Does the Semiconservative Nature of DNA Replication Facilitate Coherent Phenotypic Diversity?

It has been clear for over sixty years that the principal method whereby cells replicate and segregate their DNA is semiconservative. It is much less clear why it should be like this rather than, say, conservative. Recently, evidence has accumulated that supports the hypothesis that one of the functions of the cell cycle is to generate phenotypically different daughter cells, even in nondifferentiating bacteria such as Escherichia coli Evidence has also accumulated that the bacterial phenotype is determined by the functioning of extended assemblies of macromolecules termed hyperstructures. One class of these hyperstructures is attached dynamically to a DNA strand by the coupling of transcription and translation. Previously, we proposed in the strand segregation model that one set of hyperstructures accompanies one parental strand into one daughter cell while another set of hyperstructures accompanies the other parental strand into the other daughter cell. This epigenetic mechanism results in daughter cells having different phenotypes. Here, I propose that one of the reasons why semiconservative replication has been selected is because it allows the generation of a population containing cells with very different growth rates even in steady-state conditions.

Modeling of sensing potency of cytoskeletal systems decorated with metabolic enzymes.

The highly dynamic cytoskeleton interacts with enzymes and other proteins that are involved in metabolic or signaling pathways. These interactions can influence the structural and functional characteristics of the partners at the microscopic level of individual proteins and polymers. In this work the functional consequences of such interactions have been studied at the macroscopic level in order to evaluate the integrative and regulatory roles of the metabolic pathways associated with the microtubule cytoskeleton. Here we present mathematical models of the interactions between a hypothetical metabolic pathway and microtubule assembly, and explore for the first time the functional consequences of these interactions in distinct situations. The models include kinetic constants of the individual steps and testable, relevant parameters which allow the quantification of the coupled processes at the microscopic and macroscopic levels. For example our kinetic model for the self-assembly of microtubules reproduces the alteration of the time-dependent turbidity caused by pyruvate kinase binding. Our data reveal the power of a mechanistic description of a filamentous system to explain how cells sense the state of metabolic and other pathways.

What properties of life are universal? Substance-free, scale-free life.

One approach to answering the question of what properties of life are universal is to try to answer the question of what are the essential properties of biology's best understood model organism, Escherichia coli. One of these properties is competitive coherence whereby E. coli reconciles the generation of a coherent cell state with the generation of a coherent sequence of cell states. The second property is differentiation which occurs ineluctably when E. coli divides. The third property is dualism which is how E. coli navigates between the two main attractors of phenotypes - survival and growth - which are based on quasi-equilibrium and non-equilibrium structures, respectively. The fourth property is complementarity: the interactions between the molecules and macromolecules that constitute E. coli protect them from degradation and confer new properties. The fifth property is multi-scale existence: E. coli exists at levels extending from the bacterium to the global super-organism. The sixth property is maintenance of connectivity; growth alters connectivity and, in the case of E. coli, alters the phenotype. The seventh property is the combination of intensity sensing (the constituents can work no harder) and quantity sensing (too much unused material has been made); this combination is used by E. coli to drive its cell cycle and thereby generate an environmentally adapted population of cells. The eighth property is subjective experience which exists even at the level of a single E. coli but which only becomes important at higher levels of organisation. I propose that the search for life at other times and in other places be based on the above eight universal properties and be independent of both particular substances and spatio-temporal scales.

Perspective Strategies for Interventions in Parkinsonism: Remedying the Neglected Role of TPPP.

Neurological disorders such as Parkinsonism cause serious socio-economic problems as there are, at present, only therapies that treat their symptoms. The well-established hallmark alpha-synuclein (SYN) is enriched in the inclusion bodies characteristic of Parkinsonism. We discovered a prominent partner of SYN, termed Tubulin Polymerization Promoting Protein (TPPP), which has important physiological and pathological activities such as the regulation of the microtubule network and the promotion of SYN aggregation. The role of TPPP in Parkinsonism is often neglected in research, which we here attempt to remedy. In the normal brain, SYN and TPPP are expressed endogenously in neurons and oligodendrocytes, respectively, whilst, at an early stage of Parkinsonism, soluble hetero-associations of these proteins are found in both cell types. The cell-to-cell transmission of these proteins, which is central to disease progression, provides a unique situation for specific drug targeting. Different strategies for intervention and for the discovery of biomarkers include (i) interface targeting of the SYN-TPPP hetero-complex; (ii) proteolytic degradation of SYN and/or TPPP using the PROTAC technology; and (iii) depletion of the proteins by miRNA technology. We also discuss the potential roles of SYN and TPPP in the phenotype stabilization of neurons and oligodendrocytes.

