Interconnecting solvent quality, transcription, and chromosome folding in Escherichia coli.

All cells fold their genomes, including bacterial cells, where the chromosome is compacted into a domain-organized meshwork called the nucleoid. How compaction and domain organization arise is not fully understood. Here, we describe a method to estimate the average mesh size of the nucleoid in Escherichia coli. Using nucleoid mesh size and DNA concentration estimates, we find that the cytoplasm behaves as a poor solvent for the chromosome when the cell is considered as a simple semidilute polymer solution. Monte Carlo simulations suggest that a poor solvent leads to chromosome compaction and DNA density heterogeneity (i.e., domain formation) at physiological DNA concentration. Fluorescence microscopy reveals that the heterogeneous DNA density negatively correlates with ribosome density within the nucleoid, consistent with cryoelectron tomography data. Drug experiments, together with past observations, suggest the hypothesis that RNAs contribute to the poor solvent effects, connecting chromosome compaction and domain formation to transcription and intracellular organization.

Subcellular Organization: A Critical Feature of Bacterial Cell Replication.

Spatial organization is a hallmark of all living systems. Even bacteria, the smallest forms of cellular life, display defined shapes and complex internal organization, showcasing a highly structured genome, cytoskeletal filaments, localized scaffolding structures, dynamic spatial patterns, active transport, and occasionally, intracellular organelles. Spatial order is required for faithful and efficient cellular replication and offers a powerful means for the development of unique biological properties. Here, we discuss organizational features of bacterial cells and highlight how bacteria have evolved diverse spatial mechanisms to overcome challenges cells face as self-replicating entities.

A constant size extension drives bacterial cell size homeostasis.

Cell size control is an intrinsic feature of the cell cycle. In bacteria, cell growth and division are thought to be coupled through a cell size threshold. Here, we provide direct experimental evidence disproving the critical size paradigm. Instead, we show through single-cell microscopy and modeling that the evolutionarily distant bacteria Escherichia coli and Caulobacter crescentus achieve cell size homeostasis by growing, on average, the same amount between divisions, irrespective of cell length at birth. This simple mechanism provides a remarkably robust cell size control without the need of being precise, abating size deviations exponentially within a few generations. This size homeostasis mechanism is broadly applicable for symmetric and asymmetric divisions, as well as for different growth rates. Furthermore, our data suggest that constant size extension is implemented at or close to division. Altogether, our findings provide fundamentally distinct governing principles for cell size and cell-cycle control in bacteria.

The bacterial cytoplasm has glass-like properties and is fluidized by metabolic activity.

The physical nature of the bacterial cytoplasm is poorly understood even though it determines cytoplasmic dynamics and hence cellular physiology and behavior. Through single-particle tracking of protein filaments, plasmids, storage granules, and foreign particles of different sizes, we find that the bacterial cytoplasm displays properties that are characteristic of glass-forming liquids and changes from liquid-like to solid-like in a component size-dependent fashion. As a result, the motion of cytoplasmic components becomes disproportionally constrained with increasing size. Remarkably, cellular metabolism fluidizes the cytoplasm, allowing larger components to escape their local environment and explore larger regions of the cytoplasm. Consequently, cytoplasmic fluidity and dynamics dramatically change as cells shift between metabolically active and dormant states in response to fluctuating environments. Our findings provide insight into bacterial dormancy and have broad implications to our understanding of bacterial physiology, as the glassy behavior of the cytoplasm impacts all intracellular processes involving large components.

Replication fork passage drives asymmetric dynamics of a critical nucleoid-associated protein in Caulobacter.

