Prion Biology in the Context of Bacteria
by
Andy H. Yuan
B.A. in Biology Harvard University, 2008
Submitted to the Department of Biology in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY IN BIOLOGY
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
SEPTEMBER 2015
2015 Andy H. Yuan. All rights reserved.
The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part in any medium now known or hereaftercreated
Signature of Author: Department of Biology August 17th, 2015
Certified by: Ann Hochschild Professor of Microbiology and Immunobiology Harvard Medical School Thesis Supervisor
t, Susan L. Lindquist Professor of Biology Thesis Supervisor
Accepted by: Amy E. Keating Professor of Biology MASS~UtT NSTITUTE C1 OF TECHNOLOGY Co-Chair, Biology Graduate Committee
SEP 18?201" - 1 ILIBRARIES 2 Prion Biology in the Context of Bacteria
by
Andy H. Yuan
Submitted to the Department of Biology in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in Biology
ABSTRACT
Prions are infectious amyloid aggregates first described in the context of mammalian neurodegenerative diseases collectively known as the transmissible spongiform encephalopathies. Prions have also been uncovered in yeast, where they function as protein-based units of heredity that confer unique phenotypic traits on those cells that harbor them. To date, the discovery of prions and prion-like phenomena has been confined to the Eukarya. The work presented in this thesis seeks to explore the possibility that prion-like mechanisms are operative in the bacterial domain of life.
In what follows, we demonstrate that Escherichia coli cells can propagate a model yeast prion in a chaperone-dependent manner, establishing that the bacterial cytoplasmic milieu in general and a molecular machine in particular are poised to support protein conformation-based heredity in bacteria. Moreover, we provide evidence that a bacterial transcription termination factor exhibits prion-like behavior in E. coli and yeast. Our work thus suggests that bacterial prions exist and may function as previously unrecognized reservoirs of phenotypic diversity among the most abundant organisms on earth.
Thesis supervisor: Ann Hochschild Title: Professor of Microbiology and Immunobiology, Harvard Medical School
Thesis supervisor: Susan Lindquist Title: Professor of Biology, MIT
3 4 ACKNOWLEDGEMENTS
I am forever grateful for the love and support of my family, friends, colleagues, and mentors. I wish to acknowledge Ann Hochschild, Susan Lindquist, Alan Grossman, Tania Baker, Tony Sinksey, Mike Laub, Steve Bell, and Amy Keating for making the work presented in this thesis possible. Ann, you embody so much of what I aspire to emulate as a scientist, and perhaps more importantly, as an individual. Thank you for everything.
5 6 TABLE OF CONTENTS
Abstract 3 Acknowledgements 5 Table of Contents 7 Chapter One: An introduction to prions 9 Chapter Two: Prion propagation can occur in a prokaryote and requires 27 the ClpB chaperone Abstract 28 Introduction 29 Results 33 Discussion 43 Materials and Methods 47 References 51 Figures 57 Appendix A: A transcription-based reporter for prion propagation in E. coli 82 Results and Discussion 83 References 87 Figures 88
Appendix B: ClpB is the only non-essential chaperone or protease required 91 for prion propagation in E. coli Results and Discussion 92 References 94 Figures 95
Chapter Three: Evidence of a bacterial prion 98 Abstract 99 Introduction 100 Results 102 Discussion 109 Materials and Methods 112 References 116 Figures 121 Appendix C: A cell-based screen for amyloidogenic proteins of bacterial 131 origin Results and Discussion 132 References 136 Figures 138 Curriculum Vitae 140
7 8 CHAPTER ONE
An introduction to prions
9 History of the transmissible spongiform encephalopathies
The history of prion research represents the quintessential Kuhnian epistemological progression. While we now readily conceive of prions as infectious protein aggregates and protein-based units of heredity, this was - and perhaps is still - not always the case. Accordingly, analysis of the prion paradigm warrants a review of those normal scientific observations, anomalies, retrospections, controversies, and resolutions that led to its establishment (Kuhn, 1962).
Building upon theoretical work presented by Griffith in 1967 (Griffith, 1967),
Prusiner coined the term prion in 1982 to describe the nuclease and UV-resistant infectious agent responsible for scrapie, a member of a class of inevitably fatal neurodegenerative diseases known as the transmissible spongiform encephalopathies (TSEs) (Prusiner, 1982). Scrapie was first characterized as an affliction of sheep, with the earliest documentation of scrapie-like cognitive and motor dysfunctions common to all TSEs dating to the 18th century (Aguzzi and
Polymenidou, 2004).
It would not be until the 20th century that evidence of human TSEs would emerge. The first case study of the human prion disease known as Creutzfeldt-Jakob disease (CJD) was reported in the early 1920's (Creutzfeldt, 1920; Jakob, 1921). A second human TSE known as Kuru ("to shiver" in the Fore linguistic family) was described in 1957 as an endemic disease afflicting aboriginal societies of Papua New
Guinea (Gajdusek and Zigas, 1957).
Prion disease in humans and animals received global attention in the 1990's when a correlation between the consumption of meat from "mad cows" afflicted
10 with bovine spongiform encephalopathy (BSE) and the occurrence of a variant form of CJD (vCJD) in humans was noted (Aguzzi, 1996; Bruce et al., 1997; Hill et al.,
1997). While BSE was first documented in 1987 (Wells et al., 1987), its relationship to vCJD brought the question of transmissibility - and subsequently, the specificity of transmission between species - to the forefront of prion research and public intrigue.
Prusiner's self-proclaimed "heretical" prion hypothesis was met with criticism and challenged by alternative models of scrapie etiology. Among these models, the most notable was the "virino hypothesis", according to which the infectious scrapie agent represents a protein-associated or -encapsulated nucleic acid (Kimberlin, 1982). The difficulties inherent to experimentally distinguishing between such a virus-like particle and a prion would render the prion hypothesis controversial for several decades after its conception.
Amyloid and the PrP protein
Since Prusiner formalized the prion hypothesis, the collective work of many research groups has established that the protein culprit underlying the TSEs of animals and man is the PrP protein, the product of the PRNP gene (Prusiner et al.,
1984; Aguzzi et al., 2008). PrP is a GPI-anchored extracellular membrane protein found at the surface of neurons and glial cells. Native PrP (PrPc) undergoes a dramatic change in conformation upon conversion to the prion state (PrPsc), forming protein aggregates known as amyloids (Diaz-Espinoza and Soto, 2012).
11 Amyloids are highly-ordered fibrillar assemblies characterized by a cross-@ spine. This quaternary structure is typified by X-ray diffraction intensity maxima at
4.7 A and 10 A, the distance between adjacent beta strands and opposing beta sheets, respectively, of an amyloid fibril. PrPsc amyloid aggregates are capable of templating the conversion of PrPc and thereby mediate propagation of PrPsc in afflicted individuals as well as transmission between individuals (Legname et al.,
2004; Castilla et al., 2005; Wang et al., 2010). Critically, self-propagation is a distinguishing feature of PrPSc amyloids that sets them apart from non-infectious amyloid aggregates underlying neuropathologies like Alzheimer's, Parkinson's, and
Huntington's diseases (Chiti and Dobson, 2006). While prion disease can be initiated in animal models via intravenous, intraperitoneal, and intraocular inoculation of infectious material, the mechanistic details of PrPsc transmission, propagation, and pathogenesis - along with the native function of the PrPc protein - are poorly understood and remain the subject of ongoing PrP research (Aguzzi and
Calella, 2009).
Fungal prions
The discovery of prions in yeast vividly illustrates how scientific progress is more often than not non-linear and often necessitates the reassessment of anomalous observations that pass unnoticed under the guise of normal science
(Kuhn, 1962). Specifically, in what would lay the conceptual framework for subsequent fungal prion research, Wickner cleverly invoked prions in 1994 to
12 account for two non-chromosomal genetic elements that had been described in yeast decades earlier (Wickner, 1994).
The first of these elements (known as [PSI]) was discovered and characterized by Cox in 1965 as a heritable factor increasing the efficiency of stop codon readthrough (Cox, 1965). The second element (known as [URE3]) was uncovered by Aigle and Lacroute in 1972 as a non-chromosomal genetic element that confers upon ura2 mutant cells the ability to utilize ureidosuccinate (Aigle and
Lacroute, 1927). The cumulative work of several research groups has now elucidated the molecular biology underlying [PSI] and [URE3]. Specifically, [PSI]
(more accurately designated as [PSI+]) represents the prion form of the Sup35 protein, an essential translation release factor that - in conjunction with the Sup45 protein - mediates stop codon recognition and translation termination (Stansfield et al., 1995; Paushkin et al., 1997a). [URE3] represents the prion form of the Ure2 protein, a negative regulator of the Gln3 transcription factor, which activates transcription of genes encoding proteins involved in the uptake and catabolism of poor nitrogen sources (Masison and Wickner, 1995; Magasanik and Kaiser, 2002).
Subsequent studies provided critical experimental support for Wickner's yeast prion hypothesis. Specifically, genetic and cell biological analyses of the [PSI+] prion formally established a link between protein aggregation and the inheritance of a phenotypic trait (Patino et al., 1996; Paushkin et al., 1996). Furthermore, biochemical work illuminated the molecular basis of Sup35 aggregation and thereby accounted for the apparent self-perpetuation of a prion conformation (Glover et al.,
1997; Paushkin et al., 1997b; Tessier and Lindquist, 2009).
13 Wickner's 1994 study represented a bonafide Kuhnian paradigm shift; that is, it accounted for anomalies of the past and upended our conception of heritability all the while inaugurating a new framework for genetics that was as rigid as it was illuminating. Specifically, Wickner transformed lessons learned from [URE3] and
[PSI] into three genetic criteria for prion phenomena: (i) prions exhibit reversible curability, (ii) prion appearance can be induced by overproduction of the prion protein, and (iii) prion-dependent phenotypes resemble those associated with prion protein loss-of-function (Wickner, 1994). These genetic criteria lie at the heart of a paradigm that continues to inform an exciting period of Kuhnian "normal science" in the field of yeast prion biology (Kuhn, 1962). As is the case for any normal science, research at the peripheries of the paradigm - best represented by recent discoveries of prion-like phenomena that satisfy only a subset of Wickner's criteria (Si et al.,
2010; Hou et al., 2011; Cai et al., 2014) - continues to threaten the coherence and thereby ensures the progression of prion research.
Like PrPsc, [PSI], [URE3], and the vast majority of yeast prion proteins form amyloid in their prion states (Alberti et al., 2009) As amyloids are recalcitrant to crystallization and insoluble, atomic resolution structures of prion proteins in their amyloid state have remained, for the most part, elusive. However, solid-state NMR techniques have demonstrated success in and continue to hold great promise for defining the structure of prions (Tycko, 2011). In this regard, a prion uncovered in the filmanetous fungus Podosporaanserina known as [Het-s] is particularly notable.
[Het-s] corresponds to the prion form of the HET-s protein, which - in conjunction with the HET-S protein - regulates heterokaryon incompatibility (Saupe, 2011]. To
14 date, the solid-state NMR structure of recombinant HET-s amyloid, which can be
described as a P-solenoid, represents the only high-resolution view of a prion
domain in its amyloid conformation (Ritter et al., 2005; Wasmer et al., 2008).
One the most significant contributions of yeast prion research to our
understanding of prion biology lie in unequivocal demonstrations of so-called
"protein-only" transmission, a defining feature of the prion hypothesis that
remained experimentally elusive in mammalian models of PrPsc infectivity for
decades. Specifically, the development of protein transformation techniques permitted the delivery of recombinant Sup35 and HET-s amyloids (generated in vitro in the absence of cofactors) into Saccharomyces cerevisiae and P. anserinacells, respectively, and resulted in the emergence of "infected" [PSI+] and [Het-s] cells
(Maddelein et al., 2002; Tanaka et al., 2004; King and Diaz-Avalos, 2004).
Other landmark studies of fungal prions have established that ecologically diverse wild strains of yeast contain prions, many of which confer upon cells new phenotypic traits that can - under particular growth conditions - increase cellular fitness (True and Lindquist, 2000; True et al., 2004; Halfmann et al., 2012; Jarosz et al., 2014a; Jarosz et al., 2014b). These findings provide experimental support for the proposal that prion-dependent mechanisms represent bet-hedging strategies that enable genetically identical cells to adapt to environmental stress (True and
Lindquist, 2000; True et al., 2004; Halfmann and Lindquist, 2010; Halfmann et al.,
2010; Suzuki et al., 2012; Holmes et al., 2013; Newby and Lindquist, 2013). Future studies of yeast prions and their contributions to environmental adaptation thus
15 hold great promise for reinforcing the emerging view that prion-like processes can
be evolutionarily beneficial.
Chaperones
The stable propagation of fungal prions - and thus, the heritability of their associated phenotypes - depends on the activity of chaperone proteins (Chernoff et al., 1995). The critical role that chaperones play in prion biology has been made most apparent in studies of S. cerevisiae, where the Hsp1O0/Clp-family AAA+ disaggregase Hsp104 is strictly required for the propagation of virtually all yeast prions (Liebman and Chernoff, 2012). Hsp104 typically collaborates with Hsp70- and Hsp40-family chaperones to orchestrate the solubilization and refolding of protein aggregates (Parsell et al., 1994; Glover and Lindquist, 1998). However, in the context of prion biology, multiple lines of evidence indicate that Hsp104 functions as a regulator of amyloid fiber appositional growth by fragmenting large prion aggregates into smaller seed particles known as propagons, which can be efficiently partitioned to daughter cells during cell division (Paushkin et al., 1996;
Ness et al., 2002; Cox et al., 2003; Satpute-Krishnan et al., 2007).
While Hspl04-, Hsp70-, and Hsp40-mediated amyloid fragmentation provides a general framework for understanding prion heritability, chaperone- mediated prion propagation in yeast is remarkably complex. S. cerevisiae contains six Hsp70 paralogs and over a dozen Hsp40-like proteins, a subset of which have been shown to exhibit specific effects on the propagation of distinct prions
(Liebman and Chernoff, 2012). Moreover, an atypical yeast prion known as [GAR+],
16 which confers upon cells the ability to bypass glucose repression-associated
metabolic processes, has been shown to depend on Hsp70 chaperones - but not
Hsp104 - for its stable propagation (Ball et al., 1976; Brown and Lindquist, 2009).
As the biological complexity underlying chaperone-prion interactions has become
increasingly apparent, the study of protein folding and chaperone function remains
at the forefront of yeast prion research.
Bacterial amyloid
While prion-like epigenetic phenomena may superficially resemble bistable
and phase variable biological networks extensively characterized in bacteria
(Veening et al., 2008), to date, bonafide prion-dependent phenomena have been
discovered exclusively in the Eukarya. Furthermore, while amyloids are ubiquitous in living systems and play critical roles in extracellular physiological processes of bacteria (Schwartz and Boles, 2012), it is unclear whether or not bacteria appropriate the amyloid conformation to store intracellular protein-based heritable information.
The founding member of the so-called "functional amyloid" family of bacterial extracellular proteins is the curli fiber produced by E. coli and other
Enterobacteriaceae (Chapman et al., 2002). Composed of the secreted CsgA and
CsgB proteins, curli fibers mediate surface adhesion and have been implicated in biofilm formation as well as immune evasion (Barnhart and Chapman, 2006).
Distinct but analogous extracellular functions - particularly those concerning biofilm formation - have been assigned to the TasA and TapA proteins of Bacillus
17 subtilis (Romero et al., 2011) and the FapC protein of Pseudomonasfluorescens
(Dueholm et al., 2010).
Bacteria also deploy extracellular amyloids in the form of toxins. Microcin
E492, for example, is a bactericidal peptide secreted by Klebsiella pneumoniae capable of forming inert amyloid fibrils that, in response to changes in pH and osmolarity, dissociate into cytotoxic, pore-forming oligomers that kill neighboring bacteria (Shahnawaz and Soto, 2012). Toxic amyloid oligomers can also play a role in host-pathogen interactions, as evidenced by the plant pathogen Xanthomonas axonopodis, which secretes (via a type-III secretion system) a class of amyloidogenic proteins known as harpins into plant cells, eliciting the hypersensitive response (Oh et al., 2007).
As the ability to produce extracellular amyloid is a common feature shared among many bacteria, including those belonging to human microbiomes, bacteria- generated amyloid burdens and their impact on human physiology have begun to receive attention (Hill & Lukiw, 2015). In fact, the recent discovery that the central nervous system is connected to the lymphatic system (Louveau et al., 2015) raises the intriguing - albeit currently unexplored - possibility that amyloid burdens of bacterial origin may influence the progression of amyloid-based human neurodegenerative diseases.
Do bacterial prions exist?
It is not obvious how to go about discovering prions - should they exist - in bacteria, where the conspicuous genetic signature of non-Mendelian inheritance is
18 unobservable. Indeed, the absence of extensive cytoplasmic mixing during bacterial
genetic transactions likely prevents - or perhaps necessarily precludes - the
observation of prion-like phenomena in bacteria by classic genetic approaches that
were and remain essential for the discovery and characterization of fungal prions.
Furthermore, experimental tools analogous to cytoduction (Conde and Fink, 1976)
and protein transformation (Maddelein et al., 2002; Tanaka et al., 2004) have yet to be established in bacterial systems. These challenges necessitate alternative approaches to conceptualizing and studying prion biology in the context of bacteria, the subject of this dissertation.
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25 26 CHAPTER TWO
Prion propagation can occur in a prokaryote and requires the ClpB chaperone
A modified version of this chapter was published previously:
Yuan AH, Garrity SJ, Nako E, Hochschild A. 2014. Prion propagation can occur in a prokaryote and requires the ClpB chaperone. eLife 4:eO2949.
Specific author contributions:
I devised and performed all experiments presented in this chapter. Sean Garrity (Harvard Medical School) made preliminary findings and Entela Nako (Harvard Medical School) provided reagents.
27 ABSTRACT
Prions are self-propagating protein aggregates that are characteristically
transmissible. In mammals, the PrP protein can form a prion that causes the
inevitably fatal transmissible spongiform encephalopathies. Prions have also been
uncovered in fungi, where they act as heritable, protein-based genetic elements. We
previously showed that the yeast prion protein Sup35 can access the prion conformation in Escherichiacoli. Here we demonstrate that E. coli cells can propagate the Sup35 prion under conditions that do not permit its de novo formation. Furthermore, we show that propagation requires the ClpB chaperone.
Prion propagation in yeast requires Hsp104 (a ClpB ortholog), and several studies have come to conflicting conclusions about the ability of ClpB 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.
