Differentiating the roles of proteins and polysomes in nucleoid size homeostasis in Escherichia coli.

A defining feature of the bacterial cytosolic interior is a distinct membrane-less organelle, the nucleoid, that contains the chromosomal DNA. Although increasing experimental evidence indicates that macromolecular crowding is the dominant mechanism for nucleoid formation, it has remained unclear which crowders control nucleoid volume. It is commonly assumed that polyribosomes play a dominant role, yet the volume fraction of soluble proteins in the cytosol is comparable with that of polyribosomes. Here, we develop a free energy-based model for the cytosolic interior of a bacterial cell to distinguish contributions arising from polyribosomes and cytosolic proteins in nucleoid volume control. The parameters of the model are determined from the existing experimental data. We show that, while the polysomes establish the existence of the nucleoid as a distinct phase, the proteins control the nucleoid volume in physiologically relevant conditions. Our model explains experimental findings in Escherichia coli that the nucleoid compaction curves in osmotic shock measurements do not depend on cell growth rate and that dissociation of polysomes in slow growth rates does not lead to significant nucleoid expansion, while the nucleoid phase disappears in fastest growth rates. Furthermore, the model predicts a cross-over in the exclusion of crowders by their linear dimensions from the nucleoid phase: below the cross-over of 30-50 nm, the concentration of crowders in the nucleoid phase decreases linearly as a function of the crowder diameter, while decreasing exponentially above the cross-over size. Our work points to the possibility that bacterial cells maintain nucleoid size and protein concentration homeostasis via feedback in which protein concentration controls nucleoid dimensions and the nucleoid dimensions control protein synthesis rate.

Using conditional independence tests to elucidate causal links in cell cycle regulation in Escherichia coli.

How cells regulate their cell cycles is a central question for cell biology. Models of cell size homeostasis have been proposed for bacteria, archaea, yeast, plant, and mammalian cells. New experiments bring forth high volumes of data suitable for testing existing models of cell size regulation and proposing new mechanisms. In this paper, we use conditional independence tests in conjunction with data of cell size at key cell cycle events (birth, initiation of DNA replication, and constriction) in the model bacterium Escherichia coli to select between the competing cell cycle models. We find that in all growth conditions that we study, the division event is controlled by the onset of constriction at midcell. In slow growth, we corroborate a model where replication-related processes control the onset of constriction at midcell. In faster growth, we find that the onset of constriction is affected by additional cues beyond DNA replication. Finally, we also find evidence for the presence of additional cues triggering initiations of DNA replication apart from the conventional notion where the mother cells solely determine the initiation event in the daughter cells via an adder per origin model. The use of conditional independence tests is a different approach in the context of understanding cell cycle regulation and it can be used in future studies to further explore the causal links between cell events.

Cell Cycle-Dependent Recruitment of FtsN to the Divisome in Escherichia coli.

Cell division in Escherichia coli starts with the formation of an FtsZ protofilament network at midcell, the Z ring. However, only after a considerable lag period does the cell start to form a midcell constriction. The onset of constriction depends upon the arrival of so-called late divisome proteins, among which, FtsN is the last essential one. The timing and dependency of FtsN arrival to the divisome, along with genetic evidence, suggests it triggers cell division. In this study, we used high-throughput fluorescence microscopy to determine the arrival of FtsN and the early divisome protein ZapA to midcell at a single-cell level during the cell cycle. Our data show while the recruitment of ZapA/FtsZ is gradual in the cell cycle, recruitment of FtsN is rapid and begins at about the onset of constriction. At this time, the fraction of ZapA/FtsZ in the Z ring approaches its peak value. We also find a second increase in FtsN recruitment to the divisome, which begins once the amount of ZapA/FtsZ at midcell starts decreasing. Increasing hypermorphic FtsA* (FtsA R286W), but not FtsA, accelerates FtsN recruitment but not constriction. This finding is consistent with FtsA* recruiting FtsN with some other divisome component being rate-limiting for constriction under these conditions. Finally, our data support the recently proposed idea that ZapA/FtsZ and FtsN are part of physically separate complexes in midcell throughout the whole septation process. IMPORTANCE Cell division in most bacteria starts with the formation of an FtsZ protofilament network at midcell, the Z ring. However, cells only start to constrict after a considerable lag. A factor thought to trigger the onset of constriction in Escherichia coli is FtsN, which is the last essential protein to be recruited to the Z ring. Using a high-throughput quantitative fluorescence microscopy, we determine the cell cycle-dependent recruitment of FtsN to the Z ring. Our data show rapid accumulation of FtsN to the Z ring about a quarter of the cell cycle after the formation of the Z ring. This initial wave is followed by another increase in FtsN recruitment once the FtsZ protofilament network starts to disassemble. The presence of FtsA* accelerates FtsN recruitment to the Z ring but does not lead to earlier constrictions. Our data furthermore suggest FtsZ and FtsN are part of physically separate complexes throughout the division process.

