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.

Starving Myxococcus xanthus bacteria use short-range C-signaling to coordinate their movements and construct multicellular mounds, which mature into fruiting bodies as rods differentiate into spherical spores. Differentiation requires efficient C-signaling to drive the expression of developmental genes, but how the arrangement of cells within nascent fruiting bodies (NFBs) affects C-signaling is not fully understood. Here, we used confocal microscopy and cell segmentation to visualize and quantify the arrangement, morphology, and gene expression of cells near the bottom of NFBs at much higher resolution than previously achieved. We discovered that “transitioning cells” (TCs), intermediate in morphology between rods and spores, comprised 10 to 15% of the total population. Spores appeared midway between the center and the edge of NFBs early in their development and near the center as maturation progressed. The developmental pattern, as well as C-signal–dependent gene expression in TCs and spores, were correlated with cell density, the alignment of neighboring rods, and the tangential orientation of rods early in the development of NFBs. These dynamic radial patterns support a model in which the arrangement of cells within the NFBs affects C-signaling efficiency to regulate precisely the expression of developmental genes and cellular differentiation in space and time. Developmental patterns in other bacterial biofilms may likewise rely on short-range signaling to communicate multiple aspects of cellular arrangement, analogous to juxtacrine and paracrine signaling during animal development.

Upon depletion of nutrients, forms mounds on a solid surface. The differentiation of rod-shaped cells into stress-resistant spores within mounds creates mature fruiting bodies. The developmental process can be perturbed by the addition of nutrient medium before the critical period of commitment to spore formation. The response was investigated by adding a 2-fold dilution series of nutrient medium to starving cells. An ultrasensitive response was observed, as indicated by a steep increase in the spore number after the addition of 12.5% versus 25% nutrient medium. The level of MrpC, which is a key transcription factor in the gene regulatory network, correlated with the spore number after nutrient medium addition. The MrpC level decreased markedly by 3 h after adding nutrient medium but recovered more after the addition of 12.5% than after 25% nutrient medium addition. The difference in MrpC levels was greatest midway during the period of commitment to sporulation, and mound formation was restored after 12.5% nutrient medium addition but not after adding 25% nutrient medium. Although the number of spores formed after 12.5% nutrient medium addition was almost normal, the transcript levels of "late" genes in the regulatory network failed to rise normally during the commitment period. However, at later times, expression from a reporter gene fused to a late promoter was higher after adding 12.5% than after adding 25% nutrient medium, consistent with the spore numbers. The results suggest that a threshold level of MrpC must be achieved in order for mounds to persist and for cells within to differentiate into spores. Many signaling and gene regulatory networks convert graded stimuli into all-or-none switch-like responses. Such ultrasensitivity can produce bistability in cell populations, leading to different cell fates and enhancing survival. We discovered an ultrasensitive response of to nutrient medium addition during development. A small change in nutrient medium concentration caused a profound change in the developmental process. The level of the transcription factor MrpC correlated with multicellular mound formation and differentiation into spores. A threshold level of MrpC is proposed to be necessary to initiate mound formation and create a positive feedback loop that may explain the ultrasensitive response. Understanding how this biological switch operates will provide a paradigm for the broadly important topic of cellular behavior in microbial communities.

Photosynthetic cyanobacteria are responsible for almost half of global CO 2 fixation. Due to eutrophication, rising temperatures, and increasing atmospheric CO 2 concentrations, cyanobacteria have gained notoriety for their ability to form massive blooms in both freshwater and marine ecosystems across the globe.

Carboxysomes, the most prevalent and well-studied anabolic bacterial microcompartment, play a central role in efficient carbon fixation by cyanobacteria and proteobacteria. In previous studies, we identified the two-component system called McdAB that spatially distributes carboxysomes across the bacterial nucleoid. McdA, a ParA-like ATPase, forms a dynamic oscillating gradient on the nucleoid in response to carboxysome-localized McdB. As McdB stimulates McdA ATPase activity, McdA is removed from the nucleoid in the vicinity of carboxysomes, propelling these proteinaceous cargos toward regions of highest McdA concentration via a Brownian-ratchet mechanism. How the ATPase cycle of McdA governs its in vivo dynamics and carboxysome positioning remains unresolved. Here, by strategically introducing amino acid substitutions in the ATP-binding region of McdA, we sequentially trap McdA at specific steps in its ATP cycle. We map out critical events in the ATPase cycle of McdA that allows the protein to bind ATP, dimerize, change its conformation into a DNA-binding state, interact with McdB-bound carboxysomes, hydrolyze ATP and release from the nucleoid. We also find that McdA is a member of a previously unstudied subset of ParA family ATPases, harboring unique interactions with ATP and the nucleoid for trafficking their cognate intracellular cargos. [Media: see text] [Media: see text] [Media: see text]

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Rice is sensitive to salt stress, but its sensitivity varies among genotypes, indicating natural variation in regulatory mechanisms and genetic makeup. High-affinity potassium transporters (HKTs) that transport cations across membranes play important roles in stress responses of plants. In this study, the gene sequence polymorphisms and expression level of OsHKT1;3 which is a member of the rice HKT gene family was assessed. Sequence analysis indicated 5 single nucleotide polymorphisms (SNPs) in the coding sequence; 4 nucleotide substitutions, one nucleotide deletion and one nucleotide insertion in the promoter region of OsHKT1;3 gene. Among 5 SNPs in the coding sequence, one was nonsynonymous (C598G) which caused the change in amino acid L200V and 4 were synonymous substitutions (A798C, G2083A, T2101C, C2122T). The substituted amino acid L200V was predicted to locate in the third transmembrane segment of OsHKT1;3 protein. In the promoter region, 3 nucleotide substitutions at position-879,-453, and-202 caused the change in cis-elements with 8 deletions and 3 additions. Expression levels of OsHKT1;3 were analyzed in the leaves and the roots under 2 different salt concentrations and showed a tendency of reduction in most of the 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.