28 INTRODUCTION
Prions are infectious, self-propagating protein aggregates first described in the context of scrapie (Prusiner, 1982), an example of a class of devastating neurodegenerative diseases known as the transmissible spongiform encephalopathies (TSEs). Specifically, the prion form of a protein known as PrP is the causative agent of the TSEs, which afflict humans and other mammals. Native
PrP (PrPc) undergoes a dramatic change in conformation upon conversion to its prion form (PrPsc), forming distinctive cross-3 aggregates termed amyloids (Diaz-
Espinoza and Soto, 2012). Highly resistant to denaturation and proteolysis, PrPscis infectious and templates the conformational conversion of PrPc molecules (Caughey et al., 2009).
Prion-like phenomena have also been described in budding yeast and other fungi. Since Wickner first invoked prions to account for two examples of non-
Mendelian genetic elements in Saccharomyces cerevisiae (Wickner, 1994; Cox, 1965;
Aigle and Lacroute, 1975), the study of fungal prion proteins has resulted in profound advances in our understanding of prion biology, including the first demonstration that purified prion protein aggregates are infectious (Maddelein et al., 2002; King and Diaz-Avalos, 2004; Tanaka et al., 2004). In general, such prion proteins exist in either a native, soluble form or a self-perpetuating, amyloid form, with spontaneous conversion between forms representing a rare event (Allen et al.,
2007; Lancaster et al., 2010). However, unlike PrPsc, yeast prions do not normally cause cell death. Instead, they can act as protein-based genetic elements that confer new phenotypes on those cells that harbor them (True and Lindquist, 2000; Tuite
29 and Serio, 2010; Newby and Lindquist, 2013). Fungal prion proteins have been
found to participate in diverse cellular processes (Coustou et al., 1997; True et al.,
2004; Suzuki et al., 2012; Holmes et al., 2013). The conversion of these proteins to their prion forms typically results in a dominant loss-of-function phenotype (Cox,
1965; Aigle and Lacroute, 1975). A particularly well-characterized example
involves the essential translation release factor Sup35, which confers on cells a heritable nonsense suppression phenotype upon conversion to the prion form (True
et al., 2004).
Like other yeast prion proteins, Sup35 has a modular structure, with a distinct prion domain (PrD) that mediates conversion to the prion form, [PSI+]. In the case of Sup35, the essential prion determinants, which include a glutamine- and asparagine-rich segment and five complete copies of an imperfect oligopeptide repeat sequence, lie in the N-terminal domain (N), whereas translation release activity resides in the C-terminal domain (C) (Ter-Avanesyan et al., 1993). A highly charged middle region (M) increases the solubility of native Sup35 and enhances the mitotic stability of [PSI+] (Liu et al., 2002). Together, Sup35 N and M function as a transferable prion-forming module (NM) that maintains its prionogenic potential when fused to heterologous proteins (Li and Lindquist, 2000). A distinctive property of the Sup35 conversion process in yeast is its dependence on the presence of a pre-existing prion, referred to as a [PSI+] inducibility (PIN) factor (Derkatch et al., 1997). Thus, yeast strains containing Sup35 in the non-prion form, [psi-], support the spontaneous conversion to [PSI+] only if they contain a PIN factor, typically the prion form of the Rnq1 protein (Derkatch et al., 2000). However,
30 several other yeast prion proteins, including the Newi protein, have the capacity to
function as PIN factors in their prion forms (Derkatch et al., 2001; Osherovich and
Weissman, 2001).
Importantly, the stable propagation of yeast prions - and thus, the
heritability of their associated phenotypes - depends on the function of chaperone
proteins (Chernoff et al., 1995). Specifically, the AAA+ disaggregase Hsp104 is
strictly required for the propagation of virtually all yeast prions characterized thus
far, and several other chaperone proteins have been implicated in this process as
well (Liebman and Chernoff, 2012; Winkler et al., 2012). Various lines of evidence
support the view that the essential role of Hsp104 with respect to prion propagation
stems from its ability to fragment prion aggregates and thereby to generate smaller seed particles known as propagons that can be efficiently partitioned to daughter
cells during cell division (Paushkin et al., 1996; Ness et al., 2002; Cox et al., 2003;
Kryndushkin et al., 2003; Satpute-Krishnan et al., 2007; Higurashi et al., 2008).
Accordingly, depletion or inhibition of Hsp104 in a prion-containing cell leads to
prion loss in progeny cells.
The molecular processes underlying prion biology constitute at least two distinct phases, namely, (i) the de novo conversion of a protein from its native to prion form, and (ii) the subsequent propagation of the self-perpetuating prion form over multiple generations. While studies have demonstrated that the bacterial cytoplasm can support the de novo formation of prion-like aggregates (Sabat6 et al.,
2009; Garrity et al., 2010; Fernandes-Tresguerres et al., 2010; Espargar6 et al.,
2012; Gasset-Rosa et al., 2014), evidence for prion propagation - and thus, protein
31 conformation-dependent heredity - in bacteria has remained elusive. We
previously demonstrated that conversion of Sup35 NM to its prion form in E. coli
depends on the presence of a transplanted PIN factor derived from the Newi protein, thereby recapitulating aspects of S. cerevisiae PIN-dependent prion biology
(Garrity et al., 2010). This PIN dependence provides an experimental framework for distinguishing between the initial conversion and subsequent propagation phases of the prion cycle.
Here we show that bacteria can propagate the Sup35 NM prion in an infectious conformation over at least -100 generations under conditions that do not permit de novo prion formation. More specifically, we demonstrate maintenance of the Sup35 NM prion over multiple rounds of restreaking in E. coli cells no longer capable of synthesizing Newi protein. Furthermore, we establish that propagation of the Sup35 NM prion in E. coli requires ClpB, the bacterial ortholog of Hsp104. The striking parallel between the requirements for both prion formation and prion propagation in yeast and bacteria, which are thought to have diverged more than 2.2 billion years ago, suggests that the paradigm of protein-based heredity may be more ancient than previously inferred (DeSantis et al., 2012).
32 RESULTS
E. coli cells can propagate SDS-stable NM aggregates
Having previously shown that the prionogenic module of Sup35 (hereafter referred to as NM) can adopt an infectious amyloid conformation in the E. coli cytoplasm (Garrity et al., 2010), we wished to determine whether or not E. coli cells could stably propagate NM in its prion form. To address this question, we took advantage of the fact that conversion of NM to its prion conformation in E. coli depends on the presence of a transplanted PIN factor, mirroring the PIN dependence of Sup35 prion formation in S. cerevisiae (Figure 1A). More specifically, we envisioned inducing the formation of infectious NM aggregates in the presence of the Newi PrD (hereafter referred to as Newl), curing cells of Newl-encoding DNA, and monitoring the fate of NM over multiple generations.
To facilitate these experiments, we fused NM and Newi to two monomeric fluorescent proteins (mCherry bearing a C-terminal hexahistidine tag and mGFP, respectively). The two fusion proteins were produced from compatible plasmids under the control of IPTG-inducible promoters. The plasmid encoding New1-mGFP
(pSC101Ts-NEW1) bore a temperature-sensitive origin of replication, enabling us to cure cells of Newi-encoding DNA and thereby deplete cells of the Newi fusion protein. As an initial test of our experimental system, we introduced the plasmid encoding NM-mCherry-His6x (pBR322-NM) together with either pSC101Ts-NEW1 or an empty vector control (pSC1O1TS) into E. coli cells and induced the synthesis of the fusion proteins at the permissive temperature. After overnight growth, we detected
SDS-stable NM aggregates only in cells producing the Newi fusion protein as
33 assessed by filter retention analysis (Figure 1B, see Materials and Methods). As
Newt can independently adopt an amyloid conformation in E. coli (Garrity et al.,
2010), we also detected SDS-stable Newi aggregates in cells containing both fusion proteins (Figure 1B). Western blot analysis revealed that the intracellular levels of the NM fusion protein were comparable in the presence and absence of the Newt fusion protein (Figure IC, Supplemental Figure 1A). We also examined the cells by fluorescence microscopy. In cells containing both fusion proteins, NM formed twisted ring structures (Garrity et al., 2010) or large polar foci in a substantial fraction of cells, whereas Newt formed punctate foci in a majority of cells (Figure
1D). In contrast, NM exhibited diffuse fluorescence in cells lacking Newt (Figure
1D).
We then sought to determine whether or not E. coli cells could propagate
SDS-stable NM aggregates over multiple generations under conditions that did not permit the de novo formation of aggregates (that is, in the absence of Newt). Our experimental protocol is illustrated in Figure 2 (see also Figure 3A). We first induced fusion protein synthesis in cells transformed with pBR322-NM and either pSCt0tTs-NEW1 (experimental sample) or pSCtOtTS (control sample). These
"starter cultures" were grown overnight to allow for the formation of SDS-stable NM aggregates in the experimental sample. The cells were then plated and grown at the non-permissive temperature to cure the cells of pSCt0Ts-NEW1 or pSCtOtTS, thereby generating a set of Round 1 (R1) colonies. Twenty R1 experimental colonies and twenty RI control colonies were subsequently examined; each was (a) patched onto selective medium to test for loss of pSCt0tTs-NEW1 or pSCtOtTS, (b)
34 restreaked to generate Round 2 (R2) colonies, and (c) inoculated into liquid medium for overnight growth to test for the presence of SDS-stable NM aggregates. Four separate experimental lineages (L1E-L4E) originating from ancestral R1 experimental colonies containing detectable NM aggregates along with four separate control lineages (Lic-L4c) originating from ancestral RI control colonies were then followed through Round 3 (R3) and Round 4 (R4). For R2 and each subsequent round, ten experimental colonies and ten control colonies were analyzed.
All R1 experimental and control colonies (20 of each) had lost pSCi0iTS_
NEW1 or pSC101Ts, respectively, as assessed by patching on selective medium (data not shown). Moreover, the absence of NEW1 DNA was confirmed by PCR (Figure
3C), and the absence of Newi protein was confirmed by Western blot analysis
(Figure 3B). We detected SDS-stable NM aggregates in 8 of 20 experimental samples (Figure 3B) and none of the control samples (Figure 3D). We selected 4 of the 8 aggregate-positive clones (Figure 3B, asterisks) to establish the four experimental lineages and arbitrarily selected four aggregate-negative control clones (Figure 3D, asterisks) to establish the four control lineages. 2 of the 4 experimental lineages (LiE and L3E) retained SDS-stable NM aggregates throughout the course of the experiment (Figure 4A, Supplemental Figure 2B). Of these two lineages, one maintained aggregates in 9 of 10 R4 clones (Figure 4A), and the other maintained aggregates in 7 of 10 R4 clones (Supplemental Figure 2B). We conclude that SDS-stable NM aggregates can be propagated in E. coli for at least -100 generations in the absence of Newi (Figure 5A).
35 The remaining two experimental lineages (L2E and L4E) lost detectable SDS- stable NM aggregates at R3 and R4, respectively (Supplemental Figure 2A,
Supplemental Figure 2C, Figure 5A). Moreover, the loss of aggregates manifested itself in all ten of the selected colonies at either R3 or R4 (Supplemental Figure 2A,
Supplemental Figure 2C, Figure 5A). Curiously, the loss of SDS-stable NM aggregates from a particular lineage coincided with a loss of detectable fusion protein, as assessed by Western blot analysis (Supplemental Figure 2A, Supplemental Figure
2C), suggesting that the loss of aggregates represented an indirect consequence of a radical drop in protein levels (see Discussion). We note that the observed drop in
NM-mCherry-His 6 x fusion protein levels is not irreversible, as exemplified by L2E, which exhibited a loss of detectable NM aggregates coincident with an R3 drop in fusion protein levels and an apparent restoration of fusion protein levels by R4 in all
10 samples (Supplemental Figure 2A). Despite the presence of normal levels of fusion protein, SDS-stable NM aggregates were not recovered in R4 of L2E, consistent with the expectation that their propagation requires that protein synthesis be maintained above some threshold level (Supplemental Figure 2A,
Figure 5A). Critically, none of the samples (120 in total) from any of the four control lineages contained detectable SDS-stable NM aggregates (Figure 4B, Supplemental
Figure 3).
Fluorescence microscopy revealed that cells containing propagated NM aggregates exhibited small foci emanating from large aggregates typically localized at cell poles, a phenotype distinguished from experimental starter culture cells by the lack of twisted ring structures (Figure 5C). However, we observed one instance
36 of aggregate-positive RI cells exhibiting twisted ring structures (Supplemental
Figure 6C). Whereas we cannot definitively assign the SDS-stable NM aggregates
detected by filter retention to those structures detected by fluorescence microscopy,
we note that similar structural diversity has been observed for several yeast prions
(Derkatch et al., 2001; Zhou et al., 2001). Furthermore, cells from aggregate-
negative samples invariably exhibited diffuse fluorescence (Figure 5D).
Propagated SDS-stable NM aggregates are infectious when introduced into [psi-] yeast cells
We next sought to determine whether or not cells that maintained SDS-stable
NM aggregates in the absence of Newi contained infectious material capable of
converting [psi-] yeast cells to [PSI+]. We prepared bacterial cell extracts from both
experimental and control starter cultures, as well as aggregate-positive samples
from each of the four experimental lineages. For each experimental lineage, we
examined an arbitrarily chosen sample obtained from the last aggregate-positive round. In addition, for L2E and L4E, we examined the R2 and R3 samples (indicated
by asterisks in Figure 5E) that gave rise to aggregate-negative clones in the
subsequent round. We used these bacterial extracts to transform S. cerevisiae
spheroplasts prepared from a [pin-] [psi-] strain. The use of a [pin-] recipient strain was critical as transient overproduction of Sup35 (or NM) in [PIN+][psi-] strains significantly stimulates the conversion from [psi-] to [PSI+] (Derkatch et al., 1997), whereas conversion in a [pin-] [psi-] background requires the introduction of infectious seed material (Tanaka and Weissman, 2006).
37 The experimental starter culture yielded [PSI+] yeast transformants at a frequency of -1% (Figure 5E), consistent with our previous findings (Garrity et al.,
2010). Similarly, each of the aggregate-positive samples from the four experimental lineages yielded [PSI+] yeast transformants at a frequency of -1%; in contrast, the aggregate-negative samples yielded no [PSI+] yeast transformants (Figure 5E).
Among the [PSI+] transformants we obtained with the experimental samples, we observed both "strong" and "weak" strains (Tanaka et al., 2004; Frederick et al.,
2014) (Supplemental Figure 4). We note that -1% corresponds only to the frequency of strong [PSI+] transformants and therefore represents a conservative estimate of E. coli cell extract infectivity; we did not attempt to quantify weak [PSI+] transformants because they are difficult to distinguish from [psi-] transformants on the medium utilized to isolate transformants. We conclude that E. coli cells can propagate NM in an infectious prion conformation over at least -100 generations under conditions that do not permit de novo prion formation.
Propagation of infectious NM aggregates depends on ClpB
The propagation of [PSI+] and other prions in yeast requires Hsp104, an
Hspl00-family ATP-dependent disaggregase that functions as a ring-shaped hexamer. Specifically, Hsp104 is thought to facilitate prion propagation by fragmenting large aggregates into smaller propagons that are subsequently disseminated during cell division (Paushkin et al., 1996; Ness et al., 2002; Cox et al.,
2003; Kryndushkin et al., 2003; Satpute-Krishnan et al., 2007; Higurashi et al.,
2008). We therefore investigated whether or not the propagation of NM aggregates in E. coli requires ClpB, the bacterial ortholog of Hsp104. To address this question,
38 we sought to deplete cells of ClpB specifically during the propagation phase of our
experiments. To accomplish this, we modified pSCi0iTS-NEW1 such that it also
directed the expression of c/pB under the control of its native promoter. When
transformed into Ac/pB cells, pSC10iTs-NEW1-clpB enabled us to grow starter
cultures containing ClpB and subsequently to deplete both ClpB and Newi in cells
plated at the non-permissive temperature (Figure 6A).
As expected, we detected SDS-stable NM aggregates in Ac/pB starter culture
cells transformed with pBR322-NM and pSC1O1Ts-new1-c/pB (Supplemental Figure
5A). Furthermore, fluorescence microscopy revealed that these cells contained
visible aggregates that were nearly indistinguishable from those in wild-type cells
containing pBR322-NM and pSCi0iTS-NEW1 (Supplemental Figure 51B). After
plating AclpB starter culture cells containing pBR322-NM and pSC10Ts-NEW1-c/pB
at the non-permissive temperature to cure cells of ClpB- and Newl-encoding DNA,
we examined 60 R1 colonies for the presence of SDS-stable NM aggregates. In
parallel, we examined 60 R1 colonies derived from wild-type starter culture cells
containing pBR322-NM and pSCi0Ts-NEW1. As before, every selected colony was
patched onto selective medium to test for loss of pSC1OiTS-NEW1-clpB or pSCi0iTS-
NEW1 and inoculated into liquid medium for overnight growth to test for the
presence of SDS-stable NM aggregates. All selected colonies had lost the
appropriate temperature-sensitive vector as assessed by patching on selective
medium (data not shown), and the absence of Newi and/or ClpB was confirmed by
Western blot analysis (Figure 6B, Figure 6C). Whereas 17 of 60 (28%) wild-type Ri
samples tested aggregate-positive, all Ac/pB RI samples tested aggregate-negative
39 (Figure 6B, Supplemental Figure 6A, Figure 6C, Supplemental Figure 6B). Western blot analysis revealed that the wild-type and AcIpB R1 cells contained comparable amounts of 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 AcIpB starter culture cells transformed with pBR322-NM and pSC101Ts-NEW1-c/pB as well as in an aggregate-positive R1 clone derived from wild-type starter culture cells (Figure 6D). In contrast, a AcIpB R1 clone derived from AcIpB starter culture cells containing pBR322-NM and pSCiO1TS.
NEW1-clpB as well as an aggregate-negative RI clone derived from wild-type starter culture cells containing pBR322-NM and pSC1JOTS lacked detectable infectivity
(Figure 6D). We conclude that cells lacking CIpB cannot propagate NM in its infectious prion conformation.
C1pB disaggregase activity is required for propagation of SDS-stable Sup35 NM aggregates
To investigate the mechanistic basis for the ClpB dependence of Sup35 NM prion propagation in E. coli, we devised a strategy that enabled us to test the abilities of specific ClpB mutants to support the propagation of SDS-stable Sup35
NM aggregates after their formation in the presence of wild-type ClpB. We tested previously characterized ClpB mutants specifically defective for (i) ATP hydrolysis
(ClpB E279A/E678A) (Weibezahn et al., 2003) (ii) substrate threading through the
ClpB pore (Y653A) (Weibezahn et al., 2004), and (iii) collaboration with DnaK
(E432A) (Oguchi et al., 2012; Seffer et al., 2012; Carroni et al., 2014). Our strategy required us to construct strains in which we could induce the production of a ClpB
40 mutant specifically during the propagation phase of the experiment while providing
wild-type ClpB during the formation phase of the experiment only. To accomplish
this, we placed each of the mutant cIpB alleles (or the wild-type allele) under the
control of the anhydrotetracycline (aTc)-inducible promoter PLtetO-I (Lutz and
Bujard, 1997), integrated these constructs onto the chromosome of our AclpB strain,
and transformed the resulting strains with pBR322-NM and pSC1O1Ts-NEW1-clpB.