Coupling between DNA replication, segregation, and the onset of constriction in Escherichia coli.

Escherichia coli cell cycle features two critical cell-cycle checkpoints: initiation of replication and the onset of constriction. While the initiation of DNA replication has been extensively studied, it is less clear what triggers the onset of constriction and when exactly it occurs during the cell cycle. Here, using high-throughput fluorescence microscopy in microfluidic devices, we determine the timing for the onset of constriction relative to the replication cycle in different growth rates. Our single-cell data and modeling indicate that the initiation of constriction is coupled to replication-related processes in slow growth conditions. Furthermore, our data suggest that this coupling involves the mid-cell chromosome blocking the onset of constriction via some form of nucleoid occlusion occurring independently of SlmA and the Ter linkage proteins. This work highlights the coupling between replication and division cycles and brings up a new nucleoid mediated control mechanism in E. coli.

Distinguishing different modes of growth using single-cell data.

Collection of high-throughput data has become prevalent in biology. Large datasets allow the use of statistical constructs such as binning and linear regression to quantify relationships between variables and hypothesize underlying biological mechanisms based on it. We discuss several such examples in relation to single-cell data and cellular growth. In particular, we show instances where what appears to be ordinary use of these statistical methods leads to incorrect conclusions such as growth being non-exponential as opposed to exponential and vice versa. We propose that the data analysis and its interpretation should be done in the context of a generative model, if possible. In this way, the statistical methods can be validated either analytically or against synthetic data generated via the use of the model, leading to a consistent method for inferring biological mechanisms from data. On applying the validated methods of data analysis to infer cellular growth on our experimental data, we find the growth of length in E. coli to be non-exponential. Our analysis shows that in the later stages of the cell cycle the growth rate is faster than exponential.

All cells ­ from bacteria to humans ­ tightly control their size as they grow and divide. Cells can also change the speed at which they grow, and the pattern of how fast a cell grows with time is called 'mode of growth'. Mode of growth can be 'linear', when cells increase their size at a constant rate, or 'exponential', when cells increase their size at a rate proportional to their current size. A cell's mode of growth influences its inner workings, so identifying how a cell grows can reveal information about how a cell will behave. Scientists can measure the size of cells as they age and identify their mode of growth using single cell imaging techniques. Unfortunately, the statistical methods available to analyze the large amounts of data generated in these experiments can lead to incorrect conclusions. Specifically, Kar et al. found that scientists had been using specific types of plots to analyze growth data that were prone to these errors, and may lead to misinterpreting exponential growth as linear and vice versa. This discrepancy can be resolved by ensuring that the plots used to determine the mode of growth are adequate for this analysis. But how can the adequacy of a plot be tested? One way to do this is to generate synthetic data from a known model, which can have a specific and known mode of growth, and using this data to test the different plots. Kar et al. developed such a 'generative model' to produce synthetic data similar to the experimental data, and used these data to determine which plots are best suited to determine growth mode. Once they had validated the best statistical methods for studying mode of growth, Kar et al. applied these methods to growth data from the bacterium Escherichia coli. This showed that these cells have a form of growth called 'super-exponential growth'. These findings identify a strategy to validate statistical methods used to analyze cell growth data. Furthermore, this strategy ­ the use of generative models to produce synthetic data to test the accuracy of statistical methods ­ could be used in other areas of biology to validate statistical approaches.

Lab-on-a-chip based mechanical actuators and sensors for single-cell and organoid culture studies.

All living cells constantly experience and respond to mechanical stresses. The molecular networks that activate in cells in response to mechanical stimuli are yet not well-understood. Our limited knowledge stems partially from the lack of available tools that are capable of exerting controlled mechanical stress to individual cells and at the same time observing their responses at subcellular to molecular resolution. Several tools such as rheology setups, micropipetes, and magnetic tweezers have been used in the past. While allowing to quantify short-time viscoelastic responses, these setups are not suitable for long-term observations of cells and most of them have low throughput. In this Perspective, we discuss lab-on-a-chip platforms that have the potential to overcome these limitations. Our focus is on devices that apply shear, compressive, tensile, and confinement derived stresses to single cells and organoid cultures. We compare different design strategies for these devices and highlight their advantages, drawbacks, and future potential. While the majority of these devices are used for fundamental research, some of them have potential applications in medical diagnostics and these applications are also discussed.

