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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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ΔclpB cells containing New1 and ectopically produced ClpB support the formation of SDS-stable Sup35 NM aggregates.(A) SDS-stable Sup35 NM aggregates are detected in wild-type (WT) cells producing SDS-stable New1 aggregates as assessed by filter retention analysis. SDS-stable Sup35 NM aggregates are also detected in ΔclpB cells containing SDS-stable New1 aggregates and ectopically produced ClpB. The α-His6X antibody detects the Sup35 NM-mCherry-His6X fusion protein, and the α-GFP antibody detects the New1-mGFP fusion protein. A lane cropped from the same immunoblot is indicated by a hash mark. Intracellular levels of full-length (FL) ClpB, Sup35 NM fusion protein, and New1 fusion protein are comparable in the presence and absence of New1 and ectopically produced ClpB as assessed by Western blot analysis. The α-ClpB antibody recognizes the E. coli ClpB chaperone, the α-Sup35 antibody recognizes the Sup35 NM fusion protein, and the α-RpoA antibody recognizes the α subunit of E. coli RNA polymerase. (B) Fluorescence images of representative wild-type cells containing Sup35 NM and New1 and ΔclpB cells containing Sup35 NM, New1, and ectopically produced ClpB. The mCherry channel, GFP channel, and merged images are shown.DOI:http://dx.doi.org/10.7554/eLife.02949.013
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fig6s1: ΔclpB cells containing New1 and ectopically produced ClpB support the formation of SDS-stable Sup35 NM aggregates.(A) SDS-stable Sup35 NM aggregates are detected in wild-type (WT) cells producing SDS-stable New1 aggregates as assessed by filter retention analysis. SDS-stable Sup35 NM aggregates are also detected in ΔclpB cells containing SDS-stable New1 aggregates and ectopically produced ClpB. The α-His6X antibody detects the Sup35 NM-mCherry-His6X fusion protein, and the α-GFP antibody detects the New1-mGFP fusion protein. A lane cropped from the same immunoblot is indicated by a hash mark. Intracellular levels of full-length (FL) ClpB, Sup35 NM fusion protein, and New1 fusion protein are comparable in the presence and absence of New1 and ectopically produced ClpB as assessed by Western blot analysis. The α-ClpB antibody recognizes the E. coli ClpB chaperone, the α-Sup35 antibody recognizes the Sup35 NM fusion protein, and the α-RpoA antibody recognizes the α subunit of E. coli RNA polymerase. (B) Fluorescence images of representative wild-type cells containing Sup35 NM and New1 and ΔclpB cells containing Sup35 NM, New1, and ectopically produced ClpB. The mCherry channel, GFP channel, and merged images are shown.DOI:http://dx.doi.org/10.7554/eLife.02949.013

Mentions: As expected, we detected SDS-stable Sup35 NM aggregates in ΔclpB starter culture cells transformed with pBR322-SUP35 NM and pSC101TS-NEW1-clpB (Figure 6—figure supplement 1A). Furthermore, fluorescence microscopy revealed that these cells contained visible aggregates that were nearly indistinguishable from those in wild-type cells containing pBR322-SUP35 NM and pSC101TS-NEW1 (Figure 6—figure supplement 1B). After plating ΔclpB starter culture cells containing pBR322-SUP35 NM and pSC101TS-NEW1-clpB at the non-permissive temperature to cure cells of ClpB- and New1-encoding DNA, we examined 60 R1 colonies for the presence of SDS-stable Sup35 NM aggregates. In parallel, we examined 60 R1 colonies derived from wild-type starter culture cells containing pBR322-SUP35 NM and pSC101TS-NEW1. As before, every selected colony was patched onto selective medium to test for the loss of pSC101TS-NEW1-clpB or pSC101TS-NEW1 and inoculated into liquid medium for overnight growth to test for the presence of SDS-stable Sup35 NM aggregates. All selected colonies had lost the appropriate temperature-sensitive vector and the absence of New1 and/or ClpB was confirmed by Western blot analysis (Figure 6B, Figure 6C). Whereas 17 of 60 (28%) wild-type R1 samples tested aggregate-positive, all ΔclpB R1 samples tested aggregate-negative (Figure 6B, Figure 6—figure supplement 2A, Figure 6C, Figure 6—figure supplement 2B). Western blot analysis revealed that the wild-type and ΔclpB R1 cells contained comparable amounts of Sup35 NM fusion protein (Figure 6B, Figure 6C). Furthermore, yeast transformation assays confirmed the presence of infectious material capable of converting [psi−] yeast cells to [PSI+] in ΔclpB starter culture cells transformed with pBR322-SUP35 NM and pSC101TS-NEW1-clpB as well as in an aggregate-positive R1 clone derived from wild-type starter culture cells (Figure 6D). In contrast, a ΔclpB R1 clone derived from ΔclpB starter culture cells containing pBR322-SUP35 NM and pSC101TS-NEW1-clpB as well as an aggregate-negative R1 clone derived from wild-type starter culture cells containing pBR322-SUP35 NM and pSC101TS lacked detectable infectivity (Figure 6D). We conclude that cells lacking ClpB cannot propagate Sup35 NM in its infectious prion conformation.


