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A bacterial global regulator forms a prion

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Science  13 Jan 2017:
Vol. 355, Issue 6321, pp. 198-201
DOI: 10.1126/science.aai7776

Prions enter another domain of life

Prions are self-propagating protein aggregates, discovered in connection with the fatal transmissible spongiform encephalopathies in mammals. Prions have also been identified in fungi, where they act as protein-based elements of inheritance. Although prions have been uncovered in evolutionarily diverse eukaryotic species, it is not known whether prions exist in bacteria. Yuan and Hochschild report the identification of a bacterial protein—the transcription termination factor Rho from Clostridium botulinum—that exhibits the defining hallmarks of a prion-forming protein.

Science, this issue p. 198

Abstract

Prions are self-propagating protein aggregates that act as protein-based elements of inheritance in fungi. Although prevalent in eukaryotes, prions have not been identified in bacteria. Here we found that a bacterial protein, transcription terminator Rho of Clostridium botulinum (Cb-Rho), could form a prion. We identified a candidate prion-forming domain (cPrD) in Cb-Rho and showed that it conferred amyloidogenicity on Cb-Rho and could functionally replace the PrD of a yeast prion-forming protein. Furthermore, its cPrD enabled Cb-Rho to access alternative conformations in Escherichia coli—a soluble form that terminated transcription efficiently and an aggregated, self-propagating prion form that was functionally compromised. The prion form caused genome-wide changes in the transcriptome. Thus, Cb-Rho functions as a protein-based element of inheritance in bacteria, suggesting that the emergence of prions predates the evolutionary split between eukaryotes and bacteria.

First described as the protein-based causative agent of the fatal transmissible spongiform encephalopathies (1), prions have also been uncovered in fungi, where they act as protein-based elements of inheritance that confer new phenotypes on cells that harbor them (2, 3). Fungal prions are formed by proteins that can access alternative conformations, including a self-perpetuating amyloid fold (the prion form) that is characteristically heritable (4). At least a dozen prion-forming proteins with diverse functions have been uncovered in budding yeast (4), to which prions have been shown to confer growth advantages under specific conditions (3). Nonpathogenic, prion-like proteins have also been described in mammals (5), Aplysia (6), Drosophila (6), and, most recently, Arabidopsis (7). Although bacteria have been shown to propagate a yeast prion (8, 9), it is not known if bacterial prions exist.

We used a previously described hidden Markov model–based algorithm trained on a set of yeast prion-forming proteins (10, 11) to mine ~60,000 bacterial genomes for proteins containing candidate prion-forming domains (cPrDs) (table S1). Among the proteins identified by this analysis was the transcription termination factor Rho of Clostridium botulinum E3 strain Alaska E43 (Cb-Rho), which contains a 68–amino acid residue cPrD (residues 74 to 141, fig. S1) (12). Rho is a highly conserved hexameric helicase that loads onto nascent transcripts and couples adenosine 5′-triphosphate hydrolysis to RNA translocation, resulting in the termination of transcription by RNA polymerase (13). Phyletic analysis of bacterial Rho orthologs revealed that many Rho proteins contain an N-terminal insertion domain (NID) (14). The Cb-Rho cPrD was found to be located within an NID (Fig. 1A), and, notably, many Rho orthologs from distantly related bacteria contain similarly situated cPrDs (fig. S2).

Fig. 1 Cb-Rho contains an amyloidogenic cPrD.

(A) Cartoon of a Rho hexamer engaging RNA polymerase (RNAP) (top) and domain organization of Cb-Rho highlighting its cPrD and NID (bottom), where numbered dots indicate specific residues. (B) E. coli cells exporting the indicated protein domain were spotted on solid medium containing the amyloid-binding dye Congo Red (CR). The PrD of yeast Sup35 (Sup35NM) and its derivative (Sup35M) serve as positive and negative controls, respectively. Plate-derived material visualized by bright-field microscopy and between crossed polarizers reveals “apple-green” birefringence characteristically exhibited by CR-bound amyloid aggregates. (C) Amyloid-like aggregates (characterized by their resistance to denaturation in SDS) of the hexahistidine (His6X)–tagged protein domains were detected with His6X-specific antibody (α-His6X) in plate-derived material from (B) as assessed by filter-retention analysis. Undiluted sample and three twofold dilutions are shown. Aggregates were no longer detected once boiled (supplementary materials and methods). (D) SDS-stable aggregates were detected in cell lysates containing the indicated cPrD-containing Cb-Rho fragment fused to mYFP, but not in cell lysates containing a cPrD-lacking variant fused to mYFP. Anti-GFP antibody (α-GFP), which recognizes the mYFP tag, was used to detect aggregates. (E) SDS-stable aggregates were detected with α-His6X in cell lysates containing His6X-tagged cPrD-containing Cb-Rho, but not in cell lysates containing His6X-tagged cPrD-lacking Cb-Rho variants or His6X-tagged E. coli-Rho. (C to E) See fig. S3 for immunoblot analyses.

