SUMMARY How cells control their size and maintain size homeostasis is a fundamental open question. Cell-size homeostasis has been discussed in the context of two major paradigms: sizer, in which the cell actively monitors its size and triggers the cell cycle once it reaches a critical size, and timer, in which the cell attempts to grow for a specific amount of time before division. These paradigms, in conjunction with the “growth law” [1] and the quantitative bacterial cell cycle model [2], inspired numerous theoretical models [3-9] and experimental investigations from growth [10,11] to cell cycle and size control [12–15]. However, experimental evidence involved difficult-to-verify assumptions or population-averaged data, which allowed different interpretations [1–5,16–20] or limited conclusions [4–9]. In particular, population-averaged data and correlations are inconclusive as the averaging process masks causal effects at the cellular level. In this work, we extended a microfluidic “mother machine” [21] and monitored hundreds of thousands of Gram-negative Escherichia coli and Gram-positive Bacillus subtilis cells under a wide range of steady-state growth conditions. Our combined experimental results and quantitative analysis demonstrate that cells add a constant volume each generation irrespective of their newborn sizes, conclusively supporting the so-called constant Δ model. This model was introduced for E. coli [6,7] and recently revisited [9], but experimental evidence was limited to correlations. This “adder” principle quantitatively explains experimental data at both the population and single-cell levels, including the origin and the hierarchy of variability in the size-control mechanisms, and how cells maintain size homeostasis.

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How cells maintain a stable size has fascinated scientists since the beginning of modern biology, but has remained largely mysterious. Recently, however, the ability to analyze single bacteria in real time has provided new, important quantitative insights into this long-standing question in cell biology. In nature, cells can be as small as ∼0.2 mm (e.g., Mycoplasma gallicepticum) and as large as ∼0.1 m (e.g., Syringammina fragilissima), spanning almost six orders of magnitude. Individual organisms, however, show much narrower size distributions, and under constant conditions most single-celled microorganisms change their size by only two-fold between birth and division. For Escherichia coli, the variance of size distribution at division is ∼10% of the average [1], a strong indication that these cells know how to maintain stable size. KeywordsIn 1958, Schaechter, Maaløe, and Kjeldgaard established a general underlying principle in microbial physiology known as the 'growth law' [2]. It states that the average cell size is exponentially proportional to the average nutrient-imposed growth rate. That is, if we culture the cells in an unknown liquid medium X, we just need to measure the growth curve and we can predict the exact average size of the cells in that medium. What determines the cell size, and how do cells maintain their size under a given growth condition?Historically, cell size homeostasis has been discussed in the context of two major paradigms: sizer, in which the cell actively monitors its size and triggers the cell cycle once it reaches a critical size, and timer, in which the cell attempts to grow for a specific amount of time before division. Pinning down which model is correct poses daunting experimental challenges, because size control study requires quantitative measurements at the single-cell level with extreme precision [3] and throughput [4] under tightly controlled experimental conditions. It has only been in the past few years that the data with sufficient quantity and quality [4,5] have become available to address size maintenance in the way researchers since the 1950s dreamed of.The latest in the series of single-cell studies is the collaborative work by the Scherer and Dinner groups [5]. They studied Caulobacter crescentus, a model bacterial organism known for asymmetric cell division and cellular differentiation. Upon division, the two daughter cells of C. crescentus are distinct from each other in shape and size: the larger 'stalked' cell binds to a surface, whereas the smaller 'swarmer' cell is initially motile and differentiates into a stalked cell. This allowed the authors long-term continuous observations of growth and division of the stalked cells in a flow chamber, producing amounts of single-cell data comparable to previous work in E. coli [4]. The authors addressed two questions: (i) How do cell size and generation time change with respect to the temperature-imposed growth rate? (ii) What is the relationship between the size at birth and division?The answer to the first q...

Antimicrobial peptides (AMPs) are broad spectrum antibiotics that selectively target bacteria. Here we investigate the activity of human AMP LL37 against Escherichia coli by integrating quantitative, population and single-cell level experiments with theoretical modeling. We observe an unexpected, rapid absorption and retention of a large number of LL37 peptides by E. coli cells upon the inhibition of their growth, which increases population survivability. This transition occurs more likely in the late stage of cell division cycles. Cultures with high cell density exhibit two distinct subpopulations: a non-growing population that absorb peptides and a growing population that survive owing to the sequestration of the AMPs by others. A mathematical model based on this binary picture reproduces the rather surprising observations, including the increase of the minimum inhibitory concentration with cell density (even in dilute cultures) and the extensive lag in growth introduced by sub-lethal dosages of LL37 peptides.

Single-cell techniques have a long history of unveiling fundamental paradigms in biology. Recent improvements in the throughput, resolution, and availability of microfluidics, computational power, and genetically encoded fluorescence have led to a modern renaissance in microbial physiology. This resurgence in research activity has offered new perspectives on physiological processes such as growth, cell cycle, and cell size of model organisms such as Escherichia coli. We expect these single-cell techniques, coupled with the molecular revolution of biology’s recent half-century, to continue illuminating unforeseen processes and patterns in microorganisms, the bedrock of biological science. In this article we review major open questions in single-cell physiology, provide a brief introduction to the techniques for scientists of diverse backgrounds, and highlight some pervasive issues and their solutions.