The Rtf1/Prf1-dependent histone modification axis counteracts multi-drug resistance in fission yeast.

RNA polymerase II transcription elongation directs an intricate pattern of histone modifications. This pattern includes a regulatory cascade initiated by the elongation factor Rtf1, leading to monoubiquitylation of histone H2B, and subsequent methylation of histone H3 on lysine 4. Previous studies have defined the molecular basis for these regulatory relationships, but it remains unclear how they regulate gene expression. To address this question, we investigated a drug resistance phenotype that characterizes defects in this axis in the model eukaryote Schizosaccharomyces pombe (fission yeast). The mutations caused resistance to the ribonucleotide reductase inhibitor hydroxyurea (HU) that correlated with a reduced effect of HU on dNTP pools, reduced requirement for the S-phase checkpoint, and blunting of the transcriptional response to HU treatment. Mutations in the C-terminal repeat domain of the RNA polymerase II large subunit Rpb1 led to similar phenotypes. Moreover, all the HU-resistant mutants also exhibited resistance to several azole-class antifungal agents. Our results suggest a novel, shared gene regulatory function of the Rtf1-H2Bub1-H3K4me axis and the Rpb1 C-terminal repeat domain in controlling fungal drug tolerance.

Dynamics and quantitative contribution of the aminoglycoside 6'-N-acetyltransferase type Ib to amikacin resistance.

Aminoglycosides are essential components in the available armamentarium to treat bacterial infections. The surge and rapid dissemination of resistance genes strongly reduce their efficiency, compromising public health. Among the multitude of modifying enzymes that confer resistance to aminoglycosides, the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] is the most prevalent and relevant in the clinical setting as it can inactivate numerous aminoglycosides, such as amikacin. Although the mechanism of action, structure, and biochemical properties of the AAC(6')-Ib protein have been extensively studied, the contribution of the intracellular milieu to its activity remains unclear. In this work, we used a fluorescent-based system to quantify the number of AAC(6')-Ib per cell in Escherichia coli, and we modulated this copy number with the CRISPR interference method. These tools were then used to correlate enzyme concentrations with amikacin resistance levels. Our results show that resistance to amikacin increases linearly with a higher concentration of AAC(6')-Ib until it reaches a plateau at a specific protein concentration. In vivo imaging of this protein shows that it diffuses freely within the cytoplasm of the cell, but it tends to form inclusion bodies at higher concentrations in rich culture media. Addition of a chelating agent completely dissolves these aggregates and partially prevents the plateau in the resistance level, suggesting that AAC(6')-Ib aggregation lowers resistance to amikacin. These results provide the first step in understanding the cellular impact of each AAC(6')-Ib molecule on aminoglycoside resistance. They also highlight the importance of studying its dynamic behavior within the cell.IMPORTANCEAntibiotic resistance is a growing threat to human health. Understanding antibiotic resistance mechanisms can serve as foundation for developing innovative treatment strategies to counter this threat. While numerous studies clarified the genetics and dissemination of resistance genes and explored biochemical and structural features of resistance enzymes, their molecular dynamics and individual contribution to resistance within the cellular context remain unknown. Here, we examined this relationship modulating expression levels of aminoglycoside 6'-N-acetyltransferase type Ib, an enzyme of clinical relevance. We show a linear correlation between copy number of the enzyme per cell and amikacin resistance levels up to a threshold where resistance plateaus. We propose that at concentrations below the threshold, the enzyme diffuses freely in the cytoplasm but aggregates at the cell poles at concentrations over the threshold. This research opens promising avenues for studying enzyme solubility's impact on resistance, creating opportunities for future approaches to counter resistance.

RNA polymerase II dynamics and mRNA stability feedback scale mRNA amounts with cell size.