Hypothesis: bacteria control host appetites.

To help investigate the relationship between inflammatory and other diseases and the composition of the gut microbiota, we propose that a positive-feedback loop exists between the preferences of the host for a particular dietary regimen, the composition of the gut microbiota that depends on this regimen, and the preferences of the host as influenced by the gut microbiota. We cite evidence in support of this hypothesis and make testable predictions.

Sensor potency of the moonlighting enzyme-decorated cytoskeleton: the cytoskeleton as a metabolic sensor.

BACKGROUND: There is extensive evidence for the interaction of metabolic enzymes with the eukaryotic cytoskeleton. The significance of these interactions is far from clear. PRESENTATION OF THE HYPOTHESIS: In the cytoskeletal integrative sensor hypothesis presented here, the cytoskeleton senses and integrates the general metabolic activity of the cell. This activity depends on the binding to the cytoskeleton of enzymes and, depending on the nature of the enzyme, this binding may occur if the enzyme is either active or inactive but not both. This enzyme-binding is further proposed to stabilize microtubules and microfilaments and to alter rates of GTP and ATP hydrolysis and their levels. TESTING THE HYPOTHESIS: Evidence consistent with the cytoskeletal integrative sensor hypothesis is presented in the case of glycolysis. Several testable predictions are made. There should be a relationship between post-translational modifications of tubulin and of actin and their interaction with metabolic enzymes. Different conditions of cytoskeletal dynamics and enzyme-cytoskeleton binding should reveal significant differences in local and perhaps global levels and ratios of ATP and GTP. The different functions of moonlighting enzymes should depend on cytoskeletal binding. IMPLICATIONS OF THE HYPOTHESIS: The physical and chemical effects arising from metabolic sensing by the cytoskeleton would have major consequences on cell shape, dynamics and cell cycle progression. The hypothesis provides a framework that helps the significance of the enzyme-decorated cytoskeleton be determined.

Novel Principles and Methods in Bacterial Cell Cycle Physiology: Celebrating the Charles E. Helmstetter Prize in 2022.

This Special Issue celebrates the creation of the Charles E [...].

Open Questions about the Roles of DnaA, Related Proteins, and Hyperstructure Dynamics in the Cell Cycle.

The DnaA protein has long been considered to play the key role in the initiation of chromosome replication in modern bacteria. Many questions about this role, however, remain unanswered. Here, we raise these questions within a framework based on the dynamics of hyperstructures, alias large assemblies of molecules and macromolecules that perform a function. In these dynamics, hyperstructures can (1) emit and receive signals or (2) fuse and separate from one another. We ask whether the DnaA-based initiation hyperstructure acts as a logic gate receiving information from the membrane, the chromosome, and metabolism to trigger replication; we try to phrase some of these questions in terms of DNA supercoiling, strand opening, glycolytic enzymes, SeqA, ribonucleotide reductase, the macromolecular synthesis operon, post-translational modifications, and metabolic pools. Finally, we ask whether, underpinning the regulation of the cell cycle, there is a physico-chemical clock inherited from the first protocells, and whether this clock emits a single signal that triggers both chromosome replication and cell division.

The roles of nucleoid-associated proteins and topoisomerases in chromosome structure, strand segregation, and the generation of phenotypic heterogeneity in bacteria.

How to adapt to a changing environment is a fundamental, recurrent problem confronting cells. One solution is for cells to organize their constituents into a limited number of spatially extended, functionally relevant, macromolecular assemblies or hyperstructures, and then to segregate these hyperstructures asymmetrically into daughter cells. This asymmetric segregation becomes a particularly powerful way of generating a coherent phenotypic diversity when the segregation of certain hyperstructures is with only one of the parental DNA strands and when this pattern of segregation continues over successive generations. Candidate hyperstructures for such asymmetric segregation in prokaryotes include those containing the nucleoid-associated proteins (NAPs) and the topoisomerases. Another solution to the problem of creating a coherent phenotypic diversity is by creating a growth-environment-dependent gradient of supercoiling generated along the replication origin-to-terminus axis of the bacterial chromosome. This gradient is modulated by transcription, NAPs, and topoisomerases. Here, we focus primarily on two topoisomerases, TopoIV and DNA gyrase in Escherichia coli, on three of its NAPs (H-NS, HU, and IHF), and on the single-stranded binding protein, SSB. We propose that the combination of supercoiling-gradient-dependent and strand-segregation-dependent topoisomerase activities result in significant differences in the supercoiling of daughter chromosomes, and hence in the phenotypes of daughter cells.

