RESUMEN
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.
RESUMEN
The enteropathogen Salmonella enterica subsp. enterica sv. Typhimurium str. LT2 (hereafter S. Typhimurium) utilizes a cluster of genes encoded within the pathogenicity island 2 (SPI-2) of its genome to proliferate inside macrophages. The expression of SPI-2 is controlled by a complex network of transcriptional regulators and environmental cues, which now include a recently characterized DNA-binding protein named PagR. Growth of S. Typhimurium in low-phosphate, low-magnesium medium mimics conditions inside macrophages. Under such conditions, PagR ensures SPI-2 induction by upregulating the transcription of slyA, which encodes a known activator of SPI-2. Here, we report that PagR represses the expression of a divergently transcribed polycistronic operon that encodes the two subunits of transketolase TktC (i.e., tktD, tktE) of this bacterium. Transketolases contribute to the nonredox rearrangements of phosphorylated sugars of the pentose phosphate pathway, which provide building blocks for amino acids, nucleotides, cofactors, etc. We also demonstrate that PagR represses the expression of its own gene and define two PagR-binding sites between stm2344 and pagR.