Recent investigations in bacteria suggest that membraneless organelles play a crucial role in the subcellular organization of bacterial cells. However, the biochemical functions and assembly mechanisms of these compartments have not yet been completely characterized. This Review assesses the current methodologies used in the study of membraneless organelles in bacteria, highlights the limitations in determining the phase of complexes in cells that are typically an order of magnitude smaller than a eukaryotic cell, and identifies gaps in our current knowledge about the functional role of membraneless organelles in bacteria. Liquid-liquid phase separation (LLPS) is one proposed mechanism for membraneless organelle assembly. Overall, we outline the framework to evaluate LLPS in vivo in bacteria, we describe the bacterial systems with proposed LLPS activity, and we comment on the general role LLPS plays in bacteria and how it may regulate cellular function. Lastly, we provide an outlook for super-resolution microscopy and single-molecule tracking as tools to assess condensates in bacteria. Statement of SignificanceThough membraneless organelles appear to play a crucial role in the subcellular organization and regulation of bacterial cells, the biochemical functions and assembly mechanisms of these compartments have not yet been completely characterized. Furthermore, liquid-liquid phase separation (LLPS) is one proposed mechanism for membraneless organelle assembly, but it is difficult to determine subcellular phases in tiny bacterial cells. Thus, we outline the framework to evaluate LLPS in vivo in bacteria and we describe the bacterial systems with proposed LLPS activity in the context of these criteria.

Recent investigations in bacteria suggest that membraneless organelles play a crucial role in the subcellular organization of bacterial cells. However, the biochemical functions and assembly mechanisms of these compartments have not yet been completely characterized. This Review assesses the current methodologies used in the study of membraneless organelles in bacteria, highlights the limitations in determining the phase of complexes in cells that are typically an order of magnitude smaller than a eukaryotic cell, and identifies gaps in our current knowledge about the functional role of membraneless organelles in bacteria. Liquid-liquid phase separation (LLPS) is one proposed mechanism for membraneless organelle assembly. Overall, we outline the framework to evaluate LLPS in vivo in bacteria, we describe the bacterial systems with proposed LLPS activity, and we comment on the general role LLPS plays in bacteria and how it may regulate cellular function. Lastly, we provide an outlook for super-resolution microscopy and single-molecule tracking as tools to assess condensates in bacteria.Statement of SignificanceThough membraneless organelles appear to play a crucial role in the subcellular organization and regulation of bacterial cells, the biochemical functions and assembly mechanisms of these compartments have not yet been completely characterized. Furthermore, liquid-liquid phase separation (LLPS) is one proposed mechanism for membraneless organelle assembly, but it is difficult to determine subcellular phases in tiny bacterial cells. Thus, we outline the framework to evaluate LLPS in vivo in bacteria and we describe the bacterial systems with proposed LLPS activity in the context of these criteria.

Complex carbohydrates shape the gut microbiota, and the collective fermentation of resistant starch by gut microbes positively affects human health through enhanced butyrate production. The keystone species Ruminococcus bromii (Rb) is a specialist in degrading resistant starch; its degradation products are used by other bacteria including Bacteroides thetaiotaomicron (Bt). We analysed the metabolic and spatial relationships between Rb and Bt during potato starch degradation and found that Bt utilizes glucose that is released from Rb upon degradation of resistant potato starch and soluble potato amylopectin. Additionally, we found that Rb produces a halo of glucose around it when grown on solid media containing potato amylopectin and that Bt cells deficient for growth on potato amylopectin (∆sus Bt) can grow within the halo. Furthermore, when these ∆sus Bt cells grow within this glucose halo, they have an elongated cell morphology. This long-cell phenotype depends on the glucose concentration in the solid media: longer Bt cells are formed at higher glucose concentrations. Together, our results indicate that starch degradation by Rb cross-feeds other bacteria in the surrounding region by releasing glucose. Our results also elucidate the adaptive morphology of Bt cells under different nutrient and physiological conditions.

High-resolution imaging of biomolecular condensates in living cells is essential for correlating their properties to those observed through in vitro assays. However, such experiments are limited in bacteria due to resolution limitations. Here we present an experimental framework that probes the formation, reversibility, and dynamics of condensate-forming proteins in Escherichia coli as a means to determine the nature of biomolecular condensates in bacteria. We demonstrate that condensates form after passing a threshold concentration, maintain a soluble fraction, dissolve upon shifts in temperature and concentration, and exhibit dynamics consistent with internal rearrangement and exchange between condensed and soluble fractions. We also discovered that an established marker for insoluble protein aggregates, IbpA, has different colocalization patterns with bacterial condensates and aggregates, demonstrating its applicability as a reporter to differentiate the two in vivo. Overall, this framework provides a generalizable, accessible, and rigorous set of experiments to probe the nature of biomolecular condensates on the sub-micron scale in bacterial cells.

