Hypothesis: bacteria live on the edge of phase transitions with a cell cycle regulated by a water-clock

doi:10.21203/rs.3.rs-3930821/v1

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Hypothesis: bacteria live on the edge of phase transitions with a cell cycle regulated by a water-clock

https://doi.org/10.21203/rs.3.rs-3930821/v1

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A fundamental problem in biology is how cells obtain the reproducible, coherent phenotypes needed for natural selection to act or, put differently, how cells manage to limit their exploration of the vastness of phenotype space. A subset of this problem is how they regulate their cell cycle. Bacteria, like eukaryotic cells, are highly structured and contain scores of hyperstructures or assemblies of molecules and macromolecules. The existence and functioning of certain of these hyperstructures depend on phase transitions. Here, I propose a conceptual framework to facilitate the development of water-clock hypotheses in which cells use water to generate phenotypes by living ‘on the edge of phase transitions’. I give an example of such a hypothesis in the case of the bacterial cell cycle and show how it offers a relatively novel ‘view from here’ that brings together a range of different findings about hyperstructures, phase transitions and water and that can be integrated with other hypotheses about differentiation, metabolism and the origins of life.

Cell cycle

phase separation

assembly

DNA replication

water

crowding

There are many problems in the physiology of bacterial cells that are often considered to have been solved or, at least, nearly solved. These problems include the nature of the controls over the cell cycle and differentiation. Many of the leading players in a cast of thousands of macromolecules have been identified and the complicated circuits in which they figure have been elucidated. It is, of course, hardly surprising that very specific, regulatory macromolecules and circuits are important given that bacteria have been evolving for billions of years in vast numbers (possibly in a super-population of 10^30 cells and 10^31 phages). It is therefore likely that these regulatory systems are different from those that operated in protocells, which at some stage in evolution regulated their cell cycles. These early systems probably comprised networks of interactions between simple, universal, molecules and ions (Norris et al., 2015). It could be argued that the nature of the first system(s) regulating the cell cycle is fundamental to understanding the physiology of modern bacteria. This is because there was presumably a strong evolutionary pressure for these first regulatory systems to control many major aspects of cell physiology: the regulatory relationships between mass, growth rate, initiation of chromosome replication and cell division; to these might be added the regulation of differentiation, of phenotypic diversity and of catabolism versus anabolism. In an ideal world, discovering the principles of the first regulatory systems would allow identification of these principles across species – and even across phyla – and would lead to one or more paradigm shifts (Norris, 2019b).

The importance of phase separation and transition as an organising principle in cell structure and regulation is becoming increasingly evident (Shin & Brangwynne, 2017)(Yoo et al., 2019)(Ramm et al., 2023)(Cammarata et al., 2023). Phase transition refers to any transition from one phase of matter to another whilst phase separation refers specifically to the transition of a single liquid phase of mutually soluble components to two or more distinct liquid phases. Phase transitions come in two flavours (Ball, 2004). In a first-order phase transition, a property of the system (like density) changes sharply at the point of transition (like the melting of ice) and involves latent heat; in a second-order or continuous phase transition, a property of the system falls gradually to zero as a critical point is approached (in the case of water, the abrupt phase transition from liquid to gas disappears over 374ºC and water can go smoothly from a liquid-like density to a gas-like density by changing the pressure). Here, the critical point is known as the critical temperature and temperature is called the control parameter. In cells, phase transitions occur at the level of hyperstructures, where a hyperstructure is loosely defined as a spatially distinct group of cellular molecules and macromolecules that serves a function. Hyperstructures can be classed as either non-equilibrium or equilibrium; the former depend for their existence on a flow of energy whilst the latter do not. They can also go from one class to another as in the case of a non-equilibrium RecA hyperstructure, which consumes energy in repairing DNA damage and which, if DNA damage continues and ATP levels fall, collapses into RecA-DNA co-crystal or an equilibrium hyperstructure (Minsky et al., 2002). A bacterium contains several different types of hyperstructures, which operate at a level intermediate between the cell and the macromolecule (Norris et al., 2007a). Determining the phenotype at this intermediate level would reduce the enormous number of potential phenotypes apparently available to cells from the combinations of hundreds if not thousands of expressed genes (Kauffman, 1996)(Norris & Ripoll, 2021). Such reduction, however, may be insufficient to provide the selection of a coherent subset of hyperstructures needed for cells to have a coherent phenotype. Achieving such coherence would be facilitated by a universal principle that could act on hyperstructures so as to organise metabolism, regulate the cell cycle, orchestrate differentiation and diversity, and integrate the phenotype with the environment.

In looking for candidates for a universal principle that might operate as a ‘language’ at the level of hyperstructures and phase transitions, there is one candidate that hides in plain sight: water (Ball, 2008b). Water makes up around 70% of cellular mass and 99% of the number of the cell’s molecules. Water structures enzymes, membranes and nucleic acids (Bellissent-Funel et al., 2016). Water participates directly in many reactions as a reactant or a product (Dargaville & Hutmacher, 2022), it acts as a reactive nucleophile and proton donor and acceptor, and mediates electrostatic interactions (Ball, 2017). Water undergoes fluctuations and abrupt phase-transition–like changes in wetting and dewetting that can serve biological functions (Patel et al., 2012a). Water permits the diffusion of molecules and macromolecules within the cell. Water determines molecular and macromolecular crowding in the cytoplasm (Rivas & Minton, 2022). The water around cellular molecules has important properties that differ greatly from those of bulk water (though there is much argument about the nature and extent of this hydration or interfacial water (Hwang et al., 2018)(Elton et al., 2020)). Intracellular water reflects the physical and chemical nature of the cell’s environment. In some approaches to the origins of life, water is ascribed the primary role via cycles of wetting and drying (Hassenkam et al., 2020) whilst in the search for extra-terrestrial life (Stevenson et al., 2015), the presence of water is used as an indicator following Szent-Győrgi’s claim that there was no life without it (Ball, 2017). As regards regulation, the first known clocks are believed to have been based on water (Wikipedia, 2023). The water potential is the total thermodynamic potential energy of water, which has a maximum value of zero and which is lowered by the binding of water molecules to solutes; the result is that water moves from regions of higher water potential to regions of lower potential (i.e., osmosis). Here, I propose that this availability of water be used as the basis of a conceptual framework within which hypotheses could be developed about phenotypes, hyperstructures and life on the edge of phase shifts/transitions. To illustrate the possible use of this framework, I give a speculative example of how one such ‘water-sensor’ or ‘water-clock’ hypothesis could regulate the bacterial cell cycle.

In this simplifying, phase transition-based, hypothesis, only two sorts of water are considered: (1) free or bulk water and (2) structured water within the hydration layers of solutes (such as cellular molecules and macromolecules). Catabolism is considered as producing free water along with energy production whilst anabolism is considered as requiring it (despite catabolic enzymes requiring water and certain anabolic reactions producing water). These two sorts of water govern the functioning of hyperstructures by determining (1) the existence, size, composition and fluidity of the hyperstructures, (2) the conformations and activities of their constituents, and (3) the relationships between hyperstructures.

The water that is part of the clock is physically associated with (1) particular catabolic hyperstructures such as the glycolytic hyperstructure (which contains the glycolytic enzymes) and (2) particular anabolic hyperstructures such as the ‘nucleolar’ or ribosomal hyperstructure (which contains the nascent ribosomes) and the replication hyperstructure (which contains the enzymes needed for DNA replication). Whilst the quantity of total water in the cell may be modulated by the flux over the membrane and by ions and macromolecules, the water of the clock is essentially passed from catabolic hyperstructures to anabolic hyperstructures. The balance of all the hyperstructure-based, metabolic processes results in the quantity of available water initially decreasing during the growth of a cell. As this quantity decreases it reaches a threshold at which there is a set of phase transitions that comprise changes to percolation, molecular and macromolecular crowding and exclusion volumes. These changes alter the structure, activity and distribution of certain hyperstructures, which in turn result in the initiation of the replication of the chromosome and the initiation of the processes that lead to cell division. The alteration in the balance between water production and consumption then leads to water availability increasing as part of the resetting of the clock. This increase in water availability causes a second set of phase transitions that largely reverses the consequences of the preceding set of phase transitions. In addition, cell division entails changes to the membrane that may cause an influx of water from the environment via aequorins.