The effects of polydisperse crowders on the compaction of the Escherichia coli nucleoid.

DNA binding proteins, supercoiling, macromolecular crowders, and transient DNA attachments to the cell membrane have all been implicated in the organization of the bacterial chromosome. However, it is unclear what role these factors play in compacting the bacterial DNA into a distinct organelle-like entity, the nucleoid. By analyzing the effects of osmotic shock and mechanical squeezing on Escherichia coli, we show that macromolecular crowders play a dominant role in the compaction of the DNA into the nucleoid. We find that a 30% increase in the crowder concentration from physiological levels leads to a three-fold decrease in the nucleoid's volume. The compaction is anisotropic, being higher along the long axes of the cell at low crowding levels. At higher crowding levels, the nucleoid becomes spherical, and its compressibility decreases significantly. Furthermore, we find that the compressibility of the nucleoid is not significantly affected by cell growth rates and by prior treatment with rifampicin. The latter results point out that in addition to poly ribosomes, soluble cytoplasmic proteins have a significant contribution in determining the size of the nucleoid. The contribution of poly ribosomes dominates at faster and soluble proteins at slower growth rates.

Transient Membrane-Linked FtsZ Assemblies Precede Z-Ring Formation in Escherichia coli.

During the early stages of cytokinesis, FtsZ protofilaments form a ring-like structure, the Z-ring, in most bacterial species. This cytoskeletal scaffold recruits downstream proteins essential for septal cell wall synthesis. Despite progress in understanding the dynamic nature of the Z-ring and its role in coordinating septal cell wall synthesis, the early stages of protofilament formation and subsequent assembly into the Z-ring are still not understood. Here we investigate a sequence of assembly steps that lead to the formation of the Z-ring in Escherichia coli using high temporal and spatial resolution imaging. Our data show that formation of the Z-ring is preceded by transient membrane-linked FtsZ assemblies. These assemblies form after attachment of short cytosolic protofilaments, which we estimate to be less than 20 monomers long, to the membrane. The attachments occur at random locations along the length of the cell. The filaments treadmill and show periods of rapid growth and shrinkage. Their dynamic properties imply that protofilaments are bundled in these assemblies. Furthermore, we establish that the size of assemblies is sensitively controlled by the availability of FtsZ molecules and by the presence of ZapA proteins. The latter has been implicated in cross-linking the protofilaments. The likely function of these dynamic FtsZ assemblies is to sample the cell surface for the proper location for the Z-ring.

Does the Nucleoid Determine Cell Dimensions in Escherichia coli?

Bacillary, Gram-negative bacteria grow by elongation with no discernible change in width, but during faster growth in richer media the cells are also wider. The mechanism regulating the change in cell width W during transitions from slow to fast growth is a fundamental, unanswered question in molecular biology. The value of W that changes in the divisome and during the division process only, is related to the nucleoid complexity, determined by the rates of growth and of chromosome replication; the former is manipulated by nutritional conditions and the latter-by thymine limitation of thyA mutants. Such spatio-temporal regulation is supported by existence of a minimal possible distance between successive replisomes, so-called eclipse that limits the number of replisomes to a maximum. Breaching this limit by slowing replication in fast growing cells results in maximal nucleoid complexity that is associated with maximum cell width, supporting the notion of Nucleoid-to-Divisome signal transmission. Physical signal(s) may be delivered from the nucleoid to assemble the divisome and to fix the value of W in the nascent cell pole.

Cell cycle-dependent regulation of FtsZ in Escherichia coli in slow growth conditions.

FtsZ is the key regulator of bacterial cell division. It initiates division by forming a dynamic ring-like structure, the Z-ring, at the mid-cell. What triggers the formation of the Z-ring during the cell cycle is poorly understood. In Escherichia coli, the common view is that FtsZ concentration is constant throughout its doubling time and therefore regulation of assembly is controlled by some yet-to-be-identified protein-protein interactions. Using a newly developed functional, fluorescent FtsZ reporter, we performed a quantitative analysis of the FtsZ concentration throughout the cell cycle under slow growth conditions. In contrast to the common expectation, we show that FtsZ concentrations vary in a cell cycle-dependent manner, and that upregulation of FtsZ synthesis correlates with the formation of the Z-ring. The first half of the cell cycle shows an approximately fourfold upregulation of FtsZ synthesis, followed by its rapid degradation by ClpXP protease in the last 10% of the cell cycle. The initiation of rapid degradation coincides with the dissociation of FtsZ from the septum. Altogether, our data suggest that the Z-ring formation in slow growth conditions in E. coli is partially controlled by a regulatory sequence wherein upregulation of an essential cell cycle factor is followed by its degradation.