A characteristic of most prion-forming proteins is their ability to assemble as amyloid aggregates (3, 4). Therefore, we tested Cb-Rho for amyloidogenicity using an E. coli–based secretion assay that detects extracellular amyloid (15). Both the 68-residue cPrD of Cb-Rho and a 248-residue fragment of Cb-Rho that encompasses the cPrD in the structurally well-defined Rho N-terminal domain (NTD) (13) had amyloid-forming propensities by this test (Fig. 1, A to C, and fig. S3A). These Cb-Rho domains also formed amyloid-like material when fused to monomeric yellow fluorescent protein (mYFP) and produced in the E. coli cytoplasm, as did a truncated NTD fragment retaining the complete cPrD (NTD Δ1-73); however, an NTD variant lacking the cPrD (NTD Δ1-141) did not (Fig. 1, A and D, and fig. S3B). Similarly, full-length Cb-Rho and Cb-Rho Δ1-73 formed amyloid-like material in the E. coli cytoplasm, but excess E. coli–Rho and the three Cb-Rho variants that lacked the cPrD did not (Fig. 1, A and E, and fig. S3C). Thus, the cPrD confers amyloid-forming potential on Cb-Rho.

Next, we asked whether the Cb-Rho cPrD could functionally replace the PrD of the yeast prion-forming protein Sup35, an essential translation release factor. Yeast strains containing Sup35 in its nonprion form ([psi] strains) display normal translation termination, whereas strains containing Sup35 in its prion form ([PSI+] strains) exhibit stop codon readthrough, which is detectable as a heritable colony-color phenotype (4). Sup35 has both an N-terminal PrD (Sup35NM) that can be functionally replaced by heterologous PrDs and a C-terminal moiety (Sup35C) with translation release activity (11). We replaced Sup35NM with several Cb-Rho fragments (cPrD, NTD, or NTD Δ1-73) and then constructed three yeast strains, each containing one of the three resulting Cb-Rho–Sup35C chimeras as the sole source of translation release activity. In each case, the cells exhibited a [psi]-like phenotype that could undergo conversion to a stable [PSI+]-like phenotype (Fig. 2, A and B), the propagation of which was dependent on the chaperone Hsp104 (Fig. 2C), which mirrored the dependence of Sup35 and other yeast prions on Hsp104 (4). Cells containing any one of three Cb-Rho NTD–Sup35C chimera variants that lacked the cPrD exhibited [psi]-like phenotypes only (Fig. 2, A and B). Thus, Cb-Rho cPrD can functionally substitute for Sup35NM. Additionally, our finding that the Cb-Rho cPrD–Sup35C chimera exhibited stable [psi]-like and [PSI+]-like phenotypes in yeast enabled us to demonstrate that Cb-Rho aggregates produced in bacteria were infectious when introduced into yeast cells (fig. S5).

Fig. 2 Cb-Rho behaves as a prion in yeast.

(A) Yeast cells (containing native Sup35 or the indicated Cb-Rho fragment fused to Sup35C) were first grown in medium either containing (Gal) or lacking (Raf) galactose to induce transient overproduction of plasmid-encoded Sup35NM-YFP (in Sup35-containing cells) or Cb-Rho cPrD-GFP fusion protein (in Sup35 fusion protein-containing cells) and then spotted on adenine-lacking medium (see fig. S4, for fluorescence microscopy, and supplementary materials and methods). Adenine-lacking medium selects for [PSI+] clones, and transient overproduction of its PrD triggers conversion of a prion protein to the prion form (4). (B) Phenotypes of [psi], [PSI+], [psi]-like, and [PSI+]-like colonies from (A) restreaked on rich medium. The [psi]-like and [PSI+]-like states are designated [rho-X-C] and [RHO-X-C+], respectively, where X indicates the Cb-Rho fragment of interest. (C) Phenotypes of colonies from (B) restreaked on rich medium following passage on guanidine hydrochloride (GuHCl)–containing medium (transient inactivation of Hsp104).