The Sherpa hypothesis: Phenotype-Preserving Disordered Proteins stabilize the phenotypes of neurons and oligodendrocytes.

Intrinsically disordered proteins (IDPs), which can interact with many partner proteins, are central to many physiological functions and to various pathologies that include neurodegeneration. Here, we introduce the Sherpa hypothesis, according to which a subset of stable IDPs that we term Phenotype-Preserving Disordered Proteins (PPDP) play a central role in protecting cell phenotypes from perturbations. To illustrate and test this hypothesis, we computer-simulate some salient features of how cells evolve and differentiate in the presence of either a single PPDP or two incompatible PPDPs. We relate this virtual experiment to the pathological interactions between two PPDPs, α-synuclein and Tubulin Polymerization Promoting Protein/p25, in neurodegenerative disorders. Finally, we discuss the implications of the Sherpa hypothesis for aptamer-based therapies of such disorders.

Hyperstructure interactions influence the virulence of the type 3 secretion system in yersiniae and other bacteria.

A paradigm shift in our thinking about the intricacies of the host-parasite interaction is required that considers bacterial structures and their relationship to bacterial pathogenesis. It has been proposed that interactions between extended macromolecular assemblies, termed hyperstructures (which include multiprotein complexes), determine bacterial phenotypes. In particular, it has been proposed that hyperstructures can alter virulence. Two such hyperstructures have been characterized in both pathogenic and nonpathogenic bacteria. Present within a number of both human and plant Gram-negative pathogens is the type 3 secretion system (T3SS) injectisome which in some bacteria serves to inject toxic effector proteins directly into targeted host cells resulting in their paralysis and eventual death (but which in other bacteria prevents the death of the host). The injectisome itself comprises multiple protein subunits, which are all essential for its function. The degradosome is another multiprotein complex thought to be involved in cooperative RNA decay and processing of mRNA transcripts and has been very well characterized in nonpathogenic Escherichia coli. Recently, experimental evidence has suggested that a degradosome exists in the yersiniae as well and that its interactions within the pathogens modulate their virulence. Here, we explore the possibility that certain interactions between hyperstructures, like the T3SS and the degradosome, can ultimately influence the virulence potential of the pathogen based upon the physical locations of hyperstructures within the cell.

How did metabolism and genetic replication get married?

In addressing the question of the origins of the relationship between metabolism and genetic replication, we consider the implications of a prebiotic, fission-fusion, ecology of composomes. We emphasise the importance of structures and non-specific catalysis on interfaces created by structures. From the assumption that the bells of the metabolism-replication wedding still echo in modern cells, we argue that the functional assemblies of macromolecules that constitute hyperstructures in modern bacteria are the descendants of composomes and that interactions at the hyperstructure level control the cell cycle. A better understanding of the cell cycle should help understand the original metabolism-replication marriage. This understanding requires new concepts such as metabolic signalling, metabolic sensing and Dualism, which entails the cells in a population varying the ratios of equilibrium to non-equilibrium hyperstructures so as to maximise the chances of both survival and growth. A deeper understanding of the coupling between metabolism and replication may also require a new view of cell cycle functions in creating a coherent diversity of phenotypes and in narrowing the combinatorial catalytic space. To take these ideas into account, we propose the Accordion model in which a dynamic interface between lipid domains catalysed monomer to polymer reactions and became decorated with peptides and nucleotides that favoured their own catalysis. In this model, metabolism, replication, differentiation and division all began together at the interface between extended equilibrium structures within protocells or composomes.

The role of calcium in the recall of stored morphogenetic information by plants.