In bacteria, chromosome dynamics and gene expression are modulated by nucleoid-associated proteins (NAPs), but little is known about how NAP activity is coupled to cell cycle progression. Using genomic techniques, quantitative cell imaging, and mathematical modeling, our study in Caulobacter crescentus identifies a novel NAP (GapR) whose activity over the cell cycle is shaped by DNA replication. GapR activity is critical for cellular function, as loss of GapR causes severe, pleiotropic defects in growth, cell division, DNA replication, and chromosome segregation. GapR also affects global gene expression with a chromosomal bias from origin to terminus, which is associated with a similar general bias in GapR binding activity along the chromosome. Strikingly, this asymmetric localization cannot be explained by the distribution of GapR binding sites on the chromosome. Instead, we present a mechanistic model in which the spatiotemporal dynamics of GapR are primarily driven by the progression of the replication forks. This model represents a simple mechanism of cell cycle regulation, in which DNA-binding activity is intimately linked to the action of DNA replication.

DNA-relay mechanism is sufficient to explain ParA-dependent intracellular transport and patterning of single and multiple cargos.

Spatial ordering of macromolecular components inside cells is important for cellular physiology and replication. In bacteria, ParA/B systems are known to generate various intracellular patterns that underlie the transport and partitioning of low-copy-number cargos such as plasmids. ParA/B systems consist of ParA, an ATPase that dimerizes and binds DNA upon ATP binding, and ParB, a protein that binds the cargo and stimulates ParA ATPase activity. Inside cells, ParA is asymmetrically distributed, forming a propagating wave that is followed by the ParB-rich cargo. These correlated dynamics lead to cargo oscillation or equidistant spacing over the nucleoid depending on whether the cargo is in single or multiple copies. Currently, there is no model that explains how these different spatial patterns arise and relate to each other. Here, we test a simple DNA-relay model that has no imposed asymmetry and that only considers the ParA/ParB biochemistry and the known fluctuating and elastic dynamics of chromosomal loci. Stochastic simulations with experimentally derived parameters demonstrate that this model is sufficient to reproduce the signature patterns of ParA/B systems: the propagating ParA gradient correlated with the cargo dynamics, the single-cargo oscillatory motion, and the multicargo equidistant patterning. Stochasticity of ATP hydrolysis breaks the initial symmetry in ParA distribution, resulting in imbalance of elastic force acting on the cargo. Our results may apply beyond ParA/B systems as they reveal how a minimal system of two players, one binding to DNA and the other modulating this binding, can transform directionally random DNA fluctuations into directed motion and intracellular patterning.

The Slow Mobility of the ParA Partitioning Protein Underlies Its Steady-State Patterning in Caulobacter.

In bacteria, ParABS systems mediate intracellular transport of various cargos, including chromosomal regions in Caulobacter crescentus. Transport of the ParB/parS partition complex requires the DNA-binding activity of ParA, which transiently tethers the partition complex during translocation. In C. crescentus, the directionality of the transport is set up by a gradient of ParA whose concentration gradually increases from one end of the cell (old pole) to the other (new pole). Importantly, this ParA gradient is already observed before DNA replication and segregation are initiated when the partition complex is anchored at the old pole. How such micron-scale ParA pattern is established and maintained before the initiation of chromosome segregation has not been experimentally established. Although the stimulation of ParA ATPase activity by the localized ParB/parS partition complex is thought to be involved, this activity alone cannot quantitatively describe the ParA pattern observed inside cells. Instead, our experimental and theoretical study shows that the missing key component for achieving the experimentally observed steady-state ParA patterning is the slow mobility of ParA dimers (D ∼10(-3)µm(2)/s) due to intermittent DNA binding. Our model recapitulates the entire steady-state ParA distribution observed experimentally, including the shape of the gradient as well as ParA accumulation at the location of the partition complex. Stochastic simulations suggest that cell-to-cell variability in ParA pattern is due to the low ParA copy number in C. crescentus cells. The model also accounts for an apparent exclusion of ParA from regions with small spacing between partition complexes observed in filamentous cells. Collectively, our work demonstrates that in addition to its function in mediating transport, the conserved DNA-binding property of ParA has a critical function before DNA segregation by setting up a ParA pattern required for transport directionality.

The condensation of HP1α/Swi6 imparts nuclear stiffness.

