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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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Experimental protocol for assessing the ability of E. coli cells to propagate SDS-stable Sup35 NM aggregates.Experiments are initiated with either a starter culture (ST) of cells containing Sup35 NM and New1 (shown) or a starter culture of cells containing Sup35 NM alone (not shown). For each of the 4 lineages (L1–L4), the total number of generations over which Sup35 NM prion propagation is monitored corresponds to the number of cell divisions that occur in the absence of New1 during 4 rounds (R1–R4) of growth on solid medium and an additional round of growth in liquid medium. Growth in the absence of New1 begins at the time the starter culture cells are plated at 37°C (R1). Single R1 colonies were found to contain ∼950,000 colony forming units (CFUs), and the liquid cultures contain ∼108 CFUs per μl. Thus, prion propagation is monitored over 98.7 or ∼100 generations. We note that the presence of SDS-stable Sup35 NM aggregates in experimental starter culture cells does not represent prion propagation because the presence of New1 (i.e., [PIN+]) enables the continuing de novo conversion of newly synthesized Sup35 NM to the prion form.DOI:http://dx.doi.org/10.7554/eLife.02949.005
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fig2: Experimental protocol for assessing the ability of E. coli cells to propagate SDS-stable Sup35 NM aggregates.Experiments are initiated with either a starter culture (ST) of cells containing Sup35 NM and New1 (shown) or a starter culture of cells containing Sup35 NM alone (not shown). For each of the 4 lineages (L1–L4), the total number of generations over which Sup35 NM prion propagation is monitored corresponds to the number of cell divisions that occur in the absence of New1 during 4 rounds (R1–R4) of growth on solid medium and an additional round of growth in liquid medium. Growth in the absence of New1 begins at the time the starter culture cells are plated at 37°C (R1). Single R1 colonies were found to contain ∼950,000 colony forming units (CFUs), and the liquid cultures contain ∼108 CFUs per μl. Thus, prion propagation is monitored over 98.7 or ∼100 generations. We note that the presence of SDS-stable Sup35 NM aggregates in experimental starter culture cells does not represent prion propagation because the presence of New1 (i.e., [PIN+]) enables the continuing de novo conversion of newly synthesized Sup35 NM to the prion form.DOI:http://dx.doi.org/10.7554/eLife.02949.005

Mentions: We then sought to determine whether or not E. coli cells could propagate SDS-stable Sup35 NM aggregates over multiple generations under conditions that do not permit the de novo formation of aggregates (that is, in the absence of New1). Our experimental protocol is illustrated in Figure 2 (see also Figure 3A). We first induced fusion protein synthesis in cells transformed with pBR322-SUP35 NM and either pSC101TS-NEW1 (experimental sample) or pSC101TS (control sample). These ‘starter cultures’ were grown overnight to allow for the formation of SDS-stable Sup35 NM aggregates in the experimental sample. The cells were then plated and grown at the non-permissive temperature to cure the cells of pSC101TS-NEW1 or pSC101TS, thereby generating a set of Round 1 (R1) colonies. 20 R1 experimental colonies and 20 R1 control colonies were subsequently examined; each was (a) patched onto selective medium to test for loss of pSC101TS-NEW1 or pSC101TS, (b) restreaked to generate Round 2 (R2) colonies, and (c) inoculated into liquid medium for overnight growth to test for the presence of SDS-stable Sup35 NM aggregates. Four separate experimental lineages (L1E–L4E) originating from ancestral R1 experimental colonies containing detectable Sup35 NM aggregates along with four separate control lineages (L1C–L4C) originating from ancestral R1 control colonies were then followed through Round 3 (R3) and Round 4 (R4). For R2 and each subsequent round, 10 experimental colonies and 10 control colonies were analyzed.10.7554/eLife.02949.005Figure 2.Experimental protocol for assessing the ability of E. coli cells to propagate SDS-stable Sup35 NM aggregates.


