Spatial organization of protein-based organelles is increasingly recognized as a fundamental requirement for bacterial physiology. Carboxysomes, encapsulins, and biomolecular condensates compartmentalize metabolic reactions throughout diverse bacteria, yet their positioning within cells remains poorly understood. Mislocalization of these structures leads to heterogeneous inheritance, reduced metabolic output, and impaired growth, and poses a major challenge for efforts to engineer synthetic organelles in model hosts such as Escherichia coli. The Maintenance of Carboxysome Distribution (Mcd) system, composed of the ParA-like ATPase McdA and the adaptor McdB, directs equidistant positioning of carboxysomes along the nucleoid. Although ParA and MinD family ATPases are well studied, little is known about the molecular features that govern adaptor specificity and function. Furthermore, many adaptors, including McdB, form biomolecular condensates, suggesting that phase separation may contribute to organelle control, but the underlying sequence determinants remain unclear. This dissertation addresses these knowledge gaps by dissecting the regions and residues of McdB that mediate phase separation, interaction with McdA, and leverage control of organelle positioning, and by applying these insights to develop programmable spatial organization tools for synthetic biology.

The McdB adaptor protein undergoes phase separation. I show here that McdB condensates mature from liquid- to gel-like condensates. Given that glutamine residues are known to contribute to maturation in other condensate-forming proteins, I mutated a glutamine-rich region of McdB from Synechococcus elongatus to determine its role in McdB structure, oligomerization and phase separation activity.

Second, I generated sequence variants in Halothiobacillus neapolitanus McdB and showed that specific N-terminal basic residues, particularly K7, are necessary for full McdA engagement and proper carboxysome distribution in vivo, revealing that cargo positioning and cargo partitioning are mechanistically separable functions of the Mcd system.

Third, I translated these mechanistic insights into a synthetic, minimal positioning toolkit by engineering modular “Minimal Autonomous Positioning” (Map) tags derived from McdB. When fused to biomolecular condensates or encapsulins in Escherichia coli, Map tags conferred McdA-dependent nucleoid-associated spacing patterns, transforming immobile aggregates into dynamic and evenly distributed intracellular structures.

Together, this work identifies domain- and residue-level determinants that govern McdB-mediated organelle positioning and condensate behavior, clarifies adaptor-ATPase specificity principles in ParA/MinD-type systems, and establishes a transferable genetic strategy for engineering intracellular spatial organization in bacteria. These findings advance both fundamental understanding of bacterial cell organization and the development of spatially programmable tools for microbial synthetic biology.