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

Cell cycle progression in most organisms requires tightly regulated programs of gene expression. The transcription factors involved typically stimulate gene expression by binding specific DNA sequences in promoters and recruiting RNA polymerase. Here, we found that the essential cell cycle regulator GcrA in Caulobacter crescentus activates the transcription of target genes in a fundamentally different manner. GcrA forms a stable complex with RNA polymerase and localizes to almost all active σ 70 -dependent promoters in vivo but activates transcription primarily at promoters harboring certain DNA methylation sites. Whereas most transcription factors that contact σ 70 interact with domain 4, GcrA interfaces with domain 2, the region that binds the −10 element during strand separation. Using kinetic analyses and a reconstituted in vitro transcription assay, we demonstrated that GcrA can stabilize RNA polymerase binding and directly stimulate open complex formation to activate transcription. Guided by these studies, we identified a regulon of ∼200 genes, providing new insight into the essential functions of GcrA. Collectively, our work reveals a new mechanism for transcriptional regulation, and we discuss the potential benefits of activating transcription by promoting RNA polymerase isomerization rather than recruitment exclusively.

Bacteria use multiple sigma factors to coordinate gene expression in response to environmental perturbations. In Escherichia coli and other γ-proteobacteria, the transcription factor Crl stimulates σ Sdependent transcription during times of cellular stress by promoting the association of σ S with core RNA polymerase. The molecular basis for specific recognition of σ S by Crl, rather than the homologous and more abundant primary sigma factor σ 70 , is unknown. Here we use bacterial two-hybrid analysis in vivo and p-benzoylphenylalanine cross-linking in vitro to define the features in σ S responsible for specific recognition by Crl. We identify residues in σ S conserved domain 2 (σ S 2 ) that are necessary and sufficient to allow recognition of σ 70 conserved domain 2 by Crl, one near the promoter-melting region and the other at the position where a large nonconserved region interrupts the sequence of σ 70 . We then use luminescence resonance energy transfer to demonstrate directly that Crl promotes holoenzyme assembly using these specificity determinants on σ S . Our results explain how Crl distinguishes between sigma factors that are largely homologous and activates discrete sets of promoters even though it does not bind to promoter DNA.RNAP formation | transcription initiation | bacterial stress response | RpoS | curli fiber T ranscription initiation in bacteria requires the assembly of a sigma factor (σ) with the RNA polymerase (RNAP) catalytic core (E, composed of 2 α-subunits and one each of β, β′, and ω) to form RNAP holoenzyme (Eσ), which in turn recognizes promoter sequences (1) (reviewed in ref. 2). Multiple sigma factors compete for binding to core RNAP (reviewed in refs. 3,4), and each sigma factor controls a specific set of promoters.In Escherichia coli, which has seven sigma factors, σ 70 is the primary sigma, and σ S is important for certain stress responses and during the stationary phase of growth (5). Eσ S -dependent transcription initiation is regulated by σ S , whose concentration is itself regulated at the levels of transcription, translation, and protein stability (reviewed in ref. 6). Eσ S -dependent transcription is also activated by Crl (7), a small protein that increases expression of many stress response genes and those required for formation of amyloid curli fibers (which accounts for its name) involved in adhesion and biofilm formation (reviewed in refs. 2,6,8).The effect of Crl on σ S -dependent transcription in vivo is most pronounced during the transition into stationary phase (9). It has been proposed that Crl functions by increasing the concentration of Eσ S holoenzyme by facilitating assembly of σ S with core RNAP (10-12) because Crl's effects on transcription are greatest in vitro when the concentration of σ S is lowest, and overexpression of σ S complements a crl deletion in vivo (13). Effects of Crl have also been reported on postholoenzyme assembly steps including promoter binding (14) and open complex formation (12).Sigma factors contain several protease-resistant domains, e...

Prions are infectious, self-propagating protein aggregates that are notorious for causing devastating neurodegenerative diseases in mammals. Recent evidence supports the existence of prions in bacteria. However, the evaluation of candidate bacterial prion-forming proteins has been hampered by the lack of genetic assays for detecting their conversion to an aggregated prion conformation. Here we describe a bacteria-based genetic assay that distinguishes cells carrying a model yeast prion protein in its nonprion and prion forms. We then use this assay to investigate the prion-forming potential of single-stranded DNA-binding protein (SSB) ofCampylobacter hominis. Our findings indicate that SSB possesses a prion-forming domain that can transition between nonprion and prion conformations. Furthermore, we show that bacterial cells can propagate the prion form over 100 generations in a manner that depends on the disaggregase ClpB. The bacteria-based genetic tool we present may facilitate the investigation of prion-like phenomena in all domains of life.