Summary In model bacteria, such as E. coli and B. subtilis, regulation of cell cycle progression and cellular organization achieves consistency in cell size, replication dynamics, and chromosome positioning [1–3]. Mycobacteria elongate and divide asymmetrically, giving rise to significant variation in cell size and elongation rate among closely related cells [4, 5]. Given the physical asymmetry of mycobacteria, the models that describe coordination of cellular organization and cell cycle progression in model bacteria are not directly translatable [1, 2, 6–8]. Here we used time-lapse microscopy and fluorescent reporters of DNA replication and chromosome positioning to examine the coordination of growth, division, and chromosome dynamics at a single-cell level in Mycobacterium smegmatis (M. smegmatis) and Mycobacterium bovis Bacillus Calmette–Guérin (BCG). By analyzing chromosome and replisome localization, we demonstrated that chromosome positioning is asymmetric and proportional to cell size. Furthermore, we found that cellular asymmetry is maintained throughout the cell cycle and is not established at division. Using measurements and stochastic modeling of mycobacterial cell size and cycle timing in both slow and fast growth conditions, we found that well-studied cell size control models are insufficient to explain the mycobacterial cell cycle. Instead, we showed that mycobacterial cell cycle progression is regulated by an unprecedented mechanism involving parallel adders (i.e. constant growth increments) that start at replication initiation. Together, these adders enable mycobacterial populations to regulate cell size, growth, and heterogeneity in the face of varying environments.

DNA replication is fundamental for life, yet a detailed understanding of bacterial DNA replication is limited outside the organisms Escherichia coli and Bacillus subtilis. Many bacteria, including mycobacteria, encode no identified homologs of helicase loaders or regulators of the initiator protein DnaA, despite these factors being essential for DNA replication in E. coli and B. subtilis. In this study we discover that a previously uncharacterized protein, Rv0004, from the human pathogen Mycobacterium tuberculosis is essential for bacterial viability and that depletion of Rv0004 leads to a block in cell cycle progression. Using a combination of genetic and biochemical approaches, we found that Rv0004 has a role in DNA replication, interacts with DNA and the replicative helicase DnaB, and affects DnaB-DnaA complex formation. We also identify a conserved domain in Rv0004 that is predicted to structurally resemble the N-terminal protein-protein interaction domain of DnaA. Mutation of a single conserved tryptophan within Rv0004’s DnaA N-terminal-like domain leads to phenotypes similar to those observed upon Rv0004 depletion and can affect the association of Rv0004 with DnaB. In addition, using live cell imaging during depletion of Rv0004, we have uncovered a previously unappreciated role for DNA replication in coordinating mycobacterial cell division and cell size. Together, our data support that Rv0004 encodes a homolog of the recently identified DciA family of proteins found in most bacteria that lack the DnaC-DnaI helicase loaders in E. coli and B. subtilis. Therefore, the mechanisms of Rv0004 elucidated here likely apply to other DciA homologs and reveal insight into the diversity of bacterial strategies in even the most conserved biological processes.

Model bacteria, such as E. coli and B. subtilis, tightly regulate cell cycle progression to achieve consistent cell size distributions and replication dynamics. Many of the hallmark features of these model bacteria, including lateral cell wall elongation and symmetric growth and division, do not occur in mycobacteria. Instead, mycobacterial growth is characterized by asymmetric polar growth and division. This innate asymmetry creates unequal birth sizes and growth rates for daughter cells with each division, generating a phenotypically heterogeneous population. Although the asymmetric growth patterns of mycobacteria lead to a larger variation in birth size than typically seen in model bacterial populations, the cell size distribution is stable over time. Here, we review the cellular mechanisms of growth, division, and cell cycle progression in mycobacteria in the face of asymmetry and inherent heterogeneity. These processes coalesce to control cell size. Although Mycobacterium smegmatis and Mycobacterium bovis Bacillus Calmette-Guérin (BCG) utilize a novel model of cell size control, they are similar to previously studied bacteria in that initiation of DNA replication is a key checkpoint for cell division. We compare the regulation of DNA replication initiation and strategies used for cell size homeostasis in mycobacteria and model bacteria. Finally, we review the importance of cellular organization and chromosome segregation relating to the physiology of mycobacteria and consider how new frameworks could be applied across the wide spectrum of bacterial diversity.

All eukaryotic organisms store excess lipid in intracellular lipid droplets. These dynamic structures are associated with and regulated by numerous proteins. Perilipin 2, an abundant protein on most lipid droplets, promotes neutral lipid accumulation in lipid droplets. However, the mechanism by which perilipin 2 binds to and remains anchored on the lipid droplet surface is unknown. Here we identify features of the lipid droplet surface that influence perilipin 2 localization. We show that perilipin 2 binding to the lipid droplet surface requires both hydrophobic and electrostatic interactions. Reagents that disrupt these interactions also decrease binding. Moreover, perilipin 2 binding does not depend on other lipid droplet-associated proteins but is influenced by the lipid composition of the surface. Perilipin 2 binds to synthetic vesicles composed of dioleoylphosphatidylcholine, a phospholipid with unsaturated acyl chains. Decreasing the temperature of the binding reaction, or introducing phospholipids with saturated acyl chains, decreases binding. We therefore demonstrate a role for surface lipids and acyl chain packing in perilipin 2 binding to lipid droplets. The ability of the lipid droplet phospholipid composition to impact protein binding may link changes in nutrient availability to lipid droplet homeostasis.

Perilipin 2 binds to the surface of lipid droplets in most eukaryotic cells. However, the conditions that allow perilipin 2 to distinguish the lipid droplet surface from other intracellular organelles remain undetermined. Here, we describe the development of an in vitro assay to identify features of the lipid droplet that mediate perilipin 2 binding. We show that protease treatment of isolated lipid droplets does not prevent perilipin 2 binding, and that perilipin 2 can bind directly to synthetic liposomes (which lack proteins or a neutral lipid core). Moreover, the binding of perilipin 2 to synthetic liposomes is influenced by the phospholipid composition of these vesicles. Future experiments will further probe the characteristics of the lipid droplet surface that are required for perilipin 2 binding and determine if similar features of the lipid droplet surface mediate the binding of other lipid droplet‐associated proteins. This work was funded by the NIHGMS (LL and DB), the Research Corporation for Science Advancement (LL), and through the generous support of Dr. Elizabeth Nabel (ML).