As expected, we detected SDS-stable Sup35 NM aggregates in starter culture
cells of all strains producing plasmid-encoded Sup35 NM-mCherry-His6x, Newl-
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 Newi 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 (see 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 cIpB allele, no aggregates were detected in cells harboring the
cIpB E279A/E678A, cIpB Y653A, or cIpB 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.
41 NEW1-clpB (Supplemental Figure 8). 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
Newi.
42 DISCUSSION
Our findings establish that bacteria can propagate a prion. This is, to our knowledge, the first formal demonstration of prion propagation in a non-eukaryote.
More specifically, the PIN-dependent de novo conversion of Sup35 NM to its prion form in our E. coli system enabled us to distinguish experimentally between the initial formation phase and subsequent propagation phase of the prion cycle. Under our experimental conditions, two of four cell lineages (LiE and L3E) maintained the prion for the duration of the experiment (i.e., -100 generations), one lineage (L4E) maintained the prion for -80 generations, and one lineage (L2E) maintained the prion for -60 generations. Furthermore, our work demonstrates that prion propagation in E. coli requires ClpB, the bacterial ortholog of Hsp104. We conclude that bacteria can support a chaperone-dependent, protein-based mode of heredity and speculate that the emergence of prion-like phenomena may have predated the evolutionary split between eukaryotes and bacteria.
Stability of prion propagation in E. coli.
As only two of four lineages retained the prion for the full duration of our experiments, propagation of the Sup35 NM prion may be less stable in E. coli than in
S. cerevisiae (Cox et al., 1980; DiSalvo et al., 2011). However, as noted above, loss of the prion in two lineages was coincident with a dramatic drop in NM fusion protein levels. Furthermore, this drop was evidently reversible as NM fusion protein levels were restored in R4 of L2E without reappearance of the prion. We do not understand the mechanism underlying this reversible change in protein levels; however, we suggest that stochastic fluctuations in plasmid copy number may set
43 the stage for such an event. We note that our experiments were performed in recA cells, which should prevent plasmid rearrangements that might lead to a permanent loss of fusion protein coding capacity.
Bacterial machinery capable of remodeling NM aggregates.
The question of whether or not ClpB can substitute for Hsp104 in promoting
Sup35 prion propagation has been addressed in a number of studies yielding conflicting indications. On one hand, several in vitro studies have provided evidence that Hsp104 (Shorter and Lindquist, 2004; Shorter and Lindquist, 2006; Shorter and
Lindquist, 2008, DeSantis et al., 2012), but not ClpB (DeSantis et al., 2012), can fragment amyloid aggregates in the absence of auxiliary factors. Furthermore, whereas the presence of various combinations of S. cerevisiae Hsp70- and Hsp40- family proteins was found to modulate Hsp104 activity on amyloid substrates
(Shorter & Lindquist, 2008; DeSantis et al., 2012), ClpB appeared to remain inert even in the presence of bacterial Hsp70 (DnaK), Hsp40 (DnaJ), and nucleotide exchange factor GrpE despite exhibiting robust activity on various disordered protein aggregates in vitro (DeSantis et al., 2012).
On the other hand, the results of several in vivo studies suggest that ClpB, in the presence of appropriate co-chaperones, is competent to support Sup35 prion propagation in yeast (Tipton et al., 2008; Reidy et al., 2012). Based on an analysis of the in vivo activities of Hsp104/ClpB chimeras, Tipton et al. argue that prion replication in yeast requires that Hsp104 collaborate with its cognate Hsp70 chaperone system. A logical inference from their work is that the inability of ClpB to substitute for Hsp104 in supporting Sup35 prion propagation in S. cerevisiae is an
44 indirect consequence of the inability of ClpB to cooperate with fungal co- chaperones. More recently, Reidy et al. provided direct support for this inference.
In particular, Reidy et al. found that ClpB supported prion propagation in yeast provided that DnaK and GrpE were present. Interestingly, the activity of the bacterial disaggregase machinery in yeast was dependent on the fungal Hsp40- family SisI protein, consistent with prior work implicating Sisi as a necessary component of the chaperone network required for prion propagation in yeast
(Higurashi et al., 2008; Tipton et al., 2008). Based on these findings, Reidy et al. argue that the amyloid remodeling activity of Hsp104 is an evolutionarily conserved feature of the Hspl00-family chaperones, an inference that is strongly supported by our finding that propagation of the Sup35 NM prion in E. coli requires ClpB.
Despite the apparent prevalence of prions in the fungal kingdom, to date, no bacterial prion has been identified. Notably, the absence of cytoplasmic mixing during conjugation would preclude the discovery of prion-like phenomena by classic genetic approaches, which facilitated the discovery of prions in yeast based on the non-Mendelian inheritance of their associated phenotypes (Wickner, 1994;
Cox, 1965; Aigle and Lacroute, 1975). Nevertheless, recent bioinformatics analyses of prokaryotic proteomes have revealed that bacterial and archaeal genomes encode many proteins containing glutamine- and asparagine-rich prion-like domains resembling those found in most confirmed and putative S. cerevisiae prions (Alberti et al., 2009; Espinosa Angarica et al., 2013; A. Yuan, S. Lindquist, and A. Hochschild, unpublished data). Moreover, it is becoming increasingly clear that Q/N-richness at the level of primary amino acid sequence is neither a prerequisite for prion
45 conversion (Taneja et al., 2007) nor protein amyloidogenesis (Goldschmidt et al.,
2010). In fact, several bacteria utilize non-Q/N-rich amyloid-forming proteins to assemble extracellular appendages mediating surface attachment and biofilm formation (Chapman et al., 2002; Romero et al., 2010). These considerations - in conjunction with the work presented here - suggest that prions or prion-like proteins may exist as epigenetic reservoirs of phenotypic diversity in the bacterial domain of life.
46 MATERIALS AND METHODS
Strains, plasmids, and cell growth
Bacteria experiments were performed with E. coli strain DH5aZ1 (Lutz and
Bujard, 1997) grown in LB (Miller) medium. To construct DH5aZ1 AclpB, a
temperature-sensitive plasmid encoding the RecA protein (pSC101Ts-recA) was
constructed and transformed into DH5cZ1 cells. A AclpB::KanR allele from strain
JW2573 (Keio collection) was transferred to DH5aZ1 cells containing pSC101Ts-recA via P1 transduction. Cells were subsequently cured of pSC101Ts-recA by overnight growth and plating in the absence of antibiotic selection at the non-permissive temperature (37*C).
To construct strains harboring chromosomal PLteto--cpB alleles, plasmids pAY152, pAY154, pAY155, pAY156, and pAY157 were cloned in strain AY290 and integrated onto the chromosome of strain AY295 at attB(HK022). Single-copy integrants were selected on LB agar supplemented with kanamycin (10 tg/ml) and verified by PCR as described (Haldimann and Wanner, 2001).
Yeast experiments were performed with S. cerevisiae [pin-] [psi-] strain YJW18 grown in yeast extract peptone dextrose (YPD) medium. For yeast infectivity assays, cell extracts were co-transformed with pRS316 into YJW187 spheroplasts; [PSI+]
Ura+ transformants were identified by plating the yeast cells in top agar containing synthetic defined medium lacking uracil and adenine (SD-Ura-Ade) and supplemented with 10 mg/ml adenine hemisulfate (Sunrise Science).
Further details concerning strains and plasmids are provided in
Supplemental Table 1.
47 Propagation experiments
Cells were transformed with pBR322-NM and pSC1O ITS, pSCi0lTs-NEW1, or pSC1O1TS-NEW1-clpB and grown at 30*C on LB agar supplemented with carbenicillin (Carb, 100 pg/ml) and chloramphenicol (Cam, 12.5 pg/ml). Starter cultures were generated by growing transformants at 30'C in 6 ml of LB broth supplemented with Carb (100 pig/ml), Cam (12.5 pg/ml), and 10 .M IPTG to an
OD600 of 2.0-2.5. To cure cells of pSC101TS-derivatives and generate Round 1 (Ri) colonies, starter cultures were diluted (10-5) in pre-warmed (37'C) LB broth supplemented with Carb (100 pg/ml) and 10 pM IPTG. Diluted cells were grown at
37'C on pre-warmed (37'C) LB agar supplemented with Carb (100 pg/ml) and 10 pM IPTG. R1-R4 colonies were, (a) patched on LB agar supplemented with Cam
(12.5 pg/ml), (b) restreaked and grown at 30'C on pre-warmed (30'C) LB agar supplemented with Carb (100 pg/ml) and 10 pM IPTG, and (c) inoculated and grown at 30'C in 6 ml LB broth supplemented with Carb (100 Vg/ml) and 10 pM
IPTG. For analysis of ClpB disaggregase mutants, -1000 R1 colonies were gently scraped off LB agar plates containing Carb (100 pg/ml), 10 pM IPTG, and a range of aTc concentrations (0-500 ng/ml) in 3 ml LB broth supplemented with Carb (100 pg/ml) and 10 pM IPTG.
Cell cultures and scraped cell suspensions were normalized to 8 ml of an
OD600 of 1.0 and pelleted by centrifugation. Cell pellets were resuspended in 166 p1 STC Buffer (1 M sorbitol, 10 mM Tris-HCI [pH 7.5], 10 mM CaCl2) supplemented with 10 U of rLysozme (Novagen) and 0.1 U of OmniCleave endonuclease
(Epicentre), incubated at room-temperature for 30 min, and incubated on ice for an
48 additional 30 min. Omnicleave endonuclease was omitted from samples destined
for PCR analysis and yeast infectivity assays. Finally, samples were flash frozen and thawed on ice to yield unclarified lysates (used in filter retention assays). To generate partially clarified lysates (used in Western blot analysis and yeast transformation assays), unclarified lysates were subjected to two rounds of low- speed centrifugation, each at 500 RCF for 15 min at 4C.
Filter retention assays
25 [U of unclarified lysates were added to 375 pl of BugBuster protein extraction reagent (Novagen) supplemented with 5 U of rLysozyme and 0.1 U of
Omnicleave endonuclease and gently rocked at room-temperature for 30 min.
Samples were challenged with 100 p.l of 10% (w/v) SDS (2% SDS final concentration) and gently rocked at room-temperature for an additional 30 min.
For each sample, 100 pl of undiluted lysate and three 2-fold serial dilutions made in
PBS containing 2% SDS were filtered through a 0.2 pm cellulose acetate membrane
(Advantec) in a dot-blotting vacuum manifold. Samples on membranes were washed twice with 100 p.l of PBS containing 2% SDS and twice with 100 p.l of PBS.
Immunoblotting
Cellulose acetate membranes (used in filter retention assays) and Hybond-C
Extra nitrocellulose membranes (used in Western blot analysis) were blocked for 30 min in PBS containing 3% (w/v) milk. Membranes were probed with one of the following primary antibodies: anti-Sup35 (yS-20, Santa Cruz Biotechnology,
1:5,000), anti-His6x (His-2, Roche, 1:10,000), anti-GFP (Roche, 1:10,000), anti-RpoA
49 (NeoClone, 1:10,000), or anti-ClpB (gift from S. Wickner, 1:10,000). Membranes were washed and probed with one of the following HRP-conjugated secondary antibodies: anti-goat IgG (Santa Cruz Biotechnology, 1:10,000), anti-mouse IgG (Cell
Signaling, 1:10,000), or anti-rabbit IgG (Cell Signaling, 1:10,000). Proteins were detected with ECL Plus Western blot detection reagents (GE Healthcare) and a
ChemiDock XRS+ imaging system (Bio-Rad).
Yeast infectivity assays
Protein concentrations of partially clarified E. coli cell extracts were determined by the bicinchoninic acid (BCA) assay (ThermoFisher) and normalized to -1 mg/ml. Protein transformations were performed as previously described
(Tanaka and Weissman, 2006; Garrity et al., 2010). Each and every putative [PSI+] transformant was (a) restreaked on 1/4 YPD agar to assess the [PSI+] phenotype, (b) restreaked on YPD containing 3 mM GuHCl to cure cells of [PSI+], and (c) restreaked on 1/4 YPD to assess the [psi-] phenotype. Only those transformants exhibiting curability were scored as [PSI+].
Fluorescence Microscopy
Cells were spotted onto 1% (w/v) agarose pads consisting of Seakem LE
Agarose (Lonza) in PBS and visualized with an UplanFL N 100x/1.30 phase contrast objective mounted on an Olympus BX61 microscope. Images were captured with a
CoolSnapHQ camera (Photometrics) and the Metamorph software package
(Universal Imaging). All fluorescence images were obtained from 10 ms exposures.
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56 FIGURES A
esSup35,, U35, New1
B Sup35 + + + + C Newi + - + - Sup35 + + Boil - -+ + Newl + -
-0 kDa ~Q-FL a-Sup35 a-Sup35 a _50 kDa m-40 kDa at-GFP ~j.FL-F a-GFP - 30 kDa
a-RpoAcx-Rpo- :E-4 40 kDao
D Sup35 + Newi Sup35 alone
Sup35- New1-mGFP merge Sup35- mCherry mCherry
Figure 1. Conversion of Sup35 NM to its prion form in E. coli requires Newl.
A) Cartoon representation of how conversion of soluble Sup35 NM (Sup3Ssoluble) to 5 its amyloid conformation (Sup3 amyloid) depends on the presence of Newt in its amyloid conformation (Newlamyoid). Sup35 NM and Newt (black) are depicted as fusions to mCherry (red) and mGFP (green), respectively. (B) SDS-stable Sup35 NM aggregates are detected only in cells producing SDS-stable Newt aggregates as assessed by filter retention analysis. For each sample, undiluted lysate and three two-fold dilutions are shown (see Materials and Methods). Sup35 NM and Newt aggregates are no longer detected once boiled. The a-Sup35 antibody recognizes the Sup35 NM-mCherry-His6x fusion protein, and the cc-GFP antibody detects the Newl-mGFP fusion protein. (C) Intracellular full-length (FL) Sup35 NM fusion protein levels are comparable in the presence and absence of Newt as assessed by Western blot analysis. The a-RpoA antibody recognizes the a subunit of E. coli RNA polymerase. (D) Fluorescence images of representative cells containing Sup35 NM and Newt or Sup35 NM alone. For cells containing both fusion proteins, the mCherry channel, GFP channel, and merged images are shown.
57 :I
Sup35 + + Sup35 + + A New1 + - New1 + -
-0 kDa - 60 kDa -FL a-Sup35 F ct-HIs6, I- 50 kDa - 50 kDa
E 3 ST R1 ST R1 P N P N
Sea 1.1.5 5.1.5 I.'
a-His 0 S. * ,0~
* 1 11 1 a * - a*O |e5o| |e a-Sup35
II S l *.T ...... T R1
P N 1 P551N S S 5 5 S.SI I.I I I S11 1 E . C
a-His6x
Ca I * I * I a-Sup35 0. Ci 0' em:1 I , . . -
Supplemental Figure 1. a-Sup35 and a-His6x antibodies are interchangeable for detecting the Sup35 NM-mCherry-His6x fusion protein.
(A) Probing nitrocellulose membranes with either a-Sup35 antibody (Figure 1C) or a-His6x antibody results in the detection of similar intracellular Sup35 NM fusion protein products in the presence or absence of Newi as assessed by Western blot analysis. Full-length (FL) Sup35 NM fusion proteins are indicated by arrows. (B) Probing cellulose acetate membranes with either a-His6x antibody (Figure 3B) or a- Sup35 antibody results in the detection of similar aggregate-positive and aggregate- negative samples in starter cultures (ST) and Round 1 (Ri) experimental clones as assessed by filter retention analysis. For each sample, undiluted lysate and three two-fold dilutions are shown. Starter cultures of cells containing Sup35 NM and Newi and cells containing Sup35 NM alone serve as positive (P) and negative (N) controls, respectively. (C) Probing cellulose acetate membranes with either a-His6x antibody (Figure 3D) or a-Sup35 antibody results in the detection of similar aggregate-positive and aggregate-negative samples in starter cultures (ST) and R1 control clones as assessed by filter retention analysis.
58 I
Starter culture (ST) Cells contain Sup35 + Newi Sup35 aggregates are formed at 30"C
Cure cells of NewI-encoding plasmid by plating at 370C
R1
For each of 20 R1 colonIes: (I) inoculate liquid medium for filter retention assay and (1i) restreak on solid medium
(I)
Filter retention assay to detect SDS-stable Sup35 I -L2 aggregates ~-q1 Analyze the progeny of 4 retrospecthvely Identified aggregate-posith ve clones (L1-L4)
For each lineage, analyze the progeny of one retrospectively Identified aggregate-positive R2 clone
R4-LI.
For each lineage, analyze the progeny of one retrospectively identified aggregate-positive R3 clone X1 0 for each Ineage
59 Figure 2. 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 Newt (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 Newi during 4 rounds (R1-R4) of growth on solid medium and an additional round of growth in liquid medium. Growth in the absence of Newt begins at the time the starter culture cells are plated at 37'C (Ri). Single R1 colonies were found to contain -950,000 colony forming units (CFUs), and the liquid cultures contain ~108 CFUs per d. Thus, prion propagation is monitored over 98.7 or -100 generations.
60 A
Sup35 NM1Su3 New1lwo
B ST R1 ST R1 P N * PN ** *
a-Sup35 - -
a-GFP a-RpoA
C ST R1 P N 1.0 kb -.. - SUP35 NM 500 bp 100 bp 1.0 kb NEW1 500 bp 100 bp
D ST R1 ST R1 P N * PN ** *
a-His6 : 4WQV- No0-oe&a a-Sup35 a-RpoA
Figure 3. Converted Sup35 NM can remain in its prion conformation in E. coli cells lacking Newl.