A model of a specific water-clock

Below the critical point, the hyperstructures that constitute the clock exist in one of two phases depending on the ratio of their constituents to their water (Figures 1 and 2). The functioning of these hyperstructures depends on which phase they are in. Figure 1 shows four cases in which water availability could trigger the phase transitions of molecules and macromolecules, either between extended and condensed hyperstructures or between assembled hyperstructures and their separate constituents (i.e., disassembled hyperstructures). The critical point, above which some of the hyperstructures are in one phase and some in the other, is attained if the bacterium continues growing without entering the cell cycle. As a bacterium grows and goes through the cell cycle, it stays near the edge of phase transitions so that its hyperstructures can readily change phase (Figure 2).

Now consider a slowly growing bacterium that is born at time-step i. The proportion of available water in the clock then decreases with growth until time-step ii when this decrease reaches the threshold that causes the first set of phase transitions that result in the condensation of both catabolic and anabolic hyperstructures (Figure 3). These phase transitions trigger the replication of the chromosome. Between time-steps ii and iii, the balance between the production of water by the condensed catabolic hyperstructures and its consumption by the condensed anabolic hyperstructures is in favour of an increase in available water until it reaches a second threshold at time-step iii at which there is a phase transition of the catabolic hyperstructures into the extended state. These extended hyperstructures have a greater activity and produce still more water. A third phase transition occurs at time step iv when this increase in water (along with the rearrangements of hyperstructures due to the progression through the cell cycle) causes the anabolic hyperstructures to shift into the extended state. This corresponds to the resetting of the clock. Water availability then decreases during the replication of the chromosome and at time step v the membrane changes associated with division result in an influx of water via aequorins. This is followed by the separation at time step vi that gives the daughter cells.

Towards a water-clock interpretation of the roles of DnaA and SeqA

Sophisticated systems of regulating the cell cycle have evolved over billions of years on top of the underlying water-clock, to which they are still answerable. In the case of the initiation of chromosome replication (and, we would argue, the initiation of cell division), these hyperstructure-based systems depend on phase transitions in DNA, membranes and cytoplasm. In E. coli, these systems are based on the ‘key initiator’ DnaA and on the ‘origin sequestration protein’, SeqA. There are many highly speculative models that could be constructed to illustrate the application to modern cells of the above water-clock hypothesis. In one of these models, a decrease in water availability results in a set of phase transitions of hyperstructures containing DnaA such as the glycolytic and ribosomal hyperstructures. In addition, decreasing water availability causes anabolic systems to start to switch off the expression of DnaA-regulated genes. These phase transitions and alterations in gene expression result in the release of DnaA, which then interacts with membrane, alters its conformation, binds ATP instead of ADP, and changes its binding preferences to sites on the chromosome. The consequence is that DnaA assembles into an initiation hyperstructure that includes the origin of replication, oriC, and enzymes that supply precursors. This decrease in water availability also results in a phase transition of the ribosomal hyperstructure that causes the glycolytic enzymes to quit the ribosomal hyperstructure and join the initiation hyperstructure. This reorganisation is orchestrated in part by accompanying phase transitions in the membrane and chromosome that could modify, activate, release, and concentrate replication enzymes and Nucleoid-Associated Proteins.

After chromosome replication is initiated, the initiation hyperstructure matures into the replication hyperstructure. SeqA joins this replication hyperstructure. This is because the same change in the availability of water causes a phase transition that results in SeqA having an altered relationship with the membrane, leaving the ribosomal hyperstructure, changing its binding to the chromosome, so as to be ready to bind the newly replicated DNA (hemi-methylated GATC sequences in the case of E. coli) generated by the replication hyperstructure. This DNA can itself undergo a phase transition that facilitates the segregation of daughter chromosomes and that also releases water.

The first set of phase transitions that alters the location and activity of the glycolytic enzymes and of the translational apparatus increases the catabolic activity of the former and decreases the anabolic activity of the latter. This contributes to the availability of water increasing and, eventually, to a second set of phase transitions that changes the behaviour of DnaA as part of the resetting of the clock.

Sizer or adder?

The ‘initiation mass’ is the average cell mass per chromosomal origin of replication at which the initiation of chromosome replication occurs; this mass has been proposed to be constant at different, relatively fast, growth rates (when the size of the cells can vary greatly) (Donachie, 1968)(Pritchard et al., 1969). This proposal has been extremely influential albeit controversial (Wold et al., 1994). Recently, in an extensive analysis (four growth conditions and over 800 different mutants) of cell cycle parameters in E. coli, it was concluded that only the cell volume at the time of initiation per origin was generally constant across strains and conditions (Govers et al., 2023), as concluded for B. subtilis (Sauls et al., 2019). Such constant average cell volume per origin per initiation of replication could result from the operation of a ‘‘sizer’’ at the single-cell level, whereby individual cells initiate DNA replication at a constant volume per origin, or of an ‘‘adder,’’ whereby individual cells add, on average, a constant volume per origin between consecutive DNA replication initiation events (Ho & Amir, 2015)(Huls et al., 2018). The water-clock fits better with the adder scenario given that it would not be completely reset (i.e., with all the water molecules diffusing out of it) at the end of the cycle.

Advantages of the hypothesis

The hypothesis:

1. couples mass increase to chromosome replication. The idea of mass increasing to a threshold at which DNA replication is triggered is very popular because, in part, it provides a way to generate a distribution of cell sizes and DNA/mass ratios that is constant – that is, reproducible and hence readily selectable – for a particular environment. In the water-clock hypothesis, the availability of water is proportional to mass and therefore constitutes a good indicator of mass; moreover, it constitutes a natural trigger for the initiation of replication and cell division given the importance of water in the activity of enzymes and the structures of DNA and membrane.

2. coordinates catabolism and anabolism. If the anabolic machinery does not make enough catabolic enzymes, catabolism could fail to profit sufficiently from available nutrients and the growth rate would be slower than the maximum permitted by the environmental conditions. In the water-clock hypothesis, catabolism produces both the precursors and the water needed for anabolism; this water availability also allows catabolism to control anabolism. Hence, the water-clock couples mass increase to initiation of chromosome replication in a particularly effective way because this coupling is automatically based on the metabolic status of the cell in terms of catabolism and anabolism.

3. makes use of the sharpness of phase transitions. Abrupt, nonlinear behaviour can give a signal with a temporal precision of large amplitude (Yoo et al., 2019). This would be the case for the proposed phase transitions of hyperstructures driven by water, which would cause major, rapid changes to the cell ideal for signalling (Watson et al., 2023).

4. provides a quantity sensing mechanism for sensing the mass of the cell (Norris & Amar, 2012b). This mechanism must neither act prematurely, which might result in daughter cells that are too small to be viable, nor act tardively, which might result in a cell that is too large to retain its phenotype or identity (corresponding to a cell that has passed the critical point shown in Figure 2). Such loss of the phenotype would be particularly acute if the proportion of water relative to this mass were to increase or decrease outside critical limits.

5. provides an intensity sensing mechanism for sensing the extent to which the DNA and other cellular constituents are working (Norris & Amar, 2012b). A cell that fails to replicate its DNA before the expression of one or more genes critical for growth becomes limiting fails in consequence both to grow as fast as it could and to maintain an environmentally appropriate phenotype. Changes in the rate at which water becomes available could provide useful information about whether DNA risks becoming limiting.

6. acts as an indicator of the energy status of the cell because, during the generation of ATP, GTP and acetyl phosphate, nutrients are also converted into water.

