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.
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Polyphosphate specifically cooperates with Hfq to silence prophages and mobile genetic elements in the bacterial chromosome.
Carboxysomes are protein-based organelles essential for efficient CO2-fixation in cyanobacteria and some chemoautotrophic bacteria. We recently identified the two-component system responsible for spatially regulating carboxysomes, consisting of the proteins McdA and McdB. McdA is a member of the ParA/MinD-family of ATPases, which position a variety of cellular cargos across bacteria. McdB, however, represents a widespread but unstudied class of proteins. We previously found that McdB forms a hexamer and undergoes robust Liquid-Liquid Phase Separation (LLPS) in vitro, but the sequence and structural determinants underlying these properties are unknown. Here we define the domain architecture for McdB from the model cyanobacterium S. elongatus PCC 7942 which we use to dissect McdB oligomerization and LLPS. We identify an N-terminal Intrinsically Disordered Region (IDR), a central Q-rich dimerizing domain, and a C-terminal domain that trimerizes McdB dimers. Intriguingly, all three domains contributed to McdB LLPS. The Q-rich domain drove LLPS, the IDR tuned solubility, and the C-terminal domain provided further oligomerization to achieve full-length LLPS activity. We also identified critical basic residues in the IDR that modulate McdB LLPS, which we mutate to fine-tune condensate solubility both in vitro and in vivo. Our findings show that IDRs are not always drivers of LLPS, but can play secondary roles in modulating condensate solubility. Finally, we provide in silico evidence suggesting the N-terminal IDR of McdB acts as a MoRF, folding upon interaction with McdA. The data advance our understanding and application of carboxysomes, their positioning system, and the molecular grammar governing protein phase separation.SIGNIFICANCEThe recently characterized Maintenance of Carboxysome Distribution (Mcd) system is responsible for spatially regulating carbon-fixing organelles in bacteria called carboxysomes. Although an understanding of the Mcd system would advance our application of carboxysomes to help engineer carbon-fixing organisms and combat the climate crisis, one of its two essential components, McdB, has only recently been identified and is poorly understood. Here, we provide a thorough biochemical characterization of McdB from the model cyanobacterium S. elognatus. We define a structural model for McdB and identify how specific domains and residues contribute to its oligomerization and phase separation. Notably, we saw that a disordered region of McdB regulates phase separation in response to pH; impactful to both carboxysome regulation and protein phase separation.
The evolution of multidrug resistant pathogens and the diminishing supply of effective antibiotics are global crisis. Tiny Earth (TE) is undergraduate curriculum that encourage students to pursue science careers by engagement in authentic drug discovery research. Through the TE program, students identify environmental strains that inhibit other bacteria. Although these isolates may produce antibiotics based on the antagonistic phenotype, understanding the activity in regard to genome content remains elusive. Previously, we developed a transposon mutagenesis module for use with TE to identify genes involved in antibiotic production. Here, we extend this approach to a second semester undergraduate course to understand the origin of antagonism and genome diversity. Using a bioinformatics strategy, we identified gene clusters involved in activity, and with annotated genomes in hand, students were able to characterize strain diversity. Genomes were analyzed using different computational tools, including average nucleotide identity for species identification and whole genome comparisons. Because the focus of TE involves the evolution of drug resistance, predicted products in strains were identified and verified using a drug susceptibility assay. An application of this curriculum by TE members would assist in efforts with antibiotic discovery.
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