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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 control Lineages 2–4.(A) Control Lineage 2 (L2C). An aggregate-negative Round 1 (R1) clone (gray box) derived from a starter culture (ST) of cells containing Sup35 NM alone is identified and restreaked to yield progeny Round 2 (R2) clones (gray bracket). 0 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates as assessed by filter retention analysis. An aggregate-negative R2 clone (blue box) is restreaked to yield progeny R3 clones (blue bracket). 0 of 10 R3 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. An aggregate-negative R3 clone (green box) is restreaked to yield progeny R4 clones (green bracket). 0 of 10 R4 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. 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) Control Lineage 3 (L3C). As in L2C (A), no SDS-stable Sup35 NM aggregates are detectable in progeny R2, R3, or R4 clones. (C) Control Lineage 4 (L4C). As in L2C (A) and L3C (B), no SDS-stable Sup35 NM aggregates are detectable in progeny R2, R3, or R4 clones.DOI:http://dx.doi.org/10.7554/eLife.02949.009
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fig4s2: The fate of Sup35 NM in control Lineages 2–4.(A) Control Lineage 2 (L2C). An aggregate-negative Round 1 (R1) clone (gray box) derived from a starter culture (ST) of cells containing Sup35 NM alone is identified and restreaked to yield progeny Round 2 (R2) clones (gray bracket). 0 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates as assessed by filter retention analysis. An aggregate-negative R2 clone (blue box) is restreaked to yield progeny R3 clones (blue bracket). 0 of 10 R3 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. An aggregate-negative R3 clone (green box) is restreaked to yield progeny R4 clones (green bracket). 0 of 10 R4 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. 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) Control Lineage 3 (L3C). As in L2C (A), no SDS-stable Sup35 NM aggregates are detectable in progeny R2, R3, or R4 clones. (C) Control Lineage 4 (L4C). As in L2C (A) and L3C (B), no SDS-stable Sup35 NM aggregates are detectable in progeny R2, R3, or R4 clones.DOI:http://dx.doi.org/10.7554/eLife.02949.009

Mentions: (A) The fate of Sup35 NM in four experimental lineages (L1E-L4E) established from a starter culture of cells containing Sup35 NM and New1 is shown. Clones that maintain or lose the Sup35 NM prion are indicated by black or pink lines, respectively. Rounds 1–4 (R1–R4) are depicted as gray arcs, with R1 situated at the center of the tree. Clones are designated as aggregate-positive if they contain SDS-stable Sup35 NM aggregates that are detectable in the undiluted sample and at least 1 of the 3 two-fold serial dilutions, as analyzed by filter retention. L1E and L3E retain SDS-stable Sup35 NM aggregates for the duration of the experiment (Figure 4A, Figure 4—figure supplement 1B). L2E and L4E lose detectable SDS-stable Sup35 NM aggregates at R3 and R4, respectively. In both cases, the loss of SDS-stable aggregates coincides with a dramatic yet apparently reversible drop in fusion protein levels (Figure 4—figure supplement 1A,C; see ‘Discussion’). Cells from four aggregate-positive L1E-R4 clones visualized by fluorescence microscopy are indicated by asterisks. (B) The fate of Sup35 NM in four control lineages (L1C–L4C) established from a starter culture of cells containing Sup35 NM alone is shown. None of the 120 clones analyzed contain SDS-stable Sup35 NM aggregates (Figure 4B, Figure 4—figure supplement 2). Cells from four aggregate-negative L1C-R4 clones visualized by fluorescence microscopy are indicated by asterisks. (C) Fluorescence images of representative cells corresponding to the four aggregate-positive R4 clones indicated by asterisks in (A). (D) Fluorescence images of representative cells corresponding to the four aggregate-negative R4 clones indicated by asterisks in (B). (E) E. coli cell extracts containing propagated, SDS-stable Sup35 NM aggregates are infectious when transformed into S. cerevisiae [psi−] cells. A starter culture (ST) of cells containing Sup35 NM and New1 contain infectious SDS-stable Sup35 NM aggregates capable of converting [psi−] yeast cells to [PSI+]. In contrast, a starter culture of cells containing Sup35 NM alone lacks detectable infectivity. Progeny cell extracts transformed into yeast are identified as RX-Y, where X corresponds to a round number and Y corresponds to a clone number assigned sequentially and clockwise according to (A) and (B). Clones that gave rise to aggregate-negative progeny in the subsequent round are indicated by asterisks. Analysis of these data by Fisher's exact test indicates that the differences in the frequency of [PSI+] transformants observed with samples containing SDS-stable Sup35 NM aggregates compared with the sample containing soluble Sup35 NM are statistically significant (p < 0.0001). The percentages given refer to strong [PSI+] transformants; samples containing SDS-stable Sup35 NM aggregates (but not samples containing soluble Sup35 NM) also gave rise to weak [PSI+] transformants (Figure 5—figure supplement 1), but these were not quantified (‘Results’).


