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Prion propagation can occur in a prokaryote and requires the ClpB chaperone.

Yuan AH, Garrity SJ, Nako E, Hochschild A - Elife (2014)

Bottom Line: Here, we demonstrate that E. coli can propagate the Sup35 prion under conditions that do not permit its de novo formation.Prion propagation in yeast requires Hsp104 (a ClpB ortholog), and prior studies have come to conflicting conclusions about ClpB's ability to participate in this process.Our demonstration of ClpB-dependent prion propagation in E. coli suggests that the cytoplasmic milieu in general and a molecular machine in particular are poised to support protein-based heredity in the bacterial domain of life.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, United States Whitehead Institute for Biomedical Research, Cambridge, United States.

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Converted Sup35 NM can remain in its prion conformation in E. coli cells lacking New1.(A) Cartoon representation of how Sup35 NM can convert to its prion form in the presence of New1 and remain in the prion conformation after cells have been cured of New1-encoding DNA. Sup35 NM and New1 (black) are depicted as fusions to mCherry (red) and mGFP (green), respectively. (B) SDS-stable Sup35 NM aggregates are detected in 8 of 20 Round 1 (R1) experimental clones derived from a starter culture (ST) of cells containing Sup35 NM and New1 as assessed by filter retention analysis. Starter cultures of cells containing Sup35 NM and New1 and cells containing Sup35 NM alone serve as positive (P) and negative (N) controls, respectively. The four aggregate-positive clones selected to establish the four experimental lineages are indicated by asterisks. In all 20 R1 experimental samples, intracellular Sup35 NM fusion protein levels are comparable, and New1 fusion protein is not detectable as assessed by Western blot analysis. The α-His6X and α-Sup35 antibodies recognize the Sup35 NM-mCherry-His6X fusion protein (see Figure 1—figure supplement 1B,C), the α-GFP antibody detects the New1-mGFP fusion protein, and the α-RpoA antibody recognizes the α subunit of E. coli RNA polymerase. (C) In all 20 R1 experimental samples, DNA encoding Sup35 NM is detectable whereas DNA encoding the prionogenic module of New1 is not detectable by PCR. (D) SDS-stable Sup35 NM aggregates are not detected in any of the 20 R1 control clones derived from a starter culture of cells containing Sup35 NM alone as assessed by filter retention analysis. The four aggregate-negative clones selected to establish the four control lineages are indicated by asterisks. Intracellular Sup35 NM fusion protein levels are comparable in all 20 R1 control samples.DOI:http://dx.doi.org/10.7554/eLife.02949.006
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fig3: Converted Sup35 NM can remain in its prion conformation in E. coli cells lacking New1.(A) Cartoon representation of how Sup35 NM can convert to its prion form in the presence of New1 and remain in the prion conformation after cells have been cured of New1-encoding DNA. Sup35 NM and New1 (black) are depicted as fusions to mCherry (red) and mGFP (green), respectively. (B) SDS-stable Sup35 NM aggregates are detected in 8 of 20 Round 1 (R1) experimental clones derived from a starter culture (ST) of cells containing Sup35 NM and New1 as assessed by filter retention analysis. Starter cultures of cells containing Sup35 NM and New1 and cells containing Sup35 NM alone serve as positive (P) and negative (N) controls, respectively. The four aggregate-positive clones selected to establish the four experimental lineages are indicated by asterisks. In all 20 R1 experimental samples, intracellular Sup35 NM fusion protein levels are comparable, and New1 fusion protein is not detectable as assessed by Western blot analysis. The α-His6X and α-Sup35 antibodies recognize the Sup35 NM-mCherry-His6X fusion protein (see Figure 1—figure supplement 1B,C), the α-GFP antibody detects the New1-mGFP fusion protein, and the α-RpoA antibody recognizes the α subunit of E. coli RNA polymerase. (C) In all 20 R1 experimental samples, DNA encoding Sup35 NM is detectable whereas DNA encoding the prionogenic module of New1 is not detectable by PCR. (D) SDS-stable Sup35 NM aggregates are not detected in any of the 20 R1 control clones derived from a starter culture of cells containing Sup35 NM alone as assessed by filter retention analysis. The four aggregate-negative clones selected to establish the four control lineages are indicated by asterisks. Intracellular Sup35 NM fusion protein levels are comparable in all 20 R1 control samples.DOI:http://dx.doi.org/10.7554/eLife.02949.006

Mentions: (A) Probing nitrocellulose membranes with either α-Sup35 antibody (Figure 1C) or α-His6X antibody results in the detection of similar intracellular Sup35 NM fusion protein products in the presence or absence of New1 as assessed by Western blot analysis. Full-length (FL) Sup35 NM fusion proteins are indicated by arrows. (B) Probing cellulose acetate membranes with either α-His6X antibody (Figure 3B) or α-Sup35 antibody results in the detection of similar aggregate-positive and aggregate-negative samples in starter cultures (ST) and Round 1 (R1) experimental clones as assessed by filter retention analysis. For each sample, undiluted lysate and three twofold dilutions are shown. Starter cultures of cells containing Sup35 NM and New1 and cells containing Sup35 NM alone serve as positive (P) and negative (N) controls, respectively. (C) Probing cellulose acetate membranes with either α-His6X antibody (Figure 3D) or α-Sup35 antibody results in the detection of similar aggregate-positive and aggregate-negative samples in starter cultures (ST) and R1 control clones as assessed by filter retention analysis.