(A) Cartoon representation of how Sup35 NM can convert to its prion form in the presence of Newt and remain in the prion conformation after cells have been cured of Newl-encoding DNA. Sup35 NM and Newt (black) are depicted as fusions to mCherry (red) and mGFP (green), respectively. (B) SDS-stable Sup35 NM aggregates are detected in 8 of 20 Round 1 (Rt) experimental clones derived from a starter culture (ST) of cells containing Sup35 NM and Newt as assessed by filter retention analysis. Starter cultures of cells containing Sup35 NM and Newt and cells containing Sup35 NM alone serve as positive (P) and negative (N) controls, respectively. The four aggregate-positive clones selected to establish the four experimental lineages are indicated by asterisks. In all 20 R1 experimental samples, intracellular Sup35 NM fusion protein levels are comparable, and Newt fusion protein is not detectable as assessed by Western blot analysis. The a-His6x and a-
61 Sup35 antibodies recognize the Sup35 NM-mCherry-His6x fusion protein (see Supplemental Figure 1B, Supplemental Figure 1C), the u-GFP antibody detects the Newi-mGFP fusion protein, and the a-RpoA antibody recognizes the a subunit of E coli RNA polymerase. (C) In all 20 R1 experimental samples, DNA encoding Sup35 NM is detectable whereas DNA encoding the prionogenic module of Newi is not detectable by PCR. (D) SDS-stable Sup35 NM aggregates are not detected in any of the 20 R1 control clones derived from a starter culture of cells containing Sup35 NM alone as assessed by filter retention analysis. The four aggregate-negative clones selected to establish the four control lineages are indicated by asterisks. Intracellular Sup35 NM fusion protein levels are comparable in all 20 R1 control samples.
62 A ST R1 R2 771 P N
a-His .,
a-Sup35 pow a-RpoA
R3 R4 P N P N
a-His *
e - -- -
a-Sup35 4 a a-RpoA
B ST R1 R2 P N
a-His6X
a-Sup35 a-RpoA
R3 R4 P N P N
a-His6X0
a-Sup35 VI a-RpoA
Figure 4. E. coli cells can propagate the Sup35 NM prion over -100 generations.
(A) Experimental Lineage 1 (LiE). An aggregate-positive Round 1 (Ri) experimental clone derived from a starter culture (ST) of cells containing Sup35 NM and Newi is identified and restreaked to yield progeny Round 2 (R2) clones (gray). All ten R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. An aggregate- positive R2 clone is identified and restreaked to yield progeny Round 3 (R3) clones (blue). Again, all ten R3 clones analyzed contain SDS-stable Sup35 NM aggregates. An aggregate-positive R3 clone is identified and restreaked to yield progeny Round 4 (R4) clones (green). 9 of 10 R4 clones analyzed contain SDS-stable Sup35 NM aggregates. The filter retention assay is used to detect SDS-stable Sup3S NM aggregates. Intracellular Sup35 NM fusion protein levels are comparable in all 40 samples as assessed by Western blot analysis. Starter cultures of cells containing Sup35 NM and Newi and cells containing Sup35 NM alone serve as positive (P) and
63 negative (N) controls, respectively. The ca-His6x and u-Sup35 antibodies recognize the Sup35 NM-mCherry-His6x fusion protein, and the ca-RpoA antibody recognizes the cc subunit of E. coli RNA polymerase. (B) Control Lineage 1 (Lic). An aggregate- negative R1 control clone derived from a starter culture of cells containing Sup35 NM alone is identified and restreaked to yield progeny R2 clones (gray), an aggregate-negative R2 clone is identified and restreaked to yield progeny R3 clones (blue), and an aggregate-negative R3 clone is identified and restreaked to yield progeny R4 clones (green). No SDS-stable Sup35 NM aggregates are detectable in any sample. Intracellular Sup35 NM fusion protein levels are comparable in all 40 samples as assessed by Western blot analysis.
64 tn 110
It - CM a 04 N Cc ...... 0 0 0 a: cc ...... 0 0 0 Its ...... 14i. "a ...... V ...... be ...... ------...... i ...... 0 0 ...... 0 0 0 - z z z z z z ...... 0 CL CL CL I . a. LLJ I. o 10- 0 0 0 r ...... ------L ...... ------Tv ......
------0 0 0 V ...... co cr t- L cr(V) L cr CC z z z z a. co CL [a. 0- a. [Z a. x LO Lc) tc) w LO 24 0 Ma 0 E tv) 0 CL Mn C CL CL cl 06 U) CF cc a: ts m 8 Supplemental Figure 2. The fate of Sup35 NM in experimental Lineages 2-4.
(A) Experimental Lineage 2 (L2E). An aggregate-positive Round 1 (Ri) clone derived from a starter culture (ST) of cells containing Sup35 NM and Newt is identified and restreaked to yield progeny Round 2 (R2) clones (gray). 8 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates as assessed by filter retention analysis. An aggregate-positive R2 clone is restreaked to yield progeny Round 3 (R3) clones (blue). 0 of 10 R3 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. The apparent loss of aggregates coincides with the loss of detectable Sup35 NM fusion protein as assessed by Western blot analysis. Assaying the 10 Round 4 (R4) progeny clones derived from an aggregate-negative R3 clone (green) reveals that fusion protein levels can be restored without the recovery of SDS-stable Sup35 NM aggregates. Starter cultures of cells containing Sup35 NM and Newt and cells containing Sup35 NM alone serve as positive (P) and negative (N) controls, respectively. The ax-His6x and a-Sup35 antibodies recognize the Sup35 NM-mCherry-His6x fusion protein, and the a-RpoA antibody recognizes the c subunit of E. coli RNA polymerase. (B) Experimental Lineage 3 (L3E). 9 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates, which are retained in 8 of 10 R3 progeny clones and 7 of 10 R4 progeny clones. (C) Experimental Lineage 4 (L4E). 8 of 10 R2 clones analyzed contain detectable SDS- stable Sup35 NM aggregates, which are retained in 9 of 10 R3 progeny clones. 0 of 10 R4 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. As in R3 of L2E (A), the apparent loss of aggregates coincides with a dramatic drop in Sup35 NM fusion protein levels.
66 CM C\j Iqt CM v - ...... (r. I------cc ...... cr------...... ------......
...... ------......
...... ------
...... I-se L ...... z z z z z . . . . . CL 1z .. . . . CL El 'Li 0- ps C\j -i * 'L,2...... - ...... ------...... I------...... ------......
cf) Cf) ...... cc t7 al z z z z z a.------IL-Iii CL [ a- o m;L 0 ww 0- L6. x U) U.) x 0 21) V) 9 0 C2 CL CL CL 5 a: cr Cl) ZZ "?t5 8 ZZ Supplemental Figure 3. The fate of Sup35 NM in control Lineages 2-4.
(A) Control Lineage 2 (L2c). An aggregate-negative Round 1 (RI) clone derived from a starter culture (ST) of cells containing Sup35 NM alone is identified and restreaked to yield progeny Round 2 (R2) clones (gray). 0 of 10 R2 clones analyzed contain detectable SDS-stable Sup35 NM aggregates as assessed by filter retention analysis. An aggregate-negative R2 clone is restreaked to yield progeny R3 clones (blue). 0 of 10 R3 clones analyzed contain detectable SDS-stable Sup3S NM aggregates. An aggregate-negative R3 clone is restreaked to yield progeny R4 clones (green). 0 of 10 R4 clones analyzed contain detectable SDS-stable Sup35 NM aggregates. Starter cultures of cells containing Sup35 NM and Newi and cells containing Sup35 NM alone serve as positive (P) and negative (N) controls, respectively. The a-His6 x and u-Sup35 antibodies recognize the Sup35 NM- mCherry-His6x fusion protein, and the a-RpoA antibody recognizes the c 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.
68 -I
A B
L1E L2 E LC L2C
RI R1
L4E EL3E L4C L3C
C D Li E LiC R4-1 P- R4-1 R4-2
R4-3 R4-4 R4-3 R4-4
E Sup35 New1 % [PSI+ transformants + + 11.44% (77/5340) ST[ + I - I 0.00% (0/4928) I
Uneage Round % [PSI+] transformants Li R4-8 1.11% (56/5024) L2 R2-1 1.07% (53/4968) L3 R4-4 1.37% (66/4820) L4 R3-3 1.13% (70/6172) L2* R2-2 1.20% (69/5760) L4* R3-9 0.95% (58/6088)
Figure 5. Genealogy of E. coli cell lineages propagating Sup35 NM in an infectious prion conformation.
(A) The fate of Sup35 NM in four experimental lineages (L1E-L4E) established from a starter culture of cells containing Sup35 NM and Newi is shown. Clones that maintain or lose the Sup35 NM prion are indicated by black or orange lines,
69 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. LiE and L3E retain SDS-stable Sup35 NM aggregates for the duration of the experiment (Figure 4A, Supplemental Figure 2B). 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 (Supplemental Figure 2A, Supplemental Figure 2C; 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 (Lic-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 (Supplemental Figure 3). Cells from four aggregate-negative Lic-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 Newi 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 (Supplemental Figure 4), but these were not quantified (see Results).
70 A
B W eak Weak [PS/'] [PS/1]
[psi] nP/. psI[t/
04 0 tp
[PSI,] transformants [PSI,] transformants
Supplemental Figure 4. Bacterial cell extracts containing propagated, infectious Sup35 NM aggregates yield both strong and weak [PSI+] yeast transformants.
The phenotypes of five representative strong [PSI+] (A) and weak [PSI+] (B) strains obtained by transforming S. cerevisiae [psi-] cells with E. coli cell extracts containing propagated, SDS-stable Sup35 NM-aggregates on 1/4 YPD agar before (left) and after (right) passage on YPD agar supplemented with 3 mM GuHCl. For the purposes of comparison, untransformed [psi-], weak [PSI+], and strong [PSI+] yeast strains are shown.
71 001 Sup35, Cj Up SuSp35,u35B . New1,i -4Nes,
B WT ST R1 ST R1 P N * PN* * *
*m Sa *' *. * . a-His ::
110 kDa a-CIpB . - .. --- 80 kDa a-Sup35 * * a-GFPD - - a-RpoA ------C AcIpB pSC1 01 TS-NEW1-c/pB ST R1 ST R1 P N ****P N
-His 6x: : . .. .
110 kDa & -+ FL a-CIpB . 80 kDa a-Sup35
a-GFP a-RpoA
D ST R1 I I r Sup3 Newi CIpB % PSI+ transformants % S transformants + + + 1.68% 67/3984 .18% 49/4156) S + + 0.00% (0/5944) 0.00% (0/5324) T AcIpB pSC101 s-NEW1-cIpB + + + 1.40% (76/5440) 0.00% (0/5276)
Figure 6. Sup35 NM prion propagation in E. coli requires ClpB.
(A) Cartoon representation of how Sup35 NM can convert to its prion form in the presence of Newt and ClpB but cannot propagate in the prion conformation after cells have been cured of Newl- and ClpB-encoding DNA. Sup35 NM and Newt (black) are depicted as fusions to mCherry (red) and mGFP (green), respectively. ClpB is depicted as a purple hexamer. (B) SDS-stable Sup35 NM aggregates are detected in 5 of 20 Round 1 (R1) wild-type (WT) clones derived from a starter culture (ST) of wild-type cells containing Sup35 NM and Newt as assessed by filter retention analysis. In total, 17 of 60 R1 wild-type clones are aggregate-positive
72 (Supplemental Figure 6A). Starter cultures of cells containing Sup35 NM and Newi and cells containing Sup35 NM alone serve as positive (P) and negative (N) controls, respectively. In all 20 R1 wild-type clones shown, full-length (FL) ClpB is detectable, Sup35 NM fusion protein levels are comparable, and Newi fusion protein is not
detectable as assessed by Western blot analysis. The ca-His 6 x and u-Sup35 antibodies recognize the Sup35 NM-mCherry-His6x fusion protein, the cc-GFP antibody recognizes the Newl-mGFP fusion protein, the u-ClpB antibody recognizes the E. coli ClpB chaperone, and the u-RpoA antibody recognizes the c subunit of E. coli RNA polymerase. Cells from four aggregate-positive R1 wild-type clones visualized by fluorescence microscopy (Supplemental Figure 6C) are indicated by asterisks. (C) SDS-stable Sup35 NM aggregates are not detectable in R1 AcipB clones derived from a starter culture of AcIpB cells containing Sup35 NM, Newi, and ectopically produced ClpB as assessed by filter retention analysis. In total, 0 of 60 R1 AcipB clones are aggregate-positive (Supplemental Figure 6B). In all 20 R1 AclpB clones shown, Sup35 NM fusion protein levels are comparable, and neither ClpB nor Newi fusion protein is detectable as assessed by Western blot analysis. Cells from four aggregate-negative R1 wild-type clones visualized by fluorescence microscopy (Supplemental Figure 6D) are indicated by asterisks. (D) Extract prepared from cells lacking ClpB is not infectious when transformed into S. cerevisiae [psi-] cells. Starter cultures of wild-type cells transformed with pBR322- NM and pSC10Ts-NEW1 as well as A clpB cells transformed with pBR322-NM and pSCi0Ts-NEW1-clpB contain infectious SDS-stable Sup35 NM aggregates capable of converting [psi-] yeast cells to [PSI+]. Wild-type starter culture cells containing Sup35 NM alone lack detectable infectivity. An aggregate-positive R1 wild-type clone retains infectious Sup35 NM aggregates. In contrast, an aggregate-negative R1 AcIpB clone lacks detectable infectivity. 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 samples containing soluble Sup35 NM are statistically significant (p < 0.0001).
73 m"I
4 / B Sup35- A mCherry New1 -mGFP merqe CIpB + + + Sup35 + + + New1 + - + WT
a-Hise ------.------
AcIpB pSC1O1 TS-NEW1-dCpB
a-GFP
110 kDa a-CIpB 80 kDa
a-Sup35 Elm
a-GFP a-RpoA
Supplemental Figure 5. AcipB cells containing Newt and ectopically produced ClpB can convert Sup35 NM to its prion form.
(A) SDS-stable Sup35 NM aggregates are detected in wild-type (WT) cells producing SDS-stable Newt aggregates as assessed by filter retention analysis. SDS-stable Sup35 NM aggregates are also detected in AcipB cells containing SDS-stable Newt aggregates and ectopically produced ClpB. The a-His6 x antibody detects the Sup35 NM-mCherry-His6x fusion protein, and the a-GFP antibody detects the Newl-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 Newt fusion protein are comparable in the presence and absence of Newt and ectopically produced ClpB as assessed by Western blot analysis. The a-ClpB antibody recognizes the E. coli ClpB chaperone, the a-Sup35 antibody recognizes the Sup35 NM fusion protein, and the a-RpoA antibody recognizes the a subunit of E. coli RNA polymerase. (B) Fluorescence images of representative wild-type cells containing Sup35 NM and Newt and AclpB cells containing Sup35 NM, Newt, and ectopically produced ClpB. The mCherry channel, GFP channel, and merged images are shown.
74 A WT B Ac/pB psc101TS-NEW1-c/pB ST R1 ST R1 P N P N
*. . . . e* .0e:0 . S II f! -: f : : *. . . . W :0:e ci-His6 - -9 ct-His6 X -4 letl
-4- i : -* - *~ f I~
C Ri -6 R1-11 D R1-1 R1-2
R1-14 R1-20 R1-3 R1-4
Supplemental Figure 6. Propagation of the Sup35 NM prion in E. coli requires ClpB.
(A) SDS-stable Sup35 NM aggregates are detected in 12 of 40 Round 1 (Ri) wild- type (WT) clones derived from a starter culture (ST) of wild-type cells containing Sup35 NM and Newt as assessed by filter retention analysis. In total, 17 of 60 R1 wild-type clones are aggregate-positive (Figure 6B). Starter cultures of cells containing Sup35 NM and Newt and cells containing Sup35 NM alone serve as positive (P) and negative (N) controls, respectively. The a-His6x antibody detects the Sup35 NM-mCherry-His6x fusion protein. (B) In contrast, 0 of 40 R1 AclpB clones derived from a starter culture of AclpB cells transformed with pBR322-NM and pSC101TS-NEW1-clpB contain detectable SDS-stable Sup35 NM aggregates. In total, 0 of 60 R1 AclpB clones are aggregate-positive (Figure 6C). Cells from four aggregate-negative R1 AclpB clones visualized by fluorescence microscopy are indicated by asterisks. The observed difference in the number of aggregate-positive clones of wild-type versus AclpB cells is statistically significant (p < 0.0001 as determined by Fisher's Exact Test). (C) Fluorescence images of representative cells corresponding to the four aggregate-positive R1 wild-type clones indicated by asterisks in (Figure 6B). Notably, wild-type clone R1-14 exhibits twisted ring structures. (D) Fluorescence images of representative cells corresponding to the four aggregate-negative R1 AclpB clones indicated by asterisks in (Figure 6C).
75 A ST R1
[aTC]: .0. WT CIpB : Og II I 004 ATP CIpB E279A/ E678A ADP a-His Cip B Y653A 6X
*. . .
DnaK*)O Cip 3 E432A
no CIpB
B CIpB WT E279A/ CIpB CIpB no CIpB E678A Y653A E432A CIpB ST R1 ST R1 ST R1 ST R1 ST R1
[aTC]: a-CIpB M . J. .. A. ..
a-Sup35
a-GFP a-RpoA VIC1
Figure 7. Propagation of SDS-stable Sup35 NM aggregates in E. coli requires ClpB disaggregase activity.
(A) SDS-stable Sup35 NM aggregates are detected in starter cultures (ST) of AclpB cells containing pBR322-NM, pSClO1Ts-NEW1-clpB, and one of four aTc-inducible chromosomal cIpB alleles. Wild-type (WT) ClpB is depicted as a purple hexamer. ClpB E279A/E678A is unable to hydrolyze ATP, ClpB Y653A is pore-deficient, and ClpB E432A is unable to collaborate with DnaK. Propagated Sup35 NM aggregates are detected in scraped cell suspensions as a function of increasing aTc concentration only for Round 1 (R1) clones producing wild-type ClpB. At no aTc concentration are Sup35 NM aggregates detected in scraped cell suspensions of R1 clones producing ClpB disaggregase mutants or in R1 clones lacking ClpB. Lanes cropped from the same immunoblot are indicated by hash marks. The a-His6x antibody recognize the Sup35 NM-mCherry-His6x fusion protein. (B) Wild-type and mutant ClpB levels along with Sup35 NM fusion protein levels are comparable in R1 clones grown on solid medium supplemented with 50 ng/ml aTc as assessed by
76 Western blot analysis. The c-Sup35 antibody recognize the Sup35 NM-mCherry- His6x fusion protein, the u-GFP antibody recognizes the Newl-mGFP fusion protein, the a-ClpB antibody recognizes the E. coli ClpB chaperone, and the u-RpoA antibody recognizes the cx subunit of E. coli RNA polymerase.
77 -A
Carb + Cam + IPTG + aTC
Carb Cam
Supplemental Figure 8. All Round 1 clones producing wild-type CIpB are cured of pSC1O1TS-NEW1-clpB.