Biomolecular condensates have emerged as major drivers of cellular organization. It remains largely unexplored, however, whether these condensates can impart mechanical function(s) to the cell. The heterochromatin protein HP1α (Swi6 in Schizosaccharomyces pombe) crosslinks histone H3K9 methylated nucleosomes and has been proposed to undergo condensation to drive the liquid-like clustering of heterochromatin domains. Here, we leverage the genetically tractable S. pombe model and a separation-of-function allele to elucidate a mechanical function imparted by Swi6 condensation. Using single-molecule imaging, force spectroscopy, and high-resolution live-cell imaging, we show that Swi6 is critical for nuclear resistance to external force. Strikingly, it is the condensed yet dynamic pool of Swi6, rather than the chromatin-bound molecules, that is essential to imparting mechanical stiffness. Our findings suggest that Swi6 condensates embedded in the chromatin meshwork establish the emergent mechanical behavior of the nucleus as a whole, revealing that biomolecular condensation can influence organelle and cell mechanics.

An ESCRT grommet cooperates with a diffusion barrier to maintain nuclear integrity.

The molecular mechanisms by which the endosomal sorting complexes required for transport (ESCRT) proteins contribute to the integrity of the nuclear envelope (NE) barrier are not fully defined. We leveraged the single NE hole generated by mitotic extrusion of the Schizosaccharomyces pombe spindle pole body to reveal two modes of ESCRT function executed by distinct complements of ESCRT-III proteins, both dependent on CHMP7/Cmp7. A grommet-like function is required to restrict the NE hole in anaphase B, whereas replacement of Cmp7 by a sealing module ultimately closes the NE in interphase. Without Cmp7, nucleocytoplasmic compartmentalization remains intact despite NE discontinuities of up to 540 nm, suggesting mechanisms to limit diffusion through these holes. We implicate spindle pole body proteins as key components of a diffusion barrier acting with Cmp7 in anaphase B. Thus, NE remodelling mechanisms cooperate with proteinaceous diffusion barriers beyond nuclear pore complexes to maintain the nuclear compartment.

Mathematical modeling of a minimal protocell with coordinated growth and division.

Self-replication is an essential attribute of life but the molecular-level mechanisms involved are not well understood. Cellular self-replication requires not only duplicating all cellular components and doubling volume and membrane area, but also replicating cellular geometry. A whole-cell modeling framework is presented in which an assumed reaction network determines both concentration changes of cellular components and cell geometry. Cell shape is calculated by minimizing membrane-bending energy. Using this framework, simultaneous doubling of volume, surface area, and all components was found to be insufficient to provide mid-cell "pinching" of the parental cell to form two daughter cells. This prompted the design of a minimal protocell that includes a growing shell, a cell-cycle engine, and a contractile ring to enforce cytokinesis. Kinetic parameters were found such that the system exhibited periodic behavior with fundamental aspects of self-replication. This involved simultaneous doubling of all cellular components during a cell cycle, doubling cell volume and membrane area, achieving periodic changes in surface/volume ratio, and forming daughter cells that were geometrically equivalent to each other and to the "newborn" parental cell. The results presented here impact the design of laboratory protocells and the development of a modular strategy for constructing a comprehensive in silico whole-cell model.

Kinetic modeling of the assembly, dynamic steady state, and contraction of the FtsZ ring in prokaryotic cytokinesis.

Cytokinesis in prokaryotes involves the assembly of a polymeric ring composed of FtsZ protein monomeric units. The Z ring forms at the division plane and is attached to the membrane. After assembly, it maintains a stable yet dynamic steady state. Once induced, the ring contracts and the membrane constricts. In this work, we present a computational deterministic biochemical model exhibiting this behavior. The model is based on biochemical features of FtsZ known from in vitro studies, and it quantitatively reproduces relevant in vitro data. An essential part of the model is a consideration of interfacial reactions involving the cytosol volume, where monomeric FtsZ is dispersed, and the membrane surface in the cell's mid-zone where the ring is assembled. This approach allows the same chemical model to simulate either in vitro or in vivo conditions by adjusting only two geometrical parameters. The model includes minimal reactions, components, and assumptions, yet is able to reproduce sought-after in vivo behavior, including the rapid assembly of the ring via FtsZ-polymerization, the formation of a dynamic steady state in which GTP hydrolysis leads to the exchange of monomeric subunits between cytoplasm and the ring, and finally the induced contraction of the ring. The model gives a quantitative estimate for coupling between the rate of GTP hydrolysis and of FtsZ subunit turnover between the assembled ring and the cytoplasmic pool as observed. Membrane constriction is chemically driven by the strong tendency of GTP-bound FtsZ to self-assembly. The model suggests a possible mechanism of membrane contraction without a motor protein. The portion of the free energy of GTP hydrolysis released in cyclization is indirectly used in this energetically unfavorable process. The model provides a limit to the mechanistic complexity required to mimic ring behavior, and it highlights the importance of parallel in vitro and in vivo modeling.

