Highlightsd The adder requires accumulation of division proteins to a threshold for division d The adder requires constant production of division proteins during cell elongation d In E. coli and B. subtilis, initiation and division are independently controlled d In E. coli and B. subtilis, cell division exclusively drives size homeostasis

Nutrients—and by extension biosynthetic capacity—positively impact cell size in organisms throughout the tree of life. In bacteria, cell size is reduced three-fold in response to nutrient starvation or accumulation of the alarmone ppGpp, a global inhibitor of biosynthesis. However, whether biosynthetic capacity as a whole determines cell size or if particular anabolic pathways are more important than others remains an open question. Here we identify fatty acid synthesis as the primary biosynthetic determinant of Escherichia coli size and present evidence supporting a similar role for fatty acids as a positive determinant of size in the Gram-positive bacterium Bacillus subtilis and the single celled eukaryote, Saccharomyces cerevisiae. Altering fatty acid synthesis recapitulated the impact of altering nutrients on cell size and morphology, while defects in other biosynthetic pathways had either a negligible or fatty acid-dependent effect on size. Together our findings support a novel “outside-in” model in which fatty acid availability sets cell envelope capacity, which in turn dictates cell size. In the absence of ppGpp, limiting fatty acid synthesis leads to cell lysis, supporting a role for ppGpp as a linchpin linking expansion of cytoplasmic volume to growth of the cell envelope to preserve cellular integrity.

Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.

Multicomponent efflux complexes remove antibiotics and other toxins from Gram-negative bacteria and thereby contribute to antibiotic resistance. Multicomponent efflux complexes can shift dynamically between an assembled functional state and a disassembled non-functional state. We have previously shown that mechanical stress promotes disassembly of CusCBA, an RND family efflux complex that is responsible for removing toxic Cu þ and Ag þ from Escherichia coli. It is unclear if mechanical stress also influences other efflux complexes. Here, we examine the effect of mechanical stress on the disassembly of MacAB-TolC, an efflux complex from the ABC family that contributes to resistance to macrolide antibiotics. Mechanical stress was applied to Escherichia coli using a microfluidic device that uses fluid pressure to force bacteria into narrow tapered channels where they experience mechanical stress and deformation. The assembly of the MacAB-TolC efflux complex was examined by measuring the motion of the inner membrane pump protein MacB (fast moving MacB was not assembled) in more than 600 cells at fluid pressure levels ranging from 0 to 30 kPa. At greater magnitudes of mechanical stress, the proportion of disassembled MacAB-TolC complexes increased (p=1.7*10 À5 ). MacB diffusivity decreased at greater levels of mechanical stress (p=1.9*10 À14 ). Our findings show that mechanical stress not only influences RND family efflux complexes but also ABC family efflux complexes that enable resistance to clinically relevant antibiotics.

Objective(s) Primary human trophoblasts were previously shown to be resistant to viral infection, and able to confer this resistance to non-trophoblast cells. Can trophoblasts protect non-trophoblastic cells from infection by viruses or other intracellular pathogens that are implicated in perinatal infection? Study Design Isolated primary term human trophoblasts were cultured for 72 h. Diverse non-placental human cell lines (U2OS, HFF, TZM-bl, MeWo, and Caco-2) were pre-exposed to either trophoblast conditioned, non-conditioned medium, or miR-517-3p for 24 h. Cells were infected with several viral and non-viral pathogens known to be associated with perinatal infections. Cellular infection was defined and quantified by plaque assays, luciferase assays, microscopy, and/or colonization assays. Differences in infection were assessed by Student's t-test or ANOVA with Bonferroni's correction. Results Infection by rubella and other togaviruses, HIV-1, and varicella zoster, was attenuated in cells pre-exposed to trophoblast conditioned medium (p <0.05), and a partial effect by the Ch.19 microRNA miR-517-3p on specific pathogens. The conditioned medium had no effect on infection by Toxoplasma gondii or Listeria monocytogenes. Conclusion Our findings indicate that medium conditioned by primary human trophoblasts attenuate viral infection in non-trophoblastic cells. Our data point to a trophoblast-specific antiviral effect that may be exploited therapeutically.

Research into the mechanisms regulating bacterial cell size has its origins in a single paper published over 50 years ago. In it Schaechter and colleagues made the observation that the chemical composition and size of a bacterial cell is a function of growth rate, independent of the medium used to achieve that growth rate, a finding that is colloquially referred to as the growth law. Recent findings hint at unforeseen complexity in the growth law, and suggest that nutrients rather than growth rate are the primary arbiter of size. The emerging picture suggests that size is a complex, multifactorial phenomenon mediated through the varied impacts of central carbon metabolism on cell cycle progression and biosynthetic capacity.

HIGHLIGHTS• The adder requires accumulation of division proteins to a threshold for division.• The adder requires constant production of division proteins during cell elongation.• In E. coli and B. subtilis, initiation and division are independently controlled. • In E. coli and B. subtilis, cell division exclusively drives size homeostasis. GRAPHICAL ABSTRACT eTOC Blurb Si and Le Treut et al. show that cell-size homeostasis in bacteria is exclusively driven by accumulation of division proteins to a threshold and their balanced biosynthesis during cell elongation. This mechanistic insight allowed them to reprogram cell-size homeostasis in both E. coli and B. subtilis. Evolutionary implications are discussed.ABSTRACT Evolutionarily divergent bacteria share a common phenomenological strategy for cell-size homeostasis under steady-state conditions. In the presence of inherent physiological stochasticity, cells following this "adder" principle gradually return to their steady-state size by adding a constant volume between birth and division regardless of their size at birth. However, the mechanism of the adder has been unknown despite intense efforts. In this work, we show that the adder is a direct consequence of two general processes in biology: (1) threshold --accumulation of initiators and precursors required for cell division to a respective fixed number, and (2) balanced biosynthesis -maintenance of their production proportional to volume growth. This mechanism is naturally robust to static growth inhibition, but also allows us to "reprogram" cell-size homeostasis in a quantitatively predictive manner in both Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. By generating dynamic oscillations in the concentration of the division protein FtsZ, we were able to oscillate cell size at division and systematically break the adder. In contrast, periodic induction of replication initiator protein DnaA caused oscillations in cell size at initiation, but did not alter division size or the adder. Finally, we were able to restore the adder phenotype in slow-growing E. coli, the only known steady-state growth condition wherein E. coli significantly deviates from the adder, by repressing active degradation of division proteins. Together these results show that cell division and replication initiation are independently controlled at the gene-expression level, and that division processes exclusively drive cell-size homeostasis in bacteria.labeled replisome protein (DnaN-YPet) to image replication cycles, and a microfluidic mother machine to follow continuous lineages during steady-state growth [3, 26] (Fig. 1A, Fig. S1; STAR Methods).A major technical challenge arises in studying replication dynamics when two replisome foci spatially overlap, which makes it difficult to analyze overlapping replication cycles. To resolve this issue, we tracked multiple replication forks from initiation to termination by extending previous imaging methods [8, 19,27,28] using the "intensity weighting" techniques [29,30] develo...

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