All Round 1 (RI) clones derived from a starter culture of AclpB cells containing pBR322- NM, pSC1O1Ts-NEW]-clpB, and chromosomal aTc-inducible wild-type clpB lose pSC1O1Ts-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-NEW]-clpB confers Cam resistance.
78 Strains Alias Genotype Source and Comments (Escherichia coi) DH5aZ1 AY75 attB(k)::Iac 7-PN25- Lutz and Bujard, 1997; SpecH tetR::aadA endA 1 hsdR17(rK-mK+) ginV44 thi- 1 recA 1 gyrA96 re/A1 A(lacIZYA-argF) 169 deoR attB(p80)::/acZAM15
DH5aZ1 Ac/pB AY153 DH5aZ1 AcIpB::kan This study; SpecH and KanH
PIR2 F' Ptac-tetR AY290 Alac 169 rpoS(am) robA 1 This study; TetH creC510 hsdR514 endA recA 1 uidA(AM/u)::pir F' Ptac-tetR-/acZYAt::tetRA
DH5otZ1 Ac/pB AY295 DH5tZ1 AcIpB::FRT This study
DH5oZ1 Ac/pB AY321 DH5LZ1 Ac/pB::FRT This study; SpecH and KanH PLteto-rc/pB attB(HK022)::PLteto-rc/pB- tL3kan
DH5oZ1 Ac/pB AY322 DH5tZ1 Ac/pB::FRT This study; SpecH and KanH PLtetO-r c/pB Y653A attB(HK022)::PLteto-rc/pB Y653A-tL3 ::kan
DH5oZ1 Ac/pB AY323 DH5aZ1 AcIpB::FRT This study; SpecH and KanH PUetO-r C/PB attB(HK022)::PLteto-r c/pB E279A/E678A E279A/E678A-tL 3::kan
DH5xZ1 Ac/pB AY324 DH5aZ1 AcIpB::FRT This study; SpecH and KanH PLtetO-r C/PB E432A attB(HK022)::PLeto-rc/pB E432A-tL3::kan
DH5aZ1 Ac/pB PtetO.I AY325 DH5aZ1 Ac/pB::FRT This study; SpecH and KanH attB(HK022)::PUeto-rtL3::kan
Strains Alias Genotype Source and Comments (Saccharomyces cerevisiae) YJW187 [pin-] [psi] SG775 74D-694 MATa adel- Garrity et al., 2010; obtained by serial 14(UGA) his3 leu2 trp1 passage on YPD agar supplemented with 3 ura3 [pin'] [psi] mM GuHCI; phenotypically [pin'] [psi] YJW187 [PSI] SG862 74D-694 MATa adel- Garrity et al., 2010; phenotypically 14(UGA) his3 Ieu2 trpl strong [PSI] ura3 [PSI] YJW187 [PS[] SG863 74D-694 MATa adel- Garrity et al., 2010; phenotypically 14(UGA) his3 leu2 trp1 weak [PSI'] ura3 [PSf] Plasmids Alias Features Source and Comments pBR322-SUP35 NM pAY45 bla Ptac-SUP35 NM- This study; produces Sup35 NM
mCherry-his6 -tT 7 pMB1 ori (Sup35 residues 1-253) fused to
79 mCherry2 and a hexahistidine tag under the control of the tac promoter; AmpR pSC101 is pAY50 cat Ptac-tL3 repA " pSC101 This study; derived from pMAK700 on (Hamilton et al., 1989); CamR pSC101 '-NEW1 pAY48 cat Ptac-NEW1-mGFP-tL 3 This study; produces residues 50- repATSpSC101 ori 100 of Newi fused to mGFPmut3 under the control of the tac promoter; CamR pSC1O1 '-NEW1-c/pB pAY71 cat Ptac-NEW1-mGFP-tL 3- This study; produces residues 50- PeIP-clpB repATspSC101 100 of Newi fused to mGFPmut 3 on under the control of the tac promoter; produces CIpB under the control of its native promoter; CamR 1 pSC101 Is_-recA pAY69 cat Ptac-recA-tL 3 repA * This study; produces RecA under pSC101 on the control of the tac promoter; CamR pAH70-Pue0o0 pAY152 kan PLteto-4L3 attP(HK022) This study; derived from pAH70 yR6K on (Haldimann and Wanner, 2001); harbors the PLttO-I promoter(Lutz and Bujard, 1997); KanR pAH70-PLtetorc/pB pAY154 kan PLtetO-rC/PB-tL3 This study; produces wild-type attP(HK022) yR6K on CIpB under the control of the PLtetO- , promoter; Kan R pAH70-PLtetO-rc/pB pAY155 kan PLtetO-rc/pB Y653A-tL 3 This study; produces ClpB Y653A Y653A attP(HK022) yR6K on under the control of the PLtetoi promoter; Kan R pAH70-PLteto-rc/pB pAY156 kan PLtetO-Ic/pB This study; produces CIpB E279A/E678A E279A/E678A-tL 3 E279A/E678A under the control of attP(HK022) yR6K on the PLtetO-I promoter; KanR pAH70-PLteto-rc/pB pAY157 kan PLteto-rc/pB E432A-tL3 This study; produces CIpB E432A E432A attP(HK022) yR6K on under the control of the PLteto-i promoter; Kan R pAH69 N/A bla XPR- intHK022 Xcl857 Haldimann and Wanner, 2001; repA's R101 on produced HK022 Int under the control of the KPR promoter; Am pR pCP20 N/A bla cat XPR-f/p XcI857 Cherepanov and Wackernagel, repA Ts p15A on 1995; produced Flp recombinase under the control of the XPR promoter; AmpR and CamR pRS316 N/A URA3 bla CEN6 pMB1 ori Sikorski and Hieter, 1989; URA3 shuttle vector
Supplemental Table 1. Strains and plasmids used in this study.
80 81 APPENDIX A
A transcription-based reporter for prion propagation in E. coli
Specific author contributions:
I devised and performed all experiments presented in this appendix.
82 RESULTS AND DISCUSSION
The data presented in Chapter Two suggest that ClpB levels are elevated specifically in E. coli clones containing SDS-stable Sup35 NM prion aggregates (see
Figure 6B of Chapter Two). Taking advantage of this observation, we engineered a
PCIpB-acZ reporter (present on an F' episome) to determine whether or not increased transcription from the c/pB promoter can serve as a reliable indicator of the presence or absence of a prion in E. coli cells.
Starter cultures of cells containing F' PcpB-lacZ, pBR322-NM (encoding the
IPTG-inducible synthesis of NM-mYFP-His6x fusion protein), and either pSC1O1Ts.
NEW1 (encoding the IPTG-inducible synthesis of New1-mCFP-HA3x fusion protein; experimental sample) or pSC1OiTS (empty vector; control sample) were grown overnight to allow for the formation of the NM prion in the experimental sample
(see Figure 2 of Chapter Two for further details). The cells were plated on X-gal- containing indicator plates and grown at the non-permissive temperature to cure the cells of pSC1O1TS-NEW1 or pSC1OiTS. Dark blue Round 1 (Ri) colonies were observed at a frequency of up to -10% for the experimental sample cells. In contrast, all R1 colonies derived from control sample cells appeared pale blue
(Figure 1A). Critically, all dark blue colonies consisted of prion-containing cells whereas all pale blue colonies consisted of prion-free cells as assessed by filter retention analysis (Figure 1B).
Upon restreaking at the permissive temperature, dark blue R1 clones yielded a mixture of dark blue and white (apparently lacZ-) Round 2 (R2) progeny clones
(data not shown). An additional round of restreaking revealed that dark blue R2
83 clones yielded exclusively dark blue Round 3 (R3) clones whereas white R2 clones yielded exclusively white R3 clones (Figure 1C, sectors 1 and 2). Although we do not yet understand the appearance of white clones derived from experimental sample cells specifically in Round 2 (selection for the F' episome was maintained throughout experiments), we speculate that the presence of the NM prion may promote episomal rearrangements at the non-permissive temperature. Support for this hypothesis comes from the observation that all pale blue R1 clones derived from control sample cells yielded exclusively pale blue R2 and R3 progeny clones
(Figure IC, sectors 3, 4 and 5). Dark blue and white R3 clones that were derived from dark blue R1 colonies contained the NM prion, whereas pale blue R3 clones that were derived from pale blue R1 colonies lacked the NM prion as assessed by fluorescence microscopy (Figure 1D, R3-1, R3-2, and R3-4).
As established in Chapter Two, Sup35 NM prion propagation in E. coli requires the disaggregase activity of the ClpB chaperone. We sought to recapitulate this CIpB-dependence in the context of our transcription-based reporter. To do so, we engineered AclpB cells harboring an anhydrotetracycline (aTc)-inducible copy of cIpB on the chromosome. Experimental and control starter cultures of these ClpB- depletable cells were grown overnight in the presence of aTc and plated at the non- permissive temperature on X-gal- and aTc-containing indicator plates. Dark blue R1 colonies were observed only among colonies derived from the experimental sample.
As before, these dark blue R1 clones yielded a mixture of dark blue and white R2 progeny clones when restreaked on aTc-containing medium (data not shown).
However, dark blue R2 clones restreaked on medium lacking aTc yielded pale blue
84 R3 progeny clones (Figure 1C, sectors 6, 7, and 8) that had been cured of the NM
prion as assessed by fluorescence microscopy (Figure ID, R3-7).
Transcription in bacteria is performed by a multisubunit RNA polymerase
(RNAP). To initiate promoter-specific transcription, RNAP must associate with one of several sigma factors. In particular, E. coli contains a primary sigma factor known
70 as (5 and six alternative sigma factors, including the heat shock sigma factor ( 3 2 .
clpB transcription is controlled by a &3 2-dependent promoter and is thus upregulated during heat shock (Nonaka et al., 2006). As the activity and stability of
Cy32 are negatively regulated by DnaK (Nagai et al., 1994; Rodriguez et al., 2008), which we previously implicated in Sup35 NM prion propagation (see Figure 7A of
Chapter Two), we hypothesize that the presence of prion aggregates results in the titration of DnaK away from &32, leading to upregulation of Q32-dependent genes including cIpB (Figure 1E).
While it is unlikely that the Pc1pBI-acZ reporter discriminates between amyloid and non-amyloid protein aggregates, it nevertheless holds great potential in facilitating the discovery of prion-like phenomena. Transient production of a prion protein at high levels can facilitate the establishment of its corresponding prion form, which self-propagates even when the prion protein is subsequently produced at lower levels (Wickner, 1994). Accordingly, we are now situated to screen IPTG- inducible plasmid libraries encoding bacterial proteins for prion-like entities by (i) transiently inducing protein overproduction in library-containing E. coli cells at high
[IPTG], (ii) plating cells at low [IPTG] on appropriate X-gal and aTc-containing indicator plates, and (iii) identifying clones manifesting signs of a persistent fever
85 (i.e., dark blue colonies exhibiting a heat shock response under low [IPTG] conditions) that can be cured by CIpB-depletion as assessed by (iv) restreaking on medium lacking aTc. More generally, our ability to now reliably distinguish between prion-containing and prion-lacking E. coli clones will facilitate experimental strategies aimed at identifying environmental conditions and bacterially-encoded factors that influence the stability of prion propagation.
86 REFERENCES
Nagai H, Yuzawa H, Kanemori M, Yura T. 1994. A distinct segment of the 532 polypeptide is involved in DnaK-mediated negative control of the heat shock response in Escherichia coli. Proc Natl Acad Sci USA 91:10280-10284.
Nonaka G, Blankschien M, Herman C, Gross CA, Rhodius VA. 2006. Regulon and promoter analysis of the E. coli heat-shock factor, Q 32, reveals a multifaceted cellular response to heat stress. Genes Dev 20:1776-1789.
Rodriguez F, Arsene-Ploetze F, Rist W, Rudiger S, Schneider-Mergener 1, et al. 2008. Molecular basis for regulation of the heat shock transcription factor &32 by the DnaK and DnaJ chaperones. Mol Cell 32:347-358.
Wickner, RB. 1994. [URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae. Science 264:566-569.
87 FIGURES
3 Ric R E A R1 E C
ST P N ae e e B *
I 91.1..*i i*I'U ..ai i ..i I F* T410 a I ** m I 4 9'. 9 ? ' ' ' 9 ' ' D RA- 1 R3-2
am a mmi mmt am a a 6' 0 4 9 6 9 9 0 i
ct-His 6
a a E ii, ma ma i i
E
(&2 -
RNAP FcipB clpB
lacZ -35 -10
88 Figure 1. Transcription from the cipB promoter reports on Sup35 NM prion propagation in E. coli.
(A) Dark blue clones containing an F' Pc1pB-lacZ reporter are detected among Round 1 (R1) descendants of experimental (E) starter cultures of cells containing NM and Newi whereas all R1 descendants of control (C) starter cultures of cells containing NM alone are pale blue (i.e., 0 out of -1,000 R1c colonies are dark blue). Cells were plated and grown at the non-permissive temperature (37'C) on medium containing carbenicillin (100 [tg/ml), X-gal (40 tg/ml), 500 [tM TPEG, and 10 [M IPTG. (B) All 8 dark blue RiE clones tested contain SDS-stable NM aggregates whereas all 50 pale blue RiE clones tested lack NM aggregates as assessed by filter retention analysis. All clones were patched onto medium containing chloramphenicol (25 tg/ml) to confirm loss of the Newi-encoding plasmid. Starter cultures (ST) of cells containing NM and Newi and cells containing NM alone serve as positive (P) and negative (N) controls, respectively. For each sample, undiluted lysate and three two-fold dilutions are shown. The o-His6x antibody recognizes the Sup35 NM-mYFP-His6x fusion protein. (C) Dark blue RiE clones yield a mixture of dark blue and white Round 2 (R2) clones, which yield homogenous dark blue (sector 1) and white (sector 2) Round 3 (R3) progeny clones. Pale blue R1c clones yield pale blue R2 and R3 progeny clones (sectors 3, 4, and 5). Dark blue RiE clones that depend on aTc for ClpB production yield a mixture of dark blue and white R2 clones in the presence of aTc (100 ng/ml). Dark blue R2 clones yield pale blue R3 clones (sectors 6, 7, and 8) grown on medium lacking aTc. (D) Fluorescence images of representative cells corresponding to R3 clones identified by sector number in (C). The cells from sectors 1 and 2 contain fluorescent foci characteristic of prion-containing cells, whereas the cells from sectors 4 and 7 exhibit diffuse fluorescence characteristic of cells containing soluble NM fusion protein. (E) A working model of the transcription-based reporter for prion propagation in E. coli. In cells containing the Sup35 NM prion (red), ClpB (purple) and DnaKJE (gray) are titrated away from 32. As &2 is negatively regulated by DnaK, the presence of the prion indirectly activates transcription from the cipB promoter.
89 90 APPENDIX B
CIpB is the only non-essential chaperone or protease required for prion propagation in E. coli
Specific authorcontributions:
I devised and performed all experiments presented in this appendix.
91 RESULTS AND DISCUSSION
As discussed in Chapter One, multiple chaperone proteins play critical and complex roles in promoting prion heritability in S. cerevisiae (Liebman and Chernoff,
2012). However, the specific effects of yeast chaperones and - based on data presented in Chapter Two - bacterial chaperones on prion propagation remain controversial.
On one hand, in vitro experiments performed with purified components suggested that ClpB is unable to fragment prion amyloid aggregates even in the presence of bacterial Hsp70 (DnaK), Hsp40 (DnaJ), and the nucleotide exchange factor GrpE (DeSantis et al., 2012). On the other hand, in vivo studies indicated that
ClpB could support [PSI+] propagation in yeast provided that DnaK and GrpE were co-produced (Tipton et al., 2008; Reidy et al., 2012). Intriguingly, the ability of ClpB to propagate [PSI+] was strictly dependent on the presence of the yeast Hsp40- family protein Sisi (Reidy et al., 2012). E. coli contains three Hsp40 paralogs (DnaJ,
DjlA, and CbpA), and sequence alignment suggests that CbpA is unique in sharing homology with Sisi over its entire length, raising the possibility that CbpA rather than DnaJ is required for ClpB-dependent Sup35 NM prion propagation in bacteria.
Taken together, these preliminary considerations led us to determine
whether or not protein remodeling factors other than the ClpB disaggregase are
required for the propagation of a prion in E. coli cells. As studies performed in yeast suggest links between ubiquitin-mediated proteolysis and prion behavior (Allen et
al., 2007; Kabani et al., 2014; Yang et al., 2014), we included protein degradation
factors in our analysis as well. To perform this analysis, we systematically deleted
92 most non-essential chaperones and proteases in E. coli (we note that while dnaJ is non-essential, deletion of dnaJ in DH5uZI - the strain we use to monitor prion propagation in our E. coli system [Lutz and Bujard, 1997; Yuan et al., 2014] - renders cells too sick for analysis). Deletion strains were transformed with pBR322-NM (encoding the IPTG-inducible synthesis of NM-mCherry-His6x fusion protein) and pSC101TS-NEW1 (encoding the IPTG-inducible Newl-mGFP fusion protein). Transformants were grown overnight to allow for the formation of the
Sup35 NM prion and subsequently plated at the non-permissive temperature to cure the cells of pSC101Ts-NEW1. We prepared cell extracts from scraped Round 1
(R1) colonies (see Figure 7A and Materials and Methods of Chapter Two) and tested samples for the presence or absence of SDS-stable Sup35 NM aggregates (Figure 1).
Our results establish that ClpB is likely the only non-essential chaperone or protease required for prion propagation in E. coli cells.
Although our data confirm that ClpB is the principal mediator of Sup35 NM prion propagation in E. coli, we remain intrigued by the possibility that protein remodeling or degradation factors may alter the frequency and stability of aggregate-positive R1 clones and their progeny. With the development of the transcription-based reporter described in Appendix A, we are now situated to quantitatively assess prion propagation in our deletion strain collection.
93 REFERENCES
Allen KD, Chernova TA, Tennant EP, Wilkinson KD, Chernoff YO. 2007. Effects of ubiquitin system alterations on the formation and loss of a yeast prion. J Biol Chem 282:3004-3013.
DeSantis ME, Leung EH, Sweeny EA, Jackrel ME, Cushman-Nick M, et al. 2012. Operational plasticity enables Hsp104 to disaggregate diverse amyloid and nonamyloid clients. Cell 151:778-793.
Kabani M, Redeker V, Melki R. 2014. A role for the proteasome in the turnover of Sup35p and in [PSI+] prion propagation. Mol Microbiol 92:507-528.
Liebman SW, Chernoff YO. 2012. Prions in yeast. Genetics 191:1041-1072.