Nickel-dependent oligomerization of the alpha subunit of acetyl-coenzyme a synthase/carbon monoxide dehydrogenase.

After activation with NiCl2, the recombinant alpha subunit of the Ni-containing alpha2beta2 acetyl-CoA synthase/carbon monoxide dehydrogenase (ACS/CODH) catalyzes the synthesis of acetyl-CoA from CO, CoA, and a methyl group donated from the corrinoid-iron-sulfur protein (CoFeSP). The alpha subunit has two conformations (open and closed), and contains a novel [Fe4S4]-[Nip Nid] active site in which the proximal Nip ion is labile. Prior to Ni activation, recombinant apo-alpha contain only an Fe4S4 cluster. Ni-activated alpha subunits exhibit catalytic, spectroscopic and heterogeneity properties typical of alpha subunits contained in ACS/CODH. Evidence presented here indicates that apo-alpha is a monomer whereas Ni-treated alpha oligomerizes, forming dimers and higher molecular weight species including tetramers. No oligomerization occurred when apo-alpha was treated with Cu(II), Zn(II), or Co(II) ions, but oligomerization occurred when apo-alpha was treated with Pt(II) and Pd(II) ions. The dimer accepted only 0.5 methyl group/alpha and exhibited, upon treatment with CO and under reducing conditions, the NiFeC EPR signal quantifying to 0.4 spin/alpha. Dimers appear to consist of two types of alpha subunits, including one responsible for catalytic activity and one that provides a structural scaffold. Higher molecular weight species may be similarly constituted. It is concluded that Ni binding to the A-cluster induces a conformational change in the alpha subunit, possibly to the open conformation, that promotes oligomerization. These interrelated events demonstrate previously unrealized connections between (a) the conformation of the alpha subunit; (b) the metal which occupies the proximal/distal sites of the A-cluster; and (c) catalytic activity.

Kinetics of CO insertion and acetyl group transfer steps, and a model of the acetyl-CoA synthase catalytic mechanism.

Acetyl-CoA synthase/carbon monoxide dehydrogenase is a Ni-Fe-S-containing enzyme that catalyzes the synthesis of acetyl-CoA from CO, CoA, and a methyl group. The methyl group is transferred onto the enzyme from a corrinoid-iron-sulfur protein (CoFeSP). The kinetics of two steps within the catalytic mechanism were studied using the stopped-flow method, including the insertion of CO into a putative Ni(2+)-CH(3) bond and the transfer of the resulting acetyl group to CoA. Neither step had been studied previously. Reactions were monitored indirectly, starting with the methylated intermediate form of the enzyme. Resulting traces were analyzed by constructing a simple kinetic model describing the catalytic mechanism under reducing conditions. Besides methyl group transfer, CO insertion, and acetyl group transfer, fitting to experimental traces required the inclusion of an inhibitory step in which CO reversibly bound to the form of the enzyme obtained immediately after product release. Global simulation of the reported datasets afforded a consistent set of kinetic parameters. The equilibrium constant for the overall synthesis of acetyl-CoA was estimated and compared to the product of the individual equilibrium constants. Simulations obtained with the model duplicated the essential behavior of the enzyme, in terms of the variation of activity with [CO], and the time-dependent decay of the NiFeC EPR signal upon reaction with CoFeSP. Under standard assay conditions, the model suggests that the vast majority of active enzyme molecules in a population should be in the methylated form, suggesting that the subsequent catalytic step, namely CO insertion, is rate limiting. This conclusion is further supported by a sensitivity analysis showing that the rate is most sensitively affected by a change in the rate coefficient associated with the CO insertion step.