7. couples the phenotype with the environment. An important, open question is how cells negotiate the vastness of phenotype space so as to obtain the reproducible phenotypes on which natural selection can act (Kauffman, 1996). An answer to this question comes from the idea that cells live on the edge of phase transitions mediated by water: the state of this water is influenced by environmental conditions (such as temperature, humidity and ion concentrations) and, reciprocally, this water influences the phase transitions of hyperstructures. Given that there are scores of hyperstructures and that subsets of these hyperstructures correspond to environment-specific phenotypes, different states of water could regulate separately these subsets by causing each of them to undergo a phase transition appropriate to its constituent hyperstructures. In other words, each phenotype would depend on the phase transitions of its own subset of hyperstructures with a specific level of water availability (i.e., water potential) controlling these phase transitions so as to produce coherence and reproducibility.

8. underpins differentiation. My collaborators and I have argued that the ‘natural’ result of the processes involved in cell division, which often entail locally positive and globally negative circuits, should be to generate phenotypically different daughter cells (Norris, 1995)(Norris & Amar, 2012a)(Norris, 2019a). This scenario at its most basic level would yield one daughter with mainly equilibrium hyperstructures and the other with non-equilibrium hyperstructures appropriate for survival and growth, respectively (Norris, 2015). This scenario would fit well with the phase transitions illustrated in Figure 3, which could lead not to similar daughters but rather to one daughter with condensed, inactive, degradation-resistant hyperstructures and the other with extended, active, fragile hyperstructures.

9. makes a connection with origins of life hypotheses in which cycles of wetting and drying can generate ring-like structures of RNA (Hassenkam et al., 2020) and possibly rings of double-stranded DNA with properties that would include glycolysis and catalysing DNA replication (Norris & Demongeot, 2022). In this ‘Ring World’ hypothesis, an early cell would have contained many rings with complementary functions and the successful growth of this cell would, in the water-clock hypothesis, have led to a decrease in water availability and the simultaneous replication of all the cell’s rings. It also makes a connection with the idea that hyperstructures are the modern descendants of ‘composomes’ (Norris et al., 2015); in the scenario of a prebiotic ecology, those molecules that associated with one another were protected from degradation and accumulated in the form of composomes that gave rise to progeny with similar compositions but novel properties (Segre et al., 2000)(Hunding et al., 2006).

This section summarises the evidence consistent with the hypothesis (more details are given in the following sections). I start with the key finding that much of the cell’s water is generated by metabolism. This opens up the possibility that a proportion of this water corresponds to the water that commands the cell cycle. The availability of such water could act on physiology via molecular and macromolecular crowding and the role of crowding in hyperstructure dynamics. I then mention some of the evidence for hyperstructures and for their role in initiating both DNA replication and cell division. I focus on hyperstructures classed as condensates, which have dynamics that depend on phase separation and condensation and that have excellent signalling properties. The idea that free water is generated by catabolism is supported by evidence that metabolism fluidises the cytoplasm whilst the anabolic processes of transcription and translation do not fluidise it. To show that a reduced availability of water could initiate the cell cycle, I cite in vitro evidence that macromolecular crowding affects DNA replication. Ways in which changes in water availability could affect DNA replication are by altering the conformation of DNA or by altering the binding to DNA of proteins (e.g., via post-translational modifications) or by altering the membrane. Related effects of hydration and density include changes to protein concentration and diffusion, which affect enzymic activity, possibly in different ways depending on whether the reactions they catalyse are catabolic or anabolic. Effects of macromolecular crowding also include alterations to transcription whilst in the case of ribosomes it has been proposed that crowding coordinates growth with DNA replication. A cyclic variation in water availability is central to the operation of the water-clock; although variations in intracellular water during the cell cycle have a controversial history, there is some recent evidence in their favour. A complicating factor here is that intracellular water may exist in more than one compartment, which would strengthen the water-clock hypothesis, and for which there is also some recent evidence. This leads to other evidence in favour of channels through which water for the clock might move without an exchange with water elsewhere and in the environment. I then argue that the proposed role of the cell cycle in generating daughters with different quantities of water is consistent with the state of water reported in spores. Finally, I mention that replacing H₂O with D₂O can have a major effect, at least, on the growth rate.

Intracellular water is from metabolism

Using ¹⁸O-labelled water, 70% of the intracellular water was found to be generated by metabolism in E. coli growing at an OD₆₀₀ of around 1 whilst the quantity of this water reflected the state of the cell (Kreuzer-Martin et al., 2005). A subsequent study using ²H-labelled water showed that more than 50% of the intracellular water hydrogen atoms were isotopically distinct from the growth medium water, moreover, the ²H/¹H isotope ratio of intracellular water from log-phase cells was twice that from stationary-phase cells (Kreuzer-Martin et al., 2006). These results were supported by the results of an examination of the intracellular pool of PO₄ in Pseudomonas fluorescens, Acinetobacter ADP1, and Marinobacter aquaeolei following labelling with water enriched with ¹⁸O (Li et al., 2016). Following the labelling of activated T-cells, it has been proposed that they obtain water molecules as a byproduct of metabolic reactions, such as glycolysis, respiration, and protein synthesis (Saragovi et al., 2022).

Molecular and macromolecular crowding or water availability

The availability of water is intimately related to crowding, which decreases the volume available to water. This crowding occurs when macromolecules occupy enough of a confined volume (like a cytoplasm) to cause changes in macromolecules that include their structure, folding, conformational stability, diffusion, enzymatic activity, and their binding to one another and to small molecules (Dix & Verkman, 2008)(Kuznetsova et al., 2014)(Rivas & Minton, 2016). The water in the hydration layers surrounding molecules is more ordered and osmotically inactive compared with ‘normal’ water (Luby-Phelps, 2000)(Garlid, 2000). The crowding conditions inside cells are such that the hydration layers of the molecules overlap by several nanometers, and the fraction of interfacial water varies between 30% and 70% of the total water in a cell (Ball, 2008a). It has been estimated that macromolecules occupy 20-40% of the cytoplasm with an additional 20% being water probably in the hydration layer of these macromolecules leading to a total excluded volume in the cell of 40-60% (Cayley et al., 1991)(Zimmerman & Trach, 1991).

It may be useful here to distinguish between the terms ‘macromolecular crowding’ and ‘macromolecular confinement’: the dynamic effects of volume exclusion are caused in the former by other macromolecules and in the latter by the static shape and size of the system (Lecinski et al., 2021). It may also be useful to distinguish between different crowding agents, as shown for the compaction and DNA-binding of HU (Lin et al., 2020) and, in particular, to distinguish between molecular crowding and macromolecular crowding, which can affect water dynamics differently (Verma et al., 2018). Indeed, in highly crowded media, molecular crowding affects both the interstices between the crowder molecules and the interfacial water, whilst macromolecular crowding does not affect the bulk water and only has a small effect on the interfacial water (Verma et al., 2018).

Bacteria are highly structured

It is consistent with the water-clock hypothesis that molecular crowding is believed to promote entropically the formation of large molecular assemblies and phase-separated compartments in vitro and in vivo (for references see (Neurohr & Amon, 2020)). These structures are subsumed by the concept of hyperstructures, which are large, spatially distinct groups of cellular molecules and macromolecules that serve a function (Norris et al., 2007a, Norris et al., 2007b). There are scores of different hyperstructures in bacteria (Norris et al., 2022) but here I focus on those directly related to the hypothesis: those involving DnaA, SeqA, ribosome synthesis and glycolysis. Hyperstructures can form via direct interactions between their constituents or via indirect associations of genes, nascent RNA, and nascent proteins due to the coupling of transcription, translation, and either transertion (insertion into membrane) or transembly (assembly into cytoplasmic complexes) (Binenbaum et al., 1999)(Llopis et al., 2010).

The DnaA protein forms helical structures along the cell axis (Boeneman et al., 2009). After binding to high affinity sites scattered throughout the chromosome, it binds cooperatively in its DnaA-ATP form to low affinity sites in the region of the origin, oriC, to help unwind the double helix and initiate replication in a process modulated by proteins such as IHF, Fis and SeqA (Grimwade & Leonard, 2021)(Yoshida et al., 2023). This initiation hyperstructure (see below) is associated with the membrane (Regev et al., 2012) and probably contains some of the enzymes needed to synthesis DNA precursors, such as ribonucleotide reductase, replication enzymes and glycolytic enzymes (for references see (Kohiyama et al., 2023)).