We then asked whether Cb-Rho could interconvert between nonprion and self-perpetuating prion conformations in E. coli cells, with conversion to the prion form causing decreased Rho activity. To detect Rho activity, we used a reporter gene construct in which the Rho-dependent terminator tR1 is placed between a phage promoter and the lacZ gene (Fig. 3A). Decreased Rho activity in a strain harboring this reporter should result in increased expression of lacZ and cause the colonies to appear blue on indicator medium. Although we were unable to replace the E. coli rho gene, an essential gene, with the Cb rho gene (supplementary materials and methods), we could construct a strain with a chromosomally encoded Cb-Rho NTD–E. coli-Rho CTD (C-terminal domain) chimera in place of E. coli-Rho. This strain exhibited a slow-growth phenotype, which we could ameliorate by supplementing the chromosomally encoded Rho chimera with excess plasmid-encoded Rho chimera (Cb-Rho NTD Δ1-73–E. coli-Rho CTD) (Fig. 3A and supplementary materials and methods). Cells containing this plasmid gave rise to both pale blue and blue colonies, where pale color indicated high Rho activity and blue color indicated low Rho activity (Fig. 3B). This phenotypic heterogeneity suggested that the plasmid-encoded Rho chimera was capable of accessing alternative conformations: a soluble, nonprion conformation (pale colonies) and an aggregated, prion conformation (blue colonies).

Fig. 3 Cb-Rho exhibits prion behavior in E. coli.

(A) Schematic of E. coli reporter strain containing terminator tR1 upstream of lacZ and chromosomally encoded Rho chimera. Reporter strain growth defects were rescued by a plasmid directing the isopropyl-β-d-thiogalactopyranoside (IPTG)–inducible synthesis of His6X-tagged Rho chimera Δ1-73 (supplementary materials and methods). PR, rightward promoter of bacteriophage λ; Prho, endogenous rho gene promoter; Ptac, strong IPTG-inducible promoter; gDNA, genomic DNA. (B) A representative inducer-containing indicator plate showing blue and pale colonies of isogenic cells described in (A). (C) SDS-stable aggregates were detected with α-His6X in cell lysates of cultures inoculated with blue colonies (blue dot). Cultures inoculated with pale colonies (pale blue dot) contained little aggregated material. No aggregated material was present in cultures inoculated with naïve colonies (yellow dot) of reporter strain cells producing plasmid-encoded Rho chimera that lacks the cPrD (Δ1-141). We note that production of Rho chimera Δ1-141 was toxic and could not be evaluated for colony-color phenotype. Overproduction of ClpB, but not a catalytically inactive ClpB mutant (ClpB*), under the control of an arabinose (Ara)–inducible promoter cured cells of SDS-stable aggregates (see fig. S6 for immunoblot analyses). (D) A representative example of terminator readthrough in “blue” cells. Rockhopper-normalized counts from RNA-sequencing data aligned to sense/antisense (+/–) strands are shown for cells descended from pale colonies (pale blue tracks) or blue colonies (blue tracks). Genes exhibiting statistically significant changes in expression are identified with blue arrows. The yagNMLK genes belong to the E. coli CP4-6 cryptic prophage. Solid red line indicates a transcript with its previously inferred Rho-dependent terminator (cross bar) (20). Dashed pink line shows extension of the transcript in “blue” cells.