Mössbauer and EPR study of recombinant acetyl-CoA synthase from Moorella thermoacetica.

Mössbauer and EPR spectroscopies were used to study the electronic structure of the A-cluster from recombinant acetyl-CoA synthase (the alpha subunit of the alpha2beta2 acetyl-CoA synthase/CO dehydrogenase). Once activated with Ni, these subunits have properties mimicking those associated with the alpha2beta2 tetramer, including structural heterogeneities. The Fe4S4 portion of the A-cluster in oxidized, methylated, and acetylated states was in the 2+ core oxidation state. Upon reduction with dithionite or Ti3+ citrate, samples of Ni-activated alpha developed the ability to accept a methyl group. Corresponding Mössbauer spectra exhibited two populations of A-clusters; roughly, 70% contained [Fe4S4]1+ cubanes, while approximately 30% contained [Fe4S4]2+ cubanes, suggesting an extremely low [Fe4S4](1+/2+) reduction potential for the 30% portion (perhaps <-800 mV vs NHE). The same population ratio was observed when Ni-free unactivated alpha was used. The 70% fraction exhibited paramagnetic hyperfine structure in the absence of an applied magnetic field, excluding the possibility that it represents an [Fe4S4]1+ cluster coupled to a (proximal) Ni(p)1+. EPR spectra of dithionite-reduced, Ni-activated alpha exhibited features at g = 5.8 and g(ave) approximately 1.93, consistent with a physical mixture of {S = 3/2; S = 1/2} spin-states for A-clusters containing [Fe4S4]1+ clusters. Incubation of Ni-activated alpha with dithionite and CO converted 25% of alpha subunits into the S = 1/2 A(red)-CO state. Previous correlation of this state to functional A-clusters suggests that only the 30% fraction not reduced by dithionite or Ti3+ citrate represents functional A-clusters. Comparison of spin states in oxidized and methylated states suggests that two electrons are required for reductive activation, starting from the oxidized state containing Ni(p)2+. Refitting published activity-vs-potential data supports an n = 2 reductive activation. Enzyme starting in the methylated state exhibited catalytic activity in the absence of an external reductant, suggesting that the two electrons used in reductive activation are retained by the enzyme after each catalytic cycle and that the enzyme does not have to pass through the A(red)-CO state during catalysis. Taken together, our results suggest that a Ni(p)0 state may form upon reductive activation and reform after each catalytic cycle.

A framework for whole-cell mathematical modeling.

The default framework for modeling biochemical processes is that of a constant-volume reactor operating under steady-state conditions. This is satisfactory for many applications, but not for modeling growth and division of cells. In this study, a whole-cell modeling framework is developed that assumes expanding volumes and a cell-division cycle. A spherical newborn cell is designed to grow in volume during the growth phase of the cycle. After 80% of the cycle period, the cell begins to divide by constricting about its equator, ultimately affording two spherical cells with total volume equal to twice that of the original. The cell is partitioned into two regions or volumes, namely the cytoplasm (Vcyt) and membrane (Vmem), with molecular components present in each. Both volumes change during the cell cycle; Vcyt changes in response to osmotic pressure changes as nutrients enter the cell from the environment, while Vmem changes in response to this osmotic pressure effect such that membrane thickness remains invariant. The two volumes change at different rates; in most cases, this imposes periodic or oscillatory behavior on all components within the cell. Since the framework itself rather than a particular set of reactions and components is responsible for this behavior, it should be possible to model various biochemical processes within it, affording stable periodic solutions without requiring that the biochemical process itself generates oscillations as an inherent feature. Given that these processes naturally occur in growing and dividing cells, it is reasonable to conclude that the dynamics of component concentrations will be more realistic than when modeled within constant-volume and/or steady-state frameworks. This approach is illustrated using a symbolic whole cell model.