SeqA binds to clusters of GATC sites located in the regions around genes involved in the synthesis of the precursors of DNA and in its replication and repair. Following the initiation of chromosome replication in E. coli, SeqA binds preferentially to the newly replicated, hemi-methylated GATC sites in oriC as part of a sequestration mechanism that prevents multiple reinitiations and that involves the membrane (Ogden et al., 1988)(Shakibai et al., 1998). SeqA hyperstructures comprise SeqA multimers and SeqA-DNA complexes (which can contain 100 kb of DNA) and are close or integral to the replication hyperstructures (Helgesen et al., 2021).

Single-stranded DNA-binding proteins, SSBs, are essential to the cell cycle and interact with DNA replication proteins (Antony & Lohman, 2019)(Bianco, 2021)(Marceau et al., 2011); they can form a cooperative assembly that rapidly protects the ssDNA via an SSB hyperstructure that comprises many tetramers bound contiguously along the ssDNA (Bianco, 2021).

The synthesis of ribosomes is associated with bacterial ‘nucleoli’ or ribosomal hyperstructures, which may bring together DNA, nascent RNA and nascent proteins (Woldringh & Nanninga, 1985)(Gaal et al., 2016)(Jin et al., 2017). Significantly, ribosomal subunits have physical interactions with several replication enzymes (Morcinek-Orlowska et al., 2023) (see below).

Other hyperstructures include structures made from CTP synthase (Ingerson-Mahar et al., 2010), from EF-Tu (Mayer, 2006)(Defeu Soufo et al., 2010) and from AdhE (Kim et al., 2020); microcompartments such as the carboxysomes and metabolosomes that sequester metabolic intermediates (Herring et al., 2018)(Kirst & Kerfeld, 2019)(Sutter et al., 2021); clusters of the E1 protein of the phosphotransferase system (Govindarajan et al., 2017), "segrazones" of aerobic enzyme complexes of oxidative phosphorylation (Erhardt et al., 2014), and photosystem complexes (MacGregor-Chatwin et al., 2019). They include those containing DNA such as the protein complexes involved in DNA replication and segregation (Sunako et al., 2001)(Boeneman et al., 2009)(Duderstadt et al., 2010)(Sanchez-Romero et al., 2011)(Helgesen et al., 2015), complexes involving topoisomerases (McKie et al., 2021) along with RNA polymerase supramolecular complexes associated with topoisomerases and metabolic enzymes (Muskhelishvili et al., 2021).

In the case of the nucleoid-associated proteins in E. coli, (1) H-NS has been reported not only to form hyperstructures (Wang et al., 2011)(Kuwada et al., 2015)(Gao et al., 2017) (for artefacts see (Margolin, 2012)) but also to help create “specific mesoscale domains in chromosomal regions in response to environmental changes” (Cristofalo et al., 2020), (2) HU has been proposed to undergo a transition from a DNA bundle to a filament (depending both on the HU to DNA ratio and on environmental factors) that would alter the structure of the nucleoid (Hammel et al., 2016)(Remesh et al., 2020) (3) IHF has been proposed to bind to DNA to provide a ‘nucleation point’ for higher-order genome organisation (Purkait et al., 2021) that would involve HU and H-NS along with structural maintenance of chromosome (SMC or condensin) proteins. Both IHF and HU participate in pre-replication complexes at the E. coli oriC (Ryan et al., 2002). In the context of replication, IHF also forms a complex with DnaA and FIS on the DARS2 site on the chromosome (Kasho et al., 2014)(Yoshida et al., 2023).

A single, hyperstructure-driven, cell cycle signal

Recently, we have proposed the existence of an initiation hyperstructure that comprises the ‘key initiator protein’, DnaA, and its binding sites on the chromosome, the enzymes that supply DNA precursors (like ribonucleotide reductase), glycolytic enzymes, the membrane and part of the division hyperstructure (Kohiyama et al., 2023). There is some evidence in support of this proposal. The intimate relationship between glycolysis and DNA replication is evidenced in the interactions between catabolic enzymes responsible for glycolysis with anabolic enzymes responsible for DNA replication. For example, in Bacillud subtilis, growth was restored to replication mutants affected in the lagging strand polymerase, the replicative helicase, and the primase by mutations in genes encoding glycolytic enzymes such as the pgm, pgk, eno and pykA genes (Janniere et al., 2007); similarly, in E. coli, mutations in the DNA replication enzymes dnaA, dnaB, dnaE, dnaG and dnaN mutants can be corrected by mutations in the pta and ackA genes (Maciag-Dorszynska et al., 2012). The glycolytic enzyme, PykA, associates physically with the polymerase, DnaE, when DnaE is bound to a primed DNA template (Holland et al., 2023). Physical interactions have also been found between eight proteins involved in DNA replication in E. coli (DnaA, DnaB, Hda, SeqA, DiaA, DnaG, HolD and NrdB) and scores of proteins involved in metabolism, stress, transcription, translation, DNA structure, the membrane and so on (Morcinek-Orlowska et al., 2023). These interactions include those between DnaA, enolase and AdhE, between DnaA and many ribosomal proteins, and between DnaA and MinE; between DnaG and RpoD and RpsU (the three proteins encoded by the macromolecular synthesis operon); between DnaB, MinD, MreB and FtsZ; between SeqA and many ribosomal proteins and between SeqA, enolase and AdhE (Morcinek-Orlowska et al., 2023). Such interactions would be consistent with the proximity to a replication hyperstructure of hyperstructures for glycolysis, ribosome synthesis, cell division etc.

These and other findings have led to the proposal that changes in central carbon metabolism influence both the initiation and elongation steps of DNA replication. Some evidence consistent with the putative immediacy of initiation of replication and cell division comes from in vivo labelling studies of membranes and DNA (Fishov & Woldringh, 1999) and a possible direct relationship between initiation and division hyperstructures comes from the presence of DnaA binding sites in the genes encoding division enzymes (Masters et al., 1989)(Garrido et al., 1993) (for other references see (Kohiyama et al., 2023)). In line with a hyperstructure responding to changes in crowding to initiate DNA replication and cell division, the membrane may play a major role via the density of membrane proteins (Aranovich et al., 2015) and via surface-assisted, condensate formation (Ramm et al., 2023).

Phase separation, condensates and trajectories

The central contention of the water-clock hypothesis is that life exists on the edge of phase shifts/transitions involving hyperstructures. One class of hyperstructures is composed of membrane-less biomolecular condensates, which are proposed to play major roles in prokaryotic and eukaryotic physiology (for a review see (Gao et al., 2021) but see (Musacchio, 2022)). The phase separation that is central to the existence of condensates is due to transient, low-affinity, cohesive interactions between their constituent polymers whilst stereo-specific, macromolecular interactions only play a secondary role (Musacchio, 2022). These condensates are generally liquid-like and form via liquid-liquid demixing (like oil and water); droplets can form when multivalent proteins and intrinsically disordered proteins are concentrated; these proteins lead to phase separation via weak homotypic interactions (i.e., between the same macromolecules) or via heterotypic interactions (i.e., between different species of macromolecules). It is useful to distinguish three types of macromolecular interactions, all of which can be homotypic or heterotypic. In type 1, in the case of intrinsically disordered proteins, interactions are via ‘sticker’ regions that exhibit relatively low-affinity and poorly specific types of attractive interactions, including charge–charge, dipole–dipole, cation Π, and Π–Π stacking. In types 2 and 3, interactions are via the conformation and the detailed chemical nature of the binding interfaces; they involve one or more folded domains of a macromolecule and either a short linear segment of a target macromolecule or another folded domain, for types 2 or 3, respectively. The specificity of type 1 interactions is limited (with the possible exception of multivalent proteins) since they are due to a relatively small set of attractive or repulsive bonds whilst the specificity of types 2 and 3 interactions is much greater since they are due to the spatial and chemical complementarity of the binding interfaces as well as to steric exclusion. It has been argued recently that low-specificity, type 1 interactions between macromolecules may not constitute the primary mechanism for condensate formation in vivo but, rather, it is only after concentration of macromolecules by type 2 and 3 interactions that phase separation occurs, indeed, phase separation leading to condensates may have been overstated (Musacchio, 2022). One fundamental problem is that the cytoplasm is highly complex and crowded with a wide variety of unpredictable opportunities for competitive, short-lived, non-specific interactions (Rivas & Minton, 2018)(Musacchio, 2022). Questions that arise include “What exactly phase-separates and defines the putative condensate - a single macromolecule or its network of interactors? … How do posttranslational modifications influence this process? Are there additional energy-consuming processes that further complicate the picture?” (Musacchio, 2022).

