Intracellular phase separation is emerging as a universal principle for organizing biochemical reactions in time and space. It remains incompletely resolved how biological function is encoded in these assemblies and whether this depends on their material state. The conserved intrinsically disordered protein PopZ forms condensates at the poles of the bacterium Caulobacter crescentus, which in turn orchestrate cell-cycle regulating signaling cascades. Here we show that the material properties of these condensates are determined by a balance between attractive and repulsive forces mediated by a helical oligomerization domain and an expanded disordered region, respectively. A series of PopZ mutants disrupting this balance results in condensates that span the material properties spectrum, from liquid to solid. A narrow range of condensate material properties supports proper cell division, linking emergent properties to organismal fitness. We use these insights to repurpose PopZ as a modular platform for generating tunable synthetic condensates in human cells.

Phase separation is emerging as a universal principle for how cells use dynamic subcompartmentalization to organize biochemical reactions in time and space. Yet, whether the emergent physical properties of these biomolecular condensates are important for their biological function remains unclear. The intrinsically disordered protein PopZ forms membraneless condensates at the poles of the bacterium Caulobacter crescentus and selectively sequesters kinase-signaling cascades to regulate asymmetric cell division. By dissecting the molecular grammar underlying PopZ phase separation, we find that unlike many eukaryotic examples, where unstructured regions drive condensation, a structured domain of PopZ drives condensation, while conserved repulsive features of the disordered region modulate material properties. By generating rationally designed PopZ mutants, we find that the exact material properties of PopZ condensates directly determine cellular fitness, providing direct evidence for the physiological importance of the emergent properties of biomolecular condensates. Our work codifies a clear set of design principles illuminating how sequence variation in a disordered domain alters the function of a widely conserved bacterial condensate. We used these insights to repurpose PopZ as a modular platform for generating synthetic condensates of tunable function in human cells.

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Background Breath samples collected from patients infected with respiratory viruses are necessary for viral detection using breath analyzer devices. Given the highly transmissible nature of many of these illnesses, sample collection requires a multi-layered approach to ensure the safety of the research staff responsible for obtaining and transporting these samples. Our team established a protocol to minimize exposure to and transmission of COVID-19 when collecting breath samples. Methods We collected breath samples from 64 participants, of which 31 (48.4%) were positive for SARS-CoV-2 at the time of their visit. Before we started sample collection, biosafety inspection was conducted. We used a five-pronged approach to enhance safety and minimize transmission. First, we collected specimens in an outdoor space while the patients were seated in their vehicles. Second, we used a disposable mouthpiece and a one-way valve to fill a 1L TEDLAR bag. Third, patients were instructed to close the valve tightly before returning it to the staff. Fourth, we placed the bag in secondary containers which were placed in tertiary containers to minimize any contact with aerosols in the TEDLAR bag. In the last step, we placed a portable HEPA filter near the indoor sample processing unit to minimize exposure and air contamination with the samples. Study staff donned all forms of necessary personal protective equipment, including gloves, gowns, N95 respirators, and protective eyewear, during sample collection and transportation. Results A total of 64 breath samples were collected from 64 adult participants from February to March 2022. A total of 30 participants (46.9%) were within 7 days of their initial diagnosis. All participants were able to successfully collect samples without additional resources or attempts. All samples were able to be transported successfully into the lab. No staff contracted COVID-19 during the study period. Conclusion Layered safety measures, including protective equipment, physical barriers, and well-ventilated environments mitigated the risks associated with breath sample collections from infected participants. Disclosures Yvonne A. Maldonado, MD, Pfizer: Grant/Research Support|Pfizer: Member, DSMB, Pfizer Meningococcal Vaccine clinical trial.