Uncovering supramolecular chirality codes for the design of tunable biomaterials.

In neurodegenerative diseases, polymorphism and supramolecular assembly of ß-sheet amyloids are implicated in many different etiologies and may adopt either a left- or right-handed supramolecular chirality. Yet, the underlying principles of how sequence regulates supramolecular chirality remains unknown. Here, we characterize the sequence specificity of the central core of amyloid-ß 42 and design derivatives which enable chirality inversion at biologically relevant temperatures. We further find that C-terminal modifications can tune the energy barrier of a left-to-right chiral inversion. Leveraging this design principle, we demonstrate how temperature-triggered chiral inversion of peptides hosting therapeutic payloads modulates the dosed release of an anticancer drug. These results suggest a generalizable approach for fine-tuning supramolecular chirality that can be applied in developing treatments to regulate amyloid morphology in neurodegeneration as well as in other disease states.

The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions.

Bacterial ribonucleoprotein bodies (BR-bodies) are non-membrane-bound structures that facilitate mRNA decay by concentrating mRNA substrates with RNase E and the associated RNA degradosome machinery. However, the full complement of proteins enriched in BR-bodies has not been defined. Here, we define the protein components of BR-bodies through enrichment of the bodies followed by mass spectrometry-based proteomic analysis. We find 111 BR-body-enriched proteins showing that BR-bodies are more complex than previously assumed. We identify five BR-body-enriched proteins that undergo RNA-dependent phase separation in vitro with a complex network of condensate mixing. We observe that some RNP condensates co-assemble with preferred directionality, suggesting that RNA may be trafficked through RNP condensates in an ordered manner to facilitate mRNA processing/decay, and that some BR-body-associated proteins have the capacity to dissolve the condensate. Altogether, these results suggest that a complex network of protein-protein and protein-RNA interactions controls BR-body phase separation and RNA processing.

RNase E biomolecular condensates stimulate PNPase activity.

Bacterial Ribonucleoprotein bodies (BR-bodies) play an essential role in organizing RNA degradation via phase separation in the cytoplasm of bacteria. BR-bodies mediate multi-step mRNA decay through the concerted activity of the endoribonuclease RNase E coupled with the 3'-5' exoribonuclease Polynucleotide Phosphorylase (PNPase). In vivo, studies indicated that the loss of PNPase recruitment into BR-bodies led to a significant build-up of RNA decay intermediates in Caulobacter crescentus. However, it remained unclear whether this is due to a lack of colocalized PNPase and RNase E within BR-bodies or whether PNPase's activity is stimulated within the BR-body. We reconstituted RNase E's C-terminal domain with PNPase towards a minimal BR-body in vitro to distinguish these possibilities. We found that PNPase's catalytic activity is accelerated when colocalized within the RNase E biomolecular condensates, partly due to scaffolding and mass action effects. In contrast, disruption of the RNase E-PNPase protein-protein interaction led to a loss of PNPase recruitment into the RNase E condensates and a loss of ribonuclease rate enhancement. We also found that RNase E's unique biomolecular condensate environment tuned PNPase's substrate specificity for poly(A) over poly(U). Intriguingly, a critical PNPase reactant, phosphate, reduces RNase E phase separation both in vitro and in vivo. This regulatory feedback ensures that under limited phosphate resources, PNPase activity is enhanced by recruitment into RNase E's biomolecular condensates.

Scaffold-Scaffold Interaction Facilitates Cell Polarity Development in Caulobacter crescentus.