Flax seedlings grown in the absence of environmental stimuli, stresses and injuries do not form epidermal meristems in their hypocotyls. Such meristems do form when the stimuli are combined with a transient depletion of calcium. These stimuli include the "manipulation stimulus" resulting from transferring the seedlings from germination to growth conditions. If, after a stimulus, calcium depletion is delayed, meristem production is also delayed; in other words, the meristem-production instruction can be memorised. Memorisation includes both storage and recall of information. Here, we focus on information recall. We show that if the first transient calcium depletion is followed by a second transient depletion there is a new round of meristem production. We also show that if an excess of calcium follows calcium depletion, meristem production is blocked; but if the excess of calcium is in turn followed by another calcium depletion, again there is a new round of meristem production. The same stored information can thus be recalled repeatedly (at least twice). We describe a conceptual model that takes into account these findings.

A Defective Viral Particle Approach to COVID-19.

The novel coronavirus SARS-CoV-2 has caused a pandemic resulting in millions of deaths worldwide. While multiple vaccines have been developed, insufficient vaccination combined with adaptive mutations create uncertainty for the future. Here, we discuss novel strategies to control COVID-19 relying on Defective Interfering Particles (DIPs) and related particles that arise naturally during an infection. Our intention is to encourage and to provide the basis for the implementation of such strategies by multi-disciplinary teams. We therefore provide an overview of SARS-CoV-2 for a multi-disciplinary readership that is specifically tailored to these strategies, we identify potential targets based on the current knowledge of the properties and functions of coronaviruses, and we propose specific strategies to engineer DIPs and other interfering or therapeutic nanoparticles.

Functional taxonomy of bacterial hyperstructures.

The levels of organization that exist in bacteria extend from macromolecules to populations. Evidence that there is also a level of organization intermediate between the macromolecule and the bacterial cell is accumulating. This is the level of hyperstructures. Here, we review a variety of spatially extended structures, complexes, and assemblies that might be termed hyperstructures. These include ribosomal or "nucleolar" hyperstructures; transertion hyperstructures; putative phosphotransferase system and glycolytic hyperstructures; chemosignaling and flagellar hyperstructures; DNA repair hyperstructures; cytoskeletal hyperstructures based on EF-Tu, FtsZ, and MreB; and cell cycle hyperstructures responsible for DNA replication, sequestration of newly replicated origins, segregation, compaction, and division. We propose principles for classifying these hyperstructures and finally illustrate how thinking in terms of hyperstructures may lead to a different vision of the bacterial cell.

Combed single DNA molecules imaged by secondary ion mass spectrometry.

Studies of replication, recombination, and rearrangements at the level of individual molecules of DNA are often limited by problems of resolution or of perturbations caused by the modifications that are needed for imaging. The Combing-Imaging by Secondary Ion Mass Spectrometry (SIMS) (CIS) method helps solve these problems by combining DNA combing, cesium flooding, and quantitative imaging via the NanoSIMS 50. We show here that CIS can reveal, on the 50 nm scale, individual DNA fibers labeled with different, nonradioactive isotopes and, moreover, that it can quantify these isotopes so as to detect and measure the length of one or more short nucleic acid fragments associated with a longer fiber.

Competitive Coherence Generates Qualia in Bacteria and Other Living Systems.

The relevance of bacteria to subjective experiences or qualia is underappreciated. Here, I make four proposals. Firstly, living systems traverse sequences of active states that determine their behaviour; these states result from competitive coherence, which depends on connectivity-based competition between a Next process and a Now process, whereby elements in the active state at time n+1 are chosen between the elements in the active state at time n and those elements in the developing n+1 state. Secondly, bacteria should help us link the mental to the physical world given that bacteria were here first, are highly complex, influence animal behaviour and dominate the Earth. Thirdly, the operation of competitive coherence to generate active states in bacteria, brains and other living systems is inseparable from qualia. Fourthly, these qualia become particularly important to the generation of active states in the highest levels of living systems, namely, the ecosystem and planetary levels.

Hypothesis: nucleoid-associated proteins segregate with a parental DNA strand to generate coherent phenotypic diversity.

The generation of a phenotypic diversity that is coherent across a bacterial population is a fundamental problem. We propose here that the DNA strand-specific segregation of certain nucleoid-associated proteins or NAPs results in these proteins being asymmetrically distributed to the daughter cells. We invoke a variety of mechanisms as responsible for this asymmetrical segregation including those based on differences between the leading and lagging strands, post-translational modifications, oligomerisation and association with membrane domains.