Liquid Liquid Phase Separation (LLPS) has emerged as a mechanism for the assembly of membraneless organelles in eukaryotes, but little is known about this process in bacteria. LLPS refers to the ability of macromolecules to demix into a dilute phase and a dense phase, called a ‘biomolecular condensate’, which can be observed as clusters or foci in the cell. The major challenge for the study of LLPS in bacteria is the poor spatial resolution of foci in such tiny cells. As a result, it is difficult to demonstrate the liquid‐like nature of a focus in bacterial cells using the conventional approaches for studying large condensates in eukaryotic cells. Here, we developed a rigorous experimental framework for the characterization of LLPS in bacteria, using Escherichia coli as the host organism and the intrinsically disordered protein McdB, which robustly forms liquid‐like droplets in vitro. McdB is a protein that coats a bacterial organelle called the carboxysome. This coating demarcates the carboxysome as cargo for its positioning system, which equally distributes carboxysomes along the cell length of rod‐shaped cyanobacteria. We developed a suite of experiments to investigate the LLPS activity of McdB in vivo, based on the ability of biomolecular condensates to tune their size and shape, fuse, dissolve, and transition between phase states. We used both overexpression and tunable promoters to express fluorescent fusions of McdB and cIEP8, a well‐known aggregator protein. We found that fluorescent fusions of McdB formed nucleoid‐excluded foci in E. coli, but also maintained a soluble phase in the cytoplasm, consistent with LLPS theory. The aggregator protein cIEP8, on the other hand, lacked a soluble fraction in the cytoplasm. Condensates form at a saturation concentration threshold, called Csat. A hallmark of LLPS is that condensates will dissolve if the concentration drops below Csat, while insoluble aggregates should remain as stable foci even after dilution. We decreased protein concentration in vivo by increasing cell volume and by generational dilution via cell division. In both methods, McdB foci dissolved while cIEP8 foci remained intact as insoluble aggregates in response to decreased concentration in the cell. Finally, we also discovered that a well‐established marker for insoluble protein aggregates in vivo, IbpA, does not colocalize with McdB foci. The result suggests that the colocalization of IbpA foci can be used as a broad‐use sensor for the material state of protein complexes in bacterial cells. Our results provide multiple lines of evidence in support of LLPS of McdB in vivo. More broadly, our experimental framework for studying LLPS in bacteria overcomes current limitations in the field and can be used to assess the LLPS activity of other proteins of interest in bacterial cells.

Development of latent prints employing cyanoacrylate ester (CA) can be a multistep process including CA fuming and subsequent fluorescent staining to produce fingerprints of sufficient contrast for comparison work. To enable a single-step CA fuming-staining process, a selection of fluorophores have been developed as sublimation dyes in CA fuming. A greater array of such luminescent sublimation dyes would allow users greater flexibility in selecting a particular dye-CA combination to best suit their processing needs. Toward this end, six benzoic acid derivatives were evaluated for use as luminescent sublimation dyes under elementary CA fuming conditions using a single non-porous surface type and an inexpensive handheld UV lamp for excitation.Two benzoic acid derivatives, 2-hydroxybenzoic acid (salicylic acid) and 2-aminobenzoic acid (anthranilic acid), were identified as new potential luminescent sublimation dyes with stained fingerprints excited at 254 nm. The fluorescence intensity and stability of prints produced via the sublimation of CA with 2-hydroxybenzoic acid and 2-aminobenzoic acid were evaluated over approximately six weeks using image and statistical analysis.

Complex carbohydrates shape the gut microbiota and the collective fermentation of resistant starch by gut microbes positively affects human health through enhanced butyrate production. The keystone species Ruminococcus bromii (Rb) is a specialist in degrading resistant starch; its degradation products are used by other bacteria including Bacteroides thetaiotaomicron (Bt). We analyzed the metabolic and spatial relationships between Rb and Bt during potato starch degradation and found that Bt utilizes glucose that is released from Rb upon degradation of resistant potato starch and soluble potato amylopectin. Additionally, we found that Rb produces a halo of glucose around it when grown on solid media containing potato amylopectin and that Bt cells deficient for growth on potato amylopectin (Δsus Bt) can grow within the halo. Furthermore, when these Δsus Bt cells grow within this glucose halo, they have an elongated cell morphology. This long-cell phenotype depends on the glucose concentration in the solid media: longer Bt cells are formed at higher glucose concentrations. Together, our results indicate that starch degradation by Rb cross-feeds other bacteria in the surrounding region by releasing glucose. Our results also elucidate the adaptive morphology of Bt cells under different nutrient and physiological conditions.

High-resolution imaging of biomolecular condensates in living cells is essential for correlating their properties to those observed through in vitro assays. However, such experiments are limited in bacteria due to resolution limitations. Here we present an experimental framework that probes the formation, reversibility, and dynamics of condensate-forming proteins in Escherichia coli as a means to determine the nature of biomolecular condensates in bacteria. We demonstrate that condensates form after passing a threshold concentration, maintain a soluble fraction, dissolve upon shifts in temperature and concentration, and exhibit dynamics consistent with internal rearrangement and exchange between condensed and soluble fractions. We also discovered that an established marker for insoluble protein aggregates, IbpA, has different colocalization patterns with bacterial condensates and aggregates, demonstrating its applicability as a reporter to differentiate the two in vivo. Overall, this framework provides a generalizable, accessible, and rigorous set of experiments to probe the nature of biomolecular condensates on the sub-micron scale in bacterial cells.