Lutz R, Bujard H. 1997. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-12 regulatory elements. Nucleic Acids Res 25:1203-1210.
Reidy M, Miot M, Masison DC. 2012. Prokaryotic chaperones support yeast prions and thermotolerance and define disaggregation machinery interactions. Genetics 192:185-193.
Stansfield I, Jones KM, Kushnirov VV, Dagkesamanskaya AR, Poznyakovski Al, et al. 1995. The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae. EMBO] 14:4365-4373.
Tipton, KA, Verges, KJ, Weissman JS. 2008. In vivo monitoring of the prion replication cycle reveals a critical role for Sisi in delivering substrates to Hsp104. Mol Cell 32:584-591.
Yang Z, Stone DE, Liebman SW. 2014. Prion-promoted phosphorylation of heterologous amyloid is coupled with ubiquitin-proteasome system inhibition and toxicity. Mol Microbiol 93:1043-1056.
Yuan AH, Garrity SJ, Nako E, Hochschild A. 2014. Prion propagation can occur in a prokaryote and requires the ClpB chaperone. eLife 4:eO2949.
94 ______I
FIGURES
t
a-His6x
- - a-GFP
a-Hissx
a a a 1 S S a-GFP
a-Hisex
a-GFP
Figure 1. ClpB is the only non-essential chaperone or protease required for Sup35 NM prion propagation in E coli.
Genes encoding 15 non-essential protein remodeling factors (purple) and 11 non- essential protein degradation factors (red) were systematically deleted in strain DH5aZ1 by P1 transduction (see Materials and Methods of Chapter Two). Each deletion strain was transformed with plasmids encoding NM and Newi. Transformants were grown overnight to allow for the formation of the NM prion and plated at the non-permissive temperature (37*C) on medium containing carbenicillin (100 sg/ml) and 10 [M IPTG. For each strain, 10 Round 1 (R1) clones were patched onto medium containing chloramphenicol (25 [Lg/ml) to confirm loss of the Newi-encoding plasmid before all remaining R1 clones (-1000 colonies) were gently scraped off of plates. Filter retention analysis of cell extracts prepared from scraped cell samples indicates that ClpB is likely the only protein remodeling/degradation factor required for Sup35 NM prion propagation in E coli. Wild-type R1 clones derived from starter cultures of cells containing NM and Newi
95 and cells containing NM alone serve as positive (P) and negative (N) controls, respectively. For each sample, undiluted lysate and three two-fold dilutions are shown. The a-His 6x antibody recognizes the Sup35 NM-mCherry-His6x fusion protein, and the ca-GFP antibody recognizes the Newl-mGFP fusion protein.
96 97 CHAPTER THREE
Evidence of a bacterial prion
Specific authorcontributions:
I devised and performed all experiments and computational analyses presented in this chapter. Alex Lancaster (Whitehead Institute for Biomedical Research) generously shared code for the HMM-based algorithm used to mine all bacterial genomes for candidate prion proteins.
98 ABSTRACT
Prions are self-propagating protein aggregates. First described in mammals as the causative agent of the fatal transmissible spongiform encephalopathies, prions have also been uncovered in yeast and other fungi, where they function as non-Mendelian genetic elements. We previously demonstrated that the bacterium
Escherichiacoli can support both the formation and chaperone-dependent propagation of a fungal prion, but whether or not bacterial prion proteins exist in nature remained an open question. Here we provide preliminary evidence that transcription termination factor Rho of Clostridium botulinum may be a bacterial prion-like protein. Our findings suggest that prions exist in the bacterial domain of life and raise the possibility that the phenomenon of protein-based heredity emerged early in evolutionary history.
99 INTRODUCTION
Prions are infectious protein aggregates that are characteristically transmissible (Prusiner, 1982). A prion protein typically exists in one of two states, either a native soluble form or an aggregated amyloid form. Amyloids are highly- ordered fibrillar structures characterized by a cross-P spine and are associated with many non-transmissible neuropathologies like Alzheimer's, Parkinson's, and
Huntington's diseases (Diaz-Espinoza and Soto, 2012; Chiti and Dobson, 2006).
Despite intensive investigation, the sequence determinants of amyloidogenesis remain poorly understood, and those features that distinguish non-infectious amyloids from self-propagating prions remain mysterious.
First described in the context of scrapie, the term prion was coined by
Prusiner in reference to the nuclease- and UV-resistant particle - subsequently identified as the prion form of the PrP protein - responsible for scrapie, a disease of sheep and goats (Prusiner, 1982; Prusiner et al., 1984). Scrapie belongs to a class of inevitably fatal neurodegenerative diseases known as the transmissible spongiform encephalopathies (TSEs), which afflict animals and humans (Aguzzi, 2008). Prions have also been discovered in fungi, where they act as protein-based units of heredity that regulate a spectrum of biological processes including translation termination, nitrogen catabolism, heterokaryon incompatibility, sterol biosynthesis, and multicellular growth (Cox, 1965; True et al., 2004; Aigle and Lacroute, 1975;
Wickner, 1994; Coustou et al., 1997; Suzuki et al., 2012; Holmes et al., 2013).
To date, prion proteins and prion-like phenomena have been uncovered exclusively in eukaryotes (Aguzzi et al, 2008; Liebman and Chernoff, 2012; Si et al.,
100 2010; Hou et al., 2011; Cai et al., 2014). Here we provide evidence that transcription termination factor Rho of Clostridium botulinum E3 str. Alaska E43 exhibits characteristics of bonafide prion proteins. Our findings suggest that prion-like mechanisms may operate in the bacterial domain of life and that protein-based heredity represents a ubiquitous, ancient mode of epigenetic regulation.
101 RESULTS
A bioinformatics screen identifies bacterial prion candidates
We conducted a large-scale implementation of a hidden Markov model
(HMM)-based algorithm developed in the Lindquist lab to mine all sequenced bacterial genomes for proteins containing candidate prion domains (cPrDs) that resemble a training set of 28 glutamine/asparagine-rich yeast prion proteins
(Alberti et al., 2009; Lancaster et al., 2014). Our query returned ~ 2,000 high- scoring proteins containing a candidate bacterial prion domain of at least 60 amino acids (the cPrD "core" polypeptide) in length. The majority of computationally predicted bacterial prion proteins were annotated as membrane-associated and/or extracellular proteins, with the greatest number of proteins belonging to the
Firmicutes.
To generate a manageable list of putative cPrDs for experimental validation, we focused our attention on cytoplasmic proteins that were represented in more than one bacterial phlyum and have an unambiguous ortholog present in E. coil.
Taking into consideration the transferable modularity of yeast PrDs (Masison et al.,
1997; Li and Lindquist, 2000), we chose to pursue three proteins of known function that exhibit well-defined domain organization: translation initiation factor IF-2
(three orthologs representing two phyla), transcription termination factor Rho (five
orthologs representing four phyla), and single-stranded DNA binding protein SSB
(eight orthologs representing four phyla) (Figure 1, Supplemental Figure 1).
102 A subset of cPrDs form extracellular amyloid aggregates at the E. coli cell surface
For each candidate bacterial prion protein, we synthesized E. coli codon- optimized DNA encoding a previously defined structural domain encompassing the predicted cPrD. As a first test for prion-like properties, we asked whether or not the cPrD of each of the 16 candidate bacterial prion proteins is capable of forming extracellular amyloid at the surface of E. coli cells. To do so, we took advantage of a previously described assay (Sivanathan and Hochschild, 2012) that exploits the natural ability of E. coli cells to elaborate amyloid structures known as curli fibers at the cell surface (Chapman et al., 2002).
Curli fibers are composed of the CsgA and CsgB proteins, which are first secreted across the E. coli inner membrane via the SecYEG translocon and subsequently directed out of the cell via the outer membrane CsgG pore protein, where they assemble as surface-attached amyloid fibrils (Chapman et al., 2002;
Hammer et al., 2007; Blanco et al., 2011). CsgA and CsgB share a bipartite N- terminal signal sequence dictating secretion specificity that can be transferred to heterologous proteins (Robinson et al., 2006; Sivanathan and Hochschild, 2012).
Furthermore, secretion via the curli export pathway results in the production of extracellular amyloid fibrils specifically for heterologous proteins (such as yeast prion proteins) that have an inherent amyloid-forming propensity (Sivanathan and
Hochschild, 2012). Appropriation of the curli secretion system thus serves as the foundation for a cell-based assay of protein amyloidogenicity known as curli- dependent amyloid generator (C-DAG) (Sivanathan and Hochschild, 2013).
103 The use of C-DAG as a test of protein amyloidogenicity requires that cells be plated on solid medium, presumably to ensure a sufficiently high local concentration of the secreted protein to permit fibril assembly. Two convenient methods can be employed to detect extracellular amyloid material associated with individual colonies. The first of these methods depends on the use of the amyloid-binding dye
Congo red (CR). Like natural curli-producing E. coli cells, cells engineered to secrete an amyloidogenic polypeptide via the curli secretion pathway typically form red colonies when grown on solid medium containing CR (Sivanathan and Hochschild,
2012). Although the red colony phenotype is not in and of itself sufficiently selective (i.e., false positives are relatively frequent), CR-bound amyloid aggregates characteristically exhibit so-called "apple-green" birefringence when observed by bright-field microscopy between crossed polarizers (Howie et al., 2008; Teng and
Eisenberg, 2009). The second method depends on another characteristic of amyloid aggregates, namely, their resistance to denaturation in the presence of SDS. Thus, scraped cell suspensions can be examined for the presence of SDS-stable aggregates by means of the filter retention assay described in Chapter Two.
Of the 16 cPrDs (and 7 of the corresponding 60 amino acid core sequences; see legend to Figure 2A) tested by C-DAG, 12 yielded SDS-stable aggregates as assessed by filter retention analysis (Figure 2A, see Materials and Methods). Among these 12 aggregate-positive samples, 8 exhibited "apple-green" birefringence
(Figure 2B, Figure 2C).
104 C. botulinum Rho forms SDS-stable aggregates in the E. coli cytoplasm
To monitor the intracellular behavior of cPrDs , we fused each cPrD that tested aggregate-positive in the C-DAG assay (as well as the previously defined structural domain encompassing the corresponding cPrD) to a monomeric fluorescent protein (mYFP bearing a C-terminal hexahistidine epitope tag) and overproduced the resulting chimeras from arabinose-inducible plasmids in the E. coli cytoplasm. Among all of the YFP fusion proteins tested, we detected SDS-stable aggregates only for the 68 residue cPrD and the 183 residue N-terminal domain
(NTD) of Clostridium botulinum E3 str. Alaska E43 transcription termination factor
Rho (Cb Rho) (Figure 3A, Figure 3B). SDS-stability was assessed by two complementary methods: filter retention analysis and semidenaturing detergent- agarose gel electrophoresis (SDD-AGE), which permits the visualization of SDS- stable amyloid polymers as high molecular weight smears (Halfmann and Lindquist,
2008). Furthermore, fluorescence microscopy revealed that the Cb Rho NTD fusion protein forms non-diffuse intracellular structures similar to those formed by yeast
PrDs produced in E. coli cells (Figure 3C) (Garrity et al., 2010; Yuan et al., 2014).
Next, we synthesized E. coli codon-optimized DNA encoding full-length Cb
Rho bearing a C-terminal hexahistidine epitope tag and produced the protein in the
E. coli cytoplasm. SDD-AGE analysis revealed that full-length Cb Rho can form high molecular weight SDS-stable aggregates (Figure 4A). As discussed in Chapter Two, the ability of Sup35 to convert to its prion form in yeast depends on the presence of another prion known as a [PSI+] inducibility (PIN) factor (Derkatch et al., 1997).
Remarkably, like that of Sup35, the ability of full-length Cb Rho to form SDS-stable
105 aggregates in E. coli cells was greatly facilitated by the co-production of the PrD of the yeast PIN factor Newi (Figure 4A, Figure 4B) (Derkatch et al., 2001; Osherovich and Weissman, 2001; Garrity et al., 2010).
The C. botulinum Rho cPrD behaves as an Hsp104-dependent prion in yeast
We next asked whether or not the Cb Rho cPrD and NTD exhibit prion-like behavior in a yeast Sup35-based assay (Alberti et al., 2010). The well-characterized prion protein Sup35 is an essential translation termination factor, the non-prion and prion forms of which are known as [psi-] and [PSI+], respectively (Stansfield et al.,
1995; Paushkin et al., 1997; Cox, 1965; Wickner, 1994). In [psi-] cells, Sup35 is soluble and mediates translation termination at stop codons, whereas in [PSI+] cells,
Sup35 is aggregated, resulting in stop codon readthrough (True et al., 2004; Baudin-
Baillieu et al., 2014). Sup35 is composed of an N-terminal PrD (N), a highly-charged middle region (M), and a C-terminal domain specifying translation release activity
(C) (Ter-Avanesyan et al., 1993). Together, Sup35 NM constitutes a prionogenic moiety that is separable from Sup35 C (Li and Lindquist, 2000). The modular organization of Sup35 and other yeast prion proteins permits the characterization of Sup35 C fusion proteins, which can support [PSI+] -like states (Sondheimer and
Lindquist, 2000; Osherovich and Weissman, 2001). [PSI+] and [PSI+]-like phenotypes can manifest themselves in an adel-14 genetic background. As the adel-14 allele encodes a premature stop codon in the adenine biosynthesis gene
ADE1, [psi-] cells are defective for adenine synthesis, grow poorly on adenine-
deficient medium, and form red colonies due to the accumulation of
phosphoribosylaminoimidazole (Chernoff et al., 2002; Wickner, 2012). In contrast,
106 [PSI+] and [PSI+]-like cells contain a complete adenine biosynthesis pathway, grow
well on adenine-deficient medium, and form white colonies.
Taking advantage of these features of [PSI+], we constructed adel-14
sup35::KanMX4 yeast strains constitutively producing native Sup35 or one of the
two Cb Rho domains fused to Sup35C as the sole source of cellular translation
release activity (see Materials and Methods). As transient overproduction of a prion
protein can increase the frequency of switching to the prion state (Wickner, 1994),
we transformed these strains with galactose-inducible plasmids encoding Sup35
NM, the Cb Rho PrD, or the Cb Rho NTD fused to EGFP. Transformants were grown
in the presence or absence of galactose, visualized by fluorescence microscopy, and
plated on medium lacking adenine. Cells grown in the presence of galactose
exhibited non-diffuse intracellular structures similar to those reported previously in
studies of yeast prions (Figure 5A) (Alberti et al., 2009; Arslan et al., 2015).
Furthermore, transient overproduction of Sup35 NM and the Cb Rho PrD, but not
the Cb Rho NTD, increased the frequency of switching to [PSI+] and [PSI+]-like prion
states as inferred from the number and color of colonies grown on adenine-deficient medium (Figure 5B). As has been observed for other yeast prion protein PrD-Sup35
C chimeras, the Cb Rho NTD-Sup35 C fusion protein is sufficiently aggregation- prone to confer a [PSI+]-like state without the need for transient Cb Rho NTD-EGFP fusion protein overproduction (Alberti et al., 2009).
As bonafide prion states are self-perpetuating (Wickner, 2012), we asked whether or not the Cb Rho cPrD-Sup35 C [PSI+]-like state established upon galactose-induced overproduction of Cb Rho cPrD-EGFP fusion protein can be stably
107 propagated under non-inducing conditions. To do so, we tested the ability of Ade+ cells to remain in the [PSI+]-like state over multiple generations. Both [PSI+] and
[PSI+]-like white colony phenotypes of cells producing native Sup35 or Cb Rho cPrD-
Sup35 C, respectively, were maintained over several rounds of restreaking (Figure
5C).
The vast majority of yeast prions, including [PSI+], require Hsp104 for their stable propagation (Liebman and Chernoff, 2012). Yeast cells can therefore be cured of prions upon transient inhibition of Hsp104 activity (Ferreira et al., 2001;
Park et al., 2012). To determine whether or not the heritable [PSI+]-like state conferred by Cb Rho cPrD-Sup35 C is Hsp104-dependent, we passaged white, Ade+ cells on medium containing the Hsp104-inhibitor guanidine hydrochloride
(Kummer et al., 2013). An additional round of restreaking yielded exclusively red colonies, indicating that, as is true for [PSI+], the [PSI+]-like state specified by Cb Rho cPrD-Sup35 C can be cured (Figure 5C). These results suggest that the Cb Rho cPrD-
Sup35 C chimera behaves as an Hsp104-dependent prion in yeast.
108 DISCUSSION
Prion proteins and prion-like phenomena have been exclusively documented
in eukaryotes. Whether or not prions exist in the other two domains of life has
remained an open question. We previously demonstrated that E. coli cells can
support the formation (Garrity et al., 2010) and chaperone-dependent propagation
(Yuan et al., 2014) of a model yeast prion. Here we provide, to our knowledge, the
first evidence suggesting that a bacterial protein - the transcription termination factor Rho of Clostridium botulinum E3 str. Alaska E43 - possesses the ability to convert to a prion-like state. Our findings suggest that protein-based heredity is not a unique feature of eukaryotic cells and raise the possibility that prion-like mechanisms underlie epigenetic modes of heredity that are as ancient as they are universal.
Transcription termination in bacteria occurs at intrinsic or Rho-dependent terminators (Boudvillain et al., 2010). Transcription termination factor Rho functions as a hexameric (trimer of dimers) helicase that couples ATP binding and hydrolysis to translocation along nascent RNA associated with the RNA polymerase
(RNAP) elongation complex (Skordalakes and Berger, 2003; Gocheva et al., 2015).
Rho association with the RNAP elongation complex results in transcription termination via disruption of the RNA-DNA hybrid residing in the RNAP active site
(Kim and Patel, 2001; Epshtein et al., 2010).
The Rho NTD consists of an N-terminal basic helix bundle (NHB) and a primary RNA binding domain (PBD) (Bogden et al., 1999). The Cb Rho PrD is located within a so-called N-terminal insertion domain (NID), which lies between
109 the NHB and PBD (D'Heygere et al., 2013). The length and sequence composition of the NID are highly variable among bacteria and are particularly heterogeneous in the Firmicutes, Bacteroidetes, and Actinobacteria (D'Heygere et al., 2013). Helicase activity resides in the highly-conserved Rho CTD, which contains Walker A and B
ATP hydrolysis motifs, a secondary RNA binding site (SBS), and oligomerization determinants (Skordalakes and Berger, 2003).