With the above caveats in mind, it is significant for the water-clock hypothesis that the condensates generated by phase separation (Hondele et al., 2019) in bacteria are proposed to include the clusters of RNA polymerase and NusA (Ladouceur et al., 2020), SSB (Kozlov et al., 2022), Dps and other proteins (see (Azaldegui et al., 2021)) the RNA degradosome (Nandana et al., 2023), and division enzymes (Monterroso et al., 2019)(Ramm et al., 2023). In particular, the Nucleoid Occlusion Protein, Noc, can undergo a phase separation that depends on membrane order and composition and that results in the recruitment of FtsZ to the membrane (Babl et al., 2022). Then there is the bacterial ‘nucleolus’ (see above). In eukaryotes, membrane-less condensates include P granules, nuclear Cajal bodies, nuclear speckles, stress granules, and nucleolar bodies (arguably a multilayered biomolecular condensate formed by liquid-liquid phase separation (Lafontaine et al., 2020)).

Hyperstructures in the form of condensates may undergo major changes in their states that can be very different (e.g., liquid, gel, and solid) depending on factors that include post-translational modifications, temperature, pH, and ion concentration. For example, in the case of ions, high salt concentrations dissolve condensates or inhibit condensation in vitro because of the suppression of protein-protein interactions by electrostatic shielding thereby affecting the fluidity of condensates and the mobility of their constituents (Shin & Brangwynne, 2017)(Morishita et al., 2023). In prokaryotes, we have proposed that hyperstructures, which have different dependencies on energy-consumption, can have trajectories in which they are born, mature, respond to the environment and die (Norris et al., 2007a). In eukaryotes, the trajectory of cortical condensates involves their recruitment and dismissal of molecules that regulate the branched nucleation of F-actin (Yan et al., 2022). Hyman and collaborators have proposed that there are essentially three steps in the formation of condensates in eukaryotes: nucleation, rearrangement and supersaturation (Hyman et al., 2014); as the concentration of the molecular constituents increases, the liquid-like condensate forms and then changes into gel-like or solid states (Gao et al., 2021). All that said, whatever the exact mechanisms responsible for hyperstructure dynamics, the availability of water is going to be important in changing the existence, size and activity of hyperstructures.

Water availability and the dynamics of condensates are related

It has been proposed that the water potential drives the assembly and disassembly of protein condensates and multiprotein complexes (Zaslavsky & Uversky, 2018) and shown that such assembly releases free water whilst disassembly into the constituent macromolecules sequesters it (Watson et al., 2023): this satisfies one of the requirements of the water-clock hypothesis. Clock candidates for such assembly-disassembly include ribosomes which can go from being active in translation to being inactive in a proportion that varies with growth conditions (Dai et al., 2016)(Li et al., 2018); phase transitions involving ribosomes and ribosomal hyperstructures might therefore be expected to affect water availability given that ribosomes are in a different state (e.g., dimeric) when inactive (Wada et al., 1990).

Metabolic activity fluidises the cytoplasm

‘Glassy’ systems are characterised by a dynamic heterogeneity in which regions of high particle mobility coexist with regions of low particle mobility (Bertier, 2011). Such glassy behaviour was revealed for a wide variety of cellular constituents (crescentin, polyhydroxyalkanoate granules, plasmids with an estimated radius of gyration of 150 nm, and avian reovirus protein) in the cytoplasm of both Caulobacter crescentus and E. coli (Parry et al., 2014). This mobility, which was greatly reduced in carbon-starved cells or in stationary phase cells, was attributed to a fluidising action of metabolic activity on the cytoplasm rather than to transcription, translation, peptidoglycan synthesis or chromosome dynamics. Of the many possible, non-exclusive causes of this fluidising, one is an increase in metabolism-generated water (possibly by a particular catabolic hyperstructure).

Phase transitions are excellent candidates for signalling

Phase transitions possess a remarkable cooperativity that allows system-wide changes in response to small changes in the environment along with the ability to rearrange matter – often without energy expenditure (Yoo et al., 2019). Examples of phase separation-based sensing from eukaryotes include the sensing of thermal stress by poly(A)-binding protein, the sensing of change in intracellular pH during starvation by the translation terminator factor, Sup35, and the sensing of cytosolic DNA by cGMP–AMP synthase (for references see (Yoo et al., 2019)). In 1967, it was proposed that a phase transition can occur in a membrane at a critical concentration of a ligand that binds to that membrane (Changeux et al., 1967) and, subsequently, a phase transition in the membrane of E. coli was found to affect transport (Thilo et al., 1977). More recently, it was shown that single neuronal cells can undergo a sharp (i.e., highly nonlinear), reversible phase transition within their membrane that is accompanied by significant changes in permeability, enzyme activity, elastic, and electrical properties (Fedosejevs & Schneider, 2022); as pointed out by the authors “if a lipid membrane, for instance, suddenly softens by an order of magnitude, chances for morphological transitions as they occur during adhesion, budding, fission, and fusion are tremendously increased”. Finally, the relationship of biomolecular condensation to the water potential has been proposed to provide “cells with a thermodynamically driven rapid defence against acute fluctuations in osmotic strength, temperature and pressure that does not require specific osmosensors” (Watson et al., 2023).

Macromolecular crowding affects DNA replication

In pioneering studies, macromolecular crowding (i.e., limiting water availability) proved essential to the replication of oriC in vitro (Fuller et al., 1981) whilst in a study of the replication of the T7 bacteriophage in vitro, crowding relieved salt-inhibition of replication by gp5 (the T7 DNA polymerase) and trx (the host-encoded processivity factor), increased the activity of gp4 (the T7 helicase/primase), and increased the compaction and the activity of the complex between gp5, trx and gp4 (Akabayov et al., 2013). Against this role for water, however, molecular crowding can increase the DNA duplex stability (Singh et al., 2022) and presumably therefore decrease the probability of initiation of DNA replication.

Phase transitions and post-translational modifications

The change in the conformation of proteins with intrinsically disordered regions due to their phosphorylation is reported to play an important role in the phase transitions that result from water availability, which is attributed to the higher ratio of surface area to volume of these regions needing more hydration water (Watson et al., 2023). DnaA (which contains an intrinsically disordered region) can be acetylated at more than one residue, which affects its binding to ATP and to oriC (Li et al., 2017). SeqA can be phosphorylated though this does not appear to affect its role in preventing re-initiations (Riber et al., 2018). Nucleoid-associated proteins in bacteria undergo a wide variety of post-translational modifications that include phosphorylation, acetylation, deamidation of asparagines, succinylation, propionylation, methylation, oxidation of methionines, malonylation and the addition of short-chain poly-(R)-3-hydroxybutyrate (for references see (Norris et al., 2022)). The last class is particularly interesting given that many ribosomal subunits are also modified by the covalent addition of short-chain poly-(R)-3-hydroxybutyrate (Huang & Reusch, 1996), which occurs at sites that, it is proposed, may also be phosphorylated (Norris et al., 2015).

