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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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The fate of Sup35 NM in 40 wild-type R1 clones and 40 ΔclpB R1 clones.(A) SDS-stable Sup35 NM aggregates are detected in 12 of 40 Round 1 (R1) wild-type (WT) clones derived from a starter culture (ST) of wild-type cells containing Sup35 NM and New1 as assessed by filter retention analysis. In total, 17 of 60 R1 wild-type clones are aggregate-positive (Figure 6B). 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 antibody detects the Sup35 NM-mCherry-His6X fusion protein. (B) In contrast, 0 of 40 R1 ΔclpB clones derived from a starter culture of ΔclpB cells transformed with pBR322-SUP35 NM and pSC101TS-NEW1-clpB contain detectable SDS-stable Sup35 NM aggregates. In total, 0 of 60 R1 ΔclpB clones are aggregate-positive (Figure 6C). The observed difference in the number of aggregate-positive clones of wild-type vs ΔclpB cells is statistically significant (p < 0.0001 as determined by Fisher's Exact Test). (C) Fluorescence images of representative cells corresponding to the four aggregate-positive R1 wild-type clones indicated by asterisks in Figure 6B. Notably, wild-type clone R1-14 exhibits twisted ring structures. (D) Fluorescence images of representative cells corresponding to the four aggregate-negative R1 ΔclpB clones indicated by asterisks in Figure 6C.DOI:http://dx.doi.org/10.7554/eLife.02949.014
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fig6s2: The fate of Sup35 NM in 40 wild-type R1 clones and 40 ΔclpB R1 clones.(A) SDS-stable Sup35 NM aggregates are detected in 12 of 40 Round 1 (R1) wild-type (WT) clones derived from a starter culture (ST) of wild-type cells containing Sup35 NM and New1 as assessed by filter retention analysis. In total, 17 of 60 R1 wild-type clones are aggregate-positive (Figure 6B). 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 antibody detects the Sup35 NM-mCherry-His6X fusion protein. (B) In contrast, 0 of 40 R1 ΔclpB clones derived from a starter culture of ΔclpB cells transformed with pBR322-SUP35 NM and pSC101TS-NEW1-clpB contain detectable SDS-stable Sup35 NM aggregates. In total, 0 of 60 R1 ΔclpB clones are aggregate-positive (Figure 6C). The observed difference in the number of aggregate-positive clones of wild-type vs ΔclpB cells is statistically significant (p < 0.0001 as determined by Fisher's Exact Test). (C) Fluorescence images of representative cells corresponding to the four aggregate-positive R1 wild-type clones indicated by asterisks in Figure 6B. Notably, wild-type clone R1-14 exhibits twisted ring structures. (D) Fluorescence images of representative cells corresponding to the four aggregate-negative R1 ΔclpB clones indicated by asterisks in Figure 6C.DOI:http://dx.doi.org/10.7554/eLife.02949.014

Mentions: Fluorescence microscopy revealed that cells containing propagated Sup35 NM aggregates exhibited smaller foci emanating from large aggregates typically localized at cell poles, a phenotype distinguished from experimental starter culture cells by the lack of twisted ring structures (Figure 5C). However, we observed one instance of aggregate-positive R1 cells exhibiting twisted ring structures (see Figure 6—figure supplement 2C). Whereas we cannot definitively assign the SDS-stable Sup35 NM aggregates detected by filter retention to those structures detected by fluorescence microscopy, we note that fluorescence microscopy of prion-containing yeast cells has also revealed structural diversity (Derkatch et al., 2001; Zhou et al., 2001). Furthermore, cells from aggregate-negative samples invariably exhibited diffuse fluorescence (Figure 5D).10.7554/eLife.02949.012Figure 6.Sup35 NM prion propagation in E. coli requires ClpB.


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 40 wild-type R1 clones and 40 ΔclpB R1 clones.(A) SDS-stable Sup35 NM aggregates are detected in 12 of 40 Round 1 (R1) wild-type (WT) clones derived from a starter culture (ST) of wild-type cells containing Sup35 NM and New1 as assessed by filter retention analysis. In total, 17 of 60 R1 wild-type clones are aggregate-positive (Figure 6B). 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 antibody detects the Sup35 NM-mCherry-His6X fusion protein. (B) In contrast, 0 of 40 R1 ΔclpB clones derived from a starter culture of ΔclpB cells transformed with pBR322-SUP35 NM and pSC101TS-NEW1-clpB contain detectable SDS-stable Sup35 NM aggregates. In total, 0 of 60 R1 ΔclpB clones are aggregate-positive (Figure 6C). The observed difference in the number of aggregate-positive clones of wild-type vs ΔclpB cells is statistically significant (p < 0.0001 as determined by Fisher's Exact Test). (C) Fluorescence images of representative cells corresponding to the four aggregate-positive R1 wild-type clones indicated by asterisks in Figure 6B. Notably, wild-type clone R1-14 exhibits twisted ring structures. (D) Fluorescence images of representative cells corresponding to the four aggregate-negative R1 ΔclpB clones indicated by asterisks in Figure 6C.DOI:http://dx.doi.org/10.7554/eLife.02949.014
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fig6s2: The fate of Sup35 NM in 40 wild-type R1 clones and 40 ΔclpB R1 clones.(A) SDS-stable Sup35 NM aggregates are detected in 12 of 40 Round 1 (R1) wild-type (WT) clones derived from a starter culture (ST) of wild-type cells containing Sup35 NM and New1 as assessed by filter retention analysis. In total, 17 of 60 R1 wild-type clones are aggregate-positive (Figure 6B). 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 antibody detects the Sup35 NM-mCherry-His6X fusion protein. (B) In contrast, 0 of 40 R1 ΔclpB clones derived from a starter culture of ΔclpB cells transformed with pBR322-SUP35 NM and pSC101TS-NEW1-clpB contain detectable SDS-stable Sup35 NM aggregates. In total, 0 of 60 R1 ΔclpB clones are aggregate-positive (Figure 6C). The observed difference in the number of aggregate-positive clones of wild-type vs ΔclpB cells is statistically significant (p < 0.0001 as determined by Fisher's Exact Test). (C) Fluorescence images of representative cells corresponding to the four aggregate-positive R1 wild-type clones indicated by asterisks in Figure 6B. Notably, wild-type clone R1-14 exhibits twisted ring structures. (D) Fluorescence images of representative cells corresponding to the four aggregate-negative R1 ΔclpB clones indicated by asterisks in Figure 6C.DOI:http://dx.doi.org/10.7554/eLife.02949.014
Mentions: Fluorescence microscopy revealed that cells containing propagated Sup35 NM aggregates exhibited smaller foci emanating from large aggregates typically localized at cell poles, a phenotype distinguished from experimental starter culture cells by the lack of twisted ring structures (Figure 5C). However, we observed one instance of aggregate-positive R1 cells exhibiting twisted ring structures (see Figure 6—figure supplement 2C). Whereas we cannot definitively assign the SDS-stable Sup35 NM aggregates detected by filter retention to those structures detected by fluorescence microscopy, we note that fluorescence microscopy of prion-containing yeast cells has also revealed structural diversity (Derkatch et al., 2001; Zhou et al., 2001). Furthermore, cells from aggregate-negative samples invariably exhibited diffuse fluorescence (Figure 5D).10.7554/eLife.02949.012Figure 6.Sup35 NM prion propagation in E. coli requires ClpB.

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