A fundamental feature of cellular growth is that total protein and RNA amounts increase with cell size to keep concentrations approximately constant. A key component of this is that global transcription rates increase in larger cells. Here, we identify RNA polymerase II (RNAPII) as the limiting factor scaling mRNA transcription with cell size in budding yeast, as transcription is highly sensitive to the dosage of RNAPII but not to other components of the transcriptional machinery. Our experiments support a dynamic equilibrium model where global RNAPII transcription at a given size is set by the mass action recruitment kinetics of unengaged nucleoplasmic RNAPII to the genome. However, this only drives a sub-linear increase in transcription with size, which is then partially compensated for by a decrease in mRNA decay rates as cells enlarge. Thus, limiting RNAPII and feedback on mRNA stability work in concert to scale mRNA amounts with cell size.

Characterizing interaction of multiple nanocavity confined plasmids in presence of large DNA model nucleoid.

Bacteria have numerous large dsDNA molecules that freely interact within the cell, including multiple plasmids, primary and secondary chromosomes. The cell membrane maintains a micron-scale confinement, ensuring that the dsDNA species are proximal at all times and interact strongly in a manner influenced by the cell morphology (e.g. whether cell geometry is spherical or anisotropic). These interactions lead to non-uniform spatial organization and complex dynamics, including segregation of plasmid DNA to polar and membrane proximal regions. However, exactly how this organization arises, how it depends on cell morphology and number of interacting dsDNA species are under debate. Here, using an in vitro nanofluidic model, featuring a cavity that can be opened and closed in situ, we address how plasmid copy number and confinement geometry alter plasmid spatial distribution and dynamics. We find that increasing the plasmid number alters the plasmid spatial distribution and shortens the plasmid polar dwell time; sharper cavity end curvature leads to longer plasmid dwell times.

Dynamics and quantitative contribution of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] to amikacin resistance.

Aminoglycosides are essential components in the available armamentarium to treat bacterial infections. The surge and rapid dissemination of resistance genes strongly reduce their efficiency, compromising public health. Among the multitude of modifying enzymes that confer resistance to aminoglycosides, the aminoglycoside acetyltransferase AAC(6')-Ib is the most prevalent and relevant in the clinical setting as it can inactivate numerous aminoglycosides, such as amikacin. Although the mechanism of action, structure, and biochemical properties of the AAC(6')-Ib protein have been extensively studied, the contribution of the intracellular milieu to its activity remains unclear. In this work, we used a fluorescent-based system to quantify the number of AAC(6')-Ib per cell in Escherichia coli, and we modulated this copy number with the CRISPR interference method. These tools were then used to correlate enzyme concentrations with amikacin resistance levels. Our results show that resistance to amikacin increases linearly with a higher concentration of AAC(6')-Ib until it reaches a plateau at a specific protein concentration. In vivo imaging of this protein shows that it diffuses freely within the cytoplasm of the cell, but it tends to form inclusion bodies at higher concentrations in rich culture media. Addition of a chelating agent completely dissolves these aggregates and partially prevents the plateau in the resistance level, suggesting that AAC(6')-Ib aggregation lowers resistance to amikacin. These results provide the first step in understanding the cellular impact of each AAC(6')-Ib molecule on aminoglycoside resistance. They also highlight the importance of studying its dynamic behavior within the cell.

Confinement anisotropy drives polar organization of two DNA molecules interacting in a nanoscale cavity.

There is growing appreciation for the role phase transition based phenomena play in biological systems. In particular, self-avoiding polymer chains are predicted to undergo a unique confinement dependent demixing transition as the anisotropy of the confined space is increased. This phenomenon may be relevant for understanding how interactions between multiple dsDNA molecules can induce self-organized structure in prokaryotes. While recent in vivo experiments and Monte Carlo simulations have delivered essential insights into this phenomenon and its relation to bacteria, there are fundamental questions remaining concerning how segregated polymer states arise, the role of confinement anisotropy and the nature of the dynamics in the segregated states. To address these questions, we introduce an artificial nanofluidic model to quantify the interactions of multiple dsDNA molecules in cavities with controlled anisotropy. We find that two dsDNA molecules of equal size confined in an elliptical cavity will spontaneously demix and orient along the cavity poles as cavity eccentricity is increased; the two chains will then swap pole positions with a frequency that decreases with increasing cavity eccentricity. In addition, we explore a system consisting of a large dsDNA molecule and a plasmid molecule. We find that the plasmid is excluded from the larger molecule and will exhibit a preference for the ellipse poles, giving rise to a non-uniform spatial distribution in the cavity that may help explain the non-uniform plasmid distribution observed during in vivo imaging of high-copy number plasmids in bacteria.