Additional findings fulfilled key predictions of the hypothesis that alternative protein conformations, including a self-perpetuating prion form, were responsible for the pale and blue colony-color phenotypes: (i) Plasmid DNA originating from either blue or pale colonies revealed no sequence differences within or surrounding the chimeric rho gene, and the plasmids behaved indistinguishably when retransformed into naïve reporter strain cells (Fig. 4A); (ii) Cell lysates prepared from overnight cultures inoculated with blue colonies contained Rho protein aggregates, but those prepared from cultures inoculated with pale colonies contained little aggregated material (Fig. 3C and fig. S6); (iii) Cell cultures of blue and pale colonies produced predominantly blue and pale colonies, respectively, when plated on indicator medium, and this bias was even more pronounced when blue and pale colonies were resuspended and replated without intervening liquid growth, a procedure that enabled us to estimate the probability of spontaneous loss (<0.8% per cell per generation) and appearance (<0.2% per cell per generation) of the Rho prion (Fig. 4A and supplementary materials and methods); (iv) The blue colony-color phenotype (i.e., the Rho prion) could be propagated for ≥120 generations, and its maintenance depended on continued synthesis of the Rho chimera (Fig. 4B and supplementary materials and methods); (v) Reminiscent of the effect of Hsp104 overproduction on [PSI+] yeast cells (4), the blue colony-color phenotype was “cured” by overproduction of the disaggregase ClpB (the bacterial ortholog of Hsp104) (Figs. 3C and 4C); and (vi) Transcription profiling on cells descended from either blue or pale parent colonies revealed genome-wide readthrough of Rho-dependent terminators specifically in cells derived from blue colonies (Fig. 3D, figs. S7 and S8, and table S2) (16). Thus, Cb-Rho can undergo conversion to a self-propagating prion conformation in E. coli cells, eliciting genome-wide changes in the transcriptome.

Fig. 4 Propagation of the Cb-Rho prion occurs over ≥120 generations.

(A) Naïve, blue, or pale parent colonies of reporter strain cells producing Rho chimera Δ1-73 were either grown overnight in liquid medium (+) or simply resuspended (−) and then replated on inducer-containing indicator plates. (B) The blue colony-color phenotype was propagated over five rounds of replating without intervening growth in liquid medium (supplementary materials and methods). (C) The blue colony-color phenotype was cured upon arabinose (Ara)–inducible overproduction of ClpB, but not a catalytically inactive ClpB mutant (ClpB*). Values represent the mean ± SD of three biological replicates (supplementary materials and methods).

We also observed Cb-Rho prion behavior without protein overproduction by constructing a strain encoding Cb-Rho NTD Δ1-73–E. coli-Rho CTD at the native chromosomal rho locus. The resulting cells were healthy and yielded both blue and pale colonies when plated on indicator medium (fig. S9). Blue colonies spontaneously gave rise to pale colonies at a low frequency (fig. S9, B and C), and the blue colony-color phenotype was cured by transient ClpB overproduction (fig. S9B and supplementary materials and methods). Moreover, pale colonies gave rise to blue colonies upon transient exposure to 5% ethanol, a stress condition known to confer a fitness advantage to E. coli cells carrying a reduced-function rho allele (fig. S9B and supplementary materials and methods) (17).

The identification of Cb-Rho as a bacterial prion-forming protein establishes protein-based heredity in the bacterial domain of life, suggesting that the emergence of prion-dependent phenomena predates the divergence of Bacteria and Eukaryota. Moreover, the presence of cPrDs in Rho proteins of bacteria representing at least six phyla, including the dominant constituents of the human gut microbiota, suggests that the impact of bacterial prion-based phenomena may be far-reaching. Rho and Sup35 prion formation have an intriguing similarity. Whereas formation of the Sup35 prion triggers genome-wide changes in the proteome due to stop codon readthrough (18), formation of the Rho prion triggers genome-wide changes in the transcriptome due to terminator readthrough. Prions may represent a source of epigenetic diversity in bacteria that can contribute to bacterial fitness in a variety of settings, for example, by facilitating immune evasion in the context of infection (19) or enabling antibiotic tolerance in quasi-dormant “persister” cells (20). Moreover, because prion formation typically results in a reduced-function phenotype, it is notable that adaptive null mutations in bacteria are common, often facilitating survival in response to environmental challenge (21).

Supplementary Materials

www.sciencemag.org/content/355/6321/198/suppl/DC1

Materials and Methods

Figs. S1 to S9

Tables S1 to S3

References (2229)

References and Notes

  1. Acknowledgments: We thank A. Lancaster and the late Susan Lindquist, to whose memory this work is dedicated, for sharing the hidden Markov model–based algorithm; R. Washburn and M. Gottesman for the tR1-lacZ reporter; and S. Dove, S. Garrity, and B. Nickels for invaluable advice. RNA-sequencing data were deposited at Gene Expression Omnibus (GEO) under accession number GSE90485. Work was supported by NIH Pioneer Award OD003806 and grant GM115941 (A.H.).
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