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ABSTRACT The bacterial nucleoid is not just a genetic repository—it serves as a dynamic scaffold for spatially organizing key cellular components. ParA-family ATPases exploit this nucleoid matrix to position a wide range of cargos, yet how nucleoid compaction influences these positioning reactions remains poorly understood. We previously characterized the maintenance of carboxysome distribution (Mcd) system in the cyanobacteriumSynechococcus elongatusPCC 7942, where the ParA-like ATPase McdA binds the nucleoid and interacts with its partner protein, McdB, to generate dynamic gradients that distribute carboxysomes for optimal carbon fixation. Here, we investigate how nucleoid compaction impacts carboxysome positioning, particularly during metabolic dormancy when McdAB activity is downregulated. We demonstrate that a compacted nucleoid maintains carboxysome organization in the absence of active McdAB-driven positioning. This finding reveals that the nucleoid is not merely a passive matrix for positioning but a dynamic player in spatial organization. Given the widespread role of ParA-family ATPases in the positioning of diverse cellular cargos, our study suggests that the nucleoid compaction state is a fundamental, yet underappreciated, determinant of mesoscale organization across bacteria. IMPORTANCEBacteria can organize their internal components in specific patterns to ensure proper function and faithful inheritance after cell division. In the cyanobacteriumSynechococcus elongatus, protein-based compartments called carboxysomes fix carbon dioxide and are distributed in the cell by a two-protein positioning system. Here, we discovered that when cells stop growing or face stress, these positioning proteins stop working, yet carboxysomes remain distributed in the cell. Our study shows that the bacterial chromosome, which holds genetic information, can also act as a flexible scaffold that holds carboxysomes in place when compacted. This insight reveals that the bacterial chromosome plays a key physical role in organizing the cell. Similar positioning systems are found across many types of bacteria; therefore, our findings suggest that nucleoid compaction may be a universal and underappreciated factor in maintaining spatial order in cells that are not actively growing. 

ABSTRACT Flagella are complex, trans-envelope nanomachines that localize in species-specific patterns on the cell surface. Here, we study the localization dynamics of the earliest stage of basal body formation inBacillus subtilisusing a fluorescent fusion to the C-ring protein FliM. We find thatB. subtilisbasal bodies do not exhibit dynamic subunit exchange and are largely stationary at steady state, consistent with flagellar assembly through the peptidoglycan (PG). However, rare mobile basal bodies were observed, and the prevalence of mobile basal bodies is elevated both early in basal body assembly and when the rod is mutated. Thus, basal body mobility is a precursor to patterning, and we propose that rod polymerization probes the PG superstructure for pores of sufficient diameter to permit rod transit. Furthermore, mutation of the rod disrupts basal body patterning in a way that phenocopies mutation of the cytoplasmic flagellar patterning protein FlhF. We infer that rod synthesis and the cytoplasmic regulators coordinate flagellar assembly by interpreting a grid-like pattern of pores, pre-existent in the PG. IMPORTANCEBacteria insert flagella in a species-specific pattern on the cell body, but how patterns are achieved is poorly understood. In bacteria with a single polar flagellum, a marker protein localizes to the cell pole and nucleates the assembly of the flagellum at that site.Bacillus subtilisassembles ~25 basal bodies over the length of the cell in a grid-like pattern and lacks proteins required for their polar targeting. Here, we show thatB. subtilisbasal bodies are mobile soon after assembly and become immobilized when the flagellar rod transits the peptidoglycan (PG) wall. Moreover, defects in the flagellar rod lead to a more-random distribution of flagella and an increase in polar basal bodies. We conclude that the peritrichous patterning of flagella ofB. subtilisis different from the polar patterning of other bacteria, and we infer that theB. subtilisrod probes the PG for holes that can accommodate the machine.