E. coli Rho is a well-characterized, essential gene product (notably, rho is not essential in many bacteria and is in fact absent in some species [D'Heygere et al.,
2013]). The essentiality of Rho in E. coli can be attributed to its ability to prevent the lethal accumulation of R-loops (triple-stranded nucleic acid structures composed of nascent RNA and template DNA), which compromise genome integrity
(Washburn and Gottesman, 2011; Leela et al., 2013). Furthermore, it has been
suggested that Rho activity is required in E. coli to prevent the expression of cryptic prophage elements, many of which encode bacteriocidal toxins (Cardinale et al.,
2008); nonetheless, rho remains essential even in a strain lacking all cryptic
prophage elements (Cardinale et al., 2008; Leela et al., 2013).
Although we have yet to rigorously document prion-like behavior of Cb Rho
in E. coil, we are now well situated to do so. Specifically, our fortuitous finding that
the aggregation propensity of full-length Cb Rho is dramatically augmented in the
presence of Newi provides us with an excellent strategy to distinguish between the
de novo formation of Cb Rho aggregates in the presence of Newi and their
subsequent propagation as prion-like particles in the absence of Newi. This
experimental strategy is analogous to the one successfully implemented in Chapter
110 Two (see Figure 2 of Chapter Two) and will be greatly facilitated by the transcription-based reporter described in Appendix A.
Although we have thus far relied on E. coli as a model system for studying Cb
Rho, we have initiated experiments in a genetically tractable, non-select agent relative of C. botulinum, Clostridium difficile. In collaboration with the Shen lab
(University of Vermont), we will attempt to recapitulate Cb Rho aggregation behavior in Arho C. difficile cells in order to perform transcription profiling analyses and thereby potentially uncover evidence of transcription read-through, which may have phenotypic consequences in C. difficile that can be extrapolated to C. botulinum physiology.
111 MATERIALS AND METHODS
Computational identification of candidate bacterial prion protein
All sequenced bacterial, archaeal, and phage genomes available as of January,
2013 were downloaded and analyzed by custom Java and R scripts (Alberti et al.,
2009; Lancaster et al., 2014). Data visualization was performed with the PLAAC application available at http://plaac.wi.mit.edu (Lancaster et al., 2014).
Strains, plasmids, and cell growth
Bacteria experiments to assess intracellular behaviors of cPrDs were performed with E. coli strains BW27785 (Khlebnikov et al., 2001) and SG811
(Garrity et al., 2010) grown in LB (Miller) medium. SG811 is a derivative of
BW27785 harboring an IPTG-inducible vector encoding the Newl-mCFP-HA3x fusion protein integrated at attB(X). Plasmids directing the arabinose-inducible synthesis of cPrD-mYFP-His6x fusion proteins or Cb Rho FL were constructed using
E. coli codon-optimized DNA synthesized as gBlocks@ Gene Fragments (IDT).
Plasmids were transformed into appropriate cells, which were grown overnight in
LB broth containing carbenicillin (Carb, 100 tg/ml) and glucose (0.2%). Overnight cultures were diluted 1:100 in LB broth containing Carb (100 g/ml) and grown for
1 h prior to induction with arabinose (0.2% final concentration) and, in the case of
SG811, 1 mM IPTG. Induced cultures were grown at 37'C for 6 h, normalized to 8 ml of an OD600 of 1.0, and pelleted by centrifugation. Cell pellets were processed and lysed as previously described (see Material and Methods of Chapter Two).
112 Yeast experiments were performed with S. cerevisiae [PIN+] [psi-] strain
YRS100 grown in yeast extract peptone dextrose (YPD) medium or synthetic
defined (SD) medium. Sup35-based assays for prion behavior were performed as
previously described (Alberti et al., 2009). Plasmids pAY354 (encoding native
Sup35), pAY389 (encoding Cb Rho PrD-Sup35 C), pAY387 (encoding Cb Rho NTD-
Sup35 C) were transformed into cells and established as the sole source of cellular
translation release activity by 5-FOA-mediated plasmid shuffling. Prion induction
by transient protein overproduction was achieved by transforming galactose-
inducible plasmids pAY353 (encoding Sup35 NM-EGFP), pAY388 (encoding Cb Rho
PrD-EGFP), and pAY383 (encoding Cb Rho NTD-EGFP) into cells of corresponding
"shuffled" strains. Transformants were grown for 30 hours in SD-Trp medium
containing either 1% galactose (Gal) + 1% raffinose (Raf) (inducing condition) or
2% Raf (non-inducing condition) and spotted on solid medium lacking adenine and containing 2% glucose. Stable propagation and guanidine hydrochloride-mediated curing of resultant [PSI+] and [PSI+]-like states were monitored as previously described (Yuan et al., 2014).
C-DAG assay
The C-DAG assay was performed as previously described (Sivanathan and
Hochschild, 2013). In brief, AcsgBAC MC4100 cells constitutively overproducing
CsgG were transformed with arabinose-inducible export vectors encoding cPrDs and core sequences bearing an N-terminal CsgA signal sequence and a C-terminal hexahistidine epitope tag. Transformants were grown in LB broth containing
113 carbenicillin (Carb, 100 lg/ml) and chloramphenicol (Cam, 25 tg/ml) and spotted
onto LB agar containing arabinose (0.2%), Congo red (CR, 5 ptg/ml), Carb (100
tg/ml), and Cam (25 [Ig/ml). Cells were grown at room temperature for 4-7 days and either scraped off of plates for filter retention analysis or arrayed on glass slides
for detection of birefringence.
Filter retention assays
Filter retention assays were performed as previously described (Yuan et al.,
2014).
Congo red birefringence
Cells grown on CR-containing agar were transferred to glass slides with
pipette tips. Samples were viewed by bright-field microscopy between crossed
polarizers on a Nikon 80i microscope equipped with a Plan Apo 100x 1.4 NA
objective. Images were taken through the microscope eyepiece with an iPhone
camera.
Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE)
SDD-AGE was performed as previously described (Halfmann and Lindquist,
2008; Alberti et al., 2010).
Immunoblotting
Cellulose acetate membranes (used in filter retention assays) and Hybond-C
Extra nitrocellulose membranes (used in SDD-AGE assays and Western blot
analysis) were blocked for 30 min in PBS containing 3% (w/v) milk. Membranes
114 were probed with anti-His6x (His-2, Roche, 1:10,000), anti-GFP (Roche, 1:10,000), or anti-RpoA (NeoClone, 1:10,000) primary antibody. Membranes were washed and probed with anti-mouse IgG (Cell Signaling, 1:10,000) secondary antibody. Proteins were detected with ECL Plus Western blot detection reagents (GE Healthcare) and a
ChemiDock XRS+ imaging system (Bio-Rad).
Fluorescence microscopy
Fluorescence microscopy was performed as previously described (Yuan et al., 2014). All fluorescence images of bacteria were obtained from 5 ms exposures whereas those of yeast were obtained from 500 ms exposures.
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120 100014
FIGURES
Rho (Clostridium botulinum 53 str. Alaska 343)
-background - PrD.like
Ci- ~ MAP Vit
0 100 200 300 400 500
- FoldIndex -- -PLAAC - -4*PAPA
1 MTKQVYEGMTLVELKDIAKKLEIKNISKYKKKELIDILKSSPNTVKEGVILREKISSKVRNDNNSVISSTTTNNNRTNFNNNTVNNSGFNNNTVNNSG 101 FNNNNSNFNNNSNNNSFNNSNRFNNSNNLNSVNNNNSDNKIDAKNNENPALKQKLNNNISESNSAGILILENNNFGFLRCKNYLTSSEDIYVS 201 PSQIRRFNLRTGDEVQGKVREAKEGEKFKALLFVEQVNGENPEKAIGRKYFETLTPIYPKBRLRLETSDEKDLSSRLMDIICPIGKGQRGIIVAPPKAGK 301 TTLLKKVAQNISTYPDIKLIVLLIDERPEEVTDMKRSIKGEVIYSTFDEEPQNHAKVSRIVLERAERMVEQGKDVVILMDSITRLSRAYNLTITPTGRT 401 LSGGLDPGALIMPKKFFGAARNIEGGSLTILATALVDTGSRDDMIFEEFKGTGNMBVHLDRKLQNRRIFPAIDIYKSGTRKEDLIFSKEEKDVAYTIR 501 KVMYRDGNIEDVTENLIGMLSKTSDNKRFINVFKKVEFEKRKQNNK
Figure 1. Computational prediction of a bacterial prion candidate.
Graphical representation of data obtained from a hidden Markov model (HMM)- based prion prediction algorithm for transcription termination factor Rho of Clostridium botulinum E3 str. Alaska E43 (Cb Rho). The amino acid sequence of Cb Rho is shown in the bottom panel, with residues constituting the cPrD predicted by the prion-like amino acid composition (PLAAC) algorithm (Alberti et al., 2009; Lancaster et al., 2014) or the prion aggregation prediction algorithm (PAPA) (Toombs et al., 2012) highlighted in red or underlined in green, respectively. The top panel shows the probability of each residue belonging to the HMM state "cPrD" (red) or "background" (black). The tracks "MAP" and "Vit" illustrate the Maximum a Posteriori and Viterbi parses of the protein into these two states. Amino acid composition is represented in color, with Q and N residues shown in blue. The middle panel shows averages over a 60 amino acid sliding window of predicted intrinsic protein disorder (FoldIndex, gray) (Prilusky et al., 2005) and cPrD amino acid propensities as assessed by PLAAC (red) or PAPA (green) along with the PAPA cutoff score for predicted prion propensity (dotted green line) (Toombs et al., 2010).
121 -1
IF-2 (Enterococcus faecalis V583) r-4 - - background T - PrD.like
C - MAP Vit
0 100 200 300 400 500 600 700 80 0
------I- F l i - FoldIndex -PLAAC 77s Ii - -4*PAPA 0
1 MGKKRIYELAKEMNKASKDVVDKAHQLGMDVKNHMGAISSEQETKLRQAFGGGSTVNTOSKATNNQKQQTTQNKPANKKPHNNKPGEQRNNQNRPNNQST 101 NGQQRNNNNQNRHGQSNTQNRSNQTNTNNQNRNTQNNNGSTTNQNRTSQNNNGGNNQNRGGQNRNNNFGGGQNRNNRNNFNNQNRNRFNKKGKKGKHQQE 201 SAKPAVPARKFRELPDVLEYTEGMNVADIAKKIHREPAEIIKKLFMMGVVNQNQALDKDTIELLAVDYGMEPQEKVQVDIADIDKFFEPEAVVEENLTT 301 RPPVVTIMGHVDHGKTTLLDTLRHSRVTSGEAGGITQHIGAYLDIDGKPITFLDTPGHAAFTSMRARGASITDITILVVAADDGVMPTIEAINHAKAA 401 KVPIIVAVNKIDKPGANPDHVKQELSEHELIPEEWGGDTIFVNISAKFNQNIDELLENILLIAEVEDLKADPTOKAIGTVIEARLDKGKGPVATLLVQQG 501 TLHVGDPIVVGNTYGRVRVMTNDMGRRDKEAGPATPVEITGLNDVPQAGDRFVVFEDEKTARQAGEERAKRALLEQRSASSRVTLDNLFESLKEGELKEV 601 NIIVKADVQGSAEAVSASLQKIDVEGVRVKIVHAAVGAINESDVTLAAASNAIIIGFNVRPTPQAKQQAEQEEVDIRLHRIIYKALEEIETAMKGLLDPE 701 FEEKITGQMTVRELYKVSKVGTIAGCYVTEGFIRRDSGVRVIRDGIVIYEGKLASLKRFKDDVKEVKLGFECGAMIENFNDLRVDDAIEGFIKEEIKQ
IF-2 (Lactobacillus plantarum WCFS1)
- background - PrD.like
I] MAP Vit
0 100 2001 300 400 500 600 700 800 -1
- FoldIndex - -PLAAC - -4*PAPA 77~~
I -
1 MGKKRIYELAKELNVPSKQLIAQAQQLGFSVKNHMSTLGDSEARQLQGTTTVTSPKSQPAPAKTSRATQAVAKNVTRTANQQQSNSRHQQSGDYLNNNRN 101 NNQNNSQSRNSNGNRSNNGGNRTNSNNGNRSANASRSNSARNNGGSNTNRSNNNNNNNRSNNNNRSNTSNNRTNSTGNRTNNGGQNRNNNRTTTNNNNNN 201 SNRSNGSNNSSRNGSGRFGGSLNSNNNGGGRYRGGNNNNRRRNNRNNKSRNNKNQRIRQVNNKPAPVRKDKPLPETLVYTVGMNAQDLGKLLHREPAEII 301 KKLFMLGVAVNQNQSLDKDTIEILATDYGINAQEKVQVDVTDLDKFFDDEVNNTDNLAPRAPVVTVMGHVDHGKTTLLDQLRHTHVTEGEAGGITQAIGA 401 YQVKHDGKVITFLDTPGHAAFTEMRARGADITDITVLVVAADDGVMPQTIEAINHAKAAKVPIIVAVNKIDKPGANPNHVMEQLTEYGLIPEDWGGDTIF 501 VQISAKFGKNLDELLDMILLQSEVLELTANPKQNAAGSVLEASLDRGKGSTATLLVQQGTHVGDPIVVGNTYGKVRTMTNDHGRIKEAVPSTPIEITG 601 LNDVPDAGDRFVVFDDEKTARDAGZQRAKQALMEERKQTAHVTLDNLFDSMKQGELKEVDVIIKADVQGSVEALAGSLRKIDVEGVRVNIIHTAVGAINE 701 SDVALAEASNAIIIGFNVRPTPQAKAQADTDDVDIRLHQVIYNAIDEIESAMKGMLEPTYKEEITGQAEIREIYKVSKIGTIGGGMVTDGVIHRDSGVRV 801 IRDGVVIYDGKLASLRRFKDDVKEVKQGFELGLRIEDYNDIKVNDVIEAYVMKEVPVE
122 A
Rho (Zantho.onas oryzae pv. orysa. PZ099A)
- background - PrD.like
o -l -41MAP vit
0 100 200 300 400 500 600
Ii I FF 1 | t - FoldIndex ---TT ~ ~~I -PLAAC -4*PAPA
0 -
MSDHPSSDTGSSVDAPVEKRVRKPRVSKTAVTDEDGGAQQPNLPLPASPAPEAPRAPQAPSRAADPAPTQTSRDGAHAGPAPSANHGGDTHGESREGGQS 101 QQQRFNQAQQQQNQNQSQSQGQGQQQAQAQAQGQNQNAGNQQGQGGQGQGQNQQGQNQQGNRRDRFRNRRDRGPRDRFGNEGGGMPSSDGSNPFIARP 201 HPAVPEGFPVYSLSDLKRKPAQKLLDIADQLNIOEGVARARKQDVIPALLKVLTRHGZGVAADGVLEILPDGFGFLRAAEASYLAGPDDTYISPSQIRRF 301 NLRTGDHLSGRIRFPKDGERYFALSIVDTINGEPLEASKGKVLFENLTPLFPRRRFRLERGDGSTEDITGRILDLM4APQGKGQRALIVSPPKAGKTMMMQ 401 QVATAITTNHPEVHMIVLLIDERPEEVTE14QRTVRGEVISSTFDEPAARHVQVAENVIERAKRLVEHKXDVVILLDSITRLARAYNNVVPSSGKVLTGGV 501 DANALHRPKRFFGAARNVENGGSLTVIATALVETGSKMDEVIYEEFKGTGNSEVHLNRRITEKRVYPAIDINRSGTRREDLLIEPELLQKIWILRKLLHP 601 MDEIAAMEFLLDKMKTTKSNDEFFSSMKR
Rho (Fluvicola taffensis DSM 16823)
- background I - PrD.like I] MAP vit
0 100 200 300 400 500 600 I IU
- FoldIndex I, ~ry~,AW - -PLAAC - -4*PAPA 0 -
1 MNNKDLEGXLLPELREIAKSLGVKKVESFXKNELIDQIQANSTSTPEVEKVAAKPKAKKESEPKADRMSPTASANAQVPTADLFTNEAPKTRSVDEKPAV 101 KTEKRKRIPADRAPAPKVADAQNEVSKQETTEAKPTPAAASATETTSKGQPAPIRPFRLVNKNRPNPNQNNNANRNNPNQNKPKTNNPGENQTNS 201 TPGEAQGNSNQGNQNQPNNQNQERQNNNQRNPNQQNQNNQQRNPNONPNGNQNPNONRPQNQNSQQQEEKIDZRAYNLVGIVTANGVLEVIQDGYGrLRS 301 SDYNYLPSPDDVYVSQSQIKLFGLKTGDTVRGTIRPPREGZKFFPLVKVDSINGRDPSYIRDRVPFBYLTPLrPDRFKLTGRIZESMSTRVXDLFAPIG 401 XGQRGMIVAQPKTGKTMLLKDVANAIAANHPZVYLIVLLIDBRPRBVTDMARSVKAEVVASTFDBPADRHVKVANIVLEKAKRKVECGBDVVILLDSITR 501 LARAYNTVSPASGKVLSGGVDANALBKPKRFFGAARXIBNGGSLSILATALTBTGSKKDZVXFEEFKGTGNMZLQLDRKISNRRIYPAIDIISSGTRRED 601 LLVGKDVLQRIWLLRXFIADMNPVAMFVKGHMENTRN__FLVSMNS
123 -A
SSB (Campylobacter curvus 525.92)
- background - PrD.like
MAP I]- IIII[IIIL Vit I IIIIIIIIII
0 100 200
- FoldIndex - -PLAAC -- 4*PAPA - - - h O
1 MFNRVVLVGNLTRDIELRYTTSGSAIGNSGIAVTRKFSVNGEKREETCFIDITFFGKQAEIANQYLNKGSKLLVEGRLKFDQWTDNNGQNRSKHSISVEN 101 MEMLGGGNSQQGGYNQNNQGGGYSQGGYNQSSYNNSGYNAQSGGYNQNSQQRPQNQQSAKKSEEYYDDKVPDINVDADKYESDDTIPF
SSB (Corynebacterium glutanicum R)
- background - PrD.like
C - K I]MAP silI II I I I I Vit 7 1, mM II IIII11 1 111 1 1 o
0 100 200
- FoldIndex - -PLAAC - -4*PAPA 0 ~1 v w' I w -r
1 MAIGDTNITVVGNIVADPELRFTPSGAAVANFRIASTPRSFNRQTNQWEDGEALFLTVNVWRQAAENVAESLSKGMRVIVTGRLKQRSYETREGEKRSVF 101 EVEADEVGPSLTFAKADVQRTPRGGNSGGNYGGGNQGGGFGGNQGNQGGFSNQNSGGFGGNQGNQQQSNQGGFGGNQNQSQGNNFNQGGFGGGSPQAAP 201 DNDPWNSAPPAGSGGFGGADDEPPF
Supplemental Figure 1. Computational prediction of bacterial prion candidates.
