Limits...
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.

Show MeSH

Related in: MedlinePlus

The fate of Sup35 NM in experimental Lineages 2-4.(A) Experimental Lineage 2 (L2E). An aggregate-positive Round 1 (R1) clone (gray box) derived from a starter culture (ST) of cells containing Sup35 NM and New1 is identified and restreaked to yield progeny Round 2 (R2) clones (gray bracket). 8 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates as assessed by filter retention analysis. An aggregate-positive R2 clone (blue box) is restreaked to yield progeny Round 3 (R3) clones (blue bracket). 0 of 10 R3 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. The apparent loss of aggregates coincides with the loss of detectable Sup35 NM fusion protein as assessed by Western blot analysis. Assaying the 10 Round 4 (R4) progeny clones derived from an aggregate-negative R3 clone (green box) reveals that fusion protein levels can be restored without the recovery of SDS-stable Sup35 NM aggregates (green bracket). 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 α-His6X and α-Sup35 antibodies recognize the Sup35 NM-mCherry-His6X fusion protein, and the α-RpoA antibody recognizes the α subunit of E. coli RNA polymerase. (B) Experimental Lineage 3 (L3E). 9 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates, which are retained in 8 of 10 R3 progeny clones and 7 of 10 R4 progeny clones. (C) Experimental Lineage 4 (L4E). 8 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates, which are retained in 9 of 10 R3 progeny clones. 0 of 10 R4 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. As in R3 of L2E (A), the apparent loss of aggregates coincides with a dramatic drop in Sup35 NM fusion protein levels.DOI:http://dx.doi.org/10.7554/eLife.02949.008
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150125&req=5

fig4s1: The fate of Sup35 NM in experimental Lineages 2-4.(A) Experimental Lineage 2 (L2E). An aggregate-positive Round 1 (R1) clone (gray box) derived from a starter culture (ST) of cells containing Sup35 NM and New1 is identified and restreaked to yield progeny Round 2 (R2) clones (gray bracket). 8 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates as assessed by filter retention analysis. An aggregate-positive R2 clone (blue box) is restreaked to yield progeny Round 3 (R3) clones (blue bracket). 0 of 10 R3 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. The apparent loss of aggregates coincides with the loss of detectable Sup35 NM fusion protein as assessed by Western blot analysis. Assaying the 10 Round 4 (R4) progeny clones derived from an aggregate-negative R3 clone (green box) reveals that fusion protein levels can be restored without the recovery of SDS-stable Sup35 NM aggregates (green bracket). 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 α-His6X and α-Sup35 antibodies recognize the Sup35 NM-mCherry-His6X fusion protein, and the α-RpoA antibody recognizes the α subunit of E. coli RNA polymerase. (B) Experimental Lineage 3 (L3E). 9 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates, which are retained in 8 of 10 R3 progeny clones and 7 of 10 R4 progeny clones. (C) Experimental Lineage 4 (L4E). 8 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates, which are retained in 9 of 10 R3 progeny clones. 0 of 10 R4 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. As in R3 of L2E (A), the apparent loss of aggregates coincides with a dramatic drop in Sup35 NM fusion protein levels.DOI:http://dx.doi.org/10.7554/eLife.02949.008

Mentions: All R1 experimental and control colonies (20 of each) had lost pSC101TS-NEW1 or pSC101TS, respectively, as assessed by patching on selective medium (data not shown). Moreover, the absence of NEW1 DNA was confirmed by PCR (Figure 3C) and the absence of New1 protein was confirmed by Western blot analysis (Figure 3B). We detected SDS-stable Sup35 NM aggregates in 8 of 20 experimental samples (Figure 3B) and none of the control samples (Figure 3D). We selected 4 of the 8 aggregate-positive clones (Figure 3B, asterisks) to establish the four experimental lineages and arbitrarily selected four aggregate-negative control clones (Figure 3D, asterisks) to establish the four control lineages. 2 of the 4 experimental lineages (L1E and L3E) retained SDS-stable Sup35 NM aggregates throughout the course of the experiment (Figure 4A, Figure 4—figure supplement 1B). Of these two lineages, one maintained aggregates in 9 of 10 R4 clones (Figure 4A) and the other maintained aggregates in 7 of 10 R4 clones (Figure 4—figure supplement 1B). We conclude that SDS-stable Sup35 NM aggregates can be propagated in E. coli for at least ∼100 generations in the absence of New1 (Figure 5A).10.7554/eLife.02949.007Figure 4.E. coli can propagate the Sup35 NM prion over ∼100 generations.