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 control Lineages 2–4.(A) Control Lineage 2 (L2C). An aggregate-negative Round 1 (R1) clone (gray box) derived from a starter culture (ST) of cells containing Sup35 NM alone is identified and restreaked to yield progeny Round 2 (R2) clones (gray bracket). 0 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates as assessed by filter retention analysis. An aggregate-negative R2 clone (blue box) is restreaked to yield progeny R3 clones (blue bracket). 0 of 10 R3 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. An aggregate-negative R3 clone (green box) is restreaked to yield progeny R4 clones (green bracket). 0 of 10 R4 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. 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) Control Lineage 3 (L3C). As in L2C (A), no SDS-stable Sup35 NM aggregates are detectable in progeny R2, R3, or R4 clones. (C) Control Lineage 4 (L4C). As in L2C (A) and L3C (B), no SDS-stable Sup35 NM aggregates are detectable in progeny R2, R3, or R4 clones.DOI:http://dx.doi.org/10.7554/eLife.02949.009
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fig4s2: The fate of Sup35 NM in control Lineages 2–4.(A) Control Lineage 2 (L2C). An aggregate-negative Round 1 (R1) clone (gray box) derived from a starter culture (ST) of cells containing Sup35 NM alone is identified and restreaked to yield progeny Round 2 (R2) clones (gray bracket). 0 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates as assessed by filter retention analysis. An aggregate-negative R2 clone (blue box) is restreaked to yield progeny R3 clones (blue bracket). 0 of 10 R3 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. An aggregate-negative R3 clone (green box) is restreaked to yield progeny R4 clones (green bracket). 0 of 10 R4 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. 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) Control Lineage 3 (L3C). As in L2C (A), no SDS-stable Sup35 NM aggregates are detectable in progeny R2, R3, or R4 clones. (C) Control Lineage 4 (L4C). As in L2C (A) and L3C (B), no SDS-stable Sup35 NM aggregates are detectable in progeny R2, R3, or R4 clones.DOI:http://dx.doi.org/10.7554/eLife.02949.009
Mentions: (A) The fate of Sup35 NM in four experimental lineages (L1E-L4E) established from a starter culture of cells containing Sup35 NM and New1 is shown. Clones that maintain or lose the Sup35 NM prion are indicated by black or pink lines, respectively. Rounds 1–4 (R1–R4) are depicted as gray arcs, with R1 situated at the center of the tree. Clones are designated as aggregate-positive if they contain SDS-stable Sup35 NM aggregates that are detectable in the undiluted sample and at least 1 of the 3 two-fold serial dilutions, as analyzed by filter retention. L1E and L3E retain SDS-stable Sup35 NM aggregates for the duration of the experiment (Figure 4A, Figure 4—figure supplement 1B). L2E and L4E lose detectable SDS-stable Sup35 NM aggregates at R3 and R4, respectively. In both cases, the loss of SDS-stable aggregates coincides with a dramatic yet apparently reversible drop in fusion protein levels (Figure 4—figure supplement 1A,C; see ‘Discussion’). Cells from four aggregate-positive L1E-R4 clones visualized by fluorescence microscopy are indicated by asterisks. (B) The fate of Sup35 NM in four control lineages (L1C–L4C) established from a starter culture of cells containing Sup35 NM alone is shown. None of the 120 clones analyzed contain SDS-stable Sup35 NM aggregates (Figure 4B, Figure 4—figure supplement 2). Cells from four aggregate-negative L1C-R4 clones visualized by fluorescence microscopy are indicated by asterisks. (C) Fluorescence images of representative cells corresponding to the four aggregate-positive R4 clones indicated by asterisks in (A). (D) Fluorescence images of representative cells corresponding to the four aggregate-negative R4 clones indicated by asterisks in (B). (E) E. coli cell extracts containing propagated, SDS-stable Sup35 NM aggregates are infectious when transformed into S. cerevisiae [psi−] cells. A starter culture (ST) of cells containing Sup35 NM and New1 contain infectious SDS-stable Sup35 NM aggregates capable of converting [psi−] yeast cells to [PSI+]. In contrast, a starter culture of cells containing Sup35 NM alone lacks detectable infectivity. Progeny cell extracts transformed into yeast are identified as RX-Y, where X corresponds to a round number and Y corresponds to a clone number assigned sequentially and clockwise according to (A) and (B). Clones that gave rise to aggregate-negative progeny in the subsequent round are indicated by asterisks. Analysis of these data by Fisher's exact test indicates that the differences in the frequency of [PSI+] transformants observed with samples containing SDS-stable Sup35 NM aggregates compared with the sample containing soluble Sup35 NM are statistically significant (p < 0.0001). The percentages given refer to strong [PSI+] transformants; samples containing SDS-stable Sup35 NM aggregates (but not samples containing soluble Sup35 NM) also gave rise to weak [PSI+] transformants (Figure 5—figure supplement 1), but these were not quantified (‘Results’).

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