Dynamics of Proteins and Macromolecular Machines in Escherichia coli.

Proteins are major contributors to the composition and the functions in the cell. They often assemble into larger structures, macromolecular machines, to carry out intricate essential functions. Although huge progress in understanding how macromolecular machines function has been made by reconstituting them in vitro, the role of the intracellular environment is still emerging. The development of fluorescence microscopy techniques in the last 2 decades has allowed us to obtain an increased understanding of proteins and macromolecular machines in cells. Here, we describe how proteins move by diffusion, how they search for their targets, and how they are affected by the intracellular environment. We also describe how proteins assemble into macromolecular machines and provide examples of how frequent subunit turnover is used for them to function and to respond to changes in the intracellular conditions. This review emphasizes the constant movement of molecules in cells, the stochastic nature of reactions, and the dynamic nature of macromolecular machines.

Bound2Learn: a machine learning approach for classification of DNA-bound proteins from single-molecule tracking experiments.

DNA-bound proteins are essential elements for the maintenance, regulation, and use of the genome. The time they spend bound to DNA provides useful information on their stability within protein complexes and insight into the understanding of biological processes. Single-particle tracking allows for direct visualization of protein-DNA kinetics, however, identifying whether a molecule is bound to DNA can be non-trivial. Further complications arise when tracking molecules for extended durations in processes with slow kinetics. We developed a machine learning approach, termed Bound2Learn, using output from a widely used tracking software, to robustly classify tracks in order to accurately estimate residence times. We validated our approach in silico, and in live-cell data from Escherichia coli and Saccharomyces cerevisiae. Our method has the potential for broad utility and is applicable to other organisms.

Processive Activity of Replicative DNA Polymerases in the Replisome of Live Eukaryotic Cells.

DNA replication is carried out by a multi-protein machine called the replisome. In Saccharomyces cerevisiae, the replisome is composed of over 30 different proteins arranged into multiple subassemblies, each performing distinct activities. Synchrony of these activities is required for efficient replication and preservation of genomic integrity. How this is achieved is particularly puzzling at the lagging strand, where current models of the replisome architecture propose turnover of the canonical lagging strand polymerase, Pol δ, at every cycle of Okazaki fragment synthesis. Here, we established single-molecule fluorescence microscopy protocols to study the binding kinetics of individual replisome subunits in live S. cerevisiae. Our results show long residence times for most subunits at the active replisome, supporting a model where all subassemblies bind tightly and work in a coordinated manner for extended periods, including Pol δ, redefining the architecture of the active eukaryotic replisome.

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation.

Once described as mere "bags of enzymes," bacterial cells are in fact highly organized, with many macromolecules exhibiting nonuniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid-liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in Escherichia coli Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrient-rich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components. Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and demonstrates that this process can serve as a mechanism for intracellular organization in prokaryotes and eukaryotes alike.

Interaction with single-stranded DNA-binding protein localizes ribonuclease HI to DNA replication forks and facilitates R-loop removal.

DNA replication complexes (replisomes) routinely encounter proteins and unusual nucleic acid structures that can impede their progress. Barriers can include transcription complexes and R-loops that form when RNA hybridizes with complementary DNA templates behind RNA polymerases. Cells encode several RNA polymerase and R-loop clearance mechanisms to limit replisome exposure to these potential obstructions. One such mechanism is hydrolysis of R-loops by ribonuclease HI (RNase HI). Here, we examine the cellular role of the interaction between Escherichia coli RNase HI and the single-stranded DNA-binding protein (SSB) in this process. Interaction with SSB localizes RNase HI foci to DNA replication sites. Mutation of rnhA to encode an RNase HI variant that cannot interact with SSB but that maintains enzymatic activity (rnhAK60E) eliminates RNase HI foci. The mutation also produces a media-dependent slow-growth phenotype and an activated DNA damage response in cells lacking Rep helicase, which is an enzyme that disrupts stalled transcription complexes. RNA polymerase variants that are thought to increase or decrease R-loop accumulation enhance or suppress, respectively, the growth phenotype of rnhAK60E rep::kan strains. These results identify a cellular role for the RNase HI/SSB interaction in helping to clear R-loops that block DNA replication.

