An invariant C-terminal tryptophan in McdB mediates its interaction and positioning function with carboxysomes.

Bacterial microcompartments (BMCs) are widespread, protein-based organelles that regulate metabolism. The model for studying BMCs is the carboxysome, which facilitates carbon fixation in several autotrophic bacteria. Carboxysomes can be distinguished as type α or ß, which are structurally and phyletically distinct. We recently characterized the maintenance of carboxysome distribution (Mcd) systems responsible for spatially regulating α- and ß-carboxysomes, consisting of the proteins McdA and McdB. McdA is an ATPase that drives carboxysome positioning, and McdB is the adaptor protein that directly interacts with carboxysomes to provide cargo specificity. The molecular features of McdB proteins that specify their interactions with carboxysomes, and whether these are similar between α- and ß-carboxysomes, remain unknown. Here, we identify C-terminal motifs containing an invariant tryptophan necessary for α- and ß-McdBs to associate with α- and ß-carboxysomes, respectively. Substituting this tryptophan with other aromatic residues reveals corresponding gradients in the efficiency of carboxysome colocalization and positioning by McdB in vivo. Intriguingly, these gradients also correlate with the ability of McdB to form condensates in vitro. The results reveal a shared mechanism underlying McdB adaptor protein binding to carboxysomes, and potentially other BMCs. Our findings also implicate condensate formation as playing a key role in this association.

An experimental framework to assess biomolecular condensates in bacteria.

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 discover that an established marker for insoluble protein aggregates, IbpA, has different colocalization patterns with bacterial condensates and aggregates, demonstrating its potential 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.

Dissecting the phase separation and oligomerization activities of the carboxysome positioning protein McdB.

Across bacteria, protein-based organelles called bacterial microcompartments (BMCs) encapsulate key enzymes to regulate their activities. The model BMC is the carboxysome that encapsulates enzymes for CO2 fixation to increase efficiency and is found in many autotrophic bacteria, such as cyanobacteria. Despite their importance in the global carbon cycle, little is known about how carboxysomes are spatially regulated. We recently identified the two-factor system required for the maintenance of carboxysome distribution (McdAB). McdA drives the equal spacing of carboxysomes via interactions with McdB, which associates with carboxysomes. McdA is a ParA/MinD ATPase, a protein family well studied in positioning diverse cellular structures in bacteria. However, the adaptor proteins like McdB that connect these ATPases to their cargos are extremely diverse. In fact, McdB represents a completely unstudied class of proteins. Despite the diversity, many adaptor proteins undergo phase separation, but functional roles remain unclear. Here, we define the domain architecture of McdB from the model cyanobacterium Synechococcus elongatus PCC 7942, and dissect its mode of biomolecular condensate formation. We identify an N-terminal intrinsically disordered region (IDR) that modulates condensate solubility, a central coiled-coil dimerizing domain that drives condensate formation, and a C-terminal domain that trimerizes McdB dimers and provides increased valency for condensate formation. We then identify critical basic residues in the IDR, which we mutate to glutamines to solubilize condensates. Finally, we find that a condensate-defective mutant of McdB has altered association with carboxysomes and influences carboxysome enzyme content. The results have broad implications for understanding spatial organization of BMCs and the molecular grammar of protein condensates.

Cells contain many millions of protein molecules that must be in the right place at the right time to carry out their roles. A process called phase separation, in which a well-mixed solution separates into two phases ­ one concentrated and one dilute ­ is thought to help organize the contents of various cell types. The single-celled bacteria Synechococcus elongatus converts carbon dioxide from the air into sugars using internal reaction centers. This process depends on a protein called McdB which is crucial for spatially organizing these centers. McdB readily phase separates on its own in a test tube, raising the possibility that this phenomenon could be involved in the carbon dioxide-capturing process. To investigate, Basalla et al. identified the parts of McdB responsible for phase separation and modified them to make a version that was less able to separate. When viewed under the microscope, Synechococcus elongatus cells containing the altered McdB showed changes in the organization and structure of the reaction centers. This suggests that phase separation by McdB is required for optimal carbon capture by this bacterium. In the future, manipulation of McdB phase separation could be used to improve carbon capture technologies or enhance crop growth. Phase separation is also known to influence complex disease. Therefore, further understanding of the process could be important for developing new disease treatments.

Multiple ParA/MinD ATPases coordinate the positioning of disparate cargos in a bacterial cell.

In eukaryotes, linear motor proteins govern intracellular transport and organization. In bacteria, where linear motors involved in spatial regulation are absent, the ParA/MinD family of ATPases organize an array of genetic- and protein-based cellular cargos. The positioning of these cargos has been independently investigated to varying degrees in several bacterial species. However, it remains unclear how multiple ParA/MinD ATPases can coordinate the positioning of diverse cargos in the same cell. Here, we find that over a third of sequenced bacterial genomes encode multiple ParA/MinD ATPases. We identify an organism (Halothiobacillus neapolitanus) with seven ParA/MinD ATPases, demonstrate that five of these are each dedicated to the spatial regulation of a single cellular cargo, and define potential specificity determinants for each system. Furthermore, we show how these positioning reactions can influence each other, stressing the importance of understanding how organelle trafficking, chromosome segregation, and cell division are coordinated in bacterial cells. Together, our data show how multiple ParA/MinD ATPases coexist and function to position a diverse set of fundamental cargos in the same bacterial cell.

An experimental framework to assess biomolecular condensates in bacteria.

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.

An experimental framework to assess biomolecular condensates in bacteria.

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.