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

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

ΔclpB cells containing New1 and ectopically produced ClpB support the formation of SDS-stable Sup35 NM aggregates.(A) SDS-stable Sup35 NM aggregates are detected in wild-type (WT) cells producing SDS-stable New1 aggregates as assessed by filter retention analysis. SDS-stable Sup35 NM aggregates are also detected in ΔclpB cells containing SDS-stable New1 aggregates and ectopically produced ClpB. The α-His6X antibody detects the Sup35 NM-mCherry-His6X fusion protein, and the α-GFP antibody detects the New1-mGFP fusion protein. A lane cropped from the same immunoblot is indicated by a hash mark. Intracellular levels of full-length (FL) ClpB, Sup35 NM fusion protein, and New1 fusion protein are comparable in the presence and absence of New1 and ectopically produced ClpB as assessed by Western blot analysis. The α-ClpB antibody recognizes the E. coli ClpB chaperone, the α-Sup35 antibody recognizes the Sup35 NM fusion protein, and the α-RpoA antibody recognizes the α subunit of E. coli RNA polymerase. (B) Fluorescence images of representative wild-type cells containing Sup35 NM and New1 and ΔclpB cells containing Sup35 NM, New1, and ectopically produced ClpB. The mCherry channel, GFP channel, and merged images are shown.DOI:http://dx.doi.org/10.7554/eLife.02949.013
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fig6s1: ΔclpB cells containing New1 and ectopically produced ClpB support the formation of SDS-stable Sup35 NM aggregates.(A) SDS-stable Sup35 NM aggregates are detected in wild-type (WT) cells producing SDS-stable New1 aggregates as assessed by filter retention analysis. SDS-stable Sup35 NM aggregates are also detected in ΔclpB cells containing SDS-stable New1 aggregates and ectopically produced ClpB. The α-His6X antibody detects the Sup35 NM-mCherry-His6X fusion protein, and the α-GFP antibody detects the New1-mGFP fusion protein. A lane cropped from the same immunoblot is indicated by a hash mark. Intracellular levels of full-length (FL) ClpB, Sup35 NM fusion protein, and New1 fusion protein are comparable in the presence and absence of New1 and ectopically produced ClpB as assessed by Western blot analysis. The α-ClpB antibody recognizes the E. coli ClpB chaperone, the α-Sup35 antibody recognizes the Sup35 NM fusion protein, and the α-RpoA antibody recognizes the α subunit of E. coli RNA polymerase. (B) Fluorescence images of representative wild-type cells containing Sup35 NM and New1 and ΔclpB cells containing Sup35 NM, New1, and ectopically produced ClpB. The mCherry channel, GFP channel, and merged images are shown.DOI:http://dx.doi.org/10.7554/eLife.02949.013
Mentions: As expected, we detected SDS-stable Sup35 NM aggregates in ΔclpB starter culture cells transformed with pBR322-SUP35 NM and pSC101TS-NEW1-clpB (Figure 6—figure supplement 1A). Furthermore, fluorescence microscopy revealed that these cells contained visible aggregates that were nearly indistinguishable from those in wild-type cells containing pBR322-SUP35 NM and pSC101TS-NEW1 (Figure 6—figure supplement 1B). After plating ΔclpB starter culture cells containing pBR322-SUP35 NM and pSC101TS-NEW1-clpB at the non-permissive temperature to cure cells of ClpB- and New1-encoding DNA, we examined 60 R1 colonies for the presence of SDS-stable Sup35 NM aggregates. In parallel, we examined 60 R1 colonies derived from wild-type starter culture cells containing pBR322-SUP35 NM and pSC101TS-NEW1. As before, every selected colony was patched onto selective medium to test for the loss of pSC101TS-NEW1-clpB or pSC101TS-NEW1 and inoculated into liquid medium for overnight growth to test for the presence of SDS-stable Sup35 NM aggregates. All selected colonies had lost the appropriate temperature-sensitive vector and the absence of New1 and/or ClpB was confirmed by Western blot analysis (Figure 6B, Figure 6C). Whereas 17 of 60 (28%) wild-type R1 samples tested aggregate-positive, all ΔclpB R1 samples tested aggregate-negative (Figure 6B, Figure 6—figure supplement 2A, Figure 6C, Figure 6—figure supplement 2B). Western blot analysis revealed that the wild-type and ΔclpB R1 cells contained comparable amounts of Sup35 NM fusion protein (Figure 6B, Figure 6C). Furthermore, yeast transformation assays confirmed the presence of infectious material capable of converting [psi−] yeast cells to [PSI+] in ΔclpB starter culture cells transformed with pBR322-SUP35 NM and pSC101TS-NEW1-clpB as well as in an aggregate-positive R1 clone derived from wild-type starter culture cells (Figure 6D). In contrast, a ΔclpB R1 clone derived from ΔclpB starter culture cells containing pBR322-SUP35 NM and pSC101TS-NEW1-clpB as well as an aggregate-negative R1 clone derived from wild-type starter culture cells containing pBR322-SUP35 NM and pSC101TS lacked detectable infectivity (Figure 6D). We conclude that cells lacking ClpB cannot propagate Sup35 NM in its infectious prion conformation.

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