Small Klebsiella pneumoniae Plasmids: Neglected Contributors to Antibiotic Resistance.

Klebsiella pneumoniae is the causative agent of community- and, more commonly, hospital-acquired infections. Infections caused by this bacterium have recently become more dangerous due to the acquisition of multiresistance to antibiotics and the rise of hypervirulent variants. Plasmids usually carry genes coding for resistance to antibiotics or virulence factors, and the recent sequence of complete K. pneumoniae genomes showed that most strains harbor many of them. Unlike large plasmids, small, usually high copy number plasmids, did not attract much attention. However, these plasmids may include genes coding for specialized functions, such as antibiotic resistance, that can be expressed at high levels due to gene dosage effect. These genes may be part of mobile elements that not only facilitate their dissemination but also participate in plasmid evolution. Furthermore, high copy number plasmids may also play a role in evolution by allowing coexistence of mutated and non-mutated versions of a gene, which helps to circumvent the constraints imposed by trade-offs after certain genes mutate. Most K. pneumoniae plasmids 25-kb or smaller replicate by the ColE1-type mechanism and many of them are mobilizable. The transposon Tn1331 and derivatives were found in a high percentage of these plasmids. Another transposon that was found in representatives of this group is the bla KPC-containing Tn4401. Common resistance determinants found in these plasmids were aac(6')-Ib and genes coding for ß-lactamases including carbapenemases.

Correction: Probing the organization and dynamics of two DNA chains trapped in a nanofluidic cavity.

Correction for 'Probing the organization and dynamics of two DNA chains trapped in a nanofluidic cavity' by Xavier Capaldi et al., Soft Matter, 2018, 14, 8455-8465.

A quest for coordination among activities at the replisome.

Faithful DNA replication is required for transmission of the genetic material across generations. The basic mechanisms underlying this process are shared among all organisms: progressive unwinding of the long double-stranded DNA; synthesis of RNA primers; and synthesis of a new DNA chain. These activities are invariably performed by a multi-component machine called the replisome. A detailed description of this molecular machine has been achieved in prokaryotes and phages, with the replication processes in eukaryotes being comparatively less known. However, recent breakthroughs in the in vitro reconstitution of eukaryotic replisomes have resulted in valuable insight into their functions and mechanisms. In conjunction with the developments in eukaryotic replication, an emerging overall view of replisomes as dynamic protein ensembles is coming into fruition. The purpose of this review is to provide an overview of the recent insights into the dynamic nature of the bacterial replisome, revealed through single-molecule techniques, and to describe some aspects of the eukaryotic replisome under this framework. We primarily focus on Escherichia coli and Saccharomyces cerevisiae (budding yeast), since a significant amount of literature is available for these two model organisms. We end with a description of the methods of live-cell fluorescence microscopy for the characterization of replisome dynamics.

The bacterial cell cycle, chromosome inheritance and cell growth.

All viable bacterial cells, whether they divide symmetrically or asymmetrically, must coordinate their growth, division, cell volume and shape with the inheritance of the genome. These coordinated processes maintain genome integrity over generations as chromosomes are duplicated and segregated during each cell cycle, and include the organization of DNA into nucleoids, controlled and faithful DNA replication, chromosome unlinking and faithful segregation into daughter cells. In this Review, we explore the contributions of chromosome structure and nucleoid organization to cell cycle regulation, detail the cellular processes involved in the initiation of DNA replication and DNA segregation and explore how those processes are linked to cell growth and cell division. Furthermore, we address how the study of a growing number of bacterial species enables the search for common principles that underlie the coordination of chromosome inheritance with the cell cycle.