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

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

Experimental protocol for assessing the ability of E. coli cells to propagate SDS-stable Sup35 NM aggregates.Experiments are initiated with either a starter culture (ST) of cells containing Sup35 NM and New1 (shown) or a starter culture of cells containing Sup35 NM alone (not shown). For each of the 4 lineages (L1–L4), the total number of generations over which Sup35 NM prion propagation is monitored corresponds to the number of cell divisions that occur in the absence of New1 during 4 rounds (R1–R4) of growth on solid medium and an additional round of growth in liquid medium. Growth in the absence of New1 begins at the time the starter culture cells are plated at 37°C (R1). Single R1 colonies were found to contain ∼950,000 colony forming units (CFUs), and the liquid cultures contain ∼108 CFUs per μl. Thus, prion propagation is monitored over 98.7 or ∼100 generations. We note that the presence of SDS-stable Sup35 NM aggregates in experimental starter culture cells does not represent prion propagation because the presence of New1 (i.e., [PIN+]) enables the continuing de novo conversion of newly synthesized Sup35 NM to the prion form.DOI:http://dx.doi.org/10.7554/eLife.02949.005
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Related In: Results  -  Collection

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fig2: Experimental protocol for assessing the ability of E. coli cells to propagate SDS-stable Sup35 NM aggregates.Experiments are initiated with either a starter culture (ST) of cells containing Sup35 NM and New1 (shown) or a starter culture of cells containing Sup35 NM alone (not shown). For each of the 4 lineages (L1–L4), the total number of generations over which Sup35 NM prion propagation is monitored corresponds to the number of cell divisions that occur in the absence of New1 during 4 rounds (R1–R4) of growth on solid medium and an additional round of growth in liquid medium. Growth in the absence of New1 begins at the time the starter culture cells are plated at 37°C (R1). Single R1 colonies were found to contain ∼950,000 colony forming units (CFUs), and the liquid cultures contain ∼108 CFUs per μl. Thus, prion propagation is monitored over 98.7 or ∼100 generations. We note that the presence of SDS-stable Sup35 NM aggregates in experimental starter culture cells does not represent prion propagation because the presence of New1 (i.e., [PIN+]) enables the continuing de novo conversion of newly synthesized Sup35 NM to the prion form.DOI:http://dx.doi.org/10.7554/eLife.02949.005
Mentions: We then sought to determine whether or not E. coli cells could propagate SDS-stable Sup35 NM aggregates over multiple generations under conditions that do not permit the de novo formation of aggregates (that is, in the absence of New1). Our experimental protocol is illustrated in Figure 2 (see also Figure 3A). We first induced fusion protein synthesis in cells transformed with pBR322-SUP35 NM and either pSC101TS-NEW1 (experimental sample) or pSC101TS (control sample). These ‘starter cultures’ were grown overnight to allow for the formation of SDS-stable Sup35 NM aggregates in the experimental sample. The cells were then plated and grown at the non-permissive temperature to cure the cells of pSC101TS-NEW1 or pSC101TS, thereby generating a set of Round 1 (R1) colonies. 20 R1 experimental colonies and 20 R1 control colonies were subsequently examined; each was (a) patched onto selective medium to test for loss of pSC101TS-NEW1 or pSC101TS, (b) restreaked to generate Round 2 (R2) colonies, and (c) inoculated into liquid medium for overnight growth to test for the presence of SDS-stable Sup35 NM aggregates. Four separate experimental lineages (L1E–L4E) originating from ancestral R1 experimental colonies containing detectable Sup35 NM aggregates along with four separate control lineages (L1C–L4C) originating from ancestral R1 control colonies were then followed through Round 3 (R3) and Round 4 (R4). For R2 and each subsequent round, 10 experimental colonies and 10 control colonies were analyzed.10.7554/eLife.02949.005Figure 2.Experimental protocol for assessing the ability of E. coli cells to propagate SDS-stable Sup35 NM aggregates.

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