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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 β, which are structurally and phyletically distinct. We recently characterized the maintenance of carboxysome distribution (Mcd) systems responsible for spatially regulating α- and β-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 β-carboxysomes, remain unknown. Here, we identify C-terminal motifs containing an invariant tryptophan necessary for α- and β-McdBs to associate with α- and β-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. 

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 CO₂fixation 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 cyanobacteriumSynechococcus elongatusPCC 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. 

Abstract Curli are functional amyloids present on the outer membrane ofE. coli. CsgF is required for the proper assembly of curli. Here, we found that the CsgF phase separates in vitro and that the ability of CsgF variants to phase-separate is tightly correlated with CsgF function during curli biogenesis. Substitution of phenylalanine residues in the CsgF N-terminus both reduced the propensity of CsgF to phase-separate and impaired curli assembly. Exogenous addition of purified CsgF complementedcsgF ⁻cells. This exogenous addition assay was used to assess the ability of CsgF variants to complementcsgF ^‒cells. CsgF on the cell surface modulated the secretion of CsgA, the curli major subunit, to the cell surface. We also found that the CsgB nucleator protein can form SDS-insoluble aggregates within the dynamic CsgF condensate. We propose that these multicomponent CsgF-B condensates form a nucleation-competent complex that templates CsgA amyloid formation on the cell surface. 

Abstract Carboxysomes are protein-based organelles that are essential for allowing cyanobacteria to fix CO2. Previously, we identified a two-component system, McdAB, responsible for equidistantly positioning carboxysomes in the model cyanobacterium Synechococcus elongatus PCC 7942 (MacCready JS, Hakim P, Young EJ, Hu L, Liu J, Osteryoung KW, Vecchiarelli AG, Ducat DC. 2018. Protein gradients on the nucleoid position the carbon-fixing organelles of cyanobacteria. eLife 7:pii:e39723). McdA, a ParA-type ATPase, nonspecifically binds the nucleoid in the presence of ATP. McdB, a novel factor that directly binds carboxysomes, displaces McdA from the nucleoid. Removal of McdA from the nucleoid in the vicinity of carboxysomes by McdB causes a global break in McdA symmetry, and carboxysome motion occurs via a Brownian-ratchet-based mechanism toward the highest concentration of McdA. Despite the importance for cyanobacteria to properly position their carboxysomes, whether the McdAB system is widespread among cyanobacteria remains an open question. Here, we show that the McdAB system is widespread among β-cyanobacteria, often clustering with carboxysome-related components, and is absent in α-cyanobacteria. Moreover, we show that two distinct McdAB systems exist in β-cyanobacteria, with Type 2 systems being the most ancestral and abundant, and Type 1 systems, like that of S. elongatus, possibly being acquired more recently. Lastly, all McdB proteins share the sequence signatures of a protein capable of undergoing liquid–liquid phase separation. Indeed, we find that representatives of both McdB types undergo liquid–liquid phase separation in vitro, the first example of a ParA-type ATPase partner protein to exhibit this behavior. Our results have broader implications for understanding carboxysome evolution, biogenesis, homeostasis, and positioning in cyanobacteria. 

Abstract Carboxysomes are protein‐based organelles essential for carbon fixation in cyanobacteria and proteobacteria. Previously, we showed that the cyanobacterial nucleoid is used to equally space out β‐carboxysomes across cell lengths by a two‐component system (McdAB) in the model cyanobacteriumSynechococcus elongatusPCC 7942. More recently, we found that McdAB systems are widespread among β‐cyanobacteria, which possess β‐carboxysomes, but are absent in α‐cyanobacteria, which possess structurally and phyletically distinct α‐carboxysomes. Cyanobacterial α‐carboxysomes are thought to have arisen in proteobacteria and then horizontally transferred into cyanobacteria, which suggests that α‐carboxysomes in proteobacteria may also lack the McdAB system. Here, using the model chemoautotrophic proteobacteriumHalothiobacillus neapolitanus, we show that a McdAB system distinct from that of β‐cyanobacteria operates to position α‐carboxysomes across cell lengths. We further show that this system is widespread among α‐carboxysome‐containing proteobacteria and that cyanobacteria likely inherited an α‐carboxysome operon from a proteobacterium lacking themcdABlocus. These results demonstrate that McdAB is a cross‐phylum two‐component system necessary for positioning both α‐ and β‐carboxysomes. The findings have further implications for understanding the positioning of other protein‐based bacterial organelles involved in diverse metabolic processes. Plain language summaryCyanobacteria are well known to fix atmospheric CO₂into sugars using the enzyme Rubisco. Less appreciated are the carbon‐fixing abilities of proteobacteria with diverse metabolisms. Bacterial Rubisco is housed within organelles called carboxysomes that increase enzymatic efficiency. Here we show that proteobacterial carboxysomes are distributed in the cell by two proteins, McdA and McdB. McdA on the nucleoid interacts with McdB on carboxysomes to equidistantly space carboxysomes from one another, ensuring metabolic homeostasis and a proper inheritance of carboxysomes following cell division. This study illuminates how widespread carboxysome positioning systems are among diverse bacteria. Carboxysomes significantly contribute to global carbon fixation; therefore, understanding the spatial organization mechanism shared across the bacterial world is of great interest.