Cell polarity development is the prerequisite for cell differentiation and generating biodiversity. In the model bacterium Caulobacter crescentus, the polarization of the scaffold protein PopZ during the predivisional cell stage plays a central role in asymmetric cell division. However, our understanding of the spatiotemporal regulation of PopZ localization remains incomplete. In the current study, a direct interaction between PopZ and the new pole scaffold PodJ is revealed, which plays a primary role in triggering the new pole accumulation of PopZ. The coiled-coil 4-6 domain in PodJ is responsible for interacting with PopZ in vitro and promoting PopZ transition from monopolar to bipolar in vivo. Elimination of the PodJ-PopZ interaction impairs the PopZ-mediated chromosome segregation by affecting both the positioning and partitioning of the ParB-parS centromere. Further analyses of PodJ and PopZ from other bacterial species indicate this scaffold-scaffold interaction may represent a widespread strategy for spatiotemporal regulation of cell polarity in bacteria. IMPORTANCE Caulobacter crescentus is a well-established bacterial model to study asymmetric cell division for decades. During cell development, the polarization of scaffold protein PopZ from monopolar to bipolar plays a central role in C. crescentus asymmetric cell division. Nevertheless, the spatiotemporal regulation of PopZ has remained unclear. Here, we demonstrate that the new pole scaffold PodJ functions as a regulator in triggering PopZ bipolarization. The primary regulatory role of PodJ was demonstrated in parallel by comparing it with other known PopZ regulators, such as ZitP and TipN. Physical interaction between PopZ and PodJ ensures the timely accumulation of PopZ at the new cell pole and the inheritance of the polarity axis. Disruption of the PodJ-PopZ interaction impaired PopZ-mediated chromosome segregation and may lead to a decoupling of DNA replication from cell division during the cell cycle. Together, the scaffold-scaffold interaction may provide an underlying infrastructure for cell polarity development and asymmetric cell division.

Phase separation modulates the assembly and dynamics of a polarity-related scaffold-signaling hub.

Asymmetric cell division (ACD) produces morphologically and behaviorally distinct cells and is the primary way to generate cell diversity. In the model bacterium Caulobacter crescentus, the polarization of distinct scaffold-signaling hubs at the swarmer and stalked cell poles constitutes the basis of ACD. However, mechanisms involved in the formation of these hubs remain elusive. Here, we show that a swarmer-cell-pole scaffold, PodJ, forms biomolecular condensates both in vitro and in living cells via phase separation. The coiled-coil 4-6 and the intrinsically disordered regions are the primary domains that contribute to biomolecular condensate generation and signaling protein recruitment in PodJ. Moreover, a negative regulation of PodJ phase separation by the stalked-cell-pole scaffold protein SpmX is revealed. SpmX impedes PodJ cell-pole accumulation and affects its recruitment ability. Together, by modulating the assembly and dynamics of scaffold-signaling hubs, phase separation may serve as a general biophysical mechanism that underlies the regulation of ACD in bacteria and other organisms.

Repurposing Peptide Nanomaterials as Synthetic Biomolecular Condensates in Bacteria.

Peptide nanomaterials exhibit diverse applications in vitro, such as drug delivery. Here, we consider the utility of de novo peptide nanomaterials to organize biochemistry within the bacterial cytoplasm. Toward this goal, we discovered that ABC coiled-coil triblock peptides form gel-like biomolecular condensates with a csat of 10 µM in addition to their well-known hydrogel-forming capabilities. Expression of the coiled-coil triblock peptides in bacteria leads to cell pole accumulation via a nucleoid occlusion mechanism. We then provide a proof of principle that these synthetic biomolecular condensates could sequester clients at the cell pole. Finally, we demonstrate that triblock peptides and another biomolecular condensate, RNase E, phase-separate as distinct protein-rich assemblies in vitro and in vivo. These results reveal the potential of using peptide nanomaterials to divide the bacterial cytoplasm into distinct subcellular zones with future metabolic engineering and synthetic biology applications.

Protein engineering strategies to stimulate the functions of bacterial pseudokinases.

