Dispensability of extrinsic DnaA regulators in cell-cycle control.

Investigating a long-standing conceptual question in bacterial physiology, we examine why DnaA, the bacterial master replication initiator protein, exists in both ATP and ADP forms, despite only the ATP form being essential for initiation. We engineered the Δ4 strain, devoid of all known external elements facilitating the DnaA-ATP/ADP conversion and found that these cells display nearly wild-type behaviors under nonoverlapping replication cycles. However, during rapid growth with overlapping cycles, Δ4 cells exhibit initiation instability.

Tools and methods for high-throughput single-cell imaging with the mother machine.

Despite much progress, image processing remains a significant bottleneck for high-throughput analysis of microscopy data. One popular platform for single-cell time-lapse imaging is the mother machine, which enables long-term tracking of microbial cells under precisely controlled growth conditions. While several mother machine image analysis pipelines have been developed in the past several years, adoption by a non-expert audience remains a challenge.

Bacterial Replication Initiation as Precision Control by Protein Counting.

Balanced biosynthesis is the hallmark of bacterial cell physiology, where the concentrations of stable proteins remain steady. However, this poses a conceptual challenge to modeling the cell-cycle and cell-size controls in bacteria, as prevailing concentration-based eukaryote models are not directly applicable. In this study, we revisit and significantly extend the initiator-titration model, proposed 30 years ago, and we explain how bacteria precisely and robustly control replication initiation based on the mechanism of protein copy-number sensing.

Replication initiation in bacteria: precision control based on protein counting.

Balanced biosynthesis is the hallmark of bacterial cell physiology, where the concentrations of stable proteins remain steady. However, this poses a conceptual challenge to modeling the cell-cycle and cell-size controls in bacteria, as prevailing concentration-based eukaryote models are not directly applicable. In this study, we revisit and significantly extend the initiator-titration model, proposed thirty years ago, and explain how bacteria precisely and robustly control replication initiation based on the mechanism of protein copy-number sensing.

Tools and methods for high-throughput single-cell imaging with the mother machine.

Despite much progress, image processing remains a significant bottleneck for high-throughput analysis of microscopy data. One popular platform for single-cell time-lapse imaging is the mother machine, which enables long-term tracking of microbial cells under precisely controlled growth conditions. While several mother machine image analysis pipelines have been developed in the past several years, adoption by a non-expert audience remains a challenge.

Quantitative Examination of Five Stochastic Cell-Cycle and Cell-Size Control Models for and .

We examine five quantitative models of the cell-cycle and cell-size control in and that have been proposed over the last decade to explain single-cell experimental data generated with high-throughput methods. After presenting the statistical properties of these models, we test their predictions against experimental data. Based on simple calculations of the defining correlations in each model, we first dismiss the stochastic Helmstetter-Cooper model and the Initiation Adder model, and show that both the Replication Double Adder (RDA) and the Independent Double Adder (IDA) model are more consistent with the data than the other models.

Control of Bacillus subtilis Replication Initiation during Physiological Transitions and Perturbations.

and are evolutionarily divergent model organisms whose analysis has enabled elucidation of fundamental differences between Gram-positive and Gram-negative bacteria, respectively. Despite their differences in cell cycle control at the molecular level, the two organisms follow the same phenomenological principle, known as the adder principle, for cell size homeostasis. We thus asked to what extent and share common physiological principles in coordinating growth and the cell cycle.

Cell Boundary Confinement Sets the Size and Position of the E. coli Chromosome.

Although the spatiotemporal structure of the genome is crucial to its biological function, many basic questions remain unanswered on the morphology and segregation of chromosomes. Here, we experimentally show in Escherichia coli that spatial confinement plays a dominant role in determining both the chromosome size and position. In non-dividing cells with lengths increased to 10 times normal, single chromosomes are observed to expand > 4-fold in size.

Mechanistic Origin of Cell-Size Control and Homeostasis in Bacteria.

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.

Synthesis and degradation of FtsZ quantitatively predict the first cell division in starved bacteria.

In natural environments, microbes are typically non-dividing and gauge when nutrients permit division. Current models are phenomenological and specific to nutrient-rich, exponentially growing cells, thus cannot predict the first division under limiting nutrient availability. To assess this regime, we supplied starving with glucose pulses at increasing frequencies.

Fundamental principles in bacterial physiology-history, recent progress, and the future with focus on cell size control: a review.

Bacterial physiology is a branch of biology that aims to understand overarching principles of cellular reproduction. Many important issues in bacterial physiology are inherently quantitative, and major contributors to the field have often brought together tools and ways of thinking from multiple disciplines. This article presents a comprehensive overview of major ideas and approaches developed since the early 20th century for anyone who is interested in the fundamental problems in bacterial physiology.

Protocol for Construction of a Tunable CRISPR Interference (tCRISPRi) Strain for .

We present a protocol for construction of tunable CRISPR interference (tCRISPRi) strains for . The tCRISPRi system alleviates most of the known problems of plasmid-based expression methods, and can be immediately used to construct libraries of sgRNAs that can complement the Keio collection by targeting both essential and nonessential genes. Most importantly from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step oligo recombineering.

Isolation and Characterization of Bacterial Nucleoids in Microfluidic Devices.

We report methods for isolation of Escherichia coli nucleoids in microfluidic devices, allowing characterization of nucleoids during a controlled in vivo to in vitro transition. Biochemically, nucleoids are isolated by gentle osmotic lysis, which minimally perturbs nucleoid-associated proteins (NAPs). Biophysically, nucleoids are isolated in microfluidic chambers, which mimic confinement within the cell, as well as facilitate diffusive buffer exchange around nucleoids without subjecting them to flow.

A Fundamental Unit of Cell Size in Bacteria.

A new study clarifies a relationship between growth, gene expression, and cell size in cyanobacteria. Quite unexpectedly, cyanobacteria and Escherichia coli appear to share an invariance principle to coordinate growth and chromosome replication. This principle allows quantitative predictions of cell size across a range of growth conditions in both organisms.

Invariance of Initiation Mass and Predictability of Cell Size in Escherichia coli.

It is generally assumed that the allocation and synthesis of total cellular resources in microorganisms are uniquely determined by the growth conditions. Adaptation to a new physiological state leads to a change in cell size via reallocation of cellular resources. However, it has not been understood how cell size is coordinated with biosynthesis and robustly adapts to physiological states.

tCRISPRi: tunable and reversible, one-step control of gene expression.

The ability to control the level of gene expression is a major quest in biology. A widely used approach employs deletion of a nonessential gene of interest (knockout), or multi-step recombineering to move a gene of interest under a repressible promoter (knockdown). However, these genetic methods are laborious, and limited for quantitative study.

Adder and a coarse-grained approach to cell size homeostasis in bacteria.

Cell size control and homeostasis is a long-standing subject in biology. Recent experimental work provides extensive evidence for a simple, quantitative size homeostasis principle coined adder (as opposed to sizer or timer). The adder principle provides unexpected insights into how bacteria maintain their size without employing a feedback mechanism.

Complete Genome Sequence of Escherichia coli NCM3722.

Escherichia coli NCM3722 is a prototrophic K-12 strain with robust physiologic phenotypes. We report the complete 4,678,045-bp chromosome and 67,545-bp F-like plasmid of this unique model organism.

Single-Cell Physiology.

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.

Cell-size control and homeostasis in bacteria.

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].

Cell-size maintenance: universal strategy revealed.

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.