Prion propagation can occur in a prokaryote and requires the ClpB chaperone.

Yuan AH, Garrity SJ, Nako E, Hochschild A - Elife (2014)

Converted Sup35 NM can remain in its prion conformation in E. coli cells lacking New1.(A) Cartoon representation of how Sup35 NM can convert to its prion form in the presence of New1 and remain in the prion conformation after cells have been cured of New1-encoding DNA. Sup35 NM and New1 (black) are depicted as fusions to mCherry (red) and mGFP (green), respectively. (B) SDS-stable Sup35 NM aggregates are detected in 8 of 20 Round 1 (R1) experimental clones derived from a starter culture (ST) of cells containing Sup35 NM and New1 as assessed by filter retention analysis. Starter cultures of cells containing Sup35 NM and New1 and cells containing Sup35 NM alone serve as positive (P) and negative (N) controls, respectively. The four aggregate-positive clones selected to establish the four experimental lineages are indicated by asterisks. In all 20 R1 experimental samples, intracellular Sup35 NM fusion protein levels are comparable, and New1 fusion protein is not detectable as assessed by Western blot analysis. The α-His6X and α-Sup35 antibodies recognize the Sup35 NM-mCherry-His6X fusion protein (see Figure 1—figure supplement 1B,C), the α-GFP antibody detects the New1-mGFP fusion protein, and the α-RpoA antibody recognizes the α subunit of E. coli RNA polymerase. (C) In all 20 R1 experimental samples, DNA encoding Sup35 NM is detectable whereas DNA encoding the prionogenic module of New1 is not detectable by PCR. (D) SDS-stable Sup35 NM aggregates are not detected in any of the 20 R1 control clones derived from a starter culture of cells containing Sup35 NM alone as assessed by filter retention analysis. The four aggregate-negative clones selected to establish the four control lineages are indicated by asterisks. Intracellular Sup35 NM fusion protein levels are comparable in all 20 R1 control samples.DOI:http://dx.doi.org/10.7554/eLife.02949.006
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fig3: Converted Sup35 NM can remain in its prion conformation in E. coli cells lacking New1.(A) Cartoon representation of how Sup35 NM can convert to its prion form in the presence of New1 and remain in the prion conformation after cells have been cured of New1-encoding DNA. Sup35 NM and New1 (black) are depicted as fusions to mCherry (red) and mGFP (green), respectively. (B) SDS-stable Sup35 NM aggregates are detected in 8 of 20 Round 1 (R1) experimental clones derived from a starter culture (ST) of cells containing Sup35 NM and New1 as assessed by filter retention analysis. Starter cultures of cells containing Sup35 NM and New1 and cells containing Sup35 NM alone serve as positive (P) and negative (N) controls, respectively. The four aggregate-positive clones selected to establish the four experimental lineages are indicated by asterisks. In all 20 R1 experimental samples, intracellular Sup35 NM fusion protein levels are comparable, and New1 fusion protein is not detectable as assessed by Western blot analysis. The α-His6X and α-Sup35 antibodies recognize the Sup35 NM-mCherry-His6X fusion protein (see Figure 1—figure supplement 1B,C), the α-GFP antibody detects the New1-mGFP fusion protein, and the α-RpoA antibody recognizes the α subunit of E. coli RNA polymerase. (C) In all 20 R1 experimental samples, DNA encoding Sup35 NM is detectable whereas DNA encoding the prionogenic module of New1 is not detectable by PCR. (D) SDS-stable Sup35 NM aggregates are not detected in any of the 20 R1 control clones derived from a starter culture of cells containing Sup35 NM alone as assessed by filter retention analysis. The four aggregate-negative clones selected to establish the four control lineages are indicated by asterisks. Intracellular Sup35 NM fusion protein levels are comparable in all 20 R1 control samples.DOI:http://dx.doi.org/10.7554/eLife.02949.006
Mentions: (A) Probing nitrocellulose membranes with either α-Sup35 antibody (Figure 1C) or α-His6X antibody results in the detection of similar intracellular Sup35 NM fusion protein products in the presence or absence of New1 as assessed by Western blot analysis. Full-length (FL) Sup35 NM fusion proteins are indicated by arrows. (B) Probing cellulose acetate membranes with either α-His6X antibody (Figure 3B) or α-Sup35 antibody results in the detection of similar aggregate-positive and aggregate-negative samples in starter cultures (ST) and Round 1 (R1) experimental clones as assessed by filter retention analysis. For each sample, undiluted lysate and three twofold dilutions are shown. Starter cultures of cells containing Sup35 NM and New1 and cells containing Sup35 NM alone serve as positive (P) and negative (N) controls, respectively. (C) Probing cellulose acetate membranes with either α-His6X antibody (Figure 3D) or α-Sup35 antibody results in the detection of similar aggregate-positive and aggregate-negative samples in starter cultures (ST) and R1 control clones as assessed by filter retention analysis.

Bottom Line: Here, we demonstrate that E. coli can propagate the Sup35 prion under conditions that do not permit its de novo formation.Prion propagation in yeast requires Hsp104 (a ClpB ortholog), and prior studies have come to conflicting conclusions about ClpB's ability to participate in this process.Our demonstration of ClpB-dependent prion propagation in E. coli suggests that the cytoplasmic milieu in general and a molecular machine in particular are poised to support protein-based heredity in the bacterial domain of life.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, United States Whitehead Institute for Biomedical Research, Cambridge, United States.

Show MeSH
Related in: MedlinePlus