Enzymes orchestrate an array of concerted functions that often culminate in the chemical conversion of substrates into products. In the bacterial kingdom, histidine kinases autophosphorylate, then transfer that phosphate to a second protein called a response regulator. Bacterial genomes can encode large numbers of histidine kinases that provide surveillance of environmental and cytosolic stresses through signal stimulation of histidine kinase activity. Pseudokinases lack these hallmark catalytic functions but often retain binding interactions and allostery. Characterization of bacterial pseudokinases then takes a fundamentally different approach than their enzymatic counterparts. Here we discuss models for how bacterial pseudokinases can utilize protein-protein interactions and allostery to serve as crucial signaling pathway regulators. Then we describe a protein engineering strategy to interrogate these models, emphasizing how signals flow within bacterial pseudokinases. This description includes design considerations, cloning strategies, and the purification of leucine zippers fused to pseudokinases. We then describe two assays to interrogate this approach. First is a C. crescentus swarm plate assay to track motility phenotypes related to a bacterial pseudokinase. Second is an in vitro coupled-enzyme assay that can be applied to test if and how a pseudokinase regulates an active kinase. Together these approaches provide a blueprint for dissecting the mechanisms of cryptic bacterial pseudokinases.

Regulation of the activity of the bacterial histidine kinase PleC by the scaffolding protein PodJ.

Scaffolding proteins can customize the response of signaling networks to support cell development and behaviors. PleC is a bifunctional histidine kinase whose signaling activity coordinates asymmetric cell division to yield a motile swarmer cell and a stalked cell in the gram-negative bacterium Caulobacter crescentus. Past studies have shown that PleC's switch in activity from kinase to phosphatase correlates with a change in its subcellular localization pattern from diffuse to localized at the new cell pole. Here we investigated how the bacterial scaffolding protein PodJ regulates the subcellular positioning and activity of PleC. We reconstituted the PleC-PodJ signaling complex through both heterologous expressions in Escherichia coli and in vitro studies. In vitro, PodJ phase separates as a biomolecular condensate that recruits PleC and inhibits its kinase activity. We also constructed an in vivo PleC-CcaS chimeric histidine kinase reporter assay and demonstrated using this method that PodJ leverages its intrinsically disordered region to bind to PleC's PAS sensory domain and regulate PleC-CcaS signaling. Regulation of the PleC-CcaS was most robust when PodJ was concentrated at the cell poles and was dependent on the allosteric coupling between PleC-CcaS's PAS sensory domain and its downstream histidine kinase domain. In conclusion, our in vitro biochemical studies suggest that PodJ phase separation may be coupled to changes in PleC enzymatic function. We propose that this coupling of phase separation and allosteric regulation may be a generalizable phenomenon among enzymes associated with biomolecular condensates.

A Biosensor for Detection of Indole Metabolites.

Microbially produced indole metabolites serve as a diverse family of interspecies and interkingdom signaling molecules in the context of human health, crop production, and antibiotic resistance. We mined the protein database for sensors of indole metabolites and developed a biosensor for indole-3-aldehyde (I3A). Microbially produced I3A has been associated with reducing inflammation in diseases such as ulcerative colitis by stimulating the aryl hydrocarbon receptor pathway. We engineered an E. coli strain embedded with a single plasmid carrying a chimeric two-component system that detects I3A. Our I3A receptor characterization confirmed binding site residues that contribute to the sensor's I3A detection range of 0.1-10 µM. This new I3A biosensor opens the door to sensing indole metabolites produced at various host-microbe interfaces and provides new parts for synthetic biology applications.

The Future Potential of Biosensors to Investigate the Gut-Brain Axis.

The multifaceted and heterogeneous nature of depression presents challenges in pinpointing treatments. Among these contributions are the interconnections between the gut microbiome and neurological function termed the gut-brain axis. A diverse range of microbiome-produced metabolites interact with host signaling and metabolic pathways through this gut-brain axis relationship. Therefore, biosensor detection of gut metabolites offers the potential to quantify the microbiome's contributions to depression. Herein we review synthetic biology strategies to detect signals that indicate gut-brain axis dysregulation that may contribute to depression. We also highlight future challenges in developing living diagnostics of microbiome conditions influencing depression.