Analysis of Factors Limiting Bacterial Growth in PDMS Mother Machine Devices.

The microfluidic mother machine platform has attracted much interest for its potential in studies of bacterial physiology, cellular organization, and cell mechanics. Despite numerous experiments and development of dedicated analysis software, differences in bacterial growth and morphology in narrow mother machine channels compared to typical liquid media conditions have not been systematically characterized. Here we determine changes in E. coli growth rates and cell dimensions in different sized dead-end microfluidic channels using high resolution optical microscopy. We find that E. coli adapt to the confined channel environment by becoming narrower and longer compared to the same strain grown in liquid culture. Cell dimensions decrease as the channel length increases and width decreases. These changes are accompanied by increases in doubling times in agreement with the universal growth law. In channels 100 µm and longer, cell doublings can completely stop as a result of frictional forces that oppose cell elongation. Before complete cessation of elongation, mechanical stresses lead to substantial deformation of cells and changes in their morphology. Our work shows that mechanical forces rather than nutrient limitation are the main growth limiting factor for bacterial growth in long and narrow channels.

Kinetics of large-scale chromosomal movement during asymmetric cell division in Escherichia coli.

Coordination between cell division and chromosome replication is essential for a cell to produce viable progeny. In the commonly accepted view, Escherichia coli realize this coordination via the accurate positioning of its cell division apparatus relative to the nucleoids. However, E. coli lacking proper positioning of its cell division planes can still successfully propagate. Here, we characterize how these cells partition their chromosomes into daughters during such asymmetric divisions. Using quantitative time-lapse imaging, we show that DNA translocase, FtsK, can pump as much as 80% (3.7 Mb) of the chromosome between daughters at an average rate of 1700±800 bp/s. Pauses in DNA translocation are rare, and in no occasions did we observe reversals at experimental time scales of a few minutes. The majority of DNA movement occurs at the latest stages of cell division when the cell division protein ZipA has already dissociated from the septum, and the septum has closed to a narrow channel with a diameter much smaller than the resolution limit of the microscope (~250 nm). Our data suggest that the narrow constriction is necessary for effective translocation of DNA by FtsK.

The role of MatP, ZapA and ZapB in chromosomal organization and dynamics in Escherichia coli.

Despite extensive research over several decades, a comprehensive view of how the Escherichia coli chromosome is organized within the nucleoid, and how two daughter chromosomes segregate has yet to emerge. Here we investigate the role of the MatP, ZapA and ZapB proteins in organizing the replication terminus (Ter) region and in the chromosomal segregation process. Quantitative image analysis of the fluorescently labeled Ter region shows that the replication terminus attaches to the divisome in a single segment along the perimeter of the cell in a MatP, ZapA and ZapB-dependent manner. The attachment does not significantly affect the bulk chromosome segregation in slow growth conditions. With or without the attachment, two chromosomal masses separate from each other at a speed comparable to the cell growth. The separation starts even before the replication terminus region positions itself at the center of the nucleoid. Modeling of the segregation based on conformational entropy correctly predicts the positioning of the replication terminus region within the nucleoid. However, the model produces a distinctly different chromosomal density distribution than the experiment, indicating that the conformational entropy plays a limited role in segregating the chromosomes in the late stages of replication.

Spatial coordination between chromosomes and cell division proteins in Escherichia coli.

To successfully propagate, cells need to coordinate chromosomal replication and segregation with cell division to prevent formation of DNA-less cells and cells with damaged DNA. Here, we review molecular systems in Escherichia coli that are known to be involved in positioning the divisome and chromosome relative to each other. Interestingly, this well-studied micro-organism has several partially redundant mechanisms to achieve this task; none of which are essential. Some of these systems determine the localization of the divisome relative to chromosomes such as SlmA-dependent nucleoid occlusion, some localize the chromosome relative to the divisome such as DNA translocation by FtsK, and some are likely to act on both systems such as the Min system and newly described Ter linkage. Moreover, there is evidence that E. coli harbors other divisome-chromosome coordination systems in addition to those known. The review also discusses the minimal requirements of coordination between chromosomes and cell division proteins needed for cell viability. Arguments are presented that cells can propagate without any dedicated coordination between their chromosomes and cell division machinery at the expense of lowered fitness.

