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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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All Round 1 clones producing wild-type ClpB are cured of pSC101TS-NEW1-clpB.All Round 1 (R1) clones derived from a starter culture of ΔclpB cells containing pBR322-NM SUP35, pSC101TS-NEW1-clpB, and chromosomal aTc-inducible wild-type clpB lose pSC101TS-NEW1-clpB as assessed by replica plating from solid medium supplemented with carbenicillin (Carb), chloramphenicol (Cam), IPTG, and 50 ng/ml aTc to solid medium containing either Carb or Cam. pSC101TS-NEW1-clpB confers Cam resistance.DOI:http://dx.doi.org/10.7554/eLife.02949.016
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fig7s1: All Round 1 clones producing wild-type ClpB are cured of pSC101TS-NEW1-clpB.All Round 1 (R1) clones derived from a starter culture of ΔclpB cells containing pBR322-NM SUP35, pSC101TS-NEW1-clpB, and chromosomal aTc-inducible wild-type clpB lose pSC101TS-NEW1-clpB as assessed by replica plating from solid medium supplemented with carbenicillin (Carb), chloramphenicol (Cam), IPTG, and 50 ng/ml aTc to solid medium containing either Carb or Cam. pSC101TS-NEW1-clpB confers Cam resistance.DOI:http://dx.doi.org/10.7554/eLife.02949.016

Mentions: As expected, we detected SDS-stable Sup35 NM aggregates in starter culture cells of all strains producing plasmid-encoded Sup35 NM-mCherry-His6X, New1-mGFP, and wild-type ClpB (Figure 7A). To determine whether or not each of the mutants could support the propagation of these aggregates following the depletion of New1 and wild-type ClpB, we plated the starter culture cells at the nonpermissive temperature on solid medium lacking or containing increasing concentrations of aTc, generating sets of R1 colonies. We prepared cell extracts from scraped R1 colonies (‘Materials and methods’) and examined these extracts for the presence or absence of SDS-stable Sup35 NM aggregates. Whereas SDS-stable Sup35 NM aggregates were detected as a function of increasing aTc concentration in cells carrying the wild-type clpB allele, no aggregates were detected in cells harboring the clpB E279A/E678A, clpB Y653A, or clpB E432A allele at any concentration of aTc (Figure 7A). Western blot analysis revealed that levels of chromosomally-encoded wild-type ClpB and each of the three disaggregase mutants were comparable in cell extracts prepared from colonies scraped off of plates containing 50 ng/ml aTc (Figure 7B). Furthermore, replica plating confirmed that all colonies of R1 cells grown on medium supplemented with 50 ng/ml aTc had been cured of pSC101TS-NEW1-clpB (Figure 7—figure supplement 1). We conclude that ATP hydrolysis coupled to substrate translocation through the ClpB central pore and collaboration with DnaK are required for propagation of SDS-stable Sup35 NM aggregates in the absence of New1.10.7554/eLife.02949.015Figure 7.Propagation of SDS-stable Sup35 NM aggregates in E. coli requires ClpB disaggregase activity.


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

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

All Round 1 clones producing wild-type ClpB are cured of pSC101TS-NEW1-clpB.All Round 1 (R1) clones derived from a starter culture of ΔclpB cells containing pBR322-NM SUP35, pSC101TS-NEW1-clpB, and chromosomal aTc-inducible wild-type clpB lose pSC101TS-NEW1-clpB as assessed by replica plating from solid medium supplemented with carbenicillin (Carb), chloramphenicol (Cam), IPTG, and 50 ng/ml aTc to solid medium containing either Carb or Cam. pSC101TS-NEW1-clpB confers Cam resistance.DOI:http://dx.doi.org/10.7554/eLife.02949.016
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7s1: All Round 1 clones producing wild-type ClpB are cured of pSC101TS-NEW1-clpB.All Round 1 (R1) clones derived from a starter culture of ΔclpB cells containing pBR322-NM SUP35, pSC101TS-NEW1-clpB, and chromosomal aTc-inducible wild-type clpB lose pSC101TS-NEW1-clpB as assessed by replica plating from solid medium supplemented with carbenicillin (Carb), chloramphenicol (Cam), IPTG, and 50 ng/ml aTc to solid medium containing either Carb or Cam. pSC101TS-NEW1-clpB confers Cam resistance.DOI:http://dx.doi.org/10.7554/eLife.02949.016
Mentions: As expected, we detected SDS-stable Sup35 NM aggregates in starter culture cells of all strains producing plasmid-encoded Sup35 NM-mCherry-His6X, New1-mGFP, and wild-type ClpB (Figure 7A). To determine whether or not each of the mutants could support the propagation of these aggregates following the depletion of New1 and wild-type ClpB, we plated the starter culture cells at the nonpermissive temperature on solid medium lacking or containing increasing concentrations of aTc, generating sets of R1 colonies. We prepared cell extracts from scraped R1 colonies (‘Materials and methods’) and examined these extracts for the presence or absence of SDS-stable Sup35 NM aggregates. Whereas SDS-stable Sup35 NM aggregates were detected as a function of increasing aTc concentration in cells carrying the wild-type clpB allele, no aggregates were detected in cells harboring the clpB E279A/E678A, clpB Y653A, or clpB E432A allele at any concentration of aTc (Figure 7A). Western blot analysis revealed that levels of chromosomally-encoded wild-type ClpB and each of the three disaggregase mutants were comparable in cell extracts prepared from colonies scraped off of plates containing 50 ng/ml aTc (Figure 7B). Furthermore, replica plating confirmed that all colonies of R1 cells grown on medium supplemented with 50 ng/ml aTc had been cured of pSC101TS-NEW1-clpB (Figure 7—figure supplement 1). We conclude that ATP hydrolysis coupled to substrate translocation through the ClpB central pore and collaboration with DnaK are required for propagation of SDS-stable Sup35 NM aggregates in the absence of New1.10.7554/eLife.02949.015Figure 7.Propagation of SDS-stable Sup35 NM aggregates in E. coli requires ClpB disaggregase activity.

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