Synthetic Control of Signal Flow Within a Bacterial Multi-Kinase Network.

The signal processing capabilities of bacterial signaling networks offer immense potential for advanced phospho-signaling systems for synthetic biology. Emerging models suggest that complex development may require interconnections between what were once thought to be isolated signaling arrays. For example, Caulobacter crescentus achieves the feat of asymmetric division by utilizing a novel pseudokinase DivL, which senses the output of one signaling pathway to modulate a second pathway. It has been proposed that DivL reverses signal flow by exploiting conserved kinase conformational changes and protein-protein interactions. We engineered a series of DivL-based modulators to synthetically stimulate reverse signaling of the network in vivo. Stimulation of conformational changes through the DivL signal transmission helix resulted in changes to hallmark features of the network: C. crescentus motility and DivL accumulation at the cell poles. Additionally, mutations to a conserved PAS sensor transmission motif disrupted reverse signaling flow in vivo. We propose that synthetic stimulation and sensor disruption provide strategies to define signaling circuit organization principles for the rational design and validation of synthetic pathways.

Design of a Histidine Kinase FRET Sensor to Detect Complex Signal Integration within Living Bacteria.

Histidine kinases (HK) switch between conformational states that promote kinase and phosphatase activities to regulate diverse cellular processes. Past studies have shown that these functional states can display heterogeneity between cells in microbial communities and can vary at the subcellular level. Methods to track and correlate the kinase conformational state with the phenotypic response of living bacteria cells will offer new opportunities to interrogate bacterial signaling mechanisms. As a proof of principle, we incorporated both mClover3 (donor) and mRuby3 (acceptor) fluorescent proteins into the Caulobacter crescentus cell-cycle HK CckA as an in vivo fluorescence resonance energy transfer (FRET) sensor to detect these structural changes. Our engineered FRET sensor was responsive to CckA-specific input signals and detected subcellular changes in CckA signal integration that occurs as cells develop. We demonstrated the potential of using the CckA FRET sensor as an in vivo screening tool for HK inhibitors. In summary, we have developed a new HK FRET sensor design strategy that can be adopted to monitor in vivo changes for interrogation of a broad range of signaling mechanisms in living bacteria.

Phase-separated bacterial ribonucleoprotein bodies organize mRNA decay.

In bacteria, mRNA decay is controlled by megadalton scale macromolecular assemblies called, "RNA degradosomes," composed of nucleases and other RNA decay associated proteins. Recent advances in bacterial cell biology have shown that RNA degradosomes can assemble into phase-separated structures, termed bacterial ribonucleoprotein bodies (BR-bodies), with many analogous properties to eukaryotic processing bodies and stress granules. This review will highlight the functional role that BR-bodies play in the mRNA decay process through its organization into a membraneless organelle in the bacterial cytoplasm. This review will also highlight the phylogenetic distribution of BR-bodies across bacterial species, which suggests that these phase-separated structures are broadly distributed across bacteria, and in evolutionarily related mitochondria and chloroplasts. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Export and Localization > RNA Localization RNA Turnover and Surveillance > Regulation of RNA Stability.

BR-Bodies Provide Selectively Permeable Condensates that Stimulate mRNA Decay and Prevent Release of Decay Intermediates.

Biomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here, we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA sequencing (RNA-seq). We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved non-coding RNAs (ncRNAs) are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the buildup of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.

Manipulation of Bacterial Signaling Using Engineered Histidine Kinases.

Two-component systems allow bacteria to respond to changes in environmental or cytosolic conditions through autophosphorylation of a histidine kinase (HK) and subsequent transfer of the phosphate group to its downstream cognate response regulator (RR). The RR then elicits a cellular response, commonly through regulation of transcription. Engineering two-component system signaling networks provides a strategy to study bacterial signaling mechanisms related to bacterial cell survival, symbiosis, and virulence, and to develop sensory devices in synthetic biology. Here we focus on the principles for engineering the HK to identify unknown signal inputs, test signal transmission mechanisms, design small molecule sensors, and rewire two-component signaling networks.