Evidence for divisome localization mechanisms independent of the Min system and SlmA in Escherichia coli.

Cell division in Escherichia coli starts with assembly of FtsZ protofilaments into a ring-like structure, the Z-ring. Positioning of the Z-ring at midcell is thought to be coordinated by two regulatory systems, nucleoid occlusion and the Min system. In E. coli, nucleoid occlusion is mediated by the SlmA proteins. Here, we address the question of whether there are additional positioning systems that are capable of localizing the E. coli divisome with respect to the cell center. Using quantitative fluorescence imaging we show that slow growing cells lacking functional Min and SlmA nucleoid occlusion systems continue to divide preferentially at midcell. We find that the initial Z-ring assembly occurs over the center of the nucleoid instead of nucleoid-free regions under these conditions. We determine that Z-ring formation begins shortly after the arrival of the Ter macrodomain at the nucleoid center. Removal of either the MatP, ZapB, or ZapA proteins significantly affects the accuracy and precision of Z-ring positioning relative to the nucleoid center in these cells in accordance with the idea that these proteins link the Ter macrodomain and the Z-ring. Interestingly, even in the absence of Min, SlmA, and the putative Ter macrodomain - Z-ring link, there remains a weak midcell positioning bias for the Z-ring. Our work demonstrates that additional Z-ring localization systems are present in E. coli than are known currently. In particular, we identify that the Ter macrodomain acts as a landmark for the Z-ring in the presence of MatP, ZapB and ZapA proteins.

Robustness and accuracy of cell division in Escherichia coli in diverse cell shapes.

Cell division in typical rod-shaped bacteria such as Escherichia coli shows a remarkable plasticity in being able to adapt to a variety of irregular cell shapes. Here, we investigate the roles of the Min system and the nucleoid-occlusion factor SlmA in supporting this adaptation. We study "squeezed" E. coli in narrow nanofabricated channels where these bacteria exhibit highly irregular shapes and large volumes. Despite the severely anomalous morphologies we find that most of these bacteria maintain their ability to divide into two equally sized daughters with an accuracy comparable to that of normal rod-shaped cells (about 4%). Deletion of either slmA or minC shows that the molecular systems associated with these genes are largely dispensable for accurate cell division in these irregular cell shapes. Using fluorescence time-lapse microscopy, we determine that the functionality of the Min system is affected by the cell shape, whereas the localization of a nucleoid relative to the cell division proteins (the divisome) remains unperturbed in a broad spectrum of morphologies, consistent with nucleoid occlusion. The observed positioning of the nucleoid relative to the divisome appears not to be affected by the nucleoid-occlusion factor SlmA. The current study underscores the importance of nucleoid occlusion in positioning the divisome and shows that it is robust against shape irregularities.

Influence of electrolyte composition on liquid-gated carbon nanotube and graphene transistors.

Field-effect transistors based on single-walled carbon nanotubes (SWNTs) and graphene can function as highly sensitive nanoscale (bio)sensors in solution. Here, we compare experimentally how SWNT and graphene transistors respond to changes in the composition of the aqueous electrolyte in which they are immersed. We show that the conductance of SWNTs and graphene is strongly affected by changes in the ionic strength, the pH, and the type of ions present, in a manner that can be qualitatively different for graphene and SWNT devices. We show that this sensitivity to electrolyte composition results from a combination of different mechanisms including electrostatic gating, Schottky-barrier modifications, and changes in gate capacitance. Interestingly, we find strong evidence that the sensor response to changes in electrolyte composition is affected by a high density of ionizable groups on both the underlying substrate and the carbon surfaces. We present a model based on the (regulated) surface charge associated with these ionizable groups that explains the majority of our data. Our findings have significant implications for interpreting and optimizing sensing experiments with nanocarbon transistors. This is particularly true for complex biological samples such as cell extracts, growth media, or bodily fluids, for which the complete composition of the solution needs to be considered.

Charge noise in graphene transistors.

We report an experimental study of 1/f noise in liquid-gated graphene transistors. We show that the gate dependence of the noise is well described by a charge-noise model, whereas Hooge's empirical relation fails to describe the data. At low carrier density, the noise can be attributed to fluctuating charges in close proximity to the graphene, while at high carrier density it is consistent with noise due to scattering in the channel. The charge noise power scales inversely with the device area, and bilayer devices exhibit lower noise than single-layer devices. In air, the observed noise is also consistent with the charge-noise model.