LacZ degradation9. Sequence analysis of the identified pdt number variants showed that a majority contained multiple arginine and glutamine residues in the mutagenized region and none of them contained negatively charged residues known to disrupt mf-Lon recognition9 (Supplementary Table 1).


Tunable protein degradation in bacteria.

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

Protein degradation tag characterization(a) Schematic of the tunable protein degradation system where anhydrotetracycline (ATc) induced mf-Lon expression allows the protease to degrade constitutively expressed GFP in a pdt-dependent manner. Mutations in two pdt regions produced tag variants with altered recognition by mf-Lon (letters) or endogenous E. coli proteases (numbers). (b) Dot plot of pdt number variants that show altered steady-state levels. Fluorescence was measured by flow cytometry 6 hours after ATc induction of cells in exponential growth. As an experimental control, the pdt#3 variant was tested in a strain that did not contain the mf-Lon expression cassette (#3 con). Fluorescence units are arbitrary, with untagged GFP set to 100, and show the mean of six biological replicates. P < 0.001 for ATc induction of each pdt variant except #3 (con). (c–d) Flow cytometry measurements of GFP degradation following mf-Lon induction with 50 ng/ml ATc. Data show the geometric mean fluorescence of at least 5,000 cells as a percentage of the non-induced control for each pdt variant. (c) Pdt number variants maintain similar mf-Lon-mediated degradation dynamics. (d) Pdt letter variants display altered mf-Lon-mediated degradation rates. (e) Dot plot of hybrid pdt variants. Strains expressing the indicated GFP-pdt fusion were measured by flow cytometry 6 hours after ATc induction. Fluorescence units are arbitrary with untagged GFP set to 100, and show the mean of six biological replicates. P < 0.001 for ATc induction of each pdt variant. The data in all panels show the mean of at least three biological replicates, and the error bars show the standard deviation.
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Related In: Results  -  Collection

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Figure 1: Protein degradation tag characterization(a) Schematic of the tunable protein degradation system where anhydrotetracycline (ATc) induced mf-Lon expression allows the protease to degrade constitutively expressed GFP in a pdt-dependent manner. Mutations in two pdt regions produced tag variants with altered recognition by mf-Lon (letters) or endogenous E. coli proteases (numbers). (b) Dot plot of pdt number variants that show altered steady-state levels. Fluorescence was measured by flow cytometry 6 hours after ATc induction of cells in exponential growth. As an experimental control, the pdt#3 variant was tested in a strain that did not contain the mf-Lon expression cassette (#3 con). Fluorescence units are arbitrary, with untagged GFP set to 100, and show the mean of six biological replicates. P < 0.001 for ATc induction of each pdt variant except #3 (con). (c–d) Flow cytometry measurements of GFP degradation following mf-Lon induction with 50 ng/ml ATc. Data show the geometric mean fluorescence of at least 5,000 cells as a percentage of the non-induced control for each pdt variant. (c) Pdt number variants maintain similar mf-Lon-mediated degradation dynamics. (d) Pdt letter variants display altered mf-Lon-mediated degradation rates. (e) Dot plot of hybrid pdt variants. Strains expressing the indicated GFP-pdt fusion were measured by flow cytometry 6 hours after ATc induction. Fluorescence units are arbitrary with untagged GFP set to 100, and show the mean of six biological replicates. P < 0.001 for ATc induction of each pdt variant. The data in all panels show the mean of at least three biological replicates, and the error bars show the standard deviation.
Mentions: We renamed the mf-ssrA tag “pdt” (protein degradation tag) to minimize confusion with the E. coli ssrA tag, and incorporated it into a GFP-based test platform for inducible protein degradation in E. coli (Fig. 1a). To first engineer pdt variants that modify steady-state GFP levels in the absence of mf-Lon expression, we chose to target pdt residues 24–27 for mutagenesis due to the region’s partial homology with the ec-ssrA ClpA binding site5 and altered GFP-pdt stability in clpA, clpX, and clpP deletion strains (Supplementary Figs. 1 and 2). As seen in Figure 1b, we identified several pdt variants, denoted with numbers, that alter GFP steady-state levels and maintained near wild-type GFP degradation rates following mf-Lon expression. Importantly, untagged GFP remained largely unaffected by mf-Lon expression while the wild-type GFP-pdt fusion was reduced to 3% of its initial levels, confirming the specificity of pdt-mediated mf-Lon degradation seen by Gur and Sauer for LacZ degradation9. Sequence analysis of the identified pdt number variants showed that a majority contained multiple arginine and glutamine residues in the mutagenized region and none of them contained negatively charged residues known to disrupt mf-Lon recognition9 (Supplementary Table 1).

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