Phase transitions and DNA

DNA and the ions, molecules and macromolecules associated with it can exist in several different phases (for a review, see (King & Shakya, 2021)). DNA forms liquid crystals at high solution concentrations in the presence of polymers that induce osmotic stress as well as multivalent polycations and polymers (Livolant et al., 2006). Chromatin is proposed to have local liquid crystalline domains attributable to macromolecular crowding (Leforestier & Livolant, 1997) and, in dinoflagellates in particular, liquid crystalline DNA has been observed (Bouligand et al., 1968)(Wong, 2019). Liquid crystalline DNA has also been observed in bacteria (Reich et al., 1994)(Wolf et al., 1999) and the phase separation of liquid crystalline DNA has been invoked as a mechanism for separating daughter chromosomes (Bouligand & Norris, 2001). Such phase separation has been proposed to occur immediately after the exit from the replisome of newly replicated daughters, which would adopt immiscible structures (Woldringh, 2023).

Hydration alters DNA conformation and the binding of proteins to DNA

In the case of the nucleoid-associated proteins, HU and H-NS, macromolecular crowding increased the DNA-binding affinity of HU (Murphy & Zimmerman, 1994) and the binding of H-NS to AT-rich regions (Ramisetty et al., 2017). In the case of DNA, the sequence of hydration, dehydration and rehydration is accompanied by transitions of DNA from the B-form to the A-form to the B-form, respectively, in E. coli and other bacteria (Whelan et al., 2014) and, in the case of dehydration, by the binding to this DNA of small, acid-soluble proteins in B. subtilis (Lee et al., 2008) (for a review of crowding and DNA see (Collette et al., 2023)). This means that changes in intracellular water can indeed affect both the binding to DNA of major nucleoid-associated proteins and the conformation of the DNA itself and hence could alter the probability of strand opening and initiation of DNA replication, which would be consistent with the water-clock hypothesis.

The relationship between crowding and the structure of the nucleoid could also help explain the ‘eclipse period’ that follows the initiation of replication when the replicating chromosome becomes refractory to new initiations (Zaritsky et al., 2017). Major changes in the volume occupied by the E. coli chromosome were readily created by small forces and free energies of around 100 pN and 10^5 k_(B)T exerted by a piston at pressures a 1000-fold less than the surrounding turgor pressure inside the cell (Pelletier et al., 2012). Recently, treatments of E. coli that cause an expansion of the nucleoid and result in an increase in cytoplasmic crowding have been attributed to either the expansion of the nucleoid displacing molecules from where they are concentrated or this expanded nucleoid itself occupying a greater volume (Pittas et al., 2023); in the former case, this displacement of molecules might occur because of an alteration in the composition and structure of certain hyperstructures and the release of their molecular and macromolecular contents. In the water-clock hypothesis, this perturbation of hyperstructures and consequent alteration in water availability would accompany chromosome replication.

Water and membranes

Changes in the structure of the membrane are fundamental to the process of cell division and, arguably, to both the initiation of both chromosome replication and cell division (Kohiyama et al., 2023). In the association-induction hypothesis, a cell is a colloidal coacervate of gel proteins in water in which there is a response to metabolic events through changes in protein conformation such that proteins in an extended conformational state thereby expose groups that interact with water (Ling, 2007). This hypothesis has been combined with current ideas about membranes to explain how water interacts with part of the membrane (for example, the headgroups of phospholipids) to determine its structural dynamics; this dynamics would include the formation of both non-bilayer structures and lateral proteo-lipid domains and the generation of phase transitions (Disalvo et al., 2022). In consequence, changes in water availability should have major effects on the structure of the membrane and hence on cell cycle events.

Water and enzymic activity

The volume of cytoplasmic water and the growth rate decrease linearly as osmolarity increases, with growth predicted to stop when the volume of cytoplasmic water equals the volume of bound water (Cayley et al., 1991). One result of the decrease in the volume of cytoplasmic water with increasing osmolarity of the growth medium is that the concentration of all cytoplasmic macromolecules – alias macromolecular crowding – increases. This would be consistent with the water-clock insofar as the initiation of the cell cycle is needed to increase the availability of water for anabolism.

It has been argued that “the most direct consequence of altering cytoplasm density is that overall protein concentration changes. Because the probability of reaction partners to collide is concentration dependent, all protein-catalyzed reactions in the cell will be affected by altering total protein concentration. Changing protein concentration will also affect the assembly of protein complexes, with low affinity interactions and multimeric complexes being most sensitive to changes in protein concentration ... It follows that assembly of dynamic polymers is strongly concentration dependent” (Neurohr & Amon, 2020). More specifically, following studies of an intrinsically disordered human protein, tau, and a globular protein, maltose binding protein, it has been proposed that (1) the diffusion of water molecules on the surface of a protein (hydration water) leads to the large-amplitude motions that are needed for the activity of the protein and (2) that macromolecular activity might be modulated via a modification in the translational diffusion properties of hydration (caused, for example, by geometric confinement, macromolecular crowding or the presence of small solutes) (Schiro et al., 2015). The above changes in hydration and density are significant for the water-clock hypothesis because, in eukaryotes, an increase in cellular hydration stimulates anabolism and, in particular, protein synthesis whilst a decrease stimulates catabolism and inhibits protein synthesis (Haussinger et al., 1993)(Waldegger et al., 1997).

Finally, the oscillations of the glycolytic metabolites, ATP and NADH, in the budding yeast, Saccharomyces cerevisiae, along with oscillations in the membrane, cell volume, heat flux, and temperature, are coupled to the oscillations of intracellular water in terms of its dipolar relaxation (Bagatolli et al., 2019); this has been attributed to a polarized intracellular water network being modulated by temporal ATP fluctuations due to metabolism and the functioning of the cytoskeleton (Bagatolli et al., 2019), as envisaged in the association-induction hypothesis (Ling, 2007).

Water and ions

The general importance of inorganic ions in cell physiology has been extended to the regulation of the bacterial cell cycle. For example, we have speculated that a growth-related phase transition during the cell cycle causes ions to decondense from the chromosome and to join protein-synthesizing hyperstructures that, in this hypothesis, results in the opening of the double helix at oriC, a key step in the initiation of replication (Norris & Amar, 2012a). These sorts of ideas about ions and the cell cycle can be subsumed by the water-clock hypothesis as exemplified by SSB, which is central to DNA replication, repair and recombination. In E. coli, it exists in several states including one in which phase separation is promoted by potassium glutamate, the primary monovalent salt, and by the presence in this protein of an intrinsically disordered region (Kozlov et al., 2022). One explanation may indeed be that the ability of “SSB alone to undergo phase separation and the loss of this ability upon binding ssDNA implicates phase separation of SSB in vivo as a mechanism to sequester SSB until it is needed for DNA metabolism” (Kozlov et al., 2022). There is a clear connection here between such phase separation, ions and water given the relationship between osmotic conditions and the availability of free water (Cayley et al., 1991)(Watson et al., 2023).

Water and percolation

The addition of links or nodes to a network can reach a critical percolation threshold at which the network passes from a small, disconnected clusters to a single, giant cluster of the size of the network. It has been proposed that percolation effects may be physiologically important in biological membranes by having major effects on, for example, enzymic reactions (Thompson et al., 1995). If such a percolation threshold were indeed to exist, it is reasonable to assume that it would depend on the availability of water in proximity to the membrane as well as on the size, nature and activity of the hyperstructures that structure the membrane via transertion (Binenbaum et al., 1999), which have their own relationship to this water. Reciprocally, the state of the membrane should affect water fluxes and, in a recent investigation of lipid packing in ordered and disordered domains in model membranes, it was concluded that this packing can lead to the membrane permeability of small molecules (Tripathy & Srivastava, 2023). This would be consistent with the water-triggered phase change in hyperstructures altering membrane permeability to allow a water influx.