Replisome activity slowdown after exposure to ultraviolet light in Escherichia coli.

The replisome is a multiprotein machine that is responsible for replicating DNA. During active DNA synthesis, the replisome tightly associates with DNA. In contrast, after DNA damage, the replisome may disassemble, exposing DNA to breaks and threatening cell survival. Using live cell imaging, we studied the effect of UV light on the replisome of Escherichia coli Surprisingly, our results showed an increase in Pol III holoenzyme (Pol III HE) foci post-UV that do not colocalize with the DnaB helicase. Formation of these foci is independent of active replication forks and dependent on the presence of the χ subunit of the clamp loader, suggesting recruitment of Pol III HE at sites of DNA repair. Our results also showed a decrease of DnaB helicase foci per cell after UV, consistent with the disassembly of a fraction of the replisomes. By labeling newly synthesized DNA, we demonstrated that a drop in the rate of synthesis is not explained by replisome disassembly alone. Instead, we show that most replisomes continue synthesizing DNA at a slower rate after UV. We propose that the slowdown in replisome activity is a strategy to prevent clashes with engaged DNA repair proteins and preserve the integrity of the replication fork.

Probing the organization and dynamics of two DNA chains trapped in a nanofluidic cavity.

Here we present a pneumatically-actuated nanofluidic platform that has the capability of dynamically controlling the confinement environment of macromolecules in solution. Using a principle familiar from classic devices based on soft-lithography, the system uses pneumatic pressure to deflect a thin nitride lid into a nanoslit, confining molecules in an array of cavities embedded in the slit. We use this system to quantify the interactions of multiple confined DNA chains, a key problem in polymer physics with important implications for nanofluidic device performance and DNA partitioning/organization in bacteria and the eukaryotes. In particular, we focus on the problem of two-chain confinement, using differential staining of the chains to independently assess the chain conformation, determine the degree of partitioning/mixing in the cavities and assess coupled diffusion of the chain center-of-mass positions. We find that confinement of more than one chain in the cavity can have a drastic impact on the polymer dynamics and conformation.

Frequent exchange of the DNA polymerase during bacterial chromosome replication.

The replisome is a multiprotein machine that carries out DNA replication. In Escherichia coli, a single pair of replisomes is responsible for duplicating the entire 4.6 Mbp circular chromosome. In vitro studies of reconstituted E. coli replisomes have attributed this remarkable processivity to the high stability of the replisome once assembled on DNA. By examining replisomes in live E. coli with fluorescence microscopy, we found that the Pol III* subassembly frequently disengages from the replisome during DNA synthesis and exchanges with free copies from solution. In contrast, the DnaB helicase associates stably with the replication fork, providing the molecular basis for how the E. coli replisome can maintain high processivity and yet possess the flexibility to bypass obstructions in template DNA. Our data challenges the widely-accepted semi-discontinuous model of chromosomal replication, instead supporting a fully discontinuous mechanism in which synthesis of both leading and lagging strands is frequently interrupted.

A Replisome's journey through the bacterial chromosome.

Genome duplication requires the coordinated activity of a multi-component machine, the replisome. In contrast to the background of metabolic diversity across the bacterial domain, the composition and architecture of the bacterial replisome seem to have suffered few changes during evolution. This immutability underlines the replisome's efficiency in copying the genome. It also highlights the success of various strategies inherent to the replisome for responding to stress and avoiding problems during critical stages of DNA synthesis. Here we summarize current understanding of bacterial replisome architecture and highlight the known variations in different bacterial taxa. We then look at the mechanisms in place to ensure that the bacterial replisome is assembled appropriately on DNA, kept together during elongation, and disassembled upon termination. We put forward the idea that the architecture of the replisome may be more flexible that previously thought and speculate on elements of the replisome that maintain its stability to ensure a safe journey from origin to terminus.