α-Proteobacterial RNA Degradosomes Assemble Liquid-Liquid Phase-Separated RNP Bodies.

Ribonucleoprotein (RNP) granules play an important role in organizing eukaryotic mRNA metabolism via liquid-liquid phase separation (LLPS) of mRNA decay factors into membrane-less organelles in the cytoplasm. Here we show that the bacterium Caulobacter crescentus Ribonuclease (RNase) E assembles RNP LLPS condensates that we term bacterial RNP-bodies (BR-bodies), similar to eukaryotic P-bodies and stress granules. RNase E requires RNA to assemble a BR-body, and disassembly requires RNA cleavage, suggesting BR-bodies provide localized sites of RNA degradation. The unstructured C-terminal domain of RNase E is both necessary and sufficient to assemble the core of the BR-body, is functionally conserved in related α-proteobacteria, and influences mRNA degradation. BR-bodies are rapidly induced under cellular stresses and provide enhanced cell growth under stress. To our knowledge, Caulobacter RNase E is the first bacterial protein identified that forms LLPS condensates, providing an effective strategy for subcellular organization in cells lacking membrane-bound compartments.

Catalytic diversity in self-propagating peptide assemblies.

The protein-only infectious agents known as prions exist within cellular matrices as populations of assembled polypeptide phases ranging from particles to amyloid fibres. These phases appear to undergo Darwinian-like selection and propagation, yet remarkably little is known about their accessible chemical and biological functions. Here we construct simple peptides that assemble into well-defined amyloid phases and define paracrystalline surfaces able to catalyse specific enantioselective chemical reactions. Structural adjustments of individual amino acid residues predictably control both the assembled crystalline order and their accessible catalytic repertoire. Notably, the density and proximity of the extended arrays of enantioselective catalytic sites achieve template-directed polymerization of new polymers. These diverse amyloid templates can now be extended as dynamic self-propagating templates for the construction of even more complex functional materials.

Dynamic translation regulation in Caulobacter cell cycle control.

Progression of the Caulobacter cell cycle requires temporal and spatial control of gene expression, culminating in an asymmetric cell division yielding distinct daughter cells. To explore the contribution of translational control, RNA-seq and ribosome profiling were used to assay global transcription and translation levels of individual genes at six times over the cell cycle. Translational efficiency (TE) was used as a metric for the relative rate of protein production from each mRNA. TE profiles with similar cell cycle patterns were found across multiple clusters of genes, including those in operons or in subsets of operons. Collections of genes associated with central cell cycle functional modules (e.g., biosynthesis of stalk, flagellum, or chemotaxis machinery) have consistent but different TE temporal patterns, independent of their operon organization. Differential translation of operon-encoded genes facilitates precise cell cycle-timing for the dynamic assembly of multiprotein complexes, such as the flagellum and the stalk and the correct positioning of regulatory proteins to specific cell poles. The cell cycle-regulatory pathways that produce specific temporal TE patterns are separate from-but highly coordinated with-the transcriptional cell cycle circuitry, suggesting that the scheduling of translational regulation is organized by the same cyclical regulatory circuit that directs the transcriptional control of the Caulobacter cell cycle.

Cell fate regulation governed by a repurposed bacterial histidine kinase.

One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK∼P over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.

Kinetic intermediates in amyloid assembly.

In contrast to an expected Ostwald-like ripening of amyloid assemblies, the nucleating core of the Dutch mutant of the Aß peptide of Alzheimer's disease assembles through a series of conformational transitions. Structural characterization of the intermediate assemblies by isotope-edited IR and solid-state NMR reveals unexpected strand orientation intermediates and suggests new nucleation mechanisms in a progressive assembly pathway.