Macromolecular crowding affects transcription

In an in vitro, E. coli-based assay, macromolecular crowding enhanced the rate of late initiation and promoter clearance, which are believed to be the rate-determining steps for the transcription of many promoters (Chung et al., 2019). In modelling transcription in the eukaryotic nucleus, it was found that temporal changes in macromolecular crowding can generate significant increases in the expression of some genes and reductions in the expression of other genes; significantly, compression (i.e., increasing crowding) universally decreased gene expression (Shim et al., 2020). This is likely to be the case in bacteria too. For example, the Nucleoid-Associated Protein, HU, alters DNA structure and affects both transcription and replication; the outcome of its binding to DNA can be either compaction or extension (via HU filamentation) and these outcomes are greatly affected – and affected differently – by different crowding agents (Murphy & Zimmerman, 1994)(Lin et al., 2020).

Macromolecular crowding and ribosomes

In eukaryotes, following the use of genetically encoded, fluorescent nanoparticles in both S. cerevisiae and human cells, it has been proposed that ribosomes act as macromolecular crowders that tune phase separation, with changes in crowding regulating protein interactions, diffusion and folding (Delarue et al., 2018). In Vibrio cholerae, moving one of the principle loci encoding ribosomal proteins away from oriC decreased the growth rate and the dynamics of replication as shown by marker frequencies (Soler-Bistue et al., 2020). Despite the reduction in ribosome numbers, these effects were not due to a significant alteration of translation capacity but rather to an increase in the fluidity of the cytoplasm as shown both by the reversal of these changes in hyper-osmotic conditions and by FRAP analyses. This led to the conclusion that macromolecular crowding is needed to coordinate DNA replication with growth (Soler-Bistue et al., 2020).

Variations in intracellular water and the cell cycle

The buoyant density of cells could be considered a measure of water availability (but see below) and variations in density have long been speculated to play a role in the bacterial cell cycle (Rosenberger et al., 1978). Density over the course of the bacterial cell cycle has long been investigated using a wide variety of techniques and the results or, at least, the interpretations have varied with some authors finding significant variations (Poole, 1977)(Dicker & Higgins, 1987) and others not (Koch & Blumberg, 1976)(Woldringh et al., 1981)(Martinez-Salas et al., 1981)(Kubitschek et al., 1983). More specifically, the density of E. coli during the cell cycle was investigated using Percoll gradients and it was concluded that there were no stepwise changes in the mean buoyant density of more than 0.1% (Kubitschek et al., 1983); moreover, buoyant density was found not to vary at different growth rates and it was concluded that it is tightly regulated (Kubitschek et al., 1984). Similarly, using the dry weight of populations of different sizes, it was concluded that E. coli keeps nearly a constant ratio between dry mass and cell size, even when it produces large quantities of “useless” proteins (Basan et al., 2015). In contrast, using Spatial Light Interference microscopy to examine single cells (with the average dry-mass density for a cell being obtained via a refractive index), it was concluded that dry-mass density varies systematically during the cell cycle of E. coli and C. crescentus (essentially, dry-mass density decreasing with cell length) (Colin et al., 2021). It should be noted, however, that these results for individual cells were averages over the cell and hence did not include any putative water ‘compartments’ (see below). Importantly for the water-clock hypothesis, the refractive index and optical diffraction tomography have been used to show variations in density within the cytoplasm of individual cells of E. coli (Oh et al., 2020) and Klebsiella pneumoniae (Shin et al., 2023).

In eukaryotes, evidence consistent with the water-clock includes the finding that, during the cell cycle of S. cerevisiae, the density of the cell varies (Baldwin & Kubitschek, 1984); this density is relatively low in G1 (unbudded) cells, increases to near maximum during late G1 and S-phase entry (DNA replication and bud formation), and then decreases through mitosis (Bryan et al., 2010). One early study concluded that there are fewer free water molecules in tumour cells than in normal cells (Damadian, 1971) whilst another study concluded that the total quantity of water remained constant during the cell cycle but that the volume of osmotically active cell water is highest during S and early G2 and decreases during the mitotic phase, as cells undergo division. (DuPre & Hempling, 1978). More recently, a sensitive resonance technique was used to show that mouse cells undergoing mitosis increased their volumes by 10% (due to osmotic exchange) and decreased their density by 0.4% over a 20 minute period though it should be noted that it was assumed that all cellular water was exchangeable with extracellular water (Son et al., 2015). The fact that variation in density among human cells of a given type is 100 times smaller than variation in cell mass has been invoked as illustrating the importance of cell density (for references see (Neurohr & Amon, 2020)): put differently, small changes may have big effects.

Aquaporins

The water-clock hypothesis is grounded in the origins of life and, as presented so far, is not reliant on sophisticated macromolecules with special functions. That said, the hypothesis must accommodate the existence of such macromolecules in modern cells; in particular, aequorins are proteins responsible for allowing water to cross the membrane. In eukaryotic cells, aequorins play a central role in the cell cycle where they modulate progress through it and facilitate cell volume regulation during cell division (Galan-Cobo et al., 2016). Domains enriched in cardiolipin are found at the division sites and poles (which are former division sites) (Mileykovskaya & Dowhan, 2000)(Koppelman et al., 2001)(Kusaka et al., 2016) and it may therefore be significant that AqpZ, an aequorin from E. coli, is stabilised and modulated by cardiolipin (Laganowsky et al., 2014)(Tan & Torres, 2021); hence, following the initiation of chromosome replication by decreasing water availability, aequorins could be implicated in increasing water availability in the later step of cell division. This would be consistent with the twofold increase in the rate of water influx in activated T-cells as they approach division (Saragovi et al., 2022).

Different water compartments

Hyperosmotic shock and nutrient depletion can lead to the loss of water and plasmolysis with the shrinkage of the cytoplasm and an expansion of regions of the periplasm in bacteria such as E. coli (Shi et al., 2021). As opposed to hyperosmotic shock, membrane retraction after sudden starvation was almost entirely at the new cell pole (Shi et al., 2021). The cell volume of S. cerevisiae under glucose starvation also shrinks (without loss of mass) due to vacuolar expansion (Joyner et al., 2016), which has led to the suggestion that the bacterial periplasm may act as a vacuole-like organelle (Brauer et al., 2023). This might be consistent with the existence of vacuole-like structures in certain E. coli L-forms that lack the normal envelope (Onoda et al., 2000). These results are relevant to the water-clock hypothesis in showing that the different regions of bacteria can have different responses in terms of the influx and efflux of water and hence that the water-clock could be protected to some extent from the exchanges of water with the environment.

Differentiation and water

The state of water within spores has been proposed to play an important role in their resistance to harsh environments. In the core of spores of Bacillus species, analysis via dielectric permittivity (Carstensen et al., 1979) and electron paramagnetic resonance spectra (Johnstone et al., 1982) are consistent with the presence of immobile ions, ¹³C-NMR spectroscopy is consistent with the dipicolinic acid being in a solid-like state (Leuschner & Lillford, 2000), fluorescence shows that the GFP is over four orders of magnitude less mobile in the core than in the cytoplasm of the vegetative cell and the dormancy of these spores has been attributed to conformational changes in enzymes induced by dehydration (Cowan et al., 2003)(Sunde et al., 2009). Using deuterium solid-state NMR experiments, it was concluded that part of the water within spores is inaccessible to external water (Friedline et al., 2014). Ultrafast vibrational spectroscopy and dielectric-relaxation spectroscopy have been used to determine the random orientational motion of water molecules inside B. subtilis spores, E. coli and S. cerevisiae (Tros et al., 2017). Most of this water was similar to pure water but some water had a slower motion that was attributed to it solvating proteins and other molecules and ions (Tros et al., 2017), indeed, around 55% of the intracellular water in the spores was bulk-like compared with around 80% of the water in E. coli and S. cerevisiae (Tros et al., 2017). The proposal of a heterogeneous distribution of water in spores (Sunde et al., 2009) fits with the water-clock hypothesis.

In the case of persisters, which are metabolically inactive (Kim & Wood, 2016), there are few experiments to my knowledge on the state of their intracellular water apart from a report that an increase in the levels of intracellular solutes helps reduce the loss of intracellular water when the temperature is low (NicAogain & O'Byrne, 2016). That said, the reduction in intracellular mobility within stationary phase cells is probably due to the reduction in metabolic activity (Parry et al., 2014), which could be translated into the existence of a diversity of states of daughter cells accompanying their diversity in growth rates (Gangwe Nana et al., 2018).