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

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

The fate of Sup35 NM in experimental Lineages 2-4.(A) Experimental Lineage 2 (L2E). An aggregate-positive Round 1 (R1) clone (gray box) derived from a starter culture (ST) of cells containing Sup35 NM and New1 is identified and restreaked to yield progeny Round 2 (R2) clones (gray bracket). 8 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates as assessed by filter retention analysis. An aggregate-positive R2 clone (blue box) is restreaked to yield progeny Round 3 (R3) clones (blue bracket). 0 of 10 R3 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. The apparent loss of aggregates coincides with the loss of detectable Sup35 NM fusion protein as assessed by Western blot analysis. Assaying the 10 Round 4 (R4) progeny clones derived from an aggregate-negative R3 clone (green box) reveals that fusion protein levels can be restored without the recovery of SDS-stable Sup35 NM aggregates (green bracket). 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 α-His6X and α-Sup35 antibodies recognize the Sup35 NM-mCherry-His6X fusion protein, and the α-RpoA antibody recognizes the α subunit of E. coli RNA polymerase. (B) Experimental Lineage 3 (L3E). 9 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates, which are retained in 8 of 10 R3 progeny clones and 7 of 10 R4 progeny clones. (C) Experimental Lineage 4 (L4E). 8 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates, which are retained in 9 of 10 R3 progeny clones. 0 of 10 R4 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. As in R3 of L2E (A), the apparent loss of aggregates coincides with a dramatic drop in Sup35 NM fusion protein levels.DOI:http://dx.doi.org/10.7554/eLife.02949.008
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150125&req=5

fig4s1: The fate of Sup35 NM in experimental Lineages 2-4.(A) Experimental Lineage 2 (L2E). An aggregate-positive Round 1 (R1) clone (gray box) derived from a starter culture (ST) of cells containing Sup35 NM and New1 is identified and restreaked to yield progeny Round 2 (R2) clones (gray bracket). 8 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates as assessed by filter retention analysis. An aggregate-positive R2 clone (blue box) is restreaked to yield progeny Round 3 (R3) clones (blue bracket). 0 of 10 R3 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. The apparent loss of aggregates coincides with the loss of detectable Sup35 NM fusion protein as assessed by Western blot analysis. Assaying the 10 Round 4 (R4) progeny clones derived from an aggregate-negative R3 clone (green box) reveals that fusion protein levels can be restored without the recovery of SDS-stable Sup35 NM aggregates (green bracket). 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 α-His6X and α-Sup35 antibodies recognize the Sup35 NM-mCherry-His6X fusion protein, and the α-RpoA antibody recognizes the α subunit of E. coli RNA polymerase. (B) Experimental Lineage 3 (L3E). 9 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates, which are retained in 8 of 10 R3 progeny clones and 7 of 10 R4 progeny clones. (C) Experimental Lineage 4 (L4E). 8 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates, which are retained in 9 of 10 R3 progeny clones. 0 of 10 R4 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. As in R3 of L2E (A), the apparent loss of aggregates coincides with a dramatic drop in Sup35 NM fusion protein levels.DOI:http://dx.doi.org/10.7554/eLife.02949.008
Mentions: All R1 experimental and control colonies (20 of each) had lost pSC101TS-NEW1 or pSC101TS, respectively, as assessed by patching on selective medium (data not shown). Moreover, the absence of NEW1 DNA was confirmed by PCR (Figure 3C) and the absence of New1 protein was confirmed by Western blot analysis (Figure 3B). We detected SDS-stable Sup35 NM aggregates in 8 of 20 experimental samples (Figure 3B) and none of the control samples (Figure 3D). We selected 4 of the 8 aggregate-positive clones (Figure 3B, asterisks) to establish the four experimental lineages and arbitrarily selected four aggregate-negative control clones (Figure 3D, asterisks) to establish the four control lineages. 2 of the 4 experimental lineages (L1E and L3E) retained SDS-stable Sup35 NM aggregates throughout the course of the experiment (Figure 4A, Figure 4—figure supplement 1B). Of these two lineages, one maintained aggregates in 9 of 10 R4 clones (Figure 4A) and the other maintained aggregates in 7 of 10 R4 clones (Figure 4—figure supplement 1B). We conclude that SDS-stable Sup35 NM aggregates can be propagated in E. coli for at least ∼100 generations in the absence of New1 (Figure 5A).10.7554/eLife.02949.007Figure 4.E. coli can propagate the Sup35 NM prion over ∼100 generations.

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