Graphical representations of data obtained from a hidden Markov model-based prion prediction algorithm for a subset of candidate bacterial prion proteins characterized in this study (see legend to Figure 1 for further details).
124 A I B sA 1 2 3 4 5 6 A
'w. eoOe B
D
a-His 6
Figure 2. A subset of cPrDs form extracellular amyloid aggregates when exported to the E. coli cell surface via C-DAG.
(A) Filter retention analysis of scraped suspensions of cells engineered to export the indicated cPrD or core polypeptide to the E. coli cell surface via the curli secretion pathway. Sup35 NM serves as a positive control. For each sample, undiluted lysate and three two-fold dilutions are shown. The a-His6x antibody recognizes exported proteins, which contain a C-terminal hexahistidine epitope tag. Ef: Enterococcus faecalis V583, Pc: Pediococcus clausseniiATCC BAA-344, Lp: Lactobacillusplantarum WCFS1, Ft: Fluvicola taffensis DSM 16823, Sg: Sphaerochaetaglobus str. Buddy, Xa: Xanthomonas axonopodispv. citrumelo F1, Xo: Xanthomonas oryzae pv. oryzae PX099A, Cb E3: Clostridium botulinum E3 str. Alaska E43, Psa: Pseudoalteromonassp. SM9913, Sp: Streptococcus pneumoniae ST556, Cc: Campylobactercurvus 525.92, Ch: Campylobacter hominis ATCC BAA-381, Cb Ba4: Clostridium botulinum Ba4 str. 657, Cg: Corynebacteriumglutamicum R, Lm: Listeria monocytogenes HCC23, Pp: Propionibacteriumpropionicum F0230a. (B) Cells transformed with one of 24 C-DAG export vectors were spotted on medium containing arabinose (0.2%) and Congo red (5 [g/ml) and grown at room temperature for 72 h. The order of spots follow the order of proteins shown in (A), with the EfIF-2 core corresponding to quadrant Al and the Pp SSB cPrD corresponding to quadrant D5. Cells producing highly or moderately birefringent material are highlighted in solid or dashed green boxes, respectively. (C) Bright-field image of Cb E3 Rho PrD-secreting cells viewed between crossed polarizers.
125 A C Sup35 NM \ P? ( Cb Rho NTD alone
SDS-stable aggregates
a-His 6
monomers Sup35 NM Newi merge
B E
Cb Rho . Boil - - + +
a-His 6x ii!
Figure 3. The Cb Rho cPrD and NTD form SDS-stable aggregates in the E. coli cytoplasm.
(A) SDD-AGE analysis of cell extracts containing the indicated protein domain fused to mYFP-His6x. Sup35 NM (produced in the presence of Newl) and the Lp IF-2 cPrD serve as positive and negative controls, respectively. (B) Filter retention analysis of cell extracts containing the indicated Cb Rho domain fused to mYFP-His6x. For each sample, undiluted lysate and three two-fold dilutions are shown. Cb Rho cPrD and NTD aggregates are no longer detected once boiled. The a-His6x antibody recognizes the NM/cPrD/NTD-mYFP-His6x fusion proteins. (C) Fluorescence images of representative cells producing the Cb Rho NTD or Sup35 NM fused to mYFP-His6x (top row) and cells co-producing Sup35 NM-mYFP-His6x and Newl-mCFP-HA3x fusion proteins (bottom row). For cells containing Sup35 NM and Newt, the YFP channel, CFP channel, and merged images are shown.
126 A B Cb Rho FL Cb Rho FL + + + 1 - Newi - + + Newi - + Boil - - +
SDS-stable a-HiS6X aggregates a-His - Imonomers-GFP
C Cb Rho FL
Newi - +
SDS-stable a-Hisex - 60 koa a-GFP aggregates 40 kDa -a-GFPVI z-GF ~j 30 kDaF kt
monomers a-RpoA - 40 kDa
Figure 4. Full-length Cb Rho forms SDS-stable aggregates in the E coll cytoplasm.
(A) SDD-AGE analysis of cell extracts containing full-length (FL) Cb Rho bearing a C- terminal hexahistidine epitope tag in the absence or presence of Newi. The a-His6x antibody recognizes full-length Cb Rho-His6x protein, and the a-GFP antibody recognizes Newl-mCFP-HA3x fusion protein. (B) Filter retention analysis of the same cell extracts analyzed in (A). For each sample, undiluted lysate and three two- fold dilutions are shown. Neither full-length Cb Rho nor Newi aggregates are detected once boiled. (C) Intracellular full-length Cb Rho-His6x protein levels are comparable in the presence and absence of Newi as assessed by Western blot analyses of the same cell extracts used in (A) and (B). The a-RpoA antibody recognizes the a subunit of E coli RNA polymerase.
127 A B
Sup35 NM Cb Rho cPrD Cb Rho NTD Sup35 NM
Gal
Cb Rho cPrD
Raf
Cb Rho NTD
C
Figure 5. The Cb Rho cPrD behaves as an Hsp104-dependent prion in yeast.
(A) Fluorescence images of YRS100-derived yeast cells containing native Sup35 or the indicated Cb Rho domain fused to Sup35 C as the sole source of cellular translation release activity and overproducing Sup35 NM, the Cb Rho cPrD, or the Cb Rho NTD fused to EGFP from galactose-inducible plasmids. For each of the three EGFP fusion proteins, two representative fluorescence images are show. (B) The phenotypes of cells grown for 30 h in liquid medium containing either 1% galactose (Gal) + 1% raffinose (Rat) (top, inducing condition) or 2% Raf (bottom, non- inducing condition) and spotted on solid medium lacking adenine and containing 2% glucose. Transient overproduction of Sup35 NM and the Cb Rho PrD increases the frequency of switching to the corresponding prion state. In the case of the Cb Rho NTD, transient protein overproduction is not required for manifestation of the prion state. (C) The phenotypes of [psi-] cells containing native Sup35 (sector 1), [psi-]-like cells containing Cb Rho cPrD-Sup35 C (sector 2), [PSI+] cells isolated following overproduction of Sup35 NM-EGFP (sectors 3 and 4), and [PSI+]-like cells isolated following overproducing of Cb Rho cPrD-EGFP (sectors 5 and 6) on 1/4 YPD medium (left). The same cells were passaged once on YPD supplemented with 3
128 mM GuHCl to inhibit Hsp104 activity and restreaked on 1/4 YPD medium to reveal [psi-] and [psi-]-like progeny (right). We do not yet fully understand why the pre- GuHCl [psi-] and [psi-]-like colonies shown in sectors 1 and 2 appear less red than their corresponding post-GuHC progeny; however, we note that pre-GuHCl cells are [PIN+] whereas post-GuHCI cells are [pin-] and suggest that this distinction may, in part, account for the apparent difference in colony color.
129 130 APPENDIX C
A cell-based screen for amyloidogenic proteins of bacterial origin
Specific author contirbutions:
I devised and performed all experiments presented in this appendix. Sitara Chapman (MIT/Wellesley College) and Shelby Yuan (Harvard College) provided assistance in performing pilot screens for amyloidogenic proteins.
131 RESULTS AND DISCUSSION
As discussed in Chapter One and Chapter Two, E. coli cells are naturally capable of elaborating amyloid fibrillar structures known as curli fibers at the cell surface (Chapman et al., 2002). Curli fibers are composed of the CsgA and CsgB proteins, which are secreted via the inner membrane SecYEG translocon and the outer membrane CsgG pore (Chapman et al., 2002; Hammer et al., 2007; Blanco et al., 2011). The secretion specificity determinants of CsgA and CsgB lie within an N- terminal bipartite signal sequence, which can be transferred to heterologous proteins (Robinson et al., 2006; Sivanathan and Hochschild, 2012). A previously described E. coli cell-based curli-dependent amyloid generator (C-DAG) assay appropriates the curli secretion pathway and enables the formation of extracellular amyloid aggregates composed of known amyloidogenic protein domains
(Sivanathan and Hochschild, 2012).
We took advantage of the C-DAG assay as a methodology for identifying amyloidogenic polypeptides of bacterial origin. To do so, we first constructed high- complexity libraries encoding in-frame protein fragments derived from gDNA of five model bacteria (Escherichia coli, Pseudomonasaeruginosa, Vibreo cholerae, Bacillus subtilis, and Caulobactercrescentus). The high-resolution crystal structures of the nonameric CsgG transport complex - in conjunction with earlier studies of CsgG- mediated protein secretion - have established approximate size thresholds for substrate export (Van Gerven et al., 2014; Goyal et al., 2014). Accordingly, gDNA fragments were size-selected (100-1,000 bp) to increase the probability of efficient export through the CsgG pore.
132 Libraries were enriched for in-frame gene fragments by passage through a
so-called "ORF-filter" selection system. In brief, the ORF-filtration strategy takes
advantage of the cis-splicing activity of the yeast VMA intein and the E. coli twin-
arginine transport (TAT) secretion pathway. pInSALecT (gift of S. Lutz), a plasmid
encoding the signal sequence of the TorA protein (a TAT substrate) fused to split
VMA and P-lactamase (Bla) (Gerth et al., 2004), was modified for the purposes of
generating gDNA libraries encoding ssTorA-VMANTD-X-VMACTD-Bla chimeras, where
X represents a random gDNA fragment. Only if X is in-frame can the complete chimeric protein be produced, resulting in the excision of VMA-X fusion protein and
the formation of ssTorA-Bla fusion protein, which confers upon cells resistance to P-
lactam antibiotics. Critically, unlike its predecessors, this intein-dependent ORF-
filter technology does not select against aggregation-prone polypeptides (Gerth et
al., 2004).
ORF-filtered gDNA fragments were recovered and transferred to an
arabinose-inducible C-DAG export vector, which encodes the N-terminal signal
sequence of CsgA and a C-terminal hexahistidine epitope tag (Sivanathan and
Hochschild, 2013). In a pilot screen, we transformed a C-DAG export library
containing in-frame E. coli gDNA fragments into AcsgBA C E. coli cells harboring a
compatible multi-copy plasmid directing the constitutive overproduction of CsgG.
In parallel, we transformed pooled, C-DAG-compatible ORF libraries derived from the ASKA E. coli gene collection (Sivanathan and Hochschild, 2012; Kitagawa et al.,
2005). Transformants were plated on selective medium containing arabinose and the amyloid-binding dye Congo red (CR).
133 While amyloid-producing clones can form bright red colonies on CR-
containing medium, we have found that CR-binding is an unreliable indicator of
extracellular amyloid. In particular, when screening gDNA fragment or ORF libraries for a red colony color phenotype on CR-containing medium, large numbers
of false positives are detected. To circumvent this problem, we relied on the fact that CR-bound amyloid aggregates uniquely exhibit "apple-green" birefringence when viewed by bright-field microscopy between crossed polarizers (Howie et al.,
2008; Teng and Eisenberg, 2009). Accordingly, following four to seven days of
growth at room temperature, bright red colonies were manually picked, arrayed on
glass slides, and tested for birefringence. We screened over -4,000 individual
bright red colonies (-2,000 colonies derived from an in-frame E. coli gDNA
fragment library and -2,000 colonies derived from an E. coli pooled ORF-library) in
this manner and identified several birefringent candidates. Subsequent filter
retention analysis of scraped cell suspensions revealed that a subset of candidates produced aggregates exhibiting resistance to SDS denaturation, another
characteristic of amyloid material (Figure 1A, Figure 1B) (Alberti et al., 2010).
The amyloid conformation represents a low energy state for most
polypeptides, and many proteins can form amyloid aggregates under extreme non-
physiological conditions in vitro (Buell et al., 2014; Teng and Eisenberg, 2009).
However, the sequence determinants of amyloidogenesis in vivo remain poorly
understood. Moreover, those features that distinguish an amyloidogenic protein
from a prion protein remain mysterious. Several research groups have developed
computational tools to predict amyloidogenic and prionogenic proteins based on
134 amino acid sequence and composition (Michelitsch and Weissman, 2000; Trovato et al., 2006; Alberti et al., 2009; Maurer-Stroh et al., 2010; Toombs et al., 2012;
Espinosa Angarica V et al., 2014).
Among these computational approaches, a structure-guided algorithm developed in the Eisenberg lab is particularly notable and emerged from the observation that hexapeptides of amyloidogenic proteins can crystallize as amyloid- like fibrils in which complementary side-chains of juxtaposed n-sheets form a so- called "steric zipper" (Nelson et al., 2005; Thompson et al., 2006). These short polypeptides exhibit high fibrillation propensities and are thus referred to as "HP segments", which can be found in virtually every protein for which a structure exists
(e.g., see Figure 1C). Critically, computational analyses indicate that the position and conformational flexibility of an HP segment in the context of its corresponding native protein dictates whether or not such a protein is prone to form amyloid aggregates (Goldschmidt et al., 2010). Our ongoing identification of bacterial amyloidogenic proteins using the C-DAG assay will facilitate future experimental assessments of HP segment-based computational predictions and their implications for amyloidogenesis and prionogenesis.
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137 FIGURES
A B Protein Residues BR SDSs KdpC 32-127 +++ R YkgC 278-370 + S SrmB 219-388 + S UvrA 602-706 ++ R FabG 125-244 + S Agn43 202-316 ++ R MUtL 221-388 ++ R SufD 269-413 ++ R 01 YmjC 1-60 +++ R
C 1 121 31 41 51 NKNVLILGAGGQIARHVINQLADKOTIKOTLFAROPAKI HKPYPTNKPOTTSGKVIQDR kcafo -20,IL
Figure 1. Identification of E. coli-encoded amyloidogenic polypeptides.
(A) Table of candidate amyloidogenic polypeptides identified by C-DAG. 8 of the 9 candidates represent protein fragments that were identified by screening a C-DAG export library enriched for in-frame E. coli gDNA. YmjC was isolated from a C-DAG- compatible ORF library derived from the ASKA E. coli gene collection. Qualitative assessments of Congo red birefringence (BR) as evaluated by bright-field microscopy between crossed polarizers and resistance (R) or sensitivity (S) to 2% SDS as assessed by filter retention analyses are shown. (B) Bright-field image of YmjC-secreting cells viewed between cross polarizers (top). The 60 amino acid YmjC protein (blue) represents the N-terminus of the 212 amino acid NmrA-family protein S1416 (wheat) of Shigellaflexneri 2a str. 2457T (Protein Data Bank ID: 3QVO) (bottom). (C) ZipperDB (http://services.mbi.ucla.edu/zipperdb) analysis of YmjC reveals HP-segments exhibiting amyloid-forming potentials beyond a Rosetta threshold of -23 kcal/mol.
138 139 Contact Information: A N D Y H. Y U A N 2 Rockwell St., Unit 1 Cambridge, MA 02139 (520) 990-6567 [email protected] E D U C A T I O N
Massachusetts Institute of Technology, Cambridge, MA Ph.D. candidate in Biology (September 2009 - August 2015) M.S. in Biology (June 2012)
Harvard University, Cambridge, MA B.A. in Biology, Magna Cum Laude (June 2008)
RESEARCH EXPERI ENCE
Laboratories of Dr. Ann Hochschild & Dr. Susan Lindquist, Department of Microbiology and Immunobiology, Harvard Medical School; Whitehead Institute for Biomedical Research (June 2012 - present) Studies: a) propagation of a prion in a prokaryote b) discovery of amyloidogenic and prion-like proteins of bacterial origin
Laboratory of Dr. Michael Laub, Department of Biology, Massachusetts Institute of Technology (June 2010 - June 2012) Studies: a) characterization of GcrA, a master regulator of transcription in Caulobacter crescentus
Laboratory of Dr. Ann Hochschild, Department of Microbiology and Molecular Genetics, Harvard Medical School (February 2005 - August 2009) Studies: a) identification and analysis of protein-protein interactions between RNA polymerase and the Escherichiacoli anti-a factor Rsd b) elucidation of mechanisms underlying transcription inhibition and co-activation mediated by the bacteriophage T4 AsiA protein
Laboratory of Dr. Giovanni Bosco, Department of Molecular and Cellular Biology, Arizona Cancer Center, University of Arizona (June 2004 - September 2004) Studies: a) characterization of endoreduplication mutants in Drosophilamelanogaster follicle cells
P U B L I C A T IO N S
Baer, D.L., Yuan, A.H. & Laub, M.T. (2015). The bacterial cell cycle regulator GcrA is a 070 co-factor driving gene expression from a subset of methylated promoters. Submitted.
Yuan, A.H., Garrity, S.J., Nako, E. & Hochschild, A. (2014). Prion propagation can occur in a prokaryote and requires the ClpB chaperone. eLife. 4:e02949.
Banta, A.B., Chumanov, R.S., Yuan, A.H., Lin, H., Campbell, E.A., Burgess, R.R. & Gourse, R.L. (2013) Identification of the key features in as required for specific recognition by Cr, an RNA polymerase holoenzyme assembly factor. Proc. Natl. Acad. Sc. USA. 110(40):15955-15960.
Yuan, A.H. & Hochschild, A. (2009b) Direct activator/co-activator interaction is essential for bacteriophage T4 middle gene expression. MoL Microbiol. 74(4):1018-1030.
140 Yuan, A.H., Nickels, B.E. & Hochschild, A. (2009a) The bacteriophage T4 AsiA protein contacts the (-flap domain of RNA polymerase. Proc. Natl. Acad. Sci. USA. 106(16):6597-6602.
Yuan, A.H., Gregory, B.D., Sharp, J.S., Donovan, K.E., Dove, S.L. & Hochschild, A. (2008) Rsd family proteins make simultaneous interactions with regions 2 and 4 of the primary a factor. Mol. Microbiol. 70(5):1136-1151.
O RAL PRESENTATION S
* 2014 Molecular Genetics of Bacteria & Phages Meeting, University of Wisconsin-Madison: "Prion propagation can occur in a prokaryote and requires the ClpB chaperone."
* 2010 Molecular Genetics of Bacteria & Phages Meeting, Cold Spring Harbor Laboratory: "The bacteriophage T4 AsiA protein is the nexus of a protein interaction network at T4 middle promoters."
* 2009 Mechanism and Regulation of Prokaryotic Transcription Conference, FASEB: "The RNA Polymerase @-flap: a target for regulation during all stages of the transcription cycle."
* 2007 Mechanism and Regulation of Prokaryotic Transcription Conference, FASEB: "Multiple interactions of RNA polymerase and the Escherichiacoli anti-a factor Rsd."
ACAD EM IC AWARDS
National Science Foundation Graduate Research Fellow (2010-present) Phi Beta Kappa of Harvard College John Harvard Scholar University of Chicago "University Scholarship" University of Arizona "Provost Scholarship"
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