Ways in which a water-clock might operate

In the bacterium Shewanella oneidensis, a study using high pressures and Quasi-elastic Neutron Scattering led to the suggestion that nanoscale water channels run between macromolecular regions within a dynamically organized cytoplasm rather than a homogenous gel-like one (Foglia et al., 2019). In line with such heterogeneity of water, a FRET-based analysis of S. cerevisiae showed local increases in molecular crowding across the bud neck in the developing daughter cell (Lecinski et al., 2021). In the section ‘water and percolation’, I have raised the possibility that changes in the availability of water cause phase transitions that affect percolation in the plane of the membrane, that is, in 2-D; there is also the possibility that water could affect percolation in 3-D and, in particular, the connectivity resulting from the above water nanochannels.

Wetting and dewetting could also play a role in the dynamics of hyperstructures that leads to the initiation of chromosome replication. Bulk water in ambient conditions is near the liquid-vapour phase boundary whilst water near a weakly-interacting hydrophobic surface is pulled away from the surface to interact more strongly with other water molecules in a dewetting transition; this transition takes the form of an increase in fluctuations of the mean water density at the surface and an increase in the sensitivity of this water density to small perturbations that include conformational changes in biomolecules thereby providing them “ with the powerful ability to tune their interactions and function by manipulating the local context, for example, by confining water between them, or by changing their shape or chemistry” (Patel et al., 2012b).

Effects of heavy water

In the yeast, Schizosaccharomyces pombe, high concentrations of D₂O slowed growth, altered glucose metabolism and eventually perturbed the cytoskeleton and cell wall (Kampmeyer et al., 2020)). In E. coli, replacing H₂O with D₂0 in a glycerol growth medium halved the growth rate and altered the production of several hundred proteins but had no apparent effect on morphology (Opitz et al., 2019)(Kelpsas & von Wachenfeldt, 2021).

Predictions

Using a fluorescent stain, FM 4-64, to visualise membrane domains in E. coli and an intercalator, DAPI, to visualise its chromosome, the start of chromosome segregation coincided with the start of the formation of a membrane domain (Fishov & Woldringh, 1999), consistent with the simultaneous initiation of chromosome replication and of the process leading to cell division. This technique might be combined with refractive index and optical diffraction tomography (Oh et al., 2020)(Shin et al., 2023) so as to correlate these early events with changes in density. Moreover, such tomography combined with the localisation of GFP-labelled proteins should not only show cyclic intracellular variations in density and hence water content in individual bacteria but also help identify the hyperstructures involved. Such identification would benefit from the application of toponomics to bacteria (Schubert et al., 2012). There are two evident ways in which investigations might proceed. One way would be to revisit the experiments showing that a large proportion of intracellular water comes from metabolism (Kreuzer-Martin et al., 2005)(Kreuzer-Martin et al., 2006)(Li et al., 2016); this would entail trying to alter this proportion by using mutants defective in the assembly and activity of the hyperstructures involved. The other way would be to study this proportion of labelled water by using in vitro systems containing the enzymes involved (including in vitro transcription-translation systems) or by using a minimal cell. Treatments of E. coli that expand the nucleoid and increase cytoplasmic crowding (Mentre & Hui Bon Hoa, 2001)(Pelletier et al., 2012)(Pittas et al., 2023) might be combined with the localisation of the GFP-labelled proteins that constitute the hyperstructures in the water-clock (as identified above). If these hyperstructures were affected, there should be consequences for the regulation of the cell cycle. Finally, there are many permutations of phase transitions, and hyperstructure states and activities and simulations and mathematical modelling should prove helpful in selecting the most plausible scenario.

In physics, nonlinear behaviour often originates from phase transitions, which can resemble on/off switches insofar as they can be triggered by small variations in the system’s conditions. The phase transitions of hyperstructures invoked in the water-clock hypothesis involve spatially extensive, qualitative changes to the membrane and cytoplasm that would be perfect for the rapid and precise timing of signals needed to coordinate and integrate cell cycle events and phenotypic adaptations in general. Moreover, the selection of a manageable number of phenotypically coherent combinations of hyperstructures and phase transitions by the principal component of cells – water – means that a ‘life on the edge of phase transitions’ would solve the problem of there being apparently too many phenotypes for natural selection to be able to act (Kauffman, 1996)(Norris & Ripoll, 2021). The water-clock hypothesis also has the advantage that it is part of a conceptual framework that can bring together hypotheses about the regulation of the cell cycle, bacterial structure, differentiation and the origins of life.

One criterion for evaluating a new hypothesis is whether it can explain results that existing hypotheses cannot. For example, there has been no satisfactory explanation for how scores of minichromosomes are replicated synchronously in an integratively suppressed strain of E. coli in which the chromosome itself is replicated randomly (Eliasson & Nordstrom, 1997). In the case of the water-clock, however, the phase transition that results from the decrease in water availability would not be completely disrupted by the randomly replicating chromosome and hence would still be able to trigger the replication of the minichromosomes. Decreased water availability following a lowering of temperature (Watson et al., 2023) might also help explain the otherwise puzzling multiple initiation events that occur when a dnaA(ts) mutant is transferred from a non-permissive to a permissive temperature in the absence of protein synthesis (Eberle & Forrest, 1982).

The version of the water-clock hypothesis presented here is incomplete. It fails to invoke several fundamental, often controversial, aspects of studies into the putative properties of water. These include water activity (Nguyen & Kumar, 2022), two- or three-state water (Wiggins, 2009)(Gao et al., 2022)(Dargaville & Hutmacher, 2022), kosmotropes and chaotropes (Moelbert et al., 2004), and the long-range of the influence of a surface on water structure (Del Giudice et al., 2015)(Hwang et al., 2018). It is not therefore a question of whether the water-clock hypothesis as presented here is wrong as the extent to which it is wrong. It may, of course, be completely wrong (though hopefully not falling into Pauli’s ‘not even wrong’ class) and an enthusiastic reader might explore the opposite scenario in which the availability of water accumulates until a phase transition that triggers the cell cycle. The take-home message is that the control of bacterial cell physiology by water, phase transitions and hyperstructures is a fertile conceptual framework for the development of new hypotheses and new types of experimentation, simulation and calculation. In which case, it would constitute a Garden of Eden from which we should be neither barred nor expelled.

Author Contribution

VN had the idea and wrote the paper.

Acknowledgements

For helpful comments and/or encouragement, I thank Jie Xiao, Monika Glinkowska, Bianca Sclavi, Vladimir Uversky, Petra Schwille, German Rivas, Christine Jacobs-Wagner, Laurent Janniere, Conrad Woldringh, Marie-Claire Bellissent-Funel, Pascale Mentre, Sungchul Ji, John Helmann and Jerry Pollack.

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Hypothesis: bacteria live on the edge of phase transitions with a cell cycle regulated by a water-clock

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Abstract

Figures

Introduction

The general, water-clock hypothesis

Advantages of the hypothesis

Results relevant to the water-clock hypothesis

Intracellular water is from metabolism

Molecular and macromolecular crowding or water availability

Bacteria are highly structured

A single, hyperstructure-driven, cell cycle signal

Phase separation, condensates and trajectories

Water availability and the dynamics of condensates are related

Metabolic activity fluidises the cytoplasm

Phase transitions are excellent candidates for signalling

Macromolecular crowding affects DNA replication

Phase transitions and post-translational modifications

Phase transitions and DNA

Hydration alters DNA conformation and the binding of proteins to DNA

Water and membranes

Water and enzymic activity

Water and ions

Water and percolation

Macromolecular crowding affects transcription

Macromolecular crowding and ribosomes

Variations in intracellular water and the cell cycle

Aquaporins

Different water compartments

Differentiation and water

Ways in which a water-clock might operate

Effects of heavy water

Predictions

Discussion

Declarations

Author Contribution